View LEDGF Binding Pocket with MB36-3 Bound

FUNCTIONALIZATION OF NITROGEN-CONTAINING HETEROCYCLES IN THE

SYNTHESIS OF BIOLOGICALLY ACTIVE MOLECULES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University

Pratiq Akshay Patel

Graduate Program in Chemistry

The Ohio State University

2013

Dissertation Committee:

Professor James R. Fuchs, Advisor

Professor Craig J. Forsyth, co-Advisor

Professor Jonathan R. Parquette

Pratiq Akshay Patel

2013

Abstract

Nitrogen-containing heterocycles are commonly seen both in the pharmacophores of drug molecules and also in the cores of natural products. This dissertation explores the functionalization of nitrogen-containing heteroaromatic rings to access the highly substituted scaffolds of bioactive molecules within two distinct projects. Part I features medicinal chemistry efforts in the synthesis and derivatization of functionalized quinolines, indoles, pyridines, and pyrroles as novel HIV allosteric integrase inhibitors.

Alternatively, part II involves synthetic efforts towards the indole subunit of the bioactive natural product, sespendole.

HIV integrase (IN) plays a crucial role in the replication of the HIV virus as it catalyzes the transfer of the viral genetic code into the host genome. Raltegravir (Merck

& Co) is the first FDA drug targeting this strand transfer catalytic activity; however, rapid viral mutation has since led to drug-resistance and a renewed interest in developing new integrase inhibitors. In this regard, the host enzyme lens-epithelium derived growth factor (LEDGF)-integrase interaction served as new targets for generating novel integrase inhibitors. Recently, independent studies by other groups resulted in the identification of

2-(quinolin-3-yl)acetic acid derivatives as allosteric integrase inhibitors (ALLINIs).

Development of synthetic routes to these inhibitors and a series of structural analogues have facilitated elucidation of the novel multifunctional mechanism of action for this

ii class of compounds. Additionally, the design of novel scaffolds as ALLINIs was investigated through a scaffold hopping technique, the derivatization of leads from an in silico screen, and a fragment-based drug design approach. These drug discovery methods have resulted in the syntheses of a structurally diverse class of compounds that have provided an insight on the structural and functional requirements of the binding pocket.

Sespendole, an indolosesquiterpene alkaloid isolated from the fungus

Pseudobotrytis terrestris FKA-25, was reported as a potent inhibitor of lipid droplet synthesis. The proposed biosynthesis of sespendole, which involves a carbocation induced cyclization/rearrangement cascade, initially intrigued our group and spurred our interest in the synthesis of this complex molecule. This dissertation focuses on the synthesis of the highly substituted indole subunit of sespendole. Direct functionalization of the indole nucleus at the C4 and C5 positions via application of Bartoli’s Grignard addition and halogen-metal exchange chemistry resulted in the successful synthesis of the fully substituted indole subunit of sespendole.

iii

Dedication

To my grandparents and parents for all their love and support.

Acknowledgments

This long and arduous journey would not be accomplished without the contributions from several people, both personally and professionally. I would first like to thank my family for their constant support and encouragement. My mother has been an inspiration in my pursuit for higher education and I can only hope to live up to her accomplishments in life. My father has been there by my side whenever help was needed.

I thank my sister for sharing an interest in organic chemistry. This journey would not have been possible without my friends who made my life as a graduate student enjoyable.

I have been fortunate to work alongside colleagues who share some of the same passion towards chemistry. I would like to acknowledge my coworkers in the Fuchs lab, especially Dr. Nivedita Jena, Eric Schwartz, and John Woodard. I would like to express my gratitude to Dr. Nivedita Jena for her generosity, advice, and guidance towards becoming a better chemist; by far one of the best lab mates to work with. Eric has made the experience enjoyable with the numerous practical jokes, food runs (especially, when it’s free), and “borrowing” Milo. A special thanks to John Woodard, who has made enduring the long hours and countless failures in lab more bearable through discussions about chemistry, suggestions to “culture” me, and taking breaks outside of lab. John has become a really close friend over the years and I wish him the very best in the future.

This work would also not be possible without the research collaborations. Dr.

Mamuka Kvaratskhelia’s enthusiasm towards HIV research has pushed us to the forefront of HIV integrase research. I would also like to thank Dr. Jacques Kessl, Dr. Lei Feng, and

Alison Slaughter, who have been invaluable to the success of the project. A special thanks to Ben Naman in regards to the numerous things he has helped me with as well as the fun times spent outside lab.

I would like to acknowledge the support of the faculty and staff in the Department of Chemistry and the Division of Medicinal Chemistry who have helped me accomplish valuable research in the Fuchs lab; to that end, Drs. Pui-Kai Li and Robert S. Coleman for making it possible for me pursue valuable research as a chemistry graduate student at the College of Pharmacy. I would like to thank Professor Jon R. Parquette for serving on my dissertation committee and a special thanks to Professor Craig J. Forsyth for serving not only on my dissertation committee, but also as my co-advisor during the last year.

Lastly, I could not have asked for a better advisor than Professor James R. Fuchs.

His enormous help and direction to this day with valuable suggestions, intellectual discussions, and relentless passion towards organic chemistry has guided me through graduate school and inspired me in the lab. I am thankful to him for providing a wonderful environment for building a strong foundation in organic chemistry and developing the skills needed for what I hope will be a successful professional career.

Vita

2007...... B.S. Chemistry, Georgia Institute of Technology

2007 – 2013...... Graduate Teaching and Graduate Research Associate,

Department of Chemistry, The Ohio State University

Publications

1. Wang, H.; Jurado, K. A.; Wu, X.; Shun, M.-C.; Li, X.; Ferris, A. L.; Smith, S. J.;

Patel, P. A.; Fuchs, J. R.; Cherepanov, P.; Kvaratskhelia, M.; Hughes, S. H.;

Engelman, A. HRP2 Determines the Efficiency and Specificity of HIV-1 Integration

in LEDGF/p75 Knockout Cells but Does Not Contribute to the Antiviral Activity of a

Potent LEDGF/p75-binding Site Integrase Inhibitor. Nucleic Acids Res. 2012, 40,

11518–11530.

2. Jurado, K. A.; Wang, H.; Slaughter, A.; Feng, L.; Kessl, J. J.; Koh, Y.; Wang, W.;

Ballandras-Colas, A.; Patel, P. A.; Fuchs, J. R.; Kvaratskhelia, M.; Engelman, A.

Allosteric Integrase Inhibitor Potency Is Determined through the Inhibition of HIV-1

Particle Maturation. Proc. Natl. Acad. Sci. USA 2013, 110, 8690–8695.

vii

3. Feng, L.; Sharma, A.; Slaughter, A.; Jena, N.; Koh, Y.; Shkriabai, N.; Larue, R. C.;

Patel, P. A.; Mitsuya, H.; Kessl, J. J.; Engelman, A.; Fuchs, J. R.; Kvaratskhelia, M.

The A128T Resistance Mutation Reveals Aberrant Protein Multimerization as the

Primary Mechanism of Action of Allosteric HIV-1 Integrase Inhibitors. J. Biol.

Chem. 2013, 288, 15813–15820.

Fields of Study

Major Field: Chemistry

viii

Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vii

List of Tables ...... xiii

List of Figures ...... xiv

List of Schemes ...... xvii

Part I: Design, development, and mechanistic studies of allosteric HIV integrase inhibitors ...... 1

Chapter 1: Allosteric inhibitors of HIV-1 Integrase activity ...... 2

1.1 Active site strand transfer integrase inhibitors ...... 4

1.2 Targeting the allosteric site of integrase ...... 7

1.3 IN multimerization inhibitors ...... 11

1.4 LEDGF/p75-IN interaction inhibitors...... 13

Chapter 2: Allosteric Inhibitors of HIV Integrase ...... 20

2.1 Synthesis and elucidation of the inhibitory mechanism of known inhibitors ...... 21 ix

A. Synthesis and mechanism of inhibition with LEDGIN-6 and BI-B ...... 21

B. Synthesis and biological activity of (±)-BI-D ...... 25

C. Synthesis and mechanism of ALLINIs in HIV-1 IN A128T mutant strain ..... 29

2.2 Modification of the 2-(quinolin-3-yl)acetic acid series ...... 33

A. Scaffold hopping approach ...... 33

B. Pyridine analogues as mechanism-based selective inhibitors ...... 39

2.3 Alternate methods for the development of novel scaffolds ...... 43

A. In silico screening ...... 43

B. Fragment-based drug discovery ...... 49

2.4 Conclusion ...... 59

Part II: Synthesis of the highly substituted indole subunit of sespendole ...... 61

Chapter 3: Sespendole and Related Indoloterpenes ...... 62

3.1 Isolation and identification ...... 63

3.2 Biological activity ...... 66

3.3 Biosynthesis ...... 70

3.4 Previous approaches to indoloterpene natural products ...... 74

A. Smith’s approaches to indoloditerpenes ...... 76

B. Kuwahara’s approach to indolosesquiterpene, lecanindole D ...... 83

C. Kerr’s approach to the indole subunit of lolicines and lolitrems ...... 85

D. Nishikawa’s approach to indolosesquiterpene, sespendole ...... 88

Chapter 4: Synthesis of the Highly Substituted Indole Subunit of Sespendole...... 94

4.1 Biosynthetic inspired approach to sespendole ...... 94

4.2 Initial approach to the 4,5-disubstituted indole subunit ...... 98

4.3 Revised approach to the 4,5-disubstituted indole subunit ...... 101

A. Method development for introduction of the C4 and C5 substituents ...... 103

B. Bartoli Grignard addition of allyl/prenyl substituent ...... 105

C. Alternative strategies attempted for installation of C4 substituent ...... 107

4.5 2,3-Cycloalkyl-fused indole as a structural mimic ...... 108

A. Fischer indole synthesis ...... 109

B. Extension of the C4 methyl group to the prenyl substituent ...... 112

C. Elaboration of the C5 substituent ...... 113

D. Optimization of halogen-metal/acylation chemistry with molecular sieves .. 115

E. Unanticipated rearrangement ...... 118

F. Attenuation of indole electron density with tosyl group ...... 121

4.6 Completion of the synthesis of the fully substituted indole subunit ...... 122

A. Directed epoxidation of allylic alcohol ...... 122

B. Comparative NMR spectral analysis of the fully substituted indole subunit . 126

4.7 Conclusion ...... 132

Chapter 5: Experimental Section ...... 133

Part I: Design of allosteric HIV integrase inhibitors ...... 135

Part II: Synthesis of the highly substituted indole subunit of sespendole...... 181

References ...... 209

Appendix: NMR Spectra of Selected Compounds ...... 218

xii

List of Tables

Table 2.1. Biological activity of LEDGIN-6 and BI-B ...... 25

Table 2.2. Antiviral activity (IC50) in WT and knockout cells ...... 28

Table 2.3. Effects of A128T mutation on the biological activities of ALLINIs ...... 31

Table 2.4. LEDGF dependent integration activity of indole analogues ...... 39

Table 2.5. Inhibitory activities of BI-B, PY-1, and PB-20 ...... 42

Table 2.6. LEDGF-IN binding activity from virtual screening of drug-like compounds . 45

Table 3.1. Microbial inhibitors against lipid droplet accumulation ...... 68

Table 4.1. Optimization for methyl Grignard addition into 5-nitroindoles ...... 104

Table 4.2. Grignard addition of allyl group into 5-nitroindoles ...... 106

Table 4.3. Bartoli Grignard addition into 5-nitrotetrahydrocarbazole ...... 112

Table 4.4. Reaction conditions for halogen-metal exchange/acylation ...... 116

xiii

List of Figures

Figure 1.1. HIV-1 replication cycle in CD4+ T cells ...... 3

Figure 1.2. Integration process starting from viral RNA ...... 4

Figure 1.3. IN inhibitors for treatment of HIV ...... 6

Figure 1.4. Key metal-chelating motifs on INSTIs...... 7

Figure 1.5. Preintegration complex (PIC) with LEDGF ...... 8

Figure 1.6. Key interactions between LEDGF-IBD and IN-CCD ...... 10

Figure 1.7. Design of heavy atom-based small molecules targeting IN-CCD...... 12

Figure 1.8. Crystal structure of heavy atom-based small molecules targeting IN-CCD ...12

Figure 1.9. Acetylated bis-caffeoyl IN inhibitors ...... 13

Figure 1.10. Scaffold hopping approach to identify INSTIs and LEDGF inhibitors...... 14

Figure 1.11. Fragment screening for LEDGF inhibitors...... 15

Figure 1.12. Pharmocophore model highlighting the chemical features involved in

interactions between LEDGF-IBD and IN-CCD ...... 16

Figure 1.13. Pharmacophore-based design of small molecule integrase inhibitors ...... 17

Figure 1.14. Design of 2-(quinolin-3-yl)acetic acid inhibitor ...... 18

Figure 2.1. Known 2-(quinolin-3-yl)acetic acid derivatives ...... 21

Figure 2.2. Overlay of LEDGIN-6 and BI-B with key interactions with IN ...... 24

Figure 2.3. Systematic modification of BI-B yielded a more potent inhibitor, BI-D ...... 26

xiv

Figure 2.4. IN-CCD bound to BI-D ...... 28

Figure 2.5. Effect of BI-D on core morphology of viruses ...... 29

Figure 2.6. Overlay of BIB-II in WT and A128T ...... 32

Figure 2.7. Predicted binding of proposed indole analogue ...... 34

Figure 2.8. List of indole analogues synthesized and tested ...... 37

Figure 2.9. Indole analogue bound to allosteric site of HIV integrase ...... 38

Figure 2.10. Images of PY-1 and PB-20 binding to IN-CCD ...... 43

Figure 2.11. Flow chart showing virtual screening work flow ...... 44

Figure 2.12. Synthetic library of B22, B34, and B22 analogues ...... 46

Figure 2.13. Comparative synthetized and purchased NMR spectra of B22 and B34 ...... 48

Figure 2.14. Percent inhibition of MB36-3 and other fragments (Kvaratskhelia lab) ...... 50

Figure 2.15. Structure of MB36-3 and two active fragments ...... 51

Figure 2.16. Overlay of MB36-3 with BI-B within the LEDGF binding pocket ...... 52

Figure 2.17. View LEDGF binding pocket with MB36-3 bound ...... 53

Figure 2.18. Classification of synthesized MB59-1 analogues...... 57

Figure 2.19. Biological activity of MB59-1 analogues ...... 58

Figure 3.1. Structure of sespendole, the first reported indolosesquiterpene ...... 63

Figure 3.2. Indoloditerpenes with a similar indoloterpene framework as sespendole ...... 64

Figure 3.3. Lecanindoles A-D, structurally related fungal indolosesquiterpenes ...... 66

Figure 3.4. Effect of sespendole concentration on lipid droplet size and number ...... 67

Figure 3.5. Fungal metabolites that inhibit lipid droplet accumulation ...... 68

Figure 3.6. Lipid droplets...... 70

Figure 3.7. Comparative NMR analysis for difference in chemical shift between both

synthesized diastereomers of the indole subunit and isolated sespendole ....93

Figure 4.1. LUMO map of 5-nitroindole shows preference for nucleophilic attack ...... 102

Figure 4.2. NMR spectra of the halogen-metal exchange/acylation reaction ...... 118

Figure 4.3. Predicted dihedral angles in the stereoselective epoxidation of acyclic

allylic alcohols ...... 124

Figure 4.4. Origin of stereoselectivity for the directed epoxidation with mCPBA ...... 125

Figure 4.5. NOE correlation of N-tosylindole subunit 4.110 measured in CDCl3...... 127

Figure 4.6. Structures of the synthetic molecules and natural product, sespendole ...... 128

Figure 4.7. 1H NMR comparison between compound 4.113 and the reported syn and

anti diastereomers in C5D5N...... 129

1 Figure 4.8. H NMR (400 MHz, CDCl3) comparison between sespendole (4.1) and

compound 4.113 ...... 130

Figure 4.9. Difference in 1H and 13C NMR chemical shifts between sespendole and all

synthetic compounds ...... 131

xvi

List of Schemes

Scheme 2.1. Synthesis of LEDGIN-6 and BI-B ...... 23

Scheme 2.2. Synthetic route for the preparation of (±)-BI-D ...... 27

Scheme 2.3. Design and synthesis of BIB-II ...... 30

Scheme 2.4. Synthetic approaches for the series of indole analogues ...... 36

Scheme 2.5. Synthesis of a common intermediate for the pyridine analogues ...... 40

Scheme 2.6. Synthesis of pyridine analogues PY-1 and PB-20 ...... 41

Scheme 2.7. Synthesis of B22 and related alkyl analogues ...... 46

Scheme 2.8. Synthesis of B34...... 47

Scheme 2.9. Synthesis of MB59-1 and 2- and 3-substituted pyrroles ...... 55

Scheme 2.10. Synthesis of open-chain analogues ...... 56

Scheme 3.1. Proposed biosynthesis of indolosesquiterpene skeleton...... 71

Scheme 3.2. Tryptophan biosynthetic pathway towards indoloditerpenes ...... 72

Scheme 3.3. Overview of the proposed biosynthesis of sespendole ...... 73

Scheme 3.4. Common strategy for the synthesis of indoloterpenes ...... 75

Scheme 3.5. Common advance intermediate for synthesis of the paspalane family ...... 76

Scheme 3.6. Gassman indole synthesis, used for the paspalanes ...... 77

Scheme 3.7. Retrosynthesis of common advanced ketone intermediate ...... 78

Scheme 3.8. Nolen-Sprengeler lactone ...... 79

xvii

Scheme 3.9. Madelung indole synthesis and Smith’s modification ...... 80

Scheme 3.10. Retrosynthesis of Nolen-Sprengeler lactone ...... 81

Scheme 3.11. Smith’s retrosynthetic strategy for Penitrem D ...... 81

Scheme 3.12. Smith’s retrosynthesis of prefunctionalized aniline ...... 82

Scheme 3.13. Paspalinine and lecanindole D from a common intermediate ...... 84

Scheme 3.14. Kuwahara’s retrosynthetic strategy for the sesquiterpene subunit ...... 85

Scheme 3.15. Plieninger’s indolization sequence to 4-substituted indoles ...... 86

Scheme 3.16. Kerr’s methodology for generating 4,5-disubstituted indoles ...... 87

Scheme 3.17. Kerr’s retrosynthesis towards lolicine western half ...... 88

Scheme 3.18. Nishikawa’s synthetic strategy for sespendole ...... 89

Scheme 3.19. Nishikawa’s retrosynthesis of the sesquiterpene segment ...... 90

Scheme 3.20. Nishikawa’s retrosynthesis of the indole subunit of sespendole ...... 90

Scheme 3.21. Synthesis of both diastereomers of the indole subunit of sespendole ...... 91

Scheme 4.1. Biomimetic retrosynthetic plan ...... 95

Scheme 4.2. Attempt at trapping the carbocation as spirocyclopropylindolenines ...... 97

Scheme 4.3. Enzymatic synthesis of indoloditerpene with cyclization at C4 ...... 97

Scheme 4.4. Initial retrosynthesis for installation of C4,C5 substituents ...... 98

Scheme 4.5. Bartoli alkyl Grignard addition into nitroarenes ...... 99

Scheme 4.6. Initial synthetic scheme towards the 4,5-disubstituted indole subunit ...... 101

Scheme 4.7. Bartoli’s reaction for Grignard addition into 5-nitroindole ...... 102

Scheme 4.8. Revised strategy for the highly substituted indole subunit of sespendole .. 103

Scheme 4.9. Functionalization at C5 in the model system ...... 105

xviii

Scheme 4.10. Attempted C4-bromination based on previous reported methods ...... 107

Scheme 4.11. Attempted directed ortho-lithiation method ...... 108

Scheme 4.12. Approach for 2,3-cycloalkyl-fused 5-nitroindole ...... 109

Scheme 4.13. Fischer Indole synthesis to make 6 and 5 cycloalkyl fused indoles ...... 110

Scheme 4.14. Extension of methyl group into the prenyl chain ...... 113

Scheme 4.15. Functionalization at C5 for attempting halogen-metal/acylation ...... 114

Scheme 4.16. Other potential approaches to installation of C5 substituent ...... 115

Scheme 4.17. Functionalization at C5 to iodide needed for halogen-metal exchange ... 115

Scheme 4.18. Unanticipated rearrangement of Grignard addition reaction ...... 120

Scheme 4.19. Synthesis of the highly substituted indole subunit in sespendole ...... 122

Scheme 4.20. Stereoselective epoxidation of 4-methylpent-3-en-2-ol ...... 123

Scheme 4.21. Synthesis of the epoxyalcohol moiety ...... 126

Scheme 4.22. Completion of the highly substituted indole subunit of sespendole ...... 127

xix

Part I:

Design, development, and mechanistic studies of allosteric HIV integrase inhibitors

Chapter 1: Allosteric inhibitors of HIV-1 Integrase activity

Human immunodeficiency virus type 1 (HIV-1) is the major strain of lentivirus, which in the majority of cases, if left unchecked, infects CD4+ T cells and destroys the host immune system resulting in acquired immunodeficiency syndrome (AIDS).1

Currently, more than 30 million people worldwide live with AIDS through infection by

HIV-1 or the less prevalent type, HIV-2.2 People with HIV-2 are also susceptible to opportunistic infections, but in comparison to HIV-1, HIV-2 is characterized by lower transmissibility and slower progression to immunodeficiency.3

HIV-1 can persistently infect humans by sabotaging the adaptive immune system through a series of steps despite having a small genome.2 As illustrated in Figure 1.1, viral replication initiates with entry into the cell through interaction with a cell surface receptor and ends with particle maturation into an infectious virion. The complex replication cycle exploits various cellular factors with the aid of key retroviral enzymes such as reverse transcriptase, integrase, and protease. Over the past 15 years, however, the emergence of highly active retroviral therapy (HAART) has controlled HIV-1 replication, primarily through targeting viral reverse transcriptase and protease enzymes.

Despite the success of these therapies, they eventually become susceptible to drug- resistant viral strains or lead to complications arising from compound toxicity.1

Therefore, there is continued interest in the design and development of novel compounds

2 that target unexploited enzymes involved in the viral lifecycle. Furthermore, compounds with potential therapeutic value would facilitate the structural and mechanistic studies of the biological target in order to elucidate the mechanisms of action of the novel therapeutics.

Figure 1.1. Schematic representation of the HIV-1 replication cycle in CD4+ T cells.

Accordingly, targeting the previously understudied integration process through inhibition of the viral enzyme, HIV-1 integrase, was explored. Integrase (IN) plays a crucial role in the HIV-1 replication cycle as it is responsible for the integration of viral double-stranded complimentary DNA (cDNA) into host chromosomal DNA (Figure 1.2).

Mechanistically, integration occurs at the central catalytic core domain in two distinct processes within the preintegration complex (PIC): IN first cleaves two nucleotides from the 3’ ends of cDNA, termed 3’ processing, then catalyzes the strand transfer of the newly processed 3’ ends of cDNA into the host chromosomal DNA. The highly conserved active site residues aspartates 64 and 116 (D64, D116) as well as glutamate

152 (E152) coordinate to divalent magnesium cofactors required for the 3’ processing and strand transfer reactions.4 Exploiting this magnesium binding architecture of the IN active site has been recognized as crucial for the inhibition of IN and its catalytic processes in order to stop HIV replication.

Figure 1.2. Schematic overview of the integration process starting from viral RNA

1.1 Active site strand transfer integrase inhibitors

Equisetin (1.1) (Figure 1.3), a natural product isolated from Fusarium heterosporum, was reported by Merck as one of the first inhibitors of IN.5 The compound inhibited in vitro 3’ processing and strand transfer reactions with low micromolar

4 concentrations (IC50 = 7-15 μM and 15-20 μM, respectively). Additional screening of natural product extracts for IN inhibitory activity led to the discovery of chemically diverse natural product inhibitors, many of which possessed a -hydroxyketo group.6 A crystal structure of IN bound to 5CITEP (1.2) provided structural evidence to this group for-hydroxyketo group binding interactions in the IN active site.7 These interactions were thought to mimic viral cDNA interactions with IN during the 3’ processing reaction.

This key structural moiety, in regards to its proposed mechanism of inhibition, has been the focus for design of HIV integrase inhibitors.8

A few of these types of compounds containing similar binding units have since been further developed and approved by FDA (Figure 1.3). For example, in 2007, Merck obtained a license for the first IN inhibitor, raltegravir (RAL), which targets the strand transfer catalytic activity of IN. In addition, elvitegravir (EVG), developed by Gilead

Sciences, was recently approved by the FDA in 2012. These integrase strand transfer inhibitors (INSTIs) bind to the IN active site by displacing the reactive 3’-hydroxyl group, the product of 3’ processing, through chelation of the Mg2+ cations required for viral DNA catalysis (Figure 1.4).9 However, the emergence of HIV-1 resistant strains in patients treated with RAL and EVG, primarily containing glutamine-148 to histidine/arginine (Q148H/R) and asparagine-155 to histidine (N155H) mutations near the Mg2+ coordination site, has prompted a renewed interest in the identification and development of novel IN inhibitors.10,11 Consequently, GlaxoSmithKline licensed a novel

INSTI, dolutegravir (DTG), which is effective in patients with RAL-resistance. The significantly slower disassociation of DTG compared to RAL and EVG from IN was

5 proposed to be responsible for DTG’s effectiveness in wild type and RAL- and EVG- resistant HIV-1 IN strains.12 Although no primary INSTI resistance mutations in the clinic have yet been reported for DTG, drug design efforts towards targeting IN with a distinctive binding interaction not involving the active site should also provide an alternate mechanism for overcoming INSTI resistant strains.

Figure 1.3. First IN inhibitor, equisetin, served as a model for development of current

FDA approved IN inhibitors for treatment of HIV

Figure 1.4. Key metal-chelating motifs (red) on INSTIs that coordinate to both divalent

Mg cations in the IN active site to exhibit their inhibitory effects

1.2 Targeting the allosteric site of integrase

Research over the past decade has provided insight into the roles of virus-host cellular protein interactions involved in the integration process. Accordingly, integration is assisted by a large nucleoprotein pre-integration complex (PIC), which consists of viral cDNA bound to HIV-1 IN along with various cellular proteins such as the small DNA- binding protein Barrier-to-Autointegration Factor (BAF).13 However, the integration process in human cells is largely dictated by the interaction of IN and the principal cellular transcriptional coactivator, lens epithelium-derived growth factor (LEDGF/p75).

A study by Llano et al. has shown that LEDGF/p75 is essential not only for the integration process, but also the viral replication cycle.14 LEDGF/p75 associates with a stable IN tetramer and assists in the nuclear translocation of PIC and its tethering to host

7 chromosomal DNA for integration13 (Figure 1.5). These processes are facilitated through the N-terminal PWWP (proline-tryptophan-tryptophan-proline) chromatin binding domain and the C-terminal integrase binding domain (IBD) on LEDGF/p75.15 The

PWWP domain assists in tethering of PIC to chromatin, while the IBD interacts with the allosteric catalytic core domain (CCD) on IN. Furthermore, LEDGF/p75 is one of six members of the hepatoma-derived growth factor (HDGF) related-protein (HRP) family.

HRP2 is the only other member from this family that contains an IBD, and along with

LEDGF, independently stimulated recombinant HIV-1 IN activity in vitro.

Figure 1.5. Schematic representation of the preintegration complex (PIC) with LEDGF.

HIV-1 IN consists of three domains: the N-terminal domain, a C-terminal domain, and the catalytic core domain (CCD). The highly dynamic protein functions as a tetramer in the presence of viral DNA, with each dimer engaging the cDNA ends. Moreover,

LEDGF/p75 stabilizes this tetrameric form and interacts with IN at a cleft in the CCD dimer interface.17,18 Overexpression of the peptide sequence of IBD, which lacks the N- terminal chromatin binding domain, was shown to tightly bind to IN at the CCD and consequently inhibit HIV-1 replication in cell culture assays.17 In the absence of

LEDGF/p75, IBD shows significant increase in suppressing HIV-1 replication, which suggests that IBD effectively engages with IN and adversely affects its integration function. Furthermore, IBD also affects HIV-1 strains that are resistant to INSTIs, which confirms a different mechanism of inhibition. In addition, analysis of the co-crystal structure of IBD bound to HIV-1 IN CCD revealed the corresponding LEDGF/p75 amino acids that interact with the IN CCD subunits at the dimer interface. As illustrated in

Figure 1.6, the main interaction is the bidentate hydrogen bond between aspartate-366

(D366) on the LEDGF/p75 IBD and glutamate-170 (E170) and histidine-171 (H171) residues on one IN subunit. In addition, isoleucine-365 (I365) of IBD resides in a hydrophobic pocket containing residues leucine-102 (L102), alanine-128 (A128), and tryptophan-132 (W132) on the second subunit of the IN-CCD.

Figure 1.6. View of the key interactions between LEDGF-IBD and IN-CCD. Image

courtesy of Dr. Lei Feng (PDB 2B4J)

Further investigation by Kvaratskhelia and coworkers revealed that LEDGF stabilized additional interactions within IN and promoted its tetramerization.18 This tetrameric form is important for IN catalytic activities but fails to catalyze concerted integration in vitro. Moreover, IBD was reported to be significantly more effective at inhibiting HIV-1 replication in cells in the absence of LEDGF as compared to in the presence of LEDGF. However, the observed activity is not fully explained by the competition of IBD and LEDGF for the IN-CCD binding site. Accordingly, the authors concluded that restricting the flexibility of IN by stabilizing its multimeric form should also impair IN activity.

Overall, these studies indicate the importance of LEDGF with IN for the survival of HIV and thereby suggest a potential novel mechanism for allosteric inhibition of IN by targeting IN-CCD binding site. Therefore, development of small molecules that exploit

10 the IN-CCD to affect IN multimerization and/or disrupt LEDGF-IN interaction represents a potential avenue in the generation of a new class of antiviral drugs.

1.3 IN multimerization inhibitors

Based on the previously mentioned structural and mechanistic details of LEDGF-

IN interaction involving the dynamic interplay between IN subunits, its disruption in the assembly of the fully functional nucleoprotein complex, PIC, has gained interest for the design of IN inhibitors. Accordingly, several independent studies using chemically diverse compounds have identified inhibitor binding regions at the IN CCD dimer interface.

An X-ray crystallographic study of heavy atom small molecule-integrase complexes identified a binding site for potential IN inhibitors (Figure 1.7).19 Soaking of

IN-CCD in the presence of tetraphenylarsonium bromide (1.6) resulted in binding of the compound to the IN-CCD dimer interface (Figure 1.8). The X-ray crystallographic analysis revealed that compound 1.6 was bound to the protein through a charge-charge interaction between the carbonyl oxygen of glutamine-168 (Q168) and the electron deficient arsenic center, while one of the phenyl rings displayed π-stacking interactions with tryptophan-131 (W131) and tryptophan-132 (W132). While these compound displayed only moderate activity for affecting disintegration (IC50 > 200 µM), this study provided the first direct evidence that small molecules could effectively bind to the IN dimer interface at a site that overlaps the IBD binding pocket and affect integration.

Figure 1.7. Design of heavy atom-based small molecules targeting IN-CCD

Figure 1.8. Crystal structure showing that compound 1.6 sits between the IN dimer

interface (PDB 1HYV) (left). View of the inhibitor 1.6 bound to IN-CCD through

interaction with Gln168 (left). Images generated using UCSF Chimera20

An alternative approach to targeting the integration process has been explored using D-chicoric acid (1.9) and its derivatives (Figure 1.9).21 Tandem affinity acetylation and mass spectrometry identified the modification of lysine-173 (K173) residue with derivative 1.11.22 This compound was subjected to further studies by Kvaratskhelia and coworkers in order to further investigate its mechanism of action.23 A docking model showed that this compound was bound to a site adjacent to the LEDGF binding site at the

IN-CCD dimer interface. It also displayed extensive hydrogen bonding interactions with 12 both IN subunits of the dimer. Biochemical analysis revealed that these compounds could effectively allosterically modulate the dynamic interaction between individual IN subunits. These results further supported formation of higher order IN multimers as an allosteric mechanism for inhibiting HIV-1 IN.

Figure 1.9. Acetylated bis-caffeoyl IN inhibitors derived from chicoric acid (left).

Molecular docking of compound 1.11 with key hydrogen bonding interactions (right).

Reprinted with permission from American Society for Pharmacology and Experimental

1.4 LEDGF/p75-IN interaction inhibitors

Several independent drug discovery methods have been utilized in identifying small molecules that inhibit the LEDGF/p75-IN binding interactions. Of importance are methods that have included scaffold merging, fragment screening, and pharmacophores- based design approaches for targeting this key protein-protein interaction.

The first approach takes advantage of the known metal-chelating motifs of the β- hydroxyketo group from salicylic acid (1.12). The generation of novel scaffolds by merging the pharmacophores of salicylate and catechol (1.13) groups was predicted to provide novel dual action inhibitors against INSTI-specific resistant strains.24 This scaffold merging approach was utilized to identify an active scaffold 1.14 that was shown to not only affect IN strand transfer activity, but also disrupt binding of LEDGF/p75; the substituent on the scaffold dictated the mode of action (Figure 1.10). Accordingly, compound 1.15 was identified as an INSTI (IC50 = 5 µM) and was proposed to chelate the Mg2+ cations in the active site. On the other hand, compound 1.16 inhibited LEDGF-

IN interaction (IC50 = 8 µM) with a proposed binding mode in the LEDGF binding pocket of HIV IN.

Figure 1.10. Scaffold merging to identify strand transfer and LEDGF-IN inhibitors

In an alternate approach, a fragment-based screening approach using surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) with HIV-1 IN-CCD has led to the identification of a number of compounds.25 Soaking of several of the initial hit compounds with IN-CCD provided evidence that these compounds bound to the

LEDGF binding site of IN-CCD. Further analysis of the binding mode with compounds

1.17 and 1.18 revealed key hydrogen bonding interactions between the protein backbone at glutamate-170 (E170) and histidine-171 (H171) (Figure 1.11). Biochemical analysis demonstrated that the compounds inhibited the LEDGF-IN interaction in sub-millimolar concentrations. This relatively weak activity is common in fragment-based approaches and could be subsequently improved through optimization of the fragment hits.

Figure 1.11. Fragment screening result confirmed by X-ray crystallography: Overlay of

lactone 1.17 (PDB 3ZT4 - green) and acid 1.18 (PDB 3ZT2 - cyan)25

Drug discovery using a structure-based pharmacophore model was developed from the previously established co-crystal structure of LEDGF-IBD and IN CCD.26 The chemical features highlighted in the interactions between LEDGF-IBD residues isoleucine-365 (I635) and aspartate-366 (D366) with IN were used to generate the pharmacophore model. As shown in Figure 1.12, the obtained model contained two

15 hydrophobic groups (light yellow), one H-bond donor (green arrow), two H-bond acceptors (red arrow), and nine excluded volumes (gray).

Figure 1.12. Computational-based pharmocophore model highlighting the chemical

features involved in interactions between LEDGF-IBD and IN-CCD. Reprinted with

Using this model, an in silico screen was carried out to look for molecules which could affect inhibition of the LEDGF-IN binding interaction. This study ultimately identified the indole-based CHIBA-3002 (1.19) as a lead compound.26 Optimization of compound 1.19 led to CHIBA-3003 (1.20), which lacked the N-benzyl substituent and contained a hydroxyl group at the C4 position of the indole ring (Figure 1.13). The

16 docking analysis of compound 1.20 revealed the binding mode to be similar to IBD-IN complex, albeit, with modest affinity for disrupting LEDGF-IN interactions. Surprisingly, however, in this case the key binding elements of the CHIBA compounds resemble known INSTIs, due to the presence of the C3 β-hydroxyketo acid moiety. Consequently, these compounds were developed as dual inhibitors for affecting IN function.27

Figure 1.13. Pharmacophore-based design of small molecule integrase inhibitors for targeting LEDGF-IN interaction (left). Computation docking showing predicted binding

interactions of CHIBA-3003 in the LEDGF/p75-IN pocket (right). Reprinted with

Similarly, additional screening of a larger library of compounds by Debyser and coworkers for inhibiting the LEDGF/p75-IN interaction using the pharmocophore model was used to discover LEDGIN-1 (1.21) (Figure 1.14), which affected 36% inhibition at

100 μM of the LEDGF/p75 interaction.28 Multiple rounds of structure-activity analysis through rational design identified a series of 2-(quinolin-3-yl)acetic acids as inhibitors 17 with increased potency. The most potent compound, LEDGIN-6 (1.22), was 10x more active against LEDGF/p75-IN interaction and displayed a 20x increase in antiretroviral activity as compared to compound 1.21. In conclusion, analysis of the crystal structure revealed that compound 1.22 binds at the IN CCD, while displaying similar interactions to LEDGF-IBD peptide in the binding cavity.

Figure 1.14. Design of 2-(quinolin-3-yl)acetic acid inhibitor derived from in silico

screening hit (left). Overlay of LEDGIN-6 and IBD-IN CCD (right). Reprinted with

Overall, the examples discussed above clearly show that exploiting the LEDGF binding pocket is critical to developing compounds which display promising antiviral activity. Further development of these compounds and exploration of their activities, however, are still needed. In the next chapter, therefore, we further explore the

18 mechanism of action of LEDGIN-6 and structurally related 2-(quinolin-3-yl)acetic acids independently identified at Boehringer Ingelheim.29 These compounds will ultimately set the stage for additional drug discovery efforts directed at the discovery of novel IN inhibitors.

Chapter 2: Allosteric Inhibitors of HIV Integrase

HIV integrase (IN) plays a crucial role in the replication of the HIV virus as it catalyzes the transfer of the viral genetic code into the host genome. As a result, HIV-1 integrase has become an important target for antiretroviral therapies. As mentioned in the previous chapter, raltegravir (RAL, Merck & Co, 2007) was the first FDA approved drug to target this strand transfer catalytic activity; however, rapid viral mutation has since led to drug-resistance and renewed interest in developing new integrase inhibitors. Since then, two other drugs, elvitegravir (EVG, Gilead Sciences, 2012) and dolutegravir (DTG,

GlaxoSmithKline, 2013), have been approved by the FDA which also target the catalytic site of integrase. There is a need, however, for the development of novel integrase inhibitors with alternate mechanisms of action in order to combat potential resistance mechanisms.

In this regard, the host enzyme lens-epithelium derived growth factor (LEDGF)- integrase interaction and integrase (IN) 3’ processing catalytic activity have served as new targets for generating novel integrase inhibitors. Recently, these targets resulted in the independent identification of 2-(quinolin-3-yl)acetic acid derivatives, LEDGIN-6

(2.1) by Christ and coworkers (designated as compound 628 and LEDGIN-630 in the literature) and BI-B (2.2) by Boehringer Ingelheim (referred to as BI-100130 and

ALLINI-131 in the literature), as inhibitors of LEDGF-IN interaction (Figure 2.1).

LEDGIN-6 was reported to be highly selective for disrupting LEDGF-IN interaction

(IC50 = 1.37 μM), while exhibiting potencies of 250 μM and 19.5 μM for 3’ processing and strand transfer, respectively.28 BI-B was discovered through a high throughput screening (HTS) for inhibiting integrase 3’ processing activity.32 Structurally, both inhibitors feature a halogen at C6, an aryl substituent at C4, and an acetic acid moiety attached to the quinoline ring system. Based on their structural similarity, the synthesis of these inhibitors was initiated in our lab in order to elucidate the mechanism of action of these inhibitors in a collaborative effort with the lab of Dr. Mamuka Kvaratskhelia.

Figure 2.1. Known 2-(quinolin-3-yl)acetic acid derivatives

2.1 Synthesis and elucidation of the inhibitory mechanism of known inhibitors

A. Synthesis and mechanism of inhibition with LEDGIN-6 and BI-B

The synthetic routes for these two compounds were adapted from the synthetic schemes reported for the preparation of LEDGIN-6 by Debyser and coworkers28 and BI-

B from the patent literature.32 Accordingly, the 2-(quinolin-3-yl)acetic acid inhibitors were constructed through a Friedländer quinoline synthesis from 2-aminobenzophenones

2.4 and 2.9, with introduction of the acetic acid chain from the corresponding ketone,

21 levulinic acid in the case of LEDGIN-6 and ethyl acetopyruvate for BI-B (Scheme 2.1).30

The quinoline synthesis proceeds from condensation of the ketone with the amino group, followed by an acid-catalyzed cyclocondensation to produce the substituted quinoline rings 2.5 and 2.10. However, a potential drawback to this transformation is the regioselectivity in the cyclocondensation reaction, which could result in a regioisomeric mixture of 2,3-substituted quinolines. This regioisomeric mixture was primarily observed with ethyl acetopyruvate in the formation of quinoline 2.10 towards the synthesis of BI-

B. Further functionalization introduced the requisite α-substitution on the acetic acid chain to provide n-propyl derivative 2.7 and methyl ether 2.12, which underwent ester hydrolysis to furnish LEDGIN-6 and BI-B, respectively. With these two inhibitors in hand, our collaborators were able to examine the effects of both of these compounds on the overall integration process in an effort to elucidate their mechanism of action.

Scheme 2.1. Synthesis of LEDGIN-6 and BI-B

To that end, co-crystal structures of LEDGIN-6 and BI-B bound at the integrase dimer interface were obtained (Figure 2.2A).30 This junction of the dimer was reported to be the allosteric site of integrase, which is shown to bind to LEDGF/p75.17 An overlay of the crystallographic model provides insight to the binding modes of the two inhibitors to the protein backbone in the allosteric site (Figure 2.2B). Not surprisingly, analysis of the crystal structures indicated that the carboxylic acid moiety plays a key role in anchoring

23 of the inhibitors through hydrogen bonding interactions with the hydroxyl side chain of threonine-174 (T174) and the amide backbones corresponding to histidine-171 (H171) and glutamate-170 (E170). Furthermore, BI-B shows an additional hydrogen bonding interaction of the α-methoxy group with threonine-174 (T174), which presumably accounts for the increased observed potency of this inhibitor over LEDGIN-6. The C4- aryl substituent was shown to protrude into a hydrophobic pocket capped by tryptophan-

132 (W132), which is located deeper in the dimer interface of integrase. These residues had previously been recognized as important players for the binding of allosteric inhibitors by Cherepanov and coworkers, who published the first pharmocophore model of binding to the LEDGF-IBD site.17

Figure 2.2. Integrase bound to BI-B (a) at the dimer interface. (b) Overlay of LEDGIN-6

(green) and BI-B (yellow) with key interactions with IN30

Since both LEDGIN-6 and BI-B were shown to have similar binding modes, these compounds were analyzed in parallel experiments to elucidate the mechanism of action.

A key consideration was whether or not these compounds could allosterically modulate the dynamic interplay between integrase subunits. The results from the study suggested that these compounds potently inhibit not only LEDGF-IN interaction, but also LEDGF- independent integrase catalytic function (Table 2.1).30 Moreover, these compounds also induce aberrant multimerization. Finally, both LEDGIN-6 and BI-B exhibit a novel multifunctional cooperative mechanism for their antiviral activity, and as a result, were termed allosteric integrase inhibitors (ALLINIs). These inhibitors, however, were not potent enough to render the virus non-infectious after single round HIV infection.

Table 2.1. Biological activity of LEDGIN-6 and BI-B30

B. Synthesis and biological activity of (±)-BI-D

A recent report from Boehringer Ingelheim indicated the importance of the ether substituent adjacent to the carboxylic acid with regard to antiviral potency.29 Instead of the α-methoxy group in BI-B, the new class of compounds contained more sterically demanding t-butyl ether groups, with several of these compounds showing low nanomolar antiviral activities. One of the most potent compounds, BI-D, displayed in

Figure 2.3, was shown to be approximately one hundred times more potent than BI-B in 25 an assay of antiviral activity (EC50 = 10 nM). As a result, this compound was synthesized in our lab to continue our studies on the mechanism of action of this class of compounds.

Figure 2.3. Systematic modification of BI-B yielded a more potent inhibitor, BI-D

(±)-BI-D (2.13) was synthesized in 10 linear steps from commercially available

4-hydroxy-2-methylquinoline 2.14 (Sigma-Aldrich) via modification of a synthetic route reported by Tsantrizos and colleagues at Boehringer Ingelheim.29 The synthesis commenced with preparation of 3-bromo-4-chloro-2-methylquinoline 2.15 through a selective bromination/ chlorination sequence of the quinoline ring system at the C3 and

C4 positions, respectively (Scheme 2.2).33 The aryl bromide 2.15 was then selectively functionalized via a Stille coupling with tributyl(vinyl)tin and subsequent Upjohn dihydroxylation to provide diol 2.17. Finkelstein-type substitution of the remaining chloride group with iodide, followed by selective protection of the primary alcohol of the diol as the pivalate provided 2.18. The secondary alcohol was then converted to the t- butyl ether in the presence of perchloric acid to provide intermediate 2.19. Introduction of the chroman group through Suzuki coupling of boronic acid 2.25 with iodide 2.19 provided the biaryl adduct 2.20. Finally, hydrolysis of the pivalate group of 2.21 and

26 subsequent oxidation of the primary alcohol to the corresponding carboxylic acid furnished compound 2.13, BI-D.

Scheme 2.2. Synthetic route for the preparation of (±)-BI-D

With BI-D in hand, co-crystallization with IN CCD confirmed its binding to the

LEDGF binding pocket, while displaying similar interactions as LEDGF-IBD (Figure

2.4).34 Evaluation of its mechanism revealed that BI-D not only displays potent antiviral activity through inhibition of LEDGF-IN interaction, but more significantly, exhibited a

10-fold increase in potency in LEDGF knock out cells (Table 2.2).33 In contrast, RAL showed a decrease in potency in LEDGF knock out cells. This data indicates that LEDGF

27 competes with BI-D for the allosteric site on integrase, resulting in a decreased potency in comparison to the absence of LEDGF. A similar effect is observed with another IBD- containing host factor, hepatoma-derived growth derived factor related protein-2 (HRP2).

Consequently, these results exclude the host factor-IN interaction as the primary antiviral target of these inhibitors.

Figure 2.4. IN-CCD bound to BI-D (PDB 4ID1). Image generated by UCSF Chimera20

33 Table 2.2. Antiviral activity (IC50) in WT and knockout cells

Further studies indicated that ALLINI potency was attributed to the late phase of

HIV-1 replication as evidenced by the formation of defective electron-dense HIV-1 cores

28 of viral progeny (Figure 2.5).34 This effect resulted in the increased antiviral activity of these compounds in producer cells in comparison to target cells. Overall, this indicated the versatile nature of these allosteric inhibitors (ALLINIs) to affect viral replication at a stage that is distinct from the catalytic requirement of integrase and underscores a previously unrecognized multifunctional cooperative mechanism of action.

Figure 2.5. Frequencies of core morphology for two IN mutant viruses (ΔIN and V165A),

34 and WT HIV-1NL4-3 made in the presence of BI-D (10 μM), BI-B (50 μM), or DMSO

C. Synthesis and mechanism of ALLINIs in HIV-1 IN A128T mutant strain

Since viruses mutate under pressure from inhibitors, the ALLINIs have revealed an alanine to threonine substitution at residue 128 (A128T) in HIV-1 IN,35 located near the 6-halobenzene moiety of BI-B, as a primary mechanism of resistance. Accordingly, an additional ALLINI, proposed to be more potent than BI-B against WT HIV-1 IN, was designed in order to probe the structural and mechanistic basis of this resistance strain in

29 conjunction with BI-B. As a result, the structural features of BIB-II (referred as ALLINI-

231 in the literature) are an amalgam of key peripheral elements from both BI-B and the more potent, BI-D, namely the C6-bromine and C4-aryl group from BI-B and the sterically demanding t-butyl ether moiety from BI-D (Scheme 2.3). Accordingly, BIB-II

(2.26) was prepared in two steps from intermediate 2.11, which was previously generated in the synthesis of BI-B (Scheme 2.1). The two-step sequence involved formation of the t-butyl ether 2.27 using perchloric acid-mediated alkylation with t-butyl acetate, which underwent saponification with sodium hydroxide to obtain BIB-II (Scheme 2.3).

Scheme 2.3. Design and synthesis of BIB-II

Consequently, the A128T mutation only had a minor effect on the IC50 values for

LEDGF-IN binding, but significantly affected aberrant multimerization of higher order

IN oligomers along with 3’ processing and strand transfer in LEDGF-independent catalytic activities (Table 2.3).31 Analysis of the crystal structures of the ALLINIs bound

30 to the WT and A128T INs provide a structural basis for the resistance observed.

Although the hydrogen bonding interactions between the carboxylic acid moieties of the

ALLINIs and the protein backbone of subunit 1 still exists, a notable difference is observed in the relative binding of the compound to the WT versus A128T mutant IN

(Figure 2.6). As shown for BIB-II, the quinoline ring of the compound appears to experience a downward and inward shift relative to its binding orientation in the WT caused by the larger substituent of the A128T mutant. This result was confirmed with additional analogues of BI-B which do not possess the C6 halogen substituent. In these cases, the much smaller H-atom does not interact with the A128T residue, resulting in nearly identical potencies for the WT and mutant (data not shown).

Table 2.3. Effects of A128T mutation on the biological activities of ALLINIs31

Figure 2.6. Overlay of BIB-II in WT (yellow) and A128T (magenta)31

Overall, these studies using known compounds have facilitated the elucidation of the novel multifunctional mechanism of ALLINIs. Remarkably, these results indicate aberrant IN multimerization as the primary mechanism of HIV-1 inhibition.

Crystallographic information further revealed the binding of these 2-(quinoline-3- yl)acetic acid inhibitors at the LEDGF/p75 binding site displaying similar interactions as

LEDGF-IBD. Accordingly, the carboxylic acid moieties on these inhibitors interact with the protein backbone at E170 and H171 through hydrogen bonding interactions.

However, the existence of a C6 substituent on the quinoline ring of ALLINIs renders these compounds inactive against the A128T mutant strain. As a result, the combination of these key interactions needed for biological activity served as our guide in the design of novel scaffolds for the inhibition of HIV-1 IN.

2.2 Modification of the 2-(quinolin-3-yl)acetic acid series

A. Scaffold hopping approach

In drug discovery, the general goal of scaffold hopping, or lead hopping, requires a known biologically active structural template from which the essential features are transferred to a novel central scaffold in order to maintain similar binding interactions and thereby maintain similar biological activity.36,37 Historically, there are numerous examples in the literature which successfully demonstrate the utility of this approach for generating marketable drugs. The major reasons for scaffold hopping include swapping the polarity of scaffolds to increase solubility of the compounds, substitution to increase metabolic stability, construction of a rigid scaffold, improvement of binding affinity through an increase in the number of interactions, and generation of novel compounds that are patentable.

Generation of novel HIV-IN inhibitors using the scaffold hopping technique led us to propose the transfer of functionality from the quinoline to the indole ring, while maintaining the key binding elements of the biologically active lead compounds. A major consideration in proposing this system was the effect that the transition from the 6,6- fused quinoline ring to the 6,5-indole ring system would make on the geometry or relative orientation of the peripheral substituents in the binding pocket of integrase. The indole core, as opposed to the quinoline, would provide a potential new, synthetically versatile scaffold that could potentially be useful for targeting A128T resistant strains.

The synthetic versatility of indoles is derived in part from the numerous methods available for their preparation38 and the unique reactivity of the indole system due to its

33 electron rich nature.39 Thus, the proposed change in this system from a quinoline to an indole nucleus was predicted to affect not only the structural orientation of the essential features on the ring but also the electron density of the core ring system, thereby providing information on the electronic requirements of the core ring system for binding to the allosteric site of integrase. Fortunately, as illustrated in Figure 2.7, a computational model (provided by Guqin Shi in Dr. Cheng-long Li’s lab) carried out using AutoDock

4.0 predicted that the indole analogues would maintain a binding mode similar to BI-B.

Of note, as predicted in our preliminary structural analysis, the docking model indicates that the smaller five membered ring of the indole does affect the relative orientation of the core in the binding pocket. The acetic acid side chain and the aryl substituents are predicted to overlay well in both the quinoline and indole systems, but this ultimately results in a slight tilt of the indole aromatic ring down and away from the A128T residue

(for reference, see Figure 2.6). Encouraged by these results, we pursued the synthesis of indole analogues to confirm these predictions.

Figure 2.7. Computational model, provided by Guqin Shi in Dr. Cheng-long Li’s lab, for

the predicted binding of proposed indole analogue 34

The synthesis of the indole analogues began with the inexpensive, commercially available reagent isatin (2.29) (Scheme 2.4). The first step of the synthesis featured the introduction of the aryl ring through addition of the corresponding Grignard or aryllithium reagents into the C3 carbonyl of isatin (2.29). The indole could be generated through reduction of the 3-hydroxyoxindole with lithium aluminum hydride. Installation of the α-ether carboxylic acid moiety was then accomplished through acylation of the indole ring 2.30 with oxalyl chloride, which presumably occurs at the nucleophilic C3 position and subsequently migrates to C2. Accordingly, addition of methanol to the

Friedel Crafts reaction with oxalyl chloride resulted in formation of the desired ester

2.31. Selective reduction of the ketone with sodium borohydride resulted in alcohol 2.32.

To our surprise, however, installation of the t-butyl ether group under the previous conditions with perchloric acid in t-butyl acetate resulted exclusively in decomposition of the indole ring. Fortunately, alkylation using resin bound acid, Amberlyst H-15, successfully generated t-butyl ether 2.33. Finally, saponification of the methyl ester furnished the sodium salt of the indole analogues, referred to as INDLs. Acidification of the salt to pH ≈ 4 provided the acid, but interestingly, further lowering of the pH resulted in degradation of the compound, presumably due to the protonation of the indole ring in combination with the instability of the t-butyl group under these conditions. Due to the sensitivity of the indole system and the resulting undesired degradation, as well as the use of neutral buffer conditions for the bioassay in which the molecule exists as the

35 carboxylate anion, in a few cases the carboxylates (or sodium salts) were not protonated and directly submitted for biological evaluation.

Scheme 2.4. Synthetic approaches for the series of indole analogues

Overall, five N-methylindole analogues, shown in Figure 2.8, were tested in the homogenous time-resolved fluorescence (HTRF) based LEDGF-dependent integration assay by Dr. Jacques Kessl in Dr. Kvaratskhelia’s lab. This primary screen assay, previously used for measuring the overall activity of BI-B and BIB-II in WT and A128T

HIV-1 IN,31 measures not only the IN 3’-processing and strand transfer catalytic

36 activities, but also the IN multimerization and LEDGF/p75-IN binding interactions. The assay monitors the increase in integration products by measuring the intensity of a time- resolved FRET signal between labeled viral and target DNA substrates in the presence of

LEDGF/p75 protein. Accordingly, a decrease in the observed HTRF signal signifies inhibition by the corresponding compound of the overall integration process. The first submitted indole analogue, INDL-1, exhibited an unsatisfactory IC50 value of 45 μM, but a crystal structure of the analogue bound to integrase could still be obtained in this case.

The co-crystal structure showed that the indole analogue did bind at the integrase allosteric site and also had a similar binding mode to BI-B as predicted in the computational model (Figure 2.9 – left). The analogue was anchored to the protein through the hydrogen bonding interactions between the carboxylic acid moiety and the protein backbone with an additional interaction with the t-butyl ether.

Figure 2.8. List of indole analogues synthesized and tested

Figure 2.9. Co-crystal structure of indole analogue bound to allosteric site of HIV integrase. Left: Overlay between BI-B (magenta) and INDL-1 (yellow). Right: Binding

site of INDL-1 with area for further functionalization. Images courtesy of Dr. Lei Feng

Based on the X-ray crystallographic analysis, extension of the phenyl ring further into the pocket was anticipated to result in an increase in biological activity of these analogues (Figure 2.9 – right). While the anisole and the benzyl analogues (INDL-2S and

INDL-5, respectively) showed a minor improvement in activity as compared to INDL-1, the chroman analogue INDL-4S exhibited an IC50 of 14 μM (Table 2.4). This observed increase in potency is presumably due to the increase in the number of favorable interactions of the chroman moiety within the hydrophobic pocket, similar to the interactions of BI-D. Although this analogue shows promising biological activity similar to that of LEDGIN-6, the potency does not compare to the observed effects in the 2-

(quinolin-3-yl)acetic acid derivatives. This observation is most likely attributed to the orientation of the peripheral substituents and not the electronic effects of the different ring systems. Therefore, keeping the 6-membered core ring system with the requisite peripheral substituents, but altering the fused-ring size that sits near A128 residue in 38 subunit 2 should improve the activity in A128T resistant strains. This modest result provided proof of principle that the quinoline core of the ALLINIs could be varied.

Table 2.4. LEDGF dependent integration activity of biologically active indole analogues

B. Pyridine analogues as mechanism-based selective inhibitors

Based on the quinoline derivatives, removal of the benzene ring that interferes with the A128T substitution would potentially provide improved biological activity in the resistant strain. Recently, Boehringer Ingelheim patented various substituted pyridine analogues, some of which displayed low nanomolar antiviral activities; however, there were no reported biological activities against the A128T resistant strain.40 Accordingly, in collaboration with Dr. Nivedita Jena, a postdoctoral research in the Fuchs lab, pyridine analogues were prepared through a synthetic route adapted from the patent.

The modular route features three key steps for the installation of the essential functionality onto the pyridine core. First, the α-(t-butyl ether)acetic acid moiety was introduced onto the trihalogenated pyridine intermediate 2.41 through alkylation of benzyloxyacetaldehyde with in situ generated nucleophile followed by known formation of the t-butyl ether with t-butyl acetate and perchloric acid to furnish dichloropyridine

2.42 (Scheme 2.5). This intermediate serves as a common precursor for the step-wise

39 introduction of C2 and C4 aryl substituents en route to the generation of various pyridine analogues (Scheme 2.6). Accordingly, the second key step involves Pd-catalyzed cross- coupling selectively at the 2-chloro position for further functionalization into the C2-aryl substituent. The final key step includes installation of the 4-aryl substituent through a

Suzuki-Miyaura cross-coupling reaction. This approach facilitates facile modification for generation of functionally diverse pyridine analogues such as PY-1 (2.45) and PB-20

(2.51). Of note, however, is the formation of unanticipated dehalogenated intermediate

2.50 resulting from hydrogenation conditions applied for deprotection of the benzyl ether moiety. This problematic side-reaction was avoided by directly coupling the unsubstituted phenyl boronic ester in order to efficiently generate the dehalogenated analogue, PB-20.

Scheme 2.5. Synthesis of a common intermediate for the pyridine analogues

Scheme 2.6. Synthesis of pyridine analogues PY-1 and PB-20

With PY-1 and PB-20 in hand, elucidation of the mechanism of action of this new class of analogues was performed in Dr. Kvaratskhelia’s group. The results indicated a remarkable selectivity for affecting integrase multimerization (Table 2.5) while still exhibiting potent biological activity against the A128T resistant strain (data not shown).

Co-crystal structures of PY-1 and PB-20 both indicate that the compounds bind to IN-

CCD at the LEDGF/p75 binding site. Moreover, both aryl groups lie perpendicular to the pyridine core as a result of the high degree of substitution on the central pyridine. This effect by the C3 and C5-dimethyl groups on the pyridine core restricts the conformational/rotational freedom of the C2 and C4-diaryl substituents, thereby locking 41 these substituents orthogonal to the pyridine core. While the planar quinoline core on

BIB-II extends into the A128T substitution, the C2-aryl substituent of PY-1 is oriented away from the steric effects of the A128T substituent (Figure 2.10 – left) and, therefore, can influence its activity on the resistant strain. Furthermore, the structural data shows the presence of a strong hydrogen bonding interaction between the benzimidazole NH on PB-

20 and threonine-125 (T125) of subunit 1 (Figure 2.10 – right), which is absent in the quinoline compounds. This additional interaction gained by the C2-aryl substituent with subunit 1 is proposed to be responsible for the observed selectivity towards IN multimerization over LEDGF/p75-IN interaction. Therefore, this novel structural and its associated mechanism of action provide a useful target for the development of selective mechanism-based inhibitors.

Table 2.5. Inhibitory activities of BI-B, PY-1, and PB-20

Figure 2.10. Images of PY-1 and PB-20 binding to IN-CCD Left: Overlay of PY-1 and

BIB-II to IN-CCD in A128T mutant strain. Right: Binding interactions of PB-20 in WT

IN-CCD. Images courtesy of Dr. Lei Feng

2.3 Alternate methods for the development of novel scaffolds

With significant competition in the development of integrase allosteric inhibitors, our group was interested in moving away from the class of known quinoline and pyridine compounds to develop novel scaffolds that would also interact at the same binding site.

Accordingly, two different approaches were pursued for the identification of novel scaffolds: computer-aided and fragment based drug design.

A. In silico screening

An alternative strategy for identifying new scaffolds is through computational methods via screening of a virtual library of compounds. This in silico strategy provides access to a large number of small molecules that can be assessed for their affinity to a protein, thereby identifying drug candidates more rapidly at lower costs. Initially, a list of

43 compounds for targeting the allosteric site (IN-CCD) was generated from an in silico screen of a ZINC database, a library containing over 1.4 million commercially available drug-like molecules. As illustrated in Figure 2.11, analysis of the library of compounds through molecular docking provided a hitlist of 66 compounds, which served as a portion of Dr. Vandana Kumari’s dissertation.41

Figure 2.11. Flow chart showing virtual screening work flow using molecular docking

software, Glide and AutoDock4 (adapted from Dr. Vandana Kumari’s dissertation)41

These purchased compounds were then tested for LEDGF-IN inhibitory activity using a HTRF assay. Specifically, the fluorescence assay measured the number of

LEDGF-IN complex formations taking place, inhibition of which would lower the percentage as compared to a DMSO control. Accordingly, 4 of these purchased samples

(B22, B34, A4, B37) displayed promising activity at 200 μM while allowing less than

50% of LEDGF-IN complex formation (Table 2.6). In comparison, the known LEDGF-

IN inhibitor, LEDGIN-6, allowed only 6% of the complex formation.

Table 2.6. LEDGF-IN binding activity of the most active compounds identified from

virtual screening of drug-like compounds

Efficient and flexible synthetic routes were developed to the two most active compounds (B22 and B34) identified in this screen. Although fairly straightforward, the synthesis of B22 and its structural analogues could be divided into installation of the necessary substitution on the indole, or more accurately tetrahydrocarbazole, portion and introduction of the acyl-triazole moiety onto the indole nitrogen (Scheme 2.7). In B22, the substituted indole portion 2.56 was constructed via alkylation of the in situ generated enolate from ketone 2.53 followed by acid mediated hydrolysis of the carbamate and subsequent reduction of the resulting vinylogous amide. Introduction of the triazole 45 moiety was performed through N-acylation of indole 2.56 with bromoacetyl bromide and subsequent alkylation with thiol 2.58 to furnish B22 and its related analogues. A synthetic library of B22 structural analogues varying in indole substitution was also generated using a similar synthetic sequence (Figure 2.12).

Scheme 2.7. Synthesis of B22 and related alkyl analogues

Figure 2.12. Synthetic library of B22, B34, and B22 analogues

The synthesis of B34 was accomplished in a convergent route through acylation of benzyl alcohol 2.63 to N-methanesulfonyl acyl chloride 2.60 (Scheme 2.8). The

46 methylsulfonamide moiety in B34 was installed through sulfonylation of anthranilic acid

(2.59), while the primary amide group was introduced through amide coupling reaction between benzylamine 2.62 and carboxylic acid 2.61. After the formation of ester 2.63, a

CAN-mediated oxidation of the p-methoxybenzyl group furnished B34.

Scheme 2.8. Synthesis of B34

The synthetic B22, B34, and the B22 analogues were tested in the LEDGF-IN binding HTRF assay. Unfortunately, these synthetic compounds, including the synthetic samples of the “identical” commercially available compounds, did not show any appreciable biological activity even though the commercial compounds showed promising activity. This was surprising since the synthetic samples showed higher purity than their commercial counterparts, based on comparative analyses of the NMR spectra

(Figure 2.13) and HPLC traces. The false positives from the purchased compounds could

47 be attributed to compound degradation over time, compound aggregation in the conditions for the assay, or low-level impurities such as false positives from residual metals. Unfortunately, these occurrences are common in the screening of hits from a library of compounds.42–44 Due to this unreliability in results with this approach, we shifted the focus to a fragment-based approach for identifying new scaffolds.

Figure 2.13. Comparative synthetized and purchased NMR spectra of B22 (top) and B34

(bottom)

B. Fragment-based drug discovery

Fragment-based drug discovery (FBDD) has recently emerged as a useful alternative to high throughput screening (HTS) in the identification of novel scaffolds in the search for new lead compounds.45 In comparison to HTS, the FBDD approach requires fewer compounds to be screened and identifies fragments with low molecular weights and drug-like properties that generally bind with weak affinity for the biological target. The fragments utilized, however, are typically more amenable to structural modification and development. Optimization of fragments that form high quality interactions with the target, generally involve either linking or expanding the fragment to increase the number of interactions, termed ligand efficiency, thereby increasing the selectivity and affinity for the target. These interactions are typically visualized with the help of NMR spectroscopy, surface plasmon resonance or X-ray crystallography.

Using an FBDD approach, the lab of Dr. Edward Arnold at Rutgers University in collaboration with the Kvaratskhelia lab screened approximately 900 fragments to identify small molecules which bind to the LEDGF/p75 binding site of HIV IN. MB36-3 was the only compound found from this screen to crystallize in the desired LEDGF/p75 binding pocket. Further validation and SAR for this fragment and 15 structurally similar fragments selected by the Arnold lab were assessed in the Kvaratskhelia lab for inhibition of LEDGF/p75 dependent integration at 200 μM and 400 μM, in comparison to the known inhibitor BIB-II (Figure 2.14). Not surprisingly, MB36-3 showed weak inhibition

49 in the HTRF assay. Two of the additional compounds sent to OSU by the Arnold lab,

MB59-1 and MB13-2, also displayed promising activity.

EA Compounds 1-9 - LEDGF/p75 dependent integration Inhibition

100

40 PercentInhibition

M M

m m m m m m m m m m m m m m m m m m

μ μ

μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ 2% 4%

100

200 400 200 400 200 400 200 400 200 400 200 400 200 400 200 400 200 400 MB36- MB36- MB7-2 MB9-5 MB13-2 MB14-3 MB34-3 MB34-4 MB56-2 DMSO BI-B EDTA 3_OL 3_NL II Compound

EA Compounds 10-17 - LEDGF/p75 Dependent integration inhibition

100

40 Percent InhibitionPercent

m m m m m m m m m m m m m m m m M M

μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ

2% 4%

200 400 200 400 200 400 200 400 200 400 200 400 200 400 200 400 100 MB59-1 MB59-2 MB64-2 MB69-4 MB81-1 MB82-3 13884 SEW01838 DMSO BI-B EDTA II Compound

Figure 2.14. Percent inhibition of LEDGF/p75 dependent integration by MB36-3 and

other fragments (Kvaratskhelia lab)

Structurally, all three of these fragments possess similar functionality, a carboxylic acid moiety and a 5-membered heteroaromatic ring attached to a central aromatic/ heteroaromatic scaffold (Figure 2.15). MB13-2 (2.65), however, displays an

50 extended framework as compared to the more compact vicinal substitution of the pyrrole and carboxylic acid groups founds in both MB59-1 (2.66) and MB36-3 (2.67). Although both these compounds have the same substitution pattern, the angles between the pyrrole and carboxylic acid groups vary slightly between the 5- and 6-membered cores, even though the benzene and thiophene rings themselves are considered isosteric in medicinal chemistry. This difference in orientation was initially considered to account for their respective observed biological activities, analogous to the conclusions made in the comparison of SAR between the indole and quinoline analogues. Notably, however, we felt that the chemistry with the benzene ring would be more predictable and relatively acquiescent than the thiophene ring, suggesting that it could serve as a “model” system for similar transformations on the more expensive thiophene starting material.

Figure 2.15. Structure of crystallized fragment, MB36-3, and two active fragments

Accordingly, the design of MB59-1 analogues was based on the analysis of the crystal structure of IN bound to MB36-3. As illustrated in Figure 2.16 (left), MB36-3 occupies the same binding pocket as the reported quinoline ALLINI, BIB-II. Analysis of this binding mode indicates a similar hydrogen bonding interaction with glutamate-170

(E170), but with a distinct docking pose as compared to the quinoline based inhibitors. A

51 series of compounds was proposed to build upon the fragment structure in order to increase the number of interactions with the surrounding residues, the typical approach utilized in FBDD studies.

Figure 2.16. Overlay of MB36-3 with BI-B within the LEDGF binding pocket

An alternative approach to quickly probe the SAR was generated based on further analysis of the crystal structure, which shows that the fragment resides within a “bowl- shaped” pocket and little room to grow on the pyrrole ring as it is buried deep within the pocket. In the Arnold lab, a preliminary analysis of proposed MB36-3 derivatives using molecular docking with AutoDock Vina indicated that the 2-position of the pyrrole ring can only accommodate small substituents like amines and alcohol, while the limited flexibility of the molecule for expansion from the pyrrole ring did not allow for significant interactions with the protein. As a result, “growth” of the pyrrole ring may require the introduction of flexibility into the rigid sp2 hybridized system in order to facilitate the extension of a chain over the surrounding ridge (Figure 2.17). A preliminary study using the AlleGrow molecular modeling system (Boston De Novo Design) with the 52 assistance of Ryan Pavlovich from Dr. Cheng-long Li’s lab supported the feasibility of this approach through addition of 10 to 30 atom containing segments. With the structural similarities of MB36-3 to MB59-1, the assumption was that both of these compounds were binding to the same pocket and that the same structural modifications could be utilized in both series, despite the fact that no crystal structure had been obtained for

MB59-1 with integrase.

“Ridge”

Figure 2.17. View of the protein surface of the LEDGF binding pocket with MB36-3

bound (left). View of the residues within 5Å of MB36-3 in the binding pocket (right).

Images generated using UCSF Chimera20

The synthesis of MB59-1 analogues initially focused on introduction of 2- and 3- position pyrrole ring substituents (Scheme 2.9). Accordingly, a Paal-Knorr pyrrole synthesis with commercially available methyl anthranilate (2.68) and 2,5- dimethoxytetrahydrofuran (2.69) provided methyl ester 2.70. Subsequent saponification followed by acidic work-up furnished synthetic MB59-1. Halogenation of 2.70 with N- 53 chlorosuccimide and N-bromosuccinimide, respectively, provided the corresponding 2- halopyrrole derivatives 2.71, but saponification followed by acidic work-up caused degradation of the resulting acids, potentially due to the electron rich pyrrole ring.

Nitration and Vilsmeier-Haack formylation of the pyrrole ring of 2.70 provided the corresponding 2- and 3- substituted carboxaldehyde (2.73 and 2.74) and nitro derivatives

(2.75 and 2.76), respectively. These regioisomeric compounds could be separated through flash column chromatography to provide the pure compounds. We anticipated formation of the 2-amino derivative 2.77 through a reduction of the corresponding 2-nitro compound 2.75. Surprisingly, Pd-catalyzed hydrogenation and zinc mediated reduction of the nitro group provided the known pyrroloquinazolinone 2.78, resulting from addition of the nitrogen atom into the electrophilic ester moiety.

Scheme 2.9. Synthesis of MB59-1 and 2- and 3-substituted pyrroles

Flexible open-chain analogues based on the AlleGrow molecular modeling were synthesized either through alkylation or acylation of the aromatic amine with the corresponding α–bromoamide 2.80 or acyl chloride 2.84 (Scheme 2.10). Another class of open-chain analogues was synthesized through reductive amination of commercially available anthranilic acid (2.87) and corresponding aromatic/heteroaromatic aldehydes.

These analogues have a shorter linker length between the benzene core and the aromatic ring as compared to the previously generated open-chain analogues. Analogues with

55 heteroaromatic rings would provide a suitable site for further fragment elongation.

Finally, sulfonylation of anthranilic acid provided structurally similar sulfonamide analogues 2.89, which will alter the electron density and angle between the aromatic ring and the benzene core. Overall, a total of 18 analogues have been synthesized and submitted for testing (Figure 2.18).

Scheme 2.10. Synthesis of open-chain analogues

Figure 2.18. Classification of synthesized MB59-1 analogues

The first 9 compounds were tested in the HTRF assay to determine corresponding inhibitory effect on LEDGF dependent integration at 200 μM and 400 μM.

Unfortunately, all fragments showed little to no inhibitory effect at 400 μM (Figure 2.19).

Similar to the false positives observed in the in silico method described above, trace metal impurities or a variety of other “unknown” factors could be potentially responsible for the false positive observed in the screening of fragments.44

Figure 2.19. Biological activity of MB59-1 analogues as percent inhibition of LEDGF

dependent integration (Kvaratskhelia lab)

Although synthetic MB59-1 and its analogues do not exhibit promising biological activity, the chemistry utilized to generate these analogues can be applied to the expansion of the structurally related MB36-3 fragment. Moreover, the established crystal structure of IN-CCD bound to the MB36-3 fragment minimizes the probability towards observing false positives and serves as a promising lead for fragment derivatization.

Accordingly, the synthesis of MB36-3 has been accomplished and several additional structural analogues are currently being prepared by Janet Addae, a junior graduate student in the Fuchs lab. We were delighted that synthetic MB36-3 showed biological activity with 91% and 82% integration at 200 µM and 400 µM, respectively, in the

LEDGF-dependent integration assay, which is similar to the activity observed for the purchased fragments. In addition, three sulfonyl derivatives based on MB-59-1-13, -14, and -15 (Figure 2.18) showed slightly improved activity, supporting the validity of the approach.

2.4 Conclusion

The synthesis of a number of quinoline- and pyridine-containing inhibitors has facilitated biological studies for establishing a novel mechanism for inhibition of HIV-1 integrase. The structural and mechanistic analysis resulting from these studies with WT and A128T resistant strains strongly support higher order integrase oligomerization, not disruption of LEDGF-IN interaction, as the primary target of these inhibitors in infected cells, particularly for the PY series of compounds. Alternatively, various drug design methods have led to the identification of novel scaffolds for targeting the integrase allosteric site. The scaffold hopping technique with indole derivatives has been used to synthesize compounds with modest antiviral activity but this approach also illustrated the importance of 6-membered scaffolds for effectively inhibiting integrase. Although the computer-aided approach with B22 and B34 as well as the fragment-based approach with

MB59-1 were not successful, MB36-3 appears to provide a useful scaffold for the

59 continued development of a new class of allosteric integrase inhibitors. Lastly, sensitivity to small molecules that induce multimerization during viral egress establishes integrase as an attractive target for clinical development of allosteric integrase inhibitors.

Part II:

Synthesis of the highly substituted indole subunit of sespendole

Chapter 3: Sespendole and Related Indoloterpenes

Microorganisms, including bacteria and fungi, are important sources of secondary metabolites also known as natural products that possess diverse structural and biological properties. Based on their biosynthetic origin, these secondary metabolites are classified as terpenoids, polyketides, alkaloids, and non-ribosomal peptides. The initial discovery of penicillin sparked the interest for screening microorganisms for biologically active secondary metabolites, leading to the identification of numerous lead compounds for the treatment not only of microbial diseases, but also a variety of other disease states. Some of these isolated natural products have become commercially available for medical use such as the family of tetracyclines from Streptomyces sp. as antibiotics,46–48 lovastatin from Aspergillus terreus for treating cardiovascular disease,46,49 cyclosporine A from

Tolypocladium inflatum as an immunosuppressant,46 the family of avermectins from

Streptomyces avermitilis as antiparasitic drugs,50 and the family of ergot alkaloids from

Claviceps sp. as vasoconstrictors.51

3.1 Isolation and identification

During the course of screening various fungal extracts for leads with anti- arteriosclerotic activity, Ōmura and coworkers recently isolated a unique indolosesquiterpene alkaloid, sespendole (Figure 3.1), from the culture broth of

Pseudobotrytis terrestris FKA-25 strain. The natural product was reported to be a potent inhibitor of lipid droplet synthesis in mouse macrophages.52,53

Figure 3.1. Structure of sespendole, the first reported indolosesquiterpene

The natural product 3.1 was found to contain a 4,5-disubstituted indole ring fused to a tricyclic sesquiterpene unit.54 This structure was determined through extensive NMR studies involving 1H, 13C, DEPT, COSY, HMQC, and HMBC experiments, with corresponding fragments supported by EI-MS ion peak analyses. The relative stereochemistry of the decalin moiety (C3 through C12) was deduced from NOESY experiments. Using the (R)- and (S)-Mosher esters derived from the alcohol generated by reduction of the C7 ketone, analysis with the modified Mosher method determined the

3S, 4R, 9S, and 12S absolute stereochemistry of the sesquiterpene moiety. Unfortunately, these NMR experiments were ineffective in determining the configuration of the C30 and

C31 centers. Instead, a europium shift reagent was employed, resulting in a greater

63 downfield shift of the C30 and C31 protons by (–)-Eu(hfc)3 over (+)-Eu(hfc)3. This data was used to unambiguously determine the S configuration of the C30 benzylic alcohol.

The stereochemistry at the C31 position remained undefined in the initial disclosure of the natural product.

Figure 3.2. Indoloditerpenes with a similar indoloterpene framework (red) as sespendole

In 2006, sespendole was the first fungal metabolite reported to possess an indolosesquiterpene core. A number of other fungal metabolites, however, had previously been reported to possess a similar, yet more elaborate, indoloterpene skeleton (Figure

3.2). In this thesis, indoloterpenes is a broad term used to denote both indolosesquiterpenes and indoloditerpenes. As a class, the indoloterpenes contain an

64 indole ring fused to a 5,6,6-tricyclic terpene ring system that possesses trans vicinal quaternary methyl substituents at the C3 and C4 positions. The structurally similar indolosesquiterpene and indoloditerpene subclasses are differentiated in the terpene units which contain three or four isoprene units, respectively. The class of indoloterpenes which show both tremorgenic and non-tremorgenic activities includes paspaline, paspalicine, and paspalinine from Claviceps paspali, paxilline from Penicillum paxilli, the janthitrems from Penicillum janthinellum, the lolitrems from Neotyphodium lolli, the penitrems from Penicillium crustosum, the nodulisporic acids from Nodulisporium sp., and the terpendoles from Albophoma yamanashiensis.55 The complexity of these indoloterpenes lies not only in the substitution pattern of the terpene unit, but also in the degree of substitution on the benzene unit of the indole ring. For example, paxilline, paspalinine, and paspaline possess unsubstituted indole moieties, while janthithrem B, nodulisporic acid, penitrem, and lolicine all contain bicyclic ring systems fused to the C5-

C6 or C4-C5 positions of the indole. These bicyclic systems are presumably derived biosynthetically through cyclizations of oxidized prenyl units similar to those found at the

C4 and C5 positions of sespendole. It is also interesting to note that since the isolation and identification of sespendole, a new family of indolosesquiterpenes containing nearly identical substitution patterns in the sesquiterpene subunit was isolated as nonsteroidal progestins from the terrestrial fungus Verticillium lecanii.56 These compounds, which do not possess the complex substitution on the indole ring system, have been named lecanindoles A-D (Figure 3.3).

Figure 3.3. Lecanindoles A-D, structurally related fungal indolosesquiterpenes

3.2 Biological activity

Despite similar structural features, the reported biological activities for these related natural products are quite different. The indoloditerpenes possess a variety of intriguing biological properties including anti-insecticidal57 and mito-inhibitory activities.58 The presence of the C13 tertiary hydroxyl group in many of the indoloditerpenes is believed to be necessary for the tremorgenic neurological activity observed in domestic animals.59 Although sespendole does possess a related C9 tertiary hydroxyl group, the tremorgenic potential of this compound has not yet been reported.

Sespendole was reported, however, to exhibit a novel anti-arteriosclerotic activity through inhibition of lipid droplet synthesis.52 The anti-arteriosclerotic activity of sespendole was achieved by affecting the syntheses of cholesteryl ester (CE) and triacylglycerol (TG), the main constituents in lipid droplets with IC50 values of 4.0 µM and 3.2 µM, respectively. In preliminary studies, however, sespendole was found to reduce the number and size of cytosolic lipid droplets in mouse macrophages (Figure

3.4). In contrast, the structurally related indolosesquiterpene, lecanindole D (3.14), was reported to be a potent and selective progesterone receptor agonist.56 This observed

66 difference in activity could presumably be attributed to the additional substitution on the indole ring of sespendole.

Figure 3.4. Effect of sespendole concentration on lipid droplet size and number.

In addition to sespendole, other collaborative research efforts by Tomoda and

Ōmura have also led to the isolation and identification of numerous structurally diverse fungal metabolites through bioassay guided fractionation for inhibition of lipid droplet accumulation (Figure 3.5).60 A comparison of their inhibitory activity against lipid droplet accumulation in mouse macrophages is shown in Table 3.1. Sespendole was one of the two isolated fungal natural products that were found to affect both CE and TG syntheses towards inhibition of lipid droplet synthesis in mouse macrophages.

Figure 3.5. Fungal metabolites that inhibit lipid droplet accumulation

Table 3.1. Inhibitory activity by microbial inhibitors against lipid droplet accumulation in

mouse macrophages60

Lipid droplet accumulation has recently been implicated in various diseases.61,62

Lipid droplets were initially thought to be storage sites that provided metabolic energy through regulation of the neutral lipids, CE and TG. However, proteomic studies have recently revealed important clues about the regulation of lipid storage and lipid 68 metabolism through association of the phospholipid monolayer surface with other families of proteins.63 Further research identified these dynamic cellular organelles as key players in membrane trafficking, vesicular docking, endocytosis and exocytosis of proteins and lipids through interaction with other cellular organelles such as the mitochondria and endoplasmic reticulum (Figure 3.6). Although lipid droplets have been shown to be important in these cellular processes, more significantly, they have been implicated in various diseases including atherosclerosis, diabetes, obesity, inflammation, and cancer.62,64 Therefore, discovering inhibitors of lipid droplet formation would be beneficial for exploring novel options for treating these disease states. In normal cells,

CE and TG are stored in lipid droplets through regulation of acyl-CoA: cholesteryl acyltransferace (ACAT) and acyl-CoA synthetase (ACS), respectively.62 An over- accumulation of both neutral lipids, but primarily CE, within lipid droplets causes foam cell formation, potentially resulting in these previously mentioned disease states. The majority of natural products found to target lipid droplet formation, inhibit ACAT as a result of inhibition of CE synthesis in macrophages.60 Alternatively, another class of natural products, triacsins, prevents lipid droplet accumulation through inhibition of fatty acid synthesis through the regulation of ACS.65 While sespendole was identified to inhibit both CE and TG syntheses to similar extents as triacsins, it did not show any activity against either of those cofactors.52 Consequently, the biological target of sespendole for inhibition of lipid droplet formation by macrophages appears to be different from other natural products, although the precise mechanism of action remains to be determined.

Figure 3.6. Composition of lipid droplets (left - Reprinted with permission from Elsevier:

3.3 Biosynthesis

In addition to its complex structure and potentially novel biological activity, sespendole is also an intriguing synthetic target due to the elegant proposed biosynthetic mechanism employed in the creation of its indolosesquiterpene skeleton. As mentioned previously (Section 3.1), prenyl derived indole substituents, similar to those found on sespendole, may act as biosynthetic precursors to the complex bicyclic ring systems fused to the indole rings of nodulisporic acids, penitrems, and the lolicines. Initially, however, we were intrigued by the method through which nature assembles the indoloterpene ring system itself.

The biosynthesis of the sesquiterpene moiety was investigated through labeling studies of sespendole with [13C]acetate.67 This 13C NMR data indicated that the sesquiterpene unit attached to the indole ring appears to be derived from a farnesyl group, albeit with unusual connectivity. Formation of the indolosesquiterpene skeleton was

70 unusual in that the labeling pattern at C3, C11, C12, C13, and C21 suggested that a cascade rearrangement and cyclization occurred in the formation of the 5-membered terpene ring. As shown in Scheme 3.1, the mechanism for indolosesquiterpene formation is proposed to undergo terminal epoxidation of the farnesyl chain in 3.21 to provide intermediate 3.22 followed by acid promoted cyclization to form the 5,6-fused bicyclic secondary carbocation intermediate 3.23. This intermediate undergoes rearrangement through a 1,2-migration, resulting in the formation of the more stable tertiary carbocation, followed by cyclization with the nucleophilic indole ring to provide the indolosesquiterpene skeleton 3.24. A similar type of cascade sequence has been previously observed and reported for the biosynthesis of the indoloditerpene skeletons in penitrem A (3.9) and nodulisporic acid A (3.7).68,69

Scheme 3.1. Proposed biosynthesis of indolosesquiterpene skeleton

The biosynthetic precursor of the indole moiety in sespendole was elucidated through incorporation of [13C]tryptophan and [15N]anthranilic acid. Based on the pioneering work of Acklin and coworkers on establishing the biosynthetic precursors to 71 paspaline,70 it was believed that the indole core of related indoloterpenes would also be derived from tryptophan. However, researchers at Merck Research Laboratories were surprised to find that biosynthetic studies on the origin of nodulisporic acid A (3.7) using radiolabeled tryptophan were unsuccessful.69 Alternatively, the incorporation of isotope labeled anthranilic acid (3.25), the biosynthetic precursor to tryptophan (3.32), via indole-

3-glycerol phosphate (3.30) proved to be successful in establishing the biosynthesis of the family of nodulisporic acids, thus, suggesting a biosynthetic relationship between indoloditerpenes 3.7 and 3.9 derived from the structurally simplified anthranilic acid

(3.25), as illustrated in Scheme 3.2.

Scheme 3.2. Tryptophan biosynthetic pathway towards biosynthesis of indoloditerpenes

Accordingly, Ōmura and coworkers observed a similar outcome for identifying the biosynthetic origin of the indole moiety of sespendole via incorporation of

[15N]anthranilic acid as opposed to [13C]tryptophan.67 In 1H and 13C NMR experiments, observation of distinctive satellite signals due to 1H-15N and 13C-15N coupling further demonstrated that anthranilic acid was the biosynthetic precursor of the indole moiety of sespendole, while [13C]tryptophan showed no 13C enrichment.

Accordingly, the proposed biosynthesis of 13C enriched sespendole, shown in

Scheme 3.3, was accomplished by metabolism of [13C]acetate (3.33) into labeled farnesyl pyrophosphate (FPP) 3.36 through mevalonate 3.34 and isopentyl pyrophosphate (IPP)

3.35. Condensation of FPP with [15N]indole-3-glycerol phosphate (3.30) led to the formation of the indolosesquiterpene skeleton 3.24 through the rearrangement/cyclization cascade. Furthermore, the 13C NMR data also indicated that the two isoprenyl units attached to the indole benzene ring are derived from IPP. Further prenylation of the indole ring with IPP is proposed to provide labeled sespendole (3.37).

Scheme 3.3. Overview of the proposed biosynthesis of sespendole

3.4 Previous approaches to indoloterpene natural products

The complex structural features and interesting biological properties of indoloterpene natural products have made them attractive targets to synthetic organic chemists. Syntheses of members of this class of natural products have provided an opportunity to develop and test new methods for the construction of these complex ring systems. Extensive studies by the group of Prof. Amos Smith have provided the most significant contributions to the synthetic efforts, resulting in total syntheses of (-)- paspaline,71 (+)-paspalicine and (+)-paspalinine,72 (-)-penitrem D,73 isopentenylpaxilline,74 and (+)-nodulisporic acid F.75 The synthesis of paspalinine76 and lecanindole D77 by Kuwahara and coworkers are the only other reported total syntheses from this class of indoloterpene natural products. Synthetic efforts towards the terpene and indole subunits of these compounds, however, have also been reported by other groups.78–80

The synthesis of an inodoloterpene containing a substituted indole moiety can be viewed as containing three distinct synthetic challenges: 1) indoloterpene construction via coupling of the indole portion to the teprene unit, 2) the synthesis and stereocontrol of the terpene unit, and 3) synthesis of appropriately functionalized indole moiety. Perhaps not surprisingly, considering the common structural features present in these indoloterpene natural products, the attachment of the indole to the terpene unit has been addressed in several syntheses. The most common synthetic strategy has involved the late stage synthesis of the indole ring through union of functionalized terpene 3.40 and aniline 3.39

74 fragments (Scheme 3.4). The synthetic challenge of the terpene fragment 3.40 lies in the stereoselective installation of the trans vicinal methyl groups towards construction of the trans-anti-trans 5,6,6-tricyclic ring system, which has been tackled primarily through functionalization of the readily available Wieland-Miescher ketone (3.41). Finally, the regioselective substitution of the indole ring, particularly C5/C6 or C4/C5 functionalization, provides a unique challenge based on the structure of the desired natural product. Typically these substituted indoles have been prepared from substituted benzene rings using de novo indole syntheses that also accommodate the necessary functional groups. Unfortunately, the seemingly simple introduction of the appropriate functionality onto the indole rings has required the development of unique methods and still requires numerous synthetic steps to accomplish.

Scheme 3.4. Common strategy for the synthesis of indoloterpenes

Each of these challenges will be highlighted in the examples below. The various synthetic approaches by Prof. Amos Smith towards the indole moiety and the terpene subunit laid the groundwork for the total syntheses of related indoloditerpenes. Kerr and coworkers reported the synthesis of highly substituted indoles through a modified

Plieninger indolization and applied this method towards the indole subunit of lolicines

75 and lolitrems.81 Kuwahara and coworkers reported the first total synthesis of indolosesquiterpene, lecanindole D.77 Although there is no reported total synthesis of the structurally related indolosesquiterpene natural product, sespendole, approaches towards the terpene82 and indole83 subunits were reported by Nishikawa and coworkers.

A. Smith’s approaches to indoloditerpenes

Over the past 3 decades, the Smith research group has strived to develop versatile synthetic routes to access several members of the tremorgenic and non-tremorgenic indoloditerpene natural products. These approaches employed a unified synthetic strategy, which involved the coupling of a common advanced terpene intermediate with a suitable aniline derivative to generate the indoloterpene framework of these natural products. Construction of this framework utilized Gassman indole synthesis for coupling of common advanced ketone intermediate 3.42 with the aniline (3.43), en route to the total syntheses of paspalinine (3.3), paspalicine (3.4), and paspaline (3.5) (Scheme 3.5).

Scheme 3.5. Common advance intermediate for synthesis of the paspalane family 76

Gassman indole synthesis, developed by Gassman et al. in 1974, involves a one- pot process for the conversion of anilines and β-carbonyl sulfides to the corresponding 2-

84 substituted (R1 = H) indoles (Scheme 3.6). This method includes in situ generation of

N-chloroaniline (3.44) from aniline (3.43), followed by addition of β-carbonyl methylsulfide 3.45 to form azasulfonium ion 3.46. A tertiary amine base then deprotonates intermediate 3.46 to form an azasulfonium ylide 3.47, which undergoes a

Sommelet-Hauser type [2,3]-sigmatropic rearrangement to provide ketone 3.48. Acid- mediated condensation followed by reduction affords the indole ring system 3.50. This protocol was further developed for the preparation of 2,3-disubstituted indoles (R1 ≠ H).

Advantages with this method are that the keto sulfides are easily prepared from α- haloketones, while the aniline substrates are usually commercially available, including highly substituted rings. However, a limitation is that electron-rich anilines fail to generate the indole ring. In addition, the use of halogen sources like hypochlorite, which are strong oxidizing agents, may not be compatible with electron-rich functional groups.

Scheme 3.6. General scheme of the Gassman indole synthesis, used for the paspalines

With the Gassman indole synthesis as a late stage process in the syntheses of the paspalines, the primary efforts by the Smith group focused on the construction of common intermediate 3.42 for the terpene subunit. The challenging aspect for the synthesis of this ketone intermediate was installation of the trans vicinal quaternary methyl groups. The highly stereocontrolled approach from the (+)-Wieland-Miescher ketone ((+)-3.41) is outlined in Scheme 3.7.85 Installation of the second methyl group and the trans-fused cyclopentanone intermediate 3.42 was accomplished by conjugate addition of the methyl group into enone 3.51 followed by ring contraction. This enone was generated through Robinson annulation of cyclohexanone 3.52, which in turn was prepared via ketalization of ketone (+)-3.41. The pre-installed methyl stereocenter on ketone (+)-3.41 allowed for an efficient route to the terpene subunit (9 steps, 9.4% overall yield).

Scheme 3.7. Retrosynthesis of common advanced ketone intermediate

In the recent past, the requisite eastern hemisphere 3.53, known as the Nolen-

Sprengeler lactone, was conceived as common advanced intermediate by the Smith group in the total syntheses of penitrem D (3.54),73 and isopentenylpaxilline (3.55)74 (Scheme

3.8). This unified strategy exploited the indole synthetic protocol developed in the Smith lab, a modification of the Madelung indole synthesis.

Scheme 3.8. Total syntheses of complex indoloditerpenes with Nolen-Sprengeler lactone

The Madelung indole synthesis generally involves a strong base mediated intramolecular cyclization of N-acylated-o-toluidines 3.56 to the corresponding 2- substituted indoles 3.59 (Scheme 3.9). The Smith modification, known as the Smith indole synthesis, comprises treatment of substituted N-TMS-o-toluidines 3.60 with a strong alkyl lithium base. The in situ generated benzylic dianion 3.61 reacts with esters or lactones to afford N-lithiated-ketoanilides 3.62, which undergo intramolecular heteroatom

Peterson olefination to generate intermediate 3.63. A tautomerization then provides the corresponding 2- (R1 = H) or 2,3-subsituted (R1 ≠ H) indoles 3.64; however, reaction with non-enolizable lactones required acid to facilitate heteroatom Peterson olefination. The transformation takes place with both regio- and stereocontrol and the method is also amenable to mutli-gram scale indole formation. In constrast to the Gassman indole synthesis, this method is compatible with electron-rich functional groups and can accommodate various substitutions on the aniline ring.

Scheme 3.9. General Madelung indole synthesis (top); Smith’s modification (bottom)

The versatility of the Smith protocol in the expedient synthesis of substituted indoles from corresponding γ-lactones made it a suitable method for the syntheses of related indoloditerpenes from the key Nolen-Sprengeler lactone 3.53 intermediate. The most recent approach to this lactone, outlined in Scheme 3.10, was initiated from the (-)-

Wieland-Miescher ketone ((-)-3.41).86 Accordingly, the E ring of lactone 3.53 was constructed from a Robinson annulation of diketone 3.65, which in turn was obtained from an oxidative cleavage of trisubstituted olefin 3.66 then deprotection and lactonization. Furthermore, the installation of the α-methyl group was accomplished by a hydroxyl-directed Simmons-Smith cyclopropanation followed by a reductive ring- opening sequence from allylic alcohol 3.68. This alcohol was generated by stereoselective reduction of the enone moiety after γ-hydroxymethylation of the ketal derived from ketone (-)-3.41. This route resulted in greater stereocontrol for installation of the trans vicinal quaternary methyl groups and a higher synthetic efficiency than the

80 previous methods while being amenable to large-scale production of the lactone 3.53 (15 steps, 8.3% overall yield).

Scheme 3.10. Retrosynthesis of Nolen-Sprengeler lactone 3.53

In 2001, Smith and coworkers reported the first total synthesis of one of the most architecturally complex indoloditerpenes, (-)-penitrem D (3.54).87 The retrosynthetic disconnection for penitrem D involved eventual formation of the eight-membered cyclic ether (oxocane ring) from 2-substituted indole intermediate 3.70, which was setup to utilize the Smith indole synthesis as a late-stage method to couple the terpene subunit

3.71 and the highly substituted o-toluidine ring 3.72 (Scheme 3.11).

Scheme 3.11. Smith’s retrosynthetic strategy for penitrem D 81

A model study for preparation of the oxocane-fused indole towards penitrem D was first reported by Smith et al. in 1988, which primarily focused on the challenging construction of 4,5-disubsituted indole subunit through generation of the highly substituted o-toluidine intermediate 3.72 (Scheme 3.12).88 Accordingly, formation of 3.72 was accomplished through a Semmler-Wolff aromatization reaction from cyclohexenone

3.73, which in turn was obtained from a Robinson annulation of the 6,4-fused bicyclic ketone 3.74 with ethyl vinyl ketone. This cis-fused bicyclic ketone was generated through a [2+2] photocycloaddition from methylacrylate (3.75) and enone 3.76, which was prepared from Stork-Danheiser alkylation and subsequent reduction from enone 3.77.

Scheme 3.12. Smith’s retrosynthesis of penitrem D involving prefunctionalized aniline

In conclusion, the synthesis of penitrem D was accomplished with the highly substituted o-toluidine and the advanced Nolen-Sprengeler lactone subunit. Construction of the indole subunit of penitrem D accommodated the early construction of the complex

82 cis-6,4-fused bicyclic moiety, which would be a significant synthetic challenge if introduced at a later stage in the synthetic route. One of the drawbacks with this approach is the use of a large excess (10 equiv.) of the substituted o-toluidine coupling partner to maximize the formation of the indole ring.73 This is due to the relative rates of competing processes; generation of the second anion through deprotonation of the benzylic proton tends to be slow and competes with the addition into the electrophilic ester. Furthermore, a lengthy multistep sequence was required towards the challenging installation of the trans-anti-trans CDE ring system of the terpene subunit. Therefore, overcoming these synthetic challenges faced in the construction of both indole and terpene subunits resulted in a longest linear sequence of 43 steps for the total synthesis of (-)-penitrem D.

B. Kuwahara’s approach to indolosesquiterpene, lecanindole D

In 2012, Kuwahara and coworkers reported the total synthesis of the indoloditerpene, paspalinine (3.3) through an approach that featured not only an efficient indole ring formation but also a concise stereoselective synthesis of the terpene subunit.76

Construction of the indole moiety was accomplished via a two-step sequence involving a

Stille coupling reaction and subsequent PdII-mediated intramolecular cyclization. This approach was derived from the terpene fragment 3.78 possessing a vinyl triflate moiety for coupling with o-stannylated aniline 3.80 (Scheme 3.13). More recently, this strategy was utilized with a functionally similar terpene fragment 3.79 in the total synthesis of the indolosesquiterpene, lecanindole D (3.14).77 Furthermore, the common terpene fragment

3.81 possessed the requisite functionality and stereochemistry of lecanindole D.

Scheme 3.13. Syntheses of paspalinine and lecanindole D from a common intermediate

As outlined in Scheme 3.14, the key trans vicinal quaternary methyl group was installed through a reductive transformation of the cyclopropyl ketone intermediate 3.82, which also generated the vinyl triflate moiety required in the Stille coupling reaction.

Stereoselective installation of the tertiary hydroxyl group was attained by directed epoxidation and subsequent reductive ring opening of the epoxide from homoallylic alcohol 3.83. The gem-dimethyl group was introduced through alkylation of masked enone 3.81. The approach to this key ketal intermediate 3.81 by the Kuwahara group was analogous to the sequence performed by Saxton and coworkers in their synthetic studies towards paspalicine.78 The directed Simmons-Smith cyclopropanation of the allylic alcohol derived from enone 3.84 eventually gave rise to the trans vicinal quaternary methyl group. This tricyclic intermediate was accordingly constructed from the (±)-

Wieland-Miescher ketone (3.41).

Scheme 3.14. Kuwahara’s retrosynthetic strategy for the sesquiterpene subunit

In conclusion, the above mentioned approach to the sesquiterpene subunit of lecanindole D is applicable to the structurally related indolosesquiterpene, sespendole.

The difference between these natural products, however, exists in the complexity of the indole subunit. Therefore, approaches to synthesize an appropriately functionalized indole moiety possessing flexible groups at C4-C5 are further reviewed below.

C. Kerr’s approach to the indole subunit of lolicines and lolitrems

In 2006, Kerr and coworkers based their approach to the highly substituted indole subunit of the lolicines and lolitrems on the optimization of the Plieninger indolization method. In 1956, Plieninger developed a novel indole synthesis from dihydronaphthalenamine 3.85 to generate 4-subsituted indole 3.89 (Scheme 3.15).89 The transformation involved an oxidative cleavage of fused cyclohexene 3.86 to expose a transient dialdehyde 3.87, which underwent cyclo-condensation and dehydration to provide indolyl-4-acetaldehyde 3.88. The major drawbacks to this method are low yields on the indole formation as well as lack of flexible synthetic approaches to substituted

85 dihydronaphthalenamines. The naphthalenamine ring systems were usually obtained by

Birch reduction of 2-aminonaphthalenes, often requiring harsh conditions and resulting in low yields.90

Scheme 3.15. Plieninger’s indolization sequence to 4-substituted indoles

Accordingly, Kerr and coworkers extended the utility of Plieninger indole synthesis, employing a Diels-Alder cycloaddition reaction to obtain the requisite dihydronaphthalenamines 3.92 from quinone monoimines 3.90 (Scheme 3.16). The inherent flexibility of the Diels-Alder reaction provides access to various indoles, and more importantly, provides access to 4,5-disubstituted indoles. Major disadvantages of this strategy could be the availability of various diene/dienophiles and the potential for poor regioselectivity of the cycloaddition. However, with this methodology, they were able to obtain various 5-alkoxy-,91 5-alkyl-,92 and 5-hydroxy-93 indolyl-4-acetaldehydes

(3.93). Kerr utilized this modified Plieninger methodology in approaches towards the synthesis of various substituted indole natural products.90

Scheme 3.16. Kerr’s methodology for generating 4,5-disubstituted indoles

Accordingly, application of this methodology in the synthesis of the western half of lolicines and lolitrems was reported in a recent publication.81 The key features in

Kerr’s synthesis included the tandem conjugate addition/subsequent aldol cyclocondensation for elaboration to the 6,5-fused ring system through extension of the

Plieninger indole synthesis to generate the 4,5-disubstituted indole 3.94 (Scheme 3.17).

The tandem conjugate addition/aldol cyclo-condensation reaction required the enone and aldehyde groups at C4 and C5 in intermediate 3.95, which were formed by Horner-

Wadsworth-Emmons olefination and oxidative cleavage of olefin 3.96, respectively. The

C5 olefin moiety was installed via a Suzuki coupling reaction with the corresponding aryl triflate 3.97. Using the modified Plieninger method, both the C4 and C5 chains on the indole ring were derived from oxidative cleavage of cyclohexene 3.99 followed by acid mediated condensation. The substituted dihydronaphthaleneamine was generated from the Diels-Alder cycloaddition of the corresponding quinone imine 3.101 as the dienophile and 1-methylbutadiene 3.102. This route provided rapid access to the 4,5-disubstituted indole through the Plieninger indole synthesis, but multiple changes to the oxidation state

87 of the substituents prolonged the synthetic sequence, resulting in 20 steps and 2.4% overall yield to the western half of the lolicines and lolitrems.

Scheme 3.17. Kerr’s retrosynthesis towards lolicine western half

D. Nishikawa’s approach to indolosesquiterpene, sespendole

Nishikawa and others reported the individual synthesis of the indole and terpene subunits of sespendole (3.1).82,83 Their retrosynthetic strategy involved a late stage C-ring formation with Castro-type indole synthesis of intermediate 3.103, which was envisioned through an alkyne coupling reaction of prefunctionalized aniline 3.104 and DE ring fragment 3.105 of the terpene subunit (Scheme 3.18).

Scheme 3.18. Nishikawa’s synthetic strategy for sespendole

First, Sugino et al. focused their synthetic efforts on the stereocontrolled synthesis of the sesquiterpene subunit.82 The sesquiterpene fragment 3.105 was envisioned from

(±)-Wieland-Miescher ketone 3.41 (Scheme 3.19). The alkyne required for coupling with the functionalized aniline was obtained from one carbon homologation of aldehyde

3.106. The retrosynthetic strategy featured three key reactions for installation of the trans-anti-trans relationship of the CDE ring system in sespendole. A low-valent titanium mediated stereoselective isomerization of the spiroepoxide, generated from the exo olefin, followed by stereochemical isomerization and reduction, introduced the desired trans relationship at the C/D ring junctures. Formation of the trans vicinal methyl groups featured a stereoselective [2,3]-Wittig rearrangement of allylic alcohol 3.107. The trans decalin DE ring system containing the α-tertiary alcohol was generated from homoallylic alcohol 3.109 through epoxidation and subsequent reduction of the epoxide. Homoallylic alcohol 3.109 was prepared from Wieland-Miescher ketone 3.41 by known methods.

Scheme 3.19. Nishikawa’s retrosynthesis of the sesquiterpene segment

Recently, Nishikawa and coworkers also reported the synthesis of the indole subunit of sespendole in order to ascertain the relationship of the epoxyalcohol moiety.83

Retrosynthetically, the approach uses a Castro reaction to form the indole ring (Scheme

3.20), a strategy they proposed for coupling the two halves towards the total synthesis of sespendole. Introduction of the C4-prenyl chain was envisioned through a Claisen rearrangement, while the C5-epoxyalcohol chain was anticipated via Grignard addition and epoxidation from commercially available 3-hydroxy-4-nitrobenzaldehyde 3.114.

Scheme 3.20. Nishikawa’s retrosynthesis of the indole subunit of sespendole 90

As outlined in Scheme 3.21, the synthesis was initiated by Grignard addition of 2- methyl-1-propenylmagnesium bromide into 3-hydroxy-4-nitrobenzaldehyde (3.114).

Protection of the resulting dihydroxyl intermediate as TBS ethers followed by selective deprotection of the phenol group provided 3.115. Copper-catalyzed alkylation of phenol

3.115 with 3-chloro-3-methyl-1-butyne followed by TBS ether deprotection generated an allylic alcohol, which underwent subsequent directed epoxidation with mCPBA to furnish syn epoxyalcohol intermediate 3.112β as a single diastereomer. Inversion to the corresponding anti epoxyalcohol 3.112α diastereomer was achieved by benzylic oxidation with MnO2 and stereoselective reduction of the resulting ketone. Both diastereomers were then taken forward for the installation of the C4 prenyl substituent.

Scheme 3.21. Synthesis of both diastereomers of the indole subunit of sespendole

After protection of benzylic alcohol 3.112 with TBSCl, the alkyne moiety was reduced with Lindlar’s catalyst to afford the terminal alkene 3.116. A Claisen rearrangement under basic conditions followed by aryltriflate formation provided intermediate 3.104 containing the desired C4 and C5 functionality present in sespendole.

The indole precursor was obtained after a Stille coupling reaction with tributyl-

[(trimethylsilyl)ethynyl]stannane and nitroarene reduction to aniline 3.111. This prefunctionalized aniline was subjected to the Castro indole synthesis. Finally, deprotection of the TBS ether furnished both the 3.110β and 3.110α diastereomers of the

4,5-disubstituted indole subunit of sespendole. Overall, it took 14 steps in 3.3% yield and

16 steps in 2.3% yield to prepare the two diastereomers, respectively.

With both diastereomers in hand, the authors were able to determine the stereochemical relationship of the epoxyalcohol through differences in the 1H and 13C

NMR chemical shift of the indole subunit in comparison with the natural product. As illustrated in Figure 3.7, the smaller differences observed in the chemical shifts at the epoxyalcohol moiety (positions 30 and 31) for 3.110β diastereomer over 3.110α diastereomer led the authors to propose a syn relationship of the epoxyalcohol moiety in sespendole. Although Nishikawa has reported the synthesis of both subunits, a total synthesis of sespendole has not yet been reported by the group.

Figure 3.7. Comparative NMR analysis for difference in chemical shift between both

synthesized diastereomers of the indole subunit and isolated sespendole

Chapter 4: Synthesis of the Highly Substituted Indole Subunit of Sespendole

The structural complexity, biological activity, and proposed biosynthesis of sespendole (4.1) intrigued our group and convinced us to explore the total synthesis of this natural product. As mentioned in the previous chapter, a number of groups have previously reported syntheses of the complex ring systems of related indoloterpenes.

Notably, the significant efforts of the Smith group have provided a synthetic foundation for the construction of these natural products and influenced subsequent syntheses. Our goal was to develop a distinctive and modular synthetic route to sespendole that could ultimately provide access to analogues designed to probe the biological role of this unique indolosesquiterpene and to explore the relationship between sespendole and structurally similar indoloterpene natural products.

4.1 Biosynthetic inspired approach to sespendole

Our synthetic interest in and approach to sespendole (4.1) was initially inspired by nature’s construction of the indolosesquiterpene skeleton through a cationic cyclization cascade, ultimately terminated by the indole addition to the carbocation generated via ring expansion of the cyclopentane ring. Our group was interested in mimicking this type of indole cyclization by taking advantage of the inherent nucleophilicity of the indole C3 position to induce cyclization through an intermediate, resonance stabilized indolenine.

With this in mind, a highly convergent route was designed in order to specifically address the challenging functionality present in sespendole.

The planned route featured coupling of the prenylated 4,5-disubstituted indole 4.5 to the appropriately functionalized terpene subunit 4.6, allowing both complex subunits to be prepared separately and thereby limiting the overall length of the linear sequence

(Scheme 4.1). The key transformation to indolosesquiterpene 4.2 is the carbocation induced ring expansion/cyclization cascade initiating from an intramolecular substitution reaction of tosylate 4.4 via anchimeric assistance from the electron rich indole to form the resonance stabilized carbocation 4.3. Overall, this approach was anticipated to provide an efficient synthesis of sespendole and would also potentially be applicable to the syntheses of other related indoloterpenes.

Scheme 4.1. Biomimetic retrosynthetic plan

The applicability of this unprecedented “biomimetic” approach involving carbocation induced rearrangement/cyclization was investigated by Dr. Suresh

Narayanasamy, a postdoctoral researcher in the Fuchs lab, with structurally simplified model systems which would mask the carbocation in the form of a spirocyclopropylindolenine intermediate. Rapoport and coworkers previously observed formation of a relatively stable spirocyclopropylindolenine intermediate 4.8, which our group was able to reproduce upon treatment of 3-bromoethylindole (4.7) with sodium hydride.94 Interestingly, the cyclopropane containing compound could be purified using deactivated (i.e. triethylamine washed) silica gel. We initially studied the ability of spirocycloindolenine 4.8 to generate and trap the putative resonance stabilized carbocation intermediate 4.9 with various nucleophiles (Scheme 4.2). However, this investigation resulted in formation of undesired products, potentially due to the highly reactive carbocation intermediate generated from acid-mediated activation of the spirocycloindolenine. In addition, an intramolecular variant of this “biomimetic” rearrangement and cyclization using substituted spirocyclopropylindolenines was briefly explored using a tert-butyl substituted system. The tert-butyl substituted spirocyclopropylindolenine 4.14, upon Lewis acid mediated activation, was expected to generate a secondary carbocation 4.15, leading to migration of a methyl substituent and subsequent nucleophilic addition of the indole ring into the resulting tertiary carbocation to provide 2,3-substituted indole 4.16. However, our efforts to trap the carbocation intermediate from 4.14 also failed to produce the desired cyclization and resulted in a complex mixture of products. Although not directly observed, we were concerned that an

96 indole of this types could potentially undergo cyclization at the nucleophilic C4 position if left unsubstituted, as observed in a related enzyme catalyzed cyclization experiment

(Scheme 4.3).95 In an effort to both eliminate this potential variable and explore the synthesis of the natural product sespendole, our attention has shifted to the preparation of the C4,C5-substituted indole subunit of sespendole, which would potentially allow us to emulate the biosynthesis of sespendole.

Scheme 4.2. Attempt at trapping the carbocation as spirocyclopropylindolenines

Scheme 4.3. Enzymatic synthesis of indoloditerpene with cyclization at C4 97

4.2 Initial approach to the 4,5-disubstituted indole subunit

As highlighted in the previous chapter, there are a limited number of reported synthetic approaches for functionalization at the C4 and C5 position of the indole ring in order to introduce the functionality found in sespendole (Chapter 3.4). As compared to these previous approaches utilizing functionalized anilines, our approach to the highly substituted indole subunit involves direct functionalization of the indole nucleus for introducing the epoxyalcohol unit and the prenyl chain (Scheme 4.4). The C4 prenyl substituent was envisioned via a halogen-metal/alkylation sequence from bromide 4.20, while the C5 allylic alcohol moiety was planned from Grignard addition into known aldehyde 4.21. The key substitution at C5 would be introduced by nucleophilic addition at the ortho- position into 4-nitroindole (4.22).96

Scheme 4.4. Initial retrosynthesis for installation of C4 and C5 substituents

The nitro group, in nitroarenes, deactivates aromatic rings due to their electron withdrawing effects, and as a consequence, enables the ortho- and para- positions for nucleophilic addition. Accordingly, there have been a few known examples of nucleophilic addition into nitroarenes, such as nucleophilic aromatic substitution (SNAr),

98 vicarious nucleophilic substitution (VNS) of hydrogen,97 and Bartoli’s Grignard addition

98 (BGA) chemistry. While SNAr is generally limited to non-carbon nucleophiles, the nature of the carbanionic moiety affects the course of the reaction in VNS and BGA, which utilize stabilized carbanions and organometallic species, respectively. Moreover, both SNAr and VNS are usually assisted by a leaving group on the nitroarene and the nucleophile, respectively. The BGA method, in contrast, involves a conjugate addition to a nitroarene to form a -adduct followed by oxidative decomposition to provide the ortho- and para-substituted nitroarene. This method was also extended to the formation of substituted nitroso- and amino- arenes through use of Lewis acids or reducing agents, respectively (Scheme 4.5).98,99 Under these conditions, Bartoli observed approximately a

2:1 mixture of the ortho:para substituted ring systems.

Scheme 4.5. Bartoli alkyl Grignard addition into nitroarenes with oxidative, acidic, or

reductive workup

Accordingly, we utilized Kool’s protocol96 to introduce the aldehyde group in intermediate 4.21 from 4-nitroindole (4.22) (Scheme 4.6). This transformation involved

BGA chemistry into nitroarene 4.22 at the ortho- and para- positions of the benzene ring in the indole, followed by oxidation to yield 5-methylindole 4.23a and 7-methylindole

4.23b as a 2:1 mixture of regioisomers. Selective sulfonyl protection of the indole nitrogen on the more accessible 5-methylindole 4.23a followed by enamine formation and subsequent oxidation furnished Kool’s aldehyde 4.21. With 4.21 in hand, we performed a Grignard addition with 2,2-dimethylvinylmagnesium bromide into aldehyde

4.21 to yield allylic alcohol 4.25, which was subsequently protected as the silyl ether

4.26. Unexpectedly, sodium hydrosulfite not only reduced the nitro group to the amine, but also resulted in the surprising loss of the silyl ether oxygen to provide the undesired

4-amino-5-prenylindole 4.27 as opposed to aniline 4.28. We reasoned that the initial reduction of the nitro group generated a vinylogous aminal intermediate, which could undergo elimination and subsequent reduction of the iminium ion to provide indole 4.27.

100

Scheme 4.6. Initial synthetic scheme towards the 4,5-disubstituted indole subunit

4.3 Revised approach to the 4,5-disubstituted indole subunit

We felt that a potential solution to the unexpected elimination in 4.27 would be to initiate the synthesis with 5-nitroindole to allow for the late stage introduction of this potentially reactive benzylic alcohol en route to the indole subunit of sespendole.

Furthermore, we became confident in our decision to pursue this route, since Bartoli had previously observed regioselective alkylation at C4 with alkyl Grignard addition into 5- nitroindole (4.29), as illustrated in Scheme 4.7.100 This abnormal regioselectivity, favoring the more congested site on the ring system, was also observed with Grignard addition into related heterocycles.101 A potential argument for this observation is that the

C4 position on this nitroindole is more electrophilic than C6, making it more reactive towards nucleophiles. This preference for nucleophilic addition at the C4 position is

101 supported to some degree by the calculated LUMO map of 5-nitroindole (4.29) (Figure

4.1), which shows lower electron density at C4 over C6.102

Scheme 4.7. Bartoli’s reaction for Grignard addition into 5-nitroindole

Figure 4.1. LUMO map of 5-nitroindole shows preference for nucleophilic attack

In our revised approach, shown in Scheme 4.8, we envisioned introduction of the epoxyalcohol unit at C5 through a directed epoxidation of allylic alcohol 4.31, which can be obtained from iodide 4.32. Installation of the prenyl chain at C4 was anticipated from

BGA into 5-nitroindoles 4.34.

102

Scheme 4.8. Revised strategy for the highly substituted indole subunit of sespendole

A. Method development for introduction of the C4 and C5 substituents

With the commercially available 5-nitroindole (4.29), we first looked at the feasibility of the BGA with methylmagnesium chloride for the regioselective functionalization at C4, in our hands. For this initial investigation, the choice of reagent was based on Kool’s method and the precedent by Jeon and Gluchowski103 for the BGA chemistry. Accordingly, addition of methylmagnesium chloride provided 4-methyl-5- nitroindole (4.39) exclusively, in 40% yield (Table 4.1). Although this yield was not ideal for the synthesis of the fully substituted indole subunit, optimization of the alkyl Grignard addition into nitroindole was pursued. With a 3:1 ratio of alkyl Grignard reagent to 5- nitroindole, the initial reaction optimization using different oxidizing reagents reported

98 by Bartoli for reoxidation of the intermediate nitronate adduct (e.g. DDQ, Pb(OAc)4, and KMnO4) did not show an improvement in the yield of the reaction. It was suggested by Bartoli that because of the higher 3:1 ratio of the alkyl Grignard reagent with 5- nitroindole, as compared to 2:1 ratio with other aromatic substrates, the oxidants are not

103 as effective in decomposing the intermediate nitronate adduct potentially due to incompatibility with the excess Grignard reagent.100 The acidity of the indole N-H could also lead to competitive metalation reaction, thus resulting in a lower concentration of

Grignard reagent for addition. Consequently, protecting the indole N-H before Grignard addition improved the yield of the reaction from 40% to around 60%, while also demonstrating the tolerance of a variety of potential protecting groups for the addition reaction.

Table 4.1. Optimization for methyl Grignard addition into 5-nitroindoles

We initially decided to use the sulfonyl protected intermediate 4.41 in a model system to investigate the chemistry at C5 and the overall synthetic feasibility of this route

(Scheme 4.9). Accordingly, reduction of the nitro group with sodium dithionite provided aniline 4.44, which was subjected to diazotization and subsequent Sandmeyer reaction to provide aryl iodide 4.45. Introduction of the allylic alcohol at C5 in intermediate 4.46 104 was achieved through a lithium-halogen exchange followed by addition of the in situ generated nucleophile into 3-methylcrotonaldehyde. Finally, directed epoxidation with mCPBA generated epoxyalcohol 4.47, an intermediate containing the fully functionalized

C5 chain of sespendole.

Scheme 4.9. Functionalization at C5 in the model system

B. Bartoli Grignard addition of allyl/prenyl substituent

With the chemistry at C5 primarily established, we turned our attention back to

C4 for the BGA of the necessary prenyl substituent rather than simply a methyl group.

Although the generation of prenylmagnesium bromide from 3,3-dimethylallyl bromide in the presence of magnesium has been reported in the literature,104 this procedure gave highly variable results in our hands for the formation of the Grignard reagent. As a result, introduction of this substituent would be accomplished using commercially available allylmagnesium chloride followed by an olefin cross metathesis reaction using Grubbs’ catalyst, as demonstrated in the synthesis of garsubellin A.105 Accordingly, Grignard addition into unprotected 5-nitroindole (4.29) with 3 equivalents of allylmagnesium 105 chloride followed by reoxidation with DDQ provided 4-allyl-5-nitroindole (4.48) in only

16% yield (Table 4.2, entry 1), which is less than half the yield obtained with methylmagnesium chloride (Table 4.1, entry 1). Furthermore, Grignard addition with

TIPS protected indole which would be expected to increase the conversion to the desired product likewise yielded the 4-allylindole 4.49 in an inadequate 19% yield (Table 4.2, entry 2). Overall, this represents an approximately 3-fold drop in yield as compared to the corresponding addition of methylmagnesium chloride (Table 4.1, entry 2). In fact, Bartoli found that reaction of allylmagnesium chloride with nitroarenes primarily led to decomposition of the nitronate adduct with DDQ, but primarily formed N-allyl-N- arylhydroxylamines under reductive conditions.106,107 The observed drop in yield can be potentially attributed to the reduced reactivity of allyl Grignard reagents through delocalization of the latent carbanion. Furthermore, due to the planarity of allyl radicals, the 1,2-addition into the N=O bond predominates by exclusively linking the radical to the nitrogen atom, leading to tetrahedral intermediates that get reduced to the corresponding

N-allylhydroxylamines.99

Table 4.2. Grignard addition of allyl group into 5-nitroindoles

106

C. Alternative strategies attempted for installation of C4 substituent

Due to the less than optimal results obtained for the allyl Grignard addition into 5- nitroindoles, other alternatives for installation of the C4 prenyl substituent were briefly investigated. Halogenation at C4 from 5-aminoindoles 4.50 and 4.52 was utilized by

Boger and coworkers towards their syntheses of CC-1065 and Yatakemycin (Scheme

4.10).108,109 Our attempts at halogenations at C4 using Boger’s reported conditions failed to provide the desired C4-halogenated product 4.55, presumably due to the lack of substitution at the nucloephilic C2 and C3 positions of the indole ring.

Scheme 4.10. Attempted C4-bromination based on previous reported methods

An alternative strategy involved the use of directing groups at the C5 position of the indole for affecting a directed ortho-metallation at C4, which would generate 107 nucleophilic aryl lithium species in situ, followed by alkylation with various electrophiles to provide the ortho-substituted compounds (Scheme 4.11).110,111 Our attempts at alkylation with 3,3-dimethylallyl bromide via directed ortho-lithiation did not yield the desired results, probably due to quenching of the reactive base by moisture present in the system, or from poor regioselectivity in generating the lithiated anionic species.

Scheme 4.11. Attempted directed ortho-lithiation method

4.5 2,3-Cycloalkyl-fused indole as a structural mimic

Although 5-nitroindole (4.29) itself is commercially available, generation of a

2,3-cycloalkyl-fused 5-nitroindole moiety using a Fischer indole synthesis with para- nitrophenylhydrazine (4.64) and the corresponding cyclic ketone 4.65 provided a cost- effective alternative in order to establish and potentially optimize much of the late-stage chemistry (Scheme 4.12). In fact, the 6-nitro-tetrahydrocarbazole (4.34a) could be

108 generated on large scale for less than half the cost of the unsubstituted 5-nitroindole.

Although the presence of the cycloalkyl ring was not expected to impact the indole reactivity to any significant degree, the substitution at the C2 and C3 positions also mimics the substitution found in the natural product and would provide a useful model for a linear route to the substituted indole framework of sespendole.

Scheme 4.12. Approach to the 2,3-cycloalkyl-fused 5-nitroindole

A. Fischer indole synthesis

Practical and scalable methods for the preparation of indole ring systems are important to synthetic chemists for the synthesis of natural products and pharmaceutical agents.38 The Fischer indole synthesis is one of the oldest and most versatile methods that exist for the preparation of this ring system. The reaction involves an acid catalyzed condensation between an arylhydrazine and a ketone or aldehyde to form an intermediate arylhydrazone. The arylhydrazones then undergo tautomerization, a [3,3]-sigmatropic rearrangement, and 5-exo-trig cyclization to generate the indole ring system.112 Our attempts to carry out the Fischer indole with cyclopentanone 4.65b and 4- nitrophenylhydrazine 4.64 under various reaction conditions failed to provide the desired

5-nitrocyclopenta[b]indole 4.34b and resulted primarily in recovery of the hydrazone.

109

Moreover, evidence for forming 5-nitro substituted cyclopenta[b]indoles from the corresponding para-substituted nitrophenylhydrazine are limited; requiring a two-step process in order to complete the cyclization via a [3,3]-sigmatropic rearrangement of N- trifluoroacetyl enehydrazines.113 However, this two-step method provided the cyclopenta[b]indole ring in low yields. These data indicated that a higher activation energy is required for the [3,3]-sigmatropic rearrangement to occur with cyclopentanone.

Furthermore, electron withdrawing groups on the arylhydrazine deactivate the benzene ring, resulting in the use of harsh conditions with long reaction times to affect the [3,3]- sigmatropic rearrangement while providing the indole ring in low yields.112 Although somewhat surprising, the Fischer indole reaction of cyclopentanone is known to be somewhat more challenging than that of its homologue, cyclohexanone. Fischer indole synthesis utilizing para-nitrophenylhydrazine 4.64 and cyclohexanone 4.65a in hot acetic acid generated 6-nitro-tetrahydrocarbazole (4.34a) in high yield (Scheme 4.13 - green).

As a result, it is possible that the conformational flexibility of the cyclic ketone substrate influences the orbital alignment in the transition state of the sigmatropic rearrangement to affect the cyclization to the indole ring.

Scheme 4.13. Fischer Indole synthesis to make 6 and 5 cycloalkyl fused indoles

110

An alternative method for synthesis of 2,3-substituted cycloalkylindoles entails rearrangement of 3,3-disubstituted indolenines, which can also be generated through a

Fischer indole cyclization with the corresponding cycloalkyl carboxaldehyde.114 The spirocycloalkylindolene transition state 4.66 in this method somewhat resembles our initial attempts at a “biomimetic” approach towards 2,3-fusedindoles. Consequently, using this methodology with cyclobutane carboxaldehyde, we were able to generate the cyclopenta[b]indole 4.34b in a moderate yield (Scheme 4.13 - red). For the synthesis of the indole subunit to be used for the model studies of sespendole, however, the high yielding 6-nitro-tetrahydrocarbazole (4.34a) was chosen as a suitable starting reagent over the more costly and somewhat less efficient generation of tetrahydrocyclopenta[b]indole 4.34b.

With the 2,3-cyclohexyl-fused 5-nitroindole system in hand, we reinvestigated the efficiency of the BGA for installation of a C4 substituent. As observed previously with

2,3-unsubstituted systems (Table 4.1), methylmagnesium chloride addition into N- protected indoles provided twice as much of the alkylated product (Table 4.3) with no significant difference in reaction yield. Both electron donating benzyl (Table 4.3, entry 2) and electron withdrawing tosyl groups (Table 4.3, entry 3) were once again well tolerated in this transformation, suggesting that either system could be utilized for the synthesis of the indole subunit of sespendole. As observed in the previous system, the yield for direct introduction of the allyl group (Table 4.3, entry 5) was 3-fold lower in comparison to the methyl addition into the protected indole. With this in mind and the lack of a clear

111 alternative for the introduction of the C4 substituent, the extension of the methyl group into the prenyl chain was pursued.

Table 4.3. Bartoli Grignard addition into 5-nitrotetrahydrocarbazole

B. Extension of the C4 methyl group to the prenyl substituent

Functionalization of ortho-alkyl substituted nitroarenes has been well documented for introducing various electrophilic groups on the alkyl chain. The reactivity of the vinylogous nitromethane group has facilitated generation of aldehydes through hydrolysis of the enamine intermediate formed by addition of DMF-DMA as the electrophilic reagent. In our system, use of this reagent to affect this transformation resulted in poor conversion to the corresponding enamine. The more reactive tris(dimethylamino)methane reagent, however, improved formation of enamine 4.74, which after acidic hydrolysis provided aldehyde 4.75 (Scheme 4.14). Subsequently, formation of 4.76 with the desired

C4 prenyl chain of sespendole was accomplished with a Wittig olefination reaction. 112

Further functionalization of the C5 nitro group to the epoxyalcohol side chain was then undertaken with the previously established chemistry.

Scheme 4.14. Extension of methyl group into the prenyl chain

C. Elaboration of the C5 substituent

Accordingly, the N-benzyl-5-iodoindole 4.78 was used for the halogen- metal/acylation chemistry to introduce the epoxyalcohol chain (Scheme 4.15). The reduction of nitroarene 4.76 to aniline 4.77 followed by diazotization and subsequent

Sandmeyer reaction afforded the iodide 4.78 needed for halogen-metal/acylation chemistry. Initial attempts at iodide exchange with n-BuLi, and acylation with 3- methylcrotonaldehyde did not yield the desired allylic alcohol 4.80. This represented an unexpected result based on the previous success of this reaction. An alternate two-step process was explored through halogen-metal/acylation with DMF followed by Grignard addition into the resulting ketone. However, lithium-iodide exchange and acylation with excess DMF resulted in the undesired dehalogenated compound 4.83 as the only product. 113

The reaction with more reactive electrophiles, like ethylchloroformate, was likewise met with only limited success in generating the acylated product. In the majority of the cases, the dehalogenated compound 4.83 was obtained, indicating that halogen-metal exchange was taking place, but the in situ generated anion 4.79 was either unreactive with the respective nucleophile or was simply getting quenched under the reaction conditions.

Scheme 4.15. Functionalization at C5 for attempting halogen-metal/acylation

To circumvent the problem with the moisture sensitive halogen-metal exchange reaction, alternate methods to generate carboxyaldehyde 4.81 were investigated; such as palladium-catalyzed carbonylation and reductive hydrolysis of an aryl nitrile (Scheme

4.16). Although hydrolysis of aryl nitrile 4.85 with various reagents and temperatures was unsuccessful, the palladium-catalyzed carbonylation reaction with either the reductant, triethylsilane, or with the nucleophile, ethanol, produced the cyclic enone 4.84.

This type of cyclic acylpalladation has been previously observed by Negishi et al.,115 which is a result of intramolecular olefin coupling into the carbon monoxide-palladium 114 intermediate. Although these results were somewhat unexpected, the failure of these alternative routes suggested that the halogen-metal/acylation chemistry necessitated reinvestigation and optimization in order to obtain the desired acylation product and reduce the formation of the undesired dehalogenated product.

Scheme 4.16. Investigation of other potential approaches to installation of C5 substituent

D. Optimization of halogen-metal/acylation chemistry with molecular sieves

In order to more fully explore this reaction, previously synthesized N-tosyl-5- iodo-4-methylindole 4.88 was used as a model system to optimize the halogen- metal/acylation chemistry. The reduction of nitroarene 4.71 to aniline 4.87 followed by diazotization and subsequent Sandmeyer reaction afforded the iodide 4.88 (Scheme 4.17).

Scheme 4.17. Functionalization at C5 to iodide needed for halogen-metal exchange

115

Table 4.4. Reaction conditions for halogen-metal exchange/acylation with excess DMF

Accordingly, the halogen-metal exchange of iodide 4.88 with n-BuLi followed by acylation with DMF was investigated for generation of the desired carboxaldehyde 4.89

(Table 4.4). With both THF and DMF used after drying and distilling over lithium aluminum hydride and calcium hydride, respectively, the initial exchange and acylation provided dehalogenated compound 4.90. This result indicated that halogen-metal exchange was taking place, but the anion generated in situ was most likely getting quenched by moisture present in the reaction system. In order to reduce the exposure of moisture in the system, the time of addition of DMF was reduced from 30 minutes to 5 minutes and eventually to sequential addition of n-BuLi and DMF. These experiments demonstrated that the halogen metal exchange was extremely rapid under these 116 conditions. Unfortunately, although these modifications led to an improvement in the formation of carboxaldehyde 4.89, dehalogenation was still observed as the major product 4.90. Subsequently, absorbents, specifically molecular sieves, were utilized in the reaction to counteract any moisture that may be present in the reaction. Despite the fact that we were unaware of any previous reports using a desiccant of this type in a halogen- metal exchange reaction, we were delighted to find that addition of 4Å molecular sieves to the reaction flask prior to addition of n-BuLi and DMF, significantly enhanced formation of aldehyde 4.89 over dehalogenated compound 4.90, thus improving the yield for the reaction sequence. The formation of the desired product rather than the dehalogenated product could easily be monitored through analysis of the 1H NMR spectra of the crude reaction mixtures (Figure 4.2).

117

Figure 4.2. 1H NMR spectra of the crude reaction mixtures of the halogen-metal

exchange/acylation reaction with DMF

E. Unanticipated rearrangement

Utilizing these optimized conditions, the halogen-metal acylation sequence was applied to the N-benzyl-5-iodoindole system to generate aldehyde 4.81 in 80% yield.

Subsequently, a Grignard addition of the vinyl group into C5 aldehyde was predicted to provide the allylic alcohol necessary for epoxidation. Grignard addition with 2- 118 methylvinylmagnesium bromide into carboxaldehyde 4.89 followed by 10% aqueous

NH4Cl workup, however, did not furnish the desired allylic alcohol cleanly. Purification of the crude material using silica gel flash chromatography provided a compound containing a trans olefin, identified by two characteristic olefin protons with large coupling constants (J = 16 Hz), as the only isolate. Formation of this trans olefinic compound 4.92 is possible through activation of the benzylic alcohol 4.80 with loss of water to an azafulvene intermediate 4.91 followed by re-addition of water at the terminal olefin with concomitant re-aromatization of the indole nucleus (Scheme 4.18). This unanticipated rearrangement is activated presumably by acidic medium during either the workup or purification conditions. Formation of this product was independently confirmed via Heck coupling of iodide 4.78 with commercially available 2-methyl-3- buten-2-ol (4.93) and suggests the highly reactive nature of the benzylic alcohol. This data also made sense in the context of the loss of the silyl ether functionality observed in our earlier studies (Scheme 4.6).

119

Scheme 4.18. Unanticipated rearrangement of Grignard addition reaction and

confirmation through Heck coupling

At this stage, a number of potential approaches were considered in an effort to deal with this unanticipated result. We initially considered utilizing the rearranged product via a Payne rearrangement for formation of the epoxyalcohol with the expectation that the equilibrium would favor the more substituted epoxide.116

Alternatively, this product could be employed in a direct epoxidation reaction which would provide a “biosynthetic” intermediate that may be useful for the preparation of the complex tricyclic ring systems of the lolicines or the nodulisporic acid systems,57 although this would not move us closer to the desired goal of a synthesis of sespendole.

Ultimately we reasoned that the formation of the azafulvene intermediate 4.91 (although relatively facile) could be avoided by maintaining a neutral or basic environment for the

120 reaction mixture and product. Fortunately, using a basic workup with 50% aqueous

NaHCO3 and base washing silica gel with triethylamine for flash chromatography, we were able to obtain the desired alcohol 4.80 necessary for epoxidation.

F. Attenuation of indole electron density with tosyl group

At this stage, more careful consideration of the nature of the indole nitrogen protecting group was made. Based on the electron rich nature of the benzyl protected indole nitrogen, potential limitations for benzyl deprotection with respect to the selectivity over the prenyl group under hydrogenation conditions, and sensitivity of the allylic alcohol to protic or Lewis acid mediated removal of the benzyl group, the decision was made to switch to the N-tosyl system. Interestingly, attenuation of the electron density of the indole ring with the electron withdrawing tosyl group still failed to completely remediate the rearrangement issue. We were delighted to find, however, that workup with 10% aqueous NH4Cl was tolerated by this system. In addition, meticulous conditions were employed to limit rearrangement of the desired allylic alcohol by freshly deactivating the silica gel and using neutralized solvents (i.e. CDCl3) for spectral analysis. As a result, we focused our efforts with the N-tosyl indole derivative for the synthesis of the fully functionalized indole subunit of sespendole.

Accordingly, the N-tosyl protected indole 4.71 was subjected to the same reaction sequence as the N-benzyl system to provide aldehyde 4.97 (Scheme 4.19). Of note, however, is the change in conditions for the diazotization and Sandmeyer reaction sequence. Using the two-step protocol used in the benzyl system primarily provided the

121 reduced, dehalogenated, product. Fortunately, the one-step protocol for iodination via aprotic diazotization of aromatic and hetero-aromatic amine compounds established by

Knochel and coworkers provided iodide 4.96 exclusively.117 Finally, Grignard addition with 2-methylvinylmagnesium bromide, with the meticulous workup and purification methods mentioned above, furnished the desired allylic alcohol 4.98.

Scheme 4.19. Synthesis of the highly substituted indole subunit in sespendole

4.6 Completion of the synthesis of the fully substituted indole subunit

A. Directed epoxidation of allylic alcohol

With the desired allylic alcohol in hand, all that remained at this stage was a directed epoxidation of the allylic alcohol to provide the fully elaborated indole subunit of sespendole. Based on Nishikawa and coworkers’ proposed syn relationship of the epoxy group to the alcohol in sespendole, we explored our options for the directed epoxidation of allylic alcohol 4.98. A comparative study by Sharpless and coworkers between peracids and transition metal-hydroperoxide mediated directed epoxidation of 122 acyclic secondary allylic alcohols revealed that with 3,3-dimethylallylic alcohol 4.99, a greater stereoselectivity of the threo epoxidation product 4.100 was obtained using mCPBA over vanadium with tert-butyl hydroperoxide (Scheme 4.20).118 In addition, the proposed preferred conformations for the stereoselective epoxidation of acyclic allylic alcohols with peracids and transition-metal hydroperoxide reagents were deduced from the results. The preferred conformations, illustrated in Figure 4.3, provide a useful model for predicting the origin of stereoselectivity in epoxidations of acyclic allylic alcohols.

Scheme 4.20. Stereoselective epoxidation of 4-methylpent-3-en-2-ol118

123

Figure 4.3. Predicted dihedral angles in the stereoselective epoxidation

of acyclic allylic alcohols118

With the previous success of epoxidation in the model system and evidence in the synthesis by Adachi et al.,83 we initially attempted this transformation of allylic alcohol

4.98 with mCPBA. The proposed transition state geometry predicted a strong preference of the threo conformer 4.108, as suggested by an undesired A1,3-strain of the erythro conformer 4.109 shown in Figure 4.4. However, epoxidation with and without the addition of basic salt buffers, resulted in a complex mixture of products, potentially due to the possible elimination of the labile allylic alcohol or competitive epoxidation with the C4 prenyl chain. This lack of regioselectivity towards an allylic alcohol has also been previously shown in the epoxidation of geraniol, where mCPBA showed preference for the olefinic site (Δ6,7) furthest away from the alcohol.119

124

Figure 4.4. Predicted origin of stereoselectivity for the directed epoxidation with mCPBA

In the same study, vanadium-hydroperoxide mediated epoxidation was shown to be more regioselective for the 2,3 double bond in the directed epoxidation of geraniol.

Accordingly, in our hands the directed epoxidation of allylic alcohol 4.98 with vanadium- catalyzed tert-butyl hydroperoxide generated epoxyalcohol 4.110 (Scheme 4.21).

Analysis of the proposed transition state of the epoxidation reaction predominantly suggested formation of the threo epoxyalcohol 4.110β, due to existence of A1,3-strain between the indole ring and the methyl substituent in conformation 4.112 leading to the erythro product 4.110α. As a result, the threo product would be expected to provide the desired syn relationship of the epoxy group to the alcohol needed for the synthesis of sespendole as suggested by Nishikawa.

125

Scheme 4.21. Synthesis of the epoxyalcohol moiety through stereoselective epoxidation

B. Comparative NMR spectral analysis of the fully substituted indole subunit

With epoxyalcohol 4.110 in hand, extensive NMR experiments were performed in order to not only confirm the structure, but also to confirm the proposed syn relationship of the epoxyalcohol moiety. Spectral analyses of 1H, 13C, HSQC, HMBC, and COSY, measured in CDCl3, confirmed the structure and connectivity of compound 4.110.

Furthermore, analysis of NOESY data revealed the same through-space 1H correlations observed by Ōmura and coworkers in the structure elucidation of the indole subunit towards identification of sespendole (Figure 4.5). However, the NOESY correlation experiment could not unambiguously establish the relationship of the epoxy group to the alcohol. In addition, since our compound was structurally and electronically different 126 with the N-tosyl group on the indole ring, a direct comparison of the chemical shifts in the NMR spectra could not be made to establish the epoxyalcohol relationship.

Deprotection of the indole nitrogen atom was therefore pursued.

Figure 4.5. NOE correlation of N-tosylindole subunit 4.110 measured in CDCl3

Thus, magnesium-mediated methanolysis of the tosyl group furnished the corresponding deprotected indole subunit of sespendole, 4.113 (Scheme 4.22). The proposed syn relationship of the epoxyalcohol moiety was confirmed through NMR spectral analyses of our indole subunit 4.113 with the reported syn and anti diastereomers

4.114β and 4.114α reported by Nishikawa, and the indole subunit of sespendole as reported by Ōmura.

Scheme 4.22. Completion of the highly substituted indole subunit of sespendole

127

Comparisons of the NMR spectra of this product with the reported diastereomers revealed distinctive 1H and 13C chemical shifts at C30 and C31 indicative of the syn

1 epoxyalcohol. As illustrated in Figure 4.7, H chemical shifts measured in pyridine-d5 of the epoxyalcohol chain (H30, H31) in our compound 4.113 are in closer proximity to the syn epoxyalcohol diastereomer 4.114β as compared to the anti diastereomer 4.114α. In

1 addition, a direct comparison of the H NMR spectra measured in CDCl3 between sespendole (4.1) and the synthetic compound 4.113 also indicates the same relationship, based on the chemical shifts of 26-H, H-30, H-31, and 30-OH (Figure 4.8).

Figure 4.6. Structures of the synthetic molecules and natural product, sespendole

128

syn diastereomer 4.114 30-H 31-H

Indole subunit 4.113

anti diastereomer 4.114α

Figure 4.7. 1H NMR comparison between our compound 4.113 (middle) and Nishikawa’s

reported syn (top) and anti (bottom) diastereomers in C5D5N.

129

26-H

Indole subunit 4.113

1 Figure 4.8. H NMR (400 MHz, CDCl3) comparison between

sespendole (4.1) (top) and compound 4.113 (bottom)

Structural elucidation of compound 4.113 by NMR experiments (1H, 13C, HSQC and HMBC), facilitated the unambiguous assignment of all the protons and carbons on the indole subunit, except the cyclohexane ring. Furthermore, a comparison of selected

1H and 13C NMR chemical shift data with sespendole (4.1) against the synthetic epoxyalcohol 4.113, the previously reported syn diastereomer 4.114β and anti diastereomer 4.114α is illustrated in Figure 4.9. The shifts corresponding to the benzylic and epoxide atoms at 30, 31, and 32 are in agreement with both sespendole and 4.114β, but distinctly different from 4.114α. Overall, these results and the predicted outcome of the vanadium-catalyzed directed epoxidation comprehensively support the generation of the syn diastereomer 4.113 of the fully substituted indole subunit found in sespendole.

130

1H Chemical Shift difference with sespendole 0.50 0.40 0.30 0.20

0.10 4.113

0.00 (ppm) 4.114β

Dd -0.10 4.114α -0.20 -0.30 -0.40 -0.50 H18 H19 H25a H25b H26 H28 H29 H30 H31 H33 H34

13C Chemical Shift difference with sespendole 4.0

3.0

2.0

1.0

0.0 4.113 (ppm)

-1.0 4.114β Dd 4.114α -2.0

-3.0

-4.0

-5.0 C15 C16 C17 C18 C19 C20 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34

1 13 Figure 4.9. Difference in H and C NMR chemical shifts (400MHz, C5D5N) between

sespendole and the synthetic compounds (4.113, 4.114β, 4.114α)

131

4.7 Conclusion

The synthesis to the highly substituted indole subunit of sespendole via direct functionalization of the indole ring was accomplished in 10 steps from 5-nitroindole

4.34a to the epoxyalcohol 4.110 in 4% overall yield, while establishing a modular synthetic route to the indole subunit of sespendole. The synthetic route featured introduction of the prenyl chain through a Bartoli Grignard addition at C4 and installation of the C5 epoxyalcohol moiety through a halogen-metal/acylation sequence followed by a vanadium-catalyzed directed epoxidation. Comparison of the 1H and 13C NMR spectra of the epoxyalcohol 4.113 with both synthetic diastereomers prepared by Adachi et al. and the isolated natural product, further confirmed the presence of the syn relationship of the epoxyalcohol in the natural product.

132

Chapter 5: Experimental Section

Materials and Methods:

All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions unless otherwise stated. Oven-dried syringes were used to transfer air and moisture sensitive liquids. All commercial reagents, anhydrous solvents, and reagent-grade solvents were purchased from Sigma-Aldrich, Fisher Scientific, and VWR and used as received without further purification, unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) using aluminum backed pre-coated silica gel plates from (TLC Silica Gel F-254, 200 µm, Dynamic Adsorbents) using UV light as the visualizing agent and ceric ammonium molybdate (CAM), or iodine vapor, or acidic mixture of 2,4-dinitrophenylhydrazine (2,4-DNP), and heat as developing agents. Flash chromatography was performed using silica gel (60Å, pore size 32-63 µm, Dynamic

Adsorbents), unless otherwise stated. Silica gel was deactivated by first washing with a

10% triethylamine solution in the eluent and then washing with the eluent itself (3x).

Deuterated solvents for NMR were purchased from Cambridge Isotope Labs and used as received. However, CDCl3 stored with K2CO3 was used for acid sensitive compounds.

NMR spectra were recorded on Bruker DPX250, AV300, or DRX400 MHz spectrometers and calibrated using the residual undeuterated solvent peak (CDCl3: δ 7.26

133

1 13 1 13 ppm H NMR, 77.16 ppm C NMR; acetone-d6: δ 2.05 ppm H NMR, 206.26 ppm C

1 13 1 NMR; DMSO-d6: δ 2.50 ppm H NMR, 39.52 ppm C NMR; CD3OD: δ 3.31 ppm H

13 1 13 NMR, 49.00 ppm C NMR; Pyr-d5: δ 8.71 ppm H NMR, 149.90 ppm C NMR).

Proton (1H) NMR data is reported as follows: chemical shift in ppm (multiplicity [as: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, sept = septet, m

= multiplet, br = broad], coupling constant(s) in Hz, integration). Carbon (13C) NMR data was reported as chemical shift (δ) in ppm. Infrared (IR) data were obtained with Thermo-

Scientific Nicolet 6700 FT-IR and are reported as frequency of absorption (cm-1).

Melting points were obtained on a Thomas Hoover Uni-melt capillary melting point apparatus. High resolution mass spectra (HRMS) were recorded on Bruker microTOF

(time-of-flight) mass spectrometer by electrospray ionization (ESI) time of flight experiments and reported as m/z.

134

Part I: Design of allosteric HIV integrase inhibitors

3-Bromo-4-chloro-2-methylquinoline (2.15). 4-hydroxy-2-methylquinoline

(2.14) (1.19 g, 7.50 mmol) in acetic acid (34 mL) was treated with N-bromosuccinimide

(1.33 g, 7.50 mmol) and the resulting suspension was heated to 60 °C and stirred for 2 h.

The mixture was cooled to room temperature, cold water was added and the formed precipitate was filtered and collected. The solid was washed sequentially with water, saturated aqueous NaHCO3, and acetone, and then dried to provide 3-bromo-4-hydroxy-

2-methylquinoline (1.63 g, 91%) as a white solid. This material was taken directly into

1 the chlorination reaction with no further purification: H NMR (300 MHz, DMSO-d6) δ

12.15 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H),

13 7.36 (t, J = 7.5 Hz, 1H), 2.56 (s, 3H). C NMR (75 MHz, DMSO-d6) δ 171.0, 148.6,

138.6, 131.9, 125.3, 123.6, 122.7, 117.9, 105.9, 21.4.

A suspension of 3-bromo-4-hydroxy-2-methylquinoline (1.55 g, 6.50 mmol) in

POCl3 (5.95 mL, 65 mmol) was stirred at 80 °C for 2 h. After cooling to room temperature, the mixture was poured onto ice (~30 g) and neutralized with 50% (w/v) aqueous NaOH while maintaining the temperature at 0 °C. The resulting mixture was extracted with EtOAc (3x) and the combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to afford chloride 135

1 2.15 (1.64 g, 98%) as a white solid: H NMR (300 MHz, CDCl3) δ 8.18 (d, J = 8.4 Hz,

1H), 8.01 (d, J = 8.4 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 2.91 (s,

13 3H). C NMR (75 MHz, CDCl3) δ 158.4, 146.6, 142.2, 130.5, 129.2, 127.7, 126.1,

124.7, 119.9, 27.6.

4-Chloro-2-methyl-3-vinylquinoline (2.16). A mixture of 3-bromo-4-chloro-2- methylquinoline (2.15) (880 mg, 3.43 mmol), tributyl(vinyl)tin (1.05 mL, 3.60 mmol),

Pd(PPh3)4 (396 mg, 0.34 mmol) in DMF (17 mL) was stirred overnight at 100 °C. After cooling to room temperature, the reaction was quenched with 10% (w/v) aqueous KF (20 mL), filtered through Celite and washed with ethyl acetate. The filtrate was transferred to a separatory funnel and extracted with ethyl acetate (3x). The combined organic phases were washed with 10% aqueous KF, brine, dried over sodium sulfate, and concentrated under reduced pressure. Flash chromatography (silica gel, 5% EtOAc in hexanes)

1 afforded vinylquinoline 2.16 (459 mg, 66%) as a clear oil: H NMR (400 MHz, CDCl3) δ

8.22 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.71 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H),

7.57 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.87 (dd, J = 17.9, 11.6 Hz, 1H), 5.80 (dd, J = 11.6,

13 1.3 Hz, 1H), 5.65 (dd, J = 17.9, 1.3 Hz, 1H), 2.76 (s, 3H). C NMR (75 MHz, CDCl3) δ

157.8, 147.0, 140.4, 132.0, 130.2, 130.0, 128.9, 126.9, 125.3, 124.5, 122.9, 25.4.

136

Diol 2.17. A mixture of vinylquinoline 2.16 (305 mg, 1.50 mmol), N- methylmorpholine N-oxide (264 mg, 2.25 mmol), osmium tetroxide polymer-bound (15 mg, 0.01 g/mmol) in acetone (15 mL), tert-butanol (3 mL), and water (1.5 mL) was stirred overnight at 85 °C. The resulting yellow solution was cooled to room temperature, filtered through Celite and washed with acetone. The filtrate was concentrated under reduced pressure and suspended in CH2Cl2. The organic phase was washed with water and the resulting aqueous phase was extracted with CH2Cl2 (3x). The combined organic phases were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. Flash chromatography (silica gel, 5% MeOH in

1 CH2Cl2) provided diol 2.17 (311 mg, 77%) as a pale yellow solid: H NMR (300 MHz,

DMSO-d6) δ 8.18 (dd, J = 8.3, 0.8 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.78 (ddd, J = 8.4,

7.0, 1.4 Hz, 1H), 7.67 (ddd, J = 8.1, 7.1, 1.2 Hz, 1H), 5.73 (d, J = 4.3 Hz, 1H), 5.49 (ddd,

J = 7.2, 6.0, 4.5 Hz, 1H), 4.93 (t, J = 5.9 Hz, 1H), 3.82 (ddd, J = 11.2, 7.2, 5.7 Hz, 1H),

1 3.73 – 3.61 (m, 1H), 2.85 (s, 3H). H NMR (300 MHz, DMSO-d6-D2O wash) δ 8.18 (d, J

= 7.8 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.78 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.66 (ddd, J

= 8.2, 6.9, 1.0 Hz, 1H), 5.47 (dd, J = 7.1, 6.3 Hz, 1H), 3.80 (dd, J = 11.1, 7.3 Hz, 1H),

13 3.68 (dd, J = 6.0 Hz, 1H), 2.83 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 159.2, 146.3,

139.9, 131.8, 130.0, 128.4, 127.2, 124.4, 123.9, 71.3, 63.7, 25.1. 137

Pivalate 2.18. To a suspension of diol 2.17 (190 mg, 0.80 mmol) in THF (3.2 mL) at 0 °C was slowly added 2M HCl in Et2O (0.50 mL, 1.0 mmol). After the addition was complete, the ice bath was removed and the resulting pale yellow suspension was stirred at room temperature for 1 h before being concentrated under reduced pressure to give a pale yellow solid. The solid was suspended with NaI (600 mg, 4.0 mmol) in anhydrous acetonitrile (6.40 mL) and stirred at reflux for 18 h. After cooling to room temperature, the mixture was diluted with CH2Cl2 and H2O. The phases were separated and the aqueous phase was extracted with CH2Cl2. The combined organic phases were washed with 10 % aqueous Na2S2O3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure to give a yellow solid. The crude solid was suspended in CH2Cl2 (7.1 mL), treated sequentially with pivaloyl chloride (183 L, 1.49 mmol) and triethylamine (119 L, 0.85 mmol), and then stirred overnight at room temperature. The clear solution was diluted with water, partitioned, and extracted with

CH2Cl2 (3x). The combined organic phases were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. Flash chromatography (silica gel, 1% MeOH in CH2Cl2) afforded pivalate 2.18 (206 mg, 62 %)

1 as a pale yellow solid. 2.18a: H NMR (300 MHz, CDCl3) δ 8.09 (d, J = 8.3 Hz, 1H),

138

7.93 (d, J = 8.3 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 5.85 (dd, J =

8.3, 4.3 Hz, 1H), 4.70 – 4.60 (m, 1H), 4.35 (dd, J = 11.5, 3.8 Hz, 1H), 2.98 (s, 3H), 1.21

1 (s, 9H). 2.18b: H NMR (250 MHz, CDCl3) δ 8.21 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 8.4

Hz, 1H), 7.73 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.58 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H), 5.82 (s,

1H), 4.69 (dd, J = 11.5, 8.6 Hz, 1H), 4.36 (dd, J = 11.5, 4.9 Hz, 1H), 3.08 (s, 1H), 2.94 (s,

13 3H), 1.19 (s, 9H). C NMR (63 MHz, CDCl3) δ 179.0, 158.6, 147.5, 141.8, 130.6, 128.9,

128.7, 127.3, 125.2, 124.4, 70.2, 65.9, 39.0, 27.3, 25.4.

tert-Butyl ether 2.19. Perchloric acid (235 L, 1.65 mmol) was added to a suspension of pivalate 2.18 (206 mg, 0.50 mmol) in tert-butyl acetate (5 mL). The mixture was stirred at room temperature for 2 h, then quenched with water and neutralized with saturated NaHCO3 solution. The aqueous phase was extracted with

EtOAc (3x). The combined organic phases were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. Flash chromatography (silica gel, 1% → 5% → 10% EtOAc in hexanes) provided ether 2.19

1 (215 mg, 92%) as a pale yellow solid. 2.19a: H NMR (300 MHz, CDCl3) δ 8.09 (d, J =

8.5 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H),

5.64 (dd, J = 8.2, 5.3 Hz, 1H), 4.39 (dd, J = 10.8, 9.2 Hz, 1H), 4.22 (dd, J = 11.3, 4.8 Hz,

139

1 1H), 2.99 (s, 3H), 1.17 (s, 18H). 2.19b: H NMR (300 MHz, CDCl3) δ 8.19 (d, J = 8.1

Hz, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.70 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.56 (ddd, J = 8.1,

7.0, 0.9 Hz, 1H), 5.76 (dd, J = 7.9, 6.3 Hz, 1H), 4.42 (dd, J = 11.2, 8.0 Hz, 1H), 4.22 (dd,

J = 11.2, 6.2 Hz, 1H), 2.96 (d, J = 6.9 Hz, 3H), 1.16 (s, 9H), 1.11 (s, 9H). 13C NMR (75

MHz, CDCl3) δ 178.4, 159.6, 147.3, 141.1, 130.7, 130.2, 128.9, 127.1, 124.8, 124.6,

75.6, 69.3, 65.4, 38.9, 28.3, 27.3, 25.6.

Biaryl adduct 2.20. A mixture of t-butyl ether 2.19 (150 mg, 0.32 mmol), chroman-6-ylboronic acid 2.25 (71 mg, 0.40 mmol), potassium carbonate (133 mg, 0.96 mmol), Pd(PPh3)4 (37 mg, 0.032 mmol) in DMF (4 mL) and water (0.4 mL) was heated to 110 °C and stirred overnight. After cooling to room temperature, the mixture was diluted with cold water, filtered through Celite and washed with ethyl acetate. The filtrate was partitioned and the aqueous phase was extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Flash chromatography (silica gel, 20% EtOAc in hexanes)

1 afforded biaryl adduct 2.20 (142 mg, 93%) as a white foam: H NMR (300 MHz, CDCl3)

δ 8.04 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 9.8, 4.2 Hz, 1H), 7.42 – 7.30 (m, 2H), 7.04 –

140

6.88 (m, 2H), 6.60 (d, J = 17.9 Hz, 1H), 4.86 (ddd, J = 11.5, 8.4, 2.5 Hz, 1H), 4.35 (t, J =

9.8 Hz, 1H), 4.12 (d, J = 5.0 Hz, 1H), 3.02 (s, 3H), 2.88 – 2.79 (m, 2H), 2.72 (t, J = 6.5

Hz, 1H), 2.16 – 2.06 (m, 2H), 1.96 (dt, J = 11.7, 6.0 Hz, 1H), 1.15 (s, 9H), 0.97 (s, 9H).

Alcohol 2.21. Biaryl adduct 2.20 (142 mg, 0.30 mmol) was dissolved in methanol

(1.5 mL) and treated with 3N aqueous NaOH (0.5 mL, 1.49 mmol). After stirring for 2 h, the solution was concentrated under reduced pressure and diluted with water (10 mL).

The aqueous phase was extracted with EtOAc (3x), the combined organic phases were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. Flash chromatography (silica gel, 50% EtOAc in hexanes then flush with acetone) provided alcohol 2.21 (99 mg, 85%) as a white solid: 1H

NMR (300 MHz, CDCl3) δ 8.00 (d, J = 8.4 Hz, 1H), 7.62 (ddd, J = 8.3, 4.2, 1.5 Hz, 1H),

7.38 – 7.24 (m, 2H), 7.03 – 6.80 (m, 3H), 4.81 – 4.70 (m, 1H), 4.29 (t, J = 5.1 Hz, 2H),

3.84 (t, J = 10.5 Hz, 1H), 3.53 (t, J = 8.8 Hz, 1H), 2.98 (s, 3H), 2.94 – 2.71 (m, 2H), 2.27

(dd, J = 24.8, 10.5 Hz, 1H), 2.11 (t, J = 8.3 Hz, 2H), 1.04 (s, 9H).

141

BI-D (2.13). A mixture of alcohol 2.21 (97 mg, 0.25 mmol) and PDC (466 mg,

1.24 mmol) in DMF (0.95 mL) was stirred overnight at room temperature. The mixture was quenched with cold water and extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The concentrate was dissolved in 3N aqueous NaOH and washed with

EtOAc (3x). The aqueous phase was acidified to pH 5 and extracted with EtOAc (3x).

The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to yield 2.13 as a mixture of atropisomers (70 mg,

1 70%) as a white solid: mp 157.8-161.2 °C ; H NMR (300 MHz, CDCl3) δ 8.05 (d, J =

8.2 Hz, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 8.6 Hz, 1H), 7.44 – 7.33 (m, 2H), 7.01

(dd, J = 16.9, 7.4 Hz, 1H), 6.94 (t, J = 8.9 Hz, 1H), 5.40 (s, 1H), 4.29 (s, 2H), 2.97 – 2.69

1 (m, 5H), 2.09 (d, J = 5.3 Hz, 2H), 1.01 (s, 9H) H NMR (400 MHz, DMSO-d6) δ 12.93

(s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.49 – 7.41 (m, 1H), 7.41 –

7.34 (m, 1H), 7.14 (d, J = 13.9 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.95 (t, J = 8.8 Hz, 1H),

5.14 (d, J = 11.7 Hz, 1H), 4.24 (t, J = 4.3 Hz, 2H), 2.91 – 2.63 (m, 2H), 2.70 (d, J = 0.9

13 Hz, 3H), 2.06 – 1.90 (m, 2H), 0.91 (d, J = 2.2 Hz, 9H). C NMR (75 MHz, DMSO-d6) δ

173.6, 158.5, 154.6, 154.6, 146.1, 145.8, 145.7, 131.7, 131.3, 130.1, 129.1, 128.9, 128.8,

142

128.2, 126.8, 126.4, 125.9, 122.4, 122.2, 116.4, 116.0, 75.3, 75.2, 70.0, 66.2, 66.1, 27.8,

27.7, 27.2, 24.5, 24.4, 24.3, 21.7.IR (film): 2973, 1733, 1578, 1491, 1366, 1235, 1127,

−1 + 756 cm ; HRMS-ESI (M+H) calcd for C25H27NO4 406.2018, found 406.2015.

Chroman-6-ylboronic acid (2.25). To a solution of 6-iodochroman (2.24)1 (130 mg, 0.50 mmol) in THF (3.3 mL) at -78 °C was added nBuLi (2.5M, 250 L, 0.625 mmol), dropwise and stirred for 30 min at -78 °C. The cooled solution was treated with of B(OMe)3 (114 L, 1.0 mmol) and stirred for 30 min at -78 °C before gradually warming the reaction to room temperature over 2 h. The reaction was quenched with water and the aqueous phase was extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The concentrate was dissolved in 3N aqueous NaOH (5 mL) and washed with EtOAc (3x). The aqueous phase was acidified to pH 1 and extracted with

EtOAc (3x). The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to afford boronic acid 2.25 (75 mg, 84

1 %) as a white solid: H NMR (300 MHz, CDCl3) δ 7.95 (d, J = 8.2 Hz, 1H), 7.89 (s, 1H),

6.90 (d, J = 8.1 Hz, 1H), 4.27 (d, J = 4.8 Hz, 2H), 2.91 (t, J = 6.2 Hz, 2H), 2.13 – 2.02

(m, 2H).

143

Preparation of 3-aryl-N-methylindole 2.30:

General procedure A for 3-hydroxy-3-arylindolin-2one 2.29-1. Isatin (2.29) (1 equiv.) was dissolved in THF (0.2 M) and cooled to 0 °C. Grignard reagent (2.5 equiv.) was added slowly to the mixture and the resulting solution was gradually warmed to rt and stirred for 14 h. The reaction was quenched with the addition of 2N HCl. The resulting aqueous solution was extracted with EtOAc (3x). The combined organic layers were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure.

General procedure B for 3-arylindole 2.29-2. A solution of 3-substituted-3- hydroxyindolin-2-one (1 equiv.) in THF (0.1 M) was cooled to 0 °C. LiAlH4 (4.5 equiv.) was added portion-wise to the cooled mixture. After stirring at 0 °C for 15 min, the ice- bath was removed and the reaction stirred at 75 °C for 4 h. After cooling the reaction to 0

°C, the mixture was quenched using the Fieser work-up method: following reduction with

LiAlH4 (x grams), dropwise addition of H2O (x mL), then dropwise addition of 3N NaOH

144

(x mL), followed by addition of H2O (3x mL) and stir for 30 min. The precipitated aluminum salt was filtered out over Celite, washed with EtOAc, and the filtrate was concentrated under reduced pressure.

General procedure C for 3-aryl-N-methylindole 2.30. A solution of 3- substituted indole (1 equiv.) in DMF (0.2 M) was cooled to 0 °C before adding NaH

(60%, 1.25 equiv.). After stirring the resulting mixture at 0 °C for 30 min, iodomethane

(1. 5 equiv.) was added to the reaction. The ice-bath was removed and the reaction stirred at rt for 2 h. The reaction was quenched with cold H2O and the resulting solution was extracted with 50% EtOAc in hexanes (3x). The combined organic layers were washed with cold H2O, brine, dried over sodium sulfate, and concentrated under reduced pressure. The concentrate is dissolved in toluene, concentrated and then dried under reduced pressure to remove residual DMF.

3-Hydroxy-3-phenylindolin-2-one (2.29-1a). This reaction was performed using general procedure A with isatin (2.29) (1.03 g, 7.0 mmol), phenylmagnesium bromide

(1M in THF, 17.5 mL, 17.5 mmol), and THF (47 mL). The crude material was purified by flash column chromatography (silica gel, 30% EtOAc in hexanes) to afford 2.29-1a 145

(1.34 g, 85%) as an orange solid. Recrystallization in hot ethanol provided pure 2.29-1a

1 as pale orange crystals: H NMR (300 MHz, DMSO-d6) δ 10.38 (s, 1H), 7.36 – 7.20 (m,

6H), 7.09 (d, J = 6.9 Hz, 1H), 6.96 (td, J = 7.5, 0.6 Hz, 1H), 6.90 (d, J = 7.7 Hz, 1H),

13 6.61 (s, 1H). C NMR (75 MHz, DMSO-d6) δ 178.5, 141.9, 141.5, 133.7, 129.2, 128.1,

+ 127.4, 125.4, 124.8, 122.0, 109.8, 77.3. HRMS-ESI (M+Na) calc. for C14H11NO2Na

248.0687, found: 248.0674.

3-(Chroman-6-yl)-3-hydroxyindolin-2-one (2.29-1c). A vigorously stirred mixture of isatin (2.29) (118 mg, 0.80 mmol) and 6-iodochroman (2.24) (260 mg, 1.00 mmol) in THF was cooled to -78 °C before the dropwise addition of n-BuLi (2.5M in

THF, 0.720 mL, 1.80 mmol). The reaction was stirred at -78 °C for 30 min and then quenched with 2N HCl (3 mL). After the CO2-acetone bath was removed and the mixture was warmed to rt, the aqueous solution was extracted with EtOAc (3x). The combined organic layers were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 30% EtOAc in hexanes) to afford 2.29-1c (99

1 mg, 44%) as an orange-white solid: H NMR (250 MHz, acetone-d6) δ 9.30 (s, 1H), 7.26

146

(td, J = 7.6, 1.3 Hz, 1H), 7.20 (d, J = 7.4 Hz, 1H), 7.14 – 6.93 (m, 4H), 6.65 (d, J = 8.4

Hz, 1H), 5.36 (s, 1H), 4.12 (t, J = 5.1 Hz, 2H), 2.72 (t, J = 6.5 Hz, 2H), 2.00 – 1.88 (m,

13 2H). C NMR (63 MHz, acetone-d6) δ 179.4, 155.7, 143.0, 134.6, 133.9, 130.2, 128.3,

126.0, 125.7, 123.1, 122.8, 117.1, 110.8, 78.3, 67.1, 25.7, 23.2. HRMS-ESI (M+Na)+ calc. for C17H15NO3Na 304.0950, found: 304.0951.

3-Phenylindole (2.29-2a). This reaction was performed using general procedure

B with 3-hydroxy-3-phenylindolin-2-one intermediate 2.29-1a (0.856 mg, 3.80 mmol),

LiAlH4 (0.613 mg 16.15 mmol), and THF (38 mL). The crude material was purified by flash column chromatography (silica gel, 10% EtOAc in hexanes) to afford 2.29-2a (610

1 mg, 83%): H NMR (300 MHz, CDCl3) δ 8.24 (br s, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.69

(dd, J = 8.1, 1.0 Hz, 2H), 7.46 (t, J = 7.7 Hz, 3H), 7.38 (d, J = 2.5 Hz, 1H), 7.34 – 7.16

13 (m, 3H). C NMR (75 MHz, CDCl3) δ 136.8, 135.7, 128.9, 127.6, 126.1, 125.9, 122.6,

121.9, 120.5, 120.0, 118.5, 111.5.

147

3-Chroman-6-ylindole (2.29-2c). This reaction was performed using general procedure B with 3-hydroxy-2-indolinone intermediate 2.29-1c (133 mg, 0.47 mmol),

LiAlH4 (90 mg, 2.36 mmol), and THF (2.4 mL). The crude material was purified by flash column chromatography (silica gel, 10% EtOAc in hexanes) to afford 2.29-2c (54 mg,

1 46%): H NMR (300 MHz, CDCl3) δ 8.16 (br s, 1H, -NH), 7.90 (d, J = 8.0 Hz, 1H), 7.45

– 7.36 (m, 2H), 7.35 (s, 1H), 7.28 (d, J = 2.6 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.18 (t, J

= 7.5 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 4.24 (t, J = 5.1 Hz, 2H), 2.88 (t, J = 6.5 Hz, 2H),

13 2.12 – 2.01 (m, 2H). C NMR (75 MHz, CDCl3) δ 153.7, 136.7, 129.0, 127.7, 126.8,

126.1, 122.6, 122.4, 121.2, 120.2, 120.0, 118.4, 117.2, 111.4, 66.7, 25.2, 22.7.

3-Phenyl-N-methylindole 2.30a. This reaction was performed using general procedure C with indole 2.29-2a (350 mg, 1.81 mmol), NaH (60%, 109 mg, 2.72 mmol), iodomethane (0.282 mL, 4.53 mmol), and DMF (9 mL). The crude material was purified

148 by flash column chromatography (silica gel, 3% EtOAc in hexanes) to afford 2.30a (340

1 mg, 91%) as a pale yellow oil: IR (film): H NMR (300 MHz, CDCl3) δ 7.97 (d, J = 7.9

Hz, 1H), 7.68 (d, J = 7.3 Hz, 2H), 7.46 (t, J = 7.6 Hz, 2H), 7.39 (d, J = 8.1 Hz, 1H), 7.35

– 7.27 (m, 3H), 7.21 (t, J = 7.5 Hz, 1H), 3.87 (s, 3H).

Chroman-6-yl-N-methylindole 2.30c. This reaction was performed using general procedure C with indole 2.29-2c (124 mg, 0.49 mmol), NaH (60%, 25 mg, 0.62 mmol), iodomethane (46 µL, 0.75 mmol), and DMF (2.5 mL). The crude N-methylindole

1 was taken to the next step without further purification: H NMR (250 MHz, CDCl3) δ

7.89 (d, J = 7.9 Hz, 1H), 7.41 – 7.21 (m, 4H), 7.18 (d, J = 7.1 Hz, 1H), 7.14 (s, 1H), 6.87

(d, J = 8.2 Hz, 1H), 4.23 (t, J = 5.1 Hz, 2H), 3.83 (s, 3H), 2.87 (t, J = 6.5 Hz, 2H), 2.11 –

13 1.98 (m, 2H). C NMR (63 MHz, CDCl3) δ 153.5, 137.5, 128.8, 127.8, 126.6, 126.4,

126.0, 122.6, 121.9, 120.1, 119.7, 117.2, 116.7, 109.5, 66.7, 33.0, 25.2, 22.7.

149

General procedure D for 3-aryl-2-acyl indole 2.31. To a solution of N- alkylated-3-substituted-indole (1 equiv.) in toluene (0.2 M) was added oxalyl chloride

(1.25 equiv.). The resulting solution was heated to reflux (120 °C) and stirred for 6 h.

After cooling to rt, the mixture was diluted with methanol (0.2 M) and stirred at rt for 1 h.

The solution was concentrated under reduced pressure and re-dissolved in EtOAc. The organic layer was washed with saturated NaHCO3 solution (2x). The combined aqueous layers were extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude material by flash column chromatography (silica gel, 5% EtOAc in hexanes) yielded 2-acylated indole

3-Phenyl-2-acylindole 2.31a. This reaction was performed using general procedure D with N-methylindole 2.30a (384 mg, 1.85 mmol), oxalyl chloride (0.330 mL, 3.71 mmol), toluene (9.27 mL), and methanol (9.3 mL). Purification of the crude material by flash column chromatography (silica gel, 5% EtOAc in hexanes) yielded 2- acylated indole 2.31a (416 mg, 77%) as a bright green solid: 1H NMR (400 MHz, acetone-d6) δ 7.64 (d, J = 8.5 Hz, 1H), 7.54 – 7.45 (m, 5H), 7.43 – 7.38 (m, 2H), 7.19

(ddd, J = 7.9, 6.9, 0.7 Hz, 1H), 4.11 (s, J = 5.5 Hz, 3H), 3.20 (s, 3H). 13C NMR (101

150

MHz, acetone-d6) δ 181.4, 164.6, 140.7, 133.7, 131.8, 130.5, 129.4, 129.3, 128.9, 128.5,

+ 127.2, 122.7, 122.5, 111.8, 52.5, 32.7. HRMS-ESI (M+Na) calc. for C18H15NO3Na

316.0950, found: 316.0967.

3-Chroman-6-yl-2-acylindole 2.31c. This reaction was performed using general procedure D with crude N-methylindole 2.30c (0.49 mmol), oxalyl chloride (55 µL, 0.62 mmol), toluene (2.5 mL), and methanol (2.5 mL). Purification of the crude material by flash column chromatography (silica gel, 5% EtOAc in hexanes) yielded 2-acylated

1 indole (96 mg, 55%) as a green solid: H NMR (300 MHz, CDCl3) δ 7.62 (d, J = 8.1 Hz,

1H), 7.50 – 7.36 (m, 2H), 7.16 (t, J = 7.2 Hz, 2H), 7.05 (s, 1H), 6.89 (d, J = 8.3 Hz, 1H),

4.24 (t, J = 5.1 Hz, 2H), 4.08 (s, 3H), 3.34 (s, 3H), 2.84 (t, J = 6.4 Hz, 2H), 2.12 – 1.99

13 (m, 2H). C NMR (75 MHz, CDCl3) δ 180.6, 164.3, 155.1, 140.0, 132.6, 130.5, 129.5,

128.8, 127.7, 126.5, 124.5, 122.6, 122.3, 121.4, 116.9, 110.5, 66.8, 52.3, 32.3, 24.9, 22.4.

+ HRMS-ESI (M+Na) calc. for C21H19NO4Na 372.1212, found: 374.1228.

General procedure E for 3-aryl-2-hydroxyester 2.32. 2-Ketoester 2.31 (1.1 equiv.) was dissolved in a 4:1 mixture of THF:EtOH (0.1 M total) and cooled to 0 °C.

NaBH4 (1 equiv.) was added to the mixture and stirred at 0 °C for 1 h. The reaction was

151 quenched with slow addition of 2N HCl till the solution was neutralized. The aqueous solution was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure.

3-Phenyl-2-hydroxyester 2.32a. This reaction was performed using general procedure E with 2-ketoester 2.31a (270 mg, 0.92 mmol), NaBH4 (32 mg, 0.84 mmol),

THF (6.8 mL) and EtOH (1.7 mL). The crude material was purified by flash column chromatography (silica gel, 15% EtOAc in hexanes) to afford 2-hydroxyester 2.33a (178

1 mg, 66%) as a pale yellow solid: H NMR (300 MHz, CDCl3) δ 7.68 – 7.62 (m, 1H), 7.58

– 7.52 (m, 2H), 7.47 (t, J = 7.5 Hz, 2H), 7.40 – 7.27 (m, 3H), 7.14 (ddd, J = 8.0, 6.7, 1.3

Hz, 1H), 5.60 (d, J = 2.1 Hz, 1H), 3.77 (s, 3H), 3.75 (s, 3H), 3.37 (d, J = 2.3 Hz, 1H).

3-(Chroman-6-yl)-2-hydroxyester2.32c. This reaction was performed using general procedure E for reduction of 2-ketoester 2.31c (95 mg, 0.272 mmol), NaBH4 (9.4 mg, 0.247 mmol), THF (2.20 mL) and EtOH (0.55 mL). The crude 2-hydroxyester was

1 taken to the next step without further purification: H NMR (300 MHz, CDCl3) δ 7.64 (d,

J = 8.0 Hz, 1H), 7.37 – 7.23 (m, 3H), 7.21 (s, 1H), 7.13 (t, J = 7.3 Hz, 1H), 6.90 (d, J =

8.2 Hz, 1H), 5.60 (d, J = 1.5 Hz, 1H), 4.25 (t, J = 5.2 Hz, 2H), 3.75 (s, 6H), 3.38 (d, J =

13 2.3 Hz, 1H), 2.86 (t, J = 6.5 Hz, 2H), 2.12 – 2.01 (m, 2H). C NMR (75 MHz, CDCl3) δ

174.0, 154.1, 137.4, 131.6, 130.7, 129.3, 126.8, 126.0, 123.1, 122.4, 120.3, 119.9, 118.8,

117.0, 109.2, 66.7, 65.8, 53.5, 30.5, 25.2, 22.6.

152

General procedure F for 3-aryl-tert-butyl ether indole 2.33. A mixture of 2- hydroxyester (1 equiv.) and Amberlyst H-15 (1 mg/ mg) in t-BuOAc (0.1 M) was stirred at rt for 16 h. After reaction completion, the resin was filtered out over Celite, washed with EtOAc, and then the filtrate was concentrated to about 25% of initial volume under reduced pressure. The organic layer was washed with H2O (2x), saturated NaHCO3 solution (2x), brine, dried over sodium sulfate, and concentrated under reduced pressure.

3-Phenyl-tert-butyl ether indole 2.33a. This reaction was performed using general procedure F with 2-hydroxyester 2.32a (70 mg, 0.237 mmol), Amberlyst H-15

(70 mg) and t-BuOAc (2.4 mL). The crude material was purified by flash column chromatography (silica gel, 5% EtOAc in hexanes) to afford tert-butyl ether 2.33a (35

1 mg, 42%) as a pale yellow solid: H NMR (300 MHz, CDCl3) δ 7.65 (d, J = 7.9 Hz, 1H),

7.58 (d, J = 7.0 Hz, 2H), 7.49 (t, J = 7.5 Hz, 2H), 7.42 – 7.27 (m, 3H), 7.13 (t, J = 6.9 Hz,

13 1H), 5.50 (s, 1H), 3.90 (s, 3H), 3.80 (s, 3H), 0.98 (s, 9H). C NMR (75 MHz, CDCl3) δ

172.4, 137.6, 134.8, 133.3, 130.3, 128.7, 126.7, 126.7, 122.7, 120.0, 119.9, 116.9, 109.4,

76.3, 66.6, 52.7, 31.4, 27.8.

153

3-(Chroman-6-yl)-3-tert-butyl ether indole 2.33c. This reaction was performed using general procedure F with crude 2-hydroxyester 2.32c (0.272 mmol), Amberlyst H-

15 (96 mg) and t-BuOAc (2.72 mL). The crude material was purified by flash column chromatography (silica gel, 5% EtOAc in hexanes) to afford tert-butyl ether 2.33c (33

1 mg, 30% over 2 steps): H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 7.9 Hz, 1H), 7.37 –

7.16 (m, 4H), 7.12 (d, J = 7.7 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 5.47 (s, 1H), 4.26 (t, J =

5.3 Hz, 2H), 3.89 (s, 3H), 3.77 (s, 3H), 2.89 – 2.82 (m, 2H), 2.12 – 2.03 (m, 2H), 1.01 (s,

13 9H). C NMR (101 MHz, CDCl3) δ 172.5, 153.9, 137.6, 132.9, 131.7, 129.3, 127.0,

126.5, 122.5, 122.4, 120.1, 119.7, 116.9, 116.8, 109.3, 76.2, 66.8, 66.7, 52.6, 31.4, 27.9,

25.2, 22.6.

Phenyl indole INDL-1. To a solution of methyl ester 2.33a (30 mg, 0.085 mmol) in a 1:1 mixture of THF (0.45 mL) and MeOH (0.45 mL) was added 1N NaOH solution

(0.51 mL, 0.51 mmol). The resulting solution was stirred at rt for 2 h. After removal of

THF under reduced pressure, the remaining solution was extracted with EtOAc (3x). The combined organic layers were washed brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude solid was dissolved in H2O and treated with 2N HCl to pH ≈ 3 and the resulting aqueous layer was extracted with EtOAc (3x). The combined

154 organic layers were washed brine, dried over sodium sulfate, and concentrated under reduced pressure to provide INDL-1 (11 mg, 38%) as a pale yellow solid: 1H NMR (300

MHz, CDCl3) δ 7.66 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 7.2 Hz, 2H), 7.49 (t, J = 7.4 Hz,

2H), 7.41 – 7.26 (m, 3H), 7.14 (t, J = 7.3 Hz, 1H), 5.57 (s, 1H), 3.82 (s, 3H), 0.97 (s, 9H).

13 C NMR (101 MHz, CDCl3) δ 172.2, 137.5, 134.3, 131.6, 130.3, 128.9, 127.0, 126.4,

123.1, 120.1, 120.1, 119.0, 109.4, 78.1, 66.5, 31.0, 27.9.

4-Methoxyphenyl indole INDL-2S. To a solution of methyl ester 2.33b (100 mg,

0.262 mmol) in a 1:1 mixture of THF (1.3 mL) and MeOH (1.3 mL) was added 1N

NaOH solution (1.57 mL, 1.57 mmol). The resulting solution was stirred at rt for 2 h.

After removal of THF under reduced pressure, the remaining solution was extracted with

EtOAc (3x). The combined organic layers were washed brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting crude solid was suspended in a

50% acetone in hexanes solution, then filtered and collected. The solid was washed with additional 50% acetone in hexanes solution and then dried under reduced pressure to

1 afford INDL-2S (44 mg, 43%) as a white solid: H NMR (300 MHz, DMSO-d6) δ 7.79

(d, J = 8.5 Hz, 2H), 7.47 (d, J = 7.7 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.10 (t, J = 7.4 Hz,

1H), 7.02 (d, J = 8.4 Hz, 2H), 6.98 (t, J = 7.3 Hz, 1H), 5.08 (s, 1H), 3.87 (s, 3H), 3.80 (s,

155

13 3H), 0.83 (s, 9H). C NMR (75 MHz, DMSO-d6) δ 172.6, 157.3, 139.3, 136.5, 130.7,

127.9, 126.2, 120.7, 118.8, 118.1, 113.6, 112.7, 109.1, 73.2, 68.2, 55.0, 31.2, 27.9.

+ HRMS-ESI (M+H) calc. for C22H25NO4Na 390.1681, found: 390.1693.

Chroman-6-yl indole INDL-4S. To a solution of methyl ester 2.33c (33 mg,

0.081 mmol) in a 1:1 mixture of THF (0.40 mL) and MeOH (0.40 mL) was added 3N

NaOH solution (0.16 mL, 0.49 mmol). The resulting solution was stirred at rt for 2 h.

After removal of THF under reduced pressure, the remaining solution was extracted with

EtOAc (3x). The combined organic layers were washed brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting crude solid was triturated with a

25% CH2Cl2 in hexanes solution and then dried under reduced pressure to afford INDL-

1 4S (19 mg, 56%) as a pale yellow solid: H NMR (400 MHz, CD3OD) δ 7.50 (d, J = 7.9

Hz, 1H), 7.44 – 7.38 (m, 2H), 7.31 (d, J = 8.2 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 6.99 (t, J

= 7.5 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 5.37 (s, 1H), 4.21 (t, J = 5.2 Hz, 2H), 3.87 (s,

3H), 2.96 – 2.79 (m, 2H), 2.09 – 1.99 (m, 2H), 0.92 (s, 9H). 13C NMR (75 MHz, DMSO- d6) δ 172.7, 152.7, 138.8, 136.6, 130.7, 128.7, 127.2, 126.3, 121.8, 120.7, 118.8, 118.3,

116.0, 113.1, 109.1, 73.4, 68.1, 65.9, 31.2, 27.9, 24.5, 22.1. HRMS-ESI (M+H)+ calc. for

C24H27NO4Na 416.1838, found: 416.1826.

156

Benzyl indole INDL-5S. To a solution of methyl ester 2.33d (135 mg, 0.37 mmol) in a 1:1 mixture of THF (1.85 mL) and MeOH (1.85 mL) was added 3N NaOH solution (0.74 mL, 2.22 mmol). The resulting solution was stirred at rt for 2 h. After removal of THF under reduced pressure, the remaining solution was extracted with

EtOAc (3x). The combined organic layers were washed brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting crude solid was suspended in a

50% CH2Cl2 in hexanes solution, then filtered and collected. The solid was washed with additional 50% CH2Cl2 in hexanes solution and then dried under reduced pressure to afford INDL-5S (87 mg, 63%). Acidifying the salt to pH ≈ 3 provided INDL-5 (R = H):

1 H NMR (300 MHz, CDCl3) δ 7.40 (d, J = 7.9 Hz, 1H), 7.35 – 7.10 (m, 7H), 7.03 (t, J =

7.3 Hz, 1H), 5.47 (s, 1H), 4.25 (s, 2H), 3.83 (s, 3H), 1.16 (s, 9H). INDL-5S (R = Na) pale

1 yellow solid: H NMR (300 MHz, DMSO-d6) δ 7.41 (d, J = 7.2 Hz, 2H), 7.25 (d, J = 8.1

Hz, 1H), 7.21 – 7.14 (m, 3H), 7.12 – 7.04 (m, 1H), 7.00 (t, J = 7.7 Hz, 1H), 6.82 (t, J =

7.4 Hz, 1H), 5.10 (s, 1H), 4.14 (q, J = 15.7 Hz, 2H), 3.86 (s, 3H), 1.07 (s, 9H). 13C NMR

(75 MHz, DMSO-d6) δ 173.3, 142.2, 139.2, 136.6, 128.8, 127.8, 127.3, 125.3, 120.2,

118.3, 117.9, 109.1, 108.7, 73.4, 69.3, 31.2, 30.4, 28.1. HRMS-ESI (M+H)+ calc. for

C22H25NO3Na 374.1732, found: 374.1714.

157

N-acyl tetrahydrocarbazole. Tetrahydrocarbazole (350 mg, 2.04 mmol) and

N,N-dimethylaminopyridine (25 mg, 0.20 mmol) were dissolved in CH2Cl2 (13.6 mL) and cooled to 0 °C. Bromoacetyl bromide (0.356 mL, 4.09 mmol) was added to the solution at 0 °C before gradually warming the reaction to rt. After stirring for 14 h, the reaction was quenched with H2O. The mixture was separated and the aqueous layer was extracted with CH2Cl2 (3x). The combined organic layers were washed with saturated

NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel,

2% EtOAc in hexanes) to afford N-acyl tetrahydrocarbazole (358 mg, 60%).

Recrystallization from hot EtOAc and hexanes provided the compound as brownish-

1 yellow crystals: H NMR (300 MHz, CDCl3) δ 8.12 – 8.03 (m, 1H), 7.44 – 7.37 (m, 1H),

7.34 – 7.23 (m, 2H), 4.43 (s, 2H), 3.03 (t, J = 5.9 Hz, 2H), 2.67 (t, J = 5.9 Hz, 2H), 1.99 –

13 1.79 (m, 4H). C NMR (75 MHz, CDCl3) δ 165.7, 136.0, 134.7, 130.7, 124.7, 123.9,

119.9, 118.1, 115.9, 30.2, 26.1, 23.8, 21.9, 21.3.

158

N-acyl 6-methyltetrahydrocarbazole. 6-Methyltetrahydrocarbazole (100 mg,

0.54 mmol) and N,N-dimethylaminopyridine (7 mg, 0.054 mmol) were dissolved in

CH2Cl2 (3.6 mL) and cooled to 0 °C. Bromoacetyl bromide (0.094 mL, 1.08 mmol) was added to the solution at 0 °C before gradually warming the reaction to rt. After stirring for 14 h, the reaction was quenched with H2O. The mixture was separated and the aqueous layer was extracted with CH2Cl2 (3x). The combined organic layers were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 2% EtOAc in hexanes) to afford N-acyl 6- methyltetrahydrocarbazole (97 mg, 58%). Recrystallization from hot EtOAc and hexanes

1 provided the compound as white needles: H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.5

Hz, 1H), 7.19 (s, 1H), 7.11 (d, J = 8.5 Hz, 1H), 4.42 (d, J = 1.6 Hz, 2H), 3.02 (tt, J = 6.1,

1.9 Hz, 2H), 2.63 (tt, J = 5.9, 1.9 Hz, 2H), 2.44 (s, 3H), 1.96 – 1.79 (m, 4H). 13C NMR

(101 MHz, CDCl3) δ 165.5, 134.9, 134.1, 133.5, 130.9, 125.7, 119.8, 118.3, 115.5, 30.3,

26.1, 23.8, 21.9, 21.4, 21.3. HRMS-ESI (M+Na)+ and (M+2+Na)+ calc. for

C15H16NOBrNa 328.0313 and 330.0292, found: 328.0323 and 330.0315.

159

B22 analogues. A suspension of bromide (1 equiv.), triazole (1.5 equiv.), and acetonitrile (0.2 M) was stirred at rt for 5 min before triethylamine (1.5 equiv.) was added. The resulting suspension was stirred at reflux (80 °C) for 14 h. After cooling to rt, the mixture was diluted with H2O and the resulting aqueous solution was extracted with

(3x). The combined organic layers were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 0% → 5% MeOH in CH2Cl2) to

1 afford the thioether. B22 (R1 = H; R2 = Me) yellow solid: H NMR (400 MHz, DMSO- d6) δ 11.92 (s, 1H), 8.15 – 8.08 (m, 1H), 7.47 – 7.41 (m, 1H), 7.29 – 7.19 (m, 2H), 6.08

(s, 2H), 4.67 (q, J = 16.2 Hz, 2H), 3.10 (d, J = 16.8 Hz, 1H), 3.04 – 2.92 (m, 1H), 2.78

(dd, J = 16.2, 4.0 Hz, 1H), 2.19 (dd, J = 15.9, 9.6 Hz, 1H), 2.00 – 1.82 (m, 2H), 1.54 –

13 1.40 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 168.3, 157.5,

155.3, 135.7, 134.5, 129.5, 124.0, 123.2, 117.8, 117.4, 115.7, 38.2, 31.4, 28.9, 27.6, 25.3,

1 21.2. B22-2 (R1 = H; R2 = H) grey solid: H NMR (250 MHz, DMSO-d6) δ 11.92 (s, 1H),

8.16 – 8.05 (m, 1H), 7.49 – 7.40 (m, 1H), 7.31 – 7.19 (m, 2H), 6.08 (s, 2H), 4.67 (s, 2H),

3.01 (t, J = 5.0 Hz, 2H), 2.63 (t, J = 4.9 Hz, 2H), 1.91 – 1.71 (m, 4H). 13C NMR (63 160

MHz, DMSO-d6) δ 168.2, 157.5, 155.3, 135.5, 134.8, 129.6, 124.0, 123.1, 117.8, 117.5,

+ 115.6, 38.2, 25.7, 23.3, 21.4, 20.7. HRMS-ESI (M+H) calc. for C16H18N5OS 328.1232,

1 found: 328.1248. B22-3 (R1 = Me; R2 = H) white solid: H NMR (300 MHz, DMSO-d6)

δ 11.90 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 7.07 (d, J = 8.6 Hz, 1H), 6.05 (s,

2H), 4.64 (s, 2H), 2.99 (t, J = 4.9 Hz, 2H), 2.60 (t, J = 5.0 Hz, 2H), 2.38 (s, 3H), 1.90 –

13 1.71 (m, 4H). C NMR (75 MHz, DMSO-d6) δ 167.9, 157.4, 155.3, 134.8, 133.7, 132.1,

129.8, 125.0, 117.7, 117.4, 115.3, 38.1, 25.7, 23.3, 21.4, 20.9, 20.6. HRMS-ESI (M+H)+ calc. for C17H20N5OS 342.1389, found: 342.1402. B22-7 (R1 = H; R2 = Et) pale yellow

1 solid: H NMR (300 MHz, DMSO-d6) δ 11.90 (s, 1H), 8.15 – 8.07 (m, 1H), 7.49 – 7.42

(m, 1H), 7.29 – 7.19 (m, 2H), 6.05 (s, 2H), 4.66 (q, J = 16.2 Hz, 2H), 3.12 (d, J = 17.2

Hz, 1H), 3.04 – 2.89 (m, 1H), 2.81 (dd, J = 16.4, 4.6 Hz, 1H), 2.28 – 2.14 (m, 1H), 2.07 –

1.96 (m, 1H), 1.74 – 1.59 (m, 1H), 1.57 – 1.36 (m, 3H), 1.00 (t, J = 7.3 Hz, 3H). 13C

NMR (75 MHz, DMSO-d6) δ 168.2, 157.5, 155.3, 135.7, 134.7, 129.6, 124.0, 123.2,

117.8, 117.4, 115.7, 38.2, 34.4, 29.3, 28.1, 26.7, 25.4, 11.6. HRMS-ESI (M+H)+ calc. for

C18H22N5OS 356.1545, found: 356.1563.

Benzyl ester 2.64. To a mixture of benzylic alcohol 2.63 (150 mg, 0.56 mmol) and acyl chloride 2.60 (162 mg, 0.70 mmol) in CH2Cl2 (0.2 M) at 0 °C was added N,N-

161 dimethylaminopyridine (6 mg, 0.056 mmol) followed by triethylamine (0.097 mL, 0.697 mmol). The ice-bath was removed and the reaction was stirred at rt for 14 h. The reaction was then diluted with H2O and the resulting aqueous solution was extracted with CHCl3

(3x). The combined organic layers were washed with saturated NaHCO3 solution, 2N

HCl, brine, dried over sodium sulfate, and concentrated under reduced pressure.

Purification of the crude material by flash column chromatography (silica gel, 20%

EtOAc in hexanes) provided ester 2.64 (249 mg, 95%) as a white solid.: 1H NMR (400

MHz, CDCl3) δ 10.41 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.72 (d,

J = 8.3 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.5 Hz,

2H), 7.11 (t, J = 7.6 Hz, 1H), 6.86 (d, J = 8.5 Hz, 2H), 6.59 (t, J = 4.9 Hz, 1H), 5.37 (s,

13 2H), 4.55 (d, J = 5.5 Hz, 2H), 3.78 (s, 3H), 3.04 (s, 3H). C NMR (101 MHz, CDCl3) δ

167.7, 166.8, 159.2, 141.1, 138.7, 135.3, 134.7, 131.7, 130.3, 129.4, 128.3, 127.5, 123.0,

118.0, 115.1, 114.2, 66.7, 55.4, 43.7, 40.1.

B34. A solution of amide 2.64 (150 mg, 0.32 mmol), ceric ammonium nitrate

(439 mg, 0.80 mmol), reagent grade acetonitrile (3.2 mL), and H2O (1.6mL) was stirred at rt for 14 h. The reaction was then diluted with H2O and the resulting aqueous solution was extracted with CHCl3 (3x). The combined organic layers were washed with saturated

162

NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude material by flash column chromatography (silica gel,

5% EtOAc in CHCl3 → 1% MeOH in CHCl3) provided B34 (80 mg, 72%) as a pale yellow solid. Recrystallization from hot ethyl acetate and cold hexanes furnished B34 as

1 a white solid: H NMR (300 MHz, CDCl3) δ 10.08 (s, 1H), 8.02 (d, J = 8.2 Hz, 1H), 8.01

(s, 1H), 7.90 (d, J = 7.7 Hz, 2H), 7.72 – 7.63 (m, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.56 (d, J

= 8.2 Hz, 2H), 7.40 (s, 1H), 7.24 (t, J = 7.5 Hz, 1H), 5.42 (s, 2H), 3.17 (s, 3H). 13C NMR

(101 MHz, CDCl3) δ 167.5, 167.0, 139.7, 138.7, 134.9, 134.1, 131.3, 127.8, 127.7, 123.4,

+ 119.1, 116.9, 71.4, 66.4. HRMS-ESI (M+Na) calc. for C16H16N2O5SNa 371.0678, found: 371.0681.

MB59-1-2 (2.70). Method A – Swern oxidation of diol: To a solution of (COCl)2

(2.15 mL, 25 mmol) in CH2Cl2 (100 mL) at -78 °C was added DMSO (3.55 mL, 50 mmol). After gas evolution ceased, 1,4-butanediol (0.89 mL, 10 mmol) was added to the mixture and the reaction was stirred at -78 °C for 2 h. After addition of Et3N (13.94 mL,

100 mmol), the reaction was gradually warmed to rt over 14 h. The reaction was then cooled down to 0 °C and AcOH (10 mL) was slowly added to the mixture. Methyl anthranilate (1.30 mL, 10 mmol) was then added and the resulting mixture was stirred at

163 reflux for 20 h. After cooling to rt, the reaction was quenched by addition of saturated

NaHCO3 solution. After separating the mixture, the aqueous layer was extracted with

CH2Cl2 (3x). The combined organic layers were washed with saturated NaHCO3 solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude material by flash column chromatography (silica gel, 5% EtOAc in hexanes) provided pyrrole ester 2.70 (148 mg, 8%) as a colorless oil.

Method B – According to Mazzola et al. for the synthesis of pyrrole from dimethoxytetrahydrofurans:120 A mixture of methyl anthranilate (652 mL, 5 mmol), 2,5- dimethoxytetrahydrofuran (0.713 mL, 5.5 mmol) in glacial AcOH (20 mL) was heated at

100 °C for 2 h. After cooling the reaction to rt, the solution was diluted with cold H2O and neutralized by slow addition of solid NaHCO3. The aqueous layer was extracted with

EtOAc (3x). The combined organic layers were washed with H2O (2x), saturated

NaHCO3 (2x), brine, dried over sodium sulfate, and concentrated under reduced pressure.

Purification of the crude material by flash column chromatography (silica gel, 5% EtOAc in hexanes) provided pyrrole ester 2.70 (876 mg, 87%) as a colorless oil: 1H NMR (250

MHz, CDCl3) δ 7.80 (dd, J = 7.3, 2.0 Hz, 1H), 7.56 (td, J = 7.3, 1.7 Hz, 1H), 7.45 – 7.35

(m, 2H), 6.82 (t, J = 2.2 Hz, 2H), 6.31 (t, J = 2.2 Hz, 2H), 3.72 (s, 3H). IR (film): 3103,

2950, 1724, 1603, 1503, 1297, 1127, 765.

164

MB59-1-1. Pyrrole ester 2.70 (311 mg, 1.55 mmol) was dissolved in reagent grade MeOH (7.75 mL). After addition of 3N NaOH (3.09 mL, 9.27 mmol), the reaction was stirred at rt for 3 h. MeOH was then evaporated under reduced pressure and the resulting aqueous solution was extracted with EtOAc (3x). The aqueous layer was collected and acidified to pH ≈ 3 with dropwise addition of 6N HCl. The resulting aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure.

Recrystallization from hot hexanes yielded MB59-1 (221 mg, 76%) as a white solid: 1H

NMR (300 MHz, CDCl3) δ 7.95 (ddd, J = 7.8, 1.6, 0.4 Hz, 1H), 7.61 (td, J = 7.7, 1.5 Hz,

1H), 7.46 – 7.36 (m, 2H), 6.85 (t, J = 2.1 Hz, 2H), 6.33 (t, J = 2.1 Hz, 2H). 13C NMR (75

MHz, CDCl3) δ 170.8, 141.1, 133.4, 131.6, 127.4, 127.2, 126.3, 122.2, 110.1. IR (film):

3124, 2955, 1685, 1498, 1304, 1089, 735.

2-nitro (2.75) and 3-nitro (2.76) pyrrole ester. According to Cobb et al. for the nitration of pyrrole:121 nitric acid (0.215 mL, 4.8 mmol) was added dropwise to acetic anhydride (15 mL) at -10 °C and stirred for 5 min. The resulting mixture was added dropwise to a stirred solution of pyrrole ester 2.70 (604 mg, 3.0 mmol) in acetic 165 anhydride (15 mL) at -30 to -40 °C. The temperature was maintained for 1 h before gradually warming the reaction to rt and then stirred for 2 h. The reaction was quenched by pouring the mixture over ice. The resulting mixture was cooled to 0 °C and acetic anhydride was further hydrolyzed by slow addition of 6N NaOH until the resulting solution was neutralized. The aqueous solution was extracted with CHCl3 (3x). The combined organic layers were washed with saturated NaHCO3 (2x), brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude material by flash column chromatography (silica gel, 10% EtOAc in hexanes) provided

2-nitropyrrole ester 2.75 (293 mg, 40%) as a yellow solid and 3-nitropyrrole ester 2.76

1 (288 mg, 39%) as a white solid. 2-nitropyrrole ester 2.75: H NMR (250 MHz, CDCl3) δ

8.12 (dd, J = 7.7, 1.6 Hz, 1H), 7.66 (td, J = 7.6, 1.8 Hz, 1H), 7.57 (td, J = 7.6, 1.5 Hz,

1H), 7.40 – 7.30 (m, 2H), 6.81 (dd, J = 2.8, 2.1 Hz, 1H), 6.37 (dd, J = 4.3, 2.9 Hz, 1H),

13 3.71 (s, 3H). C NMR (63 MHz, CDCl3) δ 165.0, 139.1, 133.3, 131.6, 129.4, 129.3,

128.7, 127.9, 113.8, 109.6, 52.6. IR (film): 3127, 2950, 1722, 1521, 1338, 1301, 1175,

1 752. 3-nitropyrrole ester 2.76: H NMR (250 MHz, CDCl3) δ 8.00 (dd, J = 7.7, 1.6 Hz,

1H), 7.70 – 7.62 (m, 2H), 7.57 (td, J = 7.6, 1.4 Hz, 1H), 7.43 – 7.36 (m, 1H), 6.86 (dd, J

= 3.3, 1.8 Hz, 1H), 6.70 (dd, J = 3.3, 2.4 Hz, 1H), 3.76 (s, 3H). 13C NMR (63 MHz,

CDCl3) δ 165.7, 138.8, 138.2, 133.2, 131.7, 129.4, 127.7, 127.6, 123.4, 123.0, 106.0,

52.8. IR (film): 3162, 3127, 1725, 1532, 1320, 1094, 751.

166

MB59-1-4 and MB59-1-5. Nitropyrrole ester (2.75 or 2.76) (70 mg, 0.28 mmol) was dissolved in reagent grade THF (1.5 mL) and reagent grade MeOH (1.5 mL). After addition of 3N NaOH (0.30 mL, 0.90 mmol), the reaction was stirred at rt for 3 h. MeOH was then evaporated under reduced pressure and the resulting aqueous solution was extracted with EtOAc (3x). The aqueous layer was collected and acidified to pH ≈ 3 with dropwise addition of 6N HCl. The resulting precipitate was filtered and collected. The solid was washed with cold H2O, hexanes, and dried under reduced pressure to yield the corresponding carboxylic acid: 2-nitropyrrole MB59-1-4 (56 mg, 85%) as yellow

1 crystals: H NMR (250 MHz, CD3OD) δ 8.12 (dd, J = 7.6, 1.6 Hz, 1H), 7.71 (td, J = 7.7,

1.6 Hz, 1H), 7.61 (td, J = 7.5, 1.0 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.29 (dd, J = 4.2, 2.0

Hz, 1H), 7.02 (t, J = 2.4 Hz, 1H), 6.39 (dd, J = 4.1, 2.8 Hz, 1H). 13C NMR (63 MHz,

CD3OD) δ 167.6, 140.5, 134.1, 132.4, 131.2, 130.2, 129.8, 129.7, 114.5, 110.4. IR (film):

3133, 2981, 1701, 1466, 1357, 1172. 3-nitropyrrole MB59-1-5 (61 mg, 93%) as pink

1 crystals: H NMR (300 MHz, CDCl3) δ 8.11 (dd, J = 7.8, 1.2 Hz, 1H), 7.71 (td, J = 7.7,

1.4 Hz, 1H), 7.66 (t, J = 1.9 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H),

13 6.86 (dd, J = 3.0, 1.7 Hz, 1H), 6.72 (t, J = 2.8 Hz, 1H). C NMR (75 MHz, CDCl3) δ

167

169.2, 139.4, 138.2, 134.2, 132.5, 129.6, 128.1, 126.1, 123.5, 123.2, 106.1. IR (film):

3139, 2916, 1701, 1509, 1316.

2-pyrrolocarboxyaldehyde 2.73. According to the procedure by Lautens and coworkers for the formylation of pyrrole:122 phosphorous oxychloride (0.403 mL, 4.4 mmol) was added to a solution of DMF (0.339 mL, 4.4 mmol) in CH2Cl2 (2 mL) at 0 °C and stirred for 10 min. A solution of pyrrole ester 2.70 (805 mg, 4.0 mmol) in CH2Cl2 (18 mL) was added to the cooled solution and the resulting solution was refluxed for 2 h.

After cooling to rt, the reaction was quenched with saturated Na2CO3 solution. Once the mixture separated, the organic layer was washed with saturated Na2CO3 solution. The combined aqueous layers were extracted with CH2Cl2 (2x). The combined organic layers were washed brine, dried over sodium sulfate, and concentrated under reduced pressure.

The crude mater was purified by flash column chromatography (silica gel, 15% → 17%

→ 19% EtOAc in hexanes) provided 3-carboxaldehyde with a by-product as an inseparable mixture and 2-carboxaldehyde 2.73 (536 mg, 59%) as a red solid: 1H NMR

(250 MHz, CDCl3) δ 9.48 (s, 1H), 8.04 (dd, J = 7.6, 1.8 Hz, 1H), 7.62 (td, J = 7.6, 1.7 Hz,

1H), 7.53 (td, J = 7.5, 1.4 Hz, 1H), 7.34 (dd, J = 7.6, 1.4 Hz, 1H), 7.11 (dd, J = 4.0, 1.6

Hz, 1H), 6.98 (t, J = 2.0 Hz, 1H), 6.43 (dd, J = 3.9, 2.6 Hz, 1H), 3.68 (s, 3H). 13C NMR 168

(63 MHz, CDCl3) δ 178.9, 165.5, 139.3, 133.5, 132.7, 131.6, 131.2, 129.0, 128.9, 128.6,

123.0, 110.7, 52.4. IR (film): 2943, 1729, 1668, 1497, 1295, 1080, 783.

Quinazoline 2.78. 2-Nitropyrrole ester 2.75 (44 mg, 0.205 mmol) was dissolved in a 1:1 mixture of CH2Cl2 (2 mL) and MeOH (2 mL). 10 wt% Pd/C was added and H2

(balloon) was bubbled through the mixture for 14 h. The catalyst was filtered out over

Celite and the filtrate was concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 2% → 4% MeOH in CH2Cl2) to

1 afford 2.78 (18 mg, 41%) as a white solid: H NMR (250 MHz, CDCl3) δ 8.35 (dd, J =

8.0, 1.4 Hz, 1H), 7.77 – 7.66 (m, J = 1.5 Hz, 1H), 7.53 – 7.41 (m, 1H), 7.21 (d, J = 8.2

Hz, 1H), 4.24 (t, J = 7.3 Hz, 2H), 3.20 (t, J = 8.0 Hz, 2H), 2.41 (quint, J = 7.7 Hz, 2H).

The 1H NMR matches the literature value.121

169

N-benzylanthranilate (MB59-1-6). A suspension of anthranilic acid (2.87) (70 mg, 0.51 mmol), benzaldehyde (62 µL, 0.613 mmol), AcOH (15 µL, 0.255 mmol), and reagent grade CH2Cl2 (1.3 mL) was stirred in a closed vial at rt for 3 h. After addition of sodium triacetoxyborohydride (216 mg, 1.021 mmol), the reaction mixture was stirred for

3 h. The reaction was diluted with CH2Cl2 (2 mL) and quenched with 1N HCl (2 mL).

After the mixture separated, the aqueous layer was extracted with 5% MeOH in CH2Cl2

(3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Trituration of the crude solid with 25% CH2Cl2 in hexanes and drying under reduced pressure afforded MB59-1-6 (104 mg, 90%) as a

1 pale yellow solid: H NMR (250 MHz, CDCl3) δ 12.68 (br s, 1H), 8.33 (br s, 1H), 7.80

(d, J = 7.9 Hz, 1H), 7.31 (dd, J = 15.9, 5.7 Hz, 6H), 6.66 (d, J = 8.3 Hz, 1H), 6.55 (t, J =

13 7.2 Hz, 1H), 4.45 (s, 2H). C NMR (63 MHz, CDCl3) δ 170.0, 150.6, 139.3, 134.3,

131.6, 128.5, 127.0, 126.9, 114.4, 111.6, 110.3, 45.8. IR (film): 3381, 2917, 1655, 1575,

1234, 748.

N-(4-Methoxybenzyl)anthranilate (MB59-1-7). A suspension of anthranilic acid

(2.87) (70 mg, 0.51 mmol), anisaldehyde (74 µL, 0.613 mmol), AcOH (15 µL, 0.255

170 mmol), and reagent grade CH2Cl2 (1.3 mL) was stirred in a closed vial at rt for 3 h. After addition of sodium triacetoxyborohydride (216 mg, 1.021 mmol), the reaction mixture was stirred for 3 h. The reaction was diluted with CH2Cl2 (2 mL) and quenched with 1N

HCl (2 mL). After the mixture separated, the aqueous layer was extracted with 5%

MeOH in CH2Cl2 (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Trituration of the crude solid with 25% CH2Cl2 in hexanes and drying under reduced pressure afforded MB59-1-7 (111

1 mg, 85%) as a pale yellow solid: H NMR (250 MHz, acetone-d6) δ 10.95 (br s, 1H), 8.24

(br s, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.40 – 7.25 (m, 3H), 6.91 (d, J = 8.4 Hz, 2H), 6.74 (d,

J = 8.5 Hz, 1H), 6.59 (t, J = 7.5 Hz, 1H), 4.40 (s, 2H), 3.78 (d, J = 1.9 Hz, 3H). 13C NMR

(63 MHz, acetone-d6) δ 170.6, 159.9, 152.3, 135.5, 132.8, 132.0, 129.4, 115.4, 114.8,

112.6, 110.8, 55.5, 46.8. IR (film): 3374, 2837, 1662, 1513, 1248, 1163.

Indol-5-yl anthranilate MB59-1-8. A suspension of anthranilic acid (2.87) (70 mg, 0.51 mmol), indole-5-carboxaldehyde (89 mg, 0.613 mmol), AcOH (15 µL, 0.255 mmol), and reagent grade CH2Cl2 (1.3 mL) was stirred in a closed vial at rt for 3 h. After addition of sodium triacetoxyborohydride (216 mg, 1.021 mmol), the reaction mixture

171 was stirred for 3 h. The reaction was diluted with CH2Cl2 (2 mL) and quenched with 1N

HCl (2 mL). After the mixture separated, the aqueous layer was extracted with 5%

MeOH in CH2Cl2 (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Trituration of the crude solid with 50% CH2Cl2 in hexanes and drying under reduced pressure afforded MB59-1-8 (128

1 mg, 94%) as a pale yellow solid: H NMR (250 MHz, CDCl3) δ 10.92 (br s, 1H), 10.25

(br s, 1H), 8.27 (br s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H),

7.33 (t, J = 7.8 Hz, 2H), 7.17 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.57 (t, J = 7.5

13 Hz, 1H), 6.44 (s, 1H), 4.52 (s, 2H). C NMR (63 MHz, CDCl3) δ 170.6, 152.4, 136.6,

135.4, 132.8, 130.5, 129.3, 126.1, 122.1, 119.9, 115.1, 112.6, 112.4, 110.6, 102.3, 48.1.

IR (film): 3408, 1662, 1577, 1363, 1223, 1164, 752.

N-(4-Nitrobenzyl)anthranilate (MB59-1-9). A suspension of anthranilic acid

(2.87) (70 mg, 0.51 mmol), 4-nitrobenzaldehyde (93 mg, 0.613 mmol), AcOH (15 µL,

0.255 mmol), and reagent grade CH2Cl2 (1.3 mL) was stirred in a closed vial at rt for 3 h.

After addition of sodium triacetoxyborohydride (216 mg, 1.021 mmol), the reaction mixture was stirred for 3 h. The reaction was diluted with CH2Cl2 (2 mL) and quenched

172 with 1N HCl (2 mL). After the mixture separated, the aqueous layer was extracted with

5% MeOH in CH2Cl2 (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Trituration of the crude solid with 50% CH2Cl2 in hexanes and drying under reduced pressure afforded MB59-1-

1 9 (119 mg, 86%) as a pale yellow solid: H NMR (250 MHz, acetone-d6) δ 8.52 (br s,

1H), 8.22 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.30 (t, J

= 7.8 Hz, 1H), 6.70 – 6.56 (m, 2H), 4.73 (s, J = 17.4 Hz, 2H). 13C NMR (63 MHz, acetone-d6) δ 170.6, 151.8, 148.9, 148.1, 135.5, 133.0, 128.8, 124.5, 116.0, 112.6, 111.3,

46.6. IR (film): 1655, 1515, 1343, 1223.

2-Pyridyl anthranilate MB59-1-10. A suspension of anthranilic acid (2.87) (70 mg, 0.51 mmol), 2-pyridinecarboxaldehyde (58 µL, 0.613 mmol), AcOH (15 µL, 0.255 mmol), and reagent grade CH2Cl2 (1.3 mL) was stirred in a closed vial at rt for 3 h. After addition of sodium triacetoxyborohydride (216 mg, 1.021 mmol), the reaction mixture was stirred for 3 h. The reaction was diluted with CH2Cl2 (2 mL) and quenched with 1N

HCl (2 mL). After the mixture separated, the aqueous layer was extracted with 5%

MeOH in CH2Cl2 (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Trituration of the crude solid 173 with CH2Cl2 and drying under reduced pressure afforded MB59-1-10 (98 mg, 84%) as a

1 pale yellow solid: H NMR (250 MHz, acetone-d6) δ 8.68 (br s, 1H), 8.57 (d, J = 4.4 Hz,

1H), 7.95 (dd, J = 7.9, 1.4 Hz, 1H), 7.74 (td, J = 7.7, 1.6 Hz, 1H), 7.39 (d, J = 7.9 Hz,

1H), 7.37 – 7.29 (m, 1H), 7.29 – 7.21 (m, 1H), 6.71 (d, J = 8.5 Hz, 1H), 6.60 (t, J = 7.2

13 Hz, 1H), 4.60 (s, 2H). C NMR (63 MHz, acetone-d6) δ 170.6, 159.8, 152.1, 150.1,

137.6, 135.5, 133.0, 123.0, 122.2, 115.6, 112.6, 111.4, 49.1. IR (film): 1670, 1580, 1517,

1437, 1253, 752.

4-Pyridyl anthranilate MB59-1-11. A suspension of anthranilic acid (2.87) (70 mg, 0.51 mmol), 4-pyridinecarboxaldehyde (58 µL, 0.613 mmol), AcOH (15 µL, 0.255 mmol), and reagent grade CH2Cl2 (1.3 mL) was stirred in a closed vial at rt for 3 h. After addition of sodium triacetoxyborohydride (216 mg, 1.021 mmol), the reaction mixture was stirred for 3 h. The reaction was diluted with CH2Cl2 (2 mL) and quenched with 1N

HCl (2 mL). After the mixture separated, the aqueous layer was extracted with 5%

MeOH in CH2Cl2 (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Trituration of the crude solid with CH2Cl2 and drying under reduced pressure afforded MB59-1-11 (109 mg, 94%) as a

1 pale yellow solid: H NMR (300 MHz, DMSO-d6) δ 12.75 (s, 1H), 8.49 (d, J = 5.8 Hz, 174

2H), 8.38 (s, 1H), 7.81 (dd, J = 7.9, 1.3 Hz, 1H), 7.31 (d, J = 5.7 Hz, 2H), 7.27 (t, J = 7.0

Hz, 1H), 6.58 (d, J = 7.4 Hz, 1H), 6.55 (t, J = 7.8 Hz, 1H), 4.54 (s, 2H). 13C NMR (75

MHz, DMSO-d6) δ 170.0, 150.3, 149.7, 149.0, 134.4, 131.8, 122.0, 114.8, 111.6, 110.6,

44.7. IR (film): 1655, 1576, 1415, 1210, 1015.

5-Hydroxymethyl)furan anthranilate MB59-1-12. A suspension of anthranilic acid (2.87) (70 mg, 0.51 mmol), 5-(hydroxymethyl)furfural (77 mg, 0.613 mmol), AcOH

(15 µL, 0.255 mmol), and reagent grade CH2Cl2 (1.3 mL) was stirred in a closed vial at rt for 3 h. After addition of sodium triacetoxyborohydride (216 mg, 1.021 mmol), the reaction mixture was stirred for 3 h. The reaction was diluted with CH2Cl2 (2 mL) and quenched with 1N HCl (2 mL). After the mixture separated, the aqueous layer was extracted with 5% MeOH in CH2Cl2 (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure.

Trituration of the crude solid with CH2Cl2 drying under reduced pressure afforded

1 MB59-1-12 (103 mg, 82%) as a pale yellow solid: H NMR (300 MHz, acetone-d6) δ

8.22 (s, 1H), 7.93 (dd, J = 8.0, 1.2 Hz, 1H), 7.38 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 6.88 (d, J

= 8.4 Hz, 1H), 6.62 (t, J = 7.2 Hz, 1H), 6.23 (dd, J = 13.8, 2.9 Hz, 2H), 4.48 (s, 2H), 4.45

175

13 (s, 2H). C NMR (75 MHz, acetone-d6) δ 170.5, 156.0, 152.8, 151.8, 135.4, 132.8,

115.7, 112.3, 111.0, 108.5, 108.4, 57.2, 40.5. IR (film): 3365, 2975, 1670, 1580, 1518,

1225.

N-(4-Methoxybenzenesulfonyl)anthranilate (MB59-1-14). A suspension of anthranilic acid (2.87) (275 mg, 2.0 mmol), 4-methoxybenzenesulfonyl chloride (455 mg,

2.2 mmol), and 2M Na2CO3 (10 mL) was refluxed (110 °C) for 14 h. After cooling to rt, the mixture was filtered and collected. The solid was washed with cold H2O, hexanes, and then dried under reduced pressure to afford MB59-1-15 (249, 41%) as a white solid:

1 H NMR (300 MHz, acetone-d6) δ 12.05 (s, 1H), 10.94 (s, 1H), 8.01 (dd, J = 7.9, 1.3 Hz,

1H), 7.81 (d, J = 9.0 Hz, 2H), 7.71 (d, J = 8.4 Hz, 1H), 7.61 – 7.51 (m, 1H), 7.12 (t, J =

13 7.6 Hz, 1H), 7.03 (d, J = 8.9 Hz, 2H), 3.84 (s, 3H). C NMR (75 MHz, acetone-d6) δ

170.5, 164.3, 142.1, 135.6, 132.6, 131.8, 130.3, 123.7, 119.4, 116.5, 115.2, 56.1. IR

(film): 3152, 1683, 1596, 1493, 1262, 1156.

176

N-(4-Nitrobenzenesulfonyl)anthranilate (MB59-1-15). A suspension of anthranilic acid (2.87) (275 mg, 2.0 mmol), 4-nitrobenzenesulfonyl chloride (488 mg, 2.2 mmol), and H2O (10 mL) was refluxed (110 °C) for 14 h. After cooling to rt, the mixture was filtered and collected. The solid was washed with cold H2O, hexanes, and then dried under reduced pressure to afford MB59-1-15 (323, 50%) as a white solid: 1H NMR (250

MHz, acetone-d6) δ 12.12 (s, 1H), 11.20 (s, 1H), 8.39 (d, J = 8.6 Hz, 2H), 8.16 (d, J = 8.6

Hz, 2H), 8.03 (d, J = 7.9 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.19

13 (t, J = 7.5 Hz, 1H). C NMR (63 MHz, acetone-d6) δ 170.4, 151.5, 145.6, 140.9, 135.8,

132.8, 129.6, 125.4, 124.6, 119.7, 117.1. IR (film): 3107, 1684, 1531, 1350, 1162.

N-(4-Aminobenzenesulfonyl)anthranilate (MB59-1-16). Anthranilate MB59-1-

15 (106 mg, 0.329 mmol) was dissolved in a 3:1 mixture of MeOH (3.30 mL) and EtOAc

(1.10 mL). 10 wt% Pd/C was added and H2 (balloon) was bubbled through the mixture

177 for 14 h. The catalyst was filtered out over Celite and washed with MeOH. The filtrate was concentrated under reduced pressure and the resulting crude solid was triturated with

1 CH2Cl2 to afford amine MB59-1-16 (65 mg, 66%) as a brown solid: H NMR (300 MHz,

DMSO-d6) δ 11.45 (br s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.42 (d, J =

8.8 Hz, 2H), 7.09 – 7.00 (m, 1H), 6.52 (d, J = 8.7 Hz, 2H), 6.10 (br s, 1H), 3.36 (br s, J =

13 57.8 Hz, 2H). C NMR (75 MHz, DMSO-d6) δ 169.8, 153.3, 140.8, 134.0, 131.4, 128.9,

123.2, 122.3, 117.7, 112.6. IR (film): 3383, 1456, 1151.

Benzyloxyacetylamide antranilate MB59-1-17. 2-benzyloxyacetamide 2.85 (75 mg, 0.240 mmol) was dissolved in reagent grade THF (1.2 mL) and reagent grade MeOH

(1.2 mL). After addition of 1N LiOH (0.72 mL, 0.0.72 mmol), the reaction was stirred at rt for 14 h. The solvent was then evaporated under reduced pressure and the remaining aqueous solution was acidified to pH ≈ 4 with dropwise addition of 6N HCl. The resulting aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced

1 pressure to yield MB59-1-17 (21 mg, 73%) as a white solid: H NMR (300 MHz, CDCl3)

δ 9.59 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.57 (t, J = 7.3 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H),

7.30 – 7.15 (m, 6H), 4.50 (q, J = 11.8 Hz, 2H), 3.85 (s, J = 15.2 Hz, 2H), 3.25 (s, 3H). 178

13 C NMR (75 MHz, CDCl3) δ 170.0, 168.0, 142.2, 137.4, 134.2, 132.7, 129.7, 129.0,

128.4, 128.4, 128.1, 127.8, 73.2, 68.2, 37.5. IR (film): 3060, 2934, 1718, 1642, 1492,

1242, 1123.

2-aminoacetamide 2.81. A mixture of methyl anthranilate (2.68) (47 µL, 0.36 mmol), N-benzyl-2-bromoacetamide 2.80 (103 mg, 0.45 mmol) in DMF (1.80 mL) was heated stirred at 120 °C for 14 h. After cooling to rt, the reaction was quenched with cold

H2O. The aqueous solution was extracted with EtOAc (3x). The combined organic layers were washed with H2O, brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude material by flash column chromatography (silica gel,

20% EtOAc in hexanes) provided 2-aminoacetamide 2.81 (63 mg, 59%).

179

Benzylamide anthranilate MB59-1-18. 2-Aminoacetamide 2.81 (30 mg, 0.101 mmol) was dissolved in reagent grade THF (0.5 mL) and reagent grade MeOH (0.5 mL).

After addition of 1N LiOH (0.302 mL, 0.302 mmol), the reaction was stirred at rt for 14 h. The solvent was then evaporated under reduced pressure and the remaining aqueous solution was acidified to pH ≈ 4 with dropwise addition of 6N HCl. The resulting aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to yield

1 MB59-1-18 (21 mg, 73%) as a white solid: H NMR (300 MHz, DMSO-d6) δ 12.42 (s,

1H), 8.55 (t, J = 5.7 Hz, 1H), 8.20 (s, 1H), 7.81 (dd, J = 7.9, 1.6 Hz, 1H), 7.42 – 7.19 (m,

6H), 6.60 (t, J = 7.5 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 4.32 (d, J = 5.8 Hz, 2H), 3.90 (s,

13 2H). C NMR (75 MHz, DMSO-d6) δ 169.6, 169.1, 150.1, 139.3, 134.3, 131.6, 128.2,

127.1, 126.7, 114.7, 111.3, 110.8, 45.8, 42.0.

180

Part II: Synthesis of the highly substituted indole subunit of sespendole

N-benzenesulfonyl-4-methyl-5-iodoindole 4.45. To a 2N HCl solution (1.75 mL) at 0 °C was added aniline 4.44 (100 mg, 0.35 mmol) followed by dropwise addition of a solution of NaNO2 (60 mg, 0.87 mmol) in H2O (1.4 mL). After rapidly stirring the suspension for 30 min at 0 °C, a solution of KI (1.45 g, 8.73 mmol) in H2O (2.8 mL) was added to the cooled reaction. The ice-bath was removed and the resulting suspension was rapidly stirred at rt for 1 h, heated to 85 °C and stirred for 30 min, then cooled to rt before transferring the reaction to a separatory funnel. The aqueous solution was extracted with

CH2Cl2 (3x). The combined organic layers were washed with saturated Na2S2O3 solution

(2x) and brine, then dried over sodium sulfate and concentrated under reduced pressure.

The crude material was purified by flash column chromatography (silica gel, 35% toluene in hexanes) to provide iodide 4.45 (88 mg, 63%) as a pale white solid: 1H NMR (300

MHz, CDCl3) δ 7.86 (d, J = 7.5 Hz, 2H), 7.70 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 8.5 Hz,

1H), 7.55 – 7.51 (m, J = 3.8 Hz, 2H), 7.48 – 7.41 (m, J = 8.0 Hz, 2H), 6.69 (d, J = 3.7 Hz,

13 1H), 2.54 (s, 3H). C NMR (75 MHz, CDCl3) δ 138.2, 134.9, 134.5, 134.4, 134.1, 131.2,

129.5, 126.9, 126.5, 113.0, 108.3, 94.9, 24.4. IR (film): 3142, 2922, 1583, 1448, 1372,

+ 1171, 729, 686. HRMS-ESI (M+Na) calc. for C15H12INO2SNa 419.9531, found:

419.9106. 181

N-benzenesulfonyl-4-methyl-5-allylic alcohol 4.46. In an oven-dried flask, iodide 4.45 (100 mg, 0.25 mmol) was dissolved in THF (1.00 mL) and cooled to -78 °C. n-BuLi (2.5M in hexanes, 0.151 mL, 0.378 mmol) was added dropwise to the solution and stirred -78 °C for 1 h. 3-methylcrotonaldehyde (37 µL, 0.38 mmol) was then added and the reaction mixture warmed to rt over 1 h. The reaction was quenched with 10% aqueous NH4Cl solution and extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash column chromatography (deactivated silica gel, 15% EtOAc in hexanes) to afford alcohol 4.46 (50 mg, 56%) as a yellow gel:

1 H NMR (300 MHz, CDCl3) δ 7.94 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 7.5 Hz, 2H), 7.52 (t,

J = 7.4 Hz, 1H), 7.41 (t, J = 7.7 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 7.3 Hz,

1H), 6.68 (s, 1H), 5.89 (dd, J = 7.9, 4.4 Hz, 1H), 5.65 (d, J = 8.0 Hz, 1H), 3.38 (d, J = 4.4

13 Hz, 1H), 2.45 (s, 3H), 1.82 (s, 3H), 1.57 (s, 3H). C NMR (75 MHz, CDCl3) δ 143.0,

139.0, 137.5, 136.5, 134.0, 130.9, 129.4, 128.8, 126.5, 125.2, 124.5, 124.4, 112.4, 109.0,

+ 64.4, 25.9, 18.5, 18.5. HRMS-ESI (M+Na) calc. for C20H21NO3SNa 378.1140, found:

378.0785.

182

N-benzenesulfonyl-4-methyl-5-epoxyalcohol 4.47. To a solution of alcohol 4.46

(25 mg, 0.070 mmol) in CH2Cl2 (0.70 mL) at 0 °C was added mCPBA (77%, 17.5 mg,

0.077 mmol). The mixture was stirred at 0 °C for 1 h before quenching the reaction with a saturated aqueous solution of Na2CO3. The aqueous solution was extracted with CH2Cl2

(3x). The combined organic layers were washed with saturated Na2CO3 solution, brine, then dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash column chromatography (deactivated silica gel, 20%

EtOAc in hexanes) to furnish epoxyalcohol 4.47 (15.2 mg, 58%) as a yellow solid. 1H

NMR (300 MHz, CDCl3) δ 7.96 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.52 (t, J =

7.4 Hz, 1H), 7.40 (t, J = 7.7 Hz, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H),

6.74 (d, J = 0.6 Hz, 1H), 5.08 (d, J = 6.5 Hz, 1H), 3.45 (s, 1H, -OH), 3.35 (d, J = 6.4 Hz,

13 1H), 2.47 (s, 3H), 1.42 (s, 3H), 1.18 (s, 3H). C NMR (75 MHz, CDCl3) δ 140.1, 138.7,

137.1, 134.1, 130.9, 129.5, 128.8, 126.6, 125.5, 124.6, 112.4, 108.3, 66.3, 66.0, 59.5,

24.8, 19.0, 18.5. IR (film): 3420, 2964, 2925, 1448, 1368, 1179, 1097, 728. HRMS-ESI

+ (M+Na) calc. for C20H21NO4SNa 394.1089, found: 394.0810.

183

5-nitrocyclopenta[b]indole 4.34b. Cyclobutyl carbaldehyde (4.65c) was prepared according to Schoening et al. via Swern oxidation. To a solution of (COCl)2

(1.14 mL, 13.25 mmol) in CH2Cl2 (42.4 mL) at -78 °C was added DMSO (1.88 mL,

26.50 mmol). After gas evolution ceased, cyclobutanemethanol (1.00 mL, 10.60 mmol) was added to the mixture and was stirred at -78 °C for 15 mins before addition of Et3N

(7.39 mL, 53.00 mmol). The reaction was then gradually warmed to rt and quenched with

H2O. The aqueous layer was extracted with CH2Cl2 (3x). The combined organic layers were washed with H2O and brine then dried over sodium sulfate. Removal of CH2Cl2 by

1 distillation provided crude aldehyde 4.65c as a yellow oil: H NMR (400 MHz, CDCl3) δ

9.73 (d, J = 1.8 Hz, 1H), 3.18 (p, J = 8.1 Hz, 1H), 2.33 – 1.85 (m, 6H).

The 2,3-substituted indole was prepared based on modification of the reaction conditions by Liu et al. for rearrangement of 3,3-disubstituted indolenines: To a solution of crude cyclobutyl carboxaldehyde 4.65c (133 mg, 1.58 mmol) in glacial AcOH (2.11 mL) was added p-nitrophenylhydrazine hydrochloride (4.64) (200 mg, 1.06 mmol). The suspension was stirred at 70 °C for 1 h before addition of trifluoroacetic acid (0.244 mL,

3.17 mmol) to the reaction. The resulting mixture was heated to 115 °C and stirred for 16 h. After cooling the reaction to rt, the mixture was diluted with cold H2O and quenched with slow addition of saturated NaHCO3 solution. The aqueous layer was extracted with

EtOAc (3x), the combined organic layers were washed with H2O (2x), saturated NaHCO3 184 solution, brine and then dried over sodium sulfate and concentrated under reduced pressure. The crude solid was purified by flash column chromatography (silica gel, 15%

EtOAc in hexanes) to furnish 4.34b (96 mg, 45%) as an orange solid: 1H NMR (300

MHz, CDCl3) δ 8.38 (d, J = 2.2 Hz, 1H), 8.35 (s, 1H), 8.00 (dd, J = 9.0, 2.3 Hz, 1H), 7.31

(d, J = 9.0 Hz, 1H), 2.95 – 2.82 (m, 4H), 2.63 – 2.52 (m, 2H). 13C NMR (75 MHz,

CDCl3) δ 147.4, 144.1, 141.7, 124.1, 122.2, 116.4, 115.7, 111.1, 28.6, 26.1, 24.4. IR

+ (film): 3316, 2847, 1501, 1472, 1311, 754. HRMS-ESI (M+Na) calc. for C11H10N2O2Na

225.0640, found: 225.0650.

5-nitrotetrahydrocarbazole 4.34a. A suspension of p-nitrophenylhydrazine hydrochloride (4.64) (5.0 g, 26.37 mmol), cyclohexanone (3.42 mL, 29.01 mmol) and glacial AcOH (52.5 mL) was heated to 100 °C and stirred for 14 h. The reaction was then cooled to rt and diluted with cold H2O (50 mL). The resulting suspension was filtered and the green solid collected, washed with water and hexanes. Recrystallization with hot ethanol provided compound 4.34a (5.26 g, 92%) as yellow-green solid. 1H NMR (250

MHz, CDCl3) δ 8.41 (d, J = 1.8 Hz, 1H), 8.15 (s, 1H), 8.03 (dd, J = 8.9, 2.1 Hz, 1H), 7.28

(d, J = 8.8 Hz, 1H), 2.91 – 2.56 (m, J = 5.5 Hz, 4H), 2.01 – 1.82 (m, J = 4.5 Hz, 4H). 13C

NMR (63 MHz, CDCl3) δ 141.4, 139.0, 137.8, 127.5, 117.0, 115.1, 112.8, 110.2, 23.3,

185

23.0, 20.8. IR (film): 3350, 2930, 1474, 1318, 736. HRMS-ESI (M+Na)+ calc. for

C12H12N2O2Na 239.0796, found: 239.0695.

N-benzyl-5-nitroindole 4.67. Benzyl bromide (2.70 mL, 22.54 mmol) was added to a mixture of 5-nitrotetrahydrocarbazole 4.34a (3.25 g, 15.03 mmol) and K2CO3 (5.19 g, 37.57 mmol) in reagent grade DMF (60 mL). The reaction mixture was stirred for 14 h at rt. The reaction was diluted with cold H2O and the aqueous layer was extracted with

EtOAc (3x). The combined organic layers were washed sequentially with cold H2O, saturated NaHCO3 solution, brine, and then dried over sodium sulfate and concentrated under reduced pressure. The crude material was recrystallized with hot ethyl acetate and cold hexanes to provide 4.67 (4.41 g, 96%) as an orange solid: 1H NMR (400 MHz,

CDCl3) δ 8.46 (d, J = 1.9 Hz, 1H), 8.01 (dd, J = 9.0, 2.1 Hz, 1H), 7.32 – 7.23 (m, 3H),

7.19 (d, J = 9.0 Hz, 1H), 6.97 (d, J = 6.7 Hz, 2H), 5.28 (s, 2H), 2.78 (t, J = 5.5 Hz, 2H),

13 2.65 (t, J = 5.8 Hz, 2H), 1.98 – 1.83 (m, 4H). C NMR (101 MHz, CDCl3) δ 141.1,

139.6, 139.4, 137.0, 129.0, 127.8, 127.0, 126.1, 116.7, 115.2, 112.8, 108.7, 46.7, 22.9,

22.8, 22.2, 20.9. IR (film): 3028, 2935, 1620, 1510, 1328, 735. HRMS-ESI (M+Na)+ calc. for C19H18N2O2Na 329.1266, found: 329.1038.

186

N-benzyl-4-methyl-5-nitroindole 4.70. N-benzyl protected indole 4.67 (2.14 g,

6.97 mmol) was dissolved in THF (41 mL) and cooled to -10 °C. Methylmagnesium chloride (3.0M in THF, 4.65 mL, 13.94 mmol) was then added dropwise to the solution and the resulting mixture was stirred at -10 °C for 30 min. DDQ (1.98 g, 8.71 mmol) was then added portionwise to the reaction. After complete addition of DDQ, the ice-bath was removed and the mixture was stirred for 1 h at rt. The mixture was then diluted with H2O and the aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with a saturated NaHCO3 solution (3x). The combined saturated NaHCO3 aqueous layers were extracted once with EtOAC. The final combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure.

The crude material was purified by flash column chromatography (silica gel, 30% →

40% → 50% CH2Cl2 in hexanes) to yield N-benzyl-4-methyl-5-nitroindole 4.70 (1.32 g,

1 59%) as a yellow solid: H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 9.0 Hz, 1H), 7.31 –

7.21 (m, 3H), 7.02 (d, J = 9.0 Hz, 1H), 6.94 (d, J = 6.6 Hz, 2H), 5.24 (s, 2H), 3.06 (t, J =

4.2 Hz, 2H), 2.89 (s, 3H), 2.63 (t, J = 4.4 Hz, 2H), 1.92 – 1.83 (m, 4H). 13C NMR (101

MHz, CDCl3) δ 143.0, 138.4, 138.0, 137.1, 129.0, 128.4, 127.7, 127.0, 126.0, 118.4,

113.0, 106.9, 46.4, 24.6, 23.6, 22.5, 22.4, 16.6. IR (film): 3028, 2934, 2852, 1573, 1509,

+ 1323, 734. HRMS-ESI (M+Na) calc. for C20H20N2O2Na 343.1422, found: 343.1197.

187

N-benzyl-4-acetaldehyde-5-nitroindole 4.75. 4-methyl-5-nitroindole 4.70 (2.58 g, 8.04 mmol) was dissolved in DMF (40 mL) and heated to 105 °C.

Trisdimethylaminomethane (2.09 mL, 12.06 mmol) was added to the hot solution and the resulting mixture was stirred at 105 °C for 3 h. After complete consumption of indole

4.70, the reaction was cooled to rt and then the red solution was slowly poured into a mixture of AcOH (20 mL), H2O (100 mL), and THF (50 mL). Additional THF (50 mL) was added to rinse and transfer any remaining solution into the acidic mixture. The resulting mixture was stirred at rt for 3 h and then the aqueous layer was extracted with

EtOAc (3x). The combined organic layers were washed with H2O (3x), saturated

NaHCO3 solution (3x), and brine, then dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash column chromatography

(silica gel, 1% → 5% EtOAc with 25% CH2Cl2 in hexanes) to yield aldehyde 4.75 (2.62

1 g, 94%) as a reddish-orange solid: H NMR (400 MHz, CDCl3) δ 9.95 (s, 1H), 7.91 (d, J

= 9.0 Hz, 1H), 7.33 – 7.23 (m, 3H), 7.17 (d, J = 9.0 Hz, 1H), 6.96 (d, J = 6.6 Hz, 2H),

5.28 (s, J = 8.4 Hz, 2H), 4.45 (s, 2H), 2.93 (t, J = 3.9 Hz, 2H), 2.64 (t, J = 4.7 Hz, 2H),

13 1.95 – 1.82 (m, 4H). C NMR (101 MHz, CDCl3) δ 198.8, 142.5, 139.7, 138.8, 136.7,

129.1, 127.9, 127.7, 126.1, 122.9, 118.8, 112.3, 108.6, 46.6, 44.3, 24.2, 23.4, 22.6, 22.2.

188

IR (film): 2936, 2853, 1724, 1510, 1325, 735. HRMS-ESI (M+Na)+ calc. for

C21H20N2O3Na 371.1372, found: 371.1067.

Isopropyltriphenylphosphonium bromide: A mixture of 2-bromopropane (5.16 mL, 55 mmol) and triphenylphosphonium bromide (13.12 g, 50 mmol) was heated to 150

°C in a sealed tube. After 24 h, the reaction was cooled to rt and the resulting white solid was suspended in hot toluene. This hot suspension was filtered, the white solid collected, and dried under reduced pressure to provide the Wittig reagent (10.68g, 55%).

N-benzyl-4-prenyl-5-nitroindole 4.76. n-BuLi (2.5M in THF, 2.29 mL, 5.72 mmol) was added dropwise to a suspension of iPrPPh3 (1.84 g, 4.77 mmol) in THF (13 mL) at 0 °C. The resulting red suspension was stirred for 1 h at 0 °C. In a separate reaction flask, aldehyde 4.75 (1.11 g, 3.18 mmol) was dissolved in THF (19 mL). This solution was added, via a glass syringe, dropwise to the red suspension. After stirring the mixture at 0 °C for 15 min, the ice-bath was removed and the reaction stirred at rt for 2 h.

189

The reaction was quenched with a 10% aqueous solution of NH4Cl. The aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with H2O, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude mixture for purified by flash column chromatography (silica gel, 0% → 2.5% → 5%

EtOAc with 25% CHCl3 in hexanes) to provide olefin 4.76 (1.06 g, 89%) as a greenish-

1 yellow solid: H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.9 Hz, 1H), 7.32 – 7.22 (m,

3H), 7.05 (d, J = 8.9 Hz, 1H), 6.96 (d, J = 6.6 Hz, 2H), 5.25 (s, 2H), 5.21 (t, J = 5.8 Hz,

1H), 4.00 (d, J = 5.5 Hz, 2H), 3.01 (t, J = 4.7 Hz, 2H), 2.65 (t, J = 4.7 Hz, 2H), 1.93 –

13 1.83 (m, 4H), 1.79 (s, 3H), 1.72 (s, 3H). C NMR (101 MHz, CDCl3) δ 142.9, 138.5,

138.3, 137.1, 132.3, 131.4, 129.0, 127.7, 126.6, 126.1, 123.1, 118.6, 112.7, 107.2, 46.5,

28.3, 25.8, 23.8, 23.7, 22.6, 22.4, 18.5. IR (film): 2932, 2854, 1604, 1513, 1325, 736.

+ HRMS-ESI (M+Na) calc. for C24H26N2O2Na 397.1892, found: 397.1605.

N-benzyl-4-prenyl-aniline 4.77. Nitroindole 4.76 (875 mg, 2.34 mmol) was dissolved in reagent grade CH2Cl2 (24 mL) and cooled to 0 °C. After Zn dust (1.53 g,

23.37 mmol) and AcOH (2.34 mL) were added sequentially, the ice bath was removed and the suspension was stirred at rt for 1 h. The suspension was then filtered through a pad of Celite, washed with CH2Cl2, and concentrated under reduced pressure. The

190 concentrate was taken up in EtOAc and washed with saturated NaHCO3 solution (3x),

H2O (2x), brine, then dried over sodium sulfate and concentrated under reduced pressure.

The crude mixture can be taken to the next step without purification. Alternatively, the crude mixture can be purified by flash column chromatography (silica gel, 0% → 5% →

10% CH2Cl2 with 15% EtOAc in hexanes + 1% NH4OH) to afford amine 4.77 (622 mg,

1 77%) as a brownish-white solid: H NMR (400 MHz, CDCl3) δ 7.30 – 7.17 (m, 3H), 7.01

(d, J = 7.1 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.55 (d, J = 8.4 Hz, 1H), 5.23 (t, J = 5.0 Hz,

1H), 5.17 (s, 2H), 3.68 (d, J = 5.5 Hz, 2H), 2.99 (s, 2H), 2.61 (s, 2H), 1.90 – 1.80 (m,

13 4H), 1.86 (s, 3H), 1.74 (s, 3H). C NMR (101 MHz, CDCl3) δ 138.7, 137.1, 135.8,

132.1, 132.0, 128.8, 127.1, 126.7, 126.3, 123.7, 117.6, 112.2, 108.9, 107.5, 46.2, 27.0,

25.8, 24.1, 24.0, 22.8, 22.6, 18.2. IR (film): 3420, 3344, 2925, 1615, 1436, 1355, 729.

+ HRMS-ESI (M+Na) calc. for C24H28N2Na 367.2150, found: 367.1880.

N-benzyl-4-prenyl-iodoindole 4.78. To a solution of aniline 4.77 (620 mg, 1.80 mmol) in reagent grade THF (9 mL) at 0 °C was added 2N HCl solution (9 mL) followed by dropwise addition of a solution of NaNO2 (311 mg, 4.50 mmol) in H2O (4.5 mL).

After rapidly stirring the suspension for 1 h at 0 °C, a solution of KI (5.98 g, 36 mmol) in

H2O (4.5 mL) was added to the cooled reaction. The ice-bath was removed and the 191 resulting suspension was rapidly stirred at rt for 1 h, heated to 85 °C and stirred for 1 h, then cooled to rt before transferring the reaction to a separatory funnel. The aqueous solution was extracted with EtOAc (3x). The combined organic layers were washed with saturated Na2S2O3 solution (2x), H2O, then brine, and dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 35% toluene in hexanes) to provide iodide 4.78 (463 mg,

1 56%) as a pale white solid: H NMR (300 MHz, CDCl3) δ 7.49 (d, J = 8.6 Hz, 1H), 7.32

– 7.23 (m, 3H), 6.98 (d, J = 6.7 Hz, 2H), 6.80 (d, J = 8.5 Hz, 1H), 5.21 (s, 2H), 5.12 (t, J

= 5.1 Hz, 1H), 3.88 (d, J = 5.4 Hz, 2H), 2.97 (t, J = 3.9 Hz, 2H), 2.64 (t, J = 4.1 Hz, 2H),

13 1.87 (s, 3H), 1.93 – 1.84 (m, 4H), 1.74 (s, 3H). C NMR (75 MHz, CDCl3) δ 137.9,

136.8, 136.4, 136.0, 131.6, 131.5, 128.9, 127.4, 127.2, 126.2, 123.5, 110.1, 109.3, 90.7,

46.3, 37.2, 25.9, 23.9, 23.8, 22.7, 22.5, 19.0. IR (film): 2926, 2852, 1452, 1374, 730.

+ HRMS-ESI (M+Na) calc. for C24H26INNa 478.1008, found: 478.0684.

N-benzyl-4-prenyl-carboxaldehyde 4.81. For this reaction, THF was distilled over LiAlH4 and DMF was distilled over CaH2. In a flame-dried flask containing 4Å molecular sieves (41 mg), iodide 4.78 (92 mg, 0.202 mmol) was dissolved in THF (4.04 mL) and cooled to -78 °C. To the resulting mixture was added n-BuLi (2.5M in THF, 192

0.162 mL, 0.404 mmol) and immediately followed by addition of DMF (0.156 mL, 2.02 mmol). The reaction was stirred at -78 °C for 30 min before the CO2-acetone bath was removed to rapidly warm the reaction. The reaction was stirred at rt for 2 h before quenching the reaction with EtOAc. The molecular sieves were filtered out over a pad of

Celite and washed with EtOAc. The filtrate was concentrated under pressure to 25% of filtrate volume and then added to a 10% aqueous NH4Cl solution. The aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 2% → 4% EtOAc with 10% CH2Cl2 in hexanes) to furnish aldehyde 4.81 (60 mg, 83%) as a white solid: 1H NMR (300 MHz,

CDCl3) δ 10.33 (s, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.33 – 7.20 (m, 3H), 7.14 (d, J = 8.6 Hz,

1H), 6.99 (d, J = 6.3 Hz, 2H), 5.28 – 5.20 (m, 3H), 4.14 (d, J = 5.2 Hz, 2H), 3.08 – 2.96

(m, 2H), 2.68 – 2.58 (m, 2H), 1.93 – 1.84 (m, 4H), 1.82 (s, 3H), 1.71 (s, 3H).

N-benzyl trans olefin 4.92. Aldehyde 4.81 (30 mg, 0.084 mmol) was dissolved in

THF (0.85 mL) and cooled to -78 °C. 2-methyl-1-propenylmagnesium bromide (0.5M in

THF, 0.336 mL, 0.168 mmol) was added dropwise to the cooled solution. The solution was gradually warmed to rt and stirred for 14 h. The reaction was quenched with addition

193 of saturated aqueous NH4Cl solution and then the aqueous layer was extracted with

EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the crude mixture by flash column chromatography (silica gel, 10% EtOAc in hexanes) provided trans olefin

1 4.92: H NMR (400 MHz, CDCl3) δ 7.29 – 7.19 (m, 4H), 7.03 (d, J = 8.5 Hz, 1H), 6.99

(d, J = 6.8 Hz, 2H), 6.92 (d, J = 15.9 Hz, 1H), 6.14 (d, J = 15.9 Hz, 1H), 5.24 – 5.17 (m,

3H), 3.79 (d, J = 5.7 Hz, 2H), 3.00 (t, J = 4.0 Hz, 2H), 2.62 (t, J = 4.0 Hz, 2H), 1.89 –

1.82 (m, 4H), 1.84 (s, 3H), 1.71 (s, 3H), 1.43 (s, 6H).

N-benzyl trans olefin 4.92. In a seal tube, a mixture of iodide 4.78 (100 mg, 0.22 mmol), K2CO3 (121 mg, 0.88 mmol), 2-methyl-3-buten-2-ol (4.93) (38 µL, 0.44 mmol), in DMF (4.4 mL) was degassed with argon. Pd(OAc)2 (5 mg, 0.022 mmol) was added to the mixture and the resulting was further degassed for 5 min before sealing the reaction vessel and heating the mixture at 100 °C for 16 h. After cooling, the reaction was diluted with cold H2O, filtered over Celite and washed with EtOAc. The two layers were separated and the aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with cold H2O, saturated NaHCO3 solution, brine, then dried over

194 sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 10% EtOAc in hexanes) to provide trans

1 olefin 4.92. H NMR (300 MHz, CDCl3) δ 7.29 – 7.19 (m, 4H), 7.02 (d, J = 8.5 Hz, 1H),

6.99 (d, J = 6.2 Hz, 2H), 6.91 (d, J = 15.9 Hz, 1H), 6.14 (d, J = 15.9 Hz, 1H), 5.24 – 5.17

(m, 3H), 3.78 (d, J = 6.3 Hz, 2H), 3.05 – 2.94 (m, 2H), 2.65 – 2.57 (m, 2H), 1.84 (s, 3H),

1.89 – 1.80 (m, 4H), 1.70 (s, 3H), 1.43 (s, 6H).

N-benzyl 4,5-cyclopentanone fused indole 4.84. A solution of iodide 4.78 (1 equiv.), triethylamine (2.5 equiv.), and nucleophile (> 5 equiv.) in DMF (0.1 M) was degassed with argon. CO (balloon) was bubbled through the solution before tetrakis(triphenylphosphine)palladium(0) (10 mol %) was added to the mixture. The resulting solution, under CO balloon pressure, was heated to 80 °C and stirred for 16 h.

After cooling, the reaction mixture was diluted with H2O then filtered through Celite and washed with EtOAc. The two layers were separated and the aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 6% → 8% → 10% EtOAc in hexanes) to

1 provide enone 4.84 as a brown solid: H NMR (300 MHz, CDCl3) δ 7.53 (d, J = 8.5 Hz, 195

1H), 7.27 (d, J = 5.6 Hz, 3H), 7.17 (d, J = 8.5 Hz, 1H), 6.99 (d, J = 6.7 Hz, 2H), 5.26 (s,

2H), 3.91 (s, 2H), 3.07 – 2.92 (m, 2H), 2.71 – 2.58 (m, 2H), 2.46 (s, 3H), 2.03 (s, 3H),

13 1.98 – 1.82 (m, 4H). C NMR (75 MHz, CDCl3) δ 194.2, 146.4, 142.5, 139.5, 137.6,

136.7, 133.1, 131.1, 129.0, 127.6, 126.2, 123.5, 116.7, 112.0, 109.3, 46.7, 31.4, 24.6,

23.3, 22.9, 22.6, 22.3, 20.2. IR (film): 2931, 1680, 1434, 1304, 733. HRMS-ESI (M+Na)+ calc. for C25H25NONa 378.1834, found: 378.1581.

N-tosyl-5-nitroindole 4.68. A suspension of indole 4.34a (3.78 g, 17.50 mmol),

NaOH (1.05 g, 26.25 mmol), and 4-toluenesulfonyl chloride (3.67 g, 19.25 mmol) in reagent grade CH2Cl2 (35 mL) was stirred at rt for 2 h. The reaction was then diluted with

H2O and filtered. The solid was collected, washed with H2O and Et2O, then dried under reduced pressure to provide N-tosylindole 4.68 (6.21 g, 96%) as a yellowish-green solid:

1 H NMR (300 MHz, CDCl3) δ 8.25 (d, J = 9.1 Hz, 1H), 8.24 (d, J = 2.3 Hz, 1H), 8.14

(dd, J = 9.2, 2.2 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 3.01 (t, J =

6.0 Hz, 2H), 2.63 (t, J = 5.9 Hz, 2H), 2.36 (s, 3H), 1.96 – 1.75 (m, 4H). 13C NMR (75

MHz, CDCl3) δ 145.5, 144.2, 139.4, 138.9, 135.9, 130.4, 130.3, 126.6, 119.1, 118.8,

114.4, 24.7, 23.1, 21.9, 21.7, 21.0. IR (film): 2939, 1519, 1341, 1162, 664. HRMS-ESI

+ (M+Na) calc. for C19H18N2O4SNa 393.0885, found: 393.0621.

196

N-tosyl-4-methyl-5-nitroindole 4.71. N-tosyl protected indole 4.68 (3.52 g, 9.50 mmol) was dissolved in THF (56 mL) and cooled to -10 °C. Methylmagnesium chloride

(3.0M in THF, 3.96 mL, 11.88 mmol) was then added dropwise to the solution and the resulting mixture was stirred at -10 °C for 30 min. DDQ (2.37 g, 10.45 mmol) was then added portionwise to the reaction. After complete addition of DDQ, the ice-bath was removed and the mixture was stirred for 1 h at rt. The mixture was then diluted with H2O and the aqueous layer was extracted with CH2Cl2 (3x). The combined organic layers were washed with a saturated NaHCO3 solution (3x). The combined saturated NaHCO3 aqueous layers were extracted once with CH2Cl2. The final combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure.

The crude material was purified by flash column chromatography (silica gel, 30% →

35% → 40% CH2Cl2 in hexanes) to yield N-tosyl-4-methyl-5-nitroindole 4.71 (2.15 g,

1 59%) as a yellow solid: H NMR (300 MHz, CDCl3) δ 8.11 (d, J = 9.1 Hz, 1H), 7.72 (d, J

= 9.1 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 3.02 (t, J = 5.1 Hz, 2H),

2.91 (t, J = 5.4 Hz, 2H), 2.71 (s, 3H), 2.37 (s, 3H), 1.81 (d, J = 5.0 Hz, 4H). 13C NMR (75

MHz, CDCl3) δ 145.4, 138.1, 137.7, 135.9, 130.2, 130.0, 126.7, 126.5, 120.1, 119.5,

112.3, 25.1, 25.1, 22.7, 22.4, 21.8, 15.9. IR (film): 2940, 1517, 1355, 1181, 1090, 816,

+ 667. HRMS-ESI (M+Na) calc. for C20H20N2O4SNa 407.1041, found: 407.0705.

197

N-tosyl-4-methyl-5-iodoindole 4.88. Nitroindole 4.71 (1.02 g, 2.66 mmol) was dissolved in reagent grade CH2Cl2 (26.6 mL) and cooled to 0 °C. After Zn dust (1.74 g,

26.6 mmol) and AcOH (2.66 mL) were added sequentially, the ice bath was removed and the suspension was stirred at rt for 1 h. The suspension was then filtered through a pad of

Celite, washed with CH2Cl2, and concentrated under reduced pressure. The concentrate was taken up in EtOAc and washed with saturated NaHCO3 solution (3x), H2O (2x), brine, then dried over sodium sulfate and concentrated under reduced pressure. The crude mixture was taken to the next step without purification.

To a solution of crude aniline 4.87 (2.66 mmol) in reagent grade THF (10.5 mL) and H2O (10.5 mL) at 0 °C was added 2N HCl solution (10.5 mL) followed by dropwise addition of a solution of NaNO2 (459 mg, 6.65 mmol) in H2O (10.5 mL). After vigorously stirring the suspension for 45 min at 0 °C, a solution of KI (11.04 g, 66.53 mmol) in H2O (10.5 mL) was added to the cooled reaction. The ice-bath was removed and the resulting suspension was rapidly stirred at rt for 1 h, heated to 85 °C and stirred for 14 h, then cooled to rt before transferring the reaction to a separatory funnel. The aqueous solution was extracted with CHCl3 (3x). The combined organic layers were washed with saturated Na2S2O3 solution (2x), H2O, then brine, and dried over sodium

198 sulfate and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 0% → 1% → 2% EtOAc with 25% CH2Cl2 in hexanes) to provide iodide 4.88 (463 mg, 56%) as a white solid: 1H NMR (300 MHz,

CDCl3) δ 7.75 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.20

(d, J = 8.2 Hz, 2H), 3.00 (t, J = 5.4 Hz, 2H), 2.84 (t, J = 5.2 Hz, 2H), 2.66 (s, 3H), 2.35 (s,

13 3H), 1.88 – 1.72 (m, 4H). C NMR (75 MHz, CDCl3) δ 144.8, 136.4, 136.2, 136.0,

134.2, 133.5, 130.0, 129.9, 126.5, 119.0, 114.0, 97.0, 25.3, 25.2, 25.1, 22.9, 22.6, 21.7. IR

(film): 2937, 1597, 1366, 1178, 1091, 801, 669. HRMS-ESI (M+Na)+ calc. for

C20H20INO2SNa 488.0157, found: 487.9750.

N-tosyl-4-prenyl-5-nitroindole 4.94. 4-methyl-5-nitroindole 4.71 (1.30 g, 3.37 mmol) was dissolved in DMF (16.9 mL) and heated to 105 °C.

Trisdimethylaminomethane (0.701 mL, 4.05 mmol) was added to the hot solution and the resulting mixture was stirred at 105 °C for 3 h. After complete consumption of indole

4.71, the reaction was cooled to rt and then the red solution was slowly poured into a mixture of AcOH (10 mL), H2O (40 mL), and reagent grade THF (20 mL). Additional

THF (20 mL) was added to rinse and transfer any remaining solution into the acidic mixture. The resulting mixture was stirred at rt for 3 h and then the aqueous layer was

199 extracted with EtOAc (3x). The combined organic layers were washed with H2O (3x), saturated NaHCO3 solution (3x), and brine, then dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 5% EtOAc with 25% CHCl3 in hexanes) to provide intermediate aldehyde (1.02 g, 73%) as a yellowish-white solid: 1H NMR (300 MHz,

CDCl3) δ 9.88 (s, 1H), 8.27 (d, J = 9.3 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.68 (d, J = 8.3

Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 4.32 (s, 2H), 3.02 (t, J = 5.7 Hz, 2H), 2.77 (t, J = 5.4

13 Hz, 2H), 2.38 (s, 3H), 1.89 – 1.72 (m, 4H). C NMR (75 MHz, CDCl3) δ 197.7, 145.8,

145.7, 139.4, 138.5, 135.8, 130.5, 130.4, 126.7, 121.8, 120.8, 118.2, 113.8, 43.6, 25.1,

24.6, 22.5, 22.3, 21.8. IR (film): 2943, 1726, 1519, 1343, 1179, 666. HRMS-ESI

+ (M+Na) calc. for C21H20N2O5SNa 435.0991, found: 435.0995

The dropwise addition of n-BuLi (2.5M in THF, 0.75 mL, 1.88 mmol) to a suspension of iPrPPh3 (866 mg, 2.50 mmol) in THF (7.5 mL) at 0 °C generated a red suspension, which was stirred at 0 °C for 1 h. In a separate reaction flask, intermediate aldehyde (618 mg, 1.50 mmol) was dissolved in THF (15 mL). This solution was added, via a glass syringe, dropwise to the red suspension. After stirring the mixture at 0 °C for

15 min, the ice-bath was removed and the reaction stirred at rt for 2 h. The reaction was quenched with a 10% aqueous solution of NH4Cl. The aqueous layer was extracted with

EtOAc (3x). The combined organic layers were washed with H2O, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude mixture for purified by flash column chromatography (silica gel, 0% → 0.5% → 1% EtOAc with 30% CHCl3 in hexanes) to provide olefin 4.94 (501 mg, 76%) as a yellowish-white solid: 1H NMR

200

(300 MHz, CDCl3) δ 8.15 (d, J = 9.1 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.1

Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 5.06 (t, J = 5.9 Hz, 1H), 3.80 (d, J = 5.3 Hz, 2H), 3.02

(t, J = 5.5 Hz, 2H), 2.86 (t, J = 5.4 Hz, 2H), 2.38 (s, 3H), 1.90 – 1.74 (m, 4H), 1.70 (s,

13 3H), 1.68 (s, 3H). C NMR (75 MHz, CDCl3) δ 146.5, 145.4, 138.2, 137.9, 136.0, 133.0,

130.2, 129.9, 129.5, 126.6, 122.4, 120.2, 119.1, 112.5, 27.6, 25.7, 25.1, 24.1, 22.7, 22.4,

21.8, 18.4. IR (film): 2937, 1521, 1352, 1180, 1157, 814, 669. HRMS-ESI (M+Na)+ calc. for C24H26N2O4SNa 461.1511, found: 461.1503

N-tosyl-4-prenyl-aniline 4.95. 5-nitro-4-prenylindole 4.94 (500 mg, 1.14 mmol) was dissolved in reagent grade CH2Cl2 (11.4 mL) and cooled to 0 °C. After Zn dust (745 mg, 11.40 mmol) and AcOH (1.14 mL) were added sequentially, the ice bath was removed and the suspension was stirred at rt for 1 h. The suspension was then filtered through a pad of Celite, washed with CH2Cl2, and concentrated under reduced pressure.

The concentrate was taken up in EtOAc and washed with saturated NaHCO3 solution

(3x), H2O (2x), brine, then dried over sodium sulfate and concentrated under reduced pressure. The crude mixture can be taken to the next step without purification.

Alternatively, the crude mixture can be purified by flash column chromatography (silica gel, 0% → 2.5% EtOAc with 30% CH2Cl2 in hexanes + 1% NH4OH) to afford amine 201

1 4.95 (340 mg, 72%) as a brownish-white solid: H NMR (300 MHz, CDCl3) δ 7.87 (d, J

= 8.6 Hz, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.16 (d, J = 8.1 Hz, 2H), 6.62 (d, J = 8.8 Hz, 1H),

5.05 (t, J = 5.8 Hz, 1H), 3.49 (br s, 2H, -NH), 3.49 (d, J = 5.7 Hz, 2H), 2.97 (t, J = 5.0

Hz, 2H), 2.79 (t, J = 5.0 Hz, 2H), 2.33 (s, 3H), 1.79 (s, 3H), 1.79 (s, 4H), 1.70 (s, 3H).

13 C NMR (75 MHz, CDCl3) δ 144.2, 141.1, 136.5, 135.8, 133.0, 130.8, 129.8, 129.8,

126.5, 122.7, 119.0, 117.8, 113.7, 113.3, 26.6, 25.8, 25.3, 24.7, 22.8, 21.7, 18.2. IR

(film): 3436, 3367, 2933, 1620, 1433, 1366, 1178, 1157, 913, 811, 670. HRMS-ESI

+ (M+H) calc. for C24H29N2O2S 409.1950, found: 409.1934

N-tosyl-4-prenyl-iodoindole 4.96. Iodination was performed according to the one-pot iodination method developed by Knochel and coworkers.117 Amine 4.95 (340 mg, 0.83 mmol) was added to a solution of 4-toluenesulfonic acid monohydrate (475 mg,

2.50 mmol) in reagent grade MeCN (3.33 mL). The resulting suspension was cooled to

10-15 °C and an aqueous solution of NaNO2 (115 mg, 1.66 mmol) and KI (691 mg, 4.16 mmol), in H2O (0.50 mL) was slowly added to this suspension. The temperature was maintained for 15 min before the cold water bath was removed and the reaction was stirred vigorously at rt for 30 min. Upon reaction completion, the mixture was diluted with 50% aqueous solution of Na2S2O3 in H2O, and then basified with saturated 202

NaHCO3. The aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with saturated Na2S2O3, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified through flash column chromatography (silica gel, 25% → 30% CHCl3 in hexanes) to yield iodide 4.96

1 (227 mg, 52%) as a white solid: H NMR (300 MHz, CDCl3) δ 7.79 (d, J = 8.8 Hz, 1H),

7.66 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.1 Hz, 2H), 4.93 (t, J =

5.5 Hz, 1H), 3.74 (d, J = 4.5 Hz, 2H), 3.00 (t, J = 4.9 Hz, 2H), 2.79 (t, J = 4.9 Hz, 2H),

13 2.35 (s, 3H), 1.79 (s, 3H), 1.86 – 1.72 (m, 4H), 1.69 (s, 3H). C NMR (75 MHz, CDCl3)

δ 144.8, 136.8, 136.6, 136.2, 135.9, 134.5, 132.4, 130.0, 129.7, 126.5, 122.6, 118.4,

114.2, 96.3, 36.9, 25.8, 25.1, 24.3, 22.8, 22.6, 21.7, 18.9. IR (film): 2933, 1597, 1421,

+ 1367, 1180, 802, 670, 588. HRMS-ESI (M+Na) calc. for C24H26INO2SNa 542.0627, found: 542.0613

N-tosyl-4-prenyl-carboxaldehyde 4.97. For this reaction, THF was distilled over

LiAlH4 and DMF was distilled over CaH2. In a flame-dried flask containing 4Å molecular sieves (75 mg), iodide 4.96 (75 mg, 0.144 mmol) was dissolved in THF (2.88 mL) and cooled to -78 °C. To the resulting mixture was added n-BuLi (2.5M in THF,

0.087 mL, 0.217 mmol) and immediately followed by addition of DMF (0.111 mL, 1.44

203 mmol). The reaction was stirred at -78 °C for 30 min before the CO2-acetone bath was removed to rapidly warm the reaction to rt. The reaction was stirred at rt for 2 h before quenching the reaction with EtOAc. The molecular sieves were filtered out over a pad of

Celite and washed with EtOAc. The filtrate was concentrated under pressure to 25% of original filtrate volume and then added to a 10% aqueous NH4Cl solution. The aqueous layer was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 5% EtOAc in hexanes)

1 to furnish aldehyde 4.97 (49 mg, 82%) as a white solid: H NMR (300 MHz, CDCl3) δ

10.31 (s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 8.1 Hz, 2H),

7.23 (d, J = 8.1 Hz, 2H), 5.09 (t, J = 5.5 Hz, 1H), 4.02 (d, J = 5.5 Hz, 2H), 3.02 (t, J = 5.5

Hz, 2H), 2.88 (t, J = 5.5 Hz, 2H), 1.89 – 1.77 (m, 4H), 1.76 (s, 3H), 1.67 (s, 3H). 13C

NMR (75 MHz, CDCl3) δ 192.0, 145.1, 139.8, 139.0, 137.0, 136.3, 132.1, 130.1, 129.8,

129.3, 126.6, 125.9, 123.9, 118.9, 112.7, 26.9, 25.7, 25.1, 24.5, 22.8, 22.6, 21.7, 18.4. IR

+ (film): 2933, 1679, 1578, 1385, 1179, 669. HRMS-ESI (M+Na) calc. for C25H27NO3SNa

444.1613, found: 444.1609

204

N-tosyl allylic alcohol 4.98. Aldehyde 4.97 (40 mg, 0.0949 mmol) was dissolved in THF (1.00 mL) and cooled to 0 °C. 2-methyl-1-propenylmagnesium bromide (0.5M in

THF, 0.285 mL, 0.142 mmol) was added dropwise to the cooled solution. The solution was gradually warmed to rt and stirred for 2 h. The reaction was quenched with addition of 10% aqueous NH4Cl solution and then the aqueous layer was extracted with EtOAc

(3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude mixture was taken to the next step without purification. Alternatively, purification of the crude mixture by flash column chromatography (deactivated silica gel, 16% → 18% → 20% EtOAc in hexanes) yielded

1 allylic alcohol 4.98 (34 mg, 75%) as a white film: H NMR (300 MHz, CDCl3) δ 8.07 (d,

J = 8.8 Hz, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.8 Hz, 1H), 7.19 (d, J = 8.2 Hz,

2H), 5.67 (d, J = 8.5 Hz, 1H), 5.49 (d, J = 8.5 Hz, 1H), 5.05 (t, J = 5.6 Hz, 1H), 3.65

(ddd, J = 21.4, 16.6, 5.3 Hz, 2H), 2.34 (s, 3H), 1.88 – 1.70 (m, 13H), 1.67 (s, 3H). 13C

NMR (75 MHz, CDCl3) δ 144.5, 137.5, 136.5, 136.0, 135.5, 135.4, 131.7, 131.6, 129.9,

129.0, 127.9, 126.6, 124.5, 122.3, 118.7, 112.8, 67.2, 27.5, 26.0, 25.6, 25.2, 24.5, 23.0,

22.8, 21.7, 18.4, 18.4. IR (film): 3379, 2931, 1574, 1369, 1178, 812, 670. HRMS-ESI

+ (M+Na) calc. for C29H35NO3SNa 500.2235, found: 500.2233

205

N-tosyl epoxyalcohol (±)-4.110. tert-Butyl hydroperoxide (70%, 43 µL, 0.314 mmol) was added to the blue-green solution of VO(acac)2 (3.3 mg, 0.0126 mmol) in benzene (0.63 mL) while keeping the temperature between 10-15 °C. A solution of crude allylic alcohol 4.98 (30 mg, 0.0628 mmol) in benzene (0.63 mL) was added dropwise to the flask containing the brown colored activated vanadium-hydroperoxide solution. The temperature was gradually allowed to warm over 1 h then the reaction was stirred for 1 h at rt. The reaction was then diluted with EtOAc and poured on to a 50:50 mix of saturated

NaHCO3 and saturated Na2S2O3 solutions. The aqueous layer was extracted with EtOAc

(3x). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to give a brown film. Purification of the crude material by flash column chromatography (deactivated silica gel, 20% EtOAc in hexanes) to furnish epoxyalcohol 4.110 (16 mg, 52%) as an white solid: 1H NMR (400 MHz,

CDCl3) δ 8.09 (d, J = 8.8 Hz, 1H, H-19), 7.66 (d, J = 8.3 Hz, 2H, H-2” and H-6”), 7.37

(d, J = 8.8 Hz, 1H, H-18), 7.20 (d, J = 8.0 Hz, 2H, H-3” and H-5”), 5.03 (t, J = 5.5 Hz,

1H, H-26), 4.76 (d, J = 7.0 Hz, 1H, H-30), 3.73 (dd, J = 15.6, 5.7 Hz, 1H, H-25a), 3.65

(dd, J = 16.3, 4.9 Hz, 1H, H-25b), 3.21 (d, J = 6.9 Hz, 1H, H-31), 3.10 – 2.92 (m, 2H, H-

206

1’), 2.84 (t, J = 5.9 Hz, 2H, H-4’), 2.41 (s, 1H, -OH), 2.35 (s, 3H, H-7”), 1.86 – 1.74 (m,

4H, H-2’ and H-3’), 1.73 (s, 3H, H-28), 1.66 (s, 3H, H-29), 1.32 (s, 3H, H-33), 1.26 (s,

13 3H, H-34). C NMR (101 MHz, CDCl3) δ 144.6 (C-4”), 136.5 (C-1”), 136.4 (C-20),

135.9 (C-2), 134.1 (C-17), 132.8 (C-16), 131.9 (C-27), 130.0 (C-3” and C-5”), 129.3 (C-

15), 126.6 (C-2” and C-5”), 124.5 (C-26), 122.7 (C-18), 118.7 (C-14), 112.8 (C-19), 69.0

(C-30), 67.9 (C-31), 60.2 (C-32), 27.6 (C-25), 25.6 (C-29), 25.2 (C-1’), 24.9 (C-33), 24.4

(C-4’), 23.0 (C-3’), 22.7 (C-2’), 21.7 (C-7”), 19.3 (C-34), 18.4 (C-28). IR (film): 3424,

+ 2926, 1449, 1369, 1179, 813, 670. HRMS-ESI (M+Na) calc. for C29H35NO4SNa

516.2184, found: 516.2199

Indole subunit of sespendole (±)-4.113. According to the procedure by Lebold et al. for indole N-tosyl group deprotection:123 Epoxyalcohol 4.110 (9 mg, 18.2 µmol) was dissolved in reagent grade THF (0.40 mL) and reagent grade MeOH (0.40 mL).

Magnesium turnings (9 mg, 365 µmol) followed by NH4Cl (2 mg, 36.4 µmol) were added and the mixture stirred at rt for 14 h. The reaction was quenched with H2O and the organic layer extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated to dryness under reduced pressure. A Hitachi

207

LaChrome Elite HPLC system (Hitachi HTA) equipped with a Hitachi L-2400 ultraviolet detector (UV) and controlled by EZ Chrom Elite 3.2.1 software (Agilent Technologies), was used for the purification of 4.113. A Luna 5 µm C18(2) column (250 mm L. x 21.2 mm I.D.; Phenomenex) with an isocratic mobile phase of methanol/water (72:28, v/v) at

16.0 mL/min. for 45 min. was utilized for the preparative chromatography. Indole subunit of sespendole 4.113 was detected by observation of UV absorption at 284 nm, and had a reproducible retention time of 31.77 min using the described separation conditions.

1 Indole 4.113 was isolated as a white solid: H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H),

7.23 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 5.16 (t, J = 5.9 Hz, 1H), 4.81 (dd, J =

7.2, 3.0 Hz, 1H), 3.82 (dd, J = 16.1, 5.8 Hz, 1H), 3.75 (dd, J = 16.1, 5.8 Hz, 1H), 3.27 (d,

J = 7.2 Hz, 1H), 2.94 (t, J = 4.3 Hz, 2H), 2.73 (t, J = 4.3 Hz, 2H), 2.22 (d, J = 3.2 Hz,

1H), 1.92 – 1.81 (m, 4H), 1.78 (s, 3H), 1.67 (s, 3H), 1.31 (s, 3H), 1.26 (s, 3H). 1H NMR

(300 MHz, Pyr-d5) δ 11.51 (s, 1H, NH), 7.60 (d, J = 8.3 Hz, 1H, H-18), 7.48 (d, J = 8.3

Hz, 1H, H-19), 5.45 (t, J = 5.9 Hz, 1H, H-26), 5.27 (d, J = 7.5 Hz, 1H, H-30), 4.26 (dd, J

= 15.9, 7.1 Hz, 1H, H-25a), 4.12 (d, J = 15.9 Hz, 1H, H-25b), 3.84 (d, J = 7.6 Hz, 1H, H-

31), 3.07 (t, J = 4.3 Hz, 2H, H-1’), 2.74 (t, J = 4.7 Hz, 2H, H-4’), 1.77 (s, 3H, H-28), 1.83

– 1.69 (m, 4H, H-2’ and H-3’), 1.61 (s, 3H, H-29), 1.35 (s, 3H, H-33), 1.34 (s, 3H, H-34).

13 C NMR (75 MHz, Pyr-d5) δ 136.9 (C-20), 135.6 (identified from HMBC, C-2), 132.6

(C-16), 131.1 (C-17), 130.0 (C-27), 127.9 (C-15), 126.9 (C-26), 120.8 (C-18), 109.9 (C-

14), 109.4 (C-19), 71.1 (C-30), 69.5 (C-31), 58.6 (C-32), 28.7 (C-25), 25.6 (C-29), 25.2

(C-23), 24.5, 24.3, 23.9, 23.1, 19.8 (C-34), 18.3 (C-28).

208

References

(1) Engelman, A.; Kessl, J. J.; Kvaratskhelia, M. Curr. Opin. Chem. Biol. 2013, 17, 339–345.

(2) Engelman, A.; Cherepanov, P. Nat. Rev. Microbiol. 2012, 10, 279–290.

(3) Nyamweya, S.; Hegedus, A.; Jaye, A.; Rowland-Jones, S.; Flanagan, K. L.; Macallan, D. C. Rev. Med. Virol. 2013, 23, 221–240.

(4) Goldgur, Y.; Dyda, F.; Hickman, A. B.; Jenkins, T. M.; Craigie, R.; Davies, D. R. Proc. Natl. Acad. Sci. USA 1998, 95, 9150–9154.

(5) Singh, S. B.; Zink, D. L.; Goetz, M. A.; Dombrowski, A. W.; Polishook, J. D.; Hazuda, D. J. Tetrahedron Lett. 1998, 39, 2243–2246.

(6) Singh, S. B.; Pelaez, F.; Hazuda, D. J.; Lingham, R. B. Drugs Fut. 2005, 30, 277– 299.

(7) Goldgur, Y.; Craigie, R.; Cohen, G. H.; Fujiwara, T.; Yoshinaga, T.; Fujishita, T.; Sugimoto, H.; Endo, T.; Murai, H.; Davies, D. R. Proc. Natl. Acad. Sci. USA 1999, 96, 13040–13043.

(8) Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Stillmock, K.; Grobler, J. A.; Espeseth, A.; Gabryelski, L.; Schleif, W.; Blau, C.; Miller, M. D. Science 2000, 287, 646–650.

(9) Al-Mawsawi, L. Q.; Neamati, N. ChemMedChem 2011, 6, 228–41.

(10) Quashie, P. K.; Mesplède, T.; Wainberg, M. A. Curr. Opin. Infect. Dis. 2013, 26, 43–49.

(11) Quashie, P. K.; Sloan, R. D.; Wainberg, M. A. BMC Med. 2012, 10, 34.

(12) Hightower, K. E.; Wang, R.; Deanda, F.; Johns, B. a; Weaver, K.; Shen, Y.; Tomberlin, G. H.; Carter, H. L.; Broderick, T.; Sigethy, S.; Seki, T.; Kobayashi, M.; Underwood, M. R. Antimicrob. Agents Chemother. 2011, 55, 4552–4559. 209

(13) Cherepanov, P.; Maertens, G.; Proost, P.; Devreese, B.; van Beeumen, J.; Engelborghs, Y.; de Clercq, E.; Debyser, Z. J. Biol. Chem. 2003, 278, 372–381.

(14) Llano, M.; Saenz, D. T.; Meehan, A.; Wongthida, P.; Peretz, M.; Walker, W. H.; Teo, W.; Poeschla, E. M. Science 2006, 314, 461–464.

(15) Cherepanov, P.; Devroe, E.; Silver, P. A.; Engelman, A. J. Biol. Chem. 2004, 279, 48883–48892.

(16) Suzuki, Y.; Craigie, R. Nat. Rev. Microbiol. 2007, 5, 187–196.

(17) Cherepanov, P.; Ambrosio, A. L. B.; Rahman, S.; Ellenberger, T.; Engelman, A. Proc. Natl. Acad. Sci. USA 2005, 102, 17308–17313.

(18) McKee, C. J.; Kessl, J. J.; Shkriabai, N.; Dar, M. J.; Engelman, A.; Kvaratskhelia, M. J. Biol. Chem. 2008, 283, 31802–31812.

(19) Molteni, V.; Greenwald, J.; Rhodes, D.; Hwang, Y.; Kwiatkowski, W.; Bushman, F. D.; Siegel, J. S.; Choe, S. Acta Crystallogr. D 2001, 57, 536–544.

(20) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605–1612.

(21) Lin, Z.; Neamati, N.; Zhao, H.; Kiryu, Y.; Turpin, J. A.; Aberham, C.; Strebel, K.; Kohn, K.; Witvrouw, M.; Pannecouque, C.; Debyser, Z.; de Clercq, E.; Rice, W. G.; Pommier, Y.; Burke, T. R. J. Med. Chem. 1999, 42, 1401–1414.

(22) Shkriabai, N.; Patil, S. S.; Hess, S.; Budihas, S. R.; Craigie, R.; Burke, T. R.; Le Grice, S. F. J.; Kvaratskhelia, M. Proc. Natl. Acad. Sci. USA 2004, 101, 6894– 6899.

(23) Kessl, J. J.; Eidahl, J. O.; Shkriabai, N.; Zhao, Z.; McKee, C. J.; Hess, S.; Burke, T. R.; Kvaratskhelia, M. Mol. Pharmacol. 2009, 76, 824–832.

(24) Fan, X.; Zhang, F.-H.; Al-Safi, R. I.; Zeng, L.-F.; Shabaik, Y.; Debnath, B.; Sanchez, T. W.; Odde, S.; Neamati, N.; Long, Y.-Q. Biorg. Med. Chem. 2011, 19, 4935–4952.

(25) Peat, T. S.; Rhodes, D. I.; Vandegraaff, N.; Le, G.; Smith, J. A.; Clark, L. J.; Jones, E. D.; Coates, J. A. V; Thienthong, N.; Newman, J.; Dolezal, O.; Mulder, R.; Ryan, J. H.; Savage, G. P.; Francis, C. L.; Deadman, J. J. PloS One 2012, 7, e40147.

210

(26) De Luca, L.; Barreca, M. L.; Ferro, S.; Christ, F.; Iraci, N.; Gitto, R.; Monforte, A.-M.; Debyser, Z.; Chimirri, A. ChemMedChem 2009, 4, 1311–1316.

(27) De Luca, L.; Gitto, R.; Christ, F.; Ferro, S.; de Grazia, S.; Morreale, F.; Debyser, Z.; Chimirri, A. Antiviral Res. 2011, 92, 102–107.

(28) Christ, F.; Voet, A.; Marchand, A.; Nicolet, S.; Desimmie, B. A.; Marchand, D.; Bardiot, D.; van der Veken, N. J.; van Remoortel, B.; Strelkov, S. V; de Maeyer, M.; Chaltin, P.; Debyser, Z. Nat. Chem. Biol. 2010, 6, 442–448.

(29) Tsantrizos, Y. S.; Bailey, M. D.; Bilodeau, F.; Carson, R. J.; Coulombe, R.; Fader, L.; Halmos, T.; Kawai, S.; Landry, S.; Laplante, S.; Morin, S.; Parisien, M.; Poupart, M.-A.; Simoneau, B. Inhibitors of Human Immunodeficiency Virus Replication. International Patent Application PCT/CA2008/001611 2009, 1–174.

(30) Kessl, J. J.; Jena, N.; Koh, Y.; Taskent-Sezgin, H.; Slaughter, A.; Feng, L.; de Silva, S.; Wu, L.; Le Grice, S. F. J.; Engelman, A.; Fuchs, J. R.; Kvaratskhelia, M. J. Biol. Chem. 2012, 287, 16801–16811.

(31) Feng, L.; Sharma, A.; Slaughter, A.; Jena, N.; Koh, Y.; Shkriabai, N.; Larue, R. C.; Patel, P. A.; Mitsuya, H.; Kessl, J. J.; Engelman, A.; Fuchs, J. R.; Kvaratskhelia, M. J. Biol. Chem. 2013, 288, 15813–15820.

(32) Tsantrizos, Y. S.; Boes, M.; Brochu, C.; Fenwick, C.; Malenfant, E.; Mason, S.; Pesant, M. Inhibitors of Human Immunodeficiency Virus Replication. International Patent Application PCT/CA2007/000845 2007, 1–121.

(33) Wang, H.; Jurado, K. A.; Wu, X.; Shun, M.-C.; Li, X.; Ferris, A. L.; Smith, S. J.; Patel, P. A.; Fuchs, J. R.; Cherepanov, P.; Kvaratskhelia, M.; Hughes, S. H.; Engelman, A. Nucleic Acids Res. 2012, 40, 11518–11530.

(34) Jurado, K. A.; Wang, H.; Slaughter, A.; Feng, L.; Kessl, J. J.; Koh, Y.; Wang, W.; Ballandras-Colas, A.; Patel, P. A.; Fuchs, J. R.; Kvaratskhelia, M.; Engelman, A. Proc. Natl. Acad. Sci. USA 2013, 110, 8690–8695.

(35) Tsiang, M.; Jones, G. S.; Niedziela-Majka, A.; Kan, E.; Lansdon, E. B.; Huang, W.; Hung, M.; Samuel, D.; Novikov, N.; Xu, Y.; Mitchell, M.; Guo, H.; Babaoglu, K.; Liu, X.; Geleziunas, R.; Sakowicz, R. J. Biol. Chem. 2012, 287, 21189–21203.

(36) Böhm, H.-J.; Flohr, A.; Stahl, M. Drug Discov. Today: Technol 2004, 1, 217–224.

(37) Sun, H.; Tawa, G.; Wallqvist, A. Drug Discov. Today 2012, 17, 310–324.

(38) Humphrey, G. R.; Kuethe, J. T. Chem. Rev. 2006, 106, 2875–2911. 211

(39) Gribble, G. W. Pure Appl. Chem. 2003, 75, 1417–1432.

(40) Yoakim, C.; Bailey, M. D.; Bilodeau, F.; Carson, R. J.; Fader, L.; Kawai, S.; Laplante, S.; Simoneau, B.; Surprenant, S.; Thibeault, C.; Tsantrizos, Y. S. Inhibitors of Human Immunodeficiency Virus Replication. International Patent Application PCT/CA2010/000707 2010, 1–189.

(41) Kumari, V. Structure-Based Computer Aided Drug Design and Analysis for Different Disease Targets, The Ohio State University, 2011, p. 250.

(42) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. J. Med. Chem. 2002, 45, 1712–1722.

(43) Hermann, J. C.; Chen, Y.; Wartchow, C.; Menke, J.; Gao, L.; Gleason, S. K.; Haynes, N.-E.; Scott, N.; Petersen, A.; Gabriel, S.; Vu, B.; George, K. M.; Narayanan, A.; Li, S. H.; Qian, H.; Beatini, N.; Niu, L.; Gan, Q.-F. ACS Med. Chem. Lett. 2013, 4, 197–200.

(44) Davis, B. J.; Erlanson, D. A. Bioorg. Med. Chem. Lett. 2013, 23, 2844–2852.

(45) Murray, C. W.; Rees, D. C. Nat. Chem. 2009, 1, 187–192.

(46) Drug Discovery: Practices, Processes, and Perspectives; Li, J. J.; Corey, E. J., Eds.; 2013th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2013.

(47) Duggar, B. M. Ann. N.Y. Acad. Sci. 1948, 51, 177–181.

(48) Finlay, A. C.; Hobby, G. L.; P’an, S. Y.; Regna, P. P.; Routien, J. B.; Seeley, D. B.; Shull, G. M.; Sobin, B. A.; Solomons, I. A.; Vinson, J. W.; Kane, J. H. Science 1950, 111, 85–85.

(49) Endo, A. Atheroscler. Suppl. 2004, 5, 125–130.

(50) Burg, R. W.; Miller, B. M.; Baker, E. E.; Birnbaum, J.; Currie, S. A.; Hartman, R.; Kong, Y.-L.; Monaghan, R. L.; Olson, G.; Putter, I.; Tunac, J. B.; Wallick, H.; Stapley, E. O.; Oiwa, R.; Omura, S. Antimicrob. Agents Chemother. 1979, 15, 361–367.

(51) Schardl, C. L.; Panaccione, D. G.; Tudzynski, P. In The Alkaloids: Chemistry and Biology; Cordell, G. A., Ed.; Academic Press: San Diego, 2006; Vol. 63, pp. 45– 86.

(52) Uchida, R.; Kim, Y.-P.; Namatame, I.; Tomoda, H.; Omura, S. J. Antibiot. 2006, 59, 93–97. 212

(53) Yamaguchi, Y.; Masuma, R.; Kim, Y.-P.; Uchida, R.; Tomoda, H.; Omura, S. Mycoscience 2004, 45, 9–16.

(54) Uchida, R.; Kim, Y.-P.; Nagamitsu, T.; Tomoda, H.; Omura, S. J. Antibiot. 2006, 59, 338–344.

(55) Sings, H.; Singh, S. B. In The Alkaloids: Chemistry and Biology; Cordell, G. A., Ed.; Academic Press: San Diego, 2003; Vol. 60, pp. 51–163.

(56) Roll, D. M.; Barbieri, L. R.; Bigelis, R.; McDonald, L. A.; Arias, D. A.; Chang, L.- P.; Singh, M. P.; Luckman, S. W.; Berrodin, T. J.; Yudt, M. R. J. Nat. Prod. 2009, 72, 1944–1948.

(57) Singh, S. B.; Ondeyka, J. G.; Jayasuriya, H.; Zink, D. L.; Ha, S. N.; Dahl-Roshak, A.; Greene, J.; Kim, J. A.; Smith, M. M.; Shoop, W.; Tkacz, J. S. J. Nat. Prod. 2004, 67, 1496–1506.

(58) Nakazawa, J.; Yajima, J.; Usui, T.; Ueki, M.; Takatsuki, A.; Imoto, M.; Toyoshima, Y. Y.; Osada, H. Chem. Biol. 2003, 10, 131–137.

(59) Dorner, J. W.; Cole, R. J.; Cox, R. H.; Cunfer, B. M. J. Agric. Food Chem. 1984, 32, 1069–1071.

(60) Tomoda, H.; Omura, S. Pharmacol. Ther. 2007, 115, 375–389.

(61) Guo, Y.; Cordes, K. R.; Farese, R. V; Walther, T. C. J. Cell. Sci. 2009, 122, 749– 752.

(62) Walther, T. C.; Farese, R. V Annu. Rev. Biochem. 2012, 81, 687–714.

(63) Goodman, J. M. J. Biol. Chem. 2008, 283, 28005–9.

(64) Bozza, P. T.; Viola, J. P. B. Prostaglandins, Leukotrienes Essent. Fatty Acids 2010, 82, 243–250.

(65) Igal, R. A.; Wang, P.; Coleman, R. A. Biochem. J. 1997, 324, 529–534.

(66) Krahmer, N.; Guo, Y.; Farese, R. V; Walther, T. C. Cell 2009, 139, 1024–1024.e1.

(67) Uchida, R.; Tomoda, H.; Omura, S. J. Antibiot. 2006, 59, 298–302.

(68) De Jesus, A. E.; Gorst-Allman, C. P.; Steyn, P. S.; van Heerden, F. R.; Vleggaar, R.; Wessels, P. L.; Hull, W. E. J. Chem. Soc., Perkin Trans. 1 1983, 08, 1863– 1868. 213

(69) Byrne, K. M.; Smith, S. K.; Ondeyka, J. G. J. Am. Chem. Soc. 2002, 124, 7055– 7060.

(70) Ackin, W.; Weibel, F.; Arigoni, D. Chimia 1977, 31, 63.

(71) Smith, A. B.; Mewshaw, R. J. Am. Chem. Soc. 1985, 107, 1769–1771.

(72) Smith, A. B.; Kingery-Wood, J.; Leenay, T. L.; Nolen, E. G.; Sunazuka, T. J. Am. Chem. Soc. 1992, 114, 1438–1449.

(73) Smith, A. B.; Kanoh, N.; Ishiyama, H.; Minakawa, N.; Rainier, J. D.; Hartz, R. a; Cho, Y. S.; Cui, H.; Moser, W. H. J. Am. Chem. Soc. 2003, 125, 8228–8237.

(74) Smith, A. B.; Cui, H. Helv. Chim. Acta 2003, 86, 3908–3938.

(75) Smith, A. B.; Davulcu, A. H.; Cho, Y. S.; Ohmoto, K.; Kürti, L.; Ishiyama, H. J. Org. Chem. 2007, 72, 4596–4610.

(76) Enomoto, M.; Morita, A.; Kuwahara, S. Angew. Chem. Int. Ed. 2012, 51, 12833– 12836.

(77) Asanuma, A.; Enomoto, M.; Nagasawa, T.; Kuwahara, S. Tetrahedron Lett. 2013, 54, 4561–4563.

(78) Guile, S. D.; Saxton, J. E.; Thornton-Pett, M. J. Chem. Soc., Perkin Trans. 1 1992, 1763.

(79) Harrison, C.-A.; Jackson, P. M.; Moody, C. J.; Williams, J. M. J. J. Chem. Soc., Perkin Trans. 1 1995, 1131–1136.

(80) Rivkin, A.; González-López de Turiso, F.; Nagashima, T.; Curran, D. P. J. Org. Chem. 2004, 69, 3719–25.

(81) England, D. B.; Magolan, J.; Kerr, M. A. Org. Lett. 2006, 8, 2209–2212.

(82) Sugino, K.; Nakazaki, A.; Isobe, M.; Nishikawa, T. Synlett 2011, 647–650.

(83) Adachi, M.; Higuchi, K.; Thasana, N.; Yamada, H.; Nishikawa, T. Org. Lett. 2012, 14, 114–117.

(84) Gassman, P. G.; van Bergen, T. J.; Gilbert, D. P.; Cue, B. W. J. Am. Chem. Soc. 1974, 96, 5495–5508.

(85) Smith, A. B.; Leenay, T. L. J. Am. Chem. Soc. 1989, 111, 5761–5768. 214

(86) Smith, A. B.; Hartz, R. A.; Spoors, P. G.; Rainier, J. D. Isr. J. Chem. 1997, 37, 69– 80.

(87) Smith, A. B.; Kanoh, N.; Ishiyama, H.; Hartz, R. A. J. Am. Chem. Soc. 2000, 122, 11254–11255.

(88) Haseltine, J.; Visnick, M.; Smith, A. B. J. Org. Chem. 1988, 53, 6160–6162.

(89) Plieninger, H.; Suhr, K.; Werst, G.; Kiefer, B. Chem. Ber. 1956, 89, 270–278.

(90) Sapeta, K.; Lebold, T.; Kerr, M. A. Synlett 2011, 2011, 1495–1514.

(91) Banfield, S. C.; England, D. B.; Kerr, M. A. Org. Lett. 2001, 3, 3325–3327.

(92) Zawada, P. V.; Banfield, S. C.; Kerr, M. A. Synlett 2003, 34, 971–974.

(93) England, D. B.; Kerr, M. a J. Org. Chem. 2005, 70, 6519–6522.

(94) Johansen, J. E.; Christie, B. D.; Rapoport, H. J. Org. Chem. 1981, 46, 4914–4920.

(95) Xiong, Q.; Zhu, X.; Wilson, W. K.; Ganesan, A.; Matsuda, S. P. T. J. Am. Chem. Soc. 2003, 125, 9002–9003.

(96) Lu, H.; He, K.; Kool, E. T. Angew. Chem. Int. Ed. 2004, 43, 5834–6.

(97) Makosza, M.; Winiarski, J. Acc. Chem. Res. 1987, 20, 282–289.

(98) Bartoli, G. Acc. Chem. Res. 1984, 17, 109–115.

(99) Dalpozzo, R.; Bartoli, G. Curr. Org. Chem. 2005, 9, 163–178.

(100) Bartoli, G.; Bosco, M.; Baccolini, G. J. Org. Chem. 1980, 45, 522–524.

(101) Bartoli, G.; Leardini, R.; Medici, A.; Rosini, G. J. Chem. Soc., Perkin Trans. 1 1978, 692–696.

(102) Spartan ’10 molecular modeling software 2011.

(103) Jeon, Y. T.; Gluchowski, C. Indole and Benzothiazole Derivatives. US Patent 6,498,177 2002, 1–17.

(104) Baran, P. S.; Shenvi, R. A.; Mitsos, C. A. Angew. Chem. Int. Ed. 2005, 44, 3714– 3717.

215

(105) Spessard, S. J.; Stoltz, B. M. Org. Lett. 2002, 4, 1943–1946.

(106) Barboni, L.; Bartoli, G.; Marcantoni, E.; Petrini, M.; Dalpozzo, R. J. Chem. Soc., Perkin Trans. 1 1990, 2133–2138.

(107) Bartoli, G.; Marcantoni, E.; Bosco, M.; Dalpozzo, R. Tetrahedron Lett. 1988, 29, 2251–2254.

(108) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1988, 110, 1321–1323.

(109) Tichenor, M. S.; Trzupek, J. D.; Kastrinsky, D. B.; Shiga, F.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2006, 128, 15683–15696.

(110) Muchowski, J. M.; Venuti, M. C. J. Org. Chem. 1980, 45, 4798–4801.

(111) Griffen, E. J.; Roe, D. G.; Snieckus, V. J. Org. Chem. 1995, 60, 1484–1485.

(112) Kurti, L.; Czakó, B. Strategic Applications of Named Reactions in Organic Synthesis; Elsevier Academic Press: Burlington, MA, 2005; p. 864.

(113) Miyata, O.; Takeda, N.; Kimura, Y.; Takemoto, Y.; Tohnai, N.; Miyata, M.; Naito, T. Tetrahedron 2006, 62, 3629–3647.

(114) Liu, K. G.; Robichaud, A. J.; Lo, J. R.; Mattes, J. F.; Cai, Y. Org. Lett. 2006, 8, 5769–5771.

(115) Negishi, E.; Copéret, C.; Ma, S.; Mita, T.; Sugihara, T.; Tour, J. M. J. Am. Chem. Soc. 1996, 118, 5904–5918.

(116) Payne, G. B. J. Org. Chem. 1962, 27, 3819–3822.

(117) Krasnokutskaya, E.; Semenischeva, N.; Filimonov, V.; Knochel, P. Synthesis 2007, 2007, 81–84.

(118) Rossiter, B. E.; Verhoeven, T. R.; Sharpless, K. B. Tetrahedron Lett. 1979, 20, 4733–4736.

(119) Sharpless, K. B.; Michaelson, R. C. J. Am. Chem. Soc. 1973, 95, 6136–6137.

(120) Mazzola, V. J.; Bernady, K. F.; Franck, R. W. J. Org. Chem. 1967, 32, 486–489.

(121) Cobb, J.; Demetropoulos, I. N.; Korakas, D.; Skoulika, S.; Varvounis, G. Tetrahedron 1996, 52, 4485–4494.

216

(122) Hulcoop, D. G.; Lautens, M. Org. Lett. 2007, 9, 1761–4.

(123) Lebold, T. P.; Kerr, M. A. Org. Lett. 2007, 9, 1883–1886.

217

Appendix: NMR Spectra of Selected Compounds

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235