Novel Polycyclic Benzannulated Indole Scaffolds Via Indole

NOVEL POLYCYCLIC BENZANNULATED INDOLE SCAFFOLDS VIA INDOLE

ARYNE CYCLOADDITION, Pd (0)-CATALYZED CROSS-COUPLING AND

ROM/RCM METHODOLOGIES. THEIR APPLICATIONS TO

LIBRARY DEVELOPMENT AND DRUG DISCOVERY

A DISSERTATION IN Chemistry and Pharmaceutical Sciences

Presented to the Faculty of the University of Missouri-Kansas City in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

by LINCOLN WAMAE MAINA

B.S., University Central Missouri, 2007 M.S., University Missouri-Kansas City, 2014

Kansas City, Missouri 2014

LINCOLN WAMAE MAINA

NOVEL POLYCYCLIC BENZANNULATED INDOLE SCAFFOLDS VIA INDOLE

ARYNE CYCLOADDITION, Pd (0)-CATALYZED CROSS-COUPLING AND

ROM/RCM METHODOLOGIES, AND THEIR APPLICATION TO

LIBRARY DEVELOPMENT AND DRUG DISCOVERY

Lincoln Wamae Maina, Candidate for the Doctor of

Philosophy Degree, University of Missouri-Kansas City, 2014

ABSTRACT

Dissertation under the direction of Keith R. Buszek, Ph.D., Professor of Chemistry,

University of Missouri-Kansas City.

Heterocyclic chemistry is one of the most valuable sources of novel compounds with diverse biological activity. The indole nucleus for instance, is an important element of many natural and synthetic molecules with significant biological activity. Drug discovery and development processes highly value the utility of the ability to synthesize a diverse library based on one core scaffold which can be screened against a variety of different receptors and cell lines, yielding several active compounds. Privileged structures offer an ideal source of lead compounds for drug discovery due to their inherent affinity for diverse biological targets. Indole motifs represent one of the most prominent privileged structures and are ubiquitous in natural products and pharmaceutical compounds. A variety of fused heterocyclic structures will be designed, resulting in novel polycyclic frameworks with virtually no representation in the National Institutes of Health PubChem and Molecular

Library of Small Molecule Repository (MLSMR) databases.

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These molecular frameworks would be predicted to occupy unoccupied or sparsely occupied regions of the chemical property space, which increase their chances of finding applications as new drug leads with different modes of actions. Consequently, efficient methodologies resulting in polycyclic structures from biologically active heterocyclic templates are highly desired in the drug discovery and development programs. The synthesis of such a unique class of polycyclic benzannulated indole scaffolds is described.

The 4,6,7-tribromoindole and the 5,6,7-tribromoindole were synthesized via the

Bartoli and Leimgruber-Batcho and indole synthesis protocols respectively. Both indoles underwent selective metal-halogen exchange at C-7 and subsequent elimination to give the

6,7-indole aryne which underwent Diels-Alder cycloadditions with cyclopentadiene, furan and 2,5-dimethyl furan. These cycloadducts were N-alkylated and then subjected to Grubb’s catalyst ring opening metathesis/ring closing metathesis to afford 12 unique 6,7- benzannulated with an additional 7 or 8–membered rings fused from the benzenoid ring to the pyrrole ring of the indole nucleus.

Library development is facilitated by the extra bromine at the 4 or 5 position of the indole nucleus which serves as a diversity handle which can be subjected to several palladium catalyzed cross-couplings including but not limited to Suzuki-Miyaura, Negishi and Buchwald-Hartwig. Diversity of these scaffolds can be further enhanced via olefin functionalization: cross metathesis, hydroamination and epoxidation among others to afford pharmaceutically useful functional groups like carbamates, ureas, secondary and tertiary amines as well as sulfonamides.

The faculty listed below, appointed by the Dean of the School of Graduate Studies, have examined a dissertation titled, “Novel Polycyclic Benzannulated Indole Scaffolds via Indole Aryne Cycloaddition, Palladium (0) - Catalyzed Cross-coupling and Ring Opening Metathesis (ROM)/Ring Closing Metathesis (RCM) Methodologies, and Their Applications to Library Development and Drug Discovery” presented by Lincoln Wamae Maina, candidate for the Doctor of Philosophy degree, and certify that in their opinion it is worthy of acceptance.

Supervisory Committee

Keith R. Buszek, Ph.D., Committee Chairperson Department of Chemistry

Jerry R. Dias, Ph.D. Department of Chemistry

James R. Durig, Ph.D. Department of Chemistry

Bi Botti Youan, Ph.D. Department of Pharmaceutical Sciences

William G. Gutheil, Ph.D. Department of Pharmaceutical Sciences

CONTENTS

ABSTRACT ...... iii

SCHEMES ...... viii

ILLUSTRATIONS ...... x

SPECTRA ...... xii

ACKNOWLEDGMENTS ...... xvi

CHAPTER

1: INTRODUCTION AND BACKGROUND ...... 1

1.1: Arynes and hetarynes as emerging powerful tools for natural products

total synthesis and library development ...... 1

2: THERAPEUTICS INSPIRED BY NATURAL PRODUCTS ...... 7

2.1: Benzannulated indole alkaloid natural products as inspiration for library

development and library syntheses ...... 7

3: BENZANNULATED INDOLE ALKALOID NATURAL PRODUCTS

DISCOVERY, ISOLATION AND SYNTHESES ………………………….15

3.1: Trikentrins ...... 15

3.2: Herbindoles ...... 16

3.3: Teleocidins ...... 17

3.4: Notoamides ...... 18

3.5: Stephacidins ...... 18

3.6: Penitrems ...... 19

3.7: Nodulisporic Acids ...... 19

4: BIOLOGICAL STUDIES AND RESULTS OF A BENZANNULATED

INDOLE SCAFFOLDS LIBRARIES……………………………………….22

4.1: Synthesis of the benzannulated library cross-coupled indole library ...... 22

5: DESIGN AND SYNTHESIS OF NOVEL 4-, 5-, 6,7 BENZANNULATED

TRIBROMO SCAFFOLDS FOR CROSS-COUPLED LIBRARIES ………33

5.1: Metal halogen exchange studies ...... 33

5.2: Synthesis of 4,6,7-tribromoindole via Bartoli-Indole synthesis ...... 37

5.3: Synthesis of 5,6,7-tribromoindole via Bartoli indole synthesis ...... 38

5.4: Synthesis of 5,6,7-tribromoindole via Leimgruber-Batcho indole synthesis .39

5.5: Original synthetic scheme approach to scaffold type 1 ...... 40

5.6: N-protection of the tribromoindoles ...... 41

5.7: Second generation synthesis of type 1 scaffold ...... 43

5.8: Ring opening/ring closing metathesis scheme ...... 44

5.9: Cross-coupling reactions ...... 46

5.91: Future directions ...... 48

6: EXPERIMENTAL SECTION ...... 51

6.1: General details ...... 51

6.2: Experimental procedures ...... 52

6.3: 1H NMR and 13C NMR Spectra ...... 107

CONCLUSIONS ...... 185

REFERENCES ...... 186

VITA ...... 202

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SCHEMES

Scheme Page

Scheme 1.1. Roberts’ benzyne preparation and labelling experiment ...... 2

Scheme 1.2. Diverse 6,7-benzannulated indole library design and synthesis ...... 6

Scheme 2.1. 163-member 8-membered ring lactam library ...... 12

Scheme 4.1. Benzannulated and cross-coupled indole libraries ...... 22

Scheme 4.2. N-Methylated 4,6,7-tribromo indole synthesis via Bartoli Indole Synthesis ….23

Scheme 4.3. Metal halogen exchange studies demonstration and diimide reduction ...... 24

Scheme 5.1. Metal halogen exchange experiments with the tribromoindoles ...... 34

Scheme 5.2. Bartoli indole synthesis of the N-TBS protected 4,6,7-tribromoindole ...... 38

Scheme 5.3. Bartoli Indole Synthesis of the 5,6,7-tribromoindole……………….………….38

Scheme 5.4. Leimgruber-Batcho indole synthesis of the 5,6,7-tribromoindole ...... 39

Scheme 5.5. Original approach to polycyclic indole scaffolds of type 1 ...... 41

Scheme 5.5b. Original approach to polycyclic indole scaffolds of type 3 ...... 41

Scheme 5.6. Alternative N-alkylation of the 4- and 5-, 6,7-tribromoindoles ...... 42

Scheme 5.7. Cycloaddition reactions of the N-alkylated tribromoindoles ...... 43

Scheme 5.8. Second generation synthesis of type 1 scaffolds ...... 44

Scheme 5.9. Postulated sequence of the Grubbs catalyst ring opening metathesis (ROM)/ring closing metathesis (RCM) ...... 45

Scheme 5.91. Synthesis of type 4 benzannulated indole scaffolds ...... 46

Scheme 5.92. Specific examples of library members from scaffolds types 1 and 3 ...... 47

Scheme 5.93. Specific examples of library members from scaffolds types 2 and 4 ...... 48

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Scheme 5.94. Functionalization of olefins to yield Markovnikov and anti-Markovnikov library members ...... 49

ILLUSTRATIONS

Figure Page

Figure 1.1. Canonical representations of benzyne………………………………………….....1

Figure 1.2. Ortho-, meta-, para-benzyne…...…………………………………………………1

Figure 1.3. The first proposed heteroaryne………………………………………...………….2

Figure 1.4. Arynes and hetarynes ...... 3

Figure 1.5. Natural products synthesized from arynes and hetarynes ...... 5

Figure 2.1. Therapeutic drugs from medicinal plants ...... 8

Figure 2.2. Marine derived pharmaceuticals ...... 10

Figure 2.3. Octalactin A natural product ...... 12

Figure 2.4. Annulation pattern on the indole nucleus ...... 13

Figure 3.1. Representative members of the trikentrins ...... 16

Figure 3.2. Representative members of the herbindoles ...... 16

Figure 3.3. Representative members of the teleocidins ...... 17

Figure 3.4. Representative members of the notoamides ...... 18

Figure 3.5. Representative members of the stephacidins...... 19

Figure 3.6. Representative members of the penitrems ...... 19

Figure 3.7. Representative members of the nodulisporic acids ...... 21

Figure 4.1. Most active compounds against L1210 lymphocytic Leukemia cell line ...... 25

Figure 4.2. Benzannulated indole compounds tested against human HL-60 tumor cell line ..27

Figure 4.3. Comparison of drug concentrations of benzannulated indoles and jasplakinolide to inhibit the metabolic activity of HL60 tumor cells ...... 28

Figure 4.4. Effects of benzannulated indoles to induce mitotic and binucleated cell index ....29

Figure 4.5 (A). Comparison of the benzannulated indoles to taxol (microtubule stabilizing drug) in altering the kinetics of tubulin polymerization in absence of glycerol ...... 31

Figure 4.5 (B). Comparison of the benzannulated indoles to vincristine (microtubule de- stabilizing agent) in altering the kinetics of tubulin polymerization in presence of glycerol ..31

Figure 4.6. Comparison of in-vitro effects of the benzannulated compounds with vincristine and jasplakinolide to alter the kinetics of glycerol induced tubulin polymerization ...... 32

Figure 5.1. Polarized nature of the 6,7-indole aryne ...... 35

Figure 5.2. The proposed four basic types of polycyclic indole scaffolds ...... 37

SPECTRA Spectra Page

1H NMR for compound 4.73 ...... 109

13C NMR for compound 4.73 ...... 110

1H NMR for compound 4.74 ...... 111

13C NMR for compound 4.74 ...... 112

1H NMR for compound 3.44 ...... 113

13C NMR for compound 3.44 ...... 114

1H NMR for compound 3.46 ...... 115

13C NMR for compound 3.46 ...... 116

1H NMR for compound 1a ...... 117

13C NMR for compound 1a ...... 118

1H NMR for compound 3a ...... 119

13C NMR for compound 3a ...... 120

1H NMR for compound 6.10 ...... 121

13C NMR for compound 6.10 ...... 122

1H NMR for compound 6.11 ...... 123

13C NMR for compound 6.11 ...... 124

1H NMR for compound 6.12 ...... 125

13C NMR for compound 6.12 ...... 126

1H NMR for compound 6.13 ...... 127

13C NMR for compound 6.13 ...... 128

1H NMR for compound 1b ...... 129

13C NMR for compound 1b ...... 130

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1H NMR for compound 3b ...... 131

13C NMR for compound 3b ...... 132

1H NMR for compound 6.14 ...... 133

13C NMR for compound 6.14 ...... 134

1H NMR for compound 6.15 ...... 135

13C NMR for compound 6.15 ...... 136

1H NMR for compound 6.16 ...... 137

13C NMR for compound 6.16 ...... 138

1H NMR for compound 6.17 ...... 139

13C NMR for compound 6.17 ...... 140

1H NMR for compound 1c ...... 141

13C NMR for compound 1c ...... 142

1H NMR for compound 3c ...... 143

13C NMR for compound 3c ...... 144

1H NMR for compound 6.23 ...... 145

13C NMR for compound 6.23 ...... 146

1H NMR for compound 6.24 ...... 147

13C NMR for compound 6.24 ...... 148

1H NMR for compound 5.64 ...... 149

13C NMR for compound 5.64 ...... 150

1H NMR for compound 5.66 ...... 151

1H NMR for compound 2a ...... 152

13C NMR for compound 2a ...... 153

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1H NMR for compound 4a ...... 154

13C NMR for compound 4a ...... 155

1H NMR for compound 6.25 ...... 156

13C NMR for compound 6.25 ...... 157

1H NMR for compound 6.26 ...... 158

13C NMR for compound 6.26 ...... 159

1H NMR for compound 6.27 ...... 160

13C NMR for compound 6.27 ...... 161

1H NMR for compound 6.28 ...... 162

13C NMR for compound 6.28 ...... 163

1H NMR for compound 2b ...... 164

13C NMR for compound 2b ...... 165

1H NMR for compound 4b ...... 166

13C NMR for compound 4b ...... 167

1H NMR for compound 6.29 ...... 168

13C NMR for compound 6.29 ...... 169

1H NMR for compound 6.30 ...... 170

13C NMR for compound 6.30 ...... 171

1H NMR for compound 6.31 ...... 172

13C NMR for compound 6.31 ...... 173

1H NMR for compound 6.32 ...... 174

13C NMR for compound 6.32 ...... 175

1H NMR for compound 2c ...... 176

xiv

13C NMR for compound 2c ...... 177

1H NMR for compound 5.61 ...... 178

13C NMR for compound 5.61 ...... 179

1H NMR for compound 5.62 ...... 180

1H NMR for compound 3.41 ...... 181

13C NMR for compound 3.41 ...... 182

1H NMR for compound 3.42 ...... 183

13C NMR for compound 3.42 ...... 184

ACKNOWLEDGMENTS

Firstly, I would like to thank our gracious almighty God for unmerited favor, good health, right mind and support in my life and in pursuit of my education. I am grateful to

God for bringing people to my life that have enriched me tremendously, and whom I will forever respect and love.

I would like to extend my deepest and warmest appreciation to my advisors, Prof.

Scott E. McKay (undergraduate) and Prof. K. Richard Buszek (graduate). Prof. McKay for support, guidance and encouragement to pursue graduate school. Prof. Buszek for his steadfast support and encouragement in my development as a synthetic organic chemist. I am thankful for the academic and professional training he afforded me, which have and will continuously shape my professional career and life.

My gratitude also goes to the members of my committee: curators professors of chemistry; Jerry Dias and James Durig, professors in pharmaceutical sciences; Bi Botti

Youan and William Gutheil for their commitment in time, counsel and support towards my dissertation and overall assistance in my academic pursuit.

Former postdoctoral fellow and visiting assistant professor in the Buszek laboratories,

Dr. Neil Brown was instrumental in mentoring me as a first year graduate student. He taught, guided and offered wise counsel regarding techniques in synthetic organic chemistry. I am truly grateful.

I would like to extend my thanks to my senior predecessors; Dr. Diheng Luo, Allen

Brassfield and a good colleague and friend; Dr. Chandrasoma. I would like to also thank junior graduate students Alok Nerurkar and Anusha Bade for being great team players in our

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research projects and proof reading and critiquing a lot of presentations.

I would like to thank the department of Chemistry at the University of Missouri –

Kansas City faculty and staff for their numerous ways they contributed to my success as a graduate student. Special thanks to the department chair, Prof. Kathleen V. Kilway for her unwavering support and guidance.

Finally, I would like to thank my parents for their support and encouragement and a brave decision to allow me to pursue dreams of my choice in a foreign country. Heaps of gratitude to my dear wife Beatrice Muniu-Maina, MSN/MBA, who has been very supportive throughout my graduate school. Beatrice is a wonderful mother to our two boys; Cristian and

Kasriel and I immensely appreciate her support. In lieu of that, I would like to dedicate this dissertation to my two sons and to my dearest wife Beatrice.

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Dedicated to my wonderful family

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CHAPTER 1

INTRODUCTION AND BACKGROUND

1.1 Arynes and Hetarynes as emerging powerful tools for natural products total

synthesis and library development

Arynes and benzynes are interchangeably used terms referring to highly reactive species derived from an aromatic ring by various methodologies (Tadross and Stoltz, 2012).

Arynes are usually postulated as having a strained triple bond although they have been demonstrated to also possess some biradical character (Figure 1.1). The most frequently used representation of aryne is ortho-benzyne (benzyne, 1.1a). However, the meta (1.1f) and para

(1.1g) isomers (Figure 1.2) gained interest after the discovery of the enediyne antibiotics, “a promising new class of antitumor drugs” (Wenk, Winkler et al., 2003).

Figure 1.1: Canonical representation of benzyne.

Figure 1.2: Ortho-, meta-, para- benzyne respectively.

Benzyne was first hypothesized by Wittig in 1942 (Wittig, 1942) and a classic carbon isotopic labeling experiment (Scheme 1.1) provided the first indirect experimental evidence of benzyne’s existence (Roberts, Simmons et al., 1953).

• = 14C

Scheme 1.1: Roberts’ benzyne preparation and labeling experiment.

Hetarynes or heteroarynes are arynes with a heteroatom, usually nitrogen, oxygen or sulfur, as part of the aromatic core. The first proposed existence of a heteroaryne was reported in 1902 by Stoermer and Kahlert. They postulated the formation of the reactive intermediate; 2,3-didehydrobenzofuran (Figure 1.3, 1.15) (Stoermer and Kahlert, 1902). The existence of the intermediate has never been found to date. Many review articles on arynes prior to the year 2000 have been published (Wittig, 1957, 1965; Burnett, 1961; Heany, 1962;

Hoffman, 1967; Fields and Meyerson, 1969; Kessar, 1978) and heteroarynes (Kauffman,

1965; Den Hertog and Van der Plas, 1965, 1969; Kauffman and Wirthwein, 1971; Reinecke,

1982; Sander, 1999) as well as the years after (vide infra) which bespeaks of their relevance and importance in chemistry.

Figure 1.3: The first proposed heteroaryne.

These reactive species have emerged as versatile and effective tools in organic synthesis for the construction of complex natural products of biological importance and for the design and development of novel architecturally unique scaffolds for drug discovery

(Gampe and Carreira, 2012; Tadross and Stoltz, 2012; Goetz, Shah et al., 2015). According to Stoltz, by 2012, more than 75 individual natural products had been synthesized or prepared

using arynes to generate key synthetic intermediates (Tadross and Stoltz, 2012) which demonstrates their usefulness in organic syntheses. Figure 1.4 shows examples of hetarynes that have been reported in the literature: 2,3-pyridyne, 1.16 (Martens and den Hertog, 1962);

3,4-pyridyne, 1.17 (Kauffman and Boettcher, 1961); 2,3-thiophyne (Reinecke, Newsom et al., 1981); 1.18, 3,4-thiophyne, 1.19 (Reinecke, Newsom et al., 1981), 1-Boc-3,4-pyryne,

1.20 (Liu, Chan et al., 1999); 2,3-quinolyne, 1.21 (Fleet and Fleming, 1969); 3,4-quinolyne,

1.22 and 5,6-quinolyne, 1.23 (Kauffman and Fischer, 1967); 7,8-quinolyne, 1.24 (Collis and

Burrell, 2005); 1,2-didehydrobenzothiophene, 1.25 (Reinecke, Newsom et al., 1981); 4,5- indolyne, 1.26, 5,6-indolyne, 1.27 and 6,7-indolyne, 1.28 (Buszek, Brown et al., 2007); 4,5- didehydrobenzofuran, 1.29, 5,6-didehydrobenzofuran, 1.30, 6,7-didehydrobenzofuran, 1.31

(Brown and Buszek, 2012); 1,2-carbazolyne, 1.32 (Brown, Choi et al., 1997) and 3,4- carbazolyne, 1.33 (Goetz, Silberstein et al., 2014).

Figure 1.4: Arynes and hetarynes shown in their common triple bond canonical representation.

Arynes and hetarynes have been extensively used in the total synthesis of several important natural products as shown in figure 1.5. Among the recent examples, the powerful synthetic utility of benzyne was demonstrated by Stoltz in the total synthesis of the tetrahydroisoquinoline alkaloid, (-)-quinocarcin, 1.35 (Allan and Stoltz, 2008) and more recently in the synthesis of (-)-curvularin, 1.36, a naturally occurring benzannulated macrolactone (Tadross, Virgil et al., 2010). The 3,4-pyridynes have proved to be strategic in the concise syntheses of naturally occurring alkaloids such as (s)-macrostomine, 1.37

(Enamorado, Ondachi et al., 2010) and ellipticine, 1.38 (Diaz, Cobas et al., 2001). Most recently, the advantages of 6,7-indole aryne chemistry in particular have been successfully exploited by the Buszek laboratories to access rapidly the trikentrins (1.39-1.40) and the herbindoles (1.41-1.42). Also, there are reports of the 4,5-indole aryne being utilized as an intermediate in the total syntheses of the members of the welwitindolinone family of natural products such as N-methylwelwitindolinone C isothiocyanate, 1.43 (Huters, Quasdorf et al.,

2011), N-methylwelwitindolinone D isonitrile, 1.44 (Styduhar, Huters et al., 2013) and (-)-N- methylwewitindolinone B isothiocyanate, 1.45 (Weires, Styduhar et al., 2014). The construction of a carbazole-containing alkaloid, (+)-tubingensin A, 1.46 was carried out by efficiently employing the carbazolyne cyclization strategy (Goetz, Silberstein et al., 2014).

Figure 1.5: Examples of natural products that used the aryne and hetaryne methodologies in their total synthesis.

Significantly, the Buszek laboratories strategically utilized the 6,7-indole aryne chemistry for the first example of small molecule library synthesis, namely, an unprecedented 93-member library of polycyclic, benzannulated indoles of biological relevance (Thornton, Brown et al., 2011). The 6,7-indole aryne generated from the 4,6,7- tribromoindole precursor via a regioselective metal-halogen exchange and elimination protocol (Buszek, Luo et al., 2007), was trapped with diene partners such as furan and cyclopentadiene to achieve annulation at the 6,7-position of the indole nucleus. The cycloadduct olefin was reduced by diimide prior to subjecting the remaining 4-bromo positions to a series of cross-coupling reactions such as Suzuki-Miyaura and Buchwald-

Hartwig to introduce various adornments at the 4-position, thus enabling the development of structurally diverse library members.

Scheme 1.2: Examples of diverse library construction using a 6,7-indole aryne cycloaddition and cross-coupling tactic.

Notably, the newly synthesized benzannulated indole scaffolds exhibited anti- proliferative activities at micromolar and sub-micromolar concentrations in the L1210 leukemia and HL-60 promyelocytic leukemia cell based assays (Perchellet, Waters et al.,

2012). The proposed mechanism of action of these library members in L1210 leukemia cell lines was apoptotic DNA degradation and nuclear fragmentation. Furthermore, in a subsequent HL-60 cell based study discussed in detail in chapter 4, these compounds inhibited the proliferation of tumor cells through microtubule de-stabilization, a mode of action that resembles that of an established anti-cancer drug, vincristine (Perchellet, J.,

Perchellet, E et al., 2014). The compelling biological results obtained in the cell-based assays helped to validate the significance of 6,7-indole aryne chemistry in library development and drug discovery.

CHAPTER 2

THERAPEUTICS INSPIRED BY NATURAL PRODUCTS

2.1: Benzannulated Indole Alkaloid Natural Products as Inspiration for Library Development and Syntheses

Natural products from various sources have demonstrated unparalleled importance in drug discovery, most of which is well documented in a number of articles (Chabala, 1998;

Jack, 1998; Gurib-Fakim, 2006; Weissman and Muller, 2009; Dias, Urban et al., 2012;

Sucher, 2013). Drug discovery from medicinal plants have led to the isolation of drugs with structural and biological targets diversity (Figure 1). Some of the representative members of these medicinally derived pharmaceuticals include: reserpine (antihypertensive agent, isolated from Rauwolfia serpentine), ephedrine and later salbutamol and salmeterol (anti- asthma agents (beta agonists) isolated from Ephedra sinica), tubocurarine (muscle relaxant, isolated from Chondrodendron and Curarea species), taxol (anticancer, isolated from the stem bark of the western yew, Taxus breuifolia), vinca alkaloids: vinblastine and vincristine

(anticancer, isolated from the Madagascar periwinkle, Catharanthus roseus), cocaine

(stimulant, isolated from the coca leaves, Erythroxylum coca), morphine (centrally acting analgesic, isolated from the opium poppy Papaver somniferum), digitoxin (cardiotonic, from digitalis), aspirin (analgesic, isolated from the bark of the willow tree or the Salix species) and quinine (antimalarial, from the bark of Cinchona officinalis) (Chabala, 1998; Jack, 1998;

Gurib-Fakim, 2006; Weissman and Muller, 2009; Dias, Urban et al., 2012; Sucher, 2013).

These medicinally derived drugs have augmented the lives of many people as evidenced in humankind reliance of these drugs up to date.

Figure 2.1: Therapeutic drugs from medicinal plants

Other than medicinal plants, natural products drug discovery has also traditionally focused on environmental pharmacopeia like forests, soils and lakes. These sources have resulted in powerful antibiotic drugs such as penicillin, isolated from the filamentous fungus

Penicillium notatum) (Cragg and Newman, 2001), cephalosporins, tetracyclines, aminoglycosides, rifamycins, chloramphenicol, and lipopeptides among others

(Schwartsmann, 2000; Jeffreys, 2004; Butler, 2005; Beutler, 2009; Mishra and Tiwari, 2011).

However, marine environment sources: sponges (phylum: Porifera), corals, mollusks, ascidians, bryozoans and more recently bacterial strains that live in association with these organisms have also proved to be a rich source drug like substances. These compounds portray structural molecular diversity and unique biological targets, hence they have

consequently inspired drug development to yield therapeutically useful drugs such as: eribulin/Haloven; anticancer drug from halicondrin B (isolated from Axinella sp., Phakellia carteri Lissondendryx sp. and Halichondria okadai sp.), pseudopterosins; antiinflamatory drug isolated from the gorgonian coral Pseudopterogorgia elisabethae), trabectedin

(Yondelis); anticancer drug whose active substance is a natural product ecteinascidin 743 which was originally isolated from the Caribbean sea squirt, Ecteinascidia turbinate and

Brentuximab vedotin (Adcetris); the latest marine anticancer drug to successfully enter into the market, is based on a synthetic analog of dolastatin 10 isolated from the sea hare

Dolabella auricularia (Martins, Viera et al., 2014).

Peculiarly, most of these natural products from medicinal plants, marine sources and other natural sources portray high structural chemical diversity, biochemical specificity, binding efficiency and propensity to interact with biological targets, which make them successful pharmaceutical drugs. In fact, half of the best-selling pharmaceuticals by 1991 were actually based on a natural product precursor or pharmacophore (Cordell, 2000) and in both 2001 and 2002, approximately one quarter of the bestselling drugs worldwide were natural products or derived from natural products (Balunas and Kinghorn, 2005).

Figure 2.2: Marine derived Pharmaceuticals

In drug design and library development, many structural features common to natural products (e.g., stereogenic centers, aromatic rings, complex ring systems, degree of molecule saturation, and number and ratio of heteroatoms) have been shown to be highly relevant to drug discovery efforts (Balunas and Kinghorn, 2005). It is also noted that from a discovery perspective, the key concept in developing a library is to maximize the chemical diversity.

These programs consider the key scaffold component in medicinal chemistry as the ring systems, which are the fundamental building blocks of most drugs on the market today. The

importance of rings and their significant role in molecular properties such as the electronic distribution, three dimensionality, and scaffold rigidity is well understood (Taylor, MacCoss et al., 2014). According to Taylor, rings are often key factors in whole molecule properties such as lipophilicity or polarity and can determine molecular reactivity, metabolic stability, toxicity and overall novelty of a molecular library.

Coincidentally, combinatorial chemistry has emerged as a powerful tool in library development due to its utilization to prepare a large number of different compounds from a single scaffold or backbone. Majority of medicinal synthetic efforts in drug discovery are known to target diverse molecular skeletons than the appendices for high-throughput screening. The rationale is that this approach in compound libraries is found to achieve a high degree of skeletal diversity, and thereby effectively increasing the chances of compounds occupying unpopulated regions of the chemical property space and consequently, the hit rates for diverse biological targets in “biological space” (Zhang, Zheng et al., 2014). It is therefore prudent to design and synthesize libraries that combine the structural features of natural products with the compound-generating potential of combinatorial chemistry (Hall, Manku et al., 2001; Eldridge, Vervoot et al., 2002; Burke, Berger et al., 2004; Ganesan, 2004; Tan,

2004).

Towards these efforts, Buszek laboratory recently constructed unprecedented 163- and 43-member lactam libraries (Scheme 2.2) based on the eight-membered ring motif

(Brown, Gao et al., 2008) via ring-closing metathesis (RCM) and inspired by the potent antitumor marine polyketide octalactin A (Figure 2.3) (Buszek, Sato et al., 1994). The skeletal structure of octalactin A features a rare saturated eight-membered lactone core.

Figure 2.3: Octalactin A

Medium ring natural products are uncommon and there is a widely held view that medium rings are the most challenging and difficult systems to synthesize (Illuminati and

Mandolini, 1981). The basic scaffold was readily constructed in a convergent fashion with the major sources of diversity being commercially available protected L -amino acids, benzaldehydes, and benzyl amines and lactonization via facile Grubb’s first or second generation catalyst’s ring-closing metathesis. Derivatization of the amine R2N was achieved by use of functional groups most likely to be found in pharmacophores, namely; tertiary amines, amides, sulfonamides, carbamates, and ureas. The resultant library was a novel 163- member eight-membered lactam demonstration library, the first based on a monocyclic medium-ring platform (Scheme 1) (Brown, Gao et al., 2008).

Scheme 2.1: 163-member 8-membered ring lactam library

In preliminary anti-proliferative biological screens with murine L1210 lymphocytic leukemia, human HL-60 promyelocytic leukemia, and murine Pan02 pancreatic ductal adenocarcinoma cell lines, some of these library members exhibited low micromolar to sub-

micromolar inhibition in these assays. Moreover, pilot-scale libraries of these eight- membered medium ring lactams (MRLs) and related tricyclic compounds were screened for their ability to inhibit the catalytic activity of human recombinant 3-hydroxy-3- methylglutaryl-coenzyme A (HMG-CoA) reductase in vitro. A dozen of these synthetic compounds mimicked the inhibition of purified HMG-CoA reductase activity caused by pravastatin, fluvastatin and sodium salts of lovastatin, mevastatin and simvastatin in the studied cell-free assay, suggesting direct interaction with the rate limiting enzyme of cholesterol biosynthesis. Moreover, several MRLs inhibited the metabolic activity of L1210 tumor cells in vitro to a greater degree than fluvastatin, lovastatin, mevastatin and simvastatin, whereas pravastatin was found to be inactive (Perchellet, J., Perchellet, E et al.,

2009).

Similarly, the design and synthesis of interesting scaffolds that are based on either unknown or highly underrepresented chemotypes (unique chemical entity) in the National

Institutes of Health PubChem database and Molecular Libraries Small Molecule Repository

(MLSMR), remains an important strategy for library development and drug discovery

(Suggitt and Bibby, 2005). Annulated indoles at the benzene ring are highly underrepresented in chemical property space by comparison to structures in the PubChem database and the

National Institutes of Health Molecular Libraries Small Molecule Repository (MLSMR). By contrast, annulated indoles at the pyrrole ring (2,3-pyrrole annulation, Figure 2.4) are ubiquitous in nature and well represented in the aforementioned databases. Annulation is described as the fusion or attachment of a ring in a cyclic system as depicted in the indole nucleus (Figure 2.4).

Figure 2.4: Annulation pattern examples on the indole nucleus.

A few biologically active benzannulated compounds in this underrepresented class include the trikentrins, herbindoles, teleocidins, nodulisporic acids, stephacidins and notoamides (Chapter 3). Recently, we built the first 93-member library of polycyclic benzannulated indoles3.4 inspired by the trikentrin and herbindole family of alkaloid natural products (Brown, Luo et al., 2009; Buszek, Brown et al., 2009; Chandrasoma, Brown et al.,

2013) using our indole aryne [4+2] cycloaddition methodology (Scheme 1). The biological results, detailed in chapter 4 concluded that several of these annulated indoles mimicked the effect of vincristine, but not that of taxol, on tubulin polymerization and therefore these compounds might represent a new class of microtubule de-stabilizing agents that inhibit tubulin polymerization (Perchellet, Waters et al., 2012).

Other members of these annulated indole libraries remarkably increased the rate and level of actin polymerization in a manner similar to jasplakinolide, suggesting that they might also stabilize the cleavage furrow to block cytokinesis. Finally, these indoles appeared to interact with both tubulin to decrease microtubule assembly and actin to block cytokinesis, thereby inducing bi- and multi-nucleation resulting in genomic instability and apoptosis.

Collectively, these compelling biological results clearly helped to validate the significance of these unprecedented scaffolds.

CHAPTER 3

BENZANNULATED INDOLE ALKALOID NATURAL PRODUCTS DISCOVERY,

ISOLATION AND SYNTHESES

A few natural products exhibit the rare benzannulation. These bioactive natural products will be discussed in this chapter.

3.1: Trikentrins

The two most common members of the benzene annulated indole alkaloid natural products are the trikentrins (Figure 3.1) (Capon, Macleod et al., 1986), and the herbindoles

(Figure 3.2) (Herb, Carroll et al., 1990). In 1986 Capon and co-workers isolated five new benzannulated indole alkaloids namely, (+)-cis-trikentrin A, (+)-cis-trikentrin B, (+)-trans- trikentrin A, (-)-trans-trikentrin B and iso-trans-trikentrin B, from the marine sponge

Trikentrion flabelliforme, collected off of the Australian coast in Darwin (Capon, Macleod et al., 1986). These compounds exhibit growth inhibitory activity toward gram-positive bacteria, Bacillus subtilis, (Capon, Macleod et al., 1986) but no activity levels were provided.

This family of indole alkaloids show exclusive cyclopentenoid annulation at the 6,7-position of the benzene side of the indole nucleus with either cis- or trans-1,3-dimethyl groups and an additional carbogenic substituent at either the 4- or 5-position as shown below.

Figure 3.1: Benzannulated indole alkaloids: trikentrins

3.2: Herbindoles

In 1990 Scheuer and co-workers isolated three new structurally related benzannulated indole alkaloids from the Australian sponge Axinella sp. (Herb, Carroll et al., 1990). These compounds were named herbindole A, herbindole B and herbindole C. These compounds exhibited cytotoxicity against KB cells as well as fish antifeedant properties (Herb, Carroll et al., 1990).

Figure 3.2: Benzannulated indole alkaloids: herbindoles

Although the biosynthesis of trikentrins and herbindoles are unknown, they are apparently not derived biogenetically from tryptophan as they lack substitution at C3

position. Trikentrins and herbindoles are deceptively simple but synthetically challenging molecules.

3.3: Teleocidins

The Teleocidin B family of compounds (Figure 3.3) first isolated from Streptomyces mediocidicus (Awakawa, Zhang et al., 2014) and also produced by actinomycetes

(Takashima, Sakai et al., 1962). This class of alkaloids consists of four stereoisomers which are interesting indole alkaloids primarily because of their uncommon 6,7 cyclohexanoid benzannulation featuring a 9-membered indolactam bridging the indole 3- and 4-positions.

The 6,7-annulation is also endowed with two non-adjacent quaternary centers. In the same family, teleocidin A is structurally similar to teleocidin B as they both share the 9-membered lactam but lacks the 6,7-benzannulation. Instead, teleocidin A has a C7 tether with a stereogenic center that differs in teleocidin A1 and A2 (Xu, Zhang et al., 2011). Teleocidins exhibit tumor-promoting activity, which involves binding and activating a group of kinase receptors including protein kinase C (Xu, Zhang et al., 2011).

Figure 3.3: Teleocidin B family.

The immense synthetic challenges associated with the benzannulation in teleocidin B family are evidenced by the fact that there is no total synthesis of the teleocidin B family reported to date. This is further substantiated by fact that the total synthesis of a structurally similar indolactam V which lacks the 6,7-hetero-benzannulation (Xu, Zhang et al., 2011).

3.4: Notoamides

Other structurally similar members of these uncommon benzannulated isoprenylated indole alkaloids are the antifungal notoamides (Figure 3.4) and the antitumor stephacidins

(Figure 3.5) isolated from Aspergillus ochraceus (Kato, Yoshida et al., 2007; Greshock,

Grubbs et al., 2007; (Tsukamoto, Kato et al., 2008; J. Nat. Prod. 2008; Tsukamoto, Kato et al., 2009). These compounds exhibit a heteronuclear annulation at the 6,7-position of the indole nucleus as well as pyrrole annulation in notoamide D. Notoamides are secondary metabolites produced by the marine-derived Aspergillus species, which was isolated from the mussle, Mytilus edulis galloprovincialis, collected off the Noto Peninsula in the Sea of Japan

(Kato, Nakamura et al., 2011).

Figure 3.4: Representative members of notoamides.

3.5: Stephacidins

Stephacidins are isolated from Aspergillus ochraceus. This family is biosynthetically derived from tryptophan, a cyclic amino acid, and either one or two isoprene units. These

alkaloids show diverse bioactivities, including antitumor, insecticidal, and calmodulin inhibition (Cai, Luan et al., 2013; Finefield, Kato et al., 2011).

Figure 3.5: Representative members of stephacidin family.

3.6: Penitrems

Contrary to 6,7 annulation as shown in notoamides, herbindoles, trikentrins and teleocidins, penitrems (Figure 3.6) feature a 4,5-annulation on the benzenoid as well as pyrrole ring annulation (Wilson, B, Wilson C et al., 1968; Smith, Kuerti et al., 2007).

Additionally, they exhibit a cyclobutane moiety, an eight-membered cyclic ether ring connection between the cyclobutane ring and the secondary cyclopentanol on the pyrrole ring. Penitrems are indole-diterpene tremogens produced by ergot fungi that grow on a variety of grasses endemic to South Africa, New Zealand and the United States (Smith,

Kanoh et al., 2000).

Figure 3.6: The family of penitrems.

3.7: Nodulisporic Acids

This family of benzannulated indole alkaloids also feature both benzenoid and pyrrole annulation. This class of alkaloids features annulation at the 5,6-position of the benzene ring as well as 2,3-pyrrole annulation. Some members of this family, specifically nodulisporic acid A and B exhibit an additional ring connection to nitrogen of the pyrrole ring with the C7 position of the benzene ring.

Nodulisporic acids (Figure 3.7) are a family of potent indole alkaloid that shows potent anti-parasitic activity and were isolated by Ondeyka and co-workers at the Merck

Research Laboratories (Ondeyka, Helms et al., 1997; Ondeyka, Dahl-Roshak et al., 2002;

Ondeyka, Byrne et al., 2003; Singh, Ondeyka et al., 2004). Isolation of (+)-nodulisporic acid

A which is the most complex member of the nodulisporane class of indole diterpenoids

(Ondeyka, Helms et al., 1997) was reported and found to be particularly effective against flea and tick infestations in companion animals (Sings and Singh, 2003). Further in vitro and in vivo evaluation against the bedbug Cimex lectularius also revealed (+)-nodulisporic acid A to display a 10-fold greater systemic adulticidal efficacy (LD90 =1 ppm) over the currently prescribed flea insecticide ivermectin (LD90 = 10 ppm) (Smith, Davulcu et al., 2007). On close inspection and of particular interest to our group, other members of the nodulisporic acid family proved to be 5- to >100-fold less active (Smith, Davulcu et al., 2007). Among other differences, one of the major glaring difference in nodulisporic acid A and others was the C7 to indole N ring connection (D ring) which may signify the importance of this ring towards its biological activity. We were interested in constructing indole containing structures that feature this unique ring connection motif as shown in Nodulisporic acid A.

Figure 3.7: Representative members of nodulisporic acid class.

Importantly, (+)-nodulisporic acid A revealed no apparent toxicity to the host animal, while effectively killing the fleas subsequent to their ingestion of a blood meal. The lack of mammalian toxicity is ascribed to the observation that (+)-nodulisporic acid A is active only against glutamate-gated ion channels that are specific to invertebrates and not against glycine- and GABA-gated chloride ion channels common in mammals (Smith, Davulcu et al., 2007). It was determined that Nodulisporic acid A exerts its striking insecticidal activity by selectively modulating an invertebrate specific glutamate-gated chloride ion channel for which no mammalian homologue exists (Smith, Warren et al., 2000). As a result, (+)- nodulisporic acid A is capable of selectively killing insects, while having no adverse effect on mammals.

In synthetic efforts of the nodulisporic acids A and B, the aforementioned highly strained D ring was installed by a late-stage Hendrickson annulation tactic albeit enormous difficulties (Smith, Kuerti et al., 2007).

CHAPTER 4

BIOLOGICAL STUDIES AND RESULTS FROM A BENZANNULATED

INDOLE SCAFFOLDS LIBRARIES

4.1: Synthesis of the benzannulated cross-coupled indole library

In continuous efforts towards drug discovery, design and library development in the

Buszek group, the first 93-member benzannulated indole library inspired by the trikentrin and herbindole family of alkaloid natural products was synthesized using the group’s discovery of the indole aryne generation methodology (Buszek, Brown et al., 2007) followed by cycloaddition and cross-coupling chemistry as demonstrated in the scheme below (Brown,

Buszek et al., 2009).

Scheme 4.1: Benzannulated and cross-coupled indole libraries.

The key scaffold: 4,6,7-tribromoindole scaffold was synthesized using the Bartoli indole synthesis reaction (Bartoli, Palmieri et al., 1989; Bartoli, Bosco et al., 1991; Bosco,

Dalpozzo et al., 1991; Dalpozzo and Bartoli, 2005) and follows the group’s previously

published route (Scheme 2) (Brown, Luo et al., 2009). Synthetic efforts started with a commercially available o-nitroaniline 3.1, which was first brominated at C2 and C4 (HBr,

MeOH/CH2Cl2, 90%), followed by diazotization to afford the 2,3,5-tribromonitrobenzene.

Application of the Bartoli reaction conditions (vinylmagnesium bromide, 6.0 equiv, tetrahydrofuran (THF), -40 oC) consistently gave the desired indole in 45% yield on up to more than 50 g scale.

Scheme 4.2: Bartoli Indole Synthesis

The indole nitrogen was alkylated with methyl iodide (88%) to afford the desired N - methyl indole scaffold 1.47. Indole aryne generation was achieved by our Buszek’s group optimized conditions (n-BuLi, 1.2 equiv, toluene, -78, oC to rt) followed by cycloaddition with either cyclopentadiene or furan that gave the corresponding cycloadducts in 72% and

84% yields, respectively. Olefin reduction was achieved via diimide (generated in situ from o-nitrobenzosulfonhydrazide, 1.51 and tri-ethylamine) as the presence of the alkene resulted in sharply diminished yields in the subsequent cross-coupling reactions (Buszek and Brown,

2007). The resulting scaffolds with the bridged bicyclic system represented an uncommon structural motif in library design. A correlation study had suggested that presence of additional sp3 -hybridized content in complex library structures increased the number of hits

for activity in certain cell-based assays, particularly in the area of CNS (e.g., histone deacetylase, or HDAC, inhibition) (Marcaurelle, Comer et al., 2010)

Scheme 4.3: Metal-halogen exchange/elimination and cycloaddition with cyclopentadiene or furan followed by diimide reduction

The key scaffolds were then subjected to Suzuki-Miyaura and Buchwald-Hartwig reactions cross-coupling conditions resulting in a 93-member library reminiscent of structures as shown in scheme 4.1. The annulated library comprised of 51-member

Buchwald–Hartwig cross-coupled sub-library and 42-member Suzuki–Miyaura sub-library.

In silico profiling (Lipinski, Lombardo et al., 1997; Pearlman, Smith et al., 1999; Cruciani,

Meniconi et al., 2003) of physicochemical and ADME properties, suggested that the 6,7- indole aryne products generated from these libraries should be reasonably amenable to both biochemical and cell based assays and meant that the libraries were fully compliant with the

Lipinski Rule of Five (Lipinski, Lombardo et al., 1997) in that none of the constituent compounds had more than one violation, and nearly two-thirds of the species had no violations.

The full product manifold showed a wide range of chemical diversity, specifically 46 distinct cells in chemical space, which reflected a significant amount of chemical diversity in these library elaborations. One of the desired traits of molecular library members is to occupy unpopulated or sparsely populated chemical property space, which usually translates to these molecular entities demonstrating new modes/mechanisms of action in their molecular targets.

These compounds were then submitted to the NIH Molecular Libraries Screening Centers

Network (MLSCN) for biological evaluation with a broad range of biochemical and cell based assays and the resulting compelling biological results obtained from these various efforts helped to validate the significance of these libraries using benzannulated indole scaffolds as privileged structures in drug design and development.

From the 93-member library, a subset of 66 novel 6,7-annulated-4-substituted indole compounds were tested for their effectiveness against murine L1210 tumor cell proliferation in vitro. Most compounds inhibited the metabolic activity of L1210 lymphocytic leukemia cells in a time- and concentration dependent manner and nine of them, arbitrarily chosen between 0.5 to 4 micro-molar inhibitions (Figure 4.1) were sufficiently potent to inhibit

L1210 tumor cell proliferation by 50%. Interestingly, all nine compounds with the exception of one came from the Buchwald-Hartwig secondary amine cross-coupled compounds. The lone Suzuki – Miyaura cross- coupled bioactive compound was also found to be the weaker anti-proliferative compound in the HL-60 cell assay. However, all nine Antiproliferative compounds induced DNA cleavage at 24 h in L1210 cells that contained 3H-thymidine pre- labeled DNA, suggesting that these antitumor-annulated indoles might trigger an apoptotic pathway of DNA fragmentation.

Figure 4.1: L1210 lymphocytic leukemia cells active compounds

In another subsequent study, six of the nine synthetic 6,7-annulated-4-substituted indole compounds reported to have interesting antitumor effects in the L1210 tumor cell system (Perchellet, Waters et al., 2012) were selected for the study with human HL-60 promyelocytic leukemia cells (Figure 4.2). These six compounds were selected for the HL-60 study because they had the best anti-proliferative IC50 values in L1210 cells at day 2 in relation with their ability to inhibit DNA synthesis at 3 h and induce DNA fragmentation at

24 h. This suggested that these annulated indoles were the most effective in short-term antitumor assays and might have a more rapid mechanism of action, needing less time to interact with and be incorporated into cells, undergoing eventual metabolic activation to reach crucial cellular targets, and trigger various inhibitory pathways that finally damage and kill tumor cells (Perchellet, Waters et al., 2012).

These synthetic 6,7-annulated-4-substituted indole compounds, were tested for their ability to inhibit human HL-60 tumor cell proliferation, disrupt mitosis and cytokinesis, and interfere with tubulin and actin polymerization in vitro. The concentration-dependent inhibition of HL-60 tumor cell proliferation by the annulated indoles was found to be always more pronounced at day four rather than day two, suggesting that the efficacy of these bioactive compounds was a combination of their concentration and duration of action.

Figure 4.2: Benzannulated indole compounds tested against human HL60 tumor cell line

The ability of annulated indoles to alter the polymerizations of purified tubulin and actin were monitored in cell-free assays and were compared to the effects of drugs known to disrupt the dynamic structures of the mitotic spindle and cleavage furrow: jasplakinolide

(JAS), taxol and vincristine (VCR). Jasplakinolide is a potent inducer of actin polymerization and stabilization that interferes with actomyosin ring function and impedes cytokinesis.

Vincristine and taxol are known microtubule (MT) disrupting agents; where VCR blocks tubulin polymerization and MT assembly, and taxol lower the critical concentration (CC) of free tubulin required to promote polymerization and blocks MT disassembly.

These annulated indoles inhibited the metabolic activity of HL-60 tumor cells in the low-micromolar range in culture. However, when tested as positive controls in the same experiments, established anticancer drugs like daunorubicin and jasplakinolide (JAS), a cell- permeable F-actin probe with fungicidal, insecticidal and antitumor properties, inhibited the growth of HL-60 tumor cells at much lower nanomolar concentrations as illustrated in figure

4.3.

Reproduced with permission from [Anticancer Res 2014, 34, 1643]

Figure 4.3: Comparison of the ability of serial concentrations (logarithmic intervals) of KU- 70, KU-72 and KU-80 to inhibit the metabolic activity of HL-60 tumor cells at days 2 (open symbols) and 4 (solid symbols) in vitro. The antiproliferative activities of jasplakinolide (JAS) concentrations at days 2 and 4 are indicated for the sake of comparison. HL-60 cell proliferation results are expressed as percentage of the net absorbance of MTS/formazan after bioreduction by vehicle-treated control cells after two (A490 nm=1.179±0.048) and four (A490 nm=1.149±0.047) days in culture (100±4.1%, striped area). The blank values (A490 nm=0.370 at day 2 and 0.439 at day 4) for cell-free culture medium supplemented with MTS:PMS reagent were subtracted from the results. Data are means±SD (n=3). a Not different from respective controls; bp<0.05, cp<0.025 and dp<0.01, significantly lower than respective controls.

Antiproliferative concentrations of annulated indoles were also found to increase the mitotic index of HL-60 cells similarly to vincristine (Figure 4.4) and stimulated the formation of many non-viable cells: bi-nucleated cells, multi-nucleated cells and micronuclei (Figure

4.5), similarly to taxol and jasplakinolide. This suggested that these antitumor compounds might increase mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis. Since annulated indoles mimicked the effect of vincristine on tubulin polymerization, but not that of taxol, these compounds represented a new class of

microtubule de-stabilizing agents that inhibit tubulin polymerization. Moreover, annulated indoles remarkably increased the rate and level of actin polymerization similarly to jasplakinolide, suggesting that they might also stabilize the cleavage furrow to block cytokinesis.

Reproduced with permission from [Anticancer Res 2014, 34, 1643]

Figure 4.4: (Left). Comparison of the effects of KU-70 (10 µM), KU-72 (10 µM), KU-80 (10 µM), vincristine (VCR, 0.05 µM), taxol (0.05 µM) and jasplakinolide (JAS, 0.32 µM) on the frequency of mitotic figures (A) and bi-nucleated cells (B) in HL-60 tumor cells in vitro. HL- 60 cells were incubated in triplicate for 24 (open columns) and 48 h (closed columns) at 37˚C in the presence or absence (C: control) of the indicated drugs. The percentage of cells in each category was determined by morphological analysis, scoring an average of approximately 5,000 cells/slide to identify those containing mitotic figures or two nuclei. Results are expressed as the percentage of mitotic (A) or bi-nucleated cells (B) in drug- treated cultures relative to the mitotic (C: 1.34±0.12%) or binucleated (C: 2.66±0.23%) cells in vehicle-treated controls. Data are means±SD (n=3). aNot different from control; bp<0.005 and cp<0.0005, significantly smaller than respective controls in A and B; dp<0.025, ep<0.005 and fp<0.0005, significantly greater than respective controls in A and B.

Figure 4.5 (Right). Comparison of the effects of KU-70 (10 µM), KU-72 (10 µM), KU-80 (10 µM), vincristine (VCR, 0.05 µM), taxol (0.05 µM) and jasplakinolide (JAS, 0.32 µM) on the frequency of multi-nucleated cells (A) and micronuclei (B) in HL-60 tumor cells in vitro. HL- 60 cells were incubated in triplicate for 24 (open columns) and 48 h (closed columns) at 37˚C in the presence or absence (C: control) of the indicated drugs. The percentage of cells in each category was determined by morphological analysis, scoring an average of about 5,000 cells/slide to identify those containing 3-4 nuclei or MNi. A: Results are expressed as the percentage of multi-nucleated cells in drug-treated cultures relative to the multi- nucleated cells (C: 0.54±0.04%) in vehicle-treated controls. B: Results are expressed as the percentage of vehicle- (C: 0.04±0.002%) or drug-treated cells with MNi. Data are means±SD (n=3). ap<0.0005, significantly smaller than control; bNot different from respective controls in A and B; cp<0.025, and dp<0.0005, significantly greater than respective controls in A and B.

Glycerol and taxol stabilize tubulin and lower the critical concentration of protein required to initiate microtubule assembly (Engelborghs, 1990; Perchellet J., Perchellet E et al., 2014). The effects of anti-proliferative annulated indoles on tubulin polymerization in the presence or absence of glycerol was studied and the control curve shown in Figure 4.6 indicated a concentration of purified tubulin below 10 mg/ml which could not polymerize in the absence of 15% glycerol. However, the MT stabilizing drug taxol can easily induce the polymerization of such a low concentration of tubulin (1.15 mg/ml) in the absence of 15% glycerol. However, up to 300 µM KU-72 was not able to promote tubulin polymerization in the absence of glycerol indicating that KU-72 is not a MT-stabilizing agent but acts by blocking MT disassembly like taxol.

The control curves in Figure 4.6 shows the three typical phases of MT assembly taking place when purified tubulin (2.25 mg/ml) undergoes polymerization in the presence of

15% glycerol: a short lag phase, an exponential growth phase almost linear between 100 and

400 s, and a steady phase reaching a plateau after 520 s (Perchellet J., Perchellet E et al.,

2014). At 10 µM, compared to the control, VCR almost totally inhibited the rate and extent of glycerol-induced tubulin polymerization by 94.5% and 93.7%, respectively (Figure 4.6B).

At 300 µM, KU-72 mimics the inhibitory effect of VCR and reduces the rate and extent of glycerol-induced tubulin polymerization by 72.2% and 70.5%, respectively, suggesting that this annulated indole, which increased the mitotic cell index like VCR (Figure 4.4A), might also be a MT de-stabilizing drug that prevents MT assembly (Figure 4.6B).

Reproduced with permission from [Anticancer Res 2014, 34, 1643] Figure 4.5: Comparison of the ability of antiproliferative KU-72 and known microtubule (MT)-stabilizing (A) and MT de-stabilizing (B) anticancer drugs to respectively alter the kinetics of tubulin polymerization in the absence (A) or presence (B) of glycerol in vitro. A: Purified tubulin was diluted to a final concentration of 1.15 mg/ml in 80 mM PIPES buffer, pH 6.9, containing 10 mM MgCl2, 1 mM EGTA and 1 mM GTP. The polymerization reactions were placed in quartz microcells and incubated at 35˚C in the presence or absence (control) of 300 µM KU-72 or 10 µM taxol. B: The turbidity assay mixtures were identical to those of (A) but contained 2.25 mg tubulin/ml and 15% glycerol. The polymerization reactions were similarly incubated in the presence or absence (control) of 300 µM KU-72 or 10 µM vincristine (VCR). The rate of MT assembly was continuously monitored by scanning over 900 s the increase in turbidity (absorbance at 340 nm). Assays were performed in duplicate.

Glycerol-induced tubulin polymerization between 80 and 520 s is inhibited by 93.7% in the presence of 10 µM of the known tubulin binder VCR but remains unaltered in the

presence of 5 µM of the known actin binder JAS (Figure 4.7). In contrast, the three annulated indoles, which increased the mitotic index of HL-60 tumor cells, are also capable of inhibiting the polymerization of tubulin in the cell-free turbidity assay, albeit requiring higher concentrations.

Reproduced with permission from [Anticancer Res 2014, 34, 1643] Figure 4.6. Comparison of the ability of anti-proliferative KU-72, KU-80 and KU-70, jasplakinolide (JAS) and the known microtubule destabilizing anticancer drug vincristine (VCR) to alter the kinetics of glycerol-induced tubulin polymerization in vitro. The polymerization reactions were incubated in the presence or absence (control) of 10 µM VCR, 48, 120 or 300 µM KU-72, 300 µM KU-80, 120 µM KU-70, and 5 µM JAS. Results are expressed as the percentage of the rate of glycerol-induced tubulin polymerization based on the linear increase in turbidity between 80 and 520 s in vehicle-treated control assays (ΔA340 nm=0.190±0.012; 100±6.6%). Data are means±SD (n=2). A Not different from control; bp<0.05, significantly less than control.

The study concluded that the annulated indoles appear to interact with both tubulin to reduce microtubule assembly and actin to block cytokinesis, thereby inducing bi- and multinucleation, resulting in genomic instability and apoptosis.

CHAPTER 5

DESIGN AND SYNTHESIS OF NOVEL 4-, 5-, 6,7 BENZANNULATED

TRIBROMO SCAFFOLDS FOR CROSS-COUPLED LIBRARIES

A recent perspective by Taylor reveals that the indole nucleus is the 24th most frequently used ring system from small molecule drugs (before 2013) listed in the FDA

Orange Book (Taylor, MacCoss et al., 2014). A recent study revealed that the indole- containing natural products and derivatives provide an opportunity to control unwanted bacterial behaviors, such as virulence, biofilm formation, antibiotic resistance, and persister formation as an alternative therapeutic strategy to simply killing bacteria, and may result in a reduced evolutionary pressure for resistance development (Melander, Minvielle et al., 2014).

These reports indicate the potential and increasing relevance of the indole nucleus in therapeutic efforts. Therefore our ultimate goal is to synthesize a diverse collection of indole containing molecules for their therapeutic assessment.

5.1: Metal Halogen Exchange-Induced Indole Aryne Generation

Towards these efforts, our group developed a selective metal halogen exchange methodology (Scheme 1) on the indole nucleus of the 4-, and 5-, 6,7-tribromoindole nucleus

(Buszek et al., 2) This methodology has already demonstrated its value in the total synthesis of benzannulated indole natural products as well as in production of the first benzannulated indole libraries (Sheme 3.1). The selective metal halogen exchange was demonstrated by first

N-protecting the tribromoindoles (MeI (2.0 equiv.; NaH, 2.0 equiv.; THF, 0 °C)). The N-

Methylated 4-, and 5-, 6,7-tribromoindoles were subjected to the metal-halogen exchange/elimination protocol with n-BuLi (2.0 equiv.; PhMe, -78 °C) (Buszek, Brown et al., 2009). After 15 minutes at that temperature the reaction mixture was quenched with

water to give 5,6-dibromoindole 5.10 (55 %) and some unreacted starting material 4.84

(Scheme 5.1). The analysis by NMR revealed no cycloaddition was achieved at -78 oC and only the reduced 7-hydro indole, 5.10b was recovered, which confirmed selective metal- halogen exchange took place exclusively at C-7 position in both tribromoindoles systems.

However, upon warming the reaction from -78 oC to -40 oC under the same conditions before quenching, the metal halogen by-product (LiBr) was eliminated, consequently generating a powerful and versatile dienophile that underwent cycloaddition with cyclopentadiene and other suitable dienes as expected.

Scheme 5.1: Metal Halogen Exchange Experiment

The 6,7-indole aryne was also trapped with substituted dienes such as tert-butyl furan, and showed a remarkable regioselectivity (Brown, Luo et al., 2009; Garr, Luo et al., 2010).

These observations prompted an investigation on the fundamental electronic and steric

parameters that account for these fascinating reaction profiles. These calculations were employed by Cramer and Garr to predict the structures and reactivities of the N-methyl-4,5-,

5,6-, and 6,7-indolynes with furan and 2-alkylfurans. In this methodology, all structures were optimized using the M06-2X density functional (Zhao and Truhlar, 2008) and 6-

311+G(2df,p) basis set (Hehre, Radom et al., 1986) as implemented in MN-GFM (Zhao and

Truhlar, 2008), a locally modified version of the Gaussian03 software package (Frisch,

Trucks et al., 2004). Analytical frequency calculations were employed to characterize the nature of all gas-phase structures as minima or transition states. In some instances, the effects of diethyl ether solvation were taken into account using the SMD implicit solvation model

(Marenich, Cramer et al., 2009; Marenich, Hawkins et al., 2008). Similar calculations were also performed for the benzofuran and benzothiophene analogues of the indolynes. This theoretical and computational investigation studies showed the 6,7-aryne bond was polarized as being electrophilic at C6 and nucleophilic at C7 (Figure 5.1) (Chandrasoma, Brown et al.,

2013). The effects of indole substitutions on regioselectivity were also investigated and reported (Nerurkar et al. 2013).

Figure 5.1: Polarized nature of the 6,7-indole aryne

This revelation helped to explain the remarkable regioselectivity in the 6,7-indole aryne cycloaddition with 2-substituted furans. Based on the theoretical studies it is evident that the dipolar effects can induce regioselection with asymmetrically substituted dienes when polarization of the benzyne triple bond imparts substantial asynchronous electrophilic substitution character to the initial bond forming step.

In an effort to expand the utility of our indole aryne cycloaddition tactic and create indole-based scaffolds of increasing topological complexity, we decided to incorporate a ring-opening metathesis/ring-closing metathesis (ROM/RCM) sequence to our previous strategy in order to create a set of twelve new scaffolds (scaffold types I-IV, Scheme 1). In this manner an additional seven- or eight-membered ring is generated that effectively tethers the indole nitrogen to the benzannulated entity. The remaining Ar-Br bond at either the 4- or

5-position is then available for various Pd(0)-catalyzed cross-coupling reactions (e.g.,

Buchwald-Hartwig, Negishi, Suzuki-Miyaura) as we previously demonstrated (Brown,

Buszek et al, 2009). Furthermore, the resulting vinyl moiety might be subsequently hydroaminated by any of several attractive methods (Utsunomiya, Kuwano et al., 2003;

Utsunomiya, Hartwig et al., 2003,2004; Hartwig, Utsunomiya et al., 2005; Munro-Leighton,

Delp et al., 2007,2008; Brinkmann, Barrett et al., 2012).

Another benefit of these scaffolds is that the endocyclic olefin offers the potential for further functionalization. We now report the first examples of polycyclic, benzannulated indole scaffolds linked through the indole nitrogen, reminiscent of the ones shown in scheme 5.2 below.

Figure 5.2: The four basic types of polycyclic indole scaffolds.

The synthesis of the scaffolds began with either 4,6,7-tribromoindole 5 (for types I and III), or 5,6,7-tribromoindole 6 (for types II and IV), both of which were previously synthesized by us on a multi-gram scale via modifications of the Bartoli (Dalpozzo and

Bartoli, 2005) and Leimgruber-Batcho (Lloyd, Nichols et al., 1986; Mayer, Cassady et al.,

1990; Leze, Palusczak et al., 2008) indole synthesis methods, respectively.

5.2: Synthesis of 4,6,7-Tribromoindole

Synthetic efforts of the desired 4,6,7-tribromoindole started with a commercially available o-nitroaniline 3.1 which was first brominated at C2 and C4 (HBr , MeOH/CH2Cl2,

90%), followed by diazotization to afford the 2,3,5-tribromonitrobenzene 3.3. Application of the Bartoli reaction conditions (vinyl magnesium bromide, 6.0 equiv, tetrahydrofuran (THF),

-40 oC) consistently gave the desired indole, 3.4 in 45% yield on up to more than 50 g scale.

We needed to protect the indole nitrogen, which was achieved by reacting the 4,6,7-

tribromoindole with TBSOTf, (3.0 equiv.; NaH, 4.0 equiv.; Et3N, 2.0 equiv.; THF; 0 °C) to give the desired N-protected tribromoindole constantly in 72-78% yield.

Scheme 5.2: Bartoli Indole Synthesis of the N-TBS protected 4,6,7-tribromoindole

5.3: Synthesis of 5,6,7-Tribromoindole via Bartoli- Indole Synthesis

Due to the success and scalability of the 4,6,7-tribromoindole synthesis via Bartoli reaction conditions, we viewed that the Bartoli reaction protocol (Dalpozzo and Bartoli,

2005) would afford the shortest and easiest pathway to access the 5,6,7-tribromoindole 4.84.

Thus, our initial approach of the synthesis commenced with commercially available 2,6- dibromoaniline 4.81. Diazotization with t-BuONO (1.6 equiv.) and bromination with CuBr2

(1.3 equiv.) (Doyle, Van Lente et al., 1980), (MeCN, 60 °C) (Menzel, Fisher et al., 2006) gave 1,2,3-tribromobenzene 4.82 in 80% yield. Nitration with fuming nitric acid in 1,2- dichloroethane gave exclusively 1,2,3-tribromo-4-nitrobenzene 4.83 in 82% yield (Scheme

5.3). Unfortunately application of the Bartoli reaction conditions (CH2=CHMgBr, 6.0 equiv.;

THF, -40 °C) provided the desired 5,6,7-tribromoindole 4.84 in only 32% yield. Many attempts to optimize the conditions to improve the yield were unsuccessful.

Scheme 5.3: Bartoli Indole Synthesis of the 5,6,7-tribromoindole

5.4: Leimgruber-Batcho Indole Synthesis of the 5,6,7-Tribromoindole Alternatively, we elected to use Leimgruber-Batcho indole synthesis (Lloyd, Nichols et al., 1986; Mayer, Cassady et al., 1990; Leze, Palusczak et al., 2008). Synthetic efforts started with commercially available and inexpensive p-toluidine, 4.85 that was brominated in situ (HBr, 3.0 equiv.; H2O2, 2.0 equiv.; MeOH) (Barhate, Gajare et al., 1999) to provide 4.86 in nearly quantitative yield (Scheme 5.4). The aniline 4.86 was converted to 1,2,3-tribromo-

5-methylbenzene 4.87 in 80% yield via the diazotization and bromination protocol as shown earlier. Nitration was achieved, as before, with fuming nitric acid (2.0 equiv.) to give the

1,2,3-tribromo-5-methyl-4-nitrobenzene 4.88. Reaction of 4.88 with tri(piperidin-1- yl)methane, 4.89 (1.5 equiv., 105 °C, 15 torr) gave the enamine intermediate, 4.90 which was used without purification in the iron catalyzed reductive cyclization step to generate the desired indole 4.84 in 61% yield over 2 steps.

The Leimgruber-Batcho approach was found to be very useful as it was also highly scalable at least upto 30 g and consistently gave the desired indole in 60-61% yield. The indole nitrogen was protected as its N-TBS ether as before to afford the NTBS-protected

5,6,7-tribromoindole, 6.22.

Scheme 5.4: Leimgruber-Batcho Indole Synthesis of the N-TBS protected 5,6,7-tribromo indole

5.5: Original Approach to Synthetic Scheme of Scaffolds Type 1 and 3

With the key N-TBS protected 4,6,7-tribromoindole, 4.70 in hand, and the demonstrated selective metal halogen exchange (Buszek, Luo et al., 2007; Brown, Luo et al.,

2009; Thornton, Brown et al., 2011; Nerurkar, Chandrasoma et al., 2013). The 6,7-indole aryne was generated by reaction with n-butyllithium (1.6 M in hexanes,1.2 equiv) in toluene at -78 °C to effect a selective C-7 lithium-halogen exchange followed by elimination upon warming to -40 °C. Reaction with excess cyclopentadiene gave the corresponding cycloadduct 4.73 in 78% yield. Indole nitrogen was desilylated (TBAF, 2.0 equiv.; THF; 0 oC) to give the corresponding N-deprotected cycloadduct 4.74 in 90% yield. To achieve the desired ring size on the final scaffolds, the indole nitrogen was re-protected (allyl bromide,

3.0 equiv; NaH, 4.0 equiv; catalytic tetrabutyl ammonium iodide (TBAI) 0.5 mol%; THF, room temperature) to afford the corresponding N-allylated cycloadduct 4.75 in 90% yield

(Scheme 5.5). Ring opening metathesis (ROM)/ring closing metathesis (RCM) was achieved via reaction of 4.75 with Grubbs’ first-generation catalyst (5 mol%; PhMe; 110 °C; 0.5 h) followed by the ruthenium scavenger P(CH2OH)3, 4.0 equiv.; Et3N; MeOH; room temperature) to give the type 1 cyclopentadiene scaffold 1a in 45% yield. Scaffold type III followed similar scheme with the exception of the N-alkylation step where homoallyl bromide was used instead of allyl bromide (Scheme 5.5b).

Scheme 5.5: Original Approach to Polycyclic benzannulated Indole Scaffolds Type 1

Scheme 5.5b: Original Approach to Polycyclic benzannulated Indole Scaffolds Type 3

5.6: N-alkylation of the Tribromo Indoles

However, we were interested in investigating if the N-allylated cycloadduct 3.41 would undergo selective metal halogen exchange as demonstrated by the N-TBS protected indoles. This investigation was appealing as it would eliminate the N-TBS protection and desilylation steps in the synthesis. Thus, the tribromoindole 3.4 was first N-allylated (allyl bromide, 3.0 equiv; NaH, 4.0 equiv; catalytic tetrabutylammonium iodide, TBAI, 0.5 mol%;

THF, room temperature) to afford the corresponding product 3.41 in 90% yield (Scheme

5.6). The N-alkylation of scaffolds types III and IV proceeded in moderate yields despite numerous attempts to optimize reaction conditions such as longer reaction times, different solvents, varying catalyst loading and elevated temperatures. Starting material was recovered in all cases and required re-running the experiment multiple times. Despite this shortcoming, fewer steps to the desired scaffolds were more attractive as the overall yield of the scaffolds was synthetically useful compared to the original approach.

Scheme 5.6: Alternative N-alkylation of the 4- and 5-, 6,7-tribromo indoles Gratifyingly, these N-alkylated 4-, and 5-, 6,7-tribromoindoles demonstrated high propensity to undergo metal halogen exchange (scheme 5.7). The 6,7-indole aryne was generated by reaction with n-butyllithium (1.6 M in hexanes, 2.0 equiv) in toluene at -78 °C to effect a selective C-7 lithium-halogen exchange followed by elimination upon warming to

-40 °C.

Scheme 5.7: Cycloaddition reactions of the N-alkylated tribromoindoles

5.7: Second Generation Synthesis of Scaffold Type 1

Gratified with the successful accomplishment of the cycloadditions, we designed and executed the second generation synthesis of scaffold type I (scheme 5.8). Starting with the

4,6,7-tribromoindole 3.4, N-allylation was achieved as before (scheme 5.7) to afford the corresponding N-allylated tribromo indole product (scheme 5.8, 3.41). The 6,7-indole aryne was generated as before by reaction with n-butyllithium (1.6 M in hexanes, 1.2 equiv) in toluene at -78 °C to effect a selective C-7 lithium-halogen exchange followed by elimination upon warming to -40 °C. Reaction with cyclopentadiene gave the corresponding cycloadduct

3.44 in 67% yield. Grubb’s catalyst was used to effect ring opening of the strained cyclopentene ring. The relieve of this strain reveals two terminal olefins, one of which is in

close proximity to the N-allylated terminal olefin and sets up subsequent ring closing metathesis reaction to yield the desired scaffold of type 1a.

Scheme 5.8: Second Generation Synthesis of Type 1 benzannulated indole scaffolds via 6,7- indole aryne Cycloaddition and ROM/RCM.

5.8: Ring Opening/Ring Closing Metathesis

A one-pot ring opening metathesis (ROM)/ring closing metathesis (RCM) reaction was attractive. The versatility of the Grubbs’ catalyst had already been demonstrated in the syntheses of another library and noted that they had an advantage in their ability to tolerate a variety of functional groups in a substrate (Brown, Xie et al., 2008). We envisioned a quick access to our desired polycyclic template, by the initial ring opening driven by strain relieve of the olefinic cyclopentenoid ring. This results in another benzannulated cyclopentenoid ring possessing two cis-stereogenic centers and two terminal olefins that are essential for Grubbs’ ring closing metathesis (scheme 5.9). Upon coming into contact with the Grubb’s catalyst, the olefinic indole nitrogen tether with a terminal olefin pairs up with the terminal olefin in its vicinity to effect the ring closing metathesis as depicted below.

Scheme 5.9: Postulated sequence of the ring opening metathesis/ring closing metathesis ROM/RCM

Thus, ROM/RCM (first or second generation Grubbs’ catalyst (5 mol %); PhMe; 110 °C;

0.5 h, followed by the Ruthenium-scavenger, P(CH2OH)3, 4.0 equiv.; Et3N; MeOH, room temperature) gave the scaffold type 1a (45%). The remaining two type I scaffolds 1b and 1c were prepared in a parallel fashion using furan and 2,5-dimethylfuran respectively to give the desired products in 40% and 38% yields from their respective cycloadducts. The synthesis of the Type II scaffolds proceeded in a similar fashion starting with the 5,6,7-tribromoindole

4.84.

In the same manner, the corresponding polycyclic eight-membered scaffolds (Types 3 and

4) were obtained using the homoallyl bromide in place of allyl bromide (Scheme 5.91). Thus

3.4 and 4.84 were N-alkylated with 4-bromo-but-1-ene in 45-70% yield. Each of the N- alkylated tribromoindoles was then subjected to [4+2] cycloadditions with cyclopentadiene, furan and 2,5-dimethylfuran to yield the corresponding cycloadducts. Grubbs’ ROM/RCM sequence gave the desired scaffolds of types 3 and 4 (Scheme 5.91).

Scheme 5.91: Synthesis of the Type IV benzannulated indole scaffolds via 6,7-indole aryne cycloaddition and ROM/RCM.

5.9: Cross-coupling reactions

To diversify our scaffold types 1 to 4, and validate their application in library development, the extra bromine on the scaffolds was subjected to Suzuki-Miyaura or

Buchwald-Hartwig conditions as before (Brown, Buszek et al 2009). Thus, the polycyclic benzannulated scaffolds of types 1 to 4 were reacted with a collection of commercially available inexpensive secondary amines (Buchwald-Hartwig) and boronic acids (Suzuki-

Miyaura) reagents. With this approach, we envisioned that if we use only 4 amines and 4 boronic acids per scaffold, our target library would afford at least 192 (12 x 4 x 4) library members with enough diversity to prompt a possible Hit-to-Lead success. Schemes 5.92 and

5.93 illustrate the synthetic approach of some of the scaffold types 1 to 4 using some of the cross-coupling reagents and demonstrate the versatility of these core scaffolds.

Scheme 5.92: Specific examples of library members from scaffolds types 1 and 3.

Scheme 5.93: Specific examples of a library from scaffolds types 2 and 4.

5.91: Future Directions

Olefins are prevalent in natural products and are important functional groups in organic synthesis, mainly due to their reliability to be modified into a great diversity of other functional groups. The ubiquity of nitrogen atoms in natural and synthetic products especially drugs validates the reasons to investigate methods to functionalize the two unique olefins on these scaffolds. Literature documents existing methods for functional group interconversion of different types of olefins, specifically; un-activated, strained, electron rich and electron deficient olefins towards epoxidation, cross-metathesis and hydroamination, hydroamidation among others (Utsunomiya, Kuwano et al., 2003; Utsunomiya, Hartwig et

al., 2003,2004; Fontaine and Tilley, 2005; Karshtedt, Bell et al., 2005; Hartwig, Utsunomiya et al., 2005; Munro-Leighton, Delp et al., 2007,2008; Brinkmann, Barrett et al., 2012; Strom,

Hartwig et al., 2013). Successful adaptation of these several attractive approaches especially to selectively distinguish the endocyclic from the exocyclic olefin would be investigated in order to further diversify these scaffolds.

Construction of carbon-nitrogen bond for instance, is gaining considerable interest due to its atom efficiency (Beller, 1999). This research has led to availability of different methods for introducing carbon-nitrogen bonds including catalytic amination of olefins to yield primary, secondary and tertiary amines. Another method is intermolecular oxidative amination of aromatic olefins to give enamines (Beller, Eichberger et al., 1997). Other reagents and catalysts that selectively yield Markovnikov and anti-Markovnikov products have also been reported (Beller, Trauthwein et al., 1999; Munro-Leighton, Delp et al., 2008), in which adaptation of such catalysts would greatly diversify these scaffolds as illustrated in scheme 5.94.

Scheme 5.94: Functionalization of olefins to yield Markovnikov and anti- Markovnikov library members

Once these Markovnikov and anti-Markovnikov scaffolds are obtained, a collection of pharmaceutically useful functional groups could be obtained by functionalizing the secondary amine to afford carbamates, ureas, and sulfonamides among others. These efforts would result in very unique library members that would be expected to occupy regions of

sparsely occupied regions of chemical and biological property space; a highly desired trait in library development.

CHAPTER 6

EXPERIMENTAL SECTION

6.1 General Details

1 13 H-NMR (400 MHz) and C-NMR (101 MHz) were performed in CDCl3 unless otherwise noted with reference to residual solvent at δ 7.26 ppm and 77.0 ppm respectively.

Melting points reported are uncorrected. Unless otherwise noted, all commercially obtained starting materials were used as received. N-butyllithium was titrated against 2-butanol in anhydrous THF with 1,10-phenanthroline as an indicator prior to use. Tetrahydrofuran and diethyl ether were distilled from sodium and benzophenone under nitrogen prior to use.

Toluene was distilled from calcium hydride under nitrogen prior to use. Temperatures of -78

°C were obtained through use of a dry-ice acetone cold bath. Microwave reactions were carried out using a Biotage Initiator microwave reactor at the times and temperatures indicated.

6.2. EXPERIMENTAL PROCEDURES

6.21: Synthesis of 4,6,7-Tribromoindole Via Bartoli Indole Synthesis

1,2,3-tribromobenzene (4.82): In a 500 mL round-bottom flask was dissolved 5.78 g (25.9

o mmol) CuBr2 in 200 mL CH3CN and heated to 60 C. To the hot solution was added 3.8 mL

(32 mmol) tert-butyl nitrite followed by a solution of 5.0 g (20 mmol) 4.81 in 50 mL CH3CN.

The mixture was stirred at 60 oC for 1 h, then cooled to room temperature and poured into

200 mL 20% aqueous ammonia. The mixture was extracted with hexane (2 x 200 mL) and the combined organic phase was washed with 20% aq. NH3 (100 mL), water (100 mL), brine

(50 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was recrystallized from ethanol to give 5.12 g (82%) of 4.82 as

1 off white solid. MP: 88-90. H NMR (400MHz, CDCl3): δ 7.58 (d, J = 8.0 Hz, 2 H), 7.03 (t, J

13 = 8.0 Hz, 1 H); C NMR (CDCl3): δ 132.6, 129.2, 127.7, 126.2. The physical and spectral data is consistent with that which was previously reported for this compound (Menzel,

Fisher, et al., 2006; Teclechiel, Sundstroem et al., 2009)

1,2,3-Tribromo-4-nitrobenzene (4.83): In a 250 mL round-bottom flask was dissolved 5.02 g (15.9 mmol) of 4.82 in 100 mL 1, 2-dichloroethane and cooled to 0 oC To the vigorously

o stirred solution at 0 C was added 7.0 mL (159 mmol) fuming HNO3 dropwise over 15 minutes. The mixture was stirred at 00 C for 1 h and was poured into 500 mL ice. After 30 minutes, 100 mL of CH2Cl2 was added and the mixture was washed with water (3 x 100 mL),

NaHCO3 (3 x 100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was recrystallized from hexanes

o 1 to give 4.74g (83%) of 4.83 as an off-white solid. MP: 82-84 C H NMR (400MHz, CDCl3):

13 δ 7.76 (d, J = 8.6 Hz, 1 H), 7.51 (d, J = 8.6 Hz, 1 H); C NMR (CDCl3): δ 132.6, 132.5,

131.1, 130.1, 123.7, 118.7; HRMS (EI) m/e calcd for C6H2Br3NO2 356.7635, found

356.7633. This compound has been reported in the literature, but no spectral or physical data except melting point was given: Chen, Konstantinov et al, 2001)

4,6,7-tribromo-1H-indole (3.4): In 250 mL round-bottom flask was dissolved 2.0 g (5.6 mmol) 4.83 in 20 mL THF and cooled to -40 oC. To the vigorously stirred solution was added 16.7 mL (16.7 mmol) 1M vinyl magnesium bromide via syringe at once. The mixture was stirred for 1h. The mixture was quenched with 100 mL saturated NH4Cl. The mixture was extracted with diethyl ether (2 x 100 mL) and the combined organic phase was washed

with water (100 mL), brine (50 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 10% EtOAc in hexanes as eluent to give 0.628 g (32%) of 3.4 as a white solid. MP: 132-134oC. 1H NMR (400MHz, CDCl3): δ 8.36 (br. s., 1 H,

NH); 7.89 (s, 1 H); 7.27 (dd, J = 3.32, 3.32 Hz, 1 H); 6.57 (dd, J = 3.32, 3.32 Hz, 1 H). 13C-

NMR: δ 134.80, 128.20, 126.42, 124.01, 119.17, 115.45, 107.93, 103.54. HRMS (EI) m/e calcd for C8H4Br3N 350.7894, found.

4,6,7-tribromo-1-(tert-butyldimethylsilyl)-1H-indole (4.70): C14H18Br3NSi, Molecular

Weight: 468.10 g/mol. In a flame-dried 250 mL three-necked round bottom flask under argon was added a solution of 1.00 g (2.82 mmol) 3.4 in 50 mL dry THF. To this was added 0.271 g (11.3 mmol) of NaH followed by 0.80 mL (5.6 mmol) Et3N. The resulting suspension was cooled to 0 oC, after 15 min at 0 oC, 0.92 mL (5.6 mmol) of tert-butyldimethylsilyl trifluoromethanesulfonate diluted in 5 mL pentane was added dropwise via syringe over 5 min. The solution was stirred at 0 oC for 20 minutes. The reaction was then diluted with diethyl ether (50 mL) and quenched by addition of saturated NH4Cl (100 mL). Then the mixture was extracted with diethyl ether (3 x 100mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure.

The crude material was then purified via column chromatography on silica gel using 1%

methyl tert-butyl ether in hexanes as eluent to give 1.03 g (78%) of 4.70 as colorless oil which solidified at room temperature. 1H NMR (400MHz, CDCl3): δ 7.85 (s, 1 H); 7.41 (d, J

= 3.32 Hz, 1 H); 6.57 (d, J = 3.32 Hz, 1 H); 0.97 (s, 9 H); 0.72 (s, 6H). 13C-NMR: δ 140.47,

136.20, 133.80, 123.71, 121.31, 116.29, 109.74, 104.99, 27.37, 19.91, 2.19.

4-bromo-1-(tert-butyldimethylsilyl)-6,9-dihydro-1H-6,9-methanobenzo[g]indole (4.73):

In a flame-dried 250 mL three- necked round-bottom flask under argon was added a solution of 4.70, 0.610 g (1.30 mmol) in dry toluene (60 mL). To this was added 1.72 g (26.0 mmol) of freshly cracked cyclopentadiene. The resulting solution was cooled to -78 oC, then 1.04 mL (2.60 mmol) of a 2.5 M solution of n-butyl lithium in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl

(50 mL). After stirring for 5 minutes, the mixture was diluted with water (100 mL) and extracted with diethyl ether (3 x 100 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 1% methyl tert- butyl ether in hexanes as eluent to give 0.380 g (78%) of 4.73 as a colorless liquid. 1H NMR

(400MHz, CDCl3): δ 7.33 (s, 1 H); 7.22 (d, J = 3.37 Hz, 1 H); 6.92 (dd, J = 3.08, 5.12 Hz, 1

H); 6.83 (dd, J = 2.93, 4.83 Hz, 1 H); 6.63 (d, J = 3.37 Hz, 1 H); 4.47 (bs, 1 H); 3.98 (bs, 1

H); 2.34 (m, 1 H); 2.26 (m, 1 H); 1.03 (s, 6 H); 0.65 (s, 3 H); 0.60 (s, 3 H). 13C-NMR: δ

148.6, 145.0, 142.0, 136.9, 134.2, 132.5, 130.8, 118.6, 109.2, 105.4, 70.4, 51.0, 50.6, 26.6,

19.4, -1.4, -1.8. HRMS (EI) m/e calcd for C19H24BrNSi 373.0861, found

4-bromo-6,9-dihydro-1H-6,9-methanobenzo[g]indole (4.74): In a flame-dried 100 mL three- necked round-bottom flask under argon was added a solution of 4.73 (0.170 g, 0.460 mmol) in 5 mL dry tetrahydrofuran (THF). The solution was cooled to 0 oC for 10 mins. To this was added 1.0 M tetrabutylammonium fluoride (TBAF) (0.92 mL 0.92 mmol) dropwise.

Reaction was then followed by TLC and showed completion after 5 mins. The reaction was then quenched by addition of saturated NH4Cl (5 mL). The reaction was then extracted with diethyl ether (2 x 5 mL) and the combined organic phase dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 30% dichloromethane in hexanes as

1 eluent to give 0.116 g (97%) of 4.74 as a foamy white solid. H NMR (400 MHz, CDCl3): δ

8.05 (br. s., 1H), 7.31 (s, 1H), 7.19 (dd, J = 2.5, 3.4 Hz, 1H), 6.90 (dd, J = 2.9, 5.3 Hz, 1H),

6.83 (dd, J = 2.9, 5.3 Hz, 1H), 6.55 (dd, J = 2.0, 3.5 Hz, 1H), 4.15 (br. s., 1H), 4.01 (br. s.,

1H), 2.41 (dt, J = 1.6, 6.9 Hz, 1H), 2.33 (dt, J = 1.4, 6.7 Hz 1H). 13C-NMR: δ 148.2, 144.6,

142.0, 133.0, 131.7, 126.9, 124.6, 118.2, 109.0, 103.4, 71.7, 50.8, 47.4.

1-allyl-4-bromo-6,9-dihydro-1H-6,9-methanobenzo[g]indole (4.75): In a flame- dried 100 mL round-bottom flask under argon was added a solution of 4.74 (250 mg, 0.970 mmol) in dry tetrahydrofuran (20 mL). To the solution was added sodium hydride (92.6 mg,

3.86 mmol) and catalytic Tetrabutylammonium iodide (1.8 mg, 0.005 mmol). The solution was then cooled to 0 oC for 10 min. To this was added allyl bromide (0.25 mL, 2.91 mmol) dropwise. TLC analysis showed completion after 30 min. The reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether (2 x

10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to afford

1 4.75 (261 mg, 90%) as a yellowish oil. H NMR (400MHz, CDCl3): δ 7.37 (s, 1H), 7.04 (d,

J = 3.5 Hz, 1H), 6.93 (dd, J = 2.9, 5.3 Hz, 1H), 6.85 (dd, J = 2.9, 5.3 Hz, 1H), 6.55 (d, J = 3.2

Hz, 1H), 6.09 (tdd, J = 4.5, 10.4, 17.1 Hz, 1H), 5.25 (dd, J = 1.3, 10.4 Hz, 1H), 4.95 - 4.81

13 (m, 3H), 4.38 (br. s., 1H), 4.03 (br. s., 1H), 2.45 - 2.35 (m, 2H). C NMR (101MHz, CDCl3):

δ 148.45, 144.38, 142.80, 133.98, 132.67, 132.25, 129.21, 127.78, 117.91, 116.48, 109.18,

101.88, 71.23, 50.41, 50.22, 48.27. HRMS (EI) m/e calcd for C16H14BrN 300.20, found.

4-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-methanobenzo[g]indole (3.46): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 4.74 (250 mg,

0.970 mmol) in dry tetrahydrofuran (20 mL). To the solution was added sodium hydride

(92.6 mg, 3.86 mmol) and catalytic tetrabutylammonium iodide (1.8 mg, 0.005 mmol). The solution was then cooled to 0 oC for 10 min. To this was added Homo-allyl bromide (4- bromo-1-butene) (0.30 mL, 2.91 mmol) dropwise. TLC analysis showed completion after 1 h. The reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether (2 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert- butyl ether in hexanes as eluent to afford 3.46 (249 mg, 82%) as a clear oil that solidifies at

1 room temperature. H NMR (400MHz, CDCl3): δ 7.29 (s, 1 H), 7.01 (d, J = 3.2 Hz, 1 H),

6.89 (ddd, J = 18.1, 5.2, 3.1 Hz, 2 H), 6.44 (d, J = 3.2 Hz, 1 H), 5.80 (ddt, J = 17.1, 10.2, 6.8

Hz, 1 H), 5.15 - 5.08 (m, 2 H), 4.38 (br. s., 1 H), 4.37 - 4.22 (m, 2 H), 4.00 (br. s., 1 H), 2.61

- 2.54 (m, 2 H), 2.38 (ddt, J = 17.8, 6.7, 1.6 Hz, 2 H). 13C-NMR (125 MHz): δ 148.2, 144.8,

142.3, 134.2, 132.4, 132.0, 129.1, 127.9, 117.9, 117.6, 109.4, 101.7, 71.3, 50.5, 48.4, 47.9,

35.6.

(2S,9aR)-4-bromo-2-vinyl-1,2,7,9a-tetrahydro-6a-azacyclohepta[jkl]-as-indacene (1a):

In a 25 mL dry sealed tube was added a solution of 3.44 (0.150 g (0.500 mmol) in CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’ first generation catalyst (20.6 mg, 0.025 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris

(hydroxymethyl)-phosphine (0.248 g, 2.00 mmol) dissolved in methanol (2 mL) and Et3N

(0.5 mL) was added to the solution and stirred overnight. H2O (5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (5 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil. Flash chromatography was achieved using silica gel and eluting with 40% tert-butyl methyl ether

1 in hexanes to give 1a (0.068 g, 45%) as pale yellow oil. H NMR (400MHz, CDCl3): δ 7.04

(s, 1H), 6.99 (d, J = 2.9 Hz, 1H), 6.41 (d, J = 3.2 Hz, 1H), 6.12 - 6.07 (m, 1H), 6.04 - 5.96

(m, 1H), 5.82 (ddd, J = 8.8, 10.0, 17.0 Hz, 1H), 5.27 (ddd, J = 0.7, 1.8, 17.1 Hz, 1H), 5.20 -

5.12 (m, 2H), 4.39 (dd, J = 8.2, 14.6 Hz, 2H), 3.89 - 3.81 (m, 1H), 2.71 (td, J = 7.1, 12.1 Hz,

13 1H), 1.79 (td, J = 10.7, 12.2 Hz, 1H). C NMR (101MHz, CDCl3): δ 140.48, 140.01, 138.90,

134.11, 128.10, 127.86, 126.15, 123.60, 118.88, 116.06, 112.58, 100.85, 49.45, 45.82, 41.75,

40.53.

(2S,10aR,Z)-4-bromo-2-vinyl-2,7,8,10a-tetrahydro-1H-6a-azacycloocta[jkl]-as-indacene

(3a): In a 25 mL dry sealed tube was added a solution of 3.46 (0.060 g (0.019 mmol) in

CH2Cl2 (3 mL). The solution was stirred and purged with argon. To the solution was added

Grubbs’ second-generation catalyst (0.008 mg, 0.010 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (0.094 g, 0.760 mmol) dissolved in methanol (0.8 mL) and Et3N (0.2 mL) was added to the solution and stirred overnight. H2O (3 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (3 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil. Flash chromatography was achieved using silica gel and eluting with 40% tert-butyl methyl ether

1 in hexanes to give 3a (0.024 g, 42.0%) as pale yellow oil. H NMR (400MHz, CDCl3): δ

7.06 (s, 1H), 7.02 (d, J = 3.2 Hz, 1H), 6.48 (d, J = 3.2 Hz, 1H) 5.86 (ddd, J = 8.5, 10.0, 17.0

Hz, 1H), 5.68 (ddt, 1H), 5.50 - 5.43 (m, 1H), 5.09 (td, J = 1.3, 17.0 Hz, 1H), 5.00 (dd, J =

2.0, 10.0 Hz, 1H), 4.83 (ddd, J = 2.0, 12.6, 14.9 Hz, 1H), 4.62 (dtd, 1H), 4.13 (td, J = 4.1,

14.9 Hz, 1H), 3.85 (dt, J = 4.7, 8.8 Hz, 1H), 2.92 (td, J = 9.1, 13.5 Hz, 1H), 2.86 - 2.76 (m,

13 1H), 2.67 - 2.55 (m, 1H), 1.89 (td, J = 4.4, 13.5 Hz, 1H). C NMR (101MHz, CDCl3) δ

142.69, 139.57, 135.56, 133.46, 128.49, 128.40, 127.22, 126.81, 119.45, 113.95, 113.09,

101.92, 49.15, 46.02, 41.29, 40.55, 34.76.

4-bromo-1-(tert-butyldimethylsilyl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.10): In a flame-dried 250 mL three- necked round-bottom flask under argon was added a solution of

4.70 (1.30 g, 2.78 mmol) in dry toluene (10 mL). To this was added distilled furan (2.0 mL,

27.8 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n-butyl lithium (2.4 mL, 2.60 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature.

The reaction was then quenched by addition of saturated NH4Cl (20 mL). After stirring for 5 minutes, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 x 20 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give a colorless oil that solidifies at cold freezer temperatures 6.10 (0.816 g, 78%), MP: 135.1-136 o 1 C H NMR (400MHz, CDCl3): δ 7.35 (s, 1H), 7.23 (d, J = 3.5 Hz, 1H), 7.15 (dd, J = 5.5, 1.8

Hz, 1H), 7.11 (dd, J = 5.5, 1.8 Hz, 1H), 6.66 (d, J = 3.5 Hz, 1H), 6.22 (app. t, J = 0.8 Hz,

1H), 5.79 (dd, J = 1.6, 1.0 Hz, 1H), 1.02 (s, 9H), 0.65 (s, 3H), 0.60 (s, 3H). 13C-NMR: δ

146.1, 144.8, 142.5, 135.2, 133.3, 132.5, 131.8, 117.1, 110.3, 105.7, 83.0, 82.8, 26.4, 19.3,

2.1, 2.9.

1 H NMR (400MHz, cdcl3) δ = 7.35 (s, 1H), 7.23 (d, J=3.5 Hz, 1H), 7.16 (dd, J=1.8, 5.6 Hz,

1H), 7.11 (dd, J=1.8, 5.6 Hz, 1H), 6.65 (d, J=3.1 Hz, 1H), 6.22 (s, 1H), 5.79 (s, 1H), 1.02 (s,

9H), 0.64 (s, 3H), 0.60 (s, 3H)

4-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.11): In a flame-dried 100 mL three- necked round-bottom flask under argon was added a solution of 6.10 (0.232 g, 0.620 mmol) in dry tetrahydrofuran (8 mL). The solution was cooled to 0 oC for 10 min. To this was added

1.0 M tetrabutylammonium fluoride (TBAF) (1.25 mL, 1.24 mmol) dropwise. Reaction was then followed by TLC and showed completion after 5 min. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether

(2 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 30% dichloromethane in hexanes as eluent to

1 give 0.150 g (92%) of 6.11 as a foamy white solid. H NMR (400MHz, CDCl3) d = 8.20 (br. s., 1H), 7.35 (s, 1H), 7.21 (dd, J = 3.4 Hz, 2.5, 1H), 7.15 (dd, J = 5.4, 1.9 Hz, 1H), 7.07 (dd, J

= 5.6, 2.0 Hz, 1H), 6.57 (dd, J = 3.2, 2.0 Hz, 1H), 5.99 (dd, J = 1.6, 0.7 Hz, 1H), 5.84 (dd, J =

13 1.6, 0.7 Hz, 1H). C NMR (101MHz, CDCl3): δ 145.90, 144.66, 142.50, 131.02, 130.02,

128.06, 125.81, 116.80, 110.22, 103.59, 82.93, 80.52.

1-allyl-4-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.12): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 6.11 (250 mg, 0.970 mmol) in dry tetrahydrofuran (20 mL). To the solution was added sodium hydride (92.6 mg, 3.86 mmol) and catalytic tetrabutylammonium iodide (1.8 mg, 0.005 mmol). The solution was then cooled to 0 oC for 10 min. To this was added allyl bromide (0.25 mL, 2.91 mmol) dropwise.

TLC analysis showed completion after 30 min and therefore the reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether

o 1 afford 6.12 as a yellowish oil (261 mg, 90%), MP: 115-116 C. H NMR (400MHz, CDCl3):

δ = 7.32 (s, 1H), 7.11 (dd, J = 1.8, 5.6 Hz, 1H), 7.06 (d, J = 3.2 Hz, 1H), 7.02 (dd, J = 1.8,

5.6 Hz, 1H), 6.50 (d, J = 3.2 Hz, 1H), 6.10 - 6.08 (m, 1H), 6.08 - 6.00 (m, 1H), 5.80 (dd, J =

0.7, 1.9 Hz, 1H), 5.27 - 5.22 (m, 1H), 4.95 (dtd, J = 0.9, 1.9, 17.2 Hz, 1H), 4.90 - 4.82 (m,

13 1H), 4.76 - 4.69 (m, 1H). C NMR (101MHz, CDCl3): δ 146.01, 144.36, 143.44, 133.55,

131.13, 130.82, 130.07, 128.72, 117.10, 116.54, 110.47, 102.32, 82.61, 81.28, 50.34.

4-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.13): In a flame- dried 100 mL round-bottom flask under argon was added a solution of 6.11 (126 mg, 0.480 mmol) in dry tetrahydrofuran (8 mL). To the solution was added sodium hydride (46.1 mg,

1.92 mmol) and catalytic tetrabutylammonium iodide (8.9 mg, 0.020 mmol). The solution was then cooled to 0 oC for 10 min. To this was added homo-allyl bromide (4-bromo-1- butene) (0.63 mL, 1.44 mmol) dropwise. TLC analysis showed completion after 1 h. The reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether (2 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 50% methyl tert- butyl ether in hexanes as eluent to afford 6.13 (127 mg, 82%) as a yellowish oil. 1H NMR

(400MHz, CDCl3): δ 7.33 (s, 1H), 7.16 (dd, 1.8, 5.3 Hz, 1H), 7.12(dd, 1.8, 5.3 Hz, 1H), 7.06

(d, J = 3.2 Hz, 1H), 6.48 (d, J = 3.2 Hz, 1H), 6.16 (d, J = 0.9 Hz, 1H), 5.83 (d, J = 1.8 Hz,

1H), 5.82 - 5.72 (m, 1H), 5.15 - 5.08 (m, 2H), 4.25 (td, J = 7.2, 14.3 Hz, 1H, 1H), 4.15 (td, J

13 = 7.2, 14.3 Hz, 1H), 2.57 (tq, J = 1.4, 7.1 Hz, 2H). C NMR (101MHz, CDCl3): δ 144.67,

143.71, 143.06, 133.83, 133.04, 131.07, 130.29, 130.04, 119.52, 118.03, 106.45, 101.19,

83.51, 82.20, 47.64, 35.33.

(2S,9aR)-4-bromo-2-vinyl-7,9a-dihydro-2H-1-oxa-6a-azacyclohepta[jkl]-as-indacene

(1b): In a 25 mL dry sealed tube was added a solution of 6.12 (0.150 g (0.500 mmol) in

CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added

Grubbs’s first generation catalyst (20.6 mg, 0.025 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (0.248 g, 2.00 mmol) dissolved in methanol (2 mL) and Et3N (0.5 mL) was added to the solution and stirred overnight. H2O (5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (5 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil.

Chromatography on silica with 60% dichloromethane in hexanes as eluents afforded 1b

1 (0.061 g, 39%) as pale yellow oil. H NMR (400MHz, CDCl3): δ 7.05 (d, J = 3.2 Hz, 1H),

7.00 (s, 1H), 6.44 (d, J = 3.2 Hz, 1H), 6.40 - 6.34 (m, 1H), 5.94 - 5.82 (m, 2H), 5.69 (dd, J =

3.4, 7.8 Hz, 1H), 5.53 - 5.47 (m, 1H), 5.32 (td, J = 1.0, 10.0 Hz, 1H), 5.12 (ddd, J = 2.3, 5.3,

13 14.9 Hz, 1H), 4.44 (dd, J = 8.5, 14.9 Hz, 1H). C NMR (101MHz, CDCl3): δ 139.47,

137.38, 134.84, 131.18, 128.45, 128.26, 122.85, 120.25, 118.27, 116.27, 113.80, 101.32,

86.07, 78.89, 45.25.

(2S,10aR,Z)-4-bromo-2-vinyl-2,7,8,10a-tetrahydro-1-oxa-6a-azacycloocta[jkl]-as- indacene (3b): In a 25 mL dry sealed tube was added a solution of 6.13 (0.100 g (0.320 mmol) in CH2Cl2 (3 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’s first generation catalyst (13.0 mg, 0.016 mmol) and refluxed for 0.5 h.

TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (0.159 g, 1.28 mmol) dissolved in methanol (1.5 mL) and Et3N (0.5 mL) was added to the solution and stirred overnight. H2O

(3 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (3 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil.

Purification was achieved via silica gel chromatography and 50% dichloromethane in

1 hexanes to afford 3b (0.039 g, 39%) as yellowish oil. H NMR (400MHz, CDCl3): δ 7.08 (d,

J = 3.1 Hz, 1H), 7.02 (s, 1H), 6.54 (d, J = 3.1 Hz, 1H), 6.51 (sxt, J = 2.4 Hz, 1H), 5.94 (dq, J

= 2.4, 5.0, 13.0 Hz 1H), 5.91 - 5.85 (m, 1H), 5.67 (dd, J = 2.0, 7.8 Hz, 1H), 5.52 - 5.42 (m,

2H), 5.24 (d, J = 9.8 Hz, 1H), 4.47 (ddd, J = 1.6, 12.2, 15.1 Hz, 1H), 4.20 (ddd, J = 2.9, 5.0,

15.1 Hz, 1H), 2.81 (tddd, J = 2.0, 4.7, 7.1, 19.2 Hz, 1H), 2.59 (qddd, J = 2.4, 4.5, 12.0, 19.1

13 Hz, 1H). C NMR (101MHz, CDCl3): δ 139.01, 135.23, 134.40, 130.83, 128.96, 128.53,

125.13, 122.51, 116.79, 116.16, 114.11, 102.39, 85.81, 80.41, 46.13, 34.52.

4-bromo-1-(tert-butyldimethylsilyl)-6,9-dimethyl-6,9-dihydro-1H-6,9- epoxybenzo[g]indole (6.14): In a flame-dried 25 mL round-bottom flask under argon was added a solution of 4.70 (0.900 g, 1.92 mmol) in dry toluene (10 mL). To this was added 2,5- dimethyl furan (2.10 mL, 19.2 mmol). The resulting solution was cooled to -78 oC, then 2.5

M solution of n-butyl lithium (2.4 mL, 4.23 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl

(20 mL). After stirring for 5 minutes, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 x 20 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 10% methyl tert- butyl ether in hexanes as eluent to give 6.14 (0.557 g, 72%) as a yellowish oil, MP: 114-115 oC. 1H NMR (400MHz, CDCl3): δ 7.32 (d, J = 3.5Hz, 1 H); 7.16 (d, J = 5.3 Hz, 1 H); 6.95

(d, J = 5.3 Hz, 1 H); 6.70 (d, J = 3.5 Hz, 1 H); 2.11 (s, 3 H); 1.92 (s, 3 H); 1.10 (s, 9 H); 0.58

(s, 3 H); 0.25 (s, 3 H). 13C NMR (101MHz, CDCl3): δ 150.0, 148.0, 145.4, 138.6, 136.6,

135.2, 132.5, 115.9, 109.8, 107.3, 90.3, 87.4, 27.3, 20.0, 19.2, 15.9, -0.8, -2.0.

4-bromo-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.15): In a flame-dried

100 mL round-bottom flask under argon was added a solution of 6.14 (1.15 g, 2.83 mmol) in dry tetrahydrofuran (10 mL). The solution was cooled to 0 oC for 10 min. T8 this was added

1.0 M tetrabutylammonium fluoride (TBAF) (5.70 mL, 5.65 mmol) dropwise. Reaction was then followed by TLC and showed completion after 20 min. The reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether

(2 x 20 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 60% tert-butyl methyl ether in hexanes as eluent to give 6.15 (0.72 g, 72%) of as a foamy white solid. 1H-NMR (400 MHz, CDCl3): δ 8.12

(br. s, 1 H), 7.22 (s, 1 H); 7.20 (dd, J = 3.4, 2.5 Hz, 1 H); 6.88 (m, 2 H); 6.57 (dd, J = 3.5, 2.0

13 Hz, 1 H); 2.12 (s, 3 H); 1.94 (s, 3 H). C NMR (101 MHz, CDCl3): δ 150.0, 148.2, 146.9,

134.1, 129.8, 128.3, 126.0, 115.4, 110.0, 103.4, 89.8, 88.5, 16.9, 15.6.

1-allyl-4-bromo-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.16): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 6.15 (140 mg,

0.484 mmol) in dry tetrahydrofuran (10 mL). To the solution was added sodium hydride

(47.0 mg, 1.94 mmol) and catalytic tetrabutylammonium iodide (0.90 mg, 0.002 mmol). The solution was then cooled to 0 oC for 10 min. To this was added allyl bromide (0.13 mL, 1.45 mmol) dropwise. TLC analysis showed completion after 30 min. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether (2 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 40% methyl tert-butyl ether in hexanes as eluent to afford 6.16 (111 mg, 78%) as a yellowish oil. 1H-NMR (400 MHz,

CDCl3): δ 7.19 (s, 1 H); 7.03 (d, J = 3.2 Hz, 1 H); 6.81 (dd, J = 5.3, 19.3 Hz, 2 H); 6.54 (d, J

= 3.5 Hz, 1 H); 6.01 (tdd, J = 4.1, 10.5, 17.1 Hz, 1H); 5.21 - 5.16 (m, 1H); 4.98 - 4.91 (m,

13 1H); 4.80 - 4.73 (m, 2H); 2.15 (s, 3 H); 1.93 (s, 3H). C NMR (101 MHz, CDCl3): δ 151.0,

148.3, 147.2, 134.8, 134.0, 131.7, 131.0, 129.6, 116.6, 115.0, 110.7, 102.8, 90.2, 88.6, 51.3,

20.3, 15.5.

4-bromo-1-(but-3-en-1-yl)-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.17):

In a flame-dried 100 mL round-bottom flask under argon was added a solution of 6.15 (214 mg, 0.740 mmol) in dry tetrahydrofuran (8 mL). To the solution was added sodium hydride

(71.0 mg, 2.96 mmol) and catalytic tetrabutylammonium iodide (1.40 mg, 0.004 mmol). The solution was then cooled to 0 oC for 10 min. To this was added homo-allyl bromide (4- bromo-1-butene) (0.23 mL, 2.22 mmol) dropwise. TLC analysis showed completion after

1hr. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether (2 x 5 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert- butyl ether in hexanes as eluent to afford 6.17 (178 mg, 70%) as a yellowish oil. 1H NMR

(400MHz, CDCl3): δ 7.19 (s, 1H), 7.04 (d, J = 3.2 Hz, 1H), 6.90 (dd, J = 5.3, 7.6 Hz, 2H),

6.50 (d, J = 3.5 Hz, 1H), 5.73 (tdd, J = 6.6, 10.4, 17.1 Hz, 1H), 5.12 - 5.03 (m, 2H), 4.43

(ddd, J = 5.9, 8.2, 14.3 Hz, 1H), 4.19 - 4.10 (m, 1H), 2.53 - 2.41 (m, 2H), 2.18 (s, 3H), 1.94

13 (s, 3H). C NMR (101 MHz, CDCl3): δ 150.6, 148.8, 146.8, 134.7, 133.7, 130.8, 129.7,

117.9, 115.0, 110.8, 102.5, 90.2, 88.7, 53.4, 48.6, 35.5, 20.4, 15.6.

(2S,9aR)-4-bromo-2,9a-dimethyl-2-vinyl-7,9a-dihydro-2H-1-oxa-6a-azacyclohepta[jkl]- as-indacene (1c): In a 25 mL dry sealed tube was added a solution of 6.16 (0.120 g 0.360 mmol) in CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’s first generation catalyst (14.8 mg, 0.018 mmol) and refluxed for 0.5 h.

TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (0.178 g, 1.44 mmol) dissolved in methanol (1.5 mL) and Et3N (0.3 mL) was added to the solution and stirred overnight. H2O

(5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (5 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil.

Chromatography on silica gel with 60% dichloromethane in hexanes as eluents afforded 1c

1 (0.046 g, 38%) as pale yellow oil. H NMR (400MHz, CDCl3): δ 7.04 (d, J = 2.92 Hz, 1H),

6.95 (s, 1H), 6.54 (dd, J = 3.2, 11.7 Hz, 1H), 6.48 (d, J = 2.9 Hz, 1H), 5.98 (ddd, J = 0.9,

10.5, 17.3 Hz, 1H), 5.70 - 5.62 (m, 1H), 5.34 (apparent t, J = 1.2 Hz, 1H), 5.34 - 5.29 (m,

1H), 5.22 - 5.14 (m, 1H), 5.06 (td, J = 1.3, 10.6 Hz, 1H), 4.49 (dd, J = 8.9, 15.4 Hz, 1H), 1.89

13 (s, 3H), 1.78 (s, 3H). C NMR (101MHz, CDCl3): δ 144.7, 143.2, 138.2, 128.5, 128.52,

128.48, 125.7, 117.7, 116.0, 114.1, 112.6, 101.8, 88.2, 84.3, 45.3, 30.6, 27.7.

(2S,10aR,Z)-4-bromo-2,10a-dimethyl-2-vinyl-2,7,8,10a-tetrahydro-1-oxa-6a- azacycloocta[jkl]-as-indacene (3c): In a 25 mL dry sealed tube was added a solution of 6.17

(0.108 g 0.315 mmol) in CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’ first generation catalyst (13.0 mg, 0.016 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t.

and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (0.156 g, 1.26 mmol) dissolved in methanol (1.5 mL) and Et3N (0.3 mL) was added to the solution and stirred overnight. H2O (5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (5 mL) and dried over anhydrous

MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil. Chromatography on silica with 60% dichloromethane in hexanes as

1 eluents afforded 3c (0.044 g, 41%) as a yellow oil. H NMR (400MHz, CDCl3): δ 7.08 (d, J

= 3.6 Hz, 1H), 7.01 (s, 1H), 6.57 (d, J = 3.0 Hz, 1H), 6.20 - 6.13 (m, 1H), 6.04 (dd, J = 10.5,

17.0 Hz, 1H), 5.44 (ddd, J = 3.1, 6.5, 13.3 Hz, 1H), 5.34 - 5.26 (m, 1H), 5.04 (dd, J = 1.5,

10.5 Hz, 1H), 4.58 (ddd, J = 1.3, 11.2, 15.3 Hz, 1H), 4.26 (ddd, J = 2.3, 6.4, 15.3 Hz, 1H),

2.83 - 2.71 (m, 1H), 2.32 - 2.32 (m, 1H), 2.37 (qdd, J = 2.7, 11.2, 19.3 Hz, 1H), 1.93 (s, 3H),

13 1.68 (s, 3H). C NMR (101MHz, CDCl3): δ 143.71, 140.50, 137.97, 130.06, 129.71, 129.66,

124.24, 123.69, 116.33, 114.39, 112.18, 102.33, 86.69, 85.92, 53.42, 47.01, 36.02, 33.58,

28.33.

6.4: Alternative Syntheses

1-allyl-4,6,7-tribromo-1H-indole (3.41): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 3.4 (2.00 g, 5.65 mmol) in dry tetrahydrofuran (30 mL). To the

solution was added sodium hydride (0.900 g, 22.6 mmol) and catalytic tetrabutylammonium iodide (11.1 mg, 0.030 mmol). The solution was then cooled to 0 oC for 10 min. To this was added allyl bromide (1.50 mL, 16.95 mmol) dropwise. TLC analysis showed completion after 30 min. The reaction was then quenched by addition of saturated NH4Cl (60 mL). The reaction was then extracted with diethyl ether (3 x 60 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure.

The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to afford 3.41 (2.11 g, 95.0%) as granular white

1 solid. H NMR (400MHz, cdcl3) δ 7.56 (s, 1H), 7.11 (d, J = 3.2 Hz, 1H), 6.56 (dd, J = 1.8,

3.2 Hz, 1H), 6.10 - 5.99 (m, 1H), 5.20 - 5.13 (m, 3H), 4.7 (d, J = 17.1 Hz 1H). 13C NMR (101

MHz, CDCl3): δ 134.4, 133.4, 132.2, 130.8, 126.9, 119.6, 116.6, 114.4, 105.9, 102.8, 51.0,

31.6, 22.6, 14.1.

4,6,7-tribromo-1-(but-3-en-1-yl)-1H-indole (3.42): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 3.4 (720 mg, 2.03 mmol) in dry tetrahydrofuran

(10 mL). To the solution was added sodium hydride (326 mg, 8.14 mmol) and catalytic tetrabutylammonium iodide (3.80 mg, 0.010 mmol). The solution was then cooled to 0 oC for

10 min. To this was added homo-allyl bromide (4-bromo-1-butene) (0.60 mL, 6.10 mmol) dropwise. The reaction was monitored by TLC analysis which showed starting material even after 4 h. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether (2 x 5 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure.

The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to afford 3.42 (372.6 mg, 45.0%) as a yellowish oil and unreacted starting material (70% yield based on starting material recovered). 1H NMR

(400MHz, cdcl3) δ 7.55 (s, 1H), 7.09 - 7.06 (m, 1H), 6.51 - 6.49 (m, 1H), 5.83 - 5.70 (m, 1H),

5.07 (d, J = 12.9 Hz, 2H), 4.61 - 4.53 (m, 2H), 2.58 (d, J = 6.7 Hz, 2H). 13C NMR (101MHz,

CHLOROFORM-d) d = 133.7, 133.2, 132.3, 131.0, 126.7, 119.4, 117.9, 114.4, 105.7, 102.3,

48.6, 36.7.

1-allyl-4-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (3.44): In a flame-dried

1000 mL three- necked round-bottom flask under argon was added a solution of 3.41 (100 mg, 0.25 mmol) in dry toluene (8 mL). To this was added cyclopentadiene (0.2 mL, 2.5 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n-butyllithium

(0.20 mL, 0.51 mmol) in hexanes was added dropwise via syringe over 15 min. The solution

was stirred at -78oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (8 mL). After stirring for 5 minutes, the mixture was diluted with water (8 mL) and extracted with diethyl ether (3 x 8 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give

3.44 (60 mg, 80%) as yellowish oil that solidifies at cold freezer temperatures.

4-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (3.46): In a flame- dried 100 mL three- necked round-bottom flask under argon was added a solution of 3.42

(100 mg, 0.245 mmol) in dry toluene (10 mL). To this was added cyclopentadiene (0.20 mL,

2.45 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n- butyllithium (0.22 mL, 0.54 mmol) in hexanes was added dropwise via syringe over 15 min.

purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 3.46 (45 mg, 59%) as clear oil that solidified at cold freezer temperatures.

1-allyl-4-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.12): In a flame-dried

250 mL three- necked round-bottom flask under argon was added a solution of 3.41 (1.07 g,

2.72 mmol) in dry toluene (10 mL). To this was added distilled furan (2.0 mL, 27.2 mmol).

The resulting solution was cooled to -78 oC, then 2.5 M solution of n-butyllithium (2.2 mL,

5.44 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (20 mL). After stirring for 5 minutes, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 x 20 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 6.12 (0.816 g, 78%) as colorless oil that solidifies at cold freezer temperatures.

4-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.13): In a flame- dried 250 mL three- necked round-bottom flask under argon was added a solution of 3.42

(200 mg, 0.490 mmol) in dry toluene (10 mL). To this was added distilled furan (3.60 mL,

49.0 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n- butyllithium (0.40 mL, 0.490 mmol) in hexanes was added dropwise via syringe over 15 min.

The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (20 mL). After stirring for 5 minutes, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 x 20 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 6.13 (0.96 g, 62%) as colorless oil that solidifies at cold freezer temperatures.

1-allyl-4-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.16): In a flame-dried

250 mL three- necked round-bottom flask under argon was added a solution of 3.41 (430 mg,

1.10 mmol) in dry toluene (11 mL). To this was added 2,5-dimethyl furan (1.20 mL, 10.9

mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n-butyllithium

(0.9 mL, 2.2 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (10 mL). After stirring for 5 minutes, the mixture was diluted with water (10 mL) and extracted with diethyl ether (3 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give

6.16 (0.28 g, 77%) as yellow oil.

4-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.17): In a flame- dried 10 mL three- necked round-bottom flask under argon was added a solution of 3.42 (48 mg, 0.12 mmol) in dry toluene (5 mL). To this was added 2,5-dimethyl furan (0.13 mL, 1.18 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n-butyllithium

(0.10 mL, 0.26 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (5 mL). After stirring for 5 minutes, the mixture was diluted with water (5 mL) and extracted with diethyl ether (3 x 5 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and

concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give

6.17 (0.028 g, 68%) as yellow oil.

6.4: 5,6,7-Tribromoindole via Bartoli Route

1,2,3-Tribromobenzene (4.82): In a 500-mL round-bottom flask was dissolved 5.78 g (25.9 mmol) of CuBr2 in 200 mL of dry CH3CN and the mixture heated to 60 °C. To this warm solution was added 3.80 mL (31.9 mmol) tert-butyl nitrite, followed by a solution of 5.01 g

(19.9 mmol) of commercially available 2,6-dibromoaniline 4.81 in 50 mL CH3CN. The mixture was stirred at 60 °C for 1 h, then cooled to room temperature and poured into 200 mL 20% aqueous ammonia. The mixture was extracted with hexane (2 x 200 mL) and the combined organic phase was washed sequentially with 20% aqueous ammonia (100 mL), water (100 mL), brine (50 mL), and then dried over anhydrous magnesium sulfate. The contents were filtered and concentrated under reduced pressure. The resulting crude material was recrystallized from ethanol to give 5.12 g (80%) of 1,2,3-tribromobenzene 4.82 as an

1 off-white solid, MP: 88-90 °C. H NMR (CDCl3): δ 7.58 (d, J = 8.0 Hz, 2 H), 7.03 (t, J = 8.0

13 Hz, 1 H); C NMR (CDCl3): δ 132.6, 129.2, 127.7, 126.2. The physical and spectral data is

consistent with that which was previously reported for this compound (Menzel, Fisher et al.,

2006; Teclechiel, Sundstroem et al., 2009).

1,2,3-Tribromo-4-nitrobenzene (4.83): In a 250-mL round-bottom flask was dissolved 5.00 g (15.9 mmol) of 4.82 in 100 mL of 1,2-dichloroethane and the mixture cooled to 0 °C. To

the vigorously stirred solution at 0 °C was added 7.00 mL (159 mmol) of fuming HNO3 dropwise over 15 minutes. The mixture was stirred at 0 °C for 1 h and then poured over 500 mL of ice. After 30 minutes, 100 mL of CH2Cl2 was added and the organic layers extracted with water (3 x 100 mL), saturated NaHCO3 (3 x 100 mL), brine (100 mL), and then dried over anhydrous magnesium sulfate. The contents were filtered and concentrated under reduced pressure. The crude material was recrystallized from hexanes to give 4.74 g (82%) of

1 2,3,4-tribromonitrobenzene 4.83 as an off-white solid: MP: 82-84 °C. H NMR (CDCl3):

13 δ 7.76 (d, J = 8.6 Hz, 1 H), 7.51 (d, J = 8.6 Hz, 1 H); C NMR (101 MHz CDCl3): δ 132.6,

132.5, 131.1, 130.1, 123.7, 118.7; HRMS (EI) m/e calcd for C6H2Br3NO2 356.7635, found

356.7633. This compound has been reported in the literature, but no spectral or physical data except melting point was given (Chen, Konstantinov et al., 2001).

5,6,7-Tribromoindole (4.84) via Bartoli route: In 250-mL round-bottom flask was dissolved 2.01 g (5.55 mmol) of 4.83 in 20 mL of dry THF and cooled to -40 °C. To the vigorously stirred solution was added 16.7 mL (16.7 mmol) of 1 M vinyl magnesium bromide via syringe at once. The mixture was stirred for 1 h. The mixture was quenched with

100 mL saturated NH4Cl and then extracted with diethyl ether (2 x 100 mL) and the combined organic phase was washed sequentially with water (100 mL), brine (50 mL), and dried over anhydrous magnesium sulfate. The material was filtered and concentrated under reduced pressure. The crude material was then purified via flash column chromatography on silica gel using 10% EtOAc (ethyl acetate) in hexanes as the eluent to give 0.628 g (32%) of

1 4.84 as a white solid, MP: 132-134 °C. H-NMR (400 MHz, CDCl3): δ 8.36 (bs, 1 H, NH);

7.89 (s, 1 H); 7.27 (dd, J = 3.3, 3.3 Hz, 1 H); 6.57 (dd, J = 3.3, 3.3 Hz, 1 H). 13C NMR (101

MHz, CDCl3): δ 134.8, 128.2, 126.4, 124.0, 119.2, 115.5, 107.9, 103.5; HRMS (EI) m/e calcd for C8H4Br3N 350.7893, found 350.7891. This compound was reported in the literature, but no spectral data was provided: (Ohta, T and Somei, 1989).

2,6-Dibromo-4-methylaniline (6.19): In 500-mL round-bottom flask was dissolved 10.7 g

(100 mmol) 6.18 in 300 mL of dry methanol. To the vigorously stirred solution was carefully added 50.6 mL (300 mmol) concentrated aqueous HBr (47% wt/wt) followed by 22.7 mL

(200 mmol) 30% aqueous H2O2 dropwise over 15 minutes. The mixture was stirred

overnight. The mixture was poured into 1.0 L of 1 M NaOH and the resulting suspension was filtered and washed with water until neutral. Dried under vacuum at 50 °C gave 26.2 g (99%) of 6.19 as a purple amorphous solid, MP: 74-76 °C. Physical properties were identical to that of the commercially available compound.

3,4,5-Tribromotoluene (6.20): In a 1-L round-bottom flask was dissolved 11.6 g (52.0

° mmol) CuBr2 in 300 mL of dry CH3CN and heated to 60 C. To the warm solution was added

7.6 mL (64 mmol) tert-butyl nitrite followed by a solution of 10.6 g (40.0 mmol) of 6.19 in

100 mL of CH3CN. The mixture was stirred at 60 °C for 1 h. The solution was cooled to room temperature and poured into 400 mL of 20% aqueous ammonia. The mixture was extracted with hexane (2 x 400 mL) and the combined organic layers were washed sequentially with 20% aqueous ammonia (200 mL), water (200 mL), brine (100 mL), and then dried over anhydrous magnesium sulfate. The solution was filtered and concentrated under reduced pressure. The crude material was recrystallized from ethanol to give 11.3 g

1 (80%) of 6.20 as off-white needles, MP: 92-94 °C. H-NMR (400 MHz, CDCl3): δ 7.38 (s, 2

13 H), 2.24 (s, 3 H); C NMR (101 MHz, CDCl3): δ 139.4, 133.0, 125.4, 123.8, 20.4. This compound was consistent with the melting point and 1H NMR values previously reported

(Doyle, Van Lente, et al., 1980)

1,2,3-Tribromo-5-methyl-4-nitrobenzene (6.21): In a 250-mL round-bottom flask was dissolved 10.0 g (30.9 mmol) of 6.20 in 100 mL of 1,2-dichloroethane and cooled to 0 °C. To

° the vigorously stirred solution at 0 C was added 13.5 mL (309 mmol) of fuming HNO3 dropwise over 15 minutes. The mixture was stirred at 0 °C for 1 h and then poured into 500 mL of ice. After 30 minutes 100 mL of CH2Cl2 was added and extracted. The organic layer was washed sequentially with water (3 x 200 mL), saturated NaHCO3 (3 x 100 mL), brine

(100 mL), dried over anhydrous magnesium sulfate. The solution was filtered and concentrated under reduced pressure. The crude material was recrystallized from hexanes to

1 give 9.46 g (82%) of 6.21 as off-white solid, MP: 106-108 °C. H NMR (400 MHz, CDCl3):

13 δ 7.58 (s, 1 H), 2.26 (s, 3 H); C NMR (101 MHz, CDCl3): δ 151.6, 134.4, 130.8, 127.3,

126.7, 117.0, 17.1; HRMS (EI) m/e calcd for C7H4Br3NO2 370.7791, found 370.7790.

5,6,7-Tribromoindole (4.84) via Leimgruber-Batcho route: In a 50-mL round-bottom flask a mixture of 6.52 g (17.4 mmol) 6.21 and 6.92 g (26.1 mmol) TPM was heated to 105

°C under water-aspirator vacuum with vigorous stirring. After 3 h an aliquot of the mixture

showed complete reaction by TLC (20% dichloromethane in hexane). Cooled to room temperature and passed through a plug of silica gel eluting with 50% tert-butyl methyl ether in hexanes and concentrated under reduced pressure to give 8.15 g of crude intermediate which was then suspended in 150 mL methanol in a 250 mL round-bottom flask and heated to reflux. To the red solution was added 0.305 g (1.13 mmol) FeCl3•6H2O and 0.689 g (57.4 mmol) activated carbon. The mixture was refluxed for 5 minutes, and then 3.35 mL (68.7 mmol) NH2NH2•H2O was added via a syringe over 10 minutes and refluxed for 1 h. The mixture was cooled to room temperature and filtered through a 1-cm pad of Celite, washing with methanol. The filtrate was concentrated under reduced pressure to about 5 mL, then diluted with 1M HCl (250 mL), extracted with CH2Cl2 (3 x 75 mL). The combined organic phases were washed with water (2 x 100 mL), brine (50 mL), and dried over anhydrous magnesium sulfate. The solution was filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 10% CH2Cl2 in hexanes as eluent to give 3.68 g (61%) of 4.84 as a white solid, MP: 132-134 °C. 1H-NMR

(400 MHz, CDCl3): δ 8.36 (br. s, 1 H, NH); 7.89 (s, 1 H); 7.27 (dd, J = 3.3, 3.3 Hz, 1 H);

13 6.57 (dd, J = 3.3, 3.3 Hz, 1 H). C NMR (101 MHz, CDCl3): δ 134.8, 128.2, 126.4, 124.0,

119.2, 115.5, 107.9, 103.5. HRMS (EI) m/e calcd for C8H4Br3N 350.7893, found 350.7891.

5,6,7-Tribromo-1-(tert-butyldimethylsilyl)-1H-indole (6.22): In a flame-dried 250-mL three-neck round bottom flask under argon was added a solution of 1.00 g (2.82 mmol) 4.84 in 50 mL dry THF. To this was added 0.271 g (11.3 mmol) of NaH followed by 0.80 mL (5.6 mmol) Et3N. The resulting suspension was cooled to 0 °C for 15 min and then 0.92 mL (5.6 mmol) of tert-butyldimethylsilyl trifluoromethanesulfonate diluted in 5 mL pentane was added dropwise via syringe over 5 min. The solution was stirred at 0 °C for an additional 20 min. The reaction was then diluted with diethyl ether (50 mL) and quenched by addition of saturated NH4Cl (100 mL). Then the mixture was extracted with diethyl ether (3 x 100 mL).

The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 1% TBME in hexanes as the eluent to give 1.03 g (78%) of 6.22 as a colorless oil which solidified to a glass at room temperature. 1H-NMR (400

MHz, CDCl3): δ 7.85 (s, 1 H), 7.41 (d, J = 3.3 Hz, 1 H), 6.57 (d, J = 3.3 Hz, 1 H), 0.97 (s, 9

13 H), 0.72 (s, 6 H); C NMR (101 MHz, CDCl3): δ 140.5, 136.2, 133.8, 123.7, 121.3, 116.3,

109.7, 105.0, 27.4, 19.9, 2.2; HRMS (EI) m/e calcd for C14H18Br3NSi 464.8758, found

464.8755.

5-Bromo-1-(tert-butyldimethylsilyl)-6,9-dihydro-1H-6,9-methanobenzo[g]indole (6.23):

In a flame-dried 250 mL three- necked round-bottom flask under argon was added a solution

of 0.610 g (1.30 mmol) 6.22 in 60 mL dry toluene. To this was added 1.72 g (26.0 mmol) of freshly cracked cyclopentadiene. The resulting solution was cooled to -78 °C, then 1.04 mL

(2.60 mmol) of a 2.5 M solution of n-butyllithium in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78 °C for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl

1 solidifies to a glass below room temperature. H NMR (400 MHz, CDCl3): δ 7.37 (s, 1 H);

7.16 (d, J = 3.5 Hz, 1 H); 6.98 (dd, J = 5.3, 5.3 Hz, 1 H); 6.85 (dd, J = 5.3, 5.3 Hz, 1 H); 6.48

(d, J = 3.3 Hz, 1 H); 4.58 (bs, 1 H); 4.26 (bs, 1 H); 2.30 (m, 2 H); 1.20 (s, 9 H); 0.67 (s, 3 H);

13 0.59 (s, 3 H). C NMR (101 MHz, CDCl3): δ 145.9, 144.7, 142.3, 136.8, 136.0, 133.2,

133.1, 118.6, 109.6, 104.4, 69.8, 52.0, 51.8, 26.6, 19.4, -1.5, -1.8. HRMS (EI) m/e calcd for

C19H24BrNSi 373.0862, found 373.0864.

5-bromo-6,9-dihydro-1H-6,9-methanobenzo[g]indole (6.24). In a flame-dried 100 mL three- necked round-bottom flask under argon was added a solution of 6.23 (0.170 g, 0.460 mmol) in 5 mL dry Tetrahydrofuran (THF). The solution was cooled to 0 oC for 10 min. To

this was added 1.0 M tetrabutylammonium fluoride (TBAF) (0.92 mL 0.92 mmol) dropwise.

Reaction was then followed by TLC and showed completion after 5 min. The reaction was then quenched by addition of saturated NH4Cl (5 mL). The reaction was then extracted with diethyl ether (2 x 5 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 30% dichloromethane in hexanes as eluent to give 0.116 g (97%) of 6.24 as a foamy white solid, MP: 117-120 oC. 1H NMR

(400MHz, CDCl3) = 7.94 (br. s., 1H), 7.38 (s, 1H), 7.17 - 7.14 (apparent t, J = 2.7 Hz, 1H),

6.97 (td, J = 2.1, 4.8 Hz, 1H), 6.88 - 6.84 (m, 1H), 6.44 (td, J = 1.3, 2.9 Hz, 1H), 4.28 - 4.23

13 (m, 2H), 2.38 (s, 2H). C NMR (101 MHz, CDCl3): δ 145.67, 144.44, 142.25, 135.44,

130.73, 128.81, 125.28, 118.25, 108.96, 102.50, 71.13, 51.60, 48.80.

1-allyl-5-bromo-6,9-dihydro-1H-6,9-methanobenzo[g]indole (5.64): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 6.24 (250 mg, 0.970 mmol) in dry tetrahydrofuran (20 mL). To the solution was added sodium hydride (92.6 mg, 3.86 mmol) and catalytic tetrabutylammonium iodide (1.8 mg, 0.005 mmol). The solution was then cooled to 0 oC for 10 min. To this was added allyl bromide (0.25 mL, 2.91 mmol) dropwise. TLC analysis showed completion after 30 min. The reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether (2 x

5.64 (261 mg, 90%) as a yellowish oil that solidifies upon cooling, MP: 34-37 oC. 1H NMR

(400MHz, CDCl3): δ 7.29 (s, 1H), 7.01 (d, J = 3.2 Hz, 1H), 6.89 (ddd, J = 2.9, 5.1, 18.0 Hz,

2 H), 6.44 (d, J = 3.2 Hz, 1H), 5.80 (tdd, J = 6.8, 10.2, 17.1 Hz, 1H), 5.15 - 5.06 (m, 2H),

4.38 (br. s, 1H), 4.37 - 4.20 (m, 2H), 4.00 (br. s, 1H), 2.57 (tq, J = 1.4, 7.1 Hz, 2H), 2.38 (ddt,

13 J = 1.7, 6.7, 17.8 Hz 1H). C NMR (101 MHz, CDCl3): δ 145.94, 144.29, 143.13, 135.22,

134.12, 131.29, 129.95, 129.94, 129.86, 118.60, 116.45, 108.92, 101.04, 70.68, 51.27, 50.09,

49.71.

5-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-methanobenzo[g]indole (5.66): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 6.24 (250 mg,

0.970 mmol) in dry tetrahydrofuran (20 mL). To the solution was added sodium hydride

(92.6 mg, 3.86 mmol) and catalytic tetrabutylammonium iodide (1.8 mg, 0.005 mmol). The solution was then cooled to 0 oC for 10 min. To this was added homo-allyl bromide (4- bromo-1-butene) (0.30 mL, 2.91 mmol) dropwise. TLC analysis showed completion after 1 h. The reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether (2 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude

material was then purified via column chromatography on silica gel using 5% methyl tert- butyl ether in hexanes as eluent to afford 5.66 (249 mg,2%) as a yellowish oil. 1H NMR

(400MHz, CDCl3) δ = 7.38 (s, 1H), 7.02 (dd, J = 3.1, 5.4 Hz, 1H), 6.97 (d, J = 3.2 Hz, 1H),

6.93 (dd, J = 2.9, 5.3 Hz, 1H), 6.35 (d, J = 3.2 Hz, 1H), 5.81 (tdd, J = 6.8, 10.3, 17.1 Hz,

1H), 5.17 - 5.08 (m, 2H), 4.51 (br. s, 1H), 4.39 - 4.21 (m, 3H), 2.58 (q, J = 7.0 Hz, 2H), 2.42

(m, 2H).

(2S,9aR)-3-bromo-2-vinyl-1,2,7,9a-tetrahydro-6a-azacyclohepta[jkl]-as-indacene (2a):

In a 25 mL dry sealed tube was added a solution of 5.64 (0.150 g (0.500 mmol) in CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’ first generation catalyst (20.6 mg, 0.025 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris

(hydroxymethyl)-phosphine (0.248 g, 2.00 mmol) dissolved in methanol (2 mL) and Et3N

1 in hexanes to give 2a (0.065 g, 43%) as pale yellow oil. H NMR (400MHz, CDCl3): δ 7.57

(s, 1H), 6.95 (d, J = 3.2 Hz, 1H), 6.32 (d, J = 2.9 Hz, 1H), 6.11 (dt, J = 2.6, 10.7 Hz, 1H),

6.05 - 5.95 (m, 2H), 5.25 - 5.19 (m, 1H), 5.18 - 5.10 (m, 2H), 4.52 (t, J = 9.2 Hz, 1H), 4.39

(dd, J = 8.2, 14.9 Hz, 1H), 4.00 (q, J = 8.4 Hz, 1H), 2.74 (td, J = 8.0, 12.7 Hz, 1H), 1.80 (td,

13 J = 9.9, 12.7 Hz, 1H). C NMR (101MHz, CDCl3): δ 141.91, 140.32, 134.56, 133.32,

129.71, 128.83, 128.59, 123.52, 123.00, 114.94, 111.28, 99.78, 50.55, 45.72, 41.17, 40.82.

(2S,10aR,Z)-3-bromo-2-vinyl-2,7,8,10a-tetrahydro-1H-6a-azacycloocta[jkl]-as-indacene

(4a): In a 25 mL dry sealed tube was added a solution of 5.66 (0.145 g (0.460 mmol) in

CH2Cl2 (10 mL). The solution was stirred and purged with argon. To the solution was added

Grubbs’ first generation catalyst (19.0 mg, 0.020 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (0.199 g, 1.60 mmol) dissolved in methanol (2 mL) and Et3N (0.5 mL) was added to the solution and stirred overnight. H2O (5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (5 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil. Flash chromatography was achieved using silica gel and eluting with 5% tert-butyl methyl ether in

1 hexanes to give 4a (0.058 g, 40%) as pale yellow oil. H NMR (400MHz, CDCl3): δ 7.59 (s,

1H), 6.98 (d, J = 2.9 Hz, 1H), 6.39 (s, 1H), 6.38 (d, J = 3.2 Hz, 1H), 5.89 (ddd, J = 7.6, 10.0,

17.2 Hz, 1H), 5.84 - 5.73 (m, 1H), 5.46 - 5.36 (m, 1H), 5.03 - 4.88 (m, 3H), 4.82 - 4.70 (m,

1H), 4.14 (td, J = 4.0, 15.2 Hz, 1H), 3.99 (s, 1H), 4.03 - 3.96 (m, 1H), 2.90 - 2.74 (m, 2H),

13 2.66 - 2.43 (m, 1H), 2.04 (d, J = 13.5 Hz, 1H). C NMR (101MHz, CDCl3): δ 141.34,

135.94, 134.82, 132.20, 130.14, 129.90, 128.96, 126.56, 122.19, 114.19, 111.34, 100.70,

49.89, 45.72, 41.32, 39.34, 35.45.

5-bromo-1-(tert-butyldimethylsilyl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.25): In a flame-dried 250 mL three- necked round-bottom flask under argon was added a solution of

6.22 (1.30 g, 2.78 mmol) in dry toluene (10 mL). To this was added distilled furan (2.0 mL,

27.8 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n- butyllithium (2.4 mL, 2.60 mmol) in hexanes was added dropwise via syringe over 15 min.

The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (20 mL). After stirring for 5 minutes, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 x 20 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 6.25 (0.816 g, 78%) as a colorless oil that solidifies at cold freezer

1 temperatures. H NMR (400MHz, CDCl3): δ 7.37 (s, 1H), 7.25 - 7.22 (dd, J = 2.0, 5.3 Hz,

1H), 7.17 (d, J = 3.4 Hz, 1H), 7.13 (dd, J = 2.0, 5.3 Hz, 1H), 6.51 (d, J = 3.4 Hz, 1H), 6.31

(br. s, 1H), 5.96 (br. s, 1H), 1.01 (s, 9H), 0.64 (s, 3H), 0.58 (s, 3H). 13C NMR (101MHz,

CDCl3): δ 145.01, 144.36, 142.37, 135.22, 132.59, 132.51, 131.83, 116.96, 113.66, 105.24,

83.20, 82.81, 26.40, 19.33, -2.12, -2.96.

5-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.26): In a flame-dried 100 mL three- necked round-bottom flask under argon was added a solution of 6.25 (0.650 g, 1.73 mmol) in dry tetrahydrofuran (10 mL). The solution was cooled to 0 oC for 10 min. To this was added

1.0 M tetrabutylammonium fluoride (TBAF) (3.50 mL, 3.45 mmol) dropwise. Reaction was then followed by TLC and showed completion after 5 min. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether

1 to give 6.26 (0.372 g, 98%) of as a foamy white solid. H NMR (400MHz, CDCl3): δ 8.32

(br. s., 1H), 7.38 (s, 1H), 7.21 (dd, J = 1.7, 5.6 Hz, 1H), 7.10 - 7.07 (apparent t, J = 2.4 Hz, 1

H), 7.04 (dd, J = 1.7, 5.6 Hz, 1H), 6.44 (dd, J = 2.0, 2.9 Hz, 1H), 5.98 (m, 2H). 13C NMR

(101MHz, CDCl3): δ 144.37, 143.60, 142.67, 133.42, 130.30, 129.16, 126.67, 119.11,

106.47, 102.41, 83.80, 81.58.

1-allyl-5-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.27):. In a flame-dried 100 mL round-bottom flask under argon was added a solution of 6.26 (124 mg, 0.470 mmol) in dry tetrahydrofuran (5 mL). To the solution was added sodium hydride (46.0 mg, 1.88 mmol) and catalytic tetrabutylammonium iodide (0.90 mg, 0.002 mmol). The solution was then cooled to 0 oC for 10 min. To this was added allyl bromide (0.12 mL, 1.42 mmol) dropwise.

TLC analysis showed completion after 30 min. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether (2 x 5 mL).

The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to afford

1 6.27 (111 mg, 78%) as a yellowish oil. H NMR (400MHz, CDCl3): δ 7.36 (s, 1H), 7.20 (dd,

J = 2.2, 5.6 Hz, 1H), 7.05 (dd, J = 2.2, 5.6 Hz, 1H), 7.01 (d, J = 3.4 Hz, 1H), 6.40 (d, J = 3.4

Hz, 1H), 6.18 - 6.16 (m, 1H), 6.01 (td, J = 4.6, 10.2 Hz, 1H), 5.97 - 5.93 (m, 1H), 5.24 (d, J =

10.2 Hz, 1H), 4.97 - 4.93 (m, 1H), 4.83 (apparent. td, J = 1.9, 4.8 Hz, 1H 1H), 4.72 (td, J =

13 2.0, 4.3 Hz, 1H). C NMR (101MHz, CDCl3): δ 144.56, 144.26, 143.32, 133.97, 131.21,

130.99, 129.37, 128.82, 117.04, 116.79, 113.13, 101.88, 82.87, 81.36, 50.12.

5-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.28): In a flame- dried 100 mL round-bottom flask under argon was added a solution of 6.26 (120 mg, 0.460 mmol) in dry tetrahydrofuran (8 mL). To the solution was added sodium hydride (44.2 mg,

1.84 mmol) and catalytic tetrabutylammonium iodide (8.50 mg, 0.023 mmol). The solution was then cooled to 0 oC for 10 min. To this was added homo-allyl bromide (4-bromo-1- butene) (0.14 mL, 1.38 mmol) dropwise. TLC analysis showed completion after 2 h. The reaction was then quenched by addition of saturated NH4Cl (20 mL). The reaction was then extracted with diethyl ether (2 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 50% methyl tert- butyl ether in hexanes as eluent to afford 6.28 (104 mg, 72%) as a dark brown oil that

o 1 solidifies upon cooling, MP: 83.1-84.9 C H NMR (400MHz, CDCl3): δ 7.35 (s, 1H), 7.24

(dd, J = 1.8, 5.6 Hz, 1H), 7.15 (dd, J =1.9, 5.4 Hz, 1H), 7.01 (d, J = 3.2 Hz, 1H), 6.36 (d, J =

3.2 Hz, 1H), 6.24 (dd, J = 1.2, 1.8 Hz, 1H), 5.97 (dd, J = 1.0, 1.9 Hz, 1H), 5.83 - 5.71 (m,

1H), 5.14 - 5.07 (m, 2H), 4.29 - 4.10 (m, 2H), 2.56 (tq, J = 1.3, 7.1 Hz, 2H). 13C NMR

(101MHz, CDCl3): δ 144.7, 143.8, 143.0, 133.8, 133.1, 131.1, 130.2, 130.1, 119.5, 118.0,

106.5, 101.2, 83.5, 82.2, 47.6, 35.3.

(2S,9aR)-3-bromo-2-vinyl-7,9a-dihydro-2H-1-oxa-6a-azacyclohepta[jkl]-as-indacene

(2b): In a 25 mL dry sealed tube was added a solution of 6.27 (0.150 g (0.500 mmol) in

CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added

Grubbs’ first generation catalyst (20.6 mg, 0.025 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium

scavenger; tris (hydroxymethyl)-phosphine (0.248 g, 2.00 mmol) dissolved in methanol (2 mL) and Et3N (0.5 mL) was added to the solution and stirred overnight. H2O (5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (5 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil.

Chromatography on silica with 40% tert-butyl methyl ether (MTBE) in hexanes as eluents

1 afforded 2b (0.062 g, %) as pale yellow oil. H NMR (400MHz, CDCl3): δ 7.61 (s, 1H), 7.00

(d, J = 2.9 Hz, 1H), 6.48 - 6.39 (m, 2H), 6.37 (d, J = 2.9 Hz, 1H), 6.07 (ddd, J = 7.3, 10.1,

17.2 Hz, 1H), 5.92 - 5.83 (m, 1H), 5.77 (dd, J = 3.4, 7.3 Hz, 1H), 5.58 (td, J = 1.2, 17.1 Hz,

1H), 5.37 (td, J = 1.1, 10.5 Hz, 1H), 5.11 (ddd, J = 2.4, 5.5, 15.0 Hz, 1H), 4.45 (dd, J = 8.5,

13 14.9 Hz, 1H). C NMR (101MHz, CDCl3): δ 139.80, 136.87, 131.01, 130.31, 130.03,

128.89, 124.63, 123.68, 120.05, 118.83, 106.97, 100.28, 87.03, 79.37, 45.15.

(2S,10aR,Z)-3-bromo-2-vinyl-2,7,8,10a-tetrahydro-1-oxa-6a-azacycloocta[jkl]-as- indacene (4b): In a flame dried 25 mL sealed tube was added a solution of 6.28 (100 mg,

0.320 mmol) in CH2Cl2 (3.8 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’s first generation catalyst (13.4 mg, 0.016 mmol) and refluxed for

0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (159 mg, 1.28 mmol) dissolved in methanol (1.2 mL) and Et3N (0.3 mL) was added to the solution and stirred overnight. H2O

(4 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (2 x 4 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil. Chromatography on silica gel with 50% dichloromethane in hexanes as eluents

1 afforded 4b (42 mg, 42%) as a pale yellow oil. H NMR (400MHz, cdcl3) δ 7.66 (s, 1H),

7.04 (d, J = 2.9 Hz, 1H), 6.64 - 6.60 (m, 1H), 6.44 (d, J = 2.9 Hz, 1H), 6.09 - 5.96 (m, 2H),

5.74 (d, J = 7.6 Hz, 1H), 5.55 - 5.45 (m, 2H), 5.31 - 5.26 (m, J = 1.2, 10.2 Hz, 1H), 4.56 -

4.46 (m, 1H), 4.25 - 4.17 (m, 1H), 2.87 - 2.74 (m, 1H), 2.65 - 2.52 (m, 1H). 13C NMR

(101MHz, CDCl3): δ 137.7, 134.6, 131.9, 130.8, 129.8, 129.3, 129.4, 124.9, 123.4, 117.4,

107.0, 101.2, 86.7, 81.0, 45.9, 34.8.

5-bromo-1-(tert-butyldimethylsilyl)-6,9-dimethyl-6,9-dihydro-1H-6,9- epoxybenzo[g]indole (6.29): In a flame-dried 25 mL round-bottom flask under argon was added a solution of 6.22 (0.500 g, 1.07 mmol) in dry toluene (10 mL). To this was added 2,5- dimethyl furan (1.15 mL, 10.7 mmol). The resulting solution was cooled to -78 oC, then 2.5

M solution of n-butyl lithium (0.86 mL, 2.14 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl

material was then purified via column chromatography on silica gel using 10% methyl tert- butyl ether in hexanes as eluent to give 6.29 (0.315 g, 73%) as a yellowish oil. 1H NMR

(400MHz, CDCl3): δ 7.26 (s, 1 H), 7.17 (d, J = 3.2 Hz, 1H), 7.07 (d, J = 5.6 Hz, 1H), 6.91 (d,

J = 5.6 Hz, 1H), 6.44 (d, J = 2.9 Hz, 1H), 2.04 (s, 3H), 2.01 (s, 3H), 0.96 (s, 9H), 0.44 (s,

13 3H), 0.09 (s, 3H). C NMR (101MHz, CDCl3): δ 148.32, 145.34, 138.91, 136.57, 134.57,

132.78, 116.53, 112.33, 107.06, 90.25, 87.51, 72.79, 49.45, 27.32, 26.96, 20.01, 19.28, 16.04,

-0.88, -1.92.

5-bromo-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.30): In a flame-dried

100 mL round-bottom flask under argon was added a solution of 6.29 (0.300 g, 0.744 mmol) in dry tetrahydrofuran (8 mL). The solution was cooled to 0 oC for 10 min. To this was added

1.0 M tetrabutylammonium fluoride (TBAF) (1.50 mL, 1.49 mmol) dropwise. Reaction was then followed by TLC and showed completion after 20 min. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether

CDCl3): δ 8.01 (br. s, 1H), 7.37 (s, 1H), 7.17 (app. t, J = 2.9 Hz, 1H), 6.96 (d, J = 5.3 Hz,

1H), 6.89 (d, J = 5.3 Hz, 1H), 6.45 (dd, J = 2.0, 3.4 Hz, 1H), 2.13 (d, J = 8.8 Hz, 6H). 13C

NMR (101MHz, CDCl3): δ 148.39, 146.91, 145.05, 137.23, 130.60, 129.04, 126.85, 120.46,

106.63, 102.28, 92.17, 88.20, 18.02, 16.95.

1-allyl-5-bromo-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.31): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 6.30 (140 mg,

0.484 mmol) in dry tetrahydrofuran (10 mL). To the solution was added sodium hydride

CDCl3): δ 7.37 (s, 1H), 6.97 (d, J = 3.5 Hz, 1H), 6.92 (d, J = 5.3 Hz, 1H), 6.78 (d, J = 5.4 Hz,

1H), 6.39 (d, J = 3.1 Hz, 1H), 6.00 (tdd, J = 4.0, 10.5, 17.2 Hz, 1H), 5.17 (d, J = 10.2 Hz,

13 1H), 5.02 - 4.84 (m, 1H), 4.84 - 4.63 (m, 2H), 2.15 (s, 6H). C NMR (101MHz, CDCl3): δ

148.52, 147.22, 145.82, 138.34, 134.22, 132.36, 132.22, 130.81, 121.44, 116.46, 106.56,

101.54, 91.09, 89.65, 51.35, 20.45, 18.62.

5-bromo-1-(but-3-en-1-yl)-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.32):

In a flame-dried 50 mL round-bottom flask under argon was added a solution of 6.30 (67 mg,

0.23 mmol) in dry tetrahydrofuran (5 mL). To the solution was added sodium hydride (22.3 mg, 0.93 mmol) and catalytic tetrabutylammonium iodide (0.42 mg, 0.001 mmol). The solution was then cooled to 0 oC for 10 min. To this was added homo-allyl bromide (4- bromo-1-butene) (0.1 mL, 0.70 mmol) dropwise. TLC analysis showed starting material present even after 2 h. The reaction was then quenched by addition of saturated NH4Cl (5 mL). The reaction was then extracted with diethyl ether (2 x 5 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to afford 6.32 (46 mg, 58%) as a

1 yellow oil. H NMR (400MHz, CDCl3): δ 7.36 (s, 1H), 6.98 (d, J = 3.2 Hz, 1H), 6.97 (d, J =

5.3 Hz, 1H), 6.89 (d, J = 5.3 Hz, 1H), 6.34 (d, J = 3.2 Hz, 1H), 5.71 (tdd, J = 6.7, 10.3, 17.0

Hz, 1H), 5.11 - 5.03 (m, 2H), 4.43 (ddd, J = 5.9, 8.3, 14.3 Hz, 1H), 4.18 - 4.05 (m, 1H), 2.52

13 - 2.37 (m, 2H), 2.17 (d, J = 9.1 Hz, 6H). C NMR (101MHz, CDCl3): δ 149.03, 146.81,

145.52, 138.29, 133.75, 132.65, 131.91, 130.44, 121.53, 117.75, 106.54, 101.35, 91.18,

89.66, 48.69, 35.50, 20.62, 18.67.

(2S,9aR)-3-bromo-2,9a-dimethyl-2-vinyl-7,9a-dihydro-2H-1-oxa-6a-azacyclohepta[jkl]- as-indacene (2c): In a 25 mL dry sealed tube was added a solution of 6.31 (0.500 g 0.150 mmol) in CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’ first generation catalyst (6.2 mg, 0.008 mmol) and refluxed for 0.5 h.

TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (0.079 g, 0.64 mmol) dissolved in methanol (1mL) and Et3N (0.1 mL) was added to the solution and stirred overnight. H2O (5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (5 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil.

Chromatography on silica with 60% dichloromethane in hexanes as eluents afforded 2c

1 (0.018 g, 36%) as a yellow oil. H NMR (400MHz, CDCl3): δ 7.62 (s, 1H), 7.00 (d, J = 3.3

Hz, 1H), 6.56 (dd, J=2.9, 11.7 Hz, 1H), 6.39 (d, J = 2.9 Hz, 1H), 6.30 (dd, J = 10.5, 17.0 Hz,

1H), 5.69 (ddd, J = 4.5, 8.9, 11.7 Hz, 1H), 5.38 (dd, J = 1.6, 17.1 Hz, 1H), 5.20 - 5.12 (m,

2H), 4.50 (dd, J = 8.8, 15.5 Hz, 1H), 1.90 (br. s., 1H), 1.90 (d, J = 4.4 Hz, 6H). 13C NMR

(101MHz, CDCl3): δ 144.32, 141.45, 134.24, 130.25, 129.46, 129.24, 128.06, 124.32,

117.93, 113.80, 106.58, 100.61, 89.40, 84.01, 45.25, 30.66, 26.17.

100

(2S,10aR,Z)-3-bromo-2,10a-dimethyl-2-vinyl-2,7,8,10a-tetrahydro-1-oxa-6a- azacycloocta[jkl]-as-indacene (4c): In a flame dried 25 mL sealed tube was added a solution of 6.32 (50 mg 0.15 mmol) in CH2Cl2 (5 mL). The solution was stirred and purged with argon. To the solution was added Grubbs’ first generation catalyst (6.20 mg, 0.007 mmol) and refluxed for 0.5 h. TLC analysis showed completion of the reaction. The reaction was cooled to r.t. and the ruthenium scavenger; tris (hydroxymethyl)-phosphine (72.4 mg,

0.584 mmol) dissolved in methanol (1.2 mL) and Et3N (0.3 mL) was added to the solution and stirred overnight. H2O (5 mL) was then added and stirred vigorously for 0.5 h. With the phases separated, the organic layer was washed with brine (2 x 5 mL) and dried over anhydrous MgSO4. The solution was then filtered and concentrated under reduced pressure to afford a dark brown to black oil. Chromatography on silica with 60% dichloromethane in hexanes as eluents afforded 4c (24.6 mg, 49.2%) as a yellow oil.

Alternative Syntheses

1-allyl-5,6,7-tribromo-1H-indole (5.61): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 4.84 (1.50 g, 4.24 mmol) in dry tetrahydrofuran (30 mL). To

101

the solution was added sodium hydride (0.407 mg, 17.0 mmol) and catalytic tetrabutylammonium iodide (7.83 mg, 0.021 mmol). The solution was then cooled to 0 oC for

10 min. To this was added allyl bromide (1.10 mL, 12.7 mmol) dropwise. TLC analysis showed completion after 30 min. The reaction was then quenched by addition of saturated

NH4Cl (60 mL). The reaction was then extracted with diethyl ether (3 x 60 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to afford

o 1 5.61 (1.50 g, 90.0%) as granular white solid, MP: 107-109 C. H NMR (400MHz, CDCl3):

δ 7.86 (s, 1H), 7.10 (d, J = 3.2 Hz, 1H), 6.45 (d, J = 3.2 Hz, 1H), 6.04 (tdd, J = 4.9, 10.0,

13 17.0 Hz, 1H), 5.18 - 5.13 (m, 3H), 4.76 (d, J =17.9 Hz, 1H). C NMR (101MHz, CDCl3): δ

= 134.59, 133.10, 132.95, 131.25, 124.31, 121.57, 116.46, 115.51, 107.46, 101.57, 50.70.

5,6,7-tribromo-1-(but-3-en-1-yl)-1H-indole (5.62): In a flame-dried 100 mL round-bottom flask under argon was added a solution of 4.84 (420 mg, 1.19 mmol) in dry tetrahydrofuran

(10 mL). To the solution was added sodium hydride (114 mg, 4.75 mmol) and catalytic tetrabutylammonium iodide (2.20 mg, 0.006 mmol). The solution was then cooled to 0 oC for

10 min. To this was added homo-allyl bromide (4-bromo-1-butene) (0.36 mL, 3.57 mmol) dropwise. The reaction was monitored by TLC analysis which showed starting material even

102

after 4 h. The reaction was then quenched by addition of saturated NH4Cl (10 mL). The reaction was then extracted with diethyl ether (2 x 5 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure.

The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to afford 5.62 (233 mg, 48.0% or 78% based on recovered starting material) as yellow oil.

1-allyl-5-bromo-6,9-dihydro-1H-6,9-methanobenzo[g]indole (5.64): In a flame-dried 1000 mL three- necked round-bottom flask under argon was added a solution of 5.61 (100 mg,

0.25 mmol) in dry toluene (8 mL). To this was added cyclopentadiene (0.2 mL, 2.5 mmol).

The resulting solution was cooled to -78 oC, then 2.5 M solution of n-butyllithium (0.20 mL,

0.51 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -78oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (8 mL). After stirring for 5 minutes, the mixture was diluted with water (8 mL) and extracted with diethyl ether (3 x 8 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 5.64 (57 mg, 76%) as colorless oil that solidifies at cold freezer temperatures.

103

5-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-methanobenzo[g]indole (5.66): In a flame-dried 100 mL three- necked round-bottom flask under argon was added a solution of

5.62 (100 mg, 0.245 mmol) in dry toluene (10 mL). To this was added cyclopentadiene (0.20 mL, 2.45 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n- butyllithium (0.22 mL, 0.54 mmol) in hexanes was added dropwise via syringe over 15 min.

The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (10 mL). After stirring for 5 minutes, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 5.66 (49 mg, 64%) as colorless oil that solidifies at cold freezer temperatures.

1-allyl-5-bromo-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.27): In a flame-dried 250 mL three- necked round-bottom flask under argon was added a solution of 5.61 (200 mg, 0.510 mmol) in dry toluene (10 mL). To this was added distilled furan (2.0 mL, 27.2 mmol). The

104

resulting solution was cooled to -78 oC, then 2.5 M solution of n-butyllithium (0.41 mL, 1.02 mmol) in hexanes was added dropwise via syringe over 15 min. The solution was stirred at -

78oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (10 mL). After stirring for 5 minutes, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 6.27 (0.12 g, 78%) as colorless oil that solidified at cold freezer temperatures.

5-bromo-1-(but-3-en-1-yl)-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.28): In a flame- dried 250 mL three- necked round-bottom flask under argon was added a solution of 5.62

(200 mg, 0.490 mmol) in dry toluene (10 mL). To this was added distilled furan (3.60 mL,

49.0 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n- butyllithium (0.40 mL, 0.490 mmol) in hexanes was added dropwise via syringe over 15 min.

105

purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 6.28 (0.96 g, 62%) as colorless oil that solidifies at cold freezer temperatures.

1-allyl-5-bromo-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.31): In a flame-dried 250 mL three- necked round-bottom flask under argon was added a solution of

5.61 (430 mg, 1.10 mmol) in dry toluene (11 mL). To this was added 2,5-dimethyl furan

(1.20 mL, 10.9 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n- butyllithium (0.9 mL, 2.2 mmol) in hexanes was added dropwise via syringe over 15 min.

The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (10 mL). After stirring for 5 minutes, the mixture was diluted with water (10 mL) and extracted with diethyl ether (3 x 10 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 6.31 (0.28 g, 77%) as yellow oil.

106

5-bromo-1-(but-3-en-1-yl)-6,9-dimethyl-6,9-dihydro-1H-6,9-epoxybenzo[g]indole (6.32):

In a flame-dried 10 mL three- necked round-bottom flask under argon was added a solution of 5.62 (48 mg, 0.12 mmol) in dry toluene (5 mL). To this was added 2,5-dimethyl furan

(0.13 mL, 1.18 mmol). The resulting solution was cooled to -78 oC, then 2.5 M solution of n- butyllithium (0.10 mL, 0.26 mmol) in hexanes was added dropwise via syringe over 15 min.

The solution was stirred at -78 oC for 30 min then allowed to slowly warm to room temperature. The reaction was then quenched by addition of saturated NH4Cl (5 mL). After stirring for 5 minutes, the mixture was diluted with water (5 mL) and extracted with diethyl ether (3 x 5 mL). The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was then purified via column chromatography on silica gel using 5% methyl tert-butyl ether in hexanes as eluent to give 6.32 (0.028 g, 68%) as yellow oil.

107

Spectra

108

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111

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114

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116

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175

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182

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183

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184

CONCLUSIONS

The synthesis of unique polycyclic benzannulated scaffolds was accomplished via Bartoli indole synthesis and Leimgruber-Batcho synthesis. These scaffolds were demonstrated to possess immense potential in library development due to their several points of diversity useful in introducing other functional groups essential for library development.

Several of these scaffolds have demonstrated their ability to undergo Buchwald-Hartwig and

Suzuki-Miyaura cross-couplings on either the C4 or C5 aryl bromide bonds which bespeaks of their versatility and application in library development, these results will be included in a manuscript that is in preparation.

In summary, we have produced the first benzannulated and bridged medium ring small compound library inspired by the natural products; trikentrin and herbindole benzannulated natural products as well as a new feature of the rare medium ring inspired by the powerful antitumor agent marine natural product octalactins A.

Overall, these scaffolds features a diverse topology with stereogenic centers, unique ring systems and other functionalities commensurate with pharmaceutically desired traits, hence their success in drug development is highly anticipated.

185

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VITA

Lincoln Wamae Maina was born in the central province of Kenya, Karatina town,

Nyeri district, which is north of the capital city Nairobi. After high school, he migrated to the

United States of America to pursue higher education. He attended the first year of college at

Northwest Missouri State in Maryville, Mo and later transferred to the University of Central

Missouri, formerly Central Missouri State University, Warrensburg. Under the guidance of

Professor McKay, he obtained his bachelor’s degree in chemistry in 2007. He went on to get his master’s in organic chemistry and interdisciplinary doctorate degrees in chemistry/pharmaceutical sciences at the University of Missouri-Kansas City in 2014. He is married to Beatrice Muniu-Maina since June 2009 and has been blessed with two sons;

Cristian and Kasriel.

Prior to joining the University of Missouri-Kansas city and as an undergraduate, he held several industrial positions in local companies as a quality control lab technician in an automotive industrial company (Sika Corporation, Grandview, MO), laboratory technician in a toxicology laboratory (Clinical Reference Laboratories, Lenexa, KS) and a laboratory technician in a food processing company (Caravan Ingredients, Kansas City, KS). In his last year of graduate school, he joined the staff at the Metropolitan Community College as an adjunct chemistry instructor.

Upon joining UMKC, he joined the Buszek Research group in spring 2009. During his graduate career he was involved in several projects which included the design and synthesis of novel polycyclic benzannulated indole scaffolds and their libraries based on the

4,5-, 5,6-, and 6,7-indole arynes that were discovered and developed in these laboratories; the design and synthesis of a library of nine-membered lactams inspired by the potent antitumor

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marine natural product octalactin A; the development of indole aryne cycloaddition and related indole aryne reaction methodologies for use in natural products total synthesis and library development and the efficient construction of synthetically challenging and biologically relevant indole alkaloid natural products, specifically, the anti-parasitic nodulisporic acid E. These efforts have resulted in peer reviewed publications and manuscripts in preparation and submission stages.

Selected Peer-reviewed publications:

1. Nerurkar, A.; Chandrasoma, N.; Maina, L.; Brassfield, A.; Luo, D.; Brown, N.;

Buszek. K. R. “Further Investigations into the Regioselectivity of 6,7-Indole Aryne

Cycloadditions with 2-Substituted Furans. Remarkable Contrasteric Preference

Depends on Pyrrole and Benzene Ring Substitution” Synthesis 2013, 45, 1843.

2. McKay, S. E.; Lashlee, R. W., III; Maina, L. W.; Wheeler, K. A.; Brown, A.

“Syntheses of Bipyridine-N-Oxides and Bipyridine-N,N’-Dioxides”, Heterocycl.

Commun., 2009, 15, 181-188.

3. Maina, L. W.; Chandrasoma, N.; Buszek, K. R. “Novel Polycyclic Benzannulated

Indole Scaffolds via Indole Aryne Cycloaddition, Pd(0)-Catalyzed Cross-Coupling,

and ROM/RCM Methodologies”, (manuscript in preparation).

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