Natural Products As Lead Compounds for Drug Development. Part I: Synthesis and Biological Activity of a Structurally Diverse Library of Curcumin Analogues

Home , Berberine, Lead compound, Pregnenolone acetate

Natural Products as Lead Compounds for Drug Development. Part I: Synthesis and Biological Activity of a Structurally Diverse Library of Curcumin Analogues. Part II: Synthesis of Novel Sterol Natural Products and Related Analogues as Antileishmanial Agents.

DISSERTATION

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

Dalia Abdelhamid Sayed Abdelhamid

Graduate Program in Pharmacy

The Ohio State University

2011

Dissertation Committee:

Professor James R. Fuchs, Advisor

Professor Pui-Kai Li, Advisor

Professor Karl A. Werbovetz

Dalia Abdelhamid Sayed Abdelhamid

2011

Abstract

Natural products have served as an effective source of drugs and drug leads throughout history. In part, this is due to the unique structures and the well-defined stereochemistry found in these compounds which allow them to interact selectively with biological target molecules. Unfortunately, most natural products themselves are not suitable for administration as drugs. Chemical synthesis, however, can be employed to study and address some of these shortcomings through manipulation of pharmacological properties, structure activity relationship studies, and the preparation of compounds for mechanistic studies by molecular biologists.

In this thesis, an overview of the role of natural products is presented in order to set the stage for two current drug discovery and development studies which are based on natural product leads. The first project involves the development of a library of curcumin analogues. This effort was initially directed simply at the development of more effective anticancer agents based on the curcumin scaffold. As the project evolved, however, it became clear that a fairly comprehensive library of structurally diverse analogues may be useful for the identification of new leads which could affect other disease states or biological targets. The second project involves the isolation and development of novel compounds for the treatment of leishmaniasis, a parasitic disease. In this case, a short

ii synthetic route was developed to give access to the scaffold of the natural products isolated from a Mexican plant. Based on this strategy, a number of analogues have been produced which have helped to define the structure-activity relationship of this class of molecules.

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Dedicated to my Parents

Acknowledgments

I would like to extend my great praise for God; to fill the heavens and earth and everything in between, for blessing me with all the gifts and all the people to support me to achieve this degree.

No words can express my sincere gratitude to my parents, who I owe everything I achieved throughout my life, for their continuous love, care, and support.

I would like to express my appreciation to all the efforts and sacrifices my husband,

Haysam, has endured, and for the endless love, care, patience, understanding, and support he has been giving to me. His belief in me has always driven me forward.

Also I would like to thank my two sweet boys Yousof and Mohamad for making me stronger through their pure souls and love. I have also to acknowledge the strong emotional support I received from my sisters, brothers, mother in-law, and sister in-law

(Dr. Hala).

I would like to acknowledge my advisors Dr. James R. Fuchs and Dr. Tom Li for their genuine guidance and support. I would like to thank Dr. Fuchs for the excellent training I received in his laboratory to make me a better chemist. I want also to express my v appreciation for his inspiration and assistance and to acknowledge his dedication to reach perfection in everything. I am also grateful to Dr. Li for all his supportive care and encouragement, especially in the hard times. I would like to thank him for helping me pursue my goals.

I would like also to thank the entire faculty in the Division of Medicinal Chemistry and

Pharmacognosy for excellent and enthusiastic teaching, and for providing help and advice. I would like specifically to thank Dr. Werbovetz for being a member of my candidacy and dissertation committee, and for all the encouragement he has given to me.

I would like to sincerely thank Dr. Nivedita Jena for her continuous help, support, and all the fruitful discussions and suggestions to improve my laboratory skills.

I want to thank all my coworkers in Dr. Fuchs and Dr. Li’s laboratories (Eric Schwartz,

John Etter, John Woodard, Mike Corcoran, Pratiq Patel, Sam Boakeye, Nicholas Regan, and Deepak Bhasin) for all the help they had provided.

Finally I would like to acknowledge my former Egyptian Professors: Mohamad Alzahaby

Saber Barakat, Ehab Fetouh, Mohy Makady, and Alaa Shawky for their tremendous help, and encouragement to achieve my goals, and for their belief in me.

Vita

2000...... B.S. Pharmacy, Cairo University, Egypt

2001-2002…………………………………..Researcher; The National Organization of Drug Control and Research, Cairo, Egypt 2003-2005…………………………………..Instructor, College of Pharmacy, Minia University, Egypt 2006……………………………………… Graduate Teaching Assistant, Chemistry Department, SUNY Albany 2007-2011………………………………… Graduate Egyptian Scholar, College of Pharmacy, The Ohio State University 2009...... M.S. Pharmacy, The Ohio State University

Publications

1- Fuchs, J.R.; Pandit. B.; Bhasin, D.; Etter, J.P.; Regan, N.; Abdelhamid, D.; Li, C.; Lin, J.; Li, P.K. Structure-activity relationship studies of curcumin analogues. Bioorganic and Medicinal Chemistry Letters 2009, 19(7), 2065-2069.

2- Bill, M. A.; Fuchs, J.R.; Li, C.; Yui, J.; He, L.; Mitchell, A.P.; Bakan, C.; Benson, D. M.; Kulp, S. K.; Scwartz, E.;Abdelhamid, D.; Lin, J.; Hoyt, D. G.; Fossey, S. L.; Young, G. S.; Carson, W. E.; Li, P.K.; Lesinski, G. B. The small molecule curcumin analog FLLL32 induces apoptosis in human melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity inhibition. Molecular Cancer 2010, 9, 165.

Fields of Study

Major Field: Pharmacy

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Table of Contents

Abstract ...... ii

Dedication ...... ii

Acknowledgments...... v

Vita ...... vii

List of Tables ...... xiii

List of Figures ...... xv

List of Abbreviations ...... xxi

Chapter 1: Natural products as lead compounds for drug development ...... 1

1.1. Introduction ...... 1

1.2. Natural products-based drug discovery ...... 3

1.2.1. Historical perspective ...... 3

1.2.2. The process of drug discovery ...... 10

1.2.3. Properties of natural products ...... 13

1.2.4. Synthetic modification of natural products ...... 17

1.3. Natural products and the future of drug discovery ...... 20

Chapter 2: Curcumin: A pleiotropic natural product ...... 22 viii

2.1. Introduction ...... 22

2.2. Biological activity of curcumin ...... 25

2.2.1. Anti-inflammatory activity ...... 26

2.2.2. Antioxidant activity ...... 26

2.2.3. Cardioprotective acitvity ...... 28

2.2.4. Antiparasitic activity ...... 31

2.2.4.1. Antimalarial activity ...... 31

2.2.4.2. Antileishmanial activity ...... 32

2.2.5. Chemotherapeutic and chemopreventive activity ...... 32

2.2.5.1. Curcumin inhibits activation of numerous transcriptional factors ...... 34

2.2.5.2. Curcumin inhibits inflammatory cytokines ...... 38

2.2.5.3. Curcumin inhibits the activity of multiple protein kinases ...... 38

2.2.5.4. Curcumin modulates the activity of enzymes ...... 38

2.2.5.5. Curcumin inhibits cytokine growth factors and signaling receptors ...... 39

2.2.5.6. Curcumin inhibits anti-apoptotic proteins ...... 39

2.2.5.7. Curcumin induces expression of p53 and suppresses expression of cyclin D1 .. 40

2.2.5.8. Curcumin prevents cancer ...... 40

2.3. Therapeutic potential of curcumin ...... 40

2.4. Previously reported strucural modifications of curcumin ...... 42

2.4.1. Modifications of the methoxy phenolic units ...... 44

2.4.2. Modifications of the α,β-unsaturated ketone ...... 46

2.4.3. Modifications of the 1,3-dicarbonyl ...... 47

2.5. Conclusion ...... 49

Chapter 3: Design, synthesis, and screening of a library of diverse curcumin analogues………………………………………………………………………….. ……51

3.1. Design of the curcumin library ...... 53

3.2. Analogues with various aromatic substituents and substitution patterns...... 55

3.3 Analogues with 5 carbon linker exploring the Michael accepting property ...... 58

3.4. Analogues with different aromatic substituents on the bromopyridine/catechol ring 70

3.5. Analogues with hybrid motifs ...... 74

3.6. Analogues with larger substituents on the benzylamide portion of WP1066 ...... 75

3.7. Analogues locked in a defined tautomeric form ...... 78

3.7.1. Analogues locking curcumin into the di-keto conformation ...... 82

3.7.2. Analogues locking the curcumin into the keto-enol conformation ...... 93

3.8. Screening of the curcumin library against malaria ...... 100

3.9. Conclusions ...... 103

Chapter 4: Leishmaniasis: A neglected tropical disease ...... 105

4.1. Introduction ...... 105

4.2. Clinical manifestations...... 106

4.3 Epidemiology and health burden ...... 109

4.4. Conventional therapeutics ...... 110

4.4.1. Currently available drugs ...... 110

4.4.2. Drugs in clinical trials ...... 112

4.5. The need for new leads ...... 114

4.5.1. Screening of natural products ...... 115

4.5.2. High throughput screening of compound libraries ...... 124

4.5.3. Synthetically developed analogues ...... 125

Chapter 5: Discovery and synthesis of sterol-based antileishmanial agents ...... 129

5.1. Introduction ...... 129

5.2. Collection and screening of plant material ...... 130

5.3. Isolation and characterization of active constituents ...... 133

5.4. Antileishmanial activity of isolated compounds ...... 135

5.5. Synthesis of natural products ...... 138

5.5.1 Synthesis of PAD2F1-3-1K ...... 148

5.5.2 Synthesis of PAD2F1-3-3K and generation of compound analogues ...... 151

5.6. Biological activity of synthetic PAD2F1-3-1K ...... 159

5.7. Structure activity relationships ...... 162

5.8.Conclusion and future directions ...... 164

Experimental Section ...... 166

References ...... 218

Appendix A: MTT assay ...... 232

Appendix B: Library of compounds screened for antimalarial activity ...... 233

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List of Tables

Table 1.1. Drugs based on natural products at different stages of development ...... 12

Table 1.2. Therapeutic applications of natural product derived drugs ...... 13

Table 2.1. Aberrant activity of STAT in various tumors ...... 37

Table 3.1. Reported IC50 of curcumin in different cell lines ...... 52

Table 3.2. Proliferative activity of curcumin analogues in various cancer cell lines ...... 57

Table 3.3. Antiproliferative activity of monoketone curcumin analogues ...... 63

Table 3.4. Antiproliferative activity of representative AG490 analogues ...... 71

Table 3.5. Antiproliferative activity of representative WP1066 analogues ...... 73

Table 3.6. Antiproliferative activity of hybrid compounds ...... 75

Table 3.7. Antiproliferative activity of WP1066 analogues ...... 77

Table 3.8. Effect of different groups on the angle between the carbonyl groups of curcumin ...... 83

Table 3.9. Effect of different groups on the binding to JAK2 and STAT3 ...... 84

Table 3.10. Antiproliferative activity of alkylated derivatives ...... 93

Table 3.11. Antiproliferative activity of rigid curcumin analogues ...... 99

Table 3.12. Antimalarial activity of monoketone curcumin analogues ...... 102

Table 4.1. Various leishmanial species, their regional distribution and clinical forms . 108

Table 4.2. Different classes of antileishmanial alkaloids, their sources, and IC50 ...... 116 xiii

Table 5.1. In vitro antileishmanial activities of compounds isolated from the roots of P. andrieuxii ...... 135

Table 5.2. Effect of additives on yield of alkylation reaction ...... 146

Table 5.3. Leishmanicidal activity of synthetic PAD2F1-3-1K ...... 160

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List of Figures

Figure 1.1. The opium poppy and Cinchona bark ...... 4

Figure 1.2. Examples of (a) analgesic and (b) antimalarial drugs ...... 5

Figure 1.3. Examples of clinically useful drugs ...... 6

Figure 1.4. Examples of ‘Golden Age’ antibiotics ...... 7

Figure 1.5. Examples of marine derived drugs ...... 9

Figure 1.6. All small-molecule new chemical entities...... 10

Figure 1.7. Examples of chemotherapeutic agents ...... 11

Figure 1.8. A comparison of the molecular properties of synthetic compounds , natural products, and drug molecules ...... 16

Figure 1.9. Semisynthesis of paclitaxel ...... 17

Figure 1.10. Development of Bryostatin A ...... 18

Figure 1.11. Vancomycin and vancomycin analogue designed to overcome resistance .. 19

Figure 2.1. Structures of natural curcuminoids ...... 23

Figure 2.2. Biosynthetic pathways to curcuminoids in turmeric ...... 24

Figure 2.3. Biological targets of curcumin ...... 25

Figure 2.4. Stabilization of curcumin radicals, Degradation of curcumin in alkaline pH 28

Figure 2.5. Metabolism of arachidonic acid ...... 29

Figure 2.6. Curcumin inhibits deterioration of left ventricular contractile function ...... 30

Figure 2.7. The potential of curcumin against various tumors ...... 34 xv

Figure 2.8. α,β-Unsaturated ketone as Michael acceptor ...... 35

Figure 2.9. Metabolic pathways of curcumin ...... 41

Figure 2.10. The tautomeric structures of curcumin ...... 43

Figure 2.11. Functional groups in curcumin ...... 43

Figure 2.12. Different modifications of the methoxy phenolic units of curcumin ...... 45

Figure 2.13. Different modifications of the α,β-unsaturated ketone of curcumin ...... 47

Figure 2.14. Different modifications of the 1,3-dicarbonyl of curcumin ...... 48

Figure 3.1. Functional groups of curcumin ...... 54

Figure 3.2. Synthesis of curcumin and symmetrical analogues ...... 55

Figure 3.3. Synthesized curcumin analogues ...... 57

Figure 3.4. Synthesis of monoketone curcumin analogues ...... 59

Figure 3.5. Comparison of antiproliferative activity of curcumin analogues ...... 61

Figure 3.6. Effect of different substituents on Michael accepting property ...... 64

Figure 3.7. Synthesis of monoketone curcumin analogues containing a nitrile group ..... 65

Figure 3.8. Synthesized monoketone analogues bearing electronically different groups . 66

Figure 3.9. Structures of curcumin and related compounds ...... 68

Figure 3.10. Docking of AG490 and WP1066 in ATP binding site of JAK2 ...... 69

Figure 3.11. General procedure for synthesis of AG490 analogues ...... 71

Figure 3.12. Examples of AG490 analogues ...... 71

Figure 3.13. Analogues of WP1066 ...... 73

Figure 3.14. Scheme for synthesis of hybrid curcumin and WP1066 analogues ...... 74

Figure 3.15. Scheme for synthesis of benzyl protected phenol amines ...... 76

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Figure 3.16. Structures of synthesized WP1066 analogues ...... 77

Figure 3.17. Tautomeric structures of curcumin ...... 78

Figure 3.18. Binding mode of curcumin to DFF/CAD ...... 79

Figure 3.19. Binding of curcumin to MD-2 ...... 80

Figure 3.20. Binding of curcumin to JAK2 ...... 81

Figure 3.21. Binding modes of curcumin in JAK2 and STAT3 ...... 82

Figure 3.22. Effect of presence of cyclohexyl ring on binding to JAK2 and STAT3 ...... 86

Figure 3.23. Synthesis of symmetrical alkylated derivatives ...... 88

Figure 3.24. Synthesis of non-symmetical alkylated derivatives ...... 90

Figure 3.25. Examples of alkylated derivatives ...... 91

Figure 3.26. Synthesis of pyrazole and isoxazole derivatives ...... 94

Figure 3.27. Synthesized pyrazole and isoxazole derivatives ...... 94

Figure 3.28. Synthesis of pyrimidine derivatives ...... 95

Figure 3.29. MOM protection of vanillin and synthesis of alkyl phosphonate ...... 96

Figure 3.30. Synthesis of benzene containing derivatives ...... 97

Figure 3.31. Synthesis of pyridine phosphonate ...... 98

Figure 3.32. Synthesis of pyridine containing derivatives ...... 98

Figure 3.33. Synthesized rigid curcumin analogues ...... 98

Figure 3.34. Percent inhibiton of growth at 10 µM and 100 nM ...... 100

Figure 3.35. Structures of representative curcumin analogues screened for antimalarial activity...... 102

Figure 4.1. Life cycle of Leishmania parasites ...... 106

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Figure 4.2. Worldwide geographical distribution of various forms of leishmaniasis ..... 109

Figure 4.3. Structures of selected antileishmanial alkaloids ...... 116

Figure 4.4. Structures of selected antileishmanial chalcones, flavonoids, lignans ...... 117

Figure 4.5. Structures of selected antileishmanial quinones ...... 118

Figure 4.6. Structures of selected antileishmanial terpenoids...... 120

Figure 4.7. Structures of natural antileishmanial sterols ...... 123

Figure 4.8. Structures of antileishmanial candidates from HTS ...... 124

Figure 4.9. Structures of antileishmanial synthetic azasterols ...... 126

Figure 4.10. Pharmacophore required for antileishmanial activity of azasterols ...... 127

Figure 4.11. Structures of synthetic antileishmanial candidates ...... 128

Figure 5.1. Map showing the location of Hopelchen in Cameche, Mexico ...... 130

Figure 5.2. The air-dried roots of P. andrieuxii ...... 131

Figure 5.3. The effect of topical application of PARE in L. mexicana infection ...... 132

Figure 5.4. Structures of compounds isolated from PARE ...... 134

Figure 5.5. Chemical structures of the two most active compounds ...... 136

Figure 5.6. Flow cytometry analysis of the antileishmanial activity of PAD2F1-3-1K andPAD2F1-3-3K ...... 137

Figure 5.7. Electron microscopy of L. mexicana amastigotes ...... 138

Figure 5.8. Ring identification and numbering in sterols and the structures of lead compounds from PARE ...... 139

Figure 5.9. Retrosynthetic analysis of the PAD2F1-3-1K side chain ...... 141

Figure 5.10. Regioselective deprotonation ...... 142

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Figure 5.11. Dimethylation of pregnenolone acetate by Hanson...... 143

Figure 5.12. Negishi-enoxyborate reaction reported by Giannis and coworkers ...... 143

Figure 5.13. Silyl protection of pregnenolone ...... 144

Figure 5.14. Alkylation of silylated pregnenolone with DMAB ...... 146

Figure 5.15. Different alkylation products ...... 147

Figure 5.16. Plan for synthesis of PAD2F1-3-1K...... 148

Figure 5.17. Wittig olefination of pregnenolone ...... 149

Figure 5.18. Oxidation of pregnenolone ...... 150

Figure 5.19. Synthesis of PAD2F1-3-1K ...... 151

Figure 5.20. Grignard addition to silylated pregnenolone ...... 153

Figure 5.21. Reduction of pregnenolone...... 154

Figure 5.22. Barton-McCombie radical deoxygenation ...... 154

Figure 5.23. Structures of synthesized analogues ...... 156

Figure 5.24. Selective reduction of C-20/C-21 double bond ...... 156

Figure 5.25. Reduction of C-20/C-21 double bond using different catalysts ...... 158

Figure 5.26. Plan for synthesis of PAD2F1-3-3K...... 159

Figure 5.27. Bone marrow derived macrophages from C57BL/6 infected with RFP-L. donovani co-cultured with synthetic PAD2F1-3-1K (fluorescence microscopy images)

...... 161

Figure 5.28. Bone marrow derived macrophages from C57BL/6 infected with RFP-L. donovani co-cultured with synthetic PAD2F1-3-1K (flow cytometry data) ...... 161

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Figure 5.29. Structures of natural products and their antileishmanial activity ...... 162

List of Abbreviations

NPs Natural products

WHO World Health Organization

HTS High throughput screening

PCA Principle component analysis

SAR Structure activity relationships

IBD Inflammatory bowel disease

TNF Tumor necrosis factor-alpha

AA Arachidonic acid

PGE2 Prostiglandin E2

LOXs Lipoxygenases

COXs Cyclooxygenases

NO Nitric oxide iNOS Inducible nitric oxide syntase

SPLET Single proton loss electron transfer

HAT Histone acteyltransferase

SERCA Sarcoplasmic endoplasmic reticulum Ca2+-ATPase

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PPAR Peroxisome proliferator activated receptor

HBV Hepatitis B

HCV Hepatitis C

HPV Human papilloma virus

HIV Human immunodeficiency virus

JNK Jun N-terminal kinase

STAT Signal transducer and activator of transcription

JAK Janus kinase

IFN Interferon

EGF Epidermal growth factor

TGF Transforming growth factor

VEGF Vascular endothelial growth factor

MAPK Mitogen-activated kinase

PKC Protein kinase C cPK Protamine kinase

PhK Phosphorylase kinase

MMP Matrix metalloproteinsase

ODP Ornithine decarboxylase

TIM Tissue inhibitor of metalloproteinase

GST Glutathione-S-transferase

GCL Glutamyl cysteine ligase

PDGF Platelet-derived growth factor

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IGF Insulin growth factor

AR Androgen receptor

Cdk Cyclic dependent kinase

LPS Lipopolysaccharide

THF Tetrahydrofuran

DMF Dimethylformamide

HMPA Hexamethylphosphoramide

TMEDA Tetramethylethyelenediamine

DCM Dichloromethane

DMAB Dimethylallylbromide

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Chapter 1

Natural Products as Lead Compounds for Drug Development

1.1. Introduction

Natural products (NPs) are organic compounds produced by living organisms.

The most common sources of natural products include plants, simple marine organisms, bacteria, and fungi. These compounds are usually produced in small quantities by certain species of organisms (referred to as secondary metabolites) and do not play an obvious role in the process of growth and development of the organism itself 1. Generally natural products have an associated biological activity which is postulated to assist species survival through attracting or repelling other organisms (e.g., pheromones and defense mechanisms). Based on their biosynthetic origins, natural products are categorized into numerous broad classes including alkaloids (compounds containing at least one basic nitrogen - typically present in a heterocyclic ring), polypeptides (compounds made up of numerous amino acid residues), terpenes or terpenoids (compounds containing a hydrocarbon skeleton formed from isoprene units), and polyketides (compounds made via sequential condensations of acetyl coenzyme A and resulting in an alternating pattern of carbonyl and methylene derived groups). Many natural products have a complex molecular architecture containing polycyclic ring systems and one or more chiral centers.

1 These compounds are frequently characterized by rigid conformations which facilitate ligand binding to their specific biological targets (enzymes and receptors) through hydrogen-bonding, hydrophobic, and electrostatic interactions, ultimately resulting in their observed biological activity. In general, natural products possess relatively small molecular weights as compared to many biomolecules with most classified as small molecules (<800 Da), facilitating diffusion across cell membranes 2.

Throughout human history natural products, specifically those derived from plant sources; have played a key role in health care through the use of traditional medicine.

Plants have always served as a prolific source of folk medicines for the treatment of various ailments 3,4. Plant materials containing the active natural products were typically cut, dried, and ground into powders or extracted with water to produce teas. Herbal medications are still the most popular form of traditional medicine, and are highly profitable in the international market. According to the World Health Organization

(WHO) 80% of the population in some Asian and African countries still depend on traditional medicine for their primary health care needs. In addition about 70% - 80% of the population in many developed countries use at least some form of alternative or complementary medicine. This widespread use is reflected in the relatively high revenues derived from herbal medications in different parts of the world. For example, in Brazil a total of US$ 160 million from herbal medicine was reached in 2007. Similarly sales of herbal medications reached US$ 5 billion in Western Europe in 2003-2004 and totaled an amazing US$ 14 billion in China in 2005 5.

2 1.2. Natural Products-Based Drug Discovery

1.2.1. Historical Perspective

There is significant written evidence documenting the treatment of human ailments with medicinal plants in cultures around the world, including many of the oldest and most influential civilizations throughout history 6. Around 2600 BC, the use of various plant materials and extracts including cedar oils (Cedrus sempevirens), licorice

(Glycyrrhiza glabra), myrrh (Commiphora species), and poppy capsule juice (Papaver somniferum) for the treatment of a range of diseases including coughs, colds, inflammation, and parasitic infections was recorded on clay tablets by the Mesopotamian people. Seven hundred drugs (mostly plants) were documented in the Ebers Papyrus, an

Egyptian pharmaceutical record which dates from 1500 BC. Likewise the Chinese

Materia Medica which contains 52 prescriptions was recorded around approximately

1100 BC. Although somewhat less well-known in Western cultures, the Indian

Ayurvedic system also dates to about 1000 BC. The Greeks and the Romans also played a significant role in developing the medicinal use of plants through recording the collection, storage, use, and compound prescriptions of plants. Preservation and expansion of this knowledge was attributed to the Arab Scientists, who immensely contributed to the sciences of pharmacy and medicine and established drug stores in the eighteenth century 6.

Although traditional plant materials and extracts are still sometimes used for their medicinal properties, it is the natural products present in these extracts which are responsible for the pharmacological effects. Isolation of the natural products from 3 medicinal plants was first reported in 1816 with the isolation of the traditionally used analgesic morphine (Figure 1.2a) derived from the opium poppy (Papaver somniferum)

(Figure 1.1). Although the correct structure of the compound was not reported until 1925, this provided the foundation for the development of alkaloid chemistry and various valuable morphine-derived analgesics including oxycodone, meperidine, and methadone.

In 1820, quinine (Figure 1.2b) (the active constituent responsible for antimalarial activity) was isolated from the bark of Cinchona officinalis (Figure 1.1). Its discovery provided the starting point for the synthesis of the currently available chloroquine and mefloquine (Figure 1.2b). Similarly, artemisinin [Figure 1.2 (b)] was developed from

Artemisia annua, which was used traditionaly for management of fevers in Chinese medicine. This compound is currently used for the treatment of quinine resistant malaria species in many tropical regions.

Figure 1.1. (left) The opium poppy and (right) Cinchona bark (adapted from http://upload.wikimedia.org/wikipedia/commons/2/2a/Slaapbol_R0017601.JPG and http://en.wikipedia.org/wiki/File:Cinchona_officinalis_001.JPG).

Figure 1.2. Examples of (a) analgesic and (b) antimalarial drugs.

Thorough investigations of other traditionally useful plants also led to the isolation of their active ingredients as single chemical entities. A number of these compounds have been developed into valuable drugs for a wide range of applications such as digitoxin (cardiotonic), reserpine (antihypertensive), and tubocurarine (muscle relaxant) (Figure 1.3). These drugs are still of substantial importance in modern medicine. In addition to the natural products, semisynthetic derivatives of some natural products have been developed to overcome limitations of the parent drugs. For example, carbenoxolone sodium hemisuccinate (Figure 1.3), a drug prescribed for the treatment of gastric and duodenal ulcers, was derived from the natural product glycyrrhetic acid found

5 in licorice in order to avoid rapid acid hydrolysis. Overall, it is estimated that 80% of plant derived drugs are currently used for the same indications as the traditional application of the plant extract.

Figure 1.3. Examples of clinically useful natural product drugs.

Plants, however, are clearly not the only source of clinically useful therapeutic agents. The discovery of penicillin (Figure 1.4) from the mold Penicillium notatum in

1928 by Fleming along with its re-isolation and commercialization in 1940 initiated a great revolution in drug discovery and research which is often referred to as the “Golden

Age of Antibiotics”. This period helped to inspire drug discovery efforts from numerous microorganism cultures (bacteria and fungi), and resulted in the discovery of several key antibiotics including vancomycin, erythromycin, chloramphenicol, tetracycline, and

6 cephalosporin (Figure 1.4). The high degree of biodiversity found among these microorganisms has also led to the isolation of natural products which are used for a wide range of therapeutic applications. These include immunosuppression (the cyclosporins and rapamycin from Streptomyces species), cholesterol lowering (lovastatin and mevastatin from Penicillium species), anthelmintic (ivermectin), antidiabetic (acarbose), and chemotherapeutic (bleomycin, actinomycin, and doxorubicin) applications.

Approximately 130 currently available commercial drugs for the treatment of various diseases have been derived from microorganisms 7,8.

Figure 1.4. Examples of ‘Golden Age' antibiotics.

7 Although Phoenicians used mollusks for dying woolen cloth and seaweeds as soil fertilizers, marine organisms as a whole have not enjoyed a rich historical application in traditional medicine as compared to plants and microorganisms. Exploration of marine natural products began only about 60 years ago upon improvement of scuba diving techniques. In 1950 serendipitous isolation of spongouridine and spongothymidine from the Caribbean sponge (Cryptotheca crypta) promoted extensive investigation of this rich and significantly biodiverse resource 9. This led to the identification of two close analogues: cytosine arabinoside (anticancer) and adenine arabinoside (antiviral) (Figure

1.5) which were approved later for clinical application. Although oceans cover about

70% of earth’s surface, much of it and the diverse life forms living in it still remain unexplored or inaccessible. The burgeoning interest in this relatively novel source of natural products, however, has accelerated and extended marine drug research to involve multiple disciplines including biology, ecology, organic chemistry, biochemistry, and pharmacology 8. Although few marine natural products have yet been approved for clinical use [the peptide ziconotide from a tropical marine cone snail was approved in

2004 for chronic pain resulting from spinal cord injury and the antitumor agent trabectidin (or ecteinascidin-743) from a sea squirt was approved to treat soft-tissue sarcoma in 2007], numerous marine derived natural products are currently being evaluated in Phase I-III clinical trials for the treatment of cancer 10.

Figure 1.5. Examples of marine derived drugs.

In 2003, and 2007 two landmark reviews by D.J. Newman 11 showed that 63 % of the small-molecule new chemical entities discovered between 1980 and 2006 were inspired by natural products. These included: 6% natural products (N), 28% semisynthetically modified natural products (ND), 5% totally synthetic compounds based on a natural product pharmacophore (S*), and 24% synthetic/semisyntheic natural product mimics (S*/NM and S/NM). Therefore in total about 50% of the new small- molecule chemical entities introduced between 1980 and 2007 are derived from or based on natural products. In addition about 79% of discovered antibacterials and 74% of anticancer drugs were based on, or derived from NPs. Moreover, many of the best selling drugs currently on the market in various therapeutic fields (antibacterial, antifungal, anticoagulant, antiparasitic, immunosuppressant, and anticancer) were derived from NPs.

Figure 1.6. All small-molecule new chemical entities, 1980-2006, by source (D.J. Newman 2007).

1.2.2. The Process of Drug Discovery

Drug discovery is a time consuming process, which requires a significant workforce of trained scientists and extensive financial support. It is a process which frequently lasts

10 years or more, and costs upwards of US$ 800 million 12. The basic preclinical research process involves lead identification, lead optimization through organic synthetic and/or combinatorial chemistry efforts, and assessment and modification of pharmacokinetic and pharmacological profiles of drug candidates to move them forward into clinical trials 12.

Lead identification utilizes various methods including the screening of libraries of natural compounds isolated from medicinal plants or other natural sources, screening of synthetic compound libraries (including combinatorial libraries), mechanism-based high throughput screening (HTS), and molecular modeling-based approaches. Lead

10 identification from medicinal plants involves the identification and collection of the plant of interest, extraction of dried plant materials, testing of the crude extracts in relevant biological assays (including target based, cell-based, and in vivo bioassays), and the isolation and structural elucidation of the individual active constituents responsible for the activity. This process, referred to as bioassay-guided fractionation, has made the discovery of biologically active natural products for drug discovery efforts much more efficient. Although bioassay-guided fractionation has streamlined the process, natural products isolation still remains a highly labor intensive and extremely time-consuming process. Regardless, this approach has resulted in the discovery of a number of highly successful drug molecules over the course of the last century, including the chemotherapeutic agents paclitaxel from Taxus brevifolia and camptothecin from

Camptotheca acuminate (Figure 1.7).

Figure 1.7. Examples of chemotherapeutic agents.

11 In general, natural products have been shown to possess diverse biological activities through a wide variety of mechanisms of action including stimulation of enzymes or receptors; inhibition of protein-protein or DNA-protein interactions, allosteric modulation, and the opening of channels, leading to activity against a wide variety of disease states 13. As with paclitaxel and camptothecin, numerous other natural products have previously been approved for use as drugs or studied in clinical trials. For example, a recent paper by A.L. Harvey 14 looked at the 225 natural-product-derived compounds which were undergoing different stages of clinical trials in 2008. Most of these compounds were derived from plants and microorganisms (Table 1.1). They were principally indicated for cancer therapy and anti-infection, but other therapeutic applications (demonstrating the broad utility of natural product derivatives) were also incorporated (Table 1.2).

Development stage Plant Bacterial Fungal Animal Semisynthetic Total Preclinical 46 12 7 7 27 99 Phase I 14 5 0 3 8 30 Phase II 41 4 0 10 11 66 Phase III 5 4 0 4 13 26 Pre-registration 2 0 0 0 2 4 Total 108 25 7 24 61 225

Table 1.1. Drugs based on natural products at different stages of development.

12 Therapeutic Area Preclinical Phase I Phase II Phase III Pre- Total registration Cancer 34 15 26 9 2 86 Anti-infective 25 4 7 2 2 40 Neuropharmacological 6 3 9 4 0 22 Cardiovascular/gastrointestinal 9 0 5 6 0 20 Inflammation 6 2 9 1 0 18 Metabolic 7 3 6 1 0 17 Skin 7 1 2 0 0 10 Hormonal 3 0 2 1 0 6 Immunosuppressant 2 2 0 2 0 6 Total 99 30 66 26 4 225

Table 1.2. Therapeutic applications of natural product derived drugs at different stages of development.

1.2.3. Properties of Natural Products

Natural products as a class typically contain unique structural features not found in fully synthetic drug candidates. Frequently, natural products have a complex molecular architecture with abundant stereogenic centers, giving them increased conformational rigidity and a well defined spatial orientation. It is presumed that their complexity and conformational rigidity are responsible for eliciting their observed biological activity by enforcing key interactions with biological molecules. Hydrogen bond donors and acceptors are also routinely present in the molecules in the form of alcohols, ethers, carbonyls, and nitrogen-containing functional groups. These groups likely play a key role in the interaction of the natural products with target biomolecules and help to increase aqueous solubility 4. The overall shape and functionality of natural products have been shown to be of critical importance, as the loss of stereochemistry, introduction of greater flexibility, and decrease in the size of these molecules generally results in lower

13 selectivity and weaker activity 15. However, this is not always the case, as several structurally simple natural products (for example adrenaline, serotonin, and histamine) show good potency and specificity to their targets 4.

As with any potential lead compound, however, nature’s “engineering” of the structure of a natural product does not necessarily mean that a compound is suited for use as a drug in humans. In some cases, they may possess reactive functional groups which are not desired in drugs due to unacceptable stability, metabolism, or toxicity. In addition, the physicochemical properties of a natural product may not be optimal for solubility and absorption. In fact, many natural products violate Lipinski’s rule of 5 for oral bioavailability which is based on a comparison of drug properties and is typically considered when evaluating potential drug candidates 17. Lipinski’s rules state that orally active drug candidates should posses less than 5 hydrogen bond donors, less than 10 hydrogen bond acceptors, have a log P (partition coefficient) of less than 5, and a molecular weight under 500 Da. A corollary to this rule also states that molecules should have no more than 10 rotatable bonds and polar surface area equal to or less than 140 Å

18. Although the logP and the molecular weight of many natural products fall within the acceptable ranges (logP: 2.4–2.9 and M.W.: 360–414), most will violate one or more of the rules. Interestingly, however, although Lipinski’s rules are generally considered to be a good guideline for predicting potential success of a drug candidate, the rules are not considered to apply to natural products. The exclusion of natural products is primarily because the natural compounds often utilize transmembrane transporters (active transport mechanisms) to enter cells rather than passive diffusion. This point suggests another one of the significant differences between natural products and their synthetic counterparts. 14 These differences were further investigated by M. Feher through a rigorous analysis and comparison of the molecular properties of natural products, synthetic molecules derived from combinatorial libraries, and known drug molecules 15. In this study the authors systematically examined the differences in various simple properties which include the number of stereogenic centers, the presence of aromatic rings and/or complex ring systems, the degree of the saturation, and the number and ratios of different heteroatoms. A Principle Component Analysis (PCA) based scheme (Figure 1.8) was developed in order to visually compare these groups of compounds. It is clear from analysis of the three graphs that the synthetic compounds are most densely packed, suggesting that they are less diverse and, therefore, cover less chemical space. In contrast, the points corresponding to the natural product and drug groups are more spread out over the graph, suggesting a higher degree of structural difference. Interestingly, both of these groups seem to cover the same space as the synthetic molecules, but also share common features

Figure 1.8. A comparision of the molecular properties of a) synthetic compounds, b) natural products, and c) drug molecules.

not found in the synthetic group. The diversity observed within the drug category may in part be due to the presence of a number of natural product or natural product derived compounds.

16 1.2.4. Synthetic Modification of Natural Products

Natural products have been tightly related to organic chemistry through drug discovery and development. Their rich and complex structures have prompted drug discovery through inspiring chemical and biological research efforts. Total synthesis of these natural products has been a challenging goal for many chemists. This has inspired the invention of new chemical reactions and advanced synthetic strategies (sometimes based on insights from the biosynthetic pathways). This progress in chemistry methodologies provided an alternative for large scale production of scarce NPs. In addition, functional and structural modifications of the parent NP have generated libraries of analogues which have helped to establish mechanisms of action and structure-activity relationships (SAR). Several of these studies have led to the development of advanced leads with improved pharmacological or pharmacokinetic profiles 16. For example a renewable source of paclitaxel and several of its analogues was attained by semisynthetic conversion of naturally abundant baccatins (Figure 1.9) 19.

Figure 1.9. Semisynthesis of paclitaxel.

Moreover the power of organic synthesis is illustrated by the success in the development of Bryostatin A (Figure 1.10), a structurally simpler and more potent anticancer analogue of Bryostatin 1 which is undergoing Phase II clinical trials.

Figure 1.10. Development of Bryostatin A

18 Another example of the potential utility of synthesis was the preparation of a structurally complex vancomycin aglycon, with the aim of overcoming the emergent antibacterial resistance to vancomycin 20. Bacterial resistance arises from changing the terminal amino acid in the peptidoglycan of the cell wall from D-Ala-D-Ala to D-Ala-D-

Lac. An isosteric replacement of the amide carbonyl by a methylene in residue 4 (Figure

3 -1 1.11) resulted in a 40-fold increase in affinity to D-Ala-D-Lac (Ka= 5.2 x10 M ) and a

3 -1 35-fold reduction in affinity for D-Ala-D-Ala (Ka= 4.8 x10 M ). This synthetic analogue showed good antimicrobial activity against VanA-resistant organism (MIC =31g/ml), although it has not been advanced for clinical utility (possibly due to the complexity of the synthetic route).

Figure 1.11. Vancomycin and vancomycin analogue designed to overcome resistance.

19 1.3 Natural Products and the Future of Drug Discovery

Over the past two decades a substantial decline or even termination in the natural products research and development programs in the pharmaceutical industry has been observed. This was driven by several factors including: (1) the breakthroughs in combinatorial chemistry, high throughput-screening (HTS) of synthetic libraries, and computer-aided design of small-molecules, which provided an apparently rapid alternative for drug discovery; and (2) the significant decline in the number of approved drugs due to increasing governmental restrictions. It was thought that the rapid increase in the number of potential drug candidates through combinatorial synthetic efforts and the ability to rapidly screen the vast synthetic libraries of new compounds using HTS should lead to more hits for potential disease targets, speeding up the drug discovery process.

Nevertheless, this strategic approach did not result in the anticipated revolution in the industry. Although many more new compounds were synthesized and tested, the approach still resulted in a shortage of synthetic leads in certain therapeutic areas including metabolic disorders, immunosuppression, anti-infection, and oncology. As discussed above, this has mainly been attributed to the lack of structural diversity, chirality, and conformational rigidity in the synthetic compounds which may have resulted in the overall lack of biological activity 21, 22. Based on these shortcomings, members of the scientific community have advocated a move back to natural product isolation as a key source of drug leads 23. Recognizing the advantages of natural products in discovery and yet still hoping that this time-consuming process can be sped up, an alternative strategy referred to as Diversity Oriented Synthesis has also been adopted by some groups and companies. In this case, the focus is on the synthesis of combinatorial 20 libraries using NP pharmacophores as starting templates to make complex structures that more closely match the chemical space occupied by NPs. It is not yet clear if this approach will be more successful for the development of new drugs.

There is clearly a continued need for new drugs to combat an ever expanding number of conditions and diseases. As was the case over the course of most of the last century, natural products and their synthetic and semi-synthetic derivatives appear to be the primary candidates to fill this need. Therefore, an increase in natural products investigation is speculated primarily in academia (although possibly also in industry) over the next decade. The process will be aided by the expanding diversity of natural products sources, advances in natural products isolation techniques and structural elucidation, access to genome sequencing, identification of novel drug targets and mechanisms of action, and enhanced synthetic and combinatorial methodologies. In conclusion natural products have been and will continue to be an integral contributor to drug discovery and development for the prevention and treatment of diseases.

Chapter 2

Curcumin: A Pleiotropic Natural Product

2.1. Introduction

Turmeric is a spice and pigment derived from grinding the dried rhizomes (or rootstocks) of Curcuma longa (family Zingiberaceae) 24. In addition to its use as a spice and pigment; turmeric has a long history in traditional medicine for the treatment of various ailments (recorded in the Indian Ayruvedic medicine around 1100 BC) 25. It is characterized by the presence of turmerin (a water soluble peptide), essential oils

(tumerones, atlantones, and zingiberene), and highly conjugated polyphenolic pigments; collectively known as curcuminoids (Figure 2.1); which comprise 2–9 % of turmeric by weight 26 and are responsible for its bright yellow color. The natural curcuminoids are collectively referred to as “curcumin”, although more specifically they include curcumin

(1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione or curcumin I), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) which are present in a 77:17:3 ratio, respectively 24. These three compounds are all frequently present in commercial samples of natural curcumin, but can be separated using HPLC.

Figure 2.1. Strucures of natural curcuminoids.

The curcuminoids (Figure 2.1) were first characterized in 1910 27, however their biosynthetic pathway remained ambiguous until recently. Two synthetic pathways were proposed in 1973 by Roughley and Whiting 28 utilizing cinnamic acid, which is derived from phenylalanine, as their starting point. In 2008, T. Kita used in vitro culture systems of turmeric plants and 13C-labelled precursors to examine these pathways 29. Their results suggested that the biosynthesis of the curcuminoids used two cinnamoyl CoAs and one malonyl CoA, and that the hydroxy and methoxy functional groups on the aromatic rings were introduced after the formation of the curcuminoid scaffold (Figure 2.2).

Figure 2.2. Biosynthetic pathways to curcuminoids in turmeric. Major (thick arrows) and Minor (thin and dotted arrows) (1)phenylalanine; (2), cinnamic acid; (3), p-coumaric acid; (4), ferulic acid; (5), malonic acid; (6), putative curcuminoid skeleton intermediate: bisdeshydroxybisdesmethoxycurcumin (BDHBDMC); (7), bisdesmethoxycurcumin (BDMC); (8), desmethoxycurcumin (DMC); (9), curcumin.

Turmeric has been used in traditional medicine for the treatment of inflammation, microbial infections, indigestion, renal and urinary tract diseases, hepatic diseases

(especially jaundice), rheumatoid arthritis, and insect bites 30. Research in the second half of the 20th century identified curcumin to be responsible for most of the biological activity of turmeric. Its simple structure, broad spectrum of traditional applications, abundance, and low cost has attracted considerable research attention. Thus curcumin has been the subject of a multitude of studies against diverse diseases. These research efforts have indicated that curcumin shows a myriad of biological activities including anti- inflammatory, antioxidant, chemotherapeutic, chemopreventive, antidiabetic, antirheumatic, hypoglycemic, anthelmintic, antimalarial, antileishmanial,

24 cardioprotective, antiviral, and antibacterial activities 31. Elucidation of the mechanisms of action responsible for each observed bioactivity has also been investigated. These studies ultimately led to the discovery of numerous biological targets affected by curcumin, including various enzymes, transcription factors, and protein kinases (Figure

2.3).

Figure 2.3. Biological targets of curcumin, adapted from Anticancer Research 2003, 23, 363.

2.2.Biological Activity of Curcumin

Although our primary interest in curcumin lies in its anticancer properties

(especially as a JAK/STAT inhibitor), a relatively comprehensive discussion of the broad

25 range of biological activities reported for curcumin are discussed below. When possible, mechanisms of action are also discussed.

2.2.1. Anti-inflammatory Activity

Inflammation is an immunological response against foreign bodies which involves a series of complex reactions. Overactive inflammatory responses result in undesirable effects as represented by autoimmune diseases such as rheumatoid arthritis, inflammatory bowel diseases (IBD) such as Crohn’s disease, and ulcerative colitis.

Numerous studies have investigated the molecular mechanism of action of curcumin responsible for its ability to suppress acute and chronic inflammatory activity 32.

Curcumin has been shown to inhibit (1) production of several cytokines including tumor necrosis factor-alpha (TNF), IL-1, and IL-8; (2) activation of the transcription factor

NF-B; (3) activity of IB-kinases (IKKs); (4) cellular uptake of arachidonic acid (AA);

(5) production of prostaglandin E2 (PGE2) and leukotrienes B4 and C4; (6) lipoxygenases

(LOXs) and cyclo-oxygenases (COXs) (especially COX-2); (7) release of proteolytic enzymes from macrophages; and (8) production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) protein.

2.2.2. Antioxidant Activity

Curcumin is an efficient free radical scavenger and antioxidant 33(a). The presence of phenolic groups, methoxy groups, carbon-carbon double bonds, and the 1,3-diketone system gives curcumin exceptional antioxidant properties through the formation of resonance stabilized radicals. Curcumin can effectively scavenge hydrogen peroxide, 26 nitric oxide, and superoxide radicals. This activity protects biomembranes against lipid peroxidation. It can also enhance the activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione reductase, and stimulate heme oxygenase-1. A long debate about the source of the hydrogen atom responsible for the antioxidant activity [the phenol or central methylene (Figure 2.4)] was resolved by the theory of sequential proton loss electron transfer (SPLET) by Litwinienko and Ingold 34, which stated that in ionizing solvents curcumin reacts first at the ionized keto-enol moiety with electrophilic radicals then loses the phenolic proton to yield a phenoxy radical. However this can not take place in nonionizing solvents, where only hydrogen atom transfer from the phenolic hydroxyl group to the radical can take place. In addition it is postulated that curcumin degrades rapidly in alkaline pH into ferulic acid and vanillin 33(b); which are known antioxidants.

Figure 2.4. (a) Stabilization of curcumin radicals (b) Degradation of curcumin in alkaline pH.

2.2.3. Cardioprotective Activity

Inflammation of the walls of blood vessels and alteration of lipid metabolism associated with platelet aggregation results in atherosclerosis. Studies have shown that curcumin combats this disease through different mechanisms 35 which include (1) suppression of LDL and cholesterol oxidation; (2) reduction of plasma lipid peroxidation

(via its antioxidant properties); and (3) inhibition of platelet aggregation, thus blocking clot formation (via inhibition of platelet activating factor, arachidonic acid (AA) mediated aggregation, COXs, and thromboxane A2 (TXA2) which promote platelet

28 aggregation). The pathway showing inhibition of platelet aggregation through inhibition by curcumin is shown below.

Figure 2.5. Metabolism of arachidonic acid (red arrows show reactions inhibited by curcumin).

In addition, in vitro and in vivo studies have shown that curcumin protects against cardiac injury (ischemic or biochemical) by inhibiting the generation of reactive oxygen species; adhesion of monocytes to endothelial cells; and phosphorylation of C-Jun N-

29 terminal kinase (JNK), p38MAPK (Mitogen-activated protein kinase), signal transducer and activator of transcription (STAT3), and their downstream signals 36. This protective activity is attributed to its antioxidant properties. An alternative mechanism of cardioprotection is related to cardiomyocyte activity. Excessive stress of cardiomyocytes is known to lead to heart failure. A recent study has shown that curcumin can also control gene expression through modulation of Histone Acetyltransferase (HAT) in the nuclei of cardiomyocytes leading to protection of the heart 37.

Figure 2.6. Curcumin inhibits deterioration of ventricular contractile function. Adapted from http://en.wikipedia.org/wiki/File:Right_Ventricular_hypertrophy.svg and Circulation Journal 2010, 74(6), 1059.

30 2.2.4. Antiparasitic Activity

Curcumin was reported by different groups to have antiprotozoal activity against parasites including Plasmodium, Leishmania, and Trypanosoma species. These diseases represent a severe disease burden especially in developing countries where therapeutic options are limited by expense and toxicity. The antimalarial and antileishmanial acitivities, on which the bulk of the reports have focused, are discussed separately below.

2.2.4.1. Antimalarial Activity

Malaria is a global health problem which is in urgent need of new and cheap drugs due to the emergence of resistance to the current therapies. Curcumin and curcumin analogues revealed potent antimalarial activity against chloroquine-resistant and chloroquine-sensitive Plasmodium falciparum in a dose dependent manner 38. An alternative approach to combat the problem of resistance is the use of curcumin in combination with known antimalarial drugs. Curcumin has been shown to reduce the therapeutic dosage of primaquine and improved the survival of treated animals 39. In addition curcumin protected against the toxic effects associated with therapeutic or higher doses of chloroquine phosphate 40. Another study indicated that curcumin delayed or even prevented development of cerebral malaria, and increased the survival of treated animals 41. Also a recent study used nanoparticle formulated curcumin for treatment of malaria as adjuvant therapy 42. Computational modeling investigating the mechanism of action of curcumin as an antimalarial, indicated that curcumin interacts with the same molecular target as the artemesinins, PfATP6 [the parasite orthologue of mammalian sarcoplasmic endoplasmic reticulum Ca2+–ATPase (SERCA)], through hydrogen bonding 31 and hydrophobic interactions 43. Another in vitro study showed that the parasiticidal activity of curcumin to Plasmodium falciparum was attributed to generation of reactive oxygen species (ROS) and inhibiton of HAT leading to DNA damage 44.

2.2.4.2. Antileishmanial Activity

Recent reports also indicate antileishmanial activity 45, although there is some debate as to its efficiency and mechanism of action. Curcumin proved to be more potent than pentamidine against four Leishmania species promastigotes, but the same effect was not seen against intracellular amastigotes or in vivo 46. Also synthetic curcumin analogues showed similar and even better leishmanicidal activity against L. amazonensis promastigotes 45(a), 47. The antileishmanial activity was attributed to induction of ROS and elevation of cytosolic Ca2+ leading to mitochondrial membrane depolarization and

DNA fragmentation (of the parasite) leading to a programmed cell death-like situation 48.

When Chan et al 49 investigated the effect of curcumin’s antioxidant activity on adaptive immunity, however they concluded that long term, low dose consumption of curcumin can aggravate leishmanial infection through activation of peroxisome proliferator activated receptor- (PPAR-), and inhibition of NO synthase.

2.2.5. Chemotherapeutic and Chemopreventive Activity

Cancer is a devastating disease, characterized by abnormal proliferation of cells which grow beyond their normal boundaries. Usually it develops in one tissue which can invade adjoining tissues and spread to other parts of the body (referred to as metastasis)

50. It arises from transformation of a normal cell into a tumor cell via a progressive

32 process. This transformation is initiated by genetic mutations and exogenous agents including: (1) Physical carcinogens (such as ultraviolet and ionizing radiation); (2)

Chemical carcinogens [such as asbestos, components of tobacco smoke, aflatoxin (a food contaminant) and arsenic (a drinking water contaminant)]; and (3) Biological carcinogens

[such as infections from certain viruses including hepatitis B (HBV), hepatitis C virus

(HCV) and liver cancer, Human Papilloma Virus (HPV) and cervical cancer, and human immunodeficiency virus (HIV) and Kaposi sarcoma; bacteria (Helicobacter pylori and stomach cancer); or parasites (schistosomiasis and bladder cancer)].

Aging, alcohol consumption, tobacco smoking, and obesity are also fundamental factors which contribute to the development of cancer. Various types of cancers differ between men and women. According to WHO; lung, stomach, liver, colorectal, oesophagus and prostate cancers affect more men, whereas breast, lung, stomach, and cervical cancers are more prevalent in women 50. Cancer is a major cause of death worldwide, accounting for about 13% of all deaths (7.4 million) in 2004 50. It is estimated that the number of deaths from cancer will reach 12 million in the year 2030.

Curcumin is reported to have both chemotherapeutic (cytotoxic, antiapoptotic, antiangiogenic, antiproliferative, antimetastatic, and anti-invasion) and chemopreventive effects. This has made curcumin the subject of a huge number of studies. Curcumin was shown to be active against almost all kinds of blood and solid tumors (Figure 2.7) 51. It also affects a vast range of biochemical and molecular targets either through direct interaction with target proteins or through modulation of transcription factors, cytokines, protein kinases, enzymes, growth factors or their receptors, or gene expression 52.

Figure 2.7. The potential of curcumin against various tumors. Adapted from Cancer Letters 2008, 267, 133.

2.2.5.1. Curcumin inhibits activation of numerous transcriptional factors involved in cell proliferation, cell invasion, metastasis, angiogenesis, and resistance to chemotherapy and radiotherapy. The targets affected by curcumin include: (1) Curcumin inhibits signaling of the transcription factor NF-B which is constitutively active in almost all cancer types and suppresses apoptosis in various tumors. (2) Curcumin inhibits activation of the transcription factor AP-1 signaling which activates JNK resulting in proliferation and transformation of tumor cells. (3) Curcumin induces the expression of peroxisome proliferator activated receptor (PPAR)-c which inhibits cell proliferation, induce apoptosis, and suppress extracellular matrix gene expression. (4) Curcumin inhibits

Notch-1 signaling which is critical in maintaining the balance between cell proliferation, differentiation, and apoptosis. (5) Curcumin inhibits the Wnt/β-catenin signaling pathway which is tightly regulated and is essential for the process of development, tissue homeostasis, and regeneration. Abnormal activation of this pathway was linked to 34 initiation and progression of different cancers for example gastric, colon, and intestinal cancer. (6) Curcumin inhibits p300/CBP HAT activity (Creb-binding protein / Histone acetyltransferase). HATs represent novel molecular targets for drug

Figure 2.8. α,β-Unsaturated ketone as Michael acceptor.

development. Studies indicated that this activity is attributed to the ability of , β- unsaturated carbonyl groups in curcumin to react as Michael acceptors (Figure 2.8) 53. (7)

Curcumin inhibits activation of STAT3 signaling (which is upregulated in various tumors) and its nuclear translocation. Also it inhibits Janus kinase (JAK)-2 phosphorylation and interferon (IFN)--induced STAT1 phosphorylation but has no effect on STAT5 phosphorylation.

STAT3 is of particular importance in our research program. It is one of the six transcription factor proteins which transduce cytokine (e.g. IL-6) and growth factor

(epidermal growth factor EGF, transforming growth factor TGF-α, hepatocyte growth factor) mediated extracellular signals. These signals stimulate the phosphorylation of various kinases including cytoplasmic Janus kinases (JAKs), Src family kinases, and

35 receptor tyrosine kinases (EGFR and PDGFR) which in turn phosphorylate a critical tyrosine residue in the STAT transactivation domain. Activated STAT monomers then form a dimer which translocates to the nucleus and mediates gene expression. Normally this activation is transient to keep gene expression under tight regulation 54.

Carcinogenesis is strongly associated with constitutive activation of STAT3.

Constitutively active STAT3 is prominent in various tumors such as melanoma, lymphoma, leukemia, head and neck cancer, lung cancer, breast cancer, and prostate cancer 55 (Table 2.1). Aberrant activation of STAT3 mediates the expression of various genes which initiate cell proliferation (c-myc and cyclin D1), suppress apoptosis (Bcl-xL,

Bcl-2, Mcl-1, and survivin), and maintain angiogenesis (VEGF) thus mediating tumor survival, progression and immune system evasion 56. Suppression of apoptosis also confers resistance of cancer cells to conventional chemotherapy 57. Expression of cytokines which are essential for innate immune response against cancer cells upon suppression of STAT3 makes it an attractive target for chemoprevention and chemotherapy 58.

Table 2.1. Aberrant activity of STAT in various tumors.

Reports have shown that curcumin displayed inhibition of JAK2, Src, Erb2, and

EGFR and downregulation of the expression of Bcl-xL, cyclin D1, VEGF, and TNF, all of which are correlated with STAT3. It was also shown that curcumin could activate a protein tyrosine phosphatase SHP-2, which suppresses JAK1/2, thus inhibiting the

JAK/STAT pathway 59. Furthermore, inhibition of constitutive/and IL-6 induced STAT3 phosphorylation and abrogation of nuclear translocation of STAT3 by curcumin lead to reversible, dose and time dependant inhibition of the JAK/STAT pathway 60. Curcumin also proved to be more potent and rapid in suppressing cell proliferation, and STAT3 phosphorylation as compared to AG490, a known JAK2 inhibitor 60.

37 2.2.5.2. Curcumin inhibits inflammatory cytokines which have a considerable role in tumorigenesis. As mentioned above curcumin suppresses numerous transcription factors, cytokines, and enzymes which are involved in the inflammation process as well as in cancer. Curcumin also inhibits phoshorylation of NF-B, and thereby prevents activation of TNF which affects cellular remodeling, apoptosis, and cell survival. Finally, curcumin inhibits the activity of pro-inflammatory cytokines (interleukins IL-1, IL-2, IL-

5, IL-8, IL-12, and IL-18) which are important for induction of adhesion molecules, metalloproteinases, pro-angiogenic factors, and signaling pathways involved in tumor invasion and angiogenesis (such as NF-B and STATs). Recent studies reported that curcumin inhibited IL-6-induced STAT3 phosphorylation in myeloma cells.

2.2.5.3. Curcumin inhibits the activity of multiple protein kinases

Curcumin suppresses MAPKs activity which play a major role in response to inflammatory stimuli and environmental stresses. MAPKs activate different pathways: p44/42 MAPK, extracellular signal-regulated kinases (ERK1/ERK2), JNK, and p38

MAPK. Curcumin also suppresses the activity of protein kinase A (PKA), protein kinase

C (PKC), protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation- activated protein kinase (AK), and pp60c-src tyrosine kinases.

2.2.5.4. Curcumin modulates the activity of enzymes

Several inflammatory enzymes such as COX-2, 5-LOX, phospholipases (PLA)-2, are related various tumor cell proliferation, and suppression of apoptosis. Curcumin was reported to inhibit the expression of COX-2 matrix metalloproteinase (MMP)-9 (involved 38 in tumor metastasis) and inducible nitric oxide synthase (iNOS) (involved in inflammation and metastasis). Several enzymes are downregulated by curcumin, these include arylamine N-acetyltransferases-1, ATFase, APTase, desaturase, DNA polymerase, NAD(P)H dehydrogenase quinone (NQO)1, ornithine decarboxylase (ODC), phospholipase D, telomerase and tissue inhibitor of metalloproteinase (TIM)-3.

Nevertheless curcumin also upregulates src homology 2 domain-containing tyrosine, hemoxygenase (heme)-1, glutathione-S-transferase (GST), glutamyl cysteine ligase

(GCL), and glutamate-cysteine ligase.

2.2.5.5. Curcumin inhibits cytokine growth factors and signaling receptors

Curcumin can modulate the expression and activity of different growth factors which have a key role in proliferation of cancer cells and are upregulated in various tumors.

These include EGF (Epidermal growth factor), HER2, FGF (Fibroblast growth factor),

VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor), TNF, insulin growth factor (IGF)-1. Moreover, curcumin also suppresses androgen receptors

(AR), AR-related cofactors, and expression of estrogen receptors alpha and beta

(ER/ERβ) which greatly affect tumor progression.

2.2.5.6. Curcumin inhibits anti-apoptotic proteins

Curcumin inhibits the expression of antiapoptotic proteins including caspases, Bcl-2, Bcl- xL, X-linked inhibitors of apoptosis (XIAP), and surviving. Anti-apoptosis can result in transformation of normal cells into malignant.

39 2.2.5.7. Curcumin induces the expression of p53 and suppresses the expression of cyclin D1. p53 is a tumor suppressor transcription factor which activates downstream genes involved in regulation of cellular responses to DNA damage, gene stability, cell cycle control, and apoptosis. Cyclin D1 is an important protein involved in cell cycle progression. It is a part of the cyclin dependent kinase (Cdk)-4 and Cdk6, which is upregulated in many cancers including breast, esophagus, head and neck, and prostate.

2.2.5.8. Curcumin prevents cancer

Curcumin showed suppression of carcinogenic effects induced by various carcinogens including capsaicin, tobacco, cigarette smoke condensate, 2-AAF, benzo(a)pyrene, 12- dimethyl-benz[a] anthracene (DMBA), aflatoxin B1,and heterocyclic amines in a vast range of cancer cell lines. It is estimated that this inhibition of carcinogenicity results from inhibition of proliferation, and induction of apoptosis. Structure activity relationship

(SAR) studies indicated that presence of methoxy groups, central β-diketone moiety, and unsaturation in the side chain are responsible for this chemopreventive activity.

2.3. Therapeutic Potential of Curcumin

Currently a therapeutic trend is to combine several drugs to affect multiple targets. This multiple targeting is becoming a very desirable property in drug molecules for treatment of diseases which result from various abnormal regulation of genes, especially cancer, in which about 500 genes or gene products are not functioning normally. In addition to its effects on individual targets, the ability of curcumin to inhibit multiple targets (which accounts for much of its chemotherapeutic and chemopreventive 40 activity) makes it an intriguing lead for further research efforts for drug development and design.

The diverse bioactivity displayed by curcumin has already prompted various clinical trials which showed that curcumin has an appealing safety profile as it can be taken in a dose as high as 12 gm/day, suggesting that curcumin is a potential lead for the development of drugs for the treatment and prevention of several diseases. However curcumin could not be approved as a therapeutic agent because of its unfavorable pharmacokinetic profile. Studies have shown that curcumin has low systemic bioavailabilty due to poor absorption (based on low serum and tissue levels) and rapid metabolism into less active or inactive metabolites 61. Curcumin is rapidly conjugated to glucuronides and sulfates in the liver upon oral administration (Figure 2.9). In addition, curcumin is also reduced successively into dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin which are further glucuronidated. Traces of ferulic acid and dihydroferulic acid have also been reported in metabolic studies.

Figure 2.9. Metabolic pathways of curcumin. 41 Despite these drawbacks, scientific interest in the great potential of curcumin to prevent and treat different diseases, more specifically cancer has never stopped. Various approaches have been undertaken to understand the structural requirements for its activity, and to make analogues or derivatives that will improve its potency, selectivity, and bioavailability. Representative examples will be discussed below, but for more comprehensive reviews see Anand et al 64, and Adams et al 30 (a).

2.4. Previously Reported Structural Modifications of Curcumin

Curcumin is a bis-α,β-unsaturated β-diketone phenolic compound which exists predominantly in equilibrium with its enol tautomer both in the solid state (X-ray crystal structure) and in solution (observed via NMR) (Figure 2.10). This preference for the enol form has also been predicted by computational chemistry, which suggests that due to (1) the acidic nature of the protons on the central methylene carbon, (2) stabilization of the enol via an intramolecular hydrogen bond, and (3) the establishment of a fully conjugated system, the keto-enol tautomer is 6.7 kcal/mol lower in energy than the most stable diketone tautomer 62. In acidic and neutral aqueous solutions (pH 3-7) and in the cell membrane it also exists predominantly in the enol form, suggesting that this form would be preferred in vivo 63. Overall, this does not suggest that the diketone form which we typically invoke does not exist, but rather that the rapid equilibrium which takes place between the two tautomeric forms dramatically favors the enol tautomer in all reports. It is unclear what form curcumin exists in at basic pH, as it is unstable and rapidly degrades to ferulic acid, vanillin, feruloyl methane, and trans-6-(4’-hydroxy-3’-ethoxyphenyl)-2,4- dioxo-5-hexanal 24. Interestingly, for a compound containing an enol alcohol and several 42 oxygen substituents, curcumin displays solubility in acetone, dimethylsulfoxide, and ethanol, but is only very sparingly soluble in water. This is likely due to the intramolecular hydrogen bonding of the enol proton to the carbonyl. As in certain amino acids possessing similar intramolecular hydrogen bonding, aqeuous solubility is significantly diminished.

Figure 2.10. The tautomeric structures of curcumin.

Figure 2.11. Functional groups in curcumin.

The chemical structure of curcumin is a symmetric molecule characterized by 3 distinct structural motifs which include two methoxy phenolic units, two ,β -

43 unsaturated ketones (enones), and a 1,3- dicarbonyl unit (Figure 2.11). The olefin double bonds, while acknowledged to be important for most reported activities, are generally only considered to be a linker between the two key structural elements and have not been widely modified (other than simple hydrogenation, homologation, or truncation). Instead, synthetic efforts have primarily been directed at the most accessible structural features, variation of the aromatic rings and their substituents. It could be argued, however, that the impact of the central -diketone moiety on structure and perhaps biological activity is more significant than that of the aromatic substituents. Regardless, these 3 motifs have been the subject of various modifications, and derivatization 64. A list of the scope of these modifications for each subunit and examples of specific analogues which embody these changes are shown below.

2.4.1. Modifications of the methoxy phenolic units (Figure 2.12):

 demethylation of the methoxy group(s) to hydroxyl group(s)

 acylation, alkylation, glycosylation, and aminacylation of the hydroxyl

group(s)

 removal or exchange of hydroxyl and methoxy group(s)

 introduction of groups or atoms on aromatic rings

 replacement of aromatic ring by heteroaromatic rings or by multirings

Figure 2.12. Different modifications of the methoxy phenolic units of curcumin. Adapted from Biochemical Pharmacology 2008, 76, 1590.

Various reports have shown that:

 The phenolic hydroxyl groups are (1) Necessary for the antioxidant activity and

better antioxidant activity can be achieved in presence of more of these hydroxyl

groups. (2) Required for anti-inflammatory activity (especially inhibition of COX-

1); which is lost upon acylation and alkylation. (3) Desired for chemopreventive

45 activity to induce phase II detoxification enzymes. (4) Important to confer good

cytotoxic activity.

 The presence of ortho methoxy groups potentiates antioxidant and inflammatory

activity.

2.4.2. Modifications of the ,β - unsaturated ketone (Figure 2.13):

 Reduction of one or the two double bonds and/or carbonyl

 Restriction of conformation

 Extending conjugation (addition of one or more double bonds)

 Isolation of the 2 enones (introduction of central alkyl linkers)

 Deletion of double bonds

 Truncation into ferulic acid esters or amides

These modifications indicated that reduction of the double bonds was found to be deleterious to the antioxidant activity, decreases the anti-inflammatory activity due to suppression of NF-B, and diminishes chemotherapeutic activity.

Figure 2.13. Different modifications of the ,β-unsaturated ketone of curcumin. Adapted from Biochemical Pharmacology 2008, 76, 1590.

2.4.3. Modifications of the 1,3- dicarbonyl (Figure 2.14):

 Conformational restriction

 Heterocyclization (convert 1,3- diketone into cyclic structures) like pyrazole and

oxazole

 Central alkylated derivatives 47

Figure 2.14. Different modifications of the 1,3- dicarbonyl of curcumin. Adapted from Biochemical Pharmacology 2008, 76, 1590.

 Replace 7-carbon linked α,β-unsaturated β-diketone structure by 5-carbon α,β-

unsaturated monoketone.

48 These studies indicated that:

(1) Pyrazole derivatives provide better COX-1/COX-2 selectivity, and are more

antiangiogenic than curcumin.

(2) Analogues with five carbon linkers showed greater antioxidant activity.

Derivatives of these compounds containing a central cyclohexane and

cyclopentane derivatives were potent inhibitors of lipopolysaccharide (LPS)-

induced TNF- and IL-6 expression. They also confer potent anticarcinogenic

activity, as they retain the Michael accepting property required for induction of

phase II detoxifying enzymes such as glutathione transferase and quinone

reductase, and selective alkylation of thiols over amines and alcohols; which

provides protection against the tumorigenic effects of electrophiles and oxidative

stress 65. EF-24 30a was a superior example of these modified analogues as it

efficiently inhibited angiogenesis, arrested cell cycle, and induced apoptosis in

cancer cells.

2.5. Conclusion

A significant amount of work has been reported on the synthesis and biological evaluation of curcumin analogues. Unfortunately, however, these reports fail to provide a unified picture of the structure-activity relationships for curcumin against particular disease states or biological targets. Many of the reported studies focus on a very narrow group of compounds or report only “superficial” modifications of the curcumin scaffold.

A further concern is the fact that in most reports, analogues of curcumin are considered to possess similar biological activity as the parent compound (no matter how significant the 49 structural changes) and, therefore, operate via the same mechanism of action despite little evidence to support this assertion. These facts suggest that a more thorough analysis of the curcumin structure may be useful for future drug development.

Chapter 3

Design, Synthesis, and Screening of a Library of

Diverse Curcumin Analogues

We were initially drawn to the natural product curcumin based on the reports of its activity as an inhibitor of JAK2 and STAT3 59,60. Despite the significant limitations of curcumin with regard to stability, solubility, and bioavailability, we thought that this compound could serve as a promising lead compound for the development of novel inhibitors of the JAK/STAT pathway. As previously mentioned in Chapter 2, the relatively simple chemical structure of curcumin as compared to other complex natural products, along with its diverse bioactivity and desirable pharmacological activity, have made it an attractive lead compound for previous drug development research efforts. The relative ease of synthesis of curcumin analogues has allowed the rapid preparation of numerous analogues based on the curcumin scaffold. We believed that a similar approach, specifically the design and synthesis of a structurally diverse library of curcumin analogues, could be used to identify more potent JAK/STAT inhibitors which may overcome at least some of the limitations observed for curcumin itself.

The keys to this strategy would be the systematic modification of curcumin and a robust assay system to establish a reliable structure-activity-relationship (SAR) for these

51 analogues. Although numerous analogues had previously been reported which explored the SAR requirements of curcumin against various targets, consistent data (specifically with regard to cytotoxicity) was difficult to obtain from these highly varied literature sources. For example, a wide range of IC50 values have been reported even for curcumin itself in work published by different research groups despite using the same cancer cell lines (Table 3.1). This variability in IC50 values can be attributed to different conditions, differences in the length of time that the assay is carried out, or even to individual differences of the operator.

IC50 of Curcumin (in M)

PC-3 LNCaP MCF-7 MCF-10 Literature Source

>20 >20 Ishida et al. BMC 2002

7.7 3.8 Lin et al. J Med Chem 2006

19.8±2.1 19.6±3.7 30.1±3.7 Shi et al. Anticancer Agents in Medicinal Chemistry

17.5±1.76 55±3.53 Ramachandran et al. Breast Cancer Res Treat. 1999

Table 3.1. Reported IC50 of curcumin in different cell lines.

Therefore, in order to minimize these effects and establish an accurate baseline of activity, we planned to synthesize a highly comprehensive structural library which would be screened in one consistent assay system in the lab of Prof. Tom Li in the Division of

52 Medicinal Chemistry and Pharmacognosy here at OSU. In our case, the antiproliferative activities of all prepared compounds would be evaluated in terms of IC50 (half maximal inhibitory concentration) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds would initially be screened against various breast

(e.g., MCF-7 or MDA-MB-231) or prostate cancer (e.g., LnCAP, PC-3, or DU-145) lines. The use of multiple cell lines would provide a more accurate means of comparison of compounds and the ability to avoid cell-line specific effects (for example, the upregulation of a particular pathway in one cell type or hormone dependence). The whole cell assay system, therefore, would serve as the preliminary screen for all of the curcumin analogues. Promising or selected compounds would then be further screened in assays at the molecular level which are designed to establish JAK/STAT activity.

Unfortunately, one drawback of this strategy was that it necessitated the synthesis and evaluation of a number of known compounds in addition to the novel analogues in which we were particularly interested.

3.1. Design of the Curcumin Library

Although curcumin possesses no stereogenic centers and is C2 symmetric (plane of symmetry down the center of the molecule), the molecule is still fairly rich in diverse functionality. As mentioned in Chapter 2, analysis of the chemical structure shows three major regions of functionality (Figure 3.1): 1) the disubstituted aromatic rings; 2) the enone (or ,-unsaturated ketone) functionality; and 3) the 1,3-diketone (or -diketone) portion.

Figure 3.1. Functional groups of curcumin.

Rational modification of these groups may shed light on the role these moieties play in the interaction with or binding to a particular biological target. In addition to these groups, we also hoped to explore the role of the 7-membered “linker” region of the molecule connecting the two aromatic rings (both with regard to chain length and electronic properties) and to prepare non-symmetric analogues lacking the plane of symmetry.

This analysis led to the design of three broad classes of analogues: 1) analogues with varied substituents and substitution patterns on the aromatic rings; 2) analogues with varied linker regions; and 3) analogues locked into one specific tautomeric conformation.

Clearly the aromatic substitution is the most easily accessible modification possible for these analogues. As such, this was chosen as the first set of modifications. Many analogues prepared via subsequent modifications (classes 2 and 3) also contain variations in the aromatic substitution, but are classified according to what is considered to be the more “significant” modifications. Each class will be discussed below along with the results of the screening against various cancer cell types.

54 3.2. Analogues with Various Aromatic Substituents and Substitution Patterns.

In this series we initially investigated the requirement of the presence of phenolic hydroxyl group(s) for antiproliferative activity. In addition the number and position of these hydroxyl groups were also systematically modified. Finally the effect of protection of these hydroxyl groups with small groups was also examined. Similarly the presence, position, and number of methoxy groups were also varied.

Synthesis of these compounds followed a known precedent to make symmetrical curcumin analogues 66. In this procedure suitable benzaldehydes were condensed with acetylacetone (2,4-pentanedione) in the presence of boric oxide and tributylborate to afford the required analogues (Figure 3.2). Complexation of acetylacetone with boric acid in the first step allowed the reaction to take place only at its terminal methyl groups through generation of a cyclic boron complex which deactivates the central methylene unit. Aldol-type condensations then take place at only the terminal ends of the acetylacetone. For the most part, hydrolysis of the boron complex using AcOH in water resulted in precipitation of the desired compounds. These compounds were then typically recrystallized to provide pure materials.

Figure 3.2. Synthesis of curcumin and symmetrical analogues.

55 A number of commercially available benzaldehydes were employed in the reaction. This allowed the preparation of several symmetric curcumin-like compounds with variations at the 2-, 3-, and 4-positions of the aromatic rings (Figure 3.3).

Interestingly, we found that this procedure was not very suitable for the preparation of non-symmetric analogues. Even when equimolar amounts of two different benzaldehydes were used, a complex mixture of three products (two self-condensation products and the non-symmetric analogue) was formed. The ratio varied based on the nature of the substituents present on the rings, which ultimately affect relative reactivity. The chromatographic separation of these products was complicated further by the fact that curcumin and its derivatives tend to streak when placed on silica gel. Although pure fractions could be obtained in most cases, it was deemed to be too inefficient to pursue.

An alternative procedure was later developed (See page 90) to overcome this problem.

Regardless, the antiproliferative activities of all of the compounds prepared were evaluated using different cancer cell lines (Table 3.2). Details about the assay used are provided in Appendix A page 229.

Figure 3.3. Synthesized curcumin analogues.

IC50 in M

Compound PC-3 Ln-CAP MCF-7 MDA-231 DU-145

Curcumin 19.8 ± 2.1 19.6 ± 3.7 21.5 ± 4.7 25.6 ± 4.8 39.6±2.3

FLLL-3 27.3 ± 6.6 19.7 ± 2.9 24.3 21.9 ± 2 22.5

FLLL-4 40 ± 4.9 34.7 ± 6.6 25.9 ± 9.5 31.9 ± 11.1 15.5

FLLL-9 12.9 ± 2.3 20.2 ± 1.7 17.8 ± 5.9 12.3 ± 1 18.1

FLLL-10 5.9 ± 1.3 3.9 ± 0.6 5.4 ± 0.8 4.9 ± 0.9 7.7 ± 1.3

FLLL-17 37.2 ± 4.1 21.1 ± 4.3 37.6 ± 6.1 41.7 ± 1.2 >50

FLLL-18 40.7 ± 3.9 >40 45 ± 1.3 >40 >50

FLLL-24 >50 >50 >50 >40 >40

FLLL-25 43.6 ± 2.1 >40 14.9 ± 1.3 14.5 ± 1.9 31.8

Table 3.2. Proliferative activity of curcumin analogues in various cancer cell lines.

57 This data revealed that most of the analogues synthesized were less active than curcumin. In general, substitution at the 3- and 4-positions of the aromatic ring is necessary for activity as curcumin, FLLL-9, and FLLL-10 were the most active compounds. The lack of activity for FLLL-18, however, suggests that relative position of the free phenolic group (at the para position) is also important. Surprisingly, the trisubstituted derivative FLLL-17 also failed to show improved activity as compared to curcumin. This may demonstrate that the additional methoxy group is clashing with a potential protein target or even more likely that the electronics of the system are disrupted by introducing another electron donating ring onto the ring. This may explain in part why

FLLL-9 and FLLL-10 are the most active compounds in this series. Both of these compounds contain two donating groups, although the electron donating capacity is slightly diminished by the presence of the acetyl group in FLLL-9. In addition, the solubility and stability of both of these compounds are improved by “protecting” the free phenolic group. Although one would expect this to play a larger role in vivo as compared to in vitro, it still may play a role.

3.3. Analogues with 5 Carbon Linker Exploring the Michael Accepting Property

Synthesis of curcumin analogues with one carbonyl group instead of two

(switching from a 7-carbon linked α,β-unsaturated β-diketone to a 5-carbon linked α,β- unsaturated ketone) is a common approach which has resulted in the discovery of very potent cytotoxic analogues, specifically EF-24 30a. The anticancer activity of these analogues is sometimes considered to be due to the ability of these compounds to act as potent Michael acceptors that can form adducts with different biological targets. Dinkova 58 2001 et al 65c indicated that Michael reaction acceptors are potent inducers of Phase II detoxifying enzymes and radical scavengers which protect against the toxic and tumorigenic effects of electrophiles and free radicals. α,β-Unsaturated carbonyls (enones) are Michael acceptors that react preferentially with thiols (especially glutathione GSH) over alcohols and amines. It is suggested that curcumin forms a covalent adduct with

GSH leading to its depletion, thus resulting in elevation of Phase II enzymes 67.

We were interested in comparing this class of compounds directly to curcumin analogues containing the longer 7-carbon linker. Specifically we were interested in comparing substituent effects between the two classes to see if the same effects observed for the longer variants applied to the shorter 5-carbon analogues. In order to do this a series of “monocarbonyl” derivatives with similar variations at the 2-, 3-, and 4-positions of the aromatic rings was synthesized through a basic condensation reaction of benzaldehydes with acetone (Figure 3.4). In reactions of benzaldehydes lacking phenolic substituents, sodium hydroxide was employed as the base. When phenolic oxygen was present, however, -alanine was used as the base for the reaction, which typically also required heating to 50 °C.

Figure 3.4. Synthesis of monoketone curcumin analogues.

The antiproliferative activity of the synthesized compounds was determined using

4 cancer cell lines and the data obtained are summarized in (Table 3.3). As can be observed from (Figure 3.5), a direct comparison between similarly substituted compounds containing 5- and 7-carbon linkers shows that in all cases the monocarbonyl derivatives display significantly increased antiproliferative activity against both the prostate and breast cancer cell lines. In all cases, the observed potency is 10-20 fold greater than for curcumin itself.

Figure 3.5. Comparison of antiproliferative activity of curcumin analogues.

In the case of these analogues, substitution pattern appears to play less of a role in activity. Likewise, the presence and position of free phenolic hydroxyl groups has almost no effect on the relative activity. This is somewhat surprising as previous studies had postulated that the position of these substituents was critical for Michael activity through directing and electronic effects. For example, it was shown that the presence of ortho- hydroxyl or methoxy groups on aromatic rings of cinnamoyl moieties have led to an

61 increase in electron density at the double bond leading to dramatic enhancement of the molecule free radical scavenging ability 65b (Figure 3.6). This may be due to the ability of the oxygen atom to direct the thiol group of nucleophiles like GSH to the -position of the enone. Furthermore, Dimmock 68 showed that the positions of the substituents on the aryl rings of similar monocarbonyl compounds affect the torsional angles of the aryl ring with respect to the enone system. As the substituents on the ring begin to clash sterically with the protons on the double bond, the ring is forced to twist out of the plane to minimize this interaction. This twist means that the aryl ring is no longer able to maintain conjugation with the enone system. In these bulkier systems the enone is now isolated from the aromatic ring and, therefore, more reactive toward nucleophiles. It is not clear if these compounds exert their activity through a Michael accepting mechanism or a mechanism similar to the longer chain compounds.

Compound Substituents IC50 (µM) in various cancer cell lines

R3 R4 R5 PC-3 Ln-CAP MCF-7 MDA- MB-231 Curcumin OMe OH H 19.9 ± 19.6 ± 21.5 ± 25.6 ± 4.8 2.1 3.7 4.7 FLLL-11 OMe OH H 3.9 ± 1.1 2.7 ± 0.4 2.4 ± 0.4 2.8 ± 1

FLLL-12 OMe OMe OH 3.6 ± 1.3 2.5 ± 0.3 1.7 ± 0.3 2.7 ± 1.4

FLLL-13 OMe OMe H 2.5 ± 0.5 2.1 ± 0.9 2.7 ± 0.5 1.5 ± 0.1

FLLL-14 OMe OMe H 2.9 ± 0.6 2.2 ± 0.5 2.5 ± 0.4 1.6 ± 0.4

FLLL-15 OMe OCH2OMe H 2.7 ± 0.9 2.8 ± 0.9 2.0 ± 0.4 1.7 ± 0.5

FLLL-16 H OH H 9.5 ± 0.9 5.8 ± 0.9 6.9 ± 2.1 3.9 ± 0.6

FLLL-22 See Above 2.1 ± 1.1 0.5 ± 0.1 0.4 ± 0.1 0.6 ± 0.1

FLLL-23 See Above 4.6 ± 0.2 1.7 ± 0.6 2.4 ± 1.0 2.4 ± 0.4

Table 3.3. Antiproliferative activity of monoketone curcumin analogues.

Working on the hypothesis that the enone linker is critical to the activity of these compounds for Michael accepting capacity, we were interested in exploring the effect of modulation of electron density directly on the double bonds. We reasoned that by placing an additional electron withdrawing group on the double bond ( to the carbonyl), we would make the -position more electron deficient and, therefore, increase the reactivity of the double bond towards nucleophilic attack. Likewise, substitution of an electron donating substituent on the double bond should make the system less reactive to nucleophiles. 63

Figure 3.6. Effect of different substituents on Michael accepting property.

A series of compounds exploring the effect of different substituents on electrophilicity of the double bonds were designed and synthesized. The electron withdrawing groups included fluorine, methyl carboxylate, and nitrile while methyl substitution and cyclohexyl derivatives were used as mildly electron donating groups.

The syntheses of these compounds involved various types of condensation reactions

(Figure 3.4 and 3.7). In addition some previously reported monoketone curcumin

64 analogues were synthesized for the sake of effective comparison and to allow for more accurate interpretation of results.

Figure 3.7. Synthesis of monoketone analogues containing a nitrile group.

Figure 3.8. Synthesized monoketone analogues bearing electronically different groups.

The data obtained for these compounds (Figure 3.8) seems to go against our initial hypothesis. The most active compound is FLLL-29, which contains the piperidone ring

(an electron donating group). Interestingly, compounds containing electron withdrawing groups showed significantly lower antiproliferative activity than those which had no additional EWG’s. To reinforce this point, a comparison of compounds FLLL-35

66 (monofluoro) and FLLL-26 (difluoro) show that the compound containing only one electron withdrawing group was significantly more active than the compound with two.

During the course of this work we recognized the strong structural resemblance of the monocarbonyl compounds, especially those containing the nitrile group (FLLL-44 and FLLL-46), to AG490 a known selective inhibitor of JAK2 (Figure 3.9). AG490 was initially developed as a tyrphostin (a class of benzylidenemalononitrile protein tyrosine kinase inhibitor 69 which as a class are reported to show anticancer activity against various tumors and low toxicity to normal cells). AG490 has been shown to inhibit growth of a range of tumor cell lines and increase their sensitivity to apoptotic stimuli through selective inhibiton of JAK2 70-72. Its low potency [particularly in in vivo studies

(activity requires high concentrations in animal studies ~50 to 100 M)], however, has limited its therapeutic applicability. Obviously, the presence of a catechol moiety represents a major drawback in drug development due to its associated metabolism and toxicity (forming adducts and inducing strand breaks in DNA, uncoupling and lipid peroxidation of membranes, and deactivation of enzymes and proteins) 73. Several studies have been reported in an attempt to develop more potent and stable AG490 analogues.

These have resulted in the discovery of a family of related compounds (e.g., WP1066), which possess a bromopyridine moiety rather than the catechol 74-77.

Structurally, AG490 possesses the catechol moiety on one side and a benzylamide on the other. Obviously the sizes of AG490 and the monocarbonyl derivatives are very similar. What was more striking to us, however, was the “replacement” of one styrene unit with the benzyl amide. Instead of possessing two sp2 carbons in a double bond,

AG490 has one sp2 hybridized nitrogen and an sp3 hybridized carbon next to the 67 aromatic ring. Therefore, this compound would be expected to be fairly planar and likely adopt conformations similar to the monocarbonyl compounds. This led us to question whether the curcumin monocarbonyl analogues could be effective JAK2 inhibitors and whether or not we could learn more about targeting JAK2 by studying the structure activity relationship of AG490.

Figure 3.9. Strucures of curcumin and related compounds.

68 Molecular docking of AG490 and WP1066 in JAK2 was executed to determine how they bind to the protein (Figure 3.10). It was observed that these compounds bind in same conformation within the ATP binding pocket with the catechol ring and the bromopyridine ring both binding to the purine binding site in the hinge link region. The nitrile group is oriented into the oxyanion hole, providing the other major bonding interaction. Although difficult to see the nature of the interaction, it also appears that the

(S)-methyl substituent of WP1066 must provide additional hydrophobic interactions.

Similarly substituted derivatives of AG490 containing a methyl substituent at the benzylic carbon also showed increased activity, suggesting the importance of this group.

The benzyl aromatic ring of these compounds does not extend all the way into the substrate pocket and does not appear to play a key role in binding.

WP1066

Figure 3.10. Docking of AG490 and WP1066 in ATP binding site of JAK2.

69 We were particularly interested in exploring the relative activity of different substitutions patterns on the aromatic ring which interacts at the purine site. Our preliminary hypothesis was that introduction of substitution patterns observed to be active in the curcumin series may improve the antiproliferative activity of the AG490 compounds or conversely that introduction of functionality found in AG490 could improve the activity of the monocarbonyl compounds. The significance of the (S)-methyl group, and the effect of deeper extension into the substrate pocket of JAK2 were also investigated.

3.4. Analogues with Different Aromatic Substituents on the Bromopyridine or

Catechol Ring.

In work carried out by Michael Corcoran in the Fuchs lab, a number of AG490 derivatives were synthesized which contained curcumin-like functionality on the cinnamoyl aromatic ring. These compounds were synthesized according to the basic scheme shown in (Figure 3.11). The introduction of various substitution patterns onto the

AG490 scaffold failed to improve activity. Two of these compounds (FLLL-119 and

FLLL-118) are included in Figure 3.12 and Table 3.4 for reference.

Figure 3.11. General procedure for synthesis of AG490 analogues.

Figure 3.12. Examples of AG490 analogues.

IC50 (M) Compound HT-29 DU 145 MDA-MB-231 WP1066 3.0±0.8 7.5±1.3 5.7±0.9 FLLL-102 > 50 > 50 > 50 FLLL-103 > 50 > 50 > 50 FLLL-104 8.2±1.2 8.0±1.3 21.5±1.7 FLLL-118 26.9±1.0 27.0±1.9 > 50 FLLL-119 1.7±0.2 2.7±0.8 4.1±0.7

Table 3.4. Antiproliferative activity of representative AG490 analogues.

71 We repeatedly observed during the course of these studies that the bromopyridine containing compounds (e.g., WP1066, FLLL-104, and FLLL-119) were the only compounds which showed reasonable activity in the whole-cell assay. The molecular modeling suggested that the pyridine nitrogen may be interacting with the JAK2 protein through hydrogen bonding. In addition, the bromine atom may be forming a “halogen bond” thorough interaction with a carbonyl oxygen on the protein backbone. With this in mind, we decided to explore the effect of the individual components of the bromopyridine ring. This would be accomplished in part by synthesizing a series of pyridine compounds with variation in the position of the nitrogen atom in the pyridine ring. A series of halogenated benzene derivatives were also prepared. In this case, the bromine atom was moved around the ring and then replaced at the 3-position of the ring with other halogens (fluorine, chlorine, and iodine).

The 2-pyridine (FLLL-115) and 3-bromo compound (FLLL-113) are not quite as active as WP1066, but still show good potency. Based on the relative activities, the bromide actually appears to play a bigger role in the activity of these compounds than the pyridine nitrogen. In WP1066, these affects may be additive. Movement of the nitrogen atom around the pyridine ring in FLLL-164 and FLLL-165 results in a significant decrease in activity, suggesting that the directionality of the hydrogen bond acceptor is critical to activity. The same principle applies to the movement of the bromine atom around the benzene ring in FLLL-161 and FLLL-167. The replacement of the bromine atom, however, with other halogen atoms shows an interesting trend. Although the activity of these compounds varies from cell line to cell line, in the MDA-MB-231 line, it appears that the IC50 value decreases (indicating increased potency) as you move down 72 the periodic table from fluorine (FLLL-162) to chlorine (FLLL-163) to iodine (FLLL-

168). These results suggest that the halides do play a key role in the activity of these compounds, although further experimentation using a JAK kinase assay is necessary to confirm that observed cytotoxicity correlates to the relative JAK activity.

Figure 3.13 Analogues of WP1066.

IC50 (M) Compound HT-29 DU 145 MDA-MB-231 FLLL-113 6.5±0.7 11.6±2.4 22.9±1.8 FLLL-114 30.0±1.4 > 50 > 50 FLLL-115 17.3±1.0 23.4±2.1 16.6±2.5 FLLL-116 32.2±1.2 > 50 24.4±2.1 FLLL-161 24.5 28.4 9.2±1.2 FLLL-162 45.4 > 50 18.0±3.2 FLLL-163 24.7 31.6 6.9±1.6 FLLL-164 46.7 > 50 > 50 FLLL-165 42.5 > 50 20.6±2.5 FLLL-167 22.3 26.1 5.6±0.8 FLLL-168 21.4 33.7 4.3±0.4 Table 3.5. Antiproliferative activity of representative WP1066 analogues.

73 3.5. Analogues with hybrid motifs.

Based on the results discussed in the previous section, our initial research in this area involved synthesis of analogues of monocarbonyl curcumin analogues which have a bromopyridine or pyridine ring instead of the typical aromatic substitution patterns (i.e.,

3,4-dimethoxy aryl groups). In addition an amide was introduced on one side of the molecule rather than the ,β–unsaturated carbonyl.

Figure 3.14. Scheme for synthesis of hybrid curcumin and WP1066 analogues.

As with the previous compounds synthesized containing EWGs on the enone system (e.g. FLLL-44 and FLLL-46), these molecules (FLLL-55 and FLLL-56) failed to retain the potency observed for their unsubstituted counterparts and were much less active than WP1066 (IC50 = ~ 3 – 8 M).

74 IC50 (M) Compound HT-29 DU 145 MDA-MB-231 FLLL-44 > 50 > 50 > 50 FLLL-46 8.9±1.1 29.6±3.6 30.8±1.2 FLLL-55 32.5±1.4 > 50 > 50 FLLL-56 18.0±1.3 37.4±2.1 > 50

Table 3.6. Antiproliferative activity of hybrid compounds.

3.6. Analogues with Larger Substituents on the Benzylamide Portion of WP1066.

In addition to our interest in the bromopyridine portion of WP1066, we were also interested in exploring the benzylamine moiety of the molecule. Based on the computational modeling, we expected that extension of the benzyl ring deeper in the pocket or the addition of substituents on that ring would push these substituents into the substrate pocket of the protein. This may increase binding affinity and help to increase specificity.

The first set of compounds that were synthesized simply employed commercially available amines containing methyl substituents at the ortho (FLLL-132), meta (FLLL-

138), and para (FLLL-144) positions of the ring. We thought that a comparison of the relative activities of these compounds may give us an idea of what position to build off of in order to extend that chain further into the pocket. Based on the results with these compounds, we believed that building off of the ortho and meta positions had a higher probability of success than the para position. However, based on the availability of starting materials, we prepared compounds FLLL-170 (meta substituted) and FLLL-171

(para substituted) by alkylation of the phenol group of the benzonitrile (Figure 3.15).

Figure 3.15. Scheme for synthesis of benzyl protected phenol amines.

The nitrile was then reduced to prepare the amine which could be used in the transamidation reaction to prepare the desired products. Surprisingly, it was the para substituted compound (FLLL-171) which showed better activity in the HT-29 and MDA-

231 cell lines. We also prepared homologated analogues of WP1066 by adding additional methylene units into the benzylic chain. Compound FLLL-169, which contained two additional CH2 units, retained some degree of activity, but this was significantly diminished with respect to WP1066. The compound containing only a single additional

CH2 unit was not tested.

Figure 3.16. Strucures of synthesized WP1066 analogues.

IC50 (M) Compound HT-29 DU 145 MDA-MB-231 FLLL-132 7.2±1.0 8.9±1.2 4.9±0.8 FLLL-138 8.3±0.9 10.4±0.8 7.2±0.9 FLLL-144 14.3±1.2 9.9±0.8 33.6±2.0 FLLL-169 20.8 > 50 8.8±0.3 FLLL-170 > 50 > 50 > 50 FLLL-171 24.5 > 50 6.4±0.4

Table 3.7. Antiproliferative activity of WP1066 analogues.

3.7. Analogues Locked in a Defined Tautomeric Form

Figure 3.17. Tautomeric structures of curcumin.

The existence of the two tautomeric forms of curcumin in equilibrium (the di-keto and keto-enol forms) attracted our attention to the possible structural conformations that curcumin can adopt when binding to different biological targets (Figure 3.17). Literature consultation gave us some insights concerning these possible modes of binding. In docking studies by Sikora et al 78, it was suggested that curcumin inhibits the endonuclease activity of DNA Fragmentation Factor/Caspase Activated DNase

(DFF/CAD) by blocking the active site to prevent DNA fragmentation by binding in a bent diketone tautomeric conformation. In this model, the two aryl rings of curcumin are suggested to bind in the active site adjacent to the His260 residue (Figure 3.18).

Figure 3.18. Binding mode of curcumin to DFF/CAD.

Another computational investigation by Jerala and coworkers 79 of the binding affinity of curcumin to the myeloid differentiation protein 2 (MD-2), which is the LPS component of the endotoxin surface receptor complex MD-2/TLR4, showed that curcumin competes with LPS for its binding site in its extended and planar enol form (Figure 3.19). Other docking studies, including those done on HIV integrase indicate that both tautomeric forms may bind with nearly equal energies.

Figure 3.19. Binding of curcumin to MD-2.

These studies prompted an initial molecular docking study to examine the binding modes of curcumin to both the JAK2 and STAT3 proteins. This work was carried out by

Katryna Cisek in Dr. Chenglong Li’s research group. Both of the tautomeric forms of curcumin were used in this study, and interestingly they showed nearly identical binding energy in the JAK2 phosphotyrosine binding pocket. In the case of the diketo conformation the key binding interactions were blocking the interaction of the purine ring of ATP (in the pocket on the left of the figure) with one of the aryl rings and binding of the other aryl group to a hydrophobic pocket on the other side. One of the oxygen atoms of the carbonyl groups extends “down” in the figure to interact with the oxyanion hole in the center. On the other hand the keto-enol conformer extends in a linear fashion across the ATP binding site, with the second aryl ring extending deeper in the hinge link region.

80 In this case, there is no visible interaction of the carbonyls with the protein. There are also no residues in the active site which are predicted to interact with the olefins.

Binding energy = -9.5 Kcal/mol Binding energy = -9.4 Kcal/mol

Figure 3.20. Binding of Curcumin to JAK2.

In the case of STAT3, the enol tautomer failed to bind effectively with the protein.

The di-keto form, however, can bind in its bent conformation to the SH2 phosphorylation domain with a binding energy of -8.1Kcal/mol (Figure 5.20). An analysis of the STAT3

SH2 domain led to the identification of three key binding sites where interactions can take place: the pTyr705 site (which is similar to the phosphotyrosine pocket of JAK2), the Leu706 site, and a hydrophobic side pocket. This indicated that careful structural design of curcumin analogues could be carried out to optimize their binding affinity with these three pockets, ultimately resulting in increased potency and selectivity.

STAT3

Figure 3.21. Binding modes of curcumin in JAK2 and STAT3.

These differences in binding modes directed our attention to the possibilities of locking curcumin into each tautomeric form and exploring how this will affect activity and selectivity, not only for JAK and STAT, but potentially other targets as well.

3.7.1. Analogues Locking Curcumin into the Di-keto Conformation:

In the case of curcumin, tautomerization depends on the availability of the acidic hydrogen atoms in the center of the dicarbonyl. Replacement of these hydrogen atoms by alkyl groups was suggested as a means to eleminate these tautomerizations and effectively “lock” the compound in the diketone tautomeric form. The size and shape of these alkyl groups was expected to dramatically affect the overall size and conformation of the molecule. For example, in addition to the need of these groups to still effectively fit into the STAT3 SH2 binding site, different groups were predicted to have a profound

82 effect on the relative angle between the carbonyl groups of curcumin (Chem3D Pro). As seen in (Table 3.8), the angle between the carbonyls can be varied by approximately 10° based on the nature of the substituents (cyclobutyl: 105.5° vs. cyclopropyl: 115.6°).

Regardless, however, the introduction of dialkyl substituents at the central methylene was expected to best enforce the conformation of curcumin seen in Figure 3.21.

Table 3.8. Effect of different groups on the angle between the carbonyl groups of curcumin.

With this basic hypothesis in mind, another molecular modeling study was executed to determine how different alkyl substituents will affect binding of the curcumin-like compounds to STAT3. This study investigated the effect of size of alkyl

83 substituents on the efficiency of binding. It was predicted that substitution will generally improve the binding to JAK2 and STAT3 (with the exception of dibutyl). The most significant improvement was seen with the spiro-derivatives and was directly correlated to the number of carbon atoms in the ring. It was indicated that the best binding affinity to JAK2 and STAT3 at the molecular level was observed with the cyclopentyl and cyclohexyl derivatives where the alkyl rings interact favorably with other pockets in addition to the original ones (Figure 3.22) (Table 3.9).

Binding Energy (Kcal/mol)

Compound / (central R groups) JAK2 STAT3

Curcumin (di-keto) -9.4 -8.1

Curcumin (keto-enol) -9.5 No binding

Dimethyl -9.6 -8.1

Dibutyl No binding No binding

Cyclopropyl -9.5 -7.6

Cyclobutyl -9.7 -7.7

Cyclopentyl -10.3 -8.0

Cyclohexyl -10.3 -8.5

Table 3.9. Effect of different groups on binding to JAK2 and STAT3.

84 Interestingly, a comparison of the binding models of curcumin with the cyclohexyl derivative (named FLLL-32) show significant differences in the binding modes. With regard to the JAK2 protein binding, the cyclohexyl ring of FLLL-32 now binds to the hydrophobic pocket of the kinase, while the second aryl ring is now extended forward in the figure. Although this represents a significant conformational change, the left aryl ring and carbonyl maintain the same basic interactions with the protein as observed with curcumin. In the docking with STAT3, however, there is an even more significant change in binding mode for FLLL-32. In this case, the entire molecule is

“flipped over” in the binding pocket. This flip results in the placement of the cyclohexyl ring in the hydrophobic pocket at the bottom right of the figure. It also results in the second aryl ring being projected towards the Leu706 residue. With this change, the molecule is now able to take advantage of binding to all three key “hot spots” observed for STAT3, as opposed to only two for curcumin. This difference may at least in part explain the improved predicted binding energy for STAT3.

Figure 3.22. Effect of presence of cyclohexyl ring on binding to JAK2 and STAT3.

The synthesis of these derivatives initially utilized dimethoxycurcumin (FLLL-

10) as the starting material. This compound could be alkylated with iodoalkanes in the presence of potassium carbonate to synthesize the desired compounds, including the dimethyl and dibutyl compounds as well as the cyclopentyl and cyclohexyl compounds which utilized diiodobutane and diiopentane, respectively (Figure 3.23). In most cases, the desired products are accompanied by a small amount of the undesired O-alkylation

86 products. These products are separable by column chromatography, although the separations can be challenging at times. Fortunately, the products are readily distinguished on TLC plates using a UV lamp. This is due to the fact that the O-alkylated products are more highly conjugated and therefore are primarily visible under long wave

UV light, while the desired products are visible under short wave UV light.

Bromoalkanes failed to give the desired products cleanly, presumably due to decreased reactivity of the electrophile.

Significance of phenolic hydroxyl groups was another aspect which was carefully considered in computational modeling. It was suggested that the free phenols were important for hydrogen bonding interactions in both JAK2 and STAT3. Nevertheless methyl protection of one or more of these phenols was desirable to increase in vivo stability and bioavailability. In addition investigation of the significance of the symmetry of the molecule for activity of the molecule (since relative orientation of the molecule will not play a role in the interaction) was endeavored. This implicated the need of preparing both symmetric and non symmetrical analogues containing free hydroxyl groups in order to examine their effect on binding and activity.

The introduction of these phenolic hydroxyl groups onto these derivatives was somewhat more challenging than the synthesis of the 3,4-dimethoxy derivatives. We first approached this problem by preparing the symmetrically substituted bisphenols. Initially curcumin was protected with a Boc (t-butyloxydicarbonyl) group following the procedure of Chen and coworkers 80. This product was then alkylated with the desired alkylating reagents to provide the protected derivatives. The Boc groups could then be thermolytically deprotected at 160 °C without significant decomposition of the starting 87 materials in contrast to what was observed when using standard acidic conditions.

Eventually the use of the Boc groups gave way to MOM (methoxymethyl) protecting groups which were introduced according to the precedent of Jogireddy et al 81 to afford the bis-protected curcumin. This change was primarily due to poor solubility observed in some cases when using the Boc groups. Unlike the Boc groups, the MOM groups could be easily removed by hydrolysis under mild acidic conditions to give the desired products in good yield. It is also worth mentioning that silyl ether protecting groups (both TBS and

TIPS) were not robust enough to survive the alkylation conditions. During the course of the reaction with potassium carbonate and 1,5-diiodopentane, the TIPS group was cleaved from the phenol. This resulted exclusively in formation of the alkylated aryl ether in addition to alkylation at the central methylene carbon.

Figure 3.23. Synthesis of symmetrical alkylated derivatives.

88 With a successful protecting group strategy established, the synthesis of the monophenolic derivatives was next explored. Since the boric oxide mediated condensation failed to provide an efficient method for the synthesis of non-symmetric derivatives, a new method was developed to prepare these compounds (Figure 3.24). We believed that an alternative bond forming strategy could be the acylation of a methyl ketone in order to form the central 1,3-diketone moiety. Thus, the synthesis would require the preparation of both halves of the molecule separately, with one partner being the requisite methyl ketone and the other being an acid chloride derived from the corresponding ester. Therefore, a benzaldehyde derivative (whether commercially available or prepared) was subjected to Wittig olefination using the commercially available stabilized ylide to give a methyl ketone. Sodium hexamethyldisilazide

(NaHMDS) was used as base to generate the enolate from the methyl ketone. This was directly reacted with the 3,4-dimethoxy cinnamyl chloride (or other synthetically derived acid chloride). Optimization of the conditions of this reaction resulted in yields up to

75%. Alkylation and subsequent deprotection of the MOM protecting group afforded the desired products when appropriate. This has proven to be a highly efficient method for the production of non-symmetric curcumin analogues and is highly tolerant to variations in aryl substituents (provided no acidic protons are present).

Figure 3.24. Synthesis of non-symmetrical alkylated derivatives.

Figure 3.25. Examples of alkylated derivatives.

Many of the compounds synthesized using these methods have shown impressive activity against cancer cells and specifically against JAK2 and STAT3 (Figure 3.10). The specific JAK2 and STAT3 data, however, will not be discussed here as that forms the 91 basis for future thesis research projects by Mr. Jonathan Etter (biological assays) and Mr.

Eric Schwartz (synthetic chemistry). The introduction of small alkyl and cycloalkyl groups at the central methylene unit of curcumin increases the potency of these compounds against cancer cells by approximately 10-20 times compared to curcumin.

The most promising compound to date, FLLL-32 which contains the cyclohexyl ring shows IC50 values in the range of 0.5-3 M against a variety of cancer cells. The dimethyl (FLLL-31) and cyclopentyl (FLLL-45) variants also show significant anticancer activity. Contrary to the computational model, the introduction of free phenolic groups on the aryl rings does not significantly improve the activity of the compounds (FLLL-77).

More interestingly, however, removal of the methoxy substituents completely from one ring (FLLL-86 and FLLL-87) does not seem to affect the potency. Removal of a single double bond from these compounds, on the other hand, results in a dramatic loss of anticancer activity. There is still much to be explored with this series of compounds and this work is actively being pursued in the Fuchs lab.

Compound DU 145 IC50(M) FLLL-31 2.7±1.2 FLLL-32 1.3±0.2 FLLL-45 2.5±0.1 FLLL-49 4.6±0.8 FLLL-63 12.2±1.2 FLLL-64 15.3±1.9 FLLL-77 4.7±0.3 FLLL-78 3.1±0.01 FLLL-85 33.1±1.26

FLLL-86 2.1±0.5

FLLL-87 ± 3.1 0.6 FLLL-88 19.8±0.8

Table 3.10. Antiproliferative activity of alkylated derivatives.

3.7.2. Analogues Locking Curcumin into the Keto-enol Conformation

Having established methods for locking curcumin into the diketone tautomer, we also sought methods for enforcing the keto-enol tautomer. Our approach took advantage of cyclic derivatives of the 1,3-diketone moiety. Therefore, we considered making rigid heteroaromatic derivatives which would include heteroatoms to mimic the potential hydrogen bonding and electronic effects observed with the curcumin carbonyls.

The natural choice was making rigid five-membered pyrazole and isoxazole derivatives of curcumin and dimethoxy curcumin by essentially “connecting” the carbonyl oxygens (or in this case “heteroatoms”) into a ring. The pyrazole and isoxazole rings essentially contain an imine which mimics the ketone carbonyl and an enol ether

(isoxazole) or enamine (pyrazole) moiety which mimics the enol portion of the curcumin keto-enol structure. These derivatives were synthesized following a procedure reported by Mishra et al 38b using the previously prepared curcumin and curcumin analogues

93 which were reacted with either hydrazine hydrate, methylhydrazine, or hydroxylamine in glacial acetic acid and heated to 85˚C until the reaction was complete, to yield the pyrazole and isoxazole derivatives respectively (Figure 3.26).

Figure 3.26. Synthesis of pyrazole and isoxazole derivatives.

Figure 3.27. Synthesized pyrazole and isoxazole derivatives.

94 This concept was further extended to rigid analogues containing six-membered rings, namely pyrimidine, benzene, and pyridine rings. The aim was to prepare additional fully conjugated and completely planar structures which have different electronic properties.

(1) Pyrimidine containing derivatives: The pyrimidine ring contains two nitrogen atoms as hydrogen bond acceptors which provide two sites for hydrogen bonding interactions and presumably can lead to better binding and hence improved activity.

These compounds were synthesized using a reported procedure by Eynde et al 82 for synthesis of extended π-systems from methyl-diazines and aromatic aldehydes. Either 4- methoxy benzaldehyde or 3,4- dimethoxy benzaldehyde was refluxed with 2,6-dimethyl pyrimidine in aqueous NaOH solution in the presence of the phase transfer catalyst aliquat (a quaternary ammonium salt) to give the required products. The products could ultimately be recrystalized from ethanol.

Figure 3.28. Synthesis of pyrimidine derivatives.

95 (2) Benzene containing derivatives: The benzene derivatives are isosteric with the pyrimidines, but lack any hydrogen bond donors and/or acceptors and are very non-polar.

Therefore, these compounds instead possess a high probability for hydrophobic interactions. The Horner-Emmons strategy utilized for synthesis of these compounds was initially developed by Byeon et al 83 to prepare bis-styrylpyridine and bis-styrylbenzene derivatives as inhibitors for fibril formation to treat Alzheimer’s disease. Our compounds were prepared by using 3,4-dimethoxy benzaldehyde or 3-methoxy-4-hydroxy benzaldehyde (vanillin) after protecting the phenolic hydroxyl group with a MOM group

(Figure 3.29). The benzene unit was derived from dibromoxylene which was reacted with triethyl phosphate in an Arbuzov reaction to yield a stabilized alkyl phosphonate (Figure

3.29). The Horner-Emmons reaction then followed between the aldehyde and the phosphonate ester to produce the E-alkene predominantly. The MOM group could then subsequently be removed by acid hydrolysis in methanol (Figure 3.30).

Figure 3.29. MOM protection of vanillin and synthesis of alkyl phosphonate.

Figure 3.30. Synthesis of benzene containing derivatives.

(3) Pyridine containing analogues: The pyridine compounds contain a nitrogen atom as a hydrogen bond acceptor but the heteroatom is located in between the two side chains (at the position of the central methylene of curcumin), rather than at the positions of the carbonyl oxygens. This variation may provide a new site for hydrogen bonding, affects the electronics of the aromatic ring and reduces the lipophilicity of the central ring compared to the benzene derivatives. The procedure for their synthesis involved dibromination of 2,6-pyridine dimethanol using phosphorous tribromide in carbon tetrachloride. This product was alkylated with triethyl phosphate to give the stabilized phosphonate ester (Figure 3.31) which produced predominantly the E-product upon reacting with the corresponding benzaldehydes (Figure 3.32).

Figure 3.31. Synthesis of pyridine phosphonate.

Figure 3.32. Synthesis of pyridine containing derivatives.

Figure 3.33. Synthesized rigid curcumin analogues.

98 The compounds containing rigidfications of the central 1,3-diketone moiety were screened against DU-145 cancer cells (Table 3.11). The data indicates that the presence of two heteroatoms (i.e. the pyrazoles and pyrimidines) are necessary to mimic the positions of the carbonyl oxygens in curcumin. This may be due to electronic effects, but it is more likely that some hydrogen bonding is taking place with a protein target. The pyrazole compounds containing a free NH show significantly more activity than the corresponding isoxazoles (FLLL-67 and -69) or methylated pyrazole (FLLL-8). This difference could be due to the ability to act as a hydrogen bond donor. It may also be the result of the ability of the non-substituted pyrazoles to undergo a tautomerization, which makes the compound symmetric, allowing it to effectively bind with a target protein from either side. The compounds lacking the heteroatoms (i.e., the benzene derivatives) showed no activity in the assay.

Compound DU-145 IC50(M) FLLL-7 6.4±1.1 FLLL-8 17.8±3.1 FLLL-58 > 50 FLLL-59 16.1±3.4 FLLL-60 > 50 FLLL-67 29.6 FLLL-68 8.2±1.4 FLLL-69 > 50 FLLL-70 28.8±4.1 FLLL-98 >50 FLLL-99 >50

Table 3.11. Antiproliferative activity of rigid curcumin analogues.

99 3.8. Screening of the Curcumin Library Against Malaria

With a relatively comprehensive and structurally diverse library of curcumin analogues in hand, we felt that screening of this library may present an opportunity for the identification of novel leads for the treatment of diseases other than cancer. To that end, Dr. Mark Drew was kind enough to offer to screen these compounds against the malaria parasite Plasmodium falciparum using a Biomek. Therefore, a collection of 108 unique curcumin-like compounds was made up for testing. The structures of the compounds tested are provided in the Appendix. The actual screening and determination of IC50 values for the active compounds was carried out by Ms. Emily Cason in the Drew lab. Initially all compounds were screened at concentrations of 0.1 and 10 M to determine which compounds showed the best inhibition of parasite growth. The data from this assay is shown in Figure 3.34.

Figure 3.34. Percent inhibition of growth at 10 µM (blue) and 100 nM (red) First bars are circumin, last bars are chloroquine (both at 50 nM).

100 The actual assay for all of the compounds will be run again to verify the results, but the preliminary data obtained to date is very interesting. The most active compounds were determined to be compounds 12, 30, 63, and 64 with IC50 values of 1.2, 1.1, 1.2, and

0.5 M, respectively. A second set of assays to determine the IC50 values of the compounds in (Table 3.12) show a slight, but uniform increase in potency for all of the active compounds (data not shown). Compounds 12, 63, and 64 are all “monocarbonyl” compounds containing 3,4-dimethoxy substitution patterns on the aromatic rings. All three of these compounds are also very active in the cancer cell assays. Clearly, the enones are important for antimalarial activity, as compound 65 (which possesses only one double bond and an amide rather than a ketone) shows no activity. The only difference between compound 63 and compound 64 is the presence of the piperidone nitrogen.

Clearly, this nitrogen has an effect on the potency of this compound, as 64 shows a two- fold increase in activity. Also, increased substitution on the aryl rings results in a decrease in activity, as seen in compound 67, which also “benefits” from the presence of the piperidone nitrogen. The most unexpected result is the activity observed for compound 30. The 3,4-dimethoxy substituted pyrimidine is one of the most active compounds in the assay. Surprisingly, however, the pyrimidine which has only a 4- methoxy substituent on the aromatic rings is considered inactive in the assay. The pyrazoles and isoxazoles, which showed better activity in the cancer assays than the pyrimidine, were also considered inactive. Ultimately, further optimization of these leads could be carried out to increase the potency of the compounds against malaria. Efforts to explore the mechanism of action through which these compounds exert their effects have been discussed with Dr. Drew. These results, however, indicate that diverse curcumin 101 structures do in fact specifically affect biological targets and validate the generation of this library for drug discovery purposes.

Figure 3.35. Structures of representative curcumin analogues screened for antimalarial activity.

Compound # IC50 (µM) 95% confidence interval 1 (curcumin) 5.522 5.265-5.791 12 1.202 1.117-1.293 30 1.055 1.007-1.105 64 0.4863 0.4126-0.5730 63 1.189 1.050-1.346 65 No effect 67 3.976 3.685-4.290

Table 3.12. Antimalarial activity of curcumin analogues.

102 3.9. Conclusions

A systematic analysis of the curcumin scaffold led to the development and synthesis of a structurally diverse library of compounds. These compounds were initially screened for anticancer activity and led to the development of a number of hypotheses regarding the structure activity relationships of curcumin derivatives. Although the primary impetus for the generation of this library was the identification of novel inhibitors of the JAK/STAT pathway and optimization of compounds for this activity, the focus of the project expanded to cover a more thorough exploration of the role of functional groups in the activity of curcumin. Many of these compounds show promising activity in cancer and these specific structural subgroups will continue to be probed through structural modification, with efforts directed at improving their pharmacological properties in addition to simply affecting potency or selectivity. Although not specifically discussed in this chapter, preliminary biological data suggests that many of the curcumin analogues shown to possess anticancer activity do not show significant JAK/STAT activity. Moreover, many of those do show only JAK activity, while not affecting STAT dimerization (i.e., the inhibition is upstream of the dimerization event – either JAK inhibition or the STAT phosphorylation event). The screening necessary to determine which compounds are JAK/STAT inhibitors is still underway and will be reported in due course. Ultimately, however, this endeavor did lead to the successful generation of a library of compounds which was used for screening and the successful identification of lead compounds against malaria. This library could also be potentially screened against a number of other targets for which no leads are known or for which compounds containing cinnamoyl moieties are known to interact. The models through which we explain the 103 activity of these compounds and the mechanisms through which they inhibit diseases will continue to be refined and studied.

104

Chapter 4

Leishmaniasis: A Neglected Tropical Disease

4.1. Introduction

Leishmaniasis is a group of disorders caused by infection with various parasitic species of the genus Leishmania (protozoa) 84. It is a vector born disease transmitted by the bites of female infected sand flies (phlebotomines) during blood suction. These flies are very small in size (2-3 mm) and are typically most active at dusk. Distribution of sand flies differ by continent: Phlebotomus species are spread over Asia, Africa, and Europe, and Luzomyia species are prevalent in the Americas. Leishmania species are obligate parasites which exist as extracellular, spindle shaped, flagellate promastigotes in the fly gut where they multiply and settle. Promastigotes are the infective stage which enters the skin of the mammalian host (including humans, dogs, and rodents) through the insect bite during the blood meal (Figure 4.1). Inflammatory reactions lead to phagocytosis of promastigotes into the reticuloendothelial cells (macrophages, monocytes, and

Langerhans) where they transform into intracellular, rounded, non-flagellate amastigotes.

These actively multiply in macrophages of different tissues leading to the clinical manifestations. Upon subsequent blood meals they are ingested into the sand fly

105 gastrointestinal tract where they transform back into promastigotes in the hind and mid gut, then migrate to the upper gut to be ready for another infection (Figure 4.1).

Figure 4.1. Life cycle of Leishmania parasites. Adapted from http://upload.wikimedia.org/wikipedia/commons/thumb/e/e0/Leishmaniasis_life_cycle_diagram_en.svg/20 00px

4.2. Clinical Manifestations

Clinical manifestations of the disease depend on the geographical location (Table

4.1 and Figure 4.2), immune response of the host, and the infecting species. Three forms

of the disease are observed.

(1) Cutaneous Leishmaniasis (CL) Chiclero ulcer:

106 Different species including L. tropica, L. major, L. aethiopica, and L. mexicana are responsible for CL. It is characterised by flat, and painless ulcerous skin lesions on exposed areas of the body. In Africa, Asia, and Europe the ulcers usually heal spontaneously (if untreated) leaving scars and disfiguration, but healing is rare in the

Americas.

(2) Mucocutaneous Leishmaniasis (ML) Espundia:

ML is widespread in South America. It is caused by L. braziliensis, L. panamensis, and L. guyanesis. Nodular lesions on the mucous membranes (nasopharyngeal, pharyngeal, and laryngeal), arms and legs are obvious characteristics of this type which can exacerbate to severe disfiguration.

(3) Visceral Leishmaniais (VL) Kala-azar:

VL is the most severe form of the disease which is is usually lethal if not treated.

It is caused by L. donovani, L. infantum, and L. chagasi. It leads to infection of the lymph nodes, spleen, liver, and bone marrow, which is manifested by abdominal pain, loss of appetite, diarrhea, weakness, and febrile episodes. This can be further complicated by severe anemia, leukopenia, and thrombocytopenia.

107 Clinical Manifestation Parasite Region of distribution

Cutaneous Leishmaniasis L. tropica Mediterranean countries, Afghanistan (oriental sore, or tropical sore, uta ulcer or chiclero ulcer or Aleppo L. major Middle East, Western and Northern boil) Africa, Kenya L. aethiopica Ethiopia

L. Mexicana Central America, Amazon regions

Mucocutaneous Leishmaniasis L. braziliensis Brazil, Peru, Ecuador, Columbia, (espundia) Venezuela Visceral Leishmaniais L. donovani China, India, Iran, Sudan, Kenya, (kala-azar or dumdum fever) Ethiopia L. infantum Mediterranean countries

L. chagasi Brazil, Columbia, Venezuela, Argentina

Table 4.1. Various leishmanial species, their regional distribution, and clinical forms.

108

no data less than 10 10-20 20-30 30-40 40-50 50-60 60-80 80-100 100-120 120-150 150-200 more than 200 (DALY) rates from Leishmaniasis by country (per 100,000 inhabitants)

Figure 4.2. Worldwide geographical distribution of various forms of leishmaniasis. Adapted from http://commons.wikimedia.org/wiki/File:Leishmaniasis_world_map_-_DALY_- _WHO2004.svg

4.3. Epidemiology and Health Burden

According to WHO 85, leishmaniasis has been identified as a global neglected health problem. It is prevalent in 88 countries; 72 of which are developing. Most of the affected countries are in the tropical or subtropical regions (from rain forests in Central and South America to deserts in West Asia). 12 million people are infected worldwide, 2 million new cases are reported annually, and 350 million people are considered at risk.

Leishmaniasis represents a severe public health problem in East Africa, the Indian subcontinent, and Latin America. It is usually associated with low economic standards

(poverty), malnutrition, poor housing, illiteracy, weakness of the immune system, and lack of resources.

109 4.4. Conventional Therapeutics:

4.4.1 Currently Available Drugs:

Pentavalent antimonials have been the principal treatment for all types of leishmaniasis for more than seven decades. Widespread resistance, low patient compliance, and associated toxicity led to the introduction of newer drugs. Most of these drugs, however, still have limitations to their use including side effects and resistance.

These shortcomings necessitate further drug discovery efforts to combat the problem 86,87.

Antimonials

They are the first-line drugs for the treatment of leishmaniasis worldwide 88.

Pentavalent antimonials (for example Glucantime and Pentostam) are administered by intramuscular or intravenous injections at doses of 20 mg/kg daily for 20-30 days. A complex mode of action on multiple targets is reported for antimonials. This mechanism includes interference with the parasite bioenergetic pathways and inhibition of trypanothione reductase 89,90. Severe cardiac, hepatic, pancreatic, and renal side effects, in addition to rapid emergence of resistance are considered the main limitations for using antimonials.

110

Amphotericin B

Amphotericin B is considered another drug of choice especially in regions where antimonial resistance is prevalent 86-88. It is typically administered as an intravenous infusion at 7-20 mg/kg for up to 20 days. It is a macrolide polyene which binds to the parasite ergosterol leading to the formation of pores which increase membrane permeability to monovalent cations and small metabolites, ultimately resulting in cell lysis 91,92. Renal toxicity is the major side effect which is reduced in the liposomal formulation of Amphotericin B (2-5 mg/kg daily oral). Its high cost, however, limits its wide application in developing countries.

Pentamidine

Aromatic diamidines were initially used as second-line drugs for treatment of VL.

Due to the rapid decrease in response rate, probably due to emergence of resistance, use of pentamidine is currently restricted to CL in India 87. It is proposed to bind to the DNA minor groove thus inhibiting Topoisomerase II 93. Parenteral administration and side

111 effects including hypotension, hypoglycemia, and renal impairment limit the use of pentamidines.

Milfetosine

Hexadecylphosphocholine was the first orally available drug (100 mg daily) introduced for the treatment of leishmaniasis 86-88. It was originally developed as an antitumor agent and was reported to induce apoptotic-like cell death in L. donovani 94.

Drug resistance has been established in the laboratory, indicating the possibility of rapid emergence of resistance, especially if applied clinically as a single agent.

4.4.2. Drugs in Clinical Trials:

Paramomycin

Paramomycin is an aminoglycoside antibiotic which was developed for treatment of intestinal protozoa. It is used as an ointment for topical treatment of CL, and passed

112 phase III clinical trials for VL 86-88. It is suggested that it inhibits protein synthesis through binding to the ribosome in the parasites in the same way as in bacteria.

Azoles

Antifungal azole derivatives were used in clinical trials for CL and VL 86-88. They are potent inhibitors of ergosterol synthesis through inhibition of 14--demethylase (a target in antifungal treatment). Different azoles show different activities against various forms of Leishmania. Nevertheless, their efficacies are subject to significant controversy due to the inconsistency of the results. This may be partially due to the relatively small number of patients who have participated in the clinical trials. Different reports, however, indicate that the parasite can circumvent the inhibition of 14--demethylase through altered sterol biosynthesis 95.

Sitamaquine

113 Sitamaquine is an orally active 8-aminoquinoline analogue which was initially developed as an antimalarial. It showed activity in in vitro studies against VL, but initially failed to show the same efficacy in clinical trials in Brazil 96. A few years later sitamaquine was used in another study to assess its efficacy and safety in patients with

VL in India. They reported an 80-87% cure rate and reasonable toleration with some side effects 97. A mechanistic study concluded that sitamaquine diffuses into the parasite along an electrical gradient and concentrates in the cytoplasm by an energy and sterol

98 independent process .

4.5. The Need for New Leads:

From the previous discussion it can be concluded that there is a great demand for discovery of new drug leads to circumvent the limitations of the currently available therapeutics which include: toxicity, need for long term of adminstration, poor patient compliance, high cost of effective drugs in endemic countries, and emergence of resistance. Thus, there is a considerable necessity to discover safer, cheaper, broader spectrum agents to prevent the spread of CL, and to provide alternative treatments for

VL. Another important goal is to enable better management of the disease by the development of drugs that can be topically applied following the sand fly bite to inhibit the pathology and spread of the disease.

The search for drug entities has been assisted by the great advances in bioinformatic tools and chemical techniques which have extended the understanding of the biology, biochemical pathways, and genome of parasite. Common drug development

114 approaches for leishmaniasis include screening of natural products, high throughput screening of compound libraries, and rational drug design.

4.5.1. Screening of Natural Products

Natural products represent a valuable resource for new drug leads to treat lesihmaniasis. Although currently available drugs do not include anything derived from natural products, researchers have never stopped exploring this valuable resource which provides diverse chemical structures and potentially unique mechanisms of action.

Numerous publications demonstrated in vitro and/or in vivo antileishmanial activity of plant extracts and several chemical entities. In addition, several reviews have detailed these research efforts 87,99-103. Some of the most promising candidates will be discussed in this section in alphabetical order of the different classes of NPs.

a) Alkaloids:

An enormous number of plant alkaloids were reported to have good to excellent antileishmanial activity, however none of these compounds has reached therapeutic application or clinical trials 87,99-103. Active alkaloids comprise a large number of structurally different classes of heterocycles including indoles, isoquinolines, and quinolines. Representative examples are presented (Figure 4.3, Table 4.2) based on promising IC50 values (in the low micromolar and nanomolar range). IC50 values are reported in either micrograms/ml or in micromolar 99,100.

115

Figure 4.3. Structures of selected antileishmanial alkaloids.

Class Name of Compound Source IC50 Organism

Indole Corynantheine(s) Bark of Corynanthe pachyceras ~3 M L. major alkaloids (Rubiaceae)

Buchtienne Stem bark and leaf of Kopsia 1.56 g/ml L. donovani

griffithii (Apocyanaceae)

Tryptanthrin (4-aza-8-Cl) Strobilanthese cusia (Japanese 0.13 M L. donovani

herbal remedy)

Isoquinoline Berberine Several plant species of 10 g/ml L. major alkaloids Annonaceae, Berberifaceae

Cephaeline Stem bark of Psychotria klugii 0.03 g/ml L. donovani

(Rubiaceae)

Cocsoline Albertisia papuana 12 M L. donovani

Ancistrogriffine A Leaves and twigs of Ancistrocladus 3.1 g/ml L. donovani

griffithii (Ancistrcladaceae)

Table 4.2. Different classes of antileishmanial alkaloids, their sources, and IC50.

116 Other classes of alkaloids which showed variable activities include: 1) quinoline alkaloids from the Bolivian plant Gadipea longiflora with IC90 25 – 50 g/ml, 2) steroidal alkaloids from the leaves of Holarrhena curtisii show activity range between

0.3 g/ml to 6.25 g/ml, 3) diterpene alkaloids from plant species of the genera

Aconitum, Delphinium, and Consolida showed leishmanicidal activity against L. infantum promastigotes, 4) Canthin-6-one(s) from the bark of Zanthoxylum chilperone showed in vivo leishmanicidal activity against L.amazonensis.

Figure 4.4. Structures of selected antileishmanial chalcones, flavonoids, and lignans.

b) Chalcones

Roots of Chinese Liquorice plant contain Licochalcone A (Figure 4.4) which inhibited the growth of L. major amastigotes at 0.5 g/ml. In addition 2’,6’-dihydroxy-4- methoxychalcone, isolated from Piper aduncum (Piperaceae), displayed potent activity

87,99- against L. amazonensis promastigotes IC50 0.5 g/ml, and amastigotes IC50 24 g/ml

103.

117 c) Flavonoids

Luteolin (Figure 4.4) was reported as a potential lead for the development of antileishmanial agents 87,99-103. It showed in vitro activity against L. donovani promastigotes with an IC50 of 12.5 M. In addition, it decreased 80% of the burden of splenic parasite. This activity was attributed to induction of morphological changes leading to a loss of cellular integrity, promoting DNA linearization and inhibition of

Topoisomerase II. The glucopyranoside analogue displayed enhanced activity against L. donovani amastigotes at an IC50 of 1.1 g/ml.

d) Lignans

Moderate antileishmanial activity against L. infantum promastigotes was displayed by diphyllin (Figure 4.4); a lignan isolated from Haplophyllum bucharicum

87,99-103 with an IC50 of 14 M .

Figure 4.5. Structures of selected antileishmanial quinones.

118 e) Quinones (e.g. diospyrin, plumbagin, pendulone)

Diospyrin (Figure 4.5) is a naphthoquinone isolated from the bark of Diospyros

87,99-103 montana . It inhibited L. donovani promastigotes with an IC50 of 1.9 M.

Naphthoquinones may disrupt the mitochondrial electron transport chain due to their structural similarity with reduced coenzyme Q (ubiquinone),which is a part of the mitochondrial electron transport system.

Plumbagin (Figure 4.5) is another naphthoquinone isolated from Plumbago

87,99-103 species . It displayed a potent antileishmanial activity with an IC50 of 0.21 M against L. donovani promastigotes in vitro. In addition its administration at a dose of 5 or

2.5 mg/kg/day after infection with L. amazonensis and L. venezuelensis in BALB/c mice delayed the manifestations of the disease.

Pendulone (Figure 4.5) is an isoflavanquinone isolated from the Myanmar timber species Millettia pendula, which displayed effective antileishmanial activity with an IC50 of 0.066 g/mL against L. major promastigotes 87,99-103.

119

Figure 4.6. Structures of antileishmanial terpenoids.

f) Terpenoids

Terpenes are compounds containing a hydrocarbon skeleton formed from isoprene units. They comprise different classes which include monoterpenes, diterpenes, triterpenes, saponins, sesquiterpenes. Various active terpenoids are illustrated in Figure

4.6.

120  Monoterpenes

Linalool is an alcohol component of several essential oils. It displays an extremely potent antileishmanial activity against L. amazonensis promastigotes and amastigotes with LD50 4.3 ng/mL and 16 ng/mL respectively; without cytotoxic effects on mouse peritoneal macrophages or Vero mammalian cells. Essential oils extracted from the leaves of Croton cajucara (Euphorbiaceae) are rich in linalool 87,99-103.

 Iridoids (cyclopentan [c] monoterpene glycosides)

Amarogentin is a secoiridoid glycoside isolated from the Indian plant Swertia chirata (Gentianaceae). It inhibits Topoisomerase I from L. donovani in a dose-dependent manner 87,99-103.

Brunneogaleatoside is another iridoid isolated from Phlomis brunneogaleata

(Lamiaceae). It displayed good activity against L. donovani axenic amastigotes (IC50

4.7g/ml) with no toxicity to L6 cells at up to 90 g/ml 87,99-103.

 Sesquiterpenes

Parthenolide is a sesquiterpene lactone isolated from Tanacetum parthenium. It showed significant activity against L. amazonensis promastigotes (IC50 0.4 g/ml) and amastigotes (IC50 0.8 g/ml). In addition it was not cytotoxic to J774 macrophages and did not cause lysis of sheep erythrocytes. Appearance of large-lysosome-like structures in the cytoplasm and intense exocytic activity in the region of the flagellar pocket were

121 correlated to protein production by cells in attempt to survive. In addition cysteine protease activity was increased after treatment with parthenolide 87,99-103.

 Diterpenoids

Cryptotanshinone and 1-oximiltirone were isolated from Perovskia abrotanoides

(Lamiaceae), an Iranian medicinal herb. Both of them displayed IC50 value of 18 M against L. major promastigotes. They were also moderately toxic against proliferating phytohaemagglutinin A-stimulated human lymhocytes with IC50 values of 37 and 45 M respectively 87,99-103.

 Triterpenes

Simalikalactone D is isolated from the root bark of Simaba orinocensis

(Simaroubaceae). It exhibited antileishmanial activity against L. donovani promastigotes with an IC50 35 ng/ml and was much more potent than pentamidine and amphotericin

IC50 1.6 and 1.1 g/ml respectively. It showed good selectivity, with an IC50 2.3 g/ml towards Vero cells 87,99-103.

 Saponins

-Hederin, β-hederin and hederacolchiside A1 are three saponins extracted from ivy Hedera helix. They exhibit potent antileishmanial activity by interfering with parasitic membrane integrity and potential. -Hederin and β-hederin exhibited effective activity against intracellular amastigotes, with IC50 0.35 and 0.25 g/ml respectively. In

87,99-103 addition hederacolchiside A1 showed better activity with IC50 0.05 g/ml) . 122 Yuccasaponin MC3 is a steroidal saponin isolated from Yucca filamentosa

(agavaceae) which inhibited the growth of L. mexicana amazonensis promastigotes. In addition leishamnicidal activity was detected after only 1 hour of treatment with 10

g/ml. Cytometry experiments suggested that this activity can be attributed to interfering with parasitic membrane potential 87,99-103.

g) Sterols

Martinez et al 104 reported the isolation of epidioxysterols (Figure 4.7) from the

Colombian marine sponge Ircinia campana which showed antileishmanial activity.

Hexane extracts from the fruits of Cassia fistula showed significant antileishmanial activity against L. chagasi promastigotes (causing VL) 105. Bio-activity guided fractionation resulted in the isolation of a sterol, clerosterol (Figure 4.7), which was further analysed in different models. Clerosterol exhibited an (IC50) of 10.03 g/ml against promastigotes and 18.1 g/ml against intracellular amastigotes. In addition it was

3.6-fold less toxic than the standard drug pentamidine and had no antifungal activity.

Figure 4.7. Structures of natural antileishmanial sterols.

123 4.5.2. High Throughput Screening of Compound Libraries

Siqueira-Neto et al 106 developed an automated, high throughput, in vitro, fluorometric assay for antileishmanial candidates. A library of 26,500 structurally diverse chemical compounds with drug-like properties was screened against promastigotes of L. major. A minimum of 70% growth inhibition of the parasite identified 567 active compounds at 10 M. Only 124 compounds were then selected for further testing based on their cytotoxicity and selectivity on a human macrophage cell line. Serial dilutions of these compounds were tested against L. major amastigotes infecting THP-1 differentiated macrophages. Only 2 compounds (Figure 4.8) passed this filter with EC50 values lower than 10 M against L. major. Further characterization indicated that these 2 compounds are good candidates for drug development.

Figure 4.8. Structures of antileishmainial candidates from HTS.

124 4.5.3. Synthetically Developed Analogues

Medicinal chemistry has also taken a role in the development of antileishmanial therapies. Several classes were developed based on available therapeutics, drugs used for other conditions, and natural products. Representative examples will be discussed based on our interest in their chemical structure or therapeutic relevance, introduced in alphabetical order.

a. Azasterols

Goad and coworkers 107 were the first to investigate the effect of the antifungal

(20-piperidin-2-yl-5-pregnane-3β,20-diol) azasterol (AZA) (Figure 4.9) on leishmanial sterol biosynthesis and cell proliferation in 1995. They reported that AZA inhibited the growth of L. donovani promastigotes and affected sterol biosynthesis. 24-Alkylated sterols were replaced by 24-cholesta-type sterols which then accumulated in the cells.

Restoration of the normal 24-alkylsterols was retrieved upon removal of AZA, which indicated that 24-alkylsterols are not essential for parasite growth.

These results initiated a series of studies which further explored the effect of structural modifications and derivatizations on the antileishmanial activity. In addition explicit investigation of the mechanism of action of these sterol derivatives was also done. Magaraci et al 108(a) concluded that the presence of the free 3β-OH is essential for activity. They also concluded that the location and basicity of the nitrogen is closely correlated to the activity. They confirmed that inhibition of 24-sterol methyl transferase

(SMT) does not correlate to antileishmanial activity. Further elaboration of this work

108(b) proved that acetylation of the 3β-OH group does not affect antileishmanial activity

(likely to be hydrolytically removed by the organism), but diminishes 24-SMT inhibition. 125 Synthesis of azasterol analogues lacking the piperidine ring and with a different location of the basic nitrogen resulted in a loss of enzyme inhibition even though inhibition of parasite growth was retained 109. The effect of these compounds on ultrastrucural organelles, specifically the mitochondria, was also studied. Disruption of mitochondrial membrane potential due to changes in the inner mitochondrial membrane probably resulting from lipid composition alteration was reported as another possible mechanism of action 110.

Figure 4.9. Structures of antileishmanial synthetic azasterols.

126 Subsequent synthesis of structurally diverse azasterol analogues with the aim of establishing SAR and to derive the pharmacophore requirements of these compounds was also reported 111. These results, however, were not very conclusive and did not lead to further pharmaceutical development.

Figure 4.10. Pharmacophore required for antileishmanial activity of azasterols. Adapted from Bioorg. Med. Chem. 2009, 17(16), 5950.

b. Sterol metabolism inhibitors

ER-119884 and E5700 are two quinuclidine derivatives (Figure 4.11) which showed antileishmanial activity against intracellular amastigotes of L. amazonensis. ER-119884 displayed a very promising IC50 value of 0.5 nM, with significant selectivity against murine peritoneal macrophages at 500 nM concentrations. In addition they showed potent inhibition of sterol biosynthesis in promastigotes of L. amazonensis.

127 A synthetic series of aryloxy cyclohexyl imidazoles (Figure 4.11) were reported as inhibitors of leishmanial 14-demethylase (an important enzyme in sterol biosynthesis).

A promising analogue exhibited a potent antileishmanial activity both in vitro with IC50

1.2 µg/mL (3.6 µM) against L. donovani amasigotes and and in vivo showed 78% inhibition of parasitemia in infected hamsters 112.

Figure 4.11. Structures of synthetic antileishmanial candidates.

128

Chapter 5

Discovery and Synthesis of Sterol-Based Antileishmanial Agents

5.1. Introduction

Our own interest in the development of novel antileishmanial agents stems from an opportunity presented to us from Professors A. Douglas Kinghorn (OSU College of

Pharmacy) and Abhay Satoskar (OSU College of Medicine). Dr. Kinghorn and Dr.

Satoskar had recently initiated a program of study exploring various plants for antileishmanial activity. Screening of plant materials known to possess antileishmanial activity and isolation of the bioactive constituents by the Kinghorn lab was the first step in the approach utilized in this study. Efficient structural elucidation would then be followed up with synthetic efforts in the Fuchs lab to prepare the isolated leads on a larger scale. The synthesis of these compounds was expected to facilitate in vivo studies and an investigation of potential mechanisms of action. In addition, structural modifications would also be carried out to probe the structure-activity relationships for these leads and possibly assist in optimization of their drug properties. All compounds

(both natural and synthetic) obtained in these studies would be screened for antileishmanial activity in the Satoskar lab under the supervision of Dr. Claudio Lezama-

Davila. This work was anticipated to pave the way for further drug development based 129 upon the lead compound scaffold and could ultimately guide a rational target-based drug design approach.

5.2. Collection and Screening of Plant Material:

This project was initiated in Mexico, specifically in the Yucatan Peninsula where

CL is endemic and the availability of drugs in the rain forest is very limited. Twenty regional plants were collected from a riparian forest near the city of Hopelchen in

Campeche, a state of the Yucatan Peninsula (Figure 5.1), and screened for antileishmanial activity.

MEXICO

Hopelchen

Figure 5.1. Map showing the location of Hopelchen in Campeche, Mexico. (adapted from http://commons.wikimedia.org/wiki/File:El_Pinacate_y_Gran_Desierto_de_Altar.png)

130 The most promising of these plants was Pentalinon andreuxii. Preliminary assays of the aqueous and organic extracts of the roots of this plant showed strong in vitro antileishmanial activity. Perhaps this is not surprising, since these roots have traditionally been used by Mayan healers for the treatment of CL, snakebites, nervous disturbances, and to alleviate headaches. For topical treatment of CL, the ulcers would be cleaned carefully with an infusion of the roots and the inner parts of the roots were then fixed tightly to the lesion. This procedure would be repeated daily until the ulcer was completely healed 113.

Figure 5.2. The air-dried roots of P. andrieuxii. (Photo: courtesy of Y. Deng)

Further biological testing of this plant material revealed that the nonpolar

(hexane) Pentalinon andrieuxii root extract (PARE) exhibited broad spectrum leishmanicidal activity in vitro against L. mexicana, L. donovani, and L. major. The

131 hexane extract was the most effective in killing L. mexicana promastigotes cultured in vitro (10 μg/mL/106 promastigotes), which was more potent than the control compound, meglumine antimoniate (GlucantimeTM) 114. In addition it was not toxic to mammalian cells, and suppressed the production of anti-inflammatory cytokines. In vivo experiments using 10 weeks old male C57BL/6 mice, which were infected with L. mexicana promastigotes in the ear dermis, were also done. 10 g of PARE dissolved in 50 l of

DMSO/PBS was applied on the infected ear daily for 21 days. Sections of infected ears treated and untreated with PARE were stained and examined. PARE inhibited the growth of L. mexicana promastigotes in the ear dermis of treated sections which showed inflammatory cells including lymphocytes and macrophages containing few or no parasites. On the other hand sections from infected ears from the control mice contained macrophages full of parasites (red arrows – Figure 5.3).

Figure 5.3. The effect of topical application of PARE in L. mexicana infection.

132 5.3. Isolation and Characterization of Active Constituents

These very promising results led to an increased interest to determine the bioactive compounds responsible for this activity. Activity-guided fractionations using standard analytical techniques led to the isolation of 20 molecules from the n-hexane partition of the methanol extract of the roots of Pentalinon andrieuxii. Structure elucidation of the compounds was accomplished by interpretation of spectroscopic data.

These included a new 4-3-ketosterol [new cholesterol derivative, pentalinonsterol (1) –

Figure 5.4], a new polyoxygenated pregnane sterol glycoside, pentalinonoside (2), fourteen known sterols (3-16), three known coumarins (7, 18, 19), and a known triterpene

20.

133

Figure 5.4. Structures of compounds isolated from PARE.

134 5.4. Antileishmanial Activity of Isolated Compounds

The antileishmanial activity of the isolated compounds was evaluated in vitro

(Table 5.1). Among these compounds, 6,7-dihydroneridienone (15), was found to be the most active compound with an IC50 value 9.20 M against promastigotes of L. mexicana.

Pentalinonsterol (cholest-4,20,24-trien-3-one, 1), together with 24-methylcholest-

4,24(28)-dien-3-one (3) and neridienone (16), also exhibited significant antileishmanial activity in this same bioassay (IC50 30.00, 23.98, and 26.22 M respectively).

Compound L. mexicana b c promastigote amastigote cholestera-4,20,24-trien-3-one (1) 30.00 3.31 24-methylcholesta-4,24(28)-dien-3-one (3) 23.98 3.46 cholest-4-en-3-one (4) 80.99 0.03 cholest-5,20,24-trien-3-ol (7) > 262 14.48 6,7-dihydroneridienone (15) 9.20 1.41 neridienone (16) 26.22 3.53 pentostamd 346.1 2.66

Table 5.1. In Vitro Antileishmanial Activities of Compounds Isolated from the Roots of P. andrieuxii.a a Compounds 2, 5, 6, 8-14, and 17-20, with IC50 > 100 g/mL, were considered inactive. b Results are expressed as IC50 values (M), calculated by linear regression analysis from the KC values at the concentrations used (1, 10, 50 and 100 g/mL) at 48 h culture. c IC50 for L. mexicana in bone marrow-derived macrophages from C57Bl/6 mice (48 h experiment run in triplicate), IF RATE: average number of amastigotes/macrophage = 7 µg/106 parasites. dUsed as a positive control substance.

A flow cytometric assay was used to evaluate the antileishmanial activity of the isolated molecules against L. mexicana promastigotes. This assay depends on using propidium iodide, a dye which binds with high affinity to nucleic acids of DNA resulting

135 in fluorescence. This dye only penetrates compromised plasma membranes and, therefore, has no effect on cells with intact membranes. The assay involved staining drug- treated or control parasites with propidium iodide. The quantity of dead parasites was then determined by flow cytometry.

Figure 5.5. Chemical structures of the two most active compounds.

PAD2F1-3-1K (1) and PAD2F1-3-3K (3) (Figure 5.5) were identified as potent

6 6 antileishmanial agents with IC50 = 11.4 g/ml/10 parasites and IC50 = 9.5 g/ml/10

TM parasites, respectively, which were significantly better than Glucantime IC50 = 170

g/ml/106 parasites. Analysis of the developed histogram showed that PAD2F1-3-1K caused death of 70% of the parasites (green curve Figure 5.6), and PAD2F1-3-3K killed

58% of the parasites (blue curve Figure 5.6) which displayed higher uptake of propidium iodide and hence intensified fluorescence as compared to parasites treated with

Pentostam (black curve), DMSO (red curve), and control (purple curve). Unfortunately,

136 however, these compounds were isolated in only 1.2 and 4 mg quantities respectively from 900 grams of plant material.

Figure 5.6. Flow cytometry analysis of the antileishmanial activity of PAD2F1-3-1K, and PAD2F1-3-3K.

In addition compounds 1, 3, 4, 7, 15, and 16 showed potent antileishmanial activity against amastigotes of L. mexicana, and 4 was the most potent agent with an IC50 value of 0.03 M (Table 5.1). The cytotoxicity of these compounds was also evaluated in non-infected bone marrow-derived macrophages from C57B1/6 mice, but all compounds were inactive in this cell line. Further examination of the amastigotes treated with compounds 1, 3, 4, 15, and 16 by electron microscopy revealed morphological abnormalities and destruction of the amastigotes (red arrows Figure 5.7) as a result of the treatment with these compounds.

137

Figure 5.7. Electron microscopy of L. mexicana amastigotes.

The above data meant that these compounds have great potential for drug development efforts. Literature precedents also provided greater evidence for the potential of sterols as antileishmanial agents. Several synthetic azasterols were reported to inhibit the growth of intracellular and extracellular parasites, and some of them also displayed inhibition of sterol biosynthesis in different Leishmania species (previously discussed in chapter 4). The mechanism of action of these compounds however was not clear.

5.5. Synthesis of Natural Products

Based on the previous discussion, synthesis of the isolated natural products was determined to be necessary in order to: 1) confirm the assigned structural and

138 stereochemical assignments for the lead compounds; 2) produce sufficient quantities for in vivo and in vitro studies which are necessary for further drug development; 3) synthesize derivatives to establish the SAR and determine the pharmacophore required for activity; and 4) improve pharmacokinetic and pharmacological profiles for further therapeutic application.

An efficient synthetic (or possibly a semi-synthetic) route would need to be developed in order accomplish these goals within a reasonable time frame and to make the synthesis of structurally related compounds feasible. We initially focused our attention on the potential synthesis of two of the most active natural products, PAD2F1-

3-3K and PAD2F1-3-1K. Both of these compounds contain the same basic sterol ring

Figure 5.8. Ring identification and numbering in sterols and the structures of lead compounds from PARE.

139 system, composed of an ABCD ring system of 3 fused six-membered rings (the ABC system) and a fused five membered ring comprising the D ring (Figure 5.8). The stereochemistry of the lead compounds are the same as that found in cholesterol, although a few minor structural variations exist. Both of the lead compounds contain oxidized A ring functionality; namely, a ketone at C3 (rather than an alcohol) and conjugation of the double bond with the ketone at the C4-C5 positions (rather than the C5-C6 double bond found in cholesterol). The only other differences exist in the substitution found at the C17 side chain. In PAD2F1-3-1K, the compound contains a double bond at the C20-C21 position and another double bond at the C24-C25 position. PAD2F-3-3K (similar to cholesterol), on the other hand, contains a methyl group at C20, rather than a methylene unit. It also contains a methylene unit extending from C24 on the side chain. From a synthetic point of view, the side chain functionality (C22-C27, including various substitution and saturation patterns) could optimally be interchanged through a well planned synthetic strategy. The C20 methyl found in PAD2F1-3-3K was considered to be the most challenging structural element in a potential synthesis of this molecule. The presence of a saturated or unsaturated unit at C20, therefore, provided a major delineation between classes of compounds, as most of the other products isolated from the PARE contained one of these two functional groups. It was envisioned that introduction of these alkyl chains using a simple alkylation process would provide rapidly the two natural products and would enable synthesis of library of analogues which can help in the study of SAR (Figure 5.9). This could be done through generation of the enolate form of the pregnenolone (commercially available and inexpensive) using a suitable base which can then be reacted with the desirable alkylating reagent. In fact most sterol derivatives 140 containing C-21 functionalization (i.e., cholesterol type) have usually been used as starting materials which were further functionalized through degradation type reactions, specifically ozonolysis. In few situations ester synthesis from the 20-carboxylic acid chloride were reported 115.

Figure 5.9. Retrosynthetic analysis of the PAD2F1-3-1K side chain.

Review of the literature showed that only two reports of alkylation at the C-21 position of pregnenolone exist, although -hydroxylation of the ketone through the corresponding enolate has been reported 116. Interestingly, however, intense studies have been given to the preparation of 17α-alkyl and 16α, 17α –dialkyl pregnane derivatives in which 21-methylation was unwanted 115,117. Alkylation of the ketone will, therefore, be subject to regioselective deprotonation of the available α-protons (Figure 5.10).

141

Figure 5.10. Regioselective deprotonation.

This regioselectivity has been thoroughly studied especially with regards to the role of kinetic and thermodynamic control on the outcome of the alkylation 118.

Thermodynamic control will result in the formation of the more substituted 20(17)- enolate. On the other hand the less substituted 20(21)-enolate is the kinetic product.

Alkylation, therefore, should take place selectively at C-21 under kinetic conditions using a strong aprotic base for the generation of the enolate. Obviously, however, deprotonation at C17 is a great concern, as a deprotonation/protonation sequence may result in epimerization of the stereochemistry.

The best precedent for the reaction that we hoped to carry out was reported by

Hanson and coworkers 117. They showed that the enolate of pregnenolone acetate could be generated under kinetic control using excess NaH (10 equiv.). Trapping of the enolate with excess iodomethane resulted mainly in dialkylation which was also sometimes contaminated by a trimethylated product.

142

Figure 5.11. Dimethylation of pregnenolone acetate by Hanson.

Another interesting alkylation/coupling has been reported by Giannis and coworkers 119. They employed Negishi-enoxyborate type coupling conditions to generate the kinetic potassium enoxyborate of tertiary butyl dimethyl silyl (TBS) protected pregnenolone exclusively. This enoxyborate was reacted with the highly reactive 3- iodopropynylphosphonate to generate the phosphonate derivative which was ultimately cyclized into a furan (Figure 5.12). In this example too, kinetic conditions yielded the C-

21 alkyl product.

Figure 5.12. Negishi-Enoxyborate reaction reported by Giannis and coworkers.

143 These examples indicated the feasibility of the desired alkylation reaction. Thus initial alkylation reactions were attempted to determine the optimum conditions to accomplish this. We first protected the 3β-hydroxyl group of pregnenolone with TBSCl to form a silyl ether in presence of imidazole 120. This protection avoids interaction of the relatively acidic alcohol proton with the base necessary for enolate formation. In this case, the TBS group was chosen as a relatively robust protecting group as opposed to the potentially more reactive (-proton containing) acetate protecting group which is traditionally employed in steroid/sterol chemistry.

Figure 5.13. Silyl protection of pregnenolone.

Initial feasibility studies of the reaction involved running the alkylation reaction using different bases to generate the enolate under kinetic conditions. This enolate was then alkylated with methyl iodide. In the reaction a major concern was the possibility of intermolecular self-aldol condensation. This problem was overcome by inverse and dropwise addition of the ketone to the cooled solution of the base as reported previously

144 by Vedejs 115. This procedure was expected to reduce the possibility of self-condensation by limiting the amount of ketone present in the solution at any particular point, and thereby minimizing reaction with the enolate. The first attempt of this reaction used sodium hexamethyldisilazide as a base at -78°C, however no product was detected and starting material was recovered. Believing that ease of deprotonation might be affected by its accessibility and steric bulkiness of the base used, we moved to NaH at -10°C, following Hanson’s conditions. Partial consumption of the ketone was observed with formation of multiple products which were not separable using regular chromatographic techniques. The desired product was obtained when we switched to lithium diisopropylamide (LDA), albeit in a low yield of approximately 20% but still suggesting the feasibility of the alkylation. At this stage we switched to carrying out the alkylation with dimethyl allylbromide (DMAB) which was required for synthesis of PAD2F1-3-1K.

The alkylation reaction with DMAB proceeded successfully to give the alkylation product; however a significant amount of the starting material was recovered

(approximately 50%). To improve the yield of this alkylation reaction optimization of the conditions included addition of different co-solvents including tetramethylethylene diamine (TMEDA), and hexamethylphosphoramide (HMPA) which improved the yield significantly to 58% and 78% yield respectively (Table 5.1). Careful chromatography of the reaction mixture resulted in isolation of a minor quantity of the dialkylated product in the ratio 10:1. Fortunately, epimerization of the C17 position was not observed. Several attempts to further improve the yield of this reaction using temperature and time variations for generation of the enolate did not result in significant differences.

145 Temperature changes included warming the enolate from -78°C to -20°C and to 0°C, and the time allowed for generation of the enolate ranged from one hour to three hours. These changes did not result in any significant improvement of reaction yield.

Figure 5.14. Alkylation of silylated pregnenolone with DMAB.

Table 5.2. Effect of additives on yield of alkylation reaction.

After defining the optimum conditions needed for this reaction we explored the general applicability of this alkylation using different alkylating reagents including alkyliodides, alkylbromides, benzylbromide, geranylbromide, crotylbromide, and

146 pentylbromide (Figure 5.15). These reagents represented significant variations in terms of their reactivity and structure. The yields, however, were typically in the range of 40-65%.

Purification of the mono and dialkyl products was somewhat challenging and this directed us to explore the use of other protecting groups, which were expected to affect the relative polarity and/or visibility of the substrate and products. For example acetate protection resulted in dramatic improvement in the ease of isolation. In addition tertiary- butyldiphenylsilyl chloride was also used to facilitate the UV visualization of these products; however this approach was not useful as these compounds were still not UV active.

Figure 5.15. Different alkylation products.

147 5.5.1. Synthesis of PAD2F1-3-1K

The synthesis of PAD2F1-3-1K was planned in five overall steps from pregnenolone

(Figure 5.16). With the alkylation product in hand, all that remained was the olefination, deprotection, and oxidation reactions in order to generate the natural product.

Figure 5.16. Plan for synthesis of PAD2F1-3-1K.

The Wittig olefination was anticipated to be a straight forward method to introduce the methylene unit from the carbonyl of the sterol. A model system, however, was first attempted using pregnenolone and triphenylphosphonium methyl bromide. n-

Butyl lithium was used to generate the phosphonium ylide at -78°C which was allowed to warm to 0°C over 3 hours. This was followed by addition of the TBS protected pregnenolone to give the desired product in a modest 40% yield.

148

Figure 5.17. Wittig olefination of silyl-pregnenolone.

Unfortunately, when these conditions were applied to the -alkylated product, no reaction took place and only starting material was recovered. The reaction was then repeated with warming of the reaction mixture in order to facilitate the potentially sluggish reaction. In this case after addition of the ketone, the reaction was warmed to

50°C for 16 hours. This resulted in formation of the product in only an unsatisfactory

10% yield. It is presumed that the alkyl chain on the sterol is more sterically demanding than the methyl substituent and, therefore, the Wittig reagent is unable to react effectively with the relatively hindered ketone. Based on this result, we decided to employ the much more reactive Tebbe reagent, an organometallic reagent with the formula

(C5H5)2TiCH2ClAl(CH3)2. Use of the Tebbe reagent, however, resulted in only a 32% of the olefination product when the reaction was done in THF. When the reaction was repeated in toluene/THF in the ratio 1:3, with inverse addition of the ketone to the Tebbe reagent solution, the yield improved to a respectable 56%. The subsequent deprotection was very straightforward using tetrabutyl-ammoniumfluoride (TBAF) in THF with a respectable yield of 94%.

149 At this stage only one step remained in the synthesis of the natural product.

Unfortunately, what was anticipated to be a routine oxidation of 3β-hydroxyl group with concomitant isomerization of the ,-double bond unexpectedly required a number of experiments to complete. Initial attempts using pyridinium chlorochromate for oxidation of pregnenolone as a model system under conditions reported by Parish and Honda 121 resulted only in oxidation of the secondary alcohol without isomerization. Oppenauer oxidation using aluminium tri-isopropoxide in the presence of cyclohexanone following

Uskokvic’s 122 conditions resulted in multiple products including the oxidized product with and without olefin isomerization. Using the conditions reported by Reich and Keana

123 where they used n-methylpiperidone instead of cyclohexanone minimized the formation of the non-isomerized product significantly and resulted in 88% yield of pure product.

Figure 5.18. Oxidation of pregnenolone.

These conditions proved to be very efficient to carry out this oxidation to provide

PAD2F1-3-1K as the major product. This sequence has since been used to prepare

150 approximately 200 mg of PAD2F1-3-1K. The relatively short and efficient sequence could potentially be carried out on gram scale.

Figure 5.19. Synthesis of PAD2F1-3-1K.

5.5.2.Synthesis of PAD2F1-3-3K and Generation of Compound Analogues

As mentioned previously, the synthesis of PAD2F1-3-3K was believed to be much more challenging due to the presence of the stereocenter at C-20. With the synthesis of PAD2F-3-1K in hand, three potential synthetic routes were considered for the preparation of PAD2F-3-3K. Two of these solutions have been investigated using preliminary model systems to address the feasibility of these strategies and have ultimately led to the synthesis of valuable synthetic analogues to probe the SAR of these compounds. The third solution is considered to be a “fall-back” plan and will be utilized in the future only if necessary.

151 a) Grignard Addition/Barton-McCombie Radical Deoxygenation

Based on our success using pregnenolone as a relatively cheap and readily available starting material for the synthesis of PAD2F-3-1K, we reasoned that it may also serve as a useful starting material for the synthesis of PAD2F1-3-3K. We believed that the stereocenter at the C20 position could potentially be introduced via a Grignard addition to the TBS-protected pregnenolone ketone. This would result in the generation of a tetrasubstituted carbon containing an alcohol substituent. Good stereoselectivity has previously been observed for Grignard addition to similar sterol/steroid derivatives at the

C20 position 124. In order to convert this product to the desired PAD2F1-3-3K or analogue, the alcohol would then need then to be removed through some type of reduction or deoxygenation.

We chose to use two Grignard reagents (Figure 5.19), methylmagnesium bromide and allylmagnesium bromide, to generate model systems. Not surprisingly, addition of the methyl Grignard proceeded smoothly to generate the dimethyl alcohol. As the product of this reaction contains two methyl substituents attached to the C20 position, no stereochemical information could be gleaned from the reaction. In the case of the allyl

Grignard, however, addition resulted in the generation of two inseparable, diastereomeric products in a 10:1 ratio.

152

Figure 5.20. Grignard addition to silylated pregnenolone.

We thought that the dimethyl alcohol would serve as an excellent substrate for a potential Barton-McCombie deoxygenation reaction since the product would not be complicated by potential diastereomers. If we could show that this reaction was successful, then we would turn our attention to the allyl system and study the stereochemical outcome of the deoxygenation reaction. To our dismay, however, we were unable to cleanly generate the necessary xanthate precursor of the dimethyl alcohol.

Rather than trying to optimize this reaction, we decided to first pursue the deoxygenation of the somewhat less hindered pregnenolone reduction product. This compound was prepared through reduction with lithium aluminum hydride using reported conditions 125.

153

Figure 5.21. Reduction of pregnenolone.

The xanthate was formed from the alcohol using n-BuLi as a base and subsequent reactions with carbon disulphide and iodomethane following the procedure of

Schlessinger 126. Tributyltin hydride in presence of the free radical initiator azobisisobutyronitrile (AIBN) was used to successfully affect this deoxygenation .

Figure 5.22. Barton-McCombie radical deoxygenation.

The same procedures employed for the reduced product should be able to be repeated for the dimethyl system to get the desired product, although few Barton-

McCombie reactions have successfully been reported with tertiary alcohols. It is expected that optimization of these reactions would require more time due to the steric hindrance

154 and, therefore, different reagents and conditions may need to be adopted as well. With that in mind, we also considered the direct reduction of the dimethyl alcohol using a catalytic amount of Indium-trichloride in the presence of chlorodiphenylsilane as a hydride donor following the chemoselective reducing system for secondary and tertiary alcohols reported by Baba and coworkers 127. Attempts to apply these conditions to our compounds were not successful.

Although somewhat unsuccessful, this strategy did provide us with a number of structural analogues of the sterols at the C17 side chain. It was thought that the Grignard addition products were very structurally similar to the azasterol AZA (Chapter 4, Figure

4.9) and, therefore, may show similar activity against leishmaniasis. In addition we prepared an O-alkylated derivative bearing an isobutyl chain on the oxygen atom. This compound was suggested to be very similar to with the non-oxidized precursor of

PAD2F-3-1K. The only difference was the presence of geminal dimethyl substituents rather than the C20-C21 methylene. Interestingly all of these compounds were considered inactive in the biological assays. This suggests that these compounds, which lack the basic nitrogen found in AZA, are not working through the same mechanism. Since the most active compounds in our assays are the conjugated ketones, we oxidized the dimethyl alcohol using the Oppenauer conditions, but this compound 19 also was considered inactive.

155 Figure 5.23. Structures of synthesized analogues.

b) Stereoselective Reduction of PAD2F1-3-1K

The first potential solution to the synthesis of PAD2F1-3-3K involved the use of an alkylation/olefination strategy similar to that employed for PAD2F-3-1K, followed by the selective hydrogenation of the C20-C21 olefin. We were encouraged by the literature precendent of Seldes et al 128 who showed that the hydrogenation of the C20-C21 double bond resulted in the production of a single diastereomer (Figure 5.23) with same relative stereochemistry as that required for PAD2F-3-3K. In their case, the product was compared spectroscopically to natural material and determined to be identical.

Figure 5.24. Selective reduction of C-20/C-21 double bond.

156 Using material synthesized via our alkylation protocol, we initially investigated the effectiveness of this type of reduction of the C20-C21 double bond (vicinally disubstituted) in the presence of the C24-C25 and C5-C6 double bonds (which are both trisubstituted) using different reducing catalysts, including Willkinson’s catalyst,

Crabtree’s catalyst, and hydrogenation over Pd/C. It was not clear if the C20-C21 bond would be reduced selectively, but this “model” system was expected to at least give us some information about the stereoselectivity of the reduction.

In the first attempt to accomplish this reduction we employed hydrogen over 5%

Pd/C. We were pleased to see that the C5-C6 double bond remained intact upon hydrogenation, although both of the other two double bonds were reduced. Most importantly, however, it appeared that only one isomer was produced during the course of the reaction in accordance with the work of Seldes 128. The product was then desilylated to provide cholesterol. The 1H NMR spectrum was compared with natural cholesterol and appeared to match. This assignment, however, is tenuous at best. A search of the literature revealed that the C20 epimer of cholesterol has previously been reported 129. This paper reports the 1H NMR spectra of both cholesterol and 20- epicholesterol and shows that in CDCl3 at 270 MHz the C21 methyl peaks show up at

0.91 and 0.82 ppm, respectively. Ultimately, this reaction needs further optimization and more rigorous stereochemical assignment in order to prove that the desired isomer is being produced. The result, however, was promising enough to further investigate the selectivity of reduction.

In the case of reduction using Crabtree catalyst, both of the double bonds in the

C17 side chain were also reduced. In this case, however, two C20 isomers were formed 157 which could not be separated using regular liquid chromatographic techniques. The much less reactive, and therefore, more selective Wilkinson’s catalyst was also investigated.

This particular catalyst generally shows good selectivity for the reduction of disubstituted double bonds in the presence of those with higher degrees of substitution. Unfortunately, however, under standard reaction conditions only starting material was recovered.

Figure 5.25. Reduction of C-20/C-21 double bond using different catalysts.

Although the C20-C21 bond has not been reduced selectively, this reduction method still appears promising (pending the stereochemical outcome). A potential solution to the selectivity problem is the introduction of an alkyl side chain containing no double bonds (i.e., a protected hydroxyethyl group) in the alkylation reaction prior to the

Tebbe olefination. In this case, the reduction of the C20-C21 olefin could be carried out in the presence of the C4-C5 olefin. After reduction, the side chain could then be modified to produce the desired PAD2F-3-3K derivatives.

158

Figure 5.26. Plan for synthesis of PAD2F1-3-3K.

c) Synthesis of PAD2F1-3-3K from stigmasterol or other commercially

available starting material with the necessary C20 stereochemistry

As mentioned above a third strategy has been considered for the synthesis of

PAD2F1-3-3K, although no work has yet been carried out on this route. This would entail the use of a starting material like stigmasterol which already contains the necessary C20 stereochemistry. Chemical degradation of the stigmasterol side chain through ozonlysis would provide a means of introducing the desired substitution patterns at C24 and beyond. Su and coworkers 130 recently utilized this type of approach from stigmasterol, employing a protection of the C4-C5 olefin and subsequent ozonolysis of the side chain.

Work on this route is considered necessary only if strategy (b) above fails.

5.6. Biological Activity of Synthetic PAD2F-3-1K

Synthetic PAD2F-3-1K and the other synthesized analogues were subjected to in vitro testing against L. mexicana promatigotes. Only PAD2F1-3-1K showed activity very

159 similar to the isolated natural one (Table 5.2) and proved to be significantly potent as compared to Pentostam. All the other compounds were not active at concentration of 100

µg/ml.

Molecule IC50 Pentostam 343.7 µM Natural product PAD2F1-3-1K 25.3 µM Synthetic PAD2F1-3-1K 30 µM

Table 5.3. Leishmanicidal activity of PAD2F1-3-1K.

PAD2F1-3-1K was further tested using bone marrow derived macrophages from

C57BL/6 mice infected with RFP-L. donovani (transgenic L. donovani expressing green fluorescent protein (GFP)). As observed in the following fluorescence microscopy images (from left to right); amastigotes are abundant in the control, however their absence is recognized at concentrations of 100 and 10 µM. At 1 M the compound seemed to be ineffective.

160

Control 100 M 10 M 1 M

Figure 5.27. Bone marrow derived macrophages from C57BL/6 mice infected with RFP-L. donovani co-cultured with synthetic PAD2F-3-1K (fluorescence microscopy images)

Also a flow cytometric assay using the same transgenic L. donovani was used to confirm the observed activity. As shown in the histograms (Figure 5.28) the same trend of activity was observed at concentrations of 100 µM and 10 µM where the fluorescence is diminished. However at 1 M the compound seemed to be ineffective and expression of the GFP is obvious from the same fluorescence pattern seen in the control.

IC50%=54 mcM

0 1 2 3 4 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H FL2-H Control 100 M 10 M 1 M

Figure 5.28. Bone marrow derived macrophages from C57BL/6 mice infected with RFP-L. donovani co-cultured with synthetic PAD2F-3-1K (flow cytometry data)

161 5.7. Structure Activity Relationships

Examination of the structures of both the isolated natural products and the synthetic compounds gives some insights about the essential structural motifs and their tolerance to different functional groups. Three basic structural motifs, the C-3 ,β- conjugated carbonyl group, C20/C21 functional groups, and the C20/C22 alkyl chain substation, were considered and examined through the preliminary SAR study.

Figure 5.29. Structures of natural products and their antileishmanial activity.

162 a) C3 ,β- conjugated carbonyl group

Comparison of the biological activities of compounds 1 versus 7 and 3 versus 9 clearly shows that the presence of a C3 alcohol rather than the ,β-conjugated carbonyl group is detrimental for activity. However it is not clear whether β,γ-unsaturation can be tolerated at this position without loss of activity in the presence of the ketone. The oxidation of the alcohols using PCC generates compounds of this type, although these were not tested. Additional conjugation as an ,,,-unsaturated carbonyl is tolerated in compound 16, although the activity drops off slightly from the less conjugated ,- unsaturated compound 15. These points need further investigation.

b) The C20/C-21 functional group

C20/21 unsaturation or saturation does not significantly affect the antileishmanial activity. Instead, activity seems to be more directly tied to the substitution/unsaturation pattern on the C-22 alkyl chain. Uncertainty arises, however, when comparing their activities towards promastigotes versus amastigotes; which may ultimately be explained in terms of their mechanism of action. In addition the conserved “R” stereocenter in all the compounds isolated seems also to be important for activity as viewed in the biological activities of compounds (3) and (4). Presence of a carbonyl group instead of the C20-C21 methylene is also well tolerated in compounds (15) and (16) and progesterone

(commercially available, data not shown). To date none of the synthetic alkylated compounds have been directly oxidized and tested to explore whether alkylated ketones

(beyond the methyl ketone) are also active.

163

c) C20/C22 alkyl chain and other effects

A 5-carbon alkyl chain is seen in compounds (1) through (9), which are characterized by different degrees of saturation, and unsaturation patterns. This is best seen by comparing compounds (3) and (5) which share the same stereocenter but differ in the degree of substitution and position of double bonds. Obviously, these substitution patterns play some role in the activity of the compounds. From the known analogues it appears that while degree of substitution is not nearly very important in relation to activity, significant branching of the side chain (i.e., 5) may result in a loss of activity.

Some of these issues could be explored further through olefination and oxidation of the various alkyation products (derived from benzyl bromide, iodobutane, etc.). It is also not clear whether the presence of the C11 β-hydroxyl group (in compounds 15 and 16) affects the activity of compounds, possibly through increasing solubility or providing sites for hydrogen bonding or if unsaturation (C16-C17) of the C-20 ,β-unsaturated ketone is responsible for the observed bioactivity.

5.8. Conclusion and Future Directions

In conclusion, several isolated sterols exhibited potent antileishmanial activity; however their low quantities hindered further investigation. Therefore, synthesis of these compounds emerged as a necessity to provide sufficient quantities for in vitro and in vivo testing, and studying the mechanism of action of these compounds. In addition derivatives and analogues of the isolated compounds have been and will continue to be synthesized to determine the essential pharmacophore and the effect of different 164 functional groups on activity. It can be expected that the pharmacological properties

(absorption, distribution, metabolism, and elimination) of isolated sterols may not be optimal for therapeutic application. Therefore systematic modification of these compounds (or possibly encapsulation) might be essential to minimize toxicity and optimize their potency, solubility, etc. The structural modifications will depend on analysis of the biological information provided by in vivo and in vitro testing.

165

Experimental Section

General procedure for synthesis of monoketone carbonyl analogues.

The reaction conditions were adapted from Adeva et al. The aldehyde (1 mmol) was dissolved in 13 ml ethanol (50% aqueous), acetone (0.5 mmol) was added and stirred at rt for 5 min. A solution of aqueous NaOH (1.2 M) was added dropwise. A precipitate was formed within 1 – 3 h. The precipitate was filtered, washed with cold ethanol and recrystalized from ethanol.

(1E,4E)-1,5-bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-one, (FLLL-40). A yellow

1 solid: mp 108 °C; H NMR (CDCl3, 400 MHz) 8.06 (d, J = 16.1 Hz, 2H), 7.27 (d, J =

6.7 Hz, 2H), 7.17 (d, J = 16.1 Hz, 2H), 7.11 (t, J = 8 Hz, 2H), 6.98 (d, J = 8 Hz, 2H), 3.91

13 (s, 6H); C NMR (CDCl3, 400 MHz) 190.1, 153.5, 149.1, 138.3, 129.4, 127.3, 124.6,

119.8, 114.4, 61.8, 56.3.

166

(1E,4E)-1,5-bis(2,4-dimethoxyphenyl)penta-1,4-dien-3-one, (FLLL-41). A yellow

1 solid: mp 130 °C; H NMR (CDCl3, 400 MHz) 8.01 (d, J = 16 Hz, 2H), 7.58 (d, J = 8.6

Hz, 2H), 7.10 (d, J = 16 Hz, 2H), 6.54 (dd, J = 2.1, 8.6 Hz, 2H), 6.48 (d, J = 2.1 Hz, 2H),

3.91 (s, 6H), 3.87 (s, 6H).

(1E,4E)-1,5-bis(2,5-dimethoxyphenyl)penta-1,4-dien-3-one, (FLLL-42). A yellow

1 solid: mp 102-104 °C; H NMR (CDCl3, 400 MHz) 8.05 (d, J = 16 Hz, 2H), 7.18 (s,

2H), 7.16 (d, J = 16 Hz, 2H), 6.96 (dd, J = 2.8, 8.9 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 3.89

13 (s, 6H), 3.84 (s, 6H); C NMR (CDCl3, 400 MHz) 190.2, 153.9, 153.5, 138.4, 126.7,

124.9, 117.6, 113.5, 112.8, 56.5, 56.2.

167

(1E,4E)-1,5-bis(2,6-dimethoxyphenyl)penta-1,4-dien-3-one, (FLLL-47). A yellow

1 solid: mp 153-154 °C; H NMR (CDCl3, 400 MHz) 8.19 (d, J = 16.2 Hz, 2H), 7.61 (d,

J = 16.2 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 5.6 Hz, 2H), 6.59 (d, J = 8.3 Hz,

13 2H), 3.93 (s, 12H); C NMR (CDCl3, 400 MHz) 193.0, 160.5, 133.8, 131.4, 129.5,

113.4, 104.1, 103.9, 56.9, 56.2.

(1E,4E)-1,5-di(pyridin-2-yl)penta-1,4-dien-3-one, (FLLL-61). To a solution of 1,3- acetone dicarboxylic acid (146 mg, 1 mmol) in 1.4 ml was added 4-pyridine carboxaldehyde (213 mg, 2 mmol) dropwise with stirring. After stirring at rt for 2 h, the reaction was treated with 0.7 ml HCL then heated to 80°C for 2 h. A yellow precipitate was formed which was filtered and washed with cold ethanol. Recrystalization from

1 ethanol provided (FLLL-61) as an off-white solid: mp 140 °C; H NMR (CDCl3, 300

MHz) 8.67 (d, J = 4.6 Hz, 1H), 7.76 (d, J = 15.6 Hz, 1H), 7.44 (dt, J = 1.8, 7.8 Hz, 1H),

7.61 (d, J = 15.6 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.29 (dd, J = 4.6, 7.8 Hz, 1H); 13C

NMR (CDCl3, 300 MHz) 189.5, 153.2, 150.2, 142.1, 136.7, 128.8, 124.8, 124.3; IR

−1 + (film): 1662 cm ; HRMS-TOF m/z (M + Na) calcd for C15H12N2O 259.0847, found

259.0846.

168

(1Z)-1,5-bis(3,4-dimethoxyphenyl)-2,4-difluoropenta-1,4-dien-3-one, (FLLL-26). 3,4-

Dimethoxybenzaldehyde (335 mg, 2 mmol) was dissolved in 10 ml of ethanol. β-Alanine crystals (178 mg, 2 mmol), and difluoroacetone (92 L, 1 mmol) were added and stirred at 50°C for 6 h. Solvent was removed and the residue was dissolved in ethylacetate and washed with water then brine. The organic layer was dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography

(EtOAc-hexanes, 1:5) to give the product as a yellow solid: mp 107-109 °C; 1H NMR

(CDCl3, 400 MHz) 7.31 (m, 4H), 7.10 (m, 1H), 7.00 (m, 1H), 6.92 (d, J = 8.4 Hz, 2H),

13 3.94 (s, 6H), 3.93 (s, 6H); C NMR (CDCl3, 400 MHz) 177.9, 154.2, 151.6, 151.3,

149.4, 125.8, 125.7, 124.6, 120.7, 113.4, 114.3, 111.4, 72.2, 56.3, 56.2; IR (film): 1630

−1 + cm ; HRMS-TOF m/z (M + Na) calcd for C21H20F2O5 413.1177, found 413.1160.

(1Z,4E)-1,5-bis(3,4-dimethoxyphenyl)-2-fluoropenta-1,4-dien-3-one, (FLLL-35). 3,4-

Dimethoxybenzaldehyde (335 mg, 2 mmol) was dissolved in 10 ml of ethanol. β-Alanine crystals (178 mg, 2 mmol), and fluoroacetone (77.3 L, 1 mmol) were added and stirred at 50°C for 6 h. Solvent was removed and the residue was dissolved in ethylacetate and washed with water then brine. The organic layer was dried over sodium sulfate and 169 concentrated. The crude material was purified by silica gel column chromatography

(EtOAc-hexanes, 1:5) to give the product as a yellow solid: mp 196-198 °C; 1H NMR

(DMSO, 300 MHz) 7.48 (d, J = 2.0 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 39

Hz, 1H), 7.39 (br s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 3.85 (s, 3H),

3.82 (s, 6H), 3.81 (s, 3H); IR (film): 1638 cm−1; HRMS-TOF m/z (M + Na)+ calcd for

C21H21FO5 395.1271, found 395.1266.

(1E)-1,5-bis(3,4-dimethoxyphenyl)-2,4-dimethylpenta-1,4-dien-3-one, (FLLL-27).

FLLL-27 was synthesized using the conditions reported by Demin et al 128 where 3,4- dimethoxy benzaldehyde (332 mg, 2 mmol) was dissolved in 10 ml ethanol, dimethylacetone (108 L, 1 mmol) and NaOH (80 mg, 2 mmol) were added and stirred at rt for 5 h. A precipitate was formed within 1 – 3 h. The precipitate was filtered, washed with cold ethanol and recrystalized from ethanol to give a yellow solidas a white solid:

1 mp 61-63 °C; H NMR (CDCl3, 400 MHz) 7.41 (s, 2H), 6.99 (dd, J = 1.1, 8.3 Hz, 2H),

6.91 (s, 2H), 6.84 (d, J = 8.3 Hz, 2H), 3.84 (d, J = 2.1 Hz, 12H), 2.03 (s, 6H); 13C NMR

(CDCl3, 400 MHz) 159.3, 150.9, 150.7, 150.5, 142.5, 137.3, 129.2, 128.1, 127.5, 126.5,

126.4, 117.0, 108.3, 50.5, 22.1; IR (film): 1676 cm−1.

170

Dimethyl 2,4-bis(3,4-dimethoxybenzylidene)-3-oxopentanedioate, (FLLL-57). A

1 yellow solid: mp 122-124 °C; H NMR (CDCl3, 300 MHz) 7.87 (s, 1H), 7.41 (s, 1H),

7.09 (dd, J = 2.1, 8.4 Hz, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.98 (d, J = 2.1 Hz, 1H), 6.93 (d,

13 J = 2.1 Hz, 1H), 6.83 (dd, J = 2.1, 8.4 Hz, 2H); C NMR (CDCl3, 400 MHz) 193.9,

171.5, 168.3, 166.9, 152.5, 151.8, 149.4, 149.3, 144.9, 144.0, 132.4, 126.8, 126.3, 125.7,

125.6, 125.5, 112.2, 112.1, 111.4, 111.2, 60.7, 56.2, 53.1, 52.9, 52.8, 21.4, 14.5; IR

−1 + (film): 1727, 1647 cm ; HRMS-TOF m/z (M + Na) calcd for C10H10NBrI 493.1475, found 493.1475.

(2E,6E)-2,6-bis(3,4-dimethoxybenzylidene)cyclohexanone, (FLLL-28). The synthesis used the conditions reported by Demin et al 128 where 3,4-dimethoxy benzaldehyde (166 mg, 1 mmol) was dissolved in 10 ml ethanol, cyclohexanone (1.1 ml, 0.5 mmol) and

NaOH (40 mg, 1 mmol) were added and stirred at rt for 5 h. A precipitate was formed within 1 – 3 h. The precipitate was filtered, washed with cold ethanol and recrystalized

1 from ethanol to give a yellow solid: mp 148 °C; H NMR (CDCl3, 400 MHz) 7.76 (s,

2H), 7.11 (d, J = 8.3 Hz, 2H), 7.03 (s, 2H), 6.91 (d, J = 8.3 Hz, 2H), 3.93 (s, 6H), 3.92 (s,

171

13 6H), 2.95 (t, J = 5.3 Hz, 4H), 1.83 (m, 2H); C NMR (CDCl3, 400 MHz) 190.0, 149.5,

148.6, 136.8, 134.4, 128.9, 123.9, 113.6, 110.8, 56.9, 55.9.

(3E,5E)-3,5-bis(3,4-dimethoxybenzylidene)piperidin-4-one, (FLLL-29). Following

Dimmock et al 68, 4-piperidone hydrochloride (153 mg, 1 mmol) was suspended in 10 ml of glacial acetic acid and saturated with dry HCl gas for 30 min. 3,4-

Dimethoxybenzaldehyde (335 mg, 2 mmol) was then added and the reaction was stirred at rt for 16 h. A precipitate was formed, which was filtered, washed with cold ethanol and

1 recrystalized from ethanol to give a yellow solid: mp 168 °C; H NMR (CDCl3, 400

MHz) 7.76 (s, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 1.4 Hz, 2H), 6.92 (d, J = 8.3

13 Hz, 2H), 4.19 (s, 4H), 3.93 (s, 6H), 3.92 (s, 6H), 1.63 (m, 1H); C NMR (CDCl3, 400

MHz) 187.6, 149.9, 148.7, 135.8, 133.3, 128.2, 124.2, 113.6, 110.9, 55.9, 48.2.

(3E,5E)-3,5-bis(2,4,6-trimethoxybenzylidene)piperidin-4-one, (FLLL-34). 4-

Piperidone hydrochloride (153 mg, 1 mmol) was suspended in 10 ml of glacial acetic acid and saturated with dry HCl gas for 30 min. 2,4,6-trimethoxybenzaldehyde (392 mg,

172

2 mmol) was then added and the reaction was stirred at rt for 16 h. A precipitate was formed, which was filtered, washed with cold ethanol and recrystalized from ethanol to give an orange solid: mp 198-199 °C; 1H NMR (DMSO, 300 MHz) 7.36 (s, 2H), 6.28

(s, 4H), 3.82 (s, 6H), 3.79 (s, 12H).

(E)-N-(3,4-dimethoxybenzyl)-3-(3,4-dimethoxyphenyl)acrylamide, (FLLL-30). 3,4-

Dimethoxycinnamic acid (208 mg, 1 mmol) was dissolved in 6.6 ml of DCM. Oxalyl chloride (89 µL, 1 mmol) was added, followed by 2 drops of dimethylformamide (DMF), and then stirred for 2 h at rt. The solvent was removed and dried under vacuum for 30 min then was redissolved in 8.4 ml DCM and cooled to -10°C. 3,4-

Dimethoxybenzylamine (183 L, 1.2 mmol) was added dropwise followed by N,N-

Diisopropylethylamine (DIEA) (0.58 ml, 3.3 mmol) and stirred at rt for 16 h. Solvent was removed and the residue was dissolved in ethylacetate and washed with water then brine.

The organic layer was dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 1:5) to give the

1 product as a white solid: mp 135 °C; H NMR (CDCl3, 400 MHz) 7.61 (d, J = 15.5 Hz,

1H), 7.07 (d, J = 8.3 Hz, 1H), 7.01 (s, 1H), 6.85 (m, 4H), 6.32 (d, J = 15.5 Hz, 1H), 6.04

13 (t, J = 5.6 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.86 (s, 6H); C NMR (CDCl3, 400 MHz)

166.3, 151.0, 149.5, 148.8, 141.5, 131.2, 128.1, 122.3, 120.6, 111.6, 111.5, 111.4,

173

110.0, 56.3, 56.2, 44.1; IR (film): 3371, 1641 cm−1; HRMS-TOF m/z (M + Na)+ calcd for

C20H23NO5 380.1474, found 380.1485.

(E)-2-(3,4-dimethoxybenzylidene)-3-oxobutanenitrile. 3,4-Dimethoxybenzaldehyde

(332 mg, 2 mmol) and 5-methylisoxazole-3-carboxylic acid (127 mg, 1 mmol) were mixed and heated to 200°C for 1h. The reaction mixture was cooled then dissolved in ethylacetate and washed with water then brine. The organic layer was dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 1:9) to give the product (117.5 mg, 51%) as a yellow

1 solid: mp 113-116 °C; H NMR (CDCl3, 300 MHz) 8.07 (s, 1H), 7.80 (d, J = 2.1 Hz,

1H), 7.51 (dd, J = 2.1, 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 3.97 (s, 3H), 3.96 (s, 3H),

2.58 (s, 3H).

(2E,4E)-2-(3,4-dimethoxybenzylidene)-5-(3,4-dimethoxyphenyl)-3-oxopent-4- enenitrile, (FLLL-44).

3,4-Dimethoxybenzaldehyde (166 mg, 1 mmol) was suspended in 10 ml of glacial acetic acid and saturated with dry HCl gas for 30 min. (E)-2-(3,4-dimethoxybenzylidene)-3- 174 oxobutanenitrile (115.5 mg, 0.5 mmol) was then added and the reaction was stirred at rt for 16 h. A precipitate was formed, which was filtered, washed with cold ethanol and

1 recrystalized from ethanol to give an orange solid: mp 175 °C; H NMR (CDCl3, 300

MHz) 8.28 (s, 1H), 7.87 (d, J = 15.6 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.57 (dd, J =

2.1, 8.4 Hz, 1H), 7.41 (d, J = 15.6 Hz, 1H), 7.28 (dd, J = 2.1, 8.4 Hz, 1H), 7.17 (d, J = 2.1

Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 3.98 (s, 6H), 3.95 (s, 3H),

13 3.94 (s, 3H); C NMR (CDCl3, 400 MHz) 182.4, 154.2, 154.0, 152.5, 149.7, 149.6,

147.1, 128.7, 127.7, 125.6, 124.5, 119.1, 118.9, 112.3, 111.5, 111.4, 110.7, 107.3, 56.6,

−1 + 56.4; IR (film): 2206, 1670 cm ; HRMS-TOF m/z (M + Na) calcd for C22H21NO5

402.1317, found 402.1296.

175

Ethyl cinnamate. Cinnamic acid (305 mg, 2 mmol) was dissolved in 2 ml ethanol and 5 drops of concentrated sulphuric acid were added. The reation was refluxed for 4 h. After completion of the reaction 20 ml of cold water was added and the organic layer was separated, and the aqueous layer was extracted twice with ethylacetate. The combined organic layers were dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 3:7) to give the ester as

1 as a colorless oil: H NMR (CDCl3, 300 MHz) 7.68 (d, J = 15.9 Hz, 1H), 7.53 (m, 2H),

7.38 (m, 3H), 6.44 (d, J = 15.9 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H),

7.17 (d, J = 2.1 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 3.98 (s, 6H),

3.95 (s, 3H), 3.94 (s, 3H).

(E)-3-oxo-5-phenylpent-4-enenitrile. The reaction conditions were adapted from

Augustin et al 132 where NaH (48 mg, 2 mmol) was suspended in 1 ml of THF.

Acetonitrile (105 L, 2 mmol) was then added dropwise and stirred for 15 min at rt.

Ethylcinnamate (176 mg, 1 mmol) was dissolved in 0.2 ml of THF and added dropwise to the reaction and refluxed for 2 h. after cooling 1.2 ml of ether was added, followed by 2 ml water. The reaction mixture was then acidified with HCl to give a white precipitate which was filtered and dissolved with ethylacetate and washed with NaHCO3. The 176 organic layer was dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 2:8) to give the product

1 as a white solid: mp 96 °C;: H NMR (CDCl3, 300 MHz) 7.69 (d, J = 15.9 Hz, 1H),

7.59 (m, 2H), 7.45 (m, 3H), 6.88 (d, J = 15.9 Hz, 1H), 3.71 (s, 2H).

(E)-5-(3,4-dimethoxyphenyl)-3-oxopent-4-enenitrile. A yellowish-white solid: mp 134

1 °C;: H NMR (CDCl3, 300 MHz) 7.63 (d, J = 15.9 Hz, 1H), 7.19 (dd, J = 1.8, 8.4 Hz,

1H)), 7.08 (d, J = 8.4 Hz, 1H)), 5.75 (d, J = 15.9 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.68

(s, 2H); IR (film): 2256, 1670 cm−1;

(2E,4E)-2-((6-bromopyridin-2-yl)methylene)-5-(3,4-dimethoxyphenyl)-3-oxopent-4- enenitrile, (FLLL-56). 3-Bromo-2-pyridinecarboxaldehyde (382 mg, 2 mmol) was dissolved in 10 ml of absolute ethanol. β-alanine crystals (178 mg, 2 mmol) was added and stirred for 10 min at rt. (E)-5-(3,4-dimethoxyphenyl)-3-oxopent-4-enenitrile (462 mg,

2 mmol) was then added and stirring was continued for 16 h. Ethanol was removed and the residue was redissolved in ethylacetate, and the organic layer was washed with water then brine. The organic layer was then dried over sodium sulfate and concentrated. The

177 crude material was purified by silica gel column chromatography (EtOAc-hexanes, 1:5)

1 to give (FLLL-56) as an orange solid: mp 174-176 °C; H NMR (CDCl3, 300 MHz)

8.23 (s, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.91 (d, J = 15.3 Hz, 1H), 7.70 (d, J = 7.5 Hz,

1H), 7.43 (d, J = 15.3 Hz, 1H), 7.30 (dd, J = 2.1, 8.4 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H),

13 6.92 (d, J = 8.4 Hz, 1H), 3.96 (s, 3H), 3.95 (s, 3H); C NMR (CDCl3, 400 MHz) 181.3,

152.5, 151.3, 149.3, 148.2, 142.5, 139.1, 130.7, 127.0, 117.6, 111.1, 110.3, 56.0; IR

−1 + (film): 2215, 1672 cm ; HRMS-TOF m/z (M + Na) calcd for C19H15BrN2O3 421.0164, found 421.0163.

(2E,4E)-2-((6-bromopyridin-2-yl)methylene)-3-oxo-5-phenylpent-4-enenitrile,

(FLLL-55). 3-Bromo-2-pyridinecarboxaldehyde (382 mg, 2 mmol) was dissolved in 10 ml of absolute ethanol. β-alanine crystals (178 mg, 2 mmol) was added and stirred for 10 min at rt. (E)-3-oxo-5-phenylpent-4-enenitrile (342 mg, 2 mmol) was then added and stirring was continued for 16 h. Ethanol was removed and the residue was redissolved in ethylacetate, and the organic layer was washed with water then brine. The organic layer was then dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 1:5) to give the desired products as

1 yellow needles: mp 152 °C; H NMR (CDCl3, 400 MHz) 8.25 (s, 1H), 7.97 (d, J = 15.6

Hz, 1H), 7.93 (s, 1H), 7.71 (m, 3H), 7.63 (d, J = 8 Hz, 1H), 7.59 (d, J = 15.6, Hz, 1H),

13 7.48 (br s, 3H); C NMR (CDCl3, 400 MHz) 182.0, 151.6, 150.1, 148.4, 143.0, 139.6,

178

129.6, 125.7, 120.3, 116.4, 115.3; IR (film): 2217, 1674 cm−1; HRMS-TOF m/z (M +

+ Na) calcd for C17H11BrN2O 360.9952, found 360.9943.

General Procedure for synthesis of cyanoacetamides

Transamidation was carried out following the conditions reported by Demin et al 131.

Methylcyanoacetate (1.07 ml, 12 mmol) and the corresponding amine (8 mmol) were mixed and stirred at rt for 5 h. A white precipitate was formed, which was filtered and recrystalized from ethanol.

(S)-2-cyano-N-(1-phenylethyl)acetamide. White needles (1.58 g, 95%): 1H NMR

(DMSO, 400 MHz) 8.69 (d, J = 5.8 Hz, 1H), 7.30 (m, 4H), 7.22 (m, 1H), 4.88 (m, 1H),

13 3.65 (s, 2H), 1.34 (d, J = 5.2 Hz, 3H); C NMR (CDCl3, 400 MHz) 160.2, 142.1,

129.3, 128.3, 126.5, 72.2, 50.4, 42.0, 26.3, 21.8; IR (film): 3273, 2291, 1644 cm−1.

179

N-benzyl-2-cyanoacetamide. White needles (1.32 g, 90%): 1H NMR (DMSO, 300 MHz)

8.99 (d, J = 7.8 Hz, 1H), 8.33 (s, 1H), 7.92 (t, J = 1.8 Hz, 2H), 7.80 (d, J = 7.8 Hz, 1H),

7.32 (m, 5H), 5.16 (m, 1H), 1.46 (d, J = 7.8 Hz, 3H); IR (film): 3334, 2242, 1672 cm−1.

N-(3-(benzyloxy)benzyl)-2-cyanoacetamide. An off-white solid (prepared using the

1 general procedure for synthesis of cyanoacetamides): mp 108-109 °C; H NMR (CDCl3,

400 MHz) 7.41 (m, 4H), 7.33 (m, 1H), 7.26 (m, 1H), 6.89 (m, 3H), 6.42 (br s, 1H), 5.06

13 (s, 2H), 4.43 (dd, J = 5.6, 3.5 Hz, 2H), 3.37 (s, 2H); C NMR (CDCl3, 300 MHz)

159.1, 138.5, 136.7, 129.9, 128.6,128.1, 127.5, 120.3, 114.7, 114.5, 114.0, 70.0,

44.1, 24.9; IR (film): 3285, 2223, 1655 cm−1.

N-(4-(benzyloxy)benzyl)-2-cyanoacetamide. A white solid: mp 151 °C; 1H NMR

(CDCl3, 400 MHz) 7.40 (m, 4H), 7.33 (m, 1H), 7.21 (d, J = 8.6 Hz, 2H), 6.95 (d, J =

8.6 Hz, 2H), 6.28 (br s, 1H), 5.06 (s, 2H), 4.41 (d, J = 5.5 Hz, 2H), 3.38 (s, 2H); 13C

NMR (DMSO, 400 MHz) 157.9, 137.6, 131.2, 129.3, 128.9,128.2, 128.0, 116.7,

115.1, 69.6, 42.6, 25.7.

180

2-cyano-N-phenethylacetamide. A yellowish white solid: mp 83-85 °C; 1H NMR

(CDCl3, 400 MHz) 7.32 (m, 2H), 7.24 (m, 1H), 7.19 (m, 2H), 6.41 (br s, 1H), 3.52 (q, J

= 7 Hz , 2H), 3.29 (s, 2H), 2.84 (t, J = 7 Hz, 2H).

(S)-2-cyano-N-(1,2,3,4-tetrahydronaphthalen-1-yl)acetamide. White needles: mp 183-

1 184 °C; H NMR (CDCl3, 400 MHz) 7.19 (m, 4H), 7.12 (d, J = 7.6 Hz, 1H), 6.33 (d, J

= 7 Hz, 1H), 5.15 (m, 1H), 3.82 (s, 2H), 3.46 (s, 1H), 3.39 (s, 2H), 2.79 (m, 2H), 2.05 (m,

1H), 1.85 (m, 3H).

1 N-benzhydryl-2-cyanoacetamide. Golden needles: mp 162-164 °C; H NMR (CDCl3,

400 MHz) 7.31 (m, 6H), 7.23 (d, J = 7.2 Hz, 4H), 6.78 (d, J = 7.8 Hz, 1H), 6.20 (d, J =

13 7.8 Hz, 2H; C NMR (CDCl3, 400 MHz) 140.1, 128.8, 127.9, 127.2, 114.5,

57.8, 25.8.

181

General procedure for synthesis of WP1066 analogues

The condensation was done following the procedures reported by Demin et al 131 for synthesis of tyrenes. The desired aldehyde (2 mmol) was dissolved in 10 ml of absolute ethanol. β-alanine crystals (2 mmol) was added and stirred for 10 min at rt.

Cyanoacetamide (2 mmol) was then added and stirring was continued for 16 h. Ethanol was removed and the residue was redissolved in ethylacetate, and the organic layer was washed with water then brine. The organic layer was then dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography

(EtOAc-hexanes, 1:5) to give the desired products.

(S,E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(1-phenylethyl)acrylamide, (WP-1066). A white solid: mp 102 °C; 1H NMR (DMSO, 300 MHz) 8.99 (d, J = 7.8 Hz, 1H), 8.33 (s,

1H), 7.92 (t, J = 1.8 Hz, 2H), 7.80 (d, J = 7.8 Hz, 1H), 7.32 (m, 5H), 5.16 (m, 1H), 1.46

(d, J = 7.8 Hz, 3H).

182

(S,E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)-N-(1-phenylethyl)acrylamide,

(FLLL-102). A yellow solid: mp 151 °C; 1H NMR (DMSO, 300 MHz) 10.22 (s, 1H),

8.66 (d, J = 7.8 Hz, 1H), 8.03 (s, 1H), 7.64 (d, J = 1.8 Hz, 1H), 7.48 (dd, J = 2.1, 8.4 Hz,

1H), 7.36 (m, 4H), 7.24 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 5.05 (m, 1H), 3.81 (s, 3H),

1.47 (d, J = 7.2 Hz, 3H); IR (film): 3365, 2204, 1658 cm−1; HRMS-TOF m/z (M + Na)+ calcd for C19H18N2O3Na 345.1215, found 345.1209.

(E)-N-benzyl-2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylamide, (FLLL-103). An off-white solid: mp 158 °C; 1H NMR (DMSO, 300 MHz) 10.26 (s, 1H), 8.84 (t, J = 6

Hz, 1H), 8.09 (s, 1H), 7.66 (d, J = 2.1 Hz, 1H), 7.49 (dd, J = 1.8, 8.4 Hz, 1H), 7.31 (m,

5H), 6.94 (d, J = 8.4 Hz, 1H), 4.41 (d, J = 6 Hz, 3H), 3.81 (s, 3H); IR (film): 3367, 2209,

−1 + 1660 cm ; HRMS-TOF m/z (M + Na) calcd for C18H16N2O3Na 331.1059, found

331.1043.

(S,E)-2-cyano-N-(1-phenylethyl)-3-(pyridin-2-yl)acrylamide, (FLLL-115). Brown needles: mp 99-100 °C; 1H NMR (DMSO, 300 MHz) 8.96 (d, J = 9 Hz, 1H), 8.77 (d, J

= 6 Hz, 1H), 8.10 (s, 1H), 7.99 (t, J = 6 Hz, 2H), 7.84 (d, J = 6 Hz, 1H), 7.54 (dd, J = 6,

183

9 Hz, 1H), 7.25 (m, 1H), 5.06 (m, 1H), 1.48 (d, J = 7.2 Hz, 3H); IR (film): 3328, 2215,

1670 cm−1.

(E)-N-benzyl-2-cyano-3-(pyridin-2-yl)acrylamide, (FLLL-116). White crystalline needles: mp 135-137 °C; 1H NMR (DMSO, 300 MHz) 9.07 (t, J = 5.7 Hz, 1H), 8.77

(d, J = 6 Hz, 1H), 8.18 (s, 1H), 7.97 (dt, J = 1.8, 7.8 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H),

7.55 (m, 1H), 7.33 (m, 5H), 4.43 (d, J = 5.7 Hz, 2H); IR (film): 3334, 2201, 1672 cm−1;

+ HRMS-TOF m/z (M + Na) calcd for C16H13N3ONa 286.0956, found 286.0946.

(E)-N-benzyl-3-(3-bromophenyl)-2-cyanoacrylamide, (FLLL-114). White needles: mp

157 °C; 1H NMR (DMSO, 300 MHz) 9.07 (t, J = 5.7 Hz, 1H), 8.21 (s, 1H), 8.13 (br s,

1H), 7.95 (t, J = 9 Hz, 1H), 7.79 (d, J = 9 Hz, 1H), 7.53 (t, J = 7.8 Hz 1H), 7.33 (m, 5H),

4.42 (d, J = 5.7 Hz, 2H); IR (film): 3352, 2220, 1676 cm−1.

184

(S,E)-3-(3-bromophenyl)-2-cyano-N-(1-phenylethyl)acrylamide, (FLLL-113). White crystals: mp 96 °C; 1H NMR (DMSO, 300 MHz) 8.93 (d, J = 7.8 Hz, 1H), 8.15 (s, 1H),

8.11 (t, J = 1.8 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.8

Hz, 1H), 7.36 (m, 4H), 7.24 (m, 1H), 5.05 (m, 1H), 1.47 (d, J = 7.2 Hz, 3H); IR (film):

−1 + 3341, 2214, 1674 cm ; HRMS-TOF m/z (M + Na) calcd for C18H15BrN2ONa 377.0265, found 377.0250.

(S,E)-3-(1H-benzo[d]imidazol-5-yl)-2-cyano-N-(1-phenylethyl)acrylamide,

(FLLL-107). White solid (52%): mp 228 °C; 1H NMR (DMSO, 300 MHz) 12.87 (s,

1H), 8.88 (d, J = 6.6 Hz, 1H), 8.41 (s, 1H), 8.32 (s, 1H), 8.29 (s, 1H), 7.84 (d, J = 8.4

Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.37 (m, 4H), 7.24 (m, 1H), 5.07 (m, 1H), 1.49 (d, J =

6.9 Hz, 3H); IR (film): 3380, 2201, 1650 cm−1; HRMS-TOF m/z (M + Na)+ calcd for

C19H16N4ONa 339.1222, found 339.1209.

(S,E)-3-(2-bromophenyl)-2-cyano-N-(1-phenylethyl)acrylamide, (FLLL-161). Yellow

1 solid (85%): mp 113-114 °C; H NMR (CDCl3, 400 MHz) 8.69 (s, 1H), 8.03 (d, J = 8

Hz, 1H), 7.69 (d, J = 8 Hz, 1H), 7.37 (m, 7H), 6.55 (d, J = 6.5 Hz, 1H), 5.25 (m, 1H),

185

13 1.60 (d, J = 6.9 Hz, 3H); C NMR (CDCl3, 400 MHz) 158.9, 152.7, 142.4, 134.0,

133.6, 132.6, 130.0, 129.3, 128.3, 128.2, 126.8, 126.6, 116.6, 107.9, 72.2, 50.6, 22.0; IR

−1 + (film): 3304, 2216, 1665 cm ; HRMS-TOF m/z (M + Na) calcd for C18H15BrN2ONa

377.0265, found 377.0257.

(S,E)-3-(3-chlorophenyl)-2-cyano-N-(1-phenylethyl)acrylamide, (FLLL-163). White

1 solid (98%): mp 96-97 °C; H NMR (CDCl3, 400 MHz) 8.26 (s, 1H), 7.84 (m, 1H),

7.81 (d, J = 4 Hz, 1H), 7.50 (d, J = 8 Hz, 1H), 7.44 (d, J = 8 Hz, 1H), 7.37 (m, 4H),

7.31(m, 1H), 6.56 (d, J = 7.8 Hz, 1H), 5.24 (m, 1H), 1.60 (d, J = 6.9 Hz, 3H); 13C NMR

(CDCl3, 400 MHz) 158.7, 151.4, 141.9, 135.2, 133.3, 132.5, 130.4, 130.2, 128.8, 128.2,

127.7, 126.1, 116.4, 105.5, 50.1, 21.6; IR (film): 3344, 2216, 1665, 1202 cm−1; HRMS-

+ TOF m/z (M + Na) calcd for C18H15ClN2ONa 333.0771, found 333.0765.

(S,E)-3-(4-bromophenyl)-2-cyano-N-(1-phenylethyl)acrylamide, (FLLL-167). White

1 solid (98%): mp 81-82 °C; H NMR (CDCl3, 400 MHz) 8.26 (s, 1H), 7.77 (d, J = 8.5

Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.36 (m, 4H), 7.30 (d, J = 8 Hz, 1H), 7.37 (m, 1H),

13 6.56 (d, J = 8.0 Hz, 1H), 5.24 (m, 1H), 1.60 (d, J = 6.9 Hz, 3H); C NMR (CDCl3, 400

186

MHz) 158.9, 151.7, 142.0, 132.5, 131.8, 130.5, 128.8, 127.7, 127.6, 126.1, 116.7,

104.5, 50.1, 21.6; IR (film): 3343, 2212, 1675 cm−1; HRMS-TOF m/z (M + Na)+ calcd for

C18H15BrN2ONa 377.0265, found 377.0266.

(S,E)-2-cyano-3-(3-fluorophenyl)-N-(1-phenylethyl)acrylamide, (FLLL-162). White

1 solid (95%): mp 74-76 °C; H NMR (CDCl3, 400 MHz) 8.28 (s, 1H), 7.64 (m, 2H),

7.46 (m, 1H), 7.37 (m, 4H), 7.31(m, 1H), 7.23 (ddt, J = 0.8, 2.5, 8.2 Hz, 1H), 5.24 (m,

1H), 1.60 (d, J = 6.9 Hz, 3H); IR (film): 3344, 2217, 1670, 1232 cm−1; HRMS-TOF m/z

+ (M + Na) calcd for C18H15FN2ONa 317.1066, found 317.1073.

(S,E)-2-cyano-N-(1-phenylethyl)-3-(pyridin-3-yl)acrylamide, (FLLL-164). White

1 solid (41%): mp 84-85 °C; H NMR (CDCl3, 400 MHz) 8.92 (br s, 1H), 8.72 (dd, J =

1.4, 4.7 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.33 (s, 1H), 7.44 (m, 1H), 7.36 (m, 4H), 7.30

(m, 1H), 6.59 (d, J = 6.7 Hz, 1H), 5.24 (m, 1H), 1.61 (d, J = 6.1 Hz, 3H); 13C NMR

(CDCl3, 400 MHz) 158.4, 152.9, 152.3, 149.5, 141.8, 135.6, 128.8, 127.8, 127.7, 126.1,

123.9, 116.3, 106.4, 50.2, 21.5; IR (film): 3302, 2209, 1665 cm−1; HRMS-TOF m/z (M +

+ Na) calcd for C17H15N3ONa 300.1113, found 300.1101.

187

(S)-2-cyano-N-(1-phenylethyl)-3-(pyridin-4-yl)acrylamide, (FLLL-165). White solid

1 (45%): mp 109-110 °C; H NMR (CDCl3, 400 MHz) 8.79 (d, J = 5.0 Hz, 2H), 8.27 (br s, 1H), 6.67 (d, J = 6.0 Hz, 2H), 7.37 (m, 4H), 7.33 (m, 1H), 6.59 (d, J = 6.6 Hz, 1H),

13 5.24 (m, 1H), 1.61 (d, J = 6.9 Hz, 3H); C NMR (CDCl3, 400 MHz) 157.9, 151.0,

150.3, 141.7, 138.4, 128.9, 127.9, 126.1, 123.0, 115.7, 109.1, 71.8, 50.3, 30.9, 21.5; IR

−1 + (film): 3305, 2218, 1670 cm ; HRMS-TOF m/z (M + Na) calcd for C17H15N3ONa

300.1113, found 300.1104.

(S,Z)-2-cyano-3-(3-iodophenyl)-N-(1-phenylethyl)acrylamide, (FLLL-168). White

1 solid (50%): mp 123 °C; H NMR (CDCl3, 400 MHz) 8.24 (s, 1H), 8.18 (s, 1H), 7.91

(dd, J = 2.8,7.8 Hz, 2H), 7.34 (m, 6H), 6.59 (d, J = 6.6 Hz,, 1H), 5.27 (m, 1H), 1.63 (d, J

13 = 6.6 Hz, 3H); C NMR (CDCl3, 400 MHz) 157.9, 151.0, 150.3, 141.7, 138.4, 128.9,

127.9, 126.1, 123.0, 115.7, 109.1, 71.8, 50.3, 30.9, 21.5; IR (film): 3337, 2214, 1670,

−1 + 1199 cm ; HRMS-TOF m/z (M + Na) calcd for C18H15IN2ONa 425.0127, found

425.0128.

188

N-(3-(benzyloxy)benzyl)-3-(6-bromopyridin-2-yl)-2-cyanoacrylamide, (FLLL-170).

1 White solid (73%): mp 170 °C; H NMR (CDCl3, 400 MHz) 8.28 (s, 1H), 7.69 (m, 1H),

7.61 (m, 2H), 7.42 (m 4H), 7.32 (m, 2H), 6.95 (m, 4H), 5.09 (s, 2H), 4.61 (d, J = 4.6 Hz,

13 2H); C NMR (CDCl3, 400 MHz) 159.3, 159.1, 150.7, 148.4, 142.4, 139.1, 138.4,

136.7, 130.6, 129.9, 128.5, 127.9, 127.4, 125.5, 120.2, 115.6, 114.3, 109.1, 69.9, 44.4; IR

−1 + (film): 3343, 2221, 1671 cm ; HRMS-TOF m/z (M + Na) calcd for C23H18BrN3O2Na

470.0480, found 470.0490.

(E)-N-(4-(benzyloxy)benzyl)-3-(6-bromopyridin-2-yl)-2-cyanoacrylamide,

1 (FLLL-171). White solid (38%): mp 167 °C; H NMR (CDCl3, 400 MHz) 8.25 (s, 1H),

7.67 (m, 2H), 7.59 (m, 2H), 7.40 (m, 5H), 7.26 (m, 3H), 6.96 (m, 2H), 6.84 (br s, 1H),

13 5.06 (s, 2H), 4.54 (d, J = 5.7 Hz, 2H); C NMR (CDCl3, 400 MHz) 159.6, 158.9,

151.2, 148.7, 142.8, 139.5, 137.2, 131.1, 129.7, 129.6, 129.0, 128.4, 127.9, 125.9, 116.1,

115.6, 109.7, 72.2, 70.4, 44.5; IR (film): 3339, 2220, 1671 cm−1; HRMS-TOF m/z (M +

+ Na) calcd for C23H18BrN3O2Na 470.0480, found 470.0497.

189

(Z)-3-(6-bromopyridin-2-yl)-2-cyano-N-phenethylacrylamide, (FLLL-166). White

1 solid: mp 176-178 °C; H NMR (CDCl3, 400 MHz) 8.19 (s, 1H), 7.66 (m, 1H), 7.57 (d,

J = 7.5 Hz, 2H), 7.33 (m, 2H), 7.24 (m, 3H), 6.63 (br s, 1H), 3.68 (q, J = 7 Hz , 2H), 2.92

13 (t, J = 7 Hz, 2H); C NMR (CDCl3, 400 MHz) 159.2, 150.8, 147.9, 142.4, 139.1,

138.0, 130.6, 128.8, 128.7, 126.8, 125.5, 115.6, 109.3, 71.8, 41.8, 35.3; IR (film): 3318,

−1 + 2228, 1665 cm ; HRMS-TOF m/z (M + Na) calcd for C17H14BrN3ONa 378.0218, found

378.0210.

(E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(3-phenylpropyl)acrylamide, (FLLL-169).

1 White solid: mp 158-159 °C; H NMR (CDCl3, 400 MHz) 8.20 (s, 1H), 7.67 (m, 1H),

7.58 (d, J = 7.4 Hz, 2H), 7.28 (m, 2H), 7.20 (m, 3H), 6.61 (br s, 1H), 3.46 (dd, J = 6.8,

13 13.4 Hz , 2H), 2.70 (t, J = 7.7 Hz, 2H), 1.96 (m , 2H) ; C NMR (CDCl3, 400 MHz)

159.3, 150.8, 147.9, 142.4, 140.8, 139.1, 130.6, 128.5, 128.5, 128.3, 126.1, 125.4,

115.8, 109.3, 71.8, 40.1, 33.0, 30.7; IR (film): 3346, 2227, 1675 cm−1; HRMS-TOF m/z

+ (M + Na) calcd for C18H16BrN3ONa 392.0374, found 392.0368.

190

(S,E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(1,2,3,4-tetrahydronaphthalen-1-

1 yl)acrylamide, (FLLL-172). White solid (93%): mp 56-58 °C; H NMR (CDCl3, 400

MHz) 8.29 (s, 1H), 7.67 (m, 1H), 7.63 (dd, J = 1.1, 7.7 Hz , 1H), 7.58 (dd, J = 1.2, 7.6

Hz , 1H), 7.24 (m, 1H), 7.20 (dd, J = 1.8, 7.7 Hz , 2H), 7.14 (m, 1H), 6.74 (d, J = 8.7 Hz ,

2H), 5.32 (m , 1H), 2.13 (m, 1H), 1.91 (m , 3H).

(E)-N-benzhydryl-3-(6-bromopyridin-2-yl)-2-cyanoacrylamide, (FLLL-173). White

1 solid (76%): mp 175-176 °C; H NMR (CDCl3, 400 MHz) 8.23 (s, 1H), 7.66 (m, 1H),

7.58 (d, J = 7.5 Hz , 2H), 7.36 (m , 4H), 7.29 (m, 6H), 7.16 (d, J = 7.7 Hz , 1H), 6.36 (d, J

13 = 7.7 Hz , 1H); C NMR (CDCl3, 400MHz) 158.5, 150.7,148.6, 142.4, 140.2, 139.1,

130.7, 128.8, 127.9, 127.3, 125.5, 115.6, 109.1, 58.0.

Synthesis of 3-/4- (benzyloxy)phenyl)methanamine

191

General procedure for benzyl protection of cyanophenols

The reaction was done using the conditions reported by Lee et al 133 where the cyanophenol (3-cyanophenol or 4-cyanophenol) (10 mmol) was dissolved in 40 ml acetone. K2CO3 was added and stirred for 10 min at rt. Benzyl bromide (12 mmol) was then added and the reaction was refluxed for 6 h. The reaction was filtered, concentrated, and the crude material was purified by silica gel column chromatography (EtOAc- hexanes, 5:95) to give the desired products.

1 4-(benzyloxy)benzonitrile. White solid (99%): mp 96 °C; H NMR (CDCl3, 400 MHz)

7.58 (d, J = 8.0 Hz, 2H), 7.41 (m, 5H), 7.02 (d, J = 8.0 Hz, 2H), 5.11 (s, 2H); 13C NMR

(CDCl3, 400 MHz) 161.7, 135.5, 134.1, 133.8, 128.5, 128.4, 128.2, 127.3, 119.0, 115.4,

103.9, 70.0; IR (film): 3421, 2221, 1607 cm−1.

1 3-(benzyloxy)benzonitrile. White crystals (94%): mp 52 °C; H NMR (CDCl3, 400

13 MHz) 7.39 (m, 6H), 7.27 (m, 3H), 5.08 (s, 2H); C NMR (CDCl3, 400 MHz) 159.1,

136.2, 130.8, 129.1, 128.8, 128.1, 127.9, 125.1, 120.5, 119.1, 118.2, 113.6, 70.7; IR

(film): 2230 cm−1.

192

General Procedure for reduction of benzylprotected cyanophenols

The reduction conditions in Maslak et al 134 were used to reduce the nitrile group into primary amine. The protected cyanophenols (4 mmol) were dissolved in dry THF and cooled to 0°C. Lithium aluminium tetrahydride (6 mmol) was added portionwise at 0°C, and allowed to warm to rt for 6 h. The reation was cooled again to 0°C, then quenched with water followed by 15% NaOH then water. The reaction mixture was filtered and the solvent was removed. The crude material was used for the next reaction without further purification.

1 (3-(benzyloxy)phenyl)methanamine. Amorphous yellow solid (99%): H NMR (CDCl3,

400 MHz) 7.46 (m, 2H), 7.41 (m, 2H), 7.34 (m, 1H), 7.26 (m, 1H), 6.98 (br s, 1H), 6.92

(d, J = 8.0 Hz 1H), 6.88 (dd, J = 4.0, 8.0 Hz 2H), 5.08 (s, 2H), 3.85 (s, 2H); 13C NMR

CDCl3, 400 MHz) 158.8, 144.8, 136.8, 129.4, 128.4, 128.3, 128.1, 127.9, 127.7, 127.3,

126.6,119.4, 113.4, 113.1, 112.8, 112.5, 69.7, 46.2.

193

(4-(benzyloxy)phenyl)methanamine. White solid (93%): mp 115-118 °C; 1H NMR

(CDCl3, 400 MHz) 7.41 (m, 2H), 7.33 (m, 2H), 7.23 (d, J = 8.5 Hz, 2H), 6.95 (d, J =

13 8.5 Hz, 1H), 5.06 (s, 2H), 3.80 (s, 2H); C NMR (CDCl3, 400 MHz) 157.6, 137.0,

135.7, 128.5, 128.2, 127.8, 127.4, 114.8, 69.9, 45.8; IR (film): 3328, 2241, 1654 cm−1.

(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione. (Curcumin)

Boric oxide (35 mg, 0.5 mmol) was dissolved in 0.1 ml DMF. Acetylacetone (51 µL, 1 mmol) and tributylborate (270 µL, 2 mmol) were added and stirred at 65°C for 15 min.

Vanillin (152 mg, 2 mmol) and stirred for min. Tetrahydoquinoline (THQ) (10 µL), acetic acid (30 µL), and DMF (0.1 ml) were mixed and added to the reaction mixture and stirred for 4 h at 95°C. The reaction was cooled to 0°C and acetic acid (5 ml, 20% aqueous) was added and the reaction was heated again to 70°C for another 1 h. An orange precipitate was formed which was filtered and dried, then recrystalized from ethanol to

1 give curcumin (272 mg, 74%) as yellow crystals: mp 180-182 °C; H NMR (CDCl3, 400

MHz) 7.59 (d, J = 15.7 Hz, 2H), 7.12 (dd, J = 1.6, 8.1 Hz, 2H), 7.05 (d, J = 1.6 Hz,

2H), 6.93 (d, J = 8.1 Hz, 2H), 6.47 (d, J = 15.7 Hz, 2H), 5.86 (br s, 2H), 5.80 (s, 1H) 3.95

(s, 6H).

General procedure for synthesis of pyrazoles or izoxazoles

194

Following a procedure reported by Mishra et al 38(b), curcumin or its derivative (1.2 mmol) was dissolved in glacial acetic acid (5 ml). Hydrazine hydrate (1.5 mmol) or hydrazine hydrochloride (1.5 mmol) was added and the reaction was refluxed for 4 h. The solvent was removed and the residue was dissolved in ethylacetate and washed with water. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 1:1) to give the desired products.

4,4'-((1E,1'E)-(1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol),

(FLLL-7). An orange solid: mp 220-222 °C; 1H NMR (DMSO, 400 MHz) 12.85 (s,

1H), 9.19 (s, 2H), 7.14 (s, 2H), 7.03 (m, 2H), 6.92 (dd, J = 1.5, 8.1Hz, 4H), 6.74 (d, J =

9.4 Hz, 2H), 6.61 (s, 1H), 3.82 (s, 6H).

4,4'-((1E,1'E)-isoxazole-3,5-diylbis(ethene-2,1-diyl))bis(2-methoxyphenol),

195

(FLLL-67). Yellow solid: mp 168-169 °C; 1H NMR (DMSO, 400 MHz) 9.43 (s, 1H),

9.36 (s, 1H), 7.28 (m, 2H), 7.07 (m, 4H), 6.85 (s, 1H), 6.80 (dd, J = 2.9, 8.1 Hz, 2H), 3.83

(s, 6H); 13C NMR (DMSO, 400 MHz) 169.1, 163.1, 148.9, 148.8, 148.7, 148.6, 137.3,

135.6, 128.1, 127.8, 122.5, 122.1, 116.4, 116.3, 113.4, 111.1, 110.8, 98.7, 72.2, 56.5.

3,5-bis((E)-3,4-dimethoxystyryl)-1H-pyrazole, (FLLL-68). Off- white solid: mp 176

°C; 1H NMR (DMSO, 400 MHz) 12.89 (s, 1H), 7.19 (br s, 2H), 7.03 (m, 6H), 6.96 (br s, 2H), 6.65 (s, 1H), 3.82 (s, 6H), 3.77 (s, 6H).

3,5-bis((E)-3,4-dimethoxystyryl)isoxazole, (FLLL-69). Orange solid: mp 159-160 °C;

1 H NMR (CDCl3, 400 MHz) 7.30 (d, J = 16.4 Hz, 1H), 7.09 (m, 5H), 7.00 (d, J = 16.4

Hz, 1H), 6.88 (dd, J = 3.0, 8.2 Hz, 2H), 6.83 (d, J = 16.3 Hz, 1H), 6.43 (s, 1H),3.95 (s,

3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H).

196

4,4'-((1E,1'E)-(4-methyl-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2- methoxyphenol), (FLLL-70). Off-white solid: mp 255-258 °C; 1H NMR (DMSO, 400

MHz) 12.77 (s, 1H), 9.07 (br s, 2H), 7.17 (d, J = 1.6 Hz, 2H), 7.03 (m, 6H), 6.76 (d, J =

8 Hz, 2H), 3.83 (s, 6H), 2.24 (s, 3H); 13C NMR (DMSO, 400 MHz) 148.7, 129.1,

116.4, 112.1, 110.3, 72.2, 56.5, 9.4; IR (film): 3366 cm−1; HRMS-TOF m/z (M + Na)+ calcd for C22H22N2O4Na 401.1477, found 401.1464.

4,4'-((1E,1'E)-(4-methylisoxazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol),

1 (FLLL-100). Yellow solid: mp 184-185 °C; H NMR (CDCl3, 400 MHz) 7.31 (d, J =

16.6 Hz, 1H), 7.27 (d, J = 16.3Hz, 1H), 7.05 (m, 4H), 6.92 (dd, J = 3.3, 8.1 Hz, 2H), 6.85

(d, J = 16.3 Hz, 1H), 6.75 (d, J = 16.6Hz, 1H), 5.74 (d, J = 6 Hz, 1H), 3.95 (d, J = 3.8 Hz,

13 1H), 2.24 (s, 3H); C NMR (CDCl3, 400 MHz) 170.3, 137.8, 129.7, 129.4, 129.2, 78.3,

56.5, 53.8, 46.4, 36.0, 20.9; IR (film): 3566 cm−1. HRMS-TOF m/z (M + Na)+ calcd for

C22H21NO5Na 402.1317, found 402.1306.

General procedure for synthesis of pyrimidine analogues

197

Using the procedure reported by Dubois and coworkers 82; sodium hydroxide (12.5 ml of

5 M solution) was mixed with 125 µL of aliquat and heated to reflux. 2,6-

Dimethylpyrimidine (296 µL, 2.5 mmol) was added and stirred for 5 min, then 3- methoxybenzaldehyde or 3,4-dimethoxybenzaldehyde (5 mmol) was added portionwise and refluxed for 1 h. After cooling a precipitate was formed which was filtered, washed with ethanol, and recrystalized from ethanol.

4,6-bis((E)-3,4-dimethoxystyryl)pyrimidine,(1E,6E)-1,7-bis(4-hydroxy-3- methoxyphenyl)hepta-1,6-diene-3,5-dione (FLLL-59). Yellow solid: mp 127-129 °C;

1 H NMR (CDCl3, 300 MHz) 9.05 (d, J = 0.9 Hz, 1H), 7.84 (d, J = 15.9 Hz, 2H), 7.28

(s, 1H), 7.19 (s, 1H), 7.16 (s, 3H), 6.94 (d, J = 15.9 Hz, 2H), 6.90 (d, J = 7.8 Hz, 2H),

13 3.95 (s, 6H), 3.92 (s, 6H) ); C NMR (CDCl3, 400 MHz) 162.7, 158.5, 150.3, 149.1,

136.7, 128.6, 121.7, 115.6, 111.1, 109.3, 56.8, 56.7; IR (film): 3005 cm−1; HRMS-TOF

+ m/z (M + Na) calcd for C24H24N2O4Na 427.1634, found 427.1622.

198

4,6-bis((E)-4-methoxystyryl)pyrimidine, (FLLL-58). Yellow crystals: mp 183-185 °C;

1 H NMR (CDCl3, 300 MHz) 9.05 (s, 1H), 7.86 (d, J = 15.9 Hz, 2H), 7.58 (d, J = 9.0

Hz, 4H), 7.23 (d, J = 1.2 Hz, 1H), 6.93 (m, 6H), 3.85 (s, 6H).

Synthesis of aryl analogues

Tetraethyl (1,3-phenylenebis(methylene))bis(phosphonate). Dibromxylene (1.36 g, 5 mmol) and triethylphosphite (2.66 ml, 15 mmol) were mixed and heated to 180°C for 16 h. NMR analysis showed completion of the reaction to give colorless oil; 1H NMR

(CDCl3, 300 MHz) 7.08 (m, 4H), 3.87 (m, 8H), 2.99 (d, J = 21.9 Hz, 4H), 1.30 (m,

12H).

1,3-bis((E)-3,4-dimethoxystyryl)benzene, (FLLL-60). Prepared using the conditions reported by Kabir et al , where NaH (80 mg, 2 mmol, 60%) was suspended in dry THF (8 ml) and cooled to -10°C. 3,4-Dimethoxybenzaldehyde (332 mg, 2 mmol) was dissolved in 17 ml of dry THF and added to NaH, and the reaction was warmed to rt then refluxed for 2 h. the reaction was quenched with NH4Cl and the aqueous layer was extracted with ethylacetate. The combined organic layers were dried over sodium sulfate and

199 concentrated. The crude material was purified by silica gel column chromatography

(EtOAc-hexanes, 1:5) to give (FLLL-60) as white crystals: mp 177-178 °C; 1H NMR

(CDCl3, 300 MHz) 7.63 (s, 1H), 7.38 (m, 3H), 7.11 (d, J = 16.2 Hz, 2H), 7.09 (s, 3H),

7.06 (d, J = 2.1Hz, 1H), 6.99 (d, J = 16.2 Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 3.96 (s, 6H),

3.91 (s, 6H).

3-methoxy-4-(methoxymethoxy)benzaldehyde. Following Jogireddy et al 81, vanillin

(230 mg, 1.5 mmol) was dissolved in 4.4 ml DMF. DIEA (496 µL, 3 mmol) was added and the reaction was cooled to 0°C. Chloromethyl-methylether (MOMCl) (230 µL, 3 mmol) was added dropwise, and the reaction was allowed to warm gradually to rt and stirred for 16 h. Ice cold water was added and the reaction mixture was extracted with ether. The organic layer was washed with 5% NaOH, then brine. The combined organic layers were dried over sodium sulfate and then concentrated. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 15:85) to give white

1 solid: H NMR (CDCl3, 400 MHz) 9.79 (s, 1H), 7.35 (dd, J = 1.8, 6.7 Hz, 2H), 7.20 (d,

13 J = 6.7 Hz, 1H), 5.25 (s, 2H), 3.87 (s, 3H), 3.48 (s, 3H); C NMR (CDCl3, 400 MHz)

190.7, 151.7, 149.8, 130.8, 126.1, 114.4, 109.2, 94.7, 56.3, 55.8.

200

(E)-4-(3-methoxy-4-(methoxymethoxy)phenyl)but-3-en-2-one. 3-methoxy-4-

(methoxymethoxy)benzaldehyde (274 mg, 1.4 mmol) and 1-(Triphenyl- phosphoranylidene)-2-propanone (540 mg, 1.6 mmol) were suspended in 1.75 ml of dry toluene and heated to 40°C for 16 h. the reaction was quenched with water, and the organic layer was dried over sodium sulfate and then concentrated. The crude material was purified by silica gel column chromatography (EtOAc-hexanes, 2:8) to give white

1 needles: mp 74 °C; H NMR (CDCl3, 400 MHz) 7.45 (d, J = 16.2 Hz, 1H), 7.15 (d, J =

8.8 Hz, 1H), 7.09 (dd, J = 1.9, 9.2 Hz, 2H), 6.61 (d, J = 16.2 Hz, 1H), 5.26 (s, 2H), 3.91

13 (s, 3H), 3.50 (s, 3H), 2.36 (s, 3H),; C NMR (CDCl3, 400 MHz) 198.2, 149.7, 148.6,

143.2, 128.5, 125.6, 122.5, 115.6, 110.1, 95.0, 56.2, 55.8, 27.2.

1,3-bis((E)-3-methoxy-4-(methoxymethoxy)styryl)benzene, (FLLL-99). FLLL-99 was prepared using the same procedure for FLLL-60 to give a white solid: mp 87 °C; 1H

NMR (CDCl3, 400 MHz) 7.63 (s, 1H), 7.37 (s, 3H), 7.14 (m, 3H), 7.06(m, 7H), 5.26 (s,

4H), 3.96 (s, 6H), 3.54 (s, 6H), 5.74 (d, J = 6 Hz, 1H), 3.95 (d, J = 3.8 Hz, 1H), 2.24 (s,

13 3H); C NMR (CDCl3, 400 MHz) 149.7, 146.3, 137.7, 131.9, 128.9, 128.5, 127.1,

201

125.3, 124.3, 119.7, 116.2, 109.2, 95.4, 56.2, 55.8, 30.9; IR (film): 3445 cm−1. HRMS-

+ TOF m/z (M + Na) calcd for C28H30O6Na 485.1940, found 485.1932.

4,4'-((1E,1'E)-1,3-phenylenebis(ethene-2,1-diyl))bis(2-methoxyphenol), (FLLL-98).

(FLLL-99) (115 mg, 0.25 mmol) was dissolved in 2.1 ml of MeOH. 0.33 ml HCl (3M) was added and the reaction refluxed for 2 h. After completion of the reaction the solvent was removed and the residue was purified by silica gel column chromatography (EtOAc-

1 hexanes, 1:5) to give (FLLL-98) as an off-white solid: mp 168-169 °C; H NMR (CDCl3,

400 MHz) 7.60 (s, 1H), 7.34 (m, 3H), 7.06 (m, 6H), 6.93(m, 4H), 5.68 (s, 2H), 3.96 (s,

6H), 3.54 (s, 6H), 5.74 (d, J = 6 Hz, 1H), 3.95 (d, J = 3.8 Hz, 1H), 2.24 (s, 3H); 13C NMR

(CDCl3, 400 MHz) 146.6, 145.6, 137.8, 129.9, 128.9, 128.8, 126.3, 125.1, 124.1, 120.5,

114.5, 108.1, 55.8.

3β-((tert-butyldimethylsilyl)oxy)-5-Pregnen-20-one (1). Pregnenolone (3β-Hydroxy-5- pregnen-20-one) (3.23 g, 10 mmol) was dissolved in DMF, imidazole was added and stirred for 5 min. TBSCl was added and stirred at rt for 16 h. White precipitate was 202 formed which could be recrystalized from chloroform (CHCl3). The filtrate was also extracted with ether, dried over sodium sulfate, and concentrated. Purification by silica gel column chromatography (EtOAc-hexanes, 1:9) to give 1 (2.92 g, 68%) as white

1 crystals: mp 163 °C; H NMR (CDCl3, 400 MHz) 5.31 (d, J = 5.1 Hz, 1H), 3.47 (m,

1H), 2.52 (m, 1H), 2.21 m, 3H), 2.11 (m, 3H), 2.01 (m, 2H), 1.91 (m, 1H), 1.27 (m, 9H),

13 1.08 (m, 4H), 0.99 (s, 3H), 0.88 (s, 9H), 0.62 (s, 3H), 0.05 (s, 6H); C NMR (CDCl3, 400

MHz)  43.9, 42.7, 38.8, 37.3, 36.5, 32.0,

31.8, 31.7, 31.5, 25.9, 24.4, 22.7, 21.0, 19.4, 18.2, 13.2, -4.6; IR (film): 1701 cm−1;

+ HRMS-TOF m/z (M + Na) calcd for C27H46O2Si 453.3165, found 453.3165.

General Procedure for Alkylation

15 ml of THF was transferred to flame dried flask and cooled to -78°C. LDA (1.65 ml,

3.3 mmol) was added dropwise and cooled for 5 min. To a solution of (1) in 15 ml THF

(1.29 g, 3 mmol) was added 522 µl HMPA (3 mmol). This solution was added to the solution of LDA dropwise with stirring for 3 hr at -78°C. 1.3 Equivalent of the alkyl reagent was added then allowed to warm gradually to rt overnight. The reaction was quenched with NH4Cl and stirred for 10 min. The organic layer was separated and the aqueous layer was extracted three times with dichloromethane (DCM). The combined organic layers were dried over sodium sulfate and concentrated. The crude material was 203 purified by silica gel column chromatography (DCM-hexanes, 1:5) to give the alkylated products.

Monoalkylated Sterol (2). (1.17 g, 78 %) as a white solid: mp 120-121 °C; 1H NMR

(CDCl3, 400 MHz) 5.30 (br s, 1H), 5.06 (m, 1H), 3.47 (m, 1H), 2.50 (m, 1H), 2.38 (m,

2H), 2.21 (m, 5H), 2.00 (m, 2H), 1.81 (m, 1H), 1.66 (s, 3H), 1.60 (s, 3H), 1.48 (m, 3H),

13 0.99 (s, 3H), 0.88 (s, 9H), 0.60 (s, 3H), 0.05 (s, 6H); C NMR (CDCl3, 400 MHz)

215.4, 141.9, 133.9, 133.2, 123.2, 121.8, 121.3, 72.9, 72.2, 63.4, 57.5, 53.8, 50.5, 45.1,

43.1, 39.2, 35.0, 32.2, 31.5, 29.0, 26.3, 26.2, 26.1, 21.4, 18.6, 18.1, 14.5, 13.6, -4.1; IR

−1 + (film): 1704 cm ; HRMS-TOF m/z (M + Na) calcd for C32H54O2Si 521.3791, found

521.3814.

204

1 Bisalkylated Sterol (3). (0.10 g, 7%) as a white solid: mp 92-93 °C; H NMR (CDCl3,

400 MHz) 5.30 (d, J = 5.0 Hz, 1H), 5.04 (m, 2H), 3.46 (m, 1H), 2.56 (m, 2H), 2.14 (m,

2H), 2.89 (m, 3H), 2.01 (m, 4H), 1.67 (s, 3H), 1.64 (s, 3H), 1.59 (s, 3H), 1.55 (s, 3H),

1.38 (m, 7H), 1.25 (m, 5H), 0.98 (s, 3H), 0.95 (m, 3H), 0.88 (s, 9H), 0.59 (s, 3H), 0.05

13 (s, 6H); C NMR (CDCl3, 400 MHz) 211.3, 141.5, 132.4, 123.1, 120.8, 72.5, 62.9,

56.9, 50.0, 44.3, 44.1, 42.7, 38.9, 37.3, 36.5, 32.0, 31.8, 25.6, 24.5, 22.8, 22.4, 21.0, 19.4,

18.2, 17.6, 13.3, -4.6; IR (film): 1703 cm−1; HRMS-TOF m/z (M + Na)+ calcd for

C37H62O2Si 589.4417, found 589.4418.

1 Alkylated Sterol (4). A white solid: mp 92-93 °C; H NMR (CDCl3, 400 MHz) 5.41

(m, 1H), 5.31 (m, 2H), 3.48 (m, 1H), 2.50 (m, 1H), 2.41 (m, 2H), 2.20 (m, 5H), 2.01 (m,

2H), 1.62 (br s, 3H), 1.58 (s, 10H), 1.24 (br s, 3H), 0.98 (s, 3H), 0.88 (s, 9H), 0.6 (s, 3H),

13 0.05 (s, 6H); C NMR (CDCl3, 400 MHz) 211.3, 141.5, 130.3, 129.5, 125.1, 121.2,

72.9, 63.3, 57.4, 50.4, 44.6, 44.5, 44.3, 39.3, 37.7, 36.9, 32.4, 32.2, 31.9, 27.1, 26.3, 24.9,

23.3, 23.0, 21.6, 19.8, 18.6, 18.2, 14.5, 13.7, 13.1, -4.1; IR (film): 1704 cm−1; HRMS-

+ TOF m/z (M + Na) calcd for C31H52O2Si 507.3634, found 507.3644.

205

1 Alkylated Sterol (5). A white solid: H NMR (CDCl3, 400 MHz) 5.30 (s, 1H), 5.07 (m,

1H), 3.48 (m, 1H), 2.51 (m, 1H), 2.33 (m, 2H), 2.17 (m, 3H), 1.97 (m, 3H), 1.80 (m, 2H),

1.68 (s, 3H), 1.58 (s, 3H), 1.52 (m, 3H), 1.25 (m, 10H), 0.99 (s, 3H), 0.88 (s, 9H), 0.60 (s,

3H), 0.05 (s, 6H).

1 Alkylated Sterol (6). A white solid: mp 110 °C; H NMR (CDCl3, 400 MHz) 5.30 (br s, 1H), 3.47 (m, 1H), 2.51 (m, 1H), 2.38 (m, 1H), 2.20 (m, 3H), 1.98 (m, 2H), 1.58 (m,

12H), 1.13 (m, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.88 (s, 9H), 0.60 (s, 3H), 0.05 (s, 6H); 13C

NMR (CDCl3, 400 MHz) 212.0, 141.5, 120.8, 72.5, 71.8, 62.6, 56.9, 50.0, 44.1, 42.7,

38.8, 37.3, 36.5, 32.0, 31.8, 25.9, 24.5, 22.9, 21.0, 19.3, 18.2, 13.3, 7.7, -4.6; IR (film):

−1 + 1707 cm ; HRMS-TOF m/z (M + Na) calcd for C28H48O2Si 467.3321, found 467.3330.

206

1 Alkylated Sterol (7). A white solid: mp 104 °C; H NMR (CDCl3, 400 MHz) 5.31 (d, J

= 5.4 Hz, 1H), 3.47 (m, 1H), 2.51 (m, 1H), 2.35 (m, 2H), 2.17 (m, 3H), 1.98 (m, 2H),

1.82 (m, 1H), 1.62 (s, 12H), 1.25 (m, 10H), 0.99 (s, 3H), 0.88 (s, 9H), 0.60 (s, 3H), 0.05

(s, 6H); IR (film): 1704 cm−1.

1 Alkylated Sterol (8). A white solid: mp 111-113 °C; H NMR (CDCl3, 400 MHz) 5.30

(m, 1H), 3.47 (m, 1H), 2.61 (m, 1H), 2.34 (m, 5H), 1.95 (m, 2H), 1.54 (m, 1H), 1.52 (m,

16H), 1.25 (m, 6H), 1.11 (m, 2H), 0.99 (s, 3H), 0.88 (s, 9H), 0.66 (s, 3H), 0.05 (s, 6H);

13 C NMR (CDCl3, 400 MHz) 211.6, 141.4, 120.8, 72.4, 62.8, 56.9, 50.0, 44.5, 44.1,

42.7, 38.5, 37.3, 36.5, 32.0, 31.8, 25.6, 24.5, 22.8, 22.4, 21.0, 19.4, 18.2, 17.6, 13.3, -4.6;

−1 + IR (film): 1704 cm ; HRMS-TOF m/z (M + Na) calcd for C32H56O2Si 523.3947, found

507.3942.

207

1 Alkylated Sterol (9). A white solid: mp 127-129 °C; H NMR (CDCl3, 400 MHz) 7.29

( m, 2H), 7.21 (m, 3H), 5.30 (d, J = 5.0 Hz, 1H), 3.47 (m, 1H), 2.88 (m, 2H), 2.69 (m,

2H), 2.48 (m, 1H), 2.18 (m, 3H), 1.95 (m, 2H), 1.80 (s, 1H), 1.58 (s, 9H), 1.23 (s, 3H),

13 1.12 (m, 2H), 0.98 (s, 3H), 0.88 (s, 9H), 0.56 (s, 3H), 0.05 (s, 6H); C NMR (CDCl3,

400 MHz) 210.5, 141.5, 128.9, 128.4, 125.9, 120.8, 72.5, 71.8, 63.0 56.9, 50.0, 46.0,

44.2, 42.7, 38.9, 37.3, 36.5, 32.0, 31.8, 31.7, 29.7, 25.9, 24.4, 22.9, 21.0, 19.4, 18.2, 13.3,

−1 + -4.6; IR (film): 1704 cm ; HRMS-TOF m/z (M + Na) calcd for C34H52O2Si 543.3634, found 543.3643.

To a flame dried flask 3 ml dry THF and 3 ml dry toluene were transferred. Tebbe reagent (6 ml, 3 mmol) 0.5M solution/toluene was added dropwise. Compound (2) (498 mg, 1 mmol)was dissolved in 10 ml THF and added dropwise to the solution of Tebbe reagent at rt and stirred for 16 h. The reaction was cooled to 0°C before quenching cautiously with 1M NaOH and stirred for 10 min. The reaction mixture was filtered and

208 the filterate was extracted with DCM. The combined organic layers were dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (DCM-hexanes, 1:9) to give (10) (277 mg, 56 %) as a white solid: mp

1 86 °C; H NMR (CDCl3, 400 MHz) 5.31 (d, J = 5.1 Hz, 1H), 5.10 (m, 1H), 4.87 (s,

1H), 4.78 (s, 1H), 3.48 (m, 1H), 2.26 (m, 1H), 2.07 (m, 7H), 1.79 (m, 3H), 1.68 (s, 3H),

1.61 (s, 3H), 1.51 (s, 10H), 1.18 (m, 3H), 0.99 (s, 3H), 0.88 (s, 9H), 0.57 (s, 3H), 0.05 (s,

13 6H); C NMR (CDCl3, 400 MHz) 149.3, 141.5, 131.4, 124.3, 121.0, 72.5, 71.8, 56.6,

55.9, 50.3, 43.0, 42.7, 38.7, 37.3, 36.6, 32.2, 31.8, 27.1, 25.9, 25.8, 25.7, 21.1, 19.4, 18.2,

17.7, 12.7, 13.3, -4.5; IR (film): 2928 cm−1.

Compound (10) (124 mg, 0.25 mmol) was dissolved in 1.25 ml of dry THF. Tetrabutyl ammonium fluoride (TBAF) 1M solution (0.5 ml, 0.5 mmol) was added and stirred at rt overnight. After completion of the reaction THF was removed and the residue was purified by silica gel column chromatography (EtOAc-DCM-hexanes, 1:4:5) to give (11)

1 (94 mg, 94 %) as a white solid: mp 89-91 °C; H NMR (CDCl3, 400 MHz) 5.35 (d, J =

5.0 Hz, 1H), 5.11 (m, 1H), 4.87 (s, 1H), 4.78 (s, 1H), 3.52 (m, 1H), 2.27 (m, 2H), 2.05

(m, 6H), 1.82 (m, 4H), 1.68 (s, 3H), 1.60 (s, 3H), 1.49 (s, 7H), 1.11 (m, 6H), 1.00 (s, 3H),

13 0.57 (s, 3H); C NMR (CDCl3, 400 MHz) 149.2, 140.7, 131.4, 124.3, 121.6, 109.3,

209

71.8, 71.7, 56.6, 55.9, 50.2, 43.0, 42.2, 38.6, 37.6, 37.2, 36.5, 32.2, 31.8, 31.6, 27.1, 25.8,

+ 25.7, 24.2, 21.1, 19.4, 17.7, 12.7; HRMS-TOF m/z (M + Na) calcd for C27H42O

405.3133, found 405.3151.

General Procedure for Oppenauer Oxidation

Compound (11) (38.2 mg, 0.1 mmol) was dissolved in 2.5 ml of dry toluene, and N- methylpiperidone (1 ml, 8.4 mmol) was added and refluxed for 15 min. Aluminium tri- isopropoxide (245 mg, 1.2 mmol) was added and refluxed for 2 h. The reaction mixture was cooled and washed 4 times with 1% H2SO4, then saturated NaCl and was finally dried over Na2SO4. The organic layer was dried over sodium sulfate and concentrated.

The crude material was purified by silica gel column chromatography (DCM-hexanes,

3:7) to give the oxidized products.

1 PAD2F-3-1K (12) (33 mg, 88 %) as a white solid: mp 40 °C; H NMR (CDCl3, 400

MHz) 5.72 (s, 1H), 5.10 (br s, 1H), 4.88 (s, 1H), 4.79 (s, 1H), 2.36 (m, 3H), 2.28 (m,

1H), 2.04 (m, 6H), 1.82 (m, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.43 (m, 4H), 1.21 (m, 3H),

13 1.18 (m, 3H), 1.05 (m, 4H), 0.60 (s, 3H); C NMR (CDCl3, 400 MHz) 199.5, 171.4,

148.9, 131.4, 124.2, 123.7, 109.6, 55.8, 55.7, 53.8, 43.0, 38.5, 37.5, 25.6, 21.0, 12.8; IR

210

−1 + (film): 1677 cm ; HRMS-TOF m/z (M + Na) calcd for C27H40O 403.2977, found

403.2984.

General Procedure for Grignard addition

Compound (2) (1.29 g, 3 mmol) was dissolved in THF and cooled to -78°C. The Grignard reagent was added dropwise and the reaction was stirred for 2 h. Saturated ammonium chloride was used to quench the reaction and the organic layer was separated. The aqueous layer was extracted with CHCl3. The combined organic layers were dried over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (EthOAc-DCM-hexanes, 4:20:80) to give the addition products.

1 Methyl Grignard Adduct (13). A white solid: mp 175 °C; H NMR (CDCl3, 400 MHz)

5.31 (d, J = 5.0 Hz, 1H), 3.47 (m, 1H), 2.26 (m, 1H), 2.15 (m, 1H), 2.09 (m, 1H), 1.98

(m, 1H), 1.81 (s, 1H), 1.72 (m, 3H), 1.64 (m, 2H), 1.48 (m, 6H), 1.30 (m, 3H), 1.18 (m,

13 3H), 0.99 (s, 3H), 0.88 (s, 9H), 0.83 (s, 3H), 0.05 (s, 6H); C NMR (CDCl3, 400 MHz) 211

142.0, 121.4, 73.9, 73.0, 60.5, 57.2, 43.2, 43.1, 40.5, 37.7, 36.9, 32.4, 32.2, 31.7, 31.4,

30.4, 26.3, 24.2, 23.5, 21.3, 19.8, 18.6, 13.8, -4.1; IR (film): 3320 cm−1; HRMS-TOF m/z

+ (M + Na) calcd for C28H50O2Si 469.3478, found 469.3467.

1 Allyl Grignard Adduct (14). A white solid: mp 139-140 °C; H NMR (CDCl3, 400

MHz) 5.80 (m, 1H), 5.31 (d, J = 5.0 Hz, 1H), 5.08 (m, 2H), 3.45 (m, 1H), 2.25 (m, 1H),

2.17 (m, 3H), 2.08 (m, 1H), 1.96 (m, 1H), 1.71 (m, 5H), 1.48 (m, 6H), 1.34 (s, 1H), 1.27

(s, 3H), 1.13 (m, 2H), 1.11 (m, 2H), 0.99 (s, 3H), 0.88 (s, 9H), 0.85 (s, 3H), 0.04 (s, 6H);

13 C NMR (CDCl3, 400 MHz) 141.5, 134.4, 121.0, 118.2, 74.6, 72.5, 57.9, 56.8, 50.0,

48.1, 42.9, 42.8, 42.7, 40.0, 36.5, 32.0, 31.3, 25.9, 22.3, 20.9, 19.4, 18.2, 13.6, -4.5; IR

−1 + (film): 3548 cm ; HRMS-TOF m/z (M + Na) calcd for C30H52O2Si 495.3634, found

495.3637.

212

Alkylation of compound (13). Compound (13) (112 mg, 0.5 mmol) was dissolved in dry

THF and sodium hydride NaH 60 % (11 mg, 0.265 mmol) was added carefully. Dimethyl allylbromide (23 µl, 0.175 mmol) was added and the reaction was stirred at 50°C for 3 h.

The reaction was quenched with NH4Cl and stirred for 5 min. the organic layer was separated and the aqueous layer was extracted with DCM. The combined organic layers were dried over sodium sulfate and concentrated. The crude material was used directly for the deprotection reaction without further purification.

General procedure for TBS deprotection

Starting material was dissolved in THF (0.2 M), and TBAF (2 equivalent) was added dropwise and stirred for 16 h. The reaction was concentrated and the crude material was purified by silica gel column chromatography (EthOAc-hexanes, 2:8) to give the required products.

1 Alcohol (16). A white solid: mp 121-122 °C; H NMR (CDCl3, 400 MHz) 5.31 (d, J =

5.1 Hz, 1H), 5.26 (m, 1H), 3.86 (d, J = 6.3 Hz, 2H), 5.2 (m, 1H), 2.27 (m, 2H), 2.08 (m,

1H), 1.97 (m, 1H), 1.84 (m, 2H), 1.71 (s, 3H), 1.63 (s, 3H), 1.59 (m, 5H), 1.46 (m, 6H),

13 1.21 (s, 3H), 1.18 (s, 3H), 1.06 (m, 5H), 1.00 (s, 3H), 0.79 (s, 3H); C NMR (CDCl3, 400

MHz) 140.7, 134.1, 123.0, 121.6, 71.8, 59.1, 57.8, 56.8, 50.1, 42.5, 42.2, 40.0, 37.2,

36.5, 31.8, 31.6, 31.4, 25.7, 25.6, 25.4, 23.8, 23.2, 20.9, 19.3, 18.0, 13.9; IR (film): 3250

−1 + cm ; HRMS-TOF m/z (M + Na) calcd for C27H44O2 423.3239, found 423.3224.

213

1 Alcohol (17). A white solid: mp 190-191 °C; H NMR (CDCl3, 400 MHz) 5.34 (d, J =

5.1 Hz, 1H), 3.51 (m, 1H), 2.27 (m, 2H), 2.08 (m, 1H), 1.97 (m, 1H), 1.84 (m, 2H), 1.71

(m, 2H), 1.65 (s, 2H), 1.48 (m, 8H), 1.30 (s, 3H), 1.18 (s, 3H), 1.00 (s, 3H), 1.23 – 0.92

13 (m, 4H), 0.83 (s, 3H); C NMR (CDCl3, 400 MHz) 73.1, 71.7, 60.1, 56.7, 49.9, 42.7,

42.6, 40.0, 37.2, 36.4, 31.7, 31.6, 31.3, 30.9, 30.0, 23.8, 23.0, 20.8, 19.3, 13.4; IR (film):

−1 + 3663 cm ; HRMS-TOF m/z (M + Na) calcd for C22H36O2 355.2613, found 355.2585.

1 Alcohol (18). A white solid: mp 125-127 °C; H NMR (CDCl3, 400 MHz) 5.81 (m,

1H), 5.35 (br s, 1H), 5.09 (br s , 2H), 3.52 (m, 1H), 2.27 (m, 2H), 2.17 (m, 2H), 2.04 (m,

2H), 1.83 (m, 2H), 1.64 (m, 3H), 1.49 (s, 3H), 1.46 (m, 6H), 1.28 (s, 3H), 1.25 – 0.91 (m,

13 4H), 1.00 (s, 3H), 0.86 (s, 3H); C NMR (CDCl3, 400 MHz) 140.7, 134.1, 123.0,

121.6, 71.8, 59.1, 57.8, 56.8, 50.1, 42.5, 42.2, 40.0, 37.2, 36.5, 31.8, 31.6, 31.4, 25.7,

25.6, 25.4, 23.8, 23.2, 20.9, 19.3, 18.0, 13.9; IR (film): 3316 cm−1; HRMS-TOF m/z (M +

+ Na) calcd for C24H38O2 381.2770, found 381.2752.

214

Ketone (18). Prepared using the same procedure for Oppenauer oxidation. (18) A whit

1 solid: mp 202 °C; H NMR (CDCl3, 400 MHz) 5.72 (s, 1H), 2.35 (m, 4H), 2.12 (m,

1H), 2.02 (m, 1H), 1.84 (m, 1H), 1.64 – 1.76 (m, 4H), 1.51 - 1.41 (m, 4H), 1.30 (s, 3H),

13 1.19 (s, 3H), 1.18 (s, 3H), 1.35 – 0.92 (m, 6H), 0.87 (s, 3H); C NMR (CDCl3, 400

MHz) 124.2, 73.7, 60.4, 56.3, 54.1, 43.2, 40.3, 38.9, 36.0, 35.4, 34.3, 33.3, 32.3, 31.5,

30.4, 24.1, 23.4, 21.2, 17.7, 13.9; IR (film): 3518, 1666 cm−1; HRMS-TOF m/z (M + Na)+ calcd for C22H34O2 353.2457, found 353.2448.

Wittig olefination of (2). Triphenyl-methylphosphonium bromide (365 mg, 1 mmol) was suspended in 5 ml of dry THF and cooled to 0°C. n-BuLi was added dropwise and stirred for 1 h at 0°C. Compound (2) (215 mg, 0.5 mmol) was dissolved in 5 ml dry THF added and the reaction was warmed gradually to rt, then refluxed overnight. The reaction was quenched with NH4Cl, and stirred for 10 min. The organic layer was separated and the aqueous layer was extracted 3 times with DCM. The combined organic layers were dried

215 over sodium sulfate and concentrated. The crude material was purified by silica gel column chromatography (DCM-hexanes, 3:7) to give the olefinated product (19) (83 mg,

1 39%) as white solid: mp 127-128 °C; H NMR (CDCl3, 400 MHz) 5.32 (d, J = 5.2 Hz,

1H), 4.84 (s, 1H), 4.70 (s, 1H), 3.48 (m, 1H), 2.27 (m, 1H), 2.18 (m, 1H), 2.00 (m, 2H),

1.88 (m, 1H), 1.82 - 1.65 (m, 4H), 1.67 (s, 3H), 1.57 – 1.43 (m, 5H), 1.25 – 0.98 (m, 6H),

1.00 (s, 3H), 0.88 (s, 9H), 0.58 (s, 3H), 0.05 (s, 6H); IR (film): 2929 cm−1; HRMS-TOF

+ m/z (M + Na) calcd for C28H48OSi 451.3372, found 451.3405.

Following the general procedure for TBS deprotection compound (20) was obtained

1 as a white solid: mp 134 °C; H NMR (CDCl3, 400 MHz) 5.35 (br s, 1H), 4.84 (s, 1H),

4.70 (s , 2H), 3.52 (m, 1H), 2.25 (m, 2H), 1.97 (m, 2H), 1.84 (m, 3H), 1.75 (s, 3H), 1.67

13 (m, 2H), 1.47 (s, 7H), 1.24-0.95 (m, 6H), 1.00 (s, 3H), 0.58 (s, 3H); C NMR (CDCl3,

400 MHz) 146.0, 141.2, 122.0, 111.1, 72.1, 57.6, 56.9, 50.6, 43.5, 42.6, 39.0, 37.6,

36.9, 32.6, 32.2, 32.0, 25.8, 25.0, 24.6, 21.5, 19.8, 13.1; IR (film): 3295 cm−1.

Following the general procedure for Oppenauer oxidation compound (21) was

1 obtained as a white solid: mp 151-153 °C; H NMR (CDCl3, 400 MHz) 5.72 (s, 1H),

4.86 (s, 1H), 4.71 (s , 2H), 2.42-2.28 (m, 4H), 2.02 (m, 2H), 1.87 (m, 2H), 1.80-1.67 (m,

4H), 1.75 (s, 3H), 1.56-1.54 (m, 3H), 1.23-0.84 (m, 5H), 1.18 (m, 3H), 0.60 (s, 3H); 13C

216

NMR (CDCl3, 400 MHz) 199.6, 171.5, 145.3, 123.7, 110.9, 57.0, 55.5, 53.9, 43.0, 38.6,

38.4, 35.9, 35.6, 33.9, 32.8, 31.9, 25.3, 24.6, 24.1, 21.0, 17.3, 12.7; IR (film): 1670 cm−1;

+ HRMS-TOF m/z (M + Na) calcd for C22H32O 335.2351, found 335.2328.

217

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Appendix A: MTT Assay

All compounds were screened in two breast cancer cell lines [estrogen-dependant (MCF-

7) and estrogen-independent (MDA-MB-231)] and two prostate cancer cell lines

(androgen-dependant (LNCaP) and androgen-independent (PC-3)]. The antiproliferative activities of all prepared compounds were evaluated in terms of IC50 (half maximal inhibitory concentration) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytotoxicity bioassay was done using a modified Rubinstein method 135. Compound stock solutions were prepared in DMSO from which serial dilutions were prepared. Cancer cells were exposed to doses of the compounds for 72 hours. Afterwards MTT was added and incubated for 2 h, and then % absorbance was recorded. The IC50 values were determined from dose-response graphs.

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Appendix B: Library of Compounds Screened for Antimalarial Activity

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