Potential Natural Product Phosphodiesterase Inhibitors

University of Mississippi eGrove

Electronic Theses and Dissertations Graduate School

1-1-2013

Michael John Cunningham University of Mississippi

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Recommended Citation Cunningham, Michael John, "Potential Natural Product Phosphodiesterase Inhibitors" (2013). Electronic Theses and Dissertations. 1343. https://egrove.olemiss.edu/etd/1343

This Thesis is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. POTENTIAL NATURAL PRODUCT PHOSPHODIESTERASE INHIBITORS

A Thesis presented in partial fulfillment of requirements for the degree of Master of Science in the Department of Pharmacognosy, School of Pharmacy The University of Mississippi

Michael J. Cunningham

August 2013

Three different studies set forth to investigate the role of phosphodiesterase (PDE) enzyme inhibitors in sexual dysfunction and mood regulation. First, natural products were screened by computer modeling of PDE5A1. Second, compounds were isolated from a plant traditionally used as an aphrodisiac. Third, a new synthesis of psychoactive PDE4 inhibitor was attempted.

An in silico screen was performed using chemical structures of isolated compounds thought to have some PDE inhibitory activity based on traditional use of the plants. A collection of compounds with structures similar to known natural product inhibitors was also included in the docking library. Glide docking software (Schrödinger) was used to score compounds based on their modeled interactions with the PDE5A1 enzyme. The compounds that showed the best scores were assayed for in vitro PDE inhibitory activity. Enzymatic screening suggested some inhibition of PDE5A1 by selected compounds. Four of seven compounds showed greater than

70% inhibition at 100 μM concentration.

Tongkat ali (Eurycoma longifolia Jack) root material was subjected to chromatographic separations in an attempt to discover new PDE inhibitors. Only known terpenes, sterols and alkaloids were identified. However, one new x-ray crystal structure of a sterol previously unreported in E. lonigfolia was determined.

Mesembrine, a known alkaloidal PDE inhibitor, has been a subject of many synthetic studies. A synthetic route was initiated using a diastereoselective Michael addition reaction to generate the target quaternary center.

DEDICATION

To my family, who encouraged me to dream in a pragmatic way.

To Anne Line, who inspired me to study drug discovery.

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ACKNOWLEDGEMENTS

I want to thank my advisor Dr. Ikhlas Khan for allowing me the opportunity to work in his laboratory. His support and encouragement allowed me to complete this work. I want to express special thanks to Dr. Amar Chittiboyina for his constant guidance and countless hours of stimulating conversations. I am grateful for Dr. Nao Abe for patiently guiding me in natural product isolation chemistry. I also must thank Dr.Sateesh Rotte and Dr. Prabhakar Peddikotla for helpful discussions in and out of the laboratory. I am thankful for the guidance provided by the members of my advisory committee, Dr. Khan, Dr. Zjawiony and Dr. Ferreira, who always took time address my questions.

Much thanks to Dr. Jon Parcher for his guidance and suggestions. Henry Valle of the

Department of Chemistry graciously provided x-ray crystal analysis. Dr. Mei Wang and Dr.

Bharathi Avula provided GC-MS and LC-MS analysis. Khaled Elokely and David Watson of the Department of Medicinal Chemistry provided much appreciated instruction in setting up and explaining Schrödinger software used for docking study. Special thanks to Frank Wiggers for helping maintain the NMR facilities in the National Center for Natural Products Research.

Funding for my research was made possible by grant number P50AT006268 from the

National Center for Complementary and Alternative Medicines (NCCAM), the Office of Dietary

Supplements (ODS) and National Institutes of Health (NIH).

TABLE OF CONTENTS

ABSTRACT ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

LIST OF TABLES vi

LIST OF FIGURES vii

CHAPTER 1. IN SILICO GUIDED SCREENING OF PDE5A1 INHIBITORS 1

1.1 Introduction 2

1.2 Structure of Phosphodiesterases and the Therapeutic Role of their Inhibitors 3

1.4 Experimental Methods 23

1.5 Results and Discussion 31

CHAPTER 2. ISOLATION OF METABOLITES FROM TONGKAT ALI 33

2.1 Introduction 34

2.2 Experimental Methods 35

2.3 Results and Discussion 36

CHAPTER 3. SYNTHESIS STUDIES TOWARD MESEMBRINE-TYPE ALKALOIDS 38

3.1 Introduction 39

3.2 Experimental Approach and Results 45

LIST OF REFERENCES 50

LIST OF APPENDICES 60

VITA 90

LIST OF TABLES

1. Nucleotide specificities of phosphodiesterase family 4

2. Results of PDE5A1 inhibition assay 31

3. Alkylation of (S)-4-benzyl-3-[2-(4-methoxyphenyl)acetyl]oxazolidin-2-one 45

LIST OF FIGURES

1. GMP signaling system overview 5

2. The phosphodiesterase enzymes' core pocket with four conserved areas 7

3. Glutamine switches in various PDEs 9

4. PDE5A inhibitors marketed for erectile dysfunction 12

5. PDE4 inhibitors investigated for COPD and asthma 13

6. PDE1 and PDE4 investigated for cognitive function 14

7. Examples of natural product PDE inhibitors 15

8. Dell’Agli assay of natural products for PDE activity 17

9. Example of compounds from G. biloba used in in silico docking 20

10. Example of compounds from F. hermonis used in in silico docking 21

11. Icariside II in the catalytic fold of PDE5A1 25

12. Ligand interaction diagram of icariside II in catalytic fold of PDE5A1 26

13. RMSD of icariside II in docked pose 27

14. In silico docking results 28

15. Compounds submitted for PDE inhibition assay 29

16. Descriptive scheme of enzyme assay 30

17. Mature E. longifolia specimen 34

18. ORTEP drawing of crinosterol 37

19. Skeletal formula of crinosterol 37

20. Four classes of Sceletium alkaloids 39

21. Structures of mesembrine and rolipram 40

vii

22. Jeffs' original biosynthesis scheme of mesembrine 42

23. Starting materials and key intermediates encountered in previous mesembrine syntheses 43

24. Initial mesembrine retrosynthetic route 44

25. Reactions attempted to create the benzylic stereogenic center 46

26. Synthesis scheme of Taber (2001) 47

27. Compounds generated 49

viii

CHAPTER 1

IN SILICO GUIDED SCREENING OF PDE5A1 INHIBITORS

1.1 Introduction

The ubiquity of phosphodiester bonds in all living organisms indicates the importance of these bonds to various areas of basic biological and medical research. The highest concentrations of these bonds are used for the long-term storage and transmission of information within cells and between generations in the form of Deoxyribonucleic Acid (DNA) and

Ribonucleic Acid (RNA). In DNA and RNA, these bonds serve to link the 3ʹ and 5ʹ positions of adjacent ribose units. Phosphodiester bonds are essential to maintaining the integrity of three billion base pairs of human nuclear DNA.1 Aside from their obvious structural significance in maintaining genetic information, phosphodiester bonds are present in phospholipids, sphingolipids and cyclic nucleotides. Of all these types, it is the set of enzymes that hydrolyzes phosphodiester bonds in cyclic nucleotides that is denoted the title of “phosphodiesterases”

(PDEs).1

As primary signaling metabolites are distributed across tissues of an organism, they are differentially translated into secondary messengers when membrane-bound receptors activate internal enzymes. 1 By converting primary messengers into secondary messengers, signals are selectively transferred to different regions of the body and amplified or diminished based on the target of the secondary signal. In the case of PDE enzymes, the broadest distinguishing characteristic is their preference for cAMP or cGMP. Within the superfamily of phosphodiesterases, there are 11 families known to exist in mammals. The enzymes specific for cAMP include PDE4, 7, and 8. Those specific for cGMP include PDE 5, 6, 9. PDEs 1, 2, 3, 10 and 11 are active on both cyclic nucleotides. 2 Within these 11 PDEs there are many splice- variants, each of which potentially adopting multiple folded conformations. Taking together all

possible structural variations along with their different tissue distributions, phosphodiesterases have proved to be an alluring area for biochemists, molecular biologists and pharmaceutical researchers alike.

1.2 Structure of Phosphodiesterases and the Therapeutic Role of their Inhibitors

Signaling and Structure

Cyclic nucleotides are ubiquitous second messenger molecules, serving to integrate various intra- and extraorganismal signals (neurotransmitters, hormones, tastes, odors, and light) into a unified chemical format. cAMP and cGMP are formed by purine cyclases. In the case of adenyl cyclases (ACs), transduction of this secondary signal is initiated when G-protein coupled receptors (GPCRs) receive some primary signal. This leads to activation of heterotrimeric G- protein, with the Gαs subunit bearing responsibility for activation of all but one adenyl cyclase

(AC10) isozymes. On the other hand, guanyl cyclases (GCs) are divided into soluble and membrane-bound, receptor-coupled classes. Soluble GCs are activated by nitric oxide and receptor-bound GCs are activated by natriuretic peptide hormones. 3

Substrate PDE isoform

Both adenine/ guanine 1, 2, 3, 10, 11

cAMP only 4, 7, 8

cGMP only 5, 6, 9

Table 1: Nucleotide specificities of phosphodiesterase family.

As the concentrations of cyclic nucleotides rise within the cell, binding to their targets, protein kinase A (PKA) and protein kinase G (PKG) are enhanced. These protein kinases, in turn, phosphorylate downstream targets such as ion channels and transcription factors. The reverse of this cyclization process is hydrolysis of the PDE bond by phosphodiesterases, and the rate of formation or degradation of these cyclic nucleotides affects the overall activation of this signaling pathway.4

Molecular studies have elucidated 11 distinct families of phosphodiesterases which are grouped according to their cyclic nucleotide monophosphate substrates. PDEs 4, 7 and 8 hydrolyze cAMP whereas PDEs 5, 6 and 9 hydrolyze cGMP. PDEs 1, 2, 3, 10 and 11 are capable of hydrolyzing both substrates (Table 1). Because the cyclic nucleotide signaling system is globally distributed in organisms, there is broad potential of pharmacological investigation into the various processes including, proinflammatory response, muscle relaxation, ion channel

function, apoptosis, cellular differentiation, learning, memory and essential metabolic processes of gluconeogenesis, glycogenolysis and lipogenesis.5

Figure 1: GMP signaling system overview. 3

In the late 1970s, chemists began to produce small molecules which could inhibit kinetically distinct PDEs. Based on these findings, research into the area expanded to the present day and PDEs are considered to hold potential for treatment for various health problems including heart failure, depression, cognitive decline, asthma, COPD, inflammation and erectile

dysfunction.5-6 There are many unmet medical needs which may benefit from selective control of this critical signaling system. To date, three PDE5 inhibitors, four PDE3 inhibitors and one

PDE4 inhibitor have been approved by the FDA. There are currently two PDE4 candidates awaiting approval, with 20 other small molecule inhibitors of PDE4 in clinical studies. 7 Part of this advance in the discovery, rational design and optimization of inhibitors can be traced to the elucidation of 3D structures derived from X-ray crystallographic studies. The catalytic domains of PDE 1, 3, 4, 5, and 9 have been published and made publicly available to investigators.8

Despite their variable preference for cAMP versus cGMP, phosphodiesterases all bear the same three functional domains: the regulatory N-terminus, the conserved catalytic core and the

C-terminus. 9 The regulatory N-terminal region is adjacent to the catalytic core. Regulatory elements of N-terminal domains include an auto-inhibitory region and a targeting sequence to guide its cellular localization. Within the N-terminal region there are various other regulatory elements depending on the isozyme. In PDE1, there is a calmodulin (CaM) binding domain;

PDE2 contains multiple cyclic guanosine monophosphate binding sites; PDEs 1-5 all contain phosphorylation sites for distinct protein kinases; and PDE6 also bears a transducin binding site.10

The most studied domain of the PDE enzymes for drug discovery has been the conserved catalytic domain at the carboxylic terminus. It consists of 270 amino acids with sequence identity ranging from 18-46% across the various isoforms. 2 The catalytic domain structures of PDE5A,

PDE4B, PDE4D, PDE1B, PDE3B and PDE9A have all been reported from crystallographic studies. Within this catalytic domain are three subdomains comprised of helices: the N-terminal cyclin-fold, a linker portion and a C-terminal helical region. 8b, 8c, 8e, 8f, 8h, 8i At the junction of

these subdomains, a deep hydrophobic pocket is formed with four distinct sites. Within this pocket lies a core pocket (Q pocket), a lid region (L region), the hydrophobic pocket (H pocket) and the metal-binding site (M site). 8f

Figure 2: The phosphodiesterase enzymes' core pocket with four conserved areas 8f

Within the M site, various metal atoms are bound to fully conserved residues across the entire PDE family. Based on the coordination geometry of the coordinating ligands, the metals present in the M site are thought to be zinc and magnesium ions. The metals ions exhibit

octahedral coordination geometry. The zinc ion is coordinated by one aspartate and three histidine residues, as well as two water molecules. Magnesium is coordinated to the same aspartate residue, and five water molecules. One water molecule is shared with zinc. The metal ions are thought to aid in the stabilization and activation of hydroxide in catalysis. 8i

The Q pocket seems to interact with ligands through hydrogen-bonding with conserved glutamine residues as revealed in the PDE5A crystal structure complex with the pyrazolopyrimidinone core of sildenafil. The glutamines are present on both sides of the Q pocket providing extra stabilization in a clamp-like manner.8i

The hydrophobic H pocket can accommodate alkoxyphenyl residues such as the ethoxyphenyl, cyclopentoxyphenyl, and methoxyphenyl moieties of sildenafil, rolipram and mesembrine, respectively. It is the variation of residues within this H pocket which confers selectivity to particular phosphodiesterases. It is the flexibility of the L region of PDE5A in the

Tyr664/Met816/Ala823/Gly819 residues which allows for a conformational fluctuation between open and closed forms to control binding of different inhibitors.2, 8i

Of the many structure features observed in phosphodiesterase enzymes to differentiate between substrates, the most critical seems to be the ‘glutamine switch’ functionality.8i The proposed mechanism suggests that a conserved glutamine residue plays a critical role in PDE nucleotide selectivity. There are two potential orientations that the γ-amino group of the glutamine residue in the active site may adopt to establish different hydrogen-bonding relays. In the example of endogenous PDE substrates, one orientation will support guanine binding and preferentially process cGMP. In the opposite orientation, glutamine will establish a hydrogen- bonding network where adenine will be supported, making cAMP the preferred substrate. In 8

PDEs 1, 2, 3, 10 and 11, the side chain may adjust its orientation to accommodate both cyclic nucleotides.

Figure 3: “Glutamine switches” in various PDEs. [2]

As displayed in Figure 3, members of the phosphodiesterase family respond differently to different substrates based on a conserved glutamine residue “switch mechanism”. In images (A) and (B), PDE1B recognizes AMP and GMP, respectively. His381 and His373 allow for substrate interaction with Glu421. In images (C) and (D), Glu988 of PDE3B accommodates either cyclic nucleotide because His948 is occupied by interactions with Trp1072, allowing either Glu988 orientation. In PDE4D (E), a hydrogen-bonding relay is created between Asn321,

Tyr329, Glu369, and the exocyclic amine moiety of the adenine substrate. On the other hand, there appears to be an inversion of orientation in the Glu817 residue in PDE5A1 (F) co- crystallized with GMP. A well-ordered hydrogen-bond relay is established between Ala767,

Glu775 and Trp853 with Glu817, effectively locking it in a conformation where only the γ- 9

amide of Glu817 can interact with the exocyclic O-atom of guanine substrates. In the case of

PDE9A (G), GMP is again the preferred substrate, this time due to hydrogen-bond relay between

Gln406 and Ser486 with invariant Glu453.

The aforementioned structural features of PDE enzymes correspond to three general features of PDE ligands. First, the central rings of the purine moiety must interact with the invariant glutamine via single or dual hydrogen-bonds. Second characteristic of favorable PDE ligands is the ability of core rings to interact with the nonpolar side chains of residues in

‘hydrophobic clamp’ region. In the case of PDE5A, this feature contributed significantly to reduction in side-effects between first and second generation inhibitors. While sildenafil does interact with hydrophobic residues, its seven rotatable bonds create steric hinderance and require entropic loss to become situated in the clamp. Tadalafil, conversely, is considerably more rigid with only one rotatable bond that allows for fewer collisions as it approaches the hydrophobic clamp. The third notable feature is that substrates interact with metal ions whereas inhibitors have not demonstrated this interaction. 2

Therapeutic developments of PDE inhibitors

Sexual function

The FDA approval of Viagra (sildenafil) in 1998, followed by its market success, generated a great deal of attention for what is undoubtedly the most famous PDE inhibitor. In fact, it has become one of the most famous drugs in history. It implied that sexual dysfunction may be overcome through modern medicine. As a drug, however, it does not function as an

‘aphrodisiac’ in the traditional sense. Sildenafil action follows the initially arousing stimuli

(touch, smell, sight, fantasy, etc.), and nitric oxide (NO) is released from neurons and endothelial cells in the penis. This NO released into cells activates the soluble guanyl cyclases, leading to a

PKG-mediated phosphorylation cascade. 11 The ensuing decrease of Ca2+ leads to an arterial dilation and compression of corpus cavernosum tissue of the penis. As blood becomes trapped in the penis, intracavernosal pressure increases and erections manifest.12 In the case of sildenafil, it is not the activation of guanyl cyclase, but the inhibition of PDE5A1 that enhances cGMP concentration. The success of Viagra spawned two similar PDE5 inhibitors, Cialis (tadalafil) and Levitra (vardenafil), and aroused interest in researching whether a similar approach may work for female sexual dysfunction (see Figure 4). The clitoris and anterior vaginal wall contains erectile tissue similar to those of the penis.13 The presence of nitric oxide synthase

(NOS) and PDE5 enzyme in the clitoral corpus cavernosum suggest a common physiological mechanism of male and female arousal. 14 A double-blind, placebo-controlled crossover-design study by Caruso et al. showed that sildenafil improved sexual arousal, orgasm, frequency of sexual fantasies and frequency of sexual intercourse in premenopausal females. 15 Tadalafil, however, demonstrated no conclusive therapeutic effects compared with a placebo in a 2001 phase I clinical trial, exploring its use in women with female sexual arousal disorder. It remains to be seen whether this area will prove helpful for women suffering from sexual dysfunction.

Figure 4:PDE5A inhibitors marketed for erectile dysfunction.

Respiratory inflammation

While the majority of attention has been paid to PDE inhibitors for erectile dysfunction, there has also been active development of drugs for treatment asthma and chronic obstructive pulmonary disorder (COPD). In rabbit, guinea pig and monkey models, pretreatments with

PDE4 inhibitors diminished the level of antigen-induced bronchoconstriction and eosinophil infiltration. 16 These studies also demonstrated a reduction in both pulmonary microvascular leakage and airway reactivity.16b, 16d, 16e The perceived benefit of these compounds over existing drugs is that they are capable of relaxing airways as well as diminishing underlying airway inflammation. The PE4 inhibitor roflumilast (Daliresp) was approved by the FDA in 2011 for treatment of COPD.7 The next most promising candidate is cilomilast (Ariflo), currently in a

Phase III clinical trial.

Figure 5:PDE4 inhibitors investigated for COPD and asthma.

Central Nervous System

The last major area to be addressed is the connection of mood and cognition to phosphodiesterase signaling. The focus of research has largely involved neurodegeneration- associated cognitive decline and depression. Kakkar et al. provided evidence that brain-specific

PDE1A2 was inhibited by the antiparkinsonian drugs selegiline and amantadine, enhancing intracellular levels of cAMP. 17 This suggests that PDE1 enzymes in the brain may be a possible target of future Parkinson’s disease treatments. The semisynthetic compound vinpocetine, which inhibits PDE1 and shows potent anti-inflammatory activity through a separate mechanism has been considered as a treatment for neurodegenerative disorders.18

Figure 6: PDE1 (top row) and PDE4 (bottom row) investigated for cognitive function

Mutational analysis of cAMP response-element binding protein (CREB) has shown it to be a controlling factor in long-term memory formation.19 Transgenic Drosophila melanogaster

(fruit flies) demonstrate creation of long-term memories after less behavioral training in groups with overexpressed CREB.20 This same ‘CREB-signature’ effect was shown in animals treated with the PDE4 inhibitor rolipram. 21 Rolipram has also been investigated in animals as a treatment for depression and memory loss.22 There is also evidence that cAMP affects neuron survival, potentially through regulation of brain-derived neurotrophic factor (BDNF) upregulation.23 Although mounting evidence supports the role of cAMP in neuronal survival and memory, the side-effects of rolipram have relegated it to “probe” status. 23d-f, 24

Natural Product PDE Inhibitor Examples

Mesembrine, mesembrenone (from Sceletium tortuosum), moracin A , moracin C (from

Morus alba L.), theophylline (from Theobroma cacao).25

Figure 7:Examples of natural product PDE inhibitors

Phosphodiesterases inhibitors for erectile dysfunction

The ability to maintain satisfactory sexual function is a notable problem which can impact interpersonal relationships and quality of life in otherwise healthy individuals. The age- old quest for aphrodisiacs has taken many forms. A survey of literature on the subject turns up formulations which have been used to enhance arousal, function, stamina and fertility. While

‘arousal’ may be harder to define and test, function and fertility are slightly easier. Perhaps the easiest to quantify is the male erection, which becomes more difficult to maintain as men age.

During erection nitric oxide is released from parasympathetic nerve terminals into the smooth muscle cells of the corpus cavernosum. Activation of guanyl cyclase converts guanosine

triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). This causes relaxation of smooth muscles, dilation of blood vessels and enhanced blood flow into the penis. It is the trapping of this blood which results in erections, and the inhibition of the signaling molecule cGMP has shown to be a means of inducing erections in humans.

The most notable drug working on phosphodiesterases to be developed and marketed thus far is sildenafil citrate (Viagra), which has greatest affinity on the PDE5A enzyme. In 1985, chemists at Pfizer first began developing treatments for angina and hypertension by targeting

PDEs which were fairly unexplored at the time. Starting from Zaprinast, a known vasodilator working through PDE inhibition, Pfizer explored structure-activity relationships to enhance PDE selectivity. In this time before combinatorial synthesis, the team of five chemists impressively generated 1,600 compounds over three years for screening. Of this group compound UK-92480 proved to be most selective for PDE5, with some activity at PDE6- an enzyme known to play a role in visual signal transduction. As the compound entered into clinical trials, it was proven to not be effective for angina. However, the clinicians did notice that the majority of patients on high doses “suffered” the side effect of improved erections. Pfizer realized the opportunity to build off this effect and was able to successfully carry the drug through trials. 26 The compound

UK-92480 was labeled Viagra and would become a blockbuster drug and widely recognized by name. In the years that followed, vardenafil and tadalafil would also reach the market and share success as pro-erectile PDE inhibitors.

Despite all the success of these drugs, there remains the side-effects that where initially noticed in early development. Partial activity at PDE6 is thought to contribute to some visual disturbances such as blue and green hues in the visual field. For this reason, there is still

considerable room for improvement on these popular drugs. Many plants have been traditionally used to help attain erection. Figure 8 shows four extracts and three of the isolated components from these plants that have been screened for activity against human PDE5A1.27 Of this set, icariside II showed the greatest level of PDE inhibition. To further investigate the ability of plants to inhibit phosphodiesterases, an in silico screening project was performed. Using

Schrödinger’s Maestro interface, the Glide docking module was trained using a PDE5A1 structure co-crystallized with icariside II. Plants used for docking were Epimedium spp.,

Lepidium meyenii Walp. (maca, Ginko biloba L., and Ferula hermonis Boiss. In addition to these plants, previously isolated compounds bearing structural similarity to icariin would be included in the docking set.

Figure 8:From Dell’Agli et al. (T.t.= Tribulus terrestris, E.b.= Epimedium brevicornum, C.c. Cinnamonium cassia, F.h. = Ferula hermonis, cinn. = cinnamaldehyde) [59]

Natural Prodcucts Used

The Epimedium (Berberidaceae) genus has attracted much attention for their use as an aphrodisiac. The plant goes by many names, including Rowdy Lamb Herb, Barrenwort,

Bishop’s Hat, Fairy Winds, Horny Goat Weed, Yangheye and Yin Yang Huo.28

Out of approximately 52 species, more than 15 species in this genus have been used historically to aid kidney function and to “reinforce the Yang”. The most commonly cited species for use as a sexual tonic are E. brevicornum, E. grandifolium and E. sagittatum. The Chinese Pharmacopeia officially recognizes the five major Epimedium species as official sources of Herba Epimedii.

These recognized species are: Epimedium brevicornum Maxim, E. sagittatum (Sieb. and Zucc.)

Maxim., E. pubescens Maxim., E. wushanese T.S. Ying and E. koreanum Nakai.28 In China and

Japan, varieties have been used to treat impotence, prospermia, hyperdiuresis, osteoporosis, menopause, rheumatic arthritis, hypertension, and chronic tracheitis. In Korea, E.koreanum was also used for impotence, as well as spermatorrhoea (involuntary ejaculation) and forgetfulness.29

The chemistry of the plant has been well defined. Isolated classes include flavonoids30, lignans, phenolic glycosides, phenylethanoid glycosides and seqsuiterpenes. The greatest amount of pharmacological investigation has been in the prenylated flavonol glycosides.

Notably, icariin has been shown to have activity (IC50 value of 5.9mM) at PDE5A1, the primary target for the popular pharmaceutical proerectile drugs vardenafil, tadalafil and sildenafil.27 The structure PDE5A1 has been co-crystallized with icariside II, a derivative of icariin which lacks a rhamnose unit at the 7-O position of the flavonol structure.

Ginkgo biloba L. , also referred to as yín xìng or the “maidenhair tree” has no known living relatives, for which people consider it to be a living fossil of sorts. Originally from China, it was introduced to North America by André Michaux.31 These trees can grow to over 30 18

meters in height and is known to display erratic branching patterns. Individual trees are known to live over 1000 years and reproductive organs emit a foul odor due to its butyric acid content.32

During to the extensive relationship and the significance of the plant in East Asia, it served many roles and as a food and medicine. The plant has been used in Asia traditionally for various indications. Extracts of the ginkgo plant and isolated compounds have been assayed in humans and other animals to better understand its therapeutic potential. The characteristic constituents of the plant are ginkolides and flavonoids (See Figure 9). Whole plant material and extracts thereof have shown activity in extensive animal studies and over 120 published clinical trials.

The most common standardized extract EGb 761, or variations which contain the same proportion of flavonoid glycosides, terpene lactones and ginkgolic acids have been used in many of these trials. The most promising results were shown in anti-aggregant, anti-oxidant, neuroprotective, altitude sickness, Alzheimer’s disease, dementia, intermittent claudicating, dysmenorrheal and vertigo assays. 33 In a study investigating the memory enhancing potential of

G. biloba on geriatric patients, it was observed that many patients experienced improved erections. In a follow-up study, Cohen and Bartlik reported G.biloba had a positive effect “on all four phases of the sexual response cycle: desire, excitement (erection and lubrication), orgasm and resolution.” 34 Patients of this group had all previously “exhibited sexual dysfunction secondary to a variety of antidepressant medications including selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, monoamine oxidase inhibitors and tricyclics.”

Figure 9: Example of compounds from G. biloba used in in silico docking

Ferula hermonis Boiss. (Umbelliferae) is a plant native to mountainous regions of the

Middle East, including Lebanon and Syria. Its name refers to Mt. Hermon, upon which it grows.

The notable marker compounds of the plant are daucane sesquiterpenes. The large genus Ferula encompasses 150 species. Some species within this genus have been used traditionally for skin infections, headache, arthritis, digestive disorders, dysentery, and as an aphrodisiac and antihysteric. Claims of usefulness for impotence are supported in Arabic medical literature.35

Although somewhat rare, the root material can be found for sale, sometimes advertised as

“Lebanese Viagra” by internet retailers. Research to better understand the aphrodisiac activity of F. hermonis has demonstrated that it increases the sexual activity of male rats.36 Ferutinin has shown potent phytoestrogenic activity at the human estrogen receptor.37 In a study by Colman-

Saizarbitoria, ferutinin also exhibited the ability to “increase nitric oxide synthase activity and inositol monophosphate accumulation in the median eminence of the rat brain”.38 This suggests

a potential role in neuroendocrine regulation, but possibly activity in nitric oxide regulation in other tissues.

Figure 10: Example of compounds from F. hermonis used in in silico docking

Lepidium meyenii Walpers (Cruciferae) is widely sold as a dietary supplement and functional food under the name “maca”. The preferred plant part is the hypocotyls, which was traditionally consumed by humans and livestock largely within the Meseta de Bombon, Peru.

Although initially from an area of high altitude (3700-4500 above sea level) and intense winds, the plant has been successfully cultivated outside its native habitat. It has thus been suggested by

Chacon that the domesticated form be regarded separately as L. peruvianum Chacon.39 The plant has been held in high regard since pre-Incan times and during the Incan Empire it was consumed only by members of nobility rewarded to warriors. 40 Aside from being used in foods and 21

beverages, there have been claims that maca enhances fertility and sex-drive. Although studies have shown it does not exert its purported effects through a direct androgenic effect, other evidence shows significant alleviation of SSRI-induced sexual dysfunction in women.41

The remaining portion of the Glide screening set were compounds previously isolated from microbial transformation. In this method, organisms were selected from a group of 40 culture samples from National Center for Natural Products Research at The University of

Mississippi. Flavonoid substrates, dissolved in dimethylformamide, were added after 24 hours of initial incubation and incubated 14 days on a rotary shaker at 100 RPM. Filtrates were combined and extracted with isopropanol-ethanol (1:4) and residues were separated over silica gel or Sephadex LH-20. The isolated compounds exhibited a number of possible transformations such as hydroxylation, sulfation, glycosylation and methylation with modification occurring primarily on the A ring. The metabolites were tested for antibacterial against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Mycobacterium intracellulare. No antibacterial activity was shown against this set of bacteria. No antifungal activity was revealed against Candida albicans, C. neoformans or

Aspergillus fumigates. The compounds were also tested against chloroquine sensitive and chloroquine resistant strains of Plasmodium falciparum. Owing to the lack of established activity, structural similarity to icariin and availability for further screening, they were included with the plant metabolites.

1.3 Experimental Methods

Library generation and ligand preparation

Scifinder Chemical Abstracts Service was used to collect compounds from plants:

Epimedium spp., Lepidium meyenii Walp. (maca), Ginko biloba L., and Ferula hermonis Boiss.

Varying numbers of compounds were returned in each search. A set of filters were placed on the sets of compounds returned for each plant. Initial exclusion filters were for isotopes and metal- containing compounds. The next set of filters was based on Lipinski-type heuristics to improve chances or oral bioavailability.42 Compounds outside the range of 100-700 Daltons were excluded. Compounds with greater than 10 hydrogen-bond acceptors or greater than five hydrogen-bond donors were excluded. Compounds with logP outside -10 to 5 range or more than 10 freely rotatable bonds were also excluded. Following the application of these filters, 204 compounds remained in the set. These compounds were drawn using ChemDraw and exported into .mol2 format so they could be imported to Schrödinger software.

Within the Schrödinger software, LigPrep was used to convert chemical structures in

.mol2 format into three-dimensional models, generate alternate structures, and prepare for docking. For example, LigPrep removes any counter ions in salts and neutralizes charged groups by addition or removal of hydrogen atoms. LigPrep generates various ionization states and tautomers for the structures. LigPrep also determines chiral atoms and generates alternate isomers and generates low energy conformations for rings within compounds. 43

Preparation of protein, ligand and docking procedure

The protein structure of PDE5A1 in complex with icariside II was retrieved from

Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB).44 The protein was prepared by the Wizard procedure- removing the waters outside of catalytic domain, adding any missing hydrogens, and correcting metal ionization states. 45 Following this preparation, the collection of compounds was placed in Glide Grid for navigation and comparison of ligand interactions. A ligand interaction diagram was produced for icariside II and PDE5A1 (see figure below). Root-mean square deviation was calculated for the true

PDE5A1 crystal structure complexed with icariside II versus a simulation of the same interaction. RMSD score was acceptable at 0.7501, with the greatest variation between flexible portions, such as the prenyl side chain (see figure).

Figure 11: Icariside II in the catalytic fold of PDE5A1, containing docked icariside II molecule (generated with Schrödinger software)

Figure 12: Ligand interaction diagram of icariside II in the catalytic fold of PDE5A1.

Figure 13: RMSD of icariside II in docked pose, superimposed by atom pairs. Areas of greatest difference are in green boxes. Results of Glide Docking

Glide was used to dock the collected library into the PDE5A1 structure using basic settings. The HTVS (high-throughput virtual screening) mode was used to reduce the number of possible conformations sampled. The force field used was OPLS 2005. “Interactive optimized” mode was selected for hydrogen-bond states for the preferred tautomer. The score was a negative value based on calculated interactions. Of the top scoring hits, it was determined that all scoring lower (better) than -12 where from the in-house collection of compounds isolated from microbial transformation. The best seven of these compounds available were located and prepared to PDE5A testing by Caliper Life Sciences (Perkin Elmer).

-2

-4

-6

-8

Docking Docking Score -10

-12

-14

-16

Figure 14: In silico docking results of selected plant material: The compounds are clustered by source. From left to right: F. hermonis, G. biloba, L. meyenii, Epimedium spp. , synthetic compounds, in-house biotransformation products

Figure 15: Compounds submitted for PDE inhibition assay

Enzyme screening assay

Ten compounds were sent to PerkinElmer Discovery Services to conduct inhibition screening through phosphodiesterase PDE5A1, in an in vitro biochemical assay at concentrations

of 1.0E-4 (100 μM), 1.0E-6 (1 μM), 1.0E-8 (10 nM) in duplicate. Zaprinast, a phosphodiesterase-specific reference compound, was used with the assay.

Screening was performed using Caliper LabChip 3000 and a 12- sipper LabChip. In these assays, the product and substrate are electrophoretically separated. A microfluidic chip is used to measure conversion of fluorescence between substrate and product. The reaction mixture is introduced to chip via a capillary sipper where substrate and product are separated. Reaction is monitored by laser-induced fluorescence over time, determining the progress of the reaction.

Figure 16: Descriptive scheme of enzyme assay (from PerkinElmer Discovery Services)

Table 2: Results of PDE5A1 inhibition assay.

Percent (%) Inhibition Table, PDE5A1 Comp. ID 1.E-04 1.E-06 1.E-08 MJC 1.1 104 43 1 MJC 1.2 10 6 10 MJC 1.3 11 12 12 MJC 1.4 109 21 4 MJC 1.5 75 23 9 MJC 1.6 42 20 10 MJC 1.7 33 17 13 MJC 1.8 74 27 19 MJC 1.9 11 -3 -3 MJC 1.10 77 38 16

1.4 Results and Discussion

Mesembrine and mesembrenone were included in this screening set based on their PDE4 activity. However, no activity was demonstrated on PDE5A1. Icariin predictably showed good inhibition at 1μM concentration. Compounds 6 and 7 showed weak activity. Compounds 4, 5,

8, and 10 demonstrated dose-dependent enzyme inhibition. It was known that the four sulfated compounds may all potentially hydrolyze in DMSO at room temperature based on prior NMR experiments. In this case, activity may be attributable to the products of hydrolysis. Compound

4 showed the best activity and it was not previously reported in any phosphodiesterase assays.

Its dose-response is different from icariin but its overall inhibition warrants further investigation.

Compound 8 has been subjected to numerous assays but this is the first time to be tested for PDE enzyme inhibition. The desulfated analogue of compound 5 is luteolin, a known, nonspecific

PDE inhibitor. Compound 10 is apigenin, also known to inhibit PDE enzymes. Apigenin

previously demonstrated less than one fifth the PDE5 inhibitory activity as luteolin. 46 In this assay apigenin demonstrates slightly higher PDE inhibition than luteolin. This may be attributable to the presence of sulfate group or a preference within the PDE5 isozymes.

Compound 9 showed no inhibition. In this study we successfully applied in silico methods to screen potential PDE5A1 inhibitors.

CHAPTER 2

ISOLATION OF METABOLITES FROM TONGKAT ALI

2.1 Introduction

The popular dietary supplement “Tongkat Ali” has a history of traditional use in South

East Asian countries Malaysia, Indonesia, Vietnam and to a lesser degree in Thailand,

Cambodia, Myanmar and Singapore. Specifically, “Tongkat ali” is the Malayian name for a group for various members of the Simaroubaceae family which include Eurycoma longifolia

Jack., Entomophtora apiculata, Polythia bullata and Goniothalamus sp.. 47 The most commonly studied and used of these is E. longifolia Jack. The name “Tongkat ali” is Malaysian for

“walking stick”- because of the plant’s long roots. 48 E. longifolia is commonly referred to as

Tongkat ali or Long Jack. In Malaysia it has various names including payung ali, penawar pahit, pokok syurga and pokok jela among others.48 In Vietnam it is referred to as cây-bábinh.49 In

Indonesia it is referred to as pasak bumi.50 In Thailand it is known as ian-don and pla-lai-puek.

Figure 17: Mature E. longifolia tree (left) and root (right). 34

Traditionally, the roots of this plant are used as a bitter decoction or “coffee”- either in combination with coffee and ginseng, or alone. A native plant of South East Asia, E.longifolia has been used primarily for its fever-reducing, anti-malarial and aphrodisiac activities. There are many other secondary applications- for alleviation of general aches, swelling, and dysentery.

The plant is largely regarded as a revitalizing tonic or adaptogen to promote general healthy functioning. Non-native, western (mostly American) users of the plant have focused on two uses: muscle growth and erection enhancement.52

The phytochemistry of the plant has been examined primarily in Japan, Malaysia and

China. Characteristic chemical classes identified thus far include: squalene derivatives, canthin-

6-one and β-carboline alkaloids, quassinoids, tirucallane-type triterpenes and biphenyl neolignans.47 These compounds exhibit cytotoxic, antimalarial, anti-ulcer, antimicrobial, antiplasmodial, and antischistosomal activities in various assays. Plant extracts were also evaluated for antidiabetic and aphrodisiac activities in rats. In humans, the effects of E. longifolia extracts have been investigated. 48, 50, 53 Although limited in participants, studies have shown an increase of muscle mass, muscle strength and fat free mass and a decrease in body fat. 52

2.2 Experimental Methods

Chemical investigation of the plant was continued to better understand the basis for the broad therapeutic potential of E. longifolia’s. A water extract of Eurycoma longifolia Jack roots, purchased from Kaden Biochemicals GMBH (Porgesring 50-22113, Hamburg, Germany) served as the crude starting material. The starting material (250 g) was extracted with methanol (4x 1L) to yield a total of 91.41g of extract. 80 grams of this material was fractioned using a normal

phase silica column (12 x 120cm) starting with chloroform as mobile phase and increasing methanol content by 5% with every 4L. Fractions were collected by the liter, concentrated, and combined on the basis of their TLC profile. These subfractions were further fractioned by silica gel chromatography, leading to the isolation of previously reported compounds.

2.3 Results and Discussion

Two alkaloids were identified: canthin-6-one (2.1) and 9-methoxycanthin-6-one (2.2).

Two quassinoids were identified laurycolactone A (2.3) and dehydroeurycomalactone (2.4). The only previously unreported compound from the plant is crinosterol (2.5). The crystalline material was isolated using a “hybrid chromatography-crystallization” method to enhance the likelihood of attaining crystalline natural products from silica gel chromatography. 54 Using a chloroform/acetone gradient of increasing polarity (1 to 5%), the collected fractions were allowed to slowly dry under the laboratory ventilation hood. The fractions of this column yielded four test tubes with crystals. Three contained crinosterol. The partial structure was determined by 1D and 2D NMR spectroscopy. The X-ray crystal structure was determined by Henry Valle of the University of Mississippi Chemistry Department.

Figure 18: ORTEP drawing of crinosterol.

Figure 19: Skeletal formula of crinosterol (2.5)

CHAPTER 3

SYNTHESIS STUDIES TOWARD MESEMBRINE-TYPE ALKALOIDS

3.1 Introduction

The genus Sceletium is within the iceplant (Aizoaceae, subfamily

Mesemryanthemoideae) family. The Sceletium genus is comprised of 26 members.55 All members are endemic to South Africa, primarily within the southwestern region. These succulent plants exhibit a decumbent (creeping) growth pattern with white, yellow or pink flowers.56

The plant contains many cis-3a-aryloctahydroindole alkaloids, which have served as synthetic targets for generations of organic chemists. To date, over 30 alkaloids have been isolated from Sceletium plants. These alkaloids can be grouped into four categories: 3a-aryl-cis- octahydroindoles, C-seco mesembrine alkaloids, alkaloids bearing 2,3-disubstituted pyridine

57 moieties and C- seco A4 group (see Figure 20). Many of these compounds have served as synthetic targets over the years, with the initial syntheses of mesembrine preceding confirmation of its biological activity.

Figure 20: The four classes of Sceletium alkaloids

Although the plant is used to settle the stomach, induce bowel movements or as an oral anesthetic, the most noteworthy biological effects of the plant are psychoactive. Traditionally, fermented plant material is chewed to produce lucid euphoria which is often followed by relaxation or sedation. Kanna is often consumed in gatherings, not unlike tea or alcohol as a social lubricant. The plant is capable of exerting its effects alone, but many users have made use of the plant in combination with cannabis or alcohol. As of 2013, the plant remains popular as a euphoriant with low toxicity.56 For this reason, the plant has attracted some interest as a tool to treat depression. Although this formulation has struggled to enter the United States market via the Food and Drug Administration’s New Dietary Ingredients Notification, the plant material remains available in the US and interest in Sceletium has steadily increased.58

Sceletium alkaloids:

Figure 21: Structural similarities between mesembrine and rolipram, a known selective PDE4 inhibitor

In continuation of our efforts in identification of potential phosphodiesterase inhibitors, we approached Sceletium alkaloids as natural inhibitors due to their structural similarities with rolipram, a known PDE4 inhibitor. A brief literature survey revealed that mesembrine has been shown to be active on 5-HT transporter (Ki 1.4nM) as well as PDE4B (IC50 7.8μM).59 Its oxidative analogue, mesembrenone showed potent activity against both 5-HT (Ki 27nM) and

59 PDE4B (IC50 of 0.5nM) in the nanomolar range. Moreover, due to structural resemblance with rolipram, Napoletano et al. demonstrated structure-activity relationships of mesembrine including the enhancement of PDE4 inhibition by switching the methyl ether group with bulky cyclopentylyloxy group.60 However, further information of in vitro inhibitory activity of various alkaloids from Sceletium is sparse, but there is evidence that cross-talk between cAMP and serotonin signaling systems may be involved in depression stemming from decline in cognitive function. 61

Biosynthesis of Sceletium Alkaloids:

The biosynthesis of these alkaloids was explored by Peter Jeffs and co-workers at Duke

University from 1967-1978.62 The alkaloids were first thought to be generated from L- phenylalanine and L-tyrosine derivatives to create norbelladine which is subject to para-para oxidative coupling, generating a crinine-like heterocyclic core structure. This tetracyclic skeleton is repeatedly oxidized at C-7 adjacent to nitrogen, eventually resulting in decarboxylation, either before or after O- and N-methylations to form mesembrine. There is still some debate as to the origin of these alkaloids (see Figure 22).

Figure 22: Jeffs' original biosynthetic scheme of mesembrine

Earlier synthetic approaches for the construction of mesembrine alkaloid scaffold

Mesembrine, mesembrenol and mesembrenone are the only compounds which have been screened for CNS target activity in vitro. Only mesembrine and mesembrenone showed appreciable activity at both SERT and PDE4. Of these two alkaloids, only mesembrine has served as a synthetic target. Its sterically congested architecture with adjacent stereogenic centers has served as a ‘proving ground’ of sorts for various synthetic methodologies (see Figure

23).

Figure 23: Starting materials and key intermediates encountered in previous mesembrine syntheses.

Synthesis of mesembrine would allow for creation of starting material for further investigation of structure-activity relationships. There have been approximately 40 published total and formal syntheses of mesembrine, the first of which was demonstrated in 1965.63 To assess the potential inhibitory activities of novel analogues based on the mesembrine scaffold, we have undertaken the total synthesis of mesembrine whose retrosynthetic analysis shown below. In our proposed synthetic approach, the primary challenge posed was the establishment of

asymmetry at the all-carbon quaternary center. This route considered the possibility of using a chiral auxiliary in the early stage to induce the required chirality. The chosen auxiliary was a readily available oxazolidinone derived from phenylalanine. The use of oxazolidinone chiral auxiliaries was pioneered by Evans and co-workers in 1981. The approach was explored throughout the 1980s to provide scalable, diastereoselective Michael additions, cyclopropanations, alkylations, aldol condensations and Diels-Alder reactions.64 Despite this capacity to selectively generate diastereomers, it had not been employed to create a quaternary stereogenic center in a system similar to mesembrine.

Figure 24: Initial mesembrine retrosynthetic route

3.2 Experimental Approach and Discussion

Table 3: Alkylation of (S)-4-benzyl-3-[2-(4-methoxyphenyl)acetyl]oxazolidin-2-one

Alkyl halide Conditions Yield

Bromo acetonitrile LiHMDS/THF, 0oC to rt 80%

Allyl bromide LiHMDS/THF, 0oC to rt 30%

Methyl vinyl ketone LiHMDS/THF, 0oC to rt <1%

Methyl vinyl ketone NaHMDS/THF, 0oC to rt <1%

Methyl vinyl ketone DIPEA, TiCl4/ DCM, -40oC 67%

Alkyalation-2:

Figure 25: Reactions attempted to create benzylic stereogenic center

The initial plan was to use 4-methoxyphenylacetic acid as starting material since it was readily available from previous work in our group on the synthesis of phytoestrogens, with the second methoxy group to be added at the end by oxidation. The first alkylation was attempted using bromo acetonitrile following deprotonation by lithium bis(trimethylsilyl)amide (LiHMDS).

As anticipated, the reaction worked without any difficulty and yielded the required product in 46

good yields. Similarly, allyl bromide furnished the corresponding alkylated product in moderate yield. When the lithium enolate was subjected to Michael addition with methyl vinyl ketone, complex products were observed. Many attempts were made to create the quaternary center, using different reagents and conditions (see Table 3, alkylation 2), but none of these reactions were successful. At this point the synthetic plan was modified to effect the initial substitution with methylvinyl ketone (MVK), and then to alkylate with two carbon chain alkylating agents.

An alternate strategy would require reductive removal of the chiral auxiliary and working toward the cyclopentene synthetic intermediate of Taber et al. (Figure 26). 65

Figure 26: Synthetic scheme of Taber et al. (2001)

This reaction was based on the work of Evans in which he explored asymmetric Michael

66 additions using TiCl4 and diisopropylethylamine (Hunig’s base/ DIPEA). Initially the reaction 47

generated approximately 1% product, enough to verify by 1H-NMR. By carefully screening of various temperatures and addition sequence, the yield was improved to 67%. This improvement seemingly corresponded to slower addition of MVK, and lower enolization temperature (-40oC instead of -25oC).

Next, the ketone was protected as ketal and the resulting amide reduced to the corresponding alcohol. The goal was to then protect the resulting alcohol and subject to intramolecular alkylidene insertion reaction pioneered by Taber to cyclize into a cyclopentene ring with stereogenic quaternary center. Due to time constraints, the envisioned synthetic sequence was halted at this stage.

Figure 27: Compounds generated

LIST OF REFERENCES

1. Lehninger, A. L.; Nelson, D. L.; Cox, M. M., Lehninger principles of biochemistry. 5th ed.; W.H. Freeman: New York, 2008.

2. Jeon, Y. H.; Heo, Y. S.; Kim, C. M.; Hyun, Y. L.; Lee, T. G.; Ro, S.; Cho, J. M., Phosphodiesterase: overview of protein structures, potential therapeutic applications and recent progress in drug development. Cellular and molecular life sciences : CMLS 2005, 62 (11), 1198- 220.

3. Friebe, A.; Koesling, D., Regulation of nitric oxide-sensitive guanylyl cyclase. Circulation research 2003, 93 (2), 96-105.

4. Krebs, E. G.; Beavo, J. A., Phosphorylation-dephosphorylation of enzymes. Annual review of biochemistry 1979, 48, 923-59.

5. Perry, M. J.; Higgs, G. A., Chemotherapeutic potential of phosphodiesterase inhibitors. Current opinion in chemical biology 1998, 2 (4), 472-81.

6. (a) Mehats, C.; Andersen, C. B.; Filopanti, M.; Jin, S. L.; Conti, M., Cyclic nucleotide phosphodiesterases and their role in endocrine cell signaling. Trends in endocrinology and metabolism: TEM 2002, 13 (1), 29-35; (b) Conti, M.; Nemoz, G.; Sette, C.; Vicini, E., Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocrine reviews 1995, 16 (3), 370-89; (c) Rotella, D. P., Phosphodiesterase 5 inhibitors: current status and potential applications. Nature reviews. Drug discovery 2002, 1 (9), 674-82.

7. Drugs@FDA Database. http://www.fda.gov/Drugs/InformationOnDrugs/ucm135821.htm (accessed 06/18/2009 ).

8. (a) Huai, Q.; Liu, Y.; Francis, S. H.; Corbin, J. D.; Ke, H., Crystal structures of phosphodiesterases 4 and 5 in complex with inhibitor 3-isobutyl-1-methylxanthine suggest a conformation determinant of inhibitor selectivity. The Journal of biological chemistry 2004, 279 (13), 13095-101; (b) Huai, Q.; Wang, H.; Sun, Y.; Kim, H. Y.; Liu, Y.; Ke, H., Three- dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity. Structure 2003, 11 (7), 865-73; (c) Huai, Q.; Wang, H.; Zhang, W.; Colman, R. W.; Robinson, H.; Ke, H., Crystal structure of phosphodiesterase 9 shows orientation variation of inhibitor 3-isobutyl-1-methylxanthine binding. Proceedings of the National Academy of Sciences of the United States of America 2004, 101 (26), 9624-9; (d) Lee, M. E.; Markowitz, J.; Lee, J. O.; Lee, H., Crystal structure of phosphodiesterase 4D and inhibitor complex(1). FEBS letters 2002, 530 (1-3), 53-8; (e) Scapin, G.; Patel, S. B.; Chung, C.; Varnerin, J. P.; Edmondson, S. D.; Mastracchio, A.; Parmee, E. R.; Singh, S. B.; Becker, J. W.; Van der Ploeg, L. H.; Tota, M. R., Crystal structure of human phosphodiesterase 3B: atomic basis for substrate and inhibitor

specificity. Biochemistry 2004, 43 (20), 6091-100; (f) Sung, B. J.; Hwang, K. Y.; Jeon, Y. H.; Lee, J. I.; Heo, Y. S.; Kim, J. H.; Moon, J.; Yoon, J. M.; Hyun, Y. L.; Kim, E.; Eum, S. J.; Park, S. Y.; Lee, J. O.; Lee, T. G.; Ro, S.; Cho, J. M., Structure of the catalytic domain of human phosphodiesterase 5 with bound drug molecules. Nature 2003, 425 (6953), 98-102; (g) Xu, R. X.; Hassell, A. M.; Vanderwall, D.; Lambert, M. H.; Holmes, W. D.; Luther, M. A.; Rocque, W. J.; Milburn, M. V.; Zhao, Y.; Ke, H.; Nolte, R. T., Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Science 2000, 288 (5472), 1822-5; (h) Xu, R. X.; Rocque, W. J.; Lambert, M. H.; Vanderwall, D. E.; Luther, M. A.; Nolte, R. T., Crystal structures of the catalytic domain of phosphodiesterase 4B complexed with AMP, 8-Br-AMP, and rolipram. Journal of molecular biology 2004, 337 (2), 355-65; (i) Zhang, K. Y.; Card, G. L.; Suzuki, Y.; Artis, D. R.; Fong, D.; Gillette, S.; Hsieh, D.; Neiman, J.; West, B. L.; Zhang, C.; Milburn, M. V.; Kim, S. H.; Schlessinger, J.; Bollag, G., A glutamine switch mechanism for nucleotide selectivity by phosphodiesterases. Molecular cell 2004, 15 (2), 279-86.

9. (a) Thompson, W. J., Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function. Pharmacology & therapeutics 1991, 51 (1), 13-33; (b) Bolger, G. B., Molecular biology of the cyclic AMP-specific cyclic nucleotide phosphodiesterases: a diverse family of regulatory enzymes. Cellular signalling 1994, 6 (8), 851-9.

10. Stryer, L., Transducin and the cyclic GMP phosphodiesterase: amplifier proteins in vision. Cold Spring Harbor symposia on quantitative biology 1983, 48 Pt 2, 841-52.

11. Sausbier, M.; Schubert, R.; Voigt, V.; Hirneiss, C.; Pfeifer, A.; Korth, M.; Kleppisch, T.; Ruth, P.; Hofmann, F., Mechanisms of NO/cGMP-dependent vasorelaxation. Circulation research 2000, 87 (9), 825-30.

12. Lue, T. F., Erectile dysfunction. The New England journal of medicine 2000, 342 (24), 1802-13.

13. O'Connell, H. E.; Hutson, J. M.; Anderson, C. R.; Plenter, R. J., Anatomical relationship between urethra and clitoris. The Journal of urology 1998, 159 (6), 1892-7.

14. (a) Burnett, A. L.; Calvin, D. C.; Silver, R. I.; Peppas, D. S.; Docimo, S. G., Immunohistochemical description of nitric oxide synthase isoforms in human clitoris. The Journal of urology 1997, 158 (1), 75-8; (b) Park, K.; Moreland, R. B.; Goldstein, I.; Atala, A.; Traish, A., Sildenafil inhibits phosphodiesterase type 5 in human clitoral corpus cavernosum smooth muscle. Biochemical and biophysical research communications 1998, 249 (3), 612-7.

15. Caruso, S.; Intelisano, G.; Lupo, L.; Agnello, C., Premenopausal women affected by sexual arousal disorder treated with sildenafil: a double-blind, cross-over, placebo-controlled study. BJOG : an international journal of obstetrics and gynaecology 2001, 108 (6), 623-8.

16. (a) Gozzard, N.; el-Hashim, A.; Herd, C. M.; Blake, S. M.; Holbrook, M.; Hughes, B.; Higgs, G. A.; Page, C. P., Effect of the glucocorticosteroid budesonide and a novel phosphodiesterase type 4 inhibitor CDP840 on antigen-induced airway responses in neonatally immunised rabbits. British journal of pharmacology 1996, 118 (5), 1201-8; (b) Howell, R. E.; Sickels, B. D.; Woeppel, S. L., Pulmonary antiallergic and bronchodilator effects of isozyme- selective phosphodiesterase inhibitors in guinea pigs. The Journal of pharmacology and experimental therapeutics 1993, 264 (2), 609-15; (c) Hughes, B.; Howat, D.; Lisle, H.; Holbrook, M.; James, T.; Gozzard, N.; Blease, K.; Hughes, P.; Kingaby, R.; Warrellow, G.; Alexander, R.; Head, J.; Boyd, E.; Eaton, M.; Perry, M.; Wales, M.; Smith, B.; Owens, R.; Catterall, C.; Lumb, S.; Russell, A.; Allen, R.; Merriman, M.; Bloxham, D.; Higgs, G., The inhibition of antigen- induced eosinophilia and bronchoconstriction by CDP840, a novel stereo-selective inhibitor of phosphodiesterase type 4. British journal of pharmacology 1996, 118 (5), 1183-91; (d) Raeburn, D.; Underwood, S. L.; Lewis, S. A.; Woodman, V. R.; Battram, C. H.; Tomkinson, A.; Sharma, S.; Jordan, R.; Souness, J. E.; Webber, S. E.; et al., Anti-inflammatory and bronchodilator properties of RP 73401, a novel and selective phosphodiesterase type IV inhibitor. British journal of pharmacology 1994, 113 (4), 1423-31; (e) Turner, C. R.; Andresen, C. J.; Smith, W. B.; Watson, J. W., Effects of rolipram on responses to acute and chronic antigen exposure in monkeys. American journal of respiratory and critical care medicine 1994, 149 (5), 1153-9.

17. (a) Kakkar, R.; Raju, R. V.; Rajput, A. H.; Sharma, R. K., Amantadine: an antiparkinsonian agent inhibits bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme. Brain research 1997, 749 (2), 290-4; (b) Kakkar, R.; Raju, R. V.; Rajput, A. H.; Sharma, R. K., Inhibition of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by deprenyl. Life sciences 1996, 59 (21), PL337-41.

18. (a) Jeon, K. I.; Xu, X.; Aizawa, T.; Lim, J. H.; Jono, H.; Kwon, D. S.; Abe, J.; Berk, B. C.; Li, J. D.; Yan, C., Vinpocetine inhibits NF-kappaB-dependent inflammation via an IKK- dependent but PDE-independent mechanism. Proceedings of the National Academy of Sciences of the United States of America 2010, 107 (21), 9795-800; (b) Medina, A. E., Vinpocetine as a potent antiinflammatory agent. Proceedings of the National Academy of Sciences of the United States of America 2010, 107 (22), 9921-2; (c) Truss, M. C.; Uckert, S.; Stief, C. G.; Kuczyk, M.; Jonas, U., Cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the human detrusor smooth muscle. I. Identification and characterization. Urological research 1996, 24 (3), 123-8; (d) Truss, M. C.; Uckert, S.; Stief, C. G.; Forssmann, W. G.; Jonas, U., Cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the human detrusor smooth muscle. II. Effect of various PDE inhibitors on smooth muscle tone and cyclic nucleotide levels in vitro. Urological research 1996, 24 (3), 129-34.

19. (a) Tully, T., Regulation of gene expression and its role in long-term memory and synaptic plasticity. Proceedings of the National Academy of Sciences of the United States of America 1997, 94 (9), 4239-41; (b) Bourtchuladze, R.; Frenguelli, B.; Blendy, J.; Cioffi, D.; 53

Schutz, G.; Silva, A. J., Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein. Cell 1994, 79 (1), 59-68; (c) Kida, S.; Josselyn, S. A.; Pena de Ortiz, S.; Kogan, J. H.; Chevere, I.; Masushige, S.; Silva, A. J., CREB required for the stability of new and reactivated fear memories. Nature neuroscience 2002, 5 (4), 348-55; (d) Yin, J. C.; Wallach, J. S.; Del Vecchio, M.; Wilder, E. L.; Zhou, H.; Quinn, W. G.; Tully, T., Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 1994, 79 (1), 49-58.

20. Sanyal, S.; Sandstrom, D. J.; Hoeffer, C. A.; Ramaswami, M., AP-1 functions upstream of CREB to control synaptic plasticity in Drosophila. Nature 2002, 416 (6883), 870-4.

21. Tully, T.; Bourtchouladze, R.; Scott, R.; Tallman, J., Targeting the CREB pathway for memory enhancers. Nature reviews. Drug discovery 2003, 2 (4), 267-77.

22. (a) O'Donnell, J. M., Antidepressant-like effects of rolipram and other inhibitors of cyclic adenosine monophosphate phosphodiesterase on behavior maintained by differential reinforcement of low response rate. The Journal of pharmacology and experimental therapeutics 1993, 264 (3), 1168-78; (b) Zhang, H. T.; Crissman, A. M.; Dorairaj, N. R.; Chandler, L. J.; O'Donnell, J. M., Inhibition of cyclic AMP phosphodiesterase (PDE4) reverses memory deficits associated with NMDA receptor antagonism. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2000, 23 (2), 198-204; (c) Zhang, H. T.; O'Donnell, J. M., Effects of rolipram on scopolamine-induced impairment of working and reference memory in the radial-arm maze tests in rats. Psychopharmacology 2000, 150 (3), 311- 6.

23. (a) Fujimaki, K.; Morinobu, S.; Duman, R. S., Administration of a cAMP phosphodiesterase 4 inhibitor enhances antidepressant-induction of BDNF mRNA in rat hippocampus. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2000, 22 (1), 42-51; (b) Ghosh, A.; Carnahan, J.; Greenberg, M. E., Requirement for BDNF in activity-dependent survival of cortical neurons. Science 1994, 263 (5153), 1618-23; (c) Mamounas, L. A.; Blue, M. E.; Siuciak, J. A.; Altar, C. A., Brain-derived neurotrophic factor promotes the survival and sprouting of serotonergic axons in rat brain. The Journal of neuroscience : the official journal of the Society for Neuroscience 1995, 15 (12), 7929-39; (d) Brenneman, D. E.; Fitzgerald, S.; Litzinger, M. J., Neuronal survival during electrical blockade is increased by 8-bromo cyclic adenosine 3',5' monophosphate. The Journal of pharmacology and experimental therapeutics 1985, 233 (2), 402-8; (e) Hartikka, J.; Staufenbiel, M.; Lubbert, H., Cyclic AMP, but not basic FGF, increases the in vitro survival of mesencephalic dopaminergic neurons and protects them from MPP(+)-induced degeneration. Journal of neuroscience research 1992, 32 (2), 190-201; (f) Rydel, R. E.; Greene, L. A., cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory

neurons independently of nerve growth factor. Proceedings of the National Academy of Sciences of the United States of America 1988, 85 (4), 1257-61.

24. (a) McAfee, D. A.; Schorderet, M.; Greengard, P., Adenosine 3',5'-monophosphate in nervous tissue: increase associated with synaptic transmission. Science 1971, 171 (3976), 1156- 8; (b) Nathanson, J. A., Cyclic nucleotides and nervous system function. Physiological reviews 1977, 57 (2), 157-256; (c) Ferguson, S. M.; Phillips, P. E.; Roth, B. L.; Wess, J.; Neumaier, J. F., Direct-Pathway Striatal Neurons Regulate the Retention of Decision-Making Strategies. The Journal of neuroscience : the official journal of the Society for Neuroscience 2013, 33 (28), 11668-11676.

25. Chen, S. K.; Zhao, P.; Shao, Y. X.; Li, Z.; Zhang, C.; Liu, P.; He, X.; Luo, H. B.; Hu, X., Moracin M from Morus alba L. is a natural phosphodiesterase-4 inhibitor. Bioorganic & medicinal chemistry letters 2012, 22 (9), 3261-4.

26. Li, J. J., Laughing gas, Viagra, and Lipitor : the human stories behind the drugs we use. Oxford University Press: Oxford ; New York, 2006; p xv, 310 p.

27. Dell'Agli, M.; Galli, G. V.; Dal Cero, E.; Belluti, F.; Matera, R.; Zironi, E.; Pagliuca, G.; Bosisio, E., Potent inhibition of human phosphodiesterase-5 by icariin derivatives. Journal of natural products 2008, 71 (9), 1513-7.

28. Stearn, W. T.; Shaw, J. M. H.; Green, P. S.; Mathew, B., The genus Epimedium and other herbaceous Berberidaceae. Timber Press: Portland, Or., 2002; p xi, 342 p.

29. Ma, H.; He, X.; Yang, Y.; Li, M.; Hao, D.; Jia, Z., The genus Epimedium: an ethnopharmacological and phytochemical review. Journal of ethnopharmacology 2011, 134 (3), 519-41.

30. Liang, H.-R.; Sirén, H.; Reikkola, M.-L.; Vuorela, P.; Vuorela, H.; Hiltunen, R., Characterization of Flavonoids in Extracts from Four Species of Epimedium by Micellar Electrokinetic Capillary Chromatography with Diode-Array Detection. Journal of Chromatographic Science 1997, 35 (3), 117-125.

31. Royer, D.; Hickey, L.; Wing, S., Ecological conservatism in the ‘‘ living fossil ’’ Ginkgo. 2003, 29 (1), 84-104.

32. Plotnik, A.; Morton Arboretum., The urban tree book : an uncommon field guide for city and town. 1st ed.; Three Rivers Press: New York, 2000; p xiv, 432 p.

33. Duke, J. A.; Duke, J. A., Handbook of medicinal herbs. 2nd ed.; CRC Press: Boca Raton, FL, 2002; p 870 p.

34. Cohen, A. J.; Bartlik, B., Ginkgo Biloba for Antidepressant-Induced Sexual Dysfunction. Journal of Sex & Marital Therapy 1998, 24 (2), 139-143.

35. Watson, R. R.; Preedy, V. R., Botanical medicine in clinical practice. CABI: Wallingford, UK ; Cambridge, MA, 2008; p xviii, 915 p.

36. Hadidi, K. A.; Aburjai, T.; Battah, A. K., A comparative study of Ferula hermonis root extracts and sildenafil on copulatory behaviour of male rats. Fitoterapia 2003, 74 (3), 242-6.

37. (a) Appendino, G.; Spagliardi, P.; Cravotto, G.; Pocock, V.; Milligan, S., Daucane phytoestrogens: a structure-activity study. Journal of natural products 2002, 65 (11), 1612-5; (b) Appendino, G.; Spagliardi, P.; Sterner, O.; Milligan, S., Structure--activity relationships of the estrogenic sesquiterpene ester ferutinin. Modification of the terpenoid core. Journal of natural products 2004, 67 (9), 1557-64.

38. Colman-Saizarbitoria, T.; Boutros, P.; Amesty, A.; Bahsas, A.; Mathison, Y.; Garrido Mdel, R.; Israel, A., Ferutinin stimulates nitric oxide synthase activity in median eminence of the rat. Journal of ethnopharmacology 2006, 106 (3), 327-32.

39. .

40. Piacente, S.; Carbone, V.; Plaza, A.; Zampelli, A.; Pizza, C., Investigation of the tuber constituents of maca (Lepidium meyenii Walp.). Journal of agricultural and food chemistry 2002, 50 (20), 5621-5.

41. (a) Dording, C. M.; Fisher, L.; Papakostas, G.; Farabaugh, A.; Sonawalla, S.; Fava, M.; Mischoulon, D., A double-blind, randomized, pilot dose-finding study of maca root (L. meyenii) for the management of SSRI-induced sexual dysfunction. CNS neuroscience & therapeutics 2008, 14 (3), 182-91; (b) Bogani, P.; Simonini, F.; Iriti, M.; Rossoni, M.; Faoro, F.; Poletti, A.; Visioli, F., Lepidium meyenii (Maca) does not exert direct androgenic activities. Journal of ethnopharmacology 2006, 104 (3), 415-7.

42. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced drug delivery reviews 2001, 46 (1-3), 3-26.

43. What is the structure of LigPrep?. http://www.schrodinger.com/kb/1243 (accessed 7/20/2013).

44. Wang, H.; Liu, Y.; Huai, Q.; Cai, J.; Zoraghi, R.; Francis, S. H.; Corbin, J. D.; Robinson, H.; Xin, Z.; Lin, G.; Ke, H., Multiple Conformations of Phosphodiesterase-5: IMPLICATIONS

FOR ENZYME FUNCTION AND DRUG DEVELOPMENT. Journal of Biological Chemistry 2006, 281 (30), 21469-21479.

45. Protein Preparation Wizard. http://www.schrodinger.com/productpage/14/16/ (accessed 7/20/2013).

46. Ko, W. C.; Shih, C. M.; Lai, Y. H.; Chen, J. H.; Huang, H. L., Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships. Biochemical pharmacology 2004, 68 (10), 2087-94.

47. Chua, L. S.; Amin, N. A.; Neo, J. C.; Lee, T. H.; Lee, C. T.; Sarmidi, M. R.; Aziz, R. A., LC-MS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang). Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2011, 879 (32), 3909-19.

48. Bhat, R.; Karim, A. A., Tongkat Ali (Eurycoma longifolia Jack): a review on its ethnobotany and pharmacological importance. Fitoterapia 2010, 81 (7), 669-79.

49. (a) Miyake, K.; Tezuka, Y.; Awale, S.; Li, F.; Kadota, S., Quassinoids from Eurycoma longifolia. Journal of natural products 2009, 72 (12), 2135-40; (b) Le Van, T.; Nguyen Ngoc, S., Constituents of Eurycoma longifolia Jack. The Journal of organic chemistry 1970, 35 (4), 1104- 9.

50. Kuo, P. C.; Shi, L. S.; Damu, A. G.; Su, C. R.; Huang, C. H.; Ke, C. H.; Wu, J. B.; Lin, A. J.; Bastow, K. F.; Lee, K. H.; Wu, T. S., Cytotoxic and antimalarial beta-carboline alkaloids from the roots of Eurycoma longifolia. Journal of natural products 2003, 66 (10), 1324-7.

51. Tnah, L. H.; Lee, C. T.; Lee, S. L.; Ng, K. K. S.; Ng, C. H.; Hwang, S. S., Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), for DNA profiling. American Journal of Botany 2011, 98 (5), e130-e132.

52. An Evidence-Based Systematic Review of Tongkat Ali (Eurycoma longifolia) by the Natural Standard Research Collaboration. Journal of Dietary Supplements 2013, 10 (1), 54-83.

53. (a) Kuo, P.-C.; Damu, A. G.; Lee, K.-H.; Wu, T.-S., Cytotoxic and antimalarial constituents from the roots of Eurycoma longifolia. Bioorganic & Medicinal Chemistry 2004, 12 (3), 537-544; (b) Farouk, A. E.; Benafri, A., Antibacterial activity of Eurycoma longifolia Jack. A Malaysian medicinal plant. Saudi medical journal 2007, 28 (9), 1422-4; (c) Miyake, K.; Li, F.; Tezuka, Y.; Awale, S.; Kadota, S., Cytotoxic activity of quassinoids from Eurycoma longifolia. Natural product communications 2010, 5 (7), 1009-12; (d) Miyake, K.; Tezuka, Y.; Awale, S.; Li, F.; Kadota, S., Canthin-6-one alkaloids and a tirucallanoid from Eurycoma longifolia and their cytotoxic activity against a human HT-1080 fibrosarcoma cell line. Natural product communications 2010, 5 (1), 17-22; (e) Low, B. S.; Teh, C. H.; Yuen, K. H.; Chan, K. L., 57

Physico-chemical effects of the major quassinoids in a standardized Eurycoma longifolia extract (Fr 2) on the bioavailability and pharmacokinetic properties, and their implications for oral antimalarial activity. Natural product communications 2011, 6 (3), 337-41; (f) Kavitha, N.; Noordin, R.; Chan, K. L.; Sasidharan, S., In vitro anti-Toxoplasma gondii activity of root extract/fractions of Eurycoma longifolia Jack. BMC complementary and alternative medicine 2012, 12, 91.

54. Qu, H.; Christensen, K. B.; Fretté, X. C.; Tian, F.; Rantanen, J.; Christensen, L. P., A Novel Hybrid Chromatography− Crystallization Process for the Isolation and Purification of a Natural Pharmaceutical Ingredient from a Medicinal Herb. Organic Process Research & Development 2010, 14 (3), 585-591.

55. Tropicos Plant Database: Sceletium. http://www.tropicos.org/namesearch.aspx?name=Sceletium (accessed 5/15/2013).

56. Gericke, N.; Viljoen, A. M., Sceletium--a review update. Journal of ethnopharmacology 2008, 119 (3), 653-63.

57. Jeffs, P. W. W., Sceletium alkaloids. Part 12. Synthesis of (±)-mesembranol and (±)-O- methyljoubertiamine. Aza-ring expansion of cis-bicyclo[4.2.0]octanones. Journal of organic chemistry 1982, 47 (20), 3881-3886.

58. (a) New Dietary Ingredients Notification Process. http://www.fda.gov/Food/DietarySupplements/NewDietaryIngredientsNotificationProcess/defaul t.htm (accessed 4/15/2013); (b) Hilmas, C., Sceletium tortuosum review process. Cunningham, M. J., (Personal communication) 2013.

59. Harvey, A. L. L., Pharmacological actions of the South African medicinal and functional food plant Sceletium tortuosum and its principal alkaloids. Journal of ethnopharmacology 2011, 137 (3), 1124-1129.

60. Napoletano, M.; Fraire, C.; Santangelo, F.; Moriggi, E., Mesembrine is an inhibitor of PDE4 that follows the structure-activity relationship of rolipram. Chemistry Preprint Archive 2001, 303-308.

61. Cashman, J. R.; Voelker, T.; Zhang, H. T.; O'Donnell, J. M., Dual inhibitors of phosphodiesterase-4 and serotonin reuptake. Journal of medicinal chemistry 2009, 52 (6), 1530- 9.

62. Manske, R. H. F.; Holmes, H. L.; Rodrigo, R. G. A., The Alkaloids. Chemistry and physiology v.19. Academic Press: New York, 1981; pp 1-79.

63. Shamma, M., The total synthesis of (±)-mesembrine. Tetrahedron letters 1965, 6 (52), 4847-4851.

64. Christmann, M.; Br se, S., Asymmetric synthesis the essentials. Wiley-VCH: Weinheim, 2007; p xl, 345 p.

65. Taber, D. F.; Neubert, T. D., Enantioselective construction of cyclic quaternary centers: (-)-mesembrine. The Journal of organic chemistry 2001, 66 (1), 143-7.

66. Evans, D. A., Enantioselective Michael reactions. Diastereoselective reactions of chlorotitanium enolates of chiral N-acyloxazolidinones with representative electrophilic olefins. Journal of organic chemistry 1991, 56 (20), 5750-5752.

67. Heemstra, J. M. M., Total Synthesis of ( )-Equol. Organic letters 2006, 8 (24), 5441- 5443.

LIST OF APPENDICES

APPENDIX A:

NMR AND X-RAY DATA OF COMPOUNDS ISOLATED FROM TONGKAT ALI

Figure 17: 1H NMR spectrum of 2.1 (canthin-6-one)

Figure 18: 1H NMR spectrum of 2.2 (9-methoxycanthin-6-one)

Figure 19: 1H NMR spectrum of 2.3 (dehydroeurycomalactone)

Figure 20: 1H NMR spectrum of 2.4 (laurycolactone A)

Figure 21: Wireframe model of 2.5 (crinosterol) molecules in unit cell to clarify three-dimensional structure

Figure 22: 1H NMR spectrum of 2.5 (crinosterol)

Figure 23: 13C NMR spectrum of 2.5 (crinosterol)

APPENDIX B:

MESEMBRINE SYNTHETIC INTERMEDIATES INFORMATION

(S)-4-Benzyl-3-[2-(4-methoxyphenyl)acetyl]oxazolidin-2-one (Compound 3.1): Prepared

67 -1 according to Heemstra et al., 2006. [α]D= 63.5 (c=0.5, CHCl3), IR(cm ): 2922, 1771, 1693,

1 1 1611, 1510, 1387, 1353, 1243, 1175, 1102, 1030 H NMR: H NMR (400 MHz, CDCl3) δ 7.34

– 7.25 (m, 5H), 7.15 (dd, J = 7.3, 2.0 Hz, 2H), 6.94 – 6.88 (m, 2H), 4.68 (ddt, J = 10.2, 6.8, 3.3

Hz, 1H), 4.34 – 4.14 (m, 1H), 3.82 (s, 2H), 3.27 (dd, J = 13.4, 3.0 Hz, 1H), 2.78 (dd, J = 13.3,

13 9.4 Hz, 1H) C NMR (101 MHz, CDCl3) δ 171.56, 158.83, 153.42, 135.18, 130.84 (2C), 129.45

(2C), 128.93 (2C), 127.32, 125.54, 114.04 (2C), 66.13, 55.30, 40.70, 37.72

(R)-4-[(S)-4-benzyl-2-oxooxazolidin-3-yl]-3-(4-methoxyphenyl)-4-oxobutanenitrile

(Compound 3.2): Compound 3.1 (3.25 g, 10.0 mmol) was dissolved in 35 ml of dry tetrahydrofuran. After cooling the solution to 0oC, LiHMDS (12 ml, 1.0M in THF) was added dropwise. After 25 minutes, bromo acetonitrile (0.84 ml, 12 mmol) was added. After one hour, the reaction was quenched with saturated aqueous ammonium chloride solution. After filtration and concentration under reduced pressure, the material was purified by silica gel chromatography using hexanes and ethyl acetate (3:1). The concentrated product was an amber oil, which solidified on standing. The material was re-crystallized with warm hexane and ethyl

-1 acetate to form clear crystals. [α]D= 176.8 (c=0.5, CHCl3), IR (cm ): 2989, 2925, 2849, 1778,

1 1 1678, 1608, 1511, 1385, 1253, 1217, 1178, 1111 H NMR: H NMR (400 MHz, CDCl3) δ 7.38 –

7.18 (m, 5H), 6.86 (d, J = 8.7 Hz, 2H), 5.35 – 5.26 (m, 2H), 4.60 (ddt, J = 9.8, 5.4, 2.6 Hz, 1H),

4.17 – 3.98 (m, 2H), 3.77 (s, 3H), 3.34 (dd, J = 13.5, 3.2 Hz, 1H), 3.06 (dd, J = 16.6, 8.2 Hz, 70

13 1H), 2.88 (d, J = 9.5 Hz, 1H), 2.78 (dd, J = 16.6, 6.8 Hz, 1H). C NMR: (101 MHz, CDCl3) δ

171.09, 159.73, 152.43, 134.87, 129.44 (4C), 129.04 (2C), 127.48, 127.18, 117.83, 114.54 (2C),

+ 66.15, 55.78, 55.29, 45.01, 37.77, 22.26 ESI-HRMS: calcd. C21H21N2O4 m/z 365.1459 [M+H] ; found m/z 365.1432

(R)-1-[(S)-4-Benzyl-2-oxooxazolidin-3-yl]-2-(4-methoxyphenyl)hexane-1,5-dione

(Compound 3.3): Compound 3.2 (3.25g was dissolved in anhydrous dichloromethane at room temperature under argon an atmosphere. The solution was cooled to -50oC, and diisoproylethylamine (Hünig’s base) was added. This mixture was stirred at -50oC for 15 minutes, followed by addition of TiCl4 solution (1.0M in DCM), and stirred for one hour at -

50oC. A dilute solution of methyl vinyl ketone in anhydrous dichloromethane (1.13 ml MVK/ 40 ml dicholoromethane) was added dropwise. The reaction was quenched when bath temperature reached 0oC (~45 minutes) with saturated sodium/potassium tartrate (Rochelle salt) solution.

After filtering and concentration under reduced pressure, the material was purified by silica gel chromatography using hexanes and ethyl acetate (3:1) yielding a clear oil. [α]D= 84 (c=0.5,

-1 1 CHCl3) IR (cm ): 2930, 1773, 1691, 1607, 1509, 1450, 1367, 1244, 1178, 1106, 1029 H NMR

(500 MHz, CDCl3) δ 7.48 – 7.21 (m, 5H), 6.87 (d, J = 8.7 Hz, 2H), 5.10 – 4.88 (m, 2H), 4.60 (t,

J = 8.6 Hz, 2H), 4.34 – 4.01 (m, 7H), 3.81 (s, 5H), 3.38 (dd, J = 13.3, 3.0 Hz, 3H), 2.81 (dd, J =

13.3, 9.8 Hz, 2H), 2.54 – 2.28 (m, 12H), 2.26 – 2.00 (m, 11H), 1.28 (t, J = 7.1 Hz, 1H). 13C

NMR (126 MHz, CDCl3) δ 207.89, 173.82, 159.05, 152.90, 135.33 (2C), 129.78 (2C), 129.43

(2C), 128.98, 127.37, 114.10 (2C), 65.92, 55.81, 55.25, 46.99, 41.21, 38.05, 29.92, 27.75. ESI-

+ HRMS: calcd. C23H26NO5 m/z 396.1733 [M+H] ; found m/z 396.1739

(S)-4-Benzyl-3-[(R)-2-(4-methoxyphenyl]-4-(2-methyl-1,3-dioxolan-2- yl)butanoyl)oxazolidin-2-one (Compound 3.4): Compound 3.3 was dissolved in benzene at room temperature. To the round-bottom flask, ethylene glycol and para-toluenesulfonic acid were added. A Dean-Stark apparatus was used and reaction was refluxed for five hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution and extracted with diethyl ether. This organic material was concentrated under reduced pressure and purified by silica gel chromatography using hexanes and ethyl acetate (3:1) with triethylamine (0.2% of total volume) was added to mobile phase. The overall yield was 97% in the form of a clear oil.; [α]D=

-1 76 (c=0.5, CHCl3) IR (cm ): 2922, 2836, 1774, 1692, 1607, 1509, 1452, 1375, 1246, 1179,

1 1102, 1031 H NMR (400 MHz, CDCl3) δ 7.29 (m, 7H), 6.84 (d, J = 7.6 Hz, 2H), 4.98 (t, J = 7.4

Hz, 1H), 4.57 (t, J = 7.4 Hz, 1H), 4.16 – 3.94 (m, 6H), 3.77 (s, 3H), 3.36 (d, J = 12.5 Hz, 1H),

2.78 (dd, 1H), 2.21 (ddd, J = 17.3, 11.6, 5.5 Hz, 1H), 2.01 – 1.90 (m, 1H), 1.67 (m, 1H), 1.57

13 (1H) 1.31 (s, 3H) C NMR (101 MHz, CDCl3) δ 174.19, 158.87, 152.94, 135.41, 130.52,

129.70 (2C), 129.42 (2C), 128.99, 128.93 (2C), 127.30, 113.99 (2C), 109.71, 65.79, 64.60,

55.76, 55.21, 47.56, 38.03, 36.57, 28.54, 23.84 ESI-HRMS: calcd. C25H30NO6 m/z 440.1995

[M+H]+; found m/z 440.1982

(R)-2-(4-Methoxyphenyl)-4-(2-methyl-1,3-dioxolan-2-yl)butan-1-ol (Compound 3.5):

Compound 4.4 (0.5g, mmol) was dissolved in tetrahydrofuran at room temperature. The solution was cooled to 0oC before adding 100 mg of lithium aluminum hydride. The solution was allowed to warm to room temperature and was stirred overnight. After 12 hours, the reaction mixture was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate and

purified by column chromatography to yield alcohol 3.5 in greater than 95% yield (255 mg).

-1 [α]D= 5 (c=0.5, CHCl3) IR (cm ): 3409 (br), 2935, 2877, 1746, 1609, 1510, 1458, 1375, 1375,

1 1299, 1244, 1177, 1136, 1031 H NMR (400 MHz, CDCl3) δ 7.05 (d, J = 8.6 Hz, 2H), 6.79 (d, J

= 8.6 Hz, 2H), 3.85 – 3.74 (m, 5H), 3.71 (s, 3H), 3.66 – 3.58 (m, 2H), 2.92 (t, J = 11.3 Hz, 1H),

13 1.62 – 1.38 (m, 4H), 1.19 (s, 3H) C NMR (101 MHz, CDCl3) δ 158.38, 133.91, 128.94 (2C),

114.09 (2C), 109.95, 64.60, 64.54, 63.67, 55.23, 47.77, 36.74, 26.28, 23.79 ESI-HRMS: calcd.

+ C13H18O2 m/z 205.1234 [M-H] ; found m/z 205.1180

APPENDIX C:

MESEMBRINE SYNTHETIC INTERMEDIATES IR AND NMR SPECTRA

Figure 24: IR Spectrum (neat) of 3.1

Figure 25: 1H NMR spectrum of 3.1

Figure 26: 13C NMR spectrum of 3.1

Figure 27: IR Spectrum (neat) of 3.2

Figure 28: 1H NMR spectrum of 3.2

Figure 29: 13C NMR spectrum of 3.2

Figure 30: IR spectrum of 3.3

Figure 31: 1H NMR spectrum of 3.3

Figure 32: 13C NMR spectrum of 3.3

Figure 33: IR Spectrum of 3.4

Figure 34: 1H spectrum of 3.4

Figure 35: 13C spectrum of 3.4

Figure 36: IR Spectrum (neat) of 3.5

Figure 37: 1H NMR spectrum of 3.5

Figure 38: 13C NMR spectrum of 3.5

VITA

EDUCATION

B.S., Biochemistry & Molecular Biology May 2007 Mississippi State University, Mississippi State, MS 39762

RESEARCH POSITIONS

Graduate Research Assistant Aug. 2010 – Aug. 2013

The University of Mississippi, Department of Pharmacognosy, University, MS  Synthesis, isolation and structure elucidation of intermediates en route to natural product mesembrine  Extraction and isolation of natural products using chromatography  Structure elucidation of terpenes, alkaloids and sterols from Eurycoma longifolia Jack using IR, 1D and 2D NMR  In silico screening of natural products for phosphodiesterase 5 activity  Advisor: Dr. Ikhlas Khan

Graduate Research Assistant May 2009 – July 2010

Texas State University, San Marcos, TX  Synthesis, isolation and structure elucidation of monomers for use in construction of electroactive polymeric materials  Advisor: Dr. Jennifer Irvin

 Bacterial culture, expression and isolation of p53 protein  Thermodynamic ensemble modeling of p53 protein using Fyrestar (RedStorm) software  Advisor: Dr. Steve Whitten

Laboratory Technician Mar. 2008 – May 2008

Microbial Insights, Rockford, TN  Isolation of microbes from water and soil samples  Identification of bioremedial genes from microbial samples using qPCR

Undergraduate Research Assistant Aug. 2003 – Dec. 2006

Mississippi State University  Used microbial culture and molecular methods (PCR, blotting) studying nitrogenase fusion proteins, effects of gold nanoparticle exposure to A. vinelandii  Advisor: Dr. Nara Gavini (2003-2005) 90

 Extracted, isolated genetic material from Platanthera (Orchidaceae) species using molecular methods  Analyzed DNA sequence information using Sequencher and Mr.Bayes software to evaluate phylogenetic classifications of orchids  Advisor: Lisa Wallace (2005-2006)

TEACHING POSITIONS

Texas State University, San Marcos, TX

Department of Chemistry and Biochemistry May 2009 – July 2010  Instructor for general, analytical and organic I lab sections  Chemistry tutor for undergraduate general and organic chemistry

OTHER POSITIONS AND DISTINCTIONS

The University of Mississippi, Department of Pharmacognosy, University, MS

Director of Graduate Affairs, Graduate Student Council (GSC) 2012-2013  Graduate Student Representative to Chancellor’s Standing Committee on Libraries

President of Medicinal Chemistry Journal Club 2012-2013

Department of Pharmacognosy Senator, GSC 2010-2012

PTCB Certified to work as Pharmacy Technician (Certified in Texas) Apr. 2009