Anti-malarial Drug Discovery from Australian Flora

Author Robertson, Luke

Published 2018-09

Thesis Type Thesis (PhD Doctorate)

School School of Environment and Sc

DOI https://doi.org/10.25904/1912/3530

Copyright Statement The author owns the copyright in this thesis, unless stated otherwise.

Downloaded from http://hdl.handle.net/10072/381516

Griffith Research Online https://research-repository.griffith.edu.au

Anti-malarial Drug Discovery from Australian Flora

Luke Robertson

B. Sc (Hons)

Submitted in the fulfilment of the requirements of the degree of

Doctor of Philosophy

School of Environment and Science

Griffith University, Australia

September 2018

ABSTRACT

Malaria is a mosquito-borne disease caused by the parasitic protozoan Plasmodium that is responsible for approximately half a million deaths every year. The vast majority of these deaths are caused by P. falciparum in Sub-Saharan Africa (SSA). Although most cases of P. falciparum malaria can currently be treated effectively using artemisinin-based combination therapies (ACTs), resistance to ACTs is beginning to emerge in South-East Asia. This resistance is likely to proliferate and spread into SSA, after which a public health catastrophe is likely to follow. There is currently no drug poised to replace ACTs as the front-line treatment for malaria and there is a need for the discovery of new drugs. Historically, natural products from plants have been our best source of anti-malarial drugs: the alkaloid quinine (from the bark of the Cinchona tree) and the sesquiterpene lactone artemisinin (from the leaves of Artemisia annua) have formed the backbone of anti-malarial chemotherapeutics for centuries.

The primary goal of this thesis was to respond to the need for new anti-plasmodial compounds. This was achieved by collecting and screening a library of Australian Rutaceae species against P. falciparum, selecting species that showed high bioactivity and performing large-scale natural product purification. Isolated natural products were screened against chloroquine-resistant and sensitive P. falciparum and human embryonic kidney (HEK-293) cells to evaluate bioactivity and parasite selectivity. This forms the majority of the thesis (Chapters 2-6).

Chapter 2 reports the initial collection, screening and fingerprinting of a library of 30 Australian Rutaceae species. Chemical fingerprinting using LC-MS was used to identify species that were most likely to contain new natural products. From these results, four species were selected for investigation: Clausena brevistyla (Chapter 2) Flindersia pimenteliana (Chapters 3-4), Acronychia pubescens (Chapter 5) and Pitaviaster haplophyllus (Chapter 6). This chapter also reports the isolation of two known pyranocoumarins from C. brevistyla. One of the pyranocoumarins showed potent and selective activity against P. falciparum, with IC50 values between 466 – 822 nM.

Chapter 3 reports the chemical investigation of F. pimenteliana leaf material. From this plant, a new class of ascorbic-acid adduct indole alkaloids, pimentelamines A-C, were isolated along with one new indole alkaloid, 2-isoprenyl-N,N-dimethyltryptamine. Five known compounds were also isolated. Although the new natural products did not show strong bioactivity, three of the isolated bis-indole alkaloids, borreverine, 4-methylborreverine and dimethylisoborreverine, showed potent activity with IC50 values between 190 – 670 nM against P. falciparum.

i

Chapter 4 reports the isolation of three new isoborreverine-type alkaloids, 10,10’- dimethoxydimethylisoborreverine, 10-methoxydimethylisoborreverine and 10’- methoxydimethylisoborreverine from the bark of F. pimenteliana. Two known borreverine- type alkaloids were also isolated. The moderate anti-plasmodial activity of these alkaloids is reported, with IC50 values ranging from 959 – 2407 ng/mL. Further insights into structure- activity relationships of borreverine-type alkaloids are also discussed.

Chapter 5 reports the chemical investigation of the roots of A. pubescens, from which a highly unusual oxidized furo[2,3-c]xanthene, acrotrione, was isolated along with two known acetophenones. Acrotrione is the first natural product of its class to be isolated. Moderate anti-plasmodial activity for the natural products is reported, with IC50 values ranging from 1.7 to 4.7 µM.

Chapter 6 reports the isolation of one new quinoline alkaloid, leptanoine D, from P. haplophyllus. Nine known alkaloids were also isolated. The chemotaxonomic relationships between the monotypic Pitaviaster genus and the related Australian genera Euodia, Melicope and Acronychia are discussed.

The secondary goal of this thesis was to investigate the factors that influence diversity of natural products in Australian plants. In recent years, natural product-driven drug discovery has seen a decrease in popularity in the pharmaceutical industry, part of which has been caused by the repeated isolation of known natural products. In response to this, there is a requirement for the development of new ideas that expedite the discovery of new natural products. Some recent publications have noted that natural product diversity is positively correlated with diversity of plant-herbivore communities. This may suggest that plants in regions of high biotic stress (i.e. rainforests) should be the focal point of terrestrial plant natural product drug discovery. We aimed to validate this hypothesis by using the Australian Rutaceae genus Flindersia as a case study. Contrary to expectations, our results showed that Flindersia species growing in arid regions of central Australia produced a significantly higher number of structurally unique alkaloids than rainforest species. These unexpected results highlight the potential of the Australian arid zone as a source of new natural products.

ii

STATEMENT OF ORIGINALITY

This work has not previously been submitted for a degree or diploma at any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

X Luke P. Robertson

(Signed) (Date) 20/11/2018

iii

Table of Contents

ABSTRACT ...... i STATEMENT OF ORIGINALITY ...... iii ABBREVIATIONS ...... vi ALL PAPERS INCLUDED ARE CO-AUTHORED ...... vii ACKNOWLEDGEMENTS ...... ix Chapter 1 – Introduction ...... 1 1.1 Introduction to Malaria ...... 1 1.2 Current Malaria Chemotherapeutics ...... 4 1.2.1 Amino-alcohols (quinine, mefloquine, lumefantrine) ...... 5 1.2.2 4-Aminoquinolines (chloroquine, amodiaquine, piperaquine, pyronaridine, naphthoquine) ...... 7 1.2.3 8-Aminoquinolines (primaquine) ...... 10 1.2.4 Sesquiterpene lactones (artemisinin, dihydroartemisinin, artemether, artesunate, arteether) ...... 11 1.2.5 Diaminopyrimidines (pyrimethamine) ...... 14 1.2.6 Sulfonamides (sulfadoxine) ...... 14 1.2.7 Biguanides (proguanil) ...... 15 1.2.8 Napthoquinones (atovaquone) ...... 17 1.2.9 Antibiotics (doxycycline, tetracycline, clindamycin) ...... 18 1.3 Artemisinin Resistance ...... 19 1.4 Anti-malarial Drug Discovery...... 22 1.4.1 Current and Future Efforts ...... 22 1.4.2 The Role of Natural Products in Drug Discovery ...... 23 1.5 Natural Product Drug Discovery: Challenges ...... 26 1.5.1 Discovering Novel Structures ...... 26 1.5.2 Ecology as a Plant Selection Tool ...... 27 1.5.3 Sampling from Harsh Environments ...... 27 1.6 Natural Products from Plants ...... 28 1.6.1 The Rutaceae Family ...... 28 1.6.1.1 Anthranilic Acid Derivatives ...... 28 1.6.1.2 Tyrosine and Phenylalanine Derivatives ...... 30 1.6.1.3 Tryptophan Derivatives ...... 32 1.6.1.4 Histidine Derivatives ...... 32 1.7 Overview and Aims of Thesis ...... 33 1.8 References ...... 34

iv

Chapter 2 – Anti-plasmodial Screening and Natural Product Isolation from a Library of Australian Rutaceae Species ...... 41 2.1 Abstract ...... 41 2.2 Introduction...... 41 2.2.1 Selection of Species and Plant Parts ...... 42 2.3 Results and Discussion ...... 43 2.3.1 Anti-plasmodial Screening Results ...... 43 2.3.2 Pyranocoumarins from Clausena brevistyla ...... 46 2.3.3 Selection of Additional Species for Chemical Investigation ...... 51 2.3.3.1 Flindersia pimenteliana ...... 51 2.3.3.2 Acronychia pubescens ...... 52 2.3.3.3 Pitaviaster haplophyllus ...... 53 2.4 Conclusion ...... 54 2.5 Materials and Methods ...... 55 2.5.1 General Experimental Procedures ...... 55 2.5.2 Collection of Plant Material ...... 55 2.5.3 Preparation and Screening of Extract Library ...... 56 2.5.4 LC-MS Analysis of Bark Material ...... 57 2.5.5 Extraction and Isolation ...... 57 2.6 References ...... 58 Chapter 3 – Pimentelamines A-C, Indole Alkaloids Isolated from the Leaves of the Australian Tree Flindersia pimenteliana ...... 60 Chapter 4 – Anti-plasmodial Bis-indole Alkaloids from the Bark of the Australian Tree Flindersia pimenteliana (Rutaceae) ...... 61 Chapter 5 – Acrotrione, a new Oxidized Xanthene from the Roots of Acronychia pubescens ... 75 Chapter 6 – Quinoline Alkaloids from the Australian Tree Pitaviaster haplophyllus ...... 91 Chapter 7 – Alkaloid Diversity in the Leaves of Australian Flindersia (Rutaceae) Species Driven by Adaptation to Aridity ...... 99 Chapter 8 – Conclusion and Outlook ...... 100 8.1 Anti-plasmodial Natural Product Discovery ...... 100 8.2 New Natural Product Structure Classes ...... 101 8.3 Natural Product Diversity Across Environments ...... 102 Appendix I. Chapter 2 Supporting Information ...... 103 Appendix II. Chapter 3 Supporting Information ...... 113 Appendix III. Chapter 4 Supporting Information ...... 138 Appendix IV. Chapter 5 Supporting Information ...... 157 Appendix V. Chapter 6 Supporting Information...... 166

v

ABBREVIATIONS

ACT Artemisinin-Based Combination Therapy

CH2Cl2 Dichloromethane

CH3CN Acetonitrile COSY Correlation Spectroscopy DAD Diode Array Detector DAPI Diamidino-2-Phenylindole DMSO Dimethyl Sulfoxide ECD Electronic Circular Dichroism EtOAc Ethyl Acetate GMS Greater Mekong Subregion HEK-293 Human Embryonic Kidney Cells 293

HMBC Heteronuclear Multiple-Bond Correlation Spectroscopy

HSQC Heteronuclear Single-Quantum Correlation Spectroscopy HTS High Throughput Screening

IC50 Half Maximal Inhibitory Concentration IR Infrared K13 Kelch-13 LC/MS Liquid Chromatography/Mass Spectrometry HRESIMS High Resolution Electrospray Ionization Mass Spectrometry LRESIMS Low Resolution Electrospray Ionization Mass Spectrometry MeOH Methanol MS Mass Spectrometry NCI National Cancer Institute NMR Nuclear Magnetic Resonance PDA Photodiode Array Detector Q-TOF Quadrupole Time Of Flight RDT Rapid Diagnostic Test ROESY Rotating Frame Nuclear Overhauser Effect Spectroscopy SCX Strongly Acidic Cation Exchanger SE Standard Error

SI Selectivity Index, calculated as HEK-293 IC50 /3D7 IC50 SSA Sub-Saharan Africa TDDFT Time-Dependent Density Functional Theory TFA Trifluoroacetic Acid UV Ultraviolet WHO World Health Organization

vi

ALL PAPERS INCLUDED ARE CO-AUTHORED

Acknowledgement of papers included in this thesis

Section 9.1 of the Griffith University Code for the Responsible Conduct of Research (“Criteria for Authorship”), in accordance with Section 5 of the Australian Code for the Responsible Conduct of Research, states:

To be named as an author, a researcher must have made a substantial scholarly contribution to the creative or scholarly work that constitutes the research output, and be able to take public responsibility for at least that part of the work they contributed. Attribution of authorship depends to some extent on the discipline and publisher policies, but in all cases, authorship must be based on substantial contributions in a combination of one or more of:

 conception and design of the research project

 analysis and interpretation of research data

 drafting or making significant parts of the creative or scholarly work or critically revising it so as to contribute significantly to the final output.

Section 9.3 of the Griffith University Code (“Responsibilities of Researchers”), in accordance with Section 5 of the Australian Code, states:

Researchers are expected to:

 Offer authorship to all people, including research trainees, who meet the criteria for authorship listed above, but only those people.

 Accept or decline offers of authorship promptly in writing.

 Include in the list of authors only those who have accepted authorship

 Appoint one author to be the executive author to record authorship and manage correspondence about the work with the publisher and other interested parties.

 Acknowledge all those who have contributed to the research, facilities or materials but who do not qualify as authors, such as research assistants, technical staff, and advisors on cultural or community knowledge. Obtain written consent to name individuals.

vii

Included in this thesis are papers in Chapters 3-7, which are co-authored with other researchers. My contribution to each co-authored paper is outlined at the front of the relevant chapter. The bibliographic details (if published or accepted for publication)/status (if prepared or submitted for publication) for these papers including all authors, are:

Chapter 3: Robertson, L. P., Duffy, S., Wang, Y., Wang, D., Avery, V. M., Carroll, A. R. Pimentelamines A-C, indole alkaloids isolated from the leaves of the Australian tree Flindersia pimenteliana. Journal of Natural Products, 2017, 80, 3211-3217. Chapter 4: Robertson, L. P., Lucantoni, L., Avery, V. M., Carroll, A. R. Anti-plasmodial bis-indole alkaloids from the bark of the Australian tree Flindersia pimenteliana (Rutaceae). Organic and Biomolecular Chemistry. Under review. Chapter 5: Robertson, L. P., Lucantoni, L., Duffy, S., Avery, V. M., Carroll, A. R. Acrotrione, a new oxidised xanthene from the roots of Acronychia pubescens. Journal of Natural Products. Under review. Chapter 6: Robertson, L. P., Carroll, A. R. Quinoline alkaloids from the Australian Tree Pitaviaster haplophyllus. Tetrahedron Letters. In preparation for submission. Chapter 7: Robertson, L. P., Hall, C. R., Forster, P. I., Carroll, A. R. Alkaloid diversity in the leaves of Australian Flindersia (Rutaceae) driven by adaptation to aridity. Phytochemistry, 2018, 152, 71- 81. Appropriate acknowledgements of those who contributed to the research but did not qualify as authors are included in each paper.

X Luke P. Robertson

(Signed) (Date) 20/11/2018

X Supervisor: Anthony R. Carroll

(Signed) (Date) 20/11/2018

viii

ACKNOWLEDGEMENTS

I thank my primary supervisor, Prof. Anthony Carroll, for his support and guidance over the past several years. I also thank the Carroll lab group for support and scientific discussion over the course of my PhD.

Thanks are also extended to my associate supervisor, Prof. Vicky Avery, for guiding all biological aspects of the project and to Sandra Duffy and Leonardo Lucantoni for performing bioassays.

Paul Forster and Casey Hall are thanked for their valuable scientific contributions.

Wendy Loa-Kum-Cheung, Tanja Grkovic, Ben Matthews, Ryan Stewart, Anthony Boyle, Dan Tonzing and Jeremy Carrington are thanked for technical support.

ix

Chapter 1 – Introduction

1.1 Introduction to Malaria

Once called the “million murdering death” by Nobel Prize winner Ronald Ross,1 malaria is a mosquito-borne protozoan disease that infects more than 120 million people in the world at any one time. The World Health Organization (WHO) estimates that in 2015 it was responsible for 429,000 deaths (although the real number might be twice as high),2 over 90% of which occurred in sub-Saharan Africa (SSA). Malaria also disproportionately affects children, with those under five years of age accounting for 78% of all deaths.3 About half of the world’s population is at risk of contracting the disease4 (Figure 1) and thus malaria management is a high priority on global health agendas.

Image removed

Figure 1. Confirmed malaria cases per 1,000 population, 2013. Reproduced from WHO (2014).5

Image removed

Figure 2. Percentage of population living under US$ 2 per day, 1995-2013. Reproduced from WHO (2014).5

1 Malaria not only causes poverty but also comes because of it. From the period of 1965-1990, countries with high levels of malaria transmission had their economic growth rates stunted by 1.3%.6, 7 Because it occurs in some of the world’s poorest countries (Figure 2), many people are unable to obtain effective medicines.8 While philanthropists and charities such as the Bill and Melinda Gates Foundation contribute large amounts of money to help manage the disease, hundreds of millions of people are still without access to basic treatment and prevention tools.5 This lack of access to effective medicine means that up to 80% of some Asian and African populations turn to traditional medicine as their primary form of healthcare.9 The questionable efficacy and safety of traditional medicines and herbal remedies10, 11 means that many of those with malaria do not receive appropriate treatment. Even in cases where traditional medicines appear to be effective, the active constituents may be acting on the symptoms rather than the parasitic infection,12 providing symptomatic relief while having no effect on the cause.

The cause of malaria is the parasitic protozoan Plasmodium, of which five species are responsible for infections in humans. The most lethal and common form of malaria is caused by P. falciparum, which occurs mainly in SSA. As P. falciparum accounts for the majority of malaria cases and nearly all of the deaths,13 it is generally the focal point of research and treatment initiatives. The second most prevalent form of malaria is caused by P. vivax, which occurs predominantly in South/South-East Asia, the Eastern Mediterranean and South America. P. vivax, while not as deadly as P. falciparum,14 accounts for approximately 10% of the global malaria burden, or about 50% of cases outside of Africa.15 The wider geographic range of P. vivax when compared to P. falciparum can be ascribed to the species’ ability to develop in mosquitoes at higher altitudes and in cooler climates.16 The other three major species that cause infections in humans are P. ovale, P. malariae and P. knowlesi,17 however when compared to P. falciparum and P. vivax, these species represent an extremely small percentage of cases worldwide.18 Infection by the Plasmodium parasite in humans is caused by the bite of an infected female Anopheles mosquito. Approximately 30 species of Anopheles are known to transmit the Plasmodium parasite and spread malaria.19 The life cycle of the Plasmodium parasite is quite complex, with life development stages occurring in both the mosquito and human host (Figure 3).

2

Image removed

Figure 3. The life cycle of Plasmodium spp. Reproduced from Su et al. (2007).20

After an Anopheles mosquito takes a blood meal on a human host, Plasmodium sporozoites in the mosquito’s anti-coagulant saliva are injected into the skin of the host. The sporozoites quickly travel to the liver cells (hepatocytes) where they remain for a period of time, dividing mitotically for one to two weeks (depending on the species of Plasmodium), eventually forming merozoites that invade red blood cells (erythrocytes).21, 22 In the cases of P. vivax and P. ovale, some hypnozoites will remain dormant in liver cells. These may be activated and released weeks or months later, making patients prone to relapses after treatment.16 Once the merozoites have reached the red blood cells, they enlarge as ring trophozoites. The ring stage then grows and divides, forming a schizont, which produces more merozoites. Production of merozoites ultimately causes the erythrocyte to rupture, releasing the merozoites throughout the body along with waste and cell debris. This is the stage at which clinical symptoms emerge.23 Some of these merozoites infect new red blood cells and repeat the cycle, while others develop into male and female gametocytes.22 The male and female gametocytes are unable to produce gametes in the human body, and they must wait until they are withdrawn from the host by another Anopheles mosquito taking a blood meal. Once the gametocytes are withdrawn from the host and reach the gut of the mosquito, they form male and female gametes, further developing into diploid zygotes and eventually into oocysts. The oocysts undergo mitotic division, producing sporozoites which travel to the salivary glands into the mosquito, completing the life cycle.7

Once a patient has contracted the disease, there are three main clinical categories: severe, uncomplicated or asymptomatic.24 The WHO Guidelines for the Treatment of Malaria lists the symptoms of severe malaria as impaired consciousness, prostration, convulsions, acidosis,

3 hypoglycaemia, anaemia, renal impairment, jaundice, pulmonary oedema, significant bleeding, shock or hyperparasitaemia. Uncomplicated malaria is conversely defined as “symptoms of malaria and a positive parasitological test (microscopy or RDT) but with no features of severe malaria”.25 Approximately 10% of P. falciparum cases are severe. The other major species of Plasmodium (P. vivax, P. ovale, P. malariae) rarely lead to severe symptoms, generally only causing uncomplicated febrile illnesses.26 Asymptomatic malaria is difficult to detect due to the absence of clinical signs and often undetectable levels of parasitemia.24 In areas with high levels of malaria transmission, it is estimated that up to 40-70% of the population are carriers of asymptomatic parasitemia.27 The major problem posed by asymptomatic malaria is that it is still communicable to Anopheles mosquitoes and thus other humans. Consequently, complete elimination of the Plasmodium parasite from malaria endemic countries is very challenging.

1.2 Current Malaria Chemotherapeutics

There are many anti-malarial drugs used around the world (Table 1). Currently, the WHO recommended first-line treatment for uncomplicated P. falciparum malaria is artemisinin- based combination therapies (ACTs).25 ACTs are double or triple combination treatments in which an artemisinin-based drug is orally administered alongside a partner drug. ACTs have two major benefits:

a) They minimize the evolution of drug resistant parasites by attacking the parasite with multiple drugs possessing differing modes of action and biological half-lives;28 b) While artemisinin-based drugs rapidly reduce parasitemia, they have short elimination half-lives, leaving patients prone to recrudescence. By administering artemisinins together with a partner drug possessing a long half-life, this can be minimized.29

The current WHO recommended treatment for severe malaria varies around the world however parenteral quinine or artesunate are the most commonly administered drugs.5 The most commonly administered anti-malarial drugs are listed in Table 1 and reviewed below.

4 Table 1. Commonly used anti-malarial drugs.

Class of drug Compounds Amino-alcohols Quinine, mefloquine, lumefantrine 4-Aminoquinolines Chloroquine, amodiaquine, piperaquine, pyronaridine, naphthoquine 8-Aminoquinolines Primaquine Sesquiterpene lactones Artemisinin, artemether, artesunate, dihydroartemisinin, arteether Diaminopyrimidines Pyrimethamine Sulfonamides Sulfadoxine Biguanides Proguanil Napthoquinones Atovaquone Antibiotics Doxycycline, tetracycline, clindamycin

1.2.1 Amino-alcohols (quinine, mefloquine, lumefantrine)

Quinine (1) is a naturally occurring quinoline alkaloid. It is found in the bark of the South American genus Cinchona (Rubiaceae) and has been used to treat malaria for over 350 years.30 The bark of the Cinchona tree (customarily known as “Jesuits’ bark”) was introduced to Europe in the 17th century from Peru.31 Quinine was later isolated as a pure compound from Cinchona cordifolia by French scientists in 182032 and until the 1920s, it was the world’s most important anti-malarial, for both treatment and prevention.30 Even today, intravenous quinine is recommended by the WHO as the first line treatment for severe malaria in all but six countries in the WHO African Region.5 One of the major drawbacks of quinine is its side effects – patients often experience a range of side effects such as hypoglycaemia, hypotension and cinchonism (headaches, nausea, vomiting, diarrhoea, disturbed vision and tinnitus).33 These well-known side effects often lead to poor patient adherence to the required seven day course and thus the efficacy of quinine for the treatment of uncomplicated malaria is often limited.25, 34 Subsequently, quinine is only recommended as a treatment for uncomplicated malaria where ACTs are not available.25 Quinine has a biological half-life of eight to 14 hours35 and today it is sometimes used in a combination therapy with the antibiotics clindamycin, doxycycline or tetracycline.23 Some mild quinine resistance has been documented in South- East Asia36, 37 and South America38, 39 however it remains relatively efficacious, particularly in Africa, where quinine resistance is uncommon.40, 41

Mefloquine (2) is a semi-synthetic analogue of quinine. During the 1960s and 1970s, the Walter Reed Army Institute of Research (USA) undertook a screening process wherein 250,000+ compounds were examined for their biological activities. This led to the discovery of

5 mefloquine, a highly efficacious although expensive anti-malarial.42 Although mefloquine was formerly used in combination with sulfadoxine-pyrimethamine, today it is commonly used in combination with the artemisinin derivative artesunate.43 Mefloquine has a long biological half-life of two to four weeks44 and thus it is well suited to be used in combination with the fast-acting and quickly eliminated artesunate.45 Due to its cost, mefloquine is not suited for use in SSA. Nevertheless, mefloquine has been widely used in other parts of the world, particularly South-East Asia, where its extensive use has instigated parasite drug resistances.46, 47

Lumefantrine (3) is a synthetic phenanthrene amino-alcohol developed based on quinine by the Academy of Military Sciences in Beijing, China in the 1970s.48 Today, lumefantrine is used exclusively in combination with the artemisinin derivative artemether. Like mefloquine, lumefantrine has a long half-life (four to five days)49 and is thus well suited to be allied with an artemisinin derivative. Artemether-lumefantrine is the most frequently used anti-malarial treatment in the world.5, 50 One of the major benefits of the artemether-lumefantrine combination is that lumefantrine has never been available as a monotherapy and thus has not been subject to the parasite resistances that inevitably befall drugs used as monotherapies.51

Figure 4. Amino-alcohols: quinine (1), mefloquine (2) and lumefantrine (3).

Although quinine has been used for centuries, its mode of action (and that of the other amino- alcohols) remains debated. The most commonly accepted hypothesis is that quinine inhibits haem detoxification by the Plasmodium parasite in its intraerythrocytic life stage.52 In the food vacuole (FV), the parasite metabolises haemoglobin by proteolysis, digesting it with protease enzymes and thus releasing haem from its binding pocket.53 The haem is toxic to the parasite and must be disposed of before it reaches lethal concentrations. The parasite disposes of haem by dimerising it into the non-toxic crystal haemozoin.54 Quinine is hypothesised to accumulate in the FV, blocking the polymerisation of haem and subsequently causing a build- up of toxic haem.55 This results in cell lysis, autodigestion and eventually parasite death (Figure 5).7 The other amino-alcohols mefloquine and lumefantrine are believed to have similar modes

6 of action. Well-documented parasite cross-resistances between mefloquine, lumefantrine and quinine56-58 add credence to this hypothesis.

Parasite polymerises Parasite degrades haemoglobin in FV, haem into non- toxic haemozoin releasing toxic haem as a by-product

Quinine “caps” haem, preventing polymerisation

Parasite is unable to dispose of haem, resulting in a build-up of haem in FV and subsequent death

Figure 5. Hypothesised mode of action of quinine and other amino-alcohols.

1.2.2 4-Aminoquinolines (chloroquine, amodiaquine, piperaquine, pyronaridine, naphthoquine)

Chloroquine (4) is a synthetic 4-aminoquinoline that was designed based on quinine.59 It was first synthesised in 1934 by Hans Andersag60 and introduced into clinical practice in 1945.61 It then went on to become the most important anti-malarial drug of the 20th century. Chloroquine was used extensively worldwide as it was cheap, easily administered, well tolerated, non-toxic, well absorbed and effective against all types of malaria.62, 63 Unfortunately, the heavy use of chloroquine as a monotherapy led to widespread resistances – in 1957, chloroquine resistant P. falciparum were discovered in Colombia and on the Thai- Cambodian border.64 By the late 1980s, chloroquine resistant P. falciparum covered South America, SEA, India and Africa.65 The ensuing turmoil was a public health disaster – one study conducted on three populations in Senegal, Africa from 1984-1995 found that mortality of children aged under nine years was multiplied by 2.1, 2.5 and 5.5 after chloroquine resistant P. falciparum reached the area.66 Despite the prevalence of chloroquine resistant P. falciparum, chloroquine was still extensively used and in 1994 it was the third most widely consumed drug in the world, ranking only behind aspirin and paracetamol.67 Today, while chloroquine remains an effective treatment for P. vivax,25 it is ineffective for the treatment of P. falciparum everywhere in the world, save for Central America and the Caribbean.61 Chloroquine has a very

7 long elimination half-life of 20-60 days68 and is sometimes used in combination with the quickly eliminated 8-aminoquinoline primaquine.5

Amodiaquine (5) is a synthetic 4-aminoquinoline based on chloroquine. The side chain of chloroquine was modified to incorporate a phenolic ring, an alteration intended to enhance the lipophilicity of the molecule.23 Amodiaquine was developed shortly after chloroquine and has been in clinical use since the late 1940s as both a mono and combination therapy.69 Although its clinical use as a prophylaxis was temporarily ceased in the 1990s,70 today amodiaquine is recommended by the WHO as a prophylaxis for children aged three to 59 months in combination with sulfadoxine-pyrimethamine in some regions of SSA.5 While amodiaquine has a short half-life of five hours, in the body it is rapidly metabolised into desethylamodiaquine (6), which has a much longer half-life of six to 18 days.71, 72 Subsequently, for treatment of uncomplicated malaria, amodiaquine is commonly partnered with the artemisinin derivative artesunate. Artesunate-amodiaquine is the second most commonly used ACT in the world73 and remains effective where resistance to amodiaquine is low.16 There is some degree of cross-resistance between amodiaquine and chloroquine74 and thus the efficacy of amodiaquine is slightly lower in areas of high chloroquine resistance.75 Amodiaquine resistance has been reported in South America, Asia and Eastern Africa.69

Piperaquine (7) is a synthetic dimeric 4-aminoquinoline developed in the 1960s by Chinese scientists.23 It was developed to replace chloroquine after the emergence of chloroquine resistant parasites in Southern China and was adopted as a monotherapeutic first line treatment in 1978.76 Although its use steadily waned over the following decade as parasite resistances arose, it was reintroduced again after it was found to be an appropriate partner drug to the artemisinin derivative dihydroartemisinin. This is partly due to its long biological half-life of two to three weeks77 which, like many of the drugs discussed above, made it an ideal secondary drug for an ACT.78, 79 Today, dihydroartemisinin-piperaquine is used extensively in South-East Asia5, 76, 78 although its efficacy has recently been compromised in Cambodia.80 While there has been discussion about whether chloroquine and piperaquine resistance are linked, the debate remains unresolved.81

Pyronaridine (8) is a synthetic 4-aminoquinoline developed by Chinese scientists in 1973.82 It has since been sparsely used as a monotherapy in China.83, 84 Pyronaridine is well-tolerated and has limited cross-resistance with other 4-aminoquinlones61 however it is expensive to produce67, 84 and thus unsuitable for use in SSA. Although pyronaridine has been historically used only as a monotherapy, recent research indicating that it is an appropriate drug for use in ACTs has increased its usage.85 While drugs that have previously been used as monotherapies

8 are generally undesirable for use in ACTs,7 the sparing use of pyronaridine is likely to have minimized the evolution of drug-resistant parasites.83 Pyronaridine has an elimination half-life of two weeks86 and today is sometimes used in combination with the artemisinin derivative artesunate. Oral artesunate-pyronaridine for uncomplicated malaria is currently only recommended where other ACTs are ineffective.25 Pyronaridine is also sometimes used for the treatment of severe malaria in China.5

Naphthoquine phosphate (9) is a synthetic 4-aminoquinoline discovered by Chinese scientists in 1986.82 It is active against chloroquine-resistant Plasmodium87 and small-scale clinical trials have shown it is generally safe and well-tolerated.18 Naphthoquine is slow-acting and has a long half-life of around 12 days88 and consequently is generally used in combination with artemisinin. Despite a lack of data from large-scale clinical trials,25 artemisinin-naphthoquine is currently used as a single dose or three day regimen for the treatment of uncomplicated malaria,89 particularly in China5 and Papua New Guinea.90 Although artemisinin-naphthoquine is relatively rarely used, recent studies indicate that it may be equally or more effective than artemether-lumefantrine for the treatment of uncomplicated malaria.91, 92

Figure 6. 4-Aminoquinolines: chloroquine (4), amodiaquine (5), desethylamodiaquine (6) piperaquine (7), pyronaridine (8) and naphthoquine phosphate (9).

The mode of action of the 4-aminoquinolines (chloroquine, amodiaquine, piperaquine, pyronaridine and naphthoquine) has been hypothesised to be very similar to the amino- alcohols (Figure 5) i.e. they hyper concentrate in the FV and inhibit the polymerization of toxic haem into haemozoin.53, 74

9 1.2.3 8-Aminoquinolines (primaquine)

Primaquine (10) is a synthetic 8-aminoquinoline that was developed in the 1940s in the United States.93 It is highly useful because it is one of two currently licensed drugs capable of killing liver hypnozoites (pre-erythrocytic life stages of P. vivax and P. ovale that may lay dormant in the liver and re-activate weeks or months later, causing relapses).16, 55, 94 Primaquine also has activity against gametocytes (sexual stage parasites),95 and thus it is the only drug currently on the market that has activity against all three human life stages of Plasmodium. The major drawback of primaquine is that it can cause haemolysis in patients with a glucose-6-phosphate dehydrogenase (G6PD) deficiency96 and thus the patient’s G6PD status must be taken into account before administration of the drug. G6PD deficiencies are very common in sub-Saharan African populations97 and subsequently, primaquine is rarely used in this region.98 Primaquine has a short half-life of six hours99 and should be taken daily for 14 days in order to prevent relapse, however patient adherence to such a long course can be poor.100 Primaquine in combination with the 4-aminoquinoline chloroquine is recommended by the WHO for treatment of P. vivax malaria all around the world and for P. falciparum malaria in some areas outside of SSA.5 Quantifying resistance to primaquine has been difficult and thus it is challenging to assess the current effectiveness of the drug, although some studies have suggested that resistance is arising in South-East Asia.101 The mode of action of primaquine is not fully known, however the prevailing hypotheses indicate that it may disrupt the Plasmodium electron transport chain or produce damaging oxidative potentials.102

Figure 7. 8-Aminoquinoline: primaquine (10).

10 1.2.4 Sesquiterpene lactones (artemisinin, dihydroartemisinin, artemether, artesunate, arteether)

Artemisinin (11) is a naturally occurring sesquiterpene trioxane lactone. As a component of the wormwood Artemisia annua (Chinese: qīnghāo) (Asteraceae), artemisinin has been used to treat malaria in China for over 400 years. First isolated in 1972 from the leaves of A. annua, artemisinin (Chinese: qīnghāosu) was the progeny of a screening project conducted by the Chinese government. The project, initiated in 1967,103 aimed to identify new anti-malarial drugs through synthetic screening and scrutiny of ancient texts for traditional remedies.104 Eventual isolation of artemisinin and in vivo experiments against P. berghei in mice demonstrated 100% parasite growth inhibition.46 While artemisinin had successful human clinical trials, it was found that patients treated with artemisinin had high rates of parasite recrudescence. It also showed poor solubility and bioavailability31, 105 and as a result a number of semi-synthetic derivatives with enhanced pharmacological properties were developed over the following years.85 The half-life of artemisinin is two to three hours,85 however it is generally not used as an anti-malarial due to the favourable pharmacokinetic and anti-malarial properties of its derivatives. Artemisinin and its derivatives are well-tolerated106 and reduce parasitemia more rapidly than any other anti-malarial drug.107 They also kill P. falciparum gametocytes and subsequently reduce malaria transmission.83 Today, ACTs are recommended by the WHO as the first-line treatments for uncomplicated malaria and consequently they are the most commonly used anti-malarial drugs in the world.5

Dihydroartemisinin (12) is the first of the artemisinin derivatives. Reduction of the lactone carbonyl in artemisinin results in the formation of dihydroartemisinin,108 which is significantly more bioactive in vivo.109 It is also the major metabolite of the commercially available artemisinin derivatives in the human body (but not artemisinin itself).106, 110 While it is potently bioactive, dihydroartemisinin is poorly soluble in water and oil111 and thus it has been subject to numerous synthetic modifications to improve its pharmacokinetic properties. Dihydroartemisinin is used as an intermediate between the conversion of artemisinin to the semi-synthetic derivatives artemether, artesunate and arteether.112 Modification of dihydroartemisinin for the synthesis of artemether, artesunate and arteether occurs at the lactol carbon (C-10) (Figure 8).108 Dihydroartemisinin has a very short half-life of 40-60 minutes and despite its poor solubility, orally administered dihydroartemisinin is highly bioavailabile.112 Consequently, it is extensively used alongside the 4-aminoquinoline piperaquine as an ACT in South-East Asia.5, 76, 78 The pairing of dihydroartemisinin (and indeed all of the commercially available artemisinin derivatives) with a partner drug possessing a prolonged half-life significantly reduces patient recrudescence rates.82 11 Artemether (13) is the second of the artemisinin derivatives. Artemether is synthesised by alkylation the hemiacetal group of dihydroartemisinin.108 It is highly lipid-soluble and is usually given orally in combination with the amino-alcohol lumefantrine for uncomplicated malaria. For severe malaria, artemether is administered via intramuscular injection.113 Artemether is approximately five times more bioavailable than artemisinin and has a half-life of three to 11 hours.105 Artesunate (14) is the third of the artemisinin derivatives. Artesunate is synthetised by esterification of the hemiacetal group of dihydroartemisinin with succinic anhydride.114, 115 It is highly water-soluble and subsequently can be administered as an oral, rectal, intramuscular or intravenous formulation.116 For uncomplicated malaria, artesunate is predominantly administered orally alongside the 4-aminoquinoline amodiaquine in SSA73 or with the amino- alcohol mefloquine in SEA.47 For severe malaria, artesunate is given intravenously.117 Artesunate is eight to ten times more bioavailable than artemisinin105 and has a half-life of 20- 45 minutes.118 Arteether (15) is the fourth of the artemisinin derivatives. It synthesised similarly to artemether and it is correspondingly lipid-soluble.119 It is currently only sparingly used as an intramuscular treatment for severe malaria.120 Arteether is about three times more bioavailable than artemisinin and has a half-life of over 20 hours.105

Figure 8. Sesquiterpene lactones and chemical synthesis pathways: artemisinin (11), dihydroartemisinin (12), artemether (13), artesunate (14), and arteether (15).

12 The mode of action of the artemisinins has been widely debated. The most commonly accepted hypothesis is that the artemisinins induce oxidative stress through the production of free radicals. The bioactivity of artemisinin is directly linked to its unusual endoperoxide bridge linkage121 – if either oxygen in the bridge is removed and replaced with carbon, it is left without anti-malarial activity.82, 119 The proclivity of peroxides to act as reactive oxygen species has helped lead researchers to the conclusion that complexation of the endoperoxide bridge linkage by free iron released by ferrous-protoporphyrin IX (Fe(II)PPIX) (free haem) causes the formation of free radicals. These free radicals then alkylate various intracellular targets, eventually killing the parasite.29, 82 Observations that iron chelators and free radical scavengers antagonize the action of artemisinin122 augment the credibility of this hypothesis (Figure 9).

Artemisinin Alkylated haem alkylates haem releases free iron Fe3+ Free iron catalyses the decomposition of endoperoxide Iron chelators bridge remove iron

Free radicals alkylate intracellular targets, killing the parasite

Reactive oxygen species Free radical scavengers (ROS) are released remove ROS

Figure 9. Hypothesised mode of action of artemisinin and methods of antagonism.

13 1.2.5 Diaminopyrimidines (pyrimethamine)

Pyrimethamine (16) is a synthetic diaminopyrimidine first developed in the late 1940s. Its anti- malarial activity was discovered soon thereafter123 and it has since played an important role in anti-malarial chemotherapy and prophylaxis. Pyrimethamine has a half-life of three to four days124 and is always used in combination with the sulphonamide sulfadoxine. While sulfadoxine-pyrimethamine was originally used as a replacement for chloroquine,125 extensive resistances to the combination in nearly every major malaria-endemic region have since limited its application.126 Today, sulfadoxine-pyrimethamine is mainly used as a prophylaxis for pregnant women, infants and children in SSA and the Western Pacific. It is also occasionally used as a treatment for P. falciparum malaria in combination with the artemisinin derivative artesunate in the few areas it remains effective, such as the Eastern Mediterranean.5 Pyrimethamine inhibits the dihydrofolate reductase (DHFR) enzyme of the parasite, causing a failure of nuclear division and subsequent death (Figure 13).124

Figure 10. Diaminopyrimidine: pyrimethamine (16).

1.2.6 Sulfonamides (sulfadoxine)

Sulfadoxine (17) is a synthetic sulfonamide antibiotic. It was first observed to have synergistic activity with pyrimethamine in 1959127 and after successful clinical trials, the combination was approved for usage in 1967.57 Similar to pyrimethamine, sulfadoxine has a long half-life (five to ten days)124 and thus both of the drugs remain at low concentrations in the human body for an extended period of time following administration. As a result, sulfadoxine and pyrimethamine both have long selection windows (i.e. the window of time in which parasites can develop resistances to them).128 Likely as a result of this, parasite resistances to the combination were first detected in the same year it was introduced.57 Sulfadoxine inhibits dihydropteroate synthase (DHPS), a component of the folic acid synthesis pathway in the Plasmodium parasite (Figure 13).124 Its partner drug pyrimethamine inhibits a successive enzyme in the folic acid synthesis pathway126 and thus they exhibit outstanding synergy when used together.

14 Figure 11. Sulfonamide: sulfadoxine (17).

1.2.7 Biguanides (proguanil)

Proguanil (18) is a synthetic biguanide.129 Proguanil was first introduced as a monotherapy in 1947, however within two years of its introduction in Malaysia, single-dose proguanil failure rate had increased from 0 to 25%.130 Since the 1990s, proguanil has been used in combination with the naphthoquinone atovaquone as a prophylaxis and treatment.131 Parasite recrudescence is common after treatment with atovaquone-proguanil132 and thus today it is generally only used as a short-term prophylaxis. Atovaquone-proguanil is also expensive85, 106 and therefore it is unsuitable for use in most malaria-endemic countries. Subsequently, it is primarily marketed to travellers.131 Proguanil is metabolised in vivo to cycloguanil (19), which acts similarly to pyrimethamine, inhibiting the dihydrofolate reductase (DHFR) enzyme of the parasite (Figure 13).133 The half-life of proguanil is 11-17 hours while its in vivo metabolite, cycloguanil has a half-life of eight to 15 hours.134

Figure 12. Biguanides: proguanil (18) and cycloguanil (19).

15 GTPC

7,8 -Dihydroneopterin triphosphate

DHNA

4-Aminobenzoic 6 -Hydroxymethyl- acid 7,8 -dihydropterin

PPPK

6-Hydroxymethyl- 7,8-dihydropterin pyrophosphate

DHPS

Sulfadoxine

7,8-Dihydropterate

DHFS

7,8 -Dihydrofolate

Pyrimethamine DHFR

5,6,7,8- T etrahydrofolate Cycloguanil (proguanil)

Figure 13. Principal enzymes and substrates of tetrahydrofolic acid synthesis pathway. GTPC = guanosine triphosphate cyclohydryolase; DHNA = dihydroneopterin aldolase; PPPK = hydroxymethyldihydropterin pyrophosphokinase; DHPS = dihydropteroate synthase; DHFS = dihydrofolate synthase. Red arrows indicate antagonism of enzymes. Adapted from Hyde (2005).135 16 1.2.8 Napthoquinones (atovaquone)

Atovaquone (20) is a semi-synthetic hydroxynaphthoquinone developed in the late 1940s136 based on the natural product lapachol (21).8, 137 When used as a monotherapy, patients treated with atovaquone have high recrudescence rates.138 As a result, it is now used exclusively in combination with the biguanide proguanil,131 which increases the efficacy of atovaquone considerably.139 Atovaquone-proguanil is uniquely capable of killing liver schizonts, a trait it shares only with primaquine.95 Atovaquone acts by inhibition of the mitochondrial electron transport chain at the cytochrome bc1 complex, resulting in a loss of mitochondrial function and inhibition of the pyrimidine biosynthetic pathway (Figure 15).131, 136 When used in combination with proguanil it is proguanil, not its in vivo metabolite cycloguanil that exhibits synergy with atovaquone. It is hypothesised that proguanil synergises with atovaquone by enhancing its ability to collapse the mitochondrial membrane potential.140 Atovaquone has a half-life of two to three days.141

Figure 14. Napthoquinones: atovaquone (20) and lapachol (21).

Figure 15. Plasmodium mitochondrial electron transport chain. Adapted from Nixon et al. (2013).142

17 1.2.9 Antibiotics (doxycycline, tetracycline, clindamycin)

Doxycycline (22) is a semi-synthetic polyketide tetracycline antibiotic based on the naturally occurring oxytetracycline. First approved for use in 1967,143 doxycycline is used to treat a number of bacterial infections such as acne, rosacea, cholera, and leptospirosis.144 For the past three decades, doxycycline has also been used to treat malaria in combination with the amino- alcohol quinine.57 More recently, doxycycline is sometimes combined with artesunate25 or used as a monotherapeutic prophylaxis among travellers.145 Quinine/artesunate-doxycycline is currently only recommended as a second-line treatment for malaria.5, 25 Generally, doxycycline seems to have remained efficacious since its introduction145, 146 however reports of resistance in French Guiana may contradict this.38 Doxycycline has a half-life of around 20 hours147 and kills Plasmodium parasites very slowly.148

Tetracycline (23) is a naturally occurring polyketide tetracycline antibiotic naturally produced by Streptomyces bacteria. First approved for use in 1953,143 tetracycline has a half-life of six to 11 hours149 and is used to treat a similar suite of bacterial infections to doxycycline.150 Like doxycycline, tetracycline has been used in combination with quinine for the treatment of malaria for the past three decades.57 Doxycycline is considered to be superior to tetracycline for the prophylaxis and treatment of malaria due to its favourable pharmacokinetic properties.25

Clindamycin (24) is a semi-synthetic lincosamide antibiotic derived from the naturally occurring lincomycin.151 Clindamycin was first described in 1970152 and its anti-malarial activity was first reported in 1975.153 It has a short half-life of two to four hours154 and is often used in combination with quinine or artesunate for the treatment of severe malaria.23, 25 Clindamycin seems to have remained effective since its introduction into malaria chemotherapeutics.155 Doxycycline, clindamycin and tetracycline are hypothesised to act by impeding the function of the apicoplast of the Plasmodium parasite, leading to an inhibition of protein synthesis and eventually parasite death.25, 148

Figure 16. Antibiotics: doxycycline (22), tetracycline (23) and clindamycin (24).

18 1.3 Artemisinin Resistance

Although there have been many successes in anti-malarial drug discovery and treatment over the past century, resistances have emerged to every anti-malarial drug ever developed.126 Consequently, ACTs are our last line of defence against the multi-drug resistant Plasmodium parasites that cover all major malaria-endemic regions. While ACTs are currently effective at treating malaria, there have been multiple reports of partially and fully artemisinin-resistant P. falciparum in the Greater Mekong Subregion (GMS) (Cambodia, Laos, Myanmar, Thailand, Vietnam, and Yunnan Province, China). This is a major cause for concern – if full-blown artemisinin resistance were to spread from the GMS through India and subsequently into SSA, a public health catastrophe would be inevitable.156

Artemisinin resistance is currently being monitored by a number of measures:157-159

a) the percentage of treatment failures by 28 or 42-day follow-up after treatment (Table 2); b) the percentage of patients with detectable parasitemia on day 3 after treatment (Table 2); c) the measurement of parasite clearance half-life (Figure 17); d) the presence of Plasmodium with kelch-13 (K13) resistance-related mutations (Figure 17).

Table 2. Artemisinin resistances in the Greater Mekong Subregion. Reproduced from WHO (2014).159

Artemisinin resistance AL AS-MQ DHA-PPQ Containment Suspected activities year of Detected started D3+ TF D3+ TF D3+ TF emergence Cambodia 2001* 2006 2009 + + + + + + Laos 2013 2013 2014 + - Myanmar 2001* 2008 2011 + - + - + - Thailand 2001* 2008 2009 + + + + Vietnam 2009 2009 2011 + -

AL = artemether-lumefantrine; AS-MQ = artesunate-mefloquine; DHA-PPQ = dihydroartemisinin-piperaquine; D3+ = detectable parasitemia after day 3; TF = 28 or 42-day treatment follow-up; + = failure rates observed to be >10%; - = failure rates observed to be <10%; * = detected retrospectively using molecular markers or retrospective data; first-line treatment; no data available.

19 Image removed

Figure 17. Artemisinin resistances in South-East Asia measured by parasite clearance time and presence of K13 resistance-related mutations. Adapted from Ashley et al. (2014).160

It is difficult to estimate the impact that widespread artemisinin resistance would have on global malaria mortality. Lubell et al. (2014) estimated that in the event ACTs for uncomplicated malaria fail at a rate of 30% and treatment for severe malaria is reverted to quinine (which is 35% less effective and more poorly tolerated than artesunate),117 global mortality would increase by >116,000 deaths per year. The excess economic impact of increased medical costs and decreased productivity is estimated to be over US$ 400 million per year.156 Predictably, SSA is projected to endure the brunt of the impact (Figure 18).

20 No data available 0 to 500 500 to 1,000 1,000 to 5,000 5,000 to 15,000

Figure 18. Estimated excess annual mortality (deaths per year) per country due to artemisinin resistance (Adapted from Lubell et al. 2014).156

The figure above illustrates the need for a new anti-malarial drug to replace ACTs in the event that artemisinin resistance intensifies and spreads throughout Southern Asia and SSA. Although the WHO has initiated programs to contain artemisinin-resistant malaria in the GMS,158 follow-up reports indicate that these efforts have had limited success.157 While the intensification and spread of artemisinin resistance to South Asia and SSA is still a few years away, given the time and money it takes to develop a new drug (up to 15 years and US$ 800 million dollars),161 it is important that malaria drug discovery efforts be continued until malaria is completely eradicated.

21 1.4 Anti-malarial Drug Discovery

1.4.1 Current and Future Efforts

In the past, anti-malarial drugs that have reached the market have generally only possessed action against a single life stage of the Plasmodium parasite (usually the intra-erythrocytic stage i.e. quinine, chloroquine). Although drugs that act on this life stage are essential for symptomatic patient treatment, the intra-erythrocytic (asexual) life stage is also the stage at which the parasite is most metabolically active and thus most likely to mutate and develop drug resistances.162 Consequently, there has been a call for a redirection of efforts to develop drugs that also act on other stages of the parasite’s life cycle i.e. the liver and gametocyte (sexual) stages. The development of drugs that act on these less metabolically active life stages will not only confer the benefit of resistance mitigation – gametocytocidal drugs may reduce disease transmission while drugs that kill liver stage parasites may provide value as a prophylaxis or as a radical cure for P. vivax. Efficient high throughput screening (HTS) methods against liver parasites and gametocytes163 have recently been developed and thus we now have the tools to screen large libraries of compounds against these life stages. Today, anti- malarial drugs are expected to possess a number of qualities including action against multiple life stages, high safety, great bioavailability and rapid parasitemia clearance.164 Drugs that act against multiple stages of the Plasmodium life cycle are currently uncommon and not without liabilities (primaquine has action against liver, intra-erythrocytic and gametocyte stages however it cannot be taken by those with G6PD deficiencies; artemisinins have action against intra-erythrocytic and gametocyte life stages however emerging drug resistances may limit their usefulness).

Although there are a number of anti-malarial drugs currently in clinical development, it should be noted that a large proportion of drugs in clinical trials fail to obtain FDA approval (Table 3). Based on these average success rates, only a couple of the drugs for malaria currently in clinical trials are likely to reach the market. Thus, it is imperative that drug discovery efforts continue until we have sufficient medicines and resources to eradicate malaria completely.

Table 3. Clinical development success rates for investigational drugs.165

Phase I to II II to III III to NDA/BLA NDA/BLA to approval LOA from phase I

LOA 64% 32% 60% 83% 10.4%

BLA = biologic license application; NDA = new drug application; LOA = likelihood of approval

22 As of July 2015, the first malaria vaccine (RTS,S) was approved for use. The vaccine, which induces antibodies against the P. falciparum circumsporozoite protein (CSP),166 has been in development for over 28 years by GlaxoSmithKline and the Walter Reed Army Institute of Research.167 Although phase III clinical trials were somewhat disappointing (27% efficacy in infants and 46% in children),168 the RTS,S vaccine is well tolerated169 and will likely be used in conjunction with other prevention and treatment tools to reduce malaria mortality. Other malaria vaccines are currently in development.170

1.4.2 The Role of Natural Products in Drug Discovery

Despite the overwhelming prevalence of synthetic drugs currently in clinical development, natural products have undoubtedly played the largest role in anti-malarial chemotherapeutics over the past century (Table 4). Of the 21 drugs that currently dominate malaria chemotherapeutics, 17 have some connection to natural products. Three are natural products, eight are semi-synthetic natural product derivatives, six were designed based on the quinoline ring of quinine and four have no connection with natural products.8 It is peculiar that even though natural products have provided our most valuable source of anti-malarial chemotherapeutics, nearly every drug in clinical development for malaria is synthetic (although many are inspired by natural products).

Table 4. The role of natural products in current anti-malarial chemotherapeutics.

Natural products Quinine, artemisinin, tetracycline

Mefloquine, dihydroartemisinin, artemether, artesunate, Semi-synthetic natural product derivatives arteether, atovaquone, doxycycline, clindamycin

Chloroquine, amodiaquine, piperaquine, pyronaridine, Designed based on natural products naphthoquine, primaquine

Fully synthetic Lumefantrine, pyrimethamine, sulfadoxine, proguanil

The current predominance of synthetic drugs in development can be partially explained by the shift in the attitudes of the drug discovery community that occurred in the 1990s. After the advent of HTS in the mid-1980s,171 pharmaceutical companies suddenly acquired the ability to screen large numbers of compounds very quickly. Consequently, there became a demand for large compound libraries to make “full use” of HTS capabilities – a demand that libraries produced via combinatorial chemistry could effortlessly fulfil.172 The appeals of using combinatorial libraries for HTS were obvious. Not only is it easy to acquire, synthesise and screen hundreds of thousands of compounds, but these candidates could also be “rationally

23 designed” to hit targets.173 Conversely, natural product screening was (and still is) perceived as a troublesome endeavour due to time and effort it takes to repeatedly isolate and identify natural products.174 The consequence of these opinions was a significant shift away from the screening of natural products and towards combinatorial libraries.

Despite this lopsided distribution of efforts, combinatorial library HTS has yielded disappointing results.174 The pharmaceutical industry has seen a recent decline in productivity that has been attributed by some to the predominance of HTS in drug discovery.175 This begs the question: why has HTS failed to produce the promised results? How can it be that we now have the tools to screen millions of compounds against drug targets, yet the pharmaceutical industry has been a decrease in its productivity? The answer to these questions may lie in natural products. Despite greatly diminished attention, natural products still hugely influence drug discovery. From the period 1980-2010, 34% of approved small molecule drugs were either natural products or semi-synthetic natural product derivatives (Figure 19).

Figure 19. Sources of small molecule approved drugs, 1980-2010. NB = natural products (botanical); N = natural products; ND = natural product derivatives; S* = made by total synthesis but pharmacophore is from a natural product; NM = natural product mimic; S = fully synthetic. Adapted from Newman and Cragg (2012).172

To understand why natural products carry more than their fair share of the drug discovery burden, we must consider the benefits of natural products over combinatorial libraries. Firstly, natural products have a great deal more structural diversity than combinatorial compounds. Feher and Schmidt (2003) reported the chemical space occupied by three types of compounds: (a) compounds from combinatorial chemistry, (b) natural products and (c) drugs (Figure 20). The results show that the chemical space that natural products occupy closely mirrors that of 24 drugs, while compounds from combinatorial chemistry show a distinct lack of structural diversity (particularly a lack of chirality,176 which often influences bioactivity).177 Secondly, natural products are also generally more bioavailable than synthetic drugs – their biological geneses affords them the ability to interact with enzymes and transporter systems, often resulting in unpredictably high solubility and bioactivity in biological systems.174, 178, 179 Consequently, natural products are generally more likely to be appropriate drug candidates than compounds originating from combinatorial chemistry. Thus, the reason for the lacklustre productivity of combinatorial library HTS may be evident: it is the chemical diversity of a compound library that matters more than its size.

Image removed

Figure 20. Principal component analysis of the structural diversity of compounds from (a) combinatorial chemistry, (b) natural products and (c) drugs. Reproduced from Feher and Schmidt (2003).180

A similar trend has been seen in malaria chemotherapeutics: despite objections from natural product advocates, there has been a strong redirection of efforts away from natural products drug discovery.181 It is estimated that 90% of the Earth’s biodiversity has not been chemically evaluated182 and thus nature remains an untapped source of potential new drugs. If drugs such as quinine, artemisinin, penicillin, morphine and salicin (the precursor to aspirin)182 were discovered from only the first 10%, one can only imagine the treasures that are still to be discovered. As articulated by former NCI chief Gordon Cragg: “Mother Nature has had three billion years to refine her chemistry and we are only now scratching the surface in exploring Nature's molecular diversity”.183

25 1.5 Natural Product Drug Discovery: Challenges

Even though nature is an inconceivably rich source of drug candidates, discovering and isolating novel, bioactive substances is not a simple task. Indeed, one of the major challenges of natural product drug discovery is selecting appropriate species to chemically study. Considering vascular plants alone, there are estimated to be over 300,000 species in the world,184 the vast majority of which have not been chemically evaluated. How can we effectively select species for study, given this impossibly large smorgasbord of biodiversity? Traditionally, one of the most popular approaches has been random screening of large extract libraries, followed by bioassay-guided purification of “hits”. However, while this can be an effective tool at identifying species likely to contain natural products with activity against a particular target, this approach can have significant drawbacks. Firstly, bioassay-guided purification can be very time consuming, and the time between a “hit” in a biological screen to a purified compound can take weeks or months, even in well-equipped labs.185 Secondly, the bioactivity observed in a crude extract may be caused by synergistic effects of multiple constituents, therefore making isolation of singular, bioactive natural products impossible.186 Finally, there is no guarantee that the natural products contained within an extract will be new or novel, and this repeated re-discovery of previously known substances is one of the main drawbacks of natural product discovery.187 Unsurprisingly, with the advent of HTS and combinatorial libraries, the traditional approach of natural product drug discovery via crude extract screening and bioassay-guided purification has come under criticism.186 In response, natural products chemists have established a range of techniques to improve the efficacy of natural product drug discovery. These include dereplication methods (i.e. chemically profiling crude extracts to select those most likely to contain natural products of interest, thus minimising re-isolation of known compounds)188 and the development of large, pre- fractionated “drug-like” natural product extract libraries to expedite the process between “hit” and purified compound.185

1.5.1 Discovering Novel Structures

There are currently more than 300,000 unique natural products reported in the literature188 – and more are published every day. Even with the continual improvement of dereplication and purification techniques, it becomes increasingly difficult over time to discover novel chemical structures.187 This “diminishing returns” problem is a major reason that natural products have fallen out of favour with the drug discovery industry, wherein the perception is that the “low- hanging fruit” have already been picked.189 If natural product chemists are able to overcome this perception and accelerate the discovery of novel structures, a natural products

26 renaissance may result – but how do we achieve this? One suggestion has been that we should be sampling organisms from poorly explored, extreme environments such as remote jungles, arid regions, and deep oceans. Another suggestion has been that we should use ecological criteria to guide our search.190 These strategies are discussed briefly below.

1.5.2 Ecology as a Plant Selection Tool

Plants produce natural products for a number of reasons. They can be used as defences against herbivores/pathogens, to inhibit the growth of competing plants, or as communications tools. Broadly, many natural products have evolved in plants in response to biotic stressors. Subsequently, it has been proposed that if natural products are produced by plants in response to biotic stress, then plants growing in regions with high levels of biotic stress (e.g. rainforests) should produce a wider diversity of natural products than those growing in regions of low stress (Figure 21).190 A number of studies have offered support for this hypothesis, providing evidence that diversity of herbivore/plant communities and natural product diversity are positively correlated.191-193 It has also been reported that plants in rainforests generally contain more alkaloids than non-rainforest species.194 Thus, in order to obtain the highest number of novel structures and chemical diversity, rainforest habitats may be the most appropriate sampling locations. Observing plant-insect relationships (particularly herbivory) may also be a useful method of selecting plants likely to contain bioactive constituents.190

Figure 21. The proposed relationship between biotic stress and natural product discovery.

1.5.3 Sampling from Harsh Environments

On the other hand, natural products have evolved not only in response to biological stressors, but also to environmental (abiotic) factors. Many natural products protect plants against factors such as temperature, salt, UV radiation and drought stress.195 Therefore, sampling of plant species in regions of very high levels of abiotic stress (e.g. arid regions, cold regions or areas with very poor soil) may result in the discovery of natural products that are not found in other environments.196 This hypothesis has not been extensively tested, partially due to the obvious challenges in collecting samples from extreme locations. Harsh environments also have low levels of species diversity, resulting in expensive sampling trips yielding comparatively low numbers of specimens.

27 1.6 Natural Products from Plants

Plants have been a historically important source of drugs, particularly in the field of anti- malarial drug discovery, where the plant-derived natural products quinine and artemisinin have formed the entire backbone of anti-malarial chemotherapeutics for centuries. There is a distinct, repeating pattern in anti-malarial drug discovery: discovery of a new, bioactive natural product followed by synthetic modifications to enhance bioavailability and potency.197

1.6.1 The Rutaceae Family

In 2008, in a search for new anti-malarial drug leads, Fernandez and co-workers screened a library of 809 Australian and Papua New Guinean plant extracts against P. falciparum. One of the most interesting findings of the study was the over-representation of the Rutaceae family; while Rutaceous plants only represented 6% of the screening library, they were responsible for 38% of all hits.198 The family, which contains approximately 1800 species across 150 genera, is best known for the Citrus genus, the source of a number of commercially significant fruit. Natural product chemists know the family for its chemical diversity, which has been called “the most versatile of all the families of higher plants”.199 Its chemical constituents include a wide array of compounds such as flavonoids, terpenes, limonoids, coumarins, acetophenones and alkaloids. The most notable constituents of the family are its alkaloids, of which ten different classes have been isolated, making it one of the most diverse families in the world.200 Plants from the Rutaceae are also frequently used in traditional medicine, most notably as anti- malarials in Africa.201 Australia is also one of the family’s centres of biodiversity and is home to 43 genera (24 endemic) and 486 species (458 endemic).202 A great deal of these species remain completely chemically uninvestigated and thus the family is a potential source of new anti- plasmodial natural products. For this reason, the Rutaceae family was chosen as the focus of this thesis. Its alkaloidal constituents are reviewed briefly below.

1.6.1.1 Anthranilic Acid Derivatives

The two most common types of alkaloids found in the Rutaceae family are the anthranilic acid (25) derived203 quinoline (26) and acridone (27) alkaloids.204 Although quinoline alkaloids are common in anti-malarial chemotherapeutics, the Rutaceous quinolines differ in some of the key structural features of the anti-malarial quinolines (e.g. the amine or amino-alcohol moieties at C-4). Bioactive quinolines from the Rutaceae include chimanine B (28), chimanine D (29) and cusparine (30) (isolated from Galipea longiflora), all of which have shown anti- leishmanial activity in vivo.205 Rutaceous acridone alkaloids have shown activity against both chloroquine resistant and sensitive P. falciparum, suggesting a different mode of action to the

28 current quinoline anti-malarials. Examples of this bioactivity are four unnamed acridone alkaloids isolated from Swinglea glutinosa, which have IC50 values against chloroquine resistant P. falciparum in vitro of 7.1 ± 2.0 (31), 6.8 ± 0.9 (32), 2.3 ± 0.6 (33) and 20.5 ± 6.6 (34) µM.206

Figure 22. Anthranilic acid (25), quinoline (26) and acridone (27).

Figure 23. Anti-leishmanial quinoline alkaloids isolated from Galipea longiflora (Rutaceae): chimanine B (28), chimanine D (29) and cusparine (30).

Figure 24. Anti-plasmodial acridone alkaloids isolated from Swinglea glutinosa (Rutaceae).

Furoquinoline (35) and pyranoquinoline (36) alkaloids are similarly widespread in the family. Furoquinolines, although mostly restricted to the Rutaceae, are very abundant and have been found to possess a range of biological activities.207 Some Rutaceous furoquinoline alkaloids have been identified as active against chloroquine-resistant P. falciparum in vitro. These include haplopine (37) (8.34 µg/mL), acronycidine (38) (5.72 µg/mL) and acronydine (39) (2.18 µg/mL).208 Common pyranoquinolines from the Rutaceae include flindersine (40) and haplamine (41), both of which have shown activity against human cancer cell lines.209, 210 Tabounesinium chloride (42) (isolated from Araliopsis tabouensis)211 has also been reported to exhibit activity against chloroquine resistant and sensitive P. falciparum in vitro, with IC50 values ranging from 1.8 – 4.7 µg/mL.212

29

Figure 25. Furoquinoline (35) and pyranoquinoline (36) backbones.

Figure 26. Rutaceous furoquinoline and pyranoquinoline alkaloids: haplopine (37), acronycidine (38), acronydine (39), flindersine (40), haplamine (41) and tabounesinium chloride (42).

1.6.1.2 Tyrosine and Phenylalanine Derivatives

The quinazoline alkaloids (43) are derived from tyrosine (44) and phenylalanine (45). The Rutaceae is one of the few families from which the quinazolines have been isolated (others include Acanthaceae and Zygophyllaceae). Quinazoline alkaloids isolated from the Rutaceae include arborine (46), rutaecarpine (47) and evodiamine (48),200 all of which have reported bioactivity. Arborine has shown anti-tumour properties213 whereas rutaecarpine and evodiamine are anti-inflammatories.214 Quinazoline alkaloids are particularly prevalent in the Zanthoxylum genus, however they can be found in other genera such as Euodia.200

Figure 27. Quinazoline (43), tyrosine (44), phenylalanine (45) and other Rutaceous quinazoline- type alkaloids: arborine (46), rutaecarpine (47) and evodiamine (48). 30

The benzylisoquinoline alkaloids (49) have a narrow distribution in the Rutaceae family, being restricted to the Zanthoxylum, Phellodendron and Toddalia genera.200 Of the three, Zanthoxylum has been the richest source of benzylisoquinolines, from which a number of compounds with anti-plasmodial activity have been isolated. These include nitidine (50) (IC50 vs. chloroquine resistant P. falciparum of 0.27 µM), avicine (51) (4.3 µM) and fagaridine (52) (38.0 µM).215 Other benzylisoquinolines from Zanthoxylum include berberine (53), which has been clinically investigated as an anti-malarial partner drug for pyrimethamine.216

Figure 28. Benzylisoquinoline (49) and other anti-plasmodial benzylisoquinoline-type alkaloids isolated from Zanthoxylum species (Rutaceae): nitidine (50), avicine (51), fagaridine (52) and berberine (53).

The Rutaceous oxazole (54) alkaloids are found exclusively in the Halfordia genus. Oxazoles isolated from Halfordia species include halfordinol (55) and halfordine (56).200 Aside from a few isolated genera, oxazoles are uncommon in plants. Halfordinol has lipolytic and antiadipogenic properties and thus may be useful to help manage weight gain.217

Figure 29. Oxazole (54) and other oxazole-type alkaloids isolated from Halfordia species (Rutaceae): halfordinol (55) and halfordine (56).

31 1.6.1.3 Tryptophan Derivatives

The tryptophan (57) derived indole alkaloids (58) are another widespread constituent of the Rutaceae. While mono-indole alkaloids can be found in many species,200 their less common bis- indole counterparts are more promising anti-malarial drug leads. Of particular note are flinderole A (59), B (60) and C (61) which have been isolated from two Flindersia species. All three compounds have potent anti-plasmodial activity, with IC50 values against chloroquine resistant P. falciparum ranging of 1.42, 0.15 and 0.35 µM, respectively.218

Figure 30. Tryptophan (57), indole (58) and anti-plasmodial bis-indole alkaloids isolated from Flindersia species (Rutaceae): flinderole A (59), B (60) and C (61).

1.6.1.4 Histidine Derivatives

The imidazole alkaloids (62), derived from histidine (63), are restricted to the Pilocarpus and Casimiroa genera.200 The most common Rutaceous imidazole alkaloid pilocarpine (64) can be found in high quantities from many Pilocarpus species. Pilocarpine is a pharmaceutically important drug that is sold as treatment for both glaucoma and dry mouth. Other compounds isolated from Pilocarpus include pilocarpidine (65), pilosine (66) and isopilocarpine (67).219

Figure 31. Imidazole (62), histidine (63) and other imidazole alkaloids isolated from Pilocarpus species (Rutaceae): pilocarpine (64), pilocarpidine (65), pilosine (66) and isopilocarpine (67).

32

1.7 Overview and Aims of Thesis

The main aim of this thesis was to chemically study Australian Rutaceae species with the goal of identifying new anti-plasmodial compounds that may be used for the treatment of P. falciparum malaria. This was achieved by first conducting a screening and chemical profiling program to identify species most likely to contain new, bioactive molecules. Following this, four species were selected and chemically investigated, and their purified constituents screened against P. falciparum. This constitutes the majority of the thesis (Chapters 2-6). The secondary aim was to provide some insight into the factors that drive chemical diversity in Australian plant species. It is clear from the literature that attitudes towards natural product- driven drug discovery have been negatively impacted by the repeated re-isolation of known natural products. A number of recent publications have noted that natural product diversity is positively correlated with diversity of plant-herbivore communities, perhaps indicating that plants in regions of high biotic stress (i.e. rainforests) are the best sources of new and bioactive natural products. Conversely, others have proposed that plants growing in the understudied arid region of Central Australia are a better source of new natural products, citing the unique abiotic stresses (i.e. temperature, salinity, sunlight) faced by these plants. In chapter 7, we compare these contrasting hypotheses.

In summary, this thesis aims to make the following scientific contributions:

1. To identify species from the Rutaceae family that have bioactivity against P. falciparum (Chapter 2); 2. To chemically study these species to characterise their natural product constituents and identify new anti-plasmodial natural products (Chapters 2-6); 3. To investigate the hypothesis that rainforest plants produce a higher diversity of natural products than non-rainforest species (Chapter 7).

33

1.8 References

1. M. G. Schultz, N. Engl. J. Med., 1977, 297, 1259-1261. 2. C. J. L. Murray, L. C. Rosenfeld, S. S. Lim, K. G. Andrews, K. J. Foreman, D. Haring, N. Fullman, M. Naghavi, R. Lozano and A. D. Lopez, Lancet, 2012, 379, 413-431. 3. WHO, World Malaria Report 2016, WHO, Geneva, Switzerland, 2016. 4. T. Dinio, A. P. Gorka, A. McGinniss, P. D. Roepe and J. B. Morgan, Bioorg. Med. Chem., 2012, 20, 3292-3297. 5. WHO, World Malaria Report 2014, WHO, Geneva, Switzerland, 2014. 6. J. Sachs and P. Malaney, Nature, 2002, 415, 680-685. 7. T. N. Wells, P. L. Alonso and W. E. Gutteridge, Nat. Rev. Drug Discovery, 2009, 8, 879- 891. 8. G. Bourdy, M. L. Willcox, H. Ginsburg, P. Rasoanaivo, B. Graz and E. Deharo, Int. J. Parasitol., 2008, 38, 33-41. 9. I. Addae-Mensah, F. Fakorede, A. Holtel and S. Nwaka, Malar. J., 2011, 10, S9. 10. R. Capasso, A. A. Izzo, L. Pinto, T. Bifulco, C. Vitobello and N. Mascolo, Fitoterapia, 2000, 71, S1, S58-S65. 11. WHO, WHO Traditional Medicine Strategy 2014-2023, WHO, Geneva, Switzerland, 2013. 12. J. Bero, M. Frédérich and J. Quetin‐Leclercq, J. Pharm. Pharmacol., 2009, 61, 1401- 1433. 13. J. Liu, I. Y. Gluzman, M. E. Drew and D. E. Goldberg, J. Biol. Chem., 2005, 280, 1432- 1437. 14. R. N. Price, E. Tjitra, C. A. Guerra, S. Yeung, N. J. White and N. M. Anstey, Am. J. Trop. Med. Hyg., 2007, 77, 79-87. 15. K. Mendis, B. J. Sina, P. Marchesini and R. Carter, Am. J. Trop. Med. Hyg., 2001, 64, 97- 106. 16. B. M. Greenwood, K. Bojang, C. J. M. Whitty and G. A. T. Targett, Lancet, 2005, 365, 1487-1498. 17. N. J. White, Clin. Infect. Dis., 2008, 46, 172-173. 18. T. N. C. Wells and W. E. Gutteridge, in Neglected Diseases and Drug Discovery, eds. M. J. Palmer and T. N. C. Wells, Medicines for Malaria Venture, Geneva, Switzerland, 2012, pp. 1-32. 19. P. L. Alonso, G. Brown, M. Arevalo-Herrera, F. Binka, C. Chitnis, F. Collins, O. K. Doumbo, B. Greenwood, B. F. Hall and M. M. Levine, PLoS Med., 2011, 8, e1000406. 20. X. Su, K. Hayton and T. E. Wellems, Nat. Rev. Genet., 2007, 8, 497-506. 21. I. W. Sherman, The Malaria Genome Projects: Promise, Progress, and Prospects, Imperial College Press, London, 2012. 22. A. F. Cowman and B. S. Crabb, Cell, 2006, 124, 755-766. 23. M. Schlitzer, ChemMedChem, 2007, 2, 944-986. 24. D. D. Laishram, P. L. Sutton, N. Nanda, V. L. Sharma, R. C. Sobti, J. M. Carlton and H. Joshi, Malar. J., 2012, 11, 29. 25. WHO, Guidelines for the Treatment of Malaria, WHO, Geneva, Switzerland, 2015. 26. G. Pasvol, Br. Med. Bull., 2005, 75, 29-47. 27. N. A. V. Beare, T. E. Taylor, S. P. Harding, S. Lewallen and M. E. Molyneux, Am. J. Trop. Med. Hyg., 2006, 75, 790-797. 28. S. Yeung, W. Pongtavornpinyo, I. M. Hastings, A. J. Mills and N. J. White, Am. J. Trop. Med. Hyg., 2004, 71, 179-186. 29. S. R. Meshnick, Int. J. Parasitol., 2002, 32, 1655-1660. 30. J. Achan, A. O. Talisuna, A. Erhart, A. Yeka, J. K. Tibenderana, F. N. Baliraine, P. J. Rosenthal and U. D’Alessandro, Malar. J., 2011, 10, 1475-2875. 31. A. R. Butler and Y.-L. Wu, Chem. Soc. Rev., 1992, 21, 85-90. 32. P. J. Pelletier and J.-B. Caventou, Ann. Chim. Phys., 1820, 15, 289. 34

33. A. Corbani, P. Manousou, V. Calvaruso, I. Xirouchakis and A. K. Burroughs, Dig. Liver Dis., 2008, 40, 794-799. 34. A. Yeka, J. Tibenderana, J. Achan, U. D'Alessandro and A. O. Talisuna, PLoS ONE, 2013, 8, e53772. 35. S. Wanwimolruk, W. Kang, P. F. Coville, S. Viriyayudhakorn and S. Thitiarchakul, Br. J. Clin. Pharmacol., 1995, 40, 87-91. 36. M. Mayxay, M. Barends, A. Brockman, A. Jaidee, S. Nair, D. Sudimack, T. Pongvongsa, S. Phompida, R. Phetsouvanh and T. Anderson, Am. J. Trop. Med. Hyg., 2007, 76, 245- 250. 37. B. Pradines, L. Bertaux, C. Pomares, P. Delaunay and P. Marty, Malar. J., 2011, 10, 1-16. 38. E. Legrand, B. Volney, J.-B. Meynard, O. Mercereau-Puijalon and P. Esterre, Antimicrob. Agents Chemother., 2008, 52, 288-298. 39. M. T. Ferdig, R. A. Cooper, J. Mu, B. Deng, D. A. Joy, X. z. Su and T. E. Wellems, Mol. Microbiol., 2004, 52, 985-997. 40. W. Yavo, K. B. Bla, A. J. Djaman, S. B. Assi, L. K. Basco, A. Mazabraud and M. Kone, Afr. Health Sci, 2010, 10. 41. H. Tinto, C. Rwagacondo, C. Karema, D. Mupfasoni, W. Vandoren, E. Rusanganwa, A. Erhart, C. Van Overmeir, E. Van Marck and U. D'Alessandro, Trans. R. Soc. Trop. Med. Hyg., 2006, 100, 509-514. 42. P. Winstanley, Br. J. Clin. Pharmacol., 1996, 42, 411-413. 43. F. Nosten, M. Van Vugt, R. Price, C. Luxemburger, K. L. Thway, A. Brockman, R. McGready, F. Ter Kuile, S. Looareesuwan and N. J. White, Lancet, 2000, 356, 297-302. 44. J. Karbwang and N. J. White, Clin. Pharmacokinet., 1990, 19, 264-279. 45. R. Price, C. Luxemburger, M. van Vugt, F. Nosten, A. Kham, J. Simpson, S. Looareesuwan, T. Chongsuphajaisiddhi and N. J. White, Trans. R. Soc. Trop. Med. Hyg., 1998, 92, 207-211. 46. L. H. Miller and X. Su, Cell, 2011, 146, 855-858. 47. S. Vijaykadga, C. Rojanawatsirivej, S. Cholpol, D. Phoungmanee, A. Nakavej and C. Wongsrichanalai, Trop. Med. Int. Health, 2006, 11, 211-219. 48. L. Huang, P. S. Lizak, A. L. Jayewardene, F. Marzan, M.-N. T. Lee and F. T. Aweeka, Anal. Chem. Insights, 2010, 5, 15-23. 49. F. Ezzet, M. Van Vugt, F. Nosten, S. Looareesuwan and N. J. White, Antimicrob. Agents Chemother., 2000, 44, 697-704. 50. L. Denoeud-Ndam, A. Dicko, E. Baudin, O. Guindo, F. Grandesso, I. Sagara, E. Lasry, P. P. Palma, A. M. L. Parra and K. Stepniewska, BMC Infect. Dis., 2015, 15, 228. 51. Q. Bassat, PLoS Neglected Trop. Dis., 2011, 5, e1325-e1325. 52. M. Nightingale, P. G. Ambrose, G. L. Drusano and T. Murakawa, Antimicrobial Pharmacodynamics in Theory and Clinical Practice, Informer Healthcare, New York, NY, Second edn., 2007. 53. A. F. G. Slater, Pharmacol. Ther., 1993, 57, 203-235. 54. P. Olliaro, Pharmacol. Ther., 2001, 89, 207-219. 55. S. E. Francis, D. J. Sullivan and D. E. Goldberg, Annu. Rev. Microbiol., 1997, 51, 97-123. 56. M. B. Denis, R. Tsuyuoka, P. Lim, N. Lindegardh, P. Yi, S. N. Top, D. Socheat, T. Fandeur, A. Annerberg and E. M. Christophel, Trop. Med. Int. Health, 2006, 11, 1800-1807. 57. C. Wongsrichanalai, A. L. Pickard, W. H. Wernsdorfer and S. R. Meshnick, Lancet Infect. Dis., 2002, 2, 209-218. 58. J. E. Hyde, Trends Parasitol., 2005, 21, 494-498. 59. A. B. Oliveira, M. F. Dolabela, F. C. Braga, R. L. R. P. Jácome, F. P. Varotti and M. M. Póvoa, An. Acad. Bras. Cienc., 2009, 81, 715-740. 60. K. Krafts, E. Hempelmann and A. Skórska-Stania, Parasitol. Res., 2012, 111, 1-6. 61. J. T. Lin, J. J. Juliano and C. Wongsrichanalai, Curr. Infect. Dis. Rep., 2010, 12, 165-173. 62. N. J. White, Br. Med. Bull., 1998, 54, 703-715. 63. A. Vaziri and B. Warburton, J. Microencapsulation, 1994, 11, 641-648.

35

64. D. Payne, Parasitol. Today, 1987, 3, 241-246. 65. T. E. Wellems and C. V. Plowe, J. Infect. Dis., 2001, 184, 770-776. 66. J.-F. Trape, G. Pison, M.-P. Preziosi, C. Enel, A. D. du Loû, V. Delaunay, B. Samb, E. Lagarde, J.-F. Molez and F. Simondon, Comptes Rendus Acad. Sci., 1998, 321, 689-697. 67. A. Robert, F. Benoit-Vical, O. Dechy-Cabaret and B. Meunier, Pure Appl. Chem., 2001, 73, 1173-1188. 68. J. Ducharme and R. Farinotti, Clin. Pharmacokinet., 1996, 31, 257-274. 69. K. Beshir, C. J. Sutherland, I. Merinopoulos, N. Durrani, T. Leslie, M. Rowland and R. L. Hallett, Antimicrob. Agents Chemother., 2010, 54, 3714-3716. 70. A. R. Hasugian, E. Tjitra, A. Ratcliff, H. Siswantoro, E. Kenangalem, R. M. Wuwung, H. L. E. Purba, K. A. Piera, F. Chalfien and J. Marfurt, Antimicrob. Agents Chemother., 2009, 53, 1094-1099. 71. S. T. Mariga, J. P. Gil, C. Sisowath, W. H. Wernsdorfer and A. Björkman, Antimicrob. Agents Chemother., 2004, 48, 4089-4096. 72. C. Orrell, F. Little, P. Smith, P. Folb, W. Taylor, P. Olliaro and K. I. Barnes, Eur. J. Clin. Pharmacol., 2008, 64, 683-690. 73. J. Zwang, P. Olliaro, H. Barennes, M. Bonnet, P. Brasseur, H. Bukirwa, S. Cohuet, U. D'Alessandro, A. Djimdé and C. Karema, Malar. J., 2009, 8, 203. 74. A. B. S. Sidhu, D. Verdier-Pinard and D. A. Fidock, Science, 2002, 298, 210-213. 75. E. O. Ochong, I. V. van den Broek, K. Keus and A. Nzila, Am. J. Trop. Med. Hyg., 2003, 69, 184-187. 76. R. T. Eastman, N. V. Dharia, E. A. Winzeler and D. A. Fidock, Antimicrob. Agents Chemother., 2011, 55, 3908-3916. 77. T. Y. Hung, T. M. E. Davis, K. F. Ilett, H. Karunajeewa, S. Hewitt, M. B. Denis, C. Lim and D. Socheat, Br. J. Clin. Pharmacol., 2004, 57, 253-262. 78. T. M. Davis, T. Y. Hung, I. K. Sim, H. A. Karunajeewa and K. F. Ilett, Drugs, 2004, 65, 75- 87. 79. E. A. Ashley, R. McGready, R. Hutagalung, L. Phaiphun, T. Slight, S. Proux, K. L. Thwai, M. Barends, S. Looareesuwan and N. J. White, Clin. Infect. Dis., 2005, 41, 425-432. 80. D. L. Saunders, P. Vanachayangkul and C. Lon, N. Engl. J. Med., 2014, 371, 484-485. 81. D. Travers, J. Cren, N. Taudon, C. Rogier and D. Parzy, Malar. J., 2013, 12, 1-6. 82. L. Cui and X.-z. Su, Expert Rev. Anti-Infect. Ther., 2009, 7, 999-1013. 83. A. M. Dondorp, S. Yeung, L. White, C. Nguon, N. P. J. Day, D. Socheat and L. von Seidlein, Nat. Rev. Microbiol., 2010, 8, 272-280. 84. S. L. Croft, S. Duparc, S. J. Arbe-Barnes, J. C. Craft, C.-S. Shin, L. Fleckenstein, I. Borghini- Fuhrer and H.-J. Rim, Malar. J., 2012, 11, 270. 85. T. M. Davis, H. A. Karunajeewa and K. F. Ilett, Med. J. Aust., 2005, 182, 181-185. 86. C. A. Morris, L. Lopez-Lazaro, D. Jung, J. Methaneethorn, S. Duparc, I. Borghini-Fuhrer, R. Pokorny, C.-S. Shin and L. Fleckenstein, Am. J. Trop. Med. Hyg., 2012, 86, 489-495. 87. M. Delves, D. Plouffe, C. Scheurer, S. Meister and S. Wittlin, PLoS Med., 2012, 9, e1001169. 88. H. Y. Qu, H. Z. Gao, G. T. Hao, Y. Y. Li, H. Y. Li, J. C. Hu, X. F. Wang, W. L. Liu and Z. Y. Liu, J. Clin. Pharmacol., 2010, 50, 1310-1318. 89. R. Isba, B. Zani, M. Gathu and D. Sinclair, Cochrane Database Syst. Rev., 2015, 2, CD011547. 90. J. Benjamin, B. Moore, S. T. Lee, M. Senn, S. Griffin, D. Lautu, S. Salman, P. Siba, I. Mueller and T. M. E. Davis, Antimicrob. Agents Chemother., 2012, 56, 2465-2471. 91. M. Laman, J. M. Benjamin, B. R. Moore, M. Salib, S. Tawat, W. A. Davis, P. M. Siba, L. J. Robinson and T. M. E. Davis, Malar. J., 2015, 14, 121. 92. M. Laman, B. R. Moore, J. M. Benjamin, G. Yadi, C. Bona, J. Warrel, J. H. Kattenberg, T. Koleala, L. Manning and B. Kasian, PLoS Med., 2014, 11, e1001773. 93. N. Vale, R. Moreira and P. Gomes, Eur. J. Med. Chem., 2009, 44, 937-953.

36

94. A. U. Krettli, V. F. Andrade-Neto, M. d. G. L. Brandão and W. Ferrari, Mem. Inst. Oswaldo Cruz, 2001, 96, 1033-1042. 95. M. A. Biamonte, J. Wanner and K. G. Le Roch, Bioorg. Med. Chem. Lett., 2013, 23, 2829-2843. 96. E. Beutler and S. Duparc, Am. J. Trop. Med. Hyg., 2007, 77, 779-789. 97. C. Ruwende, S. C. Khoo, R. W. Snow, S. N. R. Yates, D. Kwiatkowski, S. Gupta, P. Warn, C. E. M. Allsopp, S. C. Gilbert, N. Peschu, C. I. Newbold, B. M. Greenwood, K. Marsh and A. V. S. Hill, Nature, 1995, 376, 246-249. 98. A. C. Eziefula, R. Gosling, J. Hwang, M. S. Hsiang, T. Bousema and L. von Seidlein, Malar. J., 2012, 11, 360. 99. G. W. Mihaly, S. A. Ward, G. Edwards, D. D. Nicholl, M. L. Orme and A. M. Breckenridge, Br. J. Clin. Pharmacol., 1985, 19, 745-750. 100. T. Leslie, M. A. Rab, H. Ahmadzai, N. Durrani, M. Fayaz, J. Kolaczinski and M. Rowland, Trans. R. Soc. Trop. Med. Hyg., 2004, 98, 168-173. 101. D. Fernando, C. Rodrigo and S. Rajapakse, Malar. J., 2011, 10, 351. 102. D. R. Hill, J. K. Baird, M. E. Parise, L. S. Lewis, E. T. Ryan and A. J. Magill, Am. J. Trop. Med. Hyg., 2006, 75, 402-415. 103. D. L. Klayman, Science, 1985, 228, 1049-1055. 104. U. S. Neill, J. Clin. Invest., 2011, 121, 3768-3773. 105. G. A. Balint, Pharmacol. Ther., 2001, 90, 261-265. 106. P. Ringwald, J. Bickii and L. K. Basco, Am. J. Trop. Med. Hyg., 1999, 61, 187-192. 107. N. J. White, Antimicrob. Agents Chemother., 1997, 41, 1413–1422. 108. P. V. Reddy, Organofluorine Compounds in Biology and Medicine Elsevier, USA, 2015. 109. C. J. Janse, A. P. Waters, J. Kos and C. B. Lugt, Int. J. Parasitol., 1994, 24, 589-594. 110. V. Navaratnam, S. M. Mansor, N.-W. Sit, J. Grace, Q. Li and P. Olliaro, Clin. Pharmacokinet., 2000, 39, 255-270. 111. M. T. Ansari, I. Iqbal and V. B. Sunderland, Arch. Pharmacal Res., 2009, 32, 155-165. 112. K. F. Ilett, K. T. Batty, S. M. Powell, T. Q. Binh, L. T. A. Thu, H. L. Phuong, N. C. Hung and T. M. E. Davis, Br. J. Clin. Pharmacol., 2002, 53, 23-30. 113. K. Silamut, P. N. Newton, P. Teja-Isavadharm, Y. Suputtamongkol, D. Siriyanonda, M. Rasameesoraj, S. Pukrittayakamee and N. J. White, Antimicrob. Agents Chemother., 2003, 47, 3795-3798. 114. H. Mehlhorn and P. M. Armstrong, Encyclopedic Reference of Parasitology: Diseases, Treatment, Therapy, Springer-Verlag, Berlin, Germany, 2001. 115. L. Chekem and S. Wierucki, Med. Trop. (Mars). 2006, 66, 602-605. 116. Q. Li and P. Weina, Pharmaceuticals, 2010, 3, 2322-2332. 117. SEAQUAMAT, Lancet, 2005, 366, 717-725. 118. C. A. Morris, S. Duparc, I. Borghini-Fuhrer, D. Jung, C.-S. Shin and L. Fleckenstein, Malar. J., 2011, 10, 10-1186. 119. S. R. Meshnick, T. E. Taylor and S. Kamchonwongpaisan, Microbiol. Rev., 1996, 60, 301- 315. 120. D. Sinclair, S. Donegan, R. Isba and D. G. Lalloo, Cochrane Database Syst. Rev., 2012, 6, CD005967. 121. P. M. O’Neill, V. E. Barton and S. A. Ward, Molecules, 2010, 15, 1705-1721. 122. S. R. Meshnick, Y. Z. Yang, V. Lima, F. Kuypers, S. Kamchonwongpaisan and Y. Yuthavong, Antimicrob. Agents Chemother., 1993, 37, 1108-1114. 123. E. A. Falco, L. G. Goodwin, G. H. Hitchings, I. M. Rollo and P. B. Russell, Br. J. Pharmacol. Chemother., 1951, 6, 185–200. 124. F. K. Dzinjalamala, A. Macheso, J. G. Kublin, T. E. Taylor, K. I. Barnes, M. E. Molyneux, C. V. Plowe and P. J. Smith, Antimicrob. Agents Chemother., 2005, 49, 3601-3606. 125. A. M. Rønn, H. A. Msangeni, J. Mhina, W. H. Wernsdorfer and I. C. Bygbjerg, Trans. R. Soc. Trop. Med. Hyg., 1996, 90, 179-181.

37

126. WHO, Global Report on Antimalarial Drug Efficacy and Drug Resistance: 2000-2010, WHO, Geneva, Switzerland, 2010. 127. I. W. Sherman, Reflections on a Century of Malaria Biochemistry, Elsevier, UK, 1st edn., 2008. 128. K. Stepniewska and N. J. White, Antimicrob. Agents Chemother., 2008, 52, 1589-1596. 129. L. Komsta, M. Waksmundzka-Hajnos and S. J., Thin Layer Chromatography in Drug Analysis, Taylor & Francis Group, LLC, USA, 2014. 130. J. F. B. Edeson and J. W. Field, Br. Med. J., 1950, 1, 147-151. 131. O. Wichmann, M. Muehlen, H. Gruss, F. P. Mockenhaupt, N. Suttorp and T. Jelinek, Malar. J., 2004, 3, 14. 132. K. P. David, M. Alifrangis, A. Salanti, L. S. Vestergaard, A. Rønn and I. B. Bygbjerg, Scand. J. Infect. Dis., 2003, 35, 898-899. 133. N. J. White, J. Clin. Invest., 2004, 113, 1084–1092. 134. M. D. Edstein, J. R. Veenendaal, H. V. Scott and K. H. Rieckmann, Chemotherapy, 1988, 34, 385-392. 135. J. E. Hyde, Acta Trop., 2005, 94, 191-206. 136. G. L. Nixon, D. M. Moss, A. E. Shone, D. G. Lalloo, N. Fisher, P. M. O'Neill, S. A. Ward and G. A. Biagini, J. Antimicrob. Chemother., 2013, 68, 977-985. 137. T. V. Shanbhag, Pharmacology: Prep Manual for Undergraduates, Elsevier, India, 2008. 138. P. A. Winstanley, Parasitol. Today, 2000, 16, 146-153. 139. P. Rasoanaivo, C. W. Wright, M. L. Willcox and B. Gilbert, Malar. J., 2011, 10, S4. 140. I. K. Srivastava and A. B. Vaidya, Antimicrob. Agents Chemother., 1999, 43, 1334-1339. 141. S. Looareesuwan, P. Wilairatana, R. Glanarongran, K. A. Indravijit, L. Supeeranontha, S. Chinnapha, T. R. Scott and J. D. Chulay, Trans. R. Soc. Trop. Med. Hyg., 1999, 93, 637- 640. 142. G. L. Nixon, C. Pidathala, A. E. Shone, T. Antoine, N. Fisher, P. M. O'Neill, S. A. Ward and G. A. Biagini, Future Med. Chem., 2013, 5, 1573-1591. 143. M. L. Nelson and S. B. Levy, Ann. N. Y. Acad. Sci., 2011, 1241, 17-32. 144. O. Aupee, D. Almeras, P. Le Garlantezec and X. Bohand, Med. Trop. (Mars). 2009, 69, 556-558. 145. K. R. Tan, A. J. Magill, M. E. Parise and P. M. Arguin, Am. J. Trop. Med. Hyg., 2011, 84, 517-531. 146. A. Ejaz, K. Haqnawaz, Z. Hussain, R. Butt, Z. I. Awan and H. Bux, JPMA, J. Pak. Med. Assoc., 57, 502. 147. C. W. Yim, N. M. Flynn and F. T. Fitzgerald, Antimicrob. Agents Chemother., 1985, 28, 347-348. 148. E. L. Dahl, J. L. Shock, B. R. Shenai, J. Gut, J. L. DeRisi and P. J. Rosenthal, Antimicrob. Agents Chemother., 2006, 50, 3124-3131. 149. K. N. Agwuh and A. MacGowan, J. Antimicrob. Chemother., 2006, 58, 256-265. 150. I. Chopra and M. Roberts, Microbiol. Mol. Biol. Rev., 2001, 65, 232-260. 151. J. Spížek and T. Řezanka, Appl. Microbiol. Biotechnol., 2004, 64, 455-464. 152. R. D. Birkenmeyer and F. Kagan, J. Med. Chem., 1970, 13, 616-619. 153. D. F. Clyde, R. H. Gilman and V. C. McCarthy, Am. J. Trop. Med. Hyg., 1975, 24, 369- 370. 154. B. Lell and P. G. Kremsner, Antimicrob. Agents Chemother., 2002, 46, 2315-2320. 155. C. O. Obonyo and E. A. Juma, Malar. J., 11, 1. 156. Y. Lubell, A. Dondorp, P. J. Guérin, T. Drake, S. Meek, E. Ashley, N. P. J. Day, N. J. White and L. J. White, Malar. J., 2014, 13, 452. 157. WHO, Emergency Response to Artemisinin Resistance in the Greater Mekong Subregion, WHO, Geneva, Switzerland, 2013. 158. WHO, Global Plan for Artemisinin Resistance Containment, WHO, Geneva, Switzerland, 2011. 159. WHO, Status Report on Artemisinin Resistance, WHO, Geneva, Switzerland, 2014.

38

160. E. A. Ashley, M. Dhorda, R. M. Fairhurst, C. Amaratunga, P. Lim, S. Suon, S. Sreng, J. M. Anderson, S. Mao and B. Sam, N. Engl. J. Med., 2014, 371, 411-423. 161. M. L. Willcox, B. Graz, J. Falquet, C. Diakite, S. Giani and D. Diallo, Malar. J., 2011, 10, S8. 162. D. Leroy, B. Campo, X. C. Ding, J. N. Burrows and S. Cherbuin, Trends Parasitol., 2014, 30, 478-490. 163. S. Duffy and V. M. Avery, Malar. J., 2013, 12, 408. 164. M. Rottmann, C. McNamara, B. K. S. Yeung, M. C. S. Lee, B. Zou, B. Russell, P. Seitz, D. M. Plouffe, N. V. Dharia and J. Tan, Science, 2010, 329, 1175-1180. 165. M. Hay, D. W. Thomas, J. L. Craighead, C. Economides and J. Rosenthal, Nat. Biotechnol., 2014, 32, 40-51. 166. A. Ajua, B. Lell, S. T. Agnandji, K. P. Asante, S. Owusu-Agyei, G. Mwangoka, M. Mpina, N. Salim, M. Tanner and S. Abdulla, Malar. J., 2015, 14, 72. 167. J. Cohen, V. Nussenzweig, J. Vekemans and A. Leach, Hum. Vaccines, 2010, 6, 90-96. 168. RTSS Clinical Trials Partnership, PLoS Med., 2014, 11, e1001685. 169. J. A. Regules, J. F. Cummings and C. F. Ockenhouse, Expert Rev. Vaccines, 2011, 10, 589-599. 170. L. Schwartz, G. V. Brown, B. Genton and V. S. Moorthy, Malar. J., 11, 11. 171. D. A. Pereira and J. A. Williams, Br. J. Pharmacol., 2007, 152, 53-61. 172. D. J. Newman and G. M. Cragg, J. Nat. Prod., 2012, 75, 311-335. 173. H. Ginsburg and E. Deharo, Malar. J., 2011, 10, 1. 174. A. L. Harvey, R. Edrada-Ebel and R. J. Quinn, Nat. Rev. Drug Discovery, 2015, 14, 111- 129. 175. R. Macarron, M. N. Banks, D. Bojanic, D. J. Burns, D. A. Cirovic, T. Garyantes, D. V. S. Green, R. P. Hertzberg, W. P. Janzen and J. W. Paslay, Nat. Rev. Drug Discovery, 2011, 10, 188-195. 176. C. M. Dobson, Nature, 2004, 432, 824-828. 177. R. A. Sheldon, Chirotechnology: Industrial Synthesis of Optically Active Compounds, Marcel Dekker, Inc., New York, 1993. 178. A. L. Harvey, Drug Discovery Today, 2008, 13, 894-901. 179. D. B. Kell, FEBS J., 2013, 280, 5957-5980. 180. M. Feher and J. M. Schmidt, J. Chem. Inf. Model., 2003, 43, 218-227. 181. V. M. Avery, personal communication. 182. D. A. Dias, S. Urban and U. Roessner, Metabolites, 2012, 2, 303-336. 183. G. M. Cragg and D. J. Newman, Biochim. Biophys. Acta, Gen. Subj., 2013, 1830, 3670- 3695. 184. M. J. M. Christenhusz and J. W. Byng, Phytotaxa, 2016, 261, 201-217. 185. M. S. Butler, F. Fontaine and M. A. Cooper, Planta Med., 2014, 80, 1161-1170. 186. F. E. Koehn and G. T. Carter, Nat. Rev. Drug Discovery, 2005, 4, 206-220. 187. D. G. Corley and R. C. Durley, J. Nat. Prod., 1994, 57, 1484-1490. 188. J. Hubert, J.-M. Nuzillard and J.-H. Renault, Phytochem. Rev., 2017, 16, 55-95. 189. G. D. Wright, Nat. Prod. Rep., 2017, 34, 694-701. 190. P. D. Coley, M. V. Heller, R. Aizprua, B. Araúz, N. Flores, M. Correa, M. Gupta, P. N. Solis, E. Ortega-Barría and L. I. Romero, Front. Ecol. Environ, 2003, 1, 421-428. 191. J. X. Becerra, K. Noge and D. L. Venable, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 18062-18066. 192. L. A. Richards, L. A. Dyer, M. L. Forister, A. M. Smilanich, C. D. Dodson, M. D. Leonard and C. S. Jeffrey, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 10973-10978. 193. T. A. Kursar, K. G. Dexter, J. Lokvam, R. T. Pennington, J. E. Richardson, M. G. Weber, E. T. Murakami, C. Drake, R. McGregor and P. D. Coley, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 18073-18078. 194. D. A. Levin, Am. Nat., 1976, 110, 261-284. 195. R. Akula and G. A. Ravishankar, Plant Signaling Behav., 2011, 6, 1720-1731.

39

196. B. S. Simpson, V. Bulone, S. J. Semple, G. W. Booker, R. A. McKinnon and P. Weinstein, Rangeland J., 2016, 38, 467-478. 197. T. N. C. Wells, Malar. J., 2011, 10, S3. 198. L. S. Fernandez, M. F. Jobling, K. T. Andrews and V. M. Avery, Phytother. Res., 2008, 22, 1409-1412. 199. J. R. Price, in Chemical Plant Taxonomy, ed. T. Swain, Academic Press, New York, 1963, ch. 15, pp. 429–452. 200. P. G. Waterman, Biochemical Systematics and Ecology, 1975, 3, 149-180. 201. A. F. K. Waffo, P. H. Coombes, N. R. Crouch, D. A. Mulholland, S. M. M. El Amin and P. J. Smith, Phytochemistry, 2007, 68, 663-667. 202. T. G. Hartley, in Flora of Australia. Volume 26, Meliaceae, Rutaceae, Zygophyllaceae, ed. A. Wilson, ABRS/CSIRO Australia, Melbourne, 2013, pp. 62-72. 203. O. H. Smith and C. Yanofsky, Methods Enzymol., 1962, 5, 794-806. 204. P. M. Dewick, Medicinal Natural Products: A Biosynthetic Approach, John Wiley & Sons, Ltd, Sussex, England, Second edn., 2009. 205. A. Fournet, A. A. Barrios, V. Munoz, R. Hocquemiller, A. Cavé and J. Bruneton, Antimicrob. Agents Chemother., 1993, 37, 859-863. 206. B. Weniger, B.-H. Um, A. Valentin, A. Estrada, A. Lobstein, R. Anton, M. Maillé and M. Sauvain, J. Nat. Prod., 2001, 64, 1221-1223. 207. H. Paulini, R. Waibel and O. Schimmer, Mutat. Res. Lett., 1989, 227, 179-186. 208. L. K. Basco, S. Mitaku, A.-L. Skaltsounis, N. Ravelomanantsoa, F. Tillequin, M. Koch and J. Le Bras, Antimicrob. Agents Chemother., 1994, 38, 1169-1171. 209. P. Varamini, K. Javidnia, M. Soltani, A. R. Mehdipour and A. Ghaderi, Planta Med., 2009, 75, 1509-1516. 210. S. Ea, S. Giacometti, J. Ciccolini, V. Akhmedjanova and C. Aubert, Planta Med., 2008, 74, 1265-1268. 211. J. P. Michael, Nat. Prod. Rep., 2008, 25, 166-187. 212. G. H. Schmezler and A. Gurib-Fakim, Plant Resources of Tropical Africa 11(2). Medicinal Plants 2., Prota Foundation, Wageningen, Netherlands, 2013. 213. C. Ito, M. Itoigawa, A. Sato, C. M. Hasan, M. A. Rashid, H. Tokuda, T. Mukainaka, H. Nishino and H. Furukawa, J. Nat. Prod., 2004, 67, 1488-1491. 214. J.-F. Liao, W.-F. Chiou, Y.-C. Shen, G.-J. Wang and C.-F. Chen, Chin. Med., 2011, 6, 6. 215. V. Jullian, G. Bourdy, S. Georges, S. Maurel and M. Sauvain, J. Ethnopharmacol., 2006, 106, 348-352. 216. W. D. Sheng, M. S. Jiddawi, X. Q. Hong and S. M. Abdulla, East Afr. Med. J., 1997, 74, 283-284. 217. M. Saravanan, P. Pandikumar, S. Saravanan, E. Toppo, N. Pazhanivel and S. Ignacimuthu, Eur. J. Pharmacol., 2014, 740, 714-721. 218. L. S. Fernandez, M. S. Buchanan, A. R. Carroll, Y. J. Feng, R. J. Quinn and V. M. Avery, Org. Lett., 2008, 11, 329-332. 219. A. C. H. F. Sawaya, B. G. Vaz, M. N. Eberlin and P. Mazzafera, Genet. Resour. Crop Evol., 2011, 58, 471-480.

40

Chapter 2 – Anti-plasmodial Screening and Natural Product Isolation from a Library of Australian Rutaceae Species

2.1 Abstract

The Rutaceae is a family of very high chemical diversity, being home to ten different classes of alkaloids and a range of other natural products including lignans, coumarins, acetophenones, terpenes and cinnamic acids. A recent study has also shown that the bark extracts of many Rutaceae species show strong activity against the malaria parasite, Plasmodium falciparum. With the aim of identifying new anti-plasmodial drug leads, a library of 30 Australian Rutaceae species was screened against P. falciparum and analysed by LC-MS. From the most active extract, Clausena brevistyla, two known pyranocoumarins (1-2) were purified and screened against chloroquine sensitive (3D7) and resistant (Dd2) P. falciparum. Compound 1 showed potent anti-plasmodial activity against both 3D7 and Dd2 parasites (IC50 = 466 nM and 822 nM, respectively). Furthermore, by using a combination of bioactivity data, LC-MS data and literature information, three additional species (Flindersia pimenteliana, Acronychia pubescens and Pitaviaster haplophyllus) were selected for further chemical investigation.

2.2 Introduction

Malaria is a mosquito-borne protozoan disease that was responsible for over 429,000 deaths in 2015. The vast majority of these (90%) occurred in Sub-Saharan Africa (SSA), of which most (78%) were children under five years of age.1 While tremendous steps have been made towards the World Health Organization’s (WHO) goal of complete global eradication of the disease, the unremitting emergence of resistance to front line drugs has consistently hampered these efforts. Our last effective treatments for Plasmodium falciparum malaria, artemisinin-based combination therapies (ACTs), are now beginning to fail in South-East Asia (SEA).2 Although the WHO has initiated programs to contain artemisinin-resistant malaria in SEA,3 follow-up reports indicate that these containment efforts have had limited success.4 It is only a matter of time until ACT resistance proliferates and spreads into SSA, after which a public health catastrophe would be inevitable.5

Currently, there is no drug poised to take the place of ACTs on the front line and there is a dire need for the discovery of new anti-malarial drugs.2 Historically, natural products from plants have been our most valuable source of anti-malarial drugs – the quinoline alkaloid quinine

41 from the bark of Cinchona cordifolia (Rubiaceae) and the sesquiterpene lactone artemisinin from the leaves of Artemisia annua (Asteraceae) have formed the backbone of antimalarial chemotherapeutics for centuries.6 However, despite the long and deeply intertwined history of anti-malarial treatments and natural products, recent drug discovery efforts have been directed away from natural products and towards synthetic libraries. The pharmaceutical industry has seen a recent decline in productivity that has been attributed by some to the shift away from natural products and towards combinatorial chemical libraries.7, 8

In 2008, in a search for new anti-malarial drug leads, Fernandez and co-workers screened a large library of Australian and Papua New Guinean bark extracts against P. falciparum. While Rutaceous plants only represented 6% of the screening library, they were responsible for 38% of all hits.9 The Rutaceae is best known commercially as the source of citrus fruits (Citrus) and the Sichuan Pepper (Zanthoxylum), however the family also contains almost unrivalled chemical diversity,10 particularly its alkaloidal constituents, of which ten different classes have been isolated.11 Australia is a centre of diversity of the family, being home to 43 genera (24 endemic) and 486 species (458 endemic).12 Many of these species have not been chemically studied and thus remain an untapped source of potential anti-plasmodial drug leads. With the goal of further characterizing the anti-plasmodial constituents of Australian Rutaceae species, a collection of Rutaceae extracts was generated and screened against P. falciparum. Chemical fingerprinting using LC-MS was also conducted on the bark extracts of the library to identify species that had the highest likelihood of containing previously undiscovered natural products.

2.2.1 Selection of Species and Plant Parts

Plants were acquired by visiting specialist rainforest nurseries in southeast Queensland and northern New South Wales (see Table 7 in section 2.5.2 for full details) where all available Rutaceae species were purchased. A brief literature review was then conducted to determine which species had been previously studied (Table 1). To do this, each species was searched in SciFinder (https://scifinder.cas.org/). Of the 30 species, more than half (16) had either no previous chemical study, or had not been studied since 1970. The unsophisticated methods used to perform these studies pre-1970 would mean that many natural products were missed and these species could almost be considered unstudied. Twelve of the specimens had been studied between 1991 and the present, while two were studied between 1971 and 1980. Initially, this project was to be focussed on the anti-plasmodial constituents from bark extracts of the selected species. This decision was made based on the work conducted by Fernandez and co-workers, whose screening project was based on bark extracts.9 The authors also postulated that bark extracts would be the best source of anti-plasmodial natural products.13

42

Ultimately however, many of the species obtained from nurseries were juvenile plant tubes, which could be harvested to obtain leaf, bark and root material for each species. Under advice that these could be screened at no extra cost, bark, leaf and root extracts were all screened against P. falciparum. However, due to cost limitations, LC-MS profiling was only performed on bark extracts, which were still considered the best source of anti-plasmodial natural products.

Table 1. Most recent chemical study of obtained Rutaceae species.

Year of most recent reported chemical study* Species (number of species)

Acronychia imperforata, Acronychia laevis, Acronychia littoralis, Acronychia oblongifolia, Acronychia pauciflora, Clausena None reported (11) smyrelliana, Coatesia paniculata, Dinosperma erythrococcum, Medicosma forsteri, Melicope rubra, Murraya ovatifoliolata

Acronychia acidula,14 Clausena brevistyla,15 Medicosma Pre-1970 (5) cunninghamii,16 Pentaceras australis17, Pitaviaster haplophyllus18

1971-1980 (2) Geijera salicifolia,19 Flindersia pimenteliana20

1981-1990 (0) --

Acronychia pubescens,21 Bosistoa floydii,22, 23 Bosistoa 1991-2000 (6) pentacocca,23, 24 Bosistoa transversa,25 Melicope hayesii,26 Melicope micrococca27

Bouchardatia neurococca,28 Glycosmis pentaphylla,29 Melicope 2001-present (6) elleryana,30 Melicope vitiflora,31 Sarcomelicope simplicifolia,32 Zanthoxylum ovalifolium33

*excluding volatile leaf oil studies

2.3 Results and Discussion

2.3.1 Anti-plasmodial Screening Results

Following the sequential extraction of dried plant samples (300 mg) in MeOH and CH2Cl2, each extract was evaporated, filtered, redissolved in DMSO and screened against P. falciparum by Dr. Sandra Duffy at the laboratory of Prof. Vicky Avery (Discovery Biology). The results of the screen are reported below (Table 2; Table 3). The highest activity was observed in the root material of Acronychia pubescens (80% growth inhibition of P. falciparum at 0.4 µg/mL), Clausena brevistyla (95% inhibition at 0.4 µg/mL) and Melicope micrococca (81% inhibition at 0.4 µg/mL). At the same dose, low cytotoxicity against HEK-293 cells was also observed in each of these three extracts (8%, 0% and 7%, respectively). As it was the extract with the highest activity, the roots of C. brevistyla were chosen as the first target for chemical investigation. 43

Table 2. Percentage inhibition of P. falciparum (3D7) of Rutaceae library.

Plant Part Leaves Bark Roots Concentration (µg/mL) 400 40 4 0.4 400 40 4 0.4 400 40 4 0.4 Acronychia acidula 100 95 39 3 97 98 98 20 95 35 4 -24 Acronychia imperforata 4 5 6 7 96 98 96 27 n.d n.d n.d n.d Acronychia laevis 97 20 14 0 98 97 91 -7 n.d n.d n.d n.d Acronychia littoralis 93 20 13 4 99 34 -6 -1 n.d n.d n.d n.d Acronychia oblongifolia 99 48 -1 1 97 96 16 -27 n.d n.d n.d n.d Acronychia pauciflora 98 8 8 14 99 65 4 -5 n.d n.d n.d n.d Acronychia pubescens 99 50 14 -4 95 98 94 -2 95 100 99 80 Bosistoa floydii 97 7 -9 -13 100 40 0 -11 n.d n.d n.d n.d Bosistoa pentacocca 99 7 -2 -10 96 99 79 -10 99 87 55 16 Bosistoa transversa 95 -21 -7 -16 100 59 -2 -18 n.d n.d n.d n.d Bouchardatia neurococca 98 10 -4 -14 96 97 95 3 n.d n.d n.d n.d Clausena brevistyla 99 3 -4 -6 97 99 72 42 97 96 97 95 Clausena smyrelliana 96 90 36 -13 98 4 -12 -33 89 82 -16 -38 Coatesia paniculata 99 65 2 -16 100 95 -15 -24 100 75 12 -17 Dinosperma erythrococcum 97 -4 -11 -11 100 40 17 -7 n.d n.d n.d n.d Geijera salicifolia 100 93 21 -2 98 98 96 37 n.d n.d n.d n.d Glycosmis pentaphylla 99 33 -12 -21 98 97 82 -2 96 70 42 -3 Medicosma cunninghamii 100 20 -11 -18 100 97 96 4 n.d n.d n.d n.d Medicosma forsteri 99 98 35 -16 98 99 100 39 97 97 96 56 Melicope elleryana 99 96 5 -7 96 99 97 8 n.d n.d n.d n.d Melicope hayesii 99 33 -12 -27 97 97 77 3 90 96 85 0 Melicope micrococca 100 67 -15 -21 98 95 53 -15 75 98 97 81 Melicope rubra 98 16 7 -7 97 82 34 -17 n.d n.d n.d n.d Melicope vitiflora 8 9 10 11 97 74 28 -20 -9 34 28 -8 Murraya ovatifoliolata 100 96 64 2 96 99 97 14 12 64 90 57 Pentaceras australis 101 96 56 24 96 97 95 31 n.d n.d n.d n.d Pitaviaster haplophyllus 97 88 53 -11 98 98 93 -3 75 65 36 -33 Sarcomelicope simplicifolia 99 68 46 -6 99 87 0 -5 96 85 13 -5 Zanthoxylum ovalifolium 99 96 95 -10 93 98 69 -6 91 97 90 61 n.d = not determined. Extracts above 80% growth inhibition are highlighted in green.

44

Table 3. Percentage inhibition of human embryonic kidney cells (HEK-293) of Rutaceae library.

Plant Part Leaves Bark Roots Concentration (µg/mL) 400 40 4 0.4 400 40 4 0.4 400 40 4 0.4 Acronychia acidula 86 45 8 -5 60 -13 -19 -5 91 -4 -2 -10 Acronychia imperforata 78 -10 2 -8 37 -38 -22 -11 n.d n.d n.d n.d Acronychia laevis 27 -42 1 -9 93 10 -11 2 n.d n.d n.d n.d Acronychia littoralis 42 -19 2 -9 11 -25 -4 -14 n.d n.d n.d n.d Acronychia oblongifolia 18 -4 -10 4 -4 -27 3 -8 n.d n.d n.d n.d Acronychia pauciflora -2 2 -6 -14 1 3 -16 1 n.d n.d n.d n.d Acronychia pubescens 29 -13 -30 -12 56 7 -23 1 99 91 13 8 Bosistoa floydii 60 -25 -16 -12 38 -26 -8 -10 n.d n.d n.d n.d Bosistoa pentacocca 22 -16 -16 -9 86 9 13 -9 79 -10 7 1 Bosistoa transversa -6 -17 -18 -12 -2 -29 -27 -18 n.d n.d n.d n.d Bouchardatia neurococca 5 -2 -9 -13 100 -11 -19 -13 n.d n.d n.d n.d Clausena brevistyla 54 -37 14 8 101 56 7 8 101 82 91 -5 Clausena smyrelliana 101 99 5 -17 99 87 -6 -3 101 97 39 1 Coatesia paniculata -1 4 -20 9 27 -15 -19 -15 78 -7 -6 6 Dinosperma erythrococcum -15 -12 -12 -6 24 -13 -13 1 n.d n.d n.d n.d Geijera salicifolia 92 -6 2 -9 99 98 24 -2 n.d n.d n.d n.d Glycosmis pentaphylla 62 -20 -14 0 60 -2 -8 -15 66 3 -2 0 Medicosma cunninghamii 22 -5 -8 -10 -54 -3 -13 -6 n.d n.d n.d n.d Medicosma forsteri 101 -11 -5 -2 101 -5 -7 -19 57 79 -2 -6 Melicope elleryana 51 -8 -10 6 70 -4 -12 -15 n.d n.d n.d n.d Melicope hayesii 18 -7 -9 8 86 5 -20 -13 -46 36 8 -5 Melicope micrococca 19 -13 -5 -1 52 -17 -14 -16 88 58 9 7 Melicope rubra 60 10 -7 -8 30 -19 -5 -5 n.d n.d n.d n.d Melicope vitiflora 49 7 -5 -8 26 -5 -6 -5 87 18 11 13 Murraya ovatifoliolata 58 -20 -26 -18 54 -2 -3 -16 -20 87 27 0 Pentaceras australis 97 73 -6 2 97 92 84 -8 n.d n.d n.d n.d Pitaviaster haplophyllus 60 -23 -9 -17 44 10 10 -11 95 7 8 4 Sarcomelicope simplicifolia 53 -7 -15 -18 24 2 1 -11 57 -4 -3 -3 Zanthoxylum ovalifolium 90 -1 6 -17 -7 2 0 -7 -20 24 14 18 n.d = not determined. Extracts above 80% growth inhibition are highlighted in red.

45

2.3.2 Pyranocoumarins from Clausena brevistyla

Figure 1. Pyranocoumarins isolated from the roots of C. brevistyla.

Extraction of the roots of C. brevistyla with MeOH and subsequent fractionation using semi- preparative HPLC resulted in the isolation of two known pyranocoumarins (1, 2) (Figure 1). Compound 1 was obtained as a brown gum. A protonated molecule in the (+)-LRESIMS at m/z 381 was confirmed as the precursor ion by the presence of a sodium adduct at m/z 403. Product ions were also observed at m/z 313 (low intensity) and m/z 245 (high intensity). These fragments, which showed sequential losses of 68 amu, indicated that the molecule contained two isoprene units. The high intensity of the m/z 245 ion relative to the m/z 313 ion indicated that the two units formed an aliphatic monoterpene. The 1H NMR spectrum (Table 4) displayed signals associated with six double bond protons at δH 8.12 (d, J = 9.8 Hz), δH 6.71 (d, J = 9.8 Hz),

δH 6.28 (dd, J = 17.5, 10.7 Hz), δH 6.11 (d, J = 9.8 Hz), δH 5.70 (d, J = 9.8 Hz) and δH 5.00 (tt, J =

6.9, 1.3 Hz). A vinyl group was also observed at δH 4.82 (dd, J = 17.5, 1.3 Hz)/4.80 (dd, J = 10.7, 1.3 Hz). The characteristic couplings (J = 9.8 Hz) exhibited by four of the double bond protons indicated that 1 is a coumarin. This was supported by literature data, which reports numerous

34-36 coumarins from Clausena. Other features of the spectra included five methyl singlets at δH

1.60, δH 1.53, δH 1.37, δH 1.36 and δH 1.35 and two methylenes at δH 2.13 (m)/1.78 (m) and δH 1 1 1.84 (m)/1.69 (m). H- H COSY correlations between δH 8.12/6.11 were used to assign their positions at H-4/H-3 in the lactone system (Figure 2). The deshielded chemical shift of H-4/C-4

(δH 8.12/δC 139.6) relative to H-3/C-3 (δH 6.11/δC 108.7) supported the assignment of H-3 as 3 vicinal to the C-2 ester. JCH HMBC correlations from H-4 into resonances associated with C-5

(δC 148.8) and C-8a (δC 154.2) revealed that C-5 was oxygenated, although no corresponding hydroxyl signal in the 1H NMR spectrum was observable. 1H-1H COSY correlations between H-

3 4 3′/H-4′ (δC 5.71/6.71) revealed these two protons to be vicinal to each other. JCH and JCH

HMBC correlations from H3-1′α/H3-1′β (δH 1.35/1.37) into C-3′/C-4′ and an additional resonance at δC 76.7 confirmed that C-1′ through C-4′ was a pyran ring containing a gem- 3 dimethyl. The pyran ring was established to be at C-6/C-7 via JCH HMBC correlations from both 3 H-4/H-4′ into C-5. A final JCH correlation from H-4′ into an unassigned resonance at δC 155.7 was used to allocate the chemical shift of C-7. Assessment of (+)-LRESIMS data revealed that 46

136 amu was unaccounted for, and the absence of any other aromatic proton resonances

3 indicated that C-8 was alkylated. This was supported by JCH HMBC correlations from δH

6.28/1.60 (H-2′′/H-3′′-Me) into a non-protonated carbon resonance at δC 112.7, which was 1 1 assigned at C-8. The vinyl group (δH 4.82/4.80) was predicted to be at H-1′′ from H- H COSY 3 correlations with H-2′′, while a JCH HMBC correlation from H-1′′ into an unassigned non- 2 protonated carbon resonance at δC 43.7 was used to assign C-3′′. JCH HMBC correlations from 3 H-2′′/H-3′′-Me into C-3′′ supported this. It was determined that C-4′′ was a methylene via JCH 1 HMBC correlations from H-4′′ (δH 2.13/1.78) into the previously assigned resonance at C-8. H- 1H COSY correlations between H-4′′/H-5′′/H-6′′ were used to assign H-4′′ through H-6′′. HMBC correlations from H3-8′′/H3-9′′ into C-8′′/C-9′′ and deshielded resonances at δC 130.5 (C-7′′) and

124.3 (C-6′′) confirmed that H3-8′′/H3-9′′ were a gem-dimethyl attached to a double bond. Assessment of (+)-LRESIMS data revealed that the structure was missing a single proton and this was assigned as the C-5 hydroxyl. Compound 1 has been previously isolated from Paramignya monophylla (Rutaceae)37, 38 and Clausena emarginata.39 Its structure was confirmed by comparison of spectral data to literature values.37

Figure 2. Key HMBC  and COSY (bold bonds) correlations of 1 and 2.

Compound 2 was isolated as a brown gum. A protonated molecule was observed in the (+)- LRESIMS at m/z 229. The 1H NMR spectrum (Table 4) displayed signals associated with six double bond protons at δH 7.91 (d, J = 9.8 Hz), δH 7.40 (s), δH 6.77 (s), δH 6.47 (d, J = 9.8 Hz), δH

6.26 (d, J = 9.8 Hz) and δH 5.84 (d, J = 9.8 Hz) and a gem-dimethyl at δH 1.42 (s). Comparison of these resonances to 1 revealed a very similar structure to 1, although 2 did not contain any aliphatic isoprene resonances in the 1H NMR spectrum nor any product ions in the (+)-LRESIMS data. It was thus concluded that 2 contains the same tricyclic backbone of 1, although it lacks any additional isoprene functionalities. The presence of two aromatic singlets in the 1H NMR

3 spectrum of 2 indicated that C-5 and C-8 were methines. This was supported by JCH HMBC correlations from H-5 into C-4/C-4′ and from H-8 into C-4a/C-6, and thus the structure of 2 was established. Compound 2 is a very common metabolite has been previously isolated from many plants.40-43 Its structure was confirmed by comparison of spectral data to literature values.44 47

1 13 Table 4. H and C NMR spectroscopic data for 1 and 2 (500 MHz, DMSO-d6, δ in ppm). 1 2 Position a b a δc δH (mult, J in Hz, int.) HMBC δc δH (mult, J in Hz, int.) 2 159.8 - - 160.1 - 3 108.7 6.11 (d, 9.8, 1H) 2, 4a 112.0 6.26 (d, 9.5, 1H) 4 139.6 8.12 (d, 9.8, 1H) 2, 5, 8a 143.7 7.91 (d, 9.5, 1H) 4a 103.7 - - 112.8 - 5 148.8 - - 125.1 7.41 (s, 1H) 6 106.0 - - 118.1 - 7 155.7 - - 156.2 - 8 112.7 - - 102.8 6.77 (s, 1H) 8a 154.2 - - 154.9 - 1′β 26.9 1.37 (s, 3H) 1′α, 2′, 3′, 4′, 7 27.9 1.42 (s, 3H) 1′α 26.9 1.35 (s, 3H) 1′β, 2′, 3′, 4′, 7 27.9 1.42 (s, 3H) 2′ 76.7 - - 77.5 - 3′ 127.9 5.71 (d, 9.8, 1H) 1′α, 1′β, 2′, 6, 7 130.8 5.84 (d, 9.5, 1H) 4′ 116.1 6.71 (d, 9.8, 1H) 1′α, 1′β, 2′, 5, 6, 7 120.0 6.48 (d, 9.5, 1H) 4.82 (dd, 17.5, 1.3, 1H) 1′′ 107.2 2′′, 3′′, 3′′-Me, 4′′, 8 - - 4.80 (dd, 10.7, 1.3, 1H) 2′′ 149.3 6.28 (dd, 17.5, 10.7, 1H) 1′′, 3′′, 3′′-Me, 4′′, 8 - - 3′′ 43.7 - - - - 2.13 (m, 1H) 2′′, 3′′, 3′′-Me, 5′′, 4′′ 39.8 - - 1.78 (m, 1H) 6′′, 8 1.84 (m, 1H) 5′′ 23.5 3′′, 4′′, 6′′, 7′′ - - 1.69 (m, 1H) 6′′ 124.3 5.00 (tt, 6.9, 1.3, 1H) 4′′, 5′′, 7′′, 8′′, 9′′ - - 7′′ 130.5 - - - - 8′′ 17.1 1.36 (s, 3H) 6′′, 7′′, 9′′ - - 9′′ 25.4 1.53 (s, 3H) 6′′, 7′′, 8′′ - - 3′′-Me 27.9 1.60 (s, 3H) 2′′, 3′′, 4′′, 8 - - a Chemical shifts determined by 2D experiments. b HMBC correlations are from proton(s) stated to the indicated carbon.

The purified pyranocoumarins 1 and 2 were tested for anti-plasmodial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum by Dr. Leonardo Lucantoni at the laboratory of Prof. Vicky Avery (Discovery Biology). Cytotoxicity using the HEK-293 mammalian cell line (Table 5) was also determined for the evaluation of parasite selectivity. Compound 2 showed weak anti-plasmodial activity, with complete inhibition of the parasite replication observed only at the highest concentration of 40 µM. The activity of 1 was interesting, as it returned submicromolar IC50 values against both 3D7 and

Dd2 parasites (IC50 = 466 nM and 822 nM). The corresponding Dd2/3D7 IC50 ratio of 1.8 suggests equal sensitivity by the drug-resistant and wild type parasite strains. Compound 1 was not devoid of cytotoxic effect, though, and it inhibited HEK293 cells by >80% at the highest concentration tested. An accurate IC50 value for HEK293 cells, and hence an accurate selectivity index, cannot be calculated given the incomplete HEK293 inhibition achieved by 2, however the selectivity index can be roughly estimated in the range of 20-40 fold, suggesting that this compound is worthy of further investigation. 48

Table 5. Anti-plasmodial activity and cytotoxicity of natural products isolated from C. brevistyla.

IC50 ± SD (µM) (n = 2) Selectivity Compound 3D7 Dd2 HEK293 Index (SI) 1 0.466 ± 0.087 0.822 ± 0.144 83.2% ± 4.2a n.d.

2 96.6% ± 1.0a 99.4% ± 4.2a 24.7% ± 13.8a n.d. artesunateb 0.0021 ± 0.0016 0.0033 ± 0.0022 62.5% ± 16.5a n.d. chloroquineb 0.0139 ± 0.0015 0.1360 ± 0.0255 53.2% ± 26.4a n.d. dihydroartemisininb 0.0009 ± 0.0002 0.0016 ± 0.0003 41.1% ± 11.5a n.d. puromycinb 0.0702 ± 0.0108 0.0747 ± 0.0188 0.6940 ± 0.183 10 pyrimethamineb 0.0077 ± 0.0017 n.d. 52.0% ± 10.2a n.d. pyronaridineb 0.0082 ± 0.0020 0.0110 ± 0.0010 1.990 ± 0.263 246 a percentage growth inhibition at 40 µM. b reference compound. SI = selectivity index (HEK293/3D7)

A number of coumarins with anti-plasmodial activity are reported in the literature (3-12), however to the best of our knowledge, only one (4) has shown potent activity (Table 6; Figure

3) against P. falciparum (IC50 = 260 nM). The majority of coumarins reported show only moderate to weak anti-plasmodial activity, ranging from 5.1 µM to inactive at the highest dose tested. Structurally, 1-9 all contain a tricyclic pyranocoumarin backbone with various substituents (hydroxys, methoxyls, phenyls and isoprenes) occuring at C-3, C-4, C-5 and C-8. In the case of 10-12, the C-6 isoprene unit has not formed a pyran. The major structural similarity shared by the two potently anti-plasmodial coumarins 1 and 4 is the presence of two isoprene units – in the case of 1, these units form a monoterpene at C-8, while in 4 single isoprene units occur at both C-3/C-8. Of the other coumarins, 9-12 show the next highest bioactivity, ranging from 5.1 – 11.1 µM. These compounds also contain two isoprene units, although they are oxidised and occur at C-4/C-8. Whether the reduced activity of 9 compared to 4 is due to isoprene oxidation or the rearrangement of the isoprene unit from C-3 to C-4 is unclear. Interestingly, the cytotoxicity (versus KB cells) of 9 is significantly lower than 10-12. This appears to be caused by the cyclisation of the C-6 isoprene unit in 10-12, which forms a pyran ring in 9. The other major structural difference between 9 and 10-12 is the presence of hydroxys at C-5/C-7, although the C-7 hydroxyl would be restrained within an intramolecular hydrogen bond and less likely to interact with other biological systems. The remaining pyranocoumarins 3, 5, 6, 7 and 8 all showed poor activity, suggesting that the presence of single isoprene and/or phenyl units is not sufficient for activity. It can be concluded that the presence of two isoprene units, notably at C-3/C-8, are likely to be important for bioactivity.

49

Non hydrogen-bound hydroxys at C-5 also appear to be a common structural feature of the anti-plasmodial coumarins, although the inactivity of 5, which contains a hydroxy at C-5 but no isoprene units, suggests isoprene units to be the more important structural feature.

Table 6. Comparison of anti-plasmodial activity 1 and 2 with those reported in the literature.

IC50 ± SE (µM) Selectivity Index (SI) Compound Ref. CQ-sensitive CQ-resistant P. Cytotoxicity (HEK-293/CQ-sensitive) P. falciparum falciparum 1 -- 0.47 ± 0.09a 0.82 ± 0.14b 83.2% ± 4.2c,d n.d. 2 -- 96.6% ± 1.0a,d 99.4% ± 4.2b,d 24.7% ± 13.8c,d n.d. 3 45 n.d. 26.0 – 37.6e n.d. n.d. 4 45 n.d. 0.26 – 1.82e n.d. n.d. 5 46 n.d. IAe IAf n.d. 6 47 130g n.d. n.d. n.d. 7 48 78.9a n.d. >100f n.d. 8 48 49.4a n.d. >100f n.d. 9 49 11.1h 10.4i 52.2f 5 10 49 9.7h 7.7i 3.5f 0.4 11 49 9.8h 9.6i 5.7f 0.6 12 49 9.5h 5.1i 6.2f 0.7 a 3D7 strain; b Dd2 strain; c HEK-293 cells; d percentage growth inhibition at 40 µM; e K1 strain; f g h i KB cells; FCMSU1/Sudan strain; D6 strain; W2 strain. IA = inactive.

Figure 3. Anti-plasmodial pyranocoumarins from the literature.

50

2.3.3 Selection of Additional Species for Chemical Investigation

Following the chemical investigation of C. brevistyla, three additional plant species were selected for further study. These species were selected using three main criteria:

1. Growth inhibition against P. falciparum (Table 2) and HEK-293 cells (Table 3); 2. Identifiable alkaloid peaks from LC-MS chromatograms; 3. Literature data. Species were considered attractive for study if they fulfilled one or more of the following criteria: a. It is chemically unstudied, or has not been studied in several decades (Table 1); b. It is a member of a small, uncommon and/or poorly studied genus; c. It comes from a genus that is previously known to produce structurally diverse and/or anti-plasmodial natural products, with a particular emphasis on alkaloids. 2.3.3.1 Flindersia pimenteliana

The culmination of the above mentioned Rutaceae screening project by Fernandez and co- workers9 was the isolation of five potently anti-plasmodial bis-indole alkaloids from the Australian/Papua New Guinean Flindersia species F. amboinensis and F. acuminata (14-18)

50 (IC50 values from 0.08 to 1.42 µM). Structurally similar bis-indoles have also been reported from F. fourneri (19-20), although these have not been screened against P. falciparum.51 All of the aforementioned Flindersia bis-indoles show m/z ions at either 509.3644 (15, 17, 18), 495.3482 (14, 20) or 481.3326 (13, 16, 19). Due to the basicity of the two nitrogen atoms on the C-3 ethylamine moieties, doubly charged m/z ions can also be observed at 255.1856 (15, 17, 18), 248.1778 (14, 20) and 241.1659 (13, 16, 19). These distinctive [M+H]+ and [M+2H]2+ ions make it very simple to detect the presence of bis-indoles in Flindersia, although it is not possible to distinguish between the three different classes (isoborreverine (13-15), flinderole (16-18) and borreverine (19-20)) (Figure 4) using LC-MS data alone. In an attempt to find new structural derivatives of the Flindersia bis-indole alkaloids, the bark extract of F. pimenteliana was subject to LC-MS analysis, which revealed it to contain many of these alkaloids in high concentrations (Figure 5). Subsequently, the extract was almost certain to display bioactivity and screening was considered unnecessary. M/z ions corresponding to bis-indoles were observed as broad peaks in the LC-MS chromatogram at 5.7 and 6.0 mins (Figure 5a). Further inspection of the bis-indole region in the LC-MS chromatogram indicated the presence of at least two undescribed bis-indole isomers. The isomers showed m/z ions at 539.3739 [M+H]+/270.1879 [M+2H]2+ and 569.3831 [M+H]+/285.1940 [M+2H]2+ (Figure 5b). These were confidently predicted to be bis-indoles based on their retention times (i.e. co-elution with

51

known bis-indoles) and distinguishing [M+2H]2+ ions. Furthermore, although eight bis-indole alkaloids are previously reported from Flindersia, only five (14-18) have been screened against P. falciparum.50 Thus, in order to further characterise the anti-plasmodial structure-activity relationships of the Flindersia bis-indole alkaloids and to identify new anti-plasmodial alkaloids, F. pimenteliana was selected as the first species for chemical investigation.

Figure 4. Bis-indole alkaloids isolated from Flindersia spp. isoborreverine (13), 4- methylisoborreverine (14) dimethylisoborreverine (15) flinderole A-C (16-18) borreverine (19) and 4-methylborreverine (20).50, 51

a)

b)

Figure 5. a) Base peak chromatogram of F. pimenteliana bark extract LC-MS. Bis-indole alkaloid peaks between 5-6 mins are circled. b) Base peak chromatogram at 5.954 mins. Peaks corresponding to undescribed bis-indole isomers are circled.

2.3.3.2 Acronychia pubescens

Additional material for the second most active extract, the roots of M. micrococca, was not available for purchase from any nearby nurseries. Subsequently, the third most active extract,

52

the roots of A. pubescens, were selected for chemical investigation (Chapter 5). The extract showed 80% growth inhibition of P. falciparum at the lowest dose (0.4 µg/mL) (Table 2). Conversely, cytotoxicity against HEK-293 was comparatively low (4% growth inhibition at 4 µg/mL) (Table 3). The only reported chemical investigation of the species was performed over 20 years ago, reporting only three simple furoquinolines (21-23).21 Other species within the genus have been a source of many diverse natural products such as alkaloids (24),52 terpenes (25), cinnamic acids (26),53 acetophenones (27-28),54 and lignans (29).55 The high chemical diversity of the Acronychia genus thus made A. pubescens a strong candidate for the discovery of new anti-plasmodial natural products.

Figure 6. Some natural products isolated from Acronychia species: dictamnine (21), kokusaginine (22), evolitrine (23), acronycine (24), bauerenol (25), an unnamed cinnamic acid (16), acronyculatin M-N (27-28) and sesamolin (29).

2.3.3.3 Pitaviaster haplophyllus

The final species selected for chemical investigation was P. haplophyllus (Chapter 6). Overall, all aerial parts of the species showed good bioactivity (93% and 53% growth inhibition at 4 µg/mL for bark and leaf extracts, respectively) (Table 2). Comparatively low cytotoxicity against HEK-293 cells was also observed (44% and 60% growth inhibition at 400 µg/mL for bark and leaf extracts, respectively) (Table 3). However, when compared to other species, the bioactivity of P. haplophyllus did not particularly stand out. Eleven other species had very similar bark extract bioactivity profiles at the same dose (within 7% activity of P. haplophyllus at 4 µg/mL) (Table 2). Comparably low cytotoxicity against HEK-293 (10% or below at 40 µg/mL) (Table 3) was observed for nine of the 11 species, making selection of a species for

53

study using bioactivity data alone quite challenging. Ultimately, the decision to chemically study P. haplophyllus was influenced by LC-MS data and taxonomy. The LC-MS chromatogram showed approximately 70 even m/z ions (i.e. alkaloids) in the bark extract (Figure 7), which was the highest of any species tested. Furthermore, P. haplophyllus is the only species in the Pitaviaster genus, and is found exclusively in north-eastern Queensland and Fraser Island. With the exception of a leaf essential oil study,56 P. haplophyllus has not been chemically investigated since 1969 (from which one acridone and two quinoline alkaloids 30-32 are reported),18, 57 making it likely to contain undiscovered natural products. In contrast, many of the other hits were from genera that have been better chemically studied, e.g. Melicope and Zanthoxylum (Table 1).

Figure 7. Base peak chromatogram of P. haplophyllus bark extract LC-MS.

Figure 8. Alkaloids isolated from P. haplophyllus: arborinine (30) acrophylline (31) and acrophyllidine (32).

2.4 Conclusion

This chapter has resulted in two major outcomes. Firstly, a library of 30 Australian Rutaceae species was screened against P. falciparum and analysed by LC-MS. Using a combination of bioactivity data, LC-MS data and literature information, three species (F. pimenteliana, A. pubescens and P. haplophyllus) were selected for further chemical investigation (Chapters 3-6). Secondly, the extract with the highest level of bioactivity against P. falciparum, the roots of C. brevistyla, was chemically studied, revealing two known pyranocoumarins (1-2). These purified natural products were screened against P. falciparum, revealing 1 to be potently active against both 3D7 and Dd2 P. falciparum with IC50 values of at 0.47 and 0.82 µM, respectively.

54

2.5 Materials and Methods

2.5.1 General Experimental Procedures

NMR spectra were recorded at 25 °C on a Bruker Avance III 500 MHz spectrometer (BBFO Smartprobe, 5mm 31P-109Ag). The 1H and 13C chemical shifts were referenced to the solvent peak for (CD3)2SO at δH 2.50 and δC 39.52. High resolution mass measurements were acquired using positive electrospray ionization on an Agilent Technologies 6530 Accurate-Mass Q-TOF LC-MS with a 1200 Series autosampler and 1290 Infinity HPLC. Oven used for drying plant material was a Contherm Thermotec 2000. C18 silica gel used to adsorb extract prior to HPLC separation was Alltech Sample Prep C18 35-75 µm, 150 Å. HPLC column used was Betasil 5 µm, 100 Å, 21.2 mm x 150 mm. A Merck Hitachi L7100 pump equipped with a Merck Hitachi L7455 PDA detector and a Merck Hitachi L7250 autosampler were used for all chromatography. Fractions were collected using a Gilson 215 liquid handler. All solvents used were Scharlau

HPLC grade and H2O was Millipore Milli-Q PF filtered. Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar. Parasite strains 3D7 and Dd2 were obtained from BEI Resources. O+ erythrocytes were obtained from the Australian Red Cross Blood Service. CellCarrier poly-D-lysine coated imaging plates were from PerkinElmer. 4′,6-Diamidino-2- phenylindole (DAPI) stain were from Invitrogen. Triton-X, saponin, chloroquine, artesunate, puromycin, pyronaridine, dihydroartemisinin and pyrimethamine were all from Sigma Aldrich. HEK293 cells were purchased from the American Tissue Culture Collection. The 384-well Falcon sterile tissue culture treated plates were from BD.

2.5.2 Collection of Plant Material

Plant material was obtained from three nurseries (Table 7). These were Torrington Nursery (346-350 Hursley Rd, Glenvale, Queensland 4350), Go Green Rainforest Nursery (2620 Steve Irwin Way, Glenview, Queensland 4553) and Burringbar Rainforest Nursery (380 Burringbar Rd, Upper Burringbar, New South Wales 2483). All tree cuttings were provided free of charge.

55

Table 7. Sources of plant material.

Species Plant parts Source Date Acronychia acidula BK, LF, RT BB 12/12/2016 Acronychia imperforata BK, LF GG 9/09/2016 Acronychia laevis BK, LF GG 9/09/2016 Acronychia littoralis BK, LF BB 12/12/2016 Acronychia oblongifolia BK, LF GG 9/09/2016 Acronychia pauciflora BK, LF BB 12/12/2016 Acronychia pubescens BK, LF, RT BB 12/12/2016 Bosistoa floydii BK, LF BB 12/12/2016 Bosistoa pentacocca BK, LF, RT BB 12/12/2016 Bosistoa transversa BK, LF BB 12/12/2016 Bouchardatia neurococca BK, LF BB 12/12/2016 Clausena brevistyla BK, LF, RT BB 12/12/2016 Clausena smyrelliana BK, LF, RT BB 12/12/2016 Coatesia paniculata BK, LF, RT BB 12/12/2016 Dinosperma erythrococcum BK, LF TN 17/09/2016 Geijera salicifolia BK, LF TN 17/09/2016 Glycosmis pentaphylla BK, LF, RT BB 12/12/2016 Flindersia pimenteliana BK, LF PL 1/5/2015 Medicosma cunninghamii BK, LF GG 9/09/2016 Medicosma forsterii BK, LF, RT BB 12/12/2016 Melicope elleryana BK, LF GG 9/09/2016 Melicope hayesii BK, LF, RT BB 12/12/2016 Melicope micrococca BK, LF, RT BB 12/12/2016 Melicope rubra BK, LF BB 12/12/2016 Melicope vitiflora BK, LF, RT BB 12/12/2016 Murraya ovatifoliolata BK, LF, RT BB 12/12/2016 Pentaceras australis BK, LF GG 9/09/2016 Pitavastier haplophylla BK, LF, RT BB 12/12/2016 Sarcomelicope simplicifolia BK, LF, RT BB 12/12/2016 Zanthoxylum ovalifolium BK, LF, RT BB 12/12/2016

BB = Burringbar Rainforest Nursery; GG = Go Green Rainforest Nursery; TN = Torrington Nursery; PL = Private land on Mt. Tamborine. BK = bark; LF = leaf; RT = root.

2.5.3 Preparation and Screening of Extract Library

All plants were removed from their pots and cleaned of dirt. Plants were then cut using secateurs and separated into leaves, bark and roots, then placed into an oven for 48 hours at 50 °C. Each plant part was ground to a powder in a coffee grinder. 300 mg of each sample was then extracted by sonication in 10 mL MeOH for 45 minutes. The supernatant was decanted and 10 mL of CH2Cl2 was added to each tube and sonicated for a further 45 minutes. MeOH and CH2Cl2 extracts were combined and evaporated, then passed through polyamide gel cartridges in MeOH under vacuum to remove tannins. Extracts were then filtered through a 0.45 µm syringe filter, then re-evaporated and weighed. For biological activity, samples were prepared to a concentration of 100mg/mL in DMSO and transferred into a 384-well microtiter 56

plate. The microtiter plate was then sealed and sent to the laboratory of Prof. Vicky Avery at Discovery Biology, Griffith Institute for Drug Discovery, where Dr. Sandra Duffy performed all biological assays using an established method.58

2.5.4 LC-MS Analysis of Bark Material

The bark samples from above were prepared to a concentration of 1.5 mg/mL. Each extract was analysed by LC-MS on an Agilent Technologies 6530 Accurate Mass Q-TOF in tandem with an Agilent 1260 LC system. Extracts were chromatographed using C18 HPLC (Kinetex 5 µm, 100

Å, 100 x 4.6 mm) using a solvent gradient from 100% H2O (0.1% FA)/5% CH3CN (0.1% FA) to

100% CH3CN (0.1% FA) over 30 minutes at a flow rate of 1 mL/min. Q-TOF MS/MS method used was positive electrospray ionisation (ESI). M/z range was set from 100-1700 at a scan rate of 3 spectra/sec (MS). Source parameters were: gas temperature 300 °C, gas flow 10 L/min, nebulizer 35 psig; and scan source parameters were: VCap 3500, nozzle voltage 1500 V, fragmentor 130, skimmer 165, and octopoleRFPeak 750. A blank was run after every third extract. Data analysis was performed using Agilent MassHunter Qualitative Analysis B.04.00 software.

2.5.5 Extraction and Isolation

Oven-dried (50 °C, 48 hours), ground C. brevistyla roots (200 g) were exhaustively extracted in

MeOH (2 L), yielding a brown gum (25 g). This extract (1 g) was adsorbed onto C18 silica gel (1 g) and the extract impregnated gel was loaded into a HPLC pre-column cartridge (10 mm x 20 mm) and connected in series to a C18 silica HPLC column (Betasil 5 µm, 100 Å, 21.2 mm x 150 mm). The column was then eluted with a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. Fractions were collected every min and UV- DAD spectroscopic analysis was conducted in tandem with the separation. Fraction 42 contained 2 (0.6 mg). Fractions 57-62 contained 1 (80 mg).

57

2.6 References

1. WHO, World Malaria Report 2016, WHO, Geneva, Switzerland, 2016. 2. D. Menard and A. Dondorp, Cold Spring Harb. Perspect. Med., 2017, a025619. 3. WHO, Global Plan for Artemisinin Resistance Containment, WHO, Geneva, Switzerland, 2011. 4. WHO, Emergency Response to Artemisinin Resistance in the Greater Mekong Subregion, WHO, Geneva, Switzerland, 2013. 5. Y. Lubell, A. Dondorp, P. J. Guérin, T. Drake, S. Meek, E. Ashley, N. P. J. Day, N. J. White and L. J. White, Malar. J., 2014, 13, 452. 6. T. N. C. Wells, Malar. J., 2011, 10, S3. 7. R. Macarron, M. N. Banks, D. Bojanic, D. J. Burns, D. A. Cirovic, T. Garyantes, D. V. S. Green, R. P. Hertzberg, W. P. Janzen and J. W. Paslay, Nat. Rev. Drug Discovery, 2011, 10, 188-195. 8. A. L. Harvey, R. Edrada-Ebel and R. J. Quinn, Nat. Rev. Drug Discovery, 2015, 14, 111- 129. 9. L. S. Fernandez, M. F. Jobling, K. T. Andrews and V. M. Avery, Phytother. Res., 2008, 22, 1409-1412. 10. J. R. Price, in Chemical Plant Taxonomy, ed. T. Swain, Academic Press, New York, 1963, ch. 15, pp. 429–452. 11. P. G. Waterman, Biochem. Syst. Ecol., 1975, 3, 149-180. 12. T. G. Hartley, in Flora of Australia. Volume 26, Meliaceae, Rutaceae, Zygophyllaceae, ed. A. Wilson, ABRS/CSIRO Australia, Melbourne, 2013, pp. 62-72. 13. L. S. Fernandez, Doctor of Philosophy Griffith University, 2009. 14. F. Dalacker and G. Adolphen, Justus Liebigs Ann. Chem. , 1966, 691, 138–141. 15. S. R. Johns, J. A. Lamberton and J. R. Price, Aust. J. Chem., 1967, 20, 2795-2797. 16. E. Bianchi, C. C. J. Culvenor and J. A. Lamberton, Aust. J. Chem., 1968, 21, 2357-2359. 17. H. F. Haynes, E. R. Nelson and J. R. Price, Aust. J. Chem. , 1952, 5, 387-400. 18. F. N. Lahey, M. McCamish and T. McEwan, Aust. J. Chem., 1969, 22, 447-453. 19. K. Padmawinata, Acta Pharmacol, 1973, 4, 1-9. 20. B. F. Bowden, L. Cleaver, P. K. Ndalut, E. Ritchie and W. C. Taylor, Aust. J. Chem., 1975, 28, 1393-1395. 21. A. N. Bissoue, F. Muyard, A. Regnier, F. Bevalot, J. Vaquette, T. G. Hartley and P. G. Waterman, Biochem. Syst. Ecol., 1996, 24, 805. 22. A. A. Auzi, A. I. Gray, T. G. Hartley, R. D. Waigh and P. G. Waterman, Phytochemistry, 1994, 36, 535-536. 23. A. A. Auzi, T. G. Hartley and P. G. Waterman, Biochem. Syst. Ecol., 1997, 25, 611-617. 24. A. A. Auzi, P. I. Forster, T. G. Hartley, R. D. Waigh and P. G. Waterman, Phytochemistry, 1997, 45, 1673-1678. 25. A. A. Auzi, T. G. Hartley, R. D. Waigh and P. G. Waterman, Phytochemistry, 1996, 42, 235-238. 26. J. Latip, T. G. Hartley and P. G. Waterman, Phytochemistry, 1999, 51, 107-110. 27. N. Sultana, T. G. Hartley and P. G. Waterman, Phytochemistry, 1999, 50, 1249-1253. 28. C. Wattanapiromsakul, P. I. Forster and P. G. Waterman, Phytochemistry, 2003, 64, 609-615. 29. T. Sripisut, T. Ritthiwigrom, T. Promgool, K. Yossathera, S. Deachathai, W. Phakhodee, S. Cheenpracha and S. Laphookhieo, Phytochem. Lett., 2012, 5, 379-381. 30. E. Wang, J. P. Bartley, G. Smith and R. C. Bott, Aust. J. Chem., 2001, 54, 739-741. 31. F. O’Donnell, V. N. Ramachandran, T. J. P. Smyth, W. F. Smyth and P. Brooks, Anal. Chim. Acta, 2009, 634, 115-120. 32. J. D. Porter and A. R. Carroll, 2017, unpublished work. 33. C. W. Halstead, P. I. Forster and P. G. Waterman, Nat. Prod. Res., 2006, 20, 940-945.

58

34. B. T. Ngadjui, J. F. Ayafor, B. L. Sondengam and J. D. Connolly, Phytochemistry, 1989, 28, 585-589. 35. Y.-S. Wang, R. Huang, N.-Z. Li and J.-H. Yang, Biosci. Biotechnol. Biochem, 2010, 74, 1483-1484. 36. Y.-S. Wang, H.-P. He, J.-H. Yang, Y.-T. Di and X.-J. Hao, Molecules, 2008, 13, 931-937. 37. V. Kumar, N. M. M. Niyaz and D. B. M. Wickramaratne, Phytochemistry, 1995, 38, 805- 806. 38. V. Kumar, N. M. M. Niyaz, S. Saminathan and D. B. M. Wickramaratne, Phytochemistry, 1998, 49, 215-218. 39. H.-M. Xia, C.-J. Li, J.-Z. Yang, J. Ma, Y. Li, L. Li and D.-M. Zhang, Phytochemistry, 2016, 130, 238-243. 40. B.-Y. Liu, C. Zhang, K.-W. Zeng, J. Li, X.-Y. Guo, M.-B. Zhao, P.-F. Tu and Y. Jiang, J. Nat. Prod., 2018, 81, 22-33. 41. Q.-G. Ma, R.-R. Wei, M. Yang, X.-Y. Huang, F. Wang, Z.-P. Sang, W.-M. Liu and Q. Yu, J. Agric. Food Chem., 2018, 66, 5540-5548. 42. T. Tahsin, J. D. Wansi, A. Al‐Groshi, A. Evans, L. Nahar, C. Martin and S. D. Sarker, Phytother. Res., 2017, 31, 1215-1219. 43. Y. Wang, H. Liang, Q. Zhang, W. Cheng and S. Yi, Biochem. Syst. Ecol., 2014, 57, 210- 215. 44. M. Nicoletti, F. Delle Monache and G. B. Marini-Bettolo, Planta Med., 1982, 45, 250- 251. 45. C. Yenjai, S. Sripontan, P. Sriprajun, P. Kittakoop, A. Jintasirikul, M. Tanticharoen and Y. Thebtaranonth, Planta Med., 2000, 66, 277-279. 46. C. Auranwiwat, S. Laphookhieo, K. Trisuwan, S. G. Pyne and T. Ritthiwigrom, Phytochem. Lett., 2014, 9, 113-116. 47. S. A. Khalid, A. Farouk, T. G. Geary and J. B. Jensen, J. Ethnopharmacol., 1986, 15, 201- 209. 48. D. A. P. dos Santos, P. A. C. Braga, M. F. G. F. da Silva, J. B. Fernandes, P. C. Vieira, A. F. Magalhães, E. G. Magalhães, A. J. Marsaioli, V. R. S. Moraes and L. Rattray, J. Pharm. Pharmacol., 2009, 61, 257-266. 49. K.-H. Lee, H.-B. Chai, P. A. Tamez, J. M. Pezzuto, G. A. Cordell, K. K. Win and M. Tin-Wa, Phytochemistry, 2003, 64, 535-541. 50. L. S. Fernandez, M. S. Buchanan, A. R. Carroll, Y. J. Feng, R. J. Quinn and V. M. Avery, Org. Lett., 2008, 11, 329-332. 51. F. Tillequin, M. Koch, M. Bert and T. Sevenet, J. Nat. Prod., 1979, 42, 92-95. 52. L. J. Drummond and F. N. Lahey, Aust. J. Chem., 1949, 2, 630-638. 53. R. H. Prager and H. M. Thredgold, Aust. J. Chem., 1966, 19, 451-454. 54. K. Miyake, A. Suzuki, C. Morita, M. Goto, D. J. Newman, B. R. O’Keefe, S. L. Morris- Natschke, K.-H. Lee and K. Nakagawa-Goto, J. Nat. Prod., 2016, 79, 2883-2889. 55. B. Cui, H. Chai, Y. Dong, F. D. Horgen, B. Hansen, D. A. Madulid, D. D. Soejarto, N. R. Farnsworth, G. A. Cordell and J. M. Pezzuto, Phytochemistry, 1999, 52, 95-98. 56. J. J. Brophy, R. J. Goldsack and P. I. Forster, JEOR, 2002, 14, 130-131. 57. F. N. Lahey and M. McCamish, Tetrahedron Lett., 1968, 9, 1525-1527. 58. S. Duffy and V. M. Avery, Am. J. Trop. Med. Hyg., 2012, 86, 84-92.

59

Chapter 3 – Pimentelamines A-C, Indole Alkaloids Isolated from the Leaves of the Australian Tree Flindersia pimenteliana

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PAPER

This chapter has been accepted as a co-authored paper to the journal Journal of Natural Products. The bibliographic details of the co-authored paper, including all authors, are:

Robertson, L. P., Duffy, S., Wang, Y., Wang, D., Avery, V. M., Carroll, A. R. Pimentelamines A-C, indole alkaloids isolated from the leaves of the Australian tree Flindersia pimenteliana. Journal of Natural Products, 2017, 80, 3211-3217.

L. P. R. carried out purifications, structure elucidations, ECD calculations, gathered physical data and wrote the manuscript. S. D. performed anti-plasmodial imaging and cytotoxicity assays. Y. W. and D.W. advised computational analysis. V. M. A. provided laboratory space and oversaw biological aspects of the project. A. R. C. oversaw all aspects of the project.

X Luke P. Robertson

(Signed) (Date) 20/11/2018

X Supervisor: Anthony R. Carroll

(Signed) (Date) 20/11/2018

60

Pages redacted Chapter 4 – Anti-plasmodial Bis-indole Alkaloids from the Bark of the Australian Tree Flindersia pimenteliana (Rutaceae)

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PAPER

This chapter has been submitted as a co-authored paper to the journal Organic and Biomolecular Chemistry. The bibliographic details of the co-authored paper, including all authors, are:

Robertson, L. P., Lucantoni, L., Avery, V. M., Carroll, A. R. Anti-plasmodial bis-indole alkaloids from the bark of the Australian tree Flindersia pimenteliana (Rutaceae). Organic and Biomolecular Chemistry.

L. P. R. carried out purifications, structure elucidations, gathered physical data and wrote the manuscript. L. L. performed anti-plasmodial imaging and cytotoxicity assays. V. M. A. provided laboratory space and oversaw biological aspects of the project. A. R. C. oversaw all aspects of the project.

X Luke P. Robertson

(Signed) (Date) 20/11/2018

X Supervisor: Anthony R. Carroll

(Signed) (Date) 20/11/2018

61 Anti-plasmodial bis-indole alkaloids from the bark of the Australian tree Flindersia pimenteliana (Rutaceae)

Luke P. Robertson,a,b Leonardo Lucantoni,b Vicky M. Avery,b and Anthony R. Carroll*,a,b aEnvironmental Futures Research Institute, Griffith University, Southport 4222, Gold Coast, Australia bGriffith Institute for Drug Discovery, Griffith University, Nathan 4111, Brisbane, Australia

62 Three new (1-3) and two known (4-5) bis-indole alkaloids were identified from the bark material of Flindersia pimenteliana. The structures of 1-3 were elucidated on the basis of their (+)-HRESIMS and 2D NMR spectroscopic data. Anti-plasmodial activity for mixtures containing 1:2 and 1:3 versus chloroquine sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum is also reported, with IC50 values ranging from 0.96 to 2.41 µg/mL. These results expand our knowledge of the structure-activity relationships of borreverine-type alkaloids, the anti-plasmodial activity of which have recently attracted significant attention in the literature.

63 Introduction

Flindersia (Rutaceae) is a genus of predominantly rainforest trees containing 17 species, 15 of which are native to Eastern Australia. The genus has considerable economic importance as a source of high-quality timbers.1 Like many other Rutaceous genera, Flindersia has historically been a source of a diverse range of natural products including coumarins, flavonoids, terpenes and most notably, alkaloids.2 The alkaloidal constituents of Flindersia have been investigated extensively, revealing an array of alkaloids of the quinoline, β-carboline and indole types.3 Of these, the most pharmacologically promising have been the bis-indoles. Currently, six bis- indole alkaloids with IC50 values below 350 nM versus chloroquine-resistant (Dd2) Plasmodium falciparum have been reported from Flindersia species.4, 5 As such, the genus is an encouraging source of anti-plasmodial alkaloids. (+)-HRESIMS analysis of the bark of several Flindersia species indicated the presence of a number of undiscovered bis-indole alkaloids in the bark of Flindersia pimenteliana. Described herein is the isolation, structure elucidation and anti- plasmodial activity testing of three new bis-indole alkaloids.

Results and Discussion

Purification of the MeOH extract of F. pimenteliana bark by preparative HPLC and selection of fractions for further purification by 1H NMR and MS analysis led to the identification of three new bis-indole alkaloids (1-3). The known alkaloids dimethylisoborreverine (4) and 4- methylborreverine (5) were also isolated (Figure 1). Although extensive efforts to purify 1-3 were attempted, including the use of size exclusion chromatography, nine different HPLC column packings (seven reversed-phased, two normal-phased) and a variety of isocratic/gradient solvent systems, ultimately these all proved unsuccessful. As a consequence, the highest level of purity achieved was a mixture of 1:2 (60:40) and 1:3. (35:65).

Fig. 1 Bis-indole alkaloids isolated from F. pimenteliana

64 10,10’-Dimethoxydimethylisoborreverine (1) was isolated as a brown gum in a 60:40 mixture with 10-methoxydimethylisoborreverine (2). Analysis of (+)-HRESIMS data at m/z 569.3858

+ allowed the molecular formula C36H49N4O2 to be assigned to the major component of the + mixture (1), while the peak at m/z 539.3744 was designated the formula C35H47N4O (2). Comparison of these formulae indicated that 1 contained one more methoxy than 2. The UV spectrum displayed an absorption maximum at 281 nm, which was indicative of an indole. The

1 H NMR spectrum (Table 1) displayed resonances associated with aromatic methoxys at δH

3.82 (3H), 3.81 (3H) and 3.69 (3H). The resonances at δH 3.81 and 3.69 were of equal intensity, and both were larger than δH 3.82, further indicating that the major component 1 contained one more methoxy than the minor component 2. The spectrum also contained resonances associated with two aromatic doublet of doublets (δH 6.74 and 6.39, 1H each), two ortho- coupled aromatic doublets (δH 7.18 and 6.12, 1H each) and two meta- coupled aromatic doublets (δH 7.16 and 7.05, 1H each). The three resonances at δH 7.18, 7.16 and 6.74 integrated to 1.0 while those at δH 7.05, 6.39 and 6.12 integrated to 0.6, suggesting the presence of two 1,2,4 trisubstituted benzene rings in 1 but only one in 2. Four additional aromatic resonances integrating to 0.4 associated with two ortho- coupled triplets (δH 6.90 and

6.73, 1H each) and two ortho- coupled doublets (δH 7.53 and 6.23, 1H each) established that 2 contained a further 1,2 disubstituted benzene ring. There were no other differences between 1 and 2 and it was confidently concluded that the molecules differed only by one aromatic methoxy. Other resonances in the spectrum were associated with a sharp deshielded NH singlet at δH 11.06 (1H), two broad deshielded NH singlets at δH 10.31 (1H) and 10.26 (1H), four deshielded methylene multiplets between δH 3.35-3.07 (8H) and two N-N-dimethyls at δH 2.93 (doublet, 6H) and 2.82 (broad singlet, 6H). Additional features of the molecule included three methyl singlets at δH 1.71 (3H), 1.04 (3H) and 0.70 (3H), one methylene at δH 2.29 (doublet, 1H) and 1.73 (doublet, 1H) and four non-aromatic methines at δH 5.47 (multiplet, 1H), 5.41 (doublet, 1H), 4.10 (multiplet, 1H) and 3.12 (multiplet, 1H). Comparison of these resonances to the 1H NMR spectrum of the known compound 4 (Table 1) revealed an identical terpenoid substructure to 4. The differences in the 1H NMR spectrum of the 1:2 mixture compared to 4 were the addition of two aromatic methoxys, the loss of four aromatic protons (associated with H-9 to H-12 in 4) and the addition of six resonances associated with two 1,2,4 trisubstituted benzene rings. Additionally, all ortho- and meta- 1H-1H coupling constants of the protons in the 1,2,4, trisubstituted benzene rings were 8.8 and 2.3 Hz, respectively. This is in contrast to 4, where all ortho- and meta- 1H-1H coupling constants were 7.5 and 1.0 Hz, respectively. This ~1.3 Hz increase in 1H-1H coupling confirmed that 1 contained an identical structure to 4 with two added aromatic methoxys. The presence of resonances coincident with

65

H-9′ to H-12′ in 4 in the 1:2 mixture 1H NMR spectrum indicated that the minor component 2 was identical to 4 at C-9′ to C-12′ and must contain an identical structure to 4 with an added methoxy at C-9 to C-12. The elucidation task was thus to assign the regiochemistry of the aromatic methoxys in 1. The 1H and 13C NMR chemical shifts of C-12/C-12′ were almost identical in 1 and 4, indicating that they were meta- to the methoxys. The multiplicity of H- 12/H-12′ methines, which were both ortho- coupled doublets, supported this. This was further

3 reinforced by JCH HMBC correlations from H-12/H-12′ to oxygenated aromatic carbon resonances at δC 153.5 and 153.0, which were assigned as C-10/C-10′ respectively. The resonances associated with methine protons at δH 6.74/6.39 were assigned as H-11/H-11′ 3 respectively from ortho- COSY correlations to H-12/H-12′. JCH HMBC correlations from methoxy resonances at δH 3.81/3.69 to carbon resonances previously assigned as C-10/C-10′ dictated their assignment as 10-OMe and 10′-OMe, respectively. The final assignment of H9/H9′ was deduced from HMBC correlations, where the resonance associated with the

3 unassigned meta- coupled doublet at δH 7.16 (H-9) showed JCH HMBC correlations to C-11 and 3 to C-13. H-11 (δH 6.74) also showed JCH HMBC correlations to C-13. The same suite of HMBC correlations was visible from δH 7.05 (H-9′) to C-11′/C-13′ and from δH 6.39 (H-11′) to C-13′.

Therefore, the resonances at δH 7.16/7.05 were correspondingly assigned as H-9/H-9′. The final piece of evidence to support the assignment of the aromatic methoxys at C-10/C-10′ was the upfield shift of the non-protonated carbon resonances at C-13/C-13′, which had moved from

δC 136.1/131.6 in 4 to δC 130.7/127.2 in 1. This typical ~5 ppm upfield shift supports their assignment as para- to the C-10/C-10′ methoxys.6 The 2D structure of 1 was therefore established. The structure of 2 was also confirmed as identical to 1 with the absence of the C- 10′ methoxy. The relative configuration of the three stereogenic centres in 1 and 2 were determined from analysis of ROESY correlations (Figure 2b). ROESY correlations between H-3′,

H-14 and H3-17 dictated their assignment as β. The α configuration of H-3 was assigned from ROESY correlations to H-16 and H-16′α. The proposed relative configuration of 1 and 2 was compared to 4 and it was concluded that both possessed the same relative configuration. The mixture of 1:2 lacked optical rotation and showed no ECD spectrum, which is congruent with observations by previous authors that borreverine-type alkaloids are racemates.7

10’-Methoxydimethylisoborreverine (3) was isolated as a brown gum in a 65:35 mixture with 1.

+ Analysis of (+)-HRESIMS data at m/z 539.3744 allowed the molecular formula C35H47N4O to be assigned to 3, indicating that it also contained one less methoxy group than 1. The 1:3 mixture contained almost identical 1H NMR resonances to the 1:2 mixture, however in the 1:3 mixture there was an additional methoxy resonance at δH 3.68 while the methoxy resonance at δH 3.82 was absent. The four aromatic resonances coincident with H-9′ to H-12′ in 4 were also absent 66

while those coincident with H-9 to H-12 in 4 were present. This confirmed that 3 lacked the C- 10 methoxy of 1 but had an otherwise identical structure.

(a)

(b)

Fig. 2 (a) Key COSY (bold bonds) and HMBC (arrows) correlations in 1 (b) Key ROESY correlations in the energy minimised conformation of 1.

1-3 were tested for anti-plasmodial activity against chloroquine sensitive (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum. Activity against HEK293 mammalian cell line was also determined to evaluate selectivity (Table 2). The mixtures of 1:2 (60:40) and 1:3

(35:65) showed IC50 values against the 3D7 strain of 0.959 and 2.407 µg/mL, respectively. Both mixtures had a very steep dose-response curve against 3D7, reaching a full inhibition plateau. A gentler slope was observed against Dd2, which led to complete inhibition of the parasite at

the highest concentration only. Because of this, only the 3D7 IC50 values could be calculated. The mixtures of 1:2 and 1:3 were both less active than the known potently anti-plasmodial

alkaloid 4 (IC50 = 0.341 µg/mL versus 3D7). 4 differs from 1-3 only by aromatic methoxys and thus the addition of C-10/C-10′ aromatic methoxys does not appear to increase the anti- plasmodial activity of isoborreverine-type alkaloids.

67

Table 1 1H and 13C NMR Data for the methoxylated dimethylisoborreverines (1-3) and

dimethylisoborreverine (4) in DMSO-d6

1 2 3 4 δ b (mult, J in δ b (mult, J in Hz, δ b (mult, J in Hz, δ b (mult, J in Hz, Position δ a H HMBC δ a H δ a H δ d H c Hz, int.) c int.) c int.) c int.) 1 N 11.06 (s, 1H) — N 11.06 (s, 1H) N 11.16 (s, 1H) N 11.23 (s, 1H) 2 134.7 — — 134.7 — 134.7 — 134.1 - 3 53.8 5.41 (d, 9.4, 15 53.8 5.41 (d, 9.4, 1H) 53.4 5.42 (d, 9.4, 1H) 53.7 5.51 (d, 9.4, 1H) 1H) 4 N 10.31 (brs, 1H) — N 10.31 (brs, 1H) N 9.68 (brs, 1H) N 10.30 (brs, 1H) 4-Me 41.8 2.93 (d, 4.7, 4-Me, 5 41.8 2.93 (d, 4.7, 6H) 41.8 2.94 (d, 4.7, 6H) 42.1 2.95 (d, 3.7, 6H) 6H) 5 56.4 3.21 (m, 1H) — 56.4 3.21 (m, 1H) 56.7 3.19 (m, 1H) 56.3 3.25 (m, 2H) 3.32 (m, 1H) 3.32 (m, 1H) 3.32 (m, 1H) 6 19.1 3.14 (m, 1H) — 19.1 3.14 (m, 1H) 19.1 3.21 (m, 1H) 19.5 3.25 (m, 1H) 3.33 (m, 1H) 3.33 (m, 1H) 3.25 (m, 1H) 3.36 (m, 1H) 7 107.4c — — 107.4c — 107.4c — 107.4 — 8 128.6 — — 128.6 — 128.6 — 127.2 — 9 100.7 7.16 (d, 2.3, 11, 13 100.7 7.16 (d, 2.3, 1H) 118.3 7.67 (d, 7.5, 1H) 118.3 7.68 (d, 7.5, 1H) 1H) 10 153.7 — — 153.5 — 118.6 7.05 (td, 7.5, 1.0, 118.9 7.05 (td, 7.5, 1.0, 1H) 1H) 10-OMe 53.8 3.81 (s, 3H) 10 53.8 3.82 (s, 3H) — — — — 11 111.0 6.74 (dd, 8.8, 9, 10, 13 111.3 6.74 (dd, 8.8, 2.3, 121.5 7.11 (td, 7.5, 1.0, 122.0 7.11 (td, 7.5, 1.0, 2.3, 1H) 1H) 1H) 1H) 12 111.1 7.18 (d, 8.8, 8, 10 111.6 7.18 (d, 8.8, 1H) 111.1 7.29 (d, 7.5, 1H) 111.6 7.29 (d, 7.5, 1H) 1H) 13 130.7 — — 130.7 — 136.0 — 136.1 — 14 57.5 3.12 (m, 1H) 3, 15, 3′, 57.2 3.12 (m, 1H) 57.2 3.12 (m, 1H) 57.4 3.16 (m, 1H) 14′, 16′ 15 32.2 — — 32.2 — 32.2 — 31.6 — 16 27.5 0.70 (s, 3H) 14, 15, 17, 27.4 0.70 (s, 3H) 27.4 0.70 (s, 3H) 27.6 0.71 (s, 3H) 16′ 17 28.3 1.04 (s, 3H) 14, 15, 16, 28.3 1.04 (s, 3H) 28.1 1.05 (s, 3H) 28.4 1.06 (s, 3H) 16′ 1′ N — — N — N — N — 2′ 143.6 — — 143.6 — 143.6 — 143.1 — 3′ 37.4 4.10 (m, 1H) — 37.4 4.10 (m, 1H) 37.4 4.09 (m, 1H) 36.5 4.13 (m, 1H) 4′ N 10.26 (brs, 1H) — N 10.26 (brs, 1H) N 9.62 (brs, 1H) N 10.18 (brs, 1H) 4’-Me 42.2 2.82 (brs, 6H) 4’-Me 42.2 2.82 (brs, 6H) 42.2 2.86 (brs, 6H) 42.3 2.84 (brs, 6H) 5′ 56.4 3.24 (m, 1H) 7′ 56.4 3.24 (m, 1H) 56.7 3.19 (m, 1H) 56.8 3.36 (m, 2H) 3.35 (m, 1H) 3.35 (m, 1H) 3.32 (m, 1H) 6′ 19.4 3.09 (m, 1H) 2′, 5′, 7′, 8′ 19.4 3.09 (m, 1H) 19.4 3.07 (m, 1H) 19.6 3.18 (m, 2H) 3.16 (m, 1H) 2′, 5′, 7′, 8′ 3.16 (m, 1H) 3.14 (m, 1H) 7′ 99.1 — — 99.1 — 99.1 — 99.2 — 8′ 132.3 — — 132.3 — 132.2 — 132.0 — 9′ 100.8 7.05 (d, 2.3, 7′, 10′, 11′, 117.8 7.53 (d, 7.5, 1H) 100.8 7.03 (d, 2.3, 1H) 118.2 7.54 (dt, 7.5, 1.0, 1H) 13′ 1H) 10′ 153.0 — — 118.1 6.90 (t, 7.5, 1H) 153.0 — 118.8 6.90 (td, 7.5, 1.0, 1H) 10′-OMe 55.5 3.69 (s, 3H) 10′ — — 55.5 3.69 (s, 3H) — — 11′ 109.2 6.39 (dd, 8.8, 9′, 10′, 13′ 120.0 6.73 (t, 7.5, 1H) 109.2 6.41 (dd, 8.8, 2.3, 120.4 6.73 (td, 7.5, 1.0, 2.3, 1H) 1H) 1H) 12′ 109.0 6.12 (d, 8.8, 8′, 10′ 108.9 6.23 (d, 7.5, 1H) 109.1 6.12 (d, 8.8, 1H) 109.2 6.23 (dt, 7.5, 1.0, 1H) 1H) 13′ 127.2 — — 131.6c — 127.2 — 131.6 — 14′ 118.7 5.47 (m, 1H) 14, 3′, 16′, 118.7 5.47 (m, 1H) 118.6 5.47 (m, 1H) 119.1 5.49 (m, 1H) 17′ 15′ 133.2 — — 132.2 — 132.7 — 132.4 — 16′α 39.7 2.29 (d, 17.4, 16, 17, 14’, 39.7 2.29 (d, 17.4, 1H) 39.7 2.28 (d, 17.4, 1H) 39.7 2.30 (d, 17.6, 1H) 1H) 15’ β 1.73 (d, 17.4, 14, 15, 16, 1.73 (d, 17.4, 1H) 1.73 (d, 17.4, 1H) 1.73 (d, 17.6, 1H) 1H) 17, 17′ 17′ 23.7 1.71 (s, 3H) 14’, 15’, 16’ 23.8 1.71 (s, 3H) 23.8 1.72 (s, 3H) 23.7 1.71 (s, 3H) aChemical shifts determined by 2D experiments. b800 MHz. cnot observed, determined by comparison to 4. d125 MHz.

68

Table 2 Antiplasmodial activity and cytotoxicity of natural products isolated from the bark of F. pimenteliana.

IC50 ± SD (µg/mL) (n = 2) Selectivity Compound 3D7 Dd2 HEK293 Index (SI) 1:2 (60:40 mixture) 0.959 ± 0.294 96.0% ± 0.8a 101.2% ± 0.3a n.d.

1:3 (35:65 mixture) 2.407 ± 0.424 94.2% ± 1.3b 75.2% ± 32.9b n.d.

4c 0.341 ± 0.214 0.096 ± 0.020 2.749 ± 0.509 8

5c 0.252 ± 0.069 0.168 ± 0.054 3.514 ± 0.643 14 artesunated 0.0021 ± 0.0016e 0.0033 ± 0.0022e 62.5% ± 16.5f n.d. chloroquined 0.0139 ± 0.0015e 0.1360 ± 0.0255e 53.2% ± 26.4f n.d. dihydroartemisinind 0.0009 ± 0.0002e 0.0016 ± 0.0003e 41.1% ± 11.5f n.d. puromycind 0.0702 ± 0.0108e 0.0747 ± 0.0188e 0.6940 ± 0.183e 10 pyrimethamined 0.0077 ± 0.0017e n.d. 52.0% ± 10.2f n.d. pyronaridined 0.0082 ± 0.0020e 0.0110 ± 0.0010e 1.990 ± 0.263e 246 apercentage growth inhibition at 13.5 µg/mL. bpercentage growth inhibition at 20 µg/mL. cbioactivity data previously reported.4 dreference compound. econcentration in µM. fpercentage growth inhibition at 40 µM. SI = selectivity index in (HEK293/3D7). n.d. = not determined.

Conclusions

In summary, three new bis-indole alkaloids were identified from the bark of F. pimenteliana. Mixtures of 1-3 displayed moderate anti-plasmodial activity against chloroquine-sensitive (3D7) P. falciparum (Table 2). 1-3 were less active than the known anti-plasmodial alkaloid 4. These results extend our knowledge of the structure-activity relationships of the Flindersia bis- indole alkaloids. Despite differing by one to two methoxy groups, 1-3 were very difficult to purify from each other due to their very low abundance, broad chromatographic peaks and lack of appreciable differences in molecular weight, polarity, and lipophilicity. Separating 1-3 from 4 was similarly challenging. As such, any further work on 1-3 should be carried out by total synthesis. The total synthesis of 4 was reported previously.8, 9 Flindersia continues to be a source of anti-plasmodial bis-indole alkaloids. Chemical profiling studies are currently ongoing to identify related alkaloids in other Flindersia species and from other closely related Australian endemic Rutaceous genera.

69

Experimental

General experimental procedures.

−1 2 OR was measured using a JASCO P-1020 polarimeter and [α]D values are given in 10 deg cm g−1. UV spectra were acquired on a Shimadzu UV-1800 spectrophotometer. CD spectra were recorded on a JASCO J-715 spectropolarimeter. IR spectra were recorded using a ThermoFisher Scientific Nicolet iS5 spectrometer in tandem with an iD5 ATR. NMR spectra were acquired at 25 °C on a Bruker Avance III 500 MHz spectrometer (BBFO Smartprobe, 5mm 31P-109Ag) and a Bruker Avance III HDX 800 MHz with a triple (TCl) resonance 5 mm cryoprobe. Chemical shifts were referenced to the solvent peak for (CD3)2SO at δH 2.50 and δC 39.52. HRESIMIS measurements were obtained in 100% ACN on an Agilent Technologies 6530 Accurate-Mass Q- TOF LC/MS with a 1200 Series autosampler and 1290 Infinity HPLC. For ion exchange, SCX resin used was Dowex 50WX8 hydrogen form. Oven used for drying plant material was a Contherm

Thermotec 2000. C18 silica gel used for HPLC was Alltech Sample Prep C18 35-75 µm, 150 Å. HPLC columns used were Betasil 5 µm, 100 Å, 21.2 mm x 150 mm; Betasil 5 µm, 100 Å, 10.0 mm x 250 mm; Zorbax SB-phenyl, 5 µm, 21.2 mm x 250mm; Gemini 5 µm, 110 Å, 21.2 mm x

250 mm; Agilent Diphenyl 5 µm, 100 Å, 21.2 mm x 250 mm; Amide C16 5 µm, 10 mm x 250 mm; YMC-pack ODS-AQ, 5 µm, 120 Å, 10 mm x 150 mm; YMC-pack diol, 5 µm, 120 Å, 21.2 mm x 150 mm and YMC-pack NH2 5 µm, 120 Å, 20 mm. MPLC was performed using a refillable HPLC column (40 mm x 100 mm) packed with Alltech Sample Prep C18 35-75 µm, 150 Å. Size exclusion was performed using a pre-packed Sephadex LH-20 column (25-100 µm, 30 mm x 900 mm). A Merck Hitachi L7100 pump with a Merck Hitachi L7455 PDA and a Merck Hitachi L7250 autosampler were used for HPLC. A Gilson 215 liquid handler was used for fraction collection. Organic solvents were Scharlau HPLC grade and H2O was Millipore Milli-Q PF filtered. Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar. Ammonium hydroxide (~25% aqueous NH3) was from VWR Chemicals. Parasite strains 3D7 and Dd2 were obtained from BEI Resources. O+ erythrocytes were obtained from the Australian Red Cross Blood Service. CellCarrier poly-D-lysine coated imaging plates were from PerkinElmer. 4′,6- Diamidino-2-phenylindole (DAPI) stain were from Invitrogen. Triton-X, saponin, chloroquine, artesunate, puromycin, pyronaridine, dihydroartemisinin and pyrimethamine were all from Sigma Aldrich. HEK293 cells were purchased from the American Tissue Culture Collection. The 384-well Falcon sterile tissue culture treated plates were from BD.

70 Collection and identification of plant material

F. pimenteliana bark material was collected from a cultivated tree growing on private land on Mt. Tamborine, South East Queensland, Australia in December 2015. The tree was grown from seed stock collected in North Queensland and obtained from Burringbar Rainforest Nursery. A voucher specimen ACRUT005 is housed within the School of Environment and Science, Gold Coast Campus, Griffith University. The plant material was identified by A.R.C.

Extraction and isolation

F. pimenteliana bark (500 g) was oven dried (50 °C, 48 hours) and ground to a powder, then exhaustively extracted in MeOH (16 L) and evaporated under vacuum to yield a brown gum (250 g). The extract was redissolved in 1 L MeOH and eluted through 200 g of strongly acidic cation exchange (SCX) resin, which was then treated with 25% aqueous NH3 (20%) and MeOH

(80%) to yield 3 g of crude alkaloid extract. This extract was bound to 30 g of C18 silica gel and loaded into a pre-packed refillable C18 MPLC column (40 mm x 100 mm) and purified using a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. Fractions were collected every min. Fractions 44-58 were recombined to yield 2 g of extract. 1 g of this extract was adsorbed onto C18 silica gel (1 g) and the extract impregnated gel was loaded into a HPLC pre-column cartridge (10 mm x 20 mm) and connected in series to a C18 silica HPLC column (Betasil 5 µm, 100 Å, 21.2 mm x 150 mm). The column was then eluted with a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. Fractions were collected every min. This process was repeated again until all 2 g of crude alkaloid extract was purified. Fractions 28-36 of both HPLC runs were recombined to yield 500 mg of extract. This extract was purified using diphenyl HPLC (Agilent Diphenyl 5

µm, 100 Å, 21.2 mm x 250 mm) using a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min. Fractions 43-45 (50 mg) were further purified by diol HPLC (YMC-pack diol, 5 µm, 120 Å, 21.2 mm x 150 mm) using isocratic hexane (1%)/DCM (99%). Fractions 18-50 (30 mg) were recombined and purified using size exclusion chromatography (Sephadex LH-20, 25-100 µm, 30 mm x 900 mm) using MeOH (100%) at a flow rate of 3 mL/min. Fractions were collected at four min intervals. Fractions 26-30 (30 mg) were further purified using diol HPLC (YMC-pack diol, 5 µm, 120 Å, 21.2 mm x 150 mm) using isocratic MeOH (3%)/DCM (97%). Fractions 7-8 contained 20 mg of a mixture of 1-5. 1 mg of fractions 7-8 was further purified using amide C16 HPLC (Amide C16 5 µm, 10 mm x 250 mm) using a gradient from H2O/0.1% TFA

(100%) to CH3CN/0.1% TFA (100%) over 60 min. Fraction 27 contained 1-5 and showed no purification. This fraction (1 mg) was recombined and purified using ODS-AQ HPLC (YMC-pack

ODS-AQ, 5 µm, 120 Å, 10 mm x 150 mm) using a gradient from H2O/0.1% TFA (100%) to 71

CH3CN/0.1% TFA (100%) over 60 min. Fraction 30 contained 1-5 as a mixture and showed no purification. 20 mg of 1-5 was further purified by diol HPLC using two columns connected in sequence (2 x YMC-pack diol, 5 µm, 120 Å, 21.2 mm x 150 mm) using isocratic MeOH (3%)/DCM (97%) at a flow rate of 6 mL/min. Fractions were collected at 0.5 min intervals. Fractions 77-80 contained 4. Fractions 71-75 (1 mg) were recombined and further purified by

C18 HPLC (Betasil 5 µm, 100 Å, 10.0 mm x 250 mm) using isocratic H2O/0.1% TFA (55%)/ MeOH/0.1% TFA (45%) at a flow rate of 4mL/min. Fractions were collected at 0.5 min intervals. Fractions 47-52 contained 5. Fraction 53-54 contained a mixture of 1:3 (0.05 mg, 1.25x10-7% dry wt.) in a 35:65 ratio and fractions 63-77 contained a mixture of 1:2 in a 60:40 ratio (0.15 mg, 3.75x10-7% dry wt.). See supplementary data (S15) for full descriptions of previous failed isolation attempts.

25 10,10’-Dimethoxydimethylisoborreverine (1) TFA salt. Brown gum, [훼]D 0 (c 0.01, MeOH); -1 UV (MeOH) 휆max (log ε) 281 (3.98), 209 (4.62) nm; IR νmax (film)/cm 3272, 2963, 1672, 1460, 1 13 1199, 1177, 1130, 1024, 1005, 746, 720; H and C NMR data ((CD3)2SO) see Table 1; (+)- + + HRESIMS m/z 569.3858 [M+H] (calcd for C36H49N4O2 , 569.3850)

25 10-Methoxydimethylisoborreverine (2) TFA salt. Brown gum, [훼]D 0 (c 0.01, MeOH); UV -1 (MeOH) 휆max (log ε) 281 (3.98), 209 (4.62) nm; IR νmax (film)/cm 3272, 2963, 1672, 1460, 1 13 1199, 1177, 1130, 1024, 1005, 746, 720; H and C NMR data ((CD3)2SO) see Table 1; (+)- + + HRESIMS m/z 539.3744 [M+H] (calcd for C35H47N4O , 539.3744)

25 10’-Methoxydimethylisoborreverine (3) TFA salt. Brown gum, [훼]D 0 (c 0.01, MeOH); UV -1 (MeOH) 휆max (log ε) 283 (4.12), 212 (4.70) nm; IR νmax (film)/cm 3272, 2963, 1672, 1460, 1 13 1199, 1177, 1130, 1024, 1005, 746, 720; H and C NMR data ((CD3)2SO) see Table 1; (+)- + + HRESIMS m/z 539.3744 [M+H] (calcd for C35H47N4O , 539.3744)

Compounds and Screening Methods. Two mixtures consisting of 1:2 (60:40) and 1:3 (35:65) were dissolved in 100% DMSO to obtain stock solutions with matching concentrations of 1 (3.5 mM) with concentrations of 2 and 3 of 2.5 mM and 5.5 mM, respectively. Chloroquine, artesunate, puromycin, pyronaridine, dihydroartemisinin and pyrimethamine were used as reference compounds. Stock solutions were prepared in 100% DMSO at 2.5 mM for dihydroartemisinin and artesunate, or 10 mM for all other reference compounds, except chloroquine and pyronaridine which were dissolved in water. Puromycin (5 µM) and 0.4% DMSO were used as positive and negative controls, respectively. Experimental compounds were tested in 16-concentrations dose-response using three concentrations per log dose against 3D7 (drug-sensitive) and Dd2 (drug-resistant) P. falciparum parasite strains and against

72 HEK293 cells for cytotoxicity assessment. Final assay concentration ranges of 20 µg/mL – 0.20 µg/mL and 13.5 µg/mL – 0.13 µg/mL were used for the 1:3 and 1:2 mixtures, respectively. A 21-concentrations dose-response range of 40 µM – 0.01 nM was used for reference anti- malarial compounds/drugs, except for dihydroartemisinin and artesunate, which were tested at a range of 10 µM to 0.003 nM.

In Vitro Anti-plasmodial Image-Based Assay. P. falciparum parasites (3D7 and Dd2 strains) were grown in RPMI 1640 supplemented with 25 mM HEPES, 5% AB human male serum, 2.5 mg/mL Albumax II, and 0.37 mM hypoxanthine. Parasites were synchronised twice using sorbitol before undergoing compound testing. Ring stage parasites were exposed to the experimental compounds in 384-wells imaging CellCarrier microplates (PerkinElmer) as

10 previously described. After incubating the parasites for 72 h at 37 °C, 90% N2, 5% CO2, 5% O2, the plates were stained using 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), and imaged using an Opera QEHS micro-plate confocal imaging system (PerkinElmer). Images were analysed as previously described.10

In Vitro Cytotoxicity Assay. Human Embryonic Kidney cells (HEK293) were cultured in DMEM medium, supplemented with 10% FBS. The cells were exposed to the compounds in TC-treated

384-wells plates (Greiner) for 72 h at 37 °C, 5% CO2. After the incubation, the media was removed from the wells and replaced with an equal volume of 44 µM resazurin. The plates were allowed to incubate for an additional 5-6 hours at standard conditions. The total fluorescence (excitation/emission: 530 nm/595 nm) was then measured using the Ensight plate reader (PerkinElmer). The positive control, puromycin, had an IC50 value of 0.69 ± 0.18 μM against the HEK293 cell line.

Biological Data Analysis. Percent inhibition data was obtained by normalising raw data using the in-plate positive and negative controls. Inhibition data was used to calculate IC50 values, by applying 4 parameter logistic curve fitting in Prism (GraphPad). The experiments were carried out in two biological replicates, each consisting of two technical repeats.

Conflicts of Interest

There are no conflicts to declare.

Acknowledgements

This work was funded by an Australian Postgraduate Award (APA) provided by the Australian Commonwealth Government. The authors thank M. Wibowo, W. Loa-Kum-Cheung, J.

73

Carrington and R. Stewart for technical assistance. We thank the Australian Red Cross Blood Service for the provision of human blood.

References

1. T. G. Hartley, in Flora of Australia. Volume 26, Meliaceae, Rutaceae, Zygophyllaceae, ed. A. Wilson, ABRS/CSIRO Australia, Melbourne, 2013, pp. 62-72. 2. P. G. Waterman, Biochem. Syst. Ecol., 1975, 3, 149-180. 3. L. P. Robertson, C. R. Hall, P. I. Forster and A. R. Carroll, Phytochemistry, 2018, 152, 71- 81. 4. L. P. Robertson, S. Duffy, Y. Wang, D. Wang, V. M. Avery and A. R. Carroll, J. Nat. Prod., 2017, 80, 3211-3217. 5. L. S. Fernandez, M. S. Buchanan, A. R. Carroll, Y. J. Feng, R. J. Quinn and V. M. Avery, Org. Lett., 2008, 11, 329-332. 6. D. S. Nunes, K. Luzia, J. J. Taveira and A. M. R. Francisco, Phytochemistry, 1992, 31, 2507-2511. 7. F. Tillequin and M. Koch, Phytochemistry, 1979, 18, 1559-1561. 8. D. H. Dethe, R. D. Erande and A. Ranjan, J. Org. Chem., 2013, 78, 10106-10120. 9. D. H. Dethe, R. D. Erande and B. D. Dherange, Org. Lett., 2014, 16, 2764-2767. 10. S. Duffy and V. M. Avery, Am. J. Trop. Med. Hyg., 2012, 86, 84-92.

74 Chapter 5 – Acrotrione, a new Oxidized Xanthene from the Roots of Acronychia pubescens

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PAPER

This chapter has been submitted as a co-authored paper to the journal Journal of Natural Products. The bibliographic details of the co-authored paper, including all authors, are:

Robertson, L. P., Lucantoni, L., Duffy, S., Avery, V. M., Carroll, A. R. Acrotrione, a new oxidised xanthene from the roots of Acronychia pubescens. Journal of Natural Products.

L. P. R. carried out purifications, structure elucidations, ECD calculations, gathered physical data and wrote the manuscript. S.D. and L. L. performed anti-plasmodial imaging and cytotoxicity assays. V. M. A. provided laboratory space and oversaw biological aspects of the project. A. R. C. oversaw all aspects of the project.

X Luke P. Robertson

(Signed) (Date) 20/11/2018

X Supervisor: Anthony R. Carroll

(Signed) (Date) 20/11/2018

75

Acrotrione, a new Oxidized Xanthene from the Roots of Acronychia pubescens

Luke P. Robertson,a,b Leonardo Lucantoni,b Sandra Duffy,b Vicky M. Avery,b and Anthony R. Carroll*,a,b aEnvironmental Futures Research Institute, Griffith University, Southport 4222, Gold Coast, Australia bGriffith Institute for Drug Discovery, Griffith University, Nathan 4111, Brisbane, Australia

76

A new oxidized xanthene, acrotrione (1) and two known acetophenones (2-3) were isolated from the methanol extract of the roots of Acronychia pubescens. The structure of 1 was elucidated on the basis of its (+)-HRESIMS, 2D NMR and ECD data. Acrotrione (1) contains an unusual oxidized furo[2,3-c]xanthene moiety that has not been previously reported. Moderate anti-plasmodial activity for the natural products against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum is also reported, with IC50 values ranging from 1.7 to 4.7 µM.

77

Introduction

Acronychia (Rutaceae) is a genus comprising 48 species of shrubs and small trees distributed throughout Southern Asia, Indonesia, New Caledonia and Australia (19 endemic).1 Historically, Acronychia species have been used in traditional medicines as anti-malarial,2 anti-fungal, anti- pyretic and anti-hemorrhagic agents.3 Chemical investigations of the genus have revealed an array of natural products including alkaloids,4 acetophenones,5 terpenes,6 lignans,7 and cinnamic acids.8 As part of our efforts to identify new anti-plasmodial drug leads,9 a library of Australian endemic Rutaceae species was screened against chloroquine-sensitive (3D7) Plasmodium falciparum. The methanol extract of the roots of Acronychia pubescens F.M.Bailey displayed 80% growth inhibition at 0.4 µg/mL, warranting a chemical investigation of the species. Commonly known as “Hairy Aspen” and growing up to 15 metres in height, A. pubescens is a rainforest tree or shrub endemic to southeast Queensland and northeast New South Wales, Australia.1 Previous chemical studies of the species have been limited, revealing only three simple furoquinolines.10 Reported herein is the isolation, structural elucidation and anti-plasmodial activity testing of a new oxidized xanthene and two known acetophenones (Figure 1).

Figure 1. Natural products isolated from the roots of Acronychia pubescens: the new oxidised xanthene acrotrione (1) and the known acetophenones acronyculatin A (2) and acronylin (3).

Results and Discussion

Exhaustive extraction of A. pubescens roots with MeOH followed by sequential purification with H2O/MeOH gradient reversed-phase and n-hexane/CH2Cl2/MeOH gradient normal-phase HPLC led to the purification of acrotrione (1), acronyculatin A (2) and acronylin (3). Acrotrione (1) was isolated as a yellow amorphous solid. A protonated molecule in the (+)-HRESIMS at m/z

+ 587.2844 allowed the molecular formula C32H43O10 to be assigned to 1. The UV spectrum had absorption maxima at 335, 290, 269 and 228 nm, which was indicative of an acetophenone.11 The 1H NMR spectrum of 1 (Table 1) displayed signals associated with six aliphatic methyl

78 groups at δH 1.67 (s), 1.57 (s), 1.16 (s), 1.13 (s), 0.99 (d, J = 6.5 Hz) and 0.90 (d, J = 6.5 Hz), two acetyl groups at δH 2.61 (s) and 2.12 (s), one aromatic methoxy at δH 3.63 (s), three methylenes at δH 3.16 (m)/3.11 (dd, J = 15.0, 5.8 Hz), 2.54 (dd, J = 13.1, 5.1 Hz)/2.21 (dd, J = 13.1, 10.8 Hz) and 2.08 (m)/1.69 (m) and five methines at δH 5.02 (t, J = 6.6 Hz), 4.78 (dd, J = 10.8, 5.1 Hz) 3.78 (dd, J = 11.8, 3.6 Hz), 3.19 (d, J = 0.8 Hz) and 1.79 (m). Resonances that could be attributed

13 to three hydroxy protons at δH 13.69 (s), 7.02 (s) and 6.57 (s) were also observed. The C NMR spectrum contained resonances associated with 13 sp2 hybridised carbons. These were three ketones (δC 203.8, 196.7, 193.2), one 2,4,6 trioxygenated aromatic ring (δC 160.8, 158.3, 155.5,

114.3, 109.8, 108.8), one double bond (δC 130.4, 123.2) and an oxygenated double bond α/β to 3 a carbonyl (δC 178.1, 111.9). Other features of the spectrum included an additional 18 sp hybridised carbons, of which four (δC 97.2, 92.9, 80.5, 69.1) were oxygenated. Comparison of the 1H and 13C NMR data and molecular formula of 1 to literature data11 suggested that 1 could be an acetophenone dimer derivative containing three isoprene units. All previously reported Acronychia-type acetophenones (AtA) dimers contain two 2,4,6 trioxygenated aromatic rings with substitution of isoprene and acetyl moieties at C-3/C-5 and C-1, respectively.11 Linkage of monomers occurs via an isopentyl group at C-3/C-5, although cyclisation of C-3/C-5 isoprene functionalities to adjacent oxygen atoms may also occur, resulting in the formation of furan or pyran rings. However, unlike previously reported AtA dimers, 1 only contained three

13 oxygenated aromatic resonances in the C NMR spectrum between δC 170.0-140.0 (δC 160.8, 158.3 and 155.5), indicating the presence of only one aromatic ring. Additional and unusual deshielded resonances at δC 193.2 and 178.1 indicated that 1 contained atypical AtA structural features. The structure of the B ring was determined from analysis of 2D NMR data and corroborated by comparison of resonances to literature values5 and those of the co-isolated compound 3. HMBC correlations from OH-6 (δH 13.70), MeCO-1 (δH 2.61), MeO-2 (δH 3.63) and

H-1′ (δH 3.11/3.16) to resonances at C-1 through C-6 (Figure 2a) confirmed this substructure. C- 4 was not assigned as a hydroxyl due to the absence of a corresponding hydroxyl peak in the

1 H NMR spectrum, and its chemical shift (δC 155.5) suggested it was an ether. The B ring in all of the known AtA dimers contains a hydroxyl at C-4.12 COSY correlations between H-1′′, H-2′′, H-3′′ and H-4′′/H-5′′ were used to assign the isopentyl unit, which was confirmed by HMBC correlations from H-4′′/H-5′′ to C-2′′, C-3′′ and C-4′′/C-5′′. H-1′′, which appeared as a methine doublet of doublets of doublets (J = 11.8, 3.6, 0.8 Hz) at δH 3.78 (δC 27.3), showed HMBC correlations to previously assigned resonances at C-3 (δC 155.5), C-4 (δC 109.8) and C-5 (δC 160.8), which supported its position as benzylic to the B ring. Additional HMBC correlations from H-1′′ to resonances at δC 193.2, 97.2 and 43.8 revealed that C-2′′′ (δC 193.2) was a ketone while C-4′′′ (δC 97.2) was doubly oxygenated. HSQC data indicated that the resonance at δC

79

43.8 was a methine and assigned at C-3′′′ (δH 3.19) from HMBC correlations to previously assigned resonances at C-2′′, C-2′′′, C-4′′′ and C-5. A small (J = 0.8 Hz) coupling between H-1′′ and H-3′′′ further supported their vicinal arrangement. The two hydroxyl singlets (δH 7.03,

6.58) both showed HMBC correlations into resonances at C-4′′′ (δC 97.2) and C-5′′′ (δC 80.5) and it was thus concluded that they are a vicinal diol at OH-4′′′ and OH-5′′′, respectively. OH-5′′′ also showed a HMBC correlation to a carbon resonance at δC 30.9 (C-1′′′′), assigned to an aliphatic methylene from HSQC correlations at δH 2.21 (J = 13.1, 5.1 Hz)/2.54 (J = 13.1, 10.8

Hz). The carbon resonance at δC 178.1 was assigned to C-6′′′ since HMBC correlations were observed between it and H-1′′′′ and OH-5′′′. The significant downfield chemical shift of C-6′′′ indicated that it is likely to be an oxygenated olefinic carbon β to a carbonyl. HMBC correlations were observed from the acetyl methyl protons at δH 2.12 (MeCO-1′′′) and H-3′′′ to an olefinic quaternary carbon at δC 111.9 (C-1′′′) and linking C-1′′′ and C-6′′′ allowed a cyclohexanone substituted at C-1′′′ by the acetyl group to be defined. COSY correlations from

H2-1′′′′ to an oxygenated methine at δH 4.78 (dd, J = 10.8, 5.1 Hz) (δC 92.9) dictated its position as C-2′′′′. HMBC correlations from the final two unassigned aliphatic methyl singlets at δH 1.13 and δH 1.16 to C-2′′′′ and an unassigned resonance at δC 69.1 revealed that they are a gem- dimethyl directly bonded to an oxygenated non-protonated carbon (C-3′′′′). The presence of only one deshielded hydrogen-bonded hydroxy resonance (OH-6) in the 1H NMR spectrum of 1 suggested that C-6′′′ is an ether and thus a linkage between either the oxygenated methine at C-2′′′′ or the oxygenated quaternary carbon C-3′′′′ and C-6′′′ could be added, generating either a furan or pyran ring. The observed 13C chemical shifts closely matched those previously reported in the literature for related furans but not pyrans, indicating a furan ring was present

5 in 1. Finally, the valence of C-4′′′ (δC 97.2) was not fulfilled, with bonds only to C-5′′′, C-3′′′ and OH-4′′′ assigned. Its deshielded chemical shift also indicated that it is a ketal and it was concluded that an ether linkage was present between C-4 and C-4′′′, although there were no HMBC correlations to support this. In the absence of relevant HMBC data, ROESY correlations between H-2′ and H-1′′′′/H-5′′′′/OH-4′′′ confirmed this assignment (Figure 2b). The relative configuration of the five stereogenic centres in 1 were determined by further analysis of ROESY data. ROESY correlations between OH-4′′′, OH-5′′′ and H-3′′′ indicated that each were β positioned. A correlation between OH-5′′′ and H-2′′′′ established the position of H-2′′′′ as β. Finally, the 0.8 Hz coupling between H-1′′ and H-3′′′ indicated that these are at 90 degrees to each other and thus H-1′′ was assigned as α. This was confirmed by a ROESY correlation observed between OH-4′′′ and H-2′′. After the relative configuration of 1 was confirmed, its absolute configuration was determined by comparison of experimental and predicted ECD spectra of the two possible enantiomers of 1, calculated using the time-dependent density

80

functional theory (TDDFT) method.13 The elucidation of the absolute configuration of 1 proved challenging, with none of the calculated ECD spectra showing a perfect match (Figure 3). Spectra calculated using the CAM-B3LYP/def2-SVP//B3LYP/def2-SVP (blue dotted line) and M062X/def2-SVP//B3LYP/def2-SVP (red dotted line) functional/basis set combinations successfully reproduced the positive cotton effects at 240 nm, 280 nm and 320 nm. However, both showed a small negative cotton effect at ~300 nm that was not observed in the experimental data. The experimental data appears to plateau at ~295 nm, indicating that the molecule may actually display a small negative cotton effect at this wavelength. Nonetheless, in both cases, the calculated ECD spectrum of (1′′R,3′′′S,4′′′S,5′′′S,2′′′′R)-1 showed better matches with the experimental data, indicating this to be the most likely absolute configuration of 1. Most of the currently known chiral AtA are racemic, with evidence coming from x-ray crystallography,14 optical rotation data5, 11 and Mosher’s acid analysis.5 To the best of our knowledge, only three AtA are reported to be optically active15, 16 although the absolute configuration of only one of these has been elucidated.16

(a)

(b)

Figure 2. (a) Key HMBC () and COSY (bold bonds) correlations of 1. (b) Key ROESY correlations in the energy-minimized conformation of 1.

81

Figure 3. Comparison of the experimental and calculated ECD spectra of (1′′R,3′′′S,4′′′S,5′′′S,2′′′′R)-1. Structures were optimised at the B3LYP/def2-SVP level. The functional/basis set combinations shown in the key are those used for the calculation of electronic transition and rational strength.

82

Table 1. NMR Spectroscopic Data for Acrotrione (1)

Acrotrione (1)

a b c position δc , type δH (J in Hz) HMBC 1 108.8, C - - 2 158.3, C - - 3 114.3, C - - 4 155.5, C - - 5 109.8, C - - 6 160.8, C - - 1′ 22.1, CH2 3.11, dd (15.0, 5.8); 2, 2′, 3, 3′, 4 3.16, m 2′ 123.2, CH 5.02, t (6.6) 3, 4′, 5′ 3′ 130.4, C - - 4′ 25.8, CH3 1.57, s 2′, 3′, 5′ 5′ 17.8, CH3 1.67, s 2′, 3′, 4′ 1′′ 27.3, CH 3.78, ddd (11.8, 3.6, 0.8) 2′′, 2′′′, 3′′′, 4, 4′′′, 5, 6 2′′ 39.8, CH2 2.08, m - 1.69, m 3′′′ 3′′ 25.1, CH 1.79, m - 4′′ 24.8, CH 0.90, d (6.5) 2′′, 3′′, 5′′ 5′′ 20.7, CH 0.99, d (6.5) 2′′, 3′′, 4′′ 1′′′ 111.9, C - - 2′′′ 193.2, C - - 3′′′ 43.8, CH 3.19, d (0.8) 1′′, 2′′, 2′′′, 4′′′, 5 4′′′ 97.2, C - - 5′′′ 80.5, C - - 6′′′ 178.1, C - - 1′′′′ 30.9, CH2 2.21, dd (13.1, 5.1) 5′′′, 6′′′ 2.54, dd (13.1, 10.8) 2′′′′, 3′′′′ 2′′′′ 92.9, CH 4.78, dd (10.8, 5.1) - 3′′′′ 69.1, C - - 4′′′′ 25.4, CH3 1.13, s 2′′′′, 3′′′′, 5′′′′ 5′′′′ 25.5, CH3 1.16, s 2′′′′, 3′′′′, 4′′′′ MeO-2 62.5, CH3 3.63, s 2 MeCO-1 31.1, CH3 2.61, s 1, MeCO-1 MeCO-1 203.8, C - - MeCO-1′′′ 31.6, CH3 2.12, s 1′′′, MeCO-1′′′ MeCO-1′′′ 196.7, C - - OH-3′′′′ OH n.o. - OH-4′′′ OH 7.02, s 2′′′, 3′′′, 4′′′, 5′′′ OH-5′′′ OH 6.57, s 1′′′′, 4′′′, 5′′′, 6′′′ OH-6 OH 13.69, s 1, 6 a125 Mhz. b800 Mhz. cHMBC correlations are from proton(s) stated to the indicated carbon. n.o. = not observed.

83 Acrotrione (1) contains a highly unusual furo[2,3-c]xanthene structure that has not been reported previously. A plausible biosynthesis could start from the known AtA dimer acrovestone14 (Figure 4) through epoxidation at C-4′′′/C-5′′′ and subsequent nucleophilic attack of C-4′′′ by OH-4, resulting in the formation of an ether linkage between C-4 and C-4′′′. Keto- tautomerism at C-2′′′ results in a ketone moiety at C-2′′′. Finally, epoxidation of the double bond in the isoprene attached at C-5′′′ and nucleophilic attack of the epoxide carbon C-2′′′′ leads to 1. The biogenetic precursor, acrovestone, is reported to be racemic;17 thus, the biosynthetic pathway leading to the formation of acrovestone may be stereoselective with subsequent racemisation within the plant. The biosynthesis of 1 may therefore take place before racemisation of acrovestone has occurred.

Figure 4. Proposed biogenetic origin of 1 from the known compound acrovestone.

All of the A. pubescens natural products isolated were tested for anti-plasmodial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum. Cytotoxicity was also determined using the HEK293 mammalian cell line (Table 3) to evaluate selectivity for the parasite (Table 2). 1 and 3 were moderately active against 3D7 P. falciparum, with IC50 values of 2.7 and 1.8 µM, respectively. Similar activity was also observed against the

Dd2 strain, and the Dd2/3D7 IC50 ratio of 1.7 for both compounds suggests equal sensitivity by both the drug resistant and wild-type parasite strains. Some cytotoxicity was also observed for both compounds, with 81% and 79% growth inhibition of HEK293 cells at 40 µM for 1 and 3, respectively. Conversely, 2 showed very weak activity, reaching only 38% and 20% growth inhibition at 40 µM against P. falciparum 3D7 and Dd2 strains, respectively. Weak cytotoxicity was also observed against HEK293 cells, showing only 7% growth inhibition at 40 µM. 84

Structurally, 2 differs from 3 only by the presence of an aldehyde at C-5, yet 3 is significantly more bioactive. This may be explained by the differences in intramolecular hydrogen bonding between the two natural products (Figure 5). Intramolecular hydrogen bonding can influence bioactivity by affecting electronic distribution and capacity to undergo intermolecular bonding.18 In the case of 2, the C-6 hydroxyl will undergo hydrogen bonding with the C-1 acetyl, while the C-4 hydroxyl will hydrogen bond with the C-5 aldehyde. Conversely, due to the presence of only a hydrogen atom at C-5, the C-4 hydroxyl in 3 will be free of intramolecular hydrogen bonds and thus is able to undergo intermolecular hydrogen bonding and interact with other molecules and enzymes, potentially leading to an increase in bioactivity.

Figure 5. Intramolecular hydrogen bonding in 2 and 3. Intramolecular hydrogen bonds are represented by dashed bonds.

Table 2. Anti-plasmodial Activity and Cytotoxicity of Natural Products Isolated from the Roots of A. pubescens

IC50 ± SD (µM) (n = 2) selectivity Dd2/3D7 Compound 3D7 Dd2 HEK293 index (SI) ratio 1 2.741 ± 0.657 4.705 ± 0.740 80.7% ± 2.6a n.d. 1.7

2 37.5% ± 22.7a 19.8% ± 11.1a 7.4% ± 4.0a n.d. n.d.

3 1.783 ± 0.310 3.112 ± 0.598 79.1% ± 3.8a n.d. 1.7

artesunateb 0.0021 ± 0.0016 0.0033 ± 0.0022 62.5% ± 16.5a n.d. 1.6

chloroquineb 0.0139 ± 0.0015 0.1360 ± 0.0255 53.2% ± 26.4a n.d. 9.8

dihydroartemisininb 0.0009 ± 0.0002 0.0016 ± 0.0003 41.1% ± 11.5a n.d. 1.8

puromycinb 0.0702 ± 0.0108 0.0747 ± 0.0188 0.6940 ± 0.183 9.9 1.1

pyrimethamineb 0.0077 ± 0.0017 n.d. 52.0% ± 10.2a n.d. n.d.

pyronaridineb 0.0082 ± 0.0020 0.0110 ± 0.0010 1.990 ± 0.263 246.1 1.3 apercentage growth inhibition at 40 µM. breference compound. Selectivity index = HEK293/3D7. n.d. = not determined.

85 Experimental

General Experimental Procedures.

Optical rotations were logged on a JASCO P-1020 polarimeter and [α]D values are reported in 10−1 deg cm2 g−1. UV spectra were recorded on a Shimadzu UV-1800 UV-Vis spectrophotometer. ECD spectra were recorded on a JASCO J-715 spectropolarimeter. IR spectra were recorded using a ThermoFisher Scientific Nicolet iS5/iD5 ATR spectrometer. NMR spectra were recorded at 25 °C on a Bruker Avance III 500 MHz spectrometer (BBFO Smartprobe, 5mm 31P-109Ag) and a Bruker Avance III HDX 800 MHz with a triple (TCl) resonance 5 mm cryoprobe. NMR spectra were referenced to the solvent peak for (CD3)2SO at

δH 2.50 and δC 39.52. High-resolution mass measurements were acquired using mobile phase 100% ACN on an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS with a 1200 Series autosampler and 1290 Infinity HPLC. Oven used for drying plant material was a Contherm

Thermotec 2000. C18 silica gel used was Alltech Sample Prep C18 35-75 µm, 150 Å. HPLC columns were Betasil 5 µm, 100 Å, 21.2 mm x 150 mm and YMC-pack diol, 5 µm, 120-NP. For HPLC, a Merck Hitachi L7100 pump in tandem with a Merck Hitachi L7455 PDA detector and a Merck Hitachi L7250 autosampler were used. A Gilson 215 liquid handler was used to collect fractions. All organic solvents used were Scharlau HPLC grade and H2O was Millipore Milli-Q PF filtered. Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar. Parasite strains 3D7 and Dd2 were obtained from BEI Resources. O+ erythrocytes were obtained from the Australian Red Cross Blood Service. CellCarrier poly-D-lysine coated imaging plates were from PerkinElmer. 4′,6-Diamidino-2-phenylindole (DAPI) stain were from Invitrogen. Triton-X, saponin, chloroquine, artesunate, puromycin, pyronaridine, dihydroartemisinin and pyrimethamine were all from Sigma Aldrich. HEK293 cells were purchased from the American Tissue Culture Collection. The 384-well Falcon sterile tissue culture treated plates were from BD.

Plant Material.

A. pubescens leaf material was purchased from Burringbar Rainforest Nursery in August 2017. A voucher specimen ACRUT007 is housed within the School of Environment and Science, Gold Coast Campus, Griffith University.

Extraction and Isolation.

Oven-dried (50 °C, 48 hours) ground A. pubescens roots (20 g) were exhaustively extracted in

MeOH (1 L), yielding a brown gum (1.5 g). The extract (1 g) was adsorbed onto C18 silica gel (1 g) and the extract-impregnated gel was loaded into a HPLC pre-column cartridge (10 mm x 20

86 mm) and connected in series to a C18-bonded silica HPLC column (Betasil 5 µm, 100 Å, 21.2 mm x 150 mm). The column was then eluted with a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. The column was then eluted with MeOH for a further 10 min. Fractions were collected every min and UV-DAD spectroscopic analysis was conducted in tandem with the separation. Fractions 53-63 (250 mg) were recombined and further purified by diol HPLC (YMC-pack diol, 5 µm, 120-NP, 21.2 mm x

150 mm) using a gradient from hexane (100%) to CH2Cl2 (100%) over 50 minutes, then to

CH2Cl2 (90%)/MeOH (10%) from minutes 51-60. Fraction 10 contained acronyculatin A (2) (2.3 mg, 2.3x10-5% dry wt), fraction 34 contained acronylin (3) (0.4 mg, 4.0x10-6% dry wt) and fraction 60 contained acrotrione (1) (1.7 mg, 1.7x10-6% dry wt).

+ + Acrotrione (1) yellow amorphous solid; HRESIMS m/z [M + H] 587.2844 (calcd for C32H43O10 , 25 587.2856); [훼]D +84 (c 0.03, MeOH); UV (MeOH) 휆max (log ε) 335 (5.93), 290 (6.43), 269

(6.55), 228 (6.51) nm; ECD (c 0.001) 휆max MeOH (Δε) 293 (+2.7), 257 (-0.1) 240 (+0.8) nm; IR -1 1 13 (film) νmax 3351, 1650, 1366, 1202, 1019 cm ; H and C NMR data, Table 1.

Compounds and Screening Methods.

Compounds were dissolved in 100% DMSO to obtain 10 mM stock solutions. Chloroquine, artesunate, puromycin, pyronaridine, dihydroartemisinin and pyrimethamine were used as reference compounds. Stock solutions were prepared at 2.5 mM (dihydroartemisinin, artesunate) or 10 mM (all other reference compounds) in 100% DMSO, except chloroquine and pyronaridine which were dissolved in water. Puromycin (5 µM) and 0.4% DMSO were used as positive and negative controls, respectively. Experimental compounds were tested in 16- concentrations dose-response using three concentrations per log dose against 3D7 (drug- sensitive) and Dd2 (drug-resistant) P. falciparum parasite strains and against HEK293 cells for cytotoxicity assessment. Final assay concentration ranges of 40 μM – 0.4 nM were used for experimental compounds. Reference anti-malarial compounds/drugs were tested in 21- concentrations dose-response range of 40 µM – 0.01 nM (10 µM – 0.003 nM for dihydroartemisinin and artesunate).

In Vitro Anti-plasmodial Image-Based Assay.

P. falciparum parasites (3D7 and Dd2 strains) were grown in RPMI 1640 supplemented with 25 mM HEPES, 5% AB human male serum, 2.5 mg/mL Albumax II, and 0.37 mM hypoxanthine. Parasites were subjected to two rounds of sorbitol synchronization before undergoing compound treatment. Ring stage parasites were exposed to the experimental compounds in

87

384-wells imaging CellCarrier microplates (PerkinElmer) as previously described.19 Plates were incubated for 72 h at 37 °C, 90% N2, 5% CO2, 5% O2, then the parasites were stained with 2-(4- amidinophenyl)-1H-indole-6-carboxamidine (DAPI), and imaged using an Opera QEHS micro- plate confocal imaging system (PerkinElmer). Images were analysed as previously described.19

In Vitro Cytotoxicity Assay.

Human Embryonic Kidney cells (HEK293) were maintained in DMEM medium supplemented with 10% FBS. HEK293 cells were exposed to the compounds in TC-treated 384-wells plates

(Greiner) for 72 h at 37 °C, 5% CO2, then the media was removed from the wells and replaced with an equal volume of 44 µM resazurin. After an additional 5-6 hours incubation at standard conditions, the total fluorescence (excitation/emission: 530 nm/595 nm) was measured using the Ensight plate reader (PerkinElmer). The positive control, puromycin, had an IC50 value of 0.69 ± 0.18 μM toward the HEK293 cell line.

Biological Data Analysis.

Raw data was normalized using the in-plate positive and negative controls to obtain normalized % inhibition data, which was then used to calculate IC50 values, through a 4 parameter logistic curve fitting in Prism (GraphPad). The experiments were carried out in two biological replicates, each consisting of two technical repeats.

Computational Methods.

The lowest energy conformers of 1 were generated using Schrödinger MacroModel 2016 by following the procedure reported by Willoughby et al.20 Initial geometry optimisations were performed on each of the 19 generated conformers using first-principles calculations based on density functional theory (DFT) at the B3LYP/6-31G(d) level using Grimme’s empirical dispersion corrections (D3).21 The conformers were then re-optimised using the B3LYP/def2SVP functional/basis set combination using empirical dispersion corrections (D3) and the Polarizable Continuum solvent Model (PCM).22 Free energy calculations were performed using the same functional/basis set combination. Electronic transition and rational strength were calculated using time-dependent DFT (TDDFT) using the CAM-B3LYP/def2-SVP and M062X/def2-SVP functional/basis set combinations with consideration of the solvent effect using the PCM. Boltzmann-weighted UV and ECD spectra were calculated using the freely available software SpecDis23 using a sigma/gamma value of 0.3 eV. The experimental ECD spectrum was processed using SDAR.24 All DFT calculations were carried out using the Gaussian 16 suite of programs.25 All TDDFT protocols were based on the method described by Pescitelli and Bruhn.26

88 Associated Content

Supporting Information Electronic supplementary information (ESI) is available free of charge on the ACS Publications website at DOI: 1D and 2D NMR spectra for acrotrione (1)

Author Information

Corresponding Author

*Tel: +61 7 55529187. Fax: +61 7 55529047. E-mail: [email protected].

ORCID Anthony R. Carroll 0000-0001-7695-8301

Notes

The authors declare no competing financial interest.

Acknowledgements

This work was funded by an Australian Postgraduate Award (APA) provided by the Australian Commonwealth Government. The authors acknowledge the support of the Griffith University eResearch Services Team and the use of the High Performance Computing Cluster “Gowonda” for TDDFT calculations. We thank W. Loa-Kum-Cheung, J. Carrington and R. Stewart for technical assistance. We thank the Australian Red Cross Blood Service for the provision of human blood. We thank L. Weber for photograph provision.

References

(1) Hartley, T. G. In Flora of Australia Volume 26—Meliaceae, Rutaceae, Zygophyllaceae; Wilson, A., Ed.; ABRS/CSIRO: Melbourne, Australia, 2013; pp 104-118. (2) Hnawia, E.; Hassani, L.; Deharo, E.; Maurel, S.; Waikedre, J.; Cabalion, P.; Bourdy, G.; Valentin, A.; Jullian, V.; Fogliani, B. Pharm. Biol. 2011, 49, 369-376. (3) Epifano, F.; Fiorito, S.; Genovese, S. Phytochem. 2013, 95, 12-18. (4) Lahey, F. N.; Thomas, W. C. Aust. J. Chem. 1949, 2, 423-426. (5) Miyake, K.; Suzuki, A.; Morita, C.; Goto, M.; Newman, D. J.; O’Keefe, B. R.; Morris-Natschke, S. L.; Lee, K. H.; Nakagawa-Goto, K. J. Nat. Prod. 2016, 79, 2883-2889. (6) Brophy, J. J.; Goldsack, R. J.; Forster, P. I. JEOR 2004, 16, 597-607. (7) Cui, B.; Chai, H.; Dong, Y.; Horgen, F. D.; Hansen, B.; Madulid, D. A.; Soejarto, D. D.; Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J. M. Phytochem. 1999, 52, 95-98. (8) Prager, R. H.; Thredgold, H. M. Aust. J. Chem. 1966, 19, 451-454. (9) Robertson, L. P.; Duffy, S.; Wang, Y.; Wang, D.; Avery, V. M.; Carroll, A. R. J. Nat. Prod. 2017, 80, 3211-3217. (10) Bissoue, A. N.; Muyard, F.; Regnier, A.; Bevalot, F.; Vaquette, J.; Hartley, T. G.; Waterman, P. G. Biochem. Syst. Ecol. 1996, 24, 805.

89 (11) Kouloura, E.; Halabalaki, M.; Lallemand, M. C.; Nam, S.; Jove, R.; Litaudon, M.; Awang, K.; Hadi, H. A.; Skaltsounis, A. L. J. Nat. Prod. 2012, 75, 1270-1276. (12) Kouloura, E.; Skaltsounis, A. L.; Michel, S.; Halabalaki, M. J. Mass Spectrom. 2015, 50, 495- 512. (13) Bringmann, G.; Bruhn, T.; Maksimenka, K.; Hemberger, Y. Eur. J. Org. Chem. 2009, 2009, 2717-2727. (14) Wu, T.-S.; Wang, M.-L.; Jong, T.-T.; McPhail, A. T.; McPhail, D. R.; Lee, K.-H. J. Nat. Prod. 1989, 52, 1284-1289. (15) Pathmasiri, W.; El‐Seedi, H. R.; Han, X.; Janson, J. C.; Huss, U.; Bohlin, L. Chem. Biodivers. 2005, 2, 463-469. (16) Su, C.-R.; Kuo, P.-C.; Wang, M.-L.; Liou, M.-J.; Damu, A. G.; Wu, T.-S. J. Nat. Prod. 2003, 66, 990-993. (17) Oyama, M.; Bastow, K. F.; Tachibana, Y.; Shirataki, Y.; Yamaguchi, S.; Cragg, G. M.; Wu, T.- S.; Lee, K.-H. Chin. Pharm. J. 2003, 55, 239-245. (18) Giordanetto, F.; Tyrchan, C.; Ulander, J. ACS Med. Chem. Lett. 2017, 8, 139-142. (19) Duffy, S.; Avery, V. M. Am. J. Trop. Med. Hyg. 2012, 86, 84-92. (20) Willoughby, P. H.; Jansma, M. J.; Hoye, T. R. Nat. Protoc. 2014, 9, 643-660. (21) Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. J. Chem. Phys. 2010, 132, 154104. (22) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. Rev. 2005, 105, 2999-. (23) Bruhn, T.; Schaumlöffel, Y.; Hemberger, Y. SpecDis, Version 1.64; University of Wuerzburg, Germany: 2015. (24) Weeratunga, S.; Hu, N.-J.; Simon, A.; Hofmann, A. BMC Bioinf. 2012, 13, 201. (25) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery, J. A. J.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 16, Revision A.03; Gaussian, Inc.: Wallingford, CT: 2016. (26) Pescitelli, G.; Bruhn, T. Chirality 2016, 28, 466-474.

90

Chapter 6 – Quinoline Alkaloids from the Australian Tree Pitaviaster haplophyllus

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PAPER

This chapter is a co-authored paper in preparation for submission to Tetrahedron Letters. The bibliographic details of the co-authored paper, including all authors, are:

Robertson, L. P., Carroll, A. R. Quinoline alkaloids from the Australian tree Pitaviaster haplophyllus. Tetrahedron Letters. In preparation for submission.

L. P. R. carried out purifications, structure elucidations, gathered physical data and wrote the manuscript. A. R. C. oversaw all aspects of the project.

X Luke P. Robertson

(Signed) (Date) 20/11/2018

X Supervisor: Anthony R. Carroll

(Signed) (Date) 20/11/2018

91 Quinoline Alkaloids from the Australian Tree Pitaviaster haplophyllus

Luke P. Robertsona,b and Anthony R. Carroll*,a,b aEnvironmental Futures Research Institute, Griffith University, Southport 4222, Gold Coast, Australia bGriffith Institute for Drug Discovery, Griffith University, Nathan 4111, Brisbane, Australia

92 One new furoquinoline alkaloid, leptanoine D (1) and nine known alkaloids (2-10) were isolated from the Australian tree Pitaviaster haplophyllus. Leptanoine D (1) was comprehensively structurally elucidated based on 2D NMR, (+)-HRESIMS and electronic circular dichroism data. Based on this analysis, the structures of the known furoquinoline alkaloids leptanoine A (11) and B (12) have been revised. The alkaloids isolated from P. haplophyllus support currently proposed taxonomic relationships between Pitaviaster with Euodia and, more distantly, Acronychia and Melicope.

93 Introduction

Pitaviaster haplophyllus (F. Muell.) (Rutaceae), the sole member of the Pitaviaster genus, is a medium sized shrub or tree growing up to 13 metres in height. It is found predominantly in the tropical rainforests of North Queensland, Australia, although a small number of specimens occur on Fraser Island, a large sand island off the coast of Eastern Queensland.1 Taxonomically, it has been proposed that the genus is most closely related to Acronychia, although recent genetic analysis has provided evidence that it may be more closely related to Euodia and Brombya.2 Chemical studies of P. haplophyllus have been limited, with the isolation of only two furoquinoline and one acridone alkaloid reported more than 50 years ago under the synonym Acronychia haplophylla.3, 4 With the exception of volatile leaf oil studies by Brophy and co- workers, which revealed mostly sesquiterpenic oils,1 no other chemical studies have been conducted on the species. As part of our ongoing efforts to identify new alkaloids,5 and to further investigate the chemotaxonomic relationships between Australian Rutaceae species,6 the bark extracts of a library of Australian endemic Rutaceae were subject to LC/MS analysis. The bark of P. haplophyllus contained the highest number of alkaloids, showing over 70 even m/z ions, indicating it to be a potential source of new natural products. Described herein is the isolation and structure elucidation of one new and nine known alkaloids.

Fig. 1. Leptanoine D (1), a new alkaloid isolated from the leaves of P. haplophyllus

Results and discussion

Extraction of P. haplophyllus material with MeOH followed by treatment with a strongly acidic cation exchange (SCX) resin and fractionation using C18 HPLC led to the isolation of one new (1) and nine known (2-10) alkaloids. Leptanoine D (1) was isolated as a yellow amorphous solid.7 A protonated molecule in the (+)-HRESIMS at m/z 300.1250 was used to assign the molecular

+ 13 formula C17H18NO4 to 1. The C NMR spectrum contained 17 resonances and these were attributed to six non-protonated carbons, eight methines, one methylene and two methyls. The 1H and HSQC NMR spectrum of 1 (Table 1) showed resonances associated with one ortho- coupled aromatic doublet (δH 8.16, J = 9.1 Hz), one meta- coupled doublet (δH 7.37, J = 2.5 Hz) and one ortho-/meta- coupled doublet of doublets (δH 7.21, J = 9.1, 2.5 Hz), suggesting the presence of a 1,2,4 trisubstituted aromatic ring. The spectra also contained resonances that could be attributed to a 4,5 disubstituted furan at δH 7.99 (doublet, J = 2.7 Hz) (δC 143.6) and 94

δH 7.45 (doublet, J = 2.7 Hz) (δC 105.6) and an aromatic methoxy at δH 4.43. These distinctive signals indicated that 1 was a C-7 substituted furoquinoline alkaloid.8, 9 The large ortho- and meta- couplings exhibited by H-5/H-6/H-8 (J = 9.1, 2.5 Hz) indicated that C-7 was oxygenated,10 and this was confirmed by HMBC correlations from H-5/H-8 to a deshielded resonance at δC 157.8. Other unassigned resonances in the 1H NMR spectrum were associated with two olefinic protons at δH 6.81 (d, J = 12.0 Hz) and δH 5.38 (dd, J = 12.0, 8.2 Hz), one oxygenated methylene at δH 3.34 (dd, J = 10.5, 6.5 Hz)/3.32 (dd, J = 10.5, 6.5 Hz), one methine at δH 2.38 (sept, J = 6.5

Hz) and one methyl at δH 1.73 (d, J = 6.7 Hz). Assessment of the molecular formula of 1 indicated that the C-7 oxygen formed an ether linkage to a hydroxylated isoprene. The olefinic proton at δH 6.81 was determined to be at H-1′ from HMBC correlations to the oxygenated aromatic carbon at C-7. Thereafter, COSY correlations between H-1′/H-2′/H-3′/H-4′ and H3-5′ were used to assign the structure of C-1′ through C-5′. The deshielded chemical shift of C-4′ (δC 66.3) indicated that it was a methylene hydroxy. This substructure was supported by HMBC correlations from H-1′ to C-3′ and from H3-5′ to C-2′/C-3′/C-4′. The configuration of H-1′/H-2′ was predicted to be (E) from the coupling constant observed (J = 12.0 Hz), which is of the intensity expected of a trans- double bond attached to an oxygen atom.10 Previously published and structurally similar compounds leptanoine A (11) and B (12) show a similar coupling at H- 1′/H-2′ (J = 12.2 Hz), however the authors report the configuration as (Z).9 In light of this, we sought additional evidence to confirm the configuration of H-1′/H-2′. Analysis of ROESY NMR data revealed a ROESY correlation between H-1′ and H-3′, while no correlation was observed between H-1′ and H-2′, indicating H-1′/H-2′ must be (E). Consequently, we also propose a structural revision to the published compounds leptanoine A (11) and B (12) and conclude that H-1′/H-2′ in both molecules is (E) and not (Z) as reported. After the elucidation of its 2D structure, the absolute configuration of 1 was determined by comparison of experimental and predicted ECD spectra, calculated using time-dependent density functional theory (TDDFT) at the B3LYP/6-31G(d)//B3LYP/6-31G(d) level (Figure 3). Although neither calculated ECD spectrum was a perfect match, the calculated spectrum of (3′S)-1 showed a much closer match with the experimental data, reproducing the negative cotton effects at ~240 and ~285 nm while also showing the positive trends at ~258 and ~340 nm. From this information, it was concluded that the most likely absolute configuration of 1 is 3′S. Other alkaloids isolated from P. haplophyllus were the acridone arborinine (2), the pyranoquinoline tabouensinium (3) and the furoquinolines melineurine (4), N-methylplatydesminium (5), 7-hydroxydictamnine (6), kokusaginine (7), evolitrine (8), pteleine (9) and acrophylline (10). The structures of 2-10 were determined by 2D NMR and confirmed by comparison of NMR spectroscopic data to literature values.8, 11-15

95

Fig. 2. Key HMBC () and COSY (bold bonds) correlations in 1.

Table 1. NMR data for leptanoine D (1) in DMSO-d6

Leptanoine D (1)

a b Position δc δH (mult, J in Hz, int.) HMBC

1 N - - 2 143.6 7.99 (d, 2.7, 1H) 3, 3a, 9a 3 105.6 7.45 (d, 2.7, 1H) 2, 3a, 9a 3a 102.3 - - 4 156.5 - - 4a 113.7 - - 5 124.0 8.16 (d, 9.1, 1H) 4, 7, 8a 6 115.9 7.21 (dd, 9.1, 2.5, 1H) 4a, 8 7 157.8 - - 8 109.3 7.37 (d, 2.5, 1H) 4a, 6, 8, 8a 8a 146.5 - - 9a 164.2 - - 1′ 140.6 6.81 (d, 12.0, 1H) 2′, 3′, 7 2′ 118.1 5.38 (dd, 12.0, 8.2, 1H) 1′, 4′, 5′ 3′ 34.8 2.38 (sept, 6.5, 1H) 1′, 2′, 4′, 5′ 4′ 66.3 3.32 (dd, 10.5, 6.5, 1H) 1′, 2′, 3′, 5′ 3.34 (dd, 10.5, 6.5, 1H) 1′, 2′, 3′, 5′ 4′-OH - n.o. - 5′ 17.3 1.04 (d, 6.7, 3H) 2′, 3′, 4′ 4-OCH3 59.5 4.43 (s, 3H) 4 a 125 Mhz. b 500 Mhz. n.o. = not observed

Fig. 3. Comparison of the experimental ECD spectrum of 1 with those calculated for (3′S)-1 and (3′R)-1

96

Fig. 4. Alkaloids isolated from P. haplophyllus.

Fig. 5. Revised structures of leptanoine A (11) and B (12).

The alkaloids isolated from P. haplophyllus (1-10) support current data about the taxonomic relationships between Pitaviaster and other Rutaceous genera. Recent molecular phylogenetic analysis by Bayly et al. concluded that the closest relatives of Pitaviaster are Euodia and Brombya.2 Molecular phylogenies of Acronychia, Melicope, Euodia and some other Rutaceous genera by Appelhans et al. also placed Pitaviaster within the Euodia clade.16 The isolation of the relatively uncommon O-prenylated furoquinolines 1 and 4 from P. haplophyllus supports this relationship. Euodia is one of the only other producers of O-prenylated furoquinoline alkaloids, which have been isolated from species including E. lepta9 and E. roxburghiana.17 A limited number of O-prenylated furoquinolines have also been isolated from Melicope species, many of which were once classified as Euodia.18-21 The pyranoquinoline tabouensinium (3) has previously only been isolated from the West African tree Araliopsis tabouensis12 and Tetradium ruticarpum (under the synonym Euodia rutaecarpa).22 Acridone alkaloids are widespread within the Rutaceae but are particularly common amongst Acronychia, Euodia and Melicope species21 and thus the isolation of 1 is also supportive of a close relationship between these genera. This relationship is also indicated by molecular data.2, 16 To the best of our knowledge, only two N-prenylated furoquinolines are known: acrophylline (10) and acrophyllidine (not isolated). Both are exclusively found in P. haplophyllus.3, 4 The other furoquinolines reported (5-9) are relatively ubiquitous throughout the Rutaceae.21

97 References

[1] Brophy JJ, Goldsack RJ, Forster PI. JEOR 2002; 14: 130-131. [2] Bayly MJ, Holmes GD, Forster PI, Cantrill DJ, Ladiges PY. PLoS One 2013; 8: e72493. [3] Lahey FN, McCamish M. Tetrahedron Lett. 1968; 9: 1525-1527. [4] Lahey FN, McCamish M, McEwan T. Aust. J. Chem. 1969; 22: 447-453. [5] Robertson LP, Duffy S, Wang Y, Wang D, Avery VM, Carroll AR. J. Nat. Prod. 2017; 80: 3211- 3217. [6] Robertson LP, Hall CR, Forster PI, Carroll AR. Phytochemistry 2018; 152: 71-81. 25 [7] Leptanoine D (1) yellow amorphous solid; [훼]D +13 (c 0.001, MeOH); UV (MeOH) 휆max (log

ε) 317 (3.30), 247 (3.84) nm; ECD (c 0.0003) 휆max MeOH (Δε) 287 (-2.6), 259 (-1.2) 223 (-2.7) -1 1 13 nm; IR (film) νmax 3383, 2936, 1621, 1454, 1371, 1207 cm ; H and C NMR data, Table 1; + + HRESIMS m/z [M + H] 300.1250 (calcd for C17H18NO4 , 300.1230). [8] Pusset J, Lopez JL, Pais M, Al Neirabeyeh M, Veillon J-M. Planta Med. 1991; 57: 153-155. [9] Sichaem J, Jirasirichote A, Sapasuntikul K, Khumkratok S, Sawasdee P, Do TML, Tip-Pyang S. Fitoterapia 2014; 92: 270-273. [10] Pretsch E, Bühlmann P, Badertscher M.Structure Determination of Organic Compounds; Springer, Berlin, 2009. pp 166, 180. [11] Bergenthal D, Mester I, Rozsa Z, Reisch J. Phytochemistry 1979; 18: 161-163. [12] Wabo HK, Tane P, Connolly JD, Okunji CC, Schuster BM, Iwu MM. Nat. Prod. Res. 2005; 19: 591-595. [13] Tillequin F, Baudouin G, Koch M. J. Nat. Prod. 1983; 46: 132-134. [14] Fish F, Meshal IA, Waterman PG. Planta Med. 1976; 29: 310-317. [15] Robertson AV. Aust. J. Chem. 1963; 16: 451-458. [16] Appelhans MS, Wen J, Wagner WL. Mol. Phylogenetics Evol. 2014; 79: 54-68. [17] McCormick JL, McKee TC, Cardellina JH, Boyd MR. J. Nat. Prod. 1996; 59: 469-471. [18] Tillequin F, Baudouin G, Ternoir M, Koch M, Pusset J, Sevenet T. J. Nat. Prod. 1982; 45: 486-488. [19] Komala I, Rahmani M, Sukari MA, Mohd Ismail HB, Cheng Lian GE, Rahmat A. Nat. Prod. Res. 2006; 20: 355-360. [20] Gell RJ, Hughes GK, Ritchie E. Aust. J. Chem. 1955; 8: 114-120. [21] Waterman PG. Biochem. Syst. Ecol. 1975; 3: 149-180. [22] Xia X, Luo J-G, Liu R-H, Yang M-H, Kong L-Y. Nat. Prod. Res. 2016; 30: 2154-2159.

98

Chapter 7 – Alkaloid Diversity in the Leaves of Australian Flindersia (Rutaceae) Species Driven by Adaptation to Aridity

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PAPER

This chapter has been accepted as a co-authored paper to the journal Phytochemistry. The bibliographic details of the co-authored paper, including all authors, are:

Robertson, L. P., Hall, C. R., Forster, P. I., Carroll, A. R. Alkaloid diversity in the leaves of Australian Flindersia (Rutaceae) driven by adaptation to aridity. Phytochemistry, 2018, 152, 71- 81.

L. P. R. carried out all laboratory work and wrote the manuscript. C. R. H. performed phylogenetic analysis and contributed to ecological and statistical analysis. P. I. F. collected the original samples in the Griffith library and contributed to botanical and ecological analysis. L. P. R. and A. R. C. designed the study. A. R. C. oversaw all aspects of the project.

X Luke P. Robertson

(Signed) (Date) 20/11/2018

X Supervisor: Anthony R. Carroll

(Signed) (Date) 20/11/2018

99 Pages redacted Chapter 8 – Conclusion and Outlook

8.1 Anti-plasmodial Natural Product Discovery

The primary aim of this thesis was to isolate anti-plasmodial natural products. To achieve this, an anti-plasmodial screening and LC-MS chemical profiling program was conducted on a library of 30 Australian Rutaceae species (Chapter 2). From the obtained results, four species were selected and chemically investigated: Clausena brevistyla (Chapter 2), Flindersia pimenteliana (Chapters 3-4), Acronychia pubescens (Chapter 5) and Pitaviaster haplophyllus (Chapter 6). A number of other species showed comparable bioactivity to the aforementioned four although they could not be studied due to time limitations. Further work continuing from this thesis may thus be chemical investigations of these hits. Acronychia acidula, Medicosma forsterii, Melicope micrococca and Zanthoxylum ovalifolium are among the most likely to yield anti- plasmodial natural products.

Of the natural products reported in chapters 2-6, four showed potent activity (IC50 values below 1 µM) against P. falciparum. These were dimethylisoborreverine (190 – 670 nM; Chapter 3), borreverine (220 – 310 nM; Chapter 3), 4-methylborreverine (340 – 510 nM; Chapter 3) and an unnamed pyranocoumarin (466 – 822 nM; Chapter 2). The anti-plasmodial activity of the latter three natural products are reported for the first time. All four had comparable activity against both wild type (3D7) and drug-resistant (Dd2) P. falciparum and showed parasite selectivity indices (SI; calculated as HEK-293/3D7) in the 8-40 range. An additional seven compounds showed moderate anti-plasmodial activity (IC50 values between 1- 5 µM), of which five are new natural products. The most selective natural product isolated, the potently active pyranocoumarin from the roots of C. brevistyla (SI = 20 – 40), is worthy of further investigation as an anti-malarial compound. The structural features of this compound leading to its bioactivity are discussed in chapter 2. This discussion lays the groundwork for further studies into the isolation, synthetic modification and anti-plasmodial testing of structurally related pyranocoumarins. If the bioactivity of this molecule can be enhanced by semi-synthetic means, mode of action studies may be worthwhile; however, this class of molecule is in its absolute infancy as potential anti-malarial drugs.

Although I am not the first to discover the anti-plasmodial activity of Flindersia bis-indole alkaloids, I report for the first time the potent activity of borreverine-type alkaloids, which showed almost identical activity to the structurally related isoborreverine-type and flinderole- type alkaloids reported by Fernandez and co-workers (Chapter 3). I also report the isolation of three new methoxylated isoborreverine-type alkaloids, which showed comparable activity to 100

previously known isoborreverine-type alkaloids (Chapter 4). These results contribute to our knowledge about the structure-activity relationships of the potently anti-plasmodial Flindersia bis-indoles. Surprisingly, since the discovery of the bioactivity of the Flindersia alkaloids by Fernandez and co-workers in 2008, there appears to be no follow-up publications that have screened any other derivatives of these molecules. As such, my report of the bioactivity of the methoxylated isoborreverine-type alkaloids (Chapter 4) and borreverine-type alkaloids (Chapter 3) is the first work in this area. The purification of borreverine/isoborreverine/flinderole-type alkaloids was extremely challenging, however, and recommend that any further studies of their structure-activity relationships be carried out on molecules produced by total synthesis. The total synthesis of all three types of Flindersia bis- indole alkaloid is already reported in the literature.

8.2 New Natural Product Structure Classes

As a by-product of my search for anti-plasmodial compounds, two new structure classes of natural products were also discovered. The first of these was the ascorbic acid-adduct indole alkaloids pimentelamines A-C from F. pimenteliana (Chapter 3). Although pimentelamine A and

B were inactive, pimentelamine C showed moderate activity (IC50 2.7 – 3.6 µM) against P. falciparum while also showing very low cytotoxicity against HEK-293 cells (inactive at 40 µM). Structurally, the pimentelamines are unlike any other natural products in the literature. They appear to be biosynthesised by reaction of an ascorbic acid radical with a prenylated dimethyltryptamine precursor. The absolute configurations of pimentelamines A-C, which were revealed by electronic circular dichroism (ECD) quantum-mechanical calculations, support this biosynthetic hypothesis.

The second new class of natural product discovered in this project was acrotrione, an oxidised furo[2,3-c]xanthene from the roots of A. pubescens (Chapter 5). Acrotrione also showed moderate anti-plasmodial activity, with IC50 values between 2.7 – 4.7 µM and comparatively low activity against HEK-293 cells (80% growth inhibition at 40 µM). However, as with the pimentelamines, the molecular structure of acrotrione is more fascinating than its bioactivity. Although there are dozens of Acronychia-type acetophenones (AtAs) reported in the literature, acrotrione is the first of the AtA dimers that has undergone oxidation and cyclisation to form a furo[2,3-c]xanthene. In this respect, it is the most unique of all AtAs. Moreover, although the vast majority of the chiral AtAs reported in the literature are racemic or have not had their absolute configurations elucidated, I have determined the absolute configuration of acrotrione via ECD calculations.

101

8.3 Natural Product Diversity Across Environments

The secondary aim of this thesis was to investigate the factors that drive natural product diversity in Australian plants (Chapter 7). It is clear from the literature that attitudes towards natural product-driven drug discovery have been negatively impacted recently by the repeated re-isolation of known natural products. Consequently, there is a need for the development of new techniques and ideas that expedite the discovery of new natural products. Although dereplication processes involving spectroscopic/spectrometric techniques and databases are becoming increasingly popular, I sought to address this issue from a different perspective: sample collection. Many recent publications have reported positive correlations between diversity of plant/herbivore communities and diversity of natural products. This may suggest that regions of high biotic stress (i.e. rainforests) are the best source of plants containing new and structurally diverse natural products. Conversely, others have proposed that plants growing in environmentally harsh environments (i.e. deserts) are most likely to contain new natural products, owing to the extreme abiotic pressures they must survive. To examine these two contrasting hypotheses, I compared the leaf alkaloid diversity of rainforest and semi- arid/arid zone adapted Australian Flindersia (Rutaceae) by LC/MS-MS and NMR spectroscopy. I found that Flindersia species predominating in drier regions produced a significantly higher number and more structurally diverse alkaloids than their rainforest-restricted congenerics. To the best of my knowledge, I am the first to report a positive correlation between aridity and natural product diversity. Additionally, although the four woodland/vinethicket restricted Flindersia are of a single evolutionary lineage, their alkaloid profiles were completely different from each other. These results outline the potential of the Australian arid zone as a source of new natural products that cannot be found in other environments. Nearly every species that was screened against P. falciparum in this thesis (Chapter 2) was a rainforest occurring species, as were all four species chemically investigated (C. brevistyla, F. pimenteliana, A. pubescens and P. haplophyllus; Chapters 2-6). While a number of potently anti-plasmodial and new natural products were successfully isolated, a future screening program and chemical profiling program focussed on species that occur in drier regions of central Australia may be a particularly fruitful source of new natural products.

102

Appendix I. Chapter 2 Supporting Information

103

Anti-plasmodial Screening and Natural Product Isolation from a Library of Australian Rutaceae Species

Table of Contents

1 Figure S1. H NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S2. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S3. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S4. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

1 Figure S5. H NMR spectrum (500 MHz) of 2 in DMSO-d6

Figure S6. COSY NMR spectrum (500 MHz) of 2 in DMSO-d6

Figure S7. HSQC NMR spectrum (800 MHz) of 2 in DMSO-d6

Figure S8. HMBC NMR spectrum (800 MHz) of 2 in DMSO-d6

104

1 Figure S1. H NMR spectrum (500 MHz) of 1 in DMSO-d6. 105

Figure S2. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6. 106

Figure S3. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6. 107

Figure S4. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d . 6 108

1 Figure S5. H NMR spectrum (500 MHz) of 2 in DMSO-d6

109

Figure S6. COSY NMR spectrum (500 MHz) of 2 in DMSO-d6 110

Figure S7. HSQC NMR spectrum (800 MHz) of 2 in DMSO-d6 111

Figure S8. HMBC NMR spectrum (800 MHz) of 2 in DMSO-d6 112

Appendix II. Chapter 3 Supporting Information

113

Pimentelamines A-C, indole alkaloids isolated from the leaves of the Australian tree Flindersia pimenteliana (Rutaceae)

Luke P. Robertson,†,‡ Sandra Duffy,‡ Yun Wang,§ Dongdong Wang,‡ Vicky M. Avery,‡ and Anthony R. Carroll*,†,‡,§

†Environmental Futures Research Institute, Griffith University, Southport 4222, Gold Coast, Australia ‡Griffith Institute for Drug Discovery, Griffith University, Nathan 4111 , Brisbane, Australia §Centre for Clean Environment and Energy, Griffith University, Southport 4222, Gold Coast, Australia

Supporting Information

114

Table of Contents

1 Figure S1. H NMR spectrum (500 MHz) of 1 in DMSO-d6

13 Figure S2. C NMR spectrum (125 MHz) of 1 in DMSO-d6

Figure S3. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S4. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S5. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S6. ROESY NMR spectrum (500 MHz) of 1 in DMSO-d6

1 Figure S7. H NMR spectrum (500 MHz) of 2 in DMSO-d6

13 Figure S8. C NMR spectrum (125 MHz) of 2 in DMSO-d6

Figure S9. COSY NMR spectrum (500 MHz) of 2 in DMSO-d6

Figure S10. HSQC NMR spectrum (500 MHz) of 2 in DMSO-d6

Figure S11. HMBC NMR spectrum (500 MHz) of 2 in DMSO-d6

Figure S12. ROESY NMR spectrum (500 MHz) of 2 in DMSO-d6

1 Figure S13. H NMR spectrum (800 MHz) of 3 in DMSO-d6

13 Figure S14. C NMR spectrum (200 MHz) of 3 in DMSO-d6

Figure S15. COSY NMR spectrum (800 MHz) of 3 in DMSO-d6

Figure S16. HSQC NMR spectrum (800 MHz) of 2 in DMSO-d6

Figure S17. HMBC NMR spectrum (800 MHz) of 3 in DMSO-d6

Figure S18. ROESY NMR spectrum (800 MHz) of 3 in DMSO-d6

1 Figure S19. H NMR spectrum (500 MHz) of 4 in DMSO-d6

Figure S20. COSY NMR spectrum (500 MHz) of 4 in DMSO-d6

Figure S21. HSQC NMR spectrum (500 MHz) of 4 in DMSO-d6

Figure S22. HMBC NMR spectrum (500 MHz) of 4 in DMSO-d6

115

DMSO-d6

1 Figure S1. H NMR spectrum (500 MHz) of 1 in DMSO-d6

116

13 Figure S2. C NMR spectrum (125 MHz) of 1 in DMSO-d6

117

Figure S3. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

118

Figure S4. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

119

Figure S5. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

120

Figure S6. ROESY NMR spectrum (500 MHz) of 1 in DMSO-d6

121

DMSO-d6

1 Figure S7. H NMR spectrum (500 MHz) of 2 in DMSO-d6

122

DMSO-d6

13 Figure S8. C NMR spectrum (125 MHz) of 2 in DMSO-d6

123

Figure S9. COSY NMR spectrum (500 MHz) of 2 in DMSO-d6

124

Figure S10. HSQC NMR spectrum (500 MHz) of 2 in DMSO-d6

125

Figure S11. HMBC NMR spectrum (500 MHz) of 2 in DMSO-d6

126

Figure S12. ROESY NMR spectrum (500 MHz) of 2 in DMSO-d6

127

1 Figure S13. H NMR spectrum (800 MHz) of 3 in DMSO-d6

128

13 Figure S14. C NMR spectrum (200 MHz) of 3 in DMSO-d6

129

Figure S15. COSY NMR spectrum (800 MHz) of 3 in DMSO-d6

130

Figure S16. HSQC NMR spectrum (800 MHz) of 2 in DMSO-d6

131

Figure S17. HMBC NMR spectrum (800 MHz) of 3 in DMSO-d6

132

Figure S18. ROESY NMR spectrum (800 MHz) of 3 in DMSO-d6

133

DMSO-d6

1 Figure S19. H NMR spectrum (500 MHz) of 4 in DMSO-d6

134

Figure S20. COSY NMR spectrum (500 MHz) of 4 in DMSO-d6

135

Figure S21. HSQC NMR spectrum (500 MHz) of 4 in DMSO-d6

136

Figure S22. HMBC NMR spectrum (500 MHz) of 4 in DMSO-d6

137

Appendix III. Chapter 4 Supporting Information

138

Anti-plasmodial bis-indole alkaloids from the bark of the Australian tree Flindersia pimenteliana (Rutaceae) Luke P. Robertson,a,b Leonardo Lucantoni,b Vicky M. Avery,b and Anthony R. Carroll*,a,b a Environmental Futures Research Institute, Griffith University, Southport 4222, Gold Coast, Australia b Griffith Institute for Drug Discovery, Griffith University, Nathan 4111 , Brisbane, Australia

Supporting Information

139

Table of Contents

1 Figure S1. H NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

13 Figure S2. C NMR spectrum (200 MHz) of 1:2 mixture in DMSO-d6

Figure S3. COSY NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

Figure S4. HSQC NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

Figure S5. HMBC NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

Figure S6. ROESY NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

1 Figure S7. H NMR spectrum (800 MHz) of 1:3 mixture in DMSO-d6

Figure S8. COSY NMR spectrum (800 MHz) of 1:3 mixture in DMSO-d6

Figure S9. HSQC NMR spectrum (800 MHz) of 1:3 mixture in DMSO-d6

1 Figure S10. H NMR spectrum (800 MHz) of 4 in DMSO-d6

13 Figure S11. C NMR spectrum (125 MHz) of 4 in DMSO-d6

Figure S12. COSY NMR spectrum (800 MHz) of 4 in DMSO-d6

Figure S13. HSQC NMR spectrum (800 MHz) of 4 in DMSO-d6

Figure S14. HMBC NMR spectrum (800 MHz) of 4 in DMSO-d6

S15. Failed isolation procedures of 1-5

140

1 Figure S1. H NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6. Picked peaks correspond to 1 and 2.

141

13 Figure S2. C NMR spectrum (200 MHz) of 1:2 mixture in DMSO-d6

142

Figure S3. COSY NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

143

Figure S4. HSQC NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

144

Figure S5. HMBC NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

145

Figure S6. ROESY NMR spectrum (800 MHz) of 1:2 mixture in DMSO-d6

146

1 Figure S7. H NMR spectrum (800 MHz) of 1:3 mixture in DMSO-d6. Picked peaks correspond to 3.

147

Figure S8. COSY NMR spectrum (800 MHz) of 1:3 mixture in DMSO-d6

148

Figure S9. HSQC NMR spectrum (800 MHz) of 1:3 mixture in DMSO-d6

149

1 Figure S10. H NMR spectrum (800 MHz) of 4 in DMSO-d6

150

13 Figure S11. C NMR spectrum (125 MHz) of 4 in DMSO-d6

151

Figure S12. COSY NMR spectrum (800 MHz) of 4 in DMSO-d6

152

Figure S13. HSQC NMR spectrum (800 MHz) of 4 in DMSO-d6

153

Figure S14. HMBC NMR spectrum (800 MHz) of 4 in DMSO-d6

154

S15. Failed isolation procedures of 1-5

Failed isolation procedure #1

F. pimenteliana bark (400 g) was exhaustively extracted in MeOH (12 L) and evaporated under vacuum to yield a brown gum (150 g). The extract was redissolved in 1 L MeOH and eluted through

100 g of strongly acidic cation exchange (SCX) resin, which was then treated with 25% aqueous NH3

(20%) and MeOH (80%) to yield 1.3 g of crude alkaloid extract. The extract was adsorbed onto C18 silica gel (1.3 g) and the extract impregnated gel was loaded into a HPLC pre-column cartridge (10 mm x 20 mm) and connected in series to a C18 silica HPLC column (Betasil 5 µm, 100 Å, 21.2 mm x

150 mm). The column was then eluted with a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. The column was then eluted in MeOH for a further 12 min. Fractions were collected every minute and UV-DAD spectroscopic analysis was conducted in tandem with the separation. Fractions 26-36 (200 mg) were recombined and further purified by phenyl HPLC (Zorbax SB-phenyl, 5 µm, 21.2 mm x 250mm) with a gradient from H2O/0.1%

TFA (65%)/ MeOH/0.1% TFA (35%) to H2O/0.1% TFA (15%)/ MeOH/0.1% TFA (85%) over 60 minutes. Fractions 34-39 (30 mg) were the further purified using diol HPLC (YMC-pack diol, 5 µm, 120 Å, 21.2 mm x 150 mm) using isocratic MeOH (6%)/ DCM (94%). Fractions 4-9 (20 mg) were recombined and further purified using diol HPLC (YMC-pack diol, 5 µm, 120 Å, 21.2 mm x 150 mm) using isocratic MeOH (3%)/ DCM (97%). Fraction 9 contained a mixture of 1-3 in a quantity that was insufficient for full 2D NMR analysis and thus additional compound was required.

Failed isolation procedure #2

F. pimenteliana bark (400 g) was re-obtained and exhaustively extracted in MeOH (16 L) and evaporated under vacuum to yield a brown gum (200 g). The extract was redissolved in 1 L MeOH and eluted through 200 g of strongly acidic cation exchange (SCX) resin, which was then treated with

25% aqueous NH3 (20%) and MeOH (80%) to yield 2.7 g of crude alkaloid extract. 0.9 g of extract was purified by C18 HPLC (Betasil 5 µm, 100 Å, 21.2 mm x 150 mm). The column was then eluted with a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min. This process was repeated twice more until all 2.7 g of crude alkaloid extract was purified. Fractions 28-32 of all three

HPLC runs were recombined (470 mg) and further purified by C18 HPLC (Gemini 5 µm, 110 Å, 21.2 mm x 250 mm) with a gradient from H2O/1% NH3 (100%) to MeOH/1% NH3 (100%) over 60 min. Fractions 48-72 were recombined (330 mg) and further purified by diphenyl HPLC (Agilent Diphenyl

5 µm, 100 Å, 21.2 mm x 250 mm) using a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min. Fractions 44-46 (120 mg) were further purified using amino HPLC (YMC-pack

155

NH2, 5 µm, 120 Å, 20 mm x 150 mm) using a gradient from hexane (100%) to isopropanol (100%) over 60 minutes. Fractions 18-76 (58 mg) were further purified by diphenyl HPLC (Agilent Diphenyl 5

µm, 100 Å, 21.2 mm x 250 mm) using isocratic H2O/0.1% TFA (45%)/ MeOH/0.1% TFA (55%). Fractions 26-30 (30 mg) were further purified using diol HPLC (YMC-pack diol, 5 µm, 120 Å, 21.2 mm x 150 mm) using isocratic MeOH (3%)/ DCM (97%). Fractions 7-8 contained 1-4 as a mixture (5 mg).

Successful isolation procedure (see manuscript).

156

Appendix IV. Chapter 5 Supporting Information

157

Acrotrione, a new Oxidized Xanthene from the Roots of Acronychia pubescens

Luke P. Robertson,a,b Leonardo Lucantoni,b Sandra Duffy,b Vicky M. Avery,b and Anthony R. Carroll*,a,b aEnvironmental Futures Research Institute, Griffith University, Southport 4222, Gold Coast, Australia bGriffith Institute for Drug Discovery, Griffith University, Nathan 4111, Brisbane, Australia

Supporting Information

158

Table of Contents

1 Figure S1. H NMR spectrum (800 MHz) of 1 in DMSO-d6

13 Figure S2. C NMR spectrum (125 MHz) of 1 in DMSO-d6

Figure S3. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S4. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S5. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S6. ROESY NMR spectrum (500 MHz) of 1 in DMSO-d6

159

1 Figure S1. H NMR spectrum (800 MHz) of 1 in DMSO-d6

160

13 Figure S2. C NMR spectrum (125 MHz) of 1 in DMSO-d6

161

Figure S3. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

162

Figure S4. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

163

Figure S5. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

164

Figure S6. ROESY NMR spectrum (500 MHz) of 1 in DMSO-d6 165

Appendix V. Chapter 6 Supporting Information

166

Quinoline alkaloids from the Australian Tree Pitaviaster haplophyllus

Luke P. Robertsona,b and Anthony R. Carroll*,a,b a Environmental Futures Research Institute, Griffith University, Southport 4222, Gold Coast, Australia b Griffith Institute for Drug Discovery, Griffith University, Nathan 4111 , Brisbane, Australia

Supporting Information

167

Table of Contents

Figure S1. Experimental section

1 Figure S2. H NMR spectrum (500 MHz) of 1 in DMSO-d6

13 Figure S3. C NMR spectrum (125 MHz) of 1 in DMSO-d6

Figure S4. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S5. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S6. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

Figure S7. ROESY NMR spectrum (500 MHz) of 1 in DMSO-d6

168

Figure S1. Experimental section

General experimental procedures.

−1 Optical rotations were recorded on a JASCO P-1020 polarimeter and [α]D values are given in 10 deg cm2 g−1. UV spectra were recorded on a Shimadzu UV-1800 spectrophotometer. ECD spectra were recorded on a JASCO J-715 spectropolarimeter. IR spectra were recorded using a ThermoFisher Scientific Nicolet iS5 spectrometer equipped with an iD5 ATR accessory. NMR spectra were recorded at 25 °C on a Bruker Avance III 500 MHz spectrometer (BBFO Smartprobe, 5mm 31P-109Ag). The 1H

13 and C NMR chemical shifts were referenced to the solvent peak for (CD3)2SO at δH 2.50 and δC 39.52. High-resolution mass measurements were acquired using positive electrospray ionization, mobile phase 1:1 CH3CN-H2O on an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS with a 1200 Series autosampler and 1290 Infinity HPLC. Oven used for drying plant material was a

Contherm Thermotec 2000. The C18 silica gel used to adsorb extracts prior to HPLC separation was

Alltech Sample Prep C18 35-75 µm, 150 Å. HPLC columns used were Thermo Betasil C18 5 µm, 100 Å,

21.2 mm x 150 mm and Phenomenex EVO C18 5 µm, 100 Å, 21.2 mm x 150 mm. A Merck Hitachi L7100 pump equipped with a Merck Hitachi L7455 PDA detector and a Merck Hitachi L7250 autosampler were used for HPLC. Fractions were collected using a Gilson 215 liquid handler. All solvents used were Scharlau HPLC grade and H2O was Millipore Milli-Q PF filtered. Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar.

Plant material.

P. haplophyllus material was purchased from Burringbar Rainforest Nursery in March 2018.

Extraction and isolation.

Oven-dried (50 °C, 48 hours) ground P. haplophyllus roots (100 g) were exhaustively extracted in methanol (2 L) and evaporated under vacuum to yield a brown gum (10 g). An aliquot (1 g) of extract was adsorbed onto C18 silica gel (1 g) and the extract-impregnated gel was loaded into a HPLC pre- column cartridge (10 mm x 20 mm) and connected in series to a C18-bonded silica HPLC column (Betasil 5 µm, 100 Å, 21.2 mm x 150 mm). The column was then eluted with a gradient from

H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. The column was then eluted with MeOH for a further 10 min. Fractions were collected every min and UV-DAD spectroscopic analysis was conducted in tandem with the separation. Fraction 22 contained N-methylplatydesminium (5) (0.9 mg, 9.0 x 10-4% dry wt.) and fraction 34 contained evolitrine (8) (2.1 mg, 2.1 x 10-3% dry wt).

169

Oven-dried (50 °C, 48 hours) ground P. haplophyllus stem bark (500 g) was exhaustively extracted in methanol (16 L) and evaporated under vacuum to yield a brown gum (100 g). The extract was redissolved in 1 L MeOH and eluted through 250 g of strongly acidic cation exchange (SCX) resin, which was then treated with 25% aqueous NH3 (20%) and MeOH (80%) to yield 1.5 g of crude alkaloid extract. This extract (1.5 g) was purified by C18 HPLC (Betasil 5 µm, 100 Å, 21.2 mm x 150 mm) using a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. The column was then eluted with MeOH for a further 10 min. Fractions 28-38 (86 mg) and 44-56 (59 mg) were separately recombined. Fractions 28-38 (86 mg) were further purified by C18 HPLC (Phenomenex EVO C18 5 µm, 100 Å, 21.2 mm x 150 mm) using a gradient from H2O/0.1%

TFA (100%) to H2O/0.1% TFA (20%)/MeOH/0.1% TFA (80%) over 60 min, then to MeOH/0.1% TFA (100%) over 6 minutes at a flow rate of 9 mL/min. The column was then eluted with MeOH for a further 10 min. Fraction 40 contained kokusaginine (7) (3.7 mg, 7.4 x 10-4% dry wt.) and fraction 42 contained tabouensinium (3) (2.7 mg, 5.4 x 10-4% dry wt.). Fractions 44-56 (59 mg) were further purified by C18 HPLC (Phenomenex EVO C18 5 µm, 100 Å, 21.2 mm x 150 mm) using a gradient from

H2O/0.1% TFA (100%) to H2O/0.1% TFA (80%)/MeOH/0.1% TFA (20%) over 6 minutes, then to MeOH/0.1% TFA (100%) over 60 minutes. The column was then eluted with MeOH for a further 10 min. Fraction 24 contained 7-hydroxydictamnine (6) (0.9 mg, 1.8 x 10-4% dry wt.) and fraction 36 contained pteleine (9) (0.2 mg, 4.0 x 10-5% dry wt.).

Oven-dried (50 °C, 48 hours) ground P. haplophyllus leaves (300 g) were exhaustively extracted in methanol (16 L) and evaporated under vacuum to yield a green gum (100 g). The extract was redissolved in 1 L MeOH and eluted through 250 g of strongly acidic cation exchange (SCX) resin, which was then treated with 25% aqueous NH3 (20%) and MeOH (80%) to yield 1.1 g of crude alkaloid extract. This extract (1.1 g) was purified by C18 HPLC (Betasil 5 µm, 100 Å, 21.2 mm x 150 mm) using a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. The column was then eluted with MeOH for a further 10 min. Fractions 41-46 (62 mg) and 47-60 (140 mg) were separately recombined. Fractions 41-46 (62 mg) were further purified by C18 HPLC (Phenomenex EVO C18 5 µm, 100 Å, 21.2 mm x 150 mm) using a gradient from H2O/0.1%

TFA (100%) to H2O/0.1% TFA (80%)/MeOH/0.1% TFA (20%) over 6 minutes, then to MeOH/0.1% TFA (100%) over 60 minutes at a flow rate of 9 mL/min. The column was then eluted with MeOH for a further 10 min. Fraction 40 contained arborinine (2) (0.3 mg, 1.0 x 10-4% dry wt.) and fraction 42 contained acrophylline (10) (2.2 mg, 7.3 x 10-4% dry wt.). Fractions 47-60 (140 mg) were also further purified by C18 HPLC (Phenomenex EVO C18 5 µm, 100 Å, 21.2 mm x 150 mm) using a gradient from

H2O/0.1% TFA (100%) to H2O/0.1% TFA (80%)/MeOH/0.1% TFA (20%) over 6 minutes, then to MeOH/0.1% TFA (100%) over 60 minutes. The column was then eluted with MeOH for a further 10

170 min. Fraction 42 contained leptanoine D (1) (0.5 mg, 1.6 x 10-4% dry wt.) and fraction 48 contained melineurine (4) (0.4 mg, 1.3 x 10-4% dry wt.).

Computational methods

The lowest energy conformers of 1 were predicted using Schrödinger MacroModel 2016 using the protocol of Willoughby et al.1 Geometry optimisations were then carried out on each of the 242 generated conformers using density functional theory (DFT) at the B3LYP/6-31G(d) level. The effect of the solvent was considered during the optimisation with the Polarizable Continuum Model (PCM).2 Single-point energy calculations were performed at the same theoretical level. Calculations of electronic transition and rotational strengths were also performed at the B3LYP/6-31G(d) level with consideration of the solvent using the PCM. The freely available software SpecDis was used to generate Boltzmann-weighted UV and ECD spectra.3 A half-bandwidth of 0.3 eV was applied to calculated spectra. The experimental ECD spectrum of 1 was smoothed using SDAR.4 All DFT calculations were performed in GAUSSIAN 16.5

171

1 Figure S2. H NMR spectrum (500 MHz) of 1 in DMSO-d6

172

13 Figure S3. C NMR spectrum (125 MHz) of 1 in DMSO-d6

173

Figure S4. COSY NMR spectrum (500 MHz) of 1 in DMSO-d6

174

Figure S5. HSQC NMR spectrum (500 MHz) of 1 in DMSO-d6

175

Figure S6. HMBC NMR spectrum (500 MHz) of 1 in DMSO-d6

176

Figure S7. ROESY NMR spectrum (500 MHz) of 1 in DMSO-d6

177

References

[1] Willoughby PH, Jansma MJ, Hoye TR. Nat. Protoc. 2014; 9: 643-660 [2] Tomasi J, Mennucci B, Cammi R. Chem. Rev. 2005; 105: 2999-3094. [3] Bruhn T, Schaumlöffel A, Hemberger Y, Bringmann G. Chirality 2013; 25: 243-249. [4] Weeratunga S, Hu N-J, Simon A, Hofmann A. BMC Bioinf. 2012; 13: 201. [5] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Petersson GA, Nakatsuji H, Li X, Caricato M, Marenich AV, Bloino J, Janesko BG, Gomperts R, Mennucci B, Hratchian HP, Ortiz JV, Izmaylov AF, Sonnenberg JL, Williams-Young D, Ding F, Lipparini F, Egidi F, Goings J, Peng B, Petrone A, Henderson T, Ranasinghe D, Zakrzewski VG, Gao J, Rega N, Zheng G, Liang W, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Throssell K, Montgomery JAJ, Peralta JE, Ogliaro F, Bearpark MJ, Heyd JJ, Brothers EN, Kudin KN, Staroverov VN, Keith TA, Kobayashi R, Normand J, Raghavachari K, Rendell AP, Burant JC, Iyengar SS, Tomasi J, Cossi M, Millam JM, Klene M, Adamo C, Cammi R, Ochterski JW, Martin RL, Morokuma K, Farkas O, Foresman JB, Fox DJ. Gaussian 16; Gaussian, Inc., Wallingford, CT, 2016.

178