Isolation and structure elucidation of cytoxotic natural products with potential anticancer activity

List of Schemes xviii Chapter 1 Introduction 1.1 The contribution of natural products to drug discovery 1 1.2 Natural products as potential anticancer agents 3 1.3 The application of “d

Isolation and Structure Elucidation of Cytotoxic Natural Products with Potential Anticancer Activity

Trong Duc Tran

- 2010 -

Isolation and Structure Elucidation of Cytotoxic Natural Products with Potential Anticancer Activity

Trong Duc Tran

(B.Eng)

Eskitis Institute for Cell and Molecular Therapies

Science, Environment, Engineering and Technology

Griffith University

Submitted in fulfilment of the requirements of the degree of

Master of Philosophy

July 2010

Declaration

This work has not previously been submitted for a degree or diploma in 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

……… ………

Acknowledgements

First of all, I would like to express my sincere appreciation to my supervisors, Professor Ronald J Quinn and Dr Ngoc B Pham, for their patience, guidance and support My skills as a scientist have definitely matured under the supervision of Prof Quinn and Dr Pham and for that I am most grateful

I would like to thank Dr Gregory Fechner for his kind supervision in training for the bioassay screening I also wish to thank Assoc Prof Anthony Carroll, Dr Rohan Davis,

Dr Yun Feng, Dr Phuc Le, Dr Hoan Vu, Dr Harish Holla, Dr Xinzhou Yang, Dr Sheng Yin and Ms Lekha Suraweera for all academic and technical discussions; Assoc Prof Andreas Hoffmann for the circular dichroism measurements; Ms B Aldred for the provision of cancer cell lines; Dr John Hooper of the Queensland Museum, Dr Paul Forster and Dr G Guymer of the Queensland Herbarium for the collection and identification of biota samples I thank all my friends Emma Barnes, Michelle Liberio and Asiah Osman for many scientific and non-scientific conversations

I acknowledge Education Australia Ltd for the provision of the “EAL Postgraduate Research Student Mobility Scholarships” which allowed me to pursue my full-time research

Abstract

This project presented a strategy to select a subset of prefractionated fractions for screening Marine and plant biota samples were chosen based on their rare taxonomies and mass spectroscopic data A method to reduce 1155 fractions generated from 105 selected samples to 330 UV active fractions was developed

Cancer cell-based screening of 330 prefractionated fractions against four cancer cell lines (A549, HeLa, LNCaP and PC3) and non-cancer cells (HEK) resulted in nineteen active fractions belonging to fourteen biota samples (two plants and twelve marine organisms) One plant and six marine animals were chosen for further investigation Subsequent mass-guided isolation led to the identification of forty-four secondary metabolites, nine of which were not previously reported Structures of these compounds were elucidated by spectroscopic methods (1D and 2D-NMR, MS, CD and specific optical rotation) and chemical methods

In the plant sample Neolitsea dealbata, a total of nine alkaloids (55-63) were

isolated A new aporphine, normecambroline (55), showed selective activity against

HeLa cells with an IC50 of 4.0 μM while its analogue, roemerine (56), displayed selective activity against all four cancer cell lines (A549, HeLa, LNCaP and PC3) and two non cancer cell lines (HEK, NFF)

non-One of the marine samples, a specimen of the Potter Reef marine sponge

Diacarnus sp., showed the presence of terpene peroxides Three known peroxide

compounds, sigmosceptrellin (27), deacarperoxide (28) and methyldiacarnoate (29), were identified Compound 27 inhibited cytotoxicity against all six cell lines with IC50

values ranging from 0.4 to 3.1 μM while the other two compounds were not active

Chemical investigation of a marine specimen from Houghton Reef, Neopetrosia

exigua, resulted in the isolation of two cytotoxic compounds, mortuporamine C (42) and

a new 3-alkylpyridinium alkaloid, dehydrocyclostellettamine A (43) These two

compounds displayed activity against four cancer cell lines with IC50 values in micromolar concentrations (3.0-13.7 μM)

Three new cyclodepsipeptides, neamphamide B (86), neamphamide C (87) and

neamphamide D (88), were found as constituents of a rare marine sponge Neamphius

huxleyi collected at Milln Reef, off Cape Grafton Their structures were elucidated by

NMR spectroscopy and multiple stages of accurate mass measurements

(ESI-FTICR-MSn) Stereochemistry of residues of the peptides was determined by the Marfey amino

acid method and J-based configurational analysis These compounds inhibited the cell

growth with IC50 values ranging from 91.3 to 366.1 nM

Two previously unreported milnamide E (116) and hemiasterlin D (117)

together with nine known small peptides were isolated from a new sponge genus,

Pipestela candelabra Compound 117 was identified as the first peptide skeleton

discovered in nature with a side chain containing 2-hydroxyacetic acid, tert-leucine and

N-methylvinylogous valine residues attached to the indole nitrogen This compound

exhibited activity against HeLa cells with an IC50 of 1.8 nM Cytotoxic results indicated all hemiasterlin derivatives were approximately 100 fold more active against cancer cell lines than the milnamide family

A series of sixteen bromotyrosine alkaloids were identified from two Australian

sponges Suberea clavate and Pseudoceratina sp Two new bromotyrosine derivatives,

pseudoceralidinone A (148) and aplysamine 7 (149) were isolated from a specimen of

the Hook Reef lagoon sponge, Pseudoceratina sp Their absolute stereostructures were

determined by synthetic methods Compound 149 inhibited moderate cytotoxicity (an

IC50 of 4.9 μM against PC3 cells) while compound 148 displayed no activity

All isolated compounds were evaluated for their physico-chemical properties Results showed that thirty-six out of forty-four compounds (81.8%) passed Lipinski’s rule and twenty-nine compounds (65.9%) displayed no violation against the requirements of both Lipinski’s and Veber’s rule

Abbreviations

HPLC High pressure liquid chromatography

C18 octadecyl bonded silica

MSn multi stage mass spectrometry

LRESIMS low resolution electrospray mass spectrum

HRESIMS high resolution electrospray mass spectrum

FTICR Fourier transform ion cyclotron resonance

m/z mass-ion ratio (z = 1)

HSQC heteronuclear single quantum coherence

HMBC heteronuclear multiple bond correlations

ROESY rotating frame overhauser effect spectroscopy

TOCSY total correlation spectroscopy

HSQMBC heteronuclear single quantum multiple bond correlation HSQC-TOCSY heteronuclear multiple bond correlation total correlation

DMSO-d 6 deuterated dimethylsylfoxide

CD3OD-d 4 deuterated methanol

CD3OH-d 3 deuterated methanol

Ro5 the Rule of Five

IC50 concentration of a compound required to inhibit 50% of the receptor

population

(Boc)2O di-tert-butyl dicarbonate

EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

HOBt Hydroxybenzotriazole

DMF Dimethylformamide

List of Schemes xviii

Chapter 1 Introduction

1.1 The contribution of natural products to drug discovery 1

1.2 Natural products as potential anticancer agents 3

1.3 The application of “drug-like” properties into natural product discovery 7

1.3.1 The decline of interest on natural products from big pharmaceutical

Chapter 2 Generation of prefractionated fractions for biological screening

2.2 Re-generation of selected drug-like fraction library 22

2.5.2 Neamphius huxleyi (QID6005333) 27

2.5.5 Diacarnus sp (QID6011588) 28

Chapter 3 Previously isolated compounds from the Australian sponges

Diacarnus sp and Neopetrosia exigua – A new 3-alkylpyridinium

alkaloid from N exigua

3.1 Chemical investigation of the sponge Diacarnus sp 37

3.2 Chemical Investigation of the Sponge Neopetrosia exigua 40

3.3 Evaluation of “drug-like” properties of the isolated compounds 46

Chapter 7 Bromotyrosine alkaloids from the Australian sponges Suberea

clavate and Pseudoceratina sp

7.2.2 Collection, Extraction and Isolation for the sponge Pseudoceratina sp 139

7.3.1 Pseudoceralidinone A (148) 141

7.3.3 Proposed biosynthesis of pseudoceralidinone A (148) and

Appendix 7 CD NMR data of isolated compounds in this research

List of Figures/Plates

Figure 1.1 All available anticancer drugs, 1940s – 06/2006 3

Figure 1.2 Structure activity relationships for the sarcodictyins 6

Figure 1.3 Lipinski violations for 546 marketed oral drugs in

Figure 1.4 Some processes generating prefractionated libraries 12

Figure 2.1 UV absorption analysis of some kinds of drugs (λ = 210-400 nm) 19

Figure 2.2 Examples of selecting fractions 22

Figure 2.3 HPLC profile of a biota sample in a prefractionation process 23

Figure 2.4 Analytical testing with simple chromophores 24

Figure 2.5 Mechanism of Alamar Blue indicator 25

Figure 2.6 Histogram of selected fractions 33

Figure 2.7 Total cancer cell-based screening results 34

Plate 3.1 Photograph of the sponge Diacarnus sp 38

Plate 3.2 Photograph of the sponge N Exigua 42

Figure 3.1 The 1H-NMR spectrum of 43 recorded at 600 MHz in DMSO-d 6 44

Figure 3.2 The partial structures A, B and C of 43 44

Figure 3.3 Key HMBC correlations to establish the structure of 43 45

Figure 4.1 Some types of aporphinoid structures 51

Figure 4.2 The special feature of the aporphine system 52

Figure 4.3 Compounds previously isolated from the plant N dealbata 53

Figure 4.4 The 1H-NMR spectrum of 55 recorded at 600 MHz in DMSO-d 6 57

Figure 4.5 The partial structures A, B and C of compound 55 58

Figure 4.6 Key HMBC and ROESY correlations to establish

Figure 4.7 CD spectra of compound 55 and 59 59

Figure 4.8 The 1H-NMR spectrum of 57 recorded at 600 MHz in DMSO-d 6 60

Figure 4.9 Key HMBC and ROESY correlations to establish

Figure 4.10 The 1H-NMR spectrum of 58 recorded at 600 MHz in DMSO-d 6 62

Figure 4.11 Key HMBC and ROESY correlations to establish

Figure 4.12 CD spectra of compound 56, 57 and 58 63

Figure 5.1 Two compounds isolated from the sponge Neamphius huxleyi 71

Figure 5.2 Four uncommon residues identified in 65 and 66 73

Plate 5.1 Photograph of the sponge Neamphius huxleyi 77

Figure 5.3 The 1H-NMR spectrum of 86 recorded at 600 MHz in MeOH-d 3 79

Figure 5.4 Key HMBC and ROESY correlations to establish 2 partial

Figure 5.5 Key HMBC and ROESY correlations to establish

Figure 5.6 FTMS2 spectrum of 86 86

Figure 5.7 Mechanism of Marfey’s reagent (FDAA) 86

Figure 5.8 Relative configurations of the β-OMeTyr, Agdha and

Figure 5.9 The 1H-NMR spectrum of 87 recorded at 600 MHz in MeOH-d 4 92

Figure 5.10 FTMS2 spectrum of 87 93

Figure 5.13 The 1H-NMR spectrum of 88 recorded at 600 MHz in MeOH-d 4 98

Figure 5.14 Htmoa unit of 88 with key COSY/TOCSY and

Figure 6.1 Three residues characterised from milnamides and hemiasterlins 105

Figure 6.2 Structure activity relationship of 89 107

Figure 6.3 Two synthetic analogues of hemiasterlin 107

Plate 6.1 Photograph of the sponge Pipestela candelabra collected at

Plate 6.2 Photograph of the sponge Pipestela candelabra collected at

Figure 6.4 The 1H-NMR spectrum of 116 recorded at 600 MHz in DMSO-d 6 113

Figure 6.5 The partial structures A, B and combined A-B of 116 114

Figure 6.6 The partial structures C, D and combined C-D of 116 115

Figure 6.7 Key HMBC and ROESY correlations to establish

Figure 6.8 CD spectrum of compounds 90 and 116 116

Figure 6.9 The 1H-NMR spectrum of 117 recorded at 600 MHz in DMSO-d 6 119

Figure 6.10 The partial structures A, B, C and combined C-A-B of 117 120

Figure 6.11 The partial structures D and E of compound 117 121

Figure 6.12 Key HMBC and COSY correlations to establish

Figure 7.1 Skeletons represented for simple bromotyrosine alkaloids 130

Figure 7.2 Skeletons represented for oxime bromotyrosine alkaloids 131

Figure 7.3 Skeletons represented for bastadin alkaloids 131

Figure 7.4 Skeletons represented for spirooxepinisoxazoline bromotyrosine

alkaloids 132

Figure 7.5 Skeletons represented for spirocyclohexadienylisoxazoline

alkaloids 133

Plate 7.1 Photograph of the sponge Suberea clavate 136

Plate 7.2 Photograph of the sponge Pseudoceratina sp 139

Figure 7.6 The 1H-NMR spectrum of 148 recorded at 600 MHz in DMSO-d 6 141

Figure 7.7 The partial structures A, B and C of 148 142

Figure 7.8 Key HMBC correlations to establish the structure of 148 142

Figure 7.9 Configurational correlation model for the (S)-MTPA and

Figure 7.10 ΔδSR

values for MTPA derivatives (155 and 156) and

their configurational correlation model 145

Figure 7.11 The 1H-NMR spectrum of 149 recorded at 600 MHz in DMSO-d 6 146

Figure 7.12 The partial structures A, B and C of 149 147

Figure 7.13 Key HMBC correlations to establish the structure of 149 148

Figure 8.1 Histogram for all isolated compounds showing physico-chemical

parameters 161

Figure 8.2 Comparison in passing Lipinski rule and combined Lipinski with

Veber rules of all isolated compounds 161

List of Tables/Lists

Table 2.1 Gradient timetable for LC/MS analysis of crude extracts 21

Table 2.2 Gradient timetable for prefractionation 23

Table 2.3 Gradient timetable for LC/MS analysis of active fractions 26

List 2.1 Taxonomic information of samples contains 19 active fractions 34

List 2.2 Chapters describing the investigated samples 34

Table 3.1 Taxonomy of sponges producing terpene peroxides 37

Table 3.2 Taxonomy of sponges producing 3-alkylpyridinium alkaloids 42

Table 3.3 NMR data for dehydrocyclostellettamine A (43) in

Table 3.4 Physico-chemical properties of isolated compounds 46

Table 3.5 Evaluation of cytotoxic potential of compounds 27-29 and 42-43 46

Table 4.1 NMR data for TFA salt of (6aR)-normecambroline (55) in

Table 4.4 NMR data for TFA salt of compounds 56, 57 and 58 in DMSO-d 6 64

Table 4.5 Physico-chemical properties of compounds 55-63 65

Table 4.6 Evaluation of cytotoxic potential of isolated compounds 66

Table 5.1 20 natural amino acids encoded by DNA 72

Table 5.2 Retention times of authentic FDAA-amino acids and

Table 5.3 NMR comparison of the β-OMeTyr residue 88

Table 5.4 NMR data for neamphamide B (86) 89

Table 5.5 NMR data for neamphamide C (87) in CD3OD 95

Table 5.6 NMR data for neamphamide D (88) in CD3OD 100

Table 5.7 Physico-chemical properties of the isolated compounds 102

Table 5.8 Biological activity of the isolated compounds 102

Table 6.1 Taxonomy of sponges producing milnamides and hemiasterlins 106

Table 6.2 NMR data for FA salt of milnamide E (116) in DMSO-d 6 117

Table 6.3 NMR data for FA salts of milnamide E (116), isolated

milnamide A (90) and referenced milnamide A in CD3CN-d 3 118

Table 6.4 NMR data for TFA salt of hemiasterlin D (117) in DMSO- d 6 123

Table 6.5 Physico-chemical properties of the isolatedcompounds 124

Table 6.6 Cytotoxicity evaluation of isolated compounds 124

Table 7.1 Taxonomy of sponges producing bromotyrosine alkaloids 129

Table 7.2 NMR data for the MTPA Esters of 154 in DMSO-d 6 144

Table 7.3 NMR data for TFA salt of 148 in DMSO-d 6 145

Table 7.4 NMR data for TFA salt of 149 in DMSO-d 6 150

Table 7.5 NMR data for the MTPA Esters of 165a and 165b in DMSO-d 6 151

Table 7.6 Physico-chemical properties of all isolated compounds 153

Table 7.7 Biological activity of some isolated compounds 154

List of Schemes

Scheme 1.1 Traditional way in natural product drug discovery 10

Scheme 1.2 Research program 13

Scheme 2.1 Diagram of selecting fractions for further investigations 20

Scheme 3.1 Extraction and Isolation Procedure for Diacarnus sp 39

Scheme 3.2 Extraction and Isolation Procedure for N exigua 43

Scheme 4.1 Extraction and Isolation Procedure for N dealbata 56

Scheme 5.1 Extraction and Isolation Procedure for Neamphius huxleyi 78

Scheme 5.2 FTMS2 fragmentations and related neutral losses of 86 85

Scheme 5.3 FTMS2,3 fragmentations and related neutral losses of 87 94

Scheme 5.4 FTMS2 fragmentations and related neutral losses of 87

Scheme 6.1 Extraction and Isolation Procedure for Pipestela candelabra

Scheme 6.2 Extraction and Isolation Procedure for Pipestela candelabra

collected at Houghton Reef, Howick Group 112

Scheme 7.1 Proposed biosynthesis of spirooxepinisoxazoline and

spirocyclohexadienylisoxazoline rings 133

Scheme 7.2 Extraction and Isolation Procedure for Suberea clavate 138

Scheme 7.3 Extraction and Isolation Procedure for Pseudoceratina sp 140

Scheme 7.4 Modification of 148 to determine its absolute stereochemistry 144

Scheme 7.5 Total synthesis of oxime-protected aplysamin 7 (165) 148 Scheme 7.6 Isolation and stereochemical determination of enantiomers

Scheme 7.7 A plausible biosynthesis of 148 and 149 152

List of Publications

1) Trong D Tran, Ngoc B Pham, Gregory Fechner and Ronald J Quinn, Chemical

Investigation of Drug-like Compounds from the Australian Tree, Neolitsea dealbata,

Bioorganic & Medicinal Chemistry Letters (approved)

2) Trong D Tran, Ngoc B Pham, Gregory Fechner and Ronald J Quinn, A Novel Cytotoxic Peptide Hemiasterlin D and Milnamide E from the Australian Sponge

Pipestela candelabra (in preparation)

3) Trong D Tran, Ngoc B Pham, Gregory Fechner and Ronald J Quinn, New

Cytotoxic Cyclic Depsipeptides from the Autralian Marine Sponge Neamphius huxleyi

(in preparation)

4) Trong D Tran, Ngoc B Pham, Gregory Fechner and Ronald J Quinn, New

Bromotyrosine Alkaloids from the Australian Marine Sponge Pseudoceratina sp (in

preparation)

Conference Poster

1) Drug-like alkaloids from the Australian tree, Neolitsea dealbata, RACI 2010 (Royal

Australian Chemical Institute's National Convention), Melbourne Convention Centre, Melbourne, Australia (4th – 8th July 2010)

Chapter 1

Introduction

1.1 The contribution of natural product to drug discovery

Natural product chemistry emerged in the 1800’s with the landmark isolation of

salicin (1) from the white willow bark, Salix alba in 1825.1 In the early stages, natural product research mainly focused on medicinal plants which had been extensively documented in written or verbal forms and passed down from generation to generation over thousands of years.2,3 For instance, turmeric and ginger, two popular spices in Asian countries, were known as effective ethnomedicines treating stomach-ache, arthritis, asthmatic and tussive in the traditional Indian and Chinese medicines for over

3000 years.4,5 Not until the twentieth century were the mysteries of the biologically active components in these spices disclosed scientifically.4 Curcumin (2) was first

isolated from turmeric in 18156 but its structure was not elucidated until 1910 as diferuloylmethane.7 Curcumin and its analogues in turmeric were found to have potential in the prevention and treatment of inflammation, cardiovascular disease, Alzheimer's disease, memory deficits, arthritis and cancer.5 The isolation and structural

characterization of ginkgolide B (3) and its derivatives in ginger were first reported in

1967.8 Biological investigation of these compounds indicated they are the antiasthmatic and antitussive components of ginger.5

H

OH

HO

OH HO H O O

Ginkolide B (3)

N

H3CO

N H

HO H

COOH O

H H

Penicillin G (7)

N S O O O

have become indispensable drugs or the core structures of specific drugs, such as

quinine (4) (antimalarial), morphine (5) (anti-analgesic), galanthamine (6) (Alzheimer’s disease), penicillins (7) (antibiotics) and cephalosporins (8) (antibiotics)

O

O

O O O O O

OH

HO

OH

H H

H

H O O O O

O

O

H O

H

O O

Marinisporolide A (11)

O O O

OH

NH2O

Discodermolide (13)

It was not until the 1950s that natural product research commenced to focus on marine sources.9 In the last 60 years, over 16,000 marine natural products were discovered from marine microbes, algae and invertebrates and this number is increasing rapidly.10 In comparison with terrestrial sources, research on marine organisms has encountered more challenges One of the most difficult tasks is deep-water sample collection as it requires high-tech deep-water collection tools The scarcity of biota, due

to the initial collection difficulty, often leads to more challenges in the following steps

of isolation, structure identification and pharmacological testing.10 For example, one

tonne of the sponge Lissodendoryx sp was collected in order to obtain only 300 mg of

halichondrin B (9) for clinical testing.10 Despite these drawbacks, the novelty in structures and interesting bioactivities of marine natural products have always appealed

to scientists in the development of new therapeutic agents.11 The discovery of

polyene-polyol and polycyclic polyketides, such as marinomycin A (10), marinisporolide (11)

and abyssomicin C (12) from the new marine genera Marinispora and Salinispora has

provided new promising antibiotic candidates and might be complementing to penicillin and its analogues discovered from fungi sources over 50 years ago.12 Some marine

products, such as halichondrin B (9), discodermolide (13), kahalalide F (17), dolastatin

10 (18) and okadaic acid (19) are now in preclinical and clinical trial for treating

cancer.13,14 According to Buss and Butler,15 over 20 drugs derived from actinomycetes, bacteria, fungi, higher plants, marine invertebrates and vertebrates were introduced to the market in the period 2003-2008 Moreover, there are 36 additional natural product-

derived compounds currently in late stage drug development.15 Natural products thus play a critical role in drug discovery programs

1.2 Natural products as potential anticancer agents

Cancer is the second leading cause of death behind cardiovascular disease in the

US.16 It is predicted that cancer will become the leading cause of death in the human population.17 According to World Health Organization (WHO),17 there were 7.9 million deaths in 2007 due to cancer, of which about 72% occurred in low- and middle-income countries Lung cancer caused the highest mortality (1.4 million deaths/year), others were stomach cancer (866,000 deaths/year), liver cancer (653,000 deaths/year), colon cancer (677,000 deaths/year) and breast cancer (548,000 deaths/year)

Figure 1.1 All available anticancer drugs, 1940s – 06/200618

• “B”: Biological; usually a large (>45 residues) peptide or protein either isolated from an organism/cell line or produced by biotechnological means in a surrogate host

Where:

• “N”: Natural product

• “ND”: Derived from a natural product and is usually a semisynthetic modification

• “S”: Totally synthetic drug, often found by random screening/modification of an existing agent

• “S*”: Total synthesis, but the pharmacophore is/was from a natural product

• “V”: Vaccine

• “NM”: Natural Product Mimic

While there are many kinds of cancer treatments such as surgery, radiation, biological therapy and chemotherapy,19 chemotherapy remains the most effective treatment for patients with solid tumours.20 Among many anticancer drugs used in chemotherapy, drugs derived from natural products including modified natural products

or synthetic products with a natural pharmacophore hold an important position by

occupying 65% of anticancer drugs approved during the 1940s-2006 (Figure 1.1).18According to the statistics of Newman et al.,18,21,22 there was an increase in the number

of anticancer drugs derived from natural products, from 54 in 1994 to 100 drugs in June

2006 Three main reasons for the success of natural products have been given.23 Firstly, there is firm evidence that substances from natural sources formed during the organism’s evolution have the capacity to correct the aberrant regulation of cellular core machinery Secondly, research on biologically active natural products can contribute to the understanding of cancer mechanisms and therefore facilitate the development of the compounds into therapies Thirdly, being trialled through many centuries, traditional therapies using natural products have selected those with no or less side effects

Beside the well-known marketed anticancer drugs9 derived from terrestrial

natural sources (taxol (14), vinblastine (15) and vicristine (16)), marine organisms are a

promising source for potential anticancer agents According to Alejandro’s survey,24-26

97 new marine secondary metabolites were reported to possess antitumor and cytotoxic properties during 2001 to 2002.25 The number of new cytotoxic natural products derived from marine sources went up to 150 and 136 in the periods 2003-200426 and 2005-

200624, respectively A small natural peptide, kahalalide F (21), isolated from the

marine mollusc Elysia rufescens, showed potent cytotoxic activities in vitro against a

panel of twenty cell lines from different tumour types (liver, ovary, breast, colon and prostate).27 The IC50 values of 21 on these cancer cell lines ranged from 0.3 to 5.3 μM

after 1-hour exposure and from 0.2 to 4 μM using continuous exposure, especially at

IC50 < 0.3μM against breast and prostate cancer cell lines This compound has recently been developed for phase I clinical trials in the US and Europe.28 Along with kahalalide

F (17), other marine natural products such as halichondrin B (9), dolastatin 10 (18) and okadaic acid (19) are also currently in phase I or II clinical trials.24 Interestingly, a

tetrahydroisoquinoline alkaloid, ecteinascidin-743 (20) (Trabectedin, Yondelis®),

identified from Ecteinascidia turbinata has recently been granted Orphan Drug

designation from the European Commission and the FDA for soft tissue sarcomas and ovarian cancer after preclinical studies over several years.24 One of the promising

anticancer drugs identified in the period from 2001 to 2006 was laurerditerpenol (21), a

novel diterpene isolated from the red alga Laurencia intricate by Kaleem and his

co-workers in 2004.29 In vitro testing showed that this compound inhibited breast tumor

cells HIF-1 with an IC50 of 0.4 μM blocking the induction of nuclear HIF-1α protein Its mechanisms of action on human cancer cells have been further investigated

O OH H

N N

O OH

O H O

H O NH O

O

O H

HN O

HN O

O HNNH O

O NH

O O OH

S O O NH AcO Me O O

HO

OMe Me H

H

H OH

Ecteinascidin 743 (20)

Besides providing drugs for diseases, natural product research also provides pharmacophores for developing better bioactive derivatives as well as designing new analogues with greater synthetic accessibility.30 So far many analogues of taxol (14)

have been synthesized, of which 6 analogues are in phase II clinical trial, 4 analogues are currently in phase I clinical trial and further 23 analogues are in preclinical development.31 Based on the core structure of the marine secondary metabolite,

sarcodictyins A (22), which was found to have taxol-like activities in tubulin

polymerization and microtubule stabilization, Nicolaou et al 32 constructed a large combinatorial library of sarcodictyin analogues and explored their structure activity

relationship (SAR) against tumour cells (Figure 1.2) The results revealed that the

activity of sarcodictyins A analogues with an ester group at C3 was higher than an amide and the reduction of the ester to give an alcohol led to the loss of its activity Furthermore, if a hydroxy group at C4 were replaced by a ketone group, the biological activity would appear more tolerable Another position found to play a crucial cytotoxicity role was an ester side chain at C8 It was found that either replacing a natural urocanic acid side chain with an acetate group or a phenyl carbamate group or changing a natural imidazole substituent for pyridine, thiazole or oxazole would result

in the complete loss of activity

N N

1 3 4 8

for activity

Both nitrogens are necessary for activity Ketal substitutions

are well tolerated

Esters have higher activity than amides, reduction of the ester to the alcohol leads to loss of activity

Sarcodictyin A (22)

Figure 1.2 Structure activity relationships for the sarcodictyins32

Bryostatin 1 (23), isolated from the marine bryozoan Bugula neritina with the

yield of 1.4x10-4 %, was found to inhibit a variety of cancer cells.33 A SAR study

indicated that the rings A and B of 23 might be the active pharmacophore and

substituents on these rings could be removed without losing activity This research also determined that the key pharmacophoric elements of bryostatin in binding protein kinase C were at C1, C19 and C26.34 A synthetic compound, bryostatin analogue A

(24), which was synthesized after 19 steps in 2% of yield, possesses a simpler structure

than the parent bryostatin.35 This analogue showed a higher affinity binding to the protein kinase C with a Ki of 0.25 nM compared with 1.35 nM for bryostatin and greater

potency in vitro against a subset of the NCI’s panel of cell lines.36 

O O

O

MeO O

O H

H HO

OAc

H

OH H

O

OMe O O

Bryostatin 1 (23)

O O

O O

OH H

H H

H O OH H

O

OMe O

OH

O

Bryostatin analogue A (24)

A B

C

1

26 19

O OH

OH

1.3 The application of “drug-like” properties into natural product discovery

1.3.1 The decline of interest on natural products from big pharmaceutical companies

Despite having made great contributions to drug discovery, in the past few years natural product research has lost financial investments from most big pharmaceutical companies These companies have either scaled down or terminated their natural product operations Reasons have been given to explain the decline of interest in natural products Natural products could not compete with other drug discovery methods in delivering large-scale compound supply within a short time frame and in delivering compounds that passed the criteria of oral bioavailability, a significant requirement in a hit-to-lead program To continue taking part in the fight against diseases, natural product research needs to address these two main problems Recent improvements in instrumentation, robotics, screening technology have shortened the hit identification period for natural products Large scale supply of natural products still requires the development of synthetic routes in many cases Oral bioavailability has been one of the big hurdles to natural products Natural product researchers need to prove to pharmaceutical companies that their novel natural product compounds can be absorbed into the human bloodstream and are worthwhile to be further developed into a drug The prediction of the amount of drug actually absorbed from a given dose into the

bloodstream can be addressed using animal models, or in silico models; logD and pKa

by chromatography; permeability profiles by artificial membrane; intestinal drug transport by Caco-2 cell membrane.37 However, these techniques require time and good

lab facility and are more suitable when the drug is in the last stage of discovery

1.3.2 Physico-chemical properties

Upon studying the solubility and permeability of 2245 drug candidates from the databases of the World Drug Index, the United States Adopted Name and International Non-proprietary Name reaching the phase II clinical process, Lipinski’s group proposed the “rule of five” (Ro5) or Lipinski’s rule as key predictors for oral bioavailability of a compound According to this rule, an oral-acting drug-like molecule should satisfy four parameters38

− The calculated logarithm of the n-octanol/water partition coefficient (ClogP) of

less than 5

− Less than 5 hydrogen bond donors (sum of OH and NH)

− Less than 10 hydrogen bond acceptors (expressed as the sum of O and N)

− Molecular weight of less than 500 Da

Lipinski’s rule also indicated that oral bioavailability does not apply to natural products or any molecules recognized by an active transport systems If a compound fails two or more parameters, there is a high probability that oral activity problems will occur

The first parameter, logP, has been considered as the lord of the “rule of five” It

is defined as the ratio of un-ionized drug distributed between octanol and water phases

at equilibrium.39 This factor plays an important role in solubility, permeability, plasma protein binding, metabolic turnover and toxicity of drugs A high logP prevents the active compounds from reaching the site of action due to poor distribution In contrast, a low logP can cause the compounds to be absorbed by active transports before they reach target cells.40,41

Hydrogen bonds increase solubility in water and must be broken in order for the compound to permeate through the lipid bilayer membrane Thus, an increasing number

of hydrogen bonds reduce partitioning from the aqueous phase into the lipid bilayer membrane Hydrogen bond donors and acceptors also participate in providing hydrogen bonding specifically between ligand and receptor.42

Molecular weight also has a significant influence on the bioavailability of a compound When molecular size increases, the solubility of the compound will reduce since a larger cavity must be formed in water to solubilize this compound Moreover, increasing the molecular weight leads to a decrease in the compound concentration at the surface of the intestinal epithelium, thus affecting the absorption Increasing the molecular size also hampers passive diffusion through the tightly packed aliphatic side chains of the bilayer membrane.43

Recently, Fotouhi and his colleagues in the Roche Research Center44 have analysed the physico-chemical properties of the currently marketed oral drugs in the

FDA Orange Book 2007 (Figure 1.3) The analysis showed over 90% of oral drugs

passed the Ro5

Veber complements Lipinski’s rule with two other parameters, polar surface area (PSA) and number of rotatable bonds (NROT) which can be considered as two more requirements of drug-like molecules from the physico-chemical property viewpoint By observing the solubility and permeability of over 1100 drug candidates in rats, Veber and co-workers45 observed that a molecule with PSA less than 140Å2and NROT less than 10 have better oral activity compared to the others Polar surface area (PSA) is a parameters affecting absorption.46 Owing to the polar surface, the molecule can form hydrogen bonds or Van der Waal bonds with other compounds A rotatable bond, indicating a chemical’s flexibility, is defined as any single non-ring bond, bound

to non-terminal heavy atoms Permeability of a molecule will be significantly reduced if

the NROT is over 10 Lipinski’s and Veber’s rules have guided drug discovery researchers in their hit-to-lead decision making process

1.3.3 Do natural products have drug-like properties?

Although natural products were cited as an exception to the Ro5, database surveys of the drug-like nature of natural products as well as their relationships with marketed drugs and synthetic molecules have been investigated using statistical analyses, computational molecular modelling The results of analysing 126,140 unique entries in the Dictionary of Natural Products (version 2005) showed that 60% of natural products had no Ro5 violations.47 In detail, natural products had median calculated logPs between 2 to 3 (reaching a peak at 2.5); median HBAs were in the range of 3-4; HBDs were maximum at 0-1 and MWs peaked at 300 Da This report also agreed with previous studies by Miklos Feher40 and Thomas Henkel48 on three natural product databases Bioactive Natural Product Database – Szenzor Management (version 1996), Available Chemicals Directory (ADC) and Dictionary of Natural Products (version

1996) In 2008, Ertl et al.42 used another database set containing 10,968 drug molecules,

670,536 combinatorial compounds and 3,287 natural products to investigate

physicochemical properties Ertl et al concluded that both drugs and natural products

had similar lipophilicity with a maximum logP at 3.0, whereas the logP of combinatorial molecules had a maximum at 4.0 A Gaussian distribution of logP indicated that compounds from natural sources were less hydrophobic than the synthetic molecules Similar result was obtained when a research group from the Novartis Institutes analysed 115,590 deglycosylation natural products and 290,000 structures from their in-house

collection of commercially available synthetic compounds.49 All of the above research shows that at least 60% of isolated natural products can be orally bioavailable, and natural products are closer to drugs than synthetic molecules However, acquiring natural products is costly and a method to build a natural product library enriched with drug-like property is vital

1.3.4 A new paradigm in building a natural products library

Chromatographic fractions Active fractions

Scheme 1.1 Traditional way in natural product drug discovery5

Traditional natural product drug discovery using crude extracts and bioassay

guided isolation (Scheme 1.1) is one of the reasons hampering the interest in natural

products from pharmaceutical companies Although this conventional routine does not require a big effort in preparing samples and supplying a high degree of chemical diversity, crude extracts have some drawbacks Firstly, non-specific interference from fatty acids, polyphenols and other salts in crude extracts can cause false-positive or false-negative results in bioassay screenings Secondly, minor components can be missed out in a complex mixture because their concentrations might be below the detection threshold of a biological screening or be masked by other compounds in a complex mixture Thirdly, chemically unattractive compounds are often isolated Fourthly, it takes a great deal of labour and time to identify and isolate bioactive components.50-52

Instead of crude extracts, pure compound libraries of natural products have been constructed.47,53 With structures in hand, compounds violating the physico-chemical properties can be eliminated before screening However, current pure compound libraries can not represent all desired chemical diversity of natural products and minor metabolites may be overlooked Time and resource for the generation of these libraries are still an issue.50,54

Prefractionated natural product libraries have been considered as a new and effective approach Several prefractionation methods have been described using single

or multi-step solid-phase extraction (SPE),55-57 flash column chromatography or preparative high performance liquid chromatography (prep-HPLC),58,59counter-current chromatography (CCC),60-62 centrifugal partition chromatography (CPC)63,64 and ultra performance liquid chromatography (UPLC).52 By using this method, extremely hydrophilic and hydrophobic components (sugars, salts, nucleotides, fatty acid…) are eliminated With 4-5 compounds per fraction, higher dose of compounds can be tested Minor components are also detected and the desired chemical diversity is still secured

On analysing the HTS results obtained from eleven screening campaigns, Merlion Pharmaceuticals reported the activity of prefractionated fractions was increased by twelve times over crude extracts They also found 80% of the primary screening hit fractions were active while their associated crude extract showed no activity even at four-time screening dose.51 The similar outcome of screening prefractionated library (79.9%) was also achieved by Wyeth Pharmaceuticals.50 These results prove that prefractionated libraries have more advantages than crude extract and pure compound libraries

Several pharmaceutical companies and research groups have reported their methods for building prefractionated libraries in natural product drug discovery.50-

52,56,58,59,65-67

Generally, these methods used reverse-phase HPLC to separate fractions cooperating with other detectors such as photodiode array (PDA), mass spectroscopy (MS) or evaporative light scattering (ELS) to identify, dereplicate and isolate biologically active constituents Wyeth Pharmaceuticals generated a 200,000 fraction

library from microorganism sources (10 fractions per extract) (Figure 1.4a).50 Also working in microbial metabolites, Merlion’s process was simpler with 4 fractions per

extract In this way, they established a library with 120,000 fractions for HTS (Figure

separation for eliminating salts and other highly hydrophilic components, followed by

reverse-phase HPLC to produce 80 fractions per sample (Figure 1.4c).67 Compared with microorganism or marine extracts, plant extracts contain tannins and other polyphenols affecting the bioassay screening results, prefractionation for these terrestrial sources is thus more sophisticated Sequoia Science presented a multi-step process for generating their 36,000 fraction library from plant extracts in which 200 fractions were created

from one sample (Figure 1.4d).58 Also from plants, Yan’s group described a less complicated method to generate their library A plant extract was treated through SPE

prior to loading on a HPLC column to separate 24 fractions (Figure 1.4e) This group

demonstrated they could provide 62,000 fractions from 2600 unique natural product samples per year.52

Marine samples

SPE

Figure 1.4 Current processes for generating prefractionated libraries

The overview shows that methods vary from collection of a small number of fractions (4-10 fractions) per sample to a larger number of fractions (80-200 fractions) per sample Although these cleaner fractions are screened, large screening points per sample will limit the number of samples being investigated A better model for the fraction generation is required

Prep-HPLC (4 fractions/extract) MeOH extracts

HTS (120,000 fractions)

HTS (15,360 fractions)

Plant samples Plant samples

EtOH extracts Aqueous extracts

SPE Flash chromatography

(24 fractions/sample)

1 fraction/extract

HTS (62,000 fractions)

HTS (36,000 fractions)

1.4 Objective – Research plan

The objective of this study is to identify drug-like natural products from both plant and marine sources which exhibit cytotoxicity on cancer cell lines The aims of the

project are (Scheme 1.2):

Aim 1: Identification of prefractionated fractions showing activity in a cancer

cell-based screening A small lead-like enhanced fraction library will be prepared 330

fractions are chosen to fit in one 384-well plate These fractions will then be screened against a panel of four cancer cells (lung cancer – A549, cervical cancer – HeLa, prostate cancer – LNCaP and PC3) and non-cancer cell line (human embryonic kidney

293 – HEK) Details are discussed in chapter 2

Aim 2: Isolation and structure elucidation of active components and their

analogues Large scale isolation and structure elucidation are performed for biota

samples whose fractions are in the hit list Active components and their analogues will

be identified for structure-activity relationship Purification and structural characterization are reported in chapters 3-7

Aim 3: Evaluations of drug-like properties and biological activity

Physico-chemical properties (MW, clogP, HBA, HBD, PSA and NROT) of all isolated compounds are calculated by using JChem 2.5.2 software The potential and selective anticancer activity of isolated compounds is also evaluated by comparing inhibitions of pure compounds against cancer cell lines (A549, HeLa, LNCaP and PC3) and non- cancer cell lines (HEK, NFF) These evaluations are reported in chapters 3-7

Samples

Generate prefractionated

fractions

Cancer cell-based screenin

Scheme 1.2 Research program

Large scale isolation

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A ntibac ter ial drugs

A ntiv iral drugs

A ntiparas itic drugs

A ntif ungal drugs

Figure 2.1 UV absorption analysis of some kinds of drugs ( λ = 210-400 nm)

An analysis on UV-absorption of drugs for cancer and infectious diseases

(excluding biological drugs and vaccines) was carried out (Figure 2.1) Using drug lists

reported by Newman and Cragg (anticancer – 164 entities from 1940s to 12/2007;1antibacteria – 98 entities, antivirus – 28 entities, antiparasite – 13 entities and antifungi – 41 entities from 01/1981 to 06/20062), the UV absorptions of these drugs were then collected from databases including Dictionary of Natural Products, SciFinder Scholar, CAPlus and National Center for Biotechnology Information (NCBI)) Results showed that over 90% of drugs related to natural products have UV activity In particular, for anticancer drugs, 75 out of 79 natural products and modified natural products (NND, 94.9%) and all 43 synthetic derived from natural products (SNM, 100%) have UV absorptions ranging from 210-400 nm, which can be detected by a photodiode array detector in HPLC These results encouraged us to choose UV absorption as a criterion