ISOLATION AND CHARACTERIZATION OF THE - USP …digilib.library.usp.ac.fj/gsdl/collect/usplibr1/index/assoc/HASHcc... · TLC Thin Layer Chromatography ... USP The University of the

ISOLATION AND CHARACTERIZATION OF THE

CYTOTOXIC COMPONENTS OF A FIJIAN SPONGE,

HIPPOSPONGIA SP.

By

Rohitesh R KUMAR

A thesis submitted in fulfillment of the requirements for the degree of

Master of Science in

Chemistry

Copyright © 2010 by Rohitesh Kumar

School of Biological and Chemical Sciences

Faculty of Science, Technology and Environment

The University of the South Pacific

June, 2010.

i

Declaration of Originality

Statement by Author

ii

Dedication

To my lovely parents and all my great teachers

iii

Acknowledgment

I extend my sincere gratitude to all those who have made this project a success but a

mere “Thank you” in acknowledgment is not enough to show my appreciation of

their support. Firstly and foremost I would like to thank my supervisor, Prof. William

Aalbersberg, the Director of Institute of Applied Sciences (IAS), USP for his kind

guidance and advice throughout this project. It was a great honor and learning

experience to work under your supervision and to be part of the Natural Products

team at IAS. Specials thanks are due to Institute of Applied Sciences and to the

Faculty of Science, Technology and Environment, USP for funding this research.

Many thanks to Prof. Jaspers Marcel and Dr Joiji Tabudravu of University of

Aberdeen, in Scotland for running NMR and LC-MS experiments for my samples,

for discussing the data with me and for their assistance. Thanks a lot.

I am grateful to Mr. Klaus Feussner, the Acting Project Manager of our team for

assisting me in choosing a sample to work on and for guiding me not only in the lab

but also during the diving trips to collect more samples. Furthermore, I would like to

thank Mr. Mukesh Kant Sharma for being a great mentor and for his support. Many

thanks are owed to the fellow colleagues for those group discussions that we had in

the lab and also for their support and ideas.

A big “Vinaka vakalevu” to the technical staff of the Natural Product Unit for their

hard work in extracting, doing bioassays and setting up experiments. I would also

like to thank the technicians of the Chemistry Department for their assistance in

using some of the instruments.

Special thanks are also due to my aunty and her family for providing me with

accommodation, hot meals and taking good care of me.

Above all, I am grateful to have such loving, caring and understanding parents who

had immense faith in me. Thank you Mum and Dad for your support financially and

emotionally through this journey in search of knowledge.

iv

I thank the lord for giving me such strength, patience, wisdom and guidance to help

me to this extent.

Thank you lord!

v

ABSTRACT

A marine sponge of the genus Hippospongia, collected near Kia Island, off Labasa,

Vanua Levu, exhibited cytotoxic activity towards brine shrimps. In order to identify

the active principles, the crude extract of this sponge was subjected to a range of

purification steps guided by brine shrimp bioassay. The purification steps which

included solvent partitioning, Flash Column Chromatography (FCC) and High

Performance Liquid Chromatography (HPLC) lead to the isolation of three known

cytotoxic compounds: epi-ilimaquinone [102], smenospongine [106] and

glycinylilimaquinone [107]

Bioactive hexane and dichloromethane (DCM) extracts from solvent partitioning

were subjected to normal phase (NP)-FCC and the fraction eluted with 4:1 ethyl

acetate-hexane was further purified on reverse phase (RP)-HPLC with 80%

acetonitrile-water and lead to the isolation of the known compound 1, epi-

ilimaquinone. Another fraction obtained from NP-FCC of the DCM extract eluted

with 1:1 ethyl acetate-hexane was subjected to RP-HPLC with the mobile phase of

80% acetonitrile-water and 0.1% trifluoroacetic acid (TFA) and yielded the second

known compound, smenospongine. Compound 3 was isolated from the methanol

fraction from solvent partitioning. This fraction was subjected to reverse phase

vacuum liquid chromatography (RP-VLC) with methanol-water. The fraction eluted

with 80% methanol-water yielded the known cytotoxic compound 3, known as

glycinylilimaquinone, upon purification on RP-HPLC with 65% acetonitrile-water

and 0.1% TFA.

LC-MS and NMR data of the compounds isolated were used to elucidate the

structures. From the 1H NMR data of the three compounds it was apparent that these

compounds are related and are analogues belonging to the group of compounds

known as sesquiterpene quinones or amino quinones. All the compounds isolated

showed cytotoxic activity towards brine shrimps. Compound 1, epi-ilimaquinone

exhibited the most cytotoxicity at 18µg/ml, compound 2, smenospongine exhibited

moderate cytotoxicity of 188µg/ml while compound 3, glycinylilimaquinone was the

least active with the cytotoxic activity of greater than 500µg/ml.

vi

ABBREVIATION

% percentage

µ micron

µg microgram 13C NMR Carbon Nuclear Magnetic Resonance 1H NMR Proton Nuclear Magnetic Resonance

AIDS Acquired Immunodeficiency Syndrome

BuOH Butanol

C Carbon

CD Circular dichroism

CD3OD Deuterated Methanol

CDCl3 Deuterated Chloroform

COSY Correlated Spectroscopy

dbe Double bond equivalence

DCM Dichloromethane

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

EMEA The European Medicines Agency

ESIMS Electron Spray Ionisation Mass Spectroscopy

EU European Union

FCC Flash Column Chromatography

FD Dichloromethane fraction

FDA Food and Drug Adminstration

FH Hexane fraction

FM Methanol fraction

FSW Filtered sea water

g Grams

H Hydrogen

H2O Water

HDAC Histone deacetylase

HIV Human immuno-deficiency virus

HMBC Hetronuclear Multiple Bond Correlation

vii

HMQC Hetronuclear Multiple Quantum Correlation

HPLC High Performance Liquid Chromatography

HSQC Hetronuclear Single Bond Correlation

Hz hertz

IAS Institute of Applied Sciences

IC50 Inhibitory concentration (IC) where 50% of a

population dies.

IR Infrared

J Coupling constant

LC-MS Liquid Chromatography-Mass Spectroscopy

LD50 Lethal dosage where 50% of a population dies.

m meter

m/z mass to charge ratio

MeCN Acetonitrile

MeOH Methanol

mg milligram

MHz Megahertz

min minute

ml milliliter

mM millimolar

mmu Molecular mass unit

MNP Marine Natural Product

Mult. Multiplicity.

NCI National Cancer Institute

NKT Natural killer T-cells lymphocytes

nm nanometer

NP Normal phase

PKC Protein kinase C

ppm parts per million

RBF round bottom flask

RDB equiv. Relative double bond equivalence

Rf Retention factor

Rho Rho-protein

viii

RP Reverse phase

Rt Retention time

sec second

sp. species

TFA Trifluoroacetic acid

TLC Thin Layer Chromatography

USA United Stated of America

USP The University of the South Pacific

UV Ultraviolet

VLC Vacuum Liquid Chromatography

WB Butanol fraction

α alpha

β beta

δ chemical shift (ppm)

λ wavelength

μl microlitre

ix

TABLE OF CONTENTS

ABSTRACT ............................................................................................................ v

ABBREVIATION .................................................................................................. vi

TABLE OF CONTENTS ........................................................................................... ix

LIST OF FIGURES .................................................................................................. xii

LIST OF TABLES ................................................................................................... xvi

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ....................... 1

1.1 Introduction ....................................................................................................... 1

1.1.1 Natural products.......................................................................................... 1

1.1.2 Marine natural products .......................................................................... 3

1.1.3 Marine natural products derived drugs in clinical trials ......................... 4

1.1.4 Morphology of marine sponges ............................................................ 15

1.2 Aim and objectives .......................................................................................... 19

1.3 Literature Review ............................................................................................ 19

1.3.1 The order Dictyoceratida ...................................................................... 20

1.3.2 The family Spongiidae .......................................................................... 23

1.3.3 Chemistry of the Genus Hippospongia ................................................. 35

CHAPTER 2 METHODOLOGY ........................................................................ 43

2.1 General Procedure ........................................................................................... 43

2.2 Isolation of bioactive compounds .................................................................... 44

x

2.2.1 Collection and identification of the specimen ....................................... 44

2.2.2 Extraction, isolation and purification .................................................... 44

2.2.3 Extraction and solvent partitioning ....................................................... 44

2.2.4 Chromatography and purification ......................................................... 45

2.2.4.1 Isolation of Compound 1............................................................... 45



2.3 Bioassay ........................................................................................................... 48

2.3.1 Brine shrimp bioassay ........................................................................... 48

2.3.2 Hatching the eggs .................................................................................. 48

2.3.3 Preparation of samples .......................................................................... 48

2.3.4 Cytotoxicity assay ................................................................................. 49

CHAPTER 3 RESULTS AND DISCUSSION ................................................... 50

3.1 Isolation of cytotoxic compounds from Hippospongia sp. .............................. 50

3.2 Isolation and purification of cytotoxic compound 1 ........................................ 51

3.2.1 Purity check ........................................................................................... 54

3.2.2 UV and IR spectroscopic analysis ........................................................ 55

3.2.3 Crystallization of the compound ........................................................... 55

3.2.4 LC-MS and NMR data of compound 1 ................................................. 57

3.2.5 Structure elucidation of the compound 1 .............................................. 60

3.2.6 Verification of the stereochemistry ....................................................... 70

3.2.7 Some reported properties of epi-ilimaquinone ...................................... 81

3.3 Isolation of cytotoxic compound 2 .................................................................. 82

3.3.1 UV and IR spectroscopic analysis ........................................................ 84

xi

3.3.2 LC-MS and NMR data of compound 2 ................................................. 86

3.3.3 Structure elucidation of compound 2 .................................................... 93

3.3.4 Some reported properties of compound 2 ............................................. 98

3.4. Isolation of cytotoxic compound 3 ................................................................. 99

3.4.1 LC-MS data of compound 3 ................................................................ 101

3.4.2 Structure elucidation of compound 3 .................................................. 102

3.5 Sesquiterpene quinones and related compounds from sponge of the order Dictyoceratida .............................................................................................. 113

CHAPTER 4 CONCLUSION ........................................................................... 114

REFERENCES .................................................................................................... 116

APPENDIX ......................................................................................................... 124

Appendix 1....................................................................................................... 124

Appendix 2....................................................................................................... 130

Appendix 3....................................................................................................... 134

Appendix 4....................................................................................................... 139

xii

LIST OF FIGURES

Figure 1-1: Distribution of marine natural products by phylum from 1965-2007. .... 14

Figure 1-2: Specialized cells of a sponge. .................................................................. 18

Figure 1-3: Taxonomic relationships within the order Dictyoceratida. ..................... 20

Figure 2-1: Isolation of secondary metabolite from the sponge. ............................... 47

Figure 3-1: NP-TLC profile of the fractions obtained from the FD fraction. ............ 52

Figure 3-2: NP-TLC (a) of the active component from FHF4H4 and FDF2H2

fraction. RP-TLC (b) of the compound isolated as yellow and violet liquid. ............ 52

Figure 3-3: Chromatogram of the active FJ05-097FDF2 fraction. ............................ 53

Figure 3-4: Chromatogram of the active HPLC fraction on a Refractive Index

Detector. ..................................................................................................................... 54

Figure 3-5: IR spectrum (KBr) of the active compound 1 ......................................... 56

Figure 3-6: Electron spray ionisation mass spectrum of compound 1. Inset: UV

profile of compound 1. ............................................................................................... 58

Figure 3-7: HMBC correlations of partial structure 1A. ............................................ 61

Figure 3-8: HMBC correlation of partial structure 1B............................................... 61

Figure 3-9: HMBC and COSY correlations of partial structure 1C. ......................... 62

Figure 3-10: All HMBC correlations of 1,4-benzoquinone chromophore. ................ 62

Figure 3-11: COSY correlation of partial structure 1D. ............................................ 65

Figure 3-12: HMBC correlations of partial structure 1E. .......................................... 65

Figure 3-13: Partial structure 1F showing COSY and HMBC correlations. ............. 66

Figure 3-14: Partial structure 1G showing COSY and HMBC correlations. ............. 66

Figure 3-15: Partial structure 1H showing COSY correlations. ................................ 67

Figure 3-16: Partial structure 1I showing HMBC correlation of the sesquiterpene

moiety. ........................................................................................................................ 68

Figure 3-17: Final structure of compound 1. ............................................................. 69

xiii

Figure 3-18: Absolute stereochemistry of epi-ilimaquinone. .................................... 71

Figure 3-19: Calculated mean δ 13C for four different 4,9-friedodrimene skeletons

found in sponges. ....................................................................................................... 72

Figure 3-20: Tautomerisation of epi-ilimaquinone and ilimaquinone. ...................... 73

Figure 3-21: 1H NMR (400MHz) spectrum of compound 1 (CDCl3). ...................... 74

Figure 3-22: 13C NMR spectrum of compound 1 (CDCl3). ....................................... 75

Figure 3-23: 1H-1H COSY spectrum of compound 1 (CDCl3). ................................. 76

Figure 3-24: HMQC spectrum of compound 1 (CDCl3). ........................................... 77

Figure 3-25: HMBC spectrum of compound 1 (CDCl3). ........................................... 78

Figure 3-26: Comparison of the 1H NMR of the two epimers. ................................. 79

Figure 3-27: NP-TLC profile of epi-ilimaquinone. ................................................... 79

Figure 3-28: HPLC chromatogram of the bioactive FJ05-097FDF4 fraction. .......... 83

Figure 3-29: RP-TLC of fraction FJ05-097FDF4H6. ................................................ 84

Figure 3-30: IR spectrum (KBr) of compound 2. Inset: UV profile of compound 2. 85

Figure 3-31: ESI-LCMS of compound 2. .................................................................. 86

Figure 3-32: 1H NMR (400MHz) of compound 2 (CD3OD). .................................... 89

Figure 3-33: 1H-1H COSY of compound 2 (CD3OD). ............................................... 90

Figure 3-34: HSQC spectrum of compound 2 (CD3OD). .......................................... 91

Figure 3-35: HMBC spectrum of compound 2 (CD3OD). ......................................... 92

Figure 3-36: HMBC correlations of partial structure 2A. .......................................... 93

Figure 3-37: HMBC correlations of partial structure 2B. .......................................... 94

Figure 3-38: HMBC correlations of compound 2. ..................................................... 95

Figure 3-39: Structure of compound 2, smenospongine. ........................................... 95

Figure 3-40: Final structure of compound 2, [106] smenospongine. ......................... 98

Figure 3-41: HPLC chromatogram of the bioactive FJ05-097FMF2 fraction. ........ 100

Figure 3-42: RP-TLC of fraction FJ05-097FMF2H7. ............................................. 100

xiv

Figure 3-43: ESI-LCMS of compound 3. Inset: UV profile of compound 3. .......... 101

Figure 3-44: HMBC correlations of partial structure 3A. ........................................ 102

Figure 3-45: 1H NMR of compound 3 (CD3OD). .................................................... 105

Figure 3-46: 13C NMR of compound 3 (CD3OD). ................................................... 106

Figure 3-47: 1H-1H COSY of compound 3 (CD3OD). ............................................. 107

Figure 3-48: HSQC spectrum of compound 3 (CD3OD). ........................................ 108

Figure 3-49: HMBC spectrum of compound 3 (CD3OD). ....................................... 109

Figure 3-50: HMBC correlations of partial structure 3B. ........................................ 110

Figure 3-51: Final structure of compound 3, [107] Glycinylilimaquinone. ............. 111

LIST OF FIGURES IN APPENDIX

Figure A 1: Expanded 1H NMR of compound 1. ..................................................... 124

Figure A 2: Expanded 1H NMR of compound 1. ..................................................... 124

Figure A 3: Expanded 13C NMR of compound 1. .................................................... 125

Figure A 4: Expanded 13C NMR of compound 1. .................................................... 125

Figure A 5: Expanded 1H-1H COSY of compound 1. .............................................. 126

Figure A 6: Expanded 1H-1H COSY of compound 1. .............................................. 126

Figure A 7: Expanded HMQC spectrum of compound 1. ....................................... 127

Figure A 8: Expanded HMQC spectrum of compound 1. ....................................... 127

Figure A 9: Expanded HMBC spectrum of compound 1. ........................................ 128

Figure A 10: Expanded HMBC spectrum of compound 1. ...................................... 128


Figure A 12: Expanded 1H NMR of compound 2. ................................................... 130


Figure A 14: Expanded 1H-1H COSY of compound 2. ............................................ 131

Figure A 15: Expanded 1H-1H COSY of compound 2. ............................................ 131

xv

Figure A 16: Expanded HSQC spectrum of compound 2. ....................................... 132






Figure A 22: Expanded 13C NMR of compound 3. .................................................. 135

Figure A 23: Expanded 13C NMR spectrum of compound 3. .................................. 135

Figure A 24: Expanded 1H-1H COSY spectrum of compound 3. ............................ 136

Figure A 25: Expanded 1H-1H COSY spectrum of compound 3. ............................ 136





Figure A 30: Plot for calculation of LD50. ............................................................... 139

xvi

LIST OF TABLES

Table 1-1: Selected Marine natural products in development in anticancer trials ..... 13

Table 3-1: Weight and bioactivity of the four fractions ............................................. 50

Table 3-2: Weight and bioactivity of FH and FD fractions ....................................... 51

Table 3-3: Weight and bioactivity of the HPLC fractions ......................................... 53

Table 3-4: Elemental composition search on mass 359.22m/z .................................. 58

Table 3-5: NMR data for the compound 1 (CDCl3) ................................................... 59

Table 3-6: NMR values of epi-ilimaquinone and ilimaquinone (CDCl3) .................. 80

Table 3-7: Weights and activity of the FJ05-097FDF4 HPLC sub-fractions ............ 83

Table 3-8: Elemental composition search on mass 344.22m/z .................................. 87

Table 3-9: NMR data of compound 2 in CD3OD ...................................................... 88

Table 3-10: Comparison of the NMR data for smenospongine ................................. 97

Table 3-11: Weights and bioactivity of the FJ05-097FMF2 HPLC sub-fractions..... 99

Table 3-12: Elemental composition search on mass 402.23m/z .............................. 101

Table 3-13: NMR data of compound 3 in CD3OD .................................................. 104

Table 3-14: Comparison of the NMR data of glycinylilimaquinone ....................... 111

Table 4-1: Summary of three compounds isolated .................................................. 114

1

CHAPTER 1 INTRODUCTION AND LITERATURE

REVIEW

1.1 Introduction

1.1.1 Natural products

Throughout the ages, Nature has catered to the basic needs of humans, not the least

of which is the provisi

on of medicines for the treatment of a wide spectrum of diseases. Plants, in

particular, have played a dominant role in the development of sophisticated

traditional medicine systems.1 Clinical, pharmacological, and chemical studies of

these traditional medicines, which were derived from plants, were the basis of most

early medicines such as quinine [1], morphine [2], aspirin [3] pilocarpine [4] and

digitoxin [5].

CO2H

OAc

[3] AsprinN

HON

H

[1] Quinine

H3CO

HO

HO

HO

NCH3

[2] Morphine

O N

N

O

[4] Pilocarpine

O

OH

HO OO

OO

O

OH OH

OO

H

H

H

OH

H

[5] Digitoxin

2

Natural products arise from a narrow selection of simple building blocks and

biosynthetic pathways, but the resulting diversity in both structure and function of

these molecules far exceeds that found in synthetic compound libraries. Natural

products are, therefore, a unique source of inspiration for chemists and biologists,

and it is not surprising that they are the lead compounds for many drug discovery and

development programs.2 Analysis of the literature from 1981-2007 shows that more

than 50% of the drugs approved since 1994 are based on natural products and in the

period 2005-2007 thirteen natural-product related drugs were approved and five of

these represented first members of new class of drugs: the peptides exenatide and

ziconotide, and the small molecules ixabepilone, retapamulin and trabectedin.

Compounds isolated from natural sources cover a range of therapeutic indications:

anti-cancer, anti-infective, anti-diabetic, among others, and they show a great

diversity of chemical structures.3 Natural products have proven to be the most

reliable single source of new and effective anticancer agents. Over the last 25 years,

63% of anticancer drugs introduced are from natural products or are derived from

natural source. Reports also state that about 79 compounds that entered the clinical

trials as anticancer agents in 2005-2007 are natural products or natural product

analogues.4

Organic compounds from terrestrial and marine organisms have extensive past and

present use in the treatment of many diseases and serve as compounds of interest

both in their natural form and as templates for synthetic modification.5 The oceans of

the planet represent a huge unexplored resource, and, as the exploitation of terrestrial

resources proceeds, the marine environment offers a new frontier for research. From

a biological perspective, the ocean is indeed a treasure; it contains perhaps 200000

organisms, all of which survive in unusual conditions of higher salt content, low or

zero light, high pressure, and unusually high or low temperatures. These properties of

the marine environment make it very likely that marine organisms, and the chemicals

they produce, will be quite different from those associated with terrestrial biology.6

3

1.1.2 Marine natural products

Natural products possess a broad diversity of structure and function, and they provide

inspiration for chemistry, biology, and medicine.2 With the ocean covering 70% of

the Earth’s surface, and with the uniqueness of the environmental conditions present

in the oceans, it is easily understandable why the ocean can be considered as a very

promising source of natural drugs or synthetic derivatives, for the future.7 The

Ocean, which is called the ‘mother of origin of life’, is also the source of structurally

unique natural products that are mainly accumulated in living organisms. Several of

these compounds show pharmacological activities and are helpful for the discovery

of bioactive compounds, primarily for deadly diseases like cancer or acquired

immuno-deficiency syndrome (AIDS), while other compounds have been developed

as analgesics or to treat inflammation.8 Natural products isolated from marine

organisms have also been shown to have a great potential in drug discovery.7 Many

marine organisms are soft bodied and have a sedentary life style necessitating

chemical means of defense; therefore, they have evolved the ability to synthesize

toxic compounds or to obtain them from marine microorganisms. These compounds

help them deter predators, keep competitors at bay or paralyze their prey. Natural

products released into the water are rapidly diluted and, therefore, need to be highly

potent to have any effect. For this reason, and because of the immense biological

diversity in the sea as a whole, it is increasingly recognized that a huge number of

natural products and novel chemical entities exist in the oceans, with biological

activities that may be useful in the quest for finding drugs with greater efficacy and

specificity for the treatment of many human diseases.9 Marine organisms have

evolved biochemical and physiological mechanisms that include the production of

bioactive compounds for such purposes as reproduction, communication, and

protection against predation, infection and competition. With the continuous

emergence of new diseases and the development of drug resistance in harmful

bacteria, viruses, and cancer cells, there is a continuous need for the development of

new drugs with novel mechanisms of action.10

In recent years, many bioactive compounds have been extracted from various marine

animals like tunicates, sponges, soft corals, sea hares, nudibranchs, bryozoans, sea

4

slugs and marine organisms8. To date, researchers have isolated more than 20000

natural products from marine flora and fauna11 and only in the year 2006, 779 new

compounds were isolated which were of marine origin.12 The sponges and

coelenterates continue to dominate as source phyla of new compounds, with

microorganisms being the other major phyla.13 Most sessile marine invertebrates

contain a primitive immune system and produce toxic chemicals as a form of

defense.

1.1.3 Marine natural products derived drugs in clinical trials

In particular, the marine environment, a rich source of structurally unique, bioactive

metabolites, has produced a number of drug candidates that are currently in clinical

trials.11 The development of clinically useful drugs is a long and tedious process that

can take many years to come to fruition.2 It has been almost five decades since the

isolation of the first leads, spongothymidine [6] and spongouridine [7], from the

marine sponge Cryptotethia crypta by Bergman that eventually led to the

development of Ara-A [8] (vidarabine™, Vidarabin Thilo™ an antiviral agent) and

Ara-C [9] (cytarabine, Alexan™, Udicil an antileukemia agent) which was approved

by FDA to be used commercially in 1969 and 1976, respectively.

O

OH

CH2OH

N

HN

O

R

O

HO

N

N N

N

OHO

OH

CH2OH

NH2

O

OH

CH2OH

HO

N

N

O

NH2

[6] R= H spongothymidine [8] Ara-A [9] Ara-C

[7] R= Me spongouridine

5

Ara A [8] along with its acetyl congener Ara U, were later isolated as natural

products from the gorgonian Eunicella cavolini.10, 14 In spite of all efforts in marine

natural product chemistry over the last few decades, the only other marine-derived

compound launched to the drug market apart from Ara A and Ara C, are Yondelis™

and Prialt™. It was not until 2004 that the next MNP, ziconotide [10] (Prialt™), was

approved for the treatment of severe chronic pain. This was soon followed by the

orphan drugi status granted to trabectedin [11] (Yondelis™) for the treatment of soft

tissue sarcomas and ovarian cancer, and its registration in 2007 in the EU for the

treatment of soft tissue sarcoma.15

Species of the cone snail genus, Conus, inject venom composed of combinatorial

libraries of several hundred peptides to stun their prey prior to capture, and the

venom may also be used for defense against predators. One component of this

combinatorial mixture has been developed as Ziconotide [10], a non-narcotic

analgesic, currently marketed as Prialt.

O

O

N

N

Me

OMe

HOH

Me

OAc

HO

O S

O

NH

HO

MeO

Me

[11] Ecteinascidin

i An orphan drug is a pharmaceutical agent that has been developed specifically to treat a rare medical condition, the condition itself being referred to as an orphan disease.

6

[10] Ziconotide

H Cys Lys Gly Lys

GlyAla

Lys

Cys Ser Arg Leu Met

Cys

Cys

CysH2N

Lys

Gly Ser Arg Cys

TyrAsp

SerGly

Thr

Ziconotide [10], known as ω-contotoxin MVIIA, selectively blocks the N-type

voltage-gated calcium channel. As a novel non-opioid analgesic, ziconotide was

developed for the treatment of severe chronic pain, and is currently used in pain

management.5 There is only one conotoxin, Xen-2174 [12], currently undergoing

clinical evaluation, as all of the other conotoxin trials have been halted or

discontinued. Xen-2174 [12] (χ-MrIA), a 13 amino acid peptide with 2 cysteine

bridges isolated from Conus marmoreus, has been found to inhibit the

norepinephrine transporter (NET), a known CNS drug target that is inhibited by the

antidepressant desipramine. Xenome are evaluating Xen- 2174 in Phase I/IIa trials

for the treatment of cancer pain.16

NGVCCGYKLCHOC

[12] Xen-2174

Trabectedin [11] (Yondelis™, ecteinascidin-743, ET-743), a tetrahydroisoquinoline

alkaloid produced by the ascidian, Ecteinascidia turbinata, was approved in

September 2007 for the treatment of advanced soft tissue sarcoma and has been

marketed by PharmaMar. Another tunicate derived natural product, Aplidin

(dehydrodidemnin B) is currently in phase II clinical trials which is also sponsored

7

by PharmaMar.17 Trabectedin [11] is also in Phase III clinical trials for the treatment

of ovarian cancer (with Johnson & Johnsons in the US) and other ongoing Phase II

trials include paediatric sarcomas, breast and prostate cancers.

Apart from trabectedin (or ET-743) isolated from Ecteinascidia turbinate (in clinical

use), bryostatin, a macrolide lactone isolated from a species of bryozoan, Bugula

neritina; kahalalide F, a cyclodepsipeptide toxin isolated from the mollusc Elysia

rubefescens; didemnin B isolated from Carribean tunicate, and the second

generation didemnin aplidine isolated from Aplidium albicans, have reached clinical

trials. More recently other compounds such as squalamine, isolated from the dogfish

shark Squalus acanthias; LAF389, a synthetic analogue of bengamide B (a

compound isolated from the Jaspis sponges of the coral reefs near the Fiji Islands

and Australia); and neovastat, a derivative of shark cartilage extract, have been

developed to the stage of clinical trials. Most of these compounds have been

recognized by the FDA and the EMEA (The European Medicines Agency) as

“orphan drugs” for the treatment of various neoplasms.18 The assignment of orphan

status to a disease and to any drugs developed to treat it is a matter of public policy

in many countries, and has resulted in medical breakthroughs that would not have

otherwise been achieved due to the economics of drug research and development.19

Didemnin B was first isolated by the Rinehart group in 1981 from the tunicate

Trididemnum solidum and displayed antiviral and in vivo cytotoxic activities at nano

molar concentrations. Early studies showed that didemnin B inhibits palmitoyl

protein thioesterase in a non competitive manner; although this low affinity target did

not fully account for the nano molar cell inhibition observed with the natural product.

Didemnin B induces inhibition of protein synthesis at a concentration that is

commensurate with cell growth inhibition. This is achieved by stabilization of

aminoacyl-tRNA and prevention of EF-2-dependent translocation on the ribosome.

However, inhibition of protein synthesis does not seem to be the primary cause of

apoptosis. Apoptosis induced by didemnin B is dependent on protein tyrosine kinases

and can be inhibited using protein tyrosine kinase inhibitors or rapamycin, possibly

through the interaction of rapamycin with the immunophilin FKBP25.20 Phase I and

Phase II clinical trials were conducted against previously treated non-small cell lung

cancer (NSClC), breast cancer, small-cell lung cancer, non-Hodgkin’s lymphoma,

8

metastatic melanoma, glioblastoma multiforme, and CNS tumors. However, these

trials resulted in significant neuromuscular toxicity and no objective responses.

Nevertheless, didemnin B showed activity in patients with advanced pretreated non-

Hodgkin’s lymphoma, but trials were suspended owing to onset of severe fatigue in

patients. Other trials showed a high incidence of anaphylaxis, and were therefore

terminated. All current trials of didemnim B are on hold.20

Aplidine (dehydrodidemnin B) was first reported in a 1991 patent by Rinehart and is

obtained from the Mediterranean tunicate Aplidium albicans.20 Aplidine differs from

didemnin B only in replacement of the N-lactyl side chain with a pyruvyl group.

Aplidine shows similar levels of antitumour activity to didemnin B in cultured tumor

cells, and has been shown to induce apoptosis by induction of oxidative stress, which

triggers the pro-apoptotic receptor Fas (CD-95) and induces mitochondrion-mediated

apoptosis. Aplidine also activates p38 mitogen-activated protein kinases (MAPKs)

and JNK and inhibits secretion of vascular endothelial growth factor (VEGF).

Aplidine has also been shown to inhibit angiogenesis in chick embryos in vivo and to

selectively disrupt β-sheet fibrils caused by prion protein sequence PrP 106–126 in a

1:1 molar ratio. Phase II clinical trials with aplidine are ongoing for metastatic

melanoma, multiple myeloma, non-Hodgkin’s lymphoma, acute lymphoblastic

leukaemia, prostate cancer and bladder cancer.20

Discodermolide was first isolated in 1990 by Gunasekera and co-workers at the

Harbor Branch Oceanographic Institute, Florida, USA, from the rare deep-water

sponge Discodermia dissoluta.20, 21 Discodermolide functions as an

immunosuppressant and induces G2/M phase cell-cycle arrest in lymphoid and non-

lymphoid cells at nanomolar concentrations, Discodermolide has further shown in

vivo activity against HCT-116 colorectal cancer xenografts in mice. Co-treatment of

mice with (+)-anhydrodiscodermolide C and bacteriolytic treatment with Clostridium

novyi-NT spores resulted in a rapid, complete cure of four out of five animals with a

single injection. A Phase I clinical study of (+)-discodermolide showed no

neuropathy or neutropaenia, and demonstrated mild-to-moderate toxicity from 0.6

mg/m2 to 19.2 mg/m2. The pharmacokinetics of (+)-discodermolide were shown to

be non-linear with recycling of (+)-discodermolide between tissues and the

9

circulatory system. At present, Novartis has discontinued Phase I trials with (+)-

discodermolide owing to lack of efficacy and toxicity problems.20, 22

The discovery of the dolastatins from Dolabella auricularia,3 had its beginnings in

1972 during an explorative expedition to the island state of Mauritius in the Western

Indian Ocean.20 Dolastatin 10 exhibited outstanding in vitro cytotoxic activity against

cancer cells. Alcohol extracts of D. auricularia showed exceptionally potent activity

against murine NCI P388 lymphocytic leukaemia cells at subnanomolar

concentrations (ED50 value of 4.6 × 10–5μg/ml).20 The dolastatins inhibit cell

proliferation and induce apoptosis in numerous malignant cell lines.22 Dolastatins

exerted profound cytotoxic effects in animals bearing intraperitoneal tumors; in

addition, they exhibited synergistic antitumor activity with vinca alkaloids and

bryostatin. Dolastatin 10 has been evaluated in various phase I clinical trials.

Preliminary data indicated that 40% of patients developed moderate peripheral

neuropathy and patients with underlying neuropathy are at increased risk for this

side-effect. Subsequent phase II studies failed to demonstrate activity in solid

tumors.22

Halichondrin B, extracted from a black-colored marine sponge Halichondria okadai,

exhibits potent in vivo antitumor activity.21 Halichondrin B was found to be most

potent among the series. It was isolated in very low yield (1.8x10-6% to 4.0x10-5%).21

Mechanistic studies conducted on halichondrin B revealed that it targeted tubulin. It

was demonstrated by the NCI that halichondrin B is a mitotic inhibitor which binds

to the vinca domain of tubulin, resulting in inhibition of microtubule formation and

tubulin-dependent GTP hydrolysis. The compound was found to be extremely

effective in vivo against human solid tumors which had been xenografted into

immune-deficient mice. It also exhibited an IC50 value of 0.3 nM against L-1210

murine leukemia cells, thereby surpassing in potency the best previously known

agents, dolastatin-10 (0.5 nM), rhizoxin (1 nM) and vinblastine (20 nM). More than

1,100 vials of Halichondrin B were distributed during 2004 for phase I and II clinical

trials.20, 21

Seasonal collections of the sacoglossan (sea slug) Elysia rufescens by the Scheuer

group from the University of Hawaii led to the isolation of the novel antitumor

10

depsipeptide kahalalide F in 1993 in addition to other analogues20, 21, 23. Kahalalide F

is a C75 cyclic tridecapeptide that contains several unusual amino-acid residues,

including the rare Z-dehydroaminobutyric acid found only in a few peptides

including the antibiotics cypemycin and hassallidin A.20 Kahalalide F was licensed

by the University of Hawaii to PharmaMar in the early 1990s.20 The mechanism of

action of kahalalide F has not yet been fully elucidated. Kahalalide F is active mainly

at the lysosomal level and induces vacuolization, which may explain its activity on

tissues that actively secrete lysosomal proteins, such as prostate cells.20 A preclinical

study of kahalalide F in rats reported renal toxicity using single doses at the

maximum tolerated dose of 1,800μg/m2 administered intravenously. However, a

multiple-dose regimen reduced drug-induced toxicity.20 A Phase I study on

androgen-refractory prostate cancer reported a maximum tolerated dose of 930μg/m2

per day, and determined a dose of 560μg/m2 per day for Phase II trials.20 Out of 32

patients, one patient treated at a dose of 80μg/m2 had a partial response with a

corresponding prostate-specific antigen reduction of at least 50%, and five patients

showed stable disease.20 Following a Phase I trial suggesting a positive therapeutic

index on advanced solid tumours, kahalalide F is currently in Phase II clinical trials

for solid tumours including melanoma, NSClC and hepatocellular carcinoma.20, 21

O

O

H

HO

HO

OH

O

O O

O O

O

O

O

O OO

O

O

H

H

H

H

H

H

H

HH

H HH

Halichondrin B

11

HN O

HN

ONH H

O

HN

OO

NH

O

N

H

H

O

HN

O

NH

O

NH

O HN

HO H

O

NH

O

HN

OO

H

H

Kahalide F

O

OH

Didemnin B R=

NH

OH

O

O

O

Me

O

NHO

NH

O O

N

O

N

OO

N

O

Me

O R

OMe

Me

O

OH

Aplidine R=

12

N

Me

MeNH

N

O

OMe

N

O

OMeHN

OS

N

Dolastatin

OH

OH

OHOO

NH2

O

OH

OH

Discodermolide

Among the previously mentioned compounds, trabectedin has received the most

extensive clinical investigation. It has shown clinical activity in a broad spectrum of

solid tumours and in September 2007, EMEA granted its marketing authorization for

the treatment of soft tissue sarcoma after failure of standard chemotherapy.18 There is

an ever-expanding list of marine natural products or synthetics inspired by marine-

derived compounds currently in or about to enter cancer clinical trials. As of 2009,

some of the selected anticancer drugs in phase I and II of the clinical trials24 are

summarized in Table 1-1.

13

Table 1-1: Selected Marine natural products in development in anticancer trials

Clinical trial Name Source Target

In clinical use ectenaisscindin 743 (Yondelis) tunicate tubulin

phase III E7389 (halichondrin B inspired) synthetic tubulin

phase II dehydrodidemnin B (Aplidine) tunicate ornithine

decarboxylase

phase II soblidotin (aka TZT1027, dola-10 insp.) synthetic tubulin

phase II Synthadotin (aka ILX651, dola-15 insp.) synthetic tubulin

phase II bryostatin 1 bryozoan PKC

phase II squalamine shark angiogenesis

phase II kahalalide F mollusk multiple

phase l PM02734 (kahalalide insp.) synthetic solid tumor

phase l Zalypsis (jorumycin insp.) synthetic DNA

phase l E7974 (hemiasterlin insp.) synthetic tubulin

phase l taltobulin (aka HTI286, hemiasterlin insp.) synthetic tubulin

phase l salinosporamide A (aka NPI0052) bacteria proteasome

phase l spisulosine (aka ES285) clam Rho

phase l KRN-7000 (agelasphin insp.) synthetic NKT

phase l NPI 2358 (halimide insp.) synthetic tubulin

phase l LBH 589 (psammaplin insp.) synthetic HDAC

Sponges, as stated by Blunt, are the largest source of new marine natural products

reported annually25 (Figure 1-1) and they have been providing a range of bioactive

compounds for the pharmaceutical industry since the 1950s, including the natural

product analogue cytosine arabinoside from the Caribbean sponge Tethya crypta,

halichondrin B from the Japanese sponge Halichondria okadai, discodermolide from

the Caribbean sponge Discodermia dissoluta and agelasphin from Agelas

mauritianus.11 Data extracted from the National Cancer Institute (NCI) preclinical

antitumor drug discovery screen showed that sponges exhibited more cytotoxic

extracts compared to plants and other marine invertebrates.17

14

Figure 1-1: Distribution of marine natural products by phylum from 1965-2007.

Sponges are extremely well represented in the marine environment, with over

700011-9000 species described and as many species not yet described26, ranging from

shallow-water to those inhabiting depths of over 8000 m, with some deep-water

species adopting carnivorous behaviour.11 Sponge metabolites, predominantly from

shallow-water species, have been reviewed previously.11

Porifera are attractive subjects for natural product chemists due to the sheer number

of secondary metabolites produced, the novelty of structures encountered, and the

therapeutic potential of these compounds in the treatment of human diseases.26 Some

proposed ecological roles for sponge metabolites include antifeedant, antifoulants,

antibiotics, antisettlememnt cues and photoprotective agents. The phylum Porifera is

comprised of three classes distinguished primarily by sketeton characteristics:

Hexactinellida, Calcarea, and Demospongiae. A few hexactinellid sponges have been

investigated by natural product chemists, but there are no reports of metabolites from

the 102 genera in this class. Sponges of the class Calcera are exclusively marine and

possess skeletons of free calcareous spicules. Within the Calcera, only the genera

Leucetta and Clathrina have reported chemistry and all 31 calcarean metabolites are

amino acid derived. Demospongia, the class which encompasses most living

sponges, are a morphologically diverse group with fibrous protein skeletons

sometimes supplemented by siliceous spicules having numerous possible

15

configurations.26 Metabolites reported from the order Dictyoceratidan are

predominantly isoprenid in origin. The distribution of isoprenoid class is specific

within the dictyocertid families: linear furano sesterterpenes (Irciniidae),

sesterterpenes with tetronic acid functional groups (Thorectidae), meroterpenoids

(Spongiidae), and sesquiterpenes (Dysideidae). It is also important to note that there

are some deviations from the general trends of the secondary metabolite distribution

within the subclass. The largest number of secondary metabolites has been reported

from Dysidae sp.26

1.1.4 Morphology of marine sponges Sponges are the simplest of the mutlicellular animals and belong to the phylum

Porifera. Their bodies consist of jelly-like mesohyl sandwiched between two thin

layers of cells. While all the animals have unspecialized cells that can transform into

specialized cells, sponges are unique in having some specialized cells that can

transform into other types, often migrating between the main cell layers and the

mesohyl in the process. Sponges do not have nervous, digestive or circulatory

systems. Instead, most rely on maintaining a constant water flow through their bodies

to obtain food and oxygen and to remove waste, and the shapes of their bodies are

adapted to maximize the efficiency of the water flow. All are sessile aquatic animals

and, although there are freshwater species, the great majorities are marine species,

ranging from tidal zones to depths exceeding 8800 meters. While most of the

approximately 7000 known species feed on bacteria and other food particles in the

water, some host photosynthesizing micro-organisms as endosymbionts and these

alliances often produce more food and oxygen than they consume.27

Sponges are known for regenerating from fragments that are broken off, although

this only works if the fragments include the right types of cells. A few species

reproduce by budding. When conditions deteriorate, for example as temperatures

drop, many freshwater species and a few marine ones produce gemmules, "survival

pods" of unspecialized cells that remain dormant until conditions improve and then

either form completely new sponges or re-colonize the skeletons of their parents.

However, most sponges use sexual reproduction by releasing sperm cells into the

16

water. In viviparousii species the cells that capture most of the adults' food capture

the sperm cells but, instead of digesting them, transport them to ova in the parent's

mesohyl. The fertilized eggs begin development within the parent and the larvae are

released to swim off in search of places to settle. In oviparous species both sperm

and egg cells are released into the water and fertilization and development take place

outside the parent's bodies.27

Sponges use various materials to reinforce their mesohyl and in some cases to

produce skeletons, and this forms the main basis for classifying sponges. Calcareous

sponges produce spicules made of calcium carbonate. Demosponges reinforce the

mesohyl with fibers of a special form of collagen called spongin, most also produce

spicules of silica, and a few secrete massive external frameworks of calcium

carbonate. Although glass sponges also produce spicules made of silica, their bodies

mainly consist of syncytia that in some ways behave like many cells sharing a single

external membrane, and in others like individual cells with multiple nuclei. Probably

because of their variety of construction methods, demosponges constitute about 90%

of all known species, including all freshwater ones, and have the widest range of

habitats. Calcareous sponges are restricted to relatively shallow marine waters where

production of calcium carbonate is easiest. The fragile glass sponges are restricted to

polar regions and the ocean depths where predators are rare, and their feeding

systems very efficiently harvest what little food is available.27

The body plan of a typical sponge is shown in Figure 1-2 below. Water (carrying

suspended plankton and other potential food) enters numerous small pores called

ostia (singular, ostium). The ostia are surrounded by donut-shaped cells called

porocytes that open and close to control water flow. For the sponge depicted in

Figure 1-2, water flows directly into an open chamber called the spongocoel (the

term "coel" refers to an open space or body cavity in an animal). Water leaves the

spongocoel by a larger opening (the osculum).28

ii A viviparous animal is an animal employing vivipary: the embryo develops inside the body of the mother, as opposed to outside in an egg (ovipary). The mother then gives live birth.

17

The interior of the spongocoel is lined with flagellated cells called choanocytes (or

collar cells; Figure 1-2). The choanocytes have a tubular collar facing the

spongocoel. A flagellum extends from the center of this collar, the movement of

which creates currents that force water through the sponge's "plumbing system".

Sponges can control the water flow by various combinations of wholly or partially

closing the osculum and ostia (the intake pores) and varying the beat of the flagella,

and may shut it down if there is a lot of sand or silt in the water.28 Suspended food

particles (plankton, larvae, etc) in the water are drawn through the collar from below,

trapped on the outside of the collar, and then phagocytized.28

18

Fi

gure

1-2

: Spe

cial

ized

cel

ls o

f a sp

onge

.

19

1.2 Aim and objectives

Aim: To isolate and structurally characterize the cytotoxic natural product from the

marine sponge, Hippospongia sp.

Objective

1. To acquire skills in extraction, isolation and purification of the active

components of the marine specimen using bioassay-guided fractionation.

2. To learn the skills of conducting Brine Shrimp Bioassay.

3. To obtain knowledge in the use of 1D and 2D NMR technique and other

spectral data for the characterization of the active compound.

4. To apply the above skills in isolation and characterization of the major

compounds of a sponge of the Genus Hippospongia.

1.3 Literature Review The sponges of the genus Hippospongia belong to the order of Dictyoceratida and

family Spongiidae. Sponges belonging to this order have four families and within

each family there are a number of genera as shown in below Figure 1-3. (Note: not

all the sponges are shown in this figure). The sponge being studied has eleven genera

and the genus to which this sponge belongs to has thirty one species.29 Sponges of

the order Dictyoceratida are often prominent members of South Pacific island coral

reefs. Biologically, they are unique as their skeleton is fibrous rather than mineral in

content.30 The Demospongiae includes three subclasses (Homoscleromorpha,

Tetractinomorpha, and Ceracinomorpha) and are composed of 12 orders of which

Halichondria, Haplosclerida, and Dictyoceratida have been reported to contain the

highest number of novel secondary metabolites.31

20

Order Dicytoceratida Family Irciniidae Spongiidae Thorectidae Dysideidae Genus Iricinia Cosinoderma Aplysinopsis Citronia Psammocina Hippospongia Cacospongia Dysidea Sarcotragus Hyattella Collospongia Euryspongia Rhopaloeides Dactylospongia Lamellodysidea Spongia Fascaplysinopsis Pleraplysilla Fasciospongia Fenestraspongia Hyrtios Luffariella Narrabeena Petrosaspongia Scalarispongia Semitaspongia Smenospongia Taonura Thorecta Thorectandra Thorectaxia

Figure 1-3: Taxonomic relationships within the order Dictyoceratida.

1.3.1 The order Dictyoceratida Many scalarane-type sesterterpenoids have been isolated from marine sponges

belonging to the order Dictyoceratida, and they showed a variety of biological

activities such as antimicrobial, cytotoxic, antifeedant, ichthyotoxic,

antiinflammatory, platelet-aggregation inhibitory and nerve growth factor synthesis-

stimulating.32 A number of tetracyclic sesterterpenes of the scalarane class have been

reported from marine sponges of the order Dictyoceratida and their predator

nudibranchs. The scalarane skeletons can vary from C24 nor-scalarane to C27

dihomo-scalarane types, the latter having methylation at C19, C20, C23, and/or C24.

These compounds have been reported to exhibit a wide spectrum of biological

activities including cytotoxicity, ichthyotoxicity, antiinflammation, erythroid

21

differentiation, anti-HIV and antimicrobial properties. The crude extract of an

Indonesian sponge, Phyllospongia sp. showed cytotoxicity and separation of the

extract gave seven new scalarane derivatives [13, 13a, 15-19] exhibiting weak

cytotoxicity. All isolated compounds [13-20] exhibited 30-95% inhibition of the

growth of KB cells at 10µg/mL.33

CHO

OR2

O

O

O

R1

R1 R2 [13] H H [13a] H Ac [13b] H R-MTPA [13c] H S-MTPA [14] Me H

OR4

O

O

R1

OR2

R3

R1 R2 R3 R4 [15] H α-OH H H [15a] H α-OAc H Ac [15b] H α-OMe H H [15c] H β-OMe H H [16] Me α-OH H H [16b] Me α-OMe H H [16c] Me β-OMe H H [17] H α- α-OMe H

22

R2

OH

O

O

O

R1

R1 R2 [18] H H [19] H OH [20] Me H

A fraction from a Carteriospongia sp. sponge activated Wnt signaling and was

subsequently shown to inhibit Glycogen Synthases Kinase (GSK-3β), which inhibits

Wnt signaling through phosphorylation of β-catenin. Three novel natural products,

carteriosulfonic acids A [21], B [22], and C [23], were identified as active

constituents. The carteriosulfonic acids contain unprecedented 4, 6, 7, 9-

tetrahydroxylated decanoic acid subunits. The Wnt signaling pathway plays major

roles in controlling cell proliferation and differentiation; therefore, misregulation of

the Wnt pathway has been implicated in a number of human diseases including

cancer and neurodegenerative diseases.34

OH

NH

O3S

O

HO

RO

HO

23

O

OH OH

[21] R=

O

O OHO

O

OH

[22] R=

[23] R=

Carteriosulfonic acid A

Carteriosulfonic acid B

Carteriosulfonic acid C

1.3.2 The family Spongiidae In search for biologically active metabolites from marine sponges, the ethanol

(EtOH) extract of an Australian marine sponge Spongia sp. (order Dictyoceratida,

family Spongiidae) which exhibited marked antimicrobial and cytotoxic activities

was investigated. From this sponge a new sesquiterpenoid quinone, named

cyclosmenospongine [24], together with the known compounds, smenospongiarine,

ilimaquinone, and smenospongine were isolated. Cyclosmenospongine [24] showed

moderate cytotoxic activity against mouse Ehrlich carcinoma cells (IC100 145µM)

and moderate hemolytic activity, inducing 50% hemolysis of mice blood

erythrocytes at a concentration of 70µM in 10 min.35

24

H

O

OO

NH2

[24] Cyclosmenospongine

In a continuing search for bioactive compounds from marine organisms, Takahashi’s

research group isolated new sesquiterpenoid quinones, metachromins J and K, from

an Okinawan sponge Spongia sp. Further investigation of extracts of this sponge

resulted in the isolation of six new sesquiterpenoid quinones with an amino acid

residue, metachromins L–Q [25-30] together with metachrominsm A [31] and C

[32].

OH

O

O

HN COOH[25] Metachromin L

OH

O

O

HN COOH[26] Metachromin M

25

OH

O

O

HN COOH[27] Metachromin N

OH OH

O

O

HN COOH[28] Metachromin O

OH OH

O

O

HN COOH[29] Metachromin P

OH OH

O

O

HN COOH[30] Metachromin Q

OH

26

OH

O

O

OMe[31] Metachromin AOH

O

O

OMe[32] Metachromin C

Metachromins L [26] and M [27] showed cytotoxicity against mouse lymphpcytic

leukemia cells (L1210) murine leukemia (IC50iii, 4.0 and 3.5mg/mL, respectively)

and KB human epidermoid carcinoma cells (IC50, 4.0 and 5.4mg/mL, respectively) in

vitro, while metachromins N [27], O [28], P [29], and Q [30] did not show such

activity (IC50>10mg/mL).36 Further investigation of the same sponge resulted in the

isolation of two new dimeric sesquiterpenoid quinones, nakijiquinones E [33] and F

[34]. These were the first dimeric sesquiterpene quinones possessing a 3-

aminobenzoate moiety. The compounds isolated did not showed any cytotoxicity

against murine leukemia P388 and L1210 and KB human epidermoid carcinoma

cells (IC50> 10µg/mL).37

iii IC50 is the inhibitory concentration (IC) where 50% of a population dies.

27

H

NH

HO

O CO2Me

OH

HOHO

[33] Nakijiquinone E

H

NH

HO

O CO2Me

OH

HOHO

[34] Nakijiquinone F

From the same sponge, Takahashi and group also isolated seven new, purple red,

sesquiterpenoid quinones, nakijiquinones J [35], K [36] and M-Q [38-42] and two

new nakijiquinones L [37] and R [43]. Nakijiquinones J-R [35-43] at 1mM were

tested for inhibitory activities against epidermal growth factor receptor (EGFR) and

human epidermal growth factor receptor (HER2) tryosine kinases. Among them,

nakijiquinones P [41] and R [43] exhibited activities against EGFR (% inhibition, 76

and >99, respectively), while nakijiquinones N [39], O [40] and R [43] showed

inhibitory activity against HER2 (66%, 59% and 52% respectively).38

28

O

O

R

OH

O

O

R

OH

[35]:R=R1[38]:R=R2[41]:R=R4[44]:R=R6

[36]:R=R1[39]:R=R2[40]:R=R3[42]:R=R4[43]:R=R5[45]:R=R6[46]:R=R7

R1=NH

NH

NH

NH

NH

SO2H

R6=OMeR7=NH2

R2=

R3=

R4=

R5=

OH

O

O

R

H

[37]:R=R1[47]:R=R6

The marine sponge Coscinoderma mathewsi Lendenfeld, which belongs to the order

Dictyoceratida and family Spongiidae has been a source of various terpenoid

compounds, as have other members of this family39. The crude ethanolic extracts of

the sponge C. mathewsi Lendenfield collected in Solomon Islands exhibited an anti-

phospholipase A2 (PLA2) activity (72% inhibition at 400mg/ml). The purification of

the polar extracts afforded two new nitrogen-containing cheilanthane

sesterterpenoids, coscinolactams A [47] and B [48], together with the known

suvanine [49]. 40

29

OSO3H

NO COOH

[47] Coscinolactam A

OSO3H

O[48] Suvanine

OSO3H

N

COOH

[49] Coscinolactam B

The in vitro pharmacological evaluation of the compounds on the inhibition of four

different secretary PLA2s (sPLA2), belonging to groups I (Naja naja venom and

porcine pancreatic enzymes), II (human synovial recombinant enzyme) and III (bee

venom enzyme), as well as on nitric oxide (NO) and prostaglandin E2 (PGE2)

production from macrophage line RAW 264.7 (mouse leukemia monocyte

macrophage cell line), showed that the suvanine aldehyde derivative exerted an

interesting anti-inflammatory profile mainly through the inhibition of inducible NO

expression40. From the same sponge, three known diterpenes spongia-13(16),14-

dien-19-oic acid [50], 15-oxospongi-13-en-19-oic acid [51], and 16-oxospongi-13-

en-19-oic acid [52] and in addition two new compounds, ent-13-norisocopalen-15-al-

18-oic acid [53] and 15ξ-hydroxy-16-oxospongi-13-en-19-oic acid [54] were

isolated. Compound [53] possesses a tricyclic skeleton bearing an aldehyde function,

while compound [54] possessed a tetracyclic lactol.39

30

O

COOHH

H

O

O

HCOOH

H

O

HCOOH

H

O

[50] spongia-13(16),14-dien-19-oic acid [51] 15-oxospongi-13-en-19-oic acid

[52] 16-oxospongi-13-en-19-oic acid

COOH

H

O

O

COOH

HH

H

O

OR

[53] ent-13-norisocopalen-15-al-18-oic acid [54] R=H 15-e-hydro-16-oxospongi-13-en-19-oic acid

The isolation of structurally unique sesquiterpenoid quinones dactyloquinones A-E,

from the Okinawan sponge Dactylospongia elegans, was reported by Mitome and

group.41 Each of these compounds has a unique cyclic-ether structure with ether

linkages between the C-1, C-8, or C-10 position of the 4,9-friedodrimane

sesquiterpene skeleton and the C-17 position of the 1,4-benzoquinone moiety.

Further examination for other sesquiterpenoid quinones and related compounds from

this sponge led to the isolation of the new sesquiterpenoid quinone

neodactyloquinone [55] and dactylolactones A-D [56-59]. Neodactyloquinone [55]

31

has a rare rearranged drimane skeleton, different from that of the dactyloquinones.

Dactylolactones A-D each possess a unique lactone structure, which is possibly

produced in the sponge by oxidative degradation of the quinone of dactyloquinone A

or D. Neodactyloquinone [55] was found to express moderate cytotoxic activity

toward HeLa cells with an IC50 of 86µM.41

HO

O OMe

[55] Neodactyloquinone

O

OO

MeO

MeO2C

[56] 18�-MeO: dactylolactone A [57] 18�-MeO: dactylolactone B

H

OO

CO2Me

OMe

[58] 18�-MeO: dactylolactone C [59] 18�-MeO: dactylolactone D

As part of the studies of pharmacologically interesting metabolites of sponges,

bioassay-monitored isolation and structure elucidation of three new cytotoxic

scalarane sesterterpenes, 12-O-deacetylscalarafuran [60], 12-O-deacetyl-12-epi-

scalarin [61], and 12-O-acetyl- 16-O-methylhyrtiolide [62], along with known

compounds, 12-epi-scalarin [63], 12-epi-deoxoscalarin [64] and sesterterpene [65],

from a marine sponge of the genus Spongia was investigated. In addition, in vivo

32

antitumor activities of three of the compounds on sarcoma-180-implanted mice were

also evaluated.

OOH

R

H

H H

[60] R=H[66] R=OAc

OR1

H

H H

H

R2

R3

[61] R1= OH,R2=OH,R3=O[63] R1=OAc,R2=OH, R3=O[64] R1=OAc, R2=OH, R3=H2[65] R1=OH, R2=H, R3=O[67] R1=OAc, R2=OAc, R3=O

O

O

OAc

H

H H

OH

OCH3

[62]

Cytotoxicity of [60-65] was tested against murine leukemia (L1210), human cervix

epithelioid carcinoma (HeLa), human lung adenocarcinoma (A549), and human oral

epidermoid carcinoma (KB) cell lines. Although sesterterpenes [61-65] exhibited

cytotoxicity against L1210, A549, and KB cells, [60] was inactive to all of the cell

lines at 50µg/ml. However, [60] was cytotoxic against HeLa cells with an IC50 value

of 19.5µg/ml. Compounds [61], [62] and [65] were also tested for in vivo mean

survival times (MST) and increases of life spans (ILS) in sarcoma-180-implanted

mice. Among these three compounds, [65] showed significant ILS: 50.3% of ILS at

5mg/kg intraperitoneal administration and this is more potent than a positive control,

5-fluorouracil (5-FU; 32.9%), at the same dose. Compound [64] also showed

comparable ILS (28.0%) to 5-FU at 10mg/ kg, and [61] was inactive at 5mg/kg.32

33

In an investigation for natural products with potential anticancer activity, an extract

of Spongia sp. was found to show inhibitory activity toward the lyase activity of

DNA polymerase β at 16.2µg/ml and was selected for bioassay-guided fractionation

using this assay. Solvent partition, followed by extensive chromatographic

fractionation, yielded two inactive sesquiterpenoids, 17-O-isoprenyldictyoceratin-C

[68] and dictyoceratin-C [69], and the active sesquiterpenoid quinone ilimaquinone

[70]. The nucleoside 2΄-deoxyuridine was also obtained.42

The chemical constituents of the genus Spongia have been extensively studied, and

polyketides, macrolides, terpenoids, sterols, alkaloids, and sesquiterpenoid quinones

have been reported as constituents of the genus. Sesquiterpenoid derivatives have

generated much interest due to their antitumor, antibacterial and anti-HIV activities.

RO

COOCH3

O

OH3CO

OH

[68] R=

[69] R=H Dictyoceratin- C

[70] ilimaquinone

17-O-isoprenyldictyoceratin-C

Purified compounds were used to determine IC50 values for inhibition of the lyase

activity of rat DNA polymerase β as well as for cytotoxicity to A2780 ovarian cancer

cells and inhibitory activity toward M-phase inducer phosphatase 2 (Cdc25B). The

eukaryotic enzyme DNA polymerase β can repair damage after exposure to DNA-

damaging agents, and inhibitors of this enzyme can potentiate cytotoxic activity by

inhibiting DNA repair. Inhibitors of DNA polymerase β may thus serve as

chemopotentiating agents in cancer treatment. Cdc25 dual specificity phosphatases

34

play central roles in cell proliferation by removing the inhibitory phosphates from the

ATP-binding site Tyr and/or Thr of the Cdk, thus activating cell cycle specific

Cdk/cyclin complexes. Inhibitors of Cdc25 are thus attractive candidates for potential

anticancer drugs. Compounds [68] and [69] and 2΄-deoxyuridine were inactive in all

three assays at the highest concentrations tested, but compound [70] was active as an

inhibitor of the lyase activity of DNA polymerase β, with an IC50 value of 45.2µM. It

was also weakly active as an inhibitor of Cdc25B, with an IC50 of 92µM, which is a

property shared by some other para quinones. Compound [70] has been reported to

have micromolar cytotoxicity to P-388, KB-16, and A-549 cells, and consistent with

this, it also showed moderate cytotoxicity to A2780 cells with an IC50 of 10.9µM.42

The first spongian diterpenoid, isogatholactone, was described by Minale and

collaborators from the Mediterranean sponge Spongia officinalis in 1974. Later a

number of other related diterpenoids were isolated from S. officinalis, S. zimocca, S.

matamata, S. arabica, and Spongia sp. Spongian and related diterpenoids isolated

from Spongia sp. have been reported to exhibit a wide spectrum of biological

activites including cytotoxicity, antibacterial properties, and toxicity against some

marine macroorganisms. The isolation of five new [71, 72, 74-76] and one known

spongian diterpenoid [73], previously found from an unidentified sponge and shown

to be an inhibitor of the lyase activity of DNA polymerase β was reported by Minale

and co-workers. Compounds [72] and [73] were tested for immunomodulatory

properties by the methods reported in the literature and demonstrated a slight

lysosomal activation (about 130% of control) of mice spleenocytes at concentrations

of 100µg/ml.43

O

H

R

OAc

H

O

H

R H[71] R=O[72] R-H, b-OAc[73] R=H,H

[74] R=b-OAc[75] R=a-OAc

35

O

H

H [76]

1.3.3 Chemistry of the Genus Hippospongia From the purple colored Okinawan marine sponge Hippospongia sp, taurospongin A

[77], a novel acetylene-containing natural product consisting of a taurine and two

fatty acid residues, was isolated. Taurospongin A [77] exhibited potent inhibitory

activity against DNA polymerase β (IC50 7.0µM, Ki 1.7µM) and c-erbB-2 kinase

(IC50 28µg/mL), but no cytotoxicity (IC50 >10µg/mL) against L1210 and KB cells

and HIV (IC50 6.5µM, Ki 1.3µM) reverse transcriptase.44, 45

RO3S

HN

O OH O O

O(CH2)13

[77] Taurospongin A R=H

In another study, it was found that an acetone-trichloromethane (CHCI3) extract of

the marine sponge Hippospongia sp. potently inhibited gastrulation of starfish

(Asterina pectinifera) embryos. Bioassay-guided purification of the crude extract

resulted in the isolation of a novel terpenoic acid, which was designated

hippospongic acid A [78].

OCO2H

[78] Hippospongic acid A

36

When fertilized starfish eggs were cultured from fertilization in the presence of

hippospongic acid A at a concentration of 14µmol/l or greater, they blastulated

normally after passing through a rapid cleavage period, and hatched on schedule; the

gastrulation was selectively inhibited, however. Furthermore, hippospongic acid A

affected neither fertilization of starfish gametes nor early embryonic development of

fertilized eggs up to the pre-gastrula stage even at the concentration of 1l0µmol/l.46

Two years after the discovery of hippospongic acid A, attempts were made to

synthetically produce the compound and was a success but the NMR spectrum of the

synthetic compound were apparently different from those of the natural product.

Careful examination of NMR spectrum led the revision of the structure of

hippospongic acid A to [79].

CO2H

O

[79] Hippospongic acid A

The structure was confirmed by the enantioselective synthesis of hippospongic acid

A.47 In investigating the chemical constituents of a purple sponge, H. metachromia

collected along the seashore area of southern Taiwan the acetone extract of the

sponge was partitioned between CHCl3 and H2O to give a CHCl3-soluble layer. The

CHCl3 solubles were subjected to a Si gel column, preparative thin layer

chromatography (TLC), high performance liquid chromatography (HPLC), and

chemical derivatization to yield nine compounds: metachromins A [31], B [80] and E

[87] two new sesquiterpene hydroquinones, hippochromins A [81] and B [82] and

their derivatives [83-86].

37

R

O

O[31] R=OH[86] R=OMe

O

R2

R1

R3

[80] R1=OH, R2 = R3=OMe[81] R1=R2=OH, R3=OMe[82] R1=R2=OH, R2=OMe[83] R1=R3=OAc,R3=OMe[84] R1=R3=OAc, R2=OMe[86] R1=OAc, R2=R3=OMe

O

OMe

O

O[87]

The cytotoxicities of the sesquiterpene hydroquinones were evaluated in vitro against

human tumor cell lines. Metachromins A [31] and B [80], hippochromin A diacetate

[84], and metachromin B monoacetate [86] exhibited potent cytotoxicities against

human colon (COLO-205) tumor cells at concentrations of 0.1, 0.26, 0.22, and

0.53µg/ml, respectively. These four sesquiterpenoids also inhibited the growth of

nasopharyngeal (KB) tumor cells with IC50 values of 1.8, 0.68, 3.06, and 1.32µg/ml,

respectively. However, compounds [84], [85], and [87] were inactive when tested on

these tumor cells.48

Bioassay guided separation of the crude extract of the sponge Hippospongia sp,

collected from the Federated States of Micronesia whose crude extract exhibited

significant inhibitory activity toward Mgnaporthe grisea isocitrate lyase (ICL),

yielded two active sesterterpene sulfates 1 and 5 respectively. These two metabolites

were previously reported as a phospholipase A2 inhibitor and antimicrobial

constituent. The IC50 value of ICL and MLS from Guy 11 by 3-nitropropionic acid

was 92.4 and 1570.8µM, respectively, which was similar to the value reported for

ICL and MLS from Aspergillus fumigatus. Compound [90] was obtained by

hydrolysis of compound [88]. Compounds [88] and [89] were found to be potent ICL

inhibitors, which inhibited appressorium formation and C2 carbon utilization in M.

grisea. Compounds [88-89] also had weak inhibitor activity against MLS, but were

38

strongly inhibitory to ICL. These results suggest that halisulfates are relatively

specific inhibitors against ICL. In addition, compounds [88] and [89] exhibited (IC50

= 12.6µM) and 6-fold (IC50 = 15.0µM) stronger ICL inhibitory activities than that of

3-nitropropionic acid (IC50 = 92.4µM), respectively. Interestingly, compound [89],

which was prepared by hydrolysis of the sulfate ester at the C-12 position of

compound [88], was found to have similar inhibitory activity compared with [88]. In

contrast, substitution of a hydroquinone moiety for furan moiety (compound [89])

resulted in a decrease in the ICL inhibitory activity (IC50 = 67.4µM). These results

suggest that the hydroquinone moiety is important for the ICL inhibitory activity of

halisulfate compounds. These compounds were the first example of ICL inhibitors

from marine natural products. Since the enzymes of the glyoxylate cycle are not

found in mammals, sesterterpene sulfates are good starting candidates for antifungal

agent design.49

OH

HO

OR

H

[88] R= SO3Na, Halisulfate 1[90] R= H, Hydrohalisulfate 1

HO

NaO3SHO

[89] Halisulfate 5

Two new sesterterpene sulfates, hipposulfates A [91] and B [92], have been isolated

from an Okinawan sponge, H. metachromia. From a species of Hippospongia, the

39

merosesquiterpenes dictyoceratins A and B and metachromins A and B were

reported, but no merosesterterpenes have been described. Hipposulfate A [91] was

cytotoxic to P388, A-549, HT-29, and MEL-28 cells, with an IC50 of 2µg/mL.50

R

NaO3SO OH

HO

R=

[91] Hipposulfate A

NaO3SO OH

HOOH

R=

[92] Hipposulfate B

Investigations into the chemistry of a southern Australian species of Hippospongia

have resulted in the isolation and identification of six new furanoterpenes,

hippospongins A-F [93-98], which are speculated to be biosynthetically related to the

C25 tetronic acids and the C21 furanoterpenes. Only hippospongin A [93] was found

to be a mild antibiotic, inhibiting the growth of Staphylococcus aureus at

concentrations of ca. 200µg/disk in a standard agar plate assay.51

40

CO2H

O O O

[93] Hippospongin A

CO2H

O O O

[94] Hippospongin B

O O O

O

NH

NH2

O O O

O

NH

O O O

O

NH2

NH

O O O

O

NH

O

According to Guo, many marine sponges of the family Spongiidae, in particular, the

genus Hippospongia frequently afford terpenoids containing 21 carbons and

displaying two β-substituted furan moieties at the end of the molecule. In

investigating the extract of the Indian sponge Hippospongia sp, a new C21

furanoterpene, named ent-untenospongin A [100], together with a known related

[95] Hippospongin C

[96] Hippospongin D

[98] Hippospongin F

[97] Hippospongin E

41

compound, tetradehydrofurspongin [99], was isolated. The activity of this compound

was not reported.52

O OH OR

R=H 1R=(R)-MTPA, 1S R=(S)-MTPA [99] tetradehydrofurspongin-1

O OH OR OH

[100] ent-untenospongin A

From the Moroccan marine sponge, H. communis, an antifungal compound, (-)-

untenospongin B [101] was isolated. (-)-Untenospongin B has shown a broad

antimicrobial activity exhibiting antibacterial activity against Escherichia coli,

Bacillus subtilis as well as against the ichthyopathogenic strain Vibrio anguillarum.

(-)-Untenospongin B showed also an in vitro antifungal activity against Candida

albicans, Aspergillus fumigatus, and some other fungi. Light microscopy

observations of the zone of fungal growth treated with (-)-untenospongin B showed

the presence of non germinated cells and hyphae presenting morphological

alterations, such as protoplast ejection and condensation of cytoplasm with empty

spaces. Comparing the antifungal activity of (-)-untenospongin B with amphotericin

B, it was found that (-)-untenospongin B was more active than amphotericin B in

inhibiting the growth of C. tropicalis (R2 CIP 1275.81) and F. oxysporum (CIP

108.74). On the other hand, (-)-untenospongin B showed the same activity as

amphotericin B in inhibiting the growth of C. albicans (ATCC 10231) and A. niger

(CIP 1082.74).53

O CH3 CH3 H HO

H OH

[101] untenospongin B

42

Many terpenoid quinones and phenols from marine sponges, H. metachromia have

exhibited interesting biological activities. From the purple-colored sponge H.

metachromia, collected at Okinawa Island, methanolic extracts were partitioned

between ethyl acetate and water. The ethyl acetate soluble fraction exhibiting

antineoplastic activity was subjected to chromatography on Sephadex LH-20

followed by a silica gel column (hexane-ethyl acetate, 4:l) to afford metachromins A

[31] and B [80] together with a known quinone compound, isospongiaquinone.

Metachromins A [31] and B [80] exhibited potent antitumor activity against L1210

murine leukemia cells in vitro with the IC50 values of 2.40 and 1.62µg/mL,

respectively. Both compounds also showed potent coronary vasodilating activity,

markedly inhibiting KCl (40mM) induced contraction of the rabbit isolated coronary

artery with an IC50 value of 3 X l0-4M each.54 Luibrand and his research team in 1979

isolated a compound which they named ilimaquinone from a bristly yellow, orange,

or brown sponge tentatively identified as Hippospongia metachromia that was

initially collected off the island of Kauai in Hawaii. The ethereal phase of an aqueous

methanol-acetone extract showed mild anti bacterial activity Staphylococcus aureus,

Candida albicans and Mycobacterium smegmatis. Preparative TLC on silica gel

furnished ilimaquinone as orange crystals. The structure was determined to be as that

of compound [102].55 In 1987, the structure of ilimaquinone was revised to [70]

following correlation with aureol [103] through a common degradation product.56

H

O

O

HO

OMeH

O

O

HO

OMe

O

OH

[102] [70] ilimaquinone [103] aureole

43

CHAPTER 2 METHODOLOGY

2.1 General Procedure The 1H NMR and 13C NMR spectra were recorded on a 400MHz and 125MHz

Varian spectrometer respectively while the 2D NMR spectra were recorded on a 400;

100MHz instrument. The NMR experiments for compound 1 were conducted in

CDCl3 and for compounds 2 and 3 they were conducted in CD3OD. The chemical

shifts are given on a δ (ppm) scale and were referenced to the residual solvent signals

(CDCl3; δH = 7.26ppm, δC = 77.0ppm and CD3OD; δH = 3.31ppm, δC = 49.0ppm).

The following abbreviations were recorded in the spectra: s = singlet, d = doublet, t =

triplet, q = quartet, m = multiplet, dd = doublet of doublets, bs = broad singlet, bd =

broad doublet. The MS spectra were recorded on a LTQ Orbitrap Discovery LC-

MS/MS spectrophotometer from Thermo Scientific. NMR measurements and LC-

MS analysis were done at University of Aberdeen, Scotland, United Kingdom while

all the other analyses were done at USP, Suva, Fiji. IR spectra were recorded with a

Perkin-Elmer spectrum 100 FT-IR spectrophotometer using KBr disc. UV spectra

were recorded with a Perkin-Elmer Lambda 25 UV/Vis spectrometer. The melting

point apparatus was used to take the melting point. Normal phase- thin layer

chromatography (TLC) was carried on Merck silica gel 60 F254 aluminum sheets

while Reverse Phase TLC was carried on Macherey-Nagel precoated TLC plates RP-

18W/UV254. TLC was visualized under the UV lamp at 254nm. Flash Column

Chromatography was carried out using Silica gel grade 922, 200-323 mesh as the

packing and n-hexane and ethyl acetate were used as the solvent system in a step-

wise gradient system for normal phase. For reverse phase vacuum liquid

chromatography (VLC), Bakerbond octadecyl (C18) 40µm prep LC packing was used

as the stationary phase with methanol-water as the solvent system. High Performance

Liquid Chromatography (HPLC) was carried on a Waters 515 pump connected to a

Waters 2487 dual λ absorbance detector set at 254nm and the signals were recorded

on a Dual channel recorder. An Alltech Econosil C18 250 x 4.6mm column was used

for analytical HPLC while an Alltech Econosil C18 250 x 10mm column was used for

semi-prep HPLC work. All the solvents used for HPLC were of HPLC grade

whereas solvents for extraction, TLC and FCC were of analytical grade.

44

2.2 Isolation of bioactive compounds

2.2.1 Collection and identification of the specimen

The animal material was collected near Kia Island off Labasa, Fiji (16° 10' 57S and

179° 03' 11E). The sample was abundant at 10-25m on coral/rock at walls. The

specimen was brown grey in color with the texture being rough and hard. It was

porous with plenty of oscules ranging from 1-7mm in diameter. The animal was

identified as a sponge, belonging to the Genus Hippospongia, by Mary Kay Harper, a

visiting taxonomist from the University of Utah, USA. A voucher specimen was also

deposited at the Marine Reference Collection; USP with the ID, FJ05-097. The

sample was stored at -20°C prior to workup.

2.2.2 Extraction, isolation and purification

This project was divided into three stages: stage 1 was extraction and solvent

partitioning, stage 2 involved the use of chromatographic techniques (such as column

chromatography, TLC and HPLC) to isolate the active compound(s) while the third

stage was to purify, check the purity and to get spectral data to characterize the active

compound(s). At each step, the activity of the extracts was tested using the Brine

Shrimp Assay as a guide to isolate the active principle. Thus this project used the

bioassay guided fractionation approach to isolate the active principles.

2.2.3 Extraction and solvent partitioning

The sponge was successively extracted three times with methanol (MeOH) and once

with dichloromethane (DCM) at room temperature. The extracts were combined and

the solvent removed under reduced pressure to yield 23g of the crude extract. A

portion of this crude extract (10g) was dissolved in MeOH and diluted with water

and partitioned (3x) with DCM in a 1L separating funnel. DCM was evaporated

45

under reduced pressure and the residue was re-dissolved in 90% aqueous MeOH and

was partitioned against n-hexane until the n-hexane layer was colorless. Hexane was

removed under reduced pressure and the residue as FH while the MeOH fraction was

adjusted to 50% aqueous MeOH and was extracted three times with DCM. MeOH

and DCM were also evaporated under reduced pressure and labeled as FM and FD

respectively. The water fraction was extracted with secondary butanol, which was

then rotovaped and the residue was named WB. Each of these fractions was tested

for Brine Shrimp Bioassay. The summary of the isolation protocol is given below

(Figure 2-1).

2.2.4 Chromatography and purification

2.2.4.1 Isolation of Compound 1

The brine shrimp active hexane fraction (FJ05-097FH) (1.59g) was subjected to

normal phase step-wise gradient flash column chromatography (Silica gel grade

922,200-325 mesh) with hexane-ethyl acetate gradient as eluant (hexane with

increasing amount of ethyl acetate) to afford 11 sub-fractions. All these fractions

were tested for activity against brine shrimps. The brine shrimp active sub-fraction 4

(FJ05-097FHF4) (500mg), eluted with 80% hexane-ethyl acetate was then subjected

to reverse phase High Performance Liquid Chromatography (RP-HPLC) on an

analytical column, Econosil C18 10µ, length 250mm and ID 4.6mm, using a mixture

of 80% acetonitrile-water as the mobile phase which gave optimum resolution. The

flow rate of the pump was set at 1ml/min. Having determined the mobile phase,

semi-prep HPLC was carried out on an Alltech Econosil C18 250 x 10mm column

with the flow rate at 3ml/min. The active principle FJ05-15-200FHF4H4 was

collected at the retention time of 15.5min.

The DCM fraction, FJ05-097 (0.497g), was also active to brine shrimp; therefore a

similar procedure as above was carried out to isolate the active principle. This

fraction was subjected to normal phase flash column chromatography and fraction 1

46

(FJ05-097FDF1) (85.7mg), eluted with 80% hexane-ethyl acetate, was further

purified on RP-HPLC (Econosil C18 10µ, length 250mm and ID 10mm) using 80%

acetonitrile-water as mobile phase. From this 17mg of the active compound FJ05-

097FDF1H2 was obtained. The active fractions collected from fractions FD and FH

had the same retention time (~15min), same color (yellow), Rf (0.58) and reacted

similarly with a base (color change from yellow to pink/violet), therefore it was

assumed to be the same compound. The major active fraction from HPLC which was

isolated as a yellow solid was sent for spectral analyses and was designated

compound 1 for further analyses.

2.2.4.2 Isolation of Compound 2 The sub-fraction FJ05-097FDF4 obtained from the DCM FCC eluted with 50%

EtOAc-hexane was also active and was further purified on RP-HPLC (Econosil C18

10µ, length 250mm and ID 10mm) using 80%MeCN-H2O + 0.1% trifluoroacetic

acid (TFA) to afford six HPLC fractions. The major fraction which was purple in

color was active and was sent for spectral analysis. HPLC fraction FJ05-097FDF4H6

was renamed compound 2 for further discussion.

2.2.4.3 Isolation of Compound 3 The purple colored methanol fraction from solvent partitioning showed weak activity

against brine shrimps. RP-VLC on Bakerbond octadecyl (C18) 40µm prep LC

packing was carried out using a step-wise gradient system of decreasing polarity of

water and methanol (100%H2O, 50%MeOH-H2O, 80%MeOH-H2O, 100%MeOH

and finally with 100% acetone). The MeOH fraction (2.69g) was dissolved in

minimum amount of MeOH and was adsorbed onto the stationary phase. The fraction

(FJ05-097FMF2) eluded with 80%MeOH-H2O, which showed weak brine shrimp

activity, was further purified on RP-HPLC (Econosil C18 10µ, length 250mm and

ID 10mm) using 65%MeCN-H2O with 0.1%TFA as mobile phase. From HPLC

seven fractions were collected. Fraction seven (FJ05-097FMF2H7), red in color, was

47

the major weakly active fraction and was submitted for spectral analysis. HPLC

fraction FJ05-097FMF2H7 was renamed compound 3 for further discussion.

Figure 2-1: Isolation of secondary metabolite from the sponge.

WB FM

Solvent partitioning

FD

Hippospongia

Total extract

FJ05-097

VLC NA NP-FCC NP-FCC

Active

Fraction FMF2

Active

Fraction FHF4

Active

Fraction FDF1

Active

Fraction FDF4

RP-HPLC RP-HPLC RP-HPLC RP-HPLC

Compound 1 Compound 2 Compound 3

FH

48

2.3 Bioassay

2.3.1 Brine shrimp bioassay

Brine shrimp bioassay is a simple method utilizing brine shrimp (Artemia salina) for

natural product research. This method is rapid, reliable, inexpensive and convenient

as an in-house general bioassay tool. Brine shrimps have been previously utilized in

various bioassay systems such as analysis of pesticide residue, mycotoxins,

morphine-like compounds and toxicants in marine environments.57 It has been

reported that the brine shrimp larvae can tolerate up to 11% of acetonitrile,

dimethylformamide, dimethylsulfoxide, ethanol, 2-propanol, methanol and

tetrahydrofuran.58

2.3.2 Hatching the eggs

The Golden fish Artemia cysts, brine shrimp eggs were obtained from Golden Ocean

Aquasupply Enterprise, Taiwan. Approximately 100mg of brine shrimp eggs were

weighed out and placed in a 250ml beaker. A volume of 200ml filtered seawater

(FSW) was added to it. An aerator was connected to the beaker to provide sufficient

aeration and to keep the eggs in circulation. The beaker was covered with aluminum

foil and was allowed to stand for 48 hours. A light source was also provided to keep

the surroundings warm. After 48 hrs the eggs hatched and were ready for the

bioassay.

2.3.3 Preparation of samples

The test samples were dissolved in appropriate amounts of FSW and if the sample

was not soluble in FSW it was dissolved in 10% of total volume of

dimethylsulfoxide (DMSO) and topped up with FSW. Once the first test

concentration was ready, serial dilutions were carried to prepare another two sets of

49

test concentrations. For instance, to prepare a first test concentration (highest

concentration of the sample in the 96-well plate) of 4000ppm, 8mg of the sample was

weighed in an eppendorf tube and dissolved in 1ml of FSW. The concentration of

this sample would be 8000ppm or 8mg/ml. To do serial dilutions, 500µl of this

sample would be transferred to another eppendorf tube which already had 500µl of

FSW diluting the sample by half (4000ppm) and so forth.

2.3.4 Cytotoxicity assay

The brine shrimp bioassay was carried out in 96-well plates and the tests were done

in triplicates. Approximately 7-14 adult shrimps, together with 100µl of filtered sea

water were pipetted out into each well. To these wells 100µl of the test sample was

added whereby further diluting the sample by 50%. Once all the samples were added

in the well, it was covered with a lid and the results were recorded after 24 hrs using

a light microscope. FSW was used as the negative control. The results were recorded

as number of dead shrimps over total number of brine shrimps in a particular well.

From these results, LD50iv of the samples were calculated to determine the toxicity of

the samples using the Reed Muench method.59 According to this method, a plot of

the number of the accumulated survivors and the number of accumulated deaths on

the same axes (number of animals vs. log dose) against log dose is constructed. The

two curves will cross at the dosage (i.e. log dose) where the number of survivors is

equal to the number of deaths. The intersection point is the dose that will kill 50% of

the animal and is known as LD50.

iv LD50: Lethal dosage where 50% of a population dies.

50

CHAPTER 3 RESULTS AND DISCUSSION

3.1 Isolation of cytotoxic compounds from Hippospongia sp. The present work investigated the isolation of the brine shrimp active components of

marine sponge belonging to the genus Hippospongia. A bioassay-guided

fractionation approach was used in order to achieve this. The combined crude

methanolic and DCM extract of this organism showed cytotoxic activity against

brine shrimps with the LD50 of 266ppm. This crude extract was subjected to solvent-

solvent partitioning which resulted in methanol (FM), DCM (FD), hexane (FH) and

butan-2-ol (WB) soluble fractions. Brine shrimp activity results showed that the

hexane and DCM soluble fractions were most active. Table 3-1 shows the weights

and the activity of each fraction obtained.

Table 3-1: Weight and bioactivity of the four fractions

Fraction ID

FJ05-097

Weight (mg) Brine shrimp Activity

LD50 (ppm)

FM 332.4 239

FH 1598 20.2

FD 497.1 56.2

WB 5763 na*

*not active

.

51

3.2 Isolation and purification of cytotoxic compound 1

The active fractions (FH and FD) were subjected to normal-phase flash column

chromatography (NP-FCC) using a step wise gradient solvent system with hexane

and ethyl acetate (EtOAc) in order of increasing polarity. From this flash column

chromatography 11 sub-fractions were collected. BSA was conducted on these

fractions and the results were recorded in Table 3-2.

Table 3-2: Weight and bioactivity of FH and FD fractions

FJ05-15-200FH FJ05-15-200FD

Sub-fraction Weight (mg) Activity LD50

(ppm)

Sub-fraction Weight (mg) Activity LD50

(ppm)

F1 254.5 87.1 F1 85.70 45.6

F2 1.300 na* F2 33.90 48.4

F3 346.5 50.1 F3 14.10 180.7

F4 512.2 44.7 F4 31.50 201.6

F5 48.00 121.1 F5 8.300 na

F6 4.900 81.1 F6 7.400 na

F7 2.700 na F7 129.5 na

F8 79.00 na F8 70.40 na

F9 12.90 na F9 29.10 na

F10 81.60 na F10 23.10 na

F11 190.3 na F11 16.40 na

* not active

Normal phase thin layer chromatography (NP-TLC), Figure 3-1, was carried on the

fractions obtained and it was noted that the brine shrimp active sub-fractions FJ05-

097FHF3 and F4 and FJ05-15-200FDF1 and F2 had a major common spot with the

solvent system of 2:3 EtOAc-hexane. The fraction FJ05-097FDF1 which looked

fairly pure based on TLC was progressed to High Performance Liquid

Chromatography (HPLC) (40mg). Semi-preparative HPLC on a 250mm x 10µ

Econosil column eluted with 80%MeCN-water as mobile phase at a flow rate of

4ml/min furnished four fractions. Table 3-3 shows the weight and activity of the

.

52

b

HPLC fractions obtained. After removing the solvents, the sub-fraction was tested

against brine shrimps. As shown in the NP-TLC profile (Figure 3-2), the same

compounds were obtained from both the hexane and DCM fraction. Figure 3-2 also

shows a RP-TLC profile of the active compound isolated using 80%MeCN-H2O and

the Rf was calculated to be 0.22. Also obvious from the two TLC profiles is that the

compound isolated has different appearance on NP and RP-TLC, that is, violet and

yellow respectively. The major fraction (17mg) (FJ05-097FDF1H2) was found to be

the only active principle which was eluted at Rt =10min as indicated by the HPLC

chromatogram in Figure 3-3. For further discussion FJ05-097FDF1H2 was

designated as compound 1.

Figure 3-1: NP-TLC profile of the fractions obtained from the FD fraction.

Figure 3-2: NP-TLC (a) of the active component from FHF4H4 and FDF2H2 fraction. RP-TLC (b) of the compound isolated as yellow and violet liquid.

a

.

53

Figure 3-3: Chromatogram of the active FJ05-097FDF2 fraction.

Table 3-3: Weight and bioactivity of the HPLC fractions

Fraction ID

FJ05-097FDF1

Weight

(mg)

Brine shrimp Activity

LD50 (ppm)

H1 3.10 na*

H2 17.2 18

H3 2.20 na

H4 0.800 na

*not active

H4

.

54

The active compound was yellow in color and was unstable at room temperature. It

was also noted that if a base was added to the compound it would change from

yellow to purple (violet). This color change indicated the possibility of a quinione

moiety in the structure.60 To protect the compound from such conditions it was

stored away from light at -20oC.

3.2.1 Purity check

This active fraction was re-injected onto an analytical RP-HPLC system connected to

a UV-VIS detector with the mobile phase as 78%MeCN-H2O to check the purity of

this peak. Fortunately from the UV-VIS HPLC profile the sample was quite pure as

indicated by the single peak. To confirm this, the sample was also injected onto an

analytical RP-HPLC system with the same mobile phase but this time connected to a

Refractive-Index Detector (RID). The RID profile also showed a single peak at the

retention time of 10.7mins indicating that the fraction indeed was pure as shown in

the chromatogram below (Figure 3-4). The peak at the Rt = 2.77mins is due to the

solvent and this was confirmed by injecting a blank sample (mobile phase) onto the

HPLC system.

Figure 3-4: Chromatogram of the active HPLC fraction on a Refractive Index Detector.

.

55

3.2.2 UV and IR spectroscopic analysis

Ultra-Violet (UV) and Infra-Red (IR) spectroscopic analyses were also conducted on

the active compound. The IR spectrum (Figure 3-5) shows absorbance at (KBr)

3649.3, 2926.7, 2857.1, 1648.8 and 1611.0 cm-1. The IR absorbance at 1648.8 and

1611.0cm-1 (literature:55 1640 and 1595cm-1) and the UV (MeOH) absorbance λmax

of 288nm (literature:55 UV λmax 288nm) indicated the presence of a 1,4-

benzoquinone chromophore, while a sharp IR absorbance at 3649.3 cm-1 indicated a

hydroxyl group.

3.2.3 Crystallization of the compound

Attempts to crystallize the isolated compound using methanol was unsuccessful as it

changed the color from yellow to violet, but using hexane proved to be fruitful. This

was achieved by dissolving the sample in minimum amount of hexane and filtering it

using a glass pasture pipette filled with cotton wool. The undissolved part (residue or

filtrand) was trapped on the cotton wool, while the filtrate was collected in a clean

vial and the solvent was allowed to evaporate slowly leaving behind nicely formed

crystals. Since the pure compound was able to crystallize, the melting point was also

checked using the melting point apparatus. The melting point of the compound was

noted to be in the range of 98-100 �C. A part of this sample was sent for LC-MS and

NMR analysis and the results are discussed below.

56

Fi

gure

3-5

: IR

spec

trum

(KB

r) o

f the

act

ive

com

poun

d 1

4000

.036

0032

0028

0024

0020

0018

0016

0014

0012

0010

0080

060

045

0.0

49.9

0

50.5

51.0

51.5

52.0

52.5

53.0

53.5

54.0

54.5

55.0

55.5

56.0

56.5

57.0

57.5

58.0

58.5

58.7

4

cm-1

%T

36

49.3

5

2926

.77

1611

.01

1380

.17

1226

.02

3840

.65

2857

.14

2362

.63

2340

.65

1648

.84

1454

.07 13

47.0

71316

.90

1201

.68

1050

.79

1034

.3389

1.6883

4.07

795.

66

718.

84

57

3.2.4 LC-MS and NMR data of compound 1

The active compound 1 (FJ05-097FDF2H2), isolated as a yellow solids, gave a

molecular ion peak [M+H+] at 359.22 m/z (Figure 3-6). Based on this mass the

molecular formulas were generated using the Xcalibur® 2.0.7 SPI software and

tabulated in Table 3-4. From Table 3-4 the most realistic molecular formula was

choosen to be the first molecular formula which was C22H31O4. Since the moelcular

ion peak at 359.22m/z is for [M+H]+ the actual mass of the compound would be

358.22 and hence the molecular formula would correspond to C22H30O4. From this

formula the double bond equivalence was calculated to be eight using Equation 3-1.

The NMR data obtained for this compound was tabulated in Table 3-5. The 1H NMR

spectrum illustrated three methyl signals: two singlet (s) tertiary methyls (3H) and

one doublet (d) secondary methyl (3H) at 0.90, 1.07 and 0.95ppm respectively, one

singlet methoxy group (OCH3) at 3.89ppm, a singlet at 5.89ppm for a olefinic proton,

a double singlet (ds) at 4.71/4.68ppm for two protons, a broad singlet (br s) for the

hydroxyl group (OH) and a AB quartet at 2.61/2.50ppm for the two methylene

protons (Figure 3-21). The carbon NMR (13C NMR) in Figure 3-22, showed two

carbonyl signals (182.8 and 182.6ppm), 2 x C=O, and six olefinic carbons (161.8,

153.6, 153.4, 117.7, 105.8, 102.1ppm), 3 x C=C, which accounted for five out of

eight degrees of unsaturation which pointed towards a tricyclic compound.

dbe= [(2(C)+2)-H]/2

Equation 3-1: Calculation of double bond equivalence.

58

[M+H]+

fj05-15-200fdf1h2_090130135037 #69 RT: 0.55 AV: 1 NL: 1.18E8T: FTMS + p ESI Full ms [100.00-1500.00]

350 400 450 500 550 600 650 700 750m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

359.22C 22 H31 O4

739.42C 35 H63 O3 N8 S 3

609.28C 9 H35 O7 N23 S437.19

H24 O4 N21 Na S

684.20C 20 H26 N20 Na 6

397.18C 17 H35 O3 N S 3

455.33C 22 H43 O4 N6

519.14C 12 H29 O19 N3

Figure 3-6: Electron spray ionisation mass spectrum of compound 1. Inset: UV

profile of compound 1.

Table 3-4: Elemental composition search on mass 359.22m/z

m/z Theoretical mass Delta (mmu) RDB equiv. Composition

359.22 359.22 -0.22 7.5 C22 H31 O4

359.22 0.62 1.0 C5 H25 O4 N15

359.22 -0.71 6.0 C6 H21 N19

359.22 -0.72 0.5 C7 H27 O5 N12

UV Profile 288

59

Table 3-5: NMR data for the compound 1 (CDCl3)

Carbon

labeled on 13C NMR

Carbon # C δ ppm H δ ppm (mult.) COSY

H - H

HMBC

C - H

A* 20 182.9 - h, o

B* 17 182.6 - h, o

C 18 161.8 - h, i

D 4 153.6 - g, p, n, j, m

E 21 153.4 7.47 (br s1 OH) h, o

F 16 117.8 - o

G 11 105.8 4.71/4.68 (d 2H) p p,p΄

H 19 102.1 5.88 (s 1H) i - shows 1J

I 22 56.9 3.89 (s 3H) - shows 1J

J 10 48.6 1.20 o, n, t

K 9 45.0 - o,j, l, u,t

L 5/8 39.6 1.18 g, n, o, j,u,t

M 6 38.0 2.02/1.11 n

N 12 33.3 1.07 (s 3H) m j

O 15 32.8 2.61/2.50 (AB

quartet 2H)

t

P 3 32.1 2.42/2.11 s/s΄, r/r΄ g

Q 7 27.9 1.50/1.21 q΄, m u

R 2 25.1 1.81/1.66 r j

S 1 22.6 2.16/1.82 s΄, j j

T 14 18.8 0.90 (s 3H) l o, l

U 13 18.4 0.95 (d 3H) l -

* Interchangeable

The 13C NMR (Figure 3-22) of the active compound showed 21 chemically distinct

carbon shifts and judging from the peak intensities, 14 of these, (G, H, I, J, M, M, O,

P, Q, R, S, T, and U) were tentatively inferred to carry directly bonded protons. This

was proven by the HMQC correlations (Figure 3-24). The molecular formula

60

(C22H30O4) when compared to the 13C NMR shows only 21 carbon signals. 1 H NMR

and 2D NMR spectra indicate that a carbon signal must be missing or is overlapping

with other closely related signals. Before the NMR data were further analyzed, the 13C NMR spectrum was labeled alphabetically using upper case letters starting from

the up field region of the NMR while the corresponding protons were labeled from

the HMQC spectra using lower case letters as stated by Crews.61

3.2.5 Structure elucidation of the compound 1

The evaluation of this compound was initiated with the assignment of the desheilded

olefinic proton resonating at 5.89ppm Hh to its corresponding olefinic carbon CH

resonating at 102.1ppm. This analysis showed the proton at 5.89ppm is attached to

the carbon H resonance at 102.1ppm. A 1H-1H COSY (Figure 3-23) correlation of

the proton Hh (5.89ppm) with the proton resonance at 3.89ppm Hi lead to the

formation of a bond between the carbons H (102.1ppm) and C (161.68ppm). A three-

bond correlation from proton Hi with the carbon resonance at C 161.8ppm and a two-

bond HMBC correlation from proton Hh with the same carbon provided the

confirmation of this bonding.

Another two-bond HMBC (Figure 3-25) correlation of the proton Hh resonance at

5.89ppm with the carbonyl carbon B resonance 181.6ppm further showed that the

carbon H (102.1ppm) was in turn also connected to this carbonyl carbon. The

second carbonyl carbon resonating at 181.6ppm displayed a three-bond HMBC

correlation to the olefinic proton h attached to carbon C, indicating a bond between

carbons A and C. These correlations resulted in the formation of the fragment shown

below in Figure 3-7.

61

OCH3

O

O

H

A

B

C

Hh

i

Figure 3-7: HMBC correlations of partial structure 1A.

Further expansion to this fragment came with a three-bond HMBC of the Hh proton

with the carbon resonance at E (153.4ppm), which had been identified as the carbon

carrying the hydroxyl group as shown below. This HMBC correlation thus gave the

indication that the carbonyl carbon was linked to the hydroxyl carbon. Due to the

presence of the E carbon (153.4ppm) resonance in the low field region of the 13C

NMR spectrum, this bond between the carbons E and A was shown to be viable

(Figure 3-8).

OCH3

O

O

H

A

B

C

Hh

i

OH

E

Figure 3-8: HMBC correlation of partial structure 1B.

A further three-bond HMBC correlation of the olefinic carbon E and a two-bond

HMBC correlation of the carbon F resonance at 117.7ppm with the benzylic

methylene protons showed that the carbon E was bonded to carbon F, which was in

turn bonded to the methylene carbon C(O) resonance at 32.8ppm. Another three-

bond HMBC correlation of the carbonyl carbon resonance at 182.6ppm with the

same benzylic methylene protons indicated that the carbon F resonance at 117.7ppm

was involved in another bonding, this time with the carbonyl carbon A. These

bondings between the carbons E/F and F/A resulted in the formation of the first ring

present in this compound, generating the partial structure 1C, a 1,4-benzoquinone

chromophore shown below.

62

OCH3

O

O

H

A

B

C

Hh

i

OH

E

F

H2C HMBC correlation

COSY correlation

Figure 3-9: HMBC and COSY correlations of partial structure 1C.

To confirm that the assignments made about the quinone moiety was correct, the

following NMR analysis was conducted. Hh is at 5.88ppm (as Hg is the two singlets

at 4.71/4.68ppm). Using the HMQC spectrum, CH is at 102ppm. Similarly H (o/o΄)/

C (O) are at 2.61/2.50ppm AB quartet/34ppm. From the HMBC spectrum H (o/o΄)

are correlated to three quaternary carbons at 117.7 (F), 153.4 (E) and 182.8ppm (A),

in the same spectrum H (h) is correlated to 153.4 (E), 161.8 (C) and 182.8ppm (A)

and the OMe at 3.89ppm is correlated to 161.8ppm (C), giving the following

situation (Figure 3-10).

O

O

OMe

Hh

Ho

Ho'

OH

117

153

161182

Figure 3-10: All HMBC correlations of 1,4-benzoquinone chromophore.

This fragment present in compound 1 is a quinone moiety. Typical quinone behavior,

as stated by Luibrand55, which includes color change in bases, and re-oxidation in air

was observed during the isolation process.

63

With five of the eight degrees of unsaturation already accounted for above in the

quinone moiety and only two olefinic carbons remaining (1 x C=C), and to fulfill the

double bond equivalence of eight, it is apparent that the compound contains one

other double bond and two more rings.

By comparison of the molecular formula, 13C NMR and HMBC spectra, it was

observed that the carbon signal CL occurring at 39.6ppm is showing HMBC

correlations to the methyl protons Hn resonance at 1.07ppm and to the exocyclic

methylene protons Hg/g΄ resonance at 4.71 and 4.68ppm while the secondary carbon

is showing HMBC correlations to the secondary methyl protons resonance at

0.95ppm, to the tertiary methyl protons resonance at 0.90ppm and to the benzylic

methylenes resonance at 2.61 and 2.50ppm. Based on these observations it was

established that the peak at 36.9ppm is due to two carbons. One of which is a

quaternary carbon while the other is a secondary carbon.

After the establishment of the partial structure 1C (Figure 3-9) and taking the above

correlations into consideration, a further extension to the structural construction came

with the analysis of the upfield proton signals. As stated earlier the proton NMR

showed two singlet tertiary and a doublet methyl proton signals at Ht 0.90, Hn 1.07

and Hu 0.95ppm respectively. The structural analysis was initiated with the

assignment of these methyl protons to their corresponding carbon resonance peaks in

the HMQC spectrum. This analysis showed that the protons at δ 0.90, 0.95, 1.07ppm

were attached to the carbon T, U and N resonances at δ 18.8, 18.4 and 33.3ppm.

Since the proton signal at 0.95ppm (Hu) is a doublet it indicated that the methyl

group is connected to a carbon which has a proton bonded to it which splits the

methyl proton signal into a doublet. The bonding of this methyl proton to a

secondary carbon CN (δ 33.3ppm) came with the two-bond HMBC correlation of the

Hu proton (δ 0.95ppm) with the carbon CL resonance at δ 39.7ppm. A 1H-1H COSY

correlation between the protons resonating at δ 0.95ppm Hu and 1.22ppm Hl

confirmed this bond.

A further extension of this structure came with the observation of the carbon

resonance at K δ 45.0ppm which according to the HMQC spectrum was quaternary.

A three-bond HMBC correlation from the proton resonance at 0.95ppm H (u) with

64

the carbon CK resonance at δ 45.0ppm and a two-bond HMBC correlation of the

proton resonance at δ 1.22ppm Hl with the carbon K were observed. Thus based on

this HMBC correlation of the Hu protons (δ 0.95ppm) with the carbon resonance at δ

45.0ppm it was assumed that the carbon L (δ 39.7ppm) was connected to the

quaternary carbon K (δ 45.0ppm).

Another three-bond HMBC correlation from the proton Hu δ 0.95ppm with a sp2

carbon resonance at CQ δ 29.8ppm was observed and lead to the formation of a bond

between carbon CL and carbon Q. Confirmation of this bonding came with a 1H-1H

COSY correlation between Hl δ 1.22ppm and Hq δ 1.51ppm.

Carbon K resonance at δ 45.0ppm being a quaternary carbon showed a two-bond

HMBC correlation to the methyl proton resonance at 0.90ppm Ht and this

observation lead to the formation of a bond between carbon K and the methyl carbon

resonance at δ 18.8ppm CT. This bonding was confirmed by a two-bond HMBC

correlation seen between the carbon resonance at CL δ 39.7ppm with the methyl

protons Ht resonance at 0.90ppm These HMBC correlations shown by the methyl

groups at δ 0.90 and 0.95ppm led to the generation of partial structure 1D (Figure

3-11). The only two remaining olefinic carbons D and G resonances at 153.6ppm

and 105.8ppm respectively are bonded together, with the carbon D being a

quaternary carbon as observed from the HMQC spectrum, Figure 3-24 and G

carrying the exocyclic methylene protons Hg/g΄at 4.71/4.68ppm. The support for this

bonding came with a two-bond HMBC correlation of the methylene proton, Hg/g΄

4.71/4.68ppm with the quaternary carbon D (153.6ppm). This fragment was further

extended with a three-bond HMBC correlation of the olefinic protons, Hg/g΄

4.71/4.68ppm with the carbon resonance at CP 32.0ppm. A strong 1H-1H COSY

correlation between the olefinic proton and the proton resonance at Hp 2.43ppm

confirmed this bonding. The olefinic methylene protons also showed a three-bond

HMBC correlation to the carbon CL΄ resonance at 39.6ppm giving the partial

structure 1E (Figure 3-12).

65

CCH3

CH

18.39

0.95

1.22

39.65

CCH3

45.01

0.90

18.77

29.77

1.51H

H1.25

Figure 3-11: COSY correlation of partial structure 1D.

.

C

CH H

CH

H

153.6

105.8

39.6

32.08

4.71 4.68

2.42

2.11

Figure 3-12: HMBC correlations of partial structure 1E.

The methyl proton Hn resonance at 1.07ppm, which gave a singlet in 1H NMR

spectrum, showed a two-bond HMBC correlation to the carbon CL΄ resonance at

39.6ppm. These methyl protons also show a three-bond HMBC correlation to the sp2

carbon CM resonance at 38.0ppm and to a sp carbon CJ resonance at 48.6ppm. The

carbon CL΄ resonance at 39.6ppm showed two-bond HMBC correlation to the proton

Hj resonance at 1.20ppm and to the methyl protons of carbon CN resonance at

33.3ppm. These correlations resulted in the partial structure 1F (Figure 3-13).

66

CC

C

CH

H

H1.20

48.6

39.6

33.3

38.2

1.11

2.02HH H

1.07

CC

C

CH3

HH

H1.20

48.6

39.6

33.338.2

1.11

2.021.07

Figure 3-13: Partial structure 1F showing COSY and HMBC correlations.

The partial structure 1F (Figure 3-13) was further extended and connected to the

partial structure 1D (Figure 3-11) with the help of 1H-1H COSY and the HMBC

correlations. A 1H-1H COSY correlation between the proton Hm resonance at

1.11ppm and the proton Hq resonance at 1.52ppm and a two-bond HMBC

correlation between the quaternary carbon CK resonance at 45.0ppm and the proton

Hj resonance at 1.20ppm led to the conclusion that the partial structures 1D and 1F

are connected through the carbons CM and CQ resonance at 38.0ppm and 27.9ppm

and CJ and CK resonance at 48.6ppm and 45.0ppm respectively. Thus these bonding

of the two partial structures led to formation of a ring system designated as partial

structure 1G (Figure 3-14) and accounted for another degree of unsaturation.

.

CC

C

CH3

HH

H1.20

48.6

39.6

33.338.2

1.11

2.021.07

CCH3

CH

18.39

0.95

1.22

39.65

CCH3

45.01

0.90

18.77

29.77

1.51H

H1.25

Figure 3-14: Partial structure 1G showing COSY and HMBC correlations.

67

After the completion of the ring, the partial structure 1E (Figure 3-12) was further

extended with a 1H-1H COSY correlation between the protons Hp resonance at

2.42/2.11ppm and the proton Hr/r’ resonance at 1.81/1.66ppm and protons Hr with

and the protons Hs/s΄ resonance at 2.16/1.82ppm .The protons Hp and Hs are also

showing a four bond (4J) W-coupling. These 1H-1H COSY correlations indicate that

the carbon CP resonance at 32.1ppm is bonded to CR resonance at 25.1ppm and

carbon CR is in turn bonded to the carbon CS resonance at 22.6ppm as shown below

(Figure 3-15)

C

CH H

C

C

HH

HCH

H H

Figure 3-15: Partial structure 1H showing COSY correlations.

The closure of the ring, which results in the fulfillment of the degree of unsaturation,

was achieved by the observation of the HMBC correlations. A two-bond HMBC

correlation of the proton Hj at 1.20ppm with the carbon resonance at 22.6ppm CS

showed that the carbons CS and CJ are connected. Confirmation of this bonding

came with a 1H-1H COSY correlation of the Hs protons (2.16/1.82ppm) with the Hj

proton resonance at 1.20ppm. Further confirmation of this bonding came with a

three-bond HMBC correlation of the carbon CK resonance at 45.0ppm with the

protons of carbon CS (22.6ppm). These correlations directed the formation of a

bicyclic partial structure 1I as shown below in Figure 3-16.

68

CH3

CH3

CH3H

D

GH H

L

MQ

L'K

JS

P

R

H H

Figure 3-16: Partial structure 1I showing HMBC correlation of the sesquiterpene moiety.

The formation of this final bicyclic sesquiterpene moiety of the tricyclic structure

accounted for all the eight degrees of unsaturation. The only task remaining was

joining the two fragments, partial structure 1C and 1I. This was accomplished by the

HMBC correlations observed between the benzylic methylene protons Ho/o΄

resonance at 2.61/2.50ppm and the carbons CJ, CK and CL resonance at 48.6ppm,

45.0ppm and 39.7ppm respectively. A series of two and three-bond HMBC

correlations of the benzylic methylene proton with the carbon resonance at 45.0, 48.6

and 39.7ppm lead to the formation of a bond between the carbons CK and CO

resonances at 32.8ppm. Confirmation of this bonding also came with the HMBC

correlations of the protons Hl and Hj resonance at 1.18 and 1.20ppm respectively to

the carbon CO. This bonding resulted in the joining of the sesquiterpene moiety to

the quinone moiety thus forming of the final structure of the natural product shown

below in Figure 3-17.

69

O

O

OMe

Hh

Ho

Ho'

OH

H

H

O

O

OCH3

HO

1

2

3 4 5

6

7

8910

11

12

13

1415

16

17

18

1920

21

22

Figure 3-17: Final structure of compound 1.

Compound 1 belongs to the class of natural products known as sesquiterpene

quinones. The structural framework resembles that of ilimaquinone and so did most

of the 1H NMR and 13C NMR except for the 1H NMR values of the exocyclic

methylene protons Hg which were resonating at 4.71 and 4.68ppm and the

quaternary carbon (L/L΄) resonating at 36.9ppm. These difference suggested that the

active compound isolated was indeed an epimerv of ilimaquinone, epi-ilimaquinone.

v Epimer are diastereomers that differ in the configuration of only one stereogenic center.

70

Compound 1 was first isolated in 1979 from a Palauan sponge of the genus

Fenestraspongia.

3.2.6 Verification of the stereochemistry

To confirm that the active compound isolated was indeed epi-ilimaquinone and not

ilimaquinone, some of its properties were compared. The properties of compound 1

were compared to those of ilimaquinone from the literature as very few details about

epi-ilimaquinone are available. Firstly and very importantly, the comparison of the 1H NMR (Figure 3-26) and 13C NMR (Table 3-6) of epi-ilimaquinone and

ilimaquinone shows that the 1H NMR signals for the exocyclic protons appear at

4.44/4.43ppm for ilimaquinone however it appears at 4.71/4.68ppm for epi-

ilimaquinone which supports and the conclusion that the isolated compound was epi-

ilimaquinone with a cis-4,9-friedodrim-4(15)-ene sketelon.

While comparing the 13C NMR data of epi-ilimaquinone to ilimaquinone a major

difference was observed in the chemical shifts of C-5 and C-8. The results showed

that for epi-ilimaquinone the chemical shifts for these carbons are very similar

(literature62 C-5 39.5ppm and C-8 39.5ppm), while that of ilimaquinone62 was at C-5

43.3ppm and C-8 36.1ppm. This was also noted from the average values calculated

for the 4,9-friedodrimene skeleton63 (Figure 3-19). The observed 13C NMR chemical

shift, C-5 (L) 39.6ppm and C-8 (L’) 39.6ppm, of the active compound are also in

agreement with this (Table 3-6). The 1H NMR and 13C NMR data corresponding to

the sesquiterpenic part closely resembles those of arenarol and other

sesquiterpene/quinone bearing the cis-4,9-friedodrim-4(15)-ene skeleton63 such as 5-

epi-smenospongine64, 5-epi-smenospongorine65 and 5-epi-smenospongidine.65 This

establishment that all the compounds possessing the same sesquiterpene moiety have

similar 13C NMR shifts (Figure 3-19) further confirms that the compound isolated

has cis-4,9-friedodrim-4(15)-ene skeleton and thus is epi-ilimaquinone and the

stereochemistry is the same as arenarol as shown below, Figure 3-18.

The second factor that verifies this was the fact that the melting point of compound 1

was in the range of 98-100°C and that of ilimaquinone is 108-110°C (pentane)66 and

71

112-113°C (hexane)55 further suggests that compound 1 is not ilimaquinone. The

melting point of epi-ilimaquinone is not reported in the literature. Lastly, the Rfvi

value of epi-ilimaquinone was calculated to be 0.44 (7:3 hexane: EtOAc) on a

normal phase TLC plate (Figure 3-27) (thin-layer analytical plates 60F254) while that

of ilimaquinone was reported to be 0.3466 (7:3 hexane: EtOAc and TLC analyses

were performed on thin-layer analytical plates 60F254). This difference in the Rf

value, while keeping all the others parameters the same, suggests that the compound

isolated was not ilimaquinone. If the Rf of two compounds are the same, it indicates

that both may be same compounds but if it is different, then the two compounds are

definitely different. The Rf values of epi-ilimaquinone is also not reported in

literature.

H

R R=

O

O

OCH3

HO

1516

17

18

1920

21

22

R=

HO

OH

[104] arenarol

[102] epi-ilimaquinone

Figure 3-18: Absolute stereochemistry of epi-ilimaquinone.

vi The retention factor, or Rf, is defined as the distance traveled by the compound divided by the

distance traveled by the solvent.

O

O

OCH3

HO

1

2

3 4 5

6

7

89

10

11

12

13

1415

16

17

18

1920

21

22

[74] ilimaquinone

72

H

R34.3

17.6

17.2

37.5

28.0

42.5

38.4

36.620.1

18.4

143.3

121.1

26.9

20.1

trans-4,9-friedodrim-3-ene

47.8

H

R32.8

16.3

19.1

37.3

29.1

42.5

38.4

37.332.3

19.7

138.9

24.4

18.2

cis-4,9-friedodrim-3-ene

46.1123.9

H

34.017.7

17.6

37.7

28.5

42.5

40.6

36.520.8

102.7

160.3

33.2

28.4

23.2

trans-4,9-friedodrim-4(15)-ene

R

49.5

H

35.119.0

18.2

39.1

28.2

42.5

40.6

38.0

33.3106.1

154.3

32.4

25.3

22.8

cis-4,9-friedodrim-4(15)-ene

R

48.1

Figure 3-19: Calculated mean δ 13C for four different 4,9-friedodrimene skeletons found in sponges.

Ilimaquinone and epi-ilimaquinone as quinones are characterized by their two

distinctive pH-dependant colours, yellow (pH<7) and violet (pH>7).67 This

phenomenon was also observed when the active component was isolated. This color

change can be explained by the keto-enol tautomerisation68 of the quinone moiety as

shown in Figure 3-20 below. The keto form is yellow while the enol form is violet.

73

H

ilimaquinone

H

epi-ilimaquinone

OH

O

O

OMe

+ H

- H OH

O

HO

OMe

-H +H

+ H - H

OH

O

HO

OMe

-H

+H

O

O

HO

OMe

H Figure 3-20: Tautomerisation of epi-ilimaquinone and ilimaquinone.

74

OH

-OC

H3

Fi

gure

3-2

1: 1 H

NM

R (4

00M

Hz)

spec

trum

of c

ompo

und

1 (C

DC

l 3).

solv

ent

=CH

-

=CH

2 H

2C

CH

3

CH

3

CH

3

75

Fi

gure

3-2

2: 13

C N

MR

spec

trum

of c

ompo

und

1 (C

DC

l 3).

76

Figu

re 3

-23:

1 H-1 H

CO

SY sp

ectr

um o

f com

poun

d 1

(CD

Cl 3)

.

77

Fi

gure

3-2

4: H

MQ

C sp

ectr

um o

f com

poun

d 1

(CD

Cl 3)

.

78

Fi

gure

3-2

5: H

MB

C sp

ectr

um o

f com

poun

d 1

(CD

Cl 3)

.

79

Solvent k Signals due to exocyclic

protons

Figure 3-26: Comparison of the 1H NMR of the two epimers.

Figure 3-27: NP-TLC profile of epi-ilimaquinone.

Ilimaquinone

Epi-ilimaquinone

80

Table 3-6: NMR values of epi-ilimaquinone and ilimaquinone (CDCl3)

Carbon

Literature NMR values 250MHz for 1H and 62.5MHz for 13C NMR

Observed 1H and 13C

NMR Epi-ilimaquinone Ilimaquinone 1H NMR69 13C

NMR62

1H NMR66, 70 13C

NMR62

1H NMR 13C

NMR

A 20 - 182.5 - 182.4 - 182.9

B 17 - 182.0 - 182.0 - 182.6

C 18 - 161.7 - 161.8 - 161.8

D 4 - 153.4 - 160.5 - 153.6

E 21 7.50 153.5 7.50 OH br 153.4 7.47 s br

1OH

153.4

F 16 117.7 - 117.4 - 117.8

G 11 4.66 105.7 4.44/4.43 ds 1H

each

1026 4.71/4.68 d

2H

105.8

H 19 5.86 102.0 5.85 s, 1H 102.1 5.88 s 1H 102.1

I 22 3.86 56.8 3.86 s, 3H 56.5 3.89 s 3H 56.9

J 10 0.76 1H dd 48.5 0.76 1H dd 50.2 1.20 48.6

K 9 44.9 - 43.3 - 45.0

L 5 39.5 39.5 - 5=43.34, - 39.7

L΄ 8 1.16 1H m 39.5 1.16 1H m 8=38.16 1.18 39.7

M 6 1.51/1.34 2H

m

37.9 1.51/1.34 2H m 36.7 2.02/1.11 38.0

N 12 1.04 33.2 1.04 s 3 H 20.6 1.07 s 3H 33.3

O 15 2.55 ABq 32.7 2.54/2.46 ABq

2H

32.4 2.61/2.50 32.8

P 3 2.32/2.08

1H

32.0 2.32/2.08 1H

ddd

33.0 2.42/2.11 32.1

Q 7 1.39 27.9 1.39 2H m 28.7 1.50/1.21 27.9

R 2 1.86/1.18 25.0 1.86/1.18 2H m 28.7 1.81/1.66 25.1

S 1 2.10/1.44 22.5 2.10/1.44 2H m 23.2 2.16/1.82 22.6

T 14 0.86 18.7 0.84 s 3H 17.9 0.90 s 3H 18.8

U 13 0.92 18.3 0.97 d 3H 17.3 0.95 d 3H 18.4

81

3.2.7 Some reported properties of epi-ilimaquinone

In the marine literature, there are many examples of sesquiterpene quinone

metabolites that have been isolated from sponges and algae. Reported biological

activities that include antimicrobial, antileukaemic, antiviral and immunomodulatory

effects have led to a wealth of metabolites possessing either the regular drimane

skeleton or the rearranged 4,9-friedodrimane skeleton.71 Epi-ilimaquinone was first

isolated form a sponge identified as a species of Fenestraspongia which was

collected from Palau. The crude extract of this sponge exhibited in vitro

antimicrobial activity and inhibited cell division in the fertilized sea urchin egg

assay. Also identified from this sponge were ilimaquinone and the inactive

metabolite furospinulosin [105]. Epi-ilimaquinone has also been previously isolated

from Dactylospongia elegans72, family Spongidae11, Polyfibrospongia australis73

and Petrosaspongia metachromia64.

O

[105] Furospinulosin

The methanolic extract was chromatographed on Sephadex LH-20 using 1:1

methanol:DCM as the eluant and separated the major inactive metabolite

furospinulosin from a 6:4 mixture of ilimaquinone and 5-epi-ilimaquinone. The

authors identified that the active mixture from this sponge contained two compounds

both having an exocyclic methylene group. The 1H NMR spectrum contained the

signals expected for ilimaquinone at δ 5.86ppm (s, 1 H), 4.45ppm (br s, 1 H),

4.43ppm (br s, 1 H), and 3.87ppm (s, 3 H) together with an additional set of signals

at δ 5.87ppm (s, 1 H), 4.70ppm (br s, 1 H), 4.67ppm (br s, 1 H), and 3.88ppm (s, 3

H) in the low-field region. These data suggest that the second compound was a

stereoisomer of ilimaquinone. Attempts to separate ilimaquinone from its isomer

were unsuccessful. The mixture was separated by acetylation of the quinones with

acetic anhydride in pyridine which produced a mixture of acetates that were

separated by LC on µ-Porasil by using 40% ether in hexane as eluant. Comparison of

82

the 1H and 13C NMR spectra of both the acetates indicated that both compounds had

the same substitution pattern about the quinone ring. The remaining 13C NMR signals

were consistent in chemical shift and multiplicity with corresponding signals of

arenarol.74 When ilimaquinone was isolated it was assigned the absolute

stereochemistry of 5R, 8R, 9S, and l0R by X-Ray analysis55 but in 1987 Capon56

reversed the absolute stereochemistry of ilimaquinone as the same as avarol 8S, 9R.

Based on these chemical correlations and NMR analysis the absolute stereochemistry

of epi-ilimaquinone was assigned the same as arenarol. The mixture of ilimaquinone

and 5-epi-ilimaquinone was tested in a number of bioassays that screen for

ecological activity. When applied to food pellets at 5µg/mg, the quinone mixture

caused significant inhibition of feeding by goldfish.74 Epi-ilimaquinone isolated in

this project showed brine shrimp activity with LD50 of 18ppm.

3.3 Isolation of cytotoxic compound 2

The fraction FDF4 from NP-FCC (Table 3-2) was also brine shrimp active with the

LD50 of 201.6ppm. This fraction was further subjected to semi-prep RP-HPLC on a

C18 250mm x 10μ Econosil column and eluted with 80% MeCN-H2O with 0.1% TFA

as the mobile phase at a flow rate of 4ml/min. With this HPLC system, six fractions

were collected over a run time of 30mins as shown in Figure 3-28. These fractions

were tested for biological activity against brine shrimps and the results show that the

major compound, FJ05-097FDF4H6, isolated was the only active compound. This

active compound which eluted at Rt of 22mins was isolated as a purple solid upon

removal of the solvents.

83

Figure 3-28: HPLC chromatogram of the bioactive FJ05-097FDF4 fraction.

Table 3-7: Weights and activity of the FJ05-097FDF4 HPLC sub-fractions

Fraction ID

FJ05-097FDF4


LD50 (ppm)

H1 4.2 na*

H2 1.9 nt**

H3 1.6 nt

H4 1.5 na

H5 1.6 na

H6 3.7 188

*not active **not tested

FDF4 H6

84

The purity of this compound was checked on RP-TLC with the solvent system of

80%MeCN-H2O on a silica gel 60 RP-18254s glass plate. As seen on Figure 3-29,

only one spot is visible indicating that the compound isolated was quite pure. No

attempt to crystallize the compound was made as the percentage yield was low.

Approximately 3mg of this compound was sent for LC-MS and NMR analysis. The

fraction F05-097FDF4H6 was designated compound 2 for further discussions.

Figure 3-29: RP-TLC of fraction FJ05-097FDF4H6.

3.3.1 UV and IR spectroscopic analysis

The IR spectrum (Figure 3-30) of compound 2 was similar to compound 1. It

showed very strong absorbance in the range of 3835-3566cm-1, indicating the

presence of either hydroxyl or animo groups. Absorbance was also observed in the

range of 1624 and 1536cm-1, indicating carbonyl (C=O) or NH functionality. The

UV (MeOH) spectrum (Figure 3-30) of compound 2 showed absorbance at λmax of

317nm. Upon the addition of a base the absorbance shifted to 327nm as this was due

to the formation of the keto-enol system. Based on these observations it can be

assumed that compound 2 has a quinone moiety and is similar in structure to

compound 1.

85

200.

3040

5060

7080

0.-

12345

6.

n

A

315.

4

265.

0

UV

pro

file

Figu

re 3

-30:

IR sp

ectr

um (K

Br)

of c

ompo

und

2. In

set:

UV

pro

file

of c

ompo

und

2.

4000

.036

0032

0028

0024

0020

0018

0016

0014

0012

0010

0080

060

045

0.0

50.6

6

51.0

51.5

52.0

52.5

53.0

53.5

54.0

54.5

55.0

5

cm-1

%T

3566

.72

1073

.98

650.

26

740.

79

828.

5889

4.42

1163

.27

1374

.51

1536

.37

1624

.15

2362

.63

2928

.57

3736

.26

3835

.16

86

3.3.2 LC-MS and NMR data of compound 2

The ESI-LCMS of compound 2 revealed a parent peak of mass to charge (m/z) ratio

of 344.22 as shown in Figure 3-31. The molecular ion peak at 344.22m/z represents

[M+H]+, therefore the actual mass of the compound is 343.22. Since the molecular

weight is an odd number, the compound contains and odd number of nitrogen.

Using the Xcalibur® 2.0.7 SPI software the LC-MS data was analyzed and the

molecular formulas for the mass 344.22 were generated. The list of molecular

formulas resulted were tabulated in Table 3-8 below. Identification of the molecular

formula for compound 2 was achieved with the aid of the NMR data in Table 3-9.

For the mass 344.22 the molecular formula was determined to be C21H30O3N which

is the first molecular formular in Table 3-8 generated using the software, but since

this formula is for the [M+H] +, the actual molecular formula of compound 2 is [M+]

C21H29O3N. Using this molecular formula the double bond equivalence (dbe) is

calculated to be 8 using Equation 3-2.

AQP392 # 2133 RT: 34.83 AV: 1 NL: 7.38E5 F: FTMS + p ESI Full ms [100.00-2000.00]

340 345 350 355 360 365 370 375 380 m/z

0 10 20 30 40 50 60 70 80 90

100 344.22

C 21 H 30 O 3 N

370.24 C 23 H 32 O 3 N 356.22

C 22 H 30 O 3 N 361.24 C 22 H 33 O 4 375.22

C 22 H 31 O 5

346.23 C 9 H 30 O 6 N 8

[M+H]+

Rel

ativ

e A

bund

ance

Figure 3-31: ESI-LCMS of compound 2.

87

Equation 3-2: Formula for calculation of double bond equivalence for molecular formula containing nitrogen.


m/z Theoretcal. mass Delta (mmu) RDB equiv. Composition

344.22 344.22 0.12 7.5 C21 H30 O3 N

344.22 1.46 8.0 C19 H28 O2 N4

344.22 -1.73 0.0 C8 H28 O5 N10

344.22 -2.56 12.0 C24 H28 N2

All the NMR data of compound 2 were taken in CD3OD. The 13C NMR of

compound 2 was not taken therefore the 13C NMR values were obtained from the

HSQC and HMBC spectra and are tabulated in Table 3-9. The 1H NMR data of

compound 2 were similar to compound 1 with the only exception of the signal due to

the methoxy group at 3.89ppm (proton i) in compound 1 (Figure 3-21) being absent

in compound 2 as seen in the 1H NMR spectrum of compound 2 in Figure 3-32. The

NMR data of compound 2 prove that the assumption made using the IR

spectroscopic and UV data about the similarity of these two compounds and the

presence of the quinone moiety are correct. Furthermore the upfield (low frequency)

region of the 1H NMR (0-2.80ppm) of compounds 1 and 2 are very similar. This

indicates that compound 2 not only has the quinone moiety but it also possesses the

same rearranged drimane, sesquiterpene bicyclic ring as ilimaquinone.

dbe= [(2C+2)-(H-N)]/2

88

Table 3-9: NMR data of compound 2 in CD3OD

Carbon

labeled

C δ ppm H δ ppm (mult.) COSY

H - H

HMBC

C - H

A 183.2 g, n/n΄

B 183.2

C 160.1 f, m/m΄, r

D 159.0 g, n/n΄

E 113.7 n/n΄, t

F 101.7 4.44 (bs 2H) r, m/m΄ m,m΄

G 93.6 5.51 (s 1H)

H 49.7 0.82 n/n΄, q΄, r, t

I 42.2 n/n΄, s, t, k, o΄, l΄, h

J 39.9 r, t, m΄, q΄, l΄, f

K 37.6 1.23 s, t, n/n΄, q΄, l΄, h

L 36.4 1.50/1.38 r

M 32.5 2.34/2.05 o/o΄, m f

N 31.6 2.47/2.40 (AB

quart. 2H)

t, h, o

O 28.3 1.23/1.82 m/m΄

P 27.5 1.39 s

Q 22.8 2.17/1.43 o/o΄ m΄, t, h

R 19.4 1.05 (s 3H) f

S 16.9 0.98 (d 3H) o΄, k n/n΄, k, o΄, t

T 16.2 0.83 (s 3H) n/n΄,

89

Fi

gure

3-3

2: 1 H

NM

R (4

00M

Hz)

of c

ompo

und

2 (C

D3O

D).

=CH

-

=CH

2

CH

3

CH

3

-CH

2-

solv

ent

CH

3

90

Fi

gure

3-3

3: 1 H

-1 H C

OSY

of c

ompo

und

2 (C

D3O

D).

91

Fi

gure

3-3

4: H

SQC

spec

trum

of c

ompo

und

2 (C

D3O

D).

92

Fi

gure

3-3

5: H

MB

C sp

ectr

um o

f com

poun

d 2

(CD

3OD

).

93

3.3.3 Structure elucidation of compound 2

Since compound 2 is an analogue of compound 1 (epi-ilimaquinone), the structure

elucidation will not be discussed in detail. The connectivities of carbons to its

corresponding protons in the molecule were determined from the 2D NMR spectrum

(HSQC spectrum) in Figure 3-34. The two olefinic protons present in the molecule

at 4.44ppm (Hf) and at 5.51ppm (Hg) were complemented by the presence of two

olefinic carbons at 101.7 and 93.6ppm repectively. It was noted that there were three

more olefinic carbons resonances at 160.1 (C), 159.0 (D), and 113.7 ppm (E). This

confirms that there are two double bond in the molecule but does not account for one

extra olefinic carbon. Therefore this point to the fact that one of the olefinic carbons

is not shown in the 2D NMRs and is assumed to be masked by other carbon signals

in similar chemical environment. The occurrence of this carbon is proven by the

molecular formula of C21H29O3N.

The presence of a rearranged drimane skeleton is confirmed by the HMBC

correlations observed. The olefinic proton Hf resonance at 4.44ppm shows a two-

bond HMBC correlation to the olefinic carbon, C, resonance at 160.1ppm, three-

bond HMBC correlations to a sp2 carbon, M, resonance at 32.5ppm, and to a

quaternary carbon, J, resonance at 39.9ppm, and a four-bond HMBC correlation to

the methyl carbon, R, at 19.4ppm. These HMBC correlations resulted in the partial

structure 2A shown in Figure 3-36.

C

CH H

CCH3H

H

160.1

101.6

39.9

32.5

4.44 4.44

2.34

2.05

19.4


As discussed earlier, UV, IR, NMR and LC-MS data confirms that compound 2 is a

six-membered quinone ring, which is also penta-substituted. This is also indicated by

94

the splitting pattern of the proton Hg which is a singlet, resonance at 5.51ppm on

carbon G, resonance at 93.6ppm. Since this proton signal is not splitting, there is no

neighbouring proton present. The proton Hg (5.51ppm) shows two and three-bond

HMBC correlations to the carbonyl carbons A and B, resonance at 183.2ppm and to

the quaternary carbon D resonance at 159.0ppm. To fulfill the carbon’s valency of

four, there should be a substitution on carbon CE. Having used up all the carbons

from the molecular formula, C21H29NO3, the only possible substitution would be the

amine, (-NH2) group. These correlations result in the partial structure 2B shown in

Figure 3-37.

O

O

NH2

OH

H

183.2

183.2

93.6

159.0

5.51E

Figure 3-37: HMBC correlations of partial structure 2B.

The two partial structures, 2A and 2B, were joined with the help of the HMBC

correlations shown by the benzylene methylene protons Hn/n΄ resonance at

2.47/2.40ppm resulting in the final structure shown below in Figure 3-38. With the

formation of this structure the double bond equivalence of eight was catered for.

From the spectral data analysis the structure of compound 2 was determined to be of

the marine natural product known as smenospongine.

This compound has been originally isolated from a bright yellow marine sponge,

Smenospongia sp. This sponge was collected in the Red Sea near Djibouti by

SCUBA diving between 20-25m. The sponge was extracted with MeOH and then

with 1:1 MeOH:chloroform mixture. After removal of the solvent the aqueous

suspension was extracted with DCM, which showed antimicrobial activity (S.

aureus) and cytotoxic activity (L 1210 leukemia cells).

95

O

OH

O

NH2

H2C

H

49.7 37.642.2

Figure 3-38: HMBC correlations of compound 2.

O

OH

O

NH2

H2C

H

Figure 3-39: Structure of compound 2, smenospongine.

Table 3-10 shows the literature NMR values for the compound smenospongine.75

The 1H NMR spectrum was recorded from a 500MHz instrument with CDCl3 as the

NMR solvent and 13C NMR was recorded from a 20MHz instrument in CDCl3. The

96

1H NMR data for smenospongine, compound 2, in this research was measured on a

400MHz instrument while the 2D NMR spectra were recorded on a 125MHz

instrument and the solvent used was CD3OD. It can be noted from Table 3-10 that

there is very minimum differences in the NMR values in the literature and in the

smenospongine isolated. These differences can be attributable to the different NMR

solvents used.

In the original report75, the absolute configuration of the compound was determined

by the direct comparison of the CD spectra (circular dichroism) of ilimaquinone and

smenospongine. It was noted that both products exhibited a Cotton effect in the

300nm region in accord with the quinone chromophore. These results suggested that

the drimane skeleton in smenospongine possessed the same absolute configuration as

ilimaquinone and thus was assigned its structure. Since no CD spectrum was

measured in this project, the absolute configuration of compound 2 was determined

by comparing the 1H NMR and 13C NMR values of compound 2 with the literature

values of smenospongine and compound 1, epi-ilimaquinone and also the mean

calculated δ 13C values for the 4,9-friedodrimene skeleton (Figure 3-19). The main

proton chemical shifts to consider in determining the stereochemistry of compound 2

or rearranged drimane system (4,9-friedodrimene skeleton) is the signals due to the

exocyclic methylene in the sesquiterpene ring, while the carbons at positions C-5 and

C-8 also play an important role in determining the stereochemistry. For a trans-4,9-

friedodrim-4(15)-ene system, the exocyclic protons resonate at 4.44/4.40ppm and the

carbons C-5 and C-8 are very distinctive as opposed to cis-4,9-friedodrim-4(15)-ene

system where these carbons are very similar in the chemical shifts.63 As seen from

Table 3-10, the 1H NMR value is 4.44/4.40ppm and the values for carbons C-5 (K)

and C-8 (L) is 40.5 and 38.2ppm indicates that compound 2 possesses a trans-4,9-

friedodrim-4(15)-ene system as shown in Figure 3-40. The isolated compound 2,

smenospongine showed moderate brine shrimp activity at 188ppm.

97

Table 3-10: Comparison of the NMR data for smenospongine

Carbon

label

No.

Literature NMR values for

smenospongine

Observed NMR values for

Compound 2 13C NMR 1H NMR 13C NMR 1H NMR

A 18 183.2 - 183.2 -

B 21 179.8 - 183.2 -

C 4 160.5 - 160.1 -

D 17 157.9 - 159.0 -

E 20 150.8 - Not shown. -

F 16 114.5 - 113.7 -

G 11 102.6 4.44/4.40 s 101.7 4.44 d

H 19 95.8 5.62 d 93.6 5.51 s

I 10 50.4 0.79 dd 49.7 0.82

J 9 43.1 - 42.1 -

K 5 40.5 - 39.9 -

L 8 38.2 1.18 m 37.6 1.23

M 3 36.8 2.33 dt/2.07 dd 36.4 1.50/1.38

N 6 33.1 1.52/ 1.45-1.36 m 32.5 2.34/2.05

O 15 32.6 2.51/2.52 dd AB

syst.

31.6 2.47/2.40

P 7 28.7 1.45-1.36 m 28.3 1.23/1.82

Q 2 28.1 1.86/ 1.45-1.36 m 27.5 1.39

R 1 23.3 2.10 dd/ 1.25 s 22.8 2.17/1.43

S 12 20.5 1.05 s 19.4 1.05 s

T 13 17.8 0.98 d 16.9 0.98 d

U 14 17.2 0.84 s 16.2 0.84 s

98

O

O

NH2

H

H OH1

4

12

813

14

10

1517

1921

11

Figure 3-40: Final structure of compound 2, [106] smenospongine.

3.3.4 Some reported properties of compound 2

Smenospongine belongs to the group of compounds known as sesquiterpene

aminoquinones. Smenospongine had originally been isolated from a Smenospongia

sponge in 198775 and after this it has been isolated from a number of sponges such as

Petrospongia metachromia64 and Dactylospongia elegans.41, 62 As reported in the

literature70, 75 smenospongine, shows a UV λmax of 209 and 317nm and IR

absorbance at 1565, 3260 and 3480cm-1, indicative of a amino quinone ring was also

observed in the compound isolated. This compound has been found to induce

differentiation of K562 cells into erythtoblasts.65 The in vitro anti-HIV activity on

HIV-1 RT of smenospongine was evaluated and it displayed moderate inhibitory

activity, with IC50 value of 176.1μM.70 Smenospongine and the 5-epi congeners (epi-

smenospongine), isolated from a Hippospongia species, promoted IL-8 production in

Chinese hamster V79 cells.13

99

3.4. Isolation of cytotoxic compound 3 Reverse phase vacuum liquid chromatography (RP-VLC) of the weakly cytotoxic

fraction, FM from solvent portioning, with water and increasing amount of MeOH

resulted in a fraction eluted with 80%MeOH-H2O retaining the original activity. This

fraction labeled FJ05-97FMF2 was further purified on RP-HPLC using the same

parameters as compound 2 but with a mobile phase of 65%MeCN-H2O and 0.1%

TFA. Seven HPLC fractions (Table 3-11) were collected (Figure 3-41) over a run

time of 30mins. Brine shrimp bioassay showed that the HPLC fraction FJ05-

97FMF2H7, eluted at Rt of 17mins, was the active principle.

Table 3-11: Weights and bioactivity of the FJ05-097FMF2 HPLC sub-fractions

Fraction ID

FJ05-097FMF2


LD50 (ppm)

H1 12.1 na*

H2 2.7 na

H3 1.3 nt**

H4 3.2 na

H5 1.1 nt

H6 5.3 na

H7 4.9 >500ppm

*not active

**not tested

RP-TLC was carried out with the solvent system of 65%MeCN-H2O to check the

purity of this compound. As seen in Figure 3-42, only one spot is visible indicating

that the compound isolated was quite pure. Since the percentage yield was low this

compound was not crystallized. Approximately 4mg of this compound was sent for

LC-MS and NMR analysis. The fraction F05-097FMF2H7 was designated

compound 3 for further discussions.

100

Figure 3-41: HPLC chromatogram of the bioactive FJ05-097FMF2 fraction.

Figure 3-42: RP-TLC of fraction FJ05-097FMF2H7.

H7

101

200. 30 40 50 60 70 800.-

1

2

3

4

5

6.

n

A

317.8270.9

UV profile

317

3.4.1 LC-MS data of compound 3

A molecular ion peak [M+H]+ at 402.23 from the ESI-LCMS (Figure 3-43)

generated a list of formulas shown in Table 3-12. Since the molecular ion peak is for

[M+H]+, the actual mass of the compound [M]+ is 401.23.

AQP387 #2019 RT: 32.81 AV: 1 NL: 1.49E6F: FTMS + p ESI Full ms [100.00-2000.00]

340 360 380 400 420 440 460 480 500 520m/z

0

10

20

30

40

50

60

70

80

90

100

402.23C 23 H32 O5 N

505.31C 17 H43 O10 N7

452.21C 16 H36 O14 487.28

C 27 H39 O6 N2416.24C 24 H34 O5 N

372.22C 22 H30 O4 N

470.42C 28 H56 O4 N

444.27C 26 H38 O5 N

388.25C 23 H34 O4 N

Figure 3-43: ESI-LCMS of compound 3. Inset: UV profile of compound 3.


m/z Theoretical mass Delta (mmu) RDB equiv. Composition

402.23 402.23 0.13 8.5 C23 H32 O5 N

402.23 0.14 14.0 C22 H26 N8

402.23 -1.21 13.5 C24 H28 O N5

402.23 1.47 9 C21 H30 O4 N4

[M+H] +

102

With the help of the NMR data (Figure 3-13), the molecular formula for compound

3 of mass 402.23 was determined to be C23H31NO5 from Table 3-12. From this

molecular formula the dbe using Equation 3-2 was calculated to be nine.

3.4.2 Structure elucidation of compound 3

The NMR data of compound 3 are tabulated in Table 3-13. On a first glance of the 1H NMR spectrum of compound 3 (Figure 3-45), it looks like compound 3 is

ilimaquinone. The presence of a rearranged drimane sesquiterpene bicyclic ring

(trans-4,9-friedodrim-4(15)-ene) is also apparent. The exocyclic methylene protons

resonances at 4.41ppm (Hh) (2H), three methyls at 1.06 s (Hu), 0.99 d (Hv) and

0.85ppm s (Hw) and the benzylic methylene protons at chemical shift of

2.55/2.40ppm (Hq/q΄) on the sesquiterpene ring were identified. The olefinic proton

(Hi) resonance at 5.28ppm as a singlet with one proton and a deshielded singlet at

3.98ppm (Hk) presumed to be due to the methoxy group on the quinone moiety were

also identified. Similar to compounds 1 and 2, this compound also showed some

common HMBC correlations shown in Figure 3-44.

CH3

CH3

CH3

O

O H

OH

X


103

With the formation of the partial structure 3A (Figure 3-44) eight out of nine degrees

of unsaturation was fulfilled. On a closer examination of the 2D NMR data, namely

the HSQC (Figure 3-48) and HMBC (Figure 3-49) spectrum, it was revealed that

the singlet at 3.98ppm, (Hk), with its corresponding carbon (CK) resonance at

44.4ppm is not due to a methoxy group as previously assumed but was due to a

methylene group (–CH2-). The deshielded chemical shift indicate that this –CH2- is

attached to an electronegative atom or group. To investigate the structure of

compound 3 further, the HMBC spectrum (Figure 3-49) was used.

104

Table 3-13: NMR data of compound 3 in CD3OD

Carbon

labeled on 13C

NMR

C δ ppm H δ ppm (mult.) COSY

H - H

HMBC

C - H

A 183.2 i, q/q΄

B 183.2

C 170.5 k

D 161.6 h, p/p΄, u

E 159.5 i, q/q΄

F 151.5 k

G 115.7 q/q΄

H 103.1 4.41 (ds 2H) p p/p΄

I 93.9 5.28 (s 3H) k

J 51.2 0.82 t΄ q/q΄, w, u

K 44.4 3.98 (s 3H)

L 43.8 q/q΄, v, w

M 41.6 h, p΄, s, u

N 39.1 1.23 q/q΄, v, w

O 37.9 1.50/1.38 t u

P 34.0 2.33/2.39 h

Q 33.2 2.53/2.40 (AB

quart. 2H)

R 29.8 1.82/1.22 p/p΄, t p

S 29.1 1.39 v

T 24.3 2.17/1.45 p΄, w

U 20.9 1.06 (s 3H) h, w

V 18.6 0.99 (d 3H) n

W 17.8 0.85 (s 3H) q/q΄, w

105

Fi

gure

3-4

5: 1 H

NM

R o

f com

poun

d 3

(CD

3OD

).

=CH

-

CH

3 -C

H2-

solv

ent

-CH

2-

=CH

2

CH

3

CH

3

106

Fi

gure

3-4

6: 13

C N

MR

of c

ompo

und

3 (C

D3O

D).

107

Fi

gure

3-4

7: 1 H

-1 H C

OSY

of c

ompo

und

3 (C

D3O

D).

108

Fi

gure

3-4

8: H

SQC

spec

trum

of c

ompo

und

3 (C

D3O

D).

solv

ent

109

Figu

re 3

-49:

HM

BC

spec

trum

of c

ompo

und

3 (C

D3O

D).

110

This methylene proton (Hk) signal showed two-bond and three-bond HMBC

correlations to two quaternary carbons, CC resonance at 170.5ppm and to CF

resonance at 151.5ppm respectively. These signals (3.97, 44.4 and 170ppm),

according to Yohei Takahashi36 are due to the glycine reisdue. The three-bond

HMBC correlation from proton Hk (3.98ppm) to the carbon, CF (170.5ppm) showed

that the carbon CK (44.4ppm) is connected to carbon CF (170.5ppm) through the

amine group (-NH-) from the glycine residue as depicted in Figure 3-50.

NH

OH

O

R

170.5

44.4/3.98

CK/k

O

OH

O

HNOH

H2C

O

H

Figure 3-50: HMBC correlations of partial structure 3B.

These correlations resulted in the final structure of compound 3, which is a glycine

derivative of ilimaquinone, known as glycinylilimaquinone Figure 3-51. To date,

there is only one report in the literature on the isolation of this compound.60 The

reported NMR data of glycinylilimaquinone is tabulated in Table 3-14.

111

O

O

OH

NH

H

H2CC

O

OH

11

1

12

8

13

14

10

1517

1921

2223

Figure 3-51: Final structure of compound 3, [107] Glycinylilimaquinone.

Table 3-14: Comparison of the NMR data of glycinylilimaquinone

Carbon

No.

Literature NMR values for

smenospongine

Observed NMR values for

Compound 2 13C NMR 1H NMR 13C NMR 1H NMR

A 18 183.7 - 183.2

B 21 179.2 - 183.2

C 23 169.9 - 170.5

D 4 160.7 - 161.6

E 17 158.0 - 159.5

F 20 150.3 - 151.5

G 16 114.4 - 115.7

H 11 103.1 4.38 103.1 4.41 d

I 19 93.4 5.37 93.9 5.28 s

J 10 50.2 N/A* 51.2 0.82 s

112

K 22 44.1 4.09 44.4 3.98 s

L 9 43.4 - 43.8 -

M 5 41.1 - 41.6 -

N 8 38.2 N/A 39.1 1.23

O 3 37.2 N/A 37.9 1.50/1.38

P 15 33.3 2.50/2.40 34.0 2.33/2.39

Q 6 32.6 N/A 33.2 2.53/2.40

R 7 29 N/A 29.8 1.82/1.22

S 1 28.4 N/A 29.1 1.39

T 2 23.6 N/A 24.3 2.17/1.45

U 12 20.6 1.04 20.9 1.06 s

V 13 18.2 0.97 18.6 0.99 d

W 14 17.5 0.84 17.8 0.85 s

*not available.

Glycinylilimaquinone was previously isolated in 1994 from a Dictyoceratid sponge

of the genus Fasciospongia collected from the Philippines. The crude extract of this

sponge was fractionated on sephadex LH-20 to obtain the bioactive compound which

was called glycinylilimaquinone, together with other known compounds.

Glycinylilimaquinone showed 1000 times more activity than the crude extract in the

in vitro HCT-116 assay.60 The UV spectrum consisted of absorptions at 492, 320 and

203nm and the IR spectrum consisted bands at 3300, 3175, 1725, 1587 and

1575cm-1 due to hydroxyl, acid and the quinone groups respectively. The IR

spectrum for compound 3 was not taken due to small amounts being isolated while

the UV spectrum (Figure 3-43) of compound 3 showed similar absorptions as

reported. On addition of a base, the absorption at 317nm underwent a bathochromic

shift to 332nm, indicating the presence of a hydroxyl group on the quinone ring.

Glycinylilimaquinone [107] showed very weak activity towards brine shrimps.

113

3.5 Sesquiterpene quinones and related compounds from sponge of

the order Dictyoceratida

The sesquiterpene quinones share a common drimane rearranged cis- or trans-

decalin ring varying at the relative position of the double bond at the C-4 carbon

and/or the stereochemical configuration about C-5. The C-9 position is decorated

with a variably hydroxylated or heteroatom-substituted benzoquinone side chain.76

Cis- or trans-decalin rings can be further divided into trans-4,9-friedodrim-3-ene,

cis-4,9-friedodrim-3-ene, trans-4,9-fridodrim-4(15)-ene and cis-4,9-fridodrim-4(15)-

ene as shown in Figure 3-19. Some examples of trans-decalin sesquiterpene

quinones/quinols similar in structure are avarol and avarone from Dysidea avara,

ilimaquinone from Hippospongia metachromia, isospongiaquinone from

Stelospongia conulata, the nakijiquinones from Spongia sp and bolinaquinone from

Dysidea sp while examples of cis-decalin sesquiterpene quinones/quinols would

include epi-smenoquinone, epi-smenospongidine, epi-smenospongorine and also epi-

ilimaquinone. All these compounds were isolated from the sponge belonging to the

order Dictyceratida, collected from different areas.77 According to Salmoun and co-

researchers the 13C chemical shifts of the carbon atoms of sesquiterpenic part are

very little influenced by the nature of the substituent at the junction of sesquiterpene

and quinone. Which lead them to calculate the mean chemical shift for all carbon

atoms for each skeleton (Figure 3-19).63 A great number of marine quinones and

hydroquinones are of considerable interest with regard to their diverse biological

activities. Furthermore, sesquiterpenes in which a decaline type unit and a quinoid

moiety are structurally associated, often exhibit pronouced cytotoxicity.78

114

CHAPTER 4 CONCLUSION

Marine natural products have been investigated predominantly for their

antimicrobial, cytotoxic, antitumor, antiviral and anti-inflammatory properties.

Success in these areas is demonstrated by the compounds now in pre-clinical trials.

This study involved the isolation, structure elucidation and biological screening of

active constituents of the marine sponge, Hippospongia sp., collected off Kia Island

in Fiji. Various chromatographic techniques (solvent partitioning, flash column

chromatography and HPLC) were employed to isolate and purify the active

components and through spectral analysis (LC-MS and NMR) the structures of these

compounds were elucidated. Brine shrimp bioassay guided purification resulted in

the isolation of three compounds. The characterizations of three cytotoxic

compounds were successfully achieved and were identified as:

Compound 1: [102] epi-ilimaquinone

Compound 2: [106] smenospongine

Compound 3: [107] glycinylilimaquinone

Table 4-1: Summary of three compounds isolated

Compound Molecular formula Bioactive

BSA (ppm)

Rf Mobile phase color

1 [102] C22H30O4 18 0.23 (80% MeCN-H2O) yellow

2 [106] C21H29O3N 188 0.22

(80% MeCN-H2O+

0.1% TFA)

purple

3 [107] C23H31O5N > 500 0.23

(65% MeCN-H2O+

0.1% TFA)

red

115

H

O

O

R

H

OH

[102] R=OMe

H

O

O

R

H

OH

[106] R=NH2[107] R=NHCH2COOH

Compound 1 belongs to the class of compounds known as sesquiterpene quinones

while compounds 2 and 3 belongs to the similar class known as sesquiterpene amino

quinones. The compound 1 isolated possess the rearranged drimane skeleton known

as cis-4,9-friedodrim-4(15)-ene while compounds 2 and 3 possess the rearranged

drimane skeleton known as trans-4,9-friedodrim-4(15)-ene. Table 4-1 summarizes

the details of the compounds isolated.

116

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124

APPENDIX

Appendix 1 Expanded NMRs of compound 1

Figure A 1: Expanded 1H NMR of compound 1.


H

O

O

OMe

H

OH

125

Figure A 3: Expanded 13C NMR of compound 1.


126

Figure A 5: Expanded 1H-1H COSY of compound 1.


127

OCH3

Figure A 7: Expanded HMQC spectrum of compound 1.

Figure A 8: Expanded HMQC spectrum of compound 1.

2 X CH3

CH3

=CH- =CH2

128

Figure A 9: Expanded HMBC spectrum of compound 1.


129


130

H

O

O

NH2

H

OH




131



132

3 X CH3

solvent

=CH2

=CH-

Figure A 16: Expanded HSQC spectrum of compound 2.


133



134

H

O

O

NHCH2COOH

H

OH




135


Figure A 23: Expanded 13C NMR spectrum of compound 3.

136

Figure A 24: Expanded 1H-1H COSY spectrum of compound 3.

Figure A 25: Expanded 1H-1H COSY spectrum of compound 3.

137

Cross-peak due to –CH2- on the glycine residue

3 x CH3’s

=CH2

=CH2-



solvent

138



139

Appendix 4

Calculation of LD50

The method for calculation of LD50 for the fraction FJ05-097FHF3 is given below as

an example. After the BSA is conducted, the results are recorded as number of dead

shrimps over total number of shrimps in a well. From this the % dead is calculated

and using the Reed Muench method59, a table as shown below is constructed (Table

A 1). From this table a plot of the number of accumulated alive and number of

accumulated dead on the same axes vs the log dosage is plotted as shown below,

Figure A 30.

Table A 1: Brine shrimp bioassy results for calculation of LD50.

dose(ppm) Dosage (log dose) % Dead % Alive Acc Dead Acc Alive

250 2.39 100 0 280 0

125 2.09 100 0 180 0

62.5 1.79 80 20 80 20

31.3 1.49 0 100 0 120

Figure A 30: Plot for calculation of LD50.

140

The point of intersection (log dose) of the two lines is where the number of survivors

is equal to the number of dead animals; hence taking antilog of this value gives the

LD50 in ppm. For fraction FJ05-097FHF3 the point of intersection is 1.7 and antilog

gives the LD50 of 50.11ppm.

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