UNIVERSITY OF NAIROBI CHEMISTRY DEPARTMENT

K ANTI PLASMODIA I. ANTH R AQL1 NONES AND BENZALDEHYIH DERIVATIVES FROM THE ROOTS OE KNIPUOFIA THOMSONU

BY

IMMACULATE CHIENG’

A THESIS SUBMITED IN PARTIAL FULFILMENT OF THE DECREE OE MASTER OE SCIENCE OF THE UNIVERSITY OF NAIROBI

2009

University ol NAIROBI Library This thesis is my original work and has never been presented for a degree in any university.

jf c IMMACULATEATE ACHIENG’

This thesis has been submitted for examination with our approval as supervisors

SUPERVISORS

SIGN...... Y / k ^ 7 T ^ : ------'

PROF. ABIY YENESEW Department of Chemistry University of Nairobi

SION

PROF. J .O. MIDIWO Department of Chemistry University of Nairobi

Department of Chemistry University of Nairobi

I! DEDICATION THIS THESIS IS DEDICATED TO MY

HUSBAND PETER AND SON LEON.

"Time is too slow for those who wait

Too swift for those who fear

Too long for those who grieve

Too short for those who rejoice

But for those who love

Time is Eternity"

Emma

in ACKNOWLEDGEMENTS

My sincere gratitude goes to my supervisors Prof. Abiy Yenesew. Prof. J. (). Midi wo and I)r. Solomon Derese for their guidance, dedication and inspiration throughout the course of this research work.

1 am greatly indebted to the University of Nairobi for giving me a partial scholarship which enabled me to complete this work on time. My sincere appreciation also goes to Prof. Martin G. Peter, University of Potsdam, the Deutsche Forschungsgemeinschaft (DFG), Germany, Grant Number Pe264/14-5 and 6 and the German Federal Ministry for Economic Cooperation and Development (BMZ) within the DFG/BMZ Programme "Research Cooperation with Developing Countries", for sponsoring part of the study. 1 am indebted to Dr. Matthias Heydenreich, University of Potsdam, for analyzing samples on high resolution NMR and MS. The United States Army Medical Research Unit-Kenya including Dr. N. C. Waters, Pamela Liyala. and Hosea Akala are acknowledged for their assistance in testing for antiplasmodial activity of the compounds isolated in this research.

I would like to sincerely thank the academic and technical staff of the Department of Chemistry. University of Nairobi for the assistance accorded to me whenever I needed it. Many thanks to the former and current members of the Natural Product research group. University of Nairobi, for their cooperation and encouragement during the course of this research work.

1 extend my heartfelt appreciation to my entire extended family and more so to my dearest husband. Peter and son. Leon for being there for me whenever I needed them for indeed “true happiness is not attained through self gratification but through fidelity to a worthy purpose". Above all I thank God for my life.

IV LIST OF ABBREVIATIONS AM) SYMBOLS hrs Broad singlet held Broad double of a doublet COSY Correlated speetroscopy d Doublet dd Double of a doublet ID One dimensional analysis 2D Two dimensional analysis ED50 Concentration of 50% Effectiveness EIMS Electron ionization mass spectroscopy F Fruit FI Flowers HMQC Heteronuclear multiple quantum coherence ( ]Jch) HMBC Heteronuclear multiple bond correlation (2JCh, Vni, V ch) Hz I lertz IC50 Concentration of 50% inhibition J Coupling constant L Leaf MS Mass spectroscopy m/z Mass to Charge ratio MHz Mega hertz m Multiplet [M]+ Molecular ion mp Melting point nm Nanometer NOESY Nuclear overhauser and exchange spectroscopy NMR Nuclear magnetic resonance PTLC Preparative thin layer chromatography Rh Rhizomes R Roots s Singlet S Stem / Triplet TLC Thin layer chromatography u v Ultra violet Maximum wavelength of absorption TABLE OF CONTENTS DEDICATION......

ACKNOWLEDGEMENTS...... LIST OF ABBREVIATIONS AND SYMBOLS ABSTRACT...... CHAPTER ONE...... 1.0 INTRODUCTION...... 1.1 General...... 1 1.2 Problem statement...... 4 1.3 Justification of the Research...... 4 1.4 Significance of the Research...... 5 1.5 Objectives of the Research...... 5 CHAPTER TWO...... 7 2.0 LITERATURE REVIEW...... 7 2.1 Malaria...... 7 2.1.1 The Disease...... 7 2.1.2 Global Malaria Situation...... 7 2.1.3 The Current Malaria Control Strategy...... 8 2.1.4 Limitations of the Current Malaria Control Strategy...... 9 2.2 Botanical Information...... 14 2.2.1 The Family Asphodelaceae...... 14 2.2.1.1 The Sub-family Asphodeloideae...... 14 2.2.1.1.1 The Genus Kniphofia...... 15 2.3 Kniphofia thomsonii...... 15 2.4 Phytochemistry of the Genus Kniphofia...... 16 2.5 Biological Activity of Anthraquinones...... 23 CHAPTER THREE...... 25 3.0 METHODOLOGY...... 25 3.1 General...... 25 3.1.1 Instrumentation...... 25 3.1.2 Collection of Plant Material...... 25

VI 3.1.3 Chromatographic conditions...... 25 3.2 Extraction and Isolation of compounds...... 26 3.3 Physical and Spectroscopic Data for the Isolated Compounds...... 27 3.3.1 Chrysophanol (1 )...... 27 3.3.2 Islandicin (2 )...... 28 3.3.3 Physcion(3)...... 28 3.3.4 Aloe-emodin (4)...... 28 3.3.5 Aloe-emodin acetate (5)...... 28 3.3.6 Knipholone (6)...... 29 3.3.7 Flavoglaucin (7)...... 29 3.3.8 3,'\4"'-Dehydroflavoglaucin (8)...... 29 3.3.9 lOJO’-Bichrysophanol anthrone (9)...... 30

3.3.10 10-Hydroxy-10-(chrysophanol-7’-yl)-chrysophanol anthrone ( 10) ...... 30 3.3.11 10-Hydroxy-10-(chrysophanol-7’-yl)-aloe-emodin anthrone (11)...... 31 3.3.12 10-Hydroxy-10-(islandicin-7’-yl)-chrysophanol anthrone (12)...... 31 3.3.13 10-Hydroxy-10-(islandicin-7,-yl)-aloe-emodin anthrone (13)...... 31 3.4 In vitro anti-plasmodial assay...... 32 CHAPTER FOUR...... 33 4.0 RESULTS AND DISCUSSION...... 33 4.1 ANTHRAQUINONE MONOMERS...... 33 4.1.1 Chrysophanol (1 )...... 33 4.1.2 Islandicin (2)...... 34 4.1.3 Physcion (3)...... 35 4.1.4 Aloe-emodin (4)...... 37 4.1.5 Aloe-emodin acetate (5)...... 38 4.1.6 Knipholone (6) ...... 40 4.2 BENZALDEHYDE DERIVATIVES...... 42 4.2.1 Flavoglaucin (7)...... 42 4.2.2 3”\4’”-Dehydroflavoglaucin (8)...... 44 4.3 ANTHRAQUINONE DIMERS...... 46 4.3.1 10,10’-Bichrysophanolanthrone (9)...... 46

vu 4.3.2 10-Hydroxy-10-(chrysophanol-7'-yl)-chrysophanol anthrone(lO)...... 49 4.3.3 10-Hydroxy-10-(chrysophanol-7'-yl)-aloe-emodin anthrone ( 11)...... 52 4.3.4 10-Hydroxy- 10-(islandicin-7’-yl)-chrysophanolanthrone (12)...... 55 4.3.5 10-Hydroxy- 10-(islandicin-7,-yl)-aloe-cmodinanthronc (13)...... 58 4.4 CHEMOTAXONOMIC SIGNIFICANCE...... 61 4.5 ANTIPLASMODIAL ACTIVITIES...... 63 CHAPTER FIVE...... 65 5.0 CONCLUSION AND RECOMMENDATION...... 65 5.1 CONCLUSION...... 65 5.2 RECOMMENDATION...... 66 REFERENCES...... 67

LIST OF TABLES

Table 1: Compounds of the genus Kniphofia...... 18 Table 2: 1H NMR (300 MHz) data for compound 1, 2 and 3 (CDCI3)...... 36 Table 3: 1H (500 MHz), ,3C (125 MHz) NMR data (acetone-d6,) along with HMBC correlation for compound 4...... 38 Table 4: !H (500 MHz), l3C (125 MHz) NMR data (CDCI3) together with HMBC correlation for compound 5...... 40 Table 5: *H NMR (200 MHz) values for compound 6 (CDCI3) ...... 41 Table 6: 'H (300 MHz), l3C (75 MHz) NMR Together with H,H-COSY and HMBC (CDCI3) data for compound 7 ...... 44 Table 7: *H NMR (300 MHz) data (CDCI3) for compound 8 and 8a ...... 45 Table 8: 'H (500 MHz), l3C (125 MHz) NMR data (CDC13) along with HMBC correlation for compound 9...... 48 Table 9: ’ll (500 MHz) and l3C (125 MHz) NMR data (acetone^) along with HMBC correlations for compound 10...... 51 Table 10: 'H (CDC13, 500 MHz) and l3C (acetone-d6, 125 MHz) NMR data for compound 11...... 54 Table 11: 'H (CDC13, 300 MHz), l3C NMR (acetone-d6+MeOH-d4. 125 MHz) data along with HMBC correlation for compound 12...... 57

vin Table 12: 'll (500 MHz. ClX’lj)and l}C (125 MHz) NMR (acetone-d*) data along with HMBC correlation for compound 13...... 60

Table 13 In vitro ICjo values against the chloroquine resistant (W2) strain of P falciparum...... 64

LIST OF FIGURES

Figure 1.1 Global malaria risks. (From 201'1 WHO Expert Committee Report on Malaria: WHO, 1998)...... 8 Figure 1.2 Reported P. falciparum drug resistance. (From 20th WHO Expert Committee Report on Malaria: WHO, 1998)...... 10 Figure 1.3: Picture of AT. thomsonii...... 15

LIST OF SPECTRA

SPECTRA FOR COMPOUND 1...... 74 SPECTRA FOR COMPOUND 2 ...... 77 SPECTRA FOR COMPOUND 3 ...... 81 SPECTRA FOR COMPOUND 4 ...... 85 SPECTRA FOR COMPOUND 5 ...... 101 SPECTRA FOR COMPOUND 6 ...... 118 SPECTRA FOR COMPOUND 7 ...... 121 SPECTRA FOR COMPOUND 8 ...... 132 SPECTRA FOR COMPOUND 9 ...... 137 SPECTRA FOR COMPOUND 10...... 151 SPECTRA FOR COMPOUND 11...... 169 SPECTRA FOR COMPOUND 12...... 180 SPECTRA FOR COMPOUND 13...... 194

IX ABSTRACT

I he roots of Knifophia tHorn.sonii (Asphodelaceae) were exhaustively extracted with dichloromethane/methanol ( 1:1) by cold percolation at room temperature. The extract showed significant antiplasmodial activity against the chloroquine-resistant (W2) strain of Plasmodium falciparum with IC50 values of 6.36 pg/ml. The extract was subjected to chromatographic separation which led to the isolation of thirteen secondary metabolites.

By the use of ID (1H and l3C) and 2D (COSY. HMBC and HMQC) NMR. MS. IIV spectroscopy and direct TLC comparison with authentic samples in some cases, these compounds were identified as the monomeric anthraquinones: chrysophanol (I), islandicin (2), physcion (3), aloe-emodin acetate (4) and aloc-emodin (5); the phenylanthraquinone: knipholone (6); the benzaldehyde derivatives: flavoglaucin (7) and 3”\4'"-dehydroflavoglaucin (8) and the dimeric anthraquinones: 10.10’- bichrysophanolanthrone (9), 10-hydroxy-10-(chrysophanol-7’-yl)-chrysophanolanthrone

( 10), 10-hydroxy-10-(chrysophanol-7'-yl)-aloe-emodinanthrone ( 11), 10-hydroxy-10- (islandicin-7’-yl)-chrysophanolanthrone (12) and 10-hydroxy-10-(islandicin-7’-yl)-aloe- emodinanthrone (13). The dimeric anthraquinone 13 is a new compound while flavoglaucin (7) and 3 " '.4 ‘"-dehydroflavoglucin (8) are reported here for the first time in higher plants. The C-6 oxygenated anthraquinone physcion (3) is reported here for the first time in the family Asphodelaceae; and this is also the first report for the occurrence of compound 9 (lO.lO'-bichrysophanolanthrone) in the genus Kniphofia.

The compounds isolated in this study were tested in vitro for anti-plasmodial activities against the chloroquine-resistant (W2) strains of Plasmodium falciparum. The monomeric anthraquinones were inactive; while the phenylanthraquinone 6 [ICso 2.50 pg/ml (W2)), the benzaldehyde derivatives 7 | IC50 2.06 pg/ml (W2)| and 8 [ICso 1.93 pg/ml (W2)J and the dimeric anthraquinones 9 (IC50 2.23 pg/ml (W2)J and 12 [ICso 3.42 pg/ml (W2)J showed good activities and appear to be partly responsible for the antiplasmodial activity of the crude extract. This investigation has showed the potential

x of dimeric anthraquinones and the benzaldehyde derivatives as lead structures for development of antimalarial drugs.

O H O O H O H O OH

R 1

R 2

1 R1 = R3= R4= H R2= CH3

2 R 1 = R4 = H R2 = C H 3, R3 = OH

3 R 1 = R3 = H, R2 = C H 3, R4 = O CH 4 R1 = R3 = R4 = H, R2 — CH 2OH

5 Ri = R3= R4= H R2= CH2OA c

CH.

CH,

xi R

c h 3

12 R = CH3 13 R = CH2OH

xn CHAPTER ONE

1.0 INTRODUCTION

1.1 General

Since prehistoric time man has gradually, through trial and error, recognized and used plants for the treatment of malaria and other ailments. Healers, elders, parents, or priests passed on orally the knowledge of efficacious traditional medicaments to some members of their family, community, and the next generation. The potential of traditional medicines in improv ing the health conditions of communities in developing countries by providing the needed medicaments at affordable costs is widely acknowledged (Akerele, 1984).

Currently, in malaria endemic tropical countries, modern medicine is not available, and when it is available, it is not affordable to most of the people living in rural areas. These people resort therefore to the use of traditional medicine as their centerpiece of primary healthcare. This has been so because traditional medicine is commonly available, culturally and socio-economically acceptable, and affordable even in remote rural areas of such populations and communities. The World Health Organization (WHO) estimated that about 80% of the world's population relies on traditional medicine for their primary healthcare needs (Farnsworth et al., 1985).

Over one third of the world's population lacks regular access to affordable drugs, such that for these people, modern medicine is unlikely to be a realistic mainstay of their primary healthcare needs. In developing countries, in Africa as well as in South America and Asia, traditional healers are still very often the only medical health care practitioners available to the majority of people living in remote areas (Akubue and Mittal. 1982).

It is estimated that there are 300-500 million cases of malaria annually with 1.75 to 2.5 million deaths (WHO, 2002). Malaria is a leading cause of death in tropical and

1 subtropical regions and is also a serious public health problem in certain regions of Southeast Asia and South America (Mishra et al., 1999). Several reasons justify the search for new and effective therapeutics for the long-term treatment of malaria. The emergence and spread of insecticide resistant mosquito vectors, and drug resistant parasites are the major reasons. For instance in Kenya, chloroquine (14) is no longer effective and the current use of sulfadoxine (15) and pyrimethamine (16) combination is jeopardized by increased resistance to this combination treatment (Sibley et al., 2001). Although artemisinin-based combination therapies (ACTs) (first line treatment in Kenya currently) have been developed to enhance clinical efficacy and to delay the development of resistance to parasites, these drugs are not yet widely available or affordable.

In this regard research on African traditional medicinal plants for their antimalarials constituents is important, first to facilitate the efficient utilization of the easily available botanical resources to the millions in need and second to provide potentially active lead anti-plasmodial compounds with new mechanisms of action. The interest on study of plants as a source of antimalarial drugs revived due to the fact that one of the most effective antimalarial compound-artemisinin (18) (at least as effective as quinine (17) and is associated with fewer serious adverse effects) is derived from the plant Artemisia annua (Asteraceae) (White et al., 1999).

Natural product research represents a major strategy for discovering and developing new drugs. The use of medicinal plants for the treatment of parasitic diseases is well known and documented since ancient times. The use of Cinchona succiruha (Rubiaceac), from which quinine (17) was discovered as an antimalarial drug, represented a milestone in the history of drugs from nature used for the treatment of parasitic diseases, especially for those caused by Plasmodium. Leishmania, and Trypanosoma spp (Oliver et al., 2000).

2 In the past decades natural products have attracted renewed interest, including from bacteria and fungi, as important sources of biologically active compounds. Recently marine organisms have also been recognized as attractive source of antiparasitic compounds. It is therefore not surprising that one of the frontiers in modern science is the study of the chemistry and biology of natural products (Oliver et al, 2000).

Exploring the untapped natural sources for novel antiplasmodial compounds remains a major challenge and a source of novelty in the era of combinatorial chemistry and genomics. Since plants contain a high variety of constituents it is often claimed that the use ot a whole plant rather than one single purified product may be more effective therapeutically (Oliver et al., 2000).

3 Earlier studies on the genus Kniphofia have led to the isolation of phenylanthraquinones and other related quinonoids which have shown biological activity against Plasmodium parasites. The study of Kniphofia thomsonii could result in the discovery of related compounds with antiprotozoal activities.

1.2 Problem statement

The resistance of P. falciparum against first-line anti-malarial drugs is the most important cause of the increasing spread of malaria. This has been due to the availability of a limited number of drugs with few structural varieties in clinical use. Other causes are unaffordable alternative drugs, collapse of vector control programs, resistance of Anopheles mosquito against DDT, migration of refugees and changes in the climate and environment (Wiesner et al., 2003). Consequently there is need for the development of efficacious and structurally diverse anti-malarial drugs which may have new modes of action to replace or augment the current drugs that are becoming increasingly ineffective (Ram et al., 2000).

1.3 Justification of the Research

I he use ol medicinal plants for the treatment of parasitic diseases is well known and documented since ancient times. One of the best examples is the use of Cinchona succiruha (Rubiaceae) and related plants as antimalarials. Quinine (17) has been identified as the active principle in this plant. This compound and its synthetic derivatives, including chloroquine (14), have been used for treatment of malaria for a long time (Oliver et al., 2000). Due to the resistance of Plasmodium falciparum to chloroquine and other drugs, there is need for alternative and more effective new drugs to treat malaria and other protozoal diseases. In this regard plants remain the principal source of lead structures.

4 Phenylanthraquinones arc a new class of antiplasmodial (antiprotozoal) compounds (Abegaz et. al., 2002); this remarkable observation was made when it was revealed that knipholone anthrone and related substances possess antiplasmodial activity comparable or only slightly lower than that of chloroquine itself (Bringmann et. al., 1999). Knipholone (6) was first reported from the African plant, Kniphofla Joliosa (Asphodelaceae) (Dagne and Steglich 1984) as the main constituent of the roots. Further work has revealed that anthraquinones including phenylanthraquinones are present in the related genera Kniphofia (Dagne and Yenesew, 1994), Bulhinella (Van Wyk et al.. 1995), and Bulhine (Bezabih and Abegaz, 1998).

Many compounds associated with the genus Kniphofia have shown several biological activities including antiplasmodial activity. Some of these compounds, especially the phenylanthraquinones and other quinonoids, also have antiprotozoal activities against leishmania parasites (Wube et al, 2006).

In Kenya the only Kniphofia species found is K. thomsonii. This study was aimed at isolation and characterization of the constituents of this plant and investigation of the isolated compounds for antiplasmodial activities.

1.4 Significance of the Research

This research is expected to contribute in the identification of lead compound(s) with antiplasmodial activities which could lead to cheap and readily available alternative drugs.

1.5 Objectives of the Research

The general objective of this research was to evaluate the antiplasmodial activities of the constituents of the roots of kniphofia thomsonii.

5 The specific objectives of the research wcre:-

1. I o establish the antiprotozoal activity of the roots extract of Kniphofia thomsonii agai nst Plasmodium falciparum.

2. To isolate and characterize the constituents of the roots extract of Kniphofia thomsonii.

3. To determine the antiplasmodial activities of the isolated compounds.

6 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Malaria

2.1.1 The Disease

Malaria in humans is caused by a protozoan of the genus Plasmodium. which is transmitted through bites by female mosquitoes of the genus Anopheles. Four subspecies, namely P. falciparum, P. vivax, P. malariae and P. ovale, are known to cause malaria in humans (WHO, 1999).

The most severe malaria fevers and about 90% of malaria deaths are caused by P falciparum, which is the predominant parasite species in Africa (WHO, 1999). It is also in Africa that the most efficient mosquito vector for malaria transmission. Anopheles gamhiae, predominates (WHO, 1999).

2.1.2 Global Malaria Situation

The global malaria situation is deteriorating faster today than at any time in the past century. The number of new cases of the disease has quadrupled in the past 10 years, such that over 2 billion people, 40% of the world population, living in about 102 countries, are at risk of being infected and half of these live in sub-Saharan Africa. I he World Health Organization estimates that between 300 and 500 million new cases occur each year. In addition to causing untold suffering and disability, malaria ranks as one ol the world s major killers, costing about 1 million people their lives annually(WHO,

2002).

Children arc especially vulnerable, as more children die from malaria than any other single disease. Pregnant women and especially primigravidae (first-time pregnant mothers) are the next highest risk group for malaria in malaria endemic areas. It is stated

7 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Malaria

2.1.1 The Disease

Malaria in humans is caused by a protozoan of the genus Plasmodium, which is transmitted through bites by female mosquitoes of the genus Anopheles. Four subspecies, namely P. falciparum, P. vivax, P. malariae and P ovale, are known to cause malaria in humans (WHO, 1999).

The most severe malaria fevers and about 90% of malaria deaths are caused by P falciparum, which is the predominant parasite species in Africa (WHO, 1999). It is also in Africa that the most efficient mosquito vector for malaria transmission. Anopheles gambiae, predominates (WHO, 1999).

2.1.2 Global Malaria Situation

The global malaria situation is deteriorating faster today than at any time in the past century. The number of new cases of the disease has quadrupled in the past 10 years, such that over 2 billion people, 40% of the world population, living in about 102 countries, are at risk of being infected and half of these live in sub-Saharan Alrica. I he World Health Organization estimates that between 300 and 500 million new cases occur each year. In addition to causing untold suffering and disability, malaria ranks as one ol the world's major killers, costing about 1 million people their lives annually(WHO,

2002).

Children are especially vulnerable, as more children die from malaria than any other single disease. Pregnant women and especially primigravidae (first-time pregnant mothers) are the next highest risk group for malaria in malaria endemic areas. It is stated

7 that malaria causes about One million deaths of children per year in Africa alone (WHO. 1999). Most malaria infections occur in Africa (Figure 1.1); with countries in tropical Africa estimated to account for 80% of all clinical cases and about 90% of all people who carry the parasite.

Global malaria risk

i— i Areas in which malaria has disappeared. been eiadicated or never existed d l Areas with limited malana risk d ] Areas where malaria transmission occurs

Figure 1.1 Global malaria risks (From 20!h WHO Expert C ommittee Report on Malaria: WHO, 1998)

2.1.3 The Current Malaria Control Strategy

The malaria control strategy aimed at malaria eradication was reoriented in 1978 to focus on the reduction of malaria to a level at which it would no longer constitute a major public health problem (WHO, 1978). This strategy was based on the combined use ol vector control measures and effective treatment of malaria patients. 1 his initial success in many tropical countries was interrupted mainly by the development ol resistant mosquitoes and chloroquine-resistant Plasmodium falciparum, as well as lack ol continous political and financial support to the program (Bradley, 1991).

The present malaria control strategy, which was adopted by the Ministerial Conference on Malaria in Amsterdam in 1992 (WHO. 1994), is based on the prevention of death.

8 reduction of illness, and reduction of social and economic loss due to malaria (WIK),

1994). The practical implementation of the strategy requires two main approaches:

1. Malaria chemotherapy for early and effective treatment of malaria cases, management of severe and complicated cases, and prophylaxis for the most susceptible population (particularly pregnant women and non-immune travelers).

2. Use of insecticide-treated nets for protection against mosquito bites. This strategy has, however, been increasingly confronted by serious setbacks due to the continued spread of insecticide-resistant species of the mosquito vectors, and political and socio-economic problems.

The malaria situation has continued to worsen as evidenced by increased frequency of malaria epidemics and levels of parasite resistance to the most affordable drug, chloroquine (WHO, 1996).

2.1.4 Limitations of the Current Malaria Control Strategy

Currently, chemotherapy and prophylaxis of malaria to those who need it most worldwide is based on nine key drugs: chloroquine, amodiaquine, primaquine, mefloquine, quinine, sulfadoxine/pyrimethamine, pyrimethamine/dapsone. halofantrine. and artemisinin derivatives (WHO, 1998). The availability o! chloroquine, amodiaquine. sulfadoxine/pyrimethamine, and quinine in Africa has considerably improved the treatment of malaria. Unfortunately, three decades ago P. falciparum was shown to have developed some resistance to most of the cheaper antimalarials. For instance resistance to the 4-aminoquinoline derivative, chloroquine (14) was First observed in the 1960s. Resistance to the antifolates pyrimethamine, cycloguanil and mefloquine, has also been reported (Ridley and Hudson, 1998). Since then, the incidence of drug resistant P. falciparum has been increasing at a faster rate than that of the efforts lor development of new drugs (Figure 1.2).

9 The most important recent discovery for the therapy of P falciparum malaria has been the identification of artemisinin (18) (a sesquiterpene lactone) from Artemisia annua (Asteraceae) which has been used to treat over 3 million cases of malaria in South East Asia (Meshnick. 1998). Another new drug, atovaquonehydroxy-naphthoquinone, identified as an antimalarial in the early 1980s, has proved to be highly effective in clinical trials but has to be used in combination with proguanil to prevent the development of resistance (Ridley and Hudson. 1998).

Reported P. Falciparum drug resistance

CD Malaria transmission areas o Chloroquine resistance • SuKadoxinepynm etha mi ne resistance * Multidrug resistance

Figure 1.2 Reported P. falciparum drug resistance. (From 20th WHO Expert Committee Report on Malaria; WHO. 1998)

Against this background of increasing drug resistance is the unfortunate situation whereby new effective antimalarial drugs coming into the market are completely unaffordable to the majority of the affected populations. Africa south of the Sahara has populations who are dying for lack of malaria treatment, not because there are no effective antimalarial drugs, but because these drugs are beyond their affordability (WHO, 1998).

10 The spread of chloroquine-resistant P falciparum malaria is a major public health problem in Africa south of the Sahara. Several countries have already abandoned chloroquine as the first-line therapy. Tanzania, Kenya, Malawi, Botswana, and South Africa have switched to sulfadoxine/pyrimethamine, while C ameroon has switched to amodiaquine. Sulfadoxine/pyrimethamine seems to have a short therapeutic life span, as resistant strains of P. falciparum are widespread in Southeast Asia and South America (WHO, 1998). Research findings from Kenya and the United Republic of Tanzania indicate a decline in parasite sensitivity to sulfadoxine/pyrimethamine in Hast Africa (WHO, 1998). P. falciparum is also reported to have developed resistance to mefloquine in the border areas of Thailand with Cambodia and Myanmar. Parasite resistance to quinine has already been reported in Southeast Asia and in the Amazon region (WHO. 1998). Furthermore, there is growing evidence that P vivax has also evolved resistance to chloroquine in Indonesia, Myanmar, Papua New Guinea, and Vanuatu (WHO. 1998).

The reemergence of malaria in areas where it had been eradicated, such as Tadjikistan, the Democratic People’s Republic of Korea, and the Republic of Korea, and the spread of malaria in countries where it was almost eradicated, such as northern Iraq. Azerbaijan, and Turkey, are indications of the increase in malaria risk areas in the world. The current malaria epidemics in the majority of these countries and in Africa are the result of a rapid deterioration of malaria prevention and control operations, due mainly to the lack ol sufficient funds for malaria control and the paucity of effective control tools (WHO. 1998).

The search for a vaccine has been plagued by a number of shortcomings. Many of the shortcomings are related to antigenic variation, antigenic diversity, and immune evasion mechanisms exhibited in various stages of the complex life cycle of malaria parasites. In addition, malaria research and the search for vaccines require large sums of money, and it can be said that malaria research has been greatly under-funded (WHO. 1998).

While efforts are being made to overcome the hurdles for vaccine development, people are dying, and the only available effective means of reducing the number of deaths is the

11 provision of affordable and effective medicines. Many young people are already dying of HIV/AIDS in Africa for lack of cure and affordable life-prolonging drugs. If, in addition to this, malaria is not controlled using effective drugs. Africa may see the loss of generations of youths and a huge economical setback to the extent that poverty eradication will remain but a dream for ages. The absence of new effective and affordable antimalarials is a formidable limitation to the current malaria control strategy, and there is an urgent need to search for traditional medicines to boost the dwindling number of treatments for malaria (WHO. 1998).

The search for new drugs through the evaluation and validation of traditional medicines offers a good opportunity and a highly credible channel for the discovery and development of better medicines. The advantage of such drugs is that their sources are plants that are often widely available in rural areas of Africa, furthermore traditional medicine research can provide information and new clues regarding the effectiveness ol combination therapy in curing malaria and preventing development ol drug resistance (WHO, 2000).

The development of traditional medicines for treatment of malaria, and of African-based pharmaceuticals, would provide the following major benefits to the poorest and worst deprived populations in the world as tar as health and economic development is concerned:

> Provision of affordable and effective drugs. > Prevention of a large number of deaths of children and pregnant women. > Alleviation of poverty by reducing the burden of malaria and offering the populations alternative commercial crops. > Creation and strengthening of capacities for drug production. > A replicable approach to the provision of effective and affordable medicines for other diseases.

12 The only fear is that if no serious actions are taken to handle the harvesting of such plants, they may soon become extinct because of over harvesting, which is already happening in many cases because of the increased cost of antimalarials (WHO. 2000).

Malaria has a history of the use of plant-based drugs that have saved humankind from many disasters. The natives of Peru discovered the first antimalarial, cinchona bark, and the bark was used in the treatment of intermittent fevers in the 16lh century. In 1920 Pelletier and Caventou working in Paris isolated quinine from cinchona bark. Quinine is still one of the best medicines for the treatment of malaria.

The second antimalarial drug is Qinghao (Artemisia annua), used in the treatment of fevers in China. The WHO supported research on A. annua and the active principle artemisinin was identified. Artemisinin formulations are used worldwide in the treatment of malaria, particularly for severe and drug-resistant cases (WHO, 1998). Both quinine (17) and artemisinin (18) are rapid-acting drugs with a short half-life, and therefore resistance against these drugs would be slow. So far there is no reported resistance against artemisinin based drugs.

In India, Azadirachta indica (neem) is widely used in the treatment of diseases, including malaria (Sharma et. al., 1993 a; 1993 b). In Africa, the use of indigenous plants still plays an important role in malaria treatment an example being Kniphqfia foliosa which has been used in Ethiopia for the treatment of malaria (Wube et al., 2006). These plants might be an interesting source for the discovery of novel antiplasmodial lead structures (Onegi et al., 2000).

Anthraquinones isolated from the tropical tree Morinda lucida (Rubiaceae) were tested for their antiplasmodial activity (Schnur et al., 1983; Obih et al.. 1985). T he most active compounds have an aldehyde group at C-2, well known as a cytotoxic moiety in other natural products. The activity may also be explained by the cyclic planar structure that makes them potential DNA-intercalators [molecules that can be reversibly occluded between two other molecules (or groups) or DNA base pairs], from the toxicological

13 point of view, some of the compounds showed moderate effects in the lymphocyte proliferation test with EC50 values over 175pM (Sittie et al., 1999).

The genus Kniphofia is known to contain phenyl anthraquinones which have been shown to possess significant activity against Leishmania, Trypanosoma, and Plasmodium (Schnur. et al., 1983: Obih et al., 1985).

2.2 Botanical Information

2.2.1 The Family Asphodelaceae

The genus Kniphofia belongs to the family Asphodelaceae which is comprised of 17 genera (10 of which occur in South Africa) and about 750 species. The family Asphodelaceae is divided into two subfamilies; the Asphodeloideae and the Alooideae. Accordingly the genera Asphodeline, Asphodelus, Bulbine, Bulbinella, Eremurus, Hemiphlacus, Jodrellia, Kniphofia, Paradisea, Simethis, and Trachandra are placed in the subfamily Asphodeloideae while Aloe, Gasteria, Haworthia, Lomatophylum, and Poellnitzia are placed in the Alooideae (Stern, 2002).

On the other hand some workers consider the above two subfamilies as distinct lamilies i.e. the Asphodelaceae and the Aloaceae. The generation of chemical information on species belonging to these two groups is believed to reveal the relationships among the various taxa and to assist in establishing taxonomic classifications at various levels (Dagne and Yenesew, 1994).

2.2.1.1 The Sub-family Asphodeloideae

This sub family Asphodeloideae comprises 11 genera with approximately 261 species. They are quite diverse in form ranging from succulent (water-retaining) through mesomorphic (robust) to xenomorphic (not well developed). They feature much less

14 prominently in collections, but some, such as the “caudiciform‘,-5w/^//ie and a number of Kniphofia genera are popular and quite hardy garden plants (Van Wyk et al., 1995).

2.2.1.1.1 The Genus Knipliofia

About 70 species of Kniphofia occur in Africa and 47 of these are found in the eastern areas of South Africa. The genus Kniphofia is very closely related to the genus Aloe. As a result, the first Kniphofia to be described, namely K uvaria, was mistakenly thought to be an Aloe and was thus initially named Aloe uvaria (Stern, 2002).

Most species of Kniphofia are evergreen while a few are deciduous and sprout again in the early summer. They bear dense, erect spikes (elongated inflorescence with stalkless flowers) above the level of the leaves in either winter or summer depending on the species. The small, tubular flowers are produced in shades of red, orange, yellow, and cream (Stern, 2002). In Kenya the genus is represented by one species Kniphofia thomsonii (‘Ted-hot poker”) seen on Mt Kenya, where it was fairly common, and is a native species (Chase et al., 1995).

2.3 Kniphofia thomsonii

Tall (1-1.5m) stems with elegant pendulous, well spaced flowers in soft orange terribly beautiful and more intolerant of soggy winter ground (Figure 1.3). This plant usually prefers damp soil, but can also grow in fairly dry conditions (Stern, 2002).

15 2.4 Phytochemistry of the Genus Knipltofia

The most common class o f compounds found in the genus Kniphofia are the anthraquinones. Previous phytochemical investigation of this genus resulted in the isolation of monomeric and dimeric anthraquinone.

2.4.1 Monomeric Anthracene Derivatives

The monomeric anthraquinones included chrysophanol (1), islandicin (2), aloe- emodinacetate (4), aloe-emodin (5), chrysophanic acid (23), chrysophanic acid dianthrone (24), emodin (25), aloesaponol III (26), and aloesaponol III 8-methyl ether (27) (Berhanu et al. 1986).

2.4.2 Dimeric Anthraquinones

The Dimeric anthraquinones isolated from various Kniphofia species include chrysalodin (11) from Kniphofia foliosa leaves (Dagne el al., 1987), chryslandicin (12) from Kniphofia foliosa roots (Yenesew el al., 1988) and asphodeline (19) from the roots of K. tysonii (Van Wyk et al., 1995).

2.4.3 Phenyl Anthraquinone and Phenylanthrones

The unique phenylanthraquinone knipholone (6) which was first isolated from kniphofia foliosa, represented the first example in which an anthraquinone is attached to acetylphloroglucinol methyl ether (30) unit hence the name phenylanthraquinones. Comparative studies on the roots of some 14 Kniphofia species showed knipholone (6) to be the major pigment in these taxa. It was therefore suggested that compound 6 may be a taxonomic marker for the genus Kniphofia (Yenesew et al., 1988). Studies on other Kniphofia species have also resulted in the isolation of a number of phenylanthraquinones and phenylanthrones including isoknipholone anthrone (21), isoknipholone (22). foliosone (28), and isofoliosone (29) (Yenesew et al, 1994).

The new phenylanthrone isolated from the stem of K. foliosa named knipholone anthrone (20), is reported to be the immediate precursor of knipholone (6) (Dagne and Yenesew,

16 1993). The presence of acetylphloroglucinol methyl ether (30) has also been detected in the same plant, a result consistent with the suggestion that knipholone-type compounds arise from oxidative coupling of 30 with a precursor ol chrysophanol (1). In support ol this, other novel pigments where acetylphloroglucinol methyl ether (30) attached to C-4 or C-10 positions of the chrysophanol (1) moiety have been characterized trom this plant (Yenesew et al., 1994). The compounds so far reported from the genus Kniphofia are summarized in fable 1.

17 Table 1: Compounds of the genus Kniphofia

Compound Source species (plant References part) Quinonoids Knipholone (6) K. acraea (R) Yenesew et al. (1988) K. caulescens (R) f t K. flammula (R) •• K. foliosa (L) Berhanu and Dagne (1984) M (R) Dagne and Steglich (1984) " (F, L, FI) Berhanu et al. ( 1986) (S) Yenesew et al. (1994) (R) Yenesew et al. (1988) K. ins ignis (Rh) Berhanu et al. ( 1986) K. isoet[folia (Rh) H K. linear ifolia (R) Yenesew et al. (1988) K. pumila (Rh, FI) Berhanu et al. ( 1986) K. reynolds (R) Yenesew et al. (1988) K. rooperi (R) M K. tysonii (R) f t K. sc him peri (Rh) Berhanu et al. ( 1986) Knipholone anthrone (20) K. folios a (S) Dagne and Yenesew ( 1993) " (S) Yenesew et al. (1994) Isoknipholone anthrone (21) " (S) Yenesew et al. (1994) Isoknipholone (22) K. foliosa (S) Yenesew et al. (1994) Folioosone (28) K. foliosa (S) Yenesew et al. (1994) Isofolioosone (29) K. foliosa (S) Yenesew et al. (1994) KEY: L = Leaf, S = Stem, R = Root, Rh = Rhizomes, FI = Flowers, F = Fruit

18 Table 1 Continued. Compound Source species (plant part) References Asphodeline (19) K. albescens (R) Van Wyk et al. (1995) K brachystachya (R) •1 •I K gracilis (R) K. brevifolia (R) •• K tysonii (R) •t Chryslandicin (12) K. caulescens (R) Yenesew et al. (1988) K. foliosa (R) 91 K. linear if alia (R) 99 Chrysophanic acid (23) K .caulescens (R) Yenesew et al. ( 1988) K. foliosa (Rh, F, L, FI) Berhanu et al. (1986) (R) Yenesew et al. (1988) " (L) Berhanu and Dagne (1984) K. ins ignis (Rh) Berhanu et al. ( 1986) K. isoetifolia (Rh. L, FI) If K. linear if alia (R) Yenesew et al. ( 1988) K. pumila (Rh) Berhanu et a\. (1986) K. reynolds (R) Yenesew et al. (1988) K. schimperi (Rh) Berhanu et al. (1986) Chrysophanic acid K. foliosa (Rh) Berhanu et al. (1985) »« dianthrone (24) K. ins ignis (Rh) •• K. isoetifolia (Rh) K. pumila (Rh) «t K. schimperi (Rh) ft Chrysalodin (11) K. foliosa ( L) Dagne et al. (1987) Aloe emodin (5) K. foliosa (F, L, FI) Berhanu et al. (1986) K. ins ignis (F) ft K. isoetifolia (FI) ft K. schimperi (FI) ft

19 Table 1 Continued. Compound Source species (plant part) References Aloe emodin acetate (4) K. foliosa (L) Berhanu and Dagne (1984) " (F, L, FI) Berhanu et al (1986) K. isoetifolia (FI) 91 Emodin (25) K. foliosa (Rh) Berhanu et al. (1986) Aloesaponol III (26) K foliosa (S) Yenesew et al. (1994) Aloesaponol III 8-methyl K. foliosa (S) Yenesew et al. (1994) ether (27) Islandicin (2) K. foliosa (F, L, FI) Berhanu et al., (1986) ” (R) Yenesew et al., (1988) K. insignis (Rh) Berhanu et al. (1986) K. isoetifolia (Rh) •9 K. linear [folia (R) Yenesew et al., (1988) K. pumila (Rh) Berhanu et al. (1986) K reynolds (R) Yenesew et al. (1988) K. schimperi (Rh) Yenesew et al. (1988) Benzenoid Acetophenone.4-6- K. foliosa (S) Yenesew et al. (1994) Dihydroxy-2-methoxy (30) Alkane to C4 Citric acid (31) K. butchellii (L) Van Rheede (1964) K. macoanii (L) 99 Malic acid (32) K. butchellii (L) Van Rheede (1964) K. macoanii (L) 99 Alicyclic Quinic acid (33) K uvaria (L) Yoshida et al. (1975) Shikimic acid (34) K. uvaria (L) Yoshida et al. (1975) KEY: L = Leaf. S = Stem, R = Root, Rh = Rhizomes, FI = Flowers, F = Fruit

20 OH O OH

R1

23 Ri=R2=R3=R4=0 2 R1 = CH3, R2 = OH, R3=H 24 R1= R2=R3= R4= H 4 R1 = CH2OAc, R2 = R3 = h 5 R1 = CH2OH, R2 = R3=h OR OH O 25 R1 = CH3, R2 = H, R3 = OH

27 R = CH.

Ch

CH.

11 R1 = H, R2= OH 12 R1 = OH, R2= H

21 20 R1 = 2H, R2 = H, R3= CH3 28 R1 = H, R2 = CH3 21 R1 = 2H, R2 = CH3, R3= H 29 R1 = CH3, R2 = H 22 R1 = O, R2=CH3, R3=H

OH OH

HO O

33 34

22 2.5 Biological Activity of Anthraquinones

During the course of screening of Ethiopian medicinal plants for their antimalarial properties, it was found that the dichloromethane extract of the roots of Kniphofiu foliosu (Asphodelaceae), which has long been used in the traditional medicine of I thiopia for the treatment of abdominal cramps and wound healing, displayed strong in vitro antiplasmodial activity against the chloroquine-sensitive 31)7 strain of Plasmodium falciparum with an IC50 value of 3.8|ig/ml and weak cytotoxic activity against KB cells with an ED50 value of 35.2 pg/ml (Wube et al., 2005).

The compounds isolated from the roots of Kniphofiu foliosu (Asphodelaceae) were evaluated for their in vitro antimalarial activity against the chloroquine-sensitive 31)7 strain of Plasmodium falciparum (Wube et al., 2005). Among the compounds tested. 10- (chrysophanol-7'-yl)-10-(xi)-hydroxychrysopanoI-9-anthronc (10) and chr>slandicin (12). showed a high inhibition of the growth of the malaria parasite, P. falciparum with ED50 values of 0.260 and 0.537 pg/m lrespectively (Wube et al., 2005).

To compare the effect on the parasite with toxicity to mammalian cells, the cytotoxic activities of the isolated compounds against the KB cell line were evaluated and 10-

(chrysophanol 7 -yl)-10-(xi)-hydroxychrysopanol-9-anthrone ( 10) and chrvslandicin ( 12) displayed very low toxicity with EDso values of 104 and 90 pg/mL, respectively (Wube et al., 2005). I his is the first report of the inhibition of the growth of P falciparum by anthraquinone-anthrone dimers and establishes them as a new class of potential antimalarial compounds with very little host cell toxicity (Wube et al., 2005)

Phenylanthraquinones such as knipholone (6) have antiplasmodial activities; whereas neither chrysophanol nor phloroacetophenone, which are its monomers, possess any significant antiplasmodial activity (Majinda et al., 2001).

Inhibition of leukotriene formation is one of the approaches for the treatment of asthma and other inflammatory diseases. Knipholone (6). isolated from the roots of Kniphofiu foliosu, (Asphodelaceae), has been investigated for inhibition of leukotriene biosynthesis

23 in an ex vivo bioassay using activated human neutrophile granulocytes. It was found to be a selective inhibitor of leukotriene metabolism in human blood assay where it showed a high dose dependent activity with an ICso value of 4.2 pM (3.6-4.9. 95% I I.) (Wube el al., 2006) being twice as active as the commercial 5-LOX (5-Lipoxygenase) inhibitor zileuton (IC50 10.4 pM. 9.0-11.7. 95% FL) (Wube el al.. 2006).

Knipholone (6) showed weak inhibition of I2(S)-III I K production using human platelets at 10 |ig/ml concentration which resulted in 28.6% inhibition. Baicalein vs as used as a positive control and exhibited 52% inhibition at 5 fig/ml. However, at concentrations of 50 |ig/ml. it produced no inhibition of other enzymes related to inflammation, such as COX-1 and -2 compared to the positive controls indomethacin which inhibits COX-1 with an ICso value of 0.9 pM and NS-398 which inhibits COX-2 with an 1( 5, value of 2.6 pM. Therefore, Knipholone showed higher affinity for the 5-LOX (enzyme involved in leukotrienes biosynthesis) pathway than for cycloxygenases and 12-LOX (Wube el al., 2006).

In an attempt to explain the mechanism of inhibition, the antioxidant activity of knipholone using various in vino assay systems including free radical scavenging (stable DPPII), non-enzymatic lipid peroxidation (inhibitory activity on bovine brain liposomes), and metal chelation (Cu \ Fe2 and Fe’+) was examined. Knipholone (6) was found to be a weak dose-independent free radical scavenger with an ICso value of 355 pM compared to positive control quercetin (ICso=3.2 pM). It was found to be a weak inhibitor of phospholipids liposomes peroxidation with an ICso value of 311 pM compared to the positive control quercetin (ICso L4 pM). However, with the addition of the metal ions, neither the absorbance nor the intensity of the UV-bands changed even with a concentration of 100 pM of metal ions. I hus Knipholone (6) is not a metal chelator. Cytotoxicity results also provided evidence that compound 6 exhibits weak toxicity for a mammalian host cell with EDs( value of 0.58 pM (Wube el al., 2006). Therefore, the ex vivo leukotriene metabolism inhibitory capacity of knipholone (6) is powerful and could be a potential candidate for a new anti-asthma drug (Wube el al., 2006).

24 CHAPTER THREE

3.0 METHODOLOGY

3.1 General

3.1.1 Instrumentation

The 13C NMR (125 or 50 MHz) and 'll NMR (300 or 200 MHz) were run on Bruker or Varian-Mercury spectrometers using IMS as the internal standard. Homonuclear correlation spectroscopy (COSY), Nuclear overhauser Enhancement spectroscopy

(NOESY), Heteronuclear correlation spectroscopy (HETCOR) including HMBC (2Jch»

Vch) and heteronuclear multiple quantum coherence ('J ch) HMQC were acquired using standard Bruker software. UV/VIS spectra were recorded using a Pye-Unicam SPS-150 Spectrophotometer. The roots were ground using willymill.

3.1.2 Collection of Plant Material

The roots of Kniphofia thomsonii was collected with the help ol botanists from the Department of Botany. University of Nairobi. The area of collection was Mt. Kenya. I he material was authenticated at the Department of Botany, University of Nairobi.

3.1.3 C hromatographic conditions

Compounds were isolated mainly from the bioactive extracts using various chromatographic techniques including column chromatography (oxalic acid impregnated) and Sephadex LH-20. Preparative thin layer chromatography (PTLC) using silica gel as the adsorbent and crystallization of solid compounds were used in the (Inal purification. Analytical TLC was done on Merck pre-coated silica gel 60 F254 plates, with UV (254 nm) and iodine vapour as detectors.

25 The spectroscopic methods used to determine the molecular structures of pure compounds isolated were ultra violet spectroscopy (UV), NMR spectroscopy and mass spectroscopy (MS) techniques.

3.2 Extraction and Isolation of compounds

Air dried and ground roots of Kniphofiu thomsonii (1.0 Kg) were extracted with dichloroniethanc/methanol (1:1) by cold percolation. The extract was evaporated under reduced pressure to yield 105 g (10.5%) crude extract. About 100 g of the extract was subjected to column chromatography on oxalic acid impregnated silica gel (400 g) eluting at gradient with hexane, dichloromethane and methanol to afford 11 combined fractions labeled 1A-1K.

Subsequent column chromatography of fraction IB (2.027 g) on oxalic acid impregnated silica (eluting with 3%, 5%, 7% 10%, 15% and 30% dichloromethane in hexane) and crystallization (from n-hexane/dichloromethane), gave chrysophanol (1) (30 mg), flavoglaucin (7) (30.3 mg) and lO.lO'-bichrysophanol anthrone (9) (29 mg) respectively. Fraction eluting with 3% dichloromethane in hexane was subjected to Sephadex LH-20, CH2CI2/CH1OII (1:1) to yield l,4,8-trihydroxy-3-methylanthraquinone, trivial name islandicin (2) (5 mg).

Combined fractions eluting with 3%, 5%. 7% and 10% dichloromethane in hexane, were subjected to Sephadex LH-20 and further purified using PTLC (preparatory thin layer chromatography). This gave L8-dihydroxy-3-melhyl-6-methoxyanthraquinone, trivial name phvscion (3) (5 mg). 3,,\ 4 '” -dehydroflavoglaucin (8) (6 mg) was achieved after subjecting the 15% dichloromethane in hexane extract from fraction IB to Sephadex LH- 20 and further purification using PTLC.

Fractions 1C-111 of the first column was combined (8Q) to yield a weight of 11 g. This was then subjected to column chromatography (200 g-oxalic acid impregnated silica gel) using n-hexane containing increasing amounts of dichloromethane and ethyl acetate. This

26 gave a total of 17 fractions labeled 10A-10Q. Crystallization (from n- hexane/dichloroincthane) of fraction I OH eluting with 100% dichloromethane gave rise to 10-hydroxy-10-(islandicin-7’-yl)-chrysophanol anthrone with the trivial name chryslandicin 12 (21 mg). The mother liquor of 1011 together with fractions 101 and 10J were combined and labeled 14A (1.06g). Subsequent column chromatography on oxalic acid impregnated silica gel (60g) gave 15A-15V. Fraction 15G eluting with 1:1 dichloromethane hexane was subjected to Sephadex LH-20, CH2CI2/CII3OH ( 1:1) and PTLC giving rise to aloe-emodin acetate 5 (5.2 mg).

Combined fractions of 10K-10P and 15M-15N (5 g) labeled 20G was subjected to column chromatography (oxalic acid impregnated silica gel-70g) using n-hexane containing increasing amounts of dichloromethane and ethyl acetate realized 19 fractions labeled 21A-21S. Fractions 21G-21I were combined and labeled 23A. Further Sephadex LH-20 [CH2CI2/CH3OH (1:1)] and PTLC on fraction 23A gave 1-(3-acetyl-2,6- dihydroxy-4-methoxyphenyl)-4,5-dihydroxy-2-methylanthraquinone. trivial name knipholone 6 (5 mg) and 10-hydroxy-10-(chrysophanol-7,-yl) -chrysophano! anthrone 10 (11 mg). Fraction 21S eluting with 50% ethyl acetate in hexane was also subjected through several Sephadex LH-20 [Cl I2CI2/CI I3OI1 (1:1 )| and PTLC' and gave rise to aloe- emodin 4 (8 mg), 10-hydroxy-10-(chrysophanol-7’-yl)-aloe-emodin anthrone (Chrysalodin) II (10 mg), and 10-hydroxy- 10-(islandicin-7’-yl)-aloe-emodin anthrone 13 (7 mg).

3.3 Physical and Spectroscopic Data for the Isolated Compounds

3.3.1 C lirysophanol (1)

Orange crystals, melting point 195-197°C; UV (>»m.lx. McOH): 270. 288 and 430 nm; 'll NMR (COCI3, 300 MHz): SH 7.08 (1H. bdd J= 7.8. 1.5 Hz, H-2), 7.62 (1H, bd J = 1.8 Hz, H-4). 7.80 ( IH. dd, ./= 7.5. 1.2 Hz, H-5), 7.65 (1H, /, J= 8.4 1 Iz, H-6), 7.28 (1H, dd. J= 7.8, 1.5 Hz. 11-7),2.45 (3H, Me-3), 11.98(011-1), 12.09(011-8).

27 3.3.2 I stand icin (2) Glittering red crystals, melting point 217-2I9°C: 'll NMR (CDCb. 3(M) VIII/): 8M 7 15 (111. i/../ 0.9 II/. 11-2). 7.89 (III. dd. J 7.5, 1.2 II/. H-5). 7.68 (III, 7.511/.. 11-6). 7.87 (111. dd ./ 7.5. 1.2 II/. 11-7). 2.38 (311. d. M e-3). 13.48 (1-OH. C-4). 12.28 (*2- OH. C-l and C-8),

3.3.3 Phvscion (3) Yellow powder: 'll NMR (CIX'lj. 300 Mil/.): 8„ 7.09 (III.

3.3.4 Aloe-emodin (4) Yellow amorphous solid; UV (/^,ax, MeOII) 271 and 429 nm: 'll NMR (CDCb, 500

MHz): 8h 7.37(1 II. J = 1.5 Hz, ,1-2), 7.80 11H. d. 1.0 Hz, H-4), 7.80 ( III. 1.0, 8.0 I Iz, 11-5), 7.83 ( IH. /../ — 8.0 I Iz. H-6) 7.37 ( III. 1.5, 8.0 I Iz. 11 7). 4.81 (211. s,CH2OH-3), 12.05 (OH-1), 1201 (OH-8); l3C NMR (ACETONE-d6, 125 Mil/.): 8( 164.1 (C-l), 117.9 (C-la), 125.8 (C-2). 155.6 (C-3). 120.1 (C-4), 138.1 (C 4a), 121.1 (C- 5), 137.9 (C-5a). 1.38.97 (C-6). 122.3 (C-7). 163.7 (C-8), ll7.6(C-8a). 194.5 (C-9), 182.9 (C-10). 64.4 (CII2OH-3).

3.3.5 Aloe-einodin acetate (5) Yellow amorphous solid: UV (/„,„„ MeOII) 268 and 429 nm; 'll NMR (CDCb, 500 MH/»: 8,1 7.27 (V. 11-2). 7.79 ( 111, v. H-4). 7.85 (111. =8.0 Hz, H-5), 7.70(111. i.J 8.0 Hz. H-6), 7.32 ( IH. d. J = 8 0 llz. H-7), 5.19 (211. ,s, CH20C(0)CH 3-3). 2.19 (3H. .r, CH ,(0)0CH 2-3). 12.06 (OH-1), 12.08 (OII-8); UC NMR (CDCb, 125 MHz): 8C 162.8 (C-l). 115.8 (C-la). 122.4 (C-2), 146.5 (C-3). 118,5 (C-4). 133.5 (C-4a), 120.2 (C-5), 133.9 (C-5a). 137.3 (C-6), 124.8 (C-7), 162.6 (C-8). 115.3 (C-8a). 192.7 (C-9), 181.5 (C- 10). 170.0 (C(())-3). 20.8 (OC(()|('lb-3). 64.7 (-C1 l20C(0)-3).

28 3.3.6 knipholonc (6) Deep red needles; UV

3.3.7 Flavo^laucin (7) Yellow amorphous solid; EIMS (m/z 304. ( 19H28O3): UV (Xmax. MeOH) 274 and 391 nm; 'H NMK (CD t’li. 300 MHz): S„ 11.93 (Oll-I), 6.90 (IH, .r. 11-5). 4.38 (v. OH-4), 10.24 (IH, v. H -l’). 3.29 (2H, d.J - 7.5 HzCH2-I”), 5.28 ( IH, l. ./= 7.5, 17 Hz, CH-2"). 1.76 (3H, bs, CH3-3” ), 1.70 (3H. bs. CM,-3”). 2.91 (211. /. CH2-1’” ), 1.58 (211. m. CH2-2’” ), 1.36 (8H.m. CH2-4), 0.90(3H, t. CHj-7’” ): l3CNMR (CDC12, 75 MHz): 0, 155.8 (CM). 117.3 (C-2). 128.6 (C-3), 144.97 (C-4). 125.7 (C-5), 128.6 (C-6), 195.5 (C-U), 27.0 (C l”), 121.2 (C-2' ). 133.6 (C-3"), 17.8 (Cllj-3"), 25.8 (CH,-3”), 23.9 (C -l" ’). 31.99 (C- 2’” ), 29.6 (C-3’"). 29.1 (C -4"’). 31.99 (5 "). 22.6 (C-6” ’). 14.1 (C-7’” ).

3.3.8 3” ’,4"’-l)ehvdrollavoj>laucin (8) Yellow amorphous solid; 'll NMR (CDCl.i. 300 Ml Iz): 8h 11.95 (OH-1), 6.90 (IH. s, II- 5). 4.38 (s,OII-4). 10.23 (III, .v. H -l’), 3.30 (211. .1=1.7 HzCH2-l”), 5.28(111.6 ./

7.2. 17.4 Hz. CH-2”), 1.76 (311. bs,CHj-3”), 1.70 (311. bs. CH,-3”). 2.96 (211. 1. Cll2- 1’” ), 2.28 (2H. bq. CH2-2’” ), 5.51 [(2H)2. /. CH-3” ’; CH-4’” ], 1.99 (211. hq, C ll2-5 "’) , 1.34 (2H, m. CH2-6 ’”) and 0.87 (311, 1.Cl lj-7’”).

3.3.9 lO.IO’-liiehrysophanol anthrone(9)

Pale-yellow amorphous powder: IIRMS | m (rel.z. hit.)]: M 479.1483 ( 100), CjolIu06; EIMS [m/z,(rel. Int.)]: M* 479 (5), 478 (15). 241 (17), 240 (85), 239 (100); UV (A™,, MeOH): 271 and 367 nm; 'll NMR (CDCIj. 300 Ml I/): 8H 2.32 (3H, s. Cl I, 3). 4.49 (III. d. J 1.5 Hz, H-10), 6.65 (111. brs. H-2). 6.15 (111. hrs. 11-4). 6.73 (III. dd ./ 7.5. 1.2

Hz, H-5), 8h 7.50 ( IH, /, J 7.5 Hz, 11-6)6.94 (111 .dd.7.5. I. 2 Hz. 11-7). 2.22 (311. s, CII3-3'), 4.45 (IH, d, J = 1.5Hz, H-10’), 6.65 (III. brs. 11-2’), 5.73 (111. brs. 11-4').

29 6.33 (III. d. J 8.0 Hz. H-5'). 7.30 (I H. /. ./=• 8.0 Hz. 11-6'). 6.87 (111. d. / 8.0 Hz. H- 7’). 11.71 (OH-I). 11.63 (OII-1 ). 11.88 (011-8). 11.79 (011-8'); l3C NMK (CDCIj. 125 Mil/): 8, 161 9(0-1), 114.0 (O-la), 117.0 (C-2). 147.5 (C-3), 121.0(0-4) 141.0 (t -4a). 117.0 (C-5). 142.1 (C-5a). 135.7(0-6), 116,8(0-7). 162.1 (0-8). 117.2 (C -8a). 191.6(0- 9), 56.3 (C-10). 22.1 (CH3-3). 161.9 (C-1), 114.5 (C-la’). 116.9 (0-2 ). 147.3 (0-3’), 121.2 (0-4’). 140,9 (0-4a’). 119.4 (C-5 ). 142.1

3.3.11) IO-llydroxy-10-(chrysophnnol-7'-yl)-chrysoplianol an throne ( 10) Yellow-red amorphous powder: IIRMS |m i, (rcl. Int.)): (|M M | 509 1253 (100). CjolhoOs); EIMS |m/z, (rcl. Int.)|: M’ 508 (21), 490 (30), 475 (26), 255 (19). 85 (55), 57 (100): UV (>.„,„. MeOll): 271. 292, 389 and 434 nm; 'll NMR (CI)013. 300 Mil/): 8„ 2.23 (3H. s,011,-3). 7.59 (III. brs H-2). 7.12 (111. 11-4), 6.77 (111. v, 11-5). 7.50 (1H, /, J = 8.0 II/. 11-6). 6.91 (111. d. J 8.0. 1.2 I Iz. H-7), 2.45 (311. ,v. (11,-3’). 6.77

(1H, bs, H-2’). 6.88 (1H. bs, 11-4’), 7.98 (IH, 11-5'), 8.81 (111. 11-6’). 12.24 (,v, OH-I), 12.32 is. OH-1’). 12.26 (,v. OH-8). 12.34 (v. OH-8 ), 5.6 (y, OH IO): l30 NMR (ACl TONE-d,,, 125 MHz): 8, 164.2(0-1). 115.4 (O-la), 122.1 (C-2), 149.2(0-3), 125.5 (C-4). 146.9 (C 4a). 118.6 (C-5), 146.9 (C-5a). 118.6 (C-6), 118.3 (C-7), 164.2 (C-8). 116.1 (C-8a). 194.9(0-9), 75.1 (C-10). 22.79 (CH,-3), 163.9(0-1’). 114.0 (O-la’). 122.3 (C-2’), 150.3 (C-3’), 120.9 (C-4 ), 132.7 (0-4a’), 120.7 (C-5’). 132.7 (C-Su’). 135.1 (0- 6’). 130.3 (C-7’). 163.7(0-8’), I 16.1 (0-8a ). 182.8 (C-9’), 202.0 (C-10’). 2 ’.8 (011,-3’).

3.3.11 10-Hydroxy-10-(chrysophanol-7'-yl)-aloe-emodin anthrone (I I) Yellow amorphous: HRMS [m,r, (rel. Im.)| ([M+l | ‘ 525.1185 (100), C,„ll3|0<,); EIMS [m/z, (rel. Int.)]: M’ 525 (5). 524 (16). 506 (15), 476 (37). 254 (21). 239 (19). 190 (45). 149 (67). 57 (79). 43 (100); UV ( \. mm MeOll): 271, 292. 385 and 494 nm; 'll NMR (CD013, 300 MHz): 8H 6.96 (111, hd.J 1.0 llz. 11-2). 6.68 (IH. J 2.5llz. 11-4). 6.96 ( 111, hd. .1 8.0 Hz, H-5). 7 5 1 (1H, /,./ = 8.0 1 Iz, 11-6). 6.92 (III, bd. J 8.0 llz. 11- 7), 4.56 (2H. bs. OH2OH-3), 7.58 (1II..V. Il-2’).and 7.11 (III. ,y. 11-4 ), 7.98 (111. d. J 7.6 llz, H-5') and 8.81 (IH, d. J 7.6- Hz. 11-6'). 2.46 (311. bs, CH3-3'). 12.29 (s. OH-1). 12.30 (.v, OH-8), 12.32 (,y, O ll-l’), 12.34 (.y. 011-8’), 5.91 (,y, OH-IO); l3C NMR

30 (AC!-TONE-cL 1^5 MHz): 6c 163.5 (C-l). U 6.7(( la), 114.6 (C-2), 153.9 (C-3). 120.3 (C-4), 143.1 (C-4a), 117.2 (C-5), 137.7 (( -6), 114.8 (C-7), 163.4 (C-8) 1 17.6 (C-8a), 194.4 (C-9), 63.8 (CH2OH-3)t 163.0 (C-l*). 114.1 (C-la), 124.8 (C-2’). 143.1 (C-3*), 117.9 (C-4’). 134.0 (C-4a’), 120.3 (C-5 ). 133.8 (C-6*). 121.5 (C-7’) 160.5 (C-8 ), 117.5 (C-8a), 182.1 (C-9*), 179.3 (C*-10*;. 22.1 (( Hi-3').

3.3.12 10-Hydroxy-10-(islandicin-7'-yl)-ehrysophanol anthrone (12) Red amorphous powder: HRMS \m/z. (rcl. lnt.)J (|M+1] 525.1211 (100). C30H20O9); EIMS \m/z, (rcl lnt.)J: M* 525 (2). 510 (5). 509 (12), 508 (22), 507 (30), 476 (7). 254 (15). 240 (32). 239 (17). 197 ( 11). 149 (35). 57 (100); UV MeOH): 271, 299. 381 and 495 nm; 'll NMR (CDCI3, >00 Mil/): oH6.78 ( 111. bs, H-2). 6.61 (111. hs\ H-4), 6.80 (1H, dd. J = 8.0. 1.2 Hz, 11-5), 7.41 (1H, /../ 8.0 11/. 11-6). 6.95 (111. dd. J 8.0. 1.2 11/.. H-7), 2.26 (311, s, CH3-3). 7.10(1 H. s, H-2 ). 8.06 (1H, d .J = 8.0 Hz, 5*). 8 66 ( 111. d. J = 8.0 Hz. H-6*). 2*.35 (3H. .y, Cl I -3’), 12.08 (OH-1, .v), 12.32 (OH-8, .v). 12.34 (OI1-1*. v). 13.56 (OH-4*, v). 12.44 (OH-8*, v); 13C NMR (ACE I ONE-df>. 125 MHz): 6< 163.5 (C-l), 114.2 (C-la). 118.5 (C-2), 150.3 (C-3), 122.2(C-4). 143.3 (C-4a). 121.1 (C-5), 149.0(C- 5a). 138.2 (C-6), 118. 2 (C-7). 163.8 (C-8), 195.0 (C-9), 24.4 (CH3-3). 158.9 (C -l’), 112.5 (C -la’), 130.4 (C-2’), 149.5 (C-3*). 158.9 (C-4’), 112.5 (C-4a*). 120.4 (C-5*), 134.6 (C-5a). 134.4(C-6’), 160.0 (C-8’), 188.2(C-9*), 188.2 (C-l O’), 24.1 (( lh-3*).

3.3.13 10-Hydroxy-10-(islandicin-7’-yl)-aloe-emodin anthrone (13) Red amorphous solid: HRMS |m/z, (rcl. Int.)] (|M + lf 541.1130, (100) C30H20O10); EIMS \m/z, (rcl. lnt.)]: M" 540 (3). 523 (5). 492 (100), 447 (3), 417 (2), 321 (3), 283 (5), 267 (7), 57 (74). 43 (85); UV (Xmax. MeOH): 271, 298. 381and 495 nm; 'll NMR (C IX*I3. 300 MI 1/): 8H 6 95 (1H, hrs, 11-2), 6.90 ( 111, hrs../ 0.6 11/. 11-4), 6.94 (III. hd. ./ 8.5 Hz. 11-5), 7.51 (111. /, J 8.0 Hz. H-6). 6.92 (HI. hd. J = 8.5 Hz, H-7). 4.56 (211. bs, CH2OH-3), 7.21 (1H, hrs. H-2’), 8.06 (HI. brs, H-5*), 8.83 (111, brs, H-6*). 2.28 (3H, .y, CH3-3’), 12.29 (5, OH-1), 12.31 (.y, OH-8). 12.33 (.v. OH-1’), 13.51 (.y, OH-4’), 12.34 (.y, OH-8’), 6.05 (.v. OH-10); l3C NMR (ACE I ONE-d„. 125 MHz): 8C 169.3 ((’ I). 114.1 ((*- la). 114.6 (C-2). 153.9 (C-3), 120.4(C-4). 148.1 (C-4a). 117.9 (C-5), 148.1 (C-5a). 137.7 (C-6), 114.7 (C-7). 163.0 (C-8). 202.8 (C-9), 63.8 (CH2OH-3), 165.9 (C -1), 111.7 (C-

31 la’). 129.9 (C-2). 144.0 (C-3’). 159.7 (C-4’), 112.6 (C-4a), 120.4 (C-5*). 133.6 (C-6’>. 163.4 (C-8'). 117.7 (C-8a), 194 4 (C-9), 183.6 (C-10’), 16.4 (CH3-3’).

3.4 In vitro anti-plasmodial assay. The crude extract and pure compounds were assayed using an automated micro-dilution technique to determine 50% growth inhibition of cultured parasites (Chula) el al.. 1983; Desjardins el al 1979). Two different strains, chloroquine-sensitive Sierra l.eone I (1)6) and chloroquine resistant Indochina I (W2). of P. falciparum were grown in a continuous culture supplemented with mixed gas (90° o nitrogen, 5% oxygen. 5% carbon dioxide), 10% human serum, and 6% hematocrit of A+ red blood cells. Once cultures reach a parasitemia of 3% with at least a 70% ring development stage present, parasites were transferred to a 96 well micro-titer plate with wells pre-coated with compound (Desjardin el al 1979). fhc samples were serially diluted across the plate to provide a range of concentrations used to determine IC50 values. Plates were incubated in a mixed gas incubator for 24 hours. Following the specified incubation time, | II|-hypo\anthine was added and parasites allowed growing for an additional 18 hours. Cells were processed with a plate harvester (TomTec) onto filter paper and washed to eliminate unincorporated isotope. Filters were then measured for activity in a micro-titer plate scintillation counter (Wallac). Data from the counter was imported into a Microsoft Excel spreadsheet, which is then imported into an oracle database/ program to determine IC50 values. A minimum of three separate determinations was carried out for each sample (Desjardin i t al., 1979).

32 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

The roots of Knifophia thomsonii (Asphodelaceae) was extracted using dichloromethanc/methanol (1:1) mixture. After removal of the solvent the extract was tested for anti-plasmodial activity against the chloroquine-sensitivc (1)6) and chloroquine-resistant (W2) strains of Plasmodium falciparum. The extract showed significant antiplasmodial activities with IC50 values of 3.2 jig/ml (1)6) and 6.4 pg/ml (W2).

The extract was subjected to chromatographic separation, which led to the isolation of eleven anthraquinone derivatives (both monomeric and dimeric) and two long alkyl chain substituted benzaldehyde derivatives. I he structures of these compounds were determined using 11) (1H and ' X ). 2D (COSY. HMBC and HMQC) NMK and MS and in some cases bv direct TLC comparison with authentic sample. I he characterization of these compounds is presented below.

4.1 ANTHRAQUINONE MONOMERS

4.1.1 Chrysophanol (1)

Compound 1 was isolated as yellowish red crystals with a melting point of 195-197°C and an R| value of 0.50 (5% EtOAc in hexane). The II NMR (Table 2) of this compound showed two downfield shifted hydroxyl protons at 8h 11.98 and 12.09. which is consistent with a 1,8-dihydrosvanthraquinone skeleton. In support of this, the lIV showed absorption bands at A.ma, 270, 288 and 430 nm which are characteristic of 1.8- dihydroxyanthraquinones.

The II NMR indicated the presence of three mutually coupled aromatic protons with an AMX spin system at 5„ 7.76 (dd, .7 -7.5, 1.2 Hz), 7.65 (/, J 8.1 Hz) and 7 27 (dd../ 8.0.

33 1.4 Hz), which arc typical of H-5, H-6 and H-7 of mono-substituted (with Oil) ring*( of 1, 8-dihydro.x>anthraquinones. respectively. The II NMR signal at 6n 2.45 is due to a methyl group attached to an aromatic ring and was placed at ( -3 of A-ring on the basis of biogenetic consideration (Wanjohi. 2006). In this ring, the proton at C-4 appeared at bn

7.62 (bd% 7=1.0 Hz), while 11-2 resonated as a broad doublet at 6h 7.08 (./ 1.0 II/). I he broadening of the signals for H-2 and H-4 resulted from long range coupling (4./) with the methyl protons. I his compound was therefore characterized as 1.8-dihydroxy-3- methylanthraquinone, triv ial name chrysophanol ( 1) I he identity of this compound was confirmed bv 1I.C comparisons with an authentic sample of chrysophanol (Wanjohi. 2006). OH O OH

1 This compound is a common metabolite in the genus Kniphofia (Van wvk el al., 1995) and also a constituent of the genera Cassia, Rumex. Rheum. Asphodelus, VJuehlenbeckia and Monilinia Jruclicola spp. h is widely distributed in plants and also found in the marine annelid l rechis unicincius. It is an antimicrobial and a purgative agent (Thomson, 1987).

4.1.2 Islandicin (2)

Compound 2 was isolated as glittering red crystals, melting point 217-219°C. TLC analysis of the compound showed a magenta spot with an Rt value of 0.50 (5% EtOAc in hexane). The spot changed to purple on exposure to NH3, a characteristic feature of 1.4.8- trihydroxy or 1,5,8-trihydroxyanthraquinones (Wanjohi, 2006).

In support of this, the 'll NMR. spectrum (fable 2) showed three chelated hydroxyl protons, 8h 12.28 (2 OH) and at 13.48. In addition, the presence of an aromatic methyl

54 group (C-3) was evident from i doublet (./ 0.9 ll/i integrating for three protons at 6u 2.3X. Also present were signals lor AMX aromatic protons. 6|t 7.8X (dd. .!■ 7.5. 1.1 II/). 7.69 (dd. J- 7.5. 0.9 Hz) and 7.30 [dd, J 7.5, 1.2 II/.) corresponding to C -ring aromatic protons (C-5. C-6 and C-7 respectively), with C-X substituted with an hydroxyl group. In A-ring. a quartei observed at 7.15 (d 0.9 Hz) is due to H-2 having a '/ coupling with the biogeneticaJl) expected methyl group at C-3. I he two remaining Indroxyl groups were then placed at C-l and C-4 of this ring. This compound was therefore identified as l,4.8-trihydro.\)-3-methylanthrai|uinone, trivial name islandicin (2).

OH O OH

CH3

The compound has been reported from the stem bark of Ventilago bombaensis (Rhamnaceae) (Pepalla et a/.. 1992); from the stem bark of Maesopsis eminii (Rhumnaceae) (Gumming and I hornson. 1970) and from rhizomes of some Knipho/ia species (Yenesew cl. a/.. 19X8).

4.1.3 Physcion (3)

Compound 3 was isolated as a yellow powder with an Rf value of 0.50 (5% HtOAc in hexane). The 'll NMR (fable 2) of this compound showed two dowi field shifted hydroxyl protons at 8h 12.13 and 12.33, which are consistent with a 1,8- dihydroxyanthraquinone skeleton. The presence of four aromatic protons at (6n 7.38. 6.70, 7.64 and 7.09), a methyl (8» 2.46) and a methoxy! (8n 3.92) groups were also evident from the 'll NMR spectrum ( fable 2).

35 The methyl is placed at C-3 of ring-A as expected biogenetically. In agreement v\ith this. H-2 and H-4 of this ring appeared at 8M 7.09 (hq. .A=1.7 Hz) and 7.64 {hj. J 1.2 Hz), respectively shoeing the characteristic me/a-coupling. These signals also showed long range ('./) coupling with C H rT In C-ring. the meta-couplcd protons at 8n 7.38 (J. ./ 2.7 Hz) and 6.70 (

This is the first report on the occurrence of C-6 oxygenation in 1.8- dihydroxyanthraquinone in the genus Kniphojia. The compound is widels distributed in lichens, e.g. Pcirmelia spp., higher plants, e g. Rumex spp. (Midiwo and Rukunga, 1085) and produced by Aspergillus and PeniciUium spp. It is also isolated from the marine annelid Urechis unicintus. Biologically, it is an antimicrobial agent and possesses purgative properties (Ulicky, et a/., 1991).

Table 2: 'fl NMR (300 MHz) data for compound 1. 2 and 3 (CDCI3)

Position Compound 1 8h m (./ in Hz) 2 8n m (./ in Hz) 3 611 m (./ in 1 Iz) 2 7.08 bdd( 1.0) 7.15 bdd (0.9) 7.09 hq (1.7) 3 2.45 s 2.38 J (0.9) 2.46 ,v 4 7.62 bdd( 1.0) 7.64 bd( 1.2) 5 7.76 dd (1.2, 7.5) 7.88 dd{ 1.1,7.5) 7.38 b d (2.1) 6 7.65 r (8.1) 7.69 dd (0.9. 7.5) 3.92 .v 7 7.27 dd (1.4. 8.0) 7.30 cW(2.0, 7.5) 6.70 d (2.4) 6-0 Me 3.92 s 1-OH 11.98 s 12.28 .v 12.33 v 4-OH 13.48 .v 8-OH 12.09 s 12.28 s 12.1 3 v

36 4.1.4 Aloe-t inoriin (4)

Compound 4 was isolated as an orange amorphous solid which on ILC analysis showed a yellow spot with an Rf value ol 0.5 (30% l .tOAc in hexane). I he spot changed to purple on exposure to Nlh. which is typical of 1.8-dihydroxyanthraquinones. In support of this, the I V spectrum showed absorption bands at 271 and 429 nm. with the 'll and l3C NMK spectra ( I able 3) showing two chelated hydroxyl protons at 12.05 and 12.01 for OH groups at C-l (6c 164.1) and C-8 (6( 163.7), an oxymethylene peak at

Three aromatic protons with an AMX spin system at 6h 7.80 d (./ 1.0. X. ) Hz), 7.83 / (.7=8.0 Hz) and 7.37 d (.7 1.5. 8.0 Hz) were assigned to H-5. 11-6 and 11-7 respectively of

ring-C of a 1.8-dihydroxyanthraquinone derivatives. Two meta coupled protons at 6h 7.37 (7=1.5 Hz, H-2) and 7.80 (.7 1.0 Hz, H-4) resonated as broad doublets. Instead of the methyl group expected biogenetically at C-3 for anthraquinones (Wanjohi, 2006).

there was an oxymethylene peak (6h 4.81. v) at position 3 of ring A. The broadening of the signals for 11-2 and 11-4 could have resulted from long range coupling ('.7) with the oxymethylene protons. This compound was therefore characterized as 1 .X-dihydroxy-3- hydroxymethylcneanthraquinone trivial name aloe-emodin (4).

The compound is common in aloes, and has also been reported from, the stem bark of Cascara sagrada. Rhamnus purshiana, Rhamnus alaternus, Chinese rhubarb Rheum palnuilum and Rheum undulalum, Rumex oriental is and leal" fruit of Cassia alula. It is also found in Asphodelus microcarpus, Asphodelus Jistulosus, Xanthorrhoea australis and Oroxylum indicum (Dictionary of Natural products, 2008). It is used as a starting material for the synthesis of anthracycline antibiotics and shows some antileukaemic.

37 antimicrobial and anti mutagenic activity as well as antibacterial ait city against methicillin resistant Staphylococcus aureus (MRSA). It is used as a purj itivc and an antiseptic agent (Dictionary of Natural products, 200X).

fable 3: 'H (500 MHz), l3C (125 MHz) NMR data along with HMBC correlation for compound 4 (acetone-d*,)

POSH ION 'll 6n m (./in 11/.) ]1C ~ HMB( 1 164.1 la 1 17.9 > 7.37 hd(\.5) 122.3 C-la. C-4 3 155.6 4 7.80 M ( 1.0) 120.1 C-la. C-2 4a 138.1 5 7.80 d (1.0, 8.0) 121.1 C-8a 5 a 137.9 6 7 83 /(8.0) 138.9 C-8a, C-5a. C-7 7 7.37 d (1.5,8.0) 125.8 C-8a. C 5 8 163.7 8a 117.6 9 194.5 10 182.9 3-Cl I2OH 4.81 64.4 C-2 l-OH 12.05 s C-la. C-2 8-OII 12.01 .v C-7

4.1.5 Aloe-einodin acetate (5)

Compound 5 was isolated as a yellow amorphous solid which on TLC anal} sis showed a yellow spot with an Rf-value ol 0.40 (5% I tOAc in hexane). The spot changed to red on exposure to NIC, a characteristic common to anthraquinones. In support ol this, the liV spectrum showed absorption bands at 268 and 429 nm. while the 'll and l3C NMR spectra (Table 4) showed peaks that were almost similar to aloe-emodin (section 4.1.4) including the carbonyl peaks at 6c 192.7 and 181.5 as well as the two hydroxyl singlets at

5h I 2.07 and 12.05. The difference is the presence ol a downfield shifted methylene at 6n

5.19 hrs (6c 64.7). an up-field shifted methy I (acetate methyl) at 6h 2.19 (6C 20.8) and an ester carbonyl at 6(- 170.0. This suggested that compound 5 is an acetate derivative of 4.

18 This compound was therefore identified as 3-acetox) methylene-1.8- dihydroxyanthraquinone. trivial name aloc-cmodin acetate (5).

This is only the third report on the occurrence of this compound in nature having been isolated earlier (Sharma and Kangaswami, 1977) from the roots of Rumex acelosa (Polygonaceae) and the leaves of Kniphofia foliosa by Berhanu and Dagnc in 1984.

39 I able 4: !H (500 MHz), l!C (125 MHz) NMR data together with HMB( correlation for compound 5 (CDCI3)

POSITION 'll oH m (./in 1 Iz) 73c IIMIU 1 162.8 L 'a . 1 15.3 2 I l l s 122.4 C-l, C-l a, C-4 3 146.5 L 4 - ...... 7.79 .v 118.5 C-la. ( l 4a 153.9 5 7.85 d (8.0) 120.2 C-6, C-7 5a 133.5 6 7.70/(8.0) 137.3 C-5, C-5a. C-8 7 7.32 d (8.0) 124.8 C-5.C-6. C-8a 8 162.6 8a 115.8 9 192.7 10 181.5 3-C(0) * 1 70.0 3-CH20( 0)CH3 2.19a- 20.8 3-C(()) 3-CH2OAc 5.19 brs 64.7 C-2, C-3, 3-C(()), C-4 1-OH 12.07 a C-2, C-la 8-OII 12.05 a C-7, C-8a * From IIMBC

4.1.6 knipholone (6)

Compound 6 was isolated as deep red needles which showed an orange spot on TLC with an R| value of 0.2 (30% EtOAc in hexane). Comparison of the *H NMR spectrum (Table 5) of this compound with chrysophanol (1) showed that it is a chrysophanol derivative.

I hus in the chr>sophanol (1) part, the two downfield shifted singlet at 8h 12 60 and 11.99 were due to the chelated hydroxyl groups at C-l and C-8 whereas the ('-ring aromatic protons at H-5. H-6 and H-7 constitute an AMX spin system at 5h 7.55 dd (J 8.0. 1.1 Hz), 7.75 / (.7 8.0 Hz), and 7.29 dd (J= 8.0, 2.0 Hz). In the ring A, the signal for 11-4 is missing with 11-2 appearing as a singlet at 6h 7.36. while the methyl at C-3 was up-field shifted at 8h 2.17 relative to that of chrysophanol (1). These NMR features suggested that this compound has an aromatic substituent at C-4 on a chrysophanol skeleton.

40 The substituent at ( -4 was identified as acetylphloroglucinol methyl ether from the II NMR (5„ 14.20 for OH-2’; 6.24 (s) for H-5'; 2.63 (.»), 3H. for the acetyl protons at C-3'; 3.98 (.v), 3H. for OMe at C-4'). I herefore. compound 6 was identified as 4-( 3-acetyl-2,6- dihydroxy-4-methoxyphenyl)-l .8-dihydroxy-3-mcthvlanthraquinone. tri\ ial name knipholone (Bezabih and Abcgaz, 1998). The identity was confirmed by direct comparison with an authentic sample (Wanjohi, 2006). OH O OH

Besides being found widely in Hulbine and Knipho/ia species, knipholone as also been reported from the pods of Senna diclymoboirya (Alemayehu, et a l 1996). Biologically, it has been reported to be a selective inhibitor of leukotriene metabolism (Wube et al., 2006) and is a potent antimalarials compound with an IC50 value of 1.49pg/mL (Wube el a l, 2005). fable 5: 'n NMR (200 Ml Iz) chemical shift values for compound 6 (( DCh)

POSI 1 ION 5m ni (./ 1 Iz) 2 7.36 5 5 7.55 c/c/ (1.1, 8.0) 6 7.75 / (8.0) 7 7.29 dd (2.0, 8.0) 5' 6.24 s 3-CH.i 2.17 .v 3’-C(())-CH3 2.63 .v 4’-OCH3 3.98 s l-OII 12.60 s 2’-011 14.20 5 8-OH 11.99 5

41 4.2 BENZACDEHYDE 1)1 KIVATIVES

4.2.1 Flavoglaucin (7)

Compound 7 was isolated as yellow crystals (mpt. I05-109°C) with an R, \alue of 0.53 (5% EtOAc in hexane). The UV spectrum showed absorption bands at Jq,iax 274 and 391 nm. EIMS analysis showed a molecular ion peak at m/z 304 corresponding to C 19H28O3

The 'll NMR (8n I 1 93 for chelated OH and 8h 10.24 for aldehydic proton) and 1 *C NMR (6c 155.8. 117.3 128.6, 144.9. 125.7 and 128.6 for aromatic carbons and 195.5 for the aldehydic carbon) 1) spectra suggested this compound is an or//?o-hydrox \ Ibenzaldchyde derivative. The highly deshielded nature of the hydroxyl group is due to chelation with the carbonyl and is consistent with its placement ortho to the aldehyde group.

From the 'll and C NMR spectra (Table 6). the presence of a prenyl substituent |6h 3.29. d(J= 7.5 Hz) and 8C27.0 forCT^-l"; 6H5.28 / (./ 7.5. 17 Hz) and 6( 121.2 for CH- 2”; a quaternary carbon resonating at 8c 133.6 for C-3”; 8H 1.76, 1.70 brs. and 8t 17.8. 8C 25.8 for (CH3)2 -3"], an alkyl substituent (C7H15, at 8H 2.91. r, 1.58. nr, 1.36. m and 0.90,

/); hydroxyl group (8h 4.38. s: 5<. 144.9) and the only aromatic singlet at 8h 6.90 is consistent with a tetra-substituted benzaldehyde dcri\ative.

The COSY spectrum showed that the aromatic proton at 8h 6.90 (11-5) correlated (allylic coupling) with a proton on the prenyl substituent at 6h 3.29 d (('112-1") implying the two are adjacent to each other. I he HMBC spectrum showed correlation between the aldehydic proton at 6h 10.24 with C-l (155.8) (verilying the aldehydic group is actually 0/7/70-substituled to the hydroxyl group), and with C-2 (117.3) and C-3 (128.6) of the benzene ring. IIMBC correlation was also observed between the methylene protons of the prenyl substituent at 8u 3.29 (CH2-1”) with C-6 (128.57), C-l (155.8) and C-5 (125.6) giving evidence that the prenyl substituent is actually attached to the benzene ring at the C-6 position.

In addition, the chelated hydroxyl group at 8u 11.93 showed HMBC correlation to C-2 (117.3), C-3 (128.6) and C-6 (128.57) of the aromatic ring. Correlations were also noted

42 between aromatic proton (11-5. PH 6.90) with C-l (155.8). C-4 (144.9) and ( ’-6 (128.57) for the aromatic ring and also with the protons on the prenyl substituent (( Ih-1”). I he methylene protons (CH2-1 ol ihe long alkyl chain at 5H 2.91 / correlated with C-2, C-3 and (-4 of the aromatic ring confirming the point of attachment of the chain to the ring is at C-3. These spectroscopic data confirm that this compound has structure 7 trivial name flavoglaucin.

7

I his compound has been isolated Irom Aspergillus Jlavus and other Aspergillus spp. and a marine derived Microsporum spp. It is biologically used as a mycotoxin (antifungal agent) (Dictionary of Natural products, 2008). This appears to be the first report on the occurrence of this compound in higher plants.

43 Fable 6 : 'll (300 MHz), MC (75 MHz) NMR together with H.H-COSY and HMBC data for compound 7 (CDC13)

POSITION 111 6h m (./in 11/) nc IIM BC(V, V) H.H-COSY 1 155.8 2 117.3 3 128.6 4 144.9 5 6.90 .y 125.6 C -l.C -P , C-3, C-4, C H rl” 6 128.6 r 10.24 s 195.5 C-l,C-2, C-3, i” 3.29 d (7.5) 27.0 C-l, C -2”, C-3”, C-5, C-6 (CH3)2-3” 2” 5.28/(7.5,17.7) 121.2 t'l I2-l”, (Cl I3)2-3” 3” 133.6 r” 2.91 / 23.9 C-2” \C-2,C-3,C-4 CH2-2” ' ^ •> * * 1.58 m 31.9 29.6 4” ’ 1.36 m 29.1 cii2-r” 5’” 31.9 6” ’ 22.6 7’” 0.90 / 14.1 C-6’” , C-5’” , 3”-CH3 1.76 brs 17.8 C-2”, C-3” CH2-1”, Cl l2-2” 3”-CH3 1.70 brs 25.8 C-2”, C-3” CH2-1”, CH2-2” 1 -OH 1 1.93 5 C-2, C-3 4-OH 4.38 5

4.2.2 3” \ 4 ’’M)chydroflavoglaucin (8)

Compound 8 was isolated as a yellow' amorphous solid with an Revalue of 0.46 (5% EtOAc in hexane). The 'H NMR data for 8 was indicative of a benzaldchyde skeleton. The 'll NMR spectrum of 8 was similar to that of compound 7 (Section 5.2.1) suggesting identical skeleton with the onl\ difference being on the nature of the long alkyl chain substituent.

In the !H NMR spectrum of compound 8, the presence of a double bond between C -3 " and C -4"w as evident from the two olellnic methine protons appearing more or less at the same frequency (due to pseudo-symmetry) as a triplet (at 6n 5.51) placed in the double bond at C-3” \ Other peaks for the straight chain were; 8h 2.96 /: 2H (C -l"), 2.28 bq\ 2H (C -2"), 1.99 bq\ 2H (C -5"). 1.34 m: 2H (C-6") and 0.87 /: 3H (C-7")

44 while the rest were similar to the ones observed tor compound 7 (Table 3). I hcreforc compound 8 was characterized as 3-(3-heptene)-1.4-dihydroxy-6-prenylbeny ildehyde.

CH3

8

In the spectrum where compound 8 is the major compound, another set of 'll NMR signals similar to those of 8 were observed. These additional peaks could he due to an additional compound in the sample with the only difference from compound 8 being on the position of the double bond on the alkene chain, which in this case is at (’-S'" (5,,',6, ’-dehydroflavoglaucin 8a). (Table 7)

8a

Table 7: 'H NMR (300 MHz) data (CDCI3) for compound 8 and 8a

POSITION 8, 'll 5h ot (Jin Hz) 8a. 'HS h ot(./in Hz) 5 6.90 .v 6.89 s r 10.23 s 10.24 i” 3.30 d{7.2) 3.30 J {1 2 ) 2” 5.28/(7.2.17.4) 5.28 / (7.3,17.4) r ” 2.96 / 2.88 / 2”’ 2.28 hq 2.28 hq 3 '” 5.51 / 1.31 hq 4” * 2.04 hq 5*” 1.99 bq 4.23 hq 6 ” 1.34 m 5.41 hq 7 '” 0.87 / 1.64 d 3”-CH3 1.76 s 1.76 v 3”-CH3 1.70 .v 1.70 .v 1-OH 11.95. v 11.93 .y 4-OH 4.38 4 38

45 4.3 ANTI IK AQUI NON K DIMKKS

4.3.1 10,10’-Bidirysophanolanthrone (9)

C ompound 9 was isolated as pale-yellow amorphous powder with an R \ alue of 0.36 (5% HtOAc in hexane). The UV spectrum exhibited absorption maxima at 271 and 367 nm consistent with the UV spectra of bianthrones (Alemayehu et al., 1992) The HRMS (|M 1J at m'z 479.1483, C30H23O6), together with the 'll and MC \M R spectra, [showing signals arising from two aromatic methyl groups (6H 2.22 and 2.32; 6C 21.9 and 22.1). two sp hybridized methine groups (6M 4.45 (./ 1.5 I lz; 8t 56.3 and 6,1 4.49 (./ 1.5 Hz: 8( 56.3)), four chelated hydroxyl groups (6H 11.71: 8c 161.9. 8M 11.88: 8C 162.1. 8,t

11.63; 8c 161.9 and 8h 11.79; 8( 162.2) and two carbonyl groups (both at i\ 191.6)] were consistent with this compound being a bianthrone derivative.

In one half ol the molecule, the NMR spectra (Table 8) suggested that it is a

chrysophanol anthrone moiety showing a methyl group 8h 2.32 (CTT-3). two meta-

oriented protons resonating at 6h 6.65 hr.\ (11-2) and 6.15 brs (H-4), together with an AMX spin system at SH 6.73 dd (J 7.5, 1.2 Hz) (H-5), SH 7.50 t (.7 7.5 II/) HI-6) and 8fl

6.94 d d (.7=7.5, 1. 2 Hz) (11-7). I he signals attributed to H-4 (5h 6.15) and 11-5 (8h 6.73) were relatively up-field compared to what is observed in chrysophanol (1). supporting the anthrone nature of this moiety in compound 9. In agreement with this, the 'll and l3C

NMR showed signals of a methine group at position 10 (6h 4.49; 8( 56.3) instead of a carbonyl as in anthraquinone such as chrysophanol (1). The methine signal (8h 4.49) appeared as a doublet (,7=1.5 Hz) suggesting that it is coupled with similar methine proton (CH-10’) from the other half of the molecule. In addition, the HMliC spectrum showed correlation of H-10 with C-4a, C-4, C-4a'. C-5a' and C-8a (Table 8) signifying that indeed the point of linkage was at 10/ 10’ of the two chrysophanol moieties.

In fact the MS (| M H ]" at nvz 479.1483. ( ’.wlfoOftand the fragment at m/z 239) and NMR

(Table 8) showing identical pattern for the other half of the molecule, is in agreement with the other half of the molecule being a chrvsophanol-anthrone linked at C-l()\ Therefore this compound was characterized as lO.lO'-bichrysophanolanlhrone (9).

46 OH O OH

I his compound has been isolated from in vitro cultures of Cassia i/iciymobotrya (Leguminosae. Caesalpinoideae) (Monache at a l. 1990), from the seeds of Cassia obstnsifolia, from the hcartwood of C. garrettinu and also from the leav es of Senna longiracemosa (Alemayehu at al.. 1992). However this appears to be the first report on its occurrence in the family Asphodelaceae.

47 Table 8: ‘H (500 MHz), l3C (125 MHz) NMR data (CDC13) along with HMBC correlation for compound 9

POSITION 'll 6h m (J in Hz) "TT HMIU’ i 161.9 la 1 14.0 2 6.65 brs 1 17.0 C-la. Cl 1 - 3, C-4 3 147.5 4 6.15 brs 121.0 C-la. C-2 4a 141.0 5 6.73 d d (7.5. 1.2) 117.0 C-7 5a 142.1 6 7.50/(7.5) 135.7 C-5a. C-8 7 6.94 dd(7.5. 1.2) 1 16.8 C-5 8 162.1 8a 117.2 9 191.6 10 4.49 z/(l .5) 56.3 C-la, C-4a. C-4. C-4a'. C-5a. C-5a'. C-8a r 161.9 la* 114.5 2’ 6.65 brs 116.9 C-la’, CH3-3\C-4’ 3’ 147.3 4’ 5.73 brs 121.2 C-la*, C-2’ 4a* 140.9 5’ 6.33 J(8.0) 119.4 C-7*. 5a* 142.1 6’ 7.30/(8.0) 135.3 C-8a*, C-8’ r 6.87 d (8.0) 116.9 C-5’ 8* 162.2 8a’ 116.7 9’ 191.6 10’ 4.45 z/ (1.5) 56.3 C -la’, C-4a\ C-4’, C-4a. C-5a, C-5a’. C-8a’ 3-CH3 2.32 5 22.1 C-2. C-3, C-4 3’-CH3 2.22 .v 21.9 C-2’, C-4’. C-3’ 1-OH 11.71 .s' C-la. C-2 r-O H 11.63 .v C-la’, C-2’ 8 OH 11.88 v C-7 8*-OH 11.79 s C-7’

48 4.3.2 IO-llvdrot)-10-(chrysophanol-7'-\ l)-chr> sophanol anthronc (10)

C ompound 10 was isolated as .ui orange amorphous powder which turned purple on exposure to ammonia. R, value of 0.32 (20% EtOAc in hexane). The HR VIS (M' at tn/z

508.1104. C30IHoOk) and the 'll and l3C NMR (Table 9) along with the l V (X ^ 271. 292. 389 and 434 nm) are indicative of an anthronc-anthraquinone dimer. I hat 10 might be a dimer based on two chr\ sophanol moieties was deduced from the presence of two aromatic methyl signals (at 6H 2.23 and 2.45). lour chelated hydroxyl signals (at 6„ 12.24. 12.26, 12.32 and 12.34) along with the corresponding oxygenated aromatic carbon atoms (5c 164.2, 164.2. 163.9. 163.7) as well as three carbonyl groups (at 5t 202.0, 194.9 and 182.8).

In one half of the molecule, the NMR signals were comparable to what w as observed for chrysophanol (1) (section 5.1.1) with methyl group (5H 2.23) located at C 3. two meta

protons assignable to H-2 and 11-4. (resonating at Sh 7.59 brs and 7.12 brs. respectively),

together with an AMX spin pattern (at 5h 6.77 brs, 6h 7.50 / (J=8.0 Hz) and 6M 6.91

respectively. Both H-4 (6h 7.12) and H-5 (5h 6.77) are shielded compared to what is

observed for these protons in chrysophanol (1) (5h 7.62 and 7.76. respectively) suggesting that C-10 in this half of the molecule is not a carbonyl. The presence of an sp' hybridized oxygenated carbon at 5( 75.1 indicated the oxanthrone nature of this half of the molecule and that C-10 is the position of attachment to the other halfol the molecule.

For the other half of the molecule, a similar pattern was observed with the 'll NMR showing a methyl group at 8h 2.45 s (C-3‘). two we/o-coupled protons assignable to 11-2' and 11-4‘ (resonating at 6h 6.7' brs and 6.88 brs respectively), whereas H 5’ and H-6" resonated as o/v/?o-coupled protons at 6h 7.98 brs and 8.81 brs respectively. In comparison with chrysophanol (1). the replacement of an AMX spin system with this AX pattern in ring ( for this half of the molecule showed that C-7’ is the point of attachment to the other half. Therefore this compound is a dimer where chrysophanol anthronc is

49 coupled at C-10 to ( -7* of a chiysophanol moiety ( I O-Hydroxy- 10-(chrysophanol-7‘-\ I) - chr> sophanol anthrone 10).

CH3

This compound has been reported from Kniphofia Joliosa (Wube el al., 2005). from the rhizomes of Aloe saponaria and Senna longiracemosa (Alemayehu el al 1992). I he antiplasmodial activity (against the chloroquine sensitive 3D7 strain of I' falciparum

with an IC50 value of 0.260 pg/mL) and a weak cytotoxic activity against KB (human epidermoid carcinoma) cell line with an ED50 value of 104 pg/mL has been reported for this compound (Wube et al., 2005).

50 Table 9: H (500 MHz) and 1V (125 MH/) NMR data (acetone-d6) along with HMBC correlations lor compound 10

POSITION 'll Ah m (Jin II/) C HMBC 1 164.2 C-la la 1 15.4 2 7.59 brs 122.1 C-la. C-4 149.2 4 7.12 brs 125.5 C-la, C-2 4a 146.9 5 6.77 brs 118.6 C-7, C-8a 5a 146.9 6 7.50/(8.0) 138.3 C-5a, C-X 7 6.91

51 4.3.3 10-Hydroxy-10-(chrysophanol-7’-yl)-aloe-cmodin an throne (11)

Compound 11 was isolated as a yellow solid with an R( value of 0.40 in M)% F.tOAc in hexane. I he I1RMS (M at m 523.5003. C30H20O9), the 'll and riC NMR (Table 10) along with the UV (^n,ax 271. 292. 385 and 494 nin), were indicative of an anthrone- anthraquinone dimer. The ‘ll NMR spectrum of compound 11 is comparable to that of compound 10 (Section 5.3.2). However, the ‘H and nC NMR spectra of 1 1 showed an

oxymethylene peak at (5m 4.56: 5C 63.83) and a methyl at (5h 2.46 hrs; 5C 22.06) while compound 10 showed two methyl groups. The rest of the peaks including four chelated

and one unchelated hydroxyl groups (5m 12.29 s, 12.30 s, 12.32 s, 12.34 and 5.91 s) along with the corresponding oxygenated aromatic carbon atoms (5c 170.26. 163.36, 163.74 and 163.03) and three carbonyl resonances (at 5c 202.60. 182.12 and 194.36) were similar with small variation in the values.

In one half of the molecule, the 'll NMR leatures were similar to those ol compound 4

(Section 5.1.4) including two /;/<7t/-couplcd broad doublets for 11-2 and 11-4 [at 5h 6.96 (<7=1.0 Hz) and 6.68 (.7=2.5 Hz), respectively], an AMX spin pattern for 11-5. H-6 and II-

7 [at 5h 6.96 bcl (./ 8.0 Hz), 5H 7.51 / (J 8.0 Hz) and 6.92 bcl (J-8.0 Hz), respectively] and an oxymethylene peak resonating as a broad singlet at 5h 4.56 (C-3). The signals attributed to H-4 (5h 6.68) and 11-5 (5m 6.96) were relatively shielded (due to the lack of anisotropic effect from the C O group) compared to what is observed for these protons in aloe-emodin (4) (5h 7.80 and 7 80, respectively) suggesting that C-10 in this half of the molecule is not a carbonyl. The presence of an sp' hybridized oxygenated carbon indicated the aloe-emodinoxanthrone nature of this half of the molecule and that C-10 is the position of attachment to the other half of the molecule.

On the other hand, the second half of the molecule exhibited values similar to chrysophanol (1) with coupling patterns among the aromatic protons indicating a broad singlet methyl proton at 5h 2.46 which is assignable to M e-3\ two meta-coupled protons assignable to H-2 and 11-4* resonating at 5m 7.58 v and 7.11 s .respectively and two

0/7/70-coupled doublets (at 5m 7.98 (.7=7.6 Hz) and 8.81 (.7=7.6 Hz)) which could be

52 assigned to H-5’ and H-6' respectively. I he de-shielded nature of the o/7/to-coupled protons and the absence of a signal for 11-7’, signified that this was the position of linkage between the two molecules. This compound was thus characterised as 10- hydroxy-10-(chrysophanol-7’-yl)-aloe-emodin anthrone (common name, chrysalodin).

CH 3

This compound has been reported from some Kniphofia foliosa (Dagne. et a/.. 1987). The compound showed borderline cytotoxic activity against in vitro growth of KB tissue culture cells with an ED50 value of 1 Opg/ml (Dagne. et a/., 1987).

53 Table 10: 'll (( IX l{, 500 Mil/.) and MC (acetone-d,,. 125 MHz) NMR data lor compound 11

POSITION *H 6h m (J in Hz) ,JC_ 1 163.5 la 116.7 2 6.96 bd (1.0) 114.6 3 153.9 4 6.68 hd{2.5) 120.3 4a 143.1 5 6.96 hd(J- 8.0) 117.2 5a 143.1 6 7.51 t(J 8.0) 137.7 7 6.92 bd(J 8.0) 114.8 8 163.4 8a 117.6 9 194.4 10 * r 163.0 la’ 114.1 2’ 7.58 s 124.8 3’ 143.1 4' 7.11 5 117.9 4a' 134.0 5’ 7.98 d(J= 7.6) 120.3 5a' 134.4 6’ 8.81 d (J 7.6) 133.8 T 121.5 8’ 160.5 8a 117.5 9’ 182.1 10’ 179.3 3-(CH2OH) 4.56 brs 63.8 3 ’ - ( C H 3 ) 2.46 brs 22.1 I-OII 12.29 s r-O H 12.32 s 8-OH 12.30 s 8’-OH 12.34 s 10-OH 5.91 5 * Not observed

54 4.3.4 10-ffydro\y-10-(islandii'in-7’-yl)-i'hrysophanolanthronc (12)

Compound 12 was isolated as a red powder with an Rf value of 0.40 in 20% EtOAc in hexane. The HRMS (M+ at m/z 524.1125. C30H20CM, ‘H and l3C NMR (Table 11) along with the UV |Xmax 271, 299. 381 and 495 nm. were indicative of in anthrone- anthraquinone dimer. Accordingly, the NMR spectra exhibited two aromatic methyl (6(t

2.26. 6c 24.4 and bn 2.35, 6c 24 1). five chelated hydroxyl groups (6h 12.08, 12.32. 12.34. 12.44 and 6H 13.56; 6c) as well as the corresponding oxygenated carbon atoms (6c 163.5. 163.8, 158.9. 160.0 and 158.9 respectively) and three carbonyl groups (6c 195.0. 188.2 and 188.2). The presence of three carbonyl groups supported that compound 12 is an anthrone-anthraquinone dimer. I he EIMS fragment ion at m/z 256 and the molecular ion (M at m/z 524.1125) indicated that indeed the compound is a dimer composed of monomers related to chrysophanol and islandicin.

The *H NMR spectrum ( l able 11) is in agreement that one half of the molecule is

chrysophanol-anthrone moiet\ showing a methyl group ( 6 h 2.26) located at C-3. two we/£/-coupled aromatic protons assignable to 11-2 and 11-4, resonating at 6, 6.78 (hrs) and 6.61 (brs) respectively, together with an AMX spin system at 6M6.80 dd (./ 8.0. 1.2 llz), 7.41 / (J-8.0 Hz) and 6.95 dd (.7=8.0. 1.2 llz) which could be assigned to 11-5, H-6 and H-7 of the chrysophanol-anthrone moiety respectively. The l3C NMR data (6c 79.5 for C- 10) and the fragment ion at m/z 256 indicated the oxanthrone nature of this moiety. The

signals attributed to H-4 (6h 6.61) and 11-5 (6h 6.80) are shielded compared to what is observed for these protons in chrysophanol (1) (6H 7.62 and 7.76. respectively) supporting that C-10 in this hall’of the molecule is not a carbonyl and the oxanthrone nature of this half of the molecule. Furthermore, the multiplicity (singlet) and the chemical shift position of C-10 (6c 79.5) indicated that this is the position of attachment to the other half of the molecule.

For the other half of the molecule, the *H NMR spectrum showed a singlet at 6h 7.10 (II-

2') and two ortho-coupled protons at 6h 8.06 d (.7=8.0 Hz) and 8.66 d (,/ 8.0 Hz) corresponding to 11-5' and H-6* respectively. These data were consistent with this half

55 being islandicin (3). The de-shielded nature of the ortho-coupled protons (6» 8.06 and 8.66) and the absence of a signal for H -7\ signified that C-7’ is the position of linkage in this half of the molecule. Thus compound 12 was identified as 10-hydroxy-10- (islandicin-7'-vl )-chrysophanol a nth rone (trivial name chryslandicin).

CH3

This compound has been reported from Kniphofia foliosa (Dagne, et a l, 1987). Mild cytotoxic activity against in vitro growth of KB tissue culture cells with an I D50 value of 20pg ml (Dagne. et al.. 1987) and a strong antiplasmodial activity against the chloroquine s e n s i t i v e 3D7 strain of P. falciparum uith an IC 50 value of 0.537pg/ml have been reported lor this compound (Wube et al., 2005).

56 Tabic 11: 'H (CD( I3, 300 MHz). 1 C NMK (acetonc-d6+MeOH-d.,. 125 Ml I/) data along with HMBC correlation for compound 12

po si n o N 'id 6 h h i (7 in Hz) HMBC 1 163.5 la 114.2 2 6.78 brs 118.5 C-la. VCH3.C -4 3 150.3 4 6.61 hrs 122.2 C-la. C-2 4a 143.3 5 6.80 77(1.2, 8.0) 121.1 C-7, C-8a 5a *149.0 6 7.41 / (8.0) 138.2 C-5a. C-8 7 6.95 77(1.2,8.0) 118.2 C-5,C-8a 8 163.8 8a *117.0 9 195.0 10 79.5 P 158.9 la’ 112.5 T 7.10 j 130.4 c - r V 149.5 4’ 1 158.9 4a’ 112.5 5’ 8.06 7(8.0) 120.4 C-8a’ 5a’ 134.6 6’ 8.66 , C-4 3’-CH3 2.35 s 24.1 C-2’, C-3’, C-4’, 1-OH 12.08 s r-0 H 12.34 s 4'-OH 13.56 s 8-OH 12.32 s 8’-OH 12.44 s 10-011 6.61 s *Detected from HMBC spectrum; ** Not Detected

57 4.3.5 10-Hydroxy-10-(islandicin-7’-yI)-aloe-emodinanthrone (13)

Compound 13 was isolated as a red solid with an Rf value of 0.47 in 4( % EitOAe in hexane. The HRMS (M at odz 540.38/2, C30ICo^io) and the 'll NMR spectrum (showing five chelated and one unchelatcd hydroxyl groups, 6n 12.29. 12.31, 12.32, 12.34, 13.51 and 3.98) suggested a dimeric anthraquinone derivative composed of 1.8- dihydroxy and 1.4.8-trihydroxyanthraquinone monomers. In agreement with this, the UV spectrum showed absorption bands lor both 1,8-dihydroxyanthrone and 1.4.8- trihydroxyanthraquinone chromophores (at ^ nax 271, 298, 381 and 495 nm). In the l3C NMR. the presence of live oxygenated sp2 hybridized carbon atoms ( I able 12) is in agreement with such skeleton. C omparison of the 'll and l3C NMR spectra of 13 with those of compounds 2 (Table 2) and 4 (Table 3), showed that one half of the molecule is aloe-emodin (4) coupled through ( -10 to C-7’ of islandicin (2).

In agreement with this, the NMR spectra for the first half showed the presence of signals

for oxymethylene (Sh 4.56 bs, 5( 63.8). two chelated and one unchelated hydroxyl groups

(5h 12.29, 5c 169.3: 5h 12.31. 5c 163.4 and 5h 6.05), an AMX spin pattern (5h 6.94 bJ (J=8.5Hz), 7.51 / (J=8.0 Hz) and 6.92 bd (,/=8.5 Hz) for H-5, H-6 and H-7 respectively)

and meta-coupled protons (5h 6.95 brs and 6.90 brs for H-2 and H-4). As in the other dimers the point of attachment is C -10.

For the other half of the molecule, the NMR (Table 12) spectra displayed signals for three chelated hydroxyl groups (SH 12.33, 5C 165.9; 5H 12.34, 5C 163.4 and 5H 13.51, 5t 159.7

tor O H -l\ O I1-8' and OI1-4’. respectively), two ortho-coupled aromatic protons ( 5 h 8.06 brs and 8.83 brs for H-5’ and H-6’), aromatic methyl group (5n 2.28 brs: 5( 16.4 for

CHi-3') and an aromatic methine (as a broad singlet at 5h 7.21 due to 11-2 having a './ coupling with the biogeneticall) expected methyl group at C-3’). This half is therefore the islandicin moiety (compound 2. Section 5.1.2). The AX spin pattern for Ring-C and the absence of 11-7'. suggested the point of attachment is at C-7’. Thus, compound 13

58 was characterized as 10-hydroxy-10-(islandicin-7'-yl)-aloe-emodin unthrone. I his compound appears to be novel.

CH3

59 Tabic 12: 'H (500 MHz, CDCIj) and ,3C (125 MHz) NMR

POSITION 'll 5h m (Jin 1 Iz) “ nc. HMBC 1 169.3 la 114.1 2 (>.95 brs 114.6 C-la, C-4 3 153.9 4 j 6.90 brs 120.4 C-la. C-2 4a 148.1 5 6 . 9 4 ^ 0 8 117.9 C-7 5a 148.1 6 7.51 ) (7=8.0) 137.7 C-5a, C-8 7 6.92 bd (7=8.5) 114.7 C-5. C-8a 8 163.0 8a 115.5 9 202.8 10 * r 165.9 la* 111.7 T 7.21 />r.9 129.9 C-la’, C-4’ V ■ J .... _ .. 144.0 4' _ 159.7 4a' 112.6 5’ 8.06 />r.v 120.4 5a' 133.8 6' 8.83 brs 133.6 T ♦ 8’ 163.4 8a' 117.7 9’ 194.4 10' 183.6 3-(C’H2OH) 4.56 brs 63.8 C-2, C-3, C-4 3’-(CH3) 2.28 s 16.4 C-2\C-3\C-4* 1-OH 12.29.9 r-0 H 12.33.9 4*-OH 13.51 .9 8-OI1 12.31 5 8’-OH 12.34 s 10-OH 6.05 5 * Not detected

60 4.4 CHEMOTAXONOMK SIGNIFICANCE

From the roots of Kniphofia thomsonii, this work has resulted in the isolation of five monomeric anthraquinones, one phenylanlhraquinone, two benzaldehyde derivatives and five dimeric anthraquinones. Most of the monomeric anthraquinones isolated here are derivatives of chrysophanol (1) and lacks oxygenation at C-6 which is t\ pical in the family Asphodelaeeae. When additional oxygenation occurs it is on the methyl carbon, as in aloe-emodin (4) and aloe-emodin acetate (5). Whereas chrysophanol il) and aloe- emodin (4) are common in many families | in aloes and has also been reported from the stem bark of ( ’ascara sagrada. Rhumnus purshiana, Rhanmus alaternus, Chinese rhubarb Rheum pal malum and Rheum undulalum. Rumex oriental is and leaf fruit of Cassia alata. and also found in Asphodelus microcarpus, Asphodelus fistu/osus, Xan/horrhoea australis and Oroxylum indicum (Dictionary of Natural products, 2008)). this is only the third report on the occurrence of aloe-emodin acetate in nature having been isolated earlier from the roots of Rumex acetosa (Sharma and Rangaswani, 1977) and the leaves of Kniphofia foliosa (Berhanu and Dagne, 1984).

The C-6 oxygenated anthraquinone physeion (3) has been isolated from this plant and this is the first concrete report the occurrence of C-6 oxygenated 1.8- dihydroxyanthraquinone in the family Asphodelaeeae. This compound is widely distributed in lichens, e.g. Parmelia spp.. higher plants, e.g. Rumex spp. (Midiwo and Rukunga, 1985) and produced by Aspergillus and Penicillium spp. It is also isolated from the marine annelid Urechis unicintus (Ulicky. et al., 1991). Islandicin (2) is an example of 1.4.8-trihydro\\-3-methyl anthraquinone and has been isolated from the stem bark of Ventilago bomhaensis (Rhamnaceae) (Pepalla et (//., 1992); from the stem bark of Maesopsis eminii (Rhamnaceae) (Gumming and fhomson. 1970) and from rhizomes of some Kniphofia species (Yenesew el. al., 1988).

The unique compound knipholone (6) in which an acetylphloroglucinol methyl ether unit (30) is attached to a chrysophanol (1) moiety, also occurs in this plant. Comparative studies on the roots of some 14 Kniphofia species showed this compound to be the major

61 pigment in these taxa. It was therefore suggested that eompound 6 may he a marker for the genus Kniphofia (Van Wyk et al., 1995). Knipholone-type compounds appear to be characteristic constituents for the genera Kniphofia (Dagne and Yencsew, 1994), Bulkinella (Van Wyk et al.. 1995). and Bulhine (Be/abih and Abegaz, 199X)

The root of this plant is a rich source of dimeric anthraquinones. Some dimeric anthraquinones have been reported from the leaves of some Kniphofia species but their occurrence in the roots is uncommon. These dimers are principally derived from chrysophanol. Two of these dimers are composed of two chrysophanol monomers vis lO.lO'-bichrysophanolanthrone (9) and l()-hydroxy-l()-(chrysophanol-7'-yl)- chrysophanol unthrone (10). I he latter compound has been reported from Kniphofia foliosa (Wube el al., 2005). from the rhizomes of Aloe saponaria and Senna longiracemosa (Alemayehu et al., 1992); whereas the former compound has been isolated from in vitro cultures of Cassia didymobolrya (Leguminosae, Caesalpinoideae) (Monache el al.. 1990), from the seeds off assia obstusifolia, from the heart wood of C. garreltina and also from the leaves of Senna longiracemosa (Alemayehu et al.. 1992). This however appears to be the lirst report on the occurrence of compound 9 in the family Asphodelaceae. The other two dimmers, 10-h\droxy-10-(chrysophanol-7’-yl)-aloe- emodin anthrone (11) and 10-hvdroxy-10-(islandicin-7'-yl)-chrysophanol anthrone (12) are composed of chrysophanol/aloe-emodin and chrysophanol/islandicin monomers respectively. Both compounds have been reported from some Kniphofia species (Dagne, et al.. 1987). Hie only new compound isolated here is 10-hydroxy-10-(islandicin-7’-yl)- aloe-emodin anthrone (13) whieh is composed of islandicin/aloe-emodin monomers. In most ol the dimers reported in the Asphodelaceae. the linkage is between C-10 of one unit to C-7' of another unit.

This appears to be the first report on the occurrence of the benzaldehvde derivative flavoglaucin (7) in higher plants. Normally for such compounds with para hydroxyl groups, the most stable structure is the quinonoid form which is not the case here. This is partly because the hydroxyl and the aldehyde group are chelated thereby forming a stable structure that does not easily convert to the otherwise stable quinonoid form. This

62 compound has been isolated from Aspergillus flavus and other Aspergillus spp. and a marine derived Microsporum spp. It is biologically used as a mycotoxin (antifungal agent) (Dictionary of Natural products. 2008). Also reported here is a derivative of flavoglaucin 3* \4'"-dehydroilavoglaucin (8) and 5” \ 6 " ,-dehydroflav(tglaucin (8a) which were in a mixture.

4.5 ANTIPLASMODIAL ACTIVITIKS

The crude Cl^Ch/MeOH (1:1) extract of the roots of Kniphofia thomsonii showed antiplasmodial activity with an IC50 value of 6.36 i 0.195 pg/ml against the chloroquine- resistant (W2) strain of Plasmodium falciparum.

The isolated compounds from this extract were tested and activities were observed for a phenylanthraquinone, two dimeric anthraquinone as well as two benzaldehyde derivatives (Table 13). These compounds appear to be partly responsible for the antiplasmodial activity of the crude extract. The phenylanthraquinone derivative knipholoiu (6) has good antiplasmodial activity with an IC’50 value of 2.5010.100 pg/ml against P falciparum (W2 strain) which is comparable (IC50 value of 1.49 jag/ml) with what has been reported for the same compound isolated from Kniphofia foliosa in literature (Wube et a/., 2005). This difference in antiplasmodial activity could be attributed to the difference in stereochemistry of this compound and that reported in literature. In addition, antiplasmodial activity against asexual erythrocytic stages of two strains <>1 Plasmodium falciparum in vitro (Kl/chloroquine-resistant and Nl 54 / chloroquine-sensitive) has also been reported (Bringmann etal.. 1999).

The dimeric anthraquinone derivative 10-hydroxy- 10-(islandicin-7’-yl)-ehrysophanol anthrone (12) showed good activity against the chloroquine resistant (W 2) strain of P falciparum with an IC50 value of 3.42^0,110 pg/ml whereas a strong antiplasmodial activity against the chloroquine sensitive 31)7 strain of P. falciparum with an IC50 value of 0.537 pg/mL has also been reported for the same compound (Wube et a/.. 2005). The difference in the strains used and the stereochemistry of the compound influences the

63 antiplasmodial activities and thus the disparities observed. The other dimeric anthraquinone derivative lO.lO’-bichrvsophanol anthrone (9) showed good antiplasmodial activity against the chloroquine resistant (W2) strain of P. falciparum with an IC'50 value of 2.23±0.022 pg/ml. This is the first report on the antiplasmodial activity of compound 9.

The benzaldehyde derivatives on the other hand, showed good antiplasmodial activities against the chloroquine resistant (W2) strain of P falciparum with compound 7 showing an IC'50 value of 2.06±0.276 pg/ml whereas its derivative 3” \4 ’” -dehydroflavoglaucin (8) showed a better activity with an ICso value of 1.93±0.784 pg/ml. An antifungal (mycotoxin) activity has been reported lor this compound (Dictionary of Natural products. 2008). however this is the first report on its antiplasmodial activity.

This investigation has showed the potential of phenylanthraquinones, dimeric anthraquinone (with an anthrone and anthraquinone dimer structure) and the benzaldehyde derivatives as lead structures for development of antimalarial drugs which could be clinically useful to combat the malaria menace.

Table 13: In vitro IC50 values against the chloroquine resistant (W2) strain ol P. falciparum

Sample IC50 (ig/ml Root extract ol Kniphofia thomsonii 6.36±0.I95 Knipholone (6) 2.50±0.100 Flavoglaucin (7) 2.06±0.276 3"' .4' ’’-Dehydro flavoglaucin (8) 1.93±0.784 lO.lO'-bichrysophanol anthrone (9) 2.23±0.022 10-hydroxy-10-(islandicin-7'-yl )-chrysophanol anthrone (12) 3.42±0.110

64 CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

> I he crude C ll >( b/MeOH (1:1) extract of the roots of Kniphofia thomsonii showed good anti pi as modi al activity against the chloroquine-resistant (W2) strain of Plasmodium falciparum and thus its use as an antimalarial agent traditionally is justified.

r f rom the roots of Kniphofiu thomsonii. a total of thirteen compounds were isolated and characterized. These include the monomeric anthraquinones: chrysophanol (1), islandicin (2). physcion (3), aloe-emodin acetate (4) and aloe-emodin (5); the phenylanthraquinone: knipholone (6); the benzaldehyde derivatives: flavoglaucin (7) and 3'’\4''-dehydroflavoglaucin (8) and the dimeric anthraquinones: 10.10’- bichrysophanolanthrone (9). 10-hydroxy-10-(chry sophanol-7’-yl) chrysophanolanthrone (10), 10-hvdroxy-10-(chrysophanol-7'-yl)-aloe- emodinanthrone (11), 10-hvdroxy- 10-(islandicin-7’-yl)-chrysophanolanthrone (12) and 10-hydroxy-10-(islandicin-7,-yl)-aloe-emodinanthrone (13). The dimeric anthraquinone 13 is a new compound while flavoglaucin (7) and 3’” .4'” - dehydroflavoglucin (8) are reported for the first time in higher plants; the C-6 oxygenated anthraquinone physcion (3) is reported for the first time in the family Asphodelaccac; and this is also the first report for the occurrence of compound 9 (10,1 (f-bichrysophanolanthrone) in the genus Kniphofia.

> The anti-plasmodia! activity of the crude ClfT h/MeOH (1:1) extract of Kniphofia thomsonii could be attributed to the phenylanthraquinone, dimeric anthraquinones and the benzaldehyde derivatives with the highest activity being attributed to the phenylanthraquinone 6 and the benzaldehyde derivative 8. Prior to this report, not much has been reported on the antiplasmodial activities of dimeric anthraquinones.

65 All the dimeric anthraquinones isolated and tested in this study showed good activities against (W2) strain

5.2 RECOMMENDATION

> It is recommended that the antiplasmodial acti\itv of the compounds isolated in this study to be tested against other strains of Plasmodium.

> It is recommended that in vivo testing of the anthraquinones be carried out.

> The structure-activity study with a wide range of anthraquinones should be done to determine the structural requirement for anti-plasmodial activity.

> The interest on the anthraquinones of the family Asphodelaceae should not be limited to antiplasmodial activities. Screening for other biological activities especially on a broader spectrum of amiprotozoal activities including anti-leishmaniasis is recommended.

> Further phytochemical work on the genus Kniphofia could lead to the isolation and characterization of more novel compounds with biological activities.

> It is recommended that studies on the stereochemistry of the dimeric anthraquinones be done.

> It is also recommended that further studies be done to establish the relationship between stereochemistry and the antiplasmodial activity of the phenylanthraquinones as well as the dimeric anthraquinones.

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82 'll NMR SPECTRUM FOR COMPOUND 3 (SOLVENT: CD 3(SOLVENT: 300Cl* COMPOUND MHz) FOR NMRSPECTRUM 'll

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85 IJC NMK SPECTRUM FOR COMPOUND 4 (SOLVENT: ACETONE-d», 125MHz)

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86 IJC NMR SPECTRUM FOR COMPOUND 4 (SOLVENT: ACETONE-^, 125MHz) ACETONE-^, 4(SOLVENT: COMPOUND FOR IJC NMRSPECTRUM

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88 [ i 5[ 4 — — 3 CM rt to CM C l ,JC NMR SPECTRUM FOR COMPOUND 4 (SOLVENT: ACETONE-d6, 125 4(SOLVENT: COMPOUND FOR MHz) ,JC NMRSPECTRUM

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92 'H NMR SPECTRUM FOR COMPOUND 4 (SOLVENT: ACETONE-d*, 5(MI MHz)

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93 IIMBC SPECTRUM FOR COMPOUND 4 (SOLVENT: ACETONE-d,„ 111-500ACETONE-d,„4(SOLVENT: COMPOUND FOR IIMBC SPECTRUM and

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95 HMBC SPECTRUM FOR COMPOUND 4 (SOLVENT: ACETONE-d,,,4(SOLVENT:' COMPOUND 11-5(10 FOR HMBCSPECTRUM and

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ppm IIMBC SPECTRUM FOR COMPOUND (SOLVENT: ACETONE-d*, (SOLVENT:111-500 and COMPOUND FOR SPECTRUM IIMBC

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__ uidd HMQC SPECTRUM FOR COMPOUND 4 (SOLVENT: ACETONE-d», 'll-5(MI and i3C-125 MHz)

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IAO-27F * 8 .3mg * gs- ,3C-125MH 100 z ) SPECTRA FOR COMPOUND 5

101 I3C NMK SPECTRUM FOR COMPOUND 5 (SOLVENT: CDCIj, I25MID)

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104 'H NMR SPECTRUM FOR COMPOUND 5 (SOLVENT: CDCIj, 500 MHz)

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H NMR SPECTRUM FOR COMPOUND 5 (SOLVENT: CDCIj, 500 Mil/) 500 CDCIj, (SOLVENT: 5 COMPOUND FOR SPECTRUM NMR H

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p p m HMBC SPECTRUM FOR COMPOUND 5 (SOLVENT: CDCIj, 'H-500 and u<-125 MHz)

111 'H NMR SPECTRUM FOR COMPOUND 5 5(l»CDCIj, (SOLVENT: FOR COMPOUND MHz) SPECTRUM NMR 'H

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p p m IQC SPECTRUM FOR COMPOUND 5 (SOLVENT: CDCI3, 111-500 and IJ( -125 MHz)

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§ SPECTRA FOR COMPOUND 6

118 m NMR SPECTRUM FOR COMPOUND 6 (SOLVENT: ACETONE-d,„ 300 Mil/)

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120 SPECTRA FOR COMPOUND 7

121 ,3C NMR SPECTRUM FOR COMPOUND 7 (SOLVENT; CDCIj, 75 MHz)

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122 IJC NMR SPECTRUM FOR COMPOUND 7 COMPOUND CDCIj,FOR (SOLVENT; 75 MHz) SPECTRUM IJCNMR

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ppm 'H NMR SPECTRUM FOR COMPOUND 7 (SOLVENT; CDCIj, 300 Mil/)

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124 'H NMR SPECTRUM FOR COMPOUND 7 (SOLVENT; CDCI3,300 MHz)

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132 'H NMR SPECTRUM FOR COMPOUND 8 (SOLVENT; CDCIj, 300 MHz)

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137 ^ NMR SPECTRUM FOR COMPOUND 9 (SOLVENT: (DC I,, 'll-SOII a„d 'V 125MHz)

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168 SPECTRA FOR COMPOUND 11

169 UC NMR SPECTRUM FOR COMPOUND 11 (SOLVENT: ACETONE-d* 125 MHz)

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179 SPECTRA FOR COMPOUND 12

180 ,JC NMR SPECTRUM FOR COMPOUND 12 (SOLVENT: ACETONE-d6+MeOH d4,125M H/)

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C u r r e n t Data Parameters NAME Febl5-2007-mh EXPNO 1 6 0 PROCNO 1

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F2 - Processing parameters SI 131072 SF 300.130CC6C MR* MDN KM

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ppm 'll NMR SPECTRUM FOR COMPOUND 12 (SOLVENT: CDC|* 300 12CDC|* MHz) COMPOUND (SOLVENT: FOR NMR SPECTRUM 'll

IAO I0H poor solubility 319 * 3 St-9 * 8 £ IfrO* 8 109*1 63£ * 1 £9£ * 1 {'£{'*! 10fr*1 T8£ * 1 880 *1 609*9 193*/ 896*9 Cfr6 *99£6 ’ 9 CI6'9 fr96 * 9 C18 * 9 908*9 T81‘9 10*8 186 3 < 4 A. 1 J A £96*0 33C' I L 1^3 * 3 61C 61C *I frgri 301*3 00C * I ao 0 0 o a. 1 'H NMR'H

IAO 10H poor solubility 980 * /.5I * 0IT*fr C26 * 6 be x * 958*2 652*2 5* —_ _ 556*2 — 656*2 9fr0 ’2 21^*2 556*0 5fr6*0 506*0 92fr*T 9Ifr*T 211*2 092*1 962*t 968*0 255*1 106*1 {'82*1 fr88 * C 126*0 SPECTRUM b t

------FOR COMPOUND 12COMPOUNDFOR 187 (SOLVENT: CDCIj,30(1Mil/)

ppm MCSETU FRCMON 12 ACETONE-d*+MeOH-d4,COMPOUND FOR(SOLVENT: HMBCSPECTRUM

IAO 10H * gs- \il\ sjillhiajImse'ssiSl!! sjillhiajImse'ssiSl!! di1-* td t t i ‘!‘ i LiHij a | t 11 a ii ; a i ; 1 i i i lal 1 ILliHHSini Iti | Hal j il- s-ss*. - p : - 5. * - * .- 5 , - ::: * p £ - .. ss-isss3* - -••* 4 5 ? * ? ■ ; i 5 I 4 I *-*••••* 'H-500 and ,JC-125 and MHz) 'H-500 ■ . - 1...... t ; = ;***‘ *=. 1...... utK ; i-s . . t \ t \ \h'jl ;•**«* «»•* |!»***«***‘»i* *:»:» !iir**» » i'!m •* ti ilillSSEiiiESS itai tlalli! ss«IS:«v r:Ssl!;j 188 j j j j

ppm HMBC SPECTRUM FOR COMPOUND 12 ACETONE-dt+MeOII-d* (SOLVENT: FORCOMPOUND HMBCSPECTRUM

1A0 10H * gs-HMBC 111-500uC-125MHz) and 189 HMQC SPECTRUM FOR COMPOUND 12 COMPOUND FOR (SOLVENT:ACETONE-dt+MeOH- HMQCSPECTRUM

IAO 10H * gs-HMQC d4, 'H-500 and ,3C-125 d4,and 'H-500 MHz) 190 HMQC SPECTRUM FOR COMPOUND 12COMPOUND FOR ACETONE-dt+NIcOH- (SOLVENT: HMQCSPECTRUM

IAO 10H * gs-HMQC j,111-500d l3C-125 and Mil/) 191

o ro CO i) O CM

a s a s s »ouppunqv »Ai»e|»a

192 MS FOR COMPOUND 12 IAO 10H HEYDENREICH 6 ESI E X M 103 (4 325) A M (Cen.5.80 00 H t5000 0.490 89 JO 00) Cm (72 108) " " " 5251211 4 0 8

m/z

193 SPECTRA FOR COMPOUND 13

194 I3C NMR SPECTRUM FOR COMPOUND 13 (SOLVENT: ACETONK-d6, 125 MHz)

u 8 a x cl x • s = u 0 y c 7 CS« 3 3Z »» r » r 3tlt *« " TO OO 9 w vfflNOr oor 300)00 i ( j in o n r. x> «n ) ' o X) Z W N Ul U f riN O P i 0 0 * 0 0 0 M f c ir iU N > CM O 'X* ■ ^ v I •— • • CM • CO (S r- vU C O O rss'd > . • Mi <*: O O )C W 'J > M> 'T' *-* OO ft* o *n > • O ^ ^ os o . • , O • 3 U )UUO o 2 C

", : c o < * a ,0 .0 C/5QJ *J X U. 01 «3 SS38* 3 i 3 % 1 i br ol x C 7 - C7. u: x Sg5lB8SSSs:?»SSP:

368’82 2 0 8 8 2 U'U'62 P8E‘629r'"6 2 S88’«2268 *62 non'or uOl •(it IrSl’OE IS2 0E 30E‘CE •OfrOE a t t o E L0S2E F8S2E 9 9 I E » lEfTE9

o 9 r 6 '* m o m 6SV90X > 9t8-/,o: < 60L‘TIT 629* 21 1 o oowxx CO 2 t s » : i 6F9‘t'jrcu m 6X6L t ' i* 'itI i . I rQ 95C-02T CM 06E-021 0X8’62X O S29EE1 < 6trfE Ei 8 36 'EE l 9 8 9 L i I EfcO'PH ran'H H ► (.6’E5l 6fr9‘6SXXfOTlX » 9 fE 9 l 9t:8's»i 22E69X 06 X' 2t X ncfr-EiX XSXKX

029

OVC’t t l ------

-c/ •7.nr------

195 l3C NMR SPECTRUM FOR COMPOUND 13 (SOLVENT:COMPOUND FOR ACF.TONE-d*, l3C NMRSPECTRUM 125

IAO 27D * 13C (AV500) t 6 F f 4 0 OZG' EBT 4 ZOZ *4/ 06 P - i E 06:2iT ZZE'691 6F9'0%: E80'8i>I 6Pf£E J>» . t itfn OTV90T bSEE9.—— F/6-E5'. EbO'm ‘iEI 9 9 9 BL'fc’Et SZ9 *EE 0i8'6Z: OO.•►.tOL I "-U 6E6‘iTT B/B'/n: I -:0 6 I 9 twti: twti: lI ’S F iS 06E'OZl 9S£'0Zt :s :-F4t —-— :-F4t :s .£0'£9 - .£0'£9 ------

------MHz) 196 3 M PCRM O OPUD 13COMPOUNDACETONF.-d* (SOLVENT: FOR l3C 125NMRSPECTRUM

IAO 27D * 13C (AV500) 9Crr r C '9 m' SECtf! ------SECtf! rj Z W r nz err z f s s i 968-6Z 68E *62ZOO'OZ 966 arc-f.z 966 * 9Z »8l'OE i)U l ■ OE ib»6Z EC60E BCE’OE tSZ 0£ »»9 109 -inn IMI’fcZ ILL

OE C ZC — ft •or ' <1 LZ

------MHz) 197 'll NMR SPECTRUM FOR COMPOUND 13 (SOLVENT: CDCI* 500 Mil/)

O O O t l> J U 0 n k t V) « SVC XXV) P 3 * H W »

» Tl O ij o ,r) co cm co cs r* -*> 0 0 0 0 0 0 U r» r-4 g O n <*> o • O o • O u O % CM I rt TiO'Jr- J) O *f M v . oo oo U r- 5. MtAO • f* © CM ^ o o o o rO vet) o «/) cn «C c^OOO U CO < m r* *2 c*> ^ r* *r o o «n ol c; 3 • — O o *- i : 2 o o • n o n • I C*i . . . c- OO

4 ) 0 7 ULU2 J > I CD x > ■* - j ; t S 04 O S W O u* o iX s r* < X x < sc pr.3CO«'-«*-CK3 3 -j £ b. m in 5 in ' C) z w o. 11 • *y) z 2 w t. < re Cj o ■ * o y. y z c. o. w

9EZS1 ppm

ISO- IS

Z219 f- in

9911 L in

SI.CZ 2622

Z89)

fOS l

6t-£P £

000'l

198 'H NRSETU FRCMON 13 COMPOUND CDCI,,(SOLVENT: FOR 500 Mllz) NMRSPECTRUM

1A0 27D * 1H (500 MHz) 6tf zzz•sorz: ?:

199 'H NMR SPECTRUM FOR COMPOUND 13COMPOUND FOR (SOLVENT:CDCIj, 500NMRMM/)SPECTRUM 'H

IAO 27D * 1H (500 MHz) SUC9 SUC9 90 E-6'9 ~n»; * <» 60S" i ots'i EfcF’ i SZS RSO'R VL L ‘ r i - — ----- 200

p p m 'H NMR SPECTRUM FOR COMPOUND 13COMPOUND FOR(SOLVENT: CDClj, 50(1NMRSPECTRUM 'H MHz)

IAO 27D * 1H (500 MHz) 656 ’656 0 ‘0 6 9 0 256" 0 St'6’0 236'0 o v a -0 6 2 9 816'0 (8'0 E 998-0 FFO-O 518'C 2 6 88:-■ S8v: 50-: S tio--: >S6'0 656'0 •.38 '0 . V 9 / ': 0 6 9 96 .' . .' 96 v z o so:-: 366'C 665' 665' . V 5 9 . 9t>- . V 1 6 v o o OPT ' ” 08 6s v . v s s otz-: . 91Z* 9IZ-:or?-: 002''. *6." :eo--. Ft'6‘0 KtrE". 060-2 - 060-2 6tr0'2 - 6tr0'2 SfrO’ Z- - V 696 256-:966• - - ’ 066 . - 6 16- . - 68C-: - 9nv 86t>-: SOO" 2 C82'2 - 282-2 8 6 :•2 - :•2 6 8 r.i-.-z 2 65 V 8t 0 * Z- 535-6 -> 086-C 6682 : r t : S60'2 - 290‘Z 890-2 2 : - 326 •: zzr. - ■: V69--. - F60-2 - 060-2 rz- tr o 86 2*2 86 2-2 862-2 - 862-2 ' - .'2 8 2 629'2 330’2 95Z-2 .62-'. .09 -: - - 2 f I . 68*0

. V . V . .

201 I AO 27D * gs-HMBC (500 MHz) 202

ppm HMBC SPECTRUM FOR COMPOUND 13 COMPOUND (SOLVENT: ACETONE-d*,FOR HMBC'H-500SPECTRUM

IAO 27D * gs-HMBC (500 MHz) and 1JC-125 and MHz)

203 udd MCSETU O OPUD 13COMPOUND (SOLVENT:ACETONE-d»,'FOR 11-500HMQCSPECTRUM

IAO 27D * gs-HMQC (500 MHz) and l3C-125 and MHz) 4 0 2

ppm HMQC SPECTRUM FOR COMPOUND 13COMPOUNDFOR (SOLVENT: ACETONE-d* 'H-500SPECTRUM HMQC

IAO 27D * gs-HMQC (500 MHz) and and i 3C-125MH 205

z

) udd IIMQC SPECTRUM FOR COMPOUND 13COMPOUND (SOLVENT:FOR SPECTRUM IIMQC

IAO 27D * g s -H M O C (500 KHz) nd d an i 3C-125 206 MH z ) MS FOR COMPOUND 13 HRMS FOR COMPOUND 13 1AO270 HEYDENREICH_9_ESI_EXM 11 (0.463) AM (Cen,5,80.00, Ht,5000 0,490.89,0 00). Cm (2:22)

208