Investigations on the antimalarial activity of alkoxylated and hydroxylated chalcones 1

INVESTIGATIONS ON THE ANTIMALARIAL ACTIVITY OF ALKOXYLATED AND HYDROXYLATED CHALCONES LIU MEI B.Eng., China Pharmaceutical University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF P

INVESTIGATIONS ON THE ANTIMALARIAL

ACTIVITY OF ALKOXYLATED AND HYDROXYLATED

CHALCONES

LIU MEI

(B.Eng., China Pharmaceutical University)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE

2003

To my parents

ACKNOWLEDGEMENTS

I am greatly indebted to my supervisor, Associate Professor Go Mei Lin, for

her constant encouragement, patient guidance, generous help and constructive criticism throughout the whole period of this research Simply speaking, this work would be mission impossible without her

I am deeply grateful to Professor Prapon Wilairat, Chair of Department of

Biochemistry in Mahidol University, Thailand, for his tremendous generosity and hospitality in providing the key screening facilities for this project

Special thanks are extended to Dr Tan May Chin, Theresa, Assistant

Professor in Department of Biochemistry, whose wise advice has been invaluable always

My gratitude also goes to the National University of Singapore for awarding

me the Research Scholarship, and, all the lecturers, technical and administrative staff, fellow students and friends in Department of Pharmacy for their great assistance and

friendship, in particular, Associate Professor Lim Lee Yong, Dr Shanti, Miss Ng Sek

Eng, Madam Oh Tang Booy, Miss Zhou Qingyu, Mr Sam Wai Johnn and Mr Wu Xiang

Sincere appreciation also goes to Dr Philip J Rosenthal in University of California, San Francisco, and Dr Simon Croft in London School of Hygiene and

Tropical Medicine for their kind help and sharing their knowledge in biochemical studies in this project

Last but not the least, I would like to express my heart-felt gratitude to my

family, especially my beloved Baba and Mama, and one anonymous close friend

Without their immeasurable understanding and sharing the joys and frustration all the way, I would never have been to this cheerful moment

1.2.1 Targets present in the digestive vacuole of the parasite 3

1.2.2 Targets involved in the synthesis of macromolecules and metabolites

8 1.2.3 Targets involved in membrane processes and signaling 13

SECTION THREE: DRUG DESIGN AND SYNTHESIS OF TARGET

3.2 Rationale of Drug Design 29

3.4.4 Method for determining 13C NMR of carbonyl group 41

SECTION FOUR: EVALUATION OF ANTIMALARIAL ACTIVITY 43-57

SECTION FIVE: STRUCTURE ACTIVITY RELATIONSHIPS 58-98

5.2.2.3 Determination of physicochemical parameters of chalcones by

5.3 Multivariate Analysis of Structure Activity Relationships (SAR) of

5.3.3.1 Principal component analysis (PCA) 65

5.3.3.2 Projection to latent structures (PLS) analysis 68

5.4 Comparison of Structure-Activity Relationships between Antileishmanial

5.4.2.1 Determination of in vitro antimalarial activity 81

5.4.2.2 Determination of in vitro antileishmanial activity 81

5.4.2.3 Determination of physicochemical properties of chalcones by

5.4.2.4 Statistical and Correlation Analyses 82

5.4.2.5 Comparative molecular field analysis (CoMFA) 83

5.4.3.1 Structural requirements for antileishmanial and antimalarial

5.4.3.2 Comparative molecular field analysis (CoMFA) of

antimalarial and antileishmanial chalcones 86

6.2.1 Degradation of [14C] methemoglobin by extracts of P falciparum

6.3 Results 102 6.3.1 Effects on the enzymatic activity of a crude plasmodial extract

catalyzing the breakdown of radiolabelled methemoglobin 102 6.3.2 Effects on falcipain-2 and associated changes in the food vacuole on

SUMMARY

The objective of this thesis is to establish structure-activity correlations for the antimalarial activity of chalcones and to investigate their possible modes of action

against Plasmodium To this end, 105 chalcones were synthesized by base-catalyzed

Claisen-Schmidt condensation and evaluated for their ability to inhibit hypoxanthine

uptake into P falciparum (K1) trophozoites Physicochemical parameters

encompassing steric, lipophilic and electronic properties were experimentally

determined or obtained in silico from molecular modeling methods Structure-activity

relationships were established using multivariate tools (principal component analysis, partial least squares projection to latent structures), multiple linear regression and comparative molecular field analysis (CoMFA)

The structural requirements for antimalarial activity vary according to the nature of ring B in the chalcone framework Among the alkoxylated chalcones,

differing requirements were found depending on whether ring B is substituted with trimethoxy, dimethoxy or methoxy groups Greater homogeneity was detected among the hydroxylated chalcones One surprising observation was that active chalcones

(defined as those with IC50 < 10 µM for alkoxylated chalcones and IC50 < 20 µM for hydroxylated chalcones) obtained from different classes share similar physicochemical characteristics, namely a preference for a large-size ring B and ring A substituted with electron withdrawing groups

The chalcones were also found to be antileishmanial in tests against

Leishmania donovani amastigotes Different structural requirements were found for

antimalarial and antileishmanial activities Antileishmanial activity was favored by hydroxylated chalcones carrying large-size ring A in contrast to antimalarial activity which is found predominantly among alkoxylated chalcones with specific requirements

for both rings A and B Despite these limitations, two chalcones (8 and 19) were found to combine good antimalarial and antileishmanial activities, with 8 being of

particular interest as it was found to increase survivability of P berghei ANKA

infected mice at 100 mg/kg

The chalcones were not cytotoxic against the KB3-1 cell line at 20 and 40 µM, indicating specificity in their antimalarial action However, there were indications of toxicity against mice macrophages revealed during the course of antileishmanial testing that require further attention

The chalcones were found to inhibit several processes involved in hemoglobin

degradation in the Plasmodium digestive vacuole, namely interference with the

enzymatic activity of a crude plasmodial extract on the breakdown of methemoglobin, inhibition of recombinant falcipain-2, and binding to hematin It appeared unlikely that these inhibitory effects contributed significantly to the antimalarial activity of

chalcones as there was no discernible trend between the various inhibitory activities and in vitro antimalarial potency It appears likely that other pathways are affected by the chalcones

PUBLICATIONS AND PRESENTATIONS

Publications:

• Liu, M.; Wilairat, P.; Go, M L

Antimalarial alkoxylated and hydroxylated chalcones: structure activity relationship analysis

Journal of Medicinal Chemistry 2001, 44: 4443-4452

Structure-Activity Relationships of Antileishmanial and Antimalarial Chalcones

Bioorganic & Medicinal Chemistry 2003, 11: 2729-2738

Conference Presentations:

• Liu, M.; Wilairat, P.; Go, M L

1 Annual meeting of American Association of Pharmaceutical Scientists

(AAPS), 2000

Antimalarial activity of chalcones, Abstract No 2724, 3232

2 Annual meeting of American Association of Pharmaceutical Scientists

(AAPS), 2002

Effect of antimalarial chalcones on parasite-induced hemoglobin

degradation, Abstract No AM02-00635

• Liu, M.; Wilairat, P.; Go, M L

61 st international congress of International Pharmaceutical Federation

SECTION ONE INTRODUCTION

1 INTRODUCTION

1.1 Malaria as a Health Problem

Malaria is undoubtedly one of the most intractable and persistent parasitic diseases confronting mankind 1 The principal causative organism in humans,

Plasmodium falciparum is responsible for the deaths of more than one million African

children annually, a staggering statistic comparable to encountering deaths on the scale

of the September 11th tragedy on a daily basis for one year The economic toll of malaria is equally tremendous It has been estimated that the African continent has forgone almost US $100 billion in lost GDP over the last 35 years due to malaria alone

Despite decades of control and treatment efforts, the mortality and morbidity associated with malaria continues unabated The experience has been that no sooner than a novel mode of control emerges, a new form of resistance will follow: DDT-resistant mosquitoes have rendered the pesticide ineffectual; The emergence of drug-resistant plasmodia has sounded the death knell of chloroquine, one of the most useful drugs available for the treatment of malaria, and possibly other drugs may lose their utility to resistance in the near future if used in an inappropriate manner Funding for malaria research is in fact increasing but does not match up to the urgency of malaria

as a global health problem The sad fact remains that profit-driven drug firms have little incentive to develop drugs or vaccines for a disease that affects mainly poor people

However, there are reasons for optimism First, there is now a better

understanding of the biochemical and metabolic processes that occur within the

parasite 2,3 Targets in the parasites that are susceptible to inhibition or interference have been identified and there is a better understanding of the mode of action of

antimalarial agents like chloroquine 4 and artemisinin 5 Secondly, the successful

sequencing of the genome of P falciparum will undoubtedly lend greater insight into

the biological roles of key parasite molecules and can be expected to have a positive impact on the development of vaccines and new drugs 6 On the socioeconomic front, there is a growing awareness that several proven, cost-effective techniques for curbing malaria (like the use of insecticide impregnated bed nets) are not as widely employed

as they should be A greater commitment on the part of governments in countries blighted with malaria can ensure control of the disease within the next decade 7

1.2 Targets for Antimalarial Drug Discovery

The conventional method for developing drugs against pathogens begins with the identification of a molecular target or process that is essential for the survival of the organism and whose inhibition would result in its demise The molecular structure and function of the target must then be elucidated if a rational basis for drug design is to be realized A promising drug target should ideally have the following characteristics: 3(i) It must be a critical feature of the organism’s life cycle, preferably involved in a rate-limiting step of a biochemical process and must be significantly different from an analogous process in the host if a “magic bullet” effect is to be realized As far as possible, the organism should not be able to circumvent this process by using

alternative routes

(ii) Effects of test compounds are readily screened against this target An added

advantage would be the strong likelihood of converting the screening process into a high throughput method

(iii) It should have a low potential for development of resistance

Such an “idealized” target has yet to be identified in the Plasmodium but may

become a reality as our understanding of essential processes in the parasite increases

Antimalarial targets may be classified into three classes, namely targets present

in the digestive vacuole of the plasmodia; enzymes involved in the synthesis of

macromolecules and metabolites; and processes involved in membrane transport As these targets have been described in excellent reviews, 2,3 they will not be discussed in detail in the following paragraphs but will be presented as summaries, with necessary updates where appropriate

1.2.1 Targets present in the digestive vacuole of the parasite

The principle targets are the processes of hemoglobin digestion and

detoxification and the oxidant defense mechanisms

Hemoglobin is ingested by the parasite and processed in the digestive vacuole

to supply essential amino acids required by the parasite for its survival This process is highly susceptible to external intervention and at least three proven antimalarial drugs (chloroquine, artemisinin, atovaquone) are postulated to exert activity by targeting heme metabolism 3 Proteolysis of hemoglobin is undertaken by four aspartic proteases (plasmepsins I, II, IV and histo-aspartic protease), 8 three cysteine proteases

(falcipains) 9 and a zinc protease (falcilysin) 10 The validity of these enzymes as chemotherapeutic targets is widely accepted 11 Most of the early inhibitors are peptidic

in nature and suffer from the usual limitations of such compounds (poor

bioavailability) In addition, selectivity against plasmodial enzymes vis-à-vis host enzymes (e.g cathepsin D) has been modest However, a recent report 12 of a non-peptidic water soluble inhibitor with good selectivity for plasmepsin II and

submicromolar antimalarial activity is encouraging and suggest that the improved inhibitors may be developed in the near future

Heme [ferrous proptoporphyrin IX, Fe(II)PPIX], the prosthetic group of

hemoglobin, is released in large amounts during the enzymatic breakdown of

hemoglobin in the acidic digestive vacuole of Plasmodium The ferrous ion in heme is

oxidized in the presence of molecular oxygen to form ferriprotoporphyrin

[Fe(III)PPIX], which is toxic to the parasite: its detergent like properties can lyse cell membranes 13 and it inhibits plasmodial enzymes 14 Thus, the disposal of heme is

central to the continued survival of the parasite To solve this problem, Plasmodium

has evolved the unique ability to detoxify heme in the food vacuole by formatting it into a chemically inert, crystalline form called hemozoin The mechanism of

hemozoin formation is still widely debated Only ferric heme [Fe(III)PPIX], not ferrous heme, forms hemozoin Hemozoin is a repeating array of coordinated dimers, with the ferric iron of each heme moiety chelated onto the carboxyl side chain of its partner The dimers are held together within a crystalline matrix by hydrogen bonds 15There is uncertainty as to whether the process is catalyzed by an enzyme (a heme polymerase) or otherwise The case for an enzyme-catalyzed process is equivocal

Hemozoin formation activities have been detected in extracts of P falciparum 16 and P

berghei 17 but these extracts appear to retain formatting activity even on heating,

indicating that they are not protein in nature On the other hand, a heat labile form of heme polymerase has been reported to coexist with a heat stimulable form of the enzyme 17

Histidine-rich proteins (HRP) have been implicated as initiators of the

hemozoin formation process 18 Two of the three known histidine rich proteins (HRPII,

HRPIII) in P falciparum bind to heme and promote hemozoin formation in vitro

Their role appears to be that of initiating the formation process rather than being involved in the extension of the dimer In another report, HRPII was noted to bind specifically to Fe (III) PPIX molecules, forming bis-histidyl complexes, with the formation occurring only after the hematin binding sites on the protein are saturated 19However, contradictory observations that a laboratory plasmodial clone (3B-D5) lacking HRPII and HRPIII grows poorly but is still capable of formatting heme, has cast some doubt on the role of the HRPs in hemozoin formation 18

The heme detoxification pathway is a unique plasmodial process with no human equivalent It is thus a highly attractive chemotherapeutic target There is widespread agreement that chloroquine (CQ) exerts its antimalarial activity by

interfering with hemozoin formation Theories on the formation of hemozoin and how quinoline antimalarials like chloroquine interfere with this process have been

reviewed 131CQ blocks hemozoin formation by binding to the µ-oxo dimer form of ferric heme, displacing the monomeric heme ⇔ heme dimer equilibrium to the left, and thus preventing formation 20 It may also interfere with hemozoin chain extension

21 It has been suggested that CQ is “chemiabsorbed” onto β-hematin (a synthetic equivalent of hemozoin), thereby inhibiting sequestration of hematin molecules

(hematin = aqua or hydroxyferriprotoporphyrin IX) and thus causing a buildup of toxic CQ-hematin complexes in the cell 21

N Cl

HN

NEt2

chloroquine (CQ)

Several investigative antiplasmodial compounds in the recent literature have been reported to exhibit heme binding, interfere with heme binding to HRPII or inhibit

β-hematin (synthetic equivalent of hemozoin) formation These in vitro observations

suggest that interference with the heme detoxification pathway may contribute

significantly to the antimalarial activity of these compounds, which includes

monoquinolines, 22 diamidines, 23 acridine derivatives, 24 phenothiazine analogues, 25hydroxyxanthones, 26 terpene isonitriles isolated from sponges, 27 and indoloquinoline alkaloids (cryptolepine, neocryptolepine and related compounds) 28

The food vacuole is also the site of free radical formation, arising in part from the release of electrons following the oxidation of heme iron from Fe2+ to Fe3+ during its degradation The electrons react with molecular oxygen to form reactive oxygen species (superoxide, hydroxyl free radical, hydrogen peroxide) that exert considerable oxidative stress on the parasite Despite this intensive free radical assault, plasmodium lacks a complete armory of oxidant defense enzymes For example, it does not possess

a classical catalase or gluthione peroxidase, and adopts most of its oxidant defense enzymes from the host However, a recent review highlights the important role of glutathione metabolism in the parasite 132 Glutathione helps to maintain a reducing environment in the plasmodial cytosol It participates in the detoxification of heme and scavenges free radicals generated in the parasite In addition, it participates in several key plasmodial metabolic processes, such as the biosynthesis of

deoxyribonucleotides and the detoxification of byproducts of glycolysis Not

surprisingly, methylene blue which inhibits plasmodial glutathione reductase and would affect the availability of glutathione, has been found to possess antiplasmodial properties Factors that enhance oxidative stress and/or reduce the efficiency of the

parasite’s oxidant defense systems would theoretically be legitimate chemotherapeutic targets

It has been widely held that artemisinin and trioxanes exert antimalarial activity

by increasing oxidative stress on the parasite 5 The peroxide bond in artemisinin is crucial to its antimalarial activity This linkage, which is a reactive oxygen species in its own right, can be cleaved homolytically by Fe2+ in heme or from non-heme sources

29 to generate oxygen-centered radicals The latter abstracts hydrogen atoms

intra-molecularly to form carbon-centered radicals, that presumably alkylates critical

plasmodial biomolecules leading to loss of function and death of the parasite The peroxide bond can also be cleaved to yield a hydroperoxide, which is in turn converted

to peroxy or hydroxyl radicals, both of which are highly reactive and can cause radical autoxidation and hydroxylation of biomolecules 5

However, a recent report proposes that artemisinin acts on a specific target, postulated to be PfATP6, a plasmodial enzyme responsible for pumping calcium ions into membrane organelles 133 On hindsight, the irreversible inhibition of this enzyme

by artemisinin may be anticipated since it shares a similar sesquiterpene backbone as thapsigargin, a known inhibitor of microsomal calcium ATPase As in the case of its general oxidant activity, iron has a role in converting artemisinin into an “activated” species for inhibition

O O

H O O

O

H H

CH3

CH3

H3C

artemisinin

In connection with drugs that enhance oxidative stress in the food vacuole, mention must be made of the well-known synergy between the antimalarial agents rufigallol and exifone 30 Often described as the “xanthone hypothesis”, the significant increase in antimalarial activity of the rufigallol-exifone combination has been

attributed to the production of free radicals from the redox cycling of rufigallol in the food vacuole These reactive oxygen species attack exifone, converting it to a

hydroxyxanthone, which is postulated to be the final antimalarial agent

Scheme 1.1 The “xanthone hypothesis”

OH HO

HO

O

OH OH

OH OH

- H2O

In keeping with this hypothesis, several xanthone derivatives have been shown

to exert antimalarial activity 26 Their mode of action appears to be linked to their ability to inhibit hemozoin formation

1.2.2 Targets involved in the synthesis of macromolecules and metabolites

The metabolism of carbohydrates, nucleic acids and phospholipids in the plasmodia differ in many ways from the corresponding pathways in the host These have been reviewed extensively in the literature 3, 31 Some of these differences have

already been exploited in chemotherapy and appropriate examples are described in the following paragraphs

As Plasmodium cannot incorporate preformed pyrimidine nucleosides, it must

rely on de novo synthesis from simpler precursors such as carbon dioxide, glutamine

and aspartic acid for its supply of pyrimidines This is in contrast to the mammalian

host, which can obtain pyrimidines from de novo synthesis as well as salvage

pathways The plasmodial enzymes [dihydroorotate dehydrogenase (DHODase), orotate phosphoribosyltransferase, ortotidine-5’-phosphate decarboxylase] involved in

de novo pyrimidine synthesis differ significantly from the host enzymes and are

potential drug targets In this context, the catalytic activity of DHODase has received much attention This enzyme catalyzes the only redox reaction in the pyrimidine synthetic pathway (dihydroorotate Æ orotate) As a major source of electrons for the mitochondrial electron transport chain of the parasite, it bridges pyrimidine synthesis and the mitochondrial electron transport system DHODase and the electron transport chain is the target of the antimalarial agent atovaquone Atovaquone, a

naphthoquinone derivative, interferes with electron transfer at the level of the

cytochrome C reductase complex, thus causing the collapse of the mitochondrial membrane potential 32, 33 The activity of DHODase is also affected because of its dependence on a functional mitochondrial electron transport chain As DHODase is a key enzyme in pyrimidine biosynthesis, its inhibition by atovaquone disrupts

plasmodial DNA synthesis and replication

Tetrahydrofolate is a key coenzyme in amino acid and nucleotide metabolism

In P falciparum, it can be synthesized de novo or acquired via a salvage pathway The folate pathway is linked to both purine (via GTP) and pyrimidine (via dTMP)

pathways In the plasmodia, dihydrofolate reductase (DHFR) and thymidylate

synthetase (TS) exists as a bifunctional enzyme that catalyzes sequential reactions in the thymidylate cycle Its inhibition prevents dTMP production and DNA synthesis

It is the target of antifolate antimalarial drugs like pyrimethamine and proguanil, but the efficiency of this class of antimalarials has been compromised by rapid resistance caused by mutations in the enzyme that prevent drug binding but still retain enzyme activity A recent report 34 on the crystal structures of PfDHFR-TS from wild type and resistant mutants in complex with pyrimethamine or the antifolate WR99210 revealed features that could be exploited to overcome drug resistance Notably, this study proposes that new inhibitors targeting parts of the enzyme other than the active site may be feasible A potential site would be the interdomain sections of the enzyme, which could result in selective inhibition of malarial dTMP synthesis

Targeting membrane biogenesis in Plasmodium has been found to be highly successful in disrupting the parasite’s life cycle One strategy focuses on the inhibition

of choline uptake into the infected erythrocyte 35 Choline is a precursor of

phosphatidylcholines, a major lipid accounting for more than half of the total

phospholipid content in plasmodial membranes Its importance is related to the fact that the rapidly growing intraerythrocytic parasites must undertake considerable

synthesis of phospholipids for their many membrane-limited barriers and organelles Factors that interfere with choline availability to infected cells would understandably undermine normal parasite growth The increased uptake of choline into infected erythrocytes is achieved (possibly) by the hyperfunctioning of the native choline

carrier present in the normal erythrocytes 36 Compounds that mimic choline and competitively bind to the choline carrier would interfere with its transport into infected cells To date, three “generations” of choline analogues have been evaluated The 1stgeneration compounds are mono and bisquaternary ammonium salts 37 Structure activity studies reveal the importance of the lipophilicity of the nitrogen substituents, the increased activity of bis-ammonium salts over mono-ammonium salts and the existence of an optimal chain length between the terminal groups The 2nd generation compounds were designed to overcome the poor bioavailability of the quaternary ammonium salts 35 This was achieved in part by substituting the quaternary

ammonium moiety by an amidine or guanidine function The 3rd generation

compounds continue to focus on the issue of poor bioavailability 35 Neutral ionic) disulfide and thioester prodrugs that are transformed to quaternary ammonium

(non-salts in vivo have been designed and promising results have been reported

Mention has been made of the plasmodial plastid organelle (“apicoplast”) as a novel target for drug discovery 38 The apicoplast is an unusual self-replicating

organelle that has its own maternally inherited DNA Replication of the apicoplast is

essential for the survival of the Plasmodium although the functions associated with this

organelle remains unclear The apicoplast is associated with enzymes that are found in plant and bacterial metabolic pathways The absence of enzymes associated with mammalian metabolic pathways suggests that the apicoplast has considerable potential

as an antimalarial drug target The fact that the apicoplast DNA encodes like proteins associated with transcription and translation may explain the

prokaryotic-susceptibility of the parasite to antibiotics such as tetracyclines, which interfere with bacterial protein synthesis The existence of another apicoplast metabolic pathway in

Plasmodium – the mevalonate-independent pathway of isoprenoid synthesis –has led

to the proposal that enzymes in this nonmevalonate pathway could be susceptible to selective inhibition 39 The antimalarial activity of the herbicide fosmidomycin lends support to this hypothesis Fosmidomycin inhibits 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an enzyme in the non-mevalonate pathway of isoprenoid biosynthesis 39 Isoprenoids are used as substrates in diverse pathways and functions (such as biosyntheses of carotenoids and terpenoids; prenylation of membrane bound proteins) However, like other agents that target a single enzyme (pyrimethamine, atovaquone), greater benefit may be obtained by using fosmidomycin in combination therapy than as a single agent 40

Cyclin dependent kinases (CDKs) play a pivotal role in controlling progression through the cell cycle and several CDK inhibitors have been developed for anticancer therapies 41 The potential of plasmodial CDKs as potential antimalarial targets have also been explored 42 CDKs are highly conserved among eukaryotic species and significant differences between human and plasmodial CDKs are necessary if specific inhibitors can be developed Fortunately, comparisons of the active sites of human and

parasite enzymes suggest that such differences do exist Xiao et al 43 reported the 1stcompound (a phenylquinolinone) to inhibit the plasmodial CDK (Pfmrk) at the

micromolar range (IC50 18 µM) The in vitro antimalarial activity of this compound has not been reported in the literature

Another target of cancer chemotherapy that has been exploited for antimalarial chemotherapy are the enzymes involved in protein prenylation, in particular farnesyl transferase (FTase) 44 FTase is responsible for activating Ras, a family of G proteins that regulates signal transduction pathways controlling cell growth and differentiation The inhibition of FTase will block the transfer of a farnesyl group from farnesyl

pyrophosphate to the thiol group of a cysteine residue near the C-terminal of Ras

When Ras is not farnesylated, it cannot mediate cell growth Thus, inhibition of FTase

is an attractive approach in halting cell proliferation Protein farnesylation has been demonstrated in several protozoa, including plasmodia A plasmodial FTase has been identified 44 and peptidomimetics based on the CaaX tetrapeptide motif have been synthesized 45 This is based on the observation that the key C-terminal sequence recognized by FTase is C-aa-X representing cysteine (c), a dipeptide sequence (a-a) and methione/glutamine or serine (X)

1.2.3 Targets involved in membrane processes and signaling

Since transport processes are a sine qua non for metabolism, interference with

such processes will invariably have far-reaching effects on the parasite’s physiology Marked changes are observed in the basic membrane transport properties of the

Plasmodium infected erythrocyte, namely (i) there is an increased flux via transport

pathways with properties similar to those present in the uninfected erythrocyte

membrane For example, choline is taken up by infected erythrocytes via a pathway with a similar Km but different V max from that of normal cells; 36, 46 (ii) The

intraerythrocytic parasite induces new transport pathways in the erythrocyte membrane which have different functional characteristics from that of normal host transporters and confers on the infected erythrocyte enhanced permeability to a wide range of solutes 47, 48 For example, endogenous pathways are incapable of handling the

increased amount of lactate produced by the parasite’s glycolytic activity This is fulfilled by a new permeability pathway (NPP), which is highly permeable to lactate and mediates its efflux from the infected cell 49, 50

Membrane transport pathways hold considerable potential as chemotherapeutic targets Besides being blocked by “inhibitors”, these pathways can also serve as

routes for administering cytotoxic agents into the intracellular parasite Examples of both applications have been reviewed in the literature The flavonoid phlorizin, 51, 52 various anion transport blockers (cinnamic acid derivatives, 53 arylaminobenzoates 54), sulphonyl ureas 55 are reported to inhibit the NPPs Inhibition of the NPP will deprive the parasite of essential nutrients or disrupt critical biochemical functions, thus leading

to their demise This appears to be the case for phlorizin, whose IC50 values for NPP inhibition and in vitro antimalarial activity are well correlated, 51 but for the other listed inhibitors, binding to serum components appear to interfere with antimalarial activity

The parasite-induced transport system has been investigated as a means of delivering cytotoxic compounds into parasite-infected cells 48 Purine nucleotides have received considerable attention in this context because unlike mammalian cells,

Plasmodium lacks the ability to synthesize purines and must acquire preformed purines

(as nucleosides) from their hosts by salvage mechanisms Following infection, a new nucleoside transport mechanism is introduced into the host cell membrane 56 This parasite-induced pathway is unique in its ability to transport L nucleosides, in contrast

to normal cells that handle only D-nucleosides This has led to the design of several nucleoside prodrugs, the rationale of which is based on the fact that the L-nucleoside functions as a “carrier” to selectively deliver a 2nd compound (e.g a cytotoxic

L-nucleotide) into the infected cell 48

Transport processes are likely to be involved in the development of chloroquine

resistance Resistant strains of Plasmodium accumulate less CQ in the acidic food

vacuole, a phenomenon that has been variously explained by alterations in the

intraerythocytic parasite that affect CQ uptake or efflux at the cytoplasmic membrane, and changes in proton or CQ concentration in the digestive vacuole 57-62 More

recently, CQ resistance has been shown to result from mutations in the novel vacuolar

transporter PfCRT (P falciparum chloroquine resistant transporter) which belongs to a

novel family of putative transporters with 10 transmembrane segments 63 The

transporter is localized at the membrane of the plasmodial digestive vacuole and

appears to have a role in maintaining the acidity of the vacuole PfCRT mutations are associated with greater acidification of the food vacuole (by 0.3 - 0.5 pH units) CQ accumulation is supposedly driven to a large extent by the binding of CQ to hematin, a process that is pH dependent 62 It is proposed that mutations in PfCRT causes

increased acidification of the digestive vacuole which leads to reduced levels of

hematin available for complex formation with CQ

An online search using “chalcone” as an entry in the Science Citation Index

199 entries (1966 to May 2003) on the biological properties of chalcones There is obviously a strong interest in the scientific properties of chalcones This may be due to the following reasons Firstly, chalcones are precursors for a vast range of flavonoid derivatives found throughout the plant kingdom Flavonoids have been widely

researched 66 They possess a wide array of biological properties and are widely used in traditional Eastern medicines, and it is not unexpected that this interest has spilled over

to chalcones Secondly, the structural simplicity of chalcones is matched by the ease

of its chemical synthesis This is an appealing feature for organic and medicinal chemists seeking to synthesize libraries of derivatives and to establish structure-

activity relationships for a particular activity

Chalcones have been reported to have a wide range of activities:

antiprotozoal (including antimalarial, antileishmanial, antitrypanocidal activities), anticancer, anti-inflammatory, antioxidant properties among others In the following section, the antimalarial properties of chalcones are reviewed in detail while

summaries are provided for the other activities, with an update of the literature where appropriate

1.3.1 Antimalarial chalcones

The antimalarial property of chalcones was reported as early as 1949, but activity was not found to be particularly impressive (1/10th the activity of quinine against avian malaria) 67 Thus, they were not pursued as lead structures It was not until the 1990s when interest was rekindled following a report on the antimalarial activity of a chalcone derivative isolated from the roots of the Chinese liquorice

(Glycyrrhizae uralensis, “Gan Cao”) 68

Figure 1.2 Gan Cao

Gan Cao as a plant is a perennial herb used in traditional Chinese medicine as a sweetening agent and as a tonic to improve the immune response of the body

The isolate from the Gan Cao root (licochalcone A) was found to inhibit the in

vitro growth of chloroquine resistant and chloroquine sensitive P falciparum and

protected mice from lethal infections of P yoelii 68 However, it was also observed to inhibit phytohemmagglutinin A-induced proliferation of human lymphocytes in vitro, which are indicative of immunosuppressive effects 69 This led to the synthesis of other oxygenated chalcones, of which 2,4-dimethoxy-4’-butoxychalcone was found to be outstanding 70 This compound is comparable to licochalcone A in terms of

antimalarial activity but is significantly less toxic than licochalcone A against human leukocytes

OCH3O

viable lead compound and it was modified in the several ways as shown in Scheme

1.2

Scheme 1.2 Lead optimization of bis-hydrazide to give acyl hydrazides and chalcones

N N H

H N O

O

N N H O

X

N N H O

a 4-atom acyl hydrazide linkage to reduce conformational flexibility; (b) incorporating nitrogen atoms into the aromatic rings of the resulting acyl hydrazide to increase water solubility; (c) replacing the hydrazide linkage with an α,β-unsaturated ketone bridge, thus converting the compound to a chalcone in an attempt to enhance stability to acid hydrolysis This was considered important because cysteine proteases are found in the

acidic food vacuole of the parasite and a potential inhibitor should be stable in such an environment

Several chalcones were identified as potential antimalarial agents based on the malarial cysteine protease model, the most active compound of which was a quinolinyl chalcone (IC50 0.23 µM, P falciparum W2) 72 The quinoline ring is considered

advantageous in conferring good antimalarial activity because its basic character will enhance accumulation in the acidic food vacuole In addition, hydrogen bonding interactions between the quinolinyl nitrogen (pKa ∼ 5) and the imidazole ring (pKa ∼ 6) in His 67, a critical amino acid in the cysteine protease active site, will enhance attraction for the active site Although widely quoted in the literature as the seminal work that correlates antimalarial activity of chalcones with cysteine protease inhibitory activity, 73 a careful inspection of the report shows that the chalcones were never tested

for cysteine protease inhibitory activity in this study Rather, they showed excellent in

silico docking onto the enzyme model that would imply good inhibitory activity, if

tested in vitro Mention was made in the report of “differential inhibition specificities exhibited by these compounds against malarial cysteine protease (unpublished results) and mammalian cysteine protease cathepsin B” but no IC50 or % inhibition data were presented The connection between antimalarial and cysteine protease activities of quinolinyl chalcones was pursued in a subsequent study by Dominguez and coworkers

74 The most active compound identified in this study had no falcipain inhibitory

activity, leading the authors to conclude that antimalarial activity was not related to enzyme inhibition

More recently, ferrocenyl chalcones have been evaluated for antimalarial activity 75 This study was prompted by various reports of ferrocene-based antimalarial agents (like ferrochloroquine) in the literature 76-78 The preliminary results showed

that ferrocenyl chalcones are less active than the corresponding “conventional”

The α,β-unsaturated linkage has also received attention as a site of metabolism and possibly, toxicity of chalcones The linker is considered as a soft electrophile The electron-withdrawing carbonyl group causes the β carbon to become electron deficient, rendering it susceptible to an attack by a soft nucleophile such as thiol

groups of cysteine or glutathione

This Michael-type reaction would deplete cells of essential thiol-containing entities In fact, this reaction has been cited as the basis of the antibacterial effects of chalcones 80

Scheme 1.3 The reaction scheme for thiolation of chalcones

Given the toxic potential of the Michael reaction, considerable attention has been paid to the occurrence of this reaction in chalcones Licochalcone is reported to form thiol conjugates with glutathione and N-acetyl-L-cysteine, 81 a factor that may account in part for its toxicity against lymphocytes On the other hand, a study on the

in vitro metabolism of 4-dimethylamino-4’-(imidazol-1-yl)-chalcone by human liver

microsomes reported an N-demethylated product and the saturated analogue as minor

and major metabolites respectively (Scheme 1.4) 82 No mention was made of thiol conjugates as metabolites from this study, but then the authors may not be looking out for its formation

Chalcones with substituents on the α or β position have comparable

antimalarial and antileishmanial activities as the unsubstituted compounds 79

Considering that such substitutions are likely to hinder reaction of nucleophiles, the undiminished activities of the α/ β substituted compounds led the authors to conclude that the alkylating properties of the α,β-linkage are probably unimportant for

H2N

4-dimethylamino-4'-(imidazol-1-yl)-chalcone

amazonensis 85 Several plant-derived chalcones are also found to be antileishmanial 86

1.3.3 Immunosuppressive activity

The immunosuppressive activity of licochalcone A was noted during

investigations of its antileishmanial activity, when low concentrations of licochalcone

A were found to inhibit proliferation of phytohemmagglutinin A-stimulated

lymphocytes 69 It was subsequently shown that the structural requirements for

antileishmanial and lymphocyte-suppressing activities were different and it would be possible to design chalcones with selective activity 64 The immune suppressing

potential of chalcones is not altogether an undesirable feature Immunosuppression reduces graft- related symptoms and is beneficial for certain autoimmune diseases

Licochalcone A and some synthetic analogues have been reported to inhibit

generalized lymphocyte proliferation that was not restricted to any particular

T-lymphocyte subset 87 The same study reported the chalcones to cause

downregulation of pro- and anti-inflammatory cytokine production from monocytes as well as to interfere with the production but not release of tumor necrosis factor - α (TNF-α) The substitution pattern on the chalcone skeleton is important to this end but

no details were revealed in the report

The effect of chalcones on the production of TNF-α may be mediated via their

effects on nitric oxide (NO) production Rojas et al reported that some fluorinated

chalcones inhibited NO production, not as a result of a direct inhibitory action on enzyme (nitric oxide synthetase) activity but through the inhibition of enzyme

expression 88 NO is a potent vasodilator that facilitates leukocyte migration, cytokine production and release of inflammatory mediators like interleukin 1β and TNF α This may account in part for the anti-inflammatory properties of chalcones reported in earlier literature 89, 90

1.3.4 Anticancer properties

The anticancer properties of chalcones have been reviewed 91 Chalcones have

been reported to be selectively cytotoxic against tumor cell lines 92-94 The

α,β-unsaturated carbonyl linkage plays an important role this respect This is because this moiety (a Michael acceptor) reacts preferentially with thiols rather than “hard”

nucleophiles like amino and hydroxyl groups that are found in nucleic acids Hence they are likely to be free from problems of genotoxicity, which are encountered with other alkylating agents used in cancer chemotherapy 95