CHAPTER 1 INTRODUCTION

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CHAPTER 1 INTRODUCTION

1.1 GENERAL BACKGROUND

In 1817, James Parkinson wrote a monograph “Essay on the Shaking Palsy,” in which he described six patients who had involuntary tremulous movements, whose muscles were weak, and who struggled to work even when supported. These patients bent their trunk forward, and had the tendency to go from a walking to a running pace. He hoped that this description would persuade nosologists that he had described an unrecognized disorder, which it did, as Jean Martin Charcot, the father of neurology, proposed that the syndrome should be called maladie de Parkinson, known today as Parkinson’s disease (Lees et al., 2009).

Parkinson’s disease is the second most common age-related neurodegenerative disease after Alzheimer’s disease. The incidence of the disease rises steeply with age, for example from 17.4 in 100 000 (between 50-59 years) to 93.1 in 100 000 (between 70-79 years), and there is a lifetime risk of 1.5% for developing the disease. The median age of onset is 60 years and the mean duration of the disease is 15 years (from diagnosis to death). Men are 1.5 times more likely to develop Parkinson’s disease when compared to women, but this may vary between different populations and countries (Lees et al., 2009, Katzenschlager et al., 2008, Twelves et al., 2003, Bower et al., 1999, de Rijk et al., 1995,).

The central pathological feature of Parkinson’s disease is the loss of neurons in the substantia nigra pars compacta (SNpc). These neurons form part of the nigrostriatal dopaminergic pathway (Lees et al., 2009, Dauer & Przedborski, 2003).

Currently there is uncertainty about the etiology and pathogenesis of Parkinson’s disease, but it seems that age, smoking, drinking coffee, environmental conditions, the use of medication, as well as genetic makeup may influence the potential for developing the disease (Lees et al., 2009, Wells et al., 2009, Elbaz & Tranchant, 2007, Ascherio et al., 2004, Dauer & Przedborski, 2003, Quik & Jeyarasasingam, 2000).

Regarding the pathogenesis, Dauer & Przedborski (2003), have two hypotheses. One hypothesis is that the misfolding and aggregation of proteins are instrumental in the death of SNpc dopaminergic neurons. The second proposes mitochondrial dysfunction and that the consequent oxidative stress (due inter alia to toxic oxidized dopamine (DA) species), is responsible for neuronal death. However, neurodegeneration can also be a result of apoptosis, neuroinflammation, a loss of trophic factors and excitotoxicity (Yacoubian & Standaert, 2009, Tansey et al., 2007, Dauer & Przedborski, 2003, Dawson & Dawson, 1996, Appel, 1981).

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Levodopa is currently the most effective drug for the treatment of Parkinson’s disease (Verhagen, 2002). Unfortunately, the development of side effects after prolonged use is almost inevitable, and is often just as disabling as the disease itself. These include involuntary movements termed “dyskinesia” as well as a loss of efficacy. Other drugs on the market include the peripheral dopa decarboxylase inhibitors, used with levodopa to reduce its peripheral metabolism, COMT inhibitors, DA agonists, anticholinergic drugs, and monoamine oxidase type B inhibitors ( Nyholm

et al., 2013, Brunton et al., 2011, Abbot, 2010, Aminoff, 2009,).

Presently, there is still no cure for Parkinson’s disease and all treatments are only symptomatic. Current research is therefore directed towards the prevention of further dopaminergic neuron degeneration, while the ultimate aim is the reversal of neurodegeneration (Dauer & Przedborski, 2003).

1.2 MONOAMINE OXIDASE AND MONOAMINE OXIDASE INHIBITORS

Monoamine oxidase (MAO) is an enzyme that oxidizes the endogenous amines DA, noradrenaline, adrenaline, 5-hydroxytryptamine and tyramine in most species (Youdim & Bakhle, 2006).

MAO exists as two isoforms, namely monoamine oxidase type A (MAO-A) and monoamine oxidase type B (MAO-B). These isoforms differ in pH optima, sensitivity, substrate and inhibitor specificity, as well as their distribution in the body. For example, MAO-B has greater activity in the basal ganglia (Collins et al., 1970).

MAO-B in the microvessels of the blood brain barrier (BBB) may act as a barrier, indicating a protective function. It is assumed that, in the peripheral- and central nervous system, intraneuronal MAO-B protects neurons from exogenous amines, terminates the actions of neurotransmitters and regulates the contents of intracellular amine stores. MAO-B occurs in astrocytes which metabolize extracellular DA in the brain, inhibition of MAO-B therefore increases the DA available for binding and consequently relieves the symptoms of Parkinson’s disease (Youdim et al., 2006).

MAO inhibitors also have neuroprotective potential and thus may slow down, halt and even reverse neurodegeneration in Parkinson’s disease (Youdim & Bakhle, 2006, Youdim et al., 2006). It is still unclear exactly how MAO inhibitors protect neurons. One theory suggests that MAO inhibition decreases oxidative stress by reducing the formation of hydrogen peroxide, a metabolic by-product of MAO oxidation of monoamines. Normally, hydrogen peroxide is inactivated by glutathione (GSH). In Parkinson’s disease, GSH levels are low resulting in the accumulation of hydrogen peroxide, which then becomes available for the Fenton reaction. In the Fenton reaction, Fe2+ _reacts with hydrogen peroxide and generates an active free radical, the hydroxyl radical. This radical depletes cellular anti-oxidants, damage lipids, proteins and deoxyribonucleic acid (DNA). MAO inhibitors reduces the formation of hydrogen peroxide, thus decreasing the formation of hydroxyl radicals and oxidative stress (Youdim & Bakhle, 2006).

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Although MAO inhibitors have potential in the treatment of Parkinson’s disease, care should be taken as non-selective and irreversible MAO-A inhibitors are associated with the cheese reaction: The cheese reaction is induced by tyramine, which is present in some foods such as certain cheeses, beer and wine. Normally tyramine is metabolized by MAO-A in the gut wall and liver and is thus prevented from entering the systemic circulation. When an irreversible MAO-A inhibitor is used, tyramine and other monoamines are not metabolized and enter the circulation. The tyramine is taken up by adrenergic neurons in the ventrolateral medulla, (MAO-A is also inhibited in the medulla), initiating the release of noradrenaline from the neurons. The release of noradrenaline stimulates the cardiovascular sympathic nervous system, which leads to hypertensive crises and even death (figure 1.1) (Youdim & Bakhle, 2006, Youdim et al., 2006).

Thus, the focus of current research has changed to developing reversible and selective inhibitors of MAO-A and MAO-B to treat depression and Parkinson’s disease, respectively (Youdim & Bakhle, 2006, Youdim et al., 2006)

Figure 1.1: The cheese reaction (Youdim et al., 2006).

Tyramine metabolism by MAO-A (80%) and MAO-B (20%) in the small intestine Tyramine metabolism by MAO-A (50%) and MAO-B (50%) in the liver Ventrolateral medullary adrenergic neuron Synaptic vesicle Amine transporter L-DOPA Dopamine Noradrenaline Adrenergic receptors MAO-A _COMT Tyramine metabolism by endothelial MAO-A Increased tyramine in blood circulation MAO-A MAO-A Noradrenaline reuptake Tyramine in gut Tyrosine MAO-A inhibitors

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1.3 RATIONALE

Chalcones are the precursors of all known flavonoids and they are abundant in edible plants (Iwashina, 2000). They are open-chain flavonoids, where the two aromatic rings are joined by a three carbon α, β- unsaturated carbonyl system e.g. as in 1,3-diphenylpropenone (figure 1.2 compound 1). Chalcones have different biological activities ranging from anticancer, anti-inflammatory, and antimalarial to antiviral activities (Chimenti et al., 2009, Trivedi et al., 2007, Dimmock et al., 1999)

The MAO inhibitory potential of both natural and synthetic chalcones has been illustrated. In 1987 for example, Tanaka et al. (1987), isolated isoliquiritigenin (2), from the roots of Glycyrrhiza uralensis (the chinese licorice plant) and also synthesized a few chalcone derivatives which were screened for MAO inhibitory activity using rat mitochondria. While an IC50 (50% inhibitory concentration) value of 17.3 µM (which is quite weak) was determined for the natural chalcone 2, the synthetic derivative 3 showed improved inhibitory activity with an IC50 value of 1.65 µM.

O

A

B

Compound 1

HO

OH

O

OH

A

B

Isoliquiritigenin (2)

HO

OH

O

A

B

Compound 3

O

HO

OH

Structure-activity relationships indicated that the presence of either a 4- or 4'-hydroxy group and the introduction of another hydroxyl group on position 2' are important for MAO inhibitory activity.

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Kinetic studies were carried out with both isoliquiritigenin (2) and compound 3, and the results indicated that they were both competitive inhibitors of rat mitochondrial MAO. Although these chalcones act as competitive inhibitors, the reversibility of inhibition was not studied in detail, and no distinction was made between MAO-A and MAO-B inhibition. Other natural chalcones (4 and 5) isolated from licorice also showed weak MAO inhibitory activity in bovine plasma (Hatano et al., 1991).

In 2000, Pan and co-workers also isolated isoliquiritigenin (2) from the medicinal plant Sinofranchetia chinensis, and evaluated this compound as an inhibitor of both rat MAO-A and B. It was shown to be more selective for the MAO-A isoform, with an IC50 value of 13.9 µM for the inhibition of MAO-A and an IC50 value of 47.2 µM for the inhibition of MAO-B. It was also shown that isoliquiritigenin was a non-competitive inhibitor of MAO-A using serotonin (5-HT) as substrate, while a mixed type of inhibition was shown for MAO-B, with [14_{C] β-phenylethylamine (β-PEA) as} substrate.

OH

O

Figure 1.3: Compounds 6 and 7 (Haraguchi et al., 2004).

In 2004, Haraguchi and co-workers isolated the dimeric chalcone 6 from the bark of Gentiana lutea, and evaluated it and its hydrolysis product, compound 7 for MAO inhibitory activity using rat brain mitochondria (figure 1.3). In this instance the chalcones were more selective for the MAO-B isoform with IC50 values for the inhibition of MAO-B IC50 of 48.7 µM and 6.2 µM, respectively. Chalcones 6 and 7 inhibited MAO-A with IC50 values of >100 µM and 12.5 µM, respectively. Kinetic studies were carried out, and it was found that these chalcones exhibited a competitive mode of inhibition.

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H

₃

CO

O

Cl

OH

Compound 8

Figure 1.4: Compound 8 (Chimenti et al., 2009).

In 2009, Chimenti and co-workers synthesized a series of chalcones and examined these compounds as MAO inhibitors using human MAO-A and –B. All the compounds in this series were selective for the MAO-B isoform with IC50 values in the micro- and nanomolar range. The most active compound, 8 (figure 1.4), inhibited MAO-B with an IC50 of 0.0044 µM. This compound was 11364-fold more selective for the MAO-B than the MAO-A isoform. An irreversible mode of binding to MAO-B was determined for selected compounds. In the present study, the usefulness of the chalcone scaffold for the design of inhibitors of MAO-B will be further examined.

As the monoamine inhibitory potential of heterocyclic substituted chalcone derivatives have not been previously explored, Robinson et al. (2013), synthesized a series of furanochalcones. Compound 9a is an example of such a furanochalcone.

Cl

O

Cl

Compound 9a

Figure 1.5: Compound 9a (Robinson et al., 2013).

For these synthesized furanochalcones, the MAO inhibitory activities, modes of binding and reversibilities of inhibition were investigated. The furanochalcones exhibited selective inhibition of the MAO-B isoform. The most active furanochalcone, compound 9a (figure 1.5), inhibited MAO-B with an IC50 value of 0.147 µM, and MAO-A with an IC50 value of 28.6 µM. Further investigation indicated that this compound was a reversible inhibitor with a competitive mode of inhibition. In summary, both natural and synthetic chalcones have displayed potential as MAO inhibitors. Most inhibition data were obtained using MAO from animal sources, however studies have indicated that the MAO inhibitory specificities differ between species. Thus, human and rat MAO

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do not necessarily exhibit the same inhibitor specificities. Extrapolation of conclusions obtained from differing species should therefore be done with caution (Novaroli et al., 2006, Hubalek et al., 2005, Krueger et al., 1995).

The effect of heterocyclic substitution, other than furan on the MAO inhibitory properties of the chalcone scaffold remains unexplored, and it is in this regard that this study will make a contribution. This study will also determine the reversibility of MAO inhibition by heterocyclic substituted chalcone derivatives.

1.4 THE HYPOTHESIS OF THIS STUDY

As discussed above, the MAO inhibitory activity of the chalcone scaffold has been validated. The effect of heterocyclic substitution of the chalcone moiety has only been determined for furanochalcones. It is thus hypothesized that substitution of 1-phenyl-propen-1-ones with other heteroaromatic groups, such as thiophenes, pyrroles and pyridines will result in selective, reversible inhibitors of MAO, with possible application in the treatment of Parkinson’s disease.

1.5 AIM AND OBJECTIVES

R

1

R

2

O

Figure 1.6: The chalcone structure: R1_{represents a heteroaromatic/aromatic group, R}2_{represents a} heteroaromatic/aromatic group.

The aim of this study is to design, synthesize, and evaluate heterocyclic chalcone analogues (figure 1.6) as inhibitors of MAO. The effect of heteroaromatic substitution with groups such as pyrrole, methylthiophene, 5-chlorothiophene and methoxypyridine on MAO inhibitory activity will be investigated. This series will be compared to the previously synthesized furanochalcones as well as to 1,3-diphenyl-2-propen-1-ones. Substitution on the phenyl ring will also be varied to investigate the effect of the different substituents and substitution patterns. The compounds that will be investigated are indicated in table 1.1 below.

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H

₃

C

Cl

10c

S

_Cl

O

F

10i

O

H

3

C

OH

Cl

8

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Ar

1

O

CH

₃

_H

O

Ar

2

Ar

1

Ar

2

O

+

a

KETONE ALDEHYDE CHALCONE

Scheme 1.1: General synthetic route for chalcone synthesis. Reactants and conditions: (a) 99%

EtOH, 40% NaOH, room temperature, 2 hours. Ar1_{= aromatic ring 1, Ar}2_{= aromatic ring 2.}

The objectives of this study are as follows:

a) To synthesize heteroaromatic substituted chalcones from commercially available aldehydes and ketones using a Claisen-Schmidt condensation reaction (scheme 1.1). (2E)-3-(4-chlorophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one (8), a chalcone with known MAO inhibitory properties, will also be synthesized for comparative purposes. b) To evaluate the synthesized chalcones as inhibitors of recombinant human MAO-A and

MAO-B. A fluorometric assay with kynuramine as substrate will be used.

c) To determine reversibility of binding to the MAO enzymes for selected compounds. d) To determine the mode of binding, by constructing a set of Lineweaver Burk plots for

selected compounds.

e) To assess the toxicity of selected compounds to cultured cells.

f) To determine possible binding orientations in the MAO-B active site, chalcone derivatives will be docked into active site models of the MAO-B using Discovery Studio 3.1 (Accelrys). Possible reasons for high or low inhibitory activity will be assessed.