Synthesis and evaluation of 1-indanone derivatives as inhibitors of monoamine oxidase

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1

Synthesis and evaluation of 1-indanone

derivatives as inhibitors of monoamine

oxidase

MS Nel

22683178

Dissertation submitted in fulfilment of the requirements for the

degree

Magister Scientiae

in

Pharmaceutical Chemistry

at the

Potchefstroom Campus of the North-West University

Supervisor:

Dr LJ Legoabe

Co-Supervisor:

Prof A Petzer

Co-Supervisor:

Prof JP Petzer

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This work is based on the research supported in part by the Medical Research Council and National Research Foundation of South Africa (Grant specific unique reference numbers (UID) 85642, 96180, 96135). The Grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by the NRF supported research are that of the authors, and that the NRF accepts no liability whatsoever in this regard.

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Preface

This dissertation is submitted in article format consisting of two articles. Both research articles presented in this dissertation were compiled for submission to Bioorganic & Medicinal Chemistry. The author guidelines have been included (see Appendix A, p. 147). All scientific research (synthesis, biology and documentation of the dissertation and articles) for the purpose of this dissertation was conducted by Miss M.S. Nel at the North-West University, Potchefstroom campus.

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Declaration

This dissertation is submitted in fulfilment of the requirements for the degree Magister Scientiae in Pharmaceutical Chemistry, at the School of Pharmacy, North-West University.

I, Magdalena Salomina Nel, hereby declare that the dissertation with the title: Synthesis

and evaluation of 1-indanone derivatives as inhibitors of monoamine oxidase is my

own work and has not been submitted at any other university either whole or in part.

_______________________ MS Nel

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Acknowledgements

I would like to thank the following people and express my gratitude to all who helped with the completion of this study:

 Dr. L.J. Legoabe for all his guidance, knowledge and patience.

 Prof. A. Petzer, for her help and advice with the MAO assays and for all her advice during the study.

 Prof. J.P. Petzer for all his help and intelligent insight.

 My parents, Cassie and Estie Nel, for their unconditional love and support throughout my life. Thank you for giving me the opportunity to pursue my dreams and for always believing in me. I will always be grateful for all that you have done for me.

 Wico du Toit, for all his love and support.

I would also like to thank the following institutions for their assistance during the study:

 North-West University for the financial support and granting me the opportunity to study at this institution.

 André Joubert and Johan Jordaan at the SASOL Centre for Chemistry, for recording NMR and MS spectra.

 Prof. Jan du Preez for assistance with HPLC analyses.

 The National Research Foundation of South Africa for financial support.

“But the Lord stood with me and gave me strength”

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Abstract

Parkinson‘s disease (PD) is a neurodegenerative disorder characterised by the death of neurons in the substantia nigra pars compacta (SNpc) in the midbrain leading to a dopamine (DA) deficiency. This deficiency is responsible for the motor symptoms in PD. One of the treatment strategies in PD is to conserve DA by inhibiting the enzyme responsible for DA catabolism. The monoamine oxidase B (MAO B) isoform catalyses the oxidation of DA in the central nervous system (CNS) and is therefore an important target for the treatment of PD. Inhibition of MAO B increases endogenous DA levels thus providing PD patients with symptomatic relief. It also enhances the levels of DA after administration of levodopa (L-dopa), the metabolic precursor of DA.

In the present study, a series of benzylidene-1-indanone derivatives and a series of 2-heteroarylidene-1-indanone derivatives were synthesised and evaluated as inhibitors of recombinant human MAO A and MAO B. These indanone derivatives are structurally related to a series of benzylidene-1-indanone derivatives, which has been reported to act as MAO B inhibitors. The 2-benzylidene-1-indanone derivatives and 2-heteroarylidene-1-indanone derivatives were successfully synthesised by reacting 1-indanone with the appropriate benzaldehyde or heteroaromatic aldehyde in either acidic (HCl) or basic (KOH or NaOH) conditions. The structures of the compounds were verified with NMR and MS analyses, while the purities were estimated by HPLC. Twenty-two 2-benzylidene-1-indanone derivatives and fifteen 2-heteroarylidene-1-indanone derivatives were synthesised.

To determine the inhibition potencies of the synthesised compounds the recombinant human MAO A and MAO B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the 2-benzylidene-1-indanone

derivatives and 2-heteroarylidene-1-indanone derivatives are highly potent and selective MAO B inhibitors and to a lesser extent inhibitors of MAO A. The most potent MAO B inhibitor was (E)-5-methoxy-2-(5-bromofuran-2-yl)methylene-2,3-dihydro-1H-inden-1-one with an IC50 value of 0.0044 µM. The results indicated that both series are potent MAO

inhibitors with most derivatives possessing higher selectivity towards the MAO B isoform compared to MAO A. In general, the 2-benzylidene-1-indanone derivatives were more potent MAO B inhibitors than the 2-heteroarylidene-1-indanone derivatives.

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ii It may thus be concluded the 2-benzylidene-1-indanone and 2-heteroarylidene-1-indanone derivatives are promising potent and selective MAO B inhibitors, and thus leads for future development of therapy for PD.

Keywords: Parkinson‘s disease; Monoamine oxidase; 2-Benzylidene-1-indanone; 2-Heteroarylidene-1-indanone; Selectivity

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iii

Uittreksel

Parkinson se siekte (PS) is ŉ neurodegeneratiewe siektetoestand wat gekenmerk word deur die afsterf van neurone in die substantia nigra pars compacta (SNpc) in die midbrein en die gevolglike tekort aan dopamien (DA) in die striatum. Hierdie tekort aan DA is verantwoordelik vir die simptome van PS. ŉ Belangrike behandelingstrategie vir PS is om die katabolisme van DA in die brein te inhibeer. Monoamienoksidase B (MAO B) is ŉ ensiem wat verantwoordelik is vir die oksidasie van DA in die sentrale senuweestelsel (SSS) en is dus ŉ belangrike teiken vir die behandeling van PS. Inhibisie van MAO B verhoog endogene DA-vlakke en lei tot simptomatiese verligting vir pasiënte met PS. Die inhibisie van MAO B verhoog ook DA-vlakke na behandeling met levodopa (L-dopa), die metaboliese voorganger van DA.

In hierdie studie is ŉ reeks bensielideen-1-indanoon derivate en ŉ reeks 2-heteroarielideen-1-indanoon derivate gesintetiseer en geëvalueer as inhibeerders van rekombinante menslike MAO A en MAO B. Hierdie indanoon derivate is struktureel verwant aan ŉ reeks bensielideen-1-indanoon derivate wat in vorige studies as MAO B inhibeerders geïdentifiseer is. Die 2-bensielideen-1-indanoon derivate en 2-heteroarielideen-1-indanoon derivate is suksesvol gesintetiseer deur 1-indanoon met ŉ geskikte bensaldehied of heteroaromatiese bensaldehied te reageer in suur (HCl) of basisomgewing (KOH of NaOH). Die chemiese strukture van die derivate is deur KMR en MS bevestig, terwyl die suiwerhede deur hoëdrukvloeistofchromotografie bepaal is. Twee-en-twintig 2-bensielideen-1-indanoon derivate en vyftien 2-heteroarielideen-1-indanoon derivate is gesintetiseer.

Die potensie waarmee die gesintetiseerde verbindings MAO A en MAO B inhibeer is bepaal deur van die rekombinante mens ensieme gebruik te maak. Die potensie van inhibisie is uitgedruk as IC50 waardes. Die resultate toon dat die 2-bensielideen-1-indanoon derivate en

2-heteroarielideen-1-indanoon derivate potente en selektiewe MAO B inhibeerders is. Sommige derivate het ook as MAO A inhibeerders opgetree. Die mees potente MAO B inhibeerder van hierdie studie is (E)-5-metoksie-2-(5-bromofuran-2-iel)metileen-2,3-dihidro-1H-indanoon met ŉ IC50 waarde van 0.0044 µM. Die resultate toon dat albei reekse potente

MAO inhibeerders is en dat meeste derivate hoër selektiwiteit vir MAO B as vir MAO A besit. Oor die algemeen was die 2-bensielideen-1-indanoon derivate egter meer potente MAO B inhibeerders as die 2-heteroarielideen-1-indanoon derivate.

Uit hierdie studie kan afgelei word dat 2-bensielideen-1-indanoon en 2-heteroarielideen-1-indanoon derivate potente en selektiewe MAO B inhibeerders is en dus kan dien as

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iv belowende leidraadverbindings vir die toekomstige ontwikkeling van geneesmiddels vir die behandeling van PS.

Sleutelwoorde: Parkinson se siekte; Monoamienoksidase; 2-Bensielideen-1-indanoon; 2-Heteroarielideen-1-2-Bensielideen-1-indanoon; Selektiwiteit

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v

List of Abbreviations

5-HT 5-Hydroxytryptamine/serotonin A α-syn Alpha-synuclein AD Alzheimer‘s disease ADH Aldehyde dehyrogenase

B

BDNF Brain-derived neurotropic factor

C

CMA Chaperone-mediated autophagy CNS Central nervous system

COMT Catechol-O-methyltransferase

Cys Cysteine

D

DA Dopamine

DOPAC 3,4-Dihydroxyphenylacetic acid

F

FAD Flavin adenine dinucleotide

G

GBA Glucocerebrosidase

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vi GPO Glutathione peroxidase

GSH Glutathione

H

HCl Hydrogen chloride H2O2 Hydrogen peroxide

HD Huntington‘s disease

HPLC High pressure liquid chromatography

K

KOH Potassium hydroxide

L

LBs Lewy bodies

L-dopa Levodopa

LN Lewy neurites

M

MAO Monoamine oxidase

MAO A Monoamine oxidase type A MAO B Monoamine oxidase type B MAPT Mitogen-activated protein kinase

MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine MS Mass spectrometry

N

NaOH Sodium hydorixide NGF Nerve growth factor

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vii NMDA N-methyl-D-aspartate

NMR Nuclear magnetic resonance

P

PD Parkinson‘s disease

R

r.m.s Root mean square

RNS Reactive nitrogen species ROS Reactive oxygen species

S

SAR Structure-activity relationship SET Single electron transfer SN Substantia nigra

SNpc Substantia nigra pars compacta

SNRIs Serotonin and noradrenaline re-uptake inhibitors SSRIs Selective serotonin re-uptake inhibitors

U

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viii

List of Figures and Tables

Chapter 1 Introduction

Table 1.1 Examples, from the literature, of chalcone derivatives with MAO activity

Table 1.2 Benzylidene-1-indanone derivatives that will be synthesised in this study

Table 1.3 2-Heteroarylidene-1-indanone derivatives that will be synthesised in this study

Chapter 2 Literature review

Figure 2.1 The structure of L-dopa

Figure 2.2 The structures of DA agonists

Figure 2.3 The structures of COMT inhibitors

Figure 2.4 The structures of MAO B inhibitors

Figure 2.5 The structures of anticholinergic drugs

Figure 2.6 The structure of amantadine

Figure 2.7 The synthesis and metabolism of DA by MAO A and MAO B

Figure 2.8 The mechanism of the cheese reaction

Figure 2.9 The structure of 5-HT

Figure 2.10 The three-dimensional structure of human MAO A

Figure 2.11 The reaction pathway of monoamine metabolism

Figure 2.12 Fenton reaction

Figure 2.13 The three-dimensional structure of human MAO B

Figure 2.14 Structure of covalent FAD in MAO

Figure 2.15 The SET mechanism of MAO catalysis

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ix

Figure 2.17 Structures of reversible MAO A inhibitors

Figure 2.18 Structure of moclobemide

Figure 2.19 Structure of clorgyline

Figure 2.20 Structure of lazabemide

Figure 2.21 Structures of non-selective MAO inhibitors

Figure 2.22 The most potent chalcone-derived MAO B inhibitor identified by Chimenti and co-workers (2009)

Figure 2.23 The most potent chalcone-derived MAO B inhibitor identified by Robinson et al. (2013)

Figure 2.24 The most potent aurone-derived MAO B inhibitor synthesised by Geldenhuys and co-workers (2012)

Chapter 3 First Article

Figure 1 The structures of isoliquiritigenin (1), chalcone 2, aurone (3) and 2-benzylidene-1-indanone (4).

Figure 2 Synthetic pathway to 2-benzylidene-1-indanone derivatives (5a–r and 6a–d).

Reagents and conditions: (a) methanol/32% HCl (1:1.5), reflux; (b) KOH, methanol, rt.

Figure 3 Sigmoidal dose-response plots for the inhibition of MAO A (open circles) and MAO B (filled circles) by 5e. The enzyme activities are given as mean ± SD.

Table 1 The IC50 values for the inhibition of recombinant human MAO A and MAO B

by 2-benzylidene-1-indanone derivatives.

Figure 4 A selected 2-benzylidene-1-indanone is a reversible MAO-A inhibitor. The test inhibitor 5g (at 4 × IC50) was preincubated with MAO-A for 15 min,

dialysed for 24 h and the residual enzyme activity was measured (5g dialysed). Similar incubation and dialysis of MAO-A in the absence inhibitor ([I] = 0, dialysed) and presence of the irreversible inhibitor, pargyline (parg,

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x dialysed), were also carried out. The residual activities of undialysed mixtures of MAO A and the test inhibitor were also recorded (5g undialysed).

Figure 5 The docked binding orientations of 2 (magenta), 5g (green) and 5j (yellow) in MAO A.

Figure 6 The docked binding orientations of 2 (magenta), 5g (green) and 5j (yellow) in MAO B.

Chapter 4 Second Article

Figure 1 The structures of known MAO inhibitors.

Figure 2 The structures of heterocyclic chalcone derivatives (8–10) that exhibit MAO

inhibition. The general structures of the 2-heteroarylidene-1-indanone derivatives (11a–o) that will be investigated in this study and that of a 2-benzylidene-1-indanone (12) are also given.

Table 1 The IC50 values for the inhibition of recombinant human MAO A and MAO B

by heteroarylidene-1-indanone derivatives.

Figure 3 The synthetic pathway to heteroarylidene-1-indanone derivatives 11a–o.

Reagents and conditions: (a) KOH, methanol, room temperature.

Figure 4 Sigmoidal plots for the inhibition of MAO A and MAO B by 11n (open circles) and 11o (filled circles). Each measurement was conducted in triplicate and is given as mean ± SD.

Figure 5 Proposed binding orientations and interactions of 11a (cyan) and 11o (yellow) in MAO A.

Figure 6 Proposed binding orientations and interactions of 11a (cyan) and 11o (yellow) in MAO B.

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xi

Chapter 5 Conclusion

Table 5.1 The synthesised 2-benzylidene-1-indanone derivatives, which were evaluated as MAO inhibitors

Table 5.2 The synthesised 2-heteroarylidene-1-indanone derivatives, which were evaluated as MAO inhibitors

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1

Chapter 1 Introduction

1.1. Introduction

Parkinson‘s disease (PD) was first described in 1817 by James Parkinson in his monograph ―Essay on the Shaking Palsy‖. In this monograph he described the core clinical features of PD (Dauer & Przedborski, 2003). He described this disorder as ―involuntary tremulous motion with lessened muscular power, in parts not in action even when supported, with a propensity to bend the trunk forward and to pass from a walking to a running pace‖ (Parkinson, 2002). PD is the second most common age-related neurodegenerative disorder, affecting 1.5% of people older than 65 and this number are expected to increase (Garbayo et al., 2013). Although PD is considered to be a sporadic disease, there are few environmental causes with age as the major known risk factor. The incidence increases steeply with age, from 17.4 in 100 000 persons between ages of 50 and 59 to 93.1 in 100 000 persons between ages of 70 and 79. The average age of onset of PD is 60 years (Lees et al., 2009). The main pathological characteristic of PD is the death of neurons in the substantia nigra pars compacta (SNpc). The SNpc neurons form the nigrostriatal dopaminergic pathway and the loss of SNpc neurons causes a dopamine (DA) deficiency in the striatum. This deficiency is the cause of the major motor symptoms of PD (Dauer & Przedborski, 2003). PD consists of four basic symptoms namely bradykinesia, rigidity, tremor at rest and postural instability (Dauer & Przedborski, 2003).

Current treatment of PD is symptomatic (Dauer & Przedborski, 2003), with the most effective therapy being levodopa (L-dopa) in combination with a peripheral dopa decarboxylase inhibitor (carpidopa, benserazide). This is recommended to be the initial treatment option (Lees et al., 2009). When using L-dopa, patients may develop motor complications which are difficult to treat and control. Many patients also develop non-motor symptoms that include anosmia, sleep disorders, autonomic impairment and cognitive impairment. The development of these symptoms is the most significant cause of disability (Yacoubian & Standaert, 2009). Monoamine oxidase (MAO) is a mitochondrial-bound flavoenzyme. The main biological role of MAO is to regulate amine levels in the brain and to metabolise amine-containing drugs in the periphery. MAO catalyses the oxidative deamination of several

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2 monoamines including 5-hydroxytryptamine (5-HT or serotonin), catecholamines (DA, noradrenaline and adrenaline) and β-phenylethylamine. The MAO catalytic reaction produces hydrogen peroxide (H2O2), a corresponding aldehyde and either ammonia or a

substituted amine as products (Bortolato et al., 2008; Edmondson et al., 2004; Ramsay, 2012; Ramsay, 2013; Youdim et al., 2006). Some of these products have the potential to be neurotoxic. For example, reactive oxygen species (ROS) can be produced from H2O2, which

may lead to mitochondrial damage and neuronal apoptosis (Bortolato et al., 2008). There are two isoenzymes of MAO, MAO A and MAO B. Both are expressed in all tissues but in different amounts. In humans, MAO A is mainly found in the intestines, placenta and heart while MAO B is mainly found in platelets, glial cells in the brain and liver (Ramsay, 2012). Patients with PD exhibit increased oxidation of DA by MAO B, which may contribute to the depletion of DA in the SNpc. MAO B inhibitors [for example (R)-deprenyl] delay the progression of symptoms by inhibiting the oxidation of DA and the associated formation of toxic species such as H2O2 (Shih et al., 1999). Currently, however, MAO B inhibitors are

considered to be useful agents in the symptomatic treatment of PD, mainly in combination with L-dopa.

As mentioned above, MAO catalyses the oxidative deamination of several monoamines forming the corresponding aldehyde, H2O2 and either ammonia or a substituted amine. Some

of these products (e,g. H2O2 and aldehydes) may contribute to neurotoxicity and

subsequently neurodegeneration in PD. As a person ages, MAO B activity in glial cells increases while MAO A activity remains constant. Therefore the inhibition of MAO B is more relevant in the treatment of PD, which mostly affects the elderly (Edmondson et al., 2004; Fowler et al., 1997). The inhibition of MAO B decreases the production of potentially neurotoxic aldehydes and H2O2, and may protect against further neurodegeneration in PD

(Lees et al., 2009).

From the above analysis it is clear that MAO B inhibitors are useful therapeutic agents for the treatment of PD because they may improve the symptoms of PD by preventing the MAO B catalysed catabolism of DA in the brain. MAO B inhibitors may also protect against neurodegeneration in PD by decreasing the formation of aldehydes and H2O2.

The MAO inhibitory potential of chalcones has been shown in previous studies. For example compound 1 (see Table 1.1) from the roots of Glycyrrhiza uralensis (also known as the Chinese licorice plant) (Tanaka et al., 1987) exhibited MAO inhibition. A few chalcone derivatives were subsequently synthesised and screened for MAO inhibitory activity using rat mitochondria. Compound 1 exhibited an IC50 value of 17.3 µM, which was improved in its

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3 synthetic derivative, compound 2 (see Table 1.1), which possesses an IC50 value of 1.65 µM

for the inhibition of MAO. With both compounds 1 and 2, kinetic studies were carried out and the results indicated that both are competitive inhibitors of rat mitochondrial MAO, but the reversibility of inhibition was not studied in detail and no distinction was made between MAO A and MAO B inhibition (Tanaka et al., 1987). Interestingly, compound 1 was also isolated from Sinofranchetia chinensis, and using rat mitochondria it was found to possess MAO inhibitory properties with IC50 values of 13.9 µM and 47.2 µM for the inhibition of MAO A and

B, respectively (Pan et al., 2000). Compound 2 is therefore more selective for the inhibition of MAO A than MAO B. A number of studies have since described the MAO inhibition properties of chalcones and heretocyclic chalcone derivatives. Examples are compounds 3 and 4 (see Table 1.1).

Table 1.1: Examples, from the literature, of chalcone derivatives with MAO activity

Structure IC50 values Reference

HO

OH O

OH

A B

Compound 1 (Isoliquiritigenin)

IC50 for non-selective MAO inhibition = 17.3 µM (Glycyrrhiza uralensis)

IC50 for MAO B inhibition = 47.2 µM (Sinofranchetia chinensis)

IC50 for MAO A inhibition = 13.9 µM (Sinofranchetia chinensis)

(Pan et al., 2000; Tanaka

et al., 1987)

HO

OH O

A B

Compound 2

IC50 for non-selective MAO inhibition = 1.65 µM (Tanaka et al., 1987) O O Cl OH A B Compound 3

IC50 for MAO B inhibition = 0.0044 µM

No inhibition observed for MAO A at 50 µM

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4 O Cl O Cl A _B Compound 4

IC50 for MAO A inhibition = 28.6 µM

(Robinson et al., 2013) O O N A C B Compound 5

(Geldenhuys et al., 2012) O O HO OH A C B Compound 6

(Geldenhuys et al., 2012) O O HO N A C B Compound 7

(Geldenhuys et al., 2012) N HO HO O A C B Compound 8

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5 N HO O O A C B Compound 9

IC50 for MAO A inhibition = 50 µM

(Huang et al., 2012) O O O O Br A C B Compound 10

IC50 for MAO A inhibition = 50 µM

(Morales-Camilo et al.,

2015)

2-Benzylidene-1-indanone, which could be considered to be a cyclic chalcone, however, has not yet been extensively investigated as a MAO inhibitor. A previous study, done by Huang and co-workers in 2012, reported the MAO inhibition activities of a series of indanone derivatives. These compounds exhibited good potential with potencies in the micromolar range. Among them, the most potent compound, compound 8 (see Table 1.1), had an IC50

value for MAO B inhibition of 7.5 µM. However, when the hydroxy group at C5 on ring A was replaced by a methoxy group to yield compound 9 (see Table 1.1), the inhibitory activity for both MAO A and MAO B decreased dramatically with IC50 values of 50 µM and 40.5 µM,

respectively. This finding highlights the importance of the hydroxy group on position 5 for inhibitory activity (Huang et al., 2012). In the reported study ring A was disubstituted with hydroxy and methoxy substituents, and different substituents and substitution patterns were explored for the benzylidene ring B.

Morales-Camilo and co-workers (2015) evaluated a total of 16 compounds which consisted of 8 chalcones and 8 aurones. In this study they found that, when the methoxy group was placed on position 4 (ring A), the methoxy group on the para position of ring B and a halogen atom on the meta position of ring B, good MAO B inhibition could be obtained. Compound 10 (see Table 1.1) was found to be the most potent compound of this study (Morales-Camilo et al., 2015).

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6

1.2. Rationale

As discussed above, MAO inhibitors have potential in the treatment of PD but great care should be taken as non-selective and irreversible MAO inhibitors (mainly MAO A inhibitors) are associated with serious adverse effects. ―The cheese reaction‖ is the most notable adverse effect of irreversible MAO A inhibition. This reaction is induced by tyramine, which is present in a variety of foods for example cheeses, beer and wine. Normally tyramine is metabolised by MAO A in the intestinal wall and the liver, thus preventing tyramine from entering the systemic circulation. When an irreversible MAO A inhibitor is present, tyramine and other monoamines cannot be metabolised and thus enter the systemic circulation. From the systemic circulation they have access to peripheral adrenergic neurons where they induce a significant release of noradrenaline. This, in turn, may lead to a potentially lethal hypertensive crisis, which is associated with cerebral haemorrhage (Bortolato et al., 2008; Youdim & Bakhle, 2006). Because the intestine contains little MAO B, and tyramine is effectively metabolised by intestinal MAO A, selective MAO B inhibitors do not elicit this effect (Youdim et al., 2006). As a result, the focus now falls on developing reversible and selective MAO A and MAO B inhibitors for the treatment of depression and PD, respectively (Youdim & Bakhle, 2006). Currently there is a growing and unmet need to improve current therapies, aimed not only at the relief of motor symptoms, but also to improve the non-motor symptoms of PD. Added to this, is the need to discover therapies that offer neuroprotection and disease-modifying effects in PD (Geldenhuys et al., 2012). MAO inhibitors not only have potential as symptomatic therapeutic agents, but may also slow the progression of PD and thus act as neuroprotective agents (Geldenhuys et al., 2012). Many scaffolds have been explored in an effort to discover potent and safe MAO inhibitors. In this study benzylidene-1-indanone derivatives will be explored as potential MAO inhibitors.

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7

Table 1.2: Benzylidene-1-indanone derivatives that will be synthesised in this study

O R R' A C B R R’ 11a 5-OH H 11b 6-OH H 11c 5-OH 3‘-F 11d 5-OH 4‘-F 11e 5-OH 3‘-Cl 11f 5-OH 4‘-Cl 11g 5-OH 3‘-Br 11h 5-OH 4‘-Br 11i 5-OH 3‘-CH3 11j 5-OH 4‘-CH3 11k 5-OH 3‘-CN 11l 5-OH 4‘-CN 11m 5-OH 4‘,5‘-Cl 11n 5-OH 3‘-OH

11o 5-OH 4‘-OH

11p 5-OH 3‘-OCH3 11q 5-OH 4‘-N(CH3)2 11r 5-OH 4‘-CH(CH3)2 12a H H 12b H 4‘-N(CH3)2 12c 5-OCH3 H 12d 5-OCH3 4‘-N(CH3)2

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8

Table 1.3: 2-Heteroarylidene-1-indanone derivatives that will be synthesised in this study

R' O R A C R R’ 13a H N 13b OCH3 N 13c H N 13d OCH3 N 13e H N Cl 13f H O 13g OCH3 O 13h H S 13i OCH3 S

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9 13j H NH 13k H S 13l H 13m OCH3 13n OCH3 O 13o OCH3 O Br

1.3. Hypothesis of this study

There are several studies that have shown that open-chain chalcones and aurones have potential as MAO inhibitors, and that substitution with appropriate groups on both rings A and B may lead to enhanced MAO inhibitory activity (Chimenti et al., 2009; Geldenhuys et al., 2012; Huang et al., 2012; Robinson et al., 2013; Tananka et al., 1987). Based on this, it may be hypothesised that benzylidene-1-indanone, which could be considered to be a cyclic chalcone, substituted with appropriate polar and lipophilic substituents will possess potent MAO inhibition activity. Such compounds will most likely exhibit selectivity for the MAO B isoform. Several studies with chalcones have demonstrated that this class of compounds is selective inhibitors of MAO B (Chimenti et al., 2009; Robinson et al., 2009).

This study will attempt to answer the following questions:

1. Is benzylidene-1-indanone a good scaffold for the design of potent MAO inhibitors?

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10 This information will contribute substantially towards the design of highly potent and selective MAO inhibitors.

1.4. Objectives of this study

The aim of this study is to explore the potential of the benzylidene-1-indanone scaffold in the design of potent and selective MAO inhibitors.

The primary objectives of this study are:

 To design and synthesise novel benzylidene-1-indanone analogues and related heterocyclic analogues with substituents on ring A and ring B.

 To evaluate the synthesised structures in vitro as inhibitors of recombinant human MAO A and MAO B

 To interpret the results of these studies with the aid of molecular docking of selected compounds into active site models of recombinant human MAO A and MAO B.

 To analyse the structure-activity relationships (SARs) of MAO inhibition by the synthesised compounds.

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1.5. References

Bortolato, M., Chen, K. & Shih, J.C. 2008. Monoamine oxidase inactivation. From pathophysiology to therapeutics. Advanced Drug Delivery Reviews, 60(13-14):1527-1533. Chimenti, F., Fioravanti, R., Bolasco, A., Chimenti, P., Secci, D., Rossi, F., Yáñez, M., Orallo, F., Ortuso, F. & Alcaro, S. 2009. Chalcones: A valid scaffold for monoamine oxidases inhibitors. Journal of Medicinal Chemistry, 52(9):2818-2824.

Dauer, W. & Przedborski, S. 2003. Parkinson's disease: Mechanisms and models. Neuron, 39(6):889-909.

Edmondson, D.E., Mattevi, A., Binda, C., Li, M. & Hubálek, F. 2004. Structure and mechanism of monoamine oxidase. Current Medicinal Chemistry, 11(15):1983-1993.

Fowler, J.S., Volkow, N.D., Wang, G.J., Logan, J., Pappas, N., Shea, C. & MacGregor, R. 1997. Age-related increases in brain monoamine oxidase B in living healthy human subjects. Neurobiology of Aging, 18(4):431-435.

Garbayo, E., Ansorena, E. & Blanco-Prieto, M.J. 2013. Drug development in Parkinson's disease: From emerging molecules to innovative drug delivery systems. Maturitas, 76(3):272-278.

Geldenhuys, W.J., Funk, M.O., Van Der Schyf, C.J. & Carroll, R.T. 2012. A scaffold

hopping approach to identify novel monoamine oxidase B inhibitors. Bioorganic & Medicinal Chemistry Letters, 22(3):1380-1383.

Huang, L., Lu, C., Sun, Y., Mao, F., Luo, Z., Su, T., Jiang, H., Shan, W. & Li, X. 2012. Multitarget-directed benzylideneindanone derivatives: Anti-ß-amyloid (Aß) aggregation, antioxidant, metal chelation, and monoamine oxidase B (MAO-B) inhibition properties against Alzheimer's disease. Journal of Medicinal Chemistry, 55(19):8483-8492.

Lees, A.J., Hardy, J. & Revesz, T. 2009. Parkinson's disease. The Lancet, 373(9680):2055-2066.

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12 Morales-Camilo, N., Salas, C.O., Sanhueza, C., Espinosa-Bustos, C., Sepu´lveda-Boza, S., Reyes-Parada, M., Gonzalez-Nilo, F., Caroli-Rezende, M. & Fierro, A. 2015. Synthesis, biological evaluation, and molecular simulation of chalcones and aurones as selective MAO-B inhibitors. Chemical MAO-Biology and Drug Design, 85(6):685-695.

Pan, X., Kong, L.D., Zhang, Y., Cheng, C.H.K. & Tan, R.X. 2000. In vitro inhibition of rat monoamine oxidase by liquiritigenin and isoliquiritigenin isolated from sinofranchetia chinensis. Acta Pharmacologica Sinica, 21(10):949-953.

Parkinson, J. 2002. An essay on the shaking palsy. 1817 (Republished). The Journal of Neuropsychiatry and Clinical Neurosciences, 14(2):223-236.

Ramsay, R.R. 2012. Monoamine oxidases: The biochemistry of the proteins as targets in medicinal chemistry and drug discovery. Current Topics in Medicinal Chemistry,

12(20):2189-2209.

Ramsay, R.R. 2013. Inhibitor design for monoamine oxidases. Current Pharmaceutical Design, 19(14):2529-2539.

Robinson, S.J., Petzer, J.P., Petzer, A., Bergh, J.J. & Lourens, A.C.U. 2013. Selected furanochalcones as inhibitors of monoamine oxidase. Bioorganic & Medicinal Chemistry Letters, 23(17):4985-4989.

Shih, J.C., Chen, K. & Ridd, M.J. 1999. Monoamine oxidase: From genes to behavior. Annual Review of Neuroscience, 22:197-217.

Tanaka, S., Kuwai, Y. & Tabata, M. 1987. Isolation of monoamine oxidase inhibitors from Glycyrrhiza uralensis roots and the structure-activity relationship. Planta Medica, 53(1):5-8.

Yacoubian, T.A. & Standaert, D.G. 2009. Targets for neuroprotection in Parkinson's disease. Biochimica et Biophysica Acta - Molecular Basis of Disease, 1792(7):676-687.

Youdim, M.B.H. & Bakhle, Y.S. 2006. Monoamine oxidase: Isoforms and inhibitors in Parkinson's disease and depressive illness. British Journal of Pharmacology, 147(Suppl. 1):S287-S296.

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13 Youdim, M.B.H., Edmondson, D. & Tipton, K.F. 2006. The therapeutic potential of

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Chapter 2 Literature Review

2.1. Parkinson’s disease

2.1.1. General background

Parkinson‘s disease (PD) is a chronic neurodegenerative disease. The main pathological characteristic of PD is the death of neurons in the substantia nigra pars compacta (SNpc) (Dauer & Przedborski, 2003; Pedrosa & Timmermann, 2013). The SNpc neurons form the nigrostriatal dopaminergic pathway and the loss of SNpc neurons leads to dopamine (DA) deficiency in this region. This deficiency is the cause of the major motor symptoms of PD (Dauer & Przedborski, 2003).

Though James Parkinson described the clinical features of PD, in his monograph ―Essay on the Shaking Palsy‖ in 1817, the cause of PD is not well understood (Dauer & Przedborski, 2003; Parkinson, 2002). During the last decades there have, however, been important advances in our understanding of the pathology of PD, most notably the identification of Lewy Bodies (LBs) in neurons of the nigrostriatal dopaminergic pathway (Pedrosa & Timmermann, 2013). LBs are one of the pathological hallmarks of PD and are described as proteinacious cytoplasmic inclusions. LBs contain oxidatively modified alpha-synuclein (α-syn) (Dauer & Przedborski, 2003). Two main hypotheses have been forwarded as potential explanation for the pathogenesis of PD. Firstly, misfolding and aggregation of proteins may lead to the death of SNpc dopaminergic neurons. The second hypothesis suggests that oxidative stress and mitochondrial dysfunction may play a leading role in the death of SNpc dopaminergic neurons. These two mechanisms will be discussed in the sections below (see

section 2.1.2) (Dauer & Przedborski, 2003).

PD is considered to be a sporadic disease, with few environmental causes. The major risk factor for PD remains age. The incidence of PD increases steeply with age, from 17.4 in 100 000 persons between the ages of 50 and 59 to 93.1 in 100 000 persons between ages of 70 and 79. The average age of onset is 60 years (Lees et al., 2009). Extensive research has shown that environmental factors such as rural living, drinking well water, and exposure to heavy metals and hydrocarbons have a small but noticeable influence on the risk of developing idiopathic PD (Goldenberg, 2008). Interestingly, cigarette smoking and caffeine

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15 consumption decreases the risk for PD (Ascherio et al., 2001; Baron, 1986; Factor et al., 2002).

The four basic symptoms of PD are tremor at rest, bradykinesia (slowness of movement), rigidity (increased resistance to passive movements of a patient‘s extremities or stiffness) and postural instability (Dauer & Przedborski, 2003). The other symptoms of PD consist of hypokinesia (the reduction in movement amplitude), akinesia (the absence of normal unconscious movements for example arm swing while walking), hypomimia (the paucity of normal facial expression), hypophonia (decreased voice volume), drooling or the failure to swallow without thinking about it, decreased size and speed of handwriting and decreased stride length during walking (Dauer & Przedborski, 2003). Patients also suffer from non-motor symptoms such as sleep disorders, neuropsychiatric issues and cognitive dysfunction (Dauer & Przedborski, 2003).

2.1.2. Mechanisms of neurodegeneration

As mentioned above, the two major mechanisms of neurodegeneration are misfolding and aggregation of proteins, and mitochondrial dysfunction and oxidative stress. There are, however, other mechanisms that also play a role in neurodegeneration namely neuroinflammation, exitotoxicity, apoptosis, the loss of trophic factors and lysosomal dysfunction. These mechanisms may act synergistically to promote neurodegeneration (Foltynie & Kahan, 2013; Yacoubian & Standaert, 2009).

2.1.2.1. Misfolding and aggregation of proteins

Protein misfolding and aggregation occurs in many neurodegenerative disorders, including PD, Alzheimer‘s disease (AD) and Huntington‘s disease (HD). Although the proteins that are involved in each of these disorders differ, it has been suggested that aggregated and misfolded proteins, in general, may be toxic to neurons (Dauer & Przedborski, 2003; Yacoubian & Standaert, 2009). α-Syn is the principal aggregating protein in PD. The link between PD and α-syn was first established in families that presented with autosomal dominant PD, which was caused by mutations in α-syn. These mutations are, however, found in only a few inherited PD cases (Athanassiadou et al., 1999; Polymeropoulos et al.,

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16 1997; Zarranz et al., 2004). Nevertheless α-syn is the main component of LBs and Lewy neurites (LN) (Mezey et al., 1998; Spillantini et al., 1997). Mutated α-syn appears to have a higher tendency to aggregate compared to unmutated α-syn (Dauer & Przedborski, 2003). The gene triplication of the α-syn locus could be considered as another cause of PD. The observation that gene multiplication leads to PD supports the central role of α-syn in the pathogenesis of PD (Singleton et al., 2003). Experimental evidence suggests that point mutations, overexpression as well as oxidative damage to α-syn may promote self-aggregation (Yacoubian & Standaert, 2009).

Although α-syn plays a central role in the development of PD, the mechanism of how aggregation of α-syn leads to neurotoxicity, is not fully understood (Yacoubian & Standeart, 2009). It has been suggested that aggregated α-syn may exert an effect on cell membranes and proteosomal function, gene transcription and regulation, and cell signalling and cell death cascades. In addition, aggregated α-syn may induce alterations in the storage and release of DA and may activate inflammatory mechanisms (Abeliovich et al., 2000; Benner et al., 2008; Cookson & van der Brug, 2008; Flower et al., 2005; Kontopoulos et al., 2006; Murphy et al., 2000; Saha et al., 2000; Smith et al., 2005; Voller & Lansbury, 2003; Yacoubian et al., 2008).

Studies done by Leroy et al. (1998) and Kitada et al. (1998) on the role of parkin and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in genetic forms of PD, support the link between protein aggregation and PD pathogenesis. Parkin is an E3 ubiquitin ligase which is involved in the degradation of misfolded proteins (Imai et al., 2000). Native α-syn does not seem to be a substrate for parkin, whereas mutated forms may be. Many studies have found that the brain tissue of patients with parkin-associated PD does not contain LBs (Farrer et al., 2001; Hayashi et al., 2000; Mori et al., 1998; Takahashi et al., 1994). HSP70 (modulate α-syn toxicity) and UCH-L1 (serves as an ubiquitin recycling enzyme in neurons) are two substrates of parkin that play a role in protein turnover and degradation. The dysfunction of these substrates encourages protein aggregation, including α-syn. From these remarks it could be said that the overproduction or impaired clearance of α-syn resulting in protein aggregation may be the fundamental mechanism of neuronal death in PD (Yacoubian & Standaert, 2009).

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2.1.2.2. Mitochondrial dysfunction and oxidative stress

Oxidative stress may occur when complex 1 of the mitochondrial respiratory chain is deficient. When complex 1 is inhibited the production of superoxide, a reactive oxygen species (ROS), by the respiratory chain is increased. Superoxide may either form the toxic hydroxyl radical or react with nitric oxide to produce peroxynitrite. The latter two molecules are highly reactive and react with nucleic acids, proteins and lipids, which in turn leads to cell damage. These reactive species may also target the electron transport chain itself, thus leading to further mitochondrial damage and enhanced production of ROS (Cohen, 2000; Dauer & Przedborski, 2003). Laboratory evidence has found oxidative damage as well as reduced levels of the antioxidant, glutathione, in the SNpc of several PD patients. The finding of reduced levels of glutathione is consistent with increased ROS and this could be an indication of a reduction of protective mechanisms against ROS. The levels of misfolded proteins may also increase as a consequence of ROS. This, in turn, would increase the demand on the ubiquitin-proteasome system to eliminate faulty proteins (Dauer & Przedborski, 2003). Based on reports that mitochondrial respiration may be impaired in PD, it can be concluded that the resulting ROS formation plays a central role in the degenerative process.

Besides impaired mitochondrial respiration, other sources of ROS exist in dopaminergic neurons, most notably DA metabolism. DA metabolism produces hydrogen peroxide (H2O2)

and superoxide radicals that may make dopaminergic neurons a fertile environment for the production of ROS. Auto-oxidation of DA produces DA-quinone, a molecule that reacts with cysteine (Cys) residues and damages proteins (Dauer & Przedborski, 2003). The products of DA metabolism may thus also contribute to neurodegeneration in PD.

As a result of mitochondrial dysfunction and oxidative stress, numerous treatment strategies have been proposed for PD. These include monoamine oxidase (MAO) inhibitors (compounds that block DA metabolism), enhancers of mitochondrial electron transport (coenzyme Q10), compounds that directly reduce free radicals (vitamin E) and molecules that can promote endogenous mechanisms to buffer free radicals (selenium) (Yacoubian & Standaert, 2009).

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2.1.2.3. Neuroinflammation

Neiroinflammation is the result of chronically activated glial cells. The role of neuroinflammation in the pathogenesis of PD has been emphasised in recent studies (Niranjan, 2014; Tansey & Goldberg, 2010). The possibility has been raised that chronic inflammation and cytokine-dependent toxicity may lead to the degeneration of dopaminergic neurons in the nigrostriatal pathway. In the microenvironment of the brain, glial cells play an important role in the homeostatic mechanisms that are responsible for neuronal survival. Microglia plays a role in immune surveillance and mediates essential immune responses to invading pathogens by secreting numerous factors such as cytokines, chemokines, prostaglandins, ROS, reactive nitrogen species (RNS) and growth factors. Several factors have neuroprotective activities while others enhance oxidative stress and triggers apoptotic cascades in neurons. Therefore, pro- and anti-inflammatory responses must be in homeostasis to prevent the potential destructive effects of prolonged or unregulated inflammation-induced oxidative stress on vulnerable neuronal populations (Tansey & Goldberg, 2010).

In postmortem PD brains of humans and the brains of animal models, activated microglia have been identified in the SN and striatum. While the activation of the microglia is poorly understood it is known that both cytokines and α-syn aggregation can stimulate activation of the microglia. The complement system is also implicated in PD pathogenesis, as elevated serum levels of complement proteins and the presence of complement proteins in LBs have been detected in PD. As a result of neuroinflammation, numerous anti-inflammatory drugs have been investigated for potential neuroprotection in PD (Yacoubian & Standaert, 2009).

2.1.2.4. Excitotoxicity

Glutamate has been identified as the major excitatory transmitter in the central nervous system (CNS), and is the main driver of the excitotoxic process. Glutamate receptors are found in high levels in dopaminergic neurons in the SN and receive glutamatergic innervation from the subthalamic nucleus and cortex. Glutamate potently activates N-methyl-D-aspartate (NMDA) receptors and the resulting high level of activated NMDA receptors may lead to increased intracellular calcium, which in turn activates cell death pathways. Calcium influx may also lead to the activation of nitric oxide synthase that produces nitric oxide, a

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19 molecule that is converted to peroxynitrite in the presence of ROS. An indicator of the formation of peroxynitrite, 3-nitrotyrosine, is found in enhanced levels in postmortem SN from PD patients. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models, NMDA receptor antagonists protect against dopaminergic neuron death. Based on these considerations it has been suggested that NMDA receptor antagonists and glutamate antagonists could be used as neuroprotective treatment in PD (Yacoubian & Standaert, 2009).

2.1.2.5. Apoptosis

Apoptosis (programmed cell death) has an effect on neural development and plays a role in neural injury. Apoptotic and autophagic cell loss has been found in the SN of PD brains, but to a limited extent. This limited extent can be explained by the slow progress of cell loss which underlies PD. Apoptosis is not likely to be the main cause of cell loss. Both apoptosis and autophagic cell loss pathways are hypothesised to be activated by oxidative stress, protein aggregation, excitotoxicity or inflammatory processes. Activation of these cell death pathways most likely represents end-stage processes in PD neurodegeneration. As a result, inhibitors of these cell loss pathways have been proposed as treatment for PD (Yacoubian & Standaert, 2009).

2.1.2.6. Loss of trophic factors

One of the contributors to cell death in PD is the loss of neurotrophic factors. Several of these factors are reduced in the SN of PD patients and include brain-derived neurotropic factor (BDNF), glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF). These agents have the ability to stimulate growth of dopaminergic neurons and it may therefore be concluded that neurotrophic factors may act as potential neuroprotective treatment (Yacoubian & Standaert, 2009). In support of this, GDNF and neurturin (an associated growth factor) have both been shown to be neuroprotective in animal models. GDNF has shown similar results in humans but neurturin is still being investigated (Yacoubian & Standaert, 2009).

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2.1.2.7. Lysosomal dysfunction

The most common Mendelian cause of PD relates to abnormalities in the glucocerebrosidase (GBA) gene. An inherited mutation in GBA, either as a homozygous or heterozygous compound state, causes Gaucher‘s disease (Sidransky & Lopez, 2012). Gaucher‘s disease is a lysosomal storage disorder and the carriers of GBA mutations have an increased risk for developing PD (Schapira, 2009). However, the mechanisms explaining the increased risk for PD in GBA patients is not well understood. Many hypotheses have been proposed to explain the association of GBA mutations and the increased risk for developing PD. Some of these hypotheses include a gain-of-function due to mutations in GBA that promotes α-syn aggregation as well as substrate accumulation due to enzymatic loss-of-function which affects α-syn processing and clearance (Sidransky & Lopez, 2012). Native α-syn is primarily degraded through lysosomal autophagic mechanisms and the accumulation of insoluble α-syn indicates lysosomal dysfunction in PD (Alvarez-Erviti et al., 2010; Dehay et al., 2012; Vogiatzi et al., 2008).

2.1.3. Treatment

The treatment of PD is mainly symptomatic (Dauer & Przedborski, 2003) and include the following agents: Levodopa (L-dopa) (Figure 2.1), DA agonists (ropinirole, pramipexole) (Figure 2.2), MAO B inhibitors (selegiline, rasagiline) (Figure 2.4), catechol-O-methyltransferase (COMT) inhibitors (entacapone, tolcapone) (Figure 2.3), amantadine (Figure 2.6) and anticholinergic drugs (trihexyphenidyl, benztropine) (Figure 2.5) (Pedrosa & Timmermann, 2013; Tarsy, 2006). Representative structures are illustrated in Figures

2.1-2.6.

The existing therapies improve the quality of life for PD patients but none of these have been shown to slow or prevent the progression of PD. Existing treatment focuses largely on the replacement of DA (L-dopa and DA agonist treatment). These treatments have been remarkably effective and have led to a significant improvement on the quality as well as duration of life of PD patients. In the advanced stages of PD, patients develop motor complications which are difficult to treat and control with the current drugs. Many patients also develop non-motor symptoms that include anosmia (the loss of the sense of smell), sleep disorders, autonomic impairment and cognitive impairment. The development of these

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21 symptoms is the most significant cause of disability in advanced PD (Yacoubian & Standaert, 2009).

L-dopa: L-Dopa (Figure 2.1) is one of the oldest and most effective therapies for the symptoms of PD. L-Dopa in combination with a peripheral dopa decarboxylase inhibitor (carbidopa or benserazide) is still considered the most effective treatment for PD and should always be the initial treatment option (Lees et al., 2009). However, after several years of use, L-dopa may lead to involuntary movements and dyskinesia (Dauer & Przedborski, 2003). Other adverse effects include nausea, anorexia, faintness and neuropsychiatric problems for example hypomania, depression and delirium (Lees et al., 2009).

HO

HO O

NH2

Figure 2.1: The structure of L-dopa

DA agonists: DA agonists are synthetic agents that directly stimulate DA receptors (Tarsy, 2006). DA agonists act at D2 receptors. Postsynaptic D2 stimulation is associated with an

improvement in motor function in PD, whereas presynaptic D2 stimulation may be associated

with a neuroprotective effect (Deleu et al., 2002). DA agonists were first used in combination with L-dopa for advanced PD complicated by motor fluctuations, but may also be used as monotherapy in early PD. When compared to L-dopa, DA agonists have a longer duration of action, they do not require metabolic conversion to an active agent, they do not compete with amino acids for intestinal and brain transport and they do not depend on neuronal uptake and release. However, DA agonists are not effective in patients who show no therapeutic response to L-dopa. Pramipexole and ropinirole (Figure 2.2) are effective monotherapies in PD patients younger than 65 years. In long-term clinical studies, patients treated with DA agonists as monotherapy exhibit a lower incidence of dyskinesia and motor fluctuations compared with those treated with L-dopa (Tarsy, 2006).

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22 NH N Cl O Ropinirole S N N H NH2 Pramipexole

Figure 2.2: The structures of DA agonists

COMT inhibitors: COMT is responsible for the catabolism of DA and L-dopa. The therapeutic action of COMT inhibitors is to reduce the peripheral conversion of L-dopa to 3-O-methyl dopa (Deleu et al., 2002). This increases the plasma half-life of L-dopa, produces more stable plasma L-dopa concentrations, and prolongs L-dopa‘s therapeutic effects (Nutt, 1998). COMT inhibitors such as tolcapone and entacapone (Figure 2.3) are used as treatment for patients displaying the wearing-off effect and are used in combination with L-dopa (Olanow et al., 2004).

Tolcapone NO₂ OH HO O O2N CN N(CH₂CH₃)₂ O HO OH Entacapone

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23 MAO B inhibitors: MAO B inhibitors are used in the treatment of PD for two reasons. The first reason is that MAO B inhibitors enhance striatal dopaminergic activity by inhibiting the metabolism of DA in the brain. This leads to an improvement in the motor symptoms of PD. The second reason is that selective MAO B inhibitors may be disease modifying agents, thus acting as neuroproctective agents (LeWitt & Taylor, 2008). The neuroproctective effect of MAO B inhibitors may be the result of the inhibition of DA oxidative metabolism which leads to the reduction of the formation of toxic metabolic by-products. Selective MAO B inhibitors are a feasible option for the treatment of PD since these compounds increase the synaptic DA concentration without affecting the activity of MAO A. However, non-selective MAO A/B inhibitors are not used in PD therapy and are contraindicated in L-dopa-treated patients. The combination of L-dopa with MAO A inhibitors may lead to an increase in blood-pressure (Figure 2.8) (Fernandez & Chen, 2007). Selective MAO B inhibitors are well tolerated in the treatment of PD (Lees et al., 2009). Selegiline [(R)-deprenyl] and rasagiline (Figure 2.4) are examples of selective irreversible MAO B inhibitors currently used for PD therapy.

N

Selegiline

N H

Rasagiline

Figure 2.4: The structures of MAO B inhibitors

Anticholinergic drugs: In PD, the therapeutic mechanism of anticholinergic drugs is to repair the disequilibria between striatal DA and acetylcholine activity (Lang & Lees, 2002). When anticholinergic drugs are used as monotherapy, they offer mild symptomatic control in PD. Anticholinergic drugs have mainly been used in tremor-predominant PD (Lees, 2005).

In patients younger than 70 years with disturbing tremor, anticholinergic drugs can be useful. Anticholinergic drugs are, however, ineffective for akinesia and gait disturbance. Anticholinergic drugs can also be useful in patients with advanced PD and who exhibit persistent tremor despite treatment with L-dopa or DA agonists (Tarsy, 2006). Anticholinergic drugs are not frequently used in PD because of their adverse effects such as impaired neuropsychiatric and cognitive function. These adverse effects are more likely to

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24 occur in older patients and those with impaired cognitive function (Lang & Lees, 2002). Other adverse effects of anticholinergic drugs are dry mouth, blurred vision, constipation, nausea, urinary retention and impaired sweating (Tarsy, 2006). Trihexyphenidyl and benztropine (Figure 2.5) are two examples of anticholinergic drugs used in the treatment of PD.

OH N Trihexyphenidyl O N Benztropine

Figure 2.5: The structures of anticholinergic drugs

Amantadine: Amantadine (Figure 2.6) was first introduced as prophylaxis for influenza A and as an antiviral drug. Its beneficial effect in PD was only discovered later (Schwab et al., 1969). The main indication of amantadine in the treatment of PD is to suppress L-dopa induced dyskinesia (Luginger et al., 2000). There are several mechanisms of actions that may account for amantadine‘s effect in PD and include the increase of DA release, the inhibition of DA reuptake, the mild anticholinergic effects and the antagonism of NMDA receptors. The NMDA antagonist activity of amantadine produces an antidyskinetic effect by reducing excessive glutamate neurotransmission in the basal ganglia. In clinical trials, two thirds of patients receiving amantadine treatment showed improved akinesia, rigidity and tremor (Tarsy, 2006).

NH2

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2.2. Monoamine oxidase

2.2.1. General background

In 1928 Mary Hare-Bernheim described an enzyme that catalyses the oxidative deamination of tyramine and named the enzyme tyramine oxidase. Hugh Blaschko found, a few years later, that tyramine oxidase, noradrenaline oxidase and aliphatic amine oxidase were the same enzyme, and is capable of metabolising primary, secondary and tertiary amines. Zeller named this enzyme mitochondrial MAO (Youdim & Bakhle, 2006). The MAOs are mitochondrial bound flavoenzymes that are situated on the outer membrane. The MAOs regulate amine levels in the brain and metabolise amine-containing drugs in the periphery. This is the main biological function of MAO. The MAOs catalyse the oxidative deamination of several monoamines including 5-hydroxytryptamine (5-HT or serotonin), catecholamines (DA, noradrenaline and adrenaline) and β-phenylethylamine. H2O2, the corresponding

aldehyde and either ammonia or a substituted amine are produced by the oxidative deamination reaction (Bortolato et al., 2008; Edmondson et al., 2004a; Ramsay, 2012; Ramsay, 2013; Youdim et al., 2006). The products of these reactions have the potential to be neurotoxic. For example ROS can be produced from H2O2. As discussed ROS may

induce mitochondrial damage and lead to neuronal apoptosis (Bortolato et al., 2008).

There are two isoenzymes of MAO, namely MAO A and MAO B (Figure 2.7). Both are expressed in all tissues but in different amounts. In humans, MAO A is mainly expressed in the intestines, placenta and heart while MAO B is mainly found in platelets, glial cells in the brain and liver (Ramsay, 2012). MAO A and MAO B were first distinguished by their sensitivities to the propargylamine-derived inhibitors, clorgyline and selegiline, as well as by their substrate specificities. MAO A is inhibited by low concentrations of clorgyline and catalyses the oxidation of 5-HT while MAO B is inhibited by low concentrations of selegiline and metabolises benzylamine and β-phenylethylamine. DA, noradrenaline, adrenaline, tryptamine and tyramine are oxidised by both forms of MAO (Youdim et al., 2006).

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Figure 2.7: The synthesis and metabolism of DA by MAO A and MAO B (Youdim et al.,

2006)

2.2.2. Genes and MAO

MAO A and MAO B are ~70% identical when comparing their amino acid sequences. The two MAOs are encoded by separate genes located on the X chromosome (XP11.23), both containing 15 exons with identical intron-exon organisation. This suggests that MAO A and MAO B are derived from the duplication of a common ancestral gene (Shih et al., 1999). The expression of the MAO A and MAO B genes differs, and is controlled by certain hormones. For example, progesterone, testosterone, corticosterone and glucocorticoids increase MAO A levels but have only a slight effect on MAO B expression. MAO B expression is regulated by a mitogen-activated protein kinase (MAPK) pathway.

As shown by gene deletion, MAO A activity is important in development. As a result of lack of MAO A activity, a compulsive-aggressive phenotype was found. Personality traits such as sensation seeking, impulsiveness, extraversion and vulnerability for substance abuse for example tobacco smoking, early onset alcoholism and gambling, are associated with low platelet MAO B activity. Lowered MAO B activity may also be associated with reduced risk of PD in smokers (Youdim et al., 2006).

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2.2.3. MAO A

2.2.3.1. Biological function

2.2.3.1.1. Cheese reaction

The ―cheese reaction‖ (Figure 2.8) is one of two serious adverse effects of MAO A inhibitors and non-selective MAO inhibitors. This reaction is induced by tyramine and other indirectly acting sympathomimetic amines that are present in food, mostly cheeses and fermented drinks such as wine and beer. Normally such dietary amines are extensively metabolised by MAO A in the intestinal wall and in the liver, thus preventing their entry into the systemic circulation. When a MAO A inhibitor is present, tyramine and other monoamines present in food cannot be metabolised and they thus enter the systemic circulation. These dietary amines can induce a significant release of noradrenaline from peripheral adrenergic neurons leading to a potentially lethal hypertensive crisis with cerebral haemorrhage (Bortolato et al., 2008; Youdim & Bakhle, 2006). Selective MAO B inhibitors do not exhibit this effect because there is little MAO B in the intestine, and tyramine is effectively metabolised by intestinal MAO A. The development of moclobemide and toloxatone, reversible MAO A inhibitors, also avoid this problem because reversible inhibitors block sufficient MAO A in the CNS to obtain an antidepressant effect, while dietary tyramine is able to displace the inhibitor from peripheral MAO A, allowing for metabolism (Youdim et al., 2006). This serious adverse effect led to a search for alternative antidepressant agents. MAO inhibitors were replaced by the tricyclic antidepressants and selective 5-HT re-uptake inhibitors (SSRIs) (Youdim & Bakhle, 2006).

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Figure 2.8: The mechanism of the cheese reaction (Youdim & Weinstock, 2004)

2.2.3.1.2. Serotonin syndrome

MAO A is a key enzyme responsible for the metabolism of 5-HT (Figure 2.9) in the brain (Youdim et al., 2006). 5-HT syndrome or toxicity is a drug-induced syndrome characterised by a group of dose related adverse effects that are the result of increased extracellular 5-HT concentrations in the CNS (Buckley et al., 2014). In the past, 5-HT syndrome has been misdiagnosed. Presently it is acknowledged as a spectrum disorder (Stanford et al., 2009) because the adverse effects that occur ranges from mild to severe (Buckley et al., 2014). The concept of 5-HT syndrome being a spectrum disorder is consistent with its severity increasing in parallel with the dose of 5-HT agonist that increases the 5-HT receptor occupancy. The only 5-HT agonists that do this directly are 5-HT2A agonists, but none are

being used clinically. However, there are drugs that increase 5-HT concentrations in the extracellular compartment and thus increase 5-HT receptor occupancy indirectly. These drugs present a greater risk for they are used to treat a great deal of CNS disorders (Stanford et al., 2009).

Severe symptoms were only detected by three classes of drugs that increase the extracellular concentration of 5-HT by either blocking the extracellular clearance or by increasing the release of 5-HT. These three classes are: SSRIs, 5HT and noradrenaline reuptake inhibitors (SNRIs) and MAO inhibitors. SNRIs and SSRIs prevent the extracellular clearance of 5-HT and MAO inhibitors prevent the metabolism of 5-HT, thus increasing the

Synthesis and evaluation of 1-indanone derivatives as inhibitors of monoamine oxidase