J. Kleynhans 21090955

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Aminopyrimidine derivatives as adenosine

antagonists

J. Kleynhans

21090955

Dissertation submitted in partial fulfillment of the requirements

for the degree Magister Scientiae

in Pharmaceutical Chemistry

at the Potchefstroom Campus of the North-West University

Supervisor:

Dr. A.C.U. Lourens

Co-supervisor:

Prof. J.J. Bergh

Co-supervisor:

Prof. G. Terre’blanche

October 2013

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The financial assistance of the National Research

Foundation (NRF) towards this research is hereby

acknowledged. Opinions expressed and

conclusions arrived at, are those of the author and

are not necessarily to be attributed to the NRF.

(3)

Abstract

Title

Aminopyrimidine derivatives as adenosine antagonists Keywords

2-Aminopyrimidines, dual adenosine A1/A2A antagonists, neuroprotection, Parkinson’s disease

Aims of this project

The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists.

Background and rationale

Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-motor symptoms, of which cognitive dysfunction is an example, also frequently occur. Current therapy provides symptomatic relief mainly by augmentation of dopaminergic signalling (levodopa, dopamine agonists, MAO and COMT enzyme inhibitors), but disease progression is not adequately addressed. New therapies that can prevent further neurodegeneration in addition to providing symptomatic relief are therefore urgently required.

Adenosine has an important function as neuromodulator in the central nervous system. The adenosine A2A receptor in particular plays an essential role in the regulation of movement. This, coupled to the fact that it is uniquely distributed in the basal ganglia, contributes to its attractiveness as non-dopaminergic target in the treatment of movement disorders, such as Parkinson’s disease. The efficacy of adenosine receptor antagonists has been illustrated in animal models of Parkinson’s disease and several adenosine receptor antagonists have also reached clinical trials. The neuroprotective properties of adenosine A2A receptor antagonists are further attributed to their ability to modulate neuro-inflammation and decrease the release of the excitatory neurotransmitter glutamate, which is implicated in neurotoxicity. While adenosine A1 receptor antagonism has a synergistic effect on the motor effects of adenosine A2A receptor antagonism, it has the additional benefit of improving cognitive

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dysfunction, a cardinal non-motor symptom of Parkinson’s disease. Dual antagonism of adenosine A1 and A2A receptors therefore offers the potential of providing symptomatic relief as well as the neuroprotection so desperately needed in the clinical environment.

Amino substituted heterocyclic scaffolds, such as those containing the 2-aminopyrimidine motif, have been shown to exhibit good efficacy as dual adenosine receptor antagonists. Since the structure activity relationships of 2-aminopyrimidines have not been comprehensively explored, it is in this regard that this study aimed to make a contribution. Results

Fourteen 2-aminopyrimidines were synthesised successfully over three steps, (although in low yields) and characterised by nuclear magnetic resonance and infrared spectroscopy, mass spectrometry, by determination of melting points and high performance liquid chromatography. Structure modifications explored included variation of the aromatic substituent on position 4, as well as variations in the substituents of the phenyl ring, present on position 6 of the pyrimidine ring.

Radioligand binding assays were performed to determine the affinities of the synthesised compounds for the adenosine A1 and A2A receptor subtypes. Several high dual affinity derivatives were identified during this study; the compound with the highest affinity was 4-(5-methylthiophen-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (39f) with K_i values of 0.5 nM and 2.3 nM for the adenosine A2A and adenosine A1 receptors, respectively.

A few general structure activity relationships were derived, which included: The effect of the aromatic substituent (position 4) on A2A affinity could be summarised (in order of declining affinity) as follows: 5-methylthiophene > phenyl > furan > pyridine > p-fluorophenyl > benzofuran. On the other hand, the effect of this substituent on A1 receptor affinity could be summarised (in order of declining affinity) as follows: phenyl > 5-methylthiophene > p-fluorophenyl > benzofuran > pyridine. The affinities as exhibited by the methylthiophene derivatives 39f, 39h – 39j, further showed that while piperidine substitution (39f) resulted in optimal A2A and A1 affinity, pyrrolidine substitution (39j) was less favourable. Substitution at the 4ʹ position of the phenyl ring, as well as thiazole substitution, generally resulted in poor adenosine A1 and A2A receptor affinity. However, 4-[2-amino-6-(5-methylfuran-2-yl)pyrimidin-4-yl]-N-(1,3-benzothiazol-2-yl)benzamide (39l) surprisingly demonstrated good affinity and selectivity for the adenosine A1 receptor.

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The results obtained during radioligand binding assays were rationalised by QSAR and molecular modelling (Discovery Studio 3.1, Accelrys) studies. The inverse relationship seen between log K_i (as indicator of affinity) and polar surface area, illustrated the importance of this physico-chemical property in the design of 2-aminopyrimidine A2A antagonists. The results from the docking study further showed that the orientation adopted by derivatives in the binding cavity (and particular hydrogen bonding to Asn 253 and Glu 169) is of importance. Results from the MTT cell viability assay indicated that none of the high affinity derivatives had a significant effect on cell viability at 1 µM, a concentration much higher than their Ki values. However, incorporation of the furan, benzofuran and p-fluorophenyl groups as aromatic substituent and a pyrrolidine as amine substituent, presented liabilities.

Lastly, the haloperidol induced catalepsy assay (in rats) was used to give a preliminary indication of adenosine receptor antagonism or agonism. Compound 39f failed to reverse catalepsy under standard conditions, but showed some reversal after an increased time period. Indications therefore exist that 39f is an adenosine receptor antagonist that suffers from bioavailability issues. Compound (39c), 4-phenyl-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine which also demonstrated promising affinity in the radioligand binding assays however showed a statistically significant reduction in catalepsy, indicating adenosine A2A receptor antagonism, and in vivo efficacy.

Highly potent, dual affinity aminopyrimidine derivatives with acceptable toxicity profiles were identified in this study, with compound 39c demonstrating in vivo activity. The aim of designing and synthesising a promising dual adenosine A1/A2A receptor antagonist is therefore realised, with compound 39c as the most favourable example.

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Opsomming

Titel

Aminopirimidienderivate as adenosienantagoniste Sleutelwoorde

2-Aminopirimidiene, dualistiese adenosien A1/A2A-antagoniste, neurobeskerming, Parkinson se siekte

Doel van die studie

Die doel van hierdie studie was om nuwe 2-aminopirimidienderivate as moontlike dualistiese adenosien A1- en A2A-reseptorantagoniste te ontwerp en te sintetiseer.

Agtergrond en motivering

Parkinson se siekte is die tweede algemeenste neurodegeneratiewe siekte (naas Alzheimer se siekte) en word gekenmerk deur die selektiewe afsterwing van die dopaminergiese neurone van die nigrostriatale senuweebaan. Kenmerkende motoriese simptome sluit bradikinesie, spierstyfheid en bewing in, terwyl nie-motoriese simptome, soos kognitiewe wanfunksie ook algemeen voorkom. Huidige terapie verskaf simptomatiese verligting hoofsaaklik deur die versterking van dopaminergiese seinoordrag (levodopa, dopamienagoniste, MAO- en KOMT-ensieminhibeerders), maar die verloop van die siekte word nie genoegsaam behandel nie. Nuwe terapieë, wat bo-en behalwe die verskaffing van simptomatiese verligting, ook verdere neurodegenerasie kan voorkom, is dus dringend nodig.

Adenosien verrig ‘n belangrike funksie as neuromoduleerder in die sentrale senuweestelsel. Die adenosien A2A reseptor veral, speel ‘n belangrike rol in die regulering van beweging. Dit, en die feit dat die reseptor uniek in die basale ganglia verspreid is, dra by tot die gunstigheid daarvan as nie-dopaminergiese teiken in die behandeling van bewegingsafwykings, soos Parkinson se siekte. Die effektiwiteit van adenosienantagoniste as beide simptomatiese sowel as neurobeskermende middels is bewys in dieremodelle van Parkinson se siekte en verskeie adenosien reseptor antagoniste het ook kliniese proewe bereik. Die neurobeskermende eienskappe van adenosien A2A-antagonisme word toegeskryf aan die vermoë van die middels om neuro-inflammasiete te moduleer en ‘n afname in die vrystelling van die eksitatoriese neuro-oordragstof glutamaat, wat geassosieer word met neurotoksisiteit, te bewerkstellig. Adenosien A1-reseptorantagonisme het bo-en behalwe die

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sinergisme van die motoriese effek van adenosien A2A-antagonisme, ook die bykomende voordeel dat dit kognitiewe wanfunksie, wat ‘n belangrike nie-motoriese simptoom van Parkinson se siekte is, verlig. Dualistiese antagonisme van adenosien A1 en A2A-reseptore bied dus die moontlikheid om beide simptomatiese verligting en neurobeskerming, wat so dringend benodig word in die kliniese omgewing, te verskaf.

Die effektiwiteit van amiengesubstitueerde heterosikliese verbindings, soos die wat die 2-aminopirimidiengroep bevat, as dualistiese adenosienreseptorantagoniste, is reeds aangetoon. Aangesien die struktuuraktiwiteitverwantskappe vir die 2-aminopirimidiene nie voorheen volledig ondersoek is nie, is daar gepoog om in die verband ‘n bydrae te lewer tydens hierdie studie.

Resultate

Veertien 2-aminopirimidiene is suksesvol in drie stappe gesintetiseer (alhoewel lae opbrengste verkry is). Die gesintetiseerde verbindings is gekarakteriseer deur gebruik te maak van kernmagnetieseresonans- en infrarooi-spektroskopie, massa spektrometrie, smeltpuntbepalings en hoë drukvloeistofchromatografie. Strukturele veranderinge wat ondersoek is sluit variasie van die aromatiese substituent op posisie-4, sowel as veranderinge in die substitusie van die fenielring, teenwoordig op posisie-6 van die pirimidienring, in.

Die affiniteit van die gesintetiseerde verbindings vir die adenosien A1- en A2A-reseptore is met radioligandbindingstudies bepaal. Verskeie hoë-affiniteit derivate is gedurende hierdie studie geïdentifiseer. Die verbinding met die hoogste affiniteit was 4-(5-metieltiofeen-2-iel)-6-[3-(piperidien-1-karboniel)feniel]pirimidien-2-amien (39f) met K_i-waardes van 0.5 nM en 2.3

nM vir die adenosien A2A- en A1-reseptore, onderskeidelik.

Verskeie struktuuraktiwiteitsverwantskappe kon afgelei word, wat die volgende insluit: Die effek van aromatiese substitusie (in posisie 4) op affiniteit kan as volg opgesom word (in volgorde van afnemende affiniteit): 5-metieltiofeen > feniel > furaan > piridien > p-fluoorfeniel > bensofuraan. Aan die ander kant, kan die effek van aromatiese substitusie op affiniteit vir die A1-reseptor as volg opgesom word (in volgorde van afnemende affiniteit): feniel > 5-metieltiofeen > p-fluoorfeniel > bensofuraan > piridien. Uit die studie van die metieltiofeenderivate 39f, 39h – 39j, het geblyk dat, terwyl piperidiensubstitusie (39f) optimale A2A en A1 affiniteit lewer, pirrolidien substitusie (39j) minder gunstig was. Substitusie op die 4' posisie (op die fenielring) sowel as substitusie met ‘n tiasoolgroep het oor die algemeen ‘n negatiewe impak op adenosien A1 en A2A-reseptoraffiniteit gehad. Goeie

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affiniteit vir die adenosien A1-reseptor is egter met 4-[2-amien-6-(5-metielfuraan-2-iel)pirimidien-4-iel]-N-(1,3-bensotiasol-2-iel)bensamied (39l) verkry.

Om die resultate van die radioligandbindingstudies te rasionaliseer, is kwantitatiewe struktuuraktiwiteitsverswantskap- en molekulêre modelleringstudies (Discovery Studio 3.1, Accelrys) gedoen. Die omgekeerde verwantskap tussen log Ki (as aanduiding van affiniteit) en polêre oppervlakarea, het die belang van dié fisiese-chemiese eienskap tydens die ontwerp van 2-aminopirimidien-adenosien-A2A-antagoniste beklemtoon. Die resultate van die passingstudie het verder aangetoon dat die die oriëntasie wat verbindings in die aktiewe setel aanneem (en veral die waterstofbindings met Asn 253 en Glu 169) van groot belang is. Resultate verkry met die MTT-seltoksisteitstoets het aangedui dat geen van die mees aktiewe verbindings ‘n merkbare effek op seloorlewing by ‘n konsentrasie van 1 µM toon nie. Hierdie konsentrasie is baie hoër as die K_i-waardes van hierdie reeks verbindings. Daar is wel waargeneem dat die insluiting van ‘n furaan-, bensofuraan- en p-fluoorfenielgroep as aromatiese substituent en pirrolidien as amiensubstituent ‘n gevaar vir seltoksitieit kan inhou. Laastens is die haloperidol-geïnduseerde katalepsiestudie (in rotte) gebruik om ‘n voorlopige aanduiding van adenosienreseptorantagonisme of –agonisme te gee. Verbinding 39f het nie katalepsie verminder tydens die standaardtoets nie, maar geringe vermindering in katalepsie is waargeneem nadat die tyd van die standaardtoets verleng is. Dit blyk dus dat 39f wel ‘n adenosienreseptorantagonis is, maar waarskynlik nie goeie breinbiobeskikbaarheid het nie. Verbinding (39c), 4-feniel-6-[3-(piperidien-1-karboniel)feniel]pirimidien-2-amien wat ook belowende affiniteit in die radioligandbindingstudies getoon het, het ‘n statistiese betekenisvolle verlaging in katalepsie teweeggebring. Hierdie verbinding is dus waarskynlik ‘n adenosien A2A-reseptorantagonis met in vivo effektiwiteit.

Hoogs potente dualistiese 2-aminopirimidienderivate met aanvaarbare toksisteitsprofiele is gedurende hierdie studie geïdentifiseer, met verbinding 39c wat in vivo aktiwiteit getoon het. Die doel van die studie, naamlik die ontwerp en sintese van moontlike adenosien A1/A2A -reseptorantagoniste is dus verwesenlik, met verbinding 39c as die mees geskikte voorbeeld.

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Acknowledgements

I would like to thank the following contributors who made this study possible: • Dr. A.C.U. Lourens, my supervisor

• Prof. J.J. Bergh and Prof. G. Terre’blanche, my co-supervisors • Prof. J.P. Petzer

• Dr. A. Petzer

• Dr. M.M. Van der Walt • Prof. D.D.N’Da

• Mnr. A. Joubert • Dr. J. Jordaan • Prof. J. Du Preez

• My fellow students: Mrs. C. Minders, Mnr. S.J. Robinson, Ms. M. Hoon, Ms. L. Meiring and Mr. F.J. Smit for help provided.

• The North-West University for facilities provided. • Financial aid provided by

o National Research Foundation o Medicine Research Council o North-West University

• My mother, father and sister for providing me my foundation in life.

• My Father in heaven for the big miracles, as well as the little ones, along the way at the most critical moments.

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List of Figures

Chapter 1

Figure 1.1 Structure activity relationships of

2-amino-5-cyano-6-(2-furayl)pyrimidine adenosine A2A receptor antagonists . . . 433

Figure 1.2 Example of a pyrimidine-4-carboxamide derivative with high affinity . . . . . 5

Figure 1.3 Examples of 4'-amide derivatives . . . . . 9

Figure 1.4 Examples of compounds with different heteroaryl and aryl groups in position 4. . . . . 9

Figure 1.5 Examples of compounds with thiazole substituents . . . 10

Chapter 2 Figure 2.1 A normal SNc on the left in comparison with depig-mented SNc of the parkinsonian brain on the right . . . 13

Figure 2.2 Electron micrograph of a Lewy body in the substiantia nigra . . . 14

Figure 2.3 Meperidine and MPP , which is metabolised to neurotoxic MPTP . . . 15

Figure 2.4 Mechanism of MPTP cytotoxicity . . . 16

Figure 2.5 Results of dysfunction of mitochondrial electron transport chain complex I 18 Figure 2.6 Catabolism of dopamine to DOPAC . . . 19

Figure 2.7 Formation of ROS . . . 19

Figure 2.8 Exogenous levodopa and metabolism in the presynaptic dopaminergic neuron . . . 21

Figure 2.9 A basic representation of the structure of the adenosine A2A receptor in cellular membrane . . . 29

Figure 2.10 Crystal structure of the adenosine A2A receptor . . . 30

Figure 2.11 The ligand binding pocket of adenosine A2A receptor with ligand ZM 241385 . . . . . . .. 30

Figure 2.12 Mechanism of action of adenosine A2Areceptor antagonists in motor function . . . . . . . 32

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Figure 2.14 Antagonism of adenosine A2A receptors leads

to reversal of haloperidol induced catalepsy . . . 40

Chapter 3

Figure 3.1 General structure of synthesised 2-aminopyrimidines . . . 42 Figure 3.2 Examples of 2-aminopyrimidines for which synthesis was

unsuccessful . . . . . . . . 47 Figure 3.3 Examples of thiazole derivatives for which synthesis failed . . . 50 Figure 3.4 Synthesis of 2-aminopyrimidine 39k . . .

. 61

Chapter 4

Figure 4.1 A sigmoidal dose-response curve illustrating adenosine A2A affinity of ZM 241385 using striata from male Sprague Dawley rats/ A sigmoidal dose-response curve illustrating adenosine A2A affinity of

ZM 241385 using striata from female Sprague Dawley rats . . . . . . . 89 Figure 4.2 A sigmoidal dose-response curve illustrating adenosine A1 affinity of

CPA using whole brains from male Sprague Dawley rats. A sigmoidal dose-response curve illustrating adenosine A1 affinity of

CPA using whole brains from female Sprague Dawley rats . . . . . . 89

Figure 4.3 General structure of the series of arylindenopyrimidines/

aminopyrimidines . . . . . . 91 Figure 4.4 The correlation between the log K_i of the synthesised

2-aminopyrimidines and their calculated polar surface area . . . 94 Figure 4.5 The docked compounds 39f and 39c in the adenosine A2A

receptor active site (docked with water of crystallisation present) . . . 95 Figure 4.6 Compound 39a and compound 39l docked into the adenosine A2A

receptor active site . . . 96 Figure 4.7 The results obtained during the catalepsy assay of known

adenosine A2A receptor antagonist istradefylline (KW 6002) . . . 98 Figure 4.8 The results obtained during the catalepsy assay of compound

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Figure 4.9 The results obtained during the catalepsy assay of compound 39c . . . 100

Figure 4.10 The original structure of ZM 241385 (violet) co-crystallised with the adenosine A2A receptor superimposed on the structure of ZM 241385 that was docked during the validation study after

preparation of the human adenosine A2A receptor for docking . . . . . . 105 Figure 4.11 The cataleptic rat with front paws on the horizontal bar in a fixed

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List of Tables

Chapter 2

Table 2.1 Comparison between apoptosis and necrosis of cells . . . 15 Table 2.2 Properties and distribution of human adenosine receptor subtypes . . 28

Chapter 3

Table 3.1 NMR data and HMBC correlations of

3-[(1E)-3-(5-methylthiophen-2-yl)-3-oxoprop-1-en-1-yl]benzoic acid (37f) . . . 53 Table 3.2 Comparison of chemical shifts of five membered

ring systems and the 2-benzofuran ring . . . . . . 54 Table 3.3 NMR data and HMBC correlation of

(2E)-1-(5-methylthiophen-2-yl)-3-[3-(piperidine-1-carbonyl)phenyl]prop-2-en-1-one (38f). . . . . . 57 Table 3.4 Chemical shifts observed for amine substituents . . . 58 Table 3.5 NMR data and HMBC correlations of

4-(5-methylthiophen-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (39f) . . . 60

Chapter 4

Table 4.1: Affinities of the synthesised 2-aminopyrimidines and reference compounds (CPA and ZM 241385) for the adenosine A2A and A1

receptor subtypes . . . . . . 90 Table 4.2 Calculated physicochemical parameters of synthesised

aminopyrimidines . . . 93 Table 4.3 Cell viability (%) after exposure to the synthesised

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List of Schemes

Chapter 1

Scheme 1.1 General synthetic route towards aminopyrimidine synthesis . . . 10

Chapter 3 Scheme 3.1 Synthesis of 2-aminopyrimidines . . . 43

Scheme 3.2 Mechanism of amide formation using CDI as coupling agent . . . 44

Scheme 3.3 Mechanism of aminopyrimidine formation . . . 45

Scheme 3.4 Alternative synthesis of 2-aminopyrimidines . . . 48

Scheme 3.5 Alternative synthesis of 4-(5-bromofuran-2-yl)-6-[3-(piperidin-1-carbonyl)phenyl]pyrimidin-2-amine . . . . . . . 49

Scheme 3.6 Proposed mechanism of dehalogenation of 2bromo -furan by sodium hydride at high temperatures . . . . . . . 63

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List of Abbreviations

[3_H]DPCPX _1,3-[3_{H]-dipropyl-8-cyclopentylxanthine} [3_H]NECA _[3_{H]5’N-ethylcarboxamide-adenosine} 6-OHDA 6-hydroxydopamine Abs Absorbance ADP Adenosine-diphosphate Asn Asparagine

ATP Adenosine- triphosphate

cAMP Cyclic adenosine-monophosphate

CDCl3 Deuterochloroform

CDI 1,1'-Carbonyldiimidazole

COMT Catechol-O-methyl-transferase

COSY Correlation spectroscopy

CPA N6_{-cyclopentyladenosine}

CPM Counts per minute

C-terminus Cytosolic carboxy terminus

DAG Diacylglycerol

DCM Dichloromethane

DEPT Distortionless enhancement by polarisation transfer

DMEM Dulbecco’s Modified Eagle Medium

DMF Dimethylformamide

DMSO Dimethyl sulfoxide

DMSO-d6 Deuterodimethyl sulfoxide

DNA Deoxyribonucleic acid

DOPAC 3,4-Dihydroxyphenylacetic acid

ECL Extracellular loops

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FDA Food and Drug Administrator USA

GABA Gamma-amino butyric acid

GDNF Glial cell derived neurotrophic factor

Gi Inhibitory G-protein

Glu Glutamic acid

GPCR Guanine nucleotide-binding protein coupled receptor G-protein Guanine nucleotide-binding protein

Gs Stimulatory G-protein

HMBC Heteronuclear multiple bond correlation

HPLC High performance liquid chromatography

HSQC Heteronuclear single quantum correlation

IC50 Half maximal inhibitory concentration

ICL Intracellular loops

IP3 Inositol triphosphate

IR Infrared spectroscopy

Kd Dissociation constant

K_i Inhibition constant

Leu Leucine

MAO Monoamine oxidase

MAO-B Monoamine oxidase isoform B

MeOH Methanol MPDP+ _{1-Methyl-4-phenyl-2,3-dihydropyridinium ion} MPP 1-Methyl-4-phenyl-4-propionoxypiperidine MPP+ 1-Methyl-4-phenylpyridinium ion MPTP 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine

mRNA Messenger ribonucleic acid

MS Mass spectrometry

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NaH Sodium hydride

NaOH Sodium Hydroxide

NBS N-Bromosuccinimide

NMDA N-methyl-D-aspartate

NMR Nuclear magnetic resonance

NSAIDs Non-steroidal anti-inflammatory drugs

N-terminus Amino-terminus

PBS Phosphate-buffered saline

PDB Protein Data Bank

Phe Phenylalanine

QSAR Quantitative structure activity relationship

ROS Reactive oxygen species

RSMD Root square mean deviation

RT Room temperature

SNc Substantia nigra pars compacta

TLC Thin layer chromatography

Trp Tryptophan

NMR:

δ delta scale indicating chemical shift

J coupling constant br d broad doublet br s broad singlet br t broad triplet d doublet dd doublet of doublets

ddd doublet of doublet of doublets

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p pentet

ppm parts per million

q quartet

s singlet

J. Kleynhans 21090955

Aminopyrimidine derivatives as adenosine

antagonists

J. Kleynhans

21090955

Dissertation submitted in partial fulfillment of the requirements

for the degree Magister Scientiae

in Pharmaceutical Chemistry

at the Potchefstroom Campus of the North-West University

Supervisor:

Dr. A.C.U. Lourens

Co-supervisor:

Prof. J.J. Bergh

Co-supervisor:

Prof. G. Terre’blanche

October 2013

The financial assistance of the National Research

Foundation (NRF) towards this research is hereby

acknowledged. Opinions expressed and

conclusions arrived at, are those of the author and

are not necessarily to be attributed to the NRF.

Abstract

Opsomming

Acknowledgements

Table of contents

List of Figures

List of Tables

List of Schemes

List of Abbreviations

NMR: