Background and rationale -
Parkinson’s disease is a neurodegenerative disorder characterised by reduced levels of
dopamine in the brain. The cause of Parkinson's disease is still unknown; however several
theories pertaining to the etiology exist. Current treatment mainly aims at dopamine
replacement, with agents such as levodopa and dopamine agonists that provide patients
with symptomatic relief. This relief is unfortunately only temporary as the progression of the
disease is not halted. Furthermore, these therapies are associated with a range of side
effects and novel approaches to the treatment are thus urgently required. Adenosine A2A
receptor antagonists recently emerged as a promising non-dopaminergic alternative, not
only as symptomatic treatment, but also as potential neuroprotective therapy.
Adenosine A2A receptors are co-localised with dopamine D2 receptors in the striatum and
other nuclei of the basal ganglia. Adenosine A2A stimulation decreases the affinity of
dopamine for the D2 receptor, and increase cyclic AMP (cAMP) levels. The stimulation of
dopamine D2 receptors, in contrast, decreases cAMP levels and therefore these receptors
(A2A and D2), act in an opposing manner. Adenosine A2A antagonism will thus have similar
effects as dopamine D2 agonism and will reduce the postsynaptic effects of dopamine
depletion to give symptomatic relief. There are also several mechanisms where by
adenosine A2A antagonists may be neuroprotective, for example by preventing glutamate
excitotoxicity, that may cause damage to dopaminergic neurons. A number of adenosine A2A
antagonists have already reached clinical trials and promising results were obtained,
especially when combined with levodopa. Consequently, A2A antagonists are realistic
prospects that have therapeutic potential in diseases with dopaminergic hypofunction, like
Parkinson's disease. Many of the current A2A antagonists contain an amino-substituted
heterocyclic scaffold, such as an aminopyrimidine. The primary aim of this study was the
design, synthesis and evaluation of 2-aminopyrimidine derivatives as adenosine A2A receptor
antagonists.
Monoamine oxidase B (MAO-B) inhibitors are also promising candidates for the symptomatic
treatment of Parkinson's disease, since MAO-B is the enzyme primarily responsible for the catabolism of dopamine in the brain. Irreversible inhibitors of MAO-B, such as selegeline and
rasagiline, have been used clinically for the treatment of Parkinson's disease. This type of
inhibition comes with certain disadvantages as it may take up to several weeks after
termination of treatment for the enzyme activity to recover. Reversible inhibitors in contrast
will have much better safety profiles seeing that they will not inactivate the enzyme
permanently and allow for competition with the substrate.
When dopamine is oxidized by MAO, toxic metabolic by-products, such as hydrogen
peroxide (H2O2) forms, and this is believed to be a possible cause of Parkinson's disease.
MAO-B inhibitors will therefore not only provide symptomatic relief but may also alter the
progression of the disease by preventing the formation of these byproducts. Promising MAOB
inhibitory activities have been reported for chalcones, and since the intermediates
obtained in the synthesis of aminopyrimidines in this study are chalcones, a secondary aim
of this study was the screening of selected chalcone intermediates as inhibitors of MAO–B.
Results -
Design and synthesis: A series of 2-aminopyrimidines were designed using known active
structures and literature pharmacophores. A molecular modelling study (Discovery Studio
3.1, Accelrys) was further done to investigate the feasibility of these compounds as potential
adenosine A2A antagonists. All of the designed aminopyrimidines were successfully docked
in the binding site of the adenosine A2A receptor. Binding orientations and observed
interactions with important residues in the active site were similar to those observed for
known A2A antagonists. It was therefore concluded that these compounds may be potential
A2A antagonists and the designed compounds were thus synthesised. Structures were
primarily confirmed with nuclear magnetic resonance spectroscopy and mass spectrometry.
MAO-B inhibition studies: Selected chalcones were evaluated using a fluorometric assay
and kynuramine as substrate. The compounds were potent and selective inhibitors of the
MAO-B enzyme with IC50 values ranging between 0.49-7.67 μM. (2E)-3-(3-Chlorophenyl)-1-
(5-methyl-2-furyl)prop-2-en-1-one (1c) was the most potent compound with an IC50 value of
0.49 μM and was approximately 60 times more selective towards MAO-B than MAO-A.
Some preliminary structure activity relationships were derived, for example, phenyl
substitution with an electron withdrawing chlorine group generally resulted in better activity
than substitution with electron donating methoxy groups. Further investigation of structure
activity relationships are however required as a very small series of chalcones were
screened.
Reversibility studies and mode of inhibition: A dilution assay was used to determine whether
compound (1c) binds reversibly or irreversibly to the MAO-B enzyme. This was done by measuring the recovery of enzymatic activity after a large dilution of the enzyme-inhibitor
complex. The results from the reversibility studies showed that the inhibition of the most
potent compound (1c) is reversible as the catalytic activities are recovered to approximately
80% and 50% respectively, compared to the control measured in the absence of an inhibitor.
For the mode of inhibition, sets of Lineweaver–Burk plots were constructed. The Lineweaver-
Burk plots intersected on the y-axis which indicates that compound 1c is a competitive inhibitor
of the MAO-B enzyme.
In vitro adenosine A2A assays: Radioligand binding assays were used to determine the
affinity of the synthesised 2-aminopyrimidines for the adenosine A2A receptor. This assay
was performed with the radioligand [3H]NECA in the presence of N6-cyclopentyladenosine
(CPA). Compounds 2a - 2h showed moderate to weak affinity in the assay, while promising
affinities were observed for compounds 2j - 2n, which all exhibited Ki values below 55 nM.
The compound with the highest affinity was 4-(5-methylfuran-2-yl)-6-[3-(piperidine-1-
carbonyl)phenyl]pyrimidin-2-amine (2m) with a Ki value of 5.76 nM, which is comparable to
the Ki value of 2.10 nM obtained for the known amino-substituted heterocyclic adenosine A2A
antagonist, ZM 241385. The higher affinities of compounds (2j – 2n) could, at least in part,
be explained by the molecular modellling studies. In the docking experiments an additional
hydrogen bond interaction was observed between the amide carbonyl and tyrosine 271
indicating that this structural feature is a major contributing factor to the improved affinity
observed for these derivatives.
In vivo adenosine A2A assays: The haloperidol induced catalepsy assay was used to
determine whether the two compounds with the highest affinity for the adenosine A2A
receptor (2m and 2k) are antagonists of the A2A receptor. These compounds caused a
statistically significant reduction in catalepsy, which clearly illustrate that they are adenosine
A2A antagonists.
The objectives of this study as set out were thus successfully realised and promising results
were obtained. During this study, several novel 2-aminopyrimidines and chalcones were
synthesised, and the respective adenosine A2A antagonistic and monoamine oxidase
inhibitory activities for all of the screened compounds were determined for the first time. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/9534 |
| Date | January 2013 |
| Creators | Robinson, Sarel Johannes |
| Publisher | North-West University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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