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 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 Ki
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 > pfluorophenyl
> 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. 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 Ki (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. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/10857 |
| Date | January 2013 |
| Creators | Kleynhans, Janke |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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