Synthesis of a series of novel 2- aminopyrimidine derivatives and their biological evaluation as adenosine receptor antagonists
Robinson, Sarel Johannes
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Parkinson’s disease (PD) is a progressive, neurodegenerative movement disorder caused by a substantial loss of dopamine in the striatum. This deficiency of dopamine in the brain results in the typical motor symptoms such as muscle rigidity, dyskinesia, tremor and impairment of postural balance. PD patients not only have to deal with the life altering motor deficits, but usually suffer from non-motor symptoms like depression and dementia. Current treatments, which mainly involve dopamine replacement therapies, are symptomatic and do not prevent the progression of PD. These treatments are also associated with numerous side effects that further complicate the lives of patients. These shortcomings have spurred a search for novel, alternative non-dopaminergic therapies. Dual antagonism of adenosine A1 and A2A receptors is a potential, promising non-dopaminergic alternative. Reports indicate that dual antagonism of A1 and A2A receptors will act synergistically to reverse the motor deficiencies of PD. Non-motor symptoms may also be addressed by dual antagonism, as adenosine A1 receptor antagonism is linked to increased cognition, whereas antagonism of the A2A receptor may improve depression symptoms. Neuroprotection, which remains the single, most elusive problem in PD, may also possibly be attained by A2A receptor antagonism in particular. The benefits of dual adenosine A1 and A2A antagonism therefore extend further than the mere symptomatic treatment of the disease and these agents have the potential to influence the progression of PD. The 2-aminopyrimidine chemotype is a privileged scaffold for antagonism of adenosine receptors as this motif frequently occurs in compounds that exhibit potent adenosine A2A and/or adenosine A1 affinity. Selected compounds from a series of 2-aminopyrimidine derivatives designed and synthesised in a previous study exhibited potent adenosine A2A affinities as well as in vivo activity in the haloperidol induced catalepsy assay in rats. The first aim of this PhD study was therefore to determine the adenosine A1 affinities of these compounds and to evaluate their potential cytotoxicity. After identification of 2- amino-4,6-diphenylpyrimidine as a feasible scaffold for the design of dual adenosine A1 and A2A antagonists, the second aim of this study was to further explore the structure-activity relationships of these aminopyrimidines with regards to their potential as dual adenosine A1 and A2A antagonists. The adenosine A1 receptor affinities of the 2-aminopyrimidines synthesised in the preceding study were determined using radioligand binding studies. 1,3-[3H]-Dipropyl-8-cyclopentylxanthine ([3H]DPCPX) was used as a radioligand to determine binding to the A1 receptors. Whole brains obtained from male Sprague-Dawley rats (NWU-0035-10-A5) were used as receptor source. These 2-aminopyrimidines illustrated moderate to good A1 receptor affinities with Ki values ranging from 9.54 nM – 650.1 nM. These compounds are therefore promising dual adenosine A1 and A2A antagonists since potent A2A receptor affinities have been illustrated in the preceding study. These compounds were also not toxic in a preliminary cytotoxicity assay as cell viability was generally still above 70% at a concentration of 10 μM, which is almost 1000 fold higher than the reported Ki values. Three novel series of amide, carbamate and ether substituted 2-amino-4,6-diphenylpyrimidines were further synthesised. The synthesis of the ether and carbamate derivatives involved firstly, the reaction of acetophenone and 3-hydroxybenzaldehyde under basic conditions to yield (2E)-3-(3- hydroxyphenyl)-1-phenylprop-2-en-1-one, as precursor. Similarly, 3-[(1E)-3-oxo-3-phenylprop-1-en- 1-yl]benzoic acid, the precursor for the amide series, was synthesised by reacting 3-formylbenzoic acid and acetophenone under basic conditions. Different carbamoyl chlorides, alkyl chlorides and amines were coupled to the respective precursors to yield chalcone intermediates for the carbamate, ether and amide series, respectively. Cyclisation of these intermediates with guanidine hydrochloride and sodium hydride in DMF afforded the desired 2-aminopyrimidines. Structures were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. The adenosine A1 and A2A receptor affinities of the newly synthesised 2-amino-4,6- diphenylpyrimidines were determined using radioligand binding studies. The non-selective radioligand, [3H]5'-N-ethylcarboxamide-adenosine ([3H]NECA) was utilised in the presence of N6- cyclopentyladenosine (CPA) to assess binding to the adenosine A2A receptor. Striata dissected from male Sprague-Dawley rats (NWU-0035-10-A5) served as receptor source. Evaluation of A1 affinities were done as described above. Compounds from both the amide and carbamate series showed moderate to potent dual adenosine A1 and A2A affinities, while compounds from the ether series were more selective towards the A1 receptor. Ki values for the A1 receptor ranged from 5.42 - 25.2 nM, 0.175 - 10.7 nM and 5.66 – 48.8 nM for the amide, carbamate and ether series, respectively. Moderate A2AKi values of 47.0 – 351 nM were observed for the ethers, whereas the amides and carbamates had superior affinities ranging from 3.37 - 106.5 nM and 1.58 - 451 nM, respectively. Molecular docking studies (C-Docker, Discovery studio 3.1), using the crystal structure of the adenosine A2A receptor (PDB 3EML) were further performed in an attempt to rationalise the results obtained in radioligand binding assays. Unfortunately, the crystal structure of the adenosine A1 receptor is not yet available. Important anchoring interactions, such as those of the exocyclic amino group and Glu169 as well as hydrophobic interactions between the tricyclic ring system and Phe168, were observed for most compounds. The amide and carbamate derivatives, however showed additional interactions between the side chain carbonyl and either Glu169 or Tyr271, located in the binding site, suggesting that this interaction is important for A2A affinity. It is postulated that the decrease in A2A affinity observed for the ether series is a result of the absence of this carbonyl group in the side chain, as this interaction is no longer possible. The compounds with the most promising dual affinities were selected for in vivo screening using the haloperidol induced catalepsy assay. This assay is often used as an indication of A2A receptor antagonism, as administration of known antagonists results in reversal of haloperidol induced catalepsy. At the same time, this assay gives a preliminary indication of bioavailability. The following compounds, [3-(2-amino-6-phenylpyrimidin-4-yl)phenyl-4-methylpiperazine-1-carboxylate, 3-(2-amino-6- phenylpyrimidin-4-yl)phenyl morpholine-4-carboxylate and 3-(2-amino-6-phenylpyrimidin-4-yl)-N- [3-(morpholin-4-yl)propyl]benzamide] were selected and illustrated in vivo activity as catalepsy was attenuated to a significant degree when compared to the control groups. One carbamate compound, 3- (2-amino-6-phenylpyrimidin-4-yl)phenyl-4-methylpiperazine-1-carboxylate had no in vivo activity. Determination of both Log D and water solubility values for this compound indicated that this derivative is highly lipophilic (Log D = 4.03), with low water solubility and it is postulated that these unfavourable physicochemical properties are responsible for the lack of in vivo activity. All objectives as set out, were met successfully as 26 novel 2-amino-4,6-diphenylpyrimidines were synthesised and evaluated as dual adenosine A1 and A2A antagonists. Promising dual adenosine A1 and A2A affinities and good in vivo results were obtained for the newly synthesised derivatives, clearly illustrating the promise of the 2-aminopyrimidines in the potential treatment of PD.
- Health Sciences 
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