Design, synthesis and pharmacological evaluation of new adenosine receptor ligands

The research reported in this Ph.D. thesis, though it has to be deepened, furthers our understanding about the ARs. New AR ligands were developed preferentially belonging to the aminopyridine-3,5-dicarbonitrile series whose structure-activity relationships have been till now poorly investigated. In fact, most of the compounds reported in the literature are included in patent documents. In this work, many derivatives were synthesized and biologically evaluated in binding and functional assays in order to assess affinity and selectivity towards a specific AR subtype, but also to evaluate their pharmacological profile. The data obtained confirmed the peculiarity of this series whose derivatives are not only characterized by a wide range of affinities but interestingly endowed with different degree of efficacies at the different ARs. Based also on molecular modelling studies two set of compounds, named DCP1 and DCP2B were designed preferentially targeting the A1 and A2B AR, respectively. Most of the compounds belonging to the DCP1 set are endowed with high affinity and good selectivity at the hA1 AR. Some selected derivatives showed also an inverse agonist profile with the only exception of compound 15 which emerged as partial agonist. Thus, further modification at the level of the thiazole moiety will be planned in order to evaluate the importance of this substituent for obtaining hA1 AR agonists. Moreover, when evaluated in an oxaliplatin-induced neuropathic pain model in mice, compound 1 (dual A1 inverse agonist/A2A antagonist) resulted to revert allodynia at very low doses after oral administration. This effect is similar to that exerted by both caffeine and other A1/A2A antagonists as well as A1 AR agonists in reverting hyperalgesia and in antinociception. Stability studies in mouse and human plasma on 1 confirmed no significant chemical modification of the compound for the whole duration of the assay. Thus, to clarify this paradoxical data, other molecules belonging to this set, endowed with similar degree of affinities and efficacies with respect to 1, will be tested in this in vivo assay, also to evaluate their potentiality for neuropathic pain treatment. The results obtained on the DCP1 set, especially in the in vivo assay, suggested that the amino-3,5-dicyanopyridine scaffold could be of interest for obtaining non-nucleoside AR agonists as to prevent two serious problems hampering the development of classical adenosine-like agonists: the low oral bioavailability and the short half-life due to the presence of the typical ribose (or ribose-mimicking) moiety. DCP2B set is composed of compounds preferentially targeting the hA2B receptor. Most of them showed to behave as partial agonist, with the best results concerning activity and selectivity of compound 37, which is three fold more potent than the reference agonist BAY60-6583. This result can be considered a real breakthrough since BAY60-6583 is absolutely the sole potent and selective hA2B AR agonist reported in literature. Moreover, it is noteworthy that derivatives 66 and 67, showing high activity at the hA2BAR but scarce selectivity, emerged as the only two amino-3,5-dicyanopyridines reported till now which are endowed with a full agonist profile. With this in mind, a study to clarify the structural requirements which are important in determining the A2B pharmacological profile of these compounds is currently on going. Thus, identification of new selective hA2B agonists could be of help in deepening the medicinal chemistry and the pharmacology related to this still unknown AR subtype. In fact, compound 37 was used as pharmacological tool in an oligodendrocyte precursor cell (OPC) differentiation assay in order to clarify whether the A2B receptor represents a promising target to modulate endogenous remyelinization process in multiple sclerosis patients. Compound 37 inhibits OPC differentiation into oligodendrocytes at lower concentration than those used for BAY60-6583 demonstrating the role of A2B AR in this important physiological process. Moreover, docking studies were performed on selected DCP1 and DCP2B derivatives at A1 and A2B homology receptor models, respectively, showing the best binding poses for compounds 15 and 37 and the most important residues involved in the binding at these two AR subtypes. It seems to clarify the role of R4 and R6 substituents, containing atoms or groups able to engage hydrogen-bonding interactions with the receptors, in modulating affinity and efficacy of the amino-3,5-diyanopyridine derivatives. Moreover, since some affinity was also achieved at the hA2A and hA3 AR with some of the compounds detailed in this thesis, it may be possible that this heterocycle core could be also manipulated to enhance affinity and selectivity for these subtypes. In fact, neither A2A nor A3 agonists belonging to the amino-3,5-dicyanopyridine series has been ever designed. Preliminary studies on the newly synthesized 7-amino-2-phenylpyrazolo[1,5-c]pyrimidine-4-carbonitrile system, derived from molecular complications of the amino-3,5-dicyanopyridine core, were reported. Biological results on a small set of compounds, that bind with good affinity the hA1 AR, indicated that this scaffold suitably decorated could generate new AR ligands.