In the late s Solvay Pharmaceuticals

In the late ‘90s, Solvay Pharmaceuticals discontinued the Phase 2 development of a promising potent and highly selective A1AR antagonist based on the pyrrolopyrimidine scaffold, SLV320 [43] (2, Chart 1, also named derenofylline; Ki hA1AR = 1 nM), for the treatment of acute heart failure [24]. In the last years, some investigations at rat cortex and striatum preparations on the activity of psoralen synthesis derivatives bearing alkyl or cycloalkyl groups in the N6,9-positions and on the activity at human ARs of 8-arylsubstituted 9-methyladenines were reported [32,44,45]. Among these molecules N-0861 (3, Chart 1) represents a prototypical example [46]. More recently, we reported the design, synthesis, and biological evaluation of AR antagonists based on the adenine moiety and presenting different substituents in 2-, 8-, and 9-position that conferred particular AR subtype selectivity [[47], [48], [49], [50], [51], [52]]. Interestingly, among these derivatives, the compound 9-ethyl-8-phenyladenine (4, Chart 1) resulted an human A1AR ligand endowed with nanomolar affinity (Ki = 31 nM) at this receptor subtype [50,53,54]. Starting from these observations and with the aim of developing potent and selective A1AR antagonists, we designed and synthesized 8-phenyladenine derivatives presenting a small alkyl (methyl, ethyl, or propyl) group in the 9-position and different cycloalkyl (c-butyl, c-pentyl, 3-tetrahydrofuryl, and c-hexyl) substituents in the N6-position. Binding studies and functional assays at Chinese hamster ovary (CHO) cells transfected with the human recombinant ARs demonstrated that the newly synthesized derivatives behave as selective A1AR ligands. Among them, the N6-cyclopentyl-9-methyl-8-phenyladenine (17, Scheme 1, Table 1) showed the highest A1AR affinity (Ki = 2.8 nM) and a good selectivity versus the other AR subtypes. This compound was hence selected as prototype to develop a second series of N6-cyclopentyl-9-methyladenine derivatives presenting in the 8-position a substituted phenyl ring or a 3-pyridyl group, with the aim to evaluate the effect of this functionalization on the A1AR affinity and selectivity. The affinities of the compounds were analyzed with the aid of molecular modeling studies performed at the recently published crystal structure of the human A1AR. Furthermore, a set of compounds selected for their good affinity/selectivity profile or chemical-physical properties was tested at functional studies on CHO cells stably transfected with hA1ARs to evaluate their antagonist activity. Based on the observation that the A1AR activation affects the intestinal motility, the same molecules were also tested at functional studies at mouse ileum preparations to evaluate their ability to counteract the inhibitory effect of A1AR activation on the tissue motility. Finally, an in-silico analysis allowed a prediction of their pharmacokinetic profile.
Results and discussion
Conclusions The design and synthesis of N6,8,9-trisubstituted adenine derivatives presenting aromatic and cycloalkyl groups, to mimic the chemical-physical properties of known A1AR antagonists, was undertaken with the aim at developing new potent and selective ligands of this AR subtype. A first series of derivatives was hence synthesized starting from the 8-phenyladenine and inserting a small alkyl chain of various length and cycloalkyl groups in the 9- and N6-position, respectively. Results of radioligand binding studies showed that the obtained compounds are endowed with high affinity for the A1AR, in some cases at low nanomolar level. The introduction of a methyl group and a cyclopentyl ring in the 9- and the N6-position, respectively (17), provided the best combination of A1AR affinity and selectivity. A second step of the work was the development of a further series of derivatives starting from compound 17 and making small modifications at the 8-aromatic moiety. The affinity for the A1AR was not improved but the selectivity versus the A2AAR was increased. Molecular modeling studies provided an interpretation of the pharmacological data and the possible binding mode of the synthesized compounds at the recently published crystal structure of the hA1AR.