A new set of amino-3,5-dicyanopyridines was synthesized and biologically evaluated at the adenosine receptors (ARs). This chemical class is particularly versatile, as small structural modifications can influence not only affinity and selectivity, but also the pharmacological profile. Thus, in order to deepen the structure–activity relationships (SARs) of this series, different substituents were evaluated at the diverse positions on the dicyanopyridine scaffold. In general, the herein reported compounds show nanomolar binding affinity and interact better with both the human (h) A₁ and A_2A ARs than with the other subtypes. Docking studies at hAR structure were performed to rationalize the observed affinity data. Of interest are compounds 1 and 5, which can be considered as pan ligands as binding all the ARs with comparable nanomolar binding affinity (A₁AR: 1, K_i = 9.63 nM; 5, K_i = 2.50 nM; A_2AAR: 1, K_i = 21 nM; 5, Ki = 24 nM; A₃AR: 1, Ki = 52 nM; 5, Ki = 25 nM; A_2BAR: 1, EC₅₀ = 1.4 nM; 5, EC₅₀ = 1.12 nM). Moreover, these compounds showed a partial agonist profile at all the ARs. This combined AR partial agonist activity could lead us to hypothesize a potential effect in the repair process of damaged tissue that would be beneficial in both wound healing and remodeling.

Protein kinase CK1δ (CK1δ) is a serine-threonine/kinase that modulates different physiological processes, including the cell cycle, DNA repair, and apoptosis. CK1δ overexpression, and the consequent hyperphosphorylation of specific proteins, can lead to sleep disorders, cancer, and neurodegenerative diseases. CK1δ inhibitors showed anticancer properties as well as neuroprotective effects in cellular and animal models of Parkinson’s and Alzheimer’s diseases and amyotrophic lateral sclerosis. To obtain new ATP-competitive CK1δ inhibitors, three sets of benzimidazole-2-amino derivatives were synthesized (1–32), bearing different substituents on the fused benzo ring (R) and diverse pyrazole-containing acyl moieties on the 2-amino group. The best-performing derivatives were those featuring the (1H-pyrazol-3-yl)-acetyl moiety on the benzimidazol-2-amino scaffold (13–32), which showed CK1δ inhibitor activity in the low micromolar range. Among the R substituents, 5-cyano was the most advantageous, leading to a compound endowed with nanomolar potency (23, IC₅₀ = 98.6 nM). Molecular docking and dynamics studies were performed to point out the inhibitor–kinase interactions.

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system characterized by oxidative stress, demyelination, and neuronal damage. Current MS therapies are unsatisfactory and new therapies are encouraged. Adenosine is highly implicated in MS as it regulates, via activation of its A_2A receptor (A_2AR), the inflammatory and immune response. The aim of this study is to investigate the therapeutic potential of new selective A_2AR antagonists, P400 and P625, in the experimental autoimmune encephalomyelitis (EAE), the principal animal model of MS. P400 and P625 administration (10 μg/mouse intranasal) for 14 days reduced motor disability (clinical score, rotarod) and thermal (hot plate) and mechanical (von Frey) hypernociceptive symptoms associated with a chronic MS model. Quantitative analysis of lymphocytes infiltration in the spinal cord sections stained with hematoxylin and eosin (H&E) showed a larger number of inflammatory cells in EAE sections that were markedly reduced by P400 and P625. P400 also reduced the immunostaining of Iba1, marker of microglia. Luxol Fast Blue (LFB) staining and myelin basic protein (MBP) immunostaining of spinal cord sections showed a robust loss of myelin that was partially restored by P400 and P625. Both treatments increased spinal neurofilament H (NfH) and GAP43 protein expression compared to untreated immunize mice. These data illustrate the efficacy of the new selective A_2AR antagonists in ameliorating EAE symptoms by attenuating neuroinflammation and demyelination. These findings further highlight A_2AR blocking as a promising perspective to control neurological disturbances in MS patients.

Adenosine receptor (AR) antagonists have attracted considerable interest due to their therapeutic potential in a wide range of pathological conditions, including neurological, cardiovascular, and inflammatory disorders. Although a large number of AR antagonists have been developed worldwide, the interest in new derivatives remains high, and achieving subtype selectivity continue to be a major challenge. This review summarizes our research on adenosine receptor antagonists, highlighting the discovery of potent and selective compounds for the diverse AR subtypes across various chemical classes. Specifically, the paper focuses on the study of the triazolo[4,3-a]quinoxalin-1-one (TQX) and pyrazolo[3,4-c]quinoline (PQ) series, along with their simplified analogues, which have yielded highly potent and selective AR antagonists. An overview of the structure–activity relationship (SAR) studies and molecular docking investigations is provided, emphasizing the structural requirements for A_2A and A₃ receptor–ligand interaction. In addition, we present pharmacological studies of selected AR antagonists, in various in vitro and in vivo models of pain, depression, neuroinflammation-related diseases, and cancer.

Background/Objective: Casein kinase 1δ (CK1δ) plays a central role in regulating circadian rhythms, DNA repair, apoptosis, and cell proliferation, and its dysregulation is associated with both cancer and neurodegenerative diseases, making CK1δ an attractive therapeutic target. This study aimed to develop novel CK1δ inhibitors with favourable drug-like properties through the synthesis and biological evaluation of benzimidazol-2-acetamide derivatives. Methods: A novel series of benzimidazol-2-acetamide derivatives was synthesized, incorporating different substituents on the benzimidazole core (5-chloro, 5,6-dichloro, 5-nitro, and 5-cyano) and various saturated cyclic amines on the acetamide moiety. Compounds were evaluated for CK1δ inhibitory activity, while molecular docking and molecular dynamics simulations were performed to investigate their binding mode. In silico ADME predictions assessed pharmacokinetic properties, and cytotoxicity was evaluated in SH-SY5Y neuroblastoma cells. Results: The 5,6-dichloro derivatives 20 and 24, bearing piperidin-4-ylmethanamine and morpholine moieties, respectively, displayed the highest CK1δ inhibitory activity (IC₅₀ = 0.172 and 0.223 μM). The 5-cyano analogue 22 also exhibited a good potency (IC₅₀ = 0.224 μM). Computational studies confirmed an ATP-competitive binding mode and highlighted the dynamic behaviour of the solvent-exposed substituents within the CK1δ binding site. Predicted pharmacokinetic properties suggested favourable oral absorption and blood–brain barrier penetration. Compounds 20, 22, and 24 showed no cytotoxicity in SH-SY5Y cells at concentrations up to 10 μM. Conclusions: The new benzimidazol-2-acetamide derivatives exhibited good inhibitor activity against CK1δ, favourable predicted ADME properties, and low cellular toxicity. The combined experimental and computational findings provide valuable insights for the further optimization of this scaffold toward the development of therapeutics for CK1δ-related diseases.