Giampiero Spalluto

Riluzole‐derived benzo[d]thiazoles were investigated as metabolite‐inspired CK1 δ inhibitors, a kinase involved in TDP‐43 phosphorylation and therefore relevant to amyotrophic lateral sclerosis (ALS). Starting from the known CK1 δ activity of riluzole, we examined whether its major hydroxylamine metabolite could preserve target recognition and provide a rational entry point for scaffold optimisation. The metabolite retained CK1 δ inhibition, but its limited chemical stability and toxicity in neuronal cells identified the hydroxylamine group as a critical liability. Replacement of this functionality with a hydrazine converted the fragile metabolite‐derived structure into a more potent and tractable CK1 δ inhibitor series. Structure–activity relationship studies revealed that the hydrazine moiety accommodates broader substitution than the hydroxylamine counterparts, with branched alkyl and benzyl groups giving the most balanced profiles. Docking and molecular dynamics suggested that the improvement could be attributed to an additional hydrogen‐bond with Gly86 in the hinge region. The best derivative combined submicromolar CK1 δ inhibition with passive blood–brain barrier permeability, short‐term cellular tolerance, ATP competitive inhibition, direct target engagement, and preliminary selectivity over GSK3β. Although this compound did not result neuroprotective in an oxidative stress model, it paves the way for a focused benzo[d]thiazole platform for probing CK1 δ biology in ALS.

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.

A series of 2,5,7‐trisubstituted [1,2,4]triazolo[1,5‐ a ][1,3,5]triazines (TTs) was developed to identify selective A ₃ AR antagonists. Guided by previous structure–activity relationship studies, the C2‐furyl substituent was replaced with phenyl or 4‐methoxyphenyl moieties to improve A ₃ AR affinity and selectivity. C2 phenyl substitution, together with appropriate C5 and C7 amino substituents, critically modulates receptor recognition. In particular, benzylamino substitution at C5 markedly enhanced A ₃ AR affinity, yielding compound 15 as the most potent antagonist of the series ( K _i = 0.485 nM), whereas incorporation of a 4‐methoxyphenyl group at C2 substantially improved subtype selectivity, exemplified by compound 30 (>2800‐fold selective for A ₃ AR). Molecular docking at the recently determined antagonist‐bound structure of the A ₃ AR rationalized these trends and identified key structural determinants of A ₃ AR preference. The biological relevance of A ₃ AR blockade was investigated in rat hippocampal slices subjected to oxygen‐glucose deprivation. Compound 15 delayed anoxic depolarization and, when administered during ischemia, prevented its onset in most slices and promoted near‐complete recovery of synaptic transmission. These findings identify TT derivatives as promising and highly selective A ₃ AR antagonists with potential neuroprotective applications in cerebral ischemia.

Adenosine plays a crucial role in various pathophysiological conditions, including neuroinflammation and neurodegeneration. Neuroinflammation can be either beneficial or detrimental to the central nervous system, depending on the intensity and duration of the inflammatory response. Across a wide range of brain disorders, neuroinflammation contributes to both the onset and progression of disease. Notably, neuroinflammation is not limited to conditions primarily classified as neuroinflammatory but is also a key factor in other neurological disorders, including life-threatening neurodegenerative diseases. All four adenosine receptor subtypes (A₁, A_2A, A_2B, and A₃) are implicated, to varying degrees, in these conditions. This review aims to summarize the roles of individual adenosine receptor subtypes in neuroinflammation and neurodegenerative diseases, emphasizing their therapeutic potential. While some therapeutic applications are well-established with clinically approved drugs, others warrant further investigation due to their promising potential.

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.

Adenosine receptors are largely distributed in our organism and are promising therapeutic targets for the treatment of many pathologies. In this perspective, investigating the structural features of the ligands leading to affinity and/or selectivity is of great interest. In this work, we have focused on a small series of pyrazolo-triazolo-pyrimidine antagonists substituted in positions 2, 5, and N8, where bulky acyl moieties at the N5 position and small alkyl groups at the N8 position are associated with affinity and selectivity at the A₃ adenosine receptor even if a good affinity toward the A_2B adenosine receptor has also been observed. Conversely, a free amino function at the 5 position induces high affinity at the A_2A and A₁ receptors with selectivity vs. the A₃ subtype. A molecular modeling study suggests that differences in affinity toward A₁, A_2A, and A₃ receptors could be ascribed to two residues: one in the EL2, E168 in human A_2A/E172 in human A₁, that is occupied by the hydrophobic residue V169 in the human A₃ receptor; and the other in TM6, occupied by H250/H251 in human A_2A and A₁ receptors and by a less bulky S247 in the A₃ receptor. In the end, these findings could help to design new subtype-selective adenosine receptor ligands.

The A₃ adenosine receptor is an interesting target whose role in cancer is controversial. In this work, a structural investigation at the 2‐position of the [1,2,4]triazolo[1,5‐c]pyrimidine nucleus was performed, finding new potent and selective A₃ adenosine receptor antagonists such as the ethyl 2‐(4‐methoxyphenyl)‐5‐(methylamino)‐[1,2,4]triazolo[1,5‐c]pyrimidine‐8‐carboxylate (20, DZ123) that showed a K_i value of 0.47 nM and an exceptional selectivity profile over the other adenosine receptor subtypes. Computational studies were performed to rationalize the affinity and the selectivity profile of the tested compounds at the A₃ adenosine receptor and the A₁ and A_2A adenosine receptors. Compound 20 was tested on both A₃ adenosine receptor positive cell lines (CHO‐A₃AR transfected, THP1 and HCT16) and on A₃ negative cancer cell lines, showing no effect in the latter and a pro‐proliferative effect at a low concentration in the former. These interesting results pave the way to further investigation on both the mechanism involved and potential therapeutic applications.

Traditionally, molecular recognition between the orthosteric site of adenosine receptors and their endogenous ligand occurs with a 1 : 1 stoichiometry. Inspired by previous mechanistic insights derived from supervised molecular dynamics (SuMD) simulations, which suggested an alternative 2 : 1 binding stoichiometry, we synthesized BRA1, a bis‐ribosyl adenosine derivative, tested its ability to bind to and activate members of the adenosine receptor family, and rationalized its activity through molecular modeling.