Development of 9-cinnamyl-9H-purine derivatives as potent anticancer agents through inactivation of NF-κb/p65 and p-STAT1 signaling pathways: Discovery of a new template for adenosine receptor

Based on the structure of resveratrol and curcumin, a novel 9-cinnamyl-9H-purine structure was designed and synthesized to avoid a pan-assay interference compound (PAINS) related to invalid metabolic pancreas activity (IMPS). In a previous study, it exhibited anti-inflammatory activities by disrupting the TLR4-MyD88 protein interaction, leading to the suppression of the NF-κB signaling pathway. In this study, further structure-activity relationship studies were conducted to evaluate 9-cinnamyl-9H-purine derivatives for their potential anticancer activities, as NF-κB inhibition is known to reduce tumor growth and increase cancer cell death. The compounds exhibited varying degrees of anti-proliferative effects in human cancer cell lines, including HT-29, Hep3B, A549, H1299, DU145, PC-3, LNCaP, PANC-1, and MIA PaCa-2. Among the tested compounds, 2,6-chloropurine-fused 5e showed the most potent anti-proliferative activity, with an IC₅₀ value of about 1 μM or lower in various human cancer cell lines. Notably, 5e was seven times more potent than 5-fluorouracil (5-FU), a commonly used anticancer drug, while demonstrating 2.6 times greater safety in normal cells. In chick chorioallantoic membrane xenograft tumor models established with Hep3B and HT-29 cells, 5e exhibited superior antitumor activity compared to 5-FU. Ligand-based target prediction using DeepZema® server identified 5e as a ligand for the A₃ Adenosine receptor (A₃AR). To further explore this interaction, in silico studies were conducted to gain insight into the protein-ligand binding interaction. Among the compounds tested in an adenosine receptor competition binding experiment, 5a was identified as an A₃AR modulator (K_i = 194.7 nM). Western blot analysis revealed that the observed anti-proliferative effect was associated with a concentration-dependent suppression of phosphorylated p65 and phosphorylated STAT1. These findings suggest that the 9-cinnamyl-9H-purine scaffold in 5e holds promise as a therapeutic candidate for various cancers, particularly inflammation-associated cancers such as Crohn's disease-related colorectal cancer and hepatitis-induced liver cancer.