Theophylline-based hybrids as acetylcholinesterase inhibitors endowed with anti-inflammatory activity: synthesis, bioevaluation, and preliminary kinetic studies.

In this study, we investigated the conjugation of theophylline with different compounds of natural origin hoping to construct new hybrids with dual activity against cholinergic and inflammatory pathways as potential agents for the treatment of Alzheimer's disease (AD). Out of 28 tested hybrids, two hybrids, acefylline-eugenol 6d and acefylline-isatin 19, were able to inhibit acetylcholinesterase (AChE) at low micromolar concentration displaying IC values of 1.8 and 3.3 μM, respectively, when compared to the galantamine standard AChE inhibitor. Moreover, the prepared hybrids exhibited a significant anti-inflammatory effect against lipopolysaccharide induced inflammation in RAW 264.7 and reduced nitric oxide (NO), tumor necrosis alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels in a dose dependent manner. These hybrids demonstrated significant reductions in nitric oxide (NO), tumor necrosis alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels in RAW 264.7 cells induced by lipopolysaccharide (LPS). The findings of this study were further explained in light of network pharmacology analysis which suggested that AChE and nitric oxide synthase were the main targets of the most active compounds. Molecular docking studies revealed their ability to bind to the heme binding site of nitric oxide synthase 3 (NOS-3) and effectively occupy the active site of AChE, interacting with both the peripheral aromatic subsite and catalytic triad. Finally, the compounds demonstrated stability in simulated gastric and intestinal environments, suggesting potential absorption into the bloodstream without significant hydrolysis. These findings highlight the possible therapeutic potential of acefylline-eugenol 6d and acefylline-isatin 19 hybrids in targeting multiple pathological mechanisms involved in AD, offering promising avenues for further development as potential treatments for this devastating disease.