Synthesis, biological evaluation, and in silico studies of pyridoxal-amiridine hybrids as multitargeting anti-Alzheimer's disease agents.

New conjugates of an anticholinesterase drug, amiridine, linked to vitamin B6 derivatives pyridoximines 3 and pyridoxamines 4 with different lengths of alkylene spacers, were synthesized and assessed as potential multifunctional anti-Alzheimer's disease (anti-AD) agents. All conjugates demonstrated high acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition (IC50: AChE, 0.386-2.53 μM; BChE, 0.031-1.45 μM), but poor activity against off-target carboxylesterase. Conjugates were mixed-type reversible inhibitors of AChE and BChE and displaced propidium from the AChE peripheral anionic site at the level of donepezil. All conjugates inhibited Aβ42 self-aggregation in the thioflavin test, wherein conjugates 3 were more active; inhibition increased with spacer elongation, being greatest for (CH2)8. Results agreed with molecular docking to AChE, BChE and Aβ42. Conjugates exhibited high ABTS•+-scavenging activity comparable to Trolox and the starting pyridoxal. Moreover, compounds 4 were three times more active than their imine analogues 3, which agreed with quantum chemical analysis. Using the example of imine 3c, the possibility of conjugates of this type to bind biogenic metal ions was shown by UV-Vis spectroscopy. Pyridoxamines 4a,b with spacers n = 4,6 were less toxic in general than imines 3a,b toward HEK293T, HepG2, and SHY5Y cell lines. Additionally, conjugates demonstrated neuroprotection in models of hydrogen peroxide and glutamate-induced oxidative stress in neuroblastoma SH-SY5Y cells, where compounds 4a,b were more active than 3a,b. Altogether, the results indicated that the new conjugates possessed potential for further development as multifunctional anti-AD drug candidates.