Abstract
Imidazo[1,2-b]pyridazine compounds are a new class of promising lead molecules to which we have incorporated polar nitro and amino moieties to increase the scope of their biological activity. Two of these substituted 3-nitro-6-amino-imidazo[1,2-b]pyridazine compounds (5c and 5h) showed potent acetylcholinesterase (AChE) inhibitory activity (IC50 40–50 nM), which we have previously reported. In this study, we wanted to test the biological efficacy of these compounds. Cytotoxicity assays showed that compound 5h mediated greater cell death with over 43% of cells dead at 100 μM and activation of caspase 3-mediated apoptosis. On the other hand, compound 5c mediated a dose-dependent decrease in cell proliferation. Both compounds showed cell cycle arrest in the G0/G1 phase and reduced cellular ATP levels leading to activation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced mitochondrial oxidative stress. It has to be noted that all these effects were observed at doses beyond 10 μM, 200-fold above the IC50 for AChE inhibition. Both compounds also inhibited bacterial lipopolysaccharide-mediated cyclooxygenase-2 and nitric oxide release in primary rat microglial cells. These results suggested that the substituted imidazo (1,2-b) pyridazine compounds, which have potent AChE inhibitory activity, were also capable of antiproliferative, anti-migratory, and anti-inflammatory effects at higher doses.
Highlights
Acetylcholinesterase (AChE) inhibitors have been commonly used to delay the progression of Alzheimer’s disease (AD), one of the most common forms of neurodegenerative disorders characterized by progressive degeneration of cholinergic neurons [1]
Of the twelve substituted imidazo[1,2-b]pyridazine compounds we synthesized [15], the two compounds with the highest AChE inhibitory activity were compounds 5c and 5h (Figure 1). Both compounds were tested for their cytotoxicity in the human neuroblastoma cell line, IMR-32, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay wherein the formation of formazan crystals by viable cells is proportional to their metabolic activity and health (Figure 2a)
At 10 μM, compound 5c did not show any appreciable reduction in cell number but compound 5h significantly reduced survival by 15% (p < 0.001), which increased to 43% at 100 μM (p < 0.001)
Summary
Acetylcholinesterase (AChE) inhibitors have been commonly used to delay the progression of Alzheimer’s disease (AD), one of the most common forms of neurodegenerative disorders characterized by progressive degeneration of cholinergic neurons [1]. Structural modifications of the coumarin heterocycle by fusing the benzofuran ring and substituting the arylamino group (anilino) with one atom linker to the fourth position of the coumarin have been tested as AChE inhibitors with additional pharmacological activities such as a decrease in β-amyloid deposition and β-secretase inhibition [10,11]. One such 4-hydroxycoumarin-1-benzotriazole hybrid compound was reported with selective inhibition of AChE and copper-induced Aβ1-42 aggregation [12]
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