Researchers discover new spiroindolin-1,2-diazepine derivatives with anti-Alzheimer therapeutic potential

By Neha MathurJul 31 2023Reviewed by Sophia Coveney

In a recent study published in Scientific Reports, researchers designed substituted spiro indolin-1,2-diazepine derivatives '5a–v' with cholinesterase (ChE) inhibitory activities as anti-Alzheimer's disease (AD) agents.

Background

Cholinergic transmission dysfunctions and reduction of acetylcholine neurotransmitters are two major molecular hallmarks of AD. The acetylcholinesterase (AChE) enzyme's catalytic action shortens the extent of acetylcholine presence in the brain cortex and hippocampus.

The increase in butyrylcholinesterase (BChE) during the later stages of AD compensates for the AChE reduction. Yet, AChE- and BChE-driven hydrolysis shortens the acetylcholine duration in the central nervous system (CNS), leading to AD-related psychological deficits. 

Thus, cholinesterase inhibitors that enhance cholinergic transmission could potentially work as AD therapeutics. Here it is important to note that as AD progresses, the routinely used drugs, donepezil, galantamine, rivastigmine, and tacrine, become less and less effective. 

About the study

In the present study, researchers used one-pot, sequential four-component multicomponent reactions (MCRs) to produce the target compounds via green chemistry, i.e., processes and technologies that generate less waste and environmental emissions as they use non-toxic solvents, such as ethanol and water.

The researchers used different spectroscopical techniques to confirm the structures of all these new derivatives. They used Ellman's method for AChE kinetic studies at five different concentrations of compound 5l and acetylthiocholine substrate.

Next, the team examined the inhibitory potential of all derivatives against AChE and BChE. Furthermore, the team performed kinetic, molecular docking, and molecular dynamic (MD) simulation studies of the most potent derivatives to get insights into their behavior against AChE and BChE enzymes. 

The MD simulations helped the researchers understand the effect and structural perturbations of 5l over the AChE enzymatically active site. So, they also analyzed the root mean square deviation (RMSD) of the AChE to evaluate the stability of the protein–ligand complex. Finally, they examined the neurotoxicity of the best-performing ChE inhibitors against the neuroblastoma cell line SH-SY5Y.

Results

The team used a molecular hybridization strategy to design dual-binding inhibitors of AChE (compound B) and different oxindole derivatives (compound C). Indolinone occupies the ChE pockets, and N-containing ring diazepine interacts with the residues of the ChE active site. In the former, the most potent derivative exhibited 32-fold more potency than donepezil; the latter showed promising AChE and BchE potencies.

They also synthesized fused triazolo[1,4]diazepines (compound D) as anti-AD agents and benzodiazepine-1,2,3-triazole derivatives (compound E) as cholinesterase inhibitors. These target compounds exhibited good AChE inhibition and selective inhibitory activities against BChE.

The team tested different solvents for sequential four-component reactions, of which ethanol emerged as the best solvent. Next, they determined optimal reaction conditions to form 5a-v. They used a range of structurally diverse 1,1-enediamines (EDAMs) and isatin derivatives to discover the scope and efficiency of the reaction.

The team synthesized 17 spiro indolin-1,2-diazepine derivatives (5a–v), which showed varying degrees of ChE inhibition in vitro. They performed structure–activity relationship (SAR) studies and categorized these into five types based on the type of substitutions at the R1 position. Kinetic study results confirmed that 5l was a mix-type AChE inhibitor.

Docking studies helped the researchers understand the mechanism through which 5l bound the target enzymes. Its binding pocket was ~20 angstroms (Å) deep and comprised a catalytic activity site (CAS) pocket.

The molecular docking analysis revealed that the derivatives exhibited higher AChE inhibiting activity, with docking scores ranging between -11.390 and -8.475 kcal/mol. Relatively, BChE-inhibiting activity was weaker, with docking scores between -8.181 and -5.272 kcal/mol. Investigating further, the researchers found that potent AChE inhibitors interacted with both critical pockets of AChE, Aspartic acid (Asp)⁷⁴ located in the peripheral anionic site (PAS) pocket and histidine (His)⁴⁴⁷ within the catalytic triad.

Compound 5l favorably interacted with the AChE binding site, thus, reducing the flexibility of the PAS amino acid residues and all residues within the CAS pocket. Thus, the overall RMSF values were lower for the AChE-5l complex than the apo form of the enzyme.

Furthermore, compound 5l showed no toxicity at the tested concentrations, even at a concentration as high as 50 µM. It exhibited a low half-maximal inhibitory concentration (IC₅₀) value against AChE equals 3.98 ± 1.07 µM.

Conclusions

The researchers used a green, efficient sequential four-component synthesis approach to design a novel series of 17 anti-AD compounds, all of which were spiro indolin-1,2-diazepine derivatives. The study protocol used readily available substrates, simple filtration, and washing techniques, which minimized solvent consumption.

In vitro, compounds 5l and 5j showed potent inhibition of AChE and AChE and BChE, respectively, with respective IC50s = 3.98 ± 1.07 µM), and 20.89 ± 2.96 µM and17.37 ± 3.29 µM. Furthermore, compound 5l showed mix-type inhibition and no neurotoxicity up to 50 µM concentration in the SH-SY5Y neuroblastoma cell line. Overall, this compound emerged as a valuable lead that merits further investigations.