Biological evaluation of synthetic α,β-unsaturated carbonyl based cyclohexanone derivatives as neuroprotective novel inhibitors of acetylcholinesterase, butyrylcholinesterase and amyloid-β aggregation

doi:10.1016/j.bmc.2016.04.015

Bioorganic & Medicinal Chemistry

Volume 24, Issue 10, 15 May 2016, Pages 2352-2359

https://doi.org/10.1016/j.bmc.2016.04.015 Get rights and content

Abstract

A series of new α,β-unsaturated carbonyl-based cyclohexanone derivatives was synthesized by simple condensation method and all compounds were characterized by using various spectroscopic techniques. New compounds were evaluated for their effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds were also screened for in vitro cytotoxicity and for inhibitory activity for self-induced Aβ_1–42 aggregation. The effect of these compounds against amyloid β-induced cytotoxicity was also investigated. The findings of in vitro experiment revealed that most of these compounds exhibited potent inhibitory activity against AChE and self-induced Aβ_1–42 aggregation. The compound 3o exhibited best AChE (IC₅₀ = 0.037 μM) inhibitory potential. Furthermore, compound 3o disassembled the Aβ fibrils produced by self-induced Aβ aggregation by 76.6%. Compounds containing N-methyl-4-piperidone linker, showed high acetylcholinesterase and self-induced Aβ aggregation inhibitory activities as compared to reference drug donepezil. The pre-treatment of cells with synthetic compounds protected them against Aβ-induced cell death by up to 92%. Collectively, these findings suggest that some compounds from this series have potential to be promising multifunctional agents for AD treatment and our study suggest the cyclohexanone derivatives as promising new inhibitors for AChE and BuChE, potentially useful to treat neurodegenerative diseases.

Graphical abstract

The pharmacological evaluation of compounds comprises BuChE and AChE inhibition, amyloid β-induced cytotoxicity in PC12 cells and effects on self-induced Aβ aggregation.

Introduction

Dementia is one of the main contributors to disability in older patients. Every health system in the world is being significantly affected by dementia, and resources and money are spent for the care of people suffering from this disease.¹ The most common form of dementia is Alzheimer’s disease (AD), characterized by continuous loss of memory and other mental functions. AD usually takes a decade before it gets on course and causes the death of patients in a totally helpless state. The long period of AD causes huge financial and emotional burden on patients and their families.²

The damage to the cholinergic function is of main significance in AD as per ‘cholinergic hypothesis’, especially hippocampus and neocortex of the brain that deal with emotional responses, behaviour, memory and learning. The most evident clinical finding in AD is brain atrophy which is characterized by decreased levels of acetylcholine owing to its fast hydrolysis by an enzyme acetylcholinesterase (AChE).³ Acetylcholinesterase inhibitors are used for the treatment of AD and they work by increasing the acetylcholine level in brain.⁴ The cholinesterase inhibitors (ChE) usually maintain the functions of brain for 10–12 months in almost half of patients.⁵ The main enzyme of central nervous system (CNS) is AChE and was regarded as the ‘cholinergic target’ for AD treatment. Hence, it has gained the attention of researchers for AD treatment owing to its involvement in cholinergic neurotransmission. Earlier, BChE was presumed not having considerable role in cholinergic neurotransmission,6, 7, 8, 9 however, recent research has shown that in addition to AChE, BChE also plays pivotal role in cholinergic neurotransmission.10, 11 Neurons, glia cells and endothelial cells express BChE.¹⁰ During CNS development, AChE and BChE play role to regulate cellular propagation and neurite growth in several species including rodents.¹⁰ Moreover, BChE is also found to have major role in normal CNS.10, 11, 12 According to ‘Amyloid hypothesis’ AChE plays role in secondary non-cholinergic functions such as elevation in the deposition of beta-amyloid (Aβ) as neurofibrillary tangles/senile plaques in the brain of suffering patients.13, 14, 15, 16 Aβ deposition has been discovered to play key role in the commencement and development of AD.¹⁷

Numerous research groups have found that the synaptic and neuronal activities are regulated by Aβ and its accumulation in brain leads to the combination of synaptic depression and aberrant network activity.¹⁸ Abnormal stimulation of glutamate receptors and injury to inhibitory interneurons leads to excitotoxicity, which seems to play key role in this pathogenic cascade.18, 19, 20 Therefore, it is considered that a drug which can dissociate that aggregate can prove to be a vital AD treatment.21, 22

The efforts to integrate the effect of AChE inhibitors and other therapeutic agents acting at numerous phases of AD pathological scenario have yielded positive results.23, 24 Inspired by these findings, the approach of using compounds that can hit multiple pharmacological targets as a single moiety, can give successful outcomes. Natural compound curcumin and its synthetic derivatives have shown the ability to hit multiple pharmacological targets. Curcumin inhibited aggregation and fibril formation of Aβ by binding to small Aβ species and is also reported to reduce amyloid pathology in Alzheimer transgenic mice.25, 26 Recently we have reviewed all reported synthetic analogues of curcumin showing effects on β-amyloid and discussed on their potential as therapeutic and diagnostic agents for AD.²⁷ We have also reported the synthesis of curcumin like α,β-unsaturated carbonyl-based compounds as potent ChE inhibitors for the treatment of AD.²⁸ In this work, we have studied the pharmacological activity of a series of novel curcumin like compounds possessing cyclohexanone moiety. The pharmacological evaluation of compounds comprises BuChE and AChE inhibition, amyloid β-induced cytotoxicity in PC12 cells and effects on self-induced Aβ aggregation.

Section snippets

Cytotoxicity of synthetic compounds in PC12 cells

MTT assay was performed to investigate the effect of compounds on cell viability using PC12 cells. The cells were treated with different concentrations (0.01–100 μM) of α,β-unsaturated carbonyl based cyclohexanone compounds for 24 h and the compounds were found to be nontoxic to PC12 cells at any concentrations tested.

Effects against Aβ-induced cell cyto-toxicity

Effects of compounds on Aβ-induced PC12 cell toxicity were investigated by MTT assay. In comparison to control cells, the Aβ-treated cells exhibited significantly reduced cell

Conclusion

A series of α,β-unsaturated carbonyl-based cyclohexanone derivatives were synthesized and evaluated as multifunctional anti-Alzheimer agents. The pretreatment of PC12 cells with synthetic compounds protected them against Aβ-induced cell death by 92%. Most compounds displayed better inhibitory potencies against Aβ aggregation than reference compounds. Compounds containing N-methyl-4-piperidone linker, showed high acetylcholinesterase and self-induced Aβ aggregation inhibitory activity as

Materials

Sigma–Aldrich, Merck and Acros Organics supplied all chemicals (above 98% purity). The chemicals were used as supplied without any modicication. BChE (E.C. 3.1.1.8, from horse serum, 1000 units), AChE (E.C.3.1.1.7 from Electric Eel, 500 units), donepezil hydrochloride, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were procured from Sigma-Aldrich (St. Louis, MO, USA). Bachem (Bubendorf, Switzerland) supplied Aβ_1–42. 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB), sodium

Notes

The authors declare no competing financial interest.

Acknowledgments

The authors gratefully acknowledge the generous support provided by the Research Incentive Fund of Universiti Kebangsaan Malaysia, Arus Perdana grant (AP-2014-023). H.Q. is grateful to Wuhan University of Technology for financial support.

References and notes (38)

Y. Huang et al.
Cell
(2012)
A. Enz et al.
Prog. Brain Res.
(1993)
E. Giacobini
Pharmacol. Res.
(2004)
F. Cerbai et al.
Eur. J. Pharmacol.
(2007)
M.M. Mesulam et al.
Neuroscience
(2002)
M. Mesulam et al.
Neurobiol. Dis.
(2002)
Q. Sun et al.
Bioorg. Med. Chem.
(2014)
A. Francioso et al.
Bioorg. Med. Chem.
(2015)
A. Manral et al.
Bioorg. Med. Chem.
(2015)
K. Ono et al.
Biochim. Biophys. Acta
(2006)

G.T. Grossberg

Curr. Ther. Res. Clin. Exp.

(2003)

F. Yang et al.

J. Biol. Chem.

(2005)

S.N.A. Bukhari et al.

Eur. J. Med. Chem.

(2014)

G.L. Ellman et al.

Biochem. Pharmacol.

(1961)

S. Rizzo et al.

Bioorg. Med. Chem.

(2010)

Q. Liu et al.

Bioorg. Med. Chem.

(2015)

L. Fang et al.

Bioorg. Med. Chem.

(2016)

W. Luo et al.

Bioorg. Med. Chem.

(2016)

H. Endo et al.

Bioorg. Med. Chem. Lett.

(2014)

Cited by (31)

Novel series of triazole containing coumarin and isatin based hybrid molecules as acetylcholinesterase inhibitors
2021, Journal of Molecular Structure
Citation Excerpt :
The technique is known as molecular hybridization [33] that already provided some bioactive molecules such as, MCB3837, Ro 23–9424, CBR-2092, and TD-1792 which are currently under clinical trials for the treatment of various disease conditions [34]. Moreover, there are also numerous reported molecules that synthesized via molecular hybridization techniques [35,36a,b]. By keeping in mind these particular findings, we also have synthesized two series of novel coumarin-triazole hybrids and isatin-triazole hybrids.
Novel series of coumarin-triazole and isatin-triazole hybrids were rationally designed, synthesized and biologically evaluated to check their inhibitory potential against acetylcholinesterase enzyme by using in vitro Ellman's method. Most of the hybrid compounds showed significant inhibition against the enzyme. Biological assay revealed that compound B-1 (among 4-hydroxycoumarin-triazole series) and compound AS-8 (from isatin-triazole series) possessed potent inhibitory activity against the AChE with the IC₅₀ values of 110 ± 1.11 nM and 155 ± 1.65 nM, respectively. These active compounds (B-1 and AS-8) exhibited mixed mode of enzyme's inhibition which was confirmed through enzyme kinetic studies. Molecular docking studies were performed to understand the binding modes of these potent compounds within the active pocket of AChE enzyme by using Discovery studio. Furthermore, to predict the stability of the most prominent compound B-1 within the catalytic cavity of AChE, molecular dynamic simulations were performed for 5 ns and was found that ligand and protein complex is stable within their dynamic system. Therefore, these hybrids could be taken as effective lead candidates for further designing, development and optimization of new acetylcholinesterase inhibitors.
Design, synthesis and biological evaluation of 2,3-dihydro-5,6-dimethoxy-1H-inden-1-one and piperazinium salt hybrid derivatives as hAChE and hBuChE enzyme inhibitors
2020, European Journal of Medicinal Chemistry
Citation Excerpt :
Indeed, AD is the most common type of dementia due to the defective central nervous system (CNS). AD begins with short-term memory loss and characterized by a progressive decrease or loss of memory, cognitive activities and severe behavioral such as irritability, anxiety, and depression [1–9]. Unfortunately, with an increase in life expectancy around the world, the number of people with AD is growing and expected to be several multiplies until 2050 [10,11].
2,3-Dihydro-5,6-dimethoxy-2-[4-(4-alkyl-4-methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one halide salt derivatives as a novel donepezil hybrid analogs with the property of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzyme inhibition were designed and synthesized via N-alkylation reaction of 2,3-dihydro-5,6-dimethoxy-2-[4-(4-methylpiperazin-1-yl)benzylidene]-1H-inden-1-one with some alkyl halides. Biological tests demonstrated that most of the synthesized compounds have moderate to good inhibitory activities effect on cholinesterase enzymes. Among them, 10e showed the best profile as a selected compound for inhibition of hAChE (IC50 = 0.32) and hBuChE (IC50 = 0.43 μM) enzymes. Kinetic analysis and molecular docking led to a better understanding of this compound. Kinetic studies disclosed that 10e inhibited acetylcholinesterase in mixed-type and butyrylcholinesterase in non-competitive type. The toxicity results showed that 10e is less toxic than donepezil and has better inhibitory activity against hBuChE when compared to donepezil or Galantamine. Other performed experiments revealed that 10e has an anti-β amyloid effect which is capable of reducing ROS, LDH and MDA also possing positive effect on TAC. On the other hand, it has shown a good anti-inflammation effect.
Cheminformatic modelling of β-amyloid aggregation inhibitory activity against Alzheimer's disease
2020, Computers in Biology and Medicine
Citation Excerpt :
The experimental IC50 values (nM) of the dataset compounds were converted to pIC50 (=-logIC50) values for model development purposes. The dataset compounds utilized in this study followed the same experimental protocol (Thioflavin T (ThT) spectrofluorometric assay method) [18–37]. All the compounds were carefully checked and filtered using different software and tools like KNIME (https://dtclab.webs.com/software-tools), MarvinView and MarvinSketch [38].
In the current research, we have developed robust two-dimensional quantitative structure-activity relationship (2D-QSAR) and pharmacophore models using a dataset of 314 heterocyclic β-amyloid aggregation inhibitors. The main purpose of this study is to determine the essential structural features which are responsible for the inhibition of β-amyloid aggregation. Prior to the development of the 2D-QSAR model, we applied a multilayered variable selection method to reduce the size of the pool of descriptors, and the final models were built by the partial least squares (PLS) regression technique. The models obtained were thoroughly analysed by applying both internal and external validation parameters. The validation metrics obtained from the analysis suggested that the developed models were significant and sufficient to predict the inhibitory activity of unknown compounds. The structural features obtained from the pharmacophore model, such as the presence of aromatic rings and hydrogen bond acceptor/donor or hydrophobic sites, are well corroborated with those of the 2D-QSAR models. Additionally, we also performed a molecular docking study to understand the molecular interactions involved in binding, and the results were then correlated with the requisite structural features obtained from the 2D-QSAR and 3D-pharmacophore models.
Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of Alzheimer's disease
2019, Bioorganic Chemistry
Cholinergic hypothesis of Alzheimer’s disease has been advocated as an essential tool in the last couple of decades for the drug development. Here in, we report de novo fragment growing strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline derivatives as acetyl cholinesterase inhibitors. Both type of scaffolds numbering forty compounds were synthesized and evaluated for their potencies against AChE, BuChE and PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE = 1.937 ± 0.066 µM; BuChE = 1.166 ± 0.088 µM; hAChE = 1.758 ± 0.095 µM; P_e = 9.491 ± 0.34 × 10⁻⁶ cm s¹) showed positive results, which on further optimization led to the development of compound 67 (AChE = 0.464 ± 0.166 µM; BuChE = 0.754 ± 0.121 µM; hAChE = 0.472 ± 0.042 µM; P_e = 13.92 ± 0.022 × 10⁻⁶ cm s¹). Compounds 44 and 67 produced significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer to MC65 cells and decreased metal induced Aβ_1-42 aggregation. Further, in-vivo behavioral studies, on scopolamine induced amnesia model, the compounds resulted in better percentage spontaneous alternation scores and were safe, had no influence on locomotion in tested animal groups at dose of 3 mg/kg. Early pharmacokinetic assessment of optimized hit molecules was supportive for further drug development.
Investigating 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole as scaffold of butyrylcholinesterase-selective inhibitors with additional neuroprotective activities for Alzheimer's disease
2019, European Journal of Medicinal Chemistry
Due to the role of butyrylcholinesterase (BChE) in acetylcholine hydrolysis in the late stages of the Alzheimer's disease (AD), inhibitors of butyrylcholinesterase (BChE) have been recently envisaged, besides acetylcholinesterase (AChE) inhibitors, as candidates for treating mild-to-moderate AD. Herein, synthesis and AChE/BChE inhibition activity of some twenty derivatives of 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (HHAI) is reported. Most of the newly synthesized HHAI derivatives achieved the inhibition of both ChE isoforms with IC₅₀s in the micromolar range, with a structure-dependent selectivity toward BChE. Apparently, molecular volume and lipophilicity do increase selectivity toward BChE, and indeed the N²-(4-phenylbutyl) HHAI derivative 15d, which behaves as a mixed-type inhibitor, resulted the most potent (IC₅₀ 0.17 μM) and selective (>100-fold) inhibitor toward either horse serum and human BChE. Moreover, 15d inhibited in vitro self-induced aggregation of neurotoxic amyloid-β (Aβ) peptide and displayed neuroprotective effects in neuroblastoma SH-SY5Y cell line, significantly recovering (P < 0.001) cell viability when impaired by Aβ_1-42 and hydrogen peroxide insults. Overall, this study highlighted HHAI as useful and versatile scaffold for developing new small molecules targeting some enzymes and biochemical pathways involved in the pathogenesis of AD.
Rational approaches, design strategies, structure activity relationship and mechanistic insights for therapeutic coumarin hybrids
2019, Bioorganic and Medicinal Chemistry
Hybrid molecules, furnished by combining two or more pharmacophores is an emerging concept in the field of medicinal chemistry and drug discovery that has attracted substantial traction in the past few years. Naturally occurring scaffolds such as coumarins display a wide spectrum of pharmacological activities including anticancer, antibiotic, antidiabetic and others, by acting on multiple targets. In this view, various coumarin-based hybrids possessing diverse medicinal attributes were synthesized in the last five years by conjugating coumarin moiety with other therapeutic pharmacophores. The current review summarizes the recent development (2014 and onwards) of these pharmacologically active coumarin hybrids and demonstrates rationale behind their design, structure-activity relationships (SAR) and mechanistic studies performed on these hybrid molecules. This review will be beneficial for medicinal chemist and chemical biologist, and in general to the drug discovery community and will facilitate the synthesis and development of novel, potent coumarin hybrid molecules serving as lead molecules for the treatment of complex disorders.

View all citing articles on Scopus

View full text

Biological evaluation of synthetic α,β-unsaturated carbonyl based cyclohexanone derivatives as neuroprotective novel inhibitors of acetylcholinesterase, butyrylcholinesterase and amyloid-β aggregation

Abstract

Graphical abstract

Introduction

Section snippets

Cytotoxicity of synthetic compounds in PC12 cells

Effects against Aβ-induced cell cyto-toxicity

Conclusion

Materials

Notes

Acknowledgments

Cell

Prog. Brain Res.

Pharmacol. Res.

Eur. J. Pharmacol.

Neuroscience

Neurobiol. Dis.

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Biochim. Biophys. Acta

Curr. Ther. Res. Clin. Exp.

J. Biol. Chem.

Eur. J. Med. Chem.

Biochem. Pharmacol.

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Bioorg. Med. Chem. Lett.