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Medicinal Chemistry
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Synthesis, Biological Evaluation and Molecular Modeling of (E)- 3-Propargylene-1, 3-Dihydro-2H-Indol-2-Ones as Acetyl- and Butyrylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease

Ying Dong1#, Xiao Ming Zha2#, Xue Ping Chu1#, Di Kang2#, Su Lan Luo1#, Tao Lu1* and Qing Fa Zhou1*

1Department of Organic Chemistry, College of Science, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China

2Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China

#These authors contributed equally to this work

*Corresponding Authors:
Tao Lu
Department of Organic Chemistry
College of Science
China Pharmaceutical University
24 Tongjiaxiang, Nanjing
210009, PR China
E-mail: [email protected]
 
Qing Fa Zhou
Department of Organic Chemistry
College of Science
China Pharmaceutical University
24 Tongjiaxiang, Nanjing
210009, PR China
Tel: +0086 025 86185172
E-mail: [email protected]

Received date: May 16, 2016; Accepted date: May 27, 2016; Published date: June 02, 2016

Citation: Dong Y, Zha XM, Chu XP, Kang D, Luo SL, et al. (2016) Synthesis, Biological Evaluation and Molecular Modeling of (E)-3-Propargylene-1, 3-Dihydro-2H-Indol-2-Ones as Acetyl- and Butyrylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease. Med chem (Los Angeles) 6:372-376. doi:10.4172/2161-0444.1000372

Copyright: © 2016 Dong Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The synthesis, pharmacological evaluation and molecular modeling of (E)-3-propargylene-1,3-dihydro-2Hindol- 2-ones, targeting both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), are described. In vitro inhibition experiments of AChE and BuChE showed that compound 2, 5 and 12 are able to inhibit the two forms of cholinesterases in the submicromolar range. The most selective inhibitor of EeAChE (acetylcholinesterase, E.C. 3.1.1.7, from Electrophorus electricus) and eqBuChE (butylcholinesterase, E.C. 3.1.1.8, from equine serum) in this series are compound 9 (IC50=0.011 ± 0.018 μM) and compound 14 (IC50=0.12 ± 0.22 μM) respectively. But the substitution at 5- or 6- position of indolones is not generally favored for eqBuChE inhibition. Kinetic studies of the BuChE inhibition suggested that compound 1 and 5 produce a mixed inhibition pattern. The molecular modeling investigation confirmed the result and indicated that π-π stacking interaction is a main contributor to the increase of inhibition efficiency.

Keywords

Indolones; Acetylcholinesterase; Butyrylcholinesterase; Kinetic analysis; Molecular modeling; Alzheimer's disease

Introduction

Alzheimer's disease (AD) is currently recognized as a complex neurodegenerative disorder which is the most common cause of late life dementia [1]. The typical pathological features of AD include cell loss, senile plaques, and neurofibrillary tangles in the neocortex, hippocampus, amygdala and the basal nucleus of Meynert [2]. The most severe and consistent biochemical change in AD is cholinergic deficit. It is seen as decreased levels of acetylcholine (ACh), choline acetyltransferase (CAT), and acetylcholinesterase (AChE). The reduced activity of both CAT and AChE could often be observed [2,3].

Since the 1970s, a great deal of research has been conducted to develop the cholinesterase inhibitors (ChEIs) for symptoms of this disease. The common mechanism of action for ChEIs is an increase in available synaptic ACh through inhibition of the catabolic cholinesterase. Clinically, approximately 50% of the AD patients show therapeutic effect of ChEIs which is to stabilize cognitive function at a steady level during 12 months of treatment as compared to placebo, this cognitive stabilizing effect can be prolonged up to 24 months in approximately 20% of the patients [4]. Meaningful symptomatic benefit supports ChEIs as the mainstay of pharmacotherapy in AD. AChE and butyrylcholinesterase (BuChE) are two major forms of cholinesterases but differ significantly in substrate specificity, enzyme kinetics, expression and activity in different regions of the brain, both of them can catalyze the hydrolysis of choline esters including ACh and play a collaborative role in cholinergic transmission [5-7]. It was pointed out that the activity of BuChE rises while the activity of AChE remains unchanged or declines in the AD brain of more severe cases [8], so dual or selective inhibitors of AChE and BuChE may hold particular benefits for the patients with different neurobiological characteristics in various stages of AD. In addition, growing evidence suggests that cholinesterase may have many non-cholinergic effects separate from its ‘classical’ function of ACh hydrolysis, which include the regulation of the activity of other proteins, regional cerebral blood flow, tau phosphorylation, and the amyloid cascade [9]. This mechanism may contribute to the patient long-term cognitive stabilization seen during ChEIs treatment [10].

Here we report the synthesis, biological evaluation, investigation on the mode of action, and the molecular modeling studies of (E)-3- propargylene-1, 3-dihydro-2H-indol-2-ones as a new class of dual or selective inhibitors of AChE and BuChE. The preliminary structure– activity relationships (SARs) are also discussed.

Results and Discussion

Chemistry

Various indolones (1~16, Table 1) were prepared by the route outlined in Scheme 1 [11,12]. The initial strategy was to synthesis N-substituted indolin-2-ones v [13]. Isonitrosoacetanilides ii were prepared by the reaction of appropriate anilines i with oxammonium hydrochloride and chloral hydrate in the yield of 55-90%. The subsequent cyclization of ii in the presence of concentrated sulfuric acid gave isatins iii in 60-85% yield. Isatins iii were subsequently alkylated with various alkyl halides to give the corresponding N-substituted isatins iv in the yield of 85-95%. Reduction of iv by using hydrazine hydrate afforded the N-substituted indolin-2-ones v with the yield of 50-91%. Then the alkyne aldehydes viii were prepared from corresponding aldehydes through a Corey– Fuchs reaction in 47-91% yield [14]. The target products (E)-3- propargylene-1, 3-dihydro-2H-indol-2-ones ix (compounds 1~16) were obtained through the condensation of N-substituted indolin-2-ones v with alkyne aldehydes viii in the presence of triethylamine in diethyl ether at room temperature with good to high yields. All these compounds gave analytical and spectroscopic data in agreement with the structures assigned. All derivatives, except 1 and 4, have threearomatic- ring backbone.

Compds. R1 R2 R3 IC50 (μM) EeAChEb IC50 (μM) eqBuChEb Selectivity BuChE/AChE
1 -Me -H -Ph 0.074 ± 0.016 1.12 ± 0.73 15.14
2 -Bn -H -Ph 0.036 ± 0.018 0.29 ± 0.24 8.06
3 -Bn 5-Me -Ph >7.5 >16  
4 -H -H p-Me-Ph >7.5 >16  
5 -Bn 5-F -Ph 0.25 ± 0.21 0.13 ± 0.25 0.52
6 -Bn 5-Cl -Ph 0.44 ± 0.30 >16 >36
7 -Bn 6-Cl -Ph >7.5 >16  
8 -Bn -H naphthalen-2-yl >7.5 >16  
9 -Bn -H 3,4-dimethoxy-Ph 0.011 ± 0.018 >16 >1455
10 -Bn -H phenylethyl 0.026 ± 0.017 >16 >615
11 -Bn -H p-Me-Ph >7.5 >16  
12 -Bn -H p-OMe-Ph 0.97 ± 0.19 0.39 ± 0.46 0.40
13 -Bn -H p-F-Ph >7.5 >16  
14 -Bn -H p-Cl-Ph 6.95 ± 1.7 0.12 ± 0.22 0.017
15 -Bn -H m-Cl-Ph 2.66 ± 0.027 0.36 ± 0.86 0.14
16 -Bn -H o-Cl-Ph >7.5 >16  
Galantamine       0.362 ± 0.31 1.42 ± 1.0 3.92
Tacrine       0.045 ± 0.028 0.0055 ± 0.0023 0.12

Table 1: Yields and inhibitory activities on EeAChE and eqBuChE of compounds 1~16a.

medicinal-chemistry-synthetic-pathway-propargylene

Scheme 1: The synthetic pathway of (E)-3-propargylene-1,3-dihydro-2H-indol-2-ones ix (compounds 1~16). Reagents and conditions: (a) Cl3CCHO, NH2OH·HCl, Na2SO4, HCl, H2O, 40-75°C; (b) concd. H2SO4, 75-80°C; (c) K2CO3, R1Cl, DMF, room temperature; (d) 85% hydrazine hydrate, reflux; (e) Ph3P, CBr4, CH2Cl2, 0°C; (f) n-BuLi, THF, DMF, KH2PO4, -60°C; (g) Et3N, viii, Et2O, room temperature.

Pharmacology

The inhibition of AChE and BuChE: The in vitro EeAChE (acetylcholinesterase, E.C. 3.1.1.7, from Electrophorus electricus) and eqBuChE (butylcholinesterase, E.C. 3.1.1.8, from equine serum) inhibitory potential of these compounds were evaluated based on the Ellman's method [15] using galantamine and tacrine as reference compounds. From Table 1, it can be seen that compound 9 exhibits a significant and selective inhibition of EeAChE (IC50=0.011 ± 0.018 μM) which is more potent than the reference compounds. Compound 14 is the most selective inhibitor of eqBuChE (IC50=0.12 ± 0.22 μM) in this series. In addition, compound 2, 5 and 12 were able to inhibit the two forms of cholinesterases in the submicromolar range.

Kinetic analysis of the BuChE inhibition: In order to explore the mechanism involved in the BuChE inhibition by these indolones a kinetic analysis of the BuChE inhibition by compound 1 and 5 was performed. The Ki, Km, and Vmax values and inhibition types were determined by fitting the kinetic data to four general models of enzyme inhibition (competitive, noncompetitive, uncompetitive and mixed models) by nonlinear regression analysis using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). The Ki and Km values represent the mean ± S.E. Statistically significant difference (F test, p<0.05) was observed between mixed inhibition and other types of inhibitions. The Lineweaver-Burk plot (Figure 1) shows increased slopes and intercepts while inhibition, indicating that the two compounds induced a mixed type of inhibition.

medicinal-chemistry-BuChE-inhibition-compound

Figure 1: BuChE inhibition kinetic profile by compound 1 (A) and 5 (B). Reciprocal plot of velocity versus butyrylthiocholine (BTCh) concentration (0.03-1.0 mg/mL). All points represent the mean of three determinations. Compound 1: Ki=0.26 ± 0.21 mM, Km=1.00 ± 0.23 mM, Vmax=0.12 ± 0.01 mmol/min. Compound 5: Ki=0.65 ± 0.90 mM, Km=1.01 ± 0.26 mM, Vmax=0.10 ± 0.01 mmol/min.

Molecular modeling: As it has suggested that the similar series of derivatives binds to the enzyme in different ways despite the high sequence homology among species, molecular modeling was used to investigate the preferred binding mode adopted by the indolone ligands (1, 2, 5, 9 and 14).

The EeAChE has a narrow, deeply buried active-site gorge. The result of docking 2 and 9 into EeAChE indicates that the binding site of the two compounds might be the peripheral anionic site (PAS) rather than the catalytic binding site (CAS) where the hydrolysis reaction takes place. The PAS is located at the entry to the active gorge and it is a very important structural element responsible for binding of many inhibitors [15]. Stabilization of the ligand-AChE complex may better account for the hydrophobic contacts and π-π interactions. No interaction with the amino acid residues of the catalytic triad (Ser203, His447 and Glu334) was detected. Figure 3 shows that the indolone scaffold of compound 2 is stacked against the aromatic rings of Trp286, and the characteristic π–π stacking for one benzene ring of compound 2 and the residue of Tyr341 is observed. The indolone scaffold of compound 9 is stacked against the aromatic rings of Trp286 and Tyr341 which located at the PAS. Moreover, additional hydrogen bond is observed between compound 9 and Phe295 (Figure 2). All these characters, together with the specific double bond configuration, contribute to the higher AChE inhibitory activity for compound 2 and 9.

medicinal-chemistry-binding-inhibitors-predicted

Figure 2: The binding mode of inhibitors (Compound 2 and 9) at the EeAChE gorge predicted by the docking simulation. The hydrogen bond is represented in dashed green line.

medicinal-chemistry-binding-eqBuChE-predicted

Figure 3: The binding mode of inhibitors (compound 1, 2, 5 and 14) at the eqBuChE gorge predicted by the docking simulation.

As depicted in Figure 3, the best ranked docking solutions revealed that BuChE can effectively accommodate the indolone ligands deeply inside the active-site gorge. Here Trp82 was found to have π-π stacking interaction with the benzene moiety of the ligands (1, 2 and 5). Tyr332 was found to allow for further π-π stacking interaction with the indolone scaffold of the ligands (2, 5 and 14). The ligand-BuChE complex was found to be stabilized mainly by the π-π stacking and hydrophobic interactions with the amino acid residues of the enzyme.

Comparing with the active site of AChE, the active-site gorge of BuChE is larger because the residues of acyl pocket (Phe295, Phe297) in AChE, which responsible for substrate specificity, are replaced by smaller aliphatic residues (Leu286, Val288) [16]. It enables the access of larger molecules to the catalytic center, but could not provide equivalent residues to establish adequately favorable π-π stacking, hydrogen bond or simple hydrophobic interactions that can stabilize the complex. It might be the reason why the molecule with increased width was found to have reduced inhibition of eqBuChE, especially for the 5- or 6-substituted indolones.

As Figure 4 shows, the top-score cluster of compound 1 and 5 that had a significantly lower FullFitness than the others is located near the central region of the active site gorge, especially near the choline binding sites, but not overlapping. According to the kinetic studies, compound 1 and 5 produce a mixed inhibition pattern which means it has a general equation that includes competitive, uncompetitive and noncompetitive inhibition as special cases. That is to say the inhibitor may bind to the eqBuChE whether or not the enzyme has already bound the substrate. As expected, there is a concordance between the results of docking and kinetic studies.

medicinal-chemistry-Superposition-cluster-ligands

Figure 4: Superposition of the top-score cluster of the ligands with butyrate (green stick) and choline (pink stick). A series of snapshots of compound 1 and 5 at the active site of eqBuChE (PDB: 1C2B), and choline, butyrate at the active site of hBuChE (PDB: 1P0M, 1P0I).

Conclusions

Sixteen derivatives of (E)-3-propargylene-1,3-dihydro-2H-indol- 2-ones were synthesized. These compounds were subjected to pharmacological evaluation as multipotent inhibitors of AChE and BuChE. Three compounds (2, 5 and 12) are able to inhibit the two forms of cholinesterases in the submicromolar range. EeAChE affinity and selectivity are maximal for compound 9 (IC50=0.011 ± 0.018 μM). Compound 14 is the most selective inhibitor of eqBuChE (IC50=0.12 ± 0.22 μM) in this series. Kinetic studies of the BuChE inhibition suggested that compound 1 and 5 produce a mixed inhibition pattern. The molecular modeling investigation confirmed the result and indicated that π-π stacking interaction is a main contributor to the increase of inhibition efficiency. Along with further research, the dual and selective inhibitors could be considered as potential drug candidates for the treatment of AD. Moreover, it can be concluded that, regardless of the electronic features, substitution at 5- or 6- position of indolones is not generally favored for eqBuChE inhibition.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant No. 21102179 and 21572271) and the project-sponsored by SRF for ROCS, SEM.

References

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    Posted on Sep 30 2016 at 3:20 pm
    is a very good work
 

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