Design, Synthesis, and Bioevaluation of Indole Core Containing 2-Arylidine Derivatives of Thiazolopyrimidine as Multitarget Inhibitors of Cholinesterases and Monoamine Oxidase A/B for the Treatment of Alzheimer Disease.

In continuation of our previous study to identify multitarget inhibitors of cholinesterases (ChEs) and monoamine oxidase (MAOs) isoforms, we synthesized and evaluated 2-arylidine derivatives of thiazolopyrimidine for the treatment of Alzheimer disease. Three series of compounds with different linker size and target-anchoring functional groups were synthesized. Compounds 34-37 showed excellent to good AChE and BChE inhibition potential at nanomolar to low micromolar concentration. While all the compounds showed excellent MAO-B inhibition and selectivity relative to MAO-A, compounds 25 and 36 emerged as the most potent MAO-B inhibitors of all the series of synthesized compounds with IC50 values of 0.13 μM and 0.10 μM, respectively. Furthermore, kinetic studies of inhibitor 35 showed mixed inhibition mode. Exploration of structure activity relationship (SAR) revealed the role of functionalities and length of linkers on potency. Acute toxicity evaluation showed the safety of tested compounds up to 2000 mg/kg dose. PAMPA-BBB evaluation showed BBB permeability of the tested compounds, while MTT assay performed on neuroblastoma SHSY5Y cells showed that all the tested compounds are non-neurotoxic in the tested concentrations. Docking studies showed a strong correlation with experimental in vitro results via binding orientations and interaction patterns of the synthesized compounds into the binding sites of target enzymes. We have successfully identified safe, non-neurotoxic, and blood brain barrier permeable multitarget lead compounds for the treatment of AD.

Introduction

Alzheimer’s disease (AD) is a complicated disease with neurodegenerative effects. Owing to a rapid increase in the reported cases of Alzheimer’s within a few years, it is expected that the number of AD patients will increase more than 350 times by about 2050.[1−9]

Various pathological hypotheses have been put forth to explain the onset and progression of AD. Cholinergic deficit, amyloid-β (Aβ) deposits, oxidative stress, tau (τ)-protein aggregation, and MAO-B hyperactivity in gliosis are considered as the molecular causes of AD.[1,10−15] Till now, inhibition of cholinesterases (acetylcholinesterase and butyrylcholinesterase) is proven as the only and effective therapeutic approach to treat AD. This was depicted by the number of FDA approved drugs. From early 1980s to 2000, four out five FDA approved drugs were classified as anticholinesterase inhibitors.[10−15] Tacrine was approved in 1993 but discontinued by FDA after 5 years due to hepatotoxicity reasons.[16]

Human monoamine oxidase (hMAO) is a flavin adenine dinucleotide (FAD) enzyme found in the outer membrane of mitochondria, hence, it is accountable for the digestion of dietary amines along with neurotic-transmitters.[15,17] There are of two distinct isoforms, MAO-A and MAO-B. Both isoenzymes are coded by distinct genes, showing clear tissue distribution, substrate, and inhibition of a definite order. Adrenaline, noradrenaline, and serotonin are preferably catalyzed by hMAO-A, whereas benzylamine and beta-phenylethylamine are catalyzed through hMAO-B. In humans, MAO-A dominates in sympathetic neuro-terminals and mucosa in the intestine, whereas MAO-B is expressed within the brain itself.[18−21]

MAO-B hyperactivity in gliosis results in higher H2O2 and oxidative free radical levels. Selegiline or deprenyl, an anti-Parkinsonian drug, has shown some effects on AD patients in clinical trials.[15,17] Hence, MAO-B inhibitors may be effective for the treatment of neurodegenerative disease including AD. In recent years, concomitant inhibition of cholinesterases and MAO-B is considered as an important strategy for the management of AD. Several dual targeting ligands have been reported in the literature as therapeutic drugs for the treatment of AD. We recently reported fluoxetine and sertraline based multitarget inhibitors of cholinesterases and monoamine oxidase-A/B for the treatment of Alzheimer’s disease.[22] In this study, several compounds possess excellent concomitant in vitro inhibitory activity against ChEs and hMAO-A/B enzymes and thus emerged as optimal multitarget hybrids. Fluoxetine derivative 1 (Figure ) exhibited IC50 values against eeAChE, eqBChE, hMAO-A, and hMAO-B of 0.010 μM, 0.203 μM, 0.181 μM, and 0.015 μM respectively, while sertraline derivative 2 (Figure ) exhibited IC50 values of 0.008 μM, 0.174 μM, 0.311 μM, and 0.031 μM, respectively.

Figure 1

Dual cholinesterases/monoamine oxidase inhibitors.

Mounting evidence showed medications that focus on a single target are ineffective in treating the multifaceted pathophysiology of neurodegenerative disorders. Various molecular scaffolds have been developed to target multiple entities concomitantly such as AChE, BChE, MAO-B, and BACE-1, to slow down the progression of neurodegenerative diseases.[23−26] Structures of the literature-reported dual ChE and MAO A/B inhibitors (3–10) are shown in Figure . Propargyl and benzyl piperidine (3–4, 8–9) containing derivatives showed excellent to good concomitant inhibition of ChEs and MAOs.[27−36]

As discussed earlier, we recently reported propargyl amine, benzylpiperidine (from donepezil) and tacrine based hybrids of fluoxetine and sertraline as multitarget inhibitors of cholinesterases and monoamine oxidase-A/B for the treatment of Alzheimer’s disease.[22] Previously, we also reported desloratadine and carbazole based tricyclic fused ring system as nanomolar concentration dual binding site inhibitors of AChE and BChE.[37] In the current research, we selected 2-aryledine derivatives of thiazolopyrimidine against target enzymes related to AD. Here, we explored the effect of a rigid double bond of the 2-aryledine core of thiazolopyrimidine which mimics the indanone part of donepezil. For a tricyclic ring system, 8-substituted 3,4-diydropyrimidine-2-thione templates were used to obtain various 2-arylidine derivatives of thiazolopyrimidine (also known as thiazolo[2,3-b]quinazoline-3,6-dione) (families A–C in Figure ). The indole ring at position 4 of the pyrimidine ring was selected to enhance the interactions with indole containing amino acid residues (Trp86 and Trp286) of human AChE. On the basis of the structural architecture of active sites of the selected molecular targets (ChEs and MAOs), effects of linkers of various length were also explored. Herein, we report the design and synthesis of 2-arylidine derivatives of thiazolopyrimidine as multitarget inhibitors of cholinesterase and monoamine oxidase A/B for the treatment of Alzheimer disease.

Figure 2

Design strategy for current research: (a) structural features of donepezil; (b) representative structural features of designed families of compounds A–C.

Results and Discussion

Chemistry

First, we synthesized aldehyde derivative by the reaction of 4-hydroxy benzaldehyde (10) and 1-bromo-2-chloroethane (11). The reaction was carried out in acetone using potassium carbonate as base under reflux conditions. The 4-(2-chloroethoxy)benzaldehyde (12) derivative obtained was further reacted with tryptamine (13) in acetonitrile (ACN) to obtain aldehyde derivative 14 with 58% yield (Scheme ).

Scheme 1

Synthesis of Substituted Aldehyde 14

The synthesis of bicyclic dihydropyrimidine-2-thione derivatives (21–24) is shown in Scheme . The synthesis was carried out by the reaction of cyclic 1,3-diketones (15–18), indole-3-carbaldehyde (19), and thiourea (20). Tin(II) chloride dihydrate was used as Lewis acid and acetonitrile (ACN) as solvent, Target bicyclic DHPM-2-thiones (21–24) were obtained in 67–73% overall yield.

Scheme 2

Synthesis of Bicyclic Dihydropyrimidine-2-thione Derivatives (21–24)

Next, we synthesized 2-arylidine derivatives of thiazolodihydropyrimidines. In the literature, there are two strategies to synthesize 2-arylidine derivatives of thiazolodihydropyrimidines.[38,40] Ashoke et al. synthesized 2-arylidine derivatives of thiazolodihydropyrimidines through a three-component reaction using DHPM-2-thione, aryl aldehydes in the presence of anhydrous sodium acetate in acetic acid and acetic anhydride medium.[38,39] Mobinikhaledi et al. reported synthesis of 8,8-dimethyl derivative of dihydropyrimidine-2-thion by the multicomponent Biginelli reaction of dimedone 5,5-dimethylcyclohexane-1,3-dione), aromatic aldehydes, and thiourea. The synthesized compounds were further reacted with ethyl chloroacetate and aromatic aldehydes to yield corresponding 8-substitued 2-arylidine derivatives.[40]

Here, we used indole-3-carbaldehyde (19), 4-(benzyloxy)benzaldehyde (29), and already synthesized 14 (from Scheme ) as aldehyde precursors for the synthesis of target thiazolodihydropyrimidines 25–28, 30–33, and 34–37 (Scheme ).

Scheme 3

Synthesis of 2-arylidine derivatives of thiazolopyrimidine 25–28, 30–33, and 34–37

Reagents and conditions: (i) AcOH/Ac2O, ClCH2COOH/NaOAc.

In Vitro Enzyme Inhibition Assays against ChEs and MAOs

In recent years, the traditional magic bullet (more precisely, one molecule–one target) strategy has experienced some failure specially for the treatment of multifactorial diseases such as Alzheimer’s disease (AD). Drug discovery scientists are now focusing on polypharmacology by modulating more than one target at the same time. In the current research, we designed a strategy to modulate cholinesterases and monoamine oxidases (MAO-A and MAO-B). Starting from the 2-indole derivatives of thiazolopyrimdines (25–28), we increased the tether length by using 4-(benzyloxy)benzaldehyde (target compounds 30–33) and 4-hydroxy derivatives (target compounds 34–37). The purpose was to obtain multitarget directed ligands (MTDLs) to inhibit our selected molecular targets concomitantly. The structures of all the synthesized compounds are shown in Figure .

Figure 3

Chemical structures of all the synthesized compounds.

All the synthesized compounds were assessed for their in vitro cholinesterases (AChE and BChE) and monoamine oxidases (MAO-A and MAO-B) inhibition activity. The results of the in vitro activities in terms of IC50 values (in μM) are presented in Table . For cholinesterases, Ellman’s method was used to assess the eeAChE and eqBChE inhibition potential of these compounds. Donepezil was used as a positive control, while for MAO-A and MAO-B assays, marketed drug safinamide was used as positive control.

Table 1

Results of In Vitro Enzyme Inhibition Studiesa

	IC50 (μM) ± SEM			IC50 (μM) ± SEM
cmpd no.	eeAChE	eqBChE	SI	hMAO-A	hMAO-B	SI
25	10.36 ± 1.03	17.21 ± 1.19	1.6	0.41 ± 0.11	0.13 ± 0.01	3.1
26	6.21 ± 0.16	19.34 ± 1.14	3.1	0.57 ± 0.08	0.24 ± 0.01	2.4
27	0.97 ± 0.10n	13.67 ± 1.21	14.1	9.36 ± 1.01	0.47 ± 0.01	19.9
28	0.89 ± 0.10	10.51 ± 1.09	11.8	36.94 ± 1.22	0.31 ± 0.01	119.2
30	4.67 ± 0.29	8.97 ± 0.77	1.9	0.48 ± 0.03	0.37 ± 0.01	1.3
31	3.71 ± 0.11	13.88 ± 0.99	3.7	0.63 ± 0.04	0.38 ± 0.01	1.6
32	0.86 ± 0.14	1.70 ± 0.11	1.9	11.23 ± 1.30	0.81 ± 0.01	13.9
33	0.79 ± 0.09	1.01 ± 0.06	1.3	48.38 ± 2.28	0.67 ± 0.01	72.2
34	0.12 ± 0.01	0.44 ± 0.01	3.7	1.50 ± 0.01	0.28 ± 0.01	5.3
35	0.042 ± 0.01	0.63 ± 0.07	15.0	1.93 ± 0.01	0.33 ± 0.01	5.8
36	0.081 ± 0.03	1.39 ± 0.06	17.2	NA	0.10 ± 0.01
37	0.069 ± 0.01	0.98 ± 0.11	14.2	NA	0.14 ± 0.01
donepezil	0.05 ± 0.01	5.4 ± 0.27	108
safinamide				8.16 ± 1.310	0.03 ± 1.075	272

All values are taken as mean ± SEM (n = 3), SI = selectivity index = IC50 of eqBChE/IC50 of eeAChE and IC50 of hMAO-A/IC50 of hMAO-B.

For AChE inhibition, compounds 25–28 and 33–37 from the first two series exhibited inhibition in the range of micromolar to submicromolar concentration, while compounds 34–37 showed inhibitory potential at nanomolar concentrations. Compounds 35–37 were found to be the more active compounds of this series with IC50 values of 0.04 μM, 0.08 μM, and 0.07 μM, respectively. On the other side, all the compounds exhibited moderate to good eqBChE inhibition. Compounds 34 and 35 from series 3 showed inhibition potential of 0.44 μM and 0.63 μM. Structure activity relationship (SAR) analysis also showed that the presence of benzyloxy benzylidene ring compounds (30–33) enhances the inhibition of cholinesterases compared with indolyl benzylidene containing compounds (25–30). Moreover, 8-phenyl and 8-(4-methoxyphenyl)-containing compounds emerged as more potent cholinesterase inhibitors. However, compound 35 with 8,8-dimethyl group, exhibited very high inhibition potential against eeAChE (IC50 = 0.042 μM). In general, increasing the bulk by increasing linker length favors the inhibition as depicted from IC50 values of compounds.

For the identification of multipotent hybrid compounds, the inhibitory activity against human MAO-A and MAO-B was also determined. The results of the activities are compared with those of Safinamide. In the literature, a number of privileged structures of heterocycles (pyrazolines, coumarins, etc.) have been used extensively as inhibitors of MAO isoforms. We are using arylidene derivatives of thiazolopyrimidine for the first time as multitarget inhibitors of monoamine oxidase A/B. Results obtained from the study are excellent. All the compounds showed excellent MAO-B inhibition relative to MAO-A. This is also depicted in a selectivity index profile presented in Table . Compounds 25, 36, and 37 emerged as the most potent compounds of all the series of synthesized compounds against MAO-B with IC50 values of 0.13 μM, 0.10 μM, and 0.14 μM, respectively. Although, a few compounds showed good MAO-A inhibition, the remaining compounds exhibited moderate to poor MAO-A inhibition. Compounds 36 and 37 were not able to show MAO-A inhibition activity at tested concentration.

Determination of Kinetic Parameters for Compound 35

The synthesized compound showed a strong inhibitory potential against acetylcholinesterase and the inhibitory effect was revealed from the calculated Vmax and Km values, and these were determined using Michaelis–Menten kinetics and further confirmed from the Lineweaver–Burk plots. The analysis of the Lineweaver–Burke double reciprocal plot of 1/velocity versus 1/substrate (Figure ) shows that the slopes are increasing at increasing concentrations of compound 35, and are intersecting above the x-axis, thus indicating a mixed-type inhibition for 35. Using the linear transformation of reciprocal enzyme rates versus inhibitor concentrations, the Ki value was calculated as 12 nM for the compound 35.

Figure 4

Lineweaver–Burke double reciprocal plot for the compound 35.

Cell Viability Assay

We evaluated compounds for their cytotoxicity potential against normal human embryonic HEK-293 cells model using MTT assay. The results presented in Figure showed that the tested compounds under study do not have any significant toxic effect on cell viability and are thus considered as safe toward this noncancerous cell line.

Figure 5

Various synthesized compounds induced concentration-dependent cytotoxicity on the cell viability of HEK-293 cells as obtained from MTT assays. Two-way ANOVA and the Bonferroni test were followed. Data were represented as mean ± S.E.M.; all the values were not-significant (ns) to that of the control group.

Acute toxicity

We selected compounds four most active (25, 30, 35, and 37) from all series as representative compounds for further acute toxicity studies. The specifications of animal grouping and dose for toxicity studies are presented in Table . We performed acute toxicity for four selected compounds at doses from 50 to 2000 mg/kg body weight on eight groups containing eight animals per group (i.e., eight animals per compound in each group). All animals were found alive, and there was no clinical sign in the central nervous system, mucous membrane, fur, skin, autonomic nervous system. Moreover, no signs of tremors/convulsions, drowsiness, and other abnormal behavior were found in tested animals in the given doses. For acute oral toxicity, doses between 300 and 2000 is in Category 1 V and is considered as safe and harmless.[41]

Table 2

Specification of the Animal Grouping and Drug Quantity Given for the Acute Toxicity Studies with Various Compounds

no. groups	animals	tested synthesized compounds (25, 30, 35, and 37)
1	8	50
2	8	100
3	8	200
4	8	300
5	8	400
6	8	500
7	8	1000
8	8	2000

PAMPA BBB Assay

Blood brain barrier (BBB) penetration is a major concern for the development of therapeutics to treat AD. Here, we performed parallel artificial membrane permeation assay (PAMPA) by using reported methods.[42,43] For this purpose, we selected compounds 25 (most active MAO-A/B inhibitor), 35, and 37 (most active AChE and BChE inhibitors). The results of PAMPA BBB evaluation are summarized in Table . All the tested compounds showed BBB penetration. These results may be attributed to the presence of hydrophobic functional groups.

Table 3

PAMPA-BBB Permeability (P) Values for the Standard Drug Donepezil, Potent Compounds, and Commercial Drugs with the Prediction of their BBB Penetration

cmpd label	permeability (PAMPA-BBB)aPe(tested) (10–6cm/s)	prediction (PAMPA-BBB) (CNS+b, CNS–c)
Evaluation of Pe (10–6 cm/s) for the Test Compounds and Standard
25	6.20 ± 0.06	CNS+
35	7.45 ± 0.23	CNS+
37	7.10 ± 0.12	CNS+
donepezil	6.50 ± 0.14	CNS+
Validation of the Model by Seven Commercial Drugs
verapamil	14.00 ± 0.20	CNS+
progesterone	8.70 ± 0.65	CNS+
diazepam	15.30 ± 0.12	CNS+
dopamine	0.18 ± 0.03	CNS-
atenolol	0.75 ± 0.10	CNS-
alprazolam	5.60 ± 0.21	CNS+
lomefloxacin	1.12 ± 0.09	CNS-

Data represent are the assay mean for the marketed drugs (n = 3).

‘CNS+’ (prediction of high BBB permeation); P (10–6 cm/s) > 4.39.

“CNS-” (prediction of low BBB permeation); P (10–6 cm/s) < 1.78.

Neurotoxicity Assay

To determine the neurotoxicity of our synthesized compounds, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on neuroblastoma SHSY5Y cells according to our previously reported procedure.[44] Cell viability was determined at concentration ranges of 1, 10, 20, and 40 μM. Donepezil was used as positive control. The results are summarized in Table . All the tested compounds were found non-neurotoxic.

Table 4

Cell Viability of the Synthesized Tested Compounds at Various Concentrations in Neuroblastoma SH-SY5Y Cell Linea

	cell viability (percent)
cmpd label	1 μM	10 μM	20 μM	40 μM
25	97.73 ± 1.03	96.81 ± 1.15	96.03 ± 1.33	95.38 ± 1.74
35	98.90 ± 1.23	95.21 ± 1.54	94.88 ± 1.01	91.61 ± 1.59
37	96.06 ± 0.98	92.13 ± 1.09	90.33 ± 0.83	88.15 ± 1.55
donepezil	96.99 ± 1.18	94.77 ± 1.29	82.41 ± 1.03	76.63 ± 1.37

Values are presented as the percent cell viability (±SD) of at least three separate experiments of SH-SY5Y cells cultured with increasing dose of synthesized test compounds.

Docking Studies

Binding orientations and interactions of synthesized compounds with amino acid residues of all selected targets were determined by using docking studies. Three-dimensional (3-D) crystal structures of all the target enzymes were downloaded from protein data bank (PDB). The PDB accession codes of the downloaded enzymes are 4EY7 for hAChE, 4BDS for BChE, 2Z5X for MAO-A, and 2 V5Z for MAO-B.

Docking protocol validation was carried out by using a redock method. All the native ligands were extracted and redocked into the binding sites of downloaded and prepared enzymes. Binding orientations and interaction patterns of redocked and experimental ligands were compared. Protocols with root-mean square deviations (RMSD) less than 2.0 Å were selected for further docking studies.

The prepared 3-D structures of all the synthesized compounds were docked into the binding sites of enzymes. The analysis of binding orientations was performed by using two-dimensional interaction plots obtained via discovery studio visualizer.

In the protein data bank (PDB), a number of three-dimensional crystal structures for AChE from different species (human, Torpedo californica and Electrophorus electricus) have been reported. For current study, we performed docking studies on human AChE (hAChE). In the binding site of hAChE, all the compounds act as dual binding site/nonclassical inhibitors by interacting with the amino acid residues present in the peripheral (PAS) and catalytic active site (CAS) residues via π–π stacking and hydrogen-bond interactions. These types of interactions may result in the prevention of Aβ-aggregation.[22,39,45−47] The representative 2-D interaction plots of most active AChE inhibitors 35 and 37 are shown in Figure . PAS residues (Tyr72, Tyr124, Trp286, Tyr33, and Tyr341) and CAS residues (Trp86, Phe338) were involved in hydrophobic as well as hydrogen-bond interactions. Compounds 35 and 37 exhibited five π–π stacking interactions (Figure a,b). Trp86, Tyr124, Trp286, Tyr341, and Phe338 interacts with aromatic rings of compounds 35 and 37 via π–π stacking interactions. Gly120 and Tyr133 form hydrogen-bond interactions with compound 35. Compound 37 showed only one hydrogen-bond interaction with Gly120. Binding orientations of compounds in the binding site of BChE were also explored.

Figure 6

2-D interaction plot of the compounds 35 and 37 into the binding site of human AChE (PDB ID = 4EY7).

Furthermore, a comparison of interaction plots of most potent (34) and less potent (28) BChE inhibitors was carried out. The 2-D interaction plots are shown in Figure . Compound 28 interacts with CAS residueTrp82 and residue present in the oxyanion hole (Gly115). Carbonyl oxygen showed hydrogen bond interaction with PAS residue Ser72 (Figure a), while compound 34 interacts with CAS residues Trp82 and Phe329 via π–π stacking interactions. PAS residue Tyr332 and oxyanion hole residues Gly115 and Gly116 form hydrophobic interactions (Figure a).

Figure 7

2-D interaction plot of the compounds 28 and 34 into the binding site of human BChE (PDB ID = 4BDS)

Docking studies were also carried out on MAO isoforms to evaluate the binding orientations and interaction pattern of experimentally tested synthesized derivatives. 2-D interaction plots of compounds 25 and 30 in the binding site of MAO-A are shown in Figure . The studied compounds interact with Gly66, Phe208, Cys323, Phe352, Cys406, Tyr407, and Tyr444. Ala68 and Tyr69 interact via hydrogen-bond interactions, while cysteine residues (Cys323 and Cys406) interact through π–sulfur interactions. The interaction plots of the compounds 25 and 36 in the binding site of MAO-B are shown in Figure . The thiazolopyrimidine ring of both compounds oriented toward the substrate cavity. Carbonyl oxygen atoms interact with Ser59, Tyr60 via hydrogen-bond interactions, while tricyclic rings establish π–π stacked interactions with Tyr398 and Tyr435. Cysteine residues Cys172 and Cys397 interact through π–sulfur interactions. The indolyl benzylidene group of compounds forms π–π stacked interactions with entrance cavity residue Tyr326, while the −NH group interacts with entrance cavity residue Ile199 via hydrogen-bond interactions.

Figure 8

2-D interaction plot of the compounds 25 and 30 into the binding site of MAO-A (PDB ID = 2Z5X).

Figure 9

2-D interaction plot of the compounds 25 and 36 into the binding site of MAO-B (PDB ID = 2V5Z)

Conclusions

The multifactorial nature of Alzheimer’s disease requires exploration of new multitargeted therapeutics due to failure of clinical drug candidates. In continuation of our previous study to identify multitarget inhibitors of ChEs and MAOs, we synthesized and evaluated 2-arylidine derivatives of thiazolopyrimidine as multitarget inhibitors of cholinesterases and monoamine oxidase A/B for the treatment of Alzheimer disease. Three series of compounds with different linker size and target-anchoring functional groups were synthesized. Compounds 25–28 and 33–37 from the first two series, exhibited eeAChE inhibition in the range of micromolar to sub-micromolar concentration, while compounds 34–37 showed inhibitory potential at nanomolar concentration. All the compounds showed excellent MAO-B inhibition and selectivity relative to MAO-A. From all the series of compounds, 25 and 36–37 emerged as the most potent inhibitors of human MAO-B with IC50 values of 0.13 μM, 0.10 μM, and 0.14 μM, respectively. Structure activity relationship (SAR) studies revealed the role of functionalities and length of linkers. The presence of benzyloxy benzylidene ring compounds (30–33) enhances the inhibition of cholinesterases compared with indolyl benzylidene containing compounds (25–30). Moreover, 8-phenyl and 8-(4-methoxyphenyl)-containing compounds emerged as more potent cholinesterase inhibitors.

Acute toxicity evaluation showed the safety of tested compounds up to 2000 mg/kg dose. PAMPA-BBB evaluation showed BBB permeability of the tested compounds, while MTT assay performed on neuroblastoma SHSY5Y cells showed that all the tested compounds are non-neurotoxic in the tested concentrations.

Docking studies were also carried out to correlate the experimental results. The binding pattern in the active site of AChE showed interaction with the amino acid residues present in peripheral (PAS) and catalytic active site (CAS) residues via π–π stacking and hydrogen-bond interactions. These dual binding sites/nonclassical types of interactions may result in the prevention of Aβ-aggregation.

Materials and Methods

General materials and methods, synthetic procedures, 1H NMR, 13C NMR, HPLC data, CHN analysis data, and experimental procedures for pharmacological evaluations (in vitro AChE/BChE, MAO-A/MAO-B inhibition, neurotoxicity and PAMPA-BBB assays) are presented in Supporting Information.

Ethical Statement

The authors have obeyed the Ethical Guidelines for the Animal Studies. All of the experimental procedures were permitted by Ethical Committee via ref No. DREC/20200405/06. After the experimental procedures, the animals were euthanized properly as per the standard procedure using AVMA Guidelines for the Euthanasia of Animals. Halothane vapors were slowly given to the animals to induce anesthesia; however, overdose for a prolonged time euthanized the animals.