Coumarin derivatives as potential inhibitors of acetylcholinesterase: Synthesis, molecular docking and biological studies

Coumarin derivatives as potential inhibitors of acetylcholinesterase: Synthesis, molecular docking and biological studies

Accepted Manuscript Coumarin derivatives as potential inhibitors of acetylcholinesterase: Synthesis, molecular docking and biological studies Shaffali...

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Accepted Manuscript Coumarin derivatives as potential inhibitors of acetylcholinesterase: Synthesis, molecular docking and biological studies Shaffali Singla, Poonam Piplani PII: DOI: Reference:

S0968-0896(16)30584-3 http://dx.doi.org/10.1016/j.bmc.2016.07.061 BMC 13174

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

3 June 2016 27 July 2016 28 July 2016

Please cite this article as: Singla, S., Piplani, P., Coumarin derivatives as potential inhibitors of acetylcholinesterase: Synthesis, molecular docking and biological studies, Bioorganic & Medicinal Chemistry (2016), doi: http:// dx.doi.org/10.1016/j.bmc.2016.07.061

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Coumarin derivatives as potential inhibitors of acetylcholinesterase: Synthesis, molecular docking and biological studies Shaffali Singlaa and Poonam Piplani a* Author affiliations: a

University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014,

India. *

Corresponding author(s) names complete affiliation/address, along with phone, fax and

email. Prof. Poonam Piplani, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014,

India.

E-mail:

09357036068, Fax No.: 91 172 2543101

[email protected],

Telephone

No.:

Abstract A series of novel hybrids has been synthesized by linking coumarin moiety through an appropriate spacer to various substituted heterocyclic amines and evaluated as dual binding site acetylcholinesterase inhibitors for the treatment of cognitive dysfunction caused by increased hydrolysis of acetylcholine and scopolamine induced oxidative stress. Anti-amnesic activity of the compounds was evaluated using morris water maze model at a dose of 1 mg/kg with reference to the standard, Donepezil. Biochemical estimation of oxidative stress markers (lipid peroxidation, superoxide dismutase, and plasma nitrite) was carried out to assess the antioxidant potential of the synthesized molecules. Among all the synthesized compounds (15a-i, 16a-d, 17a-b), compound 15a [4-[3-(4-phenylpiperazin-1-yl)propoxy]-2H-chromen2-one] displayed significant antiamnesic activity, AChE inhibitory activity (IC 50 = 2.42 µM) and antioxidant activity in comparison to donepezil (IC50 = 1.82 µM). Molecular docking study of 15a indicated that it interacts with all the crucial amino acids present at the CAS, mid-gorge and PAS of TcAChE resulting in increased inhibition of AChE enzyme.

Keywords Cognitive

dysfunction,

coumarin

moiety,

heterocyclic

acetylcholinesterase inhibition, Morris water maze

amines,

oxidative

stress,

1. Introduction The average life expectancy has increased in modern times, and with this the elderly are at an increased risk to suffer from dementia [1,2]. Cognitive dysfunction is one of the main symptoms

accompanying

ageing,

stroke,

toxicant

exposure,

head

injury

and

neurodegenerative diseases like schizophrenia, depression, Alzheimer’s disease (AD) and Parkinsonism [3]. AD is thought to be the leading cause of senile dementia affecting nearly 50 million people worldwide [4]. This complex neurodegenerative disorder affects the brain regions which are associated with higher mental functions, particularly the neocortex and the hippocampus, resulting in symptoms like delusion, disorientation, forgetfulness, mental confusion, aggression, restlessness, depression and hallucinations [5-7]. Hence, the development of an effective therapeutic treatment is of utmost importance. [8]. The past two decades have witnessed considerable research efforts devoted to unraveling the molecular, biochemical, and cellular mechanisms of cognitive deficit. Several factors both genetic and environmental e.g. reduced levels of acetylcholine, amyloid β-protein aggregation, tau-hyperphosphorylation, hypoxia etc. have been considered to play a crucial role in cognition decline [9,10]. Till date, the most promising approach for symptomatic relief is to inhibit the acetylcholinesterase (AChE) enzyme, which catalyzes the hydrolysis of acetylcholine (ACh) through its catalytic anionic site, thereby, increasing the synaptic levels of ACh in the brain. AChE inhibitors such as tacrine (1), rivastigmine (2), galanthamine (3), and donepezil (4) are the mainstay drugs for the clinical management of AD [11]. However, such drugs have limited success in alleviating the cognitive deficits, emphasizing the need for a better understanding of the way in which changes in the central cholinergic systems are related to both the AD symptomatology and the disease progression [12,13]. NH2

N

CH3 H3C

N

H3C

O

N

CH3 CH3

O

(1)

(2)

OH

O O N CH3

RO

(3)

N

H3CO H3CO

(4)

X-ray crystallographic structure of AChE has revealed (PDB ID: 1EVE) four main binding sites i.e. esteratic subsite at the bottom of the active site gorge (Ser200, His440, and Glu327); an anionic substrate (AS) binding site having Trp84, Glu199, and Phe330 amino acids; an acyl pocket (Phe288, and Phe299) and peripheral anionic site (PAS) with Trp279, Tyr70, Tyr121, Asp72 and Phe290 amino acids (Figure 1) [14,15].

Figure 1: Binding regions of the AChE enzyme From the literature findings, a new role of AChE has been suggested that it induces the amyloid fibril formation through its peripheral anionic site (PAS) by forming AChE-amyloid β complex [16-19]. The accumulation of Aβ in different areas of the brain exhibits its neurotoxic effects through inflammation, calcium dysregulation and activating microglial cells which in turn cause oxidative stress [20]. Due to the involvement of various factors in the progression of the disease, modulation of a single factor might not be sufficient to produce desired efficacy. This triggered an interest towards the design of multifunctional molecules which can interact with both the catalytic and the peripheral binding sites of AChE, thereby, increasing the synaptic availability of ACh in the brain regions and decreasing the deposition of Aβ. Dual binding site AChE inhibitors have therefore been perceived as a promising drug strategy for the effective management of cognitive dysfunction. Coumarin moiety has gained substantial interest in medicinal chemistry due to its reasonance stability and solubility in various organic solvents [21,22]. 4-Hydroxycoumarin symbolises as an important precursor in the monarchy of organic synthesis because it constitutes the structural nucleus of many natural products [23]. Coumarin analogues have shown a remarkably broad spectrum of pharmacological activities such as anticoagulant, antibacterial, antifungal, antiprotozoal, insecticidal, fungicide, antimycobacterial, antimutagenic, HIV protease inhibition, monoamine oxidase (MAO)

inhibition and anti-inflammatory activity [24-27]. Number of evidences also shows the implication of coumarin in the inhibition of AChE enzyme because the planarity and aromaticity of the coumarin ring is the key structural feature that allows its interaction easily with PAS through possible π-π stacking [28-30]. Ensaculin (5), a coumarin derivative containing a piperazine ring with a three carbon linker has shown improvement in memory and cognitive functions including lowering down of progressive neurodegeneration [31]. O

O

OCH3 O

N

OCH3 N

(5)

Piazzi et al. have designed coumarin derivatives which can bind to both the catalytic and the peripheral sites of the AChE enzyme. Coumarin has been linked to the benzylamino group (an important constituent of donepezil and galanthamine) through a phenyl ring to yield AP2238 (6) which was found to be a potent AChE inhibitor with an IC50 value of 44.5 nM [32]. The same group of scientists synthesized another compound AP2243 (7) by replacing the methyl substituent on the basic nitrogen of benzylamino group of AP2238 with an ethyl group resulting in higher potency (IC50 = 18.3 nM) than AP2238 [33]. H3CO

O

O H3CO

H3CO

CH3 N

(6)

O

O

H3CO

(7)

N

Similarly, more hybrids of coumarin have been developed by incorporating moieties like tacrine, ensaculin structural fragments and substituted piperazines. Most of these compounds showed promising acetylcholinesterase inhibitory activity [34-37]. The coumarin moiety was thus found to possess high anti-AChE potency and its choice was favoured for several other reasons like its peripheral binding ability, antioxidant activity and an excellent therapeutic profile. Furthermore, it also provides protection to neurons against Aβ-induced oxidative stress and free radicals. Easy functionalization of the aromatic ring of coumarin makes it an attractive synthetic building block for designing newer drugs.

Interactions with the residues of mid-gorge site

Coumarin moiety O

O

Heterocyclic amines Linkers

N

R'

O O

Interactions with PAS

N R

Interactions with catalytic site or acyl binding pocket

N H

O O

R = Phenyl, 2-fluorophenyl, 4-fluorophenyl,benzyl, 2-pyrimidyl, 2-pyridyl R' = H, 3, 4-OCH3

Figure 2: Designing strategy of the target compounds Besides, various literature reports have revealed the importance of different basic amino moieties in AChE inhibition due to their ability to bind to the catalytic center of AChE enzyme [38-41]. Based on these findings in the present compilation, a library of coumarin analogues has been constructed by substituting the hydroxyl group of coumarin heterocycle at fourth position with various nitrogen-containing cyclic or acyclic amino groups through a spacer. An appropriate spacer bearing a carbamate and an alkoxy group could favorably interact with some of the essential aromatic residues lining the wall of the AChE gorge. The length of the spacer linking the coumarin and the cyclic amines is an important parameter which affects the AChE inhibitory potential. Thus, a series of novel coumarin derivatives have been designed, synthesized and evaluated for their cognition improving ability, AChE inhibitory potency and antioxidant activity. Further, the hypothetical binding mode of the most active compound was proposed based on in silico docking studies by using the crystal structure of recombinant human AChE. 2.

Results and Discussion

2.1. Chemistry The synthesis of the target compounds (15a–i, 16a-d and 17a–b) was accomplished in four steps (Scheme 1). Initially, malonic acid monoester (10) was synthesized from one-pot reaction of phenol (8) and meldrum acid (9) using atom-efficient green chemistry approach in solvent-free conditions at 80 ºC [42]. 4-Hydroxycoumarin (11) was obtained by the cyclization of monoester (10) in the presence of Eaton’s reagent which is a good alternative to polyphosphoric acid as it has a much lower viscosity with no complex separation procedures involved. As hard nucleophiles preferentially attack the hydroxyl group of the

coumarin moiety, oxygen of the hydroxyl group remains the main site for attack by the acylating and the alkylating agents. Therefore, ether derivatives 12 and 13 were obtained by the alkylation of 4-hydroxycoumarin (11) with 1,3-bromochloropropane and

1,4-

dibromobutane respectively in the presence of K2CO3 in DMF. However, considerable amount of dimers were formed as byproducts during reaction. So, column chromatography technique was used to purify the reaction mixture obtained by O-Alkylation. Subsequently, Intermediate 12 was treated with various substituted secondary cyclic amines: 1-phenylpiperazine,

1-(2-fluorophenylpiperazine),

1-(4-fluorophenylpiperazine),

1-(2-

pyrimidylpiperazine), 1-(2-pyridylpiperazine) in the presence of sodium iodide and phase transfer catalyst tetrabutylammoniumhydrogen sulphate (TBAHS) to afford the analogues 15a-e respectively. But the same reaction conditions were not suitable for reaction with the primary amines. Hence, compounds 15g-i were synthesized by the fusion of 12 with 2phenylethylamine, homoveratrylamine and 4-(2-aminoethyl)morpholine respectively. While synthesizing compound 15i, a di-substituted compound containing two coumarin rings was formed instead of the expected compound with one coumarin ring. Continuing on the line of thought, compounds 16a-d were prepared by reacting intermediate 13 with 1-phenylpiperazine, 1-(2-fluorophenylpiperazine), 1-(2-pyrimidylpiperazine) and 1(2-pyridylpiperazine)

respectively

in

the

presence

of

sodium

iodide

and

tetrabutylammoniumhydrogen sulphate. But in case of reaction with the primary amines, fusion at higher temperature resulted in decomposition of the compounds with the elimination of HBr. Another series of carbamate analogues 17a-b was obtained by esterification of 4hydroxycoumarin with ethylchloroformate to afford the intermediate 14 which was treated with amines: 1-phenylpiperazine and 2-phenylethylamine to obtain the desired molecules. In this series, only two analogues were synthesized because the compounds with other amines were unstable owing to the hydrolysis of the carbamate linkage. OH

COOH

O O O O

(8)

(9)

Solvent free reaction (80°C, 5 h)

O

OH

Eaton's reagent O

(10) 54 %

(80°C, 2h)

O

O

(11) 63 %

Cl

O

(DMF, K2CO3, 70 °C, 1.5 h)

O

Br

O

O

Cl

O

OH

O

Br

O (a)

Br

(DMF, K2CO3, 70 °C, 3 h)

O

O

R

(15a-i)

(12) 60.81 %

O

O

(a,b)

Br

(13) 55.30 %

O

O

O

R

(16a-d)

(11)

Cl

O

O

O

O

O

O

R

(b) O (EMK, Cs2CO3, 80 °C, 10 h)

O

O

O (14) 78.25 %

O (17a-b) R = Cyclic amines

Scheme 1- Reagents, solvents and reaction conditions: (a) reflux with various amines at 60 °C in the presence of sodium iodide, anhydrous K2CO3, tetrabutylammoniumhydrogen sulphate, acetone (20-60 h) (b) fusion with amines at a temperature of 100-110 °C (1-13 h)

All the compounds were characterized by 1H NMR,

13

C NMR, IR, and LCMS spectral

techniques. In the infrared spectrum, a characteristic band between 1710-1740 cm-1 for >C=O stretch of the lactone ring was observed in all the compounds. The characteristic C-N stretching vibrations were observed in the range of 1105.23-1379.61 cm-1. Aromatic C-N stretching absorptions were observed at a higher frequency than the aliphatic C-N stretching vibrations because resonance increases the double bond character between the ring and the attached nitrogen. In the proton NMR spectra of the compounds 15a-e and 16a-d, the methylene protons of the piperazine ring adjacent to the propyl and the butyl chain appeared in the range of δ 2.45-2.65 ppm whereas the four methylene protons of the piperazine ring adjacent to the phenyl and heterocylic rings appeared deshielded ~ δ 3.07-3.77 ppm due to diamagnetic anisotropy. Further, proton resonance signals of the compounds possessing the o-fluorophenyl and the pfluorophenyl functionality at piperazine (15b, 15c and 16b) appeared as multiplets due to long range coupling of the aromatic proton and the fluorine atom. Aromatic carbons substituted with a fluoro group at ortho and para positions of the phenyl ring were also observed down field as compared to the other aromatic carbons because of the high electronegativity of the fluoro group. The 1H NMR spectra of the compounds 15g, 15h and 17b exhibited a quartet in

the range of δ 3.17-3.56 ppm due to coupling between the methylene protons and NH

function. The molecular ion peaks [M + H]+ of all the compounds were in agreement with the corresponding chemical structures. 2.2. Pharmacology 2.2.1. In vivo behavioral study using Morris water maze All the compounds were evaluated for their anti-amnesic activity using Morris water maze model [43]. Escape latency i.e. time required to find the hidden platform was measured, which acts as a tool to assess the spatial learning and memory capacities of the rodents. The mice received four consecutive daily training trials in 5 days. Escape latency did not vary between any of the groups on the first and the second day of testing but from the third day onwards; there was a significant difference between the escape latency of the various groups. Results were expressed as mean ± S.E.M. The intergroup variation was measured by one way analysis of variance (ANOVA) followed by Tukey’s test. The statistical analysis was done using GraphPad Prism and significance was considered at p < 0.001. On comparison with the control, the escape latency of the scopolamine treated group was significantly (p ≤ 0.001) more than that of the control indicating the efficacy of scopolamine in decreasing the ability of object recognition and spatial learning. Treatment with the various synthesized compounds (15a-i, 16a-d and 17a-b) showed a significant (p ≤ 0.001) decrease in the escape latency of the scopolamine treated mice. These compounds acted as a reversing agent on a pre-existent condition of cholinergic disruption. Compounds 15a, 15b and 15d showed an appreciable improvement in the cognitive dysfunction in comparison to both donepezil and scopolamine treated groups. Compound 15a was found to be the most active with an index of recognition (8.05 ± 0.65 s) more than that of the standard drug donepezil (11.36 ± 1.03 s). The results of memory parameters are included in Table 1 and Figure 3. Table 1: Effect of compounds on escape latency in the Morris maze test. All the compounds were administered i.p. 30 min before the training sessions. Each value represents the mean of 5 mice. S.No.

Code

Escape latency (s)

1

Control

21.75±2.34

2

Scopolamine (Sco)

49.35±2.21

3

Donepezil

11.36±1.03

4

Sco + 15a

8.05±0.65

5

Sco + 15b

11.8±2.28

6

Sco + 15d

13.98±0.52

Figure 3: Effect of various compounds (15a-i, 16a-d, 17a-b), scopolamine (0.5 mg/kg, i.p.) and reference drug (donepezil) (1 mg/kg, i.p.) on escape latency measured on Morris water maze in mice. *p ≤ 0.001 as compared to the control group, #p ≤ 0.001 as compared to the scopolamine group (ANOVA followed by Tukey’s test). n = 5, S.E.M., Standard Error of Mean. 2.2.2. Biochemical assessment of oxidative stress parameters Oxidative stress is a major factor implicated in the degeneration of cholinergic neurons because reactive oxygen species including free radicals (superoxide O 2.- and NO.) and other molecules such as H2O2 and peroxynitrite (ONOO-) generated during oxidative stress have a detrimental effect on various cellular components. Therefore, the present study was designed to investigate the influence of the synthesized compounds and donepezil on the biochemical markers of oxidative stress:

superoxide dismutase (SOD), malondialdehyde (MDA)

and plasma nitrite estimation. Scopolamine (i.p.) administration caused a significant decrease in SOD, increase in MDA and nitrite concentration as compared to the control, indicating a

state of oxidative stress in the brain of scopolamine-induced experimental model of dementia in mice. 2.2.2.1. Superoxide dismutase Superoxide dismutase (SOD) constitutes the first line of defense against reactive oxygen species. It catalyses the dismutation of superoxide ion to hydrogen peroxide which is then decomposed to water via various enzymes. Downregulation of SOD in the body is associated with an increase in the neuronal cell injury. The superoxide dismutase (SOD) activity was evaluated according to the method of Kono et al. [44]. The enzymatic activity of superoxide dismutase significantly decreased in the brain of scopolamine treated mice as compared to the control group. These alterations were significantly (p<0.05) restored by compounds 15a, 15b, 15c, 15h (1 mg/kg) and donepezil (1 mg/kg) treatment (Table 2 and Figure 4). 2.2.2.2. Lipid peroxidation As the brain tissues are rich in phospholipids, it can be attacked easily by the highly reactive oxygen species (ROS) for the initiation of lipid peroxidation. The most prominent assay currently being used as an index for lipid peroxidation is the measurement of the thiobarbituric acid reactive substances (TBARS). This assay is based upon the formation of a red adduct (absorption maxima 532 nm) between TBA and malondialdehyde (MDA), a colorless end product of lipid peroxide decomposition [45]. The groups treated with the newly synthesized compounds (15a-i, 16a-d and 17a-b) exhibited a lower level of malondialdehyde in comparison to those treated with the inducer scopolamine. Compounds 15a (0.198 ± 0.05) and 15c (0.097 ± 0.01) appreciably decreased the nmoles of brain MDA levels per mg protein as

compared to the standard donepezil (0.27 ± 0.04) (Table 2 and Figure 4). 2.2.2.3. Nitrergic stress NO prevails its toxic role by reacting with superoxide anions to form peroxynitrite (ONOO −) which in turn is able to induce neuronal cell injury mainly through nitration of tyrosine residues in the cellular proteins [46]. Nitrite levels were significantly elevated in the brain of scopolamine induced cognitive deficit animals as compared to the control group. All the compounds except 15g, 16c and 17b significantly decreased the levels of plasma nitrite scopolamine treated group, thus, suggesting the possible role of these compounds in reversal of oxidative damage induced by scopolamine. The 4-[3-(4-phenylpiperazin-1-yl)propoxy]2H-chromen-2-one (15a, 210.52 ± 19.47) exhibited better activity than scopolamine (525.49 ± 23.26) and donepezil (359.45 ± 9.38) treated groups (Table 2 and Figure 4).

Figure 4: Effect of the synthesized compounds 15a-i, 16a-d, 17a-b (1 mg/kg, i.p.), scopolamine (0.5 mg/kg, i.p.) and reference drug (donepezil) (1 mg/kg, i.p.) on (a) superoxide dismutase (SOD) (b) malondialdehyde (MDA) and (c) nitrite levels on scopolamine induced oxidative stress in mice. $p ≤ 0.001 as compared to control group, #p ≤ 0.001 as compared to scopolamine group (ANOVA followed by Tukey’s test). n = 5, S.E.M., Standard Error of Mean

Table 2: All the biochemical parameters of oxidative stress are expressed as mean ± standard error mean (SEM). Significance was determined by one-way ANOVA, followed by Tukeys test using GraphPad Prism 5

Control

Superoxide dismutase (SOD units/mg protein) 0.65 ±0.04

Lipid Peroxidation (n moles of MDA/ mg protein) 0.28 ± 0.06

Plasma nitrite concentration (µg/ml) 238.44 ± 19.88

Scopolamine

0.059 ± 0.01

1.62 ± 0.1

525.49 ± 23.26

Donepezil

0.89 ± 0.13

0.26 ±0.04

359.45 ± 9.38

15a

1.23 ± 0.18

0.19 ± 0.05

210.522 ± 19.47

15b

3.05 ± 0.49

0.26 ± 0.02

340.41 ± 47.04

15c

1.46 ± 0.21

0.096 ± 0.008

285.13 ± 21.05

15h

1.58 ± 0.12

0.29 ± 0.02

280.69 ±19.39

Compound code

2.2.3. In vitro cholinesterase inhibition In vitro inhibition study of acetylcholinesterase was determined by the spectrophotometric method of Ellman [47] and results are reported as IC50 which is a measure of the effectiveness of a compound in inhibiting the biological process, an enzyme or a cell receptor. This method is based on the principle that AChE enzyme hydrolyzes the substrate acetylthiocholine iodide into thiocholine and acetic acid. Thiocholine reacts with the oxidizing agent 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) to form a yellow coloured chromophore (5-thio-2-nitrobenzoate (TNB)), which has the absorption maxima at 412 nm. AChE inhibitory activity will be proportional to the decrease in absorption. Five different concentrations (1, 5, 10, 25 and 50 μM) of the standard drug (donepezil) and the test compounds were prepared. The concentration of compound required for 50 % inhibition of the AChE activity (IC50) was calculated by a non-linear regression of the log(concentration)response curve using GraphPad Prism 5 (Table 3). Table 3: IC50 values of all the synthesized compounds (15a-i, 16a-d and 17a-b) Compound 15a

N

IC50 (mean ± sem,

R

n

N

3

2.42 ± 0.042

3

5.20 ± 0.030

µM)

F

15b

N

N

15c

N

N

F

3

9.50 ± 0.013

3

21.63 ± 0.044

3

16.05 ±0.028

3

11.44 ± 0.029

3

66.95±3.64

3

47.07 ± 2.88

3

91.08 ± 2.62

4

14.90 ± 4.27

4

29.73 ± 0.058

4

34.81 ± 2.26

4

37.55 ± 6.26

0

74.79 ± 2.056

0

83.41 ± 1.956

N

15d

N

N N

N

15e 15f

N N

N

15g

N H

O

15h

N H

O

O

15i

N

N

16a

N

N

F

16b

N

N

N

N

N

16c

N

16d

N

17a

N

17b Donepezil

N N

N H

N

1.82 ± 0.014

As shown in Table 3, Most of the compounds showed high activities and selectivity for AChE inhibition with an IC50 value ranging from 91.08 µM of compound 15i to 2.42 µM of compound 15a, the latter showing inhibitory potency toward AChE very close to that of donepezil (1.82 µM). The potency of AChE inhibition was mainly influenced by the catalytic site interacting moieties attached at the end of the linker, as well as on the length of the alkyl

chain. Change in the length of the alkyl chain has a great affect on the AChE inhibitory potency as it influences the interaction of moieties with the catalytic and the peripheral sites. A length of three methylenes units was found to be optimal for AChE inhibition. In case of compounds having four methylenes (16a-d), there is a reduction in binding affinity towards AChE enzyme. Whereas in compounds 17a-b, even the presence of carbamate linkage does not enhance the binding of the concerned molecule to the drug receptor because decreased chain length does not allow proper interaction of the molecules. For compounds having same length of the alkyl chain (15a-i), variation of the functionality at the end of the linker led to a great change in the AChE inhibitory potency. Compound 15a showed maximum AChE inhibitory activity (IC50 = 2.42 µM) indicating that conformational and electronic changes caused by the replacement of phenyl ring of piperazine with any other heterocyclic ring might affect the interaction of the concerned molecule with the AChE enzyme and thereby, affecting its potency. Compound 15b possessing o-fluorophenyl functionality was found to be two fold more active than the compound having p-fluoro functionality (15c) because fluoro group at ortho position might favor the π-π stacking interaction via the rotation of the phenyl ring. Analogs 15g and 15h were designed to explore the involvement of freely available space around the nitrogen atom in binding to the AChE. However, they showed a decrease in inhibitory potency as compared to the analogue 15a. Limited space around the nitrogen atom leading to a decrease in conformational flexibility associated with the presence of a piperazine ring might be the possible reason for enhanced potency of 15a (IC50 = 2.42 µM) relative to 15g (IC50 = 66.95 µM) and 15h (IC50 = 47.07 µM). Further, di-substituted compound 15i showed a larger drop in AChE inhibition (IC50 = 91.08 µM) due to steric hindrance preventing proper alignment of the molecule within the receptor. 2.3. Docking study To explore the binding modes of coumarin-based hybrids at the active site of AChE, molecular docking study was performed using VLife MDS (version 4.3.30052013). Docking study of all the hybrids on recombinant human AChE (rhAChE) demonstrated that most of the compounds emerged as good dual binding site inhibitors as they interacted with the catalytic anionic site (CAS) and peripheral anionic site (PAS) through hydrophobic, Van der Waal and π–π stacking interactions. Compounds 15a and 15i afforded maximum and minimum inhibitory potencies respectively, hence, they were chosen for detailed docking simulations. A detailed view for the interaction of compound 15a with AChE is displayed in

Figure 5. The phenylpiperazine fragment of the hybrid was observed to enter the AChE gorge, adopting parallel π–π stacking interactions with the catalytic site amino acids Trp86 (4.32 Å) and His447 (4.97 Å) in a ‘sandwich’ form. In addition, the coumarin moiety also interacted with the indole and phenyl rings of Trp286 and Tyr72 located at the peripheral anionic site via aromatic π-π interactions with ring-to-ring distance of 4.5-4.7 Å. The long tether could fold with a proper conformation in the gorge to interact with Tyr341, Tyr337, Phe295, Phe338, Phe331, Gly121 and Tyr124 amino acid residues through hydrophobic and Van der Waal interactions. The interactions of 15a with all the crucial amino acids present at CAS, mid-gorge and PAS of rhAChE could be the reason for its higher inhibitory potency as compared to other analogues of series. However, in case of the compound 15i, the binding mode was drastically disturbed due to the steric clashes with the neighbouring residues. In view of the steric hindrance, a change in orientation was observed, leading to loss of essential interactions with the amino acids of the CAS (Trp86, His447, Gly121 and Tyr124). These findings could explain the reason for its decrease in inhibitory potency (Figure 6).

(a)

(b)

Figure 5: (a) 3D orientation of the best docked pose of compound 15a in the active site cavity of AChE enzyme (4EY7) (b) Hypothetical binding motif of ligand 15a within the crystal structure of AChE surrounding active amino acids. Dotted purple lines show the the ππ interaction whereas blue dotted lines depict the hydrophobic interactions.

(a)

(b) )

Figure 6: (a) 3D Orientation of the best docked pose of compound 15i at the active site cavity of AChE enzyme (4EY7) (b) Hypothetical binding motif of ligand 15i within the crystal structure of AChE surrounding active amino acids. Dotted purple lines show the π-π interaction whereas blue dotted lines depict the hydrophobic interactions. 3. Conclusion The combination of an antioxidant and PAS binding coumarin moiety with various catalytic site binding pharmacophores: substituted piperazines, benzylpiperidine, phenylethylamine, homoveratrylamine and aminoethylmorpholine yielded new hybrids (15a-i, 16a-d, 17a-b) with interesting in vitro and in vivo biological activities. The synthesized compounds inhibited the AChE enzyme in micromolar range, showed an increase in spatial memory in morris water maze and had neuroprotective effect against oxidative stress caused by scopolamine induced cognitive dysfunction. Structure activity relationship studies showed that the anti-AChE activity of the compounds was influenced by the length of linker and the catalytic site binding moieties present at the end of the linker. Further to specify, a chain of three methylene units was found to be optimal for the acetylcholinesterase inhibition. Among the fifteen coumarin-based compounds, compound 15a [4-[3-(4-phenylpiperazin-1yl)propoxy]-2H-chromen-2-one] displayed highest AChE inhibitory activity (IC50 = 2.42 µM) and antioxidant activity. Replacement of phenyl ring of piperazine (15a) with any other heterocyclic rings (15b-e) resulted in a decrease in acetylcholinesterase inhibitory potential of the compounds. On the basis of the molecular docking study of the most active compound 15a, it can be inferred that this active compound displayed significant binding interactions with both the binding pockets of the acetylcholinesterase enzyme (Trp86 and Trp286

respectively). It is thus expected that the synthesized coumarin-based hybrids can increase patient memory, decrease free radical levels and protect neurons against cognitive deficit caused by oxidative stress. This biological profile highlights the importance of these hybrids as useful dual function prototypes in the search of new protective and regenerative drugs for the potential treatment of neurodegenerative disease. 4.

Experimental

4.1. Chemistry All starting materials were purchased from Sigma Aldrich, Loba Chemie and Hi-Media Laboratories Pvt Ltd, India. Solvents used were of analytical grade and distilled prior to use according to standard procedures. Molecular sieves and anhydrous sodium sulphate were used as drying agents. Reactions were routinely monitored by thin layer chromatography (TLC). Plates for TLC were prepared with silica gel G according to Stahl’s method (E. Merck) using ethyl acetate as the solvent. Iodine was used to develop the TLC plates. Precoated silica gel aluminium plates 60 F254 were also used for TLC (E-Merck). The structures of compounds were identified using Infrared spectroscopy, Nuclear magnetic resonance studies and Mass spectrometry. Proton-NMR spectra were determined in deuterated-chloroform or deuterated-DMSO with a Bruker Avance II 400 MHz FT NMR spectrometer and signals recorded in parts per million (, ppm) downfield from tetramethylsilane (Me4Si) as internal standard and J values are given in Hz. The spin multiplicities are indicated by symbols, s (singlet), d (doublet), dd (double doublet), ddd (doublet of doublet of doublets), t (triplet), q (quartet), dt (doublet of triplets) and m (multiplet). Infrared (IR) spectra were recorded on Perkin-Elmer FT-IR spectrophotometer (Spectrum RX 1) using potassium bromide pellets (max in cm-1). Melting points were determined on a Veego melting point apparatus and were uncorrected. Mass spectra were resoluted using electron spray ionization technique on Waters, Q-TOF Micromass mass spectrometer. 4.2. Syntheses The syntheses of coumarin based hybrids (15a-i, 16a-d and 17a-b) were carried out according to Scheme 1. 4.2.1. Malonic acid monophenyl ester (10) A mixture of phenol (1 mmol) and meldrum acid (1 mmol) was reacted in solvent free conditions at 80 ºC for 5 h. The completion of the reaction was monitored by TLC. After

cooling to room temperature, the reaction mixture was dissolved in ethyl acetate and partitioned with saturated sodium bicarbonate solution. The aqueous layer was acidified to pH = 1-2 with concentrated HCl and extracted with dichloromethane thrice. The combined extracts were dried over sodium sulphate and the solvent was removed under reduced pressure to obtain the ester (10) which was used further without any purification. Yield and mp: 54 %, 66-68 °C [42]. 4.2.2. 4-Hydroxy-2H-chromen-2-one (11) A mixture of malonic acid monophenyl ester (10) (1 mmol) and Eaton’s reagent (4 ml) was refluxed at 70 ºC for 2 h. Ice-cold water was then added to this mixture with continuous stirring. The precipitates were filtered with suction, washed with water, dried and recrystallized from ethanol to afford 4-hydroxy coumarin (11). Yield and mp: 63 %, 217-18 °C [42]. 4.2.3. Synthesis of 4-(3-chloropropoxy)-2H-chromen-2-one (12) and 4-(4-bromobutoxy)2H-chromen-2-one (13) 4-Hydroxy-2H-chromen-2-one (11) (1 g, 4.18 mmol) was dissolved in dimethylformamide (DMF, 15 ml) and anhydrous potassium carbonate (0.6 g) was added to the solution. The reaction mixture was refluxed at 70 ºC with 1-bromo-3-chloropropane (0.4 ml, 4.18 mmol) and 1,4-dibromobutane respectively (0.5 ml, 4.18 mmol) with continuous stirring for 1.5-3 h. After the completion of the reaction (monitored by TLC), the mixture were filtered and diluted with water. The precipitates were filtered, washed with water, and purified through column chromatography eluting with pure chloroform to give compounds 12 and 13 respectively. 4.2.4. Synthesis of 2-Oxo-2H-chromen-4-yl propionate (14) 4-Hydroxycoumarin (1 g, 4.18 mmol) was dissolved in 60 ml of ethyl methyl ketone (EMK). Cesium carbonate (3.07 mmol) was added to the above solution. The reaction mixture was refluxed at 80 ºC for 1 h and then ethyl chloroformate (4.20 mmol) was added dropwise to the mixture with continued refluxing for 10 h. After completion of the reaction (monitored by TLC), the slurry was filtered and the solvent was removed under reduced pressure to get a solid compound which was further recrystallised from ethanol to afford 14. 4.2.5. General procedure for the synthesis of compounds 15a-e and 16a-d Compounds 12 and 13 (1 mmol) and sodium iodide (1 mmol) were dissolved in dry acetone respectively. The solutions were refluxed at 60 ºC for 1 h. The mixtures were refluxed further

for 1 h in the presence of anhydrous potassium carbonate. Subsequently, appropriate amine (1 mmol, 1-phenylpiperazine for 15a, 16a; 1-(2-fluorophenyl)piperazine for 15b; 1-(4fluorophenyl)piperazine for 15c, 16b; 1-(2-pyrimidyl)piperazine for 15d, 16c; 1-(2pyridyl)piperazine for 15e, 16d) and tetrabutylammonium hydrogen sulphate (1 mmol) were added to the mixture respectively and refluxing was continued with stirring for 20-60 h. After completion of the reaction (monitored by TLC), the slurry obtained was filtered and the solvent was removed under reduced pressure to obtain a semisolid mass. Crushed ice was added to this mass to remove any unreacted amine and yield a solid product which was filtered, dried and recrystallized from various solvents to give compounds 15a-e; 16a-d respectively. Reaction time and solvent of crystallisation of the respective compounds have been summarized in Table 4. Table 4: Reaction time and crystallisation solvent of the synthesized compounds (15a-e and 16a-d) Code

Reaction time (h)

Crystallisation solvent

15a

49

Ethanol

15b

38

Ethanol

15c

48

Ethanol

15d

58

Mixture of acetone and hexane

15e

57

Mixture of acetone and hexane

16a

30

Ethanol

16b

25

Ethanol

16c

15

Mixture of acetone and hexane

16d

22

Mixture of acetone and hexane

4.2.6. General procedure for the synthesis of compounds 15f-i and 17a-b Equimolar quantities of compounds 12 and 14 were individually fused at 100 °C and 110 °C respectively

with

various

amines

(4-benzylpipieridine,

2-phenylethylamine,

homoveratrylamine, 4-(2-aminoethyl)morpholine, 1-phenylpiperazine) for 1-13 h to obtain 15f, (15g, 17b), 15h, 15i and 17a (Table 5). The completion of reaction was monitored by TLC. Crushed ice was added to the reaction mixture to yield a solid which was recrystallized from various solvents to obtain compounds 15f-i; 17a-b. Table 5: Reaction time and solvent of crystallisation of the synthesized compounds (15f-i and 17a-b) Code

Reaction time (h)

Crystallisation solvent

15f

3.5

Ethylacetate

15g

6

Ethanol

15h

3

Acetone

15i

4

Ethanol

17a

10

Ethylacetate

17b

1

Mixture of ethylacetate and hexane

4.3. Characterization data for the synthesized compounds 4.3.1. 4-(3-Chloropropoxy)-2H-chromen-2-one (12) Yield 60.81 %, mp: 106-108 ºC; FT-IRmax (KBr): 2966.96, 1721.83, 1619.36, 1562.32, 1494.61, 1449.43, 1246.38, 920.40 and 770.13 cm-1 ; 1H NMR (CDCl3): δ 2.38 (pent, 2H, CH2-, J = 6.04 Hz), 3.78 (t, 2H, -CH2Cl, J = 6.22 Hz), 4.31 (t, 2H, -OCH2-, J = 5.86 Hz), 5.72 (s, 1H, =CH, vinylic), 7.30 (m, ArH), 7.33 (dd, ArH, Jm = 1.0 Hz, Jo = 8.36 Hz), 7.56 (ddd, ArH, Jm = 1.64 Hz, Jo = 7.34 Hz, Jo = 8.64 Hz) and 7.80 ppm (dd, ArH, Jm = 1.58 Hz, Jo = 7.90 Hz);

13

C NMR (CDCl3): δ 31.41, 40.81, 65.73, 90.81, 115.55, 116.87, 122.84, 123.94,

132.52, 153.35, 162.76 and 165.32 ppm. 4.3.2. 4-(4-Bromobutoxy)-2H-chromen-2-one (13) Yield 55.30 %, mp: 100-102 ºC; FT-IRmax (KBr): 2946.99, 2816.51, 1725.85, 1621.00, 1499.03, 1453.34, 1226.01, 930.23 and 771.48 cm-1 ; 1H NMR (CDCl3): δ 2.11 (m, 4H, (CH2)2-), 3.52 (m, 2H, -CH2Br), 4.18 (t, 2H, -OCH2-, J = 5.62 Hz), 5.67 (s, 1H, =CH, vinylic), 7.29 (m, 2H, ArH), 7.55 (ddd, ArH, Jm = 1.60 Hz, Jo = 7.36 Hz, Jo = 8.60 Hz) and 7.81 ppm (dd, ArH, Jm = 1.54 Hz, Jo = 7.94 Hz);

13

C NMR (CDCl3): δ 27.19, 29.26, 32.90,

68.42, 90.57, 115.64, 116.80, 122.93, 123.93, 132.45, 153.33, 162.82 and 165.49 ppm. 4.3.3. 2-Oxo-2H-chromen-4-yl propionate (14) Yield 78.25 %, mp: 110 ºC; FT-IRmax (KBr): 3071.1, 2960.9, 2926.0, 1764.8, 1722.8, 1617.3, 1560.6, 1427.9, 1228.0, 1202.8, 1151.8 and 1079.8 cm-1; 1H NMR (CDCl3): δ 1.44 (t, 3H, -CH3-, J = 7.14 Hz), 4.42 (q, 2H, -CH2-, J = 7.16 Hz), 6.58 (s, 1H, =CH, vinylic), 7.32 (t, ArH, J = 7.87 Hz), 7.37 (d, ArH, Jo = 8.06 Hz), 7.59 (ddd, ArH, Jm = 1.54 Hz, Jo = 7.08 Hz, Jo = 8.62 Hz) and 7.94 ppm (dd, ArH, Jm = 3.0 Hz, Jo = 7.94 Hz); 13C NMR (CDCl3): δ 14.10, 66.14, 103.53, 115.03, 117.03, 122.83, 124.40, 132.92, 150.56, 153.54, 158.40 and 161.54 ppm. 4.3.4. 4-[3-(4-Phenylpiperazin-1-yl)propoxy]-2H-chromen-2-one (15a)

Yield 45.81 %, mp: 120-122 ºC; FT-IRmax (KBr): 3066.26, 2944.46, 2823.11, 1714.32, 1619.34, 1560.57, 1495.91, 1446.61, 1368.27, 1231.61, 1184.78, 1139.72, 930.50, 811.71 and 683.46 cm-1; 1H NMR (CDCl3): δ 2.14 (pent, 2H, -CH2-, J = 6.68 Hz), 2.62 (t, 2H, CH2N-, J = 6.30 Hz), 2.65 (t, 4H, -N(CH2)2-, J = 4.94 Hz), 3.22 (t, 4H, -(CH2)2N-, J = 5.00 Hz), 4.23 (t, 2H, -OCH2-, J = 6.26 Hz), 5.70 (s, 1H, =CH, vinylic), 6.86 (t, ArH, Jo = 7.30 Hz), 6.94 (d, 2H, ArH, ortho to nitrogen function, Jo = 7.84 Hz), 7.27 (m, 3H, ArH), 7.36 (d, ArH, coumarin, Jo = 8.10 Hz), 7.55 (ddd, ArH, coumarin, Jm = 1.60 Hz, Jo = 7.38 Hz, Jo = 8.62 Hz) and 7.82 ppm (dd, ArH, coumarin, Jm = 1.54 Hz, Jo = 7.96 Hz); 13C NMR (CDCl3): δ 26.09, 49.18, 53.34, 54.75, 67.60, 90.58, 115.77, 116.13, 116.84, 119.82, 122.96, 123.89, 129.14, 132.40, 151.25, 153.37, 162.98 and 165.65 ppm; ESI-MS: 365.31 [M+H]+, 387.35 [M+Na]+ 4.3.5. 4-[3-{4-(2-Fluorophenyl)piperazin-1-yl}propoxy]-2H-chromen-2-one (15b) Yield 68.75 %, mp: 88-90 ºC; FT-IRmax (KBr): 3080.71, 2946.76, 28194.02, 1716.07, 1615.97, 1561.20, 1498.65, 1453.69, 1378.90, 1242.06, 1192.64, 1142.73, 1030.86, 931.28 and 759.95 cm-1 ; 1H NMR (CDCl3): δ 2.06 (pent, 2H, -CH2-, J = 6.70 Hz), 2.60 (t, 2H, CH2N-, J = 7.20 Hz), 2.64 (t, 4H, -N(CH2)2-, J = 4.48 Hz), 3.08 (t, 4H, -(CH2)2N-, J = 4.68 Hz), 4.16 (t, 2H, -OCH2-, J = 6.22 Hz), 5.65 (s, 1H, =CH, vinylic), 6.89 (m, 2H, ArH), 6.97 (m, 2H, ArH), 7.21 (d, ArH, Jo = 8.00 Hz), 7.26 (d, ArH, Jo = 8.24 Hz), 7.48 (ddd, ArH, coumarin, Jm = 1.56 Hz, Jo = 7.04 Hz, Jo = 8.60 Hz) and 7.76 ppm (dd, ArH, coumarin, Jm = 1.48 Hz, Jo = 7.96 Hz);

13

C NMR (CDCl3): δ 25.94, 50.39, 53.33, 54.72, 67.52, 90.59,

115.75, 116.12, 116.84, 118.98, 122.62, 122.97, 123.91, 124.51, 132.41, 139.95, 153.37, 155.74, 163.00 and 165.64 ppm; ESI-MS: 383.32 [M+H]+, 405.35 [M+Na]+ 4.3.6. 4-[3-{4-(4-Fluorophenyl)Piperazin-1-yl}propoxy]-2H-chromen-2-one (15c) Yield 25 %, mp: 142-144 ºC; FT-IRmax (KBr): 3064.13, 2938.02, 2819.74, 1712.50, 1618.01, 1560.66, 1509.04, 1446.22, 1366.56, 1229.12, 1182.95, 1139.72, 1028.82, 913.71 and 818.27 cm-1; 1H NMR (CDCl3): δ 2.06 (pent, 2H, -CH2-, J = 6.88 Hz), 2.57 [m, 6H, CH2N(CH2)2-], 3.07 (t, 4H, -(CH2)2NC6H5, J = 4.88 Hz), 4.16 (t, 2H, -OCH2-, J = 6.26 Hz), 5.64 (s, 1H, vinylic =CH), 6.82 (m, 2H, ArH), 6.89 (ddd, 2H, ArH, Jm = 2.16 Hz, Jo = 6.50 Hz, Jo = 8.66 Hz), 7.20 (t, ArH, Jo = 7.24 Hz), 7.25 (d, ArH, coumarin, Jo = 8.00 Hz), 7.48 (ddd, ArH, coumarin, Jm = 1.28 Hz, Jo = 7.22 Hz, Jo = 8.50 Hz) and 7.75 ppm (dd, ArH, coumarin, Jm = 1.44 Hz, Jo = 7.84 Hz);

13

C NMR (CDCl3): δ 26.08, 50.20, 53.32, 54.66,

67.55, 90.59, 115.42, 115.70, 116.85, 117.88, 122.94, 123.87, 132.39, 147.90, 153.38, 157.48, 162.95 and 165.63 ppm ; ESI-MS: 383.32 [M+H]+, 405.35 [M+Na]+

4.3.7. 4-[3-{4-(Pyrimidin-2-yl)piperazin-1-yl}propoxy]-2H-chromen-2-one (15d) Yield 50 %, mp: 130-132 ºC; FT-IRmax (KBr): 3041.33, 2951.51, 2821.52, 1712.05, 1618.36, 1580.42, 1477.80, 1440.39, 1364.62, 1228.45, 1183.38, 1142.59, 1027.12, 915.02 and 805.16 cm-1; 1H NMR (CDCl3): δ 2.15 (pent, 2H, -CH2-, J = 6.82 Hz), 2.54 (t, 4H, N(CH2)2-, piperazino protons adjacent to the propyl chain, J = 5.10 Hz), 2.61 (t, 2H, -CH2N-, J = 7.12 Hz), 3.85 (t, 4H, -(CH2)2N-, piperazino protons adjacent to the pyrimidine, J = 5.08 Hz), 4.24 (t, 2H, -OCH2-, J = 6.24 Hz), 5.72 (s, 1H, =CH, vinylic), 6.49 (t, ArH, pyrimidine, J = 4.74 Hz), 7.28 (m, ArH), 7.33 (d, ArH, Jo = 8.36 Hz), 7.56 (ddd, ArH, Jm = 2.00 Hz, Jo = 7.36 Hz, Jo = 8.62 Hz), 7.82 (dd, ArH, Jm = 1.56 Hz, Jo = 7.92 Hz) and 8.31 ppm (d, 2H, ArH, pyrimidine, J = 4.72 Hz);

13

C NMR (CDCl3): δ 26.06, 43.66, 53.20, 54.84, 67.56, 90.59,

109.93, 115.76, 116.85, 122.95, 123.89, 132.40, 153.37, 157.74, 161.63, 163.00 and 165.65 ppm; ESI-MS: 367.31 [M+H]+, 389.34 [M+Na]+ 4.3.8. 4-[3-{4-(Pyridin-2-yl)piperazin-1-yl}propoxy]-2H-chromen-2-one (15e) Yield 23 %, mp: 120-124 ºC; FT-IRmax (KBr): 3057.77, 2943.53, 2884.70, 1712.46, 1607.10, 1472.52, 1428.68, 1446.22, 1369.44, 1236.65, 1143.98, 1021.85, 923.53 and 767.16 cm-1; 1H NMR (CDCl3): δ 2.15 (m, 2H, -CH2-), 2.60 [m, 6H, -CH2N(CH2)2-], 3.57 (t, 4H, (CH2)2N-, piperazine, J = 5.06 Hz), 4.24 (t, 2H, -OCH2, J = 6.22 Hz), 5.72 (s, 1H, =CH, vinylic), 6.64 (m, 2H, ArH), 7.30 (m, 2H, ArH), 7.49 (ddd, ArH, Jm = 1.96 Hz, Jo = 7.14 Hz, Jo = 8.84 Hz), 7.56 (ddd, ArH, Jm = 1.60 Hz, Jo = 7.40 Hz, Jo = 8.62 Hz), 7.83 (dd, ArH, coumarin, Jm = 1.52 Hz, Jo = 7.92 Hz) and 8.20 ppm (dd, ArH, pyridine, Jm = 1.38 Hz, Jo = 4.90 Hz);

13

C NMR (CDCl3): δ 26.08, 45.22, 53.15, 54.81, 67.58, 90.59, 107.09, 113.41,

115.77, 116.85, 122.96, 123.89, 132.40, 137.51, 147.98, 153.37, 159.50, 163.00 and 165.65 ppm; ESI-MS: 366.26 [M+H]+, 388.30 [M+Na]+ 4.3.9. 4-[3-{4-Benzylpiperidin-1-yl}propoxy]-2H-chromen-2-one (15f) Yield 34.92 %, mp: 76-80 ºC; FT-IRmax (KBr): 3075.91, 2930.12, 2699.89, 1715.27, 1619.63, 1564.78, 1454.96, 1376.53, 1243.54, 1186.66, 1109.22, 933.92 and 840.97 cm-1; 1H NMR (CDCl3): δ 1.76 (m, 1H, -CH), 1.88 (d, 2H, -CH2-, J = 14.36 Hz), 2.16 (m, 2H, -CH2-), 2.66 (d, 6H, piperidino protons, J = 7.24 Hz), 3.18 (t, 2H, -CH2-, J = 7.70 Hz), 3.66 (d, 2H, CH2C6H5, J = 3.72 Hz), 4.25 (t, 2H, -OCH2-, J = 5.68 Hz), 5.67 (s, 1H, =CH, vinylic), 7.14 (d, 2H, Ar, J = 7.54 Hz), 7.23 (t, ArH, J = 7.36 Hz), 7.30 (t, 4H, Ar, J = 7.48 Hz), 7.56 (ddd, ArH, coumarin, Jm = 1.42 Hz, Jo = 7.16 Hz, Jo = 8.58 Hz) and 7.78 ppm (d, ArH, coumarin, J = 6.72 Hz);

13

C NMR (CDCl3): δ 23.50, 28.90, 30.91, 36.54, 41.94, 53.45, 54.75, 61.24,

66.38, 90.94, 115.16, 116.81, 122.89, 124.13, 126.50, 128.58 (2 × ArC), 129.00 (2 × ArC), 132.69, 138.97, 153.19, 162.61 and 165.07 ppm; ESI-MS: 378.2 [M+H]+, 400.3 [M+Na]+ 4.3.10. 4-[3-(Phenethylamino)propoxy]-2H-chromen-2-one (15g) Yield 31.76 %, mp: 210 ºC; FT-IRmax (KBr): 3398.02, 2961.20, 2703.11, 1715.78, 1618.47, 1471.76, 1416.59, 1240.17, 1183.86, 929.74 and 813.72 cm-1 ; 1H NMR (DMSO-d6): δ 2.14 (pent, 2H, -CH2-, J = 6.92 Hz), 3.00 (m, 2H, -CH2C6H5), 3.18 (m, 4H, -NH(CH2)2-), 4.31 (t, 2H, -OCH2-, J = 5.94 Hz), 5.85 (s, 1H, =CH, vinylic), 7.26 (m, 7H, ArH), 7.61 (ddd, ArH, coumarin, Jm = 1.54 Hz, Jo = 7.04 Hz, Jo = 8.58 Hz), 7.85 (dd, ArH, coumarin, Jm = 1.40 Hz, Jo = 8.32 Hz) and 9.14 ppm (s, 1H, NH); 13C NMR (DMSO-d6): δ 24.97, 31.72, 44.17, 48.17, 66.41, 90.60, 115.12, 116.22, 122.91, 123.80, 126.63, 128.44, 128.47, 132.45, 136.83, 152.72, 161.55 and 164.69 ppm; ESI-MS: 324.25 [M + H]+ 4.3.11. 4-[3-(3,4-Dimethoxyphenethylamino)propoxy]-2H-chromen-2-one (15h) Yield 25 %, mp: 172-174 ºC; FT-IRmax (KBr): 3409.66, 2945.62, 2787.11, 1710.73, 1614.89, 1513.65, 1453.05, 1385.62, 1242.97, 1147.79, 1025.39, 932.05 and 766.30 cm-1; 1H NMR (CDCl3): δ 2.21 (pent, 2H, -CH2-, J = 6.56 Hz), 2.87 (t, 2H, -CH2C6H5, J = 7.94 Hz ), 3.17 (q, 4H, -CH2NHCH2-, J = 7.22 Hz), 3.72 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 4.32 (t, 2H, -OCH2-, J = 5.84 Hz), 5.91 (s, 1H, =CH, vinylic), 6.77 (dd, ArH, Jo = 8.12 Hz, Jm = 1.84 Hz), 6.88 (d, 2H, ArH, Jo = 8.10 Hz), 7.39 (m, 2H, ArH, coumarin), 7.67 (ddd, ArH, coumarin, Jm = 1.56 Hz, Jo = 7.04 Hz, Jo = 8.60 Hz), 7.88 (dd, ArH, coumarin, Jm = 1.46 Hz, Jo = 7.90 Hz) and 8.29 ppm (s, 1H, NH) ; 13C NMR (CDCl3): δ 25.50, 32.08, 45.14, 49.95, 55.84, 55.95, 66.51, 91.11, 111.36, 111.79, 115.17, 116.70, 120.70, 122.85, 124.12, 128.46, 132.58, 148.14, 149.14, 153.03, 163.17 (C=O) and 165.34 ppm; ESI-MS: 384.30 [M + H]+ 4.3.12. 4-[3-{(2-Morpholinoethyl)(3-(2-oxo-2H-chromen-4-yloxy)propylamino}propoxy]2H-chromen-2-one (15i) Yield 31.34 %, mp: 138-140 ºC; FT-IRmax (KBr): 3085.79, 2964.52, 2827.05, 1731.46, 1621.06, 1564.85, 1458.30, 1379.61, 1250.63, 1178.73, 1111.45, 918.79 and 752.36 cm-1; 1H NMR (CDCl3): δ 2.04 (pent, 4H, 2 × -CH2-, J = 6.52 Hz), 2.43 (m, 6H, -N(CH2)3-, methylenes adjacent to N-function of morpholine), 2.65 (m, 2H, -CH2N-), 2.72 (t, 4H, 2 × NCH2-, J = 6.84 Hz), 3.67 (t, 4H, -O(CH2)2-, morpholine, J = 4.64 Hz), 4.19 (t, 4H, 2 × OCH2-, J = 6.16 Hz), 5.67 (s, 2H, 2 × =CH, vinylic), 7.24 (m, 2H, ArH, coumarin), 7.30 (d, 2H, ArH, coumarin, Jo = 8.08 Hz), 7.53 (ddd, 2H, ArH, coumarin, Jm = 1.60 Hz, Jo = 7.38 Hz, Jo = 9.22 Hz), 7.74 (dd, 2H, ArH, coumarin, Jm = 1.50 Hz, Jo = 7.94 Hz);

13

C NMR

(CDCl3): δ 26.69 (2 × CH2), 50.66 [N(CH2)2], 51.34 (NCH2), 54.17 ([2 × NCH2), 57.25

(CH2N), 66.88 [O(CH2)2, morpholine], 67.18 [2 × OCH2], 90.55 (2 × CH, vinylic), 115.62 (2 × ArC), 116.90 (2 × ArC), 122.72 (2 × ArC), 123.86 (2 × ArC), 132.44 (2 × ArC), 153.34 (2 × ArCq attached to oxygen, coumarin), 162.82 (2 × C=O) and 165.46 ppm (2 × Cq attached to oxygen, vinylic); ESI-MS: 535.38 [M + H]+ , 557.44 [M + Na]+ 4.3.13. 4-[4-(4-Phenylpiperazin-1-yl)butoxy]-2H-chromen-2-one (16a) Yield 78.94 %, mp: 124-126 ºC; FT-IRmax (KBr): 3071.38, 2942.54, 2822.90, 1729.02, 1611.70, 1497.29, 1450.19, 1372.54, 1241.22, 1181.98, 1142.50, 924.77 and 756.74 cm-1; 1H NMR (CDCl3): δ 1.77 (pent, 2H, -CH2-, J = 7.52 Hz), 1.98 (pent, 2H, -CH2-, J = 6.48 Hz), 2.50 (t, 2H, -CH2N-, J = 7.44 Hz), 2.63 (t, 4H, -N(CH2)2-, piperazino protons adjacent to the butyl chain, J = 5.02 Hz), 3.22 (t, 4H, -(CH2)N-, piperazino protons adjacent to the pyrimidine ring, J = 4.98 Hz), 4.18 (t, 2H, -OCH2-, J = 6.30 Hz), 5.69 (s, 1H, =CH, vinylic), 6.86 (t, ArH, J = 7.28 Hz), 6.94 (d, 2H, ArH, Jo = 7.88 Hz), 7.26 (m, 3H, ArH), 7.33 (d, ArH, coumarin, Jo = 8.34 Hz), 7.55 (ddd, ArH, coumarin, Jm = 1.58 Hz, Jo = 7.38 Hz, Jo = 8.62 Hz) and 7.83 ppm (dd, ArH, coumarin, Jm = 1.54 Hz, Jo = 7.94 Hz); 13C NMR (CDCl3): δ 23.39, 26.57, 49.17, 53.31, 58.01, 69.20, 90.48, 115.77, 116.08, 116.84, 119.78, 123.02, 123.88, 129.13, 132.41, 151.28, 153.37, 163.04 and 165.68 ppm; ESI-MS: 379.36 [M+H]+ 4.3.14. 4-[4-{4-(2-Fluorophenylpiperazin-1-yl)butoxy]-2H-chromen-2-one (16b) Yield 44.64 %, mp: 118 ºC; FT-IRmax (KBr): 3079.05, 2945.45, 2815.26, 1715.53, 1616.30, 1563.21, 1498.88, 1455.97, 1378.87, 1241.72, 1192.66, 934.63 and 759.16 cm-1 ; 1H NMR (CDCl3): δ 1.77 (pent, 2H, -CH2-, J = 7.53 Hz), 1.98 (m, 2H, -CH2-), 2.52 (t, 2H, -CH2N-, J = 7.42 Hz), 2.67 (t, 4H, -N(CH2)2-, piperazino methylenes adjacent to the butyl chain, J = 4.32 Hz), 3.13 (t, 4H, -(CH2)2N-, piperazino methylenes adjacent to the phenyl ring, J = 4.60 Hz), 4.19 (t, 2H, -OCH2-, J = 6.30 Hz), 5.69 (s, 1H, =CH, vinylic), 6.95 (m, 2H, ArH), 7.05 (m, 2H, ArH), 7.29 (m, 2H, ArH), 7.56 (ddd, ArH, coumarin, Jm = 1.60 Hz, Jo = 7.36 Hz, Jo = 8.62 Hz) and 7.83 ppm (dd, ArH, coumarin, Jm = 1.54 Hz, Jo = 7.94 Hz); 13C NMR (CDCl3): δ 23.36, 26.57, 50.52, 53.34, 58.03, 69.21, 90.48, 115.77, 116.11, 116.83, 118.92, 122.50, 123.02, 123.89, 124.49, 132.41, 140.09, 153.36, 156.35, 163.06 and 165.68 ppm; ESI-MS: 397.34 [M+H]+ , 419.37 [M+Na]+ 4.3.15 4-[4-{4-(Pyrimidin-2-yl)piperazin-1-yl}butoxy]-2H-chromen-2-one (16c) Yield 42.52 %, mp: 94-98 ºC; FT-IRmax (KBr): 3076.03, 2940.94, 2816.96, 1715.44, 1611.92, 1490.57, 1370.98, 1246.52, 1186.97, 929.32 and 762.00 cm-1 ; 1H NMR (CDCl3): δ 1.70 (pent, 2H, -CH2-, J = 7.49 Hz), 1.92 (pent, 2H, -CH2-, J = 6.44 Hz), 2.41 (t, 2H, -CH2N-, J = 7.38 Hz), 2.45 (t, 4H, -N(CH2)2-, piperazino protons adjacent to the butyl chain, J = 5.10

Hz), 3.77 (t, 4H, -(CH2)2N-, piperazino protons adjacent to the pyrimidine ring, J = 5.08 Hz), 4.11 (t, 2H, -OCH2-, J = 6.30 Hz), 5.61 (s, 1H, =CH, vinylic), 6.41 (t, ArH, pyrimidine, J = 4.74 Hz), 7.22 (m, 2H, ArH, coumarin), 7.48 (ddd, ArH, Jm = 1.60 Hz, Jo = 7.38 Hz, Jo = 8.60 Hz), 7.75 (dd, ArH, coumarin, Jm = 1.52 Hz, Jo = 7.96 Hz) and 8.24 ppm (dd, 2H, ArH, pyrimidine, Jm = 4.80 Hz, Jo = 7.44 Hz);

13

C NMR (CDCl3): δ 23.32, 26.55, 43.66, 53.14,

58.07, 69.18, 90.46, 109.88, 115.76, 116.81, 123.00, 123.88, 132.39, 153.35, 157.72, 161.64, 163.00 and 165.66 ppm; ESI-MS: 381.17 [M+H]+, 403.16 [M+Na]+ 4.3.16. 4-[4-{4-(Pyridin-2-yl)piperazin-1-yl}butoxy]-2H-chromen-2-one (16d) Yield 25 %, mp: 86-88 ºC; FT-IRmax (KBr): 3072.96, 2941.85, 2813.29, 1728.36, 1606.26, 1441.20, 1374.82, 1247.47, 1181.83, 928.92 and 758.39 cm-1 ; 1H NMR (CDCl3): δ 1.71 (pent, 2H, -CH2-, J = 7.51 Hz), 1.90 (pent, 2H, -CH2-, J = 6.42 Hz), 2.42 (t, 2H, -CH2N-, J = 7.42 Hz), 2.51 (t, 4H, - N(CH2)2-, piperazino protons adjacent to the butyl chain, J = 5.08 Hz), 3.48 (t, 4H, - (CH2)2N-, piperazino protons adjacent to the pyridyl ring, J = 5.08 Hz), 4.11 (t, 2H, -OCH2-, J = 6.30 Hz), 5.72 (s, 1H, =CH, vinylic), 6.55 (m, 2H, ArH), 6.58 (d, ArH, J = 8.60 Hz), 7.23 (m, 2H, ArH), 7.40 (ddd, ArH, Jm = 2.02 Hz, Jo = 7.18 Hz, Jo = 8.86 Hz), 7.48 (ddd, ArH, Jm = 1.62 Hz, Jo = 7.34 Hz, Jo = 8.62 Hz), 7.76 (dd, ArH, coumarin, Jm = 1.56 Hz, Jo = 7.92 Hz) and 8.12 ppm (m, ArH, pyridine); 13C NMR (CDCl3): δ 23.34, 26.57, 45.21, 53.10, 58.07, 69.19, 90.46, 107.10, 113.38, 115.76, 116.82, 123.01, 123.89, 132.41, 137.51, 147.96, 153.34, 159.52, 163.05 and 165.68 ppm; ESI-MS: 380.29 [M+H]+, 402.32 [M+Na]+ 4.3.17. 2-Oxo-2H-chromen-4-yl 4-phenylpiperazine-1-carboxylate (17a) Yield 43.64 %, mp: 188-190 ºC; FT-IRmax (KBr): 3075.74, 2895.31, 2837.85, 1703.29, 1601.39, 1552.88, 1494.77, 1451.49, 1391.58, 1271.12, 1188.17, 930.13 and 765.17 cm-1; 1H NMR (CDCl3): δ 3.45 (s, 8H, -N(CH2)2-, piperazine), 5.81 (s, 1H, =CH, vinylic), 6.96 (t, ArH, J = 7.32 Hz), 7.02 (d, 2H, ArH, J = 8.08 Hz), 7.32 (m, 4H, ArH), 7.54 (ddd, ArH, Jm = 1.30 Hz, Jo = 7.10 Hz, Jo = 8.40 Hz) and 7.69 ppm (dd, ArH, coumarin, Jm = 1.24 Hz, Jo = 8.0 Hz);

13

C NMR (CDCl3): δ 49.08, 51.07, 98.25, 116.27, 116.49, 117.89, 120.65, 123.53,

124.75, 129.33, 131.63, 150.82, 154.27, 161.10 and 162.40 ppm; ESI-MS: 383.38 [M+Na]+ 4.3.18. 2-Oxo-2H-chromen-4-yl phenethylcarbamate (17b) Yield 35.71 %, mp: 156-158 ºC; FT-IRmax (KBr): 3270.11, 3089.23, 2951.58, 2857.78, 1741.34, 1662.04, 1613.35, 1558.48, 1450.48, 1374.02, 1255.47, 1194.99, 1105.23, 936.48 and 750.50 cm-1 ; 1H NMR (CDCl3): δ 3.04 (t, 2H, -CH2C6H5, J = 6.88 Hz), 3.56 (m, NHCH2-), 5.14 (s, 1H, NH), 5.40 (s, 1H, =CH, vinylic), 7.22 (m, 4H, ArH), 7.32 (m, 4H,

ArH) and 7.52 ppm (ddd, ArH, Jm = 1.76 Hz, Jo = 6.96 Hz, Jo = 6.96 Hz); 13C NMR (CDCl3): δ 34.31, 43.82, 84.29, 114.16, 118.08, 119.74, 123.46, 127.12, 128.70, 129.05, 131.86, 137.78, 152.47, 153.64 and 163.05 ppm; ESI-MS: 310.26 [M+H]+ 4.4. Pharmacology 4.4.1. Animals Albino mice (LACA strain) weighing 25-35 g of either sex, (Central Animal House, Panjab University, India) were used in this study. The animals were housed in groups of five and maintained under standard laboratory conditions with natural dark and light (12:12 h) cycle and had free access to food and water. Animals were acclimatized to laboratory conditions before the test. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India. 4.4.2. Morris water maze model Spatial memory of the synthesized compounds 15a-i, 16a-d and 17a-b was determined through the Morris water maze model. Donepezil (4) (1 mg/kg i.p.) was used as the standard drug. Scopolamine (0.5 mg/kg i.p.) was used for inducing memory dysfunction. The test compounds were suspended in 0.25 % carboxymethylcellulose (CMC) and administered intraperitoneally (i.p.) to the animals. Mice received the first i.p. injection 35 min prior to the training. This injection consisted of either saline or scopolamine and was followed 5 min later by a second i.p. injection of either vehicle, donepezil or the test compounds. Morris water maze consists of a circular water tank which was filled with water to a depth of 30 cm, maintained at a temperature of 28 ± 2˚C. Using a thread this tank was divided into four quadrants and a circular black platform was placed at the center of one of the quadrants. Platform was placed in such a manner that it was not visible from the surface. This tank was placed in a large room having bright coloured clues outside the maze to help the mice spatially orient themselves. These clues remained at the same place throughout 5 days of this water maze task. After giving i.p. injections of the drugs the mice were introduced into the tank from a different position every time. The mice were held facing the wall of the tank while placing them into the water. Each mouse was given a maximum time of 90 s to locate the platform, and the same was confirmed by its ability to stay on the platform for atleast 1.5 s. Those subjects who did not stop on the platform for the stipulated

time were allowed to swim till the culmination of the trial. After the subject rested on the platform for 20 s, it was removed and placed in its home cage. In case the mouse failed to locate the platform in the allotted time, it was guided with the help of a rod to reach the platform and given a latent period of 90 s. After the completion of the training sessions, the mice were individually subjected to a probe trial in which they were allowed to swim for 90 s to search the platform. Escape latency i.e. the time required to find the platform was measured which acts as a tool to assess the spatial learning and memory capacities of mice [43]. Results were expressed as mean ± S.E.M. The intergroup variation was measured by one way analysis of variance (ANOVA) followed by Tukey’s test. Statistical significance was considered at p < 0.01-0.001. The statistical analysis was done using GraphPad Prism. 4.4.3. Antioxidant activity 4.4.3.1. Superoxide Dismutase (SOD) assay Superoxide dismutase activity was carried out by the method given by Kono et al [44]. In this method, 96 mM solution of nitro blue tetrazolium (NBT) was prepared in solution A (50 mM sodium carbonate in 0.1 mM EDTA, pH 10.8). In the cuvette, 2 ml of NBT solution was taken, and to it, 0.1 ml of brain supernatant and 0.05 ml of hydroxylamine hydrochloride (adjusted to pH 6.0 with NaOH) were added. The auto-oxidation of hydroxylamine hydrochloride was calculated by measuring the change in optical density at 560 nm for 2 min at 30/60-s intervals. 4.4.3.2. Lipid peroxidation assay The malondialdehyde content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances by the method of Wills [45]. Briefly, 0.5 ml of the brain tissue homogenate was mixed with 0.5 ml of Tris-HCl buffer (pH 7.4) and the mixture was incubated at 37 °C for 2 h. After incubation, 1 ml of the ice-cold 10 % w/v trichloroacetic acid solution was added and centrifuged at 1000×g for 10 min. To 1 ml of the supernatant, 1 ml of 0.67 % w/v thiobarbituric acid was added, and the tubes were kept in boiling water for 10 min. After cooling, 1 ml of double-distilled water was added, and the absorbance was measured at 532 nm. Tissue protein was estimated using the Biuret method, and the brain malondialdehyde content was expressed as nanomoles of malondialdehyde per milligram of the protein. 4.4.3.3. Estimation of nitrite

Nitrite was measured in the cytosolic fraction of different brain regions as it was converted to a deep purple azo compound by the addition of Griess reagent, serving as an indicator of nitric oxide production. To 200 μl of postmitochondrial supernatant, 1 ml of Greiss reagent (equal volumes of 1: 1 solution of 1 % sulphanilamide in 5 % phosphoric acid and 0.1 % naphthylethylene diamine dihydrochloric acid) and 0.8 ml of double distilled water were added and absorbance was measured spectrophotometrically at 546 nm. Nitrite concentration was calculated using a standard curve for sodium nitrite and nitrite levels were expressed as μg/ml [46]. 4.4.3.4. Statistical analysis Results were expressed as mean ± SEM. The intergroup variation was measured by two-way ANOVA and one way analysis of variance (ANOVA) followed by Tukeys test. Statistical significance was considered at p˂ 0.0001. 4.4.4. In vitro inhibition study of acetylcholinesterase Ellman’s method [47] was used to determine the inhibitory activity of compounds 15a-i, 16ad and 17a-b against acetylcholinesterase enzyme using mouse brain homogenate as a source of enzyme. Albino mice (LACA strain, 25-35 g) were sacrificed by cervical dislocation; the brains were isolated and rinsed with ice cold isotonic saline solution. Further, homogenised in a glass Teflon homogenizer (REMI MOTORS, India) containing sodium phosphate buffer of pH 7.4 and centrifuged (Research centrifuge, REMI, R-24) at 10,000 rpm for 20 min at 4 ºC to prepare supernatant. The obtained supernatant was used as a source of enzyme for the assay. Five different concentrations (1, 5, 10, 25 and 50 μM) of standard (donepezil) and test drugs were prepared by diluting their stock solutions immediately before use. AChE inhibitory activity was determined in a reaction mixture containing 50 μl of supernatant, 100 μl of a 10 mM solution of DTNB in 0.1 M phosphate buffer (pH 7.4) and 3 ml of phosphate buffer, pH 8.0. After incubation for 20 min at 25 ºC, acetylthiocholine iodide (100 μl of 14.9 mM aqueous solution) was added and AChE-catalyzed hydrolysis was measured by calculating the change in absorbance at 412 nm for 2 min using a PERKIN ELMER UV/VIS spectrophotometer. The results were expressed as nmoles of acetylthiocholine iodide hydrolyzed/min/mg protein. The concentration of the compound which determined 50 % inhibition of the AChE activity (IC50) was also calculated by non-linear regression of the log(concentration)-response curve, using GraphPad Prism 5.

Total protein content was measured using Biuret method which is based on the reduction of cupric ions (Cu2+) to cuprous ions (Cu+) during complexation with protein’s peptide bonds in an alkaline solution. The interaction of cupric ions (Cu2+) with protein produces a violetcolored chelate product which is measured by UV absorption spectroscopy at 540 nm. Therefore, protein estimation was carried out by incubating the mixture having 50 μl of supernatant, 2.9 ml of normal saline and 3 ml of Biuret reagent at room temperature for 10 min. The amount of protein content (in mg) was calculated from the standard plot of bovine serum albumin (BSA) based on the optical density. 4.5. Computational study Docking study of the synthesized compounds (15a-i, 16a-d and 17a-b) was performed against recombinant human acetylcholinesterase (rhAChE, PDB ID: 4EY7) to evaluate the interactions of the synthesized compounds with the amino acids present at the binding site of the enzyme using VLife MDS (version 4.3.30052013). X-ray crystallographic structure of 4EY7 was obtained from the Protein Data Bank. Protein was prepared by eliminating the water molecules, mutating the incomplete residues, adding the missing residues and extracting out the cocrystal ligand E20. The 3D structures of the compounds were generated and optimized by vLife engine. A systematic conformational search was performed to obtain the low energy conformations of the ligands. Docking of the low energy conformer of each molecule was done by using generalised rigid PLP docking [GRIP] method. Acknowledgment This research work was financially supported by University Grants Commission (UGC), New Delhi (File No: 41-739/2012). We are also thankful to Sophisticated Advanced Instrumentation Facilities (SAIF), Panjab University, Chandigarh for carrying out NMR and Mass spectrometric analysis. References 1.

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Highlights 

Coumarin based hybrids have been designed and synthesized.



Memory enhancing potential has been evaluated using Morris water maze model.



Biochemical assessment of acetylcholinesterase enzyme has been carried out.



Follow-up evaluation of oxidative stress biomarkers and docking study has been done.



Some of the synthesized compounds showed promising anticholinesterase activity.