Systematic in Silico Design, Synthesis, and Biological Studies of Some Novel 1,4-Benzoquinone Derivatives for the Prospective Management of Cognitive Decline

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Systematic In Silico Design, Synthesis, and Biological Studies of

Some Novel 1,4-Benzoquinone Derivatives for the Prospective

Management of Cognitive Decline

Pragati Silakari, Om Silakari, and Poonam Piplani*

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ABSTRACT: Cholinesterases are signiﬁcant biological targets for the regulation of

cholinergic neurotransmission, and their inhibitors are being exploited for the management

of cognitive decline in various neurological conditions. The 1,4-benzoquinone scaﬀold

possesses antioxidant potential along with AChE inhibition activity in various neurological

disorders. To design novel and potent selective 1,4-benzoquinone analogues as cholinesterase

inhibitors, a ligand-based drug design strategy was followed to develop a 3D quantitative

structure−selectivity relationship (QSSR) model. On the basis of the best ﬁt model, eight

novel 1,4-benzoquinone derivatives were designed and synthesized implementing appropriate

synthetic procedures and were characterized by various spectral and elemental techniques. All

the synthesized compounds were evaluated for their selective in vitro acetylcholinesterase

(AChE) and butyrylcholinesterase (BChE) inhibitory potential at diﬀerent concentrations

using mice brain homogenate as the source of the enzyme. Out of these compounds, the three

most selective compounds were further evaluated for behavioral variations using step down

passive avoidance and escape learning procedure at a dose of 0.5 mg/kg taking donepezil as

the reference drug. Biochemical estimation of the markers of oxidative stress (lipid peroxidation, superoxide dismutase, glutathione,

and catalase) has also been carried out to determine the role of the synthesized molecules on the scopolamine induced oxidative

damage. Compound 2a displayed appreciable selectivity index values as predicted through the 3D-QSSR model. Further, docked

complexes of compound 2a with AChE and BChE were subjected to molecular dynamic simulations for a period of 30 ns to study

the orientations and stable conformations of the most active molecules in the catalytic domain of these enzymes. The results

obtained from the 3D-QSSR analysis, docking, and molecular dynamic studies were found to be appreciable and provided a deep

insight into the structural features required for the selectivity of AChE inhibitors over BChE. The outcome of this study may be used

as a novel tool to design new highly selective and more potent molecules.

KEYWORDS: 3D-QSSR model, cognition, 1,4-benzoquinone, oxidative stress, acetylcholinesterase, butyrylcholinesterase

neously small sized neurotoxic peptides of β amyloid (Aβ)

which are formed via sequential proteolytic cleavages of

amyloid precursor protein (APP) by β-secretase and γ-

secretase.⁵

Cognitive decay is sprouting as one of the biggest dangers in

the ebb and ﬂow situation as it results in conditions like

weakened consideration, learning, memory, and critical

thinking inability. A portion of the populace over the age of

80 is the most noteworthy expanding area where 25% of this

age bunch experiences serious impediment in mental capacity.

1. INTRODUCTION

Cognitive decline or intellectual dysfunctions (e.g., memory,

consideration, ﬁxation, cognizance, direction) are large alerts in

a wide range of neurotic conditions, e.g., cardiovascular

diseases, inﬂammatory diseases, brain injuries, drug abuse,

age related mental retardation, and neurodegenerative brain

diseases (Alzheimer’s disease and Parkinson’s disease).¹

Alzheimer’s disease (AD) is the major cause of dementia

worldwide and is a multifaceted neurodegenerative disorder

characterized at the molecular level by oxidative stress, protein

misfolding and aggregation, mitochondrial abnormalities, and

neuronal imbalance.^2,3Diﬀerent ﬁndings have concurrently

published the relationship between dementia observed in AD

and cholinergic hypothesis, and oxidative stress.⁴On the other

hand major histopathological hallmarks of AD include

proteinaceous aggregates in the form of neuroﬁbrillary tangles

(NFTs), consisting of hyperphosphorylated tau and extrac-

ellular senile plaques. These are the deposits of heteroge-

Received: February 17, 2021

Accepted: April 1, 2021

Published: April 14, 2021

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Table 1. Molecular Structure, Biological Activity, Selectivity Index, and −log SI of Molecules Used for 3D-QSSR Model

a

Development

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Table 1. continued

a

AChE IC₅₀: dose in micromolar concentration required to produce 50% inhibition of AChE. BChE IC₅₀: dose in micromolar concentration

required to produce 50% inhibition of BChE. Selectivity index is the ratio of IC₅₀of AChE to IC₅₀of BChE. −Log SI: −log(SI) values. Pred. pSI:

predicted pSI values from the best model.

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However, cognitive dysfunctions not only occur in aged people

but can develop in persons of all ages due to various other

factors like trauma, accidents and injuries, and developmental

defects.⁶Neuroinﬂammation, neurodegradation, and oxidative

stress also lead to cognitive decline and neuronal loss.⁷

Oxidative stress is one of the key factors responsible for lipid

and protein damage resulting in neuron death and cognitive

dysfunction. Cognitive dysfunctions greatly diminish the

quality of life of the aﬀected individuals and their family and

considerably crash the health care system and society.⁸

Research in this regard has progressed to understand diﬀerent

targets involved in the cognitive decline. It is nevertheless

challenging for the researchers to come out with the selective

treatment strategy in neurological dysfunction. Consequently,

research in the ﬁeld of cognitive dysfunction targeting

acetylcholinesterase has progressed so far but still needs to

be improvised in the aspects of selectivity, eﬃcacy and reduced

toxicity.

inhibition as well antioxidant potential for the prospective

management of cognitive dysfunction.

2. RESULTS AND DISCUSSION

In the light of comprehensive description of the selective

structural and functional role of AChE and the oxidative stress

and pharmacological signiﬁcance of 1,4-benzoquinone, some

selective synthetic analogues have been designed based on

quantitative structure−selectivity relationship (QSSR) drug

designing. These designed molecules were duly synthesized

and pharmacologically evaluated for their role in the

management of cognitive decline.

2.1. Atom Based 3D-QSSR. Atom based 3D-QSSR

analysis was conducted on 43 molecules having dual inhibitory

activity against AChE and BChE as described in Table 1. The

most active molecule out of the 43 molecules was considered

for molecular alignment of the entire data set (Figure 1). The

From several studies it has been notiﬁed that butyrylcho-

linesterase (BChE) also possesses a peripheral anionic site

(PAS) region that shares some structural and physicochemical

properties with AChE. However, the BChE PAS region lacks

some aromatic residues of the AChE PAS region and displays

an inversion biochemical property like substrate activation

rather than substrate inhibition. Since BChE may have a role

inverse to that of AChE in dementia, selectivity for AChE over

BChE may be beneﬁcial for cognitive dysfunction.⁹Some FDA

approved therapeutic agents (donepezil, rivastigmine, galant-

amine, and memantine) for the treatment of AD are based on

reduction of cognitive deﬁcits by enhancing cholinergic

transmission, but only donepezil has been shown to have

selective inhibition of acetylcholinesterase over butyrylcholi-

netearse.¹⁰

Donepezil (2-[(1-benzylpiperidin-4-yl) methyl]-5,6-dime-

thoxy-2,3-dihydroinden-1-one) is a selective reversible AChE

inhibitor acquainted with the market in 1996 for the treatment

of AD. Donepezil binds to both the peripheral anionic site

(PAS) and catalytic anionic sites (CAS) of AChE. It has been

reported that the selectivity of donepezil toward AChE over

BChE was in a ratio of 1265:1 due to its interaction with the

peripheral binding site of AChE.¹¹Donepezil hydrochloride is

prescribed for the management of mild-to-moderate AD

patients, but some adverse eﬀects of donepezil have also

been reported.¹²

There are varied scaﬀolds possessing AChE inhibition

activity that have been reported in the literature for the

treatment of the cognitive decline. Out of these 1,4-

benzoquinone scaﬀold has received signiﬁcant attention by

various research groups owing to its antioxidant potential and

many other pharmacological beneﬁts. 1,4-Benzoquinone

derivatives have exibited a wide variety of pharmacological

actions like anti-Alzheimer, cognition management, anti-

inﬂammatory, anticonvulsant, anticancer, antioxidant, antima-

larial, antiviral, anthelmintic and other activities. Besides

anticholinesterase activity, the benzoquinone nucleus also

acts on diﬀerent protein targets like β-amyloids (Ab) and

NFTs.¹³The antioxidant and the neuroprotective potential of

this moiety have created a lot of research attention for the

designing of multifunctional agents eﬃcient against cognitive

decline. Some of the molecules have been reported to show

greater selectivity for AChE over BChE. On the basis of these

facts, the present study was aimed to design and synthesize

some 1,4-benzoquinone derivatives with selective targeted

Figure 1. Aligned bundle of 43 molecules used for QSSR study and

described in Table 1.

data set was randomly divided into training and test sets of 32

and 11 compounds, respectively. Statistical results of the

generated models are mentioned in Table 2. The best QSSR

Table 2. Statistical Parameters of the Developed QSSR

Models for Various PLS factors

a

b

c

d

e

f

PLS factor

SD

R²

F

RMSE

Q²

Pearson r

1

2

3

0.4025

0.2887

0.1740

0.8134

0.9073

0.9676

126.4

137.0

268.4

0.63

0.84

0.77

0.5213

0.1417

0.2814

0.8120

0.3794

0.5426

a

b

Standard deviation of regression. Coeﬃcient of determination for

c

d

training set molecules. Variance ratio. Root-mean-square error.

e

f

Coeﬃcient of determination for test set molecules. Pearson

coeﬃcient of correlation for test set molecules.

model corresponding to PLS (partial least square) factor 3

displayed acceptable values of statistical parameters, i.e.,

standard deviation (SD), 0.4025; R², 0. 8134; F-value, 126.4;

RMSE, 0.63; Q², 0.5213; Pearson r of 0.8120 where R²

corresponds to coeﬃcient of prediction for training set

molecules, RMSE to root-mean-square error, Q²to cross

validation coeﬃcient of prediction for test set molecules, and

Pearson r to Pearson coeﬃcient of correlation for the test set

molecules. Plots of predicted pSI vs actual pSI for the training

and the test sets have been reported in Figure 2. The statistical

measures of the best model clearly indicate that the generated

model is consistent and reliable and has satisfactory prediction

power for new molecules. On the basis of the 3D-QSSR model,

eight novel compounds were designed as described in Figure 3,

taking into account all the important substituted groups at

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Figure 2. Graphs of experimental and predicted pSI values: (A) for training set molecules; (B) for test set molecules (Table 1).

Figure 3. Structures of the designed and duly synthesized molecules.

various positions of benzoquinone required to obtain higher

selectivity for AChE inhibition over BChE.

generated model. In all the contour plots both the highest

selective inhibitor of AChE from the data set (molecule 12,

green colored) and the least selective (molecule 3, purple

colored) molecules are shown in the background to analyze the

properties responsible for selectivity.

Figure 4A corresponds to the contour map of electron

withdrawing property generated for best model with PLS

factor 1. This contour mapped over the aligned pair of the

molecules 2a (purple) and 12 (green color). Compound 2a

was synthesized and showed the highest selectivity for AChE,

whereas compound 12 was the highest selective molecule of

the data set library. This ﬁgure clearly indicates that the

presence of an electron withdrawing group at the ortho

position of the benzoquinone ring is favorable (2a) for

enhanced SI of the molecules. Figure 4B indicates that contour

maps of the hydrogen bond donor property of the model

where compound 2a appeared in the blue cubes region

2.2. QSSR Contour Mapping. The QSSR model displays

3D characteristics (Figure 4) as cubes that represent the model

color according to the sign of their coeﬃcient values, which by

default is blue for positive coeﬃcients and red for negative

coeﬃcients. Positive coeﬃcients indicate an increase in activity,

and negative coeﬃcients indicate an decrease in activity.

Visualization of the coeﬃcients is useful to identify character-

istics of the ligand structures that tend to increase or decrease

the activity. This might give a clue to what functional groups

are desirable or undesirable at certain positions in a molecule.

Contour plots inﬂuencing the selectivity index for the best

model (corresponding to PLS 1) were generated for diﬀerent

properties: electron withdrawing, hydrogen bond donor, and

hydrophobic properties. Other properties like negative

ionizable and positive ionizable were not signiﬁcant for the

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Docking investigations of the designed molecules on

rhAChE were carried out using CDOCKER module of

DS4.1 based on CHARMm force ﬁeld and indicated signiﬁcant

binding interactions of the molecules with the amino acid

residues at both the catalytic and the peripheral site of the

AChE enzyme. All the designed molecules were docked into

the active site of the enzyme AChE, and they demonstrated

major interaction with the key amino acid residues across the

entire length of the active site region of the enzyme. The gray

shaded region demonstrated signiﬁcant placement of the

ligands in the cavity space encompassing both the peripheral

anionic site and the catalytic site of the protein signifying a

credible elucidation of the proﬁcient ﬁt of the molecules into

the active site. To compare the selectivity toward AChE, these

designed molecules were also docked into BChE (PDB code

1P0I) protein, but no signiﬁcant binding interactions of the

molecules with this protein were observed.

Docking interactions of the most selective designed

molecules have been shown in Figure 5 with 2D and 3D

diagrams. All these ligands conﬁrmed signiﬁcant hydrogen

bonding and aromatic π−π stacking interactions with the

important amino acid residues present on the active site gorge

of AChE.

Compound 2a showed interactions similar to donepezil

(PDB code 4EY7) with AChE exhibiting hydrogen bonding

interaction with PHE295 and aromatic π−π stacking with

TRP86 and TRP286 encompassing the entire length of the

peripheral anionic site to the catalytic site of the protein. This

demonstrated a favorable position of the ligand in the binding

pocket of the AChE enzyme. This compound 2a was found to

be the most selective for AChE inhibition. The carbonyl

functional group of benzoquinone exhibited hydrogen bonding

interaction with PHE295 in the mid-gorge of the protein in the

other two designed molecules 2b and 2c. These compounds

also displayed strong aromatic interactions covering the

catalytic binding gorge by interacting with TRP86. Other

interactions like π−cationic were also displayed with HIS447

in compound 2b. 3D-QSSR model based designed molecules

displayed important interactions with amino acid residues

which were imperative for determining their selectivity proﬁle.

The results in binding interactions analysis which were

observed in docking study are in agreement with the pattern

of pharmacological activity of the compounds.

2.4. Synthesis. In view of the exhaustive outline of the

structural and functional features of the enzymes AChE and

BChE, aspects of oxidative pressure in dementia, and thorough

data about 1,4-benzoquinone, some novel analogues have been

designed based on the 3D-QSSR model. These designed

molecules were assessed by important interactions to conﬁrm

their probable ﬁtness into the cavity of the AChE protein

minimizing the tedious synthesis of unwanted molecules. After

these considerations various designed molecules were synthe-

sized by synthetic methods reported in the literature.¹⁴The

benzoquinone derivatives with various substituted piperazines

were then pharmacologically appraised for their promising

responsibility in abating the cognitive dysfunction.

Figure 4. Contour plots generated for the best model with PLS factor

1: (A) for electron withdrawing property; (B) for hydrogen bond

donor property; (C) for hydrophobic donor property. ∗ = property

for the most potent synthesized compound (2a) aligned over the least

active molecule (compound 3) of the data set. Red color is for least

active molecule (compound 3) of the data set, and green color is for

compound 2a. ∗∗ = property for the most potent synthesized

compound (2a) aligned over the most active molecule (compound

12) of the data set. Purple color is for the highest active molecule

(compound 12) of the data set, and green color is for compound 2a.

correspond to the favorable position for the placement of

hydrogen bond donor groups, whereas compound 3 of data set

molecules appearing in the red cubes region showed

unfavorable position for the placement of hydrogen bond

donor groups. Similarly, hydrophobic property’s contour map

is shown in Figure 4C. It can be identiﬁed that hydrophobic

substituents present near the pyridine ring at a distance of 4−5

carbon atoms from the core benzoquinone ring may contribute

to increased selectivity of the molecules. This eﬀect can be

seen in molecule 2a with higher selectivity index. On the other

hand, low selectivity index of the molecules 2f and 2h as

compared to the highly selective molecules may also be due to

the absence of a hydrophobic substituent at a favorable

position from the benzoquinone ring.

2.3. Docking Analysis. The most recent X-ray crystallo-

graphic record of the rhAChE (PDB code 4EY7) cocrystallized

with donepezil has revealed that it extends itself covering the

intact span of the enzymatic active site gorge. The idiosyncratic

orientation of donepezil in the active site gorge permits it to

reach out from the catalytic anionic site at the bottom near

TRP86 to the peripheral anionic site at the top near TRP286.

Since donepezil is a selective AChE inhibitor, it was used as a

reference ligand for more apparent, speciﬁc, and reliable

designing of the molecules for selective AChE inhibition.

Elicited from these speciﬁcs, a docking study was carried out

on the enzyme AChE (rhAChE with donepezil as the reference

ligand) to investigate the preferred binding mode of the

designed molecules.

2.4.1. Synthesis of 2,5-Disubstituted-1,4-benzoquinones.

The synthesized molecules comprised a 1,4-benzoquinone ring

conjugated with various substituted piperazine moieties. The

compounds 2a−2h were synthesized by the reaction of 1:2

molar ratio of hydroquinone (1a) with 1-(2-pyridyl)piperazine,

1-(2-pyrimidyl)piperazine, 1-(4-methoxyphenyl)piperazine, N-

ethylpiperazine, 1,2-phenethylpiperazine, 1-(diphenylmethyl)-

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Figure 5. Docking interactions of molecules with AChE. A1 and A2 showed 3D and 2D poses of compound 2a, respectively. B1 and B2 showed 3D

and 2D poses of compound 2b, respectively. C1 and C2 showed 3D and 2D poses of compound 2c, respectively.

piperazine, N-methylpiperazine, and 1-benzylpiperazine, re-

spectively, in methanol, at 60−70 °C temperature (Scheme 1),

with high yields and purity. The color of the reaction mixtures

turned from colorless to red. The compounds have been

characterized by elemental analyses, ¹H and ¹³C NMR spectra,

IR spectra, and mass spectroscopy.

2.5. Pharmacological Studies. 2.5.1. In Vitro Studies.

2.5.1.1. Assay of Acetylcholinesterase and Butyrylcholines-

terase. All the synthesized molecules have been assessed for

their in vitro inhibitory potential against acetylcholinesterase

and butyrylcholinesterase as stated in the spectrophotometric

method of Ellman et al.¹⁵The brains of fresh mice were

decapitated, and the hippocampus and frontal cortex were

collected as the source for the enzymes AChE and BChE. The

inhibitory activity of compounds toward AChE and BChE was

determined by quantifying the formation of the yellow anions

which were obtained by the reaction between Ellman’s reagent

and the thiocholine generated during the enzymatic hydrolysis

of acetylthiocholine iodide (ATChI) and butyrylthiocholine

iodide (BTChI), respectively. The AChE and BChE inhibitory

activities of the synthesized compounds along with the

reference standard donepezil were determined as percentage

inhibition at ﬁve diﬀerent concentrations, i.e., 10, 25, 50, 75,

and 100 μM as described in Table 3 and Table 4, respectively.

On the basis of percentage inhibitory activity of the molecules,

the IC₅₀values in micromolar range were calculated¹⁶and

converted to selectivity index (SI) of AChE inhibition over

BChE. Since the 3D-QSSR model was generated taking pSI

values, these pSI values were computed and were tabulated in

Table 5 and comparatively represented in the graph shown in

Figure 6.

The observations revealed that compound 2a having 2-

pyridylpiperazine substitution on the benzoquinone moiety

displayed the most promising selective inhibitory potential

with pSI value of 1.75 of AChE over BChE which was found to

be greater than the predicted pSI value, i.e., 1.61 from the

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moderate antioxidant activity of 30.54 0.49, 30.33 0.66,

and 29.58 0.55, respectively, while rest of the compounds

were found to be comparatively weak against hydrogen

peroxide scavenging activity.

Scheme 1. Synthesis of the Designed Compounds

2.5.1.3. DPPH Scavenging Activity. A decrease in

intellectual capacity is evident of the changes that happen in

neuronal function with age. Continuing oxidative pressure is

accepted to be one of the major contributing factors leading to

decrease in cognitive function. Various factors like free radicals,

predominantly reactive oxygen species, are implicated in the

progress of numerous neuronal degenerative diseases. The

evaluation of free radical scavenging activity for antioxidant

potential was performed using stable free radical 1,1-diphenyl-

2-picrylhydrazyl (DPPH) (Figure 7). DPPH gives a distin-

guishing deep purple color to the solution due to the existence

of an odd electron on the nitrogen of 1,1-diphenyl-2-

picrylhydrazyl radical showing a strong absorption band at

517 nm. The hydrogen atom of the antioxidants accepts the

odd electron from the nitrogen atom of DPPH. This results in

a change in color from purple to colorless when the free radical

of DPPH is scavenged by the antioxidant. The quantity of

discoloration and associated percentage of DPPH diminution

at absorbance 517 nm indicate the scavenging potency of the

compounds relating to their hydrogen donating capabil-

ity.¹⁷⁻²⁰The antioxidant eﬃcacy of the synthesized com-

pounds has been depicted at ﬁve diﬀerent concentrations (10,

25, 50, 75, 100 μM). Most of the compounds showed good

scavenging activity, while some of them exhibited moderate

activity against DPPH in a dose dependent manner. The

radical scavenging activity of all the synthesized compounds

has been tabulated in Table 7 along with the activity of

ascorbic acid which was used as a reference natural antioxidant

for DPPH radical scavenging activity. Compounds 2b, 2c, 2g,

and 2h among the synthesized library showed appreciable

percentage scavenging of DPPH radical with values of 35.01

QSSR model. Compound 2c with 2-pyrimidyl substitution also

showed appreciable selective inhibitory activity with pSI value

of 1.22. The observed pSI value (1.22) for this compound was

found to be in close proximity to the predicted pSI value

(1.70). Compounds 2b, 2f, and 2g also displayed signiﬁcant

selective inhibition of AChE with pSI values of 0.332, 0.067,

and 0.057, respectively, whereas derivatives 2d, 2e, and 2h

displayed negative pSI value indicating enhanced selectivity of

these compounds for BChE as compared to AChE.

2.5.1.2. Scavenging of Hydrogen Peroxide. Scavenging of

hydrogen peroxide is another strategy used to investigate the

antioxidant potency of the newly synthesized molecules by

employing a standard method using ascorbic acid as the

reference molecule.¹⁷The antioxidant potential as percentage

scavenging of hydrogen peroxide has been summarized in

Table 6. The results showed compounds 2a and 2c exhibited

maximum percentage scavenging of hydrogen peroxide radical

with values of 34.83 1.12 and 34.51 0.74, respectively, at

100 μM concentration as compared to ascorbic acid (89.92

0.049). Compounds 2b, 2e, and 2h at 100 μM also showed

0.70, 45.53

0.19, 39.15

0.19, and 38.48

0.78,

respectively, at 100 μM concentration. Pyridyl substituted

compound 2a displayed signiﬁcant percentage scavenging of

DPPH radical at varied concentrations (10, 25, 50, 75, and 100

μM). Investigating the eﬀectiveness of these compounds

against diﬀerent oxidative stress constraints in cognitive

dysfunction would be a further approach to determine their

neuroprotective function. These compounds thus revealed a

dual role in the management of oxidative damage and

cholinergic deﬁcit, consequently performing an auspicious

role in the management of cognitive decline.

2.5.2. In Vivo Studies. On the basis of the initial molecular

docking studies and in vitro screening assays (inhibition of

Table 3. Percentage Inhibition of AChE Activity by the Synthesized Molecules

inhibition of AChE activity by the synthesized molecules (in %) (mean SD)

compound

10 μM

25 μM

50 μM

75 μM

100 μM

2a

2b

2c

2d

2e

2f

2g

2h

35.87 0.05

15.58 0.08

22.83 0.06

17.39 0.06

28.62 0.08

17.39 0.10

09.42 0.10

32.25 0.10

81.88 0.02

44.57 0.06

26.81 0.05

23.55 0.04

16.67 0.06

33.34 0.01

21.01 0.05

13.77 0.03

38.40 0.05

81.16 0.01

47.10 0.05

31.16 0.07

26.09 0.07

30.43 0.05

44.57 0.01

25.00 0.04

25.32 0.06

40.94 0.06

82.25 0.01

51.09 0.03

32.97 0.08

28.62 0.08

34.78 0.05

45.29 0.05

27.17 0.05

37.68 0.05

46.01 0.03

85.14 0.01

60.51 0.04

32.25 0.08

32.61 0.04

36.96 0.05

48.19 0.04

32.61 0.07

39.13 0.04

53.26 0.04

88.04 0.01

donepezil

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Table 4. Percentage Inhibition of BChE Activity by the Synthesized Molecules

inhibition of BChE activity by the synthesized molecules (in %) (mean SD)

compound

10 μM

25 μM

50 μM

75 μM

100 μM

2a

2b

2c

2d

2e

2f

2g

2h

0

00.36 0.08

09.71 0.05

01.44 0.05

14.03 0.06

07.19 0.01

10.07 0.05

02.88 0.05

09.71 0.04

0

02.88 0.08

10.43 0.11

02.16 0.05

16.19 0.07

07.55 0.02

10.43 0.04

05.39 0.10

10.07 0.06

0

03.96 0.06

12.59 0.06

03.24 0.04

16.91 0.06

08.99 0.06

11.87 0.06

05.75 0.08

11.51 0.05

00.36 0.01

04.32 0.03

16.19 0.08

06.47 0.07

17.99 0.08

10.43 0.04

12.59 0.06

07.91 0.08

12.95 0.06

00.72 0.01

03.60 0.04

00.72 0.08

10.07 0.08

06.12 0.06

05.40 0.11

00.72 0.10

08.63 0.06

0

donepezil

Table 5. Inhibition of AChE and BChE by the Test Compounds and Their Selectivity Index Compared to the Predicted Value

from QSSR Model

compound

IC₅₀for AChE (μM)

IC₅₀for BChE (μM)

selectivity Index (SI)

−log SI (pSI)

predicted value (predicted pSI)

2a

2b

2c

2d

2e

2f

2g

2h

00.7895

10.7024

03.5858

15.6690

08.0550

10.0506

29.7656

06.6022

00.1216

45.0700

22.9700

60.1490

08.4980

07.5490

11.7354

33.9430

04.4700

75.0020

0.018

0.466

0.060

1.844

1.067

0.856

0.877

1.477

0.002

1.757

0.332

1.225

−0.266

−0.028

0.067

0.057

−0.169

2.790

1.6136

1.5514

1.7094

1.4714

2.2300

1.6694

1.3720

1.8354

2.1700

donepezil

acetylcholinesterase, and evaluation of oxidative stress

biomarkers.

2.5.2.1. Behavioral Studies for Memory Assessment. To

perform the behavioral studies for memory assessment, various

methods have been designed to evaluate the synthesized

molecules for their potency on memory and learning.

Evaluation of the cognitive function with respect to the

behavioral response in instant memory and aversive memory

was assessed by step down passive avoidance method with a

modiﬁcation in the standard protocol.²¹Escape latency (EL)

of the animal to locate the insulated platform is noted as the

indicator of acquisition and learning. In view of the in vitro

considerations, three benzoquinone-piperazine hybrid mole-

cules (2a, 2b, and 2c) were chosen to examine their memory

improvement capabilities utilizing step down passive avoidance

and escape learning protocol in rodent’s memory evaluator

with donepezil as the reference drug. For inducing cognitive

amnesia in the rodent, scopolamine was used to antagonize the

muscarinic receptor. After training, the mice were treated with

scopolamine (2 mg/kg) along with the newly synthesized

Figure 6. Correlation between the predicted pSI and the observed pSI

values of the synthesized compounds.

acetylcholinesterase activity and butyrylcholiesterase, scaveng-

ing of DPPH, and scavenging of H₂O₂), a total three

compounds 2a, 2b, and 2c were identiﬁed and were promoted

for their in vivo behavioral memory assessment, ex vivo assay of

Table 6. H₂O₂Radical Scavenging Activity of the Synthesized Molecules

scavenging of hydrogen peroxide (in %) (mean SD)

compound

2a

2b

2c

2d

2e

2f

2g

2h

10 μM

25 μM

50 μM

75 μM

100 μM

22.30 0.37

17.90 0.49

22.08 0.49

15.54 0.67

20.79 0.37

18.54 0.67

15.22 0.81

20.26 0.32

36.12 0.67

24.54 0.49

21.54 0.85

25.51 0.67

17.79 0.68

22.08 0.74

19.51 0.19

17.47 0.49

21.33 1.30

51.23 0.49

28.51 0.74

20.08 0.49

27.01 0.56

18.86 0.74

24.65 0.67

21.54 0.85

19.40 0.19

24.01 0.49

69.99 0.81

30.97 0.49

25.51 0.37

28.72 0.37

21.54 0.85

28.29 0.56

24.44 0.32

20.47 0.67

26.26 0.67

78.35 0.67

34.83 1.12

30.95 0.49

34.51 0.74

21.97 0.66

30.33 0.67

25.51 0.37

24.76 0.32

29.58 0.56

89.92 0.49

ascorbic acid

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step-down latency (SDL) and escape latency (EL). Further,

the number of mistakes made by the mice after 24 h of drug

administration was recorded to evaluate the retention of

memory along with step-down latency (SDL) and escape

latency (EL) to assess acquisition of memory. All the

experimental observations were expressed as mean

SD

values and have been tabulated in Table 8. This procedure

focused on the eﬀect of novel molecules on short-term and

aversive memory in an animal model. The experimental

observations showed that the escape latency in the mice group

treated with scopolamine (31.83 2.32 s) was higher than the

group treated with the control (21.33 3.78 s) in mice. In the

group that showed higher escape latency time induced due to

scopolamine, the latency decreased after the administration of

donepezil (13.17 2.14 s) at a dose of 0.5 mg/kg. Out of all

the selected compounds, analogue 2a showed lesser escape

latency time (12.33

2.33 s) to reach to the shock free

insulated wooden block as compared to the reference drug

donepezil.

Further, the experimental results showed that the step down

latency time in the group treated with scopolamine (11.00

1.41 s) was considerably shorter as compared to the group

treated with the control (154.17 8.33 s), and this short step

down latency of scopolamine was appreciably overturned by

the reference donepezil (91.17 6.11 s) at a dose of 0.5 mg/

kg. Among the tested compounds, the most appreciable

observation was noticed for compound 2a with step down

latency time (53.00

8.27 s). The memory improvement

parameter was also tested by counting the number of mistakes

done by the mice to escape from the insulated area. All the

tested compounds showed appreciable outcomes as compared

to scopolamine. The number of mistakes in compounds 2a, 2b,

and 2c was found to be 6.00 3.74, 8.33 2.06, and 9.17

1.16, respectively, which were comparable to the standard

donepezil. Investigation of the memory parameters noticeably

showed that compound 2a demonstrated appreciable proﬁ-

ciency in the management of scopolamine instigated memory

dysfunction (Table 8, Figure 8).

2.5.3. Ex Vivo Studies for the Analysis of Biological

Chemicals in the Brain Supernatant of Mice. 2.5.3.1. Ace-

tylcholinesterase. The three compounds 2a, 2b, and 2c which

were selected for behavioral studies were further subjected to

ex vivo studies, i.e., acetylcholinesterase inhibition using Ellman

method.¹⁵The mice used for previous in vivo behavioral

assessment (0.5 mg/kg body weight) were taken to assess the

brain AChE activity ((nmoles of substrate hydrolyzed/min)/

(mg of protein)) immediately after completion of the in vivo

studies. These animals were sacriﬁced by cervical dislocation,

and the acetylcholinesterase activity was calculated for control,

scopolamine, donepezil, and the tested compounds in the

respective treated groups. The AChE activity in the group of

scopolamine (30.39 0.74 (nmoles of substrate hydrolyzed/

min)/(mg of protein)) treated mice was higher than the

control (19.32 1.02 (nmoles of substrate hydrolyzed/min)/

(mg of protein) and donepezil (20.34

0.59 (nmoles of

Figure 7. Graphical representation of the synthesized compounds for

(A) inhibition of acetylcholinesterase activity, (B) inhibition of

butyrylcholinesterase, (C) % scavenging of H₂O₂, and (D) %

scavenging of DPPH at 100 μM concentration represented as the

mean SD of three individual experiments performed in triplicates.

substrate hydrolyzed/min)/(mg of protein)) treated groups

due to depletion of acetylcholine in the synaptic cleft. The

results for AChE activity of the compounds have been

tabulated in Table 9 and represented graphically in Figure 9.

These observations revealed that the AChE activities of

molecules (0.5 mg/kg) or the reference standard drug

donepezil (0.5 mg/kg) through ip route, respectively. After

training the mice, average basal readings were calculated for

compound 2a (19.71

lyzed/min)/(mg of protein)), compound 2b (22.05

(nmoles of substrate hydrolyzed/min)/(mg of protein)), and

2.17 (nmoles of substrate hydro-

2.22

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Table 7. DPPH Radical Scavenging Activity of the Synthesized Molecules

scavenging of DPPH (in %) (mean SD)

compound

2a

2b

2c

2d

2e

2f

2g

2h

10 μM

25 μM

50 μM

75 μM

100 μM

31.66 0.70

21.81 0.67

27.85 0.34

20.36 0.39

19.35 0.19

18.68 0.51

18.90 0.70

23.27 1.08

37.47 0.19

34.79 0.97

24.27 1.02

31.99 0.51

23.27 0.19

22.04 0.39

22.15 0.34

25.28 0.97

25.61 0.39

45.19 0.51

37.47 0.51

28.19 0.67

33.56 0.89

26.96 0.39

25.95 0.19

24.05 0.51

29.49 0.19

29.75 0.19

61.63 0.51

40.72 0.39

32.21 0.34

34.34 0.19

31.66 0.38

29.42 0.19

29.31 0.19

34.11 0.39

34.45 0.19

75.17 1.01

42.84 0.19

35.01 0.70

45.53 0.19

32.55 0.00

32.66 0.84

34.00 0.70

39.15 0.19

38.48 0.78

90.04 0.51

ascorbic acid

a

Table 8. Memory Parameters (Latency and the Number of Mistakes) Data of the Tested Analogues

memory parameter (mean SD)

experimental group

basal SDL

SDL

basal EL

EL

no. of mistakes

control

09.00 1.79

09.33 1.75

11.00 1.26

09.83 3.31

08.17 1.47

08.67 2.16

154.17 8.38

11.00 1.41

91.17 6.30

53.00 8.27

45.83 10.62

50.67 6.25

40.50 2.74

38.83 4.62

39.00 4.03

34.50 5.78

40.33 4.63

25.00 13.93

21.33 3.78

31.83 2.32

13.17 2.14

12.33 2.33

27.17 5.91

22.17 3.06

04.17 1.00

15.83 2.48

05.83 1.47

06.00 3.74

08.33 2.06

09.17 1.16

scopolamine

SCO + donepezil

SCO + 2a

SCO + 2b

SCO + 2c

a

All the results are expressed as the mean SD (n = 6).

compound 2c (22.09 2.56 (nmoles of substrate hydrolyzed/

min)/(mg of protein)) were in concordance with their in vivo

studies. In addition, compound 2a displayed better AChE

activity as compared to reference drug donepezil. This

suggested that the memory improving impacts of the newly

designed and synthesized compounds (2a, 2b, and 2c) may be

ascribed to the inhibition of acetylcholinesterase enzyme by

the compounds prompting increased levels of acetylcholine in

the brain. Since these results were in close proximity to the

behavioral outcomes, it was suggested that conceivable

clariﬁcation for the mechanism of the newly designed and

synthesized selective molecules was through the inhibition of

the AChE enzyme.

2.5.3.2. Lipid Peroxidation. Because of the expanded

vulnerability of the lipids to get oxidized, lipid peroxidation

is an exceptionally delicate marker to ﬁnd the degree of

oxidative pressure. The most outstanding assay presently being

used as a guide for lipid peroxidation is the assessment of the

thiobarbituric acid reactive substances (TBARS). This assay is

based on the measurement of absorbance of the production of

a red adduct (absorption maxima 532 nm) between TBA

(thiobarbituric acid) and malondialdehyde (MDA) by employ-

ing the method of Wills.^22,23

The groups treated with scopolamine at a concentration of 2

mg/kg dose demonstrated an uplift in the lipid peroxides level

(1.07 0.14 nmoles of MDA/(mg of protein)) as compared

to the control (0.46 0.01 nmoles of MDA/(mg of protein))

group. Donepezil and the tested analogues showed signiﬁcant

inhibition of the MDA levels in the brain at a concentration of

0.5 mg/kg. Compound 2c (0.72 0.10 nmoles of MDA/(mg

of protein)) was found to be the most eﬀectual. Compounds

2a, 2b, and 2c displayed appreciable inhibition of lipid

peroxide levels with the values 0.81 0.10, 0.80 0.02, and

0.72 0.10 nmoles of MDA/(mg of protein), respectively as

compared to standard drug donepezil, i.e., (0.82 0.01 nmoles

of MDA)/(mg of protein) given in Table 10 and Figure 9.

2.5.3.3. Superoxide Dismutase (SOD). Superoxide dis-

mutase (SOD) establishes a line of defense against the reactive

oxygen species. It catalyzes the dismutation of superoxide ion

into hydrogen peroxide which is then decomposed into water

through diﬀerent enzymes. Down-regulation of SOD in the

body is related to an augment in neuronal cell injury. The

superoxide dismutase (SOD) activity was calculated as per the

reported procedure²⁴which involved the reduction of

nitroblue tetrazolium (NBT, blue color) to a purple colored

chromophore (formazan). Accordingly, hydroxylamine hydro-

chloride was oxidized to nitrite and hydronium ions. SOD

enzyme declines the fall of NBT by dropping the oxidation of

hydroxylamine hydrochloride. The enzymatic activity of

superoxide dismutase drastically reduced in the brain of

scopolamine treated mice in contrast to the control group. The

selected new analogues appreciably ameliorated the unfavor-

able eﬀects of scopolamine on the SOD antioxidant protection

system in a comparable manner as seen in the lipid

peroxidation assay. The group treated with scopolamine

showed 0.34

the standard drug donepezil (05.07

comparable to compounds 2a and 2c with values of 05.21

0.94 and 04.77 0.75 SOD units/(mg of protein),

0.15 SOD units/mg which was reversed by

0.39 SOD units/mg)

respectively. This implicated that the analogues were capable

of reducing the oxidative stress induced by scopolamine (Table

11, Figure 9).

2.5.3.4. Reduced Glutathione. Glutathione (GSH) is a

signiﬁcant intracellular antioxidant agent comprising three

amino acids: cysteine, glycine, and glutamine in which the

carboxyl group of glutamate is connected through a peptide

bond to the amino group of cysteine. It is an endogenous

antioxidant agent that is primarily created by the neurons and

the ganglion cells. GSH detoxiﬁes hydroxyl ion and hydrogen

peroxide to water and atomic oxygen and helps in keeping up

the eﬃcient integrity of the cells. Consumption of glutathione

in the body creates a cascade of occasions bringing about

oxidative damage. The level of glutathione decreases with

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Figure 8. Comparison of (A) basal step-down latency and step-down latency, (B) basal escape latency and the escape latency, and (C) number of

mistakes made by mice in various treated groups. Signiﬁcance for these parameters was determined by one way ANOVA followed by Tukey’s test

to ﬁnd out intergroup variation: (@) probability value compared to control; (#) probability value compared to scopolamine; ($) probability value

compared to donepezil. A probability value of <0.05 means signiﬁcantly diﬀerent, and NS means not signiﬁcant.

increase in oxidative stress and is a clear indicator of oxidative

damage which has been seen in the scopolamine treated group

(19.42 1.40 nmoles of GSH/(mg of protein)) as compared

to the control (39.88 1.33 nmoles of GSH/(mg of protein)).

(mg of protein)). Compounds 2b and 2c also demonstrated

signiﬁcant results 23.89 0.97 and 21.38 2.97 nmoles of

GSH/(mg of protein). It can be deduced from the

observations that the tested analogues sustained antioxidant

potential (Table 12, Figure 9).

Compound 2a (28.67

protein)) treated group showed higher GSH levels as

compared to the standard (26.90 0.68 nmoles of GSH/

1.83 nmoles of GSH/(mg of

2.5.3.5. Catalase. Catalase is a tetrameric enzyme consisting

of four porphyrin heme nuclei which catalyze the conversion of

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2a, 2b, and 2c as potent inhibitors of AChE with appreciable

antioxidant potential. The outcome of ex vivo AChE inhibition

assays was also in conformity with the behavioral studies data.

Compound 2a was found to be the most potent out of the

synthesized analogues. This compound was subsequently

subjected to molecular dynamic simulation to obtain its

stability with the enzyme AChE. The graph shows that the

compound retained its stable conformation within the RMSD

value of 3 Å and conﬁrmed all the important interactions with

signiﬁcant percentage. Hence the study indicated that

compound 2a will serve as a promising lead candidate in the

area of cognitive improvement.

Table 9. Ex Vivo AChE Activity of Compounds 2a, 2b, and

2c, 0.5 mg/kg Body Weight

a

experimental group

AChE activity (mean SD)

control

19.32 1.02

30.39 0.74

20.34 0.59

19.71 2.17

22.05 2.22

22.09 2.56

scopolamine

SCO + donepezil

SCO + 2a

SCO + 2b

SCO + 2c

a

In (nmoles of substrate hydrolysed/min)/(mg of protein).

H₂O₂and glutathione into water. The H₂O₂which is formed

as a byproduct in the sequence of univalent reduction of

oxygen present in the nerve cells is converted to water. This

conversion indicates the antioxidant potential of a compound

by determining the amount of H₂O₂converted into water. This

enzyme mainly acts toward higher levels of H₂O₂. Activity of

catalase enzyme declined in dementia stimulated scopolamine

4. MATERIALS AND METHODS

4.1. Workstation and Software Considerations. Three

dimensional structures, QSSR building, and all the molecular

modeling studies were carried out on an HP Z200 workstation

running on CentOS 5.4 linex operating system. PHASE 3.2^25,26

executed in the Maestro 9.1 modeling suite (Schrodinger, LLC, New

York, NY) was explored to generate 3D QSSR models. Molecular

docking studies were carried out on a CHARMM supported docking

tool, CDOCKER, and molecular dynamics simulations were

performed with OPLS_2005 force ﬁeld in Desmond software of

treated group (0.218

0.03 (μmoles of H₂O₂decompose/

min)/(mg of protein)), whereas it was signiﬁcantly enhanced

after subsequent administration of the reference drug

donepezil (0.516

0.05 (μmoles of H₂O₂decompose/

̈

Schrodinger (Schrodinger, LLC, New York, NY).

min)/(mg of protein)). Compound 2c showed appreciable

catalase activity with a value of 0.398 0.08 (μmoles of H₂O₂

decompose/min)/(mg of protein). Two other tested com-

pounds 2a and 2b also noticeably barred the loss of catalase

activity with values of 0.371 0.05 and 0.387 0.03 of H₂O₂

decompose/min/(mg of protein), respectively, in the scopol-

amine induced oxidative pressure (Table 13, Figure 9).

2.6. Molecular Dynamics. Molecular dynamic simulation

of the highest selective molecule 2a was performed for a period

of 30 ns to identify the conformational stability of the protein−

ligand complex. The RMSD value of the protein backbone

atoms in complex with compound 2a was monitored to give an

insight into its structural conformation throughout the

simulation and was calculated to observe the overall stability

of the protein and the ligand within a period of 30 ns. RMSD

analysis indicated that the simulation has equilibrated its

ﬂuctuations toward the end of the simulation in the range of

3.1−3.5 Å (Figure 10). Simulations disclosed that the molecule

was maintaining key H-bond interactions with PHE290 amino

acid along with other interactions necessary for AChE

selectivity in a pattern similar to donepezil (Figure 11). This

disclosure established the fact that the molecule was

maintaining stable and important interactions with the protein

which is essential for the selectivity and potency of the

molecule against AChE.

4.2. Data Set. A data set of 43 twin inhibitors of AChE and BChE,

which held the same scaﬀold 1,4-benzoquinone, was selected for the

development of 3D-QSSR model.²⁷⁻²⁹Their biological activity values

corresponding to the inhibition of AChE and BChE were collected

from literature reports in the form of IC₅₀values in the μM range. For

suitable model development, all the molecules were selected with

similar bioassayed methods to draw accurate and precise structure−

activity correlation. Molecular structures and their corresponding IC₅₀

values against AChE and BChE have been summarized in Table 1

These reported IC₅₀values of the molecules were converted into the

selectivity index (SI) which was then modiﬁed into pSI values

(−log SI where SI is ratio of IC₅₀of AChE to IC₅₀of BChE) to get all

values in positive range, for normal distribution of errors, and to get

linear free energy relationship with independent variables. The

logarithmic transformation of SI values also minimizes any potential

errors resulting from minor discrepancies in bioassay conditions. A

total of 43 molecules having IC₅₀data for both enzymes were selected

for QSSR study. Out of the selected 43 dual inhibitors, 32 were

randomly selected for the training set and 11 for the test set by using

the “Automated Random Selection” option present in the PHASE

module.

4.3. 3D-QSSR Analysis. The data set for the 3D-QSSR study was

collected in the form of congeneric series; therefore atom based

QSAR has been addressed in the present study. The 3D structures of

all the molecules of the data set were sketched and cleaned in Maestro

9.1 workspace. MacroModel conformation search engine was further

employed to generate conformers of every ligand utilizing the

OPLS_2005 force ﬁeld, MCMM torsional sampling method, GB/SA

water, and no cutoﬀ for nonbonded interactions.³⁰The energy

minimization of molecules was performed using TNCG (truncated

Newton conjugate gradient) method with 5000 maximum iterations

and 0.001 gradient convergence threshold. The conformational search

was carried out using 20 kcal/mol energy window and RMSD cutoﬀ

of 0.5 Å for elimination of redundant conformers. A rectangle grid was

deﬁned during development of 3D-QSSR model in PHASE module to

encompass the space occupied by the training set of aligned

molecules. During model generation, 0/1 values were treated as

independent variables and selectivity index (−log SI) as dependent

variables in PLS regression analysis. QSSR models containing one to

three PLS factors were generated, and the models were validated by

predicting the activity of 11 test set ligands.

3. CONCLUSION

A series of novel 1,4-benzoquinone derivatives have been

designed using molecular modeling techniques. On the basis of

3D QSSR model, these analogues were docked to check their

binding aﬃnities with the target enzyme AChE. These

molecules were then synthesized and evaluated for their

selective acetylcholinesterase inhibitory activity over butyr-

ylcholinesterase activity for the management of cognitive

dysfunction. These compounds were also evaluated for their

antioxidant potential. Compounds 2a, 2b, and 2c exhibited

appreciable selective acetylcholinesterase activity and signiﬁ-

cant antioxidant potential. On the basis of in vitro studies,

compounds 2a, 2b, and 2c were selected for in vivo studies.

The results of the behavioral studies demonstrated compounds

The bioactive conformation of the highest selective molecule 12

was chosen as a reference for the alignment of molecules of the data

set after validating this compound on the basis of formation of H-

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Figure 9. Ex vivo activity of the tested compounds on the level of (A) enzyme acetylcholinesterase, (B) lipid peroxidation, (C) enzyme superoxide

dismutase, (D) glutathione, and (E) enzyme catalase in various treated groups. Signiﬁcance for these parameters was determined by one way

ANOVA followed by Tukey’s test to ﬁnd out intergroup variation: (@) probability value compared to control; (#) probability value compared to

scopolamine; ($) probability value compared to donepezil. Probability value of <0.05 means signiﬁcantly diﬀerent, and NS means not signiﬁcant.

bonds and proper orientation in the active site of AChE. Finally, the

aligned bundle of best conformations of all the molecules was put

through PHASE for the atom based QSAR module.^25,26

CHARMm force ﬁeld was employed for molecular docking studies.³³

The “deﬁne and edit binding site” tool was utilized to specify the

active site, and the prepared ligands were docked in the active site of

both AChE and BChE.

4.5. General. All chemicals and solvents were of analytical grade

and utilized as such for the synthesis without puriﬁcation. The

completion of the chemical reactions was monitored by thin layer

chromatography using silica gel 60-F254 plates acquired from E.

Merck (Darmstadt, Germany) and visualized in ultraviolet (UV) light.

Melting points were measured on Veego melting point apparatus and

were uncorrected. The percentage yields are reported for the pure

products obtained after synthesis and are not optimized. IR spectra

4.4. Docking. The 3D structures of all the designed molecules

were sketched and cleaned in the workspace of DS4.1. For the present

study, X-ray crystal structures of hAChE complexed with donepezil at

resolution of 2.35 Å and hBChE at resolution of 2.00 Å were collected

from the Protein Data Bank (PDB codes 4EY7 and 1P0I,

respectively).^31,32The proteins were prepared to generate protonation

state using the “Prepare Ligands” protocol in DS 4.1 at a pH 7.0

0.5, and subsequently, molecular geometry of the resulting compound

was calculated. The CDOCKER module of DS 4.1 based on

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Table 10. Lipid Peroxidation Levels of Compounds 2a, 2b,

and 2c

a

experimental group

lipid peroxidation level (mean SD)

control

0.46 0.01

1.07 0.14

0.82 0.01

0.81 0.10

0.80 0.02

0.72 0.10

scopolamine

SCO + donepezil

SCO + 2a

SCO + 2b

SCO + 2c

a

In nmoles of MDA/(mg of protein).

Table 11. Superoxide Dismutase Activity Data of

Compounds 2a, 2b, and 2c

a

Figure 10. RMSD plot of compound 2a complexed with AChE for a

time period of 30 ns.

experimental group

superoxide dismutase activity (mean SD)

control

10.97 1.17

00.34 0.15

05.07 0.39

05.12 0.94

04.04 076

04.77 0.75

scopolamine

SCO + donepezil

SCO + 2a

SCO + 2b

SCO + 2c

a

In SOD units/(mg of protein).

Table 12. Glutathione Level of Compounds 2a, 2b, and 2c

a

experimental group

glutathione level (mean SD)

control

39.88 1.33

19.42 1.40

26.90 0.68

28.67 1.83

23.89 0.97

21.38 2.97

scopolamine

SCO + donepezil

SCO + 2a

SCO + 2b

SCO + 2c

a

In nmoles of GSH/(mg of protein).

Table 13. Catalase Activity Data of Compounds 2a, 2b, and

2c

a

experimental group

catalase activity (mean SD)

Figure 11. Interactions of compound 2a complexed with AChE

during simulation for a time period of 30 ns.

control

0.634 0.04

0.218 0.03

0.516 0.05

0.371 0.05

0.387 0.03

0.398 0.08

scopolamine

SCO + donepezil

SCO + 2a

SCO + 2b

SCO + 2c

in methanol (15 mL) with continuous stirring. Subsequently, 1-

(pyridin-2-yl)piperazine (0.31 mL, 2.0 mmol) was added to the

reaction mixture and the contents were reﬂuxed with continuous

stirring at 60−70 °C for 10 h. The completion of the reaction was

ascertained by TLC. The slurry obtained was ﬁltered and the solvent

was removed under reduced pressure to obtain a solid residue. It was

then recrystallized from ethanol to aﬀord 2,5-bis(4-(pyridin-2-

yl)piperazin-1-yl)-1,4-benzoquinone (2a) as a red crystalline solid

(yield: 0.319 g, 74.32%). Mp: 249−251 °C. Calcd for C₂₄H₂₆N₆O₂:

C, 66.96; H, 6.09; N, 19.52%. Found: C, 66.88; H, 6.04; N, 19.42%.

¹H NMR (CDCl₃) δ: 8.20 (m, 2H), 7.50 (m, 2H), 6.65 (m, 2H), 6.61

(d, 2H, J = 8.52 Hz), 5.56 (s, 2H), 3.78 (m, 8H), 3.72 (m, 8H) ppm.

¹³CNMR (CDCl₃, 100 MHz) δ: 182.40 (CO), 158.73 (C_q), 152.09

a

In (μmoles of H₂O₂decomposed/min)/(mg of protein).

were recorded on PerkinElmer spectrum version 10.03.08 using

potassium bromide disk method for the preparation of the sample. ¹H

and ¹³C NMR spectra were recorded on a Bruker Avance II 400 MHz

spectrometer in CDCl₃or DMSO-d₆solvents at 400 and 100 MHz,

respectively. All chemical shifts (δ) are expressed in parts per million

(ppm) relative to the standard TMS, and the peak patterns are

designated as (s) singlet, (d) doublet, (t) triplet, (m) multiplet, and

(brs) broad signal peak. Mass spectra were obtained using LC−MS

spectrometer model Q-ToF Micro Waters (mass-to-charge ratio, m/

z). The CHN analysis was performed using Thermo Scientiﬁc iCE

3400 AAS. All the synthesized compounds exhibited a critical degree

of purity as evidenced by their elemental analyses.

(C_q), 148.03 (CH), 137.63 (ArCH), 113.61 (ArCH), 106.61 (ArCH),

105.57 (ArCH), 48.11 (CH), 44.26 (CH) ppm. FT-IR ν_max(KBr):

3392.96, 2966.24, 2845.82, 1639.20, 1574.66, 1502.33, 1447.81,

1335.03, 1295.87, 1038.40, 1231.94, 754.38 cm⁻¹. ESI-MS (m/z):

431.37 [M + H]⁺.

4.6. Synthesis. The designed compounds were synthesized using

previously reported procedures¹⁴with slight modiﬁcations and

characterized by the melting points, FTIR, ¹H NMR, ¹³C NMR,

and mass spectral analysis.

4.6.1. Synthesis of 2,5-Bis(4-(pyridin-2-yl)piperazin-1-yl)-1,4-

benzoquinone (2a). Hydroquinone (0.11 g, 1.0 mmol) was dissolved

4.6.2. Synthesis of 2,5-Bis(4-(pyrimidin-2-yl)piperazin-1-yl)-1,4-

benzoquinone (2b). Hydroquinone (0.11 g, 1.0 mmol) was dissolved

in methanol (20 mL) with continuous stirring. Subsequently, 2-

(piperazin-1-yl)pyrimidine (0.29 mL, 2.0 mmol) was added to the

reaction mixture and the contents were reﬂuxed with continuous

stirring at 60−70 °C temperature for 10 h. The completion of the

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reaction was ascertained by TLC. The red color mixture obtained was

ﬁltered and the solvent was removed under reduced pressure to

obtain a solid residue. It was then recrystallized from ethanol to aﬀord

2,5-bis(4-(pyrimidin-2-yl)piperazin-1-yl)-1,4-benzoquinone (2b) as a

brownish red crystalline solid (0.283 g, 65.55%). Mp: 258−260 °C

decomposed. Calcd for C₂₂H₂₄N₈O₂: C, 61.10; H, 5.59; N, 25.91%.

106.82 (CH), 60.19 (CH), 52.79 (CH), 48.72 (CH), 33.46 (CH)

ppm. FT-IR ν_max(KBr) 3029.00, 2947.49, 2823.49, 1633.34, 1568.74,

1448.91, 1367.91, 1298.76, and 744.68 cm⁻¹. ESI-MS (m/z): 485.18

[M + H]⁺.

4.6.6. Synthesis of 2,5-Bis(4-benzhydrylpiperazin-1-yl)-1,4-ben-

zoquinone (2f). Hydroquinone (0.11 g, 1.0 mmol) was dissolved in

methanol (20 mL) with continuous stirring. Subsequently, 1-

benzhydrylpiperazine (0.51 g, 2.0 mmol) was added to the reaction

mixture and the contents were reﬂuxed with continuous stirring at

60−70 °C temperature for 24 h. The completion of the reaction was

ascertained by TLC. The red color mixture obtained was ﬁltered and

the solvent was removed under reduced pressure to obtain a solid

residue. It was then recrystallized from ethanol to aﬀord 2,5-bis(4-

benzhydrylpiperazin-1-yl)-1,4-benzoquinone (2f) as a crystalline solid

(0.407 g, 66.85%). Mp: 201−203 °C decomposed. Calcd for

C₄₀H₄₀N₄O₂: C, 78.92; H, 6.62; N, 9.20. Found: C, 78.78; H, 6.56;

1

Found: C, 61.08; H, 5.57; N, 25.89%. H NMR (CDCl₃, 400 MHz):

δ 8.33 (d, 4H, J = 4.76 Hz), 6.55(t, 2H, J = 4.76 Hz), 5.58 (s, 2H),

3.97 (m, 8H), 3.72 (t, 8H) ppm. ¹³CNMR (CDCl₃, 100 MHz): δ

182.42 (CO), 157.77 (C_q), 152.25 (CH), 151.00 (C_q), 110.39 (CH),

105.94 (CH), 48.30 (CH), 43.04 (CH) ppm. FT-IR ν_max(KBr)

3026.30, 2909.40, 2870.26, 1639.05, 1585.50, 1546.71, 1444.05,

1358.46, 1255.04, 1035.42, and 793.57 cm¹. ESI-MS (m/z): 433.10

[M + H]⁺.

4.6.3. Synthesis of 2,5-Bis(4-(4-methoxyphenyl)piperazin-1-yl)-

1,4-benzoquinone (2c). Hydroquinone (0.11 g, 1.0 mmol) was

dissolved in methanol (20 mL) with continuous stirring. Sub-

sequently, 1-(4-methoxyphenyl)piperazine (0.10 mL, 2.0 mmol) was

added to the reaction mixture and the contents were reﬂuxed with

continuous stirring at 60−70 °C temperature for 30 h. The

completion of the reaction was ascertained by TLC. The red color

mixture obtained was ﬁltered and the solvent was removed under

reduced pressure to obtain a solid residue. It was then recrystallized

from ethanol to aﬀord 2,5-bis(4-(4-methoxyphenyl)piperazin-1-yl)-

1,4-benzoquinone (2c) as a crystalline solid (0.34 g, 67.51%). Mp:

229−231 °C. Calcd for C₂₈H₃₂N₄O₄: C, 68.83; H, 6.60; N, 11.47%.

1

N, 9.08%. H NMR (CHCl₃, 400 MHz): δ 7.41 (d, 9H, J = 8 Hz),

7.28 (t, 7H, J = 6 Hz), 7.19 (t, 4H, J = 7.28 Hz), 5.46 (s, 2H). 4.25 (s,

2H), 3.55 (s, 8H), 2.51 (s, 8H) ppm. ¹³C NMR (CDCl₃, 100 MHz):

δ 182.67 (CO), 152.59 (C_q), 142.98 (ArC_q), 128.67 (ArC), 127.90

(ArC), 127.90 (ArCH), 127.27 (ArCH), 106.62 (CH), 77.28 (CH),

77.03 (CH), 76.78 (CH), 51.61 (CH), 48.80 (CH) ppm. FT-IR ν_max

(KBr) 3043.30, 2955.00, 2878.50, 1618.60, 1542.00, 1447.90,

1300.70, 1212.40, and 753.20 cm⁻¹. ESI-MS (m/z): 609.41 [M +

H]⁺.

4.6.7. Synthesis of 2,5-Bis(4-methylpiperazin-1-yl)-1,4-benzoqui-

none (2g). Hydroquinone (0.11 g, 1.0 mmol) was dissolved in

methanol (15 mL) with continuous stirring. Subsequently, N-

methylpiperazine (0.22 mL, 2.0 mmol) was added to the reaction

mixture and the contents were reﬂuxed with continuous stirring at

60−70 °C temperature for 10 h. The completion of the reaction was

ascertained by TLC. The slurry obtained was ﬁltered and the solvent

was removed under reduced pressure to obtain a solid residue. It was

then recrystallized from ethanol to aﬀord a red crystalline solid 2,5-

bis(4-methylpiperazin-1-yl)-1,4-benzoquinone (2g) (0.205 g,

67.34%). Mp: 185−187 °C. Calcd for C₁₆H₂₄N₄O₂: C, 63.13; H,

7.95; N, 18.41. Found: C, 63.12; H, 7.41; N, 18.05%. ¹H NMR

(CDCl₃): δ 5.54 (s, 2H), 3.57 (t, 8H, J = 4.98 Hz), 2.52 (t, 8H, J =

5.08 Hz), 2.32 (s, 6H) ppm. ¹³C NMR (CDCl₃, 100 MHz): δ 182.69

(CO), 152.56 (C_q), 106.86 (CH), 54.64 (CH), 48.67 (CH), 45.93

(CH) ppm. FT-IR ν_max(KBr) 2937.40, 2849.00, 1636.30, 1571.50,

1447.90, 1365.50, 1236.00, and 753.20 cm⁻¹. ESI-MS (m/z): 305.95

[M + H]⁺.

4.6.8. Synthesis of 2,5-Bis(4-benzylpiperazin-1-yl)-1,4-benzoqui-

none (2h). Hydroquinone (0.11 g, 1 mmol) was dissolved in

methanol (15 mL) with continuous stirring. Subsequently, 1-

benzylpiperazine (0.34 mL, 2 mmol) was added to the reaction

mixture and the contents were reﬂuxed with continuous stirring at

60−70 °C temperature for 10 h. The completion of the reaction was

ascertained by TLC. The slurry obtained was ﬁltered and the solvent

was removed under reduced pressure to obtain a solid residue. It was

then recrystallized from ethanol to aﬀord a red crystalline solid of 2,5-

bis(4-benzylpiperazin-1-yl)-1,4-benzoquinone (2h) (0.256 g,

56.00%). Mp: 212−213 °C. Calcd for C₂₈H₃₂N₄O₂: C, 73.66; H,

7.06; N, 12.27. Found: C, 73.58; H, 7.01; N, 12.05%. ¹H NMR

(CDCl_3,400 MHz): δ 7.31 (d, 10H, J = 4.68 Hz), 5.51 (s, 2H), 3.56

(s, 12H), 2.57 (s, 8H) ppm. FT-IR ν_max(KBr) 3025.70, 2919.70,

2819.60, 1618.60, 1548.00, 1477.30, 1365.50, 1224.20, and 747.30

cm⁻¹. ESI-MS (m/z): 457.24 [M + H]⁺.

1

Found: C, 68.78; H, 6.56; N, 11.08%. H NMR (CDCl₃, 400 MHz):

7.05 (m, 2H), 6.94 (d, 4H, J = 4.4 Hz), 6.89 (d, 2H, J = 7.92 Hz),

5.62 (s, 2H), 3.88 (s, 6H), 3.76 (t, 8H, J = 4.54 Hz), 3.18 (t, 8H, J =

4.94 Hz). ¹³CNMR (CDCl₃, 100 MHz): δ 182.77 (CO), 152.65 (C_q),

152.18 (C_q), 140.40 (C_q), 123.57 (ArC), 121.06 (ArC), 118.38 (ArC),

106.86 (CH), 55.42 (CH), 50.39 (CH), 49.00 (CH) ppm. FT-IR ν_max

(KBr) 3052.38, 2987.83, 2946.48, 2834.83, 1634.21, 1573.63,

1441.09, 1301.72, 1234.43, and 735.84 cm⁻¹. ESI-MS (m/z):

489.14 [M + H]⁺.

4.6.4. Synthesis of 2,5-Bis(4-ethylpiperazin-1-yl)-1,4-benzoqui-

none (2d). Hydroquinone (0.11 g, 1.0 mmol) was dissolved in

methanol (15 mL) with continuous stirring. Subsequently 4-

ethylpiperazine (0.26 mL, 2.0 mmol) was added to the reaction

mixture and the contents were reﬂuxed with continuous stirring at

60−70 °C temperature for 17 h. The completion of the reaction was

ascertained by TLC. The red color mixture obtained was ﬁltered and

the solvent was removed under reduced pressure to obtain a solid

residue. It was then recrystallized from ethanol to aﬀord 2,5-bis(4-

ethylpiperazin-1-yl)-1,4-benzoquinone (2d) as a brown crystalline

solid (0.184 g, 55.19%). Mp: 157−159 °C. Calcd for C₁₈H₂₈N₄O₂: C,

1

65.03; H, 8.49; N, 16.85%. Found: C, 65.08; H, 8.46; N, 16.78%. H

NMR (CDCl₃, 400 MHz): δ 5.54 (s, 2H), 3.59 (t, 8H, J = 4.84 Hz),

2.58 (t, 8H, J = 4.58 Hz), 2.47 (q, 4H, J = 7.20 Hz), 1.11(t, 6H, J =

7.20 Hz) ppm. ¹³CNMR (CDCl₃, 100 MHz): δ 182.72 (CO), 152.56

(C_q), 106.80 (CH), 52.38 (CH), 52.21 (CH), 48.64 (CH), 11.81

(CH) ppm. FT-IR ν_max(KBr) 2923.79, 2826.63, 1633.26, 1569.85,

1447.98, 1372.16, 1225.04, and 755.67 cm⁻¹. ESI-MS (m/z): 333.13

[M + H]⁺.

4.6.5. Synthesis of 2,5-Bis(4-phenethylpiperazin-1-yl)-1,4-benzo-

quinone (2e). Hydroquinone (0.11 g, 1.0 mmol) was dissolved in

methanol (15 mL) with continuous stirring. Subsequently, 1-

phenethylpiperazine (0.38 mL, 2.0 mmol) was added to the reaction

mixture and the contents were reﬂuxed with continuous stirring at

60−70 °C temperature for 24 h. The completion of the reaction was

ascertained by TLC. The red color mixture obtained was ﬁltered and

the solvent was removed under reduced pressure to obtain a solid

residue. It was then recrystallized from ethanol to aﬀord 2,5-bis(4-

phenethylpiperazin-1-yl)-1,4-benzoquinone (2e) as a brown crystal-

line solid (0.64 g, 66.02%). Mp: 185−187 °C. Calcd for C₃₀H₃₆N₄O₂:

C, 74.35; H, 7.49; N, 11.56. Found: C, 74.28; H, 7.01; N, 11.45%. ¹H

NMR (DMSO-d₆): δ 7.22 (m, 4H), 7.14 (m, 6H), 5.58 (s, 2H,), 3.53

(t, 8H, J = 4.70 Hz). 2.75 (m, 6H), 2.57 (s, 12H, J = 4.54 Hz) ppm.

¹³C NMR (DMSO-d₆, 100 MHz): δ 182.72 (CO), 152.55 (C_q),

4.7. Pharmacological Activity. 4.7.1. In Vitro Studies. The in

vitro studies of the synthesized compounds were carried out to

explicate the probable eﬀectiveness of the compounds so that before

the animal studies only compounds that showed optimum values for

the target could be selected to avoid unnecessary exploitation of

animals.

4.7.1.1. Protein Estimation. The Biuret method was employed to

estimate the protein content for various in vitro and ex vivo studies.³⁴

Biuret reagent was prepared by mixing cupric sulfate pentahydrate,

sodium potassium tartrate (4.5 g in 40 mL of 0.2 N NaOH), and

potassium iodide (0.5 g). The volume of this solution was made up to

139.91 (ArC_q), 128.69 (ArC_q), 128.49 (ArCH), 126.21 (ArCH),

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100 mL by using 0.2 N NaOH solution. For protein estimation, 50 μL

of the enzyme supernatant was mixed with 3 mL of Biuret reagent and

2.9 mL of normal saline and further incubated for 10 min at room

temperature. The optical density of the incubated solution was

estimated at 540 nm using a Perkin Elmer UV/vis spectrophotometer.

The optical density was used to calculate the protein content (mg/

mL) using a standard plot of BSA (bovine serum albumin).

of pharmacological studies after the approval of the entire research

study protocol by the Institutional Animal Ethical Committee (Vide

Letter No. PU/45/99/CPCSEA/IAEC/2018/128). The handling

and maintenance of animals were directed in agreement with the

CPCSEA rules. The mice were placed in an optimum lab environment

(25 2 °C; 60 2% RH) in groups of six with proper supply of feed

and water (12 h light/dark cycle). The animals were familiarized to

the laboratory environment for 7 days prior to experimentation. All

experiments were carried out daily between 8:00 a.m. and 6:00 p.m.

The observations of the in vitro studies revealed that out of the

eight compounds 2a−h only three 2a−c showed promising in vitro

activity for the management of cognitive dysfunction. These three

compounds 2a−h were further selected for in vivo studies using step

down passive avoidance protocol. In this method, scopolamine (2.0

mg/kg) was used as an amnesia inducer in the animals and donepezil

(0.5 mg/kg) used as the standard. All the selected analogues and

donepezil were suspended in 1% Tween-80 solution and administered

intraperitoneally (ip) to the animals, respectively. Nine groups were

made with six animals in each group for the study. The group without

treatment with any compound was considered as the control.

Instantaneously after training, saline or scopolamine (2.0 mg/kg)

solution was administered to all the groups intraperitonially (ip)

followed by a second injection either the vehicle (1% Tween-80),

donepezil, or the test compound after 5 min of the ﬁrst injection,

respectively. The complete study to determine the step down and the

escape latencies was carried out using a rodent memory evaluator

(IMCORP, Ambala Cantt, India). This apparatus consist of a

fabricated box with electric grid and a centrally located wooden

platform (shock free zone). The following method was executed step

by step to carry out the behavioral study. (1) Familiarization: Each

animal was placed kindly on the wooden platform set in the center of

the grid slab. After 10 s of traveling around, it was returned to the

home cage. (2) Training: Each animal was placed kindly on the

wooden platform set in the center of the grid slab. When the mouse

stepped down from the wooden platform and placed its paws on the

grid slab, the discontinuous electric shock was distributed

continuously until the animal went back to the platform to escape

from the electric shock. The step down latency (SDL) and escape

latency (EL) were carefully noted, and the animals in the range of

criteria (SDL, 3−15 s; EL, 15−60 s) were used for the retention test.

As a general rule, mice were treated with inducer drug (scopolamine)

and standard drug (donepezil) or tested compounds straight away

after the training period, respectively. (3) Retention: Twenty-four

hours after training, once again each group was set to be placed on the

wooden platform to record SDL and EL of each treated group.

Furthermore, the number of mistakes made by the animal in 15 min

was recorded by turning on the mistake mode of the apparatus as

soon as the mouse touched the electric grid. All the data of latency

and mistakes were analyzed and the signiﬁcance was calculated by

using one way ANOVA followed by paired t-test for each compound

using GraphPad Prism 8 software.

4.7.1.2. Assay of AChE and BChE. The Ellman method was used to

calculate the percentage inhibition of AChE and BChE, utilizing brain

homogenate of mice as the enzyme source. The homogenate was

prepared from the brain of Balb/c strain mice (25−30 g) by

sacriﬁcing them by cervical dislocation. The brains were decapitated

and rinsed with ice-cold normal saline. The saline washed brain was

transferred to the glass Teﬂon homogenizer (REMI MOTORS, India)

containing sodium phosphate buﬀer of 50 volumes (pH 8.0, 0.1 M)

and then homogenized for 30−40 s. The supernatant as the source of

the enzyme was obtained after the centrifugation (Research

centrifuge, REMI, R-24) at 12 000 rpm for 20 min at 3−4 °C.

Solutions of the synthesized compounds and the standard drug

donepezil were prepared at ﬁve diﬀerent concentrations (10, 25, 50,

75, and 100 μM), respectively. The aliquot of each compound of

various concentrations was mixed separately with 2.6 mL of

phosphate buﬀer (pH 8.0), 100 μL of buﬀered Ellman’s reagent,

400 μL of supernatant, 20 μL of acetylthiocholine iodide, and 20 μL

of butyrylthiocholine iodide solution for AChE and BChE activity,

respectively. Immediately after mixing these solutions the change in

optical density for 2 min was noted at 412 nm using a Perkin Elmer

UV/vis spectrophotometer. The results of the AChE and BChE

inhibition activity were given in (nmoles of substrate hydrolyzed/

min)/(mg of protein), respectively.

4.7.1.3. Scavenging of H₂O₂. All the synthesized compounds 2a−h

were evaluated for the antiradical activity spectrophotometrically by

calculating percentage scavenging of H₂O₂. Phosphate buﬀered saline,

pH 7.4, was used to prepare 40 mM solution of H₂O₂at room

temperature. The concentration of H₂O₂was estimated using molar

extinction coeﬃcient of 81 mol/cm spectrophotometrically at 230

nm. Diﬀerent concentrations (10, 25, 50, 75, and 100 μM) of the test

compounds (2a−h) and the standard ascorbic acid were made in

DMSO and added to a hydrogen peroxide solution (0.6 mL, 40 mM)

and incubated for 10 min. After incubation, absorbance of the

solution was determined at 230 nm against a blank solution

containing only phosphate buﬀer and solution of the test compound

without hydrogen peroxide using a Perkin Elmer UV/vis spectropho-

tometer. The experiments were reproduced thrice, and the results

were calculated using the following formula:

% scavenged [H₂O₂] = [(A₀− A₁)/A₀] × 100

where A₀is the absorbance of the control and A₁is the absorbance in

the presence of the sample and standards.

4.7.1.4. Scavenging of DPPH. All the synthesized compounds 2a−

h were further evaluated for antiradical activity spectrophotometrically

by calculating percentage scavenging of DPPH. Diﬀerent concen-

trations (10, 25, 50, 75, and 100 μM) of the test compounds 2a−h

and the standard ascorbic acid were prepared in DMSO (0.1%).

DPPH solution (2 mL, 0.5 mM) was added to 1 mL solution of each

compound and incubated for 30 min at room temperature. The

absorbance was measured spectrophotometerically at 517 nm for each

solution of the synthesized molecules against the blank solution

containing only the compound solution without DPPH. The

experiments were reproduced thrice, and the results were calculated

using the following formula:

4.7.3. Ex Vivo Studies and Analysis of Biological Chemicals in

the Brain Supernatants of Mice. After the completion of in vivo

studies the treated animals of various groups were sacriﬁced by

cervical dislocation procedure. The brains of these animals were then

used for ex vivo studies to carry out the AChE assay and estimation of

biomarkers. The brains were surgically removed and rinsed with ice

cold saline solution to remove the blood. The brains of each group

were separately homogenized at 12 000 rpm for 20 min at 3−4 °C

using 0.1 M phosphate buﬀer of pH 7.4. The supernatants thus

obtained were used for the ex vivo studies.

4.7.3.1. AChE Assay. The Ellman method was used to estimate the

acetylcholinesterase activity in the whole brain of the treated groups.

The results were given as (nmoles of the substrate hydrolyzed/min)/

(mg of protein).

% scavenged [DPPH] = [(A₀− A₁)/A₀] × 100

where A₀is the absorbance of the control and A₁is the absorbance in

the presence of the sample and the standard.

4.7.2. In Vivo Studies. Animals (albino mice of strain Balb/c, 25−

30 g) were procured from CRI (Central Research Institute) Kasauli,

Himachal Pradesh, India, and Central Animal House of Panjab

University, Chandigarh, India, as per the availability during the course

4.7.3.2. Lipid Peroxidation. The brain supernatant (0.5 mL) of the

treated animals was mixed with Tris-HCl buﬀer (0.5 mL) and

incubated for 2 h at 37 °C. Later, to this mixture 1 mL of ice cold

trichloroacetic acid (10%) was added, and the mixture was

centrifuged for 10 min at 1000 rpm. After homogenization, 1 mL of

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the supernatant was mixed with 1 mL of thiobarbituric acid (0.67%)

and the test tube was kept in a boiling water bath for 10 min. The

solution was cooled, and 1 mL of double distilled water was added

further. Optical density of the solution was measured at 532 nm, and

the results were expressed as the number of moles of MDA/(mg of

protein).

Author Contributions

P.S. conceived the idea and did the in silico studies, synthesis,

characterization, pharmacological evaluation, statistical anal-

ysis, and manuscript preparation. O.S. did the in silico studies,

statistical analysis, and manuscript preparation. P.P. conceived

the idea, did the characterization, pharmacological evaluation,

statistical analysis, and manuscript preparation, and had overall

responsibility.

4.7.3.3. Superoxide Dismutase. Superoxide dismutase assay was

performed as stated in the procedure set by Kono et al.³⁶A separate

mixture was prepared by adding 24 μM of nitro-blue tetrazolium

(NBT), 0.1 mM EDTA, and 50 mM sodium carbonate solution. The

above mixed solution (2 mL) was then added to 0.05 mL of brain

homogenate and 0.05 mL of hydroxylamine hydrochloride (pH 6.0).

The change in optical density at 560 nm for 2 min at 30/60 s intervals

was measured as a parameter of auto-oxidation of hydroxylamine.

4.7.3.4. Reduced Glutathione. A mixture of 1 mL of the brain

supernatant and 1 mL of 4% sulfosalicylic acid was incubated at 4 °C

for 1 h followed by 15 min of centrifugation at 1200 rpm at 4 °C. The

supernatant from this homogenate (0.1 mL) was mixed with 2.7 mL

of phosphate buﬀer (0.1 M, pH 8) and 0.2 mL of 0.01 M DTNB.

Measurement of OD was performed at 412 nm, and the results were

expressed as nmoles of glutathione (GSH)/(mg of protein).³⁵

4.7.3.5. Catalase. The brain tissue homogenate (0.05 mL) was

mixed with 1.95 mL of phosphate buﬀer (0.05 M, pH 7.0) and 1.0 mL

of hydrogen peroxide (0.019 M). The ﬁnal volume was made up to

3.0 mL. Optical density diﬀerence was analyzed at λ_maxof 240 nm for

2 min at 30/60 s interval. The results were reported as (μmoles of

H₂O₂decomposed/min)/(mg of protein).³⁶

4.8. Molecular Dynamics (MD). Stability of the designed

molecules complexed with AChE and BChE proteins was assessed

by carrying out the molecular dynamic simulations using Desmond

software.³⁷The MD simulations were performed for a period of 30 ns

separately for the complex of the designed molecules with AChE and

BChE proteins. For carrying out the simulation studies, the “system

build” proﬁle of Desmond was utilized to generate a solvated system

with a TIP3P (transferable intermolecular potential with 3 points)

water model with an octahedral water box, physiological pH of the

system was adjusted by adding Na⁺ions, and concentration of the salt

was ﬁxed to 0.15 M. The MD simulation was carried out using the

NPT (isothermal−isobaric) ensemble and a time step of 1 ps. The

temperature was maintained at 310 K using a Nose−Hoover chain

thermostat, and pressure was maintained at 1.013 25 bar using

Martyn−Tobias−Klein barostat.³⁸The “simulation interaction

diagram” protocol of Desmond was utilized for RMSD value for

both AChE and BChE protein−ligand complexes. The simulation

interaction diagram was studied to predict most probable binding

orientation of the ligand.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors are highly thankful to University Institute of

Pharmaceutical Sciences, Panjab University, for providing the

necessary research facilities for the completion of the work and

Molecular Modeling Lab at Punjabi University for providing

various software for in silico studies. One of the authors is

thankful to ICMR, New Delhi, for providing SRF.

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AUTHOR INFORMATION

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(10) Alzheimer’s Association. FDA approved treatments for

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Corresponding Author

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Poonam Piplani − Department of Pharmaceutical Chemistry,

University Institute of Pharmaceutical Sciences, Panjab

University, Chandigarh 160014, India; orcid.org/0000-

0003-4144-5164; Phone: +91 9357036068;

Email: ppvohra28in@yahoo.co.in, ppvohra28in@pu.ac.in

Authors

Pragati Silakari − Department of Pharmaceutical Chemistry,

University Institute of Pharmaceutical Sciences, Panjab

University, Chandigarh 160014, India

Om Silakari − Department of Pharmaceutical Sciences and

Drug Research, Punjabi University, Patiala, Punjab 147002,

India

Complete contact information is available at:

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Systematic in Silico Design, Synthesis, and Biological Studies of Some Novel 1,4-Benzoquinone Derivatives for the Prospective Management of Cognitive Decline

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