Further SAR studies on natural template based neuroprotective molecules for the treatment of Alzheimer's disease

Bioorg. Med. Chem. 46 (2021) 116385

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Further SAR studies on natural template based neuroprotective molecules

for the treatment of Alzheimer’s disease

a

b

a

c

Yash Pal Singh , Gauri Shankar , Shagufta Jahan , Gourav Singh , Navneet Kumar ,

a,

1

b

a

,

2

b

d

Atanu Barik , Prabhat Upadhyay , Lovejit Singh , Kajal Kamble , Gireesh Kumar Singh ,

Sanjay Tiwari , Prabha Garg , Sarika Gupta , Gyan Modi

e

c

b

a,*

a

Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi 221005, India

National Institute of Immunology, New Delhi 110067, India

b

c

Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar 160062, India

Department of Pharmacy, School of Health Science, Central University of South Bihar, Gaya 824236, India

d

e

Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER) – Raebareli, Lucknow 226002, India

A R T I C L E I N F O

A B S T R A C T

Keywords:

In our earlier paper, we described ferulic acid (FA) template based novel series of multifunctional cholinesterase

Alzheimer’s disease

Ferulic acid

(

ChE) inhibitors for the management of AD. This report has further extended the structure–activity relationship

SAR) studies of this series of molecules in a calibrated manner to improve upon the ChEs inhibition and anti-

Cholinesterase inhibitors

Antioxidant

oxidant property to identify the novel potent multifunctional molecules. To investigate the effect of replacement

of phenylpiperazine ring with benzylpiperazine, increase in the linker length between FA and substituted phenyl

ring, and replacement of indole moiety with tryptamine on this molecular template, three series of novel mol-

ecules were developed. All synthesized compounds were tested for their acetyl and butyryl cholinestrases (AChE

and BChE) inhibitory properties. Enzyme inhibition and PAS binding studies identiﬁed compound 13b as a lead

molecule with potent inhibitor property towards AChE/BChE (AChE IC50 = 0.96 ± 0.14 µM, BChE IC50 = 1.23 ±

Iron chelator

Morris water maze

0

.23 µM) compared to earlier identiﬁed lead molecule EJMC-G (AChE IC50 = 5.74 ± 0.13

μ

M, BChE IC50

=

1

4.05 ± 0.10

μ

M, respectively). Molecular docking and dynamics studies revealed that 13b ﬁts well into the

active sites of AChE and BChE, forming stable and strong interactions with key residues Trp86, Ser125, Glu202,

Trp 286, Phe295, Tyr 337 in AChE, and with Trp 82, Gly115, Tyr128, and Ser287 in BChE. The compound, 13b

was found to be three times more potent antioxidant in a DPPH assay (IC50 = 20.25 ± 0.26 µM) over the earlier

identiﬁed EJMC-B (IC50 = 61.98 ± 0.30 µM) and it also was able to chelate iron. Co-treatment of 13b with H

2 2

O ,

2 2

signiﬁcantly attenuated and reversed H O -induced toxicity in the SH-SY5Y cells. The parallel artiﬁcial mem-

brane permeability assay-blood brain barrier (PAMPA-BBB) revealed that 13b could cross BBB efﬁciently.

Finally, the in-vivo efﬁcacy of 13b at dose of 10 mg/kg in scopolamine AD model has been demonstrated. The

present study strongly suggests that the naturally inspired multifunctional molecule 13b may behave as a po-

tential novel therapeutic agent for AD management.

Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; BuChE, butyrylcholinesterase; DPZ, donepezil; FA, ferulic acid; PAS,

peripheral anionic site; CAS, catalytic active site; ROS, reactive oxygen species; BBB, blood-brain barrier; hACh, human acetylcholine; DPPH, 2,2-diphenyl-1-pic-

rylhydrazyl; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; UV, ultraviolet; AFM, atomic force microscopy; MD, molecular dynamics; LogP,

lipophilicity; TPSA, topological polar surface area; MW, molecular weight; HBD, hydrogen bond donors; HBA, hydrogen bond acceptors; RB, Rotatable bonds; BOC,

tert-butyloxycarbonyl; DCM, dichloromethane; DIPEA, N-diisopropylethylamine; EDCI.HCl, 1-[3-(dimethyamino)-propyl]-3-ethylcarbodiimide hydrochloride; THF,

tetrahydrofuran; HOBt, N-hydroxybenzotriazole, EtOAc, ethylacetate; TFA, triﬂuoroacetic acid; TLC, thin layer chromatography; CDCl

3

, deuterated chloroform;

DMSO-d , deuterated dimethyl sulfoxide-d ; TMS, tetramethylsilane; Hz, hertz; HR-MS, high-resolution mass spectrometry; ATCI, acetylthiocholine iodide; BTCI,

6

butyrylthiocholine iodide; DTNB, 5,5’-dithiobis-2-nitrobenzoic acid.

*

Corresponding author at: Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (BHU), Rm # 23, Varanasi, UP 221005, India.

E-mail address: gpmodi.phe@itbhu.ac.in (G. Modi).

1

Current address: Evalueserve SEZ Private Limited, 8th Floor, Building No:4, Candor Gurgaon One Realty Projects Pvt. Ltd-SEZ, Sector-48, Gurgaon, Haryana

1

22001, India.

2

Current address: Mylan Laboratories Limited, Plot No. H, 12, MIDC, Industrial Area, Waluj, Aurangabad, Maharashtra 431136. India.

https://doi.org/10.1016/j.bmc.2021.116385

Received 4 April 2021; Received in revised form 2 August 2021; Accepted 23 August 2021

Available online 28 August 2021

0

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

1

. Introduction

mechanisms leading to neurodegeneration and identifying novel neu-

roprotective therapies for AD. Consequently, antioxidants therapy for

aging diseases has been of paramount importance in the last deca-

Alzheimer’s disease (AD) is a multifactorial progressive neurode-

2

6–28

generative disorder and the most common form of dementia worldwide.

The symptoms associated with AD involve impairment in memory,

language skills, personal behavior, and thinking.¹These symptoms

appear due to the neuronal damage not only limited to the brain’s region

responsible for the cognitive function but may extend to neurons in

different parts of the brain. It is worth mentioning that dementia is the

ﬁfth-leading cause of death in the United States in people older than 65.

As per 2020 AD facts and ﬁgures, the mortality rate in the last two de-

cades due to stroke, HIV, and heart disease has decreased signiﬁcantly,

while 146.2% increase in deaths from AD has been reported.²As pre-

WHO, around 50 million people worldwide live with dementia, and

de.

Antioxidant compounds curcumin and ferulic acid (FA) are

known to reduce ROS and alleviate their toxic effects.

An increased level of metals, especially iron, and copper have been

reported in the AD brain. Although metals are the utmost requirement

for normal brain functioning, however, the imbalance in metal ho-

meostasis can facilitate the overproduction of ROS and Aβ aggregation,

2

9,30

which can create metal-induced oxidative stress (OS) in the brain.

In

the presence of bio-metals like iron and copper ions, excessive produc-

•

tion of hydrogen peroxide and O

2

‾ can undergo Fenton reaction to

•

create the highly reactive (OH ), resulting in neurodegeneration in AD.

The increased level of metals also disrupts the redox system of the cell

and further creates OS. The factors such as increased metals accumu-

lation and decrease level of bodies’ antioxidants such as glutathione

peroxidase, lipid peroxidase can further contribute towards the increase

in OS.²⁵The different studies also suggested that OS is also responsible

for the increased production of malondialdehyde (MDA) and 4-hydrox-

3

every year this number is escalating at a rapid rate. The total cost for all

individuals with AD and other dementia is expected to rise to approxi-

mately $1.1 trillion by 2050. The exact molecular mechanisms respon-

sible for neurodegeneration in AD are not clear yet, however, there is a

plethora of evidence, including publications from our research group,

4

–9

31

that AD is multifactorial.

The oldest hypothesis regarding AD path-

ynonenal (4-HNE), which are the end products of lipid peroxidation.

ophysiology is the “cholinergic hypothesis”, suggesting the massive loss

Therefore, metal-induced toxicity and oxidative stress can interplay a

crucial role in AD pathogenesis and progression.

of cholinergic neurons in AD.¹

0,11

Acetylcholine (ACh) is one of the

major neurotransmitters involved in learning and memory. The brain

regions most affected by neuronal loss in AD are essentially made of

cholinergic neurons, therefore, restoring physiological ACh level has

The currently available pharmacotherapies for AD provide only

symptomatic treatment without addressing aforesaid described the

disease associated pathophysiological factors (Fig. 1). Consequently, the

progression of the disease continues with the current therapies. At

present, there is no cure for AD. Given the complex multifactorial nature

of the disease, the identiﬁcation of novel multifunctional therapeutic

agents able to act on disease associated pathophysiological factors

would be of great value to develop novel therapeutics. In our approach,

along with this line, we designed compounds with both symptomatic

and neuroprotective disease-modifying properties. Natural products

such as FA, are the major sources of therapeutic agents for diseases,

1

2,13

been considered a viable therapy for AD.

Cholinesterases (ChEs) are

responsible for the hydrolysis of ACh into choline and acetic acid. The

two main types of ChEs: (i) acetylcholinesterase (AChE), and (ii)

butyrylcholinesterase (BChE). AChE is one of the key enzymes that play

a signiﬁcant role in synaptic transmission by hydrolyzing the neuro-

transmitter ACh. The active site of AChE is a composite of the peripheral

anionic site (PAS) rich in aromatic residues located at the gorge’s entry

on the enzyme’s surface, and the active catalytic site (CAS) is located at

the bottom of a 20 Å gorge.¹⁴

32

including neurodegenerative disorders (Fig. 1). Given the signiﬁcant

Intriguingly, the amount of AChE decreases in the course of AD.

Whereas, the concentration of BChE remains unchanged or even in-

creases, BChE can compensate for AChE loss, resulting in improved

cognition.¹⁵In the AD brain, the level of BChE increases remarkably in

the cortical region. Simultaneously, AChE activity decreases, and the

ratio between BChE and AChE varies from 0.6 (normal brain) to as high

as 11 (AD brain), which further leads to the development of neuritic

plaques and neuroﬁbrillary tangles, contributing to the severity of AD

pathogenesis.¹⁶Interestingly, it has been shown in the literature that

role of FA via regulating oxidative stress in AD pathogenesis, FA is still

not druggable and suffers from several shortcomings that limit its

application as an anti-AD agent.³³In our ongoing efforts to develop

naturally inspired multifunctional therapeutics targeting, earlier we

reported a series of AChE/BChE inhibitors based on FA template that

possesses potential antioxidant, iron-chelation ability, and Aβ1-42 ag-

gregation modulation property.

To improve the cholinergic inhibition, and antioxidant property, we

have further extended out the structure–activity relationship (SAR)

studies of this series of molecules. Earlier, we have identiﬁed phenyl

piperazine derivative (PPD, EJMC-B) exhibiting promising free radical

quenching ability (IC50 = 61.98 ± 0.30 µM) in 2,2-diphenyl-1-picrylhy-

drazyl (DPPH) assay with a potency comparable to that of parent natural

compound FA (IC50 = 56.49 ± 0.62 µM). Intriguingly, majority of the

developed PPD’s were devoid of potent ChEs activity. Therefore, the ﬁrst

series of novel molecules were designed, synthesized, and evaluated for

AChE/BChE inhibitory property to investigate the effect of the

replacement of phenylpiperazine ring with benzylpiperazine. Next, the

linker length between FA and substituted phenyl ring was increased to

explore the role of linker size on enzyme inhibition and selectivity.

Finally, we also wanted to observe the effect of replacing the 5-amino-

indole moiety in our earlier reported molecule EJMC-G (Fig. 2) with a

tryptamine derivative on AChE/BChE activity. The structural modiﬁ-

cations were carried out in a calibrated manner while maintaining the

ClogP of the developed molecules in the desired range as per earlier our

1

7,18

BChE inhibitors improve cognition by increasing ACh levels.

Therefore, dual-acting AChE/BChE inhibitors can be a practical thera-

peutic approach for effective and safe AD management.

AD brains have frequently shown the signature of reactive oxygen

species (ROS) and reactive nitrogen species (RNS) mediated neuronal

injury. An imbalance between ROS/RNS and the reduction in cells’ ca-

pacity to neutralize these species creates a situation in the cells known as

oxidative stress.¹⁹^,²⁰An increase in oxidative stress level is hypothesized

to be one of the early events responsible for the disease’s pathogenesis,

which ultimately leads to neurodegeneration. Further, an imbalance in

the mitochondrial redox system is associated with the generation of

ROS. The reaction between the electrons leaked from the inner mito-

chondrial membrane and oxygen atom creates highly reactive super-

.

2

¡

oxide anions (O

). These superoxide radicals can further undergo free

radical chain reaction to generate other ROS like hydrogen peroxide

¡

(

H

2

O

2

) and hydroxyl ion (OH ). Metals, especially copper and iron, also

2

1,22

26

mediate oxidative stress.

The loss of antioxidant capacity of cells

publications. The selected compounds were evaluated for the enzyme

with age might also cause increased levels of oxidative stress. The

overproduction of ROS and simultaneously increased level of antioxi-

kinetic, and PAS binding studies. Next, we carried out detailed compu-

tational studies to corroborate our in-vitro studies and gain an insight

into the interactions between developed molecules and AChE/BChE

using molecular docking and MD simulation studies. Further antioxidant

potential and metal chelation studies were carried out. The evaluation of

the effect of the treatment with different doses of the lead molecule on

2

3

dant enzymes has been observed in the AD brain. It is evident from

recent publications that oxidative stress precedes the appearance of

senile plaques and neuroﬁbrillary tangles.²

4,25

Therefore, the oxidative

stress hypothesis has emerged as a crucial point for understanding the

2

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

reversing H

2

O

2

-induced toxicity in the neuronal N2a cells was carried

ability.²⁶One of the major goals behind the novel FA analogs’ design is

to improve the AChE inhibition, and antioxidant potential of FA-derived

compounds. One of the ﬁndings from our previous SAR studies that we

observe a loss in the free radical quenching ability of most developed

molecules compared to FA. Intriguingly, the phenyl piperazine de-

rivatives (PPD) EJMC-A-C (Fig. 2) have shown promising free radical

quenching ability (IC50 = 61.98 ± 0.30 µM for EJMC-B) in DPPH assay

with a potency comparable to that of parent natural compound FA (IC50

= 56.49 ± 0.62 µM). However, we have shown that the coupling of

substituted phenyl piperazine moiety with FA with glycinamide linker

leads to a mixed impact on AChE/BChE inhibition. The developed PPD

were found to be moderate to weak inhibitors of the target enzymes

(IC50, AChE, 9.91 ± 0.07 to 29.34 ± 0.03 µM, % BChE inhibition, 26.89

± 0.20 to 44.71 ± 0.05 ). Given the key role of OS in AD pathogenesis

and the potent antioxidant potential of the developed PPD, we planned

to improve upon the AChE/BChE while maintaining ClogP of the

out. The blood–brain barrier crossing ability of lead-developed mole-

cules was examined in parallel artiﬁcial membrane permeability assay-

blood brain barrier (PAMPA-BBB) assay. Finally, we evaluated the

treatment effect with different doses (5 and 10 mg/kg) of 13b in

reversing the scopolamine-induced memory impairment in Morris water

maze test.

2

. Results and discussion

2

.1. Chemistry

New ferulic acid-benzyl piperazine hybrids 3a-3p were obtained as

mentioned in Scheme 1. The synthesis of target compounds was

completed using a well-established synthetic method. Commercially

available benzyl halides (1e-1g, 1i and 1m-1o) underwent nucleophilic

substitution reaction (SN2) with piperazine in ethanol to give com-

pounds 2e-2g, 2i, and 2m-2o. The target compounds 3a-3p were ob-

tained by the reaction of substituted benzylpiperazine (2a-2p) with FA

using the standard amide coupling method 1-ethyl-3-(3-dimethylamino-

propyl)carbodiimide hydrochloride (EDCI.HCl), hydroxybenzotriazole

2

6

designed molecules in the desired range as per our earlier publication.

Based on our studies, we hypothesized that the nature of the long active

site of AChE/BChE and the relatively short structure of PPD is probably

the reason for their moderate inhibitory activity. Hence, in the ﬁrst se-

ries of SAR studies, we planned to introduce a spacer between phenyl

and piperazine ring in the designed molecules. Therefore, the phenyl-

piperazine ring was replaced with benzylpiperazine. Thus, compounds

3a-3p were designed and synthesized to improve the AChE/BChE in-

hibition and antioxidant potential while maintaining ClogP in the

desired range (Table 1). The addition of hydrophobic features will also

help the analogs to be able to interact with the essential hydrophobic

residues at the active site of the enzymes. Despite these structural

modiﬁcations, none of the tested compounds showed a signiﬁcantly

improvement in the enzyme inhibition studies compared to the earlier

reportedPPD. Therefore, to further improve upon the AChE/BChE in-

hibition, we planned to increase the linker length in our earlier reported

potent cholinergic inhibitors glycine amide derivatives EJMC-D, EJMC-

E, and EJMC-F (Fig. 2). In our earlier study, we have demonstrated that

our FA derivatives exhibited highest potency when the methylene linker

length is one (n = 1). In this manuscript, we rationally and selectively

synthesized only three molecules (8a-c), as a part of our SAR study, with

a linker length two to observe whether this structural modiﬁcation can

further potentiate cholinergic inhibitions. Therefore, to evaluate the

effect of a two-methylene linker size connecting FA and phenyl ring,

compounds 8a-c were designed and synthesized (Scheme 2). Intrigu-

ingly, none of the developed compounds showed an improved AChE/

BChE inhibitory property (Table 1) over the earlier reported compounds

(

HOBt), and N,N-diisopropylethylamine (DIPEA). The compound 3a has

been reported earlier in the literature as an intermediate for the devel-

3

4

opment of FA based therapeutic agents for AD. The molecular hybrids

of FA and substituted amines connected with alanine linker were syn-

thesized by following the reaction steps mentioned in Scheme 2. In the

ﬁrst step, N-Boc-propanamide derivatives (6a-6c) were obtained by

standard amide coupling of substituted amines with Boc-alanine. In the

next step, N-Boc-protected secondary amine groups were then depro-

tected by ether HCl to yield 7a-7c. Finally, the compounds 8a-8c were

synthesized by the reaction of substituted alanine, FA using standard

amide coupling. To obtain FA-tryptamine-based hybrid analogs (13a-

1

3c), we followed the synthetic route depicted in Scheme 3. First, the

Boc-glycine was reacted with a substituted tryptamine (10a-10c) to

obtain the desired intermediates 11a-11c. After Boc deprotection, the

ﬁnal compounds (13a-13c) were obtained using standard amide

coupling with FA. All newly synthesized hybrids were puriﬁed by

1

chromatographic techniques and structurally characterized by H NMR,

1

3

C-NMR, and high-resolution mass spectrometry (HRMS, Table S1).

2

.2. Design and cholinesterases inhibition studies of novel naturally

inspired FA analogs

(

EJMC-D, EJMC-E, and EJMC-F). One of the major goals of this study is

2

.2.1. Design of novel naturally inspired FA analogs

to improve upon the enzyme inhibition and antioxidant properties of our

earlier identiﬁed molecules. In our recent publication, we have identi-

ﬁed EJMC-G (Fig. 2) as a potential multifunctional agent able to provide

both symptomatic relief and neuroprotection for the management of AD.

In our continuous efforts to develop naturally inspired multifunc-

tional molecules for AD management, earlier we reported a novel series

of FA template-based AChE/BChE inhibitors that possess potential

antioxidant, Aβ1-42 aggregation modulation property, and iron-chelation

Fig. 1. Chemical structures of (A) Drugs available in the market for the treatment of AD. (B) Natural products known to modulate neurodegenerative disorders.

3

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Given the signiﬁcant role played by the presence of indole moiety in

EJMC-G in the interaction with the target enzymes and antioxidant

property, the 3rd series of compounds where FA and tryptamine moi-

eties were joined through optimized glycine amide to improve upon the

AChE/BChE inhibition and antioxidant property.

These results concur with our previous ﬁnding with the PPD where we

observed that the introduction of methoxy group could increase the

developed molecules inhibitor activity towards AChE.

We further explored various electron-withdrawing groups (EWGs) on

benzylpiperazine ring to explore the signiﬁcance of various electronic

features on the AChE/BChE inhibition. Therefore, compounds 3g-3l

were designed, synthesized and evaluated for the enzyme inhibition

assays. In this study, 3k bearing m-ﬂuoro turned out to be most potent

among all developed molecules so far [IC50, AChE (µM), 3h (m-chloro)

= 15.43 ± 0.28, 3k (m-ﬂuoro) = 13.13 ± 0.26, and 3l (p-ﬂuoro) = 14.54

± 0.65 µM]. Interestingly, all the developed molecules (3g-3l) were

found to be equipotent inhibitors of BChE [IC50, (µM), 3g (o-chloro) =

15.21 ± 0.29, 3h (m-chloro) = 14.80 ± 0.34, 3i (p-chloro) = 15.86 ±

0.31, 3j (o-ﬂuoro) = 15.89 ± 0.23, 3k (m-ﬂuoro) = 14.03 ± 0.22, and 3l

(p-ﬂuoro) = 14.85 ± 0.37 µM]. We further introduced bulky EWGs on

the benzylpiperazine fragment in order to explore their effect on en-

zymes inhibition properties. Therefore, compound 3m-3p were devel-

oped and evaluated for enzymes inhibition. Surprisingly, all developed

2

.2.2. Cholinesterases inhibition studies

Given the signiﬁcant role played by ACh and butyrylcholine (BCh) in

memory and cognitive impairment in various stages of AD, therefore,

cholinesterase’s inhibitory activity of the newly synthesized compounds

against human AChE and equine BChE was measured by using the

spectroscopic method of Ellman et al.³⁵The inhibitory activity is

expressed as IC50, i.e., the inhibitor concentration that reduces the

cholinesterase activity by 50%. In this study, DPZ served as the refer-

ence drug, and FA was used as the negative control.

In our initial attempt to explore the possible changes in enzyme in-

hibition properties due to such structural changes, compound 3a was

synthesized and evaluated for ChEs inhibitory activities. In the enzyme

inhibition studies, 3a bearing unsubstituted benzylpiperazine fragment

exhibited potent AChE inhibitory property (IC50, AChE (µM) = 13.34 ±

compounds exhibited weak inhibitory activity towards AChE (IC50

,

AChE (µM), >20 µM). In contrast, the developed molecules 3m-3p were

found to be potent inhibitors of BChE over 3a (IC50, BChE (µM), 3m =

14.89 ± 0.26, 3n = 14.73 ± 0.31, 3o = 15.58 ± 0.44, and 3p = 14.62 ±

0.16 µM, respectively).

0

.28) compared to EJMC-A (IC50, AChE (µM), = 29.34 ± 0.03). Inter-

estingly, 3a (Fig. 2) was approximately twice more potent compared to

EJMC-A). However, in the BChE inhibition assay, 3a turned out to be

equipotent compared to EJMC-A (% BChE inhibition 45.13 ± 0.61, and

7.03 ± 0.41 for 3a and EJMC-A, respectively). This result indicated

(

Despite these promising results, none of the tested compounds

showed a signiﬁcantly improved enzymes inhibition studies than earlier

reported phenylpiperazine derivatives. Therefore, to improve upon the

AChE/BChE inhibition, compounds 8a-c were designed and synthesized

(Scheme 2). To evaluate the effect of a two-methylene linker size con-

necting FA and phenyl ring, compounds 8a was designed and synthe-

sized (Scheme 2). This compound exhibited slightly more potent activity

towards AChE (IC50, AChE (µM) = 14.75 ± 0.42) while signiﬁcant in-

crease in BChE inhibition (IC50, BChE (µM) = 16.38 ± 0.37) was

observed in comparison to EJMC-F [IC50, AChE (µM) = 21.94 ± 0.81,

BChE (% inhibition) = 38.25 ± 0.25]. Our earlier enzyme inhibition

studies also demonstrated that compounds with m-methoxy and p-chloro

on the terminal phenyl ring are comparatively potent enzyme inhibitors

over the other developed FA analogs. We speciﬁcally incorporated these

groups in the development of molecules 8b-8c with increased linker

length to investigate the impact of linker length on the enzyme inhibi-

tion. Among the three compounds (8a-8c), 8b exhibited maximum in-

hibition for AChE (IC50, AChE (µM) = 2.41 ± 0.42). While 8c was found

to be most potent inhibitor towards BChE (IC50, BChE (µM) = 7.13 ±

0.37 µM). Thus, it demonstrates that the optimal methylene linker size is

one in a similarly substituted series of FA template. These results indi-

cated that the introduction of propenamide linker instead of glycine

amide did not improve upon the enzymes inhibitory property.

3

that benzylpiperazine fragment was well tolerated on the enzyme sites.

Further, the methyl group was introduced on various positions of the

phenyl ring of benzyl piperazine fragment, leading to the generation of

compounds 3b-3d. Intriguingly, compounds bearing methyl group at

different position of benzylpiperazine fragment showed weak inhibition

towards AChE over 3a (IC50, AChE (µM), >20 for 3b-3d) over the earlier

reported 4-methyl phenylpiperazine derivative EJMC-C (IC50, AChE

(

µM) = 19.48 ± 0.12, EJMC-C, Fig. 2). Intriguingly, in case of BChE

inhibition assay the developed molecules were found to be potent in-

hibitors over 3a (IC50, BChE (µM), 3b = 15.06 ± 0.38, 3c = 14.69 ±

0

.35, and 3d = 14.75 ± 0.31 µM, and % BChE inhibition 37.03 ± 0.41

for 3a). It is worth mentioning that in the case of BChE inhibition,

substitution of a methyl group on benzylpiperazine was better tolerated

over our earlier reported methyl-substituted phenylpiperazine com-

pound EJMC-C (% BChE inhibition 38.14 ± 0.12). To further validate

our previous results on the electron donating group (EDG), we intro-

duced monomethoxy group onto our parent molecule 3a, which led to

3 3

the generation of compounds 3e (o-OCH ), and 3f (p-OCH ). In the

enzyme inhibition assay, the compounds with monomethoxy group

effectively inhibited AChE and were equally potent to 3a (IC50, AChE

(

µM), 3e = 15.18 ± 0.36, and 3f = 16.39 ± 0.42). However, introduction

of monomethoxy group on the benzylpiperazine a signiﬁcant reduction

towards BChE inhibition was observed compared to 3b-3d (% BChE

inhibition 49.12 ± 0.63 and 45.13 ± 0.67, 3e and 3f, respectively).

The indole moiety played a key role in the interaction with targets

enzymes and could also reverse the loss in antioxidant property as re-

ported in the case of EJMC-G, therefore, tryptamine ring was introduced

Fig. 2. Design of the multifunctional ChEs inhibitors by integrating the structural features of ferulic acid (FA) derivatives.

4

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Scheme 1. Synthesis of compounds 3a-3p. Reagents and conditions: (i) Anhydrous piperazine, K

DIPEA, dry THF, rt, overnight, 65–75%.

2

CO

3

, ethanol, reﬂux, 4–5 h, 70–80%; (ii) FA, EDCI.HCl, HOBT,

◦

Scheme 2. Synthesis of designed compounds 8a-8c. Reagents and conditions: (i) Boc-anhydride, DCM, 0 C, overnight, 90%; (ii) aniline/3-methoxyaniline/4-

chloroaniline, EDCI.HCl, HOBT, DIPEA, dry THF, rt, overnight, 70–75%; (iii) Ether HCl, methanol, rt, 12 h, 80–85%; (iv) FA, EDCI.HCl, HOBT, DIPEA, dry THF,

rt, overnight, 70–75%.

Scheme 3. Synthetic route to compounds 13a-13c. Reagents and conditions: (i) EDCI.HCl, HOBT, DIPEA, dry THF, rt, overnight, 65–70%; (ii) Ether HCl, methanol,

rt, 12 h, 80–85%; (iii) FA, EDCI.HCl, HOBT, DIPEA, dry THF, rt, overnight, 65–70%.

in the third series of molecules. In this series, three compounds were

designed, synthesized, and evaluated for enzyme inhibition studies.

Interestingly, compounds 13a-13c bearing tryptamine functionality

showed signiﬁcant AChE/BChE inhibition over the earlier reported

compounds. The developed molecules were found to be equipotent to-

wards AChE inhibitory activity (IC50, AChE (µM), 13a = 1.42 ± 0.24,

2.3. Antioxidant activity (DPPH radical scavenging activity)

It is evident from the literature that OS plays a crucial role in the

progression of AD. Therefore, inhibition of ChEs along with the ability to

reduce OS would be a more effective approach for the treatment of AD.

The antioxidant ability of developed molecules was evaluated by 2,2-

Diphenyl-1-(2,4,6-trinitrophenyl) hydrazine (DPPH) assay. To perform

DPPH radical scavenging experiment, we have selected 13 compounds

based on ChEs inhibition property. Antioxidant activity is expressed as

IC50 value, i.e, compound concentration required to reduce 50% of the

DPPH radical concentration in a solution. All tested compounds

exhibited excellent to moderate antioxidant activity with IC50 ranging

1

3b = 0.96 ± 0.14, and 13c = 0.84 ± 0.12). Similarly, the developed

compounds were found to be potent inhibitor of BChE (IC50, BChE (µM),

1

3a = 3.14 ± 0.28, 13b = 1.23 ± 0.23, and 13c = 1.29 ± 0.22). These

results collectively suggested that both EWG or EDG on tryptamine

moiety well tolerated on enzymes (SI, Fig. S1). The SAR on this series of

all developed molecules is represented in Fig. 3.

5

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Table 1

Structures,

ChEs

inhibitory

activities,

cLogP,

and

tPSA

of

compounds

3a-3p,

8a-8c

and

13a-

1

3c.

a

b

c

Compd.

R

IC50 (µM) or % inhibition

tPSA

cLogP

hAChE

eqBChE

3

a

b

c

d

e

f

Hydrogen

2-methyl

3-methyl

4-methyl

3-methoxy

4-methoxy

2-chloro

3-chloro

4-chloro

2-ﬂuoro

3-ﬂuoro

4-ﬂuoro

2-cyano

3-cyano

4-cyano

3-nitro

13.34 ± 0.28

43.10 ± 0.84%

45.50 ± 0.86%

43.16 ± 0.69%

15.18 ± 0.36

16.39 ± 0.42

46.99 ± 0.87%

15.43 ± 0.28

37.26 ± 0.85%

34.24 ± 0.78%

13.13 ± 0.26

14.54 ± 0.65

45.39 ± 0.67%

42.41 ± 0.51%

39.40 ± 0.78%

38.76 ± 0.52%

14.75 ± 0.42

2.41 ± 0.42

45.13 ± 0.61%

15.06 ± 0.38

14.69 ± 0.35

14.75 ± 0.31

49.12 ± 0.63%

45.13 ± 0.67%

15.21 ± 0.29

14.80 ± 0.34

15.86 ± 0.31

15.89 ± 0.23

14.03 ± 0.22

14.85 ± 0.37

14.89 ± 0.26

14.73 ± 0.31

15.58 ± 0.44

53.01

62.24

53.01

76.8

3.22

3.67

3.72

3.14

3.94

3.37

2.80

2.66

2.97

2.26

2.34

3.23

2.15

2.17

3.03

1.42

4.59

g

h

3

8

1

i

j

k

l

m

n

o

76.8

p

a

14.62 ± 0.16 104.82

16.38 ± 0.37

11.34 ± 0.46

7.13 ± 0.37

104.82

87.66

96.89

87.66

99.69

108.92

99.69

66.76

38.77

Hydrogen

3-methoxy

4-chloro

Hydrogen

5-methoxy

5-chloro

–

b

c

5.32 ± 0.31

3a

3b

1.42 ± 0.24

3.14 ± 0.28

0.96 ± 0.14

1.23 ± 0.23 108.92

1.29 ± 0.22

3c

0.84 ± 0.12

d

FA

15.12 ± 0.36%

0.06 ± 0.01

17.15 ± 0.29%

2.16 ± 0.19

e

DPZ

–

a

IC50: 50% inhibitory concentration (mean ± SD of two or three independent experiments).

b

c

%

inhibition at 20 µM concentration of inhibitor.

tPSA (topological polar surface area) and cLogP values are calculated using ChemDraw.

FA (ferulic acid) = negative control.

d

e

DPZ (donepezil) = reference standard.

from 20.25 ± 0.26 to 95.59 ± 0.57 µM (Table 2). Interestingly, com-

pounds bearing m-ﬂuoro (3k), and tryptamine (13a, 13b, and 13c)

derivatives showed the maximum radical scavenging activity among all

the tested molecules in this series (IC50, 3k = 26.58 ± 0.24, 13a = 29.05

13c = 23.57 ± 0.29, respectively) the lead molecules les 3k, 13b, and

13c were selected for PAS binding studies. Next, to determine whether

3k, 13b, and 13c have an afﬁnity towards the PAS of AChE, a propidium

iodide-based competitive displacement assay was performed following

our publications.²⁶Propidium iodide is a commonly used PAS-speciﬁc

ligand of AChE, which binds selectively at its PAS, which can be

measured through the enhancement of the ﬂuorescence intensity (~10-

fold). In the presence of a ligand able to bind with strongly bind with

PAS site, a decrease in the propidium iodide ﬂuorescence is expected

due to displacement of propidium from the PAS. DPZ, which is known to

bind to the enzyme’s PAS site, served as a reference drug in this assay. To

perform this experiment, we incubated AChE (5.0 U/mL) with different

concentrations of compounds (5, 10, 20, and 50 µM) followed by pro-

pidium iodide and recorded the change in ﬂuorescence using Synergy™

HT, Bio-Tek Instruments, Inc. The results mentioned in Table 3 indicated

that 13b and 13c were found to be more efﬁcient to displace propidium

iodide in a concentration-dependent manner compared to DPZ. While,

±

0.32, 13b = 20.25 ± 0.26, and 13c = 23.57 ± 0.29 µM, respectively)

(

Fig. 4). The proposed mechanism of 13b as a radical scavenger is shown

in Fig. S2. Overall, our study suggests a signiﬁcant improvement in the

antioxidant property in the some of the newly developed molecules

compared to earlier reported phenylpiperazine derivatives (EJMC-B,

IC50 = 61.98 ± 0.30 µM), and the parent molecules FA (IC50 = 58.31 ±

0

.61).

2

.4. Kinetic studies of ChEs inhibition

The mechanism of ChEs inhibition (AChE & BChE) was evaluated for

two selected lead compounds (3k and 13b) on both human AChE

hAChE) and equine BChE (eqBChE). In this experiment, reciprocal of the

substrate [S] vs reciprocal of velocity [V] curves were plotted upon

addition of different concentrations of 3k (26, 13, and 6.5 M), and 13b

5.0, 1.0 and 0.5 M) in the presence of ﬁve different substrate [S]

concentrations (ATCI or BTCI) (0.5, 1.0, 1.5, 2.0 and 2.5 M for hAChE,

and 5, 10, 15, 20, and 25 M for eqBChE) to probe into the mechanism of

(

3

k represented weak propidium iodide displacement compared to DPZ

and other tested compounds.

μ

(

μ

2

.6. Molecular modeling studies

μ

ChEs inhibition. As depicted in Fig. 5 by Lineweaver–Burk reciprocal

plots, 3k and 13b displayed a mixed type of inhibition on both hAChE

and eqBChE (Fig. 5).

.6.1. Molecular docking

Molecular docking of selected ligands 3a, 3c, 3g-h, 3j-k, 3m, 8a, 8c,

and 13b-c were performed against the AChE and BChE using the Glide in

extra precision (XP) mode. Before performing the ﬁnal docking study of

all above mentioned molecules, the co-crystallized ligands i.e. DPZ and

Tacrine were redocked to the crystal structures of AChE and BChE,

respectively. The RMSD difference between the redocked and co-

crystallized ligands were found to be less than 1 Å. The Tables S2 and

S3 depicts the Docking score, XP GScore, glide gscore, glide emodel, and

2

.5. Peripheral anionic site (PAS) binding study

Based on the potent cholinergic inhibitory (IC50, 3k = 13.13 ± 0.26,

1

3b = 0.96 ± 0.14, and 13c = 0.84 ± 0.12

μ

M, respectively) and

antioxidant properties (IC50, 3k = 26.58 ± 0.24, 13b = 20.25 ± 0.26,

6

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Fig. 3. Brief summary of structural activity relationship (SAR).

belonging to PAS of AChE via hydrophobic and hydrogen bond in-

teractions. Similarly, the piperazine ring of 3k interacts with one of the

important CAS residue Tyr337 via both direct and water mediated

hydrogen bonds. This stable interaction of piperazine ring with CAS was

maintained during the whole period of simulation via the water bridges

and direct hydrogen bond (Figs. S3 and S4). Apart from this, one more

important interaction between the 3-ﬂuorobenzylpiperazine fragment

and CAS residue Trp86 was observed which was maintained for 74% of

Table 2

Antioxidant activity (DPPH assay) of 3a, 3c, 3f, 3h, 3i, 3k-3l, 8a-8c, 13a-13c

and FA.

Compound

DPPH assay

Radical scavenging

a

b

%

IC50 (µM)

3

8

1

a

c

38.41 ± 1.09

26.32 ± 1.13

30.49 ± 1.13

23.13 ± 1.17

32.41 ± 0.92

40.23 ± 1.21

23.14 ± 0.98

10.62 ± 0.96

21.28 ± 1.01

18.66 ± 0.96

38.55 ± 0.92

51.92 ± 1.12

47.27 ± 1.11

33.11 ± 1.30

31.34 ± 0.45

65.98 ± 0.39

46.43 ± 0.29

61.52 ± 0.42

62.40 ± 0.45

26.58 ± 0.24

51.93 ± 0.41

95.59 ± 0.57

89.43 ± 0.46

84.21 ± 0.66

29.05 ± 0.32

20.25 ± 0.26

23.57 ± 0.29

58.31 ± 0.61

f

total simulation time. Further, the residue Tyr341 makes

π

-cation

h

i

interaction while the Phe 338 makes

zylpiperazine moiety, respectively.

π- π

stacking with the 3-Fluoroben-

k

l

The ligand 13b is mainly composed of 3 moieties i.e. 5-methoxytrypt-

amine, FA and a linker glycinamide (Fig. 6B). The 5-methoxytryptamine

a

b

c

moiety of ligand 13b mainly interacts with three CAS residues Trp86 (

π

-

π

stacking), Glu202 and Tyr337 (via water mediated hydrogen bond).

3a

3b

The protein–ligand contact timeline representation depict that all of

these interactions i.e. hydrogen bond and hydrophobic interactions be-

tween the tryptamine moiety and CAS residues were maintained

throughout the simulation (Figs. S3 and S4). Further, a stable and

consistent water mediated hydrogen bond between the 5-Methoxytrypt-

amine of 13b and Ser125 was also observed. One important and inter-

esting interaction (hydrogen bond) between the carbonyl oxygen of FA

moiety and residue Phe295 was also observed. Further, the pro-

tein–ligand contact histogram and timeline (Figs. S3 and S4) depicts

that the interaction between Phe295 and FA moiety was one of the most

consistent and stable interactions, since it was maintained for 99% of

total simulation time.

3c

c

FA

a

All the values were obtained at a compound concentration of 20 µM, %

Radical scavenging; means ± SD of three independent experiments.

b

IC50: 50% inhibitory concentration (means ± SD of three independent

experiments).

c

FA (ferulic acid) = negative control.

important interactions (hydrogen bonds, hydrophobic interactions, and

salt bridges) between residues of proteins (i.e. AChE and BChE) and

small molecules.

2

.6.3. Interaction of ligands 3k and 13b to hBChE

2

.6.2. Interaction of ligands 3k and 13b with hAChE

The Fig. 6C-D shows the 2D interaction diagram of 3k and 13b with

The Fig. 6A-B shows the 2D interaction diagram of 3k and 13b with

hBChE. The FA moiety interacts with residues Tyr128, Gly115 via

hydrogen bond, and Trp82 interacts via stacking interaction.

Further, the stacking interaction between the ﬂuorobenzyl moiety of

hAChE. During the MD simulations it was observed that phenyl ring of

FA moiety of ligand 3k interacts with Trp286 via stacking interac-

π- π

π-

π

π-π

tion, and this residue is considered as one of the principal components of

PAS. This hydrophobic interaction between the Trp286 and 3k was

consistent and maintained during the entire simulation run as shown in

protein–ligand contact histogram and timeline (Figs. S3 and S4).

Similarly, the carbonyl moiety of FA exhibited water mediated hydrogen

bond with Asp74 (PAS residue) and Thr75. In short, the whole FA

fragment present in the ligand 3k mainly interacts with residues

3k and Phe329 was also observed.

The tryptamine moiety of 13b makes a stable hydrogen bond with

Ser287, and this interaction was maintained for 90% of total simulation

time. Similarly, the FA moiety of the makes a couple of hydrogen bonds

with residues Gly115 and Tyr128. The protein–ligand contact histogram

and timeline (Figs. S5 and S6) shows that the interaction between the

FA moiety of 13b and Tyr128 was found to be stable and maintained

7

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Fig. 4. DPPH scavenging activity by 3k, 13a, 13b, 13c and FA. Results are the means ± SD for the three-experiment performed in triplicate.

Fig. 5. Lineweaver-Burk double reciprocal plot showing the mechanism of hAChE and eqBChE inhibition over a range of substrate concentrations; [A, B] hAChE and

eqBChE inhibition by 3k; [C, D] hAChE and eqBChE inhibition by 13b. The experimental data are the means ± SD of two independent experiments.

during the entire simulation run. Apart from these interactions, the

Table 3

linker present between tryptamine and FA makes two hydrogen bonds

Displacement of propidium iodide from the peripheral anionic site of AChE by

with Thr120, and one water mediated hydrogen bond with Asn68.

3

k, 13b, 13c, and DPZ at the indicated concentrations.

Code

5

μM

10

μM

20

μM

50 μM

2

.6.4. Analysis of binding free energy

3

1

k

3.34 ± 0.12

10.36 ± 0.23

8.39 ± 0.26

8.87 ± 0.18

8.32 ± 0.16

13.96 ± 0.29

13.64 ± 0.32

11.65 ± 0.22

14.83 ± 0.24

21.85 ± 0.39

19.79 ± 0.42

19.10 ± 0.34

20.32 ± 0.31

33.14 ± 0.43

30.17 ± 0.45

27.96 ± 0.41

The binding free energy of complexes AChE_3k, AChE_13b, BChE_3k

3b

3c

and BChE_13b were calculated from last 25 ns of trajectory at an interval

of 50 ps using Prime/MM-GBSA (molecular mechanics/generalized born

surface area) method. The Fig. 7 shows the ﬂuctuation in binding free

energy of all four complexes with respect to simulation time. Table 4

shows the average binding free energy and its different contributing

terms for all four complexes. The Fig. 7A shows that the binding free

DPZ

a

Results are the mean ± SD for two independent experiments.

8

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Fig. 6. 2D interaction diagram of (A) 3k with AChE; (B) 13b with AChE, (C) 3k with BChE, and (D) 13b with BChE. Whereas green, red and violet colored arrows

represent the stacking, -cation interactions and hydrogen bonds, respectively.

π

-π

π

energy of complex hAChE_13b ﬂuctuates at higher values as compared

to hAChE_3k. The average binding free energy of hAChE_3k complex in

the last 25 ns was found to be ꢀ 58.46 kcal/mol while the complex

hAChE_13b was found to have ꢀ 82.17 kcal/mol.

Similarly, the BChE_13b complex was found to ﬂuctuate with higher

binding free energy as compared to BChE_3k complex (Fig. 7B). The

average binding free energy of BChE_3k complex was found to be

ꢀ

61.63 kcal/mol while BChE_13b complex has higher binding energy

75.03 kcal/mol. The calculated binding free energy for both ligand 3k

and 13b with AChE and BChE are in line with observed experimental

results.

2

.6.5. Analysis of structural stability, compactness and solvent accessbility

surface area

The structural stability of AChE and BChE and its complex with

different ligands can be explained by analyzing the RMSD and radius of

gyration (Rg) (Figs. S7 and S8). The changes in RMSD values with

respect to simulation time for all complexes (AChE/BChE) suggest that

the simulation is converged. It was observed that the RMSD ﬂuctuates at

lower values in presence of almost all ligands with respect to simulation

time for both AChE and BChE (Fig. S7). This indicates that the binding

of ligands stabilizes the AChE/BChE. The decrease in RMSD and Rg

values of AChE with respect to simulation time in presence of ligands 3k,

1

3b, and DPZ are more signiﬁcant as compared to BChE. There is very

little or no considerable changes in Rg value of BChE in presence of all

four ligands as compared to apo form of the enzyme (Fig. S8).

Fig. 7. Binding free energy of (A) AChE with ligands 3k and 13b, and (B) BChE

with ligands 3k and 13b, with respect to simulation time calculated using the

MM-GBSA method from the last 25 ns of MD simulations.

The percentage change in SASA of the residues presents 20 Å away

from the binding pocket were also calculated for both AChE and BChE in

the presence and absence of ligands. It was found that there was a

considerable decrease in SASA of active site residues of both AChE and

BChE in the presence of 3k and 13b. The Fig. S9 A-B represents the

percentage decrease in SASA of AChE and BChE in the presence of 3k,

3

.45% decrease in presence of co-crystallized ligand tacrine (Fig. S9 (B),

Fig. S10A-C).

2.7. Calculation of physicochemical parameters

1

3b and crystallized ligands (DPZ and Tacrine) with respect to simu-

lation time. The overall analysis shows that there was an average 6%

decrease in SASA values of AChE in the presence of 3k, 13b and DPZ.

Similarly, an average of around 5% decrease in SASA value was

observed for BChE in presence of 3k, 13b and DPZ, while it was only

Physicochemical properties such as molecular weight (MW), number

of hydrogen acceptors (HBA), number of hydrogen donors (HBD),

number of rotatable bonds (RB) and logBB were estimated using

admetSAR software.³⁶The values obtained were in range with the re-

ported limits of CNS + drugs (SI, Table S4). Overall, these results

9

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Table 4

The average ΔGbind (Kcal/mol) and its different contributing energy terms for 3k and 13b against hAChE and hBChE calculated from MD trajectories (last 25) ns.

Complex

Avg. ΔGb _ai nd

Avg. ΔGb_b ind

Avg. ΔGbind

Avg. ΔGbind solv

Avg. ΔGbind

ΔGbind

c

d

e

f

g

h

Coulomb

Covalent

Hbond

Lipo

Packing

GB

vdW

Total

AChE_3k

AChE_13b

BChE_3k

BChE_13b

–22.50

ꢀ 29.76

ꢀ 12.35

ꢀ 27.70

1.81

1.11

2.81

6.48

ꢀ 0.27

ꢀ 1.44

ꢀ 1.10

ꢀ 2.21

ꢀ 27.69

ꢀ 31.27

ꢀ 31.02

ꢀ 26.81

ꢀ 3.78

ꢀ 7.39

ꢀ 3.15

ꢀ 2.02

42.70

46.70

33.17

38.12

ꢀ 48.73

ꢀ 60.12

ꢀ 50.01

ꢀ 60.89

ꢀ 58.46

ꢀ 82.17

ꢀ 61.63

ꢀ 75.03

a

Coulomb energy.

b

c

d

e

f

Covalent binding energy.

Hydrogen bonding correction.

Lipophilic energy.

Pi-pi packing correction.

Generalized Born electrostatic solvation energy.

Van der Waals energy.

g

h

Total binding free energy.

collectively suggests that all the developed compounds follow Lipinski’s

spectra of the solution at pH 7.4 exhibited higher intensity at 511 nm

rule and exhibited signiﬁcant brain permeability.

than the spectra at lower pH values (4.2) (Fig. 8). This is the clear

3

+

indication of a complex formation between 13b and Fe

ion. The

binding stoichiometry of 13b with Fe³was examined by measuring the

+

2

.8. Metal chelation study

changes in absorption at 511 nm when 13b was titrated with

FeCl

3

⋅6H

2

O in a different molar ratio, as depicted in ﬁg. 8C. Finally, we

Literature studies revealed that the dyshomeostasis of biometals,

plotted the absorbance value against the mole fraction of 13b. We

observed that there is a breakage point in the plot where the mole

fraction of 13b was 50%, which reveals 1:1 binding of 13b toward

especially iron (Fe) and copper (Cu), plays a crucial role in AD’s path-

ogenesis. The extracellular accumulation of bio-metals induces the OS

and leads to the generation of highly toxic reactive free radical species.

Therefore, AD treatment through modulation of these bio-metals has

been considered a viable therapeutic approach. Therefore, based on the

AChE/BChE inhibition and antioxidant property, 13b was selected for

the iron-chelating ability. The metal chelating ability of representative

compound 13b was performed by UV–vis spectroscopy assay, and the

results are presented in the Fig. 8. To perform this experiment, equi-

3

+

Fe (Fig. 8D, Fig. S12).

2.9. In-vitro evaluation of cytotoxicity of compound 13b

The cytotoxicity studies are one of the important criteria in the drug

development process. The cytotoxicity studies were carried out prior the

in-vivo studies. The lead identiﬁed molecule 13b with potent AChE/

BChE inhibitory activity, antioxidant, and metal chelation properties

was selected to evaluate the potential for cytotoxic effect on N2a cells, a

widely used neuronal cell line. The cells were treated with different

molar concentration of 13b and FeCl

complexation study was conducted (Fig. S11). As depicted in Fig. 8, 13b

in the presence of FeCl O presented different absorption spectra

⋅6H

compared to absorption of the compound 13b alone. The absorption

3

⋅6H

2

O were mixed together, and a

3

2

Fig. 8. (A) UV absorbance spectra of 13b (300 µM) alone or in the presence of FeCl

spectra of 13b (300 µM) alone or in the presence of FeCl

with FeCl

experiment performed in duplicate.

3

(600 µM) in methanol (pH 4.2 and 7.4) from 300 to 700 nM. (B) UV absorbance

3

(1:1 equiv.) in methanol (pH 4.2 and 7.4) from 400 to 700 nm (dd-transition). (C) Metal ion titration of 13b

3

in different molar ratio (0–10 ratio). (D) Job’s plot for determining the stoichiometry of the complex between 13b and FeCl . Results are the means for the

1

0

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

concentrations of 13b for 24 hrs, and cell death was quantitatively

analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) assay. The dose-dependent effect of 13b (Fig. 9) on cell

viability indicated that the compound had no signiﬁcant effect on cell

viability and well tolerated by neuronal cells at all the tested

concentrations.

2.12. 13b ameliorates scopolamine-induced cognitive impairment in the

Morris water maze test

The improvement in cognitive impairment is the essential criteria for

the successful development of anti-AD agents. The MWM permits the

accurate and reproducible study of reference memory, cognitive maps,

place learning, spatial learning, and working memory.³

8,39

MWM is

2

.10. Neuroprotective effect on H

2

O

2

induced SH-SY5Y cell injury

highly sensitive in the assessment of damage to the hippocampus. The

robustness and reliability make this paradigm as one of the “gold stan-

dards” of behavioral neuroscience. Therefore, in the present study, we

evaluated the in-vivo efﬁcacy of 13b in MWM experiment to assess the

spatial working memory in mice (Fig. 11 A-B) . The respective drug

doses of 13b (5 and 10 mg/kg) orally, and DPZ (0.5 mg/kg) were

administered intraperitoneally 30 min prior to the administration of

scopolamine (1 mg/kg, i.p.) to the respective group of animals for

consecutively fourteen days. During the last 5 days of the treatment

period, ELTs were recorded for the animals of different experimental

groups. The escape latencies time and the track diagrams for the ﬁrst

four days of the training period are shown in the Figs. S13 and S14.

Scopolamine treatment caused a cognitive impairment and an increase

in the escape latency time compared to the control group was observed

which indicated the development of amnesia in the mice (Fig. 12A).

While DPZ signiﬁcantly reduced ELT from 33.58 ± 3.54 to 16.37 ± 5.11

s, p < 0.01, Figs. S13 and 12A) as compared to the scopolamine-treated

group. Treatment with 13b at dose of 10 mg/kg signiﬁcantly reduced

ELT from 50.11 ± 5.91 to 32.28 ± 6.37 s (Fig. S13 & 12A, p< 0.05)

which indicates signiﬁcant attenuation in the memory impairment.

Additionally, the recorded tracks of the mice from all groups clearly

further conﬁrmed these results (Fig. 12C). These outcomes collectively

suggested that the animals could retained the previous memory in the

MWM experiment. The mechanism of the spontaneous alternation

stimulation in the scopolamine model is likely mediated by the anti-

AChE activity of the compound 13b, suggesting that 13b is a potent

cholinesterase inhibitor that can cross the blood–brain barrier effec-

tively. The results suggested that molecule is orally active and signiﬁ-

cant changes in the compound treated group in comparison with the

control and the scopolamine group.

In our previous experiment, 13b has shown promising free radical

quenching ability in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which

strongly suggested that 13b is a potent antioxidant with a potency

approximate three times (IC50 = 20.25 ± 0.26 µM) compared to parent

natural compound FA (IC50 = 58.18 ± 0.52 µM). In our present study,

we carried out experiments to evaluate the effect of treatment with 13b

on protecting the neuronal N2a cells from toxicity induced by H

this experiment, we have employed various concentrations (100 to 800

M) of the pro-oxidant H (widely acceptable neurotoxin) and found

that 600 M of H can cause ~50% cell death in N2a cells (Fig. 10A).

After optimizing the concentration of H and the incubation time, we

next assessed the neuroprotection ability of 13b, to alter cytotoxicity

after co-incubating with H . The cells were incubated with various

concentrations (1, 2.5, 5, 10, and 20 µM) of 13b for 24 h followed by co-

treatment with 600 M H for an additional 24 h. As shown in ﬁg.10B

the MTT assay indicated that 13b could signiﬁcantly (*P < 0.05, ***P

0.001) protect N2a cells from H induced neurotoxicity. The sig-

niﬁcant protection conferred by 13b was exhibited in all tested con-

centration ranges of 1–20 M of the drugs. However, no signiﬁcant

difference was observed at concentration above 2.5 M. This suggest

that 2.5 M of 13b is able to negate the oxidative stress caused by 600

in cells.

2 2

O . In

μ

2 2

O

μ

2 2

O

2 2

O

2 2

O

μ

2 2

O

<

2 2

O

μ

M of H

.11. In-vitro blood–brain barrier permeation assay

Blood-brain barrier (BBB) permeability is the necessary requirement

2 2

O

2

for developing anti-AD drugs. A parallel artiﬁcial membrane permeation

assay of the blood–brain barrier (PAMPA-BBB) was performed to check

the probable BBB permeability of 3k and 13b. For this experiment,

ﬁrstly, we compared three commercial drugs’ permeability with their

3

. Conclusion

reported values to validate the experiment (Table 5). The P

and 13b are listed in Table 5. According to the limit established by Di

et al. for BBB permeation, we concluded that compounds with P > 4.0

could be considered to show high BBB permeability (CNS+), compounds

with P < 2 is expected to show low BBB permeability (CNS-). Analysis of

the developed compounds in the PAMPA-BBB assay revealed P > 4.0 for

k, and 13b showed (Table 5), demonstrating that compound ability to

e

values of 3k

In this paper, we have further extended the SAR studies on our earlier

identiﬁed FA template based novel series of molecules by using various

strategies with the aim of ﬁnely tuning the ChEs inhibition activities and

antioxidant property. All synthesized compounds were tested for their

AChE and BChE inhibitory properties. The in-vitro enzyme inhibition

studies suggested that the presence of tryptamine moiety could signiﬁ-

cantly improve the inhibitory activities of these molecules towards the

AChE and the BChE. Several compounds from this developed latest se-

ries, for example, 8a-8c, and 13a-13c, can be considered as dual acting

cholinestrases inhibitors because they were turned out to be effective

inhibitors of the both the enzymes and able to interact with the key

amino acid residues responsible for hydrolysis of cholinestrases. Com-

pound 13b-13c exhibited the highest activity for the AChE/BChE in the

current series. The enzyme kinetic studies demonstrated a mixed type of

inhibitory nature of 3k and 13b on both the AChE and BChE. The lead

molecules 3k and 13b also exhibited signiﬁcant antioxidant activity in

the DPPH assay. Interestingly, 13b was found to be three times more

potent antioxidant in a DPPH assay (IC50 = 20.25 ± 0.26 µM) over the

earlier identiﬁed EJMC-B (IC50 61.98 ± 0.30) and it also was able to

chelate iron. The PAMPA-BBB assay demonstrated that 3k, and 13b and

could effectively cross BBB and can reach to their target located in the

brain. The results from cell-based cytotoxicity studies indicated that the

compound had no signiﬁcant effect on cell viability at all the tested

concentrations. Further, in in-vitro assays, 13b exhibited neuro-

e

3

cross the BBB and can potentially reach their biological targets within

the brain.

Fig. 9. Effect of 13b on cell viability. Cells were treated with different con-

centrations of 13b (0.01, 0.1, 1, 2.5, 5, 10 and 20 µM) for 24 h. Cell viability in

%

was analyzed using MTT assay. Data represented as mean ± SE of three in-

2 2

protection property against H O induced neurotoxicity in N2a cells and

dependent experiments done in three replicates.

able to reverse the toxicity induced through OS. Compound 13b

1

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Fig. 10. Effect of 13b against H

2

O

2

mediated cell death. (A) Cell death induced by H

2

O

2

at 100, 200, 400, 600, and 800

M for 24 h and then further co-incubated with 600

2 2

H O for another 24 h. Cell death was assessed by MTT assay. The data represent the mean of three independent experiments. *P < 0.05, ***P < 0.001, compared

μ

M for 24 h. (B) Neuroprotective effect of

treatment 13b against H

2

O

2

mediated N2a cell death. Cells were pre-incubated with 13b at 1, 2.5, 5, 10, or 20

μ

M

with untreated control cells only, data expressed as mean ± SEM.

4

. Experimental section

.1. Chemistry

All the solvents required for the synthesis of the compounds were

Table 5

ꢀ

6

ꢀ 1

a

Permeability (P

e

= 10 cm s

) in the PAMPA-BBB assay for 3k, 13b, DPZ,

4

testosterone, corticosterone, and hydrocortisone with their predictive BBB

penetration.

a

b

c

S.No.

compound

P

e

(exp)

Reference value

Prediction

dried by solvent distillation techniques before use. All the chemicals and

reagents were obtained from the Sigma-Aldrich (St. Louis, MO, USA),

Alfa Aesar (Massachusetts), S.D. Fine Chemicals (India) and Avra

chemicals (India). All reactions were performed under inert atmosphere

1

2

3

4

5

6

.

Testosterone

Corticosterone

Hydrocortisone

3k

18.91 ± 1.42

10.19 ± 1.39

2.94 ± 1.12

9.57 ± 0.27

8.51 ± 0.31

15.57 ± 0.18

17.0

5.1

1.9

–

CNS (+)

CNS (-)

CNS (+)

2

(N ) unless otherwise noted. The reactions were monitored by thin-layer

13b

–

DPZ

–

chromatography (TLC) on precoated silica gel 60 F254 (MerckKGaA)

and were visualized under UV light, iodine vapors or by treatment with

ninhydrin and bromocresol green reagents. Column chromatographic

puriﬁcations were performed using silica gel 60–120 mesh size (CDH

a

b

c

Data are the mean ± SD of two independent experiments.

37

Reference values were taken from Di Li et al.

.

CNS (+), P > 4.0, high permeability; CNS (-), Pe < 2.0, low permeability;

e

d

1

CNS (±), 4.0 > P

e

> 2.0, uncertain permeability. DPZ (donepezil) reference

Laboratory Reagents, India). Proton nuclear magnetic resonance ( H

NMR) and ¹³C NMR spectra were measured on Bruker Advance, 500

MHz spectrometers with tetramethylsilane (TMS) as the internal stan-

AChE inhibitor. The data represent the mean ± SD of two independent

experiments.

dard. The NMR solvents used were CDCl

3

6

or DMSO–d as indicated.

exhibited promising in-vivo activity in the scopolamine-induced in MWM

model. . These above results conﬁrm that 13b is orally active and could

be a lead candidate for further development of potential AD therapeu-

tics. To uncover the full potential of the molecule further mechanistic

studies will be carried out in detail in AD models.

Chemical shifts were measured in ppm and coupling constants (J) were

measured in Hz. The following abbreviations are used to describe peak

splitting patterns when appropriate: d = doublet, t = triplet, q = quartet,

m = multiplet, dd = doublet of doublet, br = broad. Coupling constants J

are reported in Hertz (Hz). High resolution mass spectra (HRMS) were

recorded at Indian Institute of Technology, Ropar, India, CSIR- Indian

Fig. 11. (A) Schematic schedule of experimental design to study the anti-amnestic effect of compound 13b. (B) Outline of the Morris water maze model experiment.

I.P.; intraperitoneal, P.O; per os (orally), MWM PT-probe trial of Morris water maze test; MWM- Morris water maze.

1

2

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

Fig. 12. Scopolamine-induced memory deﬁcit mice model study. (A) Escape latency time on Day 5 (B) No. of entries in E zone. (C) Track diagram on Day 5. Data are

expressed as mean ± SEM (n = 7). ** p < 0.01, * p < 0.05 versus corresponding value in the control in Fig A and vs scopolamine in Fig. B, #p < 0.01 vs. scopolamine

(

Sco) group , $p < 0.05 vs Sco group. (Data recorded through Any Maze software). (Data recorded through Any Maze software).

Institute of Chemical Technology, Hyderabad and North East Institute of

Science & Technology (CSIR–NEIST), Jorhat. The HRMS of the haloge-

nated compounds was measured with reference to major isotope i.e.

4.1.2.1. (E)-1-(4-benzylpiperazin-1-yl)-3-(4-hydroxy-3-methoxyphenyl)

prop-2-en-1-one (3a). Into a stirring solution of compound FA (0.194 g,

1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g), HOBt (2.5

equiv., 0.37 g), 2a (0.8 equiv. 0.16 g), and DIPEA (2.5 equiv., 0.33 g)

were added at room temperature followed by the general procedure as

described above. The crude material was puriﬁed through column

chromatography using silica gel (100–200 mesh size) as a stationary

phase and EtOAc:hexane (9:1) as a mobile phase.

3

5

Cl, has been carried out and reported.

4

.1.1. General procedure for the synthesis of intermediates 2e-2g, 2i, 2m-

2

o

To a stirring solution of substituted benzyl halide (1.0 equiv, 1 mmol)

in ethanol (30 mL), K CO (3.0 equiv, 3.0 mmol), the appropriate

anhydrous piperazine (4.0 equiv) were added followed by the general

2

3

procedure in the literature.⁴

0,41

After being reﬂuxed for 4–6 h, the

ethanol was evaporated under reduced pressure, and subjected residue

was diluted with EtOAc (20 mL) and washed with ice cold water. The

aqueous layer was extracted with EtOAc (3 × 20 mL). The combined

1

White solid powder, (0.145 g, 75% yield). H NMR (DMSO‑d

6

, 500

–

OH), 7.40 (d, J = 15.2 Hz, 1H,

–

CH

–

CH^–

),

7.34–7.25 (m, 6H), 7.08–7.02 (m, 2H, Ar-H), 6.76 (d, J = 8.0 Hz, 1H, Ar-

H), 3.81 (bs, 3H, OCH of FA), 3.69 (bs, 2H, CH

), 3.54–3.50 (m, 4H,

pip-H), 2.39–2.34 (m, 4H, pip-H). C NMR (DMSO‑d , 126 MHz): δ

165.2, 148.9, 148.3, 142.7, 138.2, 129.4, 128.6, 127.4, 127.1, 122.9,

organic layer was washed with brine, dried over Na

2

4

SO , and concen-

MHz): δ 9.44 (s, 1H,

trated to yield the aforementioned compounds 2e-2g, 2i, 2m-2o, which

were used without puriﬁcation in the next step. The intermediates were

characterized with help of ¹H NMR. The NMR spectra’s of the synthe-

sized intermediates are matching with the reported values.

–

3

2

13

6

1

15.8, 114.9, 111.6, 62.3, 56.2, 53.7, 52.8, 45.4, 42.1. ESI-HRMS for

4

.1.2. General procedure for the synthesis of target compounds 3a-3p

Into a stirring solution of FA (0.2 g, 1.0 equiv.), EDCI. HCl, 0.29 g, 1.5

C

21

H

24

N

2

O

3

. (M+H)⁺calcd. 353.1865, found 353.1864.

equiv.), HOBt (0.37 g, 2.5 equiv.), and DIPEA (0.33 g, 2.5 equiv.) were

dissolved in dry THF (20 mL). The desired amount of the substituted

benzyl piperazine either commercially or synthesized derivative 2a-2p

4.1.2.2. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-(2-methylbenzyl)piper-

azin-1-yl)prop-2-en-1-one (3b.HCl). Into a stirring solution of compound

FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g),

HOBt (2.5 equiv., 0.37 g), 2b (0.8 equiv. 0.15 g), and DIPEA (2.5 equiv.,

0.33 g) were added at room temperature followed by the general pro-

cedure as described above. Thus, obtained compound was treated with

etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert into cor-

responding hydrochloride salt.

(

0.8 equiv.) was added to the reaction ﬂask and the reaction mixture was

stirred under nitrogen atmosphere at rt for 12 h. The progress of

chemical reaction was monitored using Hexane/EtOAc as mobile phase.

Upon completion of the reaction, THF was removed under reduced

pressure, and saturated NaHCO

reaction mixture was extracted with EtOAc (3 × 30 mL), and the com-

bined organic layer was dried over Na SO , and evaporated under

3

solution was slowly added into it. The

2

4

reduced pressure to obtain the crude product. Further puriﬁcation was

carried by column chromatography with the help of hexane/EtOAc.

1

3

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

White solid powder, (0.200 g, 69% yield). ¹H NMR (CDCl

, 500

), 7.24 (d, J = 7.5 Hz, 1H,

= 8.5 Hz, J = 2.0 Hz, 1H,

3

–

CH CH^–

–

MHz): δ 7.60 (d, J = 15.5 Hz, 1H,

Ar-H), 7.19–7.14 (m, 3H, Ar-H), 7.08 (dd, J

1

2

Ar-H), 6.98 (d, J = 2.0 Hz, 1H, Ar-H), 6.90 (d, J = 8.5 Hz, 1H, Ar-H),

–

CH

–

CH ), 3.91 (s, 3H, OCH

), 2.48 (t, J = 5.0 Hz, 4H, pip-

, 126 MHz): δ 171.1, 165.6, 147.3,

–

3

of FA),

6

.70 (d, J = 15.5 Hz, 1H,

3

.72–3.62 (m, 4H, pip-H), 3.48 (s, 2H, CH

2

1

White solid powder, (0.210 g, 69% yield). H NMR (DMSO‑d

6

, 500

1

3

H), 2.37 (s, 3H, CH

3

). C NMR (CDCl

3

MHz): δ 7.55–7.42 (m, 1H, Ar-H), 7.20–7.17 (m, 2H, Ar-H), 7.03–7.01

m, 1H, Ar-H), 6.92–6.85 (m, 3H, Ar-H), 6.79–6.76 (m, 2H), 3.83 (s, 3H,

1

46.7, 142.8, 137.5, 135.8, 130.3, 129.9, 127.8, 127.3, 125.5, 121.8,

(

–

14.7, 114.5, 109.9, 60.7, 60.4, 55.9, 21.0, 19.2. ESI-HRMS for

–

OCH

.41 (s, 4H, pip-H). C NMR (DMSO‑d

48.7, 147.9, 143.6, 138.7, 129, 127.2, 122.4, 121.4, 115.1, 114.6,

3

), 3.73 (s, 3H, OCH

3

), 3.67 (s, 4H, pip-H), 3.44 (s, 2H, CH

2

),

C

22

H

26

2

N O

3

. (M+H)⁺calcd. 367.2022, found 367.2031.

13

2

6

, 126 MHz): δ 166.7, 159.9,

1

4

.1.2.3. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-(3-methylbenzyl)piper-

113.4, 112.5, 110.4, 62.3, 55.1, 54.3, 53, 52.5, 47.2, 45.3. ESI-HRMS for

+

azin-1-yl)prop-2-en-1-one (3c.HCl). Into a stirring solution of compound

FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g),

HOBt (2.5 equiv., 0.37 g),2c (0.8 equiv. 0.15 g), and DIPEA (2.5 equiv.,

C₂₂H₂₆N O . (M+H) calcd. 383.1971, found 383.1968.

2

4

.1.2.6. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-(4-methoxybenzyl)

0

.33 g) were added at room temperature followed by the general pro-

piperazin-1-yl)prop-2-en-1-one (3f). Into a stirring solution of compound

FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g),

HOBt (2.5 equiv., 0.37 g), 2f (0.8 equiv. 0.16 g), and DIPEA (2.5 equiv.,

cedure as described above. Thus, obtained compound was treated with

etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert into cor-

responding hydrochloride salt.

0

.33 g) were added at room temperature followed by the general pro-

cedure as described above.

White solid powder, (0.200 g, 69% yield). ¹H NMR (CDCl

, 500

), 7.24 (t, J = 6.0 Hz, 1H,

Ar-H), 7.16–7.10 (m, 4H, Ar-H), 7.00 (d, J = 5.0 Hz, 1H, Ar-H), 6.93 (d,

3

MHz): δ 7.62 (d, J = 15.5 Hz, 1H,

CH

–

– _C_H–

–

_W_h_i_t_e_s_o_l_i_d_p_o_w_d_e_r_,₍₀_.₂₁₀_g_,₆₉_%_y_i_e_l_d₎_.1H NMR (CDCl

3

, 500

– – –

MHz): δ 7.61 (d, J = 15.3 Hz, 1H, CH

Ar-H), 7.09 (d, J = 8.2 Hz, 1H, Ar-H), 6.99 (s, 1H, Ar-H), 6.92–6.87 (m,

–

CH ), 7.25 (d, J = 8.3 Hz, 2H,

–

2

J = 7.5 Hz, 1H, Ar-H), 6.73 (d, J = 15.5 Hz, 1H,

–

CH

CH ), 3.94 (s,

), 2.50 (d, J

, 126 MHz):

3

H, OCH of FA), 3.76–3.67 (m, 4H, pip-H), 3.52 (s, 2H, CH

6.0 Hz, 4H, pip-H), 2.37 (s, 3H, CH

δ 165.5, 149.2, 148.2, 143.7, 138.5, 132.4, 130.6, 129.8, 129.1, 128.9,

3

– – –

H, Ar-H), 6.71 (d, J = 15.3 Hz, 1H, CH

–

3

CH ), 3.93 (s, 3H, OCH

and 4 pip-H merged), 3.53 (s, 2H, CH

.51 (s, 4H, pip-H). C NMR (CDCl , 126 MHz): δ 165.6, 159.0, 147.4,

46.7, 142.9, 130.5, 127.8, 121.9, 114.8, 114.4, 113.7, 109.9, 62.1,

3

of

1

3

=

3

). C NMR (DMSO‑d

6

–

FA), 3.82–3.69 (m, 7H, OCH

3

2

),

13

2

1

5

3

1

26.8, 123.1, 115.9, 114.0, 111.8, 59.5, 56.2, 21.4. ESI-HRMS for

C

22

H

26

2

N O

3

. (M+H)⁺calcd. 367.2022, found 367.2013.

26 2 4

5.9, 55.2, 53.1, 52.5, 45.6, 42.0, 29.6. ESI-HRMS for C22H N O .

(

M+H)⁺calcd. 383.1971, found 383.1970.

4

.1.2.4. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-(4-methylbenzyl)piper-

azin-1-yl)prop-2-en-1-one (3d). Into a stirring solution of compound FA

4

.1.2.7. (E)-1-(4-(2-chlorobenzyl)piperazin-1-yl)-3-(4-hydroxy-3-

(

0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g), HOBt

methoxyphenyl)prop-2-en-1-one (3g.HCl). Into a stirring solution of

compound FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv.,

2.5 equiv., 0.37 g), 2d (0.8 equiv. 0.15 g), and DIPEA (2.5 equiv., 0.33

g) were added at room temperature followed by the general procedure

as described above.

0

.29 g), HOBt (2.5 equiv., 0.37 g), 2g (0.8 equiv. 0.17 g), and DIPEA (2.5

equiv., 0.33 g) were added at room temperature followed by the general

procedure as described above. Thus, obtained compound was treated

with etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert into

corresponding hydrochloride salt.

White solid powder, (0.200 g, 68% yield). ¹H NMR (CDCl

CH^–

, 500

3

MHz): δ 7.62 (d, J = 15.5 Hz, 1H,

–

CH

–

), 7.22 (d, J = 8.0 Hz, 2H,

= 8.5 Hz, J = 2.0 Hz,

H Ar-H), 7.00 (d, J = 6.0 Hz, 1H, Ar-H), 6.92 (d, J = 8.0 Hz, 1H, Ar-H),

Ar-H), 7.16 (d, J = 8.00 Hz, 2H, Ar-H), 7.10 (dd, J

1

2

1

–

CH

–

CH ), 3.94 (s, 3H, OCH

CH

-), 2.50 (t, J = 5.0 Hz, 4H,

). C NMR (CDCl , 126 MHz): δ 165.6, 147.3,

–

3

of FA),

White solid powder, (0.214 g, 69% yield). ¹H NMR (CDCl , 500

6

.72 (d, J = 15.5 Hz, 1H,

3

.76–3.67 (m, 4H, pip-H), 3.53 (s, 2H,

–

2

–

MHz): δ 7.62 (d, J = 15.3 Hz, 1H, CH CH ), 7.49 (d, J = 7.5 Hz, 1H,

– –

1

3

pip-H), 2.36 (s,3H, CH

3

Ar-H), 7.39 (dd, J = 7.8 Hz, J = 1.3 Hz, 1H, Ar-H), 7.26–7.21 (m, 2H,

1

2

1

6

46.6, 142.8, 137.0, 129.1, 129.0, 127.8, 121.8, 114.7, 114.5, 109.8,

merged with CDCl , Ar-H), 7.12–7.10 (m, 1H, Ar-H), 7.01–7.12 (m, 1H,

3

2.6, 55.9, 31.9, 29.7, 22.6, 21.1, 14.1. ESI-HRMS for C22

H

26

2

N O

3

.

Ar-H), 6.92 (d, J = 8.2 Hz, 1H, Ar-H), 6.73 (d, J = 15.3 Hz, 1H,

(

M+H)⁺calcd. 367.2022, found 367.2018.

–

CH

–

3

CH ), 3.94 (s, 3H, OCH of FA), 3.77 (s, 4H, pip-H), 3.68 (s, 2H,

–

1

3

CH

2

), 2.58 (t, J = 5.0 Hz, 4H, pip-H). C NMR (CDCl

3

, 126 MHz): δ

4

.1.2.5. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-(3-methoxybenzyl)

165.6, 147.3, 146.7, 142.9, 135.3, 134.4, 130.8, 129.5, 128.4, 127.8,

piperazin-1-yl)prop-2-en-1-one (3e). Into a stirring solution of compound

FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g),

HOBt (2.5 equiv., 0.37 g) 2e (0.8 equiv. 0.16 g), and DIPEA (2.5 equiv.,

126.6, 121.8, 114.7, 114.5, 109.8, 59.1, 56.0, 29.7. ESI-HRMS for

C

21

H23ClN

2

O

3

. (M+H)⁺calcd. 387.1475, found 387.1471.

0

.33 g) were added at room temperature followed by the general pro-

4.1.2.8. (E)-1-(4-(3-chlorobenzyl)piperazin-1-yl)-3-(4-hydroxy-3-

methoxyphenyl)prop-2-en-1-one (3h). Into a stirring solution of com-

pound FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29

g), HOBt (2.5 equiv., 0.37 g), 2h (0.8 equiv. 0.17 g), and DIPEA (2.5

equiv., 0.33 g) were added at room temperature followed by the general

cedure as described above.

1

4

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

procedure as described above.

0.29 g), HOBt (2.5 equiv., 0.37 g), 2k (0.8 equiv. 0.15 g), and DIPEA (2.5

equiv., 0.33 g) were added at room temperature followed by the general

procedure as described above. Thus, obtained compound was treated

with etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert into

corresponding hydrochloride salt.

White solid powder, (0.214 g, 71% yield). ¹H NMR (CDCl

3

, 500

–

CH CH^–), 7.36 (s, 1H, Ar-H),

–

MHz): δ 7.61 (d, J = 15.3 Hz, 1H,

7

.28–7.21 (m, 3H, Ar-H), 7.10 (d, J = 8.0 Hz, 1H, Ar-H), 6.99 (s, 1H,

Ar-H), 6.92 (d, J = 8.2 Hz, 1H, Ar-H), 6.70 (d, J = 15.3 Hz, 1H,

CH CH ), 3.92 (s, 3H,

OCH ), 3.76–3.68 (m, 4H, pip-H), 3.52 (s,

H, CH

), 2.49 (t, J = 5.5 Hz, 4H, pip-H). C NMR (CDCl

δ 165.7, 147.4, 146.7, 143.0, 139.8, 134.3, 129.6, 129.0, 127.7, 127.5,

White solid powder, (0.216 g, 73% yield). ¹H NMR (CDCl , 500

–

3

1

3

–

2

3

, 126 MHz):

MHz): δ 7.62 (d, J = 15.5 Hz, 1H,

–

CH

–

), 7.32–7.28 (m, 1H, Ar-

H), 7.14–7.09 (m, 3H, Ar-H), 7.01–7.7.00 (m, 2H, Ar-H), 6.92 (d, J = 8.5

–

CH

–

CH ), 3.94 (s, 3H,

CH ), 2.54 (s,

, 126 MHz): δ 168.8, 165.2, 148.9,

–

1

27.1, 121.9, 114.8, 114.3, 109.9, 62.2, 55.9. ESI-HRMS for

Hz, 1H, Ar-H), 6.71 (d, J = 15.5 Hz, 1H,

OCH

of FA), 3.79–3.71 (m, 4H, pip-H), 3.58 (s, 2H,

H, pip-H). C NMR (DMSO‑d

+

C

21

H

2

23ClN O

3

. (M+H) calcd. 387.1475, found 387.1470.

–

4

3

2

1

3

6

4

.1.2.9. (E)-1-(4-(4-chlorobenzyl)piperazin-1-yl)-3-(4-hydroxy-3-

148.2, 142.7, 138, 129, 127.4, 127.1, 123, 121.5, 115.8, 114.9, 111.6,

61.8, 56.2, 49.0 ESI-HRMS for C21

found 371.1776.

2 3

H23FN O

. (M+H)⁺calcd. 371.1771,

methoxyphenyl)prop-2-en-1-one (3i). Into a stirring solution of com-

pound FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29

g), HOBt (2.5 equiv., 0.37 g), 2i (0.8 equiv. 0.17 g), and DIPEA (2.5

equiv., 0.33 g) were added at room temperature followed by the general

procedure as described above.

4.1.2.12. (E)-1-(4-(4-ﬂuorobenzyl)piperazin-1-yl)-3-(4-hydroxy-3-

methoxyphenyl)prop-2-en-1-one (3l.HCl). Into a stirring solution of

compound FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv.,

0

.29 g), HOBt (2.5 equiv., 0.37 g), 2l (0.8 equiv. 0.167 g), and DIPEA

(

2.5 equiv., 0.33 g) were added at room temperature followed by the

general procedure as described above. Thus, obtained compound was

treated with etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert

into corresponding hydrochloride salt.

White solid powder, (0.214 g, 70% yield). ¹H NMR (CDCl

, 500

), 7.32–7.27 (m, 4H, Ar-

= 2.0 Hz, 1H, Ar-H), 6.99 (d, J = 2.0 Hz,

H), 6.62 (d, J = 8.0 Hz, 1H, Ar-H), 6.71 (d, J = 15.3 Hz, 1H,

CH CH

), 3.92 (s, 3H, OCH ), 3.75–3.67 (m, 4H, pip-H), 3.51 (s, 2H,

CH ), 2.48 (t, J = 5.5 Hz, 4H, pip-H). C NMR (CDCl , 126 MHz): δ

3

MHz): δ 7.62 (d, J = 15.3 Hz, 1H,

H), 7.10 (dd, J

= 8.0 Hz, J

CH

–

– _C_H–

–

1

2

1

–

2

3

1

3

–

3

1

65.7, 147.4, 146.7, 143.0, 136.1, 133.0, 130.3, 128.5, 127.7, 121.8,

1

White solid powder, (0.210 g, 69% yield). H NMR (DMSO‑d

6

, 500

1

2 3

14.8, 114.3, 109.9, 62.0, 55.9. ESI-HRMS for C21H23ClN O

. (M+H)⁺

–

CH CH^–), 7.29–7.27 (m, 2H, Ar-

–

MHz): δ 7.59 (d, J = 15.5 Hz, 1H,

H), 7.07 (dd, J = 2.0 Hz, 1H, Ar-H), 7.08–6.97 (m, 3H, Ar-

= 8.5 Hz, J

calcd. 387.1475, found 387.1477.

1

2

H), 6.90 (d, J = 8.0 Hz, 1H, Ar-H), 6.70 (d, J = 15.0 Hz, 1H,

4

.1.2.10. (E)-1-(4-(2-ﬂuorobenzyl)piperazin-1-yl)-3-(4-hydroxy-3-

–

CH

.49 (s, 2H, CH

DMSO‑d

–

CH

–

), 3.91 (s, 3H,

–

OCH of FA), 3.65–3.73 (bs, 4H, pip-H),

methoxyphenyl)prop-2-en-1-one (3j.HCl). Into a stirring solution of

compound FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv.,

3

13

3

2

), 2.46 (t, J = 5.0 Hz, 4H, pip-H).

C NMR

(

6

, 126 MHz): δ 165.7, 163.1, 161.18, 147.4, 146.7, 143, 130.6,

0

.29 g), HOBt (2.5 equiv., 0.37 g), 2j (0.8 equiv. 0.16 g), and DIPEA (2.5

1

5

30.6, 127.8, 121.8, 115.2, 115.1, 114.7, 114.3, 109.9, 62, 55.9, 53.1,

equiv., 0.33 g) were added at room temperature followed by the general

procedure as described above. Thus, obtained compound was treated

with etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert into

corresponding hydrochloride salt.

2.7, 31.9, 29.7, 27, 19.7, 14.1. ESI-HRMS for C21

H

23FN

2

O

3

. (M+H)⁺

calcd. 371.1771, found 371.1775.

4

.1.2.13. 4.1.2.13.(E)-2-((4-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)

piperazin-1-yl)methyl) benzonitrile (3m). Into a stirring solution of

compound FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv.,

0

.29 g), HOBt (2.5 equiv., 0.37 g), 2m (0.8 equiv. 0.161 g), and DIPEA

(

2.5 equiv., 0.33 g) were added at room temperature followed by the

general procedure as described above. The crude material was puriﬁed

by column chromatography using silica gel (100–200 mesh size) as a

stationary phase with help of EtOAc:hexane(9:1) as mobile phase.

White solid powder, (0.216 g, 71% yield). ¹H NMR (CDCl

, 500

3

–

CH^–

MHz): δ 7.58 (d, J = 15.0 Hz, 1H,

–

CH

), 7.35 (td, J

1

= 7.5 Hz, J

2

=

2.0 Hz, 1H, Ar-H), 7.25–7.22 (m, 1H, Ar-H), 7.10 (td, J = 8.0 Hz, J

1

2

=

1

6

3

2

1

6

.0 1H, Ar-H), 7.05–7.00 (m, 2H, Ar-H), 6.96 (d, J = 5.5 Hz, 1H, Ar-H),

–

2

.87 (d, J = 8.0 Hz, 1H, Ar-H), 6.68 (d, J = 15.5 Hz, 1H,

–

CH

CH ),

),

, 126 MHz): δ 165.7,

.86 (s, 3H, OCH

3

of FA), 3.75–3.72 (m, 4H, pip-H), 3.62 (bs, 2H, CH

1

3

.52 (t, J = 5.0 Hz, 4H, pip-H). C NMR (CDCl

3

62.3, 160.4, 147.7, 147.0, 143.1, 131.6, 131.6, 129.1, 129.1, 127.5,

White solid powder, (0.213 g, 71% yield). ¹H NMR (CDCl

, 500

3

23.9, 123.9, 123.8, 123.7, 121.9, 115.4, 115.2, 115.0, 114.1, 110.0,

MHz): δ 7.69–7.54 (m, 4H, Ar-H), 7.40 (t, J = 8.5 Hz, 1H, Ar-H),

2 3

7.9, 55.9, 55.0, 55.0, 52.9, 52.4, 25.5. ESI-HRMS for C21H23FN O .

M+H)⁺calcd. 371.1771, found 371.1769.

7

1

3

1

.11–7.09 (m, 1H, Ar-H), 7.00 (bs, 1H, Ar-H), 6.92 (d, J = 7.75 Hz,

(

–

3

CH CH ), 3.93 (s, 3H, OCH ),

– –

–

H, Ar-H), 6.72 (d, J = 15.5 Hz, 1H,

3

CH ), 2.57 (s, 4H, pip-H). C NMR (CDCl ,

1

3

.75–3.69 (m, 6H, pip-H,

–

2

4

.1.2.11. (E)-1-(4-(3-ﬂuorobenzyl)piperazin-1-yl)-3-(4-hydroxy-3-

26 MHz): δ 165.7, 147.3, 146.7, 143.0, 141.8, 133.1, 132.6, 130.1,

methoxyphenyl)prop-2-en-1-one (3k.HCl). Into a stirring solution of

compound FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv.,

27.8, 127.8, 121.9, 117.7, 114.7, 114.4, 113.1, 109.8, 60.3, 56.0. ESI-

1

5

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

HRMS for C22

H

23

N

3

O

3

. (M+H)⁺calcd. 378.1818, found 378.1814.

127.1, 122.9, 121.6, 115.8, 114.8, 111.6, 61.8, 56.5, 56.2, 55.2, 53.6,

5

2.9, 18.9. ESI-HRMS for C21

H23ClN

2

O

3

. (M+H)⁺calcd. 398.1716,

4

1

.1.2.14. (E)-3-((4-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)piperazin

yl)methyl)benzonitrile (3n). Into a stirring solution of compound FA

found 398.1719.

(

0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g), HOBt

4.1.3. General procedure for the synthesis of 3-((tert-butoxycarbonyl)

amino)propanoic acid (5)

2.5 equiv., 0.37 g), 2n (0.8 equiv. 0.161 g), and DIPEA (2.5 equiv., 0.33

g) were added at room temperature followed by the general procedure

as described above.

Into a stirring solution alanine (0.8 g, 9.09 mmol) in DCM (30 mL),

(Boc)

2

O (2.37 g, 10.90 mmol) and NaOH (0.29 g, 7.27 mmol) were

◦

added at 0 C. The reaction mixture was stirred at room temperature for

1

2 h and was extracted with DCM (3 × 30 mL), washed with water, dried

over Na SO , ﬁltered, and concentrated in vacuo. The crude material

was puriﬁed by column chromatography over silica gel (hexane/EtOAc,

2

4

1

9

.0:1.0) to give compound 5. White solid powder, yield 80%. H NMR

Brown solid powder, (0.213 g, 70% yield). ¹H NMR (CDCl

, 500

(CDCl

3

, 500 MHz): δ 6.26 (s, 1H), 5.12 (s, 1H), 3.41 (d, J = 5.5 Hz, 2H),

3

MHz): δ 7.69–7.58 (m, 4H, Ar-H), 7.45 (t, J = 8.5 Hz, 1H, Ar-H), 7.10

2.59–2.54 (m, 2H), 1.47 (s, 9H).

(

dd, J

1

= 8.0 Hz, J

2

= 2.0 Hz, 1H, Ar-H), 7.00 (d, J = 2.0 Hz, 1H, Ar-H),

–

CH ),

4.1.4. General procedure for the synthesis of intermediates 6a-6c

Into a stirring solution of compound 5 (0.20 g, 1 mmol) in DCM (20

mL), substituted amine (0.19 g, 0.8 mmol), HOBt, (0.37 g, 2.5 mmol), 1-

Ethyl-3-EDCI.HCl (0.29 g, 1.5 mmol) and DIPEA (0.33 g, 2.5 mmol)

were added at room temperature. After the reaction mixture was stirred

for 8–10 h, a saturated brine solution was added into the reaction

mixture, and it was extracted with DCM (3 × 30 mL). The combined

organic layer was dried in vacuum, and ﬁnally puriﬁed by silica gel

column chromatography to yield compound (6a-6c).

6

3

5

1

.92 (d, J = 8.15 Hz, 1H, Ar-H), 6.71 (d, J = 15.5 Hz, 1H,

–

CH

.94 (s, 3H, OCH

3

), 3.76 (s, 4H, pip-H), 3.57 (s, 2H, CH

2

), 2.49 (t, J =

.5 Hz, 4H, pip-H) . ¹³C NMR (CDCl

3

, 126 MHz): δ 165.7, 147.4, 146.7,

43.1, 139.5, 133.3, 132.3, 131.0, 129.1, 121.8, 118.8, 114.7, 112.5,

09.9, 61.8, 55.9. ESI-HRMS for C22

H

23

3

N O

3

. (M+H)⁺calcd. 378.1818,

found 378.1816.

4

1

.1.2.15. (E)-4-((4-(3-(4-hydroxy-3-methoxyphenyl)acryloyl)piperazin-

-yl)methyl)benzonitrile (3o.HCl). Into a stirring solution of compound

FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g),

HOBt (2.5 equiv., 0.37 g) 2o (0.8 equiv. 0.161 g), and DIPEA (2.5 equiv.,

4.1.4.1. tert-butyl (3-oxo-3-(phenylamino)propyl)carbamate (6a). Into a

stirring solution of compound 5 (1 equiv. 0.189 g) in THF (20 mL), EDCI.

HCl (1.5 equiv., 0.29 g), HOBt (2.5 equiv., 0.37 g), aniline (0.8 equiv.,

0.074 g) and DIPEA (2.5 equiv., 0.33 g) were added at room temperature

followed by the general procedure as described above.

0

.33 g) were added at room temperature followed by the general pro-

cedure as described above. Thus, obtained compound was treated with

etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert into cor-

responding hydrochloride salt.

1

White solid powder, yield (0.225 g, 85%). H NMR (DMSO‑d

6

, 500

1

White solid powder, (0.195 g, 65% yield). H NMR (DMSO‑d

6

, 500

MHz): δ 10.02 (bs, 1H –NH), 09.97 (s, 1H –NH). 7.88 (d, J = 8.5 Hz, 2H,

Ar-H), 7.56 (d, J = 8.0 Hz, 2H, Ar-H), 7.33 (t, J = 6.5 Hz, 1H, Ar-H), 3.66

(t, J = 6.5 Hz, 2H, methylene protons), 3.19 (t, J = 7.0 Hz, 2H, meth-

ylene protons), 1.51 (s, 9H, Boc protons).

MHz): δ 9.52 (s, 1H, OH), 8.14 (s, 1H, Ar-H), 7.84 (d, J = 8.0 Hz, 1H, Ar-

H), 7.62 (d, J = 8.0 Hz, 1H, Ar-H), 7.50–7.46 (m, 2H), 7.33 (bs, 1H),

7

.14–7.06 (m, 2H), 6.80 (d, J = 8.0 Hz, 1H, Ar-H), 4.51–4.25 (m, 4H,

pip-H), 3.83 (m, 3H, OCH

3

of FA), 3.61–3.56 (m, 4H, pip-H), 3.17 (s, 2H,

, 126 MHz): δ 168.4, 166.1, 149.8, 149.5,

40.1, 139.6, 138.3, 129.1, 129, 128.5, 128.1, 127.2, 127.1, 113.2,

1

3

2 6

CH ). C NMR (DMSO‑d

4

.1.4.2. tert-butyl (3-((3-methoxyphenyl)amino)-3-oxopropyl)carbamate

6b). Into a stirring solution of compound 5 (1 equiv. 0.189 g) in THF

20 mL), EDCI.HCl (1.5 equiv., 0.29 g), HOBt (2.5 equiv., 0.37 g), m-

1

(

23 3 3

10.8, 67.2, 58.2, 55.9, 51.6, 47.8, 43.3. ESI-HRMS for C22H N O .

(

M+H)⁺calcd. 378.1818, found 378.1826.

anisidine (0.8 equiv. 0.098 g), and DIPEA (2.5 equiv., 0.33 g) were

added at room temperature followed by the general procedure as

described above.

4

.1.2.16. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-(3-nitrobenzyl)piper-

azin-1-yl)prop-2-en-1-one (3p.HCl). Into a stirring solution of compound

FA (0.194 g, 1 equiv.) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g),

HOBt (2.5 equiv., 0.37 g), 2p (0.8 equiv. 0.201 g), and DIPEA (2.5

equiv., 0.33 g) were added at room temperature followed by the general

procedure as described above. Thus, obtained compound was treated

with etherial HCl (2 M in diethyl ether, Sigma Aldrich) to convert into

corresponding hydrochloride salt.

1

White solid powder, yield (0.242 g, 82%). H NMR (CDCl

3

500 MHz):

δ 7.81 (q, J

bs, 1H Ar-H), 7.22 (t, J = 8.0 Hz, 2H Ar-H), 7.03 (d, J = 7.5 Hz 1H, Ar-

H), 6.68 (dd, J OCH ),

= 6.0 Hz, 2H, methylene protons), 2.62 (t, J =

.5 Hz, 2H, methylene protons), 1.44 (s, 9H, boc protons).

1

= 9.5 Hz, J

2

= 7.0 Hz, 1H), 7.48–7.45 (m, 1H, Ar-H), 7.30

(

= 6.5 Hz, J

2

= 1.5 Hz, 1H Ar-H), 3.81 (s, 3H,

–

3

1

3

6

.49 (q, J

1

= 12.0 Hz, J

2

4

.1.4.3. tert-butyl

(3-((4-chlorophenyl)amino)-3-oxopropyl)carbamate

1

Yellow solid powder, (0.251 g, 70% yield). H NMR (DMSO‑d

6

, 500

(

6c). Into a stirring solution of compound 5 (1.0 equiv. 0.180 g) in THF

MHz): δ 9.90 (bs, 1H), 9.43 (bs, 1H,

H), 7.42–7.30 (m, 4H, Ar-H), 7.08–7.02 (m, 2H), 6.77 (d, J = 8.5 Hz, 1H,

Ar-H), 3.82 (s, 3H, OCH

H), 2.53 (bs, 4H, pip-H, merged with DMSO‑d

26 MHz): δ 168.8, 165.3, 148.9, 148.2, 142.7, 137.9, 128.9, 127.5,

–

OH), 7.67 (d, J = 8.5 Hz, 2H, Ar-

(

20 mL), EDCI.HCl (1.5 equiv., 0.29 g), HOBt (2.5 equiv., 0.37 g), p-

chloroaniline (0.8 equiv. 0.10 g), and DIPEA (2.5 equiv., 0.33 g) were

added at room temperature followed by the general procedure as

described above.

–

of FA), 3.76–3.63 (m, 4H, pip-H), 3.19 (bs,

3

1

3

2

1

6 6

). C NMR (DMSO‑d ,

1

6

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

1

6

Brown solid powder, yield (0.230 g, 81%). H NMR (DMSO‑d , 500

1

White solid powder, (0.154 g, 71% yield). H NMR (DMSO‑d

6

, 500

MHz): δ 7.98 (d, J = 8.5 Hz, 1H, Ar-H), 7.72 (d, J = 8.5 Hz, 1H, Ar-H),

MHz): δ 9.44 (bs, 1H, –NH), 8.08 (s, 1H, –NH), 7.34–7.30 (m, 2H, Ar-H),

7

6

.62 (d, J = 9.0 Hz, 1H, Ar-H), 7.54 (t, J = 7.0 Hz, 1H, Ar-H), 7.41 (t, J =

.5 Hz, 1H, Ar-H), 7.33 (d, J = 8.5 Hz, 1H, Ar-H), 3.20 (t, J = 6.5 Hz, 2H,

7

.20–7.11 (m, 4H, Ar-H), 6.98 (dd, J

1

= 8.0 Hz, J

2

= 2.5 Hz, 1H, Ar-H),

6

.78 (d, J = 8.0 Hz, 1H, Ar-H), 6.61 (dd, J

1

–

= 8.0 Hz, J

2

= 3.0 Hz, 1H, Ar-

methylene protons), 3.20 (t, J = 6.0 Hz, 2H, methylene protons), 1.37 (s,

–

CH

–

CH ), 3.83–3.79 (m, 3H, OCH

–

3

),

H), 6.47 (d, J = 15.5 Hz, 1H,

9

H, boc protons).

3

.71 (s, 3H,

–

OCH

3

), 3.45 (q, J

1

= 12.5 Hz, J

2

= 6.5 Hz, 2H, methylene

1

3

protons), 2.54 (t, J = 6.5 Hz, 2H, methylene protons). C NMR

4

.1.5. General procedure for the synthesis of intermediates 7a-7c

Into a stirring solution of compound 6a-6c (0.2 g, 1 mmol) in DCM

(

DMSO‑d , 126 MHz): δ 169.2, 166.1, 148.7, 148.2, 139.8, 136.6, 127.6,

6

1

5

26.8, 123.0, 122.0, 121.3, 119.2, 118.6, 116.1, 112.1, 111.8, 111.3,

(

30 mL), TFA (15 mL) was added slowly at room temperature, and the

6.0, 42.7, 25.6. ESI-HRMS for C20

H

22

2

N O

5

. (M+H)⁺calcd. 371.1607,

reaction mixture was stirred for 6 h. The unreacted TFA and solvent

DCM were removed in vacuo, and thus formed salt was washed with

diethyl ether. To the resultant mixture saturated solution of sodium

bicarbonate was added, and it was extracted with dichloromethane (50

found 371.1605.

4

.1.6.3. 4.1.6.3. (E)-N-(3-((4-chlorophenyl)amino)-3-oxopropyl)-3-(4-

hydroxy-3-methoxyphenyl) acrylamide (8c). Into a stirring solution of

compound FA (0.3 g, 1.54 equiv.) in THF (20 mL), EDCI.HCl (2.31

equiv., 0.35 g), HOBt (3.86 equiv., 0.52 g), 7c (0.8 equiv. 0.158 g), and

DIPEA (2.5 equiv., 0.33 g) were added at room temperature followed by

the general procedure as described above.

×

2 4

3 mL). The combined organic layer was dried over Na SO , ﬁltered,

and evaporated in vacuo to provide the compound 7a-7c, which were

subjected to next reaction without further puriﬁcation.

4

.1.6. General procedure for synthesis of target compounds 8a-8c and

3a-13c

Into a stirring solution of FA (0.3 g, 1.54 mmol) in dry THF (10 mL),

HOBt (0.52 g, 3.86 mmol), EDCI.HCl (0.35 g, 2.31 mmol) and DIPEA

0.49 g, 3.86 mmol) were added. The reaction was stirred at room

1

(

temperature for 15 min. Finally, substituted amine (1.0 equiv.) was

added, and the reaction mixture was allowed to stir overnight at room

temperature. The progress of the reaction was monitored by TLC. After

1

White solid powder, (0.167 g, 74% yield). H NMR (DMSO‑d , 500

6

MHz): δ 9.97 (bs, 1H

–

OH), 9.43 (s, 1H, –NH proton), 8.08 (t, J = 5.5

completion of the reaction, the saturated NaHCO

3

solution was added

Hz, 1H, –NH proton), 7.60 (d, J = 8.0 Hz, 2H, Ar-H), 7.33–7.27 (m, 3H,

slowly into it. The mixture was extracted with ethyl acetate (3 × 50 mL),

Ar-H), 7.11–6.97 (m, 3H, Ar-H), 6.77 (d, J = 8.0 Hz, 1H Ar-H), 6.47 (d, J

and the combined organic layer was washed with brine, dried over

= 15.5 Hz, 1H,

–

CH

–

CH ), 3.79 (s, 3H,

–

OCH ), 3.45 (q, J = 8.0 Hz,

3

1

Na

2

SO

4

and concentrated under vacuum. The residue was subjected to

J = 2.5 Hz, 2H, methylene protons), 2.55 (t, J = 6.5 Hz, 2H, methylene

2

protons). ¹³C NMR (DMSO‑d , 126 MHz): δ 169.9, 166.0, 148.7, 148.2,

silica gel chromatography or crystallization to afford the aforemen-

tioned target compounds.

6

139.6, 139.4, 129.1, 126.8, 123.5, 122.0, 119.5, 119.3, 116.0, 111.1,

5

5.9, 36.8, 35.6. ESI-HRMS for

C

19

H

19ClN

2

O

4

.

(M+H)⁺calcd.

4

.1.6.1. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-(3-oxo-3-(phenylamino)

propyl) acrylamide (8a). Into a stirring solution of compound FA (0.3 g,

.54 equiv.) in THF (20 mL), EDCI.HCl (2.31 equiv., 0.35 g), HOBt (3.86

375.1112, found 375.1115.

1

4.1.7. General procedure for synthesis of intermediate 11a-11c

equiv., 0.52 g), 7a (0.8 equiv. 0.131 g), and DIPEA (2.5 equiv., 0.33 g)

were added at room temperature followed by the general procedure as

described above.

Into a stirring solution of Boc-glycine (0.3 g, 2.55 mmol) in THF (20

mL), substituted tryptamine (0.4 g, 2.55 mmol), EDCI.HCl, 0.97 g, 4.59

mmol), HOBt, 0.512 g, 3.82 mmol), and N,N-Diisopropylethylamine

(

DIPEA, 0.15 g, 2.55 mmol) were added at room temperature. After the

reaction was stirred for 12 h, a diluted solution of HCl was added into the

reaction mixture, and it was extracted with EtOAc (3 × 50 mL). The

combined organic layer was washed with brine and ﬁnally puriﬁed by

silica gel column chromatography (EtOAc/Hexane, 1:1) to get afore-

mentioned product.

1

White solid powder, yield (0.128 g, 76%). H NMR (DMSO‑d

6

, 500

MHz): δ 10.10 (s, 1H,

NH), 7.64 (d, J = 9.0 Hz, 2H, Ar-H), 7.36–7.30 (m, 3H, Ar-H), 7.11 (d, J

2.0 Hz, 1H, Ar-H), 6.97 (dd, J = 1.5 Hz, 1H, Ar-H), 6.78

= 8.0 Hz, J

d, J = 8.5 Hz, 1H), 6.48 (d, J = 15.5 Hz, 1H,

OCH ), 3.45 (q, J = 12.5 Hz, J = 6.5 Hz, 2H, methylene protons),

–

OH), 9.43 (s, 1H, –NH), 8.06 (t, J = 11.5 Hz, 1H,

–

4.1.7.1. tert-butyl (2-((2-(1H-indol-3-yl)ethyl)amino)-2-oxoethyl)carba-

mate (11a). Into a stirring solution of compound tert-butoxycarbonyl)

glycine (1 equiv. 0.175 g) in THF (20 mL), EDCI.HCl (1.5 equiv., 0.29 g),

HOBt (2.5 equiv., 0.37 g), Tryptamine (0.8 equiv. 0.128 g), and DIPEA

(2.5 equiv., 0.33 g) were added at room temperature followed by the

general procedure as described above.

=

1

2

–

CH CH ), 3.79 (s, 3H,

– –

–

(

–

2

3

1

2

1

3

.55 (t, J = 6.5 Hz, 2H, methylene protons). C NMR (DMSO‑d

6

, 126

MHz): δ 166.4, 149.4, 148.8, 148.2, 140.0, 137.2, 135.9, 129.9, 128.7,

1

26.7, 123.7, 122.2, 122.1, 118.9, 116.1, 115.4, 111.3, 56.0, 43.5. ESI-

HRMS for C21

H

23ClN

2

O

3

. (M+H)⁺calcd. 341.1501, found 341.1497.

4

.1.6.2. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-(3-((3-methoxyphenyl)

amino)-3-oxopropyl)acrylamide (8b). Into a stirring solution of com-

pound FA (0.3 g, 1.54 equiv.) in THF (20 mL), EDCI.HCl (2.31 equiv.,

1

Brown solid powder, yield (0.190 g, 75%). H NMR (DMSO‑d

6

, 500

MHz): δ 10.80 (bs, 1H –NH), 7.85 (t, J = 5.5 Hz, 1H, –NH), 7.52 (d, J =

0

.35 g), HOBt (3.86 equiv., 0.52 g), 7b (0.8 equiv. 0.155 g), and DIPEA

2.5 equiv., 0.33 g) were added at room temperature followed by the

general procedure as described above.

8

7

.0 Hz, 1H, –NH), 7.32 (d, J = 8.5 Hz, 1H, Ar-H), 7.13 (bs, 1H Ar-H),

.06 (t, J = 8.0 Hz, 1H, Ar-H), 6.97 (t, J = 7.5 Hz, 1H, Ar-H), 6.91 (t,

(

1

7

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

J = 6.0 Hz, 1H, Ar-H), 3.50 (d, J = 8.5 Hz, 2H, methylene protons), 3.34

(

q, J

1

= 7.0 Hz, J = 6.5, 2H, methylene protons), 2.80 (t, J = 6.5 Hz, 2H,

2

methylene protons), 1.38 (s, 9H, boc protons).

4

.1.7.2. tert-butyl(2-((2-(5-methoxy-1H-indol-3-yl)ethyl)amino)-2-

1

oxoethyl)carbamate (11b). Into a stirring solution of compound tert-

Brown solid powder, (0.152 g, 71% yield). H NMR (DMSO‑d , 500

6

butoxycarbonyl)glycine (1 equiv. 0.175 g) in THF (20 mL), EDCI.HCl

MHz): δ 10.78 (bs, 1H

–

OH), 9.44 (bs, 1H, –NH), 8.14 (t, J = 6.0 Hz, 1H,

(

1.5 equiv., 0.29 g), HOBt (2.5 equiv., 0.37 g), 5-methoxytryptamine

0.8 equiv. 0.152 g), and DIPEA (2.5 equiv., 0.33 g) were added at

–NH), 8.01 (t, J = 6.0 Hz, 1H, –NH), 7.53 (d, J = 8.0 Hz,

–

– –

CH CH ),

7.35–7.31 (m, 2H, Ar-H), 7.14 (t, J = 4.5 Hz, 2H, Ar-H), 7.06–6.95 (m,

3H, Ar-H), 6.79 (d, J = 8.0 Hz, 1H, Ar-H), 6.56 (d, J = 15.5 Hz, 1H,

room temperature followed by the general procedure as described

above.

–

CH

–

3

CH ), 3.80–3.78 (m, 5H, OCH and methylene protons), 3.34

– –

(

s, 2H, merge with DMSO‑d

6

methylene protons), 2.82 (t, J = 7.5 Hz, 2H,

methylene protons). C NMR (DMSO‑d , 126 MHz): δ 169.2, 166.1,

48.7, 148.2, 139.8, 136.6, 127.6, 126.8, 123.0, 122.0, 121.3, 119.2,

1

3

6

1

18.6, 116.1, 112.1, 111.8, 111.3, 56.0, 42.7, 25.6. ESI-HRMS for

C

22

H

23

N

3

O

4

. (M+H)⁺calcd. 394.1767, found 394.1773.

Brown solid powder, yield (0.202 g, 73%). ¹H NMR (CDCl

MHz): δ 10.65 (bs, 1H, –NH), 7.85 (t, J = 5.5 Hz, 1H, –NH), 7.21 (d, J =

.0 Hz, 1H, Ar-H), 7.10 (d, J = 2.0 Hz, 1H Ar-H), 7.02 (d, J = 2.0 Hz, 1H,

Ar-H), 6.92 (t, J = 7.0 Hz, 1H, Ar-H), 6.70 (dd, J = 6.0 Hz, J = 2.5 Hz,

H, Ar-H), 3.71 (s, 3H, OCH

, 500

3

4

.1.10. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-((2-(5-methoxy-1H-

indol-3-yl)ethyl)amino)-2-oxoethyl)acrylamide (13b)

9

Into a stirring solution of compound FA (0.3 g, 1.54 equiv.) in THF

1

2

(

20 mL), EDCI.HCl (2.31 equiv., 0.35 g), HOBt (3.86 equiv., 0.52 g), 12b

1

–

3

), 3.51 (d, J = 6.5 Hz, 2H, methylene

0.8 equiv. 0.197 g), and DIPEA (2.5 equiv., 0.33 g) were added at room

protons), 2.77 (t, J = 7.5 Hz, 2H, methylene protons), 1.38 (s, 9H, Boc

temperature followed by the general procedure as described above.

protons).

4

.1.7.3. tert-butyl (2-((2-(5-chloro-1H-indol-3-yl)ethyl)amino)-2-oxoeth

yl)carbamate (11c). Into a stirring solution of compound tert-butox-

ycarbonyl)glycine (1 equiv. 0.175 g) in THF (20 mL), EDCI.HCl (1.5

equiv., 0.29 g), HOBt (2.5 equiv., 0.37 g), 5-chlorotryptamine (0.8

equiv. 0.184 g), and DIPEA (2.5 equiv., 0.33 g) were added at room

temperature followed by the general procedure as described above.

1

White solid powder, (0.158 g, 66% yield). H NMR (DMSO‑d

6

, 500

MHz): δ 10.65 (bs, 1H

–

OH), 9.44 (bs, 1H, –NH), 8.15 (t, J = 6.0 Hz 1H,

–

NH), 8.03 (t, J = 6.0 Hz, 1H, –NH), 7.35 (d, J = 16.0 Hz, 1H, Ar-H),

7

.22 (d, J = 9.0 Hz, 1H, Ar-H), 7.13 (dd, J

1

= 6.5 Hz, J = 2.0 Hz,

2

H, Ar-H), 7.03–7.01 (m, 2H, Ar-H), 6.80 (d, J = 8.0 Hz, 1H, Ar-H), 6.71

(

dd, J

1

= 6.5 Hz, J

CH ), 3.81 (s, 5H, FA

), 3.36 (t, J = 6.0 Hz, 2H,

, 126 MHz): δ 169.2, 166.1, 153.4, 148.7, 148.2,

2

= 2.0 Hz, 1H, Ar-H), 6.58 (d, J = 16.0 Hz, 1H,

–

CH

–

OCH and CH -), 3.76 (s, 3H,

–

3

2

White solid powder, yield (0.254 g, 75%). ¹H NMR (CDCl

3

, 500

OCH

3

–

CH

2

-), 2.80 (t, J = 7.5 Hz, 2H, CH -

–

1

MHz): δ 11.03 (bs, 1H, –NH), 7.86 (t, J = 5.0 Hz, 1H, –NH), 7.34 (d, J =

). C NMR (DMSO‑d

6

8

1

.5 Hz, 1H, Ar-H), 7.23 (bs, 1H, Ar-H), 7.05 (dd, J

1

= 8.5 Hz, J

2

= 2.0 Hz,

139.7, 131.8, 127.9, 126.8, 123.8, 122.0, 119.2, 116.1, 112.4, 111.9,

H, Ar-H), 6.93 (t, J = 6.0 Hz, 1H, Ar-H), 3.50 (d, J = 6.0 Hz, 2H,

111.5, 111.3, 100.5, 55.9, 55.8, 42.7, 41.9, 25.7. ESI-HRMS for

methylene protons), 3.31 (t, J = 6.5 Hz, 2H, methylene protons), 2.78 (t,

J = 7.5 Hz, 2H, methylene protons), 1.38 (s, 9H, Boc protons).

C

23

H

25

N

3

O

5

. (M+H)⁺calcd. 424.1872, found 424.1874.

4

.1.11. (E)-N-(2-((2-(5-chloro-1H-indol-3-yl)ethyl)amino)-2-oxoethyl)-

4

.1.8. General procedure for synthesis of intermediate 12a-12c

3-(4-hydroxy-3-methoxyphenyl)acrylamide (13c)

Into a stirring solution of compound 11a-11c (0.3 g, 1.0 mmol) in

Into a stirring solution of compound FA (0.3 g, 1.54 equiv.) in THF

(20 mL), EDCI.HCl (2.31 equiv., 0.35 g), HOBt (3.86 equiv., 0.52 g), 12c

(0.8 equiv. 0.201 g), and DIPEA (2.5 equiv., 0.33 g) were added at room

temperature followed by the general procedure as described above.

methanol (20 mL), ether HCl (3 mL) was added slowly at room tem-

perature, and the reaction mixture was stirred for 12 h. The progress of

the reaction was monitored by TLC. After completion of the reaction, the

Et

converted to free amine by treatment with Na

extracted with EtOAc (2 × 30 mL). The combined organic layers were

dried over Na SO , and concentrated in vacuo to provide the pure free

2

O (10 mL) was added to provide HCl salt. The HCl salt was further

2

CO solution and

3

2

4

amine product 12a-12c, which were subjected to next reaction without

further puriﬁcation.

1

White solid powder, (0.167 g, 69% yield). H NMR (DMSO‑d

6

, 500

MHz): δ 11.03 (s, 1H

–

OH), 9.52 (s, 1H, –NH), 8.18 (t, J = 6.0 Hz, 1H,

–NH), 8.04 (t, J = 6.0 Hz, 1H, –NH), 7.59 (d, J = 2.0 Hz, 1H, Ar-H),

7.37–7.34 (m, 2H, Ar-H), 7.25 (d, J = 2.0 Hz, 1H, Ar-H), 7.05 (dd, J

= 6.0 Hz, J = 2.0 Hz, 2H, Ar-H), 6.80 (d, J = 8.0 Hz, 1H, Ar-H), 6.57 (d,

J = 16.0 Hz, 1H, Ar-H), 3.81 (s, 5H,

4

.1.9. (E)-N-(2-((2-(1H-indol-3-yl)ethyl)amino)-2-oxoethyl)-3-(4-

hydroxy-3-methoxyphenyl)acrylamide (13a)

Into a stirring solution of compound FA (0.3 g, 1.54 equiv.) in THF

1

(

20 mL), EDCI.HCl (2.31 equiv., 0.35 g), HOBt (3.86 equiv., 0.52 g), 12a

2

–

3

OCH and methylene protons),

0.8 equiv. 0.173 g), and DIPEA (2.5 equiv., 0.33 g) were added at room

temperature followed by the general procedure as described above. The

crude material was puriﬁed by column chromatography using silica gel

3.34–3.32 (m, 4H merged with DMSO‑d

6

methylene protons), 2.80 (t, J

= 7.5 Hz, 2H, methylene protons). ¹³C NMR (DMSO‑d

6

, 126 MHz): δ

(

100–200 mesh size) as a stationary phase with help of EtOAc:hexane

9:1) as mobile phase.

169.3, 166.2, 148.8, 148.3, 139.8, 135.1, 128.8, 126.8, 125.1, 123.4,

122.0, 121.2, 119.2, 117.9, 116.1, 113.3, 112.1, 111.3, 67.4, 56, 42.7,

2

5.4. ESI-HRMS for C21

28.1376.

H

23ClN

2

O

3

. (M+H)⁺calcd. 428.1377, found

4

1

8

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

4

.2. Biology

for 20 min with propidium iodide (50

μ

L of 20 M), and the ﬂuorescence

μ

was measured using Synergy™ HT, Bio-Tek Instruments, Inc. microplate

reader. Wavelengths of excitation and emission were 535 and 595 nm,

respectively. Each assay was repeated at least, at two different times.

.2.1. Inhibition of cholinesterase (hAChE and eqBChE)

AChE (hAChE, from human erythrocytes, CAS No. 9000–81-1),

butyrylcholinesterase (eqBChE, from equine serum, CAS No. 9001–08-

5

′

), 5, 5 -dithiobis- 2-nitrobenzoic acid (DTNB-Ellman’s reagent, CAS No.

9–78-3), acetylthiocholine iodide (ATCI, CAS No. 1866–15-5), butyr-

4.2.5. Molecular docking

6

The 3D crystal structures of hAChE in complex with DPZ (PDB ID-

ylthiocholine iodide (BTCI, CAS No. 1866–16-6) and FA (CAS No.

1

4EY7) and hBChE in complex with tacrine (PDB ID-4BDS) were

retrieved from the Brookhaven protein data bank.¹

3,42

261170–81-3) were purchased form Sigma Aldrich. Donepezil hydro-

Protein Prepa-

chloride (DPZ.HCl) (Sigma Aldrich, CAS No. 1261170–81-3) was used as

reference. The ChE inhibitory property of all the synthesized derivatives

was performed using the Ellman colorimetric method with slight

modiﬁcation.²⁶Brieﬂy, 50 µL of hAChE (0.022 U/mL) or 50 µL of BChE

ration Wizard of Schr o¨ dinger software package (Schr o¨ dinger, LLC, New

York, NY) was used to prepare both proteins. This step includes removal

of water beyond 5 Å from the HET group, addition of missing hydrogen,

optimization of orientations of hydroxyl and amino groups, assignment

of right bond orders, and the determination of ionization of amino acids

using ProtAssign utility. The resulting structures were further subjected

to restrained minimization with cutoff root mean square deviation

(RMSD) of 0.3 Å. Finally, the prepared complexes were further used for

molecular docking and MD simulation study. All the small molecules

were drawn using 2D sketcher and were subjected to ligand preparation

using the LigPrep module of Schr o¨ dinger software package

(Schr o¨ dinger, LLC, New York, NY). The different possible ionization

states for ligands were generated at the physiological pH (7.0 ± 2), and

OPLS3 force ﬁeld was used to minimize the ligands. Two different grids,

one for the AChE (4EY7) and other for the BChE (4BDS) were prepared

using the centroid of bound co-crystallized ligands. The both grids were

standardized by re-docking the co-crystallized ligands i.e. DPZ and

tacrine to AChE and BChE, respectively. Finally, docking of all ligands

was performed by the Glide module of the Schr o¨ dinger software package

(Schr o¨ dinger, LLC, New York, NY) using standard operating procedures

with the extra precision (XP) protocol.⁴³

(

0.06 U/mL) was incubated with 10 µL of test or standard compound at

◦

3

7 C for 30 min. Further, 30

μ

L of the substrate, viz. ATCI (1.5 mM) or

BTCI (15 mM) was added, and the solution was incubated for additional

0 min. Finally, 160 µL of 0.15 mM DTNB (for hAChE) or 160 µL of 1.5

3

mM DTNB (for eqBChE) were added into it and the absorbance was

measured at 415 nm wavelength using Synergy™ HT and Epoch 2, Bio-

Tek Instruments, Inc (microplate reader). Results are expressed as the

mean ± SD of at least two/three different experiments performed in

triplicate. The inhibition percent was calculated by the following

expression:%inhibition = [(Ac ꢀ Ai)/Ac]X100. Where Ai and Ac are the

absorbance obtained for AChE or BChE in the presence and absence of

inhibitors.

4

.2.2. DPPH antioxidant assay

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical method is an

antioxidant assay based on reduction of DPPH to a yellow-colored

diphenylpicrylhydrazine. 2,2-Diphenyl-1-picrylhydrazyl (DPPH; CAS

No. 1898–66-4) was purchased from Sigma-Aldrich, India. All the assays

were carried out in extra pure methanol. Brieﬂy, 75 µL of different

concentrations of test compounds were added to a 96 well plate. To this

4.2.6. Molecular dynamics simulations

All-atom MD simulations were performed using the Desmond-v6.1

module of Schr o¨ dinger Software Package (Schr o¨ dinger, LLC, New

7

5 µL of DPPH (200 µM) solution was added. Finally, 96 well microplate

◦

44

was incubated at 37 C for 25 min on thermomixer with moderate

shaking at 300 rpm. The absorbance was measured at 517 nm wave-

length using Epoch 2 microplate reader, Bio-Tek Instruments, Inc. The

activity of FA, a recognized antioxidant was used as a standard. The

radical-scavenging activity was determined by the equation. %Inhibition

York, NY). The system builder panel was used to prepare the initial

systems for MD simulations. The apo-AChE, apo-BChE and all docked

complexes were placed in a cubic box of 1.0 nm size. The boxes were

solvated with TIP3P water models and charged systems were neutralized

+

¡

45

using counter ions (Na or Cl ions). An ionic strength of 0.15 M was

+

¡

maintained by adding Na and Cl ions to all the systems. Further, the

solvated systems were minimized and equilibrated under NPT ensemble

using the default protocol of Desmond. It includes a total of nine stages,

=

[(absorbance of controlꢀ absorbance of test)absorbance of control]

100.

×

All the experiments were performed in duplicate or triplicate.

among which there are two minimization and four short simulations

equilibration phase) steps.⁴

6,47

All minimized and equilibrated systems

(

4

.2.3. Enzyme kinetic studies

were subjected to MD run with periodic boundary conditions in NPT

4

8

The enzyme kinetic studies were performed in order to investigate

ensemble using OPLS_2005 force ﬁeld parameter for 100 ns. During

the simulation, the pressure (1 atm) and temperature (300 K) of the

systems were maintained by Martyna–Tobias–Klein barostat and Nose-

the binding kinetics of ligand with the enzyme (AChE and BChE). The

kinetics study was performed as per the previously reported method.²⁶

Brieﬂy, 50 µL of enzyme (hAChE or eqBuChE) was preincubated with the

4

9,50

–Hoover Chain thermostat, respectively.

The binding energy be-

◦

test compounds at 37 C for 30 min, followed by the addition of sub-

tween the AChE/BChE and ligands (3k & 13b) was calculated using the

inbuilt script thermal_mmgbsa.py.⁵¹The binding energy was calculated

from the last 25 ns of trajectory at an interval of 50 ps for all four (AChE-

3k, AChE-13b, BChE-3k, and BChE-13b) systems. The solvent accessi-

bility surface area (SASA) of AChE/BChE in the presence of different

ligands was also calculated using the script binding_sasa.py.

strate ATCI (0.5, 1.0, 1.5, 2.0 and 2.5 mM), or BTCI (5, 10, 15, 20 and 25

′

mM). Finally, 160 µL of 5,5 -dithiobis- 2-nitrobenzoic acid (DTNB-Ell-

man’s reagent, 0.15 mM (for AChE) or 1.5 mM (for BChE) was added

into it, and the absorbance was recorded at 415 nm using Epoch 2

microplate reader, Bio-Tek Instruments, Inc. Each experiment was per-

formed in duplicate.

4

.2.7. Metal chelation

4

.2.4. PAS binding assay

The metal chelation was monitored spectrophotometrically using

The afﬁnity of compound for the PAS of AChE was examined by

Epoch 2 microplate reader, Bio-Tek Instruments, Inc. Iron (III) chloride

propidium iodide, a known PAS-speciﬁc ligand of AChE. Propidium

iodide (CAS No. 25535–16-4) was purchased from Sigma-Aldrich. All

the assays were carried out in Tris-HCl buffer (pH 8.0) and the mea-

hexahydrate (FeCl ⋅6H O, CAS No. 10025–77-1) was purchased form

3

2

Sigma Aldrich (USA). The stock solution of compound 13b (600 M) was

μ

prepared in extra pure methanol. Compound 13b was then diluted in

methanol to make a 300 M solution and the solution was kept aside for

absorbance reading on next day from 300 to 700 nm. FeCl ⋅6H O was

surements were carried out in 200 µL solution volume in 96-well plates.

μ

◦

Brieﬂy, 75

μ

L of 5 U/ml hAChE was incubated, for 6 h at 25 C, with a 75

3

2

μ

L solution of the compound (50, 20, 10 and 5 µM ﬁnal concentration) or

DPZ as positive control, respectively. Finally, the sample were incubated

next dissolved in methanol to make a 600

μ

M light orange solution. The

M)] were mixed

two solutions [13b (600

μ

M) and FeCl ⋅6H O (600

3

2

μ

1

9

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

together in equal volume, which gave a light-yellow solution. Finally,

corticosterone (CAS No. 50–22-6) were purchased from TCI chemicals.

The PAMPA kit (Cat # PAMPA 096, Lot No. BJ07A12), including the

donor microplate (PVDF membrane, pore size 0.45 mm) and acceptor

microplates, were purchased from Bioassay systems Pvt. Ltd. Porcine

brain lipid (PBL, CAS No. 86088–88-2, Lot No. 141101P-100MG-A-079)

and n-dodecane (CAS No. 112–40-3) were purchased from Avanti Polar

Lipids and Avra Synthesis, respectively. All the experiments were carried

out in phosphate buffer saline (PBS, pH 7.4).

◦

the sample solution was incubated at 25 C for 24 h on thermomixer

with vigorous shaking at 1000 rpm. After 24 h, the solution color

changed from yellow to blue, and was subjected to UV scan. Finally, the

pH of the solution was maintained to 7.4 by adding base diisopropyle-

thylamine (DIPEA) (diluted with water) which produced a deep-blue

color which was followed by UV spectra scan. Each concentration was

assayed in duplicate.

The molar ratio method was performed in order determine the

stoichiometry of the 13b-Fe (III) complex by titrating the solution of

4.2.10.1. Preparation of the stock solution. The stock solution (10 mM)

compound 13b with FeCl

3

solution (at different molar ratio). The UV

of test or standard compounds were prepared in molecular biology grade

spectra were recorded at room temperature. The absorbance spectrum

represents the formation of new absorption band, indicating that com-

pound can interact with Fe (III) ions. Thereafter, the mole fraction of Fe

DMSO (for poor solubility, MeOH was used). 500

compound was prepared via dissolving 25 L of stock solution of test

compounds in 475 L PBS (pH 7.4). Similarly, 500 L of 500 M of the

standard control was prepared in PBS (pH 7.4). 200 L of equilibrium

standard of each of the test and standard compound was prepared via

dissolving 80 L of 500 M of test and standard compound with 120

PBS. 250 L blank control was prepared via dissolving 5 L DMSO in

245 L PBS. Set aside the equilibrium standard and blank control for

analysis the next day.

μ

L of 500

μ

M test

μ

(

III) and absorbance of 13b was plotted to obtain stoichiometry of

μ

complex.

μ

μL

4

.2.8. Assessment of cell viability by MTT assay

μ

The cytotoxicity of 13b on n2a cells was determined by MTT assay

μ

following our earlier publications. Brieﬂy, Neuro2a cells were seeded at

4

a concentration of 1 × 10 cells/well in 96 well plates and were incu-

bated overnight for adherence. The compound, 13b was added in N2a

4

.2.10.2. Procedure. The acceptor plate well was ﬁlled with 300

PBS buffer. The ﬁlter membrane of the donor (top) plate was coated with

L of BBB-speciﬁc lipid solution prepared via dissolving 20 mg of PBL

in 600 L of n-dodecane. Be careful not to puncture the membrane with

the tip of pipette. After 30 min, add 200 L of each of the test (500 M)

and standard compound (500 M) to duplicate wells in donor plate. The

μ

L of

cells at a concentration of 20, 10, 5, 2.5, 1, 0.1 and 0.01 µM in triplicates

◦

and incubated for 24 h at 5% CO

2

at 37 C followed by MTT assay.

5

μ

4

.2.9. Effect of compound 13b on H

2 2

O induced cell death

μ

The H -induced cellular damage model of neurodegenerative

2 2

O

μ

disorder in n2a cell line was established to evaluate the protective effect

donor plate was kept carefully into the acceptor plate wells like a

4

of 13b. The cells were seeded at a density of 1 × 10 cells/well into 96-

◦

sandwich and incubated for 18 h at 37 C in a moistened sealed bag to

well plates and incubated for overnight. Next, the cells were treated with

prevent evaporation. Carefully remove donor plate and concentrations

of drug in the acceptor, equilibrium standard for each test compounds,

and permeability control along with blank control were determined by

UV spectroscopy (Epoch 2 micrplate reader, BioTek, USA) via placing

1

μ

2 2

00, 200, 400, 600, and 800 M of H O for 24 h, and control wells were

cultured without any treatment. Following the treatment, 3-(4,5-dime-

thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was

◦

added and incubated at 37 C for 4 h. Next, the solution in the wells was

1

00 L solution to the well of 96-well micro test plate. Read absorbance

μ

replaced by the extraction buffer (0.04 N HCl in isopropanol), followed

spectrum from 200 nm to 500 nm in 10 nm interval to determine the

absorbance of the test compound. Each experiment was performed in

◦

by additional incubation at 37 C for 1 h in dark and the optical densities

was measured at 570 nm using multimode microplate reader (Inﬁnite®

duplicate. P

1- OD /OD

is the absorbance of equilibrium standard, and if the experiment is

e

was calculated using the following expression: Pe = C × –In

2

00 PRO, TECAN.). Each experiment was repeated at least in triplicate.

The MTT assay was also used to determine the protective effect of 13b in

-induced cell damage based on the evaluation of cell viability. The

(

A

E

) cm/s, where OD

A

= absorbance of Acceptor solution, OD

E

¡

6

2 2

H O

running for 18 hrs, then C = 7.72 × 10 . The results are given as the

mean ± SD.

results were also conﬁrmed by microscopic observation. The n2a cells

4

were seeded at a density of 1 × 10 cells/well into 96-well plates and

incubated for overnight. Next, the cells were grown in the presence or

4

.2.11. In-Vivo experiments

absence of different concentrations (1, 2.5, 5, 10 or 20

μ

M) of 13b for 24

h, then further co-incubated with 600 M H for another 24 h. The

μ

2 2

O

.2.11.1. Animals. 3 months old male C57BL6 mice, 25–30 gm each,

cells in the control group were cultured without any treatment. Each

experiment was repeated at least in triplicate. Post-treatment, MTT was

were obtained from the Breeding unit of small animal Facility and

acclimatized in an experimental small animal facility for 5 days (12 h

◦

added to all of the cells seeded wells and incubated at 37 C for 4 h

_l_i_g_h_t_/_d_a_r_k_c_y_c_l_e_,_t_e_m_p_e_r_a_t_u_r_e₂₅_±₂◦C) at National Institute of

followed by a general procedure described above. Percent cell viability

was calculated by the formula

Immunology, New Delhi). Mice were supplied with commercial food

granules and sterilized water unless otherwise stated. All the methods

Mean OD value of experim ental sam ple (treated)

Percentage of viability

=

× 100

Mean OD value of experim ental control (untreated)

and procedures performed in this study were following the guidelines

approved (IAEC# 565/20) by the Institutional Animal ethical commit-

tee of National institute of Immunology, New Delhi.

4

.2.10. Parallel artiﬁcial membrane permeability assay

The blood–brain barrier penetration assay of the developed mole-

4.2.11.2. Drugs and chemicals. The compound 13b was suspended in

20 µL DMSO and volume makes up by the phosphate-buffered saline (pH

7.4), and donepezil hydrochloride & scopolamine hydrochloride was

cules was performed using the parallel artiﬁcial membrane permeation

assay (PAMPA) described by Di et al. Five commercial drugs such as

testosterone (CAS No. 58–22-0), hydrocortisone (CAS No. 50–23-7), and

2

0

Y.P. Singh et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116385

dissolved in water to carry out the in-vivo studies.

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