New coumarin-benzotriazole based hybrid molecules as inhibitors of acetylcholinesterase and amyloid aggregation

Article abstract of DOI:10.1016/j.bmcl.2020.127477

A novel series of triazole tethered coumarin-benzotriazole hybrids based on donepezil skeleton has been designed and synthesized as multifunctional agents for the treatment of Alzheimer's disease (AD). Among the synthesized compounds 13b showed most potent acetylcholinesterase (AChE) inhibition (IC₅₀ = 0.059 μΜ) with mixed type inhibition scenario. Structure-activity relationship revealed that three-carbon alkyl chain connecting coumarin and triazole is well tolerable for inhibitory potential. Hybrids obtained from 4-hydroxycoumarin and 1-benzotriazole were most potent AChE inhibitors. The inhibitory potential of all compounds against butyrylcholinesterase was also evaluated but all showed negligible activity suggesting that the hybrid molecules are selective AChE inhibitors. 13b (most potent AChE inhibitor) also showed copper-induced Aβ_1-42 aggregation inhibition (34.26% at 50 μΜ) and chelating properties for metal ions (Cu²⁺, Fe²⁺, and Zn²⁺) involved in AD pathogenesis along with DNA protective potential against degenerative actions of [rad]OH radicals. Molecular modelling studies confirm the potential of 13b in blocking both PAS and CAS of AChE. In addition, interactions of 13b with Aβ_1-42 monomer are also streamlined. Therefore, hybrid 13b can act as an effective hit lead molecule for further development of selective AChE inhibitors as multifunctional anti-Alzheimer's agents.

Full text of DOI:10.1016/j.bmcl.2020.127477

Journal Pre-proofs
New coumarin-benzotriazole based hybrid molecules as inhibitors of acetyl‐
cholinesterase and amyloid aggregation
Atamjit Singh, Sahil Sharma, Saroj Arora, Shivani Attri, Prabhjot Kaur,
Harmandeep Kaur Gulati, Kavita Bhagat, Nitish Kumar, Harbinder Singh,
Jatinder Vir Singh, Preet Mohinder Singh Bedi
PII:
S0960-894X(20)30588-6
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127477
BMCL 127477
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
12 May 2020
1 August 2020
5 August 2020
Please cite this article as: Singh, A., Sharma, S., Arora, S., Attri, S., Kaur, P., Kaur Gulati, H., Bhagat, K.,
Kumar, N., Singh, H., Vir Singh, J., Mohinder Singh Bedi, P., New coumarin-benzotriazole based hybrid
molecules as inhibitors of acetylcholinesterase and amyloid aggregation, Bioorganic & Medicinal Chemistry
Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127477
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New coumarin-benzotriazole based hybrid molecules as inhibitors of acetylcholinesterase
and amyloid aggregation
Atamjit Singha, Sahil Sharmaa, Saroj Arorab, Shivani Attrib, Prabhjot Kaurb, Harmandeep Kaur
Gulatia, Kavita Bhagata, Nitish Kumara, Harbinder Singh*,a, Jatinder Vir Singh*,a, Preet Mohinder
Singh Bedi*,a
aDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab 143005,
India
bDepartment of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar,
Punjab 143005, India
Corresponding authors: 1) Dr. Harbinder Singh
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Email id: singh.harbinder40@gmail.com
2) Jatinder Vir Singh
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Email id: jatindervirsingh1@gmail.com
3) Dr. Preet Mohinder Singh Bedi
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Email id: bedi_preet@yahoo.com
1

Abstract
A novel series of triazole tethered coumarin-benzotriazole hybrids based on donepezil skeleton
has been designed and synthesized as multifunctional agents for the treatment of Alzheimer’s
disease (AD). Among the synthesized compounds 13b showed most potent acetylcholinesterase
(AChE) inhibition (IC50 = 0.059 μΜ) with mixed type inhibition scenario. Structure-activity
relationship revealed that three-carbon alkyl chain connecting coumarin and triazole is well
tolerable for inhibitory potential. Hybrids obtained from 4-hydroxycoumarin and 1-benzotriazole
were most potent AChE inhibitors. The inhibitory potential of all compounds against
butyrylcholinesterase was also evaluated but all showed negligible activity suggesting that the
hybrid molecules are selective AChE inhibitors. 13b (most potent AChE inhibitor) also showed
copper-induced Aβ1-42 aggregation inhibition (34.26 % at 50 μΜ) and chelating properties for
metal ions (Cu2+, Fe2+, and Zn2+) involved in AD pathogenesis along with DNA protective potential
against degenerative actions of ∙OH radicals. Molecular modelling studies confirm the potential of
13b in blocking both PAS and CAS of AChE. In addition, interactions of 13b with Aβ1-42 monomer
are also streamlined. Therefore, hybrid 13b can act as an effective hit lead molecule for further
development of selective AChE inhibitors as multifunctional anti-Alzheimer’s agents.
Keywords: Alzheimer’s disease; coumarin-benzotriazole hybrids; acetylcholinesterase inhibitors;
amyloid aggregation inhibitor; molecular docking.
2

Alzheimer’s disease (AD) is the most prevalent (70 to 80 % of all dementia forms) type of
dementia in elderly people (usually 65 years and above) all around the globe characterized by
progressive memory loss, impaired linguistic efficacy, mood disturbances, behavioural changes
and cognitive impairments which finally carries toward death. It is a neurodegenerative disorder
that starts slowly and gets worse over time with lost body functions that make the life of AD patient
miserable and pose heavy monetary as well as a social burden to family and society.1 Thus, it is a
global emergency to control this disease without any delay.
Though the origin of AD is still ambiguous lifestyle, genetic and environmental factors are
suggested to be involved in the onset as well as the progression of this disease.2 Generation of
toxic amyloid-beta (Aβ) protein and its extracellular plaques, as well as neurofibrillary tangles,
originated from hyperphosphorylated tau proteins were considered as the two major pathological
hallmarks of AD and no standard treatment was available for this disease until Davies and Maloney
(in 1973) proposed cholinergic hypothesis.3 Following the same, four cholinesterases
(acetylcholinesterase and butyrylcholinesterase) inhibitors tacrine, donepezil, rivastigmine and
galantamine were approved by the FDA. These drugs offer only palliative care and unable to freeze
or reverse disease progression.4 One NMDA receptor antagonist (memantine) was also approved
but it doesn’t offer much relief similar to cholinesterase inhibitors.5 In last 20 years, apart from
cholinergic, amyloid and tau protein cascade, various novel AD mechanisms have also been
explored by the researchers that include inflammation, oxidative stress and deregulation of bio-
metals, etc. suggesting multi-targeted nature of this disease.6 Along with the hydrolysis of
acetylcholine (ACh), acetylcholinesterase enzyme (AChE) is found to promote Aβ aggregation
that interacts with its peripheral anionic site (PAS).7 Metal hemostasis in the brain of AD patient
has been observed to be disturbed by extracellular elevation of zinc and copper along with the
intracellular accumulation of iron. Copper and zinc were observed to bind with Aβ and promote
reactive oxygen species (ROS) by inducing its aggregation.8 Oxidative stress resulted from the
abundance of redox-sensitive metals (Cu, Fe, and Zn) found to induce proteolysis of amyloid
precursor protein via upregulating β-secretase that leads to an increase in Aβ. Iron was found to
delay ordered aggregation of Aβ that promotes its toxicity. Chelation of iron was observed to
provide protection from Aβ toxicity.9
3

Coumarins (benzopyran-2-ones) offer a potential class of compounds that possess a wide range of
therapeutic properties (Fig. 1). Numerous coumarin derivatives like warfarin as an anticoagulant
(1), esculin as vasoprotective (2), scopoletin as anti-inflammatory (3), hymecromone as an
antispasmodic (4), etc. are already in clinical practice. Besides this, coumarin derivatives are
reported to have a diverse range of biological activities like antioxidant, anticancer, antidepressant,
antinociceptive, hepatoprotective, antibacterial, antifungal, antiviral, antidiabetic and anti-
Alzheimer’s. Especially in Alzheimer’s drug development, multifunctional coumarin hybrids (5-
9) have also been reported with promising results.10
OH
OH
OH
O
O
O
O
O
O
O
O
OH
OH
O
OH
OH
3
4
O
O
O
O
O
N
OH OH
2
O
1
O
O
S
S
N
N
+NH
N
O
N
HN
N
O
N
O
O
NH
HN
O
O
O
O
O
Cl
N
O
N
5
6
7
8
9
Fig. 1. Various coumarin derivatives in clinical practice (1-4) and multifunctional anti-
Alzheimer’s agents (5-9).
Benzotriazole is a versatile nucleus in the field of medicinal chemistry and has long known metal
chelating properties. In past two decades, a wide range of biologically active molecules has been
designed by researchers across the globe in which benzotriazole is acting as a core nucleus itself
or modulating the activity of other biologically active pharmacophores (Fig. 2). Benzotriazole
derivatives designed so far has shown potential biological activities such as antibacterial (10),
4

antituberculosis (11), antifungal (12), antiviral (13), antiprotozoal (14), anticancer (15), antiemetic
(16), antioxidant (17), and anti-inflammatory (18), etc.11
N
N
O
N
H
Cl
O
O
N
N
N
H
NO2
N
N
N
Cl
N
N
CN
N
S
O2N
10
11
12
H
N
Br
Br
O
O
N
N
N
O
H
N
N
N
Br
N
S
N
N
N
N
N
N
O
N
Br
15
14
16
13
O
S
N
N
N
N
CH3
N
N
N
N
N
O
NH
HO
N
18
O
17
Fig. 2. Various biologically active benzotriazole derivatives (10-18).
Molecular hybridization is a well-established stratagem that involves combining two or more
pharmacophores with or without any linker and provides a single hybrid molecule having
properties of all combined bioactive substances. Molecular hybridization remains successful in
giving various potential candidates to clinical trials within the last 30 years that includes
MCB3837, Ro 23-9424, CBR-2092, and TD-1792 etc.12,13 Triazole is a well-known bioactive
moiety that has been successfully utilized for the development of potential bioactive compounds
(Fig. 3) including multifunctional anti-Alzheimer’s agents (23-28).14-17 Molecular docking studies
of these multifunctional agents suggest that triazole more fondly interacts with acetylcholine
binding site of AChE and impart higher selectivity toward AChE over butyrylcholinesterase
(BuChE).18 Triazole linker appropriately separating two active pharmacophores, was observed for
introducing anti-Aβ aggregation properties in the molecule.19
5

N
N
N
O
O
O
O
O
N
N
O
5
O
O
N
O
O
N
H
23
24
Cl
O
N
O
N
N
N
N
N
N
N
NH
O
O
N
F
O
O
25
O
O
O
26
O
O
O
O
N
N
N N
O
O
N
N
N
O
N
N
27
28
Fig. 3. Triazole linked hybrid molecules with anti-Alzheimer’s activity (23-26).
Peripheral anionic site (PAS) of AChE interacts with Aβ fragment and promotes its aggregation
while catalytic anionic site (CAS) selectively accepts ACh. Donepezil is the only drug that
interacts simultaneously with both PAS and CAS of AChE enzyme, which may be the possible
reason for its dual AChE as well as Aβ aggregation inhibitory potential. These properties makes
donepezil, a perfect template for Alzheimer’s drug development.20 The favourable skeleton of
donepezil and strong pharmacophoric features of coumarin and benzotriazole along with the
successful history of molecular hybridization with triazole linker provides a solid base for utilizing
all of them in designing novel multifunctional anti-Alzheimer agents (Fig. 4). Thus considering
the pressing need of potential multifunctional agents for the management of AD, novel triazole
linked coumarin-benzotriazole hybrids based on donepezil skeleton template has been designed
by taking account of Lipinski rule of 5 and ADME properties (Supplementary Table S1 and S2),
synthesized via click chemistry approach and evaluated for their anti-Alzheimer potential.
6

Interacts with CAS responsible
for AChE inhibition
Interacts with PAS responsible
for A aggregation inhibition
O
O
O
H
H
N
(Donepezil)
Skeleton offeres five
Optimization sites
H
B
H
C
N
D
E
A
O
O
O
O
O
O
H
H
H
H
H
H
N
N
N
N
N
N
O
N
N
n
n = 2,3,4,5,6
Interacts with PAS and has antioxidant
as well as neuroprotective action
Bioactive nucelus separated via optimum
linker enhance multitargeting efficacy
especially A aggregation inhibition and also
enhance selectivity toward AChE over BuChE
Potent metal chelator
and versatile nucleus
N
Hybridization
N
O
O
O
N
n = 2-6
N
n
N
O
O
X =
Y =
N
N
N
N
N
X
O
Y
N
N
TARGETED COMPOUNDS
O
Fig. 4. Design strategy.
Designed hybrid molecules were synthesized via a series of chemical reactions (Scheme 1).
Coumarins i.e. 4-OH coumarin (1) and 7-OH coumarin (4) were reacted with various
dibromoalkanes in the presence of K2CO3 at 25 °C in DMF yielded alkylated coumarins (2a-e and
5a-e). Alkylated coumarins were further treated with NaN3 at 25 °C in DMF to form N-azidoalkyl
coumarins (3a-e and 6a-e). Simultaneously, OH-benzotriazole (7) was reacted with propargyl
bromide in the presence of K2CO3 at 25 °C in DMF to get 1-(prop-2-yn-1-yloxy)-1H-benzotriazole
(7a). Propargylation of 1H-benzotriazole (8) was done under similar conditions as used for OH-
benzotriazole resulted in 1-(prop-2-yn-1-yl)-1H-benzotriazole (8a) with 61 % yield and 2-(prop-
2-yn-1-yl)-2H-benzotriazole (8b) with 18 % yield. Compound PBB (8a) was characterized by the
appearance of two doublets at 8.09-8.07 ppm and 7.73-7.71 ppm for two individual flagpole
protons present on the 4th and 7th position of benzene ring (Scheme 1), while in PBC (8b), these
flagpole protons are appeared as merged signals in a multiplet at 7.91-7.95 ppm as these protons
1
bear in same electromagnetic environment (refer to H NMR of 8b in supplementary data).
7

Obtained propargylated benzotriazoles were treated with prepared N-azidoalkyl coumarins in the
presence of the catalytic amount of copper sulfate and reducing agent sodium ascorbate at 25 °C
in DMF yielded various triazole linked coumarin-benzotriazole hybrids.21
HO
O
O
O
O
N
N
N
N
N
N
1
4
8
7
H
OH
OH
c
a
a
c
61%
18%
3
N
4
4
7
N3
N2
O
O
O
Br
O
n
O
O
N
5
6
5
6
N
N2
N
O
N
N
7
8a
1
1
2a-e
5a-e
7a
8b
n
Br
b
b
N
N
N
O
O
O
O
N3
n
O
O
O
N
n
N
d
N
N
O
O
7a
6a-e O
3a-e
9a-e
n
N3
N
O
N
N
n
d
N
N
O
O
8a
13a-e
N
O
O
N
N
n
N
N
d
N
O
8b
11a-e
N
O
N
N
N
N
N
O
O
O
d
n
7a
12a-e
N
N
N N
N
N
O
O
O
O
O
O
d
n
8a
10a-e
N
N
N
N
N
n
N
d
n = 2, 3, 4, 5, 6
8b
14a-e
8

Scheme 1. Synthesis of triazole linked coumarin-benzotriazole hybrids. Reagents and conditions:
(a) dibromoalkane, K2CO3, DMF, 0.5 to 2 h, stir, rt; (b) NaN3, DMF, 1 to 2 h, stir, rt; (c) propargyl
bromide, K2CO3, DMF, 0.5 to 4 h, stir, rt; (d) sodium ascorbate, CuSO4, DMF, 0.5 to 8 h, rt.
As in the hydrolysis process of acetylcholine, 80 % role is performed by AChE enzyme, while
BuChE performs only a secondary role. Therefore, all the synthesized compounds were screened
for their inhibitory potential against both AChE and BuChE enzymes using Ellman’s method and
compared with standard anticholinesterase drug donepezil. Data were obtained from triplicate
experiments and presented as the mean  SD of three independent experiments (Table 1).
Table 1. AChE, BuChE and Aβ1-42 aggregation inhibition of synthesized compounds in comparison to Donepezil.
N
n
N
N
X
Y
Comp.
X
Y
n
eeAChEb
eqBuChEc
IC50 (μΜ)a
Aβ1-42
aggregation
inhibition (%)d
16.22  1.28
≤10
IC50 (μΜ)a
9a
9b
2
3
4
5
6
2
3
4
5
6
2
3
≥10
≥10
≥10
≥10
≥10
≥10
≥10
≥10
≥10
≥10
≥10
≥10
4.322  0.056
1.324  0.012
3.233  0.027
≥10
N
N
O
O
N
9c
≤10
≤10
O
O
9d
9e
≥10
≤10
10a
10b
10c
10d
10e
11a
11b
2.112  0.029
2.924  0.016
4.329  0.031
5.429  0.038
≥10
29.36  1.69
15.86  1.48
11.36  1.89
≤10
O
O
O
N
N
N
≤10
≤10
1.890  0.023
1.670  0.014
≤10
9

O
O
O
N
N
11c
11d
4
5
6
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
≥10
≥10
≤10
≤10
≤10
2.093  0.016
4.810  0.045
6.233  0.019
2.012  0.016
1.978  0.026
≥10
N
11e
≥10
12a
≥10
11.38  1.23
≤10
N
N
O
O
O
12b
≥10
N
12c
≥10
≤10
O
12d
≥10
≥10
≤10
12e
≥10
≥10
≤10
13a
≥10
0.097  0.045
0.059  0.006
0.234  0.061
1.141  0.012
5.422  0.032
1.176  0.023
1.156  0.013
5.436  0.056
≥10
36.68  2.17
34.26  1.97
22.36  1.81
11.36  0.77
≤10
O
O
O
N
N
13b
≥10
N
13c
≥10
13d
≥10
13e
≥10
14a
≥10
≤10
O
O
O
N
14b
≥10
≤10
N
N
14c
≥10
≤10
14d
≥10
≤10
14e
≥10
≥10
≤10
Donepezil
Curcumin
0.039  0.097
8.416  0.628
62.26  2.92
aIC50: 50 % inhibitory concentration (mean  SD of three individual experiments). bAChE obtained
from electric eel. cBuChE obtained from equine serum. dPercentage Aβ1-42 inhibition estimated by
using Thioflavin T based fluorescence assay at a sample concentration of 50 µM.
In the case of anti-AChE activity, compound 13b emerged as the most potent one making 4-
hydroxycoumarin linked with 1-benzotriazole via 1,2,3-triazole, the most suitable architect for
AChE inhibition. Data from biological activity suggest that chain length of three carbons (13b) is
most suitable for AChE inhibition and as the number of carbons between triazole and coumarin
moiety increases, anti-AChE activity decreases accordingly (IC50 = 0.059 to 5.422 μΜ from 13b
to 13e respectively). A several-fold decrease in the anti-AChE activity was observed when 4-
10

hydroxycoumarin was replaced with 7-hydroxycoumarin (as X) (compare 13b with 10b, 11b with
14b) suggesting that carbonyl group of 4-hydroxycoumarin lies toward triazole linker is suitable
for anti-AChE activity as in donepezil (carbonyl group of indanone lies towards the piperidine
moiety: Fig. 4). The chain length between triazole (linker) and coumarin moieties greatly influence
the inhibitory potential of the compounds. As the chain length increases, the inhibitory potential
decreases. The chain length of three carbons was found most preferable for inhibitory activity.
Most of the compounds with long-chain (n > 4) were found inactive against the enzyme (see 9d,
9e, and 10e, 12c to 12e, 14d and 14e). 1-benzotriazole as Y and 4-hydroxycoumarin as X was
found best combination that exhibited potent AChE inhibitory potential (13a-13e) amongst which
compound 13b was found to be endowed with most prominent AChE inhibition with the IC50 value
of 0.059 µM which is comparable to that of donepezil (IC50 = 0.039 µM). While 1-hydroxy
benzotriazole as Y and 7-hydroxycoumarin as X was an unsuitable combination for AChE
inhibitory activity (12a-12e). Compounds consisting of 2-benzotriazole as Y and 4-
hydroxycoumarin as X showed moderate inhibition of the enzyme (11a-11e). Therefore, overall
preference of AChE inhibitory activity for coumarins is 4-hydroxycoumarin > 7-
hydroxycoumarin, for benzotriazoles it is 1-benzotriazole > 2-benzotriazole > 1-hydroxy
benzotriazole, and for carbon chain length it is 3 > 2 > 4 > 5 > 6. All the compounds were also
evaluated for their inhibitory potential against butyrylcholinesterase (BuChE) enzyme in a similar
manner. None of the synthetic compounds showed inhibitory activity against the BuChE (IC50
above 10 μΜ). The inhibitory potential of most potent AChE inhibitor 13e was also tested at two
more concentrations (at 50 & 100 μΜ) against BuChE enzyme while no significant enhancement
was observed in the inhibition against BuChE which suggests the significance of these hybrid
compounds towards the selective inhibition of AChE enzyme. The compound showed most potent
anti-AChE inhibition (13b) was further subjected to enzyme kinetic studies and reciprocal
Lineweaver-Burk plot was plotted with three concentration levels. In reciprocal Lineweaver-Burk
plot, increase in the slope (deacreased Vmax) as well as intercept (higher Km) was observed with
increasing concentrations of 13b (Fig. 5). Intersection in 2nd quadrant of the reciprocal
Lineweaver-Burk plot suggest that 13b followed a mixed-type inhibitory pattern against AChE.
The inhibition constant (Ki) for 13b was calculated as 0.04167 nM.22
11

Fig. 5. Overlaid Lineweaver-Burk plot derived from the kinetic study by using no and three
concentrations of 13b with varied substrate concentrations (0.025 - 0.0150 mM) depicting a mixed
type of inhibition by compound 13b.
Amyloid beta (Aβ1-40 and Aβ1-42) peptides originated from amyloid precursor protein plays an
important role in the pathogenesis of Alzheimer’s disease. Aβ1-42 has higher pathogenicity as
compare to Aβ1-40 due to its higher tendency to form fibrillar aggregates that promote neuronal
degeneration. In addition, Copper is well known to promote amyloid-beta aggregation in amyloid
pathogenesis.23 Thus, inhibition of Aβ1-42 by an anti-Alzheimer’s agent is a potential advantage.
Considering this intention synthesized compounds were screened for their inhibitory action on
copper-mediated Aβ1-42 aggregation by employing Thioflavin T based fluorescence assay using
curcumin as reference anti-amyloidogenic agent. At the concentration of 50 μΜ, most of the
compounds showed percentage inhibition below 10 % except compounds bearing 1-benzotriazole
moiety either it was attached with 4-hydroxycoumarin or with 7-hydroxycoumarin respectively.
Percentage inhibition was up to 36.68 % (Table 1) which was much lower as compare standard
curcumin (62.26 %). 13b was able to inhibit Aβ1-42 up to 34.26 %.
Amyloid plaques in AD brains are generally designated as ‘metallic sinks’ due to higher
concentrations of Fe, Cu and Zn ions in them. These metal ions trigger Fenton-type process and
are attributed to higher oxidative stress in AD.24 In vitro studies demonstrated the encouraging
effect of these metals ions in Aβ toxicity through the generation of ROS in the presence of
dioxygen.25 Thus anti-Alzheimer’s agents with metal chelating properties will be a significant
advantage. UV-Vis spectra of 13b showed an increase in absorption intensity and detectable blue
12

shifts on the addition of Cu2+, Fe2+ and Zn2+ while minor changes with Mg2+ were observed (Fig.
6A), proving the complex formation ablility of 13b with Cu2+, Fe2+ and Zn2+.26 Stoichiometry of
13b-Cu2+ complex was determined by employing a molar ratio method by titrating a solution of
13b with gradually increasing amounts of CuCl2. Absorption vs Cu2+ mole fraction plot
(Supplementary Fig. S1: 6B) indicates the 2:1 stoichiometry of the 13b-Cu2+ complex suggesting
the possible efficacy of 13b in the reduction of Cu2+ mediated Aβ aggregation and ROS generation.
A
2.0
1.9
1.8
13b + Cu2+
1.7
13b + Fe2+
13b + Zn2+
1.6
1.5
1.4
13b + Mg2+
13b
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390
400
Wavelength (nm)
Fig. 6A. Overlaid UV spectra of 13b (5 μM) itself alone and in the presence of Cu2+, Fe2+, Zn2+,
and Mg2+ (5 μM).
Ageing and Aβ induced DNA damage have significant contribution to the progression of AD.27
Increased DNA fragmentation and nicking have been detected in AD brains. Various ROS
especially H2O2 mediated oxidative stress trigger DNA damage in AD.28 Thus 13b was further
evaluated for its protective effect against Fenton-type oxidative cleavage of pBR322 plasmid DNA.
pBR322 plasmid DNA exists in three major forms namely, supercoiled circular form (I), open
13

circular (II), and linear form (III). In results, 13b was able to prevent the conversion of the
supercoiled circular form (I) of DNA to open circular (II) DNA and then linear form (III) DNA.
Thus 13b was able to protect DNA from degradative action of ∙OH radicals (Fig. 7).
1
2
3
4
5
6
7
8
Form II (Open circular DNA)
Form III (Linear DNA)
Form I (Supercoiled DNA)
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
pBR322 plasmid DNA (190 μg/mL)
3 μL
3 μL
3 μL
3 μL
3 μL
3 μL
3 μL
3 μL
Fenton’s reagent
Quercetin (1 mM)
13b (0.2 mM)
13b (0.4 mM)
13b (0.6 mM)
13b (0.8 mM)
13b (1 mM)
-
-
-
-
-
-
-
7.5 μL 7.5 μL 7.5 μL 7.5 μL 7.5 μL 7.5 μL 7.5 μL
-
-
-
-
-
-
15 μL
-
-
-
-
-
-
-
-
-
-
15 μL
-
-
-
-
-
-
-
-
15 μL
-
-
-
-
-
-
15 μL
-
15 μL
-
-
-
-
-
15 μL
Fig. 7: DNA protection properties of 13b; In lane 4 to 8 showing the capability of 13b in
preventing both open circular and linear cuts.
Cell toxicity study was performed to evaluate the safety profile of 13b on SH-SY5Y cells using
donepezil as reference. After 24 hours of incubation of 13b or donepezil with SH-SY5Y cells, cell
viability was evaluated using MTT assay. Both 13b and donepezil showed negligible toxicity in
concentrations ranging from 0.001 to 100 μM, indicating the safety of 13b in AD treatment (Fig.
8).
14

Fig. 8. Cytotoxicity study of 13b in comparison with donepezil on SH-SY5Y cells. SH-SY5Y cells
were incubated for 24 hours with increasing concentrations of both 13b and donepezil ranging
from 0.001 to 100 μM. Results are displayed as the percentage of viable cells with respect to
untreated control cells. Values are expressed as the mean  SD obtained from three individual
experiments.
Molecular modelling studies were performed to get insight into various molecular interactions
responsible for the modulation of inhibitory activity of the most potent AChE inhibitor (13b). For
that purpose, the X-ray crystallographic structure of recombinant human acetylcholinesterase
complexed with donepezil (PDB entry: 4EY7; resolution, 2.3509 Å), was employed.29 Accuracy
of docking protocol was validated by docking co-crystallized ligand donepezil into its binding site.
The program was capable to reproduce best fit confirmation of donepezil in chain B with root
mean square deviation (RMSD) value of 0.965, indicating the reliability of docking protocol. After
that 13b was docked into donepezil binding site, and best pose with -18.5818 score having ∆G
value of -38 kJ/mol was selected for discussion (Fig. 9). The overall binding mode of 13b with
residues of donepezil binding site suggest that compound is properly positioned in the cavity and
well stabilized by various electrostatic interactions. Major interactions with AChE include van der
Waals, -σ, - stacked, - T-shaped and conventional hydrogen bond interaction. The coumarin
moiety has perfectly arranged itself in a cavity formed by two polar (Tyr72 and Tyr341) and two
hydrophobic (Trp286 and Phe295) residues. The backbone -NH of Phe295 make conventional
hydrogen bond interaction with the carbonyl oxygen atom of coumarin (H-bond acceptor; d =
2.802 Å). Similar interaction of -NH in Phe295 backbone has been observed with ketonic oxygen
15

of indanone moiety in donepezil but with little higher distance (H-bond acceptor; d = 2.986 Å)
which highlight the stronghold of coumarin over indanone in the peripheral active site (PAS)
A
B
E
Trp286
Phe297
Phe295
Tyr341
Tyr124
Tyr124
Gly121
Tyr338
Ser125
Trp86
His447
C
D
F
A
B
C
PAS
E
D
CAS
Fig. 9. (A) Acetylcholinesterase (AChE) enzyme complexed with donepezil (PDB entry: 4EY7;
Resolution: 2.3509 Å); (B) 13b docked on the binding site of donepezil; (C) Donepezil positioned
in the pocket made by active site residues shown using aromatic surface interactions; (D) 13b
positioned in the pocket made by active site residues shown using aromatic surface interactions;
(E) 3D view of interactions of 13b with residues of donepezil binding site in AChE; (f) 2D view
of interactions of 13b with residues of donepezil binding site in AChE.
Both rings A and B of coumarin are showing - stacked interactions with indole ring in Trp286
backbone. Tyr72 and Tyr341 make van der Waals interactions with rings A and B of coumarin.
OH on Tyr124 backbone also showing conventional hydrogen bond interaction (H-bond acceptor;
d = 2.042 Å) with oxygen present between coumarin and three-carbon alkyl chain seems to
promote the efficiency of 13b for PAS. Triazole nucleus (Ring C) which acts as a linker between
coumarin and benzotriazole makes - T-shaped interaction with hydroxyphenyl backbone of
Tyr337 (-Orbitals to -Orbitals; d = 5.634 Å) whereas donepezil which makes -Cation
interaction with hydroxyphenyl backbone of Tyr337 with little shorter distance (-Cation; d =
4.665 Å). Additional - T-shaped interaction of triazole nucleus has been observed with
16

imidazole nucleus of His447 (-Orbitals to -Orbitals; d = 5.349 Å) which is absent on donepezil
making triazole linker an interesting replacement of piperidine moiety in donepezil skeleton for
AChE inhibition. The benzotriazole moiety is well-positioned in the cavity formed by five
hydrophobic (Gly121, Trp86, Phe338, Phe297, and Gly120) and three polar residues (Ser125,
Tyr124 and Tyr133). Nitrogen from ring D makes a conventional hydrogen bond with -OH in
Tyr133 backbone. Indole nucleus in Trp86 backbone makes similar but little closer crisscross type
- stacked interaction (To ring D; -Orbitals to -Orbitals; d = 4.435 Å) with benzotriazole
nucleus as the donepezil makes with its phenyl (To ring D; -Orbitals; d = 5.051 Å). Additional
-σ interaction (-CH- to -Orbital; d = 5.349 Å) of ring E form benzotriazole with -CH2- of Trp86
backbone has been observed which was absent in donepezil to Trp86. Apart from this, Gly121,
Ser125, Phe338, Phe297, Gly120 capture benzotriazole moiety through van der Walls interactions.
The overall study suggests that 13b is well decorated with small, rigid and planar groups making
the finest scaffold which is able to satisfy the necessary pharmacophoric requirements for AChE
inhibition.
Aβ1-42 aggregation inhibition and good interaction of 13b toward PAS observed during docking in
donepezil binding site of AChE further encourage us to study the interaction of 13b with Aβ1-42
monomer. For that purpose, the NMR elucidated structure of amyloid-beta (Aβ1-42) monomer
(PDB entry: 1IYT) was employed.30 Sphere module of LeadIT was employed for defining binding
site using a radius of 6.50 Å. Previously prepared structure of 13b was allowed to dock in prepared
monomer amino acids. Best pose with -6.7274 was selected for discussion (Fig. 10).
17

C
A
Val124
B
Asp23
Phe20
Phe19
Lys16
Gln15
Val12
Fig. 10. (A) Amyloid beta (Aβ1-42) monomer (PDB entry: 1IYT) complexed with 13b; (B) 3D
view of interactions of 13b with residues of Aβ1-42; (f) 2D view of interactions of 13b with residues
of Aβ1-42
.
The coumarin moiety of 13b positioned itself in the cavity formed by two polar (Gln15 and Lys16)
and one hydrophobic residue (Phe19). Gln15 shows conventional hydrogen bond interaction (H-
bond acceptor; d = 1.633 Å) with oxygen (-O-) present in the ring B of coumarin through -NH2 of
its backbone. Another conventional hydrogen bond interaction (H-bond acceptor; d = 1.896 Å) is
shown by a carbonyl oxygen atom of coumarin with core amino group of Lys16. These hydrogen
bonds with very short distances showing the affinity of 13b toward Aβ1-42. Phenyl ring of Phe20
backbone showing - stacked interactions with both ring A and B of coumarin. Free nitrogen
atoms of triazole moiety also showed conventional hydrogen bond interaction (H-bond acceptor;
d = 2.376 Å) with Lys16 through amine group in its backbone. Carbon hydrogen bond interaction
is also observed between Val12 and the carbonyl oxygen atom of coumarin through the core
hydrogen of Val12. Nitrogen in ring D of benzotriazole makes -lone pair interaction (lone pair to
-Orbital; d = 2.912 Å) with Phe20 through the phenyl group in its backbone. Asp23 showing van
der Walls interactions with both coumarin and benzotriazole moieties via Phe19 (H-bond acceptor;
d = 1.833 Å) and Phe20 (H-bond acceptor; d = 2.662 Å) by making conventional hydrogen bond
18

interactions with them. Aβ1-42 are susceptible to form β-pleated “hairpin” like architects that are
aggregated into fibrous form, and stabilized by hydrophobic interactions and salt bridge due to
interactions between Asp23 and Lys28 residues in them. Docking study suggests the possible
interaction of 13b with Asp 23 of Aβ1-42 monomer while strong and short distance interactions
with other residues can certainly change the conformation of Aβ1-42 monomer.
In summary, this study involved the rational design of triazole tethered coumarin-
benzotriazole hybrid molecules based on donepezil skeleton, their synthesis, and evaluation as
multifunctional agents against AD. Compounds were synthesized using click chemistry approach
and characterized by 1H, 13C NMR and elemental analysis. Among all synthesized compounds 13b
emerged as the most potent eeAChE inhibitor with mixed type of inhibitory pattern. Structure-
activity relationship revealed that distance of three-carbon alkyl chain between coumarin and
triazole linker is well tolerable for AChE inhibition. The combination of 4-hydroxycoumarin and
1-benzotriazole linked through triazole with three carbon chains was found the most suitable
combination for AChE inhibition. The insensitivity of these hybrids towards BuChE inhibition
suggesting that the compounds are selective AChE inhibitors. 13b inhibit copper-induced Aβ1-42
aggregation and have chelating properties for metal ions (Cu2+, Fe2+, and Zn2+) involved in AD
pathogenesis. DNA nicking assay confirms the ability of 13b to protect DNA from degenerative
actions of ∙OH radicals. Various binding interactions with hAChE justify the potential of 13b in
blocking both PAS and CAS. Interactions of 13b with Aβ1-42 monomer are also streamlined.
Besides having such potential multifunctional actions, coumarin and benzotriazole in 13b provide
considerable space for further improving the biological potential of the molecule and thus
providing a hit lead for further development of safer and potent selective AChE inhibitors as
multifunctional anti-Alzheimer’s agents.
Conflict of interest
The authors confirm that this article content has no conflicts of interest.
Acknowledgement
Authors are grateful to the University Grants Commission for providing funds under National
Fellowship for Other Backward Classes (NFOBC) to AS, Rajiv Gandhi National Fellowship
(RGNF) to HS, Council of Scientific and Industrial Research (CSIR: 02(0319)17/EMR-II) to JVS,
19

Department of Science and Technology for DST-Purse to HKG, WOS-A to KB and DST-FIST.
The authors are also thankful to Guru Nanak Dev University, Amritsar for providing various
facilities to carry out the research work.
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13
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 Design and synthesis of new coumarin-benzotriazole based molecular hybrids
 Ellman’s method of evaluation demonstrated selective acetylcholinesterase inhibition
 Most potent compound showed inhibition against copper-induced Aβ1-42 aggregation
 Potent compound have DNA protective properties against degenerative actions of ∙OH radicals
 Molecular modelling studies with acetylcholinesterase enzyme and Aβ1-42 monomer
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