Synthesis, biological evaluation and molecular dynamic simulations of novel Benzofuran-tetrazole derivatives as potential agents against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2018.11.005

A series of novel Benzofuran-tetrazole derivatives were successfully synthesised by integrating multicomponent Ugi-azide reaction with the molecular hybridization approach. Interestingly, a number of synthesized derivatives (5c, 5d, 5i, 5l, 5q and 5s) exhibited significant reduction of aggregation of “human” amyloid beta peptide, expressing on transgenic Caenorhabditis elegans (C. elegans) strain CL4176. Further, in silico docking results have evidenced the exquisite interaction of active compounds with the help of TcAChE–E2020 complex. These findings underscore the potential of these hybrids as lead molecules against Alzheimers's disease.

Full text of DOI:10.1016/j.bmcl.2018.11.005

Accepted Manuscript
Synthesis, biological evaluation and molecular dynamic simulations of novel-
Benzofuran-tetrazole derivatives as potential agents against Alzheimer’s disease
Pragati Kushwaha, Soobiya Fatima, Akanksha Upadhyay, Sampa Gupta, Sudha
Bhagwati, Tanvi Baghel, M.I. Siddiqi, Aamir Nazir, Koneni V. Sashidhara
PII:
S0960-894X(18)30861-8
https://doi.org/10.1016/j.bmcl.2018.11.005
BMCL 26116
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 April 2018
25 October 2018
6 November 2018
Please cite this article as: Kushwaha, P., Fatima, S., Upadhyay, A., Gupta, S., Bhagwati, S., Baghel, T., Siddiqi,
M.I., Nazir, A., Sashidhara, K.V., Synthesis, biological evaluation and molecular dynamic simulations of
novelBenzofuran-tetrazole derivatives as potential agents against Alzheimer’s disease, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.005
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Synthesis, biological evaluation and molecular dynamic simulations of
novelBenzofuran-tetrazole derivatives as potential agents against
Alzheimer’s disease
Pragati Kushwahaa, Soobiya Fatimab,# Akanksha Upadhyaya, Sampa Guptaa, Sudha
,
Bhagwatic,Tanvi Baghelb, M.I.Siddiqic, Aamir Nazirb,#, Koneni V. Sashidharaa, # *
b
aMedicinal and Process Chemistry Division, Division of Toxicology and Experimental Medicine,cMolecular
#
and Structural Biology Division, Academy of Scientific and Innovative Research (AcSIR) CSIR-Central Drug
Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, India
Abstract
A series of novel Benzofuran-tetrazole derivatives were successfully synthesised by
integrating multicomponent Ugi-azide reaction with the molecular hybridization approach.
Interestingly, a number of synthesized derivatives (5c, 5d, 5i, 5l, 5q and 5s) exhibited
significant reduction of aggregation of “human” amyloid beta peptide, expressing on
transgenic Caenorhabditis elegans (C. elegans) strain CL4176. Further, in silico docking
results have evidenced the exquisite interaction of active compounds with the help of
TcAChE–E2020 complex. These findings underscore the potential of these hybrids as lead
molecules against Alzheimers's disease.
Keywords: Benzofuran-tetrazole derivatives, molecular hybridization,Amyloid-beta
aggregation, TcAChE–E2020 complex.
*Corresponding author. Tel: +91 9919317940; fax: +91-522-2771942.
E-mail address: kv_sashidhara@cdri.res.in (K.V. Sashidhara)

According to data provided by the World health organization, an estimated 524 million
people were aged 65 or older i.e. 8% of the world's total population and these data are
expected to be nearly triple by the year 2050 due to a combination of declining fertility and
increasing life expectancy.1 The hapless result of this trend is the concomitant rise in the
number of people agonized by age-related neurodegenerative disorders. Today,
neurodegenerative diseases are the most frequent cause of death and disability worldwide.2
Alzheimer’s disease (AD), is a chronic, fatal, and most common neurodegenerative disease,
which is clinically characterized by progressive memory loss, a decline in language skills,
and progressive deficits in different cognitive domains. As per a report of the Alzheimer’s
Association (U.S.), morbidity and mortality rates of patients suffering with AD are high,
especially for elderly population over the age of 60.3 The consistent neuropathologic indicator
of the disorder is deposition of aggregated protein breakdown products, amyloid-β(Aβ)
plaques and neurofibrillary tangles, generally noted on post-mortem brain examination.
Although the primary cause of AD is still speculative due to complex etiology, Aβ plaques
are thought to be predominantly responsible for the devastating clinical effects of the
disorder.4 Some other hypotheses also been proposed to understand the pathophysiology of
AD and facilitate drug discovery programs, including low levels of acetylcholine (ACh),
aggregation of Tau protein, oxidative stress, and the accumulation of
biometals.5Acetylcholinesterase also known as AChE or acetylhydrolase, is the primary
cholinesterase in the body which catalyzes the hydrolysis of acetylcholine to acetate ion and
choline.Thus, the principal approach to cure AD has focused on acetylcholinesterase
inhibitors (AChEI) that increase the brain ACh levels. However, to date, the therapeutic
options for AD treatment are limited to four approved acetylcholinesterase (AChE) inhibitors,
tacrine (TC), donepezil, rivastigmine, and galantamine.6 However, these commercial drugs
for AD treatment are unable to meet the market demand. They ameliorate only cognition and
the degree of dementia in AD patients and do not reverse the succession of AD.7 Moreover,
high incidence of side effects including hepatotoxicity has been reported after the
administration of these drugs.8 Thus, it is necessary to develop novel anti-AD drugs that are
able to act as far upstream as possible, in the neurodegenerative cascade.
As the part of our continuous efforts in drug discovery and development, we describe herein
the synthesis and anti-Alzheimer activity of the pyrazole containing benzofuran-tetrazole
hybrids (Fig. 1). Benzofurans represent an important class of compounds found in versatile
building blocks of biologically important compounds.9-12 Further, a literature survey based on
the framework reveals that the benzofuran bearing moieties display potential anti-Alzheimer

activity by inhibiting AChE.13-15 On the other hand, tetrazole pharmacophore has also been
identified as a novel structural motif that inhibited cholinesterases.16-17 In addition, pyrazole
derivatives have the unique ability to reduce the tau and amyloid β dual aggregation.18-19
Hybrid molecules which contain multiple structural units of different nature are usually
endowed with enhanced biological activities.20-23 Inspired by the concept, we have designed
and synthesized a novel series of compounds that have benzofuran, tetrazole and pyrazole
entities in one frame and evaluated them for their anti-Alzheimer activity. This report
constitute the first combination of these three pharmacophores into a novel scaffold and their
potential anti-Alzheimer's activity.
O
N
O
O
O
O
O
O
O
O
SKF-6434614a
O
MDR-133914b
Benzofuran-chalcone hybrid
Previous lead13
R2
O
N
NH
N
H3C
CN
N
R1
O
N
N
NC
NH
N
N
R3
R4
O
N
CH3
O
N
N
H
Benzofuran-1,3,4-oxadiazole
derivative15
N
N
R5
Tetrazole linked
tetrahydronicotinonitrile16
Designed scaffold
O
O
N
N
HN
N
O
NH2
N
HN
N
H
N
O
N
OCH3
NH
N
F
Pyrano [2,3-c] pyrazole18
N
Escitalopram tetrazole17
O
Pyrazole containing curcumin
derivative19
Figure 1. Designing of Benzofuran-tetrazole hybrids.
The detailed synthetic route for the preparation of target and intermediate compounds is
outlined in Scheme 1. Initially, o-alkynation of salicyladehyde and 2-hydroxy acetophenone

with chloroacetone in the presence of K2CO3 furnished 2-acetyl benzofuran (2a-b) via
intramolecular cyclocondensation reaction. The compounds 3a-y were synthesized by the
reaction of acetyl benzofuran with different phenyl hydrazines in ethanol and catalytic
amount of acetic acid.24 These hydrazone intermediates (3a-y) were then engaged in
Vilsmeier-Haack reaction resulting in the formation of the intermediate aldehydic compounds
4a-y. Finally, these benzofuran-pyrazole aldehydes (4a-y) were subjected to Ugi azide
reaction to give the desired hybrids 5a-y.25 The atom economical synthesis of target
compounds was achieved by operationally simple, metal free, multicomponent reaction. All
1
the synthesized compounds were characterized using H NMR, 13C NMR and ESI-MS
(Supporting Information).
R2
R1
O
R1
R1
Cl
O
N
NH
PhNHNH2,AcOH
ethanol,rt,3h
O
K2CO3,ACN
reflux, 2h
O
O
OH
3a-y
1a-b
2a-b
POCl3,DMF
0-5oC,CHCl3
R1
H
R2
H
H
H
R3
R4
H
H
H
H
H
H
H
H
H
H
H
H
R5
tBu
3a,4a,5a
3b,4b,5b
3c,4c,5c
3d,4d,5d
3e,4e,5e
3f,4f,5f
3g,4g,5g
3h,4h,5h
3i,4i,5i
R2
CH3
CH3
CH3
CH3
CH3 Cl
CH3 Cl
CH3
CH3
H
tBu
R1
OCH3
CH3
OCH3
Et
tBu
N
N
tBu
tBu
O
H
H
tBu
OHC
4a-y
iPr
tBu
H
H
H
H
F
OCH3
F
tBu
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
tBu
tBu
3j,4j,5j
3k,4k,5k
3l,4l,5l
R3R4PhNH2, TMSN3, R4NC,
MeOH, rt, 6h
Cl
Br
tBu
CH3
tBu
R2
3m,4m,5m
3n,4n,5n
3o,4o,5o
3p,4p,5p
3q,4q,5q
3r,4r,5r
CH3 CH3
H
tBu
F
Me
H
iPr
Me
H
H
H
F
tBu
tBu
R1
tBu
CH3
tBu
N
N
R3
R4
H
tBu
CH3 CH3
CF3
CH3 CF3
F
tBu
O
N
tBu
3s,4s,5s
3t,4t,5t
N
H
Br
Br
H
H
H
N
tBu
H
N
N
R5
3u,4u,5u
3v,4v,5v
3w,4w,5w
3x,4x,5x
3y,4y,5y
H
H
Cl
Cl
F
CH3
OCH3
CH3
cyclohexyl
cyclohexyl
tBu
tBu
tBu
5a-y
H
CH3
OCH3
CH3
H
H
Scheme 1.Synthesis of Benzofuran-tetrazole derivatives.
AD is characterized by the aggregation of β-amyloid peptide (Aβ). The transgenic C.
elegansstrain CL4176 exhibits temperature-inducible expression of humanβ-amyloid.This

strain has been developed to express β-amyloid specifically in the muscles, thus, elevated β-
amyloid in the worm which results in early paralysis makes a clear end point to screen the
effect of compound on β-amyloid aggregation.Thus, strain CL4176 has been chosen for the
experiment. In our preliminary screening, we were interested to search for those compounds
that were effectively delaying the paralysis rate in worms. To check the effect, these
compounds were mixed in the bacterial food diet OP50 (the uracil auxotroph of E. coli) and
were fed to worms in the working concentration of 1mM, and the percent inhibition of the
worms was plotted, by taking into account 50 worms of each group , in duplicate. Among the
screened compounds, compound 5c,5d, 5i, 5l, 5q and 5s were effectively regulating the
paralysis rate of worms, with figures of 67.1%, 46.7%, 56.5%, 42.1%, 45.39% and 63.81%
respectively, suggesting effective inhibition of β-amyloid toxicity (Fig. 2).Therefore we went
further with these six compounds and tested their efficacy.
Figure 2. Amyloid-beta induced paralysis assay in the CL4176 strain of C. elegans treated
with the indicated test compounds. Data are normalized with respect to control; *p<0.05,
**p<0.01.
To further strengthen our findings for inhibitory activity of these compounds, the microscopic
technique for visualizing amyloid-beta aggregation employing Thioflavin-S has been done.26
The worms that were exposed to 5c, 5d, 5i, 5l, 5q and 5s showed significant decrease in the
fluorescent intensity as compared to control OP50 which has been shown in Fig. 3. The

figure is representative of amyloid-beta aggregation for the worms fed with the compounds
along with the control OP50 and the positive control EGCG.
Figure 3.Thioflavin-S imaging for Amyloid-beta aggregation in transgenic strain CL4176
strain of C. elegans. OP50 was used as a control and EGCG ((-)-Epigallocatechin-3-gallate)
was used as positive control . The fluorescent intensity is compared with respect to control.
All images were obtained at 63x magnification.
As increasing cholinergic neurotransmission by inhibiting the enzyme acetylcholinesterase
(AChE) still represents one of the mainstream treatment option for AD, these compounds
were investigated for their probable role as acetylcholine esterase inhibitors and studied in the
worms employing Amplex-Red®. The worms that were exposed to 5c, 5d, 5i, 5l, 5q and 5s in
the concentration of 1mM were assayed for AChE activity on their worm extract. Findings
exhibited significant inhibition of AChE levels with p-values of 0.0461, 0.0360, 0.0175 and
0.0085 particularly with compounds 5c, 5d, 5i and 5q respectively (Fig. 4). Our results
suggested that 5q has the maximum potential against acetylcholine esterase. C.elegans has
four acetyl cholinesterase genes viz. ace-1, ace-2, ace-3 and ace-4.27Amongst them, ace-3
and ace-4 have minor role and a majority of around 95% of AChE activity is encoded by ace-
1 and ace-2.28 We speculate that these compounds interact with ace-1 and ace-2, thereby
inhibiting the AChE activity in the model organism C.elegans. As we know, AChEIs are
currently approved treatment for Alzheimer’s disease (AD) type dementia. A huge success is
witnessed in this regard as these agents describes a layout for the development of other
symptomatic cognitive enhancing agents.29 In line with this finding, we regard that our
compounds could play role in enhancing the memory too, beside lowering the β-amyloid

induced toxicity and inhibiting the AChE activity as evident in our results. The activity of
these compounds to inhibit the AChE activity would result in delayed breakdown of ACh and
prolong its endogenous activity as a neurotransmitter.30
Figure 4.Effect of test compounds on acetylcholinesterase activities, as measured by the
Amplex ® Red assay in transgenic strain CL2006 strain of C. elegans. OP50 was used as a
control. Data are normalized with respect to control. All experiments were performed in
triplicate; ns-non-significant, *p<0.05, **p<0.01.
To support the above experimental results, molecular docking studies were carried out. The
structure of Torpedo californica AChE (TcAChE) with E2020 (donepezil) (PDB id- 1EVE)
has been used extensively for docking studies of AChE inhibitors,31-32 which suggests that it
can be an useful model for evaluating new chemical entities in the drug discovery program
against AD. Thus, in the present study, we have used the TcAChE–E2020 complex for the
analysis of binding mode of compounds in the active site of TcAChE. Re-docking with
E2020 resulted in a conformation close to the co-crystal structure (Fig.5), with an RMSD of
0.88 Å, thus validating our docking method adopted in this study. Residues Trp84, Trp279,
Trp290, and Phe330 play main roles in the binding of E2020.31 Trp84 and Trp279 are
predominantly significant, as both can undergo a π- π stacking interaction with E2020.13 It
has been confirmed that E2020 is an active site gorge spanning AChE inhibitor, interfering
with both the catalytic active site (CAS) and peripheral active site (PAS) simultaneously.33
Interestingly, we observed strong non-covalent interactions (H-bond) between these inhibitor
compounds and target site of AChE, which were not present in E2020 inhibitor. We also
observed π- π stacking interactions between aromatic ring of compounds and residues of the
target site. The tetrazole and pyrazole rings increase the binding strength by contributing

Figure 5.A) Superimpose co-crystallized (orange) and docked conformation (red) of E2020
in the active site TcAChE. B) Interactions between co-crystallized E2020 and TcAChE active
site residues (yellow cylindrical pattern shows π-π stacking interactions)
through H-bonds and π- π stacking interactions to the protein-ligand complex respectively.
Indole ring of Trp279 appeared to undergo π- π stacking interactions with the pyrazole rings
of all the compounds at the entrance of the active site. In the compound 5c, we observed two
H-bonds, one between –OH group of Tyr121 and N-4 of tetrazole ring, another between –NH
group of Arg289 and OCH3group. We also observed π- π stacking interactions between six-
membered & five-membered rings of Trp279 and pyrazole ring. Due to strong H-bonds and
π- π stacking interactions, this compound shows less binding energy and high binding affinity
with the target site of AChE. In all the other five compounds π- π stacking interactions and
H-bond interactions stabilizes the binding poses and affinity. We have not observed any π- π
stacking interactions with Trp84 at the bottom site of the binding pocket due to the structural
variation from the E2020. However, we observed π-π stacking interactions and hydrogen
bonding to make these inhibitor-target complexes more stable. Residues Tyr121 and Trp279
belong to the PAS, and in the docking studies, we observed that the pyrazole moieties of all
these compounds are present in close proximity to these residues. The role of PAS residues in
the formation of amyloid fibrils has been reported.34 Fig. 6 shows the interactions between
the compounds and TcAChE active site residues. Hydrogen bonds are highlighted in green
dotted lines and π-π stacking interactions are shown as yellow lined cylindrical pattern. We

hypothesize that the presence of a tetrazole moiety increases the hydrogen bond interactions
and pyrazole moiety increases the stacking interactions, allowing a better positioning into the
active site of TcAChE.
a)
b)
c)
d)
e)
f)
Figure 6. Interactions between compounds and TcAChE active site residues a) 5c, b) 5d, c)
5i, d) 5l, e) 5q and f) 5s (Green dotted lines shows H-bonding, yellow cylindrical pattern
shows π-π stacking interactions)
The preceding molecular docking studies revealed that both tetrazole and pyrazole cores are
required to show the bonding and nonbonding interactions to fit into the active site of
TcAChE, which stabilizes E2020. Initially, all the hybrids were evaluated for Amyloid-β
toxicityand the result confirmed that 5c, 5d, 5i, 5l, 5q and 5s were the most potent
compounds in the series. On the basis ofAβ aggregation results and the diverse chemical
structures of synthesized compounds, a preliminary structure activity relationship (SAR) was
established. A summary of the SAR is depicted in a pictorial representation (Fig. 7).It was
found that the compounds showing >40% inhibition of the paralysis essentially contained
tert-butyl group (R5) attached with tetrazole moiety. Further, substitution in the benzofuran
ring (R1) did not affect the activity. The presence of H or Halogen atom at R2 was favoured
over other substitutions. On the other hand, presence of the smaller atom or group (F/CH3)
was preferred over bulkier groups at R3. Additionally, H-atom at R4 was found optimal.

H/X groups
favoured
Substitution
tolerated
R2
Smaller substitutions
enhances activity
R1
N
N
R3
R4
O
N
N
N
H
N
N
R5
R4 = H is
optimal
tBu group is
essential for activity
Figure 7. Pictorial representation of SAR studies.
In conclusion, we have synthesized and characterized a novel series of hybrids by using
pharmacophore hybridization approach. Among the series compounds 5c, 5d, 5i, 5l, 5q and
5s emerged as promising agents to decrease the disease effects in transgenic C. elegans
model of AD. Studies such as effects on reducing Aβ aggregation and AChE activity confirm
and reinforce the efficacy of these compounds as a new class of anti-Alzheimer agents.
Further studies to unravel the mechanism of action and structural optimization of these
hybrids are currently underway in our laboratory. It is intended that this communication will
help to design and develop a novel class of next generation anti-Alzheimer agents.
Acknowledgments
The authors are grateful to the Director, CDRI, Lucknow, India for his constant
encouragement in the drug development program, Dr. S. P. Singh for technical support, SAIF
for NMR, IR, and Mass spectral data. P.K., S.F., A.U., S.G., T.B., and S.B. are thankful to
UGC, CSIR, and ICMR New Delhi, India for financial support. This is CSIR-CDRI
communication number 49/2018/KVS.
Supplementary data

Details for synthesis and characterization of all compounds together with protocols for
biological materials and methods can be found in the supplementary data.
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Graphical abstract
Synthesis, biological evaluation and molecular dynamic simulations of
novel Benzofuran-tetrazole derivatives as potential agents against
Alzheimer’s disease
Pragati Kushwahaa, Soobiya Fatimab,# Akanksha Upadhyaya, Sampa Guptaa, Sudha
,
Bhagwatic, Tanvi Baghelb, M.I.Siddiqic, Aamir Nazirb,#, Koneni V. Sashidharaa, # *
c
aMedicinal and Process Chemistry Division, bDivision of Toxicology and Experimental Medicine, Molecular
#
and Structural Biology Division, Academy of Scientific and Innovative Research (AcSIR) CSIR-Central Drug
Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, India
*Corresponding author. Tel: +91 9919317940; fax: +91-522-2771942.
E-mail address: kv_sashidhara@cdri.res.in (K.V. Sashidhara)

Research Highlights
Synthesis, biological evaluation and molecular dynamic simulations of
novel Benzofuran-tetrazole derivatives as potential agents against
Alzheimer’s disease
Pragati Kushwahaa, Soobiya Fatimab,#
,
Akanksha Upadhyaya, Sampa Guptaa,
Sudha Bhagwatic, Tanvi Baghelb,
M.I.Siddiqic, Aamir Nazirb,#, Koneni V.
Sashidharaa, # *
c
aMedicinal and Process Chemistry Division, bDivision of Toxicology and Experimental Medicine, Molecular
#
and Structural Biology Division, Academy of Scientific and Innovative Research (AcSIR) CSIR-Central Drug
Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow 226031, India

*Corresponding author. Tel: +91 9919317940; fax: +91-522-2771942.
E-mail address: kv_sashidhara@cdri.res.in (K.V. Sashidhara)
 A series of benzofuran-tetrazole derivatives were synthesized as potent AChE
inhibitors.
 Several compounds exhibited significant neuroprotective and Aβ aggregation
inhibitory activities.
 Docking studies corroborated well with the anticholinesterase assay results.