Naphthalene-triazolopyrimidine hybrid compounds as potential multifunctional anti-Alzheimer's agents

Article abstract of DOI:10.1016/j.bmc.2019.06.004

In an attempt to construct potential anti-Alzheimer's agents Naphthalene-triazolopyrimidine hybrids were synthesized and screened in vitro against the two cholinesterases (ChE)s, amyloid β aggregation and for antioxidation activity. Single-crystal X-ray c

Full text of DOI:10.1016/j.bmc.2019.06.004

Accepted Manuscript
Naphthalene-triazolopyrimidine hybrid compounds as potential multifunctional
anti-Alzheimer’s agents
Tarana Umar, Siddharth Gusain, Md Kausar Raza, Shruti Shalini, Jitendra
Kumar, Manisha Tiwari, Nasimul Hoda
PII:
S0968-0896(19)30223-8
https://doi.org/10.1016/j.bmc.2019.06.004
BMC 14941
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 February 2019
29 May 2019
1 June 2019
Please cite this article as: Umar, T., Gusain, S., Raza, M.K., Shalini, S., Kumar, J., Tiwari, M., Hoda, N.,
Naphthalene-triazolopyrimidine hybrid compounds as potential multifunctional anti-Alzheimer’s agents,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.06.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bioorganic & Medicinal Chemistry
Naphthalene-triazolopyrimidine hybrid compounds as potential multifunctional anti-
Alzheimer’s agents
Tarana Umar a , Siddharth Gusain b , Md Kausar Raza c , Shruti Shalini b , Jitendra Kumar d , Manisha Tiwari
b,  and Nasimul Hoda a, 
a Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi 110025, India
b Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi 110007, India
c Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
d Department of Chemistry, Sardar Vallabhbhai Patel College, Bhabua, Kaimur- 821101, V. K. S. U., Ara, Bihar- 802301, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In an attempt to construct potential anti-Alzheimer’s agents Naphthalene-triazolopyrimidine
hybrids were synthesized and screened in vitro against the two cholinesterases (ChE)s, amyloid
β aggregation and for antioxidation activity. Single-crystal X-ray crystallography was utilized
for crystal structure determination of one of the compounds. In vitro study of compounds
revealed that most of the compounds are capable of inhibiting acetylcholinesterase and
Butyrylcholinesterase activity. Particularly, the compounds 4e and 4d exhibited IC50 values
ranging from 8.6 to 14 nM against AChE lower than the standard drug Donepezil (IC50 49 nM).
Best result was found for compound 4e with IC50 of 8.6 nM (for AChE) and 150 nM (for
BuChE). Selectivity upto that of Donepezil and even more was observed for 4a, 4c and 4h.
Investigation by electron microscopy, transmission electron microscopy and ThT fluorescence
assay unveils the fact that synthesized hybrids exhibit amyloid β self-aggregation inhibition. The
compounds 4i and 4j revealed highest inhibitory potential, 85.46% and 72.77% at 50 μM
respectively; above the standard Aβ disaggregating agent, Curcumin. Their antioxidation profile
was also analyzed. Studies from DPPH free radical scavenging assay and ORAC assay depicts
molecules to possess low antioxidation profile. Results suggest that triazolopyrimidines are
potential candidate for Acetylcholinesterase (AChE), Butyrylcholinesterase (BuChE), and
amyloid β aggregation inhibition. In silico ADMET profiling indicates drug-like properties with
a very low toxic influence. Such synthesized compounds provide a strong vision for further
development of potential anti-Alzheimer’s agents.
Available online
Keywords:
Amyloid β aggregation inhibition
Acetylcholinesterase inhibitor
Butyrylcholinesterase inhibitor
Naphthalene
Triazolopyrimidine
2009 Elsevier Ltd. All rights reserved.
1. Introduction
unsolved AD therapeutics, molecules with combinatorial mode of
actions such as AChE inhibition and BuChE inhibition,
Alzheimer’s disease (AD) is a state attributed to degeneration
of distinct brain regions essentially allied with cognition
phenomenon, such as the cortex and hippocampus. This
escalating multifaceted neurodegenerative disease is the common
cause of dementia. At present proper pharmacotherapy of this
disorder is unknown and it is only possible to attenuate some
symptoms of this disease as Donepezil do by enhancement of
cholinergic neurotransmission. Deficiency of acetylcholine,
Amyloid β aggregation, free radical formation and inflammation
are amongst the known Alzheimer’s factors. In answer to the
antioxidation, metal chelation and neuroprotection are emerging
and capable of efficiently reforming the natural course of AD.[1]
The best accepted interpretation of mechanism of AD
development is the cholinergic hypothesis that directly
contributes to cognitive decline [2]. Furthermore, drugs available
today support the old and original cholinergic hypothesis, thus
cholinesterase inhibitors are of prime focus. AChE inhibition
evolves
Parkinson’s
a
strategy for treatment of Alzheimer’s disease,
disease, senile dementia and ataxia.
———
 Corresponding author. Tel.: +0091-9910200655; fax: +0091-11-26985507; e-mail: nhoda@jmi.ac.in
 Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: mtiwari07@gmail.com

Scheme 1. Molecular strategy for the hybrids of triazolopyrimidine-naphthalene 4a-j obtained from modification pathway of
reference molecule [1]
AChE is a serine hydrolase. AChE have two main subsites of
active centre along with peripheral anionic site(s) which serves
binding of acetylcholine and other quartenary ligands acting as
uncompetitive inhibitors. Cleaving it into choline and acetic acid,
it ultimately inactivates acetylcholine neurotransmitter that
signals between the central nervous system and various parts of
the body. Primarily ACh gets hydrolysed via AChE, and BChE is
the second enzymes to catalyze the hydrolysis of cholinergic
neurotransmitters, but in case of AD affected brain, AChE
exhibits lower activity and BuChE becomes more active [3]. This
makes BChE an upcoming therapeutic target that can help to
lower AD cholinergic deficit [3-5]. AChE levels in progressed
brain of AD declines upto 55-67% of normal values while
BuChE level increases to 120% of normal values.[3,6] It’s a clear
indication of the critical role that BuChE plays for ACh
hydrolysis in late stages of AD. Since reduced cholinergic
neurotransmission is most visible cause of AD, potentiation of
cholinergic system may target treatment of this disease
pathophysiology. The AChE and BChE inhibitors respectively
can be devoted to other diseases also, including myasthenia
gravis, parasitic infections, some other dementias, obstipation,
glaucoma, or to antagonize muscle relaxation[7].
pathologies like tangle formation, oxidative damage, and
neuronal apoptosis in AD.[12] Oxidative stress marks one of the
preliminary incident in AD pathogenesis [13]. Its significance in
neuronal degradation is also supported by the oxidative stress and
free radical theory of aging. As a consequence, protection of
neurons against oxidative damage could capably ward off AD.
Triazolopyrimidines a subtype of purine analogues have been
the subject of intense regard to researchers owing to versatile
biological and clinical applications. They have been reported to
display
anticancer,
antimalarial,
anti-Alzheimer’s,
antiinflammatory, antifungal, antihypertensive, leishmanicidal
cardiac stimulant, antipyretic, antimicrobial, analgesic, potential
herbicidal, and anti-HBV activities [14]. Past biological records
of triazolopyrimidine derivatives are well evident from Trapidil
[15] and Cevipabulin [16]. In recent triazolpyrimdines have been
seen to alter AChE inhibition, amyloid β aggregation inhibition
and antiinflamation altogether, proving itself as potential anti-
Alzheimer’s agent [1]. They primarily interact with AChE
through π- π interaction with residues of PAS (Peripheral anionic
site) and potentially can be used as chemotherapeutic agents.
Previously from our research group the triazolopyrimidine
scaffold was reported as multifunctional agent for the treatment
of Alzheimer's disease [1], in present paper we report an
extended study [Scheme 1] of the design, synthesis, crystal
structure and evaluation of triazolopyrimidine-naphthalene
hybrid Cholinesterase inhibitors that could exhibit potentially
better applicable bioactivities upto nM range, with the inhibition
β-amyloid aggregation inhibition is another relevant target
besides anticholinergic activity, important in Alzheimer's disease.
Aggregates of some amyloidogenic proteins have a role to play in
different organisms and protein aggregation crucially acts in the
pathogenesis of a number of human diseases. AD is one such
disease, where the excess formation and accumulation of the
amyloid β peptide[8] are considered as early critical factor.
Amyloid β is formed naturally via metabolism of a neuronal
transmembrane protein, which is amyloid precursor protein
(APP).[9] Its most abundant form Aβ1-42 has a higher tendency to
assemble into a variety of oligomeric and fibrillar complexes
compared to Aβ1–40 peptide[10] and is predicted to be the
predominant portion of Aβ plaques in AD[11]. Ongoing
accumulation of these aggregates plays a part in downstream
of AChE, BuChE, antioxidant and amyloid
β inhibition
properties.
2. Results and discussion
2.1. Chemistry
The general strategy for synthesis of multifunctional Anti-
Alzheimer’s agents naphthalen-[1,2,4]triazolo[4,3-a]pyrimidine
hybrids (4a–d) and naphthalen-5,8-dihydro-[1,2,4]triazolo[4,3-

a]pyrimidine hybrids (4e–j) is illustrated in Scheme 1. This
strategy for the synthesis of potential molecule candidates with
AChE and BuChE inhibition, Aβ aggregation inhibition, and
antioxidation properties, devised construction of molecules
derived from previously known hit molecules possessing such
properties. Triazolopyrimidine derivatives synthesized by J.
Kumar et al. that were efficient up to nano molar range towards
AChE inhibition (Scheme 1) had displayed a multitargeted
profile with cholinesterase inhibition, amyloid β aggregation
inhibition and antioxidation activity.[1] We designed to
synthesize these derivatives via a two-step convenient procedure.
The synthesis of triazolopyrimidines 4a-d was performed as
depicted in Scheme 2. Firstly the required intermediates were
prepared by Claisen-Schmidt condensation of a naphthalene
based aromatic ketone and of appropriate aromatic aldehyde
under basic conditions (quantitative yield) [17]. Followed by
synthesis of final compounds which are assumed to proceed
through 1,2-addition of aminotriazole to the various substituted
3-phenyl-1-naphthalene-1-ylpropenones affording a hydrazine
which on intramolecular conjugate addition produces cyclization
to the triazolopyrimidine ring (30-80% yield) (Scheme 3). All
products were soluble in polar solvents and the recrystallization
was carried out and also, the compounds are stable in solid state
at room temperature.
1
Structures of final compounds were established by H NMR,
13C NMR and (EI) mass spectrometry and 4a was further
characterized by X-ray crystallography (supporting information
figures S1-S32). The spectral analysis favours the purity of
compounds. The ESI mass spectra of all molecules 4a-j in
methanol showed primarily the [M+H]+ and/or [M+Na]+ peak
according to the calculated m/z values (Figures S1−S10,
1
Supporting Information). H NMR for all the final compounds
showed common signals of core naphthalene present.
Scheme 2. Synthesis of triazolopyrimidine derivatives 4a-j. Reagents and conditions: (a) NaOH, MeOH, RT; (b) 3-Amino-1,2,4-
triazole, NaOH, MeOH, Reflux, 3hs; (c) 3-Amino-1,2,4-triazole, NaOH, MeOH, Reflux, 48 hs.

Scheme 3. Plausible mechanism for the formation of triazolopyrimidine derivatives. Step 1. Formation of hydrazone. Step 2.
Intramolecular conjugate addition.
Characteristic singlet for triazole CH appeared around δ 8.7. All
the aliphatic substituents in the aryl group appeared as expected.
The 13C spectra showed characteristic peaks of the triazole-
carbon. The similar peaks for naphthalene ring moiety appeared
in the range 149-125 ppm. The different aromatic tails attached
showed their respective peaks.
system. No hydrogen bonding is present for compound 4a,
although short contact bonding with adjacent atoms was seen.
2.3. Biological evaluation and Docking Study
2.3.1. Molecular Docking Study of 4d and 4e with AChE and
BuChE
Probable binding modes of synthesized compounds with AChE
active site were studied and Donepezil and Tacrine were used as
a reference drugs. The structure of AChE used herein was
derived from the Protein Data Bank (1EVE) [18, 19]. Docked
conformations were ranked according to docking scores. The
Position of compounds 4d and 4e with respect to binding site key
residues as well as the active site interactions found via docking
are shown in Fig. 2a and 2b. As seen from figure docked
molecules mainly possess π-π interactions with dotted lines. The
naphthyl group and 4-methoxybenzene of 4e Fig. 2(b) are
engaged in π-π stacking interaction with residues Trp84 and
Trp279 (distance 3.8 Å and 4.5 Å respectively). In Fig. 2(a), the
naphthyl group of 4d involves π-π stacking with Trp279
(distance of 4.9 Å). The high binding affinity is due to the dual
binding affinity owing to π-π stacking and hydrophobic binding.
Further the preparation of the reduced bicyclic compounds 4e-
4j was done and they were obtained in 75% yield after the
purification via washing and recrystallization. The structure was
confirmed through H NMR data, which shows appearance of
NH peak (10.11-11.01) in 4e-4j that was absent in 4a-4d.
1
A similar study was done on Human BuChE[20] (PDB ID 4tpk).
Binding free energy result of compounds with BuChE is given in
Table 1. The hydrophobic interactions among key residues and
molecule depicted that compounds 4d and 4e possess better
binding interactions with Human BuChE. Figures of docking
study of 4d and 4e with BuChE are provided in Fig. 3(a, b). From
the analysis of the theoretical affinity values of ligand with
desired enzyme 4e and 4f were found to be comparable with the
gold standard drug Donepezil. In support of the molecular
docking results, it may be suggested that the triazolopyrimidine
derivatives inhibit AChE and BuChE by targeting the CAS and
PAS of AChE and CAS in BuChE.
Fig 1. An ORTEP view of the compound 4a presenting
thermal ellipsoids at 50% probability level. Hydrogen atoms have
been omitted for clarity. Color codes: C black and N green.
2.2. Single crystal structure of compound 4a
The confirmation of structurally characterized compound 7-
(naphthalen-1-yl)-5-phenyl-[1,2,4]triazolo[4,3-a]pyrimidine, 4a,
was additionally done by single-crystal X-ray crystallography
(Figure 1 (below), Table S1, Table S2, Figures S31 and S32,
Supporting Information). Molecule 4a, colorless sugar crystals
were crystallized in C 2/c space group of monoclinic crystal

Fig. 2. Docking studies of (a) 4d and (b) 4e with AChE (PDB code 1EVE). The inhibitors are presented in ball and stick structure.
The key residues are represented as sticks and are shown by different colors such as Glu199 (red), Ser200 (Cyan), and His440 (Orange)
and other residues are shown in stick representation with carbons colored yellow. Oxygen and nitrogen atoms in the complexes are
colored red and blue respectively. π-π Stacking is shown by red dashed line. Hydrogen atoms removed for clarity.
Fig. 3. Docking studies of (a) 4d and (b) 4e with Human BuChE (PDB ID 4tpk). The inhibitors are presented in ball and stick structure.
The key residues are represented in sticks and are shown by different colors such as Asn68 (Green), Asp70 (Blue), Thr120 (Graywhite),
Trp82 (Hotpink), Trp231 (Hotpink), Glu197 (Red), Ser198 (Cyan), Leu286 (lemon Green), Tyr332 (Yellow), His438 (Orange).
Hydrogen atoms removed for clarity.
2.3.2. In vitro AChE and BuChE inhibition studies
towards inhibition of AChE. The presence of naphthyl group is
one contributing factor for the good AChE inhibition activity as it
participates in the π-π stacking with the active site residues.
Along with the naphthyl group, the 4-methoxybenzene part of 4e
is expected to be engaged in π-π stacking with the active site
residues hence 4e has a very high AChE inhibition. However
lower activity was found for electron withdrawing group like
nitro and for sterically hindering dimethoxy group in compounds
4b and 4h. Molecule 4b and 4h was expected to show high
Screening of these molecules for their inhibitory activity
towards electric eel AChE and equine serum BuChE was
conducted by the method of Ellman et al.[21] and FDA approved
drug Donepezil was used as a reference drug. The IC50 values for
each ChE inhibition and their selectivity index (SI) are summed
up in Table 2. It is clear from the table that all the synthesized
compounds inhibited AChE and BuChE with IC50 values ranging
from nanomolar to micromolar and exhibiting more selectivity

b
activity according to the docking results however the
experimental result shows it to be moderately active. The
decrease in activity is assumed to be due to steric constraint that
would have developed during enzyme-ligand interaction in vitro.
Compound 4e having similar functional group aligned in a
sterically favourable manner yielded high activity results.
IC50, inhibitor concentration (mean ± SD of three
independent experiments) resulting in 50% inhibition of BuChE
from equine serum.
c AChE SI selectivity index: IC50 eqBuChE/eeIC50 AChE.
2.3.3. Self-mediated Aβ1–42 aggregation inhibition
To explore the anti-amyloidogenic function of synthesised
molecules, the experimental approach relies on the in vitro assays
with the Thioflavin-T (ThT) to monitor as well as quantify the
aggregation of Aβ1–42 peptide into fibrils. Standard disaggregating
agent curcumin was used as the reference and the results are
summarised in Table 3. According to the results, it is clear that
most molecules exhibited moderate-to-good inhibition potencies
(12-85%) as compared with curcumin (51.7%) in concentration
ratio of 2:1 [Aβ (100 M): inhibitor (50 M)]. Compounds 4i, 4j,
4c, and 4h gave the highest inhibitions with values 85.46%,
72.77%, 43.12%, and 42.14% respectively. Compounds 4a and
4g were unable to inhibit the formation of A fibrils. Compounds
(4i and 4j) with highly electronegative Fluorine group and highly
electron withdrawing nitro group were found to exhibit high
inhibition of Aβ1-42 aggregation. The highly electronegative
atoms probably provide scope for H-bonding with the fibrils and
hence disrupting aggregation formation. Furthermore compounds
4b, 4c, 4f and 4h with other interactive groups like methoxy
showed moderate inhibition owing to their lower interaction with
fibrils. Unsubstituted benzene and the furan ring showed
diminished inhibition which indicates the importance of
substituents in these synthesized compounds.
Table 1. Calculated free energies of 4a-j in molecular
docking using ParDOCK [22].
Compound
Free energy values
(kcal/mol) AChE (PDB
ID: 1EVE)
Free energy values
(kcal/mol) BuChE (PDB
ID: 4tpk)
4a
-5.09
-7.12
-6.90
-7.05
-7.53
-7.33
-6.67
-7.90
-5.89
-5.76
-6.96
-4.90
-4.54
-5.42
-4.74
-5.63
-5.82
-5.62
-6.11
-6.21
-5.82
-5.77
-6.67
-3.56
4b
4c
4d
4e
4f
4g
4h
4i
4j
Donepezil
Tacrine
Table 3. Self-induced Aβ aggregation inhibition
percentage of synthesized compounds
Compound
Self Aβ1-42 aggregation inhibitiona (%)
4a
No inhibition
36.62 ± 2.36
43.12 ± 4.49
12.07 ± 1.09
31.23 ± 3.43
40.39 ± 2.12
No inhibition
42.14 ± 1.89
85.46 ± 2.78
72.77 ± 3.97
52.70 ± 1.67
Table 2. In vitro inhibition of AChE and BuChE, and
selectivity index of the test compounds.
4b
4c
Compounds
IC50 ± SD
(M)AChEa
IC50 ± SD
(M)BuChEb
Selectivity Index
(BuChE/AChE)c
4d
4a
0.597
>100
>167.5
>33.44
166.26
37.714
17.44
1.027
2.42
4e
4b
2.997 ± 0.22
0.126 ± 0.01
0.014 ± 0.01
0.0086±0.02
0.074 ± 0.02
0.371 ± 0.01
0.602± 0.11
1.908 ± 0.12
>100
>100
4f
4c
20.95 ± 0.13
0.528 ± 0.002
0.150 ± 0.01
0.076 ± 0.02
0.900 ± 0.03
>100
4g
4h
4d
4i
4e
4j
4f
Curcumin
aInhibition of self-induced Aβ1-42 aggregation (100 μM) by
inhibitors at 50 μM by ThT based fluorescence method (means ±
SD of three experiments). Experiments have been performed in
triplicates.
4g
4h
>160
19.71
-
4i
4j
39.03 ± 0.08
>100
2.3.4. Disaggregation of Self-Induced Aβ1−42 Aggregation Fibrils
Table 4. Disaggregation percentage of self-induced Aβ1−42
aggregation fibrils for four best compounds.
Donepezil
0.049 ± 0.05
8.71 ± 1.36
166.7
Compounds
Aβ1-42a disaggregation (%)
Bolded values represent the best activity compounds 4d, 4e and
4f.
4c
4h
4i
54.51 ± 5.32
47.94 ± 2.14
62.64 ± 2.58
a
IC50, inhibitor concentration (mean
± SD of three
independent experiments) resulting in 50% inhibition of AChE
from electric eel.

Test compound was added to the sample and was incubated
for additional 24 h. Disaggregation of Aβ was measured using
ThT assay, in which the fluorescence intensity indicates the
degree of Aβ fibrillar aggregation (see Fig. 4). The ThT stains up
the amyloid deposits. Table 4 depicts the Aβ1-42 disaggregation
inhibition percentage of the tested compounds. All four
4j
58.80 ± 3.76
a
% disaggregation of Aβ1-42 (mean+ S.D) exhibited by
compounds. Experiments have been performed in triplicates.
Amyloid plaques are a result of increase in Aβ1-42 production
and accumulation. Aβ1-42 oligomerizes and deposits as senile
plaques. The compounds possessing a higher inhibition potential
towards self-mediated Aβ1-42 aggregation were further tested for
their ability to disaggregate self-induced Aβ1-42 aggregated fibrils
using the ThT assay. Compounds 4c, 4h, 4i, and 4j were selected
based on their high potency to inhibit self-induced Aβ1-42
aggregation. Fresh Aβ was incubated for 48 h at a temperature of
37 °C to facilitate formation of fibrils.
compounds showed
a
significantly high disaggregation.
Compound 4i and 4j showed the most favorable disaggregation
with 62.64% and 58.80% respectively, followed by 4c with
54.51% and 4h with 47.94%. High disaggregation displayed by
compounds 4i and 4j is in concordance with their similarly high
inhibition of Aβ fibril aggregation.
2.3.5. Concentration dependent self Aβ aggregation inhibition
study
The two compounds exhibiting the best activities against A
aggregation and disaggregation were further tested for
concentration dependent self A aggregation inhibition.
Compounds 4i and 4j were tested at five different concentrations
(12.5 µM, 25 µM, 50 µM, 75 µM and 100 µM) with Aβ at 100
µM concentration. Fluorescence intensities were measured by
adding ThT to the pre-incubated mixture of compound and Aβ. A
general trend of increase in Aβ aggregation inhibition with
increasing concentrations of both the compounds was observed.
As seen in the table S3 (supporting information) and Fig. 5, both
the compounds showed a significant inhibition at 50 µM
concentration.
Compound 4i showed high inhibitory potency even at a
concentration of 12.5 µM (70.42%) whereas compound 4j
displayed low inhibition compared to compound 4i at 12.5 µM
(42.03%). The IC50 values were calculated for both the
compounds using Graph Pad Prism software.
Fig. 4. Fluorescence intensity of Aβ 1-42 alone (dark blue), Aβ
with 4c (red), Aβ
with 4h (light blue), Aβ
with 4i
1-42
1-42
1-42
(green), Aβ 1-42 with 4j (pink) measured using ThT assay.
Fig. 5. Percent inhibition of self-induced Aβ1-42 aggregation by different concentrations of drug (12.5, 25, 50, 75 and 100 μM). The bar
graph depicts a proportional relation between increase in self-induced Aβ1-42 aggregation inhibition percentage with concentration for
compounds 4i and 4j.

Fig. 6. TEM images of self-induced Aβ1 - 4 2 aggregation inhibition; (a) Aβ1 - 4 2 (25 µM), 0 °C, 0 h; (b) Aβ1 - 4 2
alone (25 µM) was incubated at 37 °C for 48 h; (c) Aβ1 - 4 2 (25 µM) with curcumin (25 µM) were co-
incubated at 37 °C for 48 h; (d) Aβ1 - 4 2 (25 µM) and 4i (50 µM) were co-incubated at 37 °C for 48 h; (e)
Aβ1 - 4 2 (25 µM) and 4j (50 µM) were co-incubated at 37 °C for 48 h. All TEM images are at a resolution of
0.5 µ meter.
2.3.6. Transmission Electron Microscopy (TEM)
Assay
Table 5. Antioxidant ORAC assay
Compound
-Ar
Trolox
To validate the higher inhibition potency displayed by
compounds 4i and 4j we performed TEM analysis. At the
beginning (0 h), aggregation was absent in the samples of Aβ1-42
with or without test compounds (Fig. 6 (a)). Formation of Aβ
aggregates was observed in our control (Aβ alone) over the
incubation of 48 h at 37 °C, whereas significantly fewer
aggregates were observed in the positive control (curcumin) (Fig.
6 (c)). In the presence of both the test compounds, only fewer
aggregates were formed under the same experimental conditions
as compared to the standard Curcumin (Fig. 6 (c), (d) and (e))
confirming their inhibitory potencies. The TEM results were
consistent with the results obtained via ThT fluorescence assay
and hence further prove the effectiveness of the synthesized
molecules in inhibiting and slowing down the rate of Aβ1-42 fibril
formation in vitro.
(100 μM)
equivalentsa
4a
4b
0.301 ± 0.08
0.234 ± 0.11
4c
0.171 ± 0.05
2.3.7. Antioxidant Activity In Vitro Using Oxygen
Radical Absorbance Capacity (ORAC -FL) Assay
Oxidative stress plays a very significant role in the neuronal
degeneration. Multifunctional drugs with antioxidant potential
for the treatment of AD are crucial. Antioxidation potential of all
synthesized molecules was determined using the (ORAC-FL)
assay. On thermal generation of peroxyl radicals from 2,2-azobis-
(amidinopropane) dihydrochloride it was reacted with fluorescein
to give non-fluorescent products at 535 nm. Antioxidation
capability of synthesized derivatives was determined via their
competition with fluorescein in radical capturing, using
fluorescence microplate reader. Trolox (Vitamin E analog) was
used as the standard and activity of synthesized compounds was
expressed as Trolox equivalents (l mol of Trolox/l mol of tested
compound). As given in Table 5, most of the test compounds
showed a low range of ORAC values, ranging from 0.1- to 0.5-
fold of the value for Trolox. Amongst all tested molecules, 4j
gave best results with 0.508 Trolox equivalents. Compounds
possessing methoxy or nitro group showed better antioxidant
activity. Furthermore, compounds with furan tail (4d) showed
lowest antioxidant activity.
4d
4e
0.101 ± 0.10
0.446 ± 0.13
4f
0.390 ± 0.12

2.3.9. Pharmacokinetic properties and drug
likeness of compounds 4a-4j
4g
4h
0.268 ± 0.08
In the route of drug discovery and development a number of
drugs face refusal due to inadequate pharmacokinetic account. of
ADMET property determination is thus vital for lead generation.
'Drug likeness' of the synthesized inhibitors was examined using
ADMET descriptors (Accelrys Discovery studio 4.0). ADME
properties such as absorption, Blood Brain Barrier (BBB) level,
aqueous solubility, AlogP98 and PSA were studied for
triazolopyrimidine derivatives. Prediction of the human intestinal
absorption after the oral administration of synthesized inhibitors
was done by generating standard ADME model. Absorption
model comprises 95% and 99% confidence ellipses in polar
surface area (PSA) and AlogP98 plane. The ellipses in Fig. S33
describe the expected regions for well-absorbed test inhibitors.
For intestinal absorption, 99% and 95% of well-absorbed
inhibitors are assumed to lie inside the ellipses colored green and
red, respectively. Similarly, for overcoming the BBB, 95 and
99% of well absorbed test inhibitors are expected to be within the
ellipses colored magenta and aqua, respectively. All test
inhibitors were present inside all the four ellipses, thus satisfying
conditions for absorption by intestines and brain. And also there
are four prediction levels for the absorption of compounds as
good (0), moderate (1), poor (2) and very poor (3) level. In case
of our tested compounds, prediction level for the absorption was
found to be 0, so all the compounds have good absorption
properties. All the compounds had aqueous solubility logarithmic
level of 1 that suggests better aqueous solubility. As per the
criteria, PSA ˂ 60-70 Å2 and MW ˂ 450 for penetrating the BBB
[23] and all compounds were in accordance with it.
ADMET_AlogP98 and ADMET_PPB are the two that mainly
accounts for the ability of compound's distribution in body. Table
S5 (supplementary information) indexes the In silico toxicity
studies, where the inhibitors shows no Ames mutagenicity.
Weight of evidence (WOE) prediction suggests they are non-
carcinogenic. Under rodent carcinogenicity test, all synthesized
compounds were observed to be non-carcinogenic for female
mouse model however, showed single carcinogenic male rat and
male mouse model and varying carcinogenic potential in female
rat model. The LD50 values varied from 0.025 g/Kg to 0.403
g/Kg. Higher safety is suggested by higher LD50 values. The
ADMET property prediction indicates that most compounds
fulfill the criteria of drug-likeness and could be portrayed as good
drug development candidates.
0.472 ± 0.09
4i
4j
0.347 ± 0.09
0.508 ± 0.17
aData is expressed as μmol of Trolox equivalent/ μmol of test
molecule.
2.3.8. Inhibitory effects of compounds 4a-4j on
DPPH radical scavenging activities
All test compounds were assessed for their free radical
scavenging activities by using 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) assay. All compounds showed moderate anti-oxidative
activity ranging from 15.5% to 53.75% of radical scavenging.
Compound 4h displayed the most potent antioxidant activity with
53.75% of radical scavenging activities (Table 6). The radical
scavenging activity results signify that the potent cholinesterases
inhibitors and beta-amyloid inhibitors were also moderate
antioxidant agents. The activity results rose with nitro at meta of
aryl tail, dimethoxy at meta, para, and methoxy group on para
position of ring as seen from the activities of 4j, 4h and 4e,
respectively. The poor activity of 4f in DPPH scavenging and
ORAC assays could be due to the presence of chloro group on
aryl tail. Furthermore, the activity is facilitated in the presence of
fluoro group on para-position (4i).
Table 6. Antioxidant activity of test compounds through
DPPH Radical scavenging Assay
Compound (50 μM)
DPPH Scavenging abilities (%)
3. Conclusion
4a
18.75
This study aimed to gather insights into the multitargeted
efficacy of naphthalene–triazolopyrimidine hybrids in altering
the AD’s pathological events. With extension of the success of
triazolopyrimidines as potent anti-Alzheimer’s agents with a
multifunctional profile towards amyloid β disaggregation, ChE
inhibition and antioxidation, these hybrids 4a-j have been
synthesized, and evaluated as even better multiple function
therapeutic agents with higher potency. It was found that the
triazolopyrimidine derivatives had a marked inhibition capability
towards AChE and BuChE with IC50 values in nanomolar and
subnanomolar range. Most of the synthesized molecules
exhibited AChE and BuChE inhibition activity, especially
compounds 4e with inhibition value of 8.6 nM (AChE) and 150
nM (BuChE) respectively. However selectivity upto that of
Donepezil and even more was observed for 4a, 4c and 4h. In
vitro studies showed that 4i and 4j exhibit good protective action
against neurotoxicity induced via toxic Aβ aggregates. A
combination of biophysical study results involving ThT
fluorescence assay and electron microscopy revealed that 4i and
4j could inhibit or reduce β-sheet aggregation and fibril
4b
27.9
4c
21.3
4d
20.42
35
4e
4f
15.5
4g
19.2
4h
53.75
36.25
32.98
65.98
95.33
4i
4j
Gallic Acid (10 μM)
Gallic Acid (100 μM)

formation up to a significant extent making them promising
candidates for AD treatment. Altogether, the present study and
previous findings provide preclinical evidence that
triazolopyrimidine derivatives are therapeutic agents with
multiple functions and higher potency for AChE inhibition.
C23H18N4O2 (Mol. wt = 382.4146 g mol−1) (ESI, m/z): observed,
405.1329: calculated, 405.1327 [M + Na]+.
4.1.2.3. N,N-dimethyl-4-(7-(naphthalen-1-yl)-
[1,2,4] triazolo[4,3-a]pyrimidin-5-yl)aniline (4c)
1H NMR (400 MHz, CDCl3): δ (ppm) 8.59 (s, 1H, NCHN),
8.34 (d, 1H, J = 8 Hz, Ar-H), 8.23 (d, 2H, J = 8 Hz, Ar-H), 8.00
(d, 1H, J = 4Hz, Ar-H), 7.95 (d, 1H, J = 8Hz, Ar-H), 7.84 (d, 1H,
J = 8Hz, Ar-H), 7.62-7.50 (m, 4H, Ar-H), 6.86 (d, 2H, J = 8Hz,
Ar-H), 3.09 (ss, 6H, NCH3); 13C NMR ( 100 MHz, CDCl3): δ ;
164.04, 157.04, 156.24, 152.92, 148.30, 136.76, 134.40, 131.49,
131.11, 130.89, 129.06, 128.69, 127.62, 126.75, 125.77, 125.69,
116.82, 112.12, 109.38, 77.83, 77.51, 77.19, 40.62; HRMS in
MeOH for C23H19N5 (Mol. wt = 365.4305 g mol−1) (ESI, m/z):
observed, 388.1539: calculated, 388.1538 [M + Na]+.
4. Experimental
4.1. Chemistry
All reagents and solvents were purchased from commercial
suppliers including Sigma Aldrich (St. Louis, MO, USA), Merck
(Germany), Alfa Aesar (Massachusetts) and Spectrochem Pvt.
1
Ltd. (India) and were used without further purification. H NMR
spectroscopic evaluation was done on Bruker 400 MHz and 13C
NMR on Bruker 100 MHz with deuterated solvent.
Tetramethylsilane (TMS) was taken as internal standard.
4.1.2.4. 5-(furan-2-yl)-7-(naphthalen-1-yl)-
[1,2,4] triazolo[4,3-a]pyrimidine (4d)
Chemical shift values were denoted in
δ (ppm). Mass
spectrometric analyses were carried out on Agilent 6538 Ultra
High Definition Accurate Mass-Q-TOF (LC-HRMS) instrument.
Thin-layer chromatography (TLC) analysis was used to monitor
the reactions. Merck silica gel 60-120 and F254 pre-coated TLC
plates of thickness 0.25 mm were employed. TLC spots were
examined under UV light at short (254 nm) or long (365 nm)
wavelengths.
1H NMR (400 MHz, CDCl3): δ (ppm) 8.67(ss, 1H, NCHN),
8.36(d, 1H, J = 8Hz, Ar-H), 8.27(d, 1H, J = 4Hz, Ar-H), 8.02(d,
1H, J = 8Hz, Ar-H), 7.96(d, 1H, J = 8Hz, Ar-H), 7.89(ss, 1H, Ar-
H), 7.86(d, 1H, J = 8Hz, OCH), 7.74(d, 1H, Ar-H), 7.63-7.54(m,
3H, Ar-H), 6.78(dd, 1H, J = 4Hz, Ar-H); 13C NMR ( 100 MHz,
DMSO): δ ; 163.67, 157.33, 156.26, 149.09, 143.64, 137.27,
136.58, 134.36, 131.26, 130.96, 129.42, 129.24, 128.05, 127.31,
126.33, 126.13, 121.32, 114.71, 106.89, 41.08, 40.87, 40.66,
40.46, 40.25, 40.04, 39.83; HRMS in MeOH for C19H12N4O
(Mol. wt = 312.3248 g mol−1) (ESI, m/z): observed, 335.0909:
calculated, 335.0909 [M + Na]+.
4.1.1. General procedure for the preparation of
diff erent substituted 1-(1-naphthyl)-3-arylprop-2-
en-1-ones
The initial 1-(1-naphthyl)-3-arylprop-2-en-1-ones (3a-f) were
prepared by the reported method[24]. These compounds have
already been reported in the following literature.[24-27]
4.1.3. General preparation of naphthalen-5,8-
dihydro-[1,2,4]triazolo[4,3-a]pyrimidine hybrids
(4e-j)
4.1.2. General preparation of naphthalen-
[1,2,4] triazolo[4,3-a]pyrimidine hybrids (4a-d)
Respective 1,3-diaryl-propenone (20 mmol) was dissolved in
methanol. To this added 1-2 pallets of sodium hydroxide and 3-
amino-1, 2, 4-triazole (40 mmol) dissolved in methanol. The
reaction mixture was refluxed for 48 hs and was monitored via
TLC. After completion of reaction the solvent was reduced to
1/3rd and the solid was separated by filtration and washed with
methanol and recrystallized in methanol/the same to afford solid
compound in 40-70% yield.
Respective 1,3-diaryl-propenone (20 mmol) was dissolved in
methanol. To this added 1-2 pallets of sodium hydroxide and 3-
amino-1, 2, 4-triazole (40 mmol) dissolved in methanol. The
reaction mixture was refluxed for 3-4 hs and was monitored via
TLC. After completion of reaction the solvent was reduced to
1/3rd and the solid separated was filtered out and washed with
methanol and recrystallized in methanol to afford solid
compound in 40-70% yield.
4.1.3.1. 5-(4-methoxyphenyl)-7-(naphthalen-1-yl)-
5,8-dihydro-[1,2,4] triazolo[4,3-a]pyrimidine (4e)
4.1.2.1. 7-(naphthalen-1-yl)-5-phenyl-
[1,2,4] triazolo[4,3-a]pyrimidine (4a)
1H NMR (400 MHz, CDCl3): δ (ppm) 10.73 (bs, 1H, NH),
8.23 (t, 1H, Ar-H), 7.91 (m, 2H, Ar-H), 7.62 (d, 1H, Ar-H), 7.52-
7.46 (m, 3H, Ar-H), 7.38 (d, 2H, Ar-H), 6.92 (d, 2H, Ar-H), 6.55
(s, 1H, NCHN), 6.15 (d, 1H, -CH=), 5.01 (d, 1H,
triazolopyrimidine-CH), 3.79 (ss, 3H, OCH3); 13C NMR ( 100
MHz, CDCl3):δ ; 160.33, 147.28, 135.35, 134.19, 133.42, 133.24,
130.25, 129.18, 128.95, 127.42, 127.28, 126.82, 125.69, 114.76,
100.53, 77.84, 77.52, 77.20, 61.00, 55.79, 14.67; HRMS in
MeOH for C22H18N4O (Mol. wt = 354.4045 g mol−1) (ESI, m/z):
observed, 355.1557: calculated, 355.1559 [M + H]+.
1H NMR (400 MHz, CDCl3): δ (ppm) 8.61(ss, 1H), 8.36(dd,
1H, Ar-H), 8.15(m, 2H, Ar-H), 7.99(d, 1H, Ar-H), 7.94(dd, 1H,
Ar-H), 7.83(dd, 1H, Ar-H), 7.60-7.52(m, 7H, Ar-H); 13C NMR (
100 MHz, CDCl3): δ ; 164.782, 156.495, 156.402, 148.133,
135.979, 134.405, 132.418, 131.374, 130.940, 130.255, 129.863,
129.449, 129.163, 129.062, 127.864, 126.882, 125.649, 125.505,
111.796, 77.889, 77.571, 77.253; HRMS in MeOH for C21H14N4
(Mol. wt = 322.3627 g mol−1) (ESI, m/z): observed, 345.1115:
calculated, 345.1116 [M + Na]+.
4.1.2.2. 5-(3,4-dimethoxyphenyl)-7-(naphthalen-1-
yl)-[1,2,4] triazolo[4,3-a]pyrimidine (4b)
4.1.3.2. 5-(2-chlorophenyl)-7-(naphthalen-1-yl)-
5,8-dihydro-[1,2,4] triazolo[4,3-a]pyrimidine (4f)
1H NMR (400 MHz, DMSO): δ (ppm) 8.79(s, 1H, NCHN),
8.33 (d, 1H, J = 8 Hz, Ar-H), 8.15 (d, 1H, J = 4Hz, Ar-H), 8.09
(d, 2H, Ar-H), 7.92(s, 3H, Ar-H), 7.70(t, 1H, Ar-H), 7.64-
7.57(m, 2H, Ar-H), 7.23(d, 1H, Ar-H); 13C NMR ( 100 MHz,
DMSO): δ ; 164.09, 156.86, 156.68, 152.77, 149.43, 147.94,
136.71, 134.36, 131.11, 129.34, 127.99, 127.27, 126.27, 124.65,
122.64, 114.04, 112.38, 111.27, 56.76, 56.71, 41.09, 40.88,
40.67, 40.46, 40.25, 40.04, 39.84; HRMS in MeOH for
1H NMR (400 MHz, CDCl3): δ (ppm) 11.01 (bs, 1H, NH),
8.20 (t, 1H, Ar-H), 7.93 (m, 2H, Ar-H), 7.62 (d, 1H, Ar-H), 7.54-
7.48 (m, 3H, Ar-H), 7.42 (d, 1H, Ar-H), 7.31-7.24 (m, 2H, Ar-
H), 7.18-7.16 (dd, 1H, Ar-H) 6.71 (d, 1H, Ar-H), 6.62 (s, 1H, -
CH=), 5.09 (d, 1H, triazolopyrimidine-CH); 13C NMR ( 100
MHz, DMSO):δ ; 150.71, 150.61, 139.46, 136.38, 134.10,
134.01, 132.15, 131.74, 130.96, 130.60, 130.20, 130.09, 129.21,
128.75, 127.76, 127.52, 127.09, 126.29, 126.00, 98.38, 59.09,
41.10, 40.89, 40.68, 40.47, 40.27, 40.06, 39.85; HRMS in MeOH

for C21H15ClN4 (Mol. wt = 358.8236 g mol−1) (ESI, m/z):
observed, 359.1067: calculated, 359.1063 [M + H]+.
BCC procedure. Bappl (an all atom based scoring function)
scoring function[30] was used for scoring of these docked
structures. A complete protocol for docking is described
elsewhere.[31] This visualization and diagram preparation was
done using PyMOL [32].
4.1.3.3. 5-(4-chlorophenyl)-7-(naphthalen-1-yl)-
5,8-dihydro-[1,2,4] triazolo[4,3-a]pyrimidine (4g)
1H NMR (400 MHz, CDCl3): δ (ppm) 10.83 (bs, 1H, NH),
8.15 (t, 1H, Ar-H), 7.92-7.88 (m, 2H, Ar-H), 7.62 (dd, 1H, Ar-
H), 7.54-7.47 (m, 3H, Ar-H), 7.38 (d, 4H, Ar-H), 6.91 (s, 1H,
NCHN), 6.20 (d, 1H, -CH=), 5.05 (d, 1H, triazolopyrimidine-
CH); 13C NMR ( 100 MHz, CDCl3):δ ; 145.53, 139.04, 135.41,
134.21, 131.08, 130.62, 129.76, 129.26, 129.11, 127.49, 126.93,
125.70, 125.35, 100.53, 77.82, 77.51, 77.19, 61.02; HRMS in
MeOH for C21H15ClN4 (Mol. wt = 358.8236 g mol−1) (ESI, m/z):
observed, 381.0881: calculated, 381.0883 [M + Na]+.
4.3. X-ray Crystallographic Procedure
Crystal structure of compound 4a was acquired with single-
crystal X-ray diff raction method. Slow evaporation of methanol
solution of compound 4a gave the colorless rectangular block
crystals that were boarded on loops with mineral oil. Bruker
SMART APEX CCD diff ractometer provided with fine focus
1.75 kW sealed-tube Mo Kα X-ray source (λ = 0.71073 Å) with
rising ω (width of 0.3° per frame) at a scan speed 5 s frame−1,
was used to collect the geometric data and intensity data using
the ω-2θ scan mode. It was then corrected for Lorentz-
polarization as well as absorption eff ects.[33] Using WinGx
suite of programs (version 1.63.04a) the structures were solved
and refined via SHELXL-2013 method.[34] Non-hydrogen atoms
coordinates were permitted to be on their corresponding carbon
atoms and non-hydrogen atoms were refined with coefficients of
anisotropic displacement. The final refinement included atomic
positions of all the atoms, anisotropic thermal parameters for all
non-hydrogen atoms, and isotropic thermal parameters for all the
hydrogen atoms. For procuring structural views ORTEP was
used.[35] Further details of the crystal structure determination
like crystallographic parameters; bond distances, and angles data
are given in Table S1 (supplementary information). The CCDC
deposition number is 1886686.
4.1.3.4. 5-(2,5-dimethoxyphenyl)-7-(naphthalen-1-
yl)-5,8-dihydro-[1,2,4] triazolo[4,3-a]pyrimidine
(4h)
1H NMR (400 MHz, CDCl3): δ (ppm) 10.88 (s, 1H, NH), 8.25
(m, 1H, Ar-H), 7.89 (d, 2H, J=8 Hz, Ar-H), 7.60 (d, 1H, J= 8 Hz,
Ar-H), 7.51-7.47 (m, 3H, Ar-H), 6.85-6.77 (m, 2H, Ar-H), 6.69
(d, 1H, Ar-H), 6.57 (d, 1H, -CH=), 6.44 (s, 1H, NCHN), 5.01 (d,
1H, J= Hz, triazolopyrimidine-CH), 3.79 (ss, 3H, OCH3), 3.73
(ss, 3H, OCH3); 13C NMR ( 100 MHz, CDCl3):δ ; 148.33, 135.29,
134.17, 133.80, 131.38, 130.94, 129.94, 128.81, 127.30, 127.05,
126.71, 126.03, 125.67, 114.53, 114.14, 112.77, 99.65, 77.87,
77.56, 77.24, 56.74, 56.41, 56.18; HRMS in MeOH for
C23H20N4O2 (Mol. wt = 384.4305 g mol−1) (ESI, m/z): observed,
407.1484: calculated, 407.1484 [M + Na]+.
4.4. Pharmacological evaluation
4.1.3.5. 5-(4-fluorophenyl)-7-(naphthalen-1-yl)-5,8-
dihydro-[1,2,4]triazolo[4,3-a]pyrimidine (4i)
4.4.1. Inhibition of AChE and BuChE using Ellman
assay
1H NMR (400 MHz, DMSO): δ (ppm) 10.11 (s, 1H, NH), 8.12
(m, 1H, Ar-H), 7.98 (d, 2H, Ar-H), 7.64 (s, 1H, NCHN), 7.58-
7.55 (m, 4H, Ar-H), 7.47 (d, 1H, J = 8Hz, Ar-H), 7.45 (d, 1H, J =
8Hz, Ar-H), 7.24 (d, 2H, J = 12Hz, Ar-H), 6.33 (d, 1H, J = 4Hz, -
CH=), 4.90 (dd, 1H, triazolopyrimidine-CH); 13C NMR ( 100
MHz, DMSO):δ ; 163.90, 150.55, 149.89, 139.38, 135.71,
134.11, 131.75, 130.08, 130.03, 129.95, 129.24, 127.84, 127.60,
127.11, 126.32, 125.98, 116.50, 116.29, 100.31, 60.67, 60.11,
41.07, 40.86, 40.65, 40.44, 40.23, 40.03, 39.82; HRMS in MeOH
for C21H15FN4 (Mol. wt = 342.3690 g mol−1) (ESI, m/z):
observed, 365.1178: calculated, 365.1178 [M + Na]+.
The spectroscopic method by Ellman et al. [21] was utilized to
assess AChE as well as BuChE inhibitory activities of
synthesized compounds. Acetylcholinesterase (AChE, E.C.
3.1.1.7, from electric eel), Butyrylcholinesterase (BuChE, E.C.
3.1.1.8, from equine serum), acetylthiocholine (ATC) iodide,
butyrylthiocholine (BTC) iodide, 5,5’-dithiobis-(2-nitrobenzoic
acid) (DTNB, Ellman’s reagent), and Donepezil were purchased
from the Sigma Aldrich. The stock solutions were prepared in
water and DMSO by adding test compounds such as to have a
final concentration range 0.01 to 100 µM. For preparation of
enzyme solutions lyophilized powder was dissolved in
autoclaved water. Assay solution contained 195 µL of 1 M
phosphate buffer KH2PO4/K2HPO4, AChE (5 µL, 0.22 U/mL) or
BuChE (5 µL, 0.06 U/mL) and test compounds (25 µl of varying
concentrations) which were allowed to stand 5 min before 20 µL
of 0.01 M DTNB was added. Donepezil as positive control was
used in the identical range of concentrations. 4 µL of 0.075 M
substrate solution (ATC/BTC) was added to start the reaction.
After 5 min of substrate addition the absorption measurement
was done at 412 nm at a temperature of 25 °C.
4.1.3.6. 7-(naphthalen-1-yl)-5-(3-nitrophenyl)-5,8-
dihydro-[1,2,4]triazolo[4,3-a]pyrimidine (4j)
1H NMR (400 MHz, DMSO): δ (ppm) 10.26 (s, 1H, NH), 8.26
(s, 1H, Ar-H), 8.22-8.19 (m, 1H, Ar-H), 8.14-8.11 (m, 1H, Ar-
H), 8.01-7.98 (m, 2H, Ar-H), 7.88-7.86 (d, 1H, Ar-H), 7.73 (t,
1H, Ar-H), 7.70 (s, 1H, NCHN), 7.61-7.54 (m, 4H, Ar-H), 6.56
(d, 1H, J = 4Hz, -CH=), 4.98 (d, 1H, J = 4Hz, triazolopyrimidine-
CH); 13C NMR ( 100 MHz, DMSO):δ ; 150.91, 150.06, 148.89,
145.04, 136.40, 134.59, 134.11, 133.91, 131.72, 131.41, 130.18,
129.26, 127.87, 127.63, 127.14, 126.31, 125.93, 123.95, 122.48,
99.45, 60.00, 41.09, 40.88, 40.67, 40.46, 40.26, 40.05, 39.84;
HRMS in MeOH for C21H15N5O2 (Mol. wt = 369.3761 g mol−1)
(ESI, m/z): observed, 370.1361: calculated, 370.1304 [M + H]+.
4.4.2. Self-mediated Aβ1 - 4 2 Aggregation Assay
To avoid self-aggregation of peptides the commercially
brought
peptides
were
primarily
reacted
with
hexafluoroisopropanol (HFIP) at a concentration of 8 mg/ml. The
clear solution formed by dissolving the peptide was then
aliquoted in the microcentrifuge tube. HFIP present was
evaporated under a stream of nitrogen to form a clear film. To
form a stable stock solution (Aβ1-42 5mM), pretreated samples of
Aβ1-42 were dissolved in DMSO. For the inhibition of self, stock
solution was diluted using phosphate buffer (50 mM, pH 7.4) in
Aβ1-42 aggregation experiment. The mixture of peptides (10 µl,
100 µM, final concentration) with or without compound (25 µM)
4.2. Molecular docking protocol
Docking of candidate compounds with acetylcholinesterase
and butyrylcholinesterase was done with the Pardock[22]
software of Sanjeevini[28]. The ligand was used in its original
input pose for calculation. Hydrogens were added to ligand via
xleap of AMBER[29]. Rest operations like partial charge
calculations and geometry optimization were done with AM1-

was incubated for 48 h at a temperature of 37 oC. For quantifying
the fibril formation, thioflavin-T fluorescence method was
employed. Furthermore for this was done with reference solution
consisting 50 mM phosphate buffer (pH 7.4) inplace of Aβ with
or without compounds. Following incubation, dilution was done
with samples to a final volume of 200 µl using 50 mM of
glycine-NaOH buffer (at pH 8.0).
instead of the synthesized compounds and calibration solutions
with Trolox (1–8 M, final concentration) as antioxidant were
also carried out in all assays. For each sample at least three
independent runs were performed and reaction mixtures were
prepared in triplicate. The fluorescence vs time graphs
(antioxidant curves) were normalized to the curve of blank. The
following equation was used to calculate the area under the
fluorescence decay curve (AUC):
4.4.3. Disaggregation of Self-Induced Aβ1 − 4 2
Aggregation
i = 80
For disaggregation studies of self-induced Aβ1-42 aggregation
by test compounds dilution of Aβ stock solution was done with
50 mM phosphate buffer (pH 7.4). The mixture of the peptides
(10 µl, 100 µM, final concentration) was incubated for 48 h at 37
oC, following which the compounds or buffer was added and
incubated at 37 oC for 24 h. In order to measure the amyloid fibril
formation Th-T fluorescence method was employed [36,37].
Furthermore experiment with blanks containing phosphate buffer
(50 mM. pH 7.4) inplace of Aβ with or without test compounds
were also carried out. After the incubation was done the samples
were diluted with glycine-NaOH buffer (50 mM, pH 8.0) to a
final volume of 200 µl.
AUC = 1 +  (i / o)
i = 1
where, 0 is fluorescence reading at 0 min
i is fluorescence reading at time i.
Net AUC was calculated by equation:
AUCsample – AUCblank
The ORAC-FL value was calculated by the standard curve,
and this value is expressed as Trolox equivalents.
4.4.7. DPPH
4.4.4. Concentration dependent self Aβ aggregation
The radical scavenging activity of tested compounds was
determined by the reaction of stable DPPH radical, with the
method of Choi et al. [38] with some modifications. Scavenging
activity of tested compounds was determined at 50 µM
concentration. Experiment was performed in 96 well plates in
triplicates. Assay solution containing 180 µl of methanol was
added in each well, followed by 10 µl of the compounds except
in the control well. 10 µl of DPPH (0.1 mM) was added after 5
min of incubation into all wells and then the mixture was stand to
incubate for 30 min. Absorbance was measured at 517 nm by the
ultraviolet spectrophotometer. The free radicals scavenging
ability was calculated as follow:
assay
The dilution of Aβ stock solution was done with phosphate
buffer (50 mM, pH 7.4). The mixture of the peptides (at 10 µl,
100 µM, final concentration) with or without the compound (10,
15, 20 and 25 µM) was incubated at 37o C for 48 h. For
quantifying amyloid fibril formation, the Th-T fluorescence
method was utilized. Experiments for blank samples containing
phosphate buffer (50 mM, pH 7.4) inplace of Aβ with or without
test compounds were also carried out. The samples were diluted
after incubation, to a final volume of 200 µl using glycine-NaOH
buffer (50 mM, pH 8.0).
(A1-A2)/A1*100,
4.4.5. Transmission Electron Microscopy (TEM)
Assay
where A1 and A2 are the absorbance of the control treatment
and test compounds.
Incubation of Aβ1-42 (in phosphate buffer (50 mM, pH 7.4))
was done with and without synthesized compounds at 37 C.
Concentrations of Aβ1-42 and synthesized compounds were 100
and 25 µM, respectively. After the incubation of 48 h, aliquots of
samples (10 µL) were mounted on carbon grid, incubated for 1
min at RT; the grid was washed using double distilled water and
was negatively stained with sodium phosphotungstate (PTS) for 1
min. Excess staining solution was removed and the specimen was
transferred in transmission electron microscope (JEOL JEM-
1400) for imaging. All test inhibitors were solubilized in buffer
which was used for experiment.
4.4.8. ADME Property Prediction and TOPKAT
Analysis
For the prediction of ADME properties of inhibitors module
of Accelrys Discovery studio 4.0 was employed. An estimate of
bioavailability and physicochemical properties of inhibitors is
generated by this software. Calculation of parameters like polar
surface area, Predicted brain/blood partition coefficient, solvent
accessible surface area, and human oral absorption was carried
out.
The assessment of virtual toxicity risk with TOPKAT module
of Accelrys Discovery studio 4.0 was done which predicts
chemicals toxicity from 2D molecular structure rapidly and
accurately. It uses a variety of powerful, cross-validated
Quantitative Structure Toxicity Relationship (QSTR) models for
estimating different measures of toxicity. Test inhibitors were
studied regarding dose dependent toxicity parameters such as
NTP and FDA rodent carcinogen models, ocular irritancy, skin
irritancy, mutagenicity, developmental toxicity potential and
aerobic biodegradability.
4.4.6. Oxygen Radical Absorbance Capacity
(ORAC-FL) Assay
The antioxidant nature of synthesized inhibitors was
determined based on the oxygen radical absorbance capacity-
fluorescein (ORAC-FL) assay. Phosphate buffer (75 mM, pH
7.4) was used to carry out reaction and the final reaction mixture
volume was 200L. The FL (120 L; 70 nM, final concentration)
and antioxidant (20 L) solutions were placed in black 96-well
microplate (96F untreat, Nunc). The mixture was preincubated
(15 minutes) at 37 C, after which AAPH solution (60 L, 12
mM, final concentration) was rapidly added using a multichannel
pipette. Loaded microplate was kept in the reader immediately.
Every minute reading was recorded for fluorescence for
continuous 80 minutes (excitation, 485 nm; emission, 520 nm).
Measurement was done at eight individual concentrations (1–
8M). A blank (FL + AAPH) solution with phosphate buffer
Acknowledgments
One of the authors T.U. is thankful to University Grant
Commission (UGC), India for providing BSR meritorious
fellowship. Author S.S. wishes to acknowledge Ministry of
AYUSH for the (JRF) financial assistance. Furthermore M.T. is

37. Repsold, B. P.; Malan, S. F.; Joubert, J.; Oliver, D. W. SAR QSAR
Environ. Res. 2018, 29, 231-55.
38. Choi, C. W.; Jung, H. A.; Kang, S. S.; Choi, J. S.; Arch. Pharm.
Res. 2007, 30(1), 1-7.
grateful to the University of Delhi (D.U.) for sanctioning of
research funds for carrying out the study.
References and notes
1. Kumar, J.; Meena, P.; Singh, A.; Jameel, E.; Maqbool, M.;
Mobashir, M.; Shandilya, A.; Tiwari, M.; Hoda, N.; Jayaram, B.;
Eur. J. Med. Chem. 2016, 119, 260-77.
2. Umar, T.; Hoda, N.; Curr. Top. Med. Chem. 2017, 17(31), 3370 –
3389.
3. Greig, N.H.; Utsuki, T.; Yu, Q.; Zhu, X.; Holloway, H.W.; Perry,
T.; Lee, B.; Ingram, D.K.; Lahiri, D.K. Curr. Med. Res. Opin.
2001, 17, 159-165.
4. Wang, L. et al. Eur. J. Med. Chem. 2014, 80, 543–561.
5. Kamal, M. A. et al. Neurochem. Res. 2008, 33, 745–753.
6. Mushtaq, G.; Greig, N. H.; Khan, J. A.; Kamal, M. A. CNS
Neurol. Disord. Drug Targets 2014, 13, 1432-1439.
7. Pohanka, M. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc
Czech. Repub. 2011, 155, 219–29.
8. LaFerla, F.; Green, K.; Oddo, S. Nat. Rev. Neurosci. 2007, 8, 499–
509.
9. Nelson, P. T.; Alafuzoff, I.; Bigio, E. H.; et al. J. Neuropathol.
Exp. Neurol. 2012, 71, 362-81.
10. Citron, M. Nat. Rev. Drug Discov. 2010, 9, 387–398.
11. Haass, C.; Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 2007, 8, 101–
112.
12. Ricciarelli, R.; Fedele, E. Curr. Neuropharmacol. 2017, 15, 926–
935.
13. Bonda, D. J.; Wang, X.; Perry, G.; Nunomura, A.; Tabaton, M.;
Zhu, X.; Smith, M. A. Neuropharmacology 2010, 59, 290−294.
14. Gujjar, R.; Marwaha, A.; El-Mazouni, F.; White, J.; White, K. L.;
Creason, S.; Shackleford, D. M.; Baldwin, J.; Charman, W. N.;
Buckner, F. S.; Charman, S.; Rathod, P. K.; Phillips, M. A. J.
Med. Chem. 2009, 52, 1864-1872.
15. Fischer, G. Adv. Heterocycl. Chem. 2008, 95, 143-219.
16. El-Gendy, M. M. A.; Shaaban, M.; Shaaban, K. A.; El-Bondkly,
A. M.; Laatsch, H. J. Antibiot. 2008, 61, 149.
17. Kumar, C. S. C.; Loh, W. S.; Ooi, C. W.; Quah, C. K.; Fun, H. K.
Molecules. 2013, 18, 11996-12011.
18. Kryger, G.; Silman, I.; Sussman, J. L. Structure, 1999, 7, 297-307.
19. Crescenzi, O.; Tomaselli, S.; Guerrini, R.; Salvadori, S.; D’Ursi,
A. M.; Temussi, P. A.; Picone, D. Eur. J. Biochem. 2002, 269,
5642−5648.
20. Brus, B.; Kosak, U.; Turk, S.; Pislar, A.; Coquelle, N.; Kos, J.;
Stojan, J.; Colletier, J. P.; Gobec, S. J. Med. Chem. 2014, 57,
8167-79.
21. Ellman, G. L.; Courtney, K.; Andres, V.; Feather-Stone, R. M.
Biochem. Pharmacol. 1961, 7, 88-95.
22. Gupta, A.; Gandhimathi, A.; Sharma, P.; Jayaram, B. Protein
Pept. Lett. 2007, 14, 632-646.
23. Kelder, J.; Grootenhuis, P. D.; Bayada, D. M.; Delbressine, L. P.;
Ploemen, J. P. Pharm. Res. 1999, 16, 1514-9.
24. Vijayaramalingam, K.; Chandrasekaran, S.; Nagarajan, S. Pharm.
Chem. J. 2007, 41, 253-255.
25. Nepali, K.; Singh, G.; Turan, A.; Agarwal, A.; Sapra, S.; Kumar,
R.; Banerjee, U. C.; Verma, P. K.; Satti, N. K.; Gupta, M. K.; Suri,
O. P.; Dhar, K. L. Bioorg. Med. Chem. 2011, 19, 1950–1958.
26. Tripathi, A. C.; Upadhyay, S.; Paliwal, S.; Saraf, S. K. Med.
Chem. Res. 2016, 25, 390–406.
27. Ingarsal, N.; Saravanan, G.; Amutha, P.; Nagarajan, S. Eur. J.
Med. Chem. 2007, 42, 517-520.
28. Jayaram, B.; Singh, T.; Mukherjee, G.; Mathur, A.; Shekhar, S.;
Shekhar, V. BMC Bioinfo. 2012, 13, p. S7.
29. Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.;
Cheatham, T. E.; DeBolt, S.; et al. Comput. Phys. Commun. 1995,
91, 1-41.
30. Jain, T.; Jayaram, B. FEBS Lett. 2005, 579, 6659-6666.
31. Singh, T.; Biswas, D.; Jayaram, B. J. Chem. Inf. Model. 2011, 51,
2515-2527.
32. DeLano, W. L. PyMOL Reference Manual, DeLano Scientific
LLC, San Carlos, CA, USA, 2003.
33. Walker, N.; Stuart, D. Acta Crystallogr., Sect. A: Found.
Crystallogr. 1983, 39, 158−166.
34. Sheldrick, G.M. Acta Crystallogr., Sect. C: Struct. Chem. 2015,
71, 3−8.
35. Farrugia, L. J. J. Appl. Crystallogr. 2012, 45, 849−854.
36. Manral, A.; Saini, V.; Meena, P.; Tiwari, M. Bioorg. Med. Chem.
2015, 23, 6389 403.