Synthesis and screening of triazolopyrimidine scaffold as multi-functional agents for Alzheimer's disease therapies

Article abstract of DOI:10.1016/j.ejmech.2016.04.053

In present study a series of triazolopyrimidine-quinoline and cyanopyridine-quinoline hybrids were designed, synthesized and evaluated as acetylcholinesterase inhibitors (AChEIs). Molecular docking and scoring was utilized for the design of inhibitors. The molecules were synthesized via an easily accessible, convergent synthetic route. Three triazolopyrimidine based compounds showed nanomolar activity towards acetylcholinesterase. Among them, Ethyl 6-fluoro-4-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)quinoline-3-carboxylate (10d), strongly inhibited AChE with IC₅₀ value of 42 nM. Furthermore compound 10d was identified as most promising compound with 12 fold selectivity against butyrylcholinesterase (BuChE). This compound displayed a composed multitargeted profile with promising inhibition of self-induced and AChE - induced Aβ aggregation and antioxidant activity.

Full text of DOI:10.1016/j.ejmech.2016.04.053

Accepted Manuscript
Synthesis and screening of triazolopyrimidine scaffold as multi-functional agents for
Alzheimer's disease therapies
Jitendra Kumar, Poonam Meena, Anju Singh, Ehtesham Jameel, Mudasir Maqbool,
Mohammad Mobashir, Ashutosh Shandilya, Manisha Tiwari, Nasimul Hoda, B.
Jayaram
PII:
S0223-5234(16)30346-4
10.1016/j.ejmech.2016.04.053
EJMECH 8568
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2016
Revised Date: 15 April 2016
Accepted Date: 22 April 2016
Please cite this article as: J. Kumar, P. Meena, A. Singh, E. Jameel, M. Maqbool, M. Mobashir, A.
Shandilya, M. Tiwari, N. Hoda, B. Jayaram, Synthesis and screening of triazolopyrimidine scaffold as
multi-functional agents for Alzheimer's disease therapies, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.04.053.
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ACCEPTED MANUSCRIPT
Synthesis and screening of triazolopyrimidine scaffold as multi-functional agents for
Alzheimer’s disease therapies
Jitendra kumar1, Poonam Meena2, Anju1, Ehtesham Jameel3, Mudasir Maqbool1, Mohammad
Mobashir1, Ashutosh Shandilya4, Manisha Tiwari2*, Nasimul Hoda 1*, B. Jayaram4,5,6
1. Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi
110025, India.
2. Dr. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi
110007, India.
3. Department of Chemistry, B. R. Ambedkar Bihar University, Muzaffarpur 842001,
Bihar, India
4. Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New
Delhi-110016, India
5. Kusuma School of Biological Sciences, IIT Delhi, New Delhi-110016, India
6. Supercomputing facility for Bioinformatics & Computational Biology, IIT Delhi, New
Delhi-110016
*Corresponding Authors,
#Nasimul Hoda, Ph.D.
Associate Professor
$Manisha Tiwari, Ph.D.
Assistant Professor
Department of Chemistry
Jamia Millia Islamia,
Jamia Nagar, New Delhi- 110025,
India
Dr. Ambedkar Centre for Biomedical
Research, University of Delhi,
New Delhi 110007,
India
Email: nhoda@jmi.ac.in
Email: mtiwari07@gmail.com

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Highlights
•
A series of triazolopyrimidine-quinoline and cyanopyridine-quinoline hybrids were
designed and synthesized.
•
•
Good yields, high regioselectivity were registered.
The inhibiting activity of synthesized compounds on acetylcholinesterase (AChE),
butyrylcholinesterase (BuChE) were assessed. Three compounds were found to have
nanomolar activity and remaining compounds showed submicromolar to micromolar
activity.
•
•
•
AChE selectivity of most promising compound was ~ 12 fold more potent than
BuChE.
Inhibition of Aβ1-42 and AChE-induced Aβ1-42 aggregation of synthesized compounds
were more effective than curcumin.
Oxygen radical absorbance capacity of synthesized compounds was superior to that of
Trolox.

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Graphical Abstract

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Abstract
In present study a series of triazolopyrimidine-quinoline and cyanopyridine-quinoline hybrids
were designed, synthesized and evaluated as acetylcholinesterase inhibitors (AChEIs).
Molecular docking and scoring was utilized for the design of inhibitors. The molecules were
synthesized via an easily accessible, convergent synthetic route. Three triazolopyrimidine
based compounds showed nanomolar activity towards acetylcholinesterase. Among them,
Ethyl 6-fluoro-4-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)quinoline-
3-carboxylate (10d), strongly inhibited AChE with IC50 value of 42 nM. Furthermore
compound 10d was identified as most promising compound with 12 fold selectivity against
butyrylcholinesterase (BuChE). This compound displayed a composed multitargeted profile
with promising inhibition of self-induced and AChE - induced Aβ aggregation and
antioxidant activity.
Keywords:
Alzheimer’s disease, Acetylcholinesterase, triazolopyrimidine, quinoline, molecular docking,
butyrylcholinesterase.

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1. Introduction
Alzheimer’s disease (AD) is an incomprehensible neurodegenerative ailment of elderly
people with no efficacious disease modifying treatment. With remarkable increase in life
expectancy, an increase in the percentage of aging population, has resulted in a phenomenal
jump in the number of age related illness. Roughly, 40 million people worldwide are
presently tormented by AD and this number is projected to double every 20 years [1]. The
neuropathological hallmarks of Alzheimer’s brain is the augmentation and accumulation of
extracellular β-amyloid plaques and intraneuronal neurofibrillary tangles that interfere with
neurons and results in the diminution of basal forebrain cholinergic neurons. The strategy
associated with cholinergic hypothesis involves acetylcholinesterase inhibitors (AChEIs)
which are still the main pharmacological approach applied for the treatment of AD [2].
Reversible inhibitors of the enzyme AChE such as donepezil, rivastigmine, galantamine, and
tacrine (Fig. 1) [3] which diminishes the rate of decomposition of neurotransmitter
acetylcholine at synapses in the brain. These FDA approved AChEIs are the first line
medications used in the treatment of this devastating disease. The use of AChEIs slows down
the progression of memory loss and enhance cognitive and behavioural symptoms associated
with AD and analogous dementias. These drugs have modest benefits and are used for the
symptomatic treatment of the disease, however, they do not address the essential
neuropathology. Further, drawbacks of these drugs are uninvited side effects like nausea,
vomiting, and liver damage which are correlated to the abundance of excess cholinergic
neurons.
The crystal structure of Torpedo Californica AChE [4] (TcAChE) brought to light a
unique architecture, the catalytic triad Ser-His-Glu, positioned in the centre of symmetry of
the molecule at the basement of a roughly 20Å deep narrow gorge which is encrusted
primarily by aromatic residues. In the close proximity of catalytic triad Trp84 is responsible
for the cation-π interactions with the substrate ACh. While peripheral anionic site (PAS)
composed of Tyr70 and Trp279 is present at the ingress of the gorge and behave as a
molecular traffic switch and direct the substrate enroute towards the active site. Tryptophan
residues at CAS and PAS command the positioning of charged end of the substrate that enters
the active site. These two residues are also involves in another interesting feature of enzyme
where CAS and PAS cross-talk with each other. Tyr121 and Phe330 form a barrier at the
middle of the narrow hydrophobic gorge. At this point Phe330 shows high degree of
conformational change and behave as a dynamic door/lock for substrate. Characteristically,

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inhibitors which bind either site will inhibit the AChE and hence, can be used for the
symptomatic treatment of AD. Recent discoveries suggest AChE’s secondary non-classical
functions, associated to adhesion, discrimination, neurite outgrowth, and allosteric
modulation. Contemporary evidence additionally conjecture a connection between β-amyloid
fibril depositions at PAS [5,6]. Consequently, ligands which binds PAS will prevent access of
substrate to the active site, furthermore, it will also inhibit β-amyloid plaque aggregation in
AD.
In consideration of these findings and due to the collapse of β-secretase inhibitors at
clinical trial AChEIs have re-emerged at the centre of AD drug design. Development and
broadening of AChE’s ligands that attune both amyloid and cholinergic targets. Numerous
endeavours have been converged on designing dual binding site inhibitors which halts pro-
aggregating activity of PAS and elevating the AChE level in the brain. Dual binding site
inhibitors [7,8] therefore must target concurrently with Trp84 near CAS and Trp279 at PAS
for greater potency and tight binding to the enzyme [9,10]. Nevertheless, whereas AChE
levels are decreased in brains of AD, levels of butyrylcholinesterase (BuChE) are increased
[11]. However, certain findings point towards beneficial effect of butyrylcholinesterase
inhibitor (BuChEI) in the treatment of AD patients [12,13].
N
NH2
O
O
N
O
Tacrine
Donepezil
CH3
N
CH3
O
O
N
H
H3C
N
Rivastigmine
Galantamine
Fig. 1: Commercially available AChE inhibitors

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Among the multiple factors that contribute towards AD progression, Aβ plays
significant role in the pathogenesis of AD. The development of drugs which target inhibition
of Aβ fibril aggregation is presently a leading approach for the symptomatic treatment of AD
[14,15] The additional associated hypothesis, namely oxidative stress, occurs early in the
progression of AD [16]. Aging of the brain is the leading risk factor for disease development,
resulting from an imbalance between reactive oxygen species (ROS) [17,18] production and
antioxidant defences. Several evidences advise that oxidative stress improves the
manifestation of major disease hallmarks namely, the development of amyloid plaques and
neurofibrillary tangles formation in brain. Therefore, drugs that target on clearing or
preventing the formation of the free radicals in the brain would be beneficial for AD.
Combined scaffolds of quinoline and triazolopyrimidine were preferred choices due to
their various worthwhile effects. Quinoline has a great pharmacological history among which
its neuroprotective effect, AChE inhibitory activity, free radical scavenging effect, and metal-
chelating ability are important to mention. Since Aβ1–42 has a high affinity for metal ions and
forms insoluble accumulations, particularly in the presence of Cu(II), and somewhat with
Zn(II) and Fe(III). Metal-chelating capability of quinolone scaffold that acts as a bidentate
ligand was expected to dissolve amyloid aggregates of the brain, apparently by removing
metal ions from the structure and come up as a new therapeutic agent in the treatment of AD
[19-21].
Quinoline core structure was expected to interact with residues of CAS of AChE via π-π
interaction. Besides this it was supposed to intercalate between Aβ sheets and was expected
to enhance its disaggregation because of its planner structure. Additionally, AChE inhibitors
that bind to its PAS have emerged as promising AD modifying candidates [22],
triazolopyrimidine scaffold was anticipated to interact with the important residues at PAS of
AChE. Indeed, triazole scaffold has an excellent previous record in the inhibition of AChE
and BuChE [23]. Considering the above mentioned facts that both the scaffolds exhibit a vast
range of biological properties and have not been considered in one drug candidate as anti-
AD. We decided to incorporate these scaffolds in one drug candidate and investigate AChEI
activity.

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Fig. 2: Design strategy of multifactorial anti-AD agent.
Taking these features into consideration, we designed and synthesized a new series of
quinoline-triazolopyrimidine and quinoline-cyanopyridine hybrids connected through
piperazine [24] as potential multifunctional agents for the treatment of AD. All synthesized
compounds were examined for their biological activities against inhibition of AChE, BuChE,
self-induced Aβ1-42 aggregation, AChE-induced Aβ1-42 aggregation and antioxidant
properties. Furthermore, interaction of inhibition for the compounds were investigated
through molecular docking and kinetic studies.
2. Results and discussion :
2.1. Chemistry
The synthesis of the target molecules has been carried out as shown in Synthetic
Scheme 1, 2 and 3. The Synthetic route for the synthesis of 1-{5-methyl-[1,2,4]triazolo[1,5-
a]pyrimidin-7-yl} piperazine linked to Quinoline derivatives have been demonstrated in
Synthetic Scheme 1. Synthesis of 2-(piperazin-1-yl)pyridine-3-carbonitrile linked to
Quinoline derivatives have been demonstated in Synthetic Scheme 2. The synthesis of
compound 2-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)nicotinonitrile
have been demonstrated in Synthetic Scheme 3. The intermediate compounds were
synthesized according to the literature method [25-27]. In the Scheme 1, the multistep
sequence for synthesis of the compounds involved the initial preparation of substituted
diethyl 2-((phenylamino) methylene)malonate (3) from Diethyl (ethoxymethylene) malonate

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(1) and substituted aniline (2) followed by intramolecular cyclization in the presence of
excess phosphorous oxychloride to give substituted compound ethyl 4-chloroquinoline-3-
carboxylate (4). Initial preparation of 5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (7) from
condensation reaction between ethylacetoacetate with 3-amino-1,2,4-triazole. Reaction of 5-
Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (7) with an excess of POCl3 gave 7-Chloro-5-
methyl-[1,2,4]triazolo[1,5-a]pyrimidine
(8).
Reaction
of
7-Chloro-5-methyl-
[1,2,4]triazolo[1,5-a]pyrimidine (8) with piperazine to give 5-methyl-7-(piperazin-1-yl)-
[1,2,4]triazolo[1,5-a]pyrimidine (9). Finally, different substituted compound of ethyl 4-(4-(5-
methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)quinoline-3-carboxylate (10a-10e)
were synthesized through nucleophilic substitution reaction between 5-methyl-7-(piperazin-
1-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (9) with substituted compound ethyl 4-chloroquinoline-
3-carboxylate (5).
In the scheme 2, 2-(piperazin-1-yl)nicotinonitrile (12) was synthesized through
nucleophic substitution reaction of piperazine on 2-chloro-3-cyanopyridine (11). Finally,
different substituted compound of ethyl 4-(4-(3-cyanopyridin-2-yl)piperazin-1-yl)quinoline-
3-carboxylate (13a-13e) is synthesized through substitution of 2-(piperazin-1-
yl)nicotinonitrile (12) on substituted compound ethyl 4-chloroquinoline-3-carboxylate (4).
In the scheme 3, Finally, compound 2-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
yl)piperazin-1-yl)nicotinonitrile was synthesized through nucleophilic substitution reaction
of 2-chloro-3-cyano pyridine (11) on 5-methyl-7-(piperazin-1-yl)-[1,2,4]triazolo[1,5-
a]pyrimidine (9). The structure of these compounds were confirmed on the basis to their
analytical and spectroscopic data (1H,13C NMR and Mass spectra).

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Synthetic scheme 1
Synthesis of Quinoline derivatives linked to 1-{5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl}piperazine
Reagents and conditions
(i) Benzene, 83 0C; (ii) POCl3, 110 0C; (iii) Acetic acid, 110 0C; (iv) POCl3, 1100C; (v) piperazine , K2CO3, 1,4-
dioxane, 100 0C; (vi) K2CO3, 1,4-dioxane, 100 0C
Serial No.
R1
R2
R3
R4
H
H
H
H
H
CH3
OCH3
H
H
H
H
H
H
H
F
10a
10b
10c
10d
10e
CF3
H
F
H
H

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Synthetic scheme 2
Synthesis of Quinoline derivatives linked to 2-(piperazine-1-yl)pyridine-3-carbonitrile
Reagents and conditions
(i) K2CO3, 1,4-dioxane, 100 0C; (ii) K2CO3, 1,4-dioxane, 100 0C.
R1
H
R2
R3
H
R4
H
Serial No.
13a
CH3
H
H
H
H
OCH3
H
CF3
H
H
H
H
F
13b
13c
13d
13e
H
F
H
H

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Synthetic scheme 3
2-chloro-3-cyanopyridine linked with 1-{5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl}piperazine
2.2. Design of Multifunctional ligands
To cultivate a drug candidate capable of targeting manifold AD pathological factors, we
undertook structure based drug design approach. Triazolopyrimidine was selected as
preferred scaffold, because it’s planar bicyclic structure was expected to engage in π-π
stacking interaction with Trp279 present at PAS, in addition to that quinolones are known to
have neuroprotective and radical scavenging capability. Inclusion of piperazine was a natural
choice to connect two electrophilic centres. Nitrogen of piperazine was expected to be in
protonated state and hence may engage in π-cation interactions with the residues of aromatic
gorge.
Pardock [28] module of Sanjeevini (SCFBIO, IITD) a comprehensive drug design suite
was selected for docking and scoring. The computational binding free energy (ꢀGb) of
interaction of the synthesized compounds is mentioned in Table 1.
The position of compound 10d , 10e, and 10c with respect to the key residue in the
binding site is shown in Fig. 3(A, B), Fig. 3(C, D), and Fig. 3(E, F) respectively. In
Fig. 3(A, B) triazolopyrimidine interacts through π-π stacking with the indole ring of Trp279
with an average distance of 5 Å. Trp84 possessing aromatic moiety is involved in π-π
stacking with quinoline ring with aromatic-aromatic distance of 4.4 Å. Piperazine moiety is
expected to enhance its binding affinity with hydrophobic residue, such as Phe330, Phe331
and Tyr121 in the middle of the gorge. Through the Ligplot, [29] strong hydrogen bonding
was observed between the fluorine of quinoline and the hydroxyl group of Ser200 with
donor-acceptor distances of 2.90 Å. In addition, fluorine of quinolone forms hydrogen bond

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of 3.34 Å with the amine of Gly119. The piperazine ring may have van der Waals interaction
with the phenyl rings of Phe330 and Phe331. The docking study of compound 10e was also
done as illustrated in Fig. 3(C, D). Triazolopyrimidine ring forms π stacking with Trp84 with
a
separation
of
3.5 Å between the centre of mass for the aryl- aryl rings and Trp279 with the quinoline ring
with an average distance of 5.2 Å respectively. In Fig. 3(E, F), triazolopyrimidine and
quinoline rings engage in π-π stacking with Trp84 and Trp279 with distances of 3.6 Å and
Trp279 respectively. Hydrogen bond of distance 2.85 Å and 2.74 Å was observed between
oxygen of quinoline-ester and the hydroxyl group of Ser122 and triazolopyrimidine and
hydroxyl group of Tyr130 respectively. The results of docking study determine the dual
binding affinity of selected compounds due to π-π (aromatic), hydrophobic interactions and
hydrogen bonding with not only CAS and PAS but also with the aromatic gorge of AChE.
A similar docking study was performed on reported crystal structure of Human
BuChE [30] (PdB ID 4XII). The binding free energies of the synthesized compounds with the
enzyme is depicted in Table 1. Compounds 10d and 10e showed better binding interactions
with BuChE. Further docking study of the compounds 10d and 10e is provided in
supplementary material, Fig. S1(A,B) and Fig. S1(C, D) respectively.
Table 1: The calculated free energies of selected derivatives in molecular docking using Par DOCK [28]
Compound
Calculated free energy
(Kcal/mole) AChE
Calculated free energy
(Kcal/mole) BuChE
-7.83
-8.91
-8.05
-8.72
-8.70
-10.23
-9.97
-9.04
-9.65
-10.69
-7.52
-7.49
-7.55
-6.55
-7.44
-8.30
-9.58
-8.38
-8.86
-9.48
10a
10b
10c
10d
10e
13a
13b
13c
13d
13e

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-7.52
-6.30
14
Fig. 3: The inhibitors are shown in ball and stick. The key residues are shown in stick and different residues are
shown by different colour such as TYR70, TYR121, TYR279 (Green), TRP84, TRP279 (Magenta), SER122,
SER200 (Cyan), GLU199 (tint-wheat), PHE330, PHE331 (Yellow), HIS440 (Orange). Plot (A,C, E) shows

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interaction of inhibitor (10d, 10e, 10c) with key residue in the active site of TcAChE respectively. Plot (B,D,E)
shows Ligplot representation of inhibitor (10d, 10e, 10c) respectively. Hydrogen bonds are indicated with green
dashed lines and π-π stacking are shown by yellow dashed lines. Residues involved in hydrophobic interactions
with the ligand are shown with red rays.
2.3. Pharmacological evaluation
2.3.1. Cholinesterase inhibition and SAR studies
The ability of the synthesized derivatives (10a-e, 13a-e & 14) to inhibit both
cholinesterases (EeAChE and eqBuChE) was evaluated in vitro using spectrophotometric
method of Ellman et.al. [31]. Tacrine and donepezil were taken as reference compounds. The
IC50 values of all test compounds and their selectivity index for AChE over BuChE are
summarized in Table 2. It could be seen from the table that all novel target compounds are
potent ChE inhibitors with IC50 values ranging from micromolar to sub-micromolar.
Structure-activity relationship (SAR) studies indicated that the cholinesterase inhibition and
selectivity were sensitive to substitution to the quinoline ring and the variation of N-
containing heterocycles at the other terminal end of piperazine ring. Among the triazolo
pyrimidine series (10a-e), fluoro-substituted compounds 10c, 10d and 10e provided greater
ChE inhibition and selectivity toward AChE. In particular, compound 10d having fluoro
group at para position of quinoline ring was emerged as the most active compound (IC50 =
0.042 ꢁM), being ~3.2-fold more potent than the reference drug tacrine. In addition,
compound 10d also demonstrated a high selectivity for AChE over BuChE (~12-fold).
Furthermore, substitution of fluoro group at the ortho position (compound 10e) resulted in
slight decrease in AChE inhibitory activity (IC50 = 0.097 ꢁM). However, the presence of a
trifluoromethyl group at meta position on quinoline ring in compound 10c, provided
moderate AChE inhibition in comparison to the most potent compound 10d. Furthermore, it
was observed that substitution of the methyl group at para position of qunoline ring in
compound 10a provided moderate AChE inhibition (IC50 = 2.64 ꢁM). When the methyl
group was replaced with methoxy group (compound 10b), the AChE inhibitory activity
improved around ~3.5-fold. Most of compounds in this series showed slightly higher
inhibitory activity for AChE than for BuChE, indicating that these compounds were selective
inhibitors for AChE. From the triazolo pyrimidine series (10a-e), compounds 10d and 10e
provided the most potent inhibition activity for AChE. In terms of BuChE inhibition, both
these compounds (10d and 10e) were identified as equally potent BuChE inhibitors (IC50 =
0.51 and 0.18 ꢁM), being 14- and 5-fold more potent than the reference compound donepezil.

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Interestingly, IC50 values of both these compounds 10d and 10e were found to be lower than
that reported by well-known AChEIs galantamine [32,33] (~74- and 32- fold) and
rivastigmine [34] (~106- and 46-fold). In the series 13a-e, introduction of cyanopyridine
group at N-4 terminal of piperazine ring dramatically reduced the inhibition of AChE, while
the BuChE inhibition was improved slightly. Interestingly, the similar AChE inhibition
pattern was analyzed in case of fluoro substituted compounds 13d (IC50 = 0.58 ꢁM), 13e (IC50
= 0.73 ꢁM), 13c (IC50 = 0.88 ꢁM) which further emphasized that fluoro group is more
effective at ortho and para position in comparison to meta. However, the anticholinesterase
activity of these compounds was significantly reduced from 13.8- to 4.8-fold value in
comparison to the compounds of triazolo pyrimidine series. Furthermore, it was observed that
substitution of the methyl and methoxy group at para position of quinoline moiety led to the
compounds 13a (IC50 = 3.49) and 13b (IC50 = 1.1 ꢁM) with lower potencies. Replacing the
quinoline ring with triazolo pyrimidine heterocycle in compound 14 led to a decline in AChE
inhibitory potency (IC50 = 1.39) while the BuChE inhibitory activity was increased sharply
relative to compounds of cyanopyridine series. The present study suggested that the presence
of triazolo pyrimidine scaffold played a significant role in determining the inhibitory activity
for AChE. The general trend for ChE inhibition in terms of substitution at quinoline ring was
of the order: F > CF3 > OCH3 > CH3, suggesting the role of increasing electronegativity with
superior ChE inhibition.

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Table 2: Inhibition of AChE and BuChE by the test compounds, and their selectivity index.
AChEa
BuChEb
Selectivityc
Compounds
Structure
IC50 (ꢀM)
IC50 (ꢀM)
BuChE/AChE
2.64 ± 0.52
0.76 ± 0.67
0.18 ± 1.03
0.042 ± 0.79
4.09 ± 0.77
2.01 ± 2.30
0.78 ± 1.48
0.51 ± 0.91
1.55
10a
2.64
10b
10c
10d
4.33
12.14
0.097 ± 1.22
0.18 ± 1.03
1.85
0.85
10e
13a
3.49 ± 0.81
3 ± 1.33

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1.10 ± 1.09
0.88 ± 0.92
0.58 ± 0.80
0.73 ± 1.12
1.9 ± 0.96
1.72
1.11
2.12
1.21
13b
13c
13d
13e
0.98 ± 1.55
1.23 ± 2.06
0.89 ± 0.70
1.39 ± 0.45
0.78 ± 1.08
0.56
14
0.13 ± 0.21
0.038 ± 0.34
3.12 ± 0.44
4.45 ± 0.37
0.054 ± 0.85
2.58 ± 0.65
15.8 ± 3.58
1.32 ± 0.09
0.40
66.55
5.1
Tacrine
Donepezil
Galantamine
0.30
Rivastigmine
a AChE from electric eel; IC50, inhibitor concentration (mean ± SEM for these experiments) resulting in 50%
inhibition of AChE.
b BuChE from equine serum; IC50, inhibitor concentration (mean ± SEM for these experiments) resulting in 50%
inhibition of BuChE.
c Selectivity for AChE: IC50 (BuChE)/IC50 (AChE).
2.3.2. Kinetic study of AChE inhibition
To gain further insight into the mechanism of action of this family of compounds, a
kinetic study was carried out with the most potent AChE inhibitor 10d. Graphical analysis of
the reciprocal Lineweaver-Burk plot (Fig. 4) showed both increased slopes (decreased Vmax)
and intercepts (higher Km) at higher inhibitor concentration. This pattern indicated a mixed-

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type inhibition and therefore revealed that compound 10d might be able to bind to the
catalytic active site (CAS) as well as peripheral anionic site (PAS) of AChE, which were
consistent with our design strategy.
Control
24
22
20
18
16
14
12
10
8
12 nM
22 nM
42 nM
6
4
2
-10 -8 -6 -4 -2
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
1/S mM-1
Fig. 4: Lineweaver–Burk plot for the inhibition of AChE by compound 10d.
2.3.3. Inhibition of Self-Mediated Aβ1−42 Aggregation
Aβ generated from amyloid precursor protein is a critical player in the pathology of
AD. Aβ1-42 represents the predominant form found in senile plaques of AD brain, respect to
Aβ1–40. The Aβ1–42 peptide is generally considered to be more pathogenic because it has
greater propensity towards fibrillar aggregates which are neurotoxic in nature. Therefore,
preventing this peptide from aggregation could possibly be another therapeutic strategy for
AD treatment. This prompted us to investigate the ability of synthesized compounds towards
inhibition of self-assembly of Aβ1–42 oligomers through a thioflavin T-based fluorometric
assay. Curcumin, a known amyloid aggregation inhibitor, was used as a reference compound.
The results summarized in Table 3 showed that most of the compounds exhibited moderate
to good potencies (27.26% to 72.86% at 25 ꢁM) relative to that of curcumin (52.7% at 25 ꢁM)
indicating an inhibition of the Aβ1–42 self-aggregation process. Interestingly, compounds of
series 10a-e, which exhibited high inhibitory activity for AChE also displayed good
inhibitory potency against Aβ1–42 self-aggregation. The optimal Aβ1–42 aggregation inhibitory
potency was provided by compound 10d (72.8%), the most potent AChE inhibitor within this
series. Modifications of 10d with quinoline ring substituted with para-methoxy and ortho-

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fluoro groups generated 10b (61.5%) and 10e (64.1%), which provided good inhibitory
activities. In contrast, compounds 13a-c, 13e, 14 showed percentages of inhibition ranging
from 27.2 to 47.3%, nearly 1.9- to 1.1-fold lower than that of the curcumin. These results
implied that the presence of cyano pyridine heterocycle at terminal end of piperazine seemed
to be unfavourable for the inhibition of self-induced Aβ1–42 aggregation.
Table 3: Inhibition of Aβ1-42 peptide aggregation (with and without AChE) and oxygen radical absorbance
capacity (ORAC, trolox equivalents) of synthesized derivatives
Compounds
Aβ1-42 aggregation
Inhibition of AChE-induced
Trolox equivc
inhibitiona (%)
Aβ1-42 aggregationb (%)
52.54 ± 0.92
61.58 ± 0.77
43.78 ± 0.91
72.86 ± 1.20
64.16 ± 1.20
27.26 ± 0.88
39.80 ± 1.70
54.32 ± 1.44
68.66 ± 2.09
52 ± 0.99
2.13 ± 0.09
2.55 ± 0.45
2.08 ± 0.07
2.43 ± 0.61
1.96 ± 0.08
2.04 ± 0.72
2.71 ± 0.99
10a
10b
10c
10d
10e
13a
13b
80.45 ± 1.11
70.33 ± 2.63
ndd
ndd
44.19 ± 0.80
54.92 ± 0.55
47.39 ± 1.09
46.77 ± 2.11
52.79 ± 0.95
ndd
47.84 ± 0.92
2.33 ± 0.83
2.86 ± 1.04
2.66 ± 0.75
2.33 ± 1.02
ndd
13c
13d
58.95 ± 1.07
50.26 ± 2.61
49.44 ± 1.31
34.67 ± 1.50
9.3 ± 2.50
13e
14
Curcumin
Tacrine
Donepezil
ndd
ndd
25.7 ± 0.75
ndd
aThe Thioflavin-T fluorescence method was used. The maximum percentage inhibitions of aggregation (means
± SEM for three experiments) were found at the inhibitor concentration of 25 ꢁM.
b Co-aggregation inhibition of Aβ1−42 and AChE 0.02 U was detected by ThT assay. The maximum percentage
inhibitions of aggregation (means ± SEM for three experiments) were found at the inhibitor concentration of
100 ꢁM.
c Data are expressed as ꢁmol of trolox equivalent/ꢁmol of tested compound (mean ± SEM of three experiments).
d nd, not determined.

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2.3.4. Inhibition of AChE-Induced Aβ1−42 Aggregation
Accumulating evidences have indicated that AChE exerts its non-cholinergic function
by promoting the aggregation of Aβ peptide fragments. This action of AChE is primarily
mediated by the peripheral anionic site (PAS) through which it colocalizes with the Aβ
peptide and promote Aβ fibrillogenesis by forming stable AChE-Aβ complexes [5,35,36].
Therefore, compounds binding to the PAS can inhibit AChE-accelerated fibril aggregation.
The results showed that the synthesized compounds were more effective inhibitors of AChE-
induced Aβ1−42 aggregation than tacrine (9.3%) and donepezil (25.7%), with inhibition values
ranging from 47.8-80.4% at 100 ꢁM inhibitor concentration (Table 3). The results of the co-
aggregation study provided indirect evidence of ability of these compounds to interfere with
the AChE-induced Aβ aggregation. In particular, compounds 10b, 10d and 10e of triazolo
pyrimidine series were more effective than cyanopyridine series. Considering the results, the
higher Aβ anti-aggregating activity of the novel compounds seems to be indicative of their
dual binding site character.
2.3.5. Inhibition of Aβ1–42 fibril formation monitored by TEM
In order to complement the ThT binding assay, TEM analysis was performed on the
most potent Aβ1–42 aggregation inhibitors 10b, 10d and 10e to monitor the inhibitory activity
of these compounds on Aβ aggregation process. At 0h time point, there was no aggregation in
the samples containing Aβ1–42 with (Fig 5A) or without compounds (data not shown).
However, after 48 h of incubation at 37°C, the sample of Aβ1–42 alone had mostly aggregated
into dense, mature and bulky amyloid fibrils (Fig. 5B). In contrast, only small bulk
aggregates and no characteristic Aβ fibrils were observed in the electron micrographs of Aβ1–
samples incubated with 10d, 10e and 10b compounds (Fig. 5D, 5E& 5F) under same
42
experimental conditions. The TEM results were in agreement with the results of ThT binding
studies, which further proved that these compounds could effectively inhibit and slow down
the rate of Aβ1–42 fibrils formation in vitro.

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B
A
C
F
D
E
Fig. 5: TEM image analysis of Aβ1−42 aggregation in the presence of compounds 10d, 10e and 10b:(A) Aβ1−42
(25 ꢁM), 0 h (B) Aβ1−42 alone (25 ꢁM) was incubated at 37 °C for 24 h (C) Aβ1−42 (25 ꢁM) and curcumin (25
ꢁM) were incubated at 37 °C for 24 h (D) Aβ1−42 (25 ꢁM) and 10d (25 ꢁM) were incubated at 37 °C for 24 h (E)
Aβ1−42 (25 ꢁM) and 10e (25 ꢁM) were incubated at 37 °C for 24 h (F) Aβ1−42 (25 ꢁM) and 10b (25 ꢁM) were
incubated at 37 °C for 24 h.
2.2.6. Antioxidant activity in vitro
Oxidative stress is an important player in initiating a cascade of events involved in the
pathogenesis of AD [37]. Thus, its reduction is another crucial aspect in designing
multifunctional agents for AD treatment [38,39]. The antioxidant activities of all synthesized
compounds were determined by measuring the oxygen radical absorbance capacity of
fluorescein (ORAC-FL), and the results are presented in Table 3. Their ability to scavenge
radicals is expressed as Trolox (a vitamin E analog) equivalent (ꢁmol of Trolox equiv/ꢁmol
of tested compound), in a relative scale where ORAC (trolox) = 1. As shown in Table 3,
most of the target compounds demonstrated potent oxygen radical absorbance capacities,
ranging from 1.96- to 2.86-fold of the value of Trolox. Among tested compounds, compound
13d gave the best results with 2.86 Trolox equiv. interestingly, compounds 10b and 13b
which features methoxy group at para position of the quinoline ring in both the series showed
higher antioxidant activities than their corresponding methyl derivatives. Specifically, our
results implied that presence of methoxy and fluoro substituents at quinoline ring seems to be
important for radical absorbance capacity. Therefore, we further investigated the multitarget
profiles of compounds 10b, 10d and 10e which exhibited significant inhibition of AChE, Aβ
aggregation and strong antioxidant activity in vitro.
2.2.7. Pharmacokinetic properties

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Determination of ADME properties of the molecules is an essential guide for lead
generation that can avoid the failure of drug candidates in later stages of discovery and
development due to poor pharmacokinetic profile. The 'drug likeness' of newly designed
multi-targeted AChE inhibitors was analyzed using QikProp program of Schrödinger
software. About 45 physically significant descriptors and pharmacologically relevant
properties of these compounds were predicted. In the present study some of these important
properties were considered with special emphasis on the requirements of the CNS
(Supporting information, Table S1). Fundamental physiochemical features of CNS drugs
are mainly related to their ability to penetrate the blood–brain barrier (BBB) and exhibit CNS
activity. Our results indicated that all compounds showed drug like characteristics based on
Lipinski’s rule of five (mol_MW < 500, QPlogPo/w < 5, donorHB ≤ 5, accptHB ≤ 10). Based
on the predicted values for QPlogBB and CNS activity, all compounds might be able to
penetrate into the CNS. PSA is another important predictor for BBB. Generally, drugs aimed
at the CNS tends to have lower polar surface area (PSA) usually between optimal range of
60-70 Å2 [40]. The calculated theoretical PSA values for all compounds may support their
ability to penetrate the blood–brain barrier. Among calculated parameters, QPPCaco,
QPlogBB, QPLogKhsa, and QPPMDCK mainly account for the ability of the compound’s
distribution inside the body. All of these compounds showed high permeability for both in
vitro Caco-2 cells and in vitro MDCK cells. Moreover, all compounds displayed good oral
absorption. The amount of protein binding of CNS drugs is an important consideration which
tends to be rather high. The predicted values of QPlogKhsa for all compounds indicate their
strong binding with plasma protein. The predicted ADME properties revealed that all
compounds fulfil drug-like criteria and could be considered as good candidate for drug
development.
2.2.8. Cell viability and neuroprotection studies
To gain insight into the therapeutic potential of synthesized compounds, the cell
viability and neuroprotective capacity against different toxic insults (Aβ and oxidative stress)
were assayed, employing the human SH-SY5Y neuroblastoma cell line. This cell line is an
appropriate model to study neurotoxic effect of agents and frequently used for elucidating the
mechanisms of neurodegenerative diseases [41].

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Fig. 6: Effects of compounds on cell viability in SH-SY5Y cells. The cell viability was determined by the MTT
assay after 24 h of incubation with various concentrations of 10d, 10e and 10b. Data are means ± SD of three
independent experiments and expressed as percent of control value.
First, we examined the potential cytotoxic effects of compounds 10d, 10e and 10b on
the viability of SH-SY5Y cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT)-based colorimetric assay. The cells were incubated with varying
concentrations (1-50 ꢁM) of the test compounds for 24 h. As indicated in Fig. 6, test
compounds did not show significant effect on cell viability at any of the concentration tested
after 24 h of incubation.
Aβ-induced apoptotic neuronal cell death is a critical event in the pathology of AD.
Aβ1-42 is the most amyloidogenic isoform of Aβ that exhibits greater propensity towards
aggregation under different cell culture conditions [42]. Aβ1–42 induced neurotoxicity in cell
lines is considered as a reliable model in AD research because it mimic some aspects of
neurodegeneration underlying AD pathology. The neuroprotective effects of compounds 10d,
10e and 10b against Aβ1–42-induced cytotoxicity in SH-SY5Y neuroblastoma cells were
studied at three different concentrations (Fig. 7).

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150
100
50
**
**
**
**
**
*
*
###
0
10e(µM)+Aβ
10d(µM)+Aβ
10b(µM)+Aβ
Fig. 7: Protective effects of compounds 10d, 10e and 10b on Aβ1–42 -induced cytotoxicity in SH-SY5Y cells. (a)
Determination of the viability of SH-SY5Y cells by the MTT assay after treatment with (25 µM) Aβ1–42 in the
absence or presence of the indicated concentrations of compounds 10d, 10e and 10b. All data were expressed as
mean ± SD of three experiments and each included triplicate sets. ###p < 0.001 vs control; *p < 0.05, **p <
0.01, ***p < 0.001 vs Aβ1–42 alone.
Treatment of SH-SY5Y cells for 24 h with 25 ꢁM Aβ1–42 caused 57.8% reduction in
cell viability compared to control cells. On the other hand, all the compounds exhibited
neuroprotective effects and maintained efficient cell viability (~ 79 to 83%) at higher
concentration 10 and 25 ꢁM in a dose dependent manner.

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A
B
C
D
E
Fig. 8: Representative micrographs of SH-SY5Y cells morphology after treatment with Aβ1–42 in the absence or
presence of compounds 10d, 10e and 10b. (A) Control cells without Aβ1-42 treatment (B) Aβ1-42 (25 ꢁM) alone
induced neurotoxicity (C) Co-incubation of compound 10d and Aβ1-42 (25 ꢁM) for 24 h at 37ºC (D) Co-
incubation of compound 10e and Aβ1-42 (25 ꢁM) for 24 h at 37ºC (E) Co-incubation of compound 10b and Aβ1-42
(25 ꢁM) for 24 h at 37ºC (original magnification, 200).
Microscopic observation revealed marked changes in the morphology of Aβ1–42 treated
cells as compared to control cells (Fig. 8). After Aβ1–42 treatment, a large number of cells
became shrinked, rounded and vacuolated which clearly indicated apoptosis or necrosis in
these cells (Fig. 8B). However, compounds treatment significantly restored these alterations
in SH-SY5Y cells (Fig. 8C-E). These results suggested that these novel compounds exerted
marked neuroprotection against Aβ-induced neurotoxicity.
Oxidative stress plays a pivotal role in the progression of AD. Therefore, compounds
10d, 10e and 10b were investigated for their capacity to protect human SH-SY5Y
neuroblastoma cells against oxidative stress-associated death induced by H2O2, and trolox
was used as the reference compound (Fig. 9). In this study, SH-SY5Y cells were pre-treated
with different concentrations of the compounds (1-20 ꢁM) for 24 h prior to the addition of
200 ꢁM H2O2. This H2O2 concentration was selected as it represents the concentration that
showed ~ 50% loss in the ability of cells to reduce MTT (data not shown). From Fig. 9, it
could be seen that all of the compounds exhibited neuroprotective effects at concentrations
ranging from 1 to 20 ꢁM. In particular, compound 10d was able to significantly increase cell

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viability in respect to H2O2-stressed cells in the same manner as trolox at the concentration of
5 ꢁM and much better than trolox at 10 ꢁM.
Fig. 9: Neuroprotection against H2O2 toxicity. Compounds 10d, 10e and 10b were tested for
neuroprotective activity against H2O2 induced cell death in SH-SY5Y cells. Trolox was used as the
reference compound. Results are expressed as percent viability compared to cells not treated with H2O2.
Data represent the mean ± SD of three observations. Statistical analysis was performed by the one-way
ANOVA followed by Bonferroni’s test. ###p < 0.001 vs. control cells, *p < 0.05, **p < 0.05 vs. H2O2
treated cells.
We next observed morphological alterations in SH-SY5Y cells treated with different
concentrations of compounds and H2O2. As shown in Fig. 10, SH-SY5Y cells exhibited clear
morphological changes after 24 h of exposure with H2O2 (Fig. 10). Microscopic observation
showed that H2O2-treated cells were fewer in number with shrinked cell body, fading
synapses, many fragments and loss of adherence. All these morphological alterations clearly
indicated H2O2 induced neurotoxicity in SH-SY5Y cells. However, compounds treatment (at
10 ꢁM) blocked these effects and significantly prevented the cell death induced by H2O2
toxicity in these cells (Fig. 10C-E). These result implied that the neuroprotective action of
our synthesized compounds might come from radical scavenging action.

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A
B
C
D
E
C
Fig. 10: Representative micrographs of SH-SY5Y cells morphology after treatment with 200 µM of H2O2
in the absence or presence of compounds 10d, 10e and 10b. (A) Control cells without H2O2 treatment (B)
H2O2 (200 ꢁM) alone induced neurotoxicity (C) Compound 10d (10 ꢁM) was given for 24 h with H2O2
(200 ꢁM) at 37ºC (D) Compound 10e (10 ꢁM) was given for 24 h with H2O2 (200 ꢁM) at 37ºC (E)
Compound 10b (10 ꢁM) was given for 24 h with H2O2 (200 ꢁM) at 37ºC (original magnification, 200).
3. Conclusions
In conclusion, a series of novel triazolo pyrimidine (10a-e) and cyanopyridine
derivatives (13a-e, 14) have been designed, synthesized and biologically evaluated as novel
multifunctional agents against AD. Most of synthesized compounds effectively inhibited both
ChEs in the sub-micromolar range in vitro. Compounds 10b, 10d and 10e of triazolo
pyrimidine series turn out to be the most potent and selective inhibitors of AChE. In
particular, compound 10d (IC50 = 0.042 ꢁM) showed the highest AChE inhibitory activity,
which was ~3.2-fold more active than the reference compound tacrine. It also showed a
moderate BuChE inhibition with IC50 value of 0.51 ꢁM. Overall, the substitution of quinoline
ring by fluoro group and presence of triazolo pyrimidine moiety imparts strong AChE
inhibition in case of series 10a-e. Furthermore, kinetic and molecular modelling studies of
10d indicated that it was a mixed-type inhibitor binding simultaneously to active, peripheral
and mid-gorge sites of AChE. Among the synthesized compounds, compound 10d showed a
balanced multitargeted profile with significant inhibition of self-induced and AChE-induced
Aβ aggregation and antioxidant activity. In addition, compound 10d showed neuroprotective

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properties against different toxic insult (Aβ and H2O2) in SH-SY5Y cells at low micromolar
concentrations relative to trolox. According to the theoretic ADME analysis, these
compounds also exhibited appropriate drug-like properties. Altogether, the multitargeted
profile of these novel compounds highlight their potential application in modulating several key
pathological targets in the pursuit of effective treatments of AD.
4. Experimental
4.1. Chemistry
All the chemicals and reagents were purchased from Sigma-Aldrich, Alfa-Aesar,
Spectrochem and SD Fine chemicals Pvt. Ltd. India, and used as received. The reactions were
monitored and Rf values were determined using analytical thin layer chromatography (TLC)
with Merck silica gel 60-120 and F254 pre-coated plates (0.25 mm) thickness. Spot on the
TLC plates were visualized using ultraviolet light both at short (254 nm) and long wave (365
1
13
nm) UV light. H NMR spectra and C NMR were recorded on Bruker 300 spectrometers
and 400 spectrometers. hemical shifts are reported in ppm (TMS,δ 0.00) or with the solvent
C
reference relative to TMS employed as the internal standard (CDCl3 ,δ 7.26; DMSO-d6 δ
2.54) and multiplicities of NMR signals are designated as s (singlet), d (doublet), dd (double
doublet), t (triplet),q (quartet), br (broad), m (multiplet, for unresolved lines). LCMS of the
compounds were carried out on applied biosystem (absciex 2000 triple quad). Melting points
were obtained on a Kofler apparatus and are uncorrected. Elemental analyses were obtained
in an Elementar
Vario analyser.
4.1.1. General methods of preparation of compound 3
Diethyl (ethoxymethylene) malonate (25 mmol) and substituted aniline (25 mmol) were
mixed in equivalent proportions at ambient temperature, giving rise to an exotherm of 18 oC.
o
Benzene (6.5 ml) was added and the resulting solution heated under reflux (83 C) for 1.5h.
The solution was concentrated in vacuo to obtain an oil, which was crystallized on standing.
The crude product was slurried in hexane, filtered off and air dried to give the compound 3
(m.p. 47-50 oC).
4.1.2. General methods of preparation of compound 4