Design and development of 1,3,4-oxadiazole derivatives as potential inhibitors of acetylcholinesterase to ameliorate scopolamine-induced cognitive dysfunctions

Article abstract of DOI:10.1016/j.bioorg.2019.103025

The novel hybrids bearing 4-aminopyridine (4-AP) tethered with substituted 1,3,4-oxadiazole nucleus were designed, synthesized, and evaluated for their potential AChE inhibitory property along with significant antioxidant potential. The inhibitory potenti

Full text of DOI:10.1016/j.bioorg.2019.103025

Accepted Manuscript
Design and Development of 1,3,4-Oxadiazole Derivatives as Potential Inhibi-
tors of Acetylcholinesterase to Ameliorate Scopolamine-Induced Cognitive
Dysfunctions
Puja Mishra, Piyoosh Sharma, Prabhash Nath Tripathi, Sukesh Kumar Gupta,
Pavan Srivastava, Ankit Seth, Avanish Tripathi, Sairam Krishnamurthy,
Sushant Kumar Shrivastava
PII:
S0045-2068(19)30206-8
https://doi.org/10.1016/j.bioorg.2019.103025
103025
DOI:
Article Number:
Reference:
YBIOO 103025
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
8 February 2019
13 April 2019
30 May 2019
Please cite this article as: P. Mishra, P. Sharma, P. Nath Tripathi, S. Kumar Gupta, P. Srivastava, A. Seth, A. Tripathi,
S. Krishnamurthy, S. Kumar Shrivastava, Design and Development of 1,3,4-Oxadiazole Derivatives as Potential
Inhibitors of Acetylcholinesterase to Ameliorate Scopolamine-Induced Cognitive Dysfunctions, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103025
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Design and Development of 1,3,4-Oxadiazole Derivatives as Potential
Inhibitors of Acetylcholinesterase to Ameliorate Scopolamine-Induced
Cognitive Dysfunctions
Puja Mishra, Piyoosh Sharma, Prabhash Nath Tripathi, Sukesh Kumar Gupta, Pavan
Srivastava, Ankit Seth, Avanish Tripathi, Sairam Krishnamurthy, Sushant Kumar
Shrivastavaa*
Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology
(Banaras Hindu University), Varanasi – 221 005 (India)
*
Address correspondence to:
Dr. Sushant Kumar Shrivastava
Professor
Email: skshrivastava.phe@itbhu.ac.in
Phone: +91-94521-56527
1

ABSTRACT
The novel hybrids bearing 4-aminopyridine (4-AP) tethered with substituted 1,3,4-oxadiazole
nucleus were designed, synthesized, and evaluated for their potential AChE inhibitory
property along with significant antioxidant potential. The inhibitory potential (IC ) of
5
0
synthesized analogs was evaluated against human cholinesterases (hAChE and hBChE) using
Ellman’s method. Among all the compounds, 9 with 4-hydroxyl substituent showed
maximum hAChE inhibition with the non-competitive type of enzyme inhibition (IC50
=
1
.098 µM; Ki = 0.960 µM). Further, parallel artificial membrane permeation assay (PAMPA-
BBB) showed significant BBB permeability in most of the synthesized compounds.
Meanwhile, compound 9 also inhibited AChE-induced Aβ aggregation (38.2–65.9%) by
thioflavin T assay. The in vivo behavioral studies showed dose-dependent improvement in
learning and memory by compound 9. The ex vivo studies also affirmed the significant AChE
inhibition and antioxidant potential of compound 9 in brain homogenates.
Keywords: Acetylcholinesterase; Butyrylcholinesterase; 1,3,4-Oxadiazole; Molecular
hybridization; 4-Aminopyridine.
2

1
. INTRODUCTION
Severity and prevalence of neurocognitive disorders especially dementia with shocking,
demoralizing, and life-threatening consequences on the life of patients represents a
substantial challenge for the scientific community [1]. The manifestations of decline in
cognitive functions are reported in several neurodegenerative disorders like schizophrenia,
Alzheimer's disease (AD), traumatic brain injury, Parkinson’s disease, etc. [2]. AD is one of
the major neurocognitive disorders and a prominent cause of death in the elderly population
worldwide [3]. The recently published World Alzheimer’s report 2018 has emphasized to
explore new dimensions for the treatment and increase the research expenditure for the new
drug discovery as the number of dementia patients are increasing day by day [4,5].
The two widely explored therapeutic strategies for the AD are declined cholinergic
transmission and amyloid beta aggregation [6,7]. Acetylcholinesterase (AChE) is a serine
hydrolase enzyme responsible for the hydrolysis of ACh, thereby reducing the levels of ACh
which was reported to be in close association with cognitive decline [8-12]. The preceding
several years have witnessed the discovery of numerous AChE inhibitors that concreted the
way for a superior therapeutic strategy towards cognition enhancement associated with AD
[13-16]. However, the currently available pharmacotherapies are failed to address all the
pathogenic hallmarks of the disease and unable to sojourn the disease progression [17,18].
AChE has also been recognized to play a vital role in increasing the deposition of Aβ
aggregates in AD [19]. The aggregation and deposition of Aβ fibrils and oligomers leads to
disruption of cellular communication, inflammatory responses, progressive neuritic injury,
neuronal deficits, and cognitive dysfunctions [5,20]. Accumulation of Aβ could also lead to
oxidative damage in the brain by entering into mitochondria and increasing generation of free
radicals [21].
3

The synthetic versatility and broad spectrum of therapeutic potentials of oxadiazole have
been the center of attraction to pharmaceutical scientists in last few decades. Unique
structural features of oxadiazole lead to the discovery of some structurally diverse derivatives
with reported inhibitory activity against AChE as well as amyloid beta [22-24]. 4-
Aminopyridine (4-AP) is another heterocyclic nucleus reported for antiamnesic activity,
cognition enhancement and inhibition of AChE [14,25,26]. The accumulating lines of
evidence suggested that hybridization of two or more diverse bioactive molecules with
corresponding pharmacophoric functions or with different mechanisms of action render
synergistic effects contributing to the overall activity profile of the molecule [27,28]. In the
current research, a novel series of compounds were designed and synthesized by invoking the
concept of molecular hybridization. The synthesized compounds were evaluated for their in
vitro potential against AChE and butyrylcholinesterase (BChE) along with the assessment of
their permeability across the blood-brain barrier (BBB) by parallel artificial membrane
permeability assay (PAMPA-BBB) and AChE-induced amyloid beta aggregation inhibitory
potential. The outcomes of in vitro results were further corroborated using in vivo, ex vivo
and in silico studies.
1
.1. Designing Considerations
In our previous research work, we have reported 4-AP based semicarbazones (I), Schiff bases
II), and other derivatives (III and IV) for their potential antiamnesic and cognition-
(
enhancing properties with significant cholinesterase inhibition profile [13,14,26]. The 4-AP
nucleus itself was examined by dose-finding studies in the patients of the AD, but no
significant effect was observed due to the unselective release of neurotransmitters and poor
penetrability of 4-AP in CNS [29]. These studies have prompted us to select the 4-AP
pharmacophoric core group to design novel hybrids.
4

The oxadiazole nucleus reported to possess diverse pharmacological activities such as anti-
microbial [30], antimalarial [31], anticancer [32], antidepressant [33], antifungal [34],
antiviral [35], anti-inflammatory [28], analgesic [36], muscle relaxants [37] etc. Also, the
versatile nature of the 1,3,4-oxadiazole scaffold with varied substitution (V, VI, and VII) has
been reported for significant anti-cholinesterase and antioxidant activities [38,39]. At 2nd
position of 1,3,4-oxadiazole nucleus, 4-AP pharmacophoric core group was linked, and the
th
remaining 5 position was connected with aryl group substituted with variable electron
withdrawing and donating functional groups. Herein, we have designed the cyclized
analogues of semicarbazones and bioisosterically replaced it with 1,3,4-oxadizole nucleus to
impart gastric stability and improve its efficacy.
On the basis of above-mentioned conjectures, we have designed molecular hybrids of 4-AP
tethered with a substituted 1,3,4-oxadiazole nucleus (Fig. 1). These molecular hybrids were
designed as per the structural requisites for potential cholinesterase inhibitory activity with
the hypothesis that 4-AP will extend into the catalytic site, and substituted 1,3,4-oxadiazole
will efficiently interact with the peripheral anionic site residues of AChE similar to the
piperidine nucleus and indanone ring of donepezil, respectively.
5

N
N
R1
R
H
H
N
N
N
N
N
N
O
O
O
O
N
N
O
II
I
III
IV
H
N
O
Ar
N
N
N
N
N
Cl
N
O
N
H
N
R
N
N
O
O
S
O
O
F
O
O
O
O
O
V
Cl
O
VI
VII
Fig. 1. Designing of 4-AP tethered substituted 1,3,4-oxadiazole analogs.
2
2
. RESULTS AND DISCUSSION
.1. Chemistry
The designed analogs (5–19) were synthesized following the procedure mentioned in Scheme
. The 4-AP (1) was reacted with phenyl chloroformate to acquire the intermediate (3) and
1
used further without purification. Intermediate (3) was reacted with an excess of hydrazine
hydrate to get the key intermediate (4). In the final step, various aromatic aldehydes and
intermediate (4) were refluxed for 3–5 h to afford respective semicarbazones, which were in
situ proceeded with chloramine-T for cyclization to afford the target compounds (5–19).
6

O
O
NH2
Cl
O
HN
N
O
(a)
+
N
(
1)
(
2)
(
3)
(
b)
O
NH2
H
HN
N
N
H
N
O
(
c)
Ar
N
N
N
H
Ar
(
5-19)
(
4)
O
Ar : Various substituted aromatic aldehydes
Scheme 1. Synthetic scheme: (a) Pyridine (2.3 eq), dichloromethane (15 ml), stirring 0–5 °C
h (b) hydrazine hydrate (3 eq), absolute ethanol, reflux 1 h; (c) substituted aldehydes (1 eq),
8
glacial acetic acid (1–2 drops), absolute ethanol, reflux 3–5 h followed by addition of
chloramine-T, stirring 2–3 h.
The FT-IR spectra exhibited the characteristic secondary amine (—NH) stretching in the
-1
range 3496–3191 cm to confirm the cyclization of the Schiff bases into oxadiazole ring. In
1
the H NMR spectral analysis, formation N=CH bonds were characterized by the presence of
a peak between δ 8.71–8.23 ppm. The disappearance of the —NH of compound 4 and
H
2
appearance of the —NH peak of 1,3,4-oxadiazole derivatives that occurs variably around δH
1
3
9
.21–8.10 ppm confirmed the cyclization. Two aromatic groups were confirmed by C NMR
showing the peaks in the range of δ 155–104 ppm. Compound (9) having the carbon
C
attached to hydroxyl group showed the peak in the downfield region at δ 167 ppm. The
C
derivatives have a substitution at 2nd and 5 position of a 1,3,4-oxadiazole heterocyclic
moiety having pyridine ring on one end and substituted aromatic ring on the other end were
also characterized by elemental (C, H, and N) analyses and the results were found within the
th
7

range of theoretical limits (± 0.4%). The results of physicochemical characterization are
given in Table 1.
Table 1. Structural hybrids of synthesized 4-AP tethered 1,3,4-oxadiazole analogs and results
of their physicochemical characterization.
H
N
O
N
R
N
N
(5-19)
Melting
point (°C)
Compound
R-group
Rf*
LogP**
5
6
7
8
9
—H
3—Br
0.37
0.42
0.43
0.30
0.28
0.41
0.42
0.36
0.38
0.46
0.52
0.34
0.35
0.38
0.32
207-209
211-213
241-243
188-190
198-200
215-217
230-232
232-234
218-220
227-229
246-248
236-238
245-247
223-225
212-214
2.48
3.31
3.38
2.02
2.18
2.32
2.40
2.45
3.18
3.30
3.68
2.96
3.08
2.38
2.16
4—Br
2—OH
4—OH
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
2—NO2
3—NO2
4—NO2
3—Cl
4—Cl
2,3—diCl
3—CH3
4—CH3
4—OCH3
3,4,5—triOCH3
*
*
Rf values were determined using dichloromethane:methanol (9.5:0.5) as the mobile phase.
* LogP values were determined by octanol:water-based shake flask test.
2
.2. Pharmacology
2
.2.1. Cholinesterase inhibition assay
Ellman colorimetric assay was used to determine the inhibition property of compounds 5-19
against AChE and BChE using donepezil as positive reference standard [40]. The results of
IC50 values against AChE and BChE mentioned in Table 2 indicated the micromolar
8

inhibition of AChE by most of the compounds. The unsubstituted aryl ring in compound 5, p-
substituted compounds (7, 9, 12, 14, and 18), and trimethoxy substituted compound 19
showed micromolar inhibitory potential against AChE. Among the tested compounds,
compound 9 with p-hydroxy substituent showed maximum inhibitory potential against AChE
(
IC = 1.098 ± 0.140 µM). The compound 9 also showed maximum selectivity for AChE
50
inhibition as compared to other tested derivatives. Interestingly, 3-tolyl substituted derivative
showed better BChE inhibitory potential than AChE. Among other p-substituted derivatives,
only compounds 7, 9, 12, and 14 have demonstrated micromolar inhibitory potency against
BChE in the IC range of 7.794–9.220 µM. The compound 9 also exhibited the maximum
5
0
selectivity index for AChE over BChE compared to other tested derivatives.
Table 2. The results of cholinesterase inhibitory potential and prediction of brain
permeability of the synthesized compounds.
a
IC μM ± SEM
PAMPA-
BBB
hAChE permeability
PAMPA-BBB
Prediction
5
0
SI for
Comp.
Code
c
d
e
R-group
(
CNS+ , CNS- ,
hAChE
hBChE
b
-6
f
Pe(exp) (10
cm s )
CNS± )
-1
5
6
7
8
9
—H
3—Br
8.714 ± 0.255
>10
>10
>10
-
5.39 ± 0.11
6.75 ± 0.07
7.02 ± 0.06
3.62 ± 0.34
4.65 ± 0.21
3.41 ± 0.13
1.56 ± 0.12
2.64 ± 0.18
3.36 ± 0.08
4.57 ± 0.11
3.79 ± 0.03
3.20 ± 0.02
5.64 ± 0.35
4.65 ± 0.24
7.34 ± 0.14
CNS+
CNS+
CNS+
CNS±
CNS+
CNS±
CNS-
CNS±
CNS±
CNS+
CNS±
CNS±
CNS+
CNS+
CNS+
-
4—Br
2.535 ± 0.212 8.793 ± 0.474
>10 >10
1.098 ± 0.140 7.794 ± 0.084
3.47
2—OH
4—OH
2—NO2
3—NO2
4—NO2
3—Cl
-
7.10
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
>10
>10
>10
>10
-
-
1.986 ± 0.155 7.351 ± 0.329
>10 >10
4.356 ± 0.235 9.220 ± 0.238
3.70
-
4—Cl
2.12
2,3—diCl
3—CH3
4—CH3
4—OCH3
>10
>10
8.322 ± 0.231
>10
-
>10
-
6.173 ± 0.194
2.208 ± 0.233
-
-
>10
3,4,5—triOCH3 1.790 ± 0.074 7.472 ± 0.227
4.17
9

donepezil
0.043 ± 0.007 4.327 ± 0.459 100.63
nd
nd
a
b
c
All the results are reported as IC50 ± SEM (n = 3);
SI (Selectivity Index) = (IC50 BChE)/(IC50 AChE);
Data represented are the mean ± SEM of two independent experiments (n = 2).
d
-6
-1
e
‘CNS+’ (prediction of high BBB permeation); P (10 cm s ) > 4.3926.
e
-6
-1
‘
e
CNS-’ (prediction of low BBB permeation); P (10 cm s ) < 1.7766.
f‘CNS±’ (prediction of uncertain BBB permeation); P
(10 cm s ) 4.3926 to 1.7766.
-6
-1
e
nd = Not determined
2
.2.2. Enzyme kinetics
The mechanism of AChE inhibitory potential by compound 9 was investigated by enzyme
kinetics study. The reciprocal Lineweaver-Burk plot showed inhibition of hAChE through a
non-competitive mechanism in which the value of Vmax decreased, and K remained constant
m
(Ki = 0.960 µM) (Fig. 2). The intersection points on both axes of 1/Vmax and 1/[S] were
found to be higher than zero, suggesting that the inhibitor was preferentially bound to free
enzyme rather than enzyme-substrate complex.
Fig. 2. Lineweaver-Burk plot showing non-competitive inhibition of compound 9.
2
.2.3. PAMPA-BBB assay
The essential requirement of the synthesized compounds is their permeability through the
blood-brain barrier (BBB) to be efficacious in neurodegenerative disorder [41]. Therefore,
permeability of compounds was evaluated by PAMPA-BBB assay. The permeability of
10

compounds from donor microplate to acceptor microplate was most accurately determined
through a porcine brain lipid layer. The permeability of nine commercial drugs as reported in
our previously published report was used as a benchmark to establish the correlation between
experimental Pe(exp) and reference permeability Pe(ref) [42]. The CNS permeability cut-off
-6
-1
values indicated that compounds with permeability (P ) over 4.3926×10 cm.s suggest
e
-6
-1
excellent brain penetrability, whereas with P less than 1.7766×10 cm.s has poor brain
e
permeation. The permeability value in between them was considered as uncertain [43]. The
results indicated that compounds 5, 6, 7, 9, 14, 17, 18, and 19 showed excellent brain
permeability. Compounds 8, 10, 12, 13, 15, and 16 showed moderate permeation
characteristics. Only compound 11 among all the tested compounds showed very low CNS
brain permeability (Table 2).
2
.2.4 AChE-induced amyloid beta aggregation inhibition: Thioflavin T assay
The AChE was demonstrated to be involved in the promotion of amyloid beta aggregation by
specifically binding to PAS [44,45]. The role of AChEIs has been validated to prevent the
amyloid beta aggregation and deposition. Therefore, thioflavin T based fluorometric assay
was performed on compound 9 to ascertain its role in inhibiting and preventing amyloid beta
aggregation. The test was conducted at three different ratios of Aβ: compound 9 (10:5, 10:10,
and 10:20 µM). The results were reported as % Aβ aggregation inhibition (Fig. 3A) and
normalized fluorescence intensity (NFI) (Fig. 3B). The results indicated comparable anti-
aggregatory property of compound 9 (38.2–65.9%) with donepezil (39.6–68.9%). The
inhibitory potential increased with increasing concentration ratio of inhibitor.
11

[
A] Normalized fluorescence intensity
[
B] AChE-induced % aggregation inhibition
8
6
4
2
0
0
0
0
0
1.00
0.75
0.50
0.25
0.00
:
5
10
20
10:5
10
20
10:5
10
20
10:5
10
20
:
0
:
:
:
:
:
:
:
1
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
Donepezil + A
M)
Compound 9 + A
(M)
Donepezil + A
(M)
Compound 9 + A
(M)
(
Fig. 3. Effect of compound 9 and donepezil in thioflavin T assay. [A] Normalized
fluorescence intensity (NFI); [B] % aggregation inhibition. Each bar displays the mean ±
SEM of three separate experiments (n = 3).
2
.2.4. In vivo behavioral studies
Compound 9 showed promising in vitro AChE inhibitory potential and high brain
permeability through in vitro PAMPA-BBB assay. These results have prompted us to
evaluate the compound 9 through in vivo behavioral models in animals. The scopolamine-
induced models are very well validated models of learning and memory [46]. The
antiamnesic effect of compound 9 was evaluated using the Y-maze task and passive
avoidance test in mice.
2
.2.4.1 Acute toxicity study
Acute toxicity of compound 9 was determined on healthy Swiss albino mice (25-30 gm) as
per the OECD 423 guidelines. Several behavioral, cholinergic effects, and toxic reactions
were monitored such as tremors, convulsions, salivation, diarrhea, sleep, lacrimation, and
feeding behavior. There were no signs of any cholinergic side effects, any toxicity, or no
mortality was observed post administration of test compound 9. These studies suggested that
compound 9 have a significant margin of safety [42].
12

2
.2.4.1 Y-maze task
The Y-maze task is the standard animal model used for evaluation of hippocampal-dependent
short-term working memory in the rodents [47]. The percentage (%) alterations were
calculated (spontaneous alteration rate), which demonstrated the working memory capacity of
*
**
animals. The % spontaneous alternations was significantly reduced ( p < 0.001) in the
scopolamine group of animals compared to control group indicative of induction of memory
and learning impairment. Donepezil (5 mg/kg) and compound 9 showed significantly
#
##
increased ( p < 0.001) spontaneous alternations compared to scopolamine group. At the
dose of 5 and 10 mg/kg, p.o. compound 9 showed statistically non-significant difference in %
alterations compared to donepezil (5 mg/kg) (Fig. 4A). The total arm entries remained
unchanged in all groups suggested that scopolamine did not hamper the locomotor activity in
animals (Fig. 4B).
[A]
[B]
ns
8
6
4
2
0
0
0
0
0
30
20
10
0
#
##
#
##
###
#
##
*
**
l
5
e
5
5
0
tro
2
.
1
rol
5
2.5
5
0
1
n
o
e
n
o
n
t
o
D
n
C
Comp. 9 (p.o. kg -1
o
D
C
Comp. 9 (p.o. kg -1
)
)
Scopolamine (0.5 mg/kg, i.p.)
Scopolamine (0.5 mg/kg, i.p.)
Fig. 4. The effect of compound 9 at the dose of 2.5, 5, and 10 mg/kg, p.o. and donepezil (5
mg/kg, p.o.) by Y-maze test on [A] Spontaneous alteration score (% alterations); [B] Total
arm entries. All values are represented as mean ± SD (n = 6). ***p < 0.001 compared to
control; ###p < 0.001 compared to scopolamine; ns = non-significant; Done5 = donepezil.
2
.2.4.2 Passive avoidance task
This model is primarily responsible for hippocampal and cholinergic neuronal dysfunctions
48]. Transfer latency time was calculated in seconds for the transfer of mice from the light
chamber to the dark chamber. The increase in transfer latency (TL) period suggested the
[
13

improvement in learning and memory in rodents. Scopolamine-induced amnesia leads to no
significant change in the latency period between the acquisition phase and retention phase
indicated the loss of learning and memory. But, the treatment with compound 9 showed
*
significantly increased ( p < 0.05) TL in a dose dependent manner compared to the
scopolamine-treated group. Moreover, during retention test, results of compound 9 at the
dose of 5 and 10 mg/kg showed non-significant difference with donepezil (Fig. 5). The
overall results of scopolamine-induced models advocated the potential of compounds 9 in
improving spatial and immediate working memory.
Acqisition Phase
Retention Phase
3
2
1
00
00
00
0
*
*
*
*
*
l
5
.
5
5
Comp. 9 (p.o. kg-1)
0
1
o
e
n
r
2
t
n
o
o
D
C
Scopolamine (0.5 mg/kg, i.p.)
Fig. 5. The effect of compound 9 on scopolamine-induced passive avoidance task in mice.
*
All the results are represented as mean ± SD (n = 6). p < 0.05 compared to respective
acquisition test.
2
.2.5. Ex vivo and biochemical analysis
The effect of compound 9 on brain AChE level was evaluated by ex vivo study as per
Ellman’s method [40]. The results showed a significantly higher rate (Fig. 6A, ***p < 0.001)
of substrate hydrolysis or elevated levels of AChE in the scopolamine group of animals
compared to control group. The levels of AChE were remarkably attenuated (Fig. 6A, ###p <
0
.001) by treatment of compound 9 in a dose dependent manner compared to the scopolamine
group. The ex vivo brain AChE inhibitory potential of compound 9 at the dose of 10 mg/kg
14

was found to be statistically non-significant in comparison to donepezil. These results also
reflected the ability of compound 9 to permeate the BBB.
The antioxidant potential of compounds was elucidated by estimation of oxidative
biochemical markers such as malonaldehyde (MDA, lipid peroxidation by-product),
superoxide dismutase (SOD, involved in dismutation of O2– radicals), and catalase
(
decomposition of H O in water). Biochemical analysis showed that scopolamine group has
2 2
significantly elevated the levels of MDA (Fig. 6B, ***p < 0.001), while the levels of SOD
Fig. 6C, ***p < 0.001) and catalase (Fig. 6D, ***p < 0.001) were considerably reduced
(
compared to control group. However, the treatment of compound 9 attenuated the levels of
MDA (Fig. 6B, ###p < 0.001), while significantly increasing levels of SOD (Fig. 6C, ###p <
0
.001), and catalase (Fig. 6D, ###p < 0.001) compared to scopolamine group. Also, the results
suggested that donepezil has non-significant antioxidant potential. Overall, the results of ex
vivo and biochemical analysis suggested compound 9 to possess significant brain AChE
inhibitory potential along with antioxidant property.
15

[A]
[B]
2
1
1
0
5
0
5
0
2.0
1.5
1.0
0.5
0.0
***
***
ns
#
##
###
###
#
##
#
##
###
#
##
trol
5
e
.
5
5
0
l
5
.
5
5
0
2
1
tro
e
n
o
2
1
n
n
o
n
o
D
o
C
D
C
-
1
Comp. 9 (p.o. kg-1
Comp. 9 (p.o. kg
Scopolamine (0.5 mg/kg, i.p.)
)
)
Scopolamine (0.5 mg/kg, i.p.)
[C]
[D]
1
0
8
6
4
2
0
2
.0
###
#
##
#
##
#
##
1.5
#
##
###
1
.0
***
0.5
***
0
.0
l
5
.
5
5
0
1
tro
e
n
2
trol
5
e
5
5
0
n
o
.
1
o
D
n
o
D
2
C
-1
Con
Comp. 9 (p.o. kg
Scopolamine (0.5 mg/kg, i.p.)
)
-
1
Comp. 9 (p.o. kg
)
Scopolamine (0.5 mg/kg, i.p.)
Fig. 6. Effect of compound 9 on the ex vivo AChE estimation and biochemical analysis of
oxidative stress biomarkers. (A) Ex vivo AChE assay; (B) Lipid peroxidation assay; [C] SOD
assay; [D] Catalase assay. All values are expressed as mean ± SD (n = 6). ***p < 0.001
compared to control, ###p < 0.001 compared to scopolamine; ns = non-significant; Done5 =
donepezil.
2
.3. Computational Analysis
2
.3.1. In silico docking studies on AChE
To understand the binding pattern and binding ability of potential compound 9, docking
studies were performed on AChE co-crystallized with donepezil (PDB Code: 1EVE) [49]
using Glide module of Schrödinger Maestro 2018-1. The results of binding affinity and
interaction pattern were compared with co-crystallized ligand donepezil. The superposition of
docked pose of compound 9 and donepezil showed similar binding site interaction and
occupancy at the same site of AChE. The most active compound 9 with glide score of -10.6
kcal/mol exhibited several active site interactions (Fig. 7A and 7B). At the catalytic site, the
16

compound 9 interacted with Ser200 and His440 residues through polar interactions. At
anionic subsite, Glu199 residue which is very near to catalytic lining formed H-bonding
interaction with the —NH pyridine. The amino acids Trp84 and Phe330 were observed to be
involved in π-π stacking with the oxadiazole nucleus. Apart from these interactions, Trp84
also interacted with pyridine ring and —NH pyridine through π-π stacking and π-π cation
interactions, respectively. At acyl binding pocket, compound 9 was involved in hydrophobic
interactions with the aromatic amino acid residues Phe288, Phe290, and Phe331. At oxyanion
hole, Gly117 and Gly118 interacted through glycine interaction. At the peripheral anionic
site, compound 9 interacted effectively with the Tyr334, Tyr121 and Asp72 amino acid
residues through π-π stacking, hydrophobic and charged interaction, respectively. The
detailed interacting residues are also enlisted and given in Table 3.
Fig. 7. Docking of compound 9 on AChE. (A) 3D interaction image showing compound 9 in
its ligand binding surface interacting with active site residues of AChE represented in ribbon
structure; (B) 2D image showing active site interactions.
17

Table 3. Detailed active site residue interaction analysis of compound 9 and donepezil on
AChE.
Interacting residues#
Acyl
Comp.
9
Catalytic
site
Peripheral
anionic site
Anionic
subsite
Oxyanion Other interacting
binding
pocket
hole
residues
a
b
e
c
g
a
c
Ser200 ,
Tyr334 ,
Glu199 , Phe288 , Gly117 ,
Ser81 , Tyr130 ,
a
c
d
b,f
c
g
g
c
His440
Tyr121 , Asp72 Trp84 , Phe290 , Gly118
Gly441 , Ile444
c
c
Phe330 Phe331
a
c
d
c
g
c
g
Don*
Ser200 ,
Tyr334 ,
Glu199 , Phe288 , Gly117 ,
Tyr130 , Gly441 ,
a
c
d
b,f
c
g
c
c
His440
Tyr121 , Asp72 , Trp84 , Phe290 , Gly118
Tyr70 , Trp279 , Phe330 Phe331
Ile444 , Phe331 ,
c
c
f
c
d
c
Arg289 , Ile287 ,
a
c
Ser286 , Leu282 ,
#
a
All the interacting residues are within the 4Å distance with the ligand; *Don – Donepezil; polar
b
c
d
e
interaction; π-π stacking interaction; hydrophobic interaction; charged interaction; H-bonding
interaction; π-π cation interaction; glycine interaction.
f
g
2
.3.2. Molecular dynamics (MD) simulations
The dynamics simulation analysis was carried out with a docked complex of compound 9 and
AChE to find out the stability of binding pose in a flexible protein environment having the
presence of virtual water molecules. The results of RMSD (root mean square deviation)
values for protein and ligand indicated that the system is equilibrated and fluctuations are
within the range of 1–3Å [42] as compared to reference protein backbone structure. Further,
protein and ligand RMSF (root mean square fluctuations) also indicated stable trajectories of
ligand and protein residues for the complete simulation run.
The interaction analysis of protein-ligand complex was performed using stacked bar chart and
graphical representation. Stacked bar chart analysis showed the interaction fractions for four
types: H-bonds, hydrophobic, ionic and water bridge interactions. The interaction of Trp84
showed interaction fraction of more than 1.2 that suggested 120% interaction was maintained
for the total simulation run. It also showed that more than 100% interaction was due to
multiple contacts of the respective residues (Fig. 8). The graphical representation showed
18

interactions occurred for more than 30% simulation time. The results indicated that H-
bonding interactions were maintained with Glu199 and Gly119 for 35% and 38% simulation
time, respectively. The interaction with Trp84 through π-π stacking was maintained for 59%
simulation time with oxadiazole ring. Also, Tyr334 and Phe331 formed hydrophobic
interactions with the respective ligand sites (Fig. 9).
Fig. 8. The detailed interaction fraction analysis of compound 9 and AChE complex.
Fig. 9. Graphical representation of compound 9 showing interacting residues with the
interaction of more than 30% of total simulation of 30 nsec.
19

3
. CONCLUSION
The available FDA approved AChEIs could only provide the symptomatic relief to the
patient of the AD. The search to find a novel lead for the curative treatment of AD has been
failed for a long time and is still the top most priority of medicinal chemists. In our quest to
search for a novel lead, a new set of compounds bearing 4-AP nucleus with 1,3,4-oxadiazole
1
ring were designed, synthesized, and characterized by spectroscopic techniques (FT-IR, H
1
3
NMR, and C NMR) and elemental analysis. All the results were in agreement with the
structures of the synthesized compounds. The designed and synthesized analogs showed non-
competitive inhibition of AChE by compound 9 (IC =1.098 µM; Ki = 0.960 µM). The brain
5
0
permeability and penetration through BBB is the most challenging barrier for the compounds
to be effective in the AD. Most of our synthesized analogs except compound 11 showed
significant brain permeability through in vitro PAMPA-BBB assay. Compound 9 also
exhibited excellent inhibitory potential of AChE-induced Aβ aggregation as evaluated by
thioflavin T assay at three different ratios of Aβ: inhibitor. Further, in vivo behavioral studies
were observed for the most potent compound 9 at three doses through Y-maze and passive
avoidance tests in mice. The results showed improvement in learning and memory with
compound 9 in a dose dependant manner with the maximum effect was observed at the dose
of 10 mg/kg, p.o. Moreover, ex vivo studies and biochemical analysis were performed, which
showed effective AChE inhibition and antioxidant property of compound 9, respectively. In
silico molecular docking and dynamics studies displayed consensual binding interactions of
compound 9 with catalytic and peripheral anionic site residues of AChE. All these results
highlighted the potential of compound 9 to be developed as the significant lead in the
development of orally active therapeutics in the treatment of AD.
20

4
4
. EXPERIMENTAL
.1. Chemicals and instrumentation
All the reagents were commercially available and purchased from Sigma Aldrich. The
reaction progresses were monitored, and Rf values were determined using thin layer
chromatography (TLC) with precoated silica gel plates 60-F254 (Merck, Germany). Open
capillary tubes with sealed one end were used to determine the melting points on a Perfit
(India) melting point apparatus and were reported as uncorrected. FT-IR spectra were
recorded on Bruker ECO-ATR (Alpha) and reported as wavenumber Vs. % Transmittance.
1
13
H NMR (500 MHz) and C NMR (125 MHz) was recorded on a Bruker Avance FT-NMR
spectrophotometer at room temperature using TMS as an internal standard. CHN analysis
was recorded using EXETER CE-440 elemental analyzer.
4
.2 Chemistry
4
.2.1. General procedure for the synthesis of (3).
4
-AP (10 mmol) was dissolved in dichloromethane with pyridine (15 mmol), followed by
addition of phenyl chloroformate (11 mmol) at 10 °C and stirred for 24 h at room
temperature. The reaction mixture was washed with water (3 × 100 ml), brine, and dried over
sodium sulfate. The filtrate was evaporated to get the solid compound (3).
–
1
1
Yield: 1.858 g, 87%. FT-IR (ν, cm ): 3198 (—NH stretching), 1732 (C=O stretching). H
NMR (DMSO-d , ppm): δ 8.55 (d, J = 7.2 Hz, 2H, aromatic CH), 7.64 (d, J = 7.0 Hz, 2H,
6
aromatic CH), 7.28 (t, J = 6.8 Hz, 2H, aromatic CH), 7.18–7.06 (m, 3H, aromatic CH), 5.98
1
3
(
s, 1H, —NH). C NMR (DMSO-d , ppm): 156.6, 152.8, 152.7, 151.7, 151.6, 149.2, 130.1,
6
1
30.0, 126.2, 123.4, 123.2, 113.3, 113.2. Anal. calc. (%) for C H N O; C, 65.28; H, 4.71;
13 10 4
N, 13.08. Found (%): C, 65.68; H, 4.68; N, 13.16.
21

4
.2.2. General procedure for the synthesis of (4) [13].
The compound (3, 4.6 mmol) was dissolved in absolute ethanol and hydrazine hydrate (13.8
mmol) were refluxed for 2 h. The reaction mixture was then allowed to cool and solvent was
evaporated and crude residue was crystallized with absolute ethanol to afford the pure
compound (4).
–
1
Yield: 0.548 g, 78%. FT-IR (ν, cm ): 3236, 3234 (—NH stretching), 3198 (—NH
2
1
stretching). H NMR (DMSO-d , ppm): δ 8.48 (d, J = 7.4 Hz, 2H, aromatic CH), 7.56 (d, J =
6
1
3
6
.8 Hz, 2H, aromatic CH), 6.42 (s, 1H, —NH), 6.22 (s, 1H, —NH), 4.46 (s, 2H, —NH ). C
2
NMR (DMSO-d , ppm): 162.3, 153.2, 153.1, 152.7, 114.6, 114.5. Anal. calc. (%) for
6
C H N O; C, 47.36; H, 5.30; N, 36.82. Found (%): C, 47.42; H, 5.28; N, 36.78.
6
8
4
4
.2.3. General procedure for the synthesis of (5-19).
Equimolar quantities of various aldehydes and the crystallized product (4) were mixed and
refluxed for 3–5 h to get semicarbazones. Then, chloramine-T (2.3 equivalents) was added,
and stirred for 2–3 h. The reaction solution was cooled and obtained solid was filtered,
washed and recrystallized with ethanol to afford the pure 1,3,4-oxadiazole derivatives (5-19).
4
.2.3.1. 5-phenyl-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (5)
–
1
1
Yield: 0.188 g, 60.0%. FT-IR (ν, cm ): 3191 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.22 (s, 1H, —NH), 8.39–8.24 (m, 2H, aromatic CH), 7.62–7.50 (m, 4H, aromatic CH),
1
3
7
1
2
.10–6.82 (m, 3H, aromatic CH). C NMR (DMSO-d , ppm): 162.3, 160.1, 154.2, 151.2,
6
29.1, 128.3, 126.9, 126.4, 110.2. Anal. calc. (%) for C H N O; C, 65.54; H, 4.23; N,
1
3
10
4
3.52. Found (%): C, 65.58; H, 4.21; N, 23.49.
.2.3.2. 5-(3-bromophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (6)
4
–
1
1
Yield: 0.261 g, 62.8%. FT-IR (ν, cm ): 3494 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.26 (brs, 1H, —NH), 8.32-8.23 (m, 2H, aromatic CH), 7.80–7.41 (m, 3H, aromatic CH),
22

1
3
7
1
.62 (s, 1H, aromatic CH), 7.10–7.02 (m, 2H, aromatic CH). C NMR (DMSO-d , ppm):
6
62.2, 160.3, 154.2, 151.2, 133.2, 130.2, 128.4, 126.4, 122.6, 110.5. Anal. calc. (%) for
C H BrN O; C, 49.23; H, 2.86; N, 17.67. Found (%): C, 49.29; H, 2.91; N, 17.71.
1
3
9
4
4
.2.3.3. 5-(4-bromophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (7)
–
1
1
Yield: 0.256 g, 61.7%. FT-IR (ν, cm ): 3495 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.23 (brs, 1H, —NH), 8.33–8.24 (m, 2H, aromatic CH), 7.71–7.62 (m, 4H, aromatic CH),
1
3
7
.12–7.00 (m, 2H, aromatic CH). C NMR (DMSO-d , ppm): 162.4, 160.1, 154.3, 151.4,
6
1
32.7, 128.8, 125.4, 110.5. Anal. calc. (%) for C H BrN O; C, 49.23; H, 2.86; N, 17.67.
1
3
9
4
Found (%): C, 49.33; H, 2.88; N, 17.64.
4
.2.3.4. 2-(5-(pyridin-4-ylamino)-1,3,4-oxadiazol-2-yl)phenol (8)
–
1
1
Yield: 0.228 g, 68.3%. FT-IR (ν, cm ): 3460 (—OH stretching), 3191 (—NH stretching). H
NMR (DMSO-d , ppm): δ 10.5 (brs, 1H, —OH), 9.21 (s, 1H, —NH); 8.67–8.28 (m, 2H,
6
1
3
aromatic CH), 7.65–7.39 (m, 2H, aromatic CH), 7.14- 6.89 (m, 4H, aromatic CH). C NMR
DMSO-d , ppm): δ 161.9, 160.4, 158.1, 154.4, 150.9, 129.7, 126.3, 120.9, 116.5, 109.2,
(
6
1
3
11.2. Anal. calc. (%) for C H N O : C, 61.41; H, 3.96; N, 22.04. Found (%): C, 60.43; H,
13 10 4 2
.99; N, 22.01.
.2.3.5 4-(5-(pyridin-4-ylamino)-1,3,4-oxadiazol-2-yl)phenol (9)
4
–
1
1
Yield: 0.220 g, 65.9%. FT-IR (ν, cm ): 3358 (—OH stretching), 3261 (—NH stretching). H
NMR (DMSO-d , ppm): δ 12.98 (brs, 1H, —OH), 9.08 (s, 1H, —NH), 7.95 (d, J = 8.3 Hz,
6
3
H, aromatic CH), 7.62 (t, J = 7.4 Hz, 2H, aromatic CH), 7.51 (d, J = 7.8 Hz, 3H, aromatic
1
3
CH). C NMR (DMSO-d , ppm): δ 167.7, 162.3, 159.8, 154.4, 150.9, 117.3, 116.9, 118.4,
6
1
3
09.2; Anal. calc. (%) for C H N O : C, 61.41; H, 3.96; N, 22.04. Found (%): C, 60.49; H,
13 10 4 2
.92; N, 21.99.
23

4
.2.3.6. 5-(2-nitrophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (10)
–
1
1
Yield: 0.211 g, 56.7%. FT-IR (ν, cm ): 3194 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.23 (s, 1H, —NH), 8.42–8.22 (m, 2H, aromatic CH), 7.79–7.63 (m, 4H, aromatic CH),
1
3
7
1
3
.12–7.09 (m, 2H, aromatic CH). C NMR (DMSO-d , ppm): δ 162.1, 159.9, 154.2, 150.4,
6
48.2, 134.8, 131.2, 129.1, 128.2, 123.9, 109.9; Anal. calc. (%) for C H N O : C, 55.13; H,
1
3
9
5
3
.20; N, 24.73. Found (%): C, 55.19; H, 3.17; N, 24.71.
.2.3.7. 5-(3-nitrophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (11)
4
–
1
1
Yield: 0.260 g, 69.8%. FT-IR (ν, cm ): 3196 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.27 (s, 1H, —NH), 8.64–8.43 (m, 2H, aromatic CH), 8.16–7.99 (m, 2H, aromatic CH),
1
3
7
1
3
.67–7.58 (m, 4H, aromatic CH). C NMR (DMSO-d , ppm): δ 162.1, 160, 154.1, 150.1,
6
48.7, 133.4, 131.9, 129.7, 127.8, 126.0, 111.0. Anal. calc. (%) for C H N O : C, 55.13; H,
1
3
9
5
3
.20; N, 24.73. Found (%): C, 55.16; H, 3.23; N, 24.74.
.2.3.8. 5-(4-nitrophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (12)
4
–
1
1
Yield: 0.270 g, 72.4%. FT-IR (ν, cm ): 3197 (—NH stretching). H NMR (DMSO-d , ppm):
6
1
3
δ 9.21 (s, 1H, —NH), 8.68–8.27 (m, 4H, aromatic CH), 7.67–7.58 (m, 4H, aromatic CH). C
NMR (DMSO-d , ppm): δ 161.9, 160.2, 154.5, 150.4, 148.2, 132.1, 130.2, 127.9, 111.0.
6
Anal. calc. (%) for C H N O : C, 55.13; H, 3.20; N, 24.73. Found (%): C, 55.11; H, 3.19; N,
1
3
9
5
3
2
4.72.
.2.3.9. 5-(3-chlorophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (13)
4
–
1
1
Yield: 0.257 g, 71.8%. FT-IR (ν, cm ): 3195 (—NH stretching); H NMR (DMSO-d , ppm):
6
δ 9.25 (s, 1H, —NH), 8.42–8.31 (m, 2H, aromatic CH), 7.87–7.56 (m, 4H, aromatic CH),
1
3
7
1
3
.04–6.98 (m, 2H, aromatic CH); C NMR (DMSO-d , ppm): δ 161.2, 160.1, 154.9, 150.1,
6
35.1, 129.9, 128.8, 127.8, 127.1, 126.1, 111.0; Anal. calc. (%) for C H ClN O: C, 57.26; H,
1
3
9
4
.33; N, 20.55; Found (%): C, 57.27; H, 3.31; N, 20.53.
24

4
.2.3.10. 5-(4-chlorophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (14)
–
1
1
Yield: 0.260 g, 72.6%. FT-IR (ν, cm ): 3196 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.21 (s, 1H, —NH), 8.40–8.35 (m, 2H, aromatic CH), 7.71–7.53 (m, 4H, aromatic CH),
1
3
7
1
2
.01– 6.99 (m, 2H, aromatic CH). C NMR (DMSO-d , ppm): δ 161.4, 160.0, 154.2, 150.4,
6
34.9, 129.1, 128.4, 124.4, 110.9. Anal. calc. (%) for C H ClN O: C, 57.26; H, 3.33; N,
1
3
9
4
0.55. Found (%): C, 57.24; H, 3.29; N, 20.58.
.2.3.11. 5-(2,3-dichlorophenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (15)
4
–
1
1
Yield: 0.235 g, 58.4%. FT-IR (ν, cm ): 3193 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.12 (s, 1H, —NH), 8.50–8.43 (m, 1H, aromatic CH), 7.88–7.43 (m, 5H, aromatic CH),
1
3
7
1
5
.16–6.94 (m, 1H, aromatic CH). C NMR (DMSO-d , ppm): δ 158.6, 157.9, 154.2, 152.4,
6
41.8, 134.2, 133.8, 130.2, 129.8, 129.1, 124.0, 113.32. Anal. calc. (%) for C H Cl N O: C,
1
3
8
2
4
0.84; H, 2.63; N, 18.24. Found (%): C, 50.79; H, 2.59; N, 18.21.
4
.2.3.12. N-(pyridin-4-yl)-5-(m-tolyl)-1,3,4-oxadiazol-2-amine (16)
–
1
1
Yield: 0.231 g, 69.6%. FT-IR (ν, cm ): 3191 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.15 (s, 1H, —NH), 8.46-8.37 (m, 3H, aromatic CH), 7.98-7.49 (m, 3H, aromatic CH),
1
3
7
1
.17-6.99 (m, 2H, aromatic CH), 2.37 (s, 3H, CH ). C NMR (DMSO-d , ppm): δ 159.1,
3 6
57.9, 154.1, 151.2, 138.9, 130.1, 129.2, 128.9, 126.2, 124.0, 113.2, 23.1. Anal. calc. (%) for
C H N O: C, 66.65; H, 4.79; N, 22.21. Found (%): C, 66.67; H, 4.81; N, 22.19.
1
4
12
4
4
.2.3.13. N-(pyridin-4-yl)-5-(p-tolyl)-1,3,4-oxadiazol-2-amine (17)
–
1
1
Yield: 0.223 g, 67.2%. FT-IR (ν, cm ): 3194 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.06 (s, 1H, —NH), 8.44–8.31 (m, 2H, aromatic CH), 7.91–7.31 (m, 4H, aromatic CH),
1
3
7
1
.21–7.11 (m, 2H, aromatic CH), 2.34 (s, 3H, CH ). C NMR (DMSO-d , ppm): δ 159.0,
3 6
57.3, 154.7, 151.0, 130.6, 129.2, 127.1, 126.8, 123.7, 111.9, 23.4. Anal. calc. (%) for
C H N O: C, 66.65; H, 4.79; N, 22.21. Found (%): C, 66.61; H, 4.83; N, 22.24.
1
4
12
4
25

4
.2.3.14. 5-(4-methoxyphenyl)-N-(pyridin-4-yl)-1,3,4-oxadiazol-2-amine (18)
–
1
1
Yield: 0.228 g, 64.6%. FT-IR (ν, cm ): 3491 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.08 (s, 1H, —NH), 8.45–8.37 (m, 2H, aromatic CH), 7.72–7.70 (m, 2H, aromatic CH),
1
3
7
1
.19–7.03 (m, 4H, aromatic CH), 3.73 (s, 3H, OCH ). C NMR (DMSO-d , ppm): δ 163.1,
3 6
59.7, 157.2, 154.2, 151.5, 132.2, 130.5, 129.7, 126.1, 114.2, 111.0, 55.9. Anal. calc. (%) for
C H N O : C, 62.68; H, 4.51; N, 20.88. Found (%): C, 62.59, H 4.53, N, 20.92.
1
4
12
4
2
4
.2.3.15. N-(pyridin-4-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-amine (19)
–
1
1
Yield: 0.288 g, 66.7%. FT-IR (ν, cm ): 3494 (—NH stretching). H NMR (DMSO-d , ppm):
6
δ 9.08 (s, 1H, —NH), 8.41–8.36 (m, 2H, aromatic CH), 7.30 (s, 2H, aromatic CH), 7.11–7.01
1
3
(
m, 2H, aromatic CH), 3.75 (s, 3H, OCH ), 3.65 (s, 6H, OCH ×2). C NMR (DMSO-d ,
3 3 6
ppm): δ 159.2, 157.1, 154.0, 153.4, 151.2, 138.1, 120.1, 111.5, 104.5, 58.4, 55.2. Anal. calc.
(
%) for C H N O : C, 58.53; H, 4.91; N, 17.06. Found (%): C, 58.48; H, 4.94; N, 17.03.
16 16 4 4
4
.3. Determination of LogP values: Shake flask method
The lipophilic constant of all the synthesized compounds (5-19) was determined in n-octanol
and buffer (pH 7.4) by the shake flask method. The log P was calculated by correlating the
absorbance with the concentration using a standard plot [28].
4
.4. Pharmacology
4
.4.1. Cholinesterase inhibition assay [40,50]
The in vitro cholinesterase inhibitory potency against AChE and BChE was determined for
all the synthesized compounds using Ellman colorimetric assay. All the assay solutions were
prepared in 0.1M phosphate buffer solution at pH 7.4 except the test compounds, which were
prepared in biological grade DMSO (final concentration <1%). The final assay solutions
consisted of 340 µM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 2.5 units/ml of hAChE
(AChE from human erythrocytes, Sigma EC No. 3.1.1.7) or hBChE (BChE from human
serum, Sigma EC No. 3.1.1.8) at varied concentrations of test compounds (10 nM to 10 µM)
26

in biological grade DMSO, and 550 µM of acetylthiocholine iodide (ATCI) or
butyrylthiocholine iodide (BTCI) as substrate for AChE and BChE, respectively. Initially,
the test compounds were pre-incubated with enzyme for 10 min at room temperature. After
that period, DTNB and substrate were added, and absorbance was measured at 412 nm for 6
min using H1M synergy multimode microplate reader. Donepezil was used as a positive
reference standard. The reaction rates were compared, and the percentage inhibition due to
the presence of test compounds was calculated with respective IC values using graph pad
5
0
prism version 5.01. The IC values can be designed as the concentration of test inhibitors
5
0
responsible for 50% inhibition of enzyme action as compared to control (without inhibitors).
All results are determined as the mean of three independent measurements.
4
.4.2. Enzyme kinetics [51]
The Lineweaver-Burk method was used to determine the mechanism of enzyme inhibition
using the different concentrations of substrate and a fixed quantity of hAChE, in the absence
or presence of inhibitor. The concentration of inhibitor was selected by its IC50 value.
Compound 9 was used in three different concentration range (1, 1.5, and 2 µM). Each
concentration of inhibitor was evaluated with six different concentrations of ATCI. The
values of Vmax and K were calculated using Michaelis-Menten non-linear regression
m
kinetics, and the mechanism of enzyme inhibition was determined by Lineweaver-Burk plots
[
51] using Graph Pad Prism 5.01. The inhibitory constant Ki value was determined by the
Cheng‒Prusoff equation using non-competitive inhibition enzyme kinetics assay model [52].
.4.3. PAMPA-BBB assay [43,48]
4
The permeability of synthesized compounds across the blood-brain barrier (BBB) was
assessed by PAMPA-BBB assay. The protocol of PAMPA-BBB assay involved the coating
of acceptor microplate (PTFE, 0.45 µm, Merck Millipore) with 4 µl of 20 mg/ml porcine
27

brain lipid (Avanti polar lipids) in dodecane (Avra synthesis). After coating, 200 µl of pH 7.4
buffer was taken in acceptor microplates. Test compounds were initially dissolved in DMSO
and further diluted with pH 7.4 buffer to prepare 25 µg/ml concentration. 200 µl of the test
solution was taken in a donor microplate (Merck Millipore). The donor microplate placed
carefully like a sandwich over acceptor microplate and 18 h incubation period was initiated.
Spectrophotometrically using H1M synergy multimode microplate reader (Biotek, USA)
concentration of test compounds were determined in acceptor and donor microplates. The
model was validated using standard commercial drug samples.
4
.4.4. Amyloid beta aggregation inhibition: thioflavin T assay
Aβ1-42 (Sigma, India) was dissolved in 1% v/v ammonium hydroxide solution to get the 2000
µM stock solution and stored at −80 °C. The test compound was initially dissolved in DMSO,
and final dilutions were made in PBS pH 7.4. Different proportion of Aβ1-42: inhibitor was
tested (10:5, 10:10; and 10:20 µM) by thioflavin T assay [48,53,54]. All the experiments
were performed in triplicates. The mixture of Aβ1-42 (final concentration 10 µM, 2 µl) and
AChE from human erythrocytes (final concentration 230 µM, 16 µl) were incubated with or
without tested compound (5 µM, 10 µM, and 20 µM; 2 µl) at 37 °C for 48 h. The
fluorescence intensities were measured with the addition of 50 mM glycine-NaOH buffer (pH
8
.0) containing 5 µM thioflavin T at the excitation and emission wavelength of λ = 450 nm
ex
and λ = 485 nm, respectively. The percentage inhibition was calculated using the following
em
expression: 100 – (IF /IF × 100), where IF and IF are the fluorescence intensities with and
i
0
i
0
without inhibitor, respectively after subtracting the values with the blank. The results were
also reported as normalized fluorescence intensity with respect to control.
28

4
.4.5. In vivo studies
4
.4.5.1 Animals
The behavioral studies were conducted on male Albino mice (adult male, 25–30 g). These
mice were bought from Central Animal Facility, IMS (BHU), Varanasi, India. Mice were
kept in six per polyacrylic cage with semi-synthetic balanced diet, water, temperature and
relative humidity of the storage room was maintained to 25 ± 2 °C and 55 ± 10%,
respectively under 12 h light/dark rotations. Mice were maintained and treated according to
CPCSEA guidelines. The study protocols and quantity of mice were duly permitted by the
IAEC (Dean/2017/CAEC/88]. Different animals were used for each behavioral investigation.
4
.4.5.2 Experimental design
Animals were adapted to the laboratory conditions 7 days before behavioral studies.
Scopolamine hydrobromide solution was prepared by dissolving it in distilled water, and
amnesia was induced by its intraperitoneal administration of scopolamine (0.5 mg/kg) on the
7th day, 1 h after the administration of treatment. The behavioral studies were performed just
after 5 min of scopolamine administration. Donepezil hydrochloride and compound 6g were
suspended in the 0.3% sodium carboxymethyl cellulose (CMC). The animals were divided
into six groups for the behavioral experiment. Each group having six mice as following: (i)
control, (ii) scopolamine (0.5 mg/kg, i.p.), (iii) donepezil (5 mg/kg, p.o.) as a reference
standard, (iv) compound 9 (2.5 mg/kg) (v) compound 9 (5 mg/kg) (vi) compound 9 (10
mg/kg). Donepezil and compound 9 were given orally (p.o.) once daily for seven consecutive
days to all respective groups except for the control and scopolamine group. Scopolamine
group of animals were given vehicle treatment for seven days. The behavioral studies were
accomplished by Y-maze and passive avoidance test.
29