Design and development of novel N-(pyrimidin-2-yl)-1,3,4-oxadiazole hybrids to treat cognitive dysfunctions

Article abstract of DOI:10.1016/j.bmc.2019.02.031

Novel hybrids bearing a 2-aminopyrimidine (2-AP) moiety linked to substituted 1,3,4-oxadiazoles were designed, synthesized and biologically evaluated. Among the developed compounds, 28 noncompetitively inhibited human acetylcholinesterase (hAChE; pIC

Full text of DOI:10.1016/j.bmc.2019.02.031

Accepted Manuscript
Design and development of novel N-(pyrimidin-2-yl)-1,3,4-oxadiazole hybrids
to treat cognitive dysfunctions
Prabhash Nath Tripathi, Pavan Srivastava, Piyoosh Sharma, Ankit Seth, Sushant
K. Shrivastava
PII:
S0968-0896(18)31848-0
https://doi.org/10.1016/j.bmc.2019.02.031
BMC 14767
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 November 2018
14 February 2019
15 February 2019
Please cite this article as: Tripathi, P.N., Srivastava, P., Sharma, P., Seth, A., Shrivastava, S.K., Design and
development of novel N-(pyrimidin-2-yl)-1,3,4-oxadiazole hybrids to treat cognitive dysfunctions, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.031
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Design and development of novel N-(pyrimidin-2-yl)-1,3,4-oxadiazole
hybrids to treat cognitive dysfunctions
Prabhash Nath Tripathi, Pavan Srivastava, Piyoosh Sharma, Ankit Seth, Sushant K.
Shrivastava*
Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical
Engineering & Technology, Indian Institute of Technology (Banaras Hindu University),
Varanasi – 221 005 (India)
*Address correspondence to:
Dr. Sushant Kumar Shrivastava
Professor
Department of Pharmaceutical Engineering & Technology,
Indian Institute of Technology
(Banaras Hindu University), Varanasi- 221 005, India
Email: skshrivastava.phe@itbhu.ac.in
Phone: +91-542-6702725
1

ABSTRACT
Novel hybrids bearing a 2-aminopyrimidine (2-AP) moiety linked to substituted 1,3,4-
oxadiazoles were designed, synthesized and biologically evaluated. Among the developed
compounds, 28 noncompetitively inhibited human acetylcholinesterase (hAChE; pIC50
=
6.52; Ki = 0.17 µM) and showed potential in vitro antioxidant activity (60.0%) when
evaluated using the Ellman’s and DPPH assays, respectively. Compound 28 competitively
displaced propidium iodide (PI) from the peripheral anionic site (PAS) of hAChE (17.6%)
and showed high blood-brain barrier (BBB) permeability, as observed in the PAMPA-BBB
assay. Additionally, compound 28 inhibited hAChE-induced Aβ aggregation in a
concentration-dependent manner according to the thioflavin T assay and was devoid of
neurotoxic liability towards SH-SY5Y cell lines, as demonstrated by the MTT assay. The
behavioral studies of compound 28 in mice showed a significant reversal of scopolamine-
induced amnesia, as observed in Y-maze and passive avoidance tests. Furthermore,
compound 28 exhibited significant AChE inhibition in the brain in ex vivo studies. An
evaluation of oxidative stress biomarkers revealed the antioxidant potential of 28. Moreover,
in silico molecular docking and dynamics simulation studies were used as a computational
tool to evaluate the interactions of compound 28 with the active site residues of hAChE.
Keywords: Acetylcholinesterase; Aβ aggregation; Antioxidant; 2-Aminopyrimidine; 1,3,4-
Oxadiazole.
2

1. INTRODUCTION
Cognition is a combination of attention, perception, acquaintance, skills, memory,
reminiscence, decision making, planning and judgment that define the normal nature of
human beings.1,2 Memory loss and cognitive impairment are observed in various conditions,
such as aging,3 stroke,4 toxicant exposure,5 head injury6 and neurodegenerative disorders such
as schizophrenia, depression, Alzheimer’s disease (AD)7,8 and Parkinson’s disease.9,10
Neurotransmitters such as acetylcholine (ACh),11 dopamine,12 serotonin,13 and glutamate14
are responsible for regulating cognitive functions. Among these compounds, ACh, which is
hydrolyzed by acetylcholinesterase (AChE) into choline and acetic acid,15 is an important
neurotransmitter in the regulation of learning and memory processes. Low levels of ACh in
the hippocampus, cortex area and basal forebrain have been implicated in cognitive deficit
and loss of short-term memory.16,17 AChE also induces the aggregation and deposition of Aβ
fibrils by forming AChE-Aβ complexes, leading to cognitive dysfunction.18,19 Hence, the
elevation of ACh through AChE inhibition and the prevention of Aβ aggregation are the two
most promising approaches to halting the progression of dementia. Additionally, oxidative
stress is another damaging factor caused by an imbalance between the generation of reactive
oxygen species (ROS) and antioxidant enzymes. Excessive ROS release triggers protein
oxidation and lipid peroxidation, which subsequently cause oxidative damage and lead to
impaired cognitive function. Research also suggests that Aβ could enter the mitochondria,
increasing the generation of free radicals and inducing oxidative stress.20-22
Currently, there are three AChE inhibitors (donepezil, rivastigmine and galantamine)
approved by the US FDA to relieve the symptoms of AD, but these compounds have no
active role in the prevention of disease progression.23 These drugs exhibit several side effects,
such as urinary incontinence, weakness and muscle cramps, which limits their use in the
advanced stages of the disease.24,25 Therefore, the development of novel neuro-therapeutic
3

agents that will impede the metabolism of ACh by inhibiting AChE and preventing Aβ
aggregation and provide antioxidant activity to decelerate disease progression is of great
importance.26
1.1. Designing Considerations
These molecules were designed based on the bioisosteric replacement and hybrid
pharmacophore approach. Several pyrimidine-based derivatives have been explored as
attractive scaffolds because of their broad biological activities.27-30 Derivatives containing
pyrimidine-fused heterocyclic rings, such as sugar-linked 2-oxo- and 2-thioxo-pyrimidine,
5H-thiazolo[3,2-a]pyrimidine, pyrimidine-triazolopyrimidine, pyrimidine-pyridine and
thiazolo-pyrimidine hybrids (I), have been reported to show AChE inhibitory activity.31-33
These studies have suggested that 2H-thiazolo-[3,2-a]pyrimidine derivatives have a
significant binding affinity towards AChE.34 Furthermore, a [1,2,4]triazolo[1,5-a]pyrimidine
derivative (II) was designed as a multifunctional agent capable of inhibiting AChE and
AChE-induced Aβ aggregation along with showing antioxidant activity.35 A recent study also
indicated that 4,6-diarylpyrimidine derivative III exhibited dual inhibitory activities against
AChE and monoamine oxidase.36 Additionally, a 2,4-disubstituted pyrimidine derivative (IV)
has been reported to show cholinesterase (ChE) inhibitory and Aβ anti-aggregatory
activity.37-40 On the basis of these reports, a 2-aminopyrimidine (2-AP) scaffold was selected
and utilized as a template to design small molecules as potential therapeutics for the treatment
of cognitive dysfunctions.
A series of heteroaromatic/aromatic ring-containing compounds, such as imidazole, acridine,
cinnamide-dibenzylamine, p-aminobenzoic acid, triazine, and benzoxazole derivatives, were
developed to evaluate their AChE/BChE inhibitory potential and their antioxidant activities
for the treatment of dementia.7,8,10,41-43 Compounds bearing a 1,3,4-oxadiazole nucleus have
been reported to have various therapeutic applications in the field of drug discovery owing to
4

their planer aromatic core and ability to accept hydrogen bonds, which allow them to achieve
the requisite orientation of their substituents within the enzyme pocket.44-46 Additionally,
compounds containing the 2,5-substituted-1,3,4-oxadiazole nucleus, such as 2-(furanyl)-5-
(3,4,5-trimethoxystyryl)-1,3,4-oxadiazole (V), 2-(2-(benzo[d][1,3]dioxol-5-yl)vinyl)-5-(2-
chloro-4-fluorophenyl)-1,3,4-oxadiazole (VI) and 2-((benzothiazol-2-ylthio)methyl)-5-((4-
bromobenzyl)thio)-1,3,4-oxadiazole (VII), significantly inhibit AChE and AChE-induced Aβ
aggregation and show neuroprotective activity.47-49
Considering the reported literature, the 2-AP moiety was tethered to a substituted 1,3,4-
oxadiazole nucleus within a single hybrid pharmacophore skeleton with the aim of
developing multitargeted ligands with the ability to inhibit AChE and AChE-induced Aβ
aggregation and serve as an antioxidant (Fig. 1).
Fig. 1. Designing strategy for the target compounds.
5

2. RESULTS AND DISCUSSION
2.1. Chemistry
The title compounds (4-20) were prepared using the reaction sequence described in Scheme
1. In the first step, 2-AP (1) was reacted with phenyl chloroformate (PCF) and pyridine in dry
dichloromethane (DCM) to yield phenyl pyrimidin-2-ylcarbamate (2). Key intermediate 3
was synthesized in an appreciable yield (81%) by refluxing compound 2 with hydrazine
hydrate (85% v/v) in absolute ethanol. The subsequent reaction of compound 3 with
numerous aromatic aldehydes in absolute ethanol yielded the corresponding semicarbazones
(4-18). The reduction of compounds 4-18 was attempted with various reducing reagents, such
as sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3),
sodium borohydride-nickel chloride and sodium borohydride-cobalt chloride. However, only
compounds 7 and 18 reacted with sodium borohydride (NaBH4)50 in dry methanol to afford
their reduced congeners (19 and 20, respectively).
As shown in Scheme 2, compounds 4-7 and 9-18 were reacted with chloramine-T (CAT) in
absolute ethanol to obtain the corresponding 1,3,4-oxadiazole derivatives (21-24 and 26-35)
through an oxidative cyclization reaction. Compound 8 was cyclized using N-
bromosuccinimide (NBS) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base to afford
compound 25. All the synthesized compounds were preliminarily identified using
Dragendorff’s reagent on TLC (thin-layer chromatography).51 The crude products were
recrystallized from absolute ethanol to yield the title compounds. The structures of the
compounds were confirmed by spectroscopic (FT-IR, 1H NMR and 13C NMR) and elemental
analyses.
6

Scheme 1. Reagents and conditions: (a) PCF, pyridine, dry DCM, under nitrogen, 0-5 ºC, 3 h;
(b) 85% v/v hydrazine hydrate, absolute ethanol, reflux, 30 min; (c) various aromatic
aldehydes, absolute ethanol, reflux, 3 h; (d) sodium borohydride, dry methanol, 0-5 ºC, 1 h.
Scheme 2. Reagents and conditions: (a) CAT, absolute ethanol, reflux, 2 h; (b) NBS, DBU,
dry DCM, room temperature, 3-4 h.
7

1
The H NMR spectrum confirmed the formation of compound 2, as evidenced by the
appearance of a carbamate proton peak at 11.31 ppm. The diagnostic peaks of the
hydrazinecarboxamide (–NHCONHNH2) group of compound 3 appeared at 9.87, 9.65 and
4.24 ppm, respectively. Furthermore, the spectra of the semicarbazone derivatives (4-18)
indicated the presence of a distinctive imine proton (–HC=N) at 8.55-8.21 ppm, and the
amine (-NH2) peak had disappeared. Additionally, the –NHCONHN=CH group exhibited
two broad singlets between 12.55-12.23 ppm and 10.29-10.02 ppm, respectively. The 4-
methyl (–CH3), 4-methoxy (–OCH3) and 3,4-dimethoxy (–OCH3×2) groups in compounds 5,
6 and 7 displayed characteristic signals at 2.34, 3.73 and 3.82 ppm, respectively. The three
methoxy groups (–OCH3×3) of compound 8 exhibited two singlet peaks for six protons and
for three protons at 3.82 and 3.69 ppm, respectively. The formation of the 1,3,4-oxadiazole
moiety in compounds 21-35 was confirmed by the absence of the distinctive peak of the
imine proton.
13
In their C NMR spectra, compounds 4-18, with imine (–N=CH) and carbonyl (–NH–CO–
NH–) groups, showed signals at approximately 151.3 ppm and 158.1 ppm, respectively.
Compounds 5, 6 and 7, bearing 4-methyl, 4-methoxy and 3,4-dimethoxy groups, respectively,
exhibited diagnostic signals at 21.4, 55.7 and 56.0 ppm. The methoxy carbons of the 3,4,5-
trimethoxyphenyl group in compound 8 appeared as two signals at 60.5 and 56.3 ppm. The
absence of the imine (–N=CH) and carbonyl (>C=O) signals in the spectra of compounds 4-
18 and the subsequent appearance of (>C=N) signals in the spectra of 21-35 confirmed the
formation of a 1,3,4-oxadiazole ring. The purity of each intermediate and target compound
was ascertained using elemental analysis, and the results were within ± 0.4% of the
theoretical values.
8

2.2. Pharmacology
2.2.1. In vitro cholinesterase inhibition assay
All the synthesized compounds (21-35) were evaluated by Ellman’s colorimetric method on
hAChE (acetylcholinesterase from human erythrocyte) and hBChE (butyrylcholinesterase
from human serum) using donepezil as a reference standard (Table 1). With the exceptions of
compounds 27, 33 and 34, compounds 21-35 inhibited hAChE (pIC50 < 5.3). Compound 21,
with a phenyl ring at the 5 position of the 1,3,4-oxadiazole core, exhibited considerable
hAChE inhibitory activity (pIC50 = 5.69 ± 0.02). Among derivatives 22-27, compound 25,
bearing an electron-donating 3,4,5-trimethoxyphenyl group, showed significant hAChE
inhibitory potential (pIC50 = 5.91 ± 0.11). Next, of the compounds in this series, 29-35, which
possess electron-withdrawing groups on the phenyl ring, exhibited slightly diminished
hAChE inhibitory activities compared to those of compounds 22-27, which possess electron-
donating groups. Among all the evaluated derivatives (21-35), compound 28, bearing a
naphthyl ring, displayed the most significant hAChE inhibitory activity (pIC50 = 6.52 ± 0.04).
The enhanced lipophilicity of compound 28 due to its naphthyl group may be the cause of its
effective interactions with the active site residues of hAChE. Compounds 28, 31 and 32 were
the only synthesized compounds that significantly inhibited hBChE.
9

Table 1. The results of the human cholinesterase (hAChE and hBChE) and DPPH assays of
target compounds 21-35.
apIC50 ± SD
Reduction %
DPPH at 20
µM
Comp.
Code
R
bSI
hAChE
hBChE
-H
5.69 ± 0.02
5.79 ± 0.05
5.81 ± 0.03
< 4.3
< 4.3
< 4.3
-
-
-
51.4 ± 1.7
< 20
21
22
23
4-CH3
4-OCH3
45.8 ± 2.7
3,4-(OCH3)2
5.72 ± 0.12
< 4.3
-
49.5 ± 2.4
24
3,4,5-(OCH3)3
4-OH
2-OH
1-Naphthyl
4-F
5.91 ± 0.11
5.76 ± 0.06
< 5.3
< 4.3
< 4.3
-
57.7 ± 4.4
63.4 ± 1.9
58.8 ± 1.9
60.0 ± 2.3
41.9 ± 1.1
46.2 ± 2.1
47.8 ± 1.7
47.9 ± 2.6
< 20
25
26
-
< 4.3
-
27
6.52 ± 0.04
5.62 ± 0.05
5.54 ± 0.03
5.43 ± 0.03
5.48 ± 0.15
< 5.3
4.92 ± 0.05
< 4.3
39.81
28
-
29
4-Cl
< 4.3
-
30
4-Br
4.64 ± 0.07
4.69 ± 0.04
< 4.3
6.17
31
3-Br
6.17
32
2-Cl
-
33
4-NO2
2-NO2
-
< 5.3
< 4.3
-
-
< 20
34
5.47 ± 0.04
6.83 ± 0.02
< 4.3
51.8 ± 3.3
< 20
35
donepezil
5.32 ± 0.04
32.36
ascorbic
acid
-
-
-
-
64.6 ± 5.8
All the values are expressed as the mean ± S.D. (n=3); pIC50 = [-Log (IC50 × 10-6 M)]; selectivity index (S.I.)
a
b
corresponds to the antilog of ΔpIC50; (ΔpIC50 = pIC50 hAChE - pIC50 hBChE).52
2.2.2 hAChE kinetics study with compound 28
Compound 28 was subjected to an enzyme kinetics study to determine the type of hAChE
inhibition using a Lineweaver-Burk plot (1/Vmax Vs. 1/[S]), and the results suggested a
noncompetitive enzyme inhibition (Ki = 0.17 µM) (Fig. 2).
10

Fig. 2. Enzyme kinetics study of the inhibition of hAChE by compound 28.
2.2.3 DPPH (α,α-diphenyl-β-picrylhydrazyl) assay
The DPPH assay was used to determine the antioxidant activities of the synthesized
compounds (21-35). Compounds 25, 26, 27 and 28, containing hydrogen bond donating
groups (–NH and –OH), showed potential antioxidant activities (57.7, 63.4, 58.8 and 60.0%,
respectively) by scavenging the DPPH free radical at a concentration of 20 µM. The results
of the DPPH assay are shown in Table 1.
2.2.4. Propidium iodide (PI) displacement assay
Compounds 25, 26 and 28 were evaluated for PI displacement from the PAS-hAChE enzyme
complex. The results revealed that compounds 25 (17.3%) and 28 (17.6%) considerably
displaced PI from the PAS-hAChE complex. These results are comparable with those of
donepezil (18.0%) (Table 2).
2.2.5 Blood-Brain Barrier (BBB) Permeation Assay
The parallel artificial membrane permeation assay (PAMPA-BBB) was performed to assess
BBB permeability of compounds 25, 26 and 28, and the model was validated using nine
commercial drugs. The results of the PAMPA-BBB assay indicated that compound 28 had
higher permeability than compounds 25 and 26 through the BBB. Based on these results,
compound 28 was selected for further biological evaluations (Table 2).
11

Table 2. The results of PI displacement and PAMPA-BBB assays.
Comp.
Code
PAMPA-BBB permeability PAMPA-BBB Prediction
PI Displacement (%)a
Pe(exp) (10-6 cm/s)
(CNS+b, CNS-c, CNS±d)
17.3 ± 0.9
13.6 ± 0.8
17.6 ± 1.3
18.0 ± 1.2
4.21 ± 0.37
3.23 ± 0.23
5.82 ± 0.56
6.89 ± 0.61
CNS±
CNS±
CNS+
CNS+
25
26
28
donepezil
aPI displacement assay was performed on hAChE, and the values are presented as the mean ± S.D. (n=3);
b‘CNS+’ (prediction of high BBB permeation); Pe (10-6 cm/s) > 4.3926; ‘CNS-’ (prediction of low BBB
permeation); Pe (10-6 cm/s) < 1.7766; d‘CNS±’ (uncertain BBB permeation); Pe (10-6 cm/s) 4.3926 to 1.7766.
c
2.2.6 hAChE-induced Aβ aggregation assay
According to the results of the PI displacement assay, compound 28 significantly interacts
with PAS-hAChE. Therefore, its ability to inhibit hAChE-induced Aβ aggregation was
evaluated using the thioflavin T (ThT) assay. The outcome of the study revealed that its
ability to inhibit hAChE-induced Aβ aggregation was concentration dependent, and the
highest inhibition was observed at a concentration ratio of 1:2 (Aβ:inhibitor) (Fig. 3).
Normalized Fluorescence Intensity (NFI)
in hAChE-induced aggregation
1.00
***
***
0.75
***
***
***
***
0.50
0.25
0.00
)
1
2
1
2

0.5
1:
0.5
1:
1:
1:
1:
1:
A
(
ol
Donepezil
Compound 28
+ A
+ A
Contr
Fig. 3. Effects of donepezil and compound 28 on hAChE-induced Aβ aggregation. ***p <
0.001 compared to control. All values are expressed as the mean ± S.D. (n=3) of the
normalized fluorescence intensity (NFI).
12

2.2.7 Neurotoxicity assay
Neuroblastoma SH-SY5Y cells are mostly used for assessing neurotoxicity owing to their
high resemblance to mature human neurons.53 The neurotoxicity of compound 28 was
evaluated by an MTT-based colorimetric assay. The assay showed that compound 28 was not
neurotoxic (Table 3).
Table 3. The results of neurotoxicity on the SH-SY5Y cell line.
Compound
28
IC50 (µM)a
79.4 ± 5.9
84.1 ± 6.9
donepezil
aValues are expressed as the mean ± S.D. (n = 3).
2.2.8 Acute oral toxicity study
The acute toxicity of compound 28 was determined on healthy Swiss albino mice (25-30 g)
following the OECD 423 guidelines. Several behavioral changes, cholinergic effects and
toxic reactions, such as tremors, convulsions, salivation, diarrhea, sleep, lacrimation and
feeding behavior, were monitored. There were no signs of any cholinergic side effects, and
no toxicity or mortality was observed following the administration of test compound 28. The
study revealed that compound 28 has a significant safety margin.54
2.2.9 In vivo behavioral studies
Scopolamine-induced amnesia in mice is the standard model in behavioral pharmacology for
the evaluation of AChE inhibitory potential. Scopolamine is a muscarinic antagonist, and its
administration induces cognitive impairment through cholinergic deficit.55
2.2.9.1 Y-maze test in mice
Compound 28 was assessed for its ability to enhance learning and memory against a
scopolamine-induced cognitive deficit. The results showed a significantly decreased (***p <
0.001) spontaneous alternation rate in scopolamine compared to the control. Treatment with
compound 28 elicited a dose-dependent increase (2.5 mg/kg: ##p < 0.01; 5 and 10 mg/kg: ###
p
13

< 0.001) in the spontaneous alternation rate compared to scopolamine (Fig. 4A).
Additionally, the locomotive behavior of the scopolamine-treated mice did not change as
confirmed by the nonsignificant changes in total arm entries among all groups (Fig. 4B).
2.2.9.2 Passive avoidance test
The effect of treatment with compound 28 on scopolamine-induced memory impairment in
mice was evaluated by a passive avoidance test. The transfer latency time (TLT) was
calculated, and the respective groups in the acquisition and retention tests were compared.
During the retention tests, the TLT of the scopolamine group was significantly lower than
that of the control group, which signified an induction of memory impairment. There were
nonsignificant differences between the TLT of the acquisition and retention tests in the
scopolamine and vehicle groups. The TLT was significantly greater (***p < 0.001) in mice
treated with compound 28 compared to the corresponding acquisition test and the
scopolamine group in the retention test. Moreover, treatment with compound 28 also
prolonged TLT in a dose-dependent manner, which is similar to what was observed in the Y-
maze experiment (Fig. 5).
A
80
B
25
###
###
20
15
10
5
60
40
20
0
###
##
***
0
l
5
5
5
e
5
.
5
e
o
le
10
l
10
r
t
2
2.5
c
i
c
n
n
o
h
hi
o
ontrol
e
D
Done
C
C
V
Ve
28 (mg/kg)
28 (mg/kg)
Scopolamine (0.5 mg/kg)
Scopolamine (0.5 mg/kg)
Fig. 4. Effect of treatment with compound 28 (2.5, 5 and 10 mg/kg, p.o.) on scopolamine-
induced amnesia in a Y-maze test. (A) Spontaneous alternation rate; (B) total arm entries.
Bars indicate the mean ± S.D. (n = 6); ***p < 0.001 compared to the control; ###p < 0.001, ##p
< 0.01 compared to scopolamine; Done5 = donepezil.
14

Retention Test
200
150
100
50
***
***
***
***
***
Acquisition test
0
l
l
5
5
.
5
5
e
5
.
2
5
o
o
e
10
10
2
icle
icle
n
n
tr
tr
h
h
n
n
Do
Do
Ve
Ve
Co
Co
28 (mg/kg)
Scopolamine (0.5 mg/kg)
28 (mg/kg)
Scopolamine (0.5 mg/kg)
Fig. 5. Effect of treatment with compound 28 on scopolamine-induced memory impairment
in a passive avoidance test. Bars indicate the mean ± S.D. (n = 6); ***p < 0.001 compared to
the respective group in the acquisition test; Done5 = donepezil.
2.2.10 Ex vivo estimation of AChE
The ex vivo estimation of AChE was performed using the Ellman assay with mouse brain
homogenates. The results suggested that compound 28 (10 mg/kg) significantly reduced the
rate of ACh hydrolysis via the inhibition of brain AChE. It also reflected that compound 28
was able to permeate the BBB (Fig. 6A), and the results were in agreement with those of the
PAMPA-BBB assay.
2.2.11 Biochemical estimation of the oxidative stress factors
Oxidative stress is a major detrimental factor associated with neurodegenerative disorders.
Scopolamine is responsible for the induction of oxidative stress by altering the levels of
several biochemical markers, such as malondialdehyde (MDA), total nonprotein thiol, and
superoxide dismutase (SOD).21,56 Therefore, compound 28 was investigated for its
neuroprotective activity via the reduction of oxidative stress in cognitively impaired mice.
The biochemical assessment of these biomarkers was performed using mouse brain
homogenates.
15

2.2.11.1 2-Thiobarbituric acid-reactive substances (TBARS) assay
The TBARS method is widely used to estimate malondialdehyde (MDA) levels. The results
revealed that scopolamine treatment caused a significant induction (***p < 0.001) in oxidative
stress with increased levels of malondialdehyde (MDA) compared to the control. Compound
28 was not as active as scopolamine (###p < 0.001) in promoting oxidative stress (Fig. 6B).
2.2.11.2 Assay of the total nonprotein thiol content
The method reported by Sedlak and Lindsay was used to determine the total nonprotein thiol
contents in the brain samples.57,58 The results indicated that scopolamine treatment
significantly lowered (***p < 0.001) the total nonprotein thiol levels compared to the control
group, suggesting an induction of oxidative stress. Compound 28 elevated the total
nonprotein thiol levels (###p < 0.001) compared to the scopolamine-treated group (Fig. 6C).
2.2.11.3 Superoxide dismutase (SOD) assay
Compound 28 showed a higher SOD level (###p < 0.001) compared to that of the scopolamine
group, which reflects its ability to limit oxidative stress (Fig. 6D). Overall, the results
revealed that compound 28 (10 mg/kg) has significant antioxidant potential, and it can
reverse scopolamine-induced oxidative stress to improve learning and memory.
16

A
20
15
10
5
B
30
20
10
0
***
***
###
###
###
0
Control
Done
28
Control
28
Scopolamine
(0.5 mg/kg)
Scopolamine
(0.5 mg/kg)
C
D
15
10
5
8
###
6
4
2
0
###
***
***
0
Control
28
Control
28
Scopolamine
(0.5 mg/kg)
Scopolamine
(0.5 mg/kg)
Fig. 6. The results of ex vivo and biochemical analysis. (A) AChE activity; (B) MDA level; (C) total
nonprotein thiol content; (D) SOD level. The values are presented as the mean ± S.D. (n = 6); ***p <
0.001 compared to the control; ###p < 0.001 compared to scopolamine.
2.3. Computational Studies
2.3.1 In silico molecular docking studies
A molecular docking study was performed to assess the binding affinity and pose stability of
compound 28 in the active sites of hAChE (PDB Code: 4EY7) using the Glide module of
Schrödinger Maestro 2018-1. The cocrystallized Aricept (donepezil) ligand was extracted and
redocked in the hAChE grid for validation of the docking parameters. The poses of the
cocrystallized and redocked ligands were compared using a superposition tool, and the
RMSD value was found to be 0.2 Å (Supp. Fig. S2).
Compound 28 and donepezil showed docked scores of -13.8 and -14.6 kcal/mole,
respectively. The naphthyl group of compound 28 exhibited π-π stacking and polar
interactions with His447 in the catalytic active site (CAS). Additionally, compound 28
formed a polar interaction with Ser203 in the CAS. Compound 28 interacted with all PAS
17

residues through hydrophobic interactions (Tyr72, Tyr124, Trp286 and Tyr341) and
electrostatic interactions (Asp74). At the anionic subsite, compound 28 formed hydrophobic
interactions with Trp86 and Phe338 and electrostatic interactions with the Glu202 residue.
The N-atom of 4-AP interacted with the Phe295 residue in the acyl binding pocket through
H-bonding interactions. At the oxyanion site, compound 28 interacted with Gly121 and
Gly122 and formed hydrophobic interactions with Ala204 (Fig. 7A, 7B and Table 4).
Fig. 7. The docking of compound 28 in the hAChE enzyme. (A) Image of the 3D interactions
of compound 28 in the ligand binding surface (pink color) with the active site residues of
hAChE; (B) 2D image showing the active site interactions.
Table 4. Detailed analysis of the molecular docking interaction of compound 28 and
donepezil in hAChE (4EY7).
Interacting residues#
Acyl
Comp.
Anionic
subsite
Other interacting
residues
CAS
PAS
binding
pocket
Tyr72c, Asp74d,
Trp86c,
Ala204c, Val294c, Gly441c,
Leu289c, Ser293a, Tyr337c,
Arg296d, Gly121c,f, Gly122c
Ser203a,
His447a,b
Phe295c,e,
Phe297c
Tyr124c, Trp286c, Phe338b,c,
28
Tyr341c
Glu202d
Gly120c,f, Gly121c,f,
Tyr133c, Leu289c, Val294c,
Tyr337c, Gly448c, Ile451c
Ser203a, Tyr72c ,Tyr124c, Trp86b,c,g
His447a Trp286b,c,Tyr341c Phe338b,c,g
,
Phe295c,e,
Phe297c
donepezil
#All the interacting residues are within the 4 Å distance with the ligand; polar; π-π stacking; hydrophobic;
a
b
c
delectrostatic; eH-bonding; fglycine and gπ-cation interactions.
18

2.3.2 Molecular dynamics (MD) simulations
A 30 nsec MD simulation was performed to affirm the binding pose stability of the
compound 28-hAChE docked complex. The generated trajectories were utilized to produce
simulation interaction diagrams, and the results were analyzed. The stability of the docked
protein-ligand complex was determined by RMSD (root mean square deviation) and RMSF
(root mean square fluctuation) calculations. The protein-ligand RMSD showed initial
fluctuations up to 6.25 nsec before stabilizing. The docked complex was compared to the
reference protein backbone structure, which was stabilized and found to be within 1-3 Å (Fig.
8). The structural stability of the protein and compound 28 was also evaluated based on the
RMSF value, which was found to be less than 3 Å and confirmed the absence of overall local
changes along the protein chain and positions of the atoms in the ligand (Supp. Fig. S3).
Fig. 8. RMSD graph of the docked compound 28-hAChE complex from 30 nsec MD
simulations.
The results of the MD simulations were analyzed using simulation interaction diagrams. The
representation of the interaction analysis through stacked bar charts (Fig. 9A) showed the
normalized value of the interaction fractions over the course of the trajectory. For example,
the value of 0.6 suggests that a specific interaction was maintained for 60% of the simulation
period. The results suggested that the interaction between compound 28 and the PAS residues
19

remained intact throughout the simulation. The detailed analysis showed that compound 28
interacted through H bonding, hydrophobic interactions and salt-bridge formation with
Tyr72, Asp74, Tyr124, Trp286 and Tyr341 in the PAS with interaction fractions of 0.52,
0.68, 0.69, 0.74 and 0.81, respectively.
Furthermore, interaction analysis was represented graphically (Fig. 9B) to show the H-
bonding and hydrophobic interactions that existed for more than 20% of the total simulation
run time. The results showed that the nitrogen atom of 2-AP exhibited H-bonding interactions
with Asp74 (20%) and Trp286 (22%) at the PAS. Additionally, Asp74 showed H-bonding
interactions (21%) with the –NH linker. Furthermore, the results suggested a hydrophobic
interaction between compound 28 and the Tyr341 residue at the PAS.
The results of the MD simulation analysis were also analyzed as a function of time (Fig. 9C).
The top panel of a timeline representation shows the total number of specific contacts (H-
bonding, hydrophobic, ionic, and water bridge interactions) between the protein and the
ligand over the course of the simulation run. The bottom panel shows the interactions of
individual amino acid residues in each trajectory frame during the simulation run. Some
residues formed more than one specific interaction with the ligand, which was represented by
a darker shade of orange according to the scale at the right of the plot. Overall, the results of
the MD simulation analysis suggested stable and effective interactions between compound 28
and the PAS, and the results were found to be in agreement with those of the PI displacement
assay.
20

Fig. 9. MD simulation analysis of the compound 28-hAChE docked complex. (A) Stacked bar
chart showing interaction fractions; (B) graphical representation showing percentage
interactions with active site residues; (C) timeline representation.
3. CONCLUSION
Herein, we designed and synthesized a novel set of multitarget-directed molecules with both
a 2-AP moiety and a substituted 1,3,4-oxadiazole ring for the treatment of cognitive
dysfunctions. Among all the synthesized derivatives, naphthyl group-bearing compound 28
showed the highest in vitro hAChE inhibitory activity (pIC50 = 6.52) with a noncompetitive
enzyme inhibition (Ki = 0.17 µM). Among the evaluated derivatives, the antioxidant activity
of compound 28 (60.0%) was found to be comparable with that of ascorbic acid (64.6%),
which was used as a standard. Furthermore, compound 28 significantly displaced propidium
iodide (17.6%) from the PAS-hAChE complex in the PI assay and showed significant BBB
penetrability. Compound 28 also inhibited hAChE-induced Aβ aggregation in a
concentration-dependent manner based on the thioflavin T assay. Additionally, the MTT
assay with the SH-SY5Y neuroblastoma cell line suggested that compound 28 is devoid of
21

neurotoxicity. In vivo behavioral studies revealed that compound 28 (10 mg/kg) significantly
reversed the scopolamine-induced cognitive dysfunctions in mice as evaluated by Y-maze
and passive avoidance tests. The ex vivo and biochemical analysis of compound 28 showed
significant brain AChE inhibition and antioxidant activities, respectively. The computational
studies corroborated the pharmacological outcomes owing to the effective interactions
between compound 28 and the active site residues of PAS. Thus, this study indicated that
multitargeted N-(pyrimidin-2-yl)-1,3,4-oxadiazole derivatives are potential scaffolds for the
treatment of dementia with compound 28 as a promising lead for further research.
4. EXPERIMENTAL
4.1. Chemistry
4.1.1 Chemicals and Instrumentation
Chemicals and solvents were purchased from Sigma-Aldrich, TCI chemicals and Avra
Synthesis Pvt Ltd., India and were used without further purification. The progress of each
reaction was monitored by TLC. Melting points were determined using open capillary tubes
on a Stuart melting point apparatus (SMP10) and are uncorrected. FT-IR spectra were
1
recorded on an Alpha ECO-ATR Spectrophotometer (Bruker, USA). H NMR (500 MHz)
and 13C NMR (125 MHz) spectra were recorded (Bruker Avance FT-NMR
spectrophotometer, USA) in deuterated solvent (DMSO-d6) using tetramethylsilane (TMS) as
an internal standard; the chemical shifts (δ, ppm) and coupling constants (J, Hz) are reported.
Elemental analyses (C, H, N) were performed on an EXETER CE-440 elemental analyzer.
4.1.2 Synthesis of phenyl pyrimidin-2-ylcarbamate (2)
2-AP (1; 31.54 mmol) was dissolved in dry DCM in the presence of pyridine (44.16 mmol).
Following the addition of PCF (37.85 mmol) under a nitrogen blanket, the reaction mixture
was stirred for 3 h at 0-5 °C. After completion of the reaction (monitored by TLC), the
reaction mixture was concentrated in vacuo to obtain a solid. This solid was suspended in 60
22

ml of petroleum ether and 50 ml of distilled water. The resulting suspension was filtered and
washed with water (500 ml) and petroleum ether (100 ml) to yield pure compound 2 as a
yellow solid.
Yield: 79%; mp 201-203 ºC; FT-IR (ATR, ν cm-1): 3203 (-NH), 1720 (>C=O); 1H NMR (500
MHz, δH, DMSO-d6): 11.31 (s, 1H), 8.59 (d, 2H, J = 5.0), 7.45 (d, 2H, J = 7.5), 7.33-7.24
13
(m, 3H), 7.15 (t, 1H, J = 4.5 Hz); C NMR (125 MHz, δC, DMSO-d6): 158.7, 158.3, 156.3,
151.2, 149.2, 128.8, 124.5, 122.1, 114.6. Anal. C11H9N3O2: C, 61.39; H, 4.22; N, 19.53;
Found: C, 61.46; H, 4.18; N, 19.50.
4.1.3. Synthesis of N-(pyrimidin-2-yl)hydrazinecarboxamide (3)
Compound (2; 2.32 mmol) and 85% v/v hydrazine hydrate (10.45 mmol) were dissolved in
absolute ethanol and heated at 70 ºC for 30 min. The reaction mixture was cooled and
filtered, and the solid was washed with absolute ethanol and dried to afford 3 as an off-white
solid.
Yield: 81%. mp 235-237 ºC; FT-IR (ATR, ν cm-1): 3260, 3240 (-NH2), 3201 (NHCONH),
1698 (>C=O); 1H NMR (500 MHz, δH, DMSO-d6): 9.87 (s, 1H), 9.65 (s, 1H), 8.58 (d, 2H, J
13
= 5.0), 7.05 (t, 1H, J = 4.5 Hz), 4.24 (s, 2H). C NMR (125 MHz, δC, DMSO-d6): 158.5,
158.4, 156.3, 114.9. Anal. C5H7N5O: C, 39.21; H, 4.61; N, 45.73; Found: C, 39.16; H, 4.64;
N, 45.70.
4.1.4 General procedure for the synthesis of compounds 4-18
Compound 3 (4.24 mmol) and the appropriate aromatic aldehyde (8.24 mmol) were dissolved
in absolute ethanol (30 ml) and refluxed for 3 h. The reaction mixture was allowed to cool to
room temperature and then filtered. The resulting solid was washed with absolute ethanol and
dried in a rotary vacuum evaporator to yield the title compounds (4-18).
23

(E)-2-benzylidene-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (4)
Yield: 63%. mp: 207-209 ºC; FT-IR (ATR, ν cm-1): 3340, 3231 (NHCONH), 1701 (>C=O),
1578 (-C=N); 1H NMR (500 MHz, δH, DMSO- d6): 12.40 (brs, 1H), 10.15 (brs, 1H), 8.64 (d,
2H, J = 4.5 Hz), 8.27 (s, 1H), 7.72 (d, 2H, J = 7.0 Hz) 7.45- 7.40 (m, 3H), 7.13 (t, 1H, J = 7.0
13
Hz); C NMR (125 MHz, δC, DMSO-d6): 158.7, 158.0, 151.4, 134.7, 130.2, 129.2, 127.3,
115.8. Anal. C12H11N5O: C, 59.74; H, 4.60; N, 29.03; Found: C, 59.69; H, 4.63; N, 29.00.
(E)-2-(4-methylbenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (5)
Yield: 61%; mp: 200-202 ºC; FT-IR (ATR, ν cm-1): 3311, 3223 (NHCONH), 1693 (>C=O),
1
1567 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.33 (brs, 1H), 10.13 (brs, 1H), 8.64 (d,
2H, J = 4.5 Hz), 8.24 (brs, 1H), 7.61 (d, 2H, J = 7.5 Hz) 7.26 (d, 2H, J = 8.0 Hz), 7.13 (t, 1H,
13
J = 7.0 Hz), 2.34 (s, 3H); C NMR (125 MHz, δC, DMSO-d6): 157.5, 156.1, 139.3, 139.1,
132.5, 132.3, 129.6, 127.2, 127.1, 114.9, 21.4; Anal. C13H13N5O: C, 61.17; H, 5.13; N, 27.43;
Found: C, 61.25; H, 5.10; N, 27.45.
(E)-2-(4-methoxybenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (6)
Yield: 78%; mp: 188-190 ºC; FT-IR (ATR, ν cm-1): 3323, 3214 (NHCONH), 1709 (>C=O),
1
1574 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.31 (brs, 1H), 10.11 (brs, 1H), 8.63 (d,
2H, J = 4.5 Hz), 8.21 (brs, 1H), 7.66 (d, 2H, J = 8.5 Hz) 7.12 (s, 1H), 7.00- 6.83 (m, 2H),
13
3.73 (s, 3H); C NMR (125 MHz, δC, DMSO-d6): 161.0, 158.7, 158.5, 158.0, 151.4, 130.4,
128.9, 127.3, 115.5, 114.6, 110.6, 55.7; Anal. C13H13N5O2: C, 57.56; H, 4.83; N, 25.82;
Found: C, 57.50; H, 4.81; N, 25.85.
(E)-2-(3,4-dimethoxybenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (7)
Yield: 84%; mp: 216-218 ºC; FT-IR (ATR, ν cm-1): 3298, 3212 (NHCONH), 1691 (>C=O),
1
1580 (-C=N); H NMR (500 MHz, δH, DMSO-d6):12.31 (brs, 1H), 10.10 (brs, 1H), 8.65 (d,
2H, J = 4.5 Hz), 8.22 (brs, 1H), 7.34 (s, 1H) 7.21 (q, 1H, J = 1.5 Hz), 7.14 (t, 1H, J = 5.0 Hz),
13
7.03 (d, 1H, J = 8.5 Hz), 3.82 (s, 6H); C NMR (125 MHz, δC, DMSO-d6): 158.7, 158.1,
24

151.3, 150.9, 149.5, 121.7, 112.0, 56.0; Anal. C14H15N5O3: C, 55.81; H, 5.02; N, 23.24;
Found: C, 55.89; H, 4.99; N, 23.26.
(E)-2-(3,4,5-trimethoxybenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (8)
Yield: 74%; mp: 190-192 ºC; FT-IR (ATR, ν cm-1): 3312, 3239 (NHCONH), 1699 (>C=O),
1584 (-C=N); 1H NMR (500 MHz, δH, DMSO-d6): 12.42 (brs, 1H), 10.18 (brs, 1H), 8.63 (brs,
13
2H), 8.21 (brs, 1H), 7.20-7.04 (m, 3H), 3.82 (s, 6H), 3.69 (s, 3H); C NMR (125 MHz, δC,
DMSO-d6): 158.7, 158.0, 153.5, 151.5, 139.3, 130.2, 115.7, 104.6, 60.5, 56.3; Anal.
C15H17N5O4: C, 54.38; H, 5.17; N, 21.14; Found: C, 54.46; H, 5.20; N, 21.11.
(E)-2-(4-hydroxybenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (9)
Yield: 71%; mp: 225-227 ºC; FT-IR (ATR, ν cm-1): 3420 (-OH), 3322, 3231 (NHCONH),
1697 (>C=O), 1579 (-C=N); 1H NMR (500 MHz, δH, DMSO-d6): 12.23 (brs, 1H), 10.12 (brs,
1H), 9.88 (s, 1H), 8.64 (d, 2H, J = 5.0 Hz), 8.21 (brs, 1H), 7.55 (d, 2H), 7.13 (s, 1H), 6.83 (d,
13
2H); C NMR (125 MHz, δC, DMSO-d6): 159.6, 158.7, 158.1, 151.2, 129.0, 125.8, 116.1,
114.8; Anal. C12H11N5O2: C, 56.03; H, 4.31; N, 27.22; Found: C, 56.11; H, 4.27; N, 27.19.
(E)-2-(2-hydroxybenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (10)
Yield: 79%; mp: 215-217 ºC; FT-IR (ATR, ν cm-1): 3412 (-OH), 3301, 3243 (NHCONH),
1701 (>C=O), 1572 (-C=N); 1H NMR (500 MHz, δH, DMSO-d6): 12.43 (brs, 1H), 11.14 (brs,
1H), 10.29 (brs, 1H), 8.66 (d, 2H, J = 5.0 Hz), 8.54 (s, 1H), 7.29-7.26 (m, 2H), 7.15 (t, 1H, J
= 5.3 Hz), 6.91 (t, 2H, J = 7.8 Hz); 13C NMR (125 MHz, δC, DMSO-d6): 158.7, 158.0 157.2,
151.2, 131.4, 129.1, 119.7, 116.7, 115.7; Anal. C12H11N5O2: C, 56.03; H, 4.31; N, 27.22;
Found: C, 55.94; H, 4.26; N, 27.17.
(E)-2-(naphthalen-1-ylmethylene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (11)
Yield: 69%; mp: 218-220 ºC; FT-IR (ATR, ν cm-1): 3310, 3234 (NHCONH), 1699 (>C=O),
1
1579 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.55 (brs, 1H), 10.21 (brs, 1H), 8.97 (d,
2H, J = 5.0 Hz), 8.68 (d, 2H, J = 5.0 Hz), 8.00 (d, 3H, J = 4.8 Hz), 7.67-7.60 (m, 3H), 7.16 (t,
25

13
1H, J = 4.5 Hz); C NMR (125 MHz, δC, DMSO-d6): 158.7, 158.5, 158.0, 151.5, 133.9,
130.6, 130.1, 129.1, 127.6, 126.7, 124.7, 124.5, 115.7, 110.7; Anal. C16H13N5O: C, 65.97; H,
4.50; N, 24.04; Found: C, 65.91; H, 4.47; N, 24.01.
(E)-2-(4-fluorobenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (12)
Yield: 65%; mp: 205-207 ºC; FT-IR (ATR, ν cm-1): 3310, 3236 (NHCONH), 1705 (>C=O),
1
1583 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.40 (brs, 1H), 10.17 (brs, 1H), 8.67-
8.63 (m, 2H), 8.27 (s, 1H), 7.79-7.76 (m, 2H), 7.25 (t, 2H, J = 4.5 Hz), 7.13 (t, 1H, J = 4.0);
13C NMR (125 MHz, δC, DMSO-d6): 164.3, 162.4, 158.7, 158.5, 158.9, 151.5, 131.2, 129.5,
116.1, 110.7; Anal. C12H10FN5O: C, 55.60; H, 3.89; N, 27.02; Found: C, 55.53; H, 3.92; N,
26.99.
(E)-2-(4-chlorobenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (13)
Yield: 72%; mp: 220-222 ºC; FT-IR (ATR, ν cm-1): 3311, 3232 (NHCONH), 1708 (>C=O),
1
1579 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.42 (brs, 1H), 10.18 (brs, 1H), 8.63 (d,
2H, J = 4.5 Hz), 8.26 (s, 1H), 7.74 (d, 2H, J = 7.5 Hz), 7.49 (d, 2H, J = 8.0 Hz), 7.13 (s, 1H);
13C NMR (125 MHz, δC, DMSO-d6): 158.7, 157.9, 151.5, 134.6, 133.6, 130.5, 129.3, 129.0,
115.8; Anal. C12H10ClN5O: C, 52.28; H, 3.66; N, 25.40; Found: C, 52.32; H, 3.63; N, 25.37.
(E)-2-(4-bromobenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (14)
Yield: 71%; mp: 215-217 ºC; FT-IR (ATR, ν cm-1): 3301, 3239 (NHCONH), 1690 (>C=O),
1
1584 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.43 (brs, 1H), 10.19 (brs, 1H), 8.63 (d,
13
2H, J = 4.5 Hz), 8.21 (s, 1H), 8.25-8.20 (m, 4H), 7.14 (t, 1H, J = 7.5 Hz); C NMR (125
MHz, δC, DMSO-d6): 158.7, 157.9, 151.4, 134.0, 132.2, 129.2, 123.4, 114.1; Anal.
C12H10BrN5O: C, 45.02; H, 3.15; N, 21.88 Found: C, 44.96; H, 3.11; N, 21.92.
(E)-2-(3-bromobenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (15)
Yield: 74%; mp: 210-212 ºC; FT-IR (ATR, ν cm-1): 3311, 3201 (NHCONH), 1698 (>C=O),
1
1569 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.46 (brs, 1H), 10.19 (brs, 1H), 8.64 (d,
26

13
2H, J = 4.5 Hz), 8.21 (s, 1H), 7.94 (s, 1H), 7.70-6.52 (m, 4H); C NMR (125 MHz, δC,
DMSO-d6): 158.7, 158.0, 151.5, 132.7, 131.4, 129.3, 126.5, 122.5, 115.1; Anal.
C12H10BrN5O: C, 45.02; H, 3.15; N, 21.88 Found: C, 44.95; H, 3.17; N, 21.85.
(E)-2-(2-chlorobenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (16)
Yield: 82%; mp: 200-202 ºC; FT-IR (ATR, ν cm-1): 3302, 3239 (NHCONH), 1708 (>C=O),
1
1576 (-C=N); H NMR (500 MHz, δH, DMSO-d6):12.29 (brs, 1H), 10.02 (brs, 1H), 8.67 (d,
13
2H, J = 4.5 Hz), 8.55 (s, 1H), 8.06 (s, 1H), 7.52-7.41 (m, 3H), 7.16 (t, 1H, J = 8.0 Hz); C
NMR (125 MHz, δC, DMSO-d6): 158.8, 158.0, 151.2, 133.2, 132.0, 131.5, 130.3, 127.9,
127.5, 116.1; Anal. C12H10ClN5O: C, 52.28; H, 3.66; N, 25.40; Found: C, 52.36; H, 3.69; N,
25.37.
(E)-2-(4-nitrobenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (17)
Yield: 77%; mp: 279-281 ºC; FT-IR (ATR, ν cm-1): 3312, 3231 (NHCONH), 1713 (>C=O),
1581 (-C=N); 1H NMR (500 MHz, δH, DMSO-d6): 12.42 (brs, 1H), 10.11 (brs, 1H), 8.67 (m,
1H), 8.38-8.29 (m, 4H), 8.16 (d, 1H), 7.99 (d, 1H), 7.16 (d, 1H, J = 8.0 Hz); 13C NMR (125
MHz, δC, DMSO-d6): 158.7, 158.0, 151.3, 148.0, 130.0, 128.2, 124.6, 124.5, 114.1; Anal.
C12H10N6O3: C, 50.35; H, 3.52; N, 29.36; Found: C, 50.27; H, 3.55; N, 29.34.
(E)-2-(2-nitrobenzylidene)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (18)
Yield: 70%; mp: 235-237 ºC; FT-IR (ATR, ν cm-1): 3315, 3227 (NHCONH), 1702 (>C=O),
1
1578 (-C=N); H NMR (500 MHz, δH, DMSO-d6): 12.39 (brs, 1H), 10.09 (brs, 1H), 8.65 (d,
13
2H), 8.55 (s, 1H), 8.21-8.14 (m, 2H), 8.05 -7.64 (m, 3H); C NMR (125 MHz, δC, DMSO-
d6): 158.8, 158.5, 157.9, 151.4, 148.4, 134.1, 130.9, 128.9, 126.9, 124.6, 110.7; Anal.
C12H10N6O3: C, 50.35; H, 3.52; N, 29.36; Found: C, 50.31; H, 3.49; N, 29.35.
4.1.5 General procedure for the synthesis of compounds 19-20
Compounds 7 and 18 (1.87 mmol) were used for the synthesis of compounds 19 and 20,
respectively. Compound 7 or 18 was dissolved in dry methanol, sodium borohydride
27

(NaBH4) was added, and the reaction mixture was stirred for 1 h at 0-5 ºC. The solvent was
evaporated in vacuo to yield a crude product that was recrystallized from absolute ethanol to
afford the desired compound (19 or 20).
2-(3,4-dimethoxybenzyl)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (19)
Yield: 65%; mp 211-213 ºC; FT-IR (ATR, ν cm-1): 3379 (-NH), 3215 (NHCONH), 1699
(>C=O); 1H NMR (500 MHz, δH, DMSO-d6): 12.35 (brs, 1H), 10.12 (brs, 1H), 8.61-8.19 (m,
13
2H), 7.31-7.23 (m, 2H), 7.17-7.09 (m, 2H), 3.82 (s, 6H), 2.03 (s, 2H), 1.97 (brs, 1H); C
NMR (125 MHz, δC, DMSO-d6): 158.8, 158.4, 150.5, 149.4, 121.2, 112.6, 56.7, 46.71; Anal.
C14H17N5O3: C, 55.44; H, 5.65; N, 23.09; Found: C, 55.39; H, 5.62; N, 23.11.
2-(2-nitrobenzyl)-N-(pyrimidin-2-yl)hydrazine-1-carboxamide (20)
Yield: 63%; mp 239-241 ºC; FT-IR (ATR, ν cm-1): 3362 (-NH), 3219 (NHCONH), 1710
(>C=O); 1H NMR (500 MHz, δH, DMSO-d6): 12.32 (brs, 1H), 10.11 (brs, 1H), 8.65-8.55 (m,
2H), 8.19-8.11 (m, 2H), 8.02-7.81 (m, 1H), 7.62-7.58 (m, 2H), 2.12 (s, 2H), 1.94 (brs, 1H);
13C NMR (125 MHz, δC, DMSO-d6): 158.8, 158.5, 157.9, 148.4, 134.1, 130.9, 128.9, 126.9,
124.6, 110.7, 45.39; Anal. C12H12N6O3: C, 50.00; H, 4.20; N, 29.15; Found: C, 49.94; H,
4.23; N, 29.11.
4.1.6 General procedure for the synthesis of compounds 21-24 and 26-35
The appropriate substrate (4-7 and 9-18, 1.15-2.0 mmol) and CAT (2.0-3.0 mmol) were
refluxed for 2 h in absolute ethanol. The solution was concentrated to yield an oily residue,
which was mixed with ethyl acetate. The ethyl acetate layer was washed with water (2×100
ml) and passed through sodium sulfate (Na2SO4). The filtrate was concentrated in vacuo to
obtain a crude product that was recrystallized from absolute ethanol to afford the title
compounds (21-24 and 26-35).
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4.1.7 General procedure for the synthesis of compound 25
Compound 8 (0.60 mmol) and NBS (0.90 mmol) were dissolved in dry DCM (20 ml), and
then DBU (0.90 mmol) was added. The reaction mixture was stirred at room temperature for
3-4 h. After completion, the reaction mixture was washed with 1 N HCl, 5% w/v sodium
bicarbonate (NaHCO3) and brine. The DCM layer was concentrated in vacuo to give a crude
product, which was recrystallized from methanol to afford 25 as a yellow solid.
5-phenyl-N-(pyrimidin-2-yl)-1,3,4-oxadiazol-2-amine (21)
Yield: 61%; mp: 260-262 ºC; FT-IR (ATR, ν cm-1): 3240 (-NH), 1567 (-C=N); 1H NMR (500
MHz, δH, DMSO-d6): 8.62 (d, 1H, J = 5.0 Hz), 8.47 (d, 2H, J = 10.0 Hz), 7.94-7.87 (m, 2H)
13
7.53-7.35 (m, 3H), 7.07 (brs, 1H); C NMR (125 MHz, δC, DMSO-d6): 159.0, 158.0, 156.6,
131.7, 130.9, 129.8, 126.2, 125.7, 115.3. Anal. C12H9N5O: C, 60.25; H, 3.79; N, 29.27;
Found: C, 60.29; H, 3.81; N, 29.24.
5-(p-tolyl)-N-(pyrimidin-2-yl)-1,3,4-oxadiazol-2-amine (22)
Yield: 67%; mp: 265-267 ºC; FT-IR (ATR, ν cm-1): 3264 (-NH), 1556 (-C=N); 1H NMR (500
MHz, δH, DMSO-d6): 11.45 (brs, 1H), 8.61 (d, 2H, J = 5.0 Hz), 7.82-7.76 (m, 2H) 7.40-7.35
13
(m, 2H), 7.02 (brs, 1H), 2.38 (s, 3H, -CH3); C NMR (125 MHz, δC, DMSO-d6): 159.0,
158.0, 156.6, 131.7, 130.9, 129.8, 126.2, 125.7, 115.3. Anal. C13H11N5O: C, 61.65; H, 4.38;
N, 27.65; Found: C, 61.57; H, 4.41; N, 27.63.
5-(4-methoxyphenyl)-N-(pyrimidin-2-yl)-1,3,4-oxadiazol-2-amine (23)
Yield: 59%; mp: 247-249 ºC; FT-IR (ATR, ν cm-1): 3278 (-NH), 1563 (-C=N); 1H NMR (500
MHz, δH, DMSO-d6): 11.25 (brs, 1H), 8.62 (d, 2H, J = 4.5 Hz), 7.88 (d, 2H, J = 9.0 Hz), 7.14
13
(d, 2H, J = 9.0 Hz), 7.10 (t, 1H, J = 5.0 Hz), 3.84 (s, 3H); C NMR (125 MHz, δC, DMSO-
d6): 162.09, 161.03, 159.1, 158.8, 128.1, 116.5, 115.6, 115.3, 55.9; Anal. C13H11N5O2: C,
57.99; H, 4.12; N, 26.01; Found: C, 58.04; H, 4.09; N, 25.98.
29