Novel N-benzylpiperidine derivatives of 5-arylisoxazole-3-carboxamides as anti-Alzheimer's agents

Article abstract of DOI:10.1002/ardp.202000258

The complex pathophysiology of Alzheimer's disease (AD) has prompted researchers to develop multitarget-directed molecules to find an effective therapy against the disease. In this context, a novel series of N-(1-benzylpiperidin-4-yl)-5-arylisoxazole-3-ca

Full text of DOI:10.1002/ardp.202000258

Received: 21 July 2020
Revised: 5 October 2020
Accepted: 31 October 2020
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DOI: 10.1002/ardp.202000258
F U L L P A P E R
Novel N‐benzylpiperidine derivatives of
5‐arylisoxazole‐3‐carboxamides as anti‐Alzheimer's agents
Mina Saeedi1,2 | Peyman Felegari3 | Aida Iraji4
| Roshanak Hariri3
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Arezoo Rastegari2 | S. Sara Mirfazli5 | Najmeh Edraki4 | Omidreza Firuzi4
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Mohammad Mahdavi6 | Tahmineh Akbarzadeh2,3
1Medicinal Plants Research Center, Faculty of
Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
Abstract
The complex pathophysiology of Alzheimer's disease (AD) has prompted re-
searchers to develop multitarget‐directed molecules to find an effective therapy
against the disease. In this context, a novel series of N‐(1‐benzylpiperidin‐4‐yl)‐5‐
arylisoxazole‐3‐carboxamide derivatives were designed, synthesized, and evaluated
against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In vitro
2Persian Medicine and Pharmacy Research
Center, Tehran University of Medical
Sciences, Tehran, Iran
3Department of Medicinal Chemistry, Faculty
of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
4Medicinal and Natural Products Chemistry
Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
biological evaluation demonstrated that compound 4e was the best AChE (IC50
=
16.07 μM) and BuChE inhibitor (IC50 = 15.16 μM). A kinetic study of 4e was also
conducted, which presented a mixed‐type inhibition for both enzymes. Molecular
docking studies revealed that compound 4e fitted well into the active sites of AChE
and BuChE, forming stable and strong interactions with key residues Glu199, Trp84,
Asp72, Tyr121, and Phe288 in AChE and His438, Trp82, Ala328, Tyr332, Phe329,
Thr120, and Pro285 in BuChE. Besides, the inhibition of BACE1 by 4e and the
biometal chelation activity of 4e were measured. The neuroprotective assessment
revealed that 4e exhibited 23.2% protection at 50 µM toward amyloid‐beta‐induced
PC12 neuronal cells. Overall, this study exhibited that compound 4e was a pro-
mising compound targeting multiple factors associated with AD.
5Department of Medicinal Chemistry, School
of Pharmacy‐International Campus, Iran
University of Medical Sciences, Tehran, Iran
6Endocrinology and Metabolism Research
Center, Endocrinology and Metabolism
Clinical Sciences Institute, Tehran University
of Medical Sciences, Tehran, Iran
Correspondence
Tahmineh Akbarzadeh, Department of
Medicinal Chemistry, Faculty of Pharmacy,
Tehran University of Medical Sciences,
Tehran, Iran.
Email: akbarzad@tums.ac.ir
K E Y W O R D S
Alzheimer's disease, BACE1, cholinesterase, isoxazoles, multitarget compound,
neuroprotection
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1
INTRODUCTION
plaques as extracellular deposits and neurofibrillary tangles as
intracellular ones. The proteolytic cleavage of amyloid precursor
Alzheimer's disease (AD) is a progressive neurodegenerative disease
and one of the leading causes of morbidity and mortality in elderly
people with a continuous deterioration of cognition, memory, apha-
sia, behavior, and personality.[1] AD is a complex multifactorial dis-
ease with many contributing factors. The physiopathology of the
disease is characterized by the formation of amyloid‐beta (Aβ) senile
protein (APP) by β‐site APP‐cleaving enzyme 1 (BACE1) and the
γ‐secretase complex lead to the formation of Aβ plaques. The ex-
tracellular deposition of Aβ triggers a cascade of pathological events
including inflammation, mitochondrial dysfunctions, oxidative stress,
and finally cell death. There is growing evidence that BACE1 in-
hibitors could lessen APP cleavage and effectively reduce Aβ with
low synaptotoxicity.[2]
On the basis of different paths to the pathogenesis of AD, dys-
function and lack of neurotransmissions, especially acetylcholine
This paper is dedicated to the memory of our unique Chemistry and Medicinal Chemistry
teacher, Professor Abbas Shafiee (1937−2016).
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Arch Pharm. 2020;e2000258.
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SAEEDI ET AL.
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(ACh), deteriorate brain networks, which in turn results in loss of
synapses. Low amounts of ACh are mainly attributable to the en-
hanced degradation rate of ACh by two known cholinesterase (ChE)
enzymes known as acetylcholinesterase (AChE) and butyr-
ylcholinesterase (BuChE).[3] Considering the high expression of AChE
at the early stage of AD, acetylcholinesterase inhibitors (AChEIs) are
the first line of therapy in the primary‐to‐moderate stage of AD such
as rivastigmine, donepezil, and galantamine. However, due to the
increasing amount of BuChE at the late stage of AD, butyr-
ylcholinesterase inhibitors (BuChEIs) could be an ideal candidate for
moderate‐to‐severe AD.[4]
There is no fully effective cure for AD yet. Recent failures of
several AD clinical trials confirmed the complicated and multi-
factorial nature of AD pathogenesis, which leads to irreversible
neurodegeneration and neural network damage.[11]
In this regard, comprehensive pieces of evidence support the
role of multitarget direct ligands (MTDLs) as possible therapeutic
agents. In this case, simultaneous inhibition of AChE, BuChE,
and
BACE1
by
N‐(1‐benzylpiperidin‐4‐yl)‐5‐arylisoxazole‐3‐
carboxamides, combined with neuroprotective and biometal chela-
tion activities, were considered.
Inflammation is another basic mechanism of AD pathophysiology
in addition to Aβ.[5] Prolonged inflammation becomes chronic in-
flammation, which can cause detrimental effects on brain functions
due to excessive or persistent release of toxic factors.[6] It has been
demonstrated that excessive production of Aβ activates chemokine
and cytokines, releases the proinflammatory agents, and promotes
inflammation further.[7]
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2
RESULTS AND DISCUSSION
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2.1
Structure‐based design of
benzylpiperidine‐arylisoxazole‐carboxamides
Donepezil (A; Figure 1) is known as
a Food and Drug
Also, there is a large amount of evidence confirming the in-
volvement of biometals in AD pathogenesis. Metal ion dyshomeos-
tasis and metal‐induced Aβ aggregation are considered as other
biochemical factors related to AD.[8] In this regard, metal interac-
tions with Aβ and tau influence their aggregation properties and
neurotoxicity. Also, on the basis of Fenton reactions, the unregulated
interactions of metal with molecular oxygen facilitate the generation
of reactive species (reactive oxygen species and reactive nitrogen
species), which results in increased levels of lipid peroxidation
and oxidative damage to DNA and proteins.[9,10]
Administration‐approved AChEI in which the benzylpiperidine
tail interacts with the PAS residues of the AChE pocket and the
2,3‐dihydroindenone moiety occupies the CAS pocket. Many of
ChEIs typically mimic the structural feature of the benzylpiper-
idine tail. This is evidenced by the result of compound B, which
exhibited potent AChE inhibition with an IC50 value of 21.85 µM
and BuChE inhibition with an IC50 value of 76.78 µM. This com-
pound also demonstrated 22% BACE1 inhibition at 50 μM.[12] We
also recently reported ChEI (compound C; Figure 1) with an
arylisoxazole structure containing
a
benzyl‐pyridine tail.
G:
H:
Neuroprotection
FIGURE 1 The design of novel N‐benzylpiperidine derivatives of 5‐arylisoxazole‐3‐carboxamides

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This compound depicted a selective BuChEI activity (IC50 = 0.32
μM) with moderate AChE inhibition.[13]
derivatization of target compounds mainly focused on the substitution of
the arylisoxazole‐3‐carboxamides at various positions of the aryl ring.
Most of the potent BACE1 inhibitors are peptidic and pseudo-
peptidic molecules with high similarity with the structure of APP as a
substrate. One of the most efficient ones is OM99‐2 with an IC50 value
of 14.7 nM.[14] However, due to poor physicochemical and pharma-
ceutical properties, including insufficient oral bioavailability, short ser-
um half‐life, and low blood–brain barrier penetration, there is an
ongoing interest to design small‐molecule BACE1 inhibitors.[3] For the
past few years, there have been many synthetic BACE1 inhibitors;
among such compounds, there are limited examples of the isoxazole‐3‐
carboxamides molecular skeleton as the core structure. Compound D
(Figure 1) was developed according to the property and structure‐based
approach via incorporation of the dimethylisoxazole substitution into
the aminothiazine carboxamide series as potent and selective BACE1
inhibitors.[15] From literature reports, it is also stated that the amide‐
arylisoxazole core is involved in BACE1 inhibition due to its key
structural feature of heterocyclic compounds and the capability of
forming hydrogen bonds with the BACE1 active site.[3] In this respect,
aryl isoxazol‐corporating phenylpiperazine (compound E) showed
moderate BACE1 inhibitory activity (IC50 = 76.78 µM). Compound E
also showed selectivity toward AChE, compared with BuChE.[16] We
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2.2
Chemistry
The synthesis of desired compounds 4 was achieved according to
Scheme 1. The desired carboxylic acid derivatives 2 were prepared
through a three‐step reaction, as reported in our previous work.[17]
Then, the reaction of compounds 2 and 3 in the presence of hydro-
xybenzotriazole (HOBT) and 1‐ethyl‐3‐(3‐dimethylaminopropyl)
carbodiimide (EDCI) in dry acetonitrile at room temperature af-
forded the target compound 4. The structure of all compounds was
confirmed using nuclear magnetic resonance (NMR) and infrared (IR)
spectroscopy, as well as elemental analysis. It should be noted that in
the case of compounds 4a, 4c, 4g, 4h, and 4j, the 13C NMR spectra
showed the presence of two isomers probably due to restricted C–N
amide bond rotation. It is clear that the formation of rotamers is
dependent on the electronic effects and steric hindrance of sub-
stituents on the aryl ring, as they can affect the C−N rotational
barrier and planarity of the amide carbonyl group. It seems that the
presence of the electron‐withdrawing group (NO2) led to a lower
C−N rotational barrier. In the case of electron‐donating groups and
halogens, their steric effect and size as well as their positions on the
aryl ring are very important. When the aromatic ring was un-
substituted (compound 4a) or occupied by the small‐size halogen (F)
at ortho or para positions (compounds 4g and 4h), the formation of
rotamers was confirmed by 13C NMR, which can also be observed in
the case of a medium‐size halogen (Cl) at the para position (com-
pound 4j). However, a change of its position to ortho (compound 4i)
or increasing the number of Cl (compound 4k) deplanarized the
carbonyl group and stopped rotamerization. This can also be ob-
served in compound 4l with a large‐size halogen (Br). Our results
related to the electron‐donating substituted derivatives (compounds
4b‐4d) revealed that the presence of those groups only at the meta
position led to a higher C−N rotational barrier.
previously reported the discovery of
a series of arylisoxazole‐
chromenone carboxamide derivatives (compound F) with general
structure F with good BACE1 inhibitory potency.[17] Besides, the in
vitro AChE inhibitory activity was highly dependent on the presence
and position of nitro substituents. The greatest activity occurred when
the nitro group was located at the 3‐position, whereas 4‐ and 5‐
methoxy derivatives were inactive. Also, F depicted 84.9% neuropro-
tectivity at the concentration of 50 μM (Figure 1). Meanwhile, several
isoxazole‐containing motifs (F, G, H) were also reported to display
neuroprotective, anti‐inflammatory, and antioxidant activities.[18,19]
Consequently, a pharmacophoric hybridization strategy was adopted
to design and synthesize new small MTDLs possessing arylisoxazole‐
carboxamide as a core skeleton. To improve ChE inhibitory potency, the
benzylpiperidine moiety with ensured anti‐ChE properties was in-
corporated into the isoxazole‐3‐carboxamides scaffold. On the basis of
the demonstrated structure, it can be understood that the presence of
the amide moiety played an important role in inducing appropriate ChE
and BACE1 inhibitory activity. Besides, the mentioned scaffold seems to
enhance anti‐AD biological properties featured by BACE1 inhibitory,
anti‐inflammatory, metal chelation endeavors. The presence of a het-
erocyclic structure with the carboxamide linker may also amplify the
metal chelation potential of the designed scaffold. The structural
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2.3
Biology
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2.3.1
In vitro ChE inhibitory activities
All synthesized derivatives of compound 4 were evaluated for their
biological activity against AChE and BuChE, whereas donepezil was
SCHEME 1 The synthesis of compound 4. Reagents and conditions: (a) Diethyl oxalate, NaOEt/dry EtOH, 0°C‐rt, 15 h; (b) NH2OH·HCl,
EtOH, reflux, 3 h; (c) KOH, MeOH, reflux, 3 h; (d) HOBT, EDCI, dry CH3CN, rt, 24–72 h

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TABLE 1 Cholinesterase inhibitory
activity of N‐benzylpiperidine derivatives
of 5‐arylisoxazole‐3‐carboxamides 4a−la
Entry
Compound 4
4a
X
AChEI [IC50 (μM)] BuChEI [IC50 (μM)] Selectivityb
1
H
>100
>100
>100
53.29 1.65
>100
>0.5
–
2
4b
4c
4d
4e
4f
4‐Me
3
3‐OMe
47.46 1.87
>100
>0.5
>2.2
>0.9
>4.2
>0.6
–
4
3,4‐(OMe)2 45.01 0.92
5
3‐NO2
4‐NO2
2‐F
16.07 0.07
23.63 0.38
47.22 1.16
>100
15.16 0.22
>100
6
7
4g
4h
4i
26.46 0.39
>100
8
4‐F
9
2‐Cl
>100
50.88 0.21
>100
>0.5
–
10
4j
4‐Cl
>100
11
4k
4l
2,4‐Cl2
4‐Br
39.83 0.38
>100
>100
>2.5
–
12
>100
Donepezil
0.28 0.002
8.06 0.38
Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholinesterase.
aData are represented in terms of mean SD.
bSelectivity for AChE = IC50 (BuChE)/IC50 (AChE).
used as a positive control. The results are summarized in Table 1, and
the SAR of these arylisoxazole derivatives was investigated. In the
series hybrids, compound 4e bearing meta‐nitroaryl was the most
potent ChEI, whereas the highest selective inhibitor of BuChE was
compound 4c with an IC50 value of 47.46 μM. Furthermore, 4f (X =
para‐nitro) was regarded as the most selective AChEI.
properties. Our results are in accordance with previously pub-
lished data confirming the promising role of 3‐nitroarylisoxazole
in the AChE inhibitory activity.[17]
The results of anti‐BuChE inhibitory activity indicated the
following points:
In the case of AChE, the following conclusions can be drawn:
• The absence of substituents on the phenylisoxazole pendant
• The unsubstituted compound 4a displayed no AChE inhibitory
property in the whole range of concentrations studied.
(compound 4a) afforded
(IC50 = 53.29 μM).
a
moderate inhibitory activity
• Compounds 4b and 4c bearing the electron‐donating group, in-
cluding methyl and methoxy, on the arylisoxazole ring were in-
active; however, compound 4d containing 3,4‐diOMe showed a
moderate inhibitory activity with an IC50 value of 45.01 μM.
• The presence of halogen atoms at the para position of arylisox-
azole moiety led to a lack of activity. However, in the case of
fluorine, switching X from para (4h) to ortho (4g) led to the active
compound 4g (IC50 = 47.22 μM). For the series of halogen‐
substituted derivatives, the highest activities were observed when
the substitution occurred at the multiposition of the arylisoxazole.
This is obvious in compound 4k bearing 2,4‐diCl at X position with
an IC50 value of 39.83 μM.
• A comparison of the potency of the six halogenated
arylisoxazole‐benzylpiperidine hybrids (4h−l) revealed that
the activity was affected by their positions. More specifically,
the para‐substituted derivatives (4h: X = 4‐F, 4j: X = 4‐Cl, and
4l: X = 4‐Br) did not afford any BuChE inhibitory activity. Yet,
changing the position of the halogen atom from para to ortho
position of the aryl ring (4g vs. 4h and 4i vs. 4h) provided a
better BuChE inhibitory activity. This is obvious from the IC50
values for compounds 4g and 4i with 26.46 and 50.88 μM,
respectively.
• From the screening data, the best BuChE inhibitory potency
belongs to 4e (X = 3‐NO2) with an IC50 value of 15.16 while
changing meta to para (compound 4f, X = 4‐NO2, IC50 > 100 μM)
deleted BuChE inhibitory activity.
• Furthermore, compounds 4e (X = 3‐NO2, IC50 = 16.07 μM) and 4f
(X = 4‐NO2, IC50 = 23.63 μM) exhibited the best AChE inhibitory

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(a)
(b)
FIGURE 4 The absorbance change of compound 4e alone and in
the presence of Zn2+, Fe2+, and Cu2+ ions at a wavelength range of
200−600 nm
FIGURE 2 A kinetic study of inhibitor 4e against
acetylcholinesterase. The Lineweaver–Burk plot (a) and
double‐reciprocal Lineweaver–Burk plot (b) are shown
Compound 4e + Fe ions
(a)
FIGURE 5 Absorption at 258.7 nm (λmax), depending on the mole
fraction of Cu2+ toward compound 4e
moiety removed BuChE inhibitory activity (4d; X = 3,4‐diOMe,
IC50 > 100 μM).
(b)
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2.3.2
Kinetic studies
Kinetic studies were conducted to examine the mechanism of in-
hibition by compound 4e toward AChE and BuChE, respectively. A
graphical analysis of the reciprocal Lineweaver–Burk plot related to
compound 4e described a mixed‐type inhibition pattern against both
AChE and BuChE (Figures 2 and 3), indicating that 4e can bind to
both enzymes, even if it is already bound to the substrate. In addi-
tion, the Ki values were calculated using the secondary plot as 10.0
and 6.0 µM for AChE and BuChE inhibition (Figures 2b and 3b).
FIGURE 3 A kinetic study of inhibitor 4e against
butyrylcholinesterase. The Lineweaver–Burk plot (a) and
double‐reciprocal Lineweaver–Burk plot (b) are shown
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BACE1 enzymatic assay
• A comparison of inhibitory capabilities of 4a−l showed that
compound 4c bearing 3‐OMe with an IC50 value of 47.46 was
the most selective BuChEI. The results indicated that the in-
troduction of an extra methoxy group on the arylisoxazole
2.3.3
To further analyze the effect of anti‐AD activities, compound 4e with
potent inhibitory activity against ChE was selected for the BACE1

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FIGURE 6 Docking of compound 4e into the binding pocket of the 1EVE
inhibition assay. In this assay, OM99‐2 (Glu‐Val‐Asn‐Leu‐Ala‐Ala‐
Glu‐Phe) was used as a reference compound with an IC50 value of
14.7 2.8 nM. It was found that compound 4e showed 10.9% and
24.3% inhibition toward BACE1 at the concentrations of 10 and
50 µM, respectively.
change of intensity after interaction of compound 4e and the cor-
responding metal ions indicated the formation of 4e−metal complex.
However, the results depicted that the chelation between compound
4e and Cu2+ ions was not as optimally effective as that of Zn2+ and
Fe2+ ions.
The stoichiometry of complex 4e−Fe2+ was also studied
(Figure 5). The concentration of the test compound 4e was 20 μM
and the final concentration of Fe2+ ranged from 0 to 40 μM, with
4‐μM intervals at 258.7 nm. The plot was obtained by the corre-
sponding absorption against the mole fraction of Fe2+ toward ligand
(compound 4e). According to the plot, the complexation ratio of 1:1
of 4e−Fe2+ can be seen at the fracture point of the plot with the mole
fraction of 0.8.
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2.3.4
Neuroprotection effect against Aβ‐induced
damage
Neurodegeneration and neuroinflammation have been known as two
of the main complications of AD.[20] Hence, it was necessary to as-
sess the neuroprotective properties of the potent candidate. As a
result, compound 4e was evaluated on PC12 neuronal cell‐induced
Aβ
damage
using
the
3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐
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Docking study
diphenyltetrazolium bromide (MTT) viability assay, and the men-
tioned compound depicted 23.2% protection at 50 µM. Caffeic acid
as a positive control protected the PC12 cells with an IC50 value of
75.8 11.3 µM.
2.4
AutoDock software was used for the protein−ligand interaction
study. The molecular modeling simulation of compound 4e was car-
ried out in 1EVE, 1P0P, and 2QP8. The range of minimized affinity
values of the poses of the docked ligand into the AChE enzyme
(1EVE) is −12.1 to −10.5 kcal/mol. The interactions of the best‐
docked confirmation of new ligand with the active site residues of
1EVE are depicted in Figure 6.
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2.3.5
Metal chelating activity
Compound 4e was tested for its metal chelating ability toward Fe2+
,
Cu2+, and Zn2+ ions (Figure 4). The ultraviolet spectrum of metha-
nolic solution (final concentration of 20 µM) of that compound
showed a characteristic absorption peak at 258.7 nm. After the in-
teraction of compound 4e with metal ions for 30 min, as shown in
Figure 4, no significant red and blue shifts were observed, and the
The analysis of binding interactions illustrated that the nitro
substituent of the active molecule displayed substantial binding
within the active site through conventional hydrogen bond and π
stacking interactions via Glu199 and Trp84, whereas at the opposite
site, the carbonyl group and NH of amide linker formed H‐bonding

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FIGURE 7 Docking of compound 4e into the binding pocket of the 1P0P
with Asp72. Isoxazole and aryl ring enhanced energy minimization in
the active site by forming π−donor hydrogen bonds with Tyr121 and
Phe288, respectively.
The minimum affinity values for the best‐docked confirmation
of 4e in the BACE1 (2QP8) active site ranged from −10.5 to
−8.7 kcal/mol, which is related to the three conventional hydrogen
bonds with Asp289, Thr133, and Asn98, and two carbon−hydro-
gen bonds with Asp93 and Tyr132 from the surrounding residues
depicted in Figure 8. It was revealed that the specific binding
characteristics in the BACE1 active site are associated with het-
eroatoms in amide linker, isoxazole ring, and nitroaryl moiety via
hydrogen bonds.
The ligand was subjected to docking with the BuChE (1P0P), as
demonstrated in Figure 7. Affinity values of the poses of the docked
ligand ranged from −10.8 to −10.0 kcal/mol as a minimum. Similar to
1EVE active site interactions, nitroaryl moiety was fixed through
H‐bond and π stacking interactions via His438 and Trp82. The
terminal aryl ring was captured by Ala328, Tyr332, and Phe329 via π
−π interactions, π−alkyl interactions, and carbon−hydrogen bonds.
For this derivative in 1P0P, the carbon−hydrogen bond interactions
were also observed between the aliphatic part of the compound and
residues Thr120 and Pro285.
Taken together, the enzymes' inhibition results and molecular
interaction studies clearly suggested that the nitro group on the aryl
ring played a crucial role to attach strongly into the active sites of
1EVE and 2QP8, which is responsible for influencing the inhibitory
FIGURE 8 Docking of compound 4e into the binding pocket of the 2QP8

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FIGURE 9 The representation of donepezil structure (in blue) and 4e (in gray) in the 1EVE active site
activity, providing valuable information for the design of efficient
inhibitors.
metal chelation potential of 4e was also investigated and the com-
plexation ratio of 1:1 for 4e−Fe2+ was observed. The obtained results
proposed that the arylisoxazole‐benzylpiperidine‐based compounds
could be considered in the field of MTDLs against AD.
The presence of a hydrophilic group like the aryl ring was ac-
countable for the improvement of activity, particularly in the BuChE
active site.[21] In addition, the nitrogen of amide linker and oxygen of
nitro group are the biggest contributors in compound 4e, which are
involved in the binding of the ligand through the H‐bond in 1EVE
and 2QP8.
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4
EXPERIMENTAL
Chemistry
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1EVE binding site interactions for both reference ligand and
compound 4e are concurrently figured out in Figure 9. Donepezil
(blue ligand) well occupied the entire length of the AChE aromatic
gorge and formed H‐bond and aromatic stacking interactions. There
are several key binding interactions for donepezil and compound 4e
in the active site via Tyr70, Trp84, Asp72, Glu199, and Phe288,
which are identified by the binding models according to the inter-
actions with the AChE active site.
4.1
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4.1.1
General
Melting points were taken on a Kofler hot‐stage apparatus and were
uncorrected. The 1H and 13C NMR spectra were recorded on Bruker
FT‐500, using TMS as an internal standard. The IR spectra were
obtained on a Nicolet Magna FTIR 550 spectrophotometer (in KBr).
Mass spectra were determined on an Agilent Technology (HP) mass
spectrometer operating at an ionization potential of 70 eV. The
elemental analysis was performed with an Elementar Analysensys-
tem GmbH VarioEL CHNS mode. All reagents and solvents were
obtained from Merck and Aldrich and used without purification.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting
Information.
|
3
CONCLUSION
In this study,
a novel series of N‐(1‐benzylpiperidin‐4‐yl)‐5‐
arylisoxazole‐3‐carboxamides analogs were designed and synthe-
sized as MTDLs. The in vitro results revealed that compound 4e
bearing a meta‐nitro group connected to isoxazole moiety was the
best AChEI (IC50 = 16.07 μM) and BuChEI (IC50 = 15.16 μM). The
para‐nitro‐substituted analog 4f showed a selective anti‐AChE ac-
tivity (IC50 = 23.63 μM), whereas the compound 4c bearing meta‐
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4.1.2
General procedure for the synthesis of
OMe group was found to be a selective BuChE inhibitor (IC50
=
compound 2
47.46 μM). Moreover, the molecular docking analysis provided a
reasonable mechanism for the structure–activity relationship (SAR)
analysis against ChE enzymes. The in vitro kinetic assay of 4e pre-
sented a mixed type of inhibition pattern against both AChE and
BuChE. Also, compound 4e depicted 24.3% BACE1 inhibition at
50 µM, confirmed by the fluorescence resonance energy transfer
(FRET)‐based assay. More important, compound 4e showed 23.2%
neuroprotection on neuronal cell lines at 50 µM. Meanwhile, the
Here, 5‐arylisoxazole‐3‐carboxylic acid derivative 2 was exactly
prepared according to our previous report.[17] For this purpose, the
solution of sodium ethoxide was freshly prepared by the reaction of
sodium (1 mol) in dry EtOH (250 ml) in an ice bath. Then, a mixture of
diethyl oxalate (1 mol) and an appropriate acetophenone derivative
(1 mol) was added dropwise to that solution. The reaction was
stopped by the formation of a yellow pasty material, which was left

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at room temperature for 15 h. Next, it was stirred at 80°C in a water
bath for 90 min and the crude was treated with dilute sulfuric acid to
adjust to pH 2, affording an oily product that was extracted using
chloroform and washed by saturated NaHCO3 solution and brine.
The organic phase was dried over Na2SO4 and the solvent was re-
moved under vacuum to give 2,4‐dioxo‐4‐aryl butanoate derivative,
which was purified by recrystallization from chloroform and hexane.
Next, a mixture of hydroxylamine hydrochloride (0.23 mol) and 2,4‐
dioxo‐4‐aryl butanoate derivative prepared in the previous step
(0.08 mol) in ethanol (220 ml) was refluxed for 3 h. After the com-
pletion of the reaction (checking by TLC), the mixture was poured
on crushed ice, and the precipitate was extracted by di-
chloromethane and washed with water, NaOH solution (4%), and
brine, respectively. The organic phase was dried over Na2SO4
and the solvent was removed under vacuum to afford ethyl
5‐arylisoxazole‐3‐carboxylate derivative, which was then subjected
to alkaline hydrolysis. In this respect, ethyl 5‐arylisoxazole‐3‐
carboxylate (1 mmol) was treated with KOH (3 mmol) in methanol
(15 ml) under reflux conditions for 3 h. After the completion of the
reaction (checking by TLC), the mixture was poured into the water
and ice, and a white precipitated product was formed when the
concentrated HCl was added dropwise.
NH), 7.64 (d, J = 8.0 Hz, 2H, H2 H6), 7.47–7.17 (m, 7H, Ph, H3, H5),
6.86 (s, 1H, isoxazole), 4.18 (s, 2H, CH2), 3.56–3.53 (m, 1H, CH),
2.82–2.80 (m, 2H, CH2), 2.55–2.52 (m, 2H, CH2), 2.43–2.39 (m, 5H,
CH2, CH3), 2.20–2.17 (m, 2H, CH2). 13C NMR (125 MHz, CDCl3):
158.8, 158.5, 141.2, 133.4, 131.5, 130.3, 129.8, 129.4, 127.8, 125.8,
124.0, 98.3, 60.9, 51.3, 44.7, 28.6, 21.5. Anal. calc. for C23H25N3O2:
C, 73.57; H, 6.71; N, 11.19. Found: C, 73.71; H, 6.56; N, 10.91.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(3‐methoxyphenyl)isoxazole‐3‐
carboxamide (4c)
Yield: 53%, m.p. = 155–157°C. IR (KBr): 3322, 3090, 2928, 2853,
1648, 1610, 1575, 1547 cm−1. 1H NMR (500 MHz, CDCl3): 7.40–7.28
(m, 8H, Ph, H2, H5, H6), 7.00 (dd, J = 7.4, 2.0 Hz, 1H, H4), 6.94 (s, 1H,
isoxazole), 4.23–4.15 (m, 1H, CH), 4.25 (s, 2H, CH2), 3.87 (s, 3H,
OCH3), 3.61–3.58 (m, 2H, CH2), 2.96–2.93 (m, 2H, CH2), 2.27–2.25
(m, 2H, CH2), 2.06–2.02 (m, 2H, CH2). 13C NMR (125 MHz, CDCl3)
(two isomers): 171.5, 159.9, 159.8, 159.7, 146.9, 144.5, 131.4, 130.3,
129.6, 128.8, 127.7, 118.5, 116.3, 110.8, 62.0, 55.4, 51.1, 45.0, 31.2.
Anal. calc. for C23H25N3O3: C, 70.57; H, 6.44; N, 10.73. Found: C,
70.74; H, 6.58; N, 10.58.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(4‐nitrophenyl)isoxazole‐3‐
carboxamide (4d)
Yield: 70%, m.p. = 152–155°C. IR (KBr): 3308, 3084, 2940, 2848,
1658, 1606, 1548 cm−1 1H NMR (500 MHz, DMSO‐d6): 8.61
.
|
4.1.3
General procedure for the synthesis of
compounds 4a−l
(d, J = 8.0 Hz, 1H, NH), 7.48 (dd, J = 8.5, 2.0 Hz, 1H, H6), 7.44
(d, J = 2.0 Hz, 1H, H2), 7.34–7.23 (m, 6H, Ph, isoxazole), 7.10
(d, J = 8.5 Hz, 1H, H5), 3.85 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
3.81–3.79 (m, 1H, CH), 3.46 (s, 2H, CH2), 2.82–2.80 (m, 2H, CH2),
2.06–2.02 (m, 2H, CH2), 1.76–1.74 (m, 2H, CH2), 1.65–1.59 (m, 2H,
CH2). 13C NMR (125 MHz, CDCl3): 171.6, 159.3, 151.1, 149.3, 127.4,
128.4, 119.6, 119.3, 111.3, 108.5, 98.0, 96.1, 61.5, 56.1, 56.0, 52.0,
45.5, 30.5. Anal. calc. for C24H27N3O4: C, 68.39; H, 6.46; N, 9.97.
Found: C, 68.59; H, 6.28; N, 10.21.
A mixture of 5‐arylisoxazole‐3‐carboxylic acid derivative 2 (1 mmol),
HOBT (1 mmol), and EDCI (1 mmol) were stirred in dry acetonitrile
(5 ml) at room temperature for 1 h, and then 1‐benzylpiperidin‐4‐
amine (3) (1 mmol) was added to the mixture and the reaction was
continued for 24−72 h. After completion of the reaction (checking by
TLC), the solvent was evaporated under vacuum, the crude was ex-
tracted using chloroform, and washed with water, solutions of
NaHCO3 10%, saturated NaCl, and citric acid 10%, respectively.
After drying the organic phase over sodium sulfate, the solvent was
removed and the solid product was recrystallized from ethyl acetate
and petroleum ether.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(3‐nitrophenyl)isoxazole‐3‐
carboxamide (4e)
Yield: 62%, m.p. = 117–119°C. IR (KBr): 3383, 3082, 2930, 1675,
1528, 1449, 1348 cm−1 1H NMR (500 MHz, CDCl3): 12.71 (s, 1H,
.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐phenylisoxazole‐3‐carboxamide (4a)
Yield: 67%, m.p. = 239–245°C. IR (KBr): 3247, 2929, 1671, 1542,
.
1442 cm−1 1H NMR (500 MHz, DMSO‐d6): 9.06 (bs, 1H, NH),
NH), 8.61 (s, 1H, H2), 8.32 (d, J = 8.0 Hz, 1H, H6), 8.11 (d, J = 8.0 Hz,
1H, H4), 7.74–7.64 (m, 3H, H5, Ph), 7.49–7.46 (m, 3H, Ph), 7.11 (s,
1H, isoxazole), 4.22–4.18 (m, 3H, CH, CH2), 3.57–3.55 (m, 2H, CH2),
2.86–2.79 (m, 2H, CH2), 2.61–2.54 (m, 2H, CH2), 2.21–2.18 (m, 2H,
CH2). 13C NMR (125 MHz, CDCl3): 168.9, 159.1, 158.0, 148.7, 132.9,
131.3, 130.7, 130.4, 129.3, 129.1, 128.2, 125.1, 120.8, 100.8, 61.4,
51.4, 45.3, 29.4. 13C NMR (125 MHz, CDCl3): Anal. calc. for
7.95–7.90 (m, 3H, H2, H4, H6), 7.62–7.40 (m, 8H, Ph, isoxazole, H3,
H5), 4.39–4.37 (m, 1H, CH), 4.25 (s, 2H, CH2), 3.32–3.30 (m, 2H,
CH2), 3.08–3.06 (m, 2H, CH2), 2.04–1.98 (m, 4H, CH2). 13C NMR
(125 MHz, DMSO‐d6) (two isomers): 171.1, 170.3, 159.5, 159.4,
158.3, 156.9, 131.4, 131.1, 130.9, 129.4, 128.8, 126.3, 125.8, 125.7,
100.8, 99.9, 62.0, 50.5, 44.0, 30.8. Anal. calc. for C22H23N3O2: C,
73.11; H, 6.41; N, 11.63. Found: C, 73.30; H, 6.25; N, 11.45.
C22H22N4O4: C, 65.01; H, 5.46; N, 13.78. Found: C, 64.87; H, 5.66;
N, 13.54.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(4‐nitrophenyl)isoxazole‐3‐
carboxamide (4f)
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(p‐tolyl)isoxazole‐3‐carboxamide (4b)
Yield: 51%, m.p. greater than 250°C. IR (KBr): 3280, 2925, 2850,
Yield: 62%, m.p. = 117–119°C. IR (KBr): 3241, 3071, 2950, 1676,
1671, 1614, 1547 cm−1
.
1H NMR (500 MHz, CDCl3): 12.75 (s, 1H,
1607, 1560 cm−1 1H NMR (500 MHz, CDCl3): 8.35 (d, J = 8.2 Hz, 2H,
.

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H2, H6), 7.99 (d, J = 8.2 Hz, 2H, H3, H5), 7.42–7.34 (m, 5H, Ph), 7.16
(s, 1H, isoxazole), 4.13–4.09 (m, 1H, CH), 3.87 (s, 2H, CH2), 3.22–3.20
(m, 2H, CH2), 2.67–2.52 (m, 4H, CH2), 2.11–1.95 (m, 2H, CH2). 13C
NMR (125 MHz, CDCl3): 165.6, 161.5, 157.0, 147.0, 141.0, 131.3,
131.2, 129.4, 129.3, 126.7, 124.5, 101.8, 63.0, 52.5, 45.8, 30.5. Anal.
calc. for C22H22N4O4: C, 65.01; H, 5.46; N, 13.78. Found: C, 64.71; H,
5.27; N, 13.90.
(d, J = 5.8 Hz, 1H, NH), 7.94 (d, J = 8.1 Hz, 2H, H2, H6), 7.62–7.61 (m,
3H, H3, H5, Ph), 7.45–7.45 (m, 5H, Ph, isoxazole), 4.25 (s, 2H, CH2),
4.13–4.11 (m, 1H, CH2), 3.21–3.20 (m, 2H, CH2), 3.07–3.05 (m, 2H,
CH2), 2.00–1.98 (m, 2H, CH2), 1.34–1.26 (m, 2H, CH2). 13C NMR
(125 MHz, DMSO‐d6) (two isomers): 169.2, 167.0, 166.8, 159.6,
158.1, 135.5, 131.7, 131.6, 131.5, 129.5, 128.8, 127.6, 125.1, 100.5,
58.9, 50.4, 44.7, 38.1. Anal. calc. for C22H22ClN3O2: C, 66.75; H, 5.60;
N, 10.61. Found: C, 66.59; H, 5.42; N, 10.48.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(2‐fluorophenyl)isoxazole‐3‐
carboxamide (4g)
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(2,4‐dichlorophenyl)isoxazole‐3‐
carboxamide (4k)
Yield: 53%, m.p. = 222–224°C. IR (KBr): 3300, 2964, 2667, 1665,
1617, 1593, 1542 cm−1
.
1H NMR (500 MHz, CDCl3): 12.34 (s, 1H,
Yield: 62%, m.p. = 170–172°C. IR (KBr): 3290, 3078, 3029, 2927,
NH), 7.89–7.86 (m, 1H, H6), 7.67–7.65 (m, 1H, H4), 7.45–7.41 (m, 5H,
Ph), 7.24 (t, J = 8.0 Hz, 1H, H3), 7.16 (t, J = 8.0 Hz, 1H, H5), 7.07 (s,
1H, isoxazole), 4.52–4.41 (m, 1H, CH), 4.26 (s, 2H, CH2), 3.57–3.55
(m, 2H, CH2), 3.00–2.87 (m, 2H, CH2), 2.55–2.48 (m, 2H, CH2),
2.22–2.00 (m, 2H, CH2). 13C NMR (125 MHz, CDCl3) (two isomers):
169.5, 165.8, 160.0 (d, JC–F = 245.0 Hz), 158.7, 132.1 (d, JC–F = 8.7
Hz), 131.5, 131.3, 130.2, 129.9, 128.6, 128.0, 127.4, 127.3, 127.0,
126.4, 125.7, 124.7, 116.3 (d, JC–F = 21.2 Hz), 115.0 (d, JC–F = 12.5
Hz), 102.9, 102.8, 60.7, 51.2, 44.7, 28.6. Anal. calc. for C22H22FN3O2:
C, 69.64; H, 5.84; N, 11.07. Found: C, 69.48; H, 5.61; N, 10.85.
2800, 1657, 1597, 1545 cm−1 1H NMR (500 MHz, DMSO‐d6): 8.74
.
(d, J = 7.5 Hz, 1H, NH), 7.95 (d, J = 8.5 Hz, 1H, H6), 8.94 (d, J = 2.0 Hz,
1H, H3), 7.64 (dd, J = 8.5, 2.0 Hz, 1H, H5), 7.34–7.25 (m, 6H, Ph,
isoxazole). 13C NMR (125 MHz, CDCl3): 171.8, 166.7, 159.1, 157.9,
143.0, 137.0, 131.9, 130.9, 130.0, 129.3, 128.4, 127.7, 124.2, 104.1,
62.8, 52.0, 46.6, 31.6. Anal. calc. for C22H21Cl2N3O2: C, 61.40; H,
4.92; N, 9.76. Found: C, 61.65; H, 5.23; N, 9.58.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(4‐bromophenyl)isoxazole‐3‐
carboxamide (4l)
Yield: 67%, m.p. = 158–160°C. IR (KBr): 3335, 2944, 2802, 1654,
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(4‐fluorophenyl)isoxazole‐3‐
carboxamide (4h)
1605, 1541 cm−1 1H NMR (500 MHz, DMSO‐d6): 8.70 (d, J = 6.5 Hz,
.
1H, NH), 7.87 (d, J = 8.5 Hz, 2H, H2, H6), 7.76 (d, J = 8.5 Hz, 2H, H3,
H5), 7.39 (s, 1H, isoxazole), 7.34–7.27 (m, 5H, Ph), 3.90–3.70 (m, 1H,
CH), 3.52 (s, 2H, CH2), 2.88–2.85 (m, 2H, CH2), 2.60–2.55 (m, 2H,
CH2), 1.77–1.64 (m, 4H, CH2). 13C NMR (125 MHz, DMSO‐d6): 170.5,
159.3, 158.0, 143.2, 134.1, 132.4, 129.2, 128.3, 127.3, 125.6, 125.2,
99.5, 62.8, 52.0, 46.7, 31.7. Anal. calc. for C22H22BrN3O2: C, 60.01;
H, 5.04; N, 9.54. Found: C, 60.15; H, 4.81; N, 9.70.
Yield: 65%, m.p. = 248–250°C. IR (KBr): 3235, 2931, 1671, 1616,
.
1545, 1503 cm−1 1H NMR (500 MHz, CDCl3): 7.79–7.76 (m, 1H, NH),
7.63–7.61 (m, 2H, H2, H6), 7.48–7.45 (m, 5H, Ph), 7.18 (t, J = 8.5 Hz, 2H,
H3, H5), 6.87 (s, 1H, isoxazole), 4.18–4.14 (m, 3H, CH, CH2), 3.55–3.52
(m, 2H, CH2), 2.80–2.75 (m, 2H, CH2), 2.56–2.51 (m, 2H, CH2),
2.20–2.17 (m, 2H, CH2). 13C NMR (125 MHz, CDCl3) (two isomers):
176.4, 160.4 (d, JC–F = 245.6 Hz), 163.2, 162.1, 157.5, 149.6, 145.5,
144.8, 136.2, 131.7, 131.2, 129.1, 128.1, 126.2, 123.9, 117.1, 115.0,
106.8, 101.4, 60.5, 52.0, 40.1, 30.4. Anal. calc. for C22H22FN3O2: C,
69.64; H, 5.84; N, 11.07. Found: C, 69.77; H, 5.64; N, 11.22.
|
4.2
Biological activity
|
4.2.1
Anticholinesterase activity
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(2‐chlorophenyl)isoxazole‐3‐
carboxamide (4i)
All enzymes and reagents required for the assay were obtained from
Aldrich. The in vitro anticholinesterase activity of all synthesized
compounds, 4a−l, was assayed using modified Ellman's method using
a 96‐well plate reader (BioTek ELx808).[12,22] Initially, the stock so-
lutions of compounds 4 were prepared by dissolving the test com-
pound (1 mg) in dimethyl sulfoxide (DMSO; 1 ml), and then diluted
solutions at final concentrations of 1, 10, 20, and 40 μg/ml were
prepared using methanol. Each well contained 50 µl potassium
phosphate buffer (KH2PO4/K2HPO4, 0.1 M, pH 8), 25 µl sample so-
lution, and 25 µl enzyme (final concentration: 0.22 U/ml in buffer).
Control experiments were also performed under the same conditions
without enzyme. After incubation at room temperature for 15 min,
125 µL DTNB (3 mM in buffer) was added and the characterization of
enzymatic reaction was spectrometrically performed at 405 nm,
followed by the addition of substrate (ATCI 3 mM in water) after
5−10 min. The IC50 values were determined graphically from
Yield: 55%, m.p. = 158–160°C. IR (KBr): 3245, 3093, 2930, 1671,
.
1612, 1540 cm−1 1H NMR (500 MHz, CDCl3): 7.94–7.92 (m, 1H,
NH), 7.64–7.62 (m, 2H, H3, H6), 7.48–7.40 (m, 6H, Ph, H4), 7.33 (s,
1H, isoxazole), 7.19 (m, 1H, H5), 4.19–4.16 (m, 2H, CH2), 3.56–3.54
(m, 1H, CH), 2.84–2.78 (m, 2H, CH2), 2.59–2.51 (m, 2H, CH2),
2.20–2.18 (m, 2H, CH2), 1.73–1.69 (m, 2H, CH2). 13C NMR (125 MHz,
DMSO‐d6): 167.9, 159.1, 156.6, 144.2, 132.5, 131.7, 131.5, 131.0,
131.0, 130.0, 129.5, 128.0, 124.7, 104.7, 62.1, 50.1, 40.5, 30.2. Anal.
calc. for C22H22ClN3O2: C, 66.75; H, 5.60; N, 10.61. Found: C, 66.52;
H, 5.75; N, 10.78.
N‐(1‐Benzylpiperidin‐4‐yl)‐5‐(4‐chlorophenyl)isoxazole‐3‐
carboxamide (4j)
Yield: 62%, m.p. greater than 250°C. IR (KBr): 3270, 2950, 1671,
.
1604, 1544, 1489, 1443 cm−1 1H NMR (500 MHz, DMSO‐d6): 9.10

SAEEDI ET AL.
11 of 12
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inhibition curves (log inhibitor concentration vs. percent of inhibi-
tion). Also, the same method was used for the BuChE inhibition
assay.
PC12 cell damage induced by Aβ25–35. The cells were grown in
monolayer culture on collagen‐coated plates at 37°C in a humidified
atmosphere of 5% CO2. Neuronal PC12 cells were plated at a density
of 5 × 105 cells/well on 96‐well plates. The cells were preincubated
with compound 10h for 3 h before human Aβ25–35 (final concentra-
tion of 5 μM) was added. After 24 h, 90 μl medium was taken out and
20 μl of MTT (0.5 mg/ml dissolved in RPMI containing phenol red)
was added and incubated for an additional 2 h at 37°C. The absor-
bance (A570 nm) was measured using a Bio‐Rad microplate reader
(Model 680; Bio‐Rad). The details were reported in our pre-
vious work.
|
4.2.2
Kinetic studies
The kinetic study for the inhibition of AChE and BuChE by compound
4e was carried out according to Ellman's method used for the in-
hibition assay with four different concentrations of inhibitors.[22] For
the kinetic study of AChE, compound 4e was used at the con-
centrations of 0, 7.39, 29.55, and 59.11 µM. The Lineweaver–Burk
reciprocal plot was constructed by plotting 1/V against 1/[S] at
variable concentrations of the substrate acetylthiocholine (187.5,
750, 1500, 3000 µM). The inhibition constant Ki was calculated by
the plot of slopes versus the corresponding concentrations of the
compound 4e. The same method was performed for the kinetic study
of BuChE using the same concentrations of compound 4e and
butyrylthiocholine.[12,23]
|
4.2.5
Metal chelating activity
To study the metal‐binding ability of compound 4e, a mixture of
methanolic solutions of the compound (1 ml) and the related metal
(1 ml; prepared from FeSO4, ZnCl2, and CuCl2·2H2O) with the same
final concentrations (20 µM) in a quartz cuvette was incubated at
room temperature for 30 min. Then, the absorption spectra were
recorded in the range of 200–600 nm. The stoichiometry of complex
4e−Fe2+ was also studied using the molar ratio method.[28] The
concentration of compound 4e was 20 μM and the final concentra-
tion of Fe2+ ranged from 0 to 40 μM with 4‐μM intervals at 258.7 nm.
The plot was obtained by the corresponding absorption versus the
mole fraction of Fe2+ toward ligand 4e.
|
4.2.3
BACE1 enzymatic assay
A FRET‐based BACE1 enzyme assay kit was used to evaluate the
inhibitory activity of compound 4e against BACE1. The kit was
purchased from Invitrogen (former Pan Vera Corporation) and the
evaluation procedure was conducted according to the manu-
facturer's instructions.[24–26] BACE1 (purified baculovirus‐expressed
enzyme) was diluted using buffer (50 mM sodium acetate, pH 4.5) to
prepare a 3× working solution of 1 U/ml. The peptide substrate
(RhEVNLDAEFK‐Quencher) was also diluted in the same buffer to
obtain the 3× stock solution. DMSO stock solutions were diluted
with buffer to give 3× solution of test samples at different con-
centrations. The 3× solution of the BACE1 enzyme (10 µl) and each
inhibitor sample (10 µl) were placed in 96‐well plates and gently
mixed. The substrate 3× solution (10 µl) was then added to this
mixture in each well to initiate the reaction at the final reaction
volume of 30 µl. The reaction mixtures were incubated at 25°C for
90 min in the dark, and then the reaction was stopped by adding
10 µl of 2.5 mM sodium acetate. Fluorescence was monitored at
544 nm (excitation wavelength) and 590 nm (emission wavelength).
OM99‐2 was used as the reference drug, the IC50 value was calcu-
lated with CurveExpert software version 1.34 for Windows, and each
experiment was repeated three to five times.
|
4.3
Molecular docking study
To figure out the binding modes of the most active compound 4e,
the docking simulation was performed to target the crystal
structures of AChE, BuChE, and BACE1. The crystal structures of
1EVE, 1P0P, and 2QP8 were retrieved from the PDB and were
docked using the AutoDock software.[17] Flexible ligand dockings
were accomplished for the selected compound. The best posi-
tions of the selected compound in each target protein were
chosen by analyzing the interactions between the enzymes and
inhibitor. The best scoring positions, as achieved by the docking
score, were then selected and visualized using Discovery Studio
Client 2017.[17]
ACKNOWLEDGMENT
This study was financially supported by Tehran University of Medical
Sciences with project No. 99‐3‐104‐49878.
|
4.2.4
Neuroprotection effect against Aβ‐induced
CONFLICT OF INTERESTS
damage
The authors declare that there are no conflicts of interests.
The neuroprotective effect of compound 4e to protect neuronal
PC12 cells against damage induced by Aβ25–35 was examined ac-
cording to our previous report.[27] The MTT reduction assay was
used to evaluate the neuroprotectivity of compound 4e on neuronal
ORCID
Aida Iraji
S. Sara Mirfazli
Tahmineh Akbarzadeh
http://orcid.org/0000-0002-8442-2205
http://orcid.org/0000-0001-7171-683X
http://orcid.org/0000-0002-0212-7297

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How to cite this article: Saeedi M, Felegari P, Iraji A, et al.
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