Ultrasound-assisted synthesis of novel chalcone, heterochalcone and bis-chalcone derivatives and the evaluation of their antioxidant properties and as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103034

The chalcone and bis-chalcone derivatives have been synthesized under sonication conditions via Claisen-Schmidt condensation with KOH in ethanol at room temperature (20–89%). The structures were established on the basis of NMR, IR, Single-crystal XRD, and

Full text of DOI:10.1016/j.bioorg.2019.103034

Accepted Manuscript
Ultrasound-assisted synthesis of novel chalcone, heterochalcone and bis-chal-
cone derivatives and the evaluation of their antioxidant properties and as
acetylcholinesterase inhibitors
Efraín Polo, Nicol Ibarra-Arellano, Luis Prent-Peñaloza, Alejandro Morales-
Bayuelo, José Henao, Antonio Galdámez, Margarita Gutiérrez
PII:
S0045-2068(19)30318-9
https://doi.org/10.1016/j.bioorg.2019.103034
103034
DOI:
Article Number:
Reference:
YBIOO 103034
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
26 February 2019
17 May 2019
3 June 2019
Please cite this article as: E. Polo, N. Ibarra-Arellano, L. Prent-Peñaloza, A. Morales-Bayuelo, J. Henao, A.
Galdámez, M. Gutiérrez, Ultrasound-assisted synthesis of novel chalcone, heterochalcone and bis-chalcone
derivatives and the evaluation of their antioxidant properties and as acetylcholinesterase inhibitors, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103034
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Ultrasound-assisted synthesis of novel chalcone,
heterochalcone and bis-chalcone derivatives and the
evaluation of their antioxidant properties and as
acetylcholinesterase inhibitors
Efraín Polo,a Nicol Ibarra-Arellano,a Luis Prent-Peñaloza,a Alejandro Morales-Bayuelo,b José
Henao,c Antonio Galdámez,d and Margarita Gutiérrez.a*
a.
Laboratorio de Síntesis Orgánica, Instituto de Química de Recursos Naturales,
Universidad de Talca, Casilla 747, Talca 3460000, Chile.
b.
Ciencias de la Salud, Grupo de Investigaciones Básicas y Clínicas de la Universidad
del Sinú (GIBACUS), escuela de medicina, Universidad del Sinú, seccional Cartagena,
Colombia.
c.
Grupo de Investigación en Química Estructural (GIQUE), Escuela de Química, Facultad
de Ciencias, Universidad Industrial de Santander, A.A. 678, Carrera 27, Calle 9
Ciudadela Universitaria. Bucaramanga, Colombia.
d.
Departamento de Química, Facultad de Ciencias, Universidad de Chile, Santiago,
7800003, Chile.
Corresponding author: E-mail address: mgutierrez@utalca.cl (Margarita Gutierrez).
Abstract
The chalcone and bis-chalcone derivatives have been synthesized under sonication conditions via Claisen-Schmidt
condensation with KOH in ethanol at room temperature (20–89%). The structures were established on the basis of
NMR, IR, Single-crystal XRD, and MS. The best compound 3u had inhibitory activity (IC50 = 7.50 µM). The synthesis,
the antioxidative properties, chemical reactivity descriptors supported in Density Functional Theory (DFT),
acetylcholinesterase (AChE) inhibition and their potential binding modes, and affinity were predicted by molecular
docking of a number of morpholine-chalcones and quinoline-chalcone. A series of bis-chalcones are also reported.
Molecular docking and an enzyme kinetic study on compound 3u suggested that it simultaneously binds to the catalytic
active site (CAS) and peripheral anionic site (PAS) of AChE. Moreover, the pharmacokinetic profile of these compounds
was investigated using a computational method.
Keywords: Chalcones, Bis-chalcones, Alzheimer’s disease, antioxidant, Molecular modeling.
1. Introduction
Alzheimer’s disease (AD) is the major cause of dementia (1 in 8 of people
over 65 has AD in the USA). One hallmark of AD is the presence of senile
plaques (PS) and neurofibrillary tangles (NFTs), which are formed due to
the accumulation of β-amyloid peptide (Aβ) and hyperphosphorylated tau
protein (p-tau), respectively. There is also a reduction in the number of
neurons and synapses with marked cholinergic deficits.[1] Pharmacological
treatment for AD mainly involves the use of cholinesterase inhibitors
(galantamine, donepezil and rivastigmine) in the mild to moderate phases, and
glutamatergic antagonists of the N-methyl-D-aspartate receptor (NMDAR)
(memantine) in the moderate to severe phases of the disease. The use of
1

anticholinesterases, although not preventing the progression of the disease,
generates an improvement in the clinical manifestations of patients with AD,
especially in the cognitive sphere.[2,3]
Chalcones, bis-chalcones and their derivatives are attracting increasing
attention due to numerous pharmacological applications.[4] Chalcones
are the main precursors for the biosynthesis of flavonoids and
isoflavonoids and exhibit various biological activities such as anti-
tuberculosis agents,[5] antiviral, anti-Inflammatory,[6] antiplatelet,[7,8]
antimalarial,[9,10] Tubulin inhibitors,[11] Anti-Leishmanial agents,[12] and
others.[4]
On the other hand, quinolinyl chalcones have showed a wide range of
biological activities such as Antibacterial,[13] antimalarial,[14] anti-
inflammatory,[15]
anti-infective,[16]
DNA
gyrase
inhibitor,[17]
antitumor,[18] antimycotic,[19] reverse-transcriptase inhibitor,[20] and
antiulcer.[21]
A variety of methods are available for the synthesis of chalcones (figure 1).[22]
Traditionally, chalcones could be obtained via Claisen–Schmidt condensation
carried out in basic or acidic media under homogeneous conditions.[23] The
heterogeneous catalysts also have been used for Claisen–Schmidt
condensation, including Lewis acids, Brønsted acids, solid acids, and solid
bases with moderate success.[24–29]
Several research works have
demonstrated that many organic reactions have been accelerated by ultrasonic
irradiation, for example, polymer synthesis, electron transfer reaction,
cycloaddition reactions, phase transfer reactions, and others.[30] Most of them
depend on the presence of specific catalysts. The reaction of Claisen-Schmidt
to obtain chalcones has also been previously reported through the use of
specific catalysts, KF-Al2O3, the telluroxide (bis(p-methoxyphenyl)), and 2-2-
bipyridine complex of Co(OAc)2 were used as basic catalysts. [31]
O
OH
B
X
+
HO
Suzuki
O
O
HetO2S
X
O
Julia-Kocienski
Cross-coupling
+
O
+
KF3B
H
Heck
-Crafts
O
+
Friedel
O
X
X
+
HO
Figure 1. Non-conventional synthesis of chalcones.
2

1. Results and Discussion
1.1. Chemistry
We have synthetized a library of seventeen chalcones and seven
symmetric bis-chalcones with moderate to good yield (table 1) using
simple starting material and ultrasound (US) as a source of energy. US is
well known for better yields, short reaction time, and for its clean
process.[32] Hence, US methodology is considered an ecofriendly
methodology. It is worth mentioning here that reaction time was reduced
considerably with respect to the conventional methodology. That
methodology has a reported reaction time between 3 to 72 hours.[33,34]
In this work, all the compounds were prepared in a maximum time of 20
minutes. To prepare compounds 3a-l and 3m-s, the aldehyde and bis-
aldehyde components were kept, respectively, and different aromatic
ketones with electron-withdrawing groups (EWG) and electron-releasing
groups (ERG) were used. The results obtained show that there isn’t a
specific trend when the substituents are EWG or ERG. However, there is
an improvement in the yield when the number of substituents increases,
for example, compounds 3b, 3i, and 3j. In the same way, there is an
improvement in the yield when the electronegativity of halogen decreases
(compounds 3d, 3h, and 3k). Table 1 show structures of compounds
synthetized, activities, and yield. Compounds 3d, 3e, 3h, 3k, and 3l, were
previously reported as intermediate for the synthesis of
dihydropyrazole.[35,36] On the other hand, 3n-s bis-chalcones were
prepared to obtain different heterocycles.[37,38,47,48,39–46]
Table 1. Chalcones, hetero-chalcones, and bis-chalcones synthetized.
O
O
O
N
O
O
N
R1
O
O
O
R1
O
1a
R1
R1
1b
3a-l
2a-s
3m-s
O
O
O
O
O
R2
N
H
R2
+
N
H
N
N
3t-x
1c-f
2t 90%
O
O
O
O
O
N
O
N
N
O
O
O
O
3c 54%
3b 20%
AChE: 1468 µM
BuChE:166.11 ± 0.04 µM
IS: ---
3a 58%
AChE: 205.92 ± 0,04 µM
BuChE: --- µM
IS: ---
AChE: 309.49 ± 0.04 µM
BuChE: --- µM
IS: ---
O
O
O
N
CF3
N
F
N
O
O
O
O
3e 44%
3d 42%
3f 20%
AChE: 210.66 ± 0,02 µM
BuChE: >968.55 µM
IS: ---
AChE: 465.69 ± 0,04 µM
BuChE: --- µM
IS: ---
AChE: 320.38 ± 0,03 µM
BuChE: --- µM
IS: ---
3

O
O
O
O
O
O
O
O
O
O
N
Br
N
N
N
Cl
N
O
O
O
O
O
O
3g 20%
3h 77%
3i 34%
AChE: --- µM
BuChE: > 919.59 µM
IS: ---
3j 42%
3k 89%
AChE: 916.61 ± 0,03 µM
BuChE: >449.92 µM
IS: ---
AChE: 236.60 ± 0,03 µM
BuChE: >1143.96 µM
IS: ---
AChE: 91.16 ± 0,02 µM AChE: 127.22 ± 0,02 µM
BuChE: >847.58 µM
IS: ---
BuChE: --- µM
IS: ---
O
O
O
O
O
O
O
O
O
O
O
O
N
O
O
O
O
3m 68%
3n 68%
3o 58%
3l 50%
AChE: 776.79 ± 0,04 µM
BuChE: 519.10 ± 0,01µM
IS: 0.67
AChE: ? 1380.32 µM
BuChE: 259.20 ± 0,04µM
IS: ---
AChE: 49.84 ± 0,08 µM
BuChE: >1113.91 µM
IS: ---
AChE: 764.77 ± 0,04 µM
BuChE: --- µM
IS: ---
O
O
O
O
O
O
O
F
O
F
O
O
O
3s 82%
3r 68%
AChE: --- µM
3p 61%
O
3q 73%
AChE: 238.34 ± 0,01 µM
BuChE: 195.87 ± 0,03 µM
IS: 0.82
AChE: 40.48 ± 0,02 µM
BuChE: 212.92 ± 0,18 µM
IS: 5.26
AChE: 27.41 ± 0,04 µM
BuChE: 206.35 ± 0,02 µM
IS: 7.53
BuChE: 102.99 ± 0,01µM
IS: ---
O
O
O
O
O
O
O
O
O
O
N
N
Cl
N
N
H
N
N
H
H
H
N
3v 53%
N
N
3t 92%
3u 77%
3w 68%
N
AChE: 12.58 ± 0,03 µM
BuChE: >924.87 µM
IS: ---
AChE: 45.29 ± 0,004 µM
BuChE: >887.70 µM
IS: ---
AChE: 7.50 ± 0,01 µM
BuChE: --- µM
IS: ---
AChE: 214.92 ± 0,03 µM
BuChE: --- µM
IS: ---
O
O
O
N
O
N
N
N
O
3x 71%
Donepezil
H
N
AChE: --- µM
BuChE:--- µM
IS: ---
AChE: 0.02 µM
BuChE: 7.23 µM
IS: 361.5
.
The structure of 3a and 3e were further confirmed by the Single-crystal X-ray
diffraction technique. The skeleton of these molecules is depicted in Figure 3
and a summary of crystallographic data is given in Table 1S (supplementary
materials). The torsion angle of C14-C13-C12-C11 are 166.6(2)° and -177.0(4)°
in 3a and 3e, respectively. The Cremer and Pople puckering parameters of 6-
membered ring (O1/C1-C4/N1) in 3a are Q1 = 0.414(3) Å, θ = 6.1 (4)° and φ =
30.0 (5)°.[49] The six-membered ring adopts a half-chair conformation. The
torsion angle of C17-O3-C20-C18 is -15.82(5)° in 3a, N1-C2 bonds ranging
from 1.413 to 1.416 Å in 3a and 3e. All relevant structural parameters (bond
distances and angles) are as expected and in acceptable agreement with other
organic molecules.[50] Moreover, in the crystal packing of 3a, the molecules are
linked via C-H··· interactions. The C3-H3B···Cg1 symmetry code: -x,1-y,2-z
and C21-H21···Cg2symmetry code: 1-x,1-y,2-z distances are 2.97 and 2.92Å.
Crystal packing of 3e show a C19-H19···O2 intermolecular contact with a H···O
bond distance of 2.60 Å An angle of 135.00°. Crystallographic data reported in
this paper for 3a and 3e compounds have been deposited at the Cambridge
Crystallographic Data Center (CCDC), No. 1867175 and 1867174, respectively.
Copies of the data can be obtained, free of charge, on application to CCDC 12
4

Union Road, Cambridge CB2 1EZ, UK (Fax: +44-1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
1
The H-NMR coupling constant analyses indicated that all hydrogen atoms of
the olefinic carbon–carbon bond were in a trans configuration (J approximately
15 Hz). The existence of a carbonyl group conjugated with the olefinic carbon–
carbon bond was confirmed from the infrared spectra as the carbonyl peak was
observed at a lower wavenumber than a normal carbonyl peak (around 1650–
1600 cm−1). The existence of methoxy groups in 3b, 3i, and 3j was confirmed
1
by the chemical shifts near 3.9 ppm in the H-NMR spectra. The signal which
appeared as a doublet near 7.00 could be attributed to olefinic protons. In
addition, the signal which appeared in the region at d 7.6–8.7 as multiplets was
due to the aromatic protons present in the molecules. It was further supported
by the molecular ion showed in GC-MS corresponding to the molecular mass of
compounds. Similarly, all other chalcones were characterized and the data are
given in the experimental section.
1.1.
Biological evaluation
All compounds synthesized were evaluated in vitro as dual acetylcholinesterase
(AChE)-butyrylcholinesterase (BuChE) inhibitors according to the modified
Ellman’s method.[51] Galantamine, a well-known AChE inhibitor, was used as
the positive control. The concentration of the compound required for 50%
enzyme inhibition (IC50) was calculated by means of regression analysis. All
tabulated results in table 2 are expressed in µM as means ± SEM, and were
compared by using ANOVA analysis. A p value of less than 0.05 was
considered significant. Details for pharmacological experiments are described in
Materials and Methods as well as in previous works.[52,53]. The inhibit ratio
against AChE was calculated according to the absorbance of the product in the
AChE-catalyzed reaction and the results are shown in Table 2.
The preliminary investigation demonstrated that morpholine chalcones 3a-l
showed poor inhibiting effect against AChE (IC50 >50 µM). Bis-chalcone
derivatives 3m-s were found to present significant values in the range (27-1380
µM) where compound 3q, containing moiety phenyl, showed the best inhibition
(27.41 ± 0.038 µM). Electron donating groups (OCH3 and CH3) were present on
the phenyl ring of the chalcone, which are associated with the decrease in
activity. The quinoline based chalcones exhibited better inhibition values.
Among them, compound 3u contained the 3-pyridin group; it was found to have
the most potent inhibitory activity with IC50 value 7.50 ± 0.011 µM. Compounds
3o-q, 3t, and 3v have also considerable inhibitory values since their IC50 values
were lower than 50 µM.
5

Table 2. Acetyl- and butyryl-cholinesterase inhibitory activities of compounds (3a-w).
Compound
Anticholinesterase assay
Compound
Anticholinesterase assay
AChE (µM)a
BuChE (µM)a
AChE (µM)a
BuChE (µM)a
3a
3b
3c
3d
3e
3f
309.49 ± 0.04
>1468
--
3m
776.79 ± 0.04
> 1380,32
519.10 ± 0.01
259.20 ± 0.04
> 1113,91
212.92 ± 0.18
206.35 ± 0.02
102.99 ± 0.01
195.87 ± 0.03
> 887,70
166.11 ± 0.04
3n
205.92 ± 0,04
465.69± 0,03
210,66 ± 0,02
320.38± 0,03
916.61± 0,03
236,60 ± 0,03
----
--
--
3o
49.84 ± 0.08
40.48 ± 0.02
27.41 ± 0.04
--
3p
> 968.55
--
3q
3r
3g
3h
3i
> 449.9
> 1143,96
> 919.59
> 847.58
--
3s
238.34 ± 0.01
45,29 ± 0,004
7.50 ± 0.01
12,58 ± 0,03
214.92 ± 0,03
0.57 ± 0,5
3t
3u
----
3j
91,16 ± 0,02
127.2± 0,02
764.77± 0,07
3v
3w
> 924,87
3k
3l
----
--
Galantamine a
8.08 ± 0,02
a : IC50 ± SEM
Based on the obtained results in the cholinesterase inhibition, and with the aim
of assessing the kinetic mode of AChE inhibition of the target compounds, the
most active compound 3u was subjected to a kinetic study. For this purpose,
the rate of enzyme activity was measured at eight different concentrations of
substrate Acetylthiocholine iodide (ATC). The obtained Michaelis–Menten
parameters along with the Lineweaver–Burk plots (Figure 3) allowed us to
conclude that compound 3u would display a mixed-type inhibition against AChE
with variation in Km and Vmax values. The compound 3u showed Km values of
9.71x10-4, 1.89x10-4, and 4.05x10-4 μM and Vmax of 9.6x10-5, 1.89x10-5, and
2.79x10-5 mol/min, respectively.
Figure 2. The Lineweaver–Burk plot of AChE (0.02 U) with an Acetylthiocholine substrate in the absence
and presence of inhibitor 3u.
2.3. Theorical studies
Molecular docking of the most active compounds (3q, 3p, 3u, and 3v) was
performed to explain the in vitro activity. The active-site of AChE is a cylinder
cavity; Kryger G., et al.[54] have called this cavity a gorge. In the bottom of this
6

gorge, compounds show a π-π interaction between the aromatic ring of Trp84
and one of the aromatic rings at the ends. That interaction is similar to the
interaction showed by the benzyl group from donepezil (see figure 4).
In the middle of the gorge, a π-π interaction is observed between Phe330 and
the aromatic ring located in the center of each structure. The interaction of the
aromatic moiety of Phe330 is important because this connects the peripheral
site and anionic subsite in the active site. That is to say, both the orientation and
interaction of the aromatic ring of Phe330 influences the entry of compounds at
the bottom of the gorge. In the entrance of the gorge, a π-π interaction is
perceived between the aromatic ring from the other end of the molecule and the
aromatic ring of Trp279. This interaction is presented between indanone moiety
of donepezil and Trp279, too. This peripheral interaction is relevant due to the
hydrophobic environment; i.e. it can serve as a filter to allow which molecule
can enter the cavity.[54]
(a)
(b)
Docking score
-10.259
Docking score
-10.259
Donepezil
Donepezil
3q
-8.723
3p
-9.284
(c)
(D)
Docking score
Docking score
Donepezil
-10.259
-8.096
Donepezil
-10.259
-6.896
3u
3v
7

(E)
Figure 3. Molecular docking between the most active compounds against AChE binding site. Docking pose
of compound 3q (a), 3p (b), 3u (c) and 3v (D). Compound reference (donepezil) is represented in green.
Protein backbone and principal residues of interaction are in cartoon and sticker presentation. (E) 2D
representation of principal interaction of donepezil in AChE active-site.
Lipophilicity is a significant physicochemical property determining distribution,
bioavailability, metabolic activity, and elimination. Apart from the important role
of lipophilicity for the kinetics of biologically active compounds, antioxidants of
hydrophilic or lipophilic character are both needed to act as radical scavengers
in the aqueous phase or as chain-breaking antioxidants in biological
membranes. A perusal of log P and IC50 values in Tables 1, 2, and 4 revealed
that the role of lipophilicity on the inhibition of AChE wasn’t well defined in this
series of compounds. Analysing this data set, lipophilicity did not seem to affect
absolutely the AChE inhibition in compounds 3a-s. Within the chalcone
morpholine subgroup compound, the 3g lower log P value (2.920) was
correlated to higher activity (916.61± 0.033 µM). This concept was not followed
by the bis-chalcones group, compound 3q showed log P (5.372) and IC50 (27.41
± 0.038 µM). The same trend was present for quinoline-chalcones where log P
minimum was 3.772, associated to lower activity (7.50 ± 0.011 µM).
Chalcones derivatives have showed high capacity for the scavenging of free
radicals’ generation.[55] Chu, J. et. al. synthetized a set of amidochalcones
developed by the Claisen-Schmidt methodology and tested their antioxidant
activity in different in vitro systems like hydrogen peroxide scavenging activity,
DPPH radical scavenging activity, ferrous reducing power, and nitric oxide
radical scavenging activity. Their results show these compounds as potential
antioxidant agents with good activity compared with the ascorbic acid used as
reference.[56] Taking into account the previously described, we have used two
different types of assays to measure in vitro antioxidant activity: (a) the
interaction with the stable free radical DPPH and (b) the interaction with the
water-soluble azo compound ABTS+ where ascorbic acid was used as a
positive control. The effect of the fluorine atom can be analyzed by comparing
compound 3e and compound 3p. In 3e, the F contained a compound at the 4th
position of the phenyl ring, having better activity than CF3 group in the 4th
position of 3p. In the DPPH assay, the IC50 values of the compounds (3a-m)
8

were lower than 100 µg/mL. Compound 3o exhibited better DPPH free radical
scavenging activity with (IC50: 99.03 ± 3.8 µg/mL). In the ABTS assay, among
the tested compounds, 3m and 3s showed the best ABTS cation radical
scavenging activity with IC50 values of 173.59 ± 2.0 and 115.35 ± 3.3,
respectively. With the exception of compounds 3n-p, this series showed IC50
values that were higher than 200 µg/mL (Table 3).
Table 3. Antioxidant activity results for the most active compounds, DPPH and ABTS assays.
Compound
Antioxidant assay
DPPH assay IC50
ABTS+ assay IC50
(µg/mL)a
(µg/mL)a
3e
≥100
199.60 ± 3.7
3m
≥100
173.59 ± 2.0
219.66 ± 1.5
254.49 ± 3.8
115.35 ± 3.3
27.62 ± 3.5
3o
99.03 ± 3.8
131.33 ± 2.7
185.97 ± 2.4
1.5 ± 0.2
3p
3s
Ascorbic acid b
a Values expressed are mean ± SEM of three parallel measurements (p < 0.05). b Reference compound.
fk  LUMO
fk  HOMO 2
are
In Figure 5 and 6, the Fukui functions
2 and
shown for the most active compound 3u. The Fukui functions in Figure 5 and 6
represent the zones where it is possible to carry out stabilization by the retro-
donor process on the active site for the compound with the best activity. These
contours are very similar in both functions and also can be related with the
stabilization through a retro-donor process.
‒
2 for the compound 3u.
〈
〉
푓 푘 ≈ |퐻푂푀푂|
Figure 4. Fukui functions
9

+
2
〈
〉
푓 푘 ≈ |퐻푂푀푂|
Figure 5. Fukui functions
for the compound 3u.
The main zone for the stabilization with the highest surfaces is the carbonyl
groups (H- bond acceptor) and the amino groups (H-bond donor).
In Table 4, the global reactivity indices for the compounds analysed are shown.
The compounds with higher biological activity are 3q, 3u, and 3v (see Table 4).
They have chemical potential: 3q (µ= -4.2153 eV), 3u (µ= -4.4741 eV), and 3v
(µ= -4.0028 eV); chemical hardness: 3q ( = 3.4112 eV), 3u ( = 3.9516 eV),
and 3v ( = 4,0028 eV); chemical softness: 3q (S=0,2931 eV-1), 3u (S=0,2530
eV-1) and 3v (S=0,2498 eV-1 ); and electrophilicity: 3q (ω =2.6045 eV), 3u (ω
=2.5328 eV), and 3v (ω =2.3175 eV). From these results, we can see the
reactivity properties to understand the non-covalent activity in a quantitative
form. Additionally, the electrophilicity values in particular suggest stabilization
through a retro-donor process.
Table 4. Global reactivity descriptors.
Compound C. Potential (eV) C. Hardness (eV)
Softness (eV)-1
0.2791
0.2756
0.2773
0.2809
0.2925
0.2808
0.2919
0.2855
0.2770
0.2789
0.2852
0.2771
0.2771
0.2735
0.2931
0.2753
0.2931
0.2753
0.2745
0.2820
0.2530
0.2498
0.2644
0.5256
Electrophilicity (eV)
1.6925
1.6385
1.6847
1.8204
2.0875
1.7377
1.8370
1.9281
1.6355
1.6485
1.9115
1.6714
2.6169
2.5100
2.6766
2.9045
2.6045
2.7321
2.6300
2.2714
2.5328
2.3175
2.6456
2.6875
3a
3b
3c
3d
3e
3f
-3.4900
-3.4482
-3.4852
-3.5999
-3.7775
-3.5179
-3.5477
-3.6750
-3.4360
-3.4379
-3.6609
-3.4733
-4.3604
-4.2844
-4.2734
-4.5929
-4.2153
-4.4553
-4.3774
-4.0135
-4.4741
-4.3073
-4.4730
-3.1979
3.5982
3.6284
3.6050
3.5595
3.4178
3.5609
3.4256
3.5024
3.6093
3.5848
3.5056
3.6088
3.6327
3.6567
3.4115
3.6314
3.4112
3.6327
3.6428
3.5459
3.9516
4.0028
3.7813
1.9026
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
The compounds with the higher electrophilicity values are 3m and 3x. These
reactivity parameters show the stabilization of the electron cloud when it is
10

saturated by electrons from the external environment. These values obtained
give an idea about the stabilization process on the active site through non-
covalent interactions due to the presence of the EWG or ERG along with the
molecular set.
Analysis in silico was made to predict ADME/Tox properties (Table 5). This
analysis showed some interesting properties like Log P. This is an important
parameter to estimate the lipophilicity of one compound and if it is permeable
across the membrane with exception of compound 3m, compounds 3a-x are
within the range.[57,58] Another interesting parameter is the percent of human
oral adsorption because most drugs are administered via oral route. It’s known
that poor oral bioavailability is a problem in therapeutic application, particularly
when the drugs have a narrow therapeutic window. This make oral
bioavailability one of the key considerations to design and synthetize a new
compound.[59,60] Solubility in water (Log S) is an important parameter that
must be considered in the design of a possible drug because it has a significant
impact on distribution, transport, and bioavailability.[61] Another parameter is
cell permeability (PCaco), this is important due to the different barriers that a
drug has to cross when it is absorbed through intestinal mucosa.[62]
This prediction increases the probability of some compounds having better
behaviour in an in vivo ADME test, offering the possibility of achieving a
possible drug.
Table 5. Calculated physicochemical descriptors of compounds 3a-x.
Entry
3a
M.W.
Log P
Log S
PHOAa
RO5b
385.462
5.265
-6.057
100.0
100.0
1
3b
323.391
337.374
311.355
361.363
373.451
283.326
327.810
353.417
383.443
372.261
307.391
522.599
398.457
426.425
374.386
458.510
338.405
366.459
422.439
379.417
3.772
3.121
3.945
4.720
4.693
2.920
4.211
3.865
3.910
4.290
4.032
8.340
5.203
3.911
5.568
5.372
5.090
5.742
4.238
3.772
-4.314
-3.438
-4.541
-5.673
-5.538
-3.158
-4.933
-4.583
-4.561
-5.054
-4.776
-9.804
-6.072
-4.352
-6.594
-6.648
-5.845
-7.064
-5.872
-5.706
0
0
0
0
0
0
0
0
0
0
0
2
1
0
1
1
1
1
0
0
3c
3d
3e
3f
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
96.7
11

3v
3w
3x
*
378.429
463.922
382.421
273.331
379.498
4.722
5.489
3.828
1.805
4.454
-6.397
-7.818
-5.868
-1.766
-4.841
100.0
95.7
0
1
0
0
0
100.0
90.1
**
100.0
a Percent human oral absorption b Rule of Five * galantamine
**donepezil
1. Experimental
3.1. General Information
The ultrasonic irradiation was performed by using a Branson ultrasonic cleaner
bath, model 1510, 115v, 1.9 L with a mechanical timer (60 min with continuous
1
13
hold) and heater switch, 47 kHz. H and C NMR spectra (400 MHz for proton
and 100 MHz for carbon) were recorded on an AM-400 spectrometer (Bruker,
Rheinstetten, Germany) using CDCl3 and DMSO-d6 as solvents.
Tetramethylsilane (TMS) was used as an internal standard. IR spectra (KBr
pellets, 500–4000 cm−1) were recorded on a NEXUS 670 FT-IR
spectrophotometer (Thermo Nicolet, Madison, WI, USA). High-resolution mass
spectrometry ESI-MS and ESI-MS/MS analyses were conducted in a high-
resolution hybrid quadrupole (Q) and orthogonal time-of-flight (TOF) mass
spectrometer (Waters/Micromass Q-TOF micro, Manchester, UK) with a
constant nebulizer temperature of 100 °C. Melting points (uncorrected) were
measured on an Electrothermal IA9100 melting point apparatus (Stone, Staffs,
UK). Reaction progress was monitored by means of thin-layer chromatography
using silica gel 60 (Merck, Darmstadt, Germany). All reagents were purchased
from either Merck or Sigma Aldrich (St. Louis, MO, USA) and used without
further purification. Final purification of all products for analysis was carried out
by recrystallization. GC-MS analyses were performed on a model Trace 1300
GC-MS instrument (Thermo Fisher Scientific, Waltham, MA, USA) equipped
with a Rtx- 5MS on-column auto injector and a fused silica capillary column
(DB-5, 30 m × 0.25 mm ID, 0.25 μm film thickness). Operating conditions were
as follows: helium as the carrier gas with a flow rate of 1.5 mL/min; column
temperature 40°C as initial temperature, then increasing at 10°C/min to 280°C;
injector temperature, 250°C; volume injected, 1 μL; split ratio, 33.3. MS were
recorded in electron ionization (EI) mode with energy of 70 eV. The ion source
temperature was 200°C; 4.00 min solvent cut time. The compounds were
identified by comparison with the data held in the NIST 11 Library; data are
reported as m/z ratio with the relative abundance of the respective peak.
3.2. Acetylcholinesterase assay
12

The evaluation of enzyme inhibition was carried out applying the Ellman’s
spectrophotometric method.[51] AChE (from Electrophorus electricus) and
BuChE (from equine serum), 5,5’-dithio-bis-(2-nitrobenzoic acid (DTNB),
acetylthiocholine and butyrylthiocholine iodides were purchased from Sigma-
Aldrich. The stock solutions of the test compounds were prepared in 100µL of
DMSO and 900µL of phosphate buffer (8mmol/L K2HPO4, 2.3mmol/L NaH2PO4,
150mmol/L NaCl, and 0.05% Tween 20 at pH 7.6). In a 96-well plate, 50µL of
stock solutions were diluted with phosphate buffer to obtain concentrations
between 15 – 500 µg/mL for each compound. Enzyme solutions were prepared
with buffer to give 0.25 units/mL and 50µL was added to the plate. After 30 min
of incubation, the substrate solution consisting of Na2HPO4 (40mmol/L),
acetylthiocholine/butyrylthiocholine (0.24mmol/L) and DTNB (0.2mmol/L ) was
added. The mixture was incubated for another 5 min and the absorption at
405nm was determined with a Microtiter plate reader (Multiskan EX, Thermo).
Each compound concentration was tested in triplicate. The IC50 values were
calculated by means of regression analysis.
3.3. Kinetic study
For enzymatic kinetic studies of compound 3u, the enzyme solution was pre-
incubated with different substrate concentrations ranging from 9.38 x 10-4 to
0.48mM. For the determination of the type of inhibition, Vmax and Km (Michaelis
constant) as well as double reciprocal plots (1/V versus 1/[S] where V=reaction
rate and S=substrate concentration) were constructed using Lineweaver–Burk
methods. Determinations were made in the absence and presence of 3u. At
least three different concentrations of inhibitor were used in each instance and
the experiment was performed in triplicate. Data analysis was carried out with
SigmaPlot v10.0 and Enzyme Kinetic v1.3 add-on (Systat Software, Inc, CA,
US)
3.4. ADME Prediction
The absorption, distribution, metabolism, excretion properties of the
compounds, as well as the toxicity were predicted using QikProp module from
suite Schrödinger.[63]
3.5. Computational Details
All the structures included in this study were optimized at B3LYP/6-31G(d)[64]
level of theory by using the Gaussian 09 package.[65] We wish to confront the
results of model development using the conceptual of DFT (Density Functional
Theory). The DFT of chemical reactivity (called conceptual DFT) is used in
combination with the molecular quantum similarity approach introduced by
Carbo and co-workers. In this, the chemical reactivity descriptors supported in
13


DFT[66–68], like chemical potential ( )[69–72], hardness () [73–75],
electrophilicity (ω) [76,77], and local reactivity descriptors like condensed-to-
f
atom Fukui function (
),[78–80] were used to obtain new insights about the
k
stabilization process in the active site.

The global chemical reactivity like
shows the tendency of electrons to
escape from the electronic cloud. The
represents a measure of the

opposition of the electronic cloud to deformation and the
represents the
stabilization energy of the system when it is saturated by electrons from the
external environment. These global chemical descriptors can be calculated as
follows:
E
N



 
(1)


v r
 
2


 E




 
(2)
2
N

v
r
where
ω is defined as
v(r)
2 2
2 2
 

(3)

Using the finite difference approximations in equations 1 and 2,
and
can
be expressed as:
I  A
2
(4)
  
and
(5)
  I  A
Here, I and A are the ionization potential and electron affinity, respectively. I
and A were calculated through Koopmans’ theorem.
Where, I = -EHOMO
and A = -ELUMO
(6)
(7)
x
k
f
The local chemical reactivity through the Fukui functions
computed as follows:
(x = +, -) were
fk qk (N 1)qk (N)
for nucleophilic attack
(8a)
14

fk qk (N)qk (N 1)
for electrophilic attack
(8b)
In these equations, qk refers to the electron population at kth atomic site in a
compound. In this study, we adopted a natural population analysis (NPA)
scheme to evaluate atomic charge. Recently, our computational group has been
working on new methodologies to understand the non-covalent
interactions.[81,82]
3.6. Antioxidant Activity
3.6.1. Measurement of DPPH Radical Scavenging Activity
The scavenging activities of the compounds were estimated using DPPH as the
free radical model according to the method previously described and
adapted.[83] Briefly, an aliquot of 1 mL of the tested compound (10 -100 μg/mL)
and control (2% DMSO final), respectively, were mixed with 2 mL of a
methanolic solution of DPPH (0.02 mg/mL). The mixture was shaken vigorously
and left to stand at room temperature for 5 min in the absence of light. The
mixture was measured spectrophotometrically at 517nm. The free radical
scavenging activity was calculated as the percentage of DPPH decolouration
using the following equation:
%scavenging DPPH free radical = 100 × (1 − AE/AD)
where AE, is the absorbance of the solution after adding the extract and AD is
the absorbance of the blank DPPH solution. Ascorbic acid was used as
reference compounds with IC50 value of 1.5 μg/mL.
3.6.2. Measurement of ABTS Radical Scavenging Activity
The assay was performed according to a published protocol41. The stock
solution was prepared by mixing equal volumes of 7mM ABTS solution and
2.45mM potassium persulfate solution followed by incubation for 12h at room
temperature in the dark to yield a dark-colored solution containing ABTS+
radicals. A working solution was freshly prepared before each assay by diluting
the stock solution by adding 50% methanol for an initial absorbance of about
0.700 (+0.02) at 732nm at room temperature. Free radical scavenging activity
was assessed by mixing 300µL of different compounds (10-200µg/mL in
respective solvents) with 3.0mL of ABTS working standard. The decrease in
absorbance was measured exactly 1 minute after mixing the solution; the final
absorbance was noted up to 6 min. Data for each assay was recorded in
triplicate. Ascorbic acid, with an IC50 value of 28µg/mL, was used as the positive
control. The scavenging activity was estimated based on the percentage of
ABTS radicals scavenged by the following formula:
%scavenging = [(A0 − As)/A0] × 100
15

where A0 is absorption of control, As is absorption of tested compound solution.
3.7. Crystallography
XRD data of 3a were collected at room temperature using a Bruker
Kappa CCD diffractometer. Data collection, data reduction, and cell refinement
were done using Bruker Smart, Saintplus, APEX3, respectively. XRD data of 3e
were collected at room temperature using a RIGAKU XTaLAB P200
diffractometer. Data collection, data reduction and cell refinement was done
using CrystalClear 2.1.[84] The program used to refine the crystal structures
and prepare materials for publication were SHELXL, Olex2 and PLATON.[85–
87] The structures were solved by direct methods and refined with the full-
matrix least-squares method on F2. All H-atoms were positioned geometrically
and constrained to ride on their parent atoms. The 3e compound shows
disorder at C3 and F atoms. The F1, F2, and F3 atoms are disordered over two
sets of sites. The C3 atom is disordered over two sets of sites and has a refined
occupancy ratio of 0.615 (9):0.385(11).
3.8. Molecular docking
Molecular docking of the most active compound was done to check the possible
interaction modes between Ache and ligands. Molecular docking is a powerful
tool usually used in drug-design. Docking was done using grid-base ligand
docking with energetics (Glide) contained in Maestro software (Schrödinger
Release 2017-1: Maestro, Schrödinger, LLC, New York, NY, 2017). Glide
performs a meticulous search of the space available in regards to position,
orientation, and conformation for ligands. All these are made through numerous
hierarchical filters which allow finding the possible location of ligands in receptor
space previously built.[88] This conformational search is made through an
algorithm that involves systematic and stochastic methods which perform
structural variations of ligands as torsional, translational, and rotational degrees
of freedom.[89] Finally, Glide uses a modified score function based on the
ChemScore function of Eldridge et al,[90] which estimates the free binding
energy of the ligand-receptor complex.
The coordinates of protein were taken from the X-ray crystal structure of AChE-
donepezil complex (PDB code 1EVE). This structure was modified with Protein
Preparation Wizard application of the Maestro module. The grid box was
centred on the donepezil with a size of 16 Å x 16 Å x 16 Å. Exploration of large
conformational space to look for minimal energy of ligands was made with
OPLS2005 force field with a distance-dependent dielectric 2.0.[91] The Monte
Carlo procedure was used for finding the lower energy pose close to the
torsional minima.
16

3.9. Chemistry
3.9.1. N-(4-acetylphenyl)quinoline-3-carboxamide (2t)
A mixture of commercial quinoline-3-carboxylic acid and freshly distilled thionyl
chloride was warmed into reflux for 3h, then cooled to room temperature and
evaporated under vacuum to dryness to quantitatively obtain the corresponding
chloride acids. This crude material could be used without further purification. A
mixture of these chloride acids (1.0 equiv) and 4-aminoacetophenone (1.0
equiv) in toluene (10 mL) was stirred at room temperature for 2 h and then
treated with a saturated NaHCO3 solution. The biphasic solution was vigorously
stirred for 30 min, then decanted, and finally separated. The collected aqueous
phase was extracted with EtOAc (2 x 10 mL). The combined organic layer was
dried over Na2SO4 and evaporated. The solid was washed with cold water and
crude material was crystallized from ethanol.
Yield 90%, white solid; m.p. = 255-257 °C; IR (KBr, cm-1): 3327,3068, 3001,
1
1985, 1918, 1844, 1676, 1650, 1597, 1268, 1109, 799 ; H NMR (400 MHz,
DMSO-d6): 2.55 (s, 3H), 7.71 (t, J = 8.0 Hz,1H), 7.89 (t, J = 8.0 Hz, 1H), 7.99 (s,
4H), 8.12 (dd, J = 8.0, 12.3 Hz, 2H), 8.99 (s, 1H), 9.37 (s, 1H), 10.90 (bs, 1H);
13C NMR (100 MHz, DMSO-d6) δC: 26.4 (CH3), 119.5 (2 x CH), 126.3 (C),
127.3(C), 127.5 (CH), 128.8 (CH), 129.2 (CH), 129.3 (2 x CH), 131.5 (CH),
132.2 (C), 136.2 (CH), 143.4 (C), 148.5 (C), 149.0 (CH), 164.5 (C), 196.5 (C);
General procedure for the preparation and purification of chalcones 3(a–l):
A solution of equimolar amounts of 4-(4-Morpholinyl)benzaldehyde 1a (1 mmol)
and acetophenone 2a-s (1 mmol), using Potassium hydroxide in ethanol (10
mL) as a solvent in an Erlenmeyer, was placed in a water bath and sonicated at
room temperature for an appropriate time (10-20 min) until the reaction was
completed (the reaction was monitored by TLC). The reaction mixture was then
treated with a cold bath and filtered to leave a solid product, which was washed
with a hexane to yield pure products 3 (a-l). The synthesized compounds with
their physical data are listed below.
3.9.2. (E)-3-(4-morpholinophenyl)-1-(4-phenoxyphenyl)prop-2-en-1-one (3a)
Yield: 58% (223.4 mg); yellow solid; mp 202-204 °C; IR (KBr, cm-1): 3120,
2975, 1750, 1654, 1498, 1320, 1201,758, 660;1H NMR (200 MHz, CD3Cl)
3.25-3.30 (m, 4H), 3.85-3.90 (m, 4H), 6.90 (d, J = 8.0 Hz, 2H), 7.03 (s, 1H),
7.08 (bs, 2H), 7.11-7.13 (m, 1H), 7.27 (s, 1H), 7.36-7.38 (m, 1H), 7.41-7.45 (m,
2H), 7.58 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 16.0 Hz, 1H), 8.03 (d, J = 8.0, 2H);
HRMS (ESI, m/z): calculated for C25H23NO3 [M]+ 385.1678 found 385.1662
3.9.3. (E)-1-(4-methoxyphenyl)-3-(4-morpholinophenyl)prop-2-en-1-one (3b)
Yield: 20% (64.6 mg); yellow solid; mp 154-156 °C; IR (KBr, cm-1): 3097, 2950,
1
1748, 1598, 1545, 1288, 1220, 1130, 720, 554; H NMR (400 MHz, CD3Cl)
17

3.22-3.27 (m, 4H), 3.83-3.87 (m, 7H), 6.92 (dd, J = 16.1 8.9 Hz, 4H), 7.39 (d, J
= 15.5 Hz, 1H), 7.55 (d, J = 8.9 Hz, 2H), 7.75 (d, J = 15.5 Hz, 1H), 8.02 (d, J =
8.9 Hz, 2H); HRMS (ESI, m/z): calculated for C20H21NO3 [M]+ 323.1521; found
323.1514.
3.9.4. (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(4-morpholinophenyl)prop-2-en-1-one
(3c) Yield: 54% (182.0mg); yellow solid; mp 161-163 °C; IR (KBr, cm-1): 3110,
3050, 2970, 1721, 1689, 1645, 1502, 1272, 1224, 1105, 889, 572; 1H NMR (400
MHz, CD3Cl) 3.22-3.27 (m, 4H), 3.82-3.87 (m, 4H), 6.04 (s, 2H), 6.87 (d, J = 9.4
Hz, 3H), 7.34 (d, J = 15.5 Hz, 1H), 7.51-7.52 (m, 2H), 7.57 (s, 1H), 7.62 (dd, J
= 8.2,1.7Hz, 1H), 8.02 (d, J = 15.4 Hz, 1H); GC-MS m/z (rel.int. %): 164 (28),
149 (100), 121 (48), 91 (20); HRMS (ESI, m/z): calculated for C20H19NO4 [M]+
337.1314 found 337.1323.
3.9.5. (E)-1-(4-fluorophenyl)-3-(4-morpholinophenyl)prop-2-en-1-one (3d) Yield:
41% (127.6 mg); yellow solid; mp 155-157 °C; IR (KBr, cm-1): 3121, 2962, 1721,
1749, 1695, 1623, 1601, 1502, 1242, 1254, 1112, 1045, 937, 574;1H NMR (400
MHz, CD3Cl) 3.24-3.29 (m, 4H), 3.83-3.88 (m, 4H), 6.88 (d, J =8.8 Hz, 2H),
7.15 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 15.5 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H),
7.77 (d, J = 15.5 Hz, 1H), 8.02 (dd, J = 8.8, 5.4Hz, 2H); HRMS (ESI, m/z):
calculated for C19H18FNO2 [M]+ = 311.1322 found 311.1325.
3.9.6. (2E)-3-[4-(morpholin-4-yl)phenyl]-1-[4-(trifluoromethyl)phenyl]prop-2-en-
1-one (3e) Yield: 44% (158.9mg); yellow solid; mp 152-154 °C; IR (KBr, cm-1):
3113, 2987, 1704, 1651, 1532, 1343, 1207, 1003, 954, 825, 551; 1H NMR (400
MHz, CD3Cl) 3.26-3.31 (m, 4H), 3.84-3.89 (m, 4H), 6.90 (d, J = 8.8 Hz, 2H),
7.32 (d, J = 15.5 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.77 (m, 3H), 8.07 (d, J =
8.1 Hz, 2H); HRMS (ESI, m/z): calculated for C20H17F3NO2 [M-H]+ 360.1212
found 360.1205.
3.9.7.
(E)-1-(6-methoxynaphthalen-2-yl)-3-(4-morpholinophenyl)prop-2-en-1-
one (3f) Yield: 20% (74.6 mg); yellow solid; mp 192-194 °C; IR (KBr, cm-1):
3053, 2920, 1741, 1687, 1627, 1574, 1302, 1225, 1142, 1072, 937, 852, 617;1H
NMR (400 MHz, CD3Cl) 3.24-3.29 (m, 4H), 3.84-3.89 (m, 4H), 3.95 (s, 3H), 6.90
(d, J = 8.8 Hz, 2H), 7.18 (bs, 2H), 7.50 (s, 1H), 7.58-7.65 (m, 3H), 7.86 (dd, J =
8.8, 5.7 Hz, 2H), 8.07 (d, J = 8.8 Hz, 1H); 8.46 (s, 1H); HRMS (ESI, m/z):
calculated for C24H23NO3 [M-H]+ 373.1678 found 373.1698
3.9.8. (E)-1-(furan-2-yl)-3-(4-morpholinophenyl)prop-2-en-1-one (3g) Yield:
50% (141.6 mg); yellow solid; mp 172-174 °C;IR (KBr, cm-1): 3065, 2995, 1732,
1
1670,1650, 1524, 1403, 1265, 1217, 1113, 831, 761; H NMR (200 MHz,
CD3Cl) 3.23-3.27 (m, 4H), 3.82-3.87 (m, 4H), 6.55 (dd, J = 3.5, 1.7 Hz, 1H),
6.87 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 3.5 Hz, 1H), 7.33 (s, 1H), 7.54 (bs, 1H),
7.58-7.62 (m, 2H), 7.82(d, J = 16.0 Hz, 1H); HRMS (ESI, m/z): calculated for
C17H17NO3 [M]+ 283.1208 found 283.1212.
18

3.9.9. (E)-1-(4-chlorophenyl)-3-(4-morpholinophenyl)prop-2-en-1-one (3h) Yield:
77% (251.9mg); yellow solid; mp 170-172 °C; IR (KBr, cm-1): 3087, 2904, 1741,
1615, 1523, 1207, 1108, 1033, 871, 632, 584;1H NMR (200 MHz, CD3Cl) 3.24-
3.28 (m, 4H), 3.82-3.87 (m, 4H), 6.88 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 15.5 Hz,
1H), 7.44 (d, J = 8.6 Hz, 2H) 7.55 (d, J = 8.9 Hz, 2H), 7.76(d, J = 15.5 Hz, 1H),
7.93 (d, J = 8.9, 2H); GC-MS m/z (rel.int. %):191(24), 154 (22), 134 (10), 133
(100), 132 (80).
3.9.10.
(E)-1-(3,4-dimethoxyphenyl)-3-(4-morpholinophenyl)prop-2-en-1-one
(3i) Yield: 34% (120.1mg); yellow solid; mp 158-160 °C; IR (KBr, cm-1): 3101,
2983, 1733, 1678, 1662, 1587, 1492, 1304, 1254, 1197, 1073, 996, 714, 558; 1
NMR (400 MHz, CD3Cl); 3.03-3.06 (m, 4H), 3.64-3.66 (m, 4H), 3.75-3.76 (m,
6H) 6.67-6.72 (m, 3H), 7.21 (d, J = 16.0 Hz, 1H) 7.35 (d, J = 8.0 Hz, 2H), 7.42
(bs, 1H), 7.47(d, J = 8.0, 1H), 7.57(d, J = 16.0 Hz, 1H); HRMS (ESI, m/z):
calculated for C21H23NO4 [M]+ 353.1627 found 353.1626
3.9.11. (E)-3-(4-morpholinophenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(3j) Yield: 42% (160.9mg); yellow solid; mp 187-189 °C; IR (KBr, cm-1): 3091,
2961, 1733, 1667, 1652, 1498, 1230, 1175, 998, 885, 6731H NMR (200 MHz,
CD3Cl) 3.25-3.29 (m, 4H), 3.84-3.89 (m, 4H), 3.93 (s, 3H), 3.95 (s, 6H), 6.90 (d,
J = 8.0 Hz, 2H), 7.27 (s, 2H), 7.33 (d, J = 16.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 2H),
7.80 (d, J = 16.0 Hz, 1H); HRMS (ESI, m/z): calculated for C22H25NO5 [M]+
383.1733 found 383.1718
3.9.12. (E)-1-(4-bromophenyl)-3-(4-morpholinophenyl)prop-2-en-1-one (3k)
Yield: 89% (330.2mg); yellow solid; mp 182-184 °C; IR (KBr, cm-1): 3068, 3001,
1
1742, 1647, 1522, 1401, 1265, 1218, 822, 793, 672, 575; H NMR (400 MHz,
CD3Cl) 3.25-3.28 (m, 4H), 3.84-3.87 (m, 4H), 6.88 (d, J = 8.0 Hz, 2H), 7.32 (d, J
= 16.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H), 7.77(d, J =
16.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H); HRMS (ESI, m/z): calculated for
C19H18BrNO2 [M]+ 371.0521 found 371.0515
3.9.13. (E)-3-(4-morpholinophenyl)-1-(p-tolyl)prop-2-en-1-one (3l) Yield: 30%
(92.1mg); yellow solid; mp 140-142 °C; IR (KBr, cm-1): 3057, 2936, 1748, 1653,
1564, 1298, 1219, 1134, 1002, 894, 684, 581;1H NMR (400 MHz, CD3Cl) 2.42
(s, 3H), 3.24-3.26 (m, 4H), 3.84-3.86 (m, 4H), 6.88 (d, J = 8.0 Hz, 2H), 7.28 (d, J
= 8.0 Hz, 2H), 7.38 (d, J = 16.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.75 (d, J =
16.0 Hz, 1H), 7.91 (d, J = 8.0, 2H); HRMS (ESI, m/z): calculated for C20H21NO2
[M]+ 307.1572 found 307.1590
3.9.14. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(4-phenoxyphenyl)prop-2-en-1-one)
(3m) Yield: 68% (355.4mg); yellow solid; mp 183-185 °C; IR-FT (KBr, cm-1):
3064, 3038, 3007, 1661, 1591, 1489, 1416, 1337, 1225, 1168, 1024, 979, 870;
1H NMR (400 MHz, CDCl3) δ 7.19 – 7.29 (m, 8H), 7.37 (t, J = 7.9 Hz, 2H), 7.57
(t, J = 7.9 Hz, 4H), 7.73 (d, J = 15.7 Hz, 2H), 7.84 (s, 4H), 7.97 (d, J = 15.7 Hz,
19

13
2H), 8.20 (d, J = 8.7 Hz, 4H); C NMR (100 MHz, CDCl3) δ 117.4 (4 x CH),
120.1 (4 x CH), 122.7 (2 x CH), 124.6 (2 x CH), 128.8 (4 x CH), 130.0 (4 x CH),
130.8 (4 x CH), 132.5 (2 x C), 136.8 (2 x C), 143.1 (2 x CH), 155.4 (2 x C),
161.9 (2 x C), 188.4 (2 x C); HRMS (ESI, m/z): calculated for C36H26O4 [M]+
522.1831 found 522.1832.
3.9.15. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(4-methoxyphenyl)prop-2-en-1-one)
(3n) Yield: 68% (270.9mg); yellow solid; mp 245-247 °C; IR-FT (KBr, cm-1):
3077, 2981, 2931, 2834, 1659, 1609, 1570, 1507, 1424, 1340, 1288, 1262,
1228, 1175, 1113, 987, 812; 1H NMR (400 MHz, DMSO-d6) δ 3.88 (s, 6H), 7.10
(d, J = 8.8 Hz, 4H), 7.75 (d, J = 15.6, Hz, 2H), 7.98 (s, 4H), 8.04 (d, J = 15.6 Hz,
2H), 8.20 (d, J = 8.8 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) δ 54.9 (2 x CH3),
113.5 (4 x CH), 123.3 (2 x CH), 128.1 (4 x CH), 129.9 (4 x CH), 130.3 (2 x C),
136.1 (2 x C), 141.0 (2 x CH), 162.7 (2 x C), 187.2 (2 x C); HRMS (ESI, m/z):
calculated for C26H22O4 [M]+ 398.1518 found 398.1514.
3.9.16. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(benzo[d][1,3]dioxol-5-yl)prop-2-en-1-
one) (3o) Yield: 58% (247.3mg); yellow solid; mp 254-256 °C; IR-FT (KBr, cm-
1): 3088, 3033, 3921, 1661, 1599, 1502, 1444, 1332, 1251, 1115,
1
1045,925,815; H NMR (400 MHz, DMSO-d6) δ 6.17 (s, 4H), 7.11 (d, J = 8.2
Hz, 2H), 7.69 (d, J = 1.3 Hz, 2H), 7.74 (d, J = 15.6, Hz, 2H), 7.90 (d, J = 8.2 Hz,
2H), 7.98 (s, 4H), 8.02 (d, J = 15.6 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ
102.6 (2 x CH2), 108.4 (2 x CH), 125.9 (2 x CH), 129.8 (4 x CH), 130.3 (4 x CH),
137.4 (2 x C), 140.9 (2 x C), 142.2 (2 x CH), 148.5 (2 x C), 152.3 (2 x C), 193.1
(2 x C); HRMS (ESI, m/z): calculated for C26H22O4 [M]+ 426.1103 found
426.1168.
3.9.17. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(4-fluorophenyl)prop-2-en-1-one) (3p)
Yield: 61% (228.4mg); yellow solid; mp 238-240 °C; IR-FT (KBr, cm-1): 3075,
1
3028, 1660, 1598, 1504, 1416, 1346, 1164, 1024, 975; H NMR (400 MHz,
DMSO-d6) δ 7.48 (t, J = 8.6 Hz, 4H), 7.85 (d, J = 15.6 Hz, 2H), 8.06 (s, 4H),
8.12 (d, J = 15.6 Hz, 2H), 8.32 – 8.38 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ
115.9 (2 x CH), 116.1 (2 x CH), 124.4 (2 x CH), 129.5 (4 x CH), 131.7 (2 x CH),
131.7 (2 x CH), 137.3 (2 x C), 143.3 (2 x CH), 164.3 (2 x C), 166.5 (2 x C),
188.9 (2 x C); HRMS (ESI, m/z): calculated for C24H16F2O2 [M]+ 374.1118 found
374.3896.
3.9.18.
(2E,2'E)-3,3'-(1,4-phenylene)bis(1-(3,4-dimethoxyphenyl)prop-2-en-1-
one) (3q) Yield: 68% (311.8 mg); yellow solid; mp 218-220) °C; IR-FT (KBr, cm-
1): 3085, 3017, 2962, 2934, 2834, 1651, 1599, 1583, 1518, 1418,1332, 1149,
1024, 985, 815; 1H NMR (400 MHz, CDCl3) δ 3.98 (s, 12H), 6.94 (d, J = 8.4 Hz,
2H), 7.60 (d, J = 15.6 Hz, 2H), 7.64 (d, J = 1.7 Hz, 2H), 7.69 (s, 5H), 7.71 (d, J =
13
1.7 Hz, 1H), 7.81 (d, J = 15.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 56.0 (2 x
CH3), 56.1 (2 x CH3), 109.9 (2 x CH), 110.7 (2 x CH), 122.5 (2 x CH), 123.0 (2 x
20

CH), 128.8 (4 x CH), 131.1 (2 x C), 136.8 (2 x C), 142.7 (2 x CH), 149.3 (2 x C),
153.4 (2 x C), 188.2 (2 x C); HRMS (ESI, m/z): calculated for C28H26O6 [M]+
458.1729 found 458.1720.
3.9.19. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-phenylprop-2-en-1-one) (3r) Yield:
73% (247.0mg); yellow solid; mp 191-193) °C; IR-FT (KBr, cm-1): 3054, 3038,
1
1659, 1646, 1416, 1335, 1222, 1037, 1024, 979, 828, 770; H NMR (400 MHz,
CDCl3) δ 7.52 (dd, J = 10.3, 4.6 Hz, 4H), 7.55 –7.63 (m, 4H), 7.69 (s, 4H), 7.81
13
(d, J = 15.7 Hz, 2H), 8.04 (d, J = 7.1 Hz, 4H); C NMR (100 MHz, CDCl3) δ
123.0 (2 x CH), 128.5 (4 x CH), 128.6 (4 x CH), 128.9 (4 x CH), 132.9 (2 x CH),
136.8 (2 x C), 138.0 (2 x C), 143.5 (2 x CH), 190.2 (2 x C); HRMS (ESI, m/z):
calculated for C24H18O2 [M]+ 338.1307 found 338.4094.
3.9.20. (2E,2'E)-3,3'-(1,4-phenylene)bis(1-(p-tolyl)prop-2-en-1-one) (3s) Yield:
82% (300.5mg); yellow solid; mp 234-236 °C; IR-FT (KBr, cm-1): 3033, 2921,
1659, 1612, 1603, 1421, 1337, 1230, 1181, 1037, 985, 812; 1H NMR (400 MHz,
DMSO-d6) δ 2.42 (s, 6H), 7.40 (d, J = 7.4 Hz, 4H), 7.76 (d, J = 15.6 Hz, 2H),
7.98 (s, 4H), 8.03 (d, J = 15.6 Hz, 2H), 8.10 (d, J = 7.6 Hz, 4H); 13C NMR (100
MHz, DMSO-d6) δ 20.8 (2 x CH3), 124.1 (2 x C), 128.3 (4 x CH), 128.8 (4 x CH),
129.1 (4 x CH), 135.5 (2 x C), 142.2 (4 x CH), 143.1 (2 x C), 189.2 (2 x C);
HRMS (ESI, m/z): calculated for C26H22O2 [M]+ 366.1620 found 366.1649.
3.9.21.
(E)-N-(4-(3-(benzo[d][1,3]dioxol-5-yl)acryloyl)phenyl)quinoline-3-
carboxamide (3t) Yield 92% (388.6mg); yellow solid; m.p. = 237–239 °C; IR
(KBr, cm-1): 3334, 2917, 1670, 1595, 1528, 1499, 1534, 1245, 1025, 920, 819,
1
755; H NMR (400 MHz, DMSO-d6): 6.10 (s, 2H),6.99 (d, J = 8.0 Hz,1H), 7.33
(d, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.69–7.72 (m, 2H), 7.83 (s, 1H), 7.88–7.90 (m,
1H), 8.01 (d, J = 8.0 Hz, 2H), 8.13 (dd, J = 8.0, 16.8 Hz, 2H), 8.21 (d, J = 8.0
Hz, 2H), 9.00 (s, 1H), 9.39 (s, 1H), 10.93 (bs, 1H); 13C NMR (100 MHz, DMSO-
d6) δC: 102.0 (CH2), 107.3 (CH), 108.9 (CH), 120.7 (CH), 121.7 (CH), 125.9
(CH), 127.3 (C), 127.4 (CH), 129.1 (2 x CH), 129.5 (2 x CH), 130.0 (2 x CH),
130.9 (CH), 135.9 (CH), 143.0 (CH), 148.5 (C), 148.6 (C), 149.7 (C), 150.8 (C),
165.9 (C), 187.4 (C); HRMS (ESI, m/z): calculated for C26H19N2O4 [M+H]+
423.1345; found 423.1342.
3.9.22. (E)-N-(4-(3-(pyridin-3-yl)acryloyl)phenyl)quinoline-3-carboxamide (3u)
Yield 77% (292.1mg); yellow solid, mp 270-272 °C; IR (KBr, cm-1):3307, 3040,
1673, 1659, 1600, 1532, 1473, 1338, 1225, 1175, 1028, 784, 677, 576; 1H
NMR (400 MHz, DMSO-d6): δH = 7.50 (m, 1H), 7.75 (m, 2H), 7.91 (t, J = 7.6 Hz,
1H), 8.04 (d, J = 8.3 Hz, 2H), 8.14 (m, 3H), 8.26 (d, J = 8.3 Hz, 2H), 8.36 (t, J =
7.8 Hz, 1H), 8.62 (m, 1H), 9.01 (s, 1H), 9.03 (s, 1H), 9.38 (s, 1H), 10.95 (bs,
1H); 13C NMR (100 MHz, DMSO-d6) δC: 120.1 (2 x CH), 124.3 (CH), 124.4
(CH), 126.8 (C), 127.7 (C), 128.0 (CH), 129.3 (CH), 129.7 (CH), 130.4 (2 x CH),
131.1 (C), 132.0 (CH), 133.1 (C), 135.5 (CH), 136.8 (CH), 140.5 (CH), 144.1
21

(C), 149.1 (C), 149.5 (CH), 150.8 (CH), 151.4 (CH), 165.1 (C), 187.8 (C); HRMS
(ESI, m/z): calculated for C24H17N3O2 [M]+ 379.1321 found 379.1320.
3.9.23. N-(4-cinnamoylphenyl)quinoline-3-carboxamide (3v) Yield: 53%
(200.6mg); yellow solid, mp 235-237 °C; IR (KBr, cm-1): 3302, 3039, 1673,
1589, 1519, 1326, 1227, 1166, 1036, 972, 764; 1H NMR (400 MHz, DMSO-d6):
δH = 7.46 (m, 3H), 7.74 (m, 2H), 7.90 (m, 3H), 7.98 (d, J = 15.6 Hz, 1H), 8.04
(d, J = 8.0 Hz, 2H), 8.14 (dd, J = 8.0, 15.9 Hz, 1H), 8.24 (d, J = 8.0 Hz, 2H), 9.0
(s, 1H), 9.38 (s, 1H), 10.93 (bs, 1H); HRMS (ESI, m/z): calculated for
C25H18N2O2 [M]+ 378.1368 found 378.1369.
3.9.24.
(E)-N-(4-(3-(2-chloroquinolin-3-yl)acryloyl)phenyl)quinoline-3-
carboxamide (3w) Yield 68% (315.5 mg); yellow solid, mp 258-260 °C; IR (KBr,
1
cm-1): 3336, 3057, 1656, 1600, 1521, 1405, 1318, 1220, 1178, 789, 593; H
NMR (400 MHz, DMSO-d6): δH = 7.66 (t, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz,
1H), 7.83 (t, J = 8.0 Hz, 2H), 7.89 (m, 2H), 7.99 (d, J = 8.0 Hz, 1H), 8.05 (d, J =
8.0 Hz, 1H), 8.07 (m, 4H), 8.16 (d, J = 8.0 Hz, 2H), 8.22 (d, J = 15.7 Hz, 1H),
8.94 (s, 1H), 9.28 (s, 1H), 9.51 (s, 1H); HRMS (ESI, m/z): calculated for
C28H18ClN3O2 [M]+ 463.1088 found 463.1083.
3.9.25.
(E)-N-(4-(3-(1-methyl-1H-imidazol-2-yl)acryloyl)phenyl)quinoline-3-
carboxamide (3x) Yield 71% (271.5mg), yellow solid, mp 244-246 °C; IR (KBr,
cm-1):3264, 2923, 1676, 1653, 1569, 1412, 1334, 1265, 1025, 918, 793, 755;
1H-NMR (400 MHz, DMSO-d6): 6.11 (s, 1H), 6.56 (d, J = 8.6 Hz, 1H), 6.74 (s,
1H), 7.57 (m, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.72 (m, 1H), 7.82 (d, J = 8.0 Hz,
1H), 8.00 (d, J = 8.0 Hz, 2H), 8.35 (bs, 3H), 8.65 (s, 1H), 9.34 (s, 1H); HRMS
(ESI, m/z): calculated for C23H18N4O2 [M]+ 382.1430 found 382.1436.
4. Conclusion
In conclusion, a set of chalcone and bis-chalcone derivatives have been
synthesized under ultrasonication conditions via Claisen-Schmidt condensation
with KOH in ethanol at room temperature. The structures of the compounds
were characterized on the basis of NMR, IR, Single-crystal XRD, and MS.
These products were evaluated as potential AChE inhibitors. Most of these
compounds showed selectivity against AChE. Compounds 3p, 3q, 3u, and 3v
were significantly in vitro active with IC50 values of 40.48, 27.41, 7.50, and 12.58
μM, respectively. The kinetic study of compound 3u showed mixed-type
inhibition against AChE. On the other hand, compounds 3o and 3p showed a
moderated antioxidant capacity compared with the ascorbic acid used as
reference. The modelling study of compounds 3p, 3q, 3u, and 3v showed the
same interaction of Donepezil with the residues Trp84, Phe330, and Trp279.
The pharmacokinetic profile of these compounds was investigated using a
computational method, showing an interesting potential in the design of new
compounds with pharmaceutical application.
22

Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This research was supported by a FONDECYT project number 1150712, PIEI QUIMBIO,
project, Utalca. EP and MG give thanks to the CONICYT scholarship No. 63140046. LPP is
grateful for the CONICYT scholarship associated with the folio N° 63130155.
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HIGHLIGHTS

Chalcone, heterochalcone and bis-chalcone was synthetized with Ultrasound-
assisted.


Compound 3u was active as Acetylcholinesterase inhibitors.
Molecular Docking show interactions with active site.
27