Synthesis, in Silico and in vitro evaluation of some flavone derivatives for Acetylcholinesterase and BACE-1 inhibitory activity

Article abstract of DOI:10.3390/molecules25184064

Acetylcholinesterase (AChE) and β-secretase (BACE-1) have become attractive therapeutic targets for Alzheimer’s disease (AD). Flavones are flavonoid derivatives with various bioactive effects, including AChE and BACE-1 inhibition. In the present work, a series of 14 flavone derivatives was synthesized in relatively high yields (35–85%). Six of the synthetic flavones (B4, B5, B6, B8, D6 and D7) had completely new structures. The AChE and BACE-1 inhibitory activities were tested, giving pIC₅₀ 3.47–4.59 (AChE) and 4.15–5.80 (BACE-1). Three compounds (B3, D5 and D6) exhibited the highest biological effects on both AChE and BACE-1. A molecular docking investigation was conducted to explain the experimental results. These molecules could be employed for further studies to discover new structures with dual action on both AChE and BACE-1 that could serve as novel therapies for AD.

Full text of DOI:10.3390/molecules25184064

molecules
Article
Synthesis, In Silico and In Vitro Evaluation of Some
Flavone Derivatives for Acetylcholinesterase and
BACE-1 Inhibitory Activity
Thai-Son Tran 1,2, Thanh-Dao Tran 1, The-Huan Tran 2, Thanh-Tan Mai 1 , Ngoc-Le Nguyen 3,
Khac-Minh Thai 1,* and Minh-Tri Le 1,4,
*
1
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi
Minh City, Ho Chi Minh City 700000, Vietnam; ttson@huemed-univ.edu.vn (T.-S.T.);
tranthanhdao@uphcm.edu.vn (T.-D.T.); mthtan@ump.edu.vn (T.-T.M.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, College of Medicine and Pharmacy,
Hue University, Hue City 530000, Vietnam; tthuan@hueuni.edu.vn
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, South Can Tho University,
Can Tho City 640000, Vietnam; dsngocle1984@gmail.com
School of Medicine, Vietnam National University Ho Chi Minh City, Ho Chi Minh City 700000, Vietnam
Correspondence: thaikhacminh@gmail.com or thaikhacminh@uphcm.edu.vn (K.-M.T.);
leminhtri@ump.edu.vn (M.-T.L.); Tel.: +84-28-3855-2225 or +84-909-680-385 (K-M.T.);
+84-903-718-190 (M.-T.L.); Fax: +84-28-3822-5435 (K.-M.T.)
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Orazio Nicolotti
Received: 17 August 2020; Accepted: 4 September 2020; Published: 5 September 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Acetylcholinesterase (AChE) and β-secretase (BACE-1) have become attractive therapeutic
targets for Alzheimer’s disease (AD). Flavones are flavonoid derivatives with various bioactive effects,
including AChE and BACE-1 inhibition. In the present work, a series of 14 flavone derivatives was
synthesized in relatively high yields (35–85%). Six of the synthetic flavones (B4
and D7) had completely new structures. The AChE and BACE-1 inhibitory activities were tested,
giving pIC50 3.47–4.59 (AChE) and 4.15–5.80 (BACE-1). Three compounds (B3 D5 and D6) exhibited
, B5, B6, B8, D6
,
the highest biological effects on both AChE and BACE-1. A molecular docking investigation was
conducted to explain the experimental results. These molecules could be employed for further studies
to discover new structures with dual action on both AChE and BACE-1 that could serve as novel
therapies for AD.
Keywords: in silico; in vitro; QSAR; docking; flavone; acetylcholinesterase; β-secretase
1. Introduction
Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder with the defects in nervous
system and cognitive function [
complex processes; involving the production of senile plaques, neurofibrillary tangles, synapse loss
and neuroinflammation; leading to the progressive cognitive decline and memory loss [ ]. At the
present time, about 50 millions of people worldwide suffer from the disease, which creates a heavy
1]. In this ailment, there is an impairment of the cerebral cortex with
2,3
burden on the health care systems of many countries [
is an urgent task.
4–6]. Therefore, discovery of novel drugs for AD
The exact pathogenesis of AD is currently not fully know and this disease might be referred to as a
neurological disorder caused by multiple factors, such as: (i) low concentrations of acetylcholine (ACh)
in synaptic clefts; (ii) the accumulation of intracellular neurofibrillary tangles with hyperphosphorylated
Tau protein and extracellular amyloid plaques; (iii) oxidative stress; (iv) the homeostasis dysregulation
of biometals [2,7–9]. The multifactorial nature of AD has transformed the paradigm of AD drug
Molecules 2020, 25, 4064; doi:10.3390/molecules25184064
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4064
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development from single to multitarget-directed ligands [10]. Acetylcholinesterase (AChE) and
-secretase (BACE-1) are the two drug targets that have attracted much attention among others.
β
They are two critical enzymes in Alzheimer’s pathogenesis, responsible for the defects in the cholinergic
signaling pathway [11] and the formation of β-amyloid plaques [12].
During the last few years, flavonoids, including flavones, have attracted much interest because of
their diverse bioactivities [13
–
24]. Recent studies have indicated the abilities of flavones in inhibiting
32]. Studies on the bioactivity of flavone
many enzymes [25 29], especially AChE [30] and BACE-1 [31
–
,
derivatives against AChE and BACE-1 are thus promising for the discovery of novel therapeutic agents
for AD.
In this study, a series of flavone derivatives was synthesized and evaluated for AChE and BACE-1
inhibitory activities with the aim of discovering new small-molecule inhibitors with dual action on
both enzymes. The produced compounds could also be used in an external validation set to assess
the two-dimensional quantitative structure–activity relationship (2D-QSAR) models developed for
inhibitors of these enzymes. A molecular docking investigation was conducted to analyze the molecular
binding abilities of these substances with the enzymes to obtain more detailed information on these
influences on the bioactivity.
2. Results
2.1. Chemistry
Etherification (with acetone as solvent and K2CO3 as catalyst) and esterification (in pyridine)
reactions were conducted on baicalein (B1) to produce the derivatives B2
yields (48–79%, Scheme 1). A hydrolysis process was carried out with diosmin to make diosmetin
D1, yield 85%), from which the other compounds D2 D7 were synthesized via etherification and
–B8 in relatively high
(
–
esterification reactions (Scheme 2) with yields of 35–81%. The structures of the synthetic substances were
elucidated by ultraviolet (UV), infrared (IR), high-resolution mass spectrometry (HRMS), proton nuclear
magnetic resonance (1H-NMR), and carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy
as described in the Materials and Methods section and six of them (B4, B5, B6, B8, D6 and D7) were
identified as completely new compounds [33].
Scheme 1. Synthesis of flavone derivatives from baicalein.

Molecules 2020, 25, 4064
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OH
HO
HO
Me
OH
O
OH
OMe
O
OMe
HO
O
O
H2SO4 - CH3COOH - H2O
O
O
O
O
HO
HO
OH
OH
OH
Diosmin
OMe
Diosmetin (D1)
(Yield: 85%)
OMe
OEt
OMe
OMe
OMe
EtO
O
MeO
O
O
MeO
O
D2
(Yield: 56%)
OH
OEt O
OMe O
(Yield: 35%)
D6
D5
(Yield: 42%)
OH
OCH2CH=CH2
OCH3
OEt
OMe
OMe
Et2SO4
CH2=CHCH2Br
K2CO3, Aceton
HO
O
O
H2C=HCH2CO
O
EtO
O
K2CO3, Aceton
OH
OH
O
OH
O
Diosmetin
(Yield: 81%)
D4
D3 (Yield: 57%)
OBz
OMe
BzO
O
O
OH
D7 (Yield: 68%)
Scheme 2. Synthesis of diosmetin and its derivatives.
2.2. Enzyme Inhibition and 2D-QSAR Analysis
The results of the in vitro assays on AChE and BACE-1 inhibition are indicated in Table 1. From the
biological evaluation, the inhibitory activities of synthesized substances against the two enzymes
gave pIC50
values of 3.47–4.59 (IC50 340.09–25.51
Among the synthetic molecules, B3 D5 and D6 exhibited the highest bioactivities against both enzymes.
(
−
logIC50, negative common logarithm of the half maximal inhibitory concentration)
µ
M) for AChE and 4.15–5.80 (IC50 70.79–1.58 M) for BACE-1.
µ
,
Galantamine and quercetin were used as reference substances. Galantamine is a well-known AChE
inhibitor used for the treatment of cognitive decline in mild to moderate AD. Quercetin is a flavonoid
reported to have inhibitory activity against BACE-1. These two compounds have been used as
references in many studies on AChE and BACE-1 inhibition. The results showed that the biological
activities of the positive controls in this study are comparative to previously published values [34–36].
In this study, the synthesized substances were used in an external validation set for the 2D-QSAR
models published in a previous work [37] (Table 2). By using these 2D-QSAR models, the predicted
values of pIC50 for AChE and BACE-1 of the synthesized flavone derivatives were calculated and are
indicated in Table 1. Correlations between observed and predicted activities of synthesized flavones
molecules and positive controls againts AChE and BACE-1 were found (Figure 1) with good values of
the squared correlation coefficient (R2) and root-mean-square error (RMSE) (R2 = 0.83 and RMSE = 0.44
for AChE; and R2 = 0.82 and RMSE = 0.40 for BACE-1). These results indicate that the reported models
were statistically significant and sufficiently robust to predict the biological activities of new ligands.
Selected molecular descriptors used for building 2D-QSAR models are described in detail in Table S1,
and their values used in the prediction of pIC50 of the synthesized flavone derivatives are provided in
Tables S2 and S3.

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Table 1. Acetylcholinesterase (AChE) and β-secretase (BACE-1) inhibitory activities and docking scores
of the synthesized flavone derivatives.
AChE
BACE-1
Docking
Score
Docking
Score
Compound
IC50
(µM)
Obs.
pIC50
Pre.
pIC50
IC50
(µM)
Obs.
pIC50
Pre.
pIC50 *
*
(kJ·mol−1) **
(kJ·mol−1) **
B1
37.15
4.43
4.66
−27.06
69.18
4.16
3.66
−17.64
(Baicalein)
B2
B3
B4
B5
B6
B7
B8
25.51
30.01
51.81
191.47
80.32
51.29
66.24
4.59
4.52
4.29
3.72
4.10
4.29
4.18
4.59
4.55
4.44
4.32
4.32
4.52
4.56
−17.67
−17.55
−10.36
−10.49
−15.71
−17.91
−19.61
43.65
3.98
4.36
5.40
4.15
4.77
4.90
4.50
4.65
4.82
5.11
4.35
4.60
5.18
4.73
4.75
−16.37
−12.35
−10.84
−8.29
70.79
16.98
12.59
31.62
22.39
−17.35
−12.95
−10.75
D1
147.91
3.83
4.50
−22.86
43.65
4.36
3.55
−19.32
(Diosmetin)
D2
D3
D4
D5
D6
D7
87.10
128.82
151.36
73.82
75.91
340.09
4.06
3.89
3.82
4.13
4.12
3.47
5.88
-
4.42
4.37
4.26
4.39
4.31
4.35
5.14
-
−21.05
−20.90
−15.22
−21.80
−20.39
−25.35
−33.28
-
2.14
3.16
3.60
1.66
1.58
2.86
-
5.67
5.50
5.44
5.78
5.80
5.54
-
5.83
6.04
5.24
6.20
6.34
6.07
-
−19.33
−17.18
–13.31
−13.46
−12.02
−22.07
-
Galanthamine 1.31
Umibecestat
Quercetin
-
-
0.01
9.55
7.96 [38]
5.02
7.67
5.24
−47.04
-
-
-
−22.55
R2 = 0.83, RMSE =
R2 = 0.82,
RMSE = 0.40
0.44
Obs.: Observed, Pre.: Predicted, R2 (squared correlation coefficient), RMSE (root-mean-square error), IC50: the half
maximal inhibitory concentration, pIC50: -logIC50. * pIC50 values predicted using 2D-QSAR models in the previous
published work [37]. ** Molecular docking results were reported using the co-crystallized 1DX6 and 6EQM for
AChE and BACE-1, respectively. B3, D5 and D6 (highlighted in bold) exhibited the highest bioactivities against
both enzymes.
Table 2. Two-dimensional quantitative structure–activity relationship models (2D-QSAR) for AChE
and BACE-1 inhibitors reported in the previous published work [37].
AChE *
pIC50
=
−
0.928 + (2.348
×
BCUT_SLOGP_3)
−
(0.150
×
reactive)
−
(0.002
×
SlogP_VSA2)
−
(0.004
×
PEOE_VSA+1)
− (0.004 × SMR_VSA2) − (0.005 × PEOE_VSA–3)
Internal Validation External Validation
R2
0.70
Q2
0.57
R2
0.78
R2
0.78
rm2
0.65
∆r2m
0.11
rm2
0.69
N
50
RMSE
0.18
RMSELOO
0.22
N
22
RMSE
0.16
CCC
0.88
LOO
(PRED)
BACE-1 *
pIC50 = 1.268 + (6.370
×
BCUT_PEOE_1) + (3.305
×
a_ICM) + (0.870
×
petitjean) + (0.157
×
a_Nn) + (0.085
×
rings) +
(0.022 × PEOE_VSA–6) + (0.006 × PEOE_VSA–0) + (0.009 × SlogP_VSA3) + (0.009 × SlogP_VSA5) –
(0.478 × chiral_u) − (0.260 × logS)
Internal Validation
External Validation
R2
0.80
Q2
0.77
R2
0.83
R2
0.81
rm2
0.79
∆r2m
0.05
rm2
0.76
N
150
RMSE
0.37
RMSELOO
0.40
N
65
RMSE
0.41
CCC
0.91
LOO
(PRED)
N: number of compounds, RMSELOO (cross-validated root-mean-square error), Q2
(cross-validated
LOO
squared correlation coefficient), R2
(predicted R-squared), CCC (concordance correlation coefficient), and
(PRED)
rm2
, ,
r2m ∆r2m (validation metrics suggested by Roy et al. [39]). * Selected molecular descriptors used for building
2D-QSAR models are described in detail in Table S1.

Molecules 2020, 25, 4064
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(A)
(B)
Figure 1. The linear regression between observed and predicted activity of synthesized flavone
derivatives and positive control against (A) acetylcholinesterase (AChE) and (B) β-secretase (BACE-1).
2.3. Molecular Docking
The results of the molecular docking study are reported in Table 1, Figures 2 and 3, and in
the Supporting Information (Tables S4–S6). The interactions of three compounds with the highest
pIC50 (B3, D5 and D6) on both enzyme AChE and BACE-1 are indicated in Figure 3. In this
study, the co-crystallized complexes employed for AChE was 1DX6 (resolution 2.30 Å) and for
BACE-1 was 6EQM (resolution 1.35 Å). These were the complexes with high resolutions and the
co-crystallized ligands were the drugs used in clinical (1DX6: galantamine) or in clinical development
(6EQM: umibecestat). With this selection, the probability of docked compounds to be reached further
optimization would be high [40]. The results indicated that all studied substances were successfully
docked into the binding pockets of AChE and BACE-1 with the docking scores of (
−
10.36)–( 27.06)
−
kJ 8.29)–( 22.07) kJ
·
mol−1 (AChE) and ( mol−1 (BACE-1). Linear correlations between docking scores
−
−
·
and observed values of pIC50 on AChE and BACE-1 of synthesized flavone derivatives were revealed
(Figure 2). The ligand interactions between chemical molecules and their targets will be analysed more
detailed in the Discussion section.
(A)
(B)
)acetylcholinesterase
Figure 2. Thelinearregressionbetweenobservedactivityanddockingscoreagainst(
A
(AChE) (values from B2 B3, B5–8, D2, D4–6 and galantamine) and ( -secretase (BACE-1) (values
,
B) β
from B2–8, D2–4, D7, umibecestat and quercetin).

Molecules 2020, 25, 4064
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Figure 3. Interactions with AChE (left) and BACE-1 (right) of flavone derivatives B3 (A,B), D5 (C,D)
and D6 (E,F).
3. Discussion
In this study, a series of fourteen flavone molecules was synthesized in relatively high yields
(35–85%). The structures of these substances were elucidated by UV, IR, HRMS, 1H-NMR and 13C-NMR
spectroscopy, and six of them (B4
these compounds were examined with pIC50 3.47–4.59 (AChE) and 4.15–5.80 (BACE-1). The synthetic
molecules could be divided into two groups of derivatives, B2 B8 (from baicalein, B1) and D2 D7
(from diosmetin, D1). B2 B3 D2 D3 D5 and D6 had better AChE inhibiting effects than the respective
, B5, B6, B8, D6 and D7) were completely new. The bioactivities of
–
–
,
,
,
,
parent compounds. Compared to baicalein and diosmetin, the synthesized derivatives of these two
substances (except for B4) had the improved bioactivities against BACE-1. The results indicated that
the derivatives of diosmetin had the higher inhibiting effects on BACE-1 than those of baicalein. B3, D5
and D6 exhibited the highest biological effects on both enzymes among the synthesized compounds.
A molecular docking study showed that all the synthesized flavone derivatives were successfully
docked into the binding pocket of AChE (1DX6). Docking scores and pIC50 values in 12 among studied
substances (B2, B3, B5–8, D2,
D4–6 and galantamine) were quite correlated with R2 = 0.72 (Figure 2).

Molecules 2020, 25, 4064
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In addition, the differences in the observed pIC50 values could be partly explained by the analysis of
the interactions between flavone molecules with AChE. These interactions include: (i) arene–arene
interactions with Trp84, Trp279, Tyr334; (ii) arene–cation interaction with Trp84; (iii) hydrogen bonding
with Asp72, Glu199. These are residues of the active site-gorge of the enzyme where Trp84 and
Tyr334 are exposed on the surface, particularly Asp72 on the top, Glu199 on the bottom, and Trp279
is on the entrance of the active-site gorge [41]. Compared to galantamine, the flavone derivatives
were all lower in bioactivity. This could be explained by the fact that galantamine could create an
arene–cation interaction with Phe330, a strong hydrogen bond to Glu199 (score: 36%, length: 1.85 Å),
and a hydrogen bond (score: 22%, length: 3.07 Å) to Ser200 (a residue in catalytic triad). B1 and B2
had the same mode of interaction with the enzyme but the docking score of B1 was higher than that of
B2. This could be due to the hydrogen bonds made by B1 with Tyr130 (score: 68%, length: 2.61 Å) and
Gly117 (score: 20%, length: 1.82 Å) were stronger than those of B2 with these two residues (Tyr130:
score 8%, length 3.14 Å; and Gly117: score 1%, length: 3.52 Å). In addition, the interaction between
B1 and Phe330 was an arene–arene interaction (proposed to consist of van der Waals, hydrophobic,
and electrostatic forces) while this interaction seen with B2 was a van der Waals force in general.
However, the AChE inhibiting effect of B2 was higher than that of B1. This could be due to B1 lacked
the van der Waals interactions with two important residues (seen in the B2 case), including His440 of
the catalytic triad and Asp72 of the active site-gorge. The docking results also indicated that B3 could
form only a weak but important hydrogen bond with Ser200 (score: 6%, length: 3.65 Å) and van der
Waals interactions with His440. This could explain why B3 had a stronger biological activity than B1
on AChE, although the docking score of B3 is lower. In the series of diosmetin derivatives, a hydrogen
bond between the ligand molecule with either Gly119 or Ser200 could be needed for the improved
activities of these substances than the parent compound (D1).
Molecular docking results on BACE-1 expressed that all the synthesized flavone derivatives were
successfully docked into the binding pocket of this enzyme (6EQM). The interactions with catalytic
dyad residues (Asp32 and Asp228) could be necessary for high inhibiting effect against BACE-1.
The interaction models in Table S6 revealed that umibecestat could form strong hydrogen bonds with
the catalytic dyad (Asp32: score 59%, length 1.82 Å; score 68%, length 1.75 Å, and Asp228: score
13%, length 2.48 Å; score 30%, length 1.96 Å) in addition to a hydrogen bond with Gly230 (score
21%, length 1.91 Å). This mode of interaction could explain the higher bioactivity against BACE-1
of umibecestat (observed pIC50 7.96). None of the studied flavone derivatives form any hydrogen
bonds with the catalytic dyad residues. They interacted only with these amino acids through van der
Waals forces. B3, D2–D7 and quercetin (pIC50 5.02–5.80) were exposed to Asp32, Asp228, and Gly230
in the radius of 4.5–5.0 Å. Two substances with the lowest BACE-1 inhibitory activities, B1 (pIC50
4.16) and B4 (pIC50 4.15), did not produce any interactions with Asp32, Asp228, and Gly230 (B4) or
formed only a weak interaction with Gly230 at a distance >5.5 Å (B1). The remaining substances
(B2, B5–8 and D1) had pIC50 values of 4.36–4.90. These molecules could form medium van der Waals
interactions with Asp32, Asp228, and Gly230 (within a distance of 5.0–5.5 Å) or interacted only with
one residue of the catalytic dyad. D5 (pIC50 5.78) and D6 (pIC50 5.80) formed strong hydrogen bonds
with Thr232 (D5: score 89%, length 2.69 Å; D6: score 81%, length 2.49 Å; score 60%, length 2.49 Å).
These interactions could explain for the higher activities against BACE-1 of those two compounds. In
this study, a correlation between docking score and observed pIC50 for BACE-1 was found for most of
the studied substances (B2–8, D2–4,
D7, umibecestat and quercetin) with an R2 value of 0.77 (Figure 2).
However, this correlation (as in the case of AChE above) was not a conclusion but an interesting result
from the present work. In general, molecular docking (with a specified scoring function) is better in
pose (spatial orientation of a ligand in the active site of an enzyme) than affinity (free binding energy)
prediction. Free binding energy is related to the inhibition constant (Ki). The more negative the free
binding energy, the lower the Ki value. While Ki is a constant value for a given compound with an
enzyme, an IC50 (or pIC50) is a relative value, whose magnitude depends upon the concentration of

Molecules 2020, 25, 4064
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substrate used in the assay. The relation between Ki and IC50 could be obtained only if the mode of
inhibition and the concentration of substrate used in the assay are reported [42–44].
4. Materials and Methods
4.1. Materials and Instruments
All chemicals were obtained from commercial suppliers and used without further purification.
Melting points were determined on open capillary tubes and are uncorrected (using a Stuart apparatus
SMP20, Cole-Parmer, Staffordshire, UK). UV, IR, HRMS, and 1H-NMR and 13C-NMR spectra were
recorded on V-730 UV-Vis (Jasco International Co. Ltd., Tokyo, Japan), IR Prestige-21 (Shimadzu, Kyoto,
Japan), 6200 Series TOF and 6500 Series Q-TOF LC/MS (Agilent, Santa Clara, CA, USA) and AV500
(500 MHz, 125 MHz) (Bruker, Billerica, MA, USA) spectrometers, respectively. Chemical shifts were
reported in parts per million (ppm) downfield relative to tetramethylsilane as an internal standard.
Peak splitting patterns were abbreviated as m (multiplet), s (singlet), bs (broad singlet), d (doublet), bd
(broad doublet), t (triplet) and dd (doublet of doublets), respectively. All computational processes were
performed on a computer system equipped with an Intel® CoreTM i&-7700 CPU @ 3.60 Hz processor,
16.0 GB of RAM, an NVIDIA GeForce GT 1030 2GB Visual Graphics Card and the 64 bit Windows 10
operating system (Microsoft, Redmond, WA, USA).
4.2. Chemistry
A series of 14 flavone derivatives were synthesized as indicated in Schemes 1 and 2, as follows:
4.2.1. Synthesis of B2–B6
Baicalein (B1) was dissolved in anhydrous acetone. K2CO3, and a dialkyl sulfate or an alkyl
bromide were then added. The reaction mixture was heated at 40–45 ◦C and monitored by thin layer
chromatography eluting with the appropriate solvents. At the end of the reaction, the mixture was
filtered to remove insoluble solids. The obtained solution was evaporated under reduced pressure and
the crude product was purified by column chromatography with the appropriate solvent systems.
5-Hydroxy-6,7-dimethoxyflavone (B2). Yield: 65%. MP: 160–161 ◦C. UV (
λ
nm, MeOH): 210, 272, 314.
max
1
IR (
(DMSO-d6)
3H, Ar- ), 7.02 (s, 1H, Ar-
ν
cm−1, KBr): 3453, 3064, 2941, 1662, 1616, 1456, 1128. MS: [M + H]+ m/z = 299.0917. H-NMR
δ
(ppm): 12.76 (s, 1H, O
), 6.97 (s, 1H, Ar-
δ (ppm): 182.3, 163.4, 158.8, 152.7, 151.0, 132.1, 132.0, 130.6, 129.1, 126.3, 105.3, 104.9, 91.7,
H
), 8.09 (dd, J1 = 1.5 Hz, J2 = 8 Hz, 2H, Ar-
H), 7.63–7.57 (m,
H
H
H
), 3.94 (s, 3H, OCH3), 3.75 (s, 3H, OCH3). 13C-NMR
(DMSO-d6)
60.0, 56.4.
6,7-Diethoxy-5-hydroxyflavone (B3). Yield: 50%. MP: 140–141 ◦C. UV (
IR (
(DMSO-d6)
Ar- ), 7.00 (s, 1H, Ar-
(t, J = 7 Hz, 3H, CH3), 1.26 (t, J = 7 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
152.6, 152.2, 132.0, 130.9, 130.6, 129.0, 126.3, 105.2, 104.9, 92.1, 67.8, 64.6, 15.3, 14.3.
λ
max nm, MeOH): 210, 272, 315.
1
ν
cm−1, KBr): 3447, 3071, 2982, 1661, 1614, 1454, 1125. MS: [M + H]+ m/z = 327.1230. H-NMR
δ
(ppm): 12.72 (s, 1H, O
H
), 8.09 (dd, J1 = 1.5 Hz, J2 = 8 Hz, 2H, Ar-
), 4.19 (q, J = 7 Hz, 2H, C 2), 3.99 (q, J = 7 Hz, 2H, C
(ppm): 182.3, 163.3, 158.3,
H
), 7.64–7.56 (m, 3H,
H
H
), 6.94 (s, 1H, Ar-
H
H
H
2), 1.40
δ
6,7-Diallyloxy-5-hydroxyflavone (B4). Yield: 69%. MP: 132–133 ◦C. UV (
λ
nm, MeOH): 214, 273, 315.
max
1
IR (
(DMSO-d6)
Ar- ), 7.03 (s, 1H, Ar-
Hz, 1H, =CHtrans), 5.33 (dd, J1 = 1.5 Hz, J2 = 17 Hz, 1H, =CHtrans), 5.33 (dd, J1 = 1.5 Hz, J2 = 10.5 Hz,
1H, =C cis), 5.18 (dd, J1 = 1.5 Hz, J2 = 10.5 Hz, 1H, =C cis), 4.75 (d, J = 5 Hz, 2H, CH2), 4.51 (d, J = 5.5
Hz, 2H, CH2). 13C-NMR (DMSO-d6)
(ppm): 182.3, 163.4, 157.8, 152.6, 152.3, 134.4, 132.7, 132.0, 130.6,
130.5, 129.1, 126.3, 117.8, 117.5, 105.3, 104.9, 92.5, 73.0, 69.2.
ν
cm−1, KBr): 3449, 3071, 2922, 1661, 1612, 1456, 1132. MS: [M + H]+ m/z = 351.1224. H-NMR
δ
(ppm): 12.79 (s, 1H, O
H
), 8.09 (dd, J1 = 1,5 Hz, J2 = 8.5 Hz, 2H, Ar-
H), 7.63–7.57 (m, 3H,
H
H
), 6.98 (s, 1H, Ar-
H
), 6.13–6.01 (m, 2H, -C =), 5.48 (dd, J1 = 1.5 Hz, J2 = 17.5
H
H
H
δ

Molecules 2020, 25, 4064
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5,6,7-Triallyloxyflavone (B5). Yield: 48%. MP: 117–118 ◦C. UV (
λ
max nm, MeOH): 215, 264, 307. IR (
ν
δ
cm−1, KBr): 3053, 2978, 1638, 1603, 1453, 1117. MS: [M + H]+ m/z = 391.1549. H-NMR (DMSO-d6)
1
(ppm): 8.05 (dd, J1 = 2 Hz, J2 = 8 Hz, 2H, Ar-
H), 7.59–7.55 (m, 3H, Ar-H), 7.23 (s, 1H, Ar-H), 6.79 (s, 1H,
Ar- ), 6.17–6.02 (m, 3H, -C =), 5.49 (dd, J1 = 1.5 Hz, J2 = 17 Hz, 1H, =CHtrans), 5.37 (dd, J1 = 1.5 Hz, J2
H
H
= 17 Hz, 1H, =CHtrans), 5.36 (dd, J1 = 1.5 Hz, J2 = 17 Hz, 1H, =CHtrans), 5.34 (dd, J1 = 1.5 Hz, J2 = 10.5
Hz, 1H, =CHcis), 5.20 (dd, J1 = 1.5 Hz, J2 = 10.5 Hz, 2H, =CHcis), 4.76 (d, J = 5 Hz, 2H, CH2), 4.54 (d, J
= 5.5 Hz, 2H, CH2), 4.50 (d, J = 5.5 Hz, 2H, CH2). 13C-NMR (DMSO-d6)
δ (ppm): 175.7, 160.1, 156.4,
153.8, 150.4, 138.8, 134.6, 134.2, 132.6, 131.4, 130.9, 129.0, 125.9, 117.8, 117.4, 117.2, 112.4, 107.5, 98.2, 75.0,
73.8, 69.2.
5,6,7-Tribenzyloxyflavone (B6). Yield: 79%. MP: 126–127 ◦C. UV (
λ
nm, MeOH): 264, 308. IR (
ν
cm−1
(ppm):
), 7.41–7.26 (m, 10H,
), 5.33 (s, 2H, CH2), 5.03 (s, 2H, CH2), 4.97 (s, 2H, CH2). 13C-NMR (DMSO-d6)
(ppm): 175.8, 160.2, 156.7, 154.0, 150.6, 139.2, 137.3, 137.1, 135.9, 131.5, 130.9, 129.0, 128.5, 128.3, 128.2,
,
max
KBr): 3032, 2940, 1643, 1601, 1450, 1117. MS: [M + H]+ m/z = 541.2011. H-NMR (DMSO-d6)
δ
1
8.08 (dd, J1 = 2 Hz, J2 = 8 Hz, 2H, Ar-
Ar- ), 6.85 (s, 1H, Ar-
H), 7.61–7.45 (m, 8H, Ar-H), 7.43 (s, 1H, Ar-H
H
H
δ
128.0, 128.0, 127.9, 127.9, 127.8, 126.0, 112.6, 107.6, 98.6, 75.6, 74.9, 70.6.
4.2.2. Synthesis of B7 and B8
Baicalein (B1) was dissolved in pyridine and an anhydride acid or an acid chloride was then
added. The reaction mixture was heated at 70–80 ◦C and monitored by thin layer chromatography with
an appropriate solvent system. When the reaction was complete, the mixture was poured into cold
water and stirred vigorously. The precipitate was then filtered and recrystallized from an appropriate
solvent to yield final product.
5,6,7-Triacetoxyflavone (B7). Yield: 58%. MP: 196–197 ◦C. UV (
λ
max nm, MeOH): 256, 294. IR (
ν
cm−1
8.08 (dd,
), 6.94 (s, 1H, Ar- ), 2.37
175.3, 167.8, 167.5, 167.4, 162.0,
,
KBr): 3065, 2936, 1784, 1645, 1452, 1175. MS: [M + H]+ m/z = 397.0916. H-NMR (DMSO-d6)
δ
1
J1 = 1.5 Hz, J2 = 8.5 Hz, 2H, Ar-
H
), 7.83 (s, 1H, Ar-
H
), 7.62–7.57 (m, 3H, Ar-
H
H
(s, 3H, C 3), 2.37 (s, 3H, C 3), 2.3 (s, 3H, C
H
H
H
3). 13C-NMR (DMSO-d6)
δ
153.6, 146.7, 141.2, 132.6, 132.0, 130.4, 129.1, 126.4, 115.1, 111.1, 107.5, 20.4, 20.3, 19.7.
5,6,7-Tripropionoxyflavone (B8). Yield: 75%. MP: 167–168 ◦C. UV (
λ
max nm, MeOH): 256, 295. IR (
ν
cm−1
,
KBr): 3063, 2984, 2943, 1782, 1643, 1454, 1111. MS: [M + H]+ m/z = 439.1384. H-NMR (DMSO-d6)
δ
1
8.08 (dd, J1 = 1.5 Hz, J2 = 8.5 Hz, 2H, Ar-
Ar- ), 2.68 (q, J = 7.5 Hz, 2H, CH2), 2.67 (q, J = 7.5 Hz, 2H, CH2), 2.66 (q, J = 7.5 Hz, 2H, CH2), 1.18
(t, J = 7.5 Hz, 3H, C 3), 1.16 (t, J = 7.5 Hz, 3H, C 3), 1.16 (t, J = 7.5 Hz, 3H, C
3). 13C-NMR (DMSO-d6)
H), 7.83 (s, 1H, Ar-H), 7.62–7.57 (m, 3H, Ar-H), 6.93 (s, 1H,
H
H
H
H
δ 175.3, 171.0, 170.8, 170.7, 162.0, 153.6, 146.8, 141.2, 132.5, 132.0, 130.4, 129.1, 126.3, 115.1, 111.0, 107.5,
26.7, 26.6, 26.2, 9.0, 8.7, 8.6.
4.2.3. Synthesis of Diosmetin (D1)
Diosmin was placed in a two-necked flask with a solution of sulfuric acid-acetic acid-water.
The reaction mixture was heated at 95–105 ◦C and monitored by thin layer chromatography with an
appropriate solvent system. At the end of the reaction, the mixture was poured into cold water and
stirred vigorously. The precipitate formed was then filtered and recrystallized from an appropriate
solvent to yield the final product.
5,7,3’-Trihydroxy-4’-methoxyflavone (D1). Yield: 85%. MP: 277–278 ◦C. UV (
345. IR (
cm−1, KBr): 3387, 3089, 2940, 2844, 1652, 1610, 1507, 1166. MS: [M + H]+ m/z = 301.0709.
1H-NMR (DMSO-d6)
12.92 (s, 1H, O ), 10.82 (s, 1H, O ), 9.43 (s, 1H, O ), 7.52 (dd, J1 = 8.5 Hz,
J2 = 2.5 Hz, 1H, Ar- ), 7.42 (d, J = 2.5 Hz, 1H, Ar- ), 7.07 (d, J = 8.5 Hz, 1H, Ar- ), 6.73 (s, 1H, Ar- ),
6.46 (d, J = 2 Hz, 1H, Ar- ), 6.19 (d, J = 2 Hz, 1H, Ar-
), 3.86 (s, 3H, OCH3). 13C-NMR (DMSO-d6)
181.6, 164.1, 163.5, 161.4, 157.3, 151.1, 146.7, 123.0, 118.6, 112.9, 112.1, 103.7, 103.5, 98.8, 93.8, 55.7.
λmax nm, MeOH): 252, 269,
ν
δ
H
H
H
H
H
H
H
H
H
δ

Molecules 2020, 25, 4064
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4.2.4. Synthesis of D2–D6
Derivatives D2–D6 were synthesized from diosmetin (D1) through the same reaction processes as
described in the synthesis of substances B2–B6 from baicalein (B1).
5-Hydroxy-3’,4’,7-trimethoxyflavone (D2). Yield: 56%. MP: 200–201 ◦C. UV (
λmax nm, MeOH): 242, 269,
340. IR (
1H-NMR (DMSO-d6)
Ar- ), 7.14 (d, J = 8.5 Hz, 1H, Ar-
1H, Ar- ), 3.89 (s, 3H, OC 3), 3.88 (s, 3H, OC
ν
cm−1, KBr): 3444, 3001, 2951, 2835, 1662, 1589, 1502, 1168. MS: [M + H]+ m/z = 329.1015.
δ
12.92 (s, 1H, O
H
), 7.72 (dd, J1 = 8.5 Hz, J2 = 2 Hz, 1H, Ar-
H
), 7.60 (d, J = 2 Hz, 1H,
), 6.82 (d, J = 2 Hz, 1H, Ar- ), 6.45 (d, J = 2 Hz
3). 13C-NMR (DMSO-d6)
182.0,
H
H
), 7.00 (s, 1H, Ar-
H
H
,
H
H
H
3), 3.86 (s, 3H, OC
H
δ
165.2, 163.6, 161.1, 157.3, 152.2, 149.0, 122.7, 120.1, 111.7, 109.5, 104.7, 104.0, 98.0, 92.7, 56.0, 55.9, 55.7.
3’,7-Diethoxy-5-hydroxy-4’-methoxyflavone (D3). Yield: 57%. MP: 173–174 ◦C. UV (
max nm, MeOH): 269,
338. IR (
cm−1, KBr): 3452, 3057, 2920, 2835, 1654, 1622, 1510, 1168. MS: [M + H]+ m/z = 357.1327.
1H-NMR (DMSO-d6)
1H, Ar- ), 7.13 (d, J = 8.5 Hz, 1H, Ar-
6.45 (d, J = 2.5 Hz, 1H, Ar-
OCH3), 1.37 (t, J = 7 Hz, 3H, CH3), 1.36 (t, J = 7 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
163.6, 161.1, 157.3, 152.4, 148.2, 122.7, 120.1, 111.8, 110.5, 104.6, 103.9, 98.3, 93.1, 64.2, 64.1, 55.7, 14.6, 14.3.
3’,7-Diallyloxy-5-hydroxy-4’-methoxyflavone (D4). Yield: 81%. MP: 126–127 ◦C. UV (
max nm, MeOH):
cm−1, KBr): 3446, 3082, 2931, 2841, 1662, 1608, 1498, 1168. MS: [M + H]+
12.90 (s, 1H, O ), 7.71 (dd, J1 = 8.5 Hz, J2 = 2 Hz, 1H, Ar- ),
), 7.15 (d, J = 8.5 Hz, 1H, Ar- ), 6.99 (s, 1H, Ar- ), 6.82 (d, J = 2 Hz, 1H,
), 6.40 (d, J = 2 Hz, 1H, Ar- ), 6.11–6.04 (m, 2H, -C =), 5.45 (dd, J1 = 17 Hz, J2 = 1.5 Hz, 1H,
λ
ν
δ
12.90 (s, 1H, O
H
), 7.70 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 1H, Ar-
H
), 7.58 (d, J = 2.5 Hz
,
H
H
), 7.00 (s, 1H, Ar- ), 6.69 (d, J = 2.5 Hz, 1H, Ar-
H
H
),
H
), 4.16 (q, J = 7 Hz, 2H, CH2), 4.15 (q, J = 7 Hz, 2H, CH2), 3.86 (s, 3H,
181.9, 164.4,
δ
λ
243, 269, 339. IR (
m/z = 381.1333. H-NMR (DMSO-d6)
ν
1
δ
H
H
7.61 (d, J = 2 Hz, 1H, Ar-
Ar-
H
H
H
H
H
H
=CHtrans), 5.44 (dd, J1 = 17 Hz, J2 = 1.5 Hz, 1H, =CHtrans), 5.31 (dd, J1 = 10 Hz, J2 = 1.5 Hz, 1H, =CHcis),
5.29 (dd, J1 = 10 Hz, J2 = 1.5 Hz, 1H, =CHcis), 4.71–4.69 (m, 4H, CH2), 3.87 (s, 3H, OCH3). 13C-NMR
(DMSO-d6) δ 181.9, 164.0, 163.5, 161.2, 157.2, 152.5, 147.8, 133.6, 132.8, 122.7, 120.4, 118.1, 117.9, 112.0,
111.1, 104.8, 104.0, 98.4, 93.4, 69.2, 68.9, 55.8.
3’,4’,5,7-Tetramethoxyflavone (D5). Yield: 42%. MP: 196–197 ◦C. UV (
λ
nm, MeOH): 242, 265, 331.
max
1
IR (
7.61 (dd, J1 = 8.5 Hz, J2 = 2 Hz, 1H, Ar-
6.83 (d, J = 2 Hz, 1H, Ar- ), 6.74 (s, 1H, Ar-
(s, 3H, OC 3), 3.84 (s, 3H, OC 3), 3.82 (s, 3H, OC
159.0, 151.5, 148.9, 123.0, 119.2, 111.5, 109.0, 108.2, 106.9, 96.0, 93.3, 55.9, 55.8, 55.7, 55.6.
ν
cm−1, KBr): 3001, 2938, 2841, 1605, 1514, 1018. MS: [M + H]+ m/z = 343.1179. H-NMR (DMSO-d6)
δ
H
), 7.51 (d, J = 2 Hz, 1H, Ar-
), 6.48 (d, J = 2 Hz, 1H, Ar-
3). 13C-NMR (DMSO-d6)
δ
H
), 7.08 (d, J = 8.5 Hz, 1H, Ar-
), 3.90 (s, 3H, OCH3), 3.88
175.6, 163.5, 160.1, 159.5,
H),
H
H
H
H
H
H
30,5,7-Triethoxy-40-methoxyflavone (D6). Yield: 35%. MP: 167–168 ◦C. UV (
234. IR (
(DMSO-d6)
1H, Ar- ), 6.82 (d, J = 2 Hz, 1H, Ar-
2H, C 2), 4.14 (q, J = 7 Hz, 2H, C
9H, CH3). 13C-NMR (DMSO-d6)
λ
max nm, MeOH): 242, 266,
1
ν
cm−1, KBr): 3082, 2977, 2929, 1601, 1514, 1115. MS: [M + H]+ m/z = 385.1645. H-NMR
δ
7.62 (dd, J1 = 8.5 Hz, J2 = 2 Hz, 1H, Ar-
), 6.69 (s, 1H, Ar-
2), 4.08 (q, J = 7 Hz, 2H, C
175.5, 162.6, 159.4, 159.3, 159.0, 151.6, 148.1, 123.1, 119.1, 111.6, 109.9,
H
), 7.51 (d, J = 2 Hz, 1H, Ar-
), 6.45 (d, J = 2 Hz, 1H, Ar-
2), 3.85 (s, 3H, OC
H
), 7.10 (d, J = 8.5 Hz,
), 4.18 (q, J = 7 Hz,
H3), 1.37 (t, J = 7 Hz,
H
H
H
H
H
H
H
δ
108.3, 106.9, 97.1, 93.6, 64.2, 64.0, 63.9, 55.5, 14.6, 14.3, 14.3.
4.2.5. Synthesis of D7
Diosmetin (D1) was dissolved in pyridine and benzoyl chloride was then added. The reaction
mixture was heated at 40–45 ◦C and monitored by thin layer chromatography with the appropriate
solvent system. At the end of the reaction, the mixture was poured into cold water and stirred
vigorously. The precipitate formed was then filtered off and recrystallized in the appropriate solvent to
yield the final product.
3’,7-Dibenzoyloxy-5-hydroxy-4’-methoxyflavone (D7). Yield: 68%. MP: 227–228 ◦C. UV (
λ
nm, MeOH):
max
1
232, 270, 324. IR (
ν
cm–1, KBr): 3066, 1742, 1608, 1503, 1250, 1054. MS: [M + H]+ m/z = 509.1226. H-NMR

Molecules 2020, 25, 4064
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(DMSO-d6)
Ar- ), 7.39 (d, J = 9 Hz, 1H, Ar-
1H, Ar-
), 3.89 (s, 3H, OCH3). 13C-NMR (DMSO-d6)
δ
12.93 (s, 1H, O
H
), 8.16–8.11 (m, 6H, Ar-
), 7.29 (d, J = 2 Hz, 1H, Ar-
182.4, 163.8, 163.7, 163.2, 160.7, 156.1, 155.8,
H
), 7.79–7.76 (m, 2H, Ar-
H
), 7.64–7.61 (m, 4H,
H
H
H
), 7.14 (s, 1H, Ar-
H), 6.84 (d, J = 2 Hz,
H
δ
154.3, 139.6, 134.3, 134.2, 129.9, 129.8, 129.0, 128.9, 128.4, 128.3, 126.2, 122.7, 121.5, 113.3, 108.2, 105.3,
104.6, 101.7, 56.3.
All spectra of the synthesized substances are provided in the Supporting Information (Table S7).
4.3. In Vitro Assays
AChE inhibitory activities of the synthesized flavone derivatives were determined by Ellman’s
method [45]. Purified acetylcolinesterase from Electrophorus electricus (electric eel), acetylthiocholine
iodide (ATCI, substrate), and galantamine (positive control) were purchased from Sigma–Aldrich
(St. Louis, MO, USA) and used in this study. The assay was performed as described earlier [46] in
96-well microtiter plates using the EMR-500 ELISA system (Labomed, Los Angeles, CA, USA). Each
well of the plate contained a mixture of sodium phosphate buffer pH 8, sample (studied compounds or
reference), acetylcholinesterase, ATCI, and 5,50-dithio-bisnitrobenzoic acid (DTNB). The absorbance
was recorded at 405 nm. All samples were assayed in triplicate.
A fluorescence
and used to determine the effect of the synthesized flavone derivatives on
was carried out according to the manufacturer’s protocol as described earlier [47
solution was reacted with the substrate (7-methoxycumarin-4-acetyl-(Asn670, Lue671)-amyloid
β
-Secretase (BACE-1) Activity Detection Kit was purchased from Sigma–Aldrich
-secretase activity. The assay
48]. The enzyme
/A4
β
,
β
precursor protein 770 fragment 667–676-(2,4-dinitrophenyl))Lys-Arg-Arg amide trifluoroacetate salt
and sulfated polysaccharide samples in a fluorescence assay buffer in different wells. Baseline readings
were measure immediately on an F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan;
excitation: 320 nm; emission: 405 nm) and repeated after 2 h incubation at 37 ◦C. Quercetin was used
as the positive control.
4.4. Molecular Docking
Ligand molecules were prepared directly in Sybyl X 2.0 [49]. Complexes 1DX6 of AChE
(acetylcolinesterase from Electrophorus electricus in complex with ( )-galantamine, resolution 2.3 Å) and
−
6EQM of BACE-1 (human BACE-1 in complex with umibecestat, resolution 1.35 Å) were downloaded
from Protein Data Bank [50]. Docking protocols were firstly validated by the method of pose
selection [51] on three conformations of co-crystallized ligands. The root-mean-square deviation
(RMSD) value between re-docked conformations and the original bound ligand in the co-crystallized
complex which was 1.5 Å would indicate the reliability of the binding ability prediction of new ligands.
≤
In this study, FlexX program in BioSolveIT LeadIt [52] was used with default settings. This program
applied a flexible-based docking methodology, an incremental construction algorithm for the seach of
ligand conformations, and empirical scoring functions to score and rank the docking poses [53
,54].
The interactions between flavone molecules and their targets, including hydrogen bonds, , cation–π,
π
–
π
ionic and van der Waals, were rendered and analyzed in the MOE 2008.10 program [55].
5. Conclusions
In this study, 14 flavone derivatives divided into two groups were synthesized and tested for
AChE and BACE-1 inhibition, giving pIC50 values of 3.47–4.59 (AChE) and 4.15–5.80 (BACE-1).
The synthesized substances were also used in an external validation set to evaluate the developed
2D-QSAR model for AChE and BACE-1 inhibitors with relatively high correlations between the
observed and estimated bioactivities (R2 = 0.83 and RMSE = 0.44 for AChE, and R2 = 0.82 and
RMSE = 0.40 for BACE-1). Six of the synthesized derivatives (B4
The molecular docking model developed in the present work could be used to explain the biological
activity observed in the in vitro assays. Among the studied flavone derivatives, B3 D5 and D6
, B5, B6, B8, D6 and D7) were new.
,

Molecules 2020, 25, 4064
12 of 14
were the three derivatives with the strongest inhibitory activities against both AChE and BACE-1.
These substances could be used in the further studies.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/18/4064/s1,
Table S1: Selected descriptors used for building 2D-QSAR models reported in the previous published work,
Table S2: Values of selected descriptors used in the prediction of pIC50 of the synthesized flavone derivatives
(AChE), Table S3: Values of selected descriptors used in the prediction of pIC50 of the synthesized flavone
derivatives (BACE-1), Table S4: Results of re-docking (RMSD in Å), Table S5: Docking results and ligand
interactions (Co-crystallized 1DX6), Table S6: Docking results and ligand interactions (Co-crystallized 6EQM),
Table S7: Spectra of the synthesized flavone derivatives.
Author Contributions: Conceptualization, T.-D.T., K.-M.T. and M.-T.L.; Data curation, T.-S.T., T.-H.T. and T.-T.M.;
Formal analysis, T.-S.T., T.-D.T., T.-H.T. and N.-L.N.; Funding acquisition, K.-M.T.; Investigation, T.-S.T., T.-H.T.,
T.-T.M. and N.-L.N.; Methodology, T.-S.T., T.-D.T., K.-M.T. and M.-T.L.; Project administration, T.-D.T., K.-M.T.
and M.-T.L.; Resources, K.-M.T. and M.-T.L.; Software, T.-S.T. and K.-M.T.; Supervision, T.-D.T., K.-M.T. and
M.-T.L.; Validation, T.-S.T., T.-D.T., T.-T.M., N.-L.N., K.-M.T. and M.-T.L.; Visualization, T.-S.T., T.-H.T. and K.-M.T.;
Writing—original draft, T.-S.T., T.-D.T., K.-M.T. and M.-T.L.; Writing—review & editing, T.-S.T., T.-D.T., K.-M.T.
and M.-T.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research is funded by Vietnam National Foundation for Science and Technology Development
(NAFOSTED) under grant number 106-YS.05-2015.31 to Khac-Minh Thai and 108.05-2017.12 to Minh-Tri Le.
Conflicts of Interest: The authors declare no conflicts of interest.
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