2-Styrylchromone derivatives as potent and selective monoamine oxidase B inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103285

A series of eighteen 2-styrylchromone derivatives (see Chart 1) were synthesized and evaluated for their monoamine oxidase (MAO) A and B inhibitory activities. Many of the derivatives inhibited MAO-B comparable to pargyline (a positive control), and most of them inhibited MAO-B selectively. Of the eighteen derivatives, compound 9 having methoxy group at R¹ and chlorine at R⁴ showed both the best MAO-B inhibitory activity (IC₅₀ = 17 ± 2.4 nM) and the best MAO-B selectivity (IC₅₀ for MAO-A/IC₅₀ for MAO-B = 1500). The mode of inhibition of compound 9 against MAO-B was competitive and reversible. Quantitative structure–activity relationship (QSAR) analyses of the 2-styrylchromone derivatives were conducted using their pIC₅₀ values with the use of Molecular Operating Environment (MOE) and Dragon, demonstrating that the descriptors of MAO-B inhibitory activity and MAO-B selectivity were 1734 and 121, respectively, that showed significant correlations (P < 0.05). We then examined the 2-styrylchromone structures as useful scaffolds through three-dimensional-QSAR studies using AutoGPA, which is based on the molecular field analysis algorithm using MOE. The model using pIC₅₀ value indexes for MAO-B exhibited a determination coefficient (R²) of 0.873 as well as a Leave-One-Out cross-validated determination coefficient (Q²) of 0.675. These data suggested that the 2-styrylchromone structure might be a useful scaffold for the design and development of novel MAO-B inhibitors.

Full text of DOI:10.1016/j.bioorg.2019.103285

BioorganicChemistry92(2019)103285
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
2-Styrylchromone derivatives as potent and selective monoamine oxidase B
inhibitors
⁎
Koichi Takaoa, , Saki Endoa, Junko Nagaib, Hitoshi Kamauchia, Yuri Takemuraa,
Yoshihiro Uesawab, Yoshiaki Sugitaa
a Laboratory of Bioorganic Chemistry, Department of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, 1-1 Keyaki-dai,
Sakado, Saitama 350-0295, Japan
b Department of Medical Molecular Informatics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of eighteen 2-styrylchromone derivatives (see Chart 1) were synthesized and evaluated for their
monoamine oxidase (MAO) A and B inhibitory activities. Many of the derivatives inhibited MAO-B comparable
to pargyline (a positive control), and most of them inhibited MAO-B selectively. Of the eighteen derivatives,
compound 9 having methoxy group at R1 and chlorine at R4 showed both the best MAO-B inhibitory activity
2-Styrylchromone
Monoamine oxidase-A
Monoamine oxidase-B
Quantitative structure–activity relationship
Molecular Operating Environment
AutoGPA
(IC50 = 17
2.4 nM) and the best MAO-B selectivity (IC50 for MAO-A/IC50 for MAO-B = 1500). The mode of
inhibition of compound 9 against MAO-B was competitive and reversible. Quantitative structure–activity re-
lationship (QSAR) analyses of the 2-styrylchromone derivatives were conducted using their pIC50 values with the
use of Molecular Operating Environment (MOE) and Dragon, demonstrating that the descriptors of MAO-B
inhibitory activity and MAO-B selectivity were 1734 and 121, respectively, that showed significant correlations
(P < 0.05). We then examined the 2-styrylchromone structures as useful scaffolds through three-dimensional-
QSAR studies using AutoGPA, which is based on the molecular field analysis algorithm using MOE. The model
using pIC50 value indexes for MAO-B exhibited a determination coefficient (R2) of 0.873 as well as a Leave-One-
Out cross-validated determination coefficient (Q2) of 0.675. These data suggested that the 2-styrylchromone
structure might be a useful scaffold for the design and development of novel MAO-B inhibitors.
1. Introduction
distributed in plants and are scaffolds for MAO inhibitors [5–7]. Fla-
vone (2-phenylchromone) and isoflavone (3-phenylchromone) in-
Monoamine oxidase (MAO, EC 1.4.3.4) is a flavoenzyme bound to
the mitochondrial outer membranes of many types of mammalian cells.
MAO catalyzes the oxidative deamination of endogeneous and exoge-
neous amines, including neurotransmitters such as epinephrine, dopa-
mine, and serotonin [1]. Human has two types of MAO, MAO-A and
MAO-B, which share approximately 70% sequence identity at the
amino acid level and are identified based on their substrate and in-
hibitor sensitivities [2,3]. MAO-A preferentially deaminates epi-
nephrine, norepinephrine and serotonin, and is inhibited irreversibly by
clorgyline. MAO-B preferentially deaminates benzylamine, β-phe-
nethylamine and dopamine, and is inhibited irreversibly by (R)-(-)-de-
prenyl, a drug for the treatment of Parkinson’s disease. As the con-
centration change of the neurotransmitters in the brain linked to
various neurodegenerative diseases’ pathologies, many laboratories
have engaged in the development of inhibitors of the two enzymes [4].
Derivatives of chromone (4H-1-benzopyran-4-one) are widely
cluding their derivatives constitute a large group of naturally occurring
chromones, while 2-styrylchromone and its derivatives a small group
[8,9]. Synthetic 2-styrylchromones were evaluated for a number of
biological activities, such as antioxidant, anti-inflammatory, anti-al-
lergic, antitumor, and antiviral activities [8,9], but their inhibitory
activity against MAO has not been reported. On the other hand, a
natural product resveratrol (3,5,4′-trihydroxy-trans-stylbene) has been
well studied because of its numerous biological effects [10,11]. Re-
garding the inhibition of MAO, cis-resveratrol is less effective than
trans-resveratrol [12]. Hence, the related trans-stilbene-type com-
pounds such as (E)-2-styryl-2-imidazoline [13], (E)-styrylcaffeine
[14,15], (E)-2-styrylbenzimidazole [15], (E)-styrylisatin [16], (E)-styr-
ylxanthine derivatives [17], and 3-(E)-styryl-2H-chromene derivatives
[18] were reported as MAO inhibitors. These findings suggested that 2-
styrylchromones had a potential to inhibit MAO.
In order to further explore new biological activities of this family of
⁎ Corresponding author.
E-mail address: ktakao@josaiac.jp (K. Takao).
https://doi.org/10.1016/j.bioorg.2019.103285
Received 11 July 2019; Received in revised form 28 August 2019; Accepted 15 September 2019
Availableonline18September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

K. Takao, et al.
BioorganicChemistry92(2019)103285
Chart 1. Synthetic protocol for 2-styrylchromone derivatives. Reagents and conditions: (a) Na, AcOEt, rt; (b) conc. HCl, MeOH, rt; (c) NaOMe, MeOH, reflux.
compounds, we synthesized a series of 2-styrylchromone derivatives
(Chart 1). This paper also deals with their inhibitory activities against
human MAO-A and MAO-B, and from the results studies on the quan-
titative structure–activity relationship (QSAR) analyses of the deriva-
tives to MAO-B.
2.2. Biological activity
2.2.1. Inhibitory activity towards MAO-A and MAO-B
All the eighteen 2-styrylchromone derivatives were evaluated for
MAO-A and MAO-B inhibitory activities. As shown in Table 1, mod-
ifications of chromone ring (R1,R2) and of phenyl ring (R3,R4) on 2-
styrylchromone revealed several interesting structure-activity re-
lationships.
2. Results and discussion
The MAO-A inhibitory activities of the derivatives were determined.
Compounds 1, 2, 3, 4, 5, 7, 8, 10 and 11, more or less, showed in-
hibitory activities, and Compound 8 inhibited MAO-A most potently
2.1. Chemistry
2.1.1. Synthesis of 2-styrylchromone derivatives
(IC50 = 0.12
0.0060 μM). Substitution of methoxy group at R1
2-Styrylchromone derivatives (1–18) were synthesized as shown in
Chart 1. 2-Methylchromone derivatives (IIa-c) were first synthesized by
Claisen condensation of the corresponding acetophenone derivatives
(Ia-c) with ethyl acetate, followed by intramolecular cyclization as
described previously [19]. Then the resulting IIa-c reacted with each
benzaldehyde derivative (IIIa-f) in the presence of base using a mod-
ification of the previous procedure [19] to obtain corresponding 2-
styrylchromone derivatives.
seemed to intensify MAO-A inhibitory activities, such as compound 1
vs. 7, 2 vs. 8, 4 vs. 10 and 5 vs. 11, but with an exception of compound
3 vs. 9. And substitution of methoxy group at R2 weakened MAO-A
inhibitory activities without exception such as compound 1 vs. 13, 2 vs.
14, 3 vs. 15, 4 vs. 16, 5 vs. 17 and 6 vs. 18.
The MAO-B inhibitory activities of the derivatives were then de-
termined, and all the derivatives were found to inhibit MAO-B more
2

K. Takao, et al.
BioorganicChemistry92(2019)103285
Table 1
The IC50 values and selectivity of 2-styrylchromone derivatives for the inhibition of MAO-A and MAO-B. MAO-B selectivity is given as the ratio of the IC50 value for
MAO-A/IC50 value for MAO-B. All values are expressed as the mean
Compd.
standard deviation of triplicate determinations.
R4 MAO-A IC50 (μM) MAO-B IC50 (μM)
R1
R2
R3
MAO-B selectivity
1
H
H
H
H
H
H
H
F
0.95
0.59
0.29
0.33
2.3
0.030
0.032
0.025
0.0050
0.073
0.81
0.24
0.17
0.079
0.069
0.049
2.8
0.024
0.013
0.0013
0.0045
0.0013
0.32
4
4
2
3
H
H
H
Cl
4
4
H
H
H
Br
5
5
H
H
H
OMe
OMe
H
47
6
H
H
OMe
H
25
9
7
OMe
OMe
OMe
OMe
OMe
OMe
H
H
0.20
0.12
26
0.012
0.0060
3.5
0.18
0.064
0.017
0.024
0.19
0.68
0.22
0.12
0.27
0.45
1.4
0.0027
0.0025
0.0024
0.0024
0.014
0.022
0.0095
0.010
0.021
0.010
0.039
1.1
1
8
9
H
H
F
2
H
H
Cl
1500
22
10
H
H
Br
0.53
0.21
21
0.010
0.047
0.47
11
H
H
OMe
OMe
H
1
12
H
OMe
H
31
13
OMe
OMe
OMe
OMe
OMe
OMe
26
1.1
120
290
> 370
> 220
51
14
H
H
F
35
4.4
15
H
H
Cl
> 100
16
H
H
Br
> 100
72 1.3
> 100
4.6 0.27
17
H
H
OMe
OMe
18
H
OMe
10
> 10
21
Pargyline
0.22
0.024
potently than MAO-A. Compound 9 was the most potent inhibitor
(IC50 = 17 2.4 nM), showing the inhibitory activity of approxi-
mately 13-fold as much as that of pargyline (a positive control).
Compounds 3, 4, 5, 8, and 10 showed more potent inhibition than
pargyline, and compounds 1, 2, 7, 11, 12, 13, 14, 15 and 16 showed a
similar inhibition to pargyline. Substitution of methoxy group at R1
seemed to intensify MAO-B inhibitory activities, such as compound 2
vs. 8, 3 vs. 9, 4 vs. 10 and 6 vs. 12, but with an exception of compound
5 vs. 11. And substitution of methoxy group at R2 seemed to weaken
MAO-B inhibitory activities, such as compound 3 vs. 15, 4 vs. 16, 5 vs.
17 and 6 vs. 18, but with similar value of compound 1 vs. 13 or 2 vs.
14.
MAO-B selectivities of the derivatives were then calculated, defined
as the ratio of IC50 value of MAO-A to that of MAO-B for each deriva-
tive. Of the derivatives compound 9 showed a significantly high MAO-B
selectivity (1500). Thus, compound 9, the most potent MAO-B in-
hibitor, was chosen for the following kinetics study (Fig. 1). From the
Lineweaver–Burk plots, the mode of MAO-B inhibition was competitive
and reversible with Ki value of 8 nM, suggesting a tight fitting of
Fig. 2. The reversibility of inhibition of MAO-B by compound 9. MAO-B was
preincubated with compound 9 at 10 × IC50 and 100 × IC50 for 30 min and
then diluted to 0.1 × IC50 and 1 × IC50, respectively. For comparison, the ir-
reversible MAO-B inhibitor pargyline at 10 × IC50 was similarly incubated with
MAO-B and diluted to 1 × IC50. The residual activity of MAO-B was subse-
quently measured.
compound 9 to MAO-B active site. The reversibility of MAO-B inhibi-
tion by compound 9 was further confirmed in contrast to the irrever-
sibility of pargyline according to the literature method [20] (Fig. 2).
2.2.2. Computational analyses
2-Styrylchromone derivatives showed potent MAO-B inhibitory ac-
tivity and selectivity. The statistical significance (P < 0.05) of each
substituted group was computationally analyzed (Fig. 3) and showed
that the substitution of methoxy group at R2 significantly affected both
MAO-B inhibitory activity (P = 0.0440) and selectivity (P = 0.0130).
Other substitutions at R1, R2, R3 and R4 had no significant effect on
either MAO-B inhibitory activity or selectivity. These data suggested
that the 2-styrylchromone scaffold is a useful candidate structure for
the design of MAO-B selective inhibitors.
In an effort to elucidate further the structure activity relationships
between MAO-B and inhibitors, quantitative structure–activity re-
lationship (QSAR) analyses of 2-styrylchromone derivatives were con-
ducted using Molecular Operating Environment (MOE) [21] and
Dragon [22] (a total of 3106 descriptors).
Fig. 1. Lineweaver–Burk plots for the inhibition of MAO-B by compound 9. The
plots were constructed in the absence (filled circles) and presence (other sym-
bols) of various concentrations of compound 9. The inset is a graph of the slopes
of the Lineweaver–Burk plots versus inhibitor concentration (Ki = 0.0080 μM).
The rate (V) is expressed as % of control. Kynuramine was used at 30 μM.
QSAR analyses were performed for 2-styrylchromone derivatives
3

K. Takao, et al.
BioorganicChemistry92(2019)103285
Fig. 3. Effect of substitutions at R1 and R2 of 2-styrylchromone derivatives on MAO-B inhibition and selectivity.
with pIC50 values for MAO-B inhibitory activity and exhibiting MAO-B
selectivity. These analyses demonstrated that 1734 and 121 descriptors
showed significant correlations (P < 0.05) for MAO-B inhibitory ac-
tivity and selectivity, respectively. Scatter plots of the top six de-
scriptors are shown in Figs. 4, 5. The strongly correlated descriptors
indicated that properties such as molecular size, shape, and the elec-
tronic state of 2-styrylchromone derivatives are important for their
inhibitory activity and selectivity. These structural and physicochem-
ical properties suggested that the binding of 2-styrylchromone deriva-
tives to the binding site of MAO-B is stabilized by van der Waals forces
and electrostatic interactions. We also performed three-dimensional-
QSAR (3D-QSAR) studies on the eighteen compounds exhibiting MAO-B
inhibitory activity using AutoGPA based on the molecular field analysis
(CoMFA) algorithm [23] using MOE [24]. Developed models sup-
porting the prediction of pIC50 and pSelectivity for MAO-B provided
values that correlated with the corresponding experimental values, as
shown in Fig. 6. The pIC50 and pSelectivity values had determination
coefficients (R2) of 0.873 and 0.867, respectively, and Leave-One-Out
cross-validated determination coefficients (Q2) of 0.675 and 0.483,
respectively. In this analysis, Q2 > 0.5 suggested that the model is
reasonable and should have good predictive ability [25], and that the
model for MAO-B inhibition provides reasonable pIC50 values. The
contour map of the AutoGPA-based model of MAO-B pIC50 values is
shown in Fig. 7A. In the electrostatic contour map, the blue contour
suggests electropositive charge and the red contour suggests electro-
negative charge that likely help increase activity, whereas in the steric
contour map, the green contour represents bulky groups favorable for
increasing activity and the yellow contour indicates decreased activity.
A molecular docking study performed using the MOE-Dock function
was conducted to better understand the developed model (Fig. 7b, c).
The interactions between compound 9 with MAO-B protein (PDB code
4A79) were investigated using MOE. The interaction with the phar-
macophore moieties F1 and F2 in Fig. 7A corresponds to the interaction
with Ile171 and Ile199 in Fig. 7B and C, respectively. The green contour
in Fig. 7A located over the chromone ring likely corresponds to FAD in
Fig. 7B, supporting our observation that substitution with a methoxy
group at R1 increases MAO-B inhibitory activity. The 3D-QSAR model
correlated well with the molecular docking results.
A recent review [26] reported that the active site structure of
human MAO-B has a bipartite hydrophobic cavity comprising an en-
trance cavity and a substrate cavity. The structure of the cavity is
mainly determined by an entrance loop (residues 99–110) that reg-
ulates ligand access to the active site, and a cavity-shaping loop formed
by residues 200–209. The substrate cavity in MAO-B has a volume of
∼430A3 and the entrance cavity has a volume of ∼290 Å3. The com-
bined volume of the two cavities when the gating Ile199 is in its open
conformation is ∼700 Å3. Tyr326 is another important residue for
gating. Interestingly, compound 9 interacted with both Ile199 and
Tyr326, as shown in Fig. 7C. Furthermore, the above-mentioned review
reported that Tyr398 and Tyr435 stack parallel to each other and
perpendicular to the FAD ring, creating an aromatic sandwich that
stabilizes substrate binding. We also observed their interactions Tyr398
and Tyr435, as shown in Fig. 7C. The active site of MAO-A has a
4

K. Takao, et al.
BioorganicChemistry92(2019)103285
Fig. 4. Determination of the coefficient between the chemical descriptors and the pIC50 values of 2-styrylchromone derivatives for MAO-B.
monopartite cavity with a total volume of ∼550 Å3. It therefore ap-
pears that MAO-B recognizes its substrate less stringently than MAO-A.
Therefore, 2-styrylchromene derivatives might be selective for MAO-B.
Recently, our group reported the MAO-B inhibitory activities of 3-
(E)-styryl-2H-chromene derivatives [18]. Resveratrol inhibits MAO-A
more strongly than MAO-B. In contrast, the related compounds (E)-2-
styryl-2-imidazoline [13], (E)-styrylcaffeine [14] and (E)-styrylisatin
derivatives [16] showed more potent MAO-B inhibition than MAO-A
inhibition. These results suggest that the styryl moiety of these deri-
vatives prefers to bind to the substrate cavity of MAO-B compared to
the MAO-A cavity. Docking study on MAO-B has been done using (E)-
styrylisatin derivatives [16], and the isatin moiety of both C-5 and C-6
styrylisatin was located in the substrate cavity and the styryl moiety
extended to the entrance cavity of MAO-B. This was similar to the di-
rection of 2-styrylchromone derivatives in MAO-B active site.
This is the first report identifying 2-styrylchromone derivatives as
potent MAO-B inhibitors. The results suggest that the 2-styrylchromone
structure may be a useful scaffold for the design and development of
novel MAO-B inhibitors.
inhibitory activity and selectivity. Computational analyses also sug-
gested the influence of substitution by methoxy group at R1. QSAR and
3D-QSAR analyses suggested that 2-styrylchromone derivatives would
be a promising scaffold for the design and development of new MAO-B
inhibitors.
4. Experimental
4.1. Chemistry
All reagents and solvents were purchased from commercial sources.
Analytical thin-layer chromatography was performed on silica-coated
plates (silica gel 60F-254; Merck Ltd., Tokyo, Japan) and visualized
under UV light. Column chromatography was carried out using silica
gel (Wakogel C-200; Wako Pure Chemical Industry Co., Tokyo, Japan).
All melting points were determined using a Yanagimoto micro-hot stage
and are uncorrected. 1H NMR and 13C NMR spectra were recorded on a
Varian 400-MR spectrometer using tetramethylsilane as an internal
standard. MS spectra were measured using a JEOL JMS-700 spectro-
meter. Elemental analyses were carried out on a Yanaco CHN MT-6
elemental analyzer.
3. In conclusion
The present study demonstrated that 2-styrylchromone derivatives
were potent and selective MAO-B inhibitors. Of eighteen derivatives
synthesized, fifteen showed potent MAO-B inhibitory activities, and
sixteen showed MAO-B selectivity. Especially, compound 9, having a
methoxy group at R1 and chlorine at R3, exhibited the highest MAO-B
4.1.1. Synthesis of 2-methylchromones
2-Methylchromones (IIa-c) were synthesized according to previous
methods [19]. To a solution of the corresponding acetophenone (I,
20 mmol) in dry ethyl acetate (30 mL), sodium (120 mmol) was added.
The reaction mixture was stirred for 18 h at room temperature. After
5

K. Takao, et al.
BioorganicChemistry92(2019)103285
Fig. 5. Determination of the coefficient between the chemical descriptors and the MAO-B selectivity of 2-styrylchromone derivatives.
MAO-B selecꢀvity
MAO-B inhibiꢀon
4
3
2
1
0
5.5
4.5
3.5
2.5
1.5
0
1
2
3
4
1.5
2.5
3.5
4.5
5.5
pSelecꢀvity experimental
pIC50 experimental
Fig. 6. Graphs of the predicted and experimental pIC50 MAO-B inhibition and pSelectivity MAO-B selectivity values for 2-styrylchromone derivatives.
the reaction mixture was diluted with ice-water and acidified with 2 M
HCl, the aqueous layer was separated and extracted with ethyl acetate.
The combined organic layer was dried over Na2SO4 and the solvent was
evaporated under reduced pressure to obtain the crude diketone. A
solution of the crude diketone with a few drops of conc. HCl in MeOH
(50 mL) was stirred for 4 h at room temperature. MeOH was removed
under reduced pressure, ethyl acetate was added, and the solution was
washed with brine. The organic layer was dried over Na2SO4 and the
solvent was evaporated under reduced pressure. The residue was pur-
ified by silica gel column chromatography (hexane:AcOEt = 10:1) to
give the title compounds. The products (IIa-c) were identified by their
melting points and 1H NMR spectra [19,27].
4.1.2. 2-Methyl-4H-1-benzopyran-4-one (IIa)
Yield 51%. Pale brown scaly crystal. mp 71–73 °C (lit. [27]
70–72 °C). 1H NMR (CDCl3, 400 MHz) δ 8.19 (1H, dd, J = 7.9, 1.7 Hz,
6

K. Takao, et al.
BioorganicChemistry92(2019)103285
Fig. 7. AutoGPA model and docking results obtained from a set of 2-styrylchromone derivatives as MAO-B inhibitors. (a) AutoGPA steric and electrostatic contours
field plot. The position and the structure of 2-styrylchromone is superposed. Green and yellow contours indicate regions where bulky groups increase and decrease
activity, respectively. Blue and red contours indicate regions where positive and negative electrostatic groups increase activity. (b) Ligand interaction graph of the
active pocket of MAO-B with compound 9. (c) 2D interaction diagram of compound 9 with the MAO-B binding cavity.
H-5), 7.64 (1H, ddd, J = 8.4, 7.1, 1.7 Hz, H-7), 7.42 (1H, dd, J = 8.4,
1.1 Hz, H-8), 7.38 (1H, ddd, J = 7.9, 7.1, 1.1 Hz, H-6), 6.26 (1H, s, H-
methylchromone (II, 2 mmol) and benzaldehyde (III, 2.4 mmol) were
added. The mixture was refluxed until complete disappearance of the 2-
methylchromone (4–8 h). After diluting the reaction mixture with ice-
water and acidifying to pH 4 with 2 M HCl, MeOH was removed under
reduced pressure. The sample was extracted with CH2Cl2. The com-
bined organic layer was dried over Na2SO4 and the solvent was eva-
porated under reduced pressure. The residue was purified by silica gel
column chromatography (hexane:AcOEt = 10:1) to give the title com-
pounds.
3), 2.41 (3H, s, Me). MS (EI) m/z 160 [M]+
.
4.1.3. 2-Methyl-6-methoxy-4H-1-benzopyran-4-one (IIb)
Yield 48%. Pale yellow scaly crystal. mp 107–109 °C (lit. [19]
109–110 °C). 1H NMR (CDCl3, 400 MHz) δ 7.55 (1H, d, J = 3.1 Hz, H-
5), 7.36 (1H, d, J = 9.1 Hz, H-8), 7.23 (1H, dd, J = 9.1, 3.1 Hz, H-7),
6.16 (1H, s, H-3), 3.89 (3H, s, OMe), 2.38 (3H, s, Me). MS (EI) m/z 190
[M]+
.
4.1.5.1. 2-[(1E)-2-Phenylethenyl]-4H-1-benzopyran-4-one
(1). Yield
48%. White needle. mp 142–144 °C. 1H NMR (CDCl3, 400 MHz) δ
8.21 (1H, dd, J = 7.9, 1.7 Hz, H-5), 7.69 (1H, ddd, J = 8.4, 7.1, 1.7 Hz,
H-7), 7.63 (1H, d, J = 16.0 Hz, H-β), 7.62–7.59 (2H, m, H-2′, H-6′),
7.55 (1H, dd, J = 8.4, 1.1 Hz, H-8), 7.46–7.37 (4H, m, H-6, H-3′, H-4′,
H-5′), 6.81 (1H, d, J = 16.0 Hz, H-α), 6.34 (1H, s, H-3). MS (EI) m/z
248 [M]+. The 1H NMR spectrum was similar to that previously
reported [27].
4.1.4. 2-Methyl-7-methoxy-4H-1-benzopyran-4-one (IIc)
Yield 48%. Pale yellow scaly crystal. mp 109–110 °C (lit. [19]
106–108 °C). 1H NMR (CDCl3, 400 MHz) δ 8.08 (1H, d, J = 8.9 Hz, H-
5), 6.94 (1H, dd, J = 8.9, 2.4 Hz, H-6), 6.82 (1H, d, J = 2.4 Hz, H-8),
6.11 (1H, s, H-3), 3.90 (3H, s, OMe), 2.36 (3H, s, Me). MS (EI) m/z 190
[M]+
.
4.1.5. Synthesis of (E)-2-styryl-4H-1-benzopyran-4-ones (1–18)
(E)-2-Styryl-4H-1-benzopyran-4-ones (1–18) were synthesized by
modifying a previously reported procedure [19]. To a solution of
NaOMe (12 mmol) in dry MeOH (12 mL), the corresponding 2-
4.1.5.2. 2-[(1E)-2-(4-Fluorophenyl)ethenyl]-4H-1-benzopyran-4-one
(2). Yield 60%. Pale yellow needle. mp 195–198 °C. 1H NMR (CDCl3,
400 MHz) δ 8.20 (1H, dd, J = 7.9, 1.7 Hz, H-5), 7.69 (1H, ddd, J = 8.4,
7

K. Takao, et al.
BioorganicChemistry92(2019)103285
2
7.1, 1.7 Hz, H-7), 7.62–7.57 (2H, m, H-2′, H-6′), 7.58 (1H, d,
J = 16.0 Hz, H-β), 7.53 (1H, dd, J = 8.5, 1.1 Hz, H-8), 7.40 (1H, ddd,
J = 7.9, 7.1, 1.1 Hz, H-6), 7.16–7.10 (2H, m, H-3′, H-5′), 6.72 (1H, d,
J = 16.0 Hz, H-α), 6.33 (1H, s, H-3). MS (EI) m/z 266 [M]+. The 1H
NMR spectrum was similar to that previously reported [28].
(C-α), 119.2 (C-8), 116.2 (d, JC-F = 22 Hz, C-3′, C-5′), 109.9 (C-3),
105.0 (C-5), 55.9 (OMe). MS (EI) m/z 296 [M]+. Anal. Calcd for
C
18H13FO3: C, 72.97; H, 4.42. Found: C, 73.16; H, 4.56.
4.1.5.9. 2-[(1E)-2-(4-Chlorophenyl)ethenyl]-6-methoxy-4H-1-
benzopyran-4-one (9). Yield 98%. Pale yellow needle. mp 200–205 °C.
1H NMR (CDCl3, 400 MHz) δ 7.57 (1H, d, J = 3.1 Hz, H-5), 7.55 (1H, d,
J = 16.0 Hz, H-β), 7.52 (2H, d, J = 8.5 Hz, H-2′, H-6′), 7.47 (1H, d,
J = 9.1 Hz, H-8), 7.41 (2H, d, J = 8.5 Hz, H-3′, H-5′), 7.28 (1H, dd,
J = 9.1, 3.1 Hz, H-7), 6.77 (1H, d, J = 16.0 Hz, H-α), 6.33 (1H, s, H-3),
3.90 (3H, s, OMe). 13C NMR (CDCl3, 100 MHz) δ 178.3 (C-4), 161.2 (C-
2), 156.9 (C-6), 150.8 (C-9), 135.7 (C-4′), 135.3 (C-β), 133.6 (C-1′),
129.3 (C-3′, C-5′), 128.8 (C-2′, C-6′), 124.7 (C-10), 123.8 (C-7), 120.9
(C-α), 119.3 (C-8), 110.2 (C-3), 105.0 (C-5), 56.0 (OMe). MS (EI) m/z
312 [M]+. Anal. Calcd for C18H13ClO3: C, 69.13; H, 4.19. Found: C,
69.03; H, 4.23.
4.1.5.3. 2-[(1E)-2-(4-Chlorophenyl)ethenyl]-4H-1-benzopyran-4-one
(3). Yield 70%. Pale yellow needle. mp 224–226 °C. 1H NMR (CDCl3,
400 MHz) δ 8.20 (1H, dd, J = 7.9, 1.7, H-5), 7.70 (1H, dd, J = 8.4,
7.1 Hz, H-7), 7.57 (1H, d, J = 16.0 Hz, H-β), 7.54 (1H, dd, J = 8.4,
1.1 Hz, H-8), 7.53 (2H, d, J = 8.4 Hz, H-2′, H-6′), 7.41 (1H, ddd,
J = 7.9, 7.1, 1.1 Hz, H-6), 7.40 (2H, d, J = 8.4 Hz, H-3′, H-5′), 6.77
(1H, d, J = 16.0 Hz, H-α), 6.34 (1H, s, H-3). MS (EI) m/z 282 [M]+. The
1H NMR spectrum was similar to that previously reported [29].
4.1.5.4. 2-[(1E)-2-(4-Bromophenyl)ethenyl]-4H-1-benzopyran-4-one
(4). Yield 94%. Pale yellow needle. mp 212–216 °C. 1H NMR (CDCl3,
400 MHz) δ 8.20 (1H, dd, J = 7.9, 1.7 Hz, H-5), 7.69 (1H, ddd, J = 8.4,
7.1, 1.7 Hz, H-7), 7.56 (2H, d, J = 8.4 Hz, H-2′, H-6′), 7.55 (1H, d,
J = 16.0 Hz, H-β), 7.53 (1H, dd, J = 8.4, 1.1 Hz, H-8), 7.46 (2H, dd,
J = 8.4 Hz, H-3′, H-5′), 7.41 (1H, ddd, J = 7.9, 7.1, 1.1 Hz, H-6), 6.79
(1H, d, J = 16.0 Hz, H-α), 6.34 (1H, s, H-3). MS (EI) m/z 326 [M]+. The
1H NMR spectrum was similar to that previously reported [27].
4.1.5.10. 2-[(1E)-2-(4-Bromophenyl)ethenyl]-6-methoxy-4H-1-
benzopyran-4-one (10). Yield 97%. White needle. mp 204–210 °C. 1H
NMR (CDCl3, 400 MHz) δ 7.57 (1H, d, J = 3.1 Hz, H-5), 7.56 (2H, d,
J = 8.5 Hz, H-2′, H-6′), 7.53 (1H, d, J = 16.0 Hz, H-β), 7.47 (1H, d,
J = 9.1 Hz, H-8), 7.45 (2H, d, J = 8.5 Hz, H-3′, H-5′), 7.28 (1H, dd,
J = 9.1, 3.1 Hz, H-7), 6.78 (1H, d, J = 16.1 Hz, H-α), 6.33 (1H, s, H-3),
3.91 (3H, s, OMe). 13C NMR (CDCl3, 100 MHz) δ 178.3 (C-4), 161.1 (C-
2), 156.9 (C-6), 150.8 (C-9), 135.3 (C-β), 134.0 (C-1′), 132.2 (C-3′, C-
5′), 129.0 (C-2′, C-6′), 124.7 (C-10), 123.9 (C-4′), 123.8 (C-7), 121.0 (C-
α), 119.3 (C-8), 110.2 (C-3), 105.0 (C-5), 56.0 (OMe). MS (EI) m/z 356
[M]+. Anal. Calcd for C18H13BrO3: C, 60.52; H, 3.67. Found: C, 60.32;
H, 3.68.
4.1.5.5. 2-[(1E)-2-(4-Methoxyphenyl)ethenyl]-4H-1-benzopyran-4-one
(5). Yield 48%. Yellow needle. mp 139–140 °C. 1H NMR (CDCl3,
400 MHz) δ 8.20 (1H, dd, J = 7.9, 1.7 Hz, H-5), 7.68 (1H, ddd,
J = 8.4, 7.1, 1.7 Hz, H-7), 7.58 (1H, d, J = 15.9 Hz, H-β), 7.55 (2H,
d, J = 8.7 Hz, H-2′, H-6′), 7.53 (1H, dd, J = 8.4, 1.1 Hz, H-8), 7.39 (1H,
ddd, J = 7.9, 7.1, 1.1 Hz, H-6), 6.95 (2H, d, J = 8.7 Hz, H-3′, H-5′),
6.67 (1H, d, J = 15.9 Hz, H-α), 6.31 (1H, s, H-3), 3.87 (3H, s, OMe). MS
(EI) m/z 278 [M]+. The 1H NMR spectrum was similar to that
previously reported [29].
4.1.5.11. 6-Methoxy-2-[(1E)-2-(4-methoxyphenyl)ethenyl]-4H-1-
benzopyran-4-one (11). Yield 73%. Yellow needle. mp 138–140 °C. 1H
NMR (CDCl3, 400 MHz) δ 7.57 (1H, d, J = 3.1 Hz, H-5), 7.55 (1H, d,
J = 16.0 Hz, H-β), 7.54 (2H, d, J = 8.6 Hz, H-2′, H-6′), 7.46 (1H, d,
J = 9.1 Hz, H-8), 7.26 (1H, dd, J = 9.1, 3.1 Hz, H-7), 6.94 (2H, d,
J = 8.6 Hz, H-3′, H-5′), 6.66 (1H, d, J = 16.0 Hz, H-α), 6.29 (1H, s, H-
3), 3.90 (3H, s, OMe), 3.86 (3H, s, OMe). MS (EI) m/z 308 [M]+. The 1H
NMR spectrum was similar to that previously reported [30].
4.1.5.6. 2-[(1E)-2-(3,4-Dimethoxy)ethenyl]-4H-1-benzopyran-4-one
(6). Yield 58%. Yellow needle. mp 165–166 °C. 1H NMR (CDCl3,
400 MHz) δ 8.20 (1H, dd, J = 7.9, 1.7 Hz, H-5), 7.68 (1H, ddd,
J = 8.4, 7.1, 1.7 Hz, H-7), 7.57 (1H, d, J = 16.0 Hz, H-β), 7.53 (1H,
dd, J = 8.4, 1.1 Hz, H-8), 7.39 (1H, ddd, J = 7.9, 7.1, 1.1 Hz, H-6), 7.17
(1H, dd, J = 8.3, 2.0 Hz, H-6′), 7.11 (1H, d, J = 2.0 Hz, H-2′), 6.91 (1H,
d, J = 8.3 Hz, H-5′), 6.66 (1H, d, J = 16.0 Hz, H-α), 6.32 (1H, s, H-3),
3.98 (3H, s, OMe), 3.94 (3H, s, OMe). MS (EI) m/z 308 [M]+. The 1H
NMR spectrum was similar to that previously reported [29].
4.1.5.12. 2-[(1E)-2-(3,4-Dimethoxy)ethenyl]-6-methoxy-4H-1-
benzopyran-4-one (12). Yield 78%. Yellow needle. mp 159–161 °C. 1H
NMR (CDCl3, 400 MHz) δ 7.58 (1H, d, J = 3.1 Hz, H-5), 7.54 (1H, d,
J = 16.0 Hz, H-β), 7.46 (1H, d, J = 9.1 Hz, H-8), 7.27 (1H, dd, J = 9.1,
3.1 Hz, H-7), 7.17 (1H, dd, J = 8.3, 2.0 Hz, H-6′), 7.11 (1H, d,
J = 2.0 Hz, H-2′), 6.91 (1H, d, J = 8.3 Hz, H-5′), 6.65 (1H, d,
J = 16.0 Hz, H-α), 6.31 (1H, s, H-3) , 3.97 (3H, s, OMe), 3.94 (3H, s,
OMe), 3.91 (3H, s, OMe), 13C NMR (CDCl3, 100 MHz) δ 178.3 (C-4),
161.9 (C-2), 156.8 (C-6), 150.8 (C-9, C-4′), 149.3 (C-3′), 136.7 (C-β),
128.1 (C-1′), 124.7 (C-10), 123.6 (C-7), 122.0 (C-6′), 119.2 (C-8), 118.1
(C-α), 111.2 (C-5′), 109.3 (C-3, C-2′), 105.0 (C-5), 56.01 (OMe), 55.99
(OMe), 55.95 (OMe). MS (EI) m/z 338 [M]+. Anal. Calcd for C20H18O5:
C, 71.00; H, 5.36. Found: C, 71.09; H, 5.24.
4.1.5.7. 6-Methoxy-2-[(1E)-2-phenylethenyl]-4H-1-benzopyran-4-one
(7). Yield 71%. Yellow needle. mp 139–140 °C. 1H NMR (CDCl3,
400 MHz) δ 7.61–7.58 (2H, m, H-2′, H-6′), 7.60 (1H, d, J = 16.1 Hz,
H-β), 7.58 (1H, d, J = 3.1 Hz, H-5), 7.48 (1H, d, J = 9.1 Hz, H-8),
7.46–7.36 (3H, m, H-3′, H-4′, H-5′), 7.28 (1H, dd, J = 9.1, 3.1 Hz, H-7),
6.80 (1H, d, J = 16.1 Hz, H-α), 6.33 (1H, s, H-3), 3.91 (3H, s, OMe). 13
C
NMR (CDCl3, 100 MHz) δ 178.3 (C-4), 161.5 (C-2), 156.8 (C-6), 150.8
(C-9), 136.7 (C-β), 135.0 (C-1′), 129.8 (C-4′), 129.0 (C-3′, C-5′), 127.6
(C-2′, C-6′), 124.7 (C-10), 123.7 (C-7), 120.4 (C-α), 119.3 (C-8), 109.9
(C-3), 104.9 (C-5), 56.0 (OMe). MS (EI) m/z 278 [M]+. Anal. Calcd for
4.1.5.13. 7-Methoxy-2-[(1E)-2-phenylethenyl]-4H-1-benzopyran-4-one
(13). Yield 77%. Yellow prism. mp 192–194 °C. 1H NMR (CDCl3,
400 MHz) δ 8.10 (1H, d, J = 8.5 Hz, H-5), 7.61–7.57 (2H, m, H-2′, H-
6′), 7.58 (1H, d, J = 16.1 Hz, H-β), 7.46–7.36 (3H, m, H-3′, H-4′, H-5′),
6.98–6.94 (2H, m, H-6, H-8), 6.77 (1H, d, J = 16.1 Hz, H-α), 6.27 (1H,
s, H-3), 3.94 (3H, s, OMe); MS (EI) m/z 278 [M]+. The 1H NMR
spectrum was similar to that previously reported [31].
C
18H14O3: C, 77.68; H, 5.07. Found: C, 77.91; H, 5.16. MS (EI) m/z 278
[M]+
.
4.1.5.8. 2-[(1E)-2-(4-Fluorophenyl)ethenyl]-6-methoxy-4H-1-
benzopyran-4-one (8). Yield 89%. Pale yellow needle. mp 147–150 °C.
1H NMR (CDCl3, 400 MHz) δ 7.60–7.54 (3H, m, H-5, H-2′, H-6′), 7.56
(1H, d, J = 16.0 Hz, H-β), 7.47 (1H, d, J = 9.1 Hz, H-8), 7.28 (1H, dd,
J = 9.1, 3.1 Hz, H-7), 7.15–7.09 (2H, m, H-3′, H-5′), 6.71 (1H, d,
J = 16.4 Hz, H-α), 6.32 (1H, s, H-3), 3.91 (3H, s, OMe). 13C NMR
4.1.5.14. 2-[(1E)-2-(4-Fluorophenyl)ethenyl]-7-methoxy-4H-1-
benzopyran-4-one (14). Yield 66%. Pale light brown needle. mp
170–175 °C. 1H NMR (CDCl3, 400 MHz) δ 8.10 (1H, d, J = 8.8 Hz, H-
5), 7.59–7.54 (2H, m, H-2′, H-6′), 7.54 (1H, d, J = 15.9 Hz, H-β),
7.15–7.19 (2H, m, H-3′, H-5′), 6.97 (1H, dd, J = 8.8, 2.4 Hz, H-6), 6.94
1
(CDCl3, 100 MHz) δ 178.3 (C-4), 163.6 (d, JC-F = 250 Hz, C-4′), 161.3
4
(C-2), 156.9 (C-6), 150.8 (C-9), 135.4 (C-β), 131.3 (d, JC-F = 4 Hz, C-
1′), 129.5 (d, 3JC-F = 8 Hz, C-2′, C-6′), 124.7 (C-10), 123.7 (C-7), 120.1
8

K. Takao, et al.
BioorganicChemistry92(2019)103285
(1H, d, J = 2.4 Hz, H-8), 6.69 (1H, d, J = 15.9 Hz, H-α), 6.26 (1H, s, H-
3), 3.94 (3H, s, OMe). 13C NMR (CDCl3, 100 MHz) δ 177.8 (C-4), 164.2
4.3. MAO inhibitory assay
1
(C-7), 163.5 (d, JC-F = 250 Hz, C-4′), 161.2 (C-2), 157.7 (C-9), 135.0
MAO inhibitory activity was assayed using the method of Novaroli
et al. [32] with minor modifications. Briefly, 140 μL of 0.1 M potassium
phosphate buffer (pH 7.4), 8 μL of 0.75 mM kynuramine, and 2 μL of a
dimethyl sulfoxide (DMSO) inhibitor solution, were preincubated at
37 °C for 10 min. Diluted human recombinant enzyme (50 μL) was then
added to obtain a final protein concentration of 0.0075 mg/mL (MAO-
A) or 0.015 mg/mL (MAO-B) in the assay mixture. The reaction mixture
was further incubated at 37 °C and the reaction was stopped after
20 min by the addition of 75 μL of 2 M NaOH. The product generated by
MAO, 4-quinolinol, is fluorescent and was measured at Ex 310 nm/Em
400 nm using a microplate reader (Molecular Devices SPECTRA MAX
M2). Each data point was measured from triplicate samples. The sample
solution was replaced with DMSO to provide a negative control and
pargyline was used as a positive control. The IC50 values were calcu-
lated from a line through two points that sandwiched the point corre-
sponding to 50% inhibition (IC50) by plotting the remained activity (%)
related to the control (100%) versus the logarithm of the inhibitor
concentration to obtain a sigmoidal dose-response curve.
4
3
(C-β), 131.4 (d, JC-F = 4 Hz, C-1′), 129.4 (d, JC-F = 8 Hz, C-2′, C-6′),
127.1 (C-5), 120.1 (C-α), 118.0 (C-10), 116.1 (d, 2JC-F = 22 Hz, C-3′, C-
5′), 114.1 (C-6), 110.7 (C-3), 100.3 (C-8), 55.8 (OMe). MS (EI) m/z 296
[M]+. Anal. Calcd for C18H13FO3: C, 72.97; H, 4.42. Found: C, 73.11; H,
4.52.
4.1.5.15. 2-[(1E)-2-(4-Chlorophenyl)ethenyl]-7-methoxy-4H-1-
benzopyran-4-one (15). Yield 95%. Pale light brown needle. mp
199–203 °C. 1H NMR (CDCl3, 400 MHz) δ 8.10 (1H, d, J = 8.8 Hz, H-
5), 7.52 (1H, d, J = 16.0 Hz, H-β), 7.51 (2H, d, J = 8.6 Hz, H-2′, H-6′),
7.39 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.97 (1H, dd, J = 8.8, 2.4 Hz, H-6),
6.93 (1H, d, J = 2.4 Hz, H-8), 6.74 (1H, d, J = 16.0 Hz, H-α), 6.27 (1H,
s, H-3), 3.94 (3H, s, OMe). 13C NMR (CDCl3, 100 MHz) δ 177.8 (C-4),
164.2 (C-7), 161.0 (C-2), 157.7 (C-9), 135.6 (C-4′), 134.9 (C-β), 133.6
(C-1′), 129.3 (C-3′, C-5′), 128.7 (C-2′, C-6′), 127.1 (C-5), 120.9 (C-α),
118.0 (C-10), 114.1 (C-6), 111.0 (C-3), 100.3 (C-8), 55.9 (OMe). MS
(EI) m/z 312 [M]+. Anal. Calcd for C18H13ClO3: C, 69.13; H, 4.19.
Found: C, 69.00; H, 4.15.
4.4. Lineweaver–Burk plots
4.1.5.16. 2-[(1E)-2-(4-Bromophenyl)ethenyl]-7-methoxy-4H-1-
benzopyran-4-one (16). Yield 98%. White needle. mp 204–210 °C. 1H
NMR (CDCl3, 400 MHz) δ 8.10 (1H, d, J = 8.8 Hz, H-5), 7.56 (2H, d,
J = 8.5 Hz, H-2′, H-6′), 7.51 (1H, d, J = 16.0 Hz, H-β), 7.44 (2H, d,
J = 8.5 Hz, H-3′, H-5′), 6.97 (1H, dd, J = 8.8, 2.4 Hz, H-6), 6.93 (1H, d,
J = 2.4 Hz, H-8), 6.94 (1H, d, J = 2.4 Hz, H-8), 6.76 (1H, d,
J = 16.0 Hz, H-α), 6.27 (1H, s, H-3), 3.94 (3H, s, OMe). 13C NMR
(CDCl3, 100 MHz) δ 177.8 (C-4), 164.2 (C-7), 160.9 (C-2), 157.7 (C-9),
134.9 (C-β), 134.0 (C-1′), 132.2 (C-3′, C-5′), 129.0 (C-2′, C-6′), 127.1
(C-5), 123.9 (C-4′), 121.0 (C-α), 118.0 (C-10), 114.1 (C-6), 111.0 (C-3),
100.3 (C-8), 55.8 (OMe). MS (EI) m/z 356 [M]+. Anal. Calcd for
This analysis was conducted according to the method reported by
Meiring et al. [20]. The inhibition of MAO-B by compound 9 was de-
termined by constructing a set of four Lineweaver–Burk plots. The first
plot was constructed in the absence of inhibitor and the remaining three
plots were constructed in the presence of various concentrations of the
test inhibitor: 1/4 × IC50
,
1/2 × IC50
,
1 × IC50 and 2 × IC50
(IC50 = 0.017 μM). The enzyme substrate kynuramine was used at
concentrations ranging from 3.75 to 120 μM.
4.5. Analysis of the reversibility of MAO-B inhibition (dilution method).
C
18H13BrO3: C, 60.52; H, 3.67. Found: C, 60.77; H, 3.65.
This analysis was conducted according to the method reported by
Meiring et al. [20]. Briefly, compound 9 (IC50 = 0.017 μM) at con-
centrations equal to 10 × IC50 and 100 × IC50 was preincubated with
MAO-B (0.15 mg/mL) for 30 min at 37 °C. These preincubations con-
tained DMSO (4%) as co-solvent. The reaction mixtures were subse-
quently diluted 10-fold by adding a solution of kynuramine to yield
final concentrations of the test inhibitor equal to 1 × IC50 and
0.1 × IC50. After dilution, the final concentration of kynuramine was
30 μM and the final concentration of MAO-B was 0.015 mg/mL. The
reaction mixtures were incubated for a further 20 min at 37 °C, the
reactions were terminated, and the formation of 4-hydroxyquinoline
was measured as described above. For comparison, pargyline
(IC50 = 0.22 μM) at a concentration of 10 × IC50 was similarly pre-
incubated with MAO-B, diluted to 1 × IC50, and residual MAO-B ac-
tivity was measured as above. Similar reactions served as controls and
were conducted in the absence of inhibitor.
4.1.5.17. 7-Methoxy-2-[(1E)-2-(4-methoxyphenyl)ethenyl]-4H-1-
benzopyran-4-one (17). Yield 67%. Brown needle. mp 128–129 °C. 1H
NMR (CDCl3, 400 MHz) δ 8.10 (1H, d, J = 8.6 Hz, H-5), 7.54 (1H, d,
J = 16.0 Hz, H-β), 7.53 (2H, d, J = 8.9 Hz, H-2′, H-6′), 6.98–6.93 (2H, m,
H-6, H-8), 6.95 (2H, d, J = 8.9 Hz, H-3′, H-5′), 6.64 (1H, d, J = 16.0 Hz,
H-α), 6.23 (1H, s, H-3), 3.93 (3H, s, OMe), 3.86 (3H, s, OMe). 13C NMR
(CDCl3, 100 MHz) δ 177.9 (C-4), 164.1 (C-7), 161.8 (C-4′), 161.0 (C-2),
157.7 (C-9), 136.0 (C-β), 129.2 (C-2′, C-6′), 127.9 (C-1′), 127.0 (C-5),
118.04 (C-10), 117.96 (C-α), 114.5 (C-3′, C-5′), 113.9 (C-6), 109.9 (C-3),
100.3 (C-8), 55.8 (OMe), 55.4 (OMe). MS (EI) m/z 308 [M]+. Anal. Calcd
for C19H16O4: C, 74.01; H, 5.23. Found: C, 74.16; H, 5.21.
4.1.5.18. 2-[(1E)-2-(3,4-Dimethoxy)ethenyl]-7-methoxy-4H-1-
benzopyran-4-one (18). Yield 67%. Yellow needle. mp 198–199 °C. 1H
NMR (CDCl3, 400 MHz) δ 8.10 (1H, d, J = 8.7 Hz, H-5), 7.52 (1H, d,
J = 16.0 Hz, H-β), 7.17 (1H, dd, J = 8.3, 2.0 Hz, H-6′), 7.10 (1H, d,
J = 2.0 Hz, H-2′), 6.96 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.93 (1H, d,
J = 2.3 Hz, H-8), 6.91 (1H, d, J = 8.3 Hz, H-5′), 6.63 (1H, d,
J = 16.0 Hz, H-α), 6.25 (1H, s, H-3), 3.97 (3H, s, OMe), 3.93 (6H, s,
OMe). 13C NMR (CDCl3, 100 MHz) δ 177.9 (C-4), 164.1 (C-7), 161.7 (C-
2), 157.7 (C-9), 150.7 (C-4′), 149.3 (C-3′), 136.3 (C-β), 128.1 (C-1′),
127.1 (C-5), 122.0 (C-6′), 118.1 (C-α), 118.0 (C-10), 113.9 (C-6), 111.2
(C-5′), 110.0 (C-3), 109.3 (C-2′), 100.3 (C-8), 56.01 (OMe), 55.99
(OMe), 55.8 (OMe). MS (EI) m/z 338 [M]+. Anal. Calcd for C20H18O5:
C, 71.00; H, 5.36. Found: C, 71.09; H, 5.32.
4.6. Calculation of chemical descriptors
Each 3D chemical structure (Marvin Sketch ver 16; ChemAxon,
Budapest, Hungary, http://www.chemaxon.com) was optimized by
CORINA Classic (Molecular Networks GmbH, Nürnberg, Germany) with
forcefield calculations (amber-10: EHT) in Molecular Operating
Environment (MOE) version 2018.0101 (Chemical Computing Group
Inc., Quebec, Canada). The number of structural descriptors calculated
from MOE [21] and Dragon 7.0 [22] (Kode srl., Pisa, Italy) was 344 and
5255, respectively, of which 279 and 2827 (total 3106) descriptors
were used for analysis.
4.2. Biological assays
Recombinant human monoamine oxidase A (MAO-A), recombinant
human MAO-B, pargyline and kynuramine were purchased from Sigma-
Aldrich Japan Co., Tokyo, Japan.
4.7. 3D-QSAR and docking analyses
AutoGPA in MOE can automatically generate 3D-QSAR models
9

K. Takao, et al.
BioorganicChemistry92(2019)103285
based on the chemical structures and biological activities for sets of
inhibitors [24,25]. The CoMFA analysis algorithm was employed to
develop 3D-QSAR models [23]. Docking analysis was carried out with
MAO-B protein (PDB code 4A79) using MOE.
[12] M. Yáñez, N. Fraiz, E. Cano, F. Orallo, Inhibitory effects of cis- and trans-resveratrol
on noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase
activity, Biochem. Biophys. Res. Commun. 344 (2) (2006) 688–695.
[13] A. Ozaita, G. Olmos, M.A. Boronat, J.M. Lizcano, M. Unzeta, J.A. García-Sevilla,
Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine
drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat
liver, Br. J. Pharmacol. 121 (5) (1997) 901–912.
4.8. Statistical analysis
[14] J.F. Chen, S. Steyn, R. Staal, J.P. Petzer, K. Xu, C.J. Van Der Schyf, K. Castagnoli,
P.K. Sonsalla, N. Castagnoli Jr, M.A. Schwarzschild, 8-(3-Chlorostyryl)caffeine may
attenuate MPTP neurotoxicity through dual actions of monoamine oxidase inhibi-
tion and A2A receptor antagonism, J. Biol. Chem. 277 (39) (2002) 36040–36044.
[15] D. Van den Berg, K.R. Zoellner, M.O. Ogunrombi, S.F. Malan, G. Terre'Blanche,
N. Castagnoli Jr, J.J. Bergh, J.P. Petzer, Inhibition of monoamine oxidase B by
selected benzimidazole and caffeine analogues, Bioorg. Med. Chem. 15 (11) (2007)
3692–3702.
The relation between the substituents and the MAO related prop-
erties such as IC50 values and MAO-B selectivity was analyzed with
Wilcoxon signed-rank test. The relation between the chemical de-
scriptors and the MAO-B related properties was investigated using
Pearson's correlation analysis. These statistical calculations were per-
formed by JMP Pro version 14.0.0 (SAS Institute Inc., Cary, NC, USA).
The significance level was set at p < 0.05.
[16] E.M. Van der Walt, E.M. Milczek, S.F. Malan, D.E. Edmondson, N. Castagnoli Jr,
J.J. Bergh, J.P. Petzer, Inhibition of monoamine oxidase by (E)-styrylisatin analo-
gues, Bioorg. Med. Chem. Lett. 19 (9) (2009,) 2509–2513, https://doi.org/10.
1016/j.bmcl.2009.03.030.
[17] M.M. Van der Walt, G. Terre'Blanche, A. Petzer, A.C. Lourens, J.P. Petzer, The
adenosine A(2A) antagonistic properties of selected C8-substituted xanthines,
Bioorg. Chem. 49 (2013) 49–58, https://doi.org/10.1016/j.bioorg.2013.06.006.
[18] K. Takao, H. Yahagi, Y. Uesawa, Y. Sugita, 3-(E)-Styryl-2H-chromene derivatives as
potent and selective monoamine oxidase B inhibitors, Bioorg. Chem. 77 (2018)
436–442, https://doi.org/10.1016/j.bioorg.2018.01.036.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103285.
[19] A.Y. Shaw, C.Y. Chang, H.H. Liau, P.J. Lu, H.L. Chen, C.N. Yang, H.Y. Li, Synthesis
of 2-styrylchromones as a novel class of antiproliferative agents targeting carci-
noma cells, Eur. J. Med. Chem. 44 (6) (2009) 2552–2562, https://doi.org/10.1016/
j.ejmech.2009.01.034.
References
[1] A.S. Kalgutkar, N. Castagnoli Jr., Selective inhibitors of monoamine oxidase (MAO-
A and MAO-B) as probes of its catalytic site and mechanism, Med. Res. Rev. 15 (4)
(1995) 325–388.
[20] L. Meiring, J.P. Petzer, A. Petzer, Inhibition of monoamine oxidase by 3,4-dihydro-
2(1H)-quinolinone derivatives, Bioorg. Med. Chem. Lett. 23 (20) (2013)
5498–5502, https://doi.org/10.1016/j.bmcl.2013.08.071.
[2] D.E. Edmondson, L. DeColibus, C. Binda, M. Li, A. Mattevi, New insights into the
structures and functions of human monoamine oxidases A and B, J. Neural Transm.
114 (6) (2007) 703–705, https://doi.org/10.1007/s00702-007-0674-z.
[3] L. De Colibus, M. Li, C. Binda, A. Lustig, D.E. Edmondson, A. Mattevi, Three-di-
mensional structure of human monoamine oxidase A (MAO A): relation to the
structures of rat MAO A and human MAO B, Proc. Natl. Acad. Sci. U. S. A. 102 (36)
(2005) 12684–12689 g 29.
[21] Calculate Descriptors, MOE2015.10 on-line help manual, Chemical Computing
Group.
[22] https://chm.kode-solutions.net/products_dragon_descriptors.php.
[23] R.D. Cramer, D.E. Patterson, J.D. Bunce, Comparative molecular field analysis
(CoMFA). 1. Effect of shape on binding of steroids to carrier proteins, J. Am. Chem.
Soc. 110 (18) (1988) 5959–5967, https://doi.org/10.1021/ja00226a005.
[24] N. Asakawa, S. Kobayashi, J. Goto, N. Hirayama, AutoGPA: an automated 3D-QSAR
method based on pharmacophore alignment and grid potential analysis, Int. J. Med.
Chem. 2012 (2012), https://doi.org/10.1155/2012/498931.
[4] S. Schedin-Weiss, M. Inoue, L. Hromadkova, Y. Teranishi, N.G. Yamamoto,
B. Wiehager, N. Bogdanovic, B. Winblad, A. Sandebring-Matton, S. Frykman,
L.O. Tjernberg, Monoamine oxidase B is elevated in Alzheimer disease neurons, is
associated with γ-secretase and regulates neuronal amyloid β-peptide levels,
Alzheimers Res. Ther. 9 (1) (2017) 57, https://doi.org/10.1186/s13195-017-
0279-1.
[25] F.Y. Guo, Q.Y. Yan, K. Lin, W.Y. Hong, G.S. Yang, AutoGPA-based 3D-QSAR mod-
eling and molecular docking study on factor Xa inhibitors as anticoagulant agents,
MATEC Web Conf. 44 (2016) 02018.
[26] L.G. Iacovino, F. Magnani, C. Binda, The structure of monoamine oxidases: past,
present, and future, J. Neural. Transm. (Vienna) 125 (11) (2018) 1567–1579,
https://doi.org/10.1007/s00702-018-1915-z.
[5] A. Gaspar, M.J. Matos, J. Garrido, E. Uriarte, F. Borges, Chromone: a valid scaffold
in medicinal chemistry, Chem. Rev. (114(9), 2014,) 4960–4992, https://doi.org/
10.1021/cr400265z.
[27] W. Huang, Y. Ding, Y. Miao, M.Z. Liu, Y. Li, G.F. Yang, Synthesis and antitumor
activity of novel dithiocarbamate substituted chromones, Eur. J. Med. Chem. 44 (9)
(2009,) 3687–12696, https://doi.org/10.1016/j.ejmech.2009.04.004.
[28] M. Momin, D. Ramjugernath, N.A. Koorbanally, Structure elucidation of a series of
fluoro-2-styrylchromones and methoxy-2-styrylchromones using 1D and 2D NMR
spectroscopy, Magn. Reson. Chem. 52 (2014) 521–529.
[6] J. Reis, A. Gaspar, N. Milhazes, F. Borges, Chromone as a privileged scaffold in drug
discovery, Recent Adv., J. Med. Chem. 60 (19) (2017) 7941–7957, https://doi.org/
10.1021/acs.jmedchem.6b01720.
[7] A. Gaspar, T. Silva, M. Yáñez, D. Vina, F. Orallo, F. Ortuso, E. Uriarte, S. Alcaro,
F. Borges, Chromone a privileged scaffold for the development of monoamine
oxidase inhibitors, J. Med. Chem. 54 (14) (2011) 5165–5173, https://doi.org/10.
1021/jm2004267.
[29] C. Lin, P.-J. Lu, C.-N. Yang, C. Hulme, A.Y. Shaw, Structure-activity relationship
study of growth inhibitory 2-styrylchromones against carcinoma cells, Med. Chem.
Res. 22 (5) (2013) 2385–2394.
[8] C.M.M. Santos, A.M.S. Silva, An overview of 2-styrylchromones: natural occurrence,
synthesis, reactivity and biological properties, Eur. J. Org. Chem. 2017 (22) (2017)
3115–3133.
[30] Rao V. Madhava, B. Ujwala, P. Priyadarshini, Murthy P. Krishna, Synthesis, anti-
oxidant and antimicrobial activity of three new 2-styrylchromones and their ana-
logues, Der Pharma Chemica 8 (7) (2016) 1–6 ISSN 0975-413X.
[9] A. Gomes, M. Freitas, E. Fernandes, J.L. Lima, Biological activities of 2-styr-
ylchromones, Mini Rev. Med. Chem. 10 (1) (2010) 1–7.
[31] S.R. Goel, J.K. Makrandi, Microwave assisted synthesis of 1-(2-hydroxyphenyl)-5-
phenyl pent-4-ene-1,3-diones and their conversion to 2-styryl chromones, Ind. J.
Chem. 45B (5) (2006) 1278–1281.
[10] M. Koushki, N. Amiri-Dashatan, N. Ahmadi, H.A. Abbaszadeh, M. Rezaei-Tavirani,
Resveratrol: A miraculous natural compound for diseases treatment, Food Sci. Nutr.
6 (8) (2018) 2473–2490, https://doi.org/10.1002/fsn3.855 eCollection 2018 Nov.
[11] B. Salehi, A.P. Mishra, M. Nigam, B. Sener, M. Kilic, M. Sharifi-Rad, P.V.T. Fokou,
N. Martins, J. Sharifi-Rad, Resveratrol: a double-edged sword in health benefits, pii:
E91, Biomedicines 6 (3) (2018), https://doi.org/10.3390/biomedicines6030091.
[32] L. Novaroli, M. Reist, E. Favre, A. Carotti, M. Catto, P.A. Carrupt, Human re-
combinant monoamine oxidase B as reliable and efficient enzyme source for in-
hibitor screening, Bioorg. Med. Chem. 13 (22) (2005) 6212–6217.
10