Novel C7-substituted coumarins as selective monoamine oxidase inhibitors: Discovery, synthesis and theoretical simulation

Article abstract of DOI:10.3390/molecules24214003

There is a continued need to develop new selective human monoamine oxidase (hMAO) inhibitors that could be beneficial for the treatment of neurological diseases. However, hMAOs are closely related with high sequence identity and structural similarity, which hinders the development of selective MAO inhibitors. “Three-Dimensional Biologically Relevant Spectrum (BRS-3D)” method developed by our group has demonstrated its effectiveness in subtype selectivity studies of receptor and enzyme ligands. Here, we report a series of novel C7-substituted coumarins, either synthesized or commercially purchased, which were identified as selective hMAO inhibitors. Most of the compounds demonstrated strong activities with IC₅₀ values (half-inhibitory concentration) ranging from sub-micromolar to nanomolar. Compounds, FR1 and SP1, were identified as the most selective hMAO-A inhibitors, with IC₅₀ values of 1.5 nM (selectivity index (SI) < ?2.82) and 19 nM (SI < ?2.42), respectively. FR4 and FR5 showed the most potent hMAO-B inhibitory activity, with IC₅₀ of 18 nM and 15 nM (SI > 2.74 and SI > 2.82). Docking calculations and molecular dynamic simulations were performed to elucidate the selectivity preference and SAR profiles.

Full text of DOI:10.3390/molecules24214003

molecules
Article
Novel C7-Substituted Coumarins as Selective
Monoamine Oxidase Inhibitors: Discovery,
Synthesis and Theoretical Simulation
Dong Wang 1,2,†, Ren-Yuan Hong 3,†, Mengyao Guo 2, Yi Liu 2, Nianhang Chen 2, Xun Li 4,
*
and
De-Xin Kong 1,2,
*
1
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,
Wuhan 430070, China; duke.e.wang@gmail.com
Agricultural Bioinformatics Key Laboratory of Hubei Province, College of Informatics,
Huazhong Agricultural University, Wuhan 430070, China; guomengyao96@gmail.com (M.G.);
dengxue@webmail.hzau.edu.cn (Y.L.); nhchen95@gmail.com (N.C.)
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences,
Shandong University, 44 West Culture Road, Ji’nan 250012, Shandong, China; 17865132563@163.com
Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences,
No 18877, Jingshi Road, Ji’nan 250002, Shandong, China
2
3
4
*
Correspondence: tjulx2004@sdu.edu.cn (X.L.); dxkong@mail.hzau.edu.cn (D.-X.K.);
Tel.: +86-531-88382005 (X.L.); +86-27-8728 0877 (D.-X.K.)
†
The first two authors contributed equally.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Orazio Nicolotti
Received: 8 October 2019; Accepted: 31 October 2019; Published: 5 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: There is a continued need to develop new selective human monoamine oxidase (hMAO)
inhibitors that could be beneficial for the treatment of neurological diseases. However, hMAOs are
closely related with high sequence identity and structural similarity, which hinders the development
of selective MAO inhibitors. “Three-Dimensional Biologically Relevant Spectrum (BRS-3D)” method
developed by our group has demonstrated its effectiveness in subtype selectivity studies of receptor
and enzyme ligands. Here, we report a series of novel C7-substituted coumarins, either synthesized
or commercially purchased, which were identified as selective hMAO inhibitors. Most of the
compounds demonstrated strong activities with IC50 values (half-inhibitory concentration) ranging
from sub-micromolar to nanomolar. Compounds, FR1 and SP1, were identified as the most selective
hMAO-A inhibitors, with IC50 values of 1.5 nM (selectivity index (SI) <
−
2.82) and 19 nM (SI < 2.42),
−
respectively. FR4 and FR5 showed the most potent hMAO-B inhibitory activity, with IC50 of 18 nM
and 15 nM (SI > 2.74 and SI > 2.82). Docking calculations and molecular dynamic simulations were
performed to elucidate the selectivity preference and SAR profiles.
Keywords: monoamine oxidase (MAO) inhibitors; BRS-3D; subtype selectivity; molecular dynamic
simulations; in silico pharmacokinetic predictions
1. Introduction
The dynamic stability of amines is maintained by a reuptake mechanism and also the oxidation
process by monoamine oxidases (MAOs) [
an enzyme bound in the mitochondrial outer membrane by a transmembrane helix [
for regulation of biogenic and xenobiotic amines, which includes serotonin, tryptamine, dopamine,
norepinephrine, epinephrine, -phenylethylamine, tyramine and octopamine [ ]. Two MAO subtypes
were identified in mammals, namely MAO-A and MAO-B. They demonstrated different tissue distribution,
1]. MAO is a flavin adenine dinucleotide (FAD) containing
2]. It is responsible
β
3
substrate preference and inhibitor specificity [
1
,
4
]. In addition, different hMAO subtypes were associated
Molecules 2019, 24, 4003; doi:10.3390/molecules24214003
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4003
2 of 17
with different neurological diseases, such as depression, Alzheimer’s disease, Parkinson’s disease and
Huntington’s disease. Decreases in the expression level of hMAO-A with age were observed, which was
proved to be the causes of aggressive behavior [
levels with aging was thought to be an important causative factor in the etiology of neurodegenerative
diseases [ ]. Therefore, hMAO-A and hMAO-B were essential targets for elucidating the mechanism of
monoaminergic pathways and for the treatment of psychiatric disorders and neurodegenerative diseases [ ].
5]. In contrast, a four-fold increase in the hMAO-B expression
6
7
Nevertheless, high sequence identity (72.6%) and structural similarity (superimposed
root-mean-square deviation, RMSD = 0.94) between two hMAO subtypes hampered the discovery
of selective MAO inhibitors (MAOIs) [
subtype-selective MAO inhibitors over the last years [
which have many advantages such as the feasible synthesis and favorable bioavailability, were mainly
designed as potential MAO inhibitors [ 11 14]. Various substitution patterns on the coumarin skeleton
have been studied [14 15]. According to the orientation of pyrone ring of coumarin scaffold in the
4]. Efforts have been devoted for the development of
8
–
10]. For example, coumarin derivatives,
9,
–
,
binding site, most coumarins can be categorized into two groups characterized by substitution positions:
C3-substitution and C7-substitution. Of them, C3-substitution with an acyl function (e.g., carboxamide
or carbohydrazide) or an aryl group, was the most promising [16]. For instance, 3-phenyl substituted
coumarin derivatives displayed strong MAO-B inhibition [17
with other heterocyclic motifs, such as thiophenyl or indolyl, also exhibited strong activity against
MAO-B [ 20 23]. However, as shown in Figure 1, the development of hMAO-B selective inhibitors has
attracted more attention than hMAO-A. There are very few highly potent hMAO-A inhibitors (pIC50
logIC50) > 7). As deposited in ChEMBL v24, 18 coumarins were reported to be selective to hMAO-B
(pIC50 > 7 and SI > 2 (selectivity index = pIC50(MAO-B) pIC50(MAO-A)) and only three coumarins
were selective to hMAO-A with pIC50 > 7 and SI < 2 [24–28].
–19] and the replacement of 3-phenyl
9,
–
(−
−
Figure 1. Inhibition results of C3 and C7-subsituted coumarin derivatives against hMAO-A and
hMAO-B (data collected from ChEMBL v24).
Discovery or design of subtype selective inhibitors is a challenging area due to the high sequential
identity and small structural differences between the pockets of a conserved protein family (GPCRs,
kinases, etc.). In such a case that protein subtypes have small structural differences, the dynamic
profiles of the ligands’ conformation ensemble were believed to play an essential role in subtype
selectivity. Our group recently developed a shape-similarity based protocol called “Three-Dimensional

Molecules 2019, 24, 4003
3 of 17
Biologically Relevant Spectrum (BRS-3D)” [29
,30]. The method was validated in subtype selectivity
studies of receptor and enzyme ligands [31–33]. A spectrum of C7-substituted coumarin analogues
was discovered according to this protocol. The chemical space around these analogues were worthy to
be further explored owing to their interesting structure-activity relationship (SAR) profiles. To this
end, novel C7-substituted coumarin analogues were synthesized or commercially purchased and their
inhibitory potencies were experimentally tested. The results revealed two C7-substituted coumarin
compounds, FR1 and SP1, which gave the most potent hMAO-A inhibitory activities, with IC50 values
of 1.5 nM and 19 nM, respectively (SI <
−
2.82 and SI < 2.42). FR4 and FR5 showed a selective hMAO-B
−
inhibitory affinity, with IC50 value of 18 nM and 15 nM (SI > 2.74 and SI > 2.82). In addition, docking
calculations and molecular dynamics (MD) simulations were performed to a better understand this
selectivity and SAR profile. In silico pharmacokinetics predictions and drug-like properties of these
molecules were also evaluated.
2. Results and Discussion
2.1. Qualitative SAR of Previously Identified Hits
In our previous study, BRS-3D method was introduced as a 3D shape similarity profile [29,30,32,33].
In BRS-3D, 300 diverse biologically relevant ligands were used as templates and their co-crystallized
conformation was treated as the active conformation. Hence, BRS-3D was capable of reflecting the
distribution of the objective compound in the known bioactive chemical space. For example, as
shown in Figure 2, the different distribution of selective hMAO inhibitors in chemical space can be
observed on the basis of three BRS-3D features (BRS12: 1cbq_RE9, BRS43: 3i8u_18B; BRS89: 1vj5_CIU).
These characteristics make BRS-3D an important method for the study of subtype selectivity of
receptor and enzyme ligands. Recently, we applied a BRS-3D based virtual screening protocol in the
identification of selective hMAO inhibitors [31]. The experimental results demonstrated 70 compounds
with MAO inhibition higher than 70% at a concentration of 10
with IC50 values less than 1 M. Among the identified hits, several C7-substituted coumarin derivatives
were worthy to be further explored owing to their interesting SAR profile. As illustrated in Figure 3,
comparing with entries M30
M31 and M33, R5 group at C7-position was fixed, and installation of
µM, and 25 of them were potential hits
µ
,
extended alkyl side chains at C4-position induced more robust MAO-A inhibition (M30, R2 = ethyl,
56% vs. M31, R2 = propyl, 92%) than the methyl homologue M33 (R2 = methyl, 41%). Comparing with
entries M33 and M29, additional introduced methyl group at C8 position gave an improved MAO-A
inhibition (M29, 75% vs. M33, 41%). Comparing with entries M33 and M34, the introduction of a Cl
substitution at C6 position (M34) gave a nearly doubled decrease for MAO-A inhibition but a five-fold
increase for MAO-B inhibition, suggesting C6-Cl substitution might be crucial to the MAO-B selectivity
and binding affinity. It was also clear that the high MAO-B potency and selectivity of M32 was largely
attributable to the C6-Cl group and the additional methyl substitution. Comparing with entries M33
and M43, modification of C7-substituion was clearly beneficial to both MAO-A and MAO-B inhibition.
It is noteworthy that the activity of M31 and M32 against two hMAO subtypes was reversed with small
changes in 3-, 4- and 6-positions.
In addition, we can infer that hMAO-B binding site may prefer the small sized substituents
at C4-position of the coumarin nucleus, and simultaneous substitutions at C3 and C4 position was
beneficial to hMAO-B inhibition. From the SAR analysis, compound M43 with a 2-methoxy-5-
phenyl-1,3,4-thiadiazole motif at C7-position had the most potent affinity towards both MAO-A and
MAO-B subtypes, which was thereby taken as a lead to optimize its activity and selectivity by fragment
replacement at the 3-, 4- and 8-positions.

Molecules 2019, 24, 4003
4 of 17
Figure 2. The distribution of selective MAO inhibitors on the basis of three BRS-3D features (BRS12:
1cbq_RE9, BRS43: 3i8u_18B; BRS89: 1vj5_CIU).
Figure 3. Identified coumarin analogues through BRS-3D with hMAO inhibitory activities.
2.2. MM/PBSA Binding Energy and Decomposition Analysis
Three compounds, M31, M32 and M43, were selected for docking simulations, with the
co-crystallized ligand in 2V61 (C18) as reference. Based on the optimal docking models, eight systems
were then investigated through MD simulations to elucidate the selectivity mechanism, four for each
subtype. RMSD and RMSF (root mean square fluctuation) analysis was carried out to evaluate the
structural stability. The stability of hydrogen bonds interactions between hMAO and C7 substituted
coumarins, which are likely to contribute to the selectivity of MAOIs was also investigated. More details
can be found in Supporting Information (Figures S1–S5, Table S1).
To further analyze the subtype selectivity mechanism, MM/PBSA method was employed to
calculate the binding energy between hMAO subtypes and the discovered C7-substituted coumarins.
Additionally, compound C18 bound in 2V61, was used as a reference. The predicted binding free
energy and the inhibitory activity of each compound was summarized in Table 1. The calculated
binding affinities correlated well with the experimental results. For hMAO-A selective system, the
values of MAO-A-M31 complex and MAO-B-M31 complex were
127.034 kJ/mol and −113.631 kJ/mol
with respect to their pIC50 values of 6.3 and 4.2, respectively. For hMAO-B selective system, the
∆G
−

Molecules 2019, 24, 4003
5 of 17
opposite results were obtained. The binding affinities of MAO-A-M32 complex and MAO-B-M32
complex were 112.539 kJ/mol and 122.606 kJ/mol corresponding to the pIC50 values of 4.1 and 6.2,
respectively. The MM/PBSA binding energy contained four terms: the van der Waal contributions
GvdW), the electrostatic contributions ( Gele), the polar solvation contribution ( Gpol) and the apolar
solvation energy ( Gapol). Then, the key terms responsible for binding were investigated separately.
As listed in Table 2, taking M31 and M32 as an example, van der Waal contributions were the driving
force for the binding in both selective systems and non-selective systems since the result of Gele and
Gpol was positive. This demonstrated that in hMAO subtypes the unspecific binding activity was
−
−
(
∆
∆
∆
∆
∆
∆
mainly attributed to hydrophobic interactions while selectivity to a specific subtype was driven by the
above-mentioned H-bond interactions. In addition, the electrostatic terms to the solvation free energy
∆
Gpol were unfavorable for all systems. Compared to the polar solvation energy, the apolar solvation
contributions were suggested to have a positive effect upon binding.
Table 1. Experimental activities and MM/PBSA binding free energies.
MAO-A
binding free energy
MAO-B
pIC50
4
ND
4.2
6.2
ND
ND
6.4
7
Cmpd
M29
M30
M31
M32
M33
M34
M43 (7a)
C18
Inh% pIC50
Inh%
12
22
16
90
8
40
96
-
binding free energy
−109.72 ± 1.07
−102.79 ± 1.27
−113.63 ± 1.26
−122.61 ± 1.12
−105.59 ± 1.08
−111.18 ± 0.95
−135.59 ± 1.11
−144.57 ± 1.29
75
56
92
12
41
19
102
-
5.6
ND
6.3
−121.03 ± 0.92
−116.90 ± 1.09
−127.03 ± 1.28
−112.54 ± 1.12
−115.25 ± 1.01
−107.67 ± 0.99
−129.34 ± 1.10
−96.05 ± 1.31
4.1
ND
ND
6.8
4.8
*ND: the IC50 values were not determined due to the low inhibitory activity. pIC50 = −log(IC50).
Table 2. Analysis of calculated binding free energies of four systems.
Terms
MAO-A vs. M31
MAO-B vs. M31
MAO-A vs. M32
MAO-B vs. M32
∆GvdW
∆Gele
∆Gpol
∆Gnonpol
∆G
−211.794 ± 0.990
−38.523 ± 0.817
140.734 ± 0.539
−17.431 ± 0.071
−127.034 ± 1.281
−198.535 ± 1.147
−61.460 ± 0.704
164.645 ± 1.247
−18.241 ± 0.073
−113.631 ± 1.260
−214.799 ± 0.969
−31.376 ± 0.547
151.326 ± 0.787
−17.715 ± 0.066
−12.539 ± 1.116
−198.570 ± 1.187
−1.362 ± 0.727
144.800 ± 0.895
−17.418 ± 0.065
−122.598 ± 1.144
We then performed a residue-specific binding energy decomposition to identify key residues
contributing to the subtype selectivity. The detailed inspection of per-residue energy contribution of the
active site amino acids was shown in Table 3 and Table S2. Gln215 from
to be a key residue for hMAO-A selectivity. The energy contribution of Gln215 was −7.53 ± 0.24 kJ/mol
which is obviously larger than the contribution ( 1.74 0.22 kJ/mol) of the corresponding Gln206
α-helix region was calculated
,
−
±
residue in hMAO-B. Therefore, the decreased interaction with Gln206 reduced the activity to hMAO-B.
Asn181 also played a critical role in hMAO-A selectivity since it was replaced by Cys172 in hMAO-B.
Indeed, the corresponding energy contribution of Asn181 was
M31, which was larger than that of Cys172 in hMAO-B (0.2993
substitution at C6 position was thought to be crucial to hMAO-B selectivity. As can be seen in Table 3,
the energy contribution of Cys172 (which was spatially close to C6-Cl) was 2.5912 0.1582 kJ/mol,
which was apparently larger than that of compound M31 (0.2993 0.124 kJ/mol). Other residues
−
4.1478
±
0.2633 kJ/mol for compound
±
0.124 kJ/mol). For M32, the Cl
−
±
±
important for selectivity were also identified through the binding energy decomposition analysis.
For instance, the gatekeeper residue Phe208 in hMAO-A and Ile199 in hMAO-B may contribute to
the subtype selectivity. Compared with Cys323 and Leu337 in hMAO-A, the corresponding residue
(Thr314 and Leu328) in hMAO-B also displayed different energy contributions, which was supported
by the previous research of Catto and coworkers [34].

Molecules 2019, 24, 4003
6 of 17
Table 3. Free energy contributions (kJ/mol) of hMAO-A and hMAO-B pocket residues.
Residue
MAO-A vs. M31
MAO-B vs. M31
MAO-A vs. M32
MAO-B vs. M32
MAO-A
MAO-B
Tyr60
Total Energy
Total Energy
−3.5967 ± 0.0614
0.8449 ± 0.1369
0.4229 ± 0.0305
−0.0264 ± 0.0079
−0.0097 ± 0.0097
−0.6217 ± 0.0187
−0.0436 ± 0.0256
−0.4662 ± 0.026
−6.2815 ± 0.1427
0.2993 ± 0.124
Total Energy
Total Energy
Tyr69
Ala111
Phe112
Pro113
Trp128
Phe173
Leu176
Phe177
Ile180
Asn181
Tyr197
Ile207
Phe208
Gln215
Cys323
Ile325
−1.4631 ± 0.0443
0.3332 ± 0.0714
−0.3019 ± 0.0205
−0.0764 ± 0.0091
−0.0145 ± 0.0064
−0.0364 ± 0.0108
−0.0701 ± 0.0213
−0.1931 ± 0.0494
−5.3303 ± 0.1123
−4.1478 ± 0.2633
1.1053 ± 0.0829
−3.9048 ± 0.1647
−6.1625 ± 0.2403
−7.5319 ± 0.236
−0.5525 ± 0.0492
−1.7599 ± 0.0637
−4.5914 ± 0.1095
−0.4413 ± 0.0476
−3.0775 ± 0.0836
−0.168 ± 0.0143
−0.501 ± 0.0391
−5.3629 ± 0.1462
−3.919 ± 0.2074
−2.7071 ± 0.0511
0.5828 ± 0.0985
−0.1243 ± 0.0153
0.0414 ± 0.0055
0.0893 ± 0.0033
−0.0662 ± 0.0114
0.1918 ± 0.0137
0.6680 ± 0.0293
−2.4365 ± 0.2223
0.8583 ± 0.2391
−4.1025 ± 0.1387
−3.1187 ± 0.1347
−2.2755 ± 0.2184
−3.3143 ± 0.2539
−0.4392 ± 0.0685
−1.6549 ± 0.0398
−4.2492 ± 0.1381
−0.0784 ± 0.0246
−2.2824 ± 0.0758
−0.6131 ± 0.0173
−1.2125 ± 0.0482
−7.7790 ± 0.1624
−7.4496 ± 0.1530
−3.0942 ± 0.0708
−0.0127 ± 0.0890
0.0942 ± 0.0165
−0.0514 ± 0.0163
−0.1936 ± 0.0077
−0.4042 ± 0.0134
−0.2503 ± 0.0171
−0.5791 ± 0.0819
−7.2863 ± 0.1543
−2.5912 ± 0.1582
0.1502 ± 0.0949
−1.9079 ± 0.1410
0.0638 ± 0.1966
−1.3530 ± 0.2173
−2.3485 ± 0.0513
−4.0400 ± 0.0843
−5.9530 ± 0.1767
−0.5360 ± 0.0223
−1.1358 ± 0.0269
−0.3265 ± 0.0107
−2.5305 ± 0.0643
−8.5574 ± 0.1847
−1.9394 ± 0.1860
Pro102
Phe103
Pro104
Trp119
Leu164
Leu167
Phe168
Leu171
Cys172
Tyr188
Ile198
1.7647 ± 0.076
−0.9873 ± 0.1489
−1.1994 ± 0.2342
−1.7379 ± 0.2144
−2.5552 ± 0.0515
−3.1339 ± 0.0787
−4.8286 ± 0.25
Ile199
Gln206
Thr314
Ile316
Tyr326
Thr327
Leu328
Met341
Phe343
Tyr398
Tyr435
Ile335
Thr336
Leu337
Met350
Phe352
Tyr407
Tyr444
−0.3823 ± 0.0269
−1.0562 ± 0.026
−0.438 ± 0.0118
−2.0697 ± 0.1135
−6.0795 ± 0.1569
−2.1508 ± 0.1615
2.3. Discovery, Synthesis and Biological Activity
The optimization of M43 analogues was performed to further validate the above conclusions
and explore the chemical space around the hits. This series of compounds, along with others studied
in this article, were listed in Figure 4. Detailed results were listed in Table 4 (we also synthesized
several Esuprone analogs, shown in Table S3). To estimate the selectivity of inhibitors, the selectivity
index (SI) was calculated as pIC50(MAO-B)–pIC50(MAO-A) [14]. For M43 derivatives, two out of
six were experimentally identified as potent and selective hMAO-A inhibitors (FR1
values of 1.5 nM (SI < –2.82) and 19 nM (SI < –2.42), respectively. Compared with M43, all M43
analogues showed an improved MAO-A inhibition except compound 7c 7g. From an overall SAR
, SP1) with IC50
,
profiles, a direct comparison can be made between compounds M43 and SP1, since the introduction
of a methyl group at the C8 position in compound M43 increased hMAO-A selectivity. In contrast,
simply changing the substituent at C7 position of compound M43 with other groups generally resulted
in a significant decline in MAO-A selectivity (for example, compound M43 and FR5). However, the
replacement of R5 group of 5d gave the most active hMAO-A inhibitors FR1 with an IC50 value of
1.5 nM. In addition, comparison of the activity values of compounds M43 and 7g revealed the steric
hindrance at C4 position again, i.e., only substituents of small size and flexibility are tolerated in
hMAO-A inhibitors. Based on the above experimental and theoretical results, the molecular basis of
the SAR and SSR (structure-selectivity relationship) trends between hMAO subtypes and coumarin
series were summarized in Figure 5.
Compounds M29
protocol. FR1 FR5 and SP1 were purchased from Specs. Compounds 5d
synthesized in this study.
,
M31
,
M32 and M43 were obtained based on our BRS-3D virtual screening
-
,
7b 7k 7c and 7g were
,
,

Molecules 2019, 24, 4003
7 of 17
Figure 4. Strategy for the purchase or synthesis of C7 substituted coumarin analogues.
Table 4. In vitro activities against hMAO subtypes of coumarin derivatives.
Compounds R1
R2
R4
R5
R6
MAO-A a
MAO-B a
SI b
M29
M31
H
H
CH3
H
H
CH3
H
2.29
0.50
100
−1.64
−2.11
(CH2)2CH3
64.57
M32
CH3
H
CH3
CH3
Cl
H
H
H
81.28
0.16
0.60
0.36
2.13
M43(7a)
−0.35
FR1
–(CH2)3–
H
CH3
0.0015
75% (>1 uM)
<−2.82
FR2
FR3
–(CH2)3–
–(CH2)4–
H
H
CH3
CH3
2.82
0.74
12% (>10 uM)
66% (>5 uM)
<−0.55
<−0.83
FR4
–(CH2)3–
H
H
23% (>10 uM)
0.018
>2.74
FR5
SP1
5d
H
H
CH3
CH3
–(CH2)3–
H
H
H
H
H
Cl
H
H
CH3
CH3
H
28% (>10 uM)
0.019
0.015
64% (>5 uM)
2.30
>2.82
<−2.42
−1.38
0.38
0.096
7b
H
H
(CH2)2CH3
(CH2)2CH3
CH3
0.024
0.01
7k
CH3
H
0.021
0.12
−0.76
0.27
7c
CH3
H
0.81
0.44
7g
H
2.00
6.20
−0.49
a: IC50 values ( M against hMAO-A and hMAO-B. b: SI is the selectivity index
µM) or inhibition activity at 10 µ
expressed as pIC50(MAO-B)–pIC50(MAO-A).

Molecules 2019, 24, 4003
8 of 17
Figure 5. Structure-activity relationship (SAR)/ structure-selectivity relationship (SSR) profiles for the
coumarins against hMAO subtypes based on compound M43.
2.4. In Silico Pharmacokinetic Properties
Additionally, in silico pharmacokinetic properties of all the tested C7-substituted coumarin
derivatives were evaluated using ACD/percepta platform including lipophilicity logP, molecular
weight, Lipinski’s rule of five, lead-like, Caco-2 and CNS properties. Detailed pharmacokinetic
properties were presented in Table 5 [35]. According to the predicted properties, all these compounds
complied with the Lipinski’s rule without any violation, which suggested that these new C7-substituted
coumarin analogues might have good oral bioavailability. The Caco-2 permeability model was
used to predict the absorption potential of oral drugs. The theoretical results suggested that most
of these compounds possessed good intestinal permeability. A CNS penetration model was also
applied suggesting the ability of these compounds to cross the blood–brain-barrier (BBB). These results
suggested that the compounds might have the ability to pass the BBB, which is essential factor for
MAO inhibition in human brain. In addition, a CYP isozymes inhibition model was applied for these
derivatives using CypRules Server, which suggested that they do not inhibit the five CYP450 enzymes
(CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4) [36].
Table 5. Calculated physicochemical and pharmacokinetic parameters.
Compounds
logP
MW
Lipinski
Lead-like
Caco-2
CNS
M29
M31
M32
M43 (7a)
FR1
FR2
FR3
FR4
FR5
SP1
5d
1.94
2.21
2.98
3.79
3.3
2.32
4.33
4.57
3.77
3.97
4.72
4.49
4.79
4.99
275.3
289.33
309.74
336.37
284.31
301.34
328.36
354.78
296.32
350.39
376.43
364.42
378.45
384.84
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
0 violations
1 violation
1 violation
0 violations
0 violations
1 violation
1 violation
1 violation
1 violation
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Highly permeable
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Penetrant
Weak Penetrant
7b
7k
7c
3. Conclusions
An essential aspect in rational drug design or discovery against a conserved protein family is
subtype selectivity. However, the small structural difference between subtypes makes it a huge challenge.
In this study, based on our BRS-3D method and further SAR analysis, two selective hMAO-A inhibitors
(
FR1
,
SP1) and two selective hMAO-B inhibitors (FR4
,
FR5) were identified. Compound FR1 showed
2.82. FR5 was the most active
the best hMAO-A selectivity with an IC50 value of 1.5 nM and SI <
hMAO-B inhibitor with an IC50 value of 15 nM and SI > 2.82. Both docking and molecular dynamic
−
simulations further demonstrated the details of MAO subtype selectivity at molecular level. Moreover,

Molecules 2019, 24, 4003
9 of 17
to evaluate drug-like properties of these molecules, in silico pharmacokinetic evaluation was carried
out. All compounds were predicted to possess favorable pharmacokinetic profiles, and have good oral
bioavailability. This analysis provided a better understanding of hMAO subtype selectivity mechanism
using C7-subsituted coumarins as probes. In summary, the reported C7-substituted coumarins are lead
compounds for developing new drugs against depression, Alzheimer’s disease and Parkinson’s disease.
Furthermore, BRS-3D method mentioned in this paper can contribute to the discovery and rational design
of subtype selective inhibitors for other protein families, such as GPCRs and kinases.
4. Materials and Methods
4.1. Docking Simulations
In this study, GOLD 5.2.2 program was used to dock the C7-substituted coumarin analogues into
the binding site of two hMAO subtypes [37]. The 3D structures of C7-substituted coumarin analogues
were first generated by CONCORD module and then energy minimization was performed to generate
the initial conformation. The crystal structure of two hMAO subtypes was obtained from PDB (MAO-A:
2Z5X, MAO-B: 2V61), which were used for docking calculations. Each protein structure was optimized
using the Sybyl X program in order to add hydrogen, remove the co-crystallized waters and ions.
The binding area in the reference crystal structures was centered on the co-crystallized ligand with a
radius of 6 Å and the Goldscore fitness function was applied to evaluate the binding affinities scores.
For each ligand, 30 genetic algorithm runs were performed and the docking pose with the highest
score was kept for the MD simulations.
4.2. Molecular Dynamic Simulations
MD simulations were performed using GROMACS 4.5.4. The AMBER ff99SB force field was
selected to describe the MAO enzyme complexes [38]. The atomic parameters for the FAD cofactor were
derived according to the restrained electrostatic potential (RESP) protocol at the level of HF/6-31(d) [39,40].
The force field parameters for the C7-substituted coumarins were described using the GAFF in antechamber
module. The LINCS algorithm was used to keep the heavy atom H bonds in their correct lengths. Periodic
boundary conditions with a 12 Å cutoff were applied in all directions for treating the non-bonded
interaction. The long-range electrostatic interactions were calculated by the Particle–Mesh–Ewald (PME)
method. An isothermal-isobaric ensemble (300 K, 1 atm) was employed during the MD simulation using
the combination of Parrinello–Rahman method and modified Berendsen thermostat algorithm. The initial
complex structure of each system was placed in an octahedral TIP3P water box. The final production run
was performed for 20 ns with a restraint on the C-terminal of the protein. A time step of 2 fs was applied
and the generated coordinates were saved every 100 ps.
4.3. MM/PBSA Calculations and Energy Decomposition Analysis
In this study, the MM/PBSA method was employed to evaluate the binding free energy between
MAO subtypes and C7-subsituted coumarin analogues. The MM/PBSA binding energy was further
decomposed per residue to identify key residues for subtype selectivity. The binding free energy
calculations and the decomposition analysis was supported by g_mmpbsa tool [41]. For all simulated
systems, the last 10 ns MD trajectory was used for analysis. In Equation (1), the first two terms,
∆Gele and ∆Gvdw represented electrostatic interactions and van der Waals interactions, respectively.
The sum of these terms was equal to the molecular mechanics potential energy part. ∆GPolar is the
solvation energy contribution, which here was calculated according to the PB (Poisson Boltzmann)
model. ∆Gapolar term represented the apolar solvation energy, which was determined based on the
SASA model in this paper. As described in Equation (2), the SASA was surface-accessible solvent area,
γ
was set to 0.02267 kJ/mol and the fitting parameter β was set to 3.84928 kJ/mol. The last term of
Equation (1), −T∆S was not considered in g_mmpbsa. Therefore, it gave the final MM/PBSA equation
as presented in Equation (3).

Molecules 2019, 24, 4003
10 of 17
∆Gtotal = ∆Gele + ∆Gvdw + ∆Gpolar + ∆Gapolar − T∆S.
∆Gapolar = γ ∗ SASA + β.
(1)
(2)
(3)
∆GMM/PBSA = ∆Gele + ∆Gvdw + ∆Gsolv
.
4.4. Chemistry
The target 2,3-dihydrocyclopenta[c]chromen-4(1H)-one derivative (
derivatives (
5
) and 2H-chromen-2-one
7
) were prepared according to the synthetic route outlined in Schemes 1 and 2, respectively.
Scheme 1. The synthetic protocol for 2,3-dihydrocyclopenta[c]chromen-4(1H)-one derivatives (5).
Reagents and conditions: (i) Hydrazinecarbothioamide, EtOH/H2O, r.t.; (ii) FeCl3
(iii) CuBr2, isoamyl nitrite, CH3CN, r.t; (iv) Conc. H2SO4, 2-methylbenzene-1,3-diol, 0 C and (v)
K2CO3, DMF, 80 ◦C.
·
6H2O, Et◦OH, 80 ◦C;
Scheme 2. Synthetic procedures of coumarin derivatives (7). Reagents and conditions: (i) Method 1.
H2SO4, 0 ◦C and (ii) 2-Bromo-5-phenyl-1,3,4-thiadiazole (compound 3), K2CO3, DMF, 80 ◦C. Method 2.
substituted sulfuryl chloride, TEA, DCM, r.t.
4.4.1. Synthesis of (E)-2-benzylidenehydrazinecarbothioamide (1)
Benzaldehyde (0.1 g, 0.942 mmol) was dissolved in warm anhydrous alcohol (3 mL) and a mixture
of thiosemicarbazide (0.0945 g, 1.04 mmol) in warm water (3 mL) was added dropwise with continuous
stirring. The mixture was allowed to react at room temperature until TLC (Thin Layer chromatography)
showed the reaction has completed (PE/EA = 2:1). A large amount of white precipitate generated and
filtered to give white solid (0.166 g, 0.93 mmol) yield: 98%; m.p. = 142–145 ◦C; 1H-NMR (400 MHz,
DMSO-d6)
δ 11.44 (s, 1H, NH), 8.28–8.14 (m, 1H, CH=N), 8.03 (d, J = 16.5 Hz, 2H, NH2), 7.80 (dd,
J = 6.6, 2.9 Hz, 2H, ArH), 7.41 (dd, J = 5.2, 1.9 Hz, 3H, ArH); ESI-MS m/z: 180.2 [M + H]+.
4.4.2. Synthesis of 5-phenyl-1,3,4-thiadiazol-2-amine (2)
Thiosemicarbazone (0.25 g, 1.39 mmol) was suspended in 5 mL of ethanol, FeCl3 6H2O (1.13 g,
·
4.18 mmol) was slowly added with constant stirring. The resulting solution was stirred for 1.5 h at
80 ◦C until TLC showed the reaction has completed (PE/EA = 1:1). The reaction mixture was allowed
to cool to room temperature and quenched by adding citric acid (3.2 g). After neutralized with 10%
ammonia solution, the crude product was precipitated and filtered to afford a white solid (0.18 g,

Molecules 2019, 24, 4003
11 of 17
1.02 mmol). Yield: 72%; m.p. = 220–223 ◦C; 1H-NMR (400 MHz, DMSO-d6):
2H, NH2), 7.53–7.36 (m, 5H, ArH); ESI-MS m/z: 175.8 [M + H]−.
δ
7.76 (dd, J = 7.9, 1.7 Hz,
4.4.3. Preparation of 2-bromo-5-phenyl-1,3,4-thiadiazole (3)
The copper bromide (0.116 g, 0.520 mmol) and isoamyl nitrite (0.061 g, 0.520 mmol) were added
successively in CH3CN (10 mL). The resulting mixture was stirred at room temperature for 10 min,
then 5-phenyl-1,3,4-thiadiazol-2-amine (2, 0.04 g, 0.226 mmol) was added in one portion. The reaction
mixture was allowed to react at 25 ◦C for 1 h until completion, as determined by TLC. The solvent
was subsequently removed under reduced pressure, and the residue was washed with ethyl acetate
(
3 × 20 mL). The organic phase was extracted successively with 2N HCl (3
×
25 mL) and brine (25 mL).
The combined organic phase was dried over MgSO4 and filtered. All the solvent was removed under
vacuum and the resulting residue was purified by flash column chromatography (PE/EA = 1:1, v:v)
to afford light yellow solid (0.049 g, 0.20 mmol). Yield: 90.7%; m.p. = 82–84 ◦C; 1H-NMR (400 MHz,
DMSO-d6):
137.99, 131.79, 129.37, 127.85; ESI-MS m/z: 240.7 [M + H]+.
δ δ 172.16,
8.10–7.77 (m, 2H, ArH), 7.71–7.44 (m, 3H, ArH); 13C-NMR (100 MHz, CDCl3):
4.4.4. General Procedure for the Synthesis of 7-hydroxy-6-methyl-2,3-dihydrocyclopenta[c]chromen-
4(1H)-one (4)
To a solution of 2-methylbenzene-1,3-diol (0.1 g) in alkylated ethyl acetoacetate, concentrated
sulfuric acid (1 mL) was slowly added dropwise at 0 ◦C in an ice bath. The resulting solution was
allowed to react at 0 ◦C for 5–6 h until the disappearance of starting material, as monitored by TLC
(MeOH/DCM = 100:1). The reaction was quenched by adding 50 mL of ice water. The obtained crude
solid was filtered, washed with water, dried, and recrystallized by 95% ethanol to give the desired
compound (4) as a light yellow solid. Yield: 78%; m.p. = 243–244 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ
10.32 (s, 1H, OH), 7.25 (d, J = 8.0 Hz, 1H, ArH), 6.84 (d, J = 8.0 Hz, 1H, ArH), 3.00 (t, J = 6.9 Hz, 2H,
2CH2CH2), 2.71 (t, J = 6.8 Hz, 2H, CH2CH2C 2), 2.16 (s, 3H, CH3), 2.08–2.06 (m, 2H, CH2C 2CH2);
C
H
H
H
ESI-MS m/z: 215.8 [M + H]−.
4.4.5. Preparation of 7-hydroxyl Substituted Coumarin Derivatives (6a–e)
The 7-hydroxyl substituted coumarin derivatives (6a–e) were accomplished according to the
similar procedures as that of compound 4.
1Hydroxy-4-methyl-2H-chromen-2-one (6a). Light yellow solid, yield: 56%; m.p. = 180–182 ◦C; 1H-NMR
(400 MHz, DMSO-d6):
δ 10.53 (s, 1H, OH), 7.60 (d, J = 8.7 Hz, 1H, ArH), 6.81 (dd, J = 8.7, 2.4 Hz, 1H,
ArH), 6.71 (d, J = 2.4 Hz, 1H, ArH), 6.13 (d, J = 1.4 Hz, 1H, C=C
H
), 2.37 (s, 3H, CH3); ESI-MS m/z: 174.5
[M + H]−.
◦
7-Hydroxy-4-propyl-2H-chromen-2-one (6b). Light yellow solid, yield: 76.6%; m.p. = 128–130 C;
1H-NMR (400 MHz, DMSO-d6)
6.08 (s, 1H, C=CH), 2.71 (t, J = 7.8 Hz, 2H, C
(m, 3H, CH2CH2CH3); ESI-MS m/z: 203 [M + H]−.
δ
10.55 (s, 1H, OH), 7.81–7.11 (m, 1H, ArH), 6.91–6.57 (m, 2H, ArH),
2CH2CH3), 1.78–1.46 (m, 2H, CH2C 2CH3), 1.12–0.79
H
H
6-Chloro-7-hydroxy-3,4-dimethyl-2H-chromen-2-one (6c). Light yellow solid, yield: 81.1%; m.p. = 258–260 ◦C
1H-NMR (400 MHz, DMSO-d6):
11.19 (s, 1H, OH), 7.75 (s, 1H, ArH), 6.87 (s, 1H, ArH), 2.34 (s, 3H, CH3),
2.06 (s, 3H, CH3); ESI-MS m/z: 225 [M + H]+.
;
δ
◦
4-Cyclopropyl-7-hydroxy-2H-chromen-2-one (6d). Light yellow solid, yield: 86%; m.p. = 199–200 C;
1H-NMR (400 MHz, DMSO-d6):
10.54 (s, 1H, OH), 7.90 (d, J = 8.7 Hz, 1H, ArH), 6.83 (dd, J = 8.7, 2.4 Hz,
1H, ArH), 6.71 (d, J = 2.4 Hz, 1H, ArH), 5.82 (s, 1H, C=CH), 2.24 (dd, J = 8.5, 4.2 Hz, 1H, C CH2CH2),
1.17–1.00 (m, 2H, CHCH2CH2), 0.98–0.76 (m, 2H, CHCH2CH2); ESI-MS m/z: 202.9 [M + H]+.
δ
H

Molecules 2019, 24, 4003
12 of 17
◦
7-Hydroxy-8-methyl-4-propyl-2H-chromen-2-one (6e). Light yellow solid, yield: 72%; m.p. = 160 C;
1H-NMR (400 MHz, DMSO-d6):
δ 10.42 (s, 1H, OH), 7.50 (d, J = 8.7 Hz, 1H, ArH), 6.86 (d, J = 8.7 Hz,
1H, ArH), 6.08 (s, 1H, C=CH), 2.70 (t, J = 7.6 Hz, 2H, CH2CH2CH3), 2.15 (s, 3H, CH3), 1.67–1.62 (m, 2H,
CH2CH2CH3), 0.96 (t, J = 7.3 Hz, 3H, CH2CH2CH3); ESI-MS m/z: 217.8 [M + H]−.
4.4.6. General Procedure for the Synthesis of Target Compounds (5d, 7a–c, 7g and 7k)
Appropriate coumarin analogues (0.5 mmol) and 2-bromo-5-phenyl-1,3,4-thiadiazole (3, 0.8 mmol)
were dissolved in anhydrous DMF (10 mL), and K2CO3 (2.0 mmol) was added in one portion. The mixture
was then stirred at 100 ◦C for 6–7 h under an inert nitrogen atmosphere. Once the completion of the
reaction was detected by TLC, the reaction mixture was cooled to room temperature. The reaction mixture
was extracted with ethyl acetate (3
3 × 25 mL) and dried over anhydrous sodium sulfate and filtered. The solvent was removed under
vacuum and the crude product was purified by flash chromatography (silica gel, PE/EA = 10:3) to afford
the compound 5d as white powder. Yield: 56%; m.p. = 140 ◦C; 1H-NMR (400 MHz, DMSO-d6):
7.92–7.83
×
20 mL) and the combined organic layer was washed with brine
(
δ
(m, 2H, ArH), 7.62 (d, J = 8.6 Hz, 1H, ArH), 7.58–7.47 (m, 4H, ArH), 3.13 (t, J = 7.7 Hz, 2H,CH2CH2CH2),
2.81 (t, J = 7.6 Hz, 2H, CH2CH2CH2), 2.34 (s, 3H, CH3), 2.23–1.99 (m, 2H, CH2CH2CH2); 13C-NMR (100
MHz, CDCl3):
δ 173.55, 164.27, 159.83, 155.91, 155.03, 153.43, 131.11, 130.32, 129.17, 127.51, 127.21, 123.00,
118.71, 117.01, 116.18, 32.20, 30.62, 22.51, 9.38; ESI-MS m/z: 377.0 [M + H]+.
4-Methyl-7-((5-phenyl-1,3,4-thiadiazol-2-yl)oxy)-2H-chromen-2-one (7a). White powder, yield: 42.8%; m.p. =
124–125 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ
7.94–7.90 (m, 3H), 7.66 (d, J = 2.5 Hz, 1H, ArH), 7.62–7.47
(m, 4H, ArH), 6.45 (d, J = 1.4 Hz, 1H, C=CH), 2.47 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3):
δ
172.31,
164.90, 160.29, 157.08, 154.48, 151.76, 131.27, 130.19, 129.22, 127.31, 126.06, 117.93, 115.67, 114.66, 108.16,
18.81; ESI-MS m/z: 336.9 [M + H]+.
7-((5-Phenyl-1,3,4-thiadiazol-2-yl)oxy)-4-propyl-2H-chromen-2-one (7b). White powder, yield: 48.3%; m.p. =
150 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ 7.99 (d, J = 8.8 Hz, 1H, ArH), 7.95–7.88 (m, 2H, ArH), 7.66 (d, J
= 2.5 Hz, 1H, ArH), 7.60–7.53 (m, 3H, ArH), 7.50 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 6.39 (s, 1H, C=CH), 2.82
(t, J = 7.6 Hz, 2H, CH2CH2CH3), 1.72–1.63 (m, 2H, CH2CH2CH3), 1.02 (t, J = 7.3 Hz, 3H, CH2CH2CH3);
13C-NMR (100 MHz, CDCl3):
δ 172.34, 164.88, 160.55, 156.91, 155.47, 154.71, 131.27, 130.37, 129.22, 127.31,
125.84, 117.31, 115.64, 113.52, 108.36, 33.81, 21.30, 13.94; ESI-MS m/z: 364.9 [M + H]+.
6-Chloro-3,4-dimethyl-7-((5-phenyl-1,3,4-thiadiazol-2-yl)oxy)-2H-chromen-2-one (7c). White powder, yield:
51.3%; m.p. = 171–173 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ
8.12 (s, 1H, ArH), 7.94–7.88 (m, 3H, ArH),
7.60–7.52 (m, 3H, ArH), 2.45 (s, 3H, CH3), 2.15 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3):
δ
172.32,
164.93, 161.01, 151.21, 150.98, 144.32, 131.24, 130.19, 129.20, 127.29, 126.07, 123.37, 121.59, 119.96, 110.64,
15.32, 13.67; ESI-MS m/z: 385.0 [M + H]+.
4-Cyclopropyl-7-((5-phenyl-1,3,4-thiadiazol-2-yl)oxy)-2H-chromen-2-one (7g).White powder, yield: 89%;
m.p. = 158–160 ◦C; 1H-NMR (400 MHz, CDCl3):
δ
7.99 (d, J = 8.6 Hz, 1H, ArH), 7.87 (dd, J = 7.5, 2.2 Hz,
2H, ArH), 7.54–7.44 (m, 3H, ArH), 7.43–7.35 (m, 2H, ArH), 6.06 (s, 1H, C=C ), 2.14–1.97 (m, 1H, C ),
1.22–1.11 (m, 2H, CH2C 2), 0.94–0.80 (m, 2H, C 172.36, 165.43,
2CH2); 13C-NMR (100 MHz, CDCl3):
H
H
H
H
δ
160.91, 157.07, 157.06, 154.36, 131.24, 130.22, 129.21, 127.31, 126.24, 118.21, 115.61, 110.05, 108.14, 12.17,
8.07; ESI-MS m/z: 362.8 [M + H]+.
8-Methyl-7-((5-phenyl-1,3,4-thiadiazol-2-yl)oxy)-4-propyl-2H-chromen-2-one (7k). White powder, yield: 46.8%;
m.p. = 110–112 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ 7.92–7.86 (m, 2H, ArH), 7.84 (d, J = 8.9 Hz, 1H,
ArH), 7.60–7.52 (m, 3H, ArH), 7.49 (d, J = 8.8 Hz, 1H, ArH), 6.40 (s, 1H, C=CH), 2.81 (t, J = 7.6 Hz,
2H, CH2CH2CH3), 2.32 (s, 3H, CH3), 1.67 (q, J = 7.5 Hz, 2H, CH2CH2CH3), 1.00 (t, J = 7.3 Hz, 3H,
CH2CH2CH3); 13C-NMR (100 MHz, CDCl3):
δ
173.28, 164.40, 160.75, 155.89, 155.41, 153.13, 131.15, 130.28,
129.18, 127.23, 122.69, 118.95, 117.51, 116.05, 113.41, 33.93, 21.41, 13.94, 9.25; ESI-MS m/z: 377.0 [M + H]−.

Molecules 2019, 24, 4003
13 of 17
4.4.7. Synthesis of the Coumarin Sulfonates (5a–c, 7d–f and 7h–j)
A solution of the appropriate coumarin analogues (0.1 mmol) and triethylamine (0.2 mmol) in
dry dichloromethane (5 mL) was cooled in an ice bath. A solution of an appropriate sulfonyl chloride
derivative (0.12 mmol) in dry dichloromethane (2 mL) was subsequently added dropwise at the same
temperature. The reaction mixture was allowed to warm to room temperature and stirred for additional
0.5 h until the disappearance of starting material, as monitored by TLC. The reaction mixture was
extracted with ethyl acetate (3
×
20 mL), and the combined organic phase was washed with brine
(3 × 25 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced
pressure and the crude residue was purified by flash column chromatography (silica gel, PE/EA = 10:3
to 10:4, v:v) to afford the desired compounds.
6-Methyl-4-oxo-1,2,3,4-tetrahydrocyclopenta[c]chromen-7-yl ethanesulfonate (5a). White powder.
Yield: 86%; m.p. = 150–151 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
7.55 (d, J = 8.6 Hz, 1H, ArH), 7.34 (d,
J = 8.7 Hz, 1H, ArH), 3.71 (q, J = 7.3 Hz, 2H, CH3CH2), 3.10 (t, J = 8.4 Hz,2H, CH2CH2CH2), 2.79 (t,
J = 7.4 Hz, 2H, CH2CH2C 2), 2.35 (s, 3H, CH3), 2.18–2.09 (m, 2H, CH2C 2CH2), 1.43 (t, J = 7.3 Hz, 3H,
H
H
C
H3CH2); 13C-NMR (100 MHz, CDCl3):
δ 159.66, 155.75, 153.24, 148.50, 127.81, 122.56, 120.51, 118.16,
117.39, 46.37, 32.15, 30.62, 22.47, 9.89, 8.35; ESI-MS m/z: 309.4 [M + H]+.
6-Methyl-4-oxo-1,2,3,4-tetrahydrocyclopenta[c]chromen-7-yl 4-methylbenzenesulfonate (5b). White powder,
yield: 81%; m.p. = 152 ◦C; 1H-NMR (400 MHz, CDCl3):
δ 7.73 (d, J = 8.2 Hz, 2H, ArH), 7.33 (d, J = 8.1 Hz,
2H, ArH), 7.27 (d, J = 3.5 Hz, 1H, ArH), 7.13 (d, J = 8.6 Hz, 1H, ArH), 3.06 (dd, J = 8.2, 1.9 Hz, 2H,
CH2CH2CH2), 3.00–2.78 (m, 2H, CH2CH2CH2), 2.47 (s, 3H, CH3), 2.27–2.14 (m, 2H, CH2CH2CH2), 2.07
(s, 3H, CH3); 13C-NMR (100 MHz, CDCl3):
δ 159.75, 155.84, 153.00, 149.32, 145.89, 132.56, 130.01, 128.39,
127.77, 122.38, 120.67, 118.61, 117.29, 32.15, 30.59, 22.48, 21.80, 9.49; ESI-MS m/z: 370.8 [M + H]+.
6-Methyl-4-oxo-1,2,3,4-tetrahydrocyclopenta[c]chromen-7-yl 4-nitrobenzenesulfonate (5c). White powder, yield:
79%; m.p. = 192–194 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ
8.59–8.36 (m, 2H, ArH), 8.31–8.08 (m, 2H,
ArH), 7.51 (d, J = 8.6 Hz, 1H, ArH), 7.07 (d, J = 8.6 Hz, 1H, ArH), 3.07 (t, J = 7.7 Hz, 2H, CH2CH2CH2),
2.77 (t, J = 7.5 Hz, 2H, CH2CH2C 2), 2.13 (t, J = 7.6 Hz, 2H, CH2C
2CH2), 2.08 (s, 3H, CH3); 13C-NMR
(100 MHz, DMSO-d6): 158.87, 156.38, 152.87, 151.76, 148.68, 140.03, 130.47, 128.08, 125.72, 124.24, 119.84,
118.50, 117.84, 32.21, 30.71, 22.43, 9.91; ESI-MS m/z: 402.1 [M + H]+.
H
H
δ
◦
4-Cyclopropyl-2-oxo-2H-chromen-7-yl ethanesulfonate (7d). White powder, yield: 81%; m.p. = 92 C;
1H-NMR (400 MHz, DMSO-d6):
8.19 (d, J = 8.8 Hz, 1H, ArH), 6.16 (s, 1H, C=C ), 3.63 (q, J = 7.3 Hz,
2H, CH3CH2), 2.35–2.29 (m, 1H, CH), 1.40 (t, J = 7.3 Hz, 3H, CH3CH2), 1.11 (dt, J = 8.3, 3.3 Hz, 2H,
δ
H
CH2CH2), 0.99–0.84 (m, 2H, CH2CH δ 160.63, 156.90, 154.07, 150.87,
2); 13C-NMR (100 MHz, CDCl3):
126.19, 119.21, 118.19, 110.69, 110.65, 45.76, 12.17, 8.28, 8.03; ESI-MS m/z: 294.7 [M + H]+.
4-Cyclopropyl-2-oxo-2H-chromen-7-yl 4-methylbenzenesulfonate (7e). White powder, yield: 86%; m.p. =
132–134 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ
8.10 (d, J = 8.8 Hz, 1H, ArH), 7.88–7.68 (m, 2H, ArH),
7.50 (d, J = 8.1 Hz, 2H, ArH), 7.14 (d, J = 2.3 Hz, 1H, ArH), 7.09 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 6.12
(s, 1H, C=C
H
), 2.44 (s, 4H), 2.32–2.12 (m, 1H, C
H
δ
), 1.08 (dt, J = 8.3, 3.3 Hz, 2H, CH2CH2), 0.92–0.88
160.72, 157.01, 153.73, 151.52, 146.03, 132.22, 130.07,
(m, 2H, CH2CH2); 13C-NMR (100 MHz, CDCl3):
128.47, 125.91, 119.12, 118.82, 110.88, 110.48, 21.81, 12.13, 8.10; ESI-MS m/z: 356.7 [M + H]+.
4-Cyclopropyl-2-oxo-2H-chromen-7-yl 4-nitrobenzenesulfonate (7f). White powder, yield: 88%; m.p. =
192–194 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ
8.59–8.39 (m, 2H, ArH), 8.31–8.18 (m, 2H, ArH), 8.12
(d, J = 8.8 Hz, 1H, ArH), 7.26 (d, J = 2.4 Hz, 1H, ArH), 7.16 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 6.14 (s,
1H, C=C ), 2.33–2.16 (m, 1H, C ), 1.09 (dt, J = 8.3, 3.2 Hz, 2H, CH2CH2), 0.91 (dt, J = 6.8, 3.4 Hz,
H2CH2); 13C-NMR (100 MHz, CDCl3):
160.32, 156.70, 153.90, 151.23, 150.78, 140.61, 129.88, 126.27,
124.63, 119.67, 118.14, 111.01, 110.80, 12.15, 8.10; ESI-MS m/z: 388.0 [M+H]+.
H
H
C
δ

Molecules 2019, 24, 4003
14 of 17
8-Methyl-2-oxo-4-propyl-2H-chromen-7-yl ethanesulfonate (7h). White powder, yield: 71%; m.p. = 78–80 ◦C;
1H-NMR (400 MHz, DMSO-d6):
7.79 (d, J = 8.8 Hz, 1H, ArH), 7.35 (d, J = 8.8 Hz, 1H, ArH), 6.39 (s,
1H, C=C ), 3.72 (q, J = 7.3 Hz, 2H, CH3CH2), 2.79 (t, J = 7.6 Hz, 2H, CH2CH2CH3), 2.33 (s, 3H, CH3),
1.70–1.61 (m, 2H, CH2CH2CH3), 1.43 (t, J = 7.3 Hz, 3H, CH3CH2), 0.99 (t, J = 7.3 Hz, 3H, CH2CH2CH3);
δ
H
13C-NMR (100 MHz, CDCl3):
δ 160.55, 155.75, 152.95, 149.03, 122.30, 120.79, 118.05, 117.95, 113.68, 46.42,
33.87, 21.38, 13.89, 9.74, 8.34; ESI-MS m/z: 310.7[M + H]+.
8-Methyl-2-oxo-4-propyl-2H-chromen-7-yl 4-methylbenzenesulfonate (7i). White powder, yield: 83%; m.p. =
144–146 ◦C; 1H-NMR (400 MHz, DMSO-d6):
δ
7.81 (d, J = 8.2 Hz, 2H, ArH), 7.75 (d, J = 8.9 Hz, 1H, ArH),
7.52 (d, J = 8.1 Hz, 2H, ArH), 7.03 (d, J = 8.8 Hz, 1H, ArH), 6.37 (s, 1H, C=C ), 2.76 (t, J = 7.6 Hz, 2H,
2CH2CH3), 2.45 (s, 3H, C 3), 2.01 (s, 3H, C 3), 1.66–1.60 (m, 2H, CH2C 2CH3), 0.97 (t, J = 7.3 Hz, 3H,
CH2CH2CH3); 13C-NMR (100 MHz, CDCl3):
160.61, 155.77, 152.73, 149.87, 145.92, 132.66, 130.04, 128.37,
H
CH
H
H
δ
H
122.06, 120.97, 118.47, 117.86, 113.61, 33.86, 21.79, 21.34, 13.91, 9.35; ESI-MS m/z: 372.8 [M + H]+.
8-Methyl-2-oxo-4-propyl-2H-chromen-7-yl 4-nitrobenzenesulfonate (7j). White powder, yield: 86%; m.p.
◦
1
= 171–174 C; H-NMR (400 MHz, DMSO-d6):
2H, ArH), 7.75 (d, J = 8.9 Hz, 1H, ArH), 7.07 (d, J = 8.8 Hz, 1H, ArH), 6.40 (s, 1H, C=C
J = 7.6 Hz, 2H, CH2CH2CH3), 2.07 (s, 3H, CH3), 1.68–1.61 (m, 2H, CH2CH2CH3), 0.98 (t, J = 7.3 Hz,
δ
8.49 (d, J = 8.7 Hz, 2H, ArH), 8.23 (d, J = 8.6 Hz,
H), 2.77 (t,
3H, CH2CH2CH3); 13C-NMR (100 MHz, CDCl3):
124.63, 122.40, 120.90, 118.38, 117.79, 114.06, 33.84, 21.32, 13.90, 9.69; ESI-MS m/z: 402.0 [M + H]−.
δ 160.22, 155.50, 152.92, 151.21, 149.28, 141.33, 129.78,
4.5. Biological Assays
The inhibition and IC50 values of the identified C7-substituted coumarin analogues were assayed
by Shanghai ChemPartner Co. Ltd., using MAO-GloTM assay kit (Promega Corporation, V1402).
Two human MAO enzymes (Cat#31502, Cat#31503) were purchased from Active Motif. Clorgyline and
R(-)-deprenyl were purchased as reference drugs from Sigma (Cat. No. M3778) and Abcam (Cat. No.
ab120604), respectively. The MAO inhibition assays were carried out in modified HEPES buffer in light
of the manufacturer’s protocol. Firstly, the tested compounds in DMSO were transferred to a 384-well
plate by Echo. The enzyme solutions were then added into the plate and the plate was incubated for
15 min at room temperature (r.t.). Secondly, the luciferin derivative substrate was added to initiate
the reaction. After 60 min incubation at r.t., the Luciferin detection reagent was added to each well to
terminate the reaction and generate the luminescent signal. Before reading the signal, the plate was
incubated for 20 min at r.t. Finally, plate reader was used to measure and record the luminescent signal
and corresponding data were processed using GraphPad Prism 5.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/21/4003/s1,
Figure S1: The residue differences of binding pockets between hMAO-A and hMAO-B. FAD cofactor is shown
as space-filling model, MAO-A ligand as green sticks and MAO-B ligand as orange sticks. Figure S2: Predicted
binding modes of M31
space-filling model and compound as green sticks. The H-bond receptor surfaces are shown in pink and the
H-bond acceptor surfaces are shown in green. Figure S3: RMSD plots of M31 M32 M43 and C18 with different
, M32 and M43 with hMAO-A and hMAO-B active site. FAD cofactor is shown as
,
,
hMAO subtypes. Figure S4: Per-residue RMSF curves of the hMAO-A and hMAO-B systems. Figure S5: The
labeled key motifs of hMAO. Loop area is colored red. Alpha helix is colored magenta. Beta sheet is colored
orange. Figure S6: RMSD plots for 16 systems. Table S1: Hydrogen bond analysis of four systems according to
MD trajectories. Table S2: Free energy decomposition results (kJ/mol) of hMAO-A and hMAO-B pocket residues.
Table S3: pIC50 values of synthesized Esuprone derivatives.
Author Contributions: Conceptualization, D.W. and D.-X.K.; Data curation, D.W., R.-Y.H., M.G. and N.C.;
Funding acquisition, D.-X.K.; Investigation, D.W., M.G., Y.L. and N.C.; Methodology, R.-Y.H., X.L. and D.-X.K.;
Writing—original draft, D.W.; Writing—review and editing, X.L. and D.-X.K.
Funding: This research was funded by Fundamental Research Funds for the Central Universities grant number
2662017PY117, National Natural Science Foundation of China grant number 21275061, 21977033 and Natural
Science Foundation of Shandong Province grant number ZR2018MH042. The APC was funded by 21275061.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 4003
15 of 17
References
1.
2.
3.
4.
5.
Tripathi, A.C.; Upadhyay, S.; Paliwal, S.; Saraf, S.K. Privileged scaffolds as MAO inhibitors: Retrospect and
prospects. Eur. J. Med. Chem. 2018, 145, 445–497. [CrossRef] [PubMed]
Entzeroth, M.; Ratty, A.K. Monoamine Oxidase Inhibitors—Revisiting a Therapeutic Principle. Open J.
Depress. 2017, 6, 31–68. [CrossRef]
Youdim, M.B.H.; Edmondson, D.; Tipton, K.F. The therapeutic potential of monoamine oxidase inhibitors.
Nature Rev. Neurosci. 2006, 7, 295–309. [CrossRef] [PubMed]
Grimsby, J.; Chen, K.; Wang, L.J.; Lan, N.C.; Shih, J.C. Human monoamine oxidase A and B genes exhibit
identical exon-intron organization. Proc. Natl. Acad. Sci. USA 1991, 88, 3637–3641. [CrossRef]
Eisner, P.; Klasen, M.; Wolf, D.; Zerres, K.; Eggermann, T.; Eisert, A.; Zvyagintsev, M.; Sarkheil, P.;
Mathiak, K.A.; Zepf, F.; et al. Cortico-limbic connectivity in MAOA-L carriers is vulnerable to acute
tryptophan depletion. Hum. Brain Mapp. 2017, 38, 1622–1635. [CrossRef]
6.
7.
Edmondson, D.E.; Binda, C.; Wang, J.; Upadhyay, A.K.; Mattevi, A. Molecular and mechanistic properties of
the membrane-bound mitochondrial monoamine oxidases. Biochemistry 2009, 48, 4220–4230. [CrossRef]
Narayanaswami, V.; Drake, L.R.; Brooks, A.F.; Meyer, J.H.; Houle, S.; Kilbourn, M.R.; Scott, P.J.H.; Vasdev, N.
Classics in Neuroimaging: Development of PET Tracers for Imaging Monoamine Oxidases. ACS Chem.
Neurosci. 2019, 10, 1867–1871. [CrossRef]
8.
9.
Is, Y.S.; Durdagi, S.; Aksoydan, B.; Yurtsever, M. Proposing Novel MAO-B Hit Inhibitors Using Multidimensional
Molecular Modeling Approaches and Application of Binary QSAR Models for Prediction of Their Therapeutic
Activity, Pharmacokinetic and Toxicity Properties. ACS Chem. Neurosci. 2018, 9, 1768–1782. [CrossRef]
Matos, M.J.; Teran, C.; Perez-Castillo, Y.; Uriarte, E.; Santana, L.; Vina, D. Synthesis and study of a series of
3-arylcoumarins as potent and selective monoamine oxidase B inhibitors. J. Med. Chem. 2011, 54, 7127–7137.
[CrossRef]
10. Blair, H.A.; Dhillon, S. Safinamide: A Review in Parkinson’s Disease. CNS Drugs 2017, 31, 169–176. [CrossRef]
11. Pisani, L.; Farina, R.; Nicolotti, O.; Gadaleta, D.; Soto-Otero, R.; Catto, M.; Di Braccio, M.; Mendez-Alvarez, E.;
Carotti, A. In silico design of novel 2H-chromen-2-one derivatives as potent and selective MAO-B inhibitors.
Eur. J. Med. Chem. 2015, 89, 98–105. [CrossRef] [PubMed]
12. Huang, M.; Xie, S.S.; Jiang, N.; Lan, J.S.; Kong, L.Y.; Wang, X.B. Multifunctional coumarin derivatives:
Monoamine oxidase B (MAO-B) inhibition, anti-beta-amyloid (Abeta) aggregation and metal chelation
properties against Alzheimer’s disease. Bioorg. Med. Chem. Lett. 2015, 25, 508–513. [CrossRef] [PubMed]
13. Joubert, J.; Foka, G.B.; Repsold, B.P.; Oliver, D.W.; Kapp, E.; Malan, S.F. Synthesis and evaluation of
7-substituted coumarin derivatives as multimodal monoamine oxidase-B and cholinesterase inhibitors for
the treatment of Alzheimer’s disease. Eur. J. Med. Chem. 2017, 125, 853–864. [CrossRef] [PubMed]
14. Pisani, L.; Catto, M.; Nicolotti, O.; Grossi, G.; Di Braccio, M.; Soto-Otero, R.; Mendez-Alvarez, E.; Stefanachi, A.;
Gadaleta, D.; Carotti, A. Fine molecular tuning at position 4 of 2H-chromen-2-one derivatives in the search of
potent and selective monoamine oxidase B inhibitors. Eur. J. Med. Chem. 2013, 70, 723–739. [CrossRef] [PubMed]
15. Fonseca, A.; Reis, J.; Silva, T.; Matos, M.J.; Bagetta, D.; Ortuso, F.; Alcaro, S.; Uriarte, E.; Borges, F. Coumarin
versus Chromone Monoamine Oxidase B Inhibitors: Quo Vadis? J. Med. Chem. 2017, 60, 7206–7212. [CrossRef]
[PubMed]
16. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Bizzarri, B.; Granese, A.; Carradori, S.; Yanez, M.; Orallo, F.;
Ortuso, F.; et al. Synthesis, molecular modeling, and selective inhibitory activity against human monoamine
oxidases of 3-carboxamido-7-substituted coumarins. J. Med. Chem. 2009, 52, 1935–1942. [CrossRef]
17. Secci, D.; Carradori, S.; Bolasco, A.; Chimenti, P.; Yanez, M.; Ortuso, F.; Alcaro, S. Synthesis and selective
human monoamine oxidase inhibition of 3-carbonyl, 3-acyl, and 3-carboxyhydrazido coumarin derivatives.
Eur. J. Med. Chem. 2011, 46, 4846–4852. [CrossRef]
18. Matos, M.J.; Vina, D.; Picciau, C.; Orallo, F.; Santana, L.; Uriarte, E. Synthesis and evaluation of 6-methyl-3-
phenylcoumarins as potent and selective MAO-B inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 5053–5055.
[CrossRef]
19. Matos, M.J.; Vina, D.; Quezada, E.; Picciau, C.; Delogu, G.; Orallo, F.; Santana, L.; Uriarte, E. A new series of
3-phenylcoumarins as potent and selective MAO-B inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 3268–3270.
[CrossRef]

Molecules 2019, 24, 4003
16 of 17
20. Matos, M.J.; Vilar, S.; Garcia-Morales, V.; Tatonetti, N.P.; Uriarte, E.; Santana, L.; Vina, D. Insight into the
functional and structural properties of 3-arylcoumarin as an interesting scaffold in monoamine oxidase B
inhibition. ChemMedChem 2014, 9, 1488–1500. [CrossRef]
21. Matos, M.J.; Rodriguez-Enriquez, F.; Vilar, S.; Santana, L.; Uriarte, E.; Hripcsak, G.; Estrada, M.;
Rodriguez-Franco, M.I.; Vina, D. Potent and selective MAO-B inhibitory activity: Amino- versus
nitro-3-arylcoumarin derivatives. Bioorg. Med. Chem. Lett. 2015, 25, 642–648. [CrossRef] [PubMed]
22. Delogu, G.; Picciau, C.; Ferino, G.; Quezada, E.; Podda, G.; Uriarte, E.; Vina, D. Synthesis, human monoamine
oxidase inhibitory activity and molecular docking studies of 3-heteroarylcoumarin derivatives. Eur. J. Med.
Chem. 2011, 46, 1147–1152. [CrossRef] [PubMed]
23. Costas-Lago, M.C.; Besada, P.; Rodriguez-Enriquez, F.; Vina, D.; Vilar, S.; Uriarte, E.; Borges, F.; Teran, C.
Synthesis and structure-activity relationship study of novel 3-heteroarylcoumarins based on pyridazine
scaffold as selective MAO-B inhibitors. Eur. J. Med. Chem. 2017, 139, 1–11. [CrossRef] [PubMed]
24. Rendenbach-Müller, B.; Schlecker, R.; Traut, M.; Weifenbach, H. Synthesis of coumarins as subtype-selective
inhibitors of monoamine oxidase. Bioorg. Med. Chem. Lett. 1994, 4, 1195–1198. [CrossRef]
25. Santana, L.; Gonzalez-Diaz, H.; Quezada, E.; Uriarte, E.; Yanez, M.; Vina, D.; Orallo, F. Quantitative
structure-activity relationship and complex network approach to monoamine oxidase A and B inhibitors.
J. Med. Chem. 2008, 51, 6740–6751. [CrossRef]
26. Abdelhafez, O.M.; Amin, K.M.; Ali, H.I.; Abdalla, M.M.; Batran, R.Z. Synthesis of new 7-oxycoumarin derivatives
as potent and selective monoamine oxidase A inhibitors. J. Med. Chem. 2012, 55, 10424–10436. [CrossRef]
27. Abdelhafez, O.M.; Amin, K.M.; Ali, H.I.; Abdalla, M.M.; Batran, R.Z. Monoamine oxidase A and B inhibiting effect
and molecular modeling of some synthesized coumarin derivatives. Neurochem. Int. 2013, 62, 198–209. [CrossRef]
28. Gnerre, C.; Catto, M.; Leonetti, F.; Weber, P.; Carrupt, P.A.; Altomare, C.; Carotti, A.; Testa, B. Inhibition of
monoamine oxidases by functionalized coumarin derivatives: Biological activities, QSARs, and 3D-QSARs.
J. Med. Chem. 2000, 43, 4747–4758. [CrossRef]
29. Deng, Z.L.; Du, C.X.; Li, X.; Hu, B.; Kuang, Z.K.; Wang, R.; Feng, S.Y.; Zhang, H.Y.; Kong, D.X. Exploring the
biologically relevant chemical space for drug discovery. J. Chem. Inf. Model. 2013, 53, 2820–2828. [CrossRef]
30. Hu, B.; Kuang, Z.K.; Feng, S.Y.; Wang, D.; He, S.B.; Kong, D.X. Three-Dimensional Biologically Relevant
Spectrum (BRS-3D): Shape Similarity Profile Based on PDB Ligands as Molecular Descriptors. Molecules
2016, 21, 1554. [CrossRef]
31. Wang, D.; Li, Z.; Liu, Y.; Chen, M.; Chen, N.; Zuo, Z.; Kong, D.X. Identification of novel monoamine oxidase
selective inhibitors employing a hierarchical ligand-based virtual screening strategy. Future Med. Chem. 2019
,
11, 801–816. [CrossRef] [PubMed]
32. He, S.B.; Ben, H.; Kuang, Z.K.; Wang, D.; Kong, D.X. Predicting Subtype Selectivity for Adenosine Receptor
Ligands with Three-Dimensional Biologically Relevant Spectrum (BRS-3D). Sci. Rep. 2016, 6, 36595. [CrossRef]
[PubMed]
33. Kuang, Z.K.; Feng, S.Y.; Hu, B.; Wang, D.; He, S.B.; Kong, D.X. Predicting subtype selectivity of dopamine
receptor ligands with three-dimensional biologically relevant spectrum. Chem. Biol. Drug Des. 2016, 88,
859–872. [CrossRef] [PubMed]
34. Catto, M.; Nicolotti, O.; Leonetti, F.; Carotti, A.; Favia, A.D.; Soto-Otero, R.; Mendez-Alvarez, E.; Carotti, A.
Structural insights into monoamine oxidase inhibitory potency and selectivity of 7-substituted coumarins
from ligand- and target-based approaches. J. Med. Chem. 2006, 49, 4912–4925. [CrossRef] [PubMed]
35. ACD/Percepta Platform. Advanced Chemistry Development; Advanced Chemistry Development, Inc.: Toronto,
ON, Canada. Available online: www.acdlabs.com (accessed on 26 May 2019).
36. Shao, C.-Y.; Su, B.-H.; Tu, Y.-S.; Lin, C.; Lin, O.A.; Tseng, Y.J. CypRules: A rule-based P450 inhibition
prediction server. Bioinformatics 2015, 31, 1869–1871. [CrossRef] [PubMed]
37. Jones, G.; Willett, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Development and validation of a genetic algorithm
for flexible docking. J. Mol. Biol. 1997, 267, 727–748. [CrossRef]
38. Berendsen, H.J.C.; van der Spoel, D.; van Drunen, R. GROMACS: A message-passing parallel molecular
dynamics implementation. Comput. Phys. Commun. 1995, 91, 43–56. [CrossRef]
39. Bayly, C.I.; Cieplak, P.; Cornell, W.; Kollman, P.A. A well-behaved electrostatic potential based method using
charge restraints for deriving atomic charges: The RESP model. J. Phys. Chem. 1993, 97, 10269–10280. [CrossRef]

Molecules 2019, 24, 4003
17 of 17
40. Gaussian 09. Revision E. 01; Gaussian, Inc.: Wallingford, CT, USA, 2009.
41. Kumari, R.; Kumar, R.; Lynn, A. g_mmpbsa—A GROMACS Tool for High-Throughput MM-PBSA
Calculations. J. Chem. Inf. Model. 2014, 54, 1951–1962. [CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).