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L. Liu et al.
Bioorganic Chemistry 109 (2021) 104685
[29]. Five different concentrations of p-tyramine (50–1500
μ
M) were
neurotoxicity. Then their neuroprotective ability in PC12 cells was also
measured by MTT method. PC12 cells were treated with rotenone and 6-
OHDA, which is reported to damage cathecolaminergic neurons through
ROS generation to generate PD-like models in vitro [33]. Rasagiline was
used as a positive control. Represent compounds and rasagiline at 10 µM
were incubated with PC12 cells for 1 h, then 6-OHDA (200 µM) or
rotenone (1.5 µM) were added and incubated with cells for 24 h, and
finally the MTT assay was performed to assess the cell viability. As
exhibited in Table 2 and Fig. 5A, rasagiline showed a prominent pro-
tective effect (34%) at 10 µM concentration in 6-OHDA-treated PC12
cells, which was coincident with literature [11]. All the selected com-
pounds also had significant protective effects on 6-OHDA-induced cells
death (>40% protection). In particular, compound 4d exhibited the
highest protection (48% increased) on 6-OHDA-treated cells. As shown
in Fig. 5B, all compounds exhibited low neuroprotective activities in
rotenone-treated PC12 cells, which was consistent with the rasagiline.
Compounds 4d and 4h had the best neuroprotection in rotenone-
induced cell death (17% increased).
used to measure the catalytic rate, and each graph was constructed at
four different concentrations of compound 4d (0, 0.25, 0.5 and 0.75
nM). The overlapping reciprocal Lineweaver-Burk plots (Fig. 2)
exhibited that the graphs of compound 4d in different concentrations
were linear and intersected at the y-axis. This pattern suggested that
compound 4d was a competitive hMAO-B inhibitor, and this result
further proved that compound 4d was a reversible hMAO-B inhibition.
2.5. Molecular modeling studies
In order to explain the hMAO-B selectivity of 3,4-dihydrocoumarins,
a structure-based molecular modeling study was carried out using the X-
ray crystal structures of hMAO-A (PDB code 2Z5X) and hMAO-B (PDB
code 2 V61) [30]. According to the inhibition results, compound 4d was
selected as a typical ligand, and the 2D and 3D pictures of binding modes
were shown in Fig. 3. As illustrated in Fig. 3A and 3B, compound 4d
located in the well-known binding pocket of hMAO-B, with the 3,4-dihy-
drocoumarin ring interacting with Lle 198, Leu 171, Gln 206 and Cys
172 at bottom of the substrate cavity, and a hydrogen bond was also
formed between the carbonyl oxygen of the ligand and Tyr 435. More-
over, the F-substituted benzyloxy group occupied the entrance cavity,
which was a hydrophobic subunit constituted by Tyr 326, Ile 316, Pro
104, Pro 102 and Ile 199. To further prove the importance of the ben-
zyloxy to hMAO-B inhibition, compound 2 was also docked with the
hMAO-B. The result in Fig. S1 showed that compound 2 could only
locate in the substrate cavity of hMAO-B and no other interaction was
established between compound 2 and the hMAO-B, suggesting the 3,4-
dihydrocoumarin structure alone could not inhibit hMAO-B and the
benzyloxy substituent was needed for hMAO-B inhibition [20-26]. For
hMAO-A, in Fig. 3C and 3D, it showed no interaction between the
hMAO-A with the ligands [31,32]. As a result, the hMAO-B selectivity
could be owed to the hydrogen bond interaction between compound 4d
and hMAO-B.
2.7. ADMET prediction.
If a compound wants to be developed as a candidate drug, low tox-
icities and high pharmacological activities are not enough, and phar-
macokinetic profiles of new drug candidates should be assessed as early
as possible. Fortunately, the combinatorial chemistry could easily assess
the absorption, distribution, metabolism and excretion (ADME) as soon
as possible [34]. Online Molinspiration property program was used to
calculate ADME properties of compounds 4a-n [35]. The stipulation of
ADME demands that an oral drug should be no more than one violation.
Meanwhile, the capability of compounds to cross the blood–brain bar-
rier (BBB) is also essential to develop the central nervous system (CNS)
drugs [36]. Log BB was calculated for latent applications in brains and
defined by the Lipinski’s rules: the small polar surface area<90 Å2, the
calculated logarithm of the octanol–water partition coefficient (Clog P)
<5, the molecular weight (MW) <500, the number of hydrogen bond
acceptor (HBA) atoms<10 and the number of hydrogen bond donor
(HBD) atoms<5. The log BB is calculated as the following equation: log
BB = 0.0148 × PSA + 0.152 × Clog P + 0.130.
2.6. Cytotoxicity and neuroprotection assays in PC12 cells
To prove the neuroprotection of these 3,4-dihydrocoumarins, the
cytotoxicity of represent compounds 4d-f, 4 h and 4j were first test in
neuroblastoma cells (PC12). The viability was assessed by the MTT [3-
As shown in Table 3, the theoretical calculations of ADME parame-
ters (log P, molecular weight, number of hydrogen donors, topological
polar surface area (tPSA), number of rotatable bonds and volume,
number of hydrogen acceptors) were presented, and the cases violating
Lipinski’s law were listed [37]. As indicated by the data, all compounds
showed a good ability to cross the BBB and followed the Lipinski’s rule
with no more than one violate. Thus, the new compounds perhaps had a
satisfying pharmacokinetics profile, which further strengthened the
biological importance of these compounds.
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide]
assay. As exhibited in Fig. 4, the results demonstrated that these com-
pounds showed slight toxicities at 200 µM after 24 h (the viability over
75%), but most of the compounds at 50 µM and 100 µM showed no
2.8. In vitro blood–brain barrier permeation assay
The ability to permeate the BBB is vital in PD treatment. The BBB
penetration of the target compounds were determined by the parallel
artificial membrane permeability assay (PAMPA-BBB), which was
described by Pardridge et al [36]. Compared with their reported values,
the experimental permeability of 9 reference drugs was rectified
(Table 4), which presented a good linear correlation: Pe(exp) =
1.0121Pe(Bibl.) – 0.5774 (R2 = 0.9441). To permeate BBB, compounds
were classified as follows: compounds with Pe (10-6 cm sꢀ 1) > 3.47 for
high BBB permeation (CNS + ), compounds with Pe (10-6 cm sꢀ 1) < 1.45
for low BBB permeation (CNS-), and compounds with 3.47 > Pe (10-6
m
sꢀ 1) > 1.45 for uncertain BBB permeation (CNS ± ). In Table 5, the Pe
values of selected compounds showed that compounds 4d-f, 4h and 4j
might have the ability to pass BBB.
Fig. 2. Kinetic study on the mechanism of hMAO-B inhibition by compound 4d.
Overlaid Lineweaver-Burk reciprocal plots of MAO-B initial velocity at
increasing substrate concentration (50–1500 μM) in the absence of inhibitor
and in the presence of 4d are shown. Lines were derived from a weighted least-
squares analysis of the data points.
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Liu, Li
