Optimization of N-Phenylpropenoyl- l -amino Acids as Potent and Selective Inducible Nitric Oxide Synthase Inhibitors for Parkinson's Disease

Article abstract of DOI:10.1021/acs.jmedchem.1c00578

N-Phenylpropenoyl-l-amino acids (NPAs) are inducible nitric oxide synthase (iNOS) inhibitors possessing preventive effects for Parkinson's disease (PD). Here, structural modifications for improving the iNOS inhibitory activity and blood-brain barrier (BBB) permeability of NPAs were conducted, leading to 20 optimized NPA derivatives (1-20). Compound 18, with the most potent activity (IC50 = 74 nM), high BBB permeability (Pe = 19.1 × 10-6 cm/s), and high selectivity over other NOS isoforms, was selected as the lead compound. Further studies demonstrated that 18 directly binds to iNOS. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced acute PD model, the oral administration of 18 (1 and 2 mg/kg) exerted preventive effects by alleviating the loss of dopaminergic (DAergic) neurons. Notably, in the MPTP-/probenecid-induced chronic PD model, the same dose of 18 also displayed a therapeutic effect by repairing the damaged DAergic neurons. Finally, good pharmacokinetic properties and low toxicity made 18 a promising candidate for the treatment of PD.

Full text of DOI:10.1021/acs.jmedchem.1c00578

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Article
Optimization of N‑Phenylpropenoyl‑L‑amino Acids as Potent and
Selective Inducible Nitric Oxide Synthase Inhibitors for Parkinson’s
Disease
Xiao-Long Hu, Xian-Yu Lv, Rong Wang, Huan Long, Jia-Hao Feng, Bao-Lin Wang, Wei Shen, Hao Liu,
Fei Xiong,* Xiao-Qi Zhang, Wen-Cai Ye, and Hao Wang*
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ABSTRACT: N-Phenylpropenoyl-L-amino acids (NPAs) are inducible
nitric oxide synthase (iNOS) inhibitors possessing preventive effects for
Parkinson’s disease (PD). Here, structural modifications for improving the
iNOS inhibitory activity and blood−brain barrier (BBB) permeability of
NPAs were conducted, leading to 20 optimized NPA derivatives (1−20).
Compound 18, with the most potent activity (IC50 = 74 nM), high BBB
permeability (Pe = 19.1 × 10−6 cm/s), and high selectivity over other NOS
isoforms, was selected as the lead compound. Further studies
demonstrated that 18 directly binds to iNOS. In the 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP)-induced acute PD model, the oral
administration of 18 (1 and 2 mg/kg) exerted preventive effects by
alleviating the loss of dopaminergic (DAergic) neurons. Notably, in the
MPTP-/probenecid-induced chronic PD model, the same dose of 18 also
displayed a therapeutic effect by repairing the damaged DAergic neurons.
Finally, good pharmacokinetic properties and low toxicity made 18 a promising candidate for the treatment of PD.
much work has been focused on iNOS inhibitors for acute
inflammation, severe shock, and diabetes, and only a few are
related to PD due to their poor efficiency, blood−brain barrier
(BBB) permeability, and isoform selectivity.16 Our previous
efforts in achieving iNOS inhibitors with excellent potency
have led to a class of molecules bearing a scaffold of N-
phenylpropenoyl-L-amino acids (NPAs). Among these NPAs,
hit compound 4b (IC50 = 1.09 μM) has shown good efficacy
for PD prevention in vitro and in vivo.17 These results reported
that NPAs are new potent iNOS inhibitors for PD therapy;
therefore, further optimization of 4b focusing on activity,
isoform selectivity, and BBB permeability was necessary.
In this work, 20 additional optimized NPA derivatives (1−
20), which possessed enhanced iNOS inhibitory effects and
BBB permeability compared with those of 4b, were designed
and synthesized. Their structure−activity relationships (SARs)
and structure−selectivity relationships were discussed. The
preventive effects in the acute PD model and the therapeutic
INTRODUCTION
■
Parkinson’s disease (PD) is the second most common age-
related neurodegenerative disorder characterized by the loss of
dopaminergic (DAergic) neurons in the substantia nigra pars
compacta (SNpc), which decreases the interstitial dopamine
(DA) level, resulting in bradykinesia, rigidity, and tremor.1
Current therapeutics mainly focus on the enhancement of the
DA level, such as levodopa (L-DOPA), catechol-O-methyl-
transferase inhibitors, monoamine oxidase type B (MAO-B)
inhibitors, and DA receptor agonists.2−6 These available PD
drugs offer valuable symptomatic relief but are often
accompanied by serious side effects and cannot slow down
the progression of PD.7 Neuroinflammation is one of the
hallmarks of PD and contributes to DAergic neuron
degeneration.8 Targeting excessive neuroinflammation could
offer new therapeutic opportunities for PD and might slow
down the disease progression.8
Inducible nitric oxide synthase (iNOS), the key enzyme for
the synthesis process of toxic nitric oxide (NO), plays a critical
role in inflammation and neuroinflammation.9,10 NOS has
three isoforms, including endothelial NOS (eNOS), neuronal
NOS (nNOS), and iNOS, which are involved in the catalytic
production of NO and maintaining normal physiological
reactions.11 Several studies have demonstrated that iNOS is
overexpressed and overactivated in animals and patients with
PD, indicating that iNOS is a key target for PD.12−15 However,
Received: March 30, 2021
Published: May 21, 2021
https://doi.org/10.1021/acs.jmedchem.1c00578
© 2021 American Chemical Society
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Figure 1. Strategies for further structural optimization of 4b.
effects in the chronic PD model, the absorption, distribution,
metabolism, excretion, and toxicity (ADME/Tox) profiles, and
the pharmacokinetic profiles of the candidate compound were
further investigated. This study led to the identification of
highly potent compounds with significant improvement.
Among them, compound 18 proved to be particularly
promising.
Consequently, 209 compounds were designed belonging to the
19 series (a−s, Table S1). Then, molecular docking assay was
used to predict the inhibitory effects of these 209 compounds
on iNOS (PDB code: 1R35)17,18 and ADME properties. The
most important factor in identifying drugs for neurological
diseases is their ability to penetrate BBB. In the Discovery
studio software, only compounds with predicted BBB levels of
0, 1, or 2 were considered to permeate the BBB, while 3 or 4
were not. Usage of levels 0, 1, or 2 as the cutoff for
ADMET_BBB_LEVEL ensured that only the compounds that
could pass the BBB would be retained. In addition, the higher
the absolute values of -CDOCKER energy are, the more stable
the binding is. Notably, although some compounds, such as
compounds i-I, i-II, and i-III and j-I and s-I (in Table S2),
showed higher -CDOCKER energy than 4b, their BBB levels
were predicted to be level 3 or level 4 and therefore, they were
not considered. As a result, 20 of these compounds (red font in
Table S2) with the higher absolute values of -CDOCKER
energy than 4b (24.3 kcal/mol) and higher BBB permeability
(level 1 or level 2) than 4b (level 4) were screened out. Here,
in this work, these 20 NPAs (1−20) were efficiently
synthesized under mild and water-compatible conditions
(Scheme 1). Notably, compounds 1−20 can be easily obtained
in a 10 g scale without the use of column chromatography
techniques.
Inhibitory Effects of 1−20 on NO Production and
iNOS Activity. All the synthesized NPAs were tested for their
anti-neuroinflammatory effects using a lipopolysaccharide
(LPS)-induced BV-2 cell activation model. As shown in Figure
2, 1−20 exhibited stronger NO inhibitory effects than 4b,
especially 13−20 (1 μM, inhibition rate > 50%). The
inhibitory effects of these compounds and 4b on iNOS were
further tested using the iNOS enzyme activity assay. Results
showed that 13−20 also showed promising iNOS inhibitory
effects with half maximal inhibitory concentration (IC50)
values ranging from 74 to 434 nM (Table 1), consistent with
the tendency of NO inhibition. This result suggests that these
RESULTS AND DISCUSSION
■
Design and Chemistry. Our previous study has
demonstrated that NPAs, especially 4b (Figure 1), are potent
iNOS inhibitors with preventive effects in PD.17 This hit
compound matched “Lipinski’s rule of five” and it also
provided multiple diversification points for robust optimiza-
tion. With careful analysis, it was supposed that the two
phenolic hydroxy groups present in 4b might bring some
negative effects as follows: (1) the polar of the molecule is
strong, which might affect the absorption and BBB
permeability and (2) glucuronidation of multi-hydroxyl groups
may shorten the action time of the compound within the body.
Besides, 4b was not potent enough and its activity should be
improved in vitro and in vivo. Taken together, structural
modifications were conducted to increase the iNOS inhibitory
effect and BBB permeability of 4b, as well as improve its
physicochemical properties. According to the basic principles
of drug design, the following strategies were used: (1) the
structural skeleton, cinnamic acid moiety, and amino acid
moiety (L-tyrosine) of 4b were retained, and minimal
modifications were made to the benzene ring of the cinnamic
acid moiety to adjust the lipid−water partition coefficients
and/or improve its stability/or improve its activity, such as
changing the hydroxyl groups and introducing the nitrogen-
containing groups, halogen, trifluoromethyl, or aldehyde
groups (Figure 1) and (2) substantial changes were made to
the amino acid moiety by replacing the L-tyrosine with several
aliphatic amino acids, aromatic amino acids, and heterocyclic
amino acids to enhance the pharmacological effects (Figure 1).
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Scheme 1. Synthesis of Compounds 1−20
trifluoromethyl electron-withdrawing group (17−20, IC50
=
74−195 nM) can substantially increase iNOS inhibitory effects
compared with that of the NPAs substituted by methyl (13−
16, IC50 = 188−434 nM) and chlorine (5−8, IC50 = 731−945
nM) (Table 1), indicating that introducing an electronegative
group to the C-4 position might increase the inhibitory effects
of NPAs on iNOS. For the amino acid moiety, NPAs
containing an aromatic amino acid moiety were found to
exert stronger iNOS inhibitory effects than those with aliphatic
amino acid moiety, such as the IC50 values of 13 (291 nM) and
14 (188 nM) < 15 (434 nM) and 16 (421 nM), respectively,
as well as 17 (110 nM) and 18 (74 nM) < 19 (195 nM) and
20 (178 nM), respectively (Table 1). In addition, a three-
dimensional quantitative SAR (3D-QSAR) model was built by
Discovery Studio 3.0 (DS 3.0) software to evaluate the factors
in correlation with in the bioassay results, and the structural
features contributing to the corresponding activities. The
results showed that the QSAR model was acceptable because
the correlation coefficient (R2) between the observed and
predicted activities of training was 0.664, whereas that of the
Figure 2. Effects of compounds 1−20 and 4b on NO release in BV-2
cells.
compounds exerted anti-neuroinflammatory effects by target-
ing iNOS.
SAR Analysis and Three-Dimensional Quantitative
Structure−Activity Relationship Model. SARs were
discussed by structural moieties. For the cinnamic acid moiety,
the C-4 position of the NPAs functionalized with one
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Table 1. Effects of 1−20 on NOS Activity and BBB Permeability
a
IC50 (nM)
selectivity
comp.
iNOS
nNOS
eNOS
hnNOS/hiNOS
heNOS/hiNOS
Pe (10−6 cm s−1) PAMPA-BBB
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
4b
630 38
820 53
594 41
645 33
731 67
945 84
782 56
916 88
766 65
993 67
765 74
640 34
291 25
188 13
434 29
421 41
64314 5320
125618 7860
20679 1980
25814 2100
102382 9810
156987 10920
39784 3100
56719 4900
110555 9870
148510 9860
29640 2800
25613 2120
31905 2540
34210 2800
18480 1550
20250 1400
11994 980
16650 1010
6120 540
66194 5480
147301 9870
30605 2400
33142 3100
106370 8920
188770 10020
48850 3950
46610 3980
110680 9810
163860 10210
29745 1800
30560 2000
37620 2980
38000 2700
17100 1250
21050 1590
15530 1200
18500 1580
5600 380
102
153
51
105
180
52
35
51
140
166
51
146
199
62
62
51
151
149
39
144
165
39
40
48
110
182
43
129
202
39
4.61 0.78
4.32 1.02
4.88 0.56
4.97 0.71
10.42 0.97
19.02 1.32
8.05 1.00
9.01 0.89
1.23 0.46
3.18 0.95
48
50
110
74
7
6
109
225
31
45
47
141
250
29
54
52
195 12
178 14
892 45
8.4 0.4
8360 700
40330 2900
1980 140
9600 740
46590 3800
51200 4520
b
1400W
236
6095
a
b
Selectivity = IC50(human nNOS)/IC50(human iNOS); IC50(human eNOS)/IC50(human iNOS). 1400W is a selective iNOS inhibitor, used as the reference control.
Figure 3. 3D-QSAR model. (A) 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients and red
represents negative coefficients. (B) 3D-QSAR model coefficients on van der Waals grids. Green represents positive coefficients and yellow
represents negative coefficients.
test set was 0.917 (Figure S1). The molecules aligned with the
iso-surfaces of the 3D-QSAR model coefficients on van der
Waals grids (Figure 3A) and electrostatic potential grids
(Figure 3B) were also listed. Red contours in the electrostatic
map indicate the regions where a high-electron density
(negative charge) is expected to increase activity, and the
blue contours represent regions where a low-electron density
(partial positive charge) is expected to increase activity.
Likewise, the steric map indicates the regions where steric
bulk is predicted to increase (green) or decrease (yellow)
activity. According to the maps, these compounds possessing a
highly negative charge at the C-4 position and big bulk group
would show higher iNOS inhibitory effects. These results
provided a strategy for the design and optimization of NPA
derivatives as potent iNOS inhibitors.
Isoform Selectivity. The current iNOS inhibitors have not
been converted into clinically available drugs for PD due to
their poor isoform selectivity for iNOS over eNOS and
nNOS.19 These two isoforms share very similar structural
features in the active site as that of iNOS. The overinhibition
of these two NOS isoforms should be avoided because they
take part in the maintenance of normal physiological
function.16,19 First, eight compounds (13−20), with potent
iNOS inhibitory effects, were selected for the evaluation of
their NOS isoform selectivity. 13, 14, 17, and 18 exhibited
human eNOS/human iNOS selectivity, and human nNOS/
human iNOS selectivity in the 100−250 range, especially 14
and 18 (in the range of 180−250). However, 15, 16, 19, and
20 showed poor (<60-fold) isoform selectivity (Table 1). The
results indicated that the NPAs containing an aromatic amino
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Figure 4. Molecular docking and MD simulation. (A) Predicted 3D binding mode of 18 with the iNOS crystal structure (PDB 1R35). Hydrogen
bonds between inhibitors and amino acid residues are indicated by green dashed lines. (B) Predicted two-dimensional binding mode of 18 toward
iNOS. (C) RMSD plot of native iNOS and complexed with EuC in black and red colors, respectively. (D) Rg plot of native iNOS and complexed
with 18 in black and red colors, respectively.
acid moiety might have better iNOS selectivity than those with
an aliphatic amino acid moiety. The NOS isoform selectivity of
1−12 was detected as corroborative evidence to further verify
the inference. Table 1 shows that the NPAs with aromatic
amino acid moieties (1, 2, 5, 6, 9, and 10), respectively, show
better iNOS selectivity than the NPAs with aliphatic amino
acid moieties (3, 4, 7, 8, 11, and 12). These results
demonstrated that the aromatic amino acid moiety of NPAs
contributed greatly to iNOS selectivity.
inhibitory effects, high isoform selectivity, and desirable BBB
permeability, was selected as the lead compound for further
exploration.
Docking and Molecular Dynamics Simulation of 18
Bound to iNOS. We used docking and atomistic force field-
based molecular dynamic (MD) simulations to model 18
bound to iNOS. First, the crystal structure of iNOS (PDB code
1R35)18 was used for the docking studies. As seen in Figure
4A,B, the protein aids ligand’s anchoring within the pocket by
the H bond between 18 and GLU371 (2.1 Å), TRP366 (2.8
Å), GLN257 (2.8 Å), and ARG260 (2.7 Å). The binding of a
small molecule in the binding pocket of a protein can led to
large conformational changes. Root-mean square deviation
(RMSD) is one of the most important fundamental properties
used to evaluate the structural stability of a protein.21 Results
showed that the average RMSD values of the iNOS and iNOS-
18 complexes were 0.31 and 0.21 nm, respectively (Figure
4C). The binding of 18 led to lower RMSD values at several
parts and showed equilibration throughout the 100 ns MD
simulations (Figure 4C). In addition, the average fluctuations
of all residues and the root-mean square fluctuations (RMSFs)
of iNOS upon ligand binding were plotted during the
simulation as a function of the residue number to further
assess local structural flexibility (Figure 4D). The RMSF plot
reveals the residual fluctuations in iNOS at several regions of
the protein structure. These residual fluctuations were
minimized upon the binding of 18 throughout the simulations
in a region spanning from the N-terminal to the C-terminal.
These results suggested the strong binding between iNOS and
18. Therefore, 18 might directly target iNOS.
Effects of 13−20 on Artificial Membrane Perme-
ability. The poor BBB permeability of reported iNOS
inhibitors greatly limits the delivery of these compounds into
the brain.16 Therefore, 13−20 with potent inhibitory effects
and selectivity were selected to evaluate their effects on cell
membrane permeability through parallel artificial membrane
permeability for the BBB (PAMPA-BBB) assay. In the
PAMPA-BBB assay, a compound with good BBB penetration
is classified as a central neural system (CNS) (+) molecule, if
its effective permeability (Pe) is greater than 4.0 × 10−6 cm/
s.20 Results showed that 13−20 exhibited CNS (+) character-
istics, and 17−20 (Pe = 8.05 × 10−6 to 19.02 × 10−6 cm/s)
displayed higher permeability than 13−16 (Pe = 4.32 × 10−6 to
4.97 × 10−6 cm/s; Table 1). The results suggest that
introducing trifluoromethyl functionality could improve the
BBB permeability of NPAs (17−20) considerably. Replacing
methyl (13) with the trifluoromethyl group (17) resulted in an
approximately 2.5-fold increase in permeability and introduc-
ing fluorine on the aromatic ring of amino acids (18) resulted
in an approximately 4.5-fold increase in permeability (Table
1). Therefore, introducing fluorine atoms into NPAs can
increase the activity, selectivity, and BBB permeability of
NPAs. Compound 18, which had the most potent iNOS
18 Directly Binds to iNOS. Thermodynamic and kinetic
experiments were carried out to evaluate the binding character
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Figure 5. Thermodynamic and kinetic experiments of 18 toward iNOS. (A,B) CETSA and ITDRFcesta were used to evaluate the binding between
18 and iNOS at the thermodynamic level. (C) SPR was used to assess the binding between 18 and iNOS at the kinetic level. (D) Effects of 18 on
iNOS activity at different time points (n = 3).
between 18 and iNOS. The cellular thermal shift assay
(CETSA) was used in the thermodynamic test to study the
thermal stabilization of the proteins upon ligand binding.22,23
This assay has been used extensively on purified proteins to
detect the interactions between donors and ligands. Results
showed that the apparent aggregation temperatures were
obtained with either 18 or dimethyl sulfoxide (DMSO), which
could be compared, and substantial shifts demonstrated the
binding of 18 and target proteins. Figure 5A shows that the
thermal stabilization of iNOS increased compared with that of
the control group (DMSO) after 18 was bound to iNOS. This
thermal stabilization between 18 and iNOS was dose
dependent according to the isothermal dose−response finger-
print (ITDRF) in CETSA (Figure 5B). Surface plasmon
resonance, which is the most recognized method for studying
the dynamic properties between ligands and donors,24,25 was
used in the kinetic test to further confirm the binding between
18 and iNOS. Results showed a strong binding between 18
and iNOS (KD = 18 nM, Figure 5C). These results fully
demonstrated that 18 directly binds to iNOS. In addition, 18
contained a Michael-acceptor moiety (propenamide), which is
a common warhead of a number of drugs that irreversibly
inactivate their targets. Therefore, a time-dependent inhibition
study of treatment with iNOS was carried out to distinguish 18
between inhibitors and inactivators. Figure 5D shows that 18
had no time-dependent effects, suggesting 18 is an iNOS
inhibitor rather than an inactivator.
microglia treated with LPS was collected to stimulate DAergic
neurons (SH-SY5Y cells), which were used to evaluate the in
vitro anti-PD effects of 18. Results showed that the cell
viability of SH-SY5Y was decreased by 40% after 24 h of
incubation with CM. However, CM derived from BV-2 cells
with 18 (0.1, 1, and 10 μM) treatment for 6 h prior to LPS
administration reduced SH-SY5Y cell injury in a dose-
dependent manner (Figure 6A,B). Microscopic observation
(Figure 6C), Hoechst 33258 staining (Figure 6D), and
annexin V/FITC−propidium iodide (PI) double staining
(Figure 6E,F) showed that 18 can reduce cell apoptosis in a
dose-dependent manner. These results demonstrated that 18
can alleviate the injury of DAergic neurons induced by
neuroinflammatory toxicity.
Pre-treatment of 18 Alleviated the PD-like Behavior
of the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-
Induced Acute PD Model. 1-Methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) produces a L-DOPA-responsive
Parkinsonian syndrome, which is characterized by all the
cardinal symptoms of PD and represents the best PD-like
clinical picture obtainable in experimental animals.27 In this
study, traction test, rotarod test, and open-field test were used
to evaluate the in vivo anti-PD effects of 18 in the MPTP-
induced acute PD model (Figure 7A). The traction test
showed that MPTP treatment decreased strength as the hind
limb grip score of the MPTP model was lower than that of the
control group. However, oral pre-treatment with 18 (1 or 2
mg/kg) can considerably increase the traction score. There-
fore, prophylactic treatment with 18 could alleviate the
reduction in the strength and equilibrium of muscles caused
by MPTP (Figure 7B). The rotarod test showed that the time
taken by MPTP-treated mice in the rotarod apparatus was
remarkably reduced compared with the control. MPTP-treated
mice that were given 18 stayed on the apparatus significantly
18-Protected SH-SY5Y Cells from Neurotoxicity
Induced by Microglial Activation. Microglia, a brain-
resident immune cell, plays a central role in the process of PD
by neuroinflammation. Activated microglia promote the
production and activation of iNOS to generate overload NO,
which make DAergic neurons more vulnerable to degeneration
or toxicity.26 In this study, the cell medium (CM) of BV-2
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Figure 6. 18 protected DAergic neurons from neurotoxicity induced by microglial activation. SH-SY5Y cells were incubated for 24 h with
conditioned medium derived from cultures of BV-2 cells. Before collecting culture media, BV-2 cells were pre-treated with 18 (0.1, 1, or 10 μM) for
2 h and incubated with LPS (0 or 500 ng/mL). (A) Cell viability measured with the MTT assay (n = 6). (B) LDH leakage assay (n = 6). (C)
Morphological observation. (D) Cell nuclei stained with Hoechst 33258 (blue). (E) Apoptosis of SH-SY5Y cells evaluated by FCM detection of
annexin V−PI double staining [distribution of viable (lower left), necrotic (upper left), late apoptotic (upper right), and early apoptotic (lower
right) cells]. (F) Statistical analysis of three independent experiments of annexin V−PI double staining. All data are presented as means SD or
SEM. **p < 0.01 or ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, or ###p < 0.001, compared with the LPS group.
longer than MPTP mice (Figure 7C). The open-field test
showed that the total distances (Figure 7D,F) and average
speed (Figure 7E) of the control and of the MPTP model
group were substantially decreased and shortened, respectively,
and the difference was statistically significant compared with
those of the control group. The total distance and average
speed of the MPTP + 18 group were significantly higher and
longer compared with those of the model group. Although the
positive drug, L-DOPA, showed similar effects, its dose−effect
relationship was far less than that of 18. These behavioral
results suggested that 18 could effectively alleviate movement
disorders related to acute PD and had a preventive effect on
PD at low dosages.
Pre-treatment with 18 Decreased the Level of NO
and Increased the Levels of DA and Tyrosine
Hydroxylase in the MPTP-Induced Acute PD Model.
MPTP remarkably induces DAergic neuron loss by activating
the iNOS/NO/pathway in microglia cells, which results in the
reduction of DA levels.28 Results showed that MPTP
remarkably increased the NO level and decreased the DA
level in the SNpc and striatum (STR) compared with the
control group. However, pre-treatment with 18 remarkably
decreased the NO level and increased the DA level in the SNpc
and STR compared with the MPTP group (Figure 8A,B).
Tyrosine hydroxylase (TH), the rate-limiting step in the
biosynthesis of DA, is commonly regarded as a marker of
DAergic neurons to evaluate the degrees of PD. Results from
the immunohistochemistry and immunoblotting of the TH
protein showed that the number of TH positive cells (Figure
8C,D) and the expression of TH (Figure 8E) in the SNpc and
STR of the MPTP-treated mice were decreased markedly
compared with the control mice but were increased remarkably
in the 18 pre-treated mice. These results indicated that 18
elicited neuroprotective effects against DAergic neuronal cell
death in PD.
Pre-treatment of 18 Inhibited the Mitochondrial
Apoptotic Pathway of SNpc and STR in the MPTP-
Induced Acute PD Model. The apoptosis of DAergic
neurons in the SNpc was the main reason for DA loss.1 The
mitochondria of DAergic neurons are a preferential target of
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Figure 7. Prevention of 18 on MPTP-induced acute PD motor deficits. The behavioral measurements were carried out in the 4th, 24th, 48th, and
72nd h after the last injection of MPTP. (A) Experimental procedure and drug administration scheme. (B) Traction test. (C) Rotarod test. (D)
Motion trail of the first three mice in each group in the open-field test (n = 8 mice/group). (E) Total distance of mice in the open-field test (n = 8
mice). (F) Mean velocity of mice in the open-field test (n = 8 mice). All data are presented as means SD. **p < 0.01 or ***p < 0.001, compared
with the control group; #p < 0.05, ##p < 0.01, or ###p < 0.001, compared with the MPTP treatment group.
the NO induced by MPTP; thus, myriads of studies explored
mitochondrial function in PD in the subsequent decades, and
MPTP induction provides a formidable animal model of PD.
Our results showed that the Bcl-2/Bax ratios in the SNpc and
STR were remarkably decreased and the activities of caspase-
3/9 in the SNpc and STR were increased in the MPTP-treated
group compared with the control group. However, the pre-
treatment with 18 can increase the Bcl-2/Bax ratio (Figure 9A)
and decrease the activities of caspase-3 (Figure 9B) and
caspase-9 (Figure 9B) in the SNpc (Figure 9C) and the STR
(Figure 9D−F) in a dose-dependent manner. L-DOPA almost
showed no inhibitory effects on mitochondrial apoptosis-
related proteins. These results demonstrated that 18 can
decrease the DAergic neuron loss by inhibiting the
mitochondrial apoptotic pathway.
18 Showed a Therapeutic Effect in the MPTP/
Probenecid-Induced Behavior Disorder of the Chronic
PD Model. Most reported anti-PD agents were pre-treated to
mice; hence, the agents possess preventive effects on PD but
not therapeutic effects and have limitations in clinical
application. Indeed, the DAergic nigrostriatal deficits obtained
with the acute administration of MPTP (four injections over 1
day) tend to be reversible. The addition of probenecid
potentiates the effects of MPTP, which allows the gradual loss
of SNpc neurons and is associated with a substantial loss of
striatal DA and DA uptakes.29 This effect lasts for at least 6
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Figure 8. Prevention of 18 on MPTP-induced nigrostriatal DAergic injury. (A) Level of NO in SNpc and STR. (B) Level of DA in SNpc and STR.
(C) Immunohistochemical staining showing TH positive cells in the SNpc and STR (scale bar: 200 μm). (D) Stereological counting of TH
positive cells (n = 6). (E) Western blot analysis of the changes in the protein level of TH in both SNpc and STR. All data are presented as means
SEM. **p < 0.01 or ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, or ###p < 0.001, compared with MPTP treatment
group.
months after withdrawal from treatment. Therefore, in this
section, the chronic MPTP/probenecid-induced mice model of
PD (Figure 10A), which closely mimics the chronic and
progressive neurodegeneration and behavioral deficits ob-
served in human PD, was used to further investigate the
therapeutic effect of 18 on PD.29 Results showed that the mice
began to have motor disorders in several behavior tests,
namely, traction test (Figure 10B), pole test (Figure 10C), and
open-field test (Figure 10D−G), after 30 days of MPTP/
probenecid injection. However, oral treatment with 18 (1 or 2
mg/kg) considerably reversed these disorders, whereas L-
DOPA did not. These behavior tests indicated that 18 had
therapeutic effects on PD.
and DOPA decarboxylase, the two key neural markers of
DAergic neurons, was conducted to evaluate the repairing
effect of 18 on DAergic neuron injury and verify the
assumption. Results showed that the number of cells positive
for TH and DOPA decarboxylase was remarkably decreased
compared with the control group after 30 days of MPTP/
probenecid injection. This result indicates that many DAergic
neurons in the SNpc were lost. However, the number of TH-
and DOPA decarboxylase positive cells increased after 20
consecutive days of treatment with 18 (Figure 11A−C).
Western blot analysis showed that 18 can greatly increase the
expression of TH and DOPA decarboxylase compared with the
MPTP/probenecid group (Figure 11D,E). These results
indicated that 18 can repair damaged DAergic neurons in PD.
Glial cell line-derived neurotrophic factor (GDNF), which is
secreted by microglia, can promote the regeneration of
DAergic neurons in the SNpc.30,31 Therefore, the effect of
18 on GDNF secretion in glial cells was detected by
18 Can Increase the DAergic Neuron Loss Induced by
MPTP/Probenecid. Behavior tests demonstrated that 18 can
prevent and treat PD in very low dosages. The therapeutic
effects suggest that 18 might have a function in the repairing of
DAergic neuron injury in PD. Immunohistochemistry with TH
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Figure 9. 18 alleviated the apoptosis of the SNpc neuron in the MPTP-induced acute PD model. (A,D) Original bands of Bax and Bcl-2 in SNpc
and STR, β-actin served as control. (B,E) Quantitative analysis of blots. (C,F) Caspase-3 and caspase-9 activities in SNpc and STR. All data are
presented as means SEM. ***p < 0.001, compared with the control group; ##p < 0.01 or ###p < 0.001, compared with the MPTP treatment
group.
immunofluorescence histochemistry and enzyme-linked im-
munosorbent assay. Results showed that 18 can substantially
increase the level of GDNF in glial cells compared with the
control and MPTP/probenecid groups (Figure 11F). Fur-
thermore, the level of GDNF in the SNpc was also remarkably
increased by 18 in a dose-dependent manner (Figure 11G).
These data suggested that the therapeutic effect of 18 would
most likely promote the secretion of GDNF in glial cells to
repair damaged DAergic neurons in the SNpc.
biochemistry analysis (Table S4), body weight (Figure S3),
and blood routine analysis (Figure S2) also indicated that 18
can be considered for long-term administration as PD
medication.
CONCLUSIONS
■
Our previous efforts in achieving iNOS inhibitors with
excellent potency have led to a class of molecules bearing a
scaffold of NPAs. However, their iNOS inhibitory effects and
BBB levels still have much room for improvement. This study
describes the design, synthesis, and biological activities of a
series of NPA derivatives that act as potent selective iNOS
inhibitors. First, 209 NPA derivatives were designed, and
among these compounds, 20 NPA derivatives (1−20) with
both predicted potent activities, and higher BBB permeability,
were finally confirmed to be synthesized through a concise,
efficient, and gram-scale method. Some generalities about the
SARs of these compounds can be made as the following: (1)
the introduction of trifluoromethyl group into the cinnamic
acid moiety remarkably increased the activity; (2) NPAs
containing aromatic amino acid exhibited better iNOS
selectivity than those with aliphatic amino acid. The
structure−permeability relationship of these compounds
showed that the introduction of the aromatic amino acid and
trifluoromethyl group make great contributions to BBB
permeability. Notably, the oral administration of 18 at low
dosages (1 and 2 mg/kg) demonstrated preventive and
therapeutic effects in the MPTP-induced acute PD model
and MPTP/probenecid-induced chronic PD model through
iNOS inhibition and DAergic neuronal repair pathways. Our
study provided evidence supporting 18 as a potential candidate
for PD treatment because of its potency in vitro and in vivo,
impressive penetration of the BBB, and low toxicity.
In Vitro ADME/Tox and In Vivo Pharmacokinetic
Properties of 18. The drug-like properties of 18 were
examined through CYP inhibition and microsomal stability
tests. The inhibitory effects of 18 on CYP enzymes (subtypes
2C19, 2D6, 2C9, 1A2, and 3A4) were tested for the possibility
of drug−drug interactions. The results are expressed as the
percentage of CYP activity that remained after treatment with
18 at 10 μM (Table 2). The stability of 18 was determined
from the percentage of the parent compound remaining after
30 min of incubation with human liver microsomes (Table 2).
The plasma stability of 18 in mice was also excellent, with
94.2% of the parent compound remaining after 30 min of
incubation. In addition, the physicochemical properties of 18
were confirmed to be compatible with beneficial drug-like
properties (predicted pKa = 6.95; c log P = 2.75). A summary
of data related to compound 18 are shown in Table 2. In the
pharmacokinetic study, we observed that 18 dosed as an oral
solution was rapidly absorbed, and its blood and brain
concentrations reached the ideal range (Table 3) and were
higher than those of 4b. The results suggested that our
structure optimization was successful, and the optimization
method can increase the activity and improve the efficacy of 18
in vivo.
Safety Profiles of 18. In the in vivo toxicity study, 18
showed satisfactory maximum tolerated doses with a median
lethal dose of 3.45 g/kg after being orally administered to mice.
The hematoxylin and eosin (HE) staining of the main viscera
(Figure S2) showed that 18 had no detectable adverse effects
in the heart, liver, spleen, lung, and kidney at 1000 mg/kg in
the 14-day toxicity study. Organ coefficients (Table S3), blood
EXPERIMENTAL SECTION
■
General Chemistry. Reagents and solvents were purchased from
common commercial suppliers and were used without further
purification. Reaction progress was monitored using analytical thin-
layer chromatography on precoated silica gel GF254 (Qingdao
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Figure 10. Therapeutic effects of 18 on MPTP/probenecid-induced chronic PD motor deficits. (A) Experimental procedure and drug
administration scheme. (B) Traction test. (C) Rotarod test. (D) Motion trail of the first three mice in each group (no. 1, 2, and 3) in the open-field
test (n = 8 mice/group). (E) Total distance of mice in the open-field test (n = 8 mice). (F) Mean velocity of mice in the open-field test (n = 8). (F)
Mean speed of mice in the open-field test (n = 8). All data are presented as means SEM. ***p < 0.001, compared with the control group; ##p <
0.01 or ###p < 0.001, compared with the MPTP treatment group.
Haiyang Chemical Plant, Qingdao, China) plates. The melting point
was determined on a XT4MP apparatus (Taike Corp, Beijing, China).
1H and 13C NMR spectra were obtained by a BRUKER AVANCE
AV-500 (1H NMR, 500 MHz; 13C NMR, 125 MHz) or by BRUKER
AVANCE AV-300 (1H NMR, 300 MHz; 13C NMR, 75 MHz) with
DMSO-d6 as the solvents and TMS as the internal standard. High-
resolution electron impact mass spectra (HRMS) are recorded under
an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies,
USA). Optical rotations were measured on a Rudolph Autopol IV
polarimeter at 20 °C. The purities of all compounds were confirmed
to be higher than 95% by the high-performance liquid chromatog-
raphy (HPLC) method performed with an Agilent 1260 HPLC
System.
General Procedure for Synthesis of 1−20. Under a nitrogen
atmosphere, SOCl2 (1 mL, 13.75 mmol) was added in portions to a
solution of L-amino acid (6 mmol) in methanol (25 mL) at −10 °C
within 30 min, and the mixture was warmed to room temperature and
stirred for 12 h. After the solvent was removed in vacuum, 80%
aqueous ethanol (50 mL) was added to dissolve amino acid methyl
ester hydrochloride, and 5.0 mmol corresponding to the substituted
cinnamic acid (6 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methyl morpholinium chloride (1.65 g, 5.5 mol), NaHCO3 (500
mg, 6 mmol) was added. The solution was stirred at room
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Figure 11. Effects of 18 on the repair of DAergic neurons. (A) Immunohistochemical staining showing TH and DOPA decarboxylase-positive cells
in the SN (scale bar: 200 μm). (B,C) Stereological counting of TH and DOPA decarboxylase-positive cells (n = 3). (D) Western blot analysis of
the changes in the protein level of TH in SNpc. (E) Western blot analysis of the changes in the protein level of DOPA in SNpc. (F,G)
Immunofluorescence staining showing GDNF in microglia of mice. All data are presented as means SEM. ***p < 0.001, compared with the
control group; ##p < 0.01 or ###p < 0.001, compared with the MPTP treatment group.
temperature for 6 h. The mixture was concentrated and the residue
was extracted with EtOAc/H2O (50 mL × 3). The combined organic
layer was washed with a saturated NaHCO3 solution and saturated
brine solution, dried over anhydrous Na2SO4, and concentrated in
vacuum. After recrystallization in PE/EtOAc (5:1−3:1), a series of
intermediates, the methyl ester of the target product, were obtained as
colorless crystals. The mentioned intermediate was dissolved in 80%
aqueous methanol (50 mL), K2CO3 (2.075 g, 15 mmol) was added in
portions and the solution was stirred at room temperature for 2 h.
The mixture was concentrated, and KHSO4 (50 mL, 1 M) aqueous
solution was added. The residue was extracted with EtOAc (30 mL ×
5), and the combined organic layer was dried over anhydrous Na2SO4
and concentrated in vacuum to afford the final product as a white
powder.
(500 MHz, DMSO-d6): δH 12.80 (s, 1H), 8.53 (1H, d, J = 7.8 Hz),
7.72 (1H, m), 7.70 (1H, d, J = 15.8 Hz), 7.55 (1H, m), 7.44 (2H, m),
7.33−7.22 (5H, m), 6.80 (1H, d, J = 15.8 Hz), 4.61 (1H, m), 3.16
(1H, dd, J = 6.9, 2.9 Hz), 2.98 (1H, dd, J = 6.9, 5.6 Hz); 13C NMR
(125 MHz, DMSO-d6): δC 173.32, 164.88, 138.02, 134.97, 133.77,
133.08, 133.06, 131.46, 129.55 (2C), 128.71 (2C), 128.26, 128.05,
126.95, 125.21, 54.19, 37.31. HRMS (ESI) m/z: [M + H]+ calcd for
C18H16ClNO3, 330.0819; found, 330.0889.
2-Chloro-(E)-cinnamoyl]-L-4-F-phenylalanine Acid (2). White
powder in 55% yield, mp 139−140 °C, [α]D20 −15.2 (c 0.1,
1
MeOH). H NMR (300 MHz, DMSO-d6): δH 12.83 (1H, s), 8.52
(1H, d, J = 9.0 Hz), 7.73−7.67 (1H, m), 7.69 (1H, d, J = 15.7 Hz),
7.51 (1H, dd, J = 5.8, 3.5 Hz), 7.40 (2H, m), 7.29 (2H, m), 6.76 (1H,
d, J = 15.7 Hz), 4.60 (1H, m), 3.14 (1H, dd, J = 13.8, 4.9 Hz), 2.94
(1H, dd, J = 13.8, 9.3 Hz); 13C NMR (75 MHz, DMSO-d6): δC
172.67, 164.35, 162.63, 159.43, 134.49, 133.61, 133.57, 133.28,
2-Chloro-(E)-cinnamoyl]-L-phenylalanine Acid (1). White powder
in 46% yield, mp 126−127 °C, [α]2D0 −14.7 (c 0.1, MeOH). 1H NMR
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(1H, d, J = 8.2 Hz), 7.60 (2H, d, J = 8.3 Hz), 7.49 (2H, d, J = 8.3 Hz),
7.41 (1H, d, J = 15.8 Hz), 7.30 (1H, dd, J = 5.3, 3.5 Hz), 7.12 (2H, t, J
= 5.3 Hz), 6.73 (1H, d, J = 15.8 Hz), 4.59 (1H, m), 3.14 (1H, dd, J =
8.3, 2.9 Hz), 2.96 (1H, dd, J = 8.3, 5.6 Hz); 13C NMR (125 MHz,
DMSO-d6): δC 173.28, 165.15, 162.52, 160.59, 138.38, 134.46,
134.24, 134.19, 131.43, 129.75 (2C), 129.45 (2C), 122.95, 115.47,
115.30, 54.10, 36.47. HRMS (ESI) m/z: [M + H]+ calcd for
C18H15ClFNO3, 348.0724; found, 348.0797.
Table 2. Summary of Properties of 18
4-Chloro-(E)-cinnamoyl]-L-leucine Acid (7). White powder in 67%
yield, mp 158−159 °C, [α]D20 −18.5 (c 0.1, MeOH). H NMR (300
1
property
value
MHz, DMSO-d6): δH 12.58 (1H, s), 8.35 (1H, d, J = 8.0 Hz), 7.59
(2H, d, J = 8.2 Hz), 7.47 (2H, d, J = 8.2 Hz), 7.43 (1H, d, J = 15.8
Hz), 6.74 (1H, d, J = 15.8 Hz), 4.37 (1H, dd, J = 9.0, 6.0 Hz), 1.68−
1.55 (3H, m), 0.89 (6H, q, J = 6.2 Hz); 13C NMR (75 MHz, DMSO-
d6): δC 177.07, 167.76, 140.84, 136.98, 136.89, 132.62 (2C), 132.26
(2C), 125,66, 53.47, 27.45, 25.87, 24.37. HRMS (ESI) m/z: [M +
H]+ calcd for C15H18ClNO3, 296.0972; found, 296.1044.
pKa, log P (c log P)
inhibition of iNOS response
(IC50, nM)
CYP inhibition
(% of control activity, 10 μM)
4.03 (4.27)
74
77.4 (2C19), 99.7 (2D6), 96.7 (2C9),
87.2 (1A2), 97.4 (3A4)
86.3 (30 min)
a
human microsomal stability
b
(1 μM)
4-Chloro-(E)-cinnamoyl]-L-isoleucine Acid (8). White powder in
plasma stability
90.2 (60 min)
1
63% yield, mp 137−138 °C, [α]2D0 −19.3 (c 0.1, MeOH). H NMR
(% of untreated control)
PAMPA-BBB (Pe, cms−1
)
19.02 1.32 (CNS+)
(500 MHz, DMSO-d6): δH 12.58 (1H, s), 8.23 (1H, d, J = 6.8 Hz),
7.61 (2H, br), 7.51 (2H, br), 7.45 (1H, d, J = 15.8 Hz), 6.89 (1H, d, J
= 15.8 Hz), 4.37 (1H, t, J = 5.0 Hz), 1.86 (1H, m), 1.48 (1H, m), 1.27
(1H, m), 0.90 (7H, m); 13C NMR (125 MHz, DMSO-d6): δC 173.42,
165.29, 138.20, 134.42, 134.37, 129.67 (2C), 129.46 (2C), 123.30,
56.90, 37.01, 25.23, 16.11, 11.76. HRMS (ESI) m/z: [M + H]+ calcd
for C15H18ClNO3, 296.0973; found, 296.1064.
a
Cytochrome P450 (CYP) inhibition assay was performed using a
b
P450-Glo assay system (Promega). In vitro microsomal stability of
the synthesized compound; % remaining was determined after 30 min
of incubation with human microsomes. The % of parent compound
remaining is calculated by comparing peak areas.
3,4-Dichloro-(E)-cinnamoyl]-L-phenylalanine Acid (9). White
132.52, 130.93, 130.82, 129.92, 127.70, 127.51, 124.61, 115.00,
114.73, 53.64, 35.92. HRMS (ESI) m/z: [M + H]+ calcd for
C18H15ClFNO3, 348.0724; found, 348.0796.
powder in 59% yield, mp 137−138 °C, [α]D20 −14.7 (c 0.1,
1
MeOH). H NMR (500 MHz, DMSO-d6): δH 12.79 (1H, s), 8.40
(1H, d, J = 7.8 Hz), 7.86 (1H, s), 7.70 (1H, d, J = 8.3 Hz), 7.58 (1H,
d, J = 8.3 Hz), 7.39 (1H, d, J = 15.8 Hz), 7.23 (1H, m), 6.80 (1H, d, J
= 15.8 Hz), 4.62 (1H, m), 3.17 (1H, dd, J = 8.3, 3.1 Hz), 2.97 (1H,
dd, J = 8.2, 5.6 Hz); 13C NMR (125 MHz, DMSO-d6): δC 173.31,
164.87, 137.98, 137.11, 136.24, 132.17, 131.54, 129.93 (2C), 129.55
(2C), 128.70, 127.81, 126.95, 124.48, 54.11, 37.31. HRMS (ESI) m/
z: [M + H]+ calcd for C15H18ClNO3, 364.0429; found, 364.0505.
3,4-Dichloro-(E)-cinnamoyl]-L-4-F-phenylalanine Acid (10).
White powder in 80% yield, mp 153−154 °C, [α]2D0 −15.6 (c 0.1,
2-Chloro-(E)-cinnamoyl]-L-leucine Acid (3). White powder in 42%
yield, mp 85−87 °C, [α]2D0 −16.1 (c 0.1, MeOH). H NMR (300
1
MHz, DMSO-d6): δH 12.59 (1H, s), 8.45 (1H, d, J = 8.0 Hz), 7.74
(1H, d, J = 15.8 Hz), 7.71 (1H, m), 7.53 (1H, m), 7.42 (2H, m), 6.78
(1H, d, J = 15.8 Hz), 4.37 (1H, dd, J = 15.1, J = 8.7 Hz), 1.67−1.58
(3H, m), 0.90 (6H, q, J = 6.3Hz); 13C NMR (75 MHz, DMSO-d6):
δC 177.00, 167.45, 137.51, 136.36, 135.72, 134.00, 133.04, 130.84,
130.62, 127.89, 53.57, 27.46, 25.87, 24.39. HRMS (ESI) m/z: [M +
H]+ calcd for C15H18ClNO3, 296.0975; found, 296.1046.
2-Chloro-(E)-cinnamoyl]-L-isoleucine Acid (4). White powder in
53% yield, mp 66−67 °C, [α]2D0 −15.5 (c 0.1, MeOH). 1H NMR (500
MHz, DMSO-d6): δH 12.61 (1H, s), 8.34 (1H, d, J = 8.0 Hz),
7.77(1H, d, J = 15.8 Hz), 7.76 (1H, m), 7.55 (1H, br s), 7.45 (2H,
m), 6.95 (1H, d, J = 15.7 Hz), 4.37 (1H, t, J = 4.3 Hz), 1.86 (1H, m),
1.48 (1H, m), 1.26 (1H, m), 0.92 (7H, m); 13C NMR (125 MHz,
DMSO-d6): δC 173.34, 165.16, 134.88, 133.80, 133.23, 131.40,
130.48, 128.26, 127.99, 125.50, 56.98, 37.01, 25.25, 16.12, 11.77.
HRMS (ESI) m/z: [M + H]+ calcd for C15H18ClNO3, 296.0975;
found, 296.1046.
1
MeOH). H NMR (500 MHz, DMSO-d6): δH 12.83 (1H, s), 8.40
(1H, d, J = 8.3 Hz), 7.86 (1H, s), 7.70 (1H, d, J = 5.0 Hz), 7.58 (1H,
d, J = 5.0 Hz), 7.40 (1H, d, J = 15.8 Hz), 7.31 (1H, m), 7.13 (1H, m),
6.80 (1H, d, J = 15.8 Hz), 4.60 (1H, m), 3.12 (1H, dd, J = 8.3, 2.8
Hz), 2.93 (1H, dd, J = 8.3, 5.5 Hz); 13C NMR (125 MHz, DMSO-
d6): δC 175.77, 167.44, 167.44, 165.73, 139.72, 138.79, 136.70,
136.66, 134.75, 134.10, 134.03, 133.92, 132.50, 130.38, 126.99,
118.10, 117.82, 56.68, 39.04. HRMS (ESI) m/z: [M + H]+ calcd for
C18H14Cl2FNO3, 382.0335; found, 382.0409.
3,4-Dichloro-(E)-cinnamoyl]-L-leucine Acid (11). White powder in
42% yield, mp 107−109 °C, [α]2D0 −16.1 (c 0.1, MeOH). H NMR
1
4-Chloro-(E)-cinnamoyl]-L-phenylalanine Acid (5). White powder
in 64% yield, mp 221−224 °C, [α]2D0 −16.9 (c 0.1, MeOH). 1H NMR
(500 MHz, DMSO-d6): δH 12.77 (1H, s), 8.42 (1H, d, J = 7.8 Hz),
7.60 (1H, d, J = 8.0 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.42 (1H, d, J =
15.8 Hz), 7.31−7.23 (6H, m), 6.75 (1H, d, J = 15.8 Hz), 4.62 (1H,
dd, J = 7.6, J = 4.9 Hz), 3.17 (1H, dd, J = 8.3, 2.7 Hz), 2.96 (1H, dd, J
= 8.3, 5.7 Hz); 13C NMR (125 MHz, DMSO-d6): δC 173.41, 165.16,
138.35, 138.06, 134.45, 134.26, 129.73 (2C), 129.55 (2C), 129.44
(2C), 128.69 (2C), 126.93, 123.02, 54.13, 37.35. HRMS (ESI) m/z:
[M + H]+ calcd for C18H16ClNO3, 330.0819; found, 330.1046.
4-Chloro-(E)-cinnamoyl]-L-4-F-phenylalanine Acid (6). White
powder in 64% yield, mp 234−235 °C, [α]D20 −17.7 (c 0.1,
(300 MHz, DMSO-d6): δH 12.60 (1H, s), 8.35 (1H, d, J = 8.0 Hz),
7.85 (1H, br s), 7.68 (1H, d, J = 8.4 Hz), 7.57 (1H, dd, J = 8.4, 1.7
Hz), 7.42 (1H, d, J = 15.8 Hz), 6.78 (1H, d, J = 15.8 Hz), 4.37 (1H,
dd, J = 14.6, 8.1 Hz), 1.70−1.55 (3H, m), 0.89 (6H, q, J = 6.3 Hz);
13C NMR (75 MHz, DMSO-d6): δC 176.99, 167.47, 139.59, 138.89,
134.14, 132.51, 130.31, 127.17, 53.51, 27.45, 25.88, 24.38. HRMS
(ESI) m/z: [M + H]+ calcd for C15H17Cl2NO3, 330.0583; found,
330.0656.
3,4-Dichloro-(E)-cinnamoyl]-L-isoleucine Acid (12). White pow-
der in 54% yield, mp 98−101 °C, [α]2D0 −17.7 (c 0.1, MeOH). H
1
NMR (500 MHz, DMSO-d6): δH 12.61 (1H, s), 8.22 (1H, d, J = 8.5
Hz), 7.87 (1H, d, J = 2.6 Hz), 7.70 (1H, d, J = 8.5 Hz), 7.58 (1H, dd,
1
MeOH). H NMR (500 MHz, DMSO-d6): δH 12.76 (1H, s), 8.42
Table 3. Pharmacokinetic Properties of 18 and 4b
blood
brain
comp.
AUC0−∞ (h·ng/mL)
t1/2 (h)
Cmax (ng/mL)
AUC0−∞ (h·ng/mL)
t1/2 (h)
Cmax (ng/mL)
4b
18
3453.5 441.9
18977.3 1209.2
1.2 0.3
1.9 0.5
1004.3 107.2
4936.3 267.2
567.3 43.1
2100.4 100.9
0.9 0.2
1.7 0.3
40.2 5.1
578.2 43.1
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J = 8.5, 2.6 Hz), 7.44 (1H, d, J = 15.8 Hz), 6.96 (1H, d, J = 15.8 Hz),
4.35 (1H, dd, J = 5.0, 3.5 Hz), 1.87 (1H, m), 1.49 (1H, m), 1.25 (1H,
m), 0.90 (7H, m); 13C NMR (125 MHz, DMSO-d6): δC 173.37,
164.96, 136.86, 136.43, 132.17, 132.08, 131.56, 129.83, 127.75,
124.88, 57.03, 37.09, 25.22, 16.11, 11.79. HRMS (ESI) m/z: [M +
H]+ calcd for C15H17Cl2NO3, 330.0585; found, 330.0655.
6.3 Hz); 13C NMR (75 MHz, DMSO-d6): δC 177.00, 167.49, 140.52,
131.21 (2C), 128.33 (2C), 127.66, 53.53, 27.46, 25.88, 24.38. HRMS
(ESI) m/z: [M + H]+ calcd for C16H18F3NO3, 330.1239; found,
330.1311.
4-Trifluoromethyl-(E)-cinnamoyl]-L-isoleucine Acid (20). White
powder in 51% yield, mp 123−125 °C, [α]2D0 −16.4 (c 0.1, MeOH).
1H NMR (500 MHz, DMSO-d6): δH 12.63 (1H, s), 8.33 (1H, d, J =
7.8 Hz), 7.80 (4H, s), 7.53 (1H, d, J = 15.8 Hz), 7.02 (1H, d, J = 15.8
Hz), 4.37 (1H, m), 1.88 (1H, m), 1.48 (1H, m), 1.25 (1H, m), 0.92
(7H, m); 13C NMR (125 MHz, DMSO-d6): δC 173.35, 165.03,
139.53, 137.90, 129.84, 129.59, 128.61, 126.31, 126.28, 125.28, 56.95,
37.02, 25.23, 16.11, 11.76. HRMS (ESI) m/z: [M + H]+ calcd for
C16H18F3NO3, 330.1238; found, 330.1311.
4-Methyl-(E)-cinnamoyl]-L-phenylalanine Acid (13). White pow-
der in 48% yield, mp 135−137 °C, [α]2D0 −19.4 (c 0.1, MeOH). H
1
NMR (300 MHz, DMSO-d6): δH 12.76 (1H, s), 8.36 (1H, d, J = 8.0
Hz), 7.44 (2H, d, J = 7.4 Hz), 7.36 (1H, d, J = 15.8 Hz), 7.28−7.20
(7H, m), 6.65 (1H, d, J = 15.8 Hz), 4.59 (1H, m), 3.13 (1H, dd, J =
13.8, 4.8 Hz), 2.95 (1H, dd, J = 11.6, 9.5 Hz), 2.31 (3H, s); 13C NMR
(75 MHz, DMSO-d6): δC 176.07, 168.08, 142.23, 142.20, 140.68,
135.10, 132.57 (2C), 132.11 (2C), 131.25 (2C), 130.57 (2C), 129.47,
123.72, 56.67, 39.93, 23.97. HRMS (ESI) m/z: [M + H]+ calcd for
C19H19NO3, 310.1365; found, 310.1437.
Molecular Simulation Study. A docking study was conducted as
per our previous study.17 The iNOS crystal structure (PDB code:
1R35) carries an active site where the standard molecule I58 was pre-
boned as a co-crystal in IR35.18 The filtered 209 designed compounds
were subjected to docking in that active site by the CDOCKER
protocol on Discovery Studio 3.0 (DS 3.0) software. The protein
preparation protocol had been followed for preparing the protein, and
the filtered molecules were docked into the active sites on the
prepared protein. During the analysis, -CDOCKER energy for every
single conformational pose were chosen as a selection criterion. MD
simulations for 100 ns were performed on iNOS without and with
EuC at 300 K at the molecular mechanic level using the GROMOS96
43a1 force field in GROMACS. The iNOS-18 complex was solvated
in a cube box of 0.9% NaCl, and the distance between solute and the
box was 5 Å. Both the systems were minimized using 1500 steps of
the steepest descent for energy minimization. The resulting
trajectories were analyzed using rms and rmsf utilities of GROMACS.
The GROMACS 5.1.2 program was used for MD, and all graphs were
prepared using GraphPad Prism 8.0 software.
Cellular Thermal Shift Assay and Isothermal Dose−
Response Fingerprint. For cellular thermal shift assay (CETSA)
and isothermal dose−-response fingerprint (ITDRF) experiments
were conducted similar to the previous report.22,23 Briefly, for CESTA
experiments, spleen cells were seeded in 10 cm culture dishes and
then, the cells were incubated with 18 (1 μM) in fresh growth
medium) for 3 h at 37 °C with an atmosphere of 95% air and 5%
CO2. The same volume of DMSO was used for a negative control in
another 10 cm dish. Subsequently, the medium was removed, and the
cells were washed with phosphate-buffered saline (PBS) and
distributed into five different microtubes with 80 μL of cell suspension
in each tube for both 18 and DMSO-treated cells. The microtubes
were heated at the designated temperature (37 to 73 °C) for 4 min on
a heating block. After heating, the microtubes were removed and
balanced at 25 °C for another 3 min. Then, the cells were lysed to
extract the proteins, separated by sodium dodecyl-sulfate poly-
acrylamide gel electrophoresis, followed by transfer to a poly-
(vinylidene difluoride) membrane for western blot analysis. For
ITDRFCETSA experiments, all procedures are the same as CETSA, and
just the cells were incubated with 18 in different concentrations at
designated temperatures.
Surface Plasmon Resonance Assay. Surface plasmon resonance
(SPR) experiments were performed to investigate the binding affinity
between 18 and human recombinant protein iNOS. The procedures
were conducted as previously reported with some modifications.24
Briefly, purified human recombinant protein iNOS (final concen-
tration: 15 μg/mL) was loaded into the chip at 0.5 μL/s in NaAc
buffer (pH 5.5) for 20 min at 4 °C. Then, different concentrations of
18 (10 mM stock solution in DMSO) in PBS flowed through the
surface of the chip at 0.5 μL/s in PBST (pH 7.4) for 600 s at 4 °C.
Finally, the proteins were dissociated from the chip at 2 μL/s in
glycine−HCl (pH 2.0) for 360 s at 4 °C. The binding signals (RU)
were detected by SPR using a Biacore T200 (GE Healthcare, USA).
The RU responses of test compounds with purified recombinant
protein iNOS were recorded and ranked, and the binding curve and
affinity data were calculated by the system according to the Langmuir
binding model.
4-Methyl-(E)-cinnamoyl]-L-4-F-phenylalanine Acid (14). White
powder in 52% yield, mp 143−144 °C, [α]2D0 −19.7 (c 0.1, MeOH).
1H NMR (500 MHz, DMSO-d6): δH 12.75 (1H, s), 8.37 (1H, d, J =
7.8 Hz), 7.47 (1H, d, J = 8.4 Hz), 7.38 (1H, d, J = 15.8 Hz), 6.97 (1H,
d, J = 15.8 Hz), 4.60 (1H, m), 3.14 (1H, dd, J = 8.2, 2.6 Hz), 2.96
(1H, dd, J = 8.2, 5.6 Hz), 2.35 (s, 3H); 13C NMR (125 MHz, DMSO-
d6): δC 173.40, 165.52, 160.59, 139.79, 139.68, 134.26, 134.24,
132.24, 131.43, 131.36, 130.00, 128.02, 121.12, 115.46, 115.29, 54.10,
36.51, 21.39. HRMS (ESI) m/z: [M + H]+ calcd for C19H18FNO3,
328.1271; found, 328.1342.
4-Methyl-(E)-cinnamoyl]-L-leucine Acid (15). White powder in
42% yield, mp 94−96 °C, [α]2D0 −20.7 (c 0.1, MeOH). 1H NMR (500
MHz, DMSO-d6): δH 12.55 (1H, s), 8.29 (1H, d, J = 7.8 Hz), 7.48
(2H, d, J = 7.8 Hz), 7.43 (1H, d, J = 15.8 Hz), 7.25 (2H, d, J = 7.8
Hz), 6.70 (1H, d, J = 15.8 Hz), 4.40 (1H, dd, J = 8.5, 4.6 Hz), 2.35
(3H, s), 1.72−1.58 (3H, m), 0.92 (6H, q, J = 3.8Hz); 13C NMR (125
MHz, DMSO-d6): δC 174.61, 165.55, 139.72, 139.56, 132.62, 130.02
(2C), 127.97 (2C), 121.28, 50.87, 24.89, 23.31, 21.81, 21.40. HRMS
(ESI) m/z: [M + H]+ calcd for C16H21NO3, 276.1521; found,
276.1593.
4-Methyl-(E)-cinnamoyl]-L-isoleucine Acid (16). White powder in
62% yield, mp 75−78 °C, [α]2D0 −19.9 (c 0.1, MeOH). 1H NMR (500
MHz, DMSO-d6): δH 8.10 (1H, d, J = 7.2 Hz), 7.48 (2H, d, J = 7.8
Hz), 7.40 (1H, d, J = 15.8 Hz), 7.24 (2H, d, J = 7.8 Hz), 6.86 (1H, d,
J = 15.8 Hz), 4.32 (1H, dd, J = 4.3, 3.5 Hz), 1.86 (1H, m), 1.49 (1H,
m), 1.25 (1H, m), 0.90 (7H, m); 13C NMR (125 MHz, DMSO-d6):
δC 173.87, 165.37, 139.52, 139.08, 132.81, 129.97 (2C), 127.94 (2C),
121.96, 57.72, 37.40, 25.27, 21.38, 16.23, 11.90. HRMS (ESI) m/z:
[M + H]+ calcd for C16H21NO3, 276.1521; found, 276.1592.
4-Trifluoromethyl-(E)-cinnamoyl]-L-phenylalanine Acid (17).
White powder in 73% yield, mp 222−224 °C, [α]2D0 −20.7 (c 0.1,
1
MeOH). H NMR (500 MHz, DMSO-d6): δH 12.85 (1H, s), 8.49
(1H, d, J = 8.0 Hz), 7.79 (4H, s), 7.48 (1H, d, J = 15.8 Hz), 7.32−
7.23 (6H, m), 6.86 (1H, d, J = 15.8 Hz), 4.60 (1H, m) 3.14 (1H, dd, J
= 8.3, 2.9 Hz), 2.94 (1H, dd, J = 8.3, 5.6 Hz); 13C NMR (125 MHz,
DMSO-d6): δC 173.36, 164.85, 139.09, 138.07, 137.98, 129.56 (2C),
128.68 (2C), 126.92 (2C), 126.28 (2C), 125.05, 54.22, 37.35. HRMS
(ESI) m/z: [M + H]+ calcd for C19H16F3NO3, 364.1082; found,
364.1154.
4-Trifluoromethyl-(E)-cinnamoyl]-L-4-F-phenylalanine Acid (18).
White powder in 61% yield, mp 236−238 °C, [α]2D0 −19.8 (c 0.1,
1
MeOH). H NMR (300 MHz, DMSO-d6): δH 12.79 (1H, s), 8.51
(1H, d, J = 8.1 Hz), 7.77 (4H, s), 7.47 (1H, d, J = 15.9 Hz), 7.28 (2H,
m), 6.84 (1H, d, J = 15.9 Hz), 4.58 (1H, m), 3.14 (1H, dd, J = 13.9,
4.9 Hz), 2.94 (1H, dd, J = 13.9, 9.3 Hz); 13C NMR (75 MHz, DMSO-
d6): δC 175.78, 167.45, 165.72, 162.52, 141.92, 140.63, 136.69,
134.03, 133.92, 132.55, 131.25 (2C), 128.86 (2C), 127.50 (2C),
118.10, 117.82, 56.72, 39.05. HRMS (ESI) m/z: [M + H]+ calcd for
C19H15F4NO3, 382.0988; found, 382.1059.
4-Trifluoromethyl-(E)-cinnamoyl]-L-leucine Acid (19). White
powder in 46% yield, mp 236−238 °C, [α]D20 −15.7 (c 0.1,
1
MeOH). H NMR (300 MHz, DMSO-d6): δH 12.60 (1H, s), 8.44
(1H, d, J = 7.9 Hz), 7.78 (4H, s), 7.51 (1H, d, J = 15.8 Hz), 6.85 (1H,
d, J = 15.8 Hz), 4.37 (1H, m), 1.70−1.56 (3H, m), 0.90 (6H, q, J =
7773
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Cell Culture. The immortalized murine microglial cell line BV-2
cells and human neuroblastoma SH-SY5Y cells were routinely
maintained in Dulbecco’s modified Eagle’s medium (DMEM) with
10% fetal bovine serum (FBS), supplemented with 100 U/mL
penicillin and 100 μg/mL streptomycin at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. All reagents for cell culture were
purchased from Invitrogen (CA, USA).
Animals. Male C57BL/6 mice (6−8 weeks old, weighing 20−22
g) were purchased from Comparative Medicine Center (Yangzhou
University, China). Mice were acclimatized for 7 days before use. All
animals were maintained under a standard housing environment at a
temperature of 22−25 °C and kept on a 12 h light−dark cycle and
were allowed free access to food and water. All experimental
procedures were approved by the Ethical Committee of the China
Pharmaceutical University (no. SYXK2016-0011).
NO Production Assay. For the compound screening experiments,
the cells were plated in 96-well plates at a density of 5 × 103 cells/well
and 24 h later were treated with compounds 1−20 or 4b at 1 μM.
Then, 6 h later the cells were treated with LPS (500 ng/mL) and
incubated for another 24 h, and the culture media were collected for
the detection of the NO inhibition using a commercial NO detection
kit (Beyotime, China) at 540 nm (microplate reader, TECAN, Infinite
M200, Austria).
NOS Enzyme Inhibition Assay. The inhibitory effect of
compounds 1−20 on NOS isoforms were evaluated mainly according
to previous studies.32,33 Compounds 1−20 were evaluated for their
abilities to inhibit human iNOS, eNOS, and nNOS (which were
purchased from Abcam) mediated the conversion of [3H]arginine to
[3H]citrulline. Various concentrations of compounds (0.01−10 μM)
were incubated with 10 nM [3H]arginine and [14C]citrulline (80−120
nM) as an internal standard, cofactors (1.4 mM β-NADP+, 3.0 mM
glucose-6-phosphate (G6P), 3.4 mM MgCl2, 0.4 U/mL G6P
dehydrogenase, 5.0 μM FAD, 5.0 μM FMN, 5.0 μM BH4), and
enzyme in 100 μL of 50 mM HEPES buffer for 1 h at 37 °C. For
eNOS and nNOS assays, additional cofactors 25 nM calmodulin, 0.5
mM CaCl2, and 3.5 mM glutathione were included in the reactions.
The reactions were stopped by the addition of 25 μL of 1 M MES and
filtered through a AG-50W-X8 (200−400 mesh, Na form) cation
exchange resin that had been loaded onto 96-well filter plates using a
100 μL column loader (Millipore). Then, the filtrate was rinsed with
75 and 25 μL ddH2O, respectively, and the filtrate was collected and
the radioactivity in the filtrate was counted. Background activity was
determined in the presence of 10 μM AMT. Dose−response curves
were drawn according to eight test concentrations (0.01−10 μM),
and IC50 values were calculated by GraphPad Prism 8.0 (for macOS)
and were from a single experiment that was repeated three times with
similar results. The calculated standard deviations from dose−
response curves of the assays were less than 10% with all NOSs.
D-QSAR Study. Among all the 20 compounds, 80% (i.e., 16) were
utilized as a training set for QSAR modeling and the remaining 20%
(i.e., 4) were chosen as an external test subset for validating the
reliability of the QSAR model by the diverse molecules protocol in DS
3.0 software. The inhibitory activity of the compounds [IC50 (μM)]
was initially changed into the minus logarithmic scale [IC50 (μM)]
and then used for subsequent QSAR analysis as the response variable.
QSAR models were built by using the create 3D QSAR model
protocol in DS 3.0.
C
VA·VD
A(t − τss) (VA + VD)
VA + VD
(1 − R)·VD
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where Pe is the effective permeability (cm s−1); VA and VD are the
volumes of the acceptor and donor wells (0.25 cm3), respectively;
CA(t) is the concentration of the acceptor well at time t; CD(0) and
C
D(t) are the concentrations of the donor well at t0 and t, respectively;
A is the filter well area (0.21 cm2); t is the incubation time (s); τss is
the time to reach a steady state (usually very short compared with the
incubation time); and R is the retention membrane factor and was
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calculated using the following equation: R = 1 −
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Pe was reported as an average of a triplicate with a standard deviation.
Cell Medium Collection. BV-2 cells (1 × 104 cells/well in a 24-
well plate) were pre-treated with 18 (0.1, 1, and 10 μM) for 2 h and
then stimulated with LPS (500 ng/mL) for 24 h. The culture media
were collected as CM after centrifugation at 2000 rpm for 5 min.
Cell Viability and LDH Release Assays. SH-SY5Y cells (1 × 104
cells/well in a 96-well plate) were seeded 24 h before the CM test.
The CM from LPS-stimulated cells was added to SH-SY5Y cells,
which were further incubated at 37 °C for 24 h, and then the
supernatant was collected to detect the LDH release using the LDH
cytotoxicity assay kit (Beyotime, China), and cell viability was
measured using the CCK8 assay according to the kit specifications
(Dojindo Laboratories, Japan).
Hoechst 33258 Staining and Annexin V−PI Double
Staining. SH-SY5Y cells (2 × 105 cells/well in a 6-well plate) were
seeded 24 h before the CM test. The CM from LPS-stimulated cells
was added to SH-SY5Y cells, which were further incubated at 37 °C
for 24 h. For Hoechst 33258 staining (Beyotime, China), the cells
were washed with cold PBS three times, and then added Hoechst
33258 (final concentration of 5 μg/mL) for 30 min. After that, cells
were washed with cold PBS three times. Finally, images were obtained
using a fluorescence microscope (Ts-100, Nikon, Japan). For annexin
V−PI double staining, the cells were washed with cold PBS three
times and were adjusted to the density of 1 × 106 cells/mL with
binding buffer. Then, the staining assay was conducted according to
the kit (BD Pharmingen, USA). Finally, all samples were detected by
a flow cytometer (Becton Dickin-son, NJ, USA).
Establishment of the MPTP-Induced Acute PD Model and
Drug Treatment. 40 C57BL/6 mice were randomly divided into five
groups (n = 8 mice/group), including the control group, MPTP-
treated group, MPTP + L-DOPA (15 mg/kg, i.g.) group, MPTP + 18
(1 mg/kg, i.g.) group, and MPTP + 18 (2 mg/kg, i.g.) group and
allowed 3 days to acclimate before any treatments. DI water
containing 0.5% CMC-Na and 1% Tween 80 was used to dissolve
18 and L-DOPA. From day 1 to day 3, for all mice behavior training
was conducted. Then, 18 and L-DOPA were pre-administered daily
for 10 days (day 4 to day 13). The control group and model group
were administered equal volumes of the blank solvent. At day 14, four
intraperitoneally (i.p.) injections of MPTP hydrochloride (20 mg/kg)
were administered to mice at 2 h intervals a day. After the last MPTP
injection, the mice were subjected to the rotarod test, traction test,
and open-field test at different time points. 10 days later after MPTP
injection, SNpc and STR were collected to perform western blot,
immunohistochemistry, and the other biochemical criterion detection.
Establishment of the MPTP/Probenecid-Induced Chronic
PD Model and Drug Treatment. 32 C57BL/6 mice were randomly
divided into four groups (n = 8 mice/group), including control group,
MPTP/probenecid-treated group, MPTP/probenecid + 18 (1 mg/kg,
i.g.) group, and MPTP/probenecid + 18 (2 mg/kg, i.g.) group and
allowed 3 days to acclimate before any treatments. DI water
containing 0.5% CMC-Na and 1% Tween 80 was used to dissolve
18. From day 1 to day 32, the mice were administered MPTP
hydrochloride (25 mg/kg in saline) in combination with probenecid
(250 mg/kg in DMSO, ip) every three-and-a-half days. After the last
MPTP/prob injection, the mice were continually treated daily with 18
(1 or 2 mg/kg) for the next 20 days. Then, the traction test, rotarod
test, and open-field test were conducted from day 52 to day 54 to
PAMPA-BBB Assay. The PAMPA-BBB assay was performed as
per previous study.34 Briefly, the assay was conducted in 10 mM PBS
buffer (PH 7.4), and compounds were tested at a concentration of
200 μM. The donor plate was first coated with 4 μL of the porcine
brain lipid (20 mg/mL in dodecane), followed by an addition of 250
μL of a test compound. The acceptor plate was filled with 250 μL of
PBS, and the donor plate was carefully placed on top of the acceptor
plate to make a “sandwich”. The plate was incubated at 25 °C for 17 h
in a saturated humidity atmosphere with an orbital agitation at 100
rpm. After that, 150 μL of the test solution was collected from each
well from both sides (donor and acceptor) and transferred to the UV
plate for measurement. The effective permeability (Pe) was calculated
7774
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evaluate the therapeutic effects of 18 on PD. At day 55, all mice were
sacrificed to perform further biochemical detections.
reagent was added to stop the reaction and generate the luminescent
signal. After incubation for 20 min to stabilize the luminescence,
signals were detected using a microplate reader (TECAN, Infinite
M200, Austria); the inhibition of each CYP isotype by 18 was
expressed as the percentage of activity versus control.
Traction Test.35 Traction test was performed as a previous study
to assess the limb impairment. Mice were hung from a horizontal wire
by its forepaws. The mouse was scored 3 points if it grasped the wire
with both hind paws, and 2 points if it grasped the wire with one hind
paw, and 1 point if it did not grasp the wire with either hind paws.
The average time of three tests was calculated for statistical analyses.
Rotarod Test.35 The rotarod test was performed as our previous
study using a rotary rod apparatus. All mice were pre-trained for 3
days prior to drug administration. The training consisted of three
consecutive runs with a gradual increase in rpm up to a maximum 25
rpm until the mice were able to keep themselves without falling from
the rotary rod for up to 180 s. The average time of three tests was
calculated for statistical analyses.
Human Microsomal Stability. Human liver microsomes (0.5
mg/mL) were preincubated with 18 at 1 μM in 0.1 M phosphate
buffer (pH 7.4) at 37 °C for 5 min before adding the NADPH
regeneration buffer (ditto). The incubation was started by treating the
NADPH regeneration buffer and terminating with chlorpropamide in
acetonitrile after 30 min at 37 °C. Precipitated proteins were removed
by centrifugation for 5 min at 14,000g at 4 °C. The supernatant was
injected into a liquid chromatography/mass spectrometry (LC−MS)
system and analyzed using a Shimadzu Nexera XR system (Shimadzu
Corporation, Kyoto, Japan). The HPLC column was a Luna C18
column (2.0 × 5 mm, 4.6 μm), and the mobile phase was distilled
water (A) containing 0.1% formic acid and acetonitrile (B) containing
0.1% formic acid. Data analysis was performed using Xcalibur 1.6
software. The percentage of remaining 18 was calculated by
comparing the peak area.
Plasma Stability. In vitro stability of 18 was carried out with an
initial concentration of 1000 ng/mL in the rat plasma at 37 °C. A 100
μL of plasma was aliquoted from the incubation solution at 0.5 h,
followed by solid-phase extraction. 10 μL of supernatant of each
sample was injected into a LC−MS system and analyzed using a
Shimadzu Nexera XR system (Shimadzu Corporation, Kyoto, Japan).
The HPLC column was a Luna C18 column (2.0 × 5 mm, 4.6 μm),
and the mobile phase was distilled water (A) containing 0.1% formic
acid and acetonitrile (B) containing 0.1% formic acid. Data analysis
was performed using Xcalibur 1.6 software. The percentage of
remaining 18 was calculated by comparing the peak area.
Pharmacokinetic Analysis. Male BALB/c mice were randomly
divided into two groups, including the control group and the drug
treatment group. After 16 h of fasting, mice in the drug treatment
group were intragastrically administered 18 at a single dose of 30 mg/
kg, while the control group were given the same volume of the blank
solvent. Then, blood samples and brain samples were collected at 5,
10, 15, and 30 min and 1, 2, 6, 8, 12, and 24 h after dosing, following
the decapitation of the animal. The plasma was prepared by
centrifuging the blood at 5000 rpm for 10 min and the brains were
homogenized with saline, centrifuged at 10,000g for 10 min, and then
the supernatant was collected. After that, 300 μL of methyl alcohol
was added to 300 μL of plasma or brain supernatant, and vortexed 3
min, centrifuged at 10,000g for 10 min, and then the supernatant was
collected. Then, a 5 μL aliquot of the plasma or brain extract was
injected into the LC−MS system on a Shimadzu LCMS-2020 single
quadrupole system (Shimadzu Scientific Instruments Inc., Japan). For
the detection of 18 and 4b, an isocratic chromatographic procedure
was used with a mobile phase of 40% acetonitrile (0.1% formic acid)
and 65% acetonitrile (0.1% formic acid), respectively, at a flow rate of
0.5 mL/min. The chromatographic procedure was performed using
the Agilent SB-C18 column (2.1 × 50 mm, 1.8 μm, Agilent
Technologies, USA) at 30 °C.
Open-Field Test.36 Spontaneous locomotor activity was assessed
using the open-field test with an automatic recording open-field
working station (ZhongShi, Beijing, China). Mice were placed
individually in an acrylic apparatus (30 cm × 30 cm × 15 cm) with
a floor divided into 6 × 6 cm equal squares in order to explore the
arena. The arena was cleaned with 75% ethanol solution and let dry
after testing each mouse to avoid the presence of olfactory cues. Total
distance and mean velocity were analyzed over a period of 5 min.
Detection of the NO Level, DA Level, and GDNF Level.
Brains were removed to strip the SNpc and STR. Samples were
homogenized and centrifuged at 2000g for 15 min at 4 °C to separate
the supernatant. The concentrations of GDNF and DA were
measured by the enzyme-linked immunosorbent assay kits (CUSA-
BIO, Wuhan, China) and the NO level was measured by Griess
Reagent (Beyotime, China); the procedures followed the instructions.
Histochemical Analysis. The TH and DOPA decarboxylase
immunohistochemical analyses were performed as previously
described. Briefly, brains were removed and cut into 30 μm sections
using a freezing microtome (Microm, Walldorf, Germany), and the
coronal sections through the substantia nigra (SNpc) and STR were
processed. The sections were incubated with primary antibodies
against TH or DOPA decarboxylase (Abcam, MA, USA) and the
labeled proteins were visualized using 0.06% hydrogen peroxidase and
0.05% 3,3′-diaminobenzidine. After DAB staining, the sections were
observed using a light microscope (Nikon, Ts-100, Japan), and the
number of positively stained cells in each group was recorded.
Fluorescence Immunohistochemistry. The sections were
incubated with the following primary antibody GDNF: a polyclonal
rabbit anti-GDNF (Abcam, MA, USA) with a dilution of 1:100
dilution. After incubation, the sections were then incubated with the
following secondary antibodies: Alexa Fluor 488 rabbit anti-mouse
(1:1000 dilution; Abcam, MA, USA). The sections were counter-
stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI;
KeyGen, China). Each section was analyzed microscopically (Nikon,
Ts-100, Japan).
Western Blot Analysis. The western blot analysis was conducted
as per our previous study.17 Briefly, at the end of the last day, brains
were removed to strip the SNpc and STR. Then, the SNpc and STR
were adequately lysed in RIPA (100 mg tissue/mL PBS). Then, the
BCA assay (Beyotime, China) was used to adjust the concentration of
each sample protein to 3 mg/mL. Equal amounts of protein extracts
(30 μg) were subject to immunoblot analysis using primary antibodies
(all in 1:1000 dilution) followed by secondary antibodies (1:5000
dilution). All the antibodies were purchased from Abcam (MA, USA).
The exposure was conducted by a chemiluminescence apparatus
(Tanon 5200, China).
CYP Inhibition Assay. CYP inhibition was conducted using a
P450-Glo screening system (Promega, Corp.) according to the
instructions (Promega Technical Bulletin, P540-Glo). Briefly, the
CYP enzyme and the substrate were mixed in KH2PO4 buffer (100
mM, pH 7.4) with or without 18 (10 μM), and the reaction was
initiated by the addition of the NADPH regeneration system
(containing NADP+, MgCl2, G6P, and G6P dehydrogenase). After
incubation for 10−30 min at 37 °C (different incubation times
depending on the CYP isotype), the reconstituted luciferin detection
In Vivo Toxicity Assessment. Male BALB/c mice were
administered (i.g.) with 18 at 500, 1000, 2000, and 3000 mg/kg for
14 consecutive days. During the 14 days, the mortality of each group
(n = 12 mice/group) was counted. At the 15th day, the mice (1000
mg/kg group) were killed, and the blood was collected to conduct
blood biochemical analysis (Siemens, ADVIA 2120I, Germany), and
the main viscera, including the heart, liver, spleen, lung, and kidney,
were removed to perform HE staining.
HE Staining. HE staining was conducted as per a previous study.
Briefly, the heart, liver, spleen, lung, and kidney were collected to be
cut into 30 μm sections using a freezing microtome (Microm,
Walldorf, Germany). Then, the HE staining was performed on the
sections according to the kit’s specification (KeyGEN BioTECH,
China).
Statistical Analysis. The data were expressed as mean SD or
SEM and analyzed using a one-way analysis of variance (ANOVA),
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Article
followed by Dunnett’s post hoc test. p < 0.05 was considered
statistically significant.
Xiao-Qi Zhang − Institute of Traditional Chinese Medicine
and Natural Products, Jinan University, Guangzhou 510632,
People’s Republic of China; orcid.org/0000-0002-4436-
0273
Wen-Cai Ye − Institute of Traditional Chinese Medicine and
Natural Products, Jinan University, Guangzhou 510632,
People’s Republic of China; orcid.org/0000-0002-2810-
1001
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00578.
■
sı
1
Structure characterization of compounds 1−20 by H
NMR, 13C NMR, and ESI/HRMS spectroscopy; HPLC
analysis of compounds 1−20; chemical structures of 209
designed compounds; docking scores (-kcal/mol) and
BBB level prediction of 209 compounds; organ
coefficients and blood biochemistry analysis; correlation
coefficient R2; H−E staining of main viscera; and effects
of 18 on the body weight and hemocyte number (PDF)
Molecular string files for compounds 1−20 (CSV)
Compound 18 with 1R35 (PDB)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00578
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the China Postdoctoral Science
Foundation (nos. 2019M662006, 2019TQ0357), the National
Natural Science Foundation of China (nos. 81973206 and
82073804), the Major National Science and Technology
Projects of the Chinese 13th five-year plan (no.
2017ZX09309024), the Jiangsu Province Graduate Student
Training Innovation Project (no. KYLX16_1208), and the
Pharmaceutical Top-Notch Innovative Talent Training Pro-
gram of China Pharmaceutical University is acknowledged.
AUTHOR INFORMATION
Corresponding Authors
■
Fei Xiong − State Key Laboratory of Bioelectronics, Jiangsu
Laboratory for Biomaterials and Devices, Southeast
University, Nanjing 210009, People’s Republic of China;
orcid.org/0000-0002-3680-4292; Phone: +86-25-
83792496; Email: xiongfei@seu.edu.cn
Hao Wang − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China; orcid.org/0000-0003-3994-9806;
Phone: +86-25-83271328; Email: wanghao@cpu.edu.cn
ABBREVIATIONS
■
ANOVA, analysis of variance; BBB, blood−brain barrier;
CETSA, cellular thermal shift assay; CM, culture medium;
CNS, central nervous system; COMT, catechol-O-methyl-
transferase; DA, dopamine; DS, Discovery Studio; DMEM,
Dulbecco’s modified Eagle’s medium; DAPI, 4′,6-diamidino-2-
phenylindole dihydrochloride; DMTMM, 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; eNOS, en-
dothelial nitric oxide synthase; FBS, fetal bovine serum;
GDNF, glial cell line-derived neurotrophic factor; HE,
hematoxylin−eosin; iNOS, inducible nitric oxide synthase;
MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MAO-
B, monoamine oxidase type B; NPAs, N-phenylpropenoyl-L-
amino acids; NOS, nitric oxide synthase; nNOS, neuronal
nitric oxide synthase; PD, Parkinson’s disease; QSAR,
quantitative structure−activity relationship; RMSF, root-
mean-square fluctuations; RMSD, root-mean-square deviation;
STR, striatum; SNpc, substantia nigra pars compacta; SARs,
structure−activity relationships; TH, tyrosine hydroxylase
Authors
Xiao-Long Hu − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Xian-Yu Lv − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Rong Wang − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Huan Long − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Jia-Hao Feng − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Bao-Lin Wang − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Wei Shen − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Hao Liu − State Key Laboratory of Natural Medicines, School
of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
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