pubs.acs.

org/jmc Article

Development of MAO‑A and 5‑HT2AR Dual Inhibitors with Improved

Antidepressant Activity
Xiaona Sun,† Na Li,† Peisen Zhong,† Li Chen,* and Jianbo Sun*
Cite This: J. Med. Chem. 2022, 65, 13385−13400 Read Online

ACCESS Metrics & More Article Recommendations *

sı Supporting Information
Downloaded via UNIV NACIONAL AUTONOMA MEXICO on August 23, 2024 at 15:24:56 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: Designing dual-target inhibitors targeting 5-HT2AR

and MAO-A could synergistically promote interstitial 5-HT levels,
so as to exhibit a more efficient antidepressant effect. On the
premise of maintaining the original pharmacophore binding,
arylpiperazine scaffolds and 5-oxygen-substituted oxoisoaporphines
were hybridized to afford 15 dual-target inhibitors through suitable
linkers. Among all inhibitors, I14 exhibited the best inhibitory
activities against 5-HT2AR and MAO-A. In vitro cell proliferation
assays showed that most compounds were nontoxic to neuronal
cells and normal hepatocytes. I14 also significantly ameliorated the
depression-like behavior of zebrafish and mice. Further study
revealed that I14 was able to occupy the active cavity of 5-HT2AR
and MAO-A with multiple hydrogen bonding forces and π−π
stacking interaction. I14 was also able to repair the damage of mice hippocampal neuronal cells and reduce the expression of 5-
HT2AR in mice brain tissue. In conclusion, I14 could be a potential antidepressant candidate for further study.

1. INTRODUCTION symptoms has always been suggested to be important for the

Depression is a major public health problem, ranking the third clinical outcome and performance of the prototype atypical
leading cause of the global burden of disease and WHO predicts antipsychotic drug.8−10 5-HT2AR antagonists can effectively
that the disorder will rank first by 2030.1 Despite many treat dimensions of psychosis and effectively avoid the
progresses in the development of antidepressants, the clinical occurrence of the serotonin syndrome, without untoward side
use of currently available treatments for depression are limited, effects associated with the off-target binding of more
as they are not always effective and cause various adverse promiscuous antipsychotic agents.11 Therefore, designing
effects.2 Thereby, searching for new therapeutic alternatives to dual-target inhibitors that can target MAO-A to relieve 5-HT
treat this mental disorder more effectively and work faster and degradation and target 5-HT2AR to inhibit 5-HT reuptake can
safer is an urgent need. Multiple hypotheses are developed so far synergistically promote interstitial 5-HT level, which is
to explain the genesis of depression, where the monoamine conducive to more efficient antidepressant active.
neurotransmitter is the most accepted one and is the The arylpiperazine scaffolds have been studied most
cornerstone of the pathogenesis.3 The theory states that the frequently and found as selective 5-HT 2AR ligands in
deficit of monoamines, mainly 5-hidroxitriptamine (5-HT) in antidepressant clinical drugs (Figure 1A).12−14 The 5-HT2AR
the synaptic cleft is responsible for the development of antagonist contained arylpiperazine was found to exhibit a near
depression.4 “V” conformation. They followed a long-chain conformation to
Monoamine oxidase enzymes (MAOs) play a major role in 5-HT2AR (PDB: 6WGT), which was fixed by hydrogen bonding
controlling the amount of 5-HT in brain and peripheral tissues with ASP 155 and SER 159, π−π stacking with TRY 336 and
through catalyzing the oxidative deamination of them.3 MAO-A PHE 340, and hydrophobic interactions (Figure 1B).
inhibitors are one of the most widely used groups of Oxoisoaporphines are a class of nature isoquinoline alkaloids
antidepressant agents regulating the metabolism of 5-HT, and with a 1-azabenzanthrone skeleton bearing a large planar
the discovery of novel MAO-A inhibitors has important
implications for the treatment of depression.4 The serotonin
2A receptor (5-HT2AR) is a G protein-coupled receptor known Received: August 3, 2022
for its pivotal roles in cognition, behavior, and physiological Published: September 29, 2022
functions, making it a promising candidate for the treatment of
psychiatric disorders (especially depression).5−7 Indeed, pref-
erentially blocking 5-HT2AR in the presynaptic membrane by
modulating the release of 5-HT in the treatment of negative

© 2022 American Chemical Society https://doi.org/10.1021/acs.jmedchem.2c01271

13385 J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 1. Docking studies of arylpiperazine scaffolds with 5-HT2AR and 5-oxygen-substituted oxoisoaporphine with MAO-A. (A,C) Structures of
arylpiperazine scaffolds and 5-oxygen-substituted oxoisoaporphine; (B,D) arylpiperazine scaffolds and 5-OH-oxoisoaporphine interacting with
residues of 5-HT2AR (PDB: 6WGT) and MAO-A (PDB: 2Z5X), respectively.

aromatic conjugate structure.15 Studies showed that oxoisoa- HT2AR/MAO-A inhibitor design, 5-OH-oxoisoaporphine was
porphines could be better interacted with MAO-A, exerting applied as key pharmacophore element of MAO-A being
concentration-dependent and selective inhibition of the identified and analyzed, while arylpiperazines were applied as
enzymatic control activity of MAO-A.4 The 5-oxygen- scaffolds binding to 5-HT2AR. Because the binding regions of the
substituted oxoisoaporphine (Figure 1C) is a privileged two targets were not similar, on the premise of maintaining the
structure that has never been structurally modified. Molecular original pharmacophore binding, the two key pharmacophores
docking study (Figure 1D) showed the N atom, carbonyl oxygen were hybridized through suitable linkers, so that the inhibitors
in 5-oxygen-substituted oxoisoaporphine could form hydrogen
could regulate the two key proteins synergistically (Figure 2).
bond to MAO-A (PDB: 2Z5X) with TRY-407 at 3.4 Å, and the
D ring formed a π−π stacking interaction with TRY-444 (affinity The synthesis route of the target compounds is shown in
= −8.7 Kcal/mol). Scheme 1. Referring to the literature method of Prado−Prado et
Thus, by introducing selective 5-HT2AR ligands (arylpiper- al.,4 3-bromophthalide was hydrolyzed with water and heated to
azines) to the pharmacore of MAO-A (5-OH-oxoisoaporphine) reflux at 100 °C to get compound 2. 2 and 3,4-dimethoxyaniline
using alkyl linkers to design so far unexplored dual targeting
inhibitors might open a new avenue for depression treatment as
it should result in relatively improvements for low toxicity, high
efficiency, and rapid effect. As a proof of concept, here, we report
the design, synthesis, in vitro biological activities, neurotoxicity
and DMPK properties, molecular docking, and in vivo efficacy
study in zebrafish and mice model of depression of a class of dual
inhibitors of 5-HT2AR and MAO-A.

2. RESULTS AND DISCUSSION

2.1. Design and Synthesis of Dual 5-HT2AR/MAO-A
Inhibitors. The main design strategy of dual-target inhibitors is
to obtain the second activity while maintaining the original
activity or to synergistically intervene on a key regulatory factor
to obtain better drug efficacy.16 The key pharmacophore should
be able to maintain interaction with the original target while Figure 2. Design of dual 5-HT2AR/MAO-A inhibitors by the linked-
being compatible with the second target.16 In the dual 5- pharmacophore strategy.

13386 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Scheme 1. Synthetic Route of I1‑15; Reagents and Conditions: (a) H2O, 100 °C, 2 h; (b) Toluene, Reflux, 2 h; PPA, 100 °C, 10
min; (c) Pd/C, DMF, 120 °C, Overnight; (d) Zn Dust, Concentrated HCl, HOAc, Reflux, 3 h; (e) K2CO3, Acetate, Br(CH2)nBr,
50 °C, 24 h; and (f) K2CO3, KI, CH3CN, Arylpiperazine , 75 °C, 12 h

Table 1. Inhibitory Effects of Compounds I1−I15 on 5-HT2AR mRNA Expressiona

Cpd. n R1 R2 2-△△CT Cpd. n R1 R2 2-△△CT
I1 3 Cl H 1.886 ± 0.305 I9 4 F H 0.048 ± 0.014
I2 3 OCH3 H 0.200 ± 0.052 I10 4 H H 1.341 ± 0.444
I3 3 Cl Cl 0.055 ± 0.045 I11 5 Cl H 1.804 ± 0.011
I4 3 F H 0.038 ± 0.007 I12 5 OCH3 H 0.142 ± 0.007
I5 3 H H 0.069 ± 0.045 I13 5 Cl Cl 0.049 ± 0.005
I6 4 Cl H 2.575 ± 1.242 I14 5 F H 0.014 ± 0.002
I7 4 OCH3 H 0.685 ± 0.256 I15 5 H H 0.060 ± 0.031
I8 4 Cl Cl 0.578 ± 0.057 6 2.675 ± 0.434
a
Determined by the RT-qPCR assay (means ± SD, n = 3).

Table 2. Inhibitory Activities of Compounds I1−I15 on MAO-A and MAO-B

Cpd. MAO-A(IC50 μM)a MAO-B (IC50 μM) SIb Cpd. MAO-A (IC50 μM) MAO-B (IC50 μM) SI
I1 0.361 ± 0.013 15.14 ± 0.013 41.9 I10 0.049 ± 0.0062 3.42 ± 0.016 69.8
I2 0.054 ± 0.0067 3.26 ± 0.025 60.4 I11 0.086 ± 0.017 4.19 ± 0.023 48.7
I3 0.011 ± 0.0049 1.53 ± 0.018 139.1 I12 0.014 ± 0.0023 1.38 ± 0.014 98.6
I4 0.005 ± 0.0014 1.01 ± 0.021 202.0 I13 0.006 ± 0.0004 0.89 ± 0.022 148.3
I5 0.009 ± 0.0021 0.85 ± 0.004 94.4 I14 0.004 ± 0.0003 1.05 ± 0.079 262.5
I6 0.503 ± 0.06 12.72 ± 0.059 25.3 I15 0.009 ± 0.0011 1.46 ± 0.027 162.2
I7 0.091 ± 0.032 4.28 ± 0.017 47.0 6 1.26 ± 0.072 23.47 ± 0.076 18.6
I8 0.072 ± 0.024 2.16 ± 0.022 30.0 iproniazid 5.97 ± 0.64 7.81 ± 0.57 1.3
I9 0.008 ± 0.0013 1.49 ± 0.034 186.3
a
All IC50 values shown in the table are expressed as means ± SD. bSI: Selectivity index, expressed for MAO-A versus MAO-B.

(3) were then cyclized under polyphosphoric acid catalysis to (0.014−2.575) were stronger than that of the lead compound 6
obtain compound 4. 4 was catalyzed by 10% palladium/carbon (2.675); (ii) generally, when the carbon chain length was three
with DMF as the solvent and refluxed overnight at 120 °C to or five, the inhibitory effects of compounds I1−I5 and I11−I15 on
obtain 5-methoxy-1-azabenzanthrone (5). Then, 5 was 5-HT2AR (0.014−1.886) were stronger than that of four (I6−I10,
demethylated using concentrated hydrochloric acid and glacial 0.048−2.575); (iii) when R1 was replaced by F, compounds I4,
acetic acid as solvents and zinc powder as catalyst to get 5- I9, and I14 showed stronger inhibitory effects on 5-HT2AR
hydroxy-1-azabenzanthrone (6). 6 reacted with dibromoalkanes (0.038, 0.048, and 0.014) than other substituted compounds
of different lengths to obtain intermediates 7a-7c. 7a-7c reacted with the same carbon chain. Among all tested compounds, the
with arylpiperazines analogues to get the target compounds activity of I14 was the best (0.014).
I1-15. The potential inhibitory effects of I1‑15 on MAO-A and MAO-
2.2. Compounds Inhibited 5-HT2AR and MAO-A B activities were investigated by measuring their effects on the
Activities. The expression of 5-HT2AR mRNA was examined production of H2O2 from p-tyramine.4 The results in Table 2
after compounds I1‑15 and lead compound 5-hydroxy-1- showed that all derivatives exhibited potent inhibitory activities
azabenzanthrone (6) at a concentration of 1 μM. The results against MAO-A with IC50 values in the range of 0.004−0.503
in Table 1 showed that (i) most compounds reduced the μM, much lower than MAO-A privilege structure 6 (1.26 μM)
expression of 5-HT2AR mRNA, and the inhibitory effects and the reference inhibitor iproniazid (5.97 μM). Notably, I1‑15
13387 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 3. Docking studies of I14 with 5-HT2AR (PDB: 6WGT) and MAO-A (PDB: 2Z5X). (A,B) I14 (white stick) interacting with residues in the
binding site of 5-HT2AR (6WGT) and 3D docking model of compound I14 with 5-HT2AR. (C,D) I14 (gray stick) interacting with residues in the
binding site of MAO-A (2Z5X) and 3D docking model of compound I14 with MAO-A.

potently inhibited MAO-B with the IC50 values ranging from residue TRY 407 of MAO-A (binding energy was −11.2 Kcal/
0.85 to 15.14 μM and were much weaker than MAO-A. mol).
Moreover, similar to the inhibition on 5-HT2AR, the inhibitory 2.4. Interaction and Molecular Dynamics Simulation
effects of F-containing derivatives on MAO-A were stronger (I4 of I14 with 5-HT2AR and MAO-A. Based on the preliminary
0.005 μM, I9 0.048 μM, and I14 0.004 μM). Regarding selectivity docking, molecular dynamics (MD) simulations were per-
index (SI), I3-4, I9, and I13‑15 showed high values for MAO-A of formed using AmberTools 20 package18 to evaluate the stability
>139.1, while 6 and iproniazid had lower values (18.6 and 1.3, of the complex of I14 with 5-HT2AR and MAO-A. In the MD
respectively). Compound I14 had an extremely high SI at 262.5. simulation process, the appropriate force field, step length,
2.3. Molecular Docking of I14 with 5-HT2AR and MAO-A. simulation time, and sufficient stable balance of the system
Molecular docking simulations of I14 with 5-HT2AR and MAO-A dynamics process are used to obtain reliable calculation results.
were performed by Autodock.17 Pymol was used to analyze the The root-mean-square deviation (rmsd) result of the relative
docking results. As shown in Figure 3, the benzene ring of initial conformation of I14/5-HT2AR is shown in Figure 4A, and
substituent 1-(2-fluorophenyl)piperazine could be embedded in the result indicated that I14 could bind to 5-HT2AR stably within
the active cavity of 5-HT2AR to form π−π stacking interaction 20 ns. The stable conformation was extracted and shown in
with the benzene ring of amino acid residue TRP 151 (binding Figure 4B, and detailed receptor−ligand interactions were
energy was −11.3 kcal/mol). In addition, the carbonyl oxygen presented. Obviously, the nitrogen atoms of arylpiperazine
on the 5-oxygen-substituted oxoisoaporphine could formed a formed a salt bridge with ASP 155. The rmsd result of the
hydrogen bond interaction with the amino group of CYS 406 in relative initial conformation of I14/MAO-A (Figure 4C)
MAO-A at 2.5 Å; the nitrogen atom formed a hydrogen bond indicated that I14 could bind to MAO-A stably within 20 ns.
interaction with the carboxyl group of amino acid residue ASP The detailed receptor−ligand interactions showed slightly
155 at 3.2 Å; the D-ring of 5-oxygen-substituted oxoisoapor- different from the initial docking result (Figure 4D), the
phine formed a π−π stacking interaction with amino acid carbonyl oxygen of 5-oxygen-substituted oxoisoaporphine
13388 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 4. MD simulation results of I14 with 5-HT2AR and MAO-A. (A,B) rmsd result of the complex of I14 with 5-HT2AR and binding pattern of I14 in
the stable frame of MD simulation. (C,D) rmsd result of the complex of I14 with MAO-A and binding pattern of I14 in the stable frame of MD
simulation. Protein bone is shown in black, solute heavy atoms are shown in red, and small molecule is shown in blue.

Figure 5. Neurocytoprotective effect of I14 on PC12 cells. (A) PC12 cells were treated with CORT of different concentrations (1000, 750, 500, 250,
and 125 μM) for 24 h. (B) PC12 cells were treated with different concentrations of I14 (4.0, 2.0, 1.0, and 0.5 μM) and 500 μM CORT for 24 h. ####P <
0.0001 vs dimethyl sulfoxide (DMSO) group; **P < 0.01 vs model group.

formed a hydrogen bond with TRP 386 (4.0 Å). The fluorine (Figure 5A). Then, the neurocytoprotective effect of I14 on
atoms of arylpiperazine formed a hydrogen bond with SER 13. PC12 cells at different concentrations (4.0, 2.0, 1.0, and 0.5 μM)
All interactions help stabilize the conformation and combination was tested. The magnitude of the difference in cell survival
of the ligand lying in the groove of 5-HT2AR and MAO-A. between the experimental and model groups after 24 h was
2.5. Toxicity In Vitro. To investigate the toxicity of the expressed as the strength of the neurocytoprotective effect of I14
derivatives, the antiproliferative effects of compounds I1‑15 on on the depression model cells. As shown in Figure 5B,
normal hepatocytes L02, human neuroblastoma cells SH-SY5Y, compound I14 exhibited a significant neurocytoprotective effect
and rat adrenal pheochromocytoma cells PC12 were evaluated on the CORT-induced cell depression model. I14 also showed a
by the MTT assay. The results are shown in Table S1 significant protective effect on PC12 cells injury at different
(Supporting Information). All compounds exhibited low concentrations compared with the model group, where the best
proliferation inhibitory activities against L02 cells (IC50 > 100 protective effect was observed at 0.5 μM (**P < 0.01).
μM), SH-SY5Y (IC50 > 10 μM) and PC12 (IC50 > 10 μM), 2.7. Antidepressant Effect of I14 in Zebrafish. Drug-
indicating that all derivatives have a good safety profile. induced models can be established by injecting drugs to animals,
2.6. Neurocytoprotective Effects. Corticosterone and such models are simple and time-consuming to operate, and
(CORT) induces damage to PC12 cells and is a commonly can effectively simulate the occurrence of human depression.
used cellular model for screening and mechanistic studies of Among them, lisdexamfetamine is a common and classical
antidepressant drugs.19 A concentration condition with a cell depression-inducing drug.20 Zebrafish is highly sensitive to
viability of about 50% was appropriate, and cells incubated with psychotropic drugs and has become a model species for research
500 μM CORT for 24 h was chosen as the modeling condition in neuroscience fields including depression in recent years,21
13389 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 6. Effects of the treatments on the depressive-like behavior of zebrafish in the open-field test. The animals received reserpine (10 mg/L) for 40
min and they were then treated with I14 (0.1, 0.5, 1 μM) or Fluoxitine (Flu, 1 μM), respectively, for 7 days. (A) Track heat map; (B) track map; (C)
total distance traveled; (D) velocity; and (E) immobility time. Data were expressed as the mean ± SD (n = 6). #P < 0.05, ##P < 0.001 vs control group;
*P < 0.05, **P < 0.01, ****P < 0.0001 vs model group.

Figure 7. Effects of the treatments on the depressive-like behavior of zebrafish in the NTT. The animals received reserpine (10 mg/L) for 40 min and
they were then treated with I14 (0.1, 0.5, and 1 μM) or Flu (1 μM), respectively, for 7 days. (A) Track heat map; (B) track map; (C) total distance
traveled; (D) velocity; and (E) time spent in the top. Data were expressed as the mean ± SD (n = 6). #P < 0.05 vs control group; *P < 0.05, **P < 0.01,
***P < 0.001 vs model group.

which provides an important perspective for depression drug 2.7.1. Open-Field Test. As shown in Figure 6C−E, the total
screening.22 distance traveled and velocity were significantly lower (#P <
13390 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 8. Changes in the body weight of ICR mice. The animals received reserpine (1 mg/kg) for 3 consecutive days and then were treated with saline
(10 mL/kg), Flu (20 mg/kg), Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) on the 4th to 10th day. (A) Experimental schedule; (B) body weight
change of mice daily during modeling and treated.

Figure 9. Effects of the treatments on the depressive-like behavior of ICR mice in the OFT. The animals received reserpine (1 mg/kg) for 3 consecutive
days and then were treated with saline (10 mL/kg), Flu (20 mg/kg), Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) on the 4th to 10th day. (A) Track
map; (B) total distance traveled; and (C) average speed; Data were expressed as the mean ± SD (n = 6). #P < 0.05 vs control group; *P < 0.05 vs model
group.

0.05) and immobility time was significantly higher (##P < 0.001). 2.8. Efficacy Evaluation of I14 on Reserpine-Induced
The results indicated that the reserpine-induced zebrafish Depression Model in Mice. 2.8.1. Weight Changes of Mice.
depression model was successful. As seen from the heat and Depression is generally accompanied by symptoms of weight
trajectory plots in Figure 6A,B, the zebrafish in the administered loss. After 3 days of continuous modeling administration using 1
group moved significantly with more distance, faster and had mg/kg of reserpine, mice in the model group lost body weight
significantly less immobility time compared to the model group compared to the blank control group. The results showed that
(*P < 0.05, **P < 0.01, ****P < 0.0001). The results suggested the reserpine depression model mice have been successfully
that I14 improved zebrafish locomotion and the depression-like established (Figure 8). Moreover, after 1 week of continuous
behavior. drug administration, both low and high dose groups were able to
2.7.2. Novel Tank Test. Novel tank test (NTT) simulated the repair the symptoms of weight loss caused by depression in mice
compared to the model group.
open field test and the cross maze test for evaluating the
2.8.2. Open-Field Test. At the end of 1 week of continuous
exploratory behavior of zebrafish. As shown in Figure 6C−E,
treatment, the mice were tested behaviorally using open-field
zebrafish in the model group moved significantly less distance, test (OFT). As shown in Figure 9, the total distance traveled was
lower speed and spent significantly less time in the upper part of significantly reduced and the speed of movement was
the maze relative to the blank group (#P < 0.05), indicating significantly lower in the model group compared to the blank
successful modeling of reserpine. The track heat map in Figure group (#P < 0.05), suggesting the success of acute modeling of
7A,B showed that zebrafish in the I14 administered group moved reserpine. Compared to the model group, the total distance
significantly more distance, faster, and spent significantly more traveled and the movement speed were significantly increased in
time in the upper part compared to the model group (*P < 0.05, each administration group (*P < 0.05), and the trajectory plots
**P < 0.01, ***P < 0.001). The results indicated that I14 also showed that the mobility of the mice was improved after the
improved locomotion and enhanced the exploratory ability. administration of the drug. All groups showed that I 14
13391 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

significantly improved the depression-like behavior in mice, with 11B) was significantly increased in the model group compared
the low dose group (10 mg/kg) compared to the positive drug to the control group (#P < 0.05, ###P < 0.001). Mice in all
(Flu, 20 mg/kg). treatment groups showed a reduction in tail suspension and
The amount of dejection of the animals in the OFT reflected forced swimming immobility time, with the most significant
the tension level of the animals, and the more dejection, the effect in the I14 high dose group (***P < 0.001, ****P <
higher the tension level. As shown by Figure 10A, the number of 0.0001). The above experiments showed that the I 14
administration group significantly improved the behavioral
despair state of depression-like mice.
2.9. Evaluation the Effect of I14 on Chronic Unpredict-
able Mild Stress. Chronic unpredictable mild stress (CUMS)
is to simulate a series of life stress events by continuously
exposing animals to a series of unpredictable mild pressures.23 A
large number of ethological and neurobiological studies have
found that the animal symptoms caused by stress are similar to
those shown by human depression patients.24 Due to its good
validity and reliability, this model is often used to screen new
antidepressants.
2.9.1. Modeling. As shown in Figure 12, after 4 weeks of
Figure 10. Effects of the treatments on the depressive-like behavior of CUMS stimulation, mice moved significantly less distance,
ICR mice. The animals received reserpine (1 mg/kg) for 3 consecutive slower, had significantly fewer uprights and feces in OFT (#P <
days and then were treated with the saline (10 mL/kg), Flu (20 mg/kg), 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001). In FST, mice
Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) on the 4th to 10th day. spent significantly more time immobile (#P < 0.05, ##P < 0.01,
(A) Dejection amounts of mice; (B) number of rearing of mice. Data ###
P < 0.001). In TST, the immobility time of mice was
were expressed as the mean ± SD (n = 6). #P < 0.05, ##P < 0.01 vs
control group; *P < 0.05, **P < 0.01 vs model group. significantly increased (#P < 0.05, ###P < 0.001). In the sugar
preference test (SPT), the sucrose bias was reduced in each
model group relative to the control group. In summary, the
dejection of mice in the model group was higher than that in the results of all behavioral experiments indicated that the CUMS
control group, and the difference was statistically significant (##P mice depression model was established successfully.
< 0.01). The results indicated that the tension level of mice in 2.9.2. Open-Field Test. After 2 weeks of continuous
the model group was higher than that of mice in the control treatment with I14, the mice were tested behaviorally using
group. The number of dejections in each treatment group was OFT. As shown in Figure 13, the total distance traveled and the
lower than that in the model group (*P < 0.05, **P < 0.01), speed of movement were significantly increased in each
indicating that the effect of I14 was good. administration group compared to the model group (*P <
The number of rearing in the OFT responded to the mobility 0.05). The trajectory plots also showed that the mobility of mice
of the mice and their abilities to explore novelties. As shown by was improved after administration. The indicators of all groups
Figure 10B, the number of rearing was significantly lower in the could indicate that I14 significantly improved depression-like
model group compared to the control group (##P < 0.01). Mice behavior in mice, with the low dose group (10 mg/kg) being
in all treatment groups increased the number of rearing in the more potent than with the positive drug (Flu, 20 mg/kg).
OFT compared to the model group (*P < 0.05, **P < 0.01). 2.9.3. FST, TST, and SPT. After 2 weeks of continuous
The low dose group (I14, 10 mg/kg, **P < 0.01) was more administration of I14, mice were tested behaviorally using FST,
potent than the positive group (Flu, 20 mg/kg, *P < 0.05). TST, and SPT. As shown in Figure 14, in the FST and TST, the
2.8.3. Forced Swimming Test and Tail Suspension Test. As immobility time of mice was significantly reduced after the
shown in Figure 11, the immobility time in forced swimming test administration of I14 (*P < 0.05, **P < 0.01); the results of the
(FST) (Figure 11A) and tail suspension test (TST) (Figure SPT experiment showed that the glucophagy of mice was
increased after the administration of I14 for 2 weeks. Various
behavioral results showed that I14 could improve the depression-
like behavior in CUMS model mice.
2.10. Toxicity Evaluation of I14 In Vivo. The results of
pathological sections of mice organ tissues showed that the
morphology, size, and structure of liver, kidney, lung, heart, and
spleen cells were normal in all groups, without necrosis or
pathological changes (Figure 15). It showed that I14 had no
relevant toxic effects on the liver, kidney, lung, and spleen of
mice during the treatment period.
2.11. Effects of I14 on Hippocampal Tissue Morphology
in Mice. The pathological tissue section of mouse hippocampus
(Figure 16) showed that the cone cells in the blank group were
Figure 11. Effects of the treatments on the depressive-like behavior of
neatly arranged, most of them had large and round nuclei, and
ICR mice. The animals received reserpine (1 mg/kg) for 3 consecutive
days and then were treated with the saline (10 mL/kg), Flu (20 mg/kg), the nucleoli were obvious and numerous; the CA1 and CA3
Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) on the 4th to 10th day. areas of mouse hippocampus in the model group were
(A) Immobility time in FST; (B) immobility time in TST. Data were accompanied by some cone cells with solid and deep stained
expressed as the mean ± SD (n = 6). #P < 0.05, ###P < 0.001 vs control nuclei, and the granule cells in the DG area were more discrete. It
group; **P < 0.01, ***P < 0.001, ****P < 0.0001 vs model group. suggested that the neuronal cells in the hippocampal tissue of
13392 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 12. Effects of the treatments on the depressive-like behavior of ICR mice after CUMS modeling. The animals received CUMS for 4 weeks. (A)
Total distance traveled in the OFT; (B) average speed in the OFT; (C) immobility time in FST; (D) immobility time in TST; (E) number of rearing in
the open field test; (F) dejection amounts in the open field test; (G) sugar preference; and data were expressed as the mean ± SD (n = 6). #P < 0.05, ##P
< 0.01, ###P < 0.001, ###P < 0.0001 vs control group.

Figure 13. Effects of the treatments on the depressive-like behavior of ICR mice in the OFT. The animals received CUMS for 4 weeks and then were
treated with the saline (10 mL/kg), Flu (20 mg/kg), Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) for 2 weeks. (A) Track map; (B) total distance
traveled; (C) average speed; (D) dejection amounts of mice; and (E) number of rearing of mice. Data were expressed as the mean ± SD (n = 6). ###P <
0.001, ####P < 0.0001 vs control group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs model group.

mice in the model group were partially damaged. In contrast, the whole brain tissues of mice were analyzed by Western Blot. As
neuronal cells in the I14 administration group were well shown in Figure 17, after lisinopril modeling, 5-HT1AR
arranged, and only a few neuronal cells were damaged, expression was reduced and 5-HT2AR expression was increased
suggesting that the I14 administration group had a protective in brain tissues of mice in the model group compared with the
effect on the hippocampal tissue morphology of the depression- normal group, and the differences were statistically significant
like mice caused by reserpine and could improve the damage of (####P < 0.0001). While I14 administration significantly
neuronal cells. decreased 5-HT2AR expression in both low and high dose
2.12. Effects of I14 on Protein Expression in Mouse groups (****P < 0.0001), there was a trend toward increased 5-
Brain Tissue. Changes in 5-HT1AR and 5-HT2AR expression in HT1AR expression, but not statistically significant. In contrast,
13393 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 14. Effects of the treatments on the depressive-like behavior of ICR mice. The animals received CUMS for 4 weeks and then were treated with
saline (10 mL/kg), Flu (20 mg/kg), Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) for 2 weeks. (A) Immobility time in FST; (B) immobility time in
TST; and (C) sugar preference. Data were expressed as the mean ± SD (n = 6). #P < 0.05, ###P < 0.001, ####P < 0.0001 vs control group; *P < 0.05, **P
< 0.01 vs model group.

conducive to more efficient antidepressant active. Therefore, the

5-HT level in mice brain tissue was evaluated using HPLC. As
shown in Figure 18, compared with the control group, the level
of 5-HT in the hippocampal brain tissue of the mice in the model
group was significantly decreased (216.18 ng/mL). Meanwhile,
compared with the model group, the content of 5-HT in the low-
dose group (10 mg/kg) and the high-dose group (20 mg/kg) in
the I14 administration group were significantly increased (579.92
ng/mL). The above results showed that the level of 5-HT in the
brain was significantly increased after the administration of I14.
2.14. Preclinical Pharmacokinetic Evaluation of I14.
The pharmacokinetic characteristics of I14 in SD rats have been
evaluated. PK spectrum of male SD rat model (Table 3) showed
that single intragastric administration (10 mg/kg), I14 was
absorbed with Tmax of 1.33 h, Cmax of 99.67 ng/mL, and a good
half-life (T1/2) of 5.22 h. The same experiment was conducted
Figure 15. Histomorphological appearances of the organ of male mice.
Hematoxylin−eosin (H&E) staining, original magnification × 400. Flu
after intravenous injection of I14. The results showed that I14 had
(20 mg/kg), Low (I14, 10 mg/kg), and High (I14, 20 mg/kg). a good clearance rate of 345.69 mL/min/kg in rats. The results
indicated that I14 was a potential candidate drug, providing a
solid basis for further evaluation of its bioactivity in vivo.
Flu administration significantly increased the expression of 5-
HT1AR (**P < 0.01) and had a tendency to diminish the
expression of 5-HT2AR, but there was no statistically significant
3. CONCLUSIONS
difference. Therefore, the possible mechanism by which I14 Based on the potential binding affinities of arylpiperazine
improved the depression-like behavior and exerts antidepressant scaffolds and 5-oxygen-substituted oxoisoaporphines with 5-
efficacy in mice was related to the downregulation of 5-HT2AR. HT2AR and MAO-A, linked-pharmacophore strategy was
2.13. Regulation of I14 on 5-HT Level. Based on previous carried out to design and synthesize 15 dual 5-HT2AR/MAO-
assumptions, simultaneous inhibition of MAO-A and 5-HT2AR A inhibitors. RT-qPCR or H2O2 production results showed that
could synergistically promote interstitial 5-HT level, which is all hybrids showed enhanced inhibitory activities against 5-

Figure 16. Histomorphological appearances of the mice hippocampus. H&E staining, original magnification × 200. Flu (20 mg/kg), Low (I14, 10 mg/
kg), and High (I14, 20 mg/kg).

13394 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 17. Effects of I14 on the expression of 5-HT1AR and 5-HT2AR in the whole brain of mice. The animals received reserpine (1 mg/kg) for 3
consecutive days and then were treated with the saline (10 mL/kg), Flu (20 mg/kg), Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) on the 4th to 10th
day. (A) Levels of 5-HT1AR, 5-HT2AR are determined by western blot; (B) relative levels of 5-HT1AR and 5-HT2AR are determined. Data were
expressed as the mean ± SD (n = 3). ####P < 0.0001 vs control group; **P < 0.01, ****P < 0.0001 vs model group.

tissue. Overall, I14 was an antidepressant candidate worthy of in-

depth study.

4. EXPERIMENTAL SECTION
4.1. Chemistry. 4.1.1. Materials and Instruments. All reagents
were commercially available. Products were purified by silica gel
column chromatography (200−300 mesh, Qingdao, China). 1H NMR
spectra and 13C NMR spectra were recorded on Bruker AVANCE
instrument at 25 °C using TMS as the internal standard in CDCl3 or
DMSO-d6. ESI-MS spectra were recorded on a HP 1100LC/MSD
spectrometer. The purity of derivatives I1‑15 was determined by an
Figure 18. Effects of I14 on the regulation of 5-HT level in the whole Agilent Q-TOF mass spectrometer. Individual compounds with a
brain of mice. The animals received reserpine (1 mg/kg) for 3 purity of >95% were used for subsequent experiments (see the
consecutive days and then were treated with the saline (10 mL/kg), Flu Supporting Information).
(20 mg/kg), Low (I14, 10 mg/kg), and High (I14, 20 mg/kg) on the 4th 4.1.2. General Procedure for the Synthesis of I1‑15. K2CO3, KI, and
to 10th day. Data were expressed as the mean ± SD (n = 6). ****P < different arylpiperazines were first dissolved in CH3CN (15.0 mL).
0.0001 vs model group. Then, the product of 5-hydroxyl-1-azabenzanthrone with the
appropriate halide (n = 2−4) (0.27 mmol, 1 equiv) was added
dropwise. The solution was stirred for 12 h at 75 °C. The reaction
Table 3. SD Rat PK Profiles of I14 mixture was concentrated under reduced pressure, and then, the residue
parametera 2 mg/kg (i.v.) 10 mg/kg (i.g.) was purified by column chromatography with various proportion
eluents to afford I1‑15.
AUC0−24(h*ng/mL) 2230.67 ± 153.78 490.67 ± 70.43 4.1.3. Compound I1. Yellow powder; yield 70%; 1H NMR (500
AUC0‑inf(h*ng/mL) 2322.67 ± 178.02 504.00 ± 71.08 MHz, CDCl3): δH 8.88 (d, J = 7.8 Hz, 1H), 8.68 (d, J = 5.4 Hz, 1H),
MRTinf(h) 4.19 ± 0.14 4.94 ± 0.36 8.40 (d, J = 7.7 Hz, 1H), 8.29 (d, J = 1.9 Hz, 1H), 7.80 (t, J = 7.3 Hz,
t1/2(h) 6.31 ± 0.55 5.22 ± 0.79 1H), 7.63 (dd, J = 13.3, 6.4 Hz, 2H), 7.43 (d, J = 1.7 Hz, 1H), 7.36 (d, J
Tmax(h) 0.08 ± 0.00 1.33 ± 0.33 = 7.8 Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 7.06 (d, J = 7.8 Hz, 1H), 6.97 (t,
Cmax(ng/mL) 673.33 ± 25.41 99.67 ± 6.01 J = 7.2 Hz, 1H), 4.38 (t, J = 4.7 Hz, 2H), 3.14 (s, 4H), 3.03 (t, J = 4.5 Hz,
CL (mL/min/kg) 14.54 ± 1.20 345.69 ± 53.40 2H), 2.86 (s, 4H). 13C NMR (125 MHz, CDCl3): δC 183.18, 160.05,
F(%) 4.40 149.16, 148.28, 144.45, 137.41, 136.94, 134.11, 132.31, 130.89, 130.66,
a
130.21, 128.83, 127.60, 125.32, 123.77, 121.48, 120.41, 120.20, 118.87,
PK parameters in Sprague-Dawley rats (male). Data were expressed 112.52, 66.82, 57.02 (C × 2), 53.92 (C × 2), 51.14. HRMS (ESI)
as the mean ± SD (n = 3). calculated for C26H24ClN3O2 [M + H]+, 470.1630; found, 470.1623.
4.1.4. Compound I2. Yellow powder; yield 61%; 1H NMR (500
MHz, CDCl3): δH 8.88 (d, J = 7.7 Hz, 1H), 8.68 (d, J = 5.6 Hz, 1H),
8.40 (d, J = 7.2 Hz, 1H), 8.29 (d, J = 2.2 Hz, 1H), 7.80 (t, J = 7.0 Hz,
HT2AR expression and MAO-A. Most of the derivatives were not 1H), 7.63 (dd, J = 12.8, 6.3 Hz, 2H), 7.43 (d, J = 2.0 Hz, 1H), 7.01 (t, J =
significantly cytotoxic to neuronal cells SH-SY5Y, PC12, and 7.4 Hz, 1H), 6.97−6.91 (m, 2H), 6.87 (d, J = 7.6 Hz, 1H), 4.40 (t, J =
human normal hepatocytes L02. The optimal target I14 showed a 5.0 Hz, 2H), 3.88 (s, 3H), 3.17 (s, 4H), 3.04 (t, J = 5.0 Hz, 2H), 2.89 (s,
protective effect against CORT-induced PC12 cell damage at 4H). 13C NMR (125 MHz, CDCl3): δC 183.19, 160.02, 152.30, 148.26,
0.5 μM concentration, reflecting its antidepressant activity in 144.46, 141.15, 137.42, 136.95, 134.11, 132.31, 130.87, 130.20, 127.59,
vitro. I14 significantly improved the depression-like behavior of 125.32, 123.06, 121.56, 121.02, 120.21, 118.87, 118.26, 112.43, 111.24,
the acute zebrafish depression model induced by reserpine. In 66.75, 57.04, 55.39, 53.96 (C × 2), 50.51 (C × 2). HRMS (ESI)
the reserpine-induced acute depression mice model and in the calculated for C29H28N3O3 [M + H]+, 466.2125; found, 466.2116.
chronic mild unpredictable model, I14 was more efficacious than 4.1.5. Compound I3. Yellow powder; yield 58%; 1H NMR (500
MHz, CDCl3): δH 8.88 (d, J = 7.7 Hz, 1H), 8.68 (d, J = 5.5 Hz, 1H),
fluoxetine. Molecular docking showed that I14 binded better to 8.40 (d, J = 7.6 Hz, 1H), 8.29 (s, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.63 (dd,
5-HT2AR and MAO-A. H&E staining results showed no relevant J = 13.3, 6.4 Hz, 2H), 7.43 (s, 1H), 7.15 (d, J = 7.2 Hz, 2H), 6.97 (d, J =
toxic effects of I14 in vivo. Western Blot results indicated that the 5.3 Hz, 1H), 4.38 (s, 2H), 3.13 (s, 4H), 3.03 (s, 2H), 2.87 (s, 4H). 13C
possible mechanism by which I14 exerted antidepressant effects NMR (125 MHz, CDCl3): δC 183.18, 160.01, 151.11, 148.29, 144.47,
was related to downregulation of 5-HT2AR expression in brain 137.40, 136.94, 134.12, 134.08, 132.30, 130.90, 130.22, 127.59, 127.47,

13395 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

125.33, 124.71, 121.44, 120.19, 118.88, 118.64, 112.54, 66.77, 56.98, 8.40 (d, J = 7.8 Hz, 1H), 8.26 (d, J = 2.5 Hz, 1H), 7.86−7.77 (m, 1H),
53.83 (C × 2), 51.20 (C × 2). HRMS (ESI) calculated for 7.70−7.60 (m, 2H), 7.42 (d, J = 2.4 Hz, 1H), 7.32−7.27 (m, 2H),
C28H24Cl2N3O2 [M + H]+, 504.1240; found, 504.1237. 6.99−6.84 (m, 3H), 4.28 (t, J = 6.1 Hz, 2H), 3.26 (d, J = 4.0 Hz, 4H),
4.1.6. Compound I4. Yellow powder; yield 51%; 1H NMR (500 2.70 (dd, J = 11.8, 6.5 Hz, 6H), 2.22−2.12 (m, 2H). 13C NMR (125
MHz, CDCl3): δH 8.88 (d, J = 7.7 Hz, 1H), 8.68 (d, J = 5.5 Hz, 1H), MHz, CDCl3): δC 183.29, 160.29, 151.21, 148.24, 144.43, 137.47,
8.40 (d, J = 7.6 Hz, 1H), 8.29 (s, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.63 (dd, 136.96, 136.87, 134.16, 132.31, 132.23, 130.84, 130.23, 129.16, 127.61,
J = 13.3, 6.4 Hz, 2H), 7.43 (s, 1H), 7.15 (d, J = 7.2 Hz, 2H), 6.97 (d, J = 125.32, 121.56, 120.25, 119.86, 118.80, 116.15, 112.35, 66.91, 54.98,
5.3 Hz, 1H), 4.38 (s, 2H), 3.13 (s, 4H), 3.03 (s, 2H), 2.87 (s, 4H). 13C 53.31 (C × 2), 49.08 (C × 2), 26.46. HRMS (ESI) calculated for
NMR (125 MHz, CDCl3): δC 183.15, 160.02, 148.25, 144.44, 137.38, C29H28N3O2 [M + H]+, 450.2176; found, 450.2162.
136.92, 134.10, 132.29, 130.86, 130.20, 127.58, 125.31, 124.48, 122.56, 4.1.13. Compound I11. Yellow powder; yield 55%; 1H NMR (500
122.49, 121.43, 120.18, 118.97, 118.85, 116.21, 116.04, 112.50, 66.80, MHz, CDCl3): δH 8.86 (d, J = 7.8 Hz, 1H), 8.64 (d, J = 5.6 Hz, 1H),
57.02, 53.82 (C × 2), 50.50 (C × 2). HRMS (ESI) calculated for 8.38 (d, J = 7.8 Hz, 1H), 8.22 (d, J = 2.5 Hz, 1H), 7.79 (t, J = 7.0 Hz,
C28H25FN3O2 [M + H]+, 454.1925; found, 454.1920. 1H), 7.62 (t, J = 7.1 Hz, 1H), 7.58 (d, J = 5.7 Hz, 1H), 7.35 (dd, J = 8.1,
4.1.7. Compound I5. Yellow powder; yield 55%; 1H NMR (500 1.5 Hz, 2H), 7.21 (t, J = 7.7 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.96 (t, J
MHz, CDCl3): δH 8.89 (d, J = 7.7 Hz, 1H), 8.68 (d, J = 5.6 Hz, 1H), = 7.6 Hz, 1H), 4.21 (t, J = 6.2 Hz, 2H), 3.11 (s, 4H), 2.70 (s, 4H), 2.58−
8.40 (d, J = 7.4 Hz, 1H), 8.29 (d, J = 2.2 Hz, 1H), 7.81 (t, J = 7.1 Hz, 2.53 (m, 2H), 1.99−1.92 (m, 2H), 1.81 (dd, J = 14.9, 8.0 Hz, 2H). 13C
1H), 7.63 (dd, J = 12.0, 6.4 Hz, 2H), 7.44 (d, J = 2.0 Hz, 1H), 7.28 (d, J
NMR (125 MHz, CDCl3): δC 183.22, 160.30, 149.24, 148.17, 144.37,
= 8.2 Hz, 2H), 6.95 (d, J = 7.9 Hz, 2H), 6.87 (t, J = 7.1 Hz, 1H), 4.39 (t,
137.42, 136.93, 134.10, 132.29, 130.77, 130.65, 130.19, 128.77, 127.62,
J = 5.2 Hz, 2H), 3.27 (s, 4H), 3.01 (t, J = 5.2 Hz, 2H), 2.83 (s, 4H). 13C
127.58, 125.29, 123.70, 121.49, 120.38, 120.20, 118.73, 112.31, 68.51,
NMR (125 MHz, CDCl3): δC 183.20, 160.01, 151.20, 148.30, 144.48,
58.15, 53.41 (C × 2), 51.17 (C × 2), 27.11, 23.38. HRMS (ESI)
137.42, 136.95, 134.13, 132.31, 130.91, 130.22, 129.14 (C × 2), 127.60,
125.33, 121.46, 120.20, 119.87, 118.89, 116.15 (C × 2), 112.52, 66.87, calculated for C30H29ClN3O2 [M + H]+, 498.1943; found, 498.1939.
56.99, 53.77 (C × 2), 49.13 (C × 2). HRMS (ESI) calculated for 4.1.14. Compound I12. Yellow powder; yield 47%; 1H NMR (500
C28H26N3O2 [M + H]+, 436.2020; found, 436.2013. MHz, CDCl3): δH 8.85 (d, J = 7.9 Hz, 1H), 8.63 (d, J = 5.6 Hz, 1H),
4.1.8. Compound I6. Yellow powder; yield 63%; 1H NMR (500 8.37 (dd, J = 7.8, 1.0 Hz, 1H), 8.20 (d, J = 2.5 Hz, 1H), 7.82−7.76 (m,
MHz, CDCl3): δH 8.86 (1H, d, J = 8.0 Hz), 8.65 (1H, d, J = 5.6 Hz), 1H), 7.65−7.59 (m, 1H), 7.56 (d, J = 5.7 Hz, 1H), 7.34 (d, J = 2.5 Hz,
8.39 (1H, d, J = 7.9 Hz), 8.24 (1H, d, J = 2.5 Hz), 7.83−7.77 (m, 1H), 1H), 7.02−6.84 (m, 4H), 4.19 (t, J = 6.3 Hz, 2H), 3.86 (s, 3H), 3.12 (s,
7.66−7.58 (m, 2H), 7.39 (d, J = 2.5 Hz, 1H), 7.36 (dd, J = 7.9, 1.5 Hz, 4H), 2.70 (s, 4H), 2.56−2.50 (m, 2H), 1.99−1.91 (m, 2H), 1.84−1.75
1H), 7.24−7.19 (m, 1H), 7.06 (dd, J = 8.1, 1.5 Hz, 1H), 7.00−6.94 (m, (m, 2H). 13C NMR (125 MHz, CDCl3): δC 183.19, 160.29, 152.26,
1H), 4.26 (t, J = 6.3 Hz, 2H), 3.12 (s, 4H), 2.84−2.60 (m, 6H), 2.17− 148.13, 144.35, 141.30, 137.39, 136.92, 134.07, 132.28, 130.73, 130.17,
2.10 (m, 2H). 13C NMR (125 MHz, CDCl3): δC 183.23, 160.29, 127.56, 125.28, 122.95, 121.49, 120.99, 120.19, 118.69, 118.21, 112.26,
149.24, 148.18, 144.38, 137.42, 136.93, 134.11, 132.29, 130.78, 130.66, 111.12, 68.53, 58.24, 55.36, 53.50 (C × 2), 50.66 (C × 2), 27.14, 23.42.
130.20, 128.79, 127.62, 127.59, 125.29, 123.72, 121.49, 120.39, 120.21, HRMS (ESI) calculated for C31H32N3O3 [M + H]+, 494.2438; found,
118.74, 112.38, 66.97, 54.97, 53.47 (C × 2), 51.20 (C × 2), 26.56. 494.2436.
HRMS (ESI) calculated for C29H27ClN3O2 [M + H]+, 484.1786; 4.1.15. Compound I13. Yellow powder; yield 50%; 1H NMR (500
found, 484.1785. MHz, CDCl3): δH 8.86 (d, J = 7.9 Hz, 1H), 8.65 (d, J = 5.6 Hz, 1H),
4.1.9. Compound I7. Yellow powder; yield 46%; 1H NMR (500 8.38 (d, J = 7.8 Hz, 1H), 8.22 (d, J = 2.5 Hz, 1H), 7.79 (t, J = 7.6 Hz,
MHz, CDCl3): δH 8.89 (d, J = 7.7 Hz, 1H), 8.68 (d, J = 5.6 Hz, 1H), 1H), 7.65−7.57 (m, 2H), 7.36 (d, J = 2.5 Hz, 1H), 7.14 (t, J = 6.7 Hz,
8.40 (d, J = 7.4 Hz, 1H), 8.29 (d, J = 2.2 Hz, 1H), 7.81 (t, J = 7.1 Hz, 2H), 6.94 (dd, J = 7.1, 2.5 Hz, 1H), 4.21 (t, J = 6.2 Hz, 2H), 3.09 (s,
1H), 7.63 (dd, J = 12.0, 6.4 Hz, 2H), 7.44 (d, J = 2.0 Hz, 1H), 7.28 (d, J 4H), 2.70 (s, 4H), 2.58−2.53 (m, 2H), 1.99−1.92 (m, 2H), 1.81 (dd, J
= 8.2 Hz, 2H), 6.95 (d, J = 7.9 Hz, 2H), 6.87 (t, J = 7.1 Hz, 1H), 4.39 (t, = 14.9, 7.9 Hz, 2H). 13C NMR (125 MHz, CDCl3): δC 183.23, 160.29,
J = 5.2 Hz, 2H), 3.27 (s, 4H), 3.01 (t, J = 5.2 Hz, 2H), 2.83 (s, 4H). 13C 151.21, 148.18, 144.38, 137.42, 136.93, 134.12, 134.02, 132.28, 130.78,
NMR (125 MHz, CDCl3): δC 183.28, 160.29, 151.13, 148.24, 144.43, 130.20, 127.58, 127.48, 125.29, 124.62, 121.48, 120.20, 118.73, 118.61,
137.45, 136.95, 134.15, 134.06, 132.30, 130.84, 130.23, 127.61, 127.50, 112.32, 68.49, 58.09, 53.31 (C × 2), 51.25 (C × 2), 27.09, 23.35.
125.31, 124.69, 121.48, 120.23, 118.80, 118.65, 112.43, 66.89, 54.93, HRMS (ESI) calculated for C30H28Cl2N3O2 [M + H]+, 532.1553;
53.37 (C × 2), 51.18 (C × 2), 26.45. HRMS (ESI) calculated for found, 532.1544.
C30H30N3O3 [M + H]+, 480.2282; found, 480.2275. 4.1.16. Compound I14. Yellow powder; yield 60%; 1H NMR (500
4.1.10. Compound I8. Yellow powder; yield 49%; 1H NMR (500 MHz, CDCl3): δH 8.86 (d, J = 7.9 Hz, 1H), 8.65 (d, J = 5.6 Hz, 1H),
MHz, CDCl3): δH 8.88 (d, J = 8.0 Hz, 1H), 8.67 (d, J = 5.6 Hz, 1H), 8.38 (d, J = 7.7 Hz, 1H), 8.22 (s, 1H), 7.80 (t, J = 7.4 Hz, 1H), 7.66−
8.40 (d, J = 7.8 Hz, 1H), 8.26 (s, 1H), 7.84−7.78 (m, 1H), 7.66−7.60 7.57 (m, 2H), 7.36 (s, 1H), 7.08−6.91 (m, 4H), 4.21 (t, J = 6.1 Hz, 2H),
(m, 2H), 7.42 (d, J = 2.5 Hz, 1H), 7.18−7.11 (m, 2H), 6.97 (dd, J = 6.8, 3.15 (s, 4H), 2.72 (s, 4H), 2.60−2.54 (m, 2H), 2.00−1.92 (m, 2H),
2.8 Hz, 1H), 4.28 (t, J = 6.3 Hz, 2H), 3.11 (s, 4H), 2.83−2.60 (m, 6H), 1.82 (d, J = 6.8 Hz, 2H). 13C NMR (125 MHz, CDCl3): δC 183.23,
2.19−2.10 (m, 2H). 13C NMR (125 MHz, CDCl3): δC 183.28, 160.30, 160.27, 148.18, 144.38, 137.42, 136.93, 134.11, 132.29, 130.79, 130.20,
151.19, 148.23, 137.45, 136.95, 134.15, 134.06, 132.30, 130.83, 130.23,
127.58, 125.29, 124.51, 124.48, 122.60, 122.52, 121.48, 120.21, 118.96,
127.61, 127.53, 127.49, 125.31, 124.65, 121.48, 120.22, 118.79, 118.63,
118.93, 118.74, 116.22, 116.02, 112.31, 68.46, 58.09, 53.26 (C × 2),
112.43, 66.92, 54.92, 53.38 (C × 2), 51.25 (C × 2), 26.51. HRMS (ESI)
50.37, 27.06, 23.26. HRMS (ESI) calculated for C30H29FN3O2 [M +
calculated for C29H26Cl2N3O2 [M + H]+, 518.1397; found, 518.1386.
4.1.11. Compound I9. Yellow powder; yield 52%; 1H NMR (500 H]+, 482.2238; found, 482.2234.
MHz, CDCl3): δH 8.86 (d, J = 7.9 Hz, 1H), 8.65 (d, J = 5.6 Hz, 1H), 4.1.17. Compound I15. Yellow powder; yield 62%; 1H NMR (500
8.39 (d, J = 7.8 Hz, 1H), 8.24 (d, J = 2.5 Hz, 1H), 7.80 (t, J = 7.6 Hz, MHz, CDCl3): δH 8.88 (d, J = 8.0, 0.7 Hz, 1H), 8.67 (d, J = 5.6 Hz, 1H),
1H), 7.66−7.58 (m, 2H), 7.39 (d, J = 2.5 Hz, 1H), 7.09−6.92 (m, 4H), 8.40 (d, J = 7.8, 1.0 Hz, 1H), 8.26 (d, J = 2.5 Hz, 1H), 7.84−7.78 (m,
4.27 (t, J = 6.3 Hz, 2H), 3.21−3.11 (m, 4H), 2.69 (dd, J = 14.1, 6.6 Hz, 1H), 7.66−7.60 (m, 2H), 7.40 (d, J = 2.5 Hz, 1H), 7.29−7.27 (m, 1H),
6H), 2.17−2.10 (m, 2H). 13C NMR (125 MHz, CDCl3): δC 183.24, 7.25 (d, J = 2.0 Hz, 1H), 6.94 (d, J = 7.9 Hz, 2H), 6.86 (t, J = 7.3 Hz,
160.29, 148.19, 144.39, 137.42, 136.94, 134.12, 132.29, 130.79, 130.20, 1H), 4.23 (t, J = 6.2 Hz, 2H), 3.24 (t, J = 4.8 Hz, 4H), 2.68 (t, J = 4.8 Hz,
127.59, 125.30, 124.47, 122.47, 121.50, 120.21, 118.96, 118.75, 116.23, 4H), 2.55 (t, J = 7.5 Hz, 2H), 2.00−1.94 (m, 2H), 1.86−1.78 (m, 2H).
13
116.03, 112.36, 66.93, 54.97, 53.38 (C × 2), 50.51 (C × 2), 26.50. C NMR (125 MHz, CDCl3): δC 183.30, 160.32, 151.23, 148.24,
HRMS (ESI) calculated for C29H27FN3O2 [M + H]+, 468.2082; found, 144.42, 137.47, 136.97, 134.15, 132.31, 130.85, 130.22, 129.14, 127.61,
468.2073. 125.31, 121.53, 120.23, 119.82, 118.79, 116.12, 112.33, 68.49, 58.11,
4.1.12. Compound I10. Yellow powder; yield 62%; 1H NMR (500 53.23 (C × 2), 49.06 (C × 2), 27.08, 23.32. HRMS (ESI) calculated for
MHz, CDCl3): δH 8.88 (d, J = 7.9 Hz, 1H), 8.67 (d, J = 5.6 Hz, 1H), C30H30N3O2 [M + H]+, 464.2333; found, 464.2322.

13396 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

5. BIOLOGICAL EVALUATION thermostat with 1 ps coupling constant and the pressure at 1 atm
using Monte Carlo barostat with 1 ns relaxation time. Finally, 20
Cell lines including PC12 were cultured in a DMEM medium.
ns MD production was performed in NVT ensemble without any
Each medium (KeyGEN, China) was supplemented with 10%
restraint. Later, frames were extracted from 20 ns of the
fetal bovine serum (BI, KeyGEN). All the cell lines were
trajectory for analysis.
obtained from National Infrastructure of Cell Line Resource,
5.5. Cytotoxic Assay In Vitro. Compounds I1‑15 were
China. Tested compounds were dissolved in DMSO (Tansoole,
tested by the MTT method for cytotoxic activity using three cell
China) at the concentration as 10 mM for usage.
lines (PC12, SH-SY5Y, and L02). For each, in a 96-well plate,
5.1. RT-qPCR. Total RNA was extracted from zebrafish using
5.0 × 103 cells were cultured per well for 24 h. Then, different
Trizol according to the manufacturer’s protocol (Invitrogen, concentrations of compounds I1‑15, CEL, and DMSO were
USA). Then, RNA was reverse transcribed by MMLV reverse administered. After administration, the cells were cultured for 48
transcriptase (Promega, Madison, WI). The quantitative RT- h, and then, 10 μL of MTT solution (KeyGEN, China) was
PCR was performed using the SYBR Green Labeling System added to each well and incubated at 37 °C for 4 h. Next, the old
(BioRad, CA, USA). The procedure of the Quantitative RT- medium was removed, and 150 μL of DMSO was added to each
PCR included a denaturing step at 95 °C for 2 min, 40 cycles of well. Lastly, the absorbance of each well was detected at a 570
95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for real time nm wavelength with a microplate reader (POLARstar Omega,
plate read, and a final extension at 72 °C for 5 min. Primers for Germany). Cell viability was calculated by referring to the
those two genes are as follows: percentage of absorbance compared to the blank control group.
GAPDH(forward,5′-CAGCAACACAGAAGACCGTTG- IC50 values were calculated using GraphPad Prism 8.0 software
3′,reverse,5′-AGCTTGCCATTGAGCTCAGG-3′),5-HT2AR- (San Diego, CA, USA). The final value of each IC50 was the
(forward,5′-CTGCAGAACGCCACCAATTAC-3′, reverse, result of repeating these experiments three times.
5′-.GGAAAGGCCATGAGTAACCATACAC-3′). All experi-
ments were performed at least three times with similar results.
5.2. Determination of MAO-A and MAO-B Activities.
The inhibitory activities of compounds on MAO-A and MAO-B
were determined by the AmplexRed fluorescence method. The
amine substrate (p-tyramine) was oxidized and deaminated by
monoamine oxidase to generate H2O2, and the generated H2O2
converted AmplexRed into fluorescent substances resorufin 5.6. Protection of PC12 Cells Against CORT-Induced
under the action of horseradish peroxidase. Recordings were Cell Damage. The cyto-protection effect of target compounds
made at the corresponding excitation and emission wavelengths, on PC12 cells which damaged by CORT was evaluated by the
and the enzyme inhibitory activity of the compound was MTT assay. Refering to the protocol of 5.4, cells were seeded in
reflected by changes in fluorescence intensity. Briefly, different 96-well plates at the density of 5 × 104 cells/mL. After 24 h, the
concentrations of test samples (20 μL) and MAO-A or MAO-B old medium in 96-well plates were replaced with a fresh
(80 μL) were added to a 96-well plate. Incubation was carried complete medium with different compound concentrations and
out in a constant temperature incubator at 37 °C for 15 min, 500 μM CORT (DMSO less than 1%), and the culture was
then 100 μL of substrate working solution was added and continued for 24 h. The above MTT method was used to
incubated at 37 °C for 15 min, immediately detected with a measure cell viability, and the calculation formula of cell viability
multi-plate reader (EX = 545 nm, EM = 590 nm), and recorded was as follows
the fluorescence value. The blank group used 20 μL of potassium
dihydrogen phosphate buffer instead of the sample solution, the
negative control group used 20 μL of 0.1% DMSO solution to
replace the sample solution, and the positive control group used
20 μL of iproniazid instead of the sample solution.
5.3. Molecular Docking Studies. Molecular docking 5.7. Anti-Depressant Activity of I14 In Vivo. 5.7.1. Ani-
studies were performed using AutodockTools-1.5.6. The X-ray mals. The experiment used ICR male mice (8−10 weeks old,
crystallographic structure of 5-HT2AR (PDB 6WGT) and MAO- weight 18−20 g) purchased from Qinglongshan Animal
A (PDB 2Z5X) was obtained from the Protein Data Bank. All Breeding Factory in Nanjing, China and kept at a laboratory
water molecules in the PDB file were removed. I14 was docked animal barrier system with the required environment (temper-
into the active site of the protein using Autodock Vina and ature of 24 ± 1 °C, relative humidity of 45 ± 15%, and a 12 h
visualized analysis by PyMOL. light/dark cycle). Food and water were readily available
5.4. Molecular Dynamics. The MDs simulations were throughout the experiment, except where specified. The
performed by AmberTools 20 package with AMBER ff19SB and experiments began after 1 week of habituation to the housing
GAFF force fields for the complex of I14 with 5-HT2AR and conditions.
MAO-A, respectively. The system was solvated by a truncated Zebrafish (AB strain) were maintained in a fish-farming
octahedron water box using the OPC water model with a margin system at the China Pharmaceutical University. The zebrafish
of 10 Å. Periodic boundary condition was used, and the net feeding method was carried out according to The Zebrafish
charge was neutralized by 0.15 M NaCl. Briefly, two 10 000 and Book.24 The room temperature was maintained at 28.5 °C on a
10 000 steps steepest descent and conjugate gradient constant light cycle (14 h light/10 h dark), and the water (KCl
minimizations were performed to remove improper atom 0.05 g/L, NaHCO3 0.025 g/L, NaCl 3.5 g/L, and CaCl2 0.1 g/L,
contacts. Following initial optimization, the system was then purchased from Sinopharm Chemical Reagent Co., Ltd.,
equilibrated during 20 ps NVT and 200 ps NPT ensembles. The Shanghai, China) was circulated continuously. The zebrafish
temperature was maintained at 300 K using the Berendsen were fed freshly hatched brine shrimp twice daily. Less than five
13397 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

adult zebrafish were in the breeding tank. All experiments were 5.10. In Vivo PK Study. The study was conducted in the
approved by ethics Committee of China Pharmaceutical Jiangsu Ouwei Pharmaceutical Co. Ltd. The experiment meets
University. the ethical considerations. Healthy male Sprague-Dawley rats
5.7.2. Reserpine Model. After behavioral screening, the 30 (180−200 g, Zhejiang Weitong Lihua Laboratory Animal Co.
mice were divided into five groups (Control, Model, Flu, Low Ltd, China) were randomly divided into two groups (n = 3) to
and High dose group). The Model, Flu, Low, and High dose analyze PK properties of the test compound. Three rats were
groups were injected intraperitoneally with reserpine (Sigma- used for gavage (10 mg/kg), while the other three rats were used
Aldrich Trading Co., Ltd., Shanghai, China) at a dose of 1 mg/kg for intravenous administration (2 mg/kg). The compound
for 3 days. Reserpine was dissolved in glacial acetic acid (Xilong samples were dissolved in a mixture of 5% DMA+5% Solutol
Science Co., Ltd., Shenzhen, China) and diluted with distilled HS-15 in saline (i.v), 0.5% CMC-Na (i.g.) before the
water to a final concentration of 0.5% acetic acid. The volume is experiments. The animals were fasted overnight before initiating
0.2 mL. The Flu (Shanghai Huyuan Pharmaceutical Co., Ltd., the experiment but had free access to water. After admin-
Shanghai, China) group was administered at a dose of 20 mg/ istration, the blood samples were collected from the retro-orbital
kg/day, the Low group was administered at a dose of 10 mg/kg/ plexus at 10, 15, 30, 60, 120, 240, 360, 480, 600, and 1440 min
day, and the High group was administered at a dose of 20 mg/ into heparinized tubes. Blood samples were collected and placed
kg/day. in an ice bath in an EDTA-K2 anticoagulant EP tube,
Reserpine-induced zebrafish depression model was to expose centrifuged at 8000 rpm for 5 min at 4 °C, and the plasma
zebrafish to 10 mg/L reserpine solution for 40 min. According to was transferred to −20 °C as soon as possible for storage for
our previous results, Flu and I14 were given 24 h later. testing. The established LC−MS/MS method was used to
5.7.3. CUMS Model. The CUMS paradigm on mice was determine the concentration of compounds in the collected
adapted from past research. In total, 11 different stressors were plasma samples. WinNonlin 5.2 was used to calculate the
presented randomly twice a day for a total of 42 consecutive pharmacokinetic parameters.
days. Stressors include cold water swimming (5 min), room- 5.11. Statistical Analysis. All data are presented as mean ±
SD. The statistical analyses of the results were performed with
temperature swimming (5 min), tail suspension (15 min), food
one-way ANOVA and t-test, and for comparing all pairs of
deprivation (24 h), lack of water (24 h), cage tilt (30°, 24 h), wet
columns, P < 0.05, 0.01, 0.001, and 0.0001 were accepted as
litter (250 mL of water on the litter bed, 24 h), cage shaking (15
statistically significant.
min), inversion during the day and night (24 h), being restrained
(2 h), and clipped tail (2 cm from the tip of the tail, 2 min).
5.7.4. Behavior Test. The OFT, SPT, FST, and TST were
conducted as previously described. Spatial exploration behavior
■
*
ASSOCIATED CONTENT
sı Supporting Information

in mice was tested by the OFT, and the rearing and dejection The Supporting Information is available free of charge at
amounts were measured during the 5 min session. The SPT https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01271.
means the ratio of consumed 1% sucrose solution relative to that Structural characterization of compounds I1‑15 (1H NMR,
of total solution in the test and was used as a measure of 13
C NMR, and ESI/HRMS spectra), HPLC analysis of
anhedonia in mice. The duration of the FST and TST was 6 min, target compounds I1−15, and IC50 values of I1‑15 for nerve
and the immobility time was recorded during the last 4 min. The cell lines and human liver cells (PDF)
OFT and NTT were used to assess the depression response in SMILES molecular formula strings (CSV)
experimental zebrafish. Noldus software and SONY SSC-
DC578P camera were used for the automatic tracking of Binding mode of compound I14 with 5-HT2AR (PDB
zebrafish behavior. 6WGT) (PDB)
5.8. HE Staining. The embedded tissue wax blocks were Binding mode of compound I14 with MAO-A (PDB
sliced with a thickness of 4 μm, adhered to the slides, and dried at 2Z5X) (PDB)
45 °C. Tissue sections were dewaxed with xylene. After soaking
and staining with hematoxylin for 5 min, rinsing was carried out
with distilled water. Then, 75% hydrochloric acid ethanol
differentiated for 30 s; rinsed with distilled water for 10 min;
■ AUTHOR INFORMATION
Corresponding Authors
eosin staining for 2 min; dehydration; neutral resin sealing; Li Chen − State Key Laboratory of Natural Medicines,
Department of Natural Medicinal Chemistry, School of
microscope observation and photography; and reagents were
Traditional Chinese Pharmacy, China Pharmaceutical
purchased from Shenggong Biological Engineering (Shanghai)
University, Nanjing 210009, China; orcid.org/0000-
Co., Ltd., Shanghai, China.
0002-4655-9024; Phone: +86 25 83271447;
5.9. Western Blot. The proteins were obtained from mice
Email: [email protected]
brain that had been taken from different treatment groups. The
Jianbo Sun − State Key Laboratory of Natural Medicines,
proteins were quantified according to the BCA protein assay kit Department of Natural Medicinal Chemistry, School of
(Beyotime) and diluted to 3 mg/mL. Each protein sample (10 Traditional Chinese Pharmacy, China Pharmaceutical
μL) was separated by electrophoresis using SDS-PAGE (4−20% University, Nanjing 210009, China; Phone: +86 25
gel, Beyotime), and then transferred to the membrane, blocked, 83271415; Email: [email protected]
with specific binding with the primary antibody conducted
under 4 °C overnight, and later incubated with a secondary Authors
antibody at room temperature for 1 h. Finally, the membrane Xiaona Sun − State Key Laboratory of Natural Medicines,
was imaged using a Tianneng gel imaging system (Tanon-5200). Department of Natural Medicinal Chemistry, School of
The results were generated using Image J software (NIH, Traditional Chinese Pharmacy, China Pharmaceutical
Bethesda, MD, USA). University, Nanjing 210009, China
13398 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Na Li − State Key Laboratory of Natural Medicines, Department oxoisoaporphine inhibitors for MAO-A and human parasite proteins.
of Natural Medicinal Chemistry, School of Traditional Chinese Eur. J. Med. Chem. 2011, 46, 5838−5851.
Pharmacy, China Pharmaceutical University, Nanjing (5) Shelton, R. C.; Sanders-Bush, E.; Manier, D. H.; Lewis, D. A.
210009, China Elevated 5-HT 2A receptors in postmortem prefrontal cortex in major
Peisen Zhong − State Key Laboratory of Natural Medicines, depression is associated with reduced activity of protein kinase A.
Department of Natural Medicinal Chemistry, School of Neuroscience 2009, 158, 1406−1415.
(6) Barnes, N. M.; Ahern, G. P.; Becamel, C.; Bockaert, J.; Camilleri,
Traditional Chinese Pharmacy, China Pharmaceutical
M.; Chaumont-Dubel, S.; Claeysen, S.; Cunningham, K. A.; Fone, K.
University, Nanjing 210009, China
C.; Gershon, M.; Di Giovanni, G.; Goodfellow, N. M.; Halberstadt, A.
Complete contact information is available at: L.; Hartley, R. M.; Hassaine, G.; Herrick-Davis, K.; Hovius, R.; Lacivita,
https://pubs.acs.org/10.1021/acs.jmedchem.2c01271 E.; Lambe, E. K.; Leopoldo, M.; Levy, F. O.; Lummis, S. C. R.; Marin,
P.; Maroteaux, L.; McCreary, A. C.; Nelson, D. L.; Neumaier, J. F.;
Author Contributions Newman-Tancredi, A.; Nury, H.; Roberts, A.; Roth, B. L.; Roumier, A.;
† Sanger, G. J.; Teitler, M.; Sharp, T.; Villalón, C. M.; Vogel, H.; Watts, S.
X.S., N.L. and P.Z. authors contributed equally.
W.; Hoyer, D. International union of basic and clinical pharmacology.
Author Contributions
CX. Classification of receptors for 5-hydroxytryptamine; pharmacology
J.S. and L.C. conceived the project. N.L. and X.S. synthesized all and function. Pharmacol. Rev. 2021, 73, 310−520.
compounds and analyzed the results. X.S. and P.Z. interpreted (7) Zhang, G.; Stackman, R. W., Jr The role of serotonin 5-HT2A
pharmacological data. X.S., N.L., and X.S. contributed equally to receptors in memory and cognition. Front. Pharmacol. 2015, 6, 225.
this work. All of the authors reviewed and contributed to the (8) Guiard, B. P.; Giovanni, G. Central serotonin-2A (5-HT2A)
manuscript. receptor dysfunction in depression and epilepsy: the missing link?
Notes Front. Pharmacol. 2015, 6, 46.
The authors declare no competing financial interest. (9) Jin, Z.; Ling, J.; Yu, J.; He, M.; Ni, P.; Zhang, F.; Wang, Y.
Serotonin 2A receptor function and depression-like behavior in rats

■ ACKNOWLEDGMENTS
This research has been supported by the National Natural
model of hypothyroidism. Exp. Brain Res. 2021, 239, 2435−2444.
(10) Arias, B.; Fabbri, C.; Gressier, F.; Serretti, A.; Mitjans, M.; Gastó,
C.; Catalán, R.; De Ronchi, D.; Fañanás, L. TPH1, MAOA, serotonin
Science Foundation of China (82173708) and the Six-talent receptor 2A and 2C genes in citalopram response: possible effect in
Peaks Project in Jiangsu Province (SWYY-071). melancholic and psychotic depression. Neuropsychobiology 2013, 67,

■ ABBREVIATIONS USED
AUC, area under the concentration−time curve; CL, mean
41−47.
(11) Casey, A. B.; Cui, M.; Booth, R. G.; Canal, C. E. “Selective”
serotonin 5-HT2A receptor antagonists. Biochem. Pharmacol. 2022,
200, 115028.
clearance, the plasma volume of the drug cleared per unit time; (12) Bang-Andersen, B.; Ruhland, T.; Jørgensen, M.; Smith, G.;
Cmax, peak plasma concentration of a drug after administration; Frederiksen, K.; Jensen, K. G.; Zhong, H.; Nielsen, S. M.; Hogg, S.;
CORT, corticosterone; CUMS, chronic unpredictable mild Mørk, A.; Stensbøl, T. B. Discovery of 1-[2-(2,4-
stress; DMF, N,N-dimethylformamide; DMPK, drug metabo- dimethylphenylsulfanyl)phenyl]piperazine (Lu AA21004): a novel
lism and pharmacokinetic; FST, forced swimming test; H&E, multimodal compound for the treatment of major depressive disorder.
hematoxylin&eosin; 5-HT, 5-hydroxytryptamine; 5-HT2AR, 5- J. Med. Chem. 2011, 54, 3206−3221.
hydroxytryptamine 2A receptor; MAO-A, monoamine oxidase (13) Seo, H. J.; Park, E. J.; Kim, M. J.; Kang, S. Y.; Lee, S. H.; Kim, H.
A; MTT, thiazolyl blue; MRT, mean residence time; NE, J.; Lee, K. N.; Jung, M. E.; Lee, M.; Kim, M. S.; Son, E. J.; Park, W. K.;
noradrenaline; NTT, novel tank test; OFT, open-field test; PBS, Kim, J.; Lee, J. Design and Synthesis of Novel Arylpiperazine
phosphate-buffered saline; PCR, polymerase chain reaction; Derivatives Containing the Imidazole Core Targeting 5-HT2A
rmsd, root-mean-square deviation; t1/2, time required for the Receptor and 5-HT Transporter. J. Med. Chem. 2011, 54, 6305−6318.
concentration to diminish by a half; Tmax, time to reach Cmax; (14) Ostrowska, K.; Młodzikowska, K.; Głuch-Lutwin, M.; Gryboś, A.;
TST, tail suspension test Siwek, A. Synthesis of a new series of aryl/heteroarylpiperazinyl
derivatives of 8-acetyl-7-hydroxy-4-methylcoumarin with low nano-
■ REFERENCES
(1) Meng, P.; Li, C.; Duan, S.; Ji, S.; Xu, Y.; Mao, Y.; Wang, H.; Tian, J.
molar 5-HT 1A affinities. Eur. J. Med. Chem. 2017, 137, 108−116.
(15) Zhang, J. Y.; Chen, L.; Sun, J. B. Oxoisoaporphine alkaloids:
prospective anti-alzheimer’s disease, anticancer, and antidepressant
Epigenetic mechanism of 5-HT/NE/DA triple reuptake inhibitor on agents. ChemMedChem 2018, 13, 1262−1274.
adult depression susceptibility in early stress mice. Front. Pharmacol. (16) Li, X.; Li, X.; Liu, F.; Li, S.; Shi, D. J. Rational multitargeted drug
2022, 13, 848251.
design strategy from the perspective of a medicinal chemist. J. Med.
(2) Garcia, C. S.; Besckow, E. M.; da Silva Espíndola, C. N.; D’Avila
Chem. 2021, 64, 10581−10605.
Nunes, G.; Zuge, N. P.; de Azeredo, M. P.; Rocha, M. J. d.; Carraro
(17) Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and
Junior, L. R.; Penteado, F.; Gomes, C. S.; Lenardão, E. J.; Bortolatto, C.
F.; Brüning, C. A. Antidepressant-like effect of a selenoindolizine in accuracy of docking with a new scoring function, efficient optimization,
mice: in vivo and in silico evidence for the involvement of the and multithreading. J. Comput. Chem. 2010, 31, 455−461.
serotonergic 5-HT2A/C receptors. ACS Chem. Neurosci. 2022, 13, (18) Drobot, V. V.; Kirilin, E. M.; Kopylov, K. E.; Š vedas, V. K.
1746−1755. PLUMED plugin integration into high performance pmemd program
(3) Matos, M. J.; Terán, C.; Pérez-Castillo, Y.; Uriarte, E.; Santana, L.; for enhanced molecular dynamics simulations. Supercomput. Front.
Viña, D. Synthesis and study of a series of 3-arylcoumarins as potent and Innovat. 2021, 8, 94−99.
selective monoamine oxidase B inhibitors. J. Med. Chem. 2011, 54, (19) Wu, Q.; Song, J.; Meng, D.; Chang, Q. TPPU, a sEH inhibitor,
7127−7137. attenuates corticosterone-induced PC12 cell injury by modulation of
(4) Prado-Prado, F.; García-Mera, X.; Escobar, M.; Sobarzo-Sánchez, BDNF-TrkB pathway. J. Mol. Neurosci. 2019, 67, 364−372.
E.; Yañez, M.; Riera-Fernandez, P.; González-Díaz, H. 2D MI- (20) Coghill, D. R.; Caballero, B.; Sorooshian, S.; Civil, R. A
DRAGON: A new predictor for protein-ligands interactions and systematic review of the safety of lisdexamfetamine dimesylate. CNS
theoretic-experimental studies of US FDA drug-target network, Drugs 2014, 28, 497−511.

13399 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

(21) Zhang, R.; Qiao, C.; Liu, Q.; He, J.; Lai, Y.; Shang, J.; Zhong, H. A
reliable high-throughput screening model for antidepressant. Int. J. Mol.
Sci. 2021, 22, 9505.
(22) Lachowicz, J.; Niedziałek, K.; Rostkowska, E.; Szopa, A.; Świąder,
K.; Szponar, J.; Serefko, A. Zebrafish as an animal model for testing
agents with antidepressant potential. Life 2021, 11, 792.
(23) Willner, P. J. The chronic mild stress (CMS) model of
depression: History, evaluation and usage. Neurobiol. Stress 2017, 6,
78−93.
(24) Antoniuk, S.; Bijata, M.; Ponimaskin, E.; Wlodarczyk, J. Chronic
unpredictable mild stress for modeling depression in rodents: Meta-
analysis of model reliability. Neurosci. Biobehav. Rev. 2019, 99, 101−
116.

Recommended by ACS
Discovery of CVN636: A Highly Potent, Selective, and CNS
Penetrant mGluR7 Allosteric Agonist
Louise Dickson, Roland W. Bürli, et al.
MARCH 02, 2023
ACS MEDICINAL CHEMISTRY LETTERS READ

Mechanistic Insight into the Inhibition of Choline

Acetyltransferase by Proton Pump Inhibitors
Anurag T. K. Baidya, Rajnish Kumar, et al.
FEBRUARY 07, 2023
ACS CHEMICAL NEUROSCIENCE READ

SKF83959 Attenuates Memory Impairment and Depressive-

like Behavior during the Latent Period of Epilepsy via
Allosteric Activation of the Sigma-1 Receptor
Lin Guo, Yun Wang, et al.
NOVEMBER 04, 2022
ACS CHEMICAL NEUROSCIENCE READ

Discovery of Potent Cholinesterase Inhibition-Based Multi-

Target-Directed Lead Compounds for Synaptoprotection in
Alzheimer’s Disease
Bengisu Turgutalp, Mine Yarim, et al.
SEPTEMBER 09, 2022
JOURNAL OF MEDICINAL CHEMISTRY READ

Get More Suggestions >

13400 https://doi.org/10.1021/acs.jmedchem.2c01271
J. Med. Chem. 2022, 65, 13385−13400