Design, synthesis and evaluation of vilazodone-tacrine hybrids as multitarget-directed ligands against depression with cognitive impairment

Article abstract of DOI:10.1016/j.bmc.2018.04.037

Depression, a severe mental disease, is greatly difficult to treat and easy to induce other neuropsychiatric symptoms, the most frequent one is cognitive impairment. In this study, a series of novel vilazodone-tacrine hybrids were designed, synthesized and evaluated as multitarget agents against depression with cognitive impairment. Most compounds exhibited good multitarget activities and appropriate blood-brain barrier permeability. Specifically, compounds 1d and 2a exhibited excellent 5-HT_1A agonist activities (1d, EC₅₀ = 0.36 ± 0.08 nM; 2a, EC₅₀ = 0.58 ± 0.14 nM) and 5-HT reuptake inhibitory activities (1d, IC₅₀ = 20.42 ± 6.60 nM; 2a, IC₅₀ = 22.10 ± 5.80 nM). In addition, they showed moderate ChE inhibitory activities (1d, AChE IC₅₀ = 1.72 ± 0.217 μM, BuChE IC₅₀ = 0.34 ± 0.03 μM; 2a, AChE IC₅₀ = 2.36 ± 0.34 μM, BuChE IC₅₀ = 0.10 ± 0.01 μM). Good multitarget activities with goodt blood-brain barrier permeability of 1d and 2a make them good lead compounds for the further study of depression with cognitive impairment.

Full text of DOI:10.1016/j.bmc.2018.04.037

Accepted Manuscript
Design, synthesis and evaluation of vilazodone-tacrine hybrids as multitarget-
directed ligands against depression with cognitive impairment
Wenwen Liu, Huan Wang, Xiaokang Li, Yixiang Xu, Jian Zhang, Wei Wang,
Qi Gong, Xiaoxia Qiu, Jin Zhu, Fei Mao, Haiyan Zhang, Jian Li
PII:
S0968-0896(18)30236-0
https://doi.org/10.1016/j.bmc.2018.04.037
BMC 14320
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 February 2018
8 April 2018
16 April 2018
Please cite this article as: Liu, W., Wang, H., Li, X., Xu, Y., Zhang, J., Wang, W., Gong, Q., Qiu, X., Zhu, J., Mao,
F., Zhang, H., Li, J., Design, synthesis and evaluation of vilazodone-tacrine hybrids as multitarget-directed ligands
against depression with cognitive impairment, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.04.037
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Graphical Abstract
Design, synthesis and evaluation of vilazodone-tacrine hybrids as
multitarget-directed ligands against depression with cognitive
impairment
Wenwen Liua,†, Huan Wangb,d,†, Xiaokang Lia,†, Yixiang Xua, Jian Zhangb, Wei Wangb, Qi Gongb,
Xiaoxia Qiua, Jin Zhua, Fei Maoa,*, Haiyan Zhangb,c,*, Jian Lia,*
a
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of
Science and Technology, 130 Mei Long Road, Shanghai 200237, China
b CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, University of Chinese
Academy of Sciences, Chinese Academy of Sciences, Shanghai 201203, China
d University of Chinese Academy of Science, No.19A Yuquan Road, Beijing 100049, China

Bioorganic & Medicinal Chemistry
journal h omepage: www.els evier. com
Design, synthesis and evaluation of vilazodone-tacrine hybrids as multitarget-directed
ligands against depression with cognitive impairment
Wenwen Liua,†, Huan Wangb,d,†, Xiaokang Lia,†, Yixiang Xua, Jian Zhangb, Wei Wangb, Qi Gongb, Xiaoxia
Qiua, Jin Zhua, Fei Maoa,*, Haiyan Zhangb,c,*, Jian Lia,*
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai
200237, China
b CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203,
China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences, Chinese Academy of Sciences,
Shanghai 201203, China
d University of Chinese Academy of Science, No.19A Yuquan Road, Beijing 100049, China
* To whom correspondence should be addressed. jianli@ecust.edu.cn, hzhang@simm.ac.cn, maofei@ecust.edu.cn.
† These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Depression, a severe mental disease, is greatly difficult to treat and easy to induce other neuropsychiatric symptoms,
the most frequent one is cognitive impairment. In this study, a series of novel vilazodone-tacrine hybrids were
designed, synthesized and evaluated as multitarget agents against depression with cognitive impairment. Most
compounds exhibited good multitarget activities and appropriate blood-brain barrier permeability. Specifically,
compounds 1d and 2a exhibited excellent 5-HT1A agonist activities (1d, EC50 = 0.36 ± 0.08 nM; 2a, EC50 = 0.58 ±
0.14 nM) and 5-HT reuptake inhibitory activities (1d, IC50 = 20.42 ± 6.60 nM; 2a, IC50 = 22.10 ± 5.80 nM). In
addition, they showed moderate ChE inhibitory activities (1d, AChE IC50 = 1.72 ± 0.217 μM, BuChE IC50 = 0.34 ±
0.03 μM; 2a, AChE IC50 = 2.36 ± 0.34 μM, BuChE IC50 = 0.10 ± 0.01 μM). Good multitarget activities with goodt
blood-brain barrier permeability of 1d and 2a make them good lead compounds for the further study of depression
with cognitive impairment.
Available online
Keywords:
Depression
Cognitive impairment
Multitarget-directed ligand
5-HT1A agonist
2018 Elsevier Ltd. All rights reserved.
5-HT reuptake inhibition
ChE inhibition
1. Introduction
concentration, information processing and memory impairment,
executive dysfunction.12-15 Antidepressants exhibited deficiency
Depression, a common, severe mental disorder, is the primary
cause of disability, suicide, diminished productivity and
dependent care, with approximate 350 million people affected
worldwide.1 It is also a chronic and recurrent affective disease
with multifarious etiology and symptoms.2-4 Depressed people
always suffer from weak social skills, chronic stress, low
emotion, decreased energy and cognitive impairment.5 Currently,
most antidepressants take effect by noradrenergic-serotonergic
transmitter systems or inhibiting monoamine oxidase (MAO) to
reduce the degradation of serotonin (5-HT) and norepinephrine
(NE).6 It has been proved that the co-morbidity with other
in cognitive impairment despite of the successful depression
remission. Also, depressive patients with concurrent cognitive
impairment, in particular cognitive impairement, undergo a
lower antidepressant therapy response and higher recurrence.16,17
Besides, depressive patients with cognition decline are a high
risk group of Alzheimer’s disease (AD) and it also brings
negative consequences in patients and heavy burden in
caregivers.18-21 Such impaired cognition and memory processes
in depressive disorder may attribute to the dysfunction of the
cholinergic system and it could be alleviated by
acetylcholinesterase inhibitors (AChEIs).22-25 AChEIs, reducing
the hydrolysis of acetylcholine, are commonly accepted as first-
neuro-affected diseases is
a significant characteristic of
depression.7 Along with the in-depth research of depression,
antidepressant drugs based on different strategies have been
conducted.8-11
line drugs in treatment of moderate cognitive impairment.26-28
A
study has indicated that donepezil, a cholinesterase inhibitor,
improved cognition in depressed people and may delay
conversion to a diagnosis of AD.29 Therefore, therapeutic
strategies which target at cholinesterase may improve cognitive
It is recognized that depression, especially late-onset
depression, is associated with cognitive impairment, such as poor

function in depressive people and prevent depression from
recurrencing by improving executive function.28,30
We had obtained several vilazodone derivatives in previous work,
but their AChE inhibitory activities were not significantly
ehnhanced. Alternatively, pharmacophore grafting is also
considered as an efficient method to enhance AChE inhibitory
potency. Hence, grafting an AChE inhibitory pharmacophore of
known AChE inhibitors (AChEIs) into vilazodone was employed
in the current study. In view of the simple structure and
structure-modifying accessibility, tacrine was selected as the
grafting pharmacophore of AChEI.
Above all, aiming at complexed pathological mechanisms
of neuropsychiatric and neurodegenerative diseases, design of
multi-target ligands is a promising research direction.31 Several
studies have been successful in developing multi-target ligands
which acted on central nervous system (CNS), and multi-target
ligands could exhibit multiple pharmacological activities in
vivo.32,33 It is therefore understandable that the attempts to
develop a kind of drug candidate which can alleviate depressive
symptoms and cognitive impairment simultaneously based on
the multi-target design strategy have been in pressing need. Our
group devoted to the research of drug repurposing and the
secondary development of conventional drugs. Vilazodone, an
According to structure-activity relationships (SARs) of
vilazodone, the benzofuran-2-carboxamide fragment is an
appropriate site for structural modification and can tolerate a
diversity of substituents.44,45 Therefore, benzofuran-2-
carboxamide fragment of vilazodone can be substituted by
tacrine derivatives. In previous work of our group, Li et al46 have
designed and synthesized the first class of vilazodone-tacrine
hybrids targeting at ChE, 5-HT1A and 5-HT transport. In his
design strategy, piperazine ring of vilazodone was linked to
amino of tacrine. Although his design improved ChE inhibitory
activity of vilazodone, the antidepressive activity was decreased
severely. In order to enhance ChE inhibitory activity while the 5-
HT1A agonist and 5-HT reuptake inhibitory activities are
remained, a novel design strategy was conducted: piperazine ring
of vilazodone was linked to benzene ring of tacrine (Figure 1).
We aim at developing a series multifunctional compounds with
optimal antidepressive activity and adjuvant cognition promotion.
antidepressant,
was
found
possessing
moderate
acetylcholinesterase (AChE) inhibitory activity (IC50 = 21.3 ±
3.0 μM) in a high throughput screening (HTS) test with an in-
house old drug library. As reported, vilazodone is a selective
serotonin reuptake inhibitor (SSRI) and 5-HT1A receptor
agonist,34 which can increase serotonin concentration of synaptic
cleft. Serotonin, a significant neurotransmitter existing in the
brain extensively, plays a pivotal role in normal brain
function.35,36 5-HT1A receptor agonists play a significant role in
the therapeutics of major depressive disorder (MDD).37-40
Moreover, SSRI, could increase the amount of available synaptic
5-HT, also were a remarkable characteristic of antidepressants.41-
43
Based on the strategy mentioned above, a series of novel
vilazodone-tacrine hybrids were designed, synthesized and
evaluated in this article. Their AChE, BuChE, 5-HT reuptake
inhibitory activities and 5-HT1A agonist activity were evaluated
in vitro. Besides, the parallel artificial membrane permeability
assay (PAMPA) was performed to provide preliminary
predictions of the blood-brain barrier (BBB) penetration,
expecting to provide evidence for optimized selection.
In order to meet our goal in developing novel drug
candidate which can alleviate depressive symptoms and
cognitive impairment simultaneously, we attempt to enhance the
AChE inhibitory potency of vilazodone while keeping its effect
on 5-HT system by two molecular design strategies. Firstly,
traditional medicinal chemistry structural modification strategy
was employed to modify the chemical structure of vilazodone.
Figure 1. Design strategy of novel vilazodone-tacrine hybrids.
2. Results and discussion
1,2,3,4-tetrahydroacridine in four different points, was depicted
in 7a-d.
2.1. Chemistry
In Scheme 3, derivatives were synthesized from indole
intermediate and different tacrine derivatives respectively. The
synthesis of key intermediates 6b-k and 7a-g were described in
supporting information. The detailed structures of compounds
The synthesis of the target compounds was accomplished
according to the route shown in Schemes 1-3. In Scheme 1-2,
intermediate 6a (commercial available) and 6b reacted with
tacrine derivatives respectively in the presence of Et3N to obtain
1a-h. In tacrine derivatives, piperazine connected to 9-amino-

were illustrated in Table 1, and the synthesis of intermediates
were showed in supporting information.
Scheme 1. Synthesis of compounds 1a-d. Reagents and conditions: a) MeCN, KI, Et3N, reflux, 36h.
Scheme 2. Synthesis of compounds 1e-g. Reagents and conditions: a) NaBH3CN, MeOH:C2H4Cl2 = 1:1 (v:v), rt, 12 h.
Scheme 3. Synthesis of compounds 2a-b, 3a,4a-b, 5a-g. Reagents and conditions: a) MeCN, KI, Et3N, reflux, 36h.
2.2. 5-HT Agonist Activity
naphthenic ring in tacrine, the length of carbon chain and
substituent in amino of tacrine. Test results explain that 5-HT1A
agonistic activity is related with the size of naphthenic ring:
In order to evaluate the antidepressant-like activity of newly
designed and synthesized vilazodone-tacrine hybrids, 5HT1A
agonism assay was conducted by using Human Embryonic
Kidney 293 (HEK293) cells expressing correlated human 5-
HT1A receptors. The results, together with vilazodone and 8-OH-
DPAT as references, are expressed as half-maximal effective
concentration (EC50) values (Table 1).
cycloheptane cycle (2b)
< cyclopentane cycle (2a) <
cyclohexanone cycle (1d). Derivatives with short carbon chain
show poor activities (4a and 4b) and derivative with substituent
in amino (3a) also displays common activity.
Besides, series 5 were prepared for studying 5-substituent on
indole. Derivatives 5a-g were newly synthesized in order to
improve both ChE inhibition activity and 5-HT1A agonist
activity. In biological assay, although this series of compounds
display moderate ChE inhibitory activities, a large part of them
show excellent 5-HT1A agonist activities (IC50  1 nM). Among
them, compounds 1d, 2a, 5a and 5f exhibit great potency at 5-
HT1A agonist activity. Structures, 5-HT1A agonist activities, ChE
To judge the optimal chain position and linker type of
vilazodone fragments, derivatives 1a-h were provided to the
biological assay. From results of 5-HT1A agonistic activity,
piperazinyl on the position 4 is the optimal mode. Compound 1d
(EC50 = 0.36 ± 0.079 nM) turned out to be a potential derivative.
Considering that the optimal chain position is 4, derivatives 2a,
2b, 3a, 4a and 4b were tested focusing on different size of

inhibitory activities of compounds 1a-h, 2a-b, 3a, 4a-b, 5a-g
was displayed in Table 1.
Table 1. Structures, 5-HT1A agonist activities, ChE inhibitory activities of compounds 1a–h, 2a–b, 3a, 4a-b, 5a-g.
EC50 ± SDa (nM)
for 5HT1A agonist
activity
IC50 ± SDb (μM)
for mAChE
inhibitory activity
IC50 ± SDb (μM)
for mBuChE
inhibitory activity
Chain
position
Compd
Linker
R1
R2
n
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
COOCH3
F
H
H
2
2
1
2
1.55 ± 0.17
28.80 ± 1.76
14.93 ± 0.29
0.36 ± 0.079
34.98 ± 5.60
567.00 ± 64.00
1563.21 ± 324.30
62.43 ± 4.31
0.58 ± 0.14
146.01 ± 36.00
93.37 ± 15.20
>10000
2.33 ± 0.35
5.10 ± 0.88
10.24 ± 1.57
1.72 ± 0.22
3.31 ± 0.22
1.98 ± 0.26
2.91 ± 0.32
1.36 ± 0.07
2.36 ± 0.34
18.30 ± 1.46
5.61 ± 1.35
2.10 ± 0.23
1.82 ± 0.20
2.22 ± 0.27
NSc
0.31 ± 0.02
0.25 ± 0.02
0.64 ± 0.13
0.34 ± 0.03
1.85 ± 0.26
0.37 ± 0.03
0.25 ± 0.02
0.39 ± 0.02
0.10 ± 0.01
0.23 ± 0.02
2.92±0.18
1.13 ± 0.07
0.42 ± 0.02
0.41 ±0.03
NSc
1a
1b
H
2
3
1c
H
2
4
1d
H
2
1
1e
H
2
2
1f
H
2
3
1g
H
2
4
1h
H
1
4
2a
H
3
4
2b
CH3CO
H
2
4
3a
2
4
4a
H
2
4
52.00 ± 0.13
< 0.14
4b
H
2
4
5a
H
2
4
0.72 ± 0.03
0.75 ± 0.05
0.35 ± 0.09
1.20 ± 0.21
< 0.14
5b
Cl
H
2
4
NSc
NSc
5c
Br
H
2
4
NSc
NSc
5d
5e
H
H
2
4
1.44 ± 0.17
1.62 ± 0.29
1.12 ± 0.13
21.30 ± 3.00
0.13 ± 0.02
0.39 ± 0.04
0.40 ±0.02
0.36 ± 0.06
NTd
OCH3
CH3
—
H
2
4
5f
H
2
4
0.60 ± 0.01
0.12 ± 0.01
NTd
5g
—
—
—
—
—
—
—
—
Vilazodone
Tacrine
—
0.08 ± 0.01

—
—
—
—
—
NTd
0.12 ± 0.02
36.4 ± 1.838
Hup A
—Not applicable.
aResults are expressed as the mean of at least two experiments.
bResults are expressed as the mean of at least three experiments.
cNot soluble at the given concentration.
dNo test.
2.3. ChE Inhibitory Activity
compounds show good activities at nanomolar. From series 1,
we cann’t find obvious structure activity relationship (SAR) in
BuChE inhibitory activities. In series 2, compound 2a
(cyclopentane ring, IC50 = 0.10 ± 0.01 μM) shows good BuChE
inhibitory activity than 1d (cyclohexane ring) and 2b
(cycloheptane ring). Then, in series 3, 4 and 5, BuChE inhibitory
activities don’t exhibit any improvement. In general, compound
1d and 2a showed better inhibitory activities in AChE and
BuChE. According to the experimental data of ChE inhibitory
activity, compared with vilazodone, derivatives have showed
improved activity.
To evaluate the ChE inhibitory activities of the newly
synthesized vilazodone-tacrine hybrids against cognitive
impairment in depressive patients, the ChE inhibitory activities
were tested through the method of Ellman et al47 by using AChE
from mice cortex and BuChE from mice serum. Tacrine and
huperzine A (Hup A) were used as reference. Results are
presented as half-maximal inhibitory concentration (IC50) values
(Table 1).
From the whole test results as showed in Table 1, most of
these compounds have moderate AChE inhibitory activities and
considerable BuChE inhibitory activities. The IC50 values for
mAChE inhibitory activity ranged from 1.12 μM to 18.30 μM. In
initial stage, we changed different chain positions of vilazodone-
like indolebutylamine fragments, and then we found that the
optimal position is 4 (derivatives 1d and 1h). Considering
balanced AChE inhibitory activity and 5-HT1A agonist activity,
derivative 1d (IC50 = 1.72 ± 0.22 μM) is regarded as the
relatively optimal compound in the first round. Next, the change
of naphthenic ring in tacrine, the length of carbon chain and
substituent in amino of tacrine were conducted. Derivatives 2a-
4b do not show any improvements on AChE inhibitory activity.
Especially, activities of derivatives 2b declined to a great extent
and the substituted one (3a) by acetyl also shows weak activity.
In series 5, derivatives 5a-5g were synthesized aiming at
improving AChE inhibitory activities. However, derivatives 5b,
5c and 5d do not soluble at the given concentration. Derivatives
5e (IC50 = 1.44 ± 0.17 μM) and 5g (IC50 = 1.12 ± 0.13 μM)
showed slight better activities than 1d.
2.4. 5-HT Reuptake Inhibitory Activity
In order to evaluate whether these hybrids can inhibit the
process of 5-HT transporter reuptake and show antidepressant
function, 5-HT transporter reuptake inhibition (RUI) assay was
conducted. Experiment were performed by using HEK293 cell
line stably expressing the serotonin transporter protein,
vilazodone and citalopram were used as control simultaneously.
Considering the coordination of AChE inhibitory activity,
BuChE inhibitory activity and 5-HT1A agonist activity,
compounds 1d, 2a, 5a and 5f were selected to be tested in this
assay (Table 2). From the results, we can find that different
substituents in indole significantly influence the 5-HT reuptake
activities. 5-HT reuptake inhibitory activities are better when the
substituent is cyano group (1d and 2a) than ester group (5a) and
methoxy group (5f). Then, Compounds 1d (cyclohexane ring)
and 2a (cyclopentane ring) displayed almost equally good
activities. In summary, this finding explains that compounds 1d
and 2a perform well not only in 5-HT1A agonist activity and ChE
inhibitory activity but also 5-HT reuptake inhibitory activity.
The IC50 values of these hybrids for mBuChE inhibition
ranged from 0.10 ± 0.01 μM to 2.92 ± 0.18 μM. Most of these
Table 2. The 5-HT reuptake inhibitory activities of selected compounds.
IC50 ± SD (nM)
IC50 ± SD (nM)
for RUI
Compd
for RUI
Compd
20.42 ± 6.60
22.10 ± 5.80
229.30 ± 13.01
0.40 ± 0.10
1d
2a
5a
5f
Vilazodone
Citalopram
314.10 ± 77.00
6.27 ± 4.51
2.5. In Vitro Blood-Brain Barrier Permeation Assay
PBS/ethanol (70:30) through a brain lipid porcine membrane.
From the results (Table 3), we can find that most of the hybrids
(Pe > 3.08 × 10-6 cm s-1, CNS+) show the capacity of crossing the
BBB. Especially, when the chain position of vilazodone-like
indolebutylamine fragments is 4, they almost show good
permeability. Therefore, Pe values of the tested compounds can
be considered as reliable references in the screening process of
potential drug candidates.
Both anti-depression and anti-AD drugs which react on
therapeutic targets in the brain, need to cross the blood-brain
barrier. Considering the property of our derivatives, a parallel
artificial membrane permeability assay was conducted to predict
the penetration of vilazodone-tacrine hybrids preliminarily. The
assay method was conducted according to Di et al.48 In vitro
permeability (Pe) of derivatives 1a-5g was tested dissolving in
Table 3. Prediction of BBB penetration of compounds 1a–h, 2a–b, 3a, 4a-b, 5a-g.

Compd
Pe (10-6cm s-1)a
Predictionb
Compd
Pe (10-6 cm s-1)a
Predictionb
1a
1b
1c
1d
1e
1f
4.43 ± 0.56
2.99 ± 0.62
3.55 ± 0.32
5.11 ± 0.47
1.61 ± 0.38
1.68 ± 0.54
1.04 ± 0.58
1.33 ± 0.55
4.38 ± 0.41
3.97 ± 0.36
CNS+
CNS±
CNS+
CNS+
CNS±
CNS±
CNS−
CNS±
CNS+
CNS+
3a
4a
4b
5a
5b
5c
5d
5e
5f
1.92 ± 0.09
4.48 ± 0.07
5.46 ± 0.11
6.22 ± 0.46
3.76 ± 0.37
2.74 ± 0.29
3.05 ± 0.45
3.52 ± 0.45
4.53 ± 0.44
3.64 ± 0.40
CNS±
CNS+
CNS+
CNS+
CNS+
CNS±
CNS±
CNS+
CNS+
CNS+
1g
1h
2a
2b
5g
aValues are expressed as the mean ± SD of at least three independent experiments.
bCompounds with permeabilities Pe > 3.08 × 10-6 cm s-1 could cross the BBB by passive diffusion (CNS+), Pe < 1.13 × 10-6 cm s-1 could not cross the BBB
(CNS−), 1.13 × 10-6 cm s-1< Pe < 3.08 × 10-6 cm s-1 show uncertain BBB permeation (CNS±).
3. Conclusion
By a rational design strategy, a series of multifunctional
conducted on an Agilent 1100 with a quaternary pump and
diode-array detector (DAD). The peak purity was verified by UV
spectra. All final compounds showed ≥ 95% purity. (Table S1,
supporting information).
vilazodone-tacrine hybrids against depression and cognitive
impairment were designed, synthesized, and evaluated in vitro.
Most of derivatives exhibited considerable inhibitory activities
toward AChE and BuChE. Besides, most of them showed great
5-HT agonist activity, 5-HT reuptake inhibitory activity and
excellent blood-brain barrier permeability. Compounds 1d and
2a have a similar structure, being evaluated to be the most
efficient ones. The two compounds exhibited remarkable 5-HT
reuptake inhibitory activities and 5-HT agonist activities. 2a
shows stronger BuChE inhibitory activity than 1d, and the two
compounds have almost the same inhibition efficacy in AChE. In
blood-brain barrier permeation assay, which was considered as
reliable references in the screening process of potential drug
candidates, 1d and 2a all performed good penetration of BBB.
Overall, vilazodone-tacrine hybrids provide a novel thought for
drug discovery on depression with cognitive impairment and are
worthy to be further explored.
4.1.1.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-4-
yl)piperazin-1-yl)butyl)-1H-indole-5-carbonitrile (1a)
A mixture of 3-(4-chlorobutyl)-1H-indole-5-carbonitrile
(6a, 116 mg, 0.5 mmol), corresponding intermediates 7a (0.5
mmol) and KI (0.05 mmol) was added Et3N (1 mmol). The
reaction mixture was stirred at reflux temperature for 24 h. Then
removal the solvent under vacuum and the residue was purified
by column chromatography (CH3OH:CH2Cl2 = 1:20 to 1:10, v:v)
to afford compound 1a. Compound 1a was isolated as a yellow
powder, 41% yield, mp 214–216 °C. 1H NMR (400 MHz,
CD3OD) δ: 8.19–8.10 (m, 1H), 8.02 (s, 1H), 7.77 (d, J = 7.4 Hz,
1H), 7.64–7.56 (m, 1H), 7.50–7.44 (m, 1H), 7.40–7.34 (m, 1H),
7.29–7.24 (m, 1H), 3.27–2.96 (m, 8H), 2.86 (t, J = 12.7 Hz, 4H),
2.75–2.44 (m, 4H), 2.21–1.96 (m, 4H), 1.96–1.86 (m, 2H), 1.83–
1.78 (m, 2H). HRMS (EI) m/z calcd C30H33N5 (M+) 463.2736,
found 463.2737.
4. Material and Methods
4.1.2.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-3-
4.1. Chemistry
yl)piperazin-1-yl)butyl)-1H-indole-5-carbonitrile (1b)
Reagents and solvents were purchased from J&K Scientific,
Energy Chemical, Macklin, Bide Pharmatech Ltd, Adamas-beta
Target Molecule, TCI, Alfa Aesar and Acros, and they were used
without further purification. Reactions were followed by thin-
layer chromatography (TLC) on silica gel HSGF254 plates (0.2
± 0.03 mm thickness; Yantai JiangYou silicone development co.,
LTD, Yantai, China), and the spots were visualized with UV
lamp (λ = 254 nm). Yields were not optimized. Melting points
were measured in capillary tubes on a SGW X-4 melting point
apparatus without correction. Nuclear magnetic resonance
(NMR) spectroscopy was performed on a Bruker AMX-400
NMR. Chemical shifts were reported in parts per million (ppm,
δ) downfield from tetramethylsilane. Proton coupling patterns
were described as singlet (s), doublet (d), doublet of doublets
(dd), triplet (t), quartet (q), multiplet (m). High-resolution mass
spectra (HRMS) were obtained by electric ionization (EI) and
electrospray ionization (ESI) using a Waters GCT Premie and
Waters LCT. HPLC data analysis of compounds 1a-5g was
1b was synthesized by the general procedure of 1a, and 7a
was replaced by 7b. Compound 1b was isolated as a yellow
powder, 45% yield, mp 225–227 °C. 1H NMR (400 MHz,
CD3OD) δ: 8.14 (d, J = 9.5 Hz, 1H), 8.03 (d, J = 0.8 Hz, 1H),
7.51–7.47 (m, 1H), 7.40– 7.34 (m, 2H), 7.26 (s, 1H), 6.87 (d, J =
2.4 Hz, 1H), 3.58–3.45 (m, 4H), 2.99–2.73 (m, 8H), 2.71–2.62
(m, 2H), 2.59 (t, J = 5.6 Hz, 2H), 2.01– 1.92 (m, 4H), 1.85–1.77
(m, 2H), 1.76–1.67 (m, 2H). HRMS (EI) m/z calcd C30H34N6
(M+) 478.2845, found 478.2844.
4.1.3.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-2-
yl)piperazin-1-yl)butyl)-1H-indole-5-carbonitrile (1c)
1c was synthesized by the general procedure of 1a, and 7a
was replaced by 7c. Compound 1c was isolated as a yellow
powder, 39% yield, mp 208–210 °C. 1H NMR (400 MHz,
CD3OD) δ: 7.99 (d, J = 0.9 Hz, 1H), 7.70 (dd, J = 9.3, 2.4 Hz,
1H), 7.65 (s, 1H), 7.54 (d, J = 2.3 Hz, 1H), 7.46 (d, J = 8.4 Hz,

1H), 7.35 (dd, J = 8.5, 1.5 Hz, 1H), 7.26 (s, 1H), 3.64–3.40 (m,
4H), 3.25–3.01 (m, 4H), 3.01–2.88 (m, 4H), 2.85 (dd, J = 14.4,
7.7 Hz, 2H), 2.60 (t, J = 5.9 Hz, 2H), 2.03–1.86 (m, 4H), 1.84–
1.73 (m, 4H). HRMS (ESI) m/z calcd C30H34N6 [M+H]+
479.2923, found 479.2922.
1H), 3.08–2.83 (m, 8H), 2.70 (t, J = 10.3 Hz, 2H), 2.61–2.44 (m,
4H), 2.41–2.30 (m, 2H), 2.08–1.83 (m, 6H). HRMS (ESI) m/z
calcd C30H32N6O [M+H]+ 493.2716, found 493.2715.
4.1.9. 3-(4-(4-(9-Amino-2,3-dihydro-1H-cyclopenta[b]quinolin-
8-yl)piperazin-1-yl)butyl)-1H-indole-5-carbonitrile (2a)
4.1.4.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-1-
A mixture of 3-(4-chlorobutyl)-1H-indole-5-carbonitrile
(6a, 116 mg, 0.5 mmol), corresponding intermediates 7e (0.5
mmol) and KI (0.05 mmol) was added Et3N (1 mmol). The
reaction mixture was stirred at reflux temperature for 24 h. Then
removal the solvent under vacuum and the residue was purified
by column chromatography (CH3OH:CH2Cl2 = 1:20 to 1:10, v:v)
to afford compound 2a. Compound 2a was isolated as a yellow
powder, 40% yield, mp 170–171 °C. 1H NMR (400 MHz,
CD3OD) δ: 7.99 (d, J = 0.8 Hz, 1H), 7.81–7.74 (m, 1H), 7.54–
7.49 (m, 2H), 7.45 (dd, J = 8.5, 0.6 Hz, 1H), 7.37–7.32 (m, 1H),
7.24 (s, 1H), 3.20 (t, J = 7.8 Hz, 6H), 3.12–3.02 (m, 2H), 2.97–
2.91 (m, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.77–2.55 (m, 4H), 2.39–
2.26 (m, 2H), 1.83–1.75 (m, 2H), 1.74–1.63 (m, 2H). HRMS
(EI) m/z calcd C29H32N6 (M+) 464.2688, found 464.2689.
yl)piperazin-1-yl)butyl)-1H-indole-5-carbonitrile (1d)
1d was synthesized by the general procedure of 1a, and 7a
was replaced by 7d.Compound 1d was isolated as a yellow
powder, 48% yield, mp 202–205 °C.1H NMR (400 MHz,
CD3OD) δ: 7.96 (s, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.54–7.39 (m,
3H), 7.32 (d, J = 8.0 Hz, 1H), 7.22 (s, 1H), 3.16 (m, 4H), 3.03 (t,
J = 11.0 Hz, 2H), 2.92 (m, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.63 (m,
2H), 2.54 (m, 4H), 1.94 (m, 4H), 1.76 (m, 2H), 1.65 (m, 2H).
HRMS (ESI) m/z calcd C30H34N6 [M+H]+ 479.2923, found
479.2922.
4.1.5.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-4-
yl)piperazin-1-yl)butanoyl)-1H-indole-5-carbonitrile (1e)
A mixture of 3-(4-oxobutanoyl)-1H-indole-5-carbonitrile
(6b, 113 mg, 0.5 mmol) and intermediates 7a (0.5 mmol) in
CH3OH:C2H4Cl2 = 1:1 (v:v) was added NaBH3(CN) (62.8 mg, 1
mmol). The mixture was stirred at room temperature for 24 h,
and solvent was evaporated to dryness under reduced pressure.
The residue was purified on a silica gel column using mixtures
of CH3OH:CH2Cl2 as eluent, obtaining the target compounds 1e.
Compound 1e was isolated as a yellow powder, 50% yield, mp >
4.1.10.
3-(4-(4-(11-Amino-7,8,9,10-tetrahydro-6H-
cyclohepta[b]quinolin-1-yl)piperazin-1-yl)butyl)-1H-indole-5-
carbonitrile (2b)
2b was synthesized by the general procedure of 2a, and 7e
was replaced by 7f. Compound 2b was isolated as a yellow
powder, 45% yield, mp 181–183 °C. 1H NMR (400 MHz,
CD3OD) δ: 8.00 (s, 1H), 7.78 (t, J = 8.1 Hz, 1H), 7.52 (dd, J =
12.0, 7.8 Hz, 2H), 7.46 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 8.4, 1.4
Hz, 1H), 7.24 (s, 1H), 3.23–3.13 (m, 4H), 3.11–3.03 (m, 4H),
2.84 (t, J = 7.2 Hz, 4H), 2.72–2.50 (m, 4H), 1.99–1.89 (m, 2H),
1.85–1.75 (m, 4H), 1.73– 1.66 (m, 4H). HRMS (EI) m/z calcd
C31H36N6 (M+) 492.3001, found 492.3002.
1
300 °C. H NMR (400 MHz, CD3OD) δ: 8.16 (dd, J = 11.5, 6.7
Hz, 1H), 7.84 (t, J = 8.1 Hz, 1H), 7.67–7.54 (m, 2H), 7.48 (d, J =
7.4 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.09–6.99 (m, 1H), 3.47 (t,
J = 10.8 Hz, 4H), 3.30– 3.23 (m, 4H), 3.22–2.89 (m, 6H), 2.65 (t,
J = 5.1 Hz, 2H), 2.32–2.16 (m, 2H), 2.04–1.94 (m, 4H). HRMS
(ESI) m/z calcd C30H32N6O [M+H]+ 493.2716, found 493.2717.
4.1.11. N-(8-(4-(4-(5-cyano-1H-indol-3-yl)butyl)piperazin-1-yl)-
1,2,3,4-tetrahydroacridin-9-yl)acetamide (3a)
4.1.6.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-3-
yl)piperazin-1-yl)butanoyl)-1H-indole-5-carbonitrile (1f)
3a was synthesized by the general procedure of 2a, and 7e
was replaced by 7g. Compound 3a was isolated as a yellow
powder, 38% yield, mp 207–209 °C. 1H NMR (400 MHz,
CD3OD) δ: 8.01 (s, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.60 (t, J = 7.9
Hz, 1H), 7.45 (t, J = 6.9 Hz, 1H), 7.36 (dd, J = 8.5, 1.5 Hz, 2H),
7.25 (s, 1H), 3.24–3.16 (m, 6H), 3.07 (t, J = 6.1 Hz, 2H), 2.91–
2.76 (m, 6H), 2.30–2.20 (m, 2H), 2.01–1.94 (m, 2H), 1.87 (s,
3H), 1.83–1.72 (m, 4H), 1.64–1.48 (m, 2H). HRMS (EI) m/z
calcd C32H36N6O (M+) 520.2951, found 520.2949.
1f was synthesized by the general procedure of 1e, and 7a
was replaced by 7b. Compound 1f was isolated as a yellow
powder, 48% yield, mp > 300 °C. 1H NMR (400 MHz, CD3OD)
δ: 8.60 (s, 1H), 8.36 (s, 1H), 8.05 (d, J = 9.4 Hz, 1H), 7.57 (d, J
= 8.4 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H),
6.77 (s, 1H), 3.45 –3.34 (m, 4H), 3.01 (t, J = 6.6 Hz, 2H), 2.88
(m, 2H), 2.83–2.71 (m, 4H), 2.70–2.60 (m, 2H), 2.57–2.49 (m,
2H), 2.11–1.99 (m, 2H), 1.92 (m, 4H). HRMS (ESI) m/z calcd
C30H32N6O [M+H]+ 493.2716, found 493.2715.
4.1.12.
3-(2-(4-(9-Amino-5,6,7,8-tetrahydroacridin-1-
yl)piperazin-1-yl)ethyl)-1H-indole-5-carbonitrile (4a)
4.1.7.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-2-
yl)piperazin-1-yl)butanoyl)-1H-indole-5-carbonitrile (1g)
A mixture of 8-(piperazin-1-yl)-1,2,3,4-tetrahydroacridin-9-
amine (7d, 141 mg, 0.5 mmol), corresponding intermediates 6c
(0.5 mmol) and KI (0.05 mmol) was added Et3N (1 mmol). The
reaction mixture was stirred at reflux temperature for 24 h. Then
removal the solvent under vacuum and the residue was purified
by column chromatography (CH3OH:CH2Cl2 = 1:20 to 1:10, v:v)
to afford compound 4a. Compound 4a was isolated as a yellow
powder, 40% yield, mp 132–135 °C. 1H NMR (400 MHz,
CD3OD) δ: 8.08 (d, J = 16.1 Hz, 1H), 7.75 (t, J = 8.1 Hz, 1H),
7.56–7.44 (m, 3H), 7.40 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 3.29–
3.16 (m, 4H), 3.16–3.00 (m, 4H), 2.95 (t, J = 5.5 Hz, 2H), 2.86–
2.79 (m, 2H), 2.65–2.47 (m, 4H), 2.06– 1.86 (m, 4H). HRMS
(EI) m/z calcd C28H30N6 (M+) 450.2532, found 450.2533.
1g was synthesized by the general procedure of 1e, and 7a
was replaced by 7c. Compound 1g was isolated as a yellow
powder, 51% yield, mp > 300 °C. 1H NMR (400 MHz, CD3OD)
δ: 8.64 (s, 1H), 8.41 (s, 1H), 7.68–7.59 (m, 3H), 7.52–7.46 (m,
2H), 3.43–3.36 (m, 4H), 3.06 (t, J = 7.1 Hz, 2H), 3.01–2.87 (m,
6H), 2.82 –2.71 (m, 2H), 2.62 (t, J = 4.5 Hz, 2H), 2.15–2.06 (m,
2H), 2.00–1.95 (m, 4H). HRMS (ESI) m/z calcd C30H32N6O
[M+H]+ 493.2716, found 493.2715.
4.1.8.
3-(4-(4-(9-Amino-5,6,7,8-tetrahydroacridin-1-
yl)piperazin-1-yl)butanoyl)-1H-indole-5-carbonitrile (1h)
1h was synthesized by the general procedure of 1e, and 7a
was replaced by 7d. Compound 1h was isolated as a yellow
4.1.13.
3-(3-(4-(9-Amino-5,6,7,8-tetrahydroacridin-1-
1
powder, 45% yield, mp >300 °C. H NMR (400 MHz, CD3OD)
yl)piperazin-1-yl)propyl)-1H-indole-5-carbonitrile (4b)
δ: 8.61 (s, 1H), 8.37 (s, 1H), 7.61 (dd, J = 20.3, 7.6 Hz, 2H), 7.50
(d, J = 8.0 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.03 (d, J = 7.2 Hz,

4b was synthesized by the general procedure of 4a, and 6c
was replaced by 6d. Compound 4b was isolated as a yellow
powder, 42% yield, mp 94–96 °C. 1H NMR (400 MHz, CD3OD)
δ: 8.04 (s, 1H), 7.73 (t, J = 8.1 Hz, 1H), 7.54–7.43 (m, 3H), 7.37
(dd, J = 8.4, 1.3 Hz, 1H), 7.29–7.20 (m, 1H), 3.20–3.00 (m, 6H),
2.93 (t, J = 5.7 Hz, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.55 (t, J = 6.1
Hz, 4H), 2.40 (t, J = 10.7 Hz, 2H), 2.01–1.91 (m, 6H). HRMS
(EI) m/z calcd C29H32N6 (M+) 464.2688, found 464.2690.
2H), 7.47 (d, J = 7.7 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.05 (dd,
J = 14.7, 7.5 Hz, 2H), 6.98 (t, J = 7.4 Hz, 1H), 3.14 (d, J = 5.9
Hz, 4H), 3.05 (t, J = 10.9 Hz, 2H), 2.95 (t, J = 5.8 Hz, 2H), 2.82
(t, J = 7.1 Hz, 2H), 2.66–2.59 (m, 2H), 2.59–2.42 (m, 4H), 2.04–
1.89 (m, 4H), 1.84–1.77 (m, 2H), 1.72–1.63 (m, 2H). HRMS
(EI) m/z calcd C29H35N5 (M+) 453.2892, found 453.2893.
4.1.19. 8-(4-(4-(5-Methoxy-1H-indol-3-yl)butyl)piperazin-1-yl)-
1,2,3,4-tetrahydroacridin-9-amine (5f)
4.1.14. Methyl 3-(4-(4-(9-amino-5,6,7,8-tetrahydroacridin-1-
yl)piperazin-1-yl)butyl)-1H- indole-5-carboxylate (5a)
5b was synthesized by the general procedure of 4a, and 6c
was replaced by 6j. Compound 5f was isolated as a yellow
powder, 43% yield, mp 102–104 °C. 1H NMR (400 MHz,
CD3OD) δ: 7.76 (t, J = 8.1 Hz, 1H), 7.48 (dd, J = 19.5, 8.1 Hz,
2H), 7.19 (d, J = 8.7 Hz, 1H), 6.99 (s, 2H), 6.72 (dd, J = 8.7, 2.2
Hz, 1H), 3.80 (s, 3H), 3.13 (t, J = 10.3 Hz, 4H), 3.03 (t, J = 11.0
Hz, 2H), 2.93 (t, J = 5.6 Hz, 2H), 2.77 (t, J = 7.1 Hz, 2H), 2.64–
2.43 (m, 6H), 2.02–1.88 (m, 4H), 1.81–1.74 (m, 2H), 1.71–1.62
(m, 2H). HRMS (EI) m/z calcd C30H37N5O (M+) 483.2998, found
483.2999.
5a was synthesized by the general procedure of 4a, and 6c
was replaced by 6e. Compound 5a was isolated as a yellow
powder, 45% yield, mp 144–146 °C. 1H NMR (400 MHz,
CD3OD) δ: 8.31 (s, 1H), 7.76 (dd, J = 8.3, 5.2 Hz, 2H), 7.49 (dd,
J = 15.0, 8.1 Hz, 2H), 7.36 (d, J = 8.6 Hz, 1H), 7.14 (s, 1H), 3.89
(s, 3H), 3.21–3.09 (m, 4H), 3.05 (t, J = 11.1 Hz, 2H), 2.94 (t, J =
5.4 Hz, 2H), 2.85 (t, J = 7.1 Hz, 2H), 2.68–2.60 (m, 2H), 2.59–
2.44 (m, 4H), 2.02–1.89 (m, 4H), 1.86– 1.78 (m, 2H), 1.74–1.63
(m, 2H). HRMS (EI) m/z calcd C31H37N5O2 (M+) 511.2947,
found 511.2946.
4.1.20. 8-(4-(4-(5-Methyl-1H-indol-3-yl)butyl)piperazin-1-yl)-
1,2,3,4-tetrahydroacridin-9-amine (5g)
4.1.15. 8-(4-(4-(5-Fluoro-1H-indol-3-yl)butyl)piperazin-1-yl)-
1,2,3,4-tetrahydroacridin-9-amine (5b)
5g was synthesized by the general procedure of 4a, and 6c
was replaced by 6k. Compound 5g was isolated as a yellow
powder, 46% yield, mp 95–97 °C. 1H NMR (400 MHz, CD3OD)
δ: 7.76 (t, J = 8.1 Hz, 1H), 7.48 (dd, J = 22.3, 8.0 Hz, 2H), 7.29
(s, 1H), 7.18 (d, J = 8.3 Hz, 1H), 6.96 (s, 1H), 6.88 (d, J = 8.3
Hz, 1H), 3.22–3.11 (m, 4H), 3.04 (t, J = 10.9 Hz, 2H), 2.93 (t, J
= 5.8 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H), 2.68–2.61 (m, 2H), 2.60–
2.49 (m, 4H), 2.39 (s, 3H), 2.01–1.88 (m, 4H), 1.80–1.73 (m,
2H), 1.70–1.61 (m, 2H). HRMS (EI) m/z calcd C30H37N5 (M+)
467.3049, found 467.3050.
5b was synthesized by the general procedure of 4a, and 6c
was replaced by 6f. Compound 5b was isolated as a yellow
powder, 43% yield, mp 148–150 °C. 1H NMR (400 MHz,
CD3OD) δ: 7.82–7.75 (m, 1H), 7.56–7.46 (m, 2H), 7.28 (dd, J =
8.8, 4.4 Hz, 1H), 7.23–7.17 (m, 1H), 7.10 (s, 1H), 6.84 (td, J =
9.1, 2.5 Hz, 1H), 3.21– 3.01 (m, 6H), 2.96 (t, J = 5.8 Hz, 2H),
2.78 (t, J = 7.2 Hz, 2H), 2.58 (t, J = 6.0 Hz, 4H), 2.45 (t, J = 10.3
Hz, 2H), 2.06–1.91 (m, 4H), 1.83–1.73 (m, 2H), 1.71–1.62 (m,
2H). HRMS (EI) m/z calcd C29H34FN5 (M+) 471.2798, found
471.2802.
4.2 ChE Inhibitory Activity
The assay of ChE was conducted by using the method of
Ellman et al45. with slight modification. The rat cortex was
homogenized in cold 75 mM sodium phosphate buffer (pH =
7.4) with 0.4 mM Tetraisopropylpyrophosphoramide (iso-
OMPA) as the AChE source. And the rat serum was collected as
the BuChE source. The assay solution was made of 20 mM
phosphate buffer, 1 mM 5,5’-dithiobis (2-nitrobenzoic acid)
(DTNB, Sigma), 240 μM acetylthiocholine iodide (Sigma) or
320 μM butyrylthiocholine (Sigma), and 10 μL mice cortex
homogenate or 20 μL mice serum, respectively. Compounds
were concentrated appropriately and added into the assay
solution and incubated for 20 min at room temperature. Then, the
reactions were stopped by sodium-dodecyl sulfate (SDS) and the
absorption values were measured by a microplate reader (DTX
880, Beckman Coulter) at 450 nm. Last, the inhibition ratio
caused by the presence of test compounds was calculated,, and
the IC50 was defined as half-maximal inhibitory concentration
values.
4.1.16. 8-(4-(4-(5-Chloro-1H-indol-3-yl)butyl)piperazin-1-yl)-
1,2,3,4-tetrahydroacridin-9-amine (5c)
5c was synthesized by the general procedure of 4a, and 6c
was replaced by 6g. Compound 5c was isolated as a yellow
powder, 43% yield, mp 149–151 °C. 1H NMR (400 MHz,
CD3OD) δ 7.77 (t, J = 7.8 Hz, 1H), 7.61–7.43 (m, 3H), 7.28 (d, J
= 8.6 Hz, 1H), 7.09 (s, 1H), 7.02 (d, J = 8.5 Hz, 1H), 3.23–2.86
(m, 8H), 2.78 (t, J = 6.7 Hz, 2H), 2.69–2.39 (m, 6H), 1.96 (m,
4H), 1.83–1.72 (m, 2H), 1.71–1.59 (m, 2H). HRMS (EI) m/z
calcd C29H34ClN5 (M+) 487.2503, found 487.2507.
4.1.17.
8-(4-(4-(5-Bromo-1H-indol-3-yl)butyl)piperazin-1-yl)-
1,2,3,4-tetrahydroacridin-9-amine (5d)
5d was synthesized by the general procedure of 4a, and 6c
was replaced by 6h. Compound 5d was isolated as a yellow
powder, 39% yield, mp 151–153 °C. 1H NMR (400 MHz,
CD3OD) δ: 7.80–7.74 (m, 1H), 7.66 (d, J = 1.7 Hz, 1H), 7.54–
7.46 (m, 2H), 7.24 (d, J = 8.6 Hz, 1H), 7.15 (dd, J = 8.6, 1.9 Hz,
1H), 7.07 (s, 1H), 3.21– 3.00 (m, 6H), 2.95 (t, J = 5.9 Hz, 2H),
2.78 (t, J = 7.3 Hz, 2H), 2.56 (t, J = 6.1 Hz, 4H), 2.47 (t, J = 10.5
Hz, 2H), 2.03–1.89 (m, 4H), 1.83–1.72 (m, 2H), 1.70–1.61 (m,
2H). HRMS (EI) m/z calcd C29H34BrN5 (M+) 531.1998, found
531.1997.
4.3 In vitro Blood-Brain Barrier Permeation Assay
In order to evaluate the BBB penetrability of compounds
1a-5g, a parallel artificial membrane permeation assay was
conducted. Commercial drugs were purchased from Sigma and
Alfa Aesar. The porcine brain lipid (PBL) was bought from
Avanti Polar Lipids. The donor microplate (PVDF membrane,
pore size 0.45 mm) and the acceptor microplate were bought
from Millipore. The 96-well UV plate was from Corning
Incorporated. Compounds were dissolved in DMSO at 5 mg/mL
and diluted 50-fold in PBS/EtOH (7:3, v:v) to get the
concentration of 100 µg/mL. The filter membrane of the donor
96-well microplate was impregnated with 4 µL of PBL in
4.1.18.
8-(4-(4-(1H-indol-3-yl)butyl)piperazin-1-yl)-1,2,3,4-
tetrahydroacridin-9-amine (5e)
5e was synthesized by the general procedure of 4a, and 6c
was replaced by 6i. Compound 5e was isolated as a yellow
powder, 45% yield, mp 112–114 °C. 1H NMR (400 MHz,
CD3OD) δ: 7.77 (t, J = 8.1 Hz, 1H), 7.52 (dd, J = 8.1, 2.9 Hz,

dodecane (20 mg mL-1). It was added 200 µL of the tested
compounds to the donor wells and then carefully placed on the
acceptor 96-well microplate with 300 µL of PBS/EtOH (7:3, v:v)
to form a sandwich, which was left undisturbed for 12 h at 25
°C. After incubation, the donor plate was carefully removed and
the concentration of compounds in the acceptor wells was
determined using a UV plate reader (SpectraMax i3). Each
sample was analyzed at eight wavelengths, three wells and three
independent experiments, and the results are given as the mean ±
standard deviation. Pe was calculated using the following
equation.
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VD represents the volume in the donor well, and VA
represents the volume in the acceptor well. Area represents the
filter area, and time is the permeation time. [drug]acceptor means
the absorbance of the compound in the acceptor well, and
[drug]equilibrium means the theoretical equilibrium absorbance.
There were 13 quality control standards of known BBB
permeability were included to validate the analysis set (Table S2,
Supporting Information). The experimental data versus reference
values gave a strong linear correlation, Pe (exp.) = 0.974Pe (lit.)
– 0.8152 (R2 = 0.9651) (Figure S1, Supporting Information).
From this equation and the limit established by Di et al. for
blood−brain barrier permeation, we can get the conclusion that
compounds with Pe values greater than 3.08 × 10−6 cm s-1 could
cross the blood-brain barrier (CNS+), Pe values from 1.13 × 10−6
cm s-1 to 3.08 × 10−6 cm s-1 were classified as “CNS±” (BBB
permeation uncertain) and Pe values less than 1.13 × 10-6 cm s-1
could not cross the BBB (CNS−) (Table S3, supporting
information).
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Financial support for this research was provided by the
National Natural Science Foundation of China (Grants
21702061, 21672064, 81522045), Shanghai Sailing Program
(Grants 17YF1403600), and the Fundamental Research Funds
for the Central Universities.
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