Synthesis and evaluation of tetrahydroisoquinoline-benzimidazole hybrids as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.02.008

A novel series of tetrahydroisoquinoline-benzimidazole hybrids have been designed and synthesized as multifunctional agents against Alzheimer's disease (AD). These compounds were evaluated for their inhibition of neuroinflammation and human β-secretase (hBACE1), and neuroprotective activity. Among them, compound BD3 possessed significant anti-neuroinflammatory activity (IC₅₀ = 5.07 μM against nitric oxide production) through inhibiting the expression and secretion of proinflammatory cytokines in BV2 cells. Compound BD3 also exhibited moderate hBACE1 inhibitory activity (65.7% inhibition at 20 μM) and potent neuroprotective effect by increasing GSH level and reducing ROS production (91.8% cell viability at 5 μM). Parallel artificial membrane permeation assay demonstrated that BD3 could cross the blood-brain barrier (BBB). Thus, this study demonstrates that the compounds with tetrahydroisoquinoline-benzimidazole scaffold are potential anti-AD agents, and they are worth for the further development.

Full text of DOI:10.1016/j.ejmech.2019.02.008

Accepted Manuscript
Synthesis and evaluation of tetrahydroisoquinoline-benzimidazole hybrids as
multifunctional agents for the treatment of Alzheimer's disease
Yuying Fang, Huihao Zhou, Qiong Gu, Jun Xu
PII:
S0223-5234(19)30118-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.008
EJMECH 11100
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 November 2018
Revised Date: 23 January 2019
Accepted Date: 3 February 2019
Please cite this article as: Y. Fang, H. Zhou, Q. Gu, J. Xu, Synthesis and evaluation of
tetrahydroisoquinoline-benzimidazole hybrids as multifunctional agents for the treatment of
Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.02.008.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis and evaluation of tetrahydroisoquinoline-benzimidazole hybrids as
multifunctional agents for the treatment of Alzheimer’s disease
Yuying Fang, Huihao Zhou, Qiong Gu*, Jun Xu*
Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen
University, Guangzhou 510006, People’s Republic of China
*
Corresponding author
ABSTRACT
A novel series of tetrahydroisoquinoline-benzimidazole hybrids have been designed
and synthesized as multifunctional agents against Alzheimer’s disease (AD). These
compounds were evaluated for their inhibition of neuroinflammation and human
β-secretase (hBACE1), and neuroprotective activity. Among them, compound BD3
possessed significant anti-neuroinflammatory activity (IC = 5.07 µM against nitric
5
0
oxide production) through inhibiting the expression and secretion of proinflammatory
cytokines in BV2 cells. Compound BD3 also exhibited moderate hBACE1 inhibitory
activity (65.7% inhibition at 20 µM) and potent neuroprotective effect by increasing
GSH level and reducing ROS production (91.8% cell viability at 5 µM). Parallel
artificial membrane permeation assay demonstrated that BD3 could cross the
blood-brain barrier (BBB). Thus, this study demonstrates that the compounds with
tetrahydroisoquinoline-benzimidazole scaffold are potential anti-AD agents, and they
are worth for the further development.
Keywords
Tetrahydroisoquinoline-benzimidazole hybrids; Anti-neuroinflammation; BACE1
inhibition; Neuroprotection; Alzheimer’s disease
1
. Introduction
Alzheimer’s disease (AD) , a common chronic neurodegenerative disease, is
[
1]
[
2, 3]
characterized by progressive loss of memory and cognitive functions
. With the
increasing of population aging and the expanding life span, the number of AD patients
[
4]
is growing. It is estimated that there will be 74.7 million AD patients by 2030
.
Currently, only five FDA approved anti-AD drugs are clinically available. They can

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[
5]
only provide temporary and incomplete symptomatic relief . Thus, new anti-AD
agents are demanding.
[3,
Studies on AD pathogenesis demonstrate that the disease has multiple causes
6
-8]
.
Increasing evidence demonstrates that microglia-dominated neuroinflammation
[
9]
plays a significant role in AD . The chronic and sustained activation of brain
microglia cells and the subsequent overproduction of inflammatory mediators, such as
nitric oxide (NO), may cause uncontrolled neuroinflammation, elicit neurotoxicity,
[
10]
and contribute to disease pathogenesis. The amyloid cascade hypothesis
states that
amyloid β (Aβ), in particular Aβ isoform, has an early and imperative role in AD.
4
2
BACE1 (β-amyloid precursor protein cleaving enzyme 1), also known as β-secretase,
is the key rate-limiting enzyme in the process of Aβ generation. The elevated BACE1
[11]
activity has been found in the brains of patients with sporadic AD
. BACE1
[
12]
knockout mice are deficient in Aβ production and have no aberrant phenotype
.
[
13-15]
Therefore, the development of BACE1 inhibitors has been recently pursued
addition, oxidative damage with the overproduction of reactive oxygen species (ROS)
. In
[
16]
may contribute to neuron degeneration and death in this disorder
.
Since AD has multiple causes, the single targeted drug candidates have high
[17]
[18]
failure rate in clinical trials . Multi-target-directed ligands (MTDLs) , aiming to
simultaneously target multiple pathological factors (for example neuroinflammation,
oxidative damage, and Aβ formation) involved in AD etiology, can be a promising
therapeutic strategy.
Benzimidazole exists in several drug candidates, such as antiviral drug Maribavir
and antihypertensive drug Telmisartan. Some benzimidazole derivatives are also
[19]
[20]
anti-inflammatory agents
and BACE1 inhibitors
.
In addition,
[
21-24]
tetrahydroisoquinoline derivatives
have anti-AD activities, including
anti-inflammation, anti-oxidation, and neuroprotection. Therefore, we hypothesize
that compounds containing the fragments of tetrahydroisoquinoline and
benzimidazole may act as multifunctional drugs against AD. Based on the proposed
[
20]
pharmacophoric model of BACE1 inhibitors
and synthetic feasibility, benzene ring
and pyridine ring were used as the linker to assemble the two fragments (Fig. 1). This
resulted in the synthesis of compounds BD and B series. All the synthesized
compounds were evaluated for their activities of anti-neuroinflammation, inhibition of
BACE1, neuroprotective effects, and blood-brain barrier (PAMPA-BBB) permeability.
This study aims to identify novel multifunctional agents against AD.

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Fig. 1. The scheme of assembling privileged fragments for novel multifunctional
compounds against AD.
2
. Results and discussion
2
.1 Chemistry
The synthetic routes for target compounds of BD and B series are summarized in
Schemes 1 and 2. As shown in Scheme 1, the key intermediates 12 and 13 were
prepared by the reduction of 9 and 10, which were synthesized via the Aube-Schmidt
[
25]
rearrangement reaction
in the presence of NaN . The amines 11-13 were mixed
3
with commercially available ethyl 6-bromopicolinate to afford 14-16 through
[
26, 27]
Ullmann coupling reaction in the presence of CuI
. Then the intermediates 17-19,
obtained by the hydrolysis of the ester 14-16, were subjected to couple with the
diaminobenzene derivatives followed by acid catalyzed ring closure leading to
benzimidazole derivatives BD1-BD11. For B series, the reaction condition is same as
that of BD series. Twenty-two compounds including BD and B series were
synthesized.
O
R
1
R1
R1
O
R
1
Br
N
a
b
c
N
N
O
+
NH
O
NH
O
O
11 R = H
14 R = H
1
1
9
R = OCH3
12 R = OCH3
15 R1 = OCH3
1
1
1
0 R = F
13 R = F
16 R1 = F
1
1
4
5
R1
R
1
3
N
6
H N
2
d
e
R2
N
N
COOH
R
2
N
N
7
+
2
N
H
1
H N
2
1
7 R = H
BD1 ~ BD9 R = H, R2 = H, 6-OH, 6-OCH , 5,6-2F, 6-OCF ,
1
1
3
3
18 R1 = OCH3
6-F, 6-t-Bu, 6-Cl, 6-CF
3
19 R1 = F
BD10 R1 = OCH , R = 6-OCH
3 2 3
BD11 R1 = F, R = 6-F
2
Scheme 1. Synthesis of BD1-BD11. Reagents and conditions: (a) MeSO H, NaN ,
3
3
DCM, r.t., overnight; (b) AlLiH , THF, reflux, 4 h; (c) K CO , CuI, L-proline, DMSO,
4
2
3
8
0°C, 16 h; (d) LiOH, THF, reflux, 3 h; (e) TBTU, DIPEA, DMF, r.t., 6 h, then AcOH,

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reflux, overnight.
Scheme 2. Synthesis of B1-B11. Reagents and conditions: (a) K CO , CuI, L-proline,
2
3
DMSO, 80°C, 16 h; (b) LiOH, THF, reflux, 3 h; (c) TBTU, DIPEA, DMF, r.t., 6 h,
then AcOH, reflux, overnight.
2
.2 Effects of anti-neuroinflammation in LPS-induced BV2 microglial cells and SAR
study
Anti-neuroinflammatory activities of the synthesized compounds were
[
28]
determined by the Griess assay
, which detects the NO production in
lipopolysaccharide (LPS)-induced BV2 microglia cells. Resveratrol (Res) was used as
positive control. Results have been listed in Table 1. In comparison to Res, most of
the synthesized compounds exhibited potent inhibitory activities against NO
production.
For BD series, compound BD1 (R = R = H), with no substituents on
1
2
tetrahydroisoquinoline and benzimidazole moieties, possesses potent NO inhibitory
activity (IC = 7.33 µM). When substituents at R position on the benzimidazole
5
0
2
moiety, compounds BD2-BD9 (R = H) show similar NO inhibitory activity compared
1
with BD1, except for BD7 (R = t-Bu) having the IC value of more than 10 µM. This
2
50
demonstrates that the strong electron-donating group tertiary butyl (t-Bu) at R2
position is not beneficial for NO inhibitory activity. Comparing with BD3 (R = H, R
1
2
=
6-OCH ), BD10 (R = OCH , R = 6-OCH ) exhibit reduced NO inhibitory activity
3 1 3 2 3
(
IC > 10 µM), which indicates that methoxy group at R position cannot improve
50 1
inhibitory activity. Compounds BD6 (R = H, R = 6-F) and BD11 (R = F, R = 6-F)
1
2
1
2
display nearly the same inhibitory activity with IC values of 5.70 µM and 6.43 µM.
50
This indicates that the fluorine substituent at the R position has no significant impact
1

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on NO inhibitory activity.
For B series, compound B1 inhibits NO production (IC = 6.08 µM), which is
5
0
similar to that of BD1 (IC = 7.33 µM). This indicates that the effects of the linker
50
benzene and pyridine on NO inhibitory activity are similar. Compounds B2-B10 (R =
H) have different substituents at R' position on the benzimidazole moiety. The
inhibitory activities of most of these compounds do not change much compared with
B1, while compounds B3 (R' = 6-Cl), B8 (R' = 5-Cl, 6-F), and B10 (R' = 7-NH ) show
2
reduced NO inhibitory activity with IC values more than 10 µM. Comparing with
5
0
B5 (R = H, R' = 6-OCH ), compound B11 (R = OCH , R' = 6-OCH ) displays reduced
3
3
3
activity (IC >10 µM), which demonstrates that methoxy group at the R position is
5
0
not beneficial for NO inhibition.

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Table 1
Inhibitory activities against NO production and BACE1, and BBB permeability (P × 10 cm s ) of BD1-BD11 and B1-B11.
−
6
−1
e
−
6
−6
ID
R1
R2
NO IC
BACE1 inhibition Pe (×10
ID
R
R'
NO IC
(µM)
6.08 ± 0.01
5.42 ± 1.21
> 10
BACE1
inhibition (%)
1.5 ± 2.0
2.3 ± 1.7
92.7 ± 4.3
1.3 ± 0.03 µM)
Pe (×10
50
50
a
b
−1 c
a
b
−1 c
(µM)
(%)
cm s )
cm s )
BD1
BD2
BD3
H
H
H
H
7.33 ± 0.14
3.80 ± 0.42
5.07 ± 0.54
2.1 ± 1.3
2.8 ± 2.8
65.7 ± 3.5
15.9 ± 0.8
13.8 ± 0.7
16.8 ± 0.3
B1
B2
B3
H
H
H
H
6-F
6-Cl
8.5 ± 0.7
12.6 ± 0.3
4.9 ± 0.5
6-OH
6-OCH
3
d
(
BD4
BD5
H
H
5,6-2F
6.58 ± 0.16
9.74 ± 0.02
0.8 ± 1.2
98.7 ± 1.0
1.1 ± 0.02 µM)
5.9 ± 0.4
13.9 ± 0.8
10.0 ± 0.5
B4
B5
H
H
6-CH
6-OCH
3
5.70 ± 0.94
8.28 ± 1.13
2.1 ± 1.5
7.5 ± 3.3
9.6 ± 0.9
5.6 ± 0.1
6-OCF
3
3
d
(
BD6
BD7
BD8
H
H
H
6-F
6-t-Bu
6-Cl
5.70 ± 0.12
> 10
6.94 ± 0.92
14.5 ± 0.5
B6
B7
B8
H
H
H
6-CF
3
7.72 ± 0.89
7.93 ± 0.55
> 10
2.6 ± 1.4
1.8 ± 3.2
1.0 ± 2.4
8.7 ± 1.0
9.9 ± 0.3
10.1 ± 0.8
e
30.6 ± 2.6
95.3 ± 2.3
1.8 ± 0.3 µM)
n.d.
5,6-2Cl
5-Cl,6-F
16.7 ± 1.2
5.4 ± 0.5
d
(
BD9
H
6-CF
3
9.33 ± 0.67
90.2 ± 1.9
B9
H
6-OH
5.95 ± 0.01
3.0 ± 5.6
8.6 ± 0.9
d
(
3.6 ± 0.05 µM)
25.9 ± 4.2
2.7 ± 2.6
-
BD10
BD11
Res
OCH
F
-
3
6-OCH
3
> 10
6.43 ± 0.62
11.1 ± 1.3
11.5 ± 0.6
14.0 ± 0.5
1.1 ± 0.01
B10
B11
MK-8931
H
OCH
-
7-NH
6-OCH
-
2
> 10
> 10
-
4.8 ± 4.8
1.8 ± 1.5
98.6 ± 1.9
13.0 ± 1.1
7.6 ± 0.2
16.4 ± 0.3
6-F
-
3
3
d
(
20.7 ± 1.2 nM)
a
b
c
Concentration (µM) for 50% inhibition of NO release in BV2 cells. The inhibitory activity of BACE1 at the concentration of 20 µM. Compounds were dissolved in DMSO at 5 mg/mL and
-
6
−1
d
diluted with PBS/EtOH (70:30). Compounds with permeabilities Pe > 4.7 × 10 cm s could cross the BBB by passive diffusion. IC , the concentration of compounds that inhibited 50%
5
0
e
BACE1 enzymatic activity. n.d. Not determined. All values are expressed as the mean ± SD at least three independent experiments.

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2
.3 Inhibition of human BACE1 activity and SAR study
The BACE1 screening assay utilized in this work was the fluorescence resonance
[
29]
energy transfer (FRET) protocol as previously reported . The inhibitory activities of
compounds against BACE1 were tested at the concentration of 20 µM. MK-8931, one
of the most potent clinical BACE1 inhibitor, was used as the positive control. As
shown in Table 1, compounds B3, BD5, BD8, and BD9 at 20 µM displayed potent
inhibitory activities (92.7%, 98.7%, 95.3%, and 90.2%, respectively) comparing to the
positive control MK-8931 (98.6%), and the IC values at 1.3, 1.1, 1.8, and 3.6 µM,
5
0
respectively. Compound BD3 showed moderate activity with 65.7% inhibition.
Whereas other compounds showed no significant inhibitory activities.
For BD series, compounds BD1-BD9, without substituents at R position (R =
1
1
H), have different substituents at R position on the benzimidazole moiety.
2
Compounds BD5 (R = 6-OCF ), BD8 (R = 6-Cl), and BD9 (R = 6-CF ) exhibit
2
3
2
2
3
significantly higher inhibitory rate of 98.7%, 95.3%, and 90.2% at 20 µM,
respectively. It is worth to mention that BD5 with 6-OCF at R position is the most
3
2
potent compound against BACE1. However, compounds BD2 (R = 6-OH), BD3 (R
2
2
=
6-OCH ), and BD7 (R = 6-t-Bu) exhibit only 2.8%, 65.7%, and 30.6% inhibition at
3 2
2
0 µM, respectively. This indicates that electron-withdrawing groups are beneficial
for inhibitory activities comparing with electron-donating groups. Compounds BD10
and BD11 do not have improved activities, which suggests that introducing OCH or F
3
group at the R cannot improve inhibitory activities.
1
For B series, compounds B4 (R' = 6-CH ), B5 (R' = 6-OCH ), B9 (R' = 6-OH),
3
3
and B10 (R' = 7-NH ), having an electron-donating group at R' on the benzimidazole
2
moiety, display nearly no inhibitory activities (2.1%, 7.5%, 3.0%, and 4.8%,
respectively). Compounds B2 (R' = 6-F) and B6 (R' = 6-CF ) with an
3
electron-withdrawing group on the benzimidazole moiety exhibit no inhibitory
activities (2.3% and 2.6%), whereas compound B3 (R' = 6-Cl) exhibits higher activity
(92.7%). When two substitutions at the R' position on the benzimidazole moiety, the
inhibitory activities of compounds B7 (R' = 5-Cl, 6-Cl) and B8 (R' = 5-Cl, 6-F) are
not improved (1.8% and 1.0%). When the R group is OCH , the activity of compound
3
B11 nearly remains unchanged (1.8%) compared to B5 without OCH group at R
3
position (7.5%). This indicates that introducing OCH group at the R does not
3
increase inhibitory activity.

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2
.4 Neuroprotective activity against glutamate-induced cell death in HT22 cells
Neuronal death is a key characteristic in AD. Glutamate toxicity is a common
[
30, 31]
model to study oxidative stress-induced neuronal cell death
. In this model, the
HT22 cells (a mouse hippocampal neuron cell line) are exposed to high concentration
of extracellular glutamate, which suppresses cystine uptake into the cells via the
cystine/glutamate antiporter, leading to the depletion of glutathione (GSH) and
accumulation of reactive oxygen species (ROS) and then cell death. To determine
whether our compounds could protect neuronal cells from glutamate-induced
cytotoxicity, HT22 cells were treated with 5 mM glutamate (Glu) for 24 hours in the
presence or absence of different concentrations of compounds (5 µM and 1 µM). The
cell viability of neurons was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) analysis.
As shown in Figs. 2A and 2B, only 30% of HT22 cells survived after glutamate
treatment for 24 hours, whereas the tested doses of compounds showed different
degrees of protection against glutamate-induced cell death. Among BD series,
compounds BD1 (R = H), BD2 (R = 6-OH), BD3 (R = 6-OCH ), BD5 (R =
2
2
2
3
2
6
-OCF ), BD6 (R = 6-F), and BD11 (R = F, R = 6-F) showed significant protective
3 2 1 2
effect against glutamate-induced cell death at 5 µM with higher cell viability of 80.3%,
8.8%, 91.8%, 62.1%, 67.8%, and 71.1%, respectively. For compounds of B series,
9
compounds B3 (R' = 6-Cl), B8 (R' = 5-Cl, 6-F), B10 (R' = 7-NH ), and B11 (R =
2
OCH , R' = 6-OCH ) displayed potent protection against glutamate-induced
3
3
cytotoxicity at 5 µM with higher cell viability of 67.6%, 48.9%, 94.9%, and 93.1%,
respectively. It is worth noting that compound B10, which contains 7-NH on the
2
benzimidazole moiety, displayed high cell viability of 54.5% at 1 µM.

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Fig. 2. The protective effects of compounds of BD series (A) and B series (B) on
glutamate-induced cell death. HT22 cells were treated with 5 mM glutamate for 24
hours in the presence or absence of compounds. ####p < 0.0001 versus Control group；
*
***p < 0.0001 versus Glu group.
2
.5 Blood-brain barrier (BBB) permeability
As the first requirement for successful CNS drugs is to reach their therapeutic
targets, screening for the BBB penetration is of great importance. To explore whether
our compounds could penetrate into the brain, the compounds were tested with the
[
32]
parallel artificial membrane permeation assay (PAMPA)
. This method has the
advantage of predicting compounds’ passive diffusion through BBB with high
throughput and reproducibility. As listed in Table 1, all the synthesized compounds
showed good BBB permeability.
Based on the overall performance of compound BD3 (IC for NO production:
5
0

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5
.07 µM; BACE1 inhibition: 65.7%; potent protection against glutamate-induced
toxicity at 5 µM: 91.8%) and its brain BBB penetration, we decided to select
compound BD3 for further investigation.
2
.6 Compound BD3 inhibits the expression of iNOS and the secretion of
proinflammatory cytokines in LPS-induced BV2 cells
When BV2 cells are stimulated with LPS, the expression of iNOS (inducible
nitric oxide synthase) and proinflammatory cytokines such as TNF-α (tumor necrosis
factor α) and IL-1β (interleukin 1β) increases. To further evaluate the
anti-neuroinflammatory effects of BD3, we investigated the protein and mRNA
expression of iNOS, TNF-α, and IL-1β through Western blot or ELISA and qPCR.
The mRNA levels of iNOS, TNF-α, and IL-1β are generally up-regulated in
LPS-stimulated BV2 cells. When treated with various concentrations of BD3, the
mRNA expressions of iNOS, TNF-α, and IL-1β significantly decreased in a
dose-dependent manner in LPS-induced BV2 cells (Figs. 3E-3G). Western blot
analyses demonstrated that BD3 significantly decreased the expression of iNOS in
LPS-stimulated BV2 cells (Figs. 3A-3B). ELISA assays suggested that the levels of
TNF-α and IL-1β in the culture medium dramatically increased in LPS-induced BV2
cells. When incubated with different doses of BD3, the levels of TNF-α and IL-1β
reduced in a concentration-dependent manner (Figs. 3C-3D). These results indicate
that compound BD3 exhibits anti-neuroinflammatory activities by inhibiting the
protein and mRNA expression of iNOS and proinflammatory cytokines (TNF-α and
IL-1β) in LPS-induced BV2 cells.

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Fig. 3. BD3 suppressed the expression of iNOS and the production of
proinflammatory cytokines in LPS-induced BV2 cells. BV2 cells were incubated with
different concentrations of BD3 for 30 minutes, then incubated with LPS (1 µg/mL)
for 16 hours. (A) The protein level of iNOS was determined by Western blot.
α-Tubulin was used as reference. (B) Quantification of Western blot data. BV2 cells
were incubated with BD3 for 30 minutes, then incubated with LPS (1 µg/mL) for 24
hours. TNF-α (C) and IL-1β (D) in the supernatants were assayed by ELISA kits. BV2
cells were incubated with BD3 for 30 minutes, then incubated with LPS (1 µg/mL) for
1
2 hours. mRNA levels of iNOS (E), TNF-α (F) and IL-1β (G) were assayed by qPCR.
The data are presented as mean ± SEM of three independent experiments. ####p <
.0001 versus control group, ****p < 0.0001, ***p < 0.001, **p < 0.01, ns (no
significance) versus LPS group.
0

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2
.7 Compound BD3 suppresses glutamate-induced intracellular oxidative stress in
HT22 cells
As shown in Fig. 2A, 5 µM BD3 significantly increased the cell viability of
HT22 cells up to 91.8% comparing with glutamate-treated HT22 cells (30%). To
further confirm the neuroprotective effects of BD3 on glutamate-treated HT22 cells,
we performed experiments to observe the morphology of HT22 cells treated with or
without BD3. Exposed to 5 mM glutamate for 24 hours, HT22 cells lost their normal
spindle-shaped morphology, and became shrunken and rounded (Fig. 4A). While, the
cells co-treated with 5 µM BD3 stayed in normal neurite morphology, similar to the
control group. The results were consistent with the data of cell viability (Fig. 2A).
To determine whether the protective effect of BD3 against glutamate-induced
toxicity was achieved via inhibiting intracellular oxidative stress, the levels of GSH
and ROS in HT22 cells were detected. GSH is an important free radical scavenger in
the body. To some extent, the antioxidant ability of the body depends on the level of
GSH. The depletion of GSH is a key factor contributing to the glutamate toxicity.
After HT22 cells were incubated with 5 mM glutamate for 12 hours in the presence or
absence of different concentrations of BD3, the contents of GSH were measured using
the commercial assay kit. Fig. 4B indicates that 5 µM BD3 can significantly reverse
the reduction of GSH level compared to the glutamate-treated cells.
The intracellular ROS was tested using the fluorogenic probe DCFH-DA
(2′ ′-dichlorodihydrofluorescein diacetate). After treatment with glutamate in the
,7
presence or absence of BD3, HT22 cells were incubated with 10 µM DCFH-DA for
0 minutes, then the cells were photographed, and fluorescence intensity was
3
measured. As shown in Figs. 4C and 4D, ROS levels were significantly increased
after glutamate treatment for 12 hours, but greatly reduced when HT22 cells were
co-treated with 5 µM BD3. These data indicate that 5 µM BD3 can significantly
protect cells from glutamate-induced death via antioxidant mechanisms, specifically
by increasing the level of GSH and inhibiting the production of ROS.

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Fig. 4. Compound BD3 protects HT22 cells from glutamate-induced death via
antioxidant mechanisms. HT22 cells were treated with 5 mM glutamate for 24 hours
in the presence or absence of BD3. (A) The morphology of HT22 cells with different
treatments. (B) The levels of GSH were measured using the commercial assay kit.
After treated with glutamate in the presence or absence of BD3 for 12 hours, cells
were followed by incubated with 10 µM DCFH-DA for 30 minutes, then (C) the
fluorescence intensity was recorded using high content screening system, and (D)
cells were photographed. The data are presented as mean ± SEM of three independent
experiments. ####p < 0.0001, ###p < 0.001 versus Control group; ***p < 0.001, **p
<
0.01, ns (no significance) versus Glu group.
2
.8 Binding mode of BD3 with BACE1 in silico
Compound BD3 was docked into the binding pocket of hBACE1 (PDB ID:

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2
B8L) with Glide extra precision (XP). As shown in Fig. 5, the NH of the
benzimidazole moiety of the compound formed a hydrogen bond with Asp32 in the
catalytic pocket and the oxygen atom of the OCH group formed a hydrogen bond
3
with a water molecule. The pyridine ring of BD3 interacted with Phe108 via π-π
stacking and the protonation of nitrogen atom of the pyridine ring produced a
hydrogen bond between Gly230 and the hydrogen atom.
Fig. 5. The proposed binding model of BD3-BACE1 complex. The yellow dash lines
represent hydrogen bonds and the red dash lines represent π-π stacking interaction.
2
.9 ADMET properties for compound BD3
To investigate the drug-like profiles of BD3, the ADMET properties were
estimated using Discovery Studio 3.5 software package (Table 2). The results indicate
that BD3 can have high blood-brain penetration, good absorption, no liver toxicity,
and no inhibition of CYP2D6. BD3 could easily bind to carrier proteins in the blood,
however, the solubility of BD3 is low. Hence, the structure of BD3 need to be further
optimized regarding to its pharmacokinetic profile.
Table 2
ADMET properties for BD3
a
b
c
d
e
f
Structure
BBB Absorption
Solubility
Hepatotoxicity
CYP2D6
PPB

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Very low,
High
Good
Nontoxic
Non-inhibitor True
but
possible
a
b
BBB predicts blood-brain penetration after oral administration.; Absorption
c
predicts human intestinal absorption (HIA) after oral administration.; Solubility
o
d
predicts the solubility of each compound in water at 25 C.; Hepatotoxicity predicts
e
potential liver toxicity of compounds.; CYP2D6 (cytochrome P450 2D6) predicts
f
CYP2D6 enzyme inhibition using 2D chemical structure.; PPB (plasma protein
binding) predicts whether a compound is likely to be highly bound (>= 90% bound) to
carrier proteins in the blood.
3
Conclusion
This work demonstrates that multifunctional agents against AD can be
discovered by assembling the fragments benzimidazole and tetrahydroisoquinoline.
This is another example that develops lead compounds through assembling two active
[
33]
fragments
. Among the synthesized compounds, some compounds exhibit great
inhibition of neuroinflammation and hBACE1, as well as good neuroprotective effect
and BBB penetration. In vitro and in silico experiments indicate that BD3 can inhibit
BACE1 by interacting with the catalytic pocket in BACE1. BD3 showed
anti-neuroinflammatory activity through suppressing the expressions of inflammatory
proteins and genes. BD3 also possessed significant protective effects on neuronal cells
against the glutamate-induced cytotoxicity in HT22 cells by preventing the ROS
production and increasing the GSH level. Moreover, BD3 could penetrate BBB.
Therefore, BD3 could be a lead for further optimization as a multifunctional agent
against Alzheimer's disease.
4
4
4
Experimental section
.1 Chemistry
.1.1 Materials and equipment. Reagents were used without further purification
1
13
unless specified. H and C NMR spectra were recorded using tetramethylsilane
TM
TM
(
TMS) as the internal standard on a Bruker Ascend 400 or Bruker Ascend 500
spectrometer. Coupling constants are given in Hz. High-resolution mass spectra were
obtained using a Shimadzu LCMS-IT-TOF mass spectrometer. Mass spectra (MS)
were recorded on a Shimadzu LCMS-2010A instrument with an ESI source. Flash

ACCEPTED MANUSCRIPT
column chromatography was performed using silica gel (200–300 mesh) purchased
from Qingdao Haiyang Chemical Co. Ltd. All reaction progress was monitored by
thin layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang
Chemical Co. Ltd) and the spots were detected under UV light (254 nm).
4
.1.2 General procedure for the synthesis of intermediates 9 and 10
[26]
According to previously reported method
, 5-methoxy-1-indanone (4.30 g,
2
6.6 mmol) or 5-fluoro-1-indanone (4.00 g, 26.6 mmol) were dissolved in the mixed
solvent of CH Cl (40 mL) and MeSO H (methanesulfonic acid, 40 mL). NaN (3.46
2
2
3
3
g, 53.2 mmol) was added slowly in an ice-water bath. Then the mixture was stirred
overnight at room temperature. In an ice-water bath, 20% NaOH (aq) was added to
make the solution neutral. The aqueous solution was extracted with CH Cl and the
2
2
obtained organic layer was combined, washed with brine, dried over MgSO and
4
concentrated. The residue was purified by silica gel flash chromatography
(
EtOAc/hexane = 2/1).
-methoxy-3,4-dihydroisoquinolin-1(2H)-one (9). White solid (2.82 g, 60%). H NMR
400 MHz, CD OD) δ 7.93 (d, J = 8.6 Hz, 1H), 6.95 (dd, J = 8.6, 2.4 Hz, 1H), 6.89 (d,
1
6
(
3
J = 1.8 Hz, 1H), 3.91 (s, 3H), 3.54 (t, J = 6.7 Hz, 2H), 3.02 (t, J = 6.7 Hz, 2H);
-
ESI-MS: m/z 176.0 [M-H] .
1
6
-fluoro-3,4-dihydroisoquinolin-1(2H)-one (10). White solid (2.32 g, 53%). H NMR
(
400 MHz, CD OD) δ 8.03 (dd, J = 8.3, 5.9 Hz, 1H), 7.20 - 7.06 (m, 2H), 3.56 (t, J =
3
-
6
.7 Hz, 2H), 3.05 (t, J = 6.7 Hz, 2H); ESI-MS: m/z 164.0 [M-H] .
4
.1.3 General procedure for the synthesis of intermediates 12 and 13
[34]
According to previously reported method , compound 9 (1.77 g, 10 mmol) or
1
0 (1.65 g, 10 mmol) was dissolved in dry THF (50 mL). Then in an ice-water bath, it
was added dropwise to a solution of LiAlH in dry THF (1 mol/L, 20 mL) under N
4
2
environment. The mixture was stirred at reflux for 4 h, and then quenched by the
sequential addition of THF and 30% NaOH solution in the ice-water bath. The
reaction mixture was filtered through celite and washed with MeOH. The residue was
evaporated in vacuum and purified by silica gel flash chromatography (EtOAc/MeOH
=
10/1).
-methoxy-1,2,3,4-tetrahydroisoquinoline (12). White oil (0.81 g, 50%). H NMR
400 MHz, CD OD) δ 7.01 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H),
1
6
(
3
3
.99 (s, 2H), 3.79 (s, 3H), 3.17 (t, J = 6.0 Hz, 2H), 2.89 (t, J = 5.8 Hz, 2H); ESI-MS:
-
m/z 162.1 [M-H] .

ACCEPTED MANUSCRIPT
1
6
-fluoro-1,2,3,4-tetrahydroisoquinoline (13). White oil (0.95 g, 63%). H NMR (400
MHz, CD OD) δ 7.14 - 7.08 (m, 1H), 6.95 - 6.85 (m, 2H), 4.04 (s, 2H), 3.18 (t, J =
3
-
6
.1 Hz, 2H), 2.92 (t, J = 5.9 Hz, 2H); ESI-MS: m/z 150.0 [M-H] .
4
.1.4 General procedure for the synthesis of intermediates 14-16, 20, and 21
To a dried sealed vessel was added 1,2,3,4-tetrahydroisoquinoline (5 mL, 40
mmol), anhydrous K CO (8.34 g, 60 mmol), CuI (0.8 g, 4 mmol), L-Proline (0.92 g,
2
3
8
mmol), ethyl 6-bromopicolinate (4.32 g, 20 mmol), and DMSO (25 mL). Then the
mixture was stirred at 80°C for 16 h under N environment. After cooling to room
2
temperature, water (100 mL) was added and extracted with EtOAc. Then, the
combined organic layer was dried over MgSO , filtered, and concentrated. The
4
residue was purified by silica gel flash chromatography (hexane/EtOAc = 5/1) to
provide compound 14 as a white oil (3.94 g, 70%).
1
Ethyl 6-(3,4-dihydroisoquinolin-2(1H)-yl)picolinate (14). H NMR (400 MHz, CDCl )
3
δ 7.59 (t, J = 7.9 Hz, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.24 - 7.16 (m, 4H), 6.83 (d, J =
8
.6 Hz, 1H), 4.76 (s, 2H), 4.42 (q, J = 7.2 Hz, 2H), 3.94 (t, J = 5.8 Hz, 2H), 2.98 (t, J
-
=
5.8 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H) ; ESI-MS: m/z 281.1 [M-H] .
Ethyl 6-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)picolinate (15). White oil, 70%.
1
H NMR (400 MHz, CDCl ) δ 7.58 (t, J = 7.9 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.13
3
(d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 6.78 (dd, J = 8.4, 2.4 Hz, 1H), 6.72 (s,
1
H), 4.68 (s, 2H), 4.42 (q, J = 7.1 Hz, 2H), 3.92 (t, J = 5.8 Hz, 2H), 3.80 (s, 3H), 2.95
-
(
t, J = 5.8 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H); ESI-MS: m/z 311.1 [M-H] .
Ethyl 6-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)picolinate (16). Yellow oil, 72%.
1
H NMR (400 MHz, CDCl ) δ 7.60 (dd, J = 8.4, 7.4 Hz, 1H), 7.41 (d, J = 7.3 Hz, 1H),
3
7
.17 (dd, J = 8.1, 5.7 Hz, 1H), 6.93 - 6.85(m, 2H), 6.82 (d, J = 8.5 Hz, 1H), 4.72 (s,
2
H), 4.42 (q, J = 7.1 Hz, 2H), 3.91 (t, J = 5.9 Hz, 2H), 2.96 (t, J = 5.8 Hz, 2H), 1.42 (t,
-
J = 7.1 Hz, 3H); ESI-MS: m/z 299.1 [M-H] .
1
Ethyl 3-(3,4-dihydroisoquinolin-2(1H)-yl)benzoate (20).
White oil, 75%. H NMR
(
400 MHz, CDCl ) δ 7.66 (dd, J = 2.5, 1.6 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.34 (t, J
3
=
7.9 Hz, 1H), 7.22 - 7.12(m, 5H), 4.46 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 3.61 (t, J =
5
.9 Hz, 2H), 3.01 (t, J = 5.8 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H); ESI-MS: m/z 280.1
-
[
M-H] .
Ethyl 3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)benzoate (21). White oil, 70%.
1
H NMR (400 MHz, CDCl ) δ 7.68 (s, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.35 (t, J = 7.9
3
Hz, 1H), 7.25 - 7.18 (m, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.78 (dd, J = 8.4, 2.6 Hz, 1H),

ACCEPTED MANUSCRIPT
6
.71 (d, J = 2.5 Hz, 1H), 4.42 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 3.61 (t, J
=
5.9 Hz, 2H), 3.00 (t, J = 5.5 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H); ESI-MS: m/z 310.1
-
[
M-H] .
4
.1.5 General procedure for the synthesis of intermediates 17-19, 22, and 23
The intermediate 14 (2.82 g, 10 mmol) was dissolved in THF (50 mL). Then
LiOH (1.19 g, 50 mmol) and the mixture of EtOH/H O = 5:1 (5 mL) were added into
2
the round flask. After stirred at reflux for 3 h, the solvent was evaporated in vacuo. A
small amount of ice-water was added to dissolve the residue. The 1N HCl aqueous
solution was added slowly to make the solution neutral. The resulting precipitate was
isolated by filtration and dried under vacuum to afford compound 17.
1
6
-(3,4-dihydroisoquinolin-2(1H)-yl)picolinic acid (17). Light yellow solid, 92%. H
NMR (400 MHz, CD OD) δ 8.12 (dd, J = 9.1, 7.3 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H),
3
7
3
6
7
7
1
.63 (d, J = 7.1 Hz, 1H), 7.37 - 7.28 (m, 4H), 4.92 (s, 2H), 3.97 (t, J = 5.9 Hz, 2H),
-
.15 (t, J = 5.9 Hz, 2H); ESI-MS: m/z 253.1 [M-H] .
-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)picolinic acid (18). Light yellow solid,
1
0%. H NMR (400 MHz, CDCl ) δ 7.70 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 7.1 Hz, 1H),
3
.13 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.75 (s,
H), 4.65 (s, 2H), 3.86 - 3.76 (m, 5H), 2.97 (t, J = 5.5 Hz, 2H); ESI-MS: m/z 283.1
-
[
M-H] .
6
-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)picolinic acid (19). Yellow solid, 80%.
1
H NMR (400 MHz, CDCl ) δ 7.60 (dd, J = 8.4, 7.4 Hz, 1H), 7.41 (d, J = 7.3 Hz, 1H),
3
7
.17 (dd, J = 8.1, 5.7 Hz, 1H), 6.93 - 6.85(m, 2H), 6.82 (d, J = 8.5 Hz, 1H), 4.72 (s,
-
2
H), 3.91 (t, J = 5.9 Hz, 2H), 2.96 (t, J = 5.8 Hz, 2H); ESI-MS: m/z 271.1 [M-H] .
1
3
-(3,4-dihydroisoquinolin-2(1H)-yl)benzoic acid (22). Light yellow solid, 65%. H
NMR (400 MHz, CDCl ) δ 7.71 (s, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 7.9 Hz,
3
1
2
3
8
7
2
2
4
H), 7.24 - 7.17 (m, 5H), 4.48 (s, 2H), 3.64 (t, J = 5.9 Hz, 2H), 3.02 (t, J = 5.8 Hz,
-
H) ; ESI-MS: m/z 252.1 [M-H] .
-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)benzoic acid (23). Light yellow solid,
1
0%. H NMR (400 MHz, CDCl ) δ 7.67 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.36 (t, J =
3
.9 Hz, 1H), 7.18 (dd, J = 8.1, 2.3 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.78 (dd, J = 8.4,
.6 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H), 4.41 (s, 2H), 3.80 (s, 3H), 3.60 (t, J = 5.9 Hz,
-
H), 2.98 (t, J = 5.7 Hz, 2H); ESI-MS: m/z 282.1 [M-H] .
.1.6 General procedure for the synthesis of BD1-BD11 and B1-B11
To a solution of the desired carboxylic acids (17, 18, 19, 22 or 23) (1 mmol) in

ACCEPTED MANUSCRIPT
DMF (5 mL) at 0°C was added DIPEA (0.21 mL, 1.2 mmol). TBTU（385 mg, 1.2
mmol）was added and the resulting mixture were stirred at 0°C for 30 minutes. Then
the desired diaminobenzene derivative (1.1 mmol) was added. The mixture was stirred
at room temperature for 6 h and then quenched with ice-water. The precipitated solid
was filtered, washed with water, and dissolved in EtOAc. The organic layer was
washed with a 1 N HCl aqueous solution, then with a saturated NaHCO aqueous
3
solution and finally H O, dried over MgSO , and concentrated in vacuo, which was
2
4
used in the next step without further purification. To the appropriate amide was added
AcOH (10 mL), and the mixture was refluxed overnight, cooled to room temperature
and concentrated under reduced pressure. The residue was purified by flash
chromatography.
4
.1.6.1 2-(6-(1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline
BD1).
Following the general procedure above, BD1 was obtained as a white solid
(
1
(
60%). H NMR (400 MHz, CDCl ) δ 10.45 (s, 1H), 7.84 (s, 1H), 7.77 (d, J = 7.3 Hz,
3
1
H), 7.66 (t, J = 7.8 Hz, 1H), 7.55 (s, 1H), 7.32 - 7.27 (m, 2H), 7.25 - 7.17 (m, 4H),
.77 (d, J = 8.4 Hz, 1H), 4.79 (s, 2H), 3.95 (t, J = 5.9 Hz, 2H), 3.01 (t, J = 5.8 Hz, 2H).
6
1
3
C NMR (100 MHz, DMSO-d ) δ 157.7, 150.7, 145.5, 138.1, 138.0, 134.8, 134.1,
6
1
2
28.0, 127.8, 127.0, 126.0, 125.8, 125.5, 122.1, 115.0, 114.9, 109.8, 107.5, 46.3, 41.8,
+
7.8. HR-ESI-MS: [M+H] m/z = 327.1604, calcd for C H N , found 327.1585.
2
1
18
4
4
.1.6.2 2-(6-(3,4-dihydroisoquinolin-2(1H)-yl)pyridin-2-yl)-1H-benzo[d]imidazol-6-ol
(BD2).
Following the general procedure above, BD3 was obtained firstly. Then compound
BD3 was dissolved in HBr (48% in H O). The mixture was stirred at 120 °C for 3 h
2
and then concentrated in vacuo. Compound BD2 was obtained as a yellow solid
1
(
50%). H NMR (400 MHz, CD OD) δ 7.84 (dd, J = 8.7, 7.3 Hz, 1H), 7.67 (d, J = 9.1
3
Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.31 (d, J = 7.0 Hz, 1H), 7.25 - 7.12 (m, 6H), 4.90
13
(
s, 2H), 4.03 (t, J = 5.9 Hz, 2H), 3.03 (t, J = 5.9 Hz, 2H). C NMR (100 MHz,
CD OD) δ 159.9, 158.8, 148.9, 140.6, 139.9, 136.5, 135.2, 134.2, 129.3, 127.7, 127.5,
3
+
1
27.3, 126.4, 118.2, 115.8, 112.3, 112.0, 99.3, 47.8, 43.7, 29.8. HR-ESI-MS: [M+H]
m/z = 343.1553, calcd for C H N O, found 343.1522.
21
18
4

ACCEPTED MANUSCRIPT
4
2
.1.6.3
-(6-(6-methoxy-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoli
ne (BD3).
1
Following the general procedure above, BD3 was obtained as a white solid (22%). H
NMR (400 MHz, DMSO-d ) δ 7.64 (t, J = 7.9 Hz, 1H), 7.48 (d, J = 7.3 Hz, 2H), 7.25
6
(d, J = 7.4 Hz, 1H), 7.18 - 7.05 (m, 4H), 6.86 (d, J = 8.5 Hz, 1H), 6.81 (d, J = 7.9 Hz,
13
1
H), 4.79 (s, 2H), 3.93 (t, J = 5.5 Hz, 2H), 3.76 (s, 3H), 2.90 (t, J = 5.4 Hz, 2H). C
NMR (100 MHz, DMSO-d ) δ 157.8, 156.2, 150.5, 146.4, 138.5, 135.1, 134.5, 128.4,
6
1
26.5, 126.2, 125.9, 119.7, 111.5, 109.3, 107.2, 94.7, 55.3, 46.5, 41.9, 28.1.
+
HR-ESI-MS: [M+H] m/z = 357.1710, calcd for C H N O, found 357.1688.
2
2
20
4
4
2
.1.6.4
-(6-(5,6-difluoro-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquino
line (BD4).
1
Following the general procedure above, BD4 was obtained as a white solid (25%). H
NMR (400 MHz, CDCl ) δ 10.37 (s, 1H), 7.81 - 7.50 (m, 4H), 7.41 - 7.28 (m, 2H),
3
7
3
1
1
.24 (s, 3H), 6.86 - 6.73 (m, 1H), 4.80 (d, J = 11.6 Hz, 2H), 3.95 (t, J = 5.9 Hz, 2H),
13
.03 (t, J = 5.8 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 157.9, 152.9, 148.4, 148.2,
6
46.0, 145.9, 145.0, 138.6, 135.0, 134.3, 128.4, 126.4, 126.2, 126.0, 110.0, 108.3,
+
03.1, 102.8, 46.5, 41.8, 28.1. HR-ESI-MS: [M+H] m/z = 363.1416, calcd for
C H F N , found 363.1395.
21
16
2
4
4
2
.1.6.5
-(6-(6-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydro
isoquinoline (BD5).
1
Following the general procedure above, BD5 was obtained as a white solid (20%). H
NMR (400 MHz, CDCl ) δ 7.73 (d, J = 8.8 Hz, 1H), 7.69 - 7.57 (m, 2H), 7.46 (d, J =
3
8
3
1
1
.7 Hz, 1H), 7.38 (s, 1H), 7.19 - 7.07 (m, 4H), 6.74 (d, J = 8.4 Hz, 1H), 4.73 (s, 2H),
13
.89 (t, J = 5.8 Hz, 2H), 2.96 (t, J = 5.8 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ
6
57.9, 153.3, 145.5, 143.9, 139.7, 138.6, 135.0, 134.3, 128.3, 127.5, 126.5, 126.2,
25.9, 120.6, 120.4 (q, J = 255.2 Hz), 117.8, 116.2, 110.1, 108.2, 46.5, 41.9, 28.1.
+
HR-ESI-MS: [M+H] m/z = 411.1427, calcd for C H F N O, found 411.1421.
2
2
17
3
4
4
.1.6.6

ACCEPTED MANUSCRIPT
2
-(6-(6-fluoro-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline
(BD6).
1
Following the general procedure above, BD6 was obtained as a white solid (20%). H
NMR (400 MHz, CDCl ) δ 10.34 (s, 1H), 7.78 - 7.61 (m, 3H), 7.52 - 7.43 (m, 1H),
3
7
3
1
.25 – 7.19 (m, 4H), 7.09 - 7.00 (m, 1H), 6.78 (dd, J = 8.3, 3.6 Hz, 1H), 4.79 (s, 2H),
13
.96 (t, J = 5.9 Hz, 2H), 3.02 (t, J = 5.9 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ
6
60.0, 157.9, 157.7, 152.3, 145.3, 138.6 (2C), 135.0, 134.3, 128.3, 126.5, 126.2, 125.9,
+
1
11.0, 110.7, 110.0 (2C), 108.1, 46.5, 41.8, 28.1. HR-ESI-MS: [M+H] m/z =
3
45.1510, calcd for C H FN , found 345.1488.
21 17 4
4
2
.1.6.7
-(6-(6-(tert-butyl)-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquin
oline (BD7).
1
Following the general procedure above, BD7 was obtained as a white solid (50%). H
NMR (400 MHz, DMSO-d ) δ 12.50 (s, 1H), 7.74 - 7.68 (m, 1H), 7.63 - 7.52 (m, 3H),
6
7
.32 (d, J = 7.4 Hz, 2H), 7.26 - 7.17 (m, 3H), 6.96 (d, J = 8.5 Hz, 2H), 4.86 (s, 2H),
1
3
4
.01 (t, J = 5.9 Hz, 2H), 2.97 (t, J = 5.8 Hz, 2H), 1.37 (s, 9H). C NMR (100 MHz,
DMSO-d ) δ 157.66, 151.03, 146.32, 144.90, 137.94, 134.82, 134.17, 127.90, 126.06,
6
1
25.82, 125.50, 119.74, 119.69, 119.53, 109.39, 106.99, 46.36, 41.84, 34.11, 31.28
+
(
3C), 27.87. HR-ESI-MS: [M+H] m/z = 383.2230, calcd for C H N , found
2
5
26
4
3
83.2210.
4
2
.1.6.8
-(6-(6-chloro-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline
(BD8).
1
Following the general procedure above, BD8 was obtained as a white solid (40%). H
NMR (400 MHz, CDCl ) δ 7.73 (d, J = 7.3 Hz, 1H), 7.67 (t, J = 7.8 Hz, 2H), 7.63 -
3
7
4
.55(m, 1H), 7.27 (d, J = 2.5 Hz, 1H), 7.25 - 7.20 (m, 4H), 6.79 (d, J = 8.4 Hz, 1H),
1
3
.79 (s, 2H), 3.96 (t, J = 5.9 Hz, 2H), 3.03 (t, J = 5.8 Hz, 2H). C NMR (100 MHz,
DMSO-d ) δ 157.9, 152.8, 145.8, 138.6, 135.3, 135.1, 134.3, 133.4, 128.4, 127.0,
6
1
26.5, 126.2, 126.0, 122.9, 120.4, 118.4, 110.1, 108.0, 46.5, 41.8, 28.1. HR-ESI-MS:
+
[
M+H] m/z = 361.1215, calcd for C H ClN , found 361.1197.
2
1
17
4
4
.1.6.9

ACCEPTED MANUSCRIPT
2
-(6-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydrois
oquinoline (BD9).
1
Following the general procedure above, BD9 was obtained as a white solid (36%). H
NMR (400 MHz, CDCl ) δ 7.75 (d, J = 7.3 Hz, 1H), 7.67 - 7.62 (m, 2H), 7.52 (d, J =
3
8
5
1
.2 Hz, 2H), 7.24 - 7.16 (m, 4H), 6.79 (d, J = 8.4 Hz, 1H), 4.78 (s, 2H), 3.93 (t, J =
1
3
.8 Hz, 2H), 3.01 (t, J = 5.7 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 157.9, 153.7,
6
45.5, 138.6, 137.0, 135.1, 134.3, 134.0, 128.4, 126.5, 126.2, 126.0, 119.9, 119.4,
+
1
16.4, 112.9, 110.2, 109.3, 108.3, 46.5, 41.8, 28.1. HR-ESI-MS: [M+H] m/z =
3
95.1478, calcd for C H F N , found 395.1455.
22 17 3 4
4
6
.1.6.10
-methoxy-2-(6-(6-methoxy-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydr
oisoquinoline (BD10).
Following the general procedure above, BD10 was obtained as a light yellow solid
1
(
22%). H NMR (400 MHz, CDCl ) δ 7.74 - 7.61 (m, 3H), 7.15 (d, J = 8.3 Hz, 1H),
3
7
.03 (brs, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.73 (d, J = 8.4 Hz,
H), 4.71 (s, 2H), 3.93 (t, J = 5.8 Hz, 2H), 3.88 (s, 3H), 3.81 (s, 3H), 2.99 (t, J = 5.7
2
13
Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 157.9, 157.7, 156.2, 150.6, 146.4, 138.5,
6
1
36.3, 135.3, 127.5, 126.4, 119.7, 113.0, 112.3, 111.5, 109.2, 107.2, 101.3, 94.6, 55.4,
+
5
5.0, 46.1, 41.8, 28.4. HR-ESI-MS: [M+H] m/z = 387.1816, calcd for C H N O ,
2
3
22
4
2
found 387.1793.
4
6
.1.6.11
-fluoro-2-(6-(6-fluoro-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-1,2,3,4-tetrahydroiso
quinoline (BD11).
Following the general procedure above, BD11 was obtained as a light yellow solid
1
(
20%). H NMR (400 MHz, CDCl ) δ 7.78 - 7.64 (m, 3H), 7.52 - 7.45 (m, 1H), 7.25 -
3
7
.19 (m, 2H), 7.08 - 7.01 (m, 1H), 6.97 - 6.89 (m, 2H), 6.78 (dd, J = 7.9, 4.1 Hz, 1H),
13
4
.76 (s, 2H), 3.94 (t, J = 5.9 Hz, 2H), 3.02 (t, J = 5.8 Hz, 2H). C NMR (100 MHz,
DMSO-d ) δ 157.9, 153.1, 147.1 (d, JC-F = 237.4 Hz), 146.9 (d, JC-F = 237.4 Hz),
6
1
1
45.4, 138.6, 137.5, 135.0, 134.3, 130.4, 128.4, 126.4, 126.2, 125.9, 110.3, 109.9,
+
08.4, 108.1, 47.0, 46.4, 28.61. HR-ESI-MS: [M+H] m/z = 363.1416, calcd for
C H F N , found 363.1377.
21
16
2
4

ACCEPTED MANUSCRIPT
4
.1.6.12 2-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoquinoline (B1).
1
Following the general procedure above, B1 was obtained as a white solid (50%). H
NMR (400 MHz, DMSO-d ) δ 12.84 (s, 1H), 7.82 (s, 1H), 7.68 (d, J = 7.6 Hz, 1H),
6
7
5
5
1
.60-7.52 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H), 7.30 (d, J = 7.0 Hz, 1H), 7.27 - 7.17 (m,
H), 7.14 (dd, J = 8.2, 1.6 Hz, 1H), 4.52 (s, 2H), 3.66 (t, J = 5.8 Hz, 2H), 3.00 (t, J =
1
3
.7 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 151.8, 150.3, 143.7, 134.8, 134.6,
6
34.2, 130.8, 129.5, 128.3, 126.5, 126.2, 125.8, 122.3, 121.5, 118.7, 116.0, 115.9,
+
1
12.0, 111.1, 49.5, 45.4, 28.1. HR-ESI-MS: [M+H] m/z = 326.1652, calcd for
C H N , found 326.1629.
22
19
3
4
.1.6.13
2
-(3-(6-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoquinoline (B2).
1
Following the general procedure above, B2 was obtained as a white solid (40%). H
NMR (400 MHz, CDCl ) δ 7.72 (s, 1H), 7.55 (dd, J = 8.3, 4.5 Hz, 1H), 7.31-7.20 (m,
3
3
H), 7.18 - 7.10 (m, 3H), 7.09 - 7.04 (m, 1H), 6.95-6.87 (m, 2H), 4.40 (s, 2H), 3.54 (t,
1
3
J = 5.8 Hz, 2H), 2.92 (t, J = 5.7 Hz, 2H). C NMR (100 MHz, CDCl ) δ 158.6 (d, J =
3
1
95.3 Hz), 153.6, 150.3, 140.4, 134.6, 134.2, 131.6, 129.6, 128.3, 126.5, 126.3, 125.9,
119.6 (d, J = 10.3 Hz), 116.1, 116.0, 112.0, 104.2 (d, J = 23.7 Hz), 97.61 (d, J = 26.5
+
Hz), 49.5, 45.5, 28.1. HR-ESI-MS: [M+H] m/z = 344.1558, calcd for C H FN ,
22
18
3
found 344.1533.
4
.1.6.14 2-(3-(6-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-
tetrahydroisoquinoline (B3).
1
Following the general procedure above, B3 was obtained as a white solid (32%). H
NMR (400 MHz, DMSO-d ) δ 7.80 (s, 1H), 7.76 - 7.67 (m, 1H), 7.57 (d, J = 7.7 Hz,
6
2
2
1
H), 7.42 (t, J = 7.9 Hz, 1H), 7.32 - 7.14 (m, 6H), 4.53 (s, 2H), 3.67 (t, J = 5.8 Hz,
1
3
H), 3.01 (t, J = 5.7 Hz, 2H). C NMR (125 MHz, DMSO-d ) δ 153.9, 150.8, 135.1,
6
34.7, 131.0, 130.8, 130.1, 128.8, 128.7, 127.0, 126.9, 126.7, 126.4, 122.6, 116.8,
+
1
16.7, 116.5, 112.9, 112.5, 50.0, 45.9, 28.6. HR-ESI-MS: [M+H] m/z = 360.1262,
calcd for C H ClN , found 360.1242.
2
2
18
3
4
.1.6.15
-(3-(6-methyl-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoquinoline
2
(B4).

ACCEPTED MANUSCRIPT
Following the general procedure above, B4 was obtained as a light yellow solid (40%).
1
H NMR (400 MHz, DMSO-d ) δ 12.67 (d, J = 14.5 Hz, 1H), 7.76 (s, 1H), 7.52 (d, J
6
=
7.4 Hz, 1H), 7.45-7.38 (m, 1H), 7.36 (t, J = 8.0 Hz, 1H), 7.31 - 7.24 (m, 2H), 7.21 -
7
.16 (m, 3H), 7.09 (dd, J = 8.2, 2.2 Hz, 1H), 7.04-6.95 (m, 1H), 4.48 (s, 2H), 3.62 (t, J
1
3
=
5.9 Hz, 2H), 2.96 (t, J = 5.8 Hz, 2H), 2.41 (d, J = 8.4 Hz, 3H). C NMR (100 MHz,
DMSO-d ) δ 150.3 (2C), 134.6, 134.3, 130.9, 129.5, 128.3, 127.8, 127.5, 126.9, 126.6,
6
1
26.3, 125.9, 123.1, 118.3, 115.9, 115.8, 111.9, 110.9, 49.6, 45.5, 28.2, 21.3.
+
HR-ESI-MS: [M+H] m/z = 340.1808, calcd for C H N , found 340.1776.
2
3
21
3
4
.1.6.16
-(3-(6-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoquinoline
2
(B5).
1
Following the general procedure above, B5 was obtained as a white solid (30%). H
NMR (400 MHz, CD OD) δ 7.70 (s, 1H), 7.49-7.39 (m, 2H), 7.35 (t, J = 7.9 Hz, 1H),
3
7
.20 - 7.03 (m, 6H), 6.87 (dd, J = 8.8, 2.3 Hz, 1H), 4.44 (s, 2H), 3.83 (s, 3H), 3.60 (t,
1
3
J = 5.9 Hz, 2H), 2.97 (t, J = 5.8 Hz, 2H). C NMR (125 MHz, DMSO-d ) δ 155.8,
6
1
1
3
50.4, 150.3, 134.7, 134.6, 134.3, 134.3, 131.1, 131.0, 129.6, 128.3, 126.5, 126.2,
+
25.9, 115.9, 115.8, 112.0, 111.8, 55.4, 49.6, 45.5, 28.1. HR-ESI-MS: [M+H] m/z =
56.1757, calcd for C H N O, found 356.1732.
2
3
21
3
4
.1.6.17
-(3-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoquin
2
oline (B6).
Following the general procedure above, B6 was obtained as a light yellow solid (29%).
1
H NMR (400 MHz, CDCl ) δ 7.75 (s, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.42 - 7.34 (m,
3
2
2
1
H), 7.21 - 7.10 (m, 4H), 7.09 - 7.05 (m, 1H), 4.46 (s, 2H), 3.61 (t, J = 5.9 Hz, 2H),
13
.97 (t, J = 5.8 Hz, 2H). C NMR (100 MHz, CDCl ) δ 155.1, 150.9, 134.8, 134.0,
3
32.2, 132.0, 130.0 (2C), 128.4 (2C), 126.6 (2C), 126.2 (2C), 119.8, 116.9, 116.4,
+
1
16.3, 112.8 (2C), 50.5, 45.9, 29.0. HR-ESI-MS: [M+H] m/z = 394.1526, calcd for
C H F N , found 394.1514.
23
18
3
3
4
.1.6.18
-(3-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoquinoline
2
(B7).

ACCEPTED MANUSCRIPT
Following the general procedure above, B7 was obtained as a light yellow solid (32%).
1
H NMR (400 MHz, DMSO-d ) δ 13.16 (s, 1H), 7.92 (s, 1H), 7.74 (d, J = 6.0 Hz, 2H),
6
7
3
1
.53 (d, J = 7.3 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.29 - 7.08 (m, 5H), 4.48 (s, 2H),
13
.61 (d, J = 5.0 Hz, 2H), 2.95 (s, 2H). C NMR (100 MHz, DMSO- d ) δ 154.5, 150.4,
6
43.5, 134.7, 134.5, 134.2, 129.9, 129.7, 128.3, 126.6, 126.3, 125.9, 124.6, 124.1,
+
1
19.8, 116.59, 116.2, 112.6, 112.2, 49.5, 45.5, 28.2. HR-ESI-MS: [M+H] m/z =
3
94.0872, calcd for C H Cl N , found 394.0860.
22 17 2 3
4
.1.6.19
-(3-(5-chloro-6-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoquino
2
line (B8).
Following the general procedure above, B4 was obtained as a light yellow solid (40%).
1
H NMR (400 MHz, DMSO-d ) δ 7.88 (d, J = 6.9 Hz, 1H), 7.79 - 7.76 (m, 1H), 7.74 -
6
7
.68 (m, 1H), 7.58 – 7.52 (m, 1H), 7.44 - 7.38 (m, 1H), 7.29- 7.27(m, 1H), 7.23 - 7.15
1
3
(
m, 4H), 4.51 (s, 2H), 3.65 (t, J = 5.9 Hz, 2H), 2.98 (t, J = 5.8 Hz, 2H). C NMR (100
MHz, DMSO-d ) δ 154.5, 154.1, 150.4, 140.6, 134.7, 134.2, 131.7, 130.1, 129.7,
6
1
28.3, 126.6, 126.3, 125.9, 119.4, 116.4, 116.1, 112.1, 111.9, 105.6, 49.5, 45.5, 28.1.
+
HR-ESI-MS: [M+H] m/z = 378.1168, calcd for C H ClFN , found 378.1151.
2
2
17
3
4
.1.6.20
2-(3-(3,4-dihydroisoquinolin-2(1H)-yl)phenyl)-1H-benzo[d]imidazol-6-ol
(B9).
Following the general procedure above, B5 was obtained firstly and then dissolved in
HBr (48% in H O). The mixture was stirred at 120 °C for 3 h and then concentrated in
2
1
vacuo. Compound B9 was obtained as a yellow solid (50%). H NMR (400 MHz,
CD OD) δ 7.77 (s, 1H), 7.65 - 7.60 (m, 2H), 7.53 (d, J = 7.6 Hz, 1H), 7.47 (dd, J =
3
8
.2, 2.1 Hz, 1H), 7.27 - 7.20 (m, 3H), 7.14-7.10 (m, 3H), 4.63 (s, 2H), 3.79 (t, J = 6.0
13
Hz, 2H), 3.11 (t, J = 5.9 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 156.5, 150.3,
6
1
48.1, 134.6, 134.0, 132.6, 130.5, 128.5, 128.4, 126.6, 126.5, 126.1, 124.7, 123.5,
+
1
18.5, 116.2, 114.7, 112.5, 98.0, 49.3, 45.1, 27.9. HR-ESI-MS: [M+H] m/z =
3
42.1601, calcd for C H N O, found 342.1574.
22 19 3
4
.1.6.21
-(3-(3,4-dihydroisoquinolin-2(1H)-yl)phenyl)-1H-benzo[d]imidazol-7-amine (B10).
2
Following the general procedure above, the intermediate 12 (100 mg, 0.39 mmol) and

ACCEPTED MANUSCRIPT
1
,2-diamino-3-nitrobenzene (66 mg, 0.43 mmol) were reacted. The resulting product,
a catalytic amount of Ni and hydrazine hydrate in MeOH were sirred at 50°C for 2 h.
After filtration, the solvent was removed under reduced pressure and the pure
1
compound B10 was obtained as a black solid (41%). H NMR (400 MHz, CD OD) δ
3
7
7
2
.77 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.27 - 7.14 (m, 5H),
.04 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 6.58 (d, J = 7.6 Hz, 1H), 4.53 (s,
1
3
H), 3.69 (t, J = 5.8 Hz, 2H), 3.05 (t, J = 5.7 Hz, 2H). C NMR (100 MHz, DMSO-d )
6
δ 150.8, 150.5, 137.0, 135.1, 134.7, 132.0, 131.6, 130.0, 129.0, 128.8, 127.2, 127.0,
+
1
26.7, 126.4, 116.8, 116.6, 116.3, 114.6, 112.3, 50.1, 46.0, 28.7. HR-ESI-MS: [M+H]
m/z = 341.1761, calcd for C H N , found 341.1734.
22
20
4
4
.1.6.22
-methoxy-2-(3-(6-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,3,4-tetrahydroisoq
6
uinoline (B11).
Following the general procedure above, B11 was obtained as a yellow solid (42%).
1
H NMR (400 MHz, CDCl ) δ 7.72 (s, 2H), 7.41 - 7.27 (m, 3H), 7.07 (d, J = 8.4 Hz,
3
1
H), 7.03 (d, J = 8.1 Hz, 1H), 6.97 (s, 1H), 6.92 (dd, J = 8.8, 2.4 Hz, 1H), 6.77 (dd, J
8.3, 2.5 Hz, 1H), 6.71 (d, J = 2.4 Hz, 1H), 4.43 (s, 2H), 3.87 (s, 3H), 3.80 (s, 3H),
=
1
3
3
1
1
.61 (t, J = 5.8 Hz, 2H), 2.96 (t, J = 5.7 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ
6
66.9, 162.7, 148.7, 144.3, 143.9, 141.8, 141.0, 135.3, 133.8, 131.5, 130.5, 129.6,
24.8, 124.3, 123.2, 122.3, 113.5, 112.4, 106.4, 57.2, 55.9, 49.3, 48.6, 28.6.
+
HR-ESI-MS: [M+H] m/z = 386.1863, calcd for C H N O , found 386.1848.
2
4
23
3
2
4
.2 Cell culture
[
33]
As previously described
, the murine BV-2 microglial cells or HT22 cells
(
Cell Resource Center of Chinese Academy of Medical Science, Beijing, China) were
cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10 %
v/v) fetal bovine serum (Hyclone), 100 U/mL of penicillin, and 100 µg/mL of
(
streptomycin (Hyclone). These cells were cultured in a humidified incubator
containing 5% CO at 37°C and subcultured every two days.
2
4
.3 NO production in LPS-stimulated BV2 microglial cells
Microglia cell-based NO inhibitory activity assay were performed as previously
[
33]
5
described . The BV2 cells were plated in 96-well microplates at a density of 4 × 10
cells/mL (100 µL/well). After 48 h, the medium was removed, and 80 µL of DMEM

ACCEPTED MANUSCRIPT
was added. The cells were pretreated with various concentrations of compounds (10
µL) for 30 minutes, followed by stimulation with LPS (1 µg/mL, 10 µL, Sigma) for
2
4 h at 37°C. The same volume (50 µL) of Griess assay agent (Beyotime
Biotechnology) was added into the 96-well plates and mixed with the culture medium
50 µL). The mixture solution was incubated at room temperature for 10 min, and the
(
absorbance at 540 nm was measured in a multifunctional microplate reader. The
percent of inhibition of NO production was calculated with this formula: (F − F )/(F
L
L
C
−
F ) × 100%, where F = absorbance of the cells treated with the tested compound
0 C
and LPS, F = absorbance of the cells treated with LPS, and F = absorbance of the
L
0
normal cells. The IC was defined as the concentration of the compound that reduced
5
0
5
0% of NO production with LPS and was calculated by nonlinear regression. All the
experiments were repeated independently at least three times.
4
.4 Measurement of level of TNF-α and IL-1β production
The procedure is almost the same as above. BV2 cells were plated in 96-well
5
microplates at a density of 4 × 10 cells/mL. After 48 h, the medium was removed,
and 80 µL of DMEM was added. The compound solutions (10 µL) were added to the
cells firstly, and then 10 µL LPS (1 µg/mL) solution was added. The cells were
incubated at 37°C for 24 h. The levels of TNF-α and IL-1β in the culture medium
were measured using ELISA kits (Boster Biological Technology Co. Ltd) according to
the manufacturer’s instructions.
4
.5 MTT assay
The measurement
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HT22
of
the
cell
viability
was
determined
by
3
cells were plated in 96-well microplates at a density of 5000 cells/well for 24 h,
followed by treated with compounds and glutamate (5 mM) for 24 h. After incubation,
the cell culture media were removed and 20 µL MTT (5 mg/mL) solution was added
to each well and incubated for 4 h at 37°C, after that 120 µL DMSO was added to
each well. The absorbance of solubilized formazan was measured by microplate
reader at 492 nm. Controls were taken as having 100% viability.
4
.6 BACE1 enzymatic assay
[
29, 35]
According to the previously reported method
, BACE1 enzymatic activity was
determined by the enhancement of fluorescence intensity upon enzymatic cleavage of
the fluorescence resonance energy transfer substrate. The human recombinant BACE1
and the substrate peptide (Rh-EVNLDAEFK-Quencher) were purchased from

ACCEPTED MANUSCRIPT
ThermoFisher Scientific. Briefly, the BACE1 assay was conducted in 50 mM sodium
acetate buffer (pH = 4.5), in a final enzyme concentration (1U/mL). The substrate was
used at 750 nM. Compounds in DMSO were preincubated with the enzyme for 20 min
at room temperature. Subsequently, the substrate was added to initiate the reaction.
The reaction was incubated for 60 min at room temperature under dark conditions and
then stopped with 2.5 M sodium acetate. Fluorescence intensity was measured by
microplate reader at excitation and emission wavelength of 545 nm and 585 nm,
respectively.
4
.7 BBB permeation assay
The BBB penetration of compounds was evaluated using the parallel artificial
[
32]
membrane permeation assay (PAMPA) described by Di et al . Porcine brain lipid
PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF
(
membrane, pore size 0.45 mm) and acceptor microplate were both from Millipore.
The 96-well UV plate (COSTAR) was from Corning Incorporated. The acceptor
9
6-well microplate was filled with 300 µL PBS/EtOH/DMSO (68:30:2), and the filter
membrane was impregnated with 4 µL PBL in dodecane (20 mg/mL). Compounds
were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to a final
concentration of 100 µg/mL. Then, 200 µL of the solution was added to the donor
wells. The acceptor filter plate was carefully placed on the donor plate to form a
sandwich, which was left undisturbed for 10 h at 25 °C. After incubation, the donor
plate was carefully removed, and the concentration of compounds in the acceptor
wells was determined using the UV plate reader (Flexstation 3). Every sample was
analyzed at five wavelengths in four wells and in at least three independent runs. Pe
was calculated by the following expression: P = −V × V /[(V + V )A × t] × ln(1 −
e
d
a
d
a
drugacceptor/drugequilibrium), where V is the volume of donor well; V , volume in
d
a
acceptor well; A, filter area; t, permeation time; drugacceptor, the absorbance obtained in
the acceptor well; and drugequilibrium, the theoretical equilibrium absorbance. The
results are given as the mean ± standard deviation. In the experiment, 13 quality
control standards of known BBB permeability were included to validate the analysis
set. A plot of the experimental data versus literature values gave a strong linear
2
correlation, P (exp.) = 1.4574P (lit.) − 1.0773 (R = 0.9427). From this equation and
e
e
−
6
the limit established by Di et al. (P (lit.) = 4.0× 10 cm/s) for BBB permeation, we
e
−6
concluded that compounds with a permeability > 4.7 × 10 cm/s could cross the
[
36]
BBB
.