Discovery of new selective butyrylcholinesterase (BCHE) inhibitors with anti-Aβ aggregation activity: Structure-based virtual screening, hit optimization and biological evaluation

Article abstract of DOI:10.3390/molecules24142568

In this study, a series of selective butyrylcholinesterase (BChE) inhibitors was designed and synthesized from the structural optimization of hit 1, a 4-((3,4 -dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid derivative identified by virtual screening our

Full text of DOI:10.3390/molecules24142568

molecules
Article
Discovery of New Selective Butyrylcholinesterase
(BChE) Inhibitors with Anti-Aβ Aggregation Activity:
Structure-Based Virtual Screening, Hit Optimization
and Biological Evaluation
Cheng-Shi Jiang 1,
*
, Yong-Xi Ge 1, Zhi-Qiang Cheng 1, Yin-Yin Wang 1, Hong-Rui Tao 1,2
,
Kongkai Zhu 1,* and Hua Zhang 1,
*
1
School of Biological Science and Technology, University of Jinan, Jinan 250022, China
Drug Discovery and Design Center, State Key Laboratory of Drug Research,
Shanghai Institute of Meteria Medica, Chinese Academy of Sciences, Shanghai 201203, China
Correspondence: bio_jiangcs@ujn.edu.cn (C.-S.J.); bio_zhukk@ujn.edu.cn (K.Z.);
bio_zhangh@ujn.edu.cn (H.Z.); Tel.: +86-0531-8973-6199 (H.Z.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 June 2019; Accepted: 12 July 2019; Published: 15 July 2019
Abstract: In this study, a series of selective butyrylcholinesterase (BChE) inhibitors
was designed and synthesized from the structural optimization of hit a 4-((3,4
-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid derivative identified by virtual screening
our compound library. The in vitro enzyme assay results showed that compounds
1
,
9
((4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(pyrrolidin-1-yl)methanone) and 23 (N-(2
-bromophenyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide) displayed improved BChE
inhibitory activity and good selectivity towards BChE versus AChE. Their binding modes
were probed by molecular docking and further validated by molecular dynamics simulation.
Kinetic analysis together with molecular modeling studies suggested that these derivatives could
target both the catalytic active site (CAS) and peripheral anionic site (PAS) of BChE. In addition,
the selected compounds 9 and 23 displayed anti-Aβ1–42 aggregation activity in a dose-dependent
manner, and they did not show obvious cytotoxicity towards SH-SY5Y neuroblastoma cells.
Also, both compounds showed significantly protective activity against Aβ1-42-induced toxicity
in a SH-SY5Y cell model. The present results provided a new valuable chemical template for
the development of selective BChE inhibitors.
Keywords: selective BChE inhibitor; virtual screening; structural optimization; molecular dynamics;
anti-Aβ aggregation
1. Introduction
Alzheimer’s disease (AD), one of the most common neurodegenerative disorders, accounts for
about 60–80% of all cases of dementia [
worldwide have AD or a related dementia, and the number of AD patients is expected to triple
by 2050 [ ]. AD has now been the third leading cause of death, outpaced only by cardiovascular
diseases and cancer [ ]. However, there is still no successful therapy or drug to reverse or even slow
1,2]. According to the statistics, nearly 50 million people
3
4
the course of this disease [5].
Although the pathogenesis of AD is complex and not fully understood, several important clinical
hallmarks, such as low level of acetylcholine (ACh), beta-amyloid (A
β
) protein aggregation, and tau
]. In recent years,
and tau have received more attention [ ], however,
(τ)-protein phosphorylation, are involved in the occurrence and development of AD [6
therapies for anti-AD primarily focused on A
β
7
Molecules 2019, 24, 2568; doi:10.3390/molecules24142568
www.mdpi.com/journal/molecules

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various Aβ- and tau-targeting agents have failed in clinical trials [8]. Based on cholinergic dysfunction
hypothesis, increasing the level of ACh in the brain to improve cholinergic neurotransmission is still
the most effective therapy for AD treatment [ ]. The ACh in the brain can be mainly hydrolyzed
9
two types of cholinesterases (ChEs), namely acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) [10]. In the brain of healthy adults, AChE is responsible for 80% of the ACh activity,
nearly 1013 fold more active than BChE [11]. Four AChE inhibitors (tacrine, donepezil, galantamine,
and rivastigmine) have been clinically employed for the management of AD [12]. These drugs
have beneficial effects on improving the cognitive and memorial functions of AD patients at mild
and moderate stages, but tacrine was withdrawn from the market in 2013 due to its hepatotoxicity
and the others drugs cannot stop or reverse the course of AD. In addition, the side effects (e.g., nausea,
vomiting, diarrhea) of AChE inhibitors also limit the administered doses of these drugs [13].
Although BChE played a minor role in regulating the brain ACh level in the health brain,
increasing experimental evidence demonstrated that the function of BChE in termination of cholinergic
neurotransmission significantly changed in brains of patients with progressive AD. In advanced AD
patients, the activity of BChE progressively increases while the AChE activity remains unchanged
or declines [14
the experiment that AChE knockout mice could still survive with the normal level and localization of
BChE [17 18], while silent BChE mutations in humans showed a slower rate of cognitive decline [19].
–16]. Moreover, BChE was believed to compensate the function of AChE based on
,
Therefore, inhibition of BChE was also expected to contribute to ameliorating a cholinergic effect.
This hypothesis was in agreement with the experiment that selective inhibition of BChE could
significantly raise ACh levels and improve the cognitive performance of aged rats, without any adverse
parasympathetic side effects, which are known to be the defect of traditional AChE inhibitors [15,20].
Taken together, selective inhibition on BChE could represent a promising clinical treatment strategy
for AD.
However, the development of inhibitors selectively targeting BChE over AChE is a challenging
task, since the two isoforms hAChE and hBChE are highly homologous proteins [21]. Although many
structurally diverse selective BChE inhibitors, such as carbamate-based compounds [22], paltanic acid
derivatives [23], naphthamide compounds [24], phenserine-based compounds [25], and isosorbide
analogues [26], have been reported, very few of them (bisnorcymserine and eptastigmine) have entered
into clinical trials [16,27]. Thus, the discovery of effective selective BChE inhibitors with novel molecular
skeletons is urgently needed. Virtual screening has been proven to be a very powerful approach for
discovering new drug hits with desired properties and structural diversity [28], while a structure-activity
relationship (SAR) study based on enzyme or cell bioassay has emerged as the most versatile to in
provide useful information for structural modification and improvement [29,30].
In our project for discovering new ChE inhibitors [31
–36], a new selective BChE inhibitor 1
(BChE, IC50 = 17.94 1.86 M; AChE, IC50 > 50 M) (Figure 1 and Table 1) was recently identified
±
µ
µ
from our in-house compound library by performing virtual screening and in vitro BChE/AChE
assay. The follow-up molecular docking-guided chemical optimization and SAR study starting
from hit
and 23 showed improved BChE inhibitory activity by 9 and 21 folds compared with
both compounds and 23 displayed anti-A aggregation activity and nontoxicity against SH-SY5Y
cells. Herein, we describe a series of new selective BChE inhibitors with anti-A aggregation activity
1
resulted in some new potent selective BChE inhibitors. Among them, compounds
9
1. In addition,
9
β
β
using the combination of virtual screening/molecular, synthesis and in vitro biological evaluation,
as well as the molecular dynamics simulation study on the most potent compound 23.
2. Results and Discussion
2.1. Molecular Docking-Based Virtual Screening to Identify Hit 1 as Promising Selective BChE Inhibitor
The BChE:tacrine complex crystal structure (PDB entry: 4BDS) [37] was used to perform molecular
docking-based virtual screening. Residues in BChE centered on tacrine (15 Å) were defined as a binding

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pocket. An in-house database, containing 1225 compounds with different scaffold was screened in
silico to identify the active compounds against BChE. The docking score of tacrine redocked to BChE
was
−
8.498, while the docking scores of the top-100 compounds was higher than that of tacrine,
so the top-100 compounds ranked according to the docking score were obtained for the following
visual selection. After clustering the 100 compounds with “Clustering Molecules” protocols inserted
in Pipeline Plot 7.5, we finally selected 30 compounds for experimental validation. Among them,
compound 1, showed highest inhibitory activity against BChE with IC50 value of 17.94
without obvious activity towards AChE (inhibitory ratio = 26.6% at 50 M). A literature survey showed
that compound was a new compound that was structurally different from the previously reported
±
1.86 µM,
µ
1
BChE inhibitor, which highlighted that compound
structural optimization.
1 was used as a hit compound for subsequent
Figure 1. In silico screening procedure and ChE assay results of hit 1. (A) The discovery workflow of
the hit compound. (B) Chemical structure of hit 1 and its ChE inhibitory activity.
2.2. Binding mode of 1 Provides Insights for Hit Optimization
In order to get guidance for rational structural optimization of hit 1, the proposed binding mode of 1
with the active sites of BChE was explored using a molecular docking method. As depicted in Figure 2A,
compound
1 fitted well in the active-site gorge of BChE, and its tertrahydroisoquinoline fragment
was located in the catalytic active site (CAS) which had spatial overlapping with tacrine, while
the amino side-chain was oriented towards the peripheral anionic site (PAS) of BChE. Figure 2B
showed the important interactions involved between compound
including and cation- interactions of tertrahydroisoquinoline fragment with W82 (a key residue
in the CAS) and interaction of benzene fragment with Y332 (a key residue in the PAS of BChE).
1 and the residues of BChE,
π-
π
π
π-π
Since the interactions with W82 and Y332 play an important role in BChE inhibition, the two key
fragments (tertrahydroisoquinoline and benzene) should be retained before performing chemical
modifications. With this in mind, the amino side-chain was then rationally modified based on the above
molecular docking result in order to improve its inhibitory activity against BChE.

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Figure 2. Proposed binding mode of hit
magenta) in the active pocket of BChE (PDB: 4BDS) with tacrine as ligand in green color; (
interactions of with residues in the active sites of BChE. stacking and cation- interaction are
1
with BChE. (
A
) The predicted orientation of
1
(carbon in
B) The key
1
π-
π
π
displayed in green and red dashed lines, respectively.
2.3. Chemcial Synthesis
To improve the potency of hit 1 and to discuss the structure-activity relationship (SAR), a range
of amide and ester derivatives of 4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid were
designed and synthesized based on molecular docking analysis. The synthetic route outlined in
Scheme 1 started from the commercial tertrahydroisoquinoline or its derivatives A, which reacted
with 4-(bromomethyl)benzoic acid to give intermediates B. Subsequent condensation reaction of acids
B with various alkyl amines, aromatic amines, phenol, thiophenol or tryptamines smoothly yielded
the target compounds 1–30.
Scheme 1. Synthesis of compounds
1–30. (a) 4-(bromomethyl)benzoic acid or its derivatives, THF,
reflux, 8 h; ( ) agents including alkyl amines, aromatic amines, phenol, thiophenol or tryptamines,
b
HATU, DIPEA, DMF, r.t., 8 h.
2.4. Biological Evaluation and SAR Study
The Ellman’s method was used to determine all the compounds’ BChE and AChE inhibitory
activities, and tacrine was used as the reference control. The SAR investigation was mainly focused
on the amino side-chain in the first round of structural modification. From Table 1, modification of
the terminal amino group in compound
led to a significant loss of BChE inhibitory activity or selectivity towards BChE. Scaffold hopping-based
cyclization of the diethylamino group was tried to yield analogs 6–10, of which the compounds
1 using homologue (compounds 2–4) or isostere (compound 5)
7–9
possessing tetrahydropyrrole ring showed improved inhibition and selectivity towards BChE,
with the best IC50 value of 2.68 ± 0.28 µM for 9.

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Table 1. The inhibitory activities of compounds 1–10 against ChEs.
BChE
IC50 (µM)
AChE
No.
1
R
IR a(%)
IR(%)
IC50 (µM)
b
68.4
17.9 ± 1.86
9.98 ± 0.89
26.6
-
2
76.4
56.1
21.46 ± 0.98
3
4
36.9
27.2
-
-
11.4
8.8
-
-
5
56.6
38.10 ± 1.68
2.7
-
6
7
19.1
45.4
-
13.4
4.4
-
-
15.2 ± 2.33
8
9
63.0
95.1
24.98 ± 1.27
13.3
29.1
-
-
2.68 ± 0.28
10
11.2
98.4
-
3.4
-
Tacrine
0.14 ± 0.01
96.2
0.11 ± 0.03
a
IC50 values are expressed as mean
±
SD of three separate experiments. Inhibition ratio (IR) against ChE at 50
µM.
b
Not tested.
With compound
9 in hand, the substituent effect at C-6 and C-7 of tetrahydroisoquinoline on
the BChE inhibitory activity was studied through the evaluation of analogs 11–14. The results in
Table 2 indicated that both monosubstitution and disubstitution made the BChE inhibitory activity
and selectivity decrease, compared with
9, suggesting that it might not accommodate the substitution
at C-6 and C-7 of a tetrahydroisoquinoline fragment.
Table 2. The inhibitory activities of compounds 11–14 against ChEs.
No.
BChE IC50 (µM)
AChE IC50 (µM)
11
12
13
14
R1 = Br, R2 = H
R1 = H, R2 = Br
R1 = OMe, R2 = H
R1 = OMe, R2 = OMe
8.38 ± 0.65
39.44 ± 2.25
12.62 ± 0.88
-
30.61 ± 1.82
>50
12.13 ± 0.90
18.24 ± 0.86
IC50 values are expressed as mean ± SD of three separate experiments.
Next, the tetrahydroisoquinoline ring was unchanged and the tetrahydropyrrole group was later
replaced by various benzene groups. The results were shown in Table 3. Among compounds 15 18, only
one compound (16) with 2-fluoroanilino group showed BChE activity (IC50 = 6.9 M), indicating that
–
µ

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2-subsititution at benzene ring might be optimal for BChE activity and 3-/4-subsititution was inadvisable.
Notably, replacement of 2-F by 2-Br led to an obvious increase of the activity of 23 by about 3- and 8-fold
compared with that of
C-4 (25) obviously made its BChE inhibitory activity lose, which was in accordance with the SAR
obtained from compounds 16 18. Then, replacement of 2-bromoanilino moitey in compound 23 by
9 and 16, respectively. Changing the position of Br from C-2 to C-3 (24) or
–
2-bromophenol (26) or 2-bromophenylthio (27) also led to a significant loss of activity, suggesting that
the amide group takes an important role in its activity. Recently, Mahdavi et al. reported a group
of potent selective tryptamine-derived BChE inhibitors [38], thus the effect of tryptamine fragment
fused into our compounds 28
–30 on BChE activity was evaluated. Unfortunately, all these three target
compounds were inactive toward BChE.
Table 3. The inhibitory activities of compounds 15–30 against ChEs.
BChE
IC50 (µM)
AChE
IC50 (µM)
No.
15
R
IR a(%)
IR(%)
-
b
23.7
8
-
16
74.7
6.93 ± 0.48
2.1
-
17
18
19
10.0
14.1
83.9
-
7.7
7.0
2.3
-
-
-
-
14.78 ± 0.58
20
21
13.2
38.6
-
-
21.8
5
-
-
22
23
15.4
97.9
-
28.3
22.7
-
-
0.83 ± 0.08
24
25
35.1
8.1
-
-
21.6
40.6
-
-

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Table 3. Cont.
BChE
AChE
IC50 (µM)
No.
26
R
IR a(%)
IC50 (µM)
IR(%)
11
-
26.2
-
27
28
1.5
1.3
-
-
22.8
8.4
-
-
29
30
6.5
1.1
-
-
4.6
8.3
-
-
a
IC50 values are expressed as mean
Not tested.
±
SD of three separate experiments. Inhibition ratio (IR) against ChE at 50 µM.
b
2.5. Kinetic Study on BChE Inhibition
To further understand the action mechanism for BChE inhibition of this series of compounds,
the kinetic assay was performed with the optimal compound 23 as a model compound. The enzyme
kinetic result reveals that increasing slope (decreasing Vmax) and increased Kmax with increasing
concentrations of 23 (Figure 3A). This pattern clearly indicates compound 23 is mixed-type inhibitor that
simultaneously interacts with both CAS and PAS of BChE. The result was confirmed by the subsequent
molecular docking study of 23. Replots of the slope against the concentrations of 23 indicates that
the KI value is 0.52 µM, which fits its IC50 value for BChE inhibition.
Figure 3. Kinetic study of 23 on BChE inhibition. (A) The Lineweaver–Burk reciprocal plots records
enzyme velocity curves-substrate concentration in the presence of 23 at different concentrations;
(B) Plot of slopes versus the concentrations of 23.
2.6. Binding Mode Analysis
A probable binding mode of 23 with BChE was obtained by conducting molecular docking.
As illustrated in Figure 4A, compound 23 could well occupy the tacrine binding site and displayed
similar binding mode with tacrine. In addition, the bromophenyl of 23 extended outside of tacrine
binding site. As we can see from Figure 4B,C, the binding pocket of 23 was a hydrophobic pocket

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composed of D70, W82, G115, G116, Y128, E197, P285, A328, F329, Y332, and H438 residues, and the π-π
interactions of 23 with W82 and Y332 were retained. Hydrophobic interaction is the main interaction
in stabilizing 23 to the tacrine binding site.
Figure 4. Predicted binding mode of 23 with BChE. (
of 23 and tacrine. BChE, 23 and tacrine are shown as light-orange cartoon, magenta, and cyan
sticks. ( ) Detailed interactions between 23 and BChE. Compound 23 is shown as magenta sticks,
A) Superimposition of the binding modes
B
while the carbon atoms of the interacting residues are colored in white. (C) Key interactions involved
in stabilizing 23 at the tacrine binding site.
2.7. Molecular Dynamics
To further validate the binding mode of 23 with BChE, 100 ns molecular dynamics (MD) simulation
was employed on the complex model of BChE: 23 that was constructed using molecular docking.
The root-mean-square deviations (RMSDs) values for the heavy atoms of BChE and 23 (Figure S1)
were steady, which indicated that MD trajectory is reliable for further analysis. Then the binding free
energy of 23 with BChE was calculated by Molecular Mechanics Poisson-Boltzmann Surface Area
(MM-PBSA) method, as shown in Table 4, the value ( 25.93 kcal/mol) indicated a stable binding of 23
−
with BChE. Besides, based on MD trajectories, the occupancy rate of hydrophobic interactions between
23 and the active site residues was calculated and shown in Table 5. This result indicated that all
the hydrophobic interactions between 23 and BChE could be well retained during MD simulation.
Table 4. The binding free energy calculated by MM-PBSA method.
∆
Ggas represents the binding free
G = Ggas Gsolv.
energy in vacuum and Gsolv represents the solvation free energy changes.
∆
∆
∆
+
∆
The data were shown in kcal/mol.
Inhibitor
∆Ggas
−59.52 ± 10.41
∆Gsolv
∆G
−25.93 ± 5.07
23
33.59 ± 9.06

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Table 5. The hydrophobic interactions residues in BChE and their corresponding occupancy rates.
Residue
Occupancy Rate (%)
Residue
P285
Occupancy Rate (%)
D70
W82
G115
G116
Y128
E197
79
100
58
61
57
80
78
59
A328
F329
56
Y332
68
H438
55
2.8. Inhibition on Aβ Aggregation
The A peptide aggregation in the brain is known to trigger the onset of neurodegenerative disease
and considered as a key pathological hallmark of AD [39 40]. The aggregation and accumulation of
the A peptide could form oligomers and plaques, which is a step-by-step process starting from a soluble
monomer and proceeding through oligomeric structures, protofibrils, and eventually amyloid fibrils.
Of them, the soluble oligomers are the more toxic species [41 42]. BChE was found to be enriched within
plaques in brain of AD patients and to promote the formation of A fibrils, while BChE-knockout
could reduce A deposition in brain of AD mouse model [43 44]. Thus, BChE inhibitor could reduce
the A aggregation. Based on these observations, inhibitors 9 and 23 were evaluated for their in vitro
ability to inhibit A aggregation using the thioflavin T (ThT) method. Donepezil was used as a positive
control, and the results was shown in Figure 5. Both compounds exhibited anti-Aβ1-42 aggregation
activity in a dose-dependent manner from the concentration of to 10 µM. Especially, the inhibition
ratios of and 23 (at 10 M) towards Aβ1-42 aggregation are 45.5 6.2% and 44.2 3.3%, respectively,
β
,
β
,
Aβ
β
β
,
β
β
1
9
µ
±
±
indicating that the activity of both compounds are significantly more potent than that of donepezil
(25.8 ± 6.2% at 10 µM).
Figure 5. Inhibitory activity against Aβ1-42 produced by
9 (1, 5, 10 µM), 23 (1, 5, 10 µM), and donepezil
(10 µM). Data are shown as mean ± SD (n = 3), with * p < 0.05 and ** p < 0.01 versus donepezil.
2.9. Cell Viability Assay
Next, to determine the potential cytotoxic activity of selected compounds
9 and 23 towards
SH-SY5Y neuroblastoma cells, the viability of cells treated by and 23 was evaluated by MTT assay.
9
The result presented in Figure 6 revealed that both compounds did not show obvious cytotoxicity in
the range of tested concentration, even at 200 µM.

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Figure 6. The cytotoxicity of 9 and 23 against SH-SY5Y cells. Date are expressed as the percent variation
with respect to the viability recorded in control cells.
2.10. Protective Activity of 9 and 23 Against Aβ1-42-Induced Toxicity in SH-SY5Y Cells
Since compounds
9 and 23 showed anti-Aβ1-42 aggregation activity and nontoxicity towards
SH-SY5Y cells, their in vitro ability to protect SH-SY5Y cells from Aβ1-42-induced toxicity was
evaluated using the MTT method with epigallocatechin gallate (EGCG) as a positive control. As shown
in Figure 7, after the SH-SY5Y cells was exposed to 10
β1-42-treated group decreased to 63.21 1.30%, while in compound-treated groups both
could significantly protect SH-SY5Y cells from Aβ1-42-induced toxicity compared with the A
group, by increasing cell viability to 84.74 1.77% and 91.14 1.25 at 5 M, and 88.80
and 98.04 1.70 at 10 M, respectively. More importantly, the protective activity of compound 23 was
found to be significantly better than that of the positive control EGCG (87.18 1.29%) at 10 M. Based
µ
M Aβ1-42 for 24h, the cell viability of
and 23
-injured
0.81%
A
±
9
β
±
±
µ
±
±
µ
±
µ
on this observation, compound 23 might be a more promising protective agent against Aβ1-42-induced
toxicity on the SH-SY5Y cell model.
Figure 7. Protective effects of compounds
9
and 23 on Aβ1-42-induced toxicity in SH-SY5Y cells.
Data are expressed as mean
positive control EGCG.
±
SD (n = 3), *** p < 0.001 versus Aβ1–42 treated only, and ## p < 0.01 versus
3. Materials and Methods
3.1. Virtural Screening
An in-house database containing 1225 compounds was screened in silico against the BChE
protein structure (PDB code: 4BDS) [37] through the GLIDE 5.5 software [45 46]. The BChE protein
,
coordinates were first processed with the Protein Preparation Wizard Workflow inserted in Maestro,
and the default settings were used for this step. Then the docking grids were created by defining

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residues within 15 Å centered on tacrine in BChE. The parameters for the cutoff, neutralization, scaling,
etc. were adopted with default settings. All the in-house database compounds were docked into
the defined binding pocket and were ranked by G-score. The top 500 compounds were selected for
further molecular docking validation with the extra precision (XP) mode to further enrich the potential
active compounds [47]. Finally, after clustering, visual selection was made to yield the 30 candidate
compounds, which were subjected to the BChE inhibition assay.
3.2. Chemistry
3.2.1. General Methods
Commercially available reagents were used without further purification. Organic solvents
were evaporated with reduced pressure using a Büchi R-100 evaporator (Büchi, Flawil, Switzerland).
Reactions were monitored by TLC using Yantai Jingyou (Yantai, China) GF254 silica gel plates. Silica gel
column chromatography was performed on Biotage Isolera One (Biotage, Uppsala, Sweden) using
silica gel (200–300 mesh) from Qingdao Hailang (Qingdao, China). NMR spectra were measured on
Bruker Avance III 600 MHz spectrometers (Bruker, Fällanden, Switzerland). Chemical shifts were
expressed in
δ (ppm) and coupling constants (J) in Hz with solvent signals as internal standards
(CDCl3, δH 7.26 ppm and δC 77.0 ppm; DMSO-d6, δH 2.50 ppm and δC 39.5 ppm). ESI-MS (electrospray
ionization mass spectrometry) analyses were performed on an Agilent 1260–6460 Triple Quard LC-MS
instrument (Agilent, Waldbronn, Germany), and HR-ESIMS data were acquired on an Agilent Q-TOF
6520 (Agilent, Waldbronn, Germany).
3.2.2. General Procedure for the Synthesis of B
The mixtures of 4-(bromomethyl)benzoic acid (200 mg, 0.93 mmol, 1 equiv.) and corresponding
tetrahydroisoquinolines
A (1.86 mmol, 2 equiv.) in 8 mL anhydrous THF were refluxed for 8 h,
and the progress of the reaction was followed using TLC. After completion of the reaction, the reaction
mixture was cooled to room temperature and the solvent was removed under vacuo. The resultant
solid was dissolved in 1N NaOH (20 mL) and then extracted with CH2Cl2 (3
layer was acidified to pH = 1 using 10% HCl (20 mL), resulting in precipitation of the final compounds.
×
20 mL). The aqueous
1
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid (B-1). Yellow solid (yield 90%). H-NMR (600 MHz,
DMSO-d6)
4.53 (d, J = 8.5 Hz, 2H), 4.29 (d, J = 18.0 Hz, 2H), 3.63 (s, 2H), 3.03 (s, 2H). 13C-NMR (150 MHz, DMSO-d6)
166.9, 134.5, 131.8, 131.7, 131.5, 129.7, 128.6, 128.3, 127.7, 126.7, 126.6, 57.8, 51.5, 48.6, 39.5, 24.9. ESI-MS
m/z 268.1 [M + H]+.
δ 11.54 (brs, 1H, COOH), 8.02 (d, J = 8.2 Hz, 2H), 7.82 (d, J = 8.2 Hz, 2H), 7.28-7.15 (m, 4H),
δ
1
4-((6-bromo-3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid (B-2). Yellow solid (yield 92.3%). H-NMR
(600 MHz, DMSO-d6) 11.54 (brs, 1H, COOH), 8.02 (d, J = 7.9 Hz, 2H), 7.83-7.77 (m, 2H), 7.49 (s, 1H),
7.42 (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 4.52 (d, J = 18.9 Hz, 2H), 4.26 (s, 2H), 3.33-3.20 (m, 3H),
3.06 (s, 1H). 13C-NMR (150 MHz, DMSO-d6)
166.9, 134.3, 131.8, 131.6, 131.1, 129.7, 129.5, 128.9, 127.8,
120.6, 57.7, 51.0, 48.1, 39.5, 24.6. ESI-MS m/z 346.0 [M + H]+.
δ
δ
1
4-((7-bromo-3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid (B-3). White solid (yield 87.5%). H-NMR
(600 MHz, DMSO-d6) 11.66 (brs, 1H, COOH), 8.02 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 8.1 Hz, 2H), 7.50-7.42
(m, 2H), 7.20 (d, J = 8.2 Hz, 1H), 4.58-4.44 (m, 2H), 4.28 (s, 2H), 3.63 (d, J = 10.7 Hz, 2H), 3.34-3.17
δ
(m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 166.9, 134.3, 131.7, 131.6, 131.0, 130.7, 130.4, 129.6, 129.3,
119.3, 57.7, 50.8, 48.3, 39.5, 24.4. ESI-MS m/z 346.0 [M + H]+.
4-((6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid (B-4). White solid (yield, 87.7%).
1H-NMR (600 MHz, DMSO-d6)
δ
7.83 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.91-6.87 (m, 2H), 6.61
(d, J = 2.3 Hz, 1H), 3.69 (d, J = 2.2 Hz, 5H), 3.61 (s, 2H), 2.87 (t, J = 5.8 Hz, 2H), 2.78 (t, J = 5.8 Hz, 2H).

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13C-NMR (150 MHz, DMSO-d6)
δ
170.0, 157.4, 139.4, 135.3, 129.0, 127.5, 127.3, 127.0, 126.9, 113.5, 111.8,
61.9, 55.0, 54.9, 50.2, 39.5, 29.0. ESI-MS m/z 298.1 [M + H]+.
4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoic acid (B-5). White solid (yield, 79.9%).
1H-NMR (600 MHz, DMSO-d6)
δ
7.83 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 6.65 (s, 1H), 6.61
(s, 1H), 3.72 (s, 2H), 3.68 (d, J = 5.2 Hz, 8H), 2.87 (t, J = 5.8 Hz, 2H), 2.71 (d, J = 5.8 Hz, 2H). 13C-NMR
(150 MHz, DMSO-d6)
δ 170.0, 147.1, 146.9, 146.8, 129.0, 127.6, 126.7, 126.6, 125.8, 112.4, 109.7, 61.9, 55.5,
55.4, 50.2, 39.5, 28.3. ESI-MS m/z 328.1 [M + H]+.
3.2.3. General Procedure for the Synthesis of 1–30
The intermediates
B (0.374 mmol, 1 equiv.) were dissolved in 5 mL DMF in a vial.
2-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) (156.4 mg,
0.411 mmol, 1.1 equiv.) were added to the reaction mixture. A series of corresponding amines or
others ((4-fluorophenyl)hydrazine, 2-bromophenol and 2-bromobenzenethiol) (0.374 mmol, 1 equiv.)
and DIPEA (193.3 mg, 1.496 mmol, 4 equiv.) were made in a separate vial. This solution was added to
the reaction mixture drop wise and the reaction mixture was stirred overnight at room temperature.
Upon completion of the reaction as detected by TLC, water (10 mL) was added and the product
was extracted with EtOAc (3
then concentrated under vacuum, and the residues were purified by flash chromatography using
×
10 mL). The combined organic layers were dried using MgSO4,
PE:EtOAc = 2:1 as an eluent to give the target compounds 1–30.
N-(2-(diethylamino)ethyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide (
1). Yellow oil (yield 62.5%).
1H-NMR (600 MHz, DMSO-d6)
δ
8.34 (t, J = 5.8 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H),
7.13-7.06 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 3.69 (s, 2H), 3.53 (s, 2H), 3.32-3.29 (m, 2H), 2.81 (t, J = 5.8 Hz,
2H), 2.67 (t, J = 5.8 Hz, 2H), 2.57-2.50 (m, 6H), 0.97 (t, J = 7.1 Hz, 6H). 13C-NMR (150 MHz, DMSO-d6)
δ
165.9, 141.7, 134.7, 134.1, 133.4, 128.5, 127.1, 126.3, 126.0, 125.5, 61.4, 55.4, 51.5, 50.3, 46.8, 39.5, 37.5,
28.7, 11.9. ESI-MS m/z 366.2 [M + H]+. HR-ESIMS: [M + H]+ calcd for C23H32N3O+ 366.2545, found
366.2549.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-(diisopropylamino)ethyl)benzamide (
2
). Yellow oil (yield
1
54.7%). H-NMR (600 MHz, DMSO-d6)
δ 8.34 (t, J = 5.8 Hz, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2
Hz, 2H), 7.12-7.06 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 3.69 (s, 2H), 3.53 (s, 2H), 3.21-3.17 (m, 2H), 3.00-2.94
(m, 2H), 2.81 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H), 2.53-2.50 (m, 2H), 0.98 (d, J = 6.6 Hz, 12H).
13C-NMR (150 MHz, DMSO-d6) δ 165.8, 141.6, 134.7, 134.0, 133.4, 128.5, 127.1, 126.3, 126.0, 125.5, 61.4,
55.4, 50.3, 48.5, 44.2, 41.0, 39.5, 28.7, 20.7. ESI-MS m/z 394.2 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C25H36N3O+ 394.2858, found 394.2853.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-(dimethylamino)ethyl)benzamide (
3
). Yellow solid (yield
1
55.5%). H-NMR (600 MHz, DMSO-d6)
δ 8.35 (t, J = 5.7 Hz, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2
Hz, 2H), 7.13-7.06 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 3.69 (s, 2H), 3.53 (s, 2H), 2.81 (t, J = 5.8 Hz, 2H),
2.67 (t, J = 5.8 Hz, 2H), 2.50 (d, J = 1.8 Hz, 1H), 2.50 (d, J = 1.8 Hz, 1H), 2.41 (t, J = 6.8 Hz, 2H), 2.19 (s,
6H). 13C-NMR (150 MHz, DMSO-d6)
δ 166.0, 141.7, 134.7, 134.1, 133.3, 128.5, 127.2, 126.4, 126.0, 125.5,
61.4, 58.2, 55.4, 50.3, 45.2, 39.5, 37.3, 28.7. ESI-MS m/z 338.2 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C21H28N3O+ 338.2232, found 338.2236.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-(ethylamino)ethyl)benzamide (
4). Yellow solid (yield 60%).
1H-NMR (600 MHz, DMSO-d6)
δ
8.39 (t, J = 5.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H),
7.13-7.06 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 3.69 (s, 2H), 3.53 (s, 2H), 2.81 (t, J = 5.8 Hz, 2H), 2.69-2.65
(m, 4H), 2.56 (q, J = 7.1 Hz, 2H), 2.50 (d, J = 1.8 Hz, 2H), 2.50 (d, J = 1.8 Hz, 1H), 1.01 (t, J = 7.1 Hz, 3H).
13C-NMR (150 MHz, DMSO-d6)
δ 166.1, 141.7, 134.7, 134.1, 133.4, 128.5, 128.4, 127.2, 126.4, 126.0, 125.5,
61.4, 55.4, 50.3, 48.5, 43.3, 39.5, 28.7, 15.1. ESI-MS m/z 338.2 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C21H28N3O+ 338.2232, found 338.2213.

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4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2,2-dimethoxyethyl)benzamide (
5
). Yellow oil (yield 64.3%).
1H-NMR (600 MHz, DMSO-d6)
δ
8.52 ((t, J = 5.8 Hz, 1H), 7.84 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz,
2H), 7.13-7.05 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 4.52 (t, J = 5.5 Hz, 1H), 3.69 (s, 2H), 3.53 (s, 2H), 3.38-3.32
(m, 6H), 2.81 (t, J = 5.8 Hz, 2H), 2.69-2.65 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
166.2, 141.9, 134.7,
δ
134.1, 133.0, 128.5, 128.5, 127.3, 126.4, 126.0, 125.5, 101.9, 61.4, 55.4, 53.2, 53.2, 50.3, 41.1, 39.5, 28.7.
ESI-MS m/z 355.1 [M + H]+. HR-ESIMS: [M + H]+ calcd for C21H27N2O3+ 355.2022, found 355.2026.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-morpholinoethyl)benzamide (
6). Yellow solid (yield 57%).
1H-NMR (600 MHz, DMSO-d6)
δ
8.37 (t, J = 5.6 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz,
2H), 7.13-7.04 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 3.69 (s, 2H), 3.57 (t, J = 4.5 Hz, 4H), 3.53 (s, 2H), 3.38
(q, J = 6.6 Hz, 2H), 2.81 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H), 2.50 (s, 1H), 2.46 (t, J = 6.9 Hz, 2H),
2.41 (s, 3H). 13C-NMR (150 MHz, DMSO-d6)
δ 166.0, 141.8, 134.7, 134.1, 133.3, 128.5, 128.5, 127.2, 126.4,
126.0, 125.5, 66.2, 61.4, 57.4, 55.4, 53.3, 50.3, 39.5, 36.5, 28.7. ESI-MS m/z 380.2 [M + H]+. HR-ESIMS:
[M + H]+ calcd for C23H30N3O2+ 380.2338, found 380.2330.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (
7
). White solid (yield
1
60%). H-NMR (600 MHz, DMSO-d6)
δ
8.39 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2
Hz, 2H), 7.13-7.06 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 3.69 (s, 2H), 3.53 (s, 2H), 3.37 (q, J = 6.8 Hz, 2H), 2.81
(t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H), 2.56 (t, J = 7.0 Hz, 2H), 2.49-2.45 (m, 4H), 1.67 (p, J = 3.1 Hz,
4H). 13C-NMR (150 MHz, DMSO-d6)
δ 165.9, 141.7, 134.7, 134.1, 133.3, 128.5, 127.2, 126.3, 126.0, 125.5,
61.4, 55.4, 54.9, 53.7, 50.3, 39.5, 38.6, 28.7, 23.1. ESI-MS m/z 337.1 [M + H]+. HR-ESIMS: [M + H]+ calcd
for C23H30N3O+ 364.2389, found 364.2365.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-((1-ethylpyrrolidin-2-yl)methyl)benzamide (
8
). Yellow oil
1
(yield 74.5%). H-NMR (600 MHz, DMSO-d6)
δ
8.34 (t, J = 5.7 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.44
(d, J = 8.2 Hz, 2H), 7.13-7.05 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 3.69 (s, 2H), 3.53 (s, 2H), 3.43-3.38 (m, 1H),
3.08-3.00 (m, 2H), 2.87-2.82 (m, 1H), 2.81 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H), 2.57 (m, 1H), 2.27 (m,
1H), 2.15-2.08 (m, 1H), 1.77 (m, 1H), 1.67-1.55 (m, 3H), 1.04 (t, J = 7.2 Hz, 3H). 13C-NMR (150 MHz,
DMSO-d6) δ 166.1, 141.7, 134.7, 134.1, 133.4, 128.5, 127.2, 126.3, 126.0, 125.5, 62.7, 61.4, 55.4, 53.3, 50.3,
48.2, 43.2, 39.5, 28.7, 28.6, 22.4, 13.9. ESI-MS m/z 378.2 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C24H32N3O+ 378.2545, found 378.2563.
(4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(pyrrolidin-1-yl)methanone (
1H-NMR (600 MHz, DMSO-d6)
7.48 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.09 (m, 3H), 6.99
(d, J = 7.3 Hz, 1H), 3.67 (s, 2H), 3.54 (s, 2H), 3.48-3.39 (m, 4H), 2.81 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.8 Hz,
2H), 1.88-1.83 (m, 2H), 1.82-1.77 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
168.2, 140.2, 135.9, 134.7,
9). Yellow oil (yield 74%).
δ
δ
134.1, 128.5, 128.4, 127.1, 126.4, 126.0, 125.5, 61.5, 55.4, 50.3, 49.0, 45.9, 39.5, 28.7, 26.0, 23.9. ESI-MS m/z
321.1 [M + H]+. HR-ESIMS: [M + H]+ calcd for C21H25N2O+ 321.1967, found 321.1997.
(4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(morpholino)methanone (10). Yellow oil (yield 68%).
1H-NMR (600 MHz, DMSO-d6)
δ
7.43 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.13-7.06 (m, 3H),
7.00 (d, J = 7.2 Hz, 1H), 3.68 (s, 2H), 3.59 (s, 4H), 3.55 (s, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.8
Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 169.1, 140.1, 134.7, 134.2, 134.1, 128.6, 128.5, 127.1, 126.4,
126.0, 125.5, 66.1, 61.4, 55.4, 50.2, 39.5, 28.7. ESI-MS m/z 337.1 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C21H25N2O2+ 337.1916, found 337.1909.
(4-((6-bromo-3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(pyrrolidin-1-yl)methanone (11). Yellow oil
1
(yield 64%). H-NMR (600 MHz, DMSO-d6)
δ 7.48 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.32
(d, J =2.1Hz, 1H), 7.26 (dd, J = 8.2, 2.1Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 3.68 (s, 2H), 3.50 (s, 2H), 3.45
(t, J = 6.7 Hz, 2H), 3.38 (t, J = 6.7 Hz, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.66 (t, J = 5.8 Hz, 2H), 1.88-1.83
(m, 2H), 1.82-1.77 (m. 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 168.1, 140.0, 137.1, 135.9, 134.2, 131.0, 128.6,
128.4, 128.3, 127.1, 118.8, 61.2, 54.8, 49.7, 49.0, 45.9, 39.5, 28.4, 26.0, 23.9. ESI-MS m/z 399.0 [M + H]+.
HR-ESIMS: [M + H]+ calcd for C21H24BrN2O+ 399.1072, found 399.1056.

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(4-((7-bromo-3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(pyrrolidin-1-yl)methanone (12). Yellow oil
1
(yield 65%). H-NMR (600 MHz, DMSO-d6)
δ 7.48 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.29
(d, J = 8.2 Hz, 1H), 7.25 (s, 1H), 7.07 (d, J = 8.2 Hz, 1H), 3.67 (s, 2H), 3.55 (s, 2H), 3.46 (t, J = 6.7 Hz,
2H), 3.39 (t, J = 6.7 Hz, 2H), 2.77 (t, J = 5.7 Hz, 2H), 2.67 (t, J = 5.7 Hz, 2H), 1.89-1.83 (m, 2H), 1.82-1.77
(m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 168.1, 140.0, 137.5, 135.9, 133.6, 130.7, 129.0, 128.8, 128.3,
127.1, 118.3, 61.1, 54.8, 49.8, 48.9, 45.9, 39.5, 28.1, 26.0, 23.9. ESI-MS m/z 399.0 [M + H]+. HR-ESIMS:
[M + H]+ calcd for C21H24BrN2O+ 399.1072, found 399.1062.
(4-((6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(pyrrolidin-1-yl)methanone (13). Brown oil
1
(yield 71%). H-NMR (600 MHz, DMSO-d6)
δ 7.48 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 6.91
(d, J = 9.2 Hz, 1H), 6.67 (d, J = 6.6 Hz, 2H), 3.69 (s, 3H), 3.66 (s, 2H), 3.48-3.44 (m, 4H), 3.39 (t, J = 6.5
Hz, 2H), 2.79 (t, J = 5.8 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 1.88-1.83 (m, 2H), 1.82-1.77 (m, 2H). 13C-NMR
(150 MHz, DMSO-d6)
δ 168.1, 157.5, 140.2, 135.9, 135.2, 128.3, 127.3, 127.1, 126.8, 113.0, 111.9, 61.5,
54.9, 50.2, 48.9, 45.9, 39.5, 29.0, 26.0, 23.9. ESI-MS m/z 351.1 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C22H27N2O2+ 351.2073, found 351.2079.
(4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)(pyrrolidin-1-yl)methanone (14). Yellow
1
oil (yield 60.3%). H-NMR (600 MHz, DMSO-d6)
δ 7.48 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H),
6.66 (s, 1H), 6.58 (s, 1H), 3.69 (s, 3H), 3.66 (s, 5H), 3.46 (t, J = 6.9 Hz, 2H), 3.43 (s, 2H), 3.39 (t, J = 6.5
Hz, 2H), 2.73 (t, J = 5.6 Hz, 2H), 2.66 (t, J = 5.6 Hz, 2H), 1.88-1.84 (m, 2H), 1.82-1.77 (m, 2H). 13C-NMR
(151 MHz, DMSO-d6)
δ 168.1, 147.2, 146.9, 140.3, 135.9, 128.4, 127.1, 126.4, 125.7, 111.8, 109.9, 61.6, 55.5,
55.4, 55.0, 50.6, 49.0, 45.9, 28.3, 26.0, 23.9. ESI-MS m/z 381.1 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C23H29N2O3+ 381.2178, found 381.2170.
1
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-phenylbenzamide (15). White solid (yield 56%). H-NMR
(600 MHz, DMSO-d6)
(d, J = 8.2 Hz, 2H), 7.37-7.32 (m, 2H), 7.14-7.06 (m, 4H), 7.01 (s, 1H), 3.73 (s, 2H), 3.56 (s, 2H), 2.83 (t,
J = 5.9 Hz, 2H), 2.70 (t, J = 5.9 Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
165.5, 142.4, 139.2, 134.7, 134.1,
δ 10.22 (brs, 1H, NH), 7.93 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 7.6 Hz, 2H), 7.52
δ
133.7, 128.6, 128.6, 128.5, 127.8, 126.4, 126.1, 125.5, 123.7, 120.4, 61.4, 55.5, 50.3, 39.5, 28.7. ESI-MS m/z
343.1 [M + H]+. HR-ESIMS: [M + H]+ calcd for C23H23N2O+ 343.1810, found 343.1827.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-fluorophenyl)benzamide (16a). Yellow oil (yield 37%).
1H-NMR (600 MHz, DMSO-d6)
δ
10.09 (brs, 1H, NH), 7.96 (d, J = 8.2 Hz, 2H), 7.61-7.57 (m, 1H), 7.52
(d, J = 8.2 Hz, 2H), 7.32-7.25 (m, 2H), 7.24-7.2 (m, 1H), 7.15-7.06 (m, 3H), 7.01 (d, J = 7.3 Hz, 1H), 3.74
(s, 2H), 3.56 (s, 2H), 2.83 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ
165.3, 156.7, 155.0, 142.7, 134.7, 134.1, 132.7, 128.6, 128.5, 127.9, 127.2, 126.4, 126.0, 125.5, 124.3, 124.3,
115.9, 115.8, 61.4, 55.4, 50.3, 39.5, 28.7. ESI-MS m/z 361.0 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C23H22FN2O+ 361.1716, found 361.1757.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(3-fluorophenyl)benzamide (17). Yellow oil (yield 71.6%).
1H-NMR (600 MHz, DMSO-d6)
δ 10.40 (brs, 1H, NH), 7.93 (d, J = 8.2 Hz, 2H), 7.78-7.73 (m, 1H), 7.56
(dd, J = 8.1, 1.9 Hz, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.38 (q, J = 8.1 Hz, 1H), 7.13-7.07 (m, 3H), 7.00 (d, J = 7.2
Hz, 1H), 6.96-6.89 (m, 1H), 3.73 (s, 2H), 3.56 (s, 2H), 2.83 (t, J = 5.7 Hz, 2H), 2.71-2.68 (m, 2H). 13C-NMR
(150 MHz, DMSO-d6)
δ 165.7, 162.9, 161.3, 142.7, 141.1, 141.0, 134.7, 134.1, 133.4, 130.3, 130.2, 128.6,
128.5, 127.8, 126.4, 126.0, 125.5, 115.9, 115.9, 61.4, 55.5, 50.3, 39.5, 28.7. ESI-MS m/z 360.9 [M + H]+.
HR-ESIMS: [M + H]+ calcd for C23H22FN2O+ 361.1716, found 361.1722.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(4-fluorophenyl)benzamide (18). White solid (yield 60%).
1H-NMR (600 MHz, DMSO-d6)
δ
10.30 (brs, 1H, NH), 7.93 (d, J = 8.2 Hz, 2H), 7.79 (m, 2H), 7.52
(d, J = 8.2 Hz, 2H), 7.22–7.17 (m, 2H), 7.10 (m, 3H), 7.00 (d, J = 7.3 Hz, 1H), 3.73 (s, 2H), 3.56 (s, 2H), 2.83
(t, J = 5.8 Hz, 2H), 2.69 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
165.4, 159.1, 157.5, 142.5,
δ
135.6, 135.6, 134.7, 134.1, 133.6, 128.6, 128.5, 127.8, 126.4, 126.1, 125.6, 122.2, 122.1, 115.3, 115.2, 61.4, 55.5,
50.3, 39.5, 28.7. ESI-MS m/z 361.0 [M + H]+. HR-ESIMS: [M + H]+ calcd for C23H22FN2O+ 361.1716,
found 361.1707.

Molecules 2019, 24, 2568
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4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(4-fluorobenzyl)benzamide (19). Yellow solid (yield 61.2%).
1H-NMR (600 MHz, DMSO-d6)
9.03 (t, J = 6.0 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H),
δ
7.40-7.31 (m, 2H), 7.17-7.13 (m, 2H), 7.12-7.05 (m, 3H), 6.99 (d, J = 7.3 Hz, 1H), 4.46 (d, J = 6.0 Hz, 2H),
3.69 (s, 2H), 3.54 (s, 2H), 2.81 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 166.1, 161.9, 160.3, 142.0, 135.9, 135.9, 134.7, 134.0, 133.0, 129.2, 129.2, 128.5, 128.5, 127.3, 126.3, 126.0,
125.5, 115.0, 114.9, 61.4, 55.4, 50.3, 41.9, 39.5, 28.7. ESI-MS m/z 375.1 [M + H]+. HR-ESIMS: [M + H]+
calcd for C24H24FN2O+ 375.1873, found 375.1890.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N2019-(4-fluorophenyl)benzohydrazide (20). Yellow solid
(yield 50%). 1H-NMR (600 MHz, DMSO-d6)
δ 10.39 (d, J = 3.0 Hz, 1H), 7.92 (d, J = 3.0 Hz, 1H),
7.90 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.16-7.05 (m, 3H), 7.00 (t, J = 8.3 Hz, 3H), 6.84-6.73
(m, 2H), 3.71 (s, 2H), 3.55 (s, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.69 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz,
DMSO-d6) δ 166.3, 156.7, 155.1, 146.2, 142.5, 134.7, 134.1, 131.7, 128.7, 128.5, 127.4, 126.4, 126.1, 125.6,
115.3, 115.2, 113.6, 113.5, 61.4, 55.5, 50.3, 39.5, 28.7. ESI-MS m/z 376.0 [M + H]+. HR-ESIMS: [M + H]+
calcd for C23H23FN3O+ 376.1825, found 376.1845.
N-(4-chloro-2-fluorophenyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide (21). Yellow oil (yield
1
34%). H-NMR (600 MHz, DMSO-d6)
δ 8.71 (dd, J = 4.3, 1.2 Hz, 1H), 8.50 (dd, J = 8.3, 1.2 Hz, 1H), 7.92
(d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.49-7.47 (m, 1H), 7.13-7.07 (m, 3H), 7.00 (d, J = 7.3 Hz, 1H),
3.76 (s, 2H), 3.58 (s, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.73 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ
167.2, 150.7, 139.6, 134.6, 133.9, 129.6, 129.4, 128.8, 128.7, 128.5, 126.4, 126.1, 125.6, 120.5, 61.2, 55.3,
50.2, 39.5, 28.5. ESI-MS m/z 395.0 [M + H]+. HR-ESIMS: [M + H]+ calcd for C23H21ClFN2O+ 395.1326,
found 395.1301.
N-(4-bromo-2-fluorophenyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide (22). White solid (yield
1
40%). H-NMR (600 MHz, DMSO-d6)
δ 10.19 (brs, 1H, NH), 7.95 (d, J = 8.2 Hz, 2H), 7.66 (dd, J = 10.0,
2.1 Hz, 1H), 7.58 (t, J = 8.4 Hz, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.46-7.43 (m, 1H), 7.13-7.07 (m, 3H), 7.00
(d, J = 7.3 Hz, 1H), 3.73 (s, 2H), 3.56 (s, 2H), 2.83 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 5.8 Hz, 2H). 13C-NMR
(150 MHz, DMSO-d6)
δ 165.3, 156.5, 154.8, 142.9, 134.7, 134.1, 132.4, 128.7, 128.5, 128.4, 128.0, 127.5,
126.4, 126.1, 125.6, 119.4, 119.2, 117.6, 117.6, 61.4, 55.5, 50.3, 39.5, 28.7. ESI-MS m/z 439.0 [M + H]+.
HR-ESIMS: [M + H]+ calcd for C23H21BrFN2O+ 439.0821, found 439.0802.
N-(2-bromophenyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide (23). Yellow oil (yield 51%).
1H-NMR (600 MHz, DMSO-d6)
δ 8.69 (dd, J = 4.4, 1.4 Hz, 1H), 8.48 (dd, J = 8.4, 1.4 Hz, 1H), 7.93
(d, J = 8.2 Hz, 2H), 7.50 (d, J = 8.2 Hz, 2H), 7.46 (dd, J = 8.4, 4.4 Hz, 1H), 7.14-7.07 (m, 3H), 7.01 (d, J = 7.3
Hz, 1H), 3.79 (s, 2H), 3.61 (s, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.75 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz,
DMSO-d6) δ 167.21, 150.59, 139.59, 134.60, 134.10, 133.79, 129.74, 129.39, 128.92, 128.61, 128.46, 126.38,
126.16, 125.58, 120.44, 61.10, 55.14, 50.19, 39.5, 28.35. ESI-MS m/z 421.0 [M + H]+. HR-ESIMS: [M + H]+
calcd for C23H22BrN2O+ 421.0916, found 421.0910.
N-(3-bromophenyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide (24). Yellow oil (yield 61.1%).
1H-NMR (600 MHz, DMSO-d6)
δ
10.38 (brs, 1H, NH), 8.12 (d, J = 1.9 Hz, 1H), 7.95–7.92 (m, 2H), 7.76
(d, J = 7.9 Hz, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.31 (m, 2H), 7.13-7.07 (m, 3H), 7.00 (d, J = 7.2 Hz, 1H), 3.73
(s, 2H), 3.57 (s, 2H), 2.83 (t, J = 5.7 Hz, 2H), 2.70 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
165.7, 142.7,
δ
140.9, 134.7, 134.1, 133.3, 130.6, 128.7, 128.6, 128.5, 127.8, 126.4, 126.2, 125.5, 122.5, 121.4, 119.0, 61.4, 55.5,
50.3, 39.5, 28.7. ESI-MS m/z 421.0 [M + H]+. HR-ESIMS: [M + H]+ calcd for C23H22BrN2O+ 421.0916,
found 421.0915.
N-(4-bromophenyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide (25). White solid (yield 38%).
1H-NMR (600 MHz, DMSO-d6)
δ
10.36 (brs, 1H, NH), 7.93 (d, J = 8.2 Hz, 2H), 7.80-7.73 (m, 2H), 7.53
(m, 4H), 7.14-7.07 (m, 3H), 7.00 (d, J = 7.3 Hz, 1H), 3.73 (s, 2H), 3.56 (s, 2H), 2.83 (t, J = 5.8 Hz, 2H),
2.69 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 165.6, 142.6, 138.7, 134.7, 134.1, 133.5, 131.5,
128.7, 128.5, 127.8, 126.4, 126.1, 125.6, 122.2, 115.3, 61.4, 55.5, 50.3, 39.5, 28.7. ESI-MS m/z 421.0 [M + H]+.
HR-ESIMS: [M + H]+ calcd for C23H22BrN2O+ 421.0916, found 421.0922.

Molecules 2019, 24, 2568
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1
2-bromophenyl 4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzoate (26). Yellow oil (yield 64%). H-NMR
(600 MHz, DMSO-d6) 8.14 (d, J = 8.3 Hz, 2H), 7.78 (dd, J = 8.1, 1.5 Hz, 1H), 7.63 (d, J = 8.3 Hz, 2H),
δ
7.52-7.49 (m, 1H), 7.46 (dd, J = 8.1, 1.5 Hz, 1H), 7.31-7.28 (m, 1H), 7.13-7.08 (m, 3H), 7.02 (d, J = 7.3
Hz, 1H), 3.78 (s, 2H), 3.58 (s, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.72 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz,
DMSO-d6) δ 163.6, 148.0, 145.7, 134.6, 134.0, 133.2, 130.1, 129.2, 128.5, 128.1, 127.0, 126.4, 126.0, 125.5,
124.5, 115.7, 61.3, 55.4, 50.3, 39.5, 28.7. ESI-MS m/z 422.0 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C23H21BrNO2+ 422.0756, found 422.0757.
S-(2-bromophenyl) 4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzothioate (27). Yellow oil (yield 61.3%).
1H-NMR (600 MHz, DMSO-d6)
δ 7.99 (d, J = 8.3 Hz, 2H), 7.86 (dd, J = 7.9, 1.3 Hz, 1H), 7.71 (dd, J = 7.6,
1.7 Hz, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.54-7.51 (m, 1H), 7.49-7.46 (m, 1H), 7.13-7.07 (m, 3H), 7.01 (d, J = 7.3
Hz, 1H), 3.76 (s, 2H), 3.57 (s, 2H), 2.83 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 5.8 Hz, 2H). 13C-NMR (150 MHz,
DMSO-d6) δ 187.1, 145.9, 138.0, 134.6, 134.3, 134.0, 133.5, 132.1, 129.6, 129.4, 128.7, 128.5, 128.3, 127.4,
126.4, 126.0, 125.5, 61.2, 55.4, 50.3, 28.7. ESI-MS m/z 438.0 [M + H]+. HR-ESIMS: [M + H]+ calcd for
C23H21BrNOS+ 438.0527, found 438.0557.
1
N-(2-(1H-indol-3-yl)ethyl)-4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)benzamide (28). H-NMR (600 MHz,
DMSO-d6) δ 10.81 (brs, 1H, NH), 8.58 (t, J = 5.7 Hz, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.0 Hz, 1H),
7.44 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 2.2 Hz, 1H), 7.15-7.03 (m, 4H), 7.02-6.93
(m, 2H), 3.69 (s, 2H), 3.58-3.51 (m, 4H), 2.95 (t, J = 7.5 Hz, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.8 Hz,
2H). 13C-NMR (150 MHz, DMSO-d6)
δ 166.0, 141.7, 136.3, 134.7, 134.1, 133.5, 128.5, 127.3, 127.2, 126.4,
126.0, 125.5, 122.6, 120.9, 118.3, 118.2, 111.9, 111.4, 61.4, 55.5, 50.3, 40.2, 39.5, 28.7, 25.2. ESI-MS m/z 410.1
[M + H]+. HR-ESIMS: [M + H]+ calcd for C27H28N3O+ 410.2232, found 410.2250.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-(5-methyl-1H-indol-3-yl)ethyl)benzamide (29). White solid
(yield 63.2%). 1H-NMR (600 MHz, DMSO-d6)
δ 10.67 (brs, 1H, NH), 8.57 (t, J = 5.6 Hz, 1H), 7.83
(d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 7.34 (s, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.16-7.04 (m, 4H), 6.99
(d, J = 7.2 Hz, 1H), 6.89 (dd, J = 8.2, 1.2 Hz, 1H), 3.69 (s, 2H), 3.53 (d, J = 7.8 Hz, 4H), 2.92 (t, J = 7.5 Hz,
2H), 2.82 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.8 Hz, 2H), 2.35 (s, 3H). 13C-NMR (150 MHz, DMSO-d6)
δ 166.0,
141.7, 134.7, 134.6, 134.1, 133.5, 128.5, 128.4, 127.6, 127.2, 126.5, 126.3, 126.0, 125.5, 122.7, 122.5, 118.0,
111.5, 111.1, 61.4, 55.4, 50.3, 40.4, 39.5, 28.7, 25.2, 21.3. ESI-MS m/z 424.2 [M + H]+. HR-ESIMS: [M + H]+
calcd for C28H30N3O+ 424.2389, found 424.2404.
4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)benzamide (30). White
1
solid (yield 58%). H-NMR (600 MHz, DMSO-d6)
δ
10.64 (brs, 1H, NH), 8.57 (t, J = 5.7 Hz, 1H), 7.83
(d, J = 8.3 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 8.8 Hz, 1H), 7.14 (d, J = 2.3 Hz, 1H), 7.13-7.06 (m,
3H), 7.05 (d, J = 2.3 Hz, 1H), 6.99 (d, J = 7.2 Hz, 1H), 6.71 (dd, J = 8.7, 2.4 Hz, 1H), 3.71 (s, 3H), 3.69
(s, 2H), 3.57–3.50 (m, 4H), 2.92 (t, J = 7.4 Hz, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.70–2.65 (m, 2H). 13C-NMR
(150 MHz, DMSO-d6)
δ 166.0, 153.0, 141.7, 134.7, 134.1, 133.5, 131.4, 128.5, 128.5, 127.7, 127.2, 126.4,
126.0, 125.5, 123.3, 112.0, 111.8, 111.1, 100.2, 61.4, 55.4, 55.3, 50.3, 48.6, 40.2, 39.5, 28.7, 25.2. ESI-MS m/z
+
440.2 [M + H]+. HR-ESIMS: [M + H]+ calcd for C28H30N3O2 440.2338, found 440.2324.
3.3. BChE and AChE Bioassay
BChE (EC 3.1.1.8, from human serum), AChE (EC 3.1.1.8, from human erythrocyte),
butyrylthiocholine (BUC) iodide, and acetylthiocholine (ATC) iodide were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Enzyme was dissolved and pre-prepared at 2.0 U/mL. 10
enzyme, 20 L of tested compound, and 40 L of PBS
L of 0.01 M 5,50-dithiobis(2-nitrobenzoic acid), 10
were pre-incubated together for 5 min. Io initiate the reaction, 20 L of 0.01 M BUC or ATC was added.
µL of
µ
µ
µ
µ
The activity was determined by measuring the increase in absorbance at 410 nm at 37 ◦C in 2 min
intervals using Tecan Spark multimode microplate reader (Mannedorf, Switzerland). The percentage
of inhibition (I) was calculated according to the formula: I = (Ac
−
Ai)/Ac
×
100%, with Ai and Ac

Molecules 2019, 24, 2568
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representing the change in the absorbance in the presence of an inhibitor and without an inhibitor,
respectively [28,29].
3.4. Kinetic Assay
Kinetic study of BChE inhibition was performed by using Ellman’s method [28
,29] as described
in Section 3.3.
3.5. Molecular Docking
The molecular docking procedure to study the binding mode of 23 against BChE was performed
according to our previously reported protocol [28,29].
3.6. Molecular Dynamics Simulation
After the BChE:23 complex was obtained by molecular docking, MD simulation was subsequently
employed to probe this complex. The protonation states of ionizable residues in BChE were determined
using the H++ program [48]. A periodic box of TIP3P [49] water molecules that extend 10 Å from
the protein atoms was added. Then counter-ions were added to neutralize the simulation model.
The AMBER force field (parm99SB) was used on the protein. The restrained electrostatic potential
method [50] encoded in the AMBER suite of programs [51] at the RHF/6-31G* level was used to calculate
the atom charges of 23. Those already present in the AMBER force field covalent and nonbonded
parameters were assigned for 23.
All the MD simulations (at constant temperature and pressure and periodic boundary conditions)
were conducted with SANDER program (time step = 2 fs). All hydrogen atom bonds in the simulation
system were constrained by the SHAKE algorithm [52]. The particle-mesh Ewald method was used to
calculate electrostatic interactions [53].
3.7. Binding Free Energy Calculation with MM-PBSA Method
The binding free energy of 23 with BChE was calculated using the MM-PBSA method encoded
in the AMBER 10.0 program. Based on the 100 ns equilibrated dynamic trajectory, 1000 snapshots
from the trajectory were extracted every 100 ps, and were used for the binding free energy calculation.
The MMPBSA.PY module in AMBER 10.0 was employed to perform the calculation.
3.8. Determination of the Inhibitory Potency on Aβ1–42 Aggregation
The ThT-based fluorometric assay [54] was performed for the determination of compounds’
anti-Aβ1–42 aggregation potency. The detailed procedure can be found in Supplementary Materials.
3.9. Cell Viability Assay and Neuroprotective Activity Evaluation
The MTT method [55] was used to test the cytotoxicity and neuroprotective activity evaluation of
compounds. The detailed procedure was included in Supplementary Materials.
3.10. Statistical Analyses
All of the experimental values were expressed as mean
three independent experiments. GraphPad Prism 5.0 statistical software package (GraphPad Software,
San Diego, CA, USA) was used to perform all of the statistical analysis.
±
standard deviation (SD) of at least
4. Conclusions
In the present study, a series of novel selective BChE inhibitors were derived from the structural
optimization of hit 1. Compounds 9 and 23 displayed improved BChE inhibitory activity and good
selectivity towards BChE. The current preliminary SAR study mainly concerned the effects of amide
side-chain and substituents at tetrahydroisoquinoline on the BChE inhibitory activity. From the bioassay

Molecules 2019, 24, 2568
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results, it could be summarized as follows: (1) for compounds
1–
10, tetrahydropyrrole ring could
improve the BChE activity, (2) for compounds 11
tetrahydroisoquinoline led to the decrease of BChE activity and selectivity, and (3) for compounds 15
–
14, the substitution at C-6 and C-7 position of
30
–
,
the substitution at C-2 of benzene ring should be optimal for BChE activity and amide group was
found to be greatly important for the binding of inhibitor to BChE. Kinetic analysis together with
molecular modeling studies suggested that these derivatives are a mixed-type of BChE inhibitors.
Further, the selected compounds
a dose-dependent manner. Moreover, these two compounds are non-toxic towards SH-SY5Y cells even
at 200 M, indicating they are probably safe on SH-SY5Y cells. Of course, this discussion on safety
needs more in vitro and even in vivo evaluations. More importantly, compounds and 23 showed
significantly in vitro protective activity against Aβ1-42-induced toxicity in SH-SY5Y cell model at 5
and 10 M, and compound 23 even showed much better activity than the positive control EGCG
at 10 M. In summary, the present in vitro biological evaluation together with computational analyses
9 and 23 displayed inhibitory activity against Aβ1-42 aggregation by
µ
9
µ
µ
provided a new valuable chemical template, which deserves further optimization for an anti-AD
drug discovery.
Supplementary Materials: The following are available online, Figure S1, 1H, 13C, HR-ESIMS spectra for
compounds 1–30, methods for determination of the inhibitory potency on Aβ1–42 aggregation, Cell viability assay,
neuroprotective activity evaluation.
Author Contributions: Chemical synthesis, ChE inhibition and anti-Aβ aggregation test, C.-S.J. and Y.-X.G.; Cell
viability assay, Z.-Q.C.; spectroscopic measurements, Y.-Y.W.; molecular docking and analysis, K.Z. and H.-R.T.;
Writing—Original Draft Preparation, C.-S.J. and K.Z.; Writing—Review, H.Z.; Project Administration, C.-S.J.,
and K.Z. All the authors accepted the final version of the manuscript.
Funding: This research was funded by [National Natural Science Foundation of China] grant numbers [21672082,
81803438]; [Natural Science Foundation of Shandong Province] grant numbers [ZR2019YQ31, ZR2017BH038,
JQ201721, ZR2018ZC0944]; [Young Taishan Scholars Program] grant number [tsqn20161037]; and [Shandong
Talents Team Cultivation Plan of University Preponderant Discipline] grant number [10027].
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
Alzheimer’s disease
acetylcholine
beta-amyloid
(AD)
(ACh)
(Aβ)
cholinesterase
(ChE)
butyrylcholinesterase
acetylcholinesterase
catalytic active site
(BChE)
(AChE)
(CAS)
peripheral anionic site
structure-activity relationship
molecular dynamics
Molecular Mechanics Poisson-Boltzmann Surface Area
root-mean-square deviations
thioflavin T
(PAS)
(SAR)
(MD)
(MM-PBSA)
(RMSDs)
(ThT)
epigallocatechin gallate
butyrylthiocholine
acetylthiocholine
(EGCG)
(BUC)
(ATC)
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