Design, synthesis and biological evaluation of benzylisoquinoline derivatives as multifunctional agents against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2014.03.058

A novel series of benzylisoquinoline derivatives were designed, synthesized, and evaluated as multifunctional agents against Alzheimer's disease (AD). The screening results showed that most of the compounds significantly inhibited cholinesterases (ChEs),

Full text of DOI:10.1016/j.bmcl.2014.03.058

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2368–2373
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of benzylisoquinoline
derivatives as multifunctional agents against Alzheimer’s disease
⇑
Zi-Chen Xu, Xiao-Bing Wang, Wen-Ying Yu, Sai-Sai Xie, Su-Yi Li, Ling-Yi Kong
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic
of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel series of benzylisoquinoline derivatives were designed, synthesized, and evaluated as
Received 30 December 2013
Revised 1 March 2014
Accepted 18 March 2014
Available online 3 April 2014
multifunctional agents against Alzheimer’s disease (AD). The screening results showed that most of
the compounds significantly inhibited cholinesterases (ChEs), human cholinesterases (h-ChEs) and
self-induced b-amyloid (Ab) aggregation. In particular, compound 9k showed the strongest acetylcholin-
esterase (AChE) inhibitory activity, being 1000-fold and 3-fold more potent than its precursor benzyliso-
quinoline (10) and the positive control galanthamine, respectively. In addition, 9k was a moderately
Keywords:
Benzylisoquinoline derivatives
Alzheimer’s disease
Cholinesterase inhibitors
Human cholinesterase inhibitors
b-Amyloid aggregation
Low cell toxicity
potent inhibitor for h-ChEs. Compared with precursor benzylisoquinoline (36.0% at 20
lM), 9k (78.4%
at 20 M) could further inhibit Ab aggregation. Moreover, 9k showed low cell toxicity in human SH-
l
SY5Y neuroblastoma cells. Therefore, compound 9k might be a promising lead compound for AD
treatment.
Ó 2014 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD), a progressive, neurodegeneration dis-
ease, is the most common cause of dementia among the elderly.1,2
Data of 2010 reported that approximately 36 million people world-
wide suffered from AD. By 2050, it is estimated that the figure is
going to rise beyond 100 million. AD was first defined by the Ger-
man psychiatrist and neuropathologist Alois Alzheimer in 1906.
Over 100 years, the etiology of AD remains elusive. Several factors
such as low levels of acetylcholine (ACh)3,4 and amyloid b-peptide
(Ab) deposits5 play significant roles in the pathophysiology of AD.6
Cholinergic hypothesis is one of the classical hypothesis of AD,
based on which, the decline in cognitive and mental functions
associated with AD is related to the weakened cortical cholinergic
neurotransmission.7 One rational way to enhance cholinergic neu-
rotransmission is to break down the process of metabolism of ACh.
ACh can be degraded by two types of cholinesterases (ChE), namely
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).8
Compared with BuChE, AChE attracts more attention from the
pharmaceutical academics since it accounts for nearly 80% ACh
hydrolysis in normal brains.9 The crystallographic structure of
AChE indicates that it includes two separate ligand binding sites,
a peripheral cationic site (PAS) at the entrance and a catalytic ac-
tive site (CAS) at the bottom.10–12 Inhibitors binding to either site
can restrain the activity of AChE. Besides its catalytic function,
AChE can further bind to Ab and act as a promoter of Ab fibril for-
mation. PAS was associated with this action and several ligands
that bind to this site have been shown to prevent Ab aggrega-
tion.13,14 Therefore, the design of dual-site inhibitors that interact
simultaneously with both CAS and PAS appears to be a promising
therapeutic strategy. Furthermore, in healthy brains, the ability
of BuChE to hydrolyze ACh is inferior to that of AChE. While as
AD progresses, the ability of BuChE significantly increases, and that
of AChE diminishes in the hippocampus and temporal cortex.14,15
Consequently, inhibition of both enzymes is beneficial to the treat-
ment of AD.
Recent studies indicate another hypothesis, called amyloid
hypothesis, may contribute to AD pathology. The amyloid hypoth-
esis states that the accumulation and aggregation of Ab is a pivotal
factor to induce AD, as its accumulation in the brain may result in
senile plaques, neurofibrillary tangles, neuronal cell death, and
ultimately dementia.16,17 Ab is formed from a larger amyloid pre-
cursor protein (APP) via sequential proteolytic cleavage by b- and
c
-secretases.18 The cleavage of APP by b-secretase generates a sol-
uble version of APP and a resultant membrane-bound C-terminal
domain. Subsequent intramembrane proteolysis of the C-terminal
domain by c
-secretase produces Ab40 and Ab42 peptides.19 Ab42 is
more prone to self-assembly into fibrils and is the major Ab com-
ponent in amyloid plaques.20 Therefore, preventing this peptide
from aggregation is a potential therapy for AD.
⇑
Up to now, several AChE inhibitors have been launched for
treating AD including tacrine, rivastigmine, donepezil, and
Corresponding author. Tel./fax: +86 25 8327 1405.
E-mail address: cpu_lykong@126.com (L.-Y. Kong).
http://dx.doi.org/10.1016/j.bmcl.2014.03.058
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Z.-C. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2368–2373
2369
galanthamine.21 However, due to the complex nature of AD, those
drugs that modulate such a single target can only improve the
symptoms for most patients instead of reaching the etiology of
AD.22–26 Hence there is an urgent need for new, efficacious mole-
cules decorated with additional pharmacological/biochemical
properties other than ChE inhibition itself. Based on considerations
above, in this study, we attempted to explore multifunctional
agents not only inhibiting ChE but also decreasing Ab aggregation.
Isoquinoline alkaloids, a large family of natural products, have
attracted increasingly widespread attention as they possess a wide
range of pharmacological properties relevant to neurological disor-
ders, especially for AD.27–30 Many naturally occurring substances,
such as berberine and chelerythrine, exhibited inhibiting potency
of AChE and Ab aggregation, which was exerted by interacting with
PAS of AChE.31 Subsequently, a number of chemically synthesized
isoquinoline analogs were designed as AChE inhibitors.32 There-
fore, isoquinoline is a valuable scaffold for designing new effective
compounds to treat AD.
Although isoquinoline scaffolds exhibited these obvious advan-
tages in AD drug design, few studies focused on designing multi-
functional isoquinoline hybrids. In this paper, isoquinoline
hybrids were designed by combining benzylisoquinoline and a ser-
ies of different terminal amine groups as multifunctional agents for
AD treatment. Benzylisoquinoline could interact with the PAS of
AChE via aromatic stacking interactions, and the terminal amine
groups, protonated at physiological pH, could occupy the CAS via
groups, different lengths of carbon spacers were tried. Unbranched
carbon spacer was able to embed in the narrow midgorge,14 and
the length of carbon spacer was changed to obtain optional confor-
mation that could make the designed compounds interact with
both CAS and PAS of AChE.
In this study, the synthesis of a series of benzylisoquinoline hy-
brids and their biological evaluation including inhibitory activities
of AChE, BuChE and anti-Ab aggregation were described. The struc-
ture–activity relationship (SAR) was discussed based on the phar-
macological activities. Moreover, kinetic analysis and molecular
modeling were also performed to further investigate the mecha-
nism of interaction with AChE.
The synthetic strategy of target compounds was shown in
Scheme 1. The synthetic route started with the condensation of
2-(3,4-dimethoxyphenyl) ethylamine with (p-hydroxyphenyl) ace-
tic acid, which provided the amide 1 in 80.5% yield.33 The classical
Bischler–Napieralski cyclization reaction and reduction reaction
was then used to obtain compound 4 from amide 2 after the hydro-
xyl group had been conveniently protected.34 After protecting the
compound 4 with (Boc)2O, the O-benzyl protecting group of prod-
uct 5 was removed. The alkylation of 6 with different
a,x-dib-
romoalkanes in butanone provided 7a–f in 65–70% yields.35 The
compounds 8a–s were obtained by the reaction of 7 with commer-
cially available secondary amines. Finally, after removing the Boc
group, the target products 9a–s/10 were obtained.
Taking into account the cost, firstly animal enzymes were used
to determine biological activities of target compounds. The inhibi-
tory activities of target compounds 9a–s against AChE (from
cation–p interaction. Considered the distance between CAS and
PAS sites, to better connect benzylisoquinoline and terminal amine
O
CH2COOH
OH
O
NH2
HN
HN
I
II
O
O
O
O
O
O
HO
BnO
1
2
OBn
OBn
BnO
O
III
IV
V
O
O
O
O
N
NH
N
O
O
O
3
4
5
Br
R
HO
O
O
n
n
VI
O
N
O
O
VII
VIII
O
O
O
O
O
O
N
O
N
O
O
8a-8f n = 3-8
8g-8s n = 6
7a-7f n = 3-8
6
IX
IX
R
HO
O
n
O
O
O
NH
NH
O
9a-9f n = 3-8
9g-9s n = 6
10
Scheme 1. Reagents and conditions: (I) EDCI, Et3N, DCM, rt, overnight; (II) BrBn, K2CO3, EtOH, reflux, 12 h; (III) POCl3, toluene, reflux, 4 h; (IV) NaBH4, MeOH, 0 °C–rt, 6 h; (V)
(Boc)2O, DCM, rt, 2 h; (VI) Pd/C, H2, MeOH, rt, 48 h; (VII) 1, n-dibromoalkane, K2CO3, butanone, reflux, 6 h; (VIII) NHR, NaHCO3, DMF, 60 °C, 24 h (R- were showed in Table 1);
(IX) 2 M HCl, ether, rt, 3 h.

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Z.-C. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2368–2373
electric eel) and BuChE (from equine serum) were measured
according to the spectrophotometric method of Ellman et al. using
galanthamine as the reference compound.36,37 All IC50 values of
test compounds for AChE and BuChE inhibition were summarized
in Table 1. Most of benzylisoquinoline derivatives were moderately
potent inhibitors for both ChEs with IC50 values ranging from
micromolar to sub-micromolar. Among these compounds, 9k re-
The previous reports suggested that the length of the alkyl
chain between aromatic moiety and terminal amino group could
affect the ability to interact with both sites of AChE and thereby
influence the AChE inhibitory potency.38 Hence, to determine the
optimal length in present study, compounds 9a–f with linker var-
ied from three to eight carbons were synthesized. The results
showed that 9d was the most potent inhibitor for AChE, which
pointed out that the optimal length for present compounds was
six carbons.
The optimal length of the linker was confirmed as a chain of six
carbons, then different terminal amine groups were introduced to
further explore SAR. Compounds (9j–m) with a cyclic amine group
in their side chains were stronger inhibitors than those with alkyl
amine groups (9g–i). For instance, compounds 9l and 9m had low-
er IC50 values than compounds 9g and 9h for both AChE and BuChE.
Interestingly, it was found that the AChE inhibitory activities of six-
membered N-containing heterocyclic compounds 9k and 9l were
higher than five-membered N-containing heterocyclic compounds
9j and 9m, respectively, which indicated that increased lipophilic-
ity could lead to a rise in AChE inhibitory potency.39,40 Introduction
vealed the most potent inhibition for AChE (IC50 = 0.95
contrast, 9m gave the most potent inhibition of BuChE
(IC50 = 2.34 M). Most of compounds showed slightly higher inhib-
lM). By
l
itory activity for AChE than for BuChE, indicating that these com-
pounds were selective inhibitors for AChE. Moreover, the
inhibitory activities for both ChEs of all target compounds
(IC50 = 0.95–53.50
were much more potent than that of their precursor compound
benzylisoquinoline (10) (IC50 >100 M for AChE; IC50 = 39.7 lM
lM for AChE; IC50 = 2.34–15.29 lM for BuChE)
l
for BuChE), demonstrating the introduction of amino group side
chains could significantly increase the inhibitory activities of deriv-
atives. It was eventually confirmed that our molecular design is
reasonable.
Table 1
Inhibition of ChEs activity, selectivity index and inhibition of self-induced Ab42 aggregation
Compound
R
n
Formula
IC50
(lM)
Selectivity indexc
Ab42 aggregation inhibitiond(%)
AChEa
BuChEb
9a
9b
3
4
C25H36N2O3
C26H38N2O3
13.23 0.34
7.58 0.41
20.81 0.22
15.29 0.61
1.57
2.02
41.1 2.1
47.3 1.4
N
9c
9d
9e
9f
5
6
7
8
C
C
C
C
27H40N2O3
28H42N2O3
29H44N2O3
30H46N2O3
7.02 0.25
2.28 0.14
4.67 0.53
6.33 0.20
10.28 0.52
4.95 0.39
9.13 0.81
20.06 0.65
1.46
2.17
1.96
3.17
34.3 1.2
56.4 9.1
43.9 4.7
39.6 1.1
9g
9h
6
6
C26H38N2O3
C30H46N2O3
3.14 0.13
3.01 0.04
8.65 0.13
4.32 0.27
2.75
1.44
55.3 3.6
51.2 7.0
N
N
N
9i
9j
6
6
C32H50N2O3
C29H42N2O3
3.43 0.27
1.34 0.11
3.46 0.51
3.35 0.04
1.01
2.50
51.4 2.7
72.3 1.2
N
9k
6
C30H44N2O3
0.95 0.02
3.13 0.16
3.29
78.4 0.6
N
N
N
N
N
9l
6
6
6
6
C29H42N2O3
C28H40N2O3
C29H42N2O4
C28H40N2O4
1.14 0.09
1.28 0.13
13.27 0.21
28.64 0.34
2.51 0.37
2.34 0.28
14.16 1.38
13.01 0.45
1.96
2.05
1.07
0.45
76.4 2.8
69.0 1.3
62.1 0.78
57.4 1.29
9m
9n
9o
OH
O
9p
6
C36H48N2O4
41.75 0.20
12.83 1.26
0.31
62.7 3.35
N
N
OH
N
N
9q
9r
9s
6
6
6
C34H45N3O3
C35H47N3O3
C35H46N2O3
17.09 0.44
53.50 1.23
6.69 0.03
8.78 0.92
10.53 0.05
7.99 0.47
0.51
0.20
65.8 5.82
61.4 0.03
N
N
1.19
2.76
61.2 1.76
36.0 5.7
Benzylisoquinoline (10)
Galanthamine
C
18H21NO3
>100
2.67 0.15
39.7 0.54
12.7 0.20
Resveratrol
61.5 2.10
a
b
c
Inhibitor concentration (mean SEM of three experiments) required for 50% inactivation of AChE.
Inhibitor concentration (mean SEM of three experiments) required for 50% inactivation of BuChE.
Selectivity index = IC50 (BuChE)/IC50 (AChE).
d
Inhibition of self-induced Ab42 aggregation, the thioflavin-T fluorescence method was used, the mean SD of at least three independent experiments and the mea-
M compounds.
surements were carried out in the presence of 20
l

Z.-C. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2368–2373
2371
of a methyl group to 2-positon of cyclic amine group (pyrrolidinyl
h-AChE inhibitor was 9l (IC50 = 2.14
lM) rather than 9k
and piperidinyl) led to an increase in AChE inhibition but a de-
crease in BuChE inhibition. For example, AChE inhibitory activity
of compound 9k was higher than that of 9l, but its BuChE inhibi-
tory activity was lower than 9l. Moreover, introducing additional
oxygen atom or hydroxyl to terminal cyclic amino group afforded
compounds 9n and 9o, which led a sharp decrease in both AChE
and BuChE inhibition. This phenomenon can be explained by a pre-
vious report.38 Electron-withdrawing effects of oxygen atom could
reduce the electronic density of the terminal amine and further im-
(IC50 = 2.20 lM), their inhibitory activities were about the same.
This phenomenon could be due to the differences between h-AChE
and electric eel AChE.14 By contrast, 9m revealed the most potent
inhibition for both BuChEs (IC50 = 2.27
IC50 = 2.34 M for horse serum BuChE). Moreover, the inhibitory
activities for both h-ChEs of all target compounds (IC50 = 2.14 to
39.63 M for h-AChE; IC50 = 2.27 to 23.73 M for h-BuChE) were
much more potent than that of their precursor benzylisoquinoline
(10) (IC50 >100 M for h-AChE; IC50 = 46.77 M for h-BuChE), once
lM
for h-BuChE;
l
l
l
l
l
pact its protonation, which finally diminished the cation–
p
inter-
again demonstrating the introduction of amino group side chains is
action between the terminal nitrogen and the CAS of AChE.
Finally, four compounds (9p–s) with phenyl or benzyl groups at
4-positon of heterocycle (piperazinyl and piperidinyl) were also
synthesized. Unfortunately, with exception of 9s showing IC50 va-
lue in one-digit micromolar, all of them showed weak inhibitory
activity to both AChE and BuChE, which might be caused by the
steric hindrance of phenyl group.
In next step, the 6-carbon chain lead compounds (9d, 9g–s) and
10 were then evaluated as inhibitors of human ChEs (h-ChEs). The
IC50 values of test compounds for human AChE (h-AChE) and
BuChE (h-BuChE) inhibition were summarized in Table 2. All tested
derivatives showed an IC50 against h-ChEs in the low micromolar
range and they were slightly less potent efficient for inhibition of
the human enzyme than that of animal enzyme. Although the best
reasonable.
To further explore the interaction mode for AChE, molecular
docking study was performed with the most active compound 9k
by software package MOE 2008.10. The X-ray crystallographic
structure of AChE complex with bis(7)-tacrine (PDB code: 2CKM)
was obtained from the Protein Data Bank. As shown in Figure 1,
compound 9k could perfectly fit into the gorge of AChE and simul-
taneously interact with both CAS and PAS of AChE, manifesting
multiple binding modes with AChE. The aromatic ring of benzyliso-
quinoline moiety binding to the PAS site interacts with the indole
ring of Trp279 via the
gen of benzylisoquinoline group bound to the PAS was via a cat-
ion– interaction with Tyr70 and Trp279. At the bottom of the
gorge, the charged nitrogen of 2-methylpiperidine was also able
p–p stacking. Moreover, the charged nitro-
p
Table 2
Inhibition of human ChEs activity and selectivity indexa
Compound
R
n
Formula
IC50
(
lM)
Selectivity indexd
h-AChEb
h-BuChEc
N
N
9d
6
C28H42N2O3
4.99 0.23
16.70 2.71
3.35
9g
9h
6
6
C26H38N2O3
C30H46N2O3
7.13 0.31
3.46 0.16
3.43 0.45
0.48
3.23
11.17 1.39
N
9i
9j
6
6
C32H50N2O3
C29H42N2O3
3.85 0.07
4.50 0.44
6.02 1.77
6.42 2.03
1.56
1.43
N
N
9k
6
C30H44N2O3
2.20 0.12
5.83 1.43
1.49
N
N
N
N
N
9l
6
6
6
6
C29H42N2O3
C28H40N2O3
C29H42N2O4
C28H40N2O4
2.14 0.03
2.98 0.20
9.59 0.27
35.48 0.08
3.25 0.64
2.27 0.40
23.73 3.15
10.59 2.73
1.69
0.76
2.47
0.29
9m
9n
9o
OH
O
9p
6
C36H48N2O4
39.63 0.39
15.90 1.88
0.40
N
N
OH
N
N
9q
9r
6
6
6
C34H45N3O3
C35H47N3O3
C35H46N2O3
16.33 1.93
5.59 2.11
12.90 0.91
21.71 2.27
0.23
3.88
0.46
N
N
9s
9.33 0.57
>100
9.29 1.20
Benzylisoquinoline (10)
C
18H21NO3
46.77 0.36
a
b
c
AChE from human erythrocytes and BuChE from human serum were used.
Inhibitor concentration (mean SEM of three experiments) required for 50% inactivation of AChE.
Inhibitor concentration (mean SEM of three experiments) required for 50% inactivation of BuChE.
Selectivity index = IC50 (BuChE)/IC50 (AChE).
d

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Z.-C. Xu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2368–2373
Figure 1. Molecular modeling of compound 9k with AChE (A and B) generated with MOE.
to bind to the CAS via a cation–
p
interaction with Trp84. All these
inhibition values of compounds 9a–f, it seemed that the linker
results clearly indicated that compound 9k could simultaneously
bind to CAS and PAS of AChE.
length indeed played a role in determining the inhibition of Ab42
self-aggregation. The compound 9d with six-carbon length showed
better anti-Ab42 aggregation activity than the others with shorter
or longer carbon spacers. Moreover, it could be found that com-
pounds 9j–s with closed-loop groups exhibited better inhibitory
potency than compounds 9a–i with open-loop ones. Unlike the
trend of AChE inhibition, our data showed that the inhibition of
Ab42 aggregation was slightly influenced by the introduction of
electron-withdrawing groups or bulky groups. For example, the
anti-Ab42 aggregation activities of 9n–s were slightly weaker than
that of 9j–m. Consequently, these results implied that benzyliso-
quinoline was indeed a potent inhibitor of Ab42 aggregation.
On basis of the screening results above, the most potent
compound 9k was selected to further examine the potential toxic-
ity effect on the human neuroblastoma cell line SH-SY5Y.43,44 After
exposing the cells to 9k for 24 h, the cell viability was evaluated by
the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)
assay. The result indicated that 9k did not show significant effect
Kinetic study of compound 9k was further examined to investi-
gate the AChE inhibitory mechanism. The Lineweavere–Burk plots
(Fig. 2) showed both increasing slopes and increasing intercepts on
the y-axis at increasing inhibitor concentration. This pattern indi-
cated a mixed-type inhibition and therefore revealed that com-
pound 9k bound to the both sites of AChE, which were
consistent with our design.
In addition, the inhibition of self-induced Ab42 aggregation of
these compounds was evaluated by a thioflavin-T based fluoromet-
ric assay.41,42 Resveratrol (RES), a known active natural product for
the inhibition of Ab42 self-aggregation, was used as reference com-
pound and the results were showed in Table 1. From the results, it
could be seen that most compounds exhibited moderate-to-good
potencies (34.3–78.4% at 20
at 20 M) and benzylisoquinoline (36.0% at 20
indicated that 9k (78.4% at 20 M) was the most potent inhibitor
of Ab42 aggregation among the target compounds. From the
l
M) compared to that of RES (61.5%
l
lM). The result
l
on cell viability at 0.1–10 lM (0.1 lM: 96.8 10.7%; 0.5 lM:

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Figure 2. Lineweaver–Burk plots resulting from subvelocity curve of AChE activity
with different substrate concentrations (0.05–0.50 mM) in the absence and
presence of 0.47, 0.95, 1.90 lM 9k.
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98.8 13.1%;
94.5 7.3%; 10
1
l
l
M: 96.8 8.1%;
2 lM: 96.1 11.6%; 5 lM:
M: 91.1 16.3%). This suggested that 9k was non-
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toxic to SH-SY5Y cells and might be a suitable multifunctional
agent for treating AD.
In conclusion, a variety of novel benzylisoquinoline derivatives
were designed, synthesized and evaluated with the aim to prepare
multifunctional compounds for treating AD. It was observed that
most of the compounds could effectively inhibit ChEs, h-ChEs
and Ab42 aggregation in vitro, especially compound 9k exhibited
the best AChE and h-AChE inhibitory activity, good inhibition of
Ab42 aggregation activity and low cytotoxic activity. Furthermore,
molecular modeling and inhibitory kinetic analysis revealed that
9k bound simultaneously to both CAS and PAS of AChE. Overall,
the new hybrids with multifunctional effects, especially 9k, were
determined as potential anti-AD candidate.
Acknowledgments
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We acknowledge the financial supports from the Program for
Changjiang Scholars and Innovative Research Team in University
(IRT1193), A Project Founded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03.
058.
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