Simple analogues of natural product chelerythrine: Discovery of a novel anticholinesterase 2-phenylisoquinolin-2-ium scaffold with excellent potency against acetylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2020.112415

As simple analogues of the natural compound chelerythrine, a novel anti-cholinesterase 2-phenylisoquinolin-2-ium scaffold was designed by structure imitation. The activity evaluation led to the discovery of seven compounds with potent anti-acetylcholinesterase activity with IC₅₀ values of ≤0.72 μM, superior to chelerythrine and standard drugs galantamine. Particularly, compound 8y showed the excellent dual acetylcholinesterase-butyrylcholinesterase inhibition activity, superior to rivastigmine, a dual cholinesterase inhibitor drug. Furthermore, the compounds displayed a competitive anti-acetylcholinesterase mechanism with the substrate and low cytotoxicity. Molecular docking showed that the isoquinoline moiety is embedded in a cavity surrounded by four aromatic residues of acetylcholinesterase by the π-π action. Structure-activity relationship showed that the p-substituents on the C-ring can dramatically improve the anti-acetylcholinesterase activity, while 8-OMe can increase the activity against the two cholinesterases simultaneously. Thus, the title compounds emerged as promising lead compounds for the development of novel cholinesterase inhibitor agents.

Full text of DOI:10.1016/j.ejmech.2020.112415

Journal Pre-proof
Simple analogues of natural product chelerythrine: Discovery of a novel
anticholinesterase 2-phenylisoquinolin-2-ium scaffold with excellent potency against
acetylcholinesterase
Bohang Zhou, Hui Li, Zhiming Cui, Ding Li, Huiling Geng, Jinming Gao, Le Zhou
PII:
S0223-5234(20)30386-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112415
EJMECH 112415
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 March 2020
Revised Date: 16 April 2020
Accepted Date: 29 April 2020
Please cite this article as: B. Zhou, H. Li, Z. Cui, D. Li, H. Geng, J. Gao, L. Zhou, Simple analogues of
natural product chelerythrine: Discovery of a novel anticholinesterase 2-phenylisoquinolin-2-ium scaffold
with excellent potency against acetylcholinesterase, European Journal of Medicinal Chemistry (2020),
doi: https://doi.org/10.1016/j.ejmech.2020.112415.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Masson SAS.

Graphical Abstract

Full paper
Simple Analogues of Natural Product Chelerythrine: Discovery of a Novel
Anticholinesterase 2-Phenylisoquinolin-2-ium Scaffold with Excellent Potency
against Acetylcholinesterase
Bohang Zhou§, Hui Li§, Zhiming Cui, Ding Li, Huiling Geng,* Jinming Gao,* and Le Zhou*
College of Chemistry & Pharmacy, Northwest A&F University, 22 Xinong Road, Yangling, 712100, Shaanxi, China
*Corresponding author. E-mail address: zhoulechem@nwsuaf.edu.cn (L. Zhou); jinminggao@nwafu.edu.cn (J. Gao);
genghuiling5@163.com (H. Geng)
§ These authors contributed equally to this work.

ABSTRACT
As simple analogues of the natural compound chelerythrine,
a
novel anti-cholinesterase
2-phenylisoquinolin-2-ium scaffold was designed by structure imitation. The activity evaluation led to the
discovery of seven compounds with potent anti-acetylcholinesterase activity with IC50 values of ≤ 0.72 μM,
superior to chelerythrine and standard drugs galantamine. Particularly, compound 8y showed the excellent dual
acetylcholinesterase-butyrylcholinesterase inhibition activity, superior to rivastigmine, a dual cholinesterase
inhibitor drug. Furthermore, the compounds displayed a competitive anti-acetylcholinesterase mechanism with
the substrate and low cytotoxicity. Molecular docking showed that the isoquinoline moiety is embedded in a
cavity surrounded by four aromatic residues of acetylcholinesterase by the π-π action. Structure-activity
relationship showed that the p-substituents on the C-ring can dramatically improve the anti-acetylcholinesterase
activity, while 8-OMe can increase the activity against the two cholinesterases simultaneously. Thus, the title
compounds emerged as promising lead compounds for the development of novel cholinesterase inhibitor agents.
Keywords:
Isoquinoline
Benzo[c]phenanthridine
Acetylcholinesterase
Butyrylcholinesterase
Structure-activity relationship
Molecular docking

1. Introduction
Cholinesterases (ChE) include two types of enzymes, namely acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE). The most striking difference between them is that AChE preferentially hydrolyzes
acetylcholine (ACh) while BuChE hydrolyzes butyrylcholine (BuCh) more efficiently than ACh. Furthermore,
histologically, AChE is mostly of neuronal origin, while BuChE is primarily present in the blood and glial cells [1]
.
ACh is one type of ubiquitous neurotransmitter. The main function of AChE is to modulate cholinergic signal
transmission through hydrolysis of ACh. Under normal conditions, ACh is dominantly decomposed by AChE
instead of BuChE [2]. However, when the level of AChE in cholinergic transmission declines, BuChE can play a
function compensation role for AChE to some extent to maintain normal cholinergic pathways [3]. Therefore, ChE
inhibitors are very interesting candidates for development of medicines for treatment of some neurogenic
diseases related with cholinergic transmission, such as Alzheimer’s disease (AD), senile dementia, ataxia and
myasthenia gravis [4]. Additionally, AChE inhibitors can also be used for development of insecticides [5]
.
AD is one of the most common, age-related irreversible and progressive neurodegenerative disorders
characterized as dementia, memory loss, a decline in language skills and cognitive impairment with aging [6]. The
number of people with AD will be expected to reach over 131 million by 2050 [7]. Therefore, AD has been one of
the main public health issues we have to face.
The pathogenesis of AD involves different molecular events such as low level of acetylcholine (ACh) [8]
,
β-amyloid (Aβ) aggregation [9], tau-protein hyperphosphorylation [10] and oxidative stress [11], etc. Among them,
low level of ACh in different areas of the central nervous system has been proved to be associated with the
memory impairment and behavioral abnormalities in patients with AD [12]. Based on the results above, some
AChE inhibitor (AChEI) agents have been developed and used for treatment of cognitive dysfunction and
memory loss of mild-to-moderate AD patients by elevating the level of ACh in brain, such as tacrine, donepezil,
galantamine, rivastigmine, etc [13,14]. However, except for rivastigmine, a dual AChE−BuChE inhibitor, these
selective AChE inhibitor drugs are not suitable for the late stage AD since ACh hydrolysis in the late stage of AD
mainly depends on BuChE but not AChE [3]. Furthermore, some obvious adverse effects like nausea and
vomiting, decreased appetite, weight loss, hepatotoxicity, etc. were also reported for these drugs [15]. Therefore,
the search of more ideal ChE inhibitors (ChEIs), especially dual AChE−BuChE inhibitors is very necessary for
treatment of AD [16]
.

Sanguinarine (SA) and chelerythrine (CH) (Fig. 1), two most common quaternary benzo[c]phenanthridine
alkaloids (QBAs), can be considered as ideal leading compounds for development of novel ChEIs based on their
good dual AChE−BuChE inhibition [17], excellent cellular permeability [18] and high safety for mammal [19]. This
study aims to search for QBA-like cholinesterase inhibitors (ChEIs) with more simple structure and more potent
activity than SA or CH.
Figure 1. Design of the basic skeleton of the target compounds by structure imitation.
In the present study, as structurally simple analogues of QBAs, a class of novel 2-arylisoquinolin-2-ium salts
were designed by structure imitation, synthesized and evaluated for AChE and BuChE inhibition activities in
vitro. Furthermore, the kinetic action mechanism, molecular docking, structure-activity relationship (SAR) and
cytotoxicity were also investigated. To our knowledge, no reports have been found on the anti-ChE activity of
2-arylisoquinolin-2-ium compounds until now.
2. Results and discussion
2.1. Rational design of target compounds
The most remarkable structural feature of SA and CH (Fig. 1) is the presence of one benzo[c]phenanthridine
framework and a highly polar iminium moiety (C=N+). Our previous study proved that the iminium moiety is a
determinant for the bioactivities of SA and CH, including anticancer [20], acaricidal [21] and antimicrobial [22,
23]. A similar case was also found in the anti-AChE and antifungal activities of their analogues
2-aryl-β-carbolin-2-iums and their 3,4-dihydro derivatives [24, 25]. Therefore, we speculated that a similar case
may well also exist in ChE inhibition activity of SA and CH.
Based on the findings above and the principle of analogue design, we initially designed one primary target
skeleton (PTS) (Fig. 1) by deleting the C-ring of SA or CH, which is considered as a simplified QBAs-like skeleton.
Next, considering that SA or CH can be metabolized to the corresponding neutral nonactive phenanthridine
derivative by demethylation (N-Me) in cells [26], we further adjusted and simplified the structure of PTS by

deleting the (N-)methyl and changing the position of the nitrogen atom to obtain the present target molecule
framework (Fig. 1). Obviously, the framework of the target molecules possesses the similar molecular length,
width and polar iminium moiety (C=N+) as SA or CH. Therefore, theoretically, the target compounds should also
have ChE inhibition activity like SA or CH.
In aspects of substituents, almost all of the known QBAs have one alkoxy group at 7 site adjacent to its C=N+
bond. According to this characteristic, we further designed an 8-methoxy on the target skeleton to obtain the
parent compound, i.e., 2-phenyl-8-methoxyisoquinolin-2-ium (8a, Fig. 2). Meanwhile, compound 8zf
without 8-OMe was also designed to examine the effect of 8-OMe on the activity.
Figure 2. Synthesis route of the target compounds (8). (a) Br
(HCHO) , TFA, 0 °C to rt; (d) n-BuLi, dry THF, –78 °C, and then treated with water; (e) DDQ, dry MeOH,
dry DCM; (f) TMSBr, Bu NBr, dry toluene, 80 °C; (g) Ar-NH , dioxane ;(h) Pd/C, acetonitrile, reflux.
2, dry DCM; (b) NaBH
4, I
2, dry THF, 40 °C; (c)
n
4
2
Electron density distribution and stereochemistry are two important impact factors for the bioactivity of
molecules. Theoretically, the substituents on the C-ring not only can influence the electron density
distribution of the target molecules but also its stereochemistry such as the dihedral angle between the B-ring
and C-ring. With the aim of finding more potent AChE inhibitors and understanding the structure-activity
relationship, various substitution patterns were designed on the C-ring (Table 1). The substituents include

electron withdrawing groups like halogen atoms, trifluoromethyl and cyano and electron-donating groups
like methyl and methoxy. The substitution sites involve ortho-, meta- and para-position (Fig. 2).
2.2. Synthesis of compounds
The synthetic route is shown in Figure 2. According to our reported method [27], intermediates 7 were
synthesized from commercially available 2-(3-methoxyphenyl) acetic acid as a starting material via
bromination, reduction of carboxyl group, cyclization with paraformaldehyde in trifluoroacetic acid,
debromination with n-BuLi, oxidation with DDQ in the presence of methanol, ring-opening with Bu NBr in
4
the presence of TMSBr and finally reaction with aniline or substituted anilines. 7zf without 8-OMe was
synthesized from 2-phenylethanol according to the method described in literature [28]. Compounds 7 were
dehydrogenated by Pd/C in acetonitrile or toluene to provide the corresponding target compounds 8.
1
13
Compounds 8 were structurally elucidated by H NMR, C NMR and HRMS analyses. The structures of
intermediates 7 were confirmed by comparison of spectral data those reported in literature [27]. All
compounds 8 showed some similar spectroscopic characteristics due to the structural similarity. Each
compound showed a characteristic ion peak at m/z [M−Br]+ in positive HRMS spectra. The presence of
bromide anion was confirmed by ion peaks at m/z 79 and 81 in negative ESI-MS spectra. In 1H and 13C NMR
spectra, each compound 8 revealed signals of H-1 at δ
range of δ 158–160, signals of one AX system at δ ca. 8.88 (1 H, d, J = ca 6.5 Hz, H-4), ca. 8.56 (1 H, d, J = ca.
6.5 Hz, H-3), δ ca. 146 (C-4) and ca.140 (C-3). Except 8zf, all compounds 8 also showed signals of 8-OMe in
the range of δ 3.0–4.3 and δ ca. 56.5.
H
ca. 10.0 (1 H, s or d, J = ca. 1.2 Hz) and C-1 in the
C
H
C
H
C
2.3. ChE inhibition activity
According to Ellman’s method [29], compounds 8 along with intermediates 7 were initially screened for
inhibition activity on AChE at 10 μM. SA and CH as model compounds were used as reference controls.
Galantamine, a selective AChE inhibitor drug for treatment of AD, was used as a positive control.
As expected, all compounds 8 showed anti-AChE activity at 10 μM in varying degrees like SA or CH
(Figure 3). A similar case was also found for 7. However, 8 were obviously more active than the respective 7

in most cases. It is worth noting that some compounds (8g, 8j, 8p, 8y, 8zc, 8zd, 7g, 7j) displayed the excellent
activity with inhibition rates of >90%, comparable with galantamine.
Figure 3. The activity of compounds 8 and 7 against AChE at 10 μM. CH: chelerythrine; SA: sanguinarine;
Gal: galantamine.
In order to explore anti-AChE potential in more detail, selectivity to both AChE and BuChE as well as
SAR, the more active compounds with the inhibition rates of ＞50% in Figure 3 were further determined for
median inhibition concentrations (IC50) on both AChE and BuChE. The results are shown in Table 1.
Gratifyingly, seven out of 8 (8d, 8g, 8j, 8m, 8p, 8y, 8zc) gave lower IC50 values of 0.14 to 0.72 μM for AChE
than galantamine (IC50 0.79 μM) and SA and CH (IC50 1.22, 1.45 μM). A similar case was also observed for
three 7 (7h, 7j, 7v). Among them, 8g, 8j and 8y gave the highest anti-AChE activity (IC50 0.1‒0.2 μM), which
reached up to more than four-fold that of galantamine. The results above strongly supported the rationality
of our target molecular design idea. Furthermore, most of the tested compounds also showed some
anti-BuChE activity (IC50 > 20 μM), but which is much less active than the respective anti-AChE activity and
that of SA and CH (IC50 7.08, 8.58 μM). Obviously, this kind of compounds has higher selectivity to AChE
relative to BuChE. It was worth noting that 8y revealed the excellent inhibition activities on both AChE and
BuChE (IC50 0.14, 3.84 μM) simultaneously, comparable with rivastigmine (IC50 9.94, 2.86 μM), a dual

AChE−BuChE inhibitor drug. Therefore, 8y can be considered as one promising candidate compound for
development of novel dual AChE−BuChE inhibitor drug.
Table 1
Median inhibition concentrations of compounds 8 and intermediates 7 against ChEs.
IC50 (95%CI) (μM)
IC50 (95%CI) (μM)
SIa
SIa
No.
No.
AChE
BuChE
80.3 (75.9‒84.9)
80.9 (73.1‒88.9)
>250
AChE
BuChE
35.4 (30.0‒42.6)
75.0 (66.7‒83.6)
n.d.c
8a
8b
8c
8d
8e
8f
1.42 (1.23‒1.62)
2.55 (2.11‒3.10)
9.13 (7.44‒11.9)
0.67 (0.41‒0.75)
5.33 (4.84‒5.94)
9.45 (7.66‒12.3)
0.17 (0.12‒0.23)
5.23 (4.83‒5.69)
>10b
56.5
31.7
7a
7b
7c
7d
7e
7f
1.62 (1.40‒1.84)
2.94 (2.68‒3.22)
>10b
21.9
25.5
143 (126‒166)
67.2 (56.4‒77.6)
36.4 (29.4‒44.3)
105 (97.9‒114)
89.5 (80.7‒98.9)
n.d.c
213
1.99 (1.80‒2.17)
≈10b
113 (94.2‒133)
190 (172‒211)
n.d.c
56.8
19.0
12.6
3.85
618
>10b
8g
8h
8i
7g
7h
7i
0.32 (0.25‒0.38)
1.50 (1.15‒1.83)
>10b
62.1 (57.8‒67.0)
27.2 (21.8‒33.1)
n.d.c
194
17.1
18.1
8j
0.19 (0.13‒0.26)
2.06 (1.84‒2.27)
>10b
21.6 (17.7‒25.6)
36.9 (34.7‒39.2)
n.d.c
114
7j
0.28 (0.22‒0.34)
1.79 (1.58‒2.00)
>10b
20.7 (17.2‒24.4)
28.9 (26.1‒31.8)
n.d.c
73.9
16.1
8k
8l
17.9
7k
7l
8m
0.72 (0.53‒0.91)
36.1 (32.6‒39.6)
50.1
7m
1.01 (0.76‒1.24)
58.1 (43.7‒71.3)
57.5
n.d.c
n.d.c
8n
8o
>10b
>10b
n.d.c
n.d.c
7n
7o
>10b
>10b
8p
0.69 (0.62‒0.77)
1.75 (1.52‒1.94)
3.41 (3.22‒3.62)
>10b
33.5 (26.4‒41.7)
59.4 (55.7‒63.4)
>250
48.6
33.9
7p
1.94 (1.56‒2.30)
≈10b
221 (193‒256)
n.d.c
114
8q
7q
8r
7r
5.31 (4.73‒5.97)
>10b
110 (103‒119)
n.d.c
20.7
8s
n.d.c
7s
8t
2.89 (2.55‒3.23)
2.21 (1.75‒2.50)
1.08 (0.94‒1.24)
>10b
243 (224‒265)
30.6 (27.8‒33.4)
>250
37.8
13.8
7t
3.07 (2.62‒3.43)
2.89 (2.25‒3.53)
0.53 (0.42‒0.64)
>10b
133 (107‒165)
35.9 (32.4‒39.4)
47.1 (42.1‒52.4)
n.d.c
8u
7u
8.8
8v
7v
8w
n.d.c
7w
8x
6.90 (6.27‒7.62)
0.14 (0.11‒0.17)
2.70 (2.45‒2.96)
3.42 (3.02‒3.93)
>10b
98.3 (85.5‒111)
3.84 (3.17‒4.54)
48.4 (43.1‒53.6)
232 (213‒256)
n.d.c
14.2
27.4
17.9
67.8
7x
7y
5.23 (3.07‒6.05)
1.04 (0.72‒136)
1.29 (1.01‒1.62)
1.04 (0.86‒1.23)
>10b
77.3 (63.8‒91.4)
113 (93.5‒135)
105 (92.3‒120)
90.5 (80.4‒101)
n.d.c
14.8
128
8y
8z
7z
81.4
87.0
8za
7za
8zb
7zb
8zc
8zd
0.57 (0.44‒0.51)
0.90 (0.82‒0.98)
1.23 (1.05‒1.45)
2.39 (2.18‒2.62)
1.22 (1.10‒1.34)
131 (122‒139)
196 (176‒223)
89.8 (81.7‒98.4)
130 (115‒146)
7.08 (4.27‒10.2)
8.58 (5.34‒12.1)
13.7 ± 0.71
230
7zc
3.72 (3.55‒3.90)
1.29 (1.03‒1.54)
1.06 (0.87‒1.36)
10.5 (9.38‒11.9)
9.94 ± 0.83d
0.25 ± 0.01e
0.03 ± 0.01f
116 (102‒133)
69.3 (63.7‒74.8)
128 (107‒153)
63.7 (55.1‒73.8)
2.86 ± 0.22d
0.05 ± 0.00e
5.40 ± 0.27f
31.2
53.7
121
218
7zd
8ze
73.0
54.4
5.80
5.92
18.0
7ze
8zf
7zf
6.07
0.29d
0.22e
180f
Sanguinarine
Rivastigmine
Tacrine
Donepezil
Chelerythrine 1.45 (1.22‒1.71)
Galantamine
0.79 ± 0.05
a
Selectivity index = the ratio value of IC50 (BuChE)/IC50 (AChE). b Estimated values based on the results in Figure 3. c n.d.: no
determination. d The data are cited from ref. [30]. e The data are cited from ref. [31]. f The data are cited from ref. [32].
2.4. Cytotoxicity
In order to get insight into the toxicity of compounds 8 and 7, three more active compounds 8j, 8y and 8zc
along with their respective intermediates 7 were further evaluated for cytotoxic activity on three strains of

normal cells, mouse neuroblastoma N2a cells, primary cultured porcine fetal kidney cells and primary
cultured goat fetal fibroblast using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
(MTT) method [20]. Compounds 8j and 8y were chosen as the representative compounds with an
electron-withdrawing group and an electron-donating group, respectively, while 8zc was chosen as the
representative dihalogenated compound.
The results in Table 2 showed that both 8j, 8y and 8zc, especially the last two showed the much lower
cytotoxicity (IC50 values > 19 μM) on all the tested cells than their anti-AChE activity (IC50 < 0.60 μM). A
similar case was also observed for 7j, 7y and 7zc (IC50 4.26‒27.3 μM vs 0.28, 1.04, 3.72 μM). Relatively, the
cytotoxicities of 8y with 4′-OMe and 8zc with 2′,4′-diBr were lower than 8j with 4′-Br. However, compared
with the corresponding intermediates 7, compounds 8, especially 8y and 8zc (IC50 > 50 μM) showed lower
cytotoxicity on all the tested cells. The results above indicate that both aromatization of the B-ring and the
presence of 4′-OMe are beneficial for low cytotoxicity of 8. Thus, compounds 8 possess greater potential than
7 for development of new AChE inhibitor drugs in two aspects of anti-AChE activity and cytotoxicity.
Table 2
Cytotoxicity of the compounds on three strains of normal cells (48 h).
Compd.
IC50 (μM) (95% confidence interval)
Mouse neuroblastoma
N2a cells
Primary cultured porcine
fetal kidney cells
19.8 (15.8−27.0)
>50
Primary cultured goat
fetal fibroblast
>50
No.
R
8j
4
′-Br
20.8 (15.9−27.2)
>50
8y
8zc
7j
4′-OMe
>50
2
4
4
2
′,4′-diBr
′-Br
′-OMe
′,4′-diBr
>50
>50
>50
20.3 (17.0−24.2)
26.6 (21.3−30.1)
4.26 (4.09−4.44)
19.2 (16.1−23.0)
23.8 (18.4−27.5)
6.03 (5.60−6.48)
20.6 (14.4−29.5)
27.3 (23.5−32.9)
6.13 (5.22−7.20)
7y
7zc
2.5. Mechanism of AChE inhibition
As representative compounds, 8j and 7j were conducted for kinetic analysis of AChE inhibition to explore
the inhibition mechanism. The graphical analysis of the inhibition data is shown in Figure 4. Figures 4A and
4C clearly showed that the two compounds inhibited AChE in a competitive manner with the substrate
acetylthiocholine iodide (ATCh). The inhibition constants K
8.86 × 10-8 M, respectively, indicating 8j has approximately 6-fold the binding capacity of 7j with AChE.
value of 1.69 × 10-4 M of substrate ATCh, the binding capacity of 8j with AChE
i
of 8j and 7j for AChE were 1.48 × 10-8 M and
Compared with the K
m
reaches up to 11419-fold that of ATCh.

Figure 4. Mechanism of AChE inhibition by compound 8j (A) and intermediate 7j (C) respective to ATCh,
and their K determination (B, D). A and C: the reciprocals of the initial reaction rates and substrate
i
concentrations are plotted; B and D: the slope values of the lines from graph A or C are plotted versus the
inhibitor concentrations, affording an equation of linear regression. When y is 0, the equations give Ki values
of 1.48 × 10-8 M for 8j and 8.86 × 10-8 M for 7j.
2.6. Molecular docking study
As representative compounds, compounds 8 with high anti-AChE activity were conducted for molecular
docking studies to get insight into their binding interactions in the hydrolytic active site of AChE and
BuChE. The data in Table 3 show that the binding free energies (FBE) of each the compound except 8d with
the catalytic site of AChE are larger than their respective FBE with BuChE, indicating that compounds 8
have more inhibition potential to AChE than BuChE, agreement with the measured inhibition activities.
Additionally, there was an acceptable correlation between the FBE values and the pIC50 values (P < 0.3059)
for AChE (see supporting information). However, no correlation was found for BuChE, which might be
related with the lower anti-BuChE activity of the compounds and the larger measurement deviation of IC50
values.

Table 3
Estimated free energy of binding (FBE, kcal/mol) of the test compounds to the active cavities of AChE (PDB
code: 4BDT) and BuChE (PDB code: 5K5E).
FBE (kcal/mol)
IC50 (μM)
Compound
AChE
BuChE
–7.05
–7.54
–6.63
AChE
BuChE
143
–
–
–
–
–
–
–
–
8d
6.16
8.35
7.69
7.32
7.05
7.45
7.66
7.86
0.67
0.17
0.19
0.72
0.69
1.08
0.14
0.57
0.0011
8g
105
8j
21.6
36.1
33.5
>250
3.84
131
–
6.57
8m
8p
–6.63
–
6.53
–
6.49
–
7.07
/
8v
8y
8zc
huprine Wa
6QSb
10.23
/
–
–
11.49
0.443
a The inhibitor co-crystals with AChE in the crystal structure of the protein complex (PDB code: 4BDT). b The
inhibitor co-crystals with BuChE in the crystal structure of the protein complex (PDB code: 5K5E).

Figure 5. The estimated binding modes of the compounds (8j, in stick model with carbon in wheat; 8y, in
stick model with carbon in pale green) into the active site of AChE (A, C) and BuChE (B, D).
The results of molecular docking showed that 8j and 8y have very similar binding modes (Figure 5).
Figure 5A and 5C showed that the isoquinoline moiety of the compounds is embedded in a remarkable
group of aromatic rings including Trp86, Tyr337, Trp439 and Tyr449 by the π-π interactions between the A-,
B-ring of the isoquinoline and the phenyl ring of Tyr337 (distance < 4.1 Å), the B-ring and the indole ring of
Trp86 (distance = 4.3 Å), and the A-ring and the phenyl ring of Trp439 (distance = 5.0 Å). The results above
indicated that the binding region of the AChE with the compounds 8 was the cationic site (Trp86) in the
gorge of the AChE. Interestingly, the binding mode is very similar to that of 2-aryl-β-carbolin-2-iums.25 It
was worth noting that the positively charged isoquinoline moiety is poor in π-electron, whereas all the
aromatic rings of these residues are rich in π-electrons. Therefore, the compounds can form strong π-π
action with the residues to improve the overall affinity of AChE for the compounds or the activity of the
compounds. Obviously, the aromatic B-ring in 8 can give stronger π-π action than the non-aromatic B-ring
in 7, which explains why compounds 8 have larger binding free energy with AChE (Table 3) and higher
anti-AChE activity than the corresponding 7 (Table 1).
Additionally, Figure 5A and 5C also showed that 4′-Br and 4′-OMe could form an H-bond with the
hydroxyl group of Ser-125 and the amino group of Asn-87, which could increase the binding free energy of
AChE‒the compound. We speculated that similar cases should also exist in the other 4′-halogenated (8d, 8g,
8m) or 4′-CF compounds (8p). This should be the main reason why the 4′-substituted compounds showed
3
the higher anti-AChE activity than both 8a (R = H) and the 2′- or 3′-substituted isomers.
2.7. Structure-activity relationships
From the data in Table 1 and Figure 3, some important structure-activity relationship for compounds 8
and 7 can be deduced (Fig. 6). (1) In most cases, the aromatic B-ring is very important for the high
anti-AChE activity of 8 but disadvantageous for the improvement of the anti-BuChE activity. Seventy-two
percent (18/25) and sixty-eight percent (15/22) of the tested 8 showed the higher anti-AChE and lower
anti-BuChE activity than their respective 7, respectively. It was worth noting that for some substitution
patterns on the C-ring, the aromatization of the B-ring can increase or reduce the two kinds of activities at
the same time (4′-OMe, 2′-Cl, 4′-CF , 3′-Me; 2′-Br, 2′-I, 4′-Me, 3′-OMe, 2′,4′-diCl). Additionally, for
3

2′,6′-difluoro or 2′-F-4′-I substitution pattern, the aromatization of the B-ring can increase the anti-BuChE
activity but reduce the anti-AChE activity (8z vs 7z; 8ze vs 7ze). The results indicate that the effect of the
aromatization of the B-ring on the activity also depends on the substitution pattern of the C-ring in a few
cases.
(2) The substitution pattern of the C-ring dramatically impacts the anti-AChE activity of 8. Except cyano
group (8s), all the 4′-substituents can greatly enhance the anti-AChE activity of 8, whereas the 2′- or
3′-substituents cause reduction of the activity. A similar case also exists in 7. According to the results of
molecular docking above, the enhancement effect of 4′-substituents on the activity should be attributed to
the fact that it can form an H-bond with the residues Ser-125 and Asn-87 of AChE and increase the binding
free energy of AChE‒the compound. Obviously, the 4′ site should be one privileged modifiable position for
further structure optimization.
Figure 6. SAR of compounds 8.
(3) The introduction of 8-OMe can dramatically enhance both anti-AChE and anti-BuChE activities at the
same time (8j vs 8zf). This result suggests that the substituents on the A-ring can also significantly influence
the activity. Therefore, we think that it is necessary to conduct more extensive modification of the A-ring of
8 to find more potent AChE inhibitors in a follow-up study.
(4) For 4′-monohalogenated 8, the introduction of additional halogen atoms to the C-ring cannot increase
anti-AChE activity. All the 2′,4′-dihalogenated 8 were less active on both AChE and BuChE than its
respective 4′-monohalogenated compound (Table 1, 8za vs 8g; 8zc vs 8j; 8zd vs 8j; 8ze vs 8m). A similar case
was also observed in compounds 7. Furthermore, the 2′,6′-difluorinated compound and the
3′,5′-dichlorinated compound also showed the lower anti-AChE activity than the respective
monohalogenated compounds, respectively (Table 1, 8z vs 8b; 8zb vs 8f).

Interestingly, the above SAR of 8 for AChE is very similar to that of 2-aryl-β-carbolin-2-ium salts
previously reported by us [25]. The reason should be due to the structural similarity between two types of
compounds. In fact, 8 can be considered as simple analogues of 2-aryl-β-carbolin-2-niums [24].
3. Conclusions
In summary, we designed and synthesized a series of novel 2-aryl-8-methoxyisoquinol-2-iums (8) as the
structurally simple analogues of CH. All compounds 8 showed anti-AChE activity at 10 μM, and some of
which revealed the excellent activity and high selectivity for AChE, superior to SA, CH and galantamine, a
positive drug. 8y displayed dual AChE−BuChE potent inhibition action. Compounds 8j and 7j showed a
competitive manner of AChE inhibition. Furthermore, 8 also showed the lower cytotoxicity on normal cells.
SAR showed that the B-ring aromatization, the presence of 8-OMe on the A-ring and 4′-substituents on the
C-ring are very important for the high anti-AChE activity of 8. Molecular docking showed that the
isoquinoline moiety of 8 can be embedded in a remarkable group of aromatic rings of AChE by the π-π
action. These findings will be of great importance for further structural optimization design. Thus, 8 can be
considered as promising lead compounds or candidates for the development of novel ChE inhibitor agents.
4. Experimental
4.1. Materials and instruments
Chemicals.
Acetylthiocholine
iodide
(ATCh),
butyrylthiocholine
iodide
(BuTCh),
5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), and galantamine (>99%) were purchased from Sigma Chemicals
Co., Ltd. (Shanghai, China). 2-(3-Methoxyphenyl)acetic acid, 2-phenylethanol and substituted anilines were
purchased from Aladdin Industrial Inc. (Shanghai, China). Sanguinarine (SA, >98%) and chelerythrine
iodide (CH, >98%) were obtained by isolation from the plant of M. microcarpa (Maxim) Fedde in our
laboratory [33]. Other reagents and solvents were obtained locally and of analytical grade. The water used
was redistilled and ion-free.
Enzymes. AChE (E.C. 3.1.1.7) (BR, 200 u/mg) from fly’s head and BuChE (E.C. 3.1.1.8) (BR, 20 u/mg) from
horse serum were purchased from Shanghai Yuanye biological technology Co. Ltd (Shanghai, China).
1
Instruments. H and 13C NMR spectra were recorded on an Avance III 500 MHz instrument (Bruker,
Karlsruhe, Germany). Chemical shifts were measured relative to the residual solvent peaks of CD
OD (1H, δ
3

13
3.31 ppm; C, δ 49.00 ppm). Chemical shifts (δ values) and coupling constants (J values) are given in parts
per million and hertz, respectively. High-resolution mass spectra (HR-MS) were carried out with a
microTOFQ II instrument (Bruker). Molecular docking analysis was conducted with the Sybyl-X 2.0
software and Discovery Studio 2017 client.
4.2. Chemistry
4.2.1. Synthesis of 7
Intermediates 7 were synthesized from commercially available 2-(3-methoxyphenyl)acetic acid as a
starting material according to the literature method [27]. 7zf without 8-OMe was synthesized from
commercially available 2-phenylethanol according to the method described in literature [28]. The NMR and
MS data were consistent with those previously reported [28].
4.2.2. Synthesis of 8
General procedure. To a solution of 7 (0.2 mmol) in a mixture of acetonitrile (30 mL) and toluene (10 mL)
was added 5% Pd/C (wetted with ca. 55% water) (0.17 mmol, 80 mg). The mixture was refluxed at 87 °C for
about 2 days to complete the reaction. The Pd/C powders in the reaction solution was filtered off through a
sand core funnel and completely washed with methanol. The combined solution was concentrated up to
dryness under vacuum. The resulted residue was purified by column chromatography over silica
(chloroform‒methanol-40% HBr aqueous solution, 10:1:0.01) to yield the desired compounds.
4.2.2.1. 8-Methoxy-2-phenylisoquinolin-2-ium bromide (8a): Orange-red solid; yield, 67% (0.42 g, 1.34 mmol); 1H
NMR (500 MHz, CD
3
OD) δ 9.94 (s, 1H), 8.82 (d, J = 6.8 Hz, 1H), 8.58 (d, J = 6.8 Hz, 1H), 8.28 (t, J = 8.1 Hz, 1H),
13
7.97–7.93 (m, 2H), 7.91 (d, J = 8.2 Hz, 1H), 7.80–7.75 (m, 3H) 7.55 (d, J = 8.2 Hz, 1H), 4.20 (s, 3H); C NMR
(126 MHz, CD
3
OD) δ 159.8, 145.8, 144.4, 141.3, 139.6, 135.9, 132.2, 131.6, 126.9, 125.6, 121.3, 119.7, 111.2, 57.8.
14NO+ [M–Br]+ 236.1070, found 236.1069.
HRMS (ESI) m/z calcd for C16
H
4.2.2.2. 2-(2-Fluorophenyl)-8-methoxyisoquinolin-2-ium bromide (8b): Yellow solid; yield, 45% (0.30 g, 0.90 mmol);
1H NMR (500 MHz, CD
3
OD) δ 10.05 (s, 1H), 8.75 (d, J = 6.5 Hz, 1H), 8.58 (d, J = 6.7 Hz, 1H), 8.32 (t, J = 8.1 Hz,
1H), 7.97 (t, J = 7.6 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.81 (dd, J = 12.6, 7.8 Hz, 1H), 7.63‒7.55 (m, 3H), 4.21 (s,
13
3H); C NMR (126 MHz, CD OD) δ 160.2, 156.4 (d, J = 253.1 Hz), 148.2, 142.2, 140.1, 137.1, 134.8 (d, J = 8.0
3
Hz), 133.8 (d, J = 9.2 Hz), 128.7, 127.3 (d, J = 3.9 Hz), 126.7, 121.3, 120.0, 118.6 (d, J = 19.1 Hz), 111.5, 57.9.
13FNO+ [M–Br]+ 254.0976, found 254.0971.
HRMS (ESI) m/z calcd for C16
H

4.2.2.3. 2-(3-Fluorophenyl)-8-methoxyisoquinolin-2-ium bromide (8c): Brown solid; yield, 52% (0.35 g, 1.04 mmol);
1H NMR (500 MHz, CD
3OD) δ 9.97 (d, J = 4.4 Hz, 1H), 8.81 (br s, 1H), 8.58 (d, J = 6.7 Hz, 1H), 8.28 (t, J = 7.7
Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.81 (s, 2H), 7.55 (d-like, J = 7.9 Hz, 2H), 4.20 (s, 3H);
13C NMR (126 MHz, CD
3OD) δ 163.8 (d, J = 249.9 Hz), 159.9, 146.1, 145.1 (d, J = 10.1 Hz), 141.6, 139.7, 135.7,
133.4 (d, J = 8.8 Hz), 126.9, 122.0 (d, J = 3.3 Hz), 121.2, 119.7, 119.2 (d, J = 21.2 Hz), 113.7 (d, J = 26.7 Hz), 111.4,
13FNO+ [M–Br]+ 254.0976, found 254.0973.
57.9. HRMS (ESI) m/z calcd for C16
H
4.2.2.4. 2-(4-Fluorophenyl)-8-methoxyisoquinolin-2-ium bromide (8d): Yellow solid; yield, 57% (0.38 g, 1.14 mmol);
1H NMR (500 MHz, CD
3
OD) δ 9.98 (s, 1H), 8.82 (d, J = 6.6 Hz, 1H), 8.57 (d, J = 6.6 Hz, 1H), 8.27 (t, J = 8.1 Hz,
1H), 8.03 (dd, J = 8.8, 4.4 Hz, 2H), 7.90 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 8.3 Hz, 2H), 4.21
(s, 3H); 13C NMR (126 MHz, CD
3
OD) δ 165.1 (d, J = 250.9 Hz), 160.0, 146.4, 141.4, 139.8, 136.3, 128.4 (d, J = 9.3
Hz), 126.9, 121.4, 119.8, 118.4 (d, J = 24.0 Hz), 111.4, 57.8. HRMS (ESI) m/z calcd for C16H
13FNO+ [M–Br]+
254.0975, found 254.0979.
4.2.2.5. 2-(2-Chlorophenyl)-8-methoxyisoquinolin-2-ium bromide (8e): Orange-red solid; yield, 53% (0.38 g, 1.08
mmol); 1H NMR (500 MHz, CD
3
OD) δ 10.01 (s, 1H), 8.68 (d, J = 6.4 Hz, 1H), 8.63 (d, J = 6.4 Hz, 1H), 8.34 (t, J =
7.8 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H),
7.73 (t, J = 7.3 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 4.21 (s, 3H); 13C NMR (126 MHz, CD
3
OD) δ 160.1, 148.2, 142.2,
141.3, 140.0, 137.2, 134.2, 132.1, 130.2, 130.0, 129.4, 126.8, 121.1, 120.0, 111.6, 58.0. HRMS (ESI) m/z calcd for
13ClNO+ [M–Br]+ 270.0680, found 270.0683.
C16H
4.2.2.6. 2-(3-Chlorophenyl)-8-methoxyisoquinolin-2-ium bromide (8f): Brown solid; yield, 58% (0.41 g, 1.42 mmol);
1H NMR (500 MHz, CD
3
OD) δ 10.06 (s, 1H), 8.89 (d, J = 6.9 Hz, 1H), 8.59 (d, J = 6.9 Hz, 1H), 8.50 (s, 1H), 8.34
(d, J = 7.9 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.94‒7.91 (m, 2H), 7.57 (d, J = 8.1 Hz, 1H),
13
4.21 (s, 3H); C NMR (126 MHz, CD OD) δ 160.2, 146.6, 145.5, 141.7, 140.0, 136.9, 136.0, 133.0, 132.5, 126.9,
3
126.4, 124.7, 121.5, 119.9, 111.4, 57.8. HRMS (ESI) m/z calcd for C16H
13ClNO+ [M–Br]+ 270.0680, found
270.0678.
4.2.2.7. 2-(4-Chlorophenyl)-8-methoxyisoquinolin-2-ium bromide (8g): Yellow solid; yield, 61% (0.43 g, 1.50 mmol);
1H NMR (500 MHz, CD
3OD) δ 10.03 (s, 1H), 8.83 (dd, J = 6.9, 1.7 Hz, 1H), 8.55 (d, J = 6.8 Hz, 1H), 8.28 (t, J =
8.2 Hz, 1H), 7.93 (d, J = 8.9 Hz, 2H), 7.90 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 8.9 Hz, 2H), 7.56 (d, J = 8.1 Hz, 1H),

13
4.21 (s, 3H); C NMR (126 MHz, CD
3
OD) δ 160.2, 146.6, 143.3, 141.5, 140.0, 138.5, 136.1, 131.7, 127.6, 126.8,
13ClNO+ [M–Br]+ 270.0680, found 270.0660.
121.5, 119.9, 111.3, 57.6. HRMS (ESI) m/z calcd for C16
H
4.2.2.8. 2-(2-Bromophenyl)-8-methoxyisoquinolin-2-ium bromide (8h): Orange-red solid; yield, 44% (0.35 g, 0.88
1
mmol); H NMR (500 MHz, CD
3
OD) δ 9.98–9.92 (m, 1H), 8.64 (s, 2H), 8.34 (t-like, J = 7.8 Hz, 1H), 7.99–7.95
OD) δ 159.9, 147.9,
(m, 3H), 7.77–7.71 (m, 2H), 7.60 (d, J = 7.5 Hz, 1H), 4.20 (s, 3H); 13C NMR (126 MHz, CD
3
142.7, 142.2, 139.9, 137.0, 135.2, 134.4, 130.8, 129.3, 126.8, 120.9, 119.9, 119.4, 111.6, 58.1. HRMS (ESI) m/z calcd
13BrNO+ [M–Br]+ 314.0175, 316.0155, found 314.0174, 316.0154.
for C16
H
4.2.2.9. 2-(3-Bromophenyl)-8-methoxyisoquinolin-2-ium bromide (8i): Brown solid; yield, 55% (0.43 g, 1.10 mmol);1H
NMR (500 MHz, CD OD) δ 10.98 (s, 1H), 8.80 (d, J = 5.7 Hz, 1H), 8.57 (d, J = 5.9 Hz, 1H), 8.29 (t, J = 7.6 Hz,
3
1H), 8.21 (s, 1H), 7.95 (d, J = 7.4 Hz, 1H), 7.92 (t, J = 8.5 Hz, 2H), 7.70 (t-like, 1H), 7.56 (d, J = 7.6 Hz, 1H), 4.21
(s, 3H); 13C NMR (126 MHz, CD
3
OD) δ 160.1, 146.4, 145.3, 141.6, 139.9, 135.9, 135.4, 133.2, 129.0, 126.9, 125.1,
124.3, 121.4, 119.8, 111.4, 57.8. HRMS (ESI) m/z calcd for C16H
13BrNO+ [M–Br]+ 314.0175, 316.0155, found
314.0177, 316.0153.
4.2.2.10. 2-(4-Bromophenyl)-8-methoxyisoquinolin-2-ium bromide (8j): Brown solid;yield, 63% (0.50 g, 1.26 mmol);
1H NMR (500 MHz, CD
3OD) δ 10.02 (s, 1H), 8.82 (dd, J = 6.9, 1.5 Hz, 1H), 8.54 (d, J = 6.9 Hz, 1H), 8.28 (t, J =
8.1 Hz, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.1 Hz, 1H),
13
4.21 (s, 3H); C NMR (126 MHz, CD OD) δ 160.2, 146.5, 141.6, 140.0, 136.0, 134.8, 134.4, 127.8, 126.9, 126.5,
3
121.5, 119.9, 111.3, 57.6. HRMS (ESI) m/z calcd for C16H
13BrNO+ [M–Br]+ 314.0175, 316.0155, found 314.0174,
316.0158.
4.2.2.11. 2-(2-Iodophenyl)-8-methoxyisoquinolin-2-ium bromide (8k): Yellow solid; yield, 40% (0.35 g, 0.80 mmol); 1H
NMR (500 MHz, CD OD) δ 10.01 (d, J = 1.3 Hz, 1H), 8.65 (dd, J = 6.9, 1.4 Hz, 1H), 8.60 (t, J = 6.9 Hz, 1H), 8.33
3
(t, J = 8.2 Hz, 1H), 8.21 (dd, J = 8.0, 1.3 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.87 (dt, J = 8.0, 1.4 Hz, 1H), 7.76 (dt, J
= 7.7, 1.3 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.51 (dt, J = 7.8, 1.5 Hz, 1H), 4.21 (s, 3H); 13C NMR (126 MHz,
CD
3
OD) δ 160.4, 148.4, 146.9, 142.1, 141.7, 140.4, 137.3, 134.1, 131.4, 128.4, 126.8, 121.3, 120.1, 111.5, 95.1, 57.7.
13INO+ [M–Br]+ 362.0036, found 362.0014.
HRMS (ESI) m/z calcd for C16
H
4.2.2.12. 2-(3-Iodophenyl)-8-methoxyisoquinolin-2-ium bromide (8l): Orange-red solid; yield, 51% (0.45 g, 1.02
mmol); 1H NMR (500 MHz, CD
3
OD) δ 9.94 (s, 1H), 8.78 (d, J = 6.3 Hz, 1H), 8.56 (d, J = 6.6 Hz, 1H), 8.34 (s, 1H),

8.28 (t, J = 8.0 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.58–7.51 (m,
2H), 4.20 (3H, s); 13C NMR (126 MHz, CD
3
OD) δ 159.9, 146.2, 145.0, 141.5, 141.3, 139.7, 135.8, 134.4, 133.0,
126.9, 125.4, 121.3, 119.7, 111.3, 95.6, 57.9. HRMS (ESI) m/z calcd for C16H
13INO+ [M–Br]+ 362.0036, found
362.0042.
4.2.2.13. 2-(4-Iodophenyl)-8-methoxyisoquinolin-2-ium bromide (8m): Yellow solid; yield, 57% (0.50 g, 1.14 mmol);
1H NMR (500 MHz, CD
3
OD) δ 10.01 (s, 1H), 8.82 (d, J = 6.6 Hz, 1H), 8.55 (d, J = 6.8 Hz, 1H), 8.28 (t, J = 8.1 Hz,
1H), 8.13 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.1 Hz, 1H), 4.21 (s,
3H); 13C NMR (126 MHz, CD
3
OD) δ 160.2, 146.4, 144.3, 141.5, 140.9, 140.0, 136.0, 127.7, 126.9, 121.5, 119.9,
111.3, 98.2, 57.7. HRMS (ESI) m/z calcd for C16H
13INO+ [M–Br]+ 362.0036, found 362.0028.
4.2.2.14. 8-Methoxy-2-(2-trifluorophenyl)isoquinolin-2-ium bromide (8n): Brown solid; yield, 41% (0.32 g, 0.82 mmol);
1H NMR (500 MHz, CD
1H), 8.11 (d, J = 7.2 Hz, 1H), 8.06–8.00 (m, 3H), 7.98 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 4.20 (s, 3H);13C
3
OD) δ 10.15 (s, 1H), 8.75 (d, J = 6.6 Hz, 1H), 8.60 (d, J = 6.4 Hz, 1H), 8.36 (t, J = 8.1 Hz,
NMR (126 MHz, CD OD) δ 160.3, 148.7, 142.5, 140.9, 140.3 , 137.5, 135.7, 133.7, 130.3, 129.0 (q, J = 4.1 Hz),
3
126.4, 126.3, 124.0 (q, J = 272.0 Hz), 120.8, 120.1, 111.8, 56.6. HRMS (ESI) m/z calcd for C17
H13
F
NO+ [M–Br]+
3
304.0944, found 304.0940.
4.2.2.15. 8-Methoxy-2-(3-trifluorophenyl)isoquinolin-2-ium bromide (8o): Yellow solid; yield, 59% (0.45 g, 1.18
1
mmol); H NMR (500 MHz, CD
3
OD) δ 10.08 (s, 1H), 8.87 (d, J = 6.4 Hz, 1H), 8.59 (d, J = 6.5 Hz, 1H), 8.35 (s,
1H), 8.30 (t, J = 8.0 Hz, 1H), 8.25 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 8.01 (t, J = 7.7 Hz, 1H), 7.92 (d, J =
8.1 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 4.22 (s, 3H); 13C NMR (126 MHz, CD
3OD) δ 160.3, 147.0, 145.0, 141.8,
140.1, 136.2, 133.5 (q, J = 33.2 Hz), 132.9, 130.1, 129.1 (q, J = 3.2 Hz), 127.0, 124.1 (q, J = 27.0 Hz), 122.1 (q, J = 3.6
NO+ [M–Br]+ 304.0944, found 304.0945.
Hz), 121.5, 119.9, 111.4, 57.8. HRMS (ESI) m/z calcd for C17
H13F3
4.2.2.16. 8-Methoxy-2-(4-trifluorophenyl)isoquinolin-2-ium bromide (8p): Orange-red solid; yield, 61% (0.47 g, 1.22
mmol); 1H NMR (500 MHz, CD
(s-like, 2H), 8.10 (s-like, 2H), 7.91 (d, J = 7.3 Hz, 1H), 7.57 (d, J = 7.2 Hz, 1H), 4.22 (s, 3H); 13C NMR (126 MHz,
3
OD) δ 10.10 (s, 1H), 8.87 (br s, 1H), 8.57 (br s, 1H), 8.29 (t, J = 6.7 Hz, 1H), 8.15
CD OD) δ 160.3, 147.2, 147.0, 141.9, 140.2, 136.1, 134.0 (d, J = 32.9 Hz), 128.7 (d-like, J = 3.6 Hz), 127.3, 127.0,
3
124.8 (d, J = 270.6 Hz), 121.5, 120.0, 111.5, 57.8. HRMS (ESI) m/z calcd for C17
H13
F
NO+ [M–Br]+ 304.0944,
3
found 304.0940.

4.2.2.17. 2-(2-Cyanophenyl)-8-methoxyisoquinolin-2-ium bromide (8q): Yellow solid;yield, 42% (0.29 g, 0.84 mmol);
1H NMR (500 MHz, CD
3
OD) δ 10.25 (s, 1H), 8.86 (d-like, J = 5.9 Hz, 1H), 8.62 (d-like, J = 5.6 Hz, 1H), 8.35 (t, J
= 7.7 Hz, 1H), 8.15 (d, J = 7.3 Hz, 1H), 8.09 (br s, 2H), 7.95 (d-like, J = 7.5 Hz, 2H), 7.60 (d, J = 7.6 Hz, 1H), 4.22
(s, 3H); 13C NMR (126 MHz, CD
3
OD) δ 160.6, 148.4, 145.4, 142.6, 140.4, 136.8, 136.4, 135.7, 133.3, 128.9, 126.9,
121.3, 120.1, 115.4, 111.8, 111.1, 57.9. HRMS (ESI) m/z calcd for C17
H13
N
O+ [M–Br]+ 261.1022, found 261.1023.
2
4.2.2.18. 2-(3-Cyanophenyl)-8-methoxyisoquinolin-2-ium bromide (8r): Yellow solid; yield, 54% (0.37 g, 1.08 mmol);
1H NMR (500 MHz, CD OD) δ 10.10 (br s, 1H), 8.87 (s, 1H), 8.58‒7.91 (m, 7H), 7.57 (s, 1H), 4.23 (s, 3H); 13C
3
NMR (126 MHz, CD
3
OD) 13C NMR (126 MHz, CD
OD) δ 160.2, 147.5, 144.8, 144.7, 142.0, 141.7, 140.1, 140.0,
3
136.3, 135.9, 133.0, 132.1, 131.3, 130.4, 127.2, 127.1, 121.5, 120.0, 115.3, 111.6, 111.5, 58.3, 58.2. HRMS (ESI) m/z
O+ [M–Br]+ 261.1022, found 261.1018.
calcd for C17
H13N2
4.2.2.19. 2-(4-Cyanophenyl)-8-methoxyisoquinolin-2-ium bromide (8s): Yellow solid;yield, 55% (0.38 g, 1.10 mmol);
1H NMR (500 MHz, CD
3
OD) δ 10.07 (br s, 1H), 8.89 (d, J = 6.6 Hz, 1H), 8.59 (d, J = 6.6 Hz, 1H), 8.38‒8.06 (m,
5H), 7.92 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 8.1 Hz, 1H), 4.22 (s, 3H); 13C NMR (126 MHz, CD
OD) δ 160.2, 146.6,
3
145.5, 141.7, 140.0, 136.9, 136.0, 133.0, 132.5, 126.9, 126.3, 124.7, 121.5, 119.9, 111.4, 57.8. HRMS (ESI) m/z calcd
O+ [M–Br]+ 261.1022, found 261.1016.
for C17
H13
N2
1
4.2.2.20. 8-Methoxy-2-(o-toly)isoquinolin-2-ium bromide (8t): Orange red solid; yield, 40% (0.26 g, 0.80 mmol); H
NMR (500 MHz, CD OD) δ 9.94 (s, 1H), 8.67 (d, J = 6.4 Hz, 1H), 8.62 (d, J = 6.3 Hz, 1H), 8.31 (t, J = 7.9 Hz, 1H),
3
7.95 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.62‒7.52 (m, 3H), 4.21 (s, 3H), 2.24 (s,
13
3H); C NMR (126 MHz, CD
3
OD) δ 160.0, 147.4, 143.8, 141.5, 140.0, 137.2, 134.1, 133.1, 132.6, 128.9, 127.3,
16NO+ [M–Br]+ 250.1226, found 250.1229.
126.9, 121.4, 120.0, 111.4, 57.9, 17.5. HRMS (ESI) m/z calcd for C17
H
1
4.2.2.21. 8-Methoxy-2-(m-toly)isoquinolin-2-ium bromide (8u): Brown solid; yield, 52% (0.34 g, 1.04 mmol); H
NMR (500 MHz, CD OD) δ 9.92 (s, 1H), 8.82 (d, J = 5.9 Hz, 1H), 8.57 (d, J = 6.0 Hz, 1H), 8.26 (t, J = 7.7 Hz, 1H),
3
7.89 (d, J = 7.8 Hz, 1H), 7.79 (s, 1H), 7.73 (d, J = 7.1 Hz, 1H), 7.64 (t, J = 7.3 Hz, 1H), 7.56 (t-like, J = 7.2 Hz, 2H),
4.21 (s, 3H), 2.54 (s, 3H); 13C NMR (126 MHz, CD
3OD) δ 159.8, 145.8, 144.4, 142.4, 141.2, 139.6, 136.0, 132.9,
131.4, 126.9, 126.1, 122.7, 121.3, 119.8, 111.3, 57.9, 21.4. HRMS (ESI) m/z calcd for C17H
16NO+ [M–Br]+ 250.1226,
found 250.1228.

4.2.2.22. 8-Methoxy-2-(p-toly)isoquinolin-2-ium bromide (8v): Golden yellow solid;yield, 61% (0.40 g, 1.22 mmol);
1H NMR (500 MHz, CD
3
OD) δ 9.27 (s, 1H), 7.94‒7.52 (m, 5H), 7.46 (d-like, J = 5.7 Hz, 2H), 7.21 (d, J = 5.7 Hz,
1H), 7.09 (d, J = 6.1 Hz, 1H), 4.05 (s, 3H), 2.44 (s, 3H); 13C NMR (126 MHz, CD
OD) δ 163.8, 161.8, 142.72,
3
142.67, 142.3, 139.6, 132.1, 131.7, 125.6, 123.5, 121.2, 116.0, 112.4, 57.7, 21.3. HRMS (ESI) m/z calcd for
16NO+ [M–Br]+ 250.1226, found 250.1228.
C17H
4.2.2.23. 8-Methoxy-2-(2-methoxyphenyl)isoquinolin-2-ium bromide (8w): Golden yellow solid; yield, 40% (0.28 g,
0.80 mmol); H:1H NMR (500 MHz, CD
3
OD) δ 9.90 (s, 1H), 8.64 (d, J = 6.6 Hz, 1H), 8.54‒8.51 (m, 1H), 8.28 (t, J
= 8.0 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H),
7.44 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 4.19 (s, 3H), 3.92 (s, 3H). 13C NMR (126 MHz, CD
3
OD) δ 159.9,
153.7, 148.1, 141.5, 139.9, 137.9, 134.2, 133.0, 128.0, 126.3, 122.6, 121.2, 119.9, 114.4, 111.2, 57.8, 57.2. HRMS
+ [M–Br]+ 266.1176, found 266.1164.
(ESI) m/z calcd for C17
H16NO
2
4.2.2.24. 8-Methoxy-2-(3-methoxyphenyl)isoquinolin-2-ium bromide (8x): Brown solid; yield, 45% (0.31 g, 0.90 mmol);
1H NMR (500 MHz, CD
3
OD) δ 9.95 (s, 1H), 8.82 (d, J = 5.8 Hz, 1H), 8.55 (d, J = 5.8 Hz, 1H), 8.26 (t, J = 7.8 Hz,
1H), 7.89 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.46 (d, J = 7.4 Hz, 1H),
7.29 (d, J = 8.1 Hz, 1H), 4.20 (s, 3H), 3.95 (s, 3H); 13C NMR (126 MHz, CD
3
OD) δ 162.3, 160.0, 146.1, 145.6,
141.3, 139.9, 136.2, 132.5, 126.8, 121.4, 119.8, 118.3, 117.7, 111.4, 111.3, 57.7, 56.8. HRMS (ESI) m/z calcd for
+ [M–Br]+ 266.1176, found 266.1182.
C17
H16NO
2
4.2.2.25. 8-Methoxy-2-(4-methoxyphenyl)isoquinolin-2-ium bromide (8y): Brown red solid; yield, 59% (0.41 g, 1.18
mmol); 1H NMR (500 MHz, CD
3
OD) δ 9.90 (s, 1H), 8.80 (d, J = 6.2 Hz, 1H), 8.54 (d, J = 6.2 Hz, 1H), 8.24 (t, J =
7.8 Hz, 1H), 7.88 (t, J = 8.2 Hz, 3H), 7.54 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 7.5 Hz, 2H), 4.21 (s, 3H), 3.93 (s, 3H);
13C NMR (126 MHz, CD
3OD) δ 163.0, 159.8, 145.8, 141.0, 139.5, 137.6, 136.4, 127.2, 126.9, 121.5, 119.8, 116.7,
111.3, 57.9, 56.7. HRMS (ESI) m/z calcd for C17
H
16NO
+ [M–Br]+ 266.1176, found 266.1181.
2
4.2.2.26. 2-(2,6-Difluorophenyl)-8-methoxyisoquinolin-2-ium bromide (8z): Yellow solid; yield, 54% (0.38 g, 1.08
1
mmol); H NMR (500 MHz, CD
3
OD) δ 10.06 (s, 1H), 8.89 (d, J = 6.5 Hz, 1H), 8.58 (d, J = 6.6 Hz, 1H), 8.50 (s,
1H), 8.33 (d, J = 7.5 Hz, 1H), 8.29 (t, J = 8.0 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.95‒7.89 (m, 2H), 7.56 (d, J = 7.9
13
Hz, 1H), 4.21 (s, 3H); C NMR (126 MHz, CD OD) δ 160.2, 157.6 (d, J = 253.5 Hz), 149.3, 143.0, 140.1, 137.4,
3

135.3 (t, J = 9.7 Hz), 129.7, 126.9, 121.1, 120.0, 114.3 (dd, J = 18.9, 3.4, Hz), 111.8, 57.9. HRMS (ESI) m/z calcd for
NO+ [M–Br]+ 272.0881, found 272.0884.
C16
H12F2
4.2.2.27. 2-(2,4-Dichlorophenyl)-8-methoxyisoquinolin-2-ium bromide (8za): Brown solid; yield, 53% (0.41 g, 1.06
mmol); 1H NMR (500 MHz, CD
3
OD) δ 10.02 (s, 1H), 8.67 (br s, 1H), 8.63 (br s, 1H), 8.34 (t, J = 7.4 Hz, 1H), 8.03
(d, J = 7.7 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.90 (s, 1H), 7.73 (d, J = 7.3 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 4.20
(3H, s, 1H); 13C NMR (126 MHz, CD
3
OD) δ 160.0, 148.0, 142.3, 139.9, 139.7, 139.0, 136.8, 131.6, 131.2, 130.4,
130.2, 126.7, 120.9, 119.8, 111.5, 57.9. HRMS (ESI) m/z calcd for C16
H
12Cl
NO+ [M–Br]+ 304.0290, found
2
304,0294.
4.2.2.28. 2-(3,5-Dichlorophenyl)-8-methoxyisoquinolin-2-ium bromide (8zb): Orange-red solid; yield, 57% (0.44 g,
1.14 mmol); 1H NMR (500 MHz, CD
8.07 (s, 2H), 7.91 (d, J = 7.2 Hz, 1H), 7.87 (s, 1H), 7.57 (d, J = 6.9 Hz, 1H), 4.22 (s, 3H); 13C NMR (126 MHz,
3
OD) δ 10.06 (s, 1H), 8.82 (br s, 1H), 8.56 (br s, 1H), 8.29 (t, J = 7.9 Hz, 1H),
CD
3
OD) δ 160.4, 147.2, 145.8, 142.0, 140.2, 137.6, 136.0, 132.3, 126.9, 125.6, 121.4, 119.9, 111.5, 58.0. HRMS (ESI)
NO+ [M–Br]+ 304.0290, found 304,0297.
m/z calcd for C16
H12Cl
2
4.2.2.29. 2-(2,4-Dibromophenyl)-8-methoxyisoquinolin-2-ium bromide (8zc): Golden yellow solid; yield, 55% (0.52 g,
1.10 mmol); 1H NMR (500 MHz, CD
3
OD) δ 10.08 (s, 1H), 8.68 (br, s, 1H), 8.61 (br s, 1H), 8.34 (t-like, 1H), 8.21
13
(s, 1H), 7.95–7.91 (m, 3H), 7.59 (d, J = 7.0 Hz, 1H), 4.21 (s, 3H); C NMR (126 MHz, CD OD) δ 160.3, 148.5,
3
142.4, 142.3, 140.3, 137.6, 137.1, 133.9, 130.8, 127.2, 126.8, 121.2, 121.0, 120.1, 111.6, 58.0. HRMS (ESI) m/z calcd
NO+ [M-Br]+ 391.9280, found 391.9279.
for C16
H12Br
2
4.2.2.30. 2-(4-Bromo-2-fluorophenyl)-8-methoxyisoquinolin-2-ium bromide (8zd): Orange-red solid; yield, 50% (0.41 g,
1.00 mmol); 1H NMR (500 MHz, CD
7.97–7.90 (m, 2H), 7.87 (d, J = 9.4 Hz, 1H), 7.77 (br s, 1H), 7.58 (d, J = 7.0 Hz, 1H), 4.22 (s, 3H); 13C NMR (126
3
OD) δ 10.09 (s, 1H), 8.77 (br s, 1H), 8.58 (br s, 1H), 8.32 (t-like, 1H),
MHz, CD OD) δ 160.3, 156.8 (d, J = 254.8 Hz), 148.6, 142.3, 140.2, 137.1, 1231.2 (d, J = 10.9 Hz 1H), 129.3 (d, J =
3
3.7 Hz), 130.3, 127.2 (d, J = 9.0 Hz), 126.8, 122.2 (d, J = 22.0 Hz), 121.4, 120.0, 111.6, 58.0. HRMS (ESI) m/z calcd
12FBrNO+ [M–Br]+ 332.0081, found 332.0067.
for C16
H
4.2.2.31. 2-(2-Fluoro-4-iodophenyl)-8-methoxyisoquinolin-2-ium bromide (8ze): Brown solid; yield, 60% (0.55 g, 1.20
mmol); 1H NMR (500 MHz, CD
7.99 (d, J = 9.2 Hz, 1H), 7.96-7.90 (m, 2H), 7.76 (br s, 1H), 7.58 (d, J = 7.3 Hz, 1H), 4.21 (s, 3H); 13C NMR (126
3
OD) δ 10.06 (s, 1H), 8.75 (br s, 1H), 8.58 (br s, 1H), 8.32 (t-like, J = 7.8 Hz, 1H),

MHz, CD OD) δ 160.2, 155.8 (d, J = 257.1 Hz), 148.3, 142.3, 140.1, 136.9, 136.6 (d, J = 3.6 Hz), 131.6 (d, J = 12.2
3
Hz), 130.1, 127.8 (d, J = 21.4 Hz), 126.8, 121.3, 120.0, 111.6, 98.7 (d, J = 7.9 Hz), 58.0. HRMS (ESI) m/z calcd for
12FINO+ [M–Br]+ 379.9942, found 379.9936.
C16H
1
4.2.2.32. 2-(4-Bromophenyl)isoquinolin-2-ium bromide (8zf): White solid; yield, 76% (0.55 g, 1.52 mmol); H NMR
(500 MHz, CD
3OD) δ 10.25 (s, 1H), 8.91 (d, J = 6.8 Hz, 1H), 8.66 (t, J = 7.4 Hz, 2H), 8.43 (d, J = 8.3 Hz, 1H), 8.35
(t, J = 7.6 Hz, 1H), 8.15 (t, J = 7.6 Hz, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.91 (d, J = 8.7 Hz, 2H); 13C NMR (126 MHz,
CD
3
OD) δ 151.2, 143.6, 139.4, 139.3, 135.7, 134.8, 133.0, 132.5, 129.2, 128.7, 127.7, 127.6, 126.7. HRMS (ESI) m/z
11BrN+ [M–Br]+ 284.0069, found 284.0076.
calcd for C15
H
4.3. Assay of AChE and BuChE inhibition activity
The Ellman’s coupled enzyme method [29] as described previously [25], was used to assay the
inhibitory activity of compounds against AChE. Briefly, 90 μL potassium phosphate buffer (PBS, 0.1 M, pH
7.4), 10 μL the solution of AChE (2 units/mL) in 0.1 M PBS (pH 7.4) and 10 μL the solution of test
compounds (200 μM) in the mixed solution of methanol-PBS (pH 7.4) (1:9, V/V) were added to each well of a
96-well plate. The methanol-PBS solution was used as a blank control. After incubation at room temperature
for 10 min, 60 μL the solution of 10 mM DTNB in PBS was added into each the well. The plate was placed on
crushed ices and cooled for 20 min. To each the well was added 30 μL the solution of ATCh (7.5 mM) in PBS
(pH 7.4). After incubation at 37 °C for 40 min, the initial rate of the enzyme reaction was analyzed by
measuring absorbance at wavelength of 412 nm with a microplate reader (Molecular Devices Co., Ltd.).
Each test was performed in triplicate. Inhibition rates of the enzyme were calculated relative to a control
sample. The inhibitory activity against BuChE were measured as described above for AChE, using BuChE
(0.7 unit/mL) and BuTCh (4 mM) instead of AChE and ATCh as the enzyme and substrate, respectively.
The IC50 values of compounds were measured according to the same method as above. A series of
concentrations of the compound were set on the base of the screening results and used to test the inhibition
rate against AChE or BuChE. The probit value of the inhibition rate for each the concentration and the
corresponding lg[concentration] were used to conduct linear regression by the linear least-square fitting
method. IC50 values and 95% confidence intervals were calculated from the regression equations by using
PRISM software ver. 5.0 (GraphPad Software Inc., San Diego, CA, USA).
4.4. Cytotoxicity Assay

The cytotoxicity of compounds 8j, 8y, 8zc, 7j, 7y and 7zc was assayed using the MTT method. Mouse
neuroblastoma N2a cells, primary cultured porcine fetal kidney cells and primary cultured goat fetal
fibroblast were used as the test cells. The operation procedure was descripted in supporting information.
4.5. Molecular Docking Study
Molecular docking simulations were performed using the software Autodock 4.2 along with AutoDock
Tools (ADT 1.5.6) using the hybrid Lamarckian Genetic Algorithm (LGA). The three dimensional (3D) crystal
structures of AChE (PDB code: 4BDT) and BuChE (PDB code: 5K5E) were obtained from the RCSB Protein Data
Bank. The standard 3D structure (mol2 format) of all compounds were constructed by using the “SKETCH”
option function in SYBYL-X. The other details were descripted in supporting information.
Conflicts of interest
The authors state no conflict of interest.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (No. 31872008) and the Key
Research and Development Project of Shaanxi Province (2019NY-195).
Appendix A. Supplementary data
Supplementary information (1H NMR, 13C NMR and HRMS spectra, cytotoxicity assay and molecular docking study)
to this article can be found online at https://doi.org/xxx.
Author Contributions
Conceptualization: LZ.
Data curation: BZ HL.
Formal analysis: BL.
Funding acquisition: LZ.
Investigation: BZ HL ZC DL.
Methodology: LZ.
Project administration: LZ HG.
Resources: LZ.
Supervision: LZ JG HG.
Validation: LZ.
Visualization: BZ LZ DL.
Writing – original draft: BH LZ HG.
Writing – review & editing: LZ JG.

References
[1] M.M. Mesulam, A. Guillozet, P. Shaw, A. Levey, E.G. Duysen, O. Lockridge, Acetylcholinesterase knockouts establish
central cholinergic pathways and can use butyrylcholinesterase to hydrolyze acetylcholine, Neuroscience 110 (2002)
627‒639.
[2] A. Chatonnet, O. Lockridge, Comparison of butyrylcholinesterase and acetylcholinesterase, Biochem. J. 260 (1989)
625‒634.
[3] Q. Li, H. Yang, Y. Chen, H. Sun, Recent progress in the identification of selective butyrylcholinesterase inhibitors for
Alzheimer's disease, Eur. J. Med. Chem. 132 (2017) 294‒309.
[4] A. Rampa, A. Bisi, F. Belluti, S. Gobbi, P. Valenti, V. Andrisano, V. Cavrini, A. Cavalli, M. Recanatini,
Acetylcholinesterase inhibitors for potential use in Alzheimer's disease: molecular modeling, synthesis and kinetic
evaluation of 11H-indeno-[1,2-b]-quinolin-10-ylamine derivatives, Bioorg. Med. Chem. 8 (2000), 497‒506.
[5] M.A. Khorshid, A.M. Fatma Hassan, A.M. Mona Abd El-Gawad, A.K. Enab, Effect of some plants and pesticides on
acetylcholinesterase, Am. J. Food Technol. 10 (2015) 93‒99.
[6] C. Zhang, Q.Y. Du, L.D. Chen, W.H. Wu, S.Y. Liao, L.H. Yu, X.T. Liang, Design, synthesis and evaluation of novel
tacrine-multialkoxybenzene hybrids as multi-targeted compounds against Alzheimer's disease, Eur. J. Med. Chem. 116
(2016) 200‒209.
[7] M. Prince, A. Comas-Herrera, M. Knapp, M. Guerchet, M. Karagiannidou, World Alzheimer Report 2016 in
Alzheimer's Disease International, 2016.
[8] V.N. Talesa, Acetylcholinesterase in Alzheimer's disease, Mech. Ageing Dev. 122 (2001) 1961‒1969.
[9] J. Hardy, The amyloid hypothesis for Alzheimer's disease: a critical reappraisal, J. Neurochem. 110 (2009) 1129‒1134.
[10] I. Grundke-Iqbal, K. Iqbal, Y.C. Tung, M. Quinlan, H.M. Wisniewski, L.I. Binder, Abnormal phosphorylation of the
microtubule-associated protein tau (t) in Alzheimer cytoskeletal pathology, Proc. Natl. Acad. Sci. U. S. A. 83 (1986)
4913‒4917.
[11] M. Rosini, E. Simoni, A. Milelli, A. Minarini, C. Melchiorre, Oxidative stress in Alzheimer's disease: are we connecting
the dots? J. Med. Chem. 57 (2014) 2821‒2831.
[12] R.T. Bartus, On neurodegenerative diseases, models, and treatment strategies: lessons learned and lessons forgotten a
generation following the cholinergic hypothesis, Exp. Neurol. 163 (2000) 495–529.
[13] M. Singh, M. Kaur, H. Kukreja, R. Chugh, O. Silakari, D. Singh, Acetylcholinesterase inhibitors as Alzheimer therapy:
From nerve toxins to neuroprotection, Eur. J. Med. Chem. 70 (2013), 165–188.
[14] R. Cacabelos, C. Torrellas, O. Teijido, J.C. Carril, Pharmacogenetic considerations in the treatment of Alzheimer’s
disease, Pharmacogenomics 17 (2016) 1041–1074.
[15] M. Noetzli, C.B. Eap, Pharmacodynamic, pharmacokinetic and pharmacogenetic aspects of drugs used in the treatment
of Alzheimer’s disease. Clin. Pharmacokinet. 52 (2013), 225–241.
[16] R. Leon, A.G. García, J. Marco-Contelles, Recent advances in the multitarget-directed ligands approach for the
treatment of Alzheimer’s disease, Med. Res. Rev. 33 (2013) 139–189.
[17] K.-M. Cho, I.-D. Yoo, W.-G. Kim, 8-Hydroxydihydrochelerythrine and 8-hydroxydihydrosanguinarine with a
potent acetylcholinesterase inhibitory activity from Chelidonium majus L., Biol. Pharm. Bull. 29 (2006)
2317-2320.
[18] Y. Lei, L. Liu, X. Tang, D. Yang, X. Yang, F. He, Sanguinarine and chelerythrine: two natural products for
mitochondria-imaging with aggregation-induced emission enhancement and pH-sensitive characteristics. RSC

Advances 8 (2018) 3919–3927.
[19] P. Kosina, D. Walterova, J. Ulrichova, V. Lichnovsky, M. Stiborova, H. Rydlova, J. Vicar, V. Krecman, M.J.
Brabec, V. Simanek, Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding
experiment, Food Chem. Toxicol. 42 (2004) 85–91.
[20] F.-J. Cao, R. Yang, C. Lv, Q. Ma, M. Lei, H.-L. Geng, L. Zhou, Pseudocyanides of sanguinarine and chelerythrine
and their series of structurally simple analogues as new anticancer lead compounds: cytotoxic activity,
structure-activity relationship and apoptosis induction, Eur. J. Pharm. Sci. 67 (2015) 45–54.
[21] F. Miao, X.J. Yang, Y.N. Ma, F. Zheng, X.P. Song, L. Zhou, Structural modification of sanguinarine and
chelerythrine and their in vitro acaricidal activity against Psoroptes cuniculi, Chem. Pharm. Bull. 60 (2012) 1508–
1513.
[22] X.-J. Yang, F. Miao, Y. Yao, F.-J. Cao, R. Yang, Y.-N. Ma, B.-F. Qin, L. Zhou, In vitro antifungal activity of
sanguinarine and chelerythrine derivatives against phytopathogenic fungi, Molecules 17 (2012) 13026–13035.
[23] F. Miao, X.-J. Yang, L. Zhou, H.-J. Hu, F. Zheng, X.-D. Ding, D.-M. Sun, C.-D. Zhou, W. Sun, Structural
modification of sanguinarine and chelerythrine and their antibacterial activity, Nat. Prod. Res. 25 (2011) 863–875.
[24] Z. Hou, L.-F. Zhu, X.-C. Yu, M.-Q.Sun, F. Miao, L. Zhou, Design, synthesis, and structure-activity relationship of
new 2-aryl-3,4-dihydro-β-carbolin-2-ium salts as antifungal agents, J. Agric. Food Chem. 64 (2016) 2847–2854.
[25] B. Zhou, B. Zhang, X. Li, X. Liu, H. Li, D. Li, Z. Cui, H. Geng, L. Zhou, New 2-aryl-9-methyl-β-carbolinium
salts as potential acetylcholinesterase inhibitor agents: synthesis, nioactivity and structure–activity relationship,
Sci. Rep. 8 (2018) 1559.
[26] S.D. Phillips, R.N. Castle, A review of the chemistry of the antitumor benzo[c]phenanthridine alkaloids nitidine
and fagaronine and of the related anti-tumor alkaloid coralyne, J. Heterocycl. Chem. 18 (1981) 223–232.
[27] L.-F. Zhu, Z. Hou, K. Zhou, Z.-B. Tong, Q. Kuang, H.-L. Geng, L. Zhou, Synthesis, bioactivity and
structure-activity relationships of new 2-aryl-8-OR-3,4-dihydroisoquinolin-2-iums salts as potential antifungal
agents, Bioorg. Med. Chem. Lett. 26 (2016) 2413–2417.
[28] Y.-N. Ma, X.-J. Yang, L. Pan, Z. Hou, H.-L. Geng, X.-P. Song, L. Zhou, F. Miao, Synthesis of
2-aryl-3,4-dihydroisoquinolin-2-ium bromides and their in vitro acaricidal activity against Psoroptes cuniculi,
Chem. Pharm. Bull. 61(2013) 204‒211.
[29] G.L. Ellman, K.D. Courtney, J.R.V. Andres, R.-M. Featherstone, A new and rapid colorimetric determination of
acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961), 88‒95.
[30] G. Xiao, Y. Li, X. Qiang, R. Xu, Y. Zheng, Z. Cao, L. Luo, X. Yang, Z. Sang, F. Su, Y. Deng, Design, synthesis
and biological evaluation of 4′-aminochalconerivastigmine hybrids as multifunctional agents for the treatment of
Alzheimer’s disease, Bioorg. Med. Chem. 25 (2017), 1030‒1041.
[31] P. Valenti, A. Rampa, A. Bisi, V. Andrisano, V. Cavrini, L. Fin, A. Buriani, P. Giusti, Acetylcholinesterase
inhibition by tacrine analogs, Bioorg. Med. Chem. Lett. 7 (1997), 2599‒2602.
[32] N. Sultana, M. Sarfraz, S.T. Tanoli, M.S. Akram, A. Sadiq, U. Rashid, M.I. Tariq, Synthesis, crystal structure
determination, biological screening and docking studies of N1-substituted derivatives of
2,3-dihydroquinazolin-4(1H)-one as inhibitors of cholinesterases, Bioorg. Chem. 72 (2017), 256‒267.
[33] F. Miao, X.-J. Yang, L. Zhou, H.-J. Hu, F. Zheng, X.-D. Ding, D.-M. Sun, C.-D. Zhou, W. Sun. Structural
modification of sanguinarine and chelerythrine and their antibacterial activity, Nat. Prod. Res. 25 (2011) 863‒875.

Highlights
ò
ò
ò
ò
ò
Thirty-one target compounds (8) were synthesized.
Seven compounds possess the potent anti-AChE activity and low cytotoxicity.
8y has the excellent dual AChE−BuChE inhibition activity.
The compounds bind with AChE by a competitive model and the π-π action.
The 4′ site is a privileged modifiable position for the high anti-AChE activity.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: