Design, synthesis and evaluation of phthalide alkyl tertiary amine derivatives as promising acetylcholinesterase inhibitors with high potency and selectivity against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2020.115400

A series of phthalide alkyl tertiary amine derivatives were designed, synthesized and evaluated as potential multi-target agents against Alzheimer's disease (AD). The results indicated that almost all the compounds displayed significant AChE inhibitory and selective activities. Besides, most of the derivatives exhibited increased self-induced Aβ_1-42 aggregation inhibitory activity compared to the lead compound DL-NBP, and some compounds also exerted good antioxidant activity. Specifically, compound I-8 showed the highest inhibitory potency toward AChE (IC₅₀ = 2.66 nM), which was significantly better than Donepezil (IC₅₀ = 26.4 nM). Moreover, molecular docking studies revealed that compound I-8 could bind to both the catalytic active site and peripheral anionic site of AChE. Furthermore, compound I-8 displayed excellent BBB permeability in vitro. Importantly, the step-down passive avoidance test indicated that I-8 significantly reversed scopolamine-induced memory deficit in mice. Collectively, these results suggested that I-8 might be a potent and selective AChE inhibitor for further anti-AD drug development.

Full text of DOI:10.1016/j.bmc.2020.115400

Journal Pre-proofs
Design, synthesis and evaluation of phthalide alkyl tertiary amine derivatives
as promising acetylcholinesterase inhibitors with high potency and selectivity
against Alzheimer's disease
Li Luo, Qing Song, Yan Li, Zhongcheng Cao, Xiaoming Qiang, Zhenghuai
Tan, Yong Deng
PII:
S0968-0896(20)30205-4
https://doi.org/10.1016/j.bmc.2020.115400
BMC 115400
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 January 2020
17 February 2020
21 February 2020
Please cite this article as: L. Luo, Q. Song, Y. Li, Z. Cao, X. Qiang, Z. Tan, Y. Deng, Design, synthesis and
evaluation of phthalide alkyl tertiary amine derivatives as promising acetylcholinesterase inhibitors with high
potency and selectivity against Alzheimer's disease, Bioorganic & Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.bmc.2020.115400
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 Elsevier Ltd. All rights reserved.

Design, synthesis and evaluation of phthalide alkyl tertiary amine derivatives as
promising acetylcholinesterase inhibitors with high potency and selectivity against
Alzheimer's disease
a,b
a
a
a
a
c
a,*
Li Luo , Qing Song , Yan Li , Zhongcheng Cao , Xiaoming Qiang , Zhenghuai Tan , Yong Deng
aDepartment of Medicinal Chemistry, Key Laboratory of Drug-Targeting and Drug Delivery System
of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced
Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of
Pharmacy, Sichuan University, Chengdu 611041, China
bPrecision Pharmacy & Drug Development Center, Department of Pharmacy, Tangdu Hospital,
Fourth Military Medical University, Xi’an, 710038, China
cInstitute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of
Chinese Medicine Sciences, Chengdu, 610041, P. R. China
*
Corresponding Author.
E-mail: dengyong@scu.edu.cn (Yong Deng)
1

Abstract
A series of phthalide alkyl tertiary amine derivatives were designed, synthesized and evaluated
as potential multi-target agents against Alzheimer’s disease (AD). The results indicated that almost all
the compounds displayed significant AChE inhibitory and selective activities. Besides, most of the
derivatives exhibited increased self-induced Aβ1-42 aggregation inhibitory activity compared to the lead
compound DL-NBP, and some compounds also exerted good antioxidant activity. Specifically,
compound I-8 showed the highest inhibitory potency toward AChE (IC = 2.66 nM), which was
5
0
significantly better than Donepezil (IC = 26.4 nM). Moreover, molecular docking studies revealed
5
0
that compound I-8 could bind to both the catalytic active site and peripheral anionic site of AChE.
Furthermore, compound I-8 displayed excellent BBB permeability in vitro. Importantly, the step-down
passive avoidance test indicated that I-8 significantly reversed scopolamine-induced memory deficit
in mice. Collectively, these results suggested that I-8 might be a potent and selective AChE inhibitor
for further anti-AD drug development.
Keywords: Alzheimer’s disease; DL-3-n-butylphthalide derivatives; Multi-target agents;
Acetylcholinesterase inhibitors; Anti-Aβ aggregation; Antioxidant.
2

1
. Introduction
Alzheimer’s disease (AD), characterized by memory loss, language and cognitive impairment,
and severe behavioral abnormalities, is an age-related and progressive neurodegenerative disorder. To
date, an estimated 46 million people worldwide suffer from dementia, which is expected to reach 131.5
1
million by 2050. Due to the complex and unidentified etiology, many factors are thought to be related
to the occurrence and development of AD, including acetylcholine (ACh) declines, β-amyloid (Aβ)
2
,3
deposits, dyshomeostasis of biometals, oxidative stress and hyperphosphorylated τ-protein. Current
clinical drugs for AD treatment can only temporarily and modestly improve the cognitive function, but
can’t significantly modify the course and the final outcome of the disease. Thus, there is an urgent
need for new AD therapy strategies.
According to cholinergic hypothesis, the decline levels of ACh in specific regions of the brain
leads to cognitive and memory impairments of AD.4 Acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) are the two types of cholinesterases to hydrolyze ACh in the central
4
,5
nervous system (CNS), and AChE has a 10 fold hydrolytic ACh activity than BuChE. Thus, selective
inhibition of AChE is effective in AD therapy and beneficial to diminish peripheral cholinergic side
5
effects of BuChE inhibitor. Moreover, it has been known that AChE contains catalytic active site
(CAS) and peripheral anionic site (PAS), and the PAS contributes to aggregation of amyloid fibrils,
6
obtaining stable AChE-Aβ complexes, which are more toxic than single Aβ peptide. Therefore,
effective dual-site AChE inhibitors may be more valuable for the research of anti-AD drugs.
Another predominant hypothesis reveals that Aβ peptides in the brain plays a key role in AD
7
pathogenesis. The aggregation of Aβ peptides, particularly Aβ1-42, a key subtype with higher neuronal
toxicity and accumulation rate than Aβ1-40, initiates the pathogenic cascade and ultimately leads to
7
neuronal dysfunction and dementia. Additionally, the progressive accumulation of Aβ is accompanied
8
by inflammation and oxidative stress, which further leads to neurodegeneration. As a result,
prevention of Aβ1-42 aggregation could serve as a rational strategy for AD treatment.
Besides, recent evidences demonstrated that oxidative stress is a central event in AD pathology,
and associated with the formation and accumulation of both Aβ plaques and neurofibrillary tangles.9
Moreover, the biochemical activity of Monoamine oxidases (MAOs) have been shown to produce
1
0
hydrogen peroxide and cause oxidative stress. Currently, selective MAO-A inhibitors can improve
depressive symptoms, while selective MAO-B inhibitors are beneficial for the treatment of
3

1
1
neurodegenerative disorders like AD. Actually, AD patients commonly have depressive symptoms
1
2
which have been considered as risk factors for the development of AD. Thus, inhibition of MAOs
and oxidative damage can be nominated as helpful therapeutics.
Therefore, because of the multiple and complex pathogenesis of AD, molecules aimed at a single
target may be incapable of effectively altering the progression of the disease. In contrast,
multifunctional molecules with two or more complementary biological activities may exert significant
1
3
advantages against AD. Consequently, design of multi-target-directed ligands (MTDLs) which act
on multiple pathophysiological pathways of AD has recently gained special attention and might
represent an important advance in the treatment of AD.
DL-3-n-butylphthalide (Figure 1, DL-NBP) was approved for stroke treatment by the State Food
and Drug Administration of China. Studies in stroke animal models have revealed that DL-NBP could
increase the brain microcirculation, attenuate inflammation, mitochondrial dysfunction, oxidative
damage and apoptosis.14-16 Recently, DL-NBP has been shown to significantly improve cognitive
function in rats injected with Aβ peptide as well as in a mouse AD model carrying mutations in the
genes encoding amyloid precursor protein and presenilin 1.17,18 Moreover, DL-NBP could protect
against cognitive impairment in vascular dementia.19,20 These findings suggested that DL-NBP may
exert a therapeutic efficacy for AD by targeting multiple key pathways of the disease pathogenesis.
®
Donepezil (Aricept ), a clinically effective AChE inhibitor to treat mild-to-moderate AD, can bind
CAS and PAS of AChE respectively through the benzyl piperidine and indanone moieties.21
Interestingly, DL-NBP also has a structure similar to indanone backbone, and previous studies reported
by our group have shown that the tertiary amine fragments, which are derived from the ring-opening
of the benzyl piperidine group of Donepezil, are key requirement for adequate AChE inhibition.22,23
Therefore, according to the MTDLs strategy, we combined DL-NBP with appropriate secondary
amines using different lengths of carbon spacers to design a series of novel phthalide alkyl tertiary
amines derivatives (Figure 1). In this study, all designed compounds were synthesized and evaluated
for their biological activities, including AChE and BuChE, MAOs and Aβ aggregation inhibitory
activities, as well as antioxidant effects. Besides, the outstanding compounds were selected for further
evaluation of the ability to cross the blood-brain barrier (BBB) in vitro and the learning and memory
effect in vivo using mice model.
4

O
O
N
O
H CO
3
CH3
H CO
3
DL-NBP
Donepezil
MTDLs
O
H CO
3
O
E, Z and single bond
H CO
3
n = 3, 5
n
N
R1
I~III
R2
Figure 1. Design strategy for phthalide alkyl tertiary amines derivatives I~III.
2
2
. Results and discussion
.1. Chemistry
The synthetic routes for the target compounds were shown in Scheme 1 and Scheme 2. 4-bromo-
-butanol (1) was prepared according to the literature using tetrahydrofuran as starting material.24
1
Compounds 1 or 6-chloro-1-hexanol (2) were reacted with corresponding secondary amines 3a-h in
2
2
the presence of K CO and DMF to afford the aminoalkyl alcohols 4 or 5. Then, compound 4 or 5
2
3
reacted with PySO via the Parikh-Doering oxidation in the presence of dry DMSO and triethylamine
3
2
5
to give the key intermediates aminoalkylaldehydes 6 or 7 respectively. For the synthesis of target
compounds phthalide alkyl tertiary amines I-III, vanillin (8) was used as starting material to obtain
2
6
3
,4-dimethoxybenzoic acid (10) via methylation and oxidation respectively. Then compound 10
reacted with 37 % formaldehyde and conc. HCl to give 5,6-dimethoxy-2- benzofuran-1(3H)-one
2
7
(
11), and then brominated in chlorobenzene using N-bromosuccinimide and AIBN to obtain 3-
bromo-5,6-dimethoxyisobenzofuran-1(3H)-one (12).28,29 This intermediate was substituted by
triphenylphosphine to give Wittig reagent 13.29 Subsequently, compound 13 and corresponding
aminoalkylaldehydes 6 or 7 were used to give the Z-isomer compounds I and E-isomer compounds II
3
0
by Wittig reaction after separation by silica gel chromatography. The stereochemistry of each isomer
was elucidated on the basis of H NMR data and references.31,32 The protons at phthalide nucleus C4
1
of the E-isomer are observed at lower field (for CDCl solution: ca. 7.2 ppm cf. ca. 7.0 ppm for the Z-
3
5

isomer) because of the deshielding effect of the nearby alkyl group. Due to the deshielding effect
caused by the influence of the ring oxygen, the vinyl proton in the E-isomer experiences a deshielding
effect are observed at lower field than in the Z-isomer also. Finally, compounds III were obtained via
1
13
the catalytic hydrogenation reduction. All derivatives were characterized by H NMR, C NMR and
ESI-MS.
O
i
R1
R1
OH
N
nOH
N
n CHO
Br
iii
ii
1
2
R2
R2
4
5
, n = 4
, n = 6
6, n = 3
7, n = 5
OH
Cl
OCH3
OCH3
HN
HN
HN
HN
NHR R =
1
2
CH3
CH3
CH3
CH3
3a-h
a
c
d
b
N(CH3)2
HN
CH3
CH3
HN
HN
HN
CH3
CH3
N(CH3)2
g
h
e
f
Scheme 1. Synthesis of aminoalkylaldehydes 6 and 7. Reagents and conditions: (i) 40 % HBr aq.,
reflux (80%); (ii) NHR R (3a-h), K CO , DMF, r.t. (80~85%); (iii) PySO , DMSO, CH Cl , Et N,
1
2
2
3
3
2
2
3
r.t. (63~70%).
H CO
CHO
H3CO
H3CO
CHO
H3CO
H3CO
COOH
3
iv
v
vi
HO
10
8
9
O
O
Br
O
O
O
H CO
H CO
3
H CO
3
3
vii
viii
ix
O
H CO
3
H CO
3
H CO
3
+
-
P Ph Br
3
13
11
12
O
O
O
O
O
H CO
3
H CO
3
H CO
3
x
+
O
H CO
3
H CO
3
H CO
3
n
n = 3, 5
n
n = 3, 5
n = 3, 5
III
N
n
N
N
R1
R1
I (Z)
II (E)
R2
R1
R2
R2
Scheme 2. Synthesis of phthalide alkyl tertiary amines derivatives I-III. Reagents and conditions: (iv)
Me SO , K CO , acetone, reflux (92%); (v) KMnO , NaHCO , H O, 90 °C (90%); (vi) 37 % HCHO
2
4
2
3
4
3
2
aq., conc. HCl, 70 °C (43.5%); (vii) NBS, AIBN, PhCl, 85 °C (76%); (viii) PPh , Toluene, reflux
3
6

(
70%); (ix) 6 or 7, CH Cl , Et N, r.t. (I-1~9: 14.2~20.2%; II-1~10: 21.0~30.6%); (x) 10% Pd/C, H ,
2
2
3
2
CH OH, r.t. (III-1~10: 58.7~85.6%).
3
2
2
.2. Pharmacology
.2.1. Inhibition of AChE and BuChE
All synthesized derivatives were determined by modified Ellman’s method for their inhibitory
activities against AChE from Electrophorus electricus (EeAChE) and BuChE from rat serum
3
3
(
RatBuChE), using Donepezil as the reference drug. As shown in Table 1, DL-NBP exhibited no
inhibitory activity on AChE, while all target compounds showed good AChE inhibitory potency,
implying that introduction of various carbon chain length tertiary amine moieties could significantly
increase AChE inhibitory activity, which was consist with our design strategy. Among them,
compounds I-8 and II-9 displayed the most excellent efficiency to inhibit AChE (IC = 2.66 and 4.84
5
0
nM, respectively), and both of which were stronger than Donepezil (IC = 26.4 nM). In general, when
5
0
the side chain length was the same (n = 3), compounds possessing an N-benzylmethylamine group
usually displayed better AChE inhibitory activity than those with an N-benzylethylamine group (AChE
inhibitory activity: I-1  I-2, II-1 > II-2, III-1 > III-2). However, the adoption of 2-methoxy group to
N-benzylmethylamine and N-benzylethylamine showed opposite regularity except II-3 and II-4
(AChE inhibitory activity: I-3  I-4, II-3 > II-4, III-3  III-4). Moreover, the introduction of 2-
dimethylamino group on the benzylamine moiety (I-5, II-5 and III-5; IC = 0.21, 0.35 and 0.26 μM,
5
0
respectively) significantly improved AChE inhibitory potency, while the adoption of 4-dimethylamino
group dramatically decreased the activity (I-6, II-6 and III-6; IC = 23.40, 13.80 and 24.90 μM,
5
0
respectively). As a result, our results suggested that N-benzylmethylamine and N-(2-
dimethylaminobenzyl) ethylamine might be more beneficial for AChE inhibition.
In order to study the effect of the linker length on the inhibition of AChE activity, N-
benzylmethylamine and N-(2-dimethylaminobenzyl) ethylamine were fixed as tertiary amine structural
units, and then compounds I-8~9, II-9~10 and III-9~10 were synthesized by increasing the length (n
=
5). It revealed that the potency of the derivatives to inhibit AChE was enhanced with the carbon
chain length increased (except II-10 and III-10). For example, compounds I-8, II-9 and III-9 (n = 5)
were much more potent than compounds I-III~1 (n = 3). Compared with I-8 (IC = 0.00266 μM), the
5
0
AChE inhibitory activity of I-1 (IC = 0.34 μM) was about 128-fold lower, and II-1 (IC = 0.64 μM)
5
0
50
7

was almost 132-fold lower than that of II-9 (IC = 0.00484 μM). Thus, the different linker of length
5
0
was an important factor to inhibit AChE.
However, almost all compounds showed weak activity against BuChE, suggesting that these
derivatives were potent AChE inhibitors with high selectivity toward AChE. The selectivity profile
may be a limitation, but it was beneficial to diminish peripheral cholinergic side effects. For instance,
tacrine, had severe side effects due to its poor selectivity to AChE.5
Table 1. Inhibition of EeAChE and RatBuChE activities, and oxygen radical absorbance capacity
(ORAC, Trolox equivalents) by compounds I~III, DL-NBP and Donepezil.
a
% Inhibitionb
RatBuChE
IC (μM)
EeAChE
5
0
Compound
NR R
n
3
ORACc
1
2
I-1
N
0.34 ± 0.03
12.20 ± 0.25
0.32 ± 0.02
CH3
I-2
I-3
N
3
3
0.36 ± 0.02
1.64 ± 0.09
17.30 ± 0.38
16.80 ± 0.46
0.36 ± 0.03
0.44 ± 0.01
CH3
MeO
N
CH3
MeO
I-4
I-5
3
3
1.54 ± 0.07
0.21 ± 0.01
18.70 ± 0.59
40.50 ± 0.94
0.94 ± 0.05
0.78 ± 0.02
N
CH3
(
H3C)2N
N
CH3
N(CH3)2
I-6
I-7
I-8
N
3
3
5
23.40 ± 0.98
4.56 ± 0.14
13.60 ± 0.24
21.90 ± 0.83
19.10 ± 0.51
2.75 ± 0.08
0.28 ± 0.01
0.40 ± 0.02
CH3
N
N
0.00266 ± 0.0001
CH3
(H3C)2N
I-9
5
3
0.0787 ± 0.002
0.64 ± 0.04
38.08 ± 1.05
27.30 ± 0.64
1.02 ± 0.05
0.49 ± 0.02
N
CH3
II-1
N
CH3
8

II-2
II-3
N
3
3
1.40 ± 0.06
0.41 ± 0.02
11.00 ± 0.19
21.90 ± 0.47
0.52 ± 0.03
0.38 ± 0.01
CH3
MeO
N
CH3
MeO
II-4
II-5
II-6
3
3
3
0.80 ± 0.06
0.35 ± 0.03
13.80 ± 0.29
19.10 ± 0.55
43.10 ± 0.83
14.10 ± 0.23
0.53 ± 0.03
1.07 ± 0.05
2.89 ± 0.08
N
CH3
(
H3C)2N
N
CH3
N(CH3)2
N
CH3
II-7
II-8
N
3
3
0.44 ± 0.03
6.26 ± 0.34
26.90 ± 0.54
4.19 ± 0.09
0.32 ± 0.01
0.22 ± 0.01
CH3
CH3
N
II-9
II-10
III-1
III-2
N
5
5
3
3
0.00484 ± 0.0002
0.89 ± 0.05
17.55 ± 0.63
35.20 ± 0.78
11.50 ± 0.21
23.10 ± 0.56
0.43 ± 0.02
1.20 ± 0.06
0.29 ± 0.02
0.23 ± 0.01
CH3
(
H3C)2N
N
CH3
N
0.35 ± 0.03
CH3
N
0.58 ± 0.04
CH3
MeO
III-3
3
0.49 ± 0.02
21.70 ± 0.39
0.27 ± 0.01
N
CH3
MeO
III-4
III-5
3
3
0.37 ± 0.02
0.26 ± 0.01
29.80 ± 0.76
44.80 ± 0.91
0.74 ± 0.04
0.43 ± 0.02
N
CH3
(
H3C)2N
N
CH3
9

N(CH3)2
III-6
N
3
24.90 ± 0.91
6.20 ± 0.27
2.59 ± 0.07
CH3
III-7
III-8
N
3
3
15.80 ± 0.32
14.70 ± 0.48
8.14 ± 0.19
5.10 ± 0.12
0.22 ± 0.01
0.31 ± 0.02
CH3
CH3
N
III-9
N
5
5
0.16 ± 0.01
1.24 ± 0.03
13.90 ± 0.23
40.45 ± 1.10
0.25 ± 0.05
0.60 ± 0.04
CH3
(
H3C)2N
III-10
N
CH3
DL-NBP
__
__
_
_
n.a.d
2.67 ± 0.06
0.21 ± 0.01
NTe
Donepezil
0.0264 ± 0.0010
IC = 44.6 ± 0.97μM
5
0
a
AChE from Electrophorus electricus. IC50 values represent the concentration of inhibitor required to decrease
enzyme activity by 50% and are the mean of three independent experiments, each performed in triplicate. Data were
expressed as mean ± SD (SD = standard deviation).
b
BuChE from rat serum. Tested compounds were used at 50 μM.
c
The mean ± SD of the three independent experiments. Data are expressed as μM of Trolox equivalent/μM of tested
compound.
d
n.a. = no active. Compounds defined “no active” means a percent inhibition of less than 5.0% at a concentration of
5
0 μM in the assay conditions.
e
NT = not tested.
2
.2.2. Docking study with AChE
In order to clarify the interacting modes between these derivatives and Torpedo californica (Tc)
AChE (PDB: 1EVE), a molecular modeling study was performed using the docking program,
AutoDock 4.2 package with Discovery Studio 2.5.23,34 The most active compound I-8 in AChE
inhibition assay was chosen for our docking investigation. As shown in Figure 2A, compound I-8
occupied the entire enzymatic CAS, the mid-gorge sites and PAS. One hydrogen bond was generated
between the ligand I-8 and the residue Phe288 (Phe288-NH• • •O=I-8). The benzene ring of phthalein
structure and benzylamine moiety in compound I-8 could form π-π stacked interactions with Trp279
and Trp84, respectively. Moreover, the 3-substituted side chain folded in a conformation in the gorge
that allowed it to interact with Tyr334, Phe330 and Phe331 via the hydrophobic interaction. These
results indicated that I-8 could stably bind to the CAS and PAS of AChE, which provided reasonably
1
0

explanation for its potent AChE inhibitory activity.
In addition, we also selected compounds I-9, II-9 and II-10 to investigate the interacting modes
with AChE according to the results of AChE inhibition assay, to explore the effects of compounds
with different configurations and substituents. Figure 2B-D revealed that compound I-9 formed two
hydrogen bonds with Phe288 and Arg 289 (Phe288-NH, Arg 289-NH • • •O=I-8), while II-9 and II-
1
0 both formed a hydrogen bond with Phe288, which was consistent with I-8. Likewise, the phthalein
moiety in I-9 and the benzylamine moiety in II-9 and II-10 could form π-π stacked interaction with
Trp279 and Phe330, respectively. Moreover, hydrophobic interactions similar to I-8 were also
observed in compounds I-9, II-9 and II-10. By and large, compounds that share the same structural
framework act in the same way with AChE, occuping the CAS, the mid-gorge sites and PAS
simultaneously.
Figure 2. Docking pose of compounds I-8 (A), I-9 (B), II-9 (C) and II-10 (D) (colored by atom type)
into the binding site of TcAChE, highlighting the protein residues that participate in the main
1
1

interactions with the inhibitor.
2
.2.3. Evaluation for antioxidant activity
The antioxidant activity of these compounds was determined by the oxygen radical absorbance
3
5
capacity of fluorescein (ORAC-FL) method. Their ability to scavenge radicals (ORAC value) was
provided as a Trolox (a vitamin E analog) equivalent, with their relative potency at 5 μM compared
with Trolox. The results from Table 1 revealed that the antioxidant effect of all the target compounds
was improved compared with DL-NBP (ORAC-FL value of 0.21 Trolox equivalent). Among them,
I-6, II-6 and III-6 showed the most potent antioxidant activity with ORAC-FL values of 2.75, 2.89
and 2.59 Trolox equivalents, respectively, suggesting that the N-(4-dimethylaminobenzyl) ethylamine
moiety seemed to be a potent substitution pattern to improve the antioxidant activity. However, other
tertiary amine fragments and the length of the linker had no significant effect on their potency.
2
.2.4. Inhibition of self-induced Aβ1-42 aggregation and MAOs
All compounds were also tested by a thioflavin T fluorescence assay for their ability to inhibit
self-induced Aβ1-42 aggregation.36,37 Curcumin was used as the reference compound. Results
summarized in Table 2 indicated that the self-induced Aβ1-42 aggregation inhibitory effect of these
derivatives was better than that of the lead compound DL-NBP and Donepezil, but lower than curcumin
(39.0%). According to the structure-activity relationship analysis, the potency of target derivatives to
inhibit the self-aggregation of Aβ1-42 slightly enhanced (except III-10) as the length of the alkyl chain
increased, while the difference of tertiary amine group at the end of the side chain had no obvious
regularity on its activity. On the whole, III-1~10 displayed a slightly weaker inhibitory activity than
I-1~9 and II-1~10, indicating that the reduction of the double bond would destroy the large conjugated
system of the compounds and reduce their inhibitory activity of self-induced Aβ1-42 aggregation.
Subsequently, we further tested human MAO-A and -B inhibitory activity of all the compounds.23,38
Clorgyline, Iproniazid and Rasagiline were used as reference compounds. Unfortunately, as shown in
Table 2, these derivatives exhibited weak inhibitory activity against MAOs.
Table 2. Inhibition of MAOs and self-induced Aβ1-42 aggregation activities by compounds I-III, DL-
NBP and reference compounds.
Comp.
% inhibitiona,b
self-induced Aβ1-42
1
2

MAO-A
MAO-B
aggregation
11.70 ± 0.12
17.00 ± 0.53
15.10 ± 0.31
23.80 ± 0.65
15.40 ± 0.22
22.00 ± 0.48
4.90 ± 0.08
19.45 ± 0.25
16.30 ± 0.32
8.60 ± 0.11
30.30 ± 0.73
19.10 ± 0.35
19.00 ± 0.73
8.20 ± 0.12
21.70 ± 0.48
11.30 ± 0.11
15.10 ± 0.41
12.36 ± 0.22
13.00 ± 0.19
8.90 ± 0.21
15.60 ± 0.35
14.40 ± 0.29
13.80 ± 0.14
15.30 ± 0.50
< 5.0
I-1
10.90 ± 0.23
10.40 ± 0.18
2.90 ± 0.02
26.70 ± 0.54
29.10 ± 0.38
12.90 ± 0.21
1.95 ± 0.08
7.9 ± 0.17
11.90 ± 0.25
13.80 ± 0.32
10.30 ± 0.16
12.00 ± 0.29
9.90 ± 0.23
8.86 ± 0.17
32.30 ± 0.89
10.50 ± 0.20
14.70 ± 0.18
1.09 ± 0.03
15.80 ± 0.14
9.44 ± 0.12
4.80 ± 0.07
2.10 ± 0.09
17.00 ± 0.43
38.80 ± 0.85
19.70 ± 0.29
5.66 ± 0.09
7.30 ± 0.16
6.40 ± 0.24
5.51 ± 0.16
5.70 ± 0.30
10.40 ± 0.26
15.00 ± 0.50
9.47 ± 0.19
34.70 ± 0.89
21.50 ± 0.72
16.30 ± 0.35
13.45 ± 0.30
I-2
I-3
I-4
I-5
I-6
I-7
I-8
I-9
9.5 ± 0.12
II-1
II-2
II-3
II-4
II-5
II-6
II-7
II-8
II-9
II-10
III-1
III-2
III-3
III-4
III-5
III-6
III-7
III-8
III-9
III-10
17.90 ± 0.44
12.60 ± 0.18
4.90 ± 0.09
27.10 ± 0.56
30.00 ± 0.62
12.70 ± 0.27
1.52 ± 0.09
5.77 ± 0.24
22.30 ± 0.45
28.00 ± 0.70
8.00 ± 0.22
7.10 ± 0.15
0.25 ± 0.01
27.80 ± 0.42
17.80 ± 0.73
12.10 ± 0.24
1.92 ± 0.07
2.45 ± 0.05
7.88 ± 0.10
14.30 ± 0.25
< 5.0
15.00 ± 0.36
12.98± 0.21
7.66 ± 0.19
1
3

DL-NBP
11.20 ± 0.17
18.20 ± 0.46
NTc
< 5.0
39.0 ± 1.3
< 5.0
NTc
Curcumin NTc
Donepezil NTc
NTc
Clorgyline IC50 =0.00791 ± 0.0002 μM IC50 =8.85 ± 0.201 μM
NTc
Rasagiline IC50 =0.712 ± 0.021 μM
Iproniazid IC50 =1.37 ± 0.043 μM
IC50 =0.0437 ± 0.002 μM
IC50 =4.32 ± 0.174 μM
NTc
a
b
c
Inhibition of MAOs and self-induced Aβ1-42 aggregation by tested inhibitors at 10 μM and 25 μM respectively;
The mean ± SD of the three independent experiments;
NT = not tested.
2
.2.5. Evaluation of drug-likeness properties
Based on above results, compounds I-1, I-5, I-8, I-9, II~III-1, II~III-5, II~III-9 and II~III-10
were selected to analyze their drug-likeness properties. Lipinski parameters, which describe the
important molecular properties for drugs pharmacokinetics in the human body, especially their oral
absorption, are calculated using online Molinspiration Property Calculator program.39,40 The rule
requests that an oral active drug must not violate more than one of the following principles: MW <
5
00, log P < 5, hydrogen donors (nOHNH) < 5, hydrogen acceptors (nON) < 10. As shown in Table
, the chosen compounds (except III-10) were in line with Lipinski’s rule, suggesting that target
3
compounds presented drug-likeness properties.
Table 3. Drug-likeness properties of selected compounds.
Compd.
MWa
logPb
tPSAc nONd nOHNHe
Volumef
347.64
nrotbg
8
I-1
367.44
3.23
51.92
55.16
51.92
55.16
51.92
55.16
51.92
55.16
48.01
51.25
5
6
5
6
5
6
5
6
5
6
0
0
0
0
0
0
0
0
0
0
I-5
I-8
424.54
395.50
452.60
367.44
424.54
395.50
452.60
369.46
426.56
3.66
4.24
4.67
3.23
3.66
4.24
4.67
3.64
4.07
410.35
381.24
443.95
347.64
410.35
381.24
443.95
353.85
416.56
10
10
12
8
I-9
II-1
II-5
II-9
II-10
III-1
III-5
10
10
12
9
11
1
4

III-9
397.51
454.61
b
4.65
5.08
48.01
51.25
5
6
0
0
387.45
450.16
11
13
III-10
a
d
c
MW: molecular weight; log P: log octanol/water partition coefficient; tPSA: total polar surface area;
e
f
nON: number of hydrogen acceptors; nOHNH: number of hydrogen donors; Volume: molecular
g
volume; nrotb: number of rotatable bonds.
2
.2.6. In vitro blood-brain barrier permeation assay
The permeability through blood-brain barrier (BBB) is an essential element for developing anti-
AD drugs. To evaluate the in vivo BBB permeability of target derivatives, the parallel artificial
membrane permeation assay of the blood-brain barrier (PAMPA-BBB) was employed.23,41
Compounds I-8, II-9 and III-9 were selected as representatives for BBB permeation assay. Eleven
commercial drugs were chosen and evaluated for BBB permeability and compared with reported
4
1
values to validate the assay (Table S1, Supplementary Material). A plot of experimental data versus
the bibliographic values exhibited a good linear correlation: P (exp.) = 0.8792 × P (bibl.) - 0.0616
e
e
2
(
R = 0.9555) (Figure S1, Supplementary Material). According to the correlation equation and the
4
1
evaluation conditions established by Di et al. , we defined that compounds with P values above 3.46
e
-6
×
10 cm/s could pass through the BBB (Table 4). The results in Table 5 indicated that compounds
I-8, II-9 and III-9 could cross the BBB and reach their therapeutic targets in the CNS.
-6
Table 4. Ranges of permeability of PAMPA-BBB assays (P × 10 cm/s).
e
High BBB permeation predicted (CNS +)
Pe > 3.46
Uncertain BBB permeation (CNS +/-)
Low BBB permeation predicted (CNS -)
3.46 > P > 1.70
e
Pe < 1.70
-6
Table 5. Permeability results P (× 10 cm/s) from the PAMPA-BBB assay for selected compounds
e
with their predicted penetration into the CNS.
Compd.a
P (× 10 cm/s)
-6
b
Prediction
CNS +
e
I-8
10.06 ± 0.18
10.31 ± 0.31
13.58 ± 0.29
II-9
CNS +
III-9
CNS +
a
Compounds were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final concentration of
each compound was 100 μg/mL.
1
5

b
Data are the mean ± SD of three independent experiments.
2
.2.7. Step-down passive avoidance test
Considering that good AChE inhibitory activity of these derivatives in vitro, we further
investigated their effect on memory impairment induced by scopolamine in mice. The step-down
passive avoidance test is extensively used to evaluate learning and memory in mice through the latency
time in stepping down from the platform and the number of shocks, after an electric shock.22,42 The
most active compound I-8 in AChE inhibition assay was chosen for our test, with Donepezil as a
positive control. As shown in Figure 3, compared with the control group, scopolamine-injected mice
showed much shorter step-down latency time (p < 0.01), and the number of shocks was significantly
increased (p < 0.01), which indicated the memory impairment. Treatment with compound I-8 (1.0, 5.0
and 25.0 mg/kg) and Donepezil (5.0 mg/kg) obviously blocked these changes. Moreover, compared
with Donepezil group, the medium dose group (5.0 mg/kg) showed longer latency time and fewer
numbers of shocks, but the high dose (25.0 mg/kg) of compound I-8 displayed a worse effect,
suggesting that I-8 may have some neurotoxicity. These results indicated that compound I-8 could
improve cognitive deficit by increasing brain cholinergic activity due to the inhibition of AChE.
Figure 3. Effects of compound I-8 on scopolamine-induced memory deficit in the step-down passive
avoidance test. Compound I-8 (1.0, 5.0, 25.0 mg/kg, i.g.) or Donepezil (5.0 mg/kg, i.g.) were given
before scopolamine (2 mg/kg, i.p.) induced memory deficit. (A) latency time, (B) number of electric
shocks. Values are expressed as the mean ± SEM. n = 8 mice from three independent experiments; **p
#
##
<
0.01 versus control group; p < 0.05, p < 0.01 versus scopolamine treated group.
1
6

3
. Conclusion
In summary, almost all the phthalide alkyl tertiary amines derivatives exhibited significant
inhibitory activity and selectivity of AChE. Among them, compound I-8 displayed the most excellent
activity on AChE with IC value of 2.66 nM, which was significantly better than the control drug
5
0
Donepezil. Furthermore, molecular docking studies showed that compound I-8 could simultaneously
bind to the CAS and PAS of AChE, and could penetrate the BBB. More interestingly, the in vivo study
indicated that mice treated with I-8 (5.0 mg/kg, i.g.) significantly reversed scopolamine- induced
memory deficit, which was more effective than Donepezil (5.0 mg/kg, i.g.) in the step-down passive
avoidance test. In addition, compared with the lead compound DL-NBP, these derivatives exhibited
mild self-induced Aβ1-42 aggregation inhibitory activity, and some compounds also exerted good
antioxidant activity. Therefore, most derivatives had potential multi-target anti-AD properties, among
which compound I-8 could be used as a highly effective and selective AChE inhibitor for the
development of novel drugs in the treatment of AD.
4
4
. Experimental section
.1. Chemistry
All chemicals and solvents were purchased from commercial sources and used without further
purification. Reactions were monitored by thin-layer chromatography (TLC) on silica gel GF254 plates
from Qingdao Haiyang Chemical Co. Ltd. (China), and the spots were detected under UV lamp (254
1
13
nm) or in iodine chamber. H NMR and C NMR spectra were recorded on a Varian INOVA 400
NMR spectrometer or Varian INOVA 600 NMR spectrometer at room temperature using CDCl or
3
DMSO-d as the sovent. Chemical shifts were expressed in parts per millions (ppm) using
6
tetramethylsilane (TMS) as the internal standard. Coupling constants were reported in hertz. Column
chromatography was accomplished on silica gel (230-400 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd. (China). Melting points were determined on YRT-3 melting-point apparatus (China)
and uncorrected. The purity of all compounds was measured by analytical reverse phase high-
performance liquid chromatography (HPLC) with Shimadzu LC-10Avp plus system with a Kromasil
C column (4.6 mm×250 mm, 5μm). The mobile phase was a mixture of methanol and water at a flow
1
8
rate of 1.0 mL/min. All target compounds possessed purity higher than 96%. MS spectra were
performed on Agilent-627 TOF LC-MS Spectrometer.
1
7

4
.1.1. Synthesis of 4-bromo-1-butanol (1)
0% HBr aqueous solution (35 mL) was added drop-wise to refluxing tetrahydrofuran (160 mL,
.934 mol). Refluxing was then continued for 2 h. After cooling to room temperature, neutralised with
4
1
saturated Na CO aqueous solution and the resulting pale yellow organic layer was then separated. The
2
3
aqueous layer was then extracted with dichloromethane (25 mL × 4). The combined organic phases
were washed with brine, dried over sodium sulfate and filtered, concentrated under reduced pressure
to obtain the crude product 1 as a yellow liquid, which was used without further purification.
4
.1.2. General procedure for the synthesis of secondary amines (3a-f)
Compounds 3a-f were prepared as previously described.43
4
.1.3. Synthesis of aminoalkyl alcohols (4 or 5)
Anhydrous K CO (1.5 equiv.) and compounds 1 or 2 (1.5 equiv.) were added to a solution of the
2
3
corresponding secondary amines 3a-h (1.0 equiv.) in N,N-dimethylformamide (the synthesis of
compound 5 required the addition of a catalytic amount of KI). The reaction mixture was stirred at
room temperature overnight (the mixture was kept at 60 °C for the synthesis of compound 5). Upon
completion of the reaction, water was added to the mixture and then extracted with ethyl acetate. The
combined organic phases were washed with 10% HCl aqueous solution. The acid aqueous layer was
basified with 50% KOH aqueous solution to adjust the pH to 10 approximately and then extracted with
dichloromethane. The combined organic phases were washed with water and brine, dried over
anhydrous sodium sulfate and filtered, concentrated under reduced pressure to afford crude
intermediates 4 or 5.
4
.1.4. Synthesis of aminoalkylaldehydes (6 or 7)
Dry dimethyl sulfoxide (20.0 equiv.) and triethylamine (5.0 equiv.) were added to a solution of
compounds 4 or 5 (1.0 equiv.) in dry dichloromethane. When the mixture cooled to about -5°C by a
.
freezing mixture of ice and salt, Py SO solid power (3.0 equiv.) was added slowly in portions. After
3
the addition, the ice-salt bath was removed and the solution allowed to warm up to room temperature
under an argon atmosphere for 1 h. The reaction mixture was basified with 10% NaOH aqueous
solution and saturated NaHCO solution to adjust the pH to 7-8. The aqueous phase was extracted with
3
dichloromethane, then washed with brine, dried over sodium sulfate and filtered, concentrated under
reduced pressure to give the crude compounds 6 or 7.
4
.1.5. Synthesis of 3,4-dimethoxybenzaldehyde (9)
1
8

To a mixture of compound 8 (15.0 g, 98.7 mmol) and anhydrous K CO (20.5 g, 148.0 mmol) in
2
3
acetone (200 mL), dimethyl sulfate (11.3 mL, 118.41 mmol) was added. The reaction mixture was
refluxed for 3 h under an argon atmosphere. The mixture was filtered and the filtrate was concentrated
to give compound 9, which was used without further purification.
4
.1.6. Synthesis of 3,4-dimethoxybenzoic acid (10)
Compound 9 (15.15 g, 91.17 mmol) and NaHCO (9.19 g, 109.41 mmol) were suspended in H O
3
2
(
230 mL), then KMnO (17.29 g, 109.41 mmol) was added slowly in an ice-water bath. The mixture
4
was heated to 90 °C for 4 h. After the reaction mixture was cooled, filtered and the filtrate was acidified
to pH = 3 by adding conc. HCl. The precipitate was filtered, washed with water and dried at room
temperature to afford compound 10 as white solid.
4
.1.7. Synthesis of 5,6-dimethoxy-2-benzofuran-1(3H)-one (11)
7% HCHO aqueous solution (60 mL) was added to a solution of compound 10 (14.58 g, 80.06
3
mmol) in conc. HCl (250 mL). The reaction mixture was heated at 70 °C for 8 h. After the mixture
was cooled, filtered and the filtrate was extracted with dichloromethane (70 mL × 4). The combined
organic phases were washed with saturated NaHCO solution, water and brine orderly, dried over
3
anhydrous sodium sulfate and filtered, concentrated under reduced pressure to afford the crude
compound. The residue was recrystallized from ethanol to give compound 11 as a light brown solid,
4
4
yield 43.5% (6.762 g), mp 151.5-153.8 °C (Lit. 154-155 °C).
4
.1.8. Synthesis of 3-bromo-5,6-dimethoxyisobenzofuran-1(3H)-one (12)
A mixture of compound 11 (6.76 g, 34.82 mmol) and AIBN (570 mg, 3.48 mmol) in dry
chlorobenzene (100 mL) was heated to 85°C under an argon atmosphere, then NBS (6.82 g, 38.31
mmol) was added slowly in portions. After the addition, the mixture was kept at 85 °C for 6 h. After
cooling to room temperature, filtered and washed with dry chlorobenzene three times, concentrated
under reduced pressure to give the crude compound 12.
4
.1.9. Synthesis of 5,6-dimethoxy-3-triphenyl phosphonium bromide phthalide (13)
Compound 12 (9.51 g, crude product) and triphenylphosphine (10.96 g, 41.79 mmol) were
suspended in dry toluene (100 mL) and refluxed for 15 h under an argon atmosphere. The mixture was
cooled to room temperature, filtered and washed with dry toluene three times. The filter cake was
stirred in acetone uniformly and then filtered, washed with acetone and dried to afford compound 13.
4
.1.10. General procedure for the synthesis of butyphthalide tertiary amines derivatives (I-III)
1
9

A solution of compounds 6 or 7 (1.0 equiv.) and 13 (1.1 equiv.) in dry dichloromethane was
stirred and cooled in an ice bath while dry triethylamine (1.1 equiv.) was added slowly dropwise.
Stirring was continued at room temperature overnight under an argon atmosphere. The solvent was
removed, then 10% HCl aqueous solution was added to adjust the pH to 1 and stirred for 5 min. The
solution was washed with ethyl acetate and the aqueous layer was separated. The combined organic
phases were washed with 10% HCl aqueous solution twice, then the combined acid aqueous layers
were basified with 20% KOH aqueous solution and saturated NaHCO solution to adjust the pH to 8.
3
The aqueous phase was extracted with dichloromethane, then washed with brine, dried over anhydrous
sodium sulfate and then concentrated at reduced pressure. The resultant yellow oil was purified via
silica gel chromatography to give Z-isomer compounds I-1~9 and E-isomer compounds II-1~10,
respectively. Subsequently, a E/Z mixture of compounds I and II (1.0 equiv) in methanol (3 mL) and
1
0% Pd/C (0.2 equiv) was stirred under hydrogen (balloon) overnight. The mixture was filtered and
the filtrate was concentrated to obtain crude compound. The residue was purified by silica gel
chromatography to afford the desired products III-1~10.
(Z)-3-(4-(benzyl(methyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-1(3H)-one(I-1)
It was synthesized from 13 and 6a according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product I-1. 15.8%
1
yield; yellow oil. Purity: 98.8%. H NMR (400 MHz, CDCl ) δ 7.35-7.27 (m, 4H), 7.26-7.24 (m, 2H),
3
6
2
1
.96 (s, 1H), 5.46 (t, J = 7.6 Hz, 1H), 4.00 (s, 3H), 3.95 (s, 3H), 3.55 (s, 2H), 2.50 (q, J = 7.6 Hz, 2H),
1
3
.49 (t, J = 7.6 Hz, 2H), 2.21 (s, 3H), 1.81-1.75 (m, 2H); C NMR (100 MHz, CDCl ) δ 167.2, 155.1,
3
51.1, 145.7, 138.3, 134.1, 129.1 (2C), 128.2 (2C), 127.0, 116.9, 107.4, 105.2, 100.7, 62.2, 56.4, 56.3
+
(
2C), 41.9, 26.8, 23.5. HR-ESI-MS: Calcd. for C H NO [M+H] : 368.1862, found: 368.1865.
2
2
26
4
(Z)-3-(4-(benzyl(ethyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-1(3H)-one (I-2)
It was synthesized from 13 and 6b according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product I-2. 14.2%
1
yield; yellow oil. Purity: 98.9%. H NMR (400 MHz, CDCl ) δ 7.37 (d, J = 7.2 Hz, 2H), 7.32-7.27 (m,
3
2
2
H), 7.24-7.21 (m, 2H), 6.94 (s, 1H), 5.42 (t, J = 7.6 Hz, 1H), 4.00 (s, 3H), 3.95 (s, 3H), 3.63 (s, 2H),
1
3
.59-2.50 (m, 4H), 2.45 (q, J = 6.8 Hz, 2H), 1.75-1.72 (m, 2H), 1.09 (t, J = 6.8 Hz, 3H); C NMR
(
100 MHz, CDCl ) δ 167.2, 155.0, 151.1, 145.5, 138.9, 134.1, 129.0 (2C), 128.4 (2C), 126.9, 116.8,
3
2
0

1
07.5, 105.1, 100.6, 57.8, 56.3, 56.2, 52.2, 47.1, 26.5, 23.5, 11.4. HR-ESI-MS: Calcd. for C H NO
23 28 4
+
[
(
(
M+H] : 382.2018, found: 382.2016.
Z)-5,6-dimethoxy-3-(4-((2-methoxybenzyl)(methyl)amino)butylidene)isobenzofuran-1(3H)-one
I-3)
It was synthesized from 13 and 6c according to the general procedure, and then purified on silica
gel chromatography eluted with petroleum ether/acetone (2:1) to obtain the pure product I-3. 18.5%
1
yield; yellow oil. Purity: 99.2%. H NMR (400 MHz, CDCl ) δ 7.37 (d, J = 7.2 Hz, 1H), 7.26 (t, J =
3
7
.2 Hz, 1H), 7.24 (s, 1H), 6.99 (s, 1H), 6.92 (t, J = 7.2 Hz, 1H), 6.87 (d, J = 7.2 Hz, 1H), 5.50 (t, J =
.0 Hz, 1H), 4.01 (s, 3H), 3.95 (s, 3H), 3.83 (s, 3H), 3.67 (s, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.49 (q, J =
8
+
8
.0 Hz, 2H), 2.32 (s, 3H), 1.89-1.83 (m, 2H). HR-ESI-MS: Calcd. for C H NO [M+H] : 398.1967,
2
3
28
5
found: 398.1963.
(
Z)-5,6-dimethoxy-3-(4-((2-methoxybenzyl)(ethyl)amino)butylidene)isobenzofuran-1(3H)-one
I-4)
(
It was synthesized from 13 and 6d according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product I-4. 16.4%
1
yield; yellow oil. Purity: 98.5%. H NMR (400 MHz, CDCl ) δ 7.67 (d, J = 7.6 Hz, 1H), 7.33 (t, J =
3
7
8
6
.6 Hz, 1H), 7.24 (s, 1H), 7.04 (s, 1H), 6.98 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 5.53 (t, J =
.0 Hz, 1H), 4.24 (s, 2H), 4.03 (s, 3H), 3.96 (s, 3H), 3.86 (s, 3H), 3.07 (q, J = 8.0 Hz, 2H), 2.96 (t, J =
1
3
.8 Hz, 2H) , 2.48 (q, J = 7.2 Hz, 2H), 2.21-2.17 (m, 2H), 1.44 (t, J = 7.2 Hz, 3H); C NMR (100
MHz, CDCl ) δ 167.5, 158.0, 155.2, 151.4, 146.5, 133.8, 133.2, 131.4, 121.1, 117.1, 116.7, 110.8,
3
1
05.0, 104.9, 101.1, 56.5, 56.3, 55.4, 50.9, 49.8, 47.0, 23.5, 23.0, 8.8. HR-ESI-MS: Calcd. for
+
C H NO [M+H] : 412.2124, found: 412.2125.
2
4
30
5
(
Z)-3-(4-((2-(dimethylamino)benzyl)(ethyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-
(3H)-one (I-5)
It was synthesized from 13 and 6e according to the general procedure, and then purified on silica
1
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product I-5. 19.6%
1
yield; yellow oil. Purity: 98.9%. H NMR (400 MHz, CDCl ) δ 7.94 (d, J = 7.6 Hz, 1H), 7.33-7.28 (m,
3
2
3
7
H), 7.23 (s, 1H), 7.20-7.14 (m, 1H), 7.05 (s, 1H), 5.52 (t, J = 8.0 Hz, 1H), 4.31 (s, 2H), 4.03 (s, 3H),
.96 (s, 3H), 3.04-2.98 (m, 4H), 2.64 (s, 6H), 2.46 (q, J = 7.2 Hz, 2H), 2.19-2.13 (m, 2H), 1.36 (t, J =
+
.2 Hz, 3H). HR-ESI-MS: Calcd. for C H N O [M+H] : 425.2440, found: 425.2443.
2
5
33
2
4
2
1

(
Z)-3-(4-((4-(dimethylamino)benzyl)(ethyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-
(3H)-one (I-6)
It was synthesized from 13 and 6f according to the general procedure, and then purified on silica
1
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product I-6. 18.3%
1
yield; yellow oil. Purity: 99.0%. H NMR (400 MHz, CDCl ) δ 7.24 (s, 1H), 7.22 (d, J = 8.4 Hz, 2H),
3
6
2
.98 (s, 1H), 6.67 (d, J = 8.4 Hz, 2H), 5.48 (t, J = 7.6 Hz, 1H), 4.00 (s, 3H), 3.95 (s, 3H), 3.65 (s, 2H),
.92 (s, 6H), 2.63-2.60 (m, 4H), 2.46 (q, J = 7.6 Hz, 2H), 1.87-1.78 (m, 2H), 1.14 (t, J = 6.4 Hz, 3H).
+
HR-ESI-MS: Calcd. for C H N O [M+H] : 425.2440, found: 425.2442.
2
5
33
2
4
(Z)-5,6-dimethoxy-3-(4-(piperidin-1-yl)butylidene)isobenzofuran-1(3H)-one (I-7)
It was synthesized from 13 and 6g according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product I-7. 14.3%
1
yield; yellow oil. Purity: 99.4%. H NMR (400 MHz, CDCl ) δ 7.24 (s, 1H), 7.02 (s, 1H), 5.52 (t, J =
3
7
1
3
.6 Hz, 1H), 4.01 (s, 3H), 3.95 (s, 3H), 2.62-2.55 (m, 6H), 2.49 (q, J = 7.6 Hz, 2H), 1.92-1.85 (m, 2H),
+
.75-1.72 (m, 4H), 1.55-1.50 (m, 2H). HR-ESI-MS: Calcd. for C H NO [M+H] : 332.1862, found:
1
9
26
4
32.1858.
(Z)-3-(6-(benzyl(methyl)amino)hexylidene)-5,6-dimethoxyisobenzofuran-1(3H)-one (I-8)
It was synthesized from 13 and 7a according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product I-8. 17.7%
1
yield; yellow oil. Purity: 99.0%. H NMR (400 MHz, CDCl ) δ 7.38-7.24 (m, 6H), 7.02 (s, 1H), 5.49
3
(t, J = 7.6 Hz, 1H), 4.01 (s, 3H), 3.95 (s, 3H), 3.67 (s, 2H), 2.53 (t, J = 7.2 Hz, 2H), 2.46 (q, J = 7.6
Hz, 2H), 2.32 (s, 3H), 1.69-1.60 (m, 2H), 1.56-1.50 (m, 2H), 1.44-1.38 (m, 2H). HR-ESI-MS: Calcd.
+
for C H NO [M+H] : 396.2175, found: 396.2174.
2
4
30
4
(
Z)-3-(6-((2-(dimethylamino)benzyl)(ethyl)amino)hexylidene)-5,6-dimethoxyisobenzofuran-
(3H)-one (I-9)
It was synthesized from 13 and 7e according to the general procedure, and then purified on silica
1
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product I-9. 20.2%
1
yield; yellow oil. Purity: 98.0%. H NMR (600 MHz, CDCl ) δ 7.81 (d, J = 7.2 Hz, 1H), 7.28 (t, J =
3
7
8
.2 Hz, 1H), 7.23 (s, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.04 (s, 1H), 5.49 (t, J =
.4 Hz, 1H), 4.01 (s, 3H), 3.94 (s, 3H), 2.82 (s, 2H), 2.76-2.71 (m, 2H), 2.65 (s, 6H), 2.43 (q, J = 7.2
2
2

Hz, 2H), 1.76-1.72 (m, 2H), 1.55-1.50 (m, 2H), 1.42-1.35 (m, 2H), 1.30-1.22 (m, 5H). HR-ESI-MS:
+
Calcd. for C H N O [M+H] : 453.2753, found: 453.2750.
2
7
37
2
4
(E)-3-(4-(benzyl(methyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-1(3H)-one (II-1)
It was synthesized from 13 and 6a according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product II-1. 23.9%
1
yield; yellow oil. Purity: 99.5%. H NMR (400 MHz, CDCl ) δ 7.30 (s, 1H), 7.29-7.26 (m, 5H), 7.21
3
(s, 1H), 5.71 (t, J = 8.4 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 3H), 3.57 (s, 2H), 2.60 (q, J = 8.4 Hz, 2H), 2.57-
+
2
3
.53 (m, 2H), 2.26 (s, 3H), 1.84 (t, J = 7.2 Hz, 2H). HR-ESI-MS: Calcd. for C H NO [M+H] :
2
2
26
4
68.1862, found: 368.1865.
(E)-3-(4-(benzyl(ethyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-1(3H)-one (II-2)
It was synthesized from 13 and 6b according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product II-2. 25.3%
1
yield; yellow oil. Purity: 99.2%. H NMR (400 MHz, CDCl ) δ 7.33-7.24 (m, 6H), 7.17 (s, 1H), 5.68
3
(
(
(
(
t, J = 8.0 Hz, 1H), 3.97 (s, 3H), 3.92 (s, 3H), 3.64 (s, 2H), 2.60-2.54 (m, 6H), 1.83-1.79 (m, 2H), 1.09
+
t, J = 7.2 Hz, 2H). HR-ESI-MS: Calcd. for C H NO [M+H] : 382.2018, found: 382.2012.
2
3
28
4
E)-5,6-dimethoxy-3-(4-((2-methoxybenzyl)(methyl)amino)butylidene)isobenzofuran-1(3H)-one
II-3)
It was synthesized from 13 and 6c according to the general procedure, and then purified on silica
gel chromatography eluted with petroleum ether/acetone (2:1) to obtain the pure product II-3. 26.4%
1
yield; yellow oil. Purity: 98.6%. H NMR (400 MHz, CDCl ) δ 7.31 (d, J = 7.6 Hz, 1H), 7.28 (s, 1H),
3
7
.24 (s, 1H), 7.22 (t, J = 7.6 Hz, 1H), 6.89 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H), 5.73 (t, J = 8.0
Hz, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H), 3.59 (s, 2H), 2.62 (q, J = 8.0 Hz, 2H), 2.56 (t, J = 6.8
1
3
Hz, 2H), 2.26 (s, 3H), 1.90-1.83 (m, 2H); C NMR (100 MHz, CDCl ) δ 167.2, 157.8, 154.7, 151.0,
3
1
4
45.8, 132.7, 130.9, 128.5, 120.2, 118.8, 111.7, 110.3, 106.1, 105.5, 104.3, 69.1, 56.4, 56.2, 55.2, 42.3,
+
1.9, 27.0, 23.7. HR-ESI-MS: Calcd. for C H NO [M+H] : 398.1967, found: 398.1963.
2
3
28
5
(E)-5,6-dimethoxy-3-(4-((2-methoxybenzyl)(ethyl)amino)butylidene)isobenzofuran-1(3H)-one
(II-4)
It was synthesized from 13 and 6d according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product II-4. 23.7%
1
yield; yellow oil. Purity: 99.3%. H NMR (400 MHz, CDCl ) δ 7.46 (d, J = 7.6 Hz, 1H), 7.29 (s, 1H),
3
2
3

7
.25 (t, J = 7.6 Hz, 1H), 7.20 (s, 1H), 6.90 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H), 5.67 (t, J = 8.0
Hz, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.78 (s, 5H), 2.78-2.70 (m, 4H), 2.62 (q, J = 7.6 Hz, 2H), 1.98-1.88
+
(
m, 2H), 1.16 (t, J = 7.6 Hz, 3H). HR-ESI-MS: Calcd. for C H NO [M+H] : 412.2124, found:
2
4
30
5
4
12.2125.
(E)-3-(4-((2-(dimethylamino)benzyl)(ethyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-
1
(3H)-one (II-5)
It was synthesized from 13 and 6e according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product II-5.
1
3
0.6% yield; yellow oil. Purity: 99.0%. H NMR (400 MHz, CDCl ) δ 7.60 (d, J = 7.2 Hz, 1H), 7.29
3
(
(
(
s, 1H), 7.20 (t, J = 7.2 Hz, 1H), 7.17 (s, 1H), 7.08 (d, J = 7.2 Hz, 1H), 6.99 (t, J = 7.2 Hz, 1H), 5.69
t, J = 7.6 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.72 (s, 2H), 2.65 (s, 6H), 2.61-2.54 (m, 6H), 1.82-1.78
1
3
m, 2H), 1.08 (t, J = 7.6 Hz, 3H); C NMR (100 MHz, CDCl ) δ 167.5, 154.7, 153.0, 151.0, 145.6,
3
1
2
38.6, 132.6, 130.0, 127.5, 123.0, 118.9, 118.8, 111.8, 105.6, 104.2, 56.3, 52.5, 52.4, 47.6, 45.1 (2C),
+
9.6, 27.0, 23.9, 11.4. HR-ESI-MS: Calcd. for C H N O [M+H] : 425.2440, found: 425.2443.
2
5
33
2
4
(E)-3-(4-((4-(dimethylamino)benzyl)(ethyl)amino)butylidene)-5,6-dimethoxyisobenzofuran-
1
(3H)-one (II-6)
It was synthesized from 13 and 6f according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product II-6. 27.2%
1
yield; yellow oil. Purity: 98.6%. H NMR (400 MHz, CDCl ) δ 7.30 (s, 1H), 7.18 (s, 1H), 7.15 (d, J =
3
8
2
.4 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 5.72 (t, J = 8.0 Hz, 1H), 3.96 (s, 3H), 3.91 (s, 3H), 3.50 (s, 2H),
1
3
.92 (s, 6H), 2.58-2.54 (m, 6H), 1.78-1.75 (m, 2H), 1.04 (t, J = 6.8 Hz, 3H); C NMR (100 MHz,
CDCl ) δ 167.1, 154.6, 150.8, 149.6, 145.5, 132.6, 129.7 (2C), 126.4, 118.7, 112.2 (2C), 111.9, 105.4,
3
1
04.2, 57.2, 56.2 (2C), 51.8, 46.9 (2C), 40.5, 27.0, 23.8, 11.4. HR-ESI-MS: Calcd. for C H N O
25 33 2 4
+
[
M+H] : 425.2440, found: 425.2442.
(E)-5,6-dimethoxy-3-(4-(piperidin-1-yl)butylidene)isobenzofuran-1(3H)-one (II-7)
It was synthesized from 13 and 6g according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product II-7. 21.0%
1
yield; yellow oil. Purity: 99.2%. H NMR (400 MHz, CDCl ) δ 7.30 (s, 1H), 7.22 (s, 1H), 5.73 (t, J =
3
8
2
.0 Hz, 1H), 4.03 (s, 3H), 3.97 (s, 3H), 2.59 (q, J = 7.6 Hz, 2H), 2.47 (t, J = 7.2 Hz, 6H), 1.87-1.80 (m,
1
3
H), 1.64-1.60 (m, 4H), 1.48-1.45 (m, 2H); C NMR (100 MHz, CDCl ) δ 167.1, 154.7, 151.0, 145.7,
3
2
4

1
32.6, 118.9, 111.5, 105.6, 104.2, 58.3, 56.5, 56.3, 54.5 (2C), 26.5, 25.5 (2C), 24.1, 24.0. HR-ESI-MS:
+
Calcd. for C H NO [M+H] : 332.1862, found: 332.1859.
1
9
26
4
(E)-3-(4-(diethylamino)butylidene)-5,6-dimethoxyisobenzofuran-1(3H)-one (II-8)
It was synthesized from 13 and 6h according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product II-8. 23.6%
1
yield; yellow oil. Purity: 98.7%. H NMR (600 MHz, CDCl ) δ 7.30 (s, 1H), 7.25 (s, 1H), 5.72 (t, J =
3
7
1
1
.2 Hz, 1H), 4.04 (s, 3H), 3.97 (s, 3H), 2.69-2.64 (m, 6H), 2.60 (q, J = 7.2 Hz, 2H), 1.85-1.82 (m, 2H),
1
3
.09 (t, J = 7.2 Hz, 6H); C NMR (150 MHz, CDCl ) δ 167.1, 154.8, 151.1, 145.9, 132.6, 118.9, 111.1,
3
05.6, 104.3, 56.5, 56.3, 51.9, 46.8 (2C), 26.6, 23.9 (2C), 10.9. HR-ESI-MS: Calcd. for C H NO
4
1
8
26
+
[
M+H] : 320.1862, found: 320.1866.
E)-3-(6-(benzyl(methyl)amino)hexylidene)-5,6-dimethoxyisobenzofuran-1(3H)-one (II-9)
It was synthesized from 13 and 7a according to the general procedure, and then purified on silica
(
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product II-9. 26.0%
1
yield; yellow oil. Purity: 99.0%. H NMR (400 MHz, CDCl ) δ 7.32-7.23 (m, 6H), 7.16 (s, 1H), 5.74
3
(t, J = 8.0 Hz, 1H), 3.99 (s, 3H), 3.96 (s, 3H), 3.49 (s, 2H), 2.51 (q, J = 7.6 Hz, 2H), 2.39 (t, J = 7.2
1
3
Hz, 2H), 2.20 (s, 3H), 1.62-1.55 (m, 4H), 1.49-1.43 (m, 2H); C NMR (100 MHz, CDCl ) δ 167.1,
3
1
5
3
54.7, 151.0, 145.5, 138.6, 132.6, 129.0 (2C), 128.1 (2C), 126.9, 118.9, 111.9, 105.6, 104.0, 62.2, 57.0,
+
6.3 (2C), 42.1, 29.5, 27.1, 26.9, 26.0. HR-ESI-MS: Calcd. for C H NO [M+H] : 396.2175, found:
2
4
30
4
96.2172.
(E)-3-(6-((2-(dimethylamino)benzyl)(ethyl)amino)hexylidene)-5,6-dimethoxyisobenzofuran-
1
(3H)-one (II-10)
It was synthesized from 13 and 7e according to the general procedure, and then purified on silica
gel chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product II-10.
1
3
0.5% yield; yellow oil. Purity: 98.4%. H NMR (400 MHz, CDCl ) δ 7.66 (d, J = 7.6 Hz,, 1H), 7.30
3
(
(
(
s, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.16 (s, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 5.72
t, J = 8.0 Hz, 1H), 4.00 (s, 3H), 3.96 (s, 3H), 3.78 (s, 2H), 2.66 (s, 6H), 2.64-2.48 (m, 6H), 1.63-1.55
1
3
m, 4H), 1.46-1.38 (m, 2H), 1.11 (t, J = 7.6 Hz, 3H); C NMR (150 MHz, CDCl ) δ 167.1, 154.7,
3
1
5
4
53.1, 151.0, 145.5, 138.6, 132.6, 130.4, 127.9, 123.4, 119.1, 118.8, 111.9, 105.6, 104.0, 56.4, 56.2,
+
2.8, 52.0, 47.3, 45.2 (2C), 29.3, 26.9, 26.0, 25.8, 10.9. HR-ESI-MS: Calcd. for C H N O [M+H] :
2
7
37
2
4
53.2753, found: 453.2751.
2
5

3
-(4-(benzyl(methyl)amino)butyl)-5,6-dimethoxyisobenzofuran-1(3H)-one (III-1)
It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with dichloromethane/methanol (30:1) to obtain the pure product III-1. 78.1%
1
yield; yellow oil. Purity: 99.4%. H NMR (400 MHz, CDCl ) δ 7.32-7.25 (m, 6H), 6.80 (s, 1H), 5.37
3
(dd, J = 3.6, 7.6 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.52 (s, 2H), 2.41 (t, J = 6.8 Hz, 2H), 2.22 (s, 3H),
1
3
2
.04-1.99 (m, 1H), 1.73-1.69 (m, 1H), 1.62-1.58 (m, 2H), 1.54-1.47 (m, 2H); C NMR (100 MHz,
CDCl ) δ 170.8, 154.7, 150.3, 144.4, 138.1, 129.1 (2C), 128.2 (2C), 127.1, 117.9, 105.9, 103.0, 80.6,
3
+
6
2.1, 56.6, 56.3, 56.2, 41.9, 34.5, 26.7, 22.4. HR-ESI-MS: Calcd. for C H NO [M+H] : 370.2018,
2
2
28
4
found: 370.2015.
3
-(4-(benzyl(ethyl)amino)butyl)-5,6-dimethoxyisobenzofuran-1(3H)-one (III-2)
It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with dichloromethane/methanol (30:1) to obtain the pure product III-2. 74.6%
1
yield; yellow oil. Purity: 99.0%. H NMR (400 MHz, CDCl ) δ 7.42-7.27 (m, 6H), 6.81 (s, 1H), 5.36
3
(dd, J = 3.6, 8.0 Hz, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.78 (s, 2H), 2.72-2.61 (m, 4H), 2.04-2.03 (m,
1
3
1
1
5
3
5
H), 1.75-1.65 (m, 3H), 1.52-1.46 (m, 2H), 1.18 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
70.9, 154.7, 150.4, 144.5, 138.8, 129.0 (2C), 128.2 (2C), 127.0, 117.9, 106.0, 103.1, 80.6, 57.8, 56.4,
+
6.3, 52.5, 47.2, 34.5, 26.4, 22.5, 11.3. HR-ESI-MS: Calcd. for C H NO [M+H] : 384.2175, found:
2
3
30
4
84.2176.
,6-dimethoxy-3-(4-((2-methoxybenzyl)(methyl)amino)butyl)isobenzofuran-1(3H)-one (III-3)
It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with petroleum ether/acetone (2:1) to obtain the pure product III-3. 82.1%
1
yield; yellow oil. Purity: 97.6%. H NMR (400 MHz, CDCl ) δ 7.34 (d, J = 7.6 Hz, 1H), 7.28 (s, 1H),
3
7
3
3
1
5
4
5
.24 (t, J = 7.6 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.82 (s, 1H), 5.37 (dd, J =
.6, 7.6 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.83 (s, 3H), 3.62 (s, 2H), 2.49 (t, J = 7.2 Hz, 2H), 2.29 (s,
1
3
H), 2.07-2.02 (m, 1H), 1.76-1.66 (m, 3H), 1.56-1.48 (m, 2H); C NMR (100 MHz, CDCl ) δ 170.9,
3
57.8, 154.7, 150.4, 144.5, 138.1, 131.0, 128.6, 120.3, 117.9, 110.4, 106.0, 103.1, 80.6, 56.9, 56.4,
+
6.2, 55.4, 55.1, 41.9, 34.6, 26.5, 22.6. HR-ESI-MS: Calcd. for C H NO [M+H] : 400.2124, found:
2
3
30
5
00.2127.
,6-dimethoxy-3-(4-((2-methoxybenzyl)(ethyl)amino)butyl)isobenzofuran-1(3H)-one (III-4)
It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with petroleum ether/acetone (2:1) to obtain the pure product III-4. 75.8%
2
6

1
yield; yellow oil. Purity: 99.0%. H NMR (400 MHz, CDCl ) δ 7.44 (d, J = 7.6 Hz, 1H), 7.27 (s, 1H),
3
7
3
.23 (t, J = 7.6 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 6.81 (s, 1H), 5.35 (dd, J =
.6, 7.6 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.82 (s, 3H), 3.71 (s, 2H), 2.63 (q, J = 6.8 Hz, 2H), 2.55 (t,
1
3
J = 7.2 Hz, 2H), 2.04-1.99 (m, 1H), 1.73-1.64 (m, 3H), 1.56-1.47 (m, 2H), 1.12 (t, J = 6.8 Hz, 3H); C
NMR (150 MHz, CDCl ) δ 170.9, 157.7, 154.7, 150.4, 144.5, 138.1, 130.6, 128.4, 120.4, 117.9, 110.3,
3
1
06.0, 103.1, 80.6, 56.4, 56.2, 55.3, 52.7, 51.0, 47.5, 34.5, 26.1, 22.6, 11.2. HR-ESI-MS: Calcd. for
+
C H NO [M+H] : 414.2280, found: 414.2278.
2
4
32
5
3
-(4-((2-(dimethylamino)benzyl)(ethyl)amino)butyl)-5,6-dimethoxyisobenzofuran-1(3H)-one
III-5)
It was synthesized according to the general procedure, and then purified on silica gel
(
chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product III-5. 80.3%
1
yield; yellow oil. Purity: 99.5%. H NMR (400 MHz, CDCl ) δ 7.65 (d, J = 7.6 Hz, 1H), 7.27 (s, 1H),
3
7
3
.23 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.81 (s, 1H), 5.35 (dd, J =
.6, 7.6 Hz, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.81 (s, 2H), 2.66 (s, 6H), 2.65-2.58 (m, 4H), 2.01-1.99
1
3
(
m, 1H), 1.70-1.65 (m, 3H), 1.53-1.43 (m, 2H), 1.12 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl )
3
δ 170.9, 154.8, 150.4, 144.5, 138.1, 130.6, 127.7, 123.4, 119.2, 117.9, 109.6, 106.0, 103.1, 80.6, 56.4,
+
5
4
3
6.2, 52.7, 52.0, 47.4, 45.3 (2C), 34.5, 26.0, 22.5, 11.2. HR-ESI-MS: Calcd. for C H N O [M+H] :
2
5
35
2
4
27.2597, found: 427.2592.
-(4-((4-(dimethylamino)benzyl)(ethyl)amino)butyl)-5,6-dimethoxyisobenzofuran-1(3H)-one
(III-6)
It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product III-6. 85.6%
1
yield; yellow oil. Purity: 99.4%. H NMR (400 MHz, CDCl ) δ 7.28 (s, 1H), 7.18 (d, J = 8.4 Hz, 2H),
3
6
.81 (s, 1H), 6.68 (d, J = 8.4 Hz, 2H), 5.35 (dd, J = 3.6, 8.0 Hz, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.56
(
(
s, 2H), 2.93 (s, 6H), 2.56 (q, J = 6.8 Hz, 2H), 2.47 (t, J = 7.6 Hz, 2H), 2.03-1.98 (m, 1H), 1.73-1.66
1
3
m, 1H), 1.62-1.52 (m, 2H), 1.48-1.45 (m, 2H), 1.08 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl )
3
δ 170.9, 154.7, 150.4, 149.8, 144.5, 130.1 (2C), 125.7, 117.9, 112.4 (2C), 106.0, 103.2, 80.6, 56.9,
+
5
4
3
6.3, 56.2, 52.1, 46.8, 40.6, 34.5, 26.1, 22.5 (2C), 11.1. HR-ESI-MS: Calcd. for C H N O [M+H] :
2
5
35
2
4
27.2597, found: 427.2594.
-(4-((4-(dimethylamino)benzyl)(ethyl)amino)butyl)-5,6-dimethoxyisobenzofuran-1(3H)-one
(III-7)
2
7

It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product III-7. 62.1%
1
yield; yellow oil. Purity: 99.2%. H NMR (400 MHz, CDCl ) δ 7.26 (s, 1H), 6.89 (s, 1H), 5.40 (dd, J
3
=
3.2, 8.0 Hz, 1H), 4.01 (s, 3H), 3.94 (s, 3H), 2.95-2.90 (m, 4H), 2.78 (t, J = 8.0 Hz, 2H), 2.20-2.12
1
3
(
m, 1H), 1.95-1.88 (m, 6H), 1.80-1.71 (m, 1H), 1.61-1.45 (m, 4H); C NMR (100 MHz, CDCl ) δ
3
1
2
3
70.8, 154.9, 150.5, 144.2, 117.7, 105.9, 103.2, 80.2, 57.5, 56.5, 56.2, 53.5 (2C), 34.0, 24.0, 23.2 (2C),
+
2.6, 22.2. HR-ESI-MS: Calcd. for C H NO [M+H] : 334.2018, found: 334.2023.
1
9
28
4
-(4-(diethylamino)butyl)-5,6-dimethoxyisobenzofuran-1(3H)-one (III-8)
It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product III-8. 67.4%
1
yield; yellow oil. Purity: 99.3%. H NMR (400 MHz, CDCl ) δ 7.27 (s, 1H), 6.89 (s, 1H), 5.41 (dd, J
3
=
3.2, 8.0 Hz, 1H), 4.01 (s, 3H), 3.94 (s, 3H), 3.12 (q, J = 7.2 Hz, 4H), 2.95 (t, J = 6.8 Hz, 2H), 2.24-
1
3
2
.15 (m, 1H), 2.01-1.93 (m, 2H), 1.80-1.72 (m, 1H), 1.61-1.48 (m, 2H), 1.40 (t, J = 7.2 Hz, 6H); C
NMR (150 MHz, CDCl ) δ 170.7, 155.0, 150.5, 144.0, 117.6, 106.0, 103.2, 80.0, 56.5, 56.3, 51.4, 46.3
3
+
(
2C), 33.8, 23.4 (2C), 22.1, 8.4. HR-ESI-MS: Calcd. for C H NO [M+H] : 322.2018, found:
1
8
28
4
3
3
22.2015.
-(6-(benzyl(methyl)amino)hexyl)-5,6-dimethoxyisobenzofuran-1(3H)-one (III-9)
It was synthesized according to the general procedure, and then purified on silica gel
chromatography eluted with dichloromethane/methanol (40:1) to obtain the pure product III-9. 58.7%
1
yield; yellow oil. Purity: 98.9%. H NMR (600 MHz, CDCl ) δ 7.32-7.25 (m, 6H), 6.82 (s, 1H), 5.36
3
(dd, J = 3.6, 8.4 Hz, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.51 (s, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.20 (s, 3H),
1
3
2
.03-1.99 (m, 1H), 1.72-1.69 (m, 1H), 1.55-1.50 (m, 2H), 1.49-1.43 (m, 2H), 1.39−1.33 (m, 4H); C
NMR (100 MHz, CDCl ) δ 170.8, 154.6, 150.3, 144.5, 138.5, 129.0 (2C), 128.1 (2C), 126.9, 117.9,
3
1
05.9, 103.0, 80.7, 62.1, 57.1, 56.3, 56.1, 41.9, 34.6, 29.1, 27.0, 26.9, 24.6. HR-ESI-MS: Calcd. for
+
C H NO [M+H] : 398.2331, found: 398.2336.
2
4
32
4
3
-(6-((2-(dimethylamino)benzyl)(ethyl)amino)hexyl)-5,6-dimethoxyisobenzofuran-1(3H)-one
III-10)
It was synthesized according to the general procedure, and then purified on silica gel
(
chromatography eluted with dichloromethane/methanol (20:1) to obtain the pure product III-10. 63.5%
1
yield; yellow oil. Purity: 98.5%. H NMR (400 MHz, CDCl ) δ 7.69 (d, J = 7.6 Hz, 1H), 7.28 (s, 1H),
3
2
8

7
3
.24 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.85 (s, 1H), 5.36 (dd, J =
.6, 8.0 Hz, 1H), 3.99 (s, 3H), 3.94 (s, 3H), 3.88 (s, 2H), 2.74-2.67 (m, 2H), 2.66 (s, 6H), 2.65-2.58
(m, 2H), 2.04-1.98 (m, 1H), 1.72-1.60 (m, 3H), 1.48-1.42 (m, 2H), 1.37-1.25 (m, 4H), 1.15 (t, J = 6.8
1
3
Hz, 3H); C NMR (150 MHz, CDCl ) δ 170.9, 154.7, 153.2, 150.3, 144.6, 138.1, 130.7, 128.2, 123.6,
3
1
1
4
4
19.3, 117.9, 106.0, 103.1, 80.7, 56.4, 56.2, 52.7, 51.8, 47.2, 45.3 (2C), 34.6, 29.0, 26.9, 25.6, 24.6,
+
0.6. HR-ESI-MS: Calcd. for C H N O [M+H] : 455.2910, found: 455.2906.
2
7
39
2
4
.2. Biological activity
.2.1. Inhibition of AChE and BuChE
AChE from Electrophorus electricus (EeAChE, Sigma-Aldrich Co.) and BuChE from rat serum
RatBuChE) were used to determine the inhibitory activities of all the compounds according to the
(
3
3
modified Ellman’s method. For EeAChE inhibition assay, 5,5’-dithiobis-2-nitrobenzoic acid (DTNB,
.2%, 30 µL) (J&K Scientific) was added in a mixture of acetylthiocholine iodide (ATCI, 1 mM, 30
0
µL) (J&K Scientific), phosphate buffer solution (0.1 mM, pH = 8.0, 40 µL), different concentrations
of test compounds (20 µL) and EeAChE (0.05 U/mL, final concentration, 10 µL), and then incubated
at 37 °C for 15 min. The absorbance was measured at 412 nm by using a Varioskan Flash Multimode
Reader (Thermo Scientific). For BuChE inhibition assay, 25% rat serum (10 µL) and S-
butylthiocholine iodide (BTCI, 1 mM, 30 µL) (TCI Shanghai Development) were used and the assay
was performed in a phosphate buffer (0.1 mM, pH = 7.4). Changes in absorbance were detected at 405
nm.23,45 The other procedure was similar with the method of EeAChE inhibition assay described above.
IC values were calculated as the concentration of compound that produced 50% enzyme activity
5
0
inhibition. Results are expressed as the mean ± SD of at least three different experiments performed in
triplicate.
4
.2.2. Molecular Docking of AChE.
Molecular docking study of compounds I-8, I-9, II-9 and II-10 with Torpedo californica (Tc)
AChE (PDB: 1EVE) was performed using AUTODOCK 4.2 program. The crystal structure was
downloaded from the Protein Data Bank. The 3D structure of these compounds and co-crystal ligand
Donepezil was built and optimized by molecular mechanics, and then prepared by removing the
hydrogen atoms, adding Gasteiger charges and their atomic charges to skeleton atoms, assigning the
proper atomic types. By using Autodock Tools (ADT; version 1.5.6), water molecules and original
inhibitor was removed, polar hydrogen atoms were added to amino acid residues and Gasteiger charges
2
9