Design, synthesis and evaluation of scutellarein-O-acetamidoalkylbenzylamines as potential multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.04.054

A series of scutellarein-O-acetamidoalkylbenzylamines derivatives were designed based on a multitarget-directed ligands strategy for the treatment of Alzheimer's disease. Among these compounds, compound T-22 demonstrated excellent acetylcholinesterase inhibitory, moderate inhibitory effects on self-induced Aβ_1-42 aggregation, Cu²⁺-induced Aβ_1-42 aggregation, human AChE-induced Aβ_1-40 aggregation and disassembled Cu²⁺-induced aggregation of the well-structured Aβ_1-42 fibrils, and also acted as potential antioxidant and biometals chelator. Both kinetic analysis of AChE inhibition and molecular modeling study suggested that T-22 interacted with both the catalytic active site and peripheral anionic site of AChE. Moreover, compound T-22 showed a good neuroprotective effect against H₂O₂-induced PC12?cell injury and low toxicity in SH-SY5Y cells. Furthermore, the step-down passive avoidance test indicated T-22 significantly reversed scopolamine-induced memory deficit in mice. Taken together, the data showed that T-22 was an interesting multifunctional lead compound worthy of further study for AD.

Full text of DOI:10.1016/j.ejmech.2017.04.054

Accepted Manuscript
Design, synthesis and evaluation of scutellarein-O-acetamidoalkylbenzylamines as
potential multifunctional agents for the treatment of Alzheimer's disease
Zhipei Sang, Xiaoming Qiang, Yan Li, Rui Xu, Zhongcheng Cao, Qing Song, Ting
Wang, Xiaoyu Zhang, Hongyan Liu, Zhenghuai Tan, Yong Deng
PII:
S0223-5234(17)30320-3
10.1016/j.ejmech.2017.04.054
EJMECH 9405
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 January 2017
Revised Date: 27 March 2017
Accepted Date: 20 April 2017
Please cite this article as: Z. Sang, X. Qiang, Y. Li, R. Xu, Z. Cao, Q. Song, T. Wang, X. Zhang, H. Liu,
Z. Tan, Y. Deng, Design, synthesis and evaluation of scutellarein-O-acetamidoalkylbenzylamines as
potential multifunctional agents for the treatment of Alzheimer's disease, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.054.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of scutellarein-O-acetamidoalkylbenzylamines
as potential multifunctional agents for the treatment of Alzheimer’s disease
Zhipei Sanga,b,#, Xiaoming Qianga,#, Yan Lia, Rui Xua, Zhongcheng Caoa, Qing Songa, Ting Wanga,
Xiaoyu Zhanga, Hongyan Liua, Zhenghuai Tanc,*, Yong Denga,*
aDepartment of Medicinal Chemistry, Key Laboratory of Drug Targeting and Drug Delivery System
of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
bCollege of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang,
473061, China
cInstitute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of
Chinese Medicine Sciences, Chengdu, 610041, China
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; ACh, acetylcholine; Aβ,
β-amyloid; APP, amyloid precursor protein; ROS, reactive oxygen species; MTDLs,
multitarget-directed ligands; BuChE, butyrylcholinesterase; HuAChE, human AChE; EeAChE,
Electrophorus electricus AChE; PDB, Protein Data Bank; TcAChE, Torpedo californica AChE; CAS,
catalytic active site; PAS, peripheral anionic site; ORAC-FL, Oxygen Radicals Absorbance Capacity
by Fluorescence; ThT, thioflavin T; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium;
TMS, tetramethylsilane; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered
saline; DTNB, 5,5’-dithiobis-2-nitrobenzoic acid; ADT, Autodock Tools; AAPH, 2,2’-Azobis
(amidinopropane)
dihydrochloride;
HFIP,
1,1,1,3,3,3-hexafluoro-2-propanol;
HEPES,
4-(2-Hydroxyethyl)-1- piperazineethanesulfonic acid.
# These authors contributed equally to this work.
* Corresponding Author.
Phone, +86-28-85258982; E-mail: tanzhh616@163.com (Zhenghuai Tan)
Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)
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Abstract
A series of scutellarein-O-acetamidoalkylbenzylamines derivatives were designed based on a
multitarget-directed ligands strategy for the treatment of Alzheimer’s disease. Among these
compounds, compound T-22 demonstrated excellent acetylcholinesterase inhibitory, moderate
inhibitory effects on self-induced Aβ1-42 aggregation, Cu2+-induced Aβ1-42 aggregation, human
AChE-induced Aβ1-40 aggregation and disassembled Cu2+-induced aggregation of the well-structured
Aβ1-42 fibrils, and also acted as potential antioxidant and biometals chelator. Both kinetic analysis of
AChE inhibition and molecular modeling study suggested that T-22 interacted with both the catalytic
active site and peripheral anionic site of AChE. Moreover, compound T-22 showed a good
neuroprotective effect against H2O2-induced PC12 cell injury and low toxicity in SH-SY5Y cells.
Furthermore, the step-down passive avoidance test indicated T-22 significantly reversed
scopolamine-induced memory deficit in mice. Taken together, the data showed that T-22 was an
interesting multifunctional lead compound worthy of further study for AD.
Keywords: Alzheimer’s disease; Scutellarein-O-acetamidoalkylbenzylamines; Multifunctional
agents; Acetylcholinesterase inhibitors; Aβ aggregation inhibitors; Antioxidant agents.
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1. Introduction
Alzheimer’s disease (AD) is the most common age related neurodegenerative disease,
characterized by loss of cognitive ability, severe behavioral abnormalities, and ultimately death [1].
More than 47 million people worldwide suffered from AD, currently, and the figure is expecting to
reach 131.5 million in 2050 [2]. Although the etiology of AD has not been completely known today,
several factors including low levels of acetylcholine (ACh), oxidative stress, dyshomeostasis of
biometals and β-amyloid (Aβ) deposits have been considered to play significant roles in the
pathogenesis of AD [3-5].
ACh can be degraded by two types of cholinesterases, namely acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) [6]. AChE acts primarily as a regulatory enzyme at chlinergic
synapses, while BuChE functionas as a co-regulator of cholinergic neurotransmission. Both
Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) has been recognized as
critical targets for the effective management of AD by increasing the availability of synaptic ACh in
the brain regions [7]. However, BuChE is mainly localized in the peripheral tissues including plasma
and very small amount is present in the brain region. Many side effects (eg. gastrointestinal events,
nausea, vomiting, diarrhoea, dizziness) mainly associated with their peripheral AChE inhibitory
activity [8]. Therefore, development of selective AChE inhibitors, with expectation of lesser
peripheral inhibition of cholinesterase enzyme, may be a more suitable therapeutic strategy for the
treatment of AD [9].
According to the amyloid hypothesis, the production and accumulation of oligomeric aggregates
of Aβ in the brain are a central event in the pathogenesis of AD [10]. Aβ40 and Aβ42 are the main
isoforms of Aβ peptides, are primarily produced depending on cleavage of amyloid precursor protein
(APP) by β- and γ-secretases, respectively. Aβ42 is a major constituent of amyloid plaques and
displays lower solubility and more toxic and has the tendency to form protofibrils and fibrillar
aggregates at lower concentrations than Aβ40 [11]. Excess of metal ions such as Cu2+, Fe2+, Zn2+ and
Al3+ have been found in Aβ plaques of AD brains, remarkably, Cu2+ and Zn2+ have been proved to
promote Aβ aggregation as indicated previously [12]. Moreover, the abnormally high levels of
redox-active metal ions such as Cu2+ and Fe2+ in brain might contribute to the overproduction of
reactive oxygen species (ROS). Meanwhile, Aβ could enter the mitochondria where it would increase
the generation of ROS and induce oxidative stress that damages biological molecules such as
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proteins, DNA, and lipids [13]. Interestingly, oxidative stress promotes Aβ toxicity through the
production of free radicals, and increases the generation of Aβ peptides, thus forming a vicious circle
[14]. Noticeably, in accordance with “amyloid hypothesis”, AChE produces secondary
non-cholinergic functions that facilitate Aβ deposition in the form of senile plaques or neurofibrillary
tangles in the brain [9].
The multifaceted factors of the AD have encouraged the development of multitarget-directed
ligands (MTDLs) to act as agents for the treatment of this disease [15,16]. These drugs, which
possess two or more complementary biological activities, may present significant clinical
effectiveness in the future.
Scutellarin (4′,5,6-trihydroxyflavone-7-glucuronide), the major active component in breviscapine
extracted from the Chinese herb Erigeron breviscapus (vant.) Hand.-Mazz., possesses a broad range
of pharmacological properties related to neurological disorders, such as anti-inflammatory,
antioxidant, neuroprotective and metal chelating properties [17,18]. Moreover, many researchers
demonstrated that scutellarin can inhibit Aβ aggregation, attenuate Aβ-induced toxicity and reduce
senile plaques [19]. However, scutellarin’s poor solubility, low oral absorption and difficultly in
passing through the blood–brain barrier restrict its clinical uses as an anti-AD drug [20]. Donepezil is
approved by the FDA as an AChE inhibitor for the treatment of AD, the 1-benzylpiperidine fragment
of donepezil has been shown as the cholinesterase inhibitory pharmacophore [21]. In addition, in our
preliminary researches, the pharmacophore benzylamine that linked by alkylene is also required,
which has suggested that this pharmacophore was directed toward peripheral anionic site of AChE by
molecular modeling studies [22,23]. And the amide group could improve AChE inhibitory activities
[24]. Therefore, in this paper, we aimed to use a multi-target-directed drug design strategy to
combine 4′-hydroxy-5,6,7-trimethoxyflavone or 5,4′-dihydroxy-6,7-dimethoxyflavone with
appropriate benzylamine fragments using amide linkers with different lengths. These new
scutellarein-O-acetamidoalkylbenzylamines derivatives may simultaneously possess a dual binding
site for AChE inhibition, anti-oxidative, metal chelating effects, neuroprotective properties, and
inhibit Aβ aggregation.
In this study, a novel series of scutellarein-O-acetamidoalkylbenzylamines derivatives were
designed, synthesized and evaluated for their biological activities, including inhibition of AChE and
BuChE, the kinetics of enzyme inhibition, metal-chelating properties, antioxidant activities, effects
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on Aβ aggregation and disaggregation, cytotoxic effects on SH-SY5Y cells, protective effect against
H2O2-induced PC12 cell injury, and neuroprotective effects in the mouse scopolamine model of
memory impairment. The chemical design strategy for scutellarein-O-acetamidoalkylbenzylamines
derivatives is depicted in Figure. 1
Figure 1. Design strategy for the scutellarein-O-acetamidoalkylbenzylamines derivatives.
2. Result and discussion
2.1. Chemistry
The synthetic route for the target compounds was shown in Scheme 1. Firstly, the starting
material 1 was treated with excessive amounts of 1,2-dibromoethane, 1,3-dibromopropane,
1,4-dibromobutane or 1,6-dibromohexane in the presence of K2CO3 in anhydrous acetone under
reflux to give the compounds 2a-d. The key intermediates secondary amines 3a-d were obtained
according to the previous literature procedure in our lab [23]. Compounds 4-7 were obtained by the
reaction of intermediates 2a-d with 3a-d in the presence of K2CO3 in anhydrous CH3CN. Then, the
key primary amines 8-11 were synthesized through the hydrazinolysis of 4-7. On the other hand, the
p-hydroxybenzaldehyde was treated with chloromethyl methyl ether to obtain
4-(methoxymethoxy)benzaldehyde (13) which was reacted with material 12 in the presence of 50%
KOH to afford the key intermediate 14, and then hydrolyzed with 10% HCl to obtain compound 15.
Compound 16 was prepared by the reaction of compound 15 with KI in the presence of conc.H2SO4,
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removal of 5-methyl at flavonoid nucleus by anhydrous AlCl3 in CH3CN at 55–60°C gave the
corresponding 5-OH scutellarein derivative 17. Compounds 18a-b was obtained by the reaction of 16
or 17 with ethyl bromoacetate in the presence of K2CO3, and then hydrolyzed with 30% LiOH and
acidizing with 10% HCl to furnish the intermediates 19a-b. Finally, condensation of compounds
19a-b with the appropriate diamines 8-11 in the presence of EDCI in THF gave the target products
T-1~24.
Scheme 1. Synthesis of scutellarein-O-acetamidoalkylbenzylamines derivatives T-1~24. Reagents
and conditions: (a) Br(CH2)nBr, K2CO3, acetone, reflux, 7-8 h; (b) R1R2NH (3a-d)21, K2CO3, CH3CN,
.
65°C, 6-8 h; (c) H2NNH2 H2O, EtOH, reflux for 5 h; (d) p-MOM-O-PhCHO (13), 30% KOH, ethanol,
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at r.t. for 3 days; (e) 10% HCl, ethanol, 50°C, 4 h; (f) KI, conc.H2SO4, DMSO, 100°C, 3-5 h;
(g)AlCl3, anhydrous CH3CN, 60°C, 1 h; (h) BrCH2COOC2H5, K2CO3, anhydrous CH3CN, 65°C, 8 h;
(i) LiOH, THF/H2O, reflux for 2 h, then 10% HCl; (j) EDCI, THF, at r.t. for over night.
2.2. Pharmacology
2.2.1. Inhibition of AChE and BuChE
Inhibitory activities toward AChE and BuChE of the synthetic compounds were evaluated
according to the modified Ellman’s method [22]. Their activities were initially assayed on AChE
from rat cortex homogenate and BuChE from rat serum. Moreover, the compounds were evaluated
using electric eel AChE( EeAChE) (Table 1), and the representative compounds were rationally
selected and re-evaluated using human erythrocyte AChE (HuAChE) (Table 2). In the above
experiments, donepezil was used as reference compound and parent compounds 16 and 17 were also
evaluated for comparative purposes. The IC50 values and selectivity index for the inhibition of AChE
and BuChE were summarized in Table 1. As shown in Table 1, all the target compounds showed
moderate to good AChE inhibitory activity and acted as highly selective AChE inhibitors. Compound
T-22 displayed the most potent inhibitory activity for AChE with IC50 value of 0.051 ± 0.003 µM,
which was similar to donepezil (IC50 = 0.015 ± 0.002 µM). Almost all the target compounds showed
weak activity against BuChE. Among them, compound T-16 exhibited the most potent inhibitory
activity for BuChE with IC50 value of 2.4 ± 0.01 µM.
In preliminary studies, as expected, the parent compounds 16 (IC50 > 500 µM) and 17 (IC50 > 500
µM) exhibited no significant inhibition of AChE. Introduction of benzylamines group increased the
AChE inhibitory capacity and improved the selectivity for AChE over BuChE. Considering the
potential role of BuChE in AD pathogenesis, this selectivity profile may be a limitation, but this also
may be beneficial to diminish peripheral cholinergic side effects and provide lower toxicity [25].
Through analysis and comparison, we found that the properties of the substitutions at flavonoid
nucleus 5 position showed significant influence on AChE inhibitory activities: 5-methoxy substituted
derivatives (T-3, T-4, T-7, T-8, T-15 and T-16) exhibited higher activity than that with 5-hydroxyl
substituted (T-17, T-18, T-19, T-20, T-23 and T-24), except compounds T-11 and T-12 with
5-methoxy (IC50 = 8.32 ± 0.21 µM and 0.074 ± 0.006 µM, respectively) exhibited much less potent
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than corresponding compounds T-21 and T-22 with 5-hydroxyl (IC50 = 2.29 ± 0.02 µM and 0.051 ±
0.001 µM, respectively). The screening data also revealed that the structure of terminal groups
NR1R2 of side chain affected inhibitory activities. Generally, the N-2-methoxybenzyl groups
indicated stronger inhibitory potency than N-benzyl groups. And the compounds possessing a N-ethyl
group showed obviously better AChE inhibitory activity than the compounds with a N-methyl group,
which was consistent with the previously reported results in our lab [22,23]. On the other hand, we
also investigated the effects of the side chain length on inhibitory ability of AChE, the optimal chain
length was four-methylene linker. Finally, compound T-22 showed the most potent inhibition for
RatAChE with an IC50 value of 0.051 ± 0.001 µM. For the inhibition of EeAChE, it is notable that all
the tested compounds exhibited a higher inhibitory activity towards RatAChE than EeAChE (Table
1). Furthermore, compounds T-12, T-16, T-22 and T-24, which displayed the better potent inhibitory
activity for RatAChE (IC50 = 0.074 ± 0.006 µM, 0.35 ± 0.02 µM, 0.051 ± 0.001 µM and 0.37 ± 0.01
µM, respectively), were re-evaluated using HuAChE (Table 2). The inhibitory activity against
RatAChE is lower than that against HuAChE.
Table 1. Inhibition of AChE and BuChE activity, and oxygen radicals absorbance capacity (ORAC,
Trolox Equivalents) by compounds T-1~24, 16, 17 and donepezil.
Compound
R
n
-NR1R2
IC50 ± SDa (µM)
RatBuChEc
>500
ORAC f
Selectivity
indexe
>31.6
>862
76.4
RatAChEb
15.80 ± 0.46
0.58 ± 0.06
1.44 ± 0.08
2.39 ± 0.04
8.42 ± 0.32
3.28 ± 0.11
1.58 ± 0.03
0.24 ± 0.01
5.36 ± 0.22
0.76 ± 0.01
8.32 ± 0.21
EeAChEd
17.90 ± 0.56
16.20 ± 0.42
13.40 ± 0.51
6.81 ± 0.19
23.51 ± 1.05
18.56 ± 0.73
5.20 ± 0.11
6.63 ± 0.17
13.93 ± 0.32
14.38 ± 0.52
18.40 ± 0.71
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
2
0.42eq
0.39eq
0.40eq
0.42eq
0.39eq
0.38eq
0.40eq
0.33eq
0.35eq
0.36eq
0.37eq
T-1
T-2
T-3
T-4
T-5
T-6
T-7
T-8
T-9
T-10
T-11
A
B
C
D
A
B
C
D
A
B
C
2
2
2
3
3
3
3
4
4
4
>500
110.0 ± 0.56
65.4 ± 0.13
>500
27.4
>59.4
>152
>316
1442
>500
>500
346.0 ± 1.26
>500
>93.3
288
219.1 ± 2.13
>500
>60.1
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CH3
CH3
CH3
CH3
CH3
H
4
6
6
6
6
2
2
3
3
4
4
6
6
0.074 ± 0.006
4.63 ± 0.12
2.84 ± 0.09
5.54 ± 0.17
0.35 ± 0.02
1.97 ± 0.04
4.98 ± 0.22
2.59 ± 0.07
0.31 ± 0.01
2.29 ± 0.02
0.051 ± 0.001
6.63 ± 0.02
0.37 ± 0.01
>500
>500
117.7 ± 0.65
108.0 ± 0.78
130.0 ± 1.67
2.4 ± 0.01
105.5 ± 0.69
74.3 ± 0.57
>500
9.77 ± 0.34
23.44 ± 0.96
33.24 ± 1.03
16.23 ± 0.68
17.65 ± 0.47
14.03 ± 0.46
14.11 ± 0.25
15.50 ± 0.72
11.22 ± 0.11
17.13 ± 0.58
15.95 ± 0.39
12.29 ± 0.47
11.67 ± 0.26
NTg
>6757
25.3
0.38eq
0.37eq.
0.39eq.
0.41eq.
0.34eq.
1.18eq.
1.04eq.
1.14eq.
0.97eq.
0.97eq.
1.13eq.
1.01eq.
0.99eq.
1.86eq
3.07eq
NTg
T-12
T-13
T-14
T-15
T-16
T-17
T-18
T-19
T-20
T-21
T-22
T-23
T-24
16
D
A
B
C
D
C
D
C
D
C
D
C
D
38.0
23.5
6.8
53.3
H
14.9
H
>193
>1613
77.7
H
>500
H
178.0 ± 0.96
>500
H
>9804
>75.4
>1351
----
H
>500
H
>500
>500
>500
>500
NTg
----
17
Donepezil
0.015 ± 0.002
20.7 ± 1.36
0.027 ± 0.001
1380
a IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean
of 3 independent experiments.
b From 5% rat cortex homogenate.
c BuChE from rat serum.
d From electrophorus electricusc.
e Selectivity index = IC50 (RatBuChE)/IC50 (RatAChE).
f Results are expressed as µM of Trolox equivalent/µM of tested compound.
g NT = not tested.
2.2.2. Kinetic characterization of AChE inhibition.
To
investigate
the
AChE
inhibitory
mechanism
for
this
class
of
scutellarein-O-acetamidoalkylbenzylamines derivatives, compound T-22, the most potent AChE
inhibitor, was selected for further kinetic studies. Graphical analysis of the reciprocal
Lineweaver-Burk plots (Figure 2) showed both increasing of slopes (decreased Vmax) and intercepts
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(higher Km) with increasing concentration of the inhibitor. This pattern indicated a mixed-type
inhibitory type and revealed that compound T-22 could bind to both CAS and PAS of AChE.
Figure 2. Kinetic study on the mechanism of EeAChE inhibition by compound T-22. Merged
Lineweaver-Burk reciprocal plots of AChE initial velocity with increasing substrate concentration
(100-400 µM) in the absence or presence of T-22. Lines were derived from a weighted least-squares
analysis of data points.
2.2.3. Molecular modeling study
To
further
explore
the
possible
interaction
mode
of
the
scutellarein-O-acetamidoalkylbenzylamines derivatives with Torpedo californica (Tc)AChE (PDB
code: 1EVE), a molecular modeling study was performed using the docking program named
AUTODOCK 4.2 package with Discovery Studio 2.1 [26]. As shown in Figure 3, T-22 occupied the
entire enzymatic catalytic active site (CAS), the mid-gorge sites and the peripheral anionic site (PAS)
and could simultaneously bind to both CAS and PAS. In the TcAChE-T-22 complex, the benzene
ring of 4H-chromen-4-one was observed to bind to the PAS via a π-π interaction with Trp279, and
potential hydrophobic interactions with residues Trp279, Leu282, Phe288, Phe331. The 5-hydroxy
group at the flavonoid nucleus could bind to the PAS via intermolecular hydrogen bond with Ser286.
The side chain N-(2-methoxybenzyl)ethanamine moiety occupied the CAS of AChE, could bind to
the CAS via a potential hydrophobic interaction with Ser124, Tyr130, Trp84, Gly123, Ser122,
Gly118 and Gly117. The benzene ring at 2-position of 4H-chromen-4-one could bind to the
mid-gorge sites via a π-π interaction with Tyr334, and the amide group could bind to the mid-gorge
sites via one intermolecular hydrogen bond with Tyr121. In addition, the long chain of methylene,
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amide group and the benzene ring at the 2-position of 4H-chromen-4-one could fold into a
conformation in the gorge that allowed them to interact with Phe330, His440, Ser200 and Gly119
through hydrophobic interaction. Similar to the donepezil complex, water-bridged hydrogen bonding
may have occurred under biological circumstances between compound T-22 and TcAChE. Therefore,
the above results provided a reasonable explanation for its highly potent inhibitory activity against
AChE.
Figure 3. Representative compound T-22 (colored by atom type) interacting with residues in the
binding site of TcAChE (PDB code: 1EVE), highlighting the protein residues that participate in the
main interaction with the inhibitor.
2.2.4. Metal-chelating properties of compound T-22
The ability of compound T-22 to chelate biometals such as Cu2+, Zn2+, Al3+ and Fe2+ was studied
by UV-visual spectrometry [27]. As shown in Figure 4, the UV-vis spectrum of compound T-22
showed a red shift (the peak at 336 nm shifted to 343 nm) after the addition of CuCl2. The result
demonstrated that compound T-22 could interact with the Cu2+ effectively. Similar results were also
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obtained when AlCl3 was added, the peak in the UV-vis spectrum of compound T-22 at 336 nm shift
to 357 nm. However, when ZnCl2 and FeSO4 were added, the UV-vis spectrum of T-22 exhibited no
significant shift. The results showed that T-22 had the selectivity for metal chelation. It was
reasonable to estimate that the 5-hydroxyl and 4-carbonyl groups at the flavonoid nucleus
contributed to the chelation.
Figure 4. UV spectrum of compound T-22 (37.5 µM in methanol) alone or in the presence of CuCl2,
ZnCl2, AlCl3 and FeSO4 (37.5 µM for all metals in methanol).
The stoichiometry of the T-22-Cu2+ complex was determined using molar ratio method by
preparing solutions of compound T-22 with increasing amounts of CuCl2. The UV spectra was used
to obtain the absorbance of the complex of T-22 and CuCl2 at different concentrations at 386 nm.
According to the Figure 5, the absorbance linearly increased at first and then tended to be stable. The
two straight lines intersected at a mole fraction of 0.99, indicating a 1:1 stoichiometry of the
ligand-Cu2+ complex.
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Figure 5. Determination of the stoichiometry of complex-Cu2+ by using molar ratio method through
titrating the methanol solution of compound T-22 with ascending amounts of CuCl2. The final
concentration of tested compound was 37.5 µM, and the final concentration of Cu2+ ranged from 3.75
to 93.75 µM.
2.2.5. Antioxidant activity in vitro.
The antioxidant activities of the target compounds were evaluated by following the ORAC-FL
method (Oxygen Radicals Absorbance Capacity by Fluorescence) [28]. Trolox, a vitamin E analogue,
was used as a standard, and the results were expressed as Trolox equivalents. The parent compounds
16 and 17 were also tested, which had ORAC-FL values of 1.86 and 3.07 Trolox equivalents,
respectively. As shown in Table 1, all the compounds exhibited significant antioxidant activities with
ORAC-FL values of 0.33-1.18 Trolox equivalents. As expected, introduction of substitutions to
4′-hydroxyl group decreased the radical capture capacity, but the different benzylamines moieties on
the side chain had little influence on the radical capture capacity. In general, compounds (T-17~T-24)
with 5-hydroxyl at the flavonoid nucleus exhibited higher radical capture capacity than that of
5-methoxyl (T-1~T-16). It is apparent that the free hydroxyl is crucial to the radical scavenging
ability. The compound T-22 showed good antioxidant activity with an ORAC-FL value of 1.13 trolox
equivalents.
2.2.6. Inhibition of self- and HuAChE-induced Aβ aggregation.
The ability of the target compounds to inhibit self-induced Aβ1-42 aggregation was evaluated using
the thioflavin T (ThT) fluorescence assay with curcumin as reference compound [29]. Donepezil and
the parent compounds (16 and 17) were also evaluated for comparative purposes. The results were
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summarized in Table 2. All the target compounds showed potent inhibitory effect with inhibition
ratio ranging from 25.8% to 59.8%. The marketed AD drug, donepezil, did not show any significant
inhibitory activity under the same experimental conditions. The parent compounds 16 and 17 also
exhibited weak inhibitory potency with inhibition ratio of 6.7% and 9.8%, respectively. It might be
possible that the introduction of substitutions improved the inhibitory activity against self-induced
Aβ1-42 aggregation. The screening data showed that the 5-hydroxy at flavonoid nucleus (T-17~T-24)
showed remarkably higher inhibitory activity than that of 5-methoxyl (T-3, T-4, T-7, T-8, T-11, T-12,
T-15 and T-16), and different tertiary amine units on the side chain had little influence on the
inhibitory activity. The results revealed that the hydrogen bonds are crucial in the interaction between
ligand and proteins. The representative compound T-22 also showed a dose-dependent inhibitory
effect on self-induced Aβ1-42 aggregation (43.6% at 12.5 µM, 57.1% at 25 µM, 75.2% at 50 µM).
It was reported that AChE could play a crucial role in accelerating the formation of amyloid
fibrils due to the interaction of AChE with Aβ by a hydrophobic environment close to the peripheral
anionic site (PAS) [29]. Thus, inhibition of AChE, especially inhibition of PAS of AChE, might
affect Aβ aggregation. The kinetic study showed that the representative compound T-22 exhibited a
mix-type inhibition and could bind to both the CAS and PAS of AChE. To further explore the
multifunctional properties of these compounds, the better AChE inhibitors, compounds T-12, T-16,
T-22 and T-24, were selected to assess their ability to inhibit the HuAChE-induced Aβ1-40
aggregation by using a thioflavin T fluorescence method. According to the results in Table 2,
compounds T-22 and T-24 (inhibition ratio of 57.3% and 58.1% at 100 µM, respectively) indicated
much higher inhibitory potency than donepezil (inhibition ratio of 18.8%), but compounds T-12 and
T-16 (inhibition ratio of 8.8% and 6.5%, respectively) exhibited lower inhibitory potency than
donepezil under the same conditions. Compound T-22 also showed a dose-dependent inhibitory
effect on HuAChE-induced Aβ1-40 aggregation (32.7% at 25 µM, 57.3% at 50 µM, 63.8% at 100 µM).
It was apparent that compounds T-22 and T-24 with 5-methoxyl group could bond to the PAS of
AChE better than donepezil. The results also showed 5-hydroxy at flavonoid nucleus seemed to be
beneficial to inhibitory activity against HuAChE-induced Aβ1-40 aggregation, which was consistent
with the outcome of inhibitory activity against AChE.
Table 2. Inhibition of self-induced Aβ1-42 aggregation, HuAChE-induced Aβ1-40 aggregation,
Cu2+-induced Aβ1-42 aggregation and disaggregation of Cu2+-induced Aβ1-42 aggregation by target
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compounds and reference compounds.
Comp.
Inhibition of
% Inhibition of Aβ Aggregationb,g
HuAChE
Disaggregation of Aβ
HuAChE (IC50, µM) g
fibrils(%) f,g
Self-inducedc
Cu2+-inducede
-inducedd
NTh
NTh
44.8 ± 2.4
37.5 ± 1..0
29.4 ± 0.8
26.9 ± 0.6
34.2 ± 2.2
25.8 ± 0.4
27.5 ± 0.6
42.4 ± 3.2
29.6 ± 0.9
29.1 ± 0.4
41.2 ± 3.9
44.7 ± 1.5
44.3 ± 1.6
34.4 ± 1.0
36.1 ± 0.7
35.5 ± 2.1
54.7 ± 1.6
59.8 ± 0.9
53.9 ± 4.1
49.1 ± 2.0
56.9 ± 1.0
57.1 ± 1.9i
53.3 ± 3.1
56.7 ± 2.7
6.7 ± 0.3
NTh
NTh
NTh
NTh
NTh
T-1
T-2
NTh
NTh
NTh
NTh
NTh
T-3
NTh
NTh
NTh
NTh
T-4
NTh
NTh
NTh
NTh
T-5
NTh
NTh
NTh
NTh
T-6
NTh
NTh
NTh
NTh
T-7
NTh
NTh
NTh
NTh
T-8
NTh
NTh
NTh
NTh
T-9
NTh
NTh
NTh
NTh
T-10
T-11
T-12
T-13
T-14
T-15
T-16
T-17
T-18
T-19
T-20
T-21
T-22
T-23
T-24
16
NTh
NTh
NTh
NTh
0.063 ± 0.005
NTh
8.8 ± 0.2
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
1.25±0.08
NTh
6.5 ± 0.2
NTh
NTh
NTh
49.3 ± 1.2
47.5 ± 1.3
44.9 ± 0.7
45.1 ± 1.9
42.6 ± 0.7
43.4 ± 0.9
41.5 ± 0.3
42.7 ± 1.1
NTh
57.0 ± 1.0
58.9 ± 0.8
51.6 ± 1.2
54.7 ± 0.6
67.1 ± 0.6
66.3 ± 2.6
61.2 ± 2.5
65.0 ± 1.3
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
NTh
0.039 ± 0.002
NTh
57.3 ± 0.8j
NTh
1.37 ± 0.07
NTh
58.1 ± 1.9
NTh
NTh
9.8 ± 0.1
NTh
NTh
NTh
17
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Curcumin
Donepezil
NTh
43.1 ± 1.1
<5.0
NTh
58.0 ± 2.1
<5.0
56.5 ± 2.1
NTh
0.012 ± 0.002
18.8 ± 1.6
a From human erythrocytes.
b For inhibition of Aβ aggregation, the thioflavin-T fluorescence method was used.
c Inhibition of self-induced Aβ1-42 aggregation, the concentration of tested compounds and Aβ1-42 were 25µM.
d Inhibition of human AChE-induced Aβ1-40 aggregation. The concentration of tested compounds and Aβ1-40 was
100 and 230 µM, respectively, and the Aβ1-40/HuAChE ratio was equal to 100/1.
e Inhibition of Cu2+-induced Aβ1-42 aggregation produced by tested compounds at 25 µM.
f Disaggregating Cu2+-induced Aβ1-42 aggregation. The concentration of tested inhibitors and Aβ1-42 were 25 µM.
g Data are presented as the mean ± SEM of three independent experiments.
h NT=not tested.
i Compound T-22 had a dose-dependent inhibitory effect on self-induced Aβ1-42 aggregation (43.6% at 12.5 µM,
57.1% at 25 µM, 75.2% at 50 µM).
j Compound T-22 had a dose-dependent inhibitory effect on HuAChE-induced Aβ1-40 aggregation (32.7% at 25 µM,
57.3% at 50 µM, 63.8% at 100 µM).
2.2.7. Effects on Cu2+-induced Aβ1-42 aggregation and disaggregation.
In order to investigate the effects on Cu2+-induced Aβ1-42 aggregation, we carried out two
individual studies (Table 2): inhibitory activity of Cu2+-induced Aβ1-42 aggregation and
disaggregation effects on Cu2+-induced Aβ1-42 aggregation by the compounds T-17~T-24, using
Thioflavin T (ThT) method with curcumin as the reference compound [30]. The Aβ1-42 peptide (25
µM) was first treated with one equivalent of Cu2+ for 2 min at room temperature and then incubated
with or without the testing compounds for 24 h at 37°C (Figure 6). It can be seen that Cu2+ could
accelerate the aggregation of Aβ1-42. The rate of Aβ1-42 aggregation slowed when adding tested
compounds to the samples. It suggested that tested compounds could inhibit Cu2+-induced Aβ1-42
aggregation noticeably by chelating with Cu2+. As shown in Table 2, compounds T-17~T-24
(inhibition ratio ranging 41.5% to 49.3%) exhibited moderate potency compared with curcumin
(58.0±2.3%). The results indicated that the inhibitory activity decreased slightly as the length of the
side chain carbon spacer increased, for example, T-17 (49.3%) > T-19 (44.9%) > T-21 (42.6%) >
T-23 (41.5%); T-18 (47.5%) > T-20 (45.1%) > T-22 (43.4%) > T-24 (42.7%), and different
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benzylamines units on the side chain had little influence on the inhibitory activity.
Figure 6. Inhibition of Cu2+-induced aggregation by compounds T-17~24 and curcumin. The
thioflavin-T fluorescence method was used.
For the disaggregation studies, compounds T-17~T-24 were added to Aβ fibrils, which were
generated by reacting Aβ1-42 with one equivalent of Cu2+ for 24 h at 37 °C. The results (Table 2)
showed that all the tested compounds exhibited moderate to good disaggregation potency
(disaggregation ratio ranging 51.6% to 67.1%) compared with curcumin (56.5%). The disaggregation
potency decreased as the length of the side chain carbon spacer increased, which was consistent with
the results of inhibition Cu2+-induced Aβ1-42 aggregation. The compound T-22 manifested
disaggregation potency with inhibition ratio of 66.3% (Table 2). Therefore, we can conclude that
compound T-22 is capable of inhibiting Cu2+-induced Aβ1-42 aggregation and disassembling the
well-structured Aβ fibrils.
2.2.8. Cytotoxic effects of compounds T-12 and T-22 on SH-SY5Y cells
To examine the cytotoxicity of the representative compounds T-12 and T-22, human neuronal
cell line SH-SY5Y cells were exposed to the tested compounds at three different concentrations (1,
10 and 100 µM) for 24 h, and the cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium (MTT) assays [22]. As shown in Figure 7, T-12 and T-22 did not show modified
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cell viability up to the concentration of 10 µM. With increased concentration to 100 µM, T-12 and
T-22 induced a decrease of cell viability (79.4% and 82.4%, respectively), it may be considered that
the compound T-12 with 5-methoxyl at flavonoid nucleus exhibited more cytotoxicity than that of a
5-hydroxyl (T-22). These results showed that the target compounds had a wide therapeutic safety
range. Further, based on the above results, compounds T-12 and T-22 could be selected for further
studies to elucidate the neuroprotective mechanisms.
Figure 7. Effects of T-12 and T-22 on cell viability in human SH-SY5Y cells. Data are mean values
± SEM of three independent experiments.
2.2.9. Cell protective effects on H2O2-induced PC12 cell injury
The neuroprotective capacities of target compounds against oxidative stress were assayed
according to the reported protocol [31]. After 100 µM H2O2 exposure, cell viability was determined
by MTT reduction was markedly decreased to 43.9 % (p < 0.01 vs control), suggesting high
sensitivity to H2O2-induced injury. However, compounds T-12 and T-22 showed protective effects in
a dose-dependent manner against H2O2-induced PC12 cell injury (Figure 8). At concentrations of
10.0 µM, compounds T-12 and T-22 indicated significantly neuroprotective effects and the cell
viabilities were 63.3% and 82.7%, respectively. When the concentration is reduced to 1.0 µM, their
cell viabilities decreased to 60.1%, and 67.6%, respectively. It revealed that compound T-22, with
5-hydroxy on the flavonoid nucleus, had higher cell survival ratio than compound T-12 with
5-methoxy group. It is reasonable to speculate that the 5-hydroxy can capture the hydroxyl radical,
generated by H2O2.
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Figure 8. Neuroprotective effect of compound T-12 and T-22 at the concentration of 1.0 µM and
10.0 µM on cell injury induced by hydrogen peroxide (H2O2, 100 µM) in PC12 cells.
2.2.10. In vivo assay
In vitro the above effects exhibited that compound T-22 was a promising multifunctional
compound for AD treatment. However, the in vivo effect of compound T-22 in models of learning
and memory is lacking. In order to further study whether T-22 could improve memory impairment
induced by scopolamine, we carried out the step-down passive avoidance task, which had been used
extensively to evaluate potential therapeutic agents for treating AD [22,32]. As shown in Figure 9,
the step-down latency of mice treated with scopolamine alone (control group) was significantly
shorter than that of vehicle-treated mice (normal group, p < 0.01). Treatment with compound T-22
(3.67, 7.33 and 22 mg/kg) increased the latency time in a dose-dependent manner, but the medium
dose group (7.33 mg/kg) presented the longest latency time (194.3 sec., p < 0.01) of the three dose
groups. The medium dose (7.33 mg/kg) of compound T-22 also showed slightly lower latency time
than the drug control group with donepezil (5 mg/kg, 203.7 sec). Meanwhile, the latency time of the
high dose of T-22 (22 mg/kg) demonstrated slightly shorter (176.4 sec) than that of the medium dose.
It might be possible that the high dose had some neurotoxicity and the medium dose could be the
optimal dose. These results of in vivo assay indicated that compound T-22 could permeate the
blood-brain barrier and reverse cognitive deficit by increasing brain cholinergic activity due to the
inhibition of AChE.
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Figure 9. Effects of compound T-22 on scopolamine-induced memory deficit in the step-down
passive avoidance test. Compounds T-22 (3.67, 7.33 and 22.0 mg/kg, p.o.) or donepezil (5.0 mg/kg,
p.o.) were orally given 1 h before treatment with scopolamine. After 30 min, the mice were treated
with scopolamine (3 mg/kg, i.p.) and tested in the step-down passive avoidance. Values are expressed
as the mean ± SEM (n=10). # p < 0.05 vs normal group. * p < 0.05 and ** p < 0.01 vs
scopolamine-treated control group.
3. Conclusion
In conclusion, to develop effective drugs for the treatment of Alzheimer’s disease (AD), a novel
series of scutellarein-O-acetamidoalkylbenzylamines derivatives T-1~24 were designed, synthesized
and evaluated as multi-targets anti-Alzheimer agents. All of these synthetic compounds showed
potent AChE inhibitory activity in vitro and high selectivity for AChE over BuChE. Compound T-22
exhibited the best inhibitory activity toward AChE with IC50 value of 0.051 ± 0.003 µM. Moreover,
both kinetic analysis of AChE inhibition and the molecular modeling study indicated that compoound
T-22 showed mixed-type inhibition and could bind to both CAS and PAS of AChE. More importantly,
Compound T-22 exhibited good antioxidant potency (1.13eq of Trolox), metal chelating properties
and significant inhibition of Aβ aggregation. In particular, compound T-22 inhibited: self-induced
Aβ1-42 aggregation, Cu2+-induced Aβ1-42 aggregation and HuAChE-induced Aβ1-40 aggregation by
57.1%, 43.4% and 57.3%, respectively. Furthermore, compound T-22 could disassemble the
well-structured Aβ1-42 fibrils generated Cu2+-induced Aβ1-42 aggregation by 66.3%. Especially,
compound T-22 diaplayed a good neuroprotective effect against H2O2-induced PC12 cell injury and a
low toxicity in SH-SY5Y cells. More interestingly, the in vivo study indicated that mice treated with
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T-22 (7.33 mg/kg, p.o.) displayed similar step-down latency time compared with the drug control
group treated with donepezil (5.0 mg/kg, p.o.) in the step-down passive avoidance test. These
properties highlight that compound T-22 may be a promising multifunctional agent for the treatment
of AD. Further studies to evaluate compound T-22 in vivo and to develop structural refinements are
underway and will be reported in due course.
4. Experiment section
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used
without further purification. Melting points were recorded on YRT-3 melting-point apparatus (China)
and are uncorrected. 1H NMR and 13C NMR spectra were recorded at room temperature in CDCl3 or
DMSO-d6 solutions using a Varian INOVA 400 NMR spectrometer. Chemical shifts are reported in
parts per millions (ppm) relative to tetramethylsilane (TMS). Coupling constants are given in hertz,
and spin multiplicities are given as s (singlet), brs (broad singlet), d (doublet), t (triplet), or m
(multiplet). Mass spectra were recorded on Agilent-6210 TOF LC-MS Spectrometer. The purity of all
final compounds was determined by high-performance liquid chromatography (HPLC) analysis to be
over 96%. HPLC analysis was carried out on a Shimadzu LC-10Avp plus system with the use of a
Kromasil C18 column (4.6 mm × 250 mm, 5 µm). All the reactions were monitored by thin-layer
chromatography (TLC) on silica gel GF254 plates from Qingdao Haiyang Chemical Co. Ltd. (China),
and then visualized with an UV lamp (254 nm). Where appropriate, crude products were purified by
column chromatography using silica gel (230-400 mesh) purchased from Qingdao Haiyang Chemical
Co. Ltd. (China).
4.1.1. General procedures for the synthesis of compounds 2a-d.
To a suspension of the appropriate dibromoalkane (33 mmol) and potassium carbonate (18 mmol) in
anhydrous acetone (30 mL), and potassium phthalimide (15 mmol) was added slowly over a 15
minutes period, and then the reaction mixture was heated under reflux for 8-10 h. The reaction
mixture was filtered, and the acetone was evaporated in vacuo. The crude product was purified by
column chromatography on silica gel (petroleum/acetone as eluent) to obtain 2a-d as white solid.
4.1.1.1. 2-(2-Bromoethyl)-1H-isoindole-1,3(2H)-dione (2a). White solid. Yield 68.1%; mp:
151.3-152.5 °C (lit.[33] 150-153 °C).
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4.1.1.2. 2-(3-Bromopropyl)-1H-isoindoline-1,3(2H)-dione (2b). White solid. Yield 64.7%; mp:
71.0-72.5 °C (lit.[34] 72-75 °C).
4.1.1.3. 2-(4-Bromobutyl)-1H-isoindoline-1,3(2H)-dione (2c). White solid. Yield 68.3%; mp:
79.1-80.6 °C (lit. [34] 79-81 °C).
4.1.1.4. 2-(6-Bromohexyl)-1H-isoindoline-1,3(2H)-dione (2d). White solid. Yield 53.7%; mp:
56.8-58.2 °C (lit. [34] 57-58 °C).
4.1.2. General procedure for the synthesis of compounds 4-7.
A mixture of the corresponding secondary amine 3a-d [23] (5.5 mmol), anhydrous K2CO3 (6
mmol) and compounds 2a-d (5 mmol) were added in CH3CN (20 ml). The mixture was heated at
65 °C for 8-10 h. The solvent was evaporated under reduced pressure. Then water (25 mL) was
added, and the mixture was extracted with dichloromethane (20 mL×3). The combined organic
phases were washed with saturated aqueous sodium chloride (30 mL), dried over sodium sulfate, and
filtered. The solvent was evaporated under vacuum. The residue was purified on a silica gel
chromatography using mixtures of petroleum/acetone as eluent to obtain the oil products 4-7.
1
4.1.2.1. 2-[2-[Methyl(phenylmethyl)amino]ethyl]-1H-isoindole-1,3(2H)-dione (4a). Colorless oil. H
NMR (400 MHz, CDCl3) δ: 7.84-7.82 (m, 2H), 7.73-7.70 (m, 2H), 7.32-7.30 (m, 2H), 7.15-7.13 (m,
3H), 3.82 (t, J = 7.6 Hz, 2H), 3.52 (s, 2H), 2.66 (t, J = 7.6 Hz, 2H), 2.30 (s, 3H).
1
4.1.2.2. 2-[2-[Ethyl(phenylmethyl)amino]ethyl]-1H-isoindole-1,3(2H)-dione (4b). Colorless oil. H
NMR (400 MHz, CDCl3) δ: 7.81-7.79 (m, 2H), 7.72-7.69 (m, 2H), 7.17 (d, J = 6.8 Hz, 2H),
7.12-7.06 (m, 3H), 3.75 (t, J = 6.0 Hz, 2H), 3.57 (s, 2H), 2.70 (t, J = 6.0 Hz, 3H), 2.60-2.56 (m, 2H),
1.03 (t, J = 6.8 Hz, 3H).
4.1.2.3.2-[2-[[(2-Methoxyphenyl)methyl]methylamino]ethyl]-1H-isoindole-1,3(2H)-dione(4c).Colorl
1
ess oil. H NMR (400 MHz, CDCl3) δ: 7.83-7.81 (m, 2H), 7.71-7.69 (m, 2H), 7.18-7.13 (m, 2H),
6.78 (d, J = 6.4 Hz, 1H), 6.74 (t, J = 7.2 Hz, 1H), 3.85 (t, J = 6.4 Hz, 2H), 3.73 (s, 3H), 3.59 (s, 2H),
2.71 (t, J = 7.2 Hz, 2H), 2.33 (s, 3H).
4.1.2.4.2-[2-[[(2-Methoxyphenyl)methyl]ethylamino]ethyl]-1H-isoindole-1,3(2H)-dione(4d).Colorles
s oil. 1H NMR (400 MHz, CDCl3) δ 7.79-7.78 (m, 2H), 7.69-7.68 (m, 2H), 7.19 (d, J = 7.2 Hz, 1H),
7.09 (t, J = 7.2 Hz, 1H), 6.73 (t, J = 7.6 Hz, 1H), 6.67 (t, J = 7.2 Hz, 1H), 3.77 (t, J = 6.4 Hz, 2H),
3.73 (s, 3H), 3.63 (s, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.64-2.61 (m, 2H), 1.05 (t, J = 6.8 Hz, 3H).
4.1.2.5. 2-[3-[Methyl(phenylmethyl)amino]propyl]-1H-isoindole-1,3(2H)-dione (5a), colorless oil.
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1H NMR (400 MHz, CDCl3) 7.84-7.82 (m, 2H), 7.72-7.69 (m, 2H), 7.29-7.23 (m, 5H), 3.76 (t, J =
7.2 Hz, 2H), 3.48 (s, 2H), 2.45 (t, J =7.2 Hz, 2H), 2.17 (s, 3H), 1.92-1.87 (m, 2H).
1
4.1.2.6. 2-[3-[Ethyl(phenylmethyl)amino]propyl]-1H-isoindole-1,3(2H)-dione (5b).Colorless oil. H
NMR (400 MHz, CDCl3) δ: 7.84-7.80 (m, 2H), 7.73-7.70 (m, 2H), 7.32 (d, J = 6.8 Hz, 2H), 7.27 (t, J
= 6.8 Hz, 2H), 7.19 (t, J = 6.8 Hz, 1H), 3.72 (t, J = 7.2 Hz, 2H), 3.55 (s, 2H), 2.54-2.48 (m, 4H),
1.88-1.82 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H).
4.1.2.7.2-[3-[[(2-Methoxyphenyl)methyl]methylamino]propyl]-1H-isoindole-1,3(2H)-dione(5c).Colo
1
rless oil. H NMR (400 MHz, CDCl3) δ 7.85-7.83 (m, 2H), 7.73-7.71 (m, 2H), 7.30 (d, J = 7.2 Hz,
1H), 7.20 (t, J = 7.2 Hz, 1H), 6.88 (t, J = 7.2 Hz, 1H), 6.84 (d, J = 7.2 Hz, 1H), 3.82 (s, 3H), 3.77 (t, J
= 7.2 Hz, 2H), 3.51 (s, 2H), 2.50 (t, J = 7.2 Hz, 2H), 2.21 (s, 3H), 1.95-1.92 (m, 2H).
4.1.2.8. 2-[3-[[(2-Methoxyphenyl)methyl]ethylamino]propyl]-1H-isoindole-1,3(2H)-dione (5d).
1
Colorless oil. H NMR (400 MHz, CDCl3) δ 7.84-7.82 (m, 2H), 7.72-7.69 (m, 2H), 7.43 (d, J = 7.2
Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 6.90 (t, J = 7.2 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 3.82 (s, 3H), 3.72
(t, J = 6.8 Hz, 2H), 3.70 (s, 2H), 2.65-2.57 (m, 4H), 1.96-1.91 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H).
1
4.1.2.8. 2-[4-[Methyl(phenylmethyl)amino]butyl]-1H-isoindole-1,3(2H)-dione (6a).Colorless oil. H
NMR (400 MHz, CDCl3) δ 7.85-7.83 (m, 2H), 7.71-7.70 (m, 2H), 7.29-7.22 (m, 5H), 3.69 (t, J = 7.2
Hz, 2H), 3.51 (s, 2H), 2.43 (t, J = 6.8 Hz, 2H), 2.19 (s, 3H), 1.72-1.68 (m, 2H), 1.60-1.56 (m, 2H).
1
4.1.2.9. 2-[4-[Ethyl(phenylmethyl)amino]butyl]-1H-isoindole-1,3(2H)-dione (6b).Colorless oil. H
NMR (400 MHz, CDCl3) δ 7.85-7.83 (m, 2H), 7.73-7.71 (m, 2H), 7.33 (d, J = 6.8 Hz, 2H), 7.29 (t, J
= 6.8 Hz, 2H), 7.22 (t, J = 6.8 Hz, 1H), 3.67 (t, J = 7.2 Hz, 2H), 3.58 (s, 2H), 2.57-2.46 (m, 4H),
1.71-1.66 (m, 2H), 1.56-1.52 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H).
4.1.2.10. 2-[4-[[(2-Methoxyphenyl)methyl]methylamino]butyl]-1H-isoindole-1,3(2H)-dione (6c).
1
Colorless oil. H NMR (400 MHz, CDCl3) δ 7.85-7.83 (m, 2H), 7.73-7.71 (m, 2H), 7.30 (d, J = 7.2
Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.71
(t, J = 6.8 Hz, 2H), 3.49 (s, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.20 (s, 3H), 1.74-1.69 (m, 2H), 1.62-1.59
(m, 2H).
4.1.2.11. 2-[4-[[(2-Methoxyphenyl)methyl]ethylamino]butyl]-1H-isoindole-1,3(2H)-dione
(6d).
1
Colorless oil. H NMR (400 MHz, CDCl3) δ 7.84-7.81 (m, 2H), 7.72-7.69 (m, 2H), 7.39 (d, J = 7.2
Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.68
(t, J = 6.8 Hz, 2H), 3.57 (s, 2H), 2.53-2.47 (m, 4H), 1.73-1.66 (m, 2H), 1.56-1.51 (m, 2H), 1.04 (t, J
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= 7.2 Hz, 3H).
4.1.2.12. 2-[6-[Methyl(phenylmethyl)amino]hexyl]-1H-isoindole-1,3(2H)-dione (7a). Colorless oil.
1H NMR (400 MHz, CDCl3) δ 7.85-7.82 (m, 2H), 7.73-7.70 (m, 2H), 7.29-7.23 (m, 5H), 3.67 (t, J =
7.2 Hz, 2H), 3.48 (s, 2H), 2.36 (q, J = 7.2 Hz, 2H), 2.18 (s, 3H), 1.69-1.66 (m, 2H), 1.55-1.49 (m,
2H), 1.38-1.31 (m, 4H).
1
4.1.2.13. 2-[6-[Ethyl(phenylmethyl)amino]hexyl]-1H-isoindole-1,3(2H)-dione (7b). Colorless oil. H
NMR (400 MHz, CDCl3) δ 7.85-7.82 (m, 2H), 7.73-7.71 (m, 2H), 7.36 (d, J = 7.2 Hz, 2H), 7.31 (t, J
= 7.2 Hz, 2H), 7.24 (t, J = 6.8 Hz, 1H), 3.66 (t, J = 7.2 Hz, 2H), 3.63 (s, 2H), 2.56 (q, J = 7.2 Hz, 2H),
2.47 (t, J = 7.2 Hz, 2H), 1.67-1.65 (m, 2H), 1.56-1.49 (m, 2H), 1.33-1.29 (m, 4H), 1.07 (t, J = 7.2 Hz,
3H).
4.1.2.14. 2-[6-[[(2-Methoxyphenyl)methyl]methylamino]hexyl]-1H-isoindole-1,3(2H)-dione (7c).
1
Colorless oil. H NMR (400 MHz, CDCl3) δ 7.85-7.83 (m, 2H), 7.72-7.70 (m, 2H), 7.30 (d, J = 7.2
Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 6.92 (t, J = 7.2 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 3.82 (s, 3H), 3.68
(t, J = 7.2 Hz, 2H), 3.49 (s, 2H), 2.38 (q, J = 7.2 Hz, 2H), 2.20 (s, 3H), 1.70-1.65 (m, 2H), 1.55-1.51
(m, 2H), 1.39-1.35 (m, 4H).
4.1.2.15. 2-[4-[[(2-Methoxyphenyl)methyl]ethylamino]butyl]-1H-isoindole-1,3(2H)-dione (7d).
1
Colorless oil. H NMR (400 MHz, CDCl3) δ 7.85-7.83 (m, 2H), 7.72-7.70 (m, 2H), 7.40 (d, J = 8.0
Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.92 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 3.81 (s, 3H), 3.66
(t, J = 7.2 Hz, 2H), 3.58 (s, 2H), 2.52 (q, J = 6.8 Hz, 2H), 2.44 (t, J = 6.4 Hz, 2H), 1.68-1.65 (m, 2H),
1.50-1.47 (m, 2H), 1.33-1.31 (m, 4H), 1.04 (t, J = 6.8 Hz, 3H).
4.1.3. General procedure for the synthesis of compounds 8-11.
Compounds 4-7 (10 mmol) and 80% hydrazine hydrate solution (30 mmol) were added in
ethanol (50 mL). The reaction mixture was heated under reflux for 3-5 h, and then filtered, the filtrate
was concentrated under reduced pressure. The residue was dissolved in dichloromethane (50 mL),
washed with water (30 mL×2) and saturated aqueous sodium chloride (30 mL), dried over Na2SO4,
and filtered. The solvent was evaporated under reduced pressure to obtain compounds 8-11 as light
yellow oil with high purity. The products were dried over P2O5 under vacuum for 24 h and used for
further reactions directly.
4.1.3.1.
(E)-1-(6-hydroxy-2,3,4-trimethoxyphenyl)-3-(4-(methoxymethoxy)phenyl)prop-2-en-1-one
(14). To a solution of p-hydroxybenzaldehyde (10 mmol) and potassium carbonate (11 mmol) in 15
24

ACCEPTED MANUSCRIPT
mL anhydrous acetone, chloromethyl methy ether (10.5 mmol) was added slowly. The mixture was
heated at reflux for 5-6 h, cooled to room temperature and filtered, the filtered was concentrated
under reduced pressure to give compound 13 as light yellow oil, which was used in the next step
without further purification.
To a mixture of the start material 6-hydroxy-2,3,4-trimethoxyacetophenone 12 (5 mmol) with the
4-(methoxymethoxy)benzaldehyde 13 (5.5 mmol) in EtOH (7 mL). An amount of 30% aqueous
KOH (6 mmol) solution was slowly added to the reaction. The mixture was stirred for 3-4 days at
room temperature, and then poured into ice-cold water. The mixture was acidified with 10% HCl and
stirred at romm temperature for overnight. The yellow solid formed was collected by filtration and
1
recrystallized from 80% ethanol to give compound 14. Yield 90%; mp: 94.2-94.5°C; H NMR (400
MHz, CDCl3) δ 13.78 (s, 1H), 7.87 (d, J = 15.6 Hz, 1H), 7.81 (d, J = 15.6 Hz, 1H), 7.60 (d, J = 8.8
Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 6.30 (s, 1H), 5.23 (s, 2H), 3.93 (s, 3H), 3.91 (s, 3H), 3.84 (s, 3H),
3.50 (s, 3H).
4.1.3.2. 4,2′-Dihytroxy-4′,5′,6′-trimethoxychalcone (15). A mixture of compound 14 (10 mmol) and
10%HCl (6 mL) in 8 mL ethanol was heated at 50 °C for 3 h, then cooled to room temperature and
poured into ice-water. The mixture was stirred at room temperature for overnight. The yellow solid
formed was collected by filtration and recrystallized from 90% ethanol to give compound 15 as
yellow solid. Yield 91%; mp: 146.2-147.2°C; 1H NMR (400 MHz, CDCl3) δ 13.79 (s, 1H), 7.86 (d, J
= 16.0 Hz, 1H), 7.81 (d, J = 16.0 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.30 (s,
1H), 5.41 (brs, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.84 (s, 3H).
4.1.3.3. 4′-Hydroxy-5,6,7-trimethoxyflavone (16). Compound 15 (5 mmol) and potassium iodide (0.2
mmol) was added to a solution of conc. H2SO4 (0.1 mL) in 10 mL DMSO. The mixture was heated at
100°C for 3-5 h, then poured into ice-water containing sodium thiosulfate (0.21 mmol) and stirred at
room temperature for overnight. The solid formed was collected by filtration and recrystallized from
1
80% alcohol to afford yellow solid 16. Yield 91.6%; mp: 224.1-225.4°C; H NMR (400 MHz,
DMSO-d6) δ 10.25 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.19 (s, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.64 (s,
1H), 3.95 (s, 3H), 3.80 (s, 3H), 3.77 (s, 3H).
4.1.3.4. 5,4′-Dihydroxy-6,7-dimethoxyflavone (17). To a solution of compound 16 (5 mmol) in 15 mL
anhydrous acetonitrile was added aluminium chloride (10 mmol). The mixture was heat at 60 °C for
1 h, then added 10 mL10% HCl, and refluxed for 1 h. The solution was poured into ice-water,
25

ACCEPTED MANUSCRIPT
filtrating, the residue was recrystallized from methoxyethanol to afford light yellow compounds 17.
1
Yield 90.1%; mp: 261.1-262.3°C; H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 10.41 (s, 1H),
7.98 (d, J = 9.2 Hz, 2H), 6.95-6.93 (m, 3H), 6.87 (s, 1H), 3.93 (s, 3H), 3.74 (s, 3H).
4.1.4. General procedure for the synthesis of compounds 18a-b.
To a solution of compounds 16 or 17 (5 mmol) and anhydrous K2CO3 (6.0 mmol) in CH3CN, and
ethyl bromoacetate (5.5 mmol) was slowly added. The reaction mixture was heated at 65 °C and
stirred for 8-10 h under an argon atmosphere. After complete reaction, the solvent was evaporated
under reduced pressure. Then water (50 mL) was added to the residue and the mixture was extracted
with dichloromethane (30 mL×3). The combined organic phases were washed with saturated Na2CO3
aqueous solution (30 mL×3) and saturated aqueous sodium chloride (60 mL), dried over Na2SO4, and
filtered. The solvent was evaporated to dryness. The residue was purified on a silica gel
chromatography using mixtures of petroleum/acetone as eluent to obtain the compound 18a-b as
light yellow solid.
4.1.4.1. Ethyl 2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)acetate (18a). Light yellow
solid. Yield 89.5%; mp: 132.2-133.6°C; 1H NMR (400 MHz, CDCl3) δ 7.84 (d, 2H, J = 8.4 Hz), 7.02
(d, 2H, J = 8.8 Hz), 6.80 (s, 1H), 6.64 (d, J = 5.2 Hz, 1H), 4.70 (s, 2H), 4.30 (q, J1 = 14.0 Hz, J2 = 7.2
Hz, 2H), 3.99 (s, 6H), 3.92 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H).
4.1.4.2. Ethyl 2-(4-(5-hydroxy-6,7-dimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)acetate (18b). Light
yellow solid. Yield 80.7%; mp: 157.2-158.1°C; 1H NMR (400 MHz, CDCl3) δ 12.68 (brs, 1H), 7.86
(d, J = 9.2 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 6.59 (s, 1H), 6.55 (s, 1H), 4.72 (s, 2H), 4.30 (q, J= 7.2
Hz, 2H), 3.97 (s, 3H), 3.93 (s, 3H), 1.32 (t, J = 6.8 Hz, 3H).
4.1.5. General procedure for the synthesis of compounds 19a-b.
The mixture of LiOH (15 mmol) in water (15 mL) was added to a solution of compounds 18a-b in
THF (20 mL), and then heated under reflux for 2 h. The solution was concentrated in vacuo, diluted
with water (20 mL), acidified with 10% HCl. The solid formed was collected by filtration, washed
with water, dried, and recrystallized from ethanol to afford light yellow compounds 19a-b.
4.1.5.1. 2-(4-(5,6,7-Trimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)acetic acid (19a). Light yellow
solid. Yield 83.2%; mp: 234.0-235.7°C; 1H NMR (400 MHz, DMSO-d6) δ 13.15 (s, 1H), 8.02 (d, 2H,
J = 8.8 Hz), 7.23 (s, 1H), 7.09 (d, 2H, J = 9.2 Hz), 6.73 (s, 1H), 4.82 (s, 2H), 3.95 (s, 3H), 3.80 (s,
3H), 3.77 (s, 3H).
26

ACCEPTED MANUSCRIPT
4.1.5.2. 2-(4-(5-Hydroxy-6,7-dimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)acetic acid (19b).Light
1
yellow solid. Yield 88.7%; mp: 228.3-229.7°C; H NMR (400 MHz, DMSO-d6) δ 13.18 (s, 1H),
12.88 (s, 1H), 8.07 (d, 2H, J = 8.8 Hz), 7.12 (d, 2H, J = 8.8 Hz), 6.97 (s, 1H), 6.96 (s, 1H), 4.84 (s,
2H), 3.93 (s, 3H), 3.74 (s, 3H).
4.1.6. General procedure for the synthesis of compounds T-1~T-24.
To a mixture of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.45 mmol)
and compounds 19a-b (0.3 mmol) in THF (3 mL) was stirred for 0.5 h at room temperature, then
added the solution of the appropriate intermediates 8-11 (0.33 mmol) in THF (1 mL). And then was
stirred at room temperature for overnight. The solvent was evaporated in vacuo. The residue was
dissolved in dichloromethane (30 mL), washed with water (20 mL×2) and saturated aqueous sodium
chloride (30 mL), dried over sodium sulfate, and filtered. The solvent was evaporated under a
vacuum to give the crude product which was purified by column chromatography on silica gel
(CH2Cl2/CH3OH as eluent) to obtain the desired products T1-24.
4.1.6.1.N-(2-(benzyl(methyl)amino)ethyl)-2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)ac
1
etamide (T-1). Light yellow oil. Yield 41.3%; Purity: 98.2% (by HPLC); H NMR (400 MHz,
CDCl3) δ 7.88 (d, 2H, J = 8.8 Hz), 7.29-7.24 (m, 5H), 7.19 (brs, 1H), 7.09 (d, J = 8.4 Hz), 6.81 (s,
1H), 6.61 (s, 1H), 4.55 (s, 2H), 3.99 (s, 6H), 3.93 (s, 3H), 3.54 (s, 2H), 3.48-3.44 (m, 2H), 2.59-2.57
(m, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 177.1, 167.2, 160.5, 159.5, 157.6, 154.4,
152.5, 140.3, 138.6, 128.7 (2C), 128.3 (2C), 127.8 (2C), 127.2, 125.2, 115.0 (2C), 112.7, 107.4, 96.2,
67.3, 62.2, 62.1, 61.5, 56.2, 55.0, 41.6, 36.1; MS (ESI) m/z 533.3 [M + H]+.
4.1.6.2.N-(2-(benzyl(ethyl)amino)ethyl)-2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)acet
amide (T-2). Light yellow oil. Yield 49.5%; Purity: 98.8% (by HPLC); 1H NMR (400 MHz, CDCl3)
δ 7.88 (d, 2H, J = 8.8 Hz), 7.29-7.23 (m, 5H), 7.21 (brs, 1H), 7.07 (d, J = 8.0 Hz), 6.81 (s, 1H), 6.61
(s, 1H), 4.53 (s, 2H), 3.99 (s, 6H), 3.93 (s, 3H), 3.59 (s, 2H), 3.41-3.38 (m, 2H), 2.65-2.52 (m, 4H),
13
1.03 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 177.0, 167.0, 160.5, 159.5, 157.6, 154.3,
152.4, 140.2, 139.1, 128.5 (2C), 128.2 (2C), 127.8 (2C), 126.9, 125.1 114.9 (2C), 112.7, 107.3, 96.1,
67.2, 62.0, 61.4, 57.7, 56.2, 51.3, 47.0, 36.1, 11.7; MS (ESI) m/z 347.3 [M + H]+.
4.1.6.3.N-(2-((2-methoxybenzyl)(methyl)amino)ethyl)-2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl
1
)phenoxy)acetamide (T-3). Light yellow oil. Yield 41.6%; Purity: 97.6% (by HPLC); H NMR (400
MHz, CDCl3) δ 7.83 (d, J = 8.4 Hz, 2H), 7.47 (brs, 1H), 7.32 (d, J = 7.2 Hz, 1H), 7.27 (t, J = 6.8 Hz,
27

ACCEPTED MANUSCRIPT
1H), 7.04 (d, J = 8.8 Hz, 2H), 6.92-6.87 (m, 2H), 6.80 (s, 1H), 6.59 (s, 1H), 4.55 (s, 2H), 3.99 (s,
6H), 3.92 (s, 3H), 3.81 (s, 3H), 3.62 (s, 2H), 3.52-3.50 (m, 2H), 2.66-2.65 (m, 2H), 2.27 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 177.0, 167.3, 160.6, 159.6, 157.7, 157.6, 154.3, 152.4, 140.2, 130.5,
128.6, 127.7 (2C), 125.2, 125.0, 120.3, 115.1 (2C), 112.7, 110.4, 107.3, 96.2, 67.3, 62.1, 61.4, 56.2,
55.5, 55.3, 54.9, 41.8, 36.0; MS (ESI) m/z 563.2 [M + H]+.
4.1.6.4.N-(2-(ethyl(2-methoxybenzyl)amino)ethyl)-2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)ph
1
enoxy)acetamide (T-4). Light yellow oil. Yield 39.5%; Purity: 97.3% (by HPLC); H NMR (400
MHz, CDCl3) δ 7.87 (d, J = 8.8 Hz, 2H), 7.40 (brs, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.26 (t, J = 7.2 Hz,
1H), 7.04 (d, J = 8.0 Hz, 2H), 6.92-6.87 (m, 2H), 6.83 (s, 1H), 6.62 (s, 1H), 4.56 (s, 2H), 4.01 (s, 6H),
3.95 (s, 3H), 3.82 (s, 3H), 3.63 (s, 2H), 3.47-3.43 (m, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.56-2.50 (m,
2H), 1.00 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 177.1, 167.0, 160.6, 159.6, 157.6,
157.6, 154.3, 152.4, 140.2, 130.0, 128.1, 127.7 (2C), 125.0, 120.2, 114.9 (2C), 112.7, 110.3, 107.3,
96.1, 67.2, 62.1, 61.4, 56.2, 55.2, 51.5, 51.2, 47.4, 36.2, 11.7; MS (ESI) m/z 577.4 [M + H]+.
4.1.6.5.N-(3-(benzyl(methyl)amino)propyl)-2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)
1
acetamide (T-5). Light yellow oil. Yield 51.1%; Purity: 97.9% (by HPLC); H NMR (400 MHz,
CDCl3) δ 8.07 (brs, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.31-7.24 (m, 5H), 6.88 (d, J = 8.8 Hz, 2H), 6.79
(s, 1H), 6.58 (s, 1H), 4.55 (s, 2H), 3.99 (s, 6H), 3.92 (s, 3H), 3.51 (s, 2H), 3.47-3.42 (m, 2H),
13
2.52-2.49 (m, 2H), 2.17 (s, 3H), 1.75-1.72 (m, 2H); C NMR (100 MHz, CDCl3) δ 176.9, 167.1,
160.5, 159.5, 157.5, 154.2, 152.3, 140.1, 138.1, 129.0 (2C), 128.2 (2C), 127.6 (2C), 127.1, 124.8,
114.8 (2C), 112.6, 107.1, 96.1, 67.2, 62.9, 62.0, 61.4, 56.1 (2C), 41.6, 38.9, 25.4; MS (ESI) m/z
547.3 [M + H]+.
4.1.6.6.N-(3-(benzyl(ethyl)amino)propyl)-2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)phenoxy)ac
etamide (T-6). Light yellow oil. Yield 53.3%; Purity: 98.6% (by HPLC); 1H NMR (400 MHz, CDCl3)
δ 7.90 (brs, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.31-7.20 (m, 5H), 6.92 (d, J = 8.4 Hz, 2H), 6.80 (s, 1H),
6.59 (s, 1H), 4.54 (s, 2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.92 (s, 3H), 3.56 (s, 2H), 3.44-3.37 (m, 2H),
2.55-2.52 (m, 4H), 1.73-1.71 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 177.1,
167.2, 160.6, 159.5, 157.6, 154.4, 152.4, 140.3, 138.7, 129.0 (2C), 128.2 (2C), 127.7 (2C), 127.0,
125.0, 114.9 (2C), 112.7, 107.3, 96.1, 67.3, 62.1, 61.5, 58.4, 56.2, 51.8, 47.1, 39.0, 25.5, 11.1; MS
(ESI) m/z 561.3 [M + H]+.
4.1.6.7.N-(3-((2-methoxybenzyl)(methyl)amino)propyl)-2-(4-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-
28