Novel cinnamamide-dibenzylamine hybrids: Potent neurogenic agents with antioxidant, cholinergic, and neuroprotective properties as innovative drugs for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.07.077

By using fragments endowed with interesting and complementary properties for the treatment of Alzheimer's disease (AD), a novel series of cinnamamide-dibenzylamine hybrids have been designed, synthesized, and evaluated biologically. In vitro assay indicat

Full text of DOI:10.1016/j.ejmech.2017.07.077

Accepted Manuscript
Novel cinnamamide-dibenzylamine hybrids: Potent neurogenic agents with
antioxidant, cholinergic, and neuroprotective properties as innovative drugs for
Alzheimer's disease
Jin Wang, Pei Cai, Xue-Lian Yang, Fan Li, Jia-Jia Wu, Ling-Yi Kong, Xiao-Bing Wang
PII:
S0223-5234(17)30599-8
10.1016/j.ejmech.2017.07.077
EJMECH 9640
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 June 2017
Revised Date: 31 July 2017
Accepted Date: 31 July 2017
Please cite this article as: J. Wang, P. Cai, X.-L. Yang, F. Li, J.-J. Wu, L.-Y. Kong, X.-B. Wang, Novel
cinnamamide-dibenzylamine hybrids: Potent neurogenic agents with antioxidant, cholinergic, and
neuroprotective properties as innovative drugs for Alzheimer's disease, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.077.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel cinnamamide-dibenzylamine hybrids: potent neurogenic agents with
antioxidant, cholinergic, and neuroprotective properties as innovative drugs for
Alzheimer’s disease
Jin Wang, Pei Cai, Xue-Lian Yang, Fan Li, Jia-Jia Wu, Ling-Yi Kong*, Xiao-Bing
Wang*
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia
Xiang, Nanjing 210009, People’s Republic of China.
*
Corresponding authors
E-mails: cpu_lykong@126.com, xbwang@cpu.edu.cn.
Abstract
By using fragments endowed with interesting and complementary properties for
the treatment of Alzheimer’s disease (AD),
a
novel series of
cinnamamide-dibenzylamine hybrids have been designed, synthesized, and evaluated
biologically. In vitro assay indicated that most of the target compounds exhibited a
significant ability to inhibit ChEs, strong potency inhibitory of self-induced β-amyloid
(Aβ) aggregation and to act as potential antioxidants and biometal chelators. A
Lineweaver-Burk plot and molecular modeling study showed that compound 7f
targeted both the CAS and PAS of AChE. In addition, compound 7f could chelate
metal ions, reduce PC12 cells death induced by oxidative stress and penetrate the
blood–brain barrier (BBB). Overall, all of these outstanding in vitro results in
combination with promising in vivo outcomes highlighted derivative 7f as the lead
structure worthy of further investigation.
Keywords: Alzheimer’s disease; AP2238; cinnamamide; antioxidant activity;
cholinesterase
1
. Introduction
Alzheimer's disease (AD) is the most common age-related neurodegenerative
brain disorder characterized by dementia, cognitive impairment, memory loss, severe
behavioral abnormalities and ultimately death [1-3]. It afflicts more than 24 million

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people worldwide with the trend of increasing [4]. The etiology of AD is not fully
known, but some hallmarks, such as low levels of acetylcholine, β-amyloid (Aβ)
deposits, τ-protein aggregation, oxidative stress, inflammation, and dyshomeostasis of
biometals, have been implicated in the development of AD [5-7].
One of the AD cholinergic hypothesis which asserts that the decline of
acetylcholine level lead to cognitive and memory deficits, and sustaining or
recovering the cholinergic function is supposed to be clinically beneficial [8-10].
Converging lines of evidences suggest that an increased production of β-amyloid
peptide and its aggregation and accumulation in a brain lead to a neuronal cell death
[
5, 11, 12]. Another hypothesis was reported, it was suggested that oxidative stress is
involved in the early stage of the pathologic cascade and represents a key factor to
initiate the aggregation of β-amyloid and tau-protein hyperphosphorylation [13].
Considerable evidences proved that antioxidants could attenuate the syndrome of AD,
and prevent the progression of the disease [14, 15]. Thus, drugs with specifically
antioxidants activity could be useful for either the prevention or the treatment of AD
[
16, 17]. In addition, another AD hypothesis called metal hypothesis indicates the
metals (Fe, Cu and Zn) also play a role in pathogenesis of AD [18, 19]. The
abnormally high levels of metal ion in the brain is associated with the formation of Aβ
aggregates and neurofibrillary tangles (NFTs), which the promoted inflammation of
Aβ and activate neurotoxic pathways, leading to dysfunction and the death of brain
cell [19-22]. Thus, lowering of concentration of brain metals by chelating metals has
been proposed as a rational therapeutic approach for halting AD pathogenesis.
Current treatment of AD focuses on inhibiting cholinesterases (ChEs) activity
with drugs including donepezil, tacrine, rivastigmine and galantamine (Figure 1)
[
22-25]. However, due to the pathological complexity of AD, these drugs that
modulate such a single target could only enable a palliative treatment instead of
curing or preventing the neurodegeneration [7, 9, 16, 26]. Thus, multifunctional
molecules with two or more complementary biological activities may represent an
important advance against this disease [4, 15, 27, 28]. So we intend to design novel
multifunctional cholinesterase inhibitors (ChEIs) endowed with additional properties

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such as antioxidative activity, lowering Aβ the aggregation and chelating metals.
Compound AP2238 was the first published compound, designed to bind both
anionic sites of the human acetylcholinesterase (hAChE) for which the simultaneous
inhibition of the catalytic and the amyloid-β pro-aggregating activities of AChE was
verified [29]. The potency of AP2238 against AChE is comparable to that of
donepezil, while its ability to contrast Aβ aggregation is higher [30]. Cinnamamide is
a widely distributed constituent of plants [31]. Cinnamamide, a bioactive component
of Traditional Chinese Medicine, has antioxidant and anti-inflammatory effects,
inhibits Aβ fibril aggregation, and prevents Aβ-mediated toxicity both in vitro and in
vivo [32, 33].
Due to the pharmacological effects of cinnamamide, including its antioxidant
and anti-Aβ aggregation activities, are closely related to the etiology of AD, our
design principle involved the fusion of the important pharmacophores of
cinnamamide and AP2238 in a new molecule, without the major structural
modifications like those of other multi-target-directed ligands (MTDLs). The hybrid
compounds possess strong AChE inhibitory activity and many of the pharmacological
effects of multiolkoxybenzene (Figure 2).
2
.
Results and discussion
2
.1. Chemistry
New cinnamamide-dibenzylamine hybrids 5a-5n, 7a-7h ware obtained as
depicted in Scheme 1. The synthesis of target and intermediate compounds applied
readily available starting material and was completed using well-established methods.
Reaction of commercially available 1-(bromomethyl)-4-nitrobenzene and the
corresponding amine gave compounds 2b-2c (76-89% yield), which were further
reduced to corresponding amines 3b-3c (83-91% yield) in ethyl acetate using
SnCl .4H O as reducing agent [15, 34-36]. Then, these intermediate amine were
2
2
activated with (PyBOP) and subsequently coupled with commercially available
multiolkoxybenzene acid in the presence of triethylamine in dimethylformamide
solutions
at
room
temperature
overnight
to
afford
the
desired

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cinnamamide-dibenzylamine hybrids 5a-5n, 7a-7h in moderate to good yields (41-63%
yield) [37]. All new hybrids 5a-5n, 7a-7h were purified by chromatographic
techniques and structurally characterized by their analytical (HPLC and combustion
1
13
analysis) and spectroscopic data ( H NMR, C NMR, and MS).
2
.2．In vitro inhibition studies of AChE and BuChE
In clinical practice, it is well known that ChE inhibitors are effective in
improving the behavior and well-being and slowing down cognitive decline in
patients with dementia. Thus, the inhibitory activities of hybrids 5a-5n, 7a-7h against
AChE (from electric eel) and BuChE (from equine serum) were measured according
to the spectrophotometric method of Ellman et al [38]. For comparison purpose,
AP2238 and donepezil were used as reference compounds. The IC values of all
5
0
tested compounds were summarized in Table 1. Similar to the AP2238 and donepezil,
most of them showed more potent inhibitory activity for AChE than for BuChE. The
length of the alkyl spacer between benzene and AP2238 moiety could significantly
influence the AChE inhibitory activity. Compounds 5c, 5d (n = 1) exhibited the more
potent AChE inhibitory than compounds 5a, 5b (n = 0) and compounds 5e, 5f (n = 2).
Lengthening or shortening the length of alkyl spacer reduced AChE inhibition. In
contrast, the length of the alkyl spacer did not show a clear trend for BuChE inhibition.
Furthermore, a structure-activity relationship analysis suggested that the electron
density of aromatic ring played a significant role in determining the inhibitory activity
for ChEs in present work. When the aromatic ring bore more electron donating
substituents among the homogeneous class of compounds, the ChEs inhibitory
activity was increased. For instance, the homogeneous class of compounds 5g and 5h
one hydroxy substituent exhibited relatively low ChEs inhibitory activities compared
with compounds 5i and 5j, which had two hydroxyl substituents. Methoxy derivatives
(compounds 5m and 5n) showed just a little better ChEs inhibition than their hydroxy
counterparts (compounds 5i and 5j).
To further evaluate the role of the double bond between the phenyl ring and the
amide, compounds 7a-7h were synthesized and evaluated. Notably, compounds 7a
and 7b exhibited more strong ChEs inhibitory activities than compounds 5e and 5f,

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suggesting that the double bond and the conjugation system with the phenyl ring are
essential to induce inhibitory effects for these analogues. According to the above
results, substituents with varying electronic properties were introduced to the benzene
ring. The compounds 7b, 7c, and 7f-7h with electron-donating group was a slight
increase in ChEs inhibition. However, the compounds 7d and 7e with
electron-withdrawing group was a slight decrease in ChEs inhibition. These results
implied that electron-withdrawing groups seemed unbeneficial to ChEs inhibitory
activity. These results implied that electron-donating groups seemed beneficial to
ChEs inhibitory activity.
Subsequently, a selection of the compounds (5c, 5j-5n, 7c, 7f-7h) were then
evaluated as inhibitors of human ChEs (hChEs). The IC values of test compounds
5
0
for human AChE (hAChE) and BuChE (hBuChE) inhibition were listed in Table 2.
From the results, it can be seen that all compounds are also potent inhibitors of human
ChEs. For BuChE inhibition, most of these compounds showed improved inhibitory
activity for hBuChE compared to eqBuChE. Compound 7f displayed the inhibitory
activity for hBuChE with the IC value of 5.42 µM. However, no significant change
5
0
between hAChE and eeAChE was observed. Although compound 7f exhibited the
inhibitory activity for hAChE was slightly lower than for eeAChE (5.42 µM versus
4
.64 µM), it was still the more potent inhibitor of hAChE. Such an activity change
made 7f more balanced for both ChE inhibition (IC = 5.42 µM for hAChE; IC =
5
0
50
8
.01 µM for hBuChE), which would be more beneficial for AD treatment. Based on
the above assay results, we then selected the most potent AChE and BuChE inhibitor,
7
2
f, for kinetic analysis to investigate the type of inhibitor.
.3. Kinetic characterization of AChE inhibition.
Based on the in vitro results of AChE inhibition, we further elected the most
potent inhibitor 7f for kinetic analysis to investigate the type of enzyme inhibition.
Graphical analysis of the reciprocal Lineweaver–Burk plot showed both increasing
slopes (decreased Vmax) and increasing intercepts (increased Kmax) at increasing
concentration of compound 7f (Figure 3). This pattern indicated 7f was a mixed-type

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inhibitor to AChE and therefore revealed that compound 7f might be able to bind to
the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE.
2
.4. Docking study of compounds 7f with AChE.
To evaluate the binding modes of the compounds with respect to AChE,
molecular docking study was performed using software package MOE [39]. For the
docking studies, X-ray crystallographic structure of hAChE in complex with
donepezil (PDB ID: 4EY7) was utilized. The position of compound 7f with respect to
the key residues in the binding site is shown in Figure 4A and 4B. Caffee acid moiety
was located into PAS of AChE through a pep stacking interaction with Ile 287 (3.55
Å). The AP2238 moiety occupied the CAS and stacked against the benzene ring of
Phe330 with ring to ring distance of 4.53 Å. After analyzing the docking results and
taking the kinetic study into consideration, it was confirmed that compound 7f was a
dual binding site inhibitor that could interact simultaneously with the PAS and CAS of
AChE.
2
.5. Evaluation of the oxygen radical absorbance capacity.
The antioxidant activities of the above selected ware evaluated by following the
well-established ORAC-FL method (oxygen radical absorbance capacity by
fluorescence) that was recently applied by us to other compounds. Peroxyl radicals
were thermally generated from 2,2-azobis(amidinopropane) dihydrochloride and
reacted with fluorescein to form nonfluorescent products at 520 nm. The antioxidant
capacity of new hybrids was determined by their competition with fluorescein in the
radical capture, using a fluorescence microplate reader. Trolox, a vitamin E analogue,
was used as a standard, and the results were expressed as trolox equivalents (µmol of
trolox/µmol of tested compound), in a relative scale where ORAC (trolox) = 1.
Ferulic acid was also analyzed for comparison, compounds 5a-5f without a phenolic
hydroxyl had lower radical scavenging activities. Obviously, compounds 5g-5l and
7
f-7g, which contain a phenolic hydroxyl showed strong radical scavenging activities.
Hybrids bearing hydroxyl groups exhibited interesting antioxidant capacities, which
indicated hydroxyl groups is crucial for antioxidant capacities.

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2
.6. Inhibition of Aβ1-42 self-induced aggregation
All compounds tested their 0RAC-FL radical scavenging activities were also
tested for their ability to inhibit Aβ1-42 self-induced aggregation by using a
thioflavin-T based fluorometric assay [40]. Curcumin (Cur) and resveratrol (Res)
were used as reference compounds and the results were summarized in Table 1 and
showed in Figure 5. From the results, it could be seen that most hybrids exhibited
moderate-to-good potencies (33.5-58.6% at 20 µM) compared to those of curcumin
(42.5% at 25 µM) and resveratrol (48.2% at 20 µM). Noticeably, the optimal Aβ1-42
aggregation inhibition potency (58.6% at 20 µM) was provided by compound 7d.
Compounds 7a-7c, 7e-7h had good Aβ aggregation inhibition property (56.8%, 56.6%,
5
7.8%, 52.8, 56.8% and 56.6% at 20 µM). From the inhibition values of compounds
a-7h, it appeared that introduction of the double bond seemed to be beneficial to Aβ
7
self-induced aggregation inhibitory activity.
2
.7. Modulation of self- and metal-induced Aβ aggregation by compound 7f.
The regulation of self-induced Aβ aggregation by compound 7f were studied
using the ThT assay, and transmission electron microscopy (TEM) was then
performed to determine the morphological changes in the Aβ species (Figure 6) [41].
The ThT assay showed that compound 7f inhibited 56.2±1.21% of the self-induced
Aβ1-42 aggregation (Figure 6A), which were significantly higher than Curcumin
(42.5±1.12%) and resveratrol (48.2 ± 1.08%). In accord with the ThT assay, fewer and
shorter Aβ1-42 aggregates (Figure 6B, sequence 5) were observed after compound 7f
was added to the samples compared to curcumin (Figure 6B, sequence 3) and
resveratrol (Figure 6B, sequence 4). These results strongly indicated 7f could
effectively control self-induced Aβ aggregation and disassemble the preformed
self-induced Aβ aggregates, which should mainly be derived from its overall
framework rather than the individual moieties.
In addition, compound 7f also showed remarkable effects on the modulation of
2
+
2+
Cu -induced Aβ aggregation (Figure 7). Excellent Cu -induced Aβ aggregation
inhibition (87.4 ±4.5%) was obtained compared to CQ (48.8 ±4.8%). The TEM assay

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2+
showed that the Cu -treated sample of fresh Aβ produced more fibrils than did the
2
+
nontreated sample (Figure 7B, sequences 2 and 3). When compound 7f and Cu were
incubated with Aβ, fewer Aβ fibrils were detected (Figure7B, sequence 5). As
indicated, compound 7f also exhibited stronger disaggregation activity on the
2
+
preformed Cu -induced Aβ fibrils.
2
.8. Metal-chelating properties of compound 7f
A UV-Vis spectroscopy assay was performed to evaluate the ability of the
2
+
2+
2+
3+
compound 7f to chelate biometals such as Cu , Zn , Fe , and Fe (Figure 8). When
CuCl was mixed with compound 7f, the maximum absorption of 7f at 230 nm
2
exhibited a red shift to 245 nm and the minimum absorption shifted from 260 to 274
2
+
nm, indicating generation of a 7f-Cu complex. To establish the time required for the
complexation process, the spectrum of the mixture 7f plus CuSO was recorded at
4
different times, observing that the curve did not change after the first minute and the
absorbance dropped dramatically, indicating the formation of a 7f-Cu(II) complex
(
Figure 8A). Addition of ZnCl , FeCl to a solution of 7f produced a similar
2 3
maximum absorption wavelength shift, but addition of FeSO demonstrated only a
4
weak minimum absorption shift. These data suggest that 7f is a potent chelator for
2
+
3+
2+
2+
Cu 、Fe and Zn , but not for Fe .
To determine the stoichiometry of the complex 7f-Cu(II), the technique of
continuous variations (also called Job’s method) was used by preparing solutions of
compound 7f and CuSO so that the sum of concentrations of both species was
4
constant in all samples, but the proportions of both components varied between 0%
and 100%.[42]. The UV spectra were recorded and the absorbance change at 243 nm
was plotted versus the mole fraction of compound 7f, giving two straight lines whose
equations were calculated. The equations gave a solution at a mole fraction of 0.67 for
2+
compound 7f, revealing a 1:2 stoichiometry for complex 7f-Cu (Figure 8B).
2
.9. Cell toxicity and effect on H O -induced oxidative cell damage in PC12 cells
2
2
On the basis of the screening results presented above, compounds 5i, 5j, 5k, 7f
and 7g, as the multifunctional dual inhibitors against ChEs, Aβ1-42 and antioxidant,
were selected to further examine the potential toxicity effect on the PC12 cells. After

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exposing the cells to these compounds for 24 h, the cell viability was evaluated by the
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. As reported in
Figure 9, the results indicate that compounds 5i, 5j, 5k, 7f and 7g did not have a
significant effect on cell viability at 1.25-50 µM, suggesting that compounds 5i, 5j, 5k,
7
f and 7g exhibited very low toxicity to PC12 cells.
Compounds 5i, 5j, 5k, 7f and 7g were tested for their capacity to protect the rat
pheochromocytoma cell line PC12 cells against oxidative stress-associated death
induced by H O . Trolox and ferulic acid were also used as the reference compounds.
2
2
In this assay, addition of 200 µM H O to the growth medium reduced cell viability to
2
2
4
8.7% compared to control. The tested compounds were added to the media at
different concentrations immediately prior to the H O insult. As can be seen in
2
2
Figure 10, all of the compounds exhibited neuroprotective effects at concentrations
ranging from 1.25 to 25 µM. Compounds 5i, 5j and 7f had showed the highest
protective capability almost the same as trolox at the concentration of 25 µM and
much better than melatonin. Compounds 5k, 7g showed moderate protective
capabilities similar to melatonin at the concentration of 25 µM. These observations
further indicated that these new derivatives had the potential to be efficient
multifunctional agents, including antioxidant activity, for the treatment of AD.
2
.10. In vitro blood-brain barrier permeation study
Since the first requirement for successful central nervous system (CNS) drugs is
to reach their therapeutic targets, screening for the blood-brain barrier (BBB)
penetration is of great importance. To explore whether selected
cinnamamide-dibenzylamine hybrids would be able to penetrate into the brain, we
used a parallel artificial membrane permeation assay for blood-brain barrier
(PAMPA-BBB) [43]. This simple and rapid model, described by Di et al. and recently
applied by us to different compounds, has the advantage of predicting passive BBB
permeation with high success [44, 45]. The in vitro permeabilities (Pe) of the above
selected cinnamamide-dibenzylamine hybrids (5c, 5k, 5m, 5n, 7f and 7h) and 9
commercial drugs through a lipid extract of porcine brain were determined using
PBS/EtOH (70:30). Assay validation was made by comparing experimental

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permeabilities of nine commercial drugs with reported values (Table 3). A plot of
experimental data versus bibliographic values gave a good linear correlation, P (exp.)
e
2
=
0.8405 P (Bibl.) -0.0474 R =0.9529) (Figure 11). From this equation and taking
e
into account the limit established by Di et al. for blood–brain barrier permeation, we
classified compounds as follows:
-
6
-1
(
(
(
a) ‘CNS+’ (high BBB permeation predicted): P (10 cm s ) >3.73;
e
-6
-1
b) ‘CNS’ (low BBB permeation predicted): P (10 cm s ) <1.63;
e
-6
-1
c) ‘CNS±’ (BBB permeation uncertain): P (10 cm s ) from 1.63 to 3.73;
e
Finally, six compounds (5c, 5k, 5m, 5n, 7f and 7h) exhibited good activities
against ChE and Aβ1-42 aggregation were chosen as the test compounds. The results
summarized in Table 3 indicate that all of the selected compounds could penetrate the
BBB.
2
.11. Cognitive and memory improvement in a rat model of AD
Evaluation of the effect of novel cinnamamide-dibenzylamine hybrids in animal
models of learning and memory impairment is lacking. Scopolamine induces memory
deficits and has been used extensively to evaluate potential therapeutic agents for
treating AD [46-48]. Therefore, we investigated the protective effects of novel
cinnamamide-dibenzylamine hybrids against scopolamine-induced memory deficits
via the passive avoidance task. The step-down latency of mice treated with
scopolamine alone (control group) was significantly (p < 0.05) shorter than that of
vehicle-treated mice (normal group). Treatment with compound 7f (5, 10 and 15
mg/kg), increased the latency time in a dose-dependent manner. In particular, the
effect of compounds 7f (10 mg/kg) was comparable with that of donepezil (5 mg/kg)
(Figure 12). Compounds 7f inhibited AChE activity and reversed
scopolamine-induced memory deficit in the step-down passive avoidance test. These
results indicated that the synthesized Novel cinnamamide-dibenzylamine hybrids may
have reversed cognitive deficit by increasing brain cholinergic activity due to the
inhibition of AChE.
3
.
Conclusion

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In summary, this study involved the design, synthesis and biological evaluation of
a novel series of MTDLs against AD by fusing the pharmacophores of AP2238 and
multiolkoxybenzene. Most of the target compounds displayed excellent inhibition of
Aβ aggregation and ChEs activity as well as remarkable BBB penetration and
antioxidant effects. The optimal candidate compound, 7f, modulated metal-induced
Aβ aggregation, good copper and iron chelating properties, and effective inhibitory
2
+
activity against self- and Cu -induced Aβ
aggregation. Interestingly, molecular
-42
1
modeling studies, necessary to validate the results derived from the kinetic assay,
revealed that 7f binds to the enzyme in reverse fashion as compared to other
dual-binding-site inhibitors of AChE. Moreover, 7f did not show obvious cytotoxicity
against PC12cells at micromolar concentration. This unique hybrid demonstrated
significant effects in protecting neuronal cells against H O -induced oxidative cell
2
2
damage in PC12 cells. Most importantly, Under in vivo conditions, mice treated with
f displayed significantly longer step-down latency times than the control group All
7
of these results highlight compound 7f’s potential as a promising lead in the
development of orally active therapies for AD.
4
.
Experimental
4
.1. Chemistry
Reagents and solvents were purchased from common commercial suppliers and
were used without further purification. Reaction progress was monitored using
analytical thin layer chromatography (TLC) on precoated silica gel GF254 (Qingdao
Haiyang Chemical Plant, Qingdao, China) plates and the spots were detected under
UV light (254 nm). IR (KBr-disc) spectra were recorded by Bruker Tensor 27
spectrometer. Melting points (uncorrected) were determined with an XT-4
1
13
micromelting point instrument. H and C NMR spectra were recorded in CDCl or
3
DMSO solutions using a Bruker ACF-500 spectrometer at 25 °C. Chemical shifts are
reported in δ (ppm) relative to internal Me Si. J values are given in hertz, and spin
4
multiplicities are expressed as s (singlet), d (doublet), t (triplet), q (quartet), or m
(multiplet). The purity of all compounds ware confirmed to be higher than 95%

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through analytical HPLC performed with Agilent 1200 HPLC System (Supporting
information, Table S1). Mass spectra were obtained on a MS Agilent 1100 Series
LC/MSD Trap mass spectrometer (ESI-MS) and a Mariner ESI-TOF spectrometer
(HRESIMS), respectively. Column chromatography was performed on silica gel (90–
1
50 µm; Qingdao Marine Chemical Inc.)
4
.1.1. General procedure for the synthesis of compound 2b-2c
A mixture of 1-(bromomethyl)-4-nitrobenzene (10 mmol) and the corresponding
amine (30 mmol) in ethyl ether (50 mL) was refluxed for 10 h. After cooling to room
temperature, a white solid was removed by filtration, and the solvent was evaporated
to dryness under reduced pressure. The residue was purified on a silica gel column
using mixtures of PE/EtOAc as eluent, obtaining the corresponding compound 2b-2c
4
.1.1.1. N-benzyl-N-methyl-1-(4-nitrophenyl)methanamine (2b). Reagents used
were 1-(bromomethyl)-4-nitrobenzene (2.16 g, 10 mmol) and
N-methyl-1-phenylmethanmine (3.63 g, 30 mmol). Purification involved the use of
PE/EtOAc (100:1) as eluent. 2b: yellow oil (2.29 g, 89%). ESI-MS m/z: 257.08
+
1
[
(
(
M+H] ; H NMR (500 MHz, CDCl ) δ 8.24 (s, 1H), 8.22 (s, 1H), 7.61 (s, 1H), 7.59
3
s, 1H), 7.43 (s, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.38 (s, 1H), 7.34 – 7.30 (m, 1H), 3.65
s, 2H), 3.62 (s, 2H), 2.26 (s, 3H).
4
.1.1.2. N-benzyl-N-(4-nitrobenzyl)ethanamine (2c). Reagents used were
-(bromomethyl)-4-nitrobenzene (2.16 g, 10mmol and
1
N-methyl-1-phenylmethanmine (4.05 g, 30 mmol). Purification involved the use of
PE/EtOAc (100:1) as eluent. 2c: colorless syrup (2.05 g, 76%). ESI-MS m/z: 271.12
+
1
[
(
M+H] ; H NMR (500 MHz, CDCl ) δ 8.22 (s, 1H), 8.18 (s, 1H), 7.61 (s, 1H), 7.59
3
s, 1H), 7.43 (s, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.39 (s, 1H), 7.36 (s, 1H), 7.30 (d, J =
7
3
4
.2 Hz, 1H), 3.70 (s, 2H), 3.66 (s, 2H), 2.58 (q, J = 7.1 Hz, 2H), 1.15 (t, J = 7.1 Hz,
H).
.1.2 General procedure for the synthesis of compound 3b-3c
A mixture of compound 2b-2c (10 mmol) and SnCl .4H O (40 mmol) in ethyl
2
2
acetate (20 mL) was refluxed for 2 h. After cooling to room temperature, a white solid
was removed by filtration, and the solvent was evaporated to dryness under reduced

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pressure. The residue was purified on a silica gel column using mixtures of
CH Cl /MeOH as eluent, obtaining the corresponding compound 3b-3c.
2
2
4
.1.2.1. 4-((benzyl(methyl)amino)methyl)aniline (3b). Reagents used were
N-benzyl-N-methyl-1-(4-nitrophenyl)methanamine (2.2 g, 8.6 mmol) and SnCl .4H O
2
2
(
7.74 g, 34 mmol). Purification involved the use of CH Cl /MeOH (50:1) as eluent.
2 2
+
1
3
b: yellow oil (1.85 g, 91%). ESI-MS m/z: 227.05 [M+H] ; H NMR (500 MHz,
CDCl ) δ 8.26 (s, 1H), 8.15 (s, 1H), 7.71 (s, 1H), 7.55(s, 1H), 7.438(s, 1H), 7.42 (s,
3
1
3
4
H), 7.38 (s, 1H), 7.35 (s, 1H), 7.31 (t, J = 7.2 Hz, 1H), 5.72 (s, 2H), 3.75 (s, 2H),
.68(s, 2H), 2.12 (s, 3H).
.1.2.2. 4-((benzyl(ethyl)amino)methyl)aniline (3c) Reagents used were
N-benzyl-N-methyl-1-(4-nitrophenyl)methanamine (2.0 g, 7.6mmol) and SnCl .4H O
2
2
(
6.74 g, 30 mmol). Purification involved the use of CH Cl /MeOH (50:1) as eluent. 3c:
2
2
+
1
yellow oil (1.75 g, 83%). ESI-MS m/z: 241.30 [M+H] ; H NMR (500 MHz, CDCl )
3
δ 7.42 (s, 1H), 7.42 (d, J = 7.3 Hz, 1H), 7.37 (s, 1H), 7.35 (s, 1H), 7.34 (d, J = 7.3 Hz,
1H), 7.31 (s, 1H), 7.29 (s, 1H), 7.27 (s, 1H), 7.20 (d, J = 8.2 Hz, 1H), 5.72 (s, 2H),
3.62 (s, 2H), 3.56 (s, 2H), 2.54 (q, J = 7.1 Hz, 2H), 1.11 (t, J = 7.1 Hz, 3H).
4.1.3. General procedure for the synthesis of the compound 5a-5n, 7a-7h.
To a solution of the corresponding compound 4a-4g, 6a-6h (0.5 mmol),
triethylamine (1.2 mmol), and PyBOP (0.6 mmol) in DMF (8 mL) was added the
appropriated compound 3b-3c (0.5 mmol). The reaction mixture was stirred at room
temperature overnight, and DMF was then evaporated to dryness under reduced
pressure. The residue was dissolved in CH Cl (10 mL) and consecutively washed
2
2
with a 10% aqueous NaHCO solution (3 ×10 mL) and H O (10 mL). The organic
3
2
phase was dried over sodium sulfate and evaporated to dryness under reduced
pressure. The residue was purified on a silica gel column using CH Cl as eluent,
2
2
obtaining the corresponding compound.
.1.3.1. N-(4-((benzyl(methyl)amino)methyl)phenyl)benzamide （5a） Reagents
used were benzoic acid (4a) (60.5 mg, 0.5 mmol),
-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
4
4
3
2
2

ACCEPTED MANUSCRIPT
+
eluent. 5a: white solid (110mg, 75.3%). ESI-MS m/z: 331.18 [M+H] ； mp 132-136
ºC；IR (KBr) v 3345, 3028, 2939, 1654, 1597, 1517, 1447, 1406, 1359, 1316, 735, 692
-1
1
cm
H), 7.64 (d, J = 5.7 Hz, 2H), 7.60 (d, J = 6.7 Hz, 1H), 7.57 (s, 2H), 7.48 (s, 2H), 7.39
d, J = 5.7 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 3.59 (s, 2H),
;
H NMR (500 MHz, CDCl ) δ 8.53 (s, 1H), 7.95 (s, 1H), 7.91 (d, J = 6.4 Hz,
3
2
(
1
3
3
.57 (s, 2H), 2.24 (s, 3H). C NMR (125 MHz, CDCl ) δ 165.84, 136.91, 135.06,
3
1
31.80, 129.67, 129.04, 128.77, 128.30, 127.10, 127.07, 120.22, 61.78, 61.28, 42.12.
+
HRMS: calculated for C H N O [M+H] 331.1805, found 331.1812.
2
2
23
2
4
.1.3．2. N-(4-((benzyl(ethyl)amino)methyl)phenyl)benzamid e（ 5b）Reagents used
were benzoic acid (4a) (60 mg, 0.5 mmol), 4-((benzyl(ethyl)amino)methyl)aniline
120 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and Et3N (156 µL, 1.2 mmol).
Purification involved the use of CH Cl as eluent. 5b: white solid (100mg, 55.8%). mp
(
2
2
1
34-137 ºC, IR (KBr) v 3328, 3010, 2936, 2847, 1645, 1604, 1585, 1517, 1458, 1426,
-1
+ 1
1
372, 1223, 769, 705 cm ; ESI-MS m/z: 345.36 [M+H] , H NMR (500 MHz, CDCl )
3
δ 8.53 (s, 1H), 7.96 (s, 1H), 7.92(d, J = 6.9 Hz, 2H), 7.67 (d, J = 5.7 Hz, 2H), 7.66 (d,
J = 6.9 Hz, 1H), 7.58 (s, 2H), 7.53 (s, 2H), 7.46(d, J = 5.9 Hz, 1H), 7.42 (d, J = 5.9 Hz,
1
H),7.38 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 3.59 (s, 2H), 3.57 (s, 2H), 2.26
1
3
(
q, J = 6.6 Hz, 2H), 1.33 (t, J = 6.6 Hz, 3H). C NMR (125 MHz, CDCl ) δ 165.84，
3
1
1
36.91，135.33，135.06，131.06，129.67，129.04，128.77，128.30，127.10，
27.07，124.90，120.22，61.67，55.88，14.16. HRMS: calculated for C H N O
23
25
2
+
[
M+H] 331.1961, found 345.1989.
4
.1.3.3. N-(4-((benzyl(methyl)amino)methyl)phenyl)-2-phenylacetamide （5c ）
Reagents used were 2-phenylacetic acid (4b) (68 mg, 0.5 mmol),
-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
4
3
2
2
eluent. 5c: yellow oil (120mg, 70%). IR (KBr) v 3338, 3029, 2997, 2784, 1675, 1607,
-1
+ 1
1
514, 1452, 1409, 740, 698, 529 cm ; ESI-MS m/z: 345.19 [M+H] , H NMR (500
MHz, CDCl ) δ 8.22 (s, 1H), 7.44 (s, 1H), 7.42 (d, J = 6.4 Hz, 1H), 7.41 (d, J = 5.7
3
Hz, 2H), 7.40 (d, J = 6.7 Hz, 1H), 7.38 (s, 2H), 7.36 (s, 1H), 7.35(d, J = 5.7 Hz, 1H),
7
.33 (d, J = 7.9 Hz, 1H), 7.22 (s, 1H), 7.16 (d, J = 6.7 Hz, 1H), 6.88 (d, J = 8.0 Hz,

ACCEPTED MANUSCRIPT
13
2
H), 3.95 (s, 2H), 3.58 (s, 2H), 3.51 (s, 2H), 2.23 (s, 3H). C NMR (125 MHz, CDCl )
3
δ 164.47, 137.43, 129.64, 128.29, 127.85, 127.08, 122.14, 119.84, 115.02, 111.12,
+
1
09.95, 61.67, 46.36, 42.05. HRMS: calculated for C H N O [M+H] 345.1961,
2
3
25
2
found 345.1955.
4
.1.3.4. N-(4-((benzyl(ethyl)amino)methyl)phenyl)-2-phenylacetamide （ 5d ）
Reagents used were 2-phenylacetic acid (4b) (68 mg, 0.5 mmol),
-((benzyl(ethyl)amino)methyl)aniline (120 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
4
3
2
2
eluent. 5d: yellow oil (89mg, 49.4%). IR (KBr) v 3336, 3009, 2986, 2847, 1648, 1621,
-1
+ 1
1
545, 1527, 1436, 1342, 937, 751, 698 cm ; ESI-MS m/z: 359.20 [M+H] , H NMR
(
500 MHz, CDCl ) δ 8.53 (s, 1H), 7.44 (s, 1H), 7.42 (d, J = 6.4 Hz, 2H), 7.41 (d, J =
3
5
.7 Hz, 2H), 7.40 (d, J = 6.7 Hz, 1H), 7.38 (s, 1H), 7.36 (s, 2H), 7.35(d, J = 5.7 Hz,
H), 7.33 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 6.7 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H),3.79
1
(s, 2H), 3.53 (s, 2H), 3.51 (s, 2H), 2.26 (q, J = 6.8 Hz, 2H), 1.33 (t, J = 6.8Hz, 3H).
1
3
C NMR (125 MHz, CDCl ) δ 164.47, 129.64, 129.05, 128.29, 127.08, 122.14,
3
1
19.84, 119.14, 115.02, 112.75, 111.12, 109.95, 61.67, 55.88, 46.36, 14.16. HRMS:
+
calculated for C H N O [M+H] 359.2118, found 359.2114.
2
4
27
2
4
.1.3.5. N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-phenylpropanamide (5e)
Reagents used were 3-phenylpropanoic acid (4c) (75 mg, 0.5 mmol),
-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
4
3
2
2
eluent. 5e: yellow oil (75mg, 41.7%). IR (KBr) v 3321, 3027, 2943, 2836, 1678, 1628,
-1
+ 1
1
615, 1554, 1517, 1440, 1345, 954, 748, 686 cm ; ESI-MS m/z: 359.20 [M+H] , H
NMR (500 MHz, CDCl ) δ 8.85 (s, 1H), 7.44 (s, 1H), 7.42 (d, J = 6.4 Hz, 2H), 7.37 (d,
3
J = 6.7 Hz, 1H), 7.35 (s, 2H), 7.33 (s, 2H), 7.29 (d, J = 5.9 Hz, 2H), 7.19 (d, J = 7.9
Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 3.79 (d, J = 6.4 Hz, 2H), 3.53 (s, 2H), 3.51 (s, 2H),
1
3
2
1
3
.79 (d, J = 6.4 Hz, 2H), 2.20 (s, 3H). C NMR (125 MHz, CDCl ) δ 165.53, 137.33,
3
31.64, 128.49, 127.98, 122.14, 120.23, 119.84, 115.02, 111.12, 110.95, 61.67, 42.05,
+
7.36, 35.42. C H N O [M+H] 359.2112, found 359.2110.
24
27
2

ACCEPTED MANUSCRIPT
4
.1.3.6. N-(4-((benzyl(ethyl)amino)methyl)phenyl)-3-phenylpropanamide (5f)
Reagents used were 3-phenylpropanoic acid (4c) (75 mg, 0.5 mmol),
-((benzyl(ethyl)amino)methyl)aniline (120 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
4
3
2
2
eluent. 5f: yellow oil (93mg, 50%). IR (KBr) v 3248, 3046, 2936, 2927, 2845, 1675,
-1
+ 1
1
628, 1607, 1564, 1527, 1416, 1348, 735, 680 cm ; ESI-MS m/z: 373.22 [M+H] , H
NMR (500 MHz, CDCl ) δ 8.80 (s, 1H), 7.54 (s, 1H), 7.49 (d, J = 6.4 Hz, 2H), 7.39 (d,
3
J = 6.7 Hz, 2H), 7.35 (s, 1H), 7.33 (s, 1H), 7.29 (d, J = 5.9 Hz, 2H), 7.19 (d, J = 7.9
Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 3.76 (d, J = 6.4 Hz, 2H), 3.58 (s, 2H), 3.55 (s, 2H),
13
2
.79 (d, J = 6.4 Hz, 2H), 2.54 (q, J = 6.2 Hz, 2H), 1.11 (t, J = 6.2 Hz, 3H). C NMR
(
125 MHz, CDCl ) δ 165.53, 139.33, 135.64, 133.42, 130.49, 127.98, 122.64, 120.29,
3
1
19.87, 115.62, 111.69, 110.95, 61.63, 55.86, 42.05, 37.36, 35.42.14.31. C H N O
25 29 2
+
[
M+H] 373.2108, found 373.2102.
4
.1.3.7.
N-(4-((benzyl(methyl)amino)methyl)phenyl)-2-(4-hydroxyphenyl)acetamide (5g)
Reagents used were2-(4-hydroxyphenyl)acetic acid (4d) (76 mg, 0.5 mmol),
4
-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
3
2
2
eluent. 5g: yellow oil (106mg, 58.9%). IR (KBr) v 3500, 3308, 3046, 2937, 2889,
-1
2
843, 1638, 1610, 1553, 1416, 1345, 832, 745, 693 cm ; ESI-MS m/z: 361.18
+
1
[
(
M+H] , H NMR (500 MHz, CDCl ) δ 9.20 (s, 1H), 8.22 (s, 1H), 7.44 (s, 1H), 7.42
3
d, J = 6.4 Hz, 2H), 7.37 (d, J = 6.7 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 2H), 7.29 (d, J =
5
2
1
6
3
4
.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 3.79 (s, 2H), 3.53 (s,
1
3
H), 3.51 (s, 2H), 2.20 (s, 3H). C NMR (125 MHz, CDCl ) δ 164.47, 153.56, 137.83,
3
30.64, 128.69, 127.35, 127.28, 125.45, 123.14, 119.84, 115.02, 113.92, 111.95,
+
1.64, 46.35, 42.09. HRMS: calculated for C H N O [M+H] 361.1813, found
23
25
2
2
61.1807.
.1.3.8.
N-(4-((benzyl(methyl)amino)methyl)phenyl)-2-(4-hydroxyphenyl)acetamide (5h)
Reagents used were2-(4-hydroxyphenyl)acetic acid (4d) (76 mg, 0.5 mmol),

ACCEPTED MANUSCRIPT
4
-((benzyl(ethyl)amino)methyl)aniline (120 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
3
2
2
eluent. 5h: yellow oil (89mg, 48.4%). IR (KBr) v 3448, 3316, 3021, 2993, 2848, 2835,
-1
+ 1
1
632, 1604, 1543, 1421, 1348, 867, 752, 684 cm ; ESI-MS m/z: 375.20 [M+H] , H
NMR (500 MHz, CDCl ) δ 9.23 (s, 1H), 8.20 (s, 1H), 7.44 (s, 1H), 7.42 (d, J = 6.4 Hz,
3
2
H), 7.37 (d, J = 6.7 Hz, 2H), 7.35 (s, 1H), 7.33 (s, 1H), 7.29 (d, J = 5.9 Hz, 2H), 7.19
(d, J = 7.9 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 3.77 (s, 2H), 3.58 (s, 2H), 3.54 (s, 2H),
1
3
2
1
1
.53 (q, J = 6.8 Hz, 2H), 1.11 (t, J = 6.8 Hz, 3H). C NMR (125 MHz, CDCl ) δ
3
65.87, 153.54, 136.21, 130.64, 129.55, 127.29, 127.08, 125.14, 121.84, 120.68,
15.92, 113.75, 112.82, 110.65, 61.67, 55.88, 46.36, 14.16. HRMS: calculated for
+
C H N O [M+H] 375.2006, found 359.2003.
25
27
2
2
4
.1.3.9.
acetamid e（ 5i））Reagents used were 2-(3,4-dihydroxyphenyl)acetic acid (4e) (84 mg,
.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP
312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of
N-(4-((benzyl(methyl)amino)methyl)phenyl)-2-(3,4-dihydroxyphenyl)
0
(
3
CH Cl as eluent. 5i: yellow oil (106mg, 58.9%). IR (KBr) v 3378, 2924, 2783, 1621,
2
2
-1
+
1
516, 1452, 1282, 1174, 1022, 818, 742, 690, 508 cm ; ESI-MS m/z: 377.18 [M+H] ,
1
H NMR (500 MHz, CDCl ) δ 8.89 (s, 2H), 8.18 (s, 1H), 7.44 (s, 1H), 7.42 (d, J = 6.4
3
Hz, 2H), 7.37 (d, J = 6.7 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 7.29 (d, J = 5.9 Hz, 2H),
7
2
1
.19 (d, J = 7.9 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 3.76 (s, 2H), 3.56 (s, 2H), 3.53 (s,
1
3
H), 2.10 (s, 3H). C NMR (125 MHz, CDCl ) δ 165.56, 153.78. 153.42, 137.43,
3
29.64, 128.29, 127.58, 122.14, 119.84, 115.02, 111.12, 109.95, 61.67, 46.36, 42.05.
+
HRMS: calculated for C H N O [M+H] 377.1823, found 377.1819.
2
3
25
2
3
4
.1.3.10.
N-(4-((benzyl(ethyl)amino)methyl)phenyl)-2-(3,4-dihydroxyphenyl)acetamide (5j)
Reagents used were 2-(3,4-dihydroxyphenyl)acetic acid (4e) (84 mg, 0.5 mmol),,
4
-((benzyl(ethyl)amino)methyl)aniline (120 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
3
2
2
eluent. 5j: yellow oil (110mg, 55.1%). IR (KBr) v 3352, 3027, 2966, 2867, 2783,
728,, 1675, 1603, 1514, 1410, 1364, 1345, 1255, 1206,1172, 1123, 1086, 978, 912,
1

ACCEPTED MANUSCRIPT
-
1
+ 1
8
8
1
22, 741, 699, 582 cm ; ESI-MS m/z: 391.19 [M+H] , H NMR (500 MHz, CDCl ) δ
3
.82 (s, 2H), 8.20 (s, 1H), 7.44 (s, 1H), 7.42 (d, J = 6.4 Hz, 2H), 7.37 (d, J = 6.7 Hz,
H), 7.35 (s, 1H), 7.33 (s, 1H), 7.29 (d, J = 5.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.70
(d, J = 8.0 Hz, 2H), 3.77 (s, 2H), 3.58 (s, 2H), 3.53 (s, 2H), 2.52 (q, J = 6.6 Hz, 2H),
1
3
1
1
5
4
.11 (t, J = 6.6 Hz, 3H). C NMR (125 MHz, CDCl ) δ 165.51, 153.74. 153.52,
3
37.43, 129.64, 128.29, 127.58, 122.14, 119.84, 115.02, 111.12, 109.95, 61.67, 46.36,
+
5.83, 14.23. HRMS: calculated for C H N O [M+H] 391.1955, found 391.1952.
2
4
27
2
3
.1.3.11.
N-(4-((benzyl(methyl)amino)methyl)phenyl)-2-(3-hydroxy-4-methoxyphenyl)acet
amide（5k） Reagents used were 2-(3-hydroxy-4-methoxyphenyl)acetic acid (4f) (91
mg, 0.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP
(
312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of
3
CH Cl as eluent. 5j: yellow oil (91mg, 46.5%). IR (KBr) v 3368, 3027, 2783, 1621,
2
2
-1
1
516, 1452, 1365, 1278, 1172, 1129, 1075, 1022, 822, 741, 699, 510 cm ; ESI-MS
+
1
m/z: 391.19 [M+H] , H NMR (500 MHz, CDCl ) δ 9.20 (s, 1H), 8.22 (s, 1H), 7.44 (s,
3
1
H), 7.42 (d, J = 6.4 Hz, 2H), 7.37 (d, J = 6.7 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 7.29
(d, J = 5.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 3.79 (s, 2H), ,
1
3
3
1
1
.69 (s, 3H), 3.56 (s, 2H), 3.52 (s, 2H), 2.17 (s, 3H). C NMR (125 MHz, CDCl ) δ
3
65.56, 153.78. 149.42, 138.13, 132.34, 130.64, 128.76, 127.58, 122.14, 119.84,
15.02, 111.12, 109.95, 61.67, 56.26, 46.36, 42.05. HRMS: calculated for
+
C H N O [M+H] 391.1923, found 391.1919.
23
27
2
3
4
.1.3.12.
N-(4-((benzyl(ethyl)amino)methyl)phenyl)-2-(3,4-dihydroxyphenyl)acetamide (5l)
Reagents used were 2-(3-hydroxy-4-methoxyphenyl)acetic acid (4f) (91 mg, 0.5
mmol),4-((benzyl(ethyl)amino)methyl)aniline (120 mg, 0.5 mmol), PyBOP (312.2 mg,
0
.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
3 2 2
eluent. 5l: yellow oil (103mg, 50.5%). IR (KBr) v 3432, 2937, 2868, 2780, 1657,
624, 1529, 1449, 1409, 1341, 1246, 1171, 1129, 1087, 1015, 867, 784, 737, 697, 582
1
-1
+ 1
cm ; ESI-MS m/z: 405.21 [M+H] , H NMR (500 MHz, CDCl ) δ 9.16 (s, 1H), 8.24
3
(s, 1H), 7.44 (s, 1H), 7.42 (d, J = 6.4 Hz, 2H), 7.37 (d, J = 6.7 Hz, 1H), 7.35 (s, 1H),

ACCEPTED MANUSCRIPT
7
2
1
1
6
4
4
.33 (s, 1H), 7.29 (d, J = 5.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.70 (d, J = 8.0 Hz,
H), 3.79 (s, 2H), 3.69 (s, 3H), 3.53 (s, 2H), 3.51 (s, 2H), 2.54 (q, J = 7.2 Hz, 2H),
1
3
.16 (t, J = 7.2. Hz, 3H) C NMR (125 MHz, CDCl ) δ 165.54, 153.73. 149.49,
3
38.62, 135.34, 132.64, 128.76, 127.58, 122.14, 119.84, 115.02, 111.12, 109.95,
+
1.67, 56.26, 55.83, 46.36, 14.15. HRMS: calculated for C H N O [M+H]
2
5
29
2
3
05.2138, found 405.2136.
.1.3.13.
N-(4-((benzyl(methyl)amino)methyl)phenyl)-2-(3,4-dimethoxyphenyl)acetamide
5m） Reagents used were 2-(3,4-dimethoxyphenyl)acetic acid (4g) (98 mg, 0.5
（
mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP (312.2
mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl
2
3
2
as eluent. 5m: yellow oil (88mg, 43.5%). IR (KBr) v 3305, 2934, 2835, 2785, 1660,
604, 1513, 1409, 1409, 1325, 1263, 1226, 1153, 1027, 966, 820，742， 699， 512
1
-1
+ 1
cm ; ESI-MS m/z: 405.21 [M+H] , H NMR (500 MHz, CDCl ) δ 8.32 (s, 1H), 7.44
3
(s, 1H), 7.42 (d, J = 6.4 Hz, 2H), 7.37 (d, J = 6.7 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H),
7
2
.29 (d, J = 5.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 3.79 (s,
1
3
H), , 3.69 (s, 6H), 3.53 (s, 2H), 3.51 (s, 2H), 2.20 (s, 3H). C NMR (125 MHz,
CDCl ) δ 165.56, 149.78. 146.42, 138.13, 132.34, 130.64, 128.76, 127.58, 122.14,
3
119.84, 115.02, 111.12, 109.95, 61.67, 56.26, 46.36, 42.05. HRMS: calculated for
+
C H N O [M+H] 405.2138, found 405.2137
25
29
2
3
4
.1.3.14.
N-(4-((benzyl(ethyl)amino)methyl)phenyl)-2-(3,4-dimethoxyphenyl)acetamide
5n) Reagents used were 2-(3-hydroxy-4-methoxyphenyl)acetic acid (4g) (98 mg, 0.5
mmol),4-((benzyl(ethyl)amino)methyl)aniline (120 mg, 0.5 mmol), PyBOP (312.2 mg,
.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
(
0
3
2
2
eluent. 5n: yellow oil (103mg, 50.5%). IR (KBr) v 3321，2935，2847，2748，2745，
1
7
1
668， 1608， 1532， 1453， 1421， 1345， 1254， 1109， 1038，954， 831，
-1
+ 1
45， 690 cm ; ESI-MS m/z: 419.18 [M+H] , H NMR (500 MHz, CDCl ) δ 8.35 (s,
3
H), 7.44 (s, 1H), 7.42 (d, J = 6.4 Hz, 2H), 7.37 (d, J = 6.7 Hz, 1H), 7.35 (s, 1H), 7.33
(s, 1H), 7.29 (d, J = 5.9 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H),

ACCEPTED MANUSCRIPT
3
.79 (s, 2H), 3.69 (s, 6H), 3.55 (s, 2H), 3.50 (s, 2H), 2.52 (q, J = 7.0 Hz, 2H), 1.15 (t,
1
3
J = 7.0 Hz, 3H). C NMR (125 MHz, CDCl ) δ 165.54, 153.73. 149.49, 138.62,
3
1
5
4
4
35.34, 132.64, 128.76, 127.58, 122.14, 119.84, 115.02, 111.12, 109.95, 61.67, 56.26,
+
5.83, 46.36, 14.15. HRMS: calculated for C H N O [M+H] 419.1824, found
26
31
2
3
19.1821
.1.3.15. N-(4-((benzyl(methyl)amino)methyl)phenyl)cinnamamid e（ 7a）Reagents
were cinnamic acid (6a) (74 mg, 0.5 mmol),
-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
used
4
3
2
2
eluent.7a: white solid (80mg, 44.5%). mp 130-135 ºC；IR (KBr) v 3244，3027，2966，
866，2785，1685，1605，1544，1514，1452，1410，1314，1220，1175，
2
-
1
1
3
7
7
2
1
128， 1108， 1104， 912， 829， 742， 690， 582 cm ; ESI-MS m/z:
+
1
57.41[M+H] , H NMR (500 MHz, DMSO) δ 10.22 (s, 1H), 7.70 (d, J = 8.1 Hz, 2H),
.69 – 7.60 (m, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.39 – 7.34 (m, 2H), 7.36 – 7.30 (m, 2H),
.28 – 7.23 (m, 2H), 6.99 (s, 2H), 6.56 (s, 1H), 6.54 (s, 1H), 3.51 (s, 2H), 3.48 (s, 2H),
1
3
.05 (s, 3H). C NMR (125 MHz, DMSO) δ 174.75, 139.04. 136.72, 129.19, 128.58,
27.76, 127.27, 123.05, 121.33, 118.73, 118.63, 112.30, 111.80, 62.88, 42.59. HRMS:
+
calculated for C H N O [M+H] 357.1961, found 357.1963.
2
4
25
2
4
.1.3.16.
acrylamide（7b） Reagents used were (E)-3-(4-hydroxyphenyl)acrylic acid (6b)
82mg, 0.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol),
(E)-N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-(4-hydroxyphenyl)
(
PyBOP (312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved
3
the use of CH Cl as eluent. 7b: yellow oil (120mg, 63.5%). IR (KBr) v 3330， 2965，
2
2
2
9
867， 1606， 1516， 1456， 1413，1348，1208, 1167， 1127， 1086， 1014，
-1
+ 1
13，832，743，690，583 cm ; ESI-MS m/z: 373.18 [M+H] , H NMR (500 MHz,
DMSO) δ 10.21 (s, 1H), 9.15 (s, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.69 – 7.60 (m, 2H),
7
.47 (t, J = 7.2 Hz, 2H), 7.39 – 7.34 (m, 2H), 7.36 – 7.30 (m, 2H), 7.28 – 7.23 (m, 2H),
1
3
6
.99 (s, 2H), 6.56 (s, 1H), 6.54 (s, 1H), 3.51 (s, 2H), 3.48 (s, 2H), 2.05 (s, 3H).
C
NMR (125 MHz, DMSO) δ 171.42, 147.40, 137.71, 136.72，130.22, 128.54, 128.27,
27.27, 126.70, 123.07, 121.32, 118.71, 118.64, 62.42, 41.75. HRMS: calculated for
1

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+
C H N O [M+H] 373.1911, found 373.1909.
24
25
2
2
4
.1.3.17. (E)-N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-(4-methoxyphenyl)
acrylamide（7c） Reagents used were (E)-3-(4-methoxyphenyl)acrylic acid (7c) (89
mg, 0.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP
(
312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of
3
CH Cl as eluent. 7c: yellow oil (106mg, 54.8%). IR (KBr) v 3432, 2967, 2869, 1680,
2
2
1
5
7
1
604, 1554, 1513，1460, 1412, 1334, 1259, 1210，1174, 1139, 1087, 1017, 829, 743,
-1
+ 1
84 cm ; ESI-MS m/z: 387.20 [M+H] , H NMR (500 MHz, DMSO) δ 10.24 (s, 1H),
.60 (s, 2H), 7.59 (s, 2H), 7.56 (d, J = 15.7 Hz, 1H), 7.39 (s, 1H), 7.38 (s, 1H), 7.36 (s,
H), 7.34 (d, J = 6.1 Hz, 2H), 7.32 (s, 1H), 7.29 (d, J = 6.5 Hz, 1H), 7.03 (d, J = 8.6
Hz, 2H), 6.76 (d, J = 15.7 Hz, 1H), 3.83 (s, 3H), 3.58 (s, 2H), 3.53 (s, 2H), 2.12 (s,
1
3
3
1
H). C NMR (125 MHz, DMSO) δ 171.40, 153.45，137.61, 133.34, 131.89，129.92,
28.47, 128.06, 128.00, 126.41, 123.75, 112.13, 111.42, 62.81, 55.90，43.02. HRMS:
+
calculated for C H N O [M+H] 387.2083, found 387.2080.
2
5
27
2
2
4
.1.3.18. (E)-N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-(4-chlorophenyl)
acrylamide （ 7d ） Reagents used were 2-(3,4-dimethoxyphenyl)acetic acid
E)-3-(4-chlorophenyl)acrylic acid (6d) (91 mg, 0.5 mmol),
-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6
mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of CH Cl as
(
4
3
2
2
eluent. 5m: white solid (95mg, 48.7%). mp 133-137 ºC, IR (KBr) v 3371, 2770, 1665,
-1
1
625, 1597, 1523，1408, 1336, 1248, 1170, 1091, 1019, 976, 810，737，690 cm ;
+
1
ESI-MS m/z: 391.15 [M+H] , H NMR (500 MHz, CDCl ) δ 9.35(s, 1H), 7.69 (s, 1H),
3
7
.61 (d, J = 5.4 Hz, 2H), 7.42 (d, J = 7.5 Hz, 1H), 7.40 (s, 1H), 7.36 (s, 2H), 7.35 (s,
H), 7.33 (d, J = 1.6 Hz, 2H), 7.31 (d, J = 2.4 Hz, 2H), 7.30 (d, J = 3.2 Hz, 1H), 7.24
2
1
3
(
d, J = 7.1 Hz, 1H), 3.60 (s, 2H), 3.55 (s, 2H), 2.12 (s, 3H). C NMR (125 MHz, DMSO)
δ
171.40, 137.61, 133.34, 131.89，129.92, 128.47, 128.06, 128.00, 126.41, 123.75,
12.40，112.13, 111.42, 62.81, 55.90， 43.02. HRMS: calculated for C H N OCl
1
2
4
24
2
+
[
M+H] 391.1571, found 391.1576.
4
.1.3.19.
(E)-N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-(4-nitrophenyl)

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acrylamide（7e） Reagents used were(E)-3-(4-nitrophenyl)acrylic acid (6e) (97 mg,
0
.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP
(
312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of
3
CH Cl as eluent. 5m: white solid (93mg, 46.2%). mp 136-141 ºC, IR (KBr) v 3327,
2
2
2
6
7
1
848, 2793, 1664, 1605, 1595, 1523，1432, 1335, 1240, 1158，960，826，730，
-1
+ 1
86 cm ; ESI-MS m/z: 402.21 [M+H] , H NMR (500 MHz, CDCl ) δ 9.35(s, 1H),
3
.69 (s, 1H), 7.61 (d, J = 5.4 Hz, 2H), 7.42 (d, J = 6.5 Hz, 1H), 7.40 (s, 2H), 7.36 (s,
H), 7.35 (s, 2H), 7.33 (d, J = 6.6 Hz, 2H), 7.31 (d, J = 7.4 Hz, 2H), 7.30 (d, J = 3.2
1
3
Hz, 1H), 7.24 (d, J = 7.1 Hz, 1H), 3.53 (s, 2H), 3.50 (s, 2H), 2.18 (s, 3H). C NMR
125 MHz, DMSO) δ 171.40, 137.24, 132.97, 131.52，129.55, 128.10, 127.69, 127.63,
23.38, 112.03，111.76, 111.06, 62.44, 42.65. HRMS: calculated for C H N O
(
1
24
24
3
3
+
[
M+H] 402.2173, found 402.1999.
.1.3.20.
E)-N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-(3,4-dihydroxyphenyl)acryla
mide（7f） Reagents used were (E)-3-(3,4-dihydroxyphenyl)acrylic acid (6f) (90 mg,
.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol), PyBOP
312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved the use of
4
(
0
(
3
CH Cl as eluent. 7f: yellow oil (123mg,63.4%). IR (KBr) v 3337,2966，2867，2781，
2
2
1
7
2
8
1
611，1515，1452，1257，1208，1172，1128，1086，1013，912，824，
-1
+ 1
41， 582 cm ; ESI-MS m/z: 389.18[M+H] , H NMR (500 MHz, DMSO) δ 9.54 (s,
H), 8.79 (s, 1H), 7.32 (dd, J = 12.0, 4.0 Hz, 2H), 7.27 – 7.14 (m, 3H), 6.97 (d, J =
.1 Hz, 2H), 6.83 (d, J = 11.8 Hz, 2H), 6.67 (d, J = 6.4 Hz, 2H), 6.62 (d, J = 8.42Hz,
1
3
H),6.53 (d, J = 8.2 Hz, 1H), 3.42 (s, 2H), 3.31 (s, 2H), 2.03 (s, 3H). C NMR (125
MHz, DMSO) δ 171.43, 153.70, 152.14, 136.72, 130.51, 127.73， 125.84, 123.04,
1
21.33, 118.70, 118.64, 112.36 ，111.79, 60.76, 41.86. HRMS: calculated for
+
C H N O [M+H] 389.2016, found 389.1545
24
25
2
3
4
.1.3.21.
(E)-N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)
acrylamide（7g） Reagents used were (E)-3-(4-hydroxy-3-methoxyphenyl) acrylic
acid (6g) (97 mg, 0.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5

ACCEPTED MANUSCRIPT
mmol), PyBOP (312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification
3
involved the use of CH Cl as eluent. 7g: yellow oil (132mg, 56.6%). IR (KBr) v3357，
2
2
2
948，2869，2784，1729，1678，1630，1517，1454，1347,1250，1220，
-1
+ 1
1
124， 1108, 982，845， 742， 701， 584 cm ; ESI-MS m/z: 403.19 [M+H] , H
NMR (500 MHz, DMSO) δ 10.09 (s, 1H), 9.26 (s, 1H), 7.36 (d, J = 2.2 Hz, 2H), 7.36
(d, J = 3.1 Hz, 1H), 7.34 (s, 2H), 7.32 (d, J = 6.1 Hz, 3H), 7.28 – 7.24 (m, 2H), 6.99
(d, J = 8.2 Hz, 2H), 6.54 (d, J = 8.3 Hz, 2H), 3.65 (s, 3H), 3.55 (s, 4H), 2.05 (s, 3H).
1
3
C NMR (125 MHz, DMSO) δ 171.42, 153.73, 152.14, 137.39, 137.25, 131.91,
1
30.45, 129.87， 128.07, 125.78, 123.7, 112.39, 112.15, 111.42, 60.96, 55.91, 43.07.
+
HRMS: calculated for C H N O [M+H] 403.2016, found 403.2010
2
5
27
2
3
4
.1.3.22.
E)-N-(4-((benzyl(methyl)amino)methyl)phenyl)-3-(3,4-dimethoxyphenyl)
acrylamide (7h) Reagents used were (E)-3-(3,4-dimethoxyphenyl)acrylic acid (6h)
104 mg, 0.5 mmol), 4-((benzyl(methyl)amino)methyl)aniline (113 mg, 0.5 mmol),
(
(
PyBOP (312.2 mg, 0.6 mmol), and Et N (156 µL, 1.2 mmol). Purification involved
3
the use of CH Cl as eluent. 7h: yellow oil (125mg, 60%). IR (KBr) v 3240， 2965,
2
2
2
866，1679, 1610, 1512，1458， 1413, 1343，1302， 1260， 1174, 1086， 1085，
-1
+ 1
1
015， 912， 842， 582 cm ; ESI-MS m/z: 417.21 [M+H] , H NMR (500 MHz,
CDCl ) δ 8.24 (s, 1H), 7.72 (d, J = 15.5 Hz, 1H), 7.71 – 7.63 (m, 2H), 7.40 (s, 1H),
3
7
8
3
.39 (s, 1H), 7.37 (s, 2H), 7.36 (s, 2H), 7.29 (dd, J = 13.0, 5.9 Hz, 1H), 7.13 (d, J =
.3 Hz, 1H), 7.07 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.60 (d, J = 15.4 Hz, 1H), 3.93 (s,
1
3
H), 3.90 (s, 3H), 3.57 (s, 2H), 3.54 (s, 2H), 2.22 (s, 3H). C NMR (125 MHz,
DMSO) δ 171.43, 148.73, 147.14, 137.49, 136.72, 130.51, 127.73， 125.84, 123.04,
1
21.33, 118.70, 118.64, 112.36，111.79, 60.76, 53.69, 41.86. HRMS: calculated for
+
C H N O [M+H] 417.2173, found 417.2165
26
29
2
3
4
.2. In vitro inhibition experiments of ChEs
Acetylcholinesterase (AChE, E.C. 3.1.1.7) from electric eel and human
erythrocytes, butyrylcholinesterase (BuChE, E.C. 3.1.1.8) from equine serum and
human serum, S-butyrylthiocholine iodide, acetylthiocholine iodide, 5,

ACCEPTED MANUSCRIPT
′
5
-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent, DTNB) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). The inhibitory activities of test compounds
a-5n, 7a-7h were evaluated by Ellman's method [38]. The prototypes AP2238 and
5
donepezil were used as reference compounds. The compounds were dissolved in
DMSO and diluted with the buffer solution (50 mM Tris-HCl, pH = 8.0, 0.1 M NaCl,
0
.02 M MgCl .6H O) to yield corresponding test concentrations (DMSO less than
2 2
0
.01%). In each well of the plate, 160 µL of 1.5 mM DTNB, 50 µL of AChE (0.22
U/mL eeAChE or 0.05 U/mL hAChE) or 50 µL of BuChE (0.12 U/mL eqBuChE or
.024 U/mL hBuChE) were incubated with 10 µL of different concentrations of test
0
compounds (0.001-100 µM) at 37 °C for 5 min. After this period, acetylthiocholine
iodide (15 mM) or S-butyrylthiocholine iodide (15 mM) as the substrate (30 µL) was
added and the absorbance was measured with awave length of 405 nm at different
time intervals (0, 60, 120, and 180 s). The concentration of compound producing 50%
of enzyme activity inhibition (IC ) was calculated by nonlinear regression analysis of
5
0
the response–concentration (log) curve, using the Graph-Pad Prism program package
Graph Pad Software; San Diego, CA). Results are expressed as the mean ±SEM of at
least three different experiments performed in triplicate
(
4
.3. Kinetic analysis of AChE inhibition
The kinetic study of AChE was performed by Ellman's method with three
different concentrations (9.0, 4.5 and 2.25µM) of compound 7f reciprocal plots of
/velocity versus 1/substrate were constructed at different concentrations of the
1
substrate thiocholine iodide (0.05–0.5 mM) . The plots were assessed by a weighted
least-squares analysis that assumed the variance of velocity (v) to be a constant
percentage of v for the entire data set. Data analysis was performed with Graph Pad
Prism 4.03 software (San Diego, CA, USA).
4
.4. Molecular modeling studies
Molecular modeling calculations and docking studies were performed using
Molecular Operating Environment (MOE) software version 2008.10 (Chemical

ACCEPTED MANUSCRIPT
Computing Group, Montreal, Canada) [49]. The X-ray crystallographic complex of
AChE with donepezil (PDB ID: 4EY7) was obtained from the Protein Data Bank. All
water molecules in PDB files were removed and hydrogen atoms were subsequently
added to the protein. The compound 7f was built using the builder interface of the
MOE program and energy minimized using MMFF94x force field. Then the 7f was
docked into the active site of the protein by the ‘Triangle Matcher’ method, which
generated poses by aligning the ligand triplet of atoms with the triplet of alpha spheres
in cavities of tight atomic packing. The Dock scoring in MOE software was done
using ASE scoring function and Force field was selected as the refinement method.
The best 10 poses of molecules were retained and scored. After docking, the geometry
of resulting complex was studied using the MOE’s pose viewer utility.
4
.5. Metal-chelating study
The chelating studies were performed with a UV−vis spectrophotometer. The
absorption spectra of each compound (25 µM, final concentration) alone or in the
presence of CuSO , FeSO , FeCl or ZnCl (25 µM, final concentration) for 30 min in
4
4
3
2
2
0% (v/v) ethanol/buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were recorded at
room temperature in a 1 cm quartz cell [42, 50, 51]. The stoichiometry of the
2
+
compound−Cu complex was determined by employing Job’s method [52, 53].
Separate solutions of compound and CuSO were used to prepare solutions in which
4
−
6
the sum of the concentrations of both species (25 × 10 M) was constant in all
samples, 21 solutions were obtained with the condition that the sum of concentrations
of both species was a constant in all samples but the proportions of both components
varied between 0 and 100%. The UV spectra were normalized to the respective blanks.
The absorbance differences at 243 nm were plotted versus the mole fraction, showing
a maximum at 0.5 that revealed a stoichiometry of 1:2 for complex 7f-Cu (II).
4
.6. Oxygen radical absorbance capacity assay.
The ORAC method was followed. 2,2′-Azobis(amidinopropane) dihydrochloride
(AAPH), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), and

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fluorescein (FL) were purchased from Sigma-Aldrich [28, 43, 54]. All the assays were
conducted with 75 mM phosphate buffer (pH =7.4), and the final reaction mixture
was 200 µL. Antioxidant (20 µL) and fluorescein (120 µL, 70 nM final concentration)
were placed in the wells of a black 96-well plate. The mixture was pre-incubated for
1
5 min at 37 ºC, and then AAPH solution (60 µL, 12 mM final concentration) was
added rapidly using an autosampler. The plate was immediately placed in a Varioskan
Flash Multimode Reader (Thermo Scientific) and the fluorescence recorded every
minute for 120 min with excitation at 485 nm and emission at 538 nm. The plate was
automatically shaken prior to each reading. Trolox was used as standard (1–10 µM,
final concentration). A blank (FL + AAPH) using phosphate buffer instead of
antioxidant and trolox calibration were carried out in each assay. The samples were
measured at different concentration (1–10 µM). All the reaction mixture was prepared
in duplicate, and at least three independent assays were performed for each sample.
Raw data were exported from Fluostar Galaxy Software to an Excel sheet for further
calculations. Antioxidant curves (fluorescence vs time) were first normalized to the
curve of the blank corresponding to the same assay, and the area under the
fluorescence decay curve (AUC) was calculated. The net AUC corresponding to a
sample was calculated by subtracting the AUC corresponding to the blank. Regression
equations between net AUC and antioxidant concentration were calculated for all
samples. ORAC-FL values were expressed as trolox equivalents by using the standard
curve calculated for each assay, where the ORAC-FL value of trolox was taken to be
1
.0.
4
.7. ThT Assay.
Inhibition of Aβ1-42 aggregation was measured using a ThioflavinT (ThT)-binding
assay. Aβ1-42 was dissolved in HFIP to a concentration of 1 mg/mL, sonicated in a
water bath for 10 min, aliquoted into microcentrifuge tubes, dried under vacuum, and
stored in -80 ºC [55]. For the experiment of self-mediated Aβ1-42 aggregation
inhibition, the Aβ stock solution was diluted with 50 mM phosphate buffer (pH 7.4) to
5
0 µM before use. A mixture of the peptide (10 µL, 20 µM, final concentration) with

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or without the tested compound (10 µL) was incubated at 37 ºC for 48 h. Blanks using
5
0 mM phosphate buffer (pH 7.4) instead of Aβ with or without inhibitors were also
carried out. The sample was diluted to a final volume of 200 µL with 50 mM
glycine-NaOH buffer (pH 8.0) containing thioflavin T (5 µM). Then the fluorescence
intensities were recorded 5 min later (excitation, 446 nm; emission, 490 nm). The
percent inhibition of aggregation was calculated by the expression (1 - IF /IF ) ×
i
c
1
00%, in which IF and IF are the fluorescence intensities obtained for Aβ in the
i c
presence and absence of inhibitors after subtracting the background, respectively.
For the experiment of copper-mediated Aβ1-42 aggregation inhibition, the Aβ
stock solution was diluted in 20 µM HEPES (pH 6.6) with 150 µM NaCl. The mixture
of the peptide (10 µL, 20 µM, final concentration) with or without copper (10 µL, 20
µM, final concentration) and the tested compound (10 µL, 50 µM, final concentration)
was incubated at 37 ºC for 24 h. Then 20 µL of the sample was diluted to a final
volume of 200 µL with 50 mM glycineNaOH buffer (pH 8.0) containing thioflavin T
(5 µM). The detection method was the same as that of self-mediated Aβ1-42
aggregation experiment.
4
.8. Cell culture and measurement of cell viability
The toxicity effect of the tested compounds on the rat pheochromocytoma (PC12)
cells was examined according to previously reported methods [56]. The PC12 cells
routinely grown at 37 ºC in a humidified incubator with 5% CO in Dulbecco’s
2
modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum, 100
units per mL penicillin, and 100 units per mL of streptomycin. Cells were
sub-cultured in 96-well plates at a seeding density of 10000 cells per well and allowed
to adhere and grow. When cells reached the required confluence, they were placed
into serum-free medium and treated with compounds 5i, 5j, 5k, 7f, 7g. Twenty-four
hours later the survival of cells was determined by MTT assay. Briefly, after
incubation with 20 µL of MTT at 37 ºC for 4 h, living cells containing MTT formazan
crystals were solubilized in 200 µL DMSO. The absorbance of each well was
measured using a microculture plate reader with a test wavelength of 570 nm and a

ACCEPTED MANUSCRIPT
reference wavelength of 630 nm. Results are expressed as the mean ± SD of three
independent experiments.
PC 12 cells was obtained from the Cell Bank of the Chinese Academy of Sciences
(Shanghai, China) and grown in RPMI-1640 medium containing 10% (v/v) foetal
bovine serum, 100 U penicillin/mL and 100 mg streptomycin/mL under 5% CO at 37
2
ºC. The culture media were changed every other day. After pretreatment with
different concentrations of test compound (1.25, 6.25, 12.5, 25 µM) for 2 h, PC 12
cells were incubated with 100 µM of H O for 24 h. The cell viability was evaluated
2
2
using MTT assay. Briefly, the cells were treated with 10 ml MTT (5 mg/ml in PBS)
for 4 h at 37 ºC. Then, 200 µL of dimethylsulfoxide (DMSO) was added to dissolve
the dark blue formazan crystals formed in intact cells, and the absorbance at 570 nm
was detected by a microplate reader. PC12 cells were cultured without test compound
or H O as control group and the results were expressed by percentage of control.
2
2
4
.9. In vitro blood–brain barrier permeation assay
The ability of test compounds that penetrate into brain was evaluated using a
parallel artificial membrane permeation assay (PAMPA) for blood-brain barrier
according to the method established by Di et al [44]. Commercial drugs were
purchased from Sigma and Alfa Aesar. The porcine brain lipid (PBL) was obtained
from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size is 0.45
mm) and the acceptor microplate were both from Millipore. The 96-well UV plate
@
(
COSTAR ) was from Corning Incorporated. The compound was firstly dissolved in
DMSO at a concentration of 5 mg/mL. Then, it was diluted 100-fold with a mixture of
PBS/EtOH (70:30) to give a final concentration of 50 µg/mL. The filter membrane in
donor microplate was coated with PBL dissolved in dodecane (4 µL, 20 mg/mL).
After that, 200 µL of diluted solution and 300 µL of PBS/EtOH (70:30) were added to
the donor wells and the acceptor wells, respectively. The acceptor filter plate was
carefully placed on the donor plate to form a sandwich, which was left undisturbed for
1
6 h at 25 °C. After incubation, the donor plate was carefully removed and the
concentration of compound in the acceptor wells was determined using a UV plate