Discovery of novel rivastigmine-hydroxycinnamic acid hybrids as multi-targeted agents for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.09.052

A series of rivastigmine-caffeic acid and rivastigmine-ferulic acid hybrids were designed, synthesized, and evaluated as multifunctional agents for Alzheimer's disease (AD) in?vitro. The new compounds exerted antioxidant neuroprotective properties and good cholinesterases (ChE) inhibitory activities. Some of them also inhibited amyloid protein (Aβ) aggregation. In particular, compound 5 emerged as promising drug candidates endowed with neuroprotective potential, ChE inhibitory, Aβ self-aggregation inhibitory and copper chelation properties. These data suggest that compound 5 offers an attractive starting point for further lead optimization in the drug-discovery process against AD.

Full text of DOI:10.1016/j.ejmech.2016.09.052

Accepted Manuscript
Discovery of novel rivastigmine-hydroxycinnamic acid hybrids as multi-targeted
agents for Alzheimer's disease
Ziwei Chen, Maria Digiacomo, Yalin Tu, Qiong Gu, Shengnan Wang, Xiaohong Yang,
Jiaqi Chu, Qiuhe Chen, Yifan Han, Jingkao Chen, Giulia Nesi, Simona Sestito, Marco
Macchia, Simona Rapposelli, Rongbiao Pi
PII:
S0223-5234(16)30788-7
10.1016/j.ejmech.2016.09.052
EJMECH 8918
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 July 2016
Revised Date: 15 September 2016
Accepted Date: 17 September 2016
Please cite this article as: Z. Chen, M. Digiacomo, Y. Tu, Q. Gu, S. Wang, X. Yang, J. Chu, Q. Chen,
Y. Han, J. Chen, G. Nesi, S. Sestito, M. Macchia, S. Rapposelli, R. Pi, Discovery of novel rivastigmine-
hydroxycinnamic acid hybrids as multi-targeted agents for Alzheimer's disease, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.052.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of novel rivastigmine-hydroxycinnamic acid hybrids as multi-targeted agents
for Alzheimer's disease‐
Ziwei Chen a, b,c ‡, Maria Digiacomod, ‡, Yalin Tu a, c, ‡, Qiong Gua, Shengnan Wang a, c
,
Xiaohong Yang a, c, Jiaqi Chu a, c, Qiuhe Chena, c, Yifan Han c,e,Jingkao Chen a, c,Giulia Nesid,
Simona Sestitod, Marco Macchiad, Simona Rapposellid*, Rongbiao Pi a,c,f,g*
a School of Pharmaceutical Sciences, SunYat-sen University, Guangzhou 510006, China;
bZhejiang Pharmaceutical College, Ningbo, 31500, China;
c
International Joint Laboratory (SYSU-PolyU HK) of Novel Anti-dementia Drugs of
Guangdong Province, Guangzhou 510006, China;
d Dipartimento di Farmacia, Università di Pisa, Via Bonanno, 6 56126 Pisa, Italy;
e
Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic
University, Hong Kong, China.
f
National and Local United Engineering Lab of Drug ability and New Drugs Evaluation,
Sun Yat-sen University, Guangzhou 510080, China.
g Guangdong Province Key Laboratory of Brain Function and Disease, Zhongshan School of
Medicine, Sun Yat-sen University, Guangzhou 510080, China.
‡ The first three authors contributed equally.
* Corresponding authors: Prof. Rongbiao Pi (pirb@mail.sysu.edu.cn ) and Prof. Simona
Rapposelli (simona.rapposelli@farm.unipi.it ).

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Abstract:
A series of rivastigmine-caffeic acid and rivastigmine-ferulic acid hybrids were designed,
synthesized, and evaluated as multifunctional agents for Alzheimer’s disease (AD) in vitro.
The new compounds exerted antioxidant neuroprotective properties and good cholinesterases
(ChE) inhibitory activities. Some of them also inhibited amyloid protein (Aβ)
aggregation. In particular, compound 5 emerged as promising drug candidates endowed
with neuroprotective potential, ChE inhibitory, Aβ self-aggregation inhibitory and copper
chelation properties. These data suggest that compound 5 offers an attractive starting point
for further lead optimization in the drug-discovery process against AD.
Key words: rivastigmine; caffeic acid; ferulic acid; hybrid; multifunctional agents;
Alzheimer's disease

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Introduction
Alzheimer’s disease (AD) is the main cause of dementia and one of the great healthcare
challenges of the 21st century. In December, 2013, the G8 stated that dementia should be
made a global priority and their ambition that a cure or a disease-modifying therapy should
be available by 2025 [1]. It starts with mild cognitive impairment, which advances to severe
dementia and ultimately death. AD shows highly complex pathological processes that result
in the accumulation of abnormal deposits of β-amyloid peptide (Aβ) and hyper-
phosphorylated tau protein as well as massive cell death and loss of synapses, especially in
the cholinergic system [2].
Given that the pathogenesis of AD is complex and related to the dysfunction of multi-
systems, the prevention and treatment of AD have remained elusive. Currently, the
therapeutic options for the treatment of AD are limited to three acetylcholinesterase (AChE)
inhibitors donepezil, rivastigmine, galantamine and one N-methyl-D-aspartate receptor
antagonist, memantine. Unfortunately, these drugs merely stabilize some symptoms of early
to mid-stage forms of AD for a limited period of time. The complex interconnection of the
molecular events underpinning AD is thought to be one of the explanations why the single-
target directed drugs reached clinical trials failed. Thus, multi-target-directed ligands
(MTDLs) are considered as an effective way for the treatment of neurodegenerative diseases
[3-5].

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One hallmark of AD is the presence of senile plaques in the hippocampus. Aβ
oligomerization and aggregation are thought to play a key role in AD pathogenesis and
progression [6,7]. In addition, increases of damaged cellular macromolecules such as nucleic
acids, proteins, lipids has been observed in AD brains. These findings support reactive
oxygen species (ROS) plays a pivotal role in AD onset and progression. Therefore,
antioxidants should be beneficial therapeutic tools in AD treatment [8]. Small molecules
targeting Aβ, ROS and cholinesterase simultaneously might be more effective for the
treatment of AD. Actually, many multifunctional agents have been synthesized as
therapeutics that interact simultaneously with two or more targets such as AChE, Aβ, tau
protein, monoamine oxidase, metal ions, ROS and many others [9-17]. Although the
advantages of multi-target are clear, the combination of multi-target "functionalities" in one
molecule and the identification of the most appropriate therapeutic targets to select still
remain the critical points.
Previously, some group and we reported that hybrids combining hydroxycinnamic acids
and ChE inhibitory moieties have potential MTDLs’ profile for the therapy of AD [13, 18-
21]. For example, T3CA, a tacrine-caffeic acid hybrid has MTDLs’ profiles of AChE
inhibitor, free radical scavenger, inhibitor of both self- and AChE-induced Aβ aggregation,
as well as potent neuroprotectant against H2O2- and glutamate- induced cell injury with low
toxicity in HT22 cells [19]. Continuing with our interest in the design, synthesis, and

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biological evaluation of multifunctional molecules, our work are currently focused on taking
advantage of the potential anti-ROS profile of caffeic acid- and ferulic acid- based hybrids,
which are endowed with additional anticholinergic properties.
Butyrylcholinesterase (BuChE) plays several roles both in neural and non-neural
functions. Recent observations suggest that rather than selective inhibition of AChE, the
inhibition of BuChE may be more effective, especially in neurodegenerative diseases such as
AD [22, 23]. Several lines of evidence indicate that high cortical levels of BuChE are
associated with some important AD hallmarks, such as the extracellular deposition of the Aβ
and the aggregation of hyper-phosphorylated tau protein. Consistent with this suggestion, the
use of acetyl- and butyryl- cholinesterase inhibitors could improve clinical efficacy with
respect to selective AChE inhibitors. On this basis, our study describes the preparation and
in vitro activities of novel rivastigmine-hydroxycinnamic acid hybrids as inhibitors of AChE
and BuChE, ROS scavengers and inhibitors of Aβ aggregation. In addition, the toxicity as
well as the neuroprotective activity against H2O2- and glutamate-induced cytotoxicity in
HT22 cell lines were also assessed.

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Figure 1 Design of rivastigmine-hydroxycinnamic acid hybrids 1-8.
Chemistry
The small series of N-ethyl-N-methylcarbamoil derivatives 1-6 and the ureido-derivatives
7,8 were synthesized as reported in Scheme 1. The carbamate 12-14, synthesized by the
condensation of N-ethyl-methylcarbamoylchloride with the appropriate 3-substituted-phenol
9-11, was subjected to a catalytic hydrogenation to give the amine derivative 15-17. The
subsequent condensation of caffeic or ferulic acid with the amine 15-17, using
dicyclohexylcarbodiimide (DCC) as condensing reagent, gave the corresponding amide-
product 1-6. The intermediate 19 was obtained from the 3-nitro-aniline, through the
formation of the corresponding isocyanate and subsequent reaction with the N-ethyl
methylamine (Scheme 1B). The catalytic hydrogenation of 19 afforded the aniline derivative

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20, which subjected to the condensation with caffeic acid or ferulic acid in the presence of
DCC, as a condensing reagent, to give the hydroxycinnamamide hybrids 7 and 8,
respectively.
Scheme 1. Synthesis of rivastigmine-hydroxycinnamic acid hybrids 1-8. Reagents and
conditions: (i) N-ethylmethylcarbamoyl chloride,TEA, 95°C, 15h; (ii) EtOH, Pd/C, H2, rt,
12h; (iii) DCC, THF, caffeic acid or ferulic acid, 70°C, reflux, 7h; (iv)triphosgene, N-
ethylmethylamine,TEA, THF,12h.
Results and discussion
AChE and BuChE inhibitory activity. Synthesized compounds were tested for the
inhibitory activity of AChE and BuChE. Donepezil (AChE inhibitor) and rivastigmine
(BuChE inhibitor) were used as positive drugs. The data were summarized in Table 1.

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Interestingly, all the compounds synthesized have a good affinity against both
cholinesterases. Noteworthy, the compounds showed to be more potent than rivastigmine
against AChE. Within the brain, BuChE is primarily expressed and secreted by glial cells
[24]‐and it is thought to play a compensatory role in response to the decrease of AChE
activity in the brain as AD progresses [25]. Indeed, inhibition of central BuChE activity has
also been investigated as a potential therapeutic approach to ameliorate the cholinergic
deficit in moderate forms of AD [26]. As showed in Table 1, most compounds (2, 3, 4, 5,
and 6) exerted stronger inhibition against BuChE than rivastigmine at 1 ꢀM. Rivastigmine-
caffeic acid and rivastigmine-ferulic acid hybrids retained the rivastigmine ChE inhibitory
moiety, and conjunction with caffeic acid or ferulic acid seemly enhanced the cholinesterase
inhibition effect of the “native” drug (i.e. rivastigmine).
Table 1. Inhibitory effects of compounds 1-8 against AChE and BuChE activity.
Compounds Inhibition rate % Inhibition rate %
AChE
BuChE
1 ꢀM 5 ꢀM
1 ꢀM 5 ꢀM
95.19 96.81
6.77 15.56
16.96 16.75
18.56 24.62
21.24 23.3
23.83 34.28
23.42 23.99
24.43 70.66
14.78 44.56
61.04 92.99
49.62 90.96
98.04 98.34
80.85 97.00
87.86 97.86
76.32 91.95
72.30 96.59
Donepezil
Rivastigmine
1
2
3
4
5
6
7
8
18.37 16.40
12.58 14.93
13.81 24.43
19.45 36.60

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DPPH radical scavenging activities. Previous studies showed that both caffeic acid and
ferulic acid exerted radical scavenging properties and their combination with other drugs
retained the native drugs the antioxidant properties [21]. Compounds were tested for their
radical scavenging activities by using 1,1-diphenyl-2- picryl-hydrazyl (DPPH) assay. All
compounds exerted good radical scavenging activity. In particular, compound 3 and 7
showing 69.7 % and 64.7% of radical scavenging activities, respectively (Table 2).
Inhibition of Aβ1-42 self-aggregation. It is well-known that polyphenols, including
caffeic acid, inhibit the Aβ self-aggregation [27]. Recently, many evidence suggested that
the inhibitory effect on Aβ1-42 self-aggregation is strongly related to the non-cholinergic
functions of AchE [28]. Previous studies demonstrated that tacrine-caffeic acid and tacrine-
ferulic acid hybrids inhibit amyloid protein self-aggregation [13, 18, 29]. In order to evaluate
the influence of the tested compounds on amyloid fibrils, a Th-T fluorescence assay was
performed. Compounds were tested at 10 ꢀM and the results are reported in Table 2. Results
showed that compounds 5 and 7 were able to inhibit the Aβ aggregation with % values of
85.3% and 82.5% respectively, which are comparable to that of Congo red, the positive drug
(86.5%). Moreover, the compounds 1 and 3 also showed a significant inhibitory activity.
Overall, the data indicated that the rivastigmine-caffeic acid hybrids (1, 3, 5 and 7) potently
inhibited the Aβ1-42 self-aggregation, while those with ferulic acid seem to induce only a
mild effect.

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Table 2. Inhibitory effects of compounds 1-8 on DPPH radical scavenging activities
and Aβ1-42 self-aggregation.
Compouds
DPPH scavenging
Inhibition of Aβ1-42 self-
(10 ꢀM)
Caffeic acid
Ferulic acid
abilities (%)
64.6
aggregation (%)
NA
NA
73.2
13.2
77.6
3.2
85.3
2.2
82.5
2.2
19.9
37.0
19.5
69.7
17.9
51.7
18.1
64.7
1
2
3
4
5
6
7
8
24.1
Congo red
NA
86.5
NA: Not available
Molecular docking compounds 5 and 7 with Aβ. Aβ consists of a hydrophobic C-terminal
domain (residues 29-42) and an N-terminal domain (amino acids 10-24) which is responsible
for the conversion from a non-toxic α-helical form to a toxic β-sheet structure [30]. It is
well-known that molecules able to hinder this conversion could thus prevent the fibril
formation. In order to explore the binding modes of the active compounds 5 and 7 with Aβ, a
molecular docking experiment was performed. The structure of Aβ used was downloaded
from the Protein Data Bank (PDB: 1BA4) [28]. The binding modes of the active compounds
and Aβ predicted using DOCK6. As shown in Figure 2, compounds 5 and 7 were locked at
the C-terminus hydrophobic area of Aβ. Compound 5 formed five hydrogen bonds with the

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residues of Asp1, Asp 7, Asp23, Gly9, and Lys16. Compound 7 showed similar binding
modes with Aβ. It formed four hydrogen bonds with the residues Asp1, Asp23, Glu3, and
Lys16. Both compounds 5 and 7 formed the hydrophobic interactions with the residues
Phe19, Phe20 and Lys16. Moreover, the phenyl group of 5 formed π-cation interaction with
the residue of Lys16. These hydrogen bonds and hydrophobic interactions might be
favorable for the binding of Aβ and compounds 5 and 7. This might provide the molecular
basis for interpreting the biological data, and some general considerations for future design.
Figure 2 Predicted binding modes of compounds 5 and 7 with Aβ (PDB ID: 1BA4). The
blue lines represent hydrogen bonds, the gray dashed lines represent hydrophobic
interaction, the orange yellow lines stand for the π-cation interaction. Compounds 5 and 7
were locked at the -terminus hydrophobic area of Aβ (PDB: 1BA4). Compound 5 formed
five hydrogen bonds with the residues of Asp1, Asp 7, Asp23, Gly9, and Lys16. Compound
7 showed similar binding modes with Aβ. It formed four hydrogen bonds with the residues
Asp1, Asp23, Glu3, and Lys16. Both compounds 5 and 7 formed the hydrophobic
interactions with the residues Phe19, Phe20 and Lys16. Moreover, the phenyl group of 5

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formed π-cation interaction with the residue of Lys16.
Effects of compounds on the viability of HT22 cells. Compounds 1-8 (10-100 ꢀM) were
submitted to a preliminary evaluation on HT22 cell viability by using MTT reduction in
order to evaluate the cytotoxicity . Cells were exposed to compounds for 24 h and then MTT
assay was carried out. Data showed that no compound was cytotoxic at 10 ꢀM, but all of the
compounds induced cytotoxicity at 100 ꢀM (Figure 3).
Figure 3 The toxicity of novel rivastigmine-hydroxycinnamic acid hybrids in HT22
cells. HT22 cells were exposure to compounds 1-8 at the indicated concentrations (10-100
µM) for 24 h. Viability of HT22 cells were measured by MTT assay. The data were
represented as mean ± SEM, n = 6. **P< 0.01 versus glutamate-treated alone cells, ##P< 0.01
versus control).

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Neuroprotective effects against glutamate- and H2O2-induced cell death. Given that
the compounds showed DPPH radical scavenging activity, the neuroprotective effects
against ROS-induced cell death were investigated in two in vitro models in HT22 cells,
glutamate- and H2O2-induced cell death. Novel compounds were tested at 10 ꢀM and 30 ꢀM.
The results (Figure 4A) showed that compounds 1 and 7 exerted good neuroprotective
effects in glutamate-induced cell death (52.9% and 56.9% cell viability, respectively).
Interestingly, all of compounds (except compound 4) showed a significant protective effect
in HT22 cell injury induced by H2O2 (Figure 4 B). It is well know that phenolic hydroxyl has
strong anti-oxidative effect [31]. This effect may explain, at least in part, the stronger
activity of the rivastigmine-caffeic acid hybrids than rivastigmine-ferulic in DPPH test and
thus the improved capability to prevent the oxidative stress-induced cell death.

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Figure 4 The neuroprotective effects of novel rivastigmine-hydroxycinnamic acid
hybrids against glutamate- and H2O2-induced cell death in HT22 cells. (A)
Neuroprotective effects of compounds on HT22 cells against cell injury induced by
glutamate. HT22 cells were pretreated with/without compounds at the indicated
concentrations for 30 min and then incubated with/without 2 mM glutamate for 24 h. The
data were represented as mean ± SEM, n = 6. **P< 0.01 versus glutamate-treated cells, ##P<
0.01 versus control group). (B) Neuroprotective effects of compounds on HT22 cells against
H2O2 induced cell death. HT22 cells were pretreated with/without compounds at the
indicated concentrations for 30 min and then incubated with/without 500 ꢀM H2O2 for 24 h.
**
The data were represented as mean ± SEM, n = 6. P< 0.01 versus glutamate-treated cells,
##P< 0.01 versus control group).
Copper-Chelating properties of 5. Mounting evidence demonstrated that redox-active
metal ions such as Cu2+ contribute to the production of ROS [32], and elevated
concentrations of Cu2+ has been detected by spectroscopic studies in Aβ plaques [33]. Cu2+
can interact with Aβ peptide thus promoting Aβ-aggregation in in vitro studies [34].
Therefore, modulation of Cu2+ in the brain might be benefit to the treatment of AD. Hence,
the chelating ability of 5 toward Cu2+ was studied by UV–vis spectrometry [35]. As shown
in Figure 5, the maximum absorption suffered a bathochromic shift upon the addition of
Cu2+, suggesting the formation of complex 5–Cu2+. According to previous report, phenolic

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compounds can chelate with Cu2+ to inhibiting Cu2+-induced Aβ aggregation and oxidative
stress [36]. Chelating activity of Cu2+ of 5 might be due to its two phenolic hydroxyl groups .
Figure 5 The copper chelating activity of 5. UV spectra of 5 (10 ꢀM) alone (green line),
treated with CuCl2 (10 ꢀM) (red line), and treated with CuCl2 (20 ꢀM) (blue line). CuCl2
(dissolved in Milli-Q water) and 5 (dissolved in methanol and diluted in PBS), all agents
were dissolved in absolute ethanol, with a final sample volume of 200 ꢀL. The final sample
was allowed to stand at room temperature for 30 min, and then the absorption spectrum was
recorded at room temperature.
Conclusion
Developing a single molecule being able to strongly interact with structurally distinct
targets is not an easy task, even using computer-aided drug design tools [37]. Here we

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demonstrated that rivastigmine-hydroxycinnamic acid hybrids showed an interesting MTDL
profiles. In particular, the most interesting hybrid 5 was demonstrated to inhibit the
hydrolytic activity of ChE, to potentially prevent Aβ self-aggregation, to protect HT22 cells
from glutamate and H2O2 induced cell death, to scavenge free radicals and to chelate copper .
These data support that compound 5 could be an attractive lead compound for
further optimization in the drug-discovery process against AD.
Experimental Section
1
Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. H
NMR and 13C NMR spectra of all compounds were obtained with a Bruker Advance 400
spectrometer at 400 MHz in a ~2% solution of CDCl3, CD3OD-d4, Acetone-d6 and DMSO-d6.
Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane
and referenced from solvent references. Coupling constants are reported in Hz. The
elemental compositions of the compounds agreed to within ± 0.4% of the calculated value.
Chromatographic separation was performed on silica gel columns by flash (Kieselgel 40,
0.040–0.063 mm; Merck). Reactions were followed by thin-layer chromatography (TLC) on
Merck aluminum silica gel (60 F254) sheets that were visualised under a UV lamp.
Evaporation was performed in vacuo (rotating evaporator). Sodium sulphate was always
used as the drying agent. Commercially available chemicals were purchased from Sigma-
Aldrich.

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General procedure for the synthesis of amides (1-8). A solution of the appropriate
amine (0.93 mmol) in THF (5mL) was added to a mixture of the proper carboxylic acid
(0.93 mmol), and DCC (192 mg, 0.93 mmol) in THF (10 mL). The mixture was stirred at
reflux temperature for 7 h. After removal of the solvent, the crude residue was purified by
flash chromatography.
(E)-3-(3-(3,4-dihydroxyphenyl)acrylamido)phenyl-(N-ethyl(methyl))carbamate
(1)
Compound 1 was synthesized from caffeic acid (167 mg, 0.93 mmol) and 3-aminophenyl-N-
ethyl(methyl)carbamate 15 (180 mg, 0.93 mmol). The crude product was purified by flash
chromatography using CHCl3/MeOH (9.5:0.5) as the eluent, and subsequent precipitation
from AcOEt/n-hexane to give compound 1 as a yellow solid (278 mg, 0.78 mmol, 84%
1
yield). mp 193-195 °C; H NMR (Acetone-d6) δ: 1.15 (t, 1.5H, J = 7.0 Hz, CH3 rotamer),
1.24 (t, 1.5H, J = 7.0 Hz, CH3 rotamer), 2.95 (s, 1.5H, CH3 rotamer), 3.08 (s, 1.5H, CH3
rotamer), 3.37 (q, 1H, J = 7.0 Hz, CH2 rotamer), 3.49 (q, 1H, J = 7.0 Hz, CH2 rotamer), 6.59
(d, 1H, J = 15.4 Hz, CH=), 6.81-6.84 (m, 1H, Ar), 6.87 (d, 1H, J = 8.0 Hz, Ar), 6.99 (dd, 1H,
J = 8.0 , 2.1 Hz, Ar), 7.10 (d, 1H, J = 2.1 Hz, Ar), 7.28 (dd, 1H, J = 8.4, 8.0 Hz, Ar), 7.47 (d,
1H, J = 8.4 Hz, Ar), 7.54 (d, 1H, J = 15.4 Hz, CH=), 7.73 (s, 1H, Ar) ppm. 13C NMR
(Acetone-d6) δ: 164.18, 153.83, 152.21, 147.33, 145.39, 141.44, 140.52, 128.97, 127.30,
121.11, 118.61, 116.60, 115.58, 115.51, 114.10, 112.98, 43.68, 33.46 (NCH2 rotamer), 33.30
(NCH2 rotamer), 12.63 (CH3 rotamer), 11.81 (CH3 rotamer) ppm. Anal. (C19H20N2O5) Calc%:

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C 64.04, H 5.66, N 7.86; Found%: C 64.32, H 5.71, N 7.59.
(E)-3-(3-(4-hydroxy-3-methoxyphenyl)acrylamido)phenyl-(N-
ethyl(methyl))carbamate (2). Compound 2 was synthesized from ferulic acid (142 mg,
0.73 mmol) and 3-aminophenyl-N-ethyl(methyl)carbamate 15 (142 mg, 0.73 mmol) as
previously described. The crude product was purified by flash chromatography using
CHCl3/MeOH (9.5:0.5) as the eluent. The subsequent precipitation from AcOEt/n-hexane
gave compound 2 as a yellow solid. Compound 2 (124 mg, 0.36 mmol, 46% yield), mp 133-
135 °C; 1H NMR (Acetone-d6) δ: 1.15 (t, 1.5H, J = 6.8 Hz, CH3 rotamer), 1.24 (t, 1.5H, J =
6.8 Hz, CH3 rotamer), 2.95 (s, 1.5H, CH3 rotamer), 3.08 (s, 1.5H, CH3 rotamer), 3.38 (q, 1H,
J = 6.8 Hz, CH2 rotamer), 3.49 (q, 1H, J = 6.8 Hz, CH2 rotamer), 3.90 (s, 3H, CH3O), 6.66 (d,
1H, J = 15.4 Hz, CH=), 6.81-6.85 (m, 1H, Ar), 6.86 (d, 1H, J = 8.0 Hz, Ar), 7.11 (dd, 1H, J
= 8.0, 2.0 Hz, Ar), 7.21 (d, 1H, J = 2.0 Hz, Ar), 7.28 (dd, 1H, J = 8.4, 8.0 Hz, Ar), 7.48 (d,
1H, J = 8.0 Hz, Ar), 7.60 (d, 1H, J = 15.4 Hz, CH=), 7.74 (s, 1H, Ar) ppm. 13C NMR
(Acetone-d6) δ: 165.00, 154.50, 153.15, 149.56, 148.67, 142.25, 141.46, 129.86, 127.99,
122.96, 119.75, 117.50, 116.45, 116.21, 113.86, 111.47, 56.26, 44.58, 34.57 (CH2 rotamer),
34.20 (CH2 rotamer), 13.56 (CH3 rotamer), 12.80 (CH3 rotamer) ppm. Anal. (C20H22N2O5)
Calc%: C 64.85, H 5.99, N 7.56; Found%: C 64.90, H 5.87, N 7.62.
(E)-3-((3-(3,4-dihydroxyphenyl)acrylamido)methyl)phenyl-(N-
ethyl(methyl))carbamate (3). Compound 3 was synthesized from caffeic acid (86 mg, 0.48

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mmol) and 3-(aminomethyl)phenyl-N- ethyl(methyl)carbamate 16 (100 mg, 0.48 mmol).
The crude product was purified by flash chromatography using CHCl3/MeOH (9.5:0.5) as
eluent affording to compound 3 as a yellow solid (66 mg, 0.18 mmol, 37 % yield); mp 188-
1
190 °C; H NMR(CD3OD) δ: 1.17 (t, 1.5H, J = 7.0 Hz, CH3 rotamer), 1.24 (t, 1.5H, J = 7.0
Hz, CH3 rotamer), 2.96 (s, 1.5H, CH3 rotamer), 3.08 (s, 1.5H, CH3 rotamer), 3.38 (q, 1H, J =
7.0 Hz, CH2 rotamer), 3.49 (q, 1H, J = 7.0 Hz, CH2 rotamer), 4.48 (s. 2H, CH2NH), 6.40 (d,
1H, J = 15.8 Hz, CH=), 6.76 (d, 1H, J = 8.2 Hz, Ar), 6.91 (dd, 1H, J = 2.0, 8.2 Hz, Ar), 6.98-
7.02 (m, 2H, Ar), 7.06 (s, 1H, Ar), 7.18 (d, 1H, J = 7.8 Hz, Ar), 7.34 (dd, 1H, J = 7.8, 8.0 Hz,
Ar), 7.43 (d, 1H, J = 15.8 Hz, CH=) ppm. 13C NMR(CD3OD) δ: 169.18, 156.39, 153.01,
148.86, 146.74, 142.69, 141.80, 130.46, 128.25, 125.68, 122.19, 122.08, 121.72, 118.13,
116.46, 115.07, 45.15, 43.85, 34.53 (CH2 rotamer), 34.27 (CH2 rotamer), 13.40 (CH3
rotamer), 12.61 (CH3 rotamer) ppm. Anal. (C20H22N2O5) Calc%: C 64.85, H 5.99, N 7.56;
Found%: C 64.78, H 5.86, N 7.62.
(E)-3-((3-(4-hydroxy-3-methoxyphenyl)acrylamido)methyl)phenyl-(N-ethyl(methyl))
carbamate (4). Compound 4 was synthesized from ferulic acid (93 mg, 0.48 mmol) and 3-
(aminomethyl)phenyl-N-ethyl(methyl)carbamate 16 (100 mg, 0.48 mmol). The crude
product was purified by flash chromatography using CHCl3/MeOH (9.5:0.5) as the eluent
and subsequent precipitation from CHCl3/n-hexane. Coumpound 4 (yellow solid) (65 mg,
1
0.17 mmol, 35% yield); mp 138-140 °C; H NMR(CDCl3) δ: 1.19–1.26 (m, 3H, CH3), 2.98

ACCEPTED MANUSCRIPT
(s, 1.5H, CH3rotamer), 3.06 (s, 1.5H, CH3 rotamer), 3.37–3.49 (m, 2H, CH2), 3.91 (s, 3H,
OCH3), 4.56 (d, 2H, J = 5.2 Hz, CH2NH), 6.27 (d, 1H, J = 15.6 Hz, CH=), 6.90 (d, 1H, J =
8.2 Hz, Ar), 7.00–7.08 (m, 4H, Ar), 7.15 (d, 1H, J = 7.6 Hz, Ar) 7.32 (dd, 1H, J = 7.6, 8.2
Hz, Ar), 7.58 (d, 1H, J = 15.6 Hz, CH=) ppm. 13C NMR(CD3OD) δ: 169.02, 156.27, 152.97,
149.88, 149.23, 142.49, 141.73, 130.41, 128.17, 125.60, 123.23, 121.99, 121.67, 118.48,
116.45, 111.62, 56.35, 45.09, 43.82, 34.48 (CH2rotamer), 34.22 (CH2rotamer), 13.37
(CH3rotamer), 12.58 (CH3rotamer) ppm. Anal. (C21H24N2O5) Calc%: C 65.61, H 6.29, N
7.29; Found%: C 65.52, H 6.12, N 7.35.
(E)-3-(2-(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenyl-(N-
ethyl(methyl))carbamate (5). Compound 5 was synthesized from caffeic acid (81 mg, 0.45
mmol) and 3-(2-aminoethyl)phenyl-N- ethyl(methyl)carbamate 17 (100 mg, 0.45 mmol).
The crude product was purified by flash chromatography using CHCl3/MeOH (9.5:0.5) as
the eluent and subsequent precipitation from AcOEt/n-hexane to afford compound 5 as a
yellow solid (66 mg, 0.17 mmol, 38 % yield); mp 78-80 °C; 1H NMR (Acetone-d6) δ: 1.13 (t,
1.5H, J =6.7 Hz, CH3 rotamer), 1.21 (t, 1.5H, J = 6.7 Hz, CH3 rotamer), 2.85 (t, 2H, J = 7.2
Hz, ArCH2), 2.94 (s, 1.5H, CH3 rotamer), 3.06 (s, 1.5H, CH3rotamer), 3.36 (q, 1H, J = 6.7
Hz, CH2 rotamer), 3.46 (q, 1H, J = 6.7 Hz, CH2 rotamer), 3.52-3.57 (m, 2H, CH2N), 6.43 (d,
1H, J = 15.8 Hz, CH=), 6.82 (d, 1H, J = 8.4 Hz, Ar), 6.92 (dd, 1H, J = 1.6, 7.9 Hz, Ar),
6.97 (d, 1H, J = 8.4 Hz, Ar), 7.01 (s, 1H, Ar), 7.07-7.08 (m, 2H, Ar), 7.27 (t, 1H, J =7.9 Hz,

ACCEPTED MANUSCRIPT
Ar), 7.42 (d, 1H, J = 15.8 Hz, CH=) ppm.13C NMR(Acetone-d6) δ: 166.80 (CO rotamer),
166.72 (CO rotamer), 152.87, 147.94, 146.29, 141.91, 140.74, 129.82, 128.34, 126.18,
123.05, 121.54, 120.51, 119.56, 116.32, 114.89, 44.55, 41.49 (CH3 rotamer), 41.36 (CH3
rotamer), 36.25, 34.34 (CH2 rotamer), 34.02 (CH2 rotamer), 13.46 (CH3 rotamer), 12.69 (CH3
rotamer) ppm. Anal. (C21H24N2O5) Calc%: C 65.61, H 6.29, N 7.29; Found%: C 65.35, H
6.12, N 7.41.
(E)-3-(2-(3-(4-hydroxy-3-methoxyphenyl)acrylamido)ethyl)phenyl-(N-
ethyl(methyl))carbamate (6). Compound 6 was synthesized from ferulic acid (52 mg, 0.27
mmol) and 3-(2-aminoethyl)phenyl-N-(ethyl(methyl))carbamate 17 (60.00 mg, 0.27 mmol).
The crude product was purified by flash chromatography using CHCl3/MeOH (9.8:0.2) as
1
the eluent. Compoud 6 (yellow solid) (43 mg, 0.11 mmol, 40 % yield); mp 63-65 °C; H
NMR(Acetone-d6) δ:1.13 (t, 1.5H, J =6.7 Hz, CH3 rotamer), 1.21 (t, 1.5H, J = 6.7 Hz, CH3
rotamer), 2.86 (t, 2H, J = 7.2 Hz, ArCH2), 2.94 (s, 1.5 H, CH3 rotamer), 3.06 (s, 1.5 H, CH3
rotamer), 3.36 (q, 1H, J = 6.7 Hz, CH2 rotamer), 3.47 (q, 1H, J = 6.7 Hz, CH2 rotamer),
3.52-3.58 (m, 2H, CH2N), 3.87 (s, 3H, CH3O), 6.49 (d, 1H, J = 15.6 Hz, CH=), 6.83 (d, 1H,
J = 8.1 Hz, Ar), 6.99 (d, 1H, J = 8.2 Hz, Ar), 7.03 (s, 1H, Ar), 7.06 (dd, 1H, J = 1.6, 8.1 Hz,
Ar), 7.08 (d, 1H, J =7.6 Hz, Ar), 7.15 (d, 1H, J = 1.6 Hz, Ar), 7.28 (dd, 1H, J = 7.6, 8.2 Hz,
Ar), 7.44 (d, 1H, CH=, J = 15.6 Hz) ppm.13C NMR(Acetone-d6) δ: 165.47, 152.02, 148.00,
147.70, 141.20, 139.44, 128.89, 127.42, 125.23, 122.13, 121.64, 119.61, 119.18, 115.18,

ACCEPTED MANUSCRIPT
110.38, 55.33, 43.64, 40.47, 35.39, 12.60 ppm. Anal. (C22H26N2O5) Calc%: C 66.32, H 6.58,
N 7.03; Found %: C 66.07, H 6.73, N 6.95.
(2E)-3-(3,4-dihydroxyphenyl)-N-(3-{[ethyl(methyl)carbamoyl]amino}phenyl)prop-2-
enamide (7). Compound 7 was synthesized from caffeic acid (109 mg, 0.53 mmol) and 3-(3-
aminophenyl)-1-ethyl-1-methylurea 20 (111 mg, 0.53 mmol). The crude product was
purified by flash chromatography eluting with CHCl3/MeOH (9.5:0.5). Compound 7 (81 mg,
0.23 mmol, 43 % yield), yellow solid; mp 110-112 °C; 1H NMR(CD3OD) δ: 1.17 (t, 3H, J =
7.2 Hz, CH3), 3.01 (s, 3H, CH3), 3.43 (q, 1H, J = 7.2 Hz, CH2), 6.55 (d, 1H, J = 15.4 Hz,
CH=), 6.78 (d, 1H, J = 8.0 Hz, Ar), 6.95 (dd, 1H, J = 1.6, 8.0 Hz, Ar), 7.05 (d, 1H, J = 1.6
Hz, Ar), 7.11 (d, 1H, J = 8.0 Hz, Ar), 7.22 (t, 1H, J = 8.0 Hz, Ar), 7.34 (d, 1H, J = 8.0 Hz,
Ar), 7.51 (d, 1H, J = 15.4 Hz, CH=), 7.70 (s, 1H, Ar) ppm. 13C NMR (Acetone-d6) δ: 165.03,
156.18, 148.19, 146.30, 141.96, 140.62, 129.31, 128.23, 121.84, 119.79, 116.37, 115.86,
114.98, 114.10, 111.84, 43.94, 34.11, 13.19 ppm. Anal. (C19H21N3O4) Calc%: C 64.21, H
5.96, N 11.82; Found%: C 64.47, H 6.08, N 11.69
(2E)-N-(3-{[ethyl(methyl)carbamoyl]amino}phenyl)-3-(4-hydroxy-3-
methoxyphenyl)prop-2-enamide (8). Compound 8 was synthesized from ferulic acid (92
mg, 0.48 mmol) and from 3-(3-aminophenyl)-1-ethyl-1-methylurea 20 (100 mg, 0.48 mmol).
The crude product was purified by flash chromatography eluting with CHCl3/MeOH
(9.8:0.2). Compound 8 was obtained as pale yellow solid (71 mg, 0.19 mmol, 40 % yield);

ACCEPTED MANUSCRIPT
mp 112-115 °C; 1H NMR(CD3OD) δ: 1.18 (t, 3H, J = 7.2 Hz, CH3), 3.02 (s, 3H, CH3), 3.44
(q, 1H, J = 7.2 Hz, CH2), 3.91 (s, 3H, OCH3), 6.55 (d, 1H, J = 15.6 Hz, CH=), 6.82 (d, 1H, J
= 8.2 Hz, Ar), 7.09 (dd, 1H, J = 1.8, 8.2 Hz, Ar), 7.12 (d, 1H, J = 8.0 Hz, Ar), 7.17 (d, 1H, J
= 1.8 Hz, Ar), 7.22 (t, 1H, J = 8.0 Hz, Ar), 7.35 (d, 1H, J = 8.0 Hz, Ar), 7.57 (d, 1H, J = 15.6
Hz, CH=), 7.70 (s, 1H, Ar) ppm. 13C NMR (Acetone-d6) δ: 164.87, 156.00, 149.44, 148.65,
142.15, 141.75, 140.68, 129.29, 128.09, 122.77, 120.15, 116.20, 115.61, 113.88, 111.48,
56.23, 43.90, 34.08, 13.20 ppm. Anal. (C20H23N3O4) Calc%: C 65.03, H 6.28, N 11.37;
Found%: C 65.32, H 6.44, N 11.53.
General procedure for the synthesis of carbamates (12, 13, 14). To a solution of TEA
(1 mL, 7.13 mmol) were added N-ethyl-methyl carbomoyl chloride (200 mg, 1.65 mmol)
and the opportune phenol 9-11 (1.62 mmol). The mixture was then stirred at 95°C for 15 h,
then the reaction mixture was diluted with CH2Cl2 and washed with NaOH 1N, dried over
Na2SO4, and evaporated.
3-nitrophenyl-N-ethyl(methyl)carbamate (12). It was synthesized from 3-nitrophenol (9)
1
(225 mg, 1.62 mmol) affording 12 as a brown oil (272 mg, 1.21 mmol, 75 % yield); H
NMR (CDCl3) δ: 1.21 (t, 1.5H, J = 7.2 Hz, CH3 rotamer), 1.26 (t, 1.5H, J = 7.2 Hz, CH3
rotamer), 3.01 (s, 1.5H, CH3 rotamer), 3.09 (s, 1.5H, CH3 rotamer), 3.42 (q, 1H, J = 7.2 Hz,
CH2 rotamer), 3.49 (q, 1H, J = 7.2 Hz, CH2 rotamer), 7.47-7.54 (m, 2H, Ar), 8.00-8.02 (m,
1H, Ar), 8.06 (d, 1H, J = 7.2 Hz, Ar) ppm.

ACCEPTED MANUSCRIPT
3-cyanophenyl-N-ethyl(methyl)carbamate (13). It was synthesized from 3-
hydroxybenzonitrile (10) (193 mg, 1.62 mmol) affording 13 as a brown oil (215 mg, 1.05
1
mmol, 65 % yield); H NMR(CDCl3) δ: 1.20 (t, 1.5H, J = 7.2 Hz, CH3 rotamer), 1.25 (t,
1.5H, J = 7.2 Hz, CH3 rotamer), 3.00 (s, 1.5H, CH3 rotamer), 3.07 (s, 1.5H, CH3 rotamer),
3.41 (q, 1H, J = 7.2 Hz, CH2 rotamer), 3.47 (q, 1H, J = 7.2 Hz, CH2 rotamer), 7.37-7.39 (m,
1H, Ar), 7.43-7.49 (m, 3H, Ar) ppm.
3-(cyanomethyl)phenyl-N-ethyl(methyl)carbamate (14). It was synthesized from (3-
hydroxyphenyl)acetonitrile (11) (215 mg, 1.62 mmol) affording 14 as a brown oil (218 mg,
1.00 mmol, 62 % yield); 1H NMR (CDCl3) δ: 1.19 (t, 1.5H, J = 7.2 Hz, CH3 rotamer), 1.25 (t,
1.5H, J = 7.2 Hz, CH3 rotamer), 2.99 (s, 1.5H, CH3 rotamer), 3.07 (s, 1.5H, CH3 rotamer),
3.40 (q, 1H, J = 7.2 Hz, CH2 rotamer), 3.47 (q, 1H, J = 7.2 Hz,CH2 rotamer), 3.75 (s, 2H,
CH2CN), 7.08-7.11 (m, 2H, Ar), 7.16 (d, 1H, J = 7.6 Hz, Ar), 7.36 (t, 1H, J = 7.6 Hz, Ar)
ppm.
General procedure for the synthesis of amine (15, 16, 17). The appropriate nitro-
derivative 12 or cyano-derivative (13, 14) (1.45 mmol) was hydrogenated in EtOH (6 mL) in
the presence of 10% Pd-C (225 mg, 2.12 mmol) for 12 h. Then, the catalyst was filtered off,
and the solution was evaporated to dryness.
3-aminophenyl-N-ethyl(methyl)carbamate (15). It was synthesized from 3-nitrophenyl-
N-ethyl(methyl)carbamate 12 (325 mg, 1.45 mmol) to give 15 as an oil (211 mg, 1.09 mmol,

ACCEPTED MANUSCRIPT
75 % yield); 1H NMR (CDCl3) δ: 1.16–1.23 (m, 3H, CH3), 2.97 (s, 1.5H, CH3 rotamer), 3.04
(s, 1.5H, CH3 rotamer), 3.37–3.46 (m, 2H, CH2), 6.44 (s, 1H, Ar), 6.49 (dd, 2H, J = 2.2, 8.2
Hz, Ar), 7.10 (t, 1H, J = 8.2 Hz, Ar) ppm.
3-(aminomethyl)phenyl-N-ethyl(methyl)carbamate (16). It was synthesized from 3-
cyanophenyl-N-ethyl(methyl)carbamate 13 (296 mg, 1.45 mmol) to give 16 as a brown oil
1
(193 mg, 0.93 mmol, 64 % yield); H NMR (CDCl3) δ: 1.17-1.25 (m, 3H, CH3), 2.98 (s,
1.5H, CH3 rotamer), 3.06 (s, 1.5H, CH3 rotamer), 3.43 (q, 1H, J = 7.2 Hz, CH2 rotamer), 3.47
(q, 1H, J = 7.2 Hz, CH2rotamer), 3.86 (s, 2H, CH2NH2), 6.99 (d, 1H, J = 7.6 Hz, Ar), 7.08 (s,
1H, Ar), 7.13 (d, 1H, J = 7.8 Hz, Ar), 7.31 (dd, 1H, J = 7.6, 7.8 Hz, Ar) ppm.
3-(2-aminoethyl)phenyl-N-ethyl(methyl)carbamate (17). 3-(cyanomethyl)phenyl-N-
ethyl(methyl)carbamate 14 (318 mg, 1.45 mmol) was hydrogenated in EtOH (14 mL) e HCl
conc. (0.15 mL) in the presence of 10% Pd-C (112 mg, 1.06 mmol) for 4 h. Then, the
catalyst was filtered off, and the solution was evaporated to dryness. The residue was diluted
with CHCl3 and washed with NaOH 1N and H2O. The organic layer was dried over Na2SO4,
1
and evaporated, to give 17 as a brown oil (222 mg, 1.00 mmol, 69 % yield); H NMR
(CDCl3) δ: 1.17-1.25 (m, 3H, CH3), 2.73-2.77 (m, 2H, CH2), 2.93-2.99 (m, 2H, CH2), 2.98 (s,
1.5H, CH3 rotamer), 3.06 (s, 1.5H, CH3 rotamer), 3.38-3.49 (m, 2H, CH2), 6.91-6.99 (m, 2H,
Ar), 7.02 (d, 1H, J= 7.6 Hz, Ar), 7.26-7.30 (m, 1H, Ar) ppm.
1-ethyl-1-methyl-3-(3-nitrophenyl)urea (19). The 3-nitroaniline 18 (1.00 g, 7.24 mmol)

ACCEPTED MANUSCRIPT
dissolved in THF (10 ml) was dropped slowly to a stirred solution of triphosgene (2.15 g,
7.24 mmol) in THF (5 mL), then TEA (1.46 g, 15.20 mmol) was added slowly to the
reaction mixture. The solution was stirred for 1h and then evaporated. THF (10 mL) and N-
ethylmethylamine (10 mmol) were added directly to the residue. The reaction mixture was
stirred at rt for 12h, then the solvent was evaporated and the crude mixture was diluted with
AcOEt and washed with H2O. The organic layer was dried over Na2SO4 and evaporated. The
crude product was purified by precipitation from AcOEt/n-hexane to obtain 19 as a yellow
1
solid (1.32 g, 5.94 mmol, 68 % yield); H NMR (CDCl3) δ: 1.22 (t, 3H, J = 7.2 Hz, CH3),
3.04 (s, 3H, CH3), 3.44 (q, 1H, J = 7.2 Hz, CH2), 6.62 (br s, 1H, NH), 7.42 (dd, 1H, J= 8.4,
8.0 Hz, Ar), 7.83-7.86 (m, 2H, Ar), 8.22 (s, 1H, Ar) ppm.
3-(3-aminophenyl)-1-ethyl-1-methylurea (20). It was synthesized from 1-ethyl-1-
methyl-3-(3-nitrophenyl)urea 19 (323 mg, 1.45 mmol). Hydrogenation of 19 in EtOH (6 mL)
in the presence of 10% Pd-C for 12 h afforded a crude mixture which was filtered off. The
solution was evaporated to dryness to give 20 (212 mg, 1.10 mmol, 76 % yield) as a yellow
1
solid; H NMR(CDCl3) δ: 1.17-1.25 (m, 3H, CH3), 2.98 (s, 1.5H, CH3 rotamer), 3.06 (s,
1.5H, CH3 rotamer), 3.43 (q, 1H, J = 7.2 Hz, CH2 rotamer), 3.47 (q, 1H, J = 7.2 Hz, CH2
rotamer), 3.86 (s, 2H, CH2NH2), 6.99 (d, 1H, J = 7.6 Hz, Ar), 7.08 (s, 1H, Ar), 7.13 (d, 1H, J
= 7.8 Hz, Ar), 7.31 (dd, 1H, J = 7.6, 7.8 Hz, Ar) ppm.
Biological assay.

ACCEPTED MANUSCRIPT
Materials. Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovineserum (FBS)
were purchased from Gibico-BRL (Grand Island, NY, USA). Trypsin, Aβ1-42, dimethyl
sulfoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), DPPH, AChE, BuChE,
Donepezil, Rivatigamine and all other chemicals were purchased from Sigma-Aldrich (St.
Louis, MO, USA) unless stated otherwise. Test compounds were dissolved in DMSO and
stored at -20 ℃.
Cell culture and treatment. HT22 cells were maintained in DMEM supplemented with
10% (v/v) FBS and incubated at 37 °C under 5% CO2. To study the protective effect of test
compounds on glutamate or H2O2-induced neuronal death and their cell toxicity, cells were
seeded in 96-well plates (10,000 cells/well). Cells in the control group were treated with
vehicle alone.
ChE inhibitory assay. The method of Ellman et al [38] was followed. AChE stock
solution was prepared by dissolving 1000 units of lyophilized powder (Sigma Chemical) in
0.1 M phosphate buffer (pH 8.0) containing Triton X-100, 0.1%. BuChE stock solution was
prepared by dissolving 100 units of lyophilized powder (Sigma Chemical) in aqueous gelatin
solution (0.1%w/v). Enzymes stock solutions were diluted before use to reach an activity
ranging between 0.13 and 0.100 AU/min in the final assay conditions. Five different
concentrations of each compound were used to obtain inhibition of AChE or BuChE activity
between 20% and 80%. The assay solution consisted of 118 ꢀL 0.1 M phosphate buffer (pH

ACCEPTED MANUSCRIPT
8.0), with the addition of 40ꢀM DTNB (Ellman’s reagent), 2 ꢀl AChE or BuChE, 20 ꢀL
compound and 20 ꢀM acetylthiocholine iodide (ATi). The final assay volume was 200 ꢀL.
Initial rate assays were performed at 37 °C with a Jasco V-530 double beam
spectrophotometer: the rate of increase in the absorbance at 412 nm was followed for 5 min.
Test compounds were added to the assay solution and preincubated with the enzyme for 60
min, according to the kinetic type, followed by the addition of substrate. Assays were done
with a blank containing all components except AChE or BuChE to account for nonenzy-
matic reaction. The reaction rates were compared, and the percent inhibition due to the
presence of the test compound was calculated. Each concentration was analyzed in triplicate,
and IC50 values were determined graphically from the logconcentration-inhibition curves.
DPPH assay. Compounds were added into a 1 mM ethanol solution of DPPH (final
concentration was 100 ꢀM) to make final concentrations of 10 ꢀM. The reaction tubes were
wrapped in aluminum foil and kept at 25-26 °C for 60 min in dark. All measurements were
done under dim light. Spectrophotometric measurements were operated at 517 nm by
ultraviolet spectrophotometer. The capacity to scavenge free radicals was measured as
follow: capacity = (A1 – A2) / A1 *100%, where A1 and A2 are the absorbance of the
control treatment and the twelve compounds or caffeic acid in 517 nm, respectively.
Thioflavin T (Th-T) based fluorometric assay. The Th-T fluorescence method was
performed to determine that compounds can restrain amyloid fibril formation. Prepare Aβ1-42