Design, synthesis and evaluation of novel ferulic acid derivatives as multi-target-directed ligands for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2019.103413

A series of new ferulic acid derivatives were designed, synthesized and evaluated as multi-target inhibitors against Alzheimer's disease. In vitro studies indicated that most compounds showed significant potency to inhibit self-induced β-amyloid (Aβ) aggregation and acetylcholinesterase (AChE), and had good antioxidant activity. Specifically, compound 4g exhibited the potent ability to inhibit cholinesterase (ChE) (IC₅₀, 19.7 nM for hAChE and 0.66 μM for hBuChE) and the good Aβ aggregation inhibition (49.2% at 20 μM), and it was also a good antioxidant (1.26 trolox equivalents). Kinetic and molecular modeling studies showed that compound 4g was a mixed-type inhibitor, which could interact simultaneously with the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. Moreover, compound 4g could remarkably increase PC12 cells viability in hydrogen peroxide–induced oxidative cell damage and Aβ-induced cell damage. Finally, compound 4g had good ability to cross the BBB using the PAMPA–BBB assay. These results suggested that compound 4g was a promising multifunctional ChE inhibitor for the further investigation.

Full text of DOI:10.1016/j.bioorg.2019.103413

Journal Pre-proofs
Design, synthesis and evaluation of novel ferulic acid derivatives as multi-tar-
get-directed ligands for the treatment of Alzheimer’s disease
Jin-Shuai Lan, Rui-Feng Zeng, Xiao-Yi Jiang, Jian-wei Hou, Yun Liu, Zheng-
Hao Hu, Hong-Xin Li, Yin Li, Sai-Sai Xie, Yue Ding, Tong Zhang
PII:
S0045-2068(19)31262-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.103413
YBIOO 103413
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
2 August 2019
23 October 2019
28 October 2019
Please cite this article as: J-S. Lan, R-F. Zeng, X-Y. Jiang, J-w. Hou, Y. Liu, Z-H. Hu, H-X. Li, Y. Li, S-S. Xie,
Y. Ding, T. Zhang, Design, synthesis and evaluation of novel ferulic acid derivatives as multi-target-directed
ligands for the treatment of Alzheimer’s disease, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103413
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Design, synthesis and evaluation of novel ferulic acid derivatives as multi-target-
directed ligands for the treatment of Alzheimer’s disease
Jin-Shuai Lan1,2#, Rui-Feng Zeng2#, Xiao-Yi Jiang2, Jian-wei Hou1, Yun Liu1, Zheng-
Hao Hu2, Hong-Xin Li2, Yin Li4, Sai-Sai Xie3*, Yue Ding1,2*, Tong Zhang1,2*
1
Experiment Center of Teaching & Learning, Shanghai University of Traditional
Chinese Medicine, Shanghai 201203, China;
2 School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai
201203, China;
3 National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal
Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006,
China;
4 Hebei Radio & TV University, Shijiazhuang, Hebei 050080, China.
* Corresponding author: Sai-Sai Xie; Yue Ding; Tong Zhang
* E-mail: xiesaisainanchang@hotmail.com(S-S X); dingyue-2001@hotmail.com (YD);
zhangtongshutcm@hotmail.com (TZ)
# These authors contributed equally to this work.
1

Abstract
A series of new ferulic acid derivatives were designed, synthesized and evaluated as
multi-target inhibitors against Alzheimer’s disease. In vitro studies indicated that most
compounds showed significant potency to inhibit self-induced β-amyloid (Aβ)
aggregation and acetylcholinesterase (AChE), and had good antioxidant activity.
Specifically, compound 4g exhibited the potent ability to inhibit cholinesterase (ChE)
(IC50, 19.7 nM for hAChE and 0.66 μM for hBuChE) and the good Aβ aggregation
inhibition (49.2% at 20 μM), and it was also a good antioxidant (1.26 trolox
equivalents). Kinetic and molecular modeling studies showed that compound 4g was a
mixed-type inhibitor, which could interact simultaneously with the catalytic anionic site
(CAS) and the peripheral anionic site (PAS) of AChE. Moreover, compound 4g could
remarkably increase PC12 cells viability in hydrogen peroxide–induced oxidative cell
damage and Aβ-induced cell damage. Finally, compound 4g had good ability to cross
the BBB using the PAMPA−BBB assay. These results suggested that compound 4g
was a promising multifunctional ChE inhibitor for the further investigation.
Keywords
Ferulic acid deriveatives; Alzheimer’s disease; cholinesterase; Aβ (1-42) aggregation;
multi-target-directed ligands; molecular docking.
Abbreviations
AD, Alzheimer’s disease; NMDA, N-methyl-Daspartate; AChEs/BChEs,
acetylcholinesterase/butyrylcholinesterase; Ach/Buch, acetylcholine/butyrylcholine;
MTDLs, multi-target-directed ligands; FA, Ferulic acid; BBB, blood-brain barrier;
CAS, catalytic active site; PAS, peripheral anionic site; MTT, Methyl thiazolyl
tetrazolium; ADME, absorption, distribution, metabolism and excretion; tPSA,
topological polar surface area; CNS, central nervous system; PAMPA, parallel artificial
membrane permeation assay.
1. Introduction
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Alzheimer’s disease (AD), a neurodegenerative, extensive and chronic disease, is
clinically described as behavioral disturbances, continuous deterioration of
remembrance, studying and cognitive functions [1,2]. To date, there were appraised 46
million people breathing with presenile dementia over the world. Unfortunately, the
sum was predicted to approach 131.5 million till 2050 [3]. AD also induced a great
amount of economic loss, which was evaluated to cost US $818 billion on senile
psychosis in 2018 and the number will rise to 2 trillion dollar before 2030 [4]. Though
the scientific characters of AD have been identified for more than 100 years, the therapy
of AD is not known owning to its complicated pathogenesis [5]. Significant evidences
have revealed that AD is a multifactorial syndrome originated from a composite array
of neurochemical ingredient, involving the dearth of synaptic acetylcholine,
dyshomeostasis of biometals, the deposition of neurotoxic β-amyloid (Aβ) peptide,
oxidative stress, the infection of neurons and so on [6,7]. Up to date, the present clinical
treatment for presenile dementia is relieving the symptomatic aspects. There were four
kinds of drugs approved by the FDA including one N-methyl-Daspartate (NMDA)
receptor antagonist, memantine and three acetylcholinesterase inhibitors (AChEs)
including rivastigmine, donepezil and galantamine [8]. But sadly, these medicines
could only promote memory and cognitive function to a positive level, and they could
not prohibit, halt or reverse the developing of AD. Contemporary studies improved that
the ratio of butyrylcholinesterase (BuChE)/AChE gradually rose from 0.2 to 11.0 in
certain parts of the brain while AD developing. Besides, AChE and BuChE perform
similar roles in cholinergic signaling, and BuChE could hydrolyze acetylcholine (ACh)
and reimburse for AChE when the level of ACh are depleted [9,10]. Therefore, the drug
with both AChE and BuChE inhibitions could better ensure the therapy.
What’s more, the production and aggregation of Aβ, chiefly for the subtype of Aβ
(1-40) and Aβ (1-42), could endlessly cause neurodegeneration and lead AD patients to
neuronal dysfunction at last. So the inhibition of the production and accumulation of
Aβ in brain was recognized as a possible target for AD therapy [11,12]. Besides, the
progressive aggregation of Aβ is usually attached to oxidative stress, which attends to
3

be a considerable determinant in AD pathogenesis and progression. And the oxidative
damage marked by nitration, reactive carbonyls, lipid peroxidation and nucleic acid
oxidation is raised in frail neurons of AD [13,14]. So the protection of neuronal cells
from oxidative damage could probably limit the process of AD.
Considering the complex pathogenesis and progression of AD, the therapy targets on
a single path might be insufficient. Thus, based on the multi-target-directed ligands
(MTDLs) paradigm, the design of multi-target compounds against AD was proposed as
a more efficient therapeutic strategy [15-21].
Ferulic acid (4-hydroxy-3-methoxy cinnamic acid, FA) is firstly extracted form
Ferula foetida, and widely distribute in kinds of plants. FA is a nature antioxidant and
anti-inflammatory compound, and could also act as disaggregating agents of Aβ. Due
to the affirmative efficacy and low side-effects of FA, it was recognized as a potential
resistant or remedial drug for AD treatment [22]. Nonetheless, FA as an anti-AD drug
used in clinic was limited owning to its low bioavailability and poor blood–brain
transport. Thus, designing novel FA derivatives based on MTDLs is meaningful for
anti-AD [23-28]. As is well-known, AP2238 (2), whose pharmacophore was proved to
be the benzylamino group, was a highly selective AChE inhibitor for AD treatment, but
it displayed poor inhibition on BuChE with IC50 value of 48.9 μM [29]. Furthermore,
Rivastigmine (3), marketed by Novartis as Exelon, is a carbamate pseudo-irreversible
AChE inhibitor, which exhibits high selectivity in the hippocampus and cortex of brain,
and interferes both AChE and BuChE [30]. However, Rivastigmine can only benefit to
simple improvement in cognition, cannot reverse the progression of AD. Considering
the mentioned reasons, we planned to combine the FA, the benzylamino group and the
carbamyl group with different lengths as a multi-target molecule (Fig. 1). The FA
derivatives may simultaneously have the antioxidant activity, ChE inhibitory activity
and neuroprotective properties.
Continuing our works on treatment of AD [31-34], a novel series of FA derivatives
were constructed based on MTDLs (Fig. 1). They were synthesized and investigated as
the multifunctional compounds against AD, containing the inhibitory effects on AChE
4

and BuChE, protection of antioxidant damage, inhibition of the Aβ aggregation, the
capability of crossing blood-brain barrier (BBB) and prevention from H2O2-induced
and Aβ-induced PC12 cell injury in vitro. Finally, the structure-activity relationships of
the novel FA derivatives were summarized and molecular modeling studies were also
executed to achieve the binding mode.
2. Results and Discussion
2.1 Chemistry
The synthesis of target compounds 4 were shown in scheme 1 with good yields (50-
70%). Firstly, the FA derivatives 1 were purchased and pretreated in acetone solutions
with Et3N at 50 °C, and then treated with 1,2-dibromoethane, 1,3-dibromopropane, 1,4-
dibromobutane, 1,5-dibromopentane or 1,8-dibromooctane to form the necessary
intermediates 2 [35]. Next, compounds 2 were mixed with secondary amines 3 and
anhydrous K2CO3 in dry acetonitrile at 80 °C to obtain the compounds 4. At last, the
target products were clarified by chromatography, and the structures were confirmed
by analytical and spectroscopic data.
2.2 Effect of ChE inhibition activity.
To evaluate the multi-potential function of the new FA derivatives (compounds 4a–
o), we firstly measured their inhibition on both eqBuChE (equine serum) and eeAChE
(electric eel) based on the Ellman’s method [36]. FA and Donepezil were applied as
standard compounds for comparison. The selectivity index and comparable IC50 values
of ChEs were exhibited in Table 1. The target compounds exhibited wide inhibitory
activities toward eeAChE with IC50 ranging from 0.055 µM to 42.26 µM, and moderate
inhibitory activities towards eqBuChE. These results implied that these FA derivatives
performed as dual inhibitors of both eeAChE and eqBuChE, and every target
compounds was more efficient than FA on ChEs inhibition. Among the synthesized FA
derivatives, compound 4h (IC50 = 0.055 μM) displayed the best inhibitory potency for
eeAChE, while compound 4b (IC50 = 0.84 μM) exhibited the most potent inhibitory
5

activity for eqBuChE.
First of all, to select the suitable linker lengths between FA and amide groups for
ChEs inhibition, compounds 4a–e with various alkyl chains were investigated. The
inhibitory activities for eeAChE (in Table 1) changed obviously when the length of the
alkyl chains of compounds 4a–e was altered from 2 to 8. And compound 4d with five
carbon spacers connecting FA with the benzylamino group, had a better eeAChE
inhibitory activity than the other compounds. Interestingly, similar regularity was also
suitable to the inhibition of eqBuChE. The results above indicated that five carbon
atoms between the two anchoring group was the suitable linker length for the inhibition
of ChEs [37]. Next, to further promote the ChEs inhibition of the FA derivatives, we
investigated different substituents with various electronic properties and sizes (H,
OCH3, OH and NO2) on the benzene ring of FA. The results of compounds 4d and 4f-
h on eeAChE inhibition suggested that electron-withdraw substituents could promote
the inhibitory activities against eeAChE, and the presence of OCH3 also retained the
AChE inhibition. For instance, compound 4h with NO2 substituent (IC50 = 0.055 μM,
for eeAChE) was more effective than compound 4d (IC50 = 0.18 µM, for eeAChE).
However, eqBuChE inhibition was slightly lower by the substituents on FA. Moreover,
inhibitory activities against ChEs were also influenced by the different substituents on
the benzylamino group. Firstly, to better explain the importance of carbamate
pharmacophore in ChEs inhibition, compounds 4m-o without carbamate were further
synthesized. Compared with compounds 4a-h, the compounds 4m-o (without
carbamate pharmacophore) reduced ChEs inhibitory activity. For example, compound
4f (IC50 = 0.086 μM, toward eeAChE) was more effective than compound 4n (IC50 =
31.67 μM, for eeAChE), which suggested the carbamate was certainly essential to ChEs
inhibition. Lastly, the substituted groups (R2) on the carbamate also influenced the
ChEs inhibition. Compounds 4d and 4f-h possessing N-methyl groups showed higher
eeAChE inhibition than compounds 4i-k with N-ethyl groups, which implied that small
substituents were in favor of eeAChE inhibition. However, the terminal amino groups
of the carbamate moiety little affected the eqBuChE inhibitory activities, which could
6

be attributed to the conformational variation between these two different enzymes in
the preceding studies [38].
Furthermore, compounds 4f-h were selected for evaluation on hChE. As listed in
Table 2, all tested compounds showed a high selectivity for hAChE over hBuChE and
presented IC50 values in the nanomolar range for hAChE, which were slightly more
potent inhibitors for hAChE than for eeAChE. The SARs for hAChE were similar to
those drawn for eeAChE inhibition (Table 1). Compound 4h (IC50 = 13.7 nM for
hAChE) displayed the highest inhibition, which was 2.2-fold higher than that of
standard donepezil (IC50 = 30.2 nM). However, the hBuChE inhibition of the tested
derivatives was relatively low (in the micromolar range), and compound 4g (IC50 =
0.0.59 μM) exhibited the most potent inhibitory activity for hBuChE.
2.3 Effect on self-induced Aβ (1-42) aggregation
After all FA derivates were evaluated for ChEs inhibition, they were tested for their
Aβ (1-42) aggregation inhibition using a thioflavin T-based fluorometric method [39].
In the test, FA and Curcumin were used as positive references. As the results shown
(Table. 3), compounds 4a-o exhibited middle to good efficiency (37.8-51.4%, 20 μM)
compared with curcumin (58.4%, 20 μM) and FA (36.7%, 20 μM). It was notable that
compound 4j (51.4%, 20 μM) showed the best inhibition. From the inhibitive activities
of compounds 4a-e, it indicated that the linker length played a minor role in the Aβ (1-
42) aggregation inhibition.
Moreover, compared with compounds 4m-n, compounds 4f-l introducing the
carbamate pharmacophore slightly increased Aβ (1-42) inhibitory activity. And
compounds 4f-g and 4j-k with the methoxy or hydroxyl substituents on the cinnamic
acid were excellent Aβ aggregation inhibitors, ranging from 49.2 to 51.4% at 20 μM.
For instance, compound 4g (49.2% at 20 μM) was more effective than that of compound
4d (40.9% at 20 μM). This proved that the methoxy and hydroxyl groups might favor
Aβ aggregation inhibition.
7

2.4 Antioxidant activity of compounds
During the treatment of AD, the protection of nerve cells from oxidative stress is
necessary. With this understanding, the antioxidant abilities of the FA derivatives were
estimated by the ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical
scavenging analysis, and trolox (a vitamin E analog) was applied as the reference [40].
The scavenge radicals abilities of FA derivatives were valued by the trolox equivalent
(at 25 μM). The results (in Table 3) indicated the ordinary anti-oxidative abilities of FA
derivatives ranging from 0.38-1.26 trolox equivalents. Among these FA derivatives,
compound 4g exhibited the most efficient antioxidant ability with ABTS standards of
1.26 trolox equivalents, which was similar with FA (1.12 trolox equivalents). This
finding could be attributed to the hydroxy group of cinnamic acid part. Based on its
powerful inhibition of ChEs and Aβ (1-42) aggregation, compound 4g with good
antioxidant ability was selected as the most talented compound to continue the further
study.
2.5 Kinetic study for ChEs inhibition.
Based on the excellent multi-target activities of compound 4g, such as the potent
ChE inhibition (IC50, 19.7 nM for hAChE and 0.66 μM for hBuChE), the good Aβ
aggregation inhibition (49.2% at 20 μM), and the good antioxidant (1.26 trolox
equivalents), we further investigated the dual-site mechanism of compound 4g. The
Lineweaver-Burk graphics were applied to measure the type of AChE inhibition of
compound 4g [41]. The results in Fig. 2A suggested that the slopes and intercepts were
both increased with the accumulating concentration of compound 4g. The pattern
implied compound 4g was a mixed-type inhibitor for AChE and could simultaneously
react to the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE.
By comparison, a diverse plot for BuChE was picked up, exhibiting increasing slopes
and constant intercepts in diverse inhibitor concentrations (Fig. 2B). The results implied
that these compounds competed for the same binding site as the substrate acetylcholine.
8

2.6 Molecular modeling research of ChEs inhibition.
A molecular docking between compound 4g and ChEs was studied by the software
package MOE 2008.10.43. The X-ray crystal structure of the hAChE complex with
donepezil (PDB code 1EVE) was used to contribute the origin model of hAChE. As the
results in Fig. 3A and 3C, the benzylamino part of compound 4g was bound to the CAS
of hAChE, through aromatic π-π stacking interactions with the phenyl ring from Trp 84
with the ring-to-ring distance of 2.66 Å. What’s more, the charged nitrogen was also
bound to the CAS via a cation–π interaction with Tyr 334 and Phe 330. Moreover, the
FA fragment inhabited the PAS formed by Asp 285, Phe 284 and Leu 282, and a
hydrogen bond formed between the hydroxy of the ligands and Ser 286 with the
distance of 3.19 Å. These results above indicated that compound 4g could concurrently
bind to AChE via PAS and CAS.
To explain the bind model of compound 4g/hBuChE, the crystal structure of hBuChe
(PDB code 1P0I) was applied. As the result in Fig. 4B and 4D, the benzylamino part of
compound 4g stacked up against the Tyr 332 by a cation–π interaction with the distance
of 3.04 Å. Meanwhile, the phenyl ring of FA interactions with His 438 by a cation–π
interaction.
2.7 Docking studies of inhibition of Aβ (1-42)
According to the good inhibition of self-induced Aβ (1-42) aggregation, we further
explored the interaction mode between compound 4g and Aβ (1-42). Molecular docking
research was performed using the X-ray crystal structure of the protein Aβ structure
(PDB 1IYT) [41]. As suggested in Fig. 4, the benzene ring of FA combined with the
His 6 by a π-π stacking interaction with the distance of 2.63 Å. This result implied that
the π-π stacking interaction of the 4g/Aβ (1-42) complex was important in the inhibition
of Aβ (1-42).
2.8 Cell viability and neuroprotection activity assay
Based on the good antioxidant activity of FA derivatives, the neuroprotection activity
9

of the most efficient compound 4g was investigated in vitro. Firstly, neuroblastoma cell
line PC12 was exposed to compound 4g ranging from 6-100 μM. As the result in Fig.
5, compound 4g showed low cytotoxic activity at 6-100 μM by methyl thiazolyl
tetrazolium (MTT) assay.
According to the results above, the neuroprotective effects of the novel FA
derivatives against H2O2-induced oxidative stress and Aβ (1-42)-induced cell damage
were furthered explored [42]. Hydrogen peroxide (H2O2) was applied to cause oxidative
damage in PC12 cells. As the results shown in Fig. 6A, compound 4g obviously
increased the cell viability on H2O2-induced oxidative cell damage in PC12 cells.
Compound 4g exhibited a powerful protection at the dose of 5 μM or 10 μM, especially
with high cell viability of 70.17 % at 10 μM. These results indicated that the FA
derivatives had excellent antioxidant abilities in the AD treatment. And we have also
evaluated the neuroprotective effects of compound 4g against Aβ-induced neuronal
death of PC12 cells, the data were recorded after the cells were exposed to increasing
concentrations of compound 4g (6-50 µM) for 24h. As can be seen in Fig. 6B, treatment
of cells with Aβ (1–42) (25 µM) markedly reduced cell viability to 47.9% compared
with the untreated cells (control). Compound 4g exhibited neuroprotective effects at
concentrations ranging from 6 to 50 μM (6 μM: 48.8 ± 3.2%; 12.5 μM: 55.4 ± 2.1%;
25 μM: 58.9 ± 2.9%; 50 μM: 64.7 ± 3.8%). These observations further showed that
novel cinnamic acid derivatives can inhibit Aβ (1-42) self-aggregation for the treatment
of AD.
2.9 ADMET prediction and in vitro blood–brain barrier permeation assay.
Not only the good pharmacological activity is necessary, but also the
pharmacokinetic property is a key to develop a drug. Recently, vital improvement in
combinatorial chemistry does favor to the assessment of novel compounds’ absorption,
distribution, metabolism and excretion (ADME) [43]. Thus, ADME properties of the
new FA derivatives 4a-o were calculated using online Molinspiration property program
[44]. The rule requests that an orally active drug can’t be against one violation.
10

The theoretical calculations of ADME parameters (molecular weight, topological
polar surface area (tPSA), log P, number of hydrogen acceptors, number of hydrogen
donors, number of rotatable bonds and volume) were shown in Table 4, including the
violations of Lipinski’s rule. All the compounds 4a-o conformed to Lipinski’s rule
which was none violation and might penetrate into the brain. This result suggests that
target compounds may have good pharmacokinetics properties.
To develop central nervous system (CNS) drugs, the first requirement is crossing the
BBB. Therefore, to determine the BBB penetration of our present compounds, a parallel
artificial membrane permeation assay (PAMPA) was used [45]. After comparing the
experimental permeability with the reported values of 9 commercial drugs (Table 5), a
plot of experiment data versus the bibliographic values gave a good linear correlation:
Pe (exp.) = 1.0436Pe (bibl.) – 0.2325 (R2 = 0.9531). Based on this equation and
considering the limit established by Di et al. for BBB permeation, we determined that
compounds with permeabilities above 3.94*10- 6 cm s-1 could across the BBB:
(a) ‘ CNS + ’ (high BBB permeation predicted): Pe (10- 6 cm s-1) > 3.94.
(b) ‘ CNS - ’ (low BBB permeation predicted): Pe (10- 6 cm s-1) < 1.85.
(c) ‘ CNS +/- ’ (BBB permeation uncertain): Pe (10- 6 cm s-1) from 3.94 to 1.85.
Finally, compounds 4f and 4g with potent inhibitory activities against ChEs and Aβ
(1-42) aggregation were selected to evaluate the BBB penetration. It can be seen that
compounds 4f and 4g showed the Pe value higher than 3.94*10- 6 cm s-1 (Table 6),
which suggested that they were able to cross the BBB and might reach the biological
targets located in the CNS.
3. Conclusions
Finally, we designed and synthesized a series of novel FA derivatives 4a-4o. These
compounds were proved as anti-AD agents with multi-target activities, according to
their good antioxidant activity and excellent inhibitory activities on ChEs and Aβ (1-
42) aggregation. Based on the screened results, compound 4g displayed a dual
inhibitory potency on both AChE and BuChE. Furthermore, the kinetic characterization
11

indicated that compound 4g performed as a duel-site inhibitor binding to the CAS and
the PAS of AChE, which matched to the results of the molecular modelling. Compound
4g also exhibited the ability of inhibition of self-induced Aβ (1-42) aggregation and
good antioxidant activity (1.26 trolox equivalents). What’s more, the compound 4g had
the neuroprotection activity against H2O2-induced oxidative stress and Aβ (1-42)-
induced cell damage and showed high brain penetration capacity. Generally, compound
4g was a multifunctional cholinesterase inhibitor and applied as a potential clinical
theraphy medicine for AD.
4. Experimental section
4.1 Chemistry
All chemicals (reagent grade) applied during the whole experiment were obtained
from Sino pharm Chemical Reagent Co., Ltd. (China). XT-4 micromelting point
instrument and uncorrected was used to measure the melting point of novel compounds.
1H NMR and 13C NMR spectra were tested on a BRUKER AVANCE III spectrometer
at 25°C and TMS was used as the reference. Chemical shifts were recorded in ppm (δ)
using the residual solvent line as internal standard. Mass spectra were recorded on a
MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS).
4.2 General procedure for the synthesis of compounds
4.2.1. General procedure for the synthesis of intermediates 2a–h
Compounds 2a–h were easily prepared as described in the literature with some
modification [33]. Compound 1 (1 mmol) and corresponding dibromo-alkanes (2 mmol)
were added to acetone (30 ml), and the mixture was refluxed in the presence of Et3N
for 5 h. When the reaction was completed as shown by TLC, the mixture was cooled to
room temperature and evaporated under vacuum. The crude product was purified using
a silica gel column to get compound 2 with high yields.
4.2.1.1 2-Bromoethyl cinnamate (2a)
12

Cinnamic acid was reacted with 1,2-dibromoethane following the general procedure
to give compound 2a as a yellow oil (75% yield); ESI/MS m/z: 255.1 [M+H]+.
4.2.1.2 3-Bromopropyl cinnamate (2b)
Cinnamic acid was reacted with 1,3-dibromopropane following the general
procedure to give compound 2b as a yellow oil (82% yield); ESI/MS m/z: 269.2
[M+H]+.
4.2.1.3 4-Bromobutyl cinnamate (2c)
Cinnamic acid was reacted with 1,4-dibromobutane following the general procedure
to give compound 2c as a yellow oil (79% yield); ESI/MS m/z: 283.0 [M+H]+.
4.2.1.4 5-Bromopentyl cinnamate (2d)
Cinnamic acid was reacted with 1,5-dibromopentane following the general procedure
to give compound 2d as a yellow oil (80% yield); ESI/MS m/z: 297.1 [M+H]+.
4.2.1.5 8-Bromooctyl cinnamate (2e)
Cinnamic acid was reacted with 1,8-dibromooctane following the general procedure
to give compound 2e as a yellow oil (70% yield); ESI/MS m/z: 339.1 [M+H]+.
4.2.1.6 (E)-5-bromopentyl 3-(3,4-dimethoxyphenyl)acrylate (2f)
(E)-3-(3,4-dimethoxyphenyl)acrylic acid was reacted with 1,5-dibromopentane
following the general procedure to give compound 2f as a yellow oil (82% yield);
ESI/MS m/z: 357.1 [M+H]+.
4.2.1.7 (E)-5-bromopentyl 3-(4-hydroxy-3-methoxyphenyl)acrylate (2g)
(E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid was reacted with 1,5-
dibromopentane following the general procedure to give compound 2g as a yellow oil
(70% yield); ESI/MS m/z: 343.0 [M+H]+.
4.2.1.8 (E)-5-bromopentyl 3-(4-nitrophenyl)acrylate (2h)
(E)-3-(4-nitrophenyl)acrylic acid was reacted with 1,5-dibromopentane following
the general procedure to give compound 2h as a yellow oil (82% yield); ESI/MS m/z:
342.1 [M+H]+.
4.2.2. General procedure for the synthesis of intermediates 4a–o
13

Compound 2 (3 mmol) and K2CO3 (12 mmol) in CH3CN (15 ml) was stirred, then
the solution of Compound 3 (3.3 mmol) in CH3CN (5 ml) was added to the reaction
mixture. After the mixture was refluxed for 12-18 h, the reaction mixture was
evaporated under vacuum and extracted with ethyl acetate. The combined organic layer
was washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel chromatography using
CH2Cl2/MeOH as eluent to obtain target compounds 4.
4.2.2.1 2-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)ethyl cinnamate (4a)
Compound 2a was reacted with compound 3a following the general procedure to
give compound 4a as a yellow oil (45% yield); 1H NMR (400 MHz, CDCl3) δ 7.68 (d,
J = 16.0 Hz, 1H), 7.49 (dd, J = 6.6, 2.9 Hz, 2H), 7.39 (dd, J = 6.4, 3.7 Hz, 3H), 7.33 –
7.29 (m, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.12 (s, 1H), 7.01 (dd, J = 8.0, 1.6 Hz, 1H), 6.43
(d, J = 16.0 Hz, 1H), 4.28 (t, J = 6.5 Hz, 2H), 3.52 (s, 2H), 3.11 (s, 3H), 3.03 (s, 3H),
2.52 (t, J = 7.1 Hz, 2H), 2.21 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 167.03, 154.97,
151.58, 144.60, 140.58, 134.50, 130.21, 128.95, 128.88, 128.07, 125.70, 122.18,
120.35, 118.28, 62.96, 61.99, 53.75, 42.17, 36.57, 36.48; ESI-MS: 385.2 [M+H]+.
4.2.2.2 3-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)propyl cinnamate (4b)
Compound 2b was reacted with compound 3a following the general procedure to
give compound 4b as a yellow oil (52% yield); 1H NMR (400 MHz, CDCl3) δ 7.67 (d,
J = 16.0 Hz, 1H), 7.54 (dd, J = 6.6, 2.9 Hz, 2H), 7.40 (dd, J = 6.4, 3.7 Hz, 3H), 7.31 –
7.28 (m, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.13 (s, 1H), 7.01 (dd, J = 8.0, 1.6 Hz, 1H), 6.43
(d, J = 16.0 Hz, 1H), 4.30 (t, J = 6.5 Hz, 2H), 3.54 (s, 2H), 3.10 (s, 3H), 3.02 (s, 3H),
2.54 (t, J = 7.1 Hz, 2H), 2.25 (s, 3H), 1.98 – 1.90 (m, 2H); 13C NMR (100 MHz, CDCl3)
δ 167.02, 154.97, 151.58, 144.59, 140.55, 134.50, 130.21, 128.96, 128.86, 128.08,
125.69, 122.19, 120.37, 118.26, 62.86, 61.98, 53.78, 42.07, 36.68, 36.43, 26.66. ESI-
MS: 397.2 [M+H]+.
4.2.2.3 4-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)butyl cinnamate (4c)
Compound 2c was reacted with compound 3a following the general procedure to
give compound 4c as a yellow oil (50% yield); 1H NMR (400 MHz, CDCl3) δ 7.70 (d,
14

J = 16.0 Hz, 1H), 7.54 (dd, J = 6.6, 2.8 Hz, 2H), 7.42 – 7.38 (m, 3H), 7.34 – 7.30 (m,
1H), 7.16 (d, J = 7.6 Hz, 1H), 7.11 (s, 1H), 7.02 (dd, J = 8.0, 1.5 Hz, 1H), 6.46 (d, J =
16.0 Hz, 1H), 4.24 (t, J = 6.5 Hz, 2H), 3.52 (s, 2H), 3.11 (s, 3H), 3.02 (s, 3H), 2.49 –
2.42 (m, 2H), 2.22 (s, 3H), 1.76 (dd, J = 14.7, 6.4 Hz, 2H), 1.66 (dd, J = 14.8, 8.0 Hz,
2H); 13C NMR (100 MHz, CDCl3) δ 167.05, 154.97, 151.58, 144.62, 140.66, 134.49,
130.21, 128.93, 128.87, 128.06, 125.71, 122.20, 120.33, 118.28, 64.53, 61.92, 56.99,
42.07, 36.67, 36.44, 26.62, 23.86; ESI-MS: 409.3 [M+H]+.
4.2.2.4 5-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)pentyl cinnamate (4d)
Compound 2d was reacted with compound 3a following the general procedure to
give compound 4d as a yellow oil (45% yield);1H NMR (400 MHz, CDCl3) δ 7.69 (d,
J = 16.0 Hz, 1H), 7.55 – 7.52 (m, 2H), 7.41 – 7.37 (m, 3H), 7.30 – 7.27 (m, 1H), 7.17
(s, 1H), 7.13 – 7.11 (m, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.45 (d, J = 16.0 Hz, 1H), 4.21
(t, J = 6.6 Hz, 2H), 3.52 (s, 2H), 3.10 (s, 3H), 3.09 (s, 3H), 2.46 – 2.40 (m, 2H), 2.22
(s, 3H), 1.77 – 1.68 (m, 2H), 1.60 (dt, J = 15.0, 7.4 Hz, 2H), 1.49 – 1.39 (m, 2H); 13C
NMR (100 MHz, CDCl3) δ 167.02, 154.97, 151.58, 144.59, 140.55, 134.50, 130.21,
128.96, 128.86, 128.08, 125.69, 122.19, 120.37, 118.26, 62.86, 61.98, 53.78, 42.07,
36.68, 36.43, 26.66; ESI-MS: 421.3 [M+H]+.
4.2.2.5 8-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)octyl cinnamate (4e)
Compound 2e was reacted with compound 3a following the general procedure to
give compound 4e as a yellow oil (42% yield);1H NMR (400 MHz, CDCl3) δ 7.70 (d,
J = 16.0 Hz, 1H), 7.54 (dd, J = 6.6, 2.8 Hz, 2H), 7.41 – 7.36 (m, 3H), 7.29 (t, J = 3.9
Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.11 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.46 (d, J =
16.0 Hz, 1H), 4.21 (t, J = 6.7 Hz, 2H), 3.52 (s, 2H), 3.11 (d, J = 4.8 Hz, 3H), 3.01 (s,
3H), 2.46 – 2.33 (m, 2H), 2.21 (s, 3H), 1.75 – 1.67 (m, 2H), 1.54 (s, 2H), 1.41 – 1.31
(m, 8H); 13C NMR (100 MHz, CDCl3) δ 167.02, 154.97, 151.58, 144.59, 140.55, 134.50,
130.21, 128.96, 128.86, 128.08, 125.69, 122.19, 120.37, 118.26, 62.86, 61.98, 53.78,
42.07, 36.68, 36.43, 26.66. ESI-MS: 457.2 [M+H]+.
4.2.2.6 (E)-5-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)pentyl-3-(3,4-
dimethoxyphenyl)acrylate (4f)
15

Compound 2f was reacted with compound 3a following the general procedure to give
compound 4f as a yellow oil (52% yield); 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J =
15.9 Hz, 1H), 7.33–7.28 (m, 1H), 7.19 – 7.10 (m, 3H), 7.08 (d, J = 1.9 Hz, 1H), 7.02
(dd, J = 8.0, 1.6 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.34 (d, J = 15.9 Hz, 1H), 4.21 (t, J
= 6.6 Hz, 2H), 3.93 (d, J = 4.6 Hz, 6H), 3.52 (s, 2H), 3.11 (s, 3H), 3.02 (s, 3H), 2.46–
2.39 (m, 2H), 2.22 (s, 3H), 1.79–1.68 (m, 2H), 1.60 (dt, J = 14.9, 7.4 Hz, 2H), 1.51–
1.41 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 167.35, 154.99, 151.54, 151.08, 149.20,
144.53, 140.77, 128.93, 127.46, 125.74, 122.62, 122.22, 120.30, 115.96, 111.03,
109.59, 64.53, 62.00, 57.37, 55.98, 55.89, 42.22, 36.69, 36.44, 28.71, 27.06, 23.86;
ESI-MS: 485.2 [M+H]+.
4.2.2.7
(E)-5-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)pentyl-3-(4-
hydroxy-3-methoxyphenyl)acrylate (4g)
Compound 2g was reacted with compound 3a following the general procedure to
give compound 4g as a yellow oil (43% yield); 1H NMR (400 MHz, CDCl3) δ 7.73 (d,
J = 15.9 Hz, 1H), 7.39 – 7.30 (m, 1H), 7.25 – 7.15 (m, 3H), 7.10 (d, J = 1.9 Hz, 1H),
7.05 (dd, J = 8.0, 1.6 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.35 (d, J = 15.9 Hz, 1H), 4.22
(t, J = 6.6 Hz, 2H), 3.90 (d, J = 4.6 Hz, 6H), 3.52 (s, 2H), 3.12 (s, 3H), 2.46 – 2.39 (m,
2H), 2.24 (s, 3H), 1.79 – 1.65 (m, 2H), 1.63 (dt, J = 14.9, 7.4 Hz, 2H), 1.51 – 1.45 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 166.49, 154.75, 152.08, 151.35, 149.26, 144.55,
140.71, 128.72, 127.43, 124.94, 122.53, 122.13, 120.51, 116.32, 111.72, 108.43, 64.65,
62.06, 57.39, 55.38, 41.53, 36.50, 36.16, 28.70, 27.08, 23.46; ESI-MS: 471.3 [M+H]+.
4.2.2.8 (E)-5-((3-((dimethylcarbamoyl)oxy)benzyl)(methyl)amino)pentyl-3-(4-
nitrophenyl)acrylate (4h)
Compound 2h was reacted with compound 3a following the general procedure to
give compound 4h as a yellow oil (58% yield); 1H NMR (400 MHz, CDCl3) δ 8.27 (d,
J = 8.6 Hz, 2H), 7.71 (t, J = 11.9 Hz, 3H), 7.33 (d, J = 7.8 Hz, 1H), 7.19 (d, J = 7.7 Hz,
1H), 7.13 (s, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.58 (d, J = 16.1 Hz, 1H), 4.25 (t, J = 6.6
Hz, 2H), 3.60 (s, 2H), 3.11 (s, 3H), 3.02 (s, 3H), 2.54 – 2.45 (m, 2H), 2.26 (d, J = 10.4
Hz, 3H), 1.74 (dd, J = 14.5, 7.0 Hz, 2H), 1.69 – 1.59 (m, 2H), 1.47 (d, J = 7.1 Hz, 2H);
16

13C NMR (100 MHz, CDCl3) δ 166.12, 154.96, 151.55, 148.46, 141.62, 140.73, 140.59,
128.93, 128.65, 125.69, 124.15, 122.59, 122.20, 120.31, 65.09, 61.97, 57.27, 42.23,
36.68, 36.44, 28.57, 27.01, 23.78; ESI-MS: 470.2.
4.2.2.9 5-((3-((ethyl(methyl)carbamoyl)oxy)benzyl)(methyl)amino)pentyl cinnamate
(4i)
Compound 2d was reacted with compound 3b following the general procedure to
give compound 4i as a yellow oil (50% yield); 1H NMR (400 MHz, CDCl3) δ 7.70 (d,
J = 16.0 Hz, 1H), 7.55 (dd, J = 6.4, 2.8 Hz, 2H), 7.43 – 7.37 (m, 3H), 7.32 (d, J = 7.7
Hz, 1H), 7.17 (dd, J = 21.2, 13.1 Hz, 2H), 7.05 (s, 1H), 6.46 (dd, J = 16.0, 3.3 Hz, 1H),
4.23 (dd, J = 11.6, 5.0 Hz, 2H), 3.56 (s, 2H), 3.53 – 3.37 (m, 4H), 3.08 (s, 2H), 2.99 (d,
J = 8.6 Hz, 2H), 2.51 – 2.42 (m, 2H), 2.25 (s, 3H), 1.73 (dd, J = 14.7, 7.0 Hz, 2H), 1.67
– 1.58 (m, 2H), 1.46 (dd, J = 15.2, 8.1 Hz, 2H), 1.22 (d, J = 13.8 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 167.11, 151.57, 144.64, 134.47, 130.23, 129.37, 129.00, 128.88,
128.08, 125.83, 123.54, 122.37, 120.89, 120.52, 120.24, 118.26, 64.84, 64.60, 61.81,
57.19, 50.85, 44.07, 42.03, 34.24, 33.80, 29.71, 28.65, 26.83, 23.83, 13.23, 12.47; ESI-
MS: 439.3.
4.2.2.10 (E)-5-((3-((ethyl(methyl)carbamoyl)oxy)benzyl)(methyl)amino)pentyl-3-(3,4
-dimethoxyphenyl)acrylate (4j)
Compound 2f was reacted with compound 3b following the general procedure to
give compound 4j as a yellow oil (54% yield); 1H NMR (400 MHz, CDCl3) δ 7.62 (d,
J = 15.9 Hz, 1H), 7.28 (dd, J = 11.0, 4.6 Hz, 1H), 7.16 – 7.02 (m, 4H), 6.99 (d, J = 7.8
Hz, 1H), 6.85 (dd, J = 8.3, 2.8 Hz, 1H), 6.31 (d, J = 15.9 Hz, 1H), 4.19 (t, J = 6.6 Hz,
2H), 3.89 (d, J = 3.3 Hz, 7H), 3.51 – 3.34 (m, 4H), 3.01 (d, J = 30.9 Hz, 3H), 2.19 (s,
3H), 1.75 – 1.66 (m, 2H), 1.57 (dt, J = 14.9, 7.4 Hz, 2H), 1.44 (dd, J = 15.2, 8.2 Hz,
13
2H), 1.26 – 1.13 (m, 4H); C NMR (100 MHz, CDCl3) δ 167.32, 151.55, 151.06,
149.19, 144.52, 128.92, 127.44, 125.69, 122.61, 122.23, 120.34, 115.94, 111.02,
109.58, 64.50, 61.95, 57.32, 55.96, 55.88, 44.05, 42.19, 28.71, 27.01, 23.86; ESI-MS:
499.3 [M+H]+.
4.2.2.11 (E)-5-((3-((ethyl(methyl)carbamoyl)oxy)benzyl)(methyl)amino)pentyl-3-
17

(4-hydroxy-3-methoxyphenyl)acrylate (4k)
Compound 2g was reacted with compound 3b following the general procedure to
give compound 4k as a yellow oil (44% yield); 1H NMR (400 MHz, CDCl3) δ 7.63 (dd,
J = 15.9, 3.2 Hz, 1H), 7.38 – 7.30 (m, 1H), 7.16 (d, J = 7.0 Hz, 1H), 7.07 (m, 4H), 6.90
(dd, J = 20.9, 8.2 Hz, 1H), 6.31 (dt, J = 15.9, 3.4 Hz, 1H), 4.21 (t, J = 6.6 Hz, 2H), 4.13
– 3.78 (m, 5H), 3.62 – 3.35 (m, 5H), 3.08 (d, J = 6.2 Hz, 2H), 3.00 (s, 3H), 2.54 – 2.34
(m, 2H), 2.23 (s, 3H), 1.78 – 1.67 (m, 2H), 1.62 (dd, J = 15.2, 6.9 Hz, 2H), 1.46 (dd, J
= 15.2, 8.2 Hz, 2H), 1.25 – 1.16 (m, 3H). 13C NMR (100 MHz, CDCl3) δ 167.39, 151.56,
147.99, 146.83, 144.72, 128.98, 125.81, 123.07, 122.57, 122.36, 120.48, 115.59,
114.75, 112.41, 110.11, 109.35, 68.72, 64.46, 61.84, 57.25, 55.94, 50.86, 44.07, 42.07,
29.71, 28.70, 26.87, 23.86; ESI-MS: 485.3 [M+H]+.
4.2.2.12 (E)-5-((3-((ethyl(methyl)carbamoyl)oxy)benzyl)(methyl)amino)pentyl 3-(4-
nitrophenyl)acrylate (4l)
Compound 2h was reacted with compound 3b following the general procedure to
give compound 4l as a yellow oil (40% yield); 1H NMR (400 MHz, ) δ 8.24 (d, J = 7.9
Hz), 7.68 (dd, J = 16.9, 12.3 Hz), 7.28 (d, J = 8.0 Hz), 7.11 (dd, J = 30.4, 6.4 Hz), 7.00
(s), 6.56 (d, J = 16.0 Hz), 4.22 (t, J = 6.5 Hz), 3.48 (s), 3.05 (s), 2.98 (s), 2.39 (t, J = 7.1
Hz), 2.19 (s), 1.71 (dd, J = 14.4, 6.9 Hz), 1.57 (dt, J = 14.9, 7.4 Hz), 1.48 – 1.38 (m),
1.28 – 1.21 (m); 13C NMR (100 MHz, CDCl3) δ 166.15, 151.55, 148.47, 141.65, 140.60,
128.96, 128.67, 125.73, 124.18, 122.59, 122.27, 120.37, 65.11, 61.99, 57.29, 44.07,
42.24, 34.25, 33.82, 28.58, 27.00, 23.80; ESI-MS: 484.3 [M+H]+.
4.2.2.13 5-(benzyl(methyl)amino)pentyl cinnamate (4m)
Compound 2d was reacted with compound 3c following the general procedure to
give compound 4k as a yellow oil (65% yield); 1H NMR (400 MHz, CDCl3) δ 7.68 (d,
J = 16.0 Hz, 1H), 7.53 (dd, J = 6.5, 2.9 Hz, 2H), 7.43 – 7.36 (m, 3H), 7.33 (d, J = 6.2
Hz, 3H), 7.26 (s, 2H), 6.44 (d, J = 16.0 Hz, 1H), 4.20 (t, J = 6.6 Hz, 2H), 3.55 (s, 2H),
2.55 – 2.31 (m, 2H), 2.24 (s, 3H), 1.76 – 1.66 (m, 2H), 1.61 (dd, J = 14.9, 7.6 Hz, 2H),
1.49 – 1.39 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 167.09, 144.66, 134.47, 130.25,
18

129.27, 128.89, 128.34, 128.08, 127.25, 118.24, 64.54, 57.03, 41.96, 28.63, 26.71,
23.81; ESI-MS: 338.2 [M+H]+.
4.2.2.14 (E)-5-(benzyl(methyl)amino)pentyl 3-(3,4-dimethoxyphenyl)acrylate (4n)
Compound 2f was reacted with compound 3c following the general procedure to give
compound 4k as a yellow oil (68% yield); 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J =
15.9 Hz, 1H), 7.36 – 7.27 (m, 4H), 7.26 – 7.22 (m, 1H), 7.10 (dd, J = 8.3, 1.8 Hz, 1H),
7.06 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.31 (d, J = 15.9 Hz, 1H), 4.20 (t, J
= 6.6 Hz, 2H), 3.91 (d, J = 1.5 Hz, 6H), 3.51 (s, 2H), 2.46 – 2.34 (m, 2H), 2.21 (s, 3H),
1.70 (dd, J = 14.7, 7.0 Hz, 2H), 1.59 (dd, J = 15.0, 7.6 Hz, 2H), 1.48 – 1.40 (m, 2H);
13C NMR (101 MHz, CDCl3) δ 167.33, 151.09, 149.22, 144.55, 129.14, 128.26, 127.46,
127.05, 122.62, 115.94, 111.03, 109.58, 64.48, 62.25, 57.18, 55.99, 55.90, 42.13, 28.70,
26.94, 23.86; ESI-MS: 398.2 [M+H]+.
4.2.2.15 (E)-5-(benzyl(methyl)amino)pentyl 3-(4-nitrophenyl)acrylate (4o)
Compound 2h was reacted with compound 3c following the general procedure to
give compound 4k as a yellow oil (69% yield); 1H NMR (400 MHz, CDCl3) δ 8.24 (d,
J = 8.8 Hz, 2H), 7.68 (dd, J = 14.7, 12.4 Hz, 3H), 7.31 (d, J = 4.4 Hz, 4H), 7.23 (dd, J
= 5.3, 3.4 Hz, 1H), 6.56 (d, J = 16.0 Hz, 1H), 4.23 (t, J = 6.6 Hz, 2H), 3.48 (s, 2H), 2.44
– 2.34 (m, 2H), 2.18 (d, J = 9.0 Hz, 3H), 1.76 – 1.68 (m, 2H), 1.58 (dt, J = 14.7, 7.2 Hz,
13
2H), 1.48 – 1.40 (m, 2H); C NMR (101 MHz, CDCl3) δ 166.11, 148.49, 141.64,
140.59, 139.17, 129.04, 128.64, 128.21, 126.93, 124.18, 122.59, 65.11, 62.36, 57.21,
42.26, 28.59, 27.06, 23.80; ESI-MS: 383.2 [M+H]+.
4.3 Inhibition studies on ChEs in vitro
Acetylcholinesterase (AChE, E.C 3.1.1.7, from electric eel), Butyrylcholinesterase
(BuChE, E.C 3.1.1.8, from equine serum), 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB,
Ellman’s reagent), donepezil hydrochloride, acetylthiocholine iodide (ATCI) and S-
butyrylthiocholine iodide (BTCI) were obtained from Sigma-Aldrich. Ellman’s method
was applied to evaluate the inhibition activities of target compounds 4a-o towards
AChE and BuChE [34]. The tested compounds were dissolved in DMSO to 1% and
19

.
diluted by the buffer solution (0.1 M NaCl, 50 mM Tris-HCl, 0.02 M MgCl2 6H2O pH=
8.0). For ChEs inhibition test, 160 μL DTNB (1.5 mM) was added to a 96-well plate,
and mixed with 50 μL of AChE (0.22 U/mL prepared in 50 mM Tris-HCl, pH =8.0, 0.1%
w/v bovine serum albumin, BSA) or 50 μL of BuChE (0.12 U/mL prepared in 50 mM
Tris-HCl, pH=8.0, 0.1% w/v BSA). Then, the respective enzyme were treated with 10
μL various concentrations of test compounds (0.001-100 μM) at 37 °C for 6 min,
followed by the addition of substrate (30 μL), S-butyrylthiocholine iodide (15 mM)
or acetylthiocholine iodide (15 mM), and the absorbance at 405 nm was measured after
0, 1, 2, and 3 min. The concentration of FA derivatives producing 50% of enzyme
activity inhibition (IC50) was processed by the Graph-Pad Prism program package
(Graph Pad Software), according to nonlinear regression analysis of the response-
concentration (log) curve. The experiments were performed in triplicate and the results
were exhibited as the mean ± SEM.
4.4 Kinetic analysis of AChE and BuChE inhibition
To investigate the inhibition mechanism of present compounds, compound 4g was
taken for kinetic analysis by Ellman’s method [39]. Three concentrations of 4g were
selected for the study: 55, 110 and 220 nM of compound 4g were chosen for the kinetic
analysis of AChE inhibition and 1.25, 2.5 and 5 μM of compound 4g were selected for
the kinetic study of BuChE inhibition. Following a similar method mentioned above in
enzyme inhibition assay, Lineweaver–Burk reciprocal plots were constructed by
plotting 1/velocity against 1/[substrate] at varying concentrations of the substrate
acetylthiocholine (0.05 – 0.5 mM). All measurements were performed in triplicate and
data analysis was performed with Graph Pad Prism 4.03 software (San Diego, CA,
USA).
4.5 Inhibition of Aβ (1-42) self-induced aggregation
The inhibitory activities of compounds on self-induced Aβ (1-42) aggregation were
also measured by the ThT assay [37]. HFIP and ThT were purchased from TCI
20

(Shanghai) Development. Aβ (1-42) was obtained from Royobiotech Co., Ltd
(Shanghai, China). The monomeric Aβ (1-42) samples were prepared by HFIP and
diluted with a 50 mM phosphate buffer (pH 7.4) to give a 25 μM solution. The test
compound was dissolved in DMSO (250 μM) for storage and needed not to be diluted
prior to use. In each well, 1 μL of test compound at final concentration of 25 μM and 9
μL of Aβ (1-42) sample were added, and the obtained mixture was incubated in dark at
room temperature for 46-48 h with no agitation. Then, 200 μL of 5 μM ThT in 50 mM
glycine-NaOH buffer (pH 8.0) was added. Fluorescence intensity at 490 nm (emission
at 446 nm) was measured through a SpectraMax M5 (Molecular Devices, Sunnyvale,
CA, USA) with multi-mode plate reader. The fluorescence intensities of the mixture at
490 nm were collected and the percent inhibition caused by the presence of the inhibitor
was calculated based on the following formula:
Percent inhibition = 100 - (IFi / IFo × 100)
IFi and IFo were the fluorescence intensities of Aβ (1-42) incubated with inhibitor or
buffer respectively.
4.6 Study of antioxidant capability in vitro
2,2’-Azino-bis-2-ethybenz-thiazoline-6-sulfonic acid (ABTS) radical catching
method was applied to assess the antioxidant capability of the FA derivatives [38].
Firstly, ABTS (7 mM, in purified water) was treated with potassium persulfate (2.45
mM, in purified water) to form ATBS radical cation (ABTS.+), and the ABTS mixture
was placed at room temperature in dark for 24 h before use. The stock solution of ABTS
was then diluted to 100 μM in sodium phosphate buffer (50 mM, pH 7.4). Different
concentrations of trolox and compounds 4a-o (50 μL) were added to 150 μL prepared
ABTS solution (100 μM), respectively. After the complete mixing of reactants, the
absorbance of at 415 nm of the solution was measured in 6 min at 30 °C. Every
individual experiment was performed for three times.
4.7 Molecular modeling research
21

Molecular Operating Environment (MOE) 2015.10 (Montreal, Canada) was applied
to finish the molecular modeling calculations and docking research. AChE, BuChE and
Aβ (1-42) were modeled by respectively locating them in the active site according to
the published crystallographic structures (PDB code: 1EVE, code 1P0I, PDB 1IYT.)
Then, we deleted all water molecules in PDB files and added hydrogen atoms to the
protein. The compound 4g was constructed by the builder interface of the MOE and
energy minimized through MMFF94x force field. Subsequently, the compound 4g was
docked into the active site of the protein using the “Triangle Matcher” method, which
induced poses by adjusting the ligand triplet of atoms with the triplet of alpha spheres
in cavities of sealed atomic packing. The Dock scoring in MOE software was completed
by ASE scoring utility and Force field was chosed as the improved method. 10 top-
ranked conformations of compounds were recorded and stored. Finally, the geometry
of docked complex was analized by the pose viewer utility in MOE.
4.8. Blood–brain barrier permeation study in vitro
The parallel artificial membrane permeation assay (PAMPA) was a common way to
evaluate the brain penetration of molecules [43].Before the experiments, all compounds
were prepared in DMSO, and the stock solutions were diluted in PBS/EtOH (70: 30) to
make secondary stock solutions (25 μg/mL). After the pretreatment, the filter
membrane on the 96-well filtration plate (PVDF membrane, pore size 0.45 mm,
Millipore) was coated with 4 μL of PBL (Avanti Polar Lipids) in dodecane (20 mg/mL,
Sigma-Aldrich). Then, 300 μL of PBS/EtOH (70: 30) and 200μL of diluted solution
containing the corresponding drugs or test compounds were added to corresponding
acceptor well and donor well, respectively. Afterwards, the acceptor filter plate was
carefully placed on the donor plate to make the coated membrane touch both donor
solution and acceptor buffer. After incubation for 18 h at 25 °C, the concentrations of
test compounds in reference, acceptor and donor wells were determined by a UV plate
reader (SpectraMax Plus 384, Molecular Devices). All samples were analyzed in four
wells, for five wavelengths, and by three independent runs. In every experiment,
22

various quality control standards of known BBB permeability were applied to approve
the analysis set.
4.9. Cell culture and investigation of cell viability
The rat pheochromocytoma (PC12) cell was applied to test the toxicity effect of the
FA derivatives. PC 12 cells was purchased from the Cell Bank of the Chinese Academy
of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% bovine calf serum, 100 U/mL penicillin-
streptomycin and incubated at 37 °C in a humidified incubator with 5% CO2 in. Cells
were seeded in 96-well plates at 5×103 cells/well and treated for 24h. Then, the cells
were placed into serum-free medium and incubated with compound 4g. The survival of
cells was determined in 24 h by MTT assay and the absorbance at 570 nm was measured
by a microculture plate reader. Results were expressed as the percentage of the control
and repeated for three times.
The PC12 cells were selected to evaluate the protective capabilities of compounds
against the neurotoxicity induced by hydrogen peroxide (H2O2) [40]. The PC12 cells
were sub-cultured in 96-well plates at 2×104 cells/well, treated for 12 h and
subsequently incubated in serum-free DMEM containing 250 μM H2O2 and different
concentrations (1, 2.5, 5, 10 μM) of compound 4g. Cell viability was tested by MTT
assay after the incubation with 250 μM H2O2 for 24 h.
The PC12 cells were also selected to evaluate the protective capabilities of
compounds against the neurotoxicity induced by Aβ (1-42). After pretreatment with
different concentrations of compound 4g (0, 6, 12.5, 25, 50 μM) for 2h, PC 12 cells
were incubated with 25 μM of Aβ (1-42) for 24 h. The cell viability was evaluated using
MTT assay.
Acknowledgments
This work was supported by programs of the National Natural Science Foundation
of China [81872981 and 81760622]; Program of Shanghai Academic/Technology
23

Research Leader [18XD1403700]; National Scientific and Technological Major
Special Project of China (2018ZX09201008-002); Youth Talent Sail Plan from the
Shanghai Committee of Science and Technology [18YF1423600]; Projects sponsored
by the development fund for Shanghai talents [2018105]; Project of the Shanghai
Municipal Commission of Health and Family Planning [2017YQ072 and 201740152];
Research Fund for the Doctoral Program of Shanghai [B201703]; Undergraduate
innovation project from Shanghai University of traditional Chinese Medicine [grant
number 2019SHUTCM146] and Xinglin Young Talent Program.
Conflicts of Interest
The authors declare no conflict of interest.
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