Rational modification of donepezil as multifunctional acetylcholinesterase inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.07.052

A series of novel donepezil derivatives was designed, synthesized and evaluated as multifunctional acetylcholinesterase (AChE) inhibitors for the treatment of Alzheimer's disease (AD). The screening results indicated that most of the compounds exhibited potent inhibition of AChE with IC₅₀values in the nanomolar range. Moreover, these derivatives displayed good antioxidant, Aβ interaction, blood-brain barrier penetration (PAMPA-BBB+) and ADMET properties (in silico). Among them, 5c demonstrated excellent AChE inhibition (IC₅₀: 85?nM for eeAChE, 73?nM for hAChE), metal chelation, and inhibitory effects on self-induced, hAChE-induced and Cu²⁺-induced Aβ_1-42aggregation (18.5%, 72.4% and 46.3%, at 20?μM). Kinetic analysis and molecular modeling studies suggested that 5c could bind simultaneously to the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. More importantly, 5c exhibited significant neuroprotective potency against Aβ_1-42-induced PC12?cell injury. Furthermore, the step-through passive avoidance test showed 5c significantly reversed scopolamine-induced memory deficit and no hepatotoxicity in mice. These results indicated that 5c might be a promising drug candidate for AD therapy.

Full text of DOI:10.1016/j.ejmech.2016.07.052

Accepted Manuscript
Rational modification of donepezil as multifunctional acetylcholinesterase inhibitors for
the treatment of Alzheimer's disease
Zhi-Min Wang, Pei Cai, Qiao-Hong Liu, Ding-Qiao Xu, Xue-Lian Yang, Jia-Jia Wu,
Ling-Yi Kong, Xiao-Bing Wang
PII:
S0223-5234(16)30615-8
10.1016/j.ejmech.2016.07.052
EJMECH 8770
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 May 2016
Revised Date: 20 July 2016
Accepted Date: 21 July 2016
Please cite this article as: Z.-M. Wang, P. Cai, Q.-H. Liu, D.-Q. Xu, X.-L. Yang, J.-J. Wu, L.-Y. Kong,
X.-B. Wang, Rational modification of donepezil as multifunctional acetylcholinesterase inhibitors for
the treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.07.052.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Rational modification of donepezil as multifunctional
acetylcholinesterase inhibitors for the treatment of Alzheimer’s
disease
Zhi-Min Wang, Pei Cai, Qiao-Hong Liu, Ding-Qiao Xu, Xue-Lian Yang, Jia-Jia Wu,
Ling-Yi Kong *, Xiao-Bing Wang *
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, NanJing 210009,
People’s Republic of China
*
Corresponding Authors: Tel/Fax: +86-25-8327-1405. E-mail: cpu_lykong@126.com;
xbwang@cpu.edu.cn.
Abstract
A series of novel donepezil derivatives was designed, synthesized and evaluated as
multifunctional acetylcholinesterase (AChE) inhibitors for the treatment of
Alzheimer’s disease (AD). The screening results indicated that most of the
compounds exhibited potent inhibition of AChE with IC values in the nanomolar
5
0
range. Moreover, these derivatives displayed good antioxidant, Aβ interaction,
blood-brain barrier penetration (PAMPA-BBB+) and ADMET properties (in silico).
Among them, 5c demonstrated excellent AChE inhibition (IC : 85 nM for eeAChE,
5
0
7
3 nM for hAChE), metal chelation, and inhibitory effects on self-induced,
2+
hAChE-induced and Cu -induced Aβ
aggregation (18.5%, 72.4% and 46.3%, at
-42
1
2
0 ꢀM). Kinetic analysis and molecular modeling studies suggested that 5c could bind
simultaneously to the catalytic active site (CAS) and peripheral anionic site (PAS) of
AChE. More importantly, 5c exhibited significant neuroprotective potency against
Aβ1-42-induced PC12 cell injury. Furthermore, the step-through passive avoidance test
showed 5c significantly reversed scopolamine-induced memory deficit and no
hepatotoxicity in mice. These results indicated that 5c might be a promising drug
candidate for AD therapy.
Keywords: Alzheimer’s disease; Donepezil derivatives; Acetylcholinesterase
inhibitors; β-amyloid aggregation; Multifunctional agents.
1

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1
. Introduction
Alzheimer’s disease (AD), is an age-related, progressive, neurodegenerative
disorder that affects the elderly people, characterized by memory loss, decline in
language skills and other cognitive impairments [1, 2]. Due to the complexity
pathology of AD, many hypotheses have been proposed, including cholinergic
neurons system dysfunction [3], β-amyloid (Aβ) protein deposits [4], oxidative stress
[
5], τ-protein hyperphosphorylation and metal dyshomeostasis [6, 7]. The cholinergic
hypothesis suggests that the cognitive and memory deteriorations associated with AD
are caused by the selective decline of acetylcholine (ACh) in central nervous system
(CNS) [8]. The primary approach of AD therapy has therefore focused on increasing
the brain ACh levels by treating with acetylcholinesterase inhibitors (AChEIs). The
results of clinical trials have demonstrated that the treatment with these AChEIs, such
as tacrine, rivastigmine, galantamine or donepezil produces improvement of cognitive
and memory function in patients with mild, moderate or severe AD related dementia,
however their effectiveness is limited [9, 10]. In addition, the “amyloid cascade
hypothesis” of AD states that the β-amyloid peptide (Aβ) is generated via cleavage of
the transmembrane amyloid precursor protein (APP) by β- and γ-secretases [11].
Generated Aβ peptides can aggregate into oligomers, protofibrils and fibrils that adopt
the well-integrated β-sheet structure. The progressive accumulation of Aβ is
accompanied by oxidative stress and inflammation, which leads to neurodegeneration
[
12]. Two main Aβ isoforms (Aβ1-40 and Aβ1-42) are primarily generated by the
sequential cleaving of the APP. Aβ1-42 is the predominant form in senile plaques,
2

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having lower solubility and stronger neuronal toxicity than Aβ1-40. Therefore, the
inhibition of Aβ1-42 aggregation is another potential approach for the treatment of AD
[
13, 14]. The X-ray crystallographic structure of AChE reveals that it contains two
binding sites: the catalytic active site (CAS) and the peripheral anionic site (PAS)
connected by a deep hydrophobic gorge [15]. Generally, inhibitors that bind to either
one of these sites can inhibit AChE. Current studies indicate that the PAS of AChE
not only facilitates cholinergic transmission but also accelerates deposition and
aggregation of Aβ [16]. Increasing evidence supports metal ions dyshomeostasis and
miscompartmentalization which are thought to play a significant part in Aβ
production, aggregation and neurotoxicity. Aβ shows high affinity for metal ions such
2
+
2+
as Cu and Zn which are able to accelerate the formation of Aβ aggregates and
neurofibrillary tangles, resulting in neuronal dysfunction in AD patients [17, 18].
2
+
2+
Moreover, abnormally high brain levels of redox-active metal ions, like Cu and Fe ,
may contribute to the production of reactive oxygen species (ROS), which promotes
oxidative stress thus contributing to AD pathogenesis [19].
Due to the complexity of AD, drugs that modulate the activity of a single target
may not be sufficient to alter the progression of this disease. Thus, an efficient therapy
is more likely to develop multifunctional (multi-targeted) drugs that incorporate
several pharmacological effects into a single molecule [20]. Taking these facts into
account, many researchers are devoted to the investigation of multi-target-directed
ligands (MTDL) [21]. Subsequently, a variety of compounds combining the inhibitory
activities of ChEs, biometal chelation, antioxidant and Aβ aggregation inhibition have
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been developed for the treatment of AD [22-32].
Donepezil, a FDA-approved drug, is a potent, selective, uncompetitive, reversible
AChE inhibitor that is thought to enhance cholinergic function by increasing ACh
levels in the CNS [10]. However, clinical trials of this single target drug on this
disease have been modest and transient because of the multifactorial nature of AD,
and this has consequently failed to the treatment of moderate to severe AD patients
[
33]. Therefore, we focus on designing novel multifunctional agents based on rational
modification of donepezil scaffold for AD therapy. As known, the benzylpiperidine
fragment of donepezil that interacts with the AChE-CAS has been shown as the
AChE inhibitory pharmacophore. Indanone moiety can interact with the PAS of
AChE via aromatic stacking interactions and inhibit Aβ aggregation [34]. Since there
are few reports for the modification of the benzyl group of donepezil, we consider
introducing small substituents in the benzene ring to enhance the AChE inhibitory
potency [35-38]. In order to introduce antioxidant, metal chelating effects and
diversify the structures, we also replace the benzene ring with some different
fragments, such as pyridine, quinolone and hydroxyquinoline (Figure 1). These new
derivatives may simultaneously possess dual binding sites for AChE inhibition, Aβ
aggregation inhibition, antioxidant and metal chelation.
In this work, we reported the design, synthesis, biological evaluation and molecular
modeling of donepezil derivatives as multifunctional AChE inhibitors for AD therapy.
The pharmacological profiles of these novel compounds including AChE and BuChE
inhibition, metal chelation, antioxidant, self-induced, AChE-induced and
4

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2
+
Cu -induced Aβ aggregation inhibition, the kinetics and molecular modeling of
AChE inhibition, BBB permeation, neuroprotection, behavioral performance and
hepatotoxicity were evaluated in vitro and in vivo.
2
. Results and discussion
2
.1. Chemistry
The synthetic routes of donepezil derivatives 3a-o, 5a-h and 7a-c were shown in
Scheme 1-2 and Scheme 3, respectively. The key intermediates (6a-c) were obtained
by minor modifications according to the reported methods (Scheme S1, Supporting
information) [39, 40]. The commercially available starting material 1(1.0 mmol) was
suspended in acetonitrile (10 mL) containing K CO (2.0 mmol). The reaction was
2
3
treated with appropriately substituted arylalkyl derivatives 2a-o, 4a-h and
chloromethyl-quinolines 6a-c (1.2 mmol) and heated under reflux for 8 h. The
reaction progress was monitored using silica gel TLC with DCM/MeOH as mobile
phase. Upon completion, the acetonitrile was evaporated in vacuo and the mixture
was then poured into water, which was extracted with DCM, washed with brine, dried
over anhydrous Na SO and purified by chromatography (DCM/MeOH) on silica gel.
2
4
2
2
.2. Pharmacological evaluation
.2.1. Cholinesterase inhibition and SAR discussion
Inhibitory activities of the target compounds on AChE (from electric eel and human)
and BuChE (from equine serum and human) were tested by the spectrophotometric
method of Ellman et al. with donepezil and tacrine as the reference standards [41].
The IC50 values for eeAChE (hAChE) and eqBuChE (hBuChE) inhibition were
5

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summarized in Table 1. It could be seen from Table 1 that most of the synthesized
compounds showed high inhibitory activities to AChE with IC values at nanomolar
5
0
range. Similarly, all of the donepezil derivatives showed higher inhibitory potency
against the AChE than the BuChE, indicating that the introduction of different small
(3a-o) or large (5a-h and 7a-c) groups in benzylpiperidine moiety could not affect the
inhibitory capacity and selectivity for ChEs inhibition. To enhance the AChE
inhibition of donepezil, compounds of 3a-o were designed and synthesized. As shown
in Table 1, compound 3d was the best inhibitor against eeAChE (IC = 43 nМ) and
5
0
hAChE (IC = 32 nМ), revealing that a 2-F in the benzene ring was the best choice
5
0
for AChE inhibition and more active than donepezil (IC = 51 nМ for eeAChE and
5
0
IC = 48 nМ for hAChE). Compared with different substituents (CH , F, Cl, CF and
5
0
3
3
NO ), F and CH substituents were in favor of retaining and improving the inhibitory
2
3
activities of AChE, and compounds with 2- or 3- position substitutions were more
potent than those with 4-position substitutions. It is worth noting that most of the
compounds exhibited moderate to good BuChE inhibition but no clear trend could be
summarized. It could be concluded that small substituents in the benzene ring of
donepezil slightly increased AChE inhibitory potency but had no significant effect to
BuChE. The result also suggested that the small substituents could increase the
selectivity of AChE inhibitory activity. This also showed that the modification of
benzylpiperidine fragment could improve AChE inhibitory potency indeed [35, 36].
In order to further introduce additional metal chelating activities of the designed
compounds not just ChEs inhibition, we replaced the benzyl substitutions with the
6

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pyridylmethyl groups to obtain compounds 5a-h. Among the compounds, 5c (IC =
5
0
8
5 nМ for eeAChE and IC = 73 nМ for hAChE) exhibited the highest inhibitory
50
activities of eeAChE and hAChE but lower than donepezil. For these classes of
derivatives, substitutions at 6-position of pyridyl group could basically retain the
AChE inhibitory potency. In addition, compounds with electron-donating groups in
the 6-position were beneficial to AChE inhibition but no clear effect in BuChE
inhibition was observed.
Finally, some quinolylmethyl (known metal chelating fragment) substituted
compounds 7a-c were then synthesized and evaluated as inhibitors of AChE.
However, it was found that both eeAChE and hAChE inhibitory values of these
compounds were decreased, which indicated that the quinolin fragment could lead to
a significant decrease in AChE inhibitory potency. Surprisingly, these compounds
revealed moderate inhibition for eqBuChE (IC < 10 ꢀM). The previous study also
5
0
disclosed that embedding the amino functionality into a piperidine moiety led to
weaker AChE inhibitory activity [42]. In summary, all the target compounds (3a-o,
5
a-h and 7a-c) retained similar trends in the inhibition to both eeAChE and hAChE,
indicating that the rational modification of donepezil could introduce additional
activities but not remarkably affect the inhibitory potency of AChE.
2
.2.2. Kinetic study of hAChE inhibition
In order to gain insight into the mechanism of action of these derivatives on hAChE,
compounds 3d and 5c were selected for kinetic measurements because of their
superior inhibitory activities against both eeAChE and hAChE. The graphical analysis
7

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of the steady-state inhibition data of 3d and 5c for hAChE were shown in Figure 2 (A
and B). The results from the Lineweaver-Burk plots indicated that both slopes and
intercepts were increased at increasing concentration of the inhibitors and intersected
in the third- and fourth-quadrant, respectively. This pattern indicated that compounds
3
d and 5c were both mixed-type inhibitors binding to the PAS as well as the CAS of
hAChE, demonstrating rationality of our molecular design strategy [42].
2
.2.3. Molecular modeling studies
To further investigate the binding modes of these derivatives, the molecular
docking study was performed with the more promising compounds 3d and 5c by
using Molecular Operating Environment (MOE) software package. Based on the
above data, the mixed-type inhibition by 3d and 5c was shown in the
Lineweavere-Burk plots for hAChE, meaning that the binding sites might be the CAS
as well as the PAS. Accordingly, the complex of hAChE with donepezil (PDB code:
4
EY7) was chosen for modeling from the Protein Data Bank (http://www.rcsb.org)
[
43]. By analyzing Figure 3, compounds 3d and 5c could perfectly fit into the
active-site gorge of the enzyme and simultaneously interact with the PAS and CAS of
hAChE. The indanone moiety of 3d was observed to bind to the PAS and a π-π
stacking was formed with residue Trp 286. In the bottom of the gorge, the charged
nitrogen of piperidine ring and the F-substituted phenyl ring could bind to CAS by
establishing cation-π and π-π interactions with Trp 86 (Figure 3 A and C), respectively.
Interestingly, similar interactions between compound 5c and hAChE were observed in
Figure 3 B and D. Indanone moiety preferably occupied the PAS via a π-π interaction
8

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with Trp 286. The pyridylmethyl-substituted piperidine fragment was bound to the
CAS of hAChE, and formed interaction by a π-π stacking with Trp 84 and cation-π
interaction (protonated nitrogen of piperidine ring with Trp 84). The docking results
revealed that the designed compounds 3d and 5c could bind to PAS and CAS dual
binding sites, indicating mixed-type inhibitors of hAChE, which were in agreement
with kinetic analysis results. Moreover, the interacting modes of compounds 3d and
5
c with hBuChE (PDB code: 4XII) were also investigated by the modeling study [44].
As shown in Figure S2 (Supporting information), the π-π interactions were formed
between the ligands and the active site of hBuChE.
2
.2.4. Metal chelating effects
The chelating ability and selectivity of compound 5c to chelate biometals, such as
2
+
2+
2+
3+
Cu , Zn , Fe and Fe , was studied by fluorescence spectrometry [45], and the
results were shown in Figure 4. From the fluorescence emission spectra (excitation
wavelength at 210 nm), the specific fluorescence emission peak of 5c can be seen at
2
+
2+
3
90 nm with the intensity of 250 fluorescence units. After Cu and Zn were added
to the HEPES solution of 5c, the fluorescence intensity of emission spectra was
2
+
greatly enhanced, and the spectra of 5c-Zn slightly shifted to 400 nm. Interestingly,
3
+
when the same experiment was performed using 5c-Fe , the wavelength of maximum
fluorescence emission intensity was shifted to 310 nm. However, the specific peaks of
2
+
5
c and 5c-Fe overlaid mostly at 200-400 nm without the generation of new peak,
2
+
showing that there is no significant chelating effect between 5c and Fe . Overall,
these changes in the fluorescence emission spectra indicated the complexes formation
9

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2
+
2+
3+
of 5c-Cu , 5c-Zn and 5c-Fe , respectively.
2
+
The chelating effect of 5c for Cu in HEPES buffer was further investigated by
UV–vis spectrometry with wavelength ranging from 200 to 400 nm [46]. In Figure 5A,
2
+
UV–vis spectra of 5c at increasing Cu concentrations were shown (the absorbance
2
+
of Cu was subtracted). The increase in absorbance indicated that there was an
2
+
2+
interaction between Cu and compound 5c and the formation of complex 5c-Cu .
2
+
The stoichiometry of the 5c-Cu complex was investigated using the molar ratio
2
+
method, by mixing the fixed amount Cu with increasing compound 5c. The results
showed that the absorbance linearly increased initially and then plateaued (Figure 5B).
The two straight lines intersected at a mole fraction of 0.9, revealing about a 1:1
2
+
stoichiometry for complex 5c-Cu . These observations displayed that 5c could
2
+
effectively chelate Cu , and thereby could serve as metal chelator for AD therapy.
2
+
2
.2.5. Inhibition of self-induced and Cu -induced Aβ aggregation
1-42
2
+
The inhibition of self-induced and Cu -induced Aβ
aggregation of these
-42
1
selected derivatives (3b, 3d-e, 5b-c and 7b-c) was evaluated using a Thioflavin-T
based fluorometric assay [47, 48]. Inhibition activities as inhibition ratios at a test
concentration of 20 ꢀМ were listed in Table 2. Compared with the reference
compound, curcumin (57.4% for the inhibition of self-induced Aβ1-42 aggregation),
compounds 3b, 3d-e and donepezil did not show any significant inhibitory activities,
while compounds 5b-c and 7b-c exhibited moderate to good inhibition ratios from
1
8.5% to 51.1%. It was indicated that the introduction of pyridine and quinoline
fragments could enhance the inhibitory potency against Aβ1-42 self-aggregation.
1
0

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2+
From the inhibition results of Cu -induced Aβ
aggregation, compounds (5b-c
-42
1
and 7b-c) exhibited higher levels of inhibitory potency (37.2% to 76.3%) than other
compounds (3b, 3d-e, and donepezil), revealing that introduction of metal chelating
2
+
moiety could be beneficial to their activities against Cu -induced Aβ
aggregation
-42
1
2
+
indeed. In particular, compound 5c significantly inhibited Cu -induced Aβ
1
-42
aggregation in a concentration dependent manner (Figure 6).
2
.2.6. Inhibition of AChE-induced Aβ1-40 aggregation
Additionally, AChE was considered to promote in vitro the assembly of Aβ peptide
into amyloid fibrils and forming a stable AChE-Aβ complex, which could cause
neurotoxicity [34]. The concomitant inhibition of the PAS of AChE, which was
supposedly associated with the aggregation of Aβ, might turn AChE inhibitors into
potential disease modifying agents [49, 50]. It was observed that most of the selected
compounds (3b, 3d-e, 5b-c and 7b-c) showed better inhibitory activities to
hAChE-induced Aβ1-40 aggregation than the self-induced Aβ1-42 aggregation.
Moreover, compound 5c (72.4% for inhibition of hAChE-induced Aβ1-40 aggregation)
was the strongest inhibitor among the tested compounds, which was more potent than
the control donepezil (26.4%). From the inhibition values of the compounds, it
appeared that the modification of benzylpiperidine moiety did play a role in
determining the inhibition of AChE-induced Aβ1-40 aggregation, and introduction of
pyridine or quinoline fragment at the donepezil unit led to a slightly increased effect.
2
+
2
.2.7. Inhibition of Cu -Aβ H O production
1-42 2 2
2
+
As reported, binding of redox-active Cu to Aβ
species could be involved in
-42
1
11

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generation of ROS such as H O and subsequent facilitation of oxidative stress and
2
2
neurotoxicity [19]. The effects of compounds 5c and 7c on H O production by
2
2
2
+
Cu -Aβ
species was tested using the HRP/Amplex Red assay [51]. Under
-42
1
2
+
reducing conditions, the Cu -Aβ
reacted with O to generate H O . As shown in
2 2 2
1
-42
Figure 7, addition of compounds 5c and 7c to such solution significantly reduced the
2
+
production of H O by about 42% and 54% for the Cu -Aβ species. By
1-42
2
2
comparison, the strong chelator ethylenediaminetetraacetic acid (EDTA) showed an
even more pronounced effect, almost complete eliminating (>84%), and clioquinol
(CQ) showed the reducing effect by about 68%. These results indicated that the tested
2
+
compounds were capable of halting the Cu redox-active cycling involved in
oxidative stress via metal chelation, which showed promise for further application of
AD therapy.
2
.2.8. Antioxidant activity assay by ORAC
The antioxidant activities of the selected compounds were determined by
measuring the oxygen radical absorbance capacity of fluorescein (ORAC-FL) using
the vitamin E analogue, trolox, as a standard [52, 53]. The results in Table 2 indicated
that these compounds had moderate to good peroxyl radical absorbance capacities,
ranging from 0.43- to 2.85-fold of the value of trolox. Among the compounds, the
hydroxyquinoline substituted compounds 7b and 7c displayed more potent
scavenging activities than that of the reference compound trolox. However, other
derivatives (3b, 3d-e and 5b-c) showed moderate antioxidant activities and weaker
than trolox.
1
2

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2
.2.9. Cell viability and neuroprotection against Aβ1-42-induced and
2
+
Cu -Aβ1-42-induced toxicity
To investigate the therapeutic potential of these derivatives, cell viability and
2+
neuroprotection against Aβ1-42-induced and Cu -Aβ1-42-induced toxicity were assayed
using PC12 cells [54, 55]. Compounds 3c, 3d, 5b, 5c and 7c were selected as
representative compounds with different structural types. First, the colorimetric MTT
[
3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] assay was
performed to examine the potential cytotoxic effects of these compounds. As
indicated in Figure 8, compounds 3c, 3d, 5b, 5c and 7c did not show significant effect
on cell viability at 3.125-50 ꢀM after incubation for 24 h.
Compounds 5c and 7c were also tested for their capacity to protect PC12 cells
2
+
against Aβ1-42-induced and Cu -Aβ1-42-induced toxicity. Melatonin was used as the
reference compound. In the assay of coping Aβ1-42-induced toxicity, addition of 20 ꢀM
Aβ1-42 to the growth medium reduced cell viability to 68.4% compared to control. The
tested compounds were added to the media at different concentrations immediately
prior to the Aβ1-42 insult. As shown in Figure 9, all the tested compounds exhibited
significant neuroprotective effects at concentrations ranging from 10 to 20 ꢀM.
Compounds 5c and 7c displayed strong protective capability almost the same as
melatonin at the concentration of 20 ꢀM but weaker at low concentrations. From the
2
+
results of compounds 5c and 7c against Cu -Aβ1-42-induced cytotoxicity, PC12 cell
2
+
viability was substantially reduced to 54.6% after the addition of Cu -Aβ1-42.
Surprisingly, the tested compounds still exerted moderate to good neuroprotective
1
3

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2
+
activities against Cu -Aβ1-42-induced cell death. It is note that compound 7c was
2
+
more effective than 5c on both the Aβ1-42- and Cu -Aβ1-42-treated PC12 cells. Similar
observation of phase-contrast micrographs also showed Aβ1-42 and
-
2
+
Cu -Aβ1-42-induced neurotoxicity and neuroprotection in PC12 cells for the tested
compounds (Figure S4, Supporting information). These observations further indicated
that these derivatives had the potential to be efficient for the treatment of AD.
2
.2.10. In vitro blood-brain barrier (BBB) penetration assay and ADMET prediction
BBB penetration is an essential element for successful anti-AD drugs. To evaluate
the potential for these derivatives to cross the BBB, we used a parallel artificial
membrane permeation assay for BBB (PAMPA-BBB), which was described by Di et
al [56]. Assay validation was made by comparing experimental permeabilities of 9
commercial drugs with reported values (Table 3). A plot of experimental data versus
bibliographic values gave a good linear correlation, P (exp.) = 0.8754 P (bibl.) -
e
e
2
0
.1831, R = 0.9846 (Figure S2, Supporting Information). From this equation and
taking into account the limit established by Di et al. for BBB permeation, we
classified compounds as follows: (a) ‘ CNS + ’ (high BBB permeation predicted): Pe
-
6
-1
- 6
-1
(
10 cm s ) > 3.32; (b) ‘ CNS - ’ (low BBB permeation predicted): Pe (10 cm s ) <
-
6
-1
1
.57; (c) ‘ CNS +/- ’ (BBB permeation uncertain): Pe (10 cm s ) from 1.57 to 3.32.
The representative compounds (3b, 3d-e, 5b-c and 7b-c) were chosen as the tested
compounds. The results summarized in Table 4 indicated that all of the selected
compounds could penetrate the BBB. In addition, the physical properties of the
selected compounds were followed Lipinski’s rule of five (Table 5) [57], indicating
1
4

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that compounds fulfill the necessary drug-like and predicted BBB penetration criteria.
Additionally, the selected compounds with higher activities were evaluated for the
ADMET properties in silico by the Accelrys Discovery Studio (Table 5) [58]. All
these compounds showed ADMET properties similar to drug donepezil. Compare
with the standard drug, most of the compounds were predicted to have fine absorption,
high BBB penetration, good solubility and low toxicity. However, compounds 7b-c
showed a vast probability of hepatotoxicity (>0.92), which might owe to the
introduction of the hydroxlquinoline fragment. Nonetheless, the results above
indicated these compounds satisfied possible BBB penetration, containing drug-like
properties and possessed anti-AD potentials.
2
.2.11. In vivo assay of cognitive and memory improvement
The learning and memory evaluation in animal models were performed using the
passive avoidance test. Scopolamine induces memory deficits had been used
extensively to evaluate potential agents for AD therapy [59, 60]. Therefore, we
investigated the protective effects of the donepezil derivatives against
scopolamine-induced memory deficits with donepezil as the positive control
compound. The results showed that the administration of compounds 3d and 5c
during the treatment period (7 days) did not influence the mean daily body weight
profile of the mice, in particular, it did not cause any adverse or abnormal events or
affect the survival. As shown in Figure 10, the step-through latency of mice treated
with scopolamine alone (model group) was significantly shorter than that of
vehicle-treated mice (normal group). Treatment with compounds 3d or 5c (2.5, 5 and
1
5

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1
0 mg/kg) increased the latency time in a dose-dependent manner. The effects of
compounds 3d (5 mg/kg) and 5c (10 mg/kg) were comparable with that of donepezil
5 mg/kg). These results indicated that the donepezil derivatives 3d and 5c were able
(
to reverse scopolamine-induced memory deficit in the step-through passive avoidance
test by increasing brain cholinergic activity.
2
.2.12. Hepatotoxicity studies
Although donepezil was considered to be not associated with high incidence of
side-effects and general tolerability concerns from the clinical trials [62-64], these
donepezil derivatives might have definitely different pharmacokinetic profile in
animals and humans. In order to evaluate whether the preferred donepezil derivatives
had hepatotoxicity or other side effects, the experimental mice were further used for
testing hepatotoxicity [65, 66]. After being tested the passive avoidance test, the
heparinized serum of mice of different groups (normal group, model group, control
group and treatment groups) was obtained, and the levels of aspartate
aminotransferase (AST) and alanine aminotransferase (ALT) were determined (Figure
11). In comparison to the control, all groups at the experimental doses did not cause
significant hepatotoxicity, as indicated by the slightly increased activity of ALT and
AST. From the results, it appeared that 3d and 5c possessed similar safety to
donepezil. For morphological studies the liver tissue was stained with hematoxylin
and eosin (HE). As shown in Figure 12, no obvious morphological changes were
observed after the treatment with scopolamine, donepezil and its derivatives 3d and
5
c, such as no increase in the number of inflammatory cells in portal fields and no
1
6

ACCEPTED MANUSCRIPT
area of necrosis and distinct fatty degeneration of the hepatocytes were observed. All
these findings suggested that compounds 3d and 5c could be safer similar to
donepezil for the treatment of AD.
3
. Conclusions
In conclusion, a series of novel donepezil derivatives were designed, synthesized
and evaluated as multifunctional AChE inhibitors as anti-Alzheimer agents.
According to our results, most of the compounds could effectively and selectively
inhibit eeAChE and hAChE. Enzyme kinetic and the molecular modeling studies
revealed the compounds could bind simultaneously to the PAS and CAS of AChE.
Some compounds also exhibited inhibitory activities against self-induced,
2+
AChE-induced and Cu -induced Aβ
aggregation. Besides, these compounds
-42
1
behaved moderate to good antioxidant capacity by ORAC assay, and good BBB
penetration and ADMET properties for CNS activity. Moreover, the most promising
compound 5c displayed very low toxicity and significant neuroprotective effects
2+
against Aβ1-42- and Cu -Aβ1-42-induced neurotoxicity in PC12 cells assay. More
importantly, the step-through passive avoidance test showed 5c significantly reversed
scopolamine-induced memory deficit and no hepatotoxicity in mice. Altogether, the
multifunctional effects of these donepezil derivatives qualified them as potential drug
candidates for AD therapy.
4
. Experimental section
4
.1. Chemistry. General methods
All common reagents and solvents were obtained from commercial suppliers and
1
7

ACCEPTED MANUSCRIPT
used without further purification. Reaction progress was monitored using analytical
thin layer chromatography (TLC) on pre-coated silica gel GF254 plates (Qingdao
Haiyang Chemical Plant, Qingdao, China) plates and the spots were detected under
UV light (254 nm). Column chromatography was performed on silica gel (90-150 ꢀm;
Qingdao Marine Chemical Inc.) Melting point was measured on an XT-4
1
13
micromelting point instrument and uncorrected. H NMR and C NMR spectra were
measured on a Bruker ACF-500 spectrometer at 25 °C and referenced to TMS.
Chemical shifts are reported in ppm (δ) using the residual solvent line as internal
standard. Mass spectra were obtained on a MS Agilent 1100 Series LC/MSD Trap
mass spectrometer (ESI-MS) and a Mariner ESI-TOF spectrometer (HRESI-MS),
respectively.
4
.1.1. General procedures for the preparation of 3a-o and 5a-h
The commercially available starting material 1 (1.0 mmol) was suspended in
acetonitrile (10 mL) containing K CO (2.0 mmol). The reaction was treated with an
2
3
appropriately substituted arylalkyl derivatives 2a-o and 4a-4h (1.2 mmol) and heated
under reflux for 8 h. The reaction progress was monitored using silica gel TLC with
DCM/MeOH as mobile phase. Upon completion, the acetonitrile was evaporated in
vacuo and the mixture was then poured into water, which was extracted with DCM,
washed with brine, dried over anhydrous Na SO and purified by chromatography
2
4
(
DCM/MeOH) on silica gel to afford the target compounds.
.1.1.1. 5, 6-dimethoxy-2-((1-(2-methylbenzyl)piperidin-4-yl)methyl)-2, 3-dihydro-1H
inden-1-one (3a)
4
-
1
8

ACCEPTED MANUSCRIPT
1
Yield 74%, light yellow solid; m.p. 96-98 °C; H NMR (500 MHz, DMSO) δ 7.24 -
7
.17 (m, 3H), 7.11 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.40 (s, 2H), 3.25
(dd, J = 17.4, 7.9 Hz, 1H), 3.11 (d, J = 6.9 Hz, 1H), 2.81 (s, 1H), 2.68 (d, J = 14.8 Hz,
2
H), 2.34 (s, 3H), 1.96 (s, 2H), 1.73 - 1.68 (m, 3H), 1.48 (s, 1H), 1.34 - 1.26 (m, 3H);
C NMR (125 MHz, DMSO) δ 206.96, 155.75, 149.62, 149.16, 128.89, 126.06,
08.69, 104.49, 56.43, 56.11, 46.28, 45.22, 33.15, 19.42, 9.18; HRMS (ESI) m/z
1
3
1
3
4
+
94.2375 [M+H] (calcd for 394.2377, C H NO ).
2
5
32
3
.1.1.2. 5, 6-dimethoxy-2-((1-(3-methylbenzyl)piperidin-4-yl)methyl)-2, 3-dihydro-
H–inden-1-one (3b)
1
1
Yield 83%, light yellow solid; m.p. 95-97 °C; H NMR (500 MHz, DMSO) δ 7.25 -
.20 (m 1H), 7.18 - 7.03 (m, 5H), 3.88 (s, 3H), 3.80 (s, 3H), 3.44 (s, 2H), 3.23 (dd, J =
7.6, 8.1 Hz, 1H), 2.83 (s, 2H), 2.72 - 2.59 (m, 2H), 2.34-2.28 (m, 3H), 1.93 (s, 2H),
.71 (dd, J = 15.2, 6.2 Hz, 2H), 1.63 (d, J = 11.5 Hz, 1H), 1.46 (s, 1H), 1.32 – 1.21 (m,
7
1
1
3
1
3
1
3
H); C NMR (125 MHz, DMSO) δ 206.97, 155.73, 149.61, 149.14, 137.66, 130.06,
28.91, 128.50, 126.62, 108.67, 104.49, 96.24, 56.41, 56.10, 53.63, 46.22, 45.25,
+
8.60, 33.17, 21.48, 9.32; HRMS (ESI) m/z 394.2376 [M+H] (calcd for 394.2377,
C H NO ).
25
32
3
4
.1.1.3. 5, 6-dimethoxy-2-((1-(4-methylbenzyl)piperidin-4-yl)methyl)-2, 3-dihydro-
H–inden-1-one (3c)
1
1
Yield 82%, colorless solid; m.p. 223-225 °C; H NMR (500 MHz, DMSO) δ
.21-7.17 (m, 2H), 7.18 - 7.15 (m, 2H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.80 (s,
H), 3.23 (dd, J = 17.7, 8.1 Hz, 1H), 2.82 (s, 2H), 2.67 (dd, J = 11.4, 6.2 Hz, 2H),
7
3
1
9

ACCEPTED MANUSCRIPT
2
.30 (s, 3H), 1.91 (s, 2H), 1.76 - 1.70 (m, 2H), 1.63 (s, 1H), 1.46 (s, 1H), 1.37 - 0.98
1
3
(
m, 5H); C NMR (125 MHz, DMSO) δ 206.96, 155.73, 155.16, 149.61, 149.14,
1
3
29.22, 128.91, 128.09, 127.78, 108.67, 104.48, 56.41, 56.10, 53.50, 45.23, 38.64,
+
3.16, 21.18, 9.27; HRMS (ESI) m/z 394.2378 [M+H] (calcd for 394.2377,
C H NO ).
25
32
3
4
.1.1.4. 2-((1-(2-fluorobenzyl)piperidin-4-yl)methyl)-5, 6-dimethoxy-2, 3-dihydro-1H
inden-1-one (3d)
-
1
Yield 80%, light yellow solid; m.p. 119-121 °C; H NMR (500 MHz, DMSO) δ
.51 – 7.41 (m, 1H), 7.41 – 7.29 (m, 1H), 7.21 (m, 2H), 7.10 (s, 1H), 7.07 (s, 1H),
.88 (s, 3H), 3.81 (s, 3H), 3.53 (s, 1H), 3.24 (dd, J = 17.5, 7.7 Hz, 1H), 2.84 (m, 1H),
7
3
2
1
3
.67 (d, J = 14.8 Hz, 2H), 2.00 (s, 2H), 1.68 (m, 3H), 1.46 (s, 1H), 1.23 (m, 5H); C
NMR (125 MHz, DMSO) δ 206.89, 163.72, 161.41, 155.76, 149.62, 149.15, 144.16,
1
30.68, 128.88, 108.69, 104.49, 56.43, 56.11, 53.20, 46.24, 45.16, 33.13, 29.73, 9.12;
+
HRMS (ESI) m/z 398.2127 [M+H] (calcd for 398.2126, C H FNO ).
24
29
3
4
.1.1.5. 2-((1-(3-fluorobenzyl)piperidin-4-yl)methyl)-5, 6-dimethoxy-2, 3-dihydro-1H
inden-1-one (3e)
-
1
Yield 72%, light yellow solid; m.p. 99-100 °C; H NMR (500 MHz, DMSO) δ 7.46
7.38 (m, 2H), 7.21 - 7.16 (m, 2H), 7.11 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.81 (s,
H), 3.50 (s, 1H), 3.24 (dd, J = 17.5, 8.0 Hz, 1H), 3.10 (d, J = 7.0 Hz, 3H), 2.83 (s,
H), 2.67 (d, J = 14.4 Hz, 2H), 1.96 (s, 1H), 1.72 - 1.68 (m, 3H), 1.49 (s, 1H), 1.27 -
-
3
1
1
1
13
.74 (m, 3H); C NMR (125 MHz, DMSO) δ 206.89, 163.72, 161.41, 155.76, 149.62,
49.15, 144.16, 130.68, 128.88, 108.69, 104.49, 56.43, 56.11, 53.20, 46.24, 45.16,
2
0

ACCEPTED MANUSCRIPT
+
3
3.13, 29.73, 9.12; HRMS (ESI) m/z 398.2122 [M+H] (calcd for 398.2126,
C H FNO ).
24
29
3
4
.1.1.6.2-((1-(4-fluorobenzyl)piperidin-4-yl)methyl)-5, 6-dimethoxy-2, 3-dihydro-1H
inden-1-one (3f)
-
1
Yield 86%, yellow solid; m.p. 112-114 °C; H NMR (500 MHz, DMSO) δ 7.46 -
7
.31 (m, 2H), 7.24 – 7.18 (m, 2H), 7.11 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.85 - 3.81
(s, 3H), 3.47 (s, 1H), 3.24 (dd, J = 17.6, 8.1 Hz, 1H), 3.11 - 3.02 (m, 2H), 2.72 - 2.63
1
3
(
m, 2H), 2.10 - 1.86 (m, 2H), 1.84 - 1.60 (m, 3H), 1.40 - 1.16 (m, 5H); C NMR (125
MHz, DMSO) δ 206.96, 155.73, 155.16, 149.61, 149.14, 129.22, 128.91, 128.09,
1
27.78, 108.67, 104.48, 56.41, 56.10, 53.50, 45.23, 38.64, 33.16, 21.18, 9.15; HRMS
+
(
ESI) m/z 398.2124 [M+H] (calcd for 398.2126, C H FNO ).
24
29
3
4
.1.1.7. 2-((1-(2-2-chlorobenzyl)piperidin-4-yl)methyl)-5, 6-dimethoxy-2, 3-dihydro-
H -inden-1-one (3g)
Yield 77%, light yellow solid; m.p. 91-92 °C; H NMR (500 MHz, DMSO) δ 7.51
d, J = 7.2 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.38 - 7.33 (t, J = 7.3 Hz, 1H), 7.31 -
.27 (m, 1H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.55 (s, 2H), 3.25
dd, J = 17.6, 8.1 Hz, 1H), 2.85 (t, J = 11.5 Hz, 2H), 2.71 – 2.63 (m, 2H), 2.04 (t, J =
0.8 Hz, 2H), 1.81 - 1.69 (m, 2H), 1.64 (d, J = 12.5 Hz, 1H), 1.48 (s, 1H), 1.33 - 1.20
1
1
(
7
(
1
1
3
(
m, 3H); C NMR (125 MHz, DMSO) δ 207.00, 155.73, 149.61, 149.16, 136.50,
1
5
33.69, 131.18, 129.65, 128.92, 128.71, 127.44, 108.68, 104.49, 59.43, 56.41, 56.10,
+
3.90, 45.29, 38.68, 34.18, 33.18, 31.96. HRMS (ESI) m/z 414.1832 [M+H] (calcd
for 414.1830, C H ClNO ).
2
4
29
3
2
1

ACCEPTED MANUSCRIPT
4
.1.1.8. 2-((1-(2-3-chlorobenzyl)piperidin-4-yl)methyl)-5, 6-dimethoxy-2, 3-dihydro-
1H -inden-1-one (3h)
1
Yield 80%, yellow solid; m.p. 137-138 °C; H NMR (500 MHz, DMSO) δ 7.41 -
7
.35 (m, 4H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.48 (s, 2H), 3.24
(dd, J = 17.6, 8.1 Hz, 1H), 3.10 (d, J = 6.0 Hz, 2H), 2.82 (s, 2H), 2.71 - 2.63 (m, 2H),
13
1
.95 (s, 1H), 1.80 - 1.68 (m, 2H), 1.47 (s, 1H), 1.29 - 1.24 (m, 3H); C NMR (125
MHz, DMSO) δ 206.97, 175.58, 155.74, 149.61, 149.16, 133.37, 130.39, 128.91,
17.17, 108.68, 104.49, 56.42, 56.11, 53.21, 46.28, 45.24, 37.99, 33.17, 9.22. HRMS
1
+
(
ESI) m/z 414.1828 [M+H] (calcd for 414.1830, C H ClNO ).
24 29 3
4
.1.1.9. 2-((1-(2-4-chlorobenzyl)piperidin-4-yl)methyl)-5, 6-dimethoxy-2, 3-dihydro-
H -inden-1-one (3i)
1
1
Yield 71%, light yellow solid; m.p. 136-137 °C; H NMR (500 MHz, DMSO) δ
7
.48 – 7.27 (m, 4H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.80 (s, 3H), 3.24 (dd, J
=
-
17.7, 8.1 Hz, 1H), 3.07 (s, 2H), 2.81 (s, 2H), 2.71 - 2.61 (m, 2H), 1.94 (s, 2H), 1.80
13
1.68 (m, 2H), 1.63 (d, J = 9.8 Hz, 1H), 1.46 (s, 1H), 1.36 – 1.24 (m, 3H); C NMR
(125 MHz, DMSO) δ 206.98, 155.73, 149.61, 149.15, 131.36, 128.91, 128.61, 108.68,
1
4
4
04.49, 56.42, 56.10, 53.21, 46.27, 45.24, 38.65, 33.16, 9.30. HRMS (ESI) m/z
+
14.1829 [M+H] (calcd for 414.1830, C H ClNO ).
2
4
29
3
.1.1.10.
3
5, 6-dimethoxy-2-((1-(2-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)-2,
- dihydro-1H-inden-1-one (3j)
1
Yield 75%, yellow solid; m.p. 121-122 °C; H NMR (500 MHz, DMSO) δ 7.81 (d,
J = 7.7 Hz, 1H), 7.68 (dd, J = 16.9, 7.9 Hz, 2H), 7.47 (t, J = 7.6 Hz, 1H), 7.10 (s, 1H),
2
2

ACCEPTED MANUSCRIPT
7
.07 (s, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.61 (s, 2H), 3.24 (dd, J = 17.5, 7.9 Hz, 1H),
.80 (t, J = 11.9 Hz, 2H), 2.72 – 2.61 (m, 2H), 2.01 (t, J = 10.9 Hz, 2H), 1.83 – 1.68
2
13
(
m, 2H), 1.64 (d, J = 12.6 Hz, 1H), 1.49 (d, J = 2.8 Hz, 1H), 1.33 – 1.12 (m, 3H); C
NMR (125 MHz, DMSO) δ 206.97, 155.81, 149.56, 149.16, 138.46, 132.87, 130.79,
1
3
27.65, 127.23, 125.85, 123.77, 108.67, 104.48, 58.51, 56.40, 56.09, 54.00, 45.30,
+
8.31, 34.33, 33.07, 31.74. HRMS (ESI) m/z 448.2099 [M+H] (calcd for 448.2094,
C H F NO ).
25
29
3
3
4
.1.1.11.
3
5, 6-dimethoxy-2-((1-(3-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)-2,
- dihydro-1H-inden-1-one (3k)
1
Yield 82%, light yellow solid; m.p. 107-108 °C; H NMR (500 MHz, DMSO) δ
7
.69 – 7.63 (m, 3H), 7.60 – 7.53 (m, 1H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.81
(
(
(
s, 3H), 3.56 (s, 2H), 3.24 (dd, J = 17.3, 7.7 Hz, 1H), 2.80 (d, J = 11.6 Hz, 2H), 2.67
d, J = 14.7 Hz, 2H), 1.99 – 1.96 (m, 2H), 1.74 (s, 2H), 1.64 (d, J = 11.7 Hz, 1H), 1.47
1
3
s, 1H), 1.32 – 1.19 (m, 3H); C NMR (125 MHz, DMSO) δ 207.01, 155.73, 149.61,
1
6
4
4
49.16, 140.61, 133.23, 129.66, 128.79, 125.40, 124.05, 123.61, 108.68, 104.49,
2.04, 56.40, 56.10, 53.69, 45.28, 38.68, 34.28, 33.19, 31.92. HRMS (ESI) m/z
+
48.2097 [M+H] (calcd for 448.2094, C H F NO ).
2
5
29
3
3
.1.1.12.
3
5, 6-dimethoxy-2-((1-(4-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)-2,
- dihydro-1H-inden-1-one (3l)
1
Yield 81%, light yellow solid; m.p. 107-108 °C; H NMR (500 MHz, DMSO) δ
.69 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s,
H), 3.80 (s, 3H), 3.55 (s, 2H), 3.24 (dd, J = 17.7, 8.1 Hz, 1H), 2.81 (t, J = 12.9 Hz,
7
3
2
3

ACCEPTED MANUSCRIPT
2
2
H), 2.72 – 2.60 (m, 2H), 1.97 (t, J = 11.1 Hz, 2H), 1.73 (ddd, J = 13.2, 9.3, 5.0 Hz,
13
H), 1.63 (d, J = 12.6 Hz, 1H), 1.46 (d, J = 2.8 Hz, 1H), 1.31 – 1.14 (m, 3H); C
NMR (125 MHz, DMSO) δ 206.97, 155.81, 149.56, 149.16, 138.46, 132.87, 130.79,
1
3
27.65, 127.23, 125.85, 123.77, 108.67, 104.48, 58.51, 56.40, 56.09, 54.00, 45.30,
+
8.51, 34.23, 33.19, 31.92; HRMS (ESI) m/z 448.2096 [M+H] (calcd for 448.2094,
C H F NO ).
25
29
3
3
4
.1.1.13.
1H-inden-1-one (3m)
Yield 92%, yellow solid; m.p. 101-102 °C; H NMR (500 MHz, DMSO) δ 7.86 (d,
J = 8.0 Hz, 1H), 7.73 – 7.58 (m, 2H), 7.52 (t, J = 7.1 Hz, 1H), 7.09 (s, 1H), 7.07 (s,
2-((1-(2-2-nitrobenzyl)piperidin-4-yl)methyl)-5, 6-dimethoxy-2,3-dihydro
-
1
1
2
1
H), 3.88 (s, 3H), 3.80 (s, 3H), 3.71 (s, 2H), 3.23 (dd, J = 17.3, 7.8 Hz, 1H), 2.75 –
.58 (m, 4H), 1.99 (t, J = 10.6 Hz, 2H), 1.77 – 1.63 (m, 2H), 1.59 (d, J = 12.5 Hz, 1H),
13
.44 (s, 1H), 1.23 (dd, J = 16.9, 6.6 Hz, 1H), 1.17 – 1.01 (m, 2H); C NMR (125
MHz, DMSO) δ 206.97, 155.71, 150.12, 149.60, 149.14, 133.72, 133.01, 131.47,
1
3
28.90, 128.81, 124.51, 108.67, 104.48, 58.95, 56.40, 56.09, 53.81, 44.93, 38.98,
+
4.15, 33.15, 33.09, 31.94; HRMS (ESI) m/z 425.2068 [M+H] (calcd for 425.2071,
C H N O ).
24
29
2
5
4
.1.1.14.
2-((1-(2-3-nitrobenzyl)piperidin-4-yl)methyl)-5,
6-dimethoxy-2,
3-
dihydro-1H -inden-1-one (3n)
1
Yield 83%, light yellow solid; m.p. 99-101 °C; H NMR (500 MHz, DMSO) δ 8.20
8.08 (m, 2H), 7.78 (d, J = 7.5 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.10 (s, 1H), 7.07 (s,
H), 3.88 (s, 3H), 3.80 (s, 3H), 3.61 (s, 2H), 3.24 (dd, J = 17.6, 8.1 Hz, 1H), 2.83 (t, J
–
1
2
4

ACCEPTED MANUSCRIPT
=
12.5 Hz, 2H), 2.70 – 2.63 (m, 2H), 2.00 (t, J = 11.1 Hz, 2H), 1.78 – 1.69 (m, 2H),
1
3
1
.64 (d, J = 11.6 Hz, 1H), 1.47 (s, 1H), 1.30 – 1.24 (m, 3H); C NMR (125 MHz,
DMSO) δ 206.99, 167.00, 155.72, 149.61, 149.15, 141.58, 135.80, 131.73, 131.37,
1
3
30.13, 128.92, 123.40, 122.32, 108.67, 104.49, 61.65, 56.40, 56.10, 53.68, 45.27,
+
8.60, 34.24, 33.19, 33.09, 28.85, 11.27; HRMS (ESI) m/z 425.2075 [M+H] (calcd
for 425.2071, C H N O ).
2
4
29
2
5
4
.1.1.15.
2-((1-(2-4-nitrobenzyl)piperidin-4-yl)methyl)-5,
6-dimethoxy-2,
3-
dihydro-1H -inden-1-one (3o)
1
Yield 92%, yellow solid; m.p. 80-81 °C; H NMR (500 MHz, DMSO) δ 8.21 (d, J
=
8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.80
(s, 3H), 3.60 (s, 2H), 3.24 (dd, J = 17.6, 8.1 Hz, 1H), 2.81 (t, J = 12.4 Hz, 2H), 2.73 –
2
1
1
5
.61 (m, 2H), 1.99 (t, J = 11.0 Hz, 2H), 1.84 – 1.68 (m, 2H), 1.64 (d, J = 12.5 Hz, 1H),
1
3
.47 (s, 1H), 1.31 – 1.16 (m, 3H); C NMR (125 MHz, DMSO) δ 206.98, 155.72,
49.60, 149.14, 147.70, 146.93, 130.05, 128.91, 123.77, 108.66, 104.48, 61.93, 56.40,
6.10, 53.80, 45.26, 38.61, 34.19, 33.17, 33.11, 31.92; HRMS (ESI) m/z 425.2073
+
[
M+H] (calcd for 425.2071, C H N O ).
24
29
2
5
4
.1.1.16.
dihydro-1H-inden-1-one (5a)
Yield 79%, yellow solid; m.p. 131-133 °C; H NMR (500 MHz, DMSO) δ 8.50 (d,
J = 4.3 Hz, 1H), 7.78 (dd, J = 7.6, 6.3 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.30 – 7.24
5, 6-dimethoxy-2-((1-(pyridin-2-ylmethyl)piperidin-4-yl)methyl)-2, 3-
1
(m, 1H), 7.11 (s, 1H), 7.08 (s, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.60 (s, 2H), 3.27 – 3.21
(m, 1H), 2.85 (s, 2H), 2.68 (dd, J = 11.2, 6.4 Hz, 2H), 2.03 (s, 2H), 1.74 (ddd, J = 13.0,
2
5

ACCEPTED MANUSCRIPT
1
3
1
1.6, 7.7 Hz, 2H), 1.64 (d, J = 12.6 Hz, 1H), 1.47 (s, 1H), 1.31 – 1.22 (m, 3H); C
NMR (125 MHz, DMSO) δ 207.02, 155.69, 149.61, 149.19, 136.89, 128.92, 123.08,
1
22.26, 108.69, 104.50, 56.41, 56.11, 53.96, 53.76, 45.27, 38.71, 34.16, 33.17, 31.93.
+
HRMS (ESI) m/z 318.2174 [M+H] (calcd for 318.2173, C H N O ).
23
29
2
3
4
.1.1.17.
-2, 3-dihydro-1H-inden-1-one (5b)
Yield 84%, light yellow solid; m.p. 96-97 °C; H NMR (500 MHz, DMSO) δ 8.33
5,6-dimethoxy-2-((1-((6-methylpyridin-2-yl)methyl)piperidin-4-yl)methyl
)
1
(
s, 1H), 7.58 (d, J = 7.1 Hz, 1H), 7.31 – 7.15 (m, 1H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88
s, 3H), 3.80 (s, 3H), 3.66 (s, 2H), 3.24 (dd, J = 17.4, 7.9 Hz, 1H), 3.02 (s, 1H), 2.86
s, 2H), 2.67 (dt, J = 14.0, 5.9 Hz, 2H), 2.39 (s, 3H), 2.12 (s, 2H), 1.72 (ddd, J = 18.4,
(
(
1
3
1
2.8, 8.1 Hz, 2H), 1.63 (d, J = 10.5 Hz, 1H), 1.49 (s, 1H), 1.36 – 1.21 (m, 2H); C
NMR (125 MHz, DMSO) δ 206.96, 157.67, 155.75, 149.62, 149.17, 137.29, 132.21,
1
3
3
4
28.89, 122.11, 120.56, 108.69, 104.49, 56.43, 56.11, 53.67, 46.21, 45.21, 38.48,
+
3.15, 24.46, 14.33, 11.25, 9.17. HRMS (ESI) m/z 395.2331 [M+H] (calcd for
95.2329, C H N O ).
24
31
2
3
.1.1.18.
5,6-dimethoxy-2-((1-((3-methylpyridin-2-yl)methyl)piperidin-4-yl)methyl
)-2, 3-dihydro-1H-inden-1-one (5c)
1
Yield 82%, yellow solid; m.p. 96-97 °C; H NMR (500 MHz, DMSO) δ 7.68 (d, J
7.4 Hz, 1H), 7.27 (d, J = 7.4 Hz, 1H), 7.16 (d, J = 5.7 Hz, 1H), 7.11 (s, 1H), 7.07 (s,
=
1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.27 – 3.21 (m, 1H), 3.08 (d, J = 6.9 Hz, 2H), 2.98 –
2.88 (m, 1H), 2.68 (dd, J = 11.2, 6.4 Hz, 2H), 2.47 (s, 3H), 2.13 (s, 2H), 1.80 (d, J =
8.8 Hz, 1H), 1.76 – 1.63 (m, 2H), 1.52 (s, 1H), 1.31 – 1.24 (m, 2H), 1.27 - 1.12 (m,
2
6

ACCEPTED MANUSCRIPT
1
3
2
1
3
3
4
H); C NMR (125 MHz, DMSO) δ 206.96, 155.73, 149.61, 149.15, 146.16, 138.36,
33.27, 128.90, 123.00, 108.68, 104.48, 99.83, 56.42, 56.10, 53.87, 45.98, 45.24,
+
8.52, 33.93, 33.16, 18.32, 9.25. HRMS (ESI) m/z 395.2328 [M+H] (calcd for
95.2329, C H N O ).
24
31
2
3
.1.1.19.
2-((1-((3,5-dimethylpyridin-2-yl)methyl)piperidin-4-yl)methyl)-5,6-dimet
hoxy-2,3-dihydro-1H-inden-1-one (5d)
1
Yield 85%, yellow solid; m.p. 106-107 °C; H NMR (500 MHz, DMSO) δ 8.17 (s,
H), 7.41 (s, 1H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s, 3H), 3.80 (s, 3H), 3.24 (dd, J =
7.5, 7.9 Hz, 1H), 3.04 (d, J = 12.6 Hz, 1H), 2.88 (s, 2H), 2.67 (dt, J = 13.9, 5.6 Hz,
H), 2.35 (s, 3H), 2.26 (s, 3H), 1.87 – 1.56 (m, 4H), 1.50 (s, 1H), 1.33 – 1.10 (m, 5H);
C NMR (125 MHz, DMSO) δ 206.94, 155.74, 149.61, 149.14, 146.37, 142.98,
38.87, 132.59, 131.95, 128.89, 108.68, 104.48, 56.42, 56.10, 46.03, 45.22, 38.46,
1
1
2
1
3
1
3
4
4
+
3.84, 33.15, 24.39, 18.18, 17.89. HRMS (ESI) m/z 409.2484 [M+H] (calcd for
09.2686, C H N O ).
25
33
2
3
.1.1.20.
2-((1-((6-fluoropyridin-2-yl)methyl)piperidin-4-yl)methyl)-5,6-dimethoxy
-2,3-dihydro-1H-inden-1-one (5e)
1
Yield 88%, yellow solid; m.p. 95-97 °C; H NMR (500 MHz, DMSO) δ 7.97 (dd, J
=
16.0, 8.1 Hz, 1H), 7.40 (dd, J = 7.3, 1.8 Hz, 1H), 7.10 (s, 1H), 7.07 (s, 1H), 7.06 –
7
.02 (m, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.54 (s, 2H), 3.24 (dd, J = 17.7, 8.1 Hz, 1H),
.84 (t, J = 12.4 Hz, 2H), 2.72 – 2.63 (m, 2H), 2.04 (t, J = 10.9 Hz, 2H), 1.79 – 1.68
2
13
(
m, 2H), 1.64 (d, J = 12.5 Hz, 1H), 1.46 (d, J = 3.1 Hz, 1H), 1.30 – 1.16 (m, 3H); C
NMR (125 MHz, DMSO) δ 207.04, 161.90, 158.62, 155.74, 149.62, 149.18, 142.71,
2
7

ACCEPTED MANUSCRIPT
1
3
28.93, 120.83, 108.69, 107.94, 107.65, 104.50, 63.59, 56.41, 56.11, 53.89, 45.27,
+
8.70, 34.15, 33.17, 31.94. HRMS (ESI) m/z 399.2077 [M+H] (calcd for 399.2078,
C H FN O ).
23
28
2
3
4
.1.1.21.
2-((1-((6-chloropyridin-2-yl)methyl)piperidin-4-yl)methyl)-5,6-dimethox
y-2,3-dihydro-1H-inden-1-one (5f)
1
Yield 82%, light yellow solid; m.p. 114-117 °C; H NMR (500 MHz, DMSO) δ
.85 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.10 (s,
H), 7.07 (s, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.57 (s, 2H), 3.24 (dd, J = 17.6, 8.1 Hz,
H), 2.84 (t, J = 11.7 Hz, 2H), 2.73 – 2.61 (m, 2H), 2.03 (d, J = 10.7 Hz, 2H), 1.80 –
7
1
1
1
1
3
.69 (m, 2H), 1.47 (s, 1H), 1.27 (d, J = 3.2 Hz, 2H), 0.90 – 0.86 (m, 2H); C NMR
(125 MHz, DMSO) δ 207.01, 167.45, 155.75, 149.84, 149.63, 149.17, 140.58, 132.04,
1
3
29.14, 128.93, 122.89, 122.23, 108.69, 104.50, 67.92, 56.41, 56.11, 53.87, 45.26,
+
8.60, 33.17, 28.70, 22.81. HRMS (ESI) m/z 415.1780 [M+H] (calcd for 415.1783,
C H ClN O ).
23
28
2
3
4
.1.1.22.
2-((1-((6-bromopyridin-2-yl)methyl)piperidin-4-yl)methyl)-5,6-dimethox
y-2,3-dihydro-1H-inden-1-one (5g)
Yield 75%, yellow solid; m.p. 123-124 °C; H NMR (500 MHz, DMSO) δ 7.75 (d,
J = 7.7 Hz, 1H), 7.51 (dd, J = 18.2, 7.7 Hz, 2H), 7.10 (s, 1H), 7.07 (s, 1H), 3.88 (s,
1
3
H), 3.81 (s, 3H), 3.56 (s, 2H), 3.24 (dd, J = 17.7, 8.1 Hz, 1H), 2.83 (t, J = 12.6 Hz,
H), 2.68 (dd, J = 11.4, 6.1 Hz, 2H), 2.04 (t, J = 10.8 Hz, 2H), 1.73 (ddd, J = 13.0,
2
11.2, 7.5 Hz, 2H), 1.64 (d, J = 12.5 Hz, 1H), 1.47 (d, J = 3.3 Hz, 1H), 1.31 – 1.14 (m,
1
3
3
H); C NMR (125 MHz, DMSO) δ 207.00, 161.33, 155.73, 149.61, 149.16, 140.94,
2
8