Design, synthesis and biological evaluation of LX2343 derivatives as neuroprotective agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.12.080

A series of LX2343 derivatives were designed, synthesized and evaluated as neuroprotective agents for Alzheimer's disease (AD) in vitro. Most of the compounds displayed potent neuroprotective activities. Especially for compound A6, exhibited a remarkable EC₅₀ value of 0.22 μM. Further investigation demonstrated that compound A6 can significantly reduce Aβ production and increase Aβ clearance, and alleviate Tau hyperphosphorylation. Most importantly, compound A6 could ameliorate learning and memory impairments in APP/PS1 transgenic mice. The present study evidently showed that compound A6 is a potent neuroprotective agent and might serve as a promising lead candidate for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2017.12.080

Accepted Manuscript
Design, synthesis and biological evaluation of LX2343 derivatives as neuroprotective
agents for the treatment of Alzheimer's disease
Guanglong Sun, Junwei Wang, Xiaodan Guo, Min Lei, Yinan Zhang, Xiachang Wang,
Xu Shen, Lihong Hu
PII:
S0223-5234(17)31111-X
10.1016/j.ejmech.2017.12.080
EJMECH 10058
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2017
Revised Date: 12 December 2017
Accepted Date: 23 December 2017
Please cite this article as: G. Sun, J. Wang, X. Guo, M. Lei, Y. Zhang, X. Wang, X. Shen, L. Hu,
Design, synthesis and biological evaluation of LX2343 derivatives as neuroprotective agents for the
treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.080.
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Design, synthesis and biological evaluation of
LX2343 derivatives as neuroprotective
agents for the treatment of Alzheimer’s
disease
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Guanglong Sun1,2,#, Junwei Wang1,#, Xiaodan Guo2, Min Lei2, Yinan Zhang1, Xiachang Wang1, Xu Shen1, , Lihong Hu1,2,
EC50: 15.8 µM
EC50: 0.22 µM

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European Journal of Medicinal Chemistry
Journal homepage: www.elsevier.com
Design, synthesis and biological evaluation of LX2343 derivatives as neuroprotective
agents for the treatment of Alzheimer’s disease
Guanglong Sun1,2,#, Junwei Wang1,#, Xiaodan Guo2, Min Lei2, Yinan Zhang1, Xiachang Wang1, Xu Shen1, ,
Lihong Hu1,2,
1Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
Industrialization, Stake Key Laboratory Cultivation Base for TCM Quality and Efficacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing,
210023, PR China
2State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China; University of Chinese
Academy of Sciences, Beijing 100049, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of LX2343 derivatives were designed, synthesized and evaluated as neuroprotective
agents for Alzheimer's disease (AD) in vitro. Most of the compounds displayed potent
neuroprotective activities. Especially for compound A6, exhibited a remarkable EC50 value of
0.22 µM. Further investigation demonstrated that compound A6 can significantly reduce Aβ
production and increase Aβ clearance, and alleviate Tau hyperphosphorylation. Most
importantly, compound A6 could ameliorate learning and memory impairments in APP/PS1
transgenic mice. The present study evidently showed that compound A6 is a potent
neuroprotective agent and might serve as a promising lead candidate for the treatment of
Alzheimer's disease.
Available online
Keywords:
LX2343 derivatives
Neuroprotective agents
Alzheimer’s disease
structural modifications
Structure-activity relationships
2017 Elsevier Ltd. All rights reserved
.
1. Introduction
the progression. The consecutive failures in clinical trials for
dozens of anti-AD agents suggested that the agent targeting
single target might not work effectively on this complicated
pathogenesis [7,8]. Several factors including low levels of
neurotransmitter acetylcholine (ACh), oxidative stress (OS),
dyshomeostasis of biometals, the aggregation of β-amyloid
peptide and hyperphosphorylation of tau protein have been
considered to play significant roles in the pathogenesis of AD [9-
11]. Therefore, a new strategy is urgently needed for the
discovery of anti-AD drugs.
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder in the elderly population characterized by irreversible
cognitive impairment, disorientation and personality changes,
and ultimately causing death [1]. The apoptosis of neurons, the
accumulation of amyloid β (Aβ) plaques and the formation of
neurofibrillary tangles (NFTs) in the brain are the typical
hallmarks of AD [2]. There is approximately 36 million people
suffering from AD worldwide, and the figure is projected to grow
to 114 million by 2050 [3,4]. Due to complex nature and
multitude of factors potentially involved in pathogenesis, many
aspects of AD are not yet fully known, to date, an effective
treatment for this disease has not been found.
Recent evidence indicates that failing to repair the damaged
neurons may be the fundamental reason [12-15]. Neuroprotection
is a valuable tool of modern medicine to potentially combat or
slow down the progression of AD [16-22]. Therefore, the
successful protection of neuronal cells from damage may
potentially prevent AD [23-29]. In our previous work [30,31], the
preliminary high throughput screening (HTS) lead to the
discovery of a small molecule compound LX2343 which
exhibited moderate neuroprotective activities with the EC50 value
Currently, the therapeutic options for the treatment of AD are
limited to three acetylcholinesterase (AChE) inhibitors donepezil,
rivastigmine, galantamine and one N-methyl-D-aspartate receptor
antagonist, memantine [5,6]. These drugs are only able to treat
symptoms of the disease by improving cognitive, behavioral and
functional impairments. However, they do little to stop or reverse
#
Corresponding author. e-mail: xshen@simm.ac.cn (X. Shen); e-mail: lhhu@njucm.edu.cn (L. Hu); G. Sun and J. Wang contributed
equally.

2. Results and discussion
of 15.8 µM. Further investigation showed thatALXC2C34E3 PwTasEabDle MANUSCRIPT
to effectively reverse the streptozocin (STZ)-induced neuronal
apoptosis and tau hyperphosphorylation in vitro and in vivo, and
this neuroprotective effect of LX2343 was ascribed to its
function in the inhibition of OS and tauopathy. LX2343
alleviated Aβ levels by both activating its clearance and
inhibiting its production under STZ-induced pathological
conditions. Moreover, assays in APP/PS1 transgenic AD model
mice verified the amelioration of AD-relevant pathogenesis and
cognitive deficits by LX2343. These results demonstrated that
LX2343 was a multifunctional agent which exhibits potential
capability for ameliorating multi-abnormalities of AD
pathogenesis. However, the neuroprotective activity of LX2343
was a little weak and the structure-activity relationship (SAR)
has not been studied yet. In order to search novel and more
potent neuroprotective agent, LX2343 was chosen as the lead
compound for further structural optimization.
2.1. Design of novel neuroprotective agent
LX2343 was obtained through high throughput screening and
the structure-activity relationship was not clear. In this paper, we
focus on the structural modifications of LX2343 to find more
potent neuroprotective agent (Fig. 1). Firstly, considering size,
electron density and structure type, different substituent groups
were introduced to modify the part A, including aliphatic amine,
aromatic amine and heterocycle-aromatic amine (Table 1). After
the optimal R1 group was determined, modification was continue
on part B, such as introducing different substituents on the
benzene ring or replacing sulfonyl with carbonyl (Table 2).
Finally, part C was modified by introducing different substituents
on the benzene ring or replacing the aromatic ring with aliphatic
ring and chain (Table 3).
Fig. 1. Design of novel neuroprotective agents
2.2. Chemistry
afford the target compounds A1-A24 in moderate to good yields
(Scheme 1). Compounds B1-B4 were prepared according to
Scheme 2. Treatment of 3,5-dimethyl amantadine with
chloroacetyl chloride produced the corresponding amide 6 which
reacted with 2-methoxy-5-chloro aniline giving intermediate 7.
Then, 7 was coupled with different sulfonyl chloride or acryl
chlorides in the presence of DMAP to give B1-B4. The synthesis
of compounds C1-C7 is similar to that of B1-B4, coupling 6 with
different amines and then reaction with benzene sulfonyl chloride
gave the target compounds (Scheme 3). All the target compounds
were purified by chromatographic techniques (see the experiment
section for details) and structurally characterized by their
spectroscopic data (1H-NMR, 13C-NMR, MS and HRMS).
The synthetic routes for the designed thirty-five targeted
compounds were depicted in Scheme 1-3. Firstly, reaction of
commercially available ethyl bromoacetate and 5-chloro-2-
methoxyaniline gave intermediate 2. Sulfonylation of 2 with
benzenesulfonyl
chloride
in
pyridine
using
dimethylaminopyridine (DMAP) as catalytic agent provided the
product 3, which was further hydrolyzed to give the
corresponding acid 4. Then, it was activated with 2-(7-aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
phosphate (HATU) and subsequently coupled with different
hexafluoro-
amines in the presence of N,N-diisopropylethylamine (DIPEA) to
Scheme 1. Reagents and conditions: (a) 5-chloro-2-methoxyaniline, DIPEA, CH3CN, reflux, 10 h, 90%; (b) benzenesulfonyl chloride, DMAP,
Py, r.t., 6 h, 87%; (c) NaOH, H2O/THF/MeOH, r.t., 1 h, 94%; (d) amines, HATU, DIPEA, r.t., 1 h, 46-94%
.
Scheme 2. Reagents and conditions: (a) chloroacetyl chloride, TEA, CH2Cl2, r.t., 2 h, 99%; (b) 2-methoxy-5-chloro aniline, Et3N, NaI, EtOH,
reflux, 10 h, 54%; (c) sulfonyl chlorides or acryl chlorides, DMAP, Py, r.t., 10 h, 66-90%.
Scheme 3. Reagents and conditions: (a) amines, Et3N, NaI, EtOH, reflux, 10 h; (b) benzene sulfonyl chloride, DMAP, Py, r.t., 10 h, 18-68%
( two steps).

2.3. Inhibition of apoptosis in SH-SY5Y cells inAducCedCbEy PSTTZED MANUSCRIPT
A7
> 20
The protective activity of the thirty-five compounds against
STZ-induced apoptosis was evaluated in SH-SY5Y cells and the
results were shown in Table 1-3. Most of the compounds showed
O
H
N
A8
15.50 ± 1.28
potent neuroprotection against STZ-induced cells death. On the
basis of these results, following SAR has been derived to analyze
the influence of the modifications on part A、part B and part C.
When changing the R1 substituent, the amide compounds with
aliphatic groups showed similar potency to LX2343 (EC50: 15.80
µM), except for compound A6 which bearing a memantine group.
It exhibited a great increase in neuroprotection potency with the
EC50 value of 0.22 µM. Aromatic amine substituted compounds
A12 and A13 which bearing an electron-withdrawing group at
A9
17.60 ± 1.35
16.20 ± 1.10
> 20
A10
the para-position of the benzene ring displayed higher potency
than LX2343. Compounds with hetero-aromatic ring (A19-A24)
also showed potent activity, especially for compound A22, it
displayed the most potent activity with a significant EC50 value of
0.25 µM. However, the activities of B1-B4 decreased a lot by
changing the R2 substituent, which means that benzenesulfonyl is
very important for the activity and not suitable for modification
any more. The results of C1-C7 showed that the chlorine on
aromatic ring is the necessary group to maintain the activity. The
location of chlorine also affect the activity, among which, meta-
position > ortho-position > para-position (C4 > C3 > C5). In
addition, introducing a methyl on the para-position can further
improve the activity, for example, C7 showed stronger activity
than its parent compound C3. In general, the results of
modifications on part B and part C are not ideal, indicating that
these two parts are not suitable for structural modification. The
good news is that in the modification of part A, several
compounds with high potency were obtained. Considering the
potency and the structure types, compounds A3, A6, A13, A22
and A24 were selected for further investigation.
A11
A12
A13
8.21 ± 0.54
3.75 ± 0.28
> 20
O
H
N
A14
Cl
A15
A16
> 20
> 20
H
N
Table 1
The structure and the effects of compounds A1-A24 on STZ-induced
cell death in SH-SY5Y cells.
A17
A18
17.43 ± 2.06
> 20
Compd
NHR1/ R1NR1
EC50 (µM)a
> 20
A19
2.22 ± 0.25
A1
A2
A3
> 20
A20
A21
2.75 ± 0.31
1.32 ± 0.10
14.52 ± 1.08
N
H
A4
A5
A6
17.55 ± 1.45
14.26 ± 1.72
0.22 ± 0.02
A22
A23
0.25 ± 0.02
6.56 ± 0.43
N

Cl
ACCEPTED MANUSCRIPT
8.86 ± 0.49
C-7
A24
3.18 ± 0.21
aEC50 was measured by the MTT assay with six concentrations (1,
2.5, 5, 10, 15, 20 µM). Data shown are means ± SEM.
LX2343
15.80 ± 1.81
aEC50 was measured by the MTT assay with six concentrations (1,
2.5, 5, 10, 15, 20 µM). Data shown are means ± SEM.
2.4. Inhibition of Aβ production and promotion of Aβ clearance
Table 2
In order to investigate the potential to alleviate Aβ burden
under pathological conditions by LX2343 derivatives, STZ was
used as a stimulator and ELISA assays were performed in
HEK293-APPsw cells. ELISA results showed that LX2343
derivatives can effectively antagonize Aβ deposition in HEK293-
APPsw cells at the concentration of 20 µM. Among these
derivatives, compound A6 exhibited the most potent inhibition of
Aβ production (Fig. 2A). Aβ in the brain exists in a dynamic
equilibrium of Aβ production and clearance, so the potential
effect of LX2343 derivatives on exogenous Aβ clearance was
also investigated in SH-SY5Y cells. The results indicated that
compounds A6, A13, A22 and A24 can enhance exogenous Aβ
clearance, and the best effect was found for compound A6 which
was better than LX2343 (Fig. 2B). These results suggested that
our compounds could effectively inhibit STZ-induced Aβ
production and promote Aβ clearance.
The structure and the effects of compounds on STZ-induced cell
death in SH-SY5Y cells.
Compd
R2
EC50 (µM)a
> 20
B-1
> 20
> 20
> 20
B-2
B-3
B-4
aEC50 was measured by the MTT assay with six concentrations (1,
2.5, 5, 10, 15, 20 µM).
Fig. 2. LX2343 and its analogues effectively reversed STZ-induced
Aβ accumulation and promoted Aβ clearance. (A) ELISA assays of
compounds-induced Aβ decrease in HEK293-APPsw cells. (B)
ELISA results indicated that compounds increased Aβ clearance in
SH-SY5Y cells (one-way ANOVA, Dunnett’s multiple comparison
test. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 vs STZ). All data
were obtained from three independent experiments and are presented
as the mean ± SEM.
Table 3
The structure and the effects of compounds on STZ-induced cell
death in SH-SY5Y cells.
Compd
R3
EC50 (µM)a
> 20
2.5. Inhibition of Tau hyperphosphorylation
Considering the association of OS with tauopathy, we
examined the potential of compounds A3, A6, A13 and A22 (at
20 µM) to suppress Tau hyperphosphorylation at residue Ser396
in SH-SY5Y cells. As expected, the results indicated that
compounds A6 and A13 can efficiently attenuate the STZ-
induced elevation of Tau phosphorylation (Fig. 3). Among these
compounds, A6 showed the strongest activity to inhibit the Tau
hyperphosphorylation. Thus, compound A6 was selected for
further in vivo evaluation.
C-1
> 20
16.11 ± 1.88
12.30 ± 1.44
> 20
C-2
C-3
C-4
C-5
C-6
Cl
Fig. 3. LX2343 and its analogues ameliorated tau pathology. Western
blot and quantification results demonstrated that compounds reduced
tau phosphorylation at sites of serine 396 in SH-SY5Y cells (one-
way ANOVA, Dunnett’s multiple comparison test, *P < 0.05, **P <
0.01, ***P < 0.001 versus STZ). All values were presented as the
> 20

mean ± SEM (n = 3). GAPDH was used as the loading control in the
Western blot assays. Data were obtained from three independent
3. Conclusions
ACCEPTED MANUSCRIPT
In conclusion, to develop effective drugs for the treatment of
Alzheimer's disease (AD), a series of LX2343 derivatives were
designed, synthesized and evaluated. Among all the tested
compounds, compounds A6 and A22 showed very potent
neuroprotective effect with the EC50 values of 0.22 and 0.17 µM,
respectively, which were much stronger than LX2343 (EC50:
15.80 µM). ELISA assays and western blot results demonstrated
that compound A6 can significantly reduce Aβ production,
increase Aβ clearance and alleviate Tau hyperphosphorylation. In
addition, administration of compound A6 in APP/PS1 transgenic
mice also effectively improved learning and memory
impairments. Taking together, all these results suggest that
compound A6 could be considered as a potential neuroprotective
agent for the treatment of AD.
experiments.
2.6. In vivo anti-AD activity
APP/PS1 mice which express chimeric human Swedish
mutant APP and a mutant human presenilin 1 protein are widely
used as an effective animal model for AD dementia [32]. In this
model, it is evaluated the amelioration of memory impairment by
compound A6 using the Morris Water Maze (MWM) test.
As depicted in Fig. 4, the results revealed that in six-day
training trials, the path lengths and escape latencies used to find
the platform for APP/PS1 transgenic mice were remarkably
longer than those for non-transgenic mice, while 30 mg/kg
compound A6 administration obviously antagonized the
prolonged path lengths and escape latencies on the sixth day
(Figure 4A, 4B). In the probe trial assay, the compound A6-
administered transgenic mice crossed over the hidden location of
the platform more frequently compared with the vehicle-
administered transgenic mice (Figure 4C, 4D). The animal
experiments were carried out with two different doses of
compound A6, 15 and 30 mg/kg. More obvious improvement
was detected in memory and learning in the high-dose treatment
group, it indicated the dose-dependent effect of compound A6 in
vivo. Additionally, there are no apparent changes in body weight,
liver function or swimming speed of the tested mice after the
treatment of compound A6 (data not shown).
4. Experimental section
4.1. Materials and physical measurements
All cell culture reagents were purchased from Gibco
(Invitrogen,
USA).
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), STZ, wortmannin,
SB216763 and chloroquine were purchased from Sigma-Aldrich
company (USA). All chemical reagents were purchased from
Sinopharm Chemical Reagent Co., Ltd. (China), Alfa Aesar,
Adamas-beta, J&K and TCI. All solvents were used without
further purifications. Reaction progress was monitored using
analytical thin layer chromatography (TLC) on precoated silica
gel GF254 (Yantai, China) plates and the spots were detected
under UV light (254 nm). Column chromatography was
performed on silica gel (200-300 mesh) from Yantai (China).
Melting point were recorded on a WRS-1B melting point
1
apparatus and uncorrected. H NMR and 13C NMR spectra were
measured in DMSO-d6 or CDCl3 solutions using an AVANCE III
400 spectrometer. Chemical shifts are reported in δ scale (ppm)
relative to internal tetramethylsilane (TMS), J values are given in
Hertz, and spin multiplicities are expressed as s (singlet), d
(doublet), t (triplet), or m (multiplet). Low and high-resolution
mass spectra were obtained in the ESI mode.
4.2. Chemistry
4.2.1. General procedure for the synthesis of 2
To a stirred solution of ethyl bromoacetate 1 (4.0 g, 24 mmol)
and DIPEA (4.01 g, 24 mmol) in CH3CN (100 mL) was added 5-
chloro-2-methoxyaniline (3.14 g, 20 mmol) at room temperature.
The mixture was heated to reflux for 10 h, and TLC indicated
that the reaction was completed. Then the reaction mixture was
evaporated to dryness under vacuum. The obtained residue was
further purified by silica gel chromatography (petroleum ether
(PE)/ethyl acetate (EtOAc) = 5/1) to give 2 as a yellow oil (Yield
90%). 1H NMR (400 MHz, CDCl3) δ (ppm): 6.62 (s, 2H), 6.40 (s,
1H), 4.90 (t, J = 5.2 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 3.84 (d, J
= 5.2 Hz, 2H), 3.78 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H); ESI-MS
(m/z): 244.6 [M+H]+; HRMS (ESI): calcd for C11H14ClNO3
[M+H]+ 244.0735, found 244.0729.
Fig. 4. Compound A6 effectively improved learning and memory
impairments in APP/PS1 transgenic mice. Behavioral tests and
quantitative analyses of the APP/PS1 transgenic mice. (A)
Representative tracing graphs showing the training trials. (B) Escape
latency during the platform trials in the MWM tests (two-way
ANOVA with repeated measures over time: treatment, P < 0.0001;
time, P < 0.0001; treatment×time. ***P < 0.001 vs TV. n = 10). (C)
Representative tracing graphs of the probe trials. (D) Times of the
platform crossings in the probe trials (t test, *P < 0.05 vs TV. n = 10).
NV: non-transgenic mice administered vehicle, TV: Transgenic mice
administered vehicle, A6: transgenic mice administered with 15
mg.kg-1.d-1 (A6-L) and 30 mg.kg-1.d-1 (A6-H). Values are expressed
as the mean±SEM.
4.2.2. General procedure for the synthesis of 3
Benzenesulfonyl chloride (3.52 g, 20 mmol) was added
dropwise to a solution of 2 (2.43 g, 10 mmol) and DMAP (0.61 g,
5 mmol) in pyridine (50 mL) at room temperature. The mixture
was continuously stirred at room temperature for 6 h, and TLC
indicated that the reaction was completed. Then the reaction was
concentrated and diluted with CH2Cl2 (50 mL) and H2O (50 mL).
The aqueous layer was separated and the organic layer was

washed with saturated Na2CO3 solution (2 × 50 mL), brine (50
(2C), 128.7, 127.8 (2C), 125.7, 113.3, 55.8, 54.4, 41.2, 22.6, 11.3;
ESI-MS (m/z): 397.3 [M+H]+; HRMS (ESI): calcd for
C18H21ClN2O4S [M+H]+ 397.0983, found 397.0996.
ACCEPTED MANUSCRIPT
mL) and dried over anhydrous Na2SO4. After filtered, the solvent
was evaporated to dryness under vacuum. The residue was
further purified by silica gel chromatography (PE/EtOAc = 3/1)
4.2.7. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N,N-
diethylacetamide (A2)
1
to give 3 as a white solid (Yield 87%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.68 (d, J = 8.0 Hz, 2H), 7.55-7.59 (m, 2H),
7.46 (t, J = 8.0 Hz, 2H), 7.26 (dd, J = 8.8, 2.4 Hz, 1H), 6.70 (d, J
= 8.8 Hz, 1H), 4.38 (s, 2H), 4.17 (q, J = 7.2 Hz, 2H), 3.37 (s, 3H),
1.26 (t, J = 7.2 Hz, 3H); ESI-MS (m/z): 384.6 [M+H]+; HRMS
(ESI): calcd for C17H18ClNO5S [M+H]+ 384.0667, found
384.0668.
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 84%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.73-7.68 (m, 2H), 7.56 (t, J = 7.4 Hz, 1H),
7.50-7.43 (m, 3H), 7.22 (dd, J = 8.8, 2.7 Hz, 1H), 6.69 (d, J = 8.9
Hz, 1H), 4.45 (s, 2H), 3.43 (s, 3H), 3.39 (q, J = 7.1 Hz, 2H), 3.29
(q, J = 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H), 1.00 (t, J = 7.1 Hz,
3H); 13C NMR (101 MHz, DMSO-d6) δ (ppm): 166.3, 155.4,
140.2, 133.8, 133.2, 130.0, 129.3 (2C), 127.9, 127.5 (2C), 123.3,
114.1, 56.0, 50.8, 40.9, 40.6, 14.4, 13.2; ESI-MS (m/z): 411.5
[M+H]+; HRMS (ESI): calcd for C19H23ClN2O4S [M+H]+
411.1140, found 411.1152.
4.2.3. General procedure for the synthesis of 4
3 (3.83 g, 10 mmol) and NaOH (0.80 g, 20 mmol) was added
to a mixed solution of H2O/THF/MeOH (2/2/1) (20 mL) at room
temperature. The mixture was continuously stirred at room
temperature for 1 h, and TLC indicated that the reaction was
completed. Then the reaction was concentrated and diluted with
H2O (15 mL). When the solution was acidified to pH 3-4 by
hydrochloric acid, a large amount of white solid dissolve out.
After filtered, the residue was washed with H2O, and dried to
afford 4 as a white solid (Yield 94%). 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 7.63-7.69 (m, 3H), 7.56 (t, J = 7.6 Hz, 2H),
7.39-7.41 (m, 2H), 6.98 (d, J = 9.6 Hz, 1H), 4.27 (s, 2H), 3.29 (s,
3H); ESI-MS (m/z): 356.7 [M+H]+; HRMS (ESI): calcd for
C15H14ClNO5S [M+H]+ 356.0354, found 356.0350.
4.2.8. N-(5-chloro-2-methoxyphenyl)-N-(2-oxo-2-(piperidin-1-
yl)ethyl)benzenesulfonamide (A3)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 92%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.73-7.65 (m, 2H), 7.55 (t, J = 7.4 Hz, 1H),
7.50-7.41 (m, 3H), 7.22 (dd, J = 8.8, 2.7 Hz, 1H), 6.68 (d, J = 8.9
Hz, 1H), 4.46 (s, 2H), 3.52-3.42 (m, 4H), 3.38 (s, 3H), 1.68-1.60
(m, 4H), 1.55-1.45 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 165.6, 155.0, 139.6, 133.6, 132.5, 129.8, 128.5 (2C),
127.6 (2C), 127.3, 125.1, 112.4, 55.4, 51.2, 46.1, 43.1, 26.3, 25.4,
24.3; ESI-MS (m/z): 423.3 [M+H]+; HRMS (ESI): calcd for
C20H23ClN2O4S [M+H]+ 423.1140, found 423.1141.
4.2.4. General procedure for the synthesis of LX2343 and A1-24
To a stirred solution of 4 (355 mg, 1 mmol) and HATU (456
mg, 1.2 mmol) in CH2Cl2 (6 mL) was added DIPEA (155 mg, 1.2
mmol) and the appropriate amine (1 mmol) at room temperature.
The mixture was continuously stirred for 1 h, and TLC indicated
that the reaction was completed. Then the reaction mixture was
concentrated and diluted with CH2Cl2 (10 mL) and H2O (10 mL).
The aqueous layer was separated and the organic layer was
washed with saturated Na2CO3 solution (2 × 10 mL), brine (10
mL) and dried over anhydrous Na2SO4. After filtered, the solvent
was evaporated to dryness under vacuum to obtain the desired
crude products. The appropriate compounds (LX2343 and A1-24)
were obtained following purification by silica gel
chromatography.
4.2.9. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
cyclohexylacetamide (A4)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a brown solid (Yield 86%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.71-7.61 (m, 3H), 7.52 (t, J = 7.7 Hz, 2H), 7.29
(dd, J = 8.9, 2.6 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.89 (d, J =
2.6 Hz, 1H), 6.84 (d, J = 8.9 Hz, 1H), 4.08 (s, 2H), 3.78-3.68 (m,
1H), 3.66 (s, 3H), 1.92-1.82 (m, 2H), 1.78-1.68 (m, 2H), 1.66-
1.56 (m, 1H), 1.45-1.29 (m, 2H), 1.28-1.10 (m, 3H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 166.7, 155.1, 137.7, 133.3, 130.3,
130.2, 128.9 (2C), 128.8, 127.9 (2C), 125.6, 113.3, 55.8, 54.5,
48.2, 32.8 (2C), 25.5, 24.7 (2C); ESI-MS (m/z): 437.6 [M+H]+;
HRMS (ESI): calcd for C21H25ClN2O4S [M+H]+ 437.1296, found
437.1288.
4.2.5. N-(benzo[d][1,3]dioxol-5-yl)-2-(N-(5-chloro-2-
methoxyphenyl)phenylsulfonamido)acetamide (LX2343)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 92%). H NMR (400 MHz,
4.2.10. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-
CDCl3) δ (ppm): 9.08 (s, 1H), 7.74-7.68 (m, 3H), 7.57 (t, J = 8.4
Hz, 2H), 7.33 (dd, J = 8.8, 2.4 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H),
6.92 (d, J = 8.8 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 6.85 (dd, J =
8.4, 2.0 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 5.97 (s, 2H), 4.21 (s,
2H), 3.79 (s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 165.9,
155.4, 147.9, 144.6, 137.5, 133.7, 131.7, 130.6, 130.0, 129.2 (3C),
128.1 (2C), 126.1, 113.9, 113.2, 108.2, 102.8, 101.4, 56.6, 55.3;
ESI-MS (m/z): 475.5 [M+H]+; HRMS (ESI): calcd for
C22H19ClN2O6S [M+H]+ 475.0725, found 475.0729.
N,N-dicyclohexylacetamide (A5)
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a white solid (Yield 78%). H NMR (400 MHz,
1
CDCl3) δ (ppm): 7.70 (d, J = 7.6 Hz, 2H), 7.57 (t, J = 7.6 Hz, 1H),
7.44-7.49 (m, 3H), 7.24 (dd, J = 8.8, 2.4 Hz, 1H), 6.70 (d, J = 8.8
Hz, 1H), 4.41 (s, 2H), 3.76-3.79 (m, 1H), 3.39 (s, 3H), 2.91-2.95
(m, 1H), 1.72-1.89 (m, 6H), 1.12-1.56 (m, 12H); 13C NMR (101
MHz, CDCl3) δ (ppm): 165.9, 155.1, 139.6, 133.5, 132.4, 129.8,
128.4 (2C), 127.6 (2C), 127.1, 124.9, 112.4, 57.3, 56.1, 55.3,
52.5, 31.5 (2C), 29.6 (2C), 26.5 (2C), 25.8 (2C), 25.3, 25.1; ESI-
MS (m/z): 519.9 [M+H]+; HRMS (ESI): calcd for C27H35ClN2O4S
[M+H]+ 519.2079, found 519.2075.
4.2.6. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
propylacetamide (A1)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 88%). H NMR (400 MHz,
4.2.11. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)acetamide (A6)
CDCl3) δ (ppm): 7.70-7.60 (m, 3H), 7.52 (t, J = 7.7 Hz, 2H), 7.30
(dd, J = 8.9, 2.6 Hz, 1H), 6.93 (d, J = 2.6 Hz, 1H), 6.84 (d, J =
8.9 Hz, 1H), 4.10 (s, 2H), 3.63 (s, 3H), 3.24 (q, J = 6.9 Hz, 2H),
1.60-1.48 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ (ppm): 167.8, 155.0, 137.7, 133.4, 130.5, 130.2, 128.9
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 94%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.64-7.70 (m, 3H), 7.54 (t, J = 7.6 Hz, 2H), 7.31
1

55.4, 55.2; ESI-MS (m/z): 461.2 [M+H]+; HRMS (ESI): calcd for
(dd, J = 8.8, 2.4 Hz, 1H), 6.93 (d, J = 2.4 Hz,A1HC),C6.E92P(Ts,E1HD), MANUSCRIPT
6.86 (d, J = 8.8 Hz, 1H), 3.99 (s, 2H), 3.65 (s, 3H), 2.15-2.18 (m,
1H), 1.84 (d, J = 2.0 Hz, 2H), 1.67 (d, J = 11.2 Hz, 2H), 1.61 (d,
J = 11.2 Hz, 2H), 1.41 (d, J = 12 Hz, 2H), 1.31 (d, J = 12 Hz,
2H), 1.13-1.22 (m, 2H), 0.87 (s, 6H); 13C NMR (101 MHz,
CDCl3) δ (ppm): 166.7, 155.1, 137.8, 133.3, 130.4, 130.1, 128.9
(3C), 127.9 (2C), 125.6, 113.3, 55.7, 55.0, 53.5, 50.5, 47.3 (2C),
42.5 (2C), 39.8, 32.3 (2C), 30.1, 30.0 (2C); ESI-MS (m/z) 517.8
[M+H]+; HRMS (ESI): calcd for C27H33ClN2O4S [M+H]+
517.1922, found 517.1928.
C22H21ClN2O5S [M+H]+ 461.0932, found 461.0940.
4.2.16. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(4-chlorophenyl)acetamide (A11)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 74%). H NMR (400 MHz,
CDCl3) δ (ppm): 9.21 (s, 1H), 7.68-7.74 (m, 3H), 7.51-7.60 (m,
4H), 7.31-7.35 (m, 3H), 6.93 (d, J = 8.8 Hz, 1H), 6.88 (d, J = 2.8
Hz, 1H), 4.22 (s, 2H), 3.79 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ (ppm): 166.1, 155.2, 137.3, 136.0, 133.7, 130.5, 130.0, 129.5,
129.1 (2C), 129.0 (3C), 128.0 (2C), 126.0, 124.6, 121.1 (2C),
113.8, 56.4, 55.3; ESI-MS (m/z): 465.4 [M+H]+; HRMS (ESI):
calcd for C21H18Cl2N2O4S [M+H]+ 465.0437, found 465.0441.
4.2.12. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(4-methoxybenzyl)acetamide (A7)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 90%). H NMR (400 MHz,
4.2.17. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(4-nitrophenyl)acetamide (A12)
CDCl3) δ (ppm): 7.69-7.61 (m, 3H), 7.57 (t, J = 4.9 Hz, 1H), 7.52
(t, J = 7.7 Hz, 2H), 7.27 (dd, J = 8.9, 2.5 Hz, 1H), 7.14 (d, J = 8.6
Hz, 2H), 6.91-6.84 (m, 2H), 6.82 (d, J = 2.6 Hz, 1H), 6.74 (d, J =
8.9 Hz, 1H), 4.38 (d, J = 5.4 Hz, 2H), 4.15 (s, 2H), 3.81 (s, 3H),
3.30 (s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 167.6, 159.1,
155.0, 137.6, 133.4, 130.3, 130.2, 129.6, 129.1 (2C), 129.0 (2C),
128.7, 127.9 (2C), 125.6, 114.1, 113.2, 55.4, 55.3, 54.5, 43.1;
ESI-MS (m/z) 475.8 [M+H]+; HRMS (ESI): calcd for
C23H23ClN2O5S [M+H]+ 475.1089, found 475.1092.
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 70%). H NMR (400 MHz,
CDCl3) δ (ppm): 9.54 (s, 1H), 8.24 (d, J = 9.1 Hz, 2H), 7.78-7.67
(m, 5H), 7.58 (t, J = 7.7 Hz, 2H), 7.34 (dd, J = 8.9, 2.5 Hz, 1H),
6.94 (d, J = 8.9 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 4.22 (s, 2H),
3.82 (s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 166.7, 155.2,
143.8, 143.2, 137.0, 133.8, 130.7, 129.8, 129.2 (2C), 129.0, 128.0
(2C), 126.3, 125.1 (2C), 119.2 (2C), 114.1, 56.7, 55.5; ESI-MS
(m/z): 476.4 [M+H]+; HRMS (ESI): calcd for C21H18ClN3O6S
[M+H]+ 476.0678, found 476.0671.
4.2.13. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(2-methoxyphenyl)acetamide (A8)
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 76%). H NMR (400 MHz,
4.2.18. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(4-(trifluoromethyl)phenyl)acetamide (A13)
1
CDCl3) δ (ppm): 9.18 (s, 1H), 8.29 (dd, J = 7.9, 1.4 Hz, 1H),
7.71-7.65 (m, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.53 (d, J = 2.6 Hz,
1H), 7.49 (t, J = 7.7 Hz, 2H), 7.28 (dd, J = 8.9, 2.6 Hz, 1H), 7.08
(td, J = 7.8, 1.6 Hz, 1H), 6.95 (t, J = 8.5 Hz, 2H), 6.70 (d, J = 8.9
Hz, 1H), 4.36 (s, 2H), 4.04 (s, 3H), 3.32 (s, 3H); 13C NMR (101
MHz, CDCl3) δ (ppm): 166.1, 154.4, 148.4, 138.3, 133.1, 132.7,
130.3, 128.7 (2C), 127.8 (2C), 127.5, 127.0, 125.3, 124.3, 120.9,
119.9, 112.8, 110.2, 56.0, 55.2, 54.3; ESI-MS (m/z): 461.2
[M+H]+; HRMS (ESI): calcd for C22H21ClN2O5S [M+H]+
461.0932, found 461.0940.
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 50%). H NMR (400 MHz,
1
DMSO-d6) δ (ppm): 9.99 (s, 1H), 8.36 (d, J = 1.2 Hz, 1H), 8.08
(d, J = 8.0 Hz, 1H), 7.69-7.73 (m, 3H), 7.39-7.63 (m, 8H), 7.17 (t,
J = 8.0 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 4.38-4.45 (m, 4H), 3.38
(s, 3H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 166.4, 155.2, 140.5, 137.2, 133.7, 130.6, 129.8, 129.2
(2C), 129.0, 128.0 (2C), 126.3 (2C), 126.2 (2C), 126.1, 119.4
(2C), 113.9, 56.5, 55.4; ESI-MS (m/z): 499.4 [M+H]+; HRMS
(ESI): calcd for C22H18ClF3N2O4S [M+H]+ 499.0701, found
499.0707.
4.2.14. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(3-methoxyphenyl)acetamide (A9)
4.2.19. N-(5-chloro-2-methoxyphenyl)-2-(N-(5-chloro-2-
methoxyphenyl)phenylsulfonamido)acetamide (A14)
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 90%). H NMR (400 MHz,
1
CDCl3) δ (ppm): 9.12 (s, 1H), 7.72 (d, J = 7.9 Hz, 2H), 7.68 (t, J
= 7.5 Hz, 1H), 7.55 (t, J = 7.5 Hz, 2H), 7.31 (d, J = 9.0 Hz, 1H),
7.28-7.20 (m, 2H), 7.03 (d, J = 7.8 Hz, 1H), 6.93-6.84 (m, 2H),
6.69 (d, J = 7.4 Hz, 1H), 4.21 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H);
13C NMR (101 MHz, CDCl3) δ (ppm): 166.0, 160.1, 155.2, 138.6,
137.4, 133.6, 130.5, 129.9, 129.7, 129.1 (2C), 129.0, 128.0 (2C),
126.0, 113.8, 112.0, 110.4, 105.6, 56.4, 55.4, 55.3; ESI-MS (m/z):
461.2 [M+H]+; HRMS (ESI): calcd for C22H21ClN2O5S [M+H]+
461.0932, found 461.0937.
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a white solid (Yield 84%). H NMR (400 MHz,
1
CDCl3) δ (ppm): 9.21 (s, 1H), 8.39 (d, J = 2.4 Hz, 1H), 7.61-7.69
(m, 3H), 7.49-7.53 (m, 3H), 7.31 (dd, J = 8.8, 2.4 Hz, 1H), 7.05
(dd, J = 8.8, 2.4 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 6.73 (d, J =
8.8 Hz, 1H), 4.36 (s, 2H), 4.05 (s, 3H), 3.33 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 166.3, 154.3, 147.0, 138.2, 133.2,
132.6, 130.4, 128.8 (2C), 127.9, 127.8 (2C), 127.5, 126.0, 125.3,
123.7, 119.7, 112.9, 111.0, 56.3, 55.3, 54.3; ESI-MS (m/z): 495.4
[M+H]+; HRMS (ESI): calcd for C22H20Cl2N2O5S [M+H]+
495.0543, found 495.0545.
4.2.15. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(4-methoxyphenyl)acetamide (A10)
4.2.20. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(3,4-dimethoxyphenyl)acetamide (A15)
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 85%). H NMR (400 MHz,
1
CDCl3) δ (ppm): 9.03 (s, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.67 (t, J
= 7.4 Hz, 1H), 7.55 (t, J = 7.7 Hz, 2H), 7.43 (d, J = 8.9 Hz, 2H),
7.31 (dd, J = 8.9, 2.5 Hz, 1H), 6.93-6.84 (m, 4H), 4.21 (s, 2H),
3.80 (s, 3H), 3.76 (s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm):
165.8, 156.6, 155.2, 137.5, 133.6, 130.5, 130.4, 130.0, 129.1 (2C),
129.0, 128.0 (2C), 125.9, 121.6 (2C), 114.2 (2C), 113.7, 56.3,
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 82%). H NMR (400 MHz,
1
CDCl3) δ (ppm): 9.05 (s, 1H), 7.68-7.75 (m, 3H), 7.58 (t, J = 8.0
Hz, 2H), 7.32-7.35 (m, 2H), 6.91-6.96 (m, 3H), 6.84 (d, J = 8.4
Hz, 1H), 4.23 (s, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.77 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ (ppm): 165.8, 155.2, 149.0, 146.1,
137.4, 133.6, 131.0, 130.4, 130.0, 129.1 (2C), 129.0, 128.0 (2C),

1H), 8.05 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.69 (t, J
126.0, 113.7, 112.0, 111.3, 104.8, 56.3, 56.1, 5A5.9C, 5C5.E2;PETSIE-MDS MANUSCRIPT
[M+H]+ 491.1038, found 491.1029.
(m/z): 491.5 [M+H]+; HRMS (ESI): calcd for C23H23ClN2O6S
= 7.6 Hz, 1H), 7.58 (t, J = 8.0 Hz, 2H), 7.46 (dd, J = 8.4, 2.0 Hz,
1H), 7.43 (d, J = 7.6 Hz, 2H), 7.38 (d, J = 8.8 Hz, 1H), 7.33 (dd,
J = 8.8, 2.8 Hz, 1H), 7.21-7.25 (m, 1H), 6.97 (d, J = 2.8 Hz, 1H),
6.91 (d, J = 8.8 Hz, 1H), 4.31 (s, 2H), 3.77 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 166.0, 155.3, 140.2, 137.5, 136.9,
133.6, 130.4, 130.0, 129.6, 129.1 (3C), 128.0 (2C), 126.1, 125.9,
123.5, 123.1, 120.5, 119.4, 119.3, 113.7, 112.5, 110.7 (2C), 56.3,
55.3; ESI-MS (m/z): 520.9 [M+H]+; HRMS (ESI): calcd for
C27H22ClN3O4S [M+H]+ 520.1092, found 520.1099.
4.2.21. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-
N,N-diphenylacetamide (A16)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 70%). H NMR (400 MHz,
CDCl3) δ (ppm): 9.15 (s, 1H), 7.73-7.75 (m, 2H), 7.67-7.71 (m,
1H), 7.55-7.59 (m, 4H), 7.32-7.38 (m, 3H), 7.13-7.17 (m, 1H),
6.90-6.93 (m, 2H), 4.24 (s, 2H), 3.79 (s, 3H); 13C NMR (101
MHz, CDCl3) δ (ppm): 166.0, 155.3, 137.4, 133.6, 130.5, 129.9,
129.1 (3C), 129.0 (3C), 128.0 (2C), 126.0, 124.6, 119.8 (2C),
113.8, 56.4, 55.3; ESI-MS (m/z): 431.7 [M+H]+; HRMS (ESI):
calcd for C21H20ClN2O4S [M+H]+ 431.0827, found 431.0821.
4.2.26. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(9-methyl-9H-carbazol-3-yl)acetamide (A21)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 73%). H NMR (400 MHz,
CDCl3) δ (ppm): 9.29 (s, 1H), 8.34 (s, 1H), 8.08 (d, J = 7.6 Hz,
1H), 7.77 (d, J = 8.0 Hz, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.53-7.58
(m, 3H), 7.49 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.28-
7.33 (m, 2H), 7.24 (t, J = 7.6 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H),
6.88 (d, J = 8.8 Hz, 1H), 4.33 (s, 2H), 3.79 (s, 3H), 3.74 (s, 3H);
13C NMR (101 MHz, CDCl3) δ (ppm): 166.0, 155.2, 141.5, 138.4,
137.6, 133.6, 130.4, 130.2, 129.2, 129.1 (3C), 128.0 (2C), 126.0,
125.9, 122.8, 122.5, 120.5, 119.2, 118.8, 113.7, 112.5, 108.6,
108.5, 56.3, 55.3, 29.1; ESI-MS (m/z): 534.9 [M+H]+; HRMS
(ESI): calcd for C28H24ClN3O4S [M+H]+ 534.1249, found
534.1253.
4.2.22. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(naphthalen-1-yl)acetamide (A17)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 60%). H NMR (400 MHz,
CDCl3) δ (ppm): 9.65 (s, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.91 (t, J
= 6.8 Hz, 2H), 7.79-7.81 (m, 2H), 7.70-7.86 (m, 2H), 7.54-7.65
(m, 4H), 7.49 (t, J = 7.6 Hz, 1H), 7.34 (dd, J = 8.8, 2.4 Hz, 1H),
7.02 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 4.36 (s, 2H),
3.57 (s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 166.8, 155.3,
137.4, 134.1, 133.6, 131.9, 130.5, 130.1, 129.2 (3C), 128.6, 128.1
(2C), 127.3, 126.6, 126.1, 126.0, 125.9, 125.6, 121.1, 120.6,
113.6, 56.0, 55.5; ESI-MS (m/z): 481.8 [M+H]+; HRMS (ESI):
calcd for C25H21ClN2O4S [M+H]+ 481.0983, found 481.0993.
4.2.27. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(9-ethyl-9H-carbazol-3-yl)acetamide (A22)
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a light yellow solid (Yield 58%). H NMR (400
4.2.23. N-(5-chloro-2-methoxyphenyl)-N-(2-(indolin-1-yl)-2-
oxoethyl)benzenesulfonamide (A18)
1
MHz, DMSO-d6) δ (ppm): 9.99 (s, 1H), 8.36 (d, J = 1.2 Hz, 1H),
8.08 (d, J = 8.0 Hz, 1H), 7.69-7.73 (m, 3H), 7.39-7.63 (m, 8H),
7.17 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 4.38-4.45 (m,
4H), 3.38 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ (ppm): 165.8, 155.3, 140.4, 137.6, 137.4, 133.6, 130.4,
130.1, 129.2, 129.1 (3C), 128.0 (2C), 126.0, 125.9, 123.0, 122.7,
120.7, 119.2, 118.8, 113.7, 112.7, 108.5 (2C), 58.3, 55.3, 37.6,
13.8; ESI-MS (m/z): 548.7 [M+H]+; HRMS (ESI): calcd for
C29H26ClN3O4S [M+H]+ 548.1405, found 548.1416.
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a white solid (Yield 46%). H NMR (400 MHz,
1
CDCl3) δ (ppm): 8.14 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 7.6 Hz,
2H), 7.56-7.61 (m, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.24 (dd, J = 8.8,
2.8 Hz, 1H), 7.16-7.20 (m, 2H), 7.03 (t, J = 7.6 Hz, 1H), 6.72 (d,
J = 8.8 Hz, 1H), 4.56 (s, 2H), 4.18 (t, J = 8.4 Hz, 2H), 3.44 (s,
3H), 3.24 (t, J = 8.4 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ
(ppm): 166.2, 155.5, 143.1, 140.3, 133.7, 133.3, 131.9, 130.1,
129.3 (2C), 128.0, 127.5 (2C), 127.4, 125.2, 123.9, 123.4, 116.3,
114.2, 56.0, 52.6, 46.7, 28.0; ESI-MS (m/z): 457.8 [M+H]+;
HRMS (ESI): calcd for C23H21ClN2O4S [M+H]+ 457.0983, found
457.0980.
4.2.28. N-(9H-carbazol-1-yl)-2-(N-(5-chloro-2-
methoxyphenyl)phenylsulfonamido)acetamide (A23)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 57%). H NMR (400 MHz,
4.2.24. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
CDCl3) δ (ppm): 9.57 (s, 1H), 9.43 (s, 1H), 8.08 (d, J = 8.0 Hz,
1H), 7.99 (dd, J = 7.6, 0.8 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.70
(t, J = 7.6 Hz, 1H), 7.59 (t, J = 8.0 Hz, 2H), 7.41-7.49 (m, 2H),
7.35 (dd, J = 8.8, 2.4 Hz, 1H), 7.17-7.26 (m, 3H), 6.98 (d, J = 2.8
Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 4.35 (s, 2H), 3.76 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ (ppm): 166.8, 155.2, 139.9, 137.2,
133.8, 133.0, 130.6, 130.0, 129.3, 129.2 (2C), 128.0 (2C), 126.2,
126.1, 125.9, 123.3, 121.0, 120.2, 119.4, 119.2, 118.7, 118.1,
113.8, 111.5, 56.4, 55.4; ESI-MS (m/z): 520.9 [M+H]+; HRMS
(ESI): calcd for C27H22ClN3O4S [M+H]+ 520.1092, found
520.1082.
(1-ethyl-1H-indol-5-yl)acetamide (A19)
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 62%). H NMR (400 MHz,
1
CDCl3) δ (ppm): 9.12 (s, 1H), 7.83 (d, J = 1.2 Hz, 1H), 7.77 (d, J
= 8.0 Hz, 2H), 7.69 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 2H),
7.26-7.34 (m, 3H), 7.14 (d, J = 3.2 Hz, 1H), 6.95 (d, J = 2.4 Hz,
1H), 6.91 (d, J = 8.8 Hz, 1H), 6.48 (d, J = 3.2 Hz, 1H), 4.28 (s,
2H), 4.18 (q, J = 7.2 Hz, 2H), 3.77 (s, 3H), 148 (t, J = 7.2 Hz,
3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 165.8, 155.3, 137.6,
133.5, 133.4, 130.4, 130.1, 129.5, 129.1 (2C), 129.0, 128.6, 128.0
(2C), 127.9, 125.8, 115.6, 113.6, 112.9, 109.4, 101.2, 56.3, 55.3,
41.1, 15.4; ESI-MS (m/z): 498.7 [M+H]+; HRMS (ESI): calcd for
C25H24ClN3O4S [M+H]+ 498.1249, found 498.1240.
4.2.29. 2-(N-(5-chloro-2-methoxyphenyl)phenylsulfonamido)-N-
(9-methyl-9H-carbazol-1-yl)acetamide (A24)
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a yellow solid (Yield 68%). H NMR (400 MHz,
CDCl3) δ (ppm): 9.53 (s, 1H), 8.08 (d, J = 7.6 Hz, 1H), 8.02 (d, J
= 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.70 (t, J = 7.6 Hz, 1H),
7.58 (t, J = 7.6 Hz, 2H), 7.51 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 8.0
Hz, 1H), 7.37 (dd, J = 8.8, 2.0 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H),
4.2.25. N-(9H-carbazol-3-yl)-2-(N-(5-chloro-2-
methoxyphenyl)phenylsulfonamido)acetamide (A20)
1
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a yellow solid (Yield 62%). H NMR (400 MHz,
CDCl3) δ (ppm): 9.25 (s, 1H), 8.31 (s, 1H), 8.29 (d, J = 1.6 Hz,

7.26 (d, J = 7.2 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 2.0
2H), 7.33 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 6.87 (d, J = 8.4 Hz,
1H), 6.79 (s, 1H), 3.99 (s, 2H), 3.66 (s, 3H), 2.16 (s, 1H), 1.83 (s,
2H), 1.64 (d, J = 12.0 Hz, 2H), 1.60 (d, J = 12.0 Hz, 2H), 1.39 (d,
J = 12.0 Hz, 2H), 1.30 (d, J = 12.0 Hz, 2H), 1.13-1.21 (m, 2H),
0.87 (s, 6H); ESI-MS (m/z): 551 [M+H]+.
ACCEPTED MANUSCRIPT
Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 4.39 (s, 2H), 4.07 (s, 3H), 3.60
(s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 168.4, 155.2,
141.9, 137.3, 135.8, 133.7, 130.6, 130.2, 129.2 (2C), 129.0, 128.0
(2C), 126.1, 126.0, 125.6, 124.7, 122.6, 120.1, 119.6, 119.4,
119.3, 119.1, 113.7, 108.8, 56.3, 55.3, 31.3; ESI-MS (m/z): 534.9
[M+H]+; HRMS (ESI): calcd for C28H24ClN3O4S [M+H]+
534.1249, found 534.1262.
4.2.35. 2-(N-(5-chloro-2-methoxyphenyl)-4-nitrophenylsulfonam-
ido)-N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)acetamide
(B-3)
4.2.30. General procedure for the synthesis of 6
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a light yellow solid (Yield 84%). H NMR (400
To a stirred solution of 3,5-dimethyl amantadine 5 (1.79 g, 10
mmol) and Et3N (2.1 mL, 15 mmol) in CH2Cl2 (50 mL) was
added chloroacetyl chloride (0.98 mL, 13 mmol) at room
temperature. The mixture was stirred at room temperature for 2 h,
and TLC indicated that the reaction was completed. Then the
mixture was washed with saturated Na2CO3 solution (2 × 50 mL),
brine (50 mL) and dried over anhydrous Na2SO4. After filtered,
the solvent was evaporated under vacuum to give 6 as a brown
solid (Yield 99%). ESI-MS (m/z): 256 [M+H]+.
MHz, CDCl3) δ (ppm): 8.36 (t, J = 1.2 Hz, 2H), 7.88 (t, J = 1.2
Hz, 2H), 7.34-7.35 (m, 1H), 7.06 (s, 1H), 6.87-6.88 (m, 1H), 6.63
(s, 1H), 4.03 (s, 2H), 3.61 (s, 3H), 2.05-2.16 (m, 1H), 1.83 (s,
2H), 1.62 (s, 4H), 1.17-1.37 (m, 6H), 0.87 (s, 6H); ESI-MS (m/z):
562 [M+H]+.
4.2.36. N-(5-chloro-2-methoxyphenyl)-N-(2-(((1r,3R,5S,7r)-3,5-
dimethyladamantan-1-yl)amino)-2-oxoethyl)benzamide (B-4)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 90%). H NMR (400 MHz,
4.2.31. General procedure for the synthesis of 7
CDCl3) δ (ppm): 7.34 (d, J = 7.6 Hz, 2H), 7.27 (t, J = 7.2 Hz,
1H), 7.26 (d, J = 2.4 Hz, 1H), 7.18 (t, J = 7.6 Hz, 2H), 7.13-7.16
(dd, J = 8.8, 2.0 Hz, 1H), 6.65 (d, J = 8.8 Hz, 1H), 6.54 (s, 1H),
4.24-4.45 (m, 2H), 3.56 (s, 3H), 2.14 (t, J = 2.8 Hz, 1H), 1.84 (s,
2H), 1.67 (d, J = 12.0 Hz, 2H), 1.61 (d, J = 12.0 Hz, 2H), 1.39 (d,
J = 12.0 Hz, 2H), 1.29 (d, J = 12.0 Hz, 2H), 1.12-1.21 (m, 2H),
0.86 (s, 6H); ESI-MS (m/z): 481 [M+H]+.
To a stirred solution of 6 (1.27 g, 5 mmol) and Et3N (0.57 mL,
5 mmol) in EtOH (50 mL) was added 2-methoxy-5-chloro aniline
(785 mg, 5 mmol) and NaI (745 mg, 5 mmol) at room
temperature. The mixture was heated to reflux for 10 h, and TLC
indicated that the reaction was completed. Then the mixture was
filtered and the filtrate was evaporated to dryness under vacuum.
The obtained residue was dissolved in ethyl acetate (50 mL) and
washed with brine (2 × 50 mL) and dried over anhydrous Na2SO4.
After filtered, the solvent was evaporated to dryness under
vacuum. The residue was further purified by silica gel
chromatography (PE/EtOAc = 3/1) to give 7 as a white solid
(Yield 54%). ESI-MS (m/z): 377 [M+H]+.
4.2.37. General procedure for the synthesis of C1-C7
To a stirred solution of 6 (1.27 g, 5 mmol) and Et3N (0.57 mL,
5 mmol) in EtOH (50 mL) was added different amines (5 mmol)
and NaI (745 mg, 5 mmol) at room temperature. The mixture was
heated to reflux for 10 h, and TLC indicated that the reaction was
completed. Then the mixture was filtered and the filtrate was
evaporated to dryness under vacuum. The obtained residue was
dissolved in ethyl acetate (50 mL) and washed with brine (2 × 50
mL) and dried over anhydrous Na2SO4. After filtered, the solvent
was evaporated to dryness under vacuum. The residue was
further purified by silica gel chromatography (PE/EtOAc = 3/1)
to give 8 as a white solid.
4.2.32. General procedure for the synthesis of B1-B4
The appropriate sulfonyl chlorides or acryl chlorides (1 mmol)
was added dropwise to a solution of 7 (75 mg, 0.2 mmol) and
DMAP (37 mg, 0.3 mmol) in pyridine (10 mL) at room
temperature. The mixture was continuously stirred at room
temperature for 10 h, and TLC indicated that the reaction was
completed. Then the reaction was concentrated and diluted with
CH2Cl2 (10 mL) and H2O (10 mL). The aqueous layer was
separated and the organic layer was washed with saturated
Na2CO3 solution (2 × 10 mL), brine (10 mL) and dried over
anhydrous Na2SO4. After filtered, the solvent was evaporated to
dryness under vacuum to obtain the desired crude products. The
appropriate compounds (B1-B4) were obtained following
purification by silica gel chromatography.
Benzenesulfonyl chloride (1 mmol) was added dropwise to a
solution of 8 (0.2 mmol) and DMAP (37 mg, 0.3 mmol) in
pyridine (10 mL) at room temperature. The mixture was
continuously stirred at room temperature for 10 h, and TLC
indicated that the reaction was completed. Then the reaction was
concentrated and diluted with CH2Cl2 (10 mL) and H2O (10 mL).
The aqueous layer was separated and the organic layer was
washed with saturated Na2CO3 solution (2 × 10 mL), brine (10
mL) and dried over anhydrous Na2SO4. After filtered, the solvent
was evaporated to dryness under vacuum to obtain the desired
crude products. The appropriate compounds (C1-C7) were
obtained following purification by silica gel chromatography.
4.2.33. 2-(N-(5-chloro-2-methoxyphenyl)-4-methylphenylsulfona-
mido)-N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)acetamide
(B-1)
The product was purified by flash chromatography (PE/EtOAc
1
4.2.38. 2-(N-cyclohexylphenylsulfonamido)-N-((1r,3R,5S,7r)-3,5-
dimethyladamantan-1-yl)acetamide (C-1)
= 3/1) to give a white solid (Yield 72%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.57 (d, J = 6.8 Hz, 2H), 7.29-7.33 (t, 3H), 6.86-
6.95 (t, 3H), 3.96 (s, 2H), 3.69 (s, 3H), 2.46 (s, 3H), 2.15 (s, 1H),
1.81 (s, 2H), 1.65 (d, J = 10.8 Hz, 2H), 1.60 (d, J = 10.8 Hz, 2H),
1.39 (d, J = 10.8 Hz, 2H), 1.30 (d, J = 10.8 Hz, 2H), 1.15-1.16
(m, 2H), 0.86 (s, 6H); ESI-MS (m/z): 531 [M+H]+.
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 68%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.85 (d, J = 7.6 Hz, 2H), 7.62 (t, J = 7.6 Hz,
1H), 7.54 (t, J = 7.6 Hz, 2H), 6.39 (s, 1H), 3.84-3.90 (m, 1H),
3.62 (s, 2H), 2.16-2.19 (m, 1H), 1.88 (d, J = 1.6 Hz, 2H), 1.52-
1.76 (m, 10H), 1.42 (d, J = 12.0 Hz, 2H), 1.26-1.32 (m, 6H),
1.14-1.23 (m, 2H), 0.88 (s, 6H); ESI-MS (m/z): 458 [M+H]+.
4.2.34. 2-(4-chloro-N-(5-chloro-2-methoxyphenyl)phenylsulfona
mido)-N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)acetamide
(B-2)
4.2.39. N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-2-(N-
phenylphenylsulfonamido)acetamidee (C-2)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 66%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.63 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz,

4.3. Biological activity assay
4.3.1. Cell culture
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a white solid (Yield 61%). H NMR (400 MHz,
ACCEPTED MANUSCRIPT
1
CDCl3) δ (ppm): 7.64 (t, J = 7.2 Hz, 1H), 7.57 (d, J = 7.2 Hz,
2H), 7.48 (t, J = 7.6 Hz, 2H), 7.34-7.36 (m, 3H), 7.05-7.08 (m,
2H), 6.28 (s, 1H), 4.08 (s, 2H), 2.13 (t, J = 2.8 Hz, 1H), 1.77 (s,
2H), 1.60 (d, J = 12.0 Hz, 2H), 1.54 (d, J = 12.0 Hz, 2H), 1.37 (d,
J = 12.0 Hz, 2H), 1.28 (d, J = 12.0 Hz, 2H), 1.11-1.19 (m, 2H),
0.85 (s, 6H); ESI-MS (m/z): 453 [M+H]+.
SH-SY5Y cells were grown in a mixture (1:1) of Dulbecco’s
modified Eagle’s medium and Ham’s F-12 (DMEM/F12)
supplemented with 10% fetal bovine serum (FBS) and 100
unit/mL penicillin-streptomycin. HEK293 cells expressing APP
Swedish mutantK595N/M596L (HEK293-APPsw) (kindly provided by
Prof Gang PEI, Shanghai Institutes for Biological Sciences,
China) were grown in DMEM containing 10% FBS and 100
unit/mL penicillin-streptomycin. All cells were cultured in a
humidified incubator with 5% CO2 at 37 °C.
4.2.40. 2-(N-(2-chlorophenyl)phenylsulfonamido)-N-((1r,3R,5S,
7r)-3,5-dimethyladamantan-1-yl)acetamide (C-3)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a light yellow solid (Yield 18%). H NMR (400
MHz, CDCl3) δ (ppm): 7.65-7.72 (m, 3H), 7.48-7.56 (m, 3H),
7.31-7.35 (m, 1H), 7.23-7.27 (m, 1H), 6.94 (dd, J = 8.0, 1.6 Hz,
1H), 6.79 (s, 1H), 3.89-3.90 (m, 2H), 2.14-2.17 (m, 1H), 1.86 (d,
J = 2.0 Hz, 2H), 1.69 (d, J = 12.0 Hz, 2H), 1.65 (d, J = 12.0 Hz,
2H), 1.40 (d, J = 12.0 Hz, 2H), 1.30 (d, J = 12.0 Hz, 2H), 1.12-
1.22 (m, 2H), 0.86 (s, 6H); ESI-MS (m/z): 487 [M+H]+.
4.3.2. Neuroprotection activity on SH-SY5Y
Potential neuroprotective activity was evaluated in human
neuroblastoma SH-SY5Y cells treated with STZ. The compounds
were pre-dissolved in DMSO and diluted in medium to at least
five required concentrations. Viability of the cultured cells was
determined by assaying the reduction of MTT to formazan. SH-
SY5Y cells were seeded overnight in 48-well plates at a density
of 105 cells/well in 100 µL medium. Cells were then incubated
with different concentrations of compounds (5, 10 and 20 µM)
and STZ (0.8 mM for SH-SY5Y cells) for 24 h and washed twice
with PBS, followed by addition of MTT (0.5 mg/mL). After
incubation at 37 °C for 4 h, 200 µL DMSO was added to dissolve
the formazan crystals, and absorbance at 490 nm was measured
using an M5 spectrophotometer (Molecular Devices, Sunnyvale,
CA, USA).
4.2.41. 2-(N-(3-chlorophenyl)phenylsulfonamido)-N-((1r,3R,5S,
7r)-3,5-dimethyladamantan-1-yl)acetamide (C-4)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 35%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.64 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 7.6 Hz,
2H), 7.49 (t, J = 7.6 Hz, 2H), 7.27-7.33 (m, 2H), 7.10 (s, 1H),
6.97 (d, J = 7.6 Hz, 1H), 6.17 (s, 1H), 4.05 (s, 2H), 2.12 (t, J =
2.8 Hz, 1H), 1.76 (s, 2H), 1.59 (d, J = 11.6 Hz, 2H), 1.54 (d, J =
11.6 Hz, 2H), 1.37 (d, J = 11.6 Hz, 2H), 1.28 (d, J = 11.6 Hz,
2H), 1.10-1.18 (m, 2H), 0.84 (s, 6H); ESI-MS (m/z): 487
[M+H]+.
4.3.3. Intracellular Aβ production and Aβ clearance assay
ELISA assay was used to evaluate the inhibitory activity
against STZ-induced Aβ production in HEK293-APPsw cells.
Intracellular Aβ clearance in SH-SY5Y cells was detected
according to the Landreth approach. These experiments were
carried as previously described [30].
4.2.42. 2-(N-(4-chlorophenyl)phenylsulfonamido)-N-((1r,3R,5S,
7r)-3,5-dimethyladamantan-1-yl)acetamide (C-5)
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 50%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.65 (t, J = 7.2 Hz, 1H), 7.58 (d, J = 7.2 Hz,
2H), 7.49 (t, J = 7.6 Hz, 2H), 7.32 (d, J =8.8 Hz, 2H), 7.02 (d, J
=8.8 Hz, 2H), 6.17 (s, 1H), 4.05 (s, 2H), 2.13-2.15 (m, 1H), 1.77
(d, J =1.2 Hz, 2H), 1.59 (d, J = 12.4 Hz, 2H), 1.54 (d, J = 12.4
Hz, 2H), 1.37 (d, J = 12.4 Hz, 2H), 1.29 (d, J = 12.4 Hz, 2H),
1.12-1.19 (m, 2H), 0.86 (s, 6H); ESI-MS (m/z): 487 [M+H]+.
4.3.4. Immunohistochemistry assay
Immunohistochemistry assays were performed to detect the
expression of P396-Tau protein. These experiments were carried
as previously described [31].
4.3.5. Animal experiments
4.2.43. 2-(N-(3-chloro-2-methylphenyl)phenylsulfonamido)-N-
((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)acetamide (C-6)
All animal experiments were performed according to the
Institutional Ethical Guidelines of Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, on animal care. These
experiments were carried as previously described [30].
The product was purified by flash chromatography (PE/EtOAc
1
= 3/1) to give a white solid (Yield 20%). H NMR (400 MHz,
CDCl3) δ (ppm): 7.67 (d, J = 8.0 Hz, 3H), 7.54 (t, J = 8.0 Hz,
2H), 7.38 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 6.64 (d, J
= 8.0 Hz, 1H), 6.37 (s, 1H), 4.12 (d, J = 16.4 Hz, 1H), 3.83 (d, J
= 16.4 Hz, 1H), 2.37 (s, 3H), 2.17 (s, 1H), 1.84 (s, 2H), 1.67 (d, J
= 12.0 Hz, 2H), 1.62 (d, J = 12.0 Hz, 2H), 1.41 (d, J = 12.0 Hz,
2H), 1.31 (d, J = 12.0 Hz, 2H), 1.14-1.22 (m, 2H), 0.87 (s, 6H);
ESI-MS (m/z): 501 [M+H]+.
4.3.6. Statistical analysis
Data are reported as the mean ± SEM. The significant
difference between multiple treatments and the control was
analyzed using a one-way ANOVA with Dunnett’s post-test. P
values less than 0.05 were considered statistically significant.
4.2.44. 2-(N-(3-chloro-4-methylphenyl)phenylsulfonamido)-N-
((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)acetamide (C-7)
Acknowledgment
The product was purified by flash chromatography (PE/EtOAc
= 3/1) to give a white solid (Yield 36%). H NMR (400 MHz,
1
This work was supported by the National Natural Science
Foundation of China (grants 81473110, 81773596,
81561148011), Natural science foundation of Jiangsu Higher
Education Institutions (grant: 17KJA360004), Program for
Outstanding Scientific and Technological Innovation Team of
Jiangsu Higher Education Institutions, and the Priority Academic
Program Development of Jiangsu Higher Education Institutions.
CDCl3) δ (ppm): 7.66 (t, J = 7.2 Hz, 1H), 7.60 (d, J = 7.2 Hz,
2H), 7.52 (t, J = 7.2 Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.06 (d, J
= 2.0 Hz, 1H), 6.86 (dd, J = 8.0, 2.0 Hz, 1H), 6.21 (s, 1H), 4.02
(s, 1H), 2.38 (s, 3H), 2.13-2.16 (m, 1H), 1.79 (d, J = 2.0 Hz, 2H),
1.61 (d, J = 12.0 Hz, 2H), 1.56 (d, J = 12.0 Hz, 2H), 1.40 (d, J =
12.0 Hz, 2H), 1.29 (d, J = 12.0 Hz, 2H), 1.12-1.20 (m, 2H), 0.86
(s, 6H); ESI-MS (m/z): 501 [M+H]+.
Appendix A. Supplementary data

maleimides as glycogen synthase kinase-3β (GSK-3β) inhibitors,
Eur. J. Med. Chem. 68 (2013) 361-371.
Supplementary data related to this article can be found at…...
ACCEPTED MANUSCRIPT
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