Conjugation of tacrine with genipin derivative not only enhances effects on AChE but also leads to autophagy against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2020.113067

Seven tacrine/CHR21 conjugates have been designed and synthesized. Compound 8-7 was confirmed as the most active AChE inhibitor with IC₅₀ value of 5.8 ± 1.4 nM, which was 7.72-fold stronger than tacrine. It was also shown as a strong BuChE inhibitor (IC₅₀ value of 3.7 ± 1.3 nM). 8-7 was clearly highlighted not only as an excellent ChEs inhibitor, but also as a good modulator on protein expression of AChE, p53, Bax, Bcl-2, LC3, p62, and ULK, indicating its functions against programmed cell apoptosis and decrease of autophagy. 8-7 significantly reversed the glutamate-induced dysfunctions including excessive calcium influx and release from internal organelles, overproduction of nitric oxide (NO) and Aβ high molecular weight oligomer. This compound can penetrate blood?brain barrier (BBB). The in vivo hepatotoxicity assay indicated that 8-7 was much less toxic than tacrine. Altogether, these data strongly support that 8-7 is a potential multitarget-directed ligand (MTDL) for treating Alzheimer's disease (AD).

Full text of DOI:10.1016/j.ejmech.2020.113067

European Journal of Medicinal Chemistry 211 (2021) 113067
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Conjugation of tacrine with genipin derivative not only enhances
effects on AChE but also leads to autophagy against Alzheimer’s
disease
Rongtian Lin a, Shuwen Rao a, Yanbing Li a, Lei Zhang a, Liyu Xu a, Yepu He a, Zhijun Liu a,
, b, c, *
Heru Chen a
a Institute of Traditional Chinese Medicine and Natural Product, College of Pharmacy, Jinan University, Guangzhou, 510632, PR China
b Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Guangzhou, 510632, PR China
c International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education,
School of Pharmacy, Jinan University, Guangzhou, 510632, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven tacrine/CHR21 conjugates have been designed and synthesized. Compound 8-7 was confirmed as
the most active AChE inhibitor with IC50 value of 5.8 1.4 nM, which was 7.72-fold stronger than tacrine.
It was also shown as a strong BuChE inhibitor (IC50 value of 3.7 1.3 nM). 8-7 was clearly highlighted not
only as an excellent ChEs inhibitor, but also as a good modulator on protein expression of AChE, p53, Bax,
Bcl-2, LC3, p62, and ULK, indicating its functions against programmed cell apoptosis and decrease of
autophagy. 8-7 significantly reversed the glutamate-induced dysfunctions including excessive calcium
Received 8 October 2020
Received in revised form
13 November 2020
Accepted 28 November 2020
Available online 2 December 2020
influx and release from internal organelles, overproduction of nitric oxide (NO) and Ab high molecular
Keywords:
weight oligomer. This compound can penetrate bloodꢀbrain barrier (BBB). The in vivo hepatotoxicity
assay indicated that 8-7 was much less toxic than tacrine. Altogether, these data strongly support that 8-
7 is a potential multitarget-directed ligand (MTDL) for treating Alzheimer’s disease (AD).
© 2020 Elsevier Masson SAS. All rights reserved.
AChE inhibitor
Alzheimer’s disease
Autophagy
Multitarget-directed ligand
Drug design
1. Introduction
species (ROS) and reactive nitrogen species (RNS) to cause gene and
protein damage [5]. At the same time, oxidative stress factors
Due to its high incidence, slow progress and high medical costs,
Alzheimer’s disease (AD) has become a serious neurodegenerative
disease affecting human society, especially for the elderly [1]. The
occurrence and development of AD are related to the accumulation
produced by Ca2þ signal can regulate the expression of Ab1-42
protein [6], and acetylcholinesterase (AChE) can bind with Ab1-42 to
accelerate the aggregation of Ab [7].
Just like other neurodegenerative diseases, AD has long been
viewed as among the most enigmatic and problematic issues in
biomedicine [8]. The major basic processes involved are multifac-
torial in nature, caused by genetic, environmental, and endogenous
factors. For such kinds of diseases, “single-molecule, single-target”
treatment model cannot achieve good therapeutic effect [9]. On the
contrary, the “multi-target-directed ligands” (MTDLs) is a more
effective strategy in treating complex diseases because of their
ability to interact with the multiple targets thought to be respon-
sible for the disease pathogenesis [10e13].
of abnormal protein b-amyloid protein (Ab) and tau protein, syn-
aptic loss, oxidative stress, neuronal cell death and inflammation
[2]. However, the different mechanisms of AD do not exist in
isolation, they are inter-related and interact with each other. For
example, amyloid precursor protein (APP) is cleaved by
secretase and aggregated into misfolded A peptides [3], a major
a、b、g-
b
pathological hallmark of AD [4]. These misfolded Ab peptides can
act on tau protein to promote neurofibrillary tangles, Ca2þ influx to
trigger oxidative stress which produces excessive reactive oxygen
As the first AChE inhibitor approved for the treatment of AD,
tacrine emerges as a popular structure in the design of MTDLs in
recent years, because it can inhibit AChE in high potency and has a
low molecular weight suitable for modification [14,15]. So far, a lot
of MTDLs by combining tacrine with another fragment resulted in
* Corresponding author. Huangpu Avenue West 601, Institute of Traditional
Chinese Medicine and Natural Product, College of Pharmacy, Jinan University,
Guangzhou, 510632, PR China.
E-mail address: thrchen@jnu.edu.cn (H. Chen).
https://doi.org/10.1016/j.ejmech.2020.113067
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
additional activities have been designed and synthesized [15e18].
Unfortunately, serious hepatotoxicity of tacrine limits further use of
these compounds [19]. However, it has been disclosed that anti-
oxidants may probably prevent tacrine-induced hepatotoxicity
[20,21].
C7eH, respectively. The C1eH of CHR21 displayed a doublet at a
d
value of 4.81 ppm, with a coupling constant of 3.2 Hz, which in-
dicates cis to C7a-H. Since the configuration at C7a is S, the config-
uration at C1 is certainly S. On the other hand, the C7eH of CHR21
displayed a doublet at a
d value of 4.15 ppm, with a coupling con-
Interestingly, AChE may function by accelerating A
and could play a role during amyloid deposition in Alzheimer’s
brain [7]. As a matter of fact, the A deposition in the brain is the
primary influence driving AD pathogenesis [22]. This indicates that
AD represents the effects of a chronic imbalance between A pro-
duction and A clearance. Usually, proteins aggregate in the cell are
cleared by autophagy, and this mechanism is impaired in AD. It has
been demonstrated that autophagy influences the secretion of A
to the extracellular space and thereby directly affects A plaque
b
formation
stant of 9.2 Hz, which indicates trans to C7a-H, implying that the
configuration of C7 is S. Oxidation of 10-hydroxyl in 6 with Dess-
Martin periodine offered compound 7 in 85.6% yield.
Finally, all the title compounds (8-n, n ¼ 1e7) were prepared
through reductive amination of 7 with 4-n (n ¼ 1e7) in 45%e63%
yield. The reductant used was sodium borohydride. After silica gel
column chromatography, the desired tacrine conjugates were iso-
lated with a high purity degree (>97%).
b
b
b
b
b
formation [23]. Based on this cognition, we have a bold vision in
mind that an autophagy regulator will help to improve AD treat-
ment of an AChE inhibitor.
2.2. In vitro inhibition of ChEs and the structure-activity
relationship (SAR)
As an on-going project, we have been working on the possible
application of genipin (GP) and its derivatives in the treatment of
neurodegenerative diseases [24e27]. It was confirmed that the
reduction of the C6]C7 double bond in (1R)-isopropyloxygenipin
(IPRG001) or (1S)-isopropyloxygenipin led to genipin derivatives
CHR20, or CHR21, respectively, which led to increased flexibility,
stability, and enhanced neuronal protection activity [25]. The pro-
tection mechanism involved was closely related to the induction of
both NO and expressions of anti-oxidative enzymes [28,29]. On the
other hand, we noticed that NO does play an important role in
regulating autophagy [30]. Taking altogether, this is an appeal to
push us to explore whether AD treatment may benefit from the
conjugation of tacrine with either CHR20 or CHR21.
Compounds 8-n (n ¼ 1e7), as well as the free amine in-
termediates 4-n (n ¼ 1e7) were evaluated for their in vitro potential
to inhibit cholinesterases (ChEs) including acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE), using tacrine as a pos-
itive reference (Table 1). It was indicated that all compounds are
potent inhibitors of both ChEs. Just like the tacrine, most of them
showed more potent inhibitory activity to BuChE than to AChE.
Obviously, the length of the alkyl spacer between CHR21 and
tacrine moiety significantly influenced the ChEs inhibitory activity.
It can be seen from Table 1 that compounds 8-1, 8-6, and 8-7 with a
two-, seven-, and eight-carbon spacer, respectively exhibited more
potent AChE inhibitory activity with IC50 value of 8.1
1.2,
Considering that CHR21 is more active than its isomer CHR20, in
the present study, CHR21 was applied as the building scaffold in a
series of conjugates with tacrine. The chemistry and biology of
these new compounds will be described thereafter.
24.7 1.9, and 5.8 1.4 nM, which was 5.53-, 1.81-, and 7.72-fold
stronger than that of tacrine. However, compounds with carbon
spacer between 3 and 6 showed weaker AChE inhibitory activity
than tacrine itself.
Interestingly, the results of the amine intermediates (4-n,
n ¼ 1e7) showed a trend consistent with the title compounds,
implying that the CHR21 moiety might not obviously affect the
extent of ChE inhibitory activity. Amongst all the tested com-
pounds, 8-1 and 8-7 showed high activity toward both AChE and
BuChE. We ascribed the high activity of 8-1 to the enhanced
binding affinity of CHR21 to the catalytic site of ChEs. As for 8-7
with optimal spacer, it is because of the enhanced binding affinity
of CHR21 to the peripheral site of ChEs [31]. This result seems
consistent with the previous reports of lipoic acid/tacrine and
ferulic acid/tacrine [32,33].
2. Results
2.1. Chemistry
The novel conjugates consist of a tacrine moiety, a spacer, and a
genipin derivative (CHR21). Their syntheses were outlined in
Scheme 1. The strategy was to prepare the heterocycle firstly, then
to introduce the side chain, and finally to combine to CHR21 moi-
ety. Based on a previously protocol [31], 1,2,3,4-tetrahydro-9-
acridone (1) was firstly synthesized via the cyclization of anthra-
nilic acid with cyclohexanone. Due to the reason that anthranilic
acid is a controlled substance in China, methyl anthranilate was
applied as the starting material. This compound was easily hydro-
lyzed to anthranilic acid using traditional saponification. 9-Chloro-
1,2,3,4- tetrahydroacridine (2) were then obtained via chlorination
of 1 in 89% yield. In order to introduce the spacer, different alky-
lenediamines (3-n, n ¼ 1e7) reacted with 2 leading to 9-
aminoalkylamino-1,2,3,4-tetrahydroacridines (4-n, n ¼ 1e7) in
65e85% yield. It was found that addition of catalytic amount of KI
(10 mol%) shortened the reaction time from 40 h to 8 h with
comparative yield.
The synthesis of CHR21 was referred to our previous work [25].
Firstly, genipin was turned into 1-isopropylgenipin in 96% yield.
The resulted product was a mixture of (1R)- and (1S)-isomers, and
was separated by RP-HPLC (COSMOSIL 5C18-MS-II), where com-
pound 5 was obtained in 63.4% yield. Reduction of 5 with sodium
borohydride in the presence of catalytic NiCl2 at ꢀ15 ꢁC resulted in
chemo- and stereo-selective compound 6, where 80.3% yield was
observed. The absolute configurations at C1 and C7 were confirmed
by analysis of the coupling constants of C7a-H versus C1eH and
2.3. 8-7 attenuated glutamate-induced injuries to SH-SY5Y cells
In order to evaluate the potential of 8-7 as an agent against
Alzheimer disease, an excitotoxicity cell model was set up, where
SH-SY5Y cells were treated with 35 mM glutamic acid for 24 h.
Since glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system, prolonged exposure to gluta-
mate and the associated excessive influx of ions into the cell may
arise the injury and death of neurons. These disturbances of the
excitatory amino acid system may contribute to the pathogenesis of
dementia [34].
As displayed in Fig. 1A and B, treatment of 35 mM glutamate
caused cell apoptosis or death (Model group). However, pre-
treatment of SH-SY5Y cells with 1.25 mM of tacrine (TA), CHR21,
TA/CHR21 (in 1:1 mol ratio), and 8-7, respectively for 4 h attenuated
glutamate-induced cells injury. Among them, 8-7 was shown as the
most potent to make cells survive from the excitotoxicity impair-
ment, even better than the TA/CHR21 combination.
2

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
Scheme 1. Design and synthesis of tacrine-CHR21 hybrids. Reaction conditions: a. sat. NaOH, reflux, 95%; b. cyclohexone/toluent, reflux, 89%; c. POCl3, 120 ꢁC, 91%; d. diamines, KI,
1-pentanol, 160 ꢁC, 65%e85%; e. iPrOH, p-TsOH, 80 ꢁC, 3 h, 63.4%; f. NiCl2$6H2O, NaBH4, MeOH, ꢀ15 ꢁC, 2 h, 80.3%; g. DMP/DCM, r.t., 85.6%; h. NaBH4/MeOH, 0 ꢁC, 45%e63%.
Table 1
2.4. 8-7 inhibited glutamate-increased AChE level
IC50 values of 4-n and 8-n (n ¼ 1e7) against AChE and BuChE.
IC50 (nM) SEMa
SId
Recent studies have shown that glutamatergic signaling is
Compd.
compromised by A
receptors in specific brain regions, paralleling early cognitive defi-
cits [35]. Intriguingly, AChE may function by accelerating A for-
mation and can play role during amyloid deposition in
Alzheimer’s brain [7]. Therefore, we postulated that excitotoxicity
might have impact on AChE. This prediction was confirmed by data
demonstrated in Fig. 2. It can be seen that the treatment of 35 mM
glutamic acid to SH-SY5Y cells caused the up-regulation of AChE
b-induced modulation of synaptic glutamate
AChEb
BuChEc
4e1
4e2
4e3
4e4
4e5
4e6
4e7
8e1
8e2
8e3
8e4
8e5
8e6
8e7
tacrine
55.2 9.3
70.3 13.6
23.6 5.0
3.6 1.1
3.2 0.9
3.5 1.3
3.7 1.5
8.1 1.2
73.2 4.5
81.4 4.6
94.6 3.4
60.6 5.9
24.7 1.9
5.8 1.4
44.8 6.7
13.1 3.8
5.7 1.3
6.9 1.2
5.2 0.8
1.8 0.6
2.1 0.8
1.6 0.5
5.7 2.1
35.6 6.3
43.2 3.8
57.3 2.6
27.6 4.5
11.3 2.4
3.7 1.3
4.8 1.2
0.24
0.08
0.29
1.44
0.56
0.60
0.43
0.70
0.49
0.53
0.61
0.46
0.46
0.64
0.11
b
a
protein level (Model group). However, pre-treatment of 1.25 mM
tacrine alone attenuated this tendency; whilst the conjugate 8-7
showed even stronger activity than tacrine to inhibit this
glutamate-increased AChE expression. It was also noticed that pre-
treatment of CHR21 alone displayed no activity.
2.5. 8-7 decreased the glutamate-raised NO and i-NOS levels
a
Data is the mean of at least three determinations.
E.C. 3.1.1.7, type VI-S, from Electric Eel.
E.C. 3.1.1.8, from equine serum.
AChE selectivity index ¼ IC50(BuChE)/IC50(AChE).
b
c
It was demonstrated that excessive activation of glutamate re-
ceptors by excitatory amino acids leads to a number of deleterious
d
3

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
Fig. 1. Protection of 8-7 against glutamate-induced cell injury. (A) Cell survival. SH-SY5Y cells were pre-treated with 0.1% DMSO, and 1.25 mM of tacrine (TA), CHR21, TA/CHR21 (in
1:1 mol ratio), 8-7, respectively for 4 h, then were treated with 35 mM glutamic acid for 24 h. MTT assay was used to determine the cell viability; (B) Cell apoptosis rate. SH-SY5Y
cells were treated following the procedure described in (A). Then the cells were washed with PBS and stained with Annexin-V and PI. The cell apoptosis was detected by flow
cytometry. The evaluation of apoptosis is via Annexin V: FITC Apoptosis Detection Kit per manufacture’s protocol. The results represent mean SD of three separate experiments.
###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs Model group.
Excess levels of glutamate in the central nervous system (CNS) can
result in elevated intracellular Ca2þ levels, which in turn cause a
rise in the Ca2þ concentration in sensitive organelles such as
mitochondria and the endoplasmic reticulum [37]. As shown in
Fig. 4A and B, excess levels of glutamate in SH-SY5Y cells did elevate
intramolecular Ca2þ influx (Model group). More detail, 35 mM
glutamate exposure induced an increase in the fluorescence ratio
(F340/F380) from 100% in control group to 280% in model group.
When the cells were pre-treated with 1.25 mM of tacrine, CHR21,
TA/CHR21, and 8-7, F340/F380 was reduced from 280% to 145%,
132%, 126%, and 113%, respectively. The reduction of [Ca2þ]i in each
group was comparable, but the activity of 8-7 was the most potent.
2.7. 8-7 reduced the glutamate-upregulated level of high molecular
weight Ab oligomer
According to the amyloid cascade hypothesis, AD pathogenesis
is initiated by the overproduction and extracellular deposition of A
b
and the intracellular deposition of neurofibrillary tangle (NFT).
These depositions serve as initiating factors for multiple neurotoxic
pathways, which may include excitotoxicity, oxidative stress, en-
ergy depletion, inflammation and apoptosis [38]. In turn, excito-
toxicity might induce the overproduction of Ab. This deduction was
verified by the fact shown in Fig. 5. It was displayed that exposure of
Fig. 2. 8-7 inhibited glutamate-increased AChE level. SH-SY5Y cells were treated
following the procedure described in Fig. 1(A). Then the cells were washed and lysed
with RIPA buffer. The protein concentration was measured using a BCA protein assay
kit. The protein expression was analyzed using Western Blot. The density of each lane
was presented as mean standard deviation (SD) for at least three individual exper-
iments. Blots were quantified using Image J software. ###P < 0.001 vs Control group;
*P < 0.05, ***P < 0.001 vs Model group.
excessive glutamate (35 mM) raised the level of high molecular
weight A
elevated from 0.75 in Control group to 1.4 in Model group. When
the cells were pre-treated with 1.25 M of tacrine, CHR21, TA/
b oligomer (42 kD). The protein ratio to b-actin was
m
consequences, including impairment of calcium buffering, gener-
ation of free radicals, activation of the mitochondrial permeability
transition and secondary excitotoxicity [36]. The generation of free
radicals may result in overproduction of nitric oxide (NO). This
prediction was verified in Fig. 3A and B that the treatment of 35 mM
glutamic acid to SH-SY5Y cells induced the rise of intracellular NO
and inducible NO synthase (i-NOS) protein levels (Model group). As
a potential anti-AD agent, 8-7 was able to lower the intracellular NO
level as well as to down-regulate the i-NOS protein level with
statistically significant. Moreover, 8-7 was found the most potent
agent among tacrine, CHR21, and their combination.
CHR21, and 8-7, the ratio was reduced from 1.4 to 1.2, 1.1, 0.9, and
0.7, respectively. The result of tacrine group was showed without
statistically significant. Excitingly, the activity of 8-7 was better
than that of either CHR21 or the combination. It was the most
active compound in down-regulating toxic Ab oligomer.
2.8. 8-7 down-regulated the glutamate-raised p53 level
No doubt, overactivation of glutamate receptors impairs cellular
calcium homeostasis and activates NO synthesis, generates free
radicals and causes programmed cell death [39]. As an apoptotic
protein, p53 is involved during programmed cell death. It was
shown in Fig. 6 that exposure of SH-SY5Y cells to 35 mM glutamate
induced a rapid increase in p53 protein level, whereas the protein
2.6. 8-7 lowered the glutamate-elevated Ca2þ influx
Sustained Ca2þ influx through glutamate receptor channels is
thought to represent a common pathway of neuronal cell death.
ratio to
b
-actin was elevated from 0.85 in Control group to 1.28 in
M of
Model group. When the cells were pre-treated with 1.25
m
4

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
Fig. 3. 8-7 decreased glutamate-rised NO and i-NOS levels. (A) NO level. SH-SY5Y cells were treated following the procedure described in Fig. 1(A). Then the cells were washed and
lysed with RIPA buffer. The levels of intracellular NO were determined by the colorimetric assay using the nitrite colorimetric assay kit (Beyotime, China); (B) i-NOS level. The
protein concentration was measured using a BCA protein assay kit. The protein expression was analyzed using Western Blot. The density of each lane was presented as
mean standard deviation (SD) for at least three individual experiments. Blots were quantified using Image J software. ###P < 0.001 vs Control group; **P < 0.01, ***P < 0.001 vs
Model group.
Fig. 4. 8-7 lowered the glutamate-elevated Ca2þ influx. (A) Photograph of Fura-3/AM dyed cells. SH-SY5Y cells were treated following the procedure described in Fig. 1(A). Af-
terwards, the cells were trypsinized, pelleted, resuspended in medium and incubated with 5 m
M Fura-3/AM in PBS containing 1.3 mM CaCl2 for 40 min at 37 ꢁC and then washed
twice to remove any extracellular dye. Then the cells were submitted to fluorescence microscope for photograph. (B) Fluorescence intensity of intramolecular Ca2þ. All the dyed cells
were submitted to a flow cytometry (BD FACS Calibur, Franklin Lakes, CA, USA) for fluorescence detection. Excitation wavelength was 340/380 nm; emission wavelength was
510 nm. The fluorescence ratio (F340/F380) was calculated as an indicator of [Ca2þ]i. The intensity of each lane was presented as mean standard deviation (SD) for at least three
individual experiments. ###P < 0.001 vs Control group; ***P < 0.001 vs Model group.
tacrine, CHR21, TA/CHR21, and 8-7, the ratio was reduced from 1.4
to 1.1, 1.08, 1.16, and 0.72, respectively. Again, the activity of con-
jugate 8-7 was confirmed the most potent agent. Intriguingly, the
combination showed a bit less active than both monomers.
p62, which binds ubiquitin and LC3, is a selective substrate for
autophagy. It regulates the formation of protein aggregates. In the
current studies, it was disclosed that exposure of SH-SY5Y cells to
35 mM glutamate decreased LC3-II/LC3-I ratio (Fig. 7A, model
group); in the meantime, it increased p62 protein level (Fig. 7B,
model group). Both results consistently indicated the reduction of
2.9. 8-7 attenuated the glutamate-influenced LC3 and p62 protein
levels
autophagy. Interestingly, pre-treatment of 1.25
mM 8-7 reversed
this glutamate-influenced tendency. It raised the LC3-II/LC3-I ratio
and down-regulated p62 protein level, both consistently implied
the augment of autophagy.
Excessive uptake of calcium or generation of ROS induces acti-
vation of the mitochondrial permeability transition. And low-
intensity stress can cause depolarization of mitochondria leading
to autophagy, which in turn selectively removes damaged mito-
chondria as a cytoprotective mechanism [40]. As an autophago-
some marker, the microtubule-associated protein 1A/1B-light chain
3 (LC3) has been used to monitor autophagy. During autophagy, a
cytosolic form of LC3 (LC3-I) is conjugated to phosphatidyletha-
nolamine to form LC3-phosphatidyl- ethanolamine conjugate (LC3-
II), which is recruited to autophagosomal membranes. The protein
2.10. 8-7 elevated the phosphorylation levels of ULK
Since 8-7 reversed the glutamate-influenced LC3 and p62 pro-
tein levels, it may probably have an impact on Unc-51 like kinase
(ULK) 1 and 2. As we know, ULK1 and 2 are multifunctional proteins
with roles in autophagy, neurite outgrowth, and vesicle transport.
The ULK1 balance is very important in autophagy modulation.
5

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
2.11. Bloodꢀbrain barrier (BBB) permeability assay
Penetration of the bloodꢀbrain barrier (BBB) is a critical factor
for successful CNS drugs. To determine the BBB penetration of the
candidate, the parallel artificial membrane permeability (PAMPA)
assay was employed, which was initially established by Di et al.
[41]. In the current experiment, 7 commercial drugs through a lipid
extract of porcine brain were determined using a mixture of PBS
and ethanol in the ratio of 70:30 (Table S1, Supporting Information).
A plot of experimental data versus reported values produced the
linear correlation Pe (exp) ¼ 1.1719Pe(bibil.)-0.0221 (R2 ¼ 0.9404)
(Fig. S6). According to this equation and taking into account the
described limits by Di et al. for BBB permeation, we determined
that compounds with permeability above 4.67 ꢂ 10ꢀ6 cm/s pene-
trate into the CNS by passive diffusion. Based on this assay, the
permeability of 8-7 was found (4.92 0.67) ꢂ 10ꢀ6 cm/s, indicating
good permeability to penetrate BBB.
3. Discussion
Fig. 5. 8-7 reduced the glutamate-upregulated level of high molecular weight A
b
In the present investigations, we confirmed that incorporation
of CHR21 with tacrine through a propriate spacer did enhance the
binding affinity with AChE. As the most optimal candidate, 8-7
showed 7.72-fold stronger activity than tacrine itself. Furthermore,
cytotoxicity of 8-7 against SH-SY5Y cells was far less than that of
oligomer. SH-SY5Y cells were treated following the procedure described in Fig. 1(A).
Then the cells were washed and lysed with RIPA buffer. The protein concentration was
measured using a BCA protein assay kit. The protein expression was analyzed using
Western Blot. The density of each lane was presented as mean standard deviation
(SD) for at least three individual experiments. Blots were quantified using Image J
software. ###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs Model
group.
tacrine (Fig. S1). It is nontoxic even at the dose of 100 mM.
As we know, excitatory amino acids serve as the major excit-
atory neuro-transmitters in the cerebral cortex and hippocampus.
Neurons containing excitatory amino acids play crucial roles in
psychological functions such as learning and memory. Therefore,
we set up an excitotoxic cell model to evaluate their potential
against Alzheimer’s disease. It was found that exposure of SH-SY5Y
cells to 35 mM glutamate caused cell death involving excessive
calcium influx and release from internal organelles (Fig. 4), NO
generation (Fig. 3A), Ab high MW oligomer accumulation (Fig. 5),
and oxyradical production (Fig. S2). In all cases, the conjugate 8-7
showed the best activity to reverse the glutamate-elevated levels of
all these indications.
It has been recognized nowadays that acetylcholine is widely
distributed in the nervous system and has been implicated to play a
critical role in cerebral cortical development, cortical activity,
controlling of cerebral blood flow, and sleep-wake cycle as well as
in modulating cognitive performance and learning and memory
processes [42]. No doubt, overactivation of AChE causes the loss of
cholinergic neurons, consequently leading to AD. In the current
study, we noticed that exposure of SH-SY5Y cells to 35 mM gluta-
mate up-regulated AChE protein level (Fig. 2). Interestingly, as a
clinical AChE inhibitor, tacrine was capable of attenuating the level
of AChE, while the conjugate 8-7 showed more active than tacrine.
The monomer CHR21 was found no activity.
Fig. 6. 8-7 down-regulated the glutamate-raised p53 protein level. SH-SY5Y cells were
treated following the procedure described in Fig. 1(A). Then the cells were washed and
lysed with RIPA buffer. The protein concentration was measured using a BCA protein
assay kit. The protein expression was analyzed using Western Blot. The density of each
There is accumulating evidence that soluble amyloid-
b (Ab)
oligomers, rather than amyloid fibrils, are the principal pathogenic
species in AD [43,44]. Overactivation of glutamate receptors does
lane was presented as mean
standard deviation (SD) for at least three individual
experiments. Blots were quantified using Image J software. ###P < 0.001 vs Control
group; *P < 0.05, **P < 0.01, ***P < 0.001 vs Model group.
increase the level of A
relation between AD and excitotoxicity. As a clinical anti-AD agent,
tacrine showed a tendency to down-regulate the level of A high
b high MW oligomer, implying the close
b
MW oligomer without statistically significant (Fig. 5). However, 8-7
significantly decreased this oligomer level. Consistently, over-
activation of glutamate receptors also elevated the protein level of
beta-site amyloid precursor proteinecleaving enzyme 1 (BACE1)
Therefore, we investigated the influence of 8-7 on ULK. As shown in
Fig. 8, exposure of SH-SY5Y cells to 35 mM glutamate down-
regulated phospo-ULK/ULK protein ratio, implying the reduction
(Fig. S3). This enzyme is essential for the generation of Ab peptide
of autophagy. However, pre-treatment of 1.25 mM 8-7 significantly
in AD. Interestingly, 8-7 significantly down-regulated BACE1 pro-
tein level; while tacrine did not have this function. All these data
indicate that 8-7 is a more potent agent in the treatment of AD than
tacrine.
elevated the phospo-ULK/ULK protein ratio, indicating the increase
of autophagy. It can be seen from Fig. 8 that the activity of tacrine/
CHR21 combination was far less than that of 8-7.
6

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
Fig. 7. 8-7 attenuated the glutamate-influenced LC3 and p62 protein levels. (A) 8-7 up-regulated the glutamate-reduced LC3-II/LC3-I ratio; (B) 8-7 attenuated the glutamate-
increased p62 protein level. The protein expression was analyzed using Western Blot. The density of each lane was presented as mean standard deviation (SD) for at least
three individual experiments. Blots were quantified using Image J software. ###P < 0.001 vs Control group; **P < 0.01, ***P < 0.001 vs Model group.
close related to neurodegenerative diseases. Recent evidence
shows that autophagy impairment is a pathogenic role in several
major neurodegenerative diseases [45]. This provides a strong
rationale for developing therapeutics to modulate autophagy in
these disorders. In the current research, we found that exposure of
SH-SY5Y cells to 35 mM glutamate reduced autophagy, where LC3-
II/LC3-I ratio and phospo-ULK level was decreased, and p62 level
was elevated (Fig. 7A and B and Fig. 8). However, pre-treatment of
8-7 consistently reversed glutamate-induced tendencies, indi-
cating the raise of autophagy with statistically significant. Once
again, 8-7 was shown as the most active modulator to autophagy,
compared to both monomers and the combination. This implied
that 8-7 can alleviate Alzheimer’s disease by the adjustment of
autophagy.
Fortunately, incorporation of CHR21 to tacrine does reduce the
hepatotoxicity caused by tacrine. As shown in Fig. S7, tacrine
induced significant hepatotoxicity indicated by increased activity of
both aspartate aminotransferase (AST) and alanine aminotrans-
ferase (ALT) at 24, and 36 h, respectively; while compound 8-7 did
Fig. 8. 8-7 reversed the glutamate-decrased phospo-ULK/ULK ratio. SH-SY5Y cells
not alter these parameters from 0 to 72 h. In the meantime, an
were treated following the procedure described in Fig. 1(A). Then the cells were
increase in the number of inflammatory cells in portal fields and a
large area of necrosis and distinct fatty degeneration of the hepa-
tocytes were observed after tacrine treatment. However, only mi-
nor morphological changes were found after the administration
with 8-7 (Fig. S8). We attributed this to the enhanced antioxidative
capacity induced by CHR21 [25].
washed and lysed with RIPA buffer. The protein concentration was measured using a
BCA protein assay kit. The protein expression was analyzed using Western Blot. The
density of each lane was presented as mean standard deviation (SD) for at least three
individual experiments. Blots were quantified using Image J software. ###P < 0.001 vs
Control group; *P < 0.05, ***P < 0.001 vs Model group.
We did notice that programmed cell death (apoptosis) cascades
were engaged in excitotoxicity. Exposure of SH-SY5Y cells to 35 mM
glutamate induces a rapid increase in mRNA and protein levels of
p53 and Bax (Fig. 6 and Fig. S4). These apoptotic proteins induce
mitochondrial membrane permeability changes resulting in the
activation of proteases, such as caspase-3 (Fig. S5). Pre-treatment
4. Conclusion
In summary, a novel series of tacrine/CHR21 conjugates has
been designed and synthesized. Their biological activities on inhi-
bition of ChEs and protection on glutamate-induced cell impair-
ment were evaluated. It was found that when tacrine was
conjugated with CHR21 through an 8-carbon chain, the resulted
candidate (compound 8-7) showed the most active inhibitory effect
on AChE, which was 7.72-fold stronger than tacrine.
Exposure of SH-SY5Y cells to 35 mM glutamate elevated cell
death including programmed cell apoptosis, and protein levels of
AChE, i-NOS, Ab high MW oligomer, p53, Bax, and p62; Whilst in
with 1.25
mM tacrine, CHR21, TA/CHR21, and 8-7, respectively,
prevented glutamate-induced increase in p53 and Bax expression
and maintains Bcl-2 in an elevated state. The down-regulation of
p53 and Bax implied the decrease of cell apoptosis. This was
consistent with the down-regulated level of phospo-Caspase 3
(Fig. S5). Again, 8-7 was displayed as the most potent agent
amongst them.
Nowadays, it is well known that autophagy is a lysosomal
degradative process used to recycle obsolete cellular constituents
and eliminate damaged organelles and protein aggregates. It is
the meantime, down-regulated the ratios of LC3-II/LC3-I and
phospo-ULK/ULK. It also induced excessive calcium influx and
release from internal organelles, raised NO production.
7

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
Interestingly, pretreatment of 1.25
m
M 8-7 significantly reversed all
5.1.2. General procedure for the synthesis of compd. 8-n (n ¼ 1e7)
these glutamate-induced dysfunctions. It was confirmed the most
potent candidate if compared to tacrine, CHR21, and TA/CHR21
combination. Furthermore, 8-7 showed the BBB permeability by
passive diffusion, and it did not show signs of hepatotoxicity, which
was much safer than tacrine.
Altogether, these data strongly support that 8-7 is a reliable and
potent drug candidate for treating AD. It was clearly disclosed as an
excellent AChE inhibitor and protein regulator, as well as a good
protein modulator against i-NOS, p53, Bax, LC3, p62 and ULK.
To
a
25-mL round-bottom flask, compd. 4-n (n
¼
1~7)
(0.25 mmol) and compd. 7 (67.1 mg, 0.25 mmol) were dissolved in
10 mL dried methanol. After the addition of 10 mol% amount of
activated molecular sieve, the mixture was stirred at r.t. for 4 h.
Then the mixture was maintained at 0 ꢁC for 5 min. Afterwards,
NaCNBH3 (50.0 mg, 0.75 mmol) was added, and the mixture was
kept stirring overnight. To end the reaction, removal of solvent was
firstly carried on by rotate evaporation following the addition of
saturated NH4Cl solution. The mixture was then extracted with
ethyl acetate (20 mL ꢂ 3). All the extractions were combined and
washed with saturated NaCl, dried over anhydrous Na2SO4. After
filtration following removal of solvent, the residues were submitted
to RP-HPLC for purification.
5. Experimental section
5.1. General methods for chemistry
5.1.2.1. Methyl (1S,4aS,7S,7aS)-1-isopropoxy-7-(((2-((1,2,3,4-
tetrahydroacridin- 9-yl) amino)ethyl)amino)methyl)-1,4a,5,6,7,7a-
hexahydrocyclopenta[c]pyran-4- carboxylate (8-1). Compd. 4-1
(60.3 mg, 0.25 mmol) reacted with compd. 7 (67.1 mg, 0.25 mmol)
through the procedure described in general procedure. After RP-
HPLC purification, 55.5 mg of slight yellow oil 8-1 was obtained
All reagents and solvents were used as purchased from com-
mercial sources or indicated otherwise. Flash chromatography was
performed using silica gel (300 mesh). All reactions were moni-
tored by TLC, using silica gel plates with the fluorescence F254
displayed by UV light visualization. 1H NMR and 13C NMR spectra
were recorded on a Bruker AV-400 spectrometer or Bruker AV-300.
Coupling constants (J) are expressed in Hertz (Hz). Chemical shifts
in 45% yield. Purity: 97.2%. 1H NMR (300 MHz, CDCl3)
d: 8.20 (t,
J ¼ 7.8, 2H), 7.60e7.50 (m, 1H), 7.39e7.28 (m, 2H), 5.33 (d, J ¼ 3.0,
1H), 4.17 (s, 2H), 3.87e3.83 (m, 1H), 3.66 (s, 3H, eOCH3), 3.23e3.18
(m, 2H), 3.06e3.02 (m, 3H), 2.92e2.88 (m, 1H), 2.73-2.46 (m, 4H),
2.33e2.20 (m, 1H), 2.11e2.05 (m, 1H), 1.99-1.61 (m, 6H), 1.54e1.49
(m, 2H), 1.20 (s, 1H), 1.09 (d, J ¼ 6.0, 3H, eCH3), 1.06 (d, J ¼ 6.3, 3H,
(d) of NMR are reported in parts per million (ppm) units relative to
an internal control (TMS). Low resolution ESI-MS data were recor-
ded on a Finnigan LCQ Advantage MAX mass spectrometer and high
resolution ESI-MS data on an Applied Biosystems Q-STAR Elite ESI-
LC-MS/MS mass spectrometer. The purity of the compounds was
determined by reverse phase high performance liquid chroma-
tography (HPLC) analysis to be >95%. HPLC was performed on
either LC-100 liquid chromatograph equipped with a tunable LC-
100 UV detector (Shanghai Wufeng Inc., China) or Agilent 1200
series liquid chromatograph equipped with an Agilent 1200 Series
UV detector (Agilent Technologies, USA). The columns used were
Cosmosil 5C18 (Nacalai Tesque Inc., Japan) for general purification. A
flow rate of 1.0 mL/min was used with mobile phase of MeOH in
H2O with 0.1% modifier (ammonia or trifluoroacetic acid, v/v).
The synthesis of compd. 4-n (n ¼ 1~7) started from methyl
anthranilate followed a procedure reported in one of our previous
works [46]. The purity of each alkylenediamine was more than
95.0%. Whilst the preparation of compd. 6 (CHR21) was carried on
based on the process shown in another of our early works [25].
eCH3); 13C NMR (75 MHz, CDCl3)
d: 167.93, 158.18, 151.75, 151.37,
147.00, 128.41, 128.11, 123.55, 122.97, 120.18, 115.98, 110.74, 97.55,
71.45, 51.18, 50.24, 49.51, 48.17, 43.52, 41.68, 35.68, 33.59, 30.80,
26.95, 24.80, 23.66, 23.01, 22.72, 21.55. HRESIMS (m/z): calcd. for
C
29H40N3O4 ([MþH]þ) 494.2974, found: 494.3013.
5.1.2.2. Methyl (1S,4aS,7S,7aS)-1-isopropoxy-7-(((2-((1,2,3,4-
tetrahydroacridin- 9-yl) amino)propyl)amino)methyl)-1,4a,5,6,7,7a-
hexahydrocyclopenta[c]pyran-4- carboxylate (8-2). Compd. 4-2
(63.8 mg, 0.25 mmol) reacted with compd. 7 (67.1 mg, 0.25 mmol)
through the procedure described in general procedure. After RP-
HPLC purification, 60.9 mg of slight yellow oil 8-2 was obtained
in 48% yield. Purity: 98.5%. 1H NMR (300 MHz, CD3OD)
d: 8.15 (d,
J ¼ 7.8, 1H), 7.75 (dd, J ¼ 8.4, 0.9, 1H), 7.61e7.56 (m, 1H), 7.48e7.27
(m, 2H), 4.86 (d, J ¼ 9.0, 1H), 4.22e3.93 (m, 1H), 3.78e3.63 (m, 5H),
2.95 (d, J ¼ 5.7, 2H), 2.77e2.69 (m, 6H), 2.58e2.54 (m, 1H),
2.43e2.29 (m, 1H), 2.20e2.14 (m, 1H), 2.01e1.72 (m, 8H), 1.39e1.33
5.1.1. Methyl (1S,4aS,7S,7aS)-7-formyl-1-isopropoxy-1,4a,5,6,7,7a-
hexahydro- cyclopenta[c]pyran-4-carboxylate (7)
(m, 3H), 1.23e1.10 (m, 7H); 13C NMR (75 MHz, CD3OD)
d: 168.04,
156.49, 152.24, 151.74, 145.30, 129.01, 125.45, 123.58, 123.28, 119.23,
114.71, 110.41, 97.29, 71.18, 50.31, 48.48, 46.75, 46.61, 42.27, 41.67,
35.56, 32.03, 30.49, 30.06, 26.40, 24.95, 22.70, 22.55, 22.02, 20.56.
HRESIMS (m/z): calcd. for C30H42N3O4 ([MþH]þ) 508.3131, found:
508.3179.
To a 25-mL round-bottom flask, compd. 6 (121.6 mg, 0.45 mmol)
was dissolved in 8.0 mL dried dichloromethane (DCM). Then Dess-
Marin reagent (384.5 mg, 0.90 mmol) was added portion by portion
within 0.5 h. The suspension mixture was kept stirring at r. t. for 6 h,
and then was put over ice-batch for 0.5 h. Filtration was carried on.
The residues were washed with DCM. All the filtrates were com-
bined, dried over anhydrous Na2SO4. Removal of solvent was then
carried on by rotating distillation. The residues were submitted to
flash column liquid chromatography for purification applying pe-
troleum/ethyl acetate (1:1 in V:V) as eluent, offering white oil
5.1.2.3. Methyl (1S,4aS,7S,7aS)-1-isopropoxy-7-(((2-((1,2,3,4-
tetrahydroacridin- 9-yl) amino)butyl)amino)methyl)-1,4a,5,6,7,7a-
hexahydrocyclopenta[c]pyran-4- carboxylate (8-3). Compd. 4-3
(67.3 mg, 0.25 mmol) reacted with compd. 7 (67.1 mg, 0.25 mmol)
through the procedure described in general procedure. After RP-
HPLC purification, 57.4 mg of slight yellow oil 8-3 was obtained
103.4 mg in 85.6% yield. 1H NMR (300 MHz, CDCl3)
d: 9.83 (d,
J ¼ 5.1 Hz, 1H), 7.41 (s, 1H), 5.43 (d, J ¼ 3.3 Hz, 1H), 3.97e3.92 (m,
1H), 3.72 (s, 3H, eOCH3), 2.82e2.73 (q, J ¼ 8.4 Hz, 1H), 2.50e2.40
(m, 1H), 2.34e2.28 (m, 1H), 2.17 (dd, J ¼ 9.1, 3.3 Hz, 1H), 1.79e1.60
(m, 2H), 1.47e1.36 (m, 2H), 1.18e1.14 (m, 6H); 13C NMR (75 MHz,
in 44% yield. Purity: 96.6%. 1H NMR (300 MHz, CDCl3)
d: 7.91 (d,
J ¼ 8.1, 1H), 7.84 (d, J ¼ 8.1, 1H), 7.49 (t, J ¼ 7.2, 1H), 7.37 (s, 1H),
7.33e7.25 (m, 1H), 4.75 (d, J ¼ 9.0, 1H), 4.05e3.99 (m, 1H), 3.65 (s,
3H, eOCH3), 3.10e2.99 (m, 4H), 2.81e2.75 (m, 1H), 2.70e2.64 (m,
3H), 2.60e2.47 (m, 3H), 2.41e2.29 (m, 1H), 2.27e2.15 (m, 1H),
2.06e2.01 (m, 1H), 1.92e1.76 (m, 6H), 1.72e1.60 (m, 3H), 1.60e1.47
(m, 2H), 1.40e1.26 (m, 2H), 1.19 (d, J ¼ 6.3, 3H, eCH3), 1.15 (d, J ¼ 6.0,
CDCl3) d: 191.2, 167.3, 152.7, 111.6, 99.1, 70.8, 51.5, 45.2, 41.3, 34.8,
31.2, 27.3, 23.5, 21.6; ESI-MS (m/z): 291.2[MþNa]þ (calcd.
C
C
14H20O5þNa: 291.3); HRESIMS (m/z): 291.1211[MþNa]þ (calcd.
14H20O5þNa: 291.1208).
3H, eCH3); 13C NMR (75 MHz, CDCl3)
d: 168.03, 158.41, 151.80,
8

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
150.98, 147.28, 128.47, 128.35, 123.69, 122.92, 120.16, 115.85, 110.84,
97.40, 71.22, 51.41, 51.27, 49.62, 49.37, 43.22, 41.59, 35.80, 33.75,
30.80, 29.69, 27.60, 26.92, 24.92, 23.72, 23.07, 22.76, 21.50; HRE-
SIMS (m/z): calcd. for C31H44N3O4 ([MþH]þ) 522.3287, found:
522.3326.
5.1.2.7. Methyl (1S,4aS,7S,7aS)-1-isopropoxy-7-(((2-((1,2,3,4-
tetrahydroacridin-9-yl)
amino)octyl)amino)methyl)-1,4a,5,6,7,7a-
hexahydrocyclopenta[c]pyran-4-carboxylate (8-7). Compd. 4e7
(81.4 mg, 0.25 mmol) reacted with compd. 7 (67.1 mg, 0.25 mmol)
through the procedure described in general procedure. After RP-
HPLC purification, 76.6 mg of slight yellow oil 8-7 was obtained
in 53% yield. Purity: 98.5%. 1H NMR (300 MHz, CDCl3)
d: 7.89 (dd,
5.1.2.4. Methyl (1S,4aS,7S,7aS)-1-isopropoxy-7-(((2-((1,2,3,4-
tetrahydroacridin- 9-yl) amino)pentyl)amino)methyl)-1,4a,5,6,7,7a-
hexahydrocyclopenta[c]pyran-4- carboxylate (8-4). Compd. 4-4
(70.8 mg, 0.25 mmol) reacted with compd. 7 (67.1 mg, 0.25 mmol)
through the procedure described in general procedure. After RP-
HPLC purification, 68.3 mg of slight yellow oil 8-4 was obtained
J ¼ 8.4, 0.6, 1H), 7.84 (dd, J ¼ 8.4, 0.6, 1H), 7.50e7.46 (m, 1H), 7.36 (s,
1H), 7.31e7.21 (m, 1H), 4.76 (d, J ¼ 9.0, 1H), 4.10e4.03 (m, 1H),
4.01e3.90 (m, 1H), 3.64 (s, 3H, eOCH3), 3.04e2.96 (m, 2H),
2.80e2.74 (m, 1H), 2.71e2.56 (m, 4H), 2.56e2.42 (m, 3H),
2.38e2.33 (m, 1H), 2.23e2.19 (m, 1H), 2.07e2.03 (m, 1H), 1.91e1.73
(m, 6H), 1.66e1.50 (m, 2H), 1.45e1.38 (m, 2H), 1.37e1.22 (m, 11H),
1.20 (d, J ¼ 6.3, 3H, eCH3), 1.16 (d, J ¼ 6.0, 3H, eCH3); 13C NMR
in 51% yield. Purity: 96.2%. 1H NMR (300 MHz, CDCl3)
d: 7.99e7.79
(m, 2H), 7.49 (t, J ¼ 7.5, 1H), 7.36 (s, 1H), 7.28 (t, J ¼ 7.5, 1H), 4.75 (d,
J ¼ 9.0, 1H), 4.09e3.99 (m, 2H), 3.64 (s, 3H, eOCH3), 3.13e2.97 (m,
5H), 2.88e2.71 (m, 1H), 2.65e2.58 (m, 3H), 2.56e2.49 (m, 3H),
2.42e2.31 (m, 1H), 2.26e2.14 (m, 1H), 2.06e2.00 (m, 1H), 1.86e1.74
(m, 5H), 1.75e1.55 (m, 2H), 1.52e1.44 (m, 2H), 1.41e1.31 (m, 4H),
1.19 (d, J ¼ 6.3, 3H, eCH3), 1.14 (d, J ¼ 6.0, 3H, eCH3); 13C NMR
(75 MHz, CDCl3) d: 167.91, 158.34, 151.69, 150.83, 147.38, 128.57,
128.25, 123.52, 122.87, 120.15, 115.73, 110.78, 97.45, 71.16, 51.47,
51.13, 50.16, 49.49, 43.34, 41.54, 35.68, 33.93, 31.75, 30.76, 30.21,
29.49, 29.28, 27.44, 26.87, 24.75, 23.62, 23.03, 22.75, 21.44. HRE-
SIMS (m/z): calcd. for C35H52N3O4 ([MþH]þ) 578.3913, found:
578.3952.
(75 MHz, CDCl3) d: 167.90, 157.87, 151.68, 151.14, 146.71, 128.60,
127.91, 123.71, 122.92, 119.83, 115.49, 110.78, 97.33, 71.16, 51.36,
51.17, 49.80, 49.25, 43.07, 41.54, 35.68, 33.44, 31.63, 30.73, 29.76,
26.86, 24.82, 24.71, 23.63, 22.93, 22.57, 21.44. HRESIMS (m/z): calcd.
for C32H46N3O4 ([MþH]þ) 536.3444, found: 536.3483.
5.2. Cholinesterase inhibition
Acetylcholinesterase (AChE, EC 3.1.1.7) from electric eel
(600e800 units/mg, lyophilized powder), Butyrylcholinesterase
from equine serum (BuChE, EC 3.1.1.7, min. 500 units/mg protein in
buffered aqueous solution), acetylthiocholine iodide (ATCI), S-
butyrylthiocholine iodide (BTCI), 5, 50-dithiobis(2-nitrobenzoic
acid) (Ellman’s reagent, DTNB) and tacrine hydrochloride were
purchased from Sigma-Aldrich (St. Louis, MO, USA). The inhibitory
activities of test compounds 4-n (n ¼ 1e7), 7, and 8-n (n ¼ 1e7) was
evaluated by Ellman’s method [47]. The compounds were dissolved
in DMSO and diluted with 100 mM phosphate buffer (PBS, pH 8.0)
led to corresponding test concentration (DMSO less than 0.01%). In
5.1.2.5. Methyl (1S,4aS,7S,7aS)-1-isopropoxy-7-(((2-((1,2,3,4-
tetrahydroacridin- 9-yl) amino)hexyl)amino)methyl)-1,4a,5,6,7,7a-
hexahydrocyclopenta[c]pyran-4- carboxylate (8-5). Compd. 4e5
(74.4 mg, 0.25 mmol) reacted with compd. 7 (67.1 mg, 0.25 mmol)
through the procedure described in general procedure. After RP-
HPLC purification, 75.6 mg of slight yellow oil 8-5 was obtained
in 55% yield. Purity: 99.3%. 1H NMR (300 MHz, CDCl3)
d: 7.94e7.89
(m, 2H), 7.58e7.49 (m, 1H), 7.41 (s, 1H), 7.37e7.29 (m, 1H), 4.81 (d,
J ¼ 9.0, 1H), 4.11e4.04 (m, 1H), 4.03e3.95 (m, 1H), 3.70 (s, 3H,
eOCH3), 3.08e3.04 (m, 2H), 2.85e2.81 (m, 1H), 2.75e2.60 (m, 4H),
2.58e2.50 (m, 4H), 2.48e2.35 (m,1H), 2.31e2.20 (m,1H), 2.10e2.06
(m, 1H), 1.95e1.80 (m, 6H), 1.72e1.64 (m, 2H), 1.58e1.45 (m, 2H),
1.45e1.32 (m, 6H), 1.25 (d, J ¼ 6.3, 3H, eCH3), 1.20 (d, J ¼ 6.0, 3H,
each well of the plate, 150
AChE (1.0 U/ml) or 20 l of BuChE (1.0 U/ml) were incubated with
10 of different concentrations of tested compounds
(1.0e100
M) at 37 ꢁC for 6 min. After this period, ATCI (10 mM) or
BTCI (10 mM) as the substrate (10 L) was added and the absor-
ml of PBS, 10 ml of 10 mM DTNB, 20 ml of
m
mL
m
m
eCH3); 13C NMR (75 MHz, CDCl3)
d: 168.05, 158.38, 151.83, 151.05,
bance was measured with a wavelength of 412 nm at a frequency of
once per 10 s within 1 min. IC50 values were calculated as con-
centration of compound that produces 50% enzyme activity inhi-
bition, using the Graph Pad Prism 4.03 software (San Diego, CA,
USA). Results are expressed as the mean
different experiments performed in triplicate.
147.34, 128.58, 128.52, 123.76, 123.02, 120.22, 115.87, 110.94, 97.56,
71.31, 51.61, 51.30, 50.15, 49.58, 43.43, 41.68, 35.83, 33.96, 31.84,
30.90, 30.26, 27.43, 27.06, 27.03, 24.88, 23.78, 23.15, 22.85, 21.59.
HRESIMS (m/z): calcd. for C33H48N3O4 ([MþH]þ) 550.3600, found:
550.3687.
SD of at least three
5.3. Cell culture
5.1.2.6. Methyl (1S,4aS,7S,7aS)-1-isopropoxy-7-(((2-((1,2,3,4-
tetrahydroacridin- 9-yl) amino)heptyl)amino)methyl)-1,4a,5,6,7,7a-
hexahydrocyclopenta[c]pyran-4- carboxylate (8-6). Compd. 4e6
(77.7 mg, 0.25 mmol) reacted with compd. 7 (67.1 mg, 0.25 mmol)
through the procedure described in general procedure. After RP-
HPLC purification, 88.8 mg of slight yellow oil 8-6 was obtained
Human neuroblastoma SH-SY5Y cells were cultured in DMEM
medium containing 10% (v/v) fetal bovine serum (FBS), 100 g/mL
of streptomycin and 100 U/ml of penicillin in a water-jacketed
incubator at 37 ꢁC in a humidified atmosphere of 5% CO2. Cells
were fed every 3 days and sub-cultured once they reached 80e90%
confluence.
m
in 63% yield. Purity: 98.7%. 1H NMR (300 MHz, CD3OD)
d: 8.09 (d,
J ¼ 8.4, 1H), 7.77 (d, J ¼ 8.4, 1H), 7.55 (t, J ¼ 7.8, 1H), 7.43 (s, 1H), 7.36
(t, J ¼ 7.5, 1H), 5.00e4.93 (m, 1H), 4.14e4.03 (m, 1H), 3.69 (s, 3H,
eOCH3), 3.53 (t, J ¼ 6.9, 2H), 2.97 (s, 2H), 2.89e2.69 (m, 4H),
2.61e2.51 (m, 3H), 2.48e2.41 (m, 1H), 2.28e2.23 (m, 1H), 2.20e1.87
(m, 6H), 1.68e1.58 (m, 2H), 1.49e1.25 (m, 12H), 1.22e1.19 (m, 6H);
5.4. MTT assay
The viability of SH-SY5Y cells mediated by drugs was measured
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT, 570 nm) assay. The process was described below: Cells
in suspension were plated in 96-well plates at a density of
5 ꢂ 103 cells/well and were treated with either vehicle (0.1% DMSO)
or drugs for 4 h after 24 h culture. The final DMSO concentration in
all experiments was less than 0.1% in medium. The medium was
discharged and the cells were washed with PBS. Cells were treated
13C NMR (75 MHz, CD3OD)
d: 168.11, 157.49, 151.98, 151.75, 146.33,
128.45, 126.39, 123.32, 123.08, 119.80, 115.24, 110.49, 97.32, 71.16,
50.63, 50.29, 49.10, 48.31, 42.36, 41.72, 35.60, 32.67, 30.82, 30.48,
29.03, 28.89, 26.99, 26.45, 26.38, 24.75, 22.74, 22.70, 22.29, 20.56;
HRESIMS (m/z): calcd. for C34H50N3O4 ([MþH]þ) 564.3757, found:
564.3823.
9

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
with 35 mM glutamic acid for 24 h. Again, the medium was dis-
5.8. BBB permeation assay
charged and the cells were washed with PBS. Then 10 mL 5 mg/mL
MTT solutions were added to each well, and the plate was incu-
bated for an additional 4 h. The absorbance was measured at
570 nm using a microplate reader (Bio-Rad; Hercules, CA, USA)
The BBB penetration of compounds was evaluated using the
parallel artificial membrane permeation assay (PAMPA) described
by Di et al. [41]. Commercial drugs were purchased from Sigma and
Alfa Aesar. Porcine brain lipid (PBL) was obtained from Avanti Polar
Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm)
and acceptor microplate were both from Millipore. The 96-well UV
plate (COSTAR) was from Corning Incorporated. The acceptor 96-
after 150
times.
mL of DMSO added. All of the tests were repeated at least 3
5.5. Nitrite measurement in vitro
well microplate was filled with 300
filter membrane was impregnated with 4
(20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and
diluted 50-fold in PBS/EtOH (7:3) to a final concentration of 100 g/
mL. Then, 200 L of the solution was added to the donor wells. The
acceptor filter plate was carefully placed on the donor plate to form
a sandwich, which was left undisturbed for 10 h at 25 ꢁC. After
incubation, the donor plate was carefully removed, and the con-
centration of compounds in the acceptor wells was determined
using the UV plate reader (Flexstation 3). Every sample was
analyzed at five wavelengths in four wells and in at least three
independent runs. Pe was calculated by the following expression:
Pe ¼ ꢀVd ꢂ Va/[(Vd þ Va)A ꢂ t] ꢂ ln(1 ꢀ drugacceptor/drugequilibrium),
where Vd is the volume of donor well; Va, volume in acceptor well;
A, filter area; t, permeation time; drugacceptor, the absorbance ob-
tained in the acceptor well; and drugequilibrium, the theoretical
equilibrium absorbance. The results are given as the
m
L PBS/EtOH (7:3), and the
The levels of intracellular NO were determined by the colori-
metric assay using the nitrite colorimetric assay kit (Beyotime,
China), according to the manufacturer’s instructions. Briefly, SH-
SY5Y cells (2 ꢂ 105 cells/well) were treated with either vehicle
(0.1% DMSO) or drugs for 4 h after 24 h culture. The final DMSO
concentration in all experiments was less than 0.1% in medium. The
medium was discharged and the cells were washed with PBS. Cells
were treated with 35 mM glutamic acid for 24 h. Again, the medium
was discharged and the cells were washed with PBS. Subsequently,
the cells were harvested and their cell lysates were prepared and
then mixed with Griess reagent for 10 min at 37 ꢁC, followed by
measurement at 540 nm by a microplate reader. The cells treated
with 0.4% DMSO in medium were used as negative controls for the
background levels of nitrite production, while sodium nitrite at
different concentrations was prepared as the positive control for
the establishment of a standard curve. Each experiment was
repeated at least three times.
mL PBL in dodecane
m
m
mean
standard deviation. In the experiment, 7 quality control
standards (Table S1) of known BBB permeability were included to
validate the analysis set. A plot of the experimental data versus
5.6. Measurement of intracellular Ca2þ ([Ca2þ]i)
literature values gave
a
strong linear correlation, Pe
[Ca2þ]i was monitored using the fluorescent Ca2þ-sensitive dye,
Fura-3-acetoxymethy ester (Fura-3-AM). SH-SY5Y cells in the
exponential phase were plated on a 96-well cell culture plate with
2 ꢂ 103 cells/well treated with either vehicle (0.1% DMSO) or drugs
for 4 h after 24 h culture. Cells were treated with 35 mM glutamic
acid for another 24 h. Briefly, cells were trypsinized, pelleted,
(exp.) ¼ 1.1719Pe (lit.) ꢀ 0.0221 (R2 ¼ 0.9404) (Figure S6). From this
equation and the limit established by Di et al. (Pe
(lit.) ¼ 4.0 ꢂ 10ꢀ6 cm/s) for BBB permeation, we concluded that
compounds with a permeability > 4.67 ꢂ 10ꢀ6 cm/s could cross the
BBB (Table S2).
resuspended in medium and incubated with 5
mM Fura-3-AM in
5.9. Statistical analysis
PBS containing 1.3 mM CaCl2 for 40 min at 37 ꢁC and then washed
twice to remove any extracellular dye. All the dyed cells were
submitted to a flow cytometry (BD FACS Calibur, Franklin Lakes, CA,
USA) for fluorescence detection. Excitation wavelength was 340/
380 nm; emission wavelength was 510 nm. The fluorescence ratio
(F340/F380) was calculated as an indicator of [Ca2þ]i.
Experimental values were given as means
SD. Statistical
analysis of the data was performed using the SPSS 18.0 statistical
software. One-way analysis of variance (ANOVA) was applied to
analyze for difference in data of biochemical parameters among the
different groups, followed by Dunnett’s significant post-hoc test for
pairwise multiple comparisons. Differences were considered as
statistically significant at *P < 0.05, **P < 0.01.
5.7. Western blotting analysis
SH-SY5Y cells were collected and washed with PBS after the
treatment followed the procedure as described above. Then, the
cells were lysed with RIPA buffer for 45 min on ice and then
centrifuged at 12,000 g at 4 ꢁC for 15 min. Afterwards, the protein
content of the supernatant was determined with a Pierce BCA
protein assay kit (Thermo Fisher Scientific, USA) to ensure equal
sample loading. Protein lysates were separated in 12% SDS-PAGE
and blotted onto nitrocellulose membranes (Amersham Bio-
sciences, USA). Proteins were detected using the primary mono-
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
This research was financially supported by National Natural
Science Foundation of China (Nos. 81172982 and 81973190),
Guangdong Provincial Natural Science Foundation projects
(2018B030311020 and 2020A1515010857). The authors will give
heartful thanks to Prof. Dr. Hiroshi Kurihara and Prof. Dr. Rongrong
He for their equipment support and useful discussion.
clonal antibody of
b-Actin (1:3000), AChE (1:1000), BACE1
(1:2000), i-NOS (1:500), Caspase 3 (1:1000), Cleaved Caspase 3
(1:1000), p53 (1:3000), Bcl-2 (1:1000), Bax (1:1000), LC3 (1:1000),
p62 (1:1000), Ab (1:2000) with the corresponding diluted ratio
given in parentheses, respectively for at least 16 h at 4 ꢁC, and then
washed and incubated with HRP-conjugated secondary antibodies
at room temperature for 1 h. Protein bands were visualized using
enhanced chemiluminescence detection reagents (Bio-Rad, USA).
The resulting images were scanned using a scanner (Epson V330
Photo, Japan).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113067.
10

R. Lin, S. Rao, Y. Li et al.
European Journal of Medicinal Chemistry 211 (2021) 113067
stable derivatives from genipin and studies of their neuroprotective activity in
PC12 cells, ChemMedChem 7 (2012) 1661e1668.
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