Design, synthesis and biological evaluation of novel carboline-cinnamic acid hybrids as multifunctional agents for treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2020.103844

Alzheimer's disease (AD) is a complex neurodegenerative disease with multiple pathological features. Multifunctional compounds able to simultaneously interact with several pathological components have been considered as a solution to treat the complex pathologies of neurodegenerative diseases. β-carboline and cinnamic acid have been extensively studied for their widespread biological effects in treatment of AD, further application is limited due to its poor solubility and high toxicity. Herein, a series of carboline-cinnamic acid hybrids was designed and synthesized to obtain new multifunctional molecules with low toxicity and good physicochemical properties. In particular, e3 and e12 exhibited significant inhibition of Aβ aggregation (inhibitory rate at 25 μM: 65% and 72% respectively), moderate BuChE inhibition, excellent neuroprotective effects and low neurotoxicity. Furthermore, in the AD mice model, e3 and e12 could restore learning and memory function to a comparable level to that of the control and did not exhibit any acute toxicity in vivo at a relatively high dose of 600 mg/kg. Thus, these new compounds can be further studied as multifunctional molecules for AD.

Full text of DOI:10.1016/j.bioorg.2020.103844

BioorganicChemistry99(2020)103844
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel carboline-cinnamic acid
hybrids as multifunctional agents for treatment of Alzheimer’s disease
Qinghong Liaoa, Qi Lic, Yifan Zhaoa, Pan Jiangd, Yuhui Yand, Haopeng Sunc, Wenyuan Liub,
⁎
⁎
Feng Fenga,d, , Wei Qua,
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
b Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China
c Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
d Jiangsu Food and Pharmaceutical Science College, Huaian 223003, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alzheimer’s disease (AD) is a complex neurodegenerative disease with multiple pathological features.
Alzheimer’s disease
Carboline-cinnamic acid hybrid
β-amyloid
Multifunctional compounds able to simultaneously interact with several pathological components have been
considered as a solution to treat the complex pathologies of neurodegenerative diseases. β-carboline and cin-
namic acid have been extensively studied for their widespread biological effects in treatment of AD, further
application is limited due to its poor solubility and high toxicity. Herein, a series of carboline-cinnamic acid
hybrids was designed and synthesized to obtain new multifunctional molecules with low toxicity and good
physicochemical properties. In particular, e3 and e12 exhibited significant inhibition of Aβ aggregation (in-
hibitory rate at 25 μM: 65% and 72% respectively), moderate BuChE inhibition, excellent neuroprotective effects
and low neurotoxicity. Furthermore, in the AD mice model, e3 and e12 could restore learning and memory
function to a comparable level to that of the control and did not exhibit any acute toxicity in vivo at a relatively
high dose of 600 mg/kg. Thus, these new compounds can be further studied as multifunctional molecules for AD.
Neuroprotection
1. Introduction
years, Aβ hypothesis is at the center of development of anti-AD drugs,
and drugs targeting this system are greatly expected to be discovered.
Alzheimer’s disease (AD) is a neurodegenerative disease, affecting
more than 24 million people worldwide [1]. The World Alzheimer
Report 2018 showed that 46.8 million people have been suffering from
AD, and this number is expected to exceed 131.5 million in 2050 [2].
Current therapeutic options for the treatment of AD including choli-
nesterase inhibitors (donepezil, rivastigmine and galantamine) [3] and
an N-methyl-D-aspartic Acid (NMDA) receptor antagonist (memantine)
[4], have resulted in a modest improvement in memory and cognitive
function. However, they do not prevent progressive neurodegeneration
[5]. The etiology of AD remains elusive, but multiple factors, such as β-
amyloid (Aβ) deposit, τ-protein hyperphosphorylation, oxidative stress,
neuroinflammation and a low level of acetylcholine, likely play im-
portant roles in the development of AD [6–8].
Many anti-amyloid therapies were developed, such as β- and γ-secretase
inhibitors, anti-Aβ antibodies [12]. However, recent clinical trial fail-
ures cast doubt on the validity of these therapies. It has been suggested
that secretase inhibitors suppress various other pathways in the brain
and the peripheral tissues, exhibiting severe side effects [13,14]; anti-
Aβ antibodies might be trapped in the bloodstream, allowing only
limited quantities to reach the target [15]. Thus, it is promising to
develop small molecules with Aβ aggregation inhibitory activity.
On the other hand, Low levels of acetylcholine affect the transmis-
sion of information in the brain, leading to cognitive decline [16], so
donepezil is the most effective pharmacological agent for AD treatment.
However, it is effective in reversing the symptoms for only a short
period of time [17]. Studies have shown that acetylcholine (ACh) was
mainly hydrolyzed by butyrylcholinesterase (BChE) during the ad-
vanced stage of AD [18]. Therefore, compounds that are able to inhibit
BChE may offer an alternative role for the treatment of AD, especially in
its advanced stage.
The aggregation of Aβ may induce cytotoxicity by four mechanisms:
lipid membrane permeabilization, oxidative stress, endoplasmic re-
ticulum (ER) stress and mitochondrial dysfunction [9]. Meanwhile, in
the lipid membrane, activated microglia and astrocytes by extracellular
accumulation of Aβ may cause neuroinflammation [10,11]. In recent
Recent evidences had demonstrated that oxidative damage in
⁎ Corresponding authors at: Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China.
E-mail addresses: fengfeng@cpu.edu.cn (F. Feng), popoqzh@126.com (W. Qu).
https://doi.org/10.1016/j.bioorg.2020.103844
Received 7 March 2020; Accepted 8 April 2020
Availableonline13April2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Q. Liao, et al.
BioorganicChemistry99(2020)103844
Fig. 1. Several β-carboline structures with well-known pharmacological properties.
range of biological effects [28,29]. Several evidences (Fig. 1) have
suggested that β-carboline analogs function as anti-AD agents through
inhibition of cholinesterase [30], inhibition of monoamine oxidase
(MAO) [31,32], anti-inflammation [33,34] and anti-aggregation [35].
Furthermore, the studies indicated that bivalent β-carbolines analogs
exhibited more potent AChE inhibitory activities with IC50 values in the
nanomolar range [36,37]. Evidences showed that bivalent β-carboline
derivatives exhibited good multifunctional activities such as ChEs in-
hibition, Aβ1–42 aggregation inhibition and neuroprotection [38].
However, further application of bivalent β-carboline derivatives is
limited due to its poor solubility and high toxicity [38]. Other natural
compounds, such as cinnamic acids, have also been studied extensively
as anti-AD agents because of their anti-neuroinflammatory properties
[39] and ability to inhibit Aβ aggregation [40] by scavenging oxidants
[41,42]. Thus, β-carboline and cinnamic acid were considered as sui-
table multifunctional fragments.
Considering the poor druggability of carboxylic acids, we intended
to replace carboxyl with other structures. Due to the poor physico-
chemical properties and safety of bivalent β-carboline derivatives, in
the present study, we fused β-carboline with another natural product
cinnamic acid, to obtain a new kind of heterogenous dimer with low
toxicity and molecular weight, good molecular planarity and physico-
chemical properties (Fig. 2). We hypothesize that carboline-cinnamic
acid hybrids could show complementary activities of two fragments.
Fig. 2. Design strategy for β-carboline derivatives.
cellular structures is an event that precedes the appearance of other
pathological hallmarks of AD, namely, senile plaques and neurofi-
brillary tangles [19]. The inflammation response triggered by AD has
also been shown to induce the production of proinflammatory cytokines
(such as NO, TNFα, and ROS) by microglia and resident astrocytes,
which may result in neuronal cell death, and ultimately dementia [20].
Many evidences proved that antioxidants and anti-inflammatory agent
could attenuate the syndrome of AD, and prevent the progression of the
disease. Thus, drugs with specifically antioxidants and anti-in-
flammatory activity could be useful for either the prevention or the
treatment of AD.
2. Results and discussion
2.1. Chemistry
The synthetic route for the new β-carboline derivatives is shown in
Schemes 1. Using the substituted tryptamine as a raw material, the
target compounds 2a, 2b (tetrahydro-β-carboline) were obtained by
reaction with excess aldehyde (dropwisein ice bath) in a solution of
about 5% trifluoroacetic acid (pH = 2–3) in dichloromethane. Com-
mercially available substituted cinnamic acid reacted with di-
chlorosulfoxide at 80 °C to give d1–d9. Compounds d1–d9 were reacted
with different tetrahydro-β-carboline analogs in the presence of Et3N to
provide the target compounds e1–e18.
Due to the multifactorial nature of AD, multifunctional ligands are
supposed to exhibit promising therapeutic effects, therefore, it is con-
sidered to be an important strategy for the development of anti-AD
drugs [21–23]. Some researches showed that about 84% approved
drugs for CNS diseases are NPs or NP-inspired, and interestingly, 20 NP
structures provided more than 400 clinically approved CNS drugs [24].
β-carboline, a multifunctional natural products with a pyrido [3,4-b]
indoles structure [25–27], have been extensively studied for its wide
2

Q. Liao, et al.
BioorganicChemistry99(2020)103844
Scheme 1. Synthesis of e1-e18. aReagents and conditions: (a) acetaldehyde, DCM and 5% TFA, rt, 24 h; (b) SOCl2, reflux, 4 h; (c) dry THF, Et3N, rt, 12 h.
2.2. Inhibition of self-mediated Aβ1–42 aggregation
the ability to increase acetylcholine levels [47]. Research shows that
BuChE inhibitors are more durable and stable, and have unique ad-
vantages in the treatment of moderate or severe AD [48]. To further
study the multi-biological profile of the target compounds, the abilities
of the most promising compounds e3 and e12 to inhibit AChE and
BuChE were evaluated [49]. As shown in the Table 2., e3 and e12
exhibited moderate BuChE inhibitory activities and good selectivity,
which were higher than the monomeric compounds such as caffeic
acid, 1a and 1b. From the interaction mode of e12 for BuChE(PDB:
4TPK), the carboline core interacts with Tyr332 through a π-π stacking
contact. The N atom of the indole ring interacts with Asp70 through a
hydrogen bond. The phenyl ring of the cinnamic acid moiety is engaged
in π-π interactions with Trp82. Two hydroxyl group of the cinnamic
acid moiety forms two hydrogen bond with Gly115 (Fig. 3B). Therefore,
e12 can better occupy the active site of BChE, leading to its great BChE
inhibitory activities and selectivity.
The inhibitory activities of β-carboline derivatives against self-
mediated Aβ1–42 aggregation were evaluated using a thioflavin T (ThT)
fluorescence assay [43–45]. using resveratrol as the positive control
[46]. None of the test compounds exhibited interfering signals under
the experimental conditions. The data were summarized in Table 1. The
results indicated that caffeic acid and compounds 1a and 1b presented
low inhibitory activities, while e3 and e12 were the most potent in-
hibitors of Aβ1–42 aggregation among the series of compounds featuring
a 3,4-dihydroxy group on the A ring, which proved the rationality of
our designed strategy. The methoxy-substituted compounds were less
active than the hydroxy-substituted group, indicating the importance of
hydroxy substitution for inhibiting Aβ aggregation. The different posi-
tion of methoxy group has little effect on the activity of the compound.
In addition, e5 and e14, with reduced double bonds, gave slightly
weaker inhibitory activities than e3 and e12. Compounds e9 and e18
with naphthalene rings also exhibited good inhibitory activities (54.3%
and 58.2%, respectively). We next conducted docking studies of e12
with amyloid forming peptide KLVFFA (PDB code 3OVJ) by using the
CDOCKER program embedded in Discovery Studio (DS) 2019. The re-
sults showed that e12 could embedded in β-sheet structure and mainly
exhibited hydrophobic and hydrogen-bonding interactions, indicating
that the β-carboline derivatives could inhibit Aβ1–42 aggregation by
hindering the formation of the β-sheet structure (Fig. 3A). We could
draw conclusions that polyhydroxy substitution, good molecular pla-
narity and a large conjugated system are prerequisite for inhibiting Aβ
aggregation.
2.4. Cytotoxicity of synthetic compounds in PC12, SH-SY5Y, BV-2, HT22,
L02 cells
To investigate the safety profile of these synthetic compounds, we
determined the potential cytotoxic effects of compounds e3 and e12 on
five cell lines (PC12, SH-SY5Y, BV-2, HT22, L02). After exposing the
cells to these compounds for 24 h, the cell viability was evaluated by 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. All
cells could be well tolerated at a relatively high dose (Detailed IC50
values were presented in Table S1). The results demonstrated that there
was no obvious death of five cell lines at 25 μM of e3 and e12, meaning
that these compounds were not neurotoxic when applied at these
concentrations (Fig. 4).
2.3. Inhibition of cholinesterase (ChE) activities
Cholinergic dysfunction leads to cognitive decline, and a promising
approach to the treatment of AD is the usage of natural products with
3

Q. Liao, et al.
BioorganicChemistry99(2020)103844
Table 1
Table 1 (continued)
Inhibition of Aβ1–42 aggregation of β-carboline derivatives e1-
Cpd.
R1
Aβ1–42 aggregation
inhibition (%)a
e18.
Resveratrol
–
–
43.48
2.26
a
The thioflavin-T fluorescence method was used. The values are expressed
SEM of at least three independent measurements. All values
were obtained at a compound concentration of 20 μM.
n.a. means no activity.
Cpd.
R1
Aβ1–42 aggregation
inhibition (%)a
as the mean
b
e1
e2
e3
e4
Methoxyl
Methoxyl
Methoxyl
Methoxyl
18.65
32.09
65.49
20.19
5.63
1.16
2.25
4.70
2.5. Neuroprotection of compounds on H2O2-induced cell insults
Oxidative stress is an important pathogenesis of AD, which can be
induced by H2O2 and cause cell damage [50]. Therefore, we evaluated
the neuroprotective effects of the e3 and e12 on H2O2-induced cells
insults [51]. As expected, the treatment of PC12 and SH-SY5Y cells with
H2O2 for 24 h significantly reduced cell viability. Meanwhile, the sur-
vival of H2O2-treated cells obviously increased following pretreatment
with e3 or e12 in a dose-dependent manner. The compounds even
completely reversed this damage at 15 μM. These data indicated that e3
and e12 were protective agents against H2O2-induced cell death in
human PC12 and SH-SY5Y cells (Fig. 5).
e5
e6
e7
Methoxyl
Methoxyl
Methoxyl
58.64
35.33
31.15
0.68
0.70
1.17
2.6. Effect of compounds on OA-induced cytotoxicity
e8
Methoxyl
21.93
0.76
One of the pathological features of the brains of AD patients, neu-
fibrillary tangles (NFTs) are mainly caused by tau hyperpho-
sphorylation and aggregation [52,53]. Therefore, we investigated the
protective effect of these molecules on okadaic acid (OA, a tau protein
hyperphosphorylation inducer) induced cells toxicity. PC12 and SH-
SY5Y cells were incubated with OA and the selected compounds for
48 h, and the cell viability was tested using MTT [54]. As shown in
Fig. 6, OA significantly reduced the cell viability, while e3 and e12
treatment moderately increased the viability of the cells. Therefor e3
and e12 could reduce PC12 and SH-SY5Y cytotoxicity induced by OA.
e9
Methoxyl
Hydrogen
Hydrogen
Hydrogen
Hydrogen
54.30
23.72
21.99
72.51
31.87
0.68
2.45
0.72
0.84
2.33
e10
e11
e12
e13
2.7. Effect of compounds on Aβ1–42-induced cells toxicity
Senile plaque, another histopathological hallmark of AD, are mainly
due to the accumulation of amyloid β peptides [55]. We have explored
these compounds could remarkably inhibit Aβ1–42 aggregation, herein,
the neuroprotective role of compounds on Aβ1–42-dependent cell death
was investigated [56]. The results indicated that Aβ1–42 (30 μM) ob-
viously induced a sharp decline in cell viability, and compounds e3 and
e12 could protect nerve cell from Aβ1–42-mediated neurotoxicity
(Fig. 7).
e14
e15
e16
Hydrogen
Hydrogen
Hydrogen
58.41
27.03
21.45
3.21
1.80
0.29
2.8. Effect of compounds on LPS-induced ROS production
e17
Hydrogen
31.99
0.96
1.47
Microglia-mediated neuroinflammation is an important contributor
to the pathogenesis of neurodegenerative diseases [57]. Activated mi-
croglia produces toxic inflammatory mediators, such as NO, cytokines
and ROS, ultimately resulting in the loss and death of neurons [58]. To
investigate the anti-neuroinflammatory effects of hit compounds, BV-2
cells were stimulated with Lipopolysaccharide (LPS) (2.5 mg/L), and
the secretion of ROS was measured with the fluorescent dye H2DCFDA
[59,60]. The significant increasing in fluorescence intensity of BV-2
cells could be clearly observed after 24 h treatment with LPS, while that
of LPS-induced cells remarkably reduced following pretreatment with
e3 or e12 (Fig. S1). As shown in Fig. 8, the results showed that e3 and
e12 suppressed ROS released in LPS-stimulated BV-2 cells. Hence,
molecules bearing the radical scavenging phenol and β-carboline scaf-
fold could reduce ROS-induced cell death, and result in alleviating
neuroinflammatory process in the brain during AD.
e18
1a
Hydrogen
–
58.19
n.a.b
1b
–
–
n.a.b
Caffeic acid
–
31.48
0.83
4

Q. Liao, et al.
BioorganicChemistry99(2020)103844
Fig. 3. (A) Binding modes of e12 with amyloid forming peptide KLVFFA (left) and an interaction map (right) displaying the binding and interactions of compound
e12. (B) The BChE active site cavity (left) and interaction map (right) displaying the binding and interactions of compound e12. Color coding: green: hydrogen bond;
purple: π–π stacking; red: π–Alkyl; orange: π–Cation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
2.9. Predicted the ADMET and physicochemical properties of compounds
candidates
the liver and highly expressed in the central nervous system, including
substantia nigra. As reported in Table 3, e3 and e12 displayed no in-
hibition of CYP2D6 [61,62] indicating an unlikely adverse effect on
liver. Moreover, compounds exhibited moderate blood-brain barrier
permeability and high oral absorption. The selected compounds pre-
sented a favorable drug-like profile with no violation of either Lipinski’s
The ADMET and physicochemical properties of active compounds
e3 and e12 were calculated by using QikProp and Discovery Studio
2019 prior to performing animal assays. CYP2D6 is mainly expressed in
Table 2
ChE Inhibitory Activities of compounds.
Cpd.
R1
IC50
SEM (μM)a
AChEb
75.32
BuChEc
e3
Methoxyl
Hydrogen
–
9.38
3.63
6.47
4.17
0.85
e12
1a
21.29
1.32
> 100
74.81
9.45
1b
–
> 100
90.22
8.42
Caffeic acid
Donepezil
–
–
–
–
n.a.d
0.05
n.a.d
3.15
0.01
0.65
a
b
c
IC50, inhibitor concentration (means
AChE from electric eel was used.
BuChE from equine serum was used.
n.a. means no activity.
SEM of three independent experiments) for 50% inactivation of AChE and BuChE.
d
5

Q. Liao, et al.
BioorganicChemistry99(2020)103844
150
and memory functions compared to the model group (Fig. 9B). The
same conclusion was derived from the confusion degree of the trajec-
tories of the mice in each group (Fig. 9C). In additional, neither the
intragastric administration nor the hippocampal injection of the peptide
affected the body weight of mice during the two-week-therapy, showing
a good in vivo safety of the two candidates (Fig. S2).
control
PC12
SH-SY5Y
BV-2
100
50
0
HT22
L02
In summary, the representative e3 and e12 showed auspicious ef-
fects on diverse investigated behavioral parameters, and the results may
be attributed to the anti-aggregation, antioxidant or other neuropro-
tective activities of the compounds.
l
o
e3
r
t
e12
n
2.11. Assessment of acute toxicity
co
compounds(25μM)
The acute toxicity trials of e3 and e12 were carried out in adult ICR
mice (male mice, 8–10 weeks old, weight 18–20 g). All the mice re-
mained alive and abnormal behavior after intragastric administration of
e3 and e12 at doses of 200 mg/kg and 600 mg/kg (n = 5 per group).
Furthermore, from the first 4 h through 14 days after administration,
there was no obvious behavior abnormality compared to the control
group (Fig. S3). All the results support these natural product analogues
as multifunctional molecule with high efficiency, low toxicity, and good
oral bioavailability.
Fig. 4. Effects of compounds on the viability of PC12, SH-SY5Y, BV-2, HT22
and L02 cells. The cells were incubated with the indicated concentrations
(25 μM) of the test compounds for 24 h before evaluated by the MTT assay Cell
viabilities are presented as mean
SEM from three independent experiments.
rule of five or Jorgensen’s rule of three.
2.10. In vivo activity
3. Conclusion
To evaluate the in vivo activities of synthesized compounds, we used
Morris water maze and Y maze to assess learning and memory function
in an acute AD mice model, where Aβ oligomer was directly injected
into the brains of normal mice to induce acute neurotoxicity [63,64].
After orally administrating compounds e3 and e12 for two weeks, we
performed Y maze and Morris water maze to measure cognitive im-
provement of AD mice. As described in Fig. 9A, none of the blank
control groups (oral for Saline and hippocampal injection for Saline)
significantly affected the mouse cognitive function. Hippocampal in-
jection of the oligomerized Aβ1–42 peptide led to spontaneous alterna-
tion deficits compared to the sham-operation group. Donepezil showed
prevention against the Aβ1–42-induced alternation deficit at 15 mg/kg,
and e3 and e12 could also remarkably increase alternation percentage
at 15 mg/kg oral doses.
Based on the various biological activities of carboline derivatives, a
series of carboline-cinnamic acid heterogenous dimer were designed
and synthesized as multifunctional agents for the treatment of AD in
this study. Among the synthesized compounds, e3 and e12 exhibited
significant inhibition of Aβ aggregation, moderate BuChE inhibition,
excellent neuroprotective effects and low neurotoxicity. Furthermore,
orally administrating e3 and e12 to AD model mice, could restore
learning and memory function to a comparative level to that of the
control without acute toxicity in mice at doses up to 600 mg/kg. The
outcomes above verified e3 and e12 promising multifunctional agents
in the drug discovery process against AD.
Subsequently, Morris water maze test was conducted to evaluate the
learning and memory abilities. The sham-operation group remained no
difference from the control group, and it was clear that administration
of oligomerized Aβ1–42 led to a prominent delay of the latency to target,
compared to the sham-operation group. Donepezil exhibited much
improved cognitive function in the ICR mice, as the latency to target
obviously reduced compared to model. Besides, the mice treated with
e3 or e12 both demonstrated a favorable amelioration of the cognitive
4. Experimental section
4.1. Chemistry
1H and 13C NMR spectra were recorded on a Brüker 300, 400 and
500 MHz spectrometer at 298 T and referenced to TMS. MS spectra data
were obtained on an Agilent-6210 LC-MS spectrometer. Thin-layer
chromatography was carried out on silica gel GF/UV 254 supported by
B:SH-SY5Y
2.5μM
7.5μM
15μM
A:PC12
2.5μM
150
150
100
50
7.5μM
****
15μM
****
***
****
****
***
100
50
0
***
***
###
###
0
2O2
2O2
2
e3
e3
e12
e1
H
H
H2O2150μM
control
control
H2O2500μM
Fig. 5. Compounds hindered H2O2-induced cell damage in PC12 and SH-SY5Y cells. (A) PC12 cells were pre-treated with compounds (2.5–15 μM) for 2 h and then
incubated with H2O2 (500 μM) for 24 h before MTT assay was performed. (B) SH-SY5Y cells were pre-treated with compounds (2.5–15 μM) for 2 h and then incubated
with H2O2 (150 μM) for 24 h before MTT assay was performed. All data are expressed as mean
SEM of three independent experiments. ####p < 0.0001 vs.
control; ***p < 0.001, ****p < 0.0001vs·H2O2.
6

Q. Liao, et al.
BioorganicChemistry99(2020)103844
control
B:SH-SY5Y
A:PC12
control
OA
OA
2.5μM
5μM
1μM
100
90
50
2.5μM
5μM
100
90
50
15μM
***
40
30
20
10
0
40
30
20
10
0
*
*
*
*
*
**
###
###
e3
e3
OA
rol
e12
OA
e12
control
cont
OA 100nM
OA 100nM
Fig. 6. Compounds alleviates OA induced cell toxicity in PC12 and SH-SY5Y cells. (A) PC12 cells were pre-treated with compounds (1–5 μM) for 2 h and then
incubated with OA (100 nM) for 24 h before MTT assay was performed. (B) SH-SY5Y cells were pre-treated with compounds (2.5–15 μM) for 2 h and then incubated
with OA (100 nM) for 24 h before MTT assay was performed. All data are expressed as mean
SEM of three independent experiments. ####p < 0.0001 vs. control;
*p < 0.05, **p < 0.01, ***p < 0.001vs. OA.
glass plate, and the chromatograms were performed on silica gel
(200–300 mesh) visualized under UV light at 254 and 365 nm.
BV-2
40
control
model
2.5μM
5μM
4.1.1. General procedure I
####
30
20
10
0
Compounds 2a, 2b were synthesized according to Pictet-Spengler
reaction [65]. Tryptamine (1 equiv) was dissolved in CH2Cl2 and cooled
to 0 °C in an ice bath. To this solution, trifluoroacetic acid (1.5 equiv)
was added dropwise and the mixture was stirred at 0 °C for about 0.5 h.
Then acetaldehyde (4 equiv) was added to this solution and the re-
sulting mixture was stirred at room temperature for 24 h. The complete
reaction was monitored by TLC layer and the reaction mixture was then
basified with dilute NH4OH solution and extracted with CH2Cl2. The
solvents were removed under reduced pressure to obtain the crude
product, which was purified by column chromatography.
***
***
***
***
l
l
e
o
e3
r
t
e12
od
m
con
Compounds
4.1.2. General procedure Ⅱ
Fig. 8. Compounds lessened LPS-induced ROS production in BV-2 cells. BV-2
cells were pre-treated with compounds (2.5–5 μM) for 0.5 h and then incubated
with LPS (2.5 mg/L) for 24 h. Intracellular ROS production levels were then
measured with the fluorescent dye H2DCFDA. Bars correspond to the fluores-
Substituted cinnamic acid (1 equiv) was dissolved in SOCl2 (10
equiv) and refluxed for 4 h. The solvents were removed under reduced
pressure to obtain the substituted cinnamoyl chlorides.
cence intensity and are the means
SEM of three independent experiments.
####p < 0.0001 vs. control; ***p < 0.001vs. model.
4.1.3. General procedure III
2a or 2b was added to a mixture of substituted cinnamoyl chloride
A: SH-SY5Y
B: PC12
150
100
control
control
model
2.5μM
7.5μM
15μM
90
60
model
**
** **
***
***
2.5μM
7.5μM
15μM
50
40
30
20
10
0
100
*
**
**
**
###
###
50
0
e3
e3
e12
e12
model
model
control
control
Aβ1-4230μM
Aβ1-4230μM
Fig. 7. Compounds protected against Aβ1–42 -induced cell toxicity in PC12 and SH-SY5Y cells. SH-SY5Y (A) and PC12 (B) cells were pre-treated with compounds
(2.5–15 μM) for 2 h and then incubated with Aβ1–42 (30 μM, Oligomer) for 48 h before MTT assay was performed. All data are expressed as mean
SEM of three
independent experiments. ###p < 0.001 vs. control; **p < 0.01, ***p < 0.001vs. model.
7

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BioorganicChemistry99(2020)103844
Table 3
Calculated Physicochemical Properties for Selected Compounds Using QikProp and Discovery Studio 3.0.
Calculated properties
Content
e3
e12
Guideline
CYP2D6 inhibition
ADMET_BBB_Level
Percent Human Oral Absorption
QPlogPo/w
–
False
False
–
BBB permeability
2
2
Moderate
Human oral absorption
94.126
3.426
−5.236
−1.256
5
93.736
3.404
−5.25
−1.219
4
> 80%, high; < 25%, poor
Octanol/water partition coefficient
Aqueous solubility
−2–6.5
QPlogS
−6.5–0.5
QPlogBB
Brain/blood partition coefficient
Number of possible metabolic reactions
Total solvent accessible surface area
IC50 value for blockage of HERG K+ channels
Caco-2 cell permeability
−3.0–1.2
metab
1–8
SASA
251.648
−5.874
429.22
171.398
−6.185
414.993
300–1000
QPlogHERG
QPPCaco
< -5
> 500,great; < 25%, poor
A
B
80
60
40
20
0
ns
100
###
ns
****
***
80
60
40
20
0
**
**
**
##
****
C
a
b
c
d
e
f
Fig. 9. Effects of oral administration of compounds e3 and e12 (15 mg/kg) on Aβ1–42-induced induce acute neurotoxicity in ICR mice determined by the Y maze and
Morris water maze test. Donepezil was used as a positive control. (A) Effect of the compounds on Aβ1–42-induced spontaneous alternation deficits of mice in Y maze.
(B) The escape latency values to target in Morris water maze. (C) The trajectories of mice in control (a), sham-operation (b), model (c), donepezil (d), e3 (e), and e12
(f) group in the Morris water maze test. Data are presented as the mean
SEM (n = 6; ##p
<
0.01, ###p
<
0.001 vs. sham-operation; **p
< 0.01,
***p < 0.001,****p < 0.0001vs. model.)
(1 equiv) and triethylamine (4 equiv) in dry tetrahydrofuran and stirred
at room temperature for 12 h. When the substrates disappeared (as
detected by TLC), saturated sodium bicarbonate was added to neu-
tralize acid. The reaction mixture was extracted with CH2Cl2 for three
times. The organic layer was dried over anhydrous sodium sulfate,
evaporated to give the crude product, which was purified by silica
column chromatography.
4.1.3.1. 6-Methoxy-1-methyl-1,2,3,4-tetrahydro-β-carboline
(2a). Compound 2a was prepared according to general procedure I.
8

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BioorganicChemistry99(2020)103844
Yellow solid, yield: 80%. 1H NMR (300 MHz, DMSO‑d6) δ 11.19 (s, 1H),
7.47 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.17–7.08 (m, 1H),
7.07–6.97 (m, 1H), 4.06 (q, J = 6.5 Hz, 1H), 3.75 (s, 3H), 3.61 (dd,
J = 11.7, 5.6 Hz, 2H), 2.93 (d, J = 5.3 Hz, 2H), 1.6 (d, J = 7.3 Hz,3H).
19.40. MS(ESI) m/z Calcd for C25H28N2O5[M + H]+: 437.51. Found:
437.22.
4.1.3.7. 3-(3,4-Dihydroxyphenyl)-1-(6-methoxy-1-methyl-1,3,4,9-
tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propan-1-one (e5). Compound e5
was prepared according to general procedure III. Yellow solid, yield:
4.1.3.2. 1-Methyl-1,2,3,4-tetrahydro-β-carboline (2b). Compound 2b
was prepared according to general procedure I. Yellow solid, yield:
80%. 1H NMR (300 MHz, DMSO‑d6) δ 11.19 (s, 1H), 9.58 (s, 1H), 9.16
(s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.18–6.97
(m, 2H), 4.6 (q, J = 6.5 Hz, 1H), 3.61 (dd, J = 11.7, 5.6 Hz, 2H), 2.93
(d, J = 5.2 Hz, 2H), 1.65 (d, J = 7.4 Hz, 3H).
88%. 1H NMR (400 MHz, DMSO‑d6)
δ 10.60 (s, 1H), 8.57 (d,
J = 26.7 Hz, 2H), 7.09 (dd, J = 8.7, 6.4 Hz, 1H), 6.79 (dd, J = 4.5,
2.4 Hz, 1H), 6.63–6.47 (m, 3H), 6.39 (ddd, J = 7.2, 5.0, 2.1 Hz, 1H),
5.46 (q, J = 6.6 Hz, 1H), 3.64 (s, 3H), 3.26–3.09 (m, 2H), 2.65–2.49
(m, 6H), 1.27 (d, J = 6.7 Hz, 3H). 13C NMR (75 MHz, DMSO‑d6) δ
170.80, 153.58, 145.44, 143.78, 137.00, 132.73, 131.50, 127.10,
119.37, 116.31, 115.86, 112.04, 110.92, 106.53, 100.45, 55.79,
48.49, 45.10, 35.52, 30.80, 21.21, 19.31. MS(ESI) m/z Calcd for
4.1.3.3. (E)-3-(4-hydroxyphenyl)-1-(6-methoxy-1-methyl-1,3,4,9-
tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e1). Compound
e1 was prepared according to general procedure III. White solid,
yield: 87%. 1H NMR (300 MHz, DMSO‑d6) δ 10.72 (d, J = 18.0 Hz,
1H), 9.88 (s, 1H), 7.82 (t, J = 10.4 Hz, 2H), 7.62–7.51 (m, 1H), 7.39
(dd, J = 15.9, 4.5 Hz, 1H), 7.33–7.11 (m, 4H), 6.93 (q, J = 2.6 Hz, 1H),
6.70 (dq, J = 8.7, 2.2 Hz, 1H), 5.67 (q, J = 6.4 Hz, 1H), 5.67 (d,
J = 7.4 Hz, 1H), 3.74 (d, J = 6.9 Hz, 3H), 3.51–3.15 (m, 2H),
2.72–2.65 (m, 2H), 1.45 (d, J = 6.8 Hz, 3H). 13C NMR (126 MHz,
DMSO‑d6) δ 165.59, 159.43, 153.59, 142.59, 136.99, 131.40, 130.33,
129.69, 127.12, 126.71, 122.68, 116.07, 115.34, 112.10, 110.97,
106.61, 100.49, 55.81, 48.75, 45.71, 22.77, 19.39. MS(ESI) m/z
Calcd for C22H22N2O3 [M + Na]+: 385.43. Found: 385.33.
C
22H24N2O4 [M + Na]+: 403.44. Found: 403.25.
4.1.3.8. (E)-3-(3,4-dimethoxyphenyl)-1-(6-methoxy-1-methyl-1,3,4,9-
tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e6). Compound
e6 was prepared according to general procedure III. Yellow solid,
yield: 84%. 1H NMR (300 MHz, DMSO‑d6) δ 10.76 (s, 1H), 7.93–7.51
(m, 2H), 7.24 (d, J = 24.0 Hz, 2H), 7.01–6.83 (m, 2H), 6.80–6.62 (m,
1H), 5.69 (q, J = 6.9 Hz, 1H), 4.48 (d, J = 13.8 Hz, 1H), 3.84 (d,
J = 6.7 Hz, 6H), 3.76 (d, J = 5.3 Hz, 3H), 3.67–3.47 (m, 1H),
3.03–2.66 (m, 2H), 1.47 (d, J = 6.7 Hz, 3H). 13C NMR (126 MHz,
DMSO‑d6) δ 165.46, 155.17, 153.63, 152.68, 142.27, 137.01, 136.62,
131.43, 127.15, 123.22, 122.04, 117.64, 112.07, 110.96, 108.80,
106.56, 100.53, 56.43, 55.82, 55.34, 48.81, 45.77, 22.80, 19.40. MS
(ESI) m/z Calcd for C24H26N2O4 [M + Na]+: 429.48. Found: 429.24.
4.1.3.4. (E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(6-methoxy-1-methyl-
1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one
(e2). Compound e2 was prepared according to general procedure III.
Brown solid, yield: 82%. 1H NMR (400 MHz, DMSO‑d6) δ 10.74 (s, 1H),
7.56 (d, J = 2.1 Hz, 1H), 7.53–7.44 (m, 2H), 7.30–7.12 (m, 4H),
6.99–6.89 (m, 2H), 6.70 (dt, J = 8.8, 2.0 Hz, 1H), 6.09 (s, 2H), 5.66 (q,
J = 6.6 Hz, 1H), 3.76 (d, J = 1.6 Hz, 3H), 3.42 (dt, J = 14.6, 7.2 Hz,
1H), 3.18 (dd, J = 5.3, 1.8 Hz, 1H), 2.73 (d, J = 6.7 Hz, 2H), 1.45 (d,
J = 6.6 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 165.40, 153.62,
149.03, 148.42, 142.28, 133.37, 133.24, 131.42, 127.13, 124.74,
117.05, 112.11, 110.98, 108.83, 107.06, 106.63, 101.88, 100.50,
55.82, 48.78, 45.77, 22.77, 19.38. MS(ESI) m/z Calcd for C23H22N2O4
[M + Na]+: 413.44. Found: 413.24.
4.1.3.9. (E)-3-(2,6-dimethoxyphenyl)-1-(6-methoxy-1-methyl-1,3,4,9-
tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e7). Compound
e7 was prepared according to general procedure III. Yellow solid,
yield: 88%. 1H NMR (300 MHz, Chloroform-d) δ 8.80 (s, 1H), 8.03 (d,
J = 15.6 Hz, 1H), 7.27 (d, J = 6.9 Hz, 1H), 7.17–7.03 (m, 2H),
6.99–6.86 (m, 3H), 6.83 (dd, J = 8.7, 2.5 Hz, 1H), 5.95 (q, J = 6.7 Hz,
1H), 4.42–4.27 (m, 1H), 3.88 (d, J = 2.9 Hz, 6H), 3.84 (s, 3H),
3.71–3.47 (m, 1H), 2.87 (td, J = 17.9, 16.9, 12.0 Hz, 2H), 1.58 (d,
J = 6.7 Hz, 3H). 13C NMR (75 MHz, Chloroform-d) δ 166.23, 153.99,
153.48, 152.66, 138.17, 136.33, 131.34, 127.02, 125.03, 118.96,
115.76, 112.38, 111.31, 106.91, 104.32, 104.04, 100.38, 56.15,
56.01, 55.89, 53.46, 46.19, 22.56, 19.27. MS(ESI) m/z Calcd for
4.1.3.5. (E)-3-(3,4-dihydroxyphenyl)-1-(6-methoxy-1-methyl-1,3,4,9-
tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e3). Compound
e3 was prepared according to general procedure III. White solid,
yield: 86%. 1H NMR (500 MHz, DMSO‑d6) δ 10.92 (s, 1H), 9.47 (s,
1H), 9.03 (s, 1H), 7.44–7.36 (m, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.14 (d,
J = 12.0 Hz, 1H), 7.09–7.01 (m, 3H), 6.98 (q, J = 7.3, 6.6 Hz, 1H),
6.77 (d, J = 8.1 Hz, 1H), 5.68 (q, J = 6.7 Hz, 1H), 4.47 (d,
J = 14.0 Hz, 1H), 3.74 (d, J = 6.9 Hz, 3H), 3.48–3.41 (m, 1H), 2.76
(d, J = 7.6 Hz, 2H), 1.46 (d, J = 6.7 Hz, 3H). 13C NMR (126 MHz,
DMSO‑d6) δ 165.55, 153.59, 147.88, 145.90, 142.97, 137.02, 131.39,
127.20, 127.12, 121.21, 120.74, 116.07, 115.23, 112.10, 111.17,
106.60, 100.47, 55.81, 48.75, 46.17, 22.77, 19.40. MS(ESI) m/z
Calcd for C22H22N2O4 [M + Na]+: 401.43. Found: 401.29.
C
24H26N2O4[M − H]− 405.48. Found: 405.21.
4.1.3.10. (E)-1-(6-methoxy-1-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-
b]indol-2-yl)-3-(2,3,4-trimethoxyphenyl)prop-2-en-1-one
(e8). Compound a8 was prepared according to general procedure III.
Yellow solid, yield: 88%. 1H NMR (300 MHz, Chloroform-d) δ 8.60 (s,
1H), 7.91 (d, J
= 15.5 Hz, 1H), 7.40–7.16 (m, 2H), 7.08 (d,
J = 15.5 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.83 (dd, J = 8.7,
2.5 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 5.93 (q, J = 6.9 Hz, 1H), 4.33 (d,
J = 13.9 Hz, 1H), 3.94 (d, J = 9.4 Hz, 9H), 3.87 (s, 3H), 3.66–3.47 (m,
1H), 3.03–2.66 (m, 2H), 1.58 (d, J = 6.7 Hz, 3H). 13C NMR (75 MHz,
Chloroform-d) δ 165.94, 154.96, 154.04, 153.10, 142.50, 138.20,
136.05, 131.96, 131.29, 127.05, 122.37, 117.18, 111.76, 111.34,
107.56, 107.07, 100.42, 61.24, 60.96, 56.08, 56.01, 53.44, 46.13,
22.58, 19.27. MS (ESI) m/z Calcd for C25H28N2O5 [M−H]−: 435.51.
Found: 435.25.
4.1.3.6. (E)-1-(6-methoxy-1-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]
indol-2-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (e4). Compound
e4 was prepared according to general procedure III. Yellow solid,
yield: 82%. 1H NMR (400 MHz, DMSO‑d6) δ 10.78 (s, 1H), 7.55 (dd,
J = 15.2, 6.5 Hz, 1H), 7.35 (d, J = 15.3 Hz, 1H), 7.23 (d, J = 8.7 Hz,
1H), 7.10 (d, J = 10.5 Hz, 2H), 6.93 (d, J = 2.5 Hz, 1H), 6.72 (dd,
J = 8.7, 2.5 Hz, 1H), 5.77–5.70 (m, 1H), 4.49 (d, J = 15.2 Hz, 1H),3.86
(d, J = 4.9 Hz, 6H), 3.76 (s, 3H), 3.72 (s, 3H), 3.67–3.51 (m, 1H), 2.78
(d, J = 8.8 Hz, 2H), 1.49 (d, J = 6.8 Hz,3H). 13C NMR (100 MHz,
DMSO‑d6) δ 165.30, 153.64, 153.54, 153.54,142.73, 139.29, 136.95,
131.47, 131.31, 127.15, 118.33, 112.11, 110.98, 106.54, 106.15,
106.09, 100.53, 60.55, 56.52, 55.80, 55.33, 48.82, 45.76, 22.90,
4.1.3.11. (E)-1-(6-methoxy-1-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-
b]indol-2-yl)-3-(naphthalen-2-yl)prop-2-en-1-one (e9). Compound e9
was prepared according to general procedure III. Yellow solid, yield:
88%. 1H NMR (300 MHz, Chloroform-d) δ 9.01–8.75 (m, 1H), 8.63 (d,
J = 15.2 Hz, 1H), 8.31–8.20 (m, 1H), 7.97–7.84 (m, 2H), 7.81 (d,
J = 7.1 Hz, 1H), 7.56 (dtd, J = 10.6, 7.3, 3.1 Hz, 3H), 7.33 – 7.26 (m,
1H), 7.11 (d, J = 15.2 Hz, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.85 (dd,
J = 8.7, 2.5 Hz, 1H), 6.00 (q, J = 6.7 Hz, 1H), 4.37 (dd, J = 14.0,
9

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BioorganicChemistry99(2020)103844
4.6 Hz, 1H), 3.89 (s, 3H), 3.72–3.51 (m, 1H), 3.06–2.73 (m, 2H), 1.63
(d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 165.11, 153.67,
138.46, 136.90, 133.78, 132.56, 131.47, 131.36, 130.16, 129.13,
127.38, 127.19, 126.62, 126.14, 125.52, 123.58, 122.12, 112.14,
111.04, 106.63, 100.54, 55.38, 48.95, 45.95, 22.82, 19.45. MS(ESI)
m/z Calcd for C26H24N2O2 [M − H]–: 395.49. Found: 395.25.
NMR (126 MHz, DMSO‑d6) δ 170.76, 145.43, 143.77, 136.35, 135.76,
132.60, 126.75, 121.29, 119.41, 118.96, 118.14, 116.31, 115.88,
111.46, 106.66, 48.47, 45.08, 35.51, 30.81, 22.23, 19.27. MS(ESI) m/
z Calcd for C21H22N2O3 [M + Na]+: 373.42. Found: 373.20.
4.1.3.17. (E)-3-(3,4-dimethoxyphenyl)-1-(1-methyl-1,3,4,9-tetrahydro-
2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e15). Compound e15 was
prepared according to general procedure III. Yellow solid, yield: 78%.
1H NMR (300 MHz, Chloroform-d) δ 9.07 (s, 1H), 7.77 (d, J = 15.3 Hz,
1H), 7.50 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.16 (dtt,
J = 13.8, 6.5, 2.9 Hz, 4H), 6.90 (q, J = 15.5, 11.5 Hz, 2H), 5.98 (q,
J = 6.7 Hz, 1H), 4.42–4.29 (m, 1H), 4.05–3.85 (m, 6H), 3.67–3.52 (m,
1H), 2.90 (q, J = 13.1, 12.5 Hz, 2H), 1.58 (d, J = 6.7 Hz, 3H). 13C NMR
(75 MHz, DMSO‑d6) δ 165.40, 150.72, 149.38, 142.66, 136.29, 131.61,
128.48, 126.79, 122.85, 121.30, 118.96, 118.21, 116.55, 111.96,
111.49, 110.94, 106.68, 56.19, 55.98, 48.73, 45.63, 22.82, 19.38. MS
(ESI) m/z Calcd for C23H24N2O3 [M + Na]+: 399.46. Found: 399.28.
4.1.3.12. (E)-3-(4-hydroxyphenyl)-1-(1-methyl-1,3,4,9-tetrahydro-2H-
pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e10). Compound e10 was
prepared according to general procedure III. White solid, yield: 85%.
1H NMR (300 MHz, DMSO‑d6) δ 10.97 (s, 1H), 10.91 (s, 1H), 7.91 (d,
J = 8.3 Hz, 2H), 7.58 (d, J = 15.4 Hz, 1H), 7.50–7.35 (m, 5H),
7.12–6.94 (m, 3H), 5.65 (q, J = 6.4 Hz, 1H), 3.41–3.25 (m, 2H),2.78 (t,
J = 6.7 Hz, 2H), 1.48 (d, J = 6.8 Hz, 3H). 13C NMR (126 MHz,
DMSO‑d6) δ 164.99, 149.83, 140.61, 136.38, 136.07, 130.70, 126.87,
126.76, 121.74, 121.35, 120.29, 118.99, 118.19, 115.99, 111.51,
106.65, 47.68, 45.77, 22.73, 19.33. MS(ESI) m/z Calcd for
C
21H20N2O2 [M + H]+: 333.40. Found: 333.25.
4.1.3.18. (E)-3-(2,6-dimethoxyphenyl)-1-(1-methyl-1,3,4,9-tetrahydro-
2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e16). Compound e16 was
prepared according to general procedure III. Yellow solid, yield: 75%.
1H NMR (300 MHz, Chloroform-d) δ 9.33 (s, 1H), 8.11 (d, J = 15.5 Hz,
1H), 7.51 (d, J = 7.5 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.20–7.11 (m,
4H), 6.92 (h, J = 8.4, 7.9 Hz, 2H), 6.03 (q, J = 6.7 Hz, 1H), 4.50–4.16
(m, 1H), 3.88 (d, J = 10.9 Hz, 6H), 3.60 (td, J = 13.8, 5.0 Hz, 1H),
3.03–2.79 (m, 2H), 1.62 (d, J = 6.7 Hz, 3H). 13C NMR (75 MHz,
DMSO‑d6) δ 165.38, 153.69, 152.28, 136.63, 136.21, 134.00, 131.77,
126.80, 124.68, 121.30, 119.30, 118.96, 118.18, 116.74, 113.18,
111.49, 106.67, 56.49, 56.06, 48.78, 45.72, 22.79, 19.34. MS(ESI) m/
z Calcd for C23H24N2O3 [M + H]+: 377.46. Found: 377.35.
4.1.3.13. (E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(1-methyl-1,3,4,9-tetrahydro-
2H-pyrido[3,4-b] indol-2-yl)prop-2-en-1-one (e11). Compound e11 was
prepared according to general procedure III. Brown solid, yield: 80%. 1H
NMR (400 MHz, DMSO‑d6) δ 10.92 (s, 1H), 7.56 (d, J = 1.7 Hz, 1H),
7.51 (d, J = 4.1 Hz, 1H), 7.47 (s, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.33 (t,
J = 7.7 Hz, 1H), 7.10–7.02 (m, 1H), 7.01–6.90 (m, 2H), 6.09 (s, 2H),
5.69 (q, J = 6.6 Hz, 1H), 4.54 (d, J = 13.8 Hz, 1H), 3.44 (dt, J = 14.5,
8.0 Hz, 1H), 2.76 (d, J = 6.9 Hz, 2H), 1.47 (d, J = 6.7 Hz, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ 165.15, 149.03, 148.42, 142.27, 136.22, 134.58,
130.11, 126.79, 124.76, 121.34, 119.00, 118.22, 117.06, 111.50,
108.84, 107.06, 106.73, 101.88, 48.73, 45.70, 22.73, 19.34. MS(ESI)
m/z Calcd for C22H20N2O3 [M + Na]+: 383.41. Found: 383.32.
4.1.3.19. (E)-1-(1-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-
yl)-3-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (e17). Compound e17
was prepared according to general procedure III. Yellow solid, yield:
84%. 1H NMR (300 MHz, Chloroform-d) δ 9.42 (s, 1H), 8.02 (d,
J = 15.5 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.38 (dd, J = 17.0, 8.2 Hz,
2H), 7.23–7.07 (m, 4H), 6.75 (d, J = 8.8 Hz, 1H), 6.04 (q, J = 6.7 Hz,
1H), 4.37 (dd, J = 13.0, 4.2 Hz, 1H), 3.98 (s, 3H), 3.95 (d, J = 5.3 Hz,
6H), 3.62 (ddd, J = 18.6, 10.8, 5.1 Hz, 1H), 2.93 (dd, J = 13.8,
12.5 Hz, 2H), 1.62 (d, J = 6.7 Hz, 3H). 13C NMR (126 MHz, DMSO‑d6)
δ 165.45, 155.17, 152.69, 142.27, 136.59, 136.36, 136.29, 126.80,
123.22, 122.03, 121.30, 118.96, 118.18, 117.65, 111.49, 108.83,
106.68, 61.78, 60.91, 56.47, 48.74, 45.69, 22.73, 19.36. MS(ESI) m/z
Calcd for C24H26N2O4 [M + Na]+: 429.48. Found: 429.29.
4.1.3.14. (E)-3-(3,4-dihydroxyphenyl)-1-(1-methyl-1,3,4,9-tetrahydro-
2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (e12). Compound e12 was
prepared according to general procedure III. White solid, yield: 76%. 1H
NMR (500 MHz, DMSO‑d6) δ 10.92 (s, 1H), 9.47 (s, 1H), 9.03 (s, 1H),
7.44–7.36 (m, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 12.0 Hz, 1H),
7.09–7.01 (m, 3H), 6.98 (q, J = 7.3, 6.6 Hz, 1H), 6.77 (d, J = 8.1 Hz,
1H), 5.68 (q, J = 6.7 Hz, 1H), 4.47 (d, J = 14.0 Hz, 1H), 3.48–3.41 (m,
1H), 2.76 (d, J = 7.6 Hz, 2H), 1.46 (d, J = 6.7 Hz, 3H). 13C NMR
(126 MHz, DMSO‑d6) δ 165.53, 147.89, 145.92, 142.93, 136.36,
132.99, 127.24, 126.81, 121.29, 121.18, 118.95, 118.18, 116.08,
115.39, 115.32, 111.48, 106.72, 48.69, 45.62, 22.72, 19.37. MS(ESI)
m/z Calcd for C21H20N2O3 [M + H]+: 348.4. Found: 349.21.
4.1.3.15. (E)-1-(1-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-
yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (e13). Compound e13
was prepared according to general procedure III. Yellow solid, yield:
85%. 1H NMR (400 MHz, DMSO‑d6) δ 10.74 (s, 1H), 7.51 (dd, J = 15.2,
1.9 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.16 (d,
J = 15.1 Hz, 1H), 7.08–6.98 (m, 1H), 6.99–6.89 (m, 3H), 5.76 (d,
J = 7.0 Hz, 1H), 4.49 (d, J = 15.2 Hz, 1H), 3.77–3.73 (m, 3H), 3.49 (d,
J = 13.4 Hz, 1H), 2.77 (d, J = 31.2 Hz, 2H), 1.50 (d, J = 7.7 Hz, 3H).
13C NMR (75 MHz, DMSO‑d6) δ 165.31, 153.41, 153.41, 142.57,
139.24, 136.38, 136.05, 131.16, 126.69, 121.17, 118.87, 118.05,
117.94, 111.37, 106.47, 105.72, 105.72,60.54, 56.36, 56.36, 48.81,
4.1.3.20. (E)-1-(1-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-
yl)-3-(naphthalen-2-yl)prop-2-en-1-one (e18). Compound e18 was
prepared according to general procedure III. Yellow solid, yield: 88%.
1H NMR (300 MHz, Chloroform-d) δ 8.59 (d, J = 15.2 Hz, 1H), 8.42 (s,
1H), 8.26 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.78 (t,
J = 9.5 Hz, 1H), 7.55 (dq, J = 12.3, 6.5 Hz, 4H), 7.39 (d, J = 7.8 Hz,
1H), 7.25–7.03 (m, 2H), 5.99 (d, J = 6.9 Hz, 1H), 4.54 – 4.27 (m, 1H),
3.79–3.48 (m, 1H), 3.06–2.80 (m, 2H), 1.65 (t, J = 8.0 Hz, 3H). 13C
NMR (75 MHz, DMSO‑d6) δ 165.08, 153.65, 138.45, 136.15, 133.76,
132.53, 131.35, 130.15, 129.11, 127.35, 127.18, 126.60, 126.13,
125.49, 123.56, 122.08, 121.45, 118.99, 118.21, 111.52, 106.61,
45.78, 22.83, 19.28. MS(ESI) m/z Calcd for C24H26N2O4 [M + Na]+
:
48.85, 45.95, 22.74, 19.40. MS(ESI) m/z Calcd for C25H22N2O
429.48. Found: 429.29.
[M + Na]+: 389.46. Found: 389.25.
4.1.3.16. 3-(3,4-Dihydroxyphenyl)-1-(1-methyl-1,3,4,9-tetrahydro-2H-
4.2. Biological assays
4.2.1. ThT assay [66]
pyrido[3,4-b]indol-2-yl)propan-1-one (e14). Compound e14 was
prepared according to general procedure III. Yellow solid, yield: 88%.
1H NMR (300 MHz, DMSO‑d6) δ 10.93 (s, 1H), 8.73 (s, 2H), 7.37 (ddd,
J = 24.4, 7.9, 3.8 Hz, 2H), 7.03 (dt, J = 25.9, 7.3 Hz, 2H), 6.75–6.37
(m, 3H), 5.60 (q, J = 6.6 Hz, 1H), 4.09 (d, J = 13.8 Hz, 1H), 3.34 (d,
J = 13.0 Hz, 1H), 2.82–2.62 (m, 6H), 1.41 (d, J = 6.7 Hz, 3H). 13C
Aβ1–42(Beyotime) was dissolved in HFIP to give a stock solution
(1 mg/mL), which was aliquoted into small samples and stored at
−80 °C after solvent was evaporated. Solutions of test compounds were
prepared in DMSO at 30 mM for storage. For the inhibition of self-
10

Q. Liao, et al.
BioorganicChemistry99(2020)103844
induced Aβ1–42 aggregation experiment, the pretreated Aβ1–42 and
compounds were diluted with 50 mM phosphate buffer (pH 7.4) to
50 μM before use. A mixture of the peptide (20 μL, 25 μM, final con-
centration) with or without the tested compound (20 μL, 20 μM, final
concentration) was incubated at 37 °C for 24 h and used 50 mM
phosphate buffer (pH 7.4) instead of Aβ as a blank. After incubation,
160 μL of 50 mM glycine-NaOH buffer (pH 8.0) containing thioflavin T
(5 μM) was added. Fluorescence was measured on a Varioskan Flash
Multimode Reader (excitation, 450 nm; emission, 485 nm). The percent
inhibition of aggregation was calculated by the expression (1–IFi/
IFc) × 100, where IFi and IFc were the fluorescence intensities ob-
tained for Aβ in the presence and absence of inhibitors after subtracting
the background, respectively.
seeded in a 96-well plate and treated with 2.5 μM or 5 μM of
compounds and then incubated at 37 °C with 2.5 mg/ml of LPS for
24 h to induce ROS production. Then 2′,7′-dichlorodihydro fluorescein
diacetate (DCFH-DA) (15 μM) was added for 30 min at 37 °C and media
was then removed, cells were washed with DMEM for three times. The
fluorescence distribution was observed and photographed by inverted
fluorescence microscope and the fluorescence intensity was quantified
by Image J software.
4.2.4. Molecular docking study
The crystal structures of amyloid forming peptide KLVFFA (PDB ID:
3OVJ) and hBChE (PDB ID: 4TPK) were derived from the RCSB Protein
Data Bank (PDB). Docking studies were carried out using the discovery
studio (DS, version 3.0, BIOVIA, San Diego, CA) for compound e12.
Two protein structures were preprocessed by “prepare protein” module
in DS to give the structures suitable for docking. “Prepare ligands”
module in DS was applied for the structural preparation of the test
compounds. The native ligand in the crystal structure was used to de-
fine the binding site. The binding site was defined as a site sphere (in
10 Å radius) around the original ligands in the co-crystal structures. The
docking program CDOCKER encoded in DS 2019 was applied to identify
the potential binding pattern of compound e12 to the amyloid forming
peptide KLVFFA and the hBChE. Other CDOCKER parameters were set
as default.
4.2.2. Cholinesterase inhibition assay in vitro [67]
AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel), BuChE
(E.C.3.1.1.8, Type VI-S, from equine serum), 5,5′-dithiobis-2-ni-
trobenzoic acid (Ellman's reagent, DTNB), acetylthiocholine chloride
(ATC), and butyrylthiocholine chloride (BTC) were purchased form
Sigma Aldrich (Steinheim, Germany). Enzyme solutions were prepared
to give 2.5 units ml−1 in 1.4 mL aliquots. Solutions of test compounds
were prepared in DMSO at 30 mM and diluted in methanol to different
concentrations (10−3−10−9 M) before use. The assay buffer was pre-
pared from 13.6 g of potassium dihydrogen phosphate (100 mmol),
which was dissolved in 1L of water and adjusted with KOH to
pH = 7.9–8.1. Furthermore, 0.01 M DTNB solution, 0.075 M ATC and
BTC solutions were prepared with assay buffer (pH 8.0).
4.2.4.1. Predicted physicochemical properties. The ADMET properties
and physicochemical properties of compounds presented in this study
were calculated using QikProp and Discovery Studio 2019.
In 96-well plates, 40 μL of phosphate buffer, 10 μL of the test
compounds and 10 μL of enzyme, were added in turn and incubated for
2 min, followed by the addition of 0.01 M DTNB (20 μL) solution and
substrate (20 μL). Activity was determined by measuring the absor-
bance at 405 nm at 37 °C, each concentration in triplicate. Blanks
containing all components except enzyme were carried out. The con-
centration of compound producing 50% of enzyme activity inhibition
(IC50) was calculated by nonlinear regression analysis of the response-
concentration (log) curve, using the Graph-Pad Prism program package.
The in vitro BuChE assay (BuChE or BTC as the enzyme substrate)
was conducted using a method similar to that described above.
4.2.5. In vivo activity [71]
Protocols for the behavioral experiments were established pre-
viously. Briefly, the adult male ICR mice (8–10 weeks old, weight
20–25 g) were obtained from the Qinglongshan animal breeding factory
(Nanjing, China, NO.201930647). All animal handling and experi-
mental protocols were approved by the Institutional Animal Care and
Use Committee of the China Pharmaceutical University. Amyloid β
peptide (1–42) was purchased from Beyotime. Donepezil that was
gained from Energy Chemical (Shanghai, China) was used as the posi-
tive control.
4.2.3. In vitro activity
4.2.5.1. Pretreatment of Aβ1–42 and compounds. A stock solution of
Aβ1–42 was prepared by dissolving powdered Aβ peptide in saline to a
final concentration of 2 μg/μL. The stock solution incubated at 37 °C for
7 days to induce aggregation. Compounds were weighed, dissolved in
pure DMSO at a concentration of 150 mg/mL, and diluted by 100-fold
with saline to final test concentrations.
4.2.3.1. Cell culture. Rat pheochromocytoma PC12 cells, murine BV-2
microglial cells, Human SH-SY5Y neuroblastoma cells, hippocampal
neuronal HT22 cell and human L02 hepatocytes were obtained from the
Cell Bank of the Chinese Academy of Sciences (Shanghai, China). PC12,
SH-SY5Y and BV-2 cells were maintained in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/
mL penicillin, and 100 μg/mL streptomycin at 37 °C under 5% CO2.
4.2.5.2. Hippocampal injection and administration [72]. The mice were
anesthetized with 2% Pentobarbital Sodium, fixed on the stereotaxic
apparatus (Narishige, Japan), cut off the top skin of the head, wiped
with alcohol cotton, and exposed the bregma and sagittal suture. The
right hippocampus was located in 1.5 mm behind the bregma to the
posterior and 2 mm to the right the sagittal suture. A small hole was
punctured at this position, the depth of which was 2 mm below the skull
surface. Each mouse was injected 5 μL Aβ1–42 (2 μg/μL) at a constant
rate of 2.5 μL/min. In the sham operated group, 5 μL saline was injected
instead of Aβ1–42. Three days after the operation, each group was given
drug treatment. Donepezil and the synthesized compounds were orally
administered (15 mmol/kg body weight) to mice and the control group
was given normal saline. Two weeks after administration, the cognitive
function of the mice was evaluated.
4.2.3.2. Cell treatment. For cytotoxicity experiments, PC12, BV-2, SH-
SY5Y, HT22 and L02 cells were seeded in 96-well plates at a density of
104 cells per well and treated with synthetic compounds except for
blanks. For neuroprotective cell experiment [68], PC12 and SH-SY5Y
cells were subcultured in 96-well plates at a seeding density of 104 cells
per well and treated with modeling agent (H2O2, OA, Aβ1–42) in the
absence (control) or in the presence of compounds.
4.2.3.3. Cell viability assay. Cell viability was analyzed with 3-[4,5-
dimethylthylthiazol-2,5-diphenyl-tetrazolium bromide] (MTT) assay
method [69]. After incubation, the MTT reagent (15 μL/well) was
added to each well and incubated for additional 4 h followed by
solubilization of formazan crystals in DMSO (100 μL/well). The
absorbance of each well was measured at a wavelength of 492 nm by
a 1500 microplate reader (Thermo Fisher Scientific Co.). Results are
4.2.5.3. Morris water maze test [73]. The water maze system consists of
a black pool (120 cm diameter, 60 cm height) with a depth of 40 cm
water, escape platform (10 cm diameter, at the center of pool) camera
system and an Xeye animal behavior analysis system.
expressed as the mean
SEM of three independent experiments.
4.2.3.4. Measurement of intracellular ROS [70]. Briefly, 104 cells were
11

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BioorganicChemistry99(2020)103844
4.2.5.4. Acquired training. The mice were put into the water from the
opposite quadrant of the platform, with their heads facing the pool
wall. The time for the mouse to find the platform (a successful escape)
was recorded. If a mouse failed to reach the platform within 90 s, the
test was terminated and the animal was gently navigated to the
platform by hand. It was kept on the platform for 10 s. Each animal
was trained 4 times a day with a 15–20 min interval between the two
training sessions for 6 consecutive days.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103844.
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