Synthesis and biological evaluation of quinoline/cinnamic acid hybrids as amyloid-beta aggregation inhibitors

Article abstract of DOI:10.1007/s00706-020-02609-2

Abstract: The objective of the current study is to evaluate the potency of quinoline/cinnamic acid hybrids against amyloid-beta (Aβ) aggregation. In total, six new target quinoline/cinnamic acid hybrids were synthesized and screened for their in vitro anti-Aβ₄₂ aggregation activity. Some hybrids, including (E)-N-(2-cinnamamidoethyl)-6,7-dimethoxyquinoline-2-carboxamide, (E)-6,7-dimethoxy-N-[2-[3-(4-methoxyphenyl)acrylamido]ethyl]quinoline-2-carboxamide, and (E)-6,7-dimethoxy-N-[2-[3-(2-methoxyphenyl)acrylamido]ethyl]quinoline-2-carboxamide, showed significant anti-Aβ₄₂ aggregation activity. Molecular docking method was used to predict the binding modes of these compounds with Aβ₄₂. In addition, their cytotoxicity towards neuroblastoma SH-SY5Y and human normal hepatocyte LO2 cells were tested. Neuroprotective evaluation demonstrated that these compounds could attenuate Aβ₄₂-induced neurotoxicity towards SH-SY5Y cells in a dose-dependent manner. Overall, the present study provides quinoline/cinnamic acid hybrids as a new template for developing Aβ aggregation inhibitors against Alzheimer’s disease. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-020-02609-2

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-020-02609-2
ORIGINAL PAPER
Synthesis and biological evaluation of quinoline/cinnamic acid hybrids
as amyloid‑beta aggregation inhibitors
Yong‑Xi Ge1 · Zhi‑Qiang Cheng1 · Lei Zhou1 · Hong‑Xu Xie1 · Yin‑Yin Wang1 · Kongkai Zhu2 · Yang Jiao3 ·
Guangpu Liu4 · Cheng‑Shi Jiang1
Received: 6 December 2019 / Accepted: 27 April 2020
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
The objective of the current study is to evaluate the potency of quinoline/cinnamic acid hybrids against amyloid-beta (Aβ)
aggregation. In total, six new target quinoline/cinnamic acid hybrids were synthesized and screened for their in vitro anti-Aβ42
aggregation activity. Some hybrids, including (E)-N-(2-cinnamamidoethyl)-6,7-dimethoxyquinoline-2-carboxamide, (E)-
6,7-dimethoxy-N-[2-[3-(4-methoxyphenyl)acrylamido]ethyl]quinoline-2-carboxamide, and (E)-6,7-dimethoxy-N-[2-[3-(2-
methoxyphenyl)acrylamido]ethyl]quinoline-2-carboxamide, showed signifcant anti-Aβ42 aggregation activity. Molecular
docking method was used to predict the binding modes of these compounds with Aβ42. In addition, their cytotoxicity towards
neuroblastoma SH-SY5Y and human normal hepatocyte LO2 cells were tested. Neuroprotective evaluation demonstrated
that these compounds could attenuate Aβ42-induced neurotoxicity towards SH-SY5Y cells in a dose-dependent manner.
Overall, the present study provides quinoline/cinnamic acid hybrids as a new template for developing Aβ aggregation inhibi-
tors against Alzheimer’s disease.
Graphic abstract
Cl
O
OH
N
Cl
N
OH
OMe
Br
EtO
O
O
N
O
O
N
H
Cl
N
Pharmacophore hybridization
O
O
O
N
H
N
N
R
H
O
A
β
inhibitors
aggregation
Keywords Quinoline · Cinnamic acid · Aβ aggregation inhibitor · Neuroprotection · Alzheimer’s disease
Introduction
Alzheimer’s disease (AD) is the most common neurodegen-
Yong-Xi Ge and Zhi-Qiang Cheng contributed equally to this
work.
erative disorder, which ranks the third leading death cause
after cancer and heart disease. The clinic features of AD
usually involves progressive loss of memory and defcits in
cognitive functions [1, 2]. The statistic of World Alzheimer
Report 2018 shows that about 50 million AD patients were
diagnosed worldwide in 2015 and with the aging of global
population this number was projected to be 0.15 billion in
2050 [3].
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-020-02609-2) contains
supplementary material, which is available to authorized users.
* Guangpu Liu
guang_pu@hotmail.com
* Cheng-Shi Jiang
bio_jiangcs@ujn.edu.cn
The amyloid beta-peptide (Aβ) deposit is a pathologi-
cal hallmark of AD and plays a very important role in the
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Y.-X. Ge et al.
development of AD because of its high propensity for con-
version into toxic deposition [4, 5]. Aggregation of Aβ has
been identifed as a signifcant factor responsible for cogni-
tive decline and memory defcits in AD [6]. Aβ is a protein
consisting of 39–42 amino acids, which can rapidly aggre-
gate to form neurotoxic oligomers, protofbrils, and plaques,
consequently leading neuronal cell death. Among these
aggregates, the soluble oligomers consisting of Aβ42 are the
most neurotoxic [7]. Therefore, preventing the aggregation
of Aβ42 provides a potential approach for AD treatment. As
a result, discovering small molecules exhibiting dual inhibi-
tory activity against Aβ42 aggregation and neuroprotective
efect against Aβ42-induced neuronal cell death could be an
attractive strategy for R&D of anti-AD drugs [8, 9].
Molecular hybridization is widely employed as a power-
ful tool in drug design, which involves the hybridization of
diferent pharmacophores into a single molecular framework
to improve pharmaceutical profle [27]. Due to the privileges
of quinolone and cinnamic acid, some quinoline/cinnamic
acid hybrids were synthesized but mainly for the purpose of
antitumor and antimicrobic treatments [28–30], the investi-
gation on their potential for anti-AD treatment has not yet
been performed to the best of our knowledge. During our
efects to discover new anti-Aβ agents [31–34], six new qui-
noline/cinnamic acid hybrids 5a–5f (Fig. 1) were recently
achieved with chemical agents in hands based on molecular
hybridization approach. Herein, we describe the synthesis,
in vitro biological evaluation, and molecular docking of this
kind of hybrids as anti-Aβ42 aggregation and neuroprotec-
tive agents.
Quinoline is one of the most versatile pharmacophores
extensively merging in bioactive molecules against various
diseases, including neurodegenerative diseases [10, 11],
cancer [12, 13], malaria [14], and infammation [15]. Vari-
ous quinoline-based compounds have been also reported to
have potential anti-AD activity [16, 17]. One of the qui-
nolone derivatives, 8-hydroxyquinoline derivative PBT2
(a, Fig. 1), could signifcantly lower cerebrospinal fuid
levels of Aβ42 and improve cognition, and showed benefts
for AD patients in phase II clinical trials [18, 19]. Another
quinolone derivative (b, Fig. 1) reported by Hoda et al. also
showed potent inhibitory activity against self-mediated,
AChE- and Cu2+-induced Aβ42 aggregation [20]. In addition,
cinnamic acids were also the useful precursors for designing
anti-AD molecules due to their ability of endowing deriva-
tives with a variety of pharmacological activities: e.g., anti-
oxidative [21], anti-infammatory [22], anti-Aβ aggregation
[23], and enzyme inhibitory activities [24, 25]. For example,
cinnamic acid (c, Fig. 1) could remarkably reduce cerebral
Aβ42 plaque burden and improve memory of AD mice [23].
Cinnamic acid derivative (d, Fig. 1) exhibited in vitro inhibi-
tion on the aggregation of Aβ42 and neuroprotective activity
against Aβ42-induced toxicity in PC12 cells [26].
Results and discussion
Chemistry
Scheme 1 presents the synthetic approach towards these six
target hybrids 5a–5f. Briefy, 4,5-dimethoxy-2-nitrobenzoic
acid as starting material was treated with iron and acetic acid
in refuxing methanol solution to aford 2-amino-4,5-dimeth-
oxybenzoic acid (1). The cyclization reaction of 1 with pyru-
vic acid produced 6,7-dimethoxyquinoline-2-carboxylic acid
(2). The amidation reaction of cinnamic acids with tert-butyl
(2-aminoethyl)carbamate yielded (E)-tert-butyl (2-cinnama-
midoethyl)carbamates 3a–3f, which was then deprotected to
give N-(2-aminoethyl)cinnamamide 4a–4f. Finally, the target
(E)-N-(2-cinnamamidoethyl)-6,7-dimethoxyquinoline-2-car-
boxamides 5a–5f were prepared from 2 and corresponding
(E)-N-(2-aminoethyl)cinnamamide 4a–4f under amidation
condition. The structure of synthesized molecules was con-
frmed by 1H, 13C NMR, and MS data.
Fig. 1 Design strategy towards new quinoline/cinnamic acid hybrids 5a–5f
1 3

Synthesis and biological evaluation of quinoline/cinnamic acid hybrids as amyloid-beta…
Scheme 1
preliminary results indicated that the electron-donating
In vitro biological assay
group at the benzene ring of the cinnamic acid fragment
was preferred to increase the inhibitory activity towards
Aβ42 aggregation.
First, all the target compounds were evaluated for their
in vitro inhibitory activity against Aβ42 aggregation using
the thiofavin T (ThT) fuorescence method [35]. Resveratrol
was used as a positive control, and galanthamine was taken
as a negative control. Their efects on Aβ42 aggregation were
tested at the concentration of 5–100 μM. As shown in Fig. 2,
resveratrol (10 μM) efectively inhibited Aβ42 aggregation
by 29.5 2.9%, while the negative reference galanthamine
shows no signifcant activity towards Aβ42. Among these
synthetic hybrids, compounds 5a, 5b, and 5f at 10 μM
showed similar anti-Aβ42 aggregation activity compared
with resveratrol-treated group, and their anti-Aβ42 aggre-
gation potency was increased in a dose-dependent manner.
The maximal inhibition ratios of 5a, 5b, and 5f against Aβ42
aggregation at 100 μM are 67.4 2.8%, 76.3 2.6%, and
62.4 2.5%, respectively. While introducing the electron-
withdrawing groups (e.g., Cl or Br) at benzene ring in com-
pounds 5c, 5d, and 5e made the Aβ42 aggregation inhibitory
activity lose or promoted the aggregation of Aβ42. These
The Aβ42 aggregation could be promoted by the forma-
tion of β-sheet structure, thus molecules preventing the for-
mation of a β-sheet structure could have a potential efect
for anti-Aβ aggregation [36]. To probe the possible binding
modes and key interactions of these compounds with Aβ42,
the active hybrids 5a, 5b, and 5f were docked to Aβ42 mono-
mer (PDB ID: 1IYT) using AutoDock software. As shown in
Fig. 3, these compounds interact with Aβ42 mainly through
hydrophobic interactions with residues, such as Ala21,
Val24, Gyl25, Ser26, Asn27, Gly29, and Leu34. In the case
of compound 5b, an intermolecular hydrogen bond interac-
tion is formed between the O atom of carbonyl attached to
the quinoline group and the terminal NH2 group of Lys28,
at a distance of 3.12 Å (Fig. 3E). The hydrophobic interac-
tions and a salt bridge between Lys28 and Asp23/Glu22 play
an important for stabilizing β-sheet conformational changes
in the process of Aβ fbrillogenesis [37]. Based on these
Fig. 2 Inhibition on Aβ42 aggre-
gation by hybrids 5a–5f at 5, 10,
25, 50, and 100 μM. Values are
express as mean SD of three
experiments
1 3

Y.-X. Ge et al.
Fig. 3 The predicted binding
mode of hybrids 5a, 5b, and 5f
with Aβ42 (PDB code 1IYT).
a Compound 5a (colored
green) interacting with Aβ42;
b compound 5b (colored
blue) interacting with Aβ42; c
compound 5f (colored purple)
interacting with Aβ42. The rela-
tive schematic diagram (d for
5a, e for 5b, f for 5f) showed
the hydrophobic interactions
(shown as starbursts) and
H-bond interaction (denoted by
dotted green lines) (color fgure
online)
molecular docking results, it was proposed that compounds
5a, 5b, and 5f shared a similar binding mode with Aβ42 near
the stabilizing residues of the α-helix Aβ, and thus inhib-
ited Aβ42 aggregation via interfering with the formation of
β-sheets.
Table 1 Toxicity of compounds 5a, 5b, and 5f towards SH-SY5Y and
LO2 cells
Compounds
IC50/μM
SH-SY5Y
LO2
5a
5b
5f
62.3 1.2
70.2 3.4
55.8 2.1
60.4 3.1
55.7 2.5
42.9 1.4
Since compounds 5a, 5b, and 5f could inhibit the aggre-
gation of Aβ42, then their neuroprotective activities against
Aβ42 aggregation-induced toxicity towards SH-SY5Y neu-
ronal cells were tested. First, the cytotoxicity towards SH-
SY5Y cells was evaluated using MTT method [38]. The
results shown in Table 1 revealed that all these three com-
pounds have IC50 values more than 50 μM. In addition, most
of the drugs are metabolized in the liver, and the liver will
undergo damage if these drugs are toxic. Therefore, the tox-
icity of 5a, 5b, and 5f towards human hepatocyte cell line
(LO2) was also tested. As shown in Table 1, the IC50 of these
compounds toward LO2 were in the range of 42.9 1.4 to
60.4 3.1 μM.
evaluated, with EGCG as a positive reference compound.
As shown in Fig. 4, the viability of SH-SY5Y cells was
decreased to 63.1 0.8% by exposed to 10 μM Aβ42, while
after treated with 10 μM EGCG or synthetic hybrids the
viability of SH-SY5Y cells was increased to 85.9 1.6%
(EGCG), 68.2 1.2% (5a), 85.5 1.1% (5b), 79.7 0.9%
(5f), respectively, suggesting that these hybrids can stimulate
survival of neuroblastoma cells. In addition, the histogram
shows these hybrids signifcantly increasing the SH-SY5Y
cell viability in a dose-dependent manner. Among these
Then, the neuroprotective activity of 5a, 5b, and 5f
against Aβ42-induced neurotoxicity in SH-SY5Y cells was
1 3

Synthesis and biological evaluation of quinoline/cinnamic acid hybrids as amyloid-beta…
by an analytical Agilent 1260 HPLC with ZDRBAX SB-C18
column (4.6 mm × 150 mm) using parameters as follows:
H2O/MeOH, 70/30–0/100 in 15 min, plus 10 min isocratic
MeOH, fow rate at 3.0 cm3/min, λ=280 nm. ESI–MS analy-
ses were performed on an Agilent 1260-6460 Triple Quard
LC–MS instrument (Agilent, Waldbronn, Germany), and
HR-ESI–MS data were acquired on an Agilent 6520 Q-TOF
LC/MS (Agilent, Waldbronn, Germany).
2‑Amino‑4,5‑dimethoxybenzoic acid (1) To the mixture
of 3.3 g 4,5-dimethoxy-2-nitrobenzoic acid (1 equiv) and
3.5 g iron (4 equiv) in 50 cm3 CH3OH was dropwise added
4.5 cm3 CH3CO2H (5 equiv) at room temperature, then the
mixture was refuxed for 8 h. The solution was concentrated
under reduced pressure, and the obtained residues were
purifed by fash column chromatography using petroleum
ether/ethyl acetate=2:1 as eluent to give compound 1. Yield
14.2%; m.p.: 198–200 °C. The NMR analysis was in accord-
ance with previously published data [39].
Fig. 4 Neuroprotective efects of compounds 5a, 5b, and 5f against
Aβ42-induced toxicity in SH-SY5Y cells. Data are expressed as
mean SD of three experiments. *P<0.05, **P<0.01, ***P<0.001
versus Aβ42-treated group
compounds, 5b showed comparable activity to EGCG at
both 5 and 10 μM. In general, these results indicated that
these compounds could attenuate Aβ42-induced neurotoxic-
ity in SH-SY5Y cells.
6,7‑Dimethoxyquinoline‑2‑carboxylic acid (2) Compound
2 was synthesized according to earlier published report
[40]. A solution of 400 mg compound 1 (1 equiv), 124 mm3
pyruvic acid (1.1 equiv), and 486 mg NaOH (5.5 equiv)
in 20 cm3 EtOH was refuxed overnight. Then the solution
was acidifed to pH~4 by addition of 1 M aqueous HCl at
room temperature and then concentrated. The residues was
re-crystalized in MeOH to produce 2. M.p.: 213–215 °C
(Ref. [41]: 215 °C). This compound was used in the next
step without further purifcation.
Conclusion
In summary, six target (E)-N-(2-aminoethyl)cinnamamides
5a–5f were designed, prepared, and evaluated for their
Aβ42 aggregation inhibitory and neuroprotective activities.
Amongst, compounds 5a, 5b, and 5f could function as new
dual anti-Aβ42 aggregation and neuroprotective agents. On
the basis of results of anti-Aβ42 aggregation assay, the elec-
tron-donating group at the benzene ring of the cinnamic acid
fragment was benefcial for increasing anti-Aβ42 aggregation
activity. Overall, the present study supports that quinoline/
cinnamic acid hybrids could serve as a new template for
developing multifunctional anti-AD drug candidates, and
continuing investigation into its detailed SAR study and
potential for AD treatment is warranted.
Preparation of compounds 3a–3f and 4a–4f
Compounds 3a–3f and 4a–4f were synthesized according to
earlier published protocols [42, 43].
General procedure for the preparation of 5a‑5f
To a solution of the corresponding N-(2-aminoethyl)cin-
namamides 4a–4f (0.21 mmol, 1 equiv) in 5 cm3 CH2Cl2 was
added 15 mg HOBT (0.5 equiv), 61 mg EDCI (1.5 equiv),
87 mm3 Et3N (3 equiv), and 50 mg compound 2 (1 equiv).
The mixture was stirring at room temperature overnight and
then concentrated to get residue, which was subject to fash
column chromatography to yield target products 5a–5f.
Experimental
Commercially available reagents were used without fur-
ther purifcation. Organic solvents were evaporated with
reduced pressure using a Büchi R-100 evaporator (Büchi,
Switzerland). Silica gel column chromatography was per-
formed on Biotage Isolera One (Biotage, Uppsala, Sweden).
NMR spectra were measured on Bruker Avance III 600 MHz
spectrometer (Bruker, Fällanden, Switzerland). Chemical
shifts were expressed in δ (ppm) and coupling constants
(J) in Hz with residual solvent signals as standards (CDCl3,
δH = 7.26 and δC = 77.2 ppm; DMSO-d6, δH = 2.50 and
δC =39.5 ppm). The purity of the samples was determined
N‑(2‑Cinnamamidoethyl)‑6,7‑dimethoxyquinoline‑2‑car‑
boxamide (5a, C23H23N3O4) White solid; yield 7.0%; HPLC
purity: 96.7%; tR =9.60 min; 1H NMR (600 MHz, CDCl3):
δ = 8.57 (t, J = 6.0 Hz, 1H, NH), 8.14 (d, J= 8.3 Hz, 1H),
8.11 (d, J=8.3 Hz, 1H), 7.61 (d, J=15.8 Hz, 1H), 7.51–7.47
(m, 2H), 7.37 (s, 1H), 7.36–7.31 (m, 3H), 7.06 (s, 1H), 6.80
(t, J=4.4 Hz, 1H, NH), 6.45 (d, J=15.8 Hz, 1H), 4.04 (s,
1 3

Y.-X. Ge et al.
+
3H), 4.03 (s, 3H), 3.78–3.74 (m, 2H), 3.71–3.67 (m, 2H)
ppm; 13C NMR (150 MHz, CDCl3): δ=166.7, 166.6, 153.3,
151.3, 147.1, 143.8, 141.0, 135.5, 135.0, 129.7, 128.9, 128.9,
128.0, 128.0, 125.7, 120.9, 117.4, 107.9, 104.9, 56.3, 56.3,
41.4, 39.4 ppm; MS (ESI): m/z=406.0 ([M+H]+); HR-MS
HR-MS (ESI): m/z calcd for C23H22Cl2N3O4 ([M + H]+)
474.0982, found 474.0982.
(E)‑N‑[2‑[3‑(3‑Bromophenyl)acrylamido]ethyl]‑6,7‑dimeth‑
oxyquinoline‑2‑carboxamide (5e, C23H22BrN3O4) White
solid; yield 7.4%; HPLC purity: 95.2%; tR =12.44 min; 1H
NMR (600 MHz, CDCl3): δ=8.60 (t, J=6.0 Hz, 1H, NH),
8.13 (d, J=8.5 Hz, 1H), 8.09 (d, J=8.5 Hz, 1H), 7.60 (s,
1H), 7.51 (d, J = 15.5 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H),
7.38–7.35 (m, 2H), 7.19 (t, J=6.4 Hz, 1H, NH), 7.07–7.02
(m, 2H), 6.45 (d, J = 15.5 Hz, 1H), 4.04 (s, 3H), 4.02 (s,
3H), 3.78–3.73 (m, 2H), 3.71–3.67 (m, 2H) ppm; 13C NMR
(150 MHz, CDCl3): δ=166.7, 166.1, 153.3, 151.3, 147.0,
143.7, 139.3, 137.1, 135.5, 132.5, 130.4, 130.4, 126.7,
125.7, 123.0, 122.4, 117.3, 107.9, 104.9, 56.3, 56.3, 41.4,
39.4 ppm; MS (ESI): m/z=484.0, 486.0 ([M+H]+); HR-MS
(ESI): m/z calcd for C23H23BrN3O4+ ([M+H]+) 484.0866,
486.0846, found 484.0865, 486.0895.
+
(ESI): m/z calcd for C23H24N3O4 ([M + H]+) 406.1761,
found 406.1745.
(E)‑6,7‑Dimethoxy‑N‑[2‑[3‑(4‑methoxyphenyl)acrylamido]‑
ethyl]quinoline‑2‑carboxamide (5b, C24H25N3O5) White
solid; yield 20.3%; HPLC purity: 96.5%; tR = 9.57 min;
1H NMR (600 MHz, CDCl3): δ = 8.56 (brs, 1H, NH),
8.14 (d, J=8.3 Hz, 1H), 8.10 (d, J=8.3 Hz, 1H), 7.56 (d,
J = 15.3 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.37 (s, 1H),
7.06 (s, 1H), 6.86 (d, J=8.6 Hz, 2H), 6.68 (brs, 1H, NH),
6.31 (d, J=15.3 Hz, 1H), 4.04 (s, 3H), 4.03 (s, 3H), 3.81 (s,
3H), 3.77–3.72 (m, 2H), 3.70–3.66 (m, 2H) ppm; 13C NMR
(150 MHz, CDCl3): δ=166.9, 166.6, 161.0, 153.3, 151.2,
147.2, 143.8, 140.6, 135.5, 129.5, 129.5, 127.7, 125.7,
118.5, 117.4, 114.3, 114.3, 107.9, 104.9, 56.3, 56.3, 55.5,
41.3, 39.4 ppm; MS (ESI): m/z=436.1 ([M+H]+); HR-MS
(E)‑6,7‑Dimethoxy‑N‑[2‑[3‑(2‑methoxyphenyl)acrylamido]‑
ethyl]quinoline‑2‑carboxamide (5f, C24H25N3O5) White
solid; yield 6.1%; HPLC purity: 95.2%; tR =10.02 min; 1H
NMR (600 MHz, CDCl3): δ=8.56 (t, J=6.2 Hz, 1H, NH),
8.13 (d, J = 8.6 Hz, 1H), 8.10 (d, J= 8.6 Hz, 1H),7.89 (d,
J=15.8 Hz, 1H), 7.46 (dd, J=7.7, 1.6 Hz, 1H), 7.38 (s, 1H),
7.29 (dd, J=7.9, 7.4 Hz, 1H), 7.06 (s, 1H), 6.92 (dd, J=7.7,
7.4 Hz, 1H), 6.88 (d, J=7.9 Hz, 1H), 6.64 (brs, 1H, NH),
6.56 (d, J=15.8 Hz, 1H), 4.04 (s, 3H), 4.03 (s, 3H), 3.84 (s,
3H), 3.77–3.73 (m, 2H), 3.71–3.67 (m, 2H) ppm; 13C NMR
(150 MHz, CDCl3): δ=167.3, 166.4, 158.3, 153.2, 151.2,
147.2, 143.8, 136.5, 135.4, 130.9, 129.0, 125.7, 124.0,
121.6, 120.7, 117.4, 111.2, 108.0, 104.9, 56.3, 56.3, 55.5,
41.0, 39.5 ppm; MS (ESI): m/z=436.1 ([M+H]+); HR-MS
+
(ESI): m/z calcd for C24H26N3O5 ([M + H]+) 436.1867,
found 436.1876.
(E)‑N‑[2‑[3‑(4‑Chlorophenyl)acrylamido]ethyl]‑6,7‑dimeth‑
oxyquinoline‑2‑carboxamide (5c, C23H22ClN3O4) White
solid; yield 16.2%; HPLC purity: 95.6%; tR =11.84 min; 1H
NMR (600 MHz, DMSO-d6): δ = 8.94 (t, J = 5.9 Hz, 1H,
NH), 8.35–8.31 (m 2H), 7.98 (d, J = 15.8 Hz, 1H), 7.58
(d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.44–7.41
(m, 3H), 6.63 (d, J = 15.8 Hz, 1H), 3.94 (s, 3H), 3.93 (s,
3H), 3.51–3.47 (m, 2H), 3.45–3.41 (m, 2H) ppm; 13C NMR
(150 MHz, DMSO-d6): δ = 165.0, 164.6, 152.9, 150.6,
147.6, 143.0, 137.3, 135.5, 133.9, 129.3, 129.2, 129.2,
129.0, 129.0, 125.0, 123.0, 116.8, 107.4, 105.5, 55.9, 55.7,
38.9, 38.7 ppm; MS (ESI): m/z=440.1 ([M+H]+); HR-MS
(ESI): m/z calcd for C23H23ClN3O4+ ([M+H]+) 440.1372,
found 440.1377.
+
(ESI): m/z calcd for C24H26N3O5 ([M + H]+) 436.1867,
found 436.1862.
In vitro anti‑Aβ aggregation assay
1,1,1,3,3,3-Hexafuoro-2-propanol pre-treated Aβ42 (GL
Biochem Ltd, shanghai, China) was dissolved in DMSO
to make a 200 μM stock solution. The stock solution was
centrifuged at the speed of 13,500 rpm for 10 min. The
above supernatant was used for further experiments. The
compounds for testing were dissolved in DMSO at concen-
trations of 0.8 mM. A screening assay for the compounds
to inhibit Aβ aggregation was performed by measuring ThT
fuorescence emission. Compounds (2 mm3) and 2 mm3 of
200 μM Aβ42 were added into 76 mm3 of phosphate-buf-
ered saline (PBS at pH 7.4) in a 96-well microtiter plate.
After incubation for 24 h at room temperature, 80 mm3 of
5 μM ThT solution (in 50 mM glycine–NaOH at pH 8.5)
was added to the reaction solution. Fluorescence emission
was measured at 490 nm with an excitation wavelength of
( E) ‑ N‑ [ 2 ‑ [ 3 ‑ ( 3 , 4 ‑ D i c h l o r o p h e n y l ) a c r y l a m i d o ] ‑
ethyl]‑6,7‑dimethoxyquinoline‑2‑carboxamide (5d,
C23H21Cl2N3O4) White solid; yield 20.4%; HPLC purity:
1
95.0%; tR = 14.12 min; H NMR (600 MHz, DMSO-d6):
δ=8.97 (t, J=6.1 Hz, 1H, NH), 8.37–8.31 (m, 2H), 7.98 (d,
J=8.3 Hz, 1H), 7.85 (d, J=1.8 Hz, 1H), 7.67 (d, J=8.3 Hz,
1H), 7.56 (dd, J=8.3, 1.8 Hz, 1H), 7.44–7.42 (m, 2H), 7.42
(d, J=15.6 Hz, 1H), 6.69 (d, J=15.6 Hz, 1H), 3.94 (s, 3H),
3.93 (s, 3H), 3.51–3.47 (m, 2H), 3.44–3.42 (m, 2H) ppm;
13C NMR (150 MHz, DMSO-d6): δ=164.8, 164.7, 152.9,
150.7, 147.6, 143.1, 136.2, 135.9, 135.6, 131.7, 131.7,
131.1, 129.5, 127.4, 125.0, 124.5, 116.9, 107.5, 105.5, 55.9,
55.8, 38.9, 38.8 ppm; MS (ESI): m/z = 474.0 ([M + H]+);
1 3

Synthesis and biological evaluation of quinoline/cinnamic acid hybrids as amyloid-beta…
450 nm on a Tecan Spark multimode microplate reader. The
fuorescence intensities were compared and the % inhibition
was calculated by the following equation: 100−[(Fi −Fb)/
(Fo − Fb) × 100] where Fi, Fo, and Fb are the fuorescence
intensities obtained for Aβ aggregation in the presence of
inhibitors, Aβ42 and ThT; in the presence of Aβ42 and THT
but no inhibitors; and the blanks containing ThT only.
pretreatment with the compounds for 2 h, Aβ42 were added
to SH-SY5Y cell cultures in 24 h. Assays for cell viability
were performed in 24 h after cultured in fresh medium.
Acknowledgements This research work was fnancially supported
by the Natural Science Foundation of China [no. 21672082], Natu-
ral Science Foundation of Shandong Province [nos. ZR2019YQ31,
ZR2017BC101], and Shandong Talents Team Cultivation Plan of Uni-
versity Preponderant Discipline [no. 10027].
Molecular docking
The molecular docking studies was performed using Auto-
Dock 4.2, and the amyloid β42 structure fle (PDB ID: 1IYT)
[44] was saved in pdbqt format to perform the docking
analysis. The 3D structures of compounds were built and
performed MMFF94 minimization using ChemBio3D Ultra
12.0. Using Autodock Tools 1.5.6, preparation of receptor
was made by the addition of hydrogen atoms and Gasteiger
charges, and fnal assignment of atomic types as AD4 type,
and then autotorsions was used to defne the rotatable bonds
in the ligand preparation. The resulting enzyme structure
was used as an input for the AUTOGRID program. The
dimensions of the box were set to 60×60×60 with a grid
spacing of 0.375 Å. Rigid ligand docking was performed
for the compounds. Docking calculations were carried out
using the Lamarckian genetic algorithm (LGA). The pro-
posed docking complex image was created by Pymol 1.5.
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Afliations
Yong‑Xi Ge1 · Zhi‑Qiang Cheng1 · Lei Zhou1 · Hong‑Xu Xie1 · Yin‑Yin Wang1 · Kongkai Zhu2 · Yang Jiao3 ·
Guangpu Liu4 · Cheng‑Shi Jiang1
1
3
School of Biological Science and Technology, University
of Jinan, Jinan 250022, China
Shandong Institute for Food and Drug Control, Jinan 250012,
China
2
4
Shandong Key Laboratory of Biochemical Analysis,
College of Chemistry and Molecular Engineering, Qingdao
of University of Science and Technology, Qingdao 266042,
China
Department of Pharmacy, Qilu Hospital of Shandong
University, Jinan 250012, China
1 3