Preparation of 4-flexible amino-2-arylethenyl-quinoline derivatives as multi-target agents for the treatment of Alzheimer’s disease

Article abstract of DOI:10.3390/molecules23123100

Alzheimer’s disease (AD) is a complex and multifactorial neurodegenerative disorder of aged people. The development of multitarget-directed ligands (MTDLs) to act as multifunctional agents to treat this disease is the mainstream of current research. As a continuation of our previous studies, a series of 4-flexible amino-2-arylethenylquinoline derivatives as multi-target agents was efficiently synthesized and evaluated for the treatment of AD. Among these synthesized derivatives, some compounds exhibited strong self-induced Aβ1–42 aggregation inhibition and antioxidant activity. The structure-activity relationship was summarized, which confirmed that the introduction of a flexible amino group featuring a N,N-dimethylaminoalkylamino moiety at the 4-position increased the Aβ1–42 aggregation inhibition activity, with an inhibition ratio of 95.3% at 20 μM concentration. Compound 6b1, the optimal compound, was able to selectively chelate copper (II), and inhibit Cu2+-induced Aβ aggregation effectively. It also could disassemble the self-induced Aβ1–42 aggregation fibrils with a ratio of 64.3% at 20 μM concentration. Moreover, compound 6b1 showed low toxicity and a good neuroprotective effect against Aβ1–42-induced toxicity in SH-SY5Y cells. Furthermore, the step-down passive avoidance test indicated compound 6b1 significantly reversed scopolamine-induced memory deficit in mice. Taken together, these results suggested that compound 6b1 was a promising multi-target compound worthy of further study for AD.

Full text of DOI:10.3390/molecules23123100

Article
Preparation of 4-Flexible Amino-2-Arylethenyl-
Quinoline Derivatives as Multi-target Agents for the
Treatment of Alzheimer’s Disease
Xiao-Qin Wang *, Chu-Ping Zhao, Long-Cheng Zhong, De-Ling Zhu, De-Hao Mai,
Mei-Gui Liang and Ming-Hua He *
School of Pharmacy, Guangdong Medical University, Dongguan 523808, Guangdong, China;
13538678474@163.com (C.-P.Z.); 15625808258@163.com (L.-C.Z.); 15625850845@wo.cn (D.-L.Z.);
15875092028@163.com (D.-H.M.); 13071498620@163.com (M.-G.L.)
* Correspondence: wangxqgdmu@hotmail.com (X.-Q.W.); cpuhmh@163.com (M.-H.H.); Tel.: +86-769-2289-
6559 (X.-Q.W. & M.-H.H.)
Academic Editors: Diego Muñoz-Torrero & Michael Decker
Received: 30 October 2018; Accepted: 24 November 2018; Published: 27 November 2018
Abstract: Alzheimer’s disease (AD) is a complex and multifactorial neurodegenerative disorder of
aged people. The development of multitarget-directed ligands (MTDLs) to act as multifunctional
agents to treat this disease is the mainstream of current research. As a continuation of our previous
studies, a series of 4-flexible amino-2-arylethenylquinoline derivatives as multi-target agents was
efficiently synthesized and evaluated for the treatment of AD. Among these synthesized
derivatives, some compounds exhibited strong self-induced Aβ1–42 aggregation inhibition and
antioxidant activity. The structure-activity relationship was summarized, which confirmed that the
introduction of a flexible amino group featuring a N,N-dimethylaminoalkylamino moiety at the 4-
position increased the Aβ1–42 aggregation inhibition activity, with an inhibition ratio of 95.3% at 20
μM concentration. Compound 6b
(II), and inhibit Cu2+-induced Aβ aggregation effectively. It also could disassemble the self-induced
Aβ1–42 aggregation fibrils with a ratio of 64.3% at 20 μM concentration. Moreover, compound 6b
showed low toxicity and a good neuroprotective effect against Aβ1–42-induced toxicity in SH-SY5Y
cells. Furthermore, the step-down passive avoidance test indicated compound 6b significantly
reversed scopolamine-induced memory deficit in mice. Taken together, these results suggested that
compound 6b was a promising multi-target compound worthy of further study for AD.
1, the optimal compound, was able to selectively chelate copper
1
1
1
Keywords: Alzheimer’s disease; 2-arylethenylquinoline derivatives; multi-target; Aβ aggregation;
antioxidant; neuroprotection
1. Introduction
Alzheimer’s disease (AD) is a chronic and age-related neurodegenerative disorder characterized
by memory loss and cognitive impairments [1,2]. Today, the number of dementia patients is
estimated at some 46 million worldwide, and it is expected to reach 131.5 million by 2050, causing
great economic and social burdens to the patients and their families [3–5].
The histopathologic hallmarks of AD are neurofibrillary tangles and amyloid plaques [6]. Due
to its complex etiology, the pathogenesis of AD has not been completely elucidated, and multiple
factors are thought to contribute to the development of AD, including deficits of acetylcholine (ACh),
amyloid-β (Aβ) deposits, hyperphosphorylated tau protein, oxidative stress, dyshomeostasis of
biometals and neuroinflammation [7–11].
Molecules 2018, 23, 3100; doi:10.3390/molecules23123100
www.mdpi.com/journal/molecules

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Among these pathogenic factors of AD, Aβ production and aggregation in the brain play a
crucial role in AD pathogenesis [12]. It initiates the pathogenic cascade, and induces synaptic
dysfunction and causes neurotoxicity [13,14]. Aβ peptide is produced through proteolytic cleavage
of the amyloid precursor protein (APP) by α, β or γ-secretase, which can aggregate into oligomers,
protofibrils, and insoluble fibrils. These aggregates can result in the formation of senile plaques, and
ultimately lead to the neuronal loss and dementia [15,16]. Aβ aggregates can produce neurotoxicity
in many ways [17]. It can promote apoptosis, cause synaptic loss, and disrupt the cytoskeleton; It also
can disrupt cellular calcium balance and loss membrane potential; Aβ aggregates can promote the
generation of free radical via inflammatory response to cause oxidative stress; and it can disrupt
synaptic plasticity, and inhibit hippocampal long-term potentiation (LTP); It also can bind to metal
ions (especially Cu2+ and Zn2+) to form the complex of metal-Aβ, this complex can promote Aβ
aggregation and reactive oxygen species (ROS) production which lead to neuron death [18–26].
Hence, the prevention of Aβ aggregation could serves as a rational strategy for the treatment of AD
[27].
Recently, many studies show that there are high concentrations of metal ions (Cu2+, Zn2+, Fe2+) in
AD-affected brains [28]. These metal ions can bind to Aβ peptide and promote Aβ aggregation, and
the formation of Aβ plaques, and then lead to neuron death [29]. On the other hand, this interaction
also leads to the formation of ROS and causes the oxidative damage of the central nervous systems
(CNS) [30,31]. Thus, modulation of these metal ions in the brain has been proposed as a potential
therapeutic strategy for AD treatment [32].
Oxidative stress has also been linked to early events in AD pathogenesis [33]. Oxidative damage
can promote Aβ aggregation and the appearance of neurofibrillar tangles in AD [34]. Therefore, drugs
with radical scavenging activities could potentially prevent AD.
So far, there are only five drugs approved by FDA for clinical treatment of AD. They are four
acetylcholinesterase inhibitors (AChEIs) such as tacrine, donepezil, galantamine, rivastigmine, and
an N-methyl-D-aspartate (NMDA) receptor antagonist memantine. However, these drugs only
improve the memory and cognitive function of AD patients, which are unable to prevent or halt
progressive neurodegeneration of AD [35,36]. Therefore, it is urgent to develop more effective
therapeutic drugs for curing AD.
Because of the complexity of AD and identification of many potential targets, the multi-target-
directed ligand (MTDL) approach has recently attracted the attention of researchers in the pursuit of
AD drugs [37,38]. Designing of MTDL widely incorporates several different pharmacophoric
fragments into a single molecular, each fragment synergistically contributes to the overall activity
profile of the MTDL molecule[39].
Inspired by the MTDL strategy, we previously synthesized a series of 2-arylethenylquinoline
derivatives as multifunctional agents for the treatment of AD (Figure 1A) [40]. These multifunctional
agents based on the 4-rigid amino or H moiety of quinoline scaffold. However, these compounds had
unsatisfactory inhibitory activities of Aβ aggregation. On the other hand, our docking study of the
compound and Aβ showed that there is a lot of room at the 4-position of the quinoline ring, where
large substituent can be introduced to enhance the interaction of the compound with Aβ [41]. In order
to improve Aβ aggregation inhibition properties of the compound, in this work, based on the
structure-activity relationships (SAR) of our previous work, we introduced the flexible amino
substituent at the 4-position of the quinoline ring, and synthesized a series of 4-flexible amino-2-
arylethenylquinoline derivatives (Figure 1B), we also optimized the synthesis method of the target
compounds, which improved the yield and shortened the reaction time; and then evaluated their
biological activities, including inhibition of Aβ aggregation, antioxidative activity, metal chelating
property, neuroprotection and cytotoxicity. The SAR of synthesized compounds was discussed.
Furthermore, the optimal compound 6b was tested in AD mice model for the behavioral evaluations
1
in vivo.

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Figure 1. (A) The structure of previously described 2-arylethenylquinoline derivatives; (B) The
structure of newly investigated compounds.
2. Results and Discussion
2.1. Chemistry
The synthesis of 4-substituted-2-aryethenylquinoline derivatives was previously reported by
our group [40]. Herein differently substituted flexible amino-containing groups were introduced at
the 4-postion of the quinoline ring, and the synthetic method of the target compounds 6a–6e was
optimized as illustrated in Scheme 1.
Scheme 1. Synthetic route of 2-arylethenylquinoline derivatives 6a–6e. Reagents and conditions: (i)
PPA, 130 °C; (ii) POCl
3
, 120 °C; (iii) flexible amine, TsOH, MW, 120 °C, 1 h; (iv) aromatic aldehyde,
TMSCl, 150 °C, DMF.
The intermediate 4 was obtained following the procedure of our previous work [40]. The
reaction of intermediate 4 with different flexible amines (for example, 3-diethylaminopopylamine, 3-
dimethylaminopopylamine, N,N-dimethylethylenediamine, n-butyl-amine, or isobutylamine)
catalyzed by p-toluenesulfonic acid (TsOH) at 120 °C for 1 h under microwave-assisted conditions
gave the compounds 5a–5e, offered several advantages such as higher yields, shorter reaction times,
lower costs, more convenience, and higher efficiency compared to our previous synthetic method
[42]. Finally, the target compounds 6a–6e (6a
1–6a
4, 6b
1–6b
3, 6c
1–6c
2, 6d
1
–6d
2, and 6e
1–6e
2) were
obtained by the reaction of compounds 5a–5e with different substituent aromatic aldehydes in the

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presence of trimethylchlorosilane (TMSCl) at 150 °C for 24 h, The yields of compounds 6a–6e were in
range of 71%–85%. This synthetic method shortened the reaction time, and improved the product
1
yield. The structures of the target compounds were validated by using H-NMR, 13C-NMR, and
HRMS, and their purities were determined to be above 95% by using HPLC.
Table 1. Inhibition of self-induced Aβ1–42 aggregation, ORAC values of the target compounds.
ORAC b
Aβ1–42
Aggregation
Inhibition a (%)
Compound
General Structure
R1
R2
5 μM
1 μM
6a
6a
6a
6a
1
2
3
4
A
A
B
90.2 ± 1.3
88.5 ± 1.5
84.3 ± 2.1
87.9 ± 1.1
95.3 ± 1.2
92.1 ± 1.3
87.2 ± 2.1
85.1 ± 1.4
83.9 ± 2.4
80.5 ± 1.4
79.2 ± 2.3
77.9 ± 1.3
77.5 ± 1.5
83.5 ± 1.4
81.2 ± 0.8
6.85 ± 0.05
6.28 ± 0.08
1.73 ±0.07
4.52 ± 0.03
6.54 ± 0.07
5.88 ± 0.01
4.21 ± 0.08
3.24 ± 0.10
3.53 ± 0.07
1.56 ± 0.05
0.95 ± 0.09
1.92 ± 0.11
1.78 ± 0.05
3.91 ± 0.11
3.12 ± 0.10
5.92 ± 0.03
5.74 ± 0.09
1.26 ± 0.06
4.16 ± 0.04
6.23 ± 0.05
5.61 ± 0.09
3.96 ± 0.08
2.79 ± 0.04
3.28 ± 0.09
1.38 ± 0.02
0.75 ± 0.06
1.71 ± 0.05
1.68 ± 0.06
3.70 ± 0.11
2.91 ± 0.11
_
A
A
A
B
6b
6b
6b
1
2
3
_
6c
6c
1
A
A
A
A
A
A
A
A
2
6d
1
2
6d
6e
1
2
6e
4b
1
2
4b
Res
Cur
_
_
_
_
_
_
77.2 ± 1.1
49.3 ± 1.2
5.21 ± 0.50
2.61 ± 0.12
5.10 ± 0.41
2.11 ± 0.61
a
The Thioflavin T fluorescence method was used. The maximum percentage inhibitions of
aggregation (means ± SD for these experiments) were found at the inhibitors’ concentration of 20 μM.
b Data were expressed as μmol of Trolox equivalents/μmol of tested compounds. The concentration
of the tested compounds was 5 μM and 1 μM.
2.2. Inhibition of Aβ1–42 Self-Induced Aggregation

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The formation and aggregation of Aβ1–42 in the brain leads to neurotoxicity in AD. In order to
evaluate the activity of our new synthetic compounds to inhibit Aβ1–42 self-induced aggregation, the
thioflavin (ThT) fluorescence assay was performed [40,43]. Resveratrol and curcumin were used as
reference compounds. The results are shown in Table 1.
It is seen that all of the compounds exhibited strong inhibitory activity (>77% at 20 μM), which
is higher than those of curcumin (49.3% at 20 μM) and resveratrol (77.2% at 20 μM). Compound 6b
6b , and 6a showed the most potent inhibitory activities, with respective inhibition ratio of 95.3%,
92.1% and 90.2%. In order to further investigate their dose-dependent inhibition of Aβ1–42 self-induced
aggregation, the IC50 values of compound 6b , 6b , and 6a were determined. The result is shown in
Table 2, they exhibited better inhibition than resveratrol (IC50 = 11.8 μM), with the IC50 values of
compound 6b , 6b , and 6a were 4.5 μM, 6.1 μM, and 7.8 μM, respectively. The structure-activity
1,
2
1
1
2
1
1
2
1
relationship was also explored. The influence of the substituent in the benzene ring of arylethenyl
part on Aβ aggregation inhibition has been studied in our previous work [40]. Previous studies
showed that the substituent group with 4-dimethylamino or 4-diethylamino on the benzene ring is
favorable for the inhibitory activity. Here, we mainly investigated the effect of substituent at the 4-
postion of quinoline ring on Aβ aggregation inhibition.
Firstly, the diamino substitution group at the 4-postion of quinoline ring obviously increased
the inhibitory activity (the Aβ aggregation inhibitory values of the series of a, b, and c compared to
the series of d, e, respectively). Suggesting that amino substituents at 4-postion of quinoline ring
played an important role in the inhibition of Aβ aggregation, which is consistent with our previous
experimental results. Secondly, the type of diamino substituents had a great effect on the inhibitory
activity of the compounds, the substituent group featured with N,N-dimethylaminoalkylamino at 4-
postion of quinoline scaffold gave better inhibitory activity than that featured with N,N-
diethylaminoalkylamino (the Aβ1–42 aggregation inhibitory values of the series of b compared to the
series of a, respectively). This may be because N,N-diethylaminoalkylamino gives larger space
resistance.
Table 2. The IC50 values of compound 6a
1
, 6b
1
, 6b
2
, and resveratrol against self-induced Aβ1–42
aggregation.
Compound
IC50 (μM) a
7.8 ± 0.4
6a
1
6b
1
2
4.5 ± 0.7
6b
6.1 ± 0.8
Res
11.8 ± 0.2
a
The Thioflavin T fluorescence method was
used, the IC50 (μM) values shown are the
mean ± SD of three experiments.
In addition, some target compounds with different linker length between two N atoms at 4-
postion of quinoline scaffold were synthesized and evaluated. It was found that compound 6a and
6b with three-carbon atom linker exhibited better Aβ aggregation inhibition than compound 6c with
two-carbon atom linker.
Finally, we also compared the inhibitory activity of the new 4-flexible amino-2-
arylethenylquinoline derivatives with compound 4b
1
and 4b with 4-rigid amino substituents in the
2
quinoline scaffold which were previously described for the best Aβ aggregation inhibition [40]. It
was found that flexible amino at 4-postion of quinoline scaffold contributed to the increased activity.

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2.3. Antioxidant Activity In Vitro
Oxidative stress is another crucial event in AD pathogenesis. In order to determine antioxidant
activities of our synthetic compounds, the oxygen radical absorbance capacity method with
fluorescein (ORAC-FL) assay was performed [44,45]. with Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) as the standard, and their antioxidant activity was expressed
as Trolox equivalents. The data is shown in Table 1. Our data suggested that most of the compounds
demonstrated good antioxidant activities. Compounds 6a
antioxidative action, with ORAC-FL values of 6.85, 6.54 and 6.28 at a concentration of 5 μM,
respectively, which was better than that of our previously synthetic compounds 4b and 4b . In
1, 6b
1
, and 6a exhibited the most potent
2
1
2
addition, comparing the antioxidative activity of series 6d and 6e, the series of compounds 6a, 6b and
6c with a diamino substitutent at the 4-postion of the quinoline ring had better antioxidative activities.
This might be because that amino substituent is crucial for the radical scavenging ability.
2.4. Antioxidant Activity in SH-SY5Y Cells
To further investigate their antioxidant activity in SH-SY5Y cells, cellular ROS detection assay
based on dichlorofluorescein diacetate (DCFH-DA) was performed [46]. with Trolox and N-acetyl-L-
cysteine (NAC) as reference compounds. Compounds that showed higher antioxidant activity in
vitro and self-induced Aβ1–42 aggregation inhibition were selected, and the concentration of the tested
compounds was 2.5 μM which had no effect on the cell viability. As shown in Figure 2, the tested
compounds and Trolox could decrease the intensity of fluorescence differently, but NAC didn’t
remove ROS generation in SH-SY5Y cells treated with t-BuOOH. Compound 6b
1
and 6a displayed
1
more antioxidant activity than Trolox, and compound 6a and 6b exhibited a little weaker
4
3
antioxidant activity than Trolox. These results indicated that the mechanism of these compounds
abolish ROS generation might be similar to that of Trolox, which is consistent with our previous
experiment findings.
Figure 2. ROS generation in SH-SY5Y cells incubated without or with compounds measured using
DCFH-DA. The results are expressed as the percentage of control cells (without compounds).
Considering that compound 6b
1
showed more active than other compounds in inhibiting self-
was selected for further study.
induced Aβ1–42 aggregation and antioxidant activity, compound 6b
1
2.5. Metal Binding Properties of Compound 6b
1
The chelation ability of compound 6b
1
toward biometals such as Na+, K+, Cu2+, Fe2+, Mg2+, Ca2+,
and Zn2+ was studied by UV-vis spectrometry [47,48]. When Cu2+, Fe2+, and Zn2+ was added, new
optical bands were observed at about 498 nm, and the peak at about 393.5 nm which was observed
on UV spectrum of compound 6b
Cu2+, Fe2+, Zn2+ and compound 6b
the UV spectrum upon the addition of Na+, K+, Mg2+, and Ca2+ (shown in Figure 3A), which indicated
1
in ethanol alone decreased, indicative of the interaction between
1
to form the complex 6b -metal (Ⅱ). But there was little change on
1

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that compound 6b
1
may not bind to these metal ions. Interestingly, after CuSO
4
was added, the
absorption at 498 nm increased obviously, this may be because compound 6b
strongly. Its ability to selectively chelate Cu2+ versus other relevant metal ions was further
investigated by UV-vis spectrometry. Solution of compound 6b was prepared and treated with the
1
complexed with Cu2+
1
metal ion (Zn2+, Fe2+, Ca2+, Mg2+, Na+ and K+), after 10 min incubation, the optical response was
observed, followed by addition of Cu2+ and consequent analysis of further spectral changes. The
result is shown in Figure 3B. It can be seen that compound 6b had good selectivity of metal chelation
1
for Cu2+.
Figure 3. (A) UV-vis absorption spectra of compound 6b
1
(10 μM) alone or in the presence of 20 μΜ
. Relative
NaCl, KCl, MgCl , CaCl , CuSO , ZnSO , and FeSO . (B) Metal selectivity for compound 6b
2
2
4
4
4
1
changes in optical response (absorbance intensity) when the compound interacted with metal ions.
Gray bars represent normalized affinity for metal ions M (M = Zn2+, Fe2+, Ca2+, Mg2+, K+ or Na+), while
blue bars indicate affinity for Cu2+ in presence of M.
In order to further determine the stoichiometry of compound 6b
spectra was performed by titrations of compound 6b
upon stepwise additions of a Cu2+ solution.
According to Figure 4A, the absorbance at 498 nm firstly increased with ascending amount of CuSO
1
for Cu2+ binding, the UV
1
4,
and then tended to be stable, indicating that the chelation reached saturation point. The molar ratio
was calculated (Figure 4B), two straight lines were drawn with the intersection point at a mole
fraction of 0.5, revealing a 2:1 stoichiometry for complex 6b
1
-Cu2+ (compound/Cu2+; binding maybe
through N atoms of 4-flexible amine group, the chelating motif NH-N is proposed. See Scheme S1).
Figure 4. (A) UV-vis absorption spectra of compound 6b
1
(20 μM) in ethanol after addition of
ascending amount of CuSO
4
(0–20 μΜ). (B) Determination of the stoichiometry of complex 6b
1
-Cu2+
by molar ration method.

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2.6. Inhibition of Cu2+-Induced Aβ1–42 Aggregation
To investigate the ability of compound 6b
1
to inhibit Cu2+-induced Aβ1–42 aggregation, the ThT-
binding assay was performed [49]. Resveratrol and clioquinol (CQ) were used as reference
compounds. As shown in Figure 5, the fluorescence of Aβ treated with Cu2+ is 135.2% that of Aβ alone,
which indicated that Cu2+ accelerated Aβ aggregation. But treated with the compounds, the
fluorescence of Aβ treated with Cu2+ decreased differently. Compound 6b
displayed 85.8% inhibition
1
of Cu2+-induced Aβ aggregation, which was equal to CQ (83.6% inhibition of Cu2+-induced Aβ
aggregation); Resveratrol displayed weaker inhibition (71.2% inhibition of Cu2+-induced Aβ
aggregation). These results suggested that compound 6b
could inhibit Cu2+-induced Aβ aggregation
1
effectively.
Figure 5. Inhibition of Cu2+-induced Aβ1–42 aggregation by compound 6b
comparing with those of
1
resveratrol (Res) and clioquinol (CQ) ([Aβ] = 20 μM, [Cu2+] = 20 μM, [compound] = 40 μM). Values
are reported as the mean ± SD of three independent experiments. *p < 0.05 vs. Aβ, **p < 0.01 vs. Aβ
treated with Cu2+.
2.7. Cytotoxic Effect on SH-SY5Y
The cytotoxicity of our synthesized compounds was examined in human neuroblastoma SH-
SY5Y cells. The cell viability was determined by using methyl thiazolyl tetrazolium (MTT)
colorimetry, the cells were treated with different concentrations of compounds (0–100 μM) [50]. The
result is shown in the Supplementary Materials (Table S1). Our data indicated that all the compounds
have their IC50 values above 100 μM, which implied that all the compounds have low cytotoxicity. In
addition, compounds 6b
1, 6b
2
, and 6a with higher self-induced Aβ1–42 aggregation inhibition
1
exhibited lowest cytotoxicity with their IC50 values of 253.7 μM, 228.1 μM and 189.2 μM, respectively.
2.8. Effect of Compound 6b
To further complement the ThT binding assay, A transmission electron microscopy (TEM) assay
was employed to monitor and clarify the effect of compound 6b on Aβ1−42 aggregation [51,52]. As
1
on Abundance of Aβ1–42 Fibrils
1
shown in Figure 6, after 24 h incubation at 37 °C, the sample of Aβ1−42 alone had aggregated into many
amyloid fibrils (Figure 6b), while a few thick fibrils were observed for the sample of Aβ1–42 in the
presence of resveratrol (Figure 6d). Compared to resveratrol, only fewer thin fibrils and small bulk
aggregates were observed in the sample of Aβ1−42 in the presence of 6b (Figure 6c). The TEM result
1

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was well consistent with the result of ThT, which strongly proved that compound 6b
1
had better
inhibition against Aβ1−42 fibrils formation than resveratrol.
Figure 6. TEM image analysis of Aβ1–42 aggregation in the presence and absence of the compound. (a)
Aβ1–42 (20 μM), 0 h. (b) Aβ1–42 (20 μM) was incubated at 37 °C for 24 h. (c) Aβ1–42 (20 μM) and 6b (20
1
μM) were incubated at 37 °C for 24 h. (d) Aβ1–42 (20 μM) and resveratrol (20 μM) were incubated at
37 °C for 24 h.
2.9. Disaggregation of Self-Induced Aβ1–42 Aggregation Fibrils by 6b
1
The ability of compound 6b to disaggregate self-induced Aβ1–42 aggregation fibrils was also
1
investigated [52]. Aβ1–42 fibrils were prepared by incubating fresh Aβ1–42 for 24 h at 37 °C, and the
test compound was added to the sample and incubated for another 24 h at 37 °C. Then the sample
was analyzed by ThT binding assay and TEM assay. The ThT binding assay exhibited compound
6b
weaker activity with ratio of 51.8%, as shown in Figure 7A. Our TEM results exhibited compound
6b incubated with Aβ1–42 fibrils could disaggregate more Aβ1–42 fibrils than resveratrol (Figure 7B),
1
disaggregated Aβ1–42 fibrils with ratio of 64.3% at 20 μM concentration, and resveratrol showed
1
which further supported the result of the ThT binding assay.
Figure 7. (A) Results of the ThT binding assay for Aβ1–42 without and with test compound. (B) TEM
images for Aβ1–42 disaggregation. (a) Aβ1–42 (20 μM) was incubated at 37 °C for 24 h in phosphate
buffer. (b) Aβ1–42 (20 μM) was incubated with resveratrol (20 μM) at 37 °C for 24 h. (c) Aβ1–42 (20 μM)
was incubated with 6b (20 μM) at 37 °C for 24 h.
1
2.10. In vitro Protective Effect of Compound 6b
Neuroblastoma Cells
1
against Aβ1–42 Induced Toxicity in SH-SY5Y Human

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The cytoprotective effect of compound 6b
1
against Aβ1–42 damage in SH-SY5Y human
neuroblastoma cell lines was determined by MTT assay [53,54]. Our cytotoxicity study had indicated
that compound 6b does not affect cell viability at the concentration of 20 μM. However, incubation
of Aβ1–42 (20 μM) with SH-SY5Y cells for 24 h resulted in a 47.5% reduction viability, compared with
the control group (untreated group). To investigate the protective effect of compound 6b on Aβ1–42
induced cell toxicity, SH-SY5Y cells exposed to Aβ1–42 (20 μM) were incubated with different
concentrations of compound 6b (5, 10, and 20 μM) for 24 h, and the cell viability was tested. As
shown in Figure 8, treatment with compound 6b increased the cell viability by preventing Aβ1–42
induced cytotoxicity in a concentration-dependent manner. Furthermore, when the SH-SY5Y cells
were co-incubated with compound 6b and Aβ1–42 for 48 h, the cell viability increased to 89.8%. All
1
1
1
1
-
1
these results indicated that compound 6b exhibited a neuroprotective role against Aβ1–42-induced
1
cell toxicity.
Figure 8. Cytoprotective effect of 6b
measured by MTT assay after 24 and 48 h of incubation at 37 °C with Aβ1–42 (20 μM) in the absence
and presence of 6b . The percentage of MTT reduction is relative to control cells in medium. Values
1
on SH-SY5Y human neuroblastoma cells. Viability was
1
are expressed as the mean ± SEM (n = 6), #p < 0.05 vs. control; *p < 0.05 and **p < 0.01 vs. Aβ.
2.11. Step-Down Type Passive Avoidance Test
In order to determine whether compound 6b could improve the memory impairment in
1
scopolamine-induced mice, the step-down passive avoidance test was performed [55,56], donepezil
was used as positive control. The doses of the tested compounds were not toxic to the mice. As shown
in Figure 9, the model group treated with scopolamine alone exhibited much shorter latency and
more number of errors than the control group. While treatment with donepezil group (5 mg/kg)
showed longer latency time (176 s) and less number of errors (2.87) than the model group with
scopolamine, which indicated donepezil significantly reversed the cognitive impairment induced by
scopolamine. Subsequently, treatment with compound 6b
latency and reduced the number of errors in a dose-dependent manner. For the low dose group of
compound 6b (4.0 mg/kg), it didn’t exhibit significant improvement of the memory impairment
1
(4.0, 8.0 and 16.0 mg/kg) increased the
1
compared with the model group. However, the high dose group (16.0 mg/kg) presented the longest
latency time (182 s) and least number of errors (2.72), which was better than donepezil group. In
general, these results demonstrated that compound 6b could improve cognitive deficit induced by
1
scopolamine.

Molecules 2018, 23, 3100
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Figure 9. Effects of compound 6b on the (A) latency (s) and (B) number of errors in the step-down
1
test by the scopolamine-induced cognitive impairment. The data shown are mean ± SEM (n = 8). #p <
0.01 vs. control group, *p < 0.05, **p < 0.01 vs. model group.
3. Materials and Methods
3.1. General Information
All commercial reagents and solvents were purchased from commercial suppliers and used
1
without further purification. H- and 13C-NMR spectra were recorded using TMS as the internal
standard in CDCl
3
or DMSO-d with a Bruker BioSpin GmbH spectrometer (Billerica, MA, USA) at
6
400 MHz and 101 MHz, respectively. High resolution mass spectra (HRMS) were recorded using
Shimadzu LCMS-IT-TOF spectrometer (Shimadzu, Kyoto, Japan). Melting points (mp) were obtained
using a SRS-OptiMelt automated melting point instrument (Sunnyvale, CA, USA) without correction.
The purities of synthesized compounds were confirmed to be higher than 95% by analytical HPLC
performed with a dual pump Shimadzu LC-20AB (Shimadzu) system equipped with a Ultimate XB-
C18 column and eluted with methanol-water (60:40–70:30) containing 0.1% TFA at a flow rate of 0.3
mL/min. Flash column chromatography was performed with silica gel (200–300 mesh) purchased
from Qingdao Haiyang Chemical Co. Ltd. (Qingdao, China). All the reactions were monitored by thin
layer chromatography using silica gel.
3.2. Chemistry
3.2.1. Synthesis of Intermediates
The intermediates 3, 4 were prepared following the procedure shown in Scheme 1 using our
previous reported method [40]. Compounds 5a–5e were synthesized under microwave-assisted
organic synthesis (MAOS) conditions with high yield by our recent reported method [42].
3.2.2. General Procedure A for the Synthesis of Compounds 6a–6e
A mixture of compound 5a–5e (1.0 mmol), different aromatic aldehyde (1.05 mmol), and
trimethylchlorosilane (2 mL) in DMF (5 mL) was heated at 150 °C for 24 h. The reaction mixture was
allowed to cool to room temperature, and poured into ice water (40 mL), and then aqueous NaOH
was added to neutralize the solution to generate precipitate. The precipitate was filtered and washed
with water, and the crude product was purified by using flash column chromatography with
EtOAc/methanol (50:1) or CH
2
Cl /petroleum ether (3:1) elution to afford the desired products.
2
(E)-N1-(2-(4-(Diethylamino)styryl)quinolin-4-yl)-N3,N3-diethylpropane-1,3-diamine (6a
1)

Molecules 2018, 23, 3100
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Compound 5a was reacted with 4-diethylaminobenzaldehyde following the general procedure
1
A to afford product 6a
CDCl
1
as an orange solid in 72% yield. mp. 182.3–184.0 °C. H-NMR (400 MHz,
3
): δ 8.00 (s, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.60–7.53 (m, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.31 (t, J = 7.6
Hz, 1H), 7.12 (d, J = 8.8 Hz, 1H), 6.67 (d, J = 8.9 Hz, 2H), 6.57 (s, 1H), 3.49 (dd, J = 10.0, 5.5 Hz, 2H), 3.39
(q, J = 7.1 Hz, 4H), 2.75–2.69 (m, 2H), 2.66 (q, J = 7.1 Hz, 4H), 1.99–1.93 (m, 2H), 1.19 (t, J = 7.1 Hz, 6H),
1.11 (t, J = 7.1 Hz, 6H). 13C-NMR (101 MHz, CDCl
129.34, 128.78, 125.46, 125.11, 123.89, 122.87, 118.05, 112.58, 95.78, 53.66, 47.17, 44.38, 41.26, 23.88, 12.68,
3): δ 157.40, 151.21, 150.34, 148.06, 134.13, 130.28,
12.16. Purity: 99.2% by HPLC. HRMS (ESI): Cacld for [M + H]+ (C28
H
39
N ) requires m/z 431.3169, found
4
431.3188.
(E)-N1-(2-(4-(Dimethylamino)styryl)quinolin-4-yl)-N3,N3-diethylpropane-1,3-diamine (6a
Compound 5a was reacted with 4-dimethylaminobenzaldehyde following the general
procedure A to afford product 6a
as an orange-red solid in 74% yield. mp. 168.5–170.1 °C. 1H-NMR
(400 MHz, CDCl ): δ 7.96 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 3.4 Hz, 1H), 7.59–7.57 (m, 1H), 7.55–7.50 (m,
2)
2
3
3H), 7.31 (t, J = 8.8 Hz, 1H), 7.12 (d, J = 16.2 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 6.57 (s, 1H), 3.48 (dd, J =
10.1, 5.6 Hz, 2H), 3.00 (s, 6H), 2.69 (t, J = 3.4 Hz, 2H), 2.65 (q, J = 4.4 Hz, 4H), 1.99–1.91 (m, 2H), 1.11 (t,
J = 7.1 Hz, 6H). 13C-NMR (101 MHz, CDCl
128.40, 125.69, 125.25, 123.40, 120.29, 118.62, 112.29, 95.49, 53.45, 47.09, 44.55, 40.34, 24.72, 11.58. Purity:
99.7% by HPLC. HRMS (ESI): Cacld for [M + H]+ (C26
) requires m/z 403.2856, found 403.2866.
3
): δ 157.30, 150.69, 150.53, 148.58, 133.37, 130.62, 128.95,
H
35N
4
(E)-N1-(2-(2-(1H-Indol-3-yl)vinyl)quinolin-4-yl)-N3,N3-diethylpropane-1,3-diamine (6a
Compound 5a was reacted with indole-3-formaldehyde following the general procedure A to
afford product 6a ):
as a pale yellow solid in 74% yield. mp. 194.3–195.1 °C. 1H-NMR (400 MHz, CDCl
3)
3
3
δ 8.98 (s, 1H), 8.08 (d, J = 7.4 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 16.3 Hz, 1H), 7.71 (d, J = 8.3
Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.47 (s, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.33–7.26 (m, 2H), 7.22–7.15 (m,
2H), 6.53 (s, 1H), 3.48 (t, J = 3.3 Hz, 2H), 2.70 (t, J = 8.0 Hz,2H), 2.67 (q, J = 14.2 Hz, 4H), 1.98–1.91 (m,
2H), 1.11 (t, J = 7.1 Hz, 6H). 13C-NMR (101 MHz, CDCl
127.47, 126.40, 125.67, 124.85, 123.45, 122.49, 120.47, 120.42, 120.38, 118.42, 114.88, 111.78, 95.36, 53.40,
3): δ 157.37, 150.99, 147.79, 137.22, 129.28, 128.25,
47.04, 44.57, 24.60, 11.51. Purity: 97.2% by HPLC. HRMS (ESI): Cacld for [M + H]+ (C26
H
31
N ) requires
4
m/z 399.2543, found 399.2552.
(E)-N1,N1-Diethyl-N3-(2-(4-morpholinostyryl)quinolin-4-yl)propane-1,3-diamine (6a
)
4
Compound 5a was reacted with 4-morpholinyl-benzaldehyde following the general procedure
1
A to afford product 6a
CDCl
4
as an orange solid in 76% yield. mp. 172.5–174.3 °C. H-NMR (400 MHz,
3
): δ 7.97 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.61–7.52 (m, 4H), 7.32 (t, J = 11.1
Hz, 1H), 7.17 (d, J = 16.2 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 6.56 (s, 1H), 3.89–3.83 (m, 4H), 3.46 (t, J = 5.5
Hz, 2H), 3.24–3.17 (m, 4H), 2.71–2.63 (m, 6H), 1.99–1.90 (m, 2H), 1.10 (t, J = 7.1 Hz, 6H). 13C-NMR (101
MHz, CDCl
120.75, 113.36, 106.58, 96.77, 66.80, 53.67, 53.10, 47.93, 44.21, 24.54, 11.99. Purity: 98.6% by HPLC.
HRMS (ESI): Cacld for [M + H]+ (C28
O) requires m/z 445.2962, found 445.2979.
3
): δ 158.14, 151.23, 149.68, 143.61, 133.40, 129.53, 129.07, 128.34, 125.56, 124.48, 123.70,
H37
N4
(E)-N1-(2-(4-(Diethylamino)styryl)quinolin-4-yl)-N3,N3-dimethylpropane-1,3-diamine (6b
Compound 5b was reacted with 4-diethylaminobenzaldehyde following the general procedure
A to afford product 6b
as an orange-red solid in 73% yield. mp 153.9–155.2 °C. 1H-NMR (400 MHz,
CDCl ): δ 8.36 (s, 1H), 7.71 (d, J = 16.1 Hz, 1H), 7.64–7.57 (m, 2H), 7.54 (d, J = 8.9 Hz, 2H), 7.34 (t, J = 4.0
1)
1
3
Hz, 1H), 7.25 (t, J = 16.1Hz, 1H), 6.65 (d, J = 8.9 Hz, 2H), 6.49 (s, 1H), 3.57–3.50 (m, 2H), 3.40 (q, J = 8.0
Hz, 4H), 2.64 (t, J = 8.0 Hz, 2H), 2.40 (s, 6H), 2.34 (s, 1H), 2.01–1.96 (m, 2H), 1.20 (t, J = 8.0 Hz, 6H). 13C-
NMR (101 MHz, CDCl
120.78, 119.07, 117.52, 111.40, 93.95, 58.72, 45.37, 44.41, 43.66, 24.68, 12.65. Purity: 97.0% by HPLC.
HRMS (ESI): Cacld for [M + H]+ (C26
) requires m/z 403.2856, found 403.2880.
3): δ 154.47, 152.29, 148.67, 143.43, 137.10, 130.50, 129.51, 124.81, 124.56, 123.02,
H35
N4

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(E)-N1-(2-(4-(Dimethylamino)styryl)quinolin-4-yl)-N3,N3-dimethylpropane-1,3-diamine (6b
)
2
Compound 5b was reacted with 4-dimethylaminobenzaldehyde following the general
procedure A to afford product 6b
as an orange solid in 75% yield. mp 151.2–153.3 °C. 1H-NMR (400
MHz, CDCl ): δ 7.94 (d, J = 8.3 Hz, 1H), 7.56–7.48 (m, 3H), 7.45(d, J = 8.0 Hz, 2H), 7.24 (t, J = 4.4 Hz,
1H), 7.05 (d, J = 16.2 Hz, 1H), 6.63 (d, J = 8.8 Hz, 2H), 6.49 (s, 1H), 3.51–3.30 (m, 2H), 2.92 (s, 6H), 2.49
(t, J = 8.0 Hz, 2H), 2.28 (s, 6H), 2.05–1.68 (m, 2H). 13C-NMR (101 MHz, CDCl
): δ 157.22, 150.65, 149.62,
142.17, 134.07, 129.37, 128.50, 125.08, 124.28, 123.67, 121.42, 119.52, 117.95, 112.23, 94.40, 57.88, 44.71,
2
3
3
43.73, 41.54, 26.19. Purity: 97.0% by HPLC. HRMS (ESI): Cacld for [M + H]+ (C24
375.2543, found 375.2562.
H
31
N ) requires m/z
4
(E)-N1-(2-(2-(1H-Indol-3-yl)vinyl)quinolin-4-yl)-N3,N3-dimethylpropane-1,3-diamine (6b
Compound 5b was reacted with indole-3-formaldehyde following the general procedure A to
afford product 6b ):
as a pale yellow solid in 77% yield. mp. 168.2–169.5 °C. 1H-NMR (400 MHz, CDCl
3)
3
3
δ 9.30 (s, 1H), 8.03 (t, J = 8.4 Hz, 2H), 7.85 (d, J = 16.3 Hz, 1H), 7.71 (s, 1H), 7.62–7.55 (m, 2H), 7.44 (s,
1H), 7.38 (d, J = 8.4 Hz, 1H), 7.32 (t, J = 8.4 Hz, 1H), 7.25 (d, J = 16.3 Hz, 1H), 7.20–7.12 (m, 2H), 6.51 (s,
1H), 3.48–3. 41(m, 2H), 2.57 (t, J = 4.4 Hz, 2H), 2.37 (s, 6H), 1.97–1.90 (m, 2H). 13C-NMR (101 MHz,
CDCl
121.09, 120.94, 120.68, 119.76, 115.44, 112.10, 95.36, 54.70, 45.72, 44.15, 25.45. Purity: 98.5% by HPLC.
HRMS (ESI): Cacld for [M + H]+ (C24
) requires m/z 371.2230, found 371.2245.
3): δ 157.13, 151.21, 148.19, 137.22, 129.39, 128.06, 127.69, 126.97, 125.85, 124.98, 123.45, 122.62,
H27
N4
(E)-N1-(2-(4-(Diethylamino)styryl)quinolin-4-yl)-N2,N2-dimethylethane-1,2-diamine (6c
)
1
Compound 5c was reacted with 4-diethylaminobenzaldehyde following the general procedure
1
A to afford product 6c
CDCl
(d, J = 16.1 Hz, 1H), 6.75–6.59 (m, 3H), 3.40 (dd, J = 14.0, 7.1 Hz, 6H), 2.73 (t, J = 5.8 Hz, 2H), 2.33 (s,
6H), 1.20 (t, J = 7.0 Hz, 6H). 13C-NMR (101 MHz, CDCl
): δ 157.50, 149.85, 148.63, 147.95, 133.62, 129.29,
129.13, 128.72, 124.90, 124.07, 123.75, 119.66, 118.36, 111.52, 96.30, 57.24, 45.13, 44.40, 40.08, 12.70.
1
as an orange solid in 77% yield. mp. 145.2–147.1 °C. H-NMR (400 MHz,
3): δ 7.98 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.60–7.48 (m, 4H), 7.36 (t, J = 4.0 Hz, 1H), 7.10
3
Purity: 99.4% by HPLC. HRMS (ESI): Cacld for [M + H]+ (C25
389.2712.
H33
N ) requires m/z 389.2700, found
4
(E)-N1-(2-(4-(Dimethylamino)styryl)quinolin-4-yl)-N2,N2-dimethylethane-1,2-diamine (6c
Compound 5c was reacted with 4-dimethylaminobenzaldehyde following the general
procedure A to afford product 6c
as an orange-red solid in 79% yield. mp. 148.9–149.6 °C. 1H-NMR
(400 MHz, CDCl ): δ 8.02 (d, J = 8.5 Hz, 1H), 7.76 (t, J = 8.1 Hz, 1H), 7.60–7.53 (m, 2H), 7.51 (d, J = 8.7
Hz, 2H), 7.34 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 4.3 Hz, 1H), 7.12 (d, J = 16.4Hz, 1H), 6.70 (d, J = 8.2Hz, 2H),
6.59 (s, 1H), 3.39 (s, 2H), 2.99 (s, 6H), 2.71 (t, J = 5.9 Hz, 2H), 2.32 (s, 6H). 13C-NMR (101 MHz, CDCl
):
2)
2
3
3
δ 157.27, 149.60, 148.04, 147.43, 133.79, 129.68, 129.18, 128.56, 124.73, 124.48, 123.42, 119.27, 118.29,
113.76, 98.04, 56.25, 45.31, 43.21, 40.29. Purity: 96.6% by HPLC. HRMS (ESI): Cacld for [M + H]+
(C23H
29
N ) requires m/z 361.2387, found 361.2399.
4
(E)-N-Butyl-2-(4-(diethylamino)styryl)quinolin-4-amine (6d
Compound 5d was reacted with 4-diethylaminobenzaldehyde following the general procedure
A to afford product 6d ):
as a yellow solid in 80% yield. mp. 172.5–173.8 °C. 1H-NMR (400 MHz, CDCl
1)
1
3
δ 8.13 (s, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.51 (m, 3H), 7.29 (t, J = 8.2 Hz, 1H), 7.07
(d, J = 16.2 Hz, 1H), 6.64 (d, J = 8.6 Hz, 2H), 6.53 (s, 1H), 3.40–3.34 (m, 4H), 3.27 (q, J = 6.7 Hz, 2H), 1.54–
1.43 (m, 2H), 1.37–1.29 (m, 2H), 1.17 (t, J = 7.0 Hz, 6H), 0.89 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz,
CDCl
118.04, 112.25, 96.30, 43.03, 40.37, 31.15, 20.40, 14.19, 13.92. Purity: 98.8% by HPLC. HRMS (ESI): Cacld
for [M + H]+ (C25
) requires m/z 374.2591, found 374.2606.
3): δ 157.17, 150.60, 149.79, 146.44, 135.33, 133.74, 129.33, 129.20, 128.49, 125.03, 123.88, 119.06,
H
32N
3
(E)-N-Butyl-2-(4-(dimethylamino)styryl)quinolin-4-amine (6d )
2

Molecules 2018, 23, 3100
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Compound 5d was reacted with 4-dimethylaminobenzaldehyde following the general
procedure A to afford product 6d
as an orange solid in 82% yield. mp. 165.3–167.1 °C. 1H-NMR (400
MHz, CDCl ): δ 7.96 (d, J = 2.3 Hz, 1H), 7.78–7.68 (m, 1H), 7.61–7.50 (m, 3H), 7.37–7.28 (m, 1H), 7.24
2
3
(d, J = 8.0 Hz, 1H), 7.10 (d, J = 16.2 Hz, 1H), 6.70 (d, J = 8.0 Hz, 2H), 6.55 (s, 1H), 3.39 (dd, J = 12.3, 6.9
Hz, 2H), 3.02 (s, 6H), 1.53–1.48 (m, 2H), 1.35–1.28 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz,
CDCl
119.44, 118.83, 112.19, 111.74, 103.93, 51.96, 40.31, 31.25, 20.38, 13.88. Purity: 98.3% by HPLC. HRMS
(ESI): Cacld for [M + H]+ (C23
) requires m/z 346.2278, found 346.2296.
3): δ 158.12, 155.18, 147.76, 146.34, 136.29, 130.93, 130.50, 129.23, 128.78, 124.62, 124.18, 123.69,
H
28N
3
(E)-2-(4-(Diethylamino)styryl)-N-isobutylquinolin-4-amine (6e
Compound 5e was reacted with 4-diethylaminobenzaldehyde following the general procedure
A to afford product 6e
as a pale yellow solid in 85% yield. mp. 170.2–171.7 °C. 1H-NMR (400 MHz,
CDCl ): δ 7.96 (d, J = 8.3 Hz, 1H), 7.66 (t, J = 8.2 Hz, 1H), 7.61–7.55 (m, 2H), 7.53–7.48 (m, 1H), 7.35 (t, J
1)
1
3
= 7.9 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 7.08 (d, J = 16.2 Hz, 1H), 6.79 (d, J = 12.3 Hz, 1H), 6.67 (d, J = 8.8
Hz, 1H), 5.00 (s, 1H), 3.32 (q, J = 7.0 Hz, 4H), 2.83 (t, J = 6.2 Hz, 2H), 1.78–1.70 (m, 1H), 1.13 (t, J = 7.1
Hz, 6H), 0.86 (d, J = 6.4 Hz, 6H). 13C-NMR (101 MHz, CDCl
130.73, 129.49, 129.20, 128.79, 128.03, 124.19, 119.06, 117.94, 110.97, 100.09, 50.77, 44.28, 27.48, 20.30,
3): δ 158.18, 149.89, 148.93, 147.28, 134.17,
12.63 ppm. Purity: 97.7% by HPLC. HRMS (ESI): Cacld for [M + H]+ (C25
H
32
N ) requires m/z 374.2591,
3
found 374.2606.
(E)-2-(4-(Dimethylamino)styryl)-N-isobutylquinolin-4-amine (6e
Compound 5e was reacted with 4-dimethylaminobenzaldehyde following the general
procedure A to afford product 6e
as an orange solid in 83% yield. mp. 171.8–173.2 °C. 1H-NMR (400
MHz, CDCl ): δ 7.97 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.58–7.49 (m, 4H), 7.32 (t, J = 7.6 Hz,
1H), 7.14 (d, J = 16.2 Hz, 1H), 6.68 (d, J = 8.4 Hz, 2H), 6.55 (d, J = 8.6 Hz, 1H), 3.21 (t, J = 6.0 Hz, 2H),
2.98 (s, 6H), 2.14–2.03 (m, 1H), 1.08 (d, J = 6.6 Hz, 6H). 13C-NMR (101 MHz, CDCl
): δ 156.91, 150.22,
148.79, 147.57, 134.94, 129.57, 129.17, 129.01, 128.62, 127.40, 124.33, 118.98, 117.64, 112.22, 98.27, 51.21,
2)
2
3
3
43.98, 28.19, 19.95. Purity: 97.9% by HPLC. HRMS (ESI): Cacld for [M + H]+ (C23
H
28
N ) requires m/z
3
346.2278, found 346.2292.
3.3. Pharmacological Assay
3.3.1. ThT Assay
All ThT experiments were performed according to our previously published methods [40]. Aβ1–
42 (Sigma, St. Louis, MO, USA; counterion, NaOH) stock solution was prepared by dissolving Aβ1–42
peptide in ammonium hydroxide (1% v/v), and diluting with 20 mM phosphate buffer (pH 7.4) to 100
μM. The tested compounds were firstly dissolved in DMSO at a concentration of 10 mM, and then
diluted with 20 mM phosphate buffer (pH 7.4) to 40 μM.
For the inhibition of self-induced Aβ1–42 aggregation [40]. Aβ1–42 peptide (2 μL, 50 μM, final
concentration) with the tested compound (2 μL, 20 μM, final concentration) or 20 mM phosphate
buffer (2 μL) was incubated in 20 mM phosphate buffer (pH 7.4) at 37 °C for 72 h. After incubation,
the samples were diluted to a final volume of 36 μL with 50 mM glycine-NaOH buffer (pH 8.5)
containing 5 μM Thioflavin T. Fluorescence signal was measured (excitation wavelength 450 nm,
emission wavelength 485 nm, and slit widths set to 5 nm) on a monochromator-based multimode
microplate reader (INFINITE M1000, TECAN, Hombrechtikon, Switzerland), adapted for 384-well
microtiter plates. The fluorescence intensities were recorded, and the percent inhibition of Aβ1–42
aggregation was calculated with the following equation: (1 − IFi/IFc) × 100%, in which IFi and IFc are the
fluorescence intensities obtained for absorbance in the presence and absence of the compounds after
subtracting the background, respectively. Each compound was examined in triplicate.
For the disaggregation of self-induced Aβ1–42 fibrils [52]. the Aβ1–42 stock solution (2 μL, 50 μM,
final concentration) was incubated at 37 °C for 24 h. Then, the 40 μM tested compound (2 μL) or 20
mM phosphate buffer (2 μL) was added and incubated at 37 °C for another 24 h. After incubation, the

Molecules 2018, 23, 3100
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sample was diluted to a final volume of 40 μL with 50 mM glycine-NaOH buffer (pH 8.0) containing
thioflavin T (5 μM). The detection method was the same as above.
3.3.2. Antioxidant Activity Assay In Vitro
The antioxidant activities of the compounds in vitro were determined by the oxygen radical
absorbance capacity fluorescein (ORAC-FL) assay [44,45]. The tested compounds and (±)-6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were dissolved in DMSO at a concentration of
10 mM, and then diluted with 75 mM potassium phosphate buffer (pH 7.4) to 50 μM and 10 μM for
use. The fluorescein (FL) stock solution (3.4 mM) was diluted with the same buffer to 136 nM. All the
assays were conducted with 75 mM potassium phosphate bufffer (pH 7.4), and the final reaction
mixture was 200 μL.
The mixture of compounds (or buffer) (20 μL) and fluorescein (FL, 120 μL) were incubated for
15 min at 37 °C in a black 96-well plate. Then 2,2′-azobis-(amidino-propane)-dihydrochloride (AAPH)
solution (60 μL, 40 mM) was rapidly added to the reaction mixture. The fluorescence was recorded
every 5 min for 240 min at 485 nm (excitation wavelength) and 535 nm (emission wavelength)
(Varioskan Flash Multimode Reader, Thermo Scientific, Waltham, MA, USA), and each sample was
prepared and measured for three times independently. Antioxidant curves (fluorescence versus time)
were normalized to the curve of the blank (without antioxidant), and the area under the fluorescence
decay curve (AUC) was calculated by the following equation:
i120
AUC = 1 +
( f / f )
i 0
(1)

i1
where f
0
is the initial fluorescence at 0 min and f is the fluorescence at time i. The net AUC for a
i
sample was calculated using the expression net AUC = AUCsample − AUCblank, The ORAC-FL value of
each sample was calculated using the equation of net AUCsample/(AUCTrolox – AUCblank), which was
expressed as Trolox equivalents.
3.3.3. Antioxidant Activity in SH-SY5Y Cells
SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium (DEME) and
supplemented with 1 mM glutamine, 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL
streptomycin. The cultures were maintained at 37 °C in a humidified incubator containing 5% CO
and were passaged twice weekly.
2
The antioxidant activity of the compounds in SH-SY5Y cells were tested with the fluorescent
probe (2′,7′-dichlorofluorescein diacetate, DCFH-DA) as reported with some variation [46]. SH-SY5Y
cells were sub-cultured in 96-well plates at a seeding density of 1 × 104 cells per well for 24 h, then the
medium was removed, the tested compounds (2.5 μM) was added and incubated for another 24 h at
37 °C. After that, the cells were washed with PBS and incubated with 5 μM DCFH-DA in PBS at 37 °C
in 5% CO for 30 min. Then DCFH-DA was removed, the cells were washed three times and incubated
2
with 0.1 mM t-BuOOH in PBS for 30 min. After the incubation, the cell fluorescence from each well
was measured at 485 nm excitation and 535 nm emission with a monochromator based multimode
microplate reader (INFINITE M1000).
3.3.4. Metal Chelation
The metal chelation of compound 6b
(Shimadzu UV-2450PC) with wavelength ranging from 200 to 700 nm [47,48]. The absorption spectra
of compound 6b (10 μM, final concentration) alone or in the presence of metal ions (20 μM, final
1
was tested in ethanol using a UV-vis spectrophotometer
1
concentration) after incubation for 30 min at room temperature, was recorded at room temperature
in a 1 cm quartz cell. Each sample was repeated for at least three times.
Experiments for the determination of metal ion binding selectivity were carried as follows [48].
20.0 mM stock solutions of CuSO4, ZnSO4, FeSO4, CaCl
2, MgCl
2, NaCl and KCl in MQ water were
prepared and then diluted to 1.0 mM using ethanol. 2.0 mM solution of compound 6b
1, CQ and

Molecules 2018, 23, 3100
16 of 20
resveratrol in ethanol were freshly prepared prior to use. 10 μL solutions of the compounds treated
with 10 μL of ZnSO , FeSO , CaCl , MgCl , NaCl and KCl. Spectra were recorded after 10 min
incubation at 25 °C. The metal binding selectivity was assessed by then adding 10 μL of CuSO
4
4
2
2
4
solution and incubating for 10 additional min at 25 °C. Selectivity was quantified by comparing and
normalizing the absorbance values of the maximum in each case with the absorbance of the solution
at the same wavelength after addition of CuSO
4.
For binding stoichiometry assay, A fixed amount of compound 6b
1
(20 μM) was mixed with
growing amounts of copper ion (0–20 μM) and tested the difference UV-vis spectra to investigate the
ratio of ligand/metal in the complex.
3.3.5. Inhibition of Cu2+-Induced Aβ1–42 Aggregation
Aβ was diluted in 20 μM HEPES (pH 6.6) with 150 μM NaCl. The mixture of the peptide (10 μL,
20 μM, final concentration) with or without copper (10 μL, 20 μM, final concentration) and the test
compound (10 μL, 40 μM, final concentration) was incubated at 37 °C for 24 h. The 30 μL of the
sample was diluted to a final volume of 200 μL with 50 mM glycine-NaOH buffer (pH 8.0) containing
thioflavin T (5 μM) [49]. The detection method was the same as that of self-induced Aβ aggregation
experiment.
3.3.6. Cytotoxicities of the Compounds on SH-SY5Y Cells
The cytotoxicities of the compounds were evaluated using the MTT assay [50]. SH-SY5Y cells
were subcultured in 96-well plates at a seeding density of 1 × 104 cells per well. After 24 h, the medium
was removed and treated with different concentrations of tested compounds for 24 h at 37 °C. The
survival of cells was determined with MTT assay. Then 80 μL of medium and 20 μL of MTT (0.5
mg/mL, final concentration) were added to each well and incubated for another 4 h. After the removal
of MTT, the formazan crystals were dissolved in DMSO. The amount of formazan was measured
using a microculture plate reader at the wavelength of 570 nm. Each concentration was performed in
triplicate.
3.3.7. TEM Assay
Aβ1–42 peptide (Sigma) was dissolved in 20 mM phosphate buffer (pH 7.4) at 4 °C to 100 μM
before use.
For the inhibition of self-induced Aβ1–42 aggregation experiment [51,52]. it was incubated in the
presence and absence of compound 6b and resveratrol at 37 °C for 24 h; For the disaggregation of
1
self-induced Aβ1–42 fibril experiment [52]. Aβ1–42 was incubated at 37 °C for 24 h. Then, 40 μM tested
compound was added and incubated at 37 °C for another 24 h. The final concentrations of Aβ1–42 and
the compounds were 50 μM and 20 μM, respectively. After incubation, aliquots (5 μL) of the samples
were placed on a carbon-coated copper/rhodium grid for 2 min at room temperature. Each grid was
stained with uranyl acetate (1%, 5 μL) for 2 min. Excess staining solution was removed and the
specimen was transferred for imaging with transmission electron microscopy (JEOL JEM-1400, Tokyo,
Japan).
3.3.8. Aβ1–42-Induced Neurotoxicity
SH-SY5Y cells were seeded in 96-well plates (1 × 104 cells per well) and exposed to Aβ1–42 (20 μM)
in the presence of compound 6b at the increasing doses (0, 5, 10 and 20 μM). After 24 h and 48 h, cell
1
viability was measured by the MTT assay [53,54].
3.3.9. Step-Down Type Passive Avoidance Test
A step-down passive avoidance test was used to assess learning and memory in mice [55,56].
The apparatus (Shanghai XinRuan, Shanghai, China) consisted of two identically sized compartments,
with a guillotine door to separate light and dark. Illumination was available in the light box through
LED lights at 250 lux. The mice underwent two separate trials: a training trial and a test trial 24 h

Molecules 2018, 23, 3100
17 of 20
later. For training trial, mice were initially placed in the light compartment and were allowed to
explore the environment freely for 5 min so that they can be familiar with the environment. Then the
door was opened, the mice tried to enter the dark compartment since they preferred to stay in dark
place. As soon as the mice came into the dark compartment, an electrical shock was delivered through
the steel rods. The training lasted for 5 min. Mice which did not enter the dark compartment within
180 s were excluded from the test. The test trial was preformed after 24 h. The mice were placed into
the light compartment and the door was opened. The time that the mouse spent to enter the dark
compartment was recorded as the latency. The numbers that the mouse entered the dark
compartment during 5 min were measured as error numbers. If the mouse did not cross the door, the
latency was identified as 300 s.
4. Conclusions
In summary, a series of 4-flexible amino-2-arylethenylquinoline derivatives was synthesized and
characterized as multi-target anti-Alzheimer agents based on our previous study. Most compounds
displayed high effective inhibitory potencies against Aβ1–42 aggregation and antioxidant activity. The
structure-activity relationship was summarized, which confirmed the importance of diamino
substitution group at the 4-postion of the quinoline ring for Aβ1–42 aggregation inhibition. In addition,
the substituent group featuring a N,N-dimethylaminoalkylamino moiety at the 4-postion of the
quinoline scaffold displayed significantly increased activity. The optimal candidate compound 6b
1
also displayed metal-chelating ability and 85.8% inhibition of Cu2+-induced Aβ aggregation, good
disaggregation of Aβ1–42 fibrils generated by self-induced Aβ1–42 aggregation. Moreover, it exhibited
low toxicity to SH-SY5Y cells and a significant effect on the protection of neuronal cells against Aβ1–
42-induced cytotoxicity in SH-SY5Y cells. Most importantly, compound 6b could significantly
1
prolong the latency and reduce number of errors in the step-down passive avoidance test.
Unfortunately, its inhibitory activity toward AChE and BuChE was weak (data not shown). Such
excellent properties highlight compound 6b as a potential lead compound for new multitarget drug
1
development in the treatment of AD.
Supplementary Materials: Supplementary data (1H-NMR, 13C-NMR, HRMS and HPLC spectra) associated with
this article are available online.
Author Contributions: X.-Q.W. and M.-H.H. conceived of and designed the experiments. X.-Q.W., C.-P.Z., L.-
C.Z., D.-L.Z. and D.-H.M. performed the experiments. X.-Q.W., D.-H.M. and M.-G.L. analyzed the data. X.-Q.W.
wrote the paper. All authors read and approved the final manuscript.
Funding: This work was financially supported by National Natural Science Foundation of China (21502025), the
Science Foundation for the Doctoral Program of Guangdong Medical University (B2017011), the Students’
Innovative Program of Guangdong Medical University (GDMU2017096, ZYDM018), and the Students’
Innovative Program of Guangdong Province (201810571096).
Conflicts of Interest: The authors declare no conflict of interest.
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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).