Design, synthesis of N-phenethyl cinnamide derivatives and their biological activities for the treatment of Alzheimer's disease: Antioxidant, beta-amyloid disaggregating and rescue effects on memory loss

Article abstract of DOI:10.3390/molecules23102663

Gx-50 is a bioactive compound for the treatment of Alzheimer's disease (AD) found in Sichuan pepper (Zanthoxylum bungeanum). In order to find a stronger anti-AD lead compound, 20 gx-50 (1-20) analogs have been designed and synthesized, and their molecular structures were determined based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis, as well as comparison with literature data. Compounds 1-20 were evaluated for their anti-AD potential by using DPPH radical scavenging assay for considering their anti-oxidant activity, thioflavin T (ThT) fluorescence assay for considering the inhibitory or disaggregate potency of Ab, and transgenic Drosophila model assay for evaluating their rescue effect on memory loss. Finally, compound 13 was determined as a promising anti-AD candidate.

Full text of DOI:10.3390/molecules23102663

molecules
Article
Design, Synthesis of N-phenethyl Cinnamide
Derivatives and Their Biological Activities for the
Treatment of Alzheimer’s Disease: Antioxidant,
Beta-amyloid Disaggregating and Rescue Effects
on Memory Loss
Tian Chai 1, Xiao-Bo Zhao 2, Wei-Feng Wang 2, Yin Qiang 1, Xiao-Yun Zhang 1,
*
and Jun-Li Yang 2,
*
1
School of Pharmacy, Lanzhou University, Lanzhou 730000, China; chait16@lzu.edu.cn (T.C.);
qiangy@lzu.edu.cn (Y.Q.)
CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural
Medicine of Gansu Province, Lanzhou Institute of Chemical Physics (LICP),
Chinese Academy of Sciences (CAS), Lanzhou 730000, China; zhaoxb08@licp.cas.cn (X.-B.Z.);
wwf2004@126.com (W.-F.W.)
2
*
Correspondence: zhangxiaoy@lzu.edu.cn (X.-Y.Z.); yangjl@licp.cas.cn (J.-L.Y.);
Tel.: +86-931-8915686 (X.-Y.Z.); +86-931-4968385 (J.-L.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 September 2018; Accepted: 10 October 2018; Published: 16 October 2018
Abstract: Gx-50 is a bioactive compound for the treatment of Alzheimer’s disease (AD) found in
Sichuan pepper (Zanthoxylum bungeanum). In order to find a stronger anti-AD lead compound,
20 gx-50 (
determined based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis,
as well as comparison with literature data. Compounds 20 were evaluated for their anti-AD
1–20) analogs have been designed and synthesized, and their molecular structures were
1–
potential by using DPPH radical scavenging assay for considering their anti-oxidant activity,
thioflavin T (ThT) fluorescence assay for considering the inhibitory or disaggregate potency of
Aβ, and transgenic Drosophila model assay for evaluating their rescue effect on memory loss. Finally,
compound 13 was determined as a promising anti-AD candidate.
Keywords: Alzheimer’s disease; gx-50; DPPH assay; ThT assay; transgenic Drosophila model assay
1. Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder that mainly occurs in the elderly.
It is characterized by intelligence decline and memory loss, as well as changes in emotions and
personality [
double every 20 years [
explore the molecular pathogenesis of AD, which had provided robust support to develop effective
pharmacological treatments [ ]. Currently, the clinically used AD drugs mainly include cholinesterase
1
]. There are approximately 36 million AD patients worldwide, and this figure will
2]. Since the 1990s, a great deal of financial support has been invested to
3
inhibitors, including tacrine, donepezil, rivastigmine, galanthamine, and huperzine A, and the
N-methyl-D-aspartate (NMDA) receptor antagonist, that is memantine. These drugs could alleviate
cognitive symptoms in this disease. Unfortunately, there is no effective means to cure, or only slow,
the progression of this disease [4,5].
The pathogenesis of AD has not yet been fully understood. Therefore, a variety of AD hypotheses
have been proposed, such as the amyloid cascade hypothesis, the Tau phosphorylation hypothesis,
the neurovascular hypothesis, the oxidative stress hypothesis, and the immune hypothesis [4]. One of
Molecules 2018, 23, 2663; doi:10.3390/molecules23102663
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2663
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the major pathological hallmarks of AD is the aggregation of amyloid plaques in the brain. Its essence
are the fibrils of the amyloid- peptide (A ) [ ]. Oxidative stress also plays an important role in the
development and progress of AD, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay is one of the most
common methods to evaluate the anti-oxidative activity of compounds [ ]. Moreover, transgenic
β
β 6,7
8,9
Drosophila that can express AD-related proteins has been successfully used to screen the potential
anti-AD compounds [10].
N-[2-(3,4-Dimethoxyphenyl)ethyl]-3-phenyl-acrylamide (gx-50), a potential drug candidate for
the treatment of AD, was isolated from Sichuan pepper (Zanthoxylum bungeanum). Gx-50 could
penetrate the blood brain barrier (BBB) and enter the brain tissue to improve the cognitive function of
dementia mice, and meanwhile could reduce the Aβ plaques in brain tissues [11–13]. In order to find
a much stronger drug candidate for the treatment of AD, 20 analogues of gx-50 were designed and
synthesized in this study. Consequently, the DPPH assay and thioflavin T (ThT) assay were employed
to evaluate their anti-oxidative activity and their inhibition and disaggregation on Aβ aggregation.
Finally, Pavlov’s olfactory memory test was used to evaluate their rescue effects on memory loss as
analyzed on the behavior of AD model flies. Herein, we will report the design, synthesis, and anti-AD
bioactivity of these gx-50 derivatives.
2. Results and Discussion
2.1. Chemistry
The synthesis of the gx-50 derivatives is described in Schemes 1 and 2. All compounds were
synthesized by following previously reported methods with little modifications [14]. Specifically,
the cinnamic acid was synthesized firstly by the substituted aromatic aldehyde with malonic acid
through the Knoevenagel reaction. Then in the presence of 3-(ethyliminomethylideneamino)-N,N-
dimethylpropan-1-amine hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP), the cinnamic
acids with different substitutions reacted with 4-methoxyphenethylamine, 2-phenylethylamine,
3,4-dimethoxyphenethylamine, and 3-methoxyphenethylamine, respectively. Finally, one molecule
of water was removed at room temperature to produce the corresponding target compounds
1–20.
Among them, compounds 12 16, and 20 have never been reported by searching the SciFinder database.
,
Regarding the known compounds, their chemical structures were determined and confirmed by NMR
and MS data analysis as well as literature comparison. Some of them were reported to show potent
biological activities. For example, compounds
cells at 100 M [15]. Compound and 11 had an antihyperglycemic effect as inhibition percentage
of 20.1% and 30.7% in sucrose-loaded model (SLM) at a dosage of 100 mg/kg-body weight [16 17].
Compound inhibited platelet aggregation with the IC50 value of 2.6 M [18]. Compound 10 and 11
showed anti-inflammatory activity with 50% NO inhibition concentration as 14.08 M and 15.08 M,
respectively [19 20]. Compound 10 and 14 revealed 5-lipoxygenase inhibitory activity with the IC50
value of 0.12 M and 1 M, respectively [21]. Compound 15 could inhibit tyrosinase as the IC50 value
of 0.6 mM [22 23]. In this study, all compounds 20 were evaluated for their anti-AD bioactivity via
DPPH assay, ThT test, and Pavlov’s olfactory memory test for the first time.
1, 9, and 13 exhibited indution of apoptosis in U-937
µ
3
,
5
µ
µ
µ
,
µ
µ
,
1–

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Scheme 1. Synthesis of compounds
malonic acid, oil bath, 90 ◦C, 4 h; (
overnight.
1
–
16. Reagents and conditions: (
a) anhydrous pyridine, piperidine,
b
) cinnamic acid, amine, EDCI, DMAP, DMF, room temperature,
Scheme 2. Synthesis of compounds 17
–
20. Reagents and conditions: (
a) anhydrous pyridine, piperidine,
malonic acid, oil bath, 90 ◦C, 4 h; (
overnight.
b) cinnamic acid, amine, EDCI, DMAP, DMF, room temperature,
2.2. Pharmacology
2.2.1. Determination of Anti-Oxidant Activity Based on DPPH Assay
The oxidative damage to neurons is closely related to the pathogenesis of AD [24]. In this article,
the anti-oxidative activity of compounds 1–20 was evaluated by DPPH assay with Vitamin C (Vc) as the
positive control [25]. The free radical scavenging ability was evaluated based on the UV absorbance change
of the solution measured at 517 nm using a microplate reader [26]. As shown in Table 1, the compound
concentration and their anti-oxidative activity exhibited a favorable concentration-dependent relationship.
In order to discuss their structure-activity relationship, compounds
as follows. The first group contains compounds , which only contained methoxy substituents. These
compounds showed weak DPPH scavenging activity. The second group contains compounds 16
which had the substituents of hydroxyls and methoxyls. These compounds exhibited comparable DPPH
scavenging activity, compared to the positive control. Moreover, compounds 13 16 with two hydroxyls
showed much stronger activity than compounds 12 with just one hydroxyl substituent. The third group
contains compounds 17 20, which contained 3,4-(methylenedioxy)cinnamic acid moiety. These compounds
showed the weakest DPPH scavenging activity. In addition, the DPPH scavenging rate of compound
(gx-50) was less than 10%, and its derivatives 16 exhibited stronger DPPH scavenging ability ranging
from 16.69 0.46 to 60.85 0.37. As discussed above, the phenolic hydroxyl may be a functional group for
1–20 were classified into three groups
1–8
9– ,
–
9–
–
3
9–
±
±
the anti-oxidant ability of gx-50 derivatives. This summarized structure-activity relationship is consistent
with the previously reported conclusion [27].

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Table 1. The DPPH assay result for compounds 1–20.
Inhibition Ratio (%)
Compound
R1
R2
R3
R4
50 µM
100 µM
200 µM
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Vc
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OCH3
H
H
H
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
-
H
H
OCH3
OCH3
H
H
OCH3
OCH3
H
H
OCH3
OCH3
H
H
OCH3
OCH3
2.97 ± 1.67
3.1 ± 0.30
4.35 ± 2.29
6.69 ± 0.37
5.03 ± 2.01
2.91 ± 1.24
4.93 ± 2.13
9.20 ± 0.67
6.18 ± 0.65
3.13 ± 0.59
2.92 ± 1.7
H
0.99 ± 0.55
4.78 ± 2.8
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
OH
OH
OH
H
9.78 ± 0.61
5.11 ± 2.06
5.58 ± 0.78
2.81 ± 2.78
36.28 ± 3.43
34.06 ± 2.57
40.28 ± 0.21
46.93 ± 0.42
56.16 ± 1.78
55.01 ± 0.35
59.96 ± 0.79
60.85 ± 0.37
4.60 ± 0.33
1.99 ± 1.50
1.39 ± 0.85
4.09 ± 0.31
72.00 ± 1.15
11.22 ± 0.43
12.91 ± 1.57
9.58 ± 1.39
3.42 ± 0.78
46.57 ± 0.86
44.07 ± 2.20
50.19 ± 1.90
51.83 ± 1.42
56.37 ± 2.86
54.17 ± 2.62
58.33 ± 2.71
57.68 ± 0.54
4.82 ± 0.36
2.27 ± 1.52
2.02 ± 0.52
4.53 ± 0.83
78.88 ± 0.80
1.08 ± 0.70
3.61 ± 0.89
0.06 ± 2.63
30.21 ± 1.52
16.69 ± 0.46
17.43 ± 0.46
28.74 ± 0.78
54.34 ± 1.44
54.21 ± 1.21
57.85 ±0.15
58.86 ±0.43
2.13 ± 0.32
0.63 ± 0.27
1.44 ± 1.68
1.57 ± 0.13
41.42 ± 2.83
-
-
-
-
H
OCH3
OCH3
-
-
-
OCH3
H
Values are expressed as the means ± SD of at least three independent experiments.
2.2.2. Inhibition of Cu2+-Induced Aβ Aggregation and Disaggregation
The inhibitory activities of compounds
disaggregation were evaluated by using ThT assay [28
controls. As shown in Table 2, the inhibitory and disaggregate potency of compounds
than 5%. Specifically, the inhibitory and disaggregate potency of compound
(gx-50) were 1.73 ± 2.15%
and 2.39 1.35%, respectively. However, when R2 becomes methoxyl group, as in compounds 5
the inhibitory and disaggregate potency obviously increased except for compound . Therefore,
R2 may be an active position that could increase inhibitory or disaggregate potency of the test
compounds. The inhibitory and disaggregate potency of compounds 12 were less than 10%.
Compounds 13 16 exhibited equal or better inhibitory and disaggregate potency than curcumin
and resveratrol. Compounds 17 20 had little inhibitory or disaggregate potency. The most active
compound was compound 15, with 61.85 1.70% and 64.44 0.76%, respectively. As discussed,
1
–
20 on copper-mediated Aβ1–42 aggregation and
29] with resveratrol and curcumin as positive
were less
,
1–4
3
±
–8,
8
9–
–
–
±
±
the catechol part could be the bioactive part for the gx-50 derivatives to inhibit Aβ aggregation and
disaggregate Aβ aggregate.
Table 2. Thioflavin T (ThT) assay results for compounds 1–20.
Disaggregation (%) b
Compound
R1
R2
R3
R4
Aβ Aggregation (inhib. %) a
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
OCH3
H
H
H
OCH3
OCH3
H
4.83 ± 8.10
0.14 ± 1.49
1.73 ± 2.15
2.28 ± 3.24
14.47 ± 4.15
12.26 ± 7.37
7.87 ± 2.90
3.33 ± 1.26
2.72 ± 3.65
5.94 ± 3.13
4.37 ± 3.20
2.78 ± 4.27
4.40 ± 2.11
0.27 ± 0.94
2.39 ± 1.35
3.85 ± 2.22
11.92 ± 2.32
11.19 ± 1.70
9.04 ± 1.99
4.49 ± 0.49
1.05 ± 1.04
4.16 ± 1.65
2.04 ± 0.71
0.33 ± 0.45
H
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
H
OCH3
OCH3
H
H
OCH3
OCH3
10
11
12

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Table 2. Cont.
Disaggregation (%) b
Compound
R1
R2
R3
R4
Aβ Aggregation (inhib. %) a
13
14
15
16
17
18
OH
OH
OH
OH
OCH3
H
OH
OH
OH
OH
H
H
OCH3
OCH3
OCH3
H
OCH3
H
H
H
56.43 ± 1.72
60.68 ± 1.76
61.85 ± 1.70
38.47 ± 2.37
14.34 ± 2.37
3.47 ± 7.57
13.06 ± 4.58
12.93 ± 3.66
48.85 ± 1.20
60.49 ± 2.71
52.76 ± 0.33
57.28 ± 3.61
64.44 ± 0.76
51.51 ± 1.42
15.50 ± 0.07
7.74 ± 2.00
18.68 ± 1.17
0.61 ± 0.38
43.83 ± 1.22
51.27 ± 3.31
OCH3
OCH3
−
−
−
−
19
20
OCH3
H
−
−
−
−
curcumin
resveratrol
The inhibition percentage Cu2+-induced Aβ1–42 aggregation. The disaggregation percentage of Cu2+-induced
a
b
A
β1–42 aggregates. Assays were carried out in the presence of 25 M inhibitor and 25 M Aβ1–42. Values are
µ
µ
expressed as the means ± SD of at least three independent experiments.
2.2.3. Pavlovian Olfactory Aversive Immediate Memory Test
The Pavlovian olfactory memory experiment was used to observe the rescue effect of compounds on
memory loss. As analyzed from the DPPH and ThT assay results, compounds and 13 16 were selected to
explore whether they could rescue the memory loss of the Drosophila AD model. Compounds and 13 16
were administered at a concentration of 10 M. As shown in Figure 1, the PI value of the AD negative
3
–
3
–
µ
control group (P35*H29.3) was 27 (required less than 45). The PI difference between the genetic control
group (P35*2U) and the AD negative group was 28 (required bigger than 15). Also, there was a significant
difference between the memantine (MEM) 100 µM group and the AD group (p < 0.01). It demonstrated
that the model flies had an obvious defect of learning and memory. In addition, the performance index
(PI) value of compound 13 was 41.33, and there was a significant difference (p < 0.05) with the AD group.
However, the PI values of compounds
that gx-50 (compound ) and compounds 14
However, the rescue effect on memory loss of compound 13 was comparable to that of the positive control
at the same concentration. Moreover, compound 13 was also far more effective than gx-50 (compound ).
3
and 14–16 are 39.66, 37.33, 33.16, and 36.83, respectively. It proved
3
–16 have a weak rescue effect on memory loss for AD flies.
3
Overall, compound 13 should be a promising drug candidate for the treatment of AD. Figure 2 showed the
standardization results for Figure 1.
Figure 1. Differential effects of compound
Drosophila Alzheimer’s disease (AD) model at 10
by one-way ANOVA; * p <0.05; ** p < 0.01; *** p < 0.001; n ≥ 20. MEM = memantine.
3
and compounds 13
–
16 on rescuing memory deficit on a
µM. The data are shown as means
±
SD and analyzed

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Figure 2. Differential effects of compound
a Drosophila AD model at 10 M. The data are shown as means
n ≥ 20.
3
and compounds 13
–
16 on rescuing memory deficit on
SD and analyzed by Tukey’s test;
µ
±
In conclusion, twenty derivatives of gx-50 have been designed and synthesized. Among them,
compound 13 showed the potent anti-oxidative activity, the inhibition and disaggregation on Cu2+-induced
Aβ1–42 aggregation, and the rescue effect on memory loss of Drosophila AD model. Compared to gx-50
(compound ), compound 13 would be a much more potent anti-AD candidate for the development of
3
relative drugs.
3. Experimental Section
3.1. General Experimental Procedures
Benzaldehyde, p-anisaldehyde, vanillin, and protocatechualdehyde were purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Malonic acid was obtained from Xilong
Chemical Co., Ltd. (Shantou, China). The compounds 4-methoxyphenethylamine, 2-phenylethylamine,
3,4-dimethoxyphenethylamine, and 3-methoxyphenethylamine were purchased from Energy Chemical
(Shanghai, China). The compound 4-Dimethylaminopyridine was purchased from Macklin (Shanghai,
China). The compound 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was purchased
from Accela ChemBio Co. Ltd. (Shanghai, China). Column chromatography was performed with
silica gel (200–300 mesh). All other reagents and solvents were analytical grade. NMR spectra were
recorded by a Bruker AVANCE III-400 spectrometer (Bruker AXS GmbH, Karlsruhe, Germany) with
tetramethylsilane (TMS) as an internal standard. The program MestReNova 9.0 was used to handle
the NMR spectra.
3.2. General Procedure for the Preparation of Compounds 1–20
3.2.1. Synthesis of Substituted Cinnamic Acids
The synthesis of substituted cinnamic acids was performed as Reference [30] described, with slight
modifications. A mixture of substituted aromatic aldehydes (3 mmol), malonic acid (6 mmol), piperidine
(0.3 mmol), was dissolved in pyridine and stirred at 90 ◦C for 4 h. Reaction progress was monitored by

Molecules 2018, 23, 2663
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thin layer chromatography (TLC) analysis. When the reaction terminated, the pyridine was removed
under vacuum. Then the reaction mixture was poured into ice water and washed by 2N HCl. Finally the
precipitate was filtered and washed with hexane three times. After being dried under vacuum, the products
were obtained.
3.2.2. Synthesis of Gx-50 Derivatives 1–20
The synthesis of gx-50 derivatives 1–20 was performed as Reference [31] described. EDCI (0.325 g,
1.5 mmol) and DMAP (0.183 g, 1.5 mmol) were added to a solution of a substituted cinnamic acid
(1 mmol) in 10 mL of DMF. The reaction solution was stirred in an ice-water bath. After 15 min,
phenolic amine (1 mmol) was added and the solution was stirred at room temperature overnight.
When the reaction was completed as indicated by TLC analysis, the solution was washed with water
and brine. Then the organic phase was dried with anhydrous MgSO4. The solvent was then evaporated
under vacuum. The crude product was purified by silica gel column chromatography to get the target
compounds 1–20 with high yields (95.8%, 96.8%, 97.5%, 96.1%, 96.6%, 94.2%, 95.8%, 97.0%, 95.7%,
95.6%, 98.3%, 96.4%, 98.8%, 97.2%, 98.3%, 96.0%, 98.4%, 97.9%, 96.8%, 98.3%, respectively).
3.2.3. Purity Check of Compounds 1–20 by HPLC/MS Analysis
The purities of gx-50 derivatives 1–20 were analyzed by an HPLC/MS system with UV detection at
205 and 254 nm. Based on the integrity of the peak area, the purity of all compounds were determined
over 97%. The HPLC analysis was performed on an Agilent 1200 Infinity system with an INNO C18
column (5
µ
m, 120 Å, 4.6
×
150 mm, Agilent Technologies, Santa Clara, CA, USA). The temperature of
◦
column chamber was kept at 25 C, and the mobile phase at 1.0 mL/min was as follows: ACN in H2O
containing 0.1% HCO2H (0–30 min: from 0% to 100%).
Compound 12: Colorless amorphous powder; 1H-NMR (400 MHz, CDCl3)
δ 7.53 (d, J = 15.2 Hz, 1H),
7.23 (m, 1H), 7.02 (m, 1H), 6.95 (m, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.79 (m, 3H), 6.19 (d, J = 15.6 Hz,
1H), 5.74 (s, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 3.65 (m, 2H), 2.86 (t, J = 6.8 Hz, 2H). 13C-NMR (101 MHz,
CDCl3)
δ 161.50, 155.10, 142.71, 142.02, 136.35, 135.80, 124.93, 122.55, 117.38, 116.38, 113.38, 110.01,
109.76, 107.14, 104.91, 51.17, 50.46, 35.91, 30.98. ESI/MS calculated for C19H21NNaO4: 350.1363, found:
350.1365 [M + H]+.
Compound 16: yellow amorphous powder; 1H-NMR (400 MHz, DMSO-d6)
8.04 (t, J = 5.2 Hz, 1H), 7.21 (m, 2H), 6.93 (s, 1H), 6.77 (m, 5H), 6.32 (d, J = 15.6 Hz, 1H), 3.73 (s, 3H),
3.38 (m, 2H), 2.74 (t, J = 7.2 Hz, 2H). 13C-NMR (101 MHz, DMSO-d6)
170.55, 164.48, 152.47, 150.73,
δ 9.34 (s, 1H), 9.10 (s, 1H),
δ
146.33, 144.21, 134.53, 131.59, 126.09, 125.58, 123.71, 120.94, 119.44, 118.99, 116.79, 60.10, 40.45, 35.89.
ESI/MS calculated for C18H19NNaO4: 336.1206, found: 336.1201 [M + Na]+.
Compound 20: Colorless amorphous powder; 1H-NMR (400 MHz, CDCl3)
7.23 (m, 1H), 6.97 (m, 2H), 6.80 (m, 4H), 6.14 (d, J = 15.6 Hz, 1H), 5.98 (s, 2H), 5.55 (s, 1H), 3.80 (s, 3H),
3.65 (q, J = 6.6 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H). 13C-NMR (101 MHz, CDCl3)
165.96, 159.87, 149.05,
δ 7.53 (d, J = 15.0 Hz, 1H),
δ
148.22, 140.81, 140.52, 129.70, 129.21, 123.83, 121.11, 118.59, 114.49, 111.93, 108.52, 106.35, 101.43, 55.21,
40.65, 35.73. ESI/MS calculated for C19H19NNaO4: 348.1206, found: 348.1204 [M + Na]+.
3.3. Biological Assay
3.3.1. DPPH Assay
DPPH was dissolved in ethanol (0.1 mM) daily and stored in the dark. Before usage, its absorbance
was calibrated to 0.7 using ethanol by a microplate reader. Ethanol was used as a negative control and
Vitamin C (Vc) was used as a positive control. A series of sample solutions with different concentrations
in DMSO were prepared, and then 2 µL of each sample solution was removed and added into
96-well plates respectively. Then 198 µL of DPPH solution was added, and each group had 3 parallel
holes. The blank group and the positive control group had the same treatment as the sample group.

Molecules 2018, 23, 2663
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The 96-well plate was shaken in the SPH-2000 shaking incubator (Xi’an Heb Biotechnology Co., Ltd.,
◦
Shanghai, China) at 37 C for 30 min in the dark. Consequently, the absorbance was measured at
517 nm with a RT-6100 microplate reader (Rayto, Shenzhen, China). The inhibition percent of each
test compound was calculated based on the equation: DPPH inhibition percent (%) = [(AC
−
AS)/AC]
×
100, where Ac represents the absorbance of the control sample and As is the absorbance of the
test compound.
3.3.2. ThT Assay
The dried Aβ1–42 peptide powder (purity
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a concentration of 1.0 mg
was shaken at room temperature for 6 h to gain the monomeric form of Aβ1–42. Then, the HFIP/peptide
≥
95%, Aladdin, Shanghai, China) was dissolved in
mL−1. The HFIP/peptide solution
·
solution was dried under a gentle stream of nitrogen gas for 2 h. The dried peptide power was then
◦
dissolved with anhydrous DMSO and stored at
−
20 C. For the test of copper-mediated Aβ1–42
inhibition and disaggregation, the procedures were carried out according to the literature [32]. In brief,
the above Aβ1–42 stock solution was diluted in 20 M HEPES (pH 6.6) with 150 M NaCl to the desired
final concentration before use. Either 10 L of 25 M peptide solution and 10 L of test compound
µ
µ
µ
µ
µ
solution were incubated at 37 ◦C for 24 h together or the addition of peptide solution and◦compound
solution was accomplished by two steps in an interval of 24 h, it was incubated at 37 C for 24 h
continuously. Then 180 µL of 50 mM glycine-NaOH buffer (pH 8.0) containing thioflavin T (5 µM) was
added to dilute the above sample solution. After 5 min, the fluorescence intensities were measured
(excitation, 450 nm; emission, 485 nm) by a Horiba FluoroMax4 spectrophotometer (HORIBA Scientific,
Edison, NJ, USA). The percent inhibition of aggregation was calculated via the expression (1
−
IFi/IFc)
×
100%, where IFi and IFc were the fluorescence intensities for Aβ1–42 in the presence and absence of
test compounds after subtracting the background, respectively.
3.4. Pavlov’s Olfactory Memory Test
3.4.1. Fly Stocks and Culture
All flies used in this study have been backcrossed for at least five generations, which could
make them maintain the genetic background of w1118(isoCJ1). Therefore, the strain of w1118 (isoCJ1)
(“2U”) was an isogenic line used as a control in the experiments. In order to eliminate the potential
adverse effects of long-term expression of A
(“H29.3”) that carried the human A
42 gene crossed with flies of elav-GAL4c155 (“P35”) that could start
the expression of A 42 gene, its offspring could express A 42 protein in the nervous system [33 35].
However, offspring flies obtained by crossing P35 and 2U could not express A 42 protein. The learning
β42 protein on Drosophila strains, only when the flies
β
β
β
–
β
test was conducted using ◦the above flies. All flies were maintained on a 12 h light per day (light
from 07:00 to 19:00) at 24 C and 40–60% relative humidity. Control male flies (elav/Y; +/+) and
experimental male flies (elav/Y; UAS-Aβ42/+) were selected by microscope on the second day after
eclosion. The selected flies were reared at 24 ◦C and 40
meal food for subsequent drug treatment.
±
15% relative humidity with standard corn
3.4.2. Drug Intervention
All compounds and memantine (purity
in a vehicle solution, which was composed of 4% sucrose and 1% DMSO. Subsequently, the final
concentration of each compound was diluted to 10 M with 4% sucrose solution. From the second day
≥
98%, Aladdin, Shanghai, China) were dissolved
µ
to eighth day after eclosion, each group of flies were continuously administered 4 hours per day and
then rested in a fresh food environment for 20 h. For the test compound group, flies were fed with
50 µL of compound solution daily, while control group of flies were fed with the same amount of sugar
water. In this experiment, six data points were measured for each group flies, and each final data was
the average of olfactory memory of two groups [36–38].

Molecules 2018, 23, 2663
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3.4.3. Drosophila Olfactory Escape Behavior Test
The task was performed according to a previous reported method with minor modifications [36–38].
Learning and memory tests were conducted at 25 ◦C and 70% relative humidity in the dark. During the
training period, about one hundred flies were loaded into a copper-lined training tube. These flies were
successively exposed to two odors, 3-octanol (OCT) and 4-methylcyclohexanol (MCH) for 60 s. Then the
fresh air was ventilated for 45 s. Flies would receive an electric shock for 1 minute when they smelt the
first odor. To avoid the natural preference of flies for odors, one tube of flies would smell the OCT firstly
and receive an electric shock, while the other tube would smell the MCH firstly and receive an electric
shock at each data point. The experimental average value of two tubes of the flies was considered as a
data point. Then flies were immediately transferred to the choice point of a T-maze and forced to select
between the OCT and MCH containers for two minutes. Consequently, the flies in different containers
were counted and a performance index (PI) was calculated. If the flies had a 50:50 distribution in the
T-maze test, it means the PI = 0 and the flies could not remember the connection of the electric shock and
the odor. Additionally, the PI = 100 means that 100% of flies could remember the connection between the
odor and the electric shock.
4. Statistical Analysis
All data were expressed as mean
test or one-way analysis of variance (ANOVA). p < 0.05 was accepted to indicate the significance.
±
standard deviation (SD), and results were subjected to Tukey’s
Author Contributions: T.C. and X.-B.Z. contributed to the synthesis of gx-50 derivatives
1
–
20. T.C., W.-F.W.,
and Y.Q. contributed to the anti-AD bioactivity evaluation of compounds 1–20. T.C., X.-Y.Z., and J.-L.Y. contributed
to the data analysis and manuscript preparations. J.-L.Y. was the project leader and designed the experiments.
Funding: This work was financially supported by National Natural Science Foundation of China (No. 81673325,
81711540311, and 21705156) and CAS Pioneer Hundred Talents Program. We thank Suzhou Joekai Biotech for
technical support in Drosophila experiments.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–20 are not available from the authors.
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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/).