Preparation of 4-Flexible Amino-2-Arylethenyl-Quinoline Derivatives as Multi-Target Agents for the Treatment of Alzheimer’s Disease

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.


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][4][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][8][9][10][11].

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. 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, 3dimethylaminopopylamine, 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 (6a1-6a4, 6b1-6b3, 6c1-6c2, 6d1-6d2, and 6e1-6e2) were obtained by the reaction of compounds 5a-5e with different substituent aromatic aldehydes in the

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.

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. 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, 3dimethylaminopopylamine, 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 (6a1-6a4, 6b1-6b3, 6c1-6c2, 6d1-6d2, and 6e1-6e2) were obtained by the reaction of compounds 5a-5e with different substituent aromatic aldehydes in the 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 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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. C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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.

Inhibition of
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. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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.

Inhibition of Aβ1-42 Self-Induced Aggregation
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. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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.

Inhibition of Aβ1-42 Self-Induced Aggregation
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. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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. 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 yield. The structures of the target compounds were validated by using 1 H-NMR, 13 C-NMR, and HRMS, and their purities were determined to be above 95% by using HPLC. 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 1 , 6b 2 , and 6a 1 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 Aβ 1-42 self-induced aggregation, the IC 50 values of compound 6b 1 , 6b 2 , and 6a 1 were determined. The result is shown in Table 2, they exhibited better inhibition than resveratrol (IC 50 = 11.8 µM), with the IC 50 values of compound 6b 1 , 6b 2 , and 6a 1 were 4.5 µM, 6.1 µM, and 7.8 µM, respectively. The structure-activity 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.

Inhibition of Aβ1-42 Self-Induced Aggregation
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 Aβ 1-42 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. 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 2 with 4-rigid amino substituents in the 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.

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-2carboxylic 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 1 , 6b 1 , and 6a 2 exhibited the most potent 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 1 and 4b 2 . In 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.

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 Aβ 1-42 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 1 displayed more antioxidant activity than Trolox, and compound 6a 4 and 6b 3 exhibited a little weaker 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.
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.

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-Lcysteine (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 6b1 and 6a1 displayed more antioxidant activity than Trolox, and compound 6a4 and 6b3 exhibited a little weaker 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. Considering that compound 6b1 showed more active than other compounds in inhibiting selfinduced Aβ1-42 aggregation and antioxidant activity, compound 6b1 was selected for further study.

Metal Binding Properties of Compound 6b1
The chelation ability of compound 6b1 toward biometals such as Na + , K + , Cu 2+ , Fe 2+ , Mg 2+ , Ca 2+ , and Zn 2+ was studied by UV-vis spectrometry [47,48]. When Cu 2+ , Fe 2+ , and Zn 2+ 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 6b1 in ethanol alone decreased, indicative of the interaction between Cu 2+ , Fe 2+ , Zn 2+ and compound 6b1 to form the complex 6b1-metal (Ⅱ). But there was little change on the UV spectrum upon the addition of Na + , K + , Mg 2+ , and Ca 2+ (shown in Figure 3A), which indicated Considering that compound 6b 1 showed more active than other compounds in inhibiting self-induced Aβ 1-42 aggregation and antioxidant activity, compound 6b 1 was selected for further study.

Metal Binding Properties of Compound 6b 1
The chelation ability of compound 6b 1 toward biometals such as Na + , K + , Cu 2+ , Fe 2+ , Mg 2+ , Ca 2+ , and Zn 2+ was studied by UV-vis spectrometry [47,48]. When Cu 2+ , Fe 2+ , and Zn 2+ 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 1 in ethanol alone decreased, indicative of the interaction between Cu 2+ , Fe 2+ , Zn 2+ and compound 6b 1 to form the complex 6b 1 -metal (II). But there was little change on the UV spectrum upon the addition of Na + , K + , Mg 2+ , and Ca 2+ (shown in Figure 3A), which indicated 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 1 complexed with Cu 2+ strongly. Its ability to selectively chelate Cu 2+ versus other relevant metal ions was further investigated by UV-vis spectrometry. Solution of compound 6b 1 was prepared and treated with the metal ion (Zn 2+ , Fe 2+ , Ca 2+ , Mg 2+ , Na + and K + ), after 10 min incubation, the optical response was observed, followed by addition of Cu 2+ and consequent analysis of further spectral changes. The result is shown in Figure 3B. It can be seen that compound 6b 1 had good selectivity of metal chelation for Cu 2+ .
strongly. Its ability to selectively chelate Cu 2+ versus other relevant metal ions was further investigated by UV-vis spectrometry. Solution of compound 6b1 was prepared and treated with the metal ion (Zn 2+ , Fe 2+ , Ca 2+ , Mg 2+ , Na + and K + ), after 10 min incubation, the optical response was observed, followed by addition of Cu 2+ and consequent analysis of further spectral changes. The result is shown in Figure 3B. It can be seen that compound 6b1 had good selectivity of metal chelation for Cu 2+ . In order to further determine the stoichiometry of compound 6b1 for Cu 2+ binding, the UV spectra was performed by titrations of compound 6b1 upon stepwise additions of a Cu 2+ solution. According to Figure 4A, the absorbance at 498 nm firstly increased with ascending amount of CuSO4, 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 6b1-Cu 2+ (compound/Cu 2+ ; binding maybe through N atoms of 4-flexible amine group, the chelating motif NH-N is proposed. See Scheme S1).  In order to further determine the stoichiometry of compound 6b 1 for Cu 2+ binding, the UV spectra was performed by titrations of compound 6b 1 upon stepwise additions of a Cu 2+ solution. According to Figure 4A, the absorbance at 498 nm firstly increased with ascending amount of CuSO 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 -Cu 2+ (compound/Cu 2+ ; binding maybe through N atoms of 4-flexible amine group, the chelating motif NH-N is proposed. See Scheme S1).
investigated by UV-vis spectrometry. Solution of compound 6b1 was prepared and treated with the metal ion (Zn 2+ , Fe 2+ , Ca 2+ , Mg 2+ , Na + and K + ), after 10 min incubation, the optical response was observed, followed by addition of Cu 2+ and consequent analysis of further spectral changes. The result is shown in Figure 3B. It can be seen that compound 6b1 had good selectivity of metal chelation for Cu 2+ . In order to further determine the stoichiometry of compound 6b1 for Cu 2+ binding, the UV spectra was performed by titrations of compound 6b1 upon stepwise additions of a Cu 2+ solution. According to Figure 4A, the absorbance at 498 nm firstly increased with ascending amount of CuSO4, 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 6b1-Cu 2+ (compound/Cu 2+ ; binding maybe through N atoms of 4-flexible amine group, the chelating motif NH-N is proposed. See Scheme S1).
To investigate the ability of compound 6b1 to inhibit Cu 2+ -induced Aβ1-42 aggregation, the ThTbinding assay was performed [49]. Resveratrol and clioquinol (CQ) were used as reference compounds. As shown in Figure 5, the fluorescence of Aβ treated with Cu 2+ is 135.2% that of Aβ alone, which indicated that Cu 2+ accelerated Aβ aggregation. But treated with the compounds, the fluorescence of Aβ treated with Cu 2+ decreased differently. Compound 6b1 displayed 85.8% inhibition of Cu 2+ -induced Aβ aggregation, which was equal to CQ (83.6% inhibition of Cu 2+ -induced Aβ aggregation); Resveratrol displayed weaker inhibition (71.2% inhibition of Cu 2+ -induced Aβ aggregation). These results suggested that compound 6b1 could inhibit Cu 2+ -induced Aβ aggregation effectively.

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 6b1, 6b2, and 6a1 with higher self-induced Aβ1-42 aggregation inhibition exhibited lowest cytotoxicity with their IC50 values of 253.7 μM, 228.1 μM and 189.2 μM, respectively.

Effect of Compound 6b1 on Abundance of Aβ1-42 Fibrils
To further complement the ThT binding assay, A transmission electron microscopy (TEM) assay was employed to monitor and clarify the effect of compound 6b1 on Aβ1−42 aggregation [51,52]. As 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 6b1 (Figure 6c). The TEM result

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 IC 50 values above 100 µM, which implied that all the compounds have low cytotoxicity. In addition, compounds 6b 1 , 6b 2 , and 6a 1 with higher self-induced Aβ 1-42 aggregation inhibition exhibited lowest cytotoxicity with their IC 50 values of 253.7 µM, 228.1 µM and 189.2 µM, respectively.

Effect of Compound 6b 1 on Abundance of Aβ 1-42 Fibrils
To further complement the ThT binding assay, A transmission electron microscopy (TEM) assay was employed to monitor and clarify the effect of compound 6b 1 on Aβ 1−42 aggregation [51,52]. As shown in Figure 6, after 24 h incubation at 37 • C, the sample of Aβ 1−42 alone had aggregated into many amyloid fibrils (Figure 6b), while a few thick fibrils were observed for the sample of Aβ 1-42 in the presence of resveratrol (Figure 6d). Compared to resveratrol, only fewer thin fibrils and small bulk aggregates were observed in the sample of Aβ 1−42 in the presence of 6b 1 (Figure 6c). The TEM result was well consistent with the result of ThT, which strongly proved that compound 6b 1 had better inhibition against Aβ 1−42 fibrils formation than resveratrol.
Molecules 2018, 23, x 9 of 20 was well consistent with the result of ThT, which strongly proved that compound 6b1 had better inhibition against Aβ1−42 fibrils formation than resveratrol.

Disaggregation of Self-Induced Aβ1-42 Aggregation Fibrils by 6b1
The ability of compound 6b1 to disaggregate self-induced Aβ1-42 aggregation fibrils was also 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 6b1 disaggregated Aβ1-42 fibrils with ratio of 64.3% at 20 μM concentration, and resveratrol showed weaker activity with ratio of 51.8%, as shown in Figure 7A. Our TEM results exhibited compound 6b1 incubated with Aβ1-42 fibrils could disaggregate more Aβ1-42 fibrils than resveratrol ( Figure 7B), which further supported the result of the ThT binding assay.   The ability of compound 6b 1 to disaggregate self-induced Aβ 1-42 aggregation fibrils was also investigated [52]. Aβ 1-42 fibrils were prepared by incubating fresh Aβ 1-42 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 1 disaggregated Aβ 1-42 fibrils with ratio of 64.3% at 20 µM concentration, and resveratrol showed weaker activity with ratio of 51.8%, as shown in Figure 7A. Our TEM results exhibited compound 6b 1 incubated with Aβ 1-42 fibrils could disaggregate more Aβ 1-42 fibrils than resveratrol ( Figure 7B), which further supported the result of the ThT binding assay.

Disaggregation of Self-Induced Aβ1-42 Aggregation Fibrils by 6b1
The ability of compound 6b1 to disaggregate self-induced Aβ1-42 aggregation fibrils was also 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 6b1 disaggregated Aβ1-42 fibrils with ratio of 64.3% at 20 μM concentration, and resveratrol showed weaker activity with ratio of 51.8%, as shown in Figure 7A. Our TEM results exhibited compound 6b1 incubated with Aβ1-42 fibrils could disaggregate more Aβ1-42 fibrils than resveratrol ( Figure 7B), which further supported the result of the ThT binding assay.

In vitro Protective Effect of Compound 6b 1 against Aβ 1-42 Induced Toxicity in SH-SY5Y Human Neuroblastoma Cells
The cytoprotective effect of compound 6b 1 against Aβ 1-42 damage in SH-SY5Y human neuroblastoma cell lines was determined by MTT assay [53,54]. Our cytotoxicity study had indicated that compound 6b 1 does not affect cell viability at the concentration of 20 µM. However, incubation of Aβ 1-42 (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 1 on Aβ 1-42 induced cell toxicity, SH-SY5Y cells exposed to Aβ 1-42 (20 µM) were incubated with different concentrations of compound 6b 1 (5,10, and 20 µM) for 24 h, and the cell viability was tested. As shown in Figure 8, treatment with compound 6b 1 increased the cell viability by preventing Aβ 1-42 -induced cytotoxicity in a concentration-dependent manner. Furthermore, when the SH-SY5Y cells were co-incubated with compound 6b 1 and Aβ 1-42 for 48 h, the cell viability increased to 89.8%. All these results indicated that compound 6b 1 exhibited a neuroprotective role against Aβ 1-42 -induced cell toxicity. The cytoprotective effect of compound 6b1 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 6b1 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 6b1 on Aβ1-42 induced cell toxicity, SH-SY5Y cells exposed to Aβ1-42 (20 μM) were incubated with different concentrations of compound 6b1 (5, 10, and 20 μM) for 24 h, and the cell viability was tested. As shown in Figure 8, treatment with compound 6b1 increased the cell viability by preventing Aβ1-42induced cytotoxicity in a concentration-dependent manner. Furthermore, when the SH-SY5Y cells were co-incubated with compound 6b1 and Aβ1-42 for 48 h, the cell viability increased to 89.8%. All these results indicated that compound 6b1 exhibited a neuroprotective role against Aβ1-42-induced cell toxicity. Values are expressed as the mean ± SEM (n = 6), # p < 0.05 vs. control; *p < 0.05 and **p < 0.01 vs. Aβ.

Step-Down Type Passive Avoidance Test
In order to determine whether compound 6b1 could improve the memory impairment in 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 6b1 (4.0, 8.0 and 16.0 mg/kg) increased the latency and reduced the number of errors in a dose-dependent manner. For the low dose group of compound 6b1 (4.0 mg/kg), it didn't exhibit significant improvement of the memory impairment 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 6b1 could improve cognitive deficit induced by scopolamine.  (20 µM) in the absence and presence of 6b 1 . The percentage of MTT reduction is relative to control cells in medium. Values are expressed as the mean ± SEM (n = 6), # p < 0.05 vs. control; * p < 0.05 and ** p < 0.01 vs. Aβ.

Step-Down Type Passive Avoidance Test
In order to determine whether compound 6b 1 could improve the memory impairment in 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 1 (4.0, 8.0 and 16.0 mg/kg) increased the latency and reduced the number of errors in a dose-dependent manner. For the low dose group of compound 6b 1 (4.0 mg/kg), it didn't exhibit significant improvement of the memory impairment 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 1 could improve cognitive deficit induced by scopolamine. Figure 9. Effects of compound 6b1 on the (A) latency (s) and (B) number of errors in the step-down 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.

General Information
All commercial reagents and solvents were purchased from commercial suppliers and used without further purification. 1 H-and 13 C-NMR spectra were recorded using TMS as the internal standard in CDCl3 or DMSO-d6 with a Bruker BioSpin GmbH spectrometer (Billerica, MA, USA) at 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.

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].

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 CH2Cl2/petroleum ether (3:1) elution to afford the desired products.

General Information
All commercial reagents and solvents were purchased from commercial suppliers and used without further purification. 1 H-and 13 C-NMR spectra were recorded using TMS as the internal standard in CDCl 3 or DMSO-d 6 with a Bruker BioSpin GmbH spectrometer (Billerica, MA, USA) at 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.

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].

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 2 /petroleum ether (3:1) elution to afford the desired products. For the disaggregation of self-induced Aβ 1-42 fibrils [52]. the Aβ 1-42 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 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.

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: where f 0 is the initial fluorescence at 0 min and f i is the fluorescence at time i. The net AUC for a sample was calculated using the expression net AUC = AUC sample − AUC blank , The ORAC-FL value of each sample was calculated using the equation of net AUC sample /(AUC Trolox -AUC blank ), which was expressed as Trolox equivalents.

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 2 and were passaged twice weekly.
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 × 10 4 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 2 for 30 min. Then DCFH-DA was removed, the cells were washed three times and incubated 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).

Metal Chelation
The metal chelation of compound 6b 1 was tested in ethanol using a UV-vis spectrophotometer (Shimadzu UV-2450PC) with wavelength ranging from 200 to 700 nm [47,48]. The absorption spectra of compound 6b 1 (10 µM, final concentration) alone or in the presence of metal ions (20 µM, final 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 CuSO 4 , ZnSO 4 , FeSO 4 , 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 resveratrol in ethanol were freshly prepared prior to use. 10 µL solutions of the compounds treated with 10 µL of ZnSO 4 , FeSO 4 , CaCl 2 , MgCl 2 , 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 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.

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 × 10 4 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.
For the inhibition of self-induced Aβ 1-42 aggregation experiment [51,52]. it was incubated in the presence and absence of compound 6b 1 and resveratrol at 37 • C for 24 h; For the disaggregation of self-induced Aβ 1-42 fibril experiment [52]. Aβ 1-42 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 Aβ 1-42 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).

3.3.9.
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 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.

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 Aβ 1-42 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 Aβ 1-42 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 Cu 2+ -induced Aβ aggregation, good disaggregation of Aβ 1-42 fibrils generated by self-induced Aβ 1-42 aggregation. Moreover, it exhibited low toxicity to SH-SY5Y cells and a significant effect on the protection of neuronal cells against Aβ 1-42 -induced cytotoxicity in SH-SY5Y cells. Most importantly, compound 6b 1 could significantly 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 1 as a potential lead compound for new multitarget drug development in the treatment of AD.
Supplementary Materials: Supplementary data ( 1 H-NMR, 13 C-NMR, HRMS and HPLC spectra) associated with this article are available online.