Design, synthesis and evaluation of novel ferulic acid derivatives as

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Design, synthesis and evaluation of novel ferulic acid derivatives as multi-target-directed ligands for the treatment of Alzheimer’s disease Zhipei Sang, Keren Wang, Han Xue, Mengxiao Cao, Zhenghuai Tan, and Wenmin Liu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00530 • Publication Date (Web): 12 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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Design, synthesis and evaluation of novel ferulic acid derivatives as multi-target-directed ligands for the treatment of Alzheimer’s disease Zhipei Sanga,#*, Keren Wanga,#, Han Xuea, Mengxiao Caoa, Zhenghuai Tanb,*, Wenmin Liua,*. a College

of Chemistry and Pharmaceutical Engineering, Nanyang Normal University,

Nanyang, 473061, China b

Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan

Academy of Chinese Medicine Sciences, Chengdu, 610041, China

KEYWORDS. Alzheimer’s disease; Ferulic acid deriveatives; design, synthesis; multi-targets

agents;

zebrafish

AD

model;

scopalamine-induced

impairment.

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ABSTRACT: A novel series of ferulic acid derivatives were designed and synthesized based on multitarget-directed ligands (MTDLs) strategy for the treatment of Alzheimer’s disease (AD). In vitro results revealed that all the target compounds were highly effective and selective BuChE inhibitors, in particular, compound TM-10 showed the best BuChE inhibitory activity with IC50 value of 8.9 nM, remarkable MAO-A and MAO-B inhibitory potency with IC50 value of 6.3μM and 8.6μM, respectively. TM-10 could inhibit (53.9%) and disaggregate (43.8%) self-induced Aβ aggregation. In addition, compound TM-10 exhibited potent antioxidant activity (ORAC = 0.52 eq) and neuroprotective effect against Aβ1-42-mediated SH-SY5Y neurotoxicity, as well as acted as an autophagic activator. TM-10 also showed good blood-brain barrier (BBB) penetration. Furthermore, compound TM-10 exhibited favorable dyskinesia recovery rate and response efficiency on AlCl3-induced zebrafish AD model, and potent neuroprotective effect on Aβ1-40-induced zebrafish vascular inJury. Further, in vivo assay demonstrated that compound TM-10 showed low acute toxicity, and the step-down passive avoidance test indicated that this compound could improve scopolamine-induced memory deficit in mice. Therefore, the present study evidently displayed that compound TM-10 is a potent multi-functional agent against Alzheimer’s disease and could act as promising lead candidate for anti-Alzheimer drug development.

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INTRODUCTION Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive memory loss and other cognitive impairments. To date, there are over 47 million people with dementia worldwide up to now and the total estimated worldwide cost of dementia is US$818 billion, unfortunately, the number is estimated to increase to 131.5 million by 2050.1 The pathologic changes of AD are complex and elusive, however, various clinical hallmarks such as amyloid-β (Aβ) deposits, tau (τ) protein aggregation, low levels of acetylcholine (ACh) and oxidative stress are considered to play imperative roles in AD pathogenesis.2,3 AD patients have been treated with four acetylcholinesterase inhibitors (AChEIs), such as donepezil, tacrine, rivastigmine and galantamine, but unfortunately these drugs can improve memory and cognitive function to a certain extent but cannot prevent, halt or reverse the progression of the disease.4 Recent studies displayed that the ratio of butyrylcholinesterase (BuChE)/ AChE gradually increased from 0.2 up to as much as 11.0 in certain parts of the brain as the disease progressed. Moreover, it is specific that BuChE inhibition could circumvent the classical cholinergic toxicity that is a common side effect of AChE inhibition.5,6 Therefore, selective BuChE inhibitors may be of value in the treatment of the late stage of AD. Monoamine oxidase (MAO) is the enzyme responsible for the oxidative deamination of various biogenic and xenobiotic amines. MAO exists as two isoforms, MAO-A and MAO-B. Many evidences have proved that selective BuChE inhibitors such as rasagiline and its derivatives have shown the potency to improve learning and memory deficits in AD animal models and to slow down the progression of AD in patients.7 Selective MAO-A inhibitors have been shown to be effective anti-depressants. Furthermore, severe AD patients commonly present depressive symptoms that have even considered as a risk factor for the development of the disease.8,9 Therefore, the observed clinical symptoms revealed that dual inhibition of MAO-A and MAO-B, rather than MAO-B alone, may be of value for severe AD therapy. According to the amyloid hypothesis, the production and collection of oligomeric

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aggregates of Aβ in the brain likely trigger the pathogenic cascade, eventually resulting in neuronal loss and dementia.10 In addition, oxidative damage is emerge within the brain of the patients and affect every type of biological macromolecules such as proteins, nucleic acid, carbohydrates and lipids, and it could promote Aβ toxicity through the production of free radicals.11 On the other side, Aβ could enter into the mitochondria where it could efficiently produce reactive oxygen species (ROS) and cause oxidative stress.12 Thus, Aβ aggregation inhibition and antioxidant protection could play important roles in AD therapy. Moreover, autophagy is a vesicle and lysosome-mediated degradative pathway that is essential for protein homeostasis and cell health. Particularly, neurons are dependent on high basal autophagy for survival compared to non-neuronal cells. The given evidences have reported that autophagy deficits may be maJor contributors to the etiology of Alzheimer’s disease.13, 14 So, autophagy activator could be conducive to the treatment of AD. The

above

observations

have

prompted

the

search

of

Multi-Target-Directed-Ligands (MTDLs), based on the “one molecule, multiple target” paradigm.15-17 Ferulic acid (4-hydroxy-3-methoxy cinnamic acid, FA) is a widely distributed constituent of plants, and was first isolated form Ferula foetida. It is an antioxidant naturally present in plant cell walls with anti-inflammatory activities and it is able to act as a free radical scavenger, as well as act as inhibitor or disaggregating agent of amyloid structures. 18, 19 The high beneficial and low adverse effects of FA make it could be as a promising preventive or therapeutic intervention for AD patients, however, low bioavailability and poor blood–brain transport of FA restricts its clinical uses as an anti-AD drug. 19 Therefore, the development of novel ferulic acid derivatives based on MTDLs is encouraged for its clinical use anti-AD. In our previous work, compound EJMC-7f was reported as a promising multi-function agent, showing significant selective BuChE inhibitory activity with IC50 value of 0.021 μM, and moderate Aβ aggregation inhibition and antioxidant activity.20 In addition, we found that compound LDDD-3 was a high selective BuChE inhibitor with IC50 value of 3.4 μM (AChE inhibition rate, 5.6%).21 So, in order to

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further improve the BuChE inhibitory activity of EJMC-7f to develop multi-function agents against Alzheimer’s disease, we fuse lead compounds EJMC-7f and LDDD-3 to obtain novel ferulic acid derivatives. In this paper, a series of novel ferulic acid derivatives were designed, synthesized in Figure 1, and evaluated for their biological activity, including ChE inhibitory activity, MAOs inhibitory potency, antioxidant activity, inhibition and disaggregation effect on Aβ aggregation, MAOs inhibition, neuroprotective effect against Aβ1-42-mediated SH-SY5Y neurotoxicity, autophagy, and the ability of cross blood−brain barrier in vitro. Moreover, molecular modeling studies were also carried out to offer insight into the binding mode of the novel ferulic acid derivatives. Finally, the hit compound was further evaluated on zebrafish AD model and scopolamine-induced cognitive impairment in vivo. Inhibit

Antioxidant, Aβ aggregation

O MeO HO

OH Ferluic acid O

O MeO N

MeO

N

O

N

HO LDDD-3

EJMC-7f BuChE IC50 = 0.021μM AChE IC50 = 2.13μM

BuChE IC50 = 3.4 μM AChE 5.6% at 25 μM

MTDLs NR1R2 =

O O R3

N nO R4

N R2

R1

N

N

Ferulic acid derivatives

Figure 1. The design strategy for novel ferulic acid derivatives

RESULTS AND DISCUSSION Chemistry. The synthesis of ferulic acid derivatives were depicted in Scheme 1. The starting material ferulic acid 1 was treated with 4-benzylpiperidine 2a or 1,2,3,4-tetrahydroisoquinoline 2b by the condensation in the presence of EDCI and HOBt, to get the intermeditates 3, and then reacted with 1,3-dibromopropane, 1,4-dibromobutane, 1,5 –dibromopentane or 1,6-dibromohexane to obtain the key intermediates 5. Finally, the target compounds TM-1~TM-24 were obtained by the reaction of compounds 5 with secondary amines 6 in the presence of anhydrous

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K2CO3 in CH3CN at 65°C. These target compounds were purified by chromatography, and the analytical and spectroscopic data confirmed their structures, as detailed in the experimental. O MeO

O

O OH

MeO

(i)

+ NR1R2

HO

N R2

HO 1

2

R1 (ii)

MeO Br

N R2

nO

3

R1

5

O 5

+

NR3R4

MeO

(iii) R

N R2

3

N nO R4

R1

R 1R 2N =

NH

NH 2a

2b

TM-1~TM-24

Scheme 1. Synthesis of ferulic acid derivatives (TM-1~TM-24). Reagents and conditions: (i) THF, EDCI, HOBT, R1R2NH (2a~2b) room temperature, overnight. (ii) Br(CH2)nBr (4a~4d), CH3CN, reflux for 6-10 h. (iii) R3R4NH, K2CO3, CH3CN, 65°C, for 6-10 h.

Inhibition Effects on AChE and BuChE. The inhibitory activities of eeAChE (from electric eel) and eqBuChE (from equine serum) toward 24 novel ferulic acid derivatives were evaluated using the modified Ellman’s method.22,23 Donepezil, well-known cholinesterase inhibitor approved by FDA, EJMC-7f, and LDDD-3c were used as referenced compounds. The AChE and BuChE inhibitory activities of the synthesized target compounds are listed in Table 1, expressed as IC50 values. As shown in Table 1, all the target compounds TM-1~TM-24 were fascinating selective BuChE inhibitors, in particular, compound TM-10 showed the best BuChE inhibitory activity with IC50 value of 8.9 nM and SI value was 5854, which was better than that of EJMC-7f. According to the screening data, compound EJMC-7f was a significant selective BuChE inhibitor in our reported work, compound TM-1 was obtained by ring opening 1,2,3,4-tetrahydroisoquinoline of EJMC-7f, but BuChE inhibitory activity sharply decreased to 0.21 μM, and then replacing the ethyl of TM-1 with propargyl to get compound TM-2, the BuChE inhibitory potency dropped to 5.3 μM. The results displayed that propargyl group could not contribute to the BuChE inhibitory activity. In addition, we found that 1,2,3,4-tetrahydroisoquinoline group could improve BuChE inhibitory activity based on our reported papers, so, compounds TM-3~TM-24 containing 1,2,3,4-tetrahydroisoquinoline fragment were designed and synthesized. The BuChE inhibitory potencies were remarkably influenced by methylene chain length (n) and the structure of terminal groups NR1R2

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of side chain. When the terminal groups NR1R2 was 4-benzylpiperdine, the optimal n = 3, and the BuChE inhibitory activity gradually decreased (TM-3 < TM-7 < TM-21< TM-23) with the methylene chain increased. When the terminal groups NR1R2 was 1,2,3,4-tetrahydroisoquinoline and n = 3, compound TM-3 exhibited good BuChE inhibitory activity (IC

= 0.27 μM), removal of benzyl of TM-3 to get

50

compound TM-4, the inhibitory potency had a slightly decrease with IC50 value of 0.32 μM, and the compound TM-5 was obtained by ring opening piperdine of TM-4, the BuChE inhibitory activity remarkable declined (IC50 = 2.2 μM), compound TM-6 showed moderate BuChE inhibitory activity (IC50 = 4.3 μM). The structure activity relationship (SAR) was researched with the optimal methylene chain length (n = 4). Compound TM-7 with 4-benzylpiperdine was a significant BuChE inhibitor with IC50 value of 4.1 μM, removing methylene of 4-benzylpiperdine to get compound TM-8, the BuChE inhibitory activity increased to 1.7 μM. And compound TM-9 with 1,2,3,4-tetrahydroisoquinoline

group

was

obtained

through

modifying

phenylpiperidine of TM-8, TM-9 showed a satisfactory BuChE inhibitory activity with IC50 value of 0.053 μM. And then compound TM-10 was acquired by ring opening of 1,2,3,4-tetrahydroisoquinoline of TM-9, TM-10 indicated surprising BuChE inhibitory activity with IC50 value of 8.9 nM. Moreover, replacing the ethyl group with propargyl to gain compound TM-11, the BuChE inhibitory activity sharply reduced to 0.78 μM, which was consistent with TM-2. Changing ethyl group of TM-10 with isopropyl group to acquire compound TM-12, it also exhibited a remarkable decrease with IC50 value of 0.076 μM. In addition, adding methoxy group at 2 position of N-ethylbenzylamine of TM-10 to get compound TM-13, the BuChE inhibitory activity showed a noteworthy reduce with IC50 value of 0.023 μM, and the similar phenomenon was observed on compound TM-14 (IC50 = 2.8 μM). Meanwhile, replacing

1,2,3,4-tetrahydroisoquinoline

of

TM-9

with

1-methyl-1,2,3,4-tetrahydroisoquinoline we got compound TM-15 (IC 50 = 0.62 μM), and also compound TM-16 (IC 1,2,3,4-tetrahydroisoquinoline

50

= 2.1 μM) was obtained through changing of

TM-9

with

6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, both TM-15 and TM-16 showed lower

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BuChE inhibitory activity than that of TM-9. Moreover, replacing benzene ring of TM-8 with cyclohexane to obtain compound TM-17, TM-17 showed a slightly lower BuChE inhibitory activity with IC50 value of 2.9 μM, while removing the cyclohexane of TM-17 to get compound TM-19, TM-19 displayed better inhibitory activity than that of TM-17. And then compound TM-20 was got by ring opening of piperdine of TM-19, the BuChE inhibitory activity mildly declined with IC50 value of 0.41 μM. On the other hand, compound TM-18 (IC

50

= 5.8 μM) with ethyl piperazine group

showed lower BuChE inhibitory activity than that of TM-19. Therefore, according to the above results, compound TM-10 with N-ethylbenzylamine and methylene chain n was 4 showed the best BuChE inhibitory activity, and deserved to perform the further investigation. Table 1 eeAChE and eqBuChE inhibitory activity and oxygen radical absorbance capacity (ORAC, Trolox equivalents) of target compounds and referenced compounds. Compound. NR1R2 EJMC-7f LDDD-3 TM-1 TM-2 TM-3 TM-4 TM-5 TM-6 TM-7 TM-8 TM-9 TM-10 TM-11 TM-12 TM-13 TM-14 TM-15 TM-16 TM-17 TM-18 TM-19 TM-20 TM-21

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

n

4 4 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5

NR3R4

H N H N

NH

NH NH MeO H N

NH

NH

NH

H N H N H N

MeO H N MeO H N NH

MeO

NH

MeO

HN

N

HN N

NH

NH

NH

IC50 ± SD (μM)a EeAChEb

EqBuChEc

5.89±0.03 5.8%f

0.038±0.001 3.6±0.01

23.10.87 28.60.76 10.10.22 22.70.05 27.60.05 18.20.56 14.30.36 21.10.21 8.30.26 12.10.81 35.30.89 28.10.65 13.50.56 43.50.97 7.50.39 11.40.56 23.60.05 28.90.05 6.50.05 4.50.05 19.90.52

0.210.01 5.30.08 0.270.02 0.320.01 2.20.05 4.30.02 4.10.02 1.70.03 0.0530.01 0.00890.0001 0.780.08 0.0760.001 0.0230.02 2.80.06 0.620.01 2.10.02 2.90.03 5.80.05 0.330.02 0.410.01 2.60.01

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SId

ORACe

155 --110 5.4 37.4 70.9 12.5 4.2 3.5 12.4 157 1360 45.3 370 587 15.5 12.1 5.4 8.1 5.0 19.7 11.0 7.7

0.52±0.01 1.1±0.01 0.50±0.02 0.46±0.01 0.53±0.02 0.50±0.02 0.51±0.01 0.47±0.02 0.53±0.01 0.52±0.02 0.52±0.03 0.52±0.02 0.49±0.01 0.54±0.03 0.51±0.02 0.50±0.01 0.53±0.03 0.51±0.02 0.50±0.01 0.55±0.03 0.50±0.01 0.51±0.02 0.53±0.01

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TM-22 TM-23 TM-24 Donepezil

NH

NH

NH

5 6 6

NH

NH

NH

16.40.22 29.30.85 13.10.19 0.019±0.0003

0.270.02 1.80.02 0.370.03 4.76±0.02

60.7 16.3 35.4 0.004

0.51±0.02 0.52±0.01 0.50±0.02 NTg

a

IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of three independent experiments, each performed in triplicate (SD = standard deviation ). b From electrophorus electricusc. c EqBuChE from equine serum. d SI = selectivity index = IC50 (AChE)/IC50 (BuChE). e The mean ± SD of the 3 independent experiments. Data are expressed as μM of Trolox equivalent/μM of tested compound. f Inhibition rate was determined at 25 μmol/L inhibitor concentration. gNT = not tested.

Molecular modeling studies of TM-10. In order to explore possible binding mechanism to AChE and BuChE, a further computational study was performed for compound TM-10 using the docking program, AutoDock 4.2 package with Discovery Studio 2.5, based on the X-ray crystal structure of huBuChE (PDB code: 4tpk) and TcAChE (PDB code: 1EVE).20 The results of docking showed that compound TM-10 bond with BuChE via multiple sites, in the huBuChE−TM-10 complex (Figure 2), 1,2,3,4-tetrahydroisoquinoline via two π-π interactions and a cation-π interaction with key

residues

TRP430

and

TRP82,

respectively.

It

revealed

that

1,2,3,4-tetrahydroisoquinoline fragment played an important role in BuChE inhibitory activity, which was consistent with the reported reference. Moreover, hydrophobic interactions could be observed between the ligand and HIS438, ALA328, PHE329, GLY117, VAL288 and TRP231, which offered the reasonable explanation for its high BuChE inhibitory activity. On the other hand, in the TcAChE−TM-10 complex (Figure 3), compound TM-10 could simultaneously bind to both the catalytic site and the peripheral site of AChE. The benzene ring of 1,2,3,4-tetrahydroisoquinoline via π-π interaction with key residue TRP279, the benzene ring of ferulic acid via π-π interaction with key residue PHE330. Moreover, hydrophobic interactions could be observed between the TM-10 and TYR121, TYR334, TRP84, PHE331, GLU199 and SER200. This phenomenon displayed that compound TM-10 could inhibit AChE inhibitory potency. Therefore, the above observed docking results definitely explained highly effect and selective BuChE inhibitory activity of compound TM-10.

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A

B

C

Figure 2 (A) Representation of compound TM-10 (green stick) interacting with residues in the binding site of huBuChE, highlighting the protein residues that participate in the main interactions with the inhibitor. (B) 3D docking model of compound TM-10 (green stick) with huBuChE. (C) 2D schematic diagram of docking model of compound TM-10 with huBuChE.

A

B

C

Figure 3 (A) Representation of compound TM-10 (green stick) interacting with residues in the binding site of TcAChE (PDB code: 1EVE), highlighting the protein residues that participate in the main interactions with the inhibitor. (B) 3D docking model of compound TM-10 (green stick) with TcAChE. (C) 2D schematic diagram of docking model of compound TM-10 with TcAChE.

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Antioxidant Activity. The antioxidant activities of novel ferulic acid derivatives were evaluated by the ORAC-FL method (Oxygen Radicals Absorbance Capacity by Fluorescence) using vitamin E analogue Trolox as a standard.24, 25 The results were listed in Table 1, and all the target compounds exhibited moderate ORAC-FL values of 0.46−0.55 Trolox equivalents. According to the screening data, the side chain substituents moiety NR1R2, NR3R4 and methylene chain length n showed no obvious effect on the antioxidant activity. The representative compound TM-10 indicated good antioxidant activity with ORAC value was 0.52 eq. Inhibition of MAOs in Vitro. To further study the multi-potent biological profiles of the target compounds, the inhibitory activity against MAO-A and MAO-B (recombinant human enzyme) for representative compounds were carried out.26, 27 The tested results were listed in Table 2, the test compounds were potent selective huMAO-A inhibitors, expect compounds TM-7, TM-9, TM-10 and TM-24 were dual huMAO-A/huMAO-B inhibitors. Among of these compounds, compound TM-13 showed the best huMAO-A inhibitory activity with IC50 value of 0.63 μM and SI value

was

17.1.

While,

compound

TM-10

exhibited

the

best

dual

huMAO-A/huMAO-B inhibitor with IC50 values of 6.3 μM and 8.6 μM, respectively, which contributed to treatment of AD. Table 2 Inhibitory activity of MAO isoforms (MAO-A and MAO-B) and inhibition of Aβ1-42 aggregation and disaggregation of Aβ1-42 aggregation of the selected target compounds and positive comopounds. Compound TM-3 TM-7 TM-9 TM-10 TM-13 TM-23 TM-24 Clorgyline Rasagiline Donepezil EJMC-7f LDDD-3

IC50 (μM) ± SD a MAO-Ab

MAO-Bc

0.870.06 7.80.04 12.30.01 6.30.08 0.630.002 6.60.05 7.80.02 20.8 ± 0.27 0.0281±0.0068 NTi NTi NTi

12.60.29 8.60.07 15.60.02 8.60.02 10.80.21 12.10.21 10.80.36 0.0027±0.0001 0.587 ± 0.038 NTi NTi NTi

SId 14.5 1.1 1.3 1.4 17.1 1.8 1.4 0.0001 20.9 — — —

Self-induced Aβ1-42 aggregation (%)e Inhibitionf NTi NTi NTi 53.9±0.22 NTi NTi NTi NTi NTi n.a.h 51.7±0.31 62.3±0.22

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Disaggregationg NTi NTi NTi 43.8±0.31 NTi NTi NTi NTi NTi NTi 40.4±0.13 52.8±0.25

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Curcumin

NTi

NTi



56.8±0.03

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57.2±1.8

aAll

IC50 values shown in this table are the mean ± SEM from three experiments. bFrom recombinant human MAO-A. cFrom recombinant human MAO-B. d hMAO-A selectivity index = IC50 (MAO-B)/IC50 (MAO-A). eThe thioflavin-T fluorescence method was used, data are the mean ± SEM of three independent experiments. fInhibition of self-induced Aβ1-42 aggregation, the concentration of tested compounds and Aβ1-42 were 25μM. gDisaggregating of self-induced Aβ1-42 aggregation, the concentration of tested compounds and Aβ1-42 were 25μM. hn.a. = no active. Compounds defined “no active” means percent inhibition less than 5.0% at a concentration of 25 μM. iNT = not tested.

Inhibition of the Self-mediated Aβ1-42 Aggregation. Several lines of evidence indicated that high levels of BuChE are associated with the extracellular deposition of the Aβ1-42.28 The ability of representative compound TM-10 to inhibit self-induced Aβ1-42 aggregation was assessed using Thioflavin T (ThT) fluorescence method,29,30 curcumin was also used as a reference compound. As shown in Table 2, compound TM-10 showed good inhibitory potency with 53.9%. To complement the ThT binding assay, Aβ1-42 aggregation was also probed by transmission electron microscopy (TEM) (Figure 4).18 The screening phenomenon represented that Aβ1-42 had aggregated into amyloid fibrils after 24 h incubation, while only small bulk aggregates were visible and no characteristic fibrils were observed when adding TM-10 into the sample of Aβ1-42. The TEM results verified the results of ThT assess that compound TM-10 could inhibit Aβ1-42 fibrils formation.

Aβ (0h) Aβ alone (24h) Aβ + TM-10 (24h) Figure 4. TEM images analysis ofAβ1-42 aggregation in the presence and absence of 25 μM compound TM-10 after 24h of aggregation. (a) fresh Aβ1-42 (25 μM), 0 h. (b) Aβ1-42 (25 μM) alone were incubated at 37°C for 24 h. (c) Aβ1-42 (25 μM) and TM-10 were incubated at 37°C for 24 h.

Molecular Docking of Compound TM-10 with Aβ. To further explore the binding modes of the active compound TM-10 with Aβ, a molecular docking experiment was performed.21 The structure of Aβ was downloaded from the Protein Data Bank (PDB: 1BA4). As shown in Figure 5, the benzene ring of

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1,2,3,4-tetrahydroisoquinoline via π-π interaction with key residue PHE19, hydrophobic interactions were also observed between the ligand and PHE20, GLU22, LYS16, ASN27, LYS28, VAL24, ASP23, which might be favorable for the binding of Aβ and compound TM-10. A

C

B

D

Figure 5 Docking studies of compound-Aβ complex. (A) The binding pattern of compound TM-10 with Aβ1-42 (PDB ID: 1BA4). (B) 3D docking model of compound TM-10 (green stick) with Aβ. (C) Representation of compound TM-10 (green stick) interacting with residues in the binding site of Aβ, highlighting the protein residues that participate in the main interactions with the inhibitor. (D) 2D schematic diagram of docking model of compound TM-10 with Aβ.

Disaggregation of self-induced Aβ1-42 fibrils by TM-10. The ability of TM-10 to disaggregate self-induced Aβ1-42 aggregation was also investigated using ThT assay and TEM assay.20 According to the screening data in Table 2, TM-10 disaggregate Aβ1-42 fibrils at 25μM with 43.8% disaggregation rate compared with curcumin (57.2 ± 1.8%). The TEM images in Figure 6 supported the results of the ThT binding assay as well. Thus, compound TM-10 could inhibit self-induced Aβ aggregation and disassemble the well-structured Aβ fibrils.

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Disaggregation experiment Aβ (fresh)

24h, 37℃

Aβ Fibrils + TM-10

24h, 37℃

Aβ Species

Aβ (0h) Aβ alone (48h) Aβ + TM-10 (48h) Figure 6 Visualization of Aβ species from disaggregation experiments: (top) scheme of the disaggregation experiments; (bottom) TEM images of samples

Neuroprotective Effect on Aβ-42-Mediated SH-SY5Y Neurotoxicity. The neuroprotective effect against Aβ-42-mediated SH-SY5Y neurotoxicity of compound TM-10 was tested using MTT assay.31, 32 As shown in Figure 7A, compound TM-10 did not show any cytotoxicity until the concentration increased to 50 μM. It implied that compound TM-10 had a wide therapeutic safety. According to Figure 7B, 25 μM Aβ-42 exposure to SH-SY5Y cell for 48h, cell viability obviously decreased to 50.3% (p < 0.01) compared with untreated control, treatment with compound TM-10 (1 and 10 μM), the cell viability was 65.3% and 75.1% respectively. It displayed that the compound TM-10 exhibited potent neuroprotective effect against Aβ-42-mediated A SH-SY5Y neurotoxicity. A

B B

Figure 7. (A) Cytotoxicity of TM-10. (B) Aβ1-42-induced SH-SY5Y cell inJury by compound TM-10 using MTT assay. ##p < 0.01 vs.control; **p < 0.01, *p < 0.05 vs Aβ1-42 group.

TM-10 induced autophagic GFP-LC3 puncta in U87 cell. To confirm whether TM-10 is capable of inducing autophagy, we utilized U87 cells for detecting the autophagic GFP-LC3 puncta.32,33 Firstly, U87 cell were adopted in the cytotoxicity assay, TM-10 demonstrated low cytotoxic effect with IC50 value of 17.76 μM. And

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then, as shown in Figure 8, 2 μM and 4μM of TM-10 induced GFP-LC3 puncta formation in U87 cell, in addition, quantitation of the percentages of cells with autophagic puncta formation showed that U87 cell possessed significant potency for autophagy induction in response to TM-10 treatment. A

Ctrl

TM-10 2μM

TM-10 4μM

B

Figure 8. (A) U87 cells transfected with GFP-LC3 plasmids were incubated with TM-10 (2 and 4 µM) with or without the presence of 3-methyladenine (3-MA, 5 mM) for 12 h. Representative images demonstrated that the number of LC3-positive cells was smaller after 3-MA treatment. (B) Quantitation on the percentage of cells with GFP-LC3 puncta formation.

In Vitro Blood−Brain Barrier Permeation Assay. The parallel artificial membrane permeation assay of the blood−brain barrier (PAMPA-BBB) was performed to evaluate the possible BBB permeability of TM-10.34, 35 11 commercial drugs with reported values was chosen to validate the assay (Table 3), A plot of the experimental data versus the reported values produced a good linear correlation, Pe(exp) = 0.9163Pe(bibl.) − 0.2247 (R2 = 0.9558) (Figure 9). From this equation and considering the limit established by Di et al. for blood−brain barrier permeation, we determined that compounds with permeability above 3.44 × 10−6 cm/s could cross the blood−brain barrier (Table 4). According to the tested permeability (Table 5), TM-10 could cross the BBB in vitro and reach their biological targets located in the CNS. Therefore, compound TM-10 was chosen for further study. Table 3. Permeability (Pe×10-6 cm/s) in the PAMPA-BBB assay for 11 commercial drugs used in the experiment validation. Commercial drugs

Bibl a

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PBS:EtOH(70:30) b

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verapamil oxazepam diazepam clonidine Imipramine testosterone caffeine enoxacine piroxicam norfloxacin theophylline

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16 10 16 5.3 13 17 1.3 0.9 2.5 0.1 0.12

16.9 9.6 11.86 5.1 10.1 16.3 1.28 0.471 0.718 0.423 0.1

a

Taken from ref.32 b Data are the mean ± SD of three independent experiments

Figure 12. Lineal correlation between experimental and reported permeability of commercial drugs using the PAMPA-BBB assay. Pe(exp) = 0.9163Pe(bibl.) −0.2247 (R2 = 0.9558).

Table 4. Ranges of permeability of PAMPA-BBB assays (Pe×10-6 cm/s)

Compounds of high BBB permeation (CNS+)

Pe > 3.44

Compounds of uncertain BBB permeation (CNS+/-) 3.44 > Pe > 1.61 Compounds of low BBB permeation (CNS-)

Pe < 1.61

Table 5. Permeability Pe (×10−6 cm/s) in the PAMPA-BBB assay of the selected compound TM-10 and their predictive penetration in the CNS.

compounda TM-10

Pe (×10−6 cm/s)b

prediction

16.62 ± 0.28

CNS+

a

Compound TM-10 was dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final concentration of compounds was 100 μg/mL. b Values are expressed as the mean ± SD of three independent experiments.

Efficiencies of TM-10 on AlCl3-induced zebrafish AD model

36,37.

In recent

years, zebrafish models for rapid screening of anti-AD potential therapeutic agents have attracted great attention and are widely used. (1) MNLC and LC10 of compound TM-10. In order to investigate the safety profile

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of compound TM-10, the acute toxicity was tested in wild-type zerbrafish, six concentrations 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL for TM-10 were used to treat zebrafish (n = 30 per group) at 2dpf, and the untreated group and vehicle group (1% DMSO) as control group. The zebrafish were treated, counted and observed for 3 days until 5dpf. The observed phenomenon were minutely recorded in Table S1, yolk sac generation was observed at dose of 3.125 µg/mL TM-10, and the zerbrafish were all dead at given concentration 6.25 µg/mL or above. So, based on the data of concentration-death rate, the MNLC and LC10 of compound TM-10 were 3.125 and 4.0 µg/mL, respectively. (2) The phenotypes of zebrafish acute toxicity. Based on the above results of MNLC, various concentration TM-10 groups (0.35, 1.04, 3.125 and 4.0 µg/mL), untreated group and vehicle group (1% DMSO) were used to treat wild-type zerbrafish at 2dpf (n = 30 per group), and the experiment phenomenon were observed for 3 days until 5 dpf. As shown in Table S2, there were no zebrafish dead at untreated group, vehicle group, and TM-10 groups (0.35, 1.04, 3.125 and 4.0 µg/mL). Moreover, the zebrafish did not show any toxicity between the vehicle group (1% DMSO) and TM-10 group (0.35 µg/ mL) (Figure 10). However, treatment with 1.04 µg/ mL TM-10 presented 30% liver yolk sac absorption delay, 3.125 µg/mL TM-10 induced 80% liver yolk sac absorption delay and 10% heart pericardial edema, 4.0 µg/mL TM-10 emerged 93.3% liver yolk sac absorption delay and 30% heart pericardial edema, 33.3% circulation absent blood flow and 13.3% kidney edema (Figure 11).

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Figure 10. The representative phenotypes of zebrafish acute toxicity

Figure 11. Representative phenotypes of zebrafish acute toxicity, including kidney toxicity

(3) Assessing compound TM-10 in the Zebrafish AD Model. In order to further evaluate compound TM-10 in the zebrafish AD, we performed experiments to study whether

TM-10

could

improve

AlCl3-induced

dyskinesia and

reaction ../../Lenovo/AppData/Local/youdao/dict/Application/7.5.2.0/resultui/dict/?keyword=ca pacity of zebrafish AD. Firstly, The 3dpf zebrafish chosen randomly were placed in a 6-well microplate at a density of 30 zebrafish per well and treated with AlCl3, and then treated with a testing compounds TM-10 at 8 various concentrations (1 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL), respectively, and also including untreated group and model group. The survivals were counted for 2 days, and the data was displayed in Table S3, 1 μg/mL was determined as the MTC of both compound TM-10.

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Based on the MTC, three various concentrations (0.11 μg/mL, 0.33 μg/mL and 1

μg/mL) were selected for compounds TM-10 groups, respectively, meanwhile, the donepezil group (8.0 μM), untreated group and model group as the positive groups. As shown in Figure 12A, the distance of AlCl3-induced zebrafish AD (model group, 5513 mm) was significantly shorter than that of untreated zebrafish (untreated group, 7304 mm, p < 0.01). It indicated that the AlCl3-induced zebrafish AD model works well. When treatment with 8.0 μM donepezil, the distance increased to 6566 mm (DPZ group, p < 0.05) and the efficacy was 59% compared with model group, implying donepezil could improve exercise capacity. While, treatment with compound TM-10 (0.11 μg/mL, 0.33 μg/mL and 1 μg/mL) the distance gradually increased (6761 mm, 6954 mm and 6519 mm, respectively, p < 0.01, p < 0.05 & p > 0.05), and the efficacy were 70%, 80% and 56% (Figure 12B), respectively. In particular, TM-10 groups (0.11 μg/ mL and 0.33 μg/ mL) indicated the longer distance and more efficacy than that of donepezil group. In addition, the high-dose TM-10 group (1 μg/mL) showed the shortest distance in TM-10 groups, the possible reason was that the hepatotoxicity effect of TM-10. So, according to the results, compound TM-10 could improve exercise capacity of AlCl3-induced zebrafish AD model. A

B

Figure 12. Effect of TM-10 on motility distance in AD zebrafish. Compared with model,*p < 0.05,** p < 0.01.

According to Figure 13, speed change after light stimulus alteration of AlCl3-induced zebrafish AD model (model group, 1.15 mm/s, p < 0.01) was significantly shorter than that of untreated zebrafish (untreated group, 2.35 mm/s), indicating that the zebrafish AD model worked well. Treatment with 8.0 μM donepezil, the speed change increased to 1.97 mm/s (DZP group, p < 0.01) and the

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efficacy was 68%, exhibiting donepezil could improve response efficiency of AlCl3-induced zebrafish AD. When treatment with compound TM-10 (0.11 μg/mL, 0.33 μg/mL and 1 μg/mL) the speed change increased to 1.87 (p < 0.05), 1.96 (p < 0.05) and 1.52 mm/s (p > 0.05) (Figure 13A), the efficacy were 60%, 68% and 31% (Figure 13B), respectively. Among of these groups, the middle dose of TM-10 group (0.33 μg/ mL) showed the similar speed change and efficacy compared to donepezil group, but the high dose group (1 μg/mL) displayed the lowest speed change and efficacy might be the toxicity of TM-10. A

B

Figure 13. Effect of TM-10 on speed change in AD zebrafish. Compared with model,*p < 0.05, ** p < 0.01.

Based on the results of AlCl3-induced zebrafish AD, compound TM-10 could significantly improve dyskinesia recovery rate and response efficiency of zebrafish AD. Efficacy of TM-10 on Aβ1-40-induced vascular inJury in zebrafish. The 6 hpf transgenic vascular fluorescence zebrafish chosen randomly were placed in a 6-well microplate at a density of 30 zebrafish per well and treated with Aβ1-40, and then treated with testing compounds TM-10 at 3 various concentrations (0.11 μg/mL, 0.33 μg/mL and 1 μg/mL), meanwhile, untreated group and model group as control group. As shown in Table S4, the vascular inJury rate of Aβ1-40-induced vascular inJury (model group, 73%) was significantly higher than that of untreated zebrafish (untreated group, 0), meaning that Aβ1-40-induced vascular inJury model works well. When treatment with compound TM-10 (0.11 μg/mL, 0.33 μg/mL and 1 μg/mL) the vascular inJury rate was 80%, 67% and 67% (Table S4), it meant that the low dose of TM-10 did not show any protective effect on vascular inJury, while, both the middle dose TM-10 group (0.33 μg/mL, 67%) and the high dose TM-10 group (1.0 μg/mL, 67%) showed slightly lower vascular inJury rate than that of model group (73%), and

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possessed potent efficacy with 9% and 9% (Figure 14) respectively. The representative phenotype of TM-10 on vascular inJury was shown in Figure 15. Therefore, the results showed that compound TM-10 had potent protect effect on Aβ1-40-induced vascular inJury.

Figure 14. Efficacy of TM-10 on vascular inJury in zebrafish

Figure 15. Phenotype of TM-10 on vascular inJury

In vivo assay Acute toxicity. To investigate the safety profile of the derivatives, the acute toxicity of TM-10 was evaluated in Kunming mice at doses of 50, 100, 250, 500 and 1000 mg/kg (n = 10 per group) by oral administration. After administration of compound TM-10, mice were observed meticulously for the first 4h, and then were observed continuously through 7 days for any abnormal behavior and mortality changes. The results exhibited that the mice were all dead at dose of 1000 mg/kg TM-10. While, the mice did not show any acute toxicity or mortality immediately at 500 mg/kg or

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below, and then the mice were execute and dissected after 7 days, there were no abnormalities were observed in visceral organ. Therefore, the LD50 of compound TM-10 was 768.8 mg/kg. In order to further study whether TM-10 could improve scopolamine-induced memory impairment, we performed the step-down passive avoidance task, which had been used extensively to evaluate potential therapeutic agents for treating AD.38,

39

Figure 16 showed the step-down latency of mice treated with scopolamine alone (model group, 24.77 sce) was significantly shorter than that of vehicle-treated mice (untreated group, 129.09 sec, p < 0.01). Treatment with compound TM-10 (10 and 100 mg/kg) increased the latency time (42.52 and 54.37, respectively) in a dose-dependent manner, but lower than the drug control group with donepezil (5 mg/kg, 122.80 sec). It might be that the compound TM-10 had some neurotoxicity which resulted in the weak effect. These results showed that compound TM-10 could partly reversed cognitive deficit by ChE inhibition.

Figure 16. Effects of compound TM-10 on scopolamine-induced memory deficit in the step-down passive avoidance test. Compounds TM-10 (10 and 100 mg/kg, p.o.) or donepezil (5.0 mg/kg, p.o.) were orally given 1 h before treatment with scopolamine. After 30 min, the mice were treated with scopolamine (3 mg/kg, i.p.) and tested in the step-down passive avoidance. Values are expressed as the mean ± SEM (n = 6). ##P < 0.01 vs normal group. *P < 0.05 and **P < 0.01 vs scopolamine-treated control group.

CONCLUSION In conclusion, our study focused on the synthesis and pharmacological studies of novel ferulic acid derivatives as multi-functional agent for therapy AD. In vitro results revealed that these target compounds were highly effective and selective BuChE inhibitors, among of which, TM-10 showed the best BuChE inhibitory potency with IC50 value of 8.9 nM, with additional modeling analysis showing reasonable

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explanation for the effective and selectivity. Compound TM-10 displayed potent dual MAO-A and MAO-B inhibitory activity with IC50 values of 6.3μM and 8.6μM, respectively. Moreover, TM-10 could significantly inhibit (53.9%) and disaggregate (43.8%) self-induced Aβ aggregation. Compound TM-10 also exhibited potent antioxidant activity (ORAC = 0.52 eq) and neuroprotective effect against Aβ1-42-mediated SH-SY5Y neurotoxicity, as well as could serve as an autophagic activator. Importantly, compound TM-10 showed good blood-brain barrier (BBB) penetration. Further, compound TM-10 could remarkably improve dyskinesia recovery rate and response efficiency on AlCl3-induced zebrafish AD model, and showed potent neuroprotective effect on Aβ1-40-induced zebrafish vascular inJury. In vivo study revealed that compound TM-10 was low toxicity, and could improve scopolamine-induced memory deficit in mice. Therefore, compound TM-10 is a promising advanced lead compound for the development of AD, and the further in vivo study and structural refinements are underway, and will be reported in due course. Methods Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers. 1H NMR and

13C

NMR spectra were recorded using TMS as the internal

standard on a Varian INOVA at 400 and 100MHz, respectively. All the reactions were monitored by thin-layer chromatography (TLC) with an UV lamp (254 nm). Where appropriate, crude products were purified by column chromatography using silica gel (230-400 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. (China). The high-resolution mass spectra were obtained using a Shimadzu LCMS-IT-TOF mass spectrometer. The purity of all final compounds was determined by high-performance liquid chromatography (HPLC) analysis to be over 97%. HPLC analysis was carried out on a Shimadzu LC-10Avp plus system with the use of a Kromasil C18 column (4.6 mm × 250 mm, 5um), eluted with CH3CN. General Procedures for the Preparation of compounds 3a~3b. Compounds 2a and 3b were obtained referencing our previous reported work.21

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General procedure for the synthesis of intermediates 5a~5e. The appropriate dibromoalkane derivative 4a~4d (6.6 mmol) was added to a mixture of the intermediate 3a or 3b (3.0 mmol), and anhydrous K2CO3 (430 mg, 3.1 mmol) in CH3CN (30 ml). The reaction mixture was warmed to 60-65 °C and stirred for 8-10 h under an argon atmosphere. After complete reaction, the solvent was evaporated under reduced pressure. Water (30 mL) was added to the residue and the mixture was extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with saturated aqueous sodium chloride, dried over sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure. The residue was purified on a silica gel chromatography using dichloromethane/acetone (50:1) as eluent to give the intermediates 5a-5e. (E)-1-(4-benzylpiperidin-1-yl)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)prop-2-en -1-one (5a). Yield 58.5%, light yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 15.2 Hz, 1H, C=CH ), 7.30 (t, J = 7.2 Hz, 2H, 2 × Ar-H), 7.27 (d, J = 6.8 Hz, 1H, Ar-H ), 7.21 (d, J = 7.2 Hz, 1H, Ar-H ), 7.15 (d, J = 7.6 Hz, 1H, 2 × Ar-H), 7.08 (d, J = 6.4 Hz, 1H, Ar-H), 7.02 (s, 1H, Ar-H), 6.84 (d, J = 8.4 Hz, 1H, Ar-H), 6.75 (d, J = 15.6 Hz, 1H, Ar-H), 4.71 (d, J = 11.6 Hz, 1H, 1/2 phCH2) 4.14-4.10 (m, 1H, 1/2 phCH2), 4.07 (t, J = 6.0 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.50 (t, J = 6.4 Hz, 2H, BrCH2), 3.04 (t, J = 11.6 Hz, 1H, 1/2 NCH2), 2.61 (t, J = 12.0 Hz, 1H, 1/2 NCH2), 2.57 (t, J = 8.0 Hz, 2H, NCH2), 2.12-2.05 (m, 2H, CH2), 2.04-1.97 (m, 2H, CH2), 1.85-1.77 (m, 1H, CH), 1.75 (d, J = 13.6 Hz, 2H, CH2), 1.26-1.19 (m, 2H, CH2). (E)-3-(4-(3-bromopropoxy)-3-methoxyphenyl)-1-(3,4-dihydroisoquinolin-2(1H)-y l)prop-2-en-1-one (5b). Yield 56.7%, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, C=CH), 7.21-7.06 (m, 5H, 5× Ar-H), 7.06 (s, 1H, Ar-H), 6.91 (d, J = 8.4 Hz, 1H, Ar-H), 6.82 (d, J = 15.2 Hz, 1H, C=CH), 4.84 (s, 2H, phCH2), 4.19 (t, J = 6.0 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.89-3.86 (m, 2H, phCH2), 3.64 (t, J = 6.4 Hz, 2H, BrCH2), 2.94 (d, J = 21.6 Hz, 2H, NCH2), 2.41-2.34 (m, 2H, CH2). (E)-3-(4-(4-bromobutoxy)-3-methoxyphenyl)-1-(3,4-dihydroisoquinolin-2(1H)-yl) prop-2-en-1-one (5c). Yield 50.6%, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, C=CH), 7.22-7.16 (m, 4H, 4 × Ar-H), 7.12 (d, J = 8.4 Hz, 1H,

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Ar-H), 7.06 (s, 1H, Ar-H), 6.86 (d, J = 8.0 Hz, 1H, Ar-H), 6.81 (d, J = 15.2 Hz, 1H, C=CH), 4.84 (s, 2H, phCH2), 4.08 (t, J = 6.0 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.89-3.86 (m, 2H, phCH2), 3.50 (t, J = 6.4 Hz, 2H, BrCH2), 2.96-2.93 (m, 2H, NCH2), 2.09-2.06 (m, 2H, CH2), 2.02-2.00 (m, 2H, CH2). (E)-3-(4-((5-bromopentyl)oxy)-3-methoxyphenyl)-1-(3,4-dihydroisoquinolin-2(1H)yl)prop-2-en-1-one (5d). Yield 48.6%, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, C=CH), 7.19-7.12 (m, 5H, 5×Ar-H), 7.09 (d, J = 14.8 Hz, 1H, C=CH), 6.85-6.81 (m, 2H, 2×Ar-H), 4.82 (s, 2H, phCH2N), 4.03 (t, J = 6.8 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.87-3.86 (m, 2H, phCH2), 3.42 (t, J = 6.8 Hz, 2H, BrCH2), 2.93-2.88 (m, 2H, NCH2), 1.96-1.82 (m, 4H, 2×CH2), 1.65-1.57 (m, 2H, CH2). (E)-3-(4-((6-bromohexyl)oxy)-3-methoxyphenyl)-1-(3,4-dihydroisoquinolin-2(1H) -yl)prop-2-en-1-one (5e). Yield 53.7%, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, C=CH), 7.21-7.14 (m, 4H, 4×Ar-H), 7.12 (d, J = 8.4 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.86 (d, J = 8.4 Hz, 1H, Ar-H), 6.81 (s, J = 15.2 Hz, 1H, C=CH), 4.84 (s, 2H, phCH2N), 4.05 (t, J = 6.4 Hz, 2H, OCH2), 3.92 (s, 3H, OCH3), 3.90-3.86 (m, 2H, phCH2), 3.43 (t, J = 6.8 Hz, 2H, BrCH2), 2.97-2.91 (m, 2H, NCH2), 1.92-1.85 (m, 4H, 2 × CH2), 1.53-1.50 (m, 4H, 2 × CH2). General Procedures for the Synthesis of Targets Compounds TM-1~TM-24. To a mixture of the corresponding secondary amines NR3R4 (0.6 mmol) and anhydrous K2CO3 (89.7 mg, 0.65 mmol) in anhydrous CH3CN (12 ml) were added the appropriate intermediates 5a~5e (0.5 mmol). The mixture was then heated at 65 °C for 6–10 h. After complete reaction, the solvent was evaporated under reduced pressure. Water (20 ml) was added to the residue and the mixture was extracted with dichloromethane (2×30 mL). The combined organic phases were washed saturated aqueous sodium chloride (50 mL), dried over anhydrous Na2SO4 and filtered. The solvent was evaporated to dryness under reduced pressure to obtain the crude product which

was

further

purified

on

a

silica

gel

chromatography

using

dichloromethane/acetone (30:1) as eluent to give the desired target products TM-1~TM-24.

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(E)-3-(4-(4-(benzyl(ethyl)amino)butoxy)-3-methoxyphenyl)-1-(4-benzylpiperidin1-yl)prop-2-en-1-one (TM-1). Light yellow oil, 80.7% yield, 98.5% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 15.6 Hz, 1H, CH = CH), 7.34 (d, J = 7.2 Hz, 2H, 2 × Ar-H), 7.30-7.25 (m, 4H, 4 × Ar-H), 7.23 (d, J = 7.2 Hz, 1H, Ar-H), 7.21 (d, J = 7.6 Hz, 1H, Ar-H), 7.14 (d, J = 7.2 Hz, 2H, 2 × Ar-H), 7.06 (d, J = 8.0 Hz, 1H, Ar-H), 7.01 (d, J = 1.6 Hz, 1H, Ar-H), 6.80 (d, J = 8.4 Hz, 1H, Ar-H), 6.74 (d, J = 15.6 Hz, 1H, CH=CH), 4.69 (t, J = 7.6 Hz, 1H, 1/2 NCH2), 4.09 (d, J = 10.0 Hz, 1H, 1/2 phCH2), 4.00 (t, J = 6.4 Hz, 2H, OCH2), 3.87 (s, 3H, OCH3), 3.62 (s, 2H, phCH2), 3.03 (t, J = 9.6 Hz, 1H, 1/2 NCH2), 2.59-2.58 (m, 1H, 1/2 phCH2), 2.57-2.51 (m, 6H, 3 × NCH2), 1.87-1.81 (m, 4H, 2 × CH2), 1.76-1.74 (m, 1H, CH), 1.72-1.66 (m, 2H, 2 × CH2), 1.07 (t, J = 6.8 Hz, 3H, CH3).

13

C NMR (100 MHz, CDCl3) δ 165.6, 150.0,

149.5, 142.4, 140.0, 129.1, 129.0, 128.4, 128.3, 128.2, 127.0, 126.1, 121.7, 115.2, 112.7, 110.5, 68.7, 57.8, 56.1, 52.4, 47.2, 43.0, 38.4, 29.7, 26.9, 23.1, 11.5. HR-ESI-MS: Calcd. for C35H44N2O3 [M+H]+: 541.3385, found:541.3412. (E)-3-(4-(4-(benzyl(prop-2-yn-1-yl)amino)butoxy)-3-methoxyphenyl)-1-(4-benzyl piperidin-1-yl)prop-2-en-1-one (TM-2). Light yellow oil, 70.2% yield, 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 15.2 Hz, 1H, CH = CH), 7.35-7.19 (m, 8H, 8 × Ar-H), 7.13 (d, J = 7.2 Hz, 2H, 2 × Ar-H), 7.07 (dd, J1 = 6.4 Hz, J2 = 1.6 Hz, 1H, Ar-H), 7.01 (d, J = 1.6 Hz, 1H, Ar-H), 6.82 (d, J = 8.4 Hz, 1H, Ar-H), 6.74 (d, J = 15.6 Hz, 1H, CH=CH), 4.69 (t, J = 7.6 Hz, 1H, 1/2 NCH2), 4.09 (d, J = 10.0 Hz, 1H, 1/2 phCH2), 4.03 (t, J = 6.8 Hz, 2H, OCH2), 3.87 (s, 3H, OCH3), 3.64 (s, 2H, phCH2), 3.33 (d, J = 2.0 Hz, 2H, NCH2), 3.03 (t, J = 9.6 Hz, 1H, 1/2 NCH2), 2.64-2.60 (m, 3H, NCH2 + 1/2 phCH2), 2.55 (t, J = 7.2 Hz, 2H, NCH2), 2.23 (t, J = 2.0 Hz, 1H, C≡CH), 1.94-1.86 (m, 4H, 2 × CH2), 1.83-1.76 (m, 1H, CH), 1.75-1.66 (m, 4H, 2 × CH2). HR-ESI-MS: Calcd. for C36H42N2O3 [M+H]+: 551.3229, found:551.3253. (E)-3-(4-(3-(4-benzylpiperidin-1-yl)propoxy)-3-methoxyphenyl)-1-(3,4-dihydroiso quinolin-2(1H)-yl)prop-2-en-1-one (TM-3). Light yellow oil, 72.5% yield, 98.1% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 15.2 Hz, 1H, CH=CH), 7.29-7.25 (m, 2H, 2 × Ar-H), 7.19-7.10 (m, 8H, 8 × Ar-H), 7.04 (s, 1H, Ar-H), 6.87 (d,

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J = 8.4 Hz, 1H, Ar-H), 6.81 (d, J = 15.6 Hz, 1H, CH=CH), 4.83 (s, 2H, phCH2), 4.09 (t, J = 8.4 Hz, 2H, OCH2), 3.89 (s, 3H, OCH3), 3.87-3.85 (m, 2H, phCH2), 3.01 (d, J = 10.8 Hz, 2H, phCH2), 2.94-2.92 (m, 2H, NCH2), 2.59 (t, J = 7.2 Hz, 2H, NCH2), 2.54 (d, J = 6.8 Hz, 2H, NCH2), 2.09-2.05 (m, 2H, NCH2), 2.03-1.97 (m, 2H, CH2), 1.66 (d, J = 12.4 Hz, 2H, CH2), 1.58-1.51 (m, 1H, CH), 1.43-1.37 (m, 2H, CH2).

13

C NMR

(100 MHz, CDCl3) δ 166.2, 150.0, 149.5, 142.8, 140.5, 129.1 (2C), 128.6, 128.4, 128.2 (2C), 126.7, 126.6, 126.2, 125.9 (2C), 122.0, 121.8, 115.3, 112.9, 110.9, 67.4, 56.1, 55.2, 53.7 (2C), 43.0, 37.7, 31.9, 31.6 (2C), 29.7 (2C), 26.3. HR-ESI-MS: Calcd. for C34H40N2O3 [M+H]+: 525.3072, found:525.3109. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(3-methoxy-4-(3-(piperidin-1-yl)propo xy)phenyl)prop-2-en-1-one (TM-4). Light yellow oil, 79.5% yield, 98.3% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, CH=CH), 7.20-7.11 (m, 5H, 5 × Ar-H), 7.06 (s, 1H, Ar-H), 6.87-6.81 (m, 2H, Ar-H + CH=CH), 4.84 (s, 2H, phCH2), 4.06 (t, J = 6.0 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.89-3.87 (m, 2H, NCH2), 2.94 (d, J = 24.4 Hz, 2H, NCH2), 2.55-2.51 (m, 6H, 3 × NCH2), 1.89-1.84 (m, 2H, CH2), 1.79-1.77 (m, 2H, CH2), 1.70-1.67 (m, 4H, 2 × CH2), 1.49- 1.47 (m, 2H, CH2). HR-ESI-MS: Calcd. for C27H34N2O3 [M+H]+: 435.2603, found:435.2641. (E)-3-(4-(3-(diethylamino)propoxy)-3-methoxyphenyl)-1-(3,4-dihydroisoquinolin2(1H)-yl)prop-2-en-1-one (TM-5). Light yellow oil, 65.9% yield, 98.1% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 1H NMR 7.59 (d, J = 15.2 Hz, 1H, CH=CH), 7.14-7.05 (m, 5H, 5 × Ar-H), 6.98 (s, 1H, Ar-H), 6.80-6.73 (m, 2H, Ar-H + CH=CH), 4.77 (s, 2H, phCH2), 4.00 (t, J = 5.2 Hz, 2H, OCH2), 3.84-3.82 (m, 5H, OCH3 + NCH2), 2.87 (d, J = 25.2 Hz, 2H, NCH2), 2.83-2.76 (m, 6H, 3 × NCH2), 1.84-1.81 (m, 2H, CH2), 1.16 (t, J = 6.8 Hz, 6H, 2 × CH3). 13 C NMR (100 MHz, CDCl3) δ 165.1, 148.6, 148.2, 141.7, 127.4, 127.2, 125.7, 125.6, 125.3, 120.7, 114.2, 111.5, 109.4, 67.3, 54.9, 50.5, 45.4 (2C), 28.7, 25.6, 20.8, 8.8 (2C). HR-ESI-MS: Calcd. for C26H34N2O3 [M+H]+: 423.2603, found:423.2639. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(3-methoxy-4-(3-((2-methoxybenzyl)(p rop-2-yn-1-yl)amino)propoxy)phenyl)prop-2-en-1-one (TM-6). 7.67 (d, J = 15.2 Hz, 1H, CH=CH), 7.37 (d, J = 7.2 Hz, 1H, Ar-H), 7.25-7.16 (m, 6H, 6 × Ar-H), 7.11

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(d, J = 8.4 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.92 (t, J = 7.6 Hz, 1H, Ar-H), 6.87-6.79 (m, 2H, 2 × Ar-H), 6.81 (d, J = 15.6 Hz, 1H, CH=CH), 4.85 (s, 2H, phCH2), 4.07 (t, J = 6.4 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.88-3.86 (m, 2H, NCH2), 3.82 (s, 3H, OCH3), 3.69 (s, 2H, phCH2), 3.40 (s, 2H, NCH2), 2.95 (d, J = 23.2 Hz, 2H, NCH2), 2.67 (t, J = 6.8 Hz, 2H, NCH2), 2.24 (s, 1H, CH), 1.95-1.87 (m, 2H, CH2), 1.77-1.72 (m, 2H, CH2). HR-ESI-MS: Calcd. for C33H36N2O4 [M+H]+: 525.2709, found:525.2742. (E)-3-(4-(4-(4-benzylpiperidin-1-yl)butoxy)-3-methoxyphenyl)-1-(3,4-dihydroisoq uinolin-2(1H)-yl)prop-2-en-1-one (TM-7). Light yellow oil, 77.6% yield, 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 15.2 Hz, 1H, C=CH), 7.29-7.25 (m, 2H, 2 × Ar-H), 7.21-7.15 (m, 5H, 5 × Ar-H), 7.13-7.11 (m, 3H, 3 × Ar-H), 7.05 (s, 1H, Ar-H), 6.85 (d, J = 4.8 Hz, 1H, Ar-H), 6.81 (d, J = 15.6 Hz, 1H, CH=CH), 4.83 (s, 2H, phCH2), 4.05 (t, J = 4.8 Hz, 2H, OCH2), 3.89 (s, 3H, OCH3), 3.88 (s, 2H, phCH2), 3.27 (d, J = 11.2 Hz, 2H, phCH2), 2.95-2.91 (m, 2H, NCH2), 2.77 (t, J = 6.0 Hz, 2H, NCH2), 2.57 (d, J = 5.6 Hz, NCH2), 2.30 (t, J = 10.4 Hz, 2H, NCH2), 1.91-1.87 (m, 4H, 2 × CH2), 1.74-1.70 (m, 3H, CH2 + CH), 1.67-1.61 (m, 2H, CH2). 13 C NMR (100 MHz, CDCl3) δ 166.1, 149.8, 149.4, 142.7, 139.8, 129.0, 128.5, 128.3, 126.6, 126.1, 121.7, 115.4, 112.8, 110.7, 68.4, 57.5, 56.1, 53.1, 42.4, 37.0, 30.0, 29.7, 26.8, 22.0. HR-ESI-MS: Calcd. for C35H42N2O3 [M+H]+: 539.3229, found:539.3271. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(3-methoxy-4-(4-(4-phenylpiperidin-1yl)butoxy)phenyl)prop-2-en-1-one (TM-8). Light yellow oil, 70.9% yield, 98.5% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 15.2 Hz, 1H, CH=CH), 7.32-7.26 (m, 2H, 2 × Ar-H), 7.24-7.12 (m, 8H, 8× Ar-H), 7.06 (s, 1H, Ar-H), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.82 (d, J = 15.6 Hz, 1H, CH=CH), 4.84 (s, 2H, phCH2), 4.09 (t, J = 6.4 Hz, 2H, OCH2), 3.92 (s, 3H, OCH3), 3.89-3.87 (m, 2H, NCH2), 3.11 (d, J = 10.8 Hz, 2H, CH2), 2.94 (d, J = 22.4 Hz, 2H, NCH2), 2.50-2.46 (m, 3H, CH + NCH2), 2.06 (t, J = 10.8 Hz, 2H, CH2), 1.77-1.74 (m, 2H, CH2).

13

1.92-1.88 (m, 2H, CH2), 1.84-1.82 (m, 4H, 2 × CH2),

C NMR (100 MHz, CDCl3) δ 166.2, 150.0, 149.3, 146.2,

142.9, 128.5 (2C), 126.9 (2C), 126.2, 121.9, 115.0, 112.4, 110.3, 68.7, 58.5, 56.1,

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54.3 (2C), 42.6, 33.3 (2C), 27.2, 23.5. HR-ESI-MS: Calcd. for C34H40N2O3 [M+H]+: 525.3072, found:525.3098. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(4-(4-(3,4-dihydroisoquinolin-2(1H)-yl) butoxy)-3-methoxyphenyl)prop-2-en-1-one (TM-9). Light yellow oil, 70.9% yield, 98.3% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, CH=CH), 7.20-7.01 (m, 10H, 10 × Ar-H), 6.88 (d, J = 8.4 Hz, 1H, Ar-H), 6.81 (d, J = 15.6 Hz, 1H, CH=CH), 4.84 (s, 2H, phCH2), 4.09 (t, J = 6.4 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.88-3.86 (m, 2H, NCH2), 3.65 (s, 2H, phCH2), 2.96-2.89 (m, 4H, 2 × NCH2), 2.75 (t, J = 5.2 Hz, 2H, NCH2), 2.59 (t, J = 7.2 Hz, 2H, NCH2), 1.95-1.88 (m, 2H, CH2), 1.84-1.78 (m, 2H, CH2).

13

C NMR (100 MHz, CDCl3) δ 166.2, 150.1,

149.3, 142.9, 134.7, 134.3, 128.7, 128.1, 126.6 (2C), 125.6, 121.9, 112.4, 110.4, 68.7, 57.9, 56.1, 50.9, 29.0, 27.0, 23.6. HR-ESI-MS: Calcd. for C32H36N2O3 [M+H]+: 497.2759, found:497.2786. (E)-3-(4-(4-(benzyl(ethyl)amino)butoxy)-3-methoxyphenyl)-1-(3,4-dihydroisoqui nolin-2(1H)-yl)prop-2-en-1-one (TM-10). Light yellow oil, 85.3% yield, 98.7% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.6 Hz, 1H, CH = CH), 7.32 (d, J = 6.8 Hz, 2H, 2 × Ar-H), 7.29 (d, J = 7.2 Hz, 2H, 2 × Ar-H), 7.25 (d, J = 8.4 Hz, 1H, Ar-H), 7.22-7.15 (m, 4H, 4 × Ar-H), 7.11 (d, J = 8.4 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 6.83 (d, J = 4.4 Hz, 1H, Ar-H), 6.80 (d, J = 15.2 Hz, 1H, CH=CH), 4.84 (s, 2H, phCH2), 4.01 (t, J = 6.4 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.89-3.87 (m, 2H, phCH2), 3.58 (s, 2H, phCH2), 2.96-2.94 (m, 2H, NCH2), 2.55-2.48 (m, 4H, 2 × NCH2), 1.90-1.83 (m, 2H, CH2), 1.69-1.62 (m, 2H, CH2), 1.04 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (100 MHz, CDCl3) δ 166.2, 150.2, 149.5, 142.9, 139.7, 128.9, 128.2, 126.8, 126.6, 121.8, 115.1, 112.6, 110.6, 68.8, 56.1, 52.6, 47.2, 26.9, 23.4, 11.7. HR-ESI-MS: Calcd. for C32H38N2O3 [M+H]+: 499.2916, found:499.2946. (E)-3-(4-(4-(benzyl(prop-2-yn-1-yl)amino)butoxy)-3-methoxyphenyl)-1-(3,4-dihy droisoquinolin-2(1H)-yl)prop-2-en-1-one (TM-11). Light yellow oil, 82.7% yield, 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 15.2 Hz, 1H, CH=CH), 7.35-7.16 (m, 9H, 9 × Ar-H), 7.11 (d, J = 8.0 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.88-6.79 (m, 2H, Ar-H + CH=CH), 4.85 (s, 2H, phCH2), 4.06 (t, J = 6.4 Hz,

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2H, OCH2), 3.91 (s, 3H, OCH3), 3.88 (s, 2H, phCH2), 3.63 (s, 2H, phCH2), 3.33 (s, 2H, NCH2), 2.95 (d, J = 22.4 Hz, 2H, NCH2), 2.63 (t, J = 7.2 Hz, 2H, NCH2), 2.23 (s, 1H, CH), 1.96-1.90 (m, 2H, CH2), 1.72-1.66 (m, 2H, CH2). HR-ESI-MS: Calcd. for C33H36N2O3 [M+H]+: 509.2759, found:509.2793. (E)-3-(4-(4-(benzyl(isopropyl)amino)butoxy)-3-methoxyphenyl)-1-(3,4-dihydroiso quinolin-2(1H)-yl)prop-2-en-1-one (TM-12). Light yellow oil, 80.3% yield, 98.1% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 15.2 Hz, 1H, CH = CH), 7.36-7.24 (m, 5H, 5 × Ar-H), 7.22-7.15 (m, 4H, 4 × Ar-H), 7.11 (d, J = 8.0 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 6.83-6.78 (m, 2H, Ar-H + CH=CH), 4.85 (s, 2H, phCH2), 3.95 (t, J = 6.8 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.89-3.87 (m, 2H, phCH2), 3.54 (s, 2H, phCH2), 2.98-2.89 (m, 2H, NCH2), 2.87-2.83 (m, 1H, NCH), 2.46 (t, J = 6.8 Hz, 2H, NCH2), 1.89-1.82 (m, 2H, CH2), 1.59-1.50 (m, 2H, CH2), 1.11 (s, 3H, CH3), 1.10 (s, 3H, CH3). HR-ESI-MS: Calcd. for C33H40N2O3 [M+H]+: 513.3072, found:513.3106. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(4-(4-(ethyl(2-methoxybenzyl)amino)b utoxy)-3-methoxyphenyl)prop-2-en-1-one (TM-13). Light yellow oil, 85.2% yield, 98.6 % HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.6 Hz, 1H, CH=CH), 7.44 (dd, J1 = 6.4 Hz, J2 = 1.2 Hz, 1H, Ar-H), 7.25-7.10 (m, 5H, 5 × Ar-H), 7.11 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 1H, Ar-H), 7.05 (d, J =2.0 Hz, 1H, Ar-H), 6.92 (t, J = 7.2 Hz, 1H, Ar-H), 6.85 (t, J = 7.6 Hz, 2H, 2 × Ar-H), 6.81 (d, J = 15.2 Hz, 1H, CH=CH), 4.84 (s, 2H, phCH2), 4.03 (t, J = 6.4 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.88-3.86 (m, 2H, phCH2), 3.81 (s, 3H, OCH3), 3.70 (s, 2H, phCH2), 2.97-2.91 (m, 2H, NCH2), 2.65-2.58 (m, 4H, 2 × NCH2), 1.91-1.83 (m, 2H, CH2), 1.77-1.70 (m, 2H, CH2), 1.11 (t, J = 6.8 Hz, 3H, CH3). 13 C NMR (100 MHz, CDCl3) δ 166.2, 157.8, 150.1, 149.4, 142.9, 130.7, 128.2, 128.1, 126.7, 126.6, 121.8, 120.4, 115.0, 112.6, 110.6, 110.3, 68.8, 56.1, 55.4, 52.7, 51.1, 47.5, 29.7, 27.0, 23.0, 11.4. HR-ESI-MS: Calcd. for C33H40N2O4 [M+H]+: 529.3022, found: 529.3051. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(3-methoxy-4-(4-((2-methoxybenzyl)(p rop-2-yn-1-yl)amino)butoxy)phenyl)prop-2-en-1-one (TM-14). Light yellow oil, 77.2% yield, 98.4% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 15.2 Hz,

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1H, CH=CH), 7.37 (d, J = 7.2 Hz, 1H, Ar-H), 7.26-7.16 (m, 6H, 6 × Ar-H), 7.11 (d, J = 8.0 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.92 (t, J = 7.6 Hz, 1H, Ar-H), 6.89-6.79 (m, 3H, 2 × Ar-H + CH=CH), 4.84 (s, 2H, phCH2), 4.07 (t, J = 6.4 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.88-3.86 (m, 2H, NCH2), 3.84 (s, 3H, OCH3), 3.68 (s, 2H, phCH2), 3.40 (s, 2H, NCH2), 2.94 (d, J = 22.4 Hz, 2H, NCH2), 2.67 (t, J = 6.8 Hz, 2H, NCH2), 2.24 (s, 1H, CH), 1.95-1.89 (m, 2H, CH2), 1.78-1.71 (m, 2H, CH2). HR-ESI-MS: Calcd. for C34H38N2O4 [M+H]+: 539.2865, found:539.2881. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(3-methoxy-4-(4-(1-methyl-3,4-dihydro isoquinolin-2(1H)-yl)butoxy)phenyl)prop-2-en-1-one (TM-15). Light yellow oil, 68.6% yield, 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 15.6 Hz, 1H, CH=CH), 7.19-7.12 (m, 6H, 6 × Ar-H), 7.10-7.04 (m, 4H, 4 × Ar-H), 6.86 (d, J = 8.0 Hz, 1H, Ar-H), 6.80 (d, J = 15.6 Hz, 1H, CH=CH), 4.83 (s, 2H, phCH2), 4.07 (t, J = 6.0 Hz, 2H, OCH2), 3.99 (t, J = 6.8 Hz, 2H, phCH2), 3.88 (s, 3H, OCH3), 3.88-3.85 (m, 2H, phCH2), 3.17-3.11 (m, 1H, phCH), 2.99-2.84 (m, 4H, 2 × NCH2), 2.77-2.67 (m, 2H, NCH2), 1.92-1.77 (m, 4H, 2 × CH2), 1.39 (d, J = 6.4 Hz, 3H, CH3). HR-ESI-MS: Calcd. for C33H38N2O3 [M+H]+: 511.2916, found:511.2942. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(4-(4-(6,7-dimethoxy-3,4-dihydroisoqui nolin-2(1H)-yl)butoxy)-3-methoxyphenyl)prop-2-en-1-one (TM-16). Light yellow oil, 60.8% yield, 98.1% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 15.2 Hz, 1H, CH=CH), 7.21-7.17 (m, 4H, 4 × Ar-H), 7.11 (d, J = 8.0 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.82 (d, J = 15.6 Hz, 1H, CH=CH), 6.60 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 4.85 (s, 2H, phCH2), 4.10 (t, J = 6.0 Hz, 2H, OCH2), 3.92 (s, 3H, OCH3), 3.88-3.86 (m, 2H, NCH2), 3.84 (s, 6H, 2 × OCH3), 3.59 (s, 2H, phCH2), 2.95 (d, J = 23.2 Hz, 2H, NCH2), 2.84-2.82 (m, 2H, NCH2), 2.75 (t, J = 5.2 Hz, 2H, NCH2), 2.60 (t, J = 7.2 Hz, 2H, NCH2), 1.96-1.92 (m, 2H, CH2), 1.82-1.79 (m, 2H, CH2). HR-ESI-MS: Calcd. for C34H40N2O5 [M+H]+: 557.2971, found:557.2998. (E)-3-(4-(4-([1,4'-bipiperidin]-1'-yl)butoxy)-3-methoxyphenyl)-1-(3,4-dihydroisoq uinolin-2(1H)-yl)prop-2-en-1-one (TM-17). Light yellow oil, 72.1% yield, 98.5 % HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, CH=CH),

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7.21-7.15 (m, 4H, 4 × Ar-H), 7.12 (d, J = 8.0 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.86 (d, J = 8.4 Hz, 1H, Ar-H), 6.82 (d, J = 15.6 Hz, 1H, CH=CH), 4.84 (s, 2H, phCH2), 4.06 (t, J = 6.4 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.88-3.86 (m, 2H, phNCH2), 3.51-3.43 (m, 2H, NCH2), 3.03 (d, J = 11.6 Hz, 2H, NCH2), 2.97-2.91 (m, 2H, NCH2), 2.64-2.61 (m, 4H, 2 × NCH2), 2.49-2.46 (m, 1H, NCH), 2.40 (t, J = 7.2 Hz, 2H, NCH2), 1.96 (t, J = 11.6Hz, 2H, CH2), 1.90-1.82 (m, 4H, 2 × CH2), 1.72-1.64 (m, 6H, 3 × CH2), 1.49-1.46 (m, 2H, CH2).

13

C NMR (100 MHz, CDCl3) δ 166.2, 150.1,

149.4, 142.9, 128.9, 128.2, 126.7, 126.6, 121.8 (2C), 115.0, 112.5 (2C), 110.5, 68.7, 63.0, 58.0, 56.1, 53.1 (4C), 50.0 (4C), 27.1 (2C), 25.4, 24.2, 23.5. HR-ESI-MS: Calcd. for C33H45N3O3 [M+H]+: 532.3494, found: 532.3465. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(4-(4-(4-ethylpiperazin-1-yl)butoxy)-3methoxyphenyl)prop-2-en-1-one (TM-18). Light yellow oil, 63.7% yield, 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, CH=CH), 7.21-7.15 (m, 4H, 4 × Ar-H),

7.11 (d, J = 8.4 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H),

6.86 (dd, J1 = 5.2 Hz, J2 = 3.2 Hz, 1H, Ar-H), 6.82 (d, J = 15.6 Hz, 1H, CH=CH), 4.83 (s, 2H, phCH2), 4.06 (t, J = 6.4 Hz, 2H, OCH2), 4.00 (s, 3H, OCH3), 3.88-3.86 (m, 2H, phCH2), 2.96-2.92 (m, 2H, NCH2), 2.60-2.42 (m, 12H, 6 × NCH2), 1.88-1.84 (m, 2H, CH2), 1.72-1.66 (m, 2H, CH2), 1.12 (t, J = 7.2 Hz, 3H, CH3). HR-ESI-MS: Calcd. for C29H39N3O3 [M+H]+: 478.3025, found:478.3051. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(3-methoxy-4-(4-(piperidin-1-yl)butoxy)p henyl)prop-2-en-1-one (TM-19). Light yellow oil, 78.3% yield, 98.6 % HPLC purity. 1H

NMR (400 MHz, CDCl3) δ 7.58 (d, J = 15.2 Hz, 1H, CH=CH), 7.12-7.03 (m, 5H,

5 × Ar-H), 6.98 (s, 1H, Ar-H), 6.79-6.72 (m, 2H, Ar-H + CH=CH), 4.76 (s, 2H, phCH2), 3.98 (t, J = 6.4 Hz, 2H, OCH2), 3.84-3.80 (m, 5H, OCH3 + NCH2), 2.86 (d, J = 24.0 Hz, 2H, NCH2), 2.46-2.41 (m, 6H, 3 × NCH2), 1.81-1.76 (m, 2H, CH2), 1.72-1.64 (m, 2H, CH2), 1.61-1.58 (m, 4H, 2 × CH2), 1.40- 1.39 (m, 2H, CH2). 13 C NMR (100 MHz, CDCl3) δ 166.2, 149.9, 149.3, 142.9, 134.3, 133.6, 128.3, 128.2, 126.7, 121.8, 115.2, 115.0, 112.4, 110.3, 68.6, 58.5, 56.0, 54.2 (2C), 27.0, 25.2 (2C), 23.9, 22.8. HR-ESI-MS: Calcd. for C28H36N2O3 [M+H]+: 449.2759, found:449.2781. (E)-3-(4-(4-(diethylamino)butoxy)-3-methoxyphenyl)-1-(3,4-dihydroisoquinolin-2

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(1H)-yl)prop-2-en-1-one (TM-20). Light yellow oil, 66.7% yield, 98.4% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, CH=CH), 7.21-7.12 (m, 5H, 5 × Ar-H), 7.06 (s, 1H, Ar-H), 6.87-6.81 (m, 2H, Ar-H + CH=CH), 4.84 (s, 2H, phCH2), 4.07 (t, J = 5.6 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.89-3.88 (m, 2H, NCH2), 2.94 (d, J = 25.2 Hz, 2H, NCH2), 2.86-2.82 (m, 6H, 3 × NCH2), 1.92-1.82 (m, 4H, 2 × CH2), 1.97 (t, J = 7.2 Hz, 6H, 2 × CH3). HR-ESI-MS: Calcd. for C27H36N2O3 [M+H]+: 437.2759, found:437.2783. (E)-3-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)-3-methoxyphenyl)-1-(3,4-dihydr oisoquinolin-2(1H)-yl)prop-2-en-1-one (TM-21). Light yellow oil, 72.6% yield, 98.0 % HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 15.6 Hz, 1H, C=CH), 7.28-7.24 (m, 2H, 2×Ar-H), 7.18-7.06 (m, 9H, 9 × Ar-H), 6.87-6.82 (m, 2H, 1× Ar-H + C=CH), 4.83 (s, 2H, phCH2), 4.01 (t, J = 5.6 Hz, 2H, OCH2), 3.89 (s, 3H, OCH3), 3.78-3.75 (m, 2H, phCH2), 3.15 (d, J = 10.4 Hz, 2H, phCH2), 2.91 (d, J = 24.0 Hz, 2H, CH2), 2.59 (t, J = 7.2 Hz, 2H, OCH2), 2.54 (s, 2H, CH2), 2.22 (t, J = 9.2 Hz, 2H, NCH2), 1.87-1.83 (m, 2H, CH2), 1.76-1.67 (m, 5H, 1 × CH + 2 × CH2), 1.63-1.58 (m, 2H, CH2), 1.50-1.47 (m, 2H, CH2).

13

C NMR (100 MHz, CDCl3) δ 166.2, 150.0,

149.4, 142.8, 139.9, 129.1 (3C), 128.3 (4C), 128.2, 126.7, 126.6, 126.5, 126.0 (2C), 121.9, 112.5, 110.5, 68.6, 58.1, 56.1 (2C), 53.4 (2C), 42.6, 37.1, 30.5 (2C), 28.7 (2C), 25.3, 23.8 (2C). HR-ESI-MS: Calcd. for C36H44N2O3 [M+H]+: 553.3385, found: 553.3412. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(4-((5-(3,4-dihydroisoquinolin-2(1H)-yl)p entyl)oxy)-3-methoxyphenyl)prop-2-en-1-one (TM-22). Light yellow oil, 80.1% yield, 98.2 % HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, C=CH), 7.20-7.14 (m, 4H, 4 × Ar-H), 7.12-7.06 (m, 5H, 5 × Ar-H), 7.02-7.00 (m, 1H, Ar-H), 6.86 (d, J = 8.4 Hz, 1H, Ar-H), 6.81 (d, J = 15.2 Hz, 1H, C=CH), 4.83 (s, 2H, phCH2), 4.05 (t, J = 6.4 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.87 (s, 2H, phCH2), 3.65 (s, 2H, phCH2), 2.97-2.87 (m, 4H, 2 × NCH2), 2.75 (t, J = 5.6 Hz, 2H, NCH2), 2.55 (t, J = 7.6 Hz, 2H, NCH2), 1.95-1.82 (m, 2H, CH2), 1.73-1.65 (m, 2H, CH2), 1.58-1.50 (m, 2H, CH2).

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C NMR (100 MHz, CDCl3) δ 168.6, 150.1, 149.4, 142.9,

134.5, 134.2, 128.7 (2C), 128.2, 126.7, 126.6 (3C), 126.2 (2C), 125.7 (2C), 122.0,

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121.9, 112.4, 110.5, 68.8, 58.1 (2C), 56.0 (2C), 50.9 (2C), 43.9 (2C), 28.9, 26.8, 24.0. HR-ESI-MS: Calcd. for C33H38N2O3 [M+H]+: 511.2916, found: 511.2938. (E)-3-(4-((6-(4-benzylpiperidin-1-yl)hexyl)oxy)-3-methoxyphenyl)-1-(3,4-dihydroi soquinolin-2(1H)-yl)prop-2-en-1-one (TM-23). Light yellow oil, 76.1 % yield, 98.1 % HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, C=CH), 7.29-7.23 (m, 2H, 2×Ar-H), 7.18-7.11 (m, 8H, 8×Ar-H), 7.07 (s, 1H, Ar-H), 6.86-6.81 (m, 2H, Ar-H + C=CH), 4.83 (d, J = 6.0 Hz, 2H, phCH2), 4.01 (t, J = 6.8 Hz, 2H, OCH2), 3.89 (s, 3H, OCH3), 3.86-3.83 (m, 2H, phCH2), 3.08 (d, J = 8.4 Hz, 2H, CH2), 2.91 (d, J = 19.2 Hz, 2H, CH2), 2.53 (t, J = 5.6 Hz, 2H, CH2), 2.48-2.46 (m, 2H, CH2), 2.09 (t, J = 9.6 Hz, 2H, CH2), 1.86-1.80 (m, 2H, CH2), 1.67-1.61 (m, 5H), 1.57-1.54 (m, 2H), 1.50-1.45 (m, 2H), 1.38-1.34 (m, 2H). HR-ESI-MS: Calcd. for C37H46N2O3 [M+H]+: 567.3542, found: 567.3569. (E)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-(4-((6-(3,4-dihydroisoquinolin-2(1H)-yl)h exyl)oxy)-3-methoxyphenyl)prop-2-en-1-one (TM-24). Light yellow oil, 65.8 % yield, 98.2 % HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 15.2 Hz, 1H, C=CH), 7.19-7.15 (m, 4H, 4×Ar-H), 7.12-7.06 (m, 5H, 5×Ar-H), 7.01 (d, J = 4.8 Hz, 1H, Ar-H), 6.87-6.84 (m, 1H, Ar-H), 6.82 (d, J = 15.2 Hz, 1H, C=CH), 4.83 (s, 2H, phCH2), 4.04 (t, J = 6.4 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.87 (s, 2H, phCH2), 3.64 (s, 2H, phCH2), 2.94-2.92 (m, 2H, NCH2), 2.91 (t, J = 4.8 Hz, 2H, NCH2), 2.75 (t, J = 5.6 Hz, 2H, NCH2), 2.53 (t, J = 7.2 Hz, 2H, NCH2), 1.91-1.80 (m, 2H, CH2), 1.68-1.61 (m, 2H, CH2), 1.56-1.48 (m, 2H, CH2), 1.46-1.42 (m, 2H, CH2). 13 C NMR (100 MHz, CDCl3) δ 166.2, 150.2, 149.4, 142.9, 134.5, 134.2, 128.7 (2C), 128.3, 128.1, 126.7, 126.6 (2C), 126.2 (2C), 125.7 (2C), 121.9, 115.0, 112.5, 110.5, 68.9, 58.2, 56.1, 56.0, 50.9, 29.1, 28.8, 27.3, 27.0, 26.0. HR-ESI-MS: Calcd. for C34H40N2O3 [M+H]+: 525.3072, found: 525.3094. Effect on AChE and BuChE Inhibition. The inhibitory activity of AChE and BuChE for the target compounds were performed by slightly modified Ellman assay.20,

23

For AChE inhibition, a reaction mixture (100 µL) containing ATC (1

mmol/L, 30 µL), phosphate-buffered solution (0.1 M KH2PO4/K2HPO4, PH = 8.0, 40 µL), 10 µL enzyme (eeAChE 0.45 U/mL) and different concentrations (DMSO < 1%)

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of test compounds (20 µL) was incubated at 37°C for 15 min. Then 5,5’-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30 µL) was added. The activities were determined by using a Varioskan Flash Multimode Reader at 412 nm. Each concentration was assayed in triplicate. The inhibition percent was calculated by the following expression: (1−Ai/Ac) × 100, where Ai and Ac are the absorbance obtained for AChE in the presence and absence of inhibitors, respectively. In vitro BuChE assay was carried out using a similar method. Molecular modeling studies. Docking was utilized to identify the potential binding of compound TM-10 to BuChE and AChE. The crystal structure of huBuChE (PDB ID: 4tpk) and TcAChE (PDB ID: 1EVE) were obtained from the Protein Data Bank after removing the original inhibitors and water molecules.20 Docking studies were performed using the AUTODOCK 4.2.6 program. Graphic manipulations and visualizations were done by Autodock Tools or Discovery Studio 2.5 software. Recombinant human MAO-A and MAO-B inhibition studies.40 Recombinant human MAO-A and MAO-B were purchased from Sigma-Aldrich and stored at -80 °C. Tested compounds were diluted with potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM) to a final volume of 500 μL containing various concentrations of test compounds (0-100 μM) and kynuramine (45 μM for MAO-A and 30 μM for MAO-B). The reactions were initiated by the addition of the enzyme (7.5 μg/mL) and then incubated for 30 min at 37. Then 400 μL NaOH (2N) and 1000 μL water were added to terminated the enzymatic reactions and the mistures were centrifuged at 16000g for 10 min. The concentrations of the generated 4-hydroxyquinoline were determined by measuring the fluorescence of the supernatant on a Varioskan Flash Multimode Reader (PerkinElmer) with excitation and emission wavelengths at 310 nm and 400 nm, respectively. IC50 values were calculated from sigmoidal dose-response curves (graphs of the initial rate of kynuramine oxidation versus the logarithm of inhibitor concentration). Each sigmoidal curve was constructed form six different compound concentrations spanning at least three orders of magnitude. Data analyses were carried out with GraphRad Prism 5 employing the one site competition model. IC50 values were

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determined in triplicate and expressed as mean ± SD. Antioxidant activity assay. The antioxidant activity was determined by the oxygen radical absorbance capacity fluorescein (ORAC-FL) assay according to our previous work.19,37 Neuroprotective effect. The neuroprotective effects were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay based on the reported paper.32 Effects on self-induced Aβ1-42 aggregation. Thioflavin T-based flurometric assay was used to investigate the effects on self-induced Aβ1-42 aggregation.20 For the disaggregation of self-induced Aβ fibrils experiment, the Aβ1-42 stock solution was diluted in phosphate buffer solution (pH 7.4). The mixture of the Aβ1-42 was incubated 37 °C for 24 h. The tested compound was then added and incubated at 37 °C for another 24 h. After incubation, 160 μL of 5 μM thioflavin T in 50 mM glycine-NaOH buffer (pH 8.5) was added. Each assay was run in triplicate. The detailed detection method was the same as described previous work.21 Transmission Electron Microscopy (TEM) Assay. For the self-induced experiment, the Aβ stock solution was diluted with 50 mM phosphate buffer (pH 7.4). For the copper-induced experiment, the Aβ stock solution was diluted with HEPES buffer (20 mM, pH 6.6, 150 mM NaCl). The sample preparation was the same as for the ThT assay. Aliquots (10 μL) of the samples were placed on a carbon-coated copper/rhodium grid for 2 min at room temperature. Excess sample was removed using filter paper followed by washing twice with ddH2O. Each grid was negatively stained with 2% phosphomolybdic acid solution for 2 min at room temperature. After the excess of staining solution was drained off by means of a filter paper, the specimen was transferred for examination using a transmission electron microscope. In vitro blood−brain barrier permeation assay. The blood-brain barrier penetration of compounds was evaluated using the parallel artificial membrane permeation assay (PAMPA) described by Di et al.34 Commercial drugs were purchased from Sigma and Alfa Aesar. Porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and

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acceptor microplate were both from Millipore. The 96-well UV plate (COSTAR) was from Corning Incorporated. The acceptor 96-well microplate was filled with 350 μL of PBS/EtOH (70:30), and the filter membrane was impregnated with 4 μL of PBL in dodecane (20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (70:30) to a final concentration of 100 μg/ mL. Then 200 μL of the solution was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a sandwich, which was left undisturbed for 18 h at 25 °C. After incubation, the donor plate was carefully removed, and the concentration of compounds in the acceptor wells was determined using the Varioskan Flash Multimode Reader (Thermo Scientific). Every sample was analyzed at ten wavelengths in four wells and in at least three independent runs. Pe was calculated using

the

following

expression:

Pe

=

{−VdVa/[(Vd

+

Va)At]}ln(1



drugacceptor/drugequilibrium), where Vd is the volume of donor well, Va is the volume in the acceptor well, A is the filter area, t is the permeation time, drug acceptor is the absorbance obtained in the acceptor well, and drug equilibrium is the theoretical equilibrium absorbance. The results are given as the mean ± standard deviation. In the experiment, 11 quality control standards of known BBB permeability were included to validate the analysis set. A plot of the experimental data versus literature values gave a strong linear correlation, Pe(exp) = 0.9163Pe(bibl.) −0.2247 (R2 = 0.9558). From this equation and the limit established by Di et al. (Pe (bibl.) = 4.0 × 10−6 cm/s) for blood−brain barrier permeation, we concluded that compounds with a permeability greater than 3.44 ×10−6 cm/s could cross the blood−brain barrier. Effect on Zebrafish AD Model. 36,37 MNLC and LC10 of compound TM-10 determination. In order to investigate the safety profile of compound TM-10, the acute toxicity was tested in wild-type zerbrafish, six concentrations 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL for TM-10 were used to treat zebrafish (n = 30 per group) at 2dpf, and the untreated group and vehicle group (1% DMSO) as control group. The zebrafish were treated, counted and observed for 3 days until 5dpf. The observed phenomenon was minutely recorded. The MNLC and LC10 of compound TM-10 were calculated based on the data of

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concentration-death rate using OriginPro 8.0. The phenotypes of zebrafish acute toxicity. Based on the above results of MNLC, various concentration TM-10 groups (0.35, 1.04, 3.125 and 6.25 µg/mL), untreated group and vehicle group (1% DMSO) were used to treat wild-type zerbrafish at 2dpf (n = 30 per group), and the experiment phenomenon were observed and counted for 3 days until 5 dpf. After experiments completion, the toxic effect were determined under a microscope including heart, brain, the lower Jaw, eyes, liver, intestines, trunk/tail/notochord, muscle/somite/sports, the body color, circulatory system, body edema and bleeding. The representative phenotypes of zebrafish acute toxicity was photographed and recorded. Effect on compound TM-10 in the zebrafish AD model. The 3dpf zebrafish chosen randomly were placed in a 6-well microplate at a density of 30 zebrafish per well and treated with AlCl3, and then treated with a testing compound TM-10 at 8 various concentrations (1 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL), respectively, and also including untreated group and model group. The survivals were counted for 2 days, and the data was recorded. So, the MTC of compound TM-10 was determined. Based on the MTC, the 3dpf zebrafish chosen randomly were placed in a 6-well microplate at a density of 30 zebrafish per well and treated with 140 μM AlCl3, three various concentrations (0.11 μg/mL, 0.33 μg/mL and 1 μg/mL) were selected for compound TM-10 group, meanwhile, the donepezil group (8.0 μM), untreated group and model group as the positive groups. After 3 days, the distance and speed change after light stimulus alteration were analyzed and recorded for three rhythmic light within 60 minutes (10 min darkness, 10 min illumination alternately). Efficacy on motility (%) ={D(sample group)-D(model group)}/{D(untreated group)-D(model group)}×100%. Efficacy on speed change (%) ={S(sample group)-S(model group)} / { S(untreated group)-S(model group) } ×100%. All data are expressed as mean ± SEM. Differences between groups were examined for statistical significance using one-way ANOVA with Student’s t test. A P value less than 0.05 denoted the presence of a statistically significant.

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Efficacy of TM-10 on Aβ1-40-induced vascular inJury in zebrafish. The 6 hpf transgenic vascular fluorescence zebrafish chosen randomly were placed in a 6-well microplate at a density of 30 zebrafish per well and treated with Aβ1-40, and then treated with testing compound TM-10 at 3 various concentrations (0.11 μg/mL, 0.33 μg/mL and 1 μg/mL), meanwhile, untreated group and model group as control group. After 2 days, 15 zebrafish were chosen randomly, and then were observed and photographed using fluorescence microscope. Vascular integrity number was counted compared with model group. Protective efficacy of vascular (%) = (sample group – model group) / (untreated group – model group) ×100%. Differences between groups were examined for statistical significance using Fisher. A P value less than 0.05 denoted the presence of a statistically significant. Step-down Passive Avoidance Test38,39 Materials and animals Donepezil was purchased from Eisai China Inc. Scopolamine was purchase from J&K Scientific. Kunming mice at body weight of 18–22 g (six weeks old, either gender) were supplied by the Center of Experimental Animals of Sichuan Academy of Chinese Medicine Sciences (eligibility certification no. SCXK-Chuan2015-030). Mice were maintained under standard conditions with a 12 h:12 h light–dark cycle, a temperature and humidity controlled environment with access to food and water ad libitum. Acute toxicity. Prior to each experiment, mice were fasted overnight and allowed free access to water. Compound TM-10 was treated at dose of 50, 100, 250, 500 and 1000 mg/kg (n = 10 per group) by oral administration. After the administration of the compounds, the mice were observed continuously for the first 4 h for any abnormal behavior and mortality changes, intermittently for the next 24 h, and occasionally thereafter for 14 days for the onset of any delayed effects. All animals were sacrificed on the 14th day after drug administration and were macroscopically examined for possible damage to the heart, liver, and kidneys. Assay method. A modification of step-down passive avoidance test was used to assess learning and memory in mice. The apparatus consisted of a grid floor with a wooden block placed in the center. The block served as a shock free zone. The mice

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underwent two separate trials: a training trial and a test trial 24 h later. For training trial, mice were initially placed on the block and were given an electrical foot shock (0.5 mA, 2s) through the grid floor on stepping down. We used a total of 90 mice in the passive avoidance test with 10 mice were used per treatment. Compounds TM-10 and donepezil (5.0 mg/kg, p.o.) were orally given 1 h before each training trial. After 30 min, memory impairment was induced by administering scopolamine (3 mg/kg, i.p.). Twenty-four hours after the training trial, mice were placed on the block and the time for the animal to step down was measured as latency time for test trial. An upper cut-off time was set at 300 s. All data are expressed as mean ± SEM. Differences between groups were examined for statistical significance using one-way ANOVA with Student’s t test. A P value less than 0.05 denoted the presence of a statistically significant difference. ASSOCIATED CONTENT Supporting Information Table S1~S4 for AD zebrafish model experiments. Representative 1H,

13C

NMR

and HR-ESI-MS spectra for the synthesized compounds AUTHOR INFORMATION *Corresponding Author. E-mail: [email protected] (Zhipei Sang) E-mail: [email protected] (Zhenhuai Tan) E-mail: [email protected] (Wenmin Liu) ORCID Zhipei Sang: 0000-0001-7254-5518

Author Contridutions #These authors contribute equally to this work. Concept and design: Zhipei Sang, Zhenhuai Tan and Wenmin Liu. Zhipei Sang was responsible for synthesis, evaluation of in vitro, data analysis, and writing the manuscript. Zhenhuai Tan was responsible for evaluation in vivo. Keren Wang was responsible for synthesis of target compounds and evaluation in vitro. Xue Han was responsible for the synthesis of target

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compounds. Mengxiao Cao was responsible for evaluation in vitro and HPLC. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported in part by the Scientific and Technological ProJect of Henan Provincial (162300410103), the Key Scientific Research ProJect of Colleges and Universities in Henan Province (17B350002), the Special ProJect of Nanyang Normal University (ZX2016017 and STP2018004). We would like to thank Hunter Biotechnology, Inc. for the experiments on zebrafish AD model. ABBREVIATIONS USED AD, Alzheimer’s disease; Aβ, β-amyloid peptide; ACh, acetylcholine; AChEIs, acetylcholinesterase inhibitors; BuChE, butyrylcholinesterase; MAO, monoamine oxidase; MTDLs, multi-target-directed ligands; eeAChE, electric eel AChE; eqBuChE, equine serum BuChE; ORAC, Oxygen Radicals Absorbance Capacity; PDB, Protein Data Bank; ORAC-FL, Oxygen Radicals Absorbance Capacity by Fluorescence; ThT, thioflavin T; TEM, transmission electron microscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium;

PAMPA-BBB,

parallel

artificial membrane assay for the blood- brain barrier; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography;

DTNB,

5,5’-dithiobis-2-nitrobenzoic acid; PBL, Porcine brain lipid. REFERENCES (1) Price, M., Comas-Herrera, A., Knapp, M., Guerchet, M., Karagiannidou, M. (2016) World Alzheimer Report 2016. Improving healthcare for people living with dementia: Coverage, quality and costs now and in the future. Alzheimer’s Dis. Int, 1−131. (2) Kepp, K. P. (2016) Alzheimer’s disease due to loss of function: A new synthesis of the available data. Prog. Neurobiol. 143, 36. (3) Sanabria-Castro, A., Alvarado-Echeverría, I., and Monge-Bonilla, C. (2017) Molecular pathogenesis of Alzheimer’s disease: an update. Ann. Neurosci. 24 (1),

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46−54. (4) Livingston, G.; Sommerlad, A.; Orgeta, V.; Costafreda, S.G.; Huntley, J.; Ames, D.; Ballard, C.; BanerJee, S.; Burns, A.; Cohen-Mansfield, J.; Cooper, C.; Fox, N.; Gitlin, L.N.; Howard, R.; Kales, H.C.; Larson, E.B.; Ritchie, K.; Rockwood, K.; Sampson, E.L.; Samus, Q.; Schneider, L.S.; Selbæk, G.; Teri, L.; Mukadam, N. (2017) Dementia prevention, intervention, and care. Lancet, 390(10113): 2673-2734. (5) Dighe, S.N.; Deora, G.S.; De la Mora, E.; Nachon, F.; Can, S.; Parat, M.O.; Brazzolotto, X.; Ross, B.P. (2016) Discovery and structure-activity relationships

of

a

highly

selective

butyrycholinesterase

inhibitor

by

structure-based virtual screening. J. Med. Chem. 59: 7683-7689. (6) Košak, U.; Brus, B.; Knez, D.; ŽakelJ, S.; TrontelJ, J.; Pišlar, A.; Šink, R.; Jukič, M.; Živin, M.; Podkowa, A.; Nachon, F.; Brazzolotto, X.; StoJan, J.; Kos, J.; Coquelle, N.; Sałat, K.; Colletier, J.P.; Gobec, S. (2018) The magic of crystal structure-based inhibitor optimization: development of a Butyrylcholinesterase inhibitor with picomolar affinity and in vivo activity. J Med Chem. 61(1): 119-139. (7) Zheng, H., Amit, T., Bar-Am, O., Fridkin, M., Youdim, M.B., Mandel, S.A. (2012) From anti-Parkinson’s drug rasagiline to novel multitarget iron chelators with acetylcholinesterase and monoamine oxidase inhibitory and neuroprotective properties for Alzheimer’s disease. J Alzheimers Dis. 30(1); 1-16. (8) Caraci, F.; Copani, A.; Nicoletti, F.; Drago, F. (2010) Depression and Alzheimer’s disease: neurobiological links and common pharmacological targets. Eur. J. Pharmacol. 626(1), 64-71. (9) Bolea, I.; Juárez-Jiménez, J.; de Los Ríos, C.; Chioua, M.; Pouplana, R.; Luque, F. J.; Unzeta, M.; Marco-Contelles, J.; Samadi, A. (2011) Synthesis, biological evaluation,

and

molecular

modeling

of

donepezil

and

N-[(5-(benzyloxy)-1-methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine hybrids as new multipotent cholinesterase/monoamine oxidase inhibitors for the treatment of Alzheimer's disease. J. Med. Chem. 54(24), 8251-8270.

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(10)Mohamed, T.; Shakeri, A.; Rao, P.P. (2016) Amyloid cascade in Alzheimer’s disease: Recent advances in medicinal chemistry. Eur J Med Chem, 113: 258-272. (11)Wang, X., Wang, W., Li, L., Perry, G., Lee, H.G., Zhu, X. (2014) Oxidative stress and mitochondrial dysfunction in Alzheimer’s disease. Biochim Biophys Acta. 1842(8): 1240-1247. (12) Barage, S.H.; Sonawane, K.D. (2015) Amyloid cascade hypothesis: Pathogenesis and therapeutic strategies in Alzheimer's disease. Neuropeptides. 52: 1-18. (13)Zare-Shahabadi A., Masliah, E., Johnson, G.V., Rezaei, N. (2015). Autophagy in Alzheimer’s disease. Rev Neurosic. 26(4): 385-395. (14)Kumar, A., Dhawan, A., Kadam, A., Shinde, A. (2018) Autophagy and mitochondria: Targets in neurodegenerative disorders. CNS Neurol Disord Drug Targets. doi: 10.2174/1871527317666180816100203. (15)de Freitas Silva, M.; Dias, K.S.T.; GontiJo, V.S.; Ortiz, C.J.C.; Viega, Jr., C. (2018) Multi-target directed drugs as a modern approach for drug design towards Alzheimer's

disease:

an

update.

Curr

Med

Chem.

doi:

10.2174/0929867325666180111101843. (16)Umar, T.; Hoda, N. (2017) Alzheimer's disease: A systemic review of substantial therapeutic targets and the leading multi-functional molecules. Curr Top Med Chem. 17(31): 3370-3389. (17)Jones, M. R.; Mathieu, E.; Dyrager, C.; Faissner, S.; Vaillancourt, Z.; Korshavn, K. J.; Lim, M. H.; Ramamoorthy, A.; Wee Yong, V.; Tsutsui, S.; Stys, P. K.; Storr, T. (2017) Multi-target-directed phenol-triazole ligands as therapeutic agents for Alzheimer’s disease. Chem. Sci. 8, 5636−5643. (18)Sqarbossa, A.; Giacomazza, D.; di Carlo, M. (2015) Ferulic acid: A hope for Alzheimer’s disease therapy from plants. Nutrients. 7(7): 5764-5782. (19)Nabavi, S.F.; Devi, K.P.; Malar, D.S.; Sureda, A.; Daglia, M.; Nabavi, S.M. (2015) Ferulic-acid and Alzheimer’s disease: promises and pitfalls. Mini. Rev. Med. Chem. 15 (9): 776-788. (20)Sang, Z.; Pan, W.; Wang, K.; Ma, Q.; Yu, L.; Yang, Y.; Bai, P.; Leng, C.; Xu, Q.;

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Li, X.; Tan, Z.; Liu, W. (2017) Design, synthesis and evaluation of novel ferulic acid-O-alkylamine derivatives as potential multifunctional agents for the treatment of Alzheimer's disease. Eur. J. Med. Chem. 130, 379-392. (21)Li, T.; Pan, W.; Wang, K.; Liu, W.; Ma, Q.; Sang, Z. (2017) Novel ferulic aciddonepezil hybrids as multifunctional agents for the treatment of Alzheimer's disease with butyrylcholinesterase, amyloid-β, antioxidant and neuroprotective properties. Lett Drug Des Discov. 14 (8): 918-929. (22)Mao, F.; Wang, H.; Ni, W.; Zheng, X.; Wang, M.; Bao, K.; Ling, D.; Li, X.; Xu, Y.; Zhang, H.; Li, J. (2018) Design, synthesis, and biological evaluation of orally available first-generation dual-target selective inhibitors of acetylcholinesterase (AChE) and phosphodiesterase 5 (PDE5) for the treatment of Alzheimer's Disease. ACS Chem Neurosci. 9(2): 328-345. (23)Sang, Z.; Qiang, X.; Li, Y.; Yuan, W.; Liu, Q.; Shi, Y.; Ang, W.; Luo, Y.; Tan, Z.; Deng, Y. (2015) Design, synthesis and evaluation of scutellarein-O-alkylamines as multifunctional agents for the treatment of Alzheimer's disease. Eur. J. Med. Chem. 94, 348-366. (24)Davalos, A.; Gomez-Cordoves, C.; Bartolome, B. (2004) Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay. J. Agric. Food Chem. 52, 48-54. (25)Xiao, G.; Li, Y.; Qiang, X.; Xu, R.; Zheng, Y.; Cao, Z.; Luo, L.; Yang, X.; Sang, Z.; Su, F.; Deng, Y. (2017) Design, synthesis and biological evaluation of 4’-aminochalcone-rivastigmine

hybrids

as

multifunctional agents for

the

treatment of Alzheimer’s disease. Bioorg Med Chem. 25(3): 1030-1041. (26)Van der Walt, M.M., Terre Blanche, G., Petzer, J.P., Petzer, A. (2017) Benzyloxynitrostyrene analogues- A novel class of selective and highly potent inhibitors of monoamine oxidase B. Eur J Med Chem. 125: 1193-1199. (27)Joubert, J., Foka, G.B., Repsold, B.P., Oliver, D.W., Kapp, E., Malan, S.F. (2017) Synthesis and evaluation of 7-substituted coumarin derivatives as multimodal monoamine oxidase-B and cholinesterase inhibitors for the treatment of Alzheimer's disease. Eur J Med Chem. 125: 853-864.

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(28)Furukawa-Hibi, Y.; Alkam, T.; Nitta, A.; Matsuyama, A.; Mizoguchi, H.; Suzuki, K.; Moussaoui, S.; Yu, Q.S.; Greig, N.H.; Nagai, T.; Yamada, K. (2011) Butyrylcholinesterase inhibitors ameliorate cognitive dysfunction induced by amyloid-β peptide in mice. Behav Brain Res. 225(1): 222−229. (29)Deora, G.S.; Kantham, S.; Chan, S.; Dighe, S.N.; Veliyath, S.K.; McColl, G.; Parat, M.O.; McGeary, R.P.; Ross, B.P. (2017) Multifunctional analogs of kynurenic

acid

for

the

treatment

of Alzheimer's disease:

Synthesis,

pharmacology, and molecular modeling studies. ACS Chem Neurosic. 8(12): 2667-2675. (30)Mishra, C.B.; Kumari, S.; Manral, A.; Prakash, A.; Saini, V.; Lynn, A.M.; Tiwari M. (2017) Design, synthesis, in-silico and biological evaluation of novel donepezil derivatives as multi-target-directed ligands for the treatment of Alzheimer’s disease. Eur J Med Chem. 125: 736-750. (31)Amiri, M., Braidy, N., Aminzadeh, M. (2018) Protective effects of fibroblast growth factor 21 against amyloid-beta1-42-induced toxicity in SH-SY5Y cells. Neurotox Res. 34(3): 574-583. (32)Wu, A.G., Zeng, W., Wong, V.K., Zhu, Y.Z., Lo, A.C., Liu, L., Law, B.Y. (2017) Hederagenin

and

α-hederin

promote

degradation

of

proteins

in

neurodegenerative diseases and improve motor deficits in MPTP-mice. Pharmacol Res. 115: 25-44. (33)Law, B.Y., Mok, S.W., Chan, W.K., Xu, S.W., Wu, A.G., Yao, X.J., Wang, J.R., Liu, L., Wong, V.K. (2016) Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers. Oncotarget, 7(7): 8090-8104. (34)Di, L.; Kerns, E.H.; Fan, K.; McConnell, O.J.; Carter, G.T. (2003) High throughput artificial membrane permeability assay for blood-brain barrier, Eur. J. Med. Chem. 38, 223-232. (35)Hepnarova V, Korabecny J, Matouskova L, Jost P, Muckova L, Hrabinova M, Vykoukalova N, Kerhartova M, Kucera T, Dolezal R, Nepovimova E, Spilovska K, Mezeiova E, Pham NL, Jun D, Staud F, Kaping D, Kuca K, Soukup O. (2018) The concept of hybrid molecules of tacrine and benzyl quinolone carboxylic acid

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(BQCA) as multifunctional agents for Alzheimer's disease. Eur J Med Chem 150: 292-306. (36)Huang, W.; Li, C.; Shen, Z.; Zhu, X.; Xia, B.; Li, C. (2016) Development of a zebrafish model for rapid drug screening against Alzheimer’s disease. J Pharm Pharmacol. 2016, 4: 174-185. (37) Newman, M.; Ebrahimie, E.; Lardelli, M. (2014) Using the zebrafish model for Alzheimer’s disease research. Front Genet, 5: 189. (38) Abdel-Hafez AA, Meselhy MR, Nakamura N, Hattori M, Watanabe H, Murakami Y, El-Gendy MA, Mahfouz NM, Mohamed TA. (1998) Effects of paeoniflorin derivatives on scopolamine-induced amnesia using a passive avoidance task in mice; structure-activity relationship. Biol Pharm Bull. 21(11): 1174-1179. (39)Qiang X, Sang Z, Yuan W, Li Y, Liu Q, Bai P, Shi Y, Ang W, Tan Z, Deng Y. (2014) Design, synthesis and evaluation of genistein-O-alkylbenzylamines as potential multifunctional agents for the treatment of Alzheimer's disease. Eur J Med Chem 76: 314-331. (40)Xu, Y.; Zhang, J.; Wang, H.; Mao, F.; Bao, K.; Liu, W.; Zhu, J.; Li, X.; Zhang, H.; Li, J. (2018) Rational design of novel selective dual-target inhibitors of acetylcholinesterase and monoamine oxidase B as potential anti-Alzheimer's disease agents. ACS Chem Neurosci. doi:10.1021/acschemneuro.8b00357.

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Table of Contents graphic O CH3 N

O

Zebrafish AD Model N

O TM-10

EqBuChE IC50 = 0.0089 μ M;EeAChE IC50 = 12.1 μM hMAO-A IC50 = 6.3 μM ; hMAO-B IC50 = 8.6 μM Inhibit self-induced Aβ aggregation: 53.9% Disaggregate self-induced Aβ 1-42 aggregation: 43.8% ORAC = 0.55 eq; Autophagy activator Neuroprotective effect against Aβ 1-42 -mediated neurotoxicity Could cross PAMPA-BBB

Improve dyskinesia recovery rate and response efficiency on zebrafish AD Protect effect on Aβ 1-40-induced vascular injury.

In vivo assay

TM-10 could improve scopolamineinduced memory impairment

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