Accepted Manuscript Syntheses of Coumarin–Tacrine Hybrids as Dual-site Acetylcholinesterase Inhibitors and Their activity against Butylcholinesterase, Aβ aggregation, and βsecretase Qi Sun, Peng Da-Yong, Yang Sheng-Gang, Zhu Xiao-Lei, Yang Wen-Chao, Yang Guang-Fu PII: DOI: Reference:

S0968-0896(14)00503-3 http://dx.doi.org/10.1016/j.bmc.2014.06.057 BMC 11694

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

17 June 2014 30 June 2014 30 June 2014

Please cite this article as: Sun, Q., Da-Yong, P., Sheng-Gang, Y., Xiao-Lei, Z., Wen-Chao, Y., Guang-Fu, Y., Syntheses of Coumarin–Tacrine Hybrids as Dual-site Acetylcholinesterase Inhibitors and Their activity against Butylcholinesterase, Aβ aggregation, and β-secretase, Bioorganic & Medicinal Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.bmc.2014.06.057

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Syntheses of Coumarin–Tacrine Hybrids as Dual-site Acetylcholinesterase Inhibitors and Their activity against Butylcholinesterase, Aβ aggregation, and β-secretase

Sun, Qi†,a, Peng Da-Yong†,a, Yang Sheng-Ganga, Zhu Xiao-Leia, Yang Wen-Chaoa,*, Yang Guang-Fua,b,* a

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry,

Central China Normal University, Wuhan 430079, P.R. China; b

Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, P.R. China.

†

Qi Sun and Da-Yong Peng are co-first authors due to their equal contribution.

*Corresponding authors: Tel.: +86-27-67867800; fax: +86-27-67867141. Email address: [email protected] (W. C. Yang), [email protected] (G. F. Yang).

Keywords: Alzheimer’s disease, coumarin, acetylcholinesterase inhibitor, butylcholinesterase inhibitor.

Abbreviations： ： AD, Alzheimer’s disease; AChE, acetylcholinesterase; BChE, butylcholinesterase; ACh, acetylcholine; Aβ, amyloid-β; CAS, catalytic active site; PAS, peripheral active site; MD, molecular dynamics; ATC, acetylthiocholine; ESI-MS, electrospray ionisation mass spectrometry; DMF, dimethylformamide; DTNB, 5’, 5’-Dithio-bis(2-nitrobenzoic acid); LGA, Lamarkian

genetic

algorithm;

EDCI,

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride; HOBt, N-Hydroxybenzotriazole; PyBOP, benzotriazole-1-yl-oxytripyrrolidino -phosphonium hexafluorophosphate.

1

Abstract Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer’s disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine-coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (Ki = 16.7 nM) against human AChE and about 2-fold lower potency (Ki = 16.1 nM) against BChE than tacrine (Ki = 35.7 nM for AChE, Ki = 8.7 nM for BChE), respectively. In addition, some of the tacrine-coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine-coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.

2

1. Introduction

As a complex neurodegenerative disorder, Alzheimer’s disease (AD) is the most common type of dementia among people over age 65. It has been estimated that AD affected more than 37 million individuals worldwide and the population is still increasing, which makes the human cost incalculable.1 Though the specific cause of AD remains enigmatic2, most researchers support that several pathological phenomena are closely associated with AD lesions, such as a decrease of the neurotransmitter acetylcholine (ACh), formation of amyloid β-protein (Aβ) plaques, and abnormal posttranslational modifications of tau protein to yield neurofibrillary tangles.3-6 During the last twenty years, experts have done extensive research on reducing or clearing the related AD pathological phenomena with different strategies. As to the cholinergic hypothesis, two major strategies of developing acetylcholinesterase (AChE) inhibitors and designing ACh receptor agonists were used to balance the ACh level.7-9 Meanwhile, two tactics were employed to relieve the Aβ aggregation.6, 10 The first one was to design β-secretase or γ-secretase inhibitors, and non-natural amino acid or transition metal complexes for blockage of amyloid plaques. The second one was to utilize antibodies or catalytic antibodies to bind and/or degrade Aβ. Aimed to suppress the abnormal posttranslational modifications of Tau protein, various methods were carried out, such as designing antagonists to inhibit serine/threonine kinases or agonists to promote the dephosphorylation of hyper-phosphorylated Tau protein.11 Among the intensive efforts of above strategies, exploring small-molecule AChE inhibitors to slow the breakdown of ACh represents the mainstream direction for AD therapy. Among six drugs that have been approved by FDA for AD treatment, five of them are AChE inhibitors (the chemical structures are showed in Figure 1).5, 8, 11-14 However, these medicines only confer modest beneficial effects on mild to moderate AD patients. Considering the multifactorial pathogenesis of AD, design and synthesis of dual-site/multi-site AChE inhibitors that simultaneously interact with multiple subsites of the gorge-like pocket, and multifunctional AChE inhibitors targeting AChE, as well as other targets like butylcholinesterase (BChE), Aβ aggregation or β-secretase, may provide more effective candidates for AD drug discovery.15-18 Tacrine is the first AChE inhibitor permitted by the FDA to enter the medical market. 3

Although it exhibited some side effects after a long period of practical using, tacrine is still of interest due to its classical pharmacophore for potent AChE inhibition and well-known action mode. To discover new tacrine derivatives with higher activity and multiple function, a variety of hybrids have been synthesized 19, such as tacrine-melatonin hybrids20, tacrine-donepezil hybrids21, tacrine-carbazole hybrids16, tacrine-huperzine hybrids22, and tacrine-oxoisoaporphine hybrids23. As a fragrant compound, coumarin widely distributed in nature and displayed extensive biological activities, such as antioxidant, preventing asthma and antisepsis.24-29 Moreover, coumarin derivatives are usually easy to synthesize and possess good solubility, low cytotoxicity, and excellent cell permeability. Although tacrine and coumarin scaffolds showed great potency in AD drug design, few report designed coumarin-tacrine hybrids as novel AChE inhibitors, and only one report utilized piperazine as the linker at the 7th position of coumarin.30 Thus, it was expected that hybrid of tacrine-coumarin would improve the potency and spectrum compared with tacrine alone. Herein, a series of hybrids of tacrine-coumarin (1a-r) using methylene chain as linkers were designed, synthesized, and the structure and activity relationships (SARs) were evaluated. The substitution was introduced at the 3rd position of coumarin with the methylene chain as the spacer. The binding modes of representative hybrids in human AChE and BChE were further studied by molecular modeling. Based on the bioassays on multi-target, it demonstrated that some novel compounds could be multifunctional leads for AD therapy.

2. Results and discussion 2.1. Chemistry In this study, eighteen hybrids of coumarin–tacrine were synthesized. All of them were reported for the first time. The general route to the synthesis of the hybrids was illustrated in Scheme1. Starting from anthranilic acid, the related N1-(1,2,3,4-Tetrahydroacri-din-9-yl) alkane-1, n-diamines (2) were obtained in good yields by following the reported method.31 The other key intermediate, 2-oxo-2H-chromene-3-carboxylic acid (3), such as 6-methoxy-, 7-methoxy-, 6,7-dimethoxy-, 6-methyl- and 6-trifluoromethyl-2-oxo- 2H-chromene-3carboxylic acid, were synthesized according to a conventional route except 2-oxo-2H 4

-chromene-3-carboxylic acid is commercially available. In the beginning, N1-(1,2,3,4tetrahydroacridin-9-yl) heptane-1,7-diamine (2) was treated with 2-oxo-2H-chromene3-carboxylic acid (3) in the presence of 1-(3-dimethyl -aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and a catalytic amount of N-hydroxybenzotriazole (HOBt), producing the title compounds in relatively low yields. Alternatively, the title compounds were prepared in

moderate

yields

by

utilizing

benzotriazole-1-yl-oxytripyrrolidino

-phosphoniumhexafluoro-phosphate (PyBOP) as coupling agent. The chemical structures of all new compounds were fully characterized by 1 H NMR,

13

C NMR, elemental analysis, and

MS spectra.

2.2. Human AChE inhibition and structure-activity relationship study The anti-AChE activity of newly synthesized hybrids 1a-r was evaluated (Table 1). Generally, all the title compounds demonstrated inhibitory activity against AChE with inhibitory constants (Ki) at nanomolar level. As indicated in Table 1, compound 1g displayed the highest activity with Ki of 16.7 ± 3.87 nM, about 2-fold higher than tacrine and 3.5-fold higher than galanthamine, respectively. For the sake of clarity, all the compounds were divided into three groups according to the methylene number as 5 methylenes, 6 methylenes and 7 methylenes. As seen in Table 1, the linker length influences on the inhibitory activities of new compounds. The 6 methylenes linker seemed to the best suitable length for AChE inhibition. Those compounds that possessed shorter (such as 5) or longer (such as 7) methylenes linker demonstrated decreased inhibitory activity. Secondly, the substitutions on the benzene ring of coumarin at the 6 th and 7th position also influence the activity. The presence of electron-donating group at the 6th position, OCH3 and OCF3, would reduce the potency of new compounds. In addition, the presence of OCH3 substitution at the 7th position showed decreased inhibitory, which is indicating that the steric hindrance at this position is detrimental for AChE inhibition. To elucidate the correlation between the variation of the chain length and the inhibitory potency, molecular docking and molecular dynamics (MD) simulation were performed. It was observed that all the compounds shared a conserved binding mode. Compounds 1a, 1g and 1m were chosen as representatives, and their binding modes in AChE active site were 5

demonstrated in Figure 2. The tacrine part and coumarin part of the hybrids bind with catalytic active site (CAS) and peripheral active site (PAS), respectively. In more detail, the tacrine moiety was located between Trp86 and Tyr337, and the coumarin moiety between Trp286 and Tyr72, which were both apt to form π-π stacking interaction. In addition, two visible hydrogen bonds formed: one was formed between carbonyl amide of coumarin and Tyr124, the other was created between tacrine moiety and His447. It was noted that the only distinction among the binding modes of the three compounds was the length of hydrogen bonds formed by amide linkage and the surrounding residue Tyr124. The corresponding distances of these hydrogen bindings for compounds 1a, 1g and 1m were 2.3 Å, 1.9 Å and 2.6 Å, respectively, which are consistent with the order of their inhibitory activities (34.4 nM for 1a, 16.7 nM for 1g and 42.2 nM for 1m).

2.3. Human BChE inhibition and selectivity for AChE/BChE In addition to AChE, BChE has now been recognized as another target because it also regulates ACh level in brain.32, 33 It is noteworthy that the relative proportions of AChE to BChE in brain are dependent on the regions and the stages of disease progression in AD patients, which indicated that the balanced inhibitor of both AChE and BChE might result in better efficacy for AD therapy.32 Therefore, we further screened and kinetically characterized all the newly synthesized compounds against human BChE using tacrine and galanthamine as references. As expected, all new compounds showed good activity for BChE inhibition in nanomolar range, and the inhibitory constants (Ki) were summarized in Table 1. According to the inhibitory kinetic findings, compound 1e displayed about 8-fold better inhibitory activity against human AChE than that of BChE, and its selectivity for AChE over BChE increased 2-fold compared to the selectivity of tacrine. Another interesting point is that compounds 1a, 1d, 1g and 1i showed almost equivalent activity against both human AChE and BChE. Though approved by FDA as the first AChE inhibitor, tacrine actually possessed 4-fold higher activity for human BChE over AChE. However, the structural basis for the selectivity of tacrine on two cholinesterases remained undiscussed. Herein, molecular docking and MD simulation, as well as the binding free energy calculation were carried out to explore the selectivities of tacrine and compound 1g on two cholinesterases. According to the 6

superimposition of the crystal structures of human AChE (PDB ID, 1b41) and BChE (PDB ID, 1P0M), the most remarkable difference of the active sites is that, Tyr337 existed as the bottle neck of AChE cavity, while the situation for equivalent residue Ala328 was not same in active site of BChE. As showed in Figure 3A, tacrine strongly binded in CAS of AChE due to the following two major interactions: π-π stacking interaction with tow aromatic residues Trp86 and Tyr337, and the hydrogen bonding (1.6 Å) interaction with His447. In BChE, the less bulky residue Ala328 (equivalent to Tyr337 in AChE) might contribute to the orientation reversal of the amino group of tacrine. This orientation reversal abolished the hydrogen bonding with His438 (equivalent to His447 in AChE), but introduced another two hydrogen bonds (1.7 Å and 2.2 Å) with Glu197 (Figure 3B). As summarized in Table 2, the binding free energies (△Gcal) of tacrine for AChE and BChE are -11.9 and -13.4 kcal/mol, respectively, which are qualitatively in good agreement with the experimentally-derived binding free energies (△Gexp) (-10.2 and -11.0 kcal/mol). Consequently, it disclosed that the two hydrogen bonds formed by amino group of tacrine with Glu197 of BChE might account for 4-fold superior potency of tacrine against BChE. Unlike tacrine，the newly designed hybrids of tacrine-coumarin are believed to be dual site AChE inhibitors. Compound 1g was selected to illustrate the binding modes of the hybrids. Not surprisely, the tacrine part and coumarin part of 1g interacted with CAS and PAS of AChE, respectively (Figure 3C). In the simulated binding model, the binding mode of tacrine moiety for 1g in CAS was quite similar to the binding mode of tacrine alone. While the coumarin moiety additionally formed significant interactions in PAS: hydrogen bond (2.2 Å) between the amide oxygen and Tyr124, and the π-π stacking interaction associated with two aromatic residues (Trp286 and Tyr72). The overall calculated binding free energy for compound 1g (-12.7 kcal/mol) is slightly higher than that of tacrine (-11.9 kcal/mol) in AChE, which explained higher potency of compound 1g in comparison with tacrine. Inspection of the binding mode for compound 1g in BChE is helpful to understand the corresponding selectivity. In Figure 3D, compound 1g formed π-π stacking interaction with Trp82 and His438, and formed three hydrogen bonds (2.2 Å, 1.9 Å and 2.8 Å) with His438, Gln189 and Asn289, respectively. As showed in Table 2, either experimental or calculated free energies for compound 1g binding in two cholinesterases are almost identical, both 7

demonstrating equal potencies of compound 1g for two cholinesterases.

2.4. Inhibition of Aβ aggregation and β-secretase Recently, discovery of new AChE inhibitors as multi-target-directed ligands (MTDLs) addressing distinct AD-relevant targets has been an active area in AD research.20, 34, 35 Due to reported involvement of Aβ aggregation and β-secretase in AD progression, inhibitors targeting Aβ aggregation and β-secretase has attracted great research interest in discovery for disease-modifying agents.13,

36-38

We therefore tested the inhibitory activity of our newly

synthesized hybrids of tacrine-coumarin against Aβ aggregation and β-secretase, using curcumin as reference. The capability of all the title compounds was firstly evaluated at a single concentration (100 µM) against Aβ aggregation and β-secretase by fluorometric assay reported previously, followed by further kinetic characterization of promising candidates if their inhibition rates were no less than 50% percent at that screening concentration. As displayed in Table 3, compounds 1l, 1o and 1p, showed modest to high potency against both Aβ aggregation and β-secretase. More importantly, compared with curcumin (IC50 = 11.0 µM), compounds 1j and 1p revealed better activities against Aβ aggregation with IC50 values of ~5.0 µM and ~6.1 µM, respectively.

3. Conclusion

Although the long-term clinical application was hindered due to some side effects, tacrine and its derivatives have attracted extensive attention because of their high potency against AChE and good understanding of the action mode. In present work, a series of hybrids of tacrine-coumarin were designed, synthesized, and further evaluated as dual-site human AChE inhibitors. All the hybrids exhibited Ki values in the nanomolar range and some of them were more potent than tacrine. In particular, compound 1g was identified as the most potent dual-site AChE inhibitor with a Ki value of 16.7 nM, about 2-fold and 3.5-fold higher potency than tacrine and galanthamine, respectively. Moreover, the inhibitory effects toward human BChE, Aβ aggregation, and β-secretase of all the title compounds were also evaluated. Interestingly, compounds 1l and 1o-p displayed moderate to high activities for all the tested 8

targets, which indicated that they could be multifunctional lead candidates for AD therapy.

4. Experimental Section

4.1. Methods and synthesis Unless otherwise noted, all chemical reagents were commercially available and treated with standard methods before use. Solvents were dried in a routine way and redistilled before use. 1H NMR and

13

C NMR spectra were recorded on a VARIAN Mercury-Plus 600 or 400

spectrometer in CDCl3 or DMSO-d 6 with TMS as the internal reference. Mass spectral data were obtained on a ThermoFisher Mass platform DSQII by electrospray ionization (ESI-MS). Elemental analyses were performed on a Vario EL III elementary analysis instrument. Melting points were taken on a Buchi B-545 melting point apparatus and are uncorrected. All chemical yields are unoptimized and generally represent the result of a single experiment.

4.1.1. General procedure for the synthesis of tacrine-2-oxo-3H-chromene hybrids (1) To a mixture of the corresponding 2-oxo-2H-chromene-3-carboxylic acid derivative 3 (1.0 mmol) and benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 1.3 mmol) in CH2Cl2 (20 mL), then appropriate 9-alkylaminotetrahydroacridine 2 (1.0 mmol) and triethylamine (2.6 mmol) were added. The reaction system was stirred at room temperature overnight and then diluted with CH2Cl2 (50 mL). The resulting mixture was consecutively washed with 10% citric acid solution (aq, 3 × 30 mL), 10% NaHCO3 solution (aq, 3 × 30 mL), and H2O (30 mL). Then the organic phase was dried over by sodium sulfate and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using mixtures of Petrol/EtOAc/Et3N (10:3:1) to afford the corresponding hybrids of tacrine-coumarin (1).

4.1.2. 2-oxo-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-2H-chromene-3carboxamide (1a) Yellow solid; Yield 19%; m.p. 102-105 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.92 (s, 1H), 8.89 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.73 - 7.66 (m, 2H), 7.59 - 7.54 (m, 9

1H), 7.45 - 7.35 (m, 3H), 4.04 (br, 1H), 3.57 - 3.48 (m, 4H), 3.08 (t, J = 5.4Hz ,2H), 2.74 (t, J = 6.6Hz ,2H), 1.97 - 1.90 (m, 4H), 1.80 - 1.68 (m, 4H), 1.58 - 1.49 (m, 2H). 13C NMR (100 MHz, 100 MHz, CDCl3) δ 161.32, 161.29, 157.97, 154.07, 150.58, 148.10, 146.86, 133.88, 129.58, 128.14, 125.11, 123.45, 122.67, 119.80, 118.32, 118.03,116.33,115.53,49.05, 39.36, 33.57, 31.14, 28.99, 24.58, 24.13, 22.77, 22.47. MS (EI) m/z: 455.28 (M)+. Anal. Calcd. for C28H29N3O3: C, 73.82; H, 6.42; N,9.22. Found: C, 73.80; H, 6.23; N, 8.99.

4.1.3.

7-methoxy-2-oxo-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-2H-chrom

ene-3-carboxamide (1b) Yellow solid; Yield 41%; m.p. 161-164 ◦C; 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.87 (s, 1H), 8.05 - 7.91 (m, 2H), 7.62 - 7.50 (m, 2H), 7.39 - 7.29 (m, 1H), 6.94 (s, 1H), 6.86 (s, 1H), 3.98 (br, 1H), 3.92 (s, 3H), 3.59 - 3.40 (m, 4H), 3.07 (t, J =6.0 Hz ,2H), 2.71 (t, J = 5.2 Hz, 2H), 1.98 - 1.82(m, 4H), 1.79 - 1.60 (m, 4H), 1.56 - 1.40 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 164.61, 161.88, 161.73, 158.07, 156.37, 150.59, 148.07, 147.02, 130.73, 128.31, 128.17, 123.48, 122.71, 119.90, 115.62, 114.45, 113.85, 112.16, 100.04, 55.86, 49.13, 39.29, 33.72, 31.21, 29.12, 24.65, 24.20, 22.84, 22.55.MS (EI) m/z: 485.27 (M)+. Anal. Calcd. for C29H31N3O4: C, 71.73; H, 6.44; N,8.65. Found: C, 72.01; H, 6.23; N, 8.41.

4.1.4.

6-methoxy-2-oxo-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-2H-Chrom

ene-3-carboxamide (1c) Yellow solid; Yield 38%; m.p. 171-173 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.93 (s, 1H), 8.87 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.59 - 7.53(m, 1H), 7.39 - 7.34 (m, 2H), 7.28 - 7.24 (m, 1H), 7.08 (s, 1H), 4.07 (br, 1H), 3.90 (s, 3H), 3.58 - 3.46 (m, 4H), 3.09 (t, J =7.8 Hz, 2H), 2.73 (t, J = 6.0Hz, 2H), 1.97 - 1.90 (m, 4H), 1.79 - 1.67 (m, 4H), 1.56 - 1.49 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 161.47, 158.11, 156.41, 150.62, 148.77, 147.98, 147.03, 128.32，128.24, 123.54, 122.73, 122.47, 119.93, 118.78, 118.29, 117.55, 115.67, 110.41, 55.78, 49.17, 39.43, 33.74, 31.25, 29.10, 24.69, 24.24, 22.88, 22.59. MS (EI) m/z: 485.33 (M)+. Anal. Calcd. for C29H31N3O4: C, 71.73; H, 6.44; N,8.65. Found: C, 71.80; H, 6.23; N, 8.41. 4.1.5. 6-methyl-2-oxo-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-2H-chromene -3-carboxamide (1d) 10

Yellow solid; Yield 28%; m.p. 161-164 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.88 (s, 1H), 8.85 (s, 1H), 7.99 - 7.89 (m, 2H), 7.55 (m, 1H), 7.49 - 7.43 (m, 2H), 7.37 - 7.32 (m, 1H), 7.30 (d, J = 8.3 Hz, 1H), 4.03 (br, 1H), 3.56 - 3.42 (m, 4H), 3.08 (t, J = 6.6 Hz, 2H), 2.71 (t, J = 6.6 Hz, 2H), 2.44 (s, 3H), 1.95 -1.84 (m, 4H), 1.77 - 1.64 (m, 4H), 1.55 -1.48 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 161.57, 158.34, 152.43, 150.51, 148.25, 147.33, 135.16, 135.10, 129.26, 128.61, 128.15, 123.54, 122.72, 120.10, 118.24, 118.01, 116.18, 115.87, 49.24, 39.46, 33.97, 31.31, 29.14, 24.75, 24.28, 22.95, 22.68, 20.70. MS (EI) m/z: 469.20(M)+. Anal. Calcd. for C29H31N3O3: C, 74.18; H, 6.65; N,8.95. Found: C, 74.40; H, 6.93; N, 8.71.

4.1.6. 6,7-dimethoxy-2-oxo-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-2H-chro mene-3-carboxamide (1e) Yellow solid; Yield 25%; m.p. 85-87 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.87 (s, 1H), 8.82 (s, 1H), 8.08 - 7.92 (m, 3H), 7.61 - 7.52 (m, 1H), 7.37 (s, 1H), 7.01 (s, 1H), 6.91 (s, 1H), 4.00 (s, 3H), 3.96 (s, 3H), 3.66 - 3.56 (m, 4H), 3.09 (t, J = 6.6 Hz ,2H), 2.71 (t, J = 7.2 Hz ,2H), 1.95 1.86 (m, 4H), 1.79 - 1.63 (m, 4H), 1.54 - 1.48 (m, 2H). 13C NMR (100MHz, CDCl3) δ 162.73, 162.07, 157.78, 154.04, 150.68, 146.68, 141.19, 132.15, 130.71, 128.29, 127.97, 123.49, 122.70, 119.72, 115.46, 111.83, 109.27, 101.01,56.23, 55.75, 48.98, 38.45, 33.50, 31.19, 28.69, 24.54, 24.21, 22.74, 22.43.MS (EI) m/z: 515.28 (M)+. Anal. Calcd. for C30H33N3O5: C, 69.88; H, 6.45; N,8.15. Found: C, 70.10; H, 6.73; N, 8.21.

4.1.7. 2-oxo-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-6-(trifluoro methoxy) -2H-chromene-3-carboxamide (1f) Yellow solid; Yield 30%; m.p. 111-114 ◦C; 1H NMR (400 MHz, CDCl3) δ 8.86 (s, 1H), 8.76 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.59 - 7.49 (m, 3H), 7.45 (d, J = 8.9 Hz, 1H), 7.39 - 7.30 (m, 1H), 3.96 (br, 1H), 3.56 - 3.41 (m, 4H), 3.06 (t, J = 5.6 Hz, 2H), 2.72 (t, J = 5.2 Hz, 2H), 1.99 - 1.85 (m, 4H), 1.79 - 1.63 (m, 4H), 1.57 - 1.46 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 160.50, 160.44, 158.06, 151.95, 150.21, 147.05, 146.79, 145.14, 145.12, 128.37, 127.86, 126.48, 123.25, 122.46, 121.03,120.05(q, J C-F = 257 Hz), 119.86, 119.25, 118.89, 117.84, 115.62, 48.89, 39.35, 33.71, 31.01, 28.85, 24.53, 24.02, 22.71, 22.44.MS (EI)

11

m/z: 539.11(M)+. Anal. Calcd. for C29H28F3N3O3: C,64.56; H, 5.23; N,7.79. Found: C, 64.80; H, 5.23; N, 8.01.

4.1.8. 2-oxo-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-2H-chromene-3carboxamide (1g) Yellow solid; Yield 41%; m.p. 103-105 ◦C; 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 8.83 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.75 - 7.60 (m, 2H), 7.57 - 7.49 (m, 1H), 7.43 - 7.29 (m, 3H), 3.99 (br, 1H), 3.55 - 3.40 (m, 4H), 3.05 (t, J = 8.4 Hz, 2H), 2.70 (t, J = 8.4 Hz,2H), 1.98 - 1.81 (m, 4H), 1.76 - 1.59 (m, 4H), 1.53 - 1.33 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 161.14, 161.08, 158.10, 153.92, 150.29, 147.88, 147.19, 133.67, 129.41, 128.47, 127.86, 124.94, 123.23, 122.56, 119.91, 118.20, 118.01, 116.17, 115.60, 49.03, 39.35, 33.85, 31.34, 28.98, 26.44, 26.29, 24.53, 22.76, 22.53. MS (EI) m/z: 469.20 (M)+. Anal. Calcd. for C29H31N3O3: C, 74.18; H, 6.65; N, 8.95. Found: C, 74.32; H, 6.43; N, 8.71.

4.1.9. 6-methoxy-2-oxo-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-2H-chromene -3-carboxamide (1h) Yellow solid; Yield 31%; m.p. 108-111 ◦C; 1H NMR (400 MHz, cdcl3) δ 8.91 (s, 1H), 8.81 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.56 - 7.41 (m, 2H), 7.36 - 7.18 (m, 2H), 7.01 (s, 1H), 4.40 (br, 1H), 3.77 (s, 3H), 3.49 - 3.33 (m, 4H), 3.01 (t, J = 6.0 Hz, 2H), 2.65 (t, J = 6.0 Hz, 2H), 1.85 - 1.69 (m, 4H), 1.66 - 1.51 (m, 4H), 1.41 -1.26 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 161.30, 156.96, 156.21, 151.17, 148.55, 147.79, 145.72, 128.55, 126.97, 123.47, 122.84, 122.28, 119.18, 118.59, 118.08, 117.32, 114.74, 110.26, 55.61, 48.84, 39.36, 32.74, 31.24, 28.97, 26.62, 26.25, 24.37, 22.56, 22.16. MS (EI) m/z: 499.27 (M)+. Anal. Calcd. for C30H33N3O4: C, 72.12; H, 6.66; N, 8.41. Found: C, 73.91; H, 6.43; N, 8.21.

4.1.10.

7-methoxy-2-oxo-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-2H-chrom

ene-3-carboxamide (1i) Yellow solid; Yield 21%; m.p. 131-132 ◦ C; 1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.78 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.59 - 7.47 (m, 2H), 7.39 - 7.30 (m, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.85 (s, 1H), 3.94 (br, 1H), 3.91 (s, 3H), 3.53 – 3.39 (m, 4H), 12

3.05 (t, J = 6.8 Hz ,2H), 2.71 (t, J = 6.8 Hz ,2H), 1.96 -1.80 (m, 4H), 1.74 - 1.60 (m, 4H), 1.49 - 1.41 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 164.29, 161.53, 161.39, 157.89, 156.05, 150.29, 147.62, 146.96, 130.42, 128.20, 127.80, 123.14, 122.49, 119.79, 115.42, 114.27, 113.46, 111.88, 99.77, 55.59, 48.87, 39.17, 33.61, 31.21, 28.95, 26.36, 26.20, 24.44, 22.67, 22.39. MS (EI) m/z: 499.19 (M)+. Anal. Calcd. for C30H33N3O4: C, 72.12; H, 6.66; N, 8.41. Found: C, 71.95; H, 6.93; N, 8.71.

4.1.11. 6-methyl-2-oxo-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-2H-chromene -3-carboxamide (1j) Yellow solid; Yield 40%; m.p. 130-132 ◦ C; 1H NMR (600 MHz, CDCl3) δ 8.86 (s, 1H), 8.84 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.56 - 7.50 (m, 1H), 7.48 - 7.42 (m, 2H), 7.36 - 7.31 (m, 1H), 7.28 (d, J = 8.4 Hz, 1H), 3.95 (br, 1H), 3.51-3.41 (m, 4H), 3.06(t, J = 6.6 Hz, 2H), 2.72(t, J = 6.0 Hz, 2H), 2.44 (s, 3H), 1.94-1.87 (m, 4H), 1.72 - 1.60 (m, 4H), 1.49 - 1.40 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 161.57, 161.45, 158.27, 152.34, 150.52, 148.12, 147.31, 135.05, 135.00, 129.18, 128.58, 128.06, 123.42, 122.71, 120.06, 118.18, 118.00, 116.09, 115.76, 49.22, 39.48, 33.95, 31.50, 29.13, 26.60, 26.45, 24.66, 22.91, 22.65, 20.64. MS (EI) m/z: 547.15(M)+. Anal. Calcd. for C30H33N3O3: C,74.51; H, 6.88; N,8.69. Found: C, 74.80; H, 6.63; N, 8.41.

4.1.12. 6,7-dimethoxy-2-oxo-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-2H -chromene-3-carboxamide (1k) Yellow solid; Yield 29%; m.p. 134-137 ◦ C; 1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.78 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.59 - 7.47 (m,1H), 7.39 - 7.30 (m, 1H), 6.93 (s, 1H), 6.85 (s, 1H), 4.04 (br, 1H), 3.97 (s, 3H), 3.91 (s, 3H),3.53 - 3.39 (m, 4H), 3.05 (t, J = 6.8 Hz ,2H), 2.71 (t, J = 6.8 Hz ,2H), 1.96 -1.80 (m, 4H), 1.74 - 1.56 (m, 4H), 1.49 - 1.34 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 162.01, 161.95, 157.92, 154.95, 151.00, 150.93,147.95, 147.00, 128.43, 128.08, 123.60, 122.86, 122.46, 119.82, 115.46, 114.83, 111.38, 108.51, 99.20, 56.57, 56.31, 51.69, 49.23, 39.46, 33.57, 31.53, 29.23, 26.63, 26.48, 24.64, 23.84, 22.88, 22.56. MS (EI) m/z: 529.25 (M)+. Anal. Calcd. for C31H35N3O5: C, 70.30; H, 6.66; N,7.93. Found: C, 70.49; H, 6.73; N, 8.21. 13

4.1.13.

2-oxo-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-6-(trifluoromethoxy)

-2H-chromene-3-carboxamide (1l) Yellow solid; Yield 36%; m.p. 116-119 ◦C; 1H NMR (400 MHz, CDCl3) δ 8.86 (s, 1H), 8.74 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.60 - 7.48 (m, 3H), 7.43 (d, J = 8.8 Hz, 1H), 7.39 - 7.30 (m, 1H), 3.96 (br, 1H), 3.56 - 3.41 (m, 4H), 3.06 (d, J = 5.6 Hz, 2H), 2.72 (d, J = 5.2 Hz, 2H), 1.97-1.88 (m, 4H), 1.75 - 1.60 (m, 4H), 1.51-1.41 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 160.26, 160.23, 157.75, 151.74, 150.13, 146.79, 146.54, 144.85, 128.09, 127.66, 126.18, 122.99, 122.35, 120.88,119.84(q, J

C-F

= 257Hz), 119.62, 119.07, 118.73,

117.57, 115.29, 48.69, 39.22, 33.47, 31.05, 28.72, 26.20, 26.05, 24.30, 22.52, 22.25. MS (EI) m/z: 553.15(M)+. Anal. Calcd. for C30H30F3N3O3: C,65.09; H, 5.46; N,7.59. Found: C, 64.80; H, 5.23; N, 7.81.

4.1.14.

2-oxo-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-2H-chromene-3

-carboxamide (1m) Yellow solid; Yield 65%; m.p. 99-101 ◦ C; 1H NMR (600 MHz, CDCl3) δ 8.91 (s, 1H), 8.84 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.72 - 7.64 (m, 2H), 7.58 - 7.52 (m, 1H), 7.43 - 7.33 (m, 3H), 4.06 (br, 1H), 3.57 - 3.41 (m, 4H), 3.06 (t, J = 6.6 Hz ,2H), 2.70 (t, J = 6.0 Hz ,2H), 1.95 - 1.87 (m, 4H), 1.72 - 1.60 (m, 4H), 1.45 - 1.36 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 161.33, 161.33,157.45, 154.17, 151.18, 148.16, 146.24, 133.94, 129.66, 128.60, 127.52, 125.20, 123.58, 122.93, 119.50, 118.45, 118.23, 116.43, 115.05, 49.22, 39.66, 33.12, 31.49, 29.09, 28.83, 27.85, 26.66, 24.51, 22.76, 22.39. MS (EI) m/z: 483.32 (M)+. Anal. Calcd. for C30H33N3O3: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.80; H, 6.63; N, 8.41.

4.1.15.

7-methoxy-2-oxo-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-2H-chro

mene-3-carboxamide (1n) Yellow liquid; Yield 51%; 1H NMR (600 MHz, CDCl3) δ 8.81 (s, 1H), 8.76 (s, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.56 - 7.48 (m, 2H), 7.35 - 7.29 (m, 1H), 6.92 - 6.88 (m, 1H), 6.82 (s, 1H), 4.00 (br, 1H), 3.88 (s, 3H), 3.51 - 3.39 (m, 4H), 3.04 (t, J = 6.6 Hz, 2H), 2.68 (t, J = 7.2 Hz, 2H), 1.93 - 1.87 (m, 4H), 1.71 - 1.56 (m, 4H), 1.42 - 1.32 (m, 6H). 13C 14

NMR (100 MHz, CDCl3) δ 164.46, 161.69, 161.62, 157.85, 156.23, 150.66, 147.87, 146.85, 130.62, 128.10, 123.32, 122.73, 119.74, 115.33, 114.41, 113.70, 112.06, 99.92, 55.76, 49.15, 39.43, 33.55, 31.41, 29.04, 28.75, 26.60, 26.56, 24.49, 22.75, 22.46. MS (EI) m/z: 513.30 (M)+. Anal. Calcd. for C31H35N3O4: C, 72.49; H, 6.87; N, 8.18. Found: C, 72.20; H, 6.66; N, 8.41.

4.1.16. 6-methoxy-2-oxo-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-2H-chrom ene -3-carboxamide (1o) Yellow liquid; Yield 39%; 1H NMR (600 MHz, CDCl3) δ 8.89 (s, 1H), 8.85 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.57 - 7.51 (m, 1H), 7.36 - 7.29 (m, 2H), 7.24 - 7.20 (m, 1H), 7.05 (s, 1H), 4.08 (br, 1H), 3.86 (s, 3H), 3.53 - 3.42 (m, 4H), 3.05 (t, J =5.4 Hz ,2H), 2.69 (t, J = 6.6 Hz ,2H), 1.94 - 1.87 (m, 4H), 1.72 - 1.58 (m, 4H), 1.45 - 1.34 (m, 6H).

13

C

NMR (100 MHz, CDCl3) δ 161.45, 161.33, 157.55, 156.34, 151.01, 148.69, 147.88, 146.41, 128.43, 127.67, 123.49, 122.86, 122.37, 119.55, 118.73, 118.28, 117.46, 115.12, 110.36, 55.72, 49.18, 39.58, 33.24, 31.44, 29.04, 28.79, 26.66, 26.60, 24.49, 22.74, 22.39.MS (EI) m/z: 513.28 (M)+. Anal. Calcd. for C31H35N3O4: C, 72.49; H, 6.87; N, 8.18. Found: C, 72.70; H, 6.93; N, 8.21.

4.1.17. 6-methyl-2-oxo-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-2H-chromen e-3-carboxamide (1p) Yellow solid; Yield 28%; m.p. 84-87 ◦C; 1H NMR (600 MHz, CDCl3) δ 8.87-8.81 (m, 2H), 8.00 - 7.93 (m, 2H), 7.58-7.53 (m, 1H), 7.47 - 7.41 (m, 2H), 7.37 - 7.33 (m, 1H), 7.31 - 7.27 (m, 1H), 4.10 (br, 1H), 3.59-3.42 (m, 4H), 3.11 (t, J = 6.6 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H), 2.44 (s, 3H), 1.95 - 1.87 (m, 4H), 1.71 - 1.59 (m, 4H), 1.47 - 1.38 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 161.63, 161.47, 158.08, 152.40, 150.77, 148.17, 147.08, 135.10, 129.23, 128.26, 123.49, 122.82, 119.93, 118.25, 118.09, 116.15, 115.56, 49.35, 39.64, 33.78, 31.57, 29.13, 28.89, 26.74, 26.70, 24.64, 22.91, 22.62, 20.69. MS (EI) m/z: 497.23(M)+. Anal. Calcd. for C31H35N3O3: C,74.82; H, 7.09; N, 8.44. Found: C, 74.80; H, 6.23; N, 8.21.

4.1.18. 6,7-dimethoxy-2-oxo-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-2H 15

-chromene-3-carboxamide (1q) Yellow solid; Yield 45%; m.p. 124-127 ◦ C; 1H NMR (600 MHz, CDCl3) δ8.84(s, 1H), 8.81(s, 1H), 8.01 - 7.90 (m 2H), 7.59 - 7.53 (m, 1H), 7.38 - 7.32 (m, 1H), 6.99 (s, 1H), 6.88 (s, 1H), 3.99 (s, 3H), 3.95 (s, 3H), 3.56 - 3.41 (m, 4H), 3.08 (t, J = 6.0 Hz, 2H), 2.71 (t, J = 7.2 Hz, 2H), 1.95 - 1.89 (m, 4H), 1.73 - 1.57 (m, 4H), 1.46 - 1.35(m, 6H). 13C NMR (100 MHz, CDCl3) δ 161.89, 157.99, 154.86, 150.94, 150.80, 147.87, 146.97, 128.27, 123.47, 122.84, 119.87, 115.48, 114.83, 111.33, 108.46, 99.15, 56.49, 56.23, 49.31, 39.54, 33.67, 31.54, 29.16, 28.86, 26.73, 26.67,24.60, 22.87, 22.58. MS (EI) m/z: 543.29 (M)+. Anal. Calcd. for C32H37N3O5: C, 70.70; H, 6.86; N,7.73. Found: C, 70.80; H, 6.63; N, 7.55.

4.1.19.

2-oxo-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-6-(trifluoromethoxy)

-2H-chromene-3-carboxamide (1r) Yellow solid; Yield 30%; m.p. 83-86 ◦C; 1H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 8.74 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.53 (m, 3H), 7.44 (d, J = 9.0 Hz, 1H), 7.38 - 7.29 (m, 1H), 3.99 (br, 1H), 3.61 - 3.37 (m, 4H), 3.04 (t, J = 5.6 Hz, 2H), 2.71 (t, J = 5.6Hz, 2H), 1.97 - 1.87 (m, 4H), 1.76 - 1.56 (m, 4H), 1.46 - 1.34 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 160.61, 160.56, 157.82, 152.10, 150.78, 146.89, 146.75, 145.33, 145.31, 128.23, 128.01, 126.62, 123.41, 122.73, 121.11, 120.06 (q, J C-F = 269Hz), 119.75, 119.52, 119.05, 118.84, 118.01, 115.37, 49.17, 39.68, 33.48, 31.44, 28.99, 28.77, 26.64, 26.60, 24.53, 22.78, 22.47. MS (EI) m/z: 567.16 (M)+. Anal. Calcd. for C31H32F3N3O3: C,65.60; H, 5.68; N, 7.40. Found: C, 65.80; H, 5.43; N, 7.21.

4.2. AChE and BChE activity assay Our assays on the in vitro inhibition of AChE and BChE were measured according to modified methods reported previously.39, 40 Recombinant human AChE and BChE obtained from Sigma-Aldrich Company were dissolved in 0.1 M sodium phosphate, pH 7.0. Acetylthiocholine (ATC) and 5′, 5′-dithio-bis(2-nitrobenzoic acid) (DTNB) purchased from Sigma-Aldrich Company were prepared in water as stock solutions of 0.01 M and frozen at −20 °C. The reaction mixture in a total assay volume of 200 µL contained appropriate amounts of ATC, DTNB, 0.1 M sodium phosphate buffer, inhibitor and enzyme. Enzymatic 16

hydrolysis of ATC was monitored by microplate reader (SpectraMax M5, Molecular Devices) at 415 nm in the presence or absence of various concentrations of inhibitor at 30 °C. Each experiment was repeated at least three times and the values were averaged. The inhibition constant (Ki) was obtained from the Dixon plot of plotting 1/v against concentration of inhibitor at certain concentrations of substrate. After the confirm of no interference with title compounds, bovine serum albumin is usually added up to 0.5% of the total reaction volume to avoid the coating of target enzyme in our assay.

4.3. Self-mediated Aβ (1-42) aggregation assay The thioflavin T based fluorometric assay was established for measuring the self-mediated Aβ (1-42) aggregation.41, 42 Aβ (1-42) samples (GL Biochem, Shanghai) were dissolved in DMSO as stock solutions of 5 mM and frozen at −20 °C. Experiments were performed by shaking the peptide in 50 mM phosphate buffer (pH 7.4) containing 100 mM NaCl at 37 °C for 10 h (final Aβ concentration 100 µM) in the presence or absence of various concentrations of title compound. After incubation, 20 µL of the above solutions were diluted to a final volume of 200 µL with 50 mM glycine-NaOH buffer (pH 7.4) containing 10 µM thioflavin T. Then the measurement of fluorescence intensity was carried out (λexc = 450 nm, λem = 480 nm) by microplate reader (SpectraMax M5, Molecular Devices) using black microwell plates (96 wells), and IC50 value for each inhibitor was further calculated with Origin ver. 8.0 software based on triplicate measurement.

4.4. β-Secretase inhibition assay β-Secretase inhibition assay was carried out in the following procedure by employing the β-secretase fluorescence resonance energy transfer assay kit (P2985, PanVera) as described before.36, 43 The weakly fluorescent substrate becomes highly fluorescent due to the cleavage catalyzed by β-Secretase and the rate of proteolysis at the early stage would properly indicate the concentration of active enzyme. The reaction mixture consisted of various concentrations of test compound (5 µL) or DMSO (control), and certain amounts of enzyme (20 µL) and appropriate sodium acetate buffer (50 mM, pH 4.5) was preincubated at 25 °C. Then the substrate (150 nΜ Rhodamine-EVNLDAEFK-quencher) was then added to initiate the 17

reaction and incubated for 60 min at the same temperature. The fluorescence signal was recorded at λem = 585 nm (λexc = 535 nm) by microplate reader (SpectraMax M5, Molecular Devices). The inhibition percentages corresponding to the presence of different concentrations of test compound was calculated by the following equation: 100 - [(Fi/Fo) * 100], where Fi nd Fo are the fluorescence intensities obtained for β-secretase in the presence and absence of desired inhibitor, respectively. The IC50 values for tested compounds were extrapolated with Origin ver. 8.0 software after measurement of three times.

4.5. Molecular docking, MD simulation and energy calculation The chemical structures of all compounds were constructed with Sybyl7.3/Sketch (Tripos Inc.) module and the nitrogen atom fused in the tacrine ring was protonated. Energy minimizations were performed by using Powell’s method for 2000 steps with the Tripos force field at a convergence criterion of 0.05 kcal/mol.44 The human AChE and BChE crystal structures were obtained from the RCSB PDB database. The AutoDock 4.0 program was applied to prepare the complexes of cholinesterase and the inhibitors. All the ligands were added polar hydrogen and assigned for Gasteiger charges. AutoGrid 4.0 was used to create docking grids centered on the active site including all the important residues based on our previous work with 0.375 Å spacing. In the docking process, the Lamarckian genetic algorithm (LGA) was used for the conformational search. The number of generated conformation was set at 100 for each compound. The rest parameters were all set at default. Then the best one with the highest interaction binding energy was selected for the SAR study. After, the selected docking conformations were transferred into the workflow composed of MD simulation and MM/PBSA calculation applied by AMBER 9.0 package. Among all MD simulations, ff99SB force field parameters were used for amino acid residues. All the ligand structures were assigned with AM1-BCC partial atomic charges and the General AMBER force field (gaff) parameters. Then, truncated octahedral box was used to solvate each system with TIP3P water model and extended 10.0 Å away from any atoms of the complex. All the minimizations and heating processes were carried out using sander module of AMBER 9.0 package. During the energy minimization process, the receptor was first fixed and only the ligand was kept free, then the ligand and residue side chains were kept free. 18

Finally, all atoms of the system were kept free and refined to a convergence of 0.001 kcal mol-1 Å-1 was reached over 10000 steps (3000 steps of the steepest descent and 8000 steps of the conjugated gradient minimization). The refined structure was used in the final binding energy calculation with MM-PBSA module of AMBER 9.0.

Acknowledgments The research was supported in part by the National Basic Research Program of China (No. 2010CB126103), the NSFC (No. 21372094), China Postdoctoral Science Foundation funded project and the Hong Kong Scholars Program (No. XJ2013012).

References 1.

Mount, C.; Downton, C. Nat. Med. 2006, 12, 780.

2.

Schmidt, C.; Wolff, M.; Weitz, M.; Bartlau, T.; Korth, C.; Zerr, I. Arch. Neurol. 2011, 68, 1124.

3.

Cummings, J. L. Rev. Neurol. Dis. 2004, 1, 60.

4.

Lahiri, D. K.; Farlow, M. R.; Sambamurti, K.; Greig, N. H.; Giacobini, E.; Schneider, L. S. Curr. Drug Targets 2003, 4, 97.

5.

Krall, W. J.; Sramek, J. J.; Cutler, N. R. Ann. Pharmacother. 1999, 33, 441.

6.

Tomiyama, T.; Shoji, A.; Kataoka, K.; Suwa, Y.; Asano, S.; Kaneko, H.; Endo, N. J. Biol. Chem. 1996, 271, 6839.

7.

Giacobini, E. Pharmacol. Res. 2004, 50, 433.

8.

Terry, A. V., Jr.; Buccafusco, J. J. J. Pharmacol. Exp. Ther. 2003, 306, 821.

9.

Savini, L.; Gaeta, A.; Fattorusso, C.; Catalanotti, B.; Campiani, G.; Chiasserini, L.; Pellerano, C.; Novellino, E.; McKissic, D.; Saxena, A. J. Med. Chem. 2003, 46, 1.

10. Bihel, F.; Das, C.; Bowman, M. J.; Wolfe, M. S. J. Med. Chem. 2004, 47, 3931. 11. Churcher, I. Curr. Top. Med. Chem. 2006, 6, 579. 12. Wolfe, M. S. Nat. Rev. Drug Discov. 2002, 1, 859. 13. Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Melchiorre, C. Mini. Rev. Med. Chem. 2008, 8, 960. 14. Hansen, R. A.; Gartlehner, G.; Webb, A. P.; Morgan, L. C.; Moore, C. G.; Jonas, D. E. 19

Clin. Interv. Aging 2008, 3, 211. 15. Saxena, A. K.; Chaudhaery, S. S.; Roy, K. K.; Shakya, N.; Saxena, G.; Sammi, S. R.; Nazir, A.; Nath, C. J. Med. Chem. 2010, 53, 6490. 16. Rosini, M.; Simoni, E.; Bartolini, M.; Cavalli, A.; Ceccarini, L.; Pascu, N.; McClymont, D. W.; Tarozzi, A.; Bolognesi, M. L.; Minarini, A.; Tumiatti, V.; Andrisano, V.; Mellor, I. R.; Melchiorre, C. J. Med. Chem. 2008, 51, 4381. 17. Piazzi, L.; Cavalli, A.; Colizzi, F.; Belluti, F.; Bartolini, M.; Mancini, F.; Recanatini, M.; Andrisano, V.; Rampa, A. Bioorg. Med. Chem. Lett. 2008, 18, 423. 18. Rees, T.; Hammond, P. I.; Soreq, H.; Younkin, S.; Brimijoin, S. Neurobiol Aging 2003, 24, 777. 19. Romero, A.; Cacabelos, R.; Oset-Gasque, M.J. A. Bioorg. Med. Chem. Lett. 2013, 23, 1916. 20. Rodriguez-Franco, M. I.; Fernandez-Bachiller, M. I.; Perez, C.; Hernandez-Ledesma, B.; Bartolome, B. J. Med. Chem. 2006, 49, 459. 21. Camps, P.; Formosa, X.; Galdeano, C.; Gomez, T.; Munoz-Torrero, D.; Scarpellini, M.; Viayna, E.; Badia, A.; Clos, M. V.; Camins, A.; Pallas, M.; Bartolini, M.; Mancini, F.; Andrisano, V.; Estelrich, J.; Lizondo, M.; Bidon-Chanal, A.; Luque, F. J. J. Med. Chem. 2008, 51, 3588. 22. Camps, P.; El Achab, R.; Morral, J.; Munoz-Torrero, D.; Badia, A.; Banos, J. E.; Vivas, N. M.; Barril, X.; Orozco, M.; Luque, F. J. J. Med. Chem. 2000, 43, 4657. 23. Tang, H.; Zhao, L. Z.; Zhao, H. T.; Huang, S. L.; Zhong, S. M.; Qin, J. K.; Chen, Z. F.; Huang, Z. S.; Liang, H. Eur. J. Med. Chem. 2011, 46, 4970. 24. Virsdoia, V.; Shaikh, M. S.; Manvar, A.; Desai, B.; Parecha, A.; Loriya, R.; Dholariya, K.; Patel, G.; Vora, V.; Upadhyay, K.; Denish, K.; Shah, A.; Coutinho, E. C. Chem. Biol. Drug Des. 2010, 76, 412. 25. Dekic, B. R.; Radulovic, N. S.; Dekic, V. S.; Vukicevic, R. D.; Palic, R. M. Molecules,2010, 15, 2246. 26. Sashidhara, K. V.; Kumar, A.; Kumar, M.; Srivastava, A.; Puri, A. Bioorg Med Chem Lett, 2010, 20, 6504. 27.

Borges, F. F., Milhazes, Santana, N. Uriarte, L. E. Curr. Med. Chem. 2005, 12, 887. 20

28. Fonseca, F. V.; Baldissera, L., Jr.; Camargo, E. A.; Antunes, E.; Diz-Filho, E. B.; Correa, A. G.; Beriam, L. O.; Toyama, D. O.; Cotrim, C. A.; Alvin, J., Jr.; Toyama, M. H. Toxicon 2010, 55, 1527. 29. Anand, P.; Singh, B.; Singh, N. Bioorg. Med. Chem. 2012, 20, 1175. 30. Xie, S.S.; Wang, X.B.; Li, J.Y.; Yang, L.; Kang, L.J. Eur. J Med Chem, 2013, 64, 540. 31. Carlier, P. R.; Ella, S. H. C.; Han, Y.; Liu, J.; El Yazal, J.; Pang, Y. P. J Med Chem 1999, 42, 4225. 32. Dvir, H.; Silman, I.; Harel, M.; Rosenberry, T. L.; Sussman, J. L. Chem. Biol. Interact. 2010, 187, 10. 33. Lane, R. M.; Potkin, S. G.; Enz, A. International Journal of Neuropsychopharmacology 2006, 9, 101. 34. Jiang, H.; Wang, X.; Huang, L.; Luo, Z.; Su, T.; Ding, K.; Li, X. Bioorg. Med. Chem. 2011, 19, 7228. 35. Camps, P.; Formosa, X.; Galdeano, C.; Munoz-Torrero, D.; Ramirez, L.; Gomez, E.; Isambert, N.; Lavilla, R.; Badia, A.; Clos, M. V.; Bartolini, M.; Mancini, F.; Andrisano, V.; Arce, M. P.; Rodriguez-Franco, M. I.; Huertas, O.; Dafni, T.; Luque, F. J. J. Med. Chem. 2009, 52, 5365. 36. Peng, D. Y.; Sun, Q.; Zhu, X. L.; Lin, H. Y.; Chen, Q.; Yu, N. X.; Yang, W. C.; Yang, G. F. Bioorg. Med. Chem. 2012, 20, 6739. 37. Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.; Bartolini, M.; Margotti, E.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. Angew. Chem. Int. Ed. Engl. 2007, 46, 3689. 38. Bolognesi, M. L.; Banzi, R.; Bartolini, M.; Cavalli, A.; Tarozzi, A.; Andrisano, V.; Minarini, A.; Rosini, M.; Tumiatti, V.; Bergamini, C.; Fato, R.; Lenaz, G.; Hrelia, P.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. J. Med. Chem. 2007, 50, 4882. 39. Yang, W.; Xue, L.; Fang, L.; Chen, X.; Zhan, C. G. Chem. Biol. Interact. 2010, 187, 148. 38. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R. M. Biochem. Pharmacol. 1961, 7, 88. 40. Ellman, G.L.; Courtney, K.D.; Andres, V.; Feather-Stone, R.M. Biochem. Pharmacol. 1961, 7, 88. 21

41. LeVine, H., 3rd. Protein Sci. 1993, 2, 404. 42. Naiki, H.; Higuchi, K.; Nakakuki, K.; Takeda, T. Lab Invest. 1991, 65, 104. 43. Al-Tel, T. H.; Semreen, M. H.; Al-Qawasmeh, R. A.; Schmidt, M. F.; El-Awadi, R.; Ardah, M.; Zaarour, R.; Rao, S. N.; El-Agnaf, O. J. Med. Chem. 2011, 54, 8373. 44. Mizutani, M. Y.; Itai, A. J. Med. Chem. 2004, 47, 4818.

Legends 22

Table 1. Inhibitory activities and selectivities of compounds 1a-r on human AChE and BChE.

Table 2. The experimental and calculated binding free energies of compound 1g for two cholinesterases, compared with tacrine.

Table 3. Inhibitory effects of compounds 1a, 1g, 1j, 1l, 1o and 1p against Aβ aggregation and β-secretase.

Figure 1. Chemical structures of the commercial AChE inhibitors for AD treatment.

Figure 2. Simulated binding modes of compounds 1a (A), 1g (B) and 1m (C). The lengths of hydrogen bond between carbonyl oxygen of coumarin part and Tyr124 are 2.3 Å for compound 1a, 1.9 Å for compound 1g, 2.6 Å for compound 1m, respectively.

Figure 3. Simulated binding modes of tacrine and compound 1g in human AChE and BChE. (A) In CAS of AChE, tacrine formed π-π stacking interaction with Trp86 and Tyr337, and formed a hydrogen bond (1.6 Å) with His447. (B) In BChE active site, tacrine formed π-π stacking interaction with Trp82, and formed two hydrogen bonds (1.7 Å and 2.2 Å) with Glu197. (C) In addition to the hydrogen bonding (2.1 Å) with His447 and π-π stacking interaction of tacrine moiety in CAS, the coumarin moiety of compound 1g formed visible interactions with PAS in AChE: the hydrogen bond (1.9 Å) between amide oxygen and Tyr124, and the π-π stacking interaction between coumarin and two aromatic residues (Trp286 and Tyr72). (D) In BChE active site, compound 1g formed π-π stacking interaction with Trp82 and His438, and formed three hydrogen bonds (2.2 Å, 1.9 Å and 2.8 Å) with His438, Gln189 and Asn289, respectively. Scheme 1. Synthesis of hybrids of tacrine-coumarin (1).

23

Table 1． ．

a

Compound

R1

R2

n

Ki for AChE (nM)

1a

H

H

5

34.4 ± 1.59

31.7 ± 8.21

0.92

1b

H

OCH3

5

44.3 ± 4.19

29.8 ± 7.33

0.67

1c

OCH3

H

5

39.4 ± 2.57

34.0 ± 7.05

0.87

1d

CH3

H

5

35.8 ± 1.32

32.4 ± 4.13

0.91

1e

OCH3

OCH3

5

70.0 ±4.27

8.1 ± 2.99

0.11

1f

OCF3

H

5

76.1 ± 0.90

32.5 ± 3.12

0.43

1g

H

H

6

16.7 ± 3.87

16.1 ± 1.92

0.96

1h

H

OCH3

6

30.9 ± 1.96

20.2 ± 3.42

0.65

1i

OCH3

H

6

24.3 ± 7.09

23.7 ± 3.68

0.98

1j

CH3

H

6

30.1 ± 1.92

23.2 ± 1.50

0.77

1k

OCH3

OCH3

6

56.1 ± 8.01

31.2 ± 2.98

0.56

1l

OCF3

H

6

59.6 ± 4.43

17.5 ± 1.16

0.29

1m

H

H

7

42.2 ± 4.66

20.0 ± 2.34

0.47

1n

H

OCH3

7

55.2 ± 4.39

35.0 ± 6.85

0.63

1o

OCH3

H

7

50.7 ± 3.07

34.5 ± 4.93

0.68

1p

CH3

H

7

66.1 ± 7.81

50.9 ± 4.93

0.77

1q

OCH3

OCH3

7

91.1 ± 2.18

37.5 ± 6.86

0.42

1r

OCF3

H

7

78.2 ± 1.06

40.0 ± 0.47

0.51

Ki for BChE (nM)

Selectivity a

Tacrine

35.7 ± 0.59

8.7 ± 0.17

0.24

Galanthamine

61.9 ± 5.63

192.5 ± 17.57

3.11

Selectivity of inhibitor potency for AChE over BChE.

24

Table 2． ． Tacrine for AChE Ki (nM) △Gexp

a

△Gcal

1g

for BChE

for AChE

for BChE

35.7

8.7

16.7

16.1

-10.2

-11.0

-10.6

-10.6

-11.9

-13.4

-12.7

-12.4

a

∆Gexp = - RTlnKi.

Table 3. IC50 (µM) targets

Curcumin

1a

1g

1j

1l

1o

1p

Aβ

11.0 ± 1.2

>100

>100

5.0 ± 0.9

24.2 ± 3.8

13.4 ± 1.8

6.1 ± 1.1

β-secretase

5.3 ± 1.1

40.1 ± 2.2

17.2 ± 1.5

>100

22.6 ± 3.6

20.2 ± 0.6

42.8 ± 6.5

25

Figure 1. CH3 NH2

O H3C

N

O

N

O

H2N

Rivastigmine

(-)-Huprine A

Tacrine

N

OH O

O O

N

O Donepezil

H 3C

O N CH3 Galanthamine

Figure 2.

26

Figure 3.

27

Scheme 1.

Reagents and conditions: (a) cyclohexanone, toluene, reflux; (b) POCl3, reflux; (c) NH2(CH2)nNH2, n-pentanol, N2, reflux; (d) (CH2O)n, MgCl2, Et3N, CH3CN, reflux; (e) diethyl malonate, EtOH, piperidine, reflux; (f) NaOH, H2 O/EtOH, reflux; (g) EDCI, HOBt, DMF, r.t or PyBOP, Et3 N, DCM, r.t.

28

Graphic abstract

Syntheses of Coumarin–Tacrine Hybrids as Dual-site Acetylcholinesterase Inhibitors and Their activity against Butylcholinesterase, Aβ aggregation, and β-secretase

Sun, Qi,†,1 Peng Da-Yong,†,1 Yang Sheng-Gang,1 Zhu Xiao-Lei, 1 Yang Wen-Chao, 1* Yang Guang-Fu1, 2* 1

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry,

Central China Normal University, Wuhan 430079, P.R. China; 2

Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, P.R. China.

29