Design, Synthesis, and Evaluation of Acetylcholinesterase and Butyrylcholinesterase Dual-Target Inhibitors against Alzheimer’s Diseases

A series of novel compounds 6a–h, 8i–1, 10s–v, and 16a–d were synthesized and evaluated, together with the known analogs 11a–f, for their inhibitory activities towards acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The inhibitory activities of AChE and BChE were evaluated in vitro by Ellman method. The results show that some compounds have good inhibitory activity against AChE and BChE. Among them, compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and BChE (eqBChE IC50 = 0.28 μM). Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the peripheral anionic site (PAS) and the catalytic sites (CAS) of AChE and BChE. In addition, the cytotoxicity of compound 8i is lower than that of Tacrine, indicating its potential safety as anti-Alzheimer’s disease (anti-AD) agents. In summary, these data suggest that compound 8i is a promising multipotent agent for the treatment of AD.


Introduction
According to the report of the International Alzheimer's Association in the last three years, there are more than 36 million AD patients in the world. With the acceleration of the aging process of the population, the amount of AD incidence is increasing year by year, and the number of AD patients in the world will exceed 130 million by 2050 [1][2][3]. The incidence of AD is over 1.9% among people over 60 years old in China [4]. AD is the third most common disease in the elderly after cardio-cerebrovascular disease and cancer. AD can cause dementia, which is one of the six leading causes of death in the United States [5,6].
AD was initially characterized by memory loss, cognitive dysfunction, inability to take care of themselves in daily life, and subsequent exacerbations of mental and behavioral abnormalities [7,8]. AD is not only a seriously threaten of human health and life, but also brings heavy mental burden and economic pressure to the family members and friends of the patients, causing huge fluctuations in the
The structures of the new compounds were confirmed by spectral data ( 1 H-NMR, 13 C-NMR, and HRMS, see Supplementary Material).
The structures of the new compounds were confirmed by spectral data ( 1 H-NMR, 13 C-NMR, and HRMS, see Supplementary Materials).
The synthetic route of compounds 16a-d has been depicted in Scheme 4. Ethyl 2-piperidin-4-ylacetate 11 was reacted with appropriate substituted (2-Bromoethyl) benzene derivatives (12a-b) under potassium carbonate (K 2 CO 3 ) and potassium iodide (KI) conditions. Then, the obtained compounds 13a-b were used in further synthesis without purification. 4 mol/L potassium hydroxide (KOH) was added to compounds 13a-b in C 2 H 5 OH: H 2 O = 5:1. The reaction mixture was stirred at room temperature for 7 h to give compounds 14a-d. Finally, compounds 14a-d were activated with PyBOP and reacted with compounds 15a-b in DMF to give the target compounds 16a-d with moderate to good yield (30%-80%) (Scheme 4).

AChE and BChE Inhibitory Activity of the Target Molecules
Compounds 6a-h, 8i-l, 11a-f, 10s-v, and 16a-d were evaluated for their anti-ChEs activity. Tacrine and Donepezil were used as reference drugs. According to the method described by Ellman [32], the data were expressed by IC50 values. In vitro experiments showed that some of these compounds could effectively inhibit ChEs in the micromolar range (Table 1).

AChE and BChE Inhibitory Activity of the Target Molecules
Compounds 6a-h, 8i-l, 11a-f, 10s-v, and 16a-d were evaluated for their anti-ChEs activity. Tacrine and Donepezil were used as reference drugs. According to the method described by Ellman [32], the data were expressed by IC 50 values. In vitro experiments showed that some of these compounds could effectively inhibit ChEs in the micromolar range (Table 1). under potassium carbonate (K2CO3) and potassium iodide (KI) conditions. Then, the obtained compounds 13a-b were used in further synthesis without purification. 4 mol/L potassium hydroxide (KOH) was added to compounds 13a-b in C2H5OH: H2O = 5:1. The reaction mixture was stirred at room temperature for 7 h to give compounds 14a-d. Finally, compounds 14a-d were activated with PyBOP and reacted with compounds 15a-b in DMF to give the target compounds 16a-d with moderate to good yield (30%-80%) (Scheme 4).

AChE and BChE Inhibitory Activity of the Target Molecules
Compounds 6a-h, 8i-l, 11a-f, 10s-v, and 16a-d were evaluated for their anti-ChEs activity. Tacrine and Donepezil were used as reference drugs. According to the method described by Ellman [32], the data were expressed by IC50 values. In vitro experiments showed that some of these compounds could effectively inhibit ChEs in the micromolar range (Table 1). under potassium carbonate (K2CO3) and potassium iodide (KI) conditions. Then, the obtained compounds 13a-b were used in further synthesis without purification. 4 mol/L potassium hydroxide (KOH) was added to compounds 13a-b in C2H5OH: H2O = 5:1. The reaction mixture was stirred at room temperature for 7 h to give compounds 14a-d. Finally, compounds 14a-d were activated with PyBOP and reacted with compounds 15a-b in DMF to give the target compounds 16a-d with moderate to good yield (30%-80%) (Scheme 4).

AChE and BChE Inhibitory Activity of the Target Molecules
Compounds 6a-h, 8i-l, 11a-f, 10s-v, and 16a-d were evaluated for their anti-ChEs activity. Tacrine and Donepezil were used as reference drugs. According to the method described by Ellman [32], the data were expressed by IC50 values. In vitro experiments showed that some of these compounds could effectively inhibit ChEs in the micromolar range (Table 1). under potassium carbonate (K2CO3) and potassium iodide (KI) conditions. Then, the obtained compounds 13a-b were used in further synthesis without purification. 4 mol/L potassium hydroxide (KOH) was added to compounds 13a-b in C2H5OH: H2O = 5:1. The reaction mixture was stirred at room temperature for 7 h to give compounds 14a-d. Finally, compounds 14a-d were activated with PyBOP and reacted with compounds 15a-b in DMF to give the target compounds 16a-d with moderate to good yield (30%-80%) (Scheme 4).
Next, we investigated the effect of the p-Toluenesulfonamide moiety on the inhibitory activity of ChEs. Not only the amide group was replaced by Sulfonamide moiety based on the principles of bioisosterism, but also the fused nitrogen-containing bicyclic system (Indole, Indazole, Oxoindoline, Benzimidazole) in the previous compounds was replaced by Tosyl moiety. We synthesized compounds 10s-v. Except for compound 10u (eeAChE 11.61% [100 μM], eqBChE 18.31% [100 μM]) has low inhibitory activity against ChEs, the compounds 10s (eeAChE IC50 = 4.24 μM, eqBChE IC50 = 4.10 μM)), 10t (eeAChE IC50 = 5.50 μM, eqBChE IC50 = 2.01 μM), 10v (eeAChE IC50 = 7.20 μM, eqBChE IC50 = 7.14 μM) all can maintain ChEs inhibitory activity at micromolar levels, indicating that the p-Toluenesulfonamide moiety is responsible for maintaining the inhibitory activity of ChEs. On one hand, we speculate that methyl occupies the pocket of the active site and interacts with amino acid residues to increase inhibitory activity. On the other hand, Sulfonamide moiety is very important for maintaining ChEs inhibitory activity, which may be related to the bond angle between Sulfonamide and molecules [33].
In addition, we tested the number of carbon atoms between benzene and piperidine. When the number of carbon atoms becomes two (compounds 16a-d), the compounds had low inhibitory activity against the two kinds of ChEs, in particular, compound 16c has no inhibitory activity against AChE at 100 μM. We speculate that the decrease in the activity of such compounds may be that the molecular volume is too large to enter the active pocket of ChEs, indicating that the residue of N-Benzylpiperidine in the structure is an essential group for inhibiting both ChEs.
Compounds designed with a Piperazine ring instead of a piperidine ring may also have the same or higher inhibitory activity on ChEs. Luca P, Tomás Daniel, Asha H, et al. [  Then, we evaluated the effect of R on ChEs activity. We modified R with different substituent (2-Cl, 4-OCH 3 , 4-CF 3 ), compared with compound 6d, when R is 4-CF 3 (6e), 4-OCH 3 (11d), 2-Cl (11e), the ChEs inhibitory activity decreased. In particular, compound 11d showed little inhibitory activity on BChE at 100 µM. In general, with the same Y, when R is H, the compounds have the highest inhibitory activity against ChEs; when Y is substituted by 2-Cl or 4-CF 3 , the inhibitory activity of the compounds to ChEs were weakened; and when Y is substituted by 4-OCH 3 , the compounds have the worst inhibitory activity against AChEs. We speculate that enhancing the electron-withdrawing effect or the donor effect on the aromatic ring is not conducive to improving the performance of the analog, and the appropriate space may facilitate the analog to enter the CAS pocket of ChEs.
Next, we investigated the effect of the p-Toluenesulfonamide moiety on the inhibitory activity of ChEs. Not only the amide group was replaced by Sulfonamide moiety based on the principles of bioisosterism, but also the fused nitrogen-containing bicyclic system (Indole, Indazole, Oxoindoline, Benzimidazole) in the previous compounds was replaced by Tosyl moiety. We synthesized compounds 10s-v. Except for compound 10u (eeAChE 11.61% [100 µM], eqBChE 18.31% [100 µM]) has low inhibitory activity against ChEs, the compounds 10s (eeAChE IC 50 = 4.24 µM, eqBChE IC 50 = 4.10 µM)), 10t (eeAChE IC 50 = 5.50 µM, eqBChE IC 50 = 2.01 µM), 10v (eeAChE IC 50 = 7.20 µM, eqBChE IC 50 = 7.14 µM) all can maintain ChEs inhibitory activity at micromolar levels, indicating that the p-Toluenesulfonamide moiety is responsible for maintaining the inhibitory activity of ChEs. On one hand, we speculate that methyl occupies the pocket of the active site and interacts with amino acid residues to increase inhibitory activity. On the other hand, Sulfonamide moiety is very important for maintaining ChEs inhibitory activity, which may be related to the bond angle between Sulfonamide and molecules [33].
In addition, we tested the number of carbon atoms between benzene and piperidine. When the number of carbon atoms becomes two (compounds 16a-d), the compounds had low inhibitory activity against the two kinds of ChEs, in particular, compound 16c has no inhibitory activity against AChE at 100 µM. We speculate that the decrease in the activity of such compounds may be that the molecular volume is too large to enter the active pocket of ChEs, indicating that the residue of N-Benzylpiperidine in the structure is an essential group for inhibiting both ChEs.
Compounds designed with a Piperazine ring instead of a piperidine ring may also have the same or higher inhibitory activity on ChEs. Luca P, Tomás Daniel, Asha H, et al. [34] based on the structure of Donepezil, mainly the conjugation of Benzylpiperidine/ Benzylpiperazine moiety with a biologically active heterocyclic derivative (Benzimidazole or Benzofuran), which gave the compound other relevant properties. It shows good activity (IC 1/4 50 4.0-30.0 µM) for AChE inhibition, and has inhibition of Aβ peptide aggregation, antioxidant activity, and metal chelation.

Kinetic Studies of AChE and BChE Inhibition
To determine the kinetic types of AChE and BChE inhibition, compounds 8i and 10s were selected for kinetic studies. In each case, the kinetic types of enzyme inhibition were obtained by the modified Ellman's method and the Lineweaver-Burk secondary plots [35]. The Lineweaver-Burk plots showed both increasing slope (decreased Vmax) and increasing intercept (higher Km) for higher inhibitor concentrations, indicating a mixed-type inhibition, including competitive inhibition and non-competitive inhibition, which possibly was because compound 8i could bind to both CAS and PAS ( Figure 3A,B). According to the result of molecular docking study. The same inhibition type between compound 10s and ChEs was found in graphical analysis ( Figure 3C,D).
Molecules 2020, 25, x FOR PEER REVIEW 9 of 21 plots showed both increasing slope (decreased Vmax) and increasing intercept (higher Km) for higher inhibitor concentrations, indicating a mixed-type inhibition, including competitive inhibition and non-competitive inhibition, which possibly was because compound 8i could bind to both CAS and PAS ( Figure 3A,B). According to the result of molecular docking study. The same inhibition type between compound 10s and ChEs was found in graphical analysis ( Figure 3C,D).

Docking Studies
To further study the binding mode of compound 8i and ChEs, molecular docking was performed using Discovery Studio software 2016. The predicted binding mode of compound 8i is shown in Figure 4 and Figure 5. Compound 8i could interact with CAS and PAS of AChE simultaneously. For AChE (from Electrophorus electricus (electric eeAChE, Sigma-Aldrich) (Figure 4A), the N-Benzylpiperidine moiety interacted with Trp86 in CAS via aromatic π-π interaction. The Amide group formed hydrogen bond with Phe295. Moreover, 2-Oxoindoline of compound 8i bound with

Docking Studies
To further study the binding mode of compound 8i and ChEs, molecular docking was performed using Discovery Studio software 2016. The predicted binding mode of compound 8i is shown in Figures 4 and 5. Compound 8i could interact with CAS and PAS of AChE simultaneously. For AChE (from Electrophorus electricus (electric eeAChE, Sigma-Aldrich) (Figure 4A), the N-Benzylpiperidine moiety interacted with Trp86 in CAS via aromatic π-π interaction. The Amide group formed hydrogen bond with Phe295. Moreover, 2-Oxoindoline of compound 8i bound with Trp286 via π-π stacking interaction; for human AChE-huAChE (Sigma-Aldrich) (Figure 4B), the N-Benzylpiperidine moiety of compound 8i was bound to CAS, displaying a classic aromatic π-π interaction with Trp86. Moreover, 2-Oxoindoline of compound 8i bound with Trp286 and Tyr341 via π-π stacking interaction. In addition, the Amide group formed hydrogen bond with Phe295. By comparison, it was found that the compounds have similar binding patterns to eeAChE and huAChE. All these facts provide an explanation for the higher inhibitory effects of compound 8i towards AChE. interacts with Asp70 via electrostatic interaction. Moreover, the N-Benzylpiperidine moiety interacts with Trp82 in CAS by T-shaped π-π interaction, and the N-Benzylpiperidine moiety forms π-alkyl interaction with Ala328. 2-Oxoindoline moiety of compound 8i bound with Phe329 via π-π stacking interaction. These interactions increase the inhibitory activity by enhancing the binding affinity.  From the binding mode prediction of Donepezil with huAChE ( Figure 6), we found that the Molecular docking of compound 8i at the active site of human BuChE-huBChE (Sigma-Aldrich, Munich, Germany) has been shown in Figure 5. The N-Benzylpiperidine moiety of compound 8i interacts with Asp70 via electrostatic interaction. Moreover, the N-Benzylpiperidine moiety interacts with Trp82 in CAS by T-shaped π-π interaction, and the N-Benzylpiperidine moiety forms π-alkyl interaction with Ala328. 2-Oxoindoline moiety of compound 8i bound with Phe329 via π-π stacking interaction. These interactions increase the inhibitory activity by enhancing the binding affinity.  From the binding mode prediction of Donepezil with huAChE ( Figure 6), we found that the binding pattern of compound 8i is similar to Donepezil in some respects: (i) The N-Benzylpiperidine moiety was bound to CAS, displaying a classic aromatic π-π interaction with Trp86; (ii) The Oxygen atom formed hydrogen bond with Phe295; (iii) Aromatic heterocycle moiety bound with Trp286 and Tyr341 via π-π stacking interaction. In addition, the Indone moiety of Donepezil interacts with Trp286 at the center of the PAS; the piperidine moiety of Donepezil interacts with Tyr337, Phe338, Tyr341. From the binding mode prediction of Donepezil with huAChE ( Figure 6), we found that the binding pattern of compound 8i is similar to Donepezil in some respects: (i) The N-Benzylpiperidine moiety was bound to CAS, displaying a classic aromatic π-π interaction with Trp86; (ii) The Oxygen atom formed hydrogen bond with Phe295; (iii) Aromatic heterocycle moiety bound with Trp286 and Tyr341 via π-π stacking interaction. In addition, the Indone moiety of Donepezil interacts with Trp286 at the center of the PAS; the piperidine moiety of Donepezil interacts with Tyr337, Phe338, Tyr341. From the binding mode prediction of Donepezil with huBChE (Figure 7), we also found that the binding pattern of compound 8i is similar to Donepezil in some respects: (i) The N-Benzylpiperidine moiety interacts with Asp70 via electrostatic interaction. In addition, the N-Benzylpiperidine moiety of Donepezil interacts with Try332 via electrostatic interaction; (ii) the N-Benzylpiperidine moiety interacts with Trp82 in CAS by T-shaped π-π interaction, and the N-Benzylpiperidine moiety form π-alkyl interaction with Ala328. The difference is that 2-Oxoindoline of compound 8i bound with Phe329 via π-π stacking interaction while the 2-Oxoindoline moiety of Donepezil interacts with Gly116 in the center of the PAS. From the binding mode prediction of Donepezil with huBChE (Figure 7), we also found that the binding pattern of compound 8i is similar to Donepezil in some respects: (i) The N-Benzylpiperidine moiety interacts with Asp70 via electrostatic interaction. In addition, the N-Benzylpiperidine moiety of Donepezil interacts with Try332 via electrostatic interaction; (ii) the N-Benzylpiperidine moiety interacts with Trp82 in CAS by T-shaped π-π interaction, and the N-Benzylpiperidine moiety form π-alkyl interaction with Ala328. The difference is that 2-Oxoindoline of compound 8i bound with Phe329 via π-π stacking interaction while the 2-Oxoindoline moiety of Donepezil interacts with Gly116 in the center of the PAS.
From the binding mode prediction of Donepezil with huBChE (Figure 7), we also found that the binding pattern of compound 8i is similar to Donepezil in some respects: (i) The N-Benzylpiperidine moiety interacts with Asp70 via electrostatic interaction. In addition, the N-Benzylpiperidine moiety of Donepezil interacts with Try332 via electrostatic interaction; (ii) the N-Benzylpiperidine moiety interacts with Trp82 in CAS by T-shaped π-π interaction, and the N-Benzylpiperidine moiety form π-alkyl interaction with Ala328. The difference is that 2-Oxoindoline of compound 8i bound with Phe329 via π-π stacking interaction while the 2-Oxoindoline moiety of Donepezil interacts with Gly116 in the center of the PAS. Compared with Donepezil, the inhibitory activity of compound 8i on BChE is higher than Donepezil, and has similar inhibitory activity on two ChEs, so that it can exert an anti-ChEs effect in a balanced manner. Studies on compound 8i molecular docking have shown that the Compared with Donepezil, the inhibitory activity of compound 8i on BChE is higher than Donepezil, and has similar inhibitory activity on two ChEs, so that it can exert an anti-ChEs effect in a balanced manner. Studies on compound 8i molecular docking have shown that the Benzylpiperidine moiety of the compound acts on the CAS of the enzyme, while the 2-Oxoindoline moiety binds to the PAS of the enzyme, which is basically consistent with the design idea.

Cytotoxicity Studies
We focused on the cytotoxicity of the synthetic compounds. The reason for using PC12 is that our compounds act on the central nervous system, so we need to find out whether the compound has a toxic effect on normal nerve cells . Compounds 6b, 6c, 6d, 10s, 8i, and 10v were selected as representative compounds to assess their potential cytotoxic effects. Compounds 6c, 6d, 8i are less toxic than Tacrine; the toxicity of compounds 6b, 10v are similar to that of Tacrine; compound 10s is slightly more toxic than Tacrine. Among them, compound 8i has the lowest toxicity ( Figure 8).
Molecules 2020, 25, x FOR PEER REVIEW 12 of 21 Benzylpiperidine moiety of the compound acts on the CAS of the enzyme, while the 2-Oxoindoline moiety binds to the PAS of the enzyme, which is basically consistent with the design idea.

Cytotoxicity Studies
We focused on the cytotoxicity of the synthetic compounds. The reason for using PC12 is that our compounds act on the central nervous system, so we need to find out whether the compound has a toxic effect on normal nerve cells . Compounds 6b, 6c, 6d, 10s, 8i, and 10v were selected as representative compounds to assess their potential cytotoxic effects. Compounds 6c, 6d, 8i are less toxic than Tacrine; the toxicity of compounds 6b, 10v are similar to that of Tacrine; compound 10s is slightly more toxic than Tacrine. Among them, compound 8i has the lowest toxicity ( Figure 8).  of compounds 6b, 6c, 6d, 10s, 8i, and 10v on PC-12 cell line. Data were expressed as mean ± SD (n = 3).

Chemistry
All reagents were obtained from commercial suppliers and were used without any further purification unless otherwise stated. Flash column chromatography was performed with silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. Thin layer chromatography was  of compounds 6b, 6c, 6d, 10s, 8i, and 10v on PC-12 cell line. Data were expressed as mean ± SD (n = 3).

Chemistry
All reagents were obtained from commercial suppliers and were used without any further purification unless otherwise stated. Flash column chromatography was performed with silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. Thin layer chromatography was performed using silica gel 60 F254 precoated plates (purchased from Qingdao Haiyang Inc., Qingdao, China). Visualization was achieved using Ultraviolet (UV) light (254 nm and 365 nm, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China). Melting points were determined with a Mel-TEMP II melting point apparatus (Beijing Keyi Company, Beijing, China) and was uncorrected. 1 H NMR and 13 C NMR spectra were recorded with Bruker AV-600, AV-500 or AV-400 MHz instruments (Bruker, Ettlingen, Germany) using DMSO-d 6 , CD 3 OD, or CDCl 3 as solvent. Chemical shifts were reported as δ values (ppm) from internal reference tetramethylsilane (TMS). All coupling constants were reported in hertz (Hz), All chemical shifts are reported in parts per million (ppm), relative to the internal standard. In addition, proton multiplicities were labeled as br (broad), s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), and m (multiplet). HR-MS were performed on a Waters Vion IMS Q-tof (Waters, MA, USA).

General Procedure for the Synthesis of Compounds 3a-d
4-Piperidinecarboxamide (1) (3.00 g, 23.4 mmol) and substituted Benzylchloride derivatives (2a-d) (28.1 mmol) were dissolved in 20 mL acetone. Then, anhydrous K 2 CO 3 (6.47 g, 46.8 mmol) and catalytic amount KI were added. The reaction mixture was refluxed for 4 h. After completion of the reaction, acetone was concentrated, and the residue was dissolved in water (60 mL) and extracted with ethyl acetate (60 × 3 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph (DCM: methanol = 60:1-5:1) to give target compounds 3a-d.

General Procedure for the Synthesis of Compounds 4a-d
Compounds 3a-d (3 g) were dissolved in anhydrous THF (17 mL) and then LiAlH 4 (5 equiv.) was added to the above cooled solution at 0-5 • C in small portions under stirring. The reaction mixture was further stirred at room temperature for 30 min and finally refluxed for 4 h. After cooling, water and 10% NaOH solution was added at 0-5 • C. Then, the obtained white precipitate was filtered off and washed with THF. The filtrate was extracted with ethyl acetate. The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuum. The obtained compounds 4a-d were used in further synthesis without purification.

General Procedure for the Synthesis of Compounds 6a-h
CDI (1 equiv.) was added to a solution of the acids (5a-d) ((300 mg, 1 equiv.) in dry THF under nitrogen atmosphere. After 30 min, the solution of substituted 4-Amine-1-benzylpiperidines (4a-d) (1.2 equiv.) in THF were added, and the reaction mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed under reduced pressure, and then the reaction mixture was quenched with saturated NaCl solution (25 mL). The aqueous phase was extracted with DCM (25 × 3 mL). The DCM layer was combined and washed with brine solution (25 × 3 mL). The organic layer was dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph (DCM: methanol = 60:1-5:1) to give target compounds 6a-h.
N-((1-Benzylpiperidin-4-yl)methyl)-1H-indole-5-carboxamide (6a 3.5. General Procedure for the Synthesis of 8i-l Intermediates (7) (140 mg, 1.2 equiv.), PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were added to DMF and stirred at room temperature for 20 min. Then, intermediates (4a-d) (1.0 equiv.) was added and stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was quenched with saturated NaCl solution (25 mL). The aqueous phase was extracted with DCM (25 × 3 mL). The DCM layer was combined and washed with brine solution (25 × 3 mL). The organic layer was dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in DCM gradient (DCM: methanol= 60:1-5:1) yielded compounds 8i-l.    13 13  3.7. General Procedure for the Synthesis of 16a-d Ethyl 2-piperidin-4-ylacetate 11 (1 g, 5.84 mmol, 1.0 equiv.) and substituted (2-romoethyl)benzene derivatives (12a-b) (7.01 mmol, 1.2 equiv.) were dissolved in 20 mL acetone. Then, anhydrous K 2 CO 3 (11.68 mmol, 2 equiv.) and catalytic amount KI were added. The reaction mixture was refluxed for 4 h. After completion of the reaction, acetone was concentrated, and the residue was dissolved in water (60 mL) and extracted with ethyl acetate (60 × 3 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated in vacuum. The obtained oil was used in further synthesis without purification yielded compounds 13a-b (Yields were 67% and 72%). Then, 4 mol/L KOH (2.5 equiv.) was added to the solution of compounds 13a-b in C 2 H 5 OH: H 2 O = 5:1(6 mL). The reaction mixture was stirred at room temperature for 7 h. After completion of the reaction, the reaction mixture was evaporated to dryness after neutralization with dilute hydrochloric acid solution. Poured into ethyl acetate to deposit the solid, after cooling off, the mixture was filtered and washed with cold ethyl acetate to give compounds 14a-d.
Finally, intermediates (14a-b) (1.2 equiv.), PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were added to 6 mL DMF and stirred at room temperature for 20 min. Then, intermediates (15a-b) (1.0 equiv.) was added and stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was quenched with saturated NaCl solution. The aqueous phase was extracted with DCM. The DCM layer was combined and washed with brine solution. The organic layer was dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in dichloromethane gradient (DCM:methanol = 60:1-5:1) yielded compounds 16a-d.
original ligand in the co-crystal structures. The docking program CDOCKER encoded in DS 2016 was applied to identify the potential binding of compound 8i to eeAChE, huAChE, and huBChE. Other CDOCKER parameters were set to default values. Compound 8i was chosen for molecular modeling as the most active compounds in the series (Table 1). Compound 8i produced 10 poses to eeAChE, huAChE, and huBChE. These postures were visually examined, and the most appropriate docking pose was selected according to the scores and interactions with key residues of the eeAChE, huAChE, and huBChE active sites.

Conclusions
In this paper, a series of novel compounds 6a-h, 8i-1, 10s-v, and 16a-d were synthesized and evaluated, together with the known analogs 11a-f, for their inhibitory activities towards AChE and BChE. The results show that most of the compounds have AChE and/or BChE inhibitory activity. Compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC 50 = 0.39 µM) and BChE (eqBChE IC 50 = 0.28 µM). Compared with compound G801-0274, compound 8i has comparable inhibitory activity against two ChEs, so that it can exert an anti-ChEs effect in a balanced manner. Kinetic studies indicated a mixed-type inhibition of compound 8i, including competitive inhibition and non-competitive inhibition. Subsequently, molecular docking was performed to evaluate the interaction mechanism between compound 8i and enzymes. Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the PAS and the CAS of AChE and BChE. Therefore, compounds 8i may be promising scaffold for treatment, and further modifications have been made to obtain novel AChE and BChE dual-target inhibitors.

Conflicts of Interest:
The authors declare no conflict of interest.