Design, Synthesis and Molecular Modeling of Isothiochromanone Derivatives as Acetylcholinesterase Inhibitors

Background ： A series of novel isothio- and isoselenochromanone derivatives bearing N -benzyl pyridinium moiety were designed, synthesized and evaluated as acetylcholinesterase (AChE) inhibitors. Results: Most of the target compounds exhibited potent anti-AChE activities with IC 50 values in nanomolar ranges. Among them, compound 15a exhibited the most potent anti-AChE activity (IC 50 = 2.7 nM), moderate antioxidant activity and low neurotoxicity. Moreover, the kinetic and docking studies revealed that compound 15a was a mixed-type inhibitor, which bound to PAS and CAS of AChE. Conclusion: Those results suggested that compound 15a might be a potential candidate for AD treatment.

and the improvement of the cholinergic function is an effective way to overcome the occurrence, symptoms and progression of AD [6].
The crystal structure of AChE has been reported [7]. It possesses a catalytic active site (CAS) at the bottom of the deep narrow gorge and a peripheral anionic site (PAS) near the entrance of gorge which could accelerate Aβ peptide deposition and promote the formation of Aβ fibril [8]. Therefore, designing the dual-site AChE inhibitors interacting with both the CAS and PAS are meaningful for AD prevention.
It was reported that the introduction of N-benzyl pyridinium moiety into aromatic scaffolds could endow them with potent AChE inhibitory activities, such as compounds 1-4 [9][10][11][12] (Figure 1). In our previous studies, a series of novel dual-site AChE inhibitors bearing the N-benzyl pyridinium moiety derived from natural product (±)-7, 8-Dihydroxy-3-methylisochroman-4-one [(±)-XJP] were designed and synthesized ( Figure 2). The docking studies revealed that the presence of N-benzyl pyridinium moiety contributed to inhibition activities by interacting with the CAS of AChE, and the 4-isochromanone moiety formed stacking interactions with PAS of AChE [8]. To explore the effect of methoxy groups at 4-isochromanone skeleton on the AChE inhibitory activity, a series of derivatives have been further synthetized. The results showed that compound 6 exhibited the most potent anti-AChE activity and the highest AChE / BuChE selectivity with low neurotoxicity [13]. The structure-activity relationships (SARs) studies demonstrated compounds bearing methoxy group at both 6 and 7 positions exhibited more potent anti-AChE activity than those with methoxy group at other positions. Although, the preliminary SARs of the 4-isochromanone compounds bearing N-benzyl pyridinium moiety have been studied previously, the effect of the oxygen atom at the 4-isochromanone skeleton on the AChE inhibitory activity was undefined. According to previous reports, the compounds containing selenium and sulfur exhibits a wide range of bioactivity, especially for antioxidation and neuroprotection [14,15]. Therefore, with the goal of discovering new anti-AChE agents with novel skeletons as well as completing the SARs of the isochromanone hybrids, we further designed and synthesized a series of isothio or isoselenochromanone derivatives by using bioisosterism strategy ( Figure 2). Herein, we would like to report their synthesis, AChE and BuChE inhibitory activities, antioxidant activity, the kinetic and molecular modeling studies.

Chemistry
All commercially available starting materials and solvents were reagent grade and used without further purification unless otherwise noted. 1  To a solution of (3,4-dimethoxyphenyl)methanol (300 mg, 1.78 mmol) in 10 mL acetonitrile, trimethylsilyl chloride (339 μL, 2.68 mmol) and KI (444.14 mg, 2.68 mmol) were added and the mixture was stirred for 45 min at ambient temperature.
Then the mixture was extracted with ethyl acetate (3 × 25 mL). The combined organic layers were then washed with brine (25 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residues were purified by column chromatography with petroleum / ethyl acetate (10: 1) as eluent to afford 422.1 mg products 8 as yellow solids in 85.1% yields.
The synthesis of intermediate 9.
0.85 g (10.79 mmol) Se power was added into 20 mL deionized water at room temperature, and the mixture was stirred for 15 min. Sodium borohydride (612.19 mg, 16.18 mmol) in 20 mL of deionized water was slowly added into Se power suspension at room temperature. After the initial vigorous reaction had subsided (20 min), one additional equiv of Se powder (0.85 g, 10.79 mmol) was added and a brownish red solution was obtained. At room temperature, a solution of 4-(iodomethyl)-1, 2-dimethoxybenzene (3 g, 10.79 mmol) in 15 mL refined THF was added and the mixture was stirred for 30 min. Then the mixture was extracted with ethyl acetate (3 × 125 mL). The combined organic layers were then washed with brine (100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residues were purified by column chromatography with petroleum / ethyl acetate (4: 1) as eluent to afford 4.10 g 9 as yellow oil in 82.3% yields.

The synthesis of intermediates 11a and 11b.
To the solution of intermediate 10a (2 g, 7.40 mmol) or 10b (2 g, 6.30 mmol) in 20 mL MeOH, 5 mL 10% NaOH solution was dropped in and the mixture was stirred for 30 min at reflux temperature. The solvent was removed under reduced pressure. The residues were extracted with ethyl acetate (2 × 125 mL) to remove impurities. The water layer was first adjusted to pH 4, and after adding water (50 mL), it was extracted with ethyl acetate (3 × 125 mL). The combined organic layers were then washed with brine (100 mL), dried over anhydrous Na2SO4. The solvent was removed in vacuo to afford 1.55 g 11a and 1.61 g 11b as yellow oil with yields of 86.6% and 88.5%, respectively.

The synthesis of intermediates 12a and 12b.
To the solution of 11a (2 g, 8.25 mmol) or 11b (2 g, 6.92 mmol) in 20 mL dry dichloromethane, oxalyl dichloride (2 eq.) was added and the mixture was stirred for 30 min at 0 ℃. Then the solvent was removed under reduced pressure. The residues were dissolved in 10 mL chlorobenzene, then SnCl4 (2 mL) was dropped in the solutions at 0 ℃ and the mixtures were stirred for 2 h at room temperature. The solvent was removed in vacuo. The residues were washed with saturated aqueous solution of NaHCO3 and the mixtures were diluted with 125 mL ethyl acetate. After filtering, the mixtures were washed with water (3 × 75 mL). The organic layers were then washed with brine (75 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residues were purified by column chromatography with petroleum / ethyl acetate (10: 1; 4: 1) as eluent to afford 12a or 12b as white solid, yield 70.3% or 79.1%.

The synthesis of intermediates 13a and 13b.
To the solutions of 12a (300 mg, 1.34 mmol) or 12b (300 mg, 1.11 mmol) and pyridine-4-carboxaldehyde (1.2 eq.) in 2 mL DMF, 2 mL 10% K2CO3 aqueous solution was dropped in slowly at 0 ℃. After stirred for 30 s -2 min at 0 ℃, the reactions appeared the precipitates and the water was added into the reactions. Then the precipitates were collected by filtration, washed with water and dried to afford 13a and 13b in general yields.

The synthesis of intermediates 14a and 14b.
To a solution of 12a (300 mg, 1.34 mmol) or 12b (300 mg, 1.11 mg) in 10 mL THF, NaOCH3 (3 eq.) was added at room temperature. After stirred for 30 s, the N-Boc-piperidine-4-carboxaldehyde (1.2 eq.) in THF (3 mL) was dropped in and the mixtures were stirred for 30 s -2 min at room temperature. The mixtures were diluted with 10 mL water, then extracted with (3 × 25 mL) EtOAc. The combined organic layers were then washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The residues were dissolved in 10 mL DCM and CF3COOH was added. After stirred for 2 h, the mixtures were diluted with 25 mL saturated NaHCO3 solution and were extracted with DCM (3 × 25 mL). The combined organic layers were then washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The residues were purified by column chromatography with petroleum / ethyl acetate (1: 1) as eluent to afford 14a or 14b in general yields.

The synthesis of target compounds 15a -15l.
Under N2 atmosphere, to the solutions of 75 mg intermediate 13a, 13b, 14a or 14b in 8 mL acetonitrile, the proper benzyl bromide derivatives (1.1 eq.) and K2CO3 (1.5 eq.) were added and the mixtures were stirred for 1 -2 h at reflux temperature. Then the solvents were removed in vacuo and the residues were purified by column chromatography with petroleum / ethyl acetate (2: 1) as eluent to afford 15e, 15f, 15k or 15l in general yields. And the residues were purified by column chromatography with DCM / MeOH (30: 1) as eluent to afford 15a -15f in low yields or 15g -15j in general yields.

Cell viability
SH-SY5Y cells were planted in a 15 mL culture flask in Eagle's minimum essential medium (EMEM) and Ham's F-12 medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, and the culture flask was cultured in the incubator (37 ℃, 5% CO2). The cells were then grown at 10 4 cells for per well in 96-well plates. When the cells were fused, the cells were placed in serum-free medium. The different concentrations of compounds 15a and 15h (1, 10, 50 μM) were added to the serum-free medium which cultured for 24 h in the incubator, then 10 μL of MTT was added at 37 °C. After cultured for 3 h, 100 μL of DMSO was added to dissolve methanine crystals at the end, and the absorbance the mixture was determined at 490 nM by microplate reader.

Kinetic study of AChE inhibition
To obtain the mechanism of 15a, a series of experiments were performed by using

Molecular modeling
In our study, the X-ray structure of the donepezil -AChE complex was downloaded from the Protein Data Bank (PDB code: 4ey7). The protein was prepared using Schrodinger. The docking procedure was performed by employing the Glide module in the Schrodinger software package, and the structural image was obtained using PyMOL software.

In silico ADMETox study
An in silico study of synthesized compounds 15a-15l was performed for prediction of ADME properties and toxicity. For this purpose, Blood-brain barrier (BBB) permeability, human intestinal absorption (HIA), Caco2 permeability (Caco2) and some toxicity risk parameters were calculated by using online admetSAR.

Results & discussions Chemistry
The synthetic route of the target compounds was shown in Figure 3. The iodination of the starting material 7 gave the intermediate 8 [16], which was substituted by ethyl 2-mercaptoacetate in the presence of K2CO3 to afford intermediate 10a. The

In vitro inhibitory activity of cholinesterase
The inhibitory activity of all target compounds against AChE and BuChE were evaluated using the spectrophotometric method of Ellman et al. And donepezil was used as a reference [18]. The IC50 values for the inhibition of AChE and BuChE were presented in Table 1. The results revealed that all target compounds bearing N-benzyl pyridinium moiety exhibit significant inhibitory activity towards AChE with IC50 values in the nanomolar range, indicating that these compounds are potent AChE inhibitors. However, the AChE inhibitory activity of compounds 15e, 15f, 15k and 15l decreased significantly when pyridine was changed to piperidine, suggesting that the N-benzyl pyridinium moiety is crucial for AChE inhibition. The unsubstituted benzyl pyridinium compound 15a bearing an isothiochromanone moiety displayed the most potent inhibition against AChE with the IC50 value of 2.7 nM, which was 4.7-fold more potent than the positive control donepezil. The para-F substituted benzyl pyridinium compound 15h bearing isoselenochromanone moiety also displayed a potent inhibitory activity (IC50 = 5.8 nM), which was 2.2-fold more potent than donepezil. Thus, the potent AChE inhibitory activity of this class of derivatives prompted us to further investigate their kinetic and binding modes.

Kinetic study of AChE inhibition
In order to analyze the AChE inhibitory mechanism of these inhibitors, the most potent compound 15a was selected for a kinetic test. The type of inhibition was elucidated from the analysis of Lineweaver-Burk reciprocal plots (Figure 4), which showed both increasing slopes and intercepts on the y-axis with increasing concentration of inhibitor. According to this pattern, compound 15a is a mixed-type inhibitor for AChE, which showed that compound 15a might be able to simultaneously bind to the CAS and PAS of AChE.

Cell viability assay of compounds 15a and 15h
To evaluate the cytotoxic effect of target compounds against nerve cell [19], the viability of SH-SY5Y cells treated with representative compounds 15a and 15h at different concentrations was determined. As shown in Table 2, negligible cytotoxicity was noted for compounds 15a and 15h at concentrations of 1 and 10 μM. When the concentration rose to 50 μM, the survival rate only decreased slightly (> 80%) and the morphology of the SH-SY5Y cells was not affected, which indicated that compounds 15a and 15h have low neurotoxicity.

In vitro antioxidant activity
The radical scavenging activity of compounds 15a and 15h was determined by using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay and trolox was used as a reference. The results showed that compounds 15a and 15h at a concentration of 100 μM possessed moderate antioxidant activity. The free radical scavenging activity of compounds 15a and 15h were 55% and 45% compared to trolox, respectively, which were more potent than that of compound 6 (10%).

Docking studies
To further explore the binding mode of compounds 15a and 15h with AChE, a molecular docking study was performed by using the Glide module in the Schrodinger software package. The X-ray crystal structure of AChE complexed with donepezil (PDB: 4ey7) was chosen as the docking protein. As shown in Figure 5, the overlay of the best poses of docked compounds 15a and 15h with donepezil in the active site clearly demonstrated that compounds 15a and 15h adopt similar dual-binding modes to that of donepezil in the binding pocket. As presented, the methoxy groups and carbonyl group of isothio-and isoselenochromanone moieties formed three hydrogen bonds with a water molecule (H2O953) and the residue Phe295 of PAS, respectively.
Isothio-and isoselenochromanone moieties showed ππ stacking interactions with Trp286, which is another important amino acid residue of PAS. In addition, the N-benzyl pyridinium moiety of compounds 15a and 15h bound to the CAS of AChE via aromatic ππ stacking interactions and salt bridge with Trp86 and Asp74, respectively. All these results indicated that compounds 15a and 15h could simultaneously bind to the PAS and CAS of AChE, which is in accordance with the results of kinetic study.

Prediction of toxicity risks and drug-likeness properties
To evaluate the drug-likeness character of the target compounds, toxicity risks (mutagenicity, tumorigenicity, irritation, and reproduction) were predicted by Osiris calculation. Moreover, the ADME (Absorption, Distribution, Metabolism, and Excretion) properties of the target compounds, such as BBB (Blood-Brain Barrier) penetration, HIA (Human Intestinal Absorption), Caco-2 cell permeability were also calculated using admetSAR web-based application [20]. As shown in Table 3, compounds 15a-f bearing isothiochromanone moiety were predicted to be without toxicity risks, whereas compounds 15g-l bearing isoselenochromanone moiety were tumorigenic. According to the predicted values for BBB, all compounds might be able to penetrate into the central nervous system (CNS). In the case of HIA and Caco-2 permeability, all compounds might be well absorbed from intestine. represents non-toxic and red represents toxic.
b Blood Brain Barrier. The values more than 95 was considered as +.
c Human Intestinal Absorption. The values more than 90 was considered as +.
d The values more than 50 was considered as +.

Conclusion
In conclusion, on the basis of our previous studies, twelve novel AChE inhibitors bearing N-benzyl pyridinium moiety derived from natural product XJP were further was proved to be a mixed-type inhibitor binding to the PAS and CAS of AChE. All these results highlighted 15a was a promising lead compound for the development of anti-AD agents.

Future perspective
Alzheimer disease (AD), which has become a health problem worldwide, is the most common form of dementia in elderly people. However, the pathogenesis of AD still remains unclear. And the approved drug (donepezil, rivastigmine, galantamine and memantine) could only enable a palliative treatment instead of curing or preventing AD. Therefore, it is necessary to develop new and more effective anti-AD drugs.
Based on the results of our studies, most of the isothio-and isoselenochromanone derivatives were potent AChE inhibitors. Therefore, we speculate that isothio-and isoselenochromanone are desirable skeletons which could provide new insights into the development of anti-AD drugs.

Executive summary
Introduction: • Alzheimer's disease (AD), which emerged as the main cause of dementia, has affected over 46 million elderly people in the worldwide.
Experimental section: • A series of novel isothio-and isoselenochromanone derivatives were