alexa Design, Synthesis and Biological Evaluation of 1-Phenyl-Ethanone Derivatives for Multi-Targeted Treatment of Alzheimer's Disease

ISSN: 2161-0444

Medicinal Chemistry

  • Research Article   
  • Med chem (Los Angeles) 2017, Vol 7(10): 285
  • DOI: 10.4172/2161-0444.1000469

Design, Synthesis and Biological Evaluation of 1-Phenyl-Ethanone Derivatives for Multi-Targeted Treatment of Alzheimer's Disease

Meihao Liang1#, Wenhai Huang1,2#, Beibei Wang1, Wenhua Wei3, Chixiao Zhang1, Zhimin Zhang1 and Zhengrong Shen1*
1Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou, PR, China
2State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, PR, China
3Southern University of Science and Technology, Shenzhen, PR, China
#Contributed equally to this work
*Corresponding Author: Zhengrong Shen, Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou, PR, China, Tel: +086057188215506, Email: [email protected]

Received Date: Sep 28, 2017 / Accepted Date: Oct 10, 2017 / Published Date: Oct 27, 2017

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease leading to the irreversible loss of brain neurons and cognitive abilities. Multiple factors, such as acetylcholinesterase (AChE), metal ions and amyloid-β (Aβ) have been considered play an important role in the pathogenesis of AD. In this work, AChE and metal ions, both of which are also associated with the deposition of Aβ in the brain, were selected as targets simultaneously. 22 compounds were rationally designed by hybridizing AChE inhibitor rivastigmine and metal chelator 2-hydroxyacetophenone, in a hoping that these compounds could be as a substrate and inhibitor of AChE, while the subsequent enzymatic hydrolysis products by AChE could be as a metal ion chelator. Thus these 22 compounds were synthesized and their biological activities against AD were evaluated in vitro. The results showed that compound w8 presented the best inhibitory activity of AChE (IC50=31.9 μM), and the representing enzymatic hydrolysis products 7f exhibted the metal chelating function. Furthermore, both 7f and one of the targeted compound w15 could inhibit the aggregation of Aβ.

Keywords: Alzheimer’s disease; Amyloid-β; AChE inhibitor; Metal chelators; Drug design

Introduction

Alzheimer’s disease (AD), the leading cause of dementia in the elderly, is a complex neurodegenerative disorder [1,2]. Since multiple factors, such as reduced acetylcholine (ACh) level, metal dyshomeostasis, oxidative stress and amyloid-β (Aβ) aggregation, have been considered to play important roles in the pathogenesis of AD, it is difficult to shed desirable therapeutic effect for single-target strategy [3,4]. Thus, Multi- Target-Directed Ligand, which is rationally designed to hit multiple targets for a particular disease to improve pharmacological profiles, raise as a potentially more effective strategy for AD treatment [5-7].

Until now, clinical available drugs approved for AD are mainly AChE inhibitors [8,9], which improve the ACh level, one of the important neurotransmitters responsible for cognition in the central cholinergic system. Some experiments also find that the level of metal ions in AD patients are 3-7 folds higher than that of healthy individuals [10], it indicated that the dyshomeostasis of biometals (Fe, Cu, Zn) in the brain may contribute to AD pathology [11]. Clioquinol (a classic metal chelator used as antifungal drug and antiprotozoal drug), had been tested to treat AD [12,13]. The phase II clinical trials suggested that clioquinol could halt cognitive decline in AD, possibly owing to its ability to chelate copper and zinc ions. Therefore, decreasing the level of metal ions in brain by using metal chelator represents another rational therapeutic approach for the treating of AD.

Furthermore, both AChE and metal ions are associated with Aβ, which plays a central role in the pathogenesis of AD [14-18]. Recent evidence indicated certain links between Aβ and AChE [19]. AChE could form a complex with Aβ, which changes the conformation of Aβ and then promotes the aggregation Aβ [20,21]. While metal ions binding to soluble Aβ via histidine residues could greatly accelerate Aβ nucleated aggregation [22,23], which is enhanced under mild acidic conditions similar to that present in aging and AD brains. Thus, simultaneously inhibition of AChE and chelation of metal ions, both of which also contribute to the same result of decelerating the Aβ aggregation with different mechanisms, may form synergistic effect in the treatment of AD [24].

Considering the above, we focused on multi-target-directed ligands integrated AChE inhibitors and metal chelators, which not only reduce the hydrolysis of ACh and decrease the levels of metal ions in brain but also slow down the aggregation of Aβ. Rivastigmine, an AChE inhibitor approved for the treatment of AD, could be cleavaged by AChE to release metabolites NAP226-90 in brain (Figure 1) [25], which inspired us to design a molecular skeleton that could be as a substrate and inhibitor of AChE first, and then the subsequent hydrolysis products by AChE could be as a metal ion chelator. Based on this design strategy, 1-phenyl-ethanone derivatives were designed by hybridizing AChE inhibitor rivastigmine and metal chelator 2-hydroxyacetophenone (Figure 2). The 1-phenyl-ethanone derivatives were expected to have multifunctional with effects inhibiting AChE and chelating metal ion, furthermore to have the synergy functions on the inhibition of Aβ formation.

medicinal-chemistry-enzymatic-hydrolysis

Figure 1: The enzymatic hydrolysis of Rivastigmine by AChE.

medicinal-chemistry-targeted-molecular

Figure 2: The design and functions of targeted molecular skeleton.

According to the classical drug design methods such as the principle of bioisosteres and scaffold hopping, a series of 1-phenyl-ethanone derivatives had been designed by changing group R1, R2 and R3. After screening by the Rule of Five, 22 compounds were picked up (Figure 2) and then predicted the blood brain barrier (BBB) permeability by Discovery Studio 2.1. The results showed that all the compounds had good permeability through the BBB (Figure 3). Therefore, a series of 1-phenyl-ethanone derivatives were synthesized and their biological activities against AD were evaluated in vitro.

medicinal-chemistry-designed-compounds

Figure 3: The prediction of BBB permeability of designed compounds.

Results and Discussions

Chemistry

With hydroxyl acetophenone 1 as starting material, the carbonyl was reduced by sodium borohydride first to form 2, and then 2 was acetylated into 3 with acetic anhydride for the protection of phenolic hydroxyl. By chlorination of 3 with thionyl chloride and further condensation of 4 with different secondary amines, a series of compounds 5 were obtained. After acetylation of 5, the produced critical intermediates 6 through the Fries Rearrangement enabled the preparation of 7. Finally, the targeted compounds w1~w22 were synthesized by esterification of 7 with different carbamyl chlorides. The synthetic route was shown in Figure 4.

medicinal-chemistry-synthetic-route

Figure 4: The synthetic route of w1~w22.

Reagents and condition: a. NaBH4, THF, 25°C, 3 h; b. KOH, 0°C, 2 h; c. DMF, SOCl2, DCM, 25°C, 2 h; d. KI, R1, acetonitrile, reflux; e. Na2CO3, DCM, 0°C; f. AlCl3, nitrobenzene, 150°C, 5 h; g. DMAP, TEA, THF, reflux, 4 h.

In vitro biological evaluation

Metal chelating effect: The increase levels of iron, zinc and particularly copper on brain was reported to actively contribute to the formation of amyloid plaques by generating more reactive oxygen species through the Aβ (1-42) metal complex [26]. We once published a review concerning the drug-like metal chelating agents [27]. Many drug-like metal chelating agents were found to be able to reverse Aβ aggregation, dissolve amyloid plaques, and delay the AD-related cognitive impairment. In this presented work, compounds 7 were designed as active ingredients for metal chelating, so that randomly, compound 7f was picked to test the metal-chelating effect.

The metal-chelating effect of compound 7f was studied by ultra violet (UV) spectrometry with wavelength ranging from 200 to 400 nm.27 Upon the addition of Cu2+, Fe2+ and Zn2+ to the 7f methanol solution, the maximum absorption wavelength or absorption intensity happened to change, representing the formation of 7f-Cu2+, 7f-Fe2+ and 7f-Zn2+ (Figure 5). In detail, when Zn2+ was added into the solution 7f, the UV absorption intensity was no obvious changing, only the maximum absorption wavelength shifted slightly. When Fe2+ and Zn2+ were added into solution 7f respectively, the maximum absorption wavelength had no obvious change, while the UV absorption intensity increased obviously. This data revealed that metal-chelating form of 7f- Cu2+ was different from 7f-Fe2+ and 7f-Zn2+.

medicinal-chemistry-absorption-spectra

Figure 5: The absorption spectra of 7f (25 μM) chelate Copper, Iron and Zinc ions.

Aβ aggregation inhibition: Compounds 7f and w15 were tested for their ability to inhibit self-induced Aβ (1~42) aggregation by thioflavin T-based fluorometric assay, [28] with curcumin (Cur) as reference compounds. The fluorescence intensities of five tested groups, respectively 7f, w15, Aβ itself, Cur, and blank, were recorded in Table 1. The morphology of Aβ (1~42) aggregation was transferred for imaging by transmission electron microscopy with 2 μm and 0.5 μm (Figure 6). The fluorescence intensities of w15 was stronger than 7f hydrolyzed from w15 by AChE, but slightly weaker than the positive the control compound Cur. The results revealed that compounds w15 and 7f had considerable potencies inhibiting Aβ aggregation.

medicinal-chemistry-electron-micrographys

Figure 6: Transmission electron micrographys of Aβ aggregation inhibition. a. Aβ (25 μM) diluted with 10 mM phosphate buffer. b. Aβ (25 μM) mixed with Cur (20 μM) in 1% final DMSO concentration. c. Compound 7f mixed with Aβ (25 μM) in 1% final DMSO concentration. d. w15 mixed with Aβ (25 μM) in 1% final DMSO concentration.

  1 2 3 Average Absolute Inhibition%
Blank 24958 24928 24772 24886    
Aβ itself 31044 33188 35388 33206.67 8320.67 0
Cur 29563 28837 28692 29030.67 4144.67 50.19
7f 31202 31228 29030 30486.67 5600.67 32.69
w15 32061 29730 28208 29999.67 5113.67 38.55

Table 1: The results of the ability to inhibit self-induced Aβ aggregation.

AChE enzymatic hydrolysis: For confirming that the synthesized compound w1~w22 could be hydrolyzed to 7 by AChE, the enzymatic hydrolysis of compound w13 by AChE was investigated in vitro, the hydrolysis products after 0.1 h, 0.5 h, 1 h, 3 h, 6 h was detected respectively by HPLC with w13 and 7f as external standard (Figure 7). The results indicated that w13 could be hydrolyzed to 7f by AChE, moreover the hydrolysis was intensified with the increasing of hydrolysis time.

medicinal-chemistry-hydrolysis

Figure 7: HPLC profiles of w13 and 7f in the hydrolysis with AChE.

AChE inhibitory activity: The AChE inhibitory activity of 1-phenyethanone derivatives w1~w22 were tested by spectrophotometric method [29], using rivastigmine as the reference. The percentages of AChE inhibition and IC50 values of all tested compounds were summarized in Table 2.

Compound R1 R2 Inhibitiona (%) IC50 (μM)
Rivastigmine -- -- 31.0b 1.3
w1 image image 30.3 94.3
w2 image image 18.7 --
w3 image image 17.1 --
w4 image image 9.8 --
w5 image image 18.5 --
w6 image image 14.6 --
w7 image image 18.0 --
w8 image image 44.9 31.9
w9 image image 11.2 --
w10 image image 30.2 61.8
w11 image image 12.7 --
w12 image image 6.2 --
w13 image image 9.9 --
w14 image image 6.8 --
w15 image image 7.9 --
w16 image image 6.9 --
w17 image image 11.9 --
w18 image image 4.4 --
w19 image image 17.4 --
w20 image image 21.9 --
w21 image image 14.4 --
w22 image image 24.9 80.3

Table 2: Inhibition of AChE activity and value of IC50.

As shown in Table 1, compounds w1, w8, w10, w22, exhibited moderate AChE inhibitory activities in comparison with rivastigmine, wherein compound w8 displayed most potentially with an IC50 value of 31.9 μM. Unexpectedly, except the above four compounds, all the rest compounds displayed poor AChE inhibitory activities in this test. It may be attributed to that w1~w22 are harder to across the narrow aromatic gorge which linked the central site and peripheral site in AChE.[30]. According to results, it was supposed that when R1 and R2 were short or small secondary amines, such as N-dimethylamine, N-ethylmethylamine or pyrrolidine, the targeted compounds exhibited better AChE inhibitory activities.

Molecular docking study: In an attempt to understand the molecular interaction between w8 and AChE, a molecular docking study was built on previous work,[31] which was performed by Discovery Studio 4.0/CDOCKER protocol using the crystal structure of E2020/AChE complex (PDB ID: 1EVE) as the template. Docking and subsequent scoring studies were performed using default parameters. It was disclosed that the binding pattern of w8 into the AChE was similar to the crystal structure of rivastigmine/AChE (PDB ID:1GQR). As shown in Figure 8, hydrogen bond interactions were observed between amide group and Gly118/Ser200 with distance of 2.15 Å and 1.83 Å, respectively. Besides, the charged nitrogen also made a hydrogen bond interaction with Tyr121 with a distance of 2.68 Å. In addition, the benzene ring formed π-π stacking with Trp334. Moreover, same as the binding pattern of rivastigmine, the carbamate group of w8 was very close to the esteratic site (Ser200, Glu327 and His440), which was beneficial to the enzymatic hydrolysis of carbamate group, and then resulted in a ‘flattening’ of the carbamate moiety over the esteratic site, producing the prolonged inhibition of AChE.

medicinal-chemistry-molecular-docking

Figure 8: The molecular docking of representative compound w8 with AChE.

Conclusion

In summary, a series of 1-phenyl-ethanone derivatives have been successfully designed, synthesized and their biological activities were evaluated in respect to metal chelating, AChE inhibiting and Aβ aggregation inhibiting for the treatment of AD. Among which, several targeted compounds exhibited AChE inhibiting acitivity, although it was somewhat decreased compared with ravastigmine. It was also proved that 1-phenyl-ethanone derivatives could be hydrolyzed by AChE to release OH-metabolite chelating Cu2+, Fe2+ and Zn2+. Meanwhile the tested 1-phenyl-ethanone derivative and its hydrolysis product showed the ability to inhibit the Aβ aggregation. These results validated the possibility of our molecular design strategy, while improvement of AChE activities of 1-phenyl-ethanone derivatives would be the important point in the next stage of the anti-AD drug development with multi-targeted strategy.

Experimental Section

Chemical synthesis

All solvents used were analytical grade. Melting points were recorded on a Buchi apparatus without correction, IR spectra were recorded on a Bruker VECTOR 22 FTIR spectrophotometer. 1H NMR spectra were recorded on a Bruker Avance III 500M instrument (chemical shifts are expressed as δ values relative to TMS as internal standard). Mass spectra (MS) were recorded on an Esquire-LC-00075 spectrometer.

3-(1-hydroxy-ethyl)-phenol (2): A solution of 150 g compound 1 in 500 mL THF was cooled to 0°C with a salt-ice bath and sodium borohydride (51.0 g, 1.3 mol) was added in batches. After that, the reaction was stirred at room temperature for 3 h, and then was quenched with water. The aqueous solution was adjusted pH to 5~6 and extracted with ethyl acetate. The organic layer was dried over Na2SO4, and the residue was crystallized with ethyl acetate to give 2 (132.6 g, 72.5%).

3-(1-hydroxyl) ethyl benzoic acid ethyl ester (3):Compound 2 (126.6 g, 0.9 mol) was dissolved in potassium hydroxide solution (5.5 mol·L-1, 500 mL) and cooled to 0°C. Acetic anhydride (113 mL, 1.2 mol) was slowly dripped into the solution with stirring. The mixture was stirred at room temperature for 2 h and then extracted with ethyl acetate. The organic layer was successively washed with saturated sodium bicarbonate, saturated sodium chloride, water and dried over Na2SO4, at last ethyl acetate was removed to give 3 (125.5 g, 76.3%).

3-(1-chlorine) ethyl benzoic acid ethyl ester (4): Compound 3 (125.5 g, 0.7 mol) was dissolved in the solution of distilled DCM (500 mL) and DMF (123 mL, 1.6 mol). Purified SOCl2 was added slowly into the solution (67 mL, 1.1 mol) and the reaction was stirred at room temperature for 2 h. Then the mixture was slowly added into crushed ice, adjusted pH to 7~8. The organic layer was separated, and the aqueous layer was extracted three times with chloroform. The combined organic layer was washed with water and dried over Na2SO4. The organic solvent was evaporated, and then the residue was chromatographed on silica gel using petroleum ether to give compound 4 (127.0 g, 91.4%).

General procedure A for the syntheses of compounds (5a~5g): Taking 5a as an example: Compounds 4 (12.0g, 60 mmol), KI (80 mmol), K2CO3 in acetonitrile (200 mL), and N-dimethylamine hydrochloride (200 mmol) were mixed together, the mixture was stirred at 45°C for 24 h. The solvent was removed, then the residues were dissolved in water and the solution was acidified to pH 4~5. The aqueous solution was washed three times with ethyl acetate and adjusted to pH 8, then extracted with ethyl acetate. After removal of ethyl acetate, compound 5a (4.0 g, 40.1%) was obtained.

General procedure B for the synthesis of compounds (6a~6g): Taking 6a as an example: 5a (3.8 g, 23.0 mmol) and sodium carbonate (1.5 g, 14.0 mmol) were dissolved in DCM. Then acetic anhydride (2.6 mL, 28 mmol) was added slowly in the above mixture at 0°C. The reaction mixture was stirred at room temperature and monitored by TLC. When the reaction was completed, dichloromethane was removed under vacuum. The solution of the residue was dissolved in water, acidified to pH 4~5 and stirred for another 0.5 h. Followed by the aqueous solution was washed with ethyl acetate, adjusted to pH 8 with Na2CO3, and then extracted with ethyl acetate, dried over Na2SO4, at last ethyl acetate was removed to give to give compound 6a (4.3 g, 90.2%).

General procedure C for the synthesis of compounds (7a~7g): Taking 7a as an example: 6a (3.9 g, 19.0 mmol) and aluminium trichloride (12.6 g, 95.0 mmol) were dissolved in nitrobenzene (20 mL). Acetic anhydride was dissolved in nitrobenzene (20 mL) also, and added dropwise to the above reaction, stirred for 0.5 h at 150°C. Then the solution of Na2CO3 was added in slowly to adjust pH to 8. The aqueous solution was extracted with ethyl acetate, after dried over Na2SO4, ethyl acetate was removed to give compound 7a (2.3 g, 58.1%).

General procedure D for the syntheses of compounds (w1~w22): Taking w1 as an example: The compounds 7a (13 mmol), DMAP (160.0 mg, 1.3 mmol), dimethylcarbamoyl chloride and TEA (156 mmol) were dissolved in THF (100 mL). Then the mixture was stirred at reflux for 2 h. When the reaction was completed, acidified to pH 4~5 with and washed with ethyl acetate. The aqueous layer was adjusted to pH 9 and extracted with ethyl acetate. After dried over Na2SO4, ethyl acetate was removed to give compound w1. yield 90.2%. 1H NMR (400 MHz, CDCl3): δ 7.74 (1H, d, J=8.0 Hz), 7.23 (1H, dd, J1=8.0 Hz, J2=1.2 Hz), 7.11 (1H, d, J=1.2 Hz), 3.30 (1H, q, J=6.4 Hz), 3.15 (3H, s), 3.05 (3H, s), 2.55 (3H, s), 2.22 (6H, s), 1.36 (3H, d, J=6.4 Hz). MS ([M+H]+) m/z calcd for C15H15N2O3 279.2; found 279.2.

Ethyl-methyl-carbamic acid 2-acetyl-5-(1-dimethylamino-ethyl)- phenyl ester (w2): Compound w2 was obtained from compound 7a and N-ethyl-N-methylcarbamoyl chloride by general procedure D; yield 90.2%. 1H NMR (400 MHz, CDCl3): δ 7.65 (1H, d, J=8.0 Hz), 7.14 (1H, d, J=8.0 Hz), 7.02 (1H, d, J=6.8 Hz), 3.45 (1H, q, J=6.8 Hz), 3.35 (1H, q, J=6.8 Hz), 3.24 (1H, m), 3.03 (1.5H, s), 2.92 (1.5H, s), 2.47 (3H, s), 2.14 (6H, s2), 1.29 (3H, d, J=6.0 Hz), 1.21 (1.5H, t, J=7.2 Hz), 1.12 (1.5H, t, J=7.2 Hz). MS ([M+H]+) m/z calcd for C16H24NO2 293.2; found 293.2.

Diethyl-carbamic acid 2-acetyl-5-(1-dimethylamino-ethyl)- phenyl ester (w3): Compound w3 was obtained from compound 7a and diethylcarbamyl chloride by general procedure D; yield 90.4%. 1H NMR (400 MHz, CDCl3): δ 7.71 (1H, d, J=8.0 Hz), 7.21 (1H, d, J=8.0 Hz), 7.09 (1H, s), 3.50 (2H, q, J=7.2 Hz), 3.38 (2H, q, J=7.2 Hz), 3.26 (1H, q, J=6.8 Hz), 2.54 (3H, s), 2.21 (6H, s), 1.35 (3H, d, J=6.8 Hz), 1.29 (3H, t, J=7.2 Hz), 1.21 (3H, t, J=7.2 Hz). MS ([M+H]+) m/z calcd for C17H26N2O3 307.2; found 307.2.

Diethyl-carbamic acid 2-acetyl-5-(1-dimethylamino-ethyl)- phenyl ester (w4): Compound w4 was obtained from compound 7a and 1-pyrrolidinecarbonyl chloride by general procedure D; yield 89.7%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.22 (1H, d, J=8.0 Hz), 7.14 (1H, s), 3.62 (2H, t, J=6.8 Hz), 3.49 (2H, t, J=6.8 Hz), 3.29 (1H, q, J=6.8 Hz), 2.56 (3H, s), 2.21 (6H, s), 1.98 (4H, m), 1.36 (3H, d, J=6.4 Hz). MS ([M+H]+) m/z calcd for C17H24N2O3 307.2; found 305.2.

Piperidine-1-carboxylic acid 2-acetyl-5-(1-dimethylaminoethyl)- phenyl ester (w5): Compound w5 was obtained from compound 7a and piperidine-1-carbonyl chloride by general procedure D; yield 90.6%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.22(1H, dd, J1=8.0 Hz, J2=1.6 Hz), 7.10 (1H, d, J=1.6 Hz), 3.65 (2H, m), 3.52 (2H, m), 3.29 (1H, q, J=6.8 Hz), 2.55 (3H, s), 2.21 (6H, s), 1.67 (6H, m), 1.36 (3H, d, J=6.8 Hz). MS ([M+H]+) m/z calcd for C18H18N2O3 307.2; found 319.2.

Synthesis of Morpholine-4-carboxylic acid 2-acetyl-5-(1- dimethylamino-ethyl)- phenyl ester (w6): Compound w6 was obtained from compound 7a and morpholine-4-carbonyl chloride by general procedure D; yield 91.8%. 1H NMR (400 MHz, CDCl3): δ 7.75 (1H, d, J=8.0 Hz), 7.25 (1H, dd, J1=8.0 Hz, J2=1.2 Hz), 7.12 (1H, d, J=1.2 Hz), 3.78 (5H, m), 3.72 (1H, m), 3.56 (2H, t, J=4.4 Hz), 3.27 (2H, t, J=4.4 Hz), 2.55 (3H, s), 2.21 (6H, s), 1.35 (3H, d, J=6.4 Hz). MS ([M+H]+) m/z calcd for C17H24N2O4 307.2; found 321.2.

Dimethyl-carbamic acid 2-acetyl-5-(1-diethylamino-ethyl)- phenyl ester (w7): Compound w7 was obtained from compound 7c and dimethylcarbamoyl chloride by general procedure D; yield 88.6%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.30 (1H, dd, J1=8.0 Hz, J2=1.2 Hz), 7.16 (1H, d, J=1.2 Hz), 3.82 (1H, q, J=6.8 Hz), 3.15 (3H, s), 3.03 (3H, s), 2.55 (3H, s), 2.52 (4H, m), 1.32 (3H, d, J=6.8 Hz), 0.99 (6H, t, J=6.8 Hz). MS ([M+H]+) m/z calcd for C17H26N2O3 307.2; found 307.2.

Ethyl-methyl-carbamic acid 2-acetyl-5-(1-diethylamino-ethyl)- phenyl ester (w8): Compound w8 was obtained from compound 7c and N-ethyl-N-methylcarbamoyl chloride by general procedure D; yield 88.7%. 1H NMR (400 MHz, CDCl3): δ 7.72 (1H, d, J=8.0 Hz), 7.29 (1H, dd, J1=8.0 Hz, J2=1.2 Hz), 7.15 (1H, d, J=8.0 Hz), 3.81 (1H, q, J=6.4 Hz), 3.53 (q, 1H, J=6.8 Hz, -CONCH2CH3), 3.42 (q, 1H, J=6.8 Hz, -CONCH2CH3), 3.12 (s, 1.5H), 3.00 (1.5H, s), 2.55 (3H, s), 2.53 (4H, m), 1.32 (3H, d, J=6.8 Hz), 1.19 (3H, m), 0.99 (6H, t, J=6.8 Hz). MS ([M+H]+) m/z calcd for C18H28N2O3 321.2; found 321.2.

Diethyl-carbamic acid 2-acetyl-5-(1-diethylamino-ethyl)-phenyl ester (w9): w9 was obtained from compound 7c and diethylcarbamyl chloride by general procedure D; yield 89.3%. 1H NMR (400 MHz, CDCl3): δ 7.71 (1H, dd, J1=8.0 Hz, J2=2.4 Hz), 7.28 (1H, dd, J1=8.0 Hz, J2=1.6 Hz), 7.13 (1H, d, J=1.2 Hz), 3.81 (1H, m), 3.49 (2H, m), 3.38 (2H, m), 2.54 (3H, s), 2.52 (4H, m), 1.30 (6H, m), 1.21 (3H, m), 0.98 (6H, m,). MS ([M+H]+) m/z calcd for C19H30N2O3 335.2; found 335.2.

Pyrrolidine-1-carboxylic acid 2-acetyl-5-(1-diethylamino-ethyl)- phenyl ester (w10): Compound w10 was obtained from compound 7c and 1-pyrrolidinecarbonyl chloride by general procedure D; yield 87.5%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.29 (1H, dd, J1=8.0 Hz, J2=1.6 Hz), 7.14 (1H, d, J=1.2 Hz), 3.81 (1H, q, J=6.8 Hz), 3.62 (2H, t, J=6.8 Hz), 3,49 (2H, t, J=6.8 Hz), 2.55 (3H, s), 2.53 (4H, m), 1.97 (4H, m), 1.31 (3H, d, J=6.4 Hz), 0.99 (6H, t, J=7.2 Hz). MS ([M+H]+) m/z calcd for C19H28N2O3 335.2; found 335.2.

4-Methyl-piperazine-1-carboxylic acid 2-acetyl-5-(1- diethylaminoethyl)-phenyl ester (w11): Compound w11 was obtained from compound 7c and 4-Methyl-piperazine-1-carbonyl chloride by general procedure D; yield 80.2%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.32 (1H, d, J=8.0 Hz), 7.16 (1H, s), 3.82 (1H, q, J=6.8 Hz), 3.74 (2H, m), 3.60 (2H, m), 2,54 (3H, s), 2.50 (8H, m), 2.36 (s, 3H, -NCH3), 1.32 (3H, d, J=6.8 Hz), 0.99 (6H, t, J=7.2 Hz,). MS ([M+H]+) m/z calcd for C20H31N3O3 362.2; found 362.2.

Morpholine-4-carboxylic acid 2-acetyl-5-(1-diethylamino-ethyl)- phenyl ester (w12): Compound w12 was obtained from compound 7c and morpholine-4-carbonyl chloride by general procedure D; yield 90.2%. 1H NMR (400 MHz, CDCl3): δ7.74 (1H, d, J=8.0 Hz), 7.32 (1H, dd, J1=8.0 Hz, J2=1.2 Hz), 7.17 (1H, d, J=1.6 Hz), 3.78 (4H, m), 3.83 (1H, m), 3.72 (2H, m), 3.60 (2H, m), 2.54 (3H, s), 2.52 (4H, m), 1.32 (3H, d, J=6.4 Hz), 0.99 (6H, t, J=7.2 Hz). MS ([M+H]+) m/z calcd for C19H28N2O4 349.2; found 349.2.

Dimethyl-carbamic acid 2-acetyl-5-(1-morpholin-4-yl-ethyl)- phenyl ester (w13): Compound w13 was obtained from compound 7f and dimethylcarbamoyl chloride by general procedure D; yield 90.4%.1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.25 (1H, dd, J1=8.0 Hz, J2=1.6 Hz), 7.12 (1H, d, J=2.0 Hz), 3.69 (4H, m), 3.33 (1H, q, J=6.8 Hz,), 3.15 (3H, s), 3.03 (3H, s), 2.54 (3H, s), 2.49 (2H, m), 2.37 (2H, m), 1.33 (3H, d, J=6.8 Hz,). MS ([M+H]+) m/z calcd for C17H24N2O4 3321.2; found 321.2.

Ethyl-methyl-carbamic acid 2-acetyl-5-(1-morpholin-4-yl-ethyl)- phenyl ester (w14): Compound w14 was obtained from compound 7f and N-ethyl-N-methylcarbamoyl chloride by general procedure D; yield 89.9%. 1H NMR (400 MHz, CDCl3): δ 7.72 (1H, d, J=8.0 Hz), 7.25 (1H, d, J=8.0 Hz), 7.11 (1H, d, J=10.0 Hz), 3.69 (4H, m), 3.53 (1H, q, J=7.2 Hz), 3.41 (1H, q, J=7.2 Hz), 3.33 (1H, q), 3.12 (1.5H, s), 3.00 (1.5H, s), 2.54 (3H, s), 2.49 (2H, m), 2.38 (2H, m), 1.33 (3H, d, J=6.4 Hz), 1.27 (1.5H, t, J=7.2 Hz), 1.21 (1.5H, t, J=7.2 Hz). MS ([M+H]+) m/z calcd for C18H26N2O4 3321.2; found 335.2.

Diethyl-carbamic acid 2-acetyl-5-(1-morpholin-4-yl-ethyl)- phenyl ester (w15): Compound w15 was obtained from compound 7f and diethylcarbamyl chloride by general procedure D; yield 91.2%. 1H NMR (400 MHz, CDCl3): δ 7.63 (1H, d, J=8.0 Hz), 7.18 (1H, d, J=8.0 Hz), 7.02 (1H, s), 3.47 (4H, m), 3.40 (2H, q, J=7.2 Hz), 3.23 (3H, m), 2.35 (5H, m), 2.22 (2H, m), 1.19 (3H, d, J=6.8 Hz), 1.16 (3H, t, J=7.2 Hz), 1.03 (3H, t, J=6.8 Hz,). MS ([M+H]+) m/z calcd for C19H28N2O4 349.2; found 349.2.

Pyrrolidine-1-carboxylic acid 2-acetyl-5-(1-morpholin-4- yl-ethyl)-phenyl ester (w16): Compound w16 was obtained from compound 7f and 1-pyrrolidinecarbonyl chloride by general procedure D; yield 91.2%; yield 87.5%. 1H NMR (400 MHz, CDCl3): δ 7.66 (1H, d, J=8.0 Hz), 7.18 (1H, d, J=8.0 Hz), 7.08 (1H, s), 3.62 (4H, m), 3.55 (2H, t, J=6.4 Hz), 3.42 (2H, t, J=6.4 Hz), 3.22 (1H, m), 2.48 (3H, s), 2.42 (2H, m), 2.36 (2H, m), 1.90 (4H, m), 1.26 (3H, d, J=6.0 Hz,). MS ([M+H]+) m/z calcd for C19H26N2O4 347.2; found 347.2.

Piperidine-1-carboxylic acid 2-acetyl-5-(1-morpholin-4-ylethyl)- phenyl ester (w17): Compound w17 was obtained from compound 7f and piperidine-1-carbonyl chloride by general procedure D; yield 88.6%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.25 (1H, d, J=8.0 Hz), 7.11 (1H, s), 3.69 (4H, m), 3.66 (2H, m), 3.52 (2H, m), 3.33 (1H, q, J=6.4 Hz), 2.54 (3H, s), 2.49 (2H, m), 2.39(2H, m), 1.68 (6H, m), 1.33 (3H, d, J=6.4 Hz). MS ([M+H]+) m/z calcd for C20H28N2O4 360.4; found 360.4.

Morpholine-4-carboxylic acid 2-acetyl-5-(1-morpholin-4- yl-ethyl)-phenyl ester (w18): Compound w18 was obtained from compound 7f and morpholine-4-carbonyl chloride by general procedure D; yield 90.0%. 1H NMR (400 MHz, CDCl3): δ 7.75 (1H, d, J=8.0 Hz), 7.27 (1H, d, J=8.0 Hz), 7.14 (1H, s), 3.79 (4H, m), 3.69 (6H, m), 3.58 (2H, m), 3.35 (1H, q, J=6.4 Hz), 2.55 (3H, s), 2.49 (2H, m), 2.38 (2H, m), 1.34 (3H, d, J=6.4 Hz). MS ([M+H]+) m/z calcd for C19H26N2O5 363.2; found 363.2.

Ethyl-methyl-carbamic acid 2-acetyl-5-[1-(ethyl-methyl-amino)- ethyl]-phenyl ester (w19): Compound w19 was obtained from compound 7b and N-ethyl-N-methylcarbamoyl chloride by general procedure 4.7.1; yield 91.6%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J=8.0 Hz), 7.24 (1H, dd, J1=8.0 Hz, J2=1.6 Hz), 7.11 (1H, d, J=8.0 Hz), 3.54 (2H, m), 3.41 (1H, q, J=6.8 Hz,), 3.11 (1.5H, s), 3.00 (1.5H, s), 2.54 (3H, s), 2.49 (1H, m), 2.36 (1H, m), 2.19 (3H, s), 1.33 (3H, d, J=6.8 Hz), 1.28 (1.5H, t, J=6.8 Hz), 1.20 (1.5H, t, b, J=6.8 Hz), 1.02 (3H, t, J=6.8 Hz,). MS ([M+H]+) m/z calcd for C17H26N2O3 307.2; found 307.2.

Ethyl-methyl-carbamic acid 2-acetyl-5-(1-pyrrolidin-1-yl-ethyl)- phenyl ester (w20): Compound w20 was obtained from compound 7d and N-ethyl-N-methylcarbamoyl chloride by general procedure D; yield 87.7%. 1H NMR (400 MHz, CDCl3): δ 7.72 (1H, d, J=8.0 Hz), 7.25 (1H, dd, J1=8.0 Hz, J2=1.2 Hz), 7.13 (1H, d, J=6.8 Hz), 3.52 (1H, q, J=6.8 Hz), 3.41 (1H, q, J=6.8 Hz), 3.22 (1H, q, J=6.8 Hz), 3.11 (1.5H, s), 3.00 (1.5H, s), 2.54 (3H, s), 2.49 (2H, m), 2.39 (2H, m), 1.78 (4H, m), 1.38 (3H, d, J=6.8 Hz), 1.28 (1.5H, m), 1.20 (1.5H, m). MS ([M+H]+) m/z calcd for C18H26N2O3; found 319.2.

Ethyl-methyl-carbamic acid 2-acetyl-5-(1-piperidin-1-yl-ethyl)- phenyl ester (w21): Compound w21 was obtained from compound 7e and N-ethyl-N-methylcarbamoyl chloride by general procedure D; yield 86.9%. 1H NMR (400 MHz, CDCl3): δ 7.72 (d, 1H, J=8.0 Hz, ArH), 7.24 (d, 1H, J=8.0 Hz, ArH), 7.09 (d, 1H, J=8.4 Hz, ArH), 3.52 (m, 3H, -CHCH3, -CH2CH3), 3.43 (m, 3H, -NCH3), 2.54 (s, 3H, -COCH3), 2.37 (m, 4H, piperidine), 1.54 (m, 4H, piperidine), 1.40 (m, 2H, piperidine), 1.34 (d, 3H, J=6.8 Hz, -CHCH3), 1.20 (m, 3H, -CH2CH3). MS ([M+H]+) m/z calcd for C19H28N2O3; found 332.2.

Ethyl-methyl-carbamic acid 2-acetyl-5-[1-(4-methyl-piperazin- 1-yl)-ethyl]-phenyl ester (w22): Compound w22 was obtained from compound 7g and N-ethyl-N-methylcarbamoyl chloride by general procedure 4.7.1; yield 85.3%. 1H NMR (400 MHz, CDCl3): δ 7.71 (1H, d, J=8.0 Hz), 7.24 (1H, d, J=8.0 Hz), 7.10 (1H, d, J=9.2 Hz), 3.52 (1H, m), 3.39 (2H, m), 3.11 (1.5H, s), 3.00 (1.5H, s), 2.53 (3H, d, J=2.0 Hz), 2.47 (8H, m), 2.32 (3H, d, J=2.0 Hz), 1.33 (3H, dd, J1=6.4 Hz, J2=2.8 Hz), 1.29 1.5H, (m), 1.20 (1.5H, m). MS ([M+H]+) m/z calcd for C19H29N3O3; found 348.2.

Spectrophotometric measurement of complex with Cu2+ and Fe2+

All compounds were tested as metal chelators, using difference UVVis spectra recorded in methanol at 298 K with wavelength ranging from 210 nm to 400 nm. Numerical subtraction of the spectra of the metal alone and the compound alone from the spectra of the mixture obtained the difference UV-Vis spectra due to complex formation. A fixed amount of 9f (25 μmol·L-1) was mixed with growing amounts of copper ion (1.25 μmol·L-1 to 50 μmol·L-1) and tested the difference UVVis spectra to investigate the ratio of ligand/metal in the complex.

Study of AChE hydrolysis

Compound w13 20 μL (3 mol·L-1) was added into PBS buffer (pH 7.4) 160 μL, 0.05% (v/v) Triton X-100 10 μL and rat AChE homogenate, was incubated at 37°C. After 0.1 h, 0.5 h, 1 h, 3 h, and 6 h, 20 μL was taken out from the incubating mixture with a pipette gun, extracted with ethyl acetate, combined with organic layer, dried by anhydrous sodium sulfate and evaporated solvent. The residue was dissolved in acetonitrile and analyzed with HPLC. HPLC analyses was conducted on Dikma Techndogies C18 (symmetry: 250 mm × 4.6 μm) column at 35°C, eluent: acetonitrile: 0.1% ethylamine aqueous solution=6:4, flow rate: 1 mL/min. detection at 254 nm.

Conflict of Interests

The authors declare no competing financial interest.

Acknowledgements

This work was supported by Natural Science Foundation of China (2015F50015), Natural Science Foundation of Zhejiang Province (LY17H310008, LQ17H300003), Health and Family Planning Commission of Zhejiang Province (XKQ-010-001 and 2017143412), and the State Key Laboratory of Medicinal Chemical Biology.

References

Citation: Liang M, Huang W, Wang B, Wei W, Zhang C, et al. (2017) Design, Synthesis and Biological Evaluation of 1-Phenyl-Ethanone Derivatives for Multi-Targeted Treatment of Alzheimer’s Disease. Med Chem 7: 285-293. Doi: 10.4172/2161-0444.1000469

Copyright: © 2017 Liang M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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