Synthesis, in vitro acetylcholinesterase inhibitory activity evaluation and docking investigation of some aromatic chalcones

MedPharmRes, 2017, 1 15 MedPharmRes journal of University of Medicine and Pharmacy at Ho Chi Minh City homepage: and Original article Synthesis, in vitro Acetylcholinesterase Inhibitory Activity Evaluation and Docking Investigation of Some Aromatic Chalcones Tran Thanh Daoa*, Tran Thai Sonab, Nguyen Thi Cam Vic, Le Minh Tria, and Thai Khac Minha* aDepartment of Medicinal Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City 700000, Vietnam; bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, College of Medicine and Pharmacy, Hue University, Hue City 530000, Vietnam; cTon Duc Thang University, Nguyen Huu Tho St., Tan Phong Ward, Dist. 7, Ho Chi Minh City 70000, Vietnam. Received August 10, 2017: Accepted September 25, 2017: Published online December 10, 2017 Abstract: In this study, a total of twenty chalcones were synthesized via Claisen-Schmidt condensation reaction and evaluated for their in vitro acetylcholinesterase inhibitory activities using Ellman’s method. Molecular docking studies on acetylcholinesterase were performed to elucidate the interactions between these chalcone derivatives and acetylcholinesterase active site at the molecular level. From the series, six compounds (S1-5 and S17) exhibited strong acetylcholinesterase inhibitory activities with IC50 values below 100 µM compared to the parent unsubstituted chalcone. Compound S17 (4’-amino-2-chlorochalcone) showed the strongest acetylcholinesterase inhibitory activity in the investigated group with IC50 value of 36.10 µM. Molecular modeling studies were consistent with the results of in vitro acetylcholinesterase inhibitory activities, and chalcone S17 could be considered as a potential lead compound for the development of new acetylcholinesterase inhibitors. Key words: acetylcholinesterase, Alzheimer’s disease, chalcone, docking, SAR. 1. INTRODUCTION Alzheimer’s disease (AD) is a major cause of dementia in the elderly [1, 2]. This is the ailment of the central nervous system characterized by the injury of the grey matter in the cerebral cortex with a complex neurodegenerative process leading to progressive cognitive decline and memory loss [3, 4]. Nowadays, millions of people worldwide have been affected by the disease and the number of patients is which can improve this situation is an imperative task. The etiology of AD is currently not fully known, although factors including the low levels of acetylcholine (ACh), accumulation of abnormal proteins namely -amyloid ( A) and -protein, homeostasis irregularity of biometals, and the pathophysiology of AD [3, 5-7]. At the present time, clinical therapy for AD patients is primarily established upon the cholinergic hypothesis which suggests that the and drugs with ability of inhibiting acetylcholinesterase (AChE) would control symptoms of the disease [4, 8-10]. Acetylcholinesterase inhibitors (AChEIs) including rivastigmine, donepezil and galantamine have been recommended in the treatment of AD for a long period and they have created the most developed group of drugs for the disease up to now [4, 10-13]. For the last few years, a vast number of structures have been tested for acetylcholinesterase inhibitory activities. The variety of AChEI groups have provided a plentiful source of materials for in silico studies (QSAR, docking, pharmacophore). Among these structures, chalcone has been a group with great concern. pyrazolines [16], isoxazoles [17], and quinolinylpyrimidines [18]. There have been a lot of chalcone compounds with a * Address correspondence to this author at the Department of Medicinal Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, 41 Dinh Tien Hoang street, District 1, Ho Chi Minh City 700000, Vietnam; E-mails: tranthanhdao@uphcm.edu.vn and thaikhacminh@uphcm.edu.vn © 2017 MedPharmRes 16 MedPharmRes, 2017, Vol. 1, No. 1 Tran et al. diverse array of reported bioactivities such as antimalarial [19-20], antiprotozoal [21-23], antibacterial [24-27], antifungal [28-30], antiviral [31-33], antioxidant [34-36], antitumoral [37-39], and other characteristics such as anti- Recent studies on the bioactivities of chalcone compounds have also revealed their abilities in inhibiting enzymes including urease, -glucosidase [47, 48], lipoxygenase [49, [56, 57], xanthine oxidase [58], monoamine oxidase (MAO) [59-61], and -secretase [62, 63]. In addition, it was reported A aggregates in vitro, and they could serve as a useful mean for in vivo imaging of A plaques in Alzheimer’s brain [64]. The studies on bioactivities of chalcone derivatives on the for the treatment of many diseases including AD. Computational approaches have become a critical component of many drug discovery projects, and molecular docking, which was devised during the early 1980s, has been a highly active research area in this respect. This is a method for placing small molecules into the binding site of their macromolecular targets. The molecular docking can be utilised to model the interaction between a small molecule and a protein at the atomic level, which allow to characterize the behavior of small molecules in the binding site of target proteins, as well as to elucidate fundamental biochemical processes or to identify potential bioactive compounds by virtual screening processes [65]. In this study, a total of 20 chalcone compounds were synthesized and studied for their in vitro AChE inhibitory activities. The molecular binding abilities of these compounds with the enzyme were elucidated by docking procedure to provide useful information for the design and synthesis of others with better bioactivities. 2. MATERIALS AND METHODS 2.1. Material and Instruments All chemicals were obtained from commercial suppliers, determined on open capillary tubes and are uncorrected (using Gallenkamp apparatus). IR spectra were recorded on a Shimadzu FTIR 8201PC instrument. 1H-NMR spectra were recorded on a Bruker (500 MHz) spectrometer. Chemical relative to tetramethylsilane as an internal standard. Peak splitting patterns were abbreviated as m (multiplet), s (singlet), bs (broad singlet), d (doublet), bd (broad doublet), t (triplet) and dd (doublet of doublets). 2.2. Chemistry Claisen-Schmidt condensation reaction [66] was ap- plied to synthesize chalcones (Fig. 1). The reaction of acetophenone and benzaldehyde derivatives in KOH/ of concentrated HCl provided S1-S20 with satisfactory yields after re-crystallized from appropriate solvents. The by IR and 1H-NMR spectra. General procedures for the preparation of chalcone compounds S1-S20 Acetophenone and benzaldehyde derivatives (with proportion of 1:1) were dissolved in methanol with stirring. Potassium hydroxide was then added. The resulting solution was stirred at room temperature or cooled by ice as needed. The chemical reaction was monitored by thin layer crystallized from appropriate solvents to give the product. 2.3. Acetylcholinesterase inhibitory activity assay AChE inhibitory activities of chalcones were determined by Ellman’s [67] colourimetric method using and acetylthiocholine iodide (ATCI) as a substrate and galantamine as a reference. The assay was performed in 96-well microtiter plates in the same condition for both chalcones and control substance. 25 µL of 100 mM sodium phosphate buffer pH 8, 25 µL of sample and 25 µL acetylcholinesterase solution containing 0.54 U/mL were mixed in each well of the plate and allowed to incubate for 15 min at 25 °C. Subsequently, 25 µL of a solution of ATCI (15 mM, dissolved in water) and 125 µL of 3 mM DTNB (5,5’-dithio-bis-nitro benzoic acid) were added. The reaction. A control reaction which was considered to have 100% activity was carried out using the same volume of methanol/water instead of tested solutions. All samples were assayed in triplicate. Percentages (%) of AChE inhibitions of tested compounds were calculated from the absorbance values as indicated in equation (1): % I = [(A0E – A0) – (Ac – A0C)]/(A0E – A0) (1) Where I is the percent inhibition of acetylcholinesterase; A0E is the absorbance value of the control blank sample with enzyme; A0 is the absorbance value of blank sample; Ac is the absorbance value of the tested sample; A0C is the absorbance value of blank test sample. O R'3R'2 R'1 R1 R2 R3 R4 CH3 O R'3R'2 R'1 R1 R2 R3 R4 O H A B A B 1. KOH, MeOH 2. c-HCl + substituted acetophenone substituted benzaldehyde substituted chalcone Fig. 1. Claisen-Schmidt condensation reaction in chalcones synthesis 17Synthesis, In vitro Acetylcholinesterase Inhibitory Activity MedPharmRes, 2017, Vol. 1, No. 1 Table 1. The content of each sample in in vitro assay Samples ATCI DTNB Buffer Chalcone AChE Control blank sample with enzyme (A0E) + + + - + Blank sample (A0) + + + - - Tested sample (AC) + + + + + Blank test sample (A0C) + + + + - +: present; -: absent The content of each sample is indicated in Table 1. Linear recurrent equations indicating the correlation between common logarithm of the concentrations of investigated compounds (µM) and their percentages of AChE inhibition (%) were built, and from which the IC50 values (concentration that inhibits 50% AChE activity) of studied chalcones were then extrapolated. 2.4. Kinetic characterization of AChE inhibition The most active compound against AChE was selected for further kinetic investigation. Tested compound was added into the assay solution and pre-incubated with the enzyme at 25 °C for 15 minutes, followed by the addition of different concentrations (5, 10, 20, 30, 40 mM) of substrate (ATCI) mixed in the phosphate buffer (pH 8.0), DTNB (3 mM), AChE (0.54 U/mL) and studied compound (30, 40 M). Kinetic characterization of the hydrolysis of ATCI catalyzed by AChE was done spectrometrically at 405 nm. A parallel control with no inhibitor in the mixture, allowed adjusting activities to be measured at various times. 2.5. Molecular Docking Study The Protein Data Bank crystallographic structure of TcAChE(-)-Galantamine complex (pdb 1DX6) [68] was used as receptor model in this study. The 3D structure of the crystallographic complex was rendered by means of the important amino acid residues enclosed within a radius sphere of 6.5 Å centered by the bound ligand, galantamine. All unbound water molecules were eliminated and the structures of amino acid residues were checked before re- establishing the active site of the enzyme. The 2D and 3D chemical structures of 20 chalcones structures of molecules were optimized by the energy minimization and molecular dynamic modules in SYBYL-X 2.0 [71]. In the energy minimization process, Conj Grad method was chosen and the structures of molecules were optimized until converged to a minimum energy change of 0.00001 kcal.mol-1. Gasteiger-Huckel charges were assigned to the structure atoms and the maximum number of iterations to perform during minimization was set to 10,000. Molecular dynamic process was proceeded to obtain conformations with the minimum global energies. The method used in this process was Simulated Annealing. In this method, the molecules were heated at 700 K in a period of 1,000 femtoseconds, they were then cooled down to 200 K in another period of 1,000 femtoseconds to give out different necessary structures. Finally, the energy minimization process was performed one more time and the co-crystallized ligand, including: one separated from the complex (native form, not prepared), one separated from the complex and re-prepared using mentioned appropriate procedure, and one built and prepared from the beginning. The RMSD value between re-docked conformation and the original bound ligand in the co-crystal complex which was prediction of new ligands. Docking process was performed using FlexX program in BioSolveIT LeadIt. This program protein was kept rigid during docking process. FlexX used an incremental construction algorithm for the seach of ligand active site by matching interaction geometries between the ligand and protein. Then the remains were gradually utilised empirical scoring functions to score and rank the docking poses [65, 73]. In this study, the docking process was done with following options: The method in which base fragment placed in binding pocket was Triangle Matching; the maximum number of solutions per iteration was set to 1,000; the maximum number of solutions per fragmentation was set to 200; the number of poses to keep for further analysis of interaction was set to 10. The best conformation was the one with the most minus docking score. This score was the total energy emitted from the formation of binding between the molecules and the active site. The interactions between chalcone molecules and their target were rendered and analyzed in MOE 2008.10 program [74] (hydrogen bonds, - interactions, cation- interactions, ionic interactions). Van der Waals surface interactions were detected by the contact of hydrophilic and lipophilic surfaces of the ligands with those of binding points. 18 3. RESULTS AND DISCUSSION 3.1. Chemistry (E)-2-Chloro-2’-hydroxychalcone (S1) Yield: 68%. M.p: 155±1o -1, KBr): 1641 C=O C=C C=Cl). 1H-NMR (500 MHz, CDCl3 J = 15.5 Hz, H ); 7.95-7.93 (d, 1H, J1 = 8 Hz, J2 = 1.5 Hz, H6’); 7.81-7.79 (dd, 1H, J = 8 Hz, H3’); 7.69-7.66 (d, 1H, J = 15.5 Hz, H ); 7.56-7.53 (t, 1H, H5’); 7.50-7.49 (d, 1H, J1 = 7.5 Hz, J2 = 1 Hz, H6); 7.41-7.35 (m, 2H, H5 and H4’); 7.08-7.06 (dd, 1H, J1 = 8.5 Hz, J2 = 1 Hz, H3); 6.99-6.96 (t, 1H, H4). (E)-4-Chloro-2’-hydroxychalcone (S2) Yield: 74%. M.p: 152±1o -1, KBr): 1639 C=O C=C C=Cl). 1H-NMR (500 MHz, CDCl3 J = 8 Hz, H6’); 7.93-7.88 (d, 1H, J = 15.5 Hz, H ); 7.67-7.64 (d, 1H, J = 15.5 Hz, H ); 7.64-7.62 (d, 2H, J = 9 Hz, H2 and H6); 7.56-7.52 (t, 1H, H4’); 7.45-7.44 (d, 2H, J = 8.5 Hz, H3 and H5); 7.08-7.06 (d, 1H, J = 8.5 Hz, H3’); 7.00-6.97 (t, 1H, H5’). (E)-2,4-Dichloro-2’-hydroxychalcone (S3) Yield: 74%. M.p: 178±1o -1, KBr): 1641 C=O C=C C=Cl). 1H-NMR (500 MHz, CDCl3 J = 15.5 Hz, H ); 7.92-7.90 (dd, 1H, J1 = 8 Hz, J2 = 1.5 Hz, H6’); 7.74-7.72 (d, 1H, J = 7.5 Hz, H6); 7.66-7.63 (d, 1H, J = 15.5 Hz, H ); 7.56-7.53 (t, 1H, J1 = 7 Hz, J2 = 2 Hz, H5’); 7.52 (s, 1H, J = 2 Hz, H3); 7.36-7.34 (dd, 1H, J1 = 9 Hz, J2 = 2 Hz, H5); 7.08-7.06 (d, 1H, J = 7.5 Hz, H3’); 6.99-6.98 (t, 1H, H4’). (E)-2,3-Dichloro-2’-hydroxychalcone (S4) Yield: 67%. M.p: 185±1 o -1, KBr): 1684 C=O C=C C=Cl). 1H-NMR (500 MHz, CDCl3 J = 16 Hz, H ); 7.92-7.91 (d, 1H, J1 = 8.5 Hz, J2 = 1 Hz, H6’); 7.70-7.68 (d, 1H, J1 = 8 Hz, J2 = 1.5 Hz, H4); 7.65-7.62 (d, 1H, J = 15.5 Hz, H ); 7.57-7.55 (dd, 1H, J1 = 8 Hz, J2 = 1.5 Hz, H6); 7.57-7.53 (t, 1H, H5’); 7.33-7.29 (t, 1H, H4’); 7.08-7.06 (dd, 1H, J1 = 8 Hz, J2 = 1 Hz, H3’); 7.00-6.96 (t, 1H, H5). (E)-2’-Hydroxy-2,4-dimethoxychalcone (S5) Yield: 71%. M.p: 113±1oC. IR ( cm-1, KBr): 1638 ( C=O), 1558 ( C=C aromatic ring), 1171 ( C-O). 1H-NMR (500 MHz, MeOD, ppm): 8.20-8.18 (d, 1H, J = 15.5 Hz, H ); 8.06- 8.04 (d, 1H, J = 8 Hz, H6’); 7.81-7.78 (d, 1H, J = 15.5, H ); 7.75-7.73 (d, 1H, J = 9.5 Hz, H3’); 7.51-7.47 (m, 1H, H5’); 6.98-6.95 (m, 1H, H4’); 6.95-6.93 (d, 1H, J = 8 Hz, H6); 6.62-6.60 (ds, 2H, J = 7.5 Hz, H3 and H5); 3.94 (s, 3H, Ar- -OCH3); 3.86 (s, 3H, Ar-OCH3). (E)-2’-Hydroxy-2,3-dimethoxychalcone (S6) Yield: 48%. M.p: 107±1oC. IR ( cm-1, KBr): 3279 ( O-H), 1639 ( C=O), 1607 ( C=C aromatic ring), 1117 ( C-O). 1H-NMR (500 MHz, CDCl3, ppm): 12.83 (s, 1H, -OH phenol); 8.22- 8.18 (d, 1H, J = 15.5 Hz, H ); 7.92-7.91 (d, 1H, J = 1.5 Hz, 8 Hz, H6’); 7.77-7.74 (d, J = 15.5, 1H, H ); 7.51-7.48 (t, 1H, H4’); 7.29-7.27 (d, 1H, J1 = 1 Hz, J2 = 8 Hz, H6); 7.12-7.09 (t, 1H, H5); 7.04-7.02 (d, 1H, J1 = 1 Hz, J2 = 8 Hz, H6); 7.00-6.99 (d, 1H, J1 = 1 Hz, J2 = 8 Hz, H3’); 3.92 (s, 3H, -OCH3); 3.90 (s, 3H, -OCH3). (E)-2’-Hydroxy-3,4,5-trimethoxychalcone (S7) Yield: 67%. M.p: 157±1 oC. IR ( cm-1, KBr): 1636 ( C=O); 1568 ( C=C aromatic ring), 1155 ( C-O). 1H-NMR J = 8 Hz, H6’); 7.90 (s, 2H, 15 Hz, H and H ), 7.57-7.54 (t, 1H, J = 7.5 Hz, H5’), 7.16 (s, 2H, H2 and H6), 7.04-7.00 (t, 1H, J = 7.5 Hz, H4’); 7.02-7.00 (d, 1H, J = 9.5 Hz, H3’); 3.95 (s, 6H, Ar-O- CH3); 3.85 (s, 3H, Ar-OCH3). (E)-2’-Hydroxy-4-dimethylaminochalcone (S8) Yield: 87%. M.p: 180±1 oC. IR ( cm-1, KBr): 1622 ( C=O), 1599 ( C=C aromatic ring), 1155 ( C-O). 1H-NMR (500 MHz, CDCl3, ppm): 13.17 (s, 1H, -OH phenol); 7.93- 7.90 (d, 1H, J = 15.5 Hz, H ); 7.93-7.91 (d, 1H, J1 = 1.5 Hz, J2 = 7.5 Hz, H6); 7.58-7.56 (d, 2H, J = 8 Hz, H2’ and H6’); 7.47-7.44 (d, 1H, J = 15 Hz, H ); 7.47-7.44 (t, 1H, H4); 7.02- 7.00 (d, 1H, J = 8 Hz, H3); 6.94-6.90 (t, 1H, H4’); 6.71-6.69 (d, 2H, J1 = 2 Hz, J2 = 8 Hz, H3 and H5); 3.06 (s, 6H, -CH3). (E)-2’-Hydroxy-2,3,4’-trimethoxychalcone (S9) Yield: 58%. M.p: 133±1 oC. IR ( cm-1, KBr): 1638 ( C=O), 1582 ( C=C aromatic ring), 1269 ( C-O). 1H-NMR (500 MHz, CDCl3, ppm): 13.44 (s, 1H, -OH phenol); 8.17- 8.14 (d, 1H, J = 15,5 Hz, H ); 7.83-7.81 (d, 1H, J1 = 2 Hz, J2 = 9 Hz, H6’); 7.68-7.65 (d, 1H, J = 16 Hz, H ); 7.27-7.26 (d, 1H, J1 = 1.5 Hz, J2 = 8 Hz, H6); 7.11-7.08 (t, 1H, J = 8 Hz, H5); 7.00-6.97 (d, 1H, J1 = 1,5 Hz, J2 = 8 Hz, H5’); 6.49-6.48 (d, 1H, J1 = 2.5 Hz, J2 = 8 Hz, H3); 6.47 (s, 1H, H3’); 3.91 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3); 3.86 (s, 3H, -OCH3). (E)-2’-Hydroxy-3,4,4’-trimethoxychalcone (S10) Yield: 62%. M.p: 170±1 oC. IR ( cm-1, KBr): 1634 ( C=O), 1565 ( C=C aromatic ring), 1126 ( C-O). 1H-NMR (500 MHz, CDCl3, ppm): 13.50 (s, 1H, -OH phenol); 7.86-7.82 (d, 1H, J = 15.5 Hz, H ); 7.84-7.82 (d, 1H, J = 8 Hz, H6’); 7.44-7.41 (d, 1H, J = 15.5 Hz, H ); 7.26-7.24 (d, 1H, J1 = 2 Hz, J2 = 8.5 Hz, H6); 7.16 (d, 1H, J = 2 Hz, H3’); 6.91-6.90 (d, 1H, J = 8 Hz, H5); 6.50-6.47 (d, 1H, J1 = 2.5 Hz, J2 = 8.5 Hz, H5’); 6.47 (s, 1H, J = 2 Hz, H2); 3.96 (s, 3H, -OCH3); 3.93 (s, 3H, -OCH3), 3.86 (s, 3H, -OCH3). (E)-2’-Hydroxy-3,4,4’,5-tetramethoxychalcone (S11) Yield: 63%. M.p: 123±1 oC. IR ( cm-1, KBr): 1643 ( C=O), 1574 ( C=C aromatic ring), 1202 ( C-O). 1H-NMR (500 MHz, CDCl3, ppm): 13.43 (s, 1H, -OH phenol); 7.84- MedPharmRes, 2017, Vol. 1, No. 1 Tran et al. 19Synthesis, In vitro Acetylcholinesterase Inhibitory Activity MedPharmRes, 2017, Vol. 1, No. 1 7.82 (d, 1H, J1 = 2 Hz, J2 = 9 Hz, H6’); 7.82-7.78 (d, 1H, J = 16 Hz, H ); 7.46-7.43 (d, 1H, J = 16 Hz, H ); 6.87 (s, 2H, H2 and H6); 6.50-6.48 (d, 1H, J1 = 2.5Hz, J2 = 9 Hz, H5); 6.48 (s, 1H, H3); 3.93 (s, 6H, -OCH3 x 2); 3.91 (s, 3H, -OCH3), 3.86 (s, 3H, -OCH3). (E)-4-Chloro-2’-hydroxy-4’-methoxychalcone (S12) Yield: 68%. M.p: 143±1 oC. IR ( cm-1, KBr): 1635 ( C=O), 1566 ( C=C aromatic ring), 1204 ( C-O). 1H-NMR (500 MHz, CDCl3, ppm): 13.33 (s, 1H, -OH phenol); 7.84- 7.80 (d, 1H, J = 15.5 Hz, H ); 7.81-7.79 (d, 1H, J = 8.5 Hz, H6); 7.58-7.56 (d, 2H, J1 = 2 Hz, J2 = 8.5 Hz, H2’ and H6’); 7.55-7.52 (d, 1H, J = 15.5 Hz, H ); 7.41-7.39 (d, 2H, J1 = 2.5 Hz, J2 = 8.5 Hz, H3’ and H5’); 6.50-6.47 (d, 1H, J1 = 2,5 Hz, J2 = 8,5 Hz, H5); 6.48 (s, 1H, H3); 3.86 (s, 3H, -OCH3). (E)-2’-Hydroxy-2,4,4’,6’-tetramethoxychalcone (S13) Yield: 55%. M.p: 132±1 oC. IR ( cm-1, KBr): 1549 ( C=O), 1504 ( C=C aromatic ring), 1119 ( C-O). 1H-NMR 13.79 (s, 1H, -OH phenol); 7.91-7.88 (d, 1H, J = 15.5 Hz, H ); 7.80-7.77 (d, 1H, J = 16 Hz, H ); 7.65-7.63 (d, 1H, J = 8.5 Hz, H6); 6.65 (d, 1H, J = 2 Hz, H3); 6.63- 6.61 (dd, 1H, J1 = 2,5 Hz, J2 = 8.5 Hz, H5); 6.14 (d, 1H, J = 2 Hz, H3’); 6.11 (d, 1H, J = 2 Hz, H5’); 3.90 (s, 6H, -OCH3); 3.84 (s, 3H, -OCH3); 3.82 (s, 3H, -OCH3). (E)-2’-Hydroxy-3,4,4’,6’-tetramethoxychalcone (S14) Yield: 66%. M.p: 130±1 oC. IR ( cm-1, KBr): 1622 ( C=O), 1549 ( C=C aromatic ring), 1304 ( C-O). 1H-NMR 7.67-7.63 (d, 1H, J = 15.5 Hz, H ); 7.62-7.59 (d, 1H, J = 15.5 Hz, H ); 7.30-7.28 (dd, 2H, J1 = 7 Hz, J2 = 2 Hz, H2 and H6); 7.03-7.02 (d, 1H, J = 8.5 Hz, H5); 6.16-6.15 (d, 1H, J = 2.5 Hz, H3’); 6.12 (d, 1H, J = 2.5 Hz, H5’); 3.89 (s, 3H, -OCH3); 3.83 (s, 3H, -OCH3); 3.82 (s, 6H, -OCH3). (E)-2’-Hydroxy-2,3,4,4’,6’-pentamethoxychalcone (S15) Yield: 72%. M.p: 134±1 oC. IR ( cm-1, KBr): 1638 ( C=O), 1593 ( C=C aromatic ring), 1113 ( C-O). 1H-NMR 7.82-7.78 (d, 1H, J = 16 Hz, H ); 7.76-7.73 (d, 1H, J = 15.5 Hz, H ); 7.50 (d, 1H, J = 8.5 Hz, H6); 6.92-6.91 (d, 1H, J = 9 Hz, H5); 6.16-6.15 (d, 1H, J = 2.5 Hz, H3’); 6.12 (d, 1H, J = 2.5 Hz, H5’); 3.89 (s, 3H, -OCH3); 3.86 (s, 3H, -OCH3); 3.85 (s, 3H, -OCH3); 3.82 (s, 3H, -OCH3); 3.78 (s, 3H, -OCH3). (E)-4-Chloro-2’-hydroxy-4’,6’-dimethoxychalcone (S16) Yield: 69%. M.p: 138±1 oC. IR ( cm-1, KBr): 1630 ( C=O), 1568 ( C=C aromatic ring), 1290 ( C-O). 1H-NMR 7.77-7.73 (t, 3H, J1 = 8.5 Hz, J2 = 7.5 Hz, J3 = 16 Hz, H , H3, H4); 7.64-7.61 (d, 1H, J = 16 Hz, H ); 7.52-7.50 (d, 2H, J = 8.5 Hz, H2 and H5); 6.17-6.16 (d, 1H, J = 2 Hz, H3’); 6.14-6.13 (d, 1H, J = 2.5 Hz, H5’); 3.90 (s, 3H, -OCH3); 3.83 (s, 3H, -OCH3). (E)-4’-Amino-2-chlorochalcone (S17) Yield: 66%. M.p: 135±1 o -1 NH2); C=O C=C C-Cl). 1H-NMR (500 MHz, CDCl3 J = 8.5 Hz, H3); 7.96-7.94 (d, 2H, J1 = 9 Hz, J2 = 2 Hz, H2’ and H6’); 7.77-7.75 (m, 1H, H5); 7.57-7.54 (d, 1H, J = 15.5 Hz, H ); 7.53-7.46 (m, 1H, H4); 7.46-7.44 (d, 1H, J = 8 Hz, H6); 7.34-7.31 (d, 1H, J = 15.5 Hz, H ); 6.73-6.71 (dd, 2H, J1 = 7 Hz, J2 = 2 Hz, H3’ and H5’); 4.23 (s, 2H, -NH2). (E)-4’-Amino-4-chlorochalcone (S18) Yield: 70%. M.p: 114±1 o -1 NH2); C=O C=C C -Cl). 1H-NMR (500 MHz, CDCl3 J1 = 7 Hz, J2 = 2 Hz, H2’ and H6’); 7.76-7.73 (d, 1H, J = 15.5 Hz, H ); 7.59 (dd, 2H, J1 = 6.5 Hz, J2 = 1.5 Hz, H3’ and H5’); 7.55-7.52 (dd, 1H, J = 15.5 Hz, H ); 7.61-7.52 (d, 2H, J1 = 7 Hz, J2 = 2 Hz, H2 and H6); 7.41-7.39 (dd, 2H, J1 = 7 Hz, J2 = 2 Hz, H3 and H5); 4.17 (s, 2H, -NH2). (E)-4’-Amino-4-nitrochalcone (S19) Yield: 76%. M.p: 174±1o -1, KBr): 3489 NH2 C = O C=C aromatic ring). 1H-NMR J = 9 Hz, H3 and H5); 8.13-8.12 (d, 2H, J = 8.5 Hz, H2 and H6); 8.08-8.05 (d, 1H, J = 15.5 Hz, H ); 7.96-7.95 (d, 2H, J = 8.5 Hz, H2’ and H6’); 7.70-7.67 (d, 1H, J = 15.5 Hz, H ); 6.64-6.62 (d, 2H, J = 9 Hz, H3’ and H5’); 6.24 (s, 2H, -NH2). (E)-3’,4-Dinitrochalcone (S20) Yield: 60%. M.p: 185±1 o -1 C=O); C=C aromatic ring). 1 8.88-8.87 (m, 1H, H4’); 8.64-8.62 (d, 1H, H6’); 8.54-8.52 (m, 1H, H2’); 8.32-8.30 (d, 2H, J = 7.5 Hz, H3 and H5); 8.24 (d, 2H, J = 8, H2 and H6); 8.22-8.21 (d, 1H, J = 15.5, H ), 7.94-7.92 (d,1H, J = 16 Hz, H ), 7.91-7.89 (d, 1H, H5’). 3.2. In vitro Assay Inhibitory activities against AChE of the chalcone de- rivatives S1-S20 were determined by the Ellman’s method, with galantamine as a reference compound. The IC50 val- ues for AChE inhibition are indicated in Table 2. Results of AChE inhibitory assay showed that in comparision with the control substance galantamine, synthesized chalcone com- pounds had weaker activities. And based on their activities, they could be divided into 3 groups: those with strong ac- tivities including S1, S2, S3, S4, S5, S17 whose IC50 values were from 36.10 µM (S17) to 92.42 µM (S1), those with average activities including S9, S10, S16 whose IC50 values were from 129.90 (S10) to 213.14 (S16), and the rest with weak activities. The results also indicated that the derivative S17 was the most potent compound. 20 Table 2. Acetylcholinesterase inhibitory activities of studied chalcone compounds O R'3R'2 R'1 R1 R2 R3 R4 Compounds A-ring B-ring IC50 R’1 R’2 R’3 R1 R2 R3 R4 (µg/ml) (µM) r2* S1 OH H H Cl H H H 23.81 92.42 0.97 S2 OH H H H H Cl H 13.62 52.71 0.93 S3 OH H H Cl H Cl H 14.91 51.01 0.89 S4 OH H H Cl Cl H H 18.32 62.37 0.85 S5 OH H H OMe H OMe H 24.63 86.45 0.84 S6 OH H H OMe OMe H H > 100 ND ND S7 OH H H H OMe OMe OMe > 100 ND ND S8 OH H H H H N(Me)2 H > 100 ND ND S9 OH OMe H OMe OMe H H 60.12 190.98 0.90 S10 OH OMe H H OMe OMe H 40.80 129.90 0.99 S11 OH OMe H H OMe OMe OMe > 100 > 250 ND S12 OH OMe H H H Cl H > 100 > 250 ND S13 OH OMe OMe OMe H OMe H > 100 > 250 ND S14 OH OMe OMe H OMe OMe H > 100 > 250 ND S15 OH OMe OMe OMe OMe OMe H > 100 > 250 ND S16 OH OMe OMe H H Cl H 67.88 213.14 0.95 S17 H NH2 H Cl H H H 9.32 36.10 0.88 S18 H NH2 H H H Cl H > 100 > 250 ND S19 H NH2 H H H NO2 H > 100 > 250 ND S20 H NO2 H H H N(Me)2 H > 100 > 250 ND Chalcone H H H H H H H > 100 > 250 ND Galantamine** 1.36 0.4 0.93 between common logarithm of the concentrations of investigated compounds (µM) and their percentages of AChE inhibition (%); **: used as control substance; ND: Not detected 3.3. Enzyme Kinetics The Lineweaver-Burk plots (Fig. 2) describing the AChE inhibition by S17 showed both increasing slopes and increasing intercepts with higher inhibitor concentrations. This pattern indicated a mixed-type inhibition of enzyme and it was in agreement with the results of studies carried out by Liu et al [51, 52, 55] on the chalcone analogs of S17. These results also revealed that compound S17 and the an- alogs bound to both catalytic active site and the peripheral anionic site of AChE, which was elucidated by molecular modeling studies. 3.4. Molecular Simulation 3.4.1. Binding mode of galantamine in crystallographic structure of TcAChE(-)-Galantamine complex (pdb 1DX6) The rather tight binding of galantamine to TcAChE came from many interactions with the protein. The model of interaction between galantamine and its target (Fig. 3) indicated that this compound could make 3 interaction with the catalytic site, including 2 hydrogen bonds with Ser220 and His440 of the catalytic triad, 1 strong hydrogen bond between the hydroxyl group with Glu199 of the ‘anionic’ subsite with the distance of about 1.5 Å. In addion there was one arene-cation interaction between galantamine and res- idue Phe330 of the ‘anionic’ subsite in the enzyme’s active site. These were important bonds which contributed to the potent activity of galantamine and could be considered in the interactions of studied chalcones. MedPharmRes, 2017, Vol. 1, No. 1 Tran et al. 21Synthesis, In vitro Acetylcholinesterase Inhibitory Activity MedPharmRes, 2017, Vol. 1, No. 1 22 Table 4. Docking scores of studied chalcone compounds Compound Score (kJ.mol-1) pIC50 Compound Score (kJ.mol-1) pIC50 S1 -27.33 4.03 S11 -19.41 3.13 S2 -34.46 4.28 S12 -19.41 3.05 S3 -29.98 4.29 S13 -20.27 3.46 S4 -23.47 4.21 S14 -15.40 3.14 S5 -26.43 4.06 S15 -18.37 3.20 S6 -16.50 2.86 S16 -20.54 3.67 S7 -17.50 2.85 S17 -36.29 4.44 S8 -18.30 3.13 S18 -14.53 3.23 S9 -24.28 3.72 S19 -19.41 3.25 S10 -25.90 3.89 S20 -23.55 3.16 Galantamine -31.47 5.87 MedPharmRes, 2017, Vol. 1, No. 1 Tran et al. Table 3. Results of re-docking processes with co-crystallized ligands Ligand RMSD (Å) 1 0.4912 2 0.5184 3 0.5021 - Ligand 1: separated from the complex (native form, not prepared). - Ligand 2: separated from the complex and re-prepared using mentioned appropriate procedure. - Ligand 3: built and prepared from the beginning. 3.4.2. Re-docking results of co-crystallized ligand Re-docking results of galantamine showed that interac- tions made by re-docked conformations with the active site were resemble those of the original bound ligand in 1DX6 (Fig. 4). The RMSD values of re-docked conformations were < 1.5 Å (Table 3) indicated that the molecular model could be applied to explain the interactions of new ligands with the active site. 3.4.3. Docking results of chalcone compounds The docking process was performed successfully with all 20 chalcone compounds. Docking scores are listed in Table 4. There was a good correlation between docking scores and bioactivities of studied chalcone compounds. This correlation is indicated in Fig. 5. Among the studied compounds, S17 showed the strongest interaction with its target. This compound bound to both catalytic active site and the peripheral anionic site of AChE. This was similar to chalcone analogs in the studies of Liu et al [51, 52, 55], but S17 interacted with the different residues. S17 made 4 interactions with the catalytic site, including 1 hydrogen bond with Ser200 of the catalytic triad, 2 hydrogen bonds with the “oxyanion” pocket (Gly118 and Gly119), and 1 arene-arene interaction with Phe330 of anionic subsite. Moreover, S17 could make 1 strong hydrogen bond with the important residue of the peripheral site Trp121 (Fig. 6). The docking score of S17 is -36.29 kJ.mol-1, which was the most minus value of the chalcone compounds in the docking process. All of these could give the explanation for the highest AChE inhibitory activity of S17 in the studied structures. However, this compound was less potent than galantamine, and the reason for this could arise from the fact that galantamine interacted with catalytic site of AChE more effectively with 2 hydrogen bonds with catalytic triad and 1 strong hydrogen bond with “oxyanion” pocket. In addition, a low entropy cost for binding due to the rigid nature of the galantamine structure which did not occur with S17 molecule also gave a further explanation. Fig. 5. The correlation between docking scores and bioactivities of studied chalcone compounds (A) with and (B) without galantamine included 23Synthesis, In vitro Acetylcholinesterase Inhibitory Activity MedPharmRes, 2017, Vol. 1, No. 1 Fig. 6. (A) 2D and and (B) 3D interactions between S17 and the active site of AChE 4. CONCLUSIONS By applying Claisen-Schmidt condensation method, 20 chalcone derivatives were synthesized. These compounds parameters including IR and 1H-NMR spectra. Among those which were determined for in vitro acetylcholinesterase inhibitory activities, six had IC50 values below 100 µM; three had the IC50 values in the range of 100 – 250 µM and all the rest had IC50 values above 250 µM or not detected. Compound S17 (4’-amino-2-chlorochalcone) showed the highest AChE inhibitory activity with IC50 value of 36.10 µM. It could be considered to use as a lead compound for the studies on AChE inhibitory activity of chalcone compounds. Together with the results of acetylcholinesterase inhibitory assay, the molecular docking studies gave some elucidation for the binding modes of chalcones to the active site of AChE. The information could be valuable for further investigation and would be useful in later research on the design and synthesis new acetylcholinesterase inhibitors. ACKNOWLEDGEMENT This work was supported by the Vietnam’s National Foundation for Science and Technology Development - NAFOSTED (Grand # 106-YS.05-2015.31 to Khac-Minh Thai). REFERENCES Alzheimers Dement. 2015; 11(3):332-84. 2. A. Wimo, M. Guerche, G.C. Ali, Y.T. Wu, A.M. Prina, B. Winblad, et al. The worldwide costs of dementia 2015 and comparisons with 2010. Alzheimers Dement. 2016. 3. J.M. Ringman. Update on Alzheimer’s and the Dementias: Introduction. Neurol Clin. 2017;35(2):171-4. 4. W.V. Graham, A. Bonito-Oliva, T.P. Sakmar. Update on Alzheimer’s Disease Therapy and Prevention Strategies. Annu Rev Med. 2017;68: 413-30. 5. L.L. Blazer, R.R. Neubig. Small molecule protein-protein interaction inhibitors as CNS therapeutic agents: current progress and future hurdles. Neuropsychopharmacology. 2009;34(1):126-41. 6. H.W. Querfurth, F.M. LaFerla. Alzheimer’s disease. N Engl J Med. 2010;362(4):329-44. 7. V. Tumiatti, A. Minarini, M.L. Bolognesi, A. Milelli, M. Rosini, C. Melchiorre. Tacrine derivatives and Alzheimer’s disease. Curr Med Chem. 2010;17(17):1825-38. 8. Q. Xie, H. Wang, Z. Xia, M. Lu, W. Zhang, X. Wang, et al. Bis-(-) -nor- meptazinols as novel nanomolar cholinesterase inhibitors with high inhibitory potency on amyloid-beta aggregation. J Med Chem. 2008; 51(7):2027-36. 9. R.T. Bartus, R.L. Dean, B. Beer, A.S. Lippa. The cholinergic hypothesis of geriatric memory dysfunction. Science. 1982;217(4558):408-14. 10. P.N. Tariot, H.J. Federoff. Current treatment for Alzheimer disease and future prospects. Alzheimer Dis Assoc Disord. 2003;17(4):S105-13. 11. D.A. Drachman, P. Leber. Treatment of Alzheimer’s disease -- searching for a breakthrough, settling for less. N Engl J Med. 1997; 336 (17):1245-7. 12. E. Giacobini. Cholinesterase inhibitors: new roles and therapeutic alternatives. Pharmacol Res. 2004;50(4):433-40. 13. J.G. Evans, G. Wilcock, J. Birks. Evidence-based pharmacotherapy of Alzheimer’s disease. Int J Neuropsychopharmacol. 2004; 7(3):351-69. 14. W. Baker, J. Chadderton, J.B. Harborne, W.D. Ollis. A new synthesis 1953; 1852-60. 15. E. Ono, M. Fukuchi-Mizutani, N. Nakamura, Y. Fukui, K. Yonekura- expression of the aurone biosynthetic pathway. Proc Natl Acad Sci U S A. 2006; 103(29):11075-80. 16. R. Sasikala, K. Thirumurthy, P. Mayavel, G. Thirunarayanan. Eco- friendly synthesis and antimicrobial activities of some 1-phenyl-3 (5-bromothiophen-2-yl)-5-(substituted phenyl)-2-pyrazolines. Org Med Chem Lett. 2012;2:20. 17. R. Kalirajan, S. Sivakumar, S. Jubie, B. Gowramma, B. Suresh. Synthesis and biological evaluation of some heterocyclic derivatives of chalcones. International Journal of ChemTech Research. 2009;1(1):27-34. 18. M.A. Munawar, M. Azad, H.L. Siddiqui, F.-U.-H. Nasim. Synthesis and Antimicrobial Studies of Some Quinolinylpyrimidine Derivatives. Journal of the Chinese Chemical Society. 2008; 55(2):394-400. 19. D. Kumar, M. Kumar, A. Kumar, S.K. Singh. Chalcone and curcumin derivatives: a way ahead for malarial treatment. Mini Rev Med Chem. 2013;13(14):2116-33. 20. P. Singh, A. Anand, V. Kumar. Recent developments in biological activities of chalcones: a mini review. Eur J Med Chem. 2014;85:758-77. 24 21. F. Lunardi, M. Guzela, A.T. Rodrigues, R. Corrêa, I. Eger-Mangrich, M. Steindel, et al. Trypanocidal and Leishmanicidal Properties of Substitution-Containing Chalcones. Antimicrob Agents Chemother. 2003;47(4):1449-51. 22. T.F. de Mello, B.M. Cardoso, S.N. Lopes, H.R. Bitencourt, E.M. Voltarelli, L. Hernandes, et al. Activity of synthetic chalcones in hamsters experimentally infected with Leishmania (Viannia) braziliensis. Parasitol Res. 2015;114(10):3587-600. 23. T.G. Passalacqua, L.A. Dutra, L. de Almeida, A.M. Velasquez, F.A. Torres, P.R. Yamasaki, et al. Synthesis and evaluation of novel prenylated chalcone derivatives as anti-leishmanial and anti-trypanosomal compounds. Bioorg Med Chem Lett. 2015;25(16):3342-5. 24. M.A. Shakhatreh, M.L. Al-Smadi, O.F. Khabour, F.A. Shuaibu, E.I. Hussein, K.H. Alzoubi. Study of the antibacterial and antifungal activities of synthetic benzyl bromides, ketones, and corresponding chalcone derivatives. Drug Des Devel Ther. 2016;10:3653-60. 25. Z.Y. Wei, K.Q. Chi, Z.K. Yu, H.Y. Liu, L.P. Sun, C.J. Zheng, et al. Synthesis and biological evaluation of chalcone derivatives containing aminoguanidine or acylhydrazone moieties. Bioorg Med Chem Lett. 2016;26 (24):5920-5. 26. B. Evranos-Aksoz, F.K. Onurdag, S.O. Ozgacar. Antibacterial, antifungal and antimycobacterial activities of some pyrazoline, hydrazone and chalcone derivatives. Z Naturforsch C. 2015;70(7-8):183-9. 27. T.D. Tran, T.H. Do, N.C. Tran, T.D. Ngo, T.N. Huynh, C.D. Tran, et al. Synthesis and anti Methicillin resistant Staphylococcus aureus activity of substituted chalcones alone and in combination with non-beta- lactam antibiotics. Bioorg Med Chem Lett. 2012;22(14):4555-60. 28. B.T. Yin, C.Y. Yan, X.M. Peng, S.L. Zhang, S. Rasheed, R.X. Geng, et al. Synthesis and biological evaluation of alpha-triazolyl chalcones as a new type of potential antimicrobial agents and their interaction with calf thymus DNA and human serum albumin. Eur J Med Chem. 2014; 71:148-59. 29. Y.H. Wang, H.H. Dong, F. Zhao, J. Wang, F. Yan, Y.Y. Jiang, et al. The synthesis and synergistic antifungal effects of chalcones against drug resistant Candida albicans. Bioorg Med Chem Lett. 2016;26 (13):3098-102. 30. K.L. Lahtchev, D.I. Batovska, S.P. Parushev, V.M. Ubiyvovk, A.A. Sibirny. Antifungal activity of chalcones: a mechanistic study using various yeast strains. Eur J Med Chem. 2008;43(10):2220-8. 31. N. Mateeva, S.V.K. Eyunni, K.K. Redda, U. Ononuju, T.D. Hansberry, chalcones for potential antiviral and anticancer properties. Bioorg Med Chem Lett. 2017;27(11):2350-6. novel 5,6-dimethoxyindan-1-one derivatives as antiviral agents. Med Chem. 2017. 33. J.S. Lee, S.N. Bukhari, N.M. Fauzi. Effects of chalcone derivatives on players of the immune system. Drug Des Devel Ther. 2015; 9:4761-78. 34. R.J. Anto, K. Sukumaran, G. Kuttan, M.N. Rao, V. Subbaraju, R. Kuttan. Anticancer and antioxidant activity of synthetic chalcones and related compounds. Cancer Lett. 1995;97(1):33-7. 35. P.M. Sivakumar, P.K. Prabhakar, M. Doble. Synthesis, antioxidant evaluation, and quantitative structure–activity relationship studies of chalcones. Medicinal Chemistry Research. 2011;20(4):482-92. 36. M. Mojarrab, R. Soltani, A. Aliabadi. Pyridine Based Chalcones: Synthesis and Evaluation of Antioxidant Activity of 1-Phenyl-3- (pyridin-2-yl)prop-2-en-1-one Derivatives. Jundishapur J Nat Pharm Prod. 2013;8(3):125-30. 37. C. Yamali, H.I. Gul, D.O. Ozgun, S.S. Hiroshi, N. Umemura, C. Kazaz, Chalcones. Anticancer Agents Med Chem. 2017. 38. H. Sakagami, Y. Masuda, M. Tomomura, S. Yokose, Y. Uesawa, N. Ikezoe, et al. Quantitative Structure-Cytotoxicity Relationship of Chalcones. Anticancer Res. 2017;37(3):1091-8. 39. C. Echeverria, J.F. Santibanez, O. Donoso-Tauda, C.A. Escobar, R. Ramirez-Tagle. Structural antitumoral activity relationships of synthetic chalcones. Int J Mol Sci. 2009;10(1):221-31. MedPharmRes, 2017, Vol. 1, No. 1 Tran et al. 40. A. Ozdemir, M.D. Altintop, G. Turan-Zitouni, G.A. Ciftci, I. Ertorun, O. Alatas, et al. Synthesis and evaluation of new indole-based chalcones as 41. I. Jantan, S.N.A. Bukhari, O.A. Adekoya, I. Sylte. Studies of synthetic chalcone derivatives as potential inhibitors of secretory phospholipase Drug Des Devel Ther. 2014;8:1405-18. 42. K.R. Abdellatif, H.A. Elshemy, S.A. Salama, H.A. Omar. Synthesis, agents. J Enzyme Inhib Med Chem. 2015;30(3):484-91. 43. M.R. Heidari, A. Foroumadi, H. Noroozi, A. Samzadeh-Kermani, B.S. rigid benzofuran-3, 4-dihydroxy chalcone by formalin, hot-plate and carrageenan tests in mice. Pak J Pharm Sci. 2009;22(4):395-401. 44. N.I. Ismail, L. Ming-Tatt, N. Lajis, M.N. Akhtar, A. Akira, E.K. Perimal, et al. Antinociceptive Effect of 3-(2,3-Dimethoxyphenyl)-1-(5-methylfuran- 2-yl)prop-2-en-1-one in Mice Models of Induced Nociception. Molecules. 2016;21(8). 45. S.M. Dhiyaaldeen, Z.A. Amin, P.H. Darvish, I.F. Mustafa, M.M. Jamil, E. Rouhollahi, et al. Protective effects of (1-(4-hydroxy-phenyl)-3-m-tolyl- propenone chalcone in indomethacin-induced gastric erosive damage in rats. BMC Vet Res. 2014;10:961. 46. K.V. Sashidhara, S.R. Avula, V. Mishra, G.R. Palnati, L.R. Singh, N. antiulcer agents. Eur J Med Chem. 2015;89:638-53. 47. F.L. Ansari, S. Umbreen, L. Hussain, T. Makhmoor, S.A. Nawaz, M.A. Lodhi, et al. Syntheses and biological activities of chalcone and 1,5-benzothiazepine derivatives: promising new free-radical scavengers, and esterase, urease, and alpha-glucosidase inhibitors. Chem Biodivers. 2005;2(4):487-96. 48. W.D. Seo, J.H. Kim, J.E. Kang, H.W. Ryu, M.J. Curtis-Long, H.S. Lee, et al. Sulfonamide chalcone as a new class of alpha-glucosidase inhibitors. Bioorg Med Chem Lett. 2005;15(24):5514-6. 49. I. Jantan, S.N. Bukhari, O.A. Adekoya, I. Sylte. Studies of synthetic chalcone derivatives as potential inhibitors of secretory phospholipase Drug Des Devel Ther. 2014;8:1405-18. 50. A. Hasan, K.M. Khan, M. Sher, G.M. Maharvi, S.A. Nawaz, M.I. Choudhary, et al. Synthesis and inhibitory potential towards acetylcholinesterase, butyrylcholinesterase and lipoxygenase of some variably substituted chalcones. J Enzyme Inhib Med Chem. 2005; 20(1):41-7. 51. H.R. Liu, X.J. Liu, H.Q. Fan, J.J. Tang, X.H. Gao, W.K. Liu. Design, synthesis and pharmacological evaluation of chalcone derivatives as acetylcholinesterase inhibitors. Bioorg Med Chem. 2014;22(21):6124-33. 52. H.R. Liu, X.Q. Huang, D.H. Lou, X.J. Liu, W.K. Liu, Q.A. Wang. Synthesis and acetylcholinesterase inhibitory activity of Mannich base derivatives 53. M.S. Shah, S.U. Khan, S.A. Ejaz, S. Afridi, S.U. Rizvi, M. Najam-Ul- Haq, et al. Cholinesterases inhibition and molecular modeling studies of piperidyl-thienyl and 2-pyrazoline derivatives of chalcones. Biochem Biophys Res Commun. 2017;482(4):615-24. 54. S. Bag, S. Ghosh, R. Tulsan, A. Sood, W. Zhou, C. Schifone, et al. Design, synthesis and biological activity of multifunctional alpha,beta-unsaturated carbonyl scaffolds for Alzheimer’s disease. Bioorg Med Chem Lett. 2013; 23(9):2614-8. 55. H. Liu, H. Fan, X. Gao, X. Huang, X. Liu, L. Liu, et al. Design, synthesis and preliminary structure-activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: a further study based on Flavokawain B Mannich base derivatives. J Enzyme Inhib Med Chem. 2016;31(4):580-9. Larijani. Trans-chalcone: a novel small molecule inhibitor of mammalian alpha-amylase. Mol Biol Rep. 2011;38(3):1617-20. 57. E. Kashani-Amin, B. Larijani, A. Ebrahim-Habibi. Neohesperidin dihydrochalcone: presentation of a small molecule activator of mammalian alpha-amylase as an allosteric effector. FEBS Lett. 2013; 587(6):652-8. 25 58. E. Hofmann, J. Webster, T. Do, R. Kline, L. Snider, Q. Hauser, et al. Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers. Bioorg Med Chem. 2016;24(4):578-87. 59. B. Mathew, G.E. Mathew, G. Ucar, M. Joy, E.K. Nafna, J. Suresh. Monoamine oxidase inhibitory activity of methoxy-substituted chalcones. Int J Biol Macromol. 2017. 60. B. Mathew, G.E. Mathew, G. Ucar, I. Baysal, J. Suresh, S. Mathew, et al. Potent and Selective Monoamine Oxidase-B Inhibitory Activity: Chem Biodivers. 2016;13(8):1046-52. 61. C. Minders, J.P. Petzer, A. Petzer, A.C. Lourens. Monoamine oxidase inhibitory activities of heterocyclic chalcones. Bioorg Med Chem Lett. 2015;25(22):5270-6. 62. J.E. Kang, J.K. Cho, M.J. Curtis-Long, H.W. Ryu, J.H. Kim, H.J. Kim, et al. Inhibitory evaluation of sulfonamide chalcones on β-secretase and acylcholinesterase. Molecules. 2012;18(1):140-53. 63. L. Ma, Z. Yang, C. Li, Z. Zhu, X. Shen, L. Hu. Design, synthesis and SAR study of hydroxychalcone inhibitors of human beta-secretase (BACE1). J Enzyme Inhib Med Chem. 2011;26(5):643-8. 64. M. Ono, M. Haratake, H. Mori, M. Nakayama. Novel chalcones as probes for in vivo imaging of beta-amyloid plaques in Alzheimer’s brains. Bioorg Med Chem. 2007;15(21):6802-9. 65. D.B. Kitchen, H. Decornez, J.R. Furr, J. Bajorath. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat Rev Drug Discov. 2004;3(11):935-49. 66. Z. Wang. Comprehensive Organic Name Reactions and Reagents. John Wiley & Sons, Inc.: New York. 2010;3:660-4. 67. G.L. Ellman, K.D. Courtney, V. Andres, Jr., R.M. Feather-Stone. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961; 7:88-95. 68. RCSB Protein Data Bank - RCSB PDB - 1DX6: Structure of acetylcholinesterase complexed with (-)-galanthamine at 2.3Å resolution - [accessed on 2016 Dec 29]. Available from: rcsb.org/pdb/explore.do?structureId=1dx6. 69. LeadIT. 2.0.2 edition. BioSolveIT GmbH, An der Ziegelei 79, 53757 St. Augustin, Germany - [accessed on 2016 Dec 29]. Available from: www.biosolveit.de. - [accessed on 2016 Dec 29]. Available from: https://www. 71. Sybyl X 2.0. - [accessed on 2016 Dec 29]. Available from: www.tripos. com. 72. K.M. Thai, T.D. Ngo, T.V. Phan, T.D. Tran, N.V. Nguyen; T.H. Nguyen, pump inhibitors from natural products. Med Chem. 2015; 11 (2):135-55. 73. N.S. Pagadala, K. Syed, J. Tuszynski. Software for molecular docking: a review. Biophys Rev. 2017; 9(2):91-102. 74. MOE. 2008.10 edition. Chemical Computing Group Inc., 1010 Sherbrooke St. W, Suite 910, Montreal, Quebec, Canada H3A 2R7 - [accessed on 2016 Dec 29]. Available from: www.chemcomp.com. Synthesis, In vitro Acetylcholinesterase Inhibitory Activity MedPharmRes, 2017, Vol. 1, No. 1