Design, synthesis and evaluation of acetylcholinesterase inhibitory activity of chalcone derivatives for the discovery of new anti - Alzheimer drugs

TP Hồ Chí Minh - Năm 2017 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ----------------------------- Nguyen Thi Cam Vi DESIGN, SYNTHESIS AND EVALUATION OF ACETYLCHOLINESTERASE INHIBITORY ACTIVITY OF CHALCONE DERIVATIVES FOR THE DISCOVERY OF NEW ANTI-ALZHEIMER DRUGS Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF ORGANIC CHEMISTRY DOCTORAL THESIS Ho Chi Minh – 2018 Công trình được hoàn thành tại Viện Công Nghệ Hóa Học Viện Khoa Học và Công Nghệ Việt Nam Người hướng dẫn khoa học 1. PGS. TS. TRẦN THÀNH ĐẠO 2. PGS. TS. THÁI KHẮC MINH Phản biện 1: TS. Nguyễn Thụy Việt Phương Phản biện 2: GS. TS. Phan Thanh Sơn Nam Luận án sẽ được bảo vệ trước Hội đồng đánh giá luận án cấp cơ sở họp tại Viện Công Nghệ Hóa Học, Viện Khoa Học và Công Nghệ Việt Nam. Vào hồi.. giờ ngày . tháng . năm 2017 Có thể tìm hiểu luận án tại: Viện Công Nghệ Hóa Học và Thư Viện quốc gia. The doctoral thesis was finished at: Graduate University Science and Technology - Vietnam Academy of Science and Technology. The 1st supevisor: Ass c. Prof. Dr. Tran Thanh Dao The 2nd supevisor: Assoc. Prof. Dr. Thai Khac Minh The 1st doctoral thesis reviewer: The 2nd doctoral thesis reviewer: The 3rd doctoral thesis reviewer: . The doctoral thesis will be protected at the evaluation coucil of PhD dissertation (Academy degree), meeted at Graduate University Science and Technology - Vietnam Academy of Science and Technology, at am (pm), day month year 201. Read the doctoral thesis: - Graduate University Science and Technology Library - National Library of Vietnam 1 INTRODUCTION 1. The urgency of the thesis Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is affecting millions of people worldwide. The ailment is characterized by a complex neurodegenerative process occurring in the central nervous system which leads to progressive cognitive decline and memory loss. [1] The etiology of AD is not fully known, although factors including the low levels of acetylcholine (ACh), accumulation of abnormal proteins namely -amyloid and -protein, homeostasis irregularity of biometals, and oxidative stress are considered to play significant roles in the pathophysiology of AD.[2] At the present , clinical therapy for AD patients is primarily established upon the cholinergic hypothesis which suggests that the decline of the ACh level might lead to cognitive and memory deficits, and drugs with the ability of inhibiting acetylcholinesterase (AChE) would control symptoms of the disease. [1] Chalcone is a sub-group of flavonoid and is the intermediary in the synthesis process of other flavonoids, pyrazoline, isoxazole, and quinolinylpyrimidine. There are a lot of chalcone compounds which are reported to have a diverse array of bioactivities such as antibacterial, antifungal, antiviral, antioxidant, antitumoral, and other characteristics such as anti-inflammatory, analgesic, antiulce. Recent studies on the bioactivities of chalcone compounds have also revealed their abilities in inhibiting enzymes including urease, - glucosidase, lipoxygenase, acetylcholinesterase, mammalian alpha- amylase, xanthine oxidase 58 , monoamine oxidase (MAO), and - secretase. In addition, it was reported that chalcone derivatives exhibit high binding affinity to A aggregates in vitro, and they 2 could serve as a useful mean for in vivo imaging of A plaques in Alzheimer’s brain.[2-4] The studies on bioactivities of chalcone derivatives on the function of human brain promise the finding of new drugs for the treatment of many diseases including AD. From the above scientific bases, the research project "Design, synthesis and acetylcholinesterase inhibitory activity evaluation of chalcone derivatives for the discovery of new anti-alzheimer drugs" was conducted. 2. The objectives of the thesis Molecular docking studies on acetylcholinesterase were performed to predict the chalcone structure has good in silico AChE acetylcholinesterase inhibitory activity. The potential chalcone compounds were synthesized and studied for their in vitro and in vivo AChE inhibitory activities. 3 . The main contents of the thesis - The molecular binding abilities of chalcone derivatives with ACHE were elucidated by docking procedure to predict the chalcone structure has good in silico AChE acetylcholinesterase inhibitory activity. - The potential chalcone compounds were synthesized and studied for their in vitro and in vivo AChE inhibitory activities. Chapter 1. OVERVIEW 1.1. Alzheimer disease Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is affecting millions of people worldwide. The ailment is characterized by a complex neurodegenerative process occurring 3 in the central nervous system which leads to progressive cognitive decline and memory loss. [1] The etiology of AD is not fully known, although factors including the low levels of acetylcholine, accumulation of abnormal proteins namely -amyloid and -protein, homeostasis irregularity of biometals, and oxidative stress are considered to play significant roles in the pathophysiology of AD.[12] At the present , clinical therapy for AD patients is primarily established upon the cholinergic hypothesis which suggests that the decline of the ACh level might lead to cognitive and memory deficits, and drugs with the ability of inhibiting acetylcholinesterase (AChE) would control symptoms of the disease. [1] 1.2. Acetylcholinesterase (AChE) Acetylcholinesterase (acetycholine acetylhydrolase, E.C. 3.1.1.7) [11] is involved in the hydrolysis of acetylcholine, an essential neurotransmitter of the central nervous system, into choline. This enzyme catalyzes the hydrolysis of the neurotransmitter acetylcholine at neuronal synapses, and at neuromuscular junctions, at the end of the signaling process. In certain neurological disorders such as Alzheimer’s disease, acetylcholinesterase is overactivated in the synapses so that levels of acetylcholine in the brains is significantly diminished, which leads to weakened neurotransmission and thereby memory loss and other adverse effects. 1.3. Chalcone Chalcones (1,3-diphenyl-2-propen-1-one) are open chain flavonoids with a 15-carbon structure arranged in a C6-C3-C6 configuration. They consist in two phenolic rings (A and B rings) connected by a 3C bridge with a double bond between α- and β-positions, which confers them a particularly singular structure. [16] 4 Figure 1.7. Structure and numbering of chalcone 1.4. Molecular Docking Molecular docking is an attractive scaffold to understand drugbiomolecular interactions for the rational drug design and discovery, as well as in the mechanistic study by placing a molecule (ligand) into the preferred binding site of the target specific region of the DNA/protein (receptor) mainly in a non-covalent fashion to form a stable complex of potential efficacy and more specificity. The information obtained from the docking technique can be used to suggest the binding energy, free energy and stability of complexes. At present, docking technique is utilized to predict the tentative binding parameters of ligand-receptor complex beforehand. [21] 1.5. In vitro screening for acetylcholinesterase inhibition AChE inhibitory activity was determined spectrophotometrically using the Ellman's colorimetric method. ACHE hydrolyzes the substrate ATCI to thiocholine and acetic acid. Thiocholine is allowed to react with DTNB, and this reaction resulted in the development of a yellow color. The color intensity of the product is measured at 405 nm, and it is proportional to the enzyme activity. [27] 1.6. Short-term memory impairment models Loss of memory is among the first symptoms reported by patients suffering from Alzheimer's disease (AD) and by their caretakers. 5 Currently, short-term memory impairment models are widely used in the treatment of AD. [27] The Y-maze model and Novel Object Recognition model are quick and useful initial tests to study short-term memory. Chapter 2. CONDITION AND EXPERIMENTAL METHOD 2.1. Time and place of study Time: 1 11 2011 01/05/2017 Place: Labs of Department of Pharmacology, Department of Pharmaceutical, Department of Microbiology, Faculty of Pharmacy, Ho Chi Minh City Medicine and Pharmacy University. 2.2. Experimental content and method 2.2.1. Experimental content The molecular binding abilities of chalcone derivatives with ACHE were elucidated by docking procedure to predict the chalcone structure has strong in silico AChE acetylcholinesterase inhibitory activity. The potential chalcone compounds were synthesized by Claisen-Schmidt condensation reaction. These chalcone compounds are studied for their in vitro and in vivo AChE inhibitory activities. 2.2.2. Experimental method Molecular Docking Study The Protein Data Bank crystallographic structure of TcAChE(-)- Galantamine complex (pdb 1DX6) 67 was used as receptor model in this study. The 3D structure of the crystallographic complex was rendered by means of BioSolveIT LeadIt. The active site was defined as all 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 6 amino acid residues were checked before re-establishing the active site of the enzyme. Docking process of 107 chalcone derivatives (35 normal chalcone derivatives, 24 heterocyclic chalcone derivatives, 32 benzylaminochalcone derivatives and 16 promazine chalcone derivatives) was performed in BioSolveIT LeadIt with the 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 is the one that has the most minus docking score. This score was the total energy emitted from the formation of binding between the molecules and the active site. General Procedures for the Preparation of chalcone derivatives Claisen-Schmidt condensation reaction was applied to synthesize chalcone derivatives (Scheme 2.1). The reaction of acetophenone and benzaldehyde derivatives in KOH/MeOH was followed by an acidification with concentrated HCl provided chalcone derivatives with satisfactory yields after recrystallized from appropriate solvents. The structures and purities of the target compounds were confirmed by UV, MS, IR, 1 H-NMR and 13 C-NMR spectra. Scheme 2.1. Claisen-Schmidt condensation reaction in chalcones synthesis [18] 7 In vitro Acetylcholinesterase inhibitory activity assay AChE inhibitory activities of chalcones were determined using purified acetylcolinesterase from electric eel (Sigma, Type VI) and acetylthiocholine iodide (Sigma) as a substrate with the colourimetric method of Ellman66. Galantamine, ATCI (acetylthiocholin iodide), and DTNB (5,5’-dithio-bis-nitro benzoic acid) were purchased from Sigma. This assay was performed in 96-well microtiter plates in the same condition for both chalcones and control substance (galantamine). In vivo Acetylcholinesterase inhibitory activity assay The best ACHE inhibitory chalcone derivative is tested for their ability to improve memory dysfunction in mice using two short-term memory impairment models: Y - maze model and Novel Object Recognition model based on Tran Phi Hoang Yen model (2007). [28] Chapter 3. RESULTS AND DISCUSSION 3.1. Molecular Docking Study 3.1.1. Re-docking result of co-crystallized ligand Re-docking results of galantamine showed that interactions made by re-docked conformations with the active site were resemble those of the original bound ligand in 1DX6. The RMSD values of re- docked conformations were < 1.5 Å (Table 3.1) indicated that the molecular model could be applied to explain the interactions of new ligands with the active site. 8 Table 3.1. Results of re-docking processes with co-crystallized ligands Ligand RMSD (Å) (1) separated from the complex (native form, not prepared). 0,4912 (2) separated from the complex and re-prepared using mentioned appropriate procedure. 0,5184 (3) built and prepared from the beginning. 0,5021 3.1.2 Docking results of chalcone derivatives 3.1.2.1 Docking results of 35 normal chalcone derivatives The docking process was performed successfully with all chalcone derivatives. The ways of change which are beneficial for the binding ability to acetylcholinesterase of chalcones are summarized and displayed in Fig 3.5. Fig 3.5. The ways of change which are beneficial for the binding ability to acetylcholinesterase of chalcones 9 The molecular docking studies elucidated the binding modes of chalcones to the active site of AChE quite precisely, and from which a structure – activity relationship was then drawn out. Thenceforward, we have the direction to design and synthesize new compounds that have high acetylcholinesterase inhibitory activities. 3.1.2.2. Docking results of 24 heterocyclic chalcone derivatives The docking results showed that chalcones containing thiophen moiety may increase the acetylcholinesterase inhibitory activity compaire with other heterochalcone. Beside, the substitution methoxy group(s) on B-ring (benzen ring) also lead to improve the bioactivity of the heterochalcone. Hình 3.11. The ways of change which are beneficial for the binding ability to acetylcholinesterase of heterocyclic chalcones This study was published in "Evaluation of acetylcholinesterase inhibitory activity of heterochalcones derivaties" in Journal of Medicine, Ho Chi Minh city, 2015. 3.1.2.3 Docking results of 32 benzylaminochalcone derivatives The docking process was performed successfully with all benzylaminochalcone derivatives. The ways of change which are beneficial for the binding ability to acetylcholinesterase of benzylaminochalcone derivatives are summarized and displayed in Fig 3.8. X: thiophen moiety more beneficial than pyridin, furan moiety. -OCH3 groups 10 Fig 3.18. The ways of change which are beneficial for the binding ability to acetylcholinesterase of benzylamino chalcones From the docking results as fig 3.18, we have the direction to design and synthesize new benzylamino chalcones that have high acetylcholinesterase inhibitory activities. 3.1.2.4. Docking results of promazine chalcone derivatives Promazine chalcones are chalcone derivatives that ring A is replaced acepromazine. The docking process was performed with 16 promazine chalcone derivatives by BioSovelIT LeadIT. The ways of change which are beneficial for the binding ability to acetylcholinesterase of promazine chalcone derivatives are summarized and displayed in Fig 3.22. - OH (necessary for a high activity) - OH at position 2 or 3 g tốt N or O heterocyclic -OCH3 or -NO2 group on ring B affect the binding orientation to the target. 11 Fig 3.22. The ways of change which are beneficial for the binding ability to acetylcholinesterase of promazine chalcone derivatives 3.2. Synthesis of chalcone derivatives 3.2.1. Synthesis of normal chalcone derivatives 20 Normal chalcone derivatives based on the orientation of docking results are synthesized by Claisen-Schmidt condensation reaction. Derivatives Name of derivatives Yield (%) ST1 (E)-2-chloro-2’-hydroxychalcone 68 ST2 (E)-4-chloro-2’-hydroxychalcone 74 ST3 (E)-2,4-dichloro-2’-hydroxychalcone 74 ST4 (E)-2,3-dichloro-2’-hydroxychalcone 67 ST5 (E)-2’-hydroxy-2,4-dimethoxychalcone 71 ST6 (E)-2’-hydroxy-2,3-dimethoxychalcone 48 ST7 (E)-2’-hydroxy-3,4,5-trimethoxychalcone 67 ST8 (E)-2’-hydroxy-4-dimethylaminochalcone 87 ST9 (E)-2’-hydroxy-2,3,4’-trimethoxychalcone 58 ST10 (E)-2’-hydroxy-3,4,4’-trimethoxychalcone 62 -Cl group -Br group -F group which have -Cl at position ortho -OCH3 group 12 ST11 (E)-2’-hydroxy-3,4,4’,5-tetramethoxychalcone 63 ST12 (E)-4-chloro-2’-hydroxy-4’-methoxychalcone 68 ST13 (E)-2’-hydroxy-2,4,4’,6’-tetramethoxychalcone 55 ST14 (E)-2’-hydroxy-3,4,4’,6’-tetramethoxychalcone 66 ST15 (E)-2’-hydroxy-2,3,4,4’,6’- pentamethoxychalcone 72 ST16 (E)-4-chloro-2’-hydroxy-4’,6’- dimethoxychalcone 69 ST17 (E)-4’-amino-2-chlorochalcone 66 ST18 (E)-4’-amino-4-chlorochalcone 70 ST19 (E)-4’-amino-4-nitrochalcone 76 ST20 (E)-3’,4-dinitrochalcone 60 Structure of all synthesized chalcone derivatives were confirmed by UV, IR, 1H-NMR spectra and showed in addendum 6. 3.2.2. Synthesis of heterocyclic chalcone derivatives 24 heterocyclic chalcone derivatives are synthesized by Claisen- Schmidt condensation reaction. Derivatives Name of derivatives Yield (%) D1 (E)-1-(pyridin-2-yl)-3-[2-(hydroxy)phenyl]-2- propen-1-one 56 D2 (E)-1-(pyridin-2-yl)-3-[4-(hydroxy)phenyl]-2- propen-1-one 65 D3 (E)-1-(pyridin-2-yl)-3-[3-(hydroxy)phenyl]-2- propen-1-one 62 D4 (E)-1-(pyridin-2-yl)-3-[4- (dimethylamino)phenyl]-2-propen-1-one 58 D5 (E)-1-(pyridin-2-yl)-3-[3,4-(dimethoxy)phenyl]-2- propen-1-one 52 D6 (E)-1-(pyridin-2-yl)-3-[3,4,5-(trimethoxy)phenyl]- 2-propen-1-one 76 13 D7 (E)-1-(pyridin-2-yl)-3-[2,4-(dimethoxy)phenyl]-2- propen-1-one 63 D8 (E)-1-(furan-2-yl)-3-[3,4-(dimethoxy)phenyl]-2- propen-1-one 51 D9 (E)-1-(furan-2-yl)-3-[4-(methoxy)phenyl]-2- propen-1-one 64 D10 (E)-1-(furan-2-yl)-3-[3,4,5-(trimethoxy)phenyl]-2- propen-1-one 68 D11 (E)-1-(furan-2-yl)-3-[4-(hydroxy)phenyl]-2- propen-1-one 50 D12 (E)-1-(furan-2-yl)-3-[3-(hydroxy)phenyl]-2- propen-1-one 54 D13 (E)-1-(furan-2-yl)-3-[2-(hydroxy)phenyl]-2- propen-1-one 52 D14 (E)-1-(furan-2-yl)-3-[3-(nitro)phenyl]-2-propen-1- one 54 D15 (E)-1-(furan-2-yl)-3-[4-(dimethylamino)phenyl]- 2-propen-1-one 62 D16 (E)-1-(thiophen-2-yl)-3-[4-(hydroxy)phenyl]-2- propen-1-one 56 D17 (E)-1-(thiophen-2-yl)-3-[3-(hydroxy)phenyl]-2- propen-1-one 52 D18 (E)-1-(thiophen-2-yl)-3-[2-(hydroxy)phenyl]-2- propen-1-one 54 D19 (E)-1-(thiophen-2-yl)-3-[4-(methoxy)phenyl]-2- propen-1-one 66 D20 (E)-1-(thiophen-2-yl)-3-[2,4-(dimethoxy)phenyl]- 2-propen-1-one 52 D21 (E)-1-(thiophen-2-yl)-3-[3,4,5- (trimethoxy)phenyl]-2-propen-1-one 74 D22 (E)-1-(thiophen-2-yl)-3-[3-(nitro)phenyl]-2- propen-1-one 52 D23 (E)-1-(thiophen-2-yl)-3-[3-(nitro)phenyl]-2- propen-1-one 56 14 D24 (E)-1-(thiophen-2-yl)-3-[4- (dimethylamino)phenyl]-2-propen-1-one 60 3.2.3. Synthesis of benzylaminochalcone derivatives The Claisen-Schmidt condensation reaction of 4'- aminoacetophenone and benzaldehyde derivatives provided 10 benzylaminochalcones. Derivatives Name of derivatives Yield (%) A1 (E)-1-(4-((2-hydroxylbenzyl)amino)phenyl)-3- phenyl)prop-2-ene-1-one 80,88 A2 (E)-3-(2-chlorophenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one 88 A3 (E)-3-(4-chlorophenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one 81,60 A4 (E)-3-(4-nitrophenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one 60 A5 (E)-3-(2,3-dimethoxyphenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one 81,51 A6 (E)-3-(3,4-dimethoxyphenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one 58,85 A7 (E)-3-(2,4-dimethoxyphenyl)-1-(4-((2- hydroxylbe