Tổng hợp luận án Tiến sĩ Hóa hữu cơ: Thiết kế, tổng hợp và đánh giá hoạt tính ức chế acetylcholinesterase của các dẫn xuất chalcone để khám phá thuốc chống Alzheimer mới

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-----------------------------

TP Hồ Chí Minh - Năm 2017

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

The doctoral thesis was finished at: Graduate University Science and

Công trình được hoàn thành tại Viện Công Nghệ Hóa Học

Technology - Vietnam Academy of Science and Technology.

Viện Khoa Học và Công Nghệ Việt Nam

The 1st supevisor: Assoc. Prof. Dr. Tran Thanh Dao

Người hướng dẫn khoa học

1. PGS. TS. TRẦN THÀNH ĐẠO

2. PGS. TS. THÁI KHẮC MINH

The 2nd supevisor: Assoc. Prof. Dr. Thai Khac Minh

The 1st doctoral thesis reviewer: …

Phản biện 1: TS. Nguyễn Thụy Việt Phương

The 2nd doctoral thesis reviewer: …

The 3rd doctoral thesis reviewer: ….

Phản biện 2: GS. TS. Phan Thanh Sơn Nam

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….

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

Read the doctoral thesis:

Có thể tìm hiểu luận án tại: Viện Công Nghệ Hóa Học và Thư Viện quốc gia.

- Graduate University Science and Technology Library

- National Library of Vietnam

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 oxidase58, monoamine oxidase (MAO), and - secretase. In addition, it was reported that chalcone derivatives exhibit high binding afﬁnity to A aggregates in vitro, and they

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

the chalcone structure has good

Molecular docking studies on acetylcholinesterase were performed to in silico AChE predict 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 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

including low the

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 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]

in involved

catalyzes the hydrolysis of

1.2. Acetylcholinesterase (AChE) Acetylcholinesterase (acetycholine acetylhydrolase, E.C. 3.1.1.7) [11] is the hydrolysis of acetylcholine, an essential neurotransmitter of the central nervous system, into choline. This enzyme 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]

Figure 1.7. Structure and numbering of chalcone

1.4. Molecular Docking

is an attractive scaffold

Molecular docking 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.

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

amino acid residues were checked before re-establishing the active site of the enzyme.

24 chalcone derivatives, heterocyclic

Docking process of 107 chalcone derivatives (35 normal 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, 1H-NMR and 13C-NMR spectra.

Scheme 2.1. Claisen-Schmidt condensation reaction in chalcones synthesis[18]

for both chalcones and control

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 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.

Table 3.1. Results of re-docking processes with co-crystallized ligands

RMSD (Å)

0,4912

0,5184

0,5021

Ligand (1) separated from the complex (native form, not prepared). (2) separated from the complex and re-prepared using mentioned appropriate procedure. (3) built and prepared from the beginning. 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

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.

X: thiophen moiety -OCH3 more beneficial than groups pyridin, furan

moiety.

Hình 3.11. The ways of change which are beneficial for the binding ability to acetylcholinesterase of heterocyclic chalcones

study was published in "Evaluation inhibitory activity of heterochalcones of This acetylcholinesterase derivaties" in Journal of Medicine, Ho Chi Minh city, 2015.

3.1.2.3 Docking results of 32 benzylaminochalcone derivatives

the binding ability

The docking process was performed successfully with all benzylaminochalcone derivatives. The ways of change which are beneficial for to acetylcholinesterase of benzylaminochalcone derivatives are summarized and displayed in Fig 3.8.

- OH at position 2 or 3

N or O heterocyclic

-OCH3 or -NO2 group on

g tốt

ring B affect the binding - OH (necessary for a high activity) orientation to the target.

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.

-Cl group

-OCH3 group

-Br group

-F group which have -Cl at position ortho

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

ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 ST10 (E)-2-chloro-2’-hydroxychalcone (E)-4-chloro-2’-hydroxychalcone (E)-2,4-dichloro-2’-hydroxychalcone (E)-2,3-dichloro-2’-hydroxychalcone (E)-2’-hydroxy-2,4-dimethoxychalcone (E)-2’-hydroxy-2,3-dimethoxychalcone (E)-2’-hydroxy-3,4,5-trimethoxychalcone (E)-2’-hydroxy-4-dimethylaminochalcone (E)-2’-hydroxy-2,3,4’-trimethoxychalcone (E)-2’-hydroxy-3,4,4’-trimethoxychalcone Yield (%) 68 74 74 67 71 48 67 87 58 62

ST11 ST12 ST13 ST14 63 68 55 66

ST15 72

ST16 69

ST17 ST18 ST19 ST20 (E)-2’-hydroxy-3,4,4’,5-tetramethoxychalcone (E)-4-chloro-2’-hydroxy-4’-methoxychalcone (E)-2’-hydroxy-2,4,4’,6’-tetramethoxychalcone (E)-2’-hydroxy-3,4,4’,6’-tetramethoxychalcone (E)-2’-hydroxy-2,3,4,4’,6’- pentamethoxychalcone (E)-4-chloro-2’-hydroxy-4’,6’- dimethoxychalcone (E)-4’-amino-2-chlorochalcone (E)-4’-amino-4-chlorochalcone (E)-4’-amino-4-nitrochalcone (E)-3’,4-dinitrochalcone 66 70 76 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 56

D2 65

D3 62

D4 58

D5 52

D6 76 (E)-1-(pyridin-2-yl)-3-[2-(hydroxy)phenyl]-2- propen-1-one (E)-1-(pyridin-2-yl)-3-[4-(hydroxy)phenyl]-2- propen-1-one (E)-1-(pyridin-2-yl)-3-[3-(hydroxy)phenyl]-2- propen-1-one (E)-1-(pyridin-2-yl)-3-[4- (dimethylamino)phenyl]-2-propen-1-one (E)-1-(pyridin-2-yl)-3-[3,4-(dimethoxy)phenyl]-2- propen-1-one (E)-1-(pyridin-2-yl)-3-[3,4,5-(trimethoxy)phenyl]- 2-propen-1-one

D7 63

D8 51

D9 64

D10 68

D11 50

D12 54

D13 52

D14 54

D15 62

D16 56

D17 52

D18 54

D19 66

D20 52

D21 74

D22 52

D23 56 (E)-1-(pyridin-2-yl)-3-[2,4-(dimethoxy)phenyl]-2- propen-1-one (E)-1-(furan-2-yl)-3-[3,4-(dimethoxy)phenyl]-2- propen-1-one (E)-1-(furan-2-yl)-3-[4-(methoxy)phenyl]-2- propen-1-one (E)-1-(furan-2-yl)-3-[3,4,5-(trimethoxy)phenyl]-2- propen-1-one (E)-1-(furan-2-yl)-3-[4-(hydroxy)phenyl]-2- propen-1-one (E)-1-(furan-2-yl)-3-[3-(hydroxy)phenyl]-2- propen-1-one (E)-1-(furan-2-yl)-3-[2-(hydroxy)phenyl]-2- propen-1-one (E)-1-(furan-2-yl)-3-[3-(nitro)phenyl]-2-propen-1- one (E)-1-(furan-2-yl)-3-[4-(dimethylamino)phenyl]- 2-propen-1-one (E)-1-(thiophen-2-yl)-3-[4-(hydroxy)phenyl]-2- propen-1-one (E)-1-(thiophen-2-yl)-3-[3-(hydroxy)phenyl]-2- propen-1-one (E)-1-(thiophen-2-yl)-3-[2-(hydroxy)phenyl]-2- propen-1-one (E)-1-(thiophen-2-yl)-3-[4-(methoxy)phenyl]-2- propen-1-one (E)-1-(thiophen-2-yl)-3-[2,4-(dimethoxy)phenyl]- 2-propen-1-one (E)-1-(thiophen-2-yl)-3-[3,4,5- (trimethoxy)phenyl]-2-propen-1-one (E)-1-(thiophen-2-yl)-3-[3-(nitro)phenyl]-2- propen-1-one (E)-1-(thiophen-2-yl)-3-[3-(nitro)phenyl]-2- propen-1-one

D24 60 (E)-1-(thiophen-2-yl)-3-[4- (dimethylamino)phenyl]-2-propen-1-one

The Claisen-Schmidt condensation reaction of

3.2.3. Synthesis of benzylaminochalcone derivatives 4'- aminoacetophenone and benzaldehyde derivatives provided 10 benzylaminochalcones.

Derivatives Name of derivatives Yield (%)

A1 80,88

A2 88

A3 81,60

A4 60

A5 81,51

A6 58,85

A7 80

A8 82,71

A9 69,23

A10 76,30 (E)-1-(4-((2-hydroxylbenzyl)amino)phenyl)-3- phenyl)prop-2-ene-1-one (E)-3-(2-chlorophenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one (E)-3-(4-chlorophenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one (E)-3-(4-nitrophenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one (E)-3-(2,3-dimethoxyphenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one (E)-3-(3,4-dimethoxyphenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one (E)-3-(2,4-dimethoxyphenyl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one (E)-1-(4-((2-hydroxylbenzyl)amino)phenyl)-3- (pyridin-2-yl)prop-2-ene-1-one (E)-1-(4-((2-hydroxylbenzyl)amino)phenyl)-3- (pyridin-4-yl)prop-2-ene-1-one (E)-3-(furan-2-yl)-1-(4-((2- hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one

10 Benzylaminochalcone were

10 promazine chalcones (AC1-AC10) based on

recognised as new compounds based on Scifinder (2016). This study was published in "Synthesis of novel chalcones as acetylcholinesterase inhibitors" in Applied Sciences, 2016.[44] 3.2.4. Synthesis of promazine chalcone derivatives the orientation of the docking results are synthesized by Claisen-Schmidt condensation reaction.

Derivatives Name of derivatives Yield (%)

AC1 81%

AC2 78%

AC3 62%

AC4 57%

AC5 51%

AC6 43%

AC7 58% (E)-3-(2-chlorophenyl)-1-(10-(3- (dimethylamino)propyl)-10H-phenothiazin-2-yl)- 3-phenylprop-2-en-1-one (E)-3-(4-chlorophenyl)-1-(10-(3- (dimethylamino)propyl)-10H-phenothiazin-2-yl)- 3-phenylprop-2-en-1-one (Z)-3-(2,4-dichlorophenyl)-1-(10-(3- (dimethylamino)propyl)-10H-phenothiazin-2-yl)- 3-phenylprop-2-en-1-one (E)-1-(10-(3-(dimethylamino)propyl)-10H- phenothiazin-2-yl)-3-(4-fluorophenyl)prop-2-en-1- one (E)-3-(3-bromophenyl)-1-(10-(3- (dimethylamino)propyl)-10H-phenothiazin-2-yl)- 3-phenylprop-2-en-1-one (E)-3-(2-chloro-6-fluorophenyl)-1-(10-(3- (dimethylamino)propyl)-10H-phenothiazin-2-yl)- 3-phenylprop-2-en-1-one (E)-1-(10-(3-(dimethylamino)propyl)-10H- phenothiazin-2-yl)-3-(2-

AC8 43%

AC9 41%

AC10 51% trifluoromethyl)phenyl)prop-2-en-1-one (E)-1-(10-(3-(dimethylamino)propyl)-10H- phenothiazin-2-yl)-3-(3-methoxyphenyl)prop-2- en-1-one (E)-1-(10-(3-(dimethylamino)propyl)-10H- phenothiazin-2-yl)-3-(4-methoxyphenyl)prop-2- en-1-one (E)-1-(10-(3-(dimethylamino)propyl)-10H- phenothiazin-2-yl)-3-(3,4,5- trimethoxyphenyl)prop-2-en-1-one 9 Promazine chalcones (AC1-AC8 and AC10) were recognised as new compounds based on Scifinder (2017).

3.3. In vitro Acetylcholinesterase inhibitory activity assay

3.3.1. In vitro Acetylcholinesterase inhibitory activity assay of normal chalcone derivatives

The IC50 values of 20 normal chalcone derivatives for AChE inhibition are indicated in Table 3.13.

Bảng 3.13. The IC50 values (%) of 20 normal chalcone derivatives for AChE inhibition

Derivatives Derivatives IC50 (µM) IC50 (µM)

92,42 52,71 51,01 62,37 86,45 Docking score (kJ/mol) -27,33 -34,46 -29,98 -23,47 -26,43 Docking score (kJ/mol) -19,41 -19,41 -20,27 -15,40 -18,37 ST11 ST12 ST13 ST14 ST15 > 500 > 500 349,09 > 500 > 500 ST1 ST2 ST3 ST4 ST5

ST6 ST7 ST8 ST9 ST10 > 500 > 500 > 500 190,98 129,90 -16,50 -17,50 -18,30 -24,28 -25,90 ST16 ST17 ST18 ST19 ST20 213,14 36,10 > 500 > 500 > 500 -20,54 -36,29 -14,53 -19,41 -21,25

There was a good correlation between docking scores and bioactivities of studied chalcone compounds. Among the studied compounds, S17 showed the strongest interaction with its target.

Hình 3.35. 2D interactions between S17 and the active site of AChE (pdb id: 1dX6)

4.3.2 In vitro Acetylcholinesterase inhibitory activity assay of heterocyclic chalcone derivatives

The results showed that heterocyclic chalcones containing thiophen moiety may increase the acetylcholinesterase inhibitory activity compaire with other heterochalcone. Among the studied heterocyclic chalcones, D21 containing thiophen moiety and 3 - OCH3 groups has the best IC50 (114,8 µM). This study was published in "Evaluation of acetylcholinesterase inhibitory activity of heterochalcones derivaties" in Journal of Medicine, Ho Chi Minh

city, 2015.[43]

4.2.3 Khảo sát khả năng kháng acetylcholinesterase của các dẫn chất benzylaminochalcone

The IC50 values of 10 benzylaminochalcone derivatives for AChE inhibition are indicated in Table 3.18.

Table 3.18. The IC50 values of 10 benzylaminochalcone derivatives for AChE inhibition

Derivatives A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 Galantamine IC50 (µM) 160.33 121.91 23.71 31.57 121.61 23.02 147.84 116.34 38.97 89.19 1.27 pIC50 -2.21 -2.09 -1.37 -1.50 -2.08 -1.36 -2.17 -2.07 -1.59 -1.95 0.10 Docking score (kJ/mol) −18.23 −20.34 −20.55 −21.89 −20.56 −19.32 −18.34 −20.07 −21.42 −21.22 −23.1

exhibit inhibitory significant activities

them, of (A6) has

Compounds with ring B bearing pyridin-4-yl, 4-nitrophenyl, 4-chlorophenyl and 3,4-dimethoxyphenyl moieties were discovered to against acetylcholinesterase, with IC50 values ranging from 23 to 39 µM. (E)-3-(3,4-dimethoxyphenyl)-1-(4-((2- Among hydroxylbenzyl)amino) phenyl)prop-2-ene-1-one a strongest bioactivity as acetyl-cholinesterase inhibitors (IC50 23,02 µM). This result was published in "Synthesis of novel chalcones as acetylcholinesterase inhibitors" in Applied Sciences, 2016.[44]

3.3.4 In vitro Acetylcholinesterase inhibitory activity assay of promazine chalcone derivatives

The IC50 values of 10 promazine chalcone derivatives for AChE inhibition are indicated in Table 3.19.

Table 3.19. Giá trị IC50 của 10 promazine chalcone đối với AChE

IC50 (µM) pIC50 Docking score (kJ/mol)

Derivatives AC1 AC2 AC3 AC4 AC5 AC6 AC7 AC8 AC9 AC10 Galantamine 35.96 160.35 50.21 90.09 24.39 40.37 347.34 93.10 33.50 120.64 1,27 -1.56 -2.21 -1.70 -1.95 -1.39 -1.61 -2.54 -1.97 -1.53 -2.08 0,10 -22,072 -18,455 -21,271 -18,926 -24,261 -21,891 -17,721 -20,026 -22,567 not -23,1

Compounds with ring B bearing 3-bromophenyl (AC5), 2- chlorophenyl (AC1) and 4-methoxyphenyl (AC9 were discovered to exhibit significant inhibitory activities against acetylcholinesterase, with IC50 values ranging from 24,39 to 35,96 µM. Among of them, (E)-3-(3-bromophenyl)-1-(10-(3-(dimethylamino)propyl)-10H- phenothiazin-2-yl)-3-phenylprop-2-en-1-on (AC5 has a strongest bioactivity as acetyl-cholinesterase inhibitors (IC50 24,39 µM). 3.4 In vivo Acetylcholinesterase inhibitory activity assay of benzylaminochalcone A6 The best ACHE inhibitory chalcone derivative (A6) 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. The mice received three dose of A6 derivative: 20 mg/kg, 15 mg/kg and 10 mg/kg three days before the intraperitoneal (i.p) injection of 2.4 mg/kg trimethyltin (TMT); and three days later, the mice were tested on the models. Results on both models showed that A6 derivative dose 15 mg/kg could improve memory impairment in mice similar to galanthamine dose 10,0 mg/kg.

This result was published in "Experimental antioxidant and memory-improving property of benzylaminochalcon in mice" in Pharmaceutical journal, 2017.[47]

CONCLUSION

The new results of the thesis "Design, synthesis and acetylcholinesterase inhibitory activity evaluation of chalcone derivatives for the discovery of new anti-alzheimer drugs" are indicated as followings: 1. The molecular binding abilities of 107 chalcone derivatives (35 normal chalcone derivatives, 24 heterocyclic chalcone derivatives, 32 benzylaminochalcone derivatives and 16 promazine chalcone derivatives) with ACHE were elucidated by docking procedure to predict the chalcone structure has strong in silico AChE acetylcholinesterase inhibitory activity. 2. By applying Claisen-Schmidt condensation method, 64 (20 normal chalcone derivatives, 24 chalcone derivatives derivatives, 10 benzylaminochalcone heterocyclic chalcone derivatives and 10 promazine chalcone derivatives) based on the orientation of the docking results were synthesized sucessfully with them, 10 yield to 88 %. Among of from 40 ranging

studied chalcone

benzylaminochalcone derivatives and 9 promazine chalcone derivatives were recognised as new compounds. 3. Invitro AChE inhibitory activities of synthesized chalcones were determined. There was a good correlation between docking scores and bioactivities of compounds. Benzylaminochalcones with ring B bearing 3,4-dimethoxyphenyl (A6), 4 - chlorophenyl (A3) and promazine chalcone with ring B bearing 3-bromophenyl (AC5) were discovered to exhibit significant inhibitory activities against acetylcholinesterase, with IC50 values ranging from 23,02 to 24,39 µM. 4. The best AChE inhibitory chalcone derivative (A6) 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. A6 derivative dose 15 mg/kg could improve memory impairment in mice similar to galanthamine dose 10,0 mg/kg.

The discovered results may be regarded as efficient

candidates for further developments of new anti-alzheimer drugs. NEW FINDINGS 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. This result supported to synthesize chalcone derivatives more effectively and economically.

activities. Among of inhibitory

- 64 chalcone derivativess were synthesized and studied for their in vitro AChE them, 10 benzylaminochalcone derivatives and 9 promazine chalcone derivatives were recognised as new compounds.

- Many new chalcone derivatives were discovered to exhibit significant inhibitory activities against acetylcholinesterase, with IC50 < 50 µM. - Experimental memory-improving property of benzylaminochalcon A6 discovered that A6 derivative dose 15 mg/kg could improve memory impairment in mice similar to galanthamine dose 10,0 mg/kg.

LIST OF WORKS HAS BEEN PUBLISHED

1. Thanh-Dao Tran, Thi-Cam-Vi Nguyen, Ngoc-Son Nguyen, Dai- Minh Nguyen, Thi-Thu-Ha Nguyen, Minh-Tri Le, and Khac-Minh Thai, Synthesis of novel chalcones as acetylcholinesterase inhibitors, Applied Sciences, 2016, 6(7), 198.

2. Thanh-Dao Tran, Thai-Son Tran, Thi-Cam-Vi Nguyen, Minh-Tri Le and Khac-Minh Thai, Synthesis, In vitro Acetylcholinesterase Inhibitory Activity Evaluation and Docking Investigation of Some Aromatic Chalcones, MedPharmRes, 2017, Volume 1, Issue 1.

3. Nguyen Thi Cam Vi, Trinh Quynh Dieu,Tran Phi Hoang Yen,Thai Khac Minh, Tran Thanh Dao, Experimental antioxidant and memory-improving property of benzylaminochalcon in mice, Pharmaceutical journal, 2017, 494, 17-21.

4. Minh Dai Nguyen, Vi Thi Cam Nguyen, Dat Van Truong, Ha Tuong Do and Dao Thanh Tran, Synthesis and cytotoxic activities of some heterocyclic chalcones, The 19th International Electronic Conference on Synthetic Organic Chemistry, 2015.

acetylcholinesterase inhibitory activity of

5. Nguyen Thi Cam Vi, Tran Thi Kieu Diem, Tran Thanh Dao, Evaluation of heterochalcones derivaties, Journal of Medicine, 2015, 19(3), 744- 750.

6. Nguyen Thi Cam Vi, Mai Hoang Yen, Tran Thanh Dao, Evaluation of acetylcholinesterase inhibitory activity of isoflavone derivatives by molecular docking model, Journal of Medicine, 2015, 19(3), 751-760.

7. Tran Hong Thoai Nga, Nguyen Thi Cam Vi, Tran Cat Dong, Tran Thanh Dao, Investigation of antimicrobial activities of some combinations of heterocyclic chalcone and antibiotic against samonella and shigella, Journal of Medicine, 2011, 15, 431-437.

8. Tran Thi Kim Thoa, Do Tuong Ha, Nguyen Thi Cam Vi, Tran Cat Dong, Tran Thanh Dao, Effects on methicillin-resistant Staphylococcus aureus of flavonoids separately and in combination with ciprofloxacin, Pharmaceutical journal, 2011, 417, 24-30.