Summary of organic chemistry doctoral thesis: Design, synthesis and evaluation of acetylcholinesterase inhibitory activity of chalcone derivatives for the discovery of new anti-alzheimer

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

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

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: Assoc 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 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

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]

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

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 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

TcAChE(-)-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

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]

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 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

(2) separated from the complex and re-prepared using

(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

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

-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’-

H-NMR spectra and showed in addendum 6

3.2.2 Synthesis of heterocyclic chalcone derivatives

24 heterocyclic chalcone derivatives are synthesized by Schmidt condensation reaction

Claisen-Derivatives Name of derivatives Yield

D6

D23

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 aminoacetophenone and benzaldehyde derivatives provided 10 benzylaminochalcones

4'-Derivatives Name of derivatives Yield

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-

hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one 80 A8 (E)-1-(4-((2-hydroxylbenzyl)amino)phenyl)-3-

(pyridin-2-yl)prop-2-ene-1-one 82,71 A9 (E)-1-(4-((2-hydroxylbenzyl)amino)phenyl)-3-

(pyridin-4-yl)prop-2-ene-1-one 69,23 A10 (E)-3-(furan-2-yl)-1-(4-((2-

hydroxylbenzyl)amino)phenyl)prop-2-ene-1-one 76,30