Rational Design of sym-Triazines For Multitarget-Directed ... · Rational Design of sym-Triazines For Multitarget-Directed Modulation of ... For Multitarget-Directed Modulation of

Rational Design of sym-Triazines For Multitarget-Directed Modulation of Cholinesterases and Amyloid-Beta in

Alzheimer’s Disease

By

Devjani Dhar

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

© Copyright by Devjani Dhar (2013)

ii

Rational Design of sym-Triazines For Multitarget-Directed

Modulation of Cholinesterases and Amyloid-Beta in Alzheimer’s

Disease

Devjani Dhar

Master of Science

Department of Chemistry

University of Toronto

2013

Abstract

Alzheimer’s disease (AD), a progressive age-related neurodegenerative disorder is characterized

by impairments in memory and cognitive functions. The two main pathogenic hallmarks

associated with the progression of this multifactorial disease include accumulation of amyloid-

beta (Aβ) plaques and the deterioration of the cholinergic system in the brain. Using cost-

effective synthetic procedures, mono-, di-, and tri- substituted sym-triazine derivatives

incorporating acetylcholine substrate analogues and aromatic phenyl moieties were synthesized

for the targeted modulation of Aβ aggregation and acetylcholinesterase (AChE) activity. A

subset of these sym-triazines demonstrated dual inhibition of Aβ fibrillization and AChE

hydrolytic activity in vitro studies. These highly effective compounds were also shown to be well

tolerated by differentiated human neuronal cells in cell viability tests. These novel compounds

have the potential to undergo future in vivo pharmaceutical analysis and have a positive impact

on the quality of life of the people living with this devastating disease and their caretakers.

iii

Acknowledgments

I would like to thank my supervisor, Professor Kagan Kerman for being very supportive and

considerate throughout my graduate studies. I appreciate all his help and guidance for my

research project. His knowledge and enthusiasm in organic and analytical chemistry has been a

great inspiration to me. He has taught me a great deal and I can’t thank him enough for his

mentorship.

It was a great pleasure working with my lab mates in the Kerman group-Anthony, Vinci, Nan,

and Xavier. My graduate experience would not have been the same without the feedback and

advice of all these amazing and intelligent people. I am very grateful for getting to know them

and have found great friends in them.

I would like to thank Dr. Lana Mikhaylichenko for her kind support during my graduate studies.

Her advice, feedback and motivation have been very helpful at all times.

Furthermore, I would like to thank the Department of Chemistry and especially the Graduate

administration for their patience and help throughout my graduate studies.

I would also like to thank Professor Heinz-Bernhard Kraatz for his time and commitment in

serving as a second reader and reviewing my thesis. I really appreciate his help.

Finally, I am very grateful to my parents and my sisters, Riya and Anu, for their support and

motivation. I would like to thank Nick for his constant encouragement and support throughout

my time as a graduate student.

iv

Table of Contents

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Figures ............................................................................................................................... vii

List of Tables .................................................................................................................................. x

List of Appendices ......................................................................................................................... xi

List of Abbreviation ..................................................................................................................... xiii

Copyright Acknowledgements...................................................................................................... xv

Chapter 1 ......................................................................................................................................... 1

1 Introduction ............................................................................................................................. 1

1.1 Amyloid-Beta (Aβ) .......................................................................................................... 2

1.1.1 β- and γ- Secretase Enzymes..................................................................................... 2

1.1.2 Aβ1-40 and Aβ1-42 Peptides ......................................................................................... 3

1.1.3 Aβ Fibrillogenesis Pathway ...................................................................................... 4

1.2 The Cholinergic System ................................................................................................... 4

1.2.1 Acetylcholine (ACh) Production .............................................................................. 5

1.2.2 Acetycholinesterase (AChE) Structure and its Mechanism of Action ...................... 7

1.2.3 Cholinesterase Inhibitors (ChEIs) ............................................................................. 9

1.3 AChE induced Aβ aggregation ...................................................................................... 10

1.4 Other Pathologies in AD ................................................................................................ 11

1.4.1 Oxidative Stress ...................................................................................................... 11

1.4.2 Calcium (Ca2+

) Dyshomeostasis ............................................................................. 11

1.4.3 Formation of Neurofibrillary Tangles (NFTs) ........................................................ 12

1.4.4 Chronic Activation of Microglia ............................................................................. 12

1.5 Conclusion ...................................................................................................................... 13

Chapter 2 ....................................................................................................................................... 14

2 Introduction ........................................................................................................................... 14

2.1 Dual Binding ChEIs ....................................................................................................... 15

2.2 Dual Inhibition of AChE and Other Neurotransmitter Systems .................................... 16

v

2.3 Multi-Target Inhibition of AChE, AChE-Induced Aβ Aggregation and Oxidative Stress

18

2.4 Multi-Target Inhibition of AChE, BuChE and Ca2+

Channels ...................................... 18

2.5 Multi-Target Inhibition of AChE, AChE-induced Aβ aggregation, Oxidative stress and

N-Methyl-D-Aspartate (NMDA) Receptor ............................................................................... 20

2.6 Conclusion ...................................................................................................................... 21

Chapter 3 ....................................................................................................................................... 23

3 Introduction ........................................................................................................................... 23

3.1 Reaction Scheme ............................................................................................................ 26

3.2 Experimental Procedure ................................................................................................. 26

3.2.1 Procedure for sym-Triazine Based Methoxy Compounds ...................................... 27

3.2.2 Procedure for sym-Triazine Based Acids ................................................................ 27

3.2.3 Procedure for sym-Triazine Based Acyl Chlorides ................................................. 28

3.2.4 Procedure for sym-Triazine Based Esters ............................................................... 30

3.2.5 Procedure for sym-Triazine Based Iodine-Ester Salts ............................................ 33

3.3 Discussion ...................................................................................................................... 35

3.4 Inhibition of AChE, BuChE, and Aβ Aggregation by sym-Triazine Derivatives and their

Cell Viability Studies ................................................................................................................ 40

3.4.1 Cholinesterase inhibition studies ............................................................................ 42

3.4.2 Inhibition of Aβ1-40 Aggregation ............................................................................. 42

3.4.3 Cell Viability Studies .............................................................................................. 44

3.5 Conclusion ...................................................................................................................... 44

Chapter 4 ....................................................................................................................................... 46

4 Future Directions and Conclusion ......................................................................................... 46

References ..................................................................................................................................... 50

Appendices .................................................................................................................................... 60

Appendix A: 1H-Nuclear Magnetic Resonance (NMR) Spectra of sym-Triazine Derivatives .....60

A.1. 1H-NMR spectrum of 3.18 .................................................................................................60





vi



Appendix B: 13

C-Nuclear Magnetic Resonance (NMR) Spectra of sym-Triazine Derivatives .....67

B.1. 13

C-NMR spectrum of 3.12 ................................................................................................67

B.2. 13

C-NMR spectrum of 3.21 ................................................................................................68

B.3. 13

C-NMR spectrum of 3.13 ................................................................................................69

B.4. 13

C-NMR spectrum of 3.19 ................................................................................................70

B.5. 13

C-NMR spectrum of 3.11 ................................................................................................71

B.6. 13

C-NMR spectrum of 3.14 ................................................................................................72

B.7. 13

C-NMR spectrum of 3.17 ................................................................................................73

B.8. 13

C-NMR spectrum of 3.23 ................................................................................................74

Appendix C: Mass Spectra (MS) of sym-Triazine Derivatives .....................................................75

C.1. MS of 3.18 ..........................................................................................................................75

C.2. MS of 3.21 ..........................................................................................................................76

C.3. MS of 3.19 ..........................................................................................................................77

C.4. MS of 3.14 ..........................................................................................................................78

C.5. MS of 3.17 ..........................................................................................................................79

C.6. MS of 3.20 ..........................................................................................................................80

C.7. MS of 3.23 ..........................................................................................................................81

Appendix D: Infrared (IR) spectra of sym-Triazine Derivatives ...................................................82

D.1. IR spectrum of 3.18 ............................................................................................................82

D.2. IR spectrum of 3.21 ............................................................................................................83

D.3. IR spectrum of 3.11 ............................................................................................................84

D.4. IR spectrum of 3.14 ............................................................................................................85

D.5. IR spectrum of 3.17 ............................................................................................................86

D.6. IR spectrum of 3.20 ............................................................................................................87

D.7. IR spectrum of 3.23 ............................................................................................................88

vii

List of Figures

Fig. 1.1: Amino acid sequences of Aβ1-40 and Aβ1-42. Aβ1-42 incorporates two hydrophobic

amino acid residues, Ile and Ala in the C-terminal that are absent in Aβ1-40 and make it

more fibrillogenic than Aβ1-40 (adapted from Jarrett et al, 1993 (43)). .............................. 3

Fig. 1.2: Fibrillogenesis pathway of Aβ in AD. The Aβ monomers can form amorphous

aggregates and small oligomers, which are in equilibrium with each other. The large

oligomers formed from the small ones can form the metastable protofibril that eventually

form the fibril structure responsible for the development of senile plaques. The

oligomeric species have been shown to be toxic to neuronal cells (adapted from Stains et

al, 2007 (54)). ..................................................................................................................... 4

Fig. 1.3: Chemical structure of acetylcholine (ACh) (adapted from Milkani et al, 2011 (64)).

............................................................................................................................................. 5

Fig. 1.4: Schematic representation of the biosynthesis and action of ACh. ACh can be

produced from acetyl CoA and choline by the action of choline acetyltransferase. It is

released to the synaptic cleft upon cell depolarization upon which it can either bind to

presynaptic receptors for its further release or bind to postsynaptic receptors for synaptic

transmission. Acetylcholinesterase (AChE) breaks down ACh into choline and acetate,

which are then taken up by the presynaptic cell (adapted from Lleo et al, 2006 (7)). ....... 7

Fig. 1.5: Structural features of the AchE enzyme. X-ray crystallography has identified the

active site found in the deep narrow gorge of AChE and the peripheral anionic site (PAS)

found near the lip of the active site (adapted from Soreq et al, 2001 (63)). ....................... 8

Fig. 1.6: FDA approved AChE inhibitors and the first generation of AD drugs - Tacrine

(Cognex), Donepezil (Aricept), Rivastigmine (Exelon), and Galanthamine (Reminyl) -

used for the symptomatic treatment of AD (adapted from Palmer et al, 2002 (87)). ....... 10

Fig. 2.1: Possible molecular factors involved in the pathology of AD and the potential

inhibitors to target these etiologies. The multifactorial nature of the disease make it

challenging to treat and requires a multi-target-directed approach for therapy (adapted

from Cavalli et al, 2008 (29)). .......................................................................................... 15

Fig. 2.2: Design strategy for development of compounds with AChE inhibition and AChE-

induced Aβ aggregation inhibition activities. Compounds 2.2 was derived through the

octamethylene linker from the diamine diamide compound 2.1 (adapted from Cavalli et

al, 2008 (29)). ................................................................................................................... 16

Fig. 2.3: Design strategy for the development of compounds with AChE inhibition and

monoamine oxidase (MAO) inhibition activities. Compounds 2.5 incorporates the

proparyglamine moiety residing the MAO inhibition property and a carbamate moiety

shown to have AChE inhibition property in the FDA approved AChE inhibitor

rivastagmine (2.3) (adapted from Cavalli et al, 2008 (29)). ............................................. 17

Fig. 2.4: Design strategy for the formation of 2.8 derived from the organosulphur compound

2.6 residing the antioxidant properties and the tetrahydroacridine compound 2.7

viii

possessing the inhibition properties of AChE and AChE-induced Aβ aggregation

(adapted from Cavalli et al, 2008 (29)). ........................................................................... 18

Fig. 2.5: Design strategy for the development of compounds with AChE, and

butyrylcholinesterase (BuChE) inhibition properties and voltage-gated calcium channel

(VDCC) blockade activity. Compound 2.10 was derived from 2.7 by altering the

cyclohexane ring size and replacing the benzene ring with a heterocyclic moiety.

Compound 2.15 includes the dihydropyridine ring from compound 2.9 that resides the

Ca2+

channel inhibition property (adapted from Cavalli et al, 2008 (29)). ....................... 19

Fig. 2.6: Design strategy for the development of compounds with AChE, AChE-induced Aβ

aggregation, oxidative stress and N-methyl-D-aspartate (NMDA) inhibition activities.

Compounds 2.13, 2.14, 2.15, and 2.16 possessing these inhibition properties incorporate

the carbazole moiety residing the Aβ inhibiton and antioxidant properites from 2.11. It

also contains the tetrahydroacridine moiety from 2.7 which has shown to possess AChE

inhibition and low affinity NMDA antagonist properties (adapted from Rosini et al, 2008

(111))................................................................................................................................. 21

Fig. 3.1: Chemical structures of sym-triazine (3.1) and cyanuric chloride (CC) (3.2) (adapted

from Mur, 1964 (145)). ..................................................................................................... 23

Fig. 3.2: Chemical structure of the sym-triazine molecule 3.3 that demonstrated beta-sheet

breaking activity in Aβ1-42 in the fluorescent-based screen and was chosen for further in

vitro studies (Adapted from Kim et al, 2006 (156)). ........................................................ 25

Fig. 3.3: Synthetic routes for new ester and ester-salt sym-triazine derivatives. sym-Triazine

based methoxy compounds, acids, acyl chlorides were formed as intermediates iReactions

involving formation of sym-triazine acids 3.6, 3.7, and 3.8. iiReactions involving

formation of sym-triazine acyl chloride 3.9, 3.10, and 3.11. iii

Reactions involving

formation of sym-triazine esters 3.12, 3.15, 3.13, 3.16, 3.14, and 3.17. iv

Reactions

involving formation of sym-triazine ester salts 3.18, 3.21, 3.19, 3.22, 3.20, and 3.23 (6).

........................................................................................................................................... 26

Fig. 3.4: Synthesis of sym-triazine based methoxy compounds 3.4 and 3.5 from CC (3.2).

Reactions were carried out using methanol (CH3OH) as the reagent and sodium

bicarbonate (NaHCO3) as the base. .................................................................................. 27

Fig. 3.5: Synthesis of sym-triazine based acids 3.6, 3.7, and 3.8. iReactions were run using

oxybenzoic acid, sodium hydroxide (NaOH) and water in acetone. Acidification was

performed using concentrated hydrochloric acid (HCl) to yield 3.6, 3.7, and 3.8. .......... 27

Fig. 3.6: Synthesis of sym-triazine based acyl chlorides 3.9, 3.10, and 3.11. iiReactions were

run at 60ºC in thionyl chloride (SOCl2) using anhydrous chloroform (CHCl3) as solvent

and pyridine as catalyst. .................................................................................................... 28

Fig. 3.7: Synthesis of sym-triazine based esters 3.12, 3.15, 3.13, 3.16, 3.14, and 3.17. iii

Reactions were carried out using a: 3-dimethylamino-1-propanol, b: 1-dimethylamino-

2-propanol and c: 2-dimethylaminoethanol in anhydrous tetrahydrofuran (THF) in the

presence of triethylamine ((C2H5)3N) as a base. ............................................................... 30

ix

Fig. 3.8: Synthesis of sym-triazine based ester salt compounds 3.18, 3.21, 3.19, 3.22, 3.20,

and 3.23. iv

Reactions were carried out at 40ºC using iodomethane (CH3I) in anhydrous

THF. .................................................................................................................................. 33

Fig. 3.9: Reaction mechanism for the formation of sym-triazine based methoxy compound

(adapted from Bruice, 2001 (165)). .................................................................................. 36

Fig. 3.10: Reaction mechanism for the formation of mono-substituted sym-triazine based

acid (adapted from Bruice, 2001 (165)). ........................................................................... 37


acyl chloride (adapted from Fox et al, 2008 (166)). ......................................................... 38


ester (adapted from Bruice, 2001 (165)). .......................................................................... 39


ester-salt (adapted from Bruice, 2001 (165)). ................................................................... 40

Fig. 3.14: sym-Triazine esters, ester-salt compounds and a negative control (3.24) tested for

their activity as AChE, BuChE, and Aβ aggregation inhibitors (6).................................. 41

Fig. 4.1: Structure of 4.2 to be developed in order to possess AChE, BuChE, Aβ aggregation

and tau protein aggregation inhibitor properties. .............................................................. 46

Fig. 4.2: Synthesis of 4.1 using diphenylamine. Reaction was carried out at 0ºC and sodium

carbonate (Na2CO3) was employed as a base and acetone as solvent. ............................. 47

Fig. 4.3: Structure of 4.4 to be developed in order to possess AChE, BuChE, MAO and Aβ

aggregation inhibitor properties. ....................................................................................... 47

Fig. 4.4: Synthesis of 4.3 using propargylamine. Reaction was carried out at 0ºC and

triethylamine ((C2H5)3N) was employed as a base and acetone as solvent. ..................... 48

Fig. 4.5: Structure of 4.5 to be developed in order to possess AChE, BuChE, Aβ

aggregation, and VDCC inhibition properties. ................................................................. 48

x

List of Tables

Table 1: Analysis of AChE, BuChE, and Aβ inhibition in vitro by sym-triazine derivatives. aMeasured with 100 µM Aβ1-40 incubated with 200 µM (2x) or 100 µM (1x) inhibitor

([i]) at 37 oC for 72 h in 50 mM PBS (pH 7.4) ................................................................ 43

xi

List of Appendices

Appendix A: 1H-Nuclear Magnetic Resonance (NMR) Spectra of sym-Triazine Derivatives .....60







A.7. 1H-NMR spectrum of 3.23 .....................................................................................................66

Appendix B: 13

C-Nuclear Magnetic Resonance (NMR) Spectra of sym-Triazine Derivatives .....67

B.1. 13

C-NMR spectrum of 3.12 ................................................................................................67

B.2. 13

C-NMR spectrum of 3.21 ................................................................................................68

B.3. 13

C-NMR spectrum of 3.13 ................................................................................................69

B.4. 13

C-NMR spectrum of 3.19 ................................................................................................70

B.5. 13

C-NMR spectrum of 3.11 ................................................................................................71

B.6. 13

C-NMR spectrum of 3.14 ................................................................................................72

B.7. 13

C-NMR spectrum of 3.17 ................................................................................................73

B.8. 13

C-NMR spectrum of 3.23 ................................................................................................74

Appendix C: Mass Spectra (MS) of sym-Triazine Derivatives .....................................................75

C.1. MS of 3.18 ..........................................................................................................................75

C.2. MS of 3.21 ..........................................................................................................................76

C.3. MS of 3.19 ..........................................................................................................................77

C.4. MS of 3.14 ..........................................................................................................................78

C.5. MS of 3.17 ..........................................................................................................................79

C.6. MS of 3.20 ..........................................................................................................................80

C.7. MS of 3.23 ..........................................................................................................................81

Appendix D: Infrared (IR) spectra of sym-Triazine Derivatives ...................................................82

D.1. IR spectrum of 3.18 ............................................................................................................82

D.2. IR spectrum of 3.21 ............................................................................................................83

xii

D.3. IR spectrum of 3.11 ............................................................................................................84

D.4. IR spectrum of 3.14 ............................................................................................................85

D.5. IR spectrum of 3.17 ............................................................................................................86

D.6. IR spectrum of 3.20 ............................................................................................................87

D.7. IR spectrum of 3.23 ............................................................................................................88

xiii

List of Abbreviations

Aβ Amyloid-beta

APP Aβ precursor proteins

ACh Acetylcholine

AChE Acetylcholinesterase

AD Alzheimer’s disease

BuChE Butyrylcholinesterase

Ca2+

Calcium

CC Cyanuric chloride

ChEIs Cholinesterase inhibitors

CNS Central nervous system

E. coli Escherichia coli

ER Endoplasmic reticulum

GFP Green fluorescent protein

IR Infrared

LA Lipoic acid

MAO Monoamine oxidase

MS Mass spectra

MTDL Multi-target-directed ligand

MRI Magnetic resonance imaging

NFTs Neurofibrillary tangles

NMDA N-methyl-D aspartate

NMR Nuclear magnetic resonance

PAS Peripheral anionic site

xiv

ROS Reactive oxygen species

SAR Structure-activity relationship

SOCCs Store-operated calcium channels

THF Tetrahydrofuran

TLC Thin layer chromatography

VDCCs Voltage-dependent calcium channels

xv

Copyright Acknowledgements

Parts of Chapters 1, 2 and 3:

Reprinted (adapted) with permission from (Veloso, A. J., Dhar, D., Chow, A. M., Zhang, B.,

Tang, D. W. F., Ganesh, H. V. S., Mikhaylichenko, S., Brown, I. R., and Kerman, K. (2012)

sym-Triazines for Directed Multitarget Modulation of Cholinesterases and Amyloid-β in

Alzheimer’s Disease, ACS Chem. Neurosci. (Available online 30 October 2012, DOI:

10.1021/cn300171c)). Copyright (2012) American Chemical Society.

Fig. 2.1 in Chapter 2:

Reprinted (adapted) with permission from (Cavalli, A., Bolognesi, M. L., Minarini, A., Rosini,

M., Tumiatti, V., Recanatini, M., and Melchiorre, C. (2008) Multi-target-directed ligands to

combat neurodegenerative diseases, J. Med. Chem. 51, 2326-2326.). Copyright (2008) American

Chemical Society.

Fig. 2.6 in Chapter 2:

Reprinted (adapted) with permission from (Rosini, M., Simoni, E., Bartolini, M., Cavalli, A.,

Ceccarini, L., Pascu, N., McClymont, D. W., Tarozzi, A., Bolognesi, M. L., Minarini, A.,

Tumiatti, V., Andrisano, V., Mellor, I. R., and Melchiorre, C. (2008) Inhibition of

acetylcholinesterase, beta-amyloid aggregation, and NMDA receptors in Alzheimer's disease: A

promising direction for the multi-target-directed ligands gold rush, J. Med. Chem. 51, 4381-

4384.). Copyright (2008) American Chemical Society.

1

Chapter 1 Alzheimer’s Disease and Its Pathologies

1 Introduction

Alzheimer’s disease (AD), a complex neurodegenerative disorder prevalent in the aging

population (1), is characterized by progressive deterioration in memory (2, 3) and cognitive

functions (4-6). 25 million cases of AD were reported worldwide in the year 2000 and it is

expected to increase to 114 million by 2050 if new therapies do not emerge in the near future (7).

Cross-sectional (8-10) and longitudinal (11-16) neuroimaging data in AD research have

indicated that AD patients experience a decrease in total brain volume and expansion in

ventricular system compared to individuals without AD. A typical magnetic resonance imaging

(MRI) of the brain of a control (normal volunteer) and a patient suffering from AD can

demonstrate the atrophy in certain regions of the brain, mainly the hippocampus and cerebral

cortex in AD patients along with an increase in size of the AD brain (8, 9, 15-17).

Pathological deterioration of AD patients has been strongly associated with the overproduction

and disregulation of the amyloid-beta (Aβ) peptide (6, 18). Aβ has been implicated in a number

of neurotoxic pathways related to the formation of reactive oxygen species (ROS) (19), the

disruption of calcium (Ca2+

) homeostasis (20), formation of neurofibrillary tangles (NFTs) (21)

and chronic activation of microglia (22). One prominent strategy to reduce neurodegeneration

has thus emphasized the removal of neurotoxic Aβ oligomers by implementing small-molecule

aggregation modulators that disrupt π-π stacking between β-sheets in order to impede Aβ self-

association (23, 24). Although the development and commercialization of Aβ-modulating agents

are underway (25, 26), currently available pharmacological treatments have targeted more distal

pathways of neurodegeneration, which are limited to two classes of compounds: N-methyl-D

aspartate (NMDA) receptor antagonists and cholinesterase inhibitors (ChEIs) (27). The aim of

such drug therapies is not to impede the proposed aetiopathologies, but rather to ameliorate

behavioral and cognitive dysfunctions, which have significantly delayed - and in some cases

avoided – the need for institutionalization (6, 28).1 In order to develop drugs for the efficient

1 Reprinted (adapted) with permission from (Veloso, A. J., Dhar, D., Chow, A. M., Zhang, B.,


2

therapy of AD, it is vital to understand the multiple pathologies that contribute to the etiology of

this complex disease (29).

1.1 Amyloid-Beta (Aβ)

Aβ, the peptide responsible for the formation of the senile plaques in the brain of AD patients, is

one of main pathological factors of AD (29). The peptide was first sequenced twenty years ago

from the meningeal blood vessels of patients suffering from AD as well as individuals with

Down syndrome (30, 31). The following year, Aβ peptide was recognized as the main

component of senile plaques of brain tissues in AD patients (32). The amyloid hypothesis, which

states that the Aβ accumulation in the brain is the primary driving force of AD pathology, has

become a significant focus in AD research in the past 10 years since the discovery of

amyloidogenic Aβ derived from mutations in Aβ precursor proteins (APP) (33). Although this

hypothesis has its strengths and weakness, Aβ pathology has been a strong focus for AD therapy

(34). Aβ aggregation has been considered as the triggering event for further neurotoxic effects

such as, mitochondrial dysfunction (35), induction of apoptotic genes (36), activation of protein-

kinases that may form toxic neurofibrillary tangles (NFTs) (37), stimulation of microglia cells,

oxidative stress, and ultimately neuronal damage and cell death (29, 38).

1.1.1 β- and γ- Secretase Enzymes

Aβ peptide is generated by endoproteolytic cleavages of the APP, a cell surface glycoprotein

consisting of 770 amino acids and involved in neural signaling. The APP gene is abundantly

expressed in several tissues to form APP, a fraction of which undergoes endoproteolysis by three

proteases called α-, β-, and γ- secretases. This generates the Aβ peptide that can deposit and

aggregate (or vice versa) in extracellular insoluble plaques (39).

The β-secretase pathway involves the formation of the amyloidogenic form of Aβ peptide and is

initiated with the first cleavage of APP, which is carried out by the β-site APP cleaving enzyme




3

or BACE (40, 41). BACE generates the N-terminal of the APP (βAPPs) and the C-terminal

fragment (C99) by cleaving APP at the N-terminal of the Aβ sequence (39, 42). The C99 protein

intermediate is then subsequently cleaved by the γ-secretase to produce the amyloidogenic

peptide, which is a heterogenous event that leads to the generation of Aβ with different C termini

(39, 42). The most common forms of the Aβ peptide are the Aβ1-40 and Aβ1-42, which are

generated when γ-secretase cleaves the C99 fragment at Val at position 40 and Ala at position

42, respectively (39). Alternatively, the α-secretase causes the release of the non-amyloidogenic

form of Aβ, a soluble N-terminal portion of APP (αAPPs) and a C-terminal fragment (C83), by

cleaving the APP inside the Aβ sequence (39).

1.1.2 Aβ1-40 and Aβ1-42 Peptides

Aβ1-42 is the most dominant form of Aβ peptide in senile plaques and more neurotoxic than Aβ1-

40 (39). Aβ1-42 differs in its amino acid sequence from Aβ1-40 by the addition of two amino acids-

Ile in position 41 and Ala in position 42-in the C-terminus (43) (amino acid sequence shown in

Fig. 1.1). Recent studies have indicated that these two predominant Aβ isoforms demonstrate

different oligomeric pathways (44, 45) during fibrillization of the peptide with kinetic studies

indicating that Aβ1-42 is more fibrillogenic than Aβ1-40 (46, 47). Soluble Aβ can be converted to

high β-sheet content following a conformational change; thus, making it prone to aggregation

into soluble oligomers and larger insoluble fibrils in senile plaques. This can direct the

fibrillogenetic Aβ1-42 isoform to stimulate the misfolding of other Aβ species (1). Aβ1-42

deposition can increase the accumulation of Aβ1-40 and trigger the amyloid cascade processes

(43).

Fig. 1.1: Amino acid sequences of Aβ1-40 and Aβ1-42. Aβ1-42 incorporates two hydrophobic

amino acid residues, Ile and Ala in the C-terminal that are absent in Aβ1-40 and make it more

fibrillogenic than Aβ1-40 (adapted from Jarrett et al, 1993 (43)).

4

1.1.3 Aβ Fibrillogenesis Pathway

Both in vitro (48, 49) and in vivo (50, 51) studies have strongly indicated that soluble oligomeric

species of Aβ incorporating a β-sheet secondary structure are the main factors causing toxicity in

neuronal cells (51, 52). Haas has argued that these soluble oligomers provide a larger surface

area for interaction with neuronal cells compared to Aβ aggregates; thus, allowing it to rapidly

diffuse into the synaptic cleft and cause dysfunction in neuronal transmission (53, 54).

Bitan et al, 2003 demonstrated that oligomerization of Aβ monomers are initiated with the

formation of pentamer/hexamer units called paranuclei. These minimal units can oligomerize to

form larger oligomers, protofibrils and fibrils (39, 45, 54). These monomeric, paranuclei and

large oligomeric species are mainly unstructured and consists of short β-sheet/β-turn and helical

elements (Fig. 1.2) (39).

Fig. 1.2: Fibrillogenesis pathway of Aβ in AD. The Aβ monomers can form amorphous

aggregates and small oligomers, which are in equilibrium with each other. The large oligomers

formed from the small ones can form the metastable protofibril that eventually form the fibril

structure responsible for the development of senile plaques. The oligomeric species have been

shown to be toxic to neuronal cells (adapted from Stains et al, 2007 (54)).

1.2 The Cholinergic System

The cholinergic system has received considerable attention in AD disease research due to the

early and selective dysfunction in cholinergic transmission associated with this

neurodegenerative disease (55). The cholinergic hypothesis that stems from pathological,

biochemical, and pharmacological observations, suggests that the cognitive and behaviour

5

deficits experienced in AD patients arise from the impairment in cholinergic neurotransmission

(56-58). The pathological changes such as, depletion of cholinergic neurons due to decrease in

neurotransmission usually have implications in biochemical functions. These biochemical

disruptions in cholinergic functions are associated with severe dementia (7). Pharmacological

studies done in animal models such as, rats and rodents further supported the hypothesis

indicating the close relation between cognition and cholinergic neurotransmission (7, 59, 60).

Cholinergic antagonists such as, antimuscarinic agents, have shown to impair performances in

memory during behavioural experiments including spatial learning tests, operant tasks, and

avoidance procedures (60).

Because cholinergic function plays a major role in neuronal plasticity in the adult brain,

impairment in the cholinergic function in AD patients not only affects neuronal transmission but

also deteriorates the brain’s ability to compensate for progressive neurodegeneration (55).

Cognitive dysfunction associated with deterioration of cholinergic neurons of the basal forebrain

and neocortex leads to profound deficits in the production of the neurotransmitter, acetylcholine

(ACh) (Fig. 1.3) (61). The gradual loss of ACh can become lethal as in AD and not only cause

impairment in cognitive function but also in autonomic and neuromuscular functions (62, 63).

Fig. 1.3: Chemical structure of acetylcholine (ACh) (adapted from Milkani et al, 2011 (64)).

1.2.1 Acetylcholine (ACh) Production

As a result of all the research studies and findings, the primary approach for AD therapy to date

involves the cholinergic replacement strategy that includes the use of acetylcholinesterase

inhibitors (ChEIs) (65). ChEIs act upon the catabolic enzyme, acetylcholinesterase (AChE) in

order to increase the availability of acetylcholine in the synaptic cleft (7).

Being the most biologically potent neurotransmitter in the brain, ACh is generally strictly

regulated in the synaptic cleft (55). It is synthesized in the cholinergic neurons from acetyl CoA

and choline with the aid of the enzyme, choline acetyltransferase (7, 55). Once produced, it is

6

released from the presynaptic cell into the synaptic cleft following depolarization of the cell,

which allows for its binding to muscarinic and nicotinic cholinergic receptors in the pre- and

post-synaptic cells. In the case of presynaptic cells, this interaction leads to the further release of

Ach in the synaptic cleft. However, when Ach acts upon the postsynaptic receptors, it leads to

the activation of the cell’s biochemical pathways regulating neurotransmission. For the

controlled regulation of ACh in the synaptic cleft, AChE can inactivate the neurotransmitter in

the synaptic cleft by hydrolyzing it into choline and acetate. Choline is taken up into the

presynaptic cell for further production of ACh (Fig. 1.4) (7). Due to decreased levels of ACh in

patients suffering from AD, inhibiting AChE to prolong the time ACh remains in the synaptic

cleft for postsynaptic neurotransmission, can be a useful therapy in AD (7, 55).

7

Fig. 1.4: Schematic representation of the biosynthesis and action of ACh. ACh can be produced

from acetyl CoA and choline by the action of choline acetyltransferase. It is released to the

synaptic cleft upon cell depolarization upon which it can either bind to presynaptic receptors for

its further release or bind to postsynaptic receptors for synaptic transmission.

Acetylcholinesterase (AChE) breaks down ACh into choline and acetate, which are then taken up

by the presynaptic cell (adapted from Lleo et al, 2006 (7)).

1.2.2 Acetycholinesterase (AChE) Structure and its Mechanism of

Action

Crystal structures of AChE from mouse (66), Drosphila (67), and human beings (68) were

derived and found to be very similar to one another. The two sites of AChE include the active

site and the peripheral anionic site (PAS) (Fig. 1.5). The active site of AChE has been shown to

be located in a deep narrow gorge of about 20 Å long and located more than halfway into the

enzyme (63, 69). Early kinetic studies have demonstrated that the active site contains two

subsites, the ‘esteratic’ and ‘anionic’ subsites that correspond to the catalytic site and choline-

binding site, respectively (70). 40% of the surface of the active-site gorge consists of 14 aromatic

8

amino acid residues. The gorge contains only few acidic residues including aspartic acid,

glutamic acid, and tyrosine (69). Ser 203, His 447 and Glu 334 compose the catalytic triad in the

‘esteratic’ site of mammalian AChE (71, 72). AChE can be classified as a serine hydrolase due to

the presence of this catalytic triad that is similar to that of other catalytic sites in serine proteases

such as, trypsin and blood clot factors. These serine proteases usually have Asp in the catalytic

triad; however, in AChE, Glu is substituted for Asp as the acidic residue (63). The anionic site

consists of the amino acid residues Trp 86, Tyr 133, Tyr 337 and Phe 338, involved in binding to

the quaternary trimethylammonium group of ACh while positioning the ester of ACh optimally

at the catalytic or acylation site (72, 73).

Fig. 1.5: Structural features of the AchE enzyme. X-ray crystallography has identified the active

site found in the deep narrow gorge of AChE and the peripheral anionic site (PAS) found near

the lip of the active site (adapted from Soreq et al, 2001 (63)).

While the anionic active site is responsible for binding the quartenary trimethylammonium

moiety, the esteratic active site consists of the nucleophilic Ser residue responsible for the

hydrolysis of the ester bond in ACh (74, 75). The imidazole proton from a His residue causes a

concerted protonation of the carbonyl oxygen and the hydroxyl group of Ser attacks the partial

positive carbon of the ester carbonyl moiety of ACh. This results in the formation of a tetrahedral

intermediate that collapses due to its instability, releasing choline and acetylated AChE. This

9

acetylated complex undergoes subsequent hydrolysis with water to release the active form of

AChE and acetic acid (74, 75).

Site-directed mutagenesis studies have also indicated the presence of the PAS on the surface of

AChE, which is about 20 Å distant from the active site (69, 71). It is involved in the allosteric

modulation of the protein and binds ACh during the first step of the catalytic pathway (76-78). It

also has non-cholinergic functions such as, cell adhesion and neutrite outgrowth in neuronal cells

(77-80). Tyr 72, Asp 74, Tyr 124, Trp 286 and Tyr 341 include the five residues of the

mammalian PAS found near the entrance of the active site gorge (81-83).

1.2.3 Cholinesterase Inhibitors (ChEIs)

ChEIs have been implemented effectively to restore pathologically reduced neurotransmitter

levels by modulating native hydrolytic degradation catalyzed by the enzyme, AChE (6, 84)

Currently, four ChEIs have been approved by the FDA for clinical treatment of mild to severe

stage AD. These include: Galanthamine (REMINYL ®), Rivastigmine (EXELON ®), Tacrine

(COGNEX ®) and Donepezil (ARICEPT ®) (Fig. 1.6) (85, 86). Tacrine is rarely used due to its

hepatotoxicity effects (86, 87). In view of the limited number of commercially available drug

therapies for AD, continued progression towards more biologically compatible ChEI is

imperative (6).2






10

Fig. 1.6: FDA approved AChE inhibitors and the first generation of AD drugs - Tacrine

(Cognex), Donepezil (Aricept), Rivastigmine (Exelon), and Galanthamine (Reminyl) - used for

the symptomatic treatment of AD (adapted from Palmer et al, 2002 (87)).

1.3 AChE induced Aβ aggregation

Further motivation for the synthesis of novel ChEI has stemmed from the recent identification of

AChE as an accelerant of Aβ aggregation via interaction with the PAS of the enzyme (6). This

has been further confirmed by studies with butyrylcholinesterase (BuChE), a non-specific

cholinesterase variant that effectively lacks a PAS, which is unable to induce such accelerated

growth (88). Rivastigmine is a ‘psuedo-irreversible’ inhibitor that binds to the catalytic active

site region forming a carbamylated enzyme intermediate that dissociates slowly. Conversely,

Donepezil, Tacrine and Galanthamine are rapidly reversible inhibitors capable of only short-term

interaction with the AChE PAS.(89, 90) Notably, certain PAS-binding ChEI, such as Donepezil,

have been shown to impede AChE-induced Aβ aggregation, thereby prompting the development

of a novel class of ChEI capable of mediating long-term beneficial changes (91).3






11

1.4 Other Pathologies in AD

Although AChE plays a pivotal role in neuronal dysfunction in AD and research findings have

suggested that Aβ is the triggering event in AD pathology, there are other secondary events

involved that make this multifaceted disease very challenging to treat (29). Some of these factors

include oxidative stress (19), imbalance in calcium (Ca2+

) homeostasis (20), formation of NFTs

(21), and activation of the microglia cells (22, 29).

1.4.1 Oxidative Stress

Many scientists have debated for years whether oxidative stress was a cause or a consequence of

neurodegenerative disorders but have come to a consensus of intracellular oxidative imbalance

being an early event in the neurodegenerative cascade (92). It is therefore considered to play a

significant role in the pathology of neurodegenerative diseases (93). Imbalance in the

homeostasis in pro-oxidant versus antioxidant in the central nervous system (CNS) can lead to

the generation of toxic radical and non-radical ROS that are involved in initiating and/or

propagating radical chain reactions. Damage caused by ROS is commonly observed within the

brain of AD patients as neuronal tissues are extremely sensitive to oxidative stress (29). It is seen

within every class of biological macromolecules: nucleic acids, proteins, lipids, and

carbohydrates (94, 95). Aβ-induced free radicals can also cause secondary to excessive oxidative

stress (29, 96). All the multifactorial biological pathways involved in AD seem to share oxidative

stress as a common factor; hence, suggesting it to play a causative role in AD pathogenesis (29).

1.4.2 Calcium (Ca2+

) Dyshomeostasis

Khachaturian (97) was the first to propose the Ca2+

hypothesis in AD, which basically states that

Ca2+

dyshomeostasis is a proximal cause in the pathogenesis of AD. This hypothesis was initially

not supported by experimental evidence; however, evidence for its role in AD has now emerged

(98). Studies have shown that disregulation of Ca2+

have implications on Aβ accumulation and

tau hyperphosphorylation in the brain. Experimental evidence from human subjects have

suggested that disruptions in the signaling of Ca2+

occur during the early phases of AD (99).

However, there has been an ongoing debate as to whether this phenomena is correct or does Ca2+

dyshomeostasis precede Aβ in the cascade because it has been suggested that disregulation in

12

Ca2+

signaling is one of the ways through which Aβ toxicity is manifested (100, 101). Many

researchers have argued that Aβ can exert a toxic effect on Ca2+

regulation (102, 103).

Ca2+

entry, including ligand-gated channels, voltage-dependent calcium channels (VDCCs) that

permit calcium entry after membrane depolarization, and store-operated calcium channels

(SOCCs), are regulated in many ways by neurons (104). An influx of extracellular Ca2+

into the

cytosol through SOCCs on the plasma membrane triggers the decrease of calcium stores in the

endoplasmic reticulum (ER). The ER membrane then pumps Ca2+

through the sarco-

/endoplasmic reticulum Ca2+

ATPase located in the ER membrane to replenished the Ca2+

load in

the ER. This mechanism of store-dependent entry of Ca2+

is known as capacitative calcium entry,

and has been shown to contribute in the pathogenesis of Alzheimer’s disease (103, 105-108).

1.4.3 Formation of Neurofibrillary Tangles (NFTs)

NFTs consist of paired helical filaments that are composed of tau proteins in its abnormal

hyperphosphorylated form (Fig. 1.12). Tau proteins are generally involved in the stability of

cytoskeletal microtubule assembly, which gets hindered during phosphorylation within the

microtubule binding domain of the protein (21). The tau and tangle hypothesis is AD states that

the normal activity of tau proteins is impaired in AD and the microtubules are replaced with

NFTs in the neurons (109). This can cause disruption in the neuronal transmission and ultimately

lead to cell death. Isolating tau protein from NFTs and its subsequent dephosphorylation can

restore its activity in stabilizing microtubules, which suggests that

phosphorylation/dephosphorylation kinetics greatly contributes to the neurodegeneration in AD

(21), (29). Recent studies have shown that inhibition of the neurofibrillary degeneration in AD

can be achieved by phosphoseryl/phosphothreonyl protein phosphatase-2A activation or

glycogen synthase kinase-3β and cyclin-dependent protein kinase inhibition (21), (29).

1.4.4 Chronic Activation of Microglia

Microglia cells, macrophages found in the brain and spinal cord, have received considerable

attention in AD because they have been recognized in contributing to the neuroinflammation in

the CNS (29). They produce cytokines, chemokines, and neurotoxins when activated by Aβ

proteins. These are potentially toxic to neurons and ultimately lead to neuronal degeneration

13

(29). Therefore, modulating the neuroinflammation of CNS is considered a therapeutic target for

AD (29, 110).

1.5 Conclusion

AD, a multifactorial syndrome, is triggered by a cascade of molecular events such as, Aβ

aggregation (18), depression of the cholinergic system, formation of toxic NFTs in neurons (21),

oxidative stress (19), Ca2+

dyshomeostasis (20) and activation of the microglial cells (22).

Although this disease is involved in multiple pathologies, ChEIs are the only drugs marketed for

AD therapy. Hence, current therapeutic approaches should focus on a multi-targeted approach

for drug development for the efficient treatment of such a complex disorder (29, 111). The

objective of the research conducted in our laboratory involved the synthesis of potential drugs

that could act as multi-target-directed ligands (MTDLs) for AD.

14

Chapter 2 Multi-Target-Directed Approach in Alzheimer’s Disease Therapy

2 Introduction

Combination targeting of distinct AD pathologies using multiple, independently acting drug

therapies has emerged as a highly potent strategy to address the complex, degenerative nature of

AD (Fig. 2.1) (6). Combination studies of ChEI have a synergistic enhancement of therapeutic

effects compared to single drug therapies (112). However, the implementation of multiple single-

drug entities also raises a potential for conflicting bioavailabilities, pharmacokinetics and

metabolism between compounds (113, 114). More recent innovations have sought to address

these concerns through incorporation of multiple pharmacophores into single drug entities, which

still retain the ability of each individual component to interact with its intended target (29).

Multi-targeted therapies are also expected to simplify therapeutic regiments for patients leading

to improved applicability for AD care. An approach in multi-target drug therapy development for

AD is to integrate a ChEI pharmacophore with a second unit capable of targeting a separate AD

pathology (6, 111, 115-117).4






15

Fig. 2.1: Possible molecular factors involved in the pathology of AD and the potential inhibitors

to target these etiologies. The multifactorial nature of the disease make it challenging to treat and

requires a multi-target-directed approach for therapy (adapted from Cavalli et al, 2008 (29)).5

2.1 Dual Binding ChEIs

ChEIs that are able to simultaneously interact with both the active and peripheral sites of AChE

can not only help restore the cholinergic system in AD patients but also target Aβ aggregation

(29). Such a compound with the ability to have dual inhibition properties was demonstrated in

Melchiorre et al, 2003. This compound named caproctamine (2.1) was synthesized from a

tetraamine disulfide benextramine compound, which was previously shown to act as a reversible

inhibitor of AChE (Fig. 2.2) (118). Docking studies to analyze the binding mode of compound

5 Reprinted (adapted) with permission from (Cavalli, A., Bolognesi, M. L., Minarini, A., Rosini,

M., Tumiatti, V., Recanatini, M., and Melchiorre, C. (2008) Multi-target-directed ligands to

combat neurodegenerative diseases, J. Med. Chem. 51, 2326-2326.). Copyright (2008) American

Chemical Society.

16

2.1 indicated that it can bind to both AChE sites; however, this property was not verified through

experimental studies (29). Structure-activity relationship (SAR) studies of 2.1 were elaborated by

replacing the octamethylene spacer separating the two amide moieties with dianiline moieties to

form compound 2.2 (Fig. 2.2). Compound 2.2 demonstrated the most potent inhibition of AChE

(pIC50 = 8.48 ± 0.02) in vitro. (29, 119).

Fig. 2.2: Design strategy for development of compounds with AChE inhibition and AChE-

induced Aβ aggregation inhibition activities. Compounds 2.2 was derived through the

octamethylene linker from the diamine diamide compound 2.1 (adapted from Cavalli et al, 2008

(29)).

2.2 Dual Inhibition of AChE and Other Neurotransmitter Systems

Behavioural disturbances and changes experienced in AD patients are not only related to severity

in loss of ACh but also deterioration in seretoninergic and noradrenergic systems that have been

linked to depression (29, 120, 121). Neurotransmitters including serotonin, noradrenalin, and

dopamine undergo deamination during the catalytic activity of the enzyme, monoamine oxidase

(MAO). This results in the production of hydrogen peroxide that is a potential source of

oxidative stress to neurons in the brains of AD patients (122). Hence, MAO inhibition property

17

incorporated in a hybrid molecule designed to be a multi-target-directed ligand (MTDL), can be

a very useful property in AD therapy (29). Research studies to develop compounds with both

MAO and AChE inhibition activities resulted in a series of compounds shown in Fig. 2.3. These

compounds retained the propargylamine pharmacophore that have shown to exhibit irreversible

MAO inhibition properties (123). Rasagiline (2.4) has previously demonstrated MAO inhibition

along with neuroprotective effects in vitro and in vivo studies (29). The potential MAO/AChE

inhibitor 2.5 with an indane scaffold was rationally designed by incorporating a carbamate group

seen in rivastigmine (2.3) (an FDA approved ChEI) in order to exhibit AChE inhibition

properties (124). Compound 2.5, named ladostigil is currently under phase II clinical trials for

treatment in dementia (29, 125).

Fig. 2.3: Design strategy for the development of compounds with AChE inhibition and

monoamine oxidase (MAO) inhibition activities. Compounds 2.5 incorporates the

proparyglamine moiety residing the MAO inhibition property and a carbamate moiety shown to

have AChE inhibition property in the FDA approved AChE inhibitor rivastagmine (2.3) (adapted

from Cavalli et al, 2008 (29)).

18

2.3 Multi-Target Inhibition of AChE, AChE-Induced Aβ

Aggregation and Oxidative Stress

Oxidative stress is considered a significant factor in AD pathology due to the neuronal damage

caused by ROS (29). Lipoic acid (LA) (2.6), an antioxidant has previously shown to exert

neuroprotective effects (126). Combining this antioxidant feature with a pharmacophore

exhibiting a biological property such as, AChE inhibition can be an optimal design strategy for a

MTDL (127). The disulfide cyclic moiety of LA was also argued to have the potential to bind to

the PAS of AChE and inhibit AChE-induced Aβ aggregation. Based on these properties of LA, it

was combined with the tetrahydroacridine compound, tacrine (2.7) (an FDA approved AChE

inhibitor) in order to develop the compound lipocrine (2.8) for restoring cholinergic

transmission, inhibit Aβ aggregation, and decrease damage caused by oxidative stress (Fig. 2.4).

2.8 demonstrated nanomolar affinity against AChE and kinetic studies confirmed its interaction

with PAS of AChE. It was highly efficient in inhibiting AChE-induced Aβ aggregation and

protecting cells against ROS formation compared to its parent compound, 2.6 (29, 128).

Fig. 2.4: Design strategy for the formation of 2.8 derived from the organosulphur compound 2.6

residing the antioxidant properties and the tetrahydroacridine compound 2.7 possessing the

inhibition properties of AChE and AChE-induced Aβ aggregation (adapted from Cavalli et al,

2008 (29)).

2.4 Multi-Target Inhibition of AChE, BuChE and Ca2+

Channels

Ca2+

dyshomeostasis is another factor contributing to the pathology of AD as it can ultimately

cause neuronal cell injury and death (29). This may occur due to overactivation of NMDA

receptors that leads to the influx of excessive Ca2+

through the receptor’s ion channel (111).

19

Compounds that can posses Ca2+

channel blockage activity by inhibiting VDCC in dorsal root

ganglionic cells (129) along with AChE inhibition property would have a pivotal impact in AD

research (130, 131). A series of tacrine derivatives were explored for their properties as

AChE/BuChE inhibitors and VDCC modulators (132-137). Modifications of the structure of

tacrine was achieved by replacement of the benzene ring with various substituted heterocyclic

moieties and by changing the size of the cyclohexane ring to generate two series of compounds

labeled 2.15 and 2.16 in Fig. 2.5. Compound 2.17, a 1-dihydropyridine hybrid from compound

2.15 series termed tacripyrine demonstrated a 4-fold higher AChE inhibition (IC50 = 45 nM)

compared to tacrine (2.11) and exhibited a high selectivity towards BuChE in vitro studies.

Furthermore, it displayed efficient Ca2+

blockage activity and provided neuroprotection in cells

with Ca2+

overload. It also acted as a neuroprotectant to cells exposed to hydrogen peroxide with

an activity 1.5 fold higher than that of its prototype 2.14 (29, 137).

Fig. 2.5: Design strategy for the development of compounds with AChE, and

butyrylcholinesterase (BuChE) inhibition properties and voltage-gated calcium channel (VDCC)

blockade activity. Compound 2.10 was derived from 2.7 by altering the cyclohexane ring size

and replacing the benzene ring with a heterocyclic moiety. Compound 2.15 includes the

20

dihydropyridine ring from compound 2.9 that resides the Ca2+

channel inhibition property

(adapted from Cavalli et al, 2008 (29)).

2.5 Multi-Target Inhibition of AChE, AChE-induced Aβ

aggregation, Oxidative stress and N-Methyl-D-Aspartate

(NMDA) Receptor

Because blocking the NMDA receptor can also modulate Ca2+

dyshomeostasis, incorporating a

pharmacophore with NMDA receptor antagonist activity in a hybrid molecule with other

inhibition properties can generate a significantly potent AD drug (111). Rosini et al, 2008

reported the rational development of a potential AD drug incorporating AChE inhibition,

vasodilating beta-blocker, antioxidant, and low-affinity NMDA receptor antagonist activities

(111). They chose the carbazole moiety of carvedilol (2.11) as the building block for the design

of their MTDL because it has been previously shown to possess antioxidant properties (138) and

inhibition of Aβ aggregation (139). In order for the carbazole compound to act as an effective

AChE inhibitor, they selected the chloro-substituted tetrahydroacridine moiety of 6-

chlorotacrine, which was previously used as a starting point for the formation of lipocrine (5-

[1,2] dithiolan-3-ylpentanoic acid [3-(6-chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-propyl]

amide) (2.12) , a promising lead as a MTDL for AD (127). Lipocrine’s non-cytotoxicity and

bis(7)-tacrine’s (N,N’-bis-1,2,3,4-tetrahydroacridin-9-yl)heptanes-1, 7-diamine) neuroprotective

property through the moderate NMDA receptor blockade (140, 141) supported the selection of

the tetrahydroacridine core for the synthesis of the series of compounds 2.13, 2.14, 2.15, and

2.16, shown in Fig. 2.6 (111).

Compounds 2.13, 2.14, 2.15, and 2.16 showed AChE and BuChE inhibitory activities on human

recombinant AchE and Lineweaver-Burk plots derived for AChE inhibition indicated that all the

compounds demonstrated a mixed-type inhibition suggesting that they can interact with the

catalytic site and PAS of AChE. These compounds also exhibited a good inhibitory potency on

AChE-induced Aβ aggregation that was assessed through a Thioflavin T (ThT)-based

fluorometric assay (111, 142). Activity profiles of 2.13, 2.14, 2.15, and 2.16 tested on Ca2+

-

permeable NR1/NR2A NMDA receptors expressed in Xenopus laevis revealed that all the

compounds had NMDA receptor antagonist effects comparable to that of memantine, a popular

NMDA receptor blocker. Compound 2.13 presented greater NMDA receptor blocking effect than

21

memantine and was able to counteract the production of ROS in human neuronal SH-SY5Y cells

when treated with the oxidative damage inducer tert-butyl hydroperoxide (111).

Fig. 2.6: Design strategy for the development of compounds with AChE, AChE-induced Aβ

aggregation, oxidative stress and N-methyl-D-aspartate (NMDA) inhibition activities.

Compounds 2.13, 2.14, 2.15, and 2.16 possessing these inhibition properties incorporate the

carbazole moiety residing the Aβ inhibiton and antioxidant properites from 2.11. It also contains

the tetrahydroacridine moiety from 2.7 which has shown to possess AChE inhibition and low

affinity NMDA antagonist properties (adapted from Rosini et al, 2008 (111)).6

2.6 Conclusion

In summary, MTDL are better candidates for AD therapy due to their ability to hit multiple

targets at once compared to single-target drugs whose efficacy is questionable for such a

complex multifaceted disease. The compounds under investigation mentioned above for the

6 Reprinted (adapted) with permission from (Rosini, M., Simoni, E., Bartolini, M., Cavalli, A.,

Ceccarini, L., Pascu, N., McClymont, D. W., Tarozzi, A., Bolognesi, M. L., Minarini, A.,

Tumiatti, V., Andrisano, V., Mellor, I. R., and Melchiorre, C. (2008) Inhibition of

acetylcholinesterase, beta-amyloid aggregation, and NMDA receptors in Alzheimer's disease: A

promising direction for the multi-target-directed ligands gold rush, J. Med. Chem. 51, 4381-

4384.). Copyright (2008) American Chemical Society.

22

treatment of AD explore multifunctional mechanisms to target the different biological pathways

for a desired therapeutic agent for AD patients (29).

23

Chapter 3 Rational design of sym-Triazine Compounds for Multitarget Inhibition of Cholinesterases and Aβ in Alzheimer’s Disease

3 Introduction

sym-Triazines (3.1), a six-membrane ring with a closed system of π electrons comprise of

alternating nitrogen and carbon atoms with alternating single and double bonds (Fig. 3.1). They

differ from the aromatic system of the benzene ring by the presence of the nitrogen atoms

containing six unpaired electrons. The electron symmetry is destroyed in the triazine ring due to

the greater electron density on the nitrogen atoms compared to that of the carbon (143-145).

The decrease in the stability of the sym-triazine compared to benzene allows for addition

reactions such as, with hydrogen chloride to yield 2C3H3N3.3HCl and with silver to yield

AgNO3.2C3H3N3 and AgNO.C3H3N3 (144). It can also undergo substituted reactions for C-

halogenated triazines such as, 2, 4, 6-trichloro-1, 3, 5-triazine (cyanuric chloride, CC) (3.2) (Fig.

3.1). However, unlike benzene, triazine is incapable of electrophilic substitutions including

nitration and sulphonation because of the decreased electron density on the carbon atoms. Due to

this, it is not possible to synthesize C-nitro derivatives by substituting a chlorine atom in

cyanuric chloride by a nitro group or by oxidizing amino derivatives of triazine (145, 146).

Fig. 3.1: Chemical structures of sym-triazine (3.1) and cyanuric chloride (CC) (3.2) (adapted

from Mur, 1964 (145)).

24

Hydrolysis reactions of unsubstituted symmetrical sym-triazine demonstrates its instability,

especially involving mineral acids, which generates formic acid and7ammonia during

instantaneous reactions (143, 144). Ring opening of the triazine ring also occurs when it is

treated with primary or secondary amine, which can result in the formation of substituted

formamidine. Aluminium chloride can be used to weaken or break the C-N bond in triazine (145,

147).

Most reactions of sym-triazines are nucleophilic substitutions of chloro-triazines, in particular,

CC (148, 149). CC is often used as a starting material due to the facile displacement of its

chlorine atoms, which has been demonstrated using a number of nucleophiles in the presence of

hydrochloride acceptors such as sodium carbonate, sodium bicarbonate and sodium hydroxide

(145). CC is especially advantageous as it decreases in reactivity with successive substitutions

(150, 151). Consequently, the number of arm substitutions can be controlled by various factors

including reaction duration, solvent and base strength, the nucleophilic structure, steric factors

and substituents already present in the sym-triazine ring. Temperature is a primary factor

governing CC substitutions. Generally mono- substitutions were performed at approximately

4ºC, di-substitutions at room temperature and tri-substitutions above 60ºC. The commercial

availability and low cost of CC render it a practical choice for the synthesis of sym-triazine

derivatives (150, 152).

sym-Triazine derivatives have shown to possess various biological activities (150). Previous

studies have indicated its properties as anti-cancer (153, 154), anti-microbial (155) as well as

potential AD drugs (156-159). An efficient sym-triazine based inhibitor of Aβ aggregation or a

beta-sheet breaker was discovered through a high-throughput fluorescent based screen conducted

by Kim and her co-workers (156). The compound was selected based on its ability to prevent

misfolding of the green fluorescent protein (GFP) when fused with Aβ1-42 (156). Previous studies

Major parts of this chapter is reprinted (adapted) with permission from (Veloso, A. J., Dhar, D.,

Chow, A. M., Zhang, B., Tang, D. W. F., Ganesh, H. V. S., Mikhaylichenko, S., Brown, I. R.,

and Kerman, K. (2012) sym-Triazines for Directed Multitarget Modulation of Cholinesterases

and Amyloid-β in Alzheimer’s Disease, ACS Chem. Neurosci. (Available online 30 October

2012, DOI: 10.1021/cn300171c)). Copyright (2012) American Chemical Society.

25

done by the group demonstrated the inability of GFP to fluoresce in the presence of Aβ1-42 and

Aβ1-42-GFP fusions expressed in Escherichia coli (E.coli) generated non-fluorescent colonies.

Mutations in the Aβ1-42 sequence were able to retard aggregation in the fusion and hence, caused

proper folding of the GFP to yield green fluorescent colonies (160, 161).

This artificial genetic system in E.coli was used to search for small molecule inhibitors that can

retard Aβ1-42 aggregation in the Aβ1-42-GFP fusion (156). Based on this study, the compound 3.3

was found to be an effective Aβ1-42 aggregation inhibitor (Fig. 3.2). Further in vitro studies

utilizing the ThT-based fluorescent assay proved 3.3 compound’s property in inhibiting the beta-

sheet structure of Aβ1-42. Furthermore, electron microscopy studies showed modulation of the

Aβ1-42 structure when conjugated with this sym-triazine compound due to the absence of the fibril

structure of Aβ1-42 (156). 3.3 also demonstrated in further studies to have protective effects

against hydrogen peroxide (158) and Aβ (157) induced damages in human SK-N-MC

neuroblastoma cell lines. Moreover, cytotoxicity analysis in SK-N-MC cells indicated that the

compound’s interaction with the hydrophobic region of Aβ1-42 fibrils (KLVFF amino acid

sequence) decreases the toxicity of Aβ aggregates (159).

Fig. 3.2: Chemical structure of the sym-triazine molecule 3.3 that demonstrated beta-sheet

breaking activity in Aβ1-42 in the fluorescent-based screen and was chosen for further in vitro

studies (Adapted from Kim et al, 2006 (156)).

Considering the promising Aβ inhibition properties seen in the studies mentioned above, our

laboratory has developed a library of novel sym-triazine-derived compounds capable of

paralleled modulation Aβ aggregation and AChE/BuChE hydrolytic activity.

26

3.1 Reaction Scheme

Using an efficient synthetic route, sym-triazine based- methoxy compounds, acids, acyl

chlorides, esters, and ester-salt compounds were developed using CC (3.2) as a starting material

(Fig. 3.3).

Fig. 3.3: Synthetic routes for new ester and ester-salt sym-triazine derivatives. sym-Triazine

based methoxy compounds, acids, acyl chlorides were formed as intermediates iReactions

involving formation of sym-triazine acids 3.6, 3.7, and 3.8. iiReactions involving formation of

sym-triazine acyl chloride 3.9, 3.10, and 3.11. iii

Reactions involving formation of sym-triazine

esters 3.12, 3.15, 3.13, 3.16, 3.14, and 3.17. iv

Reactions involving formation of sym-triazine ester

salts 3.18, 3.21, 3.19, 3.22, 3.20, and 3.23 (6).

3.2 Experimental Procedure

Thin-layer chromatography (TLC) was performed on Merck Silica Gel 60 F254, 20 x 20 cm plates

and visualized using a 254 nm UV lamp. 1H-Nuclear Magnetic Resonance (NMR) spectra (400

MHz) (See Appendices) and 13

C-NMR (100 MHz) spectra (See Appendices) were recorded on

Bruker Avance III using solvents DMSO-d6, D2O , and CDCl3 and are internally referenced to

residual protic solvent signals (δ 2.50, 4.79, and 7.26 δ, respectively). Data for 1

H-NMR are

reported as chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m

= multiplet, dd = doublet of doublets), J coupling constant (Hz) and assignment. Data for 13

C-

27

NMR are reported in as chemical shift (δ ppm). Infrared (IR) spectra (See Appendices) were

recorded on a Bruker Alpha-P spectrometer with ATR attachment and reported in terms of

frequency of absorption (cm-1

). Mass spectra (MS) (See Appendices) were obtained using an

AB/Sciex QStar mass spectrometer (ESI-TOF) in positive ion mode. Melting points were

recorded on a melting point apparatus (Fisher Scientific). Reagents were obtained from

commercial vendors and used as received unless otherwise noted (6).

3.2.1 Procedure for sym-Triazine Based Methoxy Compounds

2-chloro-4,6-dimethoxy-1,3,5-triazine (3.4) 2,4-dichloro-6-methoxy-1,3,5-triazine (3.5) (Fig.

3.4): 3.4 and 3.5 can be synthesized following the known procedure by Dudley (6, 162).

Fig. 3.4: Synthesis of sym-triazine based methoxy compounds 3.4 and 3.5 from CC (3.2).

Reactions were carried out using methanol (CH3OH) as the reagent and sodium bicarbonate

(NaHCO3) as the base.

3.2.2 Procedure for sym-Triazine Based Acids

Fig. 3.5: Synthesis of sym-triazine based acids 3.6, 3.7, and 3.8. iReactions were run using

oxybenzoic acid, sodium hydroxide (NaOH) and water in acetone. Acidification was performed

using concentrated hydrochloric acid (HCl) to yield 3.6, 3.7, and 3.8.

28

4-((4,6-dimethoxy-1,3,5-triazin-2-yl)oxy)benzoic acid (3.6) and4,4'-((6-methoxy-1,3,5-

triazine-2,4-diyl)bis(oxy))dibenzoic acid (3.7) (Fig. 3.5): 3.6 and 3.7 can be synthesized

following the known procedure by Pogosyan (6, 163).

2,4,6-tris (4’-Chlorocarbonylphenoxy )-1,3,5- triazine (3.8) (Fig. 3.5): 3.8 can be produced in

one step by CC (3.2) and 4-oxybenzoic acid; applying the known method by Sklyarskii (6, 164).

3.2.3 Procedure for sym-Triazine Based Acyl Chlorides

Fig. 3.6: Synthesis of sym-triazine based acyl chlorides 3.9, 3.10, and 3.11. iiReactions were run

at 60ºC in thionyl chloride (SOCl2) using anhydrous chloroform (CHCl3) as solvent and pyridine

as catalyst.

4-((4,6-dimethoxy-1,3,5-triazin-2-yl)oxy)benzoyl chloride (3.9) (Fig. 3.6) (Procedure A): The

mixture of 11 g of 3.6 (0.04 mol), 8.82 mL of thionyl chloride (SOCl2) (0.12 mol) and one drop

of pyridine in 100 mL of dry chloroform was refluxed and boiled for 6 hours. The reaction was

monitored by thin layer chromatography (TLC) for completion. Chloroform was distilled off at

60 °C until the solution started to turn light yellow. 120 mL of petroleum ether was added to the

solution and allowed to precipitate for an hour. The white precipitate was washed with petroleum

ether. The white solid had a melting point of 105 °C - 107 °C with a 60.6 % final yield (6).

IR (neat) v = 3072.00, 1771.45, 1746.00, 1564.14, 1468.49, 1199.02, 806.35 cm-1

; 1H-NMR (400

MHz, CDCl3) δ 8.21-8.19 (d, J = 8.9 Hz, 2 H), 7.36-7.34 (d, J = 8.9 Hz, 2 H), 4.02 (s, 6 H); 13

C-

29

NMR (100.42 MHz, CDCl3) δ 174.1, 173.0, 167.9, 157.1, 133.4, 121.5, 55.9; Calculated

C12H10N3O4Cl ([M +H]+): 295.68, found: 296.0.

4,4'-((6-methoxy-1,3,5-triazine-2,4 diyl)bis(oxy))dibenzoyl chloride (3.10) (Fig. 3.6): 5g (0.01

mol) of 3.7, 11.25mL (moles) of SOCl2, one drop of pyridine in 100 mL of dry chloroform was

heated until boiling for 6 hours. Procedure A was followed to provide 3.10 in 79.8 % yield as a

yellow powder with a melting range of 133 °C - 135 °C (6).

IR (neat) v = 3076.93, 1744.13, 1560.86, 1496.04, 1197.93, 815.30 cm-1

; 1

H-NMR (400 MHz,

CDCl3) δ 8.21-8.19 (d, J = 8.8 Hz, 4 H), 7.35-7.33 (d, J = 8.8 Hz, 4 H), 3.98 (s, 3 H); 13

C-NMR

(100.42 MHz, CDCl3) δ 172.8, 169.0, 167.8, 157.0, 133.8, 131.7, 121.4, 56.5, Calculated

C18H11N3O5Cl2 ([M +H]+): 420.2, found: 420.1.

2,4,6-tris(4’-Chlorocarbonylphenoxy)-1,3,5-triazine (3.11) (Fig. 3.6): The mixture of 4.894 g

(0.01 mol) of 3.8 and 43.5 mL (0.60 mol) of SOCl2 in 80 mL of dry chloroform and 3 drops of

dry pyridine (catalyst) was refluxed under dry conditions. Procedure A was followed to provide

3.11, light yellow powder with 185 - 188 оС melting point and final yield of 88.7 % (6).

IR (neat) v: 3107.63, 3074.38, 1778.23, 1737.83, 1605.77,1560.62, 1494.60, 1209.28, 1193.44,

1166.72,1085.76,1016.90, 850.96; 1H-NMR (400 MHz, CDCl3) δ 8.19-8.17 (d, J = 8.8 Hz, 6H),

7.32-7.29 (d, J = 8.8 Hz, 6H); 13

C-NMR (100.42 MHz, CDCl3) δ 173.4, 167.0, 158.3, 133.5,

131.8, 122.0; Calculated С24Н12N3O6Cl3 ([M +H]+): 544.8, found: 545.0.

30

3.2.4 Procedure for sym-Triazine Based Esters

Fig. 3.7: Synthesis of sym-triazine based esters 3.12, 3.15, 3.13, 3.16, 3.14, and 3.17.

iiiReactions were carried out using a: 3-dimethylamino-1-propanol, b: 1-dimethylamino-2-

propanol and c: 2-dimethylaminoethanol in anhydrous tetrahydrofuran (THF) in the presence of

triethylamine ((C2H5)3N) as a base.

3-(dimethylamino)propyl-4-((4,6-dimethoxy-1,3,5 triazin-2-yl)oxy)benzoate (3.12) (Fig. 3.7)

(Procedure B): 2.0 g of 3.9 was dissolved in 40 mL of dry tetrahydrofuran (THF). The solution

of 6.76 mmol (0.94 mL) of dry triethylamine and 6.76 mmol (0.80 mL) of 3-dimethylamino-1-

propanol in 10 mL of dry THF was added drop wise to the 3.9 solution. The reaction was

refluxed and temperature maintained at 40 ºC. Reaction was monitored by TLC for completion.

The ester slurry was gravity filtered and washed with 3 х 6 mL of dry THF. The combined

filtrate was evaporated under vacuum conditions. 50 mL of dichloromethane was used to

dissolve the ester residue. The dichloromethane layer was washed with 6 x 50 mL water and the

organic layer was collected. The ester solution was dried with anhydrous sodium sulphate

(Na2SO4) overnight. The resulting residue was washed with small portions of petroleum ether

(40 - 60 oC) resulting in the formation of a precipitate. Petroleum ether was evaporated 3.12 was

collected in 68.6 % yield as a white powder with melting point of 60 °C - 64 °C (6).

IR (neat) v = 2953.87, 2822.95, 2764.52, 1709.96, 1567.03, 1468.08, 1264.86, 1220.38, 806.66

cm-1; 1

H-NMR (400 MHz, CDCl3) δ 8.11-8.09 (d, J = 8.8 Hz, 2H), 7.27-7.25 (d, J = 8.8 Hz, 2H),

31

4.40-4.37 (t, J = 6.4 Hz, 2H), 4.00 (s, 6H), 2.48-2.45 (t, J = 7.4 Hz, 2H), 2.29 (s, 6H), 2.00-1.93

(m, 2 H); 13

C-NMR (100.42 MHz, DMSO-d6) δ 174.5, 172.9, 165.8, 155.8, 131.8, 127.8, 122.9,

63.6, 56.4, 56.1, 45.5, 26.5; Calculated C17H22N4O5 ([M +H]+): 362.4, found: 363.0.

1-(dimethylamino)propan-2-yl-4-((4,6-dimethoxy-1,3,5-triazin-2-yl)oxy)benzoate (3.15) (Fig.

3.7): 2.0 g of 3.9 was dissolved in 40 mL of dry THF. The solution of 6.76 mmol (0.94 mL) of

dry triethylamine and 6.76 mmol (0.83 mL) of 1-dimethylamino-2-propanol in 10 mL of dry

THF was added drop wise to the 3.9 solution. Procedure B was followed to provide 3.15 in 41.6

% yield as a white powder with melting point of 48 °C – 52 °C (6).

IR (neat) v = 2979.50, 2820.82, 2765.94, 1715.25, 1566.16, 1467.85, 1267.09, 1217.91, 806.42

cm-1

; 1

H-NMR (400 MHz, CDCl3) δ 8.11-8.09 (d, J = 8.8 Hz, 2 H), 7.27-7.25 (d, J = 8.8 Hz, 2

H), 5.38-5.30 (m, 1 H), 4.00 (s, 6H), 2.74-2.69 (dd, 1H), 2.49-2.44 (dd, 1H), 2.28 (s, 6 H), 1.38-

1.36 (d, J = 6.4 Hz, 3H); 13

C-NMR (100.42 MHz, DMSO-d6) δ 174.0, 172.5, 165.6, 155.8, 131.6,

128.1, 122.8, 70.0, 64.4, 56.2, 46.8, 19.0; Calculated C17H22N4O5 ([M +H]+): 362.4, found: 363.0

Bis(3-(dimethylamino)propan-yl)4,4'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(oxy))dibenzoate

(3.13) (Fig. 3.7): 2.0 g (0.0048 mol) of 3.10 was dissolved in 55 mL of THF. 1.32mL of

triethylamine and 0.0095 moles (1.13 mL) of 3-dimethylamino-1-propanol in 18.96 mL of THF

was added to the 3.10 solution in THF. Procedure B was followed to provide 3.13 in 53.5 %

yield as a yellow powder with melting point of 110 °C - 125 °C (6).

IR (neat) v = 2948.39, 2856.39, 2768.09, 1710.28, 1578.14, 1466.51, 1268.23, 1207.71, 818.89

cm-1

; 1

H-NMR (400 MHz, DMSO-d6) δ 8.01-7.98 (d, J = 8.8 Hz, 4H), 7.26-7.24 (d, J = 8.8 Hz,

4H), 4.35-4.32 (t, 4H), 3.50-3.45 (t, 4H), 3.08 (s, 3H), 2.17 (s, 12H), 1.88-1.81 (m, 4H); 13

C-

NMR (100.42 MHz, DMSO-d6) δ 172.8, 170.0, 166.0, 157.7, 131.5, 126.5, 123.0, 62.6, 56.0,

53.0, 45.5, 26.8, Calculated C28H35N5O7 ([M +H]+): 553.7, found: 554.3.

Bis(1-(dimethylamino)propan-2-yl)4,4'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(oxy))dibenzoate

(3.16) (Fig. 3.7): 2.0 g (0.0048 mol) of 3.10 was dissolved in 55 mL of THF. 1.32 mL of

triethylamine and 0.009519 moles (1.126mL) of 1-dimethylamino-2-propanol in 18.96 mL of

THF was added to the 3.10 solution in THF. Procedure B was followed to provide 3.16 in 34.2 %

yield as a yellow powder with melting point of 77 °C - 82 °C (6).

32

IR (neat) v = 2976.79, 2822.66, 2766.79, 1710.21, 1556.42, 1469.03, 1265.37, 1208.40, 814.06

cm-1

; 1

H-NMR (400 MHz, DMSO-d6) δ 8.05-8.03 (d, J = 8.8 Hz, 4H), 7.29-7.27 (d, J = 8.8 Hz,

4H), 5.55-5.50 (m, 2H), 3.96-3.92 (dd, 2H), 3.69-3.66 (dd, 2H), 3.16 (s, 3H), 2.09 (s, 12H), 1.38-

1.36 (d, J = 6.4 Hz, 6H); 13

C-NMR (100.42 MHz, DMSO-d6) δ 184.05, 180.47, 159.79, 157.57,

131.21, 125.87, 122.77, 66.24, 53.42, 45.90, 31.01, 19.06; Calculated C28H35N5O7 ([M +H]+):

553.7, found: 554.3.

2,4,6-tris[(2’-dimethylamino-1’-ethoxy)-4’-]-1,3,5-triazine (3.14) (Fig. 3.7): 3.0 g (5.51 mmol)

of 3.11 was dissolved in 60 mL of dry THF at room temperature conditions. The solution of 2.3

mL of 2-dimethylaminoethanol and 2.5 mL of triethylamine in 10 mL of dry THF was added

dropwise to the 3.11 solution. Procedure B was followed however with the temperature for

reaction maintained at 15 оС. Compound 3.14 formed had a melting point of 85

oC - 87

oC and

final yield of 17 % (6).

IR (neat) v: 2968.62, 2947.08, 2821.10, 2769.13, 1715.83, 1605.70, 1569.88, 1503.57, 1464.05,

1412.61, 1360.34, 1269.70, 1211.83, 1115.89, 1017.03, 863.99; 1H-NMR (400 MHz, DMSO-d6)

δ 8.01-7.99 (d, J = 8.8 Hz, 6H); 7.42-7.40 (d, J = 8.8 Hz, 6H), 4.38-4.36 (t, J = 5.6 Hz, 6H), 2.67-

2.65 (t, J = 5.6 Hz, 6H), 2.24 (s, 18H); 13

C-NMR (100.42 MHz, DMSO-d6) δ 173.3, 166.1,

155.0, 131.8, 128.2, 122.6, 62.9, 57.5, 46.0; Calculated С36Н42N6O9 ([M +H]+): 702.9, found:

704.3.

2,4,6-tris[(1’-dimethylamino-2’-propoxy)-4’-carbonylphenoxy]-1,3,5-triazine (3.17) (Fig. 3.7):

1.3 g (2.39 mmol) of 3.11 was dissolved in 60 mL of dry THF at room temperature conditions.

The solution of 0.725 g (7.16 mmol) of 1-dimethylamino-2-propanol and 0.725 g (7.16 mmol) of

triethylamine in 10 mL of dry THF was added dropwise to the 3.11 solution under anhydrous

conditions. Procedure B was followed with temperature maintained at 40 ºC to provide 3.17 in

64.4 % yield as a white powder with melting point of 76 oC - 82

оС (6).

IR (neat) v: 2978.12, 2938.02, 2822.64, 2769.07, 1711.82, 1593.67, 1566.82, 1502.47, 1461.03,

1412.42, 1360.07, 1265.59, 1210.33, 1160.89, 1014.13, 812.56 cm -1

, 1H-NMR (400 MHz,

DMSO-d6) δ 8.01-7.99 (d, J = 8.8 Hz, 6H), 7.42-7.40 (d, J = 8.8 Hz, 6H), 5.5-5.19 (m, 3H), 2.67-

2.63 (dd, 3H), 2.44-2.44 (dd, 6H), 2.24 (s, 18H), 1.30-1.28 (d, J = 6.3, 9H); 13

C-NMR (100.42

MHz, DMSO-d6) δ 172.6, 164.7, 153.4, 131.3, 128.4, 122.3, 69.5, 63.7, 46.0, 19.1; Calculated

С39Н48N6O9 ([M +H]+): 745.0, found: 746.3.

33

3.2.5 Procedure for sym-Triazine Based Iodine-Ester Salts

Fig. 3.8: Synthesis of sym-triazine based ester salt compounds 3.18, 3.21, 3.19, 3.22, 3.20, and

3.23. iv

Reactions were carried out at 40ºC using iodomethane (CH3I) in anhydrous THF.

3-((4-((4,6-dimethoxy-1,3,5-triazin-2-yl)oxy)benzoyl)oxy)-N,N,N-trimethylpropan-1-aminium

iodide (3.18) (Procedure C) (Fig. 3.8): 2.06 mL of iodomethane (0.033 mol) was added drop

wise to the solution of 1.20 g (3.3 mmol) of 3.12 in 40 mL of dry THF. The mixture was stirred

two hours at 40 оС and left overnight under dry conditions. The resulting precipitate was filtered,

washed by 2 х 6 mL of dry THF and 2 x 10 mL of petroleum ether to provide 3.18 in 88.0 %

yield as a white powder with a melting point of 176 °C - 179 °C (6).

IR (neat) v = 3002.94, 2954.31, 1717.40, 1554.68, 1459.01, 1266.75, 1215.76, 815.41 cm-1

; 1

H-

NMR (400 MHz, D2O) δ 8.02-8.01 (d, J = 8.9 Hz, 2H), 7.27-7.25 (d, J = 8.9 Hz, 2H), 4.37-4.35

(t, 2H), 3.88 (s, 6H), 3.46-3.43 (t, 2 H), 3.06 (s, 6H), 2.28-2.20 (m, 2 H); 13

C-NMR (100.42

MHz, D2O) δ 173.6, 172.7, 167.6, 155.5, 131.6, 126.7, 122.2, 64.3, 62.5, 56.4, 53.3, 22.7;

Calculated C18H25N4O5 ([M +H]+): 377.3, found: 377.2.

3-((4-((4,6-dimethoxy-1,3,5-triazin-2-yl)oxy)benzoyl)oxy)-N,N,N,2-trimethylpropan-1-aminium

iodide (3.21) (Fig. 3.8): 1.60 mL of iodomethane (0.025 mol) was added drop wise to the

solution of 0.92 g (0.0025 mol) of 3.15 in 40 mL of dry THF. Procedure C was followed to

provide 3.21 in 74.2 % yield as a white powder with a melting point of 188 °C - 191 °C (6).

34

IR (neat) v = 2997.32, 2952.72, 1721.29, 1558.83, 1467.22, 1256.47, 1220.57, 810.91 cm-1

; 1H-


(m, 1H), 3.93-3.89 (dd, 1H), 4.65 (s, 6H), 3.47-3.42 (dd, 1H), 3.11 (s, 9H), 1.36-1.34 (d, J = 6.43

Hz, 3H); 13

C-NMR (100.42 MHz, D2O) δ 173.6, 172.7, 166.2, 155.8, 132.0, 127.5, 122.2, 67.6,

56.2, 54.2, 54.1, 19.0; Calculated C18H25N4O5 ([M +H]+): 377.3, found: 377.2.

3,3’-((4,4’-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(oxy))bis(benzoyl))bis(oxy))bis(N,N,N-

trimethylpropan-1-aminium) iodide (3.19) (Fig. 3.8): 0.38 mL (6.1mmol) of iodomethane was

added drop wise to the solution of 1.3 mmol of 3.13 in 34.6 mL of THF. Procedure C was

followed to provide 3.19 in 38.7 % yield as a yellow powder with melting point of 180 °C - 185

°C (6).

IR (neat) v = 3014.25, 2956.28, 1710.56, 1559.66, 1476.14, 1271.12, 1208.75, 821.05 cm-1

; 1H-


(t, 4H), 3.8 (s, 3H), 3.65-3.61 (t, 4H), 3.01 (s, 18H), 1.78-1.74 (m, 2H) ; 13

C-NMR (100.42 MHz,

D2O) δ 181.2, 180.1, 167.3, 155.7, 131.6, 127.1, 122.2, 68.0, 64.0, 53.3, 53.0, 22.5, Calculated

C30H41N5O7 ([M + 2H]2+

): 291.9, found: 291.7.

2,2’-((4,’4-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(oxy))bis(benzoyl))bis(oxy))bis(N,N,N-

trimethylpropan-1-aminium) iodide (3.22) (Fig. 3.8): 0.38 mL (6.1mmol) of iodomethane was

added drop wise to the solution of 1.3 mmol of 3.16 in 34.6 mL of THF. Procedure C was

followed to provide 3.22 in 44.4 % yield as a yellow powder with melting point of 205 °C - 210

°C (6).

IR (neat) v = 3007.65, 2958.47, 1709.58, 1560.48, 1498.38, 1264.01, 1210.11, 765.47 cm-1

; 1H-

NMR (400 MHz, DMSO-d6) δ 8.13-8.11 (d, J = 8.5 Hz, 4H), 7.48-7.46 (d, J = 8.5 Hz, 4H), 5.56-

5.50 (m, 2H), 3.98-3.94 (dd, 2H), 3.72-3.69 (dd, 2H), 3.18 (s, 3H), 2.09 (s, 18H), 1.39-1.37 (d, J

= 6.3 Hz, 6H); 13

C-NMR (100.42 MHz, DMSO-d6) δ 180.06, 176.57, 174.34, 164.18, 131.60,

127.17, 121.33, 68.47 ,66.71, 53.43, 19.50; Calculated C30H41N5O7 ([M + 2H]2+

): 291.9, found:

291.7.

2,4,6-tris[iodomethylate(2’-trimethylamino-1’-ethoxy)-4’-carbonylphenoxyl 1,3,5-triazine (3.20)

(Fig. 3.8): 0.62 g (0.88 mmol) of purified 3.14 was dissolved in 45 mL of dry THF at room

temperature conditions. Separately, 0.4 mL (6.2 mmol) of methyl iodide was dissolved in 15 mL

35

of THF and stirred for 3 h. Procedure C was followed to provide 3.20 with a melting point >185

oC with decomposition and a final yield of 68.0 % (6).

IR (neat) v: 3005.09, 2960.27, 1716.52, 1605.43, 1568.43, 1502.64, 1267.58, 1211.31, 1164.68,

1114.20, 1013.18 cm -1

; 1H-NMR (400 MHz, DMSO-d6) δ 8.10-8.08 (d, J = 8.8 Hz, 6H); 7.47-

7.45 (d, J = 8.8 Hz, 6H), 4.74-4.73 (t, 6H); 3.85-3.83 (t, 6H), 3.23 (s, 27H); 13

C-NMR (100.42

MHz, DMSO-d6) δ 173.06, 165.08, 155.43, 131.66, 127.24, 122.32, 64.48, 59.21, 53.47;

Calculated С39Н51N6O9 ([M +H]3+

): 249.3, found: 249.5.

2,4,6-tris[iodomethylate(1’-trimethylamino-2’-propoxy)-4’-carbonylphenoxyl 1,3,5-triazine

(3.23) (Fig. 3.8): 0.65 mL (10.6 mmol) of methyl iodine was added drop wise into 1.16 g (1.56

mmol) solution of 3.17 in 40 mL of dry THF at room temperature conditions. Procedure C was

followed to provide 3.23 as light yellow powder with 189 oC - 191

оС melting point and final

yield of 89.1 % (6).

IR (neat) v: 3003.32, 2956.57, 1711.91, 1604.65, 1567.36, 1501.60, 1264.14, 1210.76, 1162.85,

1092.82, 1013.12; 1H-NMR (400 MHz, DMSO-d6) δ 8.12-8.10 (d, J = 8.8 Hz, 6H), 7.47-7.45 (d,

J = 8.8 Hz, 6H); 5.56-5.50 (m, 3H), 3.98-3.93 (dd, 3H), 3.72-3.69 (dd, 3H), 3.30 (s, 27H); 1.39-

1.37 (d, J = 6.0 Hz, 9H); 13

C-NMR (100.42 MHz, DMSO-d6) δ 173.09, 164.23, 155.59, 131.69,

127.62, 122.68, 68.38, 66.92, 53.69, 19.03; Calculated С42Н57N6O9 ([M +H]3+

): 263.4, found:

263.5.

3.3 Discussion

Initial derivatization of CC (3.2) involved the incorporation of methoxy groups (3.4 and 3.5) via

nucleophilic aromatic substitution of chlorine with methanol, following procedures established

by Dudley (6, 162). The predicted reaction mechanism is shown in Fig. 3.9 (165). The

nucleophile, methanol can attack the carbon attached to the leaving group, chloride ion. This

nucleophilic attack results in a carbanian intermediate that is resonance-stabilized. The chloride

ion then leaves, which regains the aromaticity of the ring (165). Methanol substitution was

integral for controlling the number of Aβ and AChE-targeted substitutions within the triazine

core. Accordingly, the synthesis of 3.5 was performed at 0ºC in order to displace only a single

chlorine unit in the triazine ring of CC, leaving two chlorine units available for further

derivatization forming the targeted di- substituted species. Similarly, the synthesis of 3.4

36

required reaction temperatures of 60ºC for the displacement of two chorine units, leaving a single

unit available for derivitization of a mono-substituted triazine. Temperature was a key factor in

this reaction as synthesis of 3.5 resulted in a mixture of mono- and di-substituted sym-triazine

methoxy compounds when temperature conditions were over 0ºC. The resulting products (3.4

and 3.5) were washed with water to eliminate inorganic salts formed during the reaction (6).

Fig. 3.9: Reaction mechanism for the formation of sym-triazine based methoxy compound

(adapted from Bruice, 2001 (165)).

The sym-triazine-based carboxylic acids, 3.6 and 3.7, were subsequently synthesized from the

methoxy derivatives, 3.4 and 3.5 respectively, using 4-oxybenzoic acid reported by Pogosyan et

al (6, 163). The predicted reaction mechanism is shown in Fig. 3.10 (165). The base, sodium

hydroxide (NaOH) can deprotonate the two oxygen molecules on 4-oxybenzoic acid, which can

then cause this nucleophile to attack the carbon bearing the chlorine (leaving group) to

eventually form the sym-triazine based acids. The tri-substituted acid, 3.8, was directly

synthesized from a reaction of 4-oxybenzoic acid with CC (3.2) following conditions and

procedures developed by Sklyarskii et al.(164). Sodium hydroxide was employed as a base to

synthesize oxybenzoate salts of 3.6, 3.7 and 3.8 followed by the addition of dilute hydrochloric

acid (HCl) to the salt solution. This resulted in the precipitation of compounds 3.6, 3.7 and 3.8

that were washed thoroughly with water to yield the final sym-triazine acids. Notably, we have

observed that Aβ aggregation could be severely impaired in vitro by the presence of hydrophobic

poly aromatic compounds, which are capable of disrupting or preventing π-π stacking. Thus, the

addition of multiple aromatic functional units by substitution with oxybenzoate salts was

performed for rational targeting of Aβ aggregation (6).

37

Fig. 3.10: Reaction mechanism for the formation of mono-substituted sym-triazine based acid


The synthesis of the acyl chloride compounds 3.9, 3.10 and 3.11 from 3.6, 3.7 and 3.8,

respectively, by nucleophilic acyl substitution reaction involved replacement of the hydroxy

groups of the carboxylic acids with chlorine functional units (6). To accomplish this, the

carboxylic acid derivatives were treated with thionyl chloride using anhydrous chloroform as the

solvent with a pyridine catalyst. The predicted reaction mechanism is shown in Fig. 3.11 (166).

The nucleophile, carbonyl oxygen can attack the sulphur of thionyl chloride to form a tetravalent

sulphur intermediate. Subsequently, the chloride ion is displaced from the intermediate, which

results in an acyl chlorosulphite intermediate and at the same time, also reestablishing the

sulphur-oxygen π bond. The chloride ion that was lost from the intermediate can then attack the

activated carbonyl group, which eventually causes the displacement of the thionyl chloride group

and formation of the sym-triazine based acyl chloride (166). IR spectroscopic data indicated

formation of the acyl chloride by a shift of carbonyl and carboxyl absorption peaks as well as the

disappearance of the hydroxyl peak. The resulting acyl chlorides were unstable hygroscopic

species that could be easily converted back to their corresponding carboxylic acid derivatives

simply by exposure to air. Due to this reason, anhydrous chloroform was employed and a drying

tube was used to ensure no exposure to air and water (6).

38

Fig. 3.11: Reaction mechanism for the formation of mono-substituted sym-triazine based acyl

chloride (adapted from Fox et al, 2008 (166)).

Due to the instability of acyl chlorides, esterification was performed immediately following the

formation of the acyl chlorides by nucleophilic addition-elimination or nucleophilic acyl

substitution with various combinations of the following compounds: 1-dimethylamino-2-

propanol, 3-dimethylamino-1-propanol and 2-dimethylaminoethanol (6). The predicted reaction

mechanism is shown in Fig. 3.12 (165). In order to convert the acyl chloride to an ester, the

amino alcohol (1-dimethylamino-2-propanol, 3-dimethylamino-1-propanol or 2-

dimethylaminoethanol) can attack the carbonyl carbon of the sym-triazine based acyl chloride. A

tetrahedral intermediate is formed followed by the expulsion of the chloride ion as it is a weaker

base compared to the alkoxide ion (165). This can then result in the formation of the sym-triazine

based ester compounds. These compounds were designed as analogues of the native enzyme

substrate, acetylcholine, and were thus incorporated into the triazine structure for functional

targeting of the AChE active site (6).

All ester compounds demonstrated moderate to high conversion to the esters except for one due

to the different temperature conditions employed (6). 3.11 reacted with 2-dimethylaminoethanol

at a temperature maintained at 15 oC, which generated the ester, 3.14, in a poor isolated yield of

17%. Hence, reaction conditions were optimized for the formation of esters 3.12, 3.15, 3.13, 3.16

and 3.17 by carrying out their synthesis at 40oC. The triethylamine hydrochloride salt formed

during these esterification reactions was eliminated from the solvent THF comprising the ester.

39

For each esterification reaction, brown, viscous oil resulted after evaporation of THF, which was

washed several times with water to allow the esters to precipitate out. Water was eliminated to

obtain the solid esters, 3.12, 3.15, 3.13, 3.16, 3.14 and 3.17 in 68.6, 41.6, 53.5, 34.2, 17.0 and

64.4% yields, respectively (6).

Fig. 3.12: Reaction mechanism for the formation of mono-substituted sym-triazine based ester


Further electrophilic substitution of the ester products with iodomethane (CH3I) was used to

generate a series of novel iodine-ester salts, 3.18 in 88.0% yield, 3.21 in 74.2% yield, 3.19 in

38.7% yield, 3.22 in 44.4% yield, 3.20 in 68.0% yield and 3.23 in 89.1% yields (6). The

predicted reaction mechanism is shown in Fig. 3.13 (165). The tertiary amide of the sym-triazine

based esters can attack the methyl group of iodomethane and result in a protonated quartenary

ammonium iodide (165). The compounds 3.21, 3.22 and 3.23 were isolated as stereoisomers.

These compounds initially precipitated out in THF during the reaction upon addition of CH3I and

had to be washed off with THF and petroleum ether to get rid of any impurities and yield the

final solid ester-salt compounds. They were initially tested for their solubility in water to confirm

that they were salts and not their corresponding esters that are insoluble in water (6).

40

Fig. 3.13: Reaction mechanism for the formation of mono-substituted sym-triazine based ester-

salt (adapted from Bruice, 2001 (165)).

Reactions for all compounds synthesized were monitored using thin layer chromatography

(TLC) in 1:1 acetone and hexane solution and structures were confirmed using IR, MS, 1H NMR

and 13

C NMR (See Appendices) (6).

3.4 Inhibition of AChE, BuChE, and Aβ Aggregation by sym-Triazine Derivatives and their Cell Viability Studies

The sym-triazines esters and ester-salt compounds containing the hydrophobic aromatic phenyl

were analyzed for their activity as Aβ fibrillogenesis inhibitors (6) (Fig. 3.14). The esters and

ester-salt compounds were also tested for their inhibition activities against AChE and BuChE.

The ester-salt sym-triazines differed from their corresponding esters by the positive charge on the

nitrogen atom in the ester chain and by an additional methyl group on the positively charged

nitrogen. It would be interesting to observe how this affects the in vitro AChE inhibition studies

as the ester-salt component of the ester-salt sym-triazines resemble the structure of ACh (6).

41

Fig. 3.14: sym-Triazine esters, ester-salt compounds and a negative control (3.24) tested for

their activity as AChE, BuChE, and Aβ aggregation inhibitors (6).

42

3.4.1 Cholinesterase inhibition studies

The inhibitory effects of 12 novel sym-triazine derivatives (Fig. 3.14) on AChE activity was

characterized using Ellman's colorimetric assay (6, 91, 116, 167, 168), in which AChE-catalyzed

hydrolysis was initiated in the presence of chromogenic agent, 5,5'-dithiobis (2-nitrobenzoic

acid) (DTNB). Enzyme activity was determined by comparing the rate of substrate hydrolysis

(ΔOD410/min) in the presence or absence of inhibitor. The AChE IC50 values for all sym-triazines

are reported in Fig. 3.15, and correspond to the concentration of inhibitor that resulted in a 50%

reduction of enzyme activity. Clinically established cholinesterase inhibitor (ChEI), Donepezil,

was implemented as a positive control for AChE inhibition. The AChE IC50 for Donepezil was

determined to be approximately 0.02 µM, in agreement with previously reported values (116,

169).

All quaternary amines exhibited drastic improvements to inhibition with increasing arm

substitutions, with tri-substituted compounds, 3.20 and 3.23, resulting in the most profound

inhibition of AChE activity (IC50 0.3 µM and 2.8 µM, respectively) (6). A negative control

lacking the acetylcholine-like substitutions, 3.24 (Fig. 3.14), showed lower activity (IC50 of

153.3 µM) than the least effective quaternary-amine-based sym-triazine, 3.18 (IC50 38.8 µM),

thus emphasizing the relevance of positive charge in addition to acetylcholine-like structures for

enhancing targeted inhibition of AChE. Analysis of BuChE IC50 (Table 1) indicated that all

compounds possessed substantially higher AChE selectivity (i.e. lower IC50), suggesting that

interaction with the PAS was critical to sym-triazine-mediated cholinesterase inhibition. The

most effective BuChE inhibitors, 3.20 and 3.23, exhibited similar activity (BuChE IC50 3.9 µM

and 15.3 µM, respectively) to Donepezil (BuChE IC50 4.0 µM) (6).

3.4.2 Inhibition of Aβ1-40 Aggregation

Targeting of Aβ aggregation was achieved primarily through the addition of multiple aromatic

phenyl groups proximal to the triazine core (6). Thus, a significant reduction of Aβ1-40

aggregation was observed by ThT fluorescence, as the number of phenyl-containing substitutions

was increased between mono- and tri-substituted derivatives. This measured activity was

substantially greater than established penta-peptide-based β-sheet breaker, iAβ5p (58.5 %). ThT

studies also confirmed that Donepezil exhibited a near negligible effect on amyloid aggregation

43

(6.9%), further highlighting the significance of rational sym-triazine functionalization for β-sheet

targeting (6).

Transmission electron microscopy (TEM) studies confirmed the inhibition of fibril formation

using quaternary amine-salt converted sym-triazines 3.19, 3.22, 3.20, 3.23 as well as iAβ5p,

measured after 4 days of incubation (6). Aβ1-40 controls showed distinct fibrillar networks of

defined elongated structure in the absence of sym-triazines. As in fluorescence studies, Donepezil

appeared to demonstrate no observable effect on the fibril elongation process. While in the

presence of known inhibitor, iAβ5p, a mixture of both globular aggregates and fibrils was

observed, suggesting incomplete inhibition concurrent with fluorescence data. Conversely, Aβ1-40

samples containing sym-triazines exhibited only globular features lacking a defined fibrillar

component (6).

Table 1: Analysis of AChE, BuChE, and Aβ inhibition in vitro by sym-triazine derivatives.

aMeasured with 100 µM Aβ1-40 incubated with 200 µM (2x) or 100 µM (1x) inhibitor ([i]) at 37

oC for 72 h in 50 mM PBS (pH 7.4) (6).

Compound

Yield

(%)

IC50

AChE

(µM)

IC50

BuChE

(µM)

IC50 ratio

BuChE/AChE

%

Inhibition

Aβ fibrils

2x[i]a

%

Inhibition

Aβ fibrils

1x[i]a

3.12 68.6 89.8 ± 2.9 > 200 - 48.3 5.5

3.15 41.6 56.4 ± 1.7 > 200 - 46.6 6.6

3.18 88.0 38.8 ± 1.4 > 200 - 28.8 34.2

3.21 74.2 18.4 ± 1.3 > 200 - 25.2 31.3

3.13 53.5 171.5 ± 8.6 > 200 - 71.3 42.3

3.16 34.2 110.5 ± 4.3 > 200 - 88.2 58.4

3.19 38.7 20.3 ± 1.5 > 200 - 75.2 41.8

3.22 44.4 9.7 ± 0.4 > 200 - 95.2 65.6

3.14 17.0 9.8 ± 0.2 13.9 ± 0.3 1.4 67.9 53.2

3.17 64.4 80.8 ± 2.9 163.8±

7.7

- 87.4 68.5

3.20 68.0 0.3 ± 0.02 3.9 ± 0.2 13.0 70.2 50.9

3.23 89.1 2.8 ± 0.1 15.3 ± 0.9 5.5 89.9 72.1

3.24 82.1 153.2 ± 12.7 > 200 - - -

Donepezil 23.0 0.02 ± 0.004 4.0 ± 0.4 200.0 - -

iAβ5p - - - - 58.5 28.6

44

3.4.3 Cell Viability Studies

Collective analysis of all tested sym-triazine compounds revealed that 3.20 and 3.23 distinctly

exhibited high activity in regards to multi-targeted inhibition of both AChE and Aβ pathologies

(6). 3.20 and 3.23 possess the maximum number of quaternary amine-derivatized acetylcholine

substitutions and thus served as effective structures to be used for comprehensive assessment of

the sym-triazines impact to live cells. Propidium iodide (PI) exclusion assay demonstrated that

the viability of differentiated SH-SY5Y human neuronal cells was not affected with treatment of

up to 400 µM of 3.20 and 3.23, demonstrating a tolerable threshold well above the dosages

required in this study for effective inhibition of AChE activity. Fluorescence microscopy

demonstrated the absence of cell death morphology in differentiated human neuronal cells

treated with 400 µM of 3.20 and 3.23 compared to controls in which cell death was induced by

toxic levels of celastrol (170). The results further emphasized the applicability of these sym-

triazines to biological samples, and demonstrating a promising potential for further preclinical

investigations (6).

3.5 Conclusion

Multi-targeted drug therapies are an innovative approach to address the complex and inter-

related nature of AD pathologies (6). We presented the structure-activity studies of a novel class

of sym-triazine-based therapies for AD capable of multi-targeted inhibition of both AChE

activity and Aβ aggregation in vitro. Rational optimization of triazines resulted in progressive

enhancement of activity through successive derivitization. Improved targeting of AChE was

achieved through direct incorporation of structural analogues of the native substrate, ACh.

Additional directed inhibition of Aβ self-assembly was achieved via integration of multiple,

hydrophobic phenyl units that disrupted π-π stacking. Several di-substituted and tri-substituted

sym-triazine derivatives possessed comparable or greater activity compared to several

commercially available inhibitors in regards to the modulation of both AChE and Aβ activity.

Compounds 3.22, 3.14, 3.20 and 3.23 demonstrated a mixed-type mechanism of inhibition and

high PAS affinity, which has been associated with the capacity to impede AChE-accelerated Aβ

fibril formation. Cell viability studies showed that 3.20 and 3.23 were well tolerated by

differentiated human neuronal cells. Our design of sym-triazines with ACh-like substitutions has

45

generated hybrid molecules that possess multiple beneficial activities to be used as potential

candidates for AD therapy (6).

46

Chapter 4 Future Directions and Conclusion

4 Future Directions and Conclusion

We have achieved promising results in our lab as a starting point for the development of multi-

target-directed compounds for AD. Our sym-triazine ester-salt compounds 3.20 and 3.23 were

the most potent inhibitors of Aβ aggregation, AChE and BuChE overall. These compounds can

be further analyzed for its antioxidant properties and its ability to inhibit AChE-induced Aβ

aggregation. Moreover, we hope to conduct in vivo studies on these compounds to test their

blood brain barrier permeability in animal models.

In order to develop compounds that could hit multiple biological targets involved in AD

pathology, other pharmacophores can be incorporated into the sym-triazine scaffold. Chemical

moieties that can target the issues with tau hyperphosphorylation, the MAO system, and Ca2+

dyshomeostasis will have a significant potential in AD therapy (29).

N-phenylamine compounds have been shown previously to inhibit toxic tau aggregates in vitro

studies (171) and can be a moiety to be incorporated in the sym-triazine core along with the

ester-salt chains. This novel compound 4.2 can potentially have multi inhibition properties

against AChE, BuChE, Aβ aggregation and tau protein aggregation (Fig. 4.1). 4,6-dichloro-N,N-

diphenyl-1,3,5-triazin-2-amine (4.1) was synthesized in our lab (Fig. 4.2) using similar procedure

used to synthesize compound 3.5 although a stronger base, sodium carbonate was employed

instead of sodium bicarbonate. The compound can be further derivatized to form 4.2 via sym-

triazine based acid, acyl chloride, and ester intermediates.

Fig. 4.1: Structure of 4.2 to be developed in order to possess AChE, BuChE, Aβ aggregation

and tau protein aggregation inhibitor properties.

47

Fig. 4.2: Synthesis of 4.1 using diphenylamine. Reaction was carried out at 0ºC and sodium

carbonate (Na2CO3) was employed as a base and acetone as solvent.

The propargylamine moiety discussed in Chapter 2 was shown in research studies to reside MAO

inhibition properties (29, 123). Adding this phamacophore to the sym-triazine core that also

consists of the ester-salt chains, can help target other neurotransmitter systems in addition to

ACh, which are involved in AD pathology (29). This novel compound 4.4 can potentially have

multi inhibition properties against AChE, BuChE, MAO and Aβ aggregation (Fig. 4.3). The

novel compound 4.3 was synthesized using similar procedure used for the synthesis of 3.5

although the base employed was triethylamine instead of sodium bicarbonate. 4.3 will be used to

undergo acidification, acylation, and esterification intermediate reactions to synthesize the final

4.4 compound for inhibition studies.

Fig. 4.3: Structure of 4.4 to be developed in order to possess AChE, BuChE, MAO and Aβ

aggregation inhibitor properties.

48

Fig. 4.4: Synthesis of 4.3 using propargylamine. Reaction was carried out at 0ºC and

triethylamine ((C2H5)3N) was employed as a base and acetone as solvent.

Dihydropyrdine compounds have demonstrated neurprotective effects by inhibiting VDCCs (29,

137). Hence, adding the dihydropyridine core in the sym-triazine core that also incorporates the

ester-salt chains can be synthesized (compound 4.5) in order to develop a MTDL for AChE,

BuChE, Aβ aggregation, and VDCC inhibitor (Fig. 4.5).

Fig. 4.5: Structure of 4.5 to be developed in order to possess AChE, BuChE, Aβ aggregation,

and VDCC inhibition properties.

MTDLs are the best approach for the treatment of AD (29). The multifactorial nature of AD,

which includes biological factors such as the cholinergic system (55), Aβ aggregation (18),

formation of toxic NFTs (21), imbalance in Ca2+

homeostasis (20), oxidative stress (19), and

activation of the microglia cells (22), make it challenging to treat through single-drug therapy

(29). Moreover, the one-molecule-one target paradigm in single-drug therapy involve side effects

arising from different bioavailabilites, metabolism, and pharmacokinetics of different drugs that

can be obviated with the use of MTDLs (29, 113, 114). Our lab has synthesized novel sym-

triazine ester-salt compounds incorporating ACh-like substrate analogues and hydrophobic

phenyl moieties through cost-efficient synthetic procedures. These compounds have shown to act

as efficient AChE, BuChE and Aβ aggregation inhibitors in vitro making it a MTDL for AD.

49

They were also well tolerated in human neuronal cell lines indicating that they are non-toxic. We

believe these novel compounds have the potential to be lead drug candidates for pharmaceutical

analysis in the near future. These compounds can be tested further for its AChE-induced Aβ

inhibition activity, antioxidant properties and blood brain barrier permeability. Further

modulation of these sym-triazine compounds will be conducted in our lab to incorporate different

pharmacophores in order to target different biological factors. We hope to develop and analyze a

library of novel sym-triazine compounds that can have a huge impact in AD research and

improve the quality of life of AD patients and their caretakers.

50

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Appendices

Appendix A: 1H-Nuclear Magnetic Resonance (NMR) Spectra of

sym-Triazine Derivatives

A.1. 1H-NMR spectrum of 3.18

61


62


63


64


65


66


67

Appendix B: 13

C-Nuclear Magnetic Resonance (NMR) Spectra of

sym-Triazine Derivatives

B.1. 13

C-NMR spectrum of 3.12

68

B.2. 13


69

B.3. 13


70

B.4. 13


71

B.5. 13


72

B.6. 13


73

B.7. 13


74

B.8. 13


75

Appendix C: Mass Spectra (MS) of sym-Triazine Derivatives

C.1. MS of 3.18

76

C.2. MS of 3.21

77

C.3. MS of 3.19

78

C.4. MS of 3.14

79

C.5. MS of 3.17

80

C.6. MS of 3.20

81

C.7. MS of 3.23

82

Appendix D: Infrared (IR) spectra of sym-Triazine Derivatives

D.1. IR spectrum of 3.18

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