“Design, Synthesis and Biological Evaluation of Novel Coumarin Derivatives Targeting Acetylcholinesterase As Neuroprotective Against Alzheimer's Disease”

“Design, synthesis and biological evaluation of novel coumarin derivatives targeting acetylcholinesterase as neuroprotective against Alzheimer's disease”

Thesis Presented by Haidy Hany Abdel Momen Abdel Hamid El-Zoheiry B.Sc., Faculty of Pharmacy, Cairo University (2013) Submitted in Partial Fulfillment of Master Degree In Pharmaceutical Sciences (Pharmaceutical Chemistry)

Under the supervision of

Prof. Dr. Kamilia Mahmoud Amin Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University

Prof. Dr. Doaa Ezzat Abdel Rahman Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University

Dr. Heba Abdel-Rasheed Allam Lecturer of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University

Cairo University (2019)

Abstarct Design, synthesis and biological evaluation of novel coumarin derivatives targeting acetylcholinesterase as neuroprotective against Alzheimer's disease

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by loss of memory and cognitive abilities. According to cholinergic hypothesis, reduced levels of acetylcholine (ACh) is contributed significantly to the cognitive symptoms associated with AD and advanced age. Accordingly, the mainstream direction for AD therapy is slowing the breakdown of ACh by the use of acetylcholinesterase inhibitors (AChEIs).

Primarily, AChEIs have been considered only as a symptomatic therapy for AD, however recent studies have suggested that AChEIs can act as disease- modifying agents by inhibition of the amyloid cascade.

Coumarin is an interesting bioactive scaffold eliciting a wide range of biological activities including anticancer, antibacterial, anti-inflammatory and anticoagulant. A structural survey of AChEIs revealed that a number of naturally occurring and synthetic coumarin analogues exhibited potent AChE inhibitory activity. Among these inhibitors AP2238 and ensaculin which have distinct scaffold with a coumarin moiety. Moreover, it has been demonstrated that AChEIs with coumarin moiety primarily interact with the peripheral anionic site (PAS) of AChE. These findings prompt medicinal chemists to design dual inhibitors of AChE by incorporating a catalytic site interacting moiety with coumarin through an appropriate linker. In this regard, we highlighted on the AChE inhibitory activity of the coumarin nucleus notably 7-benzyloxycoumarin derivatives.

In this work, novel 7-benzyloxycoumarin based compounds were synthesized with a variety of bioactive chemical fragments which possess AChE

2 inhibitory activity. Moreover, in vitro acetylcholinesterase inhibition study was conducted according to modified Ellman’s method besides scopolamine induced dementia in vivo assay on the most active compounds. Furthermore, in silico studies were performed on the synthesized compounds which included molecular docking study at the active site of recombinant human acetylcholinesterase enzyme (rhAChE) as well as prediction of ADMET and pharmacokinetic parameters.

The thesis includes the following sections: 1. Introduction This section presents a brief literature review about Alzheimer’s disease, its pathogenesis and treatment besides a review on acetylcholinesterase and its inhibitors. In addition, a survey on the different biological activities of coumarin compounds with special emphasis on their acetylcholinesterase inhibitory activity is included.

2. Aim of the work This section illustrates the scientific basis upon which the synthesized compounds were designed.

3. Theoretical discussion This section deals with the discussion of the experimental methods adopted for the synthesis of the designed compounds in addition to a summarized data for the characterization and verification of the new compounds structures using different spectroscopic methods.

4. Experimental part This section describes the practical procedures used for the synthesis of the published compounds, the new intermediates and the new final compounds. In

3 addition, physical, elemental analyses and spectral data (IR, 1H-NMR, 13C-NMR and mass spectroscopy) are cited.

Published compounds:  7-Hydroxy-4-methyl-2H-chromen-2-one (I)  7-Benzyloxy-4-methyl-2H-chromen-2-one (II)  7-Benzyloxy-2-oxo-2H-chromene-4-carbaldehyde (III)  2-(7-Hydroxy-2-oxo-2H-chromen-4-yl)acetic acid (X)  Methyl-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (XI)  2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)acetohydrazide (XIII)

New intermediate compounds:  1-[(7-Benzyloxy-2-oxo-2H-chromen-4-yl)methylene]thiosemicarbazide (IV)  Methyl 2-(7-benzyloxy-2-oxo-2H-chromen-4-yl)acetate (XII)

New final compounds:  7-Benzyloxy-4-{[(4-methylthiazol-2(3H)-ylidene)hydrazono]methyl}-2H- chromen-2-one (V)  7-Benzyloxy-4-{[(4-phenylthiazol-2(3H)-ylidene)hydrazono]methyl}-2H- chromen-2-one (VIa)  7-Benzyloxy-4-({[4-(4-bromophenyl)thiazol-2(3H)-ylidene]hydrazono} methyl)-2H-chromen-2-one (VIb)  7-Benzyloxy-4-({[4-(4-methoxyphenyl)thiazol-2(3H)-ylidene]hydrazono} methyl)-2H-chromen-2-one (VIc)  Ethyl-2-{2-[(7-benzyloxy-2-oxo-2H-chromen-4-yl)methylene]hydrazono} -4-methyl-2,3-dihydrothiazole-5-carboxylate (VII)  2-{2-[(7-Benzyloxy-2-oxo-2H-chromen-4-yl)methylene]hydrazono} thiazolidin-4-one (VIII)  2-(2-{[(7-Benzyloxy-2-oxo-2H-chromen-4-yl)methylene]hydrazono}-4- oxothiazolidin-5-yl)acetic acid (IX)

 2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)-N-(2,5-dioxopyrrolidin-1- yl)acetamide (XIVa)  2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)-N-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)acetamide (XIVb)  2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)-N-(1,3-dioxoisoindolin-2- yl)acetamide (XIVc)  1-[2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)acetyl]-4-methyl thiosemicarbazide (XVa)  1-[2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)acetyl]-4-phenyl thiosemicarbazide (XVb)  2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)-N’-(3-methyl-4-oxothiazolidin-2- ylidene)acetohydrazide (XVI)  2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)-N-(2-methylimino-4- phenylthiazol-3(2H)-yl)acetamide (XVIIa)  2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)-N-[4-(4-methoxyphenyl)-2- methyliminothiazol-3(2H)-yl]acetamide (XVIIb)  4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]-7- benzyloxy-2H-chromen-2-one (XVIII)  1-[2-(7-Benzyloxy-2-oxo-2H-chromen-4-yl)acetyl]-3-methyl-1,2- dihydropyrazol-5-one (XIX)  7-Benzyloxy-4-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethyl]-2H-chromen- 2-one (XX)  5-Amino-1-[2-(7-benzyloxy-2-oxo-2H-chromen-4-yl)acetyl]-1H-pyrazole-4- carbonitrile (XXI)

5. Biological evaluation This section includes the procedures and discussion of the results of the in vitro acetylcholinesterase inhibitory activity against donepezil as a reference drug

5 in addition to scopolamine induced dementia in vivo assay on the most active compounds VIa, XVIIa and XXI to investigate their behavior study.

6. Molecular modeling studies 6.1. Molecular docking study This section includes molecular docking of the synthesized compounds on the active site of acetylcholinesterase to explore their binding mode of interaction as well as their amino acids binding interactions.

6.2. Physicochemical, ADMET and pharmacokinetic properties prediction This section includes prediction of distribution and toxicity parameters of the synthesized compounds where most of the compounds explored good pharmacokinetic profile.

7. References This part includes 221 references covering the period of 1904 to 2019.

8. Arabic summary

Introduction 1.1. Alzheimer’s disease: Alzheimer’s disease (AD) was identified more than 100 years ago when Alois Alzheimer first published a case of "presenile dementia" in a female patient named Auguste Deter who died in a completely demented state. AD is a neurodegenerative brain disease which is the most common cause of dementia and is ultimately fatal.(1)

Dementia is characterized by a decline in memory, language, problem solving and other cognitive skills that affects a person’s ability to perform everyday activities. This decline occurs because nerve cells in parts of the brain involved in cognitive function have been damaged. The damage of neurons eventually affects other parts of the brain including those that enable a person to carry out basic bodily functions such as walking and swallowing. Unfortunately, people in the final stages of this disease are bed-bound and require around-the- clock care.(2)

It is well known that, the primary risk factors of AD are aging, family history and genetics. According to Alzheimer’s Association annual report in 2018, 10% of people older than 65 years old and 50% of people older than 85 years old are easily affected by AD. Apart from these statistics, people younger than 65 years old may be affected by Familial Alzheimer’s disease (FAD) in which gene mutations are the foremost cause.(3)

1.2. Pathology of Alzheimer’s disease: The two most common hypotheses used to describe the pathology of AD are known as "amyloid hypothesis" and "cholinergic hypothesis".(4)

1.2.1. Cholinergic hypothesis: The cholinergic hypothesis was formulated approximately 30 years ago. It stated that a serious loss of cholinergic function in the central nervous system

(CNS) contributed significantly to the cognitive symptoms associated with AD and advanced age. This loss in cholinergic function is due to deficiency in choline acetyltransferase enzyme (ChAT) which is responsible for the synthesis of acetylcholine (ACh) in addition to the reduction of choline uptake and ACh release. Thus, a sharp decrease in the neurotransmitter ACh in brain synapses specifically in amygdala and hippocampus was the most consistent neurochemical finding in diseased brains that lead to the cholinergic hypothesis.(11,12) It is well known that ACh has an important role in the enhancement of different types of encoding of new memories in many cortical structures. This role is mediated by muscarinic and nicotinic ACh receptors. Thus, ACh plays crucial role in cognitive impairment in memory and intellectual ability to perform basic activities of daily living associated with AD.

Owing to the aforementioned role, restoring ACh levels in brain synapses is considered the most effective symptomatic treatment for AD that can be achieved by the use of acetylcholinesterase inhibitors (AChEI).(15) Acetylcholinesterase (AChE) is a hydrolase enzyme involved in the termination of impulse transmission at cholinergic synapses by rapid hydrolysis of ACh in the central and peripheral nervous systems. AChEIs inhibit the hydrolysis of ACh improving both the level and duration of the neurotransmitter.(15) It is clear from 3D crystallographic structure of AChE that it possesses two binding sites. First, a regulatory site called the peripheral anionic binding site (PAS) that is rich in hydrophobic residues and is located at the rim of a 20 Å deep narrow gorge on the surface of the enzyme.(18,19) Second, the active center site called catalytic active binding site (CAS) or primary site contains the catalytic triad of three amino acids: Ser203, His447, and Glu334 where acetylcholine is

8 hydrolyzed to acetate and choline. This active site is located at 4 Å from the bottom of the enzyme. Most of the reported AChEI interact with PAS or CAS or both. Compounds such as tacrine 1 act exclusively at the CAS, whereas others such as propidium 2 act at the PAS only. On the other hand dual binding AChEIs, that are molecules able to interact simultaneously with both the PAS and CAS of the enzyme such as donepezil 3.(22,23)

1.3. Current therapeutic strategies: There are two main strategies in the symptomatic treatment of AD and slowing its progression. The first strategy includes restoring the levels of neurotransmitters in brain synapses by using acetylcholinesterase inhibitors (AChEI), monoamine oxidase inhibitors (MAOI) and glutaminergic N-methyl-D-aspartate receptor (NMDAR) antagonists which are helpful in relieving the symptoms and improving cognitive functions.(27) On the contrary, the second one includes the inhibition of Aβ aggregates by using β-secretase and γ-secretase inhibitors and the use of α-secretase promoters or the inhibition of tau tangles aggregation or hyperphosphorylation which are helpful in abolishing the processes responsible for disease progression.(28) Up to date studies in AD therapy has been at least partly successful in developing symptomatic treatments but has also had several failures in developing disease-modifying therapies.(8)

1.3.1. NMDA receptor antagonists: Glutamate is the most abundant excitatory neurotransmitter in the mammalian CNS. One subgroup of ligand-gated ionotropic glutamate receptors is selectively gated by specific agonist known as N-methyl-D-aspartate (NMDA). Excitatory glutamatergic neurotransmission via NMDAR is critical for synaptic plasticity and survival of neurons. However, excessive NMDAR activity causes excitotoxicity and promotes cell death that occurred in AD which can be blocked by an NMDAR antagonist.(29)

Memantine 4 is a FDA approved drug acting as NMDAR antagonist. It was thought to reduce NMDAR mediated neurotoxicity so it is indicated for moderate to severe AD treatment. Memantine has a statistically significant effect on cognition, behaviour and ability to perform activities of daily living. Although memantine provides symptomatic relief, it does not modify disease progression.(26)

1.3.2. Monoamine oxidase inhibitors (MAOIs): Monoamine oxidases (MAOs) are a protein family of flavin-containing amine oxidoreductases that play an important role in the regulation and metabolism of biogenic amines by oxidative deamination. There are two MAO isoenzymes, MAO-A; the primary type in fibroblasts preferentially degrades serotonin, norepinephrine and dopamine and MAO-B; found in platelets but also in the brain which preferentially degrades phenylethylamine and benzylamine.(31)

MAOIs could be useful in the treatment of psychiatric and neurological diseases such as AD by restoring neurotransmitters level in the brain. In addition, part of the biochemical activity of MAO results in higher levels of H2O2 and oxidative free radicals which are a possible source of oxidative stress for

10 vulnerable neurons affected by AD. Thus MAO-B inhibitors could be used in the treatment of AD and other neurological disorders such as Parkinson’s disease while MAO-A inhibitors could be used as antidepressants and antianxiety agents.(32) Recently, selegiline or L-deprenyl 5 and rasagiline 6, irreversible selective human MAO-B inhibitors were used in the treatment of Parkinson’s disease, are used as a part of multitarget therapy of AD.(33)

1.3.3. Anti-amyloid drugs: 1.3.3.1. β-secretase inhibitors:

The druggability of BACE-1 was first demonstrated with the development of substrate-based pseudopeptide inhibitors and subsequent determination of the X-ray structure of inhibitors bound BACE-1 which provided important drug design templates as well as a wealth of information regarding the enzyme and inhibitors interactions in the BACE-1 active site.(6) CNP-520 7, a small molecule BACE-1 inhibitor suitable for AD prevention. It has the ability to reduce brain and cerebrospinal fluid (CSF) Aβ in rats and dogs in addition to Aβ plaque deposition in APP-transgenic mice. It also has in vitro

IC50= 11 nM on human BACE-1. Animal toxicology studies of CNP-520 demonstrated sufficient safety margins with no signs of serious side effects.(35)

Similarly, AZD3839 8, a small-molecule BACE-1 inhibitor with Ki= 26 nM. Phase I clinical trials with healthy volunteers after a single oral dose of up to 300 mg showed rapid absorption of AZD3839 followed by reduction by 57.8% for Aβ (1-40) and 39.3% for Aβ (1-42). This drug showed no deaths or serious adverse effects.(36) 1.3.3.2. γ-Secretase inhibitors:

A literature review revealed compound NGP555 9 which was designed and evaluated as a γ-secretase inhibitor. NGP555 has good oral absorption, brain penetration and CNS activity. NGP555 demonstrates in vivo efficacy for lowering the biomarker Aβ (1-42) in rodent studies for brain, CSF and plasma. Additionally, chronic administration of NGP555 lead to significant prevention of cognitive deficits in Y-maze performance and in the Morris water maze test in a transgenic mouse model.(40)

1.3.3.3. α-Secretase activators:

In 2008, Marcade et al. (45) discovered that etazolate (EHT-0202) 10, a selective gamma-aminobutyric acid (GABAA) receptor modulator, stimulates neuronal α-secretase and increases sAPPα production. Etazolate is orally bioavailable and has been recently tested in a phase II clinical trials in patients with mild-to-moderate AD. In vivo and in vitro data showed 20% reduction in total Aβ (1-40).

1.3.4. Acetylcholinesterase inhibitors (AChEIs): The first successful development of AChEIs was tacrine 1, 9-amino-1,2,3,4- tetrahydroacridine, a reversible nonselective AChE/BuChE inhibitor with IC50= 590 nM against AChE. It was the first drug approved by FDA in 1993 for the treatment of AD. Unfortunately, it was withdrawn soon after reports of liver toxicity. Tacrine opened up a pathway to design cholinergic drugs for AD treatment.(3)

Similarly, edrophonium 11, a 3-hydroxy-N,N-dimethylanilino derivative, was emerged as a very short-acting reversible inhibitor of AChE interacting with CAS only. Because of its short term effects, it is primarily used in a diagnostic test for the confirmation of myasthenia gravis.(53)

On the other hand, donepezil 3 was the first of a new class of noncovalent reversible AChEI approved by FDA for treatment of dementia associated with AD. It is a piperidine-based AChEI which act as a dual binding site inhibitor that binds to PAS and CAS sites in the enzyme. Donepezil exhibits a relatively high degree of tissue selectivity because it inhibits AChE in the brain at doses that exert little effect on intestinal or cardiac AChE. It also displays a significant selectivity for AChE over BuChE.(54)

Aim of the work Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to loss of memory and cognitive ability. It was estimated that more than 18 million people suffer from AD worldwide and the number of patients will be increased to 70 million by 2050.(96) Moreover, AD is officially listed as the sixth- leading cause of death in the United States according to Alzheimer’s association report in 2018.(97)

Since a cholinergic hypothesis has been postulated as a crucial element in AD symptoms etiology, AChE inhibitors (AChEIs) have constituted until today the main drugs used against AD. It seems however necessary today to complete this symptomatic approach with a more disease-modifying one, these two activities being able to be associated in a single chemical compound such as multi-target directed ligands (MTDL). Among them, dual binding site AChEIs appear as promising anti-AD agents. In fact, they have the potential to restore the cholinergic deficit by inhibiting the CAS of AChE and at the same time to reduce the Aβ deposition and aggregation through their interaction with the PAS of the enzyme.(98) Therefore, the design of selective dual binding site AChEIs could represent a successful therapeutic strategy for the symptomatic treatment of AD and its progression.

Coumarin, 1H-benzopyran-2-one, is a core structure in various natural products and synthetic compounds displaying a diverse array of biological activities including acetylcholinesterase inhibitory activity. Moreover, the fact that chemical substitutions can occur at many positions of this core structure, has made coumarins interesting molecules for drug discovery in the field of AChEIs.(99)

Based on the above cited findings and with the aim to identify compounds with potent AChE inhibitory activity and safety profile for AD, it was considered valuable to present new compounds as promising potent AChEIs, having the 7-

14 benzyloxycoumarin substituent with different distinct heterocycles at position 4 connected together with an appropriate linker which stabilize the molecule inside the gorge of AChE enzyme and adjust the distance between PAS and CAS binding moieties. 1.4. AD and AChE inhibitory activity of thiosemicarbazone and acyl substituted thiosemicarbazide derivatives of coumarins: In search for the functional moieties of anti-AD importance, literature review revealed the contribution of some thiosemicarbazide fragments to the AChE inhibitory activity of compounds bearing them. For example, compound 12. So, it was of interest to design and synthesize thiosemicarbazone derivative IV and acyl substituted thiosemicarbazide derivatives XVa,b as potential anti-AChE.

1.5. AD and AChE inhibitory activity of thiazolyl coumarins: Thiazole ring is an important pharmacologically active heterocylic ring whose mono-, di-, tri-substituted, reduced analogues (thiazoline, thiazolidine) and thiazolidinone analogues have been widely studied in medicinal chemistry. It is worthy to mention that various thiazolines as compounds 13 and 14 with %

(105) (106) inhibition= 49.92 μg/mL and IC50= 6.62 μM, respectively, demonstarated

15 an efficient AChE inhibitory activity.

Moreover, from structural point of view versatile linkers especially nitrogenous linkers were incorporated in potent AChEIs. Among these linkers hydrazonomethyl linker as compound 15 (107), acetamide linker as compound 16.(88)

Consequently, it was thought worthwhile to incorporate thiazoline or thiazolidin-4-one moieties with hydrazonomethyl, acetamide or acetohydrazide linkers in compounds V, VIa-c, VII, VIII, IX, XVI and XVIIa,b, hoping to enahance the total biological activity as AChEIs.

Y R1 R2

V CH=N-N H CH3

VIa CH=N-N H C6H5

VIb CH=N-N H C6H4Br

Y R1 R2 VIII CH=N-N H H

IX CH=N-N H CH2COOH

XVI CH2CONHN CH3 H

1.6. AD and AChE inhibitory activity of cyclic imides of coumarins: In the same vein, literature review indicated that cyclic imide moieties such as phthalimide and succinimide heterocycles have a significant effect on AChE inhibitory activity owing to their interaction with Trp86 via π-π stacking

(109) (110) interaction as compounds 17 and 18 with IC50= 1.35 μM and <0.1 μg/mL, respectively.

Accordingly, it deemed of interest to design compounds combining cyclic imides as derivatives XIVa-c and evaluate their AChE inhibitory activity.

Review This thesis includes literature review about 7-benzyloxycoumarins and their mechanisms related to acetylcholinesterase inhibition from previous studies. The present study deals with the synthesis of some 7-benzyloxy-4-substituted coumarins aiming to obtain new AChEIs. This was based on previously reported similar compounds reported in different studies. The newly synthesized compounds were afforded through different schemes, all illustrated through the thesis. The newly synthesized compounds were confirmed by their spectral data. Finally, the newly synthesized compounds were evaluated for their AChE inhibition assay method and the most active ones were evaluated for in vivo scopolamine induced dementia model. Moreover, a molecular modeling study was performed to figure out the interaction of some selected new compounds with the active site of AChE and predict their physicochemical, ADME and pharmacokinetic properties.

Summary The present study deals with the synthesis of some 7-benzyloxycoumarins starting which were converted into thiosemicarbazide IV and acetohydrazide XIII intermediates. The key intermediate thiosemicarbazone IV reacts with chloroactone, phenacyl bromides and ethyl 2-chloroacetoacetate to yield thiazoline derivatives V, VIa-c and VII while reactions with ethyl bromoacetate and maleic anhydride give thiazolidinone derivatives VIII and IX. Moreover, the acetohydrazide intermediate XIII reacts with cyclic anhydrides to yied Pyrollidine, dihydro pyrollidine 2,5-dione and isoindoline-1,3,-dione derivatives XIVa-c and with ethyl acetoacetate, acetylacetone and ethoxymethylene malononitrile to give derivatives XIX, XX and XXI, respectively.

The target compounds were evaluated for their AChE inhibition assay compared to donepezil. Candidates showing the lowest IC50 values were further

18 screened for their in vivo scopolamine-induced dementia model. Molecular modelling study was performed to some most active new compounds. Furthermore, ADMET study assigned their pharmacokinetic data.

Conclusion  A novel series of twenty coumarin derivatives were screened for their in vitro anticholinesterase activities and in vivo model using Y-maze and passive avoidance tests.

 In vitro assay revealed that compounds VIa (IC50= 0.451 μM), XXI (IC50=

0.487 μM) and XVIIa (IC50= 0.505 μM) exhibited promising AChE inhibitory

activity even better than donepezil (IC50= 0.668 μM).  In vivo evaluation of compounds VIa, XVIIa and XXI confirmed significant memory improvement in scopolamine-induced impairment model.  Docking of the synthesized compounds into AChE active site reproduced binding interactions like that of the native ligand donepezil.

 ADMET prediction results revealed that the final target compounds possess good lipophilicity, moderate penetration to the BBB and good CNS

permeability values.