investigating the synthesis of a sulphur analogue of 4-benzyloxyphenylacetic acid as a potential...

Investigating the Synthesis of a Sulphur Analogue

of 4-benzyloxyphenylacetic acid as a Potential

Inhibitor of hBCAT in Alzheimer’s Disease

Dennie Sebastian

BSc (Hons) Biomedical Sciences 2016

13008271

Department of Biological, Biomedical and Analytical Sciences,

University of the West of England, Bristol

2016

USSJ73-40-3

This submitted project is my work, it contains no unreferenced or unacknowledged verbatim extracts from the works of others and it has not (either in whole or in part) been submitted towards the award of any other award either at UWE or elsewhere. Signed ....................................................... Date 17/03/16

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I give permission for UWE Library Services to hold and make available an electronic copy of this project /dissertation. Signed: Date: 17/03/16 E-mail address: [email protected]

mailto:[email protected]

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Acknowledgments

I would like to express my deepest gratitude to my supervisor Dr Annabelle Hodson

for her guidance, encouragement and her continued support throughout this year; for

without your help I couldn’t have done it. Thank you for your patience and it has been

an honour undertaking your project this year. I would also like to take this opportunity

to sincerely thank all the technical staff for their constant support in the lab.

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Contents

CONTENTS 4

ABBREVIATIONS 5

ABSTRACT 7

1. Introduction 8

2. Experimental section 20

3. Results 25

4. Discussion 57

5. Further work 62

6. Conclusion 63

7. References 64

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Abbreviations

4-HAP: 4- Hydroxyacetophenone

Aβ: Amyloid beta

ACh: Aceytylcholine

AChE: Acetylcholinesterase

AChEI: Acetylcholinesterase inhibitor

AD: Alzheimer’s disease

aKG: a-ketoglutarate

APP: Amyloid precursor protein

APoE: Apolipoprotein E

BBB: Blood brain barrier

BCAT: Branched chain amino transferases

BCKA: Branched chain keto-acids

CNS: Central nervous system

COOH: carboxylic acid group

DABCO: 1,4-Diazabicyclo[2.2.2]octane

DMTC: N,N-dimethylthiocarbamoyl chloride

EAAT: Excitatory amino acid transporter

GC-MS: Gas chromatography- mass spectrometry

hBCATc: Human branched chain amino transferases (cytosolic isoform)

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hBCATm: Human branched chain amino transferases (mitochondrial isoform)

HCl: Hydrochloric acid

HSPG: Heparin sulphate proteoglycan moieties

IR: Infrared

L1- Large neutral amino acid transporter 1

NaOH: Sodium hydroxide

N-K rearrangement: Newman-Kwart rearrangement

NMDA: N-methyl-D-aspartic acid

PS: Pear shaped flask

PSEN1: Presenilin 1

PSEN2: Presenilin 2

QSAR: Quantitative Structure activity relationships

RB: Round bottom flask

RT: Retention time

SAR: Structure activity relationships

SA: V ratio: Surface area: volume ratio

TEBA: Triethyl benzyl ammonium chloride

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Abstract

Alzheimer’s disease is a chronic neurodegenerative disease that accounts for over

80% of senile dementia cases in the elderly worldwide, and is characterised by the

gradual and progressive deterioration in cognitive function including behavioural,

mental, and functional decline that impairs daily activities. A strong genetic

predisposition to the APOE gene has been linked by which an impairment of Aβ

clearance contributes to the pathophysiology of AD. A disruption to the glutamate

pathways involving the prolonged activation of the NMDA receptors, and the cascade

of events involving the influx of Ca2+ ions into the postsynaptic neurons leads to a

loss in synaptic function promoting excitotoxicity hence cause cell death. The

multifactorial characteristics of AD has made this difficult with current treatments only

providing symptomatic relief and not alleviate the disease itself. A distribution of

BCAT in the human brain has revealed that an overexpression of hBCATm in the

endothelial layer causing a disruption to glutamate homeostasis. Studies of 4-

benzyloxyphenylacetic acids investigated the effects of chemical inhibitors on the

hBCAT active site and computational studies proposed an alternative structure using

a sulphur analogue of the compound. 4-hydroxyacetophenone was reacted with N,N-

dimethylthiocarbamoyl chloride to yield an O-aryl thiocarbamate. This underwent the

Newman-Kwart rearrangement to produce the S-aryl thiocarbamate and further

hydrolysis reactions were used to yield 4-acetylthiophenol. These intermediates were

characterised using accurate analytical techniques to characterise functional groups

and to determine the molecular weight of structures. Further testing is now needed to

couple the second aromatic ring onto the ring, and saponification reactions to bind

the carboxylic acid group in place of the ketone to yield the final target compound.

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1. Introduction

Alzheimer’s disease is a chronic neurodegenerative disease that accounts for over

80% of senile dementia cases in the elderly worldwide (Anand et al., 2014). It is

characterised clinically by the gradual and progressive deterioration (fig 1.1) in

cognitive function including behavioural, mental, and functional decline that impairs

daily activities (Imbimbo et al., 2005). Morphologically, brain atrophy as well as

enlarged cerebral ventricles are observed in AD sufferers and Braak staging uses

histology to show extracellular deposits of cerebral plaques composed of dense

proteinaceous core containing Amyloid Beta (Aβ) peptides and intracellular

neurofibrillary tau tangles (fig 1.2) (Imbimbo et al., 2005). Certain risk factors such as

head trauma, positive family history, depression, and diabetes mellitus,

hyperlipidemia and hypertension in midlife are found to increase the onset of AD in

later life (Kivilepto et al., 2001).

Figure 1.1. Graph to show the progression of Alzheimer’s disease symptoms over years. MMSE: Mini-Mental State Examination, MCI: Mild Cognitive Impairment, BADL: Basic activities of daily living

(Feldman and Woodward, 2005)

Graph to show the progression of symptoms present in Alzheimer’s disease sufferers

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1.1 Epidemiology of Alzheimer’s disease

The incidence and prevalence of AD increases with age and are higher in women

than men (Prince et al., 2013). The World Health Organisation (WHO) has reported

47.5 million cases of dementia worldwide with 60% of these cases being of AD

sufferers. Almost 7.7 million new cases are reported every year, equating to 14 new

cases approximately every minute. The incidence of AD is expected to increase to

135.5 million cases by 2050 (WHO, 2015). In the US alone, the mortality attributable

to AD is an estimated 700,000 in aged 65 or older signifying the need for the

development of medical breakthroughs to prevent or cure the disease (Alzheimer’s

Association, 2015).

Figure 1.2. Diagram to show the deposition of tau in Alzheimer’s disease using the Braak staging system.

A: Illustrations show the regional distribution of tau deposition using Braak staging. Neurofibrillary tangles found at the transentorhinal region of the brain (stage I-II) spread into the limbic areas (stage III-IV) and the isocortical regions of the brain (stage V-V1). Cognitive function deteriorates as the tau pathology increases. B: A sequential diagram of the neurocognitive changes that occur with the progression of tau deposition to named regions of the brain

(Villemagne et al., 2015)

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1.2 Genetic predisposition to Alzheimer’s disease

AD pedigrees have identified autosomal dominant mutations that have been found in

three genes including APP, PSEN1 and PSEN2 that collectively explain 5-10% of the

occurrence of early- onset AD (<60 years) (Cauwenberghe et al., 2015).

APOE is a widely studied gene found to have a strong genetic predisposition in AD.

APOE encodes for a polymorphic glycoprotein present in chromosome 19. The

APOE e4 allele has been associated with an increased risk of familial and sporadic

early-onset and late-onset AD (>60 years). Whilst only 20-25% of the general

population carries one or more of these alleles, an estimated 40-65% of AD patients

are carriers of e4 (Cauwenberghe et al., 2015).

APOE is predominantly found at astrocytes and microglial cells that covalently bond

to form lipoprotein particles (fig 1.3). These bind to isoform-dependent patterns to

form parenchymal amyloid plaques within the CNS. LDLR and LRP1 receptors aid

the cellular uptake of these plaques through the process of endocytosis of an APOE

complex bound to Aβ peptides. These peptides can retain within the CNS by binding

to HSPG moieties. Soluble Aβ from the interstitial fluid of the neuron is transported

into the blood stream where it crosses the blood brain barrier via P-glycoprotein and

LRP1. The impairment of Aβ clearance aids in the pathogenesis of AD (Barage and

Sonawane, 2015).

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1.3 Pathophysiology of Alzheimer’s disease

The aetiology of AD is multifactorial that includes genetic, environmental, behavioural

and developmental components that together contribute to the pathophysiology of

the disease (Anand et al., 2014). Theories of AD, such as amyloid cascade, tau and

cholinergic hypothesis, have been extensively studied but new studies have

highlighted the role of Aβ oligomers that cause synaptic impairment that destroy the

integrity of the brain (Kumar et al., 2015). These oligomers have been found to

interact with glutamatergic receptors of the NMDA receptors and with proteins

involved in glutamate homeostasis (Danysz and Parsons, 2012).

Figure 1.3. A schematic showing the role of APOE metabolism and plaque formation

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1.31 Glutamatergic system involvement in Alzheimer’s disease

Glutamate is a major excitatory neurotransmitter of cortical and hippocampal

pyramidal neurones that is involved in cognitive function. Glutamate and its receptors

are important processes involved in learning and memory (Francis, 2003). Under

normal physiological conditions, glutamate concentrations are found at low

micromolar ranges. Stimulation of a glutamatergic neuron will cause a transient

release of extracellular glutamate into the synapse increasing the glutamate

concentrations to a few milliseconds. This also stimulates the activation of

metabotropic and ionotropic glutamate receptors (fig 1.4) (Vandenberg and Ryan,

2013). This process is regulated by controls that re-uptake glutamate to glutamine

using the glutamine synthetase enzyme. For example, transporters (EAATs) that are

found in glial cells recycle synaptic glutamate into glutamine using glutaminase

enzyme and release it into the presynaptic neuron by passive diffusion in order to

maintain low and non-toxic glutamate concentrations (Danysz and Parsons, 2012;

Danbolt, 2001).

Figure 1.4. Diagram to illustrate the glutamatergic CA1 synapses located at the hippocampus and the

cerebellar purkinje cell synapses showing the distribution of the EAAT subtypes, astrocytes and glial cells

(Vandenberg and Ryan, 2013)

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The transport of glutamate is carried out by EAAT 1, 2 and 3. EAAT 3 couples to the

cotransport of 3 Na+, 1H+ in exchange for 1K+. The transmembrane potential across

the cell membrane can be worked out using the Goldman-Hodgkin-Katz voltage

equation (fig 1.5) which calculates the concentrating capacities of the EAAT

transporter. The ion flux coupling supports a 106 fold gradient of glutamate across the

cell membrane at equilibrium (Vandenberg and Ryan, 2013).

In the pathogenesis of AD, these reuptake mechanisms are impaired due to an

impaired expression of glutamate transporters (Danbolt, 2001). This can be caused

by several mechanisms such as a reduced energy supply to the brain or due to ion

gradient homeostasis imbalances changing the capacity of EAATs to clear

extracellular glutamate at which point glutamate can exceed the Km of the EAAT

transporters.

Evidence from biochemical tests have identified both pre- and postsynaptic

disruptions in glutamate pathways. Prolonged activation of the NMDA receptors

leads to chronic excitotoxicity; the prolonged depolarisation of the cell plasma

membrane causes an influx of Mg2+ that block the NMDA receptors triggering an

influx of Ca2+ ions into the postsynaptic neurons. Ca2+ overload leads to a loss in

synaptic function that promote excitotoxicity This causes cell death corresponding to

the characteristic appearances of senile plaques and neurofibrillary tangles (fig 1.6)

(Danysz and Parsons, 2012).

[X]o and [X]i = outside and inside concentrations of the various ionic species R is the gas constant T is temperature (in oK) F is Faraday's constant Z = 2 Figure 1.5. Goldman-Hodgkin-Katz equation used to estimate the concentrating capacity of the EAAT

transporter

[Glu]o/[Glu]i = RT/ZF ln {([Na+]o/[Na+]i)3([H+]o/[H+]i)([K+]i/[K+]o)}

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1.4 Therapeutics of Alzheimer’s disease: Past, present and future

A variety of therapeutic strategies such as immunological, chemical and gene

therapy have been extensively studied. However, the multifactorial characteristic of

AD have made this difficult, with current treatments only providing symptomatic relief

and they do not alleviate the disease itself (Anand et al., 2014).

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Tacrine was a drug that aimed to alleviate cognitive function impairments such as

memory loss and intellectual decline in AD patients. However, trials found no

statistically significant difference between tacrine and its placebo. Gastrointestinal

side effects including abdominal pain, diarrhoea and hepatotoxicity caused by a

significantly increased ALT levels seen in 49% of 1203 patients saw the withdrawal of

this drug (Watkins et al., 1994).

Currently, mematine is an NMDA receptor antagonist that works by blocking the

prolonged NMDA activation that leads to excitotoxicity. Evidence also shows

mematine to have a protective role from Aβ toxicity, improve spatial learning and

restore synaptic degeneration in rat studies (Miguel-Hidalgo et al., 2012). Acetyl

cholinesterase inhibitors, such as donepezil, rivastigmine and galantamine, are

therapeutics that inhibit AChE hence preventing the breakdown of ACh (McGleenon

et al., 1999). The increased level of ACh have been found to temporarily improve

cognition but these drugs have been found to have other pharmacological roles too

(Wilkinson et al., 2004).

1.5 Human branched chain aminotransferases in glutamate regulation

An investigation into the distribution of BCAT in the human brain has revealed that an

overexpression of hBCATm in the endothelial layer catalyses the transamination of

BCAA’s in astrocytes, causing a disruption to glutamate homeostasis via the

glutamate-glutamine cycle leading to a cascade of reactions involving increased

intracellular Ca2+. hBCAT has an initial neuroprotective role but as the products

(glutamate and BCKA’s) of its metabolism are toxic, the role turns from

neuroprotective to neurotoxic, thus contributing to glutamate excitotoxicity and hence

neuronal death (Danysz and Parsons, 2012; Hull et al., 2012). Futhermore, a greater

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expression of hBCATm is seen in AD patients with Braak staging (fig 1.2) and a

decrease in brain weight correlating with the underlying pathology of AD (Hull, 2013).

The BBB regulates the complex interface involving the exchange between blood and

brain compartments; thus regulating the brain homeostasis (fig 1.7) (Cardoso et al.,

2010).

Figure 1.7. A proposed model showing hBCAT signalling in the human brain during the disease state. The BBB allows for the direct entry of BCAAs via L-facilitative transporters on both the abluminal and luminal sides (Hull et al., 2012). However, the luminal side of the BBB only allows for the minimal entry of glutamate protecting the brain from peripheral glutamate (Chaudhry et al., 1995). EAATs on the astrocytes associate with the endothelial membrane on the BBB when the brain is exposed to excess glutamate. This and a decrease in glutamate recycling cause glutamate to be transported out into the luminal blood (Hull et al., 2012).

(Hull, 2013)

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1.5 Future treatment: drug development

Hodson and Conway (UWE, Bristol) have investigated the effects of a series of

substituted phenylacetic acids which is a known inhibitor of hBCAT. A lipophilic

pocket on hBCATm was identified by a PhD student using computational studies

helping to discriminate hBCATm over hBCATc. Studies of phenylacetic acids helped

to identify the pharmacophore that helped to synthesis a series of potential inhibitors

using an extension strategy. SAR studies have been used to determine the potencies

of 4-benzyloxyphenylacetic acids on activity and its ability to discriminate hBCATm

over hBCATc. An alternative structure using a sulphur analogue to link the two

adjacent aromatic rings was proposed using computational studies (Hodson and

Forshaw, 2015).

Bioisosteres are substituents that have similar chemical and biological properties. It

is a common approach used to alter the pKa, binding, selectivity, potency, drug

duration and in reducing toxicity. The hybrid displacement law describes the

similarities between groups sharing the same number of valence electrons but

slightly different number of atoms. Sulphur is a classical bioisostere that is commonly

used in drug development that have the same number of valence electrons as

oxygen (Silverman, 2004). Structurally specific drugs often have potencies that are

susceptible to structural changes. However, sulphur and oxygen share similar

chemical properties but as sulphur is slightly larger, it is therefore less

electronegative and therefore undergoes little hydrogen bonding. The binding of a

drug to its active sites may involve hydrogen bonding; therefore an altered inhibition

of hBCAT to its active site can help to identify the method of binding of the sulphur

analogue to the enzyme active site (Hodson and Forshaw, 2015).

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The Willgerodt- Kindler reaction (fig 1.8) is a method used in the synthesis of

phenylacetic acids using aryl ketone as a starting compound. The reaction is

commonly carried out using TEBA; a phase transfer catalyst. However this method of

reaction is not preferred due to its reaction conditions, of prolonged reaction times

and hazardous reaction conditions such as the use of microwave ovens (Alam and

Adapa, 2003).

The Newman-Kwart rearrangement (fig 1.9) is a preferred method used in the

synthesis of S-aryl thiocarbamates from O-aryl thiocarbamates by nucleophilic

substitution in the presence of an acidic phenol and N,N-dimethylthiocarbamoyl

chloride (Aslam and Davenport, 1988).

Figure 1.9. Schematic showing the rearrangement of O-aryl thiocarbamates to form S-aryl thiocarbamates

using the Newman-Kwart rearrangement

(Aslam and Davenport, 1988)

Figure 1.8. Schematic illustrating the Willgerodt- Kindler reaction whereby thiomorpholides are hydrolysed using TEBA to yield phenylacetic acids

(Alam and Adapa, 2003).

http://www.tandfonline.com/doi/full/10.1081/SCC-120015559

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Aims of the project

The first aim of this project was to synthesise 4-benzyloxyphenylacetic acid to

confirm that the methodology used provided a successful route to synthesise these

compounds. The second aim was to synthesise a sulphur analogue of 4-

benzyloxyphenylacetic acid as a potential inhibitor of hBCAT using the Newman-

Kwart rearrangement and hydrolysis methods in the treatment of Alzheimer’s

disease. Due to time implications, this project will strictly aim to synthesis target one

which is 4-acetylthiophenol. Each of the intermediates formed and target one were

characterised by GC-MS and IR spectroscopy.

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2. Experimental section

All of the experimental work was carried out in the fume cupboard.

A COSHH risk assessment form (No. R1003) was carried out before experimental

work was carried out which was written by Dr Annabelle Hodson and endorsed by

Paul Bowdler.

The methodology used in this study was acquired from a patent (US4794205 A) that

provides a method for the synthesis of alkenylthiophenols; which was modified to

alter reaction conditions and the quantities of chemicals used (Aslam and Davenport,

1988).

2.1. Materials and chemicals

A PerkinElmer spectrometer (FT-IR, spectrum two) was the spectrometer of choice in

this project. The data collected were analysed using PerkinElmer spectrum software

ran on a Hewlett Packard desktop. The GC-MS system used was manufactured by

Agilent Technologies 6890N, (serial number CN10309011) with a 5973network mass

selective detector. The rotary evaporator used was manufactured by Buchi

Switzerland R-210, B-491 heating bath, V-801 easyvac vacuum module and V-700

vacuum pump.

4- hydroxyacetophenone, 1,4-Diazabicyclo[2.2.2]octane, N,N-dimethylthiocarbamoyl

chloride. Other chemicals include dichloromethane, diethyl ether, methanol,

potassium hydroxide, 2M hydrochloric acid, sodium bicarbonate, anhydrous

magnesium sulphate, ethylene glycol, de-ionised water. Chemicals were purchased

from Sigma- Aldrich and used without further purification.

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2.2. Synthesis of methyl 4-hydroxyphenylacetate

A 50ml RB flask containing a plastic stirrer bar was charged with 4-

hydroxyphenylacetic acid (4.9741g), methanol (50ml) and concentrated sulphuric

acid (0.5ml). This solution was refluxed under heat for 60 minutes and allowed to

cool. Next, 0.1M sodium hydrogen carbonate (2.093g) was added to neutralise the

mixture, and the product was extracted using ethyl acetate (2x30ml). The combined

organic extracts was washed for impurities using de-ionised water (3x20ml) followed

by sodium chloride (20ml) and dried using magnesium sulphate. The drying agent

was removed using a Buchner funnel and flask, and the solvent was removed from

the product using a rotary evaporator to yield 3.7782g (69.6% yield) methyl 4-

hydroxyphenylacetate.

2.3. Synthesis of methyl 4-(benzyloxy) phenylacetate

A 100ml RB flask was charged with methyl 4-hydroxyphenylacetate (3.328g,

13mmol), benzyl bromide (1.6ml), dimethylformamide (50ml), potassium carbonate

(6.5g) and the mixture was heated at 120-140oC for 240 minutes using a rotamantle.

The resulting solution was cooled to room temperature and diluted using de-ionised

water (50ml) and the organic layer was extracted using ethyl acetate. The combined

organic layers were washed in a separating funnel with de-ionised water (4x50ml)

and sodium chloride (50ml). The organic layers were dried using anhydrous

magnesium sulphate and the drying agent was removed using vacuum filtration. The

solvent was removed from the product using a rotary evaporator to yield 2.7231g

(81.8% yield) of methyl 4-(benzyloxy) phenylacetate.

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2.4. Synthesis of [4-(benzyloxy)phenyl]acetic acid

A 100ml RB flask was charged with methyl 4-(benzyloxy) phenylacetate (2.4988g,

10mmol), sodium hydroxide (4.1024g) and de-ionised water (8ml). This mixture was

refluxed under heat for 15 minutes using a rotamantle and the resulting solution was

allowed to cool. Next, 1M hydrochloric acid (35ml) was added until solution no longer

precipitated and the product was filtered from solution using a Buchner funnel and

flask. This solution was washed for impurities using de-ionised water (3x20ml) and

dried in an oven (24h, 50 oC) on a watch glass to establish a constant weight to afford

1.785g (71.4% yield) of [4-(benzyloxy)phenyl]acetic acid.

2.5. Synthesis of O-(4-acetophenyl) N,N-dimethylthiocarbamate

A 50 ml RB flask containing a plastic stirrer bar was charged with 1,4-

diazabicyclo[2.2.2]octane (2.47g, 22mmol) and methanol (15 ml). This solution was

cooled in an ice-water bath. 4-Hydroxyacetophenone (2.744g, 20mmol)) was added

to the mixture and stirred for 15 minutes using a rotamantle. Next, N,N-

dimethylthiocarbamoyl chloride (2.7466g, 22mmol) was slowly added to the mixture

and heated to room temperature (heat evolved). The resulting mixture was further

stirred (1 h) after which the flask was cooled under an ice-bath. Water (40 ml) was

added to the flask and the solid precipitate that formed was collected by vacuum

filtration using a Buchner funnel and flask. The solid precipitate that collected was

washed for impurities using de-ionised water (2x30 ml) and dried in an oven (24 h,

56oC) on a watch glass to establish a constant weight to afford 3.3917g (72.8% yield)

of O-(4-acetophenyl) N,N-dimethylthiocarbamate which was characterised using IR

spectroscopy and GC-MS.

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2.6. Synthesis of S-(4-acetophenyl) N,N-dimethylthiocarbamate by Newman-

Kwart rearrangement

Two samples of O-(4-acetophenyl) N,N-dimethylthiocarbamate was investigated

using a 50 ml RB (0.7829g) and 50 ml PS (0.7847g) flask equipped with a plastic

stirrer bar. These flasks are heated at 200-220oC for 80 minutes under reflux using a

rotamantle and under a constant flow of dinitrogen gas atmosphere. The apparatus

was also lagged using glass wool. A water:methanol mixture (3:1, 30 ml) was added

respectively to the individual flasks whilst the mixtures are still hot and was stirred

using a rotamantle. The resulting precipitates are vacuum filtered using a Buchner

funnel and flask, and washed for impurities using de-ionised water (2x30 ml). The

solids are dried in a vacuum oven (24h, 56oc) on a watch glass to establish a

constant weight to yield 0.281g and 0.521 g (35.8 and 66.3% yield) of intermediate 2;

S-(4-acetophenyl) N,N-dimethylthiocarbamate and was characterised using IR


Figure 2. Reaction schematic of 4-hydroxyacetophenone and N,N-dimethylthiocarbamoyl chloride to form O-(4-acetophenyl) N,N-dimethylthiocarbamate


Figure 3. Reaction schematic showing the Newman-Kwart rearrangement of O-(4-acetophenyl) N,N-dimethylthiocarbamate to form S-(4-acetophenyl) N,N-dimethylthiocarbamate


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2.7. Synthesis of 4-acetylthiphenol by hydrolysis

In the final step, S-(4-acetophenyl) N,N-dimethylthiocarbamate (0.440g and 0.431g)

from the different flasks are added respectively to separate 50 ml RB and PS flasks

equipped with a plastic stirrer. A solution containing potassium hydroxide (0.123g) in

a ethylene glycol:water mixture (3:1, 20 ml) are added to flasks lagged using glass

wool, stirred using a rotamantle and under reflux for 180 minutes. The mixtures are

cooled under an ice-bath down to room temperature, transferred to separating

funnels and washed with de-ionised water (100 ml). The aqueous layers are washed

with dichloromethane (50 ml) and was acidified to pH 6.0 using 2M hydrochloric acid.

To the RB flask containing mixture, 2M hydrochloric acid (10 ml) was added and

steep increases in pH was compensated for using 1M sodium bicarbonate (26 ml)

and 2M sodium bicarbonate (9 ml). 2M hydrochloric acid (2 ml) was added to the PS

flask containing mixture and increases in pH (>6.0) was compensated with 2M

sodium bicarbonate (0.7 ml). Diethyl ether (3x50 ml) was added to the organic layer

and the combined organic extracts are dried using anhydrous magnesium sulphate.

The drying agent was removed from solution by vacuum filtration and the solvent was

removed from the product using a rotary evaporator to yield 0.082g and 0.0965g

(18.6 and 22.4% yield) 4-acetylthiophenol which was characterised using IR


Figure 4. Reaction schematic showing the hydrolysis of S-(4-acetophenyl) N,N-dimethylthiocarbamate to form

4-acetylthiophenol


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3. Results

(Note: GC-MS readings top = A, bottom= B)

Reaction of 4-hydroxyphenylacetic acid, methanol and concentrated sulphuric acid

under reflux, and the product as described in section 2.2 was characterised by IR

and GC-MS methods (fig 3.1).

3.1.

Infrared spectroscopy of methyl 4-hydroxyphenylacetate

Graph to show the IR spectra of methyl 4-hydroxyphenylacetate (cm-1)

Figure 3.5. Graph to show the IR spectra illustrating the functional groups present on methyl 4-hydroxyphenylacetate.

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Gas chromatogram of methyl 4-hydroxyphenylacetate

Graph to show the retention time at 7.333 minutes with its subsequent fragmentation patterns of

methyl 4-hydroxyphenylacetate using GC-MS

Figure 3.2. Graph to show the fragmentation pattern at different retention times of preliminary intermediate 1

using GC-MS. (A) Gas chromatogram showing a peak with a retention time of 7.33 minutes. (B) Mass spectrum

of preliminary intermediate 1 showing a molecular mass at 166, corresponding to the molecular mass of

intermediate 1 at 166.0gmol-1

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Next, intermediate 1 was reacted with benzyl bromide, dimethylformamide,

potassium carbonate and the mixture was heated, and the organic layer extracted

using ethyl acetate to yield methyl 4-(benzyloxy) phenylacetate (see section 2.3).

3.2.

Infrared spectroscopy of 4-(benzyloxy) phenylacetate

Graph to show the IR spectrum of 4-(benzyloxy) phenylacetate (cm-1)

Figure 3.3. Graph to show the IR spectrum illustrating the functional groups present on 4-(benzyloxy) phenylacetate.

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Gas chromatogram of 4-(benzyloxy) phenylacetate

Graph to show the retention time at 15.655 minutes with its subsequent fragmentation patterns of 4-

(benzyloxy) phenylacetate using GC-MS


using GC-MS. (A) Gas chromatogram showing a peak with a retention time of 15.665 minutes. (B) Mass

spectrum of preliminary intermediate 2 showing a molecular mass at 256, corresponding to the molecular mass

of intermediate 2 at 256.0gmol-1

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In the final reaction, intermediate 2 was reacted with NaOH and de-ionised water;

was heated and HCl was added (see section 2.4). The product was characterised by

IR and GC-MS (fig 3.3).

3.3.

IR spectra of [4-(benzyloxy)phenyl]acetic acid

Graph to show the IR spectra of [4-(benzyloxy)phenyl]acetic acid (cm-1)

Figure 3.5. Graph to show the IR spectra illustrating the functional groups present of [4-(benzyloxy)phenyl]acetic acid.

(Albreej, 2016)

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Gas chromatogram of [4-(benzyloxy)phenyl]acetic acid

3.4.

Graph to show the retention time at 16.093 minutes with its subsequent fragmentation patterns of [4-

(benzyloxy)phenyl]acetic acid using GC-MS


using GC-MS. (A) Gas chromatogram showing a peak with a retention time of 16.093 minutes. (B) Mass

spectrum of preliminary intermediate 3 showing a molecular mass at 242, corresponding to the molecular mass

of intermediate 3 at 242.0gmol-1

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The spectra of the starting material for the synthesis of the sulphur analogue was

recorded for comparison (see fig 3.4).

Infrared spectroscopy of 4-hydroxyacetophenone

3.5.

Graph to show the IR spectra of 4-hydroxyacetophenone (cm-1)

Figure 3.7. Graph to show the IR spectra illustrating the functional groups present on 4-hydroxyacetophenone

(see fig 2). The presence of the aromatic ring is seen at 1282.07 or 1581.40 cm-1. The graph also shows the C=O functional group of the ketone at 1642.93 cm-1 and the O-H aromatic group at 3107.92 cm-1

Figure 3.8. Structural diagram of 4-hydroxyacetophenone

1581.40

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Infrared spectroscopy of O-(4-acetophenyl) N,N-dimethylthiocarbamate

DABCO was reacted with 4-HAP and DMTC to form a yellow powder of intermediate

1 (see section 2.5 methodology) and characterised (fig 3.5).

Table to highlight the absorption frequencies

characteristic of the IR spectra of O-(4-acetophenyl)

N,N-dimethylthiocarbamate

Major functional group Absorption frequency

region (cm-1)

Aromatic ring 1204.02

C-O 1127.63

C=O 1677.83

1531.88

Table 1. Table to show the absorption frequencies matched with the corresponding functional groups present on the IR spectra of intermediate 1

Graph to show the IR spectra of O-(4-acetophenyl) N,N-dimethylthiocarbamate (cm-1)

Figure 3.9. Graph to show the functional groups present on intermediate 1; O-(4-acetophenyl) N,N-dimethylthiocarbamate. (see table 1 for corresponding functional groups identifications)

Figure 3.10. Structural diagram of O-(4-acetophenyl) N,N-dimethylthiocarbamate

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Gas chromatogram of O-(4-acetophenyl) N,N-dimethylthiocarbamate

The molecular ion at 223 corresponds to the molecular mass of intermediate 1; 223.0

gmol-1 confirming that intermediate 1 was synthesised (fig 3.10). The fragmentation

pattern also shows a smaller molecular ion at 88 which is the molecular mass of

DMTC after the chloride ion has been broken off (fig 3.12).

Graph to show the retention times (minutes) of O-(4-acetophenyl) N,N-dimethylthiocarbamate using

GC-MS

Figure 3.11. Chromatogram of intermediate 1 showing various retention times at 14.58 and 14.704 minutes

Figure 3.12. Structural diagram of unreacted N,N-dimethylthiocarbamoyl chloride

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Graph to show the retention time at 14.575 minutes with its subsequent fragmentation patterns of O-

(4-acetophenyl) N,N-dimethylthiocarbamate using GC-MS

Figure 3.13. Graph to show the fragmentation pattern at different retention times of intermediate 1 using GC-MS.

(A) Gas chromatogram showing a single peak with a retention time of 14.58 minutes. (B) Mass spectrum of

intermediate 1 at RT 14.575 minutes showing the molecular ion furthest to the right at 223 and unreacted DMTC

at 88

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Graph to show the retention time at 14.704 minutes with its subsequent fragmentation patterns of O-


Figure 3.14. Graph to show the fragmentation pattern at different retention times of intermediate 1 using GC-MS.

(A) Gas chromatogram showing a peak with a retention time of 14.704 minutes. (B) Mass spectrum of

intermediate 1 at RT 14.704 minutes showing a small peak of the molecular ion at 208 and unreacted DMTC at

88

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The unreacted DMTC identified using the mass

spectrometer highlights the structure that is

represented in figure 3.12. The molecular ion at 208

represents a fragment of intermediate 1 of 208gmol-1

whereby the methyl group on the ketone side chain

has been lost (fig 3.15).

Figure 3.15. Structural diagram of a fragment of O-(4-acetophenyl) N,N-dimethylthiocarbamate

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3.6.

Intermediate 1 was heated in a RB flask under a dinitrogen gas atmosphere to

undergo Newman- Kwart rearrangement and to yield intermediate 2; S-(4-

acetophenyl) N,N-dimethylthiocarbamate (see section 2.6) and its characterisation

(fig 3.6).

Infrared spectra of S-(4-acetophenyl) N,N-dimethylthiocarbamate (RB flask)


characteristic of the IR spectra of S-(4-acetophenyl)



region (cm-1)


C=O 1671.34

C=S 1090.84

C-N 1257.34


Graph to show the IR spectra of S-(4-acetophenyl) N,N-dimethylthiocarbamate (cm-1)

Figure 3.16. Graph to show the functional groups present on intermediate 2; S-(4-acetophenyl) N,N-dimethylthiocarbamate. (see table 2 for corresponding functional groups identifications)

Figure 3.17 Structural diagram of S-(4-acetophenyl) N,N-dimethylthiocarbamate

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Gas chromatogram of S-(4-acetophenyl) N,N-dimethylthiocarbamate (RB flask)

Graph to show the retention times (minutes) of S-(4-acetophenyl) N,N-dimethylthiocarbamate using

GC-MS

Figure 3.18. GC-MS graph of the chromatogram of intermediate 2 showing various retention times at 14.281 and 14.371 minutes

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Graph to show the retention time at 14.281 minutes with its subsequent fragmentation patterns of S-


Figure 3.19. Graph to show the fragmentation pattern at different retention times of intermediate 2 using GC-MS. (A) Gas chromatogram showing a small peak with a retention time of 14.28 minutes. (B) Mass spectrum of intermediate 2 at RT 14.281 minutes showing a molecular ion furthest to the right at 223 and fragments of DMTC at 88 and 72

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The molecular ion at 223 corresponds to the molecular mass of intermediate 2

therefore confirming the synthesis of S-(4-

acetophenyl) N,N-dimethylthiocarbamate. The

peak at 88 corresponds to unreacted DMTC (fig

3.12) and the peak at 72 is a fragment of

intermediate 2.

Figure 3.20. Structural diagram of the fragment of intermediate 2 observed in the GC-MS producing a molecular ion at 72

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Figure 3.21. Graph to show the fragmentation pattern at different retention times of intermediate 2 using GC-MS. (A) Gas chromatogram showing a small peak with a retention time of 14.37 minutes. (B) Mass spectrum of intermediate 2 at RT 14.371 minutes showing a molecular ion furthest to the right at 223 and fragments of intermediate 2 at 72

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3.7.

In comparison, a PS flask was also used in the synthesis of intermediate 2 to establish the

better synthetic method.

Infrared spectra of S-(4-acetophenyl) N,N-dimethylthiocarbamate (PS flask)


characteristic of the IR spectra of S-(4-acetophenyl)



region (cm-1)


C=O 1670.13

C=S 1091.21

C-N 1257.95


Graph to show the IR spectra of S-(4-acetophenyl) N,N-dimethylthiocarbamate (cm-1)

Figure 3.22. Graph to show the functional groups present on intermediate 2; S-(4-acetophenyl) N,N-dimethylthiocarbamate. (see table 3 for corresponding functional groups identifications)

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characteristic of the IR spectrum of S-(4-

acetophenyl) N,N-dimethylthiocarbamate (crystal

formation)


region (cm-1)


C=O 1671.64

C=S 1091.07

C-N 1257.60

Table 4. Table to show the absorption frequencies matched with the corresponding functional groups present on the IR spectra of the crystals formed in intermediate 2

Graph to show the IR spectra of S-(4-acetophenyl) N,N-dimethylthiocarbamate (cm-1) crystals using IR

spectroscopy

Figure 3.23. Graph to show the functional groups present in the crystals that formed from intermediate 2; S-(4-acetophenyl) N,N-dimethylthiocarbamate. (see table 4 for corresponding functional groups identifications)

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Gas chromatogram of S-(4-acetophenyl) N,N-dimethylthiocarbamate (PS flask)

Graph to show the IR spectra of the synthesis of S-(4-acetophenyl) N,N-dimethylthiocarbamate (cm-1)

using different flask types

Figure 3.24. Graph to show the functional groups present on intermediate 2; S-(4-acetophenyl) N,N-dimethylthiocarbamate. (see table 1,2 and 3 for corresponding functional groups identifications). Green: round bottom flask, Orange: pear shaped flask and purple: pear shaped flask crystal formation

Graph to show the retention times (minutes) of S-(4-acetophenyl) N,N-dimethylthiocarbamate using

GC-MS

Figure 3.25. Chromatogram of intermediate 2 showing various retention times at 14.281 and 14.359 minutes

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Figure 3.26. Graph to show the fragmentation pattern at different retention times of intermediate 2 using GC-

MS. (A) Gas chromatogram showing a small peak with a retention time of 14.28 minutes. (B) Mass spectrum of intermediate 2 at RT 14.281 minutes showing a molecular ion furthest to the right at 223 and fragments of DMTC at 72 and 88

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Figure 3.29. Graph to show the fragmentation pattern at different retention times of intermediate 2 using GC-

MS. (A) Gas chromatogram showing a small peak with a retention time of 14.36 minutes. (B) Mass spectrum of intermediate 2 at RT 14.359 minutes showing a molecular ion furthest to the right at 223 and fragments of DMTC at 72

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GC-MS of S-(4-acetophenyl) N,N-dimethylthiocarbamate (crystal formation)

Graph to show the retention times (minutes) of S-(4-acetophenyl) N,N-dimethylthiocarbamate crystals

using GC-MS

Figure 3.30. GC-MS graph of the chromatogram of intermediate 2 crystals showing various retention times at 14.365 minutes

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(4-acetophenyl) N,N-dimethylthiocarbamate crystals using GC-MS

Figure 3.31. Graph to show the fragmentation pattern at different retention times of intermediate 2 crystals using GC-MS. (A) Gas chromatogram showing a small peak with a retention time of 14.37 minutes. (B) Mass spectrum of intermediate 2 at RT 14.365 minutes showing a molecular ion furthest to the right at 223 and a fragment of starting material at 72

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3.8

S-(4-acetophenyl) N,N-dimethylthiocarbamate underwent hydrolysis; and the product was

extracted from organic layers that were combined to afford product; 4-acetylthiophenol.

Infrared spectroscopy of 4-acetylthiophenol (RB flask)

Table 5. Table to show the absorption frequencies matched with the corresponding functional groups present on the IR spectra of product

Note* Product didn’t dry sufficiently hence

the presence of the O-H group at 3301.13 is

observed


characteristic of the IR spectra of 4-acetylthiophenol


region (cm-1)

O-H* 3301.13

C-H aliphatic 2946.99

S-H 2548.7

C=O 1671.21


C-C stretch 1037.88

Graph to show the IR spectra of 4-acetylthiophenol (cm-1)

Figure 3.32. Graph to show the functional groups present on intermediate 3; 4-acetylthiophenol. (See table 5 for corresponding functional groups identifications)

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Gas chromatogram of 4-acetylthiophenol (RB flask)

Figure 3.33. GC-MS graph of the chromatogram of product showing various retention times at 6.025 minutes

Graph to show the retention times (minutes) of 4-acetylthiophenol using GC-MS

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acetylthiophenol using GC-MS

Figure 3.34. Graph to show the fragmentation pattern at different retention times of product using GC-MS. (A) Gas chromatogram showing a peak with a retention time of 6.025 minutes. (B) Mass spectrum of product at RT 6.025 minutes showing a molecular ion at 152 of product, and fragments of product at 137 and 109

Figure 3.35. Structural diagram of target 1

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3.9

PS flask was also used in the synthesis of intermediate 3 to establish the better synthetic

method.

Infrared spectrum of 4-acetylthiophenol (PS flask)


characteristic of the IR spectra of 4-acetylthiophenol


region (cm-1)

O-H* 3293.42

C-H aliphatic 2943.32

C=O 1647.95


C-C stretch 1034.56

Table 6. Table to show the absorption frequencies matched with the corresponding functional groups present on the IR spectra of product

Note* Product did not dry sufficiently hence the

presence of the O-H group at 3293.42 is observed

Graph to show the IR spectra of 4-acetylthiophenol (cm-1)

Figure 3.36. Graph to show the functional groups present on intermediate 3; 4-acetylthiophenol. (see table 6 for corresponding functional groups identifications)

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Graph to show the IR spectrum of two 4-acetylthiophenol (cm-1) samples

Figure 3.37. Graph to show the functional groups present on intermediate 3; 4-acetylthiophenol. (See table 5&6 for corresponding functional groups identifications) Black: round bottom flask and red: pear shaped flask

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Gas chromatogram of 4-acetylthiophenol (PS flask)

Graph to show the retention times (minutes) of 4-acetylthiophenol using GC-MS

Figure 3.38. GC-MS graph of the chromatogram of product showing various retention times at 6.011 minutes

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acetylthiophenol using GC-MS

Figure 3.39. Graph to show the fragmentation pattern at different retention times of product using GC-MS. (A) Gas chromatogram showing a peak with a retention time of 6.011 minutes. (B) Mass spectrum of product at RT 6.011 minutes showing a molecular ion of product at 152 and its fragments at 137 and 109

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3.10

The IR spectra’s of all three intermediates were analysed.

Infrared spectroscopy of the two intermediates and product (RB flask)

Infrared spectroscopy of the two intermediates and product (PS flask)

Graph to show the IR spectra of intermediates and final product collected in the round bottom flask

method (cm-1)

Figure 3.41. Graph to show the functional groups present on intermediate 1, 2 and final product. (See table 1, 2 and 5 for corresponding functional groups identifications) Pink: intermediate 1, black: intermediate 2 and red: product

Figure 3.40. Graph to show the functional groups present on intermediate 1, 2 and final product. (See table 1, 3 and 6 for corresponding functional groups identifications) Pink: intermediate 1, black: intermediate 2 and red: product

Graph to show the IR spectras of intermediates and final product collected in the pear shaped flask

method (cm-1)

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4. Discussion

The general aims of the project were to synthesise a sulphur analogue of 4-

benzyloxyphenylacetic acids as a potential inhibitor of hBCAT in AD. The findings of

the preliminary experiments confirm that the route of synthesising 4-

benzyloxyphenylacetic acids works. This is confirmed by the IR spectroscopy (fig 3.5)

and GC-MS (fig 3.6). The key findings from the sulphur analogue investigation was

the introduction of the thiol group onto the aromatic ring by optimising an established

method from a patent (US4794205 A). The resulted (sections 3.5-3.10) identified

molecular ions at specific retention times that correlate with the molecular masses of

intermediates 1 (223.0gmol-1), 2 (223.0gmol-1) and 3 (152.0gmol-1). Furthermore, the

predicted functional groups present on these compounds were observed using the IR

spectrums (fig 3.9, 3.16 and 3.32 and tables 1-6) show the identification of specific

functional groups that correspond with the structures of intermediates (fig 3.10, 3.17)

and product, confirming the synthesis of intermediates target one; 4-acetylthiophenol

(fig 3.35). It is worth noting that the IR spectra’s of intermediates 2 and 3 show traces

of water suggesting that products needed to be further dried. In reaction two (section

2.2), N-K rearrangement involves the removal of the phenol proton using DMTC.

Electron withdrawing groups on the ring structure undergo nucleophilic attack with

the sulphur analogue at the α-carbon (C1) of the ring structure. The ketone group of

4-HAP was therefore of advantage in this rearrangement. The gas chromatograms

often showed the presence of unreacted starting materials; fragments of DMTC at 88

and 72 were commonly observed suggesting either a reaction that didn’t reach

completion or due to using excess quantities of DMTC.

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The methodology acquired from the patent was significant in this project as it

provided the synthetic route which was quantified to smaller amounts to provide

sufficient yields of each intermediate. However as the results section 2.7 show, the

yield of 4-acetylthiophenol using the RB flask was little. This could be due to

experimental and human error including poor extraction techniques and accidental

spillages.

The use of RB and PS flasks was a parameter that was introduced in the synthesis of

intermediates 2 and 3 to determine whether the differences in surface area could

influence the reaction. Interestingly, the synthesis of intermediates 2 and 3 using the

PS flask synthesised a higher yield and crystallised product (section 2.6, 2.7 and fig

3.23, 3.31). The differences in yield may be attributed to the difference in surface

area and isothermal conditions. Whilst the RB flask had a larger SA: V ratio speeding

up the thermodynamic process to drive the reaction to completion sooner (Geyer et

al., 2006; Wirth, 2013), the RB flask does not retain heat and hence the reason for

the apparatus being lagged using glass wool. In contrast, the lower SA: V ratio of the

PS flask provided a steadier reaction temperature hence reducing the risk of product

degradation.

A key limitation of this study however was that targets two (1-[4-

(Benzylsulfanyl)phenyl]ethanone) and three (4-(Benzylsulfanyl)benzoic acid) was not

investigated, therefore not fulfilling the true aims of this project.

Previous studies (Hodson and Forshaw, 2015) have tested the potencies of 4-

benzyloxyphenylacetic acids to discriminate between hBCATm over hBCATc.

Whether these sulphur analogues are any more significant than the oxygen analogue

is unconfirmed and qualitative SARs and quantitative QSAR modelling will be needed

to determine the affinity and efficacy (Emax) of the synthesised compound to bind to

the hBCAT’s active site (McKinney et al., 2000). The sulphur analogue is expected to

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alter physical and chemical properties which influences the biological system and

hence the biological activity of a drug (fig 4.1).

Importance of hydrogen bonding in target-drug interactions

The inhibition of the enzyme active site involves two important processes involving

reversible and irreversible reactions. Reversible inhibition of an enzyme involves a

combination of electrostatic, hydrogen bonding, VdW and hydrophobic forces of

attraction partially block the enzyme active site. These inhibitors bind through

competitive, non-competitive or by a combination of inhibition methods (Wermuth,

2008). Hydrogen bonding has large influences on the maintenance of the secondary

and tertiary structure of the active site and within the target-drug interactions.

Sulphur is slightly larger than oxygen and is therefore less electronegative, exerting

less hydrogen bonding. In theory, this should show an altered inhibition of binding of

the drug with its active site.

The active site of hBCAT has been mapped in previous studies (Goto et al., 2005)

that provide the three dimensional structure of hBCATc. This model has been

Figure 4.1. A diagram to illustrate the interaction of Chemistry, Biology

and Statistics in SAR studies

(McKinney et al., 2000)

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previously used to explain how the anticonvulsant gabapentin binds to the active site

of hBCAT; providing the points of hydrogen bonding and salt bridge interactions of

the active site residues. Using this data, it is possible to predict the mode of

mechanism of inhibition involved in the binding of 4-(Benzylsulfanyl) benzoic acid to

hBCAT in an attempt to control the excitotoxicity processes involved in the disease

state of AD (fig 4.2, 4.3). Based on the proposed mechanism of action (Hull, 2013),

the effect of a non-specific inhibition of both hBCAT cytosolic and mitochondrial

isoforms is explained in figure 4.4 Here, the inhibion of hBCATc causes a ‘shutdown’

of the glutamine cycle hence decreases the brain glutamate pool. The inhibition of

hBCATm limits the conversion of BCAAs and a-KG in producing glutamate (fig 4.4).

Figure 4.2. Mapping of the active site of hBCATc- ox.

gabapentin complex. The hBCAT enzyme encapsulates the gabapentin (pink) within its active site that is composed of carboxylate and aminomethyl groups. Green and blue: A secondary structure of the small and large domains of one homodimer; Yellow: disulphide bond of the CXCC motif; Red circles: water molecules; Red loops: loops consisting of Tyr-90*, Leu-173*, and Val-

175* of the small domain of other subunits and interdomain loop

(Gota et al., 2005)

Figure 4.3. Diagram to illustrate hydrogen bonding and salt-bridge interactions involved in the hBCATc-ox and gabapentin complex. Interactions in which acceptors and donors are <3.5Å are represented by the dotted lines. Hydrogen bonding is also observed between Y227 complex and PLP

(Gota et al., 2005)

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Effects of thiols

Thiols are almost always associated with a high chemical reactivity and are therefore

not commonly used as substituents in QSAR studies. Thiol group containing drugs

are sometimes exploited for its strong affinity of the thiolate anion with heavy metals.

For example, zofenopril; an Angiotensin-Converting-Enzyme inhibitor is used in the

treatment of hypertension. The lipophilicity of these drugs means that they can

permeate into cardiac tissues easier. Based on these findings, it might be possible

that the sulphur analogue inhibitor of hBCAT possess these high lipophilic properties

allowing the drugs to permeate through the BBB (Wermuth, 2008).

Figure 4.4. A proposed mechanism of action showing the effects of a non-specific inhibition of hBCAT cystolic and mitochondrial isoform

(Hull, 2013)

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5. Further work

This project aimed to synthesise target 1 compound; a stepping stone towards

synthesising the final product.

Firstly, target one will need to undergo a coupling reaction involving benzyl chloride to

yield target 2; (1-[4-(Benzylsulfanyl)phenyl]ethanone) (fig 4.5) which can be determined

by IR and GC-MS. Saponification of target two replaces the ketone group with a

COOH group to yield the final target of 4-(Benzylsulfanyl)benzoic acid (fig 4.6) which is

therefore the sulphur analogue of the 4-benzyloxyphenylacetic acid which could

potential inhibit hBCAT (fig 4.4).

Figure 4.5. Chemical structure of (1-[4-(Benzylsulfanyl)phenyl]ethanone)

Figure 4.6. Chemical structure of 4-(Benzylsulfanyl)benzoic acid.

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6. Conclusion

To summarise, the patent therefore provided an excellent route of in the synthesis of

the thiol group and analytical tools aided in the characterisation of products. In

reaction one, 4- HAP was reacted with DMTC in the presence of DABCO to yield

intermediate one at 72.8% yield. Secondly, the N-K rearrangement provided a

successful route in the introduction of the thiol group onto 4-HAP to yield target two.

The use of RB and PS flasks was introduced at this stage as a parameter in an

attempt to determine how certain reaction conditions alter the chemical reaction.

Interestingly, the PS flask yielded higher amount of product in intermediate two and

target one. In the final reaction, a hydrolysis reaction was used to synthesise the final

product.

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investigating the synthesis of a sulphur analogue of 4-benzyloxyphenylacetic acid as a potential...

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