Synthesis, Characterisation and Antibacterial Activities of Tris-thiourea Derivatives

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Synthesis, Characterisation and Antibacterial Activities of Tris-thiourea Derivatives

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Siti Afifah Binti Rosli (44042)

Bachelor of Science with Honours

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Acknowledgement

My deepest gratitude and appreciation goes to my supervisor, Assoc. Prof. Dr. Zainab Ngaini and my co-supervisor, Miss Wan Sharifatun Handayani Wan Zullkiplee whose gave constant encouragement, guidance, advice, useful critiques and support in developing understanding on the project thus, allowing the possibility to complete my degree's thesis.

Futhermore, special thanks I would like to offer to Miss Ainaa Nadiah and all postgraduates of Organic Chemistry's laboratory for guiding me patiently, proficient assistance to complete the analysis and for their valuable feedback and involvement.

My gratitude is extended to Madam Nurhayati and Mr. Wahab whose helps me in handling NMR and FTIR spectroscopy. I would like to thank all laboratory assistant for providing the apparatus to be used in my research.

Lastly, I offer my regards to all of those who supported me for their cooperation and encouragement throughout this study.

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Declaration

I hereby declare this thesis submitted is my own research work in support of an application for another degree of qualification of this or any other university or institution of higher learning. The sources used in the thesis are text with declaration that clearly marked as such .

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Resource Chemistry Programme Department of Chemistry

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

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Table of Contents

Acknowledgement. ... i

Declaration ... ii

Table of Contents ... iii

List of Abbreviations ... vi

List of Figures ... vii

List of Schemes ... ix

Abstract. ... I Chapter I: Introduction ... 2

Problem Statement. ... 4

Objectives... 5

Chapter 2: Literature Review ... 6

Thiourea ... 6

Synthesis of Thiourea ... 7

The choice of acyl chloride compounds ... 7

The choice ofthiols ... 8

The choice of amines ... 9

The choice of solvents ... 11

Biological properties of thiourea ... 14

Thiourea derivatives with antifungal activity ... 14

Thiourea derivatives as antioxidant. .... .. .... ... 15

Thiourea derivatives with anticancer activity ... 16

Thiourea derivatives with antiviral activity ... 17

Thiourea derivatives with antibacterial activity ... 18

Introduction to Escherichia coli and Staphylococcus aureus ... 19

Chapter 3: Materials and Method ... 21

Materials ... 21

Physical Measurement. ... 21

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Methodology... 22

Synthesis of N', N³, N5-tris«4-bromophenyl)carbamothioyl) Synthesis of N', N³, ~-tris((4-fluorophenyl)carbamothioyl) Synthesis of N', N³^,~-tris((2-fluorophenyl)carbamothioyl) Synthesis of N', N³^,~-tris((3-fluorophenyl)carbamothioyl) Synthesis of N', N³, ~-tris((3-bromophenyl)carbamothioyl) Synthesis of N', N³, ~-tris((2-bromophenyl)carbamothioyl) benzene-l,3,5-tricarboxamide (21) ... 22

benzene-l,3,5-tricarboxamide (22) ... ,... 23

benzene-l ,3,5-tricarboxamide (23) ... 24

benzene-l,3,5-tricarboxamide (24) ... 25

benzene-l,3,5-tricarbozamide (25) ... 26

benzene-l ,3,5-tricarbozamide (26) ... '" 27 Antibacterial screening ... 28

Chapter 4: Result and Discussion ... , ... 30

Synthesis of N', N³, ~-tris((4-bromophenyl)carbamothioyl) Synthesis of N', N³^,~-tris((4-fluorophenyl)carbamothioyl) Synthesis of N', N³^,~-tris((2-fluorophenyl)carbamothioyl) Synthesis of N', N³, ~-tris((3-fluorophenyl)carbamothioyl) Synthesis of N', N³^,~-tris((3-bromophenyl)carbamothioyl) Synthesis of N', N³^,~-tris((2-bromophenyl)carbamothioyl) benzene-l,3,5-tricarboxamide (21) ... 30

benzene-l ,3,5-tricarboxamjk (23) ... 38

Antibacterial Activities ... 54

Chapter 5: Conclusion and Recommendations ... 57

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Chapter 6: References .. ... 58 Appendix A ... ... 63 Appendix B.... ... 64

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I3C NMR Spectroscopy d

DMF DMSO-d6 DPPH FTIR GJso

IH NMR Spectroscopy

ICso J m

MH MHz MIC mmol m.p ppm rpm s SRSA

TGI

~g Vmax

List of Abbreviations

Chemical shift in parts per million Carbon-I 3 Nuclear Magnetic Resonance Doublet

N,N-dimethylformamide Deutarated Dimethyl sulfoxide

I,I-diphenyl-picrylhydrazyl

Fourier Transform Infrared Spectroscopy Growth Inhibitory Concentration of 50%

Hydrogen Nuclear Magnetic Resonance Inhibitory Concentration of 50%

Coupling constant in Hertz Multiplet

Mueller Hinton Mega Hertz

Minimum Inhibitory Concentration milli mol

melting point part per million

rev~ions per minute Singlet

Superoxide Radical Scavenging Activity Triplet

Total Growth Inhibition microgram

maximum vibrational frequency

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List of Figures

Figure 1: Structure of thiourea ... 2

Figure 2: Tautomeric forms ofthiourea ... 2

Figure 3: Isophthaloyl dichloride 3 and Terepthaloyl dichloride 4 ... 8

Figure 4: Benzoyl chloride 5 ..." ... 8

Figure 5: Potassium thiocyanate ... 8

Figure 6: Ammonium thiocyanate ... 9

Figure 7: L-alanine 6 ... 9

Figure 8: Aromatic amine (4-ethylaniline) 7 ..." ... 10

Figure 9: 4-(aminophenyl) acetic acid 8 ... '" 10 Figure 10: Toluene 9 ... 11

Figure 11: Tetrahydrofuran (THF) 10 ... 11

Figure 12: Acetone (propanone) 11 ... 12

Figure 13: Acetonitrile 12 ... 12

Figure 14: I-(2-fluorophenethyl)-3-(5-chloro-l ,3-dimethyl-I H-pyrazole I-carbonyl) thiourea 13 ... 14

Figure 15: I-benzyl-3-(5-chloro-3-methyl-2-oxo-3 H benzoxazole-6-yl)thiourea 14... 15

Figure 16: 2-«2-amino-6-oxo-l ,6-dihydro-9H-purin-9-yl)methoxy)ethyl (phenylcarbamothioyl)valinate 15 ... [5

Figure 17: 4-(3-heptylthioureido)i~enesulfonamide 16... 16

Figure 18: N-[ 1-( 4R)-( 4-isopropyl-1-methylcyclohexyl)]-N' -[2-(phenyl)] thiourea 17... .. ... 16

Figure 19: N-(3-bromophenyl)-4-[2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazole 5-carbonyl]piperazine-l-carbothioamide 18 ... 17

Figure 20: [N,N '-bis(N,N '-benzoyltricarbamoyl)-N,N '-bisbenzyl] ethane-l,2-diamine 19 ... 18

Figure 21: NI,N4-bis (phenylcarbamothioyl)terephthalamide 20 ... 19

Figure 22: FTIR spectrum for compound 21. ... 31

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,...

Figure 23: IHNMR spectrum for compound 21. ... 32

Figure 24: I3CNMR spectrum for compound 21. ... , ... 33

Figure 25: FTIR spectrum for compound 22 ... 35

Figure 26: IHNMR spectrum for compound 22 ... 36

Figure 27: I3CNMR spectrum for compound 22 ... , ... 37

Figure 30: I3CNMR spectrum for compound 23 ... 41

Figure 39: I3CNMR spectrum for compound 26 ... 53 Figure 40: Antibacterial assay ofE. coli on compound 21, 25 and 26 ... '" 54

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List of Schemes

Scheme 1: Reaction pathway of synthesis thiourea derivatives ... 5

Scheme 2: Geneml mechanism of synthesis thiourea derivatives ... 7

Scheme 3: Synthesis of compound 21. ... 30

Scheme 4: Synthesis of compound 22 ... 34

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ABSTRACT

A novel series of tris-thiourea derivatives were successfully synthesized by reaction of benzene-l,3,5-tricarbonyl isothiocyanate intermediates 2 in acetonitrile with aromatic amine yielded optically active tris-thiourea 21-26. The structure of the synthesized compounds were established by Fourier Transform Infra Red spectroscopy (FTIR) , 'H and l3C Nuclear magnetic resonance (NMR) spectroscopy. The antibacterial activities of tris-thiourea 21-26 were investigated against Gram negative bacteria, Escherichia coli and Gram positive bacteria, Staphylococcus Aureus using Kirby-Bauer disc diffusion method and the diameter of inhibition zone were determined. The result of all six tris-thiourea compounds showed poor antibacterial activities.

Keywords: tris-thiourea derivatives, amine, spectroscopy, antibacterial activities

ABSTRAK

Sam sir; novel derivatiftris-thiourea dan terbitannya telah berjaya disintesis oleh tindak balas sebatian pertengahan benzena-i,3,5-trikarbonil isotiosianat 2 dalam asetonitril dengan amina aromatik untuk menghasilkan optik aktif tris-thiourea 21-26. Struktur sebatian yang disintesis telah dikenalpasti dengan menggun~f!n Fourier Transform infra red !.pektroskopi (FTIR), JH dan

lJe

Nuklear resonans magnet (NMR) spectroskopi. Aktiviti anti-bakteria tris-thiourea 21

26 terhadap pertumbuhan bakteria Gram-negatif, Escherichia coli dan bakteria Gram-positif, Staphylococcus Aureus telah dikenalpasti menggunakan ujian Kirby-Bauer kaedah cakera penyebaran dan diameter zon perencatan telah ditentukan. Hasil daripada semua enam sebatian tris-thiourea menunjukkan aktiviti anti-bakteria lemah.

Kata kunci: tris-thiourea derivatif, amine, spektroskopi, aktiviti antibakteria

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Chapter 1

Introduction

ThioUTea is a compound with the chemical fonnula of CSN2H4 which consists of sulphur and nitrogen. The basic structure of thiourea is shown in Figure 1. Thiourea is extracted from urea, where oxygen atom in urea structure is replaced with sulphur, and thiourea has been successfully used in many diseases (Edrah, 2010). Thiouea derivatives are useful in biological activities for example, as antimicrobial (Abdel-rahman et al., 2007), anti-HIV (Venkatachalam e/ ai., 2001) and antitumor (Shusheng et al., 2008).

It consists of three different functional groups which are amino, imino and thiol and it can occur in tautomeric fonns as shown in Figure 2. Thiourea can be fonned by reacting amino group and thiocyanato group in a possible solvent such as acetone and THF (Saeed et al., 2009, Katla et al., 2012). In 2005, Yonova et al. have reported that thiazolylthiourea derivatives displayed fungicidal and nematocidal activity including acaricidal and insecticidal activity.

Figure 1: Structure of thiourea

Figure 2: Tautomeric forms of thiourea

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Song et al. (2011) reported a common method used in synthesis of thiourea derivatives by a reaction of isothiocyanate intermediates with chiral amines. In applying such method, it is required to use solvents such as benzene, toluene, acetonitrile, dimethylformamide (DMF), dichloromethane and THF (Song et a/., 2011).

Thiourea derivatives are widely used in pharmaceutical industry due to its biological activities such as antifungal (Madhava et a/., 2012), anti-HIV (Lucero et a/., 2006) and antiviral (Tabarrini et a/., 2008). In 2011, De Souza et al. reported that thiourea derivatives have a crucial role in inhibition of HIV reverse transcriptase and can act as a strong anti-tuberculosis activity. Thiourea derivatives have shown various biological activities as stated. In this study, the antibacterial activity of thiourea derivatives against Escherichia coli will be studied.

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1.2 Problem Statement

1biourea is a unique compound having three different functional groups which are amino, imino and thiol. There are many studies in the synthesis of mono- and bis-thiourea where bis

thiourea derivatives reported to show better antibacterial activity compared to mono-thiourea (Asha et 01. , 2008). Tris-thiourea group are seldom to be reported. In 2007, Zhong et al.

reported that synthesized thiourea compound with C=S, N-H and C=O groups to give good antibacterial activities, therefore synthesized bis- and tris-thiourea are believed to give better antibacterial activities as they consist more thiourea functional groups compared to the synthesized mono-thiourea. It is also believed that presence of substituent groups in thiourea such as bromo, nitro and chloro are able to enhance the antibacterial activity of the compounds (Ibrahim et

at.,

2009). Hence, in this study, compounds with three thiourea groups containing different substituents were synthesized with the aim to possess better antibacterial activities.

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1.2 Objectives

1- To synthesize new tristhiourea derivatives by utilizing benzene-I,3,5-tricarbonyl trichloride 1 as a starting material.

acetonitrile

-

^X

Q

N

x

H H

X= Orlho-, mcta-, para- Br and F

Scheme 1: Reaction pathway of synthesis thiourea derivatives

2- To characterize the synthesized compounds using FfIR, IH and l3C NMR spectroscopy.

3- To study the antibacterial activity of the synthesized compounds against Escherichia coli and Staphylococcus aureus.

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Chapter 2

Literature Review

2.1 Thiourea

A white crystalline solid compound known as thiourea has a chemical formula ofCSN2H4 and molecular weight of 76.12 glmol. Thiocarbamide is also referred to thiourea and it is found to be soluble in water but insoluble in non-polar solvents. In addition, thiourea compound is also soluble in protic and aprotic polar solvents (Wiley, 2004).

Thiourea can be synthesized using amino group and thiocyanato group with a suitable solvent for example, acetone (Saeed et al., 2009) and tetrahydrofuran (THF) (Katla et al., 2012).

Thiourea derivatives play a role in developing useful agrochemicals and pharmacological agents. For instance, compound 3-(3,4-dimethylphenyl)-5-( 4-methoxyphenyl)-4,5-dihydro

I H-pyrazole-1-carbothoamide displayed good antiproliferative activity against MCF -7 with ICso of O.08/JM which is therefore possible to be an anticancer agent (Peng et al., 2010). In 2015, Kee et al. reported that thiourea derivatives (thioamides) are the drugs that is used to block thyroid hormone action.

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2.1.1 Synthesis of Thiourea

Thiourea derivatives can be synthesized by direct reaction of isothiocyanate ion with an amine. The reaction involved nucleophilic attack from amine to the electrophilic carbon of thiocyanate ion to form thiourea (McEwen, 1991). The general mechanism is shown in Scheme 2.

y y s

I I

R H

Scheme 2: General mechanism of synthesis thiourea derivatives

The choice of reagents such as acyl chlorides, thiols, amines and solvents are very important to synthesize desirable thiourea derivatives.

2.1.1.1 The choice of acyl chloride compounds

Wan Zullkiplee et al. (2014) reported that 2-[[3

carboxymethylcarbamothioylcarbamoyl)benzoyl] carbamothioylamino] acetic acid was successfuUy synthesized by using_yl chloride of isophthaloyl dichloride 3 as a starting material in acetone and acyl chloride of terepthaloyl dichloride 4 was used in synthesizing 3

[[ 4-(2-carboxyethylcarbamothioylcarbamoyl)benzoyl]carbamothioylamino] propanoic acid.

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CI Cl

3 4

Figure 3: IsophthaJoyl dichloride 3 and TerepthaloyJ dichloride 4

A thiourea derivative of I-phenyl-3-benzoyl-2-thiourea was successfully synthesized by a reaction of benzoyl isothiocyanate with aniline in benzene. Benzoyl isothiocyanate was produced from a starting material of benzoyl chloride 5 (Alkherraz et aI. , 2014).

5

Figure 4: Benzoyl chloride 5

2.1.1.2 Tbe cboice of tbiols

A reaction mixture using thiol of potassium thiocyanate (Figure 5) and starting material of 4

fluorobenzoyl chloride in acetone was allowed to cooi before secondary amine is added to the mixture in yielding 88% ofN,N-diet~-N'-4-fluorobenzoyl thiourea (Binzet et al., 2013).

K-S =:=N Figure 5: Potassium thiocyanate

In addition, Saeed et al. (2010) have reported the synthesis of novel 1-(3,4,5

trimethoxybenzoyl)-3-aryl thiourea derivative. The reaction was performed by a solution of 1

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(3,4,5-trimethoxy)benzoyl chloride mixed with potassium thiocyanate (Figure 5) to obtain intennediates which is then to be treated with a solution of substituted aniline.

Another type of reported thiols used in synthesis of thiourea derivatives was ammonium thiocyanate. Preparation of aroyl isothiocyanate was done from a mixture of aroyl chloride and ammonium thiocyanate (Figure 6). The aroyl isothiocyanate was treated with arene diamine by using a catalyst to yield arene-bis-aroyl thiourea of 1,4-phenylene-bis-benzoyl thiourea (Zhang et al., 2000).

H

w~

I H

Figure 6: Ammonium thiocyanate

2.1.1.3 The choice of amines

Various types of amines have been reported in the synthesis of thiourea. Wan Zullkiplee et al.

(2014) reported the synthesis of bis-thiourea derivatives by utilizing amino acid compound as a source of amines. One of the product synthesized was 2-[[4-[(2-hydroxy-1-methyl-2-oxo

ethyl)carbamothioylcarbamoyl]benzoyl]carbamothioylamino] propanoic. It was prepared by treating 4-acetylbenzoyl isothiocyaniJ,.. with amine group of L-alanine 6 (Figure 7).

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Figure 7: L-alanine 6

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Another type of amines used in the synthesis of thiourea derivatives is aromatic amines. For instance, 4-ethylaniline 7 was used to react with ethyl isothiocyanate to afford 80% yield of 1

ethyl-3-(4-ethylphenyl)thiourea (Yahyazadeh et at., 2013).

7

Figure 8: Aromatic amine (4-ethylaniline) 7

Aside from amino acids and aromatic amines, acetic acids have been reported to be used as the source of amines in the synthesis of thiourea. A study reported that the used of 4

(aminophenyl) acetic acid 8 treated with 4-fluorophenyl isothiocyanate to yield 67% of (4

{[(4-fluorophenyl)thiocarbamoyl]amino}phenyl) acetic acid (Celen et aI., 2011).

.. -

⁸

Figure 9: 4-(aminophenyl) acetic acid 8

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1.1.1.4 Tbe cboice of solvent

Solvent plays important role in synthesis of thiourea. There are few types of sol vent that have been reported to be used in the synthesis of thiourea. For instance, a study by Katritzky et al.

(2004) reported that refluxing I-benzotriazole-l-carbothioamide with p-anisidine in toluene 9 for 18 hours successfully yielded about 54% ofp-methoxyphenylthiourea. Structure of toluene is shown in Figure 3.

9

Figure 10: Toluene 9

In 2012, Katla et al. successfully synthesized thiourea derivative 2-«2-amino-6-oxo-1H

purine-9(6H)-yl)methoxy)ethyl-3-methyl-2-(3-phenylthioureido )butanoate in dry THF 10 with yield of 82%. The compound was reported to exhibit high Tobacco Mosaic Virus (TMV) inhibition (Katla et al .• 2012).

Q

10

Figure 11: Tetrahydrofuran (THF) 10

One of the common solvent used in the synthesis of thiourea derivatives is acetone (Patel et al., 2007, Saeed et aI., 2010). A reaction of 4-nitrobenzoyl chloride and ammonium

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thiocyanate with tetrabutyl ammonium bromide (TBAB) in acetone 11 successfully obtained 93% yield of 1-(4-nitrobenzyl)-3-(thiazol-2-yl) thiourea (Saeed et al., 2009).

A ^o

11

Figure 12: Acetone (propanone) 11

In 2014, Shoaib et al. reported that a reaction of benzoyl chloride and potassium thiocyanate

in acetone has been successfully synthesized to produce l-benzoyl-3-methyl thiourea in high yield of 91 %. I-benzoyl-3-methyl thiourea showed excellent analgesic activity as at 30 mg/kg oral dose, it is more potent with 100% analgesic activity compared to the standard analgesic drug diclofenac with only 91.08% activity.

Syamala. M. (2009) reported the used of acetonitrile 12 (Figure 13) as another common solvent in the synthesis ofthiourea derivatives.

12

tt)lgure 13: acetonitrile 12

Acetonitrile is used as a solvent in synthesizing thiourea because it produces higher yield compared to other solvents such as toluene and THF. Acetonitrile is one of the common solvent used in bimolecular nucleophilic substitution SN2 reaction because it is a polar aprotic solvent. It is highly polar due to the presence of nitrogen atom which is high in electronegativity.

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Synthesis, Characterisation and Antibacterial Activities of Tris-thiourea Derivatives