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SYNTHESIS AND CHARACTERISATION OF MULTI-SUBSTITUTED
THIOUREA DERIVATIVES AND THEIR ANTIBACTERIAL
ACTIVITIES
WAN SHARIFATUN HANDAYANI BINTI WAN ZULLKIPLEE
Master of Science
(Organic Chemistry)
2014
Faculty of Resource Science and Technology
SYNTHESIS AND CHARACTERISATION OF MULTI-SUBSTITUTED THIOUREA
DERIVATIVES AND THEIR ANTIBACTERIAL ACTIVITIES
WAN SHARIFATUN HANDAYANI BINTI WAN ZULLKIPLEE
A thesis submitted in fulfillment of the requirement for the degree of Master of Science
Faculty of Resource Science and Technology
UNIVERSITI MALAYSIA SARAWAK
2014
i
Declaration
I hereby declare that no portion of the work referred to in this dissertation has been submitted
in support of an application for another degree of qualifications of this or any other university
or institution of higher learning.
____________________
Wan Sharifatun Handayani Binti Wan Zullkiplee
Department of Chemistry
Faculty of Resource Science and Technology
University Malaysia Sarawak
ii
List of Publications
1. Wan Zullkiplee, W. S. H., Ngaini, Z., Mohd Arif, M. A., Hussain, H. & Abdul Halim, A. N.
(2014). Bis-thiourea bearing aryl and amino acids side chains and application on antibacterial
activities. Phosphorus, Sulfur, and Silicon and the Related Elements, 189(6), 832-838.
2. Ngaini Z., Mohd Arif, M. A., Wan Zullkiplee, W. S. H., Hussain, H., Rosli, M. M. (2013).
N,N0-Bis(phenylcarbamothioyl)benzene-1,3-dicarboxamide. Acta Crystallographica, E69,
1374-1375.
iii
Acknowledgements
I would like to express my biggest gratitude and appreciation to my supervisor, Madam Maya
Asyikin Mohd Arif and my co-supervisor, Assoc. Prof. Dr. Zainab Ngaini for their constant
guidance, generous supervision, encouragement and advices throughout the completion of this
research.
I would like to thank Assoc. Prof. Dr. Wan Mohd Khairul Wan Mohamed Zin from Universiti
Malaysia Terengganu for providing CHNS elemental analysis services. I also thank Prof.
Abdul Razak Ibrahim from Universiti Sains Malaysia for providing facilities used for x-ray
crystallography study.
My warmest thank to my parents (Wan Zullkiplee and Hasiah Baseri) for their abundance
love, supports and inspirations. Great deals appreciated go to all my lab mates especially
Vannessa Lawai, Asyilla Nordin, Nurul Qhalila, Farid Noh, Farra Sharom, Farahen Jamil,
Carolynne Sie and Noorien Qaseeda for their helpful cooperation and cheerful
encouragement.
Finally, I would like to convey my appreciation toward Yayasan Tunku Abdul Rahman
Cawangan Sarawak for the scholarship and thanks to Universiti Malaysia Sarawak and
Ministry of Science, Technology and Innovation for the financial support through
FRGS/01(22)/754/2010(40).
iv
Synthesis and characterisation of multi-substituted thiourea derivatives and their
antibacterial activities
Wan Sharifatun Handayani
ABSTRACT
Two series of bis-substituted thiourea derivatives 40a-e and tri-substituted thiourea
derivatives 42a-i have been successfully synthesised by reaction of isothiocyanate
intermediates with series of amines. 1,3-benzenedicarbonyl chloride 9 and 1,3,5-
benzenetricarbonyl chloride 46 were utilized as a scaffold to prepare the isothiocyanate
intermediates and formed 40a-e and 42a-i respectively. The synthesis of proposed hexa-
substituted thiourea derivatives was done by reacting hexachlorocyclotriphosphazene 4 as a
precursor and formed six unexpected thiourea derivatives 43a-f. The preparation of
unexpected thiourea products was triggered by the formation of HCl in the reaction system
due to existence of free chlorine atoms which mildly dissociated from 4. The synthesis of the
proposed hexa-substituted thiourea derivatives was continued via aminolysis reaction of 4
with single thiourea compounds 43a-i but the reaction was unsuccessful. All synthesised
products were characterised using CHNS elemental analysis, FTIR, 1H NMR,
13C NMR and
31P NMR spectroscopic techniques.
Antibacterial assay of all synthesised products was carried out using turbidimetric method
against the growth of Escherichia Coliform sp. (E. coli). The results indicated that only
compounds 42a-i which bear three thiourea moieties showed significant antibacterial
activities. The effects of constituents and structure of the investigated compounds on the
antibacterial performance were discussed.
v
Sintesis dan pencirian sebatian multi-terrganti tiourea dan aktiviti anti-bakteria
Wan Sharifatun Handayani
ABSTRAK
Dua siri tiourea dan terbitannya iaitu bis-penukarganti tiourea 40a-e dan tri-penukarganti
tiourea 42a-i telah berjaya dihasilkan melalui tindakbalas diantara sebatian pertengahan
isotiosianat dan kumpulan amina. 1,3-benzendikarbonil klorida 9 dan 1,3,5-benzendikarbonil
klorida 46 telah digunakan sebagai reaktan untuk membentuk sebatian pertengahan
isotiosianat dan seterusnya menghasilkan sebatian terbitan 40a-e dan 42a-i. Penghasilan
sebatian cadangan heksa-penukarganti tiourea telah dijalankan dengan menggunakan
heksaklorotrifosfazena 4 sebagai reaktan dan telah menghasilkan enam sebatian terbitan
tiourea 43a-f yang tidak dijangkakan. Pembentukan sebatian terbitan tiourea 43a-f ini
adalah disebabkan oleh pembentukan HCl semasa tidakbalas tersebut yang disebabkan oleh
kehadiran ion klorin yang dilepaskan daripada 4 secara perlahan. Penghasilan sebatian
cadangan heksa-penukarganti tiourea telah diteruskan melalui tindakbalas aminolisis
diantara sebatian 4 dan sebatian terbitan tiourea 43a-i tetapi tindakbalas tersebut juga tidak
berjaya. Semua struktur sebatian yang telah berjaya dihasilkan dikenalpasti dengan
menggunakan analisa CHNS, spektroskopi FTIR, 1H NMR,
13C NMR dan
31P NMR.
Ujian saringan anti-bakteria untuk semua sebatian ke atas tahap pertumbuhan bakteria
Escherichia Coliform sp. (E. coli) telah dijalankan menggunakan kaedah turbidimetrik.
Keputusan ujian menunjukkan bahawa hanya sebatian terbitan 42a-i yang mengandungi tiga
moieti tiourea telah mempamerkan aktiviti anti-bakteria yang memberangsangkan. Oleh itu,
pengaruh kandungan dan struktur sebatian hasilan terhadap tahap aktiviti anti-bakteria telah
dibincangkan.
vi
Table of Contents
Page
Declaration i
List of Publications ii
Acknowledgement iii
Abstract / Abstrak iv
Table of Contents vi
List of Abbreviations x
List of Tables xii
List of Figure xiii
List of Schemes
Xv
Chapter 1: Introduction 1
1.1 Thiourea background and its applications 1
1.2 Multi-substituted thiourea derivatives 2
1.2.1 Acyl Chloride Compounds 3
1.2.2 Phosphazenes 3
1.3 Objectives of study
5
Chapter 2: Literature Review 6
2.1 Preparation of thiourea 6
2.1.1 The choice of reagents 7
2.1.1.1 Acyl chloride compounds 7
vii
2.1.1.2 Phosphazenes as scaffold mimicking acyl chlorides 9
2.1.2 The choice of thiols 12
2.1.3 The choice of amines 14
2.1.4 The choice of solvents 17
2.2 Application of thiourea derivatives 17
2.2.1 Thiourea derivatives with antibacterial activity 17
2.2.2 Thiourea derivatives with ion-selective activity 19
2.2.3 Thiourea derivatives with antitumor activity 20
2.2.4 Thiourea derivatives with antiviral activity 21
2.2.5 Thiourea derivatives with anticancer activity 22
2.3 Inroduction of E. coli 23
Chapter 3: Experimental 24
3.1 General methods 24
3.1.1 Reagents, solvents and reaction conditions 24
3.1.2 Physical measurement 25
3.2 Synthesis of 1,3-bis carbamothioyl derivatives (40a-e) 26
3.3 Synthesis of 1,3,5-tricarbamothioyl derivatives (42a-i) 31
3.4 Attempted synthesis of 1-(4-methylcyclohexa-1,5-dien-1-yl)-3-
[2,4,4,6,6-pentakis(phenylcarbamothioylamino)-1,3,5-triaza-
triphosphacyclohexa-1,3,5-trien-2-yl]thiourea
40
3.5 Synthesis of aromatic thiourea and carbamothioylamino acid
derivatives (43a-f)
41
3.6 Synthesis of chlorophenyl thiourea series (43g-i) 47
3.7 Antibacterial screening 50
viii
Chapter 4: Results and Discussion 51
4.1 Synthesis of 1,3-bis carbamothioyl derivatives 40a-e as a model
Reaction
51
4.1.1 Preparation of 1,3-bis carbamothioyl derivatives 40a-e 52
4.2 Synthesis of 1,3,5-tricarbamothioyl derivatives 42a-i 57
4.2.1 Preparation of 1,3,5-tricarbamothioyl derivatives 42a-i 58
4.3 Synthesis of hexa-substituted cyclotriphosphazenes-based thiourea
derivatives 43a-f
63
4.3.1 Attempted synthesis of cyclotriphosphazenes-based thiourea
43a-f
64
4.3.1.1 Strategy 1 64
4.3.1.2 Strategy 2 65
4.4 Synthesis of hexa-substituted cyclotriphosphazenes based thiourea
derivativesvia aminolysis reactions.
75
4.4.1 Preparation of compounds 43 via reaction of amino and
thiocyanato groups in the presence of HCl (Venkatesh &
Pandeya, 2009)
76
4.4.2 Attempted synthesis of hexa-substituted cyclotriphosphazenes-
based thiourea derivatives via aminolysis reaction
80
4.5 Biological Studies 83
4.5.1 Antibacterial activities of compounds 43a-i 84
4.5.2 Antibacterial activities of compounds 40a-e 88
4.5.3 Antibacterial activities of compounds 42a-i 92
ix
Chapter 5: Conclusion 97
Recommendations
98
Chapter 6: References
99
Appendix A: FTIR spectra of synthesised compounds 104
Appendix B: 1H NMR spectra of synthesised compounds 116
Appendix C: 13
C NMR spectra of synthesised compounds 128
Appendix D: 31
P NMR spectra of synthesised compounds 140
Appendix E: Antibacrerial data and inhibition growth graphs 141
Appendix F: Publications 165
x
List of Abbreviations
δ Chemical shift in parts per million
b.p boiling point
13C Carbon Nuclear Magnetic Resonance
CHNS Carbon Hydrogen Nitrogen Sulfur
Elemental Analysis
d Douplet
DMSO-d6 Deutarated Dimethyl sulfoxide
EtOH Ethanol
FTIR Fourier Transform Infrared
h Hour
HCl Hydrochloric acid
1H Proton Nuclear Magnetic Resonance
Hz Hertz
J Coupling constant in Hertz
KBr Potassium bromide
KCl Potassium chloride
m Multiplet
m Meta
mHz mega Hertz
mmol mili mol
xi
m.p melting point
nm nano meter
NMR Nuclear Magnetic Resonance
o Ortho
31P Phosphorus Nuclear Magnetic Resonance
ppm parts per million
p Para
rt room temperature
s Singlet
t Triplet
THF Tetrahydrofuran
TLC Thin Layer Chromatography
UV Ultra-violet
νmax Maximum vibrational frequency
xii
List of Tables
Page
Table 4.1: Physical properties and analytical data for compound 40a-e 52
Table 4.2: CHN microelemental analysis data for compound 40a-e 53
Table 4.3: Physical properties and analytical data for compound 42a-i 58
Table 4.4: CHN microelemental analysis data for compound 42a-i 59
Table 4.5: Physical properties and analytical data for compounds 43a-f 67
Table 4.6: CHN microelemental analysis data for compounds 43a-f 67
Table 4.7: Physical properties and analytical data for compounds 43g-i 77
Table 4.8: CHN microelemental analysis data for compound 43g-i 77
Table 4.9: Minimum inhibitory concentration (MIC) for compounds 42a-i 95
xiii
List of Figures
Page
Figure 1.1: General structure of thiourea 1
Figure 1.2: Tautomeric forms of thiourea 1
Figure 1.3: Basic unit for phosphazenes 4
Figure 1.4: Hexachlorocyclophosphazenes 4
Figure 2.1: The molecular structure of cycloalkylamines 11
Figure 2.2: The thiocarbonyl transfer reagents 13
Figure 2.3: The molecular structures of piperidine 27 and N-ethylethanamine
28
15
Figure 2.4: Compound 33 with antibacterial activity 18
Figure 2.5: Bis thiourea with antimycobacterial properties 18
Figure 2.6: The molecular structure of 35 as neutral ionophore 19
Figure 2.7: Compound 36 with antitumor properties 20
Figure 2.8: Complex of 37 with antitumor properties 21
Figure 4.1: FTIR spectrum of 40c 54
Figure 4.2: 1H NMR spectrum of 40a 55
Figure 4.3: 13
C NMR spectrum of 40c 56
Figure 4.4: FTIR spectrum of compound 42a 60
Figure 4.5: The 1H NMR spectrum of compound 42e 61
Figure 4.6: The 13
C NMR spectrum of compound 42c 62
Figure 4.7: FTIR spectrum of compound 43c 69
Figure 4.8: 1H NMR spectrum of compound 43c 70
xiv
Figure 4.9: 13
C NMR spectrum for compounds 43e
71
Figure 4.10: Single x-ray crystallography structure of compound 43d and 43e 72
Figure 4.11: FTIR spectrum of (4-chlorophenyl) thiourea 43i 78
Figure 4.12: 1H
NMR spectrum of (2-chlorophenyl) thiourea43g 79
Figure 4.13: 13
C NMR spectrum of (3-chlorophenyl) thiourea 43h 80
Figure 4.14: Growth of E. coli in media containing compound 43b 84
Figure 4.15: Growth of E. coli in media containing compound 43d 85
Figure 4.16: Growth of E. coli in media containing compound 43g 85
Figure 4.17: Growth of E. coli in media containing compound 43h 86
Figure 4.18: Minimum inhibitory concentration of compounds 43a-i 87
Figure 4.19: Growth of E. coli in media containing compound 40a 88
Figure 4.20: Growth of E. coli in media containing compound 40c 89
Figure 4.21: Growth of E. coli in media containing compound 40d 89
Figure 4.22: Growth of E. coli in media containing compound 40e 90
Figure 4.23: Minimum inhibitory concentration of compounds 40a-e 91
Figure 4.24: Growth of E. coli in media containing compound 42b 92
Figure 4.25: Growth of E. coli in media containing compound 42d 93
Figure 4.26: Growth of E. coli in media containing compound 42g 93
Figure 4.27: Growth of E. coli in media containing compound 42h 94
Figure 4.28: Minimum inhibitory concentration of compounds 42a-i 95
xv
List of Schemes
Page
Scheme 2.1: The general reaction for the synthesis of thiourea compounds 6
Scheme 2.2: The preparation of thiourea derivative 8 from mono acyl
chloride compound
8
Scheme 2.3: The preparation of thiourea derivative 10 from diacyl
chloride compound
9
Scheme 2.4: The synthesis of cyclic trimeric isothiocyanato phosphazenes 11 10
Scheme 2.5: Aminolysis reaction of 4 by aromatic primary amines 11
Scheme 2.6: The synthesis of 22 from primary amine compound 14
Scheme 2.7: The synthesis of thiourea derivatives from simple aliphatic
Amines
15
Scheme 2.8: The synthesis of aspirin-thiourea derivatives from the reaction
with amino acids
16
Scheme 2.9: The preparation of compound 38 22
Scheme 2.10: Bis thiourea derivatives with anticancer activity 22
Scheme 4.1: A proposed pathway to synthesise 40a-e 51
Scheme 4.2: The synthetic pathways of 1,3,5-tricarbamothioyl derivatives
42a-i
57
Scheme 4.3: A proposed synthetic pathway of hexa-substituted
cyclotriphosphazenes-based thiourea 43a-f
63
Scheme 4.4: The proposed attempt of the synthesis of 43a-f 64
xvi
Scheme 4.5: The proposed attempt of the synthesis of 43a-f via heating
method
66
Scheme 4.6: The synthesis of unexpected 43a-f 73
Scheme 4.7: Mechanism on the formation of 43a-f triggered by HCl
moieties
74
Scheme 4.8: The reaction pathway for the formation of the adduct 75
Scheme 4.9: Planned synthesis route for aminolysis reaction of 4 75
Scheme 4.10: Synthesis of compounds 43g-i 76
Scheme 4.11: Alternative pathway to synthesise hexa-substituted cyclo
triphosphazenes-based thiourea compounds
81
1
CHAPTER 1
INTRODUCTION
1.1 Thiourea background and its applications
Thiourea 1 is an organic compound that has three main functional groups which are amine,
imine and thiol with the general molecular structure of (H2N)(H2N)C=S. It is also known as
thiocarbamide or sulfourea. It usually exhibits as white solid compound with molecular
weight of 76.12 g/mol. Thiourea can be synthesised by reacting amino and thiocyanato groups
in a suitable solvent. Figure 1.1 shows the basic molecular structure of thiourea. Thiourea
occurs as a mixture of two tautomers, S=C(NH₂)₂ (thiourea) 1 and HS=CNHNH₂ (isothiourea)
2, as shown in Figure 1.2.
1
Figure 1.1: General structure of thiourea
+SH
H2N NH -
Thiourea Isothiourea
1 2
Figure 1.2: Tautomeric forms of thiourea
2
Thiourea is a well known compound which is used as herbicides, pesticides, rodenticides,
vulcanization accelerator, and scaffold in organic synthesis reaction (Mohanta et al., 1999).
Thiourea moiety consists of two points of reactivity which are amines (NH) and thiol group
(C=S) (Zhong et al., 2008). In the field of biochemistry, thiourea derivatives have been
reported to possesss biological activities such as antibacterial (Saeed et al., 2009), antitumor
(Manjula et al., 2009), and antiviral (Küçükgüzel et al., 2008). Thiourea derivatives were also
reported for their ion-selective ability (Nishizawa et al., 1998), and widely used in the
production of ion electrodes and receptors.
1.2 Multi-substituted thiourea derivatives
Synthesis of thiourea derivatives have been widely explored by researchers due to their useful
and unique properties. Research has been focused on multi-substituted thiourea has become a
focus as they display a wide range of pharmacological properties. Multi-substituted thiourea
could be prepared by utilizing a scaffold via acyl chlorides. Bis-substituted thiourea
derivatives are the example of compounds which contain two thiourea moieties and reported
to possesss antimicrobial activities (Fernandez et al., 2005, Sharma et al., 2010). In the
synthesis of multi-substituted thiourea, acyl chlorides compounds are commonly used as
starting materials for preparation of thiocyanate intermediates (Arslan et al., 2009). There is
also a report on the usage of phosphazenes compounds as a precursor in the preparation of
multi-substituted thiourea (Allcock et al., 1991).
3
1.2.1 Acyl Chloride Compounds
Acyl chloride, a compound with general structure R(C=O)Cl, is the most reactive among acyl
derivatives. The chlorine atom is a very good leaving group and can be easily replaced by
nucleophiles to produce various types of desirable acid derivatives (Solomon & Fryhle, 2008).
Due to this property, acyl chloride derivatives are preferable in the synthesis of thiol
intermediates that are vital in the production of thiourea compounds.
1.2.2 Phosphazenes
Phosphazenes are well known phosphorus-nitrogen containing compounds with various
interesting properties and has been extensively studied (Bissessur et al., 2003). Phosphazenes
can exist as linear or cyclic forms which are soluble in organic solvents. Phosphazenes contain
phosphorus-nitrogen group with two substituents connected to the phosphorus atom. The
types of substituent groups that attached to the phosphorus atom could influence the physical
and chemical behaviors of the phosphazenes compound (Skaggs et al., 1995). General
phosphazenes unit 3 is shown in Figure 1.3, where substituent R can be either halogen groups
such as chlorine, fluorine and bromine or other organic groups such as alkyl, aryl, alkylamino
and alkoxy (Allcock, 1972). Thomas et al. (1995) also reported the use of pyrozolyl group as
a substituent on phosphorus atoms.
4
R
R
3
Figure 1.3: Basic unit for phosphazenes
Hexachlorocyclotriphosphazenes 4 (Figure 1.4) is an example of well known phosphazenes
compounds. It exists as white crystalline powder that is also known as phosphonitrilic
chloride trimer with molecular weight of 347.66 g/mole.
4
Figure 1.4: Hexachlorocyclophosphazenes
Research on hexachlorocyclotriphosphazenes have emerged rapidly over time and greatly
contributes to wide range of applications (Gleria & Jaeger, 2004, Ngaini & Abdul Rahman,
2010). One interesting chemical property of hexachlorocyclotriphosphazenes is the presence
of six reactive P-Cl bonds, which are multi-functional peripheries that easily weaken by
nucleophilic substitution and applicable for hexa-substituted thiourea derivatives (Allcock,
1972).
5
1.3 Objectives of study
Many studies have been reported on the synthesis and charactherisation of mono thiourea
compounds for pharmacological properties. Based on the literature precedent, the presence of
many thiourea moieties could further enhance the biological properties of thiourea derivatives
(Fernandez et al., 2005).
Therefore, this study embarked on the following objectives:
a) To synthesise di and tri-substituted thiourea derivatives using diacyl chloride and
triacyl chloride respectively.
b) To synthesise hexa-substituted thiourea using hexachlorocyclotriphosphazenes as a
precursor.
c) To characterise the synthesised compounds using CHNS elemental analysis, FT-IR,
1H NMR,
13C NMR and
31P NMR.
d) To study the biological activity of the synthesised compounds against Escherichia coli
sp. (ATCC 8739).
6
CHAPTER 2
LITERATURE RIVIEW
2.1 Preparation of thiourea
There are various methods on the synthesis of thiourea derivatives have been reported. The
common route of synthesising thiourea is by reacting amines 6 with isothiocyanates group 5
in an appropriate solvent. The strong electrophilicity of 5 enables these heterocumulenes to
take part in addition reaction which is favorable in the preparation of thiourea compound
(Lopez et al., 2003). The general reaction for the synthesis of thiourea compounds is shown in
Scheme 2.1.
5 6
NH2-RK+SCN- RR
R R
Scheme 2.1: The general reaction for the synthesis of thiourea compounds
The initial stage on the synthesis of thiourea compounds is the preparation of isothiocyanates
intermediate. The acyl chloride compounds were commonly utilized as starting materials for
the preparation of the intermediates due to the reactivity of the chlorine atom which is easily
replaced by nucleophiles attack. Different types of acyl chloride compounds were reported to
be used such as aliphatic and aromatic acyl chloride compounds (Arslan et al., 2009). The
backbone structure for the final thiourea compounds is based on the structure of acyl chloride
used as a scaffold.