ABSTRACT
Palladium(II)thiosemicarbazone complexes are known to be good anticancer agents. However, their stability is a challenge during biological application because of faster ligand exchange kinetics. This research focused on synthesis of palladium complexes with bulky ligands to improve their stability. Thiosemicarbazone (TSC) ligands were synthesized via condensation reaction of aldehydes and respective amines. The following ligands were synthesized; (E)-N,N’- dimethyl-2-((5’-methyl-[2,2’-bithiophen]-5-yl)methylene)hydrazine-1-carbothioamide (L1), (E)- N-ethyl-2-((5-phenylthiophen-2-yl)methylene)hydrazine-1-carbothioamide (L2)and (E)-2- ((5’methyl)-[2,2’-bithiophen]-5-yl)methylene)-N-phenylhydrazine-1-carbothioamide (L3). Corresponding Pd(II) complexes were synthesized by reacting equimolar amounts of ligands and Pd(cod)Cl2. Complexes synthesized were; (E)-N,N’-dimethyl-2-((5’-methyl-[2,2’-bithiophen]-5- yl)methylene)hydrazine-1-carbothioamide palladium(II)chloride complex (C1), (E)-N-ethyl-2- ((5-phenylthiophen-2-yl)methylene)hydrazine-1-carbothioamide palladium(II)chloride complex (C2) and (E)-2-((5’methyl)-[2,2’-bithiophen]-5-yl)methylene)-N-phenylhydrazine-1- carbothioamide palladium(II)chloride complex (C3).Stability test for ligands and complexes were done using 1HNMR in DMSO-d6 within a period of 72 hours at an interval of 6 hours monitoring any peak change. Both ligands and complexes were characterized by UV-Vis, FTIR, 1H NMR, 13C NMR, elemental analysis and single crystal X-ray diffraction for L2. Single crystal X-ray crystallography revealed that the prepared ligand had E conformationand existed as thione tautomer. Elemental analysis result indicated that L1 and L2 coordinated to palladium metal in N S bidentate fashion while L3 in SNS tridentate fashion. UV-Vis studies were done on ligand to confirm formation of imine around 300nm (π→π* transition). UV-Visalso revealed Ligand to metal charge transfer (LMCT) transition confirmed successful coordination of ligands through S- M bond. The anticancer activities of ligands and complexes were screened against Caco-2, HT- 29, HeLa and KMST cell lines using cisplatin as a positive control. The ligands displayed low anticancer potency compared to corresponding complexes except for L1. L3 had the lowest inhibition to all cell lines with IC50> 100 µg/mL.However after complex formation, C3 showed inhibition; Caco-2 (IC50=84.32 µg/mL), HT-29 (IC50=49.10 µg/mL), HeLa (IC50=0.73 µg/mL) and KMST (IC50>100 µg/mL). All the cell lines displayed high susceptibility to L1; Caco-2 (IC50=6.814 µg/mL), HT-29 (IC50=6.449 µg/mL), HeLa (IC50=0.2619 µg/mL) and KMST (IC50=10.7900 µg/mL). Among the ligands and complexes C3 had selective anticancer properties. Caco-2 cell displayed some resistance to inhibition to all the compounds. The complexes had enhanced anticancer activities compared with the respective ligands except for L1. Synthesized palladium(II) complexes with bulky ligands were stable and were cytotoxic towards cancer cell lines.
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
LIST OF FIGURES x
LIST OF SCHEMES xii
LIST OF TABLES xiii
LIST OF ABREVIATIONS xiv
CHAPTER ONE
INTRODUCTION
1.0 Coordination compounds 1
1.1 Platinum Group Metals 2
1.2 Thiosemicarbazone palladium complexes 3
1.2.5 Heterocyclic thiosemicarbazone complexes of palladium (II) 3
1.4 Cancer 5
1.5 Statement of the problem 5
1.6 Objectives 6
1.6.1 General Objective 6
1.6.2 Specific Objectives 6
1.7 Justification of the study 6
CHAPTER TWO
LITERATURE REVIEW
2.0 Transition metal complexes 8
2.1 Stability of transition complexes 9
2.2 Transition metal complexes in catalysis 10
2.2.1 Iridium complexes 11
2.2.2 Copper complexes 13
2.2.3 Palladium complexes 13
2.3 Metal complexes in medicine 15
2.3.1 Gold complexes 15
2.3.2 Ruthenium complexes 16
2.3.3 Nickel complexes 17
2.4 Metals complexes in cancer treatment 19
2.5 Schiff base ligands 20
2.5. 1 Thiosemicarbazones 21
2.5.2 Heterocyclic thiosemicarbazones 24
2.5.3 Thiophene substituted thiosemicarbazones 26
2.6 Anticancer activities of palladiumcomplexes 27
CHAPTER THREE
MATERIALS AND METHODS
3.0 instrumentation 31
3.1 Chemicals 31
3.2 Synthesis of ligands 32
3.2.1 (E)-N,N’-dimethyl-2-((5’-methyl-[2,2’-bithiophen]-5-yl)methylene)hydrazine-1- carbothioamide (L1) 32
3.2.2 (E)-N-ethyl-2-((5-phenylthiophen-2-yl)methylene)hydrazine 1-carbothioamide (L2)32
3.2.3 (E)-2-((5’methyl)-[2,2’-bithiophen]-5-yl)methylene)-N-phenylhydrazine-1- carbothioamide (L3) 32
3.3 Synthesis ofthiosemicarbazone complexes of palladium 33
3.3.1 Synthesis of cis-cyclooctadienepalladium(II) chloride precursor 33
3.3.2 Synthesis of (E)-N,N’-dimethyl-2-((5’-methyl-[2,2’-bithiophen]-5- yl)methylene)hydrazine-1-carbothioamide palladium(II)chloride (C1) 33
3.3.3 Synthesis of (E)-N-ethyl-2-((5-phenylthiophen-2-yl)methylene)hydrazine-1- carbothioamide palladium(II)chloride (C2) 34
3.3.4 Synthesis of (E)-2-((5’methyl)-[2,2’-bithiophen]-5-yl)methylene)-N-phenylhydrazine- 1-carbothioamide palladium(II)chloride (C3) 34
3.4 Characterization of ligands and complexes 34
3.4.1 Melting point 34
3.4.2 Fourier Transform Infrared spectroscopy 34
3.4.3 UV-Visible spectroscopy 35
3.4.4 Nuclear Magnetic Resonance 35
3.4.5 Elemental analysis 35
3.4.6 Single X-ray crystallography 35
3.5 Determination of stability 36
3.6 Determination of anticancer activities 36
CHAPTER FOUR
RESULTS AND DISCUSSION
4.0 Thiosemicarbazones ligands 37
4.2 Physical properties for ligand L1 37
4.3 Characterization and structural confirmation of ligand L1 38
4.4 Physical properties for ligand L2 40
4.5 Characterization Structural confirmation of ligand L2 40
4.6 Physical properties for ligand L3 47
4.7 Characterization andstructural confirmation of ligand L3 47
4.8. Palladium (II) thiosemicarbazone complexes 48
4.9 Physical properties for complex C1 51
4.10 Characterization and structural confirmation of complex C1 52
4.11 Physical properties for complex C2 54
4.12 Characterization and structural confirmation of complex C2 55
4.13 Physical properties for complex C3 56
4.14 Characterization and structural confirmation of complex C3 56
4.15 Cytotoxicity studies 58
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.0 Conclusion 63
5.1 Recommendations 64
REFERENCES 65
APPENDICES 83
LIST OF FIGURES
Figure 2.1: Bonding modes of thiosemicarbazone 24
Figure 4.1: FT-IR spectrum for L1 38
Figure 4.2: UV-Vis spectrum for L1 39
Figure 4.3: 1H NMR spectra for L2 41
Figure 4.4: 13C NMR spectrum for ligand L2 42
Figure 4.5: DEPT spectra for L2 43
Figure 4.6: Crystal structure for L2 45
Figure 4.7: Tortion angles in thiophene and intramolecular H-bonding 46
Figure 4.8: FTIR spectra of prepared Pd(cod)Cl2 49
Figure 4.9: FTIR spectra of Pd(cod)Cl2 from the authentic source 50
Figure 4.10: FTIR spectra for complex C1 52
Figure 4.11: UV-Vis spectrum for complex C1 53
Figure 4.12:1H NMR spectrum for complex C1 54
Figure 4.13: Viability of HeLa cell line to compounds 59
Figure 4.14: Response of KMST cell line to treatment 59
Figure 4.15: Response of colon cancer (HT-29) upon treatment 60
Figure 4.16: Response of Caco-2 upon treatment 61
Figure 1: UV-Vis spectra for C2 83
Figure 2: UV-Vis spectra for C3 83
Figure 3: UV-Vis spectra for L2 84
Figure 4: UV-Vis spectra for L3 84
Figure 5: FTIR spectra for L3 85
Figure 6: FTIR spectra for C3 85
Figure 7: FTIR spectra for L2 86
Figure 8: FTIR spectra for C2 86
Figure 9: 1H NMR for L1 87
Figure 10: 13C NMR spectra for L1 87
Figure 11: 1H NMR spectra for C2 88
Figure 12: 13C NMR spectra for L3 88
Figure 13: 1H NMR spectra for L3 89
Figure 14: 1H NMR spectra for C3 89
LIST OF SCHEMES
Scheme 1.1: Neutral and Anionic forms 3
Scheme 2.1: Diels alder catalysis using copper (I) oxide 11
Scheme 2.2: Methoxytrifluoromethylation of styrene 12
Scheme 2.3: Mechanism of Suzuki coupling 14
Scheme 2.4: Reaction in thiosemicarbazone synthesis 22
Scheme 2.5: Reaction mechanism for aldehyde and primary amines 22
Scheme 3.1: Synthesis of Pd(cod)Cl2 33
Scheme 4.1: Synthesis of thiosemicarbazone ligands 37
Scheme 4.2: Synthesis of the complexes 51
LIST OF TABLES
Table 4. 1: 1D and 2D NMR data for L2 43
Table 4.2: Single-Crystal Data and Structure Refinement Parameters for L2 44
Table 4.3: Geometric parameters for ligand L2 47
Table 4.4. IC50 values (µg/mL) of compounds 61
LIST OF ABREVIATIONS
DCM Dichloromethane
DMF Dimethylformamide
DMSO Dimethylsulfoxide
DNA Dioxyribonucleic acid
EWG Electron withdrawing group
FTIR Fourier-transform infrared spectroscopy
HIV Human Immunodeficiency Virus
HOMO High Occupied Molecular Orbital
LUMO Low Unoccupied Molecular Orbital
MDR Multidrug resistance
NMR Nuclear magnetic resonance
PMG Platinum Group Metal
ppm parts per million
THF Tetrahydrofuran
TLC Thin Layer Chromatography
TSC Thiosemicarbazone
UV-Vis Ultraviolet-visible
CHAPTER ONE
INTRODUCTION
1.0 Coordination compounds
Coordination compounds have proved to be important in various aspects of life based on their wide application. These compounds are formed when ligands (electron donors) coordinate to the central metal through coordinate bonds. The chemistry of coordination compounds is naturally witnessed in hemoglobin (1) whichis a coordination complex of polyporphyrin and iron. Iron is coordinated to polyporphyrin compounds via nitrogen because of the presence of more labile lone pair of electrons for donation to form coordinate bonds.
1
It is responsible for oxygen transportation from the lungs to other parts of the body and carbon (IV)oxide from the body to the lungs. Other naturally available compounds include chlorophyll which is responsible for photosynthesis and metalloenzymes for biocatalysis (Helland et al., 2019).
Transition elements like iridium have versatile applications due to their ability to accept lone pairs of electronsinto their empty d-orbitals. Iridium ion exists in three stable oxidation states; Ir+, Ir3+ and Ir4+. Iridium ion is considered good catalyst in industrial application because they are resistant to corrosion; they are used in both acidic and basic medium. besides corrosion resistant, iridium complexes are used as industrial green catalyst because they are not environmental pollutants (Singh, 2016). For instance triscyclometalated iridium(III)2-(1-naphthyl)pyridine is used as a catalyst in methoxytrifluoromethylation of styrene (Njogu et al., 2019). Other catalytic applications includehydrogenation of benzene, amination of primary alcohols and 1,3-dipolar cycloaddition (Bayram et al., 2010).
Other reactions catalyzed by transition elements include Wacker process where PdCl2 is used in the conversion of olefins to aldehyde and Suzuki coupling where C-C bonds are formed. Palladium is used in Suzuki coupling and the reaction proceeds via Pd0-Pd2+ route (Lennox & Lloyd-Jones, 2014).
1.1 Platinum Group Metals
Platinum group metals, also known as platinum metals are elements of group VIIIB. They consist osmium, palladium, platinum, iridium, rhodium and ruthenium. The electronic configurations of these elements are Ru (4d7 5s1); Rh (4d8 5s1); Pd (4d10/ 4d85s2);Os (5d6 6s2); Ir (5d7 6s2); Pt (5d9 5s1). They have close nuclear sizes making them to have similar chemical and physical characteristics. Platinum group metals have various oxidation states however there stable oxidation states are; Ru2+/Ru3+/Ru4+,Rh3+, Pd2+/ Pd4+,Os3+/ Os4+, Ir4+/Ir3+, Pt2+,/Pt4+, and (Jain & Chauhan, 2016). These group of metals form sigma bonds with electron donors (ligands) like H2O, Cl-, NH3 and sulphur. They also bond with donors in pi-systems like aromatic heterocyclics, azometine, thiocarbonyls and carbonyls. They have wide industrial applications because of their high heat resistance, resistance to corrosion, high melting point and catalytic activities. This group of metals have wide application in medicinalfieldexample is cisplatin (2), platinum complexes used to treat cancer.
2
Palladium(II) complexes are also emerging as anticancer drugs as a result of platinum (II) complexes setbacks. One of the major setbacks of platinum(II) complexes as anticancer drug is that they are kinetically inert. Palladium(II) chelates especially with N,S donor ligands (for example thiosemicarbazones) are thermodynamically stable, they have high lability in bringing metal towards DNA and allowing it to interact with it. For these reasons, they are used as anticancer, antimalarial and antiviral drugs. Their mode of action is to prevent conversion of ribonucleotides to deoxyribonucleotides through inhibition of enzyme ribonucleotide reductase. (Munikumari et al., 2019).
1.2 Thiosemicarbazone palladium complexes
Thiosemicarbazones are Schiff base ligands with majorly sulfur and nitrogen as donor atoms. They are derived from a condensation reaction of aldehydes/ketones and thiosemicarbazides. Bidentate thiosemicarbazone ligands coordinate with the central metal forming five membered chelates. Their applications are due to their selectivity in coordination and good coordination tendency. They can interact with the metal either in anionic or in neutral form. The neutral forms are its two tautomers; thione and thiol and anionic after deprotonation of thiol as shown in Scheme1.1 below. The Rs can be alkyl group or H (Pal et al.,2002).
Scheme 1.1: Neutral and Anionic forms
1.2.5 Heterocyclic thiosemicarbazone complexes of palladium (II)
Pd2+thiosemicarbazone complexes with heterocyclic substituents such as thiophene have attracted interest in bioinorganic chemistry due to their potential biological activities such as antifungal, antimalarial, antiviral and anticancer. Pd2+ coordinates with ligands in the same mode as Pt2+ hence they are likely to form square complexes with thiosemicarbazone ligands. However, Pt2+complexes are more kinetically and thermodynamically stable than the corresponding Pd2+ complexes. Ligand exchange rate for Pd2+ complexes are 104 times quicker than platinum because it readily forms 5-coordinate intermediate complex. Faster kinetic property of palladium is related to its small atomic size compared to platinum, causing steric repulsion of ligands(Ali et al., 2017). Many Pd2+complexes have been synthesized using the standard condensation method, characterized and investigated to be potent towards cancer cells.
Some of these complexes include 5-nitroisatin thiosemicarbazone complex of palladium (3)
which proved to be active towards colorectal cancer (Munikumari et al., 2019).
3
The biological activities of thiosemicarbazone ligands which improves when coordinated with a metal make this group of compounds to be used as anticancer agents. Triapine (4) (3- aminopyridine-2-carboxaldehyde thiosemicarbazone) is one of thiosemicarbazone group of compounds currently on clinical trials against breast, leukemia, and cervical cancer (Nunes et al., 2020).
4
Its combination with cisplatin has improved the effectiveness of cisplatin as far as Multi Drug Resistance (MRD) is concerned. Triapine-cisplatin combination is currently showing exemplary anticancer activities on vaginal and cervical cancers (Enyedy et al., 2020). Thiophene substituted thiosemicarbazones have also proved to be cytotoxic against a number of cancer cell lines Mbugua et al(2020) found that (E)-1-((5-bromothiophen-2- yl)methylene)thiosemicarbazidepalladium(II) chloride (5) had anticancer activities against human cervical (HeLa), human colon (Caco-2) and breast cancer (MCF-7). The cytotoxicity of the complex was more enhanced compared to free ligand.
5
1.4 Cancer
Cancer is one of the leading causes of deaths word wide. Cancer incidences and mortality cases are rapidly increasing due to aging and population growth. The rate at which population increases, cancer is likely to be the leading cause of deaths replacing coronary heart diseases and stroke (Wilson et al., 2019). The malady is a burden to many nations because a lot of funds have to be allocated to curb it. The increasing cases of cancer are related to exposure to carcinogenic agents, some due to poverty and westernized lifestyles in developed nations according Bray et al. (2018). According to world health organization (WHO) 2018 statistics, lung cancer is the leading cause of deaths followed by breast cancer(Bray et al., 2018; Wilson et al., 2019).
In 2012, 14 million new cases of cancer are reported annually all over the world with 8.2 million deaths registered; 8.8 million deaths registered in 2015. The deaths are projected to 13 million by 2030 as a result of population growth, lack of exercise, poor lifestyle as well as increase in the rate of infection (Atieno et al., 2018).
Most of the existing anticancer drugs such as cisplatin face the challenge of multidrug resistance (MRD). MRD on chemotherapeutically potent drugs are associated with numerous mechanisms that include; increased drug efflux, decreased uptake of drug, activated DNA repair mechanism, activation of detoxifying systems, and drug induced apoptosis evasion among many more. Most of palladium thiosemicarbazone complexes are hydrophobic and insoluble in most organic solvents other than DMF and DMSO. This reason limits the biological applications of the ligands and complexes (Hosseini-Yazdi et al., 2017).
In this work various thiosemicarbazone ligands and their palladium complexes have been synthesized, characterized and their anticancer activities in various cell lines investigated. This work focuses on synthesis of stable thiosemicarbazone ligands and their palladium(II) complexes.
1.5 Statement of the problem
For a very long time, cisplatin has been the most potent metal-based drug towards tumor cells; lung, ovarian, neck, bladder and brain cancers. Other platinum based drugs that are active towards cancer cells include carboplatin (6) and oxaliplatin (7) (Prajapati &Patel, 2019).
6 7
Cisplatin and other existing cancer drugs have shortcomings including multidrug resistance and severe side effects like nephrotoxicity, nausea, neurotoxicity and ototoxicity. Non-selectivity is also a major shortcoming of the available cancer drugs since they kill non-cancerous cells too (Prajapati &Patel, 2019).
The chemistry of thiosemicarbazones and their transition metal complexes are of interest due to their modes of coordination and numerous biological activities such as anti-tumor, anti- microbial, anti-malarial and antibacterial activities. Thiosemicarbazone complexes of palladium are active towards tumor cells however, activity problem arising from instability is a challenge in drug development, for this reason research on new biologically active metal complexes with bulky ligands, which enhance stability is necessary is necessary (Matsinha et al., 2015).
1.6 Objectives
1.6.1 General Objective
Synthesize and determine the anticancer activities of thiosemicarbazones and their palladium complexes.
1.6.2 Specific Objectives
1. Synthesize thiosemicarbazone ligands.
2. Synthesize palladium (II) thiosemicarbazone complexes.
3. Determine the anticancer activities of the thiosemicarbazone ligands and palladium(II) complexes.
1.7 Justification of the study
Stability of palladium complexes is a challenge in biological application despite their anticancer activities. Palladium complexes have been reported to be cytotoxic towards breast and colorectal cancer cells by Munikumariet al., (2019). Palladium(II) complexes are square planar complexes where planarity is necessary for interaction with DNA and inhibition of replication. Therefore synthesis of thiosemicarbazone complexes of palladium(II), which are five-membered bulky chelates will improve the stability to act on pharmacological target without being structurally compromised (Munikumari et al., 2019). Palladium(II) complexes have also been reported to have anticancer activities same as those of cisplatin with reduced side effects like nephrotoxicity, neurotoxicity and ototoxicty (Qin et al., 2018).
Thiosemicarbazones derivatives of thiophene moiety are interesting owing to the structural and pharmacological activities such as anti-malarial, antifungal, antibacterial and antitumor (Munikumari et al., 2019). Nyawade et al.(2021) established that thiophene based thiosemicarbazones had effective anticancer activities towards cervical cancer cell lines (HeLa), human colon carcinoma cell lines (Caco-2), human colorectal adenocarcinoma cells (HT-29) and non-cancerous immortalized human cells (KMST). The anticancer activities of the ligands were enhanced after coordination with palladium(II).The presence of thiophene ring in the structure increases the flexibility of denticity due to presence of sulfur (donor atom). In this research work new thiophene based thiosemicarbazone ligands and palladium complexes were prepared and anticancer activities determined.
This research is aimed at reducing the cancer burdens all over the world by synthesizing anticancer palladium(II) complexes which are stable with bulky ligands. The prepared compounds will also be useful in pharmaceutical industries and manufacturing industries to enhance efficiency of production, contribution to the Kenya’s big four agenda (Universal Health Care and manufacturing) and realization of the good health and well-being, one of Sustainable Development Goals (SDG) of vision 2030.
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