ABSTRACT
Coordination compounds have great potential as drug molecules. However, their instability, poor water solubility, and multi-drug resistance limit their use. Unstable coordination compounds may undergo unnecessary ligand exchange reactions when introduced into biological systems. While literature reports that bulky ligands can help stabilize the metal center, their use with platinum metal centers has not been explored extensively. This work involved the synthesis of stable metal complexes to be used as anticancer agents. Four thiosemicarbazone ligands; 2-((5-ethylthiophen- 2-yl)methylene-N-phenylhydrazine carbothiomide (L1), 2-((5-ethylthiophene-2-yl)methylene)-1- methylhydrazine carbothiomide (L2), N,N–dimethyl–2-((4-nitrophene-2-yl)methylene)hydrazine carbothiomide (L3), and 2-([2,2’-bithiophen]-5-ylmethylene-1-methylhydrazine carbothiomide (L4) were synthesized through condensation reactions of aldehydes and amines. Their respective platinum(II) complexes 2-((5-ethylthiophen-2-yl)methylene-N-phenylhydrazine carbothiomide platinum(II) chloride (C1), 2-((5-ethylthiophene-2-yl) methylene)-1-methylhydrazine carbothiomide platinum(II) chloride (C2), N,N–dimethyl–2-((4-nitrophene-2- yl)methylene) hydrazine carbothiomide platinum(II) chloride (C3), and 2-([2,2’-bithiophen]-5- ylmethylene-1-methylhydrazine carbothiomide platinum(II) (C4) were synthesized by reacting the ligands with K2PtCl4. The synthesis reactions were all performed under mild conditions, and refluxing was the preferred reaction technique. The yields of the thiosemicarbazone ligands ranged between 89% and 99% while the yields of the complexes were between 77% and 88%. The yields of the complexes were significantly lower than those of the ligands, which can be attributed to the steric shielding of the heavy ligands to the platinum metal center. The compounds exhibited sharp melting points. The characterization techniques employed were: FTIR, UV-Vis, 1H NMR, 13C NMR, elemental analysis, and XRD. The ligands were bidentate and coordinated via sulphur and nitrogen atoms. The mode of coordination was established by the disappearance of the thioamide proton and changes in chemical shifts observed in the NMR spectra of the complexes. The anti-cancer activities of the thiosemicarbazone ligands and complexes were performed in vitro on four human cell lines; three cancerous cell lines (HeLa, Caco-2 and HT-29) and the non-cancerous KMST-6, and cisplatin was used as the positive control. The results revealed that three ligands (L1, L2, and L4) had less anti-cancer activity compared to their complexes. L3 was lethal to Caco-2 (IC50 = 3.412 µg/mL), HT-29 (IC50 = 0.6886 µg/mL) and HeLa (IC50 = 0.107 µg/mL) cell lines at lower concentrations than cisplatin. However, L3 was also lethal to KMST-6 (IC50 = 2.1 µg/mL). Complexes exhibited varying anti- cancer activity on the different cell lines. C1 had a low inhibition value of (IC50 = 45.42 µg/mL) for the Caco-2 cell line. C2 showed inhibition values of; (IC50 = 31.63 µg/mL) for HT-29 and (IC50 = 41.82 µg/mL) for Caco-2 cell line, suggesting that it had better activity than cisplatin, which had inhibition values (IC50 = 35.5 µg/mL) for HT-29 and (IC50 = 48.83 µg/mL) for Caco-2 cell lines. C3 showed inhibition values of (IC50 = 0.0001973 µg/mL) for HeLa, suggesting better activity than cisplatin, whose inhibition was (IC50 = 0.1099 µg/mL). Altogether, this study shows that these some of these thiosemicarbazone (II) complexes offer a promising alternative to other platinum complexes in cancer therapy.
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
LIST OF FIGURES xi
LIST OF SCHEMES xii
LIST OF TABLES xiii
LIST OF ABBREVIATIONS xiv
CHAPTER 1
INTRODUCTION
1.1 Background Information 1
1.1.1 Application of Metal Complexes 1
1.1.2 The Cancer Burden 2
1.1.3 Coordination Complexes Used in Cancer Treatment 2
1.1.4 Thiophene-Based Platinum Thiosemicarbazone Complexes as the Future of Cancer Therapies 4
1.2 Statement of the Problem 6
1.3 Objectives 6
1.3.1 General Objective 6
1.3.2 Specific Objectives 6
1.4 Justification and Significance of the Study 7
CHAPTER 2
LITERATURE REVIEW
2.1 Applications of Metal Complexes 8
2.2 Cancer 11
2.2.1 Cancer Statistics 11
2.2.2 Cancer drug resistance 12
2.3 The Use of Platinum Compounds in Cancer Treatment 13
2.3.1 The Development of Cisplatin Analogs 15
2.3.2 Chiral platinum complexes 17
2.3.4 New Platinum Drugs Under Pre-Clinical Development and Clinical Trials 17
2.4 Thiophene-based Compounds and their Applications 19
2.4.1 Thiophene Derivatives as Anticancer Agents 20
2.5 Thiosemicarbazone Ligands and Complexes 20
2.5.1 Applications of Thiosemicarbazone Ligands and Complexes 23
2.5.2 The Use of Thiosemicarbazone ligands and Complexes in Cancer Treatment 23
2.6 Synthesis of thiosemicarbazone ligands and their complexes 25
CHAPTER 3
MATERIALS AND METHODS
3.1 Chemicals 28
3.2 Equipment 28
3.3 Synthesis of Thiosemicarbazone Ligands (L1-L4) 28
3.3.1 Synthesis of 2-((5-ethylthiophen-2-yl)methylene-N-phenylhydrazinecarbothiomide (L1) 29
3.3.2 Synthesis of 2-((5-ethylthiophene-2-yl) methylene)-1-methylhydrazine carbothiomide (L2) 29
3.3.3 Synthesis of N,N–dimethyl–2-((4-nitrophene-2-yl)methylene)hydrazinecarbothiomide (L3) 30
3.3.4 Synthesis of 2-([2,2’-bithiophen]-5-ylmethylene-1-methylhydrazinecarbothiomide (L4) 31
3.4 Synthesis of Thiosemicarbazone Complexes (C1-C4) 32
3.4.1 Synthesis of 2-((5-ethylthiophen-2-yl)methylene-N-phenylhydrazinecarbothiomide platinum(II) chloride (C1) 32
3.4.2 Synthesis of 2-((5-ethylthiophene-2-yl) methylene)-1-methylhydrazine carbothiomide platinum(II) chloride (C2) 33
3.4.3 Synthesis of N,N–dimethyl–2-((4-nitrophene-2-yl)methylene)hydrazinecarbothiomide platinum(II) chloride (C3) 33
3.4.4 Synthesis of 2-([2,2’-bithiophen]-5-ylmethylene-1-methylhydrazine carbothiomide platinum(II) (C4) 34
3.5. Characterization of the Ligands and Complexes 35
3.5.1 Fourier Transform Infrared Spectroscopy (FTIR) 35
3.5.2 UV-Visible Spectroscopy (UV-Vis) 35
3.5.3 Nuclear Magnetic Resonance Spectroscopy 35
3.5.4 Elemental Analysis (C, H, N, S) 36
3.5.5 Single Crystal X-ray Crystallography 36
3.6 Anticancer Screening of Ligands and Complexes 36
3.6.1 Preparation of Solutions for the Ligands and Complexes 36
3.6.2 Cell culture 36
3.6.3 Morphological Evaluation 37
3.6.4 MTT Assay 37
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Background 38
4.2 Characterization of Ligands 38
4.2.1 Characterization and Structure of 2-((5-ethylthiophen-2-yl)methylene-N- phenylhydrazine carbothiomide (L1) 39
4.2.2 Characterization and Structure of 2-((5-ethylthiophene-2-yl) methylene)-1- methylhydrazinecarbothiomide (L2) 52
4.2.3 Characterization and Structure of N,N–dimethyl–2-((4-nitrophene-2- yl)methylene)hydrazinecarbothiomide (L3) 56
4.2.4 Characterization and Structure of L4 58
4.2.5 Summary of the Ligands 60
4.3 Characterization of Complexes 61
4.3.1 Characterization and Structure of 2-((5-ethylthiophen-2-yl)methylene-N- phenylhydrazinecarbothiomide platinum(II) chloride (C1) 61
4.3.2 Characterization and Structure of 2-((5-ethylthiophene-2-yl) methylene)-1- methylhydrazine carbothiomide platinum(II) chloride (C2) 64
4.3.3 Characterization and Structure of N,N–dimethyl–2-((4-nitrophene-2- yl)methylene) hydrazinecarbothiomide platinum(II) chloride (C3) 65
4.3.4 Characterization and Structure of 2-([2,2’-bithiophen]-5-ylmethylene-1- methylhydrazine carbothiomide platinum(II) (C4) 67
4.3.5 Summary of the Complexes 68
4.4 Anticancer Studies 69
4.4.1 Cytotoxicity Assays 69
4.4.2 Cell Viability Graphs 71
4.4.3 Morphological Changes Observed in the Cells 73
CHAPTER 5
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion 76
5.2 Recommendations 77
REFERENCES 79
APPENDICES 85
Appendix (I) – FTIR Spectra 85
Appendix (II) - UV-Vis Spectra 88
Appendix (III) – 1H NMR Spectra 92
Appendix (IV) - 13C NMR Spectra 98
Appendix (V) - XRD data for L2 100
LIST OF FIGURES
Figure 2.1: Thione-thiol tautomerism of thiosemicarbazones 21
Figure 4.1: ATR-IR spectrum of 5-ethyl-2-thiophenecarboxyaldehyde 39
Figure 4.2: FTIR spectrum for L1 40
Figure 4.3: UV-Vis peaks for L1 41
Figure 4.4: 1H NMR spectrum for 5-ethyl-2-thiophenecarboxyaldehyde 42
Figure 4.5: 1H NMR spectrum for L1 43
Figure 4.6: 13C NMR peaks for L1 45
Figure 4.7: DEPT spectra for L1 46
Figure 4.8: HSQC peaks for L1 47
Figure 4.9: HMBC peaks for L1 48
Figure 4.10: COSY peaks for L1 50
Figure 4.11: XRD structure for L2 55
Figure 4.12: Crystal packing 55
Figure 4.13: FTIR peaks for C1 61
Figure 4.14: UV-Vis peaks for C1 62
Figure 4.15: 1H NMR peaks for C1 63
Figure 4.16: HeLa viability graph 71
Figure 4.17: Caco-2 viability graph 72
Figure 4.18: KMST-6 viability graph 73
Figure 4.19: Illustration of observed morphological changes 74
LIST OF SCHEMES
Scheme 3.1: Synthesis of L1 29
Scheme 3.2: Synthesis of L2 30
Scheme 3.3: Synthesis of L3 31
Scheme 3.4: Synthesis of L4 31
Scheme 3.5: Synthesis of C1 32
Scheme 3.6: Synthesis of C2 33
Scheme 3.7: Synthesis of C3 34
Scheme 3.8: Synthesis of C4 35
LIST OF TABLES
Table 4.1: Summary of the 2D NMR data for L1 49
Table 4.2: Peak assignments made from the COSY spectrum 51
Table 4.3: Crystal data and refinement 54
Table 4.4: IC50 (µg/mL) values for ligands and complexes 70
LIST OF ABBREVIATIONS
ATR-IR Attenuated Total Reflectance Infrared
Caco-2 Colon Carcinoma (Human Colorectal Adenocarcinoma Cells)
CFSE Crystal Field Stabilization Energy
Cisplatin Cis-dichlorodiammineplatinum
COSY Correlated Spectroscopy
DEPT Distortionless Enhancement by Polarization Transfer
DMF Dimethylformamide
DMSO Dimethyl Sulfoxide
DNA Deoxyribonucleic Acid
EDTA Ethylenediaminetetraacetic Acid
FTIR Fourier Transform Infrared Spectroscopy
Hela Henrietta Lacks (Cervical Cancer Cell Line)
HMBC Heteronuclear Multiple Bond Correlation
HOMO Highest Occupied Molecular Orbital
HSQC Heteronuclear Single Quantum Coherence Spectroscopy
HT-29 Human Colorectal Adenocarcinoma Cell Line
IC50 Half-Maximal Inhibitory Concentration
IR Infrared Spectroscopy
KBr Potassium Bromide
KMST-6 Human Fibroblast Cell Line
LUMO Lowest Unoccupied Molecular Orbital
MLCT Metal to Ligand Charge Transfer
NMR Nuclear Magnetic Resonance
TMS Tetramethylsilane
TSC Thiosemicarbazone
UV-Vis Ultraviolet - Visble
XRD X-Ray Diffraction
CHAPTER 1
INTRODUCTION
1.1 Background Information
1.1.1 Application of Metal Complexes
Coordination compounds have been studied for several decades now, emphasizing their synthesis, characterization, and applications. Their unique properties, such as varying oxidation states, distinct colours, magnetic susceptibility, and volatility, have made the compounds attractive in many fields. One of these areas is catalysis, like in the case of triscyclomatalated homoleptic iridium(III) complexes (Njogu et al., 2019). The iridium complexes can be installed in equipment that utilizes solar energy, thus helping to conserve energy. Metal complexes have also been used in environmental control, such as crown ether chelates (Odhiambo et al., 2018), which can detect the heavy metals they have a high affinity to.
Another use of coordination metal complexes is in biological applications, where they have been used to treat several ailments. For instance, the gold complex, auranofin (Jurca et al., 2017), treats rheumatoid arthritis. Lanthanum carbonate is a compound used as a phosphate binder in clinical practice and has been used on patients with severe kidney diseases. The biological activity of the compounds has been found to increase with chelation, which proves the importance of using complexes and not only the ligands on their own.
Coordination compounds have been widely used as anticancer drug agents such as cisplatin, nedaplatin, and carboplatin (Parkin et al., 2021). Although many cancer treatments are available, they are prescribed based on the cancer type and how advanced it is. Many patients use a combination of therapies to achieve the best results. While newer therapies like stem cell therapy and immunotherapy are coming up, chemotherapy remains a superior treatment method because it is a systematic approach to treating cancer (Jurca et al., 2017). This means that the drug travels to all body parts and kills all metastasized cells, making it ideal for cure, palliation, or control purposes (Dasari & Tchounwou 2014).
1.1.2 The Cancer Burden
In GLOBCAN survey of 2020, as summarized by Sung et al. (2021), cancer was responsible for ten million deaths in 2020 alone. An estimated nine million new cancer cases were reported in 2020. In the same year, female breast cancer was reported to have been the most commonly diagnosed cancer type, not lung cancer as reported in the previous year (Sung et al., 2021). Out of the 183 countries involved in the GLOBCAN study, cancer was the primary cause of death for people under 70 years old in 112 nations. The statistics indicate that cancer is a prominent cause of death and is a considerable impediment to increasing life expectancy across the globe. As the world’s population continues to increase, it is expected that the global cancer burden will continue to rise. Therefore, it is no doubt that the need for speedy solutions and effective cancer therapies is a priority.
1.1.3 Coordination Complexes Used in Cancer Treatment
The earliest complex used in cancer treatment was Cis-dichlorodiammineplatinum (cisplatin) (1), which had chloro and amino ligands and a platinum metal center (Pitt et al., 2016).
Before its introduction, scientists had not managed to control the toxic effects of metal-based drugs while harnessing their benefits (Dasari & Tchounwou 2014). This made cisplatin a significant success. When cisplatin is introduced into a biological system, the chlorine atoms are quickly replaced by water molecules to bind with DNA since they are more labile than the amino groups (Kellinger et al., 2013). However, sometimes the amino ligands on the cisplatin molecule are replaced by Sulphur-containing bioligands, abundant in the body. When this happens, the drug changes its form and cannot treat cancer as it was intended to. This usually causes drug resistance, and the patient may not get better.
The challenges posed by cisplatin prompted the development of cisplatin analogs known as platins (Zalba & Garrido, 2013). Their mode of action requires them first to undergo aquation. One or more ligands is replaced with water molecules in the body, allowing the compound to bind to DNA and prevent replication and transcription. This causes the cancer cells to die. One of the first analogs was the nedaplatin (2) (Shimada et al., 2013), which has amine carrier ligands like cisplatin, but the leaving group is a bulky five-membered ring compound.
2
Its mechanism is similar to cisplatin because it prevents DNA duplication, but nedaplatin is more stable and has a shorter elimination life than cisplatin (Niioka et al., 2007). Another analog is carboplatin (3) (Liu et al., 2014), which has good chemical stability, due to its large ligands that increase steric hindrance.
Fewer doses of carboplatin are required to treat cancer than what is needed when using cisplatin (Zalba & Garrido, 2013). Oxaliplatin (4) (Lévi et al., 2012), a second-generation cisplatin analog, has a diamino cyclohexane ligand and a platinum metal center, increasing the compound’s chemical stability.
4
Oxaliplatin is a significant upgrade from cisplatin because it doesn’t have a high resistance profile like cisplatin (Graham et al., 2004). Several platinum-based drugs are under clinical trials like dicyloplatin (5) (Yu et al., 2014), a carboplatin analog under evaluation.
5
Oxaliplatin has three ligands, and the extra ligand slows down the rate of the molecule’s aquation in the body, thus stabilizing it (Yu et al., 2014). The LA-12 compound (6) is a platinum (IV) drug under clinical trial (Bouchal et. al., 2011).
The chloro ligands of LA-12 undergo aquation for it to bind to DNA. Interestingly, this molecule's acetate groups are released in the biological system; the compound is reduced to platinum(II). Another drug under clinical examination is phenanthriplatin (7) (Kellinger et al., 2013), 40 times more efficient than cisplatin (Johnstone et al., 2014).
7
1.1.4 Thiophene-Based Platinum Thiosemicarbazone Complexes as the Future of Cancer Therapies
From the literature, it is clear that platinum-based drugs have superior biological properties, especially when treating cancer. As cancer becomes a global problem, platinum metal complexes to treat cancer could help reduce the cancer burden. Unfortunately, out of the thousands of newly synthesized platinum complexes, only a few make it to clinical trials because many are unstable (El-Saied et al, 2019). In addition, many complexes are only effective on specific cancer types, hence the need to synthesize new compounds and test them for their anti-cancer properties to ease the cancer challenge.
While complexation plays a crucial role in developing drug molecules, the ligands used have a significant impact, such as the compound’s stability. For instance, steric hindrance is desired when designing molecules for anti-cancer agents because it prevents unnecessary ligand substitution reactions (Kayed et al., 2016). It allows improved selectivity when the compound is binding to DNA. That is why thiosemicarbazone ligands are the desired option when designing potential anti-cancer compounds.
Previous use of thiophene-based compounds in biological applications such as anti-cancer, anti- inflammatory, anti-anxiety, anti-mitotic, anti-psychotic, anti-fungal, anti-arrhythmic, and anti- microbial shows great success. Substituting the thiophene ring (8) with bulky substituents shows improved anti-proliferative activity (Oliveira et al., 2015).
8
While substituting the thiophene ring on positions 2 and 3 has been widely studied, there is limited research on substitution on position 5, which requires some attention. Research by Mbugua et al. (2020) emphasizes the potential of using thiosemicarbazone ligands with a substituted thiophene ring as anticancer agents. From the study, the introduction of bromine to the thiophene ring showed better activity.
The high steric hindrance offered by thiosemicarbazone ligands, coupled with the anti- proliferative activity of thiophene and the success of platinum-based complexes, is likely to yield stable compounds to be used drug molecules. The use of thiophene-based thiosemicarbazone platinum complexes for anti-cancer application is yet to be well studied, despite showing great potential. That is what this study seeks to achieve.
1.2 Statement of the Problem
Coordination complexes have great potential for use as drug molecules, such as anticancer agents, with statistics showing that almost half of all cancer patients receive a platinum-based drug (Sava & Dyson, 2006). Although countless complexes have been synthesized, many are unstable. This has led to issues with resistance because the compounds undergo unnecessary ligand exchange reactions and do not reach the target cells in their intended state. The challenge has prevented the full utilization of metal complexes as medicinal agents.
Literature suggests that using bulky ligands increases steric hindrance and consequently reduces the ligand substitution reaction rate. In this case, the ease of displacing the ligands reduces significantly, and the compound can get to the target cells with minimal alterations. However, the use of bulky ligands has not been studied extensively.
Therefore, it is essential to synthesize new platinum complexes with better stability to make them more useful, particularly for biological applications. Thiosemicarbazone complexes could offer a suitable solution. TSC ligands are highly sterically hindered and good anti-cancer agents with anti-proliferative properties. Therefore, newly synthesized thiosemicarbazone platinum(II) complexes are likely to be stable enough for use as anticancer agents.
1.3 Objectives
1.3.1 General Objective
To synthesize and screen for the anti-cancer activities of novel platinum(II) thiosemicarbazone complexes.
1.3.2 Specific Objectives
1. To synthesize thiosemicarbazone ligands
2. To synthesize platinum(II) thiosemicarbazone complexes
3. To screen for the anti-cancer activities of the thiosemicarbazone ligands and their platinum complexes against HeLa, Caco-2, HT-29, and KMST-6 cell lines.
1.4 Justification and Significance of the Study
Cancer is a leading cause of death and it contributes to disease and economic burden globally. While chemotherapy is a desired approach to fighting the disease, multi-drug resistance and undesired side effects decrease its effectiveness.
Platinum complexes, such as thiosemicarbazone complexes, have shown potential as good anti- cancer agents. Since thiosemicarbazone ligands are bulky, they are unlikely to undergo unnecessary ligand exchange reactions. The steric hindrance TSC ligands exhibit may also increase their selectivity when used as drugs. Platinum(II) is suitable for biological applications because it is relatively inert and has a low ligand exchange rate. Therefore, when thiosemicarbazone platinum(II) complexes are introduced in a biological system, the ligands aren’t replaced easily, which means that the drug is likely to get to the target cells in its intended state.
It is expected that this research will contribute to Universal Health care which is one of the big four agendas because of their medicinal application. Since the study contributes to cancer development, it will also enhance the achievement of good health and well-being, which is anchored in the Sustainable Development Goals (SDGs).
Research, innovation and scientific research is one of the sectors that Vision 2030 seeks to reform to achieve a prosperous nation and improve the quality of life. Therefore, this research will contribute positively to the achievement of innovation goals as stated in this plan
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