Dissertations / Theses on the topic 'Antimalarials'

The rise in resistance to current antimalarial drugs has led researchers to consider drug repositioning as a quicker alternative for drug development and discovery. Preliminary drug repositioning screens carried out at the University of Salford identified calcium channel blockers (CCBs) as potential antimalarial agents. A growing body of evidence has demonstrated the importance of calcium within the Plasmodium life cycle. Studies have shown CCBs and calmodulin inhibitors to exhibit antimalarial activity. The research carried out in this project aims to evaluate the antimalarial efficacy, safety profile, mode of action and drug interactivity of the commercially available CCB and calmodulin inhibitor fendiline, and a range of its synthetic analogues. Initial screening of fendiline alone and in combination with commercially available drugs was carried out using a SYBR Green (SG) plate reader assay. Both CalcuSyn-based combination studies and a chloroquine potentiation assay were carried out. This was succeeded by the synthesis of fendiline analogues, which were carried out via a two-step synthetic route starting with a palladium catalysed coupling reaction followed by a reductive amination. Both the antimalarial activity and the cytotoxicity of the synthesised compounds were evaluated which led to a lead candidate to be selected (the hydroxy fendiline analogue, 4c). Further investigations into the activity, stage specificity and the effect compound 4c has on the hERG channel was carried out to develop a preliminary understanding of the mode of action of the compound. Finally, optimisation experiments to develop a flow cytometry-based assay that would detect fluctuations in calcium levels within infected red blood cells (RBCs) were performed. The conducted research showed the commercially available fendiline to have activity towards the multi-drug resistant Plasmodium falciparum K1 strain within the micromolar range (IC₅₀ = 3.74 ± 0.64 μM). CalcuSyn-based combinations studies showed fendiline to have either an antagonistic or additive effect with currently available drugs. Interestingly, fendiline was found to reverse chloroquine resistance, similar to verapamil, however at half the concentration required for verapamil. Furthermore, the range of synthesised fendiline analogues identified several compounds that exhibited more activity towards the P. falciparum infected RBCs. The 2’ hydroxyl fendiline analogue (4c) was 5.6-fold more potent than fendiline itself (IC₅₀ = 0.67 ± 0.21 μM) on the P. falciparum K1 strain, with an almost one-hundred-fold difference between antimalarial activity and cytotoxicity. The compound was found to be slow acting that targets the schizont stages of the parasite blood stages. The hERG channel inhibition assay gave an IC₅₀ of 4.03 ± 0.52 μM, which is within the range that most small compounds fall within (1-10 μM). Finally, the optimisation experiments showed the developed method was only sensitive to dramatic calcium changes within RBCs and not within the parasites themselves. Further work is required to improve the sensitivity of the assay. In conclusion, the hydroxy fendiline compound provides an interesting candidate to investigate further as a combinatory partner with other antimalarials, and as a scaffold to synthesise other potentially more potent fendiline analogues.

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Neste trabalho estudamos dois grupos de moléculas: as anilinoquinolinas e as sulfonamidas, inibidores do Plasmodium causador da malária, com o objetivo de correlacionar a estrutura eletrônica com a atividade antimalarial. Em nossas buscas utilizamos métodos empíricos e semi-empíricos para o estudo conformacional e obtenção dos descritores eletrônicos. Também aplicamos vários métodos estatísticos como: Regressão Linear Simples e Múltipla, Análise de Componentes Principais (PCA) e Análise Discriminante Linear (LDA), para verificar uma possível correlação estrutura-atividade dessas moléculas. Os resultados apontaram os descritores eletrônicos mais relevantes na classificação das moléculas antimalariais.
In this work we study two groups of antimalarial compounds: the anilinoquinolines and sulfonamides, aiming the correlation of the electronic structure with the antimalarial activity. In our studies we employ empirical and semi empirical quantum chemistry methods for the geometry optimization and calculation of the electronic descriptors. Also we employed the statistical methods Simple and Multiple Linear Regression, Principal Component Analysis (PCA) and Linear Discriminating Analysis (LDA), to verify the existence of a possible structure-activity correlation for these compounds. The results of this work have pointed out the best electronic descriptors in the classification of the active compounds.

Introduction. Age-based dosing of antimalarials is a widely used alternative to weight-based dosing practices. However, it is rarely taken into account in the drug development phase and standardized methods to devise age-based proxies that optimize the effectiveness and safety of antimalarial drugs do not exist. This has contributed to the variation in existing age-based regimens for antimalarials, at times resulting in poor, but widely-used regimens. Inaccurate dosing poses a threat to the individual (treatment failure, adverse effects) and the population (emergence and spread of resistance). Objectives. To address the lack of standardization and optimization in the development of age- based dosing regimens for antimalarials through the creation of regional weight-for-age growth ,,;,. .... references and the development and testing of a tool that uses these r~rer;JCes to calculate optimized age-based dosing regimens to inform drug developers and policy makers. Methods. To meet the objectives the following three steps were taken. First, a population representative reference data set was compiled. Secondly this data set was used to model regionally representative growth references. Finally these references were applied in a modelling tool to optimise age-based dosing regimens. Together with a team of expert statisticians, I developed a method to model multi-source anthropometric data to generate regional weight-for-age reference curves. I compiled a weight-for-age reference database from malaria-endemic countries using population representative data from health surveys and individual studies. An extension of the new generalized additive model for location, scale and shape (GAMLSS) was developed to generate smoothed country level curves, interpolating missing data from adjacent age categories and neighbouring countries with similar weigh-for- age distributions. These were combined in a finite mixture model weighted by population size or by population at risk of malaria to obtain regional weight-for-age growth references. In combination with drug specific parameters such as the therapeutic dose range and tablet strength, and regimen specific criteria such as the number of age categories and the use of tablet fractions, I determined age cut-offs that would result in regimens with the lowest number of patients receiving drugs outside the therapeutic range. The tool was initially used to support the development of age-based dosing regimens for the new fixed-dose combination of artesunate+mefloquine for Africa, Latin America and the Asia-Pacific regions and for specific countries (i.e. Cambodia and Brazil). I then evaluated the predicted dosing accuracy of all age- based ACT regimens recommended in the 2nd edition of the WHO malaria treatment guidelines; artemether+lumefantrine (AL), dihydroartemisinin+piperaquine (DHA+PPQ) and artesunate (AS) plus amodiaquine (AQ), mefloquine (MQ) or sulfadoxine-pyrimethamine (SP). Alternative age- thresholds were evaluated to further optimize the regimens. Results. Robust region specific weight-for-age references were created using >900.000 measurements from 167 data sources and 67 countries. The models accurately predicted observed regional weight-far-age distributions. Significant inter-regional variation existed in growth patterns (e.g. delayed growth spurt in Asia compared to Africa and Latin America) and attained growth. Dosing accuracies varied greatly by age and drug. ACT regimens with narrow therapeutic ranges (including MQ, AL, PPQ,) likely result in considerable dosing outside WHO recommended dose ranges. Suboptimal drug ratios in fixed-dose drug combinations contribute to low dose accuracies. Conclusions. Our reference and regimen modelling tools for the design of age-based dosing recommendations provide drug developers and policy makers with a powerful decision-support tool to optimize the safety and effectiveness of antimalarials dosed by age. The methods can further be extrapolated to other drugs from the WHO essential medicines list, and can be improved by incorporating pharmacokinetic data and safety data from post marketing surveillance when they become available. This work was supported by grants from the Liverpool School of Tropical Medicine, the British Medical Research Council, the European and Developing Countries Clinical Trials Partnership and the Drugs for Neglected Disease initiative.

Includes bibliographical references (leaves 118-124).
Based on a previous study, arylpiperazines (2-chlorophenylpiperazine, 2-ethoxyphenylpiperazine and phenylpiperazine) were found to be significantly more potent against the chloroquine-resistant (K1) strain than against the chloroquine-sensitive(DIO) strain. In other studies, 8-hydroxy-2-(di-n-propylamino)tetralin (8-0H-DPAT) has been identified as a potential antimalarial agent for the inhibition of the 5-hydroxytryptamine type 1A receptor in Plasmodium falciparum. A number of arylpiperazines are also known to target this receptor in other systems. Coupled with the potential role of arylpiperazines as replacements for the antimalarial 8-OH-OPA T, these results prompted a further investigation into the antiplasmodial properties of a broader range of simple un substituted and substituted arylpiperazines against a broader range of chloroquine-sensitive and chloroquine-resistant strains of PlasmodiumJalciparum.

Aurein is a cationic antimicrobial peptide, rich in phenylalanine residues. Although the peptide has been extensively studied, its mechanism of action is not fully understood and has not been established. This project is focused on studying the interactions of aurein with model biological membranes and antimalarials using Fourier Transform Infrared (FTIR), fluorescence, dynamic light scattering (DLS), atomic force microscopy (AFM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. FTIR data revealed conformational changes to the secondary structure of the peptide in the presence of the model membranes. The strongest interactions of aurein were found with DOPC and lipid raft systems. Fluorescence data revealed some differences in the mechanism of interaction between aurein and lipid rafts. Topographical analysis was performed using Atomic force microscopy (AFM). AFM images of the peptide with its lipid rafts showed a change in surface roughness suggesting a different mechanism of interaction. DLS data in agreement with FTIR confirmed that aurein interacts differently with the lipid rafts. The results gathered from this study provided new insights on the interaction of aurein. On the other hand, drug-drug interaction issues continue to present a major dilemma for the clinician caring for complex patients such as those infected with infectious disease. This study has examined the interaction of aurein with quinine, primaquine, and chloroquine. Significant interactions between aurein and antimalarials occured at a higher concentration of antimalarials. Interactions between aurein and antimalarials reveal a strong interaction between aurein and primaquine. Interactions between aurein and quinine or chloroquine were found to be weak and negligible. FTIR, TGA, and DSC may be used in a complementary way to gain insights into the possible drug-drug interactions involving aurein. These studies are needed to initiate in vivo controlled interaction studies between antibiotics and antimalarials.

Malaria is an infectious, often fatal disease that afflicts nearly 200 million people every year. The disease, characterized by recurring and extreme flu-like symptoms, is caused by the protozoan parasite Plasmodium falciparum. Victims usually contract the disease through a mosquito vector. Chloroquine is a chemotherapeutic that was introduced in the 1940s. For many years the drug was the foremost treatment of malaria, being effective and producing few side effects. Unfortunately, tolerance to chloroquine developed when the parasite evolved a resistance mechanism. Newer drugs have been developed and implemented, but these medicines also show a decreasing effect with continued administration. It is imperative that a new pipeline of drugs be developed in order to combat the disease and anticipated resistance. Reversed chloroquines are a new class of multiple-ligand compounds that are active against chloroquine-sensitive and chloroquine-resistance malaria species. This thesis describes research targeted at the modification of lead reversed chloroquine molecules to discover new and effective moieties, as well as to improve pharmacokinetic-related properties. An especial emphasis of this project is the addition of a sulfonamide functional group to a reversed chloroquine. Preliminary evidence indicates that this is a promising direction for this line of research. Brief discussions of some reversed chloroquine characterization studies are included in appendices.

The development of Plasmodium falciparum malarial resistance to the current armoury of anti-malarial drugs requires the development of new treatments to help combat this disease. The marine environment is a well established source of potential pharmaceuticals. Of interest to us are isonitrile, isocyanate and isothiocyanate compounds isolated from marine sponges and molluscs which have exhibited nano-molar anti-plasmodial activities. Through quantitative structure-activity relation studies (QSAR), a literature precedent exists for a pseudoreceptor model from which a pharmacophore for the design of novel anti-malarial agents was proposed. The current theory suggests that these marine compounds exert their inhibitory action through interfering with the heme detoxification pathway in P. falciparum. We propose that the computational methods used to draw detailed conclusions about the mode of action of these marine compounds were inadequate. This thesis addresses this problem using contemporary computational methodologies and seeks to propose a more robust method for the rational design of new anti-malarial drug compounds that inhibit heme polymerization to hemozoin. In order to investigate the interactions of the marine compounds with their heme targets, a series of modern computational procedures were formulated, validated and then applied to theoretical systems. The validations of these algorithms, before their application to the marine compound-heme systems, were achieved through two case studies. The first was used to investigate the applicability of the statistical docking algorithm AutoDock to be used for the exploration of conformational space around the heme target. A theoretical P. falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) enzyme model, constructed by the Biochemistry Department at Rhodes University, provided the ideal model to validate the AutoDock program. The protein model was accordingly subjected to rigorous docking simulations with over 30 different ligand molecules using the AutoDock algorithm which allowed for the docking algorithm’s limitations to be ascertained and improved upon. This investigation facilitated the successful validation of the protein model, which can now be used for the rational design of new PfDXR-inhibiting anti-plasmodial compounds, as well as enabling us to propose an improvement of the docking algorithm for application to the heme systems. The second case study was used to investigate the applicability of an ab initio molecular dynamics algorithm for simulation of bond breaking/forming events between the marine compounds and their heme target. This validation involved the exploration of intermolecular interactions in a naturally occurring nonoligomeric zipper using the Car-Parrinello Molecular Dynamics (CPMD) method. This study allowed us to propose a model for the intermolecular forces responsible for zipper self-assembly and showcased the CPMD method’s abilities to simulate and predict bond forming/breaking events. Data from the computational analyses suggested that the interactions between marine isonitriles, isocyanates and isothiocyanates occur through bond-less electrostatic attractions rather than through formal intermolecular bonds as had been previously suggested. Accordingly, a simple bicyclic tertiary isonitrile (5.14) was synthesized using Kitano et al’s relatively underutilized isonitrile synthetic method for the conversion of tertiary alcohols to their corresponding isonitriles. This compound’s potential for heme detoxification inhibition was then explored in vitro via the pyridine-hemochrome assay. The assay data suggested that the synthesized isonitrile was capable of inhibiting heme polymerization in a similar fashion to the known inhibitor chloroquine. Attempts to synthesize tricyclic analogues of 5.14 were unsuccessful and highlighted the limitation of Kitano et al’s isonitrile synthetic methodology.

Thesis (Ph. D.)--West Virginia University, 2002.
Title from document title page. Document formatted into pages; contains viii, 146 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 125-142).

The following work investigates two mechanistic proposals for the metal-catalysed reactions of allylic hydroperoxides. A heterolytic mechanism, involving bond migration, and a radical mechanism, involving a peroxy radical intermediate were considered. A study of the cleavage reaction of cyclic allylic hydroperoxides indicated that the outcome was substrate and catalyst dependent. Treatment of the six membered cyclic allylic hydroperoxide 129 with various metal catalysts led to the formation of the aldol 131 as the major product. However, similar treatment of the five-membered cyclic allylic hydroperoxide 130 led to the formation of a variety of indiscriminate products. Further, treatment of the tertiary hydroperoxide 144 with various metal catalysts led to the formation of the epoxy alcohol 155. The results obtained from reactions involving the cleavage of simple cyclic allylic hydroperoxides bear significant similarities with metal-catalysed reactions of hydroperoxides derived from fatty acids and the methyl ester of qinghao acid. An examination of the involvement of the heterolytic pathway in metal catalysed reactions of qinghao acid methyl ester hydroperoxide 77 was also undertaken. Metal-catalysed reactions involving a presumed enol ether intermediate of the heterolytic pathway were examined under both nitrogen and oxygen. The reactions yielded a variety of products which were found to be solvent dependent. The aldol products 169 were efficiently formed as a result of treatment of the enol ether 158 with copper(II) trifluoromethane sulfonate in dichloromethane/acetonitrile (5:1). In acetonitrile, under prolonged exposure to oxygen, oxygenation products 160 and 178 were observed. Further, it was found that one of the aldol diastereomers 169a underwent oxygenation in the presence of copper(II) trifluoromethane sulfonate to yield the peroxyhemiaceta l 161. A mechanistic pathway for the oxygenation of the aldol 169 via the formation of an oxetanol intermediate was best able to account for the observation that only one aldol diastere omer underwent oxygenat ion. Low temperature NMR experiments of the cleavage reaction of qinghao acid methyl ester hydroperoxide 77 to the dicarbonyl compound 81 revealed the intermediacy of two distinct intermediates which were identified as the triflyl enol ether acetal 213 resulting from bond migration and, the aldol 207. An oxetanol intermediate was proposed to account for the transformation from the triflyl enol ether acetal 213 to the aldol 207. Further, an investigatory metal catalysed, intermolecular reaction with ethyl vinyl ether and cyclohexanone led to the formation of a proposed trioxane 200. The formation of such a product implicates the involvement of an electron transfer reaction to generate a radical cation intermediate.

Malaria can be regarded as one of the world's worst health problems and its incidence is rising inexorably. It already accounts for the deaths of approximately three children every minute. This situation is exacerbated by the increased frequency of parasite resistance to current antimalarial agents and necessitates the development of new drugs to combat this disease P. falciparum possesses a plastid-like organelle, termed the apicoplast, which contains a small, highly reduced 35kb genome encoding tRNA, DNA polymerases and ribosomal proteins. Nuclear proteins are targeted to the apicoplast using clearly defined N-terminal signal and target peptide sequences. This led to the discovery that the apicoplast may be the site of at least two anabolic pathways isoprenoid synthesis and Type II fatty acid synthesis (FAS). This system is also present in bacteria and plants and differs significantly from the Type I FAS system found in humans. This makes the pathway an attractive target for novel antimalarials. Furthermore, the antibiotic thiolactomycin, inhibits the growth of bacteria via inhibition of Type II FAS. This project describes the characterisation of pfFabF and pfFabH, the p-ketoacyl-ACP synthase enzymes in the Type II FAS pathway of P. falciparum. One of these proteins, pfFabF, was shown to be the target of thiolactomycin inhibition. This enzyme was also capable of catalysing the final step of the unsaturated FAS pathway. Thiolactomycin derivatives synthesised in this project were tested against cultures of the parasite and both the condensing enzymes of P. falciparum. Significant improvements in the inhibitory activity against parasite cultures and one of the condensing enzymes were achieved compared to thiolactomycin. Although the inhibitory effects of these compounds was only in the micromolar range, this study demonstrates that thiolactomycin derivatives may provide lead compounds for the inhibition of P. falciparum by targeting either of the p-ketoacyl-ACP synthase proteins.

Malaria is arguably one of the main medical concerns worldwide because of the numbers of people affected, the severity of the disease, and the complexity of the life cycle of its causative agent, the protozoan Plasmodium sp. The clinical, social and economic burden of malaria has led for the last 100 years to several waves of serious efforts to reach its control and eventual eradication, without success to this day. At present, administration methods of antimalarial drugs release the free compound in the blood stream, from where it can be significantly removed by many tissues and organs, thus reducing its availability for Plasmodium-infected erythrocytes. Due to this lack of specificity regarding the target cells, current oral or intravenous delivery approaches for most antimalarial drugs require high doses. However, unspecificity of toxic drugs demands low concentrations to minimize undesirable side-effects, thus incurring the risk of sublethal doses favouring the appearance of resistant pathogen strains. Targeted nanovector systems can fulfill the objective of achieving the intake of total doses sufficiently low to be innocuous for the patient but that locally are high enough to be lethal for the malaria parasite. With the advent of nanoscience, renewed hopes have appeared of finally obtaining the long sought-after magic bullet against malaria in the form of a nanovector for the targeted delivery of antimalarial drugs exclusively to Plasmodium-infected cells. We work on the development of antimalarial drug-carrying nanovectors specifically targeted to Plasmodium-infected red blood cells (pRBCs). Our first immunoliposomal prototype delivers its contents exclusively to pRBCs containing the P. falciparum late forms trophozoites and schizonts, and improves on average tenfold the efficacy of the antimalarial drugs chloroquine and fosmidomycin. Using chloroquine concentrations well below its IC50, and by modifying parameters such as liposome size, density of targeting antibodies on the liposome surface, targeted antigen, and intraliposomal drug concentration, we approach 100% of parasitemia reduction both in vitro and in vivo using a murine model for P. falciparum malaria. We are working in the improvement of the nanovector through modification of (i) the targeting element: better antibodies, non-protein molecules such as DNA aptamers and polysaccharides, (ii) the encapsulated drug(s), and (iii) the type of nanocapsule, making special emphasis on polymeric structures. Our objective in the short term is the design of a nanostructure adequate to enter the preclinical pipeline as an economically affordable new antimalarial therapy.
Desarrollo de nanovectores para la liberación dirigida de antimaláricos Los métodos actuales de administración oral o intravenosa requieren dosis elevadas que a menudo desencadenan efectos secundarios perniciosos. Por el contrario, el riesgo de suministrar dosis subletales a causa de dichas concentraciones terapéuticas críticas o por razones de inestabilidad del compuesto, favorece la aparición de cepas resistentes de Plasmodium. La liberación dirigida de antimaláricos es una aproximación prometedora para evitar ese riesgo. El trabajo presentado en esta tesis doctoral tiene como objetivo principal el desarrollo de un nanovector para la mejora de la eficacia de los antimaláricos existentes y la comprensión de los parámetros fundamentales de su diseño que determinan la eficacia de dicho nanovector. Liposomas con quantum dots en su interior y que han sido funcionalizados con hemi-anticuerpos contra formas tardías del parásito se unen en menos de 90 minutos a eritrocitos infectados por Plasmodium y liberan su contenido en el interior de las células diana. Cuando se encapsulan fármacos antimaláricos en el modelo inmunoliposomal, se incrementa hasta diez veces la eficacia de los fármacos. La formulación para administración oral de anticuerpos y liposomas es complicada, nanovectores adecuados para esta vía de administración serían una contribución valiosa para el tratamiento de la malaria en zonas endémicas, alejadas de centros de salud. Durante la última parte de esta tesis, nos hemos centrado en el desarrollo de nuevos nanovectores poliméricos que liberen de forma específica los fármacos a pRBCs, ya que las nanopartículas poliméricas pueden ser formuladas para administración oral más fácilmente que los liposomas. Las diferentes partes de futuros nanovectores (moléculas direccionalizadoras, formulación liposomal, recubrimiento exterior, fármaco encapsulado) están diseñadas de tal manera que puedan ser sustituidas por nuevos elementos para su utilización contra diferentes especies del parásito o para reconocer diferentes dianas intracelulares.

The association between side effects to antimalarial chemoprophylaxis and noncompliance was assessed in travellers visiting five pre-travel clinics in the greater Montreal area between February and August 1996. Participants completed pre and post-travel questionnaires to ascertain frequency and severity of side effects and compliance with malaria chemoprophylaxis. A participation rate of 83% was achieved. Of 157 travellers prescribed mefloquine and 132 travellers prescribed chloroquine, proportions experiencing symptoms or side effects were high (7% and 83%, respectively), but not statistically significantly different. Gastrointestinal side effects were more common in chloroquine users (68%) than in mefloquine users (56%) (p = 0.04). No difference was observed in proportions of psychological side effects between groups; however, they were more commonly reported in the mefloquine group. Between 26% and 33% of noncompliance was directly attributed to side effects; however, logistic regression analysis showed no significant impact of side effects on noncompliance, after controlling for other determinants.

Malaria is a vector-borne disease that is still responsible for high human morbidity and mortality. Of the five Plasmodium species that can cause malaria in humans, Plasmodium falciparum is regarded the most virulent species. The most fundamental component of sustained control and eradication efforts is the development of effective drugs for malaria treatment and prophylaxis. Plasmodium falciparum’s sexual stages (gametocytes) are not associated with malarial pathogenesis or the clinical symptoms, but they are responsible for the transmission of the disease from human hosts to mosquitos. As such, the development of gametocytocidal interventions that targets the transmission stage to break the disease’s lifecycle forms the basis of efforts towards malaria elimination and eradication. However, despite the importance of this developmental stage, the biology and pharmacology of gametocytes are still very poorly understood. This thesis has set out to gain a better understanding of the identity of gametocyte-active antimalarials and a deeper understanding of the mechanisms underpinning the activity. Using a newly generated luciferase-reporting transgenic line, pharmacodynamic gametocyte studies could be performed to help characterise the activity of selected known reference antimalarials, new potential gametocyte inhibitors in pre-clinical development as well as newly developed fully synthetic compounds designed against the sexual stages. This novel assay revealed that the efficacy of active tested compounds is highly stage-specific. Of all the tested reference antimalarial drugs, MB and DHA were the most potent antimalarial across all gametocyte stages and importantly they were active at clinically relevant levels. These observations were progressed further, developing a time- dependent killing assay that was performed with different concentrations of targeted drug over discrete time intervals to determine the drug’s kill rate. These parameters were then used to simulate the PK/PD relationship of the drug in order to estimate gametocyte clearance profiles during the human treatment period (Chapter 3 and 4). A main focus of the thesis was conducted to better understand the mechanism of drug activity of the 8-aminoquinolines against gametocytes. The ability of a series of 8-aminoquinolines (primaquine as the parent drug, synthesised metabolites in chapter 5, three novel analogues and tafenoquine in (chapter 6) to interact with CYP2D6 was tested by measuring their ability to specifically inhibit the metabolism of fluorescently-tagged tracer substrate by recombinant human CYP 2D6. Reaction products from the CYP metabolites and HLM were then used to test firstly their ability to kill gametocytes, and then to establish their ability to generate hydrogen peroxides and finally measure their haemolytic toxicity. At 10 µM, primaquine CYP metabolites showed activity against the gametocytes that was higher than that of the parent drugs, with the exception of tafenoquine which, interestingly, demonstrated good activity and haemolytic toxicity as a parent drug. These analyses are presented and discussed in the context of strategies that aim at the discovery and development of new transmission-reducing antimalarial drugs.

Malaria is a leading cause of morbidity and mortality, causing more than 400,000 deaths per year. Malaria is caused by parasites of the Plasmodium genus with most deaths due to P. falciparum infection. The control of malaria is complicated by the lack of a widely effective vaccine, the spread of mosquito resistance to insecticides and Plasmodium parasite resistance to available drugs, including the gold standard artemisinin-combination therapies. Thus, there is an urgent requirement for the development of new antimalarials, in particular those with different modes of action to existing drugs to limit potential problems of cross-resistance. Plasmodium species have a complex lifecycle that includes transmission from the female Anopheles mosquito vector to a human host requiring significant morphological changes. These morphological changes are associated with stage-specific changes in transcription regulated by epigenetic mechanisms. The proteins involved in these processes are potential new therapeutic targets for malaria. This includes histone deacetylases (HDACs), which together with histone acetyltransferases (HATs), are involved in reversible posttranslational acetylation of histone and non-histone proteins, regulating transcription and other cellular processes. To date, over 650 HDAC inhibitors have been investigated for in vitro activity against malaria parasites. Some inhibitors, particularly those with a hydroxamic acid zinc-binding group that targets inhibitors to the HDAC active site, have demonstrated low nM in vitro potency against P. falciparum and selectivity for the parasite over human cells. However, antiplasmodial HDAC inhibitor drug development has been hindered by factors including the lack of recombinant P. falciparum HDACs (only one available and purity is low), the lack of HDAC crystal structures (none available) and low throughput activity assays that are largely indirect measures of HDAC inhibition. Without these tools, mode of action studies, the rational design of new and improved inhibitors and the prioritisation of compounds for preclinical testing remains difficult. To address some of these challenges and further progress the development of antimalarial HDAC inhibitors, the current study employed a multi-pronged approach, including: (i) investigating the in vitro and in vivo activity of new HDAC inhibitors; (ii) establishing a higher throughput ELISA method to analyse P. falciparum lysine acetylation alterations and; (iii) developing a quantitative structure-activity relationship (QSAR) model based on classification algorithms. HDAC inhibitors typically have a pharmacophore comprising a zinc-binding group that interacts with the zinc ion in the active site of the enzyme, a linker unit and a cap group promoting hydrophobic interaction with amino acid residues at the entry of the active site. Here, a set of 26 new HDAC inhibitors with a peptoid-based scaffold was tested in vitro against drug sensitive asexual intraerythrocytic-stage P. falciparum 3D7 parasites. The set are analogues of compounds that have previously shown in vitro dual-stage antiplasmodial activity against asexual intraerythrocytic and exoerythrocytic stages and includes 16 compounds with a hydroxamic acid zinc-binding group and 10 prodrugs of this compound class. The unprotected hydroxamate-based inhibitors demonstrated growth inhibition of P. falciparum 3D7 asexual intraerythrocytic-stage parasites in the nanomolar to micromolar range (50% growth inhibition values (PfIC50) 0.008-1.04 μM) and up to 1,250-fold selectivity (selectivity indices (SI; PfIC50/human cell IC50): 10-1,250) for the parasite compared to human cells. Structure-activity relationship (SAR) analysis of cap region residues (carbonyl region, carboxylic region and isocyanide region) indicated that benzyl groups in the isocyanide region and alkyl groups in the para position of the carboxylic region are associated with increased antiplasmodial activity. In addition, methyl groups in the carbonyl region of the cap group demonstrated reduced cytotoxicity against neonatal foreskin fibroblasts (NFF), however, also somewhat reduced activity against asexual blood-stage parasites. Work by collaborators demonstrated micromolar in vitro activity of several compounds of this set against exoerythrocytic P. berghei parasite forms indicating dual-stage activity. The compound with the greatest dual-stage activity displayed an IC50 of 8 nM against asexual blood-stage P. falciparum and an IC50 of 60 nM against exoerythrocytic P. berghei in vitro. Compounds with PfIC50 of 100 nM or lower were tested against the multi-drug resistant P. falciparum Dd2 line (resistant to chloroquine, pyrimethamine, mefloquine, and other antimalarial drugs), and demonstrated a resistance index (RI) <1 indicating a lack of cross-resistance by this parasite line. The same subset of compounds was investigated for their ability to hyperacetylate P. falciparum histone H4; differential effects were observed with some compounds causing up to ~2.5-fold hyperacetylation compared to untreated controls. 10 prodrug peptoid-based HDAC inhibitors were also investigated. The prodrug strategy seeks to make the hydroxamic acid-based inhibitors more stable and bioavailable for in vivo applications as they are prone to degradation processes such as hydrolysis or reduction. These compounds were synthesised with masked hydroxamate functionalities that may undergo activation in vitro. Preliminary data demonstrated in vitro PfIC50 of 0.014-1.75 μM and 6-642-fold selectivity for the parasite over human fibroblasts. Three of these compounds displayed PfIC50 <0.1 μM and SI >100 and may therefore be of interest in further studies. Based on the in vitro antiplasmodial activity, selectivity and chemical diversity in the cap region, five peptoid-based compounds (3a, 3c, 3f, 3m, 3n, Pf3D7 IC50 0.008-0.034 μM, SI 97-625) were further investigated for in vivo efficacy against Plasmodium parasites. In addition, four analogues of the tethered phenylbutyrate-based HDAC inhibitor AR42 (Pf3D7 IC50 0.02 μM, SI 39) were also investigated in vivo (JT21b, JT83, JT92a, JT94; Pf3D7 IC50 0.005-0.21 μM, SI 55-118, (data generated by Dr MJ Chua, personal communication)). AR42 is currently in phase 1 clinical trials against various types of cancer and demonstrates an improved pharmacokinetic profile compared to a number of clinically approved HDAC inhibitors (e.g. AR42 Cmax 14.7 μM compared to vorinostat Cmax 1.9 μM, AR42 t1/2 11.1 h compared to vorinostat t1/2 0.75 h; tested in mice). AR42 analogues were of interest as AR42 has previously been shown to cure Plasmodium infections in mice (Dr MJ Chua, Griffith Institute for Drug Discovery; unpublished). While the two analogue sets differ significantly in linker and cap group, both bear a hydroxamic acid zinc-binding group. Compounds were tested in groups of two female BALB/c mice infected with P. berghei ANKA infected erythrocytes. Dosing was via oral gavage at 25 mg/kg twice daily with four hours between dosing (beginning 2 h post infection) for four consecutive days. Peripheral blood parasitemia was monitored by microscopic evaluation of stained thin blood films from day four post infection. None of the peptoid-based HDAC inhibitors attenuated P. berghei growth in BALB/c mice by more than 33% (3f (31%) and 3n (33%) on day 6 post infection). Data from collaborators demonstrated 3n to have the best metabolic stability (t1/2 271 min, Clint 6 μL/min/mg in mice; Prof Finn Hansen, University of Bonn, Germany) which may have contributed to this compound’s improved activity compared to some other analogues. In comparison, AR42 and two if its analogues cured mice of infection (AR42, 1 of 2 mice; JT21b, 2 of 2 mice; JT83 2 of 2 mice), up until day 24 post infection, at which point the mice were euthanised. AR42 and analogues are the first demonstration of oral cures in mice with a HDAC inhibitor (manuscript in preparation) and these data will be pursued in future work to further develop this HDAC inhibitor chemotype for malaria. One of the current limitations in the field is the lack of recombinant P. falciparum HDACs and the need to rely on low throughput assays to demonstrate HDAC inhibitor action via reduced total deacetylase activity or in situ lysine acetylation alterations. While deacetylase assays do not allow the differentiation of compound effects, Western blot using different acetyl-lysine antibodies can reveal compound specific acetylation profiles. Here, two higher throughput methods, dot blot and ELISA, were investigated to assess the effects of HDAC inhibitors on lysine acetylation. Using the control hydroxamate HDAC inhibitor vorinostat (first HDAC inhibitor clinically approved for cancer), the ELISA method was demonstrated to be more reliable than dot blot in detecting acetylation changes in protein lysates from P. falciparum trophozoites exposed to compound for 3 h. ELISA was therefore used to investigate histone H3 and H4 lysine acetylation alterations following exposure of P. falciparum to six commercially available anti-cancer HDAC inhibitors (vorinostat, panobinostat, trichostatin A, romidepsin, entinostat and tubastatin A). All compounds have in vitro activity against asexual intraerythrocytic P. falciparum parasites (Pf3D7), with tubastatin A activity reported for the first time here (PfIC50 0.15 ± 0.03 μM). All compounds were also shown to inhibit >84% deacetylase activity using P. falciparum protein lysates in an in vitro assay at 1 μM, with the exception of entinostat (~50% inhibition at 1 μM); this compound was also the least active against the parasite (PfIC50 11.5 μM). Using ELISA, vorinostat, panobinostat, trichostatin A, romidepsin and entinostat were all found to cause a ~3-fold increase in the signal detected using an anti-tetra-acetyl-lysine antibody. In comparison, the only human HDAC6-specific inhibitor tested, tubastatin A, caused 1.8-fold histone H4 hyperacetylation compared to the control. Further investigations of the individual N-terminal H4 lysine residues using antibodies specific to acetylated lysine 5, 8, 12 or 16 revealed that all compounds, except tubastatin A, caused hyperacetylation using each antibody. No differential effect was observed for histone H3 acetylation, with all compounds causing an ~1.8-fold increased signal using an acetyl-H3 antibody. The new ELISA method developed here provides a higher throughput way to assess differential compound induced lysine acetylation alterations in P. falciparum and therefore represents a valuable new tool to aid the investigation of HDAC inhibitors for malaria. As discussed above, the lack of tools, such as recombinant P. falciparum HDAC proteins, crystal structures and homology models, has meant that the identification of antiplasmodial HDAC inhibitors has been limited to whole-cell screening approaches which can be time-consuming and costly. To begin to address this problem, quantitative structure-activity relationship (QSAR) models were developed based on logistic algorithms with the aim of providing a new tool to triage compounds for in vitro testing. A database of 457 antiplasmodial HDAC inhibitors was assembled with published data on PfIC50 and, for 292 of those compounds with data on plasmodial selectivity. Two independent prediction algorithms based on logistic regression were developed to classify (1) antiplasmodial activity or (2) selectivity of hydroxamate-based HDAC inhibitors. Seven different activity and five different selectivity models were built, each with individual decision cut-offs defining active/selective and non-active/unselective compounds (e.g. PfIC50: active compound <0.1 μM> non-active compound; SI: selective compound >100< unselective compound). Activity model A7 revealed the highest prediction performance by predicting 93% of the training compound set and 87% of the external test compound set correctly. Cross validation revealed a prediction accuracy of 91%. The most accurate selectivity model S4 demonstrated a slightly poorer prediction performance due to a much smaller initial data set as not all the HDAC inhibitors had reported selectivity information (64%). Despite this, the selectivity model demonstrated an internal prediction accuracy of 91%, a cross-validated (internal) prediction accuracy of 82% and an external prediction accuracy of moderate 72%. To validate the prediction performance of the activity model further, they were applied to a set of 22 experimentally untested compounds (validation set) and the prediction performance compared to their experimental antiplasmodial activity. Applying prediction model A7 to this compound set predicted three hit compounds (two of which were confirmed by experimental assay data) and 12 non-actives (confirmed for 11 based on experimental assay data). The experimental PfIC50 assessment revealed asexual blood-stage PfIC50s for the whole set in the nanomolar to micromolar range (PfIC50 0.006-8.45 μM; data from Dr MJ Chua), with the correctly predicted hits (S2_E10 and LD016) having PfIC50 <0.008 μM. Overall, virtual screen using QSAR model A7 identified 87% of the validation compounds correctly and revealed high prediction specificity, identifying 92% of the non-active compounds correctly. Due to a lack of available data sets with selectivity index information (and time constraints for this project), the selectivity models were not able to be tested with an external set. These activity and selectivity QSAR models are the first generated for antiplasmodial HDAC inhibitors. These models will aid the in silico assessment of antiplasmodial activity and selectivity of hydroxamate-based HDAC inhibitors and therefore represent useful new tools in the investigation of HDAC inhibitors for malaria. In summary, data presented in this thesis include the identification of novel antiplasmodial HDAC inhibitors with activity against asexual intraerythrocytic-stage P. falciparum parasites, in vivo data demonstrating oral cures in mice for two analogues of the anti-cancer HDAC inhibitor AR42, a new ELISA method to allow higher throughput assessment of HDAC inhibitor induced changes to histone lysine residues and the first antiplasmodial HDAC inhibitor QSAR models. HDAC inhibitors identified in this study with promising in vitro and in vivo antiplasmodial activity profiles are new starting points for further development of HDAC inhibitors for malaria. In addition, the in vitro and in silico approaches developed in this study are useful new tools to facilitate the discovery of HDAC inhibitors and the understanding of their biological effects on the parasite.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

An estimated 500 million cases of malaria occur each year. The increasing prevalence of drug resistant strains of Plasmodium in most malaria endemic areas has significantly reduced the efficacy of current antimalarial drugs for prophylaxis and treatment of this disease. Therefore, discovery of new, inexpensive, and effective drugs are urgently needed to combat this disease. Marine biodiversity is an enormous source of novel chemical entities and has been barely investigated for antimalarial drug discovery. In an effort to discover novel therapeutics for malaria, we studied the antimalarial activities of a unique marine-derived peak fraction library provided by Harbor Branch Oceanographic Institute (HBOI). Within this unique library, we have screened 2,830 marine natural product (MNP) peak fractions through a medium throughput screening effort utilizing the SYBR Green-I fluorescence based assay, and have identified 253 fractions that exhibit antimalarial activity. From those inhibiting fractions we have identified twenty species of marine organisms that inhibit Plasmodium falciparum growth, from which thirty-five fractions were selected for further study. Among those thirty-five, eighty-three percent were also found to inhibit the chloroquine resistant strain of P. falciparum, Dd2. The most potent inhibitors were then screened for their cytotoxic properties using the MTT cell viability assay. Among the samples that exhibited potent inhibition of P. falciparum growth were fractions derived from a sponge of the genus Spongosorites sp.. This genus of sponge has been reported to contain the nortopsentin and topsentin class of bis-indole imidazole alkaloids. Nortopsentin A inhibited the parasite growth at the trophozoite stage with an IC[sub]50 value of 1.6micrometer]. This is the first report of antimalarial activity for this class of compound.
ID: 030423269; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.S.)--University of Central Florida, 2010.; Includes bibliographical references (p. 56-61).
M.S.
Masters
Burnett School of Biomedical Sciences
Medicine

Malaria is one of the most deadly parasitic diseases known to man. Although the number of malaria cases reported each year is decreasing, this disease continues to pose health and economic problems mainly in developing countries. Significant progress has been made in the fight against this disease. This includes the discovery and development of potent antimalarial agents. However, the development of resistance to most of these potent antimalarials has made the development of new antiplasmodial agents of paramount importance. Several promising antiplasmodial agents have been found from the marine environment. Amongst these are the tetraprenylated toluquinols from the brown alga: Sargassum heterophyllum. These metabolites have been reported to exhibit a range of antiplasmodial activity; however, the mechanisms by which these compounds bring about their antiplasmodial activity and the pharmacophoric groups responsible for such activity are unknown. Two species of Sargassum algae were encountered during the course of this project. From the investigation of the geographical and seasonal variation of metabolites of S. heterophyllum and S. elegans we established that there were no significant intra and inter site variations amongst metabolite profiles of both species both within and between the sampled seasons. These results enabled us to establish that the collection of both species from three different sites on the eastern coast of South Africa namely; Kenton on Sea, Port Alfred and Noordhoek in autumn, winter or spring would qualitatively yield the same metabolites. A comparison of metabolite profiles of both species also revealed no qualitative differences between metabolites of S. heterophyllum and S. elegans. The quantities of selected prenylated metabolites extracted from S. heterophyllum using four different extraction techniques was also assessed using qNMR as the method of quantification. This led to the identification of optimal extraction techniques and conditions for the extraction of sargahydroquinoic acid (1.38), sargaquinoic aid (1.39) and sargachromenol (2.10) from S. heterophyllum. From this study, the extraction of algae by soxhlet extraction using EtOH as the extraction solvent led to the extraction of the highest quantities of sargahydroquinoic acid. The potential of other extraction techniques such as microwave assisted extraction, to yield high quantities of the selected metabolites were also identified. With gram quantities of sargahydroquinoic acid (1.38) in hand, this compound was modified by oxidation, reduction, acetylation, methylation and cyclization reactions to yield nine derivatives. The derivatives and four naturally occurring prenylated toluquinols were assessed for antiplasmodial and cytotoxic activity against the FCR-3 Gambian Chloroquine resistant strain of P. falciparum and the MDA-MB-231 breast carcinoma cell line respectively. Comparison of antiplasmodial data for all twelve compounds showed that the hydroquinone moeity of sargahydroquinoic acid (1.38) is important for antiplasmodial activity while esterification of the carboxylic acid group in 1.38 resulted in more potent antiplasmodial compounds. Of all twelve compounds, compound 5.2, the hydroquinone methyl ester of 1.38 was found to be the most potent antiplasmodial compound with an IC₅₀ value of 1.94 μM and a selectivity index of 22.68.

The aim of this thesis was to develop analytical methods for measuring antimalarial drugs in biological fluids. Solid phase extraction (SPE) was used for the enrichment and purification of the drugs. Automatic extraction procedures using a SPE robot were developed to reduce the workload for the analyst and to minimize variations in the extraction procedure. Liquid chromatography (LC) with either UV or mass spectrometric (MS) detection was used to determine sample concentrations. Determination of Pyronaridine in whole blood utilised a weak cation exchanger to extract Pyronaridine from blood. To improve LC separation between Pyronaridine and the internal standard, ion-pairing was utilized. For the simultaneous quantification of the highly lipophilic Atovaquone and the strong basic drug Proguanil with metabolites, a novel mixed mode solid phase extraction column was used. It combines the properties of a carboxylic acid (CBA) column and a non-polar octyl-silica (C8) column to extract the compounds from plasma; it also required a gradient LC separation. Stability is an important factor when developing new methods. A new approach was used to evaluate the stability of Amodiaquine in blood and plasma. This included the use of a stability marker, a stable compound which was added together with Amodiaquine when preparing the stability samples. This eliminated between-run variations and variations associated with preparation of new stock solutions. Lumefantrine (LF) is one of the active components in a new drug combination recommended by the World Health Organization as a replacement for older drugs which have lost their effect. The first of the two methods described for this compound is the determination of LF and a possible metabolite in plasma with a calibration range suitable for pharmacokinetic studies. In the second method, a capillary sampling technique is used where the blood is dried on a sampling paper and sent to the laboratory where the extraction and determination of LF concentrations take place. This method facilitates sample collection and will enable drug efficacy studies conducted in rural settings. To monitor a current change in treatment policy and self medication, a screening assay was developed. Its purpose is to be a complement to interviewing patients about their previous medication (in the previous few weeks) and to detect some of the more common drugs which might have been used.
Paper 6. as Manuscript

Chloroquine (CQ) is a well-known antimalarial drug acting through the inhibition of the formation of haemozoin crystals in the Plasmodium parasite’s digestive vacuole within human infected red blood cells. The formation of haemozoin is considered a defence strategy of the parasite in order to decrease the levels of toxic ferriprotoporphyrin (Fe(III)PPIX) present in the DV. Chloroquine is supposed to be adsorbed onto the fastest growing face of the crystal, inhibiting the growth and increasing the amount of toxic Fe(III)PPIX; however, the mechanism of this interaction is still largely unproven. The study of the interactions of CQ with both Fe(III)PPIX and β-haematin, haemozoin’s synthetic crystalline equivalent, can be used to clarify the mechanism of antimalarial action of CQ. In this project a CQ derivative was synthesized and labelled with quantum dots (QDs) in order to exploit their particular fluorescence properties during the investigation of the interaction. The synthesis of a chloroquine derivative was used in order to insert a primary amino function in the drug suitable for the coupling with QDs. The formation of this derivative (N 1 -(2-aminoethyl)-N 4 - (7-chloroquinolin-4-yl)-N 1 -methylpentane-1,4-diamine) was achieved through a four step reaction. After the extraction of chloroquine as a free base from diphosphate salt, it was desethylated. The insertion of the new group was obtained through a N-alkylation using a Boc protected aldehyde, leading to the formation of the final product by cleaving the Boc group with TFA. CdSe/ZnS quantum dots with carboxylic acid functionalized ligands (QD-COOHs) were obtained from Cytodiagnostics. Thanks to their dimension, they presented a fluorescence emission peak at 630 nm. During the characterization, the optimal fluorimeter conditions were found: excitation wavelength was set at 400 nm; photo multiplier voltage (PMT) of 1000 V; slow scan speed and slit widths of 10 nm for excitation and 5 nm for emission. Quantum dots were visualized using TEM technique and their dimension was confirmed. Thanks to previous studies carried out in this lab, a 0.01 M HEPES (pH 7.5) buffer was used for all the experiments in order to afford a good solubility of Fe(III)PPIX and a minimal QD-COOH emission quenching. In order to use QD-COOHs to study the interactions of CQ with Fe(III)PPIX and β-haematin it was necessary to attach the CQ derivative to QD-COOH. The carboxylate ligand coating allows the formation of covalent bonds, coupling the primary amine-function of chloroquine derivative (CQ- NH2) and QD-COOH. The reaction was completed using a two-step, one-pot process with 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) as a coupling agent. The product of the reaction, chloroquine derivative-labelled CdSe/ZnS quantum dot (QD-CQ), was characterized, recording the fluorescence emission spectrum with maximum intensity peak at 630 nm. In order to verify the achieved functionalization, SEM with EDS was used to detect the presence of Cl on the nanoparticles surface. TEM was also used to visualise the physical appearance of the QD-CQ. Labelled nanoparticles showed an enhanced tendency to aggregate than QD-COOH. The aggregation caused a decreasing of the fluorescence emission spectrum intensity. Sonication was shown to partially reduce QD-CQ aggregation and other experiments were done by adding a little quantity of different solvents (ethanol, methanol, acetone and 0.01 M HEPES pH 4.2) without significant improvements in enhancing fluorescence intensity. Fluorescence emission spectra of the interactions of the QD-COOHs with CQ and Fe(III)PPIX were then evaluated for comparison with those of the QD-CQ with both compounds. The final interaction studied was between β-haematin and the QDs. β-haematin was synthesised in 9.7 M acetate. TEM was used to visualise the interaction between QD-COOHs and QD-CQs with βhaematin. The QD-COOHs showed no selectivity in binding to the β-haematin crystals, whereas the QD-CQs bound primarily to the (001) face. This evidence supports theory that CQ adsorbs to the fastest growing face of β-haematin.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Malaria is a pestilent disease associated with massive socioeconomic burden of sub-Saharan Africa. This disease is caused by a blood infection of the single cellular parasite of the Plasmodium genus. Two enzymes of this parasite have been identified to be essential to the survival of this parasite, notably Spermidine Synthase and Protein Farnesyltransferase. The goal of this dissertation was to search for and synthesise novel inhibitors of these two enzymes with a strong focus towards understanding their structure/activity relationships. To achieve the first goal, molecular modelling was employed. An in-depth discussion is presented to describe the underlying principles relevant to this branch of computational chemistry. This ensures that the experiments using these methods are set-up correctly and results are interpreted within context. Two virtual high-throughput screens were then performed using prepared crystallographic structures of Spermidine Synthase. The first was pharmacophore based method and the second based on LibDock. The database used, containing 7.1 million compounds, was filtered using a custom developed tool prior to screening. Finally, CDOCKER was then used to investigate the activity of potential hit compounds. Spermidine Synthase has a natural affinity for adenosine and this trait was exploited by derivatising analogues to synthesise potential inhibitors of the enzyme. This was to be achieved by the incorporation of both electrophilic and nucleophilic moieties at selected positions, including the use of a high yielding Mitsunobu reaction. A number of additional residues were then synthesised and joined to the adenosine which were proposed to increase the active site occupancy and increase affinity to the enzyme. For the second enzyme targeted for inhibition, Protein Farnesyltransferase, indole was used as a starting scaffold to synthesise potential hits de novo. It was aimed to derivatise the indole at the Nʹ and 3ʹ positions. The crystal structure of one of the intermediates was published. Furthermore, a synthetic sequence which culminated in a palladium catalysed Suzuki coupling was performed.
AFRIKAANSE OPSOMMING: Malaria is ‘n peslike siekte wat geassosieer word met beduinde sosio-ekonomiese implikasies vir sub-Sahara Afrika. Die siekte word veroorsaak deur ‘n bloed infeksie van die enkel sellulêre parasiet van die Plasmodium genus. Twee ensieme, naamlik Spermidien Sintetase en Protein Farnesieltransferase, is geïdentifiseer om noodsaaklik te wees vir die oorlewing van die parasiet. Die doelwit van hierdie verhandeling is die soektog en sintese van oorspronklike inhibeerders van hierdie twee ensieme met ‘n sterk fokus daarop om struktuur/aktiwiteit interaksies te verstaan. Om die eerste doelwit te bereik is molekulêre modellering toegepas. ‘n Indiepte ondersoek word voorgestel om die onderliggende beginsels relevant tot hierdie tak van berekenkundige chemie te beskryf. Dit verseker dat eksperimente wat op hierdie tegnieke berus korrek opgestel word en dat die resultate binne konteks geïnterpreteer word. Twee virtuele hoë-deurset skerms was deurgevoer op voorbereide kristallografiese strukture van Spermidien Sintetase. Die eerste het berus op ‘n pharmakoforiese metode en die tweede op LibDock. ‘n Self-ontwikkelde sagteware gereedskap stuk is gebruik om a databasis van 7.1 miljoen verbindings te filtreer voor dit gebruik is in hoë-deurset skerms. Uiteindelik is CDOCKER gebruik om die potensiele aktiwiteit van “treffer” verbindings te beraam. Spermidien syntetase het ‘n natuurlike affiniteit vir adenosien en hierdie eienskap is benut deur analoeë af te lei na potensiële inhibeerders teen die ensiem. Dit is bewerkstellig deur die insluiting van beide elektrofiliese asook nukleifielese funksionele groepe op gekose posisies. Dit het die gebruik van ‘n hoë opbrengs Mitsunobu reaksie ingesluit. ‘n Aantal ander addisionele residueë is toe gesintetiseer en geheg aan die afgeleide adenosien om die ensiem setel te vul en sodoende die affinitieit te verhoog. Vir die tweede ensiem wat geteiken is vir inhibisie, Protein Farnesieltransferase, is indool benuttig as ‘n begin steier te dien om potensiële treffers de novo te sintetiseer. Dit is geteiken om die indool af te lei op die Nʹ en 3ʹ posisies en die kristal struktuur van een van hierdie tussengangers is gepubliseer. Verder is ‘n sintetiese weg, wat uitgeloop het op ‘n palladium gekataliseerde Suzuki koppeling, uitgevoer.

The overall aim of this project was to investigate the synthesis and activity of a range of compounds based on the benzo[b]thiophene-2-carboxamide structural motif as potential new antimalarial agents, and to a lesser extent as antibacterial agents.In order to subsequently explore any structure-biological activity relationships, the first part of the project involved the systematic synthesis of some 39 non-fused substituted benzo[b]thiophene amide derivatives including tetrahydroisoquinolines, tetrahydro-β-carbolines, dihydropyrroles, piperazines, piperidines and other bridged ring systems as part of the amide-nitrogen component. Methods for the synthesis of the new amide derivatives were developed based on benzo[b]thiophene acid chloride and amine reactions, or on dicyclohexylcarbodiimide-mediated carboxylic acid amine coupling reactions.Further substitution reactions were also undertaken on tetrahydro-β-carboline amides (55, 56, and 57) with the introduction of an N-benzyl group and an N-onitrobenzyl group in the case of 55. The N-boc protected piperazine amide 63 also served as a precursor for other N-substituted piperazine amide derivatives. A single crystal X-ray structure on amides 63 and 73 confirmed the amide rotamer geometry in the solid state with these compounds.The second part of the project incorporated the synthesis of fused analogues which were more conformationally restricted while still retaining the benzo[b]thiophene amide structural motif. A new free radical cyclisation approach to the benzo[b]thieno[2,3-c]pyridin-1-one system in compound 92 was developed, together with the corresponding model isoquinolinone analogue 95. The reaction was based on the use of tributyltin hydride and AIBN to produce the required free radical intermediate from an arylbromide precursor.The free radical cyclisation reaction was extended to synthesise N-benzyl and substituted N-benzyl analogues of 92. The dihydroxylation of the N-allyl substituent in 92 was achieved using potassium osmate and N-methylmorpholine-N-oxide (NMO). A further free radical cyclisation route to the N-benzo[b]thien-2-oyl derivatives 112 was also achieved.The synthesis of the novel 9-membered ring containing fused derivatives 120 and 121 was also achieved. This synthesis involved ring closing metathesis methodology using the bis-allyl amides 118 and 119 and Grubbs’ I ruthenium catalyst. The polymer supported version of this catalyst gave better yields of the cyclisation products. A single crystal X-ray structure of 120 confirmed the cis geometry of the double bond in the 9-membered ring. In the course of preparing the required precursor 119 for the 9-membered ring synthesis, a new imine allylation reagent combination was discovered involving zinc, allyltributyltin and boron trifluoride etherate. This reaction is worthy of further investigation to determine its wider synthetic utility.Ring closing metathesis reactions also afforded the dihydropyrrole amides 113 and 114 in good yields.A palladium-mediated cyclisation approach to the new benzo[b]thieno[2,3- c]pyrrolo[2,3-a]indol-11-one system in 126 was also accomplished based on the Nacylindoles 124 and 125.A number of the compounds were tested for their possible in vitro antimalarial activity against two strains of Plasmodium falciparum (K1 CB1 and TM4/ 8.2) and two active leads, the benzo[b]thienoquinolinone derivatives 95 and 102, were discovered. Some potential structure-activity trends for the tested benzo[b]thiophene derivatives were observed (Chapter 4).Antibacterial testing of a few benzo[b]thiophene compounds against Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains was also done, and the benzo[b]thienoquinolinone derivative 92 exhibited promising activity against Staphylococcus aureus.