Malaria is a parasitic disease that is caused by the plasmodium parasite. Plasmodium infection has affected man for thousands of years. With advances in drug discovery over the past century, malaria has evolved to possess resistance to most mainline therapeutics. This war of drug discovery vs plasmodium evolution continues to be fought to this very day, with attempts to eradicate malaria worldwide. Frontline treatments such as chloroquine, artemisinin, and atovaquone/proguanil have all seen parasitic resistance in strains of P. vivax as well as P. falciparum. While plasmodium possesses resistance to most classes of anti-malarials, the 8-aminoquinoline (8-AQ) class has seen minimal resistance development. 8-AQs have been shown to be effective against erythrocytic and exo-erythrocytic forms of plasmodium, and are often given in combination with a blood schizonticide such as chloroquine or artemisinin. These combinations clear all forms of plasmodium infection. With 8-AQs unique set of anti-malarial properties and the advent of increased drug resistance to other drugs, much research is being done to understand 8-AQs mechanism of action and toxicity. 8-AQ use is limited due to inducing extreme hemolytic anemia in those with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Primaquine is the only 8-AQ molecule available on the market with tafenoquine, an analog primaquine, currently in phase III clinical trials. It is believed that if the mechanism of action and toxicity of the 8-AQs are understood, then we can create new generation anti-malarials that will maintain the unique action of 8-AQs while reducing their toxicity. Studies have shown that 8-AQ mechanism of action has been attributed to the generation of unstable metabolites that induce ROS production in the parasite, as well as mitochondrial swelling. While there is some evidence suggesting molecular targets of 8-AQs, the actual target is still unknown. When 8-AQs is given in combination with chloroquine, a synergistic effect is observed. While chloroquine has no activity against liver stages, it still somehow potentiates primaquine’s activity in those stages. This mechanism of synergy in liver stages is not well understood, and its understanding can give us increased understanding of basic plasmodium biology in the liver. Additionally, more information about the mechanisms of action of both chloroquine and primaquine could be elucidated. Tagging drugs with fluorescent probes is a technique that can give much information about the drug’s pharmacological activity in vitro, and sometimes in vivo as well. Such an approach has been used for various disease states such as HIV and cancer. Malaria is no exception; fluorescent probes of artemisinin and chloroquine have been used to examine resistance mechanisms to both molecules. In addition to 8-AQs, there are other older antimalarials that have received attention recently due to increases in resistance. Menoctone, a hydroxynapthoquinone that subsequently lead to the discovery of atovaquone, has recently gained increased attention because of its similarities to atovaquone. Research surrounding menoctone was abandoned due to the discovery of more efficacious compounds. Similar to 8-AQs, understanding the mechanisms of action and resistance to menoctone could give us much more information about plasmodium responses to this class of compounds. This understanding could potentially lead to the discovery of novel therapeutics. To understand mechanisms of action and synergy of 8-AQs, we report the creation of novel fluorescent probes of the 8-AQ molecules primaquine and tafenoquine. The organic synthesis was designed and characterization was confirmed by NMR and high resolution mass spectra, and the fluorescent properties were examined using absorbance and steady-state emission experiments. We found that the anti-malarial, anti-leishmaniasis, and cytotoxic properties of these novel probes were similar to the parent compounds. These probes localized in the cytoplasm of infected parasites in vitro. We also attempted to view their localization in liver stage infection, and investigated the synergistic combination of 8-AQs with chloroquine and quinine. Menoctone resistance was induced in vivo to determine mechanisms of resistance. Cross resistance to atovaquone was observed, and the mutation responsible for resistance was also found.

Structure-based drug design and virtual screening are areas of computational medicinal chemistry that use 3D models of target proteins. It is important to develop better methods in this field with the aim of increasing the speed and quality of early stage drug discovery. The first part of this thesis focuses on the application of structure-based drug design in the search for inhibitors for the protein 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), one of the enzymes in the DOXP/MEP synthetic pathway. This pathway is found in many bacteria (such as Mycobacterium tuberculosis) and in the parasite Plasmodium falciparum. In order to evaluate and improve current virtual screening methods, a benchmarking data set was constructed using publically available high-throughput screening data. The exercise highlighted a number of problems with current data sets as well as with the use of publically available high-throughput screening data. We hope this work will help guide further development of well designed benchmarking data sets for virtual screening methods. Conformal prediction is a new method in the computer-aided drug design toolbox that gives the prediction range at a specified level of confidence for each compound. To demonstrate the versatility and applicability of this method we derived models of skin permeability using two different machine learning methods; random forest and support vector machines.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21716号 / 薬科博第107号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 高須 清誠, 教授 竹本 佳司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Coronaviruses have been responsible for several global disease outbreaks over the last 20 years, including the “Severe Acute Respiratory Syndrome” in 2002/2003, the “Middle East Respiratory Syndrome” in 2012, and the “Coronavirus Disease of 2019 (COVID19)”. These viruses are highly contagious and can cause multiple medical disorders upon contraction, such as common cold or lower respiratory infections. SARS-CoV-2, the newly emerged coronavirus variant of 2019, has been confirmed as the cause of the ongoing COVID19 pandemic, which infected over 167 million people worldwide and, by the end of May 2021, has a death toll of over 3 million people. Even though several SARS-CoV-2 vaccines have made it to the market, no proven options have yet been discovered for treating COVID19 infections. The aim of this project is, therefore, to improve the potency of two active SARS-CoV-2 main protease (Mpro) inhibitors (ML188 and X77) by performing a structure-activity-relationship study where two specific sites of the inhibitors are altered. The inhibition activity of these compounds is then tested on isolated SARS-CoV-2 Mpro. The four-component Ugi reaction was utilized to synthesize the ML188 and X77 analogs, which were purified by column chromatography before testing. During this project, six pure analogs were successfully synthesized and will be sent shortly for testing. Inhibitors with good activity against SARS-CoV-2 Mpro will be further tested for their antiviral activity in cell-based infection assays. The results obtained from this study will later be used to perform a second structure-activity-relationship study to further improve the potency of the two inhibitors by developing a 2nd generation library.

Les fluoroalcènes, du fait de leurs propriétés physico-chimiques singulières, sont des composés intéressants présents dans de nombreux domaines comme les matériaux polymères, l'agrochimie ou encore la chimie médicinale. Les mono-fluoroalcènes présentent des similarités électroniques et géométriques avec la liaison amide et sont donc de très bons mimes de la liaison peptidique. Néanmoins, la synthèse et la fonctionnalisation de ces composés représente toujours à l'heure actuelle un enjeu synthétique important. Au cours de cette thèse deux briques moléculaires ont été préparées pour développer de nouvelles méthodes d'accès aux fluoroalcènes : les gem-bromofluoroalcènes et les α-fluoroacrylates. Les gem-bromofluoroalcènes ont été employés dans deux méthodologies. La première consiste en une mono-catalyse au cuivre pour réaliser la fluoroalcénylation d'hétéroraryles par fonctionnalisation catalytique de la liaison C-H de dérivés de type azoles. La seconde consiste en la phosphination des gem-bromofluoroalcènes par des phosphines boranes, dans le but d'obtenir de nouvelles phosphines originales. Les α-fluoroacrylates et les acides α-fluoroacryliques ont été utilisés dans deux réactions complémentaires. Une réaction de Heck a été employée pour synthétiser des α-fluoroacrylates tri- et tétrasubstitués, offrant une nouvelle voie d'accès à ces composés. Quant aux acides α-fluoroacryliques, ils ont été utilisés dans une méthode innovante de couplage décarboxylant/déshydrogénant avec des azoles. En effet, il s'agit du premier exemple de couplage décarboxylant/déshydrogénant sur des alcènes. / The fluoroalkenes are compounds with relevant physico-chemical properties and are used in various fields as polymers, agrochemistry or medicinal chemistry. Mono-fluoroalkenes can be used as an effective peptidic bond mimic because of their electronic and geometric similarity with the amide bond. Nevertheless, this interesting moiety still suffers from difficulty of synthesis which constitute a synthetic challenge. For that purpose, two build-blocks (gem-bromofluoroalkenes and α-fluoroacrylates) were used during this thesis to develop new access to mono-fluoroalkenes.The first one is the gem-bromofluoroalkenes moiety were used for the development of two methodologies. First, a copper-catalysed fluoroalkenylation via C-H bond functionalisation of heteroaryles has been reported. Then, the second methodology is the phosphination of gem-bromofluoroalkenes using phosphines boranes, in the goal to get new kind of phosphines.The second building-blocks used are the α-fluoroacrylates and α-fluoroacrylic acids which have been involved in two complementary reactions. A Heck reaction allowed the synthesis of tri- and tetrasubstitued α-fluoroacrylates, giving a new access to these compounds. α-Fluoroacrylic acids were used in a decarboxylative/deshydrogenative cross-coupling with azoles. Indeed, it is the first example of decarboxylative/deshydrogenative cross-coupling with alkenes.

Les récepteurs couplés aux protéines G (RCPGs), constituent la plus grande famille de protéines membranaires dans le génome humain. Les RCPGs sont des protéines de signalisation, qui exercent leur action à la surface des cellules, en réponse à une grande variété de stimuli extérieurs. Ils jouent un rôle primordial dans de nombreuses fonctions physiologiques et sont donc impliqués dans une multitude de pathologies comme les maladies cardiovasculaires, métaboliques, neurodégénératives, psychiatriques et oncologiques. L'attribution du prix Nobel de chimie 2012 aux professeurs Robert Lefkowitz et Brian Kobilka pour leurs travaux et avancées spectaculaires dans le champ de recherches des RCPGs, souligne encore leur importance. Les RCPGs constituent également la plus importante cible thérapeutique, avec 30% des médicaments actuellement disponibles sur le marché qui exercent leur action via un RCPG. Cependant, la découverte de nouveaux ligands reste un chalenge. Le but est de développer des approches basées sur la RMN à l’état liquide, qui auront un impact positif sur la recherche de ligand de RCPGs, grâce à l’étude et la caractérisation de récepteur pleine taille, solubilisés en micelles de détergents ou enchâssés en bicouches lipidiques natives / G protein-coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome. GPCRs act as cell surface signalling proteins and respond to a variety of external signals. They play a pivotal role in many physiological functions and are therefore associated with a multitude of diseases, including cardiovascular, metabolic, neurodegenerative, psychiatric, and oncologic diseases. The 2012 noble Prize in Chemistry was awarded jointly to Robert J. Lefkowitz and Brian K. Kobilka for studies of GPCRs, highlighting the importance of this protein superfamily. GPCRs constitute also the most important family of drug targets in the human body, with 30% of current drugs acting on GPCRs. However, drug discovery targeting GPCRs remains difficult, owing to the restricted structural information on GPCRs related to the instability of these proteins when isolated from their cell membrane environments. There is also a lack of knowledge for the structural and functional consequences of the interactions of small-molecule compounds with GPCR. The aim is to develop methods to study and characterize a full GPCR solubilized in detergents or in native lipid bilayers, both in its free form and in small molecule bound forms, using liquid-state NMR experiments. The aim is to develop NMR-based approaches that will strongly impact the structure-based drug discovery process for the GPCR family

Ce projet d’interface Chimie/Biologie repose sur les expertises complémentaires de deux équipes. Il concerne la conception et le développement d’inhibiteurs dirigés contre les Kinétoplastidés (trypanosomes, leishmanies). Il est en effet urgent de développer de nouvelles stratégies thérapeutiques pour répondre à la chimiorésistance et à la toxicité des médicaments actuellement utilisés contre ces parasites. Le métabolisme et le transport des polyamines étant essentiel chez les parasites, ils constituent des cibles thérapeutiques d’intérêt contre les Kinétoplastidés. Le projet intègre la synthèse de nouveaux dérivés polyamines spécifiques des parasites, l’évaluation sur des modèles in vitro de leishmaniose et de trypanosomose africaine, ainsi qu’une évaluation sur trypanothione réductase. La mise au point d’une méthode de quantification du transport de polyamine a également été initiée. Cinquante-quatre composés, répartis en trois séries chimiques, ont été synthétisés et évalués. Un grand nombre d’entre eux présentent des activités antiparasitaires de l’ordre du micromolaire et des évaluations in vivo sont actuellement en cours avec le composé le plus prometteur. / This project is at the interface of chemistry and biology and relies on the expertise of two different teams. This thesis involves the design and development of inhibitors directed against Kinetoplastids. It is urgent to develop new therapeutic strategies to respond to drug resistance and toxicity of currently used drugs against these parasites. Polyamine metabolism and transporter have been demonstrated as essential for parasite growth. Therefore, these systems are potential drug targets for development of antikinetoplastid compounds. We chose to synthesize polyamine derivatives and evaluate their biological activity against Kinetoplatids. Fifty-four compounds, divided into three chemical series, have been synthesized and evaluated. Many have shown a micromolar biological activity in vitro against parasite. In vivo evaluation is foreseen for the most promising derivative.

As they make up DNA and RNA, nucleosides are considered the key to life. Synthetic nucleosides also constitute many drugs that treat viral infections and cancer. As a result, more efficient methods to access these crucial molecules would have implications that extend beyond a synthetic chemist's benchtop and into medicinal chemistry and medical research. One of the most challenging steps in the synthesis of nucleosides is the glycosylation step between the acceptor heterocycle (nucleobase) and the saccharide-based donor. Often to obtain satisfactory yield of this step with good regio- and stereochemical control the extensive use of protecting groups must be employed to squelch reactivity at unwanted reactive groups. Consequently, this process of protection−glycosylation−deprotection is laborious, inefficient, and often requires the use of toxic reagents. It would be, therefore, highly welcomed if new methodology to effect this glycosylation step was designed that reduces or removes the need to use protecting groups, but would still provide nucleosides in good yield, regio- and stereoselectively. Herein, this thesis presents my efforts into achieving this end. By employing modified Mitsunobu conditions, I determined that it is possible to directly glycosylate a nucleobase with D-ribose to afford...

This dissertation describes the density functional theory (DFT) computational modelling of reactions between organolithium nucleophiles and various substituted quinoxalines. These reactions result in the functionalisation of the C (sp2)–H bond, thus substituting the sigma-hydrogen. The reactions are known as nucleophilic substitution of hydrogen (SNH) and are used by experimental chemists to form new C–C bonds. The SNH reactions are very important in various industries, e.g. in designing and manufacturing of pharmaceuticals. Quinoxaline is widely used in medicinal chemistry due to its various biological activities; these reactions play a crucial role in the synthesis of new classes of compounds. The reactions of 2-phenyl- (A), 2-butyl- (B), and 6-nitro-2-phenyl- (C) quinoxaline with lithiofuran (a) and lithiothiophene (b) involves a direct (1) nucleophilic attack on an activated electron-deficient system, leading to the intermediate sigma^H-complex. This is followed by hydrolysis (2), where an sp2-type nitrogen is changed to an sp3 while forming Li---OH as a by-product. The presence of Li---OH then allows the departure of an sigma-proton via oxidation reaction, concomitantly forming H2O2 as the second by-product. All approaches to functionalise the C(sp2)–H bond involve elimination of a proton, and an oxidant is needed for the departure of the sigma-hydrogen. Although the sequence of steps and mechanisms of these C–H transformations are the same, various factors have shown to affect the reactions differently. The theoretical study of this catalytic-free transformation, shows that the formation of sigma^H-adducts is not easily reversible, and that their formation is spontaneous. The reaction does not just require an oxidant to eliminate the sigma-hydrogen with the pair of electrons, but rather requires the presence of water for hydrolysis prior to oxidation. We must stress the crucial role of the oxidant since the key problem of the SNH reactions is associated with the elimination of sigma-hydrogen. However, the main objective of this study is to present a correct and complete mechanistic picture of oxidative nucleophilic substitution of hydrogen (ONSH). Previous reports indicated that the presence of an electron donating/withdrawing group on the quinoxaline ring had a significant influence on the yield and selectivity. This is between reactions A+a, A+b, and B+a. These experimental observations correlated well with the modelling results when the potential energy surfaces (PES) of the reactions were compared. / Dissertation (MSc)--University of Pretoria, 2020. / National Research Foundation (NRF) / Chemistry / MSc / Unrestricted

The biopharmaceutical industry is constantly developing biological drugs, resulting in increased levels of product related impurities having similar characteristics as the target. The aim of the ligand project was to address future challenging purifications by developing new ligands for future resins for the biopharmaceutical industry. The purpose of this study was to develop a high-throughput screening method and use it to compare 15 novel multimodal anionic exchange ligand analogues with two reference ligands, for future polishing steps in the downstream process. The protein binding behavior of the ligands were studied with alkaline phosphatase, human serum albumin, α-chymotrypsinogen A and a monoclonal antibody as model proteins, at various pH values and salt concentrations. The selection process of the model proteins was based on stability studies, a study of their adsorption to the 96 well plate, and their binding behavior on three of the ligand analogues and one reference ligand. The percent protein bound to the ligands at the various conditions was calculated and presented in plots in order to study their binding behaviors. The calculated values were also used in order to evaluate the results in principal component analysis, creating chromatographic diversity maps. The maps were used to get an overview of the differences and similarities of the ligand analogues compared to the reference resins, which can be used for selecting ligands for future research and biomanufacturing. Four analogues and one reference ligand were also studied in a column format where different gradients were used, which confirmed the obtained results in the plate experiments.