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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Le développement de composés de coordination biologiquement actifs (antimicrobiens, les anti-inflammatoires, les antifongiques, les anti-oxydants et les anticancéreux) est un domaine de la chimie inorganique en évolution rapide, susceptible d'avoir un impact direct sur l'amélioration de la qualité de la vie. Les complexes métallo-antibiotique tirent parti de l'effet synergique pour aboutir à une activité pharmacologique améliorée. La reconnaissance du rôle des ions métalliques dans les systèmes biologiques et dans le traitement de diverses maladies attire l'attention sur les avantages d'étudier l'interaction des ions métalliques avec les molécules de médicaments organiques. Dans la continuité avec les travaux précédents de l’équipe, nous nous intéressons à la synthèse de nouvelles familles de complexes métaux-antibiotiques associant l’activité antiseptique d’un ion métallique à un ou deux types de molécules bioactives. Leurs actions additives doivent avoir un effet synergique et conduire à des traitements plus efficaces et devraient fortement minimiser les risques d'apparition de bactéries mutantes. Au cours de ce travail, nous avons réussi à synthétiser le premier complexe métal-antibiotique associant deux types d'antibiotiques différents comme ligands du Zn(II). Des études comparatives montrent qu'il a une meilleure activité antibactérienne contre E. Coli, E. Aureus, E. Feacalis que les antibiotiques parents et les complexes ne contenant qu'un seul de ces antibiotiques. Cela ouvre un nouveau concept appelé « Assemblage de Biomolécules Multi-actifs, ABM ». De plus, nous décrivons la synthèse et la caractérisation de nouveaux ligands antimicrobiens trifluorométhylés / Development of novel coordination complexes with diverse biological activities (antimicrobial, anti-inflammatory, antifungal, antioxidant and anticancer) is a rapidly evolving field of inorganic chemistry with potential direct impact on quality of life. Metal–drug complexes are of increasing interest in bioinorganic chemistry, leveraging the synergistic effect to lead to compounds with improved pharmacological activity. The recognition of the role of metal ions in biological systems and in treatment of various diseases calls attention to the benefits of studying the interaction of metal ions with organic drug molecules. In continuation with previous works of team, we focus here on the synthesis of new families of metal-antibiotic complexes associating, on one single-molecule, the antiseptic activity of a metal ion with the bioactivity of one or two type of bioactive molecules. Their additive actions have a synergetic effect and lead to more effective and shorter treatments and should strongly minimize the risks for appearance of bacteria mutants. In this work, we succeeded to synthesis the first metal-antibiotic complex associating two types of different antibiotic as ligands with Zn(II). The structure in the solid state of this new complex was established together with the studies of the chemical-physical properties. Comparative studies show it has a better antibacterial activity against (E.Coli, E,Aureus, E.Feacalis ) than parent antibiotics and complexes with only one of the antibiotic. This open a new concept named as Multi-Active Biomolecule Assembly. Moreover, the synthesis and characterisation of new trifluorométhylated antimicrobial ligands are described

Thesis submitted in fulfilment of the requirements for the Degree of Masters of Technology: Organic Chemistry, Durban University of Technology, 2011. / One of the greatest medical challenges facing mankind is the Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) which has now become a major epidemic with more than 40 million people infected worldwide. Of equal concern is its implication in high mortality and the onset of a number of opportunist mycobacterial infections, principally tuberculosis. In spite of the discovery of some relatively effective antiretroviral (ARV) drugs such as Azido Thymidine (AZT), Nevirapine (NVP) and Efavirenz (EFV), its’ application as either a single or combinational form causes side effects by harming the bone marrow. Drug resistance is a key cause of failure for treatment of HIV infection. Hence greater interdisciplinary efforts, involving both natural and social sciences, are needed urgently to combat this HIV/AIDS pandemic. Heterocyclic nitrogen based compounds, obtained from either natural sources or synthesis are adequately documented to have increased biological activity against several diseases. Recently a study of drugs containing the naphthyridine scaffold has acquired increasing attention because of its potential against HIV/AIDS. Generally, naphthyridines demonstrate good potency in both the enzyme and cellular systems and this prompted our interest in the synthesis of naphthyridine derivatives from simple and readily available starting compounds. Furthermore we wanted to form an intermediate quinolone moiety since it has good biological potential. In this study we report the synthesis of three naphthyridine derivatives, i.e. 6-phenyl-dibenzo [b, h] [1, 6] naphthyridine, 4-methyl-6-phenyl-dibenzo [b, h] [1, 6] naphthyridine and 2- methyl-6-phenyl-dibenzo [b, h] [1, 6] naphthyridine from easily available chemicals such as aniline, ortho-toludine, para-toluidine and ethyl benzoylacetate via a five step reaction scheme using either conventional reflux, microwave irradiation or both methodologies. It was found that microwave irradiation was several folds faster than conventional reflux methodology and the yield of the product was higher. The first step of the reaction scheme is a simple condensation reaction: three acrylate derivatives, viz. ethyl-3-aniline-3-phenyl acrylate, ethyl-3-phenyl-3-(ortho-tolylamino) acrylate and ethyl-3-phenyl-3-(para-tolylamino) acrylate were synthesized by refluxing ethyl benzoylacetate in an acidified ethanolic solution with aniline, ortho-toluidine and paratoluidine respectively for three hours; the yields were 95, 87.5 and 80 % respectively. Page v In the second step, thermal cyclisation was achieved for the synthesis of three quinoline derivatives, viz. 2-phenylquinoline-4(1H)-one, 8-methyl-2-phenylquinoline-4(1H)-one and 6- methyl-2-phenylquinoline-4(1H)-one from their respective acrylates under microwave irradiation for 5 minutes at 180 °C and 250 watts; the yields were 92, 84 and 80 % respectively. In the third step of the reaction, synthesis of 4-chloro-2-phenylquinoline, 4- chloro-8-methyl- 2-phenylquinoline and 4- chloro-6-methyl-2-phenylquinoline was achieved from a mixture of POCl3 and their respective quinolines via microwave irradiation for 3 minutes at 75 °C and 150 watts and via conventional reflux for 5 hours. It was found that under microwave irradiation, the reaction occurred nearly 100 fold faster but the % yield of the product was marginally higher. The fourth step of the reaction resulted in the formation of three schiff’s base, viz. 4-(Nphenyl)- 2-phenyl-4-aminoquinoline, 8-methyl-4-(N-phenyl)-2-phenyl-4-aminoquinoline and 6-methyl-4-(N-phenyl)-2-phenyl-4-aminoquinoline from their respective quinolines via microwave irradiation for 20 minutes at 180 °C and 180 watts and via conventional reflux for 2 hours. It was found that under microwave irradiation, the reaction occurred nearly 6 fold faster and the % yield of the product was over 10 % higher. The final step of the reaction was achieved by a Vilsmeir Haack reaction and in situ base catalyzed thermal cyclisation: 6-phenyl-dibenzo [b, h] [1, 6] naphthyridine, 4-methyl-6- phenyl-dibenzo [b, h] [1, 6] naphthyridine and 2-methyl-6-phenyl-dibenzo [b, h] [1, 6] naphthyridine were synthesized from their respective schiffs base via microwave irradiation for 20 minutes at 75 °C at 120 watts and via conventional reflux for 21 hours. It was found that under microwave irradiation, the reaction occurred over 60 fold faster and the % yield of the product was over 20 % higher.The outline for the five step synthesis of the three naphthyridines is presented graphically below: Page vi Key: (a) R1= H; R2=H (b) R1 = H; R2 = CH3 (c) R1 = CH3; R2 =H Reaction Conditions: 1) conc.HCl, EtOH, 3hrs, 50 °C; 2) conc. HCl, hand stirring 10 min; 3) 180 °C, MWI, 250 watts, 5 min; 4) POCl₃, MWI, 75 °C, 150 watts, 2 min; 5) POCl₃, 100 oC, 5 hrs; 6) aniline, t-BuOH, MWI, 180 °C, 180 watts, 20 min; 7) aniline, t-BuOH, 80 °C, 3 hrs; 8) DMF, POCl₃, MWI, 75 °C,120 watts 20 minute; 9) DMF, POCl3, 100 oC, 21 hrs.

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Infectious diseases are the second leading cause of deaths in the world with malaria being responsible for approximately the same amount of deaths as cancer in 2012. Despite the success in malaria prevention and control measures decreasing the disease mortality rate by 45% since 2000, the development of single-dose therapeutics with radical cure potential is required to completely eradicate this deadly disease. Targeting multiple stages of the malaria parasite is becoming a primary requirement for new candidates in antimalarial drug discovery and development. Recently, 4(1H)-pyridone, 4(1H)-quinolone, 1,2,3,4-tetrahydroacridone, and phenoxyethoxy-4(1H)-quinolone chemotypes have been shown to be antimalarials with blood stage activity, liver stage activity, and transmission blocking activity. Advancements in structure-activity relationship and structure-property relationship studies, biological evaluation in vitro and in vivo, as well as pharmacokinetics of the 4(1H)-pyridone and 4(1H)-quinolone chemotypes is discussed in the first chapter of the dissertation. Convenient synthetic approaches to 3-aryl-4(1H)-quinolones via metal-catalyzed and metal-free arylation of β-keto carbonyl compounds is addressed in Chapter 2. A clean arylation protocol of ethyl acetoacetate was developed by using hypervalent diaryl iodonium salts under mild and metal-free conditions. The scope of the reaction, using symmetric and unsymmetric iodonium salts varying in sterics and electronics was examined. This method has been applied for the synthesis of antimalarial compound ELQ-300, which is currently in preclinical development. Additionally, a first gram scale synthesis of ELQ-300 and its structurally related 4(1H)-quinolone P4Q-391 using operationally simple and highly yielding metal-catalyzed conditions have been shown. Despite of 3-aryl-4(1H)-quinolone chemotypes displaying potent antimalarial activities against Plasmodium species in vitro and in vivo, their development is also associated with risks. 4(1H)-quinolones are known to be poorly soluble and thus represent challenging drug candidates for pharmacokinetic and bioavailability reasons. Disrupting of molecular crystal packing and prodrug approaches were employed to overcome solubility and bioavailability issues in current series. Quantum mechanics torsion profile calculations, 13C T1 spin-lattice relaxation experiments as well as X-ray studies were conducted with the objective to determine possible effects improving key physicochemical properties such as solubility and stability. As a backup strategy, a prodrug approach was developed enabling the 4(1H)-quinolone scaffold to be functionalized at the quinolone's oxygen. In order to avoid any enzymatic dependences, an approach was developed in which the prodrug moiety was removed via a pH-triggered decay. Additionally, phosphate prodrugs regenerating the active compound via extrahepatic enzymes such as the ubiquitous alkaline phosphatase were investigated. The development of orally bioavailable prodrugs enabled an advance overcoming in vivo efficacy limitations and has been confirmed by pharmacokinetic profiling studies. The herein presented approaches present viable options for any pyridone quinolone antimalarial chemotype which are currently studied.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen often infecting the lungs of individuals with the heritable genetic disease cystic fibrosis and the peritoneum of those undergoing continuous peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this dissertation I used a rat dialysis membrane peritoneal model to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo. Included in this analysis are genes important for iron acquisition and growth in lowoxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in the environment. This lysis was shown to be dependent on antimicrobial quinolones produced by P. aeruginosa. I demonstrate that these quinolones are present in outer membrane vesicles (MVs). Not only were these quinolones present in MVs, but the quorum sensing molecule; 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal; PQS) was also packaged into MVs and was necessary for MV formation. These findings illustrate that a prokaryote possesses a signal trafficking system with features common to those used by higher organisms and outlines a novel mechanism for delivery of a signal critical for coordinating group behaviors in P. aeruginosa. Although MVs are involved in important processes besides signaling, the molecular mechanism is unknown. To provide insight into the molecular mechanism of MV formation, I examined the interaction of PQS with bacterial lipids. In this work, I demonstrated that PQS interacts strongly with the acyl chains and 4’-phosphate of bacterial lipopolysaccharide. The results of my studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signaling functions. Finally, I propose a model of PQSmediated MV formation where PQS interacts with specific outer membrane components to allow the necessary curvature for MV formation. / text

The global emergence of drug resistance is posing increasing difficulties in the public health control and treatment of tuberculosis (TB). Fluoroquinolones (FQs) are regarded as having a pivotal role among the antimicrobial agents in multidrug regimens against multidrug-resistant tuberculosis (MDR-TB). Thus, early diagnosis of fluoroquinolone-resistant (FQr) MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) by molecular tests has predictive value for the guidance of TB therapy. The pharmacokinetic (PK) and pharmacodynamic (PD) indices are valuable parameters to evaluate the activity and efficacy of fluoroquinolones (FQs) based upon the bactericidal effect and prevention of the emergence of resistance. In the first part of this study, the potencies of ofloxacin (OFX) and moxifloxacin (MXF) against clinical isolates of MDR-TB in terms of their PK/PD indices (Cmax/MIC90, AUC/MIC90, Cmax/MPC90 and AUC/MPC90) were investigated and compared. The results revealed that MXF displays higher ratios of PK/PD in vitro and could serve as a promising agent for the treatment of MDR-TB. Molecular tests on resistance genes are reliable and rapid technology for diagnosis of drug-resistant TB which facilitates timely patient management and public health control of TB. In the second part of the study, the feasibility of a PCRsequencing assay for the examination of mutations in the quinolone-resistance-determining- region (QRDR) of the gyrase A (gyrA) gene in FQ-resistant (FQr) Mycobacterium tuberculosis in direct clinical specimens was evaluated. As determined by gyrA QRDR DNA sequencing analysis, complete concordance of phenotypic and genotypic outcomes was demonstrated. The results indicate that the molecular assay is an accurate and effective method for the diagnosis of FQr TB and allows identification of mixed resistant variants in the same patient. GyrA mutations that associated with FQr in clinical isolates of M. tuberculosis were clustered in hotspot codons 88, 90, 91 and 94, corroborating other reports. We also detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from the respiratory specimens by using the PCR-DNA sequencing assay. In the third part of this study, the functional effect of the Ala74Ser mutant was verified through study of the DNA supercoiling inhibitory activities of OFX and MXF against the recombinant DNA gyrase. Fifty percent inhibitory concentrations (IC50) of FQs against the DNA supercoiling activities of the recombinant DNA gyrase complex reconstituted with gyrA Ala74Ser were eight-fold and 14-fold greater than the wild-type H37Rv reference strain, and results correlated well with their phenotypic drug susceptibilities. Besides, a combination of gyrA mutations (Glu21Gln, Ser95Thr and Gly668Asp) was also characterized to be non-functional polymorphisms. The impact of the gyrA Ala74Ser mutation on drug binding affinity was elucidated through a crystal structure model of the gyrA-MXF-DNA cleavage complex. Alanine at position 74 of gyrA in M. tuberculosis, which corresponds to the alanine at position 67 of gyrA in Escherichia coli, is an amino acid lying in the α3 helix domain which forms a hydrophobic interface between the gyrA-gyrA dimer. Perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative drug binding pocket, and leads to the reduction of the binding affinity of FQ due to the distance effect. This is the first report verifying that gyrA Ala74Ser mutation alone is responsible for FQr in M. tuberculosis. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy