A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Masters of Science in Medicine (Pharmacology) Johannesburg,2015 / Breast cancer, is the most frequently diagnosed cancer in women and is associated with high mortality rates in South Africa. There is a high prevalence of metastatic breast cancer and triple negative tumours, which are associated with poor prognosis. In this study, the response of two breast cancer cell lines, MCF-7 and MDA-MB-231, were evaluated when treated with novel imidazo[1 ,2-a]pyridine amines. The compounds were synthesized by the School of Chemistry of the University of the Witwatersrand using the Groebke-Blackburn-Bienayme multicomponent reaction and tested for purity by elemental analysis. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay was used to determine the cytotoxic effects of test compounds on breast cancer cells and the toxic effect of compounds on non-tumorigenic unstimulated peripheral leukocytes. IC50 values of test compounds were calculated from sigmoidal dose response curves. The morphology of cells exposed to test compounds was assessed by fluorescent microscopy using Hoechst 33342, acridine orange and ethidium bromide. The ability of test compounds to induce apoptosis was measured by a colorimetric caspase-3 assay and a fluorometric Annexin-V-FITC assay. Monodansylcadaverine was used to determine if autophagic vacuoles were formed after exposure to test compounds. Three imidazo[1,2-a]pyridine amines, JD88, JD253 and JD256, were more cytotoxic to MCF-7 than to MDA-MB-231 cells. MCF-7 cells showed morphological features associated with apoptosis, and proteolysis by caspase-3/7 was observed after MCF-7 cells were exposed to JD88 for two hours. Vacuole formation induced by these compounds was not autophagic in since they did not co-localize with MDC florescent clusters. This together with the exposure of phosphatidylserine to the outer surface of MCF-7 cells suggests that apoptosis is induced in these cells. There was no evidence of cytochrome c translocation to the cytoplasm, which indicates that the intrinsic pathway of apoptosis is not activated. MDA-MB-231 cells treated with JD88, JD253 and JD256 were large with multiple nuclei and decondensed chromatin, morphological features associated with mitotic catastrophe. The cells also showed morphological features associated with necrosis and apoptosis, which include loss of cell membrane integrity and cell membrane blebbing respectively. MDA-MB-231 cells exposed to JD88 showed marked exposure of phosphatidylserine and this was observed to a minor extent in cells exposed to JD253 and JD256. Proteolysis by caspase-3/7 was activated in MDA-MB-231 cells exposed to JD88 as early as 2 hours after exposure. In conclusion three compounds; JD88, JD253 and JD256 were able to induce apoptosis in MCF-7 cells. These compounds were selectively toxic against MCF-7 cells compared to MDA-MB-231 cells and JD256 in particular was less toxic to leukocytes, which may translate to fewer serious adverse effects. Addition of a copper dioxygen complex to these compounds increases activity against both breast cancer cells. JD88 in particular has shown effective induction of apoptosis and this merits further investigation into its potential as a lead compound in breast cancer therapy. / AC2016

L’hépatite C est une maladie silencieuse, souvent asymptomatique, mais qui entraîne des lésions du foie et peut évoluer vers une cirrhose et, dans certains cas, vers un cancer. Le carcinome hépatocellulaire engendré par l’hépatite C constitue la première cause de transplantation hépatique. Les virus de l’hépatite C (VHC) et de la diarrhée virale bovine (VDVB) sont deux pestivirus possédant un ARN monocaténaire, de la famille des Flaviviridae. Bien qu’ayant des génomes différents, ils présentent une organisation structurelle et des processus de développement de l’enveloppe cellulaire comparables. Le screening de la chimiothèque du laboratoire a permis d’identifier cinq composés chefs de files, actifs à l’encontre du virus de l’hépatite C. Deux de ces composés de la série imidazo[1,2-a]pyridine ont fait l’objet d’un travail de pharmacomodulation dans le cadre des thèses de Jean-Baptiste Véron et Nicolas Henry. La première partie de mon travail de recherche a donc consisté à poursuivre ces travaux de pharmacomodulation afin de tenter d’améliorer l’activité de cette série chimique à l’égard du VHC ainsi que son index thérapeutique. La synthèse convergente de ces molécules a été effectuée grâce à des couplages métallo-catalysés.La seconde partie de mon projet de recherche a porté sur l’étude de la bifonctionnalisation des positions 7 et 8 du noyau imidazo[1,2-a]pyridine. Ces travaux ont permis de développer de nouvelles méthodologies pour introduire une diversité fonctionnelle sur ces positions. Ces molécules ont également été évaluées à l’encontre du VHC et l’une d’entre elle a montré une activité intéressante à l’encontre de ce virus. L’activité à l’encontre du VHC et l’index thérapeutique ont été améliorés pour deux molécules, analogues du BPIP. / Hepatitis C is a silent disease, often asymptomatic, responsible for hepatic lesions which may lead to cirrhosis and in some cases, to cancer. Hepatocellular carcinoma caused by hepatitis C virus is the leading cause of liver transplantation. Bovine viral diarrhoea (BVDV) and hepatitis C (HCV) viruses are two pestiviruses from the Flaviviridae family that have a single-stranded RNA. Despite having different genomes, they present a similar structural organization and processes of development of the cell envelope.The laboratory’s chemical library screening has identified five hits, active against the HCV. Two of these compounds from the imidazo[1,2-a]pyridine serie were pharmacomodulated as part of the Ph.D. thesis of Jean-Baptiste Véron and Nicolas Henry.The first part of my research work was therefore to continue the pharmacomodulation study of these chemical series to improve their activity against HCV and their therapeutic index. To do so, the convergent synthesis of these molecules was performed using metal-catalyzed couplings.The second part of my project has focused on the study of the difunctionalization of positions 7 and 8 of the imidazo[1,2-a]pyridine nucleus. This work helped to develop new methodologies for introducing a functional diversity on these positions. The antiviral activity of these molecules was also assessed against HCV and one of them has shown interesting activity against this virus.In conclusion, activity against HCV and therapeutic index have been improved for two molecules, analogues of BPIP.

Les motifs imidazo[1,2-a]pyridines et imidazo[1,2-b]pyridazines sont des noyaux très étudiés par la communauté scientifique, notamment dans le domaine thérapeutique. Ceci s’explique en partie par les progrès récents réalisés dans le domaine de la métallocatalyse qui ont permis une fonctionnalisation plus simple de ces molécules. Cependant, les dérivés tricycliques de ces structures sont restés assez peu étudiés malgré le fait que certains de leurs isostères présentent des propriétés biologiques intéressantes. Les travaux de cette thèse ont porté sur deux projets distincts : -La synthèse d’imidazo[1,2-b]pyridazines présentant un troisième cycle diazoté entre les positions 7 et 8, dans le cadre d’un contrat conclu avec la société Sanofi-Aventis. Ces composés, totalement originaux, représentent un véritable défi chimique et leur synthèse a nécessité d’importants travaux de mise au point. Nous avons ainsi employé différentes méthodes de couplages métallocatalysés. -La synthèse d’imidazo[1,2-a]pyridines un troisième cycle pyridinique entre les positions 2 et 3. Ces molécules, peu décrites dans la littérature, n’ont fait l’objet d’aucune évaluation biologique. Dans le but de synthétiser efficacement une chimiothèque intéressante pour ces structures, j’ai développé une méthode d’hétérocyclisation qui nous permet d’obtenir en deux étapes, gràce à des produits de départ très accessible, une importante variété de tricycles. / The imidazo[1,2-a]pyridines and imidazo[1,2-b]pyridazines moeities are very studied by scientific community, specially in therapeutic field. This is mostly due to recent progress in metallocatalyzed couplings which allow easier functionnalization of these structures. However, the tricyclic derivatives of these compounds remained not very studied despite important biological properties of some of there isosters. This thesis is divided in two parts : -The synthesis of imidazo[1,2-b]pyridazines with a dinitrogenated third cycle between the positions 7 and 8 in collaboration with Sanofi-Aventis. These new compounds were a real chemical challenge and their synthesis required important works of development. We used various metallocatalyzed couplings methods. -The synthesis of imidazo[1,2-a]pyridines with a pyridinic cycle between the positions 2 and 3. These molecules, poorly described in the literature, have never been subject to biological study. In order to effectively synthesize an interesting range of these structures, I have developed a new heterocyclization method which allows us to obtain in two steps, starting from commercialy available starting materials, some original tricyclics compounds.

Les préparations de composés comportant un noyau imidazo[1,2-a]pyridinique constituent un thème de recherche important en synthèse organique, compte tenu des nombreuses activités biologiques qu’ils peuvent présenter. Dans la première partie, nous nous sommes concentrés sur le développement de nouvelles stratégies rapides et efficaces basées sur l’utilisation de cuivre et de fer pour fonctionnaliser la position 6 du cycle imidazo[1,2-a]pyridine avec diverses amines et divers thiols. Ensuite, nous avons appliqué avec succès cette procédure pour la préparation de thioéthers symétriques et dissymétriques en utilisant le 2-mercaptobenzooxasole, réactif économique qui ne présente aucun risque chimique. Dans le dernier volet de ce travail, nous nous sommes intéressés au développement de nouvelles réactions multicomposants en vue de synthétiser divers pyrrolo[3',2':4,5]imidazo[1,2-a]pyridines et 5-aminopyrido[2’,1’:2,3]imidazo[4,5-c]isoquinoléines. / The preparations of imidazo[1,2-a]pyridine is one of important research topic in organic synthesis, This entitie present some interesting biological activities. First, we devoleped a new rapid and efficient strategies to functionalize position 6 of the imidazo[1,2-a]pyridine with various amines and thiols catalysed by copper and iron. Then we applied this procedure to the preparation of symmetric and asymmetric thioethers using 2 mercaptobenzooxasole, which is an economical reagent and which presents no chemical risk. The last part of this work concerns the development of new multicomponent reactions for the synthesis of various pyrrolo[3',2':4,5]imidazo[1,2-a]pyridine and 5-amino pyrido[2’,1’:2,3] imidazo [4,5-c]isoquinoléines.