SYNTHESIS AND BIOLOGICAL ACTIVITY OF CHLOROQUINE FERROCENYL CONJUGATES FOR THE TREATMENT OF MALARIA by PALOMA SALAS FERNANDEZ Hons. B. Sc. Pontificia Universidad Catolica del Peru, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2012 © Paloma Salas Fernandez, 2012 ABSTRACT Malaria is one of the main causes of mortality and morbidity in the world, endangering billions and affecting millions of people each year. Resistance to common antimalarial drugs has proven to be a challenging problem in malaria control. In an attempt to develop an effective and affordable treatment for malaria, ferrocenyl conjugates incorporating a common antimalarial drug such as chloroquine have been developed. Based on the previous successes of organometallic derivatives of classical pharmacophores, a series of chloroquine-bridged ferrocenyl derivatives was synthesized. These novel compounds present an unprecedented binding mode of chloroquine to the ferrocene moiety, through the bridging of the two Cp rings. The structural effects of this type of conjugation of chloroquine and ferrocene were studied by NMR spectroscopy and crystal structure determination. These compounds were studied along with the monosubstituted ferrocenyl analogs and the organic components in order to compare the effects of the substitution on their biological response. The antiplasmodial activity of these sets of compounds was evaluated against the chloroquine-sensitive (D10) and the chloroquine-resistant (D2d and K1) malaria parasite (Plasmodium falciparum) strains. Additionally, their biological activity was assessed using a number of in vitro assays. Biological and physical properties were correlated to the antimalarial activity. All compounds were active against the tested parasite strains. The presence of the ferrocene significantly improved the antiplasmodial action, when compared to chloroquine, against the drug-resistant parasite strains. While the chloroquine-bridged ferrocenyl derivatives were in general less active than the monosubstituted ferrocenyl analogs, they ii retained activity in the drug-resistant strain to a greater extent. Their particular conformation, compact size and lipophilicity/hydrophilicity balance could be providing them with the structural characteristics needed to escape the mechanisms responsible for resistance. Additionally, two strategies for drug design were applied: multiple-loading and multifunctional therapy approaches. Ferrocenyl compounds loaded with two molecules of chloroquine and mefloquine were synthesized and characterized. Similarly, ferrocenyl derivatives of chloroquine and mefloquine were further derivatized with a monossacharide molecule. The double-loaded compounds are the first few examples of their kind. The multifunctional conjugates improved the antimalarial action of the ferrocenyl quinoline derivatives. iii PREFACE The majority of the material in Chapter 1 has been submitted for publication: P. F. Salas, C. Herrmann, C. Orvig. Metalloantimalarials. Portions of the introductory part of Chapters 2 and 3 are also part of this manuscript. I was responsible for the researching and writing of this review article. Preparation of figures and tables and formatting of the manuscript was done by me and Dr. Herrmann. Editing was performed by me, Dr. Herrmann and Dr. Orvig. Portions of the content in Chapter 2 pertaining to the antimalarial activity of compounds 4a, 4b and 4e have been published: C. Herrmann, P. F. Salas, B. O. Patrick, C. de Kock, P. J. Smith, M. Adam, C. Orvig. 1,2-Disubstituted Ferrocenyl Carbohydrate Chloroquine Conjugates as Potential Antimalarial Agents. Dalton Transactions, 2012, 41, 6431-6442. The majority of the content in Chapters 2 and 3 will be submitted for publication: P. F. Salas, C. Herrmann, J. Cawthray, C. de Kock, P. J. Smith, J. Chen, E. Polishchuk, B. O. Patrick, M. J. Adam, C. Orvig. Synthesis and Biological Activity Evaluation of Chloroquine- Bridged Ferrocenyl Conjugates as Potential Antimalarial Agents. I was responsible for the outline and design of this project. I performed all synthetic experiments and structural characterization, with the help of Dr. Herrmann. All solid-state structures were obtained by X-ray diffraction by Dr. Patrick at UBC X-Ray Crystallography services. The in vitro antiplasmodial activity assay was performed by Dr. De Kock and Dr. Smith at the University of Cape Town. The in vitro antitumor and cytotoxicity assays were performed by me, Dr. Herrmann and Jessie Chen from the Biological Services Laboratory at the UBC. The qualitative evaluation of inhibition of β-hematin formation was performed by Dr. Cawthray with my help. The electron density surface maps and polar surface area values were obtained iv by Dr. Cawthray. I was solely responsible for the rest of the assays performed and for the structure-activity correlations established. The work in Chapter 4 will be submitted for publication: P. F. Salas, C. Herrmann, C. Nimphius, A. Kenkel, M. J. Adam, C. Orvig. Synthesis and Characterization of Bischloroquine and Bismefloquine Ferrocenyl Derivatives as Potential Antimalarials. I was responsible for the outline and design of this project. C. Nimphius and A. Kenkel (under my instruction and supervision) performed part of the synthetic experiments. I was responsible for the remaining synthetic work and structural characterization. The work in Chapter 5 will be submitted for publication: P. F. Salas, C. Herrmann, B. O. Patrick, M. J. Adam, C. Orvig. Synthesis and Characterization of Ferrocenyl Carbohydrate Conjugates of Chloroquine and Mefloquine. I was responsible for the outline and design of this project. I performed all synthetic experiments and structural characterization, with the help of Dr. Herrmann. All solid state structures were obtained by X-ray diffraction by Dr. Patrick UBC X-Ray Crystallography services. The in vitro antitumor and cytotoxicity assays were performed by me, Dr. Herrmann and Jessie Chen from the Biological Services Laboratory at the UBC. This work is covered under an international patent: Adam, M. J., Orvig, C., Salas, P., Herrmann, C. Carbohydrate-metallocene-antimalarial conjugates. U.S. Pat. Appl. US2010216727, 2010. v TABLE OF CONTENTS ABSTRACT ........................................................................................................................................... ii PREFACE ............................................................................................................................................. iv TABLE OF CONTENTS ...................................................................................................................... vi LIST OF TABLES ................................................................................................................................ ix LIST OF FIGURES................................................................................................................................ x LIST OF SCHEMES ........................................................................................................................... xiii LIST OF ABBREVIATIONS .............................................................................................................. xv ACKNOWLEDGEMENTS .............................................................................................................. xviii DEDICATION ..................................................................................................................................... xx Chapter 1 Introduction ........................................................................................................................... 1 1.1 Malaria ............................................................................................................................ 1 1.2 Prevention, diagnosis and treatment of malaria .............................................................. 5 1.3 Traditional antimalarial therapy ...................................................................................... 9 1.3.1 Quinoline-containing antimalarial drugs....................................................................... 11 1.3.2 Mode of action of quinoline-containing drugs .............................................................. 15 1.3.3 Resistance ..................................................................................................................... 20 1.4 Metalloantimalarials...................................................................................................... 22 1.4.1 Metal chelators .............................................................................................................. 23 1.4.2 Metal complexes ........................................................................................................... 28 1.4.3 Bioorganometallic compounds ..................................................................................... 37 1.5 Thesis overview ............................................................................................................ 53 Chapter 2 Synthesis and Characterization of Chloroquine-Bridged Ferrocenyl Conjugates ............... 55 2.1 Introduction ................................................................................................................... 55 2.2 Experimental ................................................................................................................