New Drugs against Trypanosomatid Parasites: Rediscovery of Fexinidazole INAUGURALDISSERTATION zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Marcel Kaiser aus Obermumpf, Aargau Basel, 2014 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Dieses Werk ist unter dem Vertrag „Creative Commons Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 3.0 Schweiz“ (CC BY-NC-ND 3.0 CH) lizenziert. Die vollständige Lizenz kann unter creativecommons.org/licenses/by-nc-nd/3.0/ch/ eingesehen werden. 1 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel auf Antrag von Prof. Reto Brun, Prof. Simon Croft Basel, den 10. Dezember 2013 Prof. Dr. Jörg Schibler, Dekan 2 3 Table of Contents Acknowledgement .............................................................................................. 5 Summary ............................................................................................................ 6 Zusammenfassung .............................................................................................. 8 CHAPTER 1: General introduction ................................................................. 10 CHAPTER 2: Fexinidazole - A New Oral Nitroimidazole Drug Candidate Entering Clinical Development for the Treatment of Sleeping Sickness ........ 26 CHAPTER 3: Anti-trypanosomal activity of Fexinidazole – A New Oral Nitroimidazole Drug Candidate for the Treatment of Sleeping Sickness ....... 66 CHAPTER 4: 1-Aryl-4-nitro-1H-imidazoles, a new promising series for the treatment of Human African Trypanosomiasis .................................... 84 CHAPTER 5: Novel 3-Nitro-1H-1,2,4-triazole-based Aliphatic and AromaticAmines as anti-Chagasic Agents .................................................... 119 CHAPTER 6: Novel 3-Nitro-1H-1,2,4-triazole-based Amides and Sulfonamides as Potential anti-Trypanosomal Agents .................................. 145 CHAPTER 7: Novel 3-nitro-1H-1,2,4-triazole-based compounds as potential anti-Chagasic drugs: in vivo studies .............................................................. 175 CHAPTER 8: Agrochemicals against Malaria, Sleeping Sickness, Leishmaniasis and Chagas Disease ................................................................ 200 CHAPTER 9: Antiprotozoal activity profiling of approved drugs: a starting point toward drug repositioning ..................................................... 222 CHAPTER 10: General Discussion ............................................................... 263 4 Acknowledgement This thesis would not have been possible without the help and support of a number of people whom I would like to acknowledge. My first and foremost gratitude goes to my supervisor Reto Brun and the Swiss TPH director Marcel Tanner who encouraged me to my PhD. They gave me the roots for my fruits, to us the words of Marcel Tanner. I owe a debt of gratitude to Reto Brun for his constant trust and support since I joined the parasite chemotherapy group 13 years ago. I particular thank him for the trips to Africa and the introduction into the drug screening network. Swiss TPH is a great place to work and I would like to thank Marcel Tanner for his positive spirit, his outstanding enthusiasm and leadership. My thanks go also to Pascal for his openness and advice. I thank Simon Croft (LSHTM) for joining my thesis committee. My gratitude is also addressed to all former and present Parasite Chemotherapy Unit members. Particularly I thank Monica Cal for their hard work, our fruitful discussions and the implementation of my ideas. A big thank you to Sonja Keller, Sibylle Sax, Christiane Braghiroli and Guy Riccio, for your expertise and support in carrying out all the in vitro and in vivo studies. I thank Christina Kunz for sharing her knowledge of parasite cultivation and our good discussions. Many thanks to DNDi especially to Jean-Robert Ioset, for the constant support and exchange. My thanks go to Els Torreele and Bernadette Bourdin Trunz, who initiated the project of nitroimidazoles by researching the literature and collecting all the compounds. My warmest thanks go to my wife, Erika and to my sons Simon and Oliver; you are my oasis of peace and a place of inspiration. 5 Summary Neglected tropical diseases (NTDs) are a group of communicable diseases mostly affecting people in developing countries. These diseases are responsible for a major part of the global morbidity, mortality and poverty. There is no doubt that the well-being of people in the developing world can only be improved if the NTDs are controlled. An important tool for disease control is the drug treatment. The few available drugs are unsatisfactory because of the limited efficacy, adverse effects and the high price. Chagas disease, leishmaniasis and human African trypanosomiasis belong to this group of NTDs. They are caused by infections with protozoa of the family Trypanosomatidae. For these three diseases new drugs are urgently needed. By definition there is no commercial market for drugs against NTDs. Drug research and development (R&D) for NTDs is mainly driven by the public sector, the so-called product development partnerships (PDPs). Drug R&D is a very long (10-15 years), risky and therefore expensive process. Three different series of compounds (agrochemicals, marketed drugs and nitro-heterocyclic compounds) were tested for their antiparasitic effects, with the aim to identify new lead compounds or even clinical candidates against leishmaniasis, sleeping sickness, and Chagas disease. Agrochemicals are used worldwide on a large scale in food production. They undergo a rigorous toxicological testing prior to launch. Over 600 compounds were screened for their antiparasitic activity. Agrochemicals are not optimized for use in mammals, yet a significant number of molecules were found with good and selective in vitro activity. Some of them showed also efficacy in the corresponding rodent model. These results indicate that agrochemicals can provide very interesting starting structures for drug research against parasitic diseases. Drugs or drug-like compounds are an ideal starting point for antiparasitic drug discovery, because very often pharmacokinetic and toxicological data are available. A number of drugs, including antibiotics, antivirals, antifungals, and anti-psychotics were assayed for antiparasitic activity. Some of the drugs tested showed selective antiparasitic activity. These compounds can be regarded as new lead structures and should be further investigated. 6 Nitroheterocycles belong to a well- known class of compounds with the stigma of being mutagenic or genotoxic. Over 700 compounds, mainly nitroimidazoles, have been systematically tested for their antiparasitic activity, and their pharmacokinetics and mutagenicity was investigated. A number of effective, non-mutagenic and non- genotoxic compounds was identified. So fexinidazole was rediscovered, a drug that had been in clinical development already in the 70’s as a broad-spectrum antimicrobial drug. Fexinidazole is rapidly metabolized to fexinidazole-sulfoxide and -sulfone. The parent compound and the two principle metabolites showed in vitro trypanocidal activity against all (sensitive and resistant) tested T. brucei strains (IC50 of 0.2 - 0.9 g / ml). Fexinidazole cured the first stage mouse model with a 4-day oral treatment of 100 mg/kg/day and the 2nd stage mouse model with a 5-day oral treatment of 200 mg/kg/day. The two metabolites are mainly responsible for the good efficacy in animal models. Both reach very high concentrations in blood and brain tissue. Fexinidazole has successfully completed preclinical development and Phase I clinical trials and is currently in a clinical phase II / III study. With the approach of phenotypic screening of compounds that have been developed for other purposes, new leads for drug R&D against Chagas’ disease, leishmaniasis and human African trypanosomiasis were identified. Fexinidazole is the first drug candidate in clinical Phase II / III trials since decades. It would be the first oral drug for the treatment of stage 1 and 2 of human African sleeping sickness. If fexinidazole overcomes all obstacles, this would be a major breakthrough in the fight against African sleeping sickness. With a well tolerated, orally active drug like Fexinidazole the elimination of sleeping sickness seems finally tangible. 7 Zusammenfassung Vernachlässigte Tropenkrankheiten („Neglected Tropical Diseases“ NTDs) sind eine Gruppe von übertragbaren Krankheiten, welche vor allem die Bevölkerung der Entwicklungsländer betreffen. Diese Krankheiten verursachen einen großen Teil der weltweiten Morbidität, Mortalität und Armut. Es besteht kein Zweifel, dass das Wohlergehen der Menschen in Entwicklungsländern nur verbessert werden kann, wenn diese Krankheiten unter Kontrolle gebracht werden. Ein wichtiges Instrument dafür ist die medikamentöse Behandlung. Die wenigen verfügbaren Medikamente sind unbefriedigend, aufgrund ihrer beschränkten Wirksamkeit, Nebenwirkungen und des hohen Preises. Chagas-Krankheit, Leishmaniose und die menschliche afrikanische Schlafkrankheit gehören zu dieser Gruppe von NTDs. Sie werden durch eine Infektion mit Protozoen aus der Familie Trypanosomatidae verursacht. Für diese drei Krankheiten werden dringend neue Medikamente benötigt. Es gibt keinen kommerziell interessanten Markt für Medikamente gegen vernachlässigten Krankheiten. Die Medikamentenforschung