QUALITY ASSESSMENT OF ANTIMALARIAL MEDICINES SOLD IN THE DEMOCRATIC REPUBLIC OF THE CONGO AND PHYTOCHEMICAL INVESTIGATIONS ON A CONGOLESE ANCISTROCLADUS LIANA DISSERTATION zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Jean-Pierre Mufusama Koy Sita aus Kinshasa, Demokratische Republik Kongo Würzburg 2019 Eingereicht bei der Fakultät für Chemie und Pharmazie am: ………………………………. Gutachter der schriftlichen Arbeit: 1. Gutachter ………………………………….. 2. Gutachter ………………………………….. Prüfer des öffentlichen Promotionskolloquiums: 1. Prüfer ………………………………….. 2. Prüfer ………………………………….. 3. Prüfer ………………………………….. Datum des öffentlichen Promotionskolloquiums: ………………………………….. Doktorurkunde ausgehändigt am: Die vorliegende Arbeit wurde in der Zeit von Januar 2014 bis Oktober 2018 am Institut für Organische Chemie der Universität Würzburg angefertigt. Herrn Prof. Dr. Dr. h.c. mult. Gerhard Bringmann danke ich für die hervorragende Unterstützung bei dieser Arbeit, die freundliche Atmosphäre und die exzellenten Arbeitsbedingungen. Teile der im Rahmen dieser Arbeit erzielten Ergebnisse waren bereits Gegenstand von Publikationen[196,235,243,275] sowie von Posterpräsentationen und Vorträgen. Dedicated to my late parents Contents Contents Contents ....................................................................................................................................... I Abbreviations ........................................................................................................................... IV Introduction ................................................................................................................................ 3 I.1 Malaria and Poor-Quality Medicines .................................................................................. 11 I.1.1 Malaria ......................................................................................................................... 11 I.1.2 Poor-Quality Medicines ............................................................................................... 13 I.2 Capillary Electrophoresis .................................................................................................... 16 I.3 Naphthylisoquinoline Alkaloids ......................................................................................... 18 II.1 Quality of the Antimalarial Medicine Artemether - Lumefantrine in Eight Cities of the Democratic Republic of the Congo .......................................................................................... 25 II.1.1 Introduction ................................................................................................................. 25 II.1.2 Sample Collection ....................................................................................................... 26 II.1.3 Visual Inspection ........................................................................................................ 27 II.1.4 Analysis by Thin-Layer Chromatography .................................................................. 28 II.1.5 Analysis by High-Performance Liquid Chromatography ........................................... 30 II.1.6 Analysis by Liquid Chromatography - Mass Spectrometry ....................................... 34 II.1.7 Comparison with Other Studies .................................................................................. 35 II.1.8 Limitations of the Study .............................................................................................. 36 II.1.9 Conclusion .................................................................................................................. 36 II.2 Capillary Zone Electrophoresis for the Determination of Amodiaquine and Three of its Synthetic Impurities in Pharmaceutical Formulations ............................................................. 37 II.2.1 Introduction ................................................................................................................. 37 II.2.2 Method Development .................................................................................................. 39 II.2.2.1 Influence of Buffer Concentration and pH Value Changes ................................. 39 II.2.2.2 Influence of Voltage and Separation Temperature............................................... 41 II.2.3 Method Validation ...................................................................................................... 43 II.2.3.1 Content Determination of the Main Component .................................................. 43 II.2.3.2 Impurity Profiling ................................................................................................. 45 II.2.4 Analysis of Antimalarial Drug Samples ..................................................................... 47 II.2.5 Conclusion .................................................................................................................. 49 II.3 Phytochemical Investigations on a Congolese Ancistrocladus Liana ............................... 50 II.3.1 Introduction ................................................................................................................. 50 II.3.2 Results and Discussion ............................................................................................... 51 II.3.3 Biological Evaluation .................................................................................................. 61 I Contents II.3.4 Conclusion .................................................................................................................. 64 II.4 (R)-Tonkafuranone and Related Compounds: Improved Synthesis, Stereochemical Purity in Nature, and Bioactivities of the Pure Enantiomers .............................................................. 66 II.4.1 Introduction ................................................................................................................. 66 II.4.2 Results and Discussion ............................................................................................... 67 II.4.3 Conclusion .................................................................................................................. 73 III.1 Summary .......................................................................................................................... 77 III.2 Zusammenfassung ............................................................................................................ 83 IV.1 General Aspects ............................................................................................................. 91 IV.1.1 Analytical Instruments .............................................................................................. 91 IV.1.2 Chemicals and Cell Lines .......................................................................................... 93 IV.2 Quality of the Antimalarial Medicine Artemether - Lumefantrine in Eight Cities of the Democratic Republic of the Congo .......................................................................................... 95 IV.2.1 Sample Collection ..................................................................................................... 95 IV.2.2 Visual Inspection ....................................................................................................... 95 IV.2.3 Testing by Thin‐Layer Chromatography ................................................................... 96 IV.2.4 Analysis by High‐Performance Liquid Chromatography ......................................... 96 IV.2.5 Analysis by Liquid Chromatography - Mass Spectrometry ...................................... 97 IV.3 Capillary Zone Electrophoresis (CZE) for the Determination of Amodiaquine (13) and Three of its Synthetic Impurities in Pharmaceutical Formulations ........................................ 106 IV.3.1 Buffers and Background Electrolyte (BGE)............................................................ 106 IV.3.2 Sample Solution Preparation ................................................................................... 106 IV.3.2.1 Content Determination ...................................................................................... 106 IV.3.2.2 Impurity Profiling ............................................................................................. 106 IV.3.3 Method Validation ................................................................................................... 107 IV.3.3.1 Content Determination ...................................................................................... 107 IV.3.3.2 Impurity Profiling ............................................................................................. 108 IV.3.4 Content Determination Using the HPLC Method Described in The International Pharmacopoeia ................................................................................................................... 109 IV.4 Phytochemical Investigation on a Congolese Ancistrocladus Liana ............................. 110 IV.4.1 Plant Material .......................................................................................................... 110 IV.4.2 Extraction and Isolation ........................................................................................... 110 IV.4.3 Newly Isolated Alkaloids ........................................................................................ 111 IV.4.3.1 Mbandakamine B3 (38) ....................................................................................