http://researchcommons.waikato.ac.nz/ Research Commons at the University of Waikato Copyright Statement: The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). The thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: Any use you make of these documents or images must be for research or private study purposes only, and you may not make them available to any other person. Authors control the copyright of their thesis. You will recognise the author’s right to be identified as the author of the thesis, and due acknowledgement will be made to the author where appropriate. You will obtain the author’s permission before publishing any material from the thesis. A Reinvestigation of Salvarsan and Related Arsenic Chemistry. A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at The University of Waikato by Nicholas Lloyd 2011 i “Life is a chemical incident, A normal oxidation, A fluorescence of the brain” Paul Ehrlich 1872 ii Abstract In 1910 the first deliberately targeted search for a new chemotherapeutic agent came to fruition when Paul Ehrlich introduced Salvarsan for the treatment of syphilis. This thesis presents a detailed review of the history and literature leading up to and following on from Ehrlich‟s discovery and thoroughly investigates the chemistry of Salvarsan and related species. A series of arylarsonic acids was prepared and fully characterised by electrospray mass spectrometry, NMR and X-ray crystallography for six examples. A detailed analysis of the hydrogen bonding in crystals of these molecules showed that they adopt several characteristic motifs which govern the packing in the crystals. Two of the examples containing NH2 groups crystallised as zwitterions while one NH2 containing example containing other bulky groups was is its molecular form. Salvarsan (cyclo 3-amino-4-hydroxyphenylarsenic(I)) was prepared by several different methods and analysed in detail using high resolution electrospray mass spectroscopy. This showed that Salvarsan consists of small cyclic species of the type (RAs)n where R is 3-NH2-4-OHC6H3 and n is three or greater. The dominant species in an aqueous solution of Salvarsan were found to be (RAs)3 and (RAs)5 . A detailed analysis is presented of impurities in Salvarsan prepared by different methods and also in a sample of original commercial Salvarsan. Mixtures of (RAs)n and (R‟As)n exchange R groups in aqueous solution at room temperature, as shown by ESI-MS. ESI-MS studies are reported for the oxidation product of Salvarsan, RAs(OH)2 (commercially known as Mapharsen) and related As(III) compounds. Oligomers iii involving As-O-As linkages were found in solution and one tetrameric example (R = 3- NO2-4-OHC6H3) was isolated and structurally characterised. Preliminary ESI-MS studies showed that As(III) species bind to thioredoxin, a possible target for the pharmaceutical activity of Salvarsan and its derivatives. iv Acknowledgments Thanks to everyone who has supported me throughout this project, especially Professor Brian Nicholson for pushing me through to completion and supplying me with seedling leeks for my garden. Big thanks also to Wendy Jackson and Pat Gread for technical assistance, support and friendly conversation throughout this project. Thanks to The Marsden Fund administered by the Royal Society of New Zealand for financial support. Thanks to Professor Steve Reithmiller for visiting to assist with the project, information on Paul Ehrlich and sourcing original samples of Salvarsan. Thanks to Pfizer for their generous donation of Mapharsen from the Parke Davis Archives Many thanks to Professor Hugh Morgan and Dr. Ron Ronimus for assistance and many helpful suggestions in understanding the biological aspects of this project. Thanks to all the other students who i have shared a lab with over the years, especially Carol, Bevan, Steve, Narendra, Kelly and Megan for assistance with Microsoft word. Thanks to everyone else who has offered advice, support, encouragement and conversation over the years I have been at Waikato. Finally thanks to Mum and Dad for constant support and encouragement. v Table of Contents Abstract ......................................................................................................................... iii Acknowledgments ......................................................................................................... v Table of Contents ......................................................................................................... vi List of Figures .............................................................................................................. xii List of Tables ............................................................................................................... xv 1 Chapter One - Introduction .................................................................................. 17 1.1 Some background on arsenic ........................................................................ 17 1.1.1 Pre-alchemy ........................................................................................... 17 1.1.2 Discovery of elemental arsenic.............................................................. 18 1.1.3 Geber or Pseudo-Geber? ........................................................................ 20 1.1.4 Chinese alchemy .................................................................................... 20 1.1.5 Problems ................................................................................................ 21 1.1.6 Albertus Magnus ................................................................................... 21 1.2 Syphilis ......................................................................................................... 22 1.2.1 Early medicine ....................................................................................... 22 1.2.2 A new disease ........................................................................................ 23 1.2.3 Origins ................................................................................................... 25 1.2.4 A complicated disease ........................................................................... 27 1.2.5 New disease needs new cure ................................................................. 30 1.2.6 Competing cures .................................................................................... 31 1.2.7 Arsenic ................................................................................................... 33 1.3 More on syphilis ........................................................................................... 34 1.3.1 More confusion. ..................................................................................... 35 vi 1.3.1.1 Many diseases – one name ............................................................. 36 1.3.1.2 Different stages .............................................................................. 36 1.3.1.3 Syphilis – the great imitator ........................................................... 38 1.3.2 Other interesting points on syphilis ....................................................... 39 1.3.3 Advances in treatment ........................................................................... 40 1.4 Paul Ehrlich ................................................................................................... 42 1.4.1 Beginnings ............................................................................................. 42 1.4.2 Chemistry in Ehrlich‟s time ................................................................... 44 1.4.3 Paul Ehrlich and Robert Koch ............................................................... 44 1.4.4 The Japanese link................................................................................... 46 1.4.5 Trypanosomes and arsenic..................................................................... 49 1.4.6 Atoxyl .................................................................................................... 50 1.4.7 Ehrlich and atoxyl .................................................................................. 53 1.4.8 Enter Alfred Bertheim ........................................................................... 54 1.4.9 Ehrlich‟s numbering system .................................................................. 58 1.5 Salvarsan ....................................................................................................... 59 1.5.1 Discovery of Salvarsan .......................................................................... 59 1.5.2 The Salvarsan wars ................................................................................ 64 1.5.3 Administration of Salvarsan .................................................................. 67 1.5.4 After Salvarsan ...................................................................................... 71 1.5.5 Commercial preparation of Salvarsan ................................................... 73 1.5.6 The end of Ehrlich ................................................................................. 74 1.6 Literature ....................................................................................................... 77 1.6.1 The Salvarsan investigations ................................................................. 79 1.6.2 Neosalvarsan .........................................................................................