L Antim icFolDial activities of nitrofiiFantoin and some novel nitFofiiFan componnds X lie s i tm itte i io T lie University of Lon ty Catlierine A nn Skarpe £ Jegree of D octor of Pkilosopky Microbiology Section, Department of Pbarmaceutics, TLe School of Pharmacy, Brunswick Square, London 1 ProQuest Number: 10104255 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104255 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Dedicated to my parents M^argaret and. A llan Sliarpe Declaration and Acknowledgments The work presented in this thesis was carried out in the Microbiology Section of the Department of Pharmaceutics and in the Department of Pharmaceutical and Biological Chemistry at The School of Pharmacy, University of London between November 1994 and November 1997. During this period ten days were spent at laboratories of the Genetic and Reproductive Toxicology Department, Medicine Safety Evaluation Division of GlaxoWellcome in Ware, Hertfordshire. I would like to thank my supervisors Dr. R. J. Pinney and Dr. R. A. Watt for their substantial support and encouragement throughout this endeavour. I should also like to thank: Marianne and Marion for their technical help and suggestions. Wilf for achieving such marvellous nuclear magnetic resonance spectra from the most contrary of compounds. Members of the GlaxoWellcome laboratories, especially Rob, Julia, Dave, Steve, Brian and Dr. Gatehouse, for providing facilities and guidance for the Salmonella and SOS/umu mutagenicity testing. Dr. Kazuei Igarashi of the Faculty of Pharmaceutical Sciences, Chiba University, Japan for the gift of Escherichia coli polyamine transport-deficient mutants. Proctor & Gamble Pharmaceuticals Inc., Norwich, NY, USA for their gift of semicarbazide acetic acid, used for the synthesis of a novel nitrofuran compound. The School of Pharmacy, University of London for their financial support. DECLARATION AND ACKNOWLEDGMENTS The mechanism of action of nitrofurantoin against the Escherichia coli K12 strain AB1157 was investigated. Like quinoione antibacterials, nitrofurans damage DNA and induce SOS error-prone DNA repair. However, unlike the quinolones, which exhibit three separate mechanisms of action depending on whether RNA or protein synthesis or cell division is functioning, the activity of nitrofurantoin was shown not to be dependent on protein or RNA synthesis or on cell division. The resistance of Escherichia co//AB1157, which is wild-type with respect to DNA repair, and some DNA repair-deficient mutants to nitrofurantoin decreased in the order TK702 umuC > wild-type AB1157 > AB2494 lexA > AB2470 recB > AB2463 recA > JG138 pci A > TK501 uvrB umuC > JC3890 uvrB > AB1886 uvrA. Plasmid R46, which confers mutagenic error-prone DNA repair on its host, increased the minimum inhibitory concentration (MIC) of nitrofurantoin against uvrB- deficient strains but did not affect the MICs of the wild-type or other DNA repair- deficient strains. R46 conferred some protection against bactericidal concentrations of nitrofurantoin to AB1157 and the umuC, uvrA, uvrB and uvrB umuC mutants but not to the lexA, recA, recB or po/A-deficient strains. Other mutator plasmids pGW12, pGW16, pKM101, pYD1, R16, R124, R144, R391, R446b, R621a and R805a, from a variety of incompatibility groups, were also tested. None affected the MIC of nitrofurantoin against E. coli strain AB1157. However, pKM101, R446b, R621a and R805a conferred some protection whereas pGW12, pGW16, R144 and R391 sensitized AB1157 to a bactericidal concentration of nitrofurantoin. E. coli AB1157 showed low spontaneous mutation to nitrofurantoin resistance. Mutation frequencies were not increased in cells exposed to an inhibitory concentration of nitrofurantoin. R46 was the only plasmid of a variety of incompatibility groups to be eliminated from E. coli AB1157 by nitrofurantoin. Nine novel nitrofuran compounds were synthesized. The general method involved condensation of a polyamine (putrescine, spermidine or spermine) with a nitrofuran and isolation of the required products by extraction and other techniques. The products were characterized by UV spectroscopy, ^H nuclear magnetic resonance and mass spectroscopy. The inhibitory activities of the novel nitrofuran compounds were determined on a range of Gram-positive and Gram-negative ABSTRACT 4 bacteria and fungi. Bis-5-nitrofurfurylidenylputrescine (compound I), bis-5- nitrofurfurylidenylspermine (compound II) and bis-5-nitrofurfurylidenylspermidine (compound III) showed excellent broad spectrum antibacterial activity and good antifungal activity. Bis-5-nitrofurfurylidenylethylspermidine (compound IX ) also showed good antifungal activity and 5-nitrofurfurylidenylglucosamine (compound V I) good antipseudomonal activity, neither of which are exhibited by nitrofurantoin. Compounds I, II, III and 4-(N-5-nitrofurfurylidenyl)aminobenzoyl-2-acetylamino- ethylamide (compound IV ) were screened for mutagenicity and genotoxicity using the Ames Salmonella mutagenicity assay and the SOSIumu genotoxicity test. Nitrofurantoin was not mutagenic against S. typhimuhum strain TA98, which detects frameshift mutations, but was highly mutagenic against strain TA100, which detects base-pair substitutions. All four novel nitrofuran compounds tested were less mutagenic than nitrofurantoin in strain TA100. Similarly, all four novel nitrofuran compounds were shown to be considerably less genotoxic than nitrofurantoin in the SOSIumu test. The activities of nitrofurantoin and the four novel nitrofuran compounds I - IV were also compared in wild-type and polyamine transport- deficient mutants of E. coli K12. The novel compounds were active against transport-deficient mutants suggesting they would enter the cell via uptake mechanisms other than those determined by polyamine transport pathways. ABSTRACT a Page Line Corrigendum 27 8 (Smith, 1980) should read (Smith, 1984) 50 2 (Lewin, 1994) should read (Lewin, 1994; Wang, 1985) 50 3/4 Insert “such as topoisomerases II and IV ” to read “Type 2 topoisomerases, such as topoisomerases II and IV, produce a transient...” 50 8 Insert “Topoisomerase IV is necessary for chromosome segregation (Kato at a!., 1990; Kato at a/., 1992)” to read “...(Srivenugopal at a/., 1984). Topoisomerase IV is necessary for chromosome segregation (Kato at a/., 1990; Kato at a!., 1992). Type 1...” 58 15 (Ferrrante ef a/., 1995) should read (Ferrante ef a/., 1995) 54 18 Replace (Lewin, 1994) with (Hardy, 1988; Jacob ef a/., 1963) 8 6 8 Insert “using strains of E. coli 343/113 thy arg lys (Materials & Apparatus Table 2.1.1)” to read “Plasmids were transferred by conjugation in nutrient broth using R^ strains of E. coli 343/113 thy arg lys (Materials & Apparatus Table 2.1.1).” 8 8 9 Replace “Bactericidal” with “Bacteriostatic” to read “Bacteriostatic activity was determined from the zones of inhibition.” 112 N/A Figure 4.1.19. legend should read Survival of E. coli AB1886 uvrA and uvrA(R46) in 20xMIC of nitrofurantoin. # = uvrA] O = uvrA(R46) 125 32 Replace “Quinolones target DNA as their site of action (Goss at a/., 1964)...” with “Quinolones bind to DNA;topoisomerase II (DNA gyrase) complexes blocking DNA replication (Chen at ai, 1996)...” CORRIGENDA 169 last Replace “thanthose” with “than those” 170 13 Replace “polyamine-deficient” with “polyamine uptake- deficient” 178 27 Replace “stevens” with “Stevens” 184 25/26 After “...exposed to clinically-attainable concentrations of nitrofurantoin.” insert “Nitrofurantoin-induced mutants may have been observed had the concentration of nitrofurantoin to which the cells were exposed been less lethal. Mutants may also have been observed had the cells been given a chance to recover and express resistance in nitrofurantoin- free medium after exposure to nitrofurantoin and before plating onto nutrient agar containing nitrofurantoin.” 194 26 Replace “antimicrobialsmight” with “antimicrobials might” Figures Error bars on all figures show standard error of the mean (SEM) R e fe re n c e s Chen, C.-R., Malik, M., Snyder, M. & Driica, K. (1996) DNA gyrase and topoisomerase IV on the bacterial chromosome: quinolone-induced DNA cleavage. Journal of Molecular Biology 258:627-637 Jacob, P., Brenner, S. & Cuzin, F (1963) On the regulation of DNA replication in bacteria. Cold Spring Harbor Symposium on Quantitative Biology 2B\ 329- 348 Kato, J., Nishimura, Y., Imamura, R., Niki, H., Hiraga,8 . & Suzuki, H. (1990) New topoisomerase essential for chromosome segregation in E. coll. Cell 63: 393-404 Kato, J., Suzuki, H. & Ikeda, H. (1992) Purification and characterization of DNA topoisomerase IV in Escherichia coli. Joumal of Biological Chemistry 2S7: 25676-25684 Wang, J. 0. (1985) DNA topoisomerases. Annual Review of Biochemistry 54: 665-697 Appeniis A (pages 232-235) makes up part of this corrigenda CORRIGENDA Page Declaration and Acknowledgements 3 Abstract 4 Contents 6 Abbreviations and Symbols 14 List of Figures 18 List of Tables 24 List of Chemical Schemes 26 Aims of This Thesis 27 PART ONE