DISTRIBUTION, METABOLISM AND. EXCRETION OF BIFLÜRANOL, A NOVEL BIBENZYL A thesis submitted "for the degree of Doctor of Philosoohy in the University of Surrey by DEREK JOHN POPE July 1978 ,^C] o ' I ■' ProQ uest Number: 27750268 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent on 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 27750268 Published by ProQuest LLC (2019). Copyright of the Dissertation is held by the Author. Ail 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 SUMMARY The distribution, metabolism■and excretion of bifluranol, a novel bibenzyl, has been determined in mouse, rat, ferret and dog and compared to the fate of diethylstilboestrol. Bifluranol is extensively absorbed, but systemic tissue levels are low due to hepatic uptake and biliary excretion. After enterohepatic recirculation the dose is excreted in the faeces and only small amounts appear in urine. This pattern is similar to that reported for diethylstilboestrol. The metabolic fate of bifluranol was found to differ considerably from that of diethylstilboestrol. In rat oxidative metabolism of bifluranol (4-8%) was considerably lower than that reported for diethylstilboestrol (309o). Bifluranol was extensively excreted as diconjugates with glucuronic acid and sulphate, in contrast to the major conjugate of diethylstilboestrol, the monoglucuronide. Preliminary data indicate conjugation of bifluranol with phosphate in ferret and dog. Glucuronyl- and sulpho-transferase enzyme systems were shown to conjugate both bifluranol and diethylstilboestrol. The rate of bifluranol conjugation in both systems was > ten-fold higher than with diethylstilboestrol and may explain in part the extensive diconjugate formation of bifluranol. The differences in metabolism of bifluranol and diethylstilboestrol are discussed in terms of the aromatic fluorine substituents of bifluranol. II ACKNOWLEDGEMENTS I would like to thank the Directors of Biorex Laboratories Ltd, for placing their laboratory facilities at my disposal, I . am very grateful to Dr. Gottfried for making this study possible and for his constant encouragement, I am indebted to Professor Parke for his excellent overall supervision of the project and to Drs. Rhodes, Mervyn and Easter for their help and advice during these studies, I would like to thank Dr, Chan of Biorex Laboratories for the ' 5 synthesis of the intermediate for H labelling of bifluranol and the preparation of the hydroxylated derivatives of bifluranol. I would like to thank Professor Foster and Dr. Jarman of the Chester Beatty Institute for providing the mass spectra of various compounds. I would also like to thank Dr. Jones of Surrey University for his invaluable help with the isolated rat liver preparations. I am very grateful to Miss Lenora Thompson for her considerable help in the preparation and typing of this manuscript. Finally I would like to thank all my colleagues at Biorex Laboratories and Surrey University for their help in ways too numerous to mention. Ill CONTENTS CHAPTER ONE Page Bifluranol - historical aspects and metabolic 1 considerations CHAPTER TWO Distribution and excretion of bifluranol 21 CHAPTER THREE Metabolism of bifluranol 53 CHAPTER FOUR Oxidation of bifluranol 80 CHAPTER FIVE Conjugation of bifluranol 102 CHAPTER SIX Final discussion 128 REFERENCES 157 CHAPTER 1 BIFLURANOL - HISTORICAL ASPECTS AND METABOLIC CONSIDERATIONS 1.1 Historical Aspects 1.2 Metabolism of Hexoestrol and Diethylstilboestrol 1.5 Aromatic Halogen Substitution and Metabolism Mediated Toxicity 1.1 Historical Aspects Sex, until comparatively recent times in mankind*s history was a state of affairs to be accepted and enjoyed with little systematic consideration of the reasons for the basic differences between male and female. The advent of the scientific method and its application to the study of mans (and womans) chemical makeup has hov/ever changed this. It is now apparent that these fundamental differences in anatomical structure can arise from small differences in the structures and relative amounts of a few molecules within the body. Previously it had been observed that extracts from testes and ovaries were capable of restoring certain secondary sexual characteristics in castrated experimental animals. The successful isolation of the compounds became possible with the development of the vaginal cornification test for oestrogens (Allen and Doisy, 1923) and the growth of the comb in capons for androgens (Gallagher and Koch, 1929)* Oestrone was the first material to be isolated (Doisy, et al, 1929; Butenandt, 1929) and this was followed by oestriol (Marrian, 1930)^ androsterone (Butenandt, 1931)t progesterone (Butenandt and Westphal, 1934), ©estradiol (MacCorquodale, et al, 1935) and testosterone (David, et al , 1935)* Pure samples of the steroids were characterised chemically and their structures determined shortly after their isolation (Butenandt, 1932; Wintersteiner and Allen, 1934; Butenandt and Hanisch, 1935), although the absolute configuration of ©estradiol was not confirmed for some time (Turner, 1950; Gallagher and Kritchev.sky, 1950). The oestrogenic hormones isolated were potentially useful as contraceptive agents, but it was found that they lacked oral activity and had to be administered parenterally. A search for synthetic oestrogens started and, following the observation that 1-keto 1 ,2,3,4 tetrahydrophenanthrene was a weak oestrogen (Cook, et a l , 1933) and that all the oestrogenic hormones had a cyclopentenohexahydrophenanthrene ring structure (Dodds, 1937) , attempts were made to see how far one could depart from the structure of natural oestrogens and still retain oestrogenic activity. The development of syntheses had led to 4,4 - dihydroxystilbene and p-hydroxypropylbenzene was prepared as a simplification of this structure and found to be oestrogenic. Subsequently it was shown that this activity was due to a highly oestrogenic contaminant which proved to be the stilbene diethylstilboestrol (Dodds, 1969). The production of this and the two structurally similar compounds hexoestrol and dienestrol (Figure 1.1) provided materials of comparable or greater activity than oestradicl. Their most important feature however was that, unlike the natural oestrogens, they were active by the oral route (Dodds, et al, 1938a). Several analogues were produced subsequently and it was shown that the phenanthrene ring base was not essential for oestrogenicity, but none of these later compounds were as potent as diethylstilboestrol (Dodds, et al, 1958b,c). Many analogues of stilbene and bibenzyl structures have been produced since this early work and their pharmacological activity reviewed (Emraens, 1970). Figure 1.1 Structures of oestfadiol, diethylstilboestrol, hexoestrol, dienoestrol and bifluranol OH Oejstradiol Diethylstilboestrol Hexoestrol OH HO Dienoestrol Bifluranol (erythro-3,3 - difiuoro-4,4 -dihydroxy- OC-methyl-a -ethyl bibenzyl ) However, none of these compounds have to date been able to compete successfully with the original discoveries. It is of importance at this stage to note the difference between stilbene structures such as diethylstilboestrol, which are based on phenanthrene, and the bibenzyl structures, such as hexoestrol, where two phenols are joined by a saturated carbon chain. The central double bond of stilbenes not only imparts a different conformation to the molecule, but also allows electron delocalisation throughout the structui'e and provides an additional > site for oxidative metabolism in vivo (Metzler, 1976). Syntheses of steroids and their derivatives continued during this period, but in the field of sex steroids the only notable immediate success was with» ethynyloestradiol and mestranol (Inhoffen, et al, 1938), which were highly active orally. Despite extensive modifications to the basic steroid structure, the next major advance in this fiej.d did not come until the discovery of the norsteroids (Djerassi, et a l , 1934; McGinty and Djerassi, 1958), which produced many orally active progestational compounds and opened the way for the development of an oral steroidal contraceptive pill. The major novel departure in modifying steroid structures w a s achieved with the corticosteroids by the introduction of halogens, notably fluorine, into the steroid nucleus. Side effects in the treatment of rheumatoid arthritis with cortisone had limited the drugs usefulness and modifications to the structures were being made in an attempt to improve the drug. These had been largely unsuccessful until a 9-bromo derivative was found to possess unexpected cortisol like activity (Fried, et al, 1955)» The substitution of other halogen atoms quickly followed and the relative activities for the 9-halogenated cortisol acetate derivatives were 0 .1 (iodo-), 0 .2 8 (bromo-), 4,7 (chloro-) and 10.7 (fluoro-). Unfortunately, whilst the 9-fluoro- derivative was effective at one tenth the dose of cortisol acetate, it also possessed high mineralocorticoid activity which limited the drugs usefulness. Despite this initial disappointment, subsequent work produced;potent fltiorocorticosteroids with high anti-inflammatory activity and low mineralocorticoid