STUDIES RELATED TO THE SYNTHESIS OF TETRACYCLINE a thesis presented by NORMAN JAMES ALBERT GUTTERIDGE in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chemistry Department, Imperial College, LONDON June, 1966. To Jo ABSTRACT A review of some important reactions of tetracycline are presented. An account of the attempts to synthesise tetracycline follows with a summary of the progress contributed by workers in these laboratories. The new contribution to this work commences with a study of the reduction of the 9-oxo function in 2-phenyl- 3-cyano-3-tetrahydropyranyloxy-9-oxo-2a18,8a-trihydro- naphthacene[4,4a$5,bc]furan (A). 0 (A) An attempted conversion of 2-phenyl-3,8a-(N-phenyl- isoxazolidine)-9-oxo-2a1 3,8,8a-tetrahydronaphthaceno[4,4a,- 5,b0]-furan (B) into 2-phenyl-3/9-dioxo-2a0,8a-trihydro- naphthaceno[4,4a,5,bc]furan (C) is then described together with a re-examination of previous work on these derivatives. (B) (C) To assist studies in this series of compounds, 1-N- phenylamino-1,2,3,4-tetrahydronaphthalene was synthesised. A preparation of 2-phenyl-4-(31 15'-diacetoxy-21- ethylene-acetal-4'-carbomethoxybenzy1)-5-oxo-naphtho- [4,10,5,bc]furan (D), a suitable precursor for the recently described photocyclisation process, is described in detail. (D) 0 — C Ph Finally, studies in the 2 -phenyl -4,6 -dihydroxy-5 -carbo - methoxy -3,8a -(N -phanylisoxazolidine)-9 -oxo -2a,3,8,8a - tetrahydronaphthaceno[4,4a,5,bc]furan series (E) were commenced. (E) ACKNOWLEDGEMNTS thank Professor D.H.R. Barton, F.R.S., for the privilege of working for him and for his constant interest, guidance and encouragement. I also thank Dr. J. E. Baldwin for his helpful advice and enthusiasm at all times. I wish also to thank other members of the Laboratory (1963-1966) for helpful discussions. Thanks are due to the technical staff for their assistance; in particular to Mr. D. Aldrich, Mr. P.R. Boshoff, Mrs. 1. Boston, Mr. E. Pilch and the analytical staff of the department. A Science Research Council grant is gratefully acknowledged. CONTENTS Page Introduction 1 Some reactions of the tetracyclines 8 1.Reduction 10 2.Oxidation 14 3.Aromatisation 16 4.Epimerisation 20 5.Reactions on nitrogen atoms 21 6.Reactions on oxygen atoms 24 7.6-Methylenetetracyclines 27 8.Halogenation 29 9.Nitration 30 10.Photolyses 32 11.Chelation 33 Synthesis of tetracyclines 36 References 52 Outline of previous work 62 Studies related to the synthesis of tetracyclines 78 Experimental 184 References 290 1 INTRODUCTION The name 'tetracycline, has been assigned(1) to a group of antibiotics characterised by their common hydronaphthacene skeleton. These pale yellow crystalline antibiotics possess broad spectra of antibacterial activity and are produced by strains of streptomyces. The first example of this class of compound, aureomycin (1), was isolated in 1948 by Duggar(2) from streptomyces aureofaciens. A similar compound, terramycin (2), was isolated from streptomyces rimosus in 1950 in Chas. Pfizer and Co. Laboratories:(3) In 1952, the structures of these complex natural products were elucidated by Woodward and his co-workersS4) The parent compound of the family, tetracycline (3), was isolated from streptomyces albo-niger(5) and shown to be identical to the hydrogenolysis product of aureomycin.(6,7) Terramycin and aureomycin have thus the generic names oxytetracycline and chlorotetracycline respectively. The accepted numbering system for the carbon skeleton of tetracycline is as depicted in (4). These antibiotics have been extensively used in the chemotherapy of infectious diseases on account of their 2 — Cl OH\ Tie N(Me) 2 OH (1) CONH2 O H OH OH 0 ( 3 ) ( 4 ) powerful activity against a broad spectrum of pathogenic organisms. Tetracycline itself has largely replaced the other two tetracycline antibiotics in clinical practice and now is one alternative to penicillin for treating acute throat infections and is standard treatment for chronic bronchitis.(8) Tetracycline is readily absorbed from the gastro intestinal tract and the blood stream, is highly stable and well tolerated by any route of administration.(9) The low side effects are mainly due to the effect of the drug on the alimentary tract and its flora. The tetracyclines have also found favour as preser- vatives of fish and meat.(10) Studies on the biosynthesis of 7-chlorotetracycline have demonstrated that [1-14C]- and [2-14C]acetate,(11) [2-140]glycine and [CH3-140]methionine(12) all produced high levels of 14a incorporation. The skeleton is largely built up from ahead to tail' linkage of acetate units. Birch has shown that the methyl and chlorine atoms were introduced directly and suggested that whereas the greater part of the molecule was derived from the poly-3-carbonyl framework (5), a large part of ring A was derived from glutamic acid.(13) Gatenbeck's results, on the other hand, indicate that the hydronaphthacene ring system was - 4 — [Mei [Me] [0] 0 o o o ( 5 ) *-[CO 2H] 0 [0 ] 0 Me 1 OH (6) CONH2 0H ( 7 ) Me H OH H OH (9) CONH2 OH derived entirely from acetate units and the carboxamido group was formed from carbon dioxide produced in the fermentation. Support for the later theory comes from the demonstration that compound (6) is a direct precursor to tetracycline. McCormick has established that oxidative hydroxylation of 5a,6-anhydrotetracycline (7) at C-6, succeeded by reduction of the resulting 5a,11a-dehydrotetracycline (8), were the final steps in the biogenesis of these anti- biotics.(15) Chemical and X-ray studies(16,17) as well as the recent application of N.M.R. (18,19,20) have confirmed the relative stereochemistry depicted in structures (1,2,3). In all probability the other naturally occurring tetracyclines(21) (e.g. 9, 10, 11) possess the same con- figurations at all common asymmetric centres. In 5- hydroxytetracycline, however, differences in interpretation of the X-ray data for the 0-5 configuration has led to an N.M.R. study. The protons at C-5a and C-4a have been shown in 4-dedimethylamino-5-hydroxy-12a-22175a16- anhydrotetracycline (12) to be trans-diaxial. The con- formations of several 5-hydroxytetracyclines in solution appear to be different from that derived tentatively by X-ray studies.(17) Cl CH 1.1 NMe2 (10) CONH OH 2 OH OH Bt OH Me 1IMe2 (12) Shemyakin(22) has shown that structure (1) also expresses the absolute configuration of these antibiotics by O.R.D. studies on 7-chlorotetracycline. Tetracyclines have recently been subjected to treat- ment by mass spectrometry(23) as well as some related compounds .(24) All the derivatives of tetracycline examined were reasonably volatile without extensive thermal decomposition except for 5-hydroxytetracycline which was partially cleaved before entry into the ion chamber. Strong molecular ion peaks were observed and almost all cleavages occurred in rings A and B, since the aromatic ring D and the ease of aromatisation of ring C conferred stability on the molecule. - 8 - Some reactions of the tetrac clines The reactions may be divided into a) skeletal re- arrangements, including reactions causing extensive decomposition of the molecule and b) transformations involving the existing functional groups. In the former group the reactions have been used primarily for structure elucidation. Reaction conditions to be controlled with care in synthetic work include the use of both acids and bases. In mildly alkaline (pH 8) solution the C ring of the tetracyclines are opened to yield the biologically inactive iso-tetracyclines (13). This reaction is markedly accelerated by the presence of substituents at C-7.(25) Acid catalysed dehydration occurs readily as expected of a t-benzyl alcohol. In the tetra- cyclic series the 5a,6-anhydrotetracyclines are formed but the 5-hydroxy-5a,6-anhydrotetracyclines react further under these conditions to give the ado-terramycins (14).(9) In the latter category numerous compounds have been prepared in order to provide a base for a detailed insight into structure-activity relationships. Some of these important reactions will be reviewed here and procedures that might be used to complete a total synthesis will be noted. Me (me)2 ( 13 ) H (14) ONH2 N(1,1e)2 H (15) ONH2 a) X = 01 ID) X =Br -10 - The reactions presented are classified as far as possible according to general types of reaction although it is recognised, however, that in such a classification many reactions affect the total molecule. A consideration of separated functional groups is not possible in some cases and instead their mutual interactions are discussed. 1. Reduction Many functional groups of the tetracyclines have been reduced quite specifically, so that a study of the effect of these groups on antibacterial activity can be made. The reductive removal of halogen in 7-.halogenotetracyclines to give tetracycline illustrates the non-essential role of halogen for hiological activity. This reduction can be carried out in neutral solution(25) with a Pd/C catalyst, but it was usual to add a bse e.g. triethylamine or triethanolamine.(6) In a similar way,(26,27,28) ila-halogenoderivatives (15) may be hydrogenolysed. An alternative means of replacement of the lla-halogen substituent was treatment with zinc in acetic acid or with aqueous sodium hydro- sulphite.(28) The lla-halogen thus offers a convenient way of protecting the 11,12-P-dicarbonyl system In syn- thetic work. — 11 - Zinc in glacial acetic acid also effected removal of the 4-dimethylamino and the 12a-hydroxyl functions.(25) A superior method for the reductive removal of the 4- dimethylamino group was the reduction of the methiodide (P.23) with zinc in aqueous acetic acid. All 4-dedi- methylamino derivatives appear to have little biological activity.