"Science, Faculty of"@en . "Chemistry, Department of"@en . "DSpace"@en . "UBCV"@en . "Yalpani, Mohamed"@en . "2011-09-29T20:33:32Z"@en . "1965"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "In part I of this thesis are described some studies toward the total synthesis of tetracycline.\r\nAttempts to transform the key aromatic compounds, terrarubein and 6-methylpretetramid into actual or hypothetical biosynthetic intermediates, failed to yield useful non-aromatic products. However, several of the transformations further along the route have been achieved. Thus, the conversion of 12a-deoxy-5a,6-anhydrotetracycline into 7-chloro-5a,6-anhydrotetracycline was successfully carried out.\r\nIn a different approach it was attempted to convert the synthetic tetracycline derivative 7-chloro-4-dedimethylamino-5a,6-anhydrotetracycline to 7-chloro-5a,6-anhydrotetracycline via a series of bromination-aminatipn experiments. Chromatographic evidence is presented for the formation, in trace amounts, of 7-chloro-5a,6-anhydro-4-epi-tetracycline.\r\nPart II is concerned with the study of the possible precursor activity of triacetic acid lactone, a potential polyketomethylene chain intermediate in the biosynthesis of aromatic compounds. (3,5-\u00C2\u00B9\u00E2\u0081\u00B4 C) Triacetic acid lactone was fed to P. patulum and labelled griseofulvin was isolated and degraded. It was found that radioactivity is incorporated into griseofulvin a non-specific way. In one strain of the mould used two new metabolites were found as a result of the addition of triacetic acid lactone. Addition of triacetic acid lactone to the mould also causes an unexplained enhancement of metabolite formation."@en . "https://circle.library.ubc.ca/rest/handle/2429/37686?expand=metadata"@en . "The University of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of MOHAMED YALPANI. B.Sc . j University of Washington, 1961 WEDNESDAY, SEPTEMBER 8, 1965, at 10:00 A.M. IN ROOM 261, CHEMISTRY BUILDING COMMITTEE IN CHARGE Chairman: I. McT, Cowan L. Bo Hayward T. Money J. P. Kutney R. E, Pincock F. McCapra R. Stewart G. H. N. Towers External Examiner: Professor W. A. Ayer Department of Chemistry University of Alber t a Edmonton, Albe r t a SOME ASPECTS OF NATURAL PRODUCTS CHEMISTRY ABSTRACT In part I of t h i s thesis are described some studies toward the t o t a l synthesis of t e t r a c y c l i n e . Attempts to transform the key aromatic compounds terrarubein and 6-methylpretetramid into actual or hypothetical biosynthetic intermediates, f a i l e d to y i e l d useful non-aromatic products. However, several of the transformations further along the route have been achieved. Thus, the conversion of 12a-deoxy-5a, 6-anhydrotetracycline into 7-chloro-5a,6-anhydrotetra c y c l i n e was su c c e s s f u l l y c a r r i e d out. In a d i f f e r e n t approach i t was attempted to con-vert the synthetic t e t r a c y c l i n e d e r i v a t i v e 7-chloro-4 dedimethylamino-5a,6-anhydrotetracycline to 7-chloro-5a,6-anhydrotetracycline via a series of bromination-amination experiments. Chromatographic evidence i s presented for the formation, i n trace amounts, of 7-chloro-5a,6-anhydro-4-epi-tetracycline. Part IT i s concerned with the study of the possible precursor a c t i v i t y of t r i a c e t i c acid lactone a potential polyketomethylene chain intermediate i n the biosynthesis of aromatic compounds. (3,5-^C) T r i a c e t i c acid lactone was fed to P.patulum and l a b e l l e d g r i s e o f u l v i n was i s o l a t e d and degraded. I t was found that r a d i o a c t i v i t y i s incorporated into g r i s e o f u l v i n a non-specific way. In one s t r a i n of th mould two new metabolites were found as a r e s u l t of the addition of t r i a c e t i c acid lactone. Addition of t r i a c e t i c acid lactone to the mould also causes an un-explained enhancement of metabolite formation. GRADUATE STUDIES F i e l d of Study: Organic Chemistry Topics i n Physical Chemistry Seminar i n Chemistry Topics i n Inorganic Chemistry Chemical Ki n e t i c s Topics i n Organic Chemistry Organic Stereochemistry Physical Organic Chemistry Organic Reaction Mechanisms Recent Synthetic Methods i n Organic Chemistry A. Bree J. A. R. Coope J. P, Kutney N. B a r t l e t t W. R. Cullen D. G, L. James E. A. Ogryzlo J. P. Kutney D. E. McGreer R. E. Pincock L. D, Hayward R. Stewart A. I. Scott G. G. S. Dutton A. Rosenthal PUBLICATION A. Rosenthal and M. Yalpani. Reaction of A r a l k y l Ketone Oximes with Carbon Monoxide and Hydrogen to Y i a l d Formamides and Secondary Amines. Can. J . Chem. 43 (1965) SOME ASPECTS OF NATURAL PRODUCTS CHEMISTRY by Mohamed Yalpani\" B.Sc., U n i v e r s i t y of Washington, 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Chemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE DEPARTMENT OF CHEMISTRY UNIVERSITY OF BRITISH COLUMBIA August, 1965 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s representatives\u00E2\u0080\u009E I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^-fr***?*? The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada Date 5- C) T r i a c e t i c acid lactone was fed to P 3patulum and l a b e l l e d g r i s e o f u l v i n was i s o l a t e d and degradedo It was found that r a d i o a c t i v i t y i s incorporated into g r i s e o f u l v i n a non-specific way. In one s t r a i n of the mould used two new metabolites were found as a r e s u l t of the i v a ddition of t r i a c e t i c acid lactone 0 Addition of t r i a c e t i c acid lactone to the mould also causes an unexplained enhancement of metabolite formation 0 V CONTENTS PART I : Approaches t o the T o t a l S y n t h e s i s of T e t r a c y c l i n e I n t r o d u c t i o n \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 ~ \u00C2\u00BB - \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 1 S y n t h e t i c Attempts \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094- \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 5 The B i o g e n e s i s of T e t r a c y c l i n e \u00E2\u0080\u0094 \u00E2\u0080\u0094 ~ ~ \u00E2\u0080\u0094 10 D i s c u s s i o n I\u00E2\u0080\u009E '^Biogenet ic Type1* Approach \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094-.\u00E2\u0080\u0094 \u00E2\u0080\u0094 >\u00E2\u0080\u0094 17 (a) T r a n s f o r m a t i o n o f T e r r a r u b e i n \u00E2\u0080\u00A2\u00E2\u0080\u0094 \u00E2\u0080\u0094 2 1 (b) T r a n s f o r m a t i o n o f 6 - M e t h y l p r e t e t r a m i d \u00E2\u0080\u0094 3 5 I I 0 Non B i o g e n e t i c Type T o t a l S y n t h e s i s of T e t r a c y c l i n e \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u00A2\u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 42 E x p e r i m e n t a l \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 ~ \u00E2\u0080\u0094\u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 54 References \u00E2\u0080\u0094- \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 84 PART I l s S t u d i e s i n R e l a t i o n t o the Acetate Pathway t o Aromatic Compounds I n t r o d u c t i o n \u00C2\u00AB=- \u00E2\u0080\u0094 \u00C2\u00AB - \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094' 89 D i s c u s s i o n \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 =- \u00E2\u0080\u0094 \u00E2\u0080\u0094 99 E x p e r i m e n t a l \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 123 References ~ - \u00E2\u0080\u0094 ^ - \u00E2\u0080\u0094 139 S y n t h e t i c Approaches to the T e t r a c y c l i n e s \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 appendix v i LIST OF FIGURES AND TABLES PART Is F i g u r e 1 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 4 3 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 m- \u00E2\u0080\u009E \u00E2\u0080\u0094 io 4 \u00E2\u0080\u009E \u00E2\u0080\u0094 \u00E2\u0080\u0094 ... \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u009E 14 5 . 20 6 \u00E2\u0080\u0094 \u00E2\u0080\u0094 22 7 . 37 T a b l e 1 ~ - \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 29 PART I I F i g u r e 1 ~ \u00E2\u0080\u0094 ~ \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 -- \u00E2\u0080\u0094 91 2 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 . \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 94 3 \u00E2\u0080\u009E \u00E2\u0080\u0094 \u00E2\u0080\u009E .\u00E2\u0080\u009E \u00E2\u0080\u0094 97 4 \u00E2\u0080\u0094 __ \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 101 5 \u00E2\u0080\u009E \u00E2\u0080\u009E \u00E2\u0080\u0094 \u00E2\u0080\u0094. \u00E2\u0080\u009E \u00E2\u0080\u0094. \u00E2\u0080\u0094 101 6 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 116 7 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 -- .-- \u00E2\u0080\u0094 \u00E2\u0080\u0094 ~ 120 8 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094. 120 viz. ACKNOWLEDGEMENTS I wish t o express my thanks t o P r o f e s s o r A d o S c o t t n D r e To Money p and D r 0 F\u00C2\u00BB McCapra f o r the p r i v i l e d g e o f working w i t h them 9 and f o r t h e i r constant h e l p and p a t i e n c e d u r i n g the course of my re s e a r c h 0 I wish a l s o t o thank Mr\u00C2\u00BB P 0 S a l i s b u r y f o r h i s expert and i n v a l u a b l e a s s i s t a n c e d u r i n g the i n v e s t i g a t i o n o f mould m e t a b o l i t e S o Thanks are a l s o due t o the L e d e r l e L a b o r a t o r i e s f o r the g i f t o f the many samples of t e t r a c y c l i n e d e r i v a t i v e s 9 and a l s o t o D r 0 A o Rhodes of the Glaxo L a b o r a t o r i e s f o r the g i f t o f Popatulum c u l t u r e S o PART Io APPROACHES TO THE TOTAL SYNTHESIS OF TETRACYCLINE I N T R O D U C T I O N A n t i b i o t i c s a r e m e t a b o l i c p r o d u c t s o f m i c r o o r g a n i s m s w h i c h p o s s e s s t h e c a p a c i t y ^ i n low c o n c e n t r a t i o n ^ o f i n h i b i t -i n g t h e growth o f s o r d e s t r o y i n g o t h e r m i c r o o r g a n i s m s . They a r e e l a b o r a t e d m a i n l y by f u n g i , b a c t e r i a and a c t i n o m y c e t e s g a l t h o u g h m a t e r i a l s e x h i b i t i n g a n t i b i o t i c p r o p e r t i e s a r e a l s o produced by l i c h e n s 9 i n s e c t s and h i g h e r p l a n t s , , C e r t a i n s u b s t a n c e s d e r i v e d f r o m t h e f i r s t t h r e e c a t e g o r i e s have a t t a i n e d ? d u r i n g t h e p a s t two decades, c o n s i d e r a b l e s u c c e s s a s t h e r a p e u t i c a g e n t s j p e n i c i l l i n , s t reptomycin;, b a c l t e r a c i n j t e r r a m y c i n p a u r e o m y c i n , C h l o r o m y c e t i n and e r y t h r o m y c i n a r e well-known examples,, A n t i b i o t i c s , i n a d d i t i o n t o b e i n g i m p o r t a n t f o r r e a s o n s o f p u b l i c h e a l t h , a r e o f g r e a t i n t e r e s t t o t h e o r g a n i c c h e m i s t f o r i n no o t h e r a r e a o f t h e n a t u r a l p r o d u c t f i e l d has he c o n f r o n t e d such n o v e l t y and c o m p l e x i t y o f s t r u c t u r e S o The developments i n t h e a n t i b i o t i c f i e l d a r e o r d i n a r i l y t r a c e d back t o 1929 p when F l e m i n g n o t e d t h a t a p r i n c i p l e p r oduced by a m o u l d P e n i c i l l i u m n o t a t u m a p o s s e s s e d p o t e n t b a c t e r i o s t a t i c p r o p e r t i e s . T h i s f o r t u n a t e o b s e r v a t i o n was c a p i t a l i z e d upon d u r i n g t h e e a r l y p a r t o f 1940s when commer-c i a l p r o d u c t i o n by f e r m e n t a t i o n commenced,, G h e m i c a l i n v e s t i ~ g a t i o n on t h e c o n s t i t u t i o n o f t h i s r e m a r k a b l e s u b s t a n c e c u l m i n a t e d d u r i n g t h i s p e r i o d <> A t t e n t i o n d u r i n g ;more r e c e n t y e a r s has s h i f t e d t o the a n t i b i o t i c materials produced by the actinomycetes group of microorganisms, As a consequence of these e f f o r t s the knowledge of natural product chemistry has been enriched correspondingly\u00C2\u00BB ISOLATION AND STRUCTURE OF THE TETRACYCLINES The t e t r a c y c l i n e s are a family of broadspectrum a n t i b i o t i c s produced i n the course of the metabolism of various Streptomyces species, or by simple chemical modifi-cations of naturally occuring members of the c l a s s . The f i r s t of these y e l l o w - c r y s t a l l i n e compounds/ aureomycin ( 1 , R ^ G l j R2r=H)iJ was is o l a t e d by Duggar 1 from Streptomyces aureofacienSo The discovery of i t s powerful a c t i v i t y against a broad spectrum of pathogenic microorganisms was followed i n 1950 by the I s o l a t i o n of the second member of the series, terramycin^ < l p R|=Hj R2=0H).\u00E2\u0080\u009E a metabolite of Streptomyces rimosus 0 (1) This stimulated an intense e f f o r t toward the s t r u c t u r a l e l u c i d a t i o n of these complex natural products,, The p r e l i m i -nary studies early indicated two c l o s e l y r e l a t e d compounds^; as well as affording similar degradation products3 t h e i r u l t r a v i o l e t spectra were nearly superimposable* and t h e i r orange-yellow c r y s t a l l i n e hydrochloride s a l t s were isomor-phous o The pu b l i c a t i o n ^ of the structure of aureomycin and terramycin i n 1953 confirmed the pre d i c t i o n as to t h e i r s i m i l a r i t i e s and the name t e t r a c y l i n e was proposed f o r the structure of ( i * R^=R2=H), 5 ~ 1 0 This prototype t e t r a c y c l i n e was obtained by c a t a l y t i c hydrogenolysis of 7-chlorotetracyclineHj, and was l a t e r discovered i n a metabolite of S o aureofaciens- 1- 2; i t t o o 3 showed high a n t i b i o t i c activity\u00C2\u00BB In a search f o r f u r t h e r a n t i b i o t i c s a number of other t e t r a c y c l i n e s were found some of which w i l l be discussed below i n the section on the biosynthesis of the t e t r a c y l i n e s * The problem of the t o t a l synthesis i s rendered formi-dable by the complex array of functional groups located upon the c h a r a c t e r i s t i c hydroriaphthacene r i n g system. Imposed upon the t e t r a c y c l i c framework i s a variety of substituents which r e s u l t i n a minimum of f i v e asymmetric carbon atom within the molecule p thereby imposing a major stereochemical challenge to the t o t a l synthesls 0 The s t e r e o c h e m i s t r y has been examined by X - r a y s t r u c -t u r a l a n a l y s l s ^ - l S and has e s t a b l i s h e d the r e l a t i v e c o n f i g u r a t i o n o f the s i x asymmetric carbon atoms of t e r r a -mycin* (see f i g u r e 1) F i g u r e 1\u00C2\u00BB I t appears e s t a b l i s h e d t h a t the presence o f the d imethy1-amino group a t G-4 i n the c o r r e c t n a t u r a l c o n f i g u r a t i o n i s i. e s s e n t i a l f o r f u l l b i o l o g i c a l activity<> Thus e p i m e r i s a t i o n a t p o s i t i o n kg which t a k e s p l a c e r e a d i l y a t i n t e r m e d i a t e pH's- , r e s u l t s i n a sharp f a l l i n the a n t i b a c t e r i a l a c t i v i t y o f a l l t e t r a c y c l i n e s ^ ' * ^ \u00C2\u00BB On the o t h e r hand removal of the G-6 h y d r o x y l and C-6 methyl has no e f f e c t upon the a c t i v i t i e s o f these compounds\u00E2\u0080\u009E Thus the compound ^ - d e o x y - S - d e m e t h y l -7 -ch loro te t raeyc l ine'p ( 2 ) 9 d e r i v e d from the n a t u r a l l y o c c u r i n g 6 - d e m e t h y i - 7 - c h l o r 6 t e t r a c y c l i n e by s e l e c t i v e r e d u c t i o n of the C-6 h y d r o x y l group e x h i b i t s the p o w e r f u l a n t i b a c t e r i a l a c t i -v i t y a s s o c i a t e d w i t h 7 - c h l o r o t e t r a c y c l i n e ^ - ? but a l s o has the d e s i r a b l e f e a t u r e f o r s y n t h e s i s of hav ing o n l y f o u r asymmetric c e n t e r s x o 0 Removal o f the 12a h y d r o x y l produces complete l o s s o f a c t i v i t y and c o n v e r s i o n of the 2-carboxamido s u b s t i t u e n t t o a n i t r i l e r e s u l t s i n a s i m i l a r d e c r e a s e ^ d (2) A comprehensive r e v i e w o f the chemica l p r o p e r t i e s of the t e t r a c y c l i n e B 0 c o v e r i n g the l i t e r a t u r e up t o 1963 * has 20 been p u b l i s h e d by M u x f e l d t and Bangert \u00C2\u00B0 Another i n c l u d e s a l u c i d summary of the v a r i o u s approaches w h i c h have been made t o the t o t a l s y n t h e s i s of the t e t r a c y c l i n e s 2 : i % Other r e v i e w s have appeared s t r e s s i n g e i t h e r the c h e m i s t r y or t h e i r g e n e r a l p h a r m a c o l o g i c a l p r o p e r t i e s and u s e , 2 2 < ~ 2 ^ Q \u00C2\u00A3 the t e t r a c y c l i n e So A r e c e n t r e v i e w by S c o t t and Money 2/* d i s c u s s e s the chemis t ry and b i o s y n t h e s i s o f the t e t r a c y c l i n e s 0 SYNTHETIC ATTEMPTS Attempts towards the t o t a l s y n t h e s i s o f the t e t r a c y -c l ines^ , w i t h t h e i r asymmetric c e n t e r s and t h e i r complex a r r a y o f s u b s t i t u e n t s of d i f f e r e n t k inds . , ga ined impetus i n 1957 w i t h the announcement by s e v e r a l r e s e a r c h groups of t h e i r p r o j e c t e d r o u t e s ^ t o g e t h e r w i t h t h e i r p r e l i m i n a r y e x p e r i m e n t a l r e s u i t S o - 6 -While a number o f workers have indeed been ab le t o s y n t h e s i z e t e t r a c y c l i n e d e r i v a t i v e s , no s y n t h e s i s of a n a t u r a l l y o c c u r r i n g t e t r a c y c l i n e has as yet been reported , , Two r e s e a r c h g r o u p s , those of F i e l d s , Kende and B o o t h e 2 ^ i n the U n i t e d S t a t e s and M u x f e l d t 2 ^ i n Germany, announced i n 1959 the obtent id 'n of ( i ) dedimethylamino-6 , 1 2 a - d e o x y - 7 - c h l o r o t e t r a c y c l i n e (3) and (zt) dedimethylamino-1 2 a - d e o x y - 5 a , 6 - a n h y d r o - 7 - c h l o r o t e t r a c y c l i n e (4) r e s p e c t i v e l y . R e c e n t l y each group has been a b l e to i n t r o d u c e the 12a h y d r o x y l groups M u x f e l d t by* the a c t i o n of m o l e c u l a r oxygen i n the presence of l i g h t and c a t a l y s t , and the American 29 group by a e r o b i c o x i d a t i o n i n the presence of sodium n i t r i t e \u00C2\u00AB (3) (4) Employing a m o d i f i e d approach i n which the i n t r o d u c t i o n of the dimethylamino group was accompl ished p r i o r t o the con-s t r u c t i o n o f the r i n g . A , Woodward e t 0 a l were ab le i n 1962 t o announce the t o t a l s y n t h e s i s of a f u l l y b i o l o g i c a l l y 30 a c t i v e t e t r a c y c l i n e , (\u00C2\u00B1) 6 - d e m e t h y l - 6 - d e o x y t e t r a c y c l i n e ( 5 ) A d e t a i l e d o u t l i n e of these and o ther a t tempts toward the t o t a l - 7 -s y n t h e s i s of the t e t r a c y c l i n e s i s g i v e n i n appendix !\u00E2\u0080\u009E More r e c e n t l y (February, 1965) Muxfeldt and R o g a l s k i ^ announced the t o t a l s y n t h e s i s of 6-deoxy - 6-demethyl - 7-chloro-t e t r a c y c l i n e ( 2 ) (the d e s c h l o r o analog of which had p r e v i o u s l y been s y n t h e s i z e d by Woodward e t 0 a l o ^ \u00C2\u00B0 ) by a method which\u00E2\u0080\u009E f o r the f i r s t t i me, a l l o w s b i o l o g i c a l l y a c t i v e t e t r a c y c l i n e d e r i v a t i v e s to be prepared on a l a r g e scale\u00C2\u00BB The s t a r t i n g m a t e r i a l f o r t h i s s y n t h e s i s was l-chloro ^ 2-bromomethyl - 4 -methoxybenzene ( 6 ) . T h i s compound was transformed i n f o u r s t e p s t o the aldehyde ( 7 ) \u00C2\u00B0 R e a c t i o n of the aldehyde (7) w i t h h i p p u r i c a c i d f o l l o w e d by condensation w i t h methyl-N-t e r t i a r y b u t y l - 3 - o x o g l u t a r a m a t e ( 3 ) , i n t o the G - 4 epimeric t e t r a c y c l i c compound (9 )\u00C2\u00BB Removal o f the p r o t e c t i n g g r o u p s a f o l l o w e d by N - f o r m y l a t i o n and 1 2 a - h y d r o x y l a t i o n produced (2)o These steps are shown i n f i g u r e 2 0 The importance of t h i s new method i s w e l l worth empha-s i s i n g , s i n c e i n p r i n c i p l e i t has solved the problem of the l a b o r a t o r y n o n - b i o g e n e t i c s y n t h e s i s of tetracycline\u00E2\u0080\u00A2<>. There - 8 -F i g u r e 2 - 9 -seems t o be no o b s t a c l e i n the way of p r e p a r a t i o n o f a 6 -methylated d e r i v a t i v e ( 10 ) 0 T h i s compound c o u l d then be brominated a t G - l l a and dehydrobrominated by c o n v e n t i o n a l methods t o g i v e 7 - c h l o r o - 5 a \u00C2\u00AB 6 - a n h y d r o t e t r a c y c l i n e (11) which has a l r e a d y been t ransformed t o 7 - c h l o r o t e t r a c y c l i n e (laR-pGlgR^H) by S c o t t and Bedford32 0 (1) (11) The exact d e t a i l s o f t h i s l a t t e r sequence of r e a c t i o n s w i l l be d i s c u s s e d l a t e r 0 I t should be s t r e s s e d t h a t no s y n t h e t i c t e t r a c y c l i n e p o s s e s s i n g a C\u00C2\u00B06 h y d r o x y l has been c o n s t r u c t e d and i t seems t h a t any f u t u r e t e t r a c y c l i n e s y n t h e s i s w i l l have t o u t i l i z e t h i s G-6 h y d r o x y l a t i o n method\u00C2\u00BB 10 -THE BIOGENESIS OF THE TETRACYCLINES In recent y e a r s the b i o s y n t h e s i s of the t e t r a c y c l i n e s have been the sub jec t o f c o n s i d e r a b l e s p e c u l a t i o n and e x p e r i m e n t a l i n v e s t i g a t i o n , , I n s p e c t i o n o f the formulae o f the t e t r a c y c l i n e s ^ eogo t e r r a m y e l n (1, R-j=HgR2=0H), r e v e a l s a t y p i c a l oxygenat ion p a t t e r n f o r a compound d e r i v e d from acetate*. T r a c e r s t u d i e s ^ u s i n g 2-\u00C2\u00B1l*G a c e t a t e and ( M e - ^ C ) m e t h i o n i n e , suggested t h a t the molecule c o n t a i n s C-methyl and N-iaethyl groups d e r i v e d from the l a t t e r s o u r c e , and t h a t the main s k e l e t o n i s l a r g e l y , but not e n t i r e l y , b u i l t up by the head t o t a i l l i n k a g e of a c e t i c a c i d u n i t s 0 I t was a t the t ime suggested t h a t carbon atoms 203s>kok& a n d the carboxamido carbon were d e r i v e d from glutamate (broken l i n e s f i g u r e 3)\u00E2\u0080\u009E F i g u r e 3c - 11 -Later studies revealed that glutamate was incorporated i n a non-specific way34 e i n addition^'-feeding experiments w i t h \"^G-sodium bicarbonate le d to incorporation of the t o t a l a c t i v i t y into the carboxamido group of terramycin-^,, Feeding the organism carboxyl-labelled malonic a c i d showed that the r a d i o a c t i v i t y of the carboxamido group was approx-imately 10-20$ of the t o t a l r a d i o a c t i v i t y of terramycin 0 This suggests that the carboxylatloni reaction involves the wintermediate formation of malonate and s furthermore 9 that malonate constitutes the condensing p r i n c i p l e f o r the formation of the whole carbon skeleton of terramycin. Consequently s the p r i n c i p l e of biosynthesis of the t e t r a -c y c l i n e s i s r e l a t e d to f a t t y a c i d biosynt ! hes i s35 a n Q t o the formation of 6-methylsalieylic i acid and o r s e l l i n i c ac id36 which have been shown to involve malonate. In those cases condensation i s thought to star t with acetyl-coenzyme Ap reacting with a malonyl-coenzyme A to form a B-polyketo-methylene chain of ten C 2 u n i t s which then c y c l i z e 0 In the te t r a c y c l i n e s the! s t a r t i n g unit could be malonyl-coenzyme A (see figure 4 K This i s supported by co-occurrence^, i n S 0 a u r e o f a c i e n s 3 7 g of 2-aeetyl~2-decarboxamid\u00C2\u00A9-terramycin37 (12) i n which the primer unit i s presumably a c e t y l Coenzyme Ao Although unusual$ maXpnyl\u00E2\u0080\u0094coenzyme A has been demonstrated to be a chain s t a r i e r unit i n the polyacetate derived cyclo-hexamid ( 1 3 ) ^ , ' - 12 These r e s u l t s l e d t o the s u g g e s t i o n t h a t naphthacenic compounds of, the type (14) c o u l d be i n t e r m e d i a t e s i n the b i o s y n t h e s i s of the t e t r a c y c l i n e s , , McGormick et<,aro39-4Q prepared and f e d p r e t e t r a m i d \u00E2\u0080\u00A2';(-14p R i = i * 2 = * ^ v 6 - m e t h y l p r e -t e t r a m i d (14p R-^CH^Rg\u00E2\u0080\u0094H), and t e r r a r u b e i n (14D R - ^ H ^ J R2=NMe2) t o s u i t a b l e mutants o f S 0 a u r e o f a e i e n s and were a b l e t\u00C2\u00A9 demonstrate t h a t p r e t e t r a m i d and 6-methylpretetramid were normal i n t e r m e d i a t e s i n the b i o s y n t h e s i s of 6=demethyl and 6 - m e t h y l t e t r a e y c l i n e S o However, t e r r a r u b e i n , although i t appears t o be a reasonable i n t e r m e d i a t e , d i d not show s i g n i f i c a n t ' p r e c u r s o r activity\u00C2\u00A9 Experiments, t\u00C2\u00A9 be d i s c u s s e d l a t e r , seem t\u00C2\u00A9 i n d i c a t e t h a t N=>methylati@n \u00C2\u00A9csiars l a t e r ^ I n the biosynthe't.ic sequence, -As a re.sult of these- experiments MeCormlck etoal - 15 -and d) res p e c t i v e l y . These r e s u l t s showed that the terminal step of the biosynthesis may be represented as i n the conver-sion of (20) to (1) i n figure 4\u00C2\u00B0 The existence of the reduction step was indicated by the i s o l a t i o n of 7-chloro-5a s 11a dehydrotetracycline from a mutant of S\u00E2\u0080\u009E aureofaciens 9 17 blocked f o r the reduction of (21) , It was further found i n these and add i t i o n a l experiments that 4-dedimethylamino-5a s6-anhydrotetracycline (18) and 4-epianhydrotetracyclines (19) f a i l to exhibit any precursor a c t i v i t y and that when-ever 7-chlorinated t e t r a c y c l i n e s were used i n a terramycin producing mutant n S, rimosus,, no incorporation was observed and s t a r t i n g material was i s o l a t e d . This explains why no chlorinated terramycin d e r i v a t i v e s have ever'been i s o l a t e d ^ - . (18) \u00E2\u0080\u00A2 (19)'^ Although i t seems that the presence of the 12a hydroxyl group in. the anhydro precursors i s not necessary f o r hydroxy-l a t i o n at C-6P i t i s nevertheless a fact that the 12a hydroxyl cannot be introduced at a l a t e r s t a g e ^ ^ Furthermore;, since recently the i s o l a t i o n and precursor a c t i v i t y of a series of N-dedimethyl-5a,6-anhydrotetracyclines (22) have been repor-t e d ^ 2 , i t would seem that 12a-hydroxylation precedes the - 16 -development o f t h e N-dimethylamino group ( a l t h o u g h not n e c e s s a r i l y t h e amino group) a t C -4\u00C2\u00B0 More r e c e n t l y McCormick e t . a l o ^ i s o l a t e d 4-hydroxy-m e t h y l - p r e t e t r a m i d (17) and demonstrated i t s i n t e r m e d l a c y i n t h e b i o s y n t h e s i s o f t h e t e t r a c y c l i n e s . The e v i d e n c e p r e s e n t e d so f a r l i m i t s the number of ways i n w h i c h 4 - h y d r o x y m e t h y l p r e t e t r a m i d (17) can be t r a n s -formed t o N ~ d e d i m e t h y l - $ a s 6 - a n h y d r o t e t r a c y c l i n e (22 ) . These a r e o u t l i n e d i n f i g u r e 4\u00C2\u00BB and w i l l be d i s c u s s e d i n a l a t e r s e c t i o n o - 17 -DISCUSSION (I) \"Biogenetic Type** Approach, In recent years the t o t a l synthesis of many complex naturally occurring organic substances has been accomplished using what has been c a l l e d the biogenetic type s y n t h e s i s ^ . The term \"biogenetic type\" has been selected to describe an organic synthesis which has; been desighed to follow, at l e a s t i n i t s major aspects, 1 the proven or postulated bio-synthesis of the p a r t i c u l a r natural product, A recent r e v i e w ^ demonstrates the wide a p p l i c a t i o n of t h i s approach and sets out c e r t a i n general guide l i n e s by which these syntheses have been accomplished. Generally, i n the laboratory duplication of the bio-l o g i c a l steps, any conditions or reagents, necessary f o r the completion of the reaction, may be used. Thus i t i s not required to simulate the s p e c i f i c conditions which supposedly approximate the environment i n the l i v i n g c e l l o This type of approach i s i n general more a e s t h e t i c a l l y pleasing and s a t i s f y i n g , as i t often leads to a shorter, neater, and more e f f i c i e n t synthesis, than those i n which no attention i s paid to the natural processes. The synthesis of d , l chimonathine (23), completed e a r l i e r i n our own l a b o r a t o r i e s ^ by the dramatically simple dimerisation of the natural product d i p t e r i n (N-methyltryptamine), serves to - 1 8 -i l l u s t r a t e the usefulness of t h i s approach, and we f e e l that the design of a synthesis i n the t e t r a c y c l i n e f i e l d i s also possible using information from the known biosynthesis of t h i s molecule. (23) However, i t i s recognised that i n the t e t r a c y c l i n e f i e l d the usual one-or two step construction of the large and complex molecules,is not f e a s i b l e , neither i s the actual natural pathway to t e t r a c y c l i n e a short one , a s described i n the introduction. Thus the design f o r the present synthesis i s to follow, i n general, a stepwise buildup of the various functional groups of the t e t r a c y c l i n e moleculeo _ 1 9 -In the t e t r a c y c l i n e b i o s y n t h e s i s , a s o u t l i n e d i n f i g u r e 4 , the f i r s t a v a i l a b l e b i o l o g i c a l p r e c u r s o r i s the p o l y a c e t a t e condensat ion p r o d u c t , the aromat ic substance 6 - m e t h y l p r e t e t r a m i d (14a)o I f we are t o use t h e knowledge o f the b i o s y n t h e t i c s t e p s , i t i s d e s i r a b l e t o t r a n s f o r m , i n the l a b o r a t o r y , t h i s compound t o i t s 4 -hydroxy analog (17 ) , and from t h e r e v i a e i t h e r of the s teps shown i n f i g u r e 4 t o N - d e m e t h y l - 5 a , 6 - a n h y d r o t e t r a e y c l i n e (22) . F u r t h e r t r a n s f o r m a t i o n o f t h i s compound t o t e t r a c y c l i n e has been ach ieved e l s e w h e r e ^ * 32 oWhen1 the present i n v e s t i g a t i o n was s t a r t e d , the o n l y known i n t e r m e d i a t e s i n the b i o l o g i c a l pathway t o the t e t r a -c y c l i n e s were 6 - m e t h y l p r e t e t r a m i d (14a) and 5 a , 6 - a n h y d r o -t e t r a c y c l i n e (20) \u00E2\u0080\u009E S i n c e t h e n the i n t e r m e d i a c y of 4 - h y d r o x y -6 -methyIpre te t ramid (17) and t h a t o f N - d e m e t h y l - 5 a , 6 - a n h y d r o -t e t r a c y c l i n e have been demonstra ted ,4 2 f . 43 A s o l u t i o n t o the s y n t h e t i c problem t h u s appears t o i n v o l v e the t r a n s f o r m a t i o n o f 6 - m e t h y l p r e t e t r a m i d (14a) t o t e r r a r u b e i n (14b) j> r e d u c t i o n of the l a t t e r t o d i h y d r o t e r r a -r u b e i n ( 2 4 ) , f o l l o w e d by 12a and 6 - o x y g e n a t i o n t o t e t r a c y c l i n e (see f i g u r e 5p p a t h a)\u00C2\u00AB, For the present i n v e s t i g a t i o n s no attempt i s made t o s y n t h e s i s e 6 - m e t h y l p r e t e t r a m i d or t e r r a -r u b e i n f rom s m a l l e r u n i t S p n e i t h e r i s the s y n t h e s i s of t e r r a r u b e i n f rom 6 - m e t h y l p r e t e t r a m i d c o n s i d e r e d . F i g u r e 5\u00C2\u00B0 - 21 -Both t e r r a r u b e i n and 6-methylpretetramid are a v a i l a b l e from d e g r a d a t i o n s o f t e t r a c y c l i n e (see f i g u r e 6)\u00E2\u0080\u00A2 As showh p both have been obtained from two s o u r c e s 1 0 * ^ ' 4 7 ^ r n the pre sent experiments p t e r r a r u b e i n prepared by formate p y r r o l y s i s , and 6-methylpretetramid by dehydrobromination method are usede (a) T r a n s f o r m a t i o n of T e r r a r u b e i n Examination of t h e , t e r r a r u b e i n (14b) s t r u c t u r e shows t h a t w i t h r i n g B i n the keto form, r i n g A i s i s o l a t e d as a s u b s t i t u t e d r e s o r c i n o l o (14b) The i n f r a r e d spectrum o f t e r r a r u b e i n shows a band a t 1660 cm\" 1 and one a t 1620 cm\" 1 D The 1620 cm\" 1 band i s as s i g n e d t o the s t r o n g l y hydrogen-bonded amide a t C-2 and to the hydrogen-bonded c a r b o n y l a t C-120 The weaker 1660 em - 1 band i s a s s i g n e d t o the non-hydrogen bonded c a r b o n y l a t G-12.-T h i s i s analogous t o the 1665 cm\"\"-*- band i n anthrone (25) and t o the naphthacene ( 2 6 ) ^ which i s r e p o r t e d t o have a I6l3cm~ 22 F i g u r e 6 a - 23 -and a 1675 cm band, due t o the hydrogen-bonded and non-bonded c a r b o n y l r e s p e c t i v e l y a t 0-12, A l s o , the u l t r a -v i o l e t s p e c t r a of t e r r a r u b e i n and the o t h e r members of t h i s s e r i e s o f aromat ic t e t r a c y c l i n e s a l l show c h a r a c t e r i s t i c acy lnaphtha lene a b s o r p t i o n bands (the u l t r a v i o l e t spectrum of the acy lnaphtha lene (27) , i s r e p o r t e d t o be 270, 450 mu^ H (27) The presence o f the ke to form i n t e r r a r u b e i n would expose the double bond at 4,4a t o h y d r o g e n a t i o n . S e v e r a l model compounds which i n c o r p o r a t e a l l the f e a t u r e s o f an i s o l a t e d r i n g A have been s u c c e s s f u l l y reduced ^\"^3 6 Thus the r e s o r c i n o l d e r i v a t i v e s (20) have been conver ted t o the d i h y d r o compounds (29)\u00E2\u0080\u009E In a d d i t i o n 9 r e d u c t i o n of a number - 24 -of 4a\u00E2\u0080\u009E12a -dehydrote t racyc l ines have been d e s c r i b e d ^ f and p r o v i d e a very c l o s e analogy f o r the case a t hand a For example, the r e d u c t i o n of (30) y i e l d s 12a - d e o x y t e t r a c y -c l i n e (31)\u00C2\u00AB COlMH, Rl~H; R 2=NH 2 5 1 R j = H ; R 2=NMe| 2 RjfMe; R2\"=H53 C O N H z O O H O H ( 3 D M ( C H 3 ) , OH s-)H O (30) F a r t h e r a long the proposed s y n t h e t i c pathway i t would be necessary t o i n t r o d u c e the h y d r o x y l groups a t C-6 and C-12a and t o a f f e c t the r e d u c t i o n of the r e s u l t i n g 5 a , l l a double b o n d . The i n t r o d u c t i o n o f the h y d r o x y l a t G~12a has 30 28 been ach ieved by Woodward e t \u00C2\u00BB a l and by M u x f e l d t on t h e i r s y n t h e t i c t e t r a c y c l i n e s < - 25 -Thus , Woodwards f i n a l product (5) was o b t a i n e d by the a c t i o n o f oxygen i n the presence of cesium c h l o r i d e\u00E2\u0080\u009E Mux-f e l d t completed h i s s y n t h e s i s o f (4) by u s i n g p l a t i n u m c a t a l y z e d o x y g e n a t i o n Other reagents which have been found u s e f u l f o r 12a h y d r o x y l a t i o n i n c l u d e sodium n i t r i t e and m - c h l o r o p e r -benzo ic a c i d 2 ^ 0 \" ^ On the o ther hand, the oxygen a t C-6 had been i n t r o d u c e d by S c o t t e t.alo-^, i n c l o s e analogy t o the b i o s y n t h e t i c s t e p s , by p a s s i n g oxygen through an i r r a d i a t e d s o l u t i o n of 7 - c h l o r o -5a,6 - a n h y d r o t e t r a c y c l l n e (11) t o g i v e 7 - c h l o r o-6 - h y d r o p e r o x y -- 26 5 a , l l a - a n h y d r o t e t r a c y c l i n e (32), which subsequently absorbed two moles o f hydrogen t o form 7 - c h l o r o t e t r a c y c l i n e ( 1 ) 0 T h i s r e a c t i o n was r e p o r t e d t o be s p e c i f i c f o r the 7 - c h l o r i -nated anhydro compound (11)\u00C2\u00AB Thus f o r the purposes of the present s y n t h e t i c sequence i t became necessary t o c h l o r i n a t e 5 a , 6 - a n h y d r o t e t r a c y c l i n e (20) i n the Cr-7 p o s i t i o n On t h i s b a s i s a p r e l i m i n a r y experiment by D r D To Money of t h i s d e p a r t -ment h a d , i t seemed, t rans formed t e r r a r u b e i n v i a the s teps shown on f i g u r e 5* p a t h (b) t o t e t r a c y c l i n e . Thus , a s m a l l sample o f t e r r a r u b e i n ^ which had been sub jec ted to h y d r o -g e n a t i o n w i t h p a l l a d i u m on c h a r c o a l as c a t a l y s t , was oxygen-a ted under d i r e c t i r r a d i a t i o n by f l u o r e s c e n t l i g h t s t o a compound which had an u l t r a v i o l e t spectrum n e a r l y i d e n t i c a l t o t h a t o f 5 a , l l a - a n h y d r o t e t r a c y c l i n e (20) e T h i s sample, which a t t h i s stage was a v a i l a b l e i n s p e c t r o s c o p i c q u a n t i t i e s o n l y , was f u r t h e r hydrogenated t o g i v e a compound whose spectrum was i d e n t i c a l b o t h i n wave- length and i n t e n s i t y to t h a t o f t e t r a c y c l i n e o When, however, repeat exper iments were a t t e m p t e d , the above o b s e r v a t i o n s c o u l d not be r e p r o d u c e d , Thus i t became necessary to proceed i n a stepwise f a s h i o n w i t h i s o l a t i o n and i d e n t i f i c a t i o n of the i n d i v i d u a l i n t e r m e d i a t e S o A c c o r d i n g l y , the i n t r o d u c t i o n of the 12a-h y d r o x y l In to d i h y d r o t e r r a r u b e i n (24) was a c h i e v e d u s i n g an 2# a d a p t a t i o n of the method d e s c r i b e d by M u x f e l d t * When - 27 -oxygen was, passed through an i r r a d i a t e d benzene s o l u t i o n of a s p e c i a l l y p u r i f i e d sample of d i h y d r o t e r r a r u b e i n f r e e base (prepared by the method of Boothe et oai,* 5- 5}, the u l t r a -v i o l e t spectrum of the s o l u t i o n changed s l o w l y over a p e r i o d of 7 days from the c h a r a c t e r i s t i c d i h y d r o t e r r a r u b e i n spectrum t o t h a t o f a n h y d r o t e t r a c y c l i n e (20) , The change was a l s o f o l l o w e d by paper chromatography, u n t i l no s t a r t i n g m a t e r i a l c o u l d be d e t e c t e d . The r e a c t i o n product was f u r t h e r p u r i f i e d from a f r o n t - r u n n i n g m a t e r i a l (probably l2a~ e p i a n h y d r o t e t r a c y c l i n e ) by e l a t i o n o f the anhydro spot f rom the papergram. The i n f r a r e d spectrum of t h i s m a t e r i a l was i d e n t i c a l t o t h a t of a u t h e n t i c a n h y d r o t e t r a -c y c l i n e (20), G h l o r i n a t i o n a t the C-7 p o s i t i o n was ach ieved by the a c t i o n of s u l f u r y l c h l o r i d e on a s o l u t i o n of a n h y d r o t e t r a -c y c l i n e (Id) i n g l a c i a l a c e t i c a c i d . The product was i d e n t i f i e d as 7 - c h l o r \u00C2\u00A9 a n h y d r o t e t r a c y c l i n e (11) by comparison of i t s i n f r a r e d and NMR s p e c t r a w i t h those o f a u t h e n t i c m a t e r i a l . P r e v i o u s h a l o g e n a t i o n a t tempts on the t e t r a c y -c l i n e s had l e d t o s u b s t i t u t i o n a t the G-9 p o s i t i o n 1 ^ . Having succeeded i n i n t r o d u c i n g oxygen i n t o the r e q u i r e d p o s i t i o n s u s i n g d i h y d r o t e r r a r u b e i n (24) as s u b s t r a t e , i t r e m a i n e d , as an e s s e n t i a l s t e p , t o prepare (24 ) t A l l a t tempts t o c a r r y out t h i s key s tep i n the s y n t h e t i c r o u t e , namely, the r e d u c t i o n of t e r r a r u b e i n to i t s d i h y d r o d e r i v a t i v e , have so f a r remained u n s u c c e s s f u l , A v a r i e t y of c o n d i t i o n s were used (see t a b l e 1.), Of t h e s e , o n l y h y d r o g e n a t i o n u s i n g a p l a t i n u m c a t a l y s t a l t e r e d the t e r r a r u b e i n chromophore. The appearance o f a 350 mu band i n p l a c e o f t h e u s u a l 450 mu band suggested t h a t t h e a c y l n a p h t h a l e n e chromophore had been d i s r u p t e d . T h i s r e a c t i o n p r o d u c t was not f u r t h e r i n v e s t i -gated,, T h i s d i f f i c u l t y i n t h e r e d u c t i o n o f t e r r a r u b e i n c a s t s doubt on t h e e x a c t sequence o f t h e p r e l i m i n a r y t e r r a r u b e i n r e d u c t i o n - o x y g e n a t i o n e x p e r i m e n t s , e s p e c i a l l y s i n c e i n t h a t c a s e , r e d u c t i o n o f t e r r a r u b e i n had not r e s u l t e d i n t h e f o r m a t i o n o f t h e c h a r a c t e r i s t i c d i h y d r o t e r r a r u b e i n u l t r a v i o l e t s pectrum, b u t showed an unchanged t e r r a r u b e i n spectrum,, T h i s was t h e sample t h a t had r e s u l t e d i n t h e f o r m a t i o n of t h e 5a, l l a - d e h y d r o t e t r a c y c l i n e spectrum (255-370 mu) on exposure t o l i g h t and oxygen. I t was t h u s suggested t h a t t h e o b s e r v e d change i n t h e spectrum of t e r r a r u b e i n had been due t o s i m u l -t a n e o u s o x y g e n a t i o n a t C-6 and a t C-12a t o y i e l d (33)\u00C2\u00B0 T h i s compound i s e x p e c t e d t o have t h e o b s e r v e d u l t r a v i o l e t spectrum. I t s h o u l d a l s o be mentioned a t t h i s p o i n t t h a t 12a hydroxy-l a t i o n a t t h e a r o m a t i c stage has been p o s t u l a t e d a s one o f t h e p o s s i b l e b i o g e n e t i c r o u t e s t o t e t r a c y c l i n e 2 - * \u00E2\u0080\u009E An a n a l o g y f o r t h i s t y p e o f b i o l o g i c a l c o n v e r s i o n can be seen i n the m e t a b o l i s m , by r a t l i v e r microsomes, of 6 - h y d r o x y t e t r a l i n (34) - 29 -TABLE'I S e l e c t e d Reductions of T e r r a r u b e i n C o n d i t i o n s used Product Palladium on c h a r c o a l E t h a n o l No r e a c t i o n P a l l a d i u m on c h a r c o a l E t h a n o l / sodium b i c a r b o n a t e No r e a c t i o n P a l l a d i u m on c h a r c o a l E t h a n o l / sodium hydroxide No r e a c t i o n P a l l a d i u m on c h a r c o a l E t h a n o l / t r l e t h y l a m i n e No r e a c t i o n Platinum/ e t h a n o l Over r e d u c t i o n of the moleculeo P a l l a d i u m on c h a r c o a l Pressure s 40 p e s e i o E t h a n o l No r e a c t i o n P a l l a d i u m on c h a r c o a l Pressures 600 p e s , i 0 E t h a n o l / sodium b i c a r b o n a t e No r e a c t i o n - 3 0 -to i t s c o r r e s p o n d i n g p - q u i n o l e ( 3 5 ) . \u00C2\u00B0 OH (34) (35) In the case o f t e r r a r u b e i n , h y d r o x y l a t i o n should be even more f a c i l e due t o the presence of two a c t i v a t i n g h y d r o x y l groups on r i n g A at C-2 and C-4\u00C2\u00BB At tempts t o oxygenate t e r r a r u b e i n i n benzene, d i m e t h y l -formamide, or d i m e t h y l s u l f o x i d e f a i l e d t o g i v e a compound h a v i n g a 2 5 5 - 3 7 0 mu u l t r a v i o l e t spectrum, as had been observed i n the p r e l i m i n a r y experiment\u00C2\u00AB The r e p o r t t h a t 4 - h y d r o x y m e t h y l p r e t e t r a m i d e (17) was c o v e r t i b l e t o l , 4 j \u00C2\u00BB 6 , l l - t e t r a h y d r o - 3 , 6 , 1 0 , 1 2 - t e t r a h y d r o x y - 6 -- 3 1 -m e t h y l - l i , 4 s l l - t r i o x o n a p h t h a c e n e - 2 - c a r b o x a m i d e {36) i n d i m e t h y l s u l f o x i d e c o n t a i n i n g 1% magnesium a c e t a t e t e t r a -57 h y d r a t e when exposed t o a i r , prompted an i n v e s t i g a t i o n o f t h e r e a c t i o n o f t e r r a r u b e i n i n t h i s s o l v e n t system. I t was hoped t h a t t h e p o w e r f u l c h e l a t i n g a c t i o n o f t h e added magnesium a c e t a t e would f a v o u r the h y d r o x y l a t i o n a t t h e 12a p o s i t i o n . (17) (36) T e r r a r u b e i n d i s s o l v e d r e a d i l y i n t h i s s o l v e n t and r a p i d l y a b s o r b e d one mole o f oxygeno N i t r o g e n a n a l y s i s o f t h i s compound showed t h a t one n i t r o g e n atom o n l y was p r e s e n t i n the m o l e c u l e . I t s u l t r a v i o l e t spectrum had r e m a i n e d unchanged. I t r e a c t e d w i t h sodium h y d r o s u l f i t e t o g i v e a m a t e r i a l h a v i n g an u l t r a v i o l e t spectrum i d e n t i c a l w i t h t h a t o f 4 - h y d r o x y m e t h y l p r e t e t r a m i d (17)0 F u r t h e r exposure o f t h i s \u00C2\u00BBoxyterrarubein* t o oxygen r e s u l t e d i n t h e a b s o r p t i o n o f a n o t h e r mole o f oxygen and an u l t r a v i o l e t spectrum i d e n t i c a l t o t h a t o f (36). T h i s compound on r e d u c t i o n w i t h h y d r o i o d i c a c i d i n p h e n o l was c o n v e r t e d t o - 32 ~ 4 - h y d r o x y m e t h y l p r e t e t r a m i d e ; sodium h y d r o s u l f i t e f a i l e d t o a f f o r d (1?)o On the b a s i s of the above f i n d i n g s i t i s suggested t h a t t e r r a r u b e i n i s i n i t i a l l y converted t o the r i n g A quinone (37) f o l l o w e d by oxygenat ion at C\u00E2\u0080\u009E6 t o g i v e (36) , (37) An a l t e r n a t i v e method of a c h i e v i n g 12a h y d r o x y l a t i o n can be based on the use o f common phenol o x i d i z i n g agent Lead t e t r a a c e t a t e i s a reasonable f i r s t c h o i c e , and o t h e r s such as C a r o ' s a c i d , F r e m y ' s s a l t , and p e r a c i d s have a l s o been usedc O x i d a t i o n o f p - e r e s o l (3&) t o p - t o l u q u i n o l (39) u s i n g C a r o * s a c i d ^ 0 , and of oesterone (40) t o the p - q u i n o l - m e t h y l e ther (4 l ) u s i n g l e a d t e t r a a c e t a t e i n methanol i n the presence o f boron t r i f l u o r i d e ^ , are t y p i c a l examples of a n g u l a r o x y g e n a t i o n , o f a l k y l - s u b s t i t u t e d p h e n o l s . However, b o t h l e a d t e t r a a c e t a t e and C a r o ' s a c i d have p r e v i o u s l y been u n s u c c e s s f u l l y a p p l i e d to oxygenat ions i n v o l v i n g the C-12a and the C-6 p o s i t i o n of the t e t r a c y -d i n e s , ' o Fremy 's s a l t and C a r o * s a c i d were a l s o used - 33 -i n a d i f f e r e n t c o n n e c t i o n on 6 - m e t h y l p r e t e t r a m i d g i v i n g m u l t i p l e p r o d u c t s (v ide i n f r a ) . F o r the present experiments the cho ice f e l l on p e r a c i d s as oxidants<> Of t h e s e , m - c h l o r o -p e r b e n z o i c a c i d i s commerc ia l ly a v a i l a b l e . (3\u00C2\u00AB) (39) (40) (41) A s o l u t i o n o f t e r r a r u b e i n i n dioxane a f t e r two days of exposure t o the p e r a c i d began t o show, s i m u l t a n e o u s l y , s e v e r a l spots on paper chromatograms. As an a u t h e n t i c sample of the d e s i r e d product (33) was not a v a i l a b l e , the t a s k o f s e p a r a t i n g and i d e n t i f y i n g the v a r i o u s components o f r e a c t i o n mix ture was a f o r m i d a b l e one , and t h i s method was abandoned. The p r i m a r y d i f f i c u l t y i n w o r k i n g w i t h t e r r a r u b e i n i s 34 i t s extreme i n s o l u b i l i t y i n common o r g a n i c s o l v e n t s . I n g e n e r a l i t d i s s o l v e s i n s o l v e n t s t o g i v e a c o n c e n t r a t i o n r a n g i n g f r om 1-2 mg/10 m l . Dimethylformamide and d i m e t h y l -s u l f o x i d e were the o n l y s o l v e n t s .found c a p a b l e o f d i s s o l v i n g t h e substance t o any p r a c t i c a l e x t e n t . A second c o m p l i c a t i n g f a c t o r was t h e d i f f i c u l t y o f o b t a i n i n g a r e l i a b l y pure sample. The sample s u p p l i e d by L e d e r l e Co. n o r m a l l y showed a y e l l o w ( f a d i n g ) spot c o r r e s -p o n d i n g t o d i h y d r o t e r r a r u b e i n (24). A pure sample i s c h a r a c t e r i z e d by t h e i n a b i l i t y of any o f t h e chrom a t o g r a p h i c s o l v e n t s used t o move i t f r o m the o r i g i n . Such a sample was p r e p a r e d by r e p e a t e d w a s h i n g s w i t h d i m e t h y l f o r m a m i d e and acetone\u00C2\u00AB F u r t h e r , i t was f o u n d t h a t s o l u t i o n s o f t e r r a r u b e i n a r e e x t r e m e l y oxygen l a b i l e . Thus\" w h i l e i t remained unchanged under an atmosphere o f n i t r o g e n , exposure of i t s s o l u t i o n t o a i r , e s p e c i a l l y i n t h e p r e s e n c e o f m e t a l c a t a l y s t s , r a p i d l y changed i t s u l t r a v i o l e t spectrum f r o m 440,270 mu t o 3#0,262 mu. T h i s u l t r a v i o l e t spectrum, a l t h o u g h s i m i l a r t o t h a t o f 5 a , l l a - d e h y d r o t e t r a c y c l i n e (32), remained u n a f f e c t e d by h y d r o g e n a t i o n . I n c o n t r a s t , (32) can be smoothly c o n v e r t e d t o t e t r a c y c l i n e by c a t a l y t i c h y d r o g e n a t i o n - ^ 2 . 35 -(b) T r a n s f o r m a t i o n s o f 6 - M e t h y l p r e t e t r a m i d \ S i n c e t h e t r a n s f o r m a t i o n of t e r r a r u b e i n t o a u s e f u l i n t e r m e d i a t e had met w i t h l i t t l e s u c c e s s , a t t e n t i o n was f o c u s s e d on a second r o u t e w h i c h was c o n c e i v e d a s a r e s u l t o f t h e a n n o u n c e d ^ p r e c u r s o r a c t i v i t y o f 4-hydroxy-6-methyl-p r e t e t r a m i d (17) and N - d e r a e t h y l - 5 a , 6 - a n h y d r o t e t r a c y c l i n e (22, i n f i g u r e 4) \u00C2\u00B0 T h i s new r o u t e was d e s i g n e d t o f o l l o w more c l o s e l y t h e proposed b i o s y n t h e t i c pathway t o t h e t e t r a c y c l i n e s (see f i g u r e 4) \u00C2\u00AB> I t r e q u i r e d , f o r i t s i n i t i a l s t e p , t h e s u c c e s s f u l h y d r o x y l a t i o n o f 6 - m e t h y l p r e t e t r a m i d t o i s 4-hydroxy d e r i v a t i v e (17)\u00C2\u00BB T h i s compound c o u l d c o n c e i v a b l y be t r a n s f o r m e d i n t o N - d e m e t h y l - 5 a , 6 - a n h y d r o t e t r a c y c l i n e (22) by a sequence o f r e a c t i o n s c l o s e l y s i m i l a r t o t h a t e n v i s i o n e d i n t h e b i o s y n t h e t i c p r o c e s s (see f i g u r e 4)\u00C2\u00AB A t t e n t i o n was m a i n l y d i r e c t e d t o t h e r o u t e i n v o l v i n g t h e 12a h y d r o x y l a t i o n o f (17) a s t h i s was c o n s i d e r e d t o be t h e more l i k e l y b i o -25 l o g i c a l pathway . F u r t h e r t r a n s f o r m a t i o n s o f N-demethyl 31 32 5 a , 6 - a n h y d r o t e t r a c y c l i n e have a l r e a d y been r e p o r t e d ' \u00E2\u0080\u009E As seen i n f i g u r e 4 the f o l l o w i n g i n t e r m e d i a t e s were r e q u i r e d f o r t h e c o m p l e t i o n o f t h i s r o u t e : 4-hydroxy-6-m e t h y l p r e t e t r a m i d (17), 4-keto-4-desdimethylamino-5a,6-anhy-d r o t e t r a c y c l i n e (42,figure 7), and 4 - o x i m i n o - 4 - d e s d i m e t h y l -a m i n o - 5 a p 6 - a n h y d r o t e t r a c y c l i n e ( 4 3 ) f i g u r e 7). These were p r e p a r e d by d e g r a d a t i v e p r o c e d u r e s a t t h e o u t s e t o f the - 36 -i n v e s t i g a t i o n , s i n c e t h e y were r e q u i r e d i n some q u a n t i t y f o r the f a c i l e i d e n t i f i c a t i o n o f s u c c e s s f u l r e a c t i o n p r o d u c t s and a l s o a s s t a r t i n g m a t e r i a l s f o r some o f t h e d e s c r i b e d t r a n s f orma11ons\u00E2\u0080\u009E I n i t i a l l y , a t t e m p t s were made t o p r e p a r e t h e ketone (42) and t h e oxime (43) u s i n g methods r e c e n t l y r e p o r t e d by B l a c k -wood e t o a l . ^ and by E s s e et 0al.\u00E2\u0080\u0094^\u00C2\u00BB T h i s i n v o l v e d t h e p r e p a r a t i o n o f the t e t r a c y c l o x i d e (44) f r o m t e t r a c y c l i n e by th e a c t i o n o f N - c h l o r o s u c c i n i m i d e i n aquous medium, f o l l o w e d by l i q u i d hydrogen f l u o r i d e d e h y d r a t i o n t o t h e 4-keto com-pound (see f i g u r e 7)o The t e t r a c y c l o x i d e (44) was p r e p a r e d a s d e s c r i b e d ^ . A l l a t t e m p t s t o d e h y d r a t e t h i s compound w i t h hydrogen f l u o -r i d e f a i l e d t o produce t h e d e s i r e d p r o d u c t . We were l a t e r i n f o r m e d t h a t t h e o r i g i n a l w o r k e r s had a l s o e n c o u n t e r e d 66 d i f f i c u l t i e s i n r e p e a t i n g t h i s r e a c t i o n , An a l t e r n a t i v e method was d e v i s e d i n o r d e r t o y i e l d t h e d e s i r e d ketone d i r e c t l y . Thus, r e a c t i o n o f N - c h l o r o s u c -c i n i m i d e on 5 a , 6 r - a n h y d r o t e t r a c y c l i n e (20) r e s u l t e d i n a compound h a v i n g a l l t h e p r e d i c t e d c h a r a c t e r i s t i c s ( i n f r a r e d , u l t r a v i o l e t and a n a l y t i c a l d a t a ) o f t h e r e q u i r e d 4-keto compound. - 37 -(43) (51) F i g u r e 7* - 3 8 -OH OH ( 2 0 ) ( 4 2 ) The p r e p a r a t i o n o f 4 - h y d r o x y - 6 - m e t h y l p r e t e t r a m i d f r o m d e g r a d a t i o n o f t e r r a r u b e i n has a l r e a d y been d e s c r i b e d ( v i d e supra)\u00E2\u0080\u009E\" (A method r e c e n t l y d e v e l o p e d i s more conven-i e n t f o r t h e p r e p a r a t i o n o f t h i s compound a s i t employs the more r e a d i l y a v a i l a b l e t e t r a c y c l i n e a s a s t a r t i n g m a t e r i a l ') Of t h e , s e v e r a l p o s s i b l e p o s i t i o n s open t o p a r a q u i n o l f o r m a t i o n i n 6 - m e t h y l p r e t e t r a m i d (14a) ( C - 4 , C-$\u00C2\u00AB C-6, C-7 and C-12a);j, r i n g A was c o n s i d e r e d t o o f f e r the most a c t i v a -t e d s i t e s a C-4 and C-12*a w i t h p o s i t i o n C-4 the l e a s t h i n d e r e d and more l i k e l y s i t e 0 - 39 -Oxygenation of 6 - m e t h y l p r e t e t r a m i d was attempted u s i n g c o n d i t i o n s s i m i l a r t o t h a t used f o r t e r r a r u b e i n oxygenat ion a t C -4 and C-6 0 I t was hoped to o b t a i n (36) as i n the t e r r a -r u b e i n o x y g e n a t i o n A c c o r d i n g l y , 6 - m e t h y l p r e t e t r a m i d was d i s s o l v e d i n d i m e t h y l s u l f o x i d e c o n t a i n i n g 1$ magnesium a c e t a t e and s t i r r e d i n a i r , A c o l o u r change from amber to r e d was observed a f t e r one d a y 0 The u l t r a v i o l e t a b s o r p t i o n changed d u r i n g t h i s p e r i o d from 480 mu t o 520 mu- A f t e r s e v e r a l weeks o f exposure b o t h the c o l o u r o f the s o l u t i o n and i t s u l t r a v i o l e t spectrum remained unchanged at 520 mu. I s o -l a t i o n of the r e a c t i o n product a f t e r 2 weeks and r e d u c t i o n of the r e s u l t a n t brown s o l i d w i t h h y d r o i o d i c a c i d i n phenol d i d not r e s u l t i n the f o r m a t i o n o f 4-hydroxy-6 -methylpre-t e t r a m i d o Had the i n i t i a l oxygenat ion o c c u r r e d at G-4* the r e s u l t -i n g spectrum should have been i d e n t i c a l t o t h a t of (37)\u00C2\u00BB the f i r s t oxygenat ion product o:f t e r r a r u b e i n ; and subsequent ly , the s o l u t i o n should have turned deep p u r p l e i n c o l o u r and should have shown a 550 mu v i s i b l e spectrum as was obser -ved f o r (36), On the o ther hand, oxygenat ion a t G-12a should have r e s u l t e d i n a breakup of the naphthacene chromophore, w i t h a b lue s h i f t i n the v i s i b l e r e g i o n . T h i s compound was, t h e r e f o r e , not a u s e f u l i n t e r m e d i a t e . Oxygenat ion u s i n g C a r o l s a c i d r e s u l t e d i n complete disappearance of the a b s o r p t i o n i n the v i s i b l e r e g i o n of the - 40 spectrum and t h i s r e a c t i o n was abandoned. Using Fremy f ls s a l t 0K2S\u00C2\u00B03^2 N 0 - J a d i m e t n y l s ^ f o x i d e / m ^ 6 n e s i u m a c e t a t e s o l u t i o n o f 6-methylpretetramid became deep blue a f t e r 7 hours of s t i r r i n g i n a i r , On a c i d i f i c a t i o n of the s o l u t i o n , a deep bl u e s o l i d was o b t a i n e d having a 550 mu band i n the v i s i b l e .spectrum with e t h a n o l as s o l v e n t , In d i m e t h y l -s u l f o x i d e the spectrum was s h i f t e d to 500 mu. Oxygenation of 4-hydroxy-6-methylpretetramid i n t h i s s o l v e n t l e a d s t o the same s p e c t r a l c h a r a c t e r i s t i c s as d e s c r i b e d above f o r the r e a c t i o n w i t h 6-methylpretetramide, The same s p e c t r a were a l s o o btained when the f i n a l oxygenation product o f t e r r a -r u b e i n (36) was s u b j e c t e d t o t h i s r e a c t i o n , , The above r e s u l t s i n d i c a t e d t h a t Fremy's s a l t converted .6-methylpretetramide beyond the d e s i r e d stage, A l l attempts at .stopping the r e a c t i o n a t an e a r l i e r stage f a i l e d t o pro-duce the d e s i r e d compound, Sim u l t a n e o u s l y w i t h the above C-4 oxygenation e x p e r i -ment s a an attempt was made t o achieve the t r a n s f o r m a t i o n of the o t h e r i n t e r m e d i a t e s i n t h i s sequence, T h u s s sevef*al\u00E2\u0080\u009E -attempts a t the oxygenation i n the 12a p o s i t i o n of 4-hydroxy-6-methyl p r e t e t r a m i d u s i n g m-chloroperbenzoic a c i d as oxidant it r e s u l t e d i n a multi-component r e a c t i o n product* None of the r e q u i r e d 4-keto-4-dedimethylamino-5a s6-anhydrotetracyqline (42 c o u l d be d e t e c t e d . - 41 -S i m i l a r l y p s e v e r a l attempts t o prepare the oxime o f the 4-keto compound,(43)9 f a i l e d t o produce a d e r i v a t i v e a n a l y s i n g f o r the expected number of n i t r o g e n atoms i n the m o l e c u l e 9 a l t h o u g h i t was r e a d i l y formed from the t e t r a -c y c l o x i d e (44)<> The present i n v e s t i g a t i o n on the b i o g e n t i e - t y p e syn-t h e s i s of the t e t r a c y c l i n e molecule was t e r m i n a t e d at t h i s stageo I t i s f e l t t h a t a l t h o u g h a l a r g e number of r e a c t i o n s and c o n d i t i o n s were t r i e d , , the number of p o s s i b i l i t i e s were not n e c e s s a r i l y exhausted Q However^ no s t r a i g h t f o r w a r d s o l u t i o n can be suggested, The key s t e p s i n these v a r i o u s r o u t e s , which s t i l l remain unsolved^, are hydrogenation of t e r r a r u b e i n ^ oxygenation of t e r r a r u b e i n a t C-12a9 4-hydroxy-l a t i o n o f 6-methylpretetramid s and 12a h y d r o x y l a t i o n of the l a t t e r . A l l remaining s t e p s were e i t h e r s u c c e s s f u l l y completed or do not seem to present a major s y n t h e t i c problem. - 42 I I , Non B i o g e n e t i c Type T o t a l S y n t h e s i s o f T e t r a c y c l i n e In a l l the t o t a l syntheses so f a r attempted, i n t r o d u c -t i o n of n i t r o g e n a t G-4 has p resented a major problem. T h i s was overcome i n the s y n t h e s i s of 6-deoxy-6-demethyltetra-c y c l i n e (5) by Woodward and r e c e n t l y i n the s y n t h e s i s of the c h l o r o d e r i v a t i v e o f (5) by Muxfeldt\" 3 1, In both of these syntheses the n i t r o g e n was i n c o r p o r a t e d i n t o the mole-c u l e b e f o r e the f i n a l c l o s u r e o f r i n g A, Although i t appears that the C-6 methylated d e r i v a t i v e o f (5) can p o t e n t i a l l y be converted to t e t r a c y c l i n e (see i n t r o d u c t i o n ) , the s y n t h e s i s of the l a t t e r has not y e t been r e p o r t e d . (4) (11) An examination of the a v a i l a b l e s y n t h e t i c t e t r a c y c l i n e d e r i v a t i v e s showed t h a t 7-chloro - 4-dedimethylamino-5a 96-anhy-d r o t e t r a c y c l i n e (4) possessed a p o t e n t i a l l y a c t i v a t e d methylene - 43 group a t C - 4 , F u n c t i o n a l ! s a t i o n o f t h i s p o s i t i o n c o u l d p r o v i d e an e n t r y i n t o the dimethylamirio-5 a g 6-anhydr\u00C2\u00A9tetra-c y c l i n e s such a s ( 1 1 ) p w h i c h can be c o n v e r t e d t o t e t r a -c y c l i n e . D u r i n g t h e p r e s e n t s t u d y t h e f o l l o w i n g t r a n s f o r m a t i o n s a t C - 4 were attempteds (a) B r o m i n a t i o n a f o l l o w e d by d i s p l a c e m e n t w i t h d i m e t h y 1 -amine 0 (b) O x y g e n a t i o n t o t h e 4 - k e t o compound f o l l o w e d by e i t h e r oxime f o r m a t i o n o r r e d u c t i v e - a m i n a t i o n and N - f o r m y l a -t i o n Q (c) N i t r o s a t i o n t o g i v e e i t h e r t h e oxime or t h e k e t o n e , B r o m i n a t i o n , n i t r o s a t i o n and o x y g e n a t i o n o f a l i p h a t i c k e t o n e s o r a l l y l i c c a r b o n atoms a r e g e n e r a l r e a c t i o n s and Art \"7C\ a r e w e l l r e v i e w e d elsewhere , A s i n g l e example o f a b r o m i n a t i o n o f a t e t r a c y c l i n e r i n g A model i s w o r t h 71 m e n t i o n i n g h e r e . Thus Arakawa and I r i e ' r e a c t e d s u b s t i -t u t e d l ; ? 3 - c y c l o h e x a n e d i o n e monoenolates ( 4 5 ) w i t h N-bromo-s u c c i n i m i d e and o b t a i n e d C - 2 b r o m i n a t e d compounds (45&-\"-Ri=H5 o r krol) R 2=H) o When the d i a l k y l a t e d d e r i v a t i v e C45p R^R^On^) was b r o m i n a t e d 0 t h e bromine e n t e r e d t h e G-6 p o s i t i o n . The l a t t e r ( 4 6 a \"R.-^-'K^\"^^) w a s f o u n d t o r e a c t smoothly with amine s, - 4 4 -( 4 6 ) In the wider a p p l i c a t i o n of these r e a c t i o n s i t was r e a l i z e d t h a t the t e t r a c y c l i n e s o f f e r e d a more complex problem t h a n the examples c i t e d , or those found i n the above mentioned reviews,, In the case of the 4-dedimethyl-amino- 5 a j , 6-anhydrotetracycline framework'the number of p o s s i b l e r e a c t i v e p o s i t i o n s are numerous,, Foremost i s the C - 2 p o s i t i o n which i s f l a n k e d by two c a r b o n y l groups\u00E2\u0080\u009E However p i n a l l h a l o g e n a t i o n s r e p o r t e d f o r t h e t e t r a c y c l i n e s , none had l e d t o G-2 halogenated producto Other p o t e n t i a l l y r e a c t i v e c e n t e r s i n ( 4 ) are C - 5 p C-Me and the aromatic p o s i t i o n s a t C - 6 and 0 - 9 = No h a l o g e n a t i o n had been r e p o r t e d a t G -6 a l t h o u g h oxygenation of 5 a s 6 - a n h y d r o t e t r a c y c l i n e s r e s u l t e d i n the i n t r o d u c t i o n 45 -of a h y d r o x y l group at t h i s p o s i t i o n c B r o m i n a t i o n of 4 - d e d i m e t h y l a m i n o - 5 a 0 6 ~ a n h y d r o t e t r a - . . c y c l i n e (18) i s r e p o r t e d t o y i e l d the 9~bromo d e r i v a t i v e ^ (47)o An i d e n t i c a l compound has been o b t a i n e d by b r o m i -n a t i o n of 4 - d e d l m e t h y l a m i n o t e t r a c y c l i n e (48) i n the 11a p o s i t i o n (49) f o l l o w e d by rearrangement of bromine t o the C-9 p o s i t i o n i n the presence of hydrogen bromide^?\u00E2\u0080\u009E However, the p o s i t i o n of the bromine i n (47) has not been u n e q u i v o c a l l y e s t a b l i s h e d . (13) (47) For the b r o m i n a t i o n - a m i n a t i o n experiments^ which were chosen as the f i r s t of the three suggested r o u t e s f o r t e t r a -c y c l i n e s y n t h e s i s ^ i t was necessary a t the outse t t o e s t a b l i s h the p o s i t i o n of the bromine i n the above bromo compound0 - 46 -I t was hoped t h a t the presence of the c h a r a c t e r i s t i c r i n g D aromatic p r o t o n ( s ) i n the n,m,r, would i n d i c a t e the presence^ or absence of bromine i n the suggested C-9 position<> R e a c t i o n of (4)p prepared from 4-dedimethylamino-7-c h l o r o - t e t r a c y c l i n e g ( k i n d l y s u p p l i e d by the L e d e r l e Co,) w i t h one molar e q u i v a l e n t of N-bromosuccinimide r e s u l t e d i n a compound whose carbon, hydrogen and n i t r o g e n a n a l y s i s i n d i c a t e d the i n c o r p o r a t i o n of 1 mole of bromine 0 As t h i s sample was r a t h e r i n s o l u b l e i n common nomor0 s o l v e n t s s i t was a c e t y l a t e d to i n c r e a s e i t s s o l u b i l i t y . The a c e t a t e was f r e e l y s o l u b l e i n c h l o r o f o r m 0 I t s n,m,ro showed a s i n g l e t a t 2 o l T f o r 1 hydrogeno The a c e t a t e of the non-brominated compound ( 4 ) showed two d o u b l e t s . a t 2,1 and 2,9.T* f o r the AB system of C -8 and G-9 hydrogens. I t was t h u s e s t a b l i s h e d t h a t b r o m i n a t i o n of ( 4 ) with N-bromosuccinimide r e s u l t s i n s u b s t i -t u t i o n of the bromine at C-9\u00C2\u00B0 T h i s method of b r o m i n a t i o n c o u l d now be r e j e c t e d f o r the purposes of these experiments. I t was hoped t o a v o i d C-9 as w e l l as C-5, C-6 and C-methyl b r o m i n a t i o n by u s i n g i o n i c r e a c t i o n c o n d i t i o n s . Thus a sample of ( 4 ) was brominated u s i n g one molar e q u i v a l e n t o f bromine i n g l a c i a l a c e t i c a c i d . The product a n a l y s e d f o r the i n c o r p o r a t i o n of one atom o f bromine i n the molecule ( f o r the sake of c l a r i t y i n subsequent d i s c u s s i o n s t h i s sample i s d e s i g -nated as bromo compound A ) , The i n f r a r e d spectrum of t h i s - 47 bromo compound was d i f f e r e n t from the C-9 brominated compound 0 When i t was t r e a t e d w i t h about one molar e q u i v a l e n t o f d i m e t h y l -amine i n methanol f o r 4 days and subsequently a n a l y s e d by paper chromatography u s i n g a butanol-phosphate system, a green-y e l l o w spot was observed a t Rf 0,3. In the same system 7-chl o r o - 5 a , 6 - a n h y d r o t e t r a c y c l i n e (11), the expected p r o d u c t , gave an orange-red spot a t Rf 0 o$, while 4-epi-5a,6-anhydro-t e t r a c y c l i n e (19) showed a yellow spot a t Rf 002S and the 5a, 6 - a n h y d r o t e t r a c y c l i n e (20) had a yellow spot a t Rf 0,45\u00C2\u00B0 (50) (53) Esse e t , a l , ^ and Blackwood e t 0 a l o ^ ^ r e p o r t e d the i s o -l a t i o n of N-demethyl-4-epi-5a,6-anhydrotetracycline (50) as the major isomer i n the r e d u c t i o n of the hydrazone (51? see f i g u r e 7), the oxime (52, see f i g u r e 7) and from r e d u c t i v e -amination o f the t e t r a c y c l o x i d e (44) \u00E2\u0080\u00A2> I t t h e r e f o r e appears t h a t the 4-epi-isomer i s formed more e a s i l y , i n these reduc-t i o n s i Thus the p o s s i b i l i t y was r a i s e d t h a t the above green y e l l o w spot a t Rf 0=3 was a l s o due to the f o r m a t i o n o f the epimer o f the expected product (53)\u00C2\u00B0 - 48 -In another bromination attempt, which simulated the above c o n d i t i o n s , a product was obtained which had an i n f r a -r e d spectrum i n d i c a t i v e of a mixture of the C-9 bromo com-pound (A) o In a l l subsequent brominations on (4) i t was noted t h a t b r o m inations t h a t were l e f t t o r e a c t f o r l o n g e r than 8 hours, showed i n f r a r e d s p e c t r a i d e n t i c a l to the C -9 bromo-compound (47)*> while i n some experiments, stopped a t in t e r m e d i a t e r e a c t i o n t i m e s , some of the bands a s s o c i a t e d w i t h bromo compound (A) c o u l d be detected\u00E2\u0080\u009E I t i s b e l i e v e d t h a t t h i s o b s e r v a t i o n i s p a r a l l e l t o t h a t o f the aforementioned f a c i l e bromine rearrangement i n 11a-brorao - 4-dedimethylaminotetracycline (49) * I t i s suggested t h a t the i n i t i a l b r o m i n a t i o n t a k e s p l a c e a t a d i f f e r e n t s i t e , p o s s i b l y at C - 4 , f o l l o w e d by rearrangement t o C - 9 \u00C2\u00B0 A l l attempts t o repeat the p r e p a r a t i o n of bromo compound (A) l e d t o e i t h e r C -9 or incomplete brominationSo When some of these incomplete b r o m i n a t i o n p r o d u c t s were r e a c t e d w i t h l i q u i d dimethylamine f o r a s h o r t time, a compound was produced which d i s s o l v e d i n d i l u t e h y d r o c h l o r i c a c i d and which c o u l d be e x t r a c t e d w i t h c h l o r o f o r m from the a c i d s o l u t i o n a t a pH of 5\u00C2\u00B05\u00C2\u00B0 Paper chromatographic a n a l y s i s o f the c h l o r o -form e x t r a c t showed a green-yellow spot at Rf 0*3 as i n the - 4 9 -f i r s t experimento In b u t a n o l - a c e t i c acid-water (12s5S3) solv e n t system, the above green-yellow spot had an Rf 0 . 5 , i d e n t i c a l t o t h a t of 7 - c h l o r o - a n h y d r o t e t r a c y c l i n e (11) and i t s d e s c h l o r o and4-epi d e s c h l o r o d e r i v a t i v e s . Both 4-epi a n h y d r o t e t r a c y c l i n e and the dimethylamine-r e a c t i o n product showed an Rf a t 0 .9 on TLC p l a t e s u s i n g polyamide powder as adsorbent and b u t a n o l - a c e t i c acid-water (12s5 ;3) as s o l v e n t . In the same system 7 - c h l o r o anhydro-t e t r a c y c l i n e and a n h y d r o t e t r a c y c l i n e had RfS of 0 . 4 and 0 .2 r e s p e c t i v e l y . They a l s o showed t r a c e s of s p o t s a t Rf 0 .9 c o r r e s p o n d i n g , both i n qolour and i n Rf, t o 4 - e p l anhydro-t e t r a c y c l i n e and t o the r e a c t i o n p r o d u c t . A s i m i l a r chroma-togram was obtained when the s o l v e n t system was changed to ethanol-acetone-water ( 2 s 2 s l ) . These r e s u l t s i n d i c a t e d s t r o n g l y t h a t the b r o m i n a t i o n - a m i n a t i o n product was the 4-epimer of the d e s i r e d 7 - c h l o r o t e t r a c y c l i n e . S e v e r a l months l a t e r when an a u t h e n t i c sample of 7-c h l o r o - 4 - e p i - t e t r a c y c i i n e became a v a i l a b l e ( s u p p l i e d by L e d e r l e Co 'o) i t was dehydrated to the anhydro compound and a n a l y s e d chromat\u00C2\u00A9graphically g i v i n g a green-yellow spot at Rf 0 .9 on polyamide t h i n l a y e r p l a t e s . The bromination-amination experiments which o r i g i n a l l y had been reporodueed t w i c e , c o u l d not be repeated when an / - 50 -a u t h e n t i c sample o f (53) became a v a i l a b l e . F o l l o w i n g t h e f a i l u r e o f the above mentioned method t o reproduce the o r i g i n a l r e s u l t s i t was thought t h a t p o s s i b l y the successes had been due t o the presence of t r a c e amounts of hydrobromic a c i d i n the b r o m i n a t i o n m i x t u r e . I t i s known t h a t hydrogen bromide c a t a l y s e d b r o m i n a t i o n of p - h y d r o x y -acetophenone i n a c e t i c a c i d had r e s u l t e d i n a l i p h a t i c b r o m i -n a t i o n w h i l e i n the absence of hydrogen bromide aromat ic b r o m i n a t i o n o c c u r s e x c l u s i v e l y ^ . I t seemed probable t h a t s i m i l a r c o n d i t i o n s would be of va lue i n the present c a s e . B r o m i n a t i o n of (4) i n the presence of added t r a c e s o f hydrogen bromide gas gave a produce which produced only t r a c e s of the g r e e n i s h - y e l l o w spot when t r e a t e d w i t h d i m e t h y l -am ine . Having a s c e r t a i n e d t h a t b r o m i n a t i o n at C-9 i s more f a c i l e i t was d e c i d e d to prepare a d i b r o m i n a t e d compound, i n the hope o f t h u s i n t r o d u c i n g the second bromine at C - 4 . The C-9 bromine was to be subsequent ly removed by h y d r o g e n o l y s i s a f t e r the r e a c t i o n w i t h d i m e t h y l a m i n e . A l l a t tempts a t b r o m i n a t i o n of the C-9 bromo compound w i t h bromine i n g l a c i a l a c e t i c a c i d f a i l e d t o g i v e a compound a n a l y s i n g f o r 2 atoms of bromine and r e a c t i n g w i t h d i m e t h y l a m i n e . B r o m i n a t i o n o f the C-9 bromo compound w i t h N-bromosuccinimide r e s u l t e d i n - 51 -the d i s r u p t i o n o f the c h a r a c t e r i s t i c a c y l n a p h t h a l e n e chromo-phore o The new u l t r a v i o l e t spectrum was s i m i l a r t o t h a t o f 5 a j l l a - d e h y d r o t e t r a c y c l i n e ( 2 1 ) . B r o m i n a t i o n a t C-6 was su g g e s t e d , F o l l o w i n g the f a i l u r e of t h e above a t t e m p t s , i t was d e c i d e d t o bro m i n a t e an a c e t y l a t e d sample o f (4) a s i t had been r e p o r t e d ^ t h a t b r o m i n a t i o n o f p-acetoxyacetophenone had r e s u l t e d i n a l i p h a t i c b r o m i n a t i o n . A sample o f (4) was t h u s a c e t y l a t e d * I t showed 3 a c e t a t e peaks i n t h e n.m.r. I t i s b e l i e v e d t h a t a c e t y l a t i o n had r e s u l t e d i n t h e f o r m a t i o n o f C-10, C - l l , C-3 t r i a c e t a t e (54). (54) T h i s a c e t a t e f a i l e d t o r e a c t w i t h N-bromo-succinimide* but r e a c t e d w i t h bromine i n g l a c i a l a c e t i c a c i d t o g i v e a compound showing t h e C-8 and 9 a r o m a t i c p r o t o n s , i n the n.m.r,,, a s i n the s t a r t i n g m a t e r i a l , but seemed t o have l o s t one o f t h e a c e t a t e g r o u p s , A d e a c e t y l a t e d sample ana-l y s e d f o r t h e i n c o r p o r a t i o n o f e x a c t l y one atom o f bromine - 52 -i n the molecule and showed a strong 1 7 2 0 cm\"**' band i n the i n f r a r e d suggesting the f o r m a t i o n of a non-hydrogen bonded c a r b o n y l o I t s u l t r a v i o l e t spectrum was unchanged from the s t a r t i n g n o n - a c e t y l a t e d compound. Mass s p e c t r o s c o p i c a n a l y -s i s showed, the parent peak at 4 9 5 - 6 (poor r e s o l u t i o n ) . The above r e s u l t s suggest b r o m i n a t i o n at C - 2 j ( 5 5 ) \u00C2\u00BB T h i s i s the f i r s t i n s t a n c e of h a l o g e n a t i o n a t C - 2 of the t e t r a c y c l i n e s . ( 5 5 ) Simultaneous with the above bromination experiments, s e v e r a l attempts to oxygenate ( 4 ) u s i n g selenium d i o x i d e , or potassium t e r t i a r y - b u t o x i d e - o x y g e n f a i l e d t o b r i n g about any change i n ( 4 ) \u00C2\u00BB In a d d i t i o n attempts t o i n t r o d u c e oxygen using c a t a l y t i c p rocedures'(e.g. f i n e l y d i v i d e d platinum) were a l s o u n s u c c e s s f u l , S i m i l a r l y j n i t r o s a t i o n r e a c t i o n s w i t h n - b u t y l n i t r i t e a n d p - n i t r o s o - d i m e t h y l a n i l i n e f a i l e d t o y i e l d d e r i v a t i v e s a n a l y s i n g f o r two n i t r o g e n atoms. - 53 -The above r e s u l t s s t r o n g l y i n d i c a t e t h a t the C -4 p o s i t i o n of (4) i s h i g h l y u n r e a c t i v e . N e v e r t h e l e s s , i t i s b e l i e v e d t h a t success had been achieved i n s e v e r a l of the o r i g i n a l b romination-amination r e a c t i o n s . The f a c t t h a t i n those experiments an a c i d s o l u b l e c o l o u r e d m a t e r i a l could , be e x t r a c t e d from a c h l o r o f o r m s o l u t i o n of the r e a c t i o n pro-duct and t h a t t h i s m a t e r i a l c o u l d be r e - e x t r a c t e d from the a c i d s o l u t i o n at a p a r t i c u l a r pH supports the presence of an amine a Furthermore, the chromatographic behaviour o f the r e a c t i o n product and t h a t of 4 - e p i a n h y d r o t e t r a c y c l i n e both i n . c o l o u r and Rf were i d e n t i c a l i n the separate experiments t h a t were performed on them. I t i s b e l i e v e d t h e r e f o r e , t h a t with the e x c e p t i o n o f the f i r s t b r o m i n a t i o n experiments which had r e s u l t e d i n a bromo-compound w i t h c h a r a c t e r i s t i c s p e c t r a l d a t a , a l l other brominations had y i e l d e d o n l y t r a c e s of the d e s i r e d bromo: compound. No e x p l a n a t i o n o f t h i s f a i l u r e i n r e p r o d u c i b i l i t y i s a v a i l a b l e . - 54 -EXPERIMENTAL Because o f t h e g e n e r a l l y n o n - c h a r a c t e r i s t i c n a t u r e o f t h e m e l t i n g o r d e c o m p o s i t i o n p o i n t s o f t e t r a c y c l i n e d e r i v a -t i v e s , none were r e c o r d e d . U l t r a v i o l e t s p e c t r a were measured w i t h a Cary 1 1 s p e c t r o p h o t o m e t e r , f o r s o l u t i o n s i n e t h a n o l u n l e s s o t h e r w i s e mentioned. Where no q u a n t i t a t i v e s p e c t r a were measured, t h e peak r a t i o s a r e g i v e n . I n f r a r e d s p e c t r a were measured w i t h a P e r k i n E l m e r 2 1 s p e c t r o p h o t o m e t e r . N u c l e a r magnetic resonance s p e c t r a were t a k e n w i t h a V a r i a n model A 6 0 , For paper chromatography Whatman no. 1 p a p e r , and f o r t h i n l a y e r chromatography Woelm polyamide powder, was used. The f o l l o w i n g s o l v e n t s were commonly a p p l i e d : ( 1 ) B u t a n o l s a t u r a t e d w i t h 0 . 1 M sodium d i h y d r o g e n p h o s p h a t e (pH 3 ) (paper s t r i p s were impregnated w i t h t h e aqueous phase b e f o r e u s i n g ) \u00E2\u0080\u00A2 ( 2 ) B u t a n o l - a c e t i c a c i d - w a t e r ( 1 2 s 3 s 5 ) (3) E t h a n o l - a c e t o n e - w a t e r ( 2 s 2 s l ) In t h e i r r a d i a t i o n e x p e r i m e n t s , t h e samples were exposed d i r e c t l y t o the l i g h t o f f o u r f l o u r e s c e n t lamps. Samples o f t e t r a c y c l i n e , and some o f i t s d e r i v a t i v e s , were k i n d l y sup-- 55 -p l i e d by the Lederle Co., Pearl River, N.J., U.S.A. Attempted Transformations of Terrarubein (a) Oxidation of Terrarubein with Platinum Terrarubein (8 mg), was dissolved i n 100 ml of benzene and 20 mg of pre-reduced platinum oxide catalyst was added. Oxygen was passed through the solution f o r 3 days. The u l t r a v i o l e t spectrum of the product showed features at \max 450, 272. .250 mu. (1:4:5) (sh) Paper chromatography, using solvent 1, d i d not move the compound from the o r i g i n . (b) Attempted Oxidation of Terrarubein with I r r a d i a t i o n Terrarubein (8 mg) was dissolved i n 100 ml of benzene. Oxygen was passed through while being d i r e c t l y i r r a d i -ated f o r 24 hours. The benzene was evaporated. The residue showed features at Amax: 450(&), 380, 270 mu (1:2:5) Papergram (solvent 2) \u00E2\u0080\u00A2 Rf 0.5 (yellow, fading) (c) Oxidation of Terrarubein with Pd/C and I r r a d i a t i o n A benzene solution of terrarubein (8 mg), to which 10 mg of palladium on charcoal (5%) had been added, was ir r a d i a t e d and oxygen was passed through the solution - 56 -f o r 12 hourSo The p r o d u c t was c o l l e c t e d by re m o v a l o f solvento I t showed /max? 450 ^ g h ) ,380 , 262 mu ( 1 : 2 : 5 ) . Papergram ( s o l v e n t 2)? R f 0 O 9 ( p i n k , under U o V . l i g h t ) o Attempted H y d r o g e n a t i o n o f O x y d i z e d T e r r a r u b e i n (c) The benzene s o l u t i o n f r o m (c) was e v a p o r a t e d t o d r y n e s s , t h e r e s i d u e d i s s o l v e d i n e t h a n o l and t h e s o l u t i o n h ydrogenated f o r 4 h o u r s , u s i n g 5 mg o f p a l l a d i u m on c h a r c o a l (5^)o The p r o d u c t a b s o r b e d i n t h e u l t r a v i o l e t a t Xmax 4 5 0 | s ^ \u00C2\u00BB , 3 8 0 , 26? mu ( 1 : 2 : 5 )o Papergram ( s o l v e n t 2)s R f 0\u00C2\u00AB9 ( y e l l o w , f a d i n g ) d i h y d r o t e r r a r u b e i n R f 0\u00C2\u00AB5 ( y e l l o w ) o A t tempted H y d r o g e n a t i o n o f T e r r a r u b e i n w i t h P a l l a d i u m on C h a r c o a l T e r r a r u b e i n (30 mg) was d i s s o l v e d i n 200 ml o f e t h a n o l and hydrogenated f o r & h o u r s u s i n g 5$ p a l l a d i u m on c h a r c o a l c a t a l y s t (20 mg) o The p r o d u c t had an u l t r a -v i o l e t spectrum a t Xmax 450\u00C2\u00BB 295. .272 mu (Is4s5) (sh) Papergram ( s o l v e n t 2) R f 0\u00C2\u00BB9 (yellow-brown) 57 -T . L . G , ( p o l y a m i d e , s o l v e n t 2) R f 0,9 ( y e l l o w , f a d i n g ) d i h y d r o t e r r a r u b e i n R f 0,7 ( o r a n g e ) 0 ( f ) A t t empted H y d r o g e n a t l o n o f T e r r a r u b e i n i n a B a s i c Medium (Sodium b i c a r b o n a t e ) T e r r a r u b e i n (40 mg) was suspended i n 50 ml o f e t h a n o l c o n t a i n i n g 5 ml o f s a t u r a t e d aqueous s o l u t i o n o f sodium bicarbonate o P a l l a d i u m on c h a r c o a l (3Q#p 60 mg) was added and hydrog e n a t e d f o r 8 h o u r s . The r e a c t i o n p r o -d u c t was a c i d i f i e d w i t h d i l u t e h y d r o c h l o r i c a c i d and e x t r a c t e d w i t h c h l o r o f o r m . The u l t r a v i o l e t spectrum o f t h e p r o d u c t showed f e a t u r e s a t ^max 435p 290, 265 mu (ls4 \u00C2\u00A7 5 ) T o L o G , (polyamide s o l v e n t 2) R f 0,4 ( y e l l o w , f a d i n g ) Papergrams ( s o l v e n t 2) R f 0,5 ( y e l l o w - b r o w n ) (g) Attempted H y d r o g e n a t l o n o f T e r r a r u b e i n i n a B a s i c Medium ( T r i e t h y l a m i n e ) T e r r a r u b e i n (40 mg) was d i s s o l v e d i n water-glyme m i x t u r e ( l s i , 20 ml) c o n t a i n i n g about 2% t r i e t h y l a m i n e and was hydroge n a t e d w i t h p a l l a d i u m on c h a r c o a l (30#p 50 mg) a t room t e m p e r a t u r e f o r 20 h o u r s . The s o l u t i o n was t h e n f i l t e r e d and e x t r a c t e d w i t h c h l o r o f o r m , The e x t r a c t \u00E2\u0080\u0094 58 \u00E2\u0080\u0094 was washed w i t h N h y d r o c h l o r i c a c i d u n t i l n e u t r a l t o l i t m u s , t h e n d r i e d and t h e c h l o r o f o r m removed, The r e s i d u e had a b s o r p t i o n bands a t Xmax 435? 2 9 5( s njp 270, ( I s 4 s 5 ) 260, .mu, l shJ Papergram ( s o l v e n t 2) R f 0o5 ( y e l l o w , f a d i n g ) T,L,C, (polyamide s o l v e n t 2) R f 0,5 ( y e l l o w , f a d i n g ) The above r e a c t i o n was r e p e a t e d u s i n g r u t h e n i u m on c h a r c o a l (2%) a s c a t a l y s t w i t h s i m i l a r r e s u l t s . A t t e mpted H y d r o g e n a t i o n o f T e r r a r u b e i n i n B a s i c Medium (Sodium h y d r o x i d e ) T e r r a r u b e i n (10 mg) i n 100 ml o f 6N sodium h y d r o x i d e -e t h a n o l (1?1) and p a l l a d i u m on c h a r c o a l 5% (10 mg) was hydroge n a t e d a t room t e m p e r a t u r e a t a t m o s p h e r i c p r e s s u r e f o r 24 h o u r s . The s o l u t i o n was n e u t r a l i z e d w i t h 6N h y d r o c h l o r i c a c i d and e x t r a c t e d w i t h c h l o r o f o r m a A max? 450, 390, 273 mu (ls2,5) Papergram ( s o l v e n t 2) R f 0,8 ( y e l l o w s f a d i n g ) R e d u c t i o n o f T e r r a r u b e i n w i t h P l a t i n u m T e r r a r u b e i n (10 mg) was d i s s o l v e d i n 100 ml o f e t h a n o l and p r e r e d u c e d p l a t i n u m (10 mg) was added and hydrogen-- 59 -a t e d f o r 4 h o u r s . A l i q u o t s were t a k e n every \u00C2\u00A7 hour and t h e i r u l t r a v i o l e t spectrum examined. T h i s showed the g r a d u a l disappearance o f the 450 mu band and the consor ted appearance of a 350 mu band. A f t e r 4 hours i t showed Xmaxs 350, 271 mpL (1?3) Pressure Hydrogenat lon of T e r r a r u b e i n (40 p , s , i , ) T e r r a r u b e i n (10 mg) i n 100 ml of e t h a n o l and 7 mg of p a l l a d i u m on c h a r c o a l (10$) were shaken a t a pressure o f 40 p , s , i , of hydrogen f o r 5 h o u r s , ^maxi 450, 270, 260 mu (Is5s5) Papergram? ( s o l v e n t 2) R f 0,4 ( y e l l o w , f a d i n g ) Pressure Hydrogenat lon of T e r r a r u b e i n (600 p , S o i , ) T e r r a r u b e i n ($0 mg) i n 250 m l of s a t u r a t e d e t h a n o l i c sodium b i c a r b o n a t e was hydrogenated at 600 p , s , i , hydrogen pressure and room temperature f o r 14 h o u r s . The s o l u t i o n was a c i d i f i e d w i t h d i l u t e h y d r o c h l o r i c a c i d , d i l u t e d w i t h water and e x t r a c t e d w i t h c h l o r o f o r m . The u l t r a v i o l e t spectrum of t h e product remained unchanged from s t a r t i n g m a t e r i a l . Oxygenat ion o f T e r r a r u b e i n (Dimethyl S u l f o x i d e ) T e r r a r u b e i n (50 mg) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e - 60 -(50 ml) and f r e s h l y reduced p l a t i n u m c a t a l y s t (50 mg) was added. The s o l u t i o n was exposed t o s o f t l i g h t and oxygen was passed through t h e s o l u t i o n f o r 3 d a y s . F r e s h c a t a l y s t was added at s u i t a b l e i n t e r v a l s . The u l t r a v i o l e t spectrum of the p r o d u c t ^ i s o l a t e d by d i s -t i l l a t i o n o f the so lvent under reduced p r e s s u r e , showed f e a t u r e s a t Xmaxs 382,; 275 W\u00C2\u00B0 (2s5) Attempted R e d u c t i o n of O x i d i z e d T e r r a r u b e i n (1) The o x i d i z e d t e r r a r u b e i n (1, 10 mg) was d i s s o l v e d i n dioxane (25 m l , p a l l a d i u m on c h a r c o a l , 5$) c a t a l y s t was added and hydrogenated f o r 5 h o u r s , No change i n the u l t r a v i o l e t spectrum c o u l d be d e t e c t e d . Oxygenat ion of T e r r a r u b e i n (Dimethyl S u l f o x i d e / M a g n e s - ium Ace ta te ) T e r r a r u b e i n (0 ,5 gm) was d i s s o l v e d i n 100 ml o f d i m e t h y l s u l f o x i d e c o n t a i n i n g 1% magnesium a c e t a t e t e t r a h y d r a t e , The s o l u t i o n was oxygenated i n a hydrogenat lon a p p a r a t u s . A f t e r 1 m o l e - e q u i v a l e n t of oxygen had been absorbed (1/2 hour) an a l i q u o t (20 ml) was w i t h d r a w n . T h i s a l i -quot was added t o 50 ml of 0 .1N h y d r o c h l o r i c a c i d and the r e s u l t i n g dark brown s o l i d c o l l e c t e d by c e n t r i f u -g a t i o n . I t was washed w i t h water (2 x 10 ml) and acetone - 61 -(3 x 10 ml) and d r i e d (80 mg), I t showed Amax ( d i o x -ane) 450, 272 mu (7 9100s Hp500) A n a l , C a l c , f o r C ^ I i ^ O . ^ N , 3\u00C2\u00BB7 ; found 3\u00C2\u00BB3\u00C2\u00BB I R ( K B r ) : 3300, 1700, 'g 1640, 1600 P 1220, 1100, 860, \ 3) 740 cm\" The remainder of the oxygenat ion mix ture was f u r t h e r oxygenated f o r I S h o u r s . Dur ing t h a t t ime another m o l e - e q u i v a l e n t o f oxygen was absorbed , Worked up as above , i t p r o v i d e d 250 mg o f a brown s o l i d . I t showed X max(DMS0/MgAc2) I R ( K B r ) : s i m i l a r t o above. R e d u c t i o n of Mono-oxygenated T e r r a r u b e i n (37) (sodium h y d r o s u l f i t e ) , The mono-oxygenated, t e r r a r u b e i n (37* 50 mg) was d i s s o l -ved i n d i m e t h y l s u l f o x i d e (5 ml) and sodium h y d r o s u l f i t e s o l u t i o n was added u n t i l the r e d d i s h c o l o u r o f the quinone had changed t o orange 0 The s o l u t i o n was a c i d i -f i e d and the s o l i d t h a t was f o r m e d , separated by c e n t r i -f u g a t i o n . I t was washed s e v e r a l t i m e s w i t h water f o l l o w e d by acetone and d r i e d t o y i e l d 30 mg of a brown s o l i d Amax ( 9 7 ^ 2 3 0 ^ 1% ^ B ^ O y ) ; 520, 470, 317, 280 mu (76,000s 155OOOs 42,000s 2 2 , 3 0 0 ) , - 62 -R e d u c t i o n of the Dioxygenated T e r r a r u b e i n (36) (sodium h y d r o s u l f i t e ) , Dioxygenated t e r r a r u b e i n (36, 50 mg) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e (5 ml) and sodium h y d r o s u l f i t e s o l u -t i o n was added u n t i l the c o l o u r o f the quinone had changed t o orange . A f t e r a c i d i f i c a t i o n the s o l i d was i s o l a t e d a s i n ( o ) , I t showed A max.(DM50) .400, 285 mu (1:3) (4-hydroxy m e t h y l p r e t e t r a m i d had Xmax (DMSO): 451* 363 , 305, 266 mu (2 :1 :1 :5) ) R e d u c t i o n of the Dioxygenated T e r r a r u b e i n (36) i n H i / Phenol 0 Dioxygenated t e r r a r u b e i n (20 mg) was d i s s o l v e d i n phenol (4 m l ) , To i t was added h y d r o i o d i c a c i d (3 ml) and the mixture kept a t 120\u00C2\u00B0 f o r 4-5 m i n u t e s s I t was t h e n added t o i c e water c o n t a i n i n g sodium hydrogen s u l f i t e . The aqueous m i x t u r e was e x t r a c t e d w i t h e t h e r and the remainder c e n t r i f u g e d t o g i v e a dark-brown s o l i d , I t showed Xmax (97$ HgSO^- 156 N a 2 B ^0 7): 520, 470, 317> 280 mu (1 .1 :3 :2 ) A c t i o n of m -Chloroperbenzoic A c i d on T e r r a r u b e i n T e r r a r u b e i n (50 mg) was d i s s o l v e d i n t e t r a h y d r o f u r a n (250 ml) and m-ch loroperbenzo ic a c i d , (30 mg) was - 63 -added. The s o l u t i o n was a l l o w e d t o stand a t room t e m p e r a t u r e . A l i q u o t s were removed d a i l y . A f t e r 2 days a papergram ( s o l v e n t 3) showed s e v e r a l spots a t R f s 0 ,2 ( b l u e - f l u o r e s c e n t ) ; 0 ,4 (brown); 0 ,6 (ye l low) and 0 ,9 (brown). I t showed Amax(Dioxane)s 402, 273 mu ( ls4) v C h l o r l n a t i o n of A n h y d r o t e t r a c y c l i n e (20) A n h y d r o t e t r a c y c l i n e (100 mg) and f r e s h l y d i s t i l l e d s u l f u r y l c h l o r i d e (32 mg) were d i s s o l v e d i n g l a c i a l a c e t i c a c i d (25 m l ) . The s o l u t i o n was l e f t a t room temperature i n the dark f o r 12 hours* The a c e t i c a c i d was removed under reduced p r e s s u r e and the b l a c k s o l i d o b t a i n e d was d i s s o l v e d i n water and the pH a d j u s t e d t o 4 \u00C2\u00B0 0 w i t h 6N sodium hydrox-i d e , ; The s o l i d t h a t formed was f i l t e r e d o f f and t r i t u r a t e d w i t h b o i l i n g benzene. On c o o l i n g the benzene e x t r a c t depos-i t e d a r e d amorphous s o l i d (52 mg), ^ ( K B r ) 1 l 6 k Q ( a ) \u00C2\u00BB l62G* 1 5 7 \u00C2\u00B0 9 1 4 3 5 \u00C2\u00BB 1 3 8 \u00C2\u00B0 \u00C2\u00BB 1 2 6 5 \u00C2\u00BB 1215> 1120, 1090, 820 c m \" 1 . I R ( K B r ) o : C A n n y d r o t e t r a c y c l i n e s 1645, 1620 <> 1570, 1445;> 1375, 1335, 1320, 1245* 1225, 1175 9 1110, 754 c m \" 1 , NMR (CDC1 )s 2 ,9 ( d o u b l e t ) , 3.7 (doublet) X (Area l s i ) . - 64 -P r e p a r a t i o n o f 1 2 a - D e o x y t e t r a c y c l i n e (10) A mix ture o f t e t r a c y c l i n e h y d r o c h l o r i d e (2 gm) and z i n c dust (4 gm) i n 15% aqueous ammonia (50 ml) was s t i r r e d f o r 2 hours i n a stoppered f l a s k o The r e a c t i o n mix ture was f i l t e r e d and the f i l t r a t e n e u t r a l i z e d w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d , care be ing t a k e n t o keep the tempera-t u r e below 20\u00C2\u00B0 . At pH 7,0 the s o l i d was removed by f i l t r a -t i o n and r e d i s s o l v e d i n 450 ml of water by a c i d i f y i n g t o pH of lo& w i t h 6N h y d r o c h l o r i c ac ldo Adjustment t o pH 4*2 w i t h d i l u t e sodium h y d r o x i d e , caused the p r e c i p i t a t i o n of a f l o c u l e n t y e l l o w s o l i d which was f i l t e r e d o f f , The f i l -t r a t e was e x t r a c t e d i n a constant e t h e r e x t r a c t o r f o r 50 h o u r s . The e x t r a c t y i e l d e d 500 mg o f s o l i d w h i c h was r e c r y s t a l U s e d f rom methanol , I t showed Xmax .(O.OlN'HCl.) s 457* 432, 325* 263 mu ( 3 : 3 : 1 : 4 ) P r e p a r a t i o n o f D i h y d r o t e r r a r u b e i n (21) 1 2 a - D e o x y t e t r a c y c l i n e (10, 42 mg) was heated w i t h 3,5 ml of g l a c i a l a c e t i c a c i d and 1 ml of $1% hydrobromic acid i n g l a c i a l a c e t i c a c i d added, The s o l u t i o n was heated f o r a f u r t h e r 15 minutes on steam b a t h , c o o l e d and f i l t e r e d t o y i e l d 44 mg of a brown s o l i d . I t showed Xmax: 440j> 380, 328, 273 mu ( 3 : 2 : 2 : 4 ) - 65 -The hydrobromide s a l t ( 4 0 mg) was suspended i n 1 0 0 ml of water and the pH a d j u s t e d t o 7 \u00C2\u00BB 5 w i t h d i l u t e sodium h y d r o x i d e , t a k i n g care not t o i n c r e a s e the pH above 3 . 0 . E x t r a c t i o n i n a constant c h l o r o f o r m e x t r a c t o r f o r 2 4 hours y i e l d e d 2 2 mg of - s o l i d w h i c h gave a n e g a t i v e Be 1 1 s t e i n t e s t . I t showed Xmaxs 4 4 0 , 330, 3 2 5 , 2 7 1 mu ( 3 ? 2 j 2 : 4 ) Papergram ( s o l v e n t 1 ) R f 0 . 1 (ye l low) ( s o l v e n t 2 ) R f 0 . 5 (ye l low) 1 2 & - 0 x y g e n a t i o n of D i h y d r o t e r r a r u b e i n ( 2 1 ) A s o l u t i o n o f d i h y d r o t e r r a r u b e i n ( 3 2 mg i n benzene ( 2 0 0 m l ) , i n the presence o f f r e s h l y reduced p l a t i n u m , was i r r a d i a t e d w h i l e a stream of oxygen was passed t h r o u g h the s o l u t i o n . A d d i t i o n a l c a t a l y s t ( 2 x 2 5 mg) was added a t s u i t a b l e i n t e r v a l s . U l t r a v i o l e t s p e c t r a were o b t a i n e d p e r i o d i c a l l y u n t i l the s t a r t i n g m a t e r i a l spectrum had c o m p l e t e l y d i sappeared and the spectrum had become i d e n t i c a l t o t h a t o f a n h y d r o t e t r a c y c l i n e . Paper chromatograms a t t h i s stage showed no t r a c e of d i h y d r o t e r r a r u b e i n but showed two s p o t s , one a t R f 0 . 5 (ye l low) the o ther a t R f 0.3 (ye l low-brown) ( so lvent 1 ) . The R f 0 . 5 spot was e l u t e d f rom the papergram and showed the same i n f r a r e d spectrum a s a n h y d r o t e t r a c y c l i n e . 66 -P r e p a r a t i o n o f 6 - M e t h y l p r e t e t r a m i d (14a) T e r r a r u b e i n (0,15 gm] was suspended i n a m i x t u r e o f hot p h e n o l (12 ml) and h y d r o i o d i c a c i d (10 m l ) . The mix-t u r e was heated i n an o i l b a t h a t 120\u00C2\u00B0 f o r 4 h o u r s . The s o l u t i o n was added t o i c e water t o w h i c h sodium hydrogen s u l f i t e had been added. I t was r e p e a t e d l y washed w i t h e t h e r . The aqueous m i x t u r e was t h e n c e n t r i f u g e d and t h e s o l i d r e s i d u e was washed s e v e r a l t i m e s w i t h acetone and e t h e r t o y i e l d 0,12 gm o f a brown s o l i d . I t showed Xmax 448, 355, 300, 272 mu ( i s i , 3 , 5 ) O x i d a t i o n of 6 - M e t h y l p r e t e t r a m i d w i t h C a r o l s A c i d 6 - M e t h y l p r e t e t r a m i d (31 mg) was d i s s o l v e d i n a m i x t u r e of p y r i d i n e (2 ml) and p o t a s s i u m h y d r o x i d e ( 5 % * 3 m l ) . The orange s o l u t i o n was c o o l e d t o 0 G and p o t a s s i u m p e r s u l f a t e (40 mg, i n 2 ml o f w a t e r ) was added dro p w i s e d u r i n g 1 ho u r . I t was kept a t 0\u00C2\u00B0 f o r 5 h o u r s . A c i d i f i c a t i o n t o pH 3 produced a b l a c k s o l i d (22 mg), T h i s s o l i d was washed w i t h acetone a n d e t h e r , I t showed \ma.x 500, 370, 240 mu (.1 s5S7) On papergrams i t d i d not move from t h e o r i g i n . O x i d a t i o n o f 6 - M e t h y l p r e t e t r a m i d ( D i m e t h y l s u l f o x i d e / Magne-sium a c e t a t e ) , 6 - M e t h y l p r e t e t r a m i d (20 mg) was d i s s o l v e d i n d i m e t h y l - 67 -s u l f o x i d e (15 ml) c o n t a i n i n g 1% magnesium a c e t a t e t e t r a -h y d r a t e . The s o l u t i o n changed from orange t o r e d when oxygen was passed t h r o u g h i t f o r 2 d a y s . I t showed Xmax (DMS0/MgAc 2) ; 520, 278 mu (2s5) ( 6 - M e t h y l p r e t e t r a m i d Xmax(DJ60 /MgAc 2 ) s'. 485, 280 mu (1 ;3) A f t e r s e v e r a l more days o f exposure the spectrum d i d n o t change 0 The p r o d u c t was p r e c i p i t a t e d by d i l u t i o n w i t h 0,1N h y d r o c h l o r i c a c i d 0 I t was washed w i t h w a t e r and acetone t o y i e l d 5 mg o f a brown solid o I t showed Xmax! 452 9 380, 269* 250 mu ( 2:ls6 : 5 ) R e d u c t i o n o f O x i d i s e d 6 - M e t h y l p r e t e t r a m i d The brown s o l i d o b t a i n e d above (4 mg) was d i s s o l v e d i n p h e n o l (1 m l ) . To i t was added h y d r o i o d i c a c i d (1 ml) and the m i x t u r e h eated a t 120\u00C2\u00B0 f o r 4-5 m i n u t e s . I t was t h e n added t o i c e water c o n t a i n i n g sodium hydrogen s u l f i t e . The m i x t u r e was e x t r a c t e d w i t h e t h e r and t h e aqueous l a y e r cen-t r i f u g e d t o g i v e a d a r k s o l i d (1 mg). I t showed Xmax (97$ H 2 S 0 4 ~ ^ N a 2 B 4 0 7 * 5 5 0 p * * 9 \u00C2\u00B0 * 3 G \u00C2\u00B0 3 2 8 0 W ( l s l s 2 s / f ) A c t i o n o f Fremy's S a l t on 6 - M e t h y l p r e t e t r a m i d 6 - M e t h y l p r e t e t r a m i d (5 mg) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e c o n t a i n i n g 1% magnesium a c e t a t e t e t r a h y d r a t e (3 m l ) , Fremy*s s a l t (8,5 mg) was added and t h e s o l u t i o n s t i r r e d i n - 68 a i r f o r two days. I t s c o l o u r changed f r o m orange t o g r e e n t o b l u e . I t showed A max(DMS0/MgAc 2): 650, 600. 280 mu. The s o l u t i o n upon a c i d i f i c a t i o n w i t h 0.1N h y d r o c h l o r i c a c i d a f f o r d e d a b l u e s o l i d which a f t e r washing w i t h water and acetone showed /Xmax: 550, 280 mu (1:4) A c t i o n o f Fremy's S a l t on 4 - H y d r o x y - 6 - M e t h y l p r e t e t r a m i d 4-Hydroxy m e t h y l p r e t e t r a m i d (35 mg) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e c o n t a i n i n g 1% magnesium a c e t a t e t e t r a -h y d r a t e (10 m l ) . E x c e s s Fremy's s a l t (50 mg) was added. The m i x t u r e was l e f t a t room t e m p e r a t u r e f o r 2 h o u r s . The s o l u t i o n became d a r k b l u e . I t showed Xmax(DMS0/MgAc2): 650, 600, 280 mu (1:1:9) A c t i o n of m - C h l o r o p e r b e n z o i c A c i d on 4-hydroxy-6-methyl-p r e t e t r a m i d 4 - h y d r o x y - 6 - m e t h y l p r e t e t r a r a i d (60 mg) was d i s s o l v e d i n t e t r a h y d r o f u r a n (30 m l ) , m - c h l o r o p e r b e n z o i c a c i d (25 mg) was added and t h e s o l u t i o n k e p t a t 0\u00C2\u00B0 f o r 72 h o u r s . The t e t r a -h y d r o f u r a n was removed under r e d u c e d p r e s s u r e and t h e r e s i -due t r i t u r a t e d w i t h e t h a n o l . The r e s i d u e o f t h i s t r i t u r -a t i o n showed an u l t r a v i o l e t spectrum i d e n t i c a l t o t h e s t a r t -i n g m a t e r i a l , w h i l e the e t h a n o l s o l u t i o n (orange-red) showed A m a x(dioxane): 4 4 0 ( s j , 380, 279 mu (1:3:5)\u00C2\u00B0 - 69 -Papergram ( s o l v e n t 3) showed a y e l l o w spot Rf 0,7 ( s t a r t -i n g m a t e r i a l ) and a l o n g s t r e a k from o r i g i n (pink) <> B r ominations of Dedimethylaminoanhydro-7-chlorotetracy- c l i n e (4)\u00C2\u00B0 (a) To a s o l u t i o n o f 46 mg of (4) i n 40 ml of g l a c i a l a c e t i c a c i d , bromine (1.2 molar e q u i v a l e n t s ) i n g l a c i a l a c e t i c a c i d (2 ml) were added. The s o l u t i o n allowed t o stand a t room temperature f o r 9 hours. D i l u t i o n w i t h water f o l l o w e d by e x t r a c t i o n w i t h c h l o r o f o r m y i e l d e d 40 mg o f a brown s o l i d . I t showed Xmax: 451* 350* 272 mu (2,1,5). 1 1 1 (KBr) l 6 6 0 p l 6 3 0 p 1 5 5 \u00C2\u00B0 s ^50/1215* 1105* 1050 cm\"\"1 A n a l . C a l c . f o r CgQH-^NOyBrGl C, 48,38, H, 3.03 = Found ( f o r crude s o l i d ) : C, 47=29; H.3\u00C2\u00BB80\u00E2\u0080\u009E 47 \u00E2\u0080\u00A2(b) A s o l u t i o n of 1,08 gm of (4) i n 20 ml of g l a c i a l a c e t i c a c i d was c o o l e d and s t i r r e d j u s t above i t s f r e e z i n g p o i n t . A s o l u t i o n of 0,4 gm of bromine i n 1 ml of a c e t i c a c i d was added dropwise s w i t h i n a few minutes p r e c i p i t a t i o n commenced. A f t e r 2 hours the s o l u t i o n was f i l t e r e d and the p r e c i p i t a t e washed thoroughly With e t h e r and d r i e d to g i v e 0,85 gm of a brown s o l i d . I t showed Xmax: 445* 335* 275 mu (2:1:6). - 70 -I R ( K B r y : 1660, 1630, 1575* 1420, 1215, 1060, 1035* 1000 cm\" 1. A n a l , C a l c , f o r CggH^NQyBrCls . C, .48.32; H, 3=03, Found C, 47o90\u00C2\u00BB H, 3\u00C2\u00BB25, in \u00E2\u0080\u00A2(c) A mixture o f 38 mg o f (4) was suspended i n 15 ml o f c h l o r o f o r m and N-bromosuccinimide (18 mg) was allowed t o stand a t room temperature f o r 3 hours, A c l e a r orange s o l u t i o n was o b t a i n e d a f t e r a few minutes. P r e c i p i t a t i o n of a yellow-brown s o l i d commenced a f t e r an hour of s t a n d i n g . The s o l i d upon completion o f the r e a c t i o n was f i l t e r e d and washed wi t h e t h e r . Y i e l d 35 mg, /\maxi 440* 335* 275 mu (2;ls6), \" ( K B r ) 8 A S l n ( b ) A n a l , c a l c , f o r C ^ H ^ N O y B r C l s C s 48.22; H p 3 = 03, Founds C, 48,38; H, 3,53\u00C2\u00BB \"(d) To a s o l u t i o n o f 100 mg of (4) i n 50 mg of g l a c i a l a c e t i c a c i d was added slowly 1 molar e q u i v a l e n t of bromine i n 2 ml of a c e t i c a c i d . The s o l u t i o n was l e f t to stand a t room temperature f o r 8 h o u r s a d i l u t e d w i t h water and e x t r a c t e d w i t h c h l o r o f o r m . A f t e r d r y i n g , the c h l o r o f o r m was removed t o y i e l d 80 mg o f a brown s o l i d , A maxs 440 p 332* 273 mu (2sls6), - 71 -I R / \u00E2\u0084\u00A2 s i m i l a r t o (b) IKBrJ A n a l , CalCo f o r C^oH^NOyBrCls 4S\u00C2\u00B038\u00C2\u00A7 H , 3,03 o Found ( f o r crude s o l i d ) ? C , 50 .01 ; 3<>92, (e) Hydrogen bromide gas was passed through a s o l u t i o n of 150 mg of (4) i n 80 ml of g l a c i a l a c e t i c a c i d f o r a few m i n u t e s s the bromine (1 ,2 molar e q u i v a l e n t s ) i n 2 ml of a c e t i c a c i d was added, A f t e r 5 hours the r e a c t i o n mixture was worked up i n the u s u a l way t o y i e l d 100 mg of a brown s o l i d , Amaxs 440* 3 3 2 P 272 mu (2 :1 :6 ) \u00E2\u0080\u00A2 ^ ( K B r ) 5 S i m i l a r *o Co) A n a l , C a l c , f o r C ^ H ^ N O y B r C l s C , 48 ,38 ; H s 3=03, Found ( f o r crude s o l i d ) : Cg 50,9; H & 4,2, R e a c t i o n s of the Monobrominated Compounds ( a g b9 d and e) w i t h Dimethylamine , (a) Monobrorao compound ( a , 20 mg) was d i s s o l v e d i n 100 ml of methanol c o n t a i n i n g 1,0 ml of d i m e t h y l a m i n e . A f t e r 5 days s tanding a t room temperature a g r e e n i s h - y e l l o w spot (Rf 0,3) developed on a papergram ( s o l v e n t 1 ) , 7 - C h l o r o a n h y d r o t e t r a c y e l i n e Rf 0 ,5 ( y e l l o w ) . S t a r t i n g m a t e r i a l R f 0,9 (brown). 72 (b) Monobromo compound ( b p 10 mg) i n 2 ml of l i q u i d dimethylamine was heated g e n t l y u n t i l a l l the amine had evaporated, c h l o r o f o r m added and the s o l u t i o n r e f l u x e d f o r a few minutes. H y d r o c h l o r i c a c i d 10% (5 ml) was addedo No c o l o u r was e x t r a c t e d i n t o the a c i d l a y e r , (c) Monobromo compound (d, 20 mg) was t r e a t e d w i t h l i q u i d dimethylamine a s above, H y d r o c h l o r i c a c i d e x t r a c t e d a y e l l o w compound. The a c i d s o l u t i o n was a d j u s t e d t o pH 5\u00C2\u00AB5 w i t h d i l u t e sodium hydroxide and e x t r a c t e d w i t h c h l o r o f o r m . The p a l e y e l l o w c h l o r o f o r m e x t r a c t was reduced i n bul k and examined c h r o m a t o g r a p h i c a l l y . Papergrams; (1) S o l v e n t (1) Rf 0 ,3 (green-yellow) 7 - c h l o r o a n h y d r o t e t r a c y c l i n e Rf 0 ,5 ( r e d d i s h - y e l l o w ) A n h y d r o t e t r a c y c l i n e Rf 0,4# (yellow) 4 - E p i a n h y d r o t e t r a c y c l i n e Rf 0 ,28 (yellow) (2) S o l v e n t (2) Rf 0 ,5 (green-yellow) 7 - C h l o r o - a n h y d r o t e t r a c y c l i n e Rf 0 ,5 ( r e d d i s h - y e l l o w ) A n h y d r o t e t r a c y c l i n e R f 0 ,5 (yellow) 4 - E p i a n h y d r o t e t r a c y c l i n e R f 0 ,5 (yellow) - 73 Polyamide T 0 L 0 C 0 : (1) S o l v e n t (2)i Rf 0 o8 (yellow-brown) 7-ChloroanhydrotetracyGline Rf 0 02 (yellow) t r a c e Rf,0 o8 (yellow-brown) A n h y d r o t e t r a c y c l i n e R f 0 34 (yellow) t r a c e Rf Qo8 (yellow-brown) 4 - E p i a n h y d r o t e t r a c y c l i n e Rf 0c8 (yellow-brown) (2) S o l v e n t (3)*. Rf 0.9 (yellow-brown) 7 - C h l o r o a n h y d r o t e t r a c y c l i n e R f 0 ,3 ( y e l l o w ) t r a c e R f 0 09 (yellow-brown) A n h y d r o t e t r a c y c l i n e Rf 0=4 (yellow) t r a c e R f 0 09 (yellow-brown) 4 - E p i a n h y d r o t e t r a c y c l i n e R f 0,8 (yellow-brown) The u l t r a v i o l e t spectrum of the product showed f e a t u r e s a t 4289 3409 270 mu0 Monobromo compound (e) was t r e a t e d as above 0 A very weak spot a t Rf 0o3 ( s o l v e n t 1) d e v e l o p e d 0 7-Chloro-4 e p i - a n h y d r o t e t r a c y c l i n e Rf 0o3 ( g r e e n i s h -yellow) Brominations of 9-Bromo-dedimethylaminoanhydroaureomycin (47) (a) To a s o l u t i o n of 48 mg of (47) i n 5 ml of warm c h l o r o f o r m - 74 -was added 20 mg of N-bromosuccinimide, A f t e r 24 hours carbon t e t r a c h l o r i d e was added and the r e s u l t i n g p r e c i -p i t a t e washed wi t h carbon t e t r a c h l o r i d e and with e t h y l e t h e r t o y i e l d 30 mg o f a brown s o l i d Xmax: 390, 254 mu (1:2), I R ( K B r ) s 1775* 1710, 1635* 1575* 1410, 1135 cm\" 1. A n a l , c a l c , f o r C 2 0 H 1 / f N 0 7 C l B r 2 : C, 41 = 7; H, 2,44* Founds. C, 42,5p H, 3c09<> (b) A s o l u t i o n of 50 mg of (47) i n 25 ml of a c e t i c a c i d was t r e a t e d w i t h 1 e q u i v a l e n t of bromine f o r 43 hours a t room temperature. At the end o f t h i s time iodo-m e t r i c t i t r a t i o n gave negative r e s u l t f o r f r e e bromine. The s o l u t i o n was d i l u t e d w i t h water and e x t r a c t e d w i t h c h l o r o f o r m . E v a p o r a t i o n of the c h l o r o f o r m y i e l d e d 50 mg of a s o l i d A max, 444p 338, 273 mu (2:1:6), (KBr) s i m H a r t o s t a r t i n g m a t e r i a l . A n a l , C a l c , f o r C ^ H - ^ N O y B r ^ l s C, 41,7; H 9 2,41; N, 2o41; B r , 27,2, C I , 6,09, Found: C, 43,33; H, 3,13; N, 3-27; B r , 19=11; CI, 8,31, R e a c t i o n s of Dibrominate Compound (b) w i t h Dimethylamine (a) Dibromo compound (b, 10 mg) was d i s s o l v e d i n dimethyl \u00E2\u0080\u009E .75 -amine (2 ml) and t r e a t e d as b e f o r e . No c o l o u r d e v e l -oped i n the a c i d l a y e r d u r i n g e x t r a c t i o n , (b) S e a l e d Tube R e a c t i o n The dibromo compound (10 mg) was d i s s o l v e d i n d i m e t h y l -amine (2 ml) i n a s e a l e d tube. The tube was heated a t 50\u00C2\u00B0, The s o l u t i o n became b l a c k a f t e r 1 hour. The r e a c t i o n was stopped and t r e a t e d w i t h c h l o r o f o r m and a c i d , as b e f o r e . No c o l o u r appeared i n the a c i d l a y e r , A c e t y l a t i o n of 7-chloro-dedimethylaminoanhydrotetracyeline (4) A s o l u t i o n of 0,5 gm o f (4) i n 5 ml o f p y r i d i n e and 2,5 ml of a c e t i c anhydride was allowed t o stand a t room tempera-t u r e f o r 20 hours. The r e a c t i o n mixture was poured i n t o about 500 ml o f s t i r r e d water and the yellOw s o l i d t h a t ap-peared f i l t e r e d , washed w i t h water and d r i e d t o g i v e 0,2 gm of a p a l e y e l l o w s o l i d . Amax: 400, 342, 330, 272 mu (2:3: 4:8) T,L.C, ( s o l v e n t 3)s two y e l l o w spots Rf 0,6 and 0,8 N.M.R,: 2,2 - f ( d o u b l e t ) ; 2,9 T ( d o u b l e t ) (Area 1:1); a l s o 3 A c e t a t e and 1 methyl a t 7-8 X \u00C2\u00B0 Bromination of A c e t a t e Mixture Brominations w i t h N-bromosuccinimide i n c a r b o n t e t r a -- 76 -chloride f a i l e d to change the compound, Acetate mixture of ( 4 ) ( 1 0 0 mg) was dissolved in. 1 ml of g l a c i a l acetic acid and bromine 1 0 0 mg i n 1 , 5 ml of acetic acid was added, The solution was l e f t to stand f o r 7 days at room temperature i n the dark. The acetic acid was removed i n vacuum and replaced with 5 ml of chloroform and 4 0 ml of petroleum ether (b.p 30-60\u00C2\u00B0) was added slowly causing the p r e c i p i t a t i o n of 80 mg of an orange yellow s o l i d ToL'.Co ( s o l v e n t ' 3 ) ; Rf, 0 , 7 (yellow), NoM,R, Showed l o s s of one acetate p otherwise no change from st a r t i n g material, IoR, Showed acetates present. Hydrolysis of Brominated Acetate Brominated acetate (40 mg) was suspended i n 10 ml of ethanol and f i l t e r e d , 1 0 ml of 6N hydrochloric acid was added to the f i l t r a t e and the mixture was l e f t on steam bath f o r 5 hours allowing the ethanol to evaporate, A brown s o l i d ( 3 0 mg) separated, Xmax? 4 3 9 \u00C2\u00BB 3 3 0 ^ , 2 6 4 mu ( 8 ? 3 0 0 p 3 * 4 0 0 9 2 6 p 3 0 0 ) \u00E2\u0080\u009E I R ( K B r ) : 1 7 3 5 p 1 6 5 5 , 1 6 1 5 , 1 5 8 0 , 1 4 1 0 , 1 2 2 0 / 8 2 5 cm\"\"1. Anal, c a l c , f o r C 2QH 1 5 N 0 y B r C l : C, 4 8 , 3 8 ; H s 3 , 0 3 ; . N, 2 . 8 4 ; CI, 7=14; Br, 1 6 . 1 0 , 0 , 2 2 . 4 7 = - 77 -Found: C, 50.10;.H, 3=22; N , 2.80; CI, 7\u00C2\u00BB25; Br, 16.57; 0, 23.94. Molo w t o (found by mass s p e c t r o m e t r i c a n a l y s i s ) : 495-6. A c e t y l a t i o n of 9-Bromo-dedimethylaminoanhydroaureomycine (47) A s o l u t i o n o f 200 mg of (47) i n 2 ml of p y r i d i n e and 1 ml o f a e e t i c anhydride was allowed t o stand o v e r n i g h t . The s o l u t i o n was then poured i n t o s t i r r e d water and the r e -s u l t i n g p r e c i p i t a t e f i l t e r e d , washed t h o r o u g h l y w i t h water and d r i e d t o y i e l d 130 mg o f a p a l e y e l l o w s o l i d . T h i s s o l i d was r e p r e c i p i t a t e d by d i s s o l v i n g i t i n c h l o r o f o r m and t r i t u r -a t i n g with petroleum e t h e r (b.p. 30-60\u00C2\u00B0) Xmax: 390, 333s 321, 273 mu (2:3:4:8) T.L.C.: ( s o l v e n t 3) Rf 0 o6, 0.8 (yellow) N . M o R . : s i n g l e t 2.1 T . Attempted Selenium d i o x i d e O x i d a t i o n of 7-Chloro-5a,6 anhydro- 4-dedimethylamino t e t r a c y c l i n e (4) The t e t r a c y c l i n e (4* 50 mg) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (50 m l ) . Selenium d i o x i d e (100 mg) was added and the mixture r e f l u x e d f o r 24 hours. The r e a c t i o n product was c o o l e d , d i l u t e d w i t h water and e x t r a c t e d w i t h c h l o r o f o r m . - 78 -The s o l i d r e m a i n i n g a f t e r t h e s o l v e n t was e v a p o r a t e d was shown t o be u n r e a c t e d s t a r t i n g m a t e r i a l , . T h i s e xperiment was r e p e a t e d u s i n g 70% a c e t i c a c i d , e t h a n o l and d i o x a n e a s s o l v e n t w i t h s i m i l a r r e s u l t s . A t t empted O x y g e n a t i o n o f 7-chioro-5a,6-anhydro-4-dedimethyl- amino t e t r a c y c l i n e (4) w i t h P o t a s s i u m t e r t i a r y b u t o x i d e . P o t a s s i u m m e t a l (200 mg) was added t o 10 ml o f d r y t -b u t y l a l c o h o l . To t h i s s o l u t i o n was added (4* 80 mg) and d i m e t h y l formamide (10 m l ) . Oxygen was p a s s e d t h r o u g h the s o l u t i o n f o r 10 h o u r s . I t was t h e n a c i d i f i e d w i t h g l a c i a l a c e t i c a c i d , c o n c e n t r a t e d i n vacuo, w a t e r added and e x t r a c -t e d w i t h e t h e r . I t showed an u l t r a v i o l e t spectrum and c h r o m a t o g r a p h i c b e h a v i o u r i d e n t i c a l t o t h e s t a r t i n g m a t e r i a l . A t t empted N i t r o s a t i o n of 7-Chloro-5ag6-anhydro-4-dedimethyl-. amino t e t r a c y c l i n e (4), (a) The t e t r a c y c l i n e (4* 0,5 gm) d i s s o l v e d i n e t h a n o l (40 ml) and p y r i d i n e (8 m l ) . Sodium c a r b o n a t e (10%, 10 ml) was added f o l l o w e d by p - n i t r o s o d i m e t h y l a n i l i n e h y dro-c h l o r i d e (1.0 gm), The s o l u t i o n was r e f l u x e d f o r 6 h o u r s , c o o l e d , a c i d i f i e d w i t h d i l u t e h y d r o c h l o r i c a c i d , r e f l u x e d a g a i n f o r 1/2 h o u r , d i l u t e d w i t h w a t e r and e x t r a c t e d w i t h e t h e r . The e t h e r was s t r i p p e d , The - 79 -brown r e s i d u e showed Xmax; 402, 267 ( l s3)o A n a l y s i s showed no i n c o r p o r a t i o n of n i t r o g e n . (b) 7 - C h l o r o - 4 - d e d i m e t h y l a m i n o - 5 a , 6 - a n h y d r o t e t r a c y c l i n e ( 4 * 100 mg) was d i s s o l v e d i n 15 ml of dry diglyme i n a three necked f l a s k * A slow stream of h y d r o c h l o r i c a c i d gas was passed t h r o u g h the s t i r r e d s o l u t i o n . A s o l u t i o n of 0.03 ml of n - b u t y l n i t r i t e i n 3 ml of diglyme was added i n s m a l l p o r t i o n s . The c o l o u r of the s o l u t i o n darkened p r o g r e s s i v e l y . S t i r r i n g and the passage of h y d r o c h l o r i c a c i d gas was cont inued f o r an h o u r . A f t e r t h i s the i n t r o d u c t i o n of h y d r o c h l o r i c a c i d gas was stopped and the s o l u t i o n l e f t over n i g h t . The s o l v e n t was evaporated t o y i e l d a dark brown s o l i d . T h i s was r e p r e c i p i t a t e d from i t s d i o x a n s o l u t i o n by a d d i t i o n of petro leum e t her ( b . p . 30-60$) . I n f r a r e d and u l t r a -v i o l e t spectrum o f t h i s sample showed no change f rom s t a r t i n g m a t e r i a l . P r e p a r a t i o n of 4 O x o - 4 - d e d i m e t h y l amino t e t r a c y c l i n e - 4 a 6 - h e m i k e t a l (44) T e t r a c y c l i n e h y d r o c h l o r i d e (5 gm) was d i s s o l v e d i n 250 ml of water c o n t a i n i n g concentra ted h y d r o c h l o r i c (2 ml) a c i d . To the s o l u t i o n , s t i r r i n g at room tempera ture 9 was added 3 c 5 gm of powdered N - c h l o r o s u c c i n i m i d e . The crude product began 80 t o p r e c i p i t a t e w i t h i n a few m i n u t e s . A f t e r 30 minutes i t was f i l t e r e d and washed r e p e a t e d l y w i t h w a t e r . The s o l i d was d i s s o l v e d i n e t h e r (200 ml) and washed w i t h water u n t i l the aqueous l a y e r remained c o l o u r l e s s . The ether phase was s t r i p p e d t o dryness t o y i e l d 3*5 gm (60$) of a l i g h t y e l l o w s o l i d , Amax(Me0H - 0.01NHG1): 346, 268 mu (1:5). I R ( K B r ) : 1775, 1710, 1650, 1450, 1180, 1020, 920 c m - 1 . P r e p a r a t i o n of 4 -hydrox imino-4 -dedimethylamino t e t r a c y c l i n e (52) 4-0xo-4-dedimethyl amino t e t r a c y c l i n e 4,6 h e m i k e t a l (44, 2 gm) was d i s s o l v e d i n 10 ml of methanol . Potass ium b i c a r -bonate (5 gm) and hydroxylamine h y d r o c h l o r i d e (1 .0 gm) was added. The r e s u l t i n g deep p u r p l e s o l u t i o n was s t i r r e d f o r 20 m i n u t e s , f i l t e r e d and the f i l t r a t e a c i d i f i e d to pH2 w i t h d i l u t e h y d r o c h l o r i c acid*, The a c i d s o l u t i o n was e x t r a c t e d w i t h e ther (4 x 100 m l ) . The s o l i d o b t a i n e d on e v a p o r a t i o n of the e ther was washed w i t h c o l d benzene and the crude product was c r y s t a l l i s e d from t o l u e n e - i s o p r o p y l a l c o h o l m i x t u r e (3:1), The y i e l d o f the product was 0.5 gm (40$) . I t showed A max (MeOH 0,01 NHC1) 359, 308, 276 mu (3 : 4 : 2 ) . I R ( K B r ) : 1 6 6 0 \u00C2\u00BB l o3\u00C2\u00B0> 1590, 1470, 1235p 1040, 870 c m - 1 . P r e p a r a t i o n of 5a,6 -Anhydro-4 -hydroxyimino-4 -dedimethyl- a m i n o t e t r a c y c l i n e (43)\u00C2\u00B0 4 - h y d r o x y i m i n o-4 - d e d i m e t h y l a m i n o t e t r a c y c l i n e (52, 200 mg) - 31 was d i s s o l v e d i n 2 ml o f 4$ m e t h a n o l i c hydrogen c h l o r i d e . The s o l u t i o n was b o i l e d f o r 3 m i n u t e s and l e f t t o c r y s t a l l i s e o v e r n i g h t a t room t e m p e r a t u r e . The p r o d u c t (110 mg, 49$) was r e c o v e r e d by f i l t r a t i o n and washed w i t h m e t h a n o l . I t showed Amax(MeOH - 0,01 NHC1): 421, 303, 264 my (1:2:4), I R ( K B r ) : 1650, 1620, 1570, 1440, 1240 cm\" 1. P r e p a r a t i o n o f 4-keto-4-dedimethylamino-5a p6-anhydro t e t r a - c y c l i n e (42) 5 a , 6 - a n h y d r o t e t r a c y c l i n e (500 mg) ..was d i s s o l v e d i n wa t e r (350 ml) and c o n c e n t r a t e d h y d r o c h l o r i c a c i d (10 m l ) . N - C h l o r o s u c c i n i m i d e (150 mg) was added i n p o r t i o n s d u r i n g 10 m i n u t e s t o t h e w e l l s t i r r e d s o l u t i o n . The s t i r r i n g was c o n t i n u e d f o r 40 m i n u t e s a t room t e m p e r a t u r e . I t was t h e n e x t r a c t e d w i t h water u n t i l t h e w a s h i n g s were n e u t r a l t o l i t m u s . The e t h e r was s t r i p p e d t o y i e l d a brown s o l i d (240 mg, 45$) w h i c h was r e p r e c i p i t a t e d f r o m t e t r a h y d r o f u r a n - p e t r o l e u m e t h e r (30-60\u00C2\u00B0). I t showed Xmax: 437, 273 mu (17,000; 75*000). I t showed one spo t a t R f 0.3 on paper u s i n g M e t h a n o l - A c e t o n e - w a t e r (3:1:1), A n a l , G a l e , f o r C 20H 1 5O 7H: C, 64,75; H, 4\u00C2\u00AB04; 0, 30,18, Found: C, 64.51? H, 4=25; 0, 30.49= I R ( K B r ) : 3400, 1721, 1655, 1625, 1525* 1450, 1240, 980 cm\" 1. - 82 -Attempted F o r m a t i o n o f Oxime of (42) (a) 4-keto-4-dedimethylamino-5a,6-anhydro t e t r a c y c l i n e (42, 20 mg) was d i s s o l v e d i n methanol (2 m l ) . P o t a s -sium b i c a r b o n a t e (30 mg) and h y d r o x y l a m l n e hydro-c h l o r i d e (20 mg) was added. The s o l u t i o n was s t i r r e d f o r 20 m i n u t e s . I t was t h e n d i l u t e d w i t h w a t e r , a c i -d i f i e d t o pH2 and e x t r a c t e d w i t h e t h e r . Paper chroma-t o g r a p h y o f t h e e t h e r e x t r a c t showed t h e p r e s e n c e of a t l e a s t 5 compounds. (b) 4-keto-4-dedimethylamino-5a,6-anhydro t e t r a c y c l i n e (42, 20 mg) was d i s s o l v e d i n 1 ml o f a c e t i c a c i d and h y d r o x y l a m i n e h y d r o c h l o r i d e (20 mg) was added. The s o l u t i o n was a l l o w e d t o s t a n d f o r 20 minute So Water was added and e x t r a c t e d w i t h e t h e r . The e t h e r was s t r i p p e d t o y i e l d 15 mg of a brown s o l i d . T h i s mater-i a l was shown t o be i d e n t i c a l t o s t a r t i n g compound c Attempted R e d u c t i v e a m i n a t i o n o f (42) To a s o l u t i o n o f (42, 24 mg) i n monoglyme (20 ml) and aqueous amonia (28%, 2 ml) was added p a l l a d i u m on c h a r c o a l (10%s 40 mg) and shaken under hydrogen g a s a t 36 p . s . i . f o r 1 hour. The p r o d u c t was f i l t e r e d and a c i d i f i e d w i t h d i l u t e h y d r o c h l o r i c a c i d t o pH o f 2, washed w i t h e t h e r (3 x 20 ml) and i t s pH adjusted to 5.5\u00C2\u00BB with dilute sodium hydroxide. I t was then extracted with chloroform. The chloroform extract was colourless. The aqueous solution showed A max: 444, 270 mu, (1:6)^ (authentic 4-dedimethyl 5a, 6-anhydro showed Amax(H20): 273, 430 mu (1:6)} - 84 -REFERENCES BoM0 Duggar, Ann, N , Y . A c a d . S c i . , j j l , 177 (1948) , A.C. F i n e l y , G , L , Hobby, S . Y . P\u00C2\u00BBan, P . P , Regna, J . B . R o u t i e n , D . B , S e e l e y , G , M , S h u l l , B . A , S o b i n , I . A . Solomons, J . W , V i n s o n and J , H . Kane, Sc ience I I I , 85 (1950), G,R. Stephens , L , H , Conover , F , A , H o c h s t e i n , P . P . ' Regna, F \u00E2\u0080\u009E J , P i l g r i m , K , J . B r u n i n g s , R , B , Woodward, J , Am, Chem, Soc , 2Jks> 4976 (1952) = L . H . 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K r e u t z e r , Ber., 2^, 892 (1961)c 63. C o B e d f o r d , Ph.D. t h e s i s , Glasgow, (1963). 64\u00C2\u00B0 R o K . Blackwood, C R . Stephens, J . Am. Chem. S o c , 86, 2736 (1964). \" ~~. 65. R 0 C 0 E s s e , J.A. Lowery, C 0 R o Tamorria and G.M. S i e g e r , i b i d p 3875 (1964)o 66. R . K o Blackwood, P r i v a t e Communication. 67o J o J 0 Hlarkap P. B i t h a and J.H. Boothe, J . Am. Chem, S o c , 8Zp 1795 (1965)o - 88 -68 0 P o A o Hart* i n \" S t e r o i d R e a c t i o n s 0 An O u t l i n e f o r Organic Chemists\", edo C D j e r a s s i , Holden-Day I r i C o , San F r a n c i s c o , 1963* p 0179\u00C2\u00B0 69o 0 a T o u s t e r , i n \"Organic R e a c t i o n s \" Volo V I I , John Wiley & Sons l n c ; , New York, 1953* P<> 327, 70 o No Rabjon 5 i n Organic R e a c t i o n s , Volo V, John Wiley & Sons Inco, New York, 1949* p> 331= 71, K 0 Arakawa and M0 I r i e , PharnioBull (Tokyo), 5, 531* (1957)o PART I I o STUDIES IN RELATION TO THE ACETATE PATHWAY TO AROMATIC COMPOUNDS 89 INTRODUCTION D u r i n g the l a s t decade a l a r g e amount of e x p e r i m e n t a l and t h e o r e t i c a l work has been done on the b i o s y n t h e s i s of n a t u r a l products , , As a r e s u l t of these s t u d i e s many c l a s s e s of n a t u r a l l y o c c u r r i n g organic compounds have been shown t o be d e r i v e d from simple s t r u c t u r a l u n i t s 0 T h i s has l e d t o the d e r i v a t i o n of c e r t a i n r u l e s which have c o n s i d e r a b l y s i m p l i f i e d the t a s k of s t r u c t u r e a n a l y s i s and s t r u c t u r e c o r r e l a t i o n f o r the organic c h e m i s t 0 The f i r s t of t h e s e , the \" i s o p r e n e r u l e \" was proposed by R u z i c k a 1 as a r e s u l t o f h i s s t u d i e s i n the terpene f l e l d 0 The b a s i c b i o g e n e t i c h y p o t h e s i s was l a t e r e n l a r g e d and I t was found t h a t mevalonic a c i d l a c t o n e 0 w h i c h i s formed f rom condensa t ion of 3 a c e t i c a c i d u n i t s a i s a d i r e c t p r e c u r s o r of t e r p e n o i d p r o d u c t s 0 The c a r b o h y d r a t e 9 or the s h i k i m i c a c i d pa thway 2 , l e a d s t o another major c l a s s of n a t u r a l l y o c c u r r i n g compounds, such as aromat ic amino a c i d s p i n d o l e a l k a l o i d s , f l a v a n o i d s , coumar ins , s t l l b e n e s p l i g n a n s and l i g n i n s 0 They are r e a d i l y d i s t i n g u i s h e d from aromat ic r i n g s a r i s i n g by p o l y a c e t a t e c v c l i z a t i o n , ( v i d e i n f r a ) n since, t h e ' l a t t e r - p o s s e s s the c h a r a c t e r i s t i c r e s o ' r c i n o l p a t t e r n of oxygenat ion on the r i n g s o \u00E2\u0080\u00A2 . - 90 -The involvement of acetic a c i d , another building block of natural phenolic compounds, was f i r s t proposed by C o l l l e 3 0 On the basis of some in t e r e s t i n g t ransfor-mations that he had observed with polyaeetyl compounds and the s i m i l a r i t y of the products to natural phenolic and naphthalenie compounds. C o l l i e postulated that such compounds might be formed i n nature from condensation of several \"head to t a i l \" l inked a c e t i c acid u n i t s and t h e i r subsequent cycllssationo For example, i t was found that in, weak a l k a l i diacetylacetone (1) dimerized via a l d o l condensations to the naphthalene derivative (2) and dehydroacetic a c i d (3) s i m i l a r l y treated produced o r c i n o l (4K - 91 .-^CHf02H combustion 0,04 c/min/mg BaCOo F i g u r e !<> - 92 . Many years later\u00E2\u0080\u009E a f t e r examination of the structure of naturally occurring phenols, pyronesp quinones p flavones, Birch^^^ and Robinson^, independently of C o l l i e , l a i d down most of the modern view of the acetate hypothesis and pointed out i t s broad scope and usefulness. The key step i n the acetate pathway i s the formation, of a l i n e a r B-polyketoacid chain, which can i :cycllze to either of the; two f a m i l i e s of aromatic, compounds, the acylphlorpglucinols (\u00E2\u0080\u00A2\u00C2\u00A3) and the o r s e l l i n i c acid d e r i v a t i v e s (6) as shown below p or i f l e s s than the required number of C 2 u n i t s are present 0 by M0M c y c l i z a t i o h to pyrones (7JU - 93 -The actual involvement of acetic acid i n aromatic natural products was f i r s t demonstrated by Birch and h i s eolleagues7 0 They fed l a b e l l e d acetate to micro-organisms, is o l a t e d the natural product of i n t e r e s t and located the po s i t i o n of the l a b e l by knowndegradatlve procedures,, Thus they demonstrated the incorporation of (1- 1 40) ac e t i c a c i d into 6-methylsalicylie a c i d (8) by P e n i c i l l i u m grlseo-fulvW? g i v i n g the d i s t r i b u t i o n pattern shown i n figure '1, Subsequently tracer methods using 1 4 G l a b e l l e d acetate, have been applied to the study of the biosynthesis of many natural products, such as g r l s e o f u l v l n (9)^p a l t e r n a r i o l (10)\u00C2\u00B0% the t e t r a c y c l i n e s ( l l ) 1 0 and such aromatic derived compounds as p a t u l l n (12) 1 1, p e n i c i l l i c acid ( 1 3 ) 1 2 and the tropoionic acids (14)13<> (see f i g u r e 2) While of great i n t e r e s t these t r a c e r studies alone did not throw much l i g h t on the mechanism of the I n i t i a l stages of the formation of the B-polyketomethylene chainSo It was early recognized that any a c i d found i n nature might conceivably i n i t i a t e a chain by adding t o the methyl end of a C 2 u n i t . Among such acids which do so are f a t t y acids of varying chain length ( C 2 , 03,0^, 05, Gg, G l o , G ^ ) , branched chain acids such as i s o b u t y r i c , cx -methyIbutyric, i s o v a l e r i c a c i d , cinnamic a c i d and n i c o t i n i c a c i d 1 4 , . - :94 -\u00E2\u0080\u00A2(14) F i g u r e 2, 95 -I n some e x p e r i m e n t s i t was f o u n d t h a t t h e l a b e l l i n g o f t h e f i r s t a c e t i c a c i d u n i t i n c o r p o r a t e d i n t o t h e c h a i n by f e e d i n g a c e t i c a c i d showed an a c t i v i t y 8% h i g h e r t h a n t h e o t h e r u n i t s 1 ' * \u00E2\u0080\u009E T h i s was l a t e r e x p l a i n e d by the i n v o l v e m e n t o f malonate r a t h e r t h a n a c e t a t e i n the c h a i n b u i l d i n g p r o c e s s a s d i s t i n c t f r o m c h a i n i n i t i a t i o n , , Gatenbeck and Mosbach 1^ i n c o r p o r a t e d 1 8 0 f r o m ( l - ^ G p ^ O ) a c e t i c a c i d i n t o o r s e l l i n i c a c i d . They were a b l e t o show t h a t 0 c o n t e n t o f t h e c a r b o x y l group i s h a l f t h a t i n e a c h p h e n o l i c h y d r o x y l 0 T h i s i s t h e r e s u l t e x p e c t e d i f id t h e 0 marker a s a c e t y l coenzyme A goes d i r e c t l y t h r o u g h t h e s y n t h e s i s w i t h o u t random d i s t r i b u t i o n ^ e n d i n g i n o r s e l l i n i c coenzyme A ( v i d e i n f r a ) s t h e h y d r o l y s i s o f the l a t t e r w i t h u n l a b e l l e d w a t e r d i l u t e s t h e t o t a l c a r b o x y l x o 0 p e r oxygen by h a l f o Thus i t i s c l e a r t h a t the i n i t i a l a c e t y l oxygens s t a y on a l i n e a r p o l y a c e t y l i n t e r m e d i a t e o f f o u r a c e t a t e s i n l e n g t h and t h e r e b y a r e d i r e c t l y i n c o r p o r a t e d 16 i n t o o r s e l l i n i c a c i d 0 F i n a l l y B a s s e t t and Tanenbaum 1? and L y n e n 1 8 showed t h a t a c e t y l coenzyme A i s t h e most d i r e c t c o n d e n s i n g agent and i s I n c o r p o r a t e d i n t o 6 - m e t h y l s a l i c y l i c a c i d w i t h o u t f i r s t r e v e r t i n g t o f r e e a c e t a t e * F o l l o w i n g t h e e l e g a n t work on f a t t y a c i d b i o s y n t h e s i s by L y n e n 1 ^ W a k i l 2 0 and B r a d y 2 1 w h i c h had e s t a b l i s h e d - 96 -m a l o n y l coenzyme A as a b i o s y n t h e t i e p r e c u r s o r of the f a t t y a c i d s , B e n t l e y and K e i l 2 2 and Bu^Lock and h i s c o l l e a g u e s 2 ^ , a lmost s i m u l t a n e o u s l y r e p o r t e d the i n c o r -p o r a t i o n o f malonic a c i d i n t o p e n i c i l l i c a c i d (13) and 6 - m e t h y l s a l i c y l l c a c i d . I t was suggested t h a t as i n the f a t t y a c i d s malonyl-coenzyme A i s the a c t u a l u n i t i n v o l v e d i n c h a i n bui ld ing\u00E2\u0080\u009E F u r t h e r , l a c k o f s i g n i f i c a n t amounts o f l a b e l i n the methyl group of p e n i c i l l i c a c i d 2 2 demons-t r a t e d t h a t malonate i s not r e c o n v e r t e d t o a c e t a t e t o any a p p r e c i a b l e ex tent and so I s a v a l i d d i r e c t i n t e r m e d i a t e , I t i s assumed t h a t as I n the f a t t y a c i d case m a l o n y l coenzyme A i s formed by c a r b o x y l a t i o n of a c e t y l coenzyme A 2 ^ ; t h i s same c a r b o x y l b e i n g l o s t d u r i n g the c h a i n b u i l d i n g s t e p s , No a t tempts have succeeded i n showing i n c o r p o r a t i o n o f a p p r o p r i a t e l y l a b e l l e d f o u r carbon p r e c u r s o r s such as b u t y r a t e or a c e t \u00C2\u00A9 a c e t a t e . Thus B i r k e n s h a w 2 ^ found t h a t (1 - ^ * 0 ) sodium b u t y r a t e , f e d t o P e n i c i l l i u m m a d r i t i n produced o r s e l l i n i c a c i d w h i c h was l a b e l l e d . The c a r b o x y l group had one q u a r t e r o f the t o t a l a c t i v i t y , t h u s showing t h a t b u t y r a t e had s p l i t i n t o two C ^ . u n i t s be fore i n c o r p o r -a t i o n . T h i s p r e c l u d e s u t i l i z a t i o n of t h e i n t a c t c h a i n . S i m i l a r r e s u l t s were r e p o r t e d f o r a c e t o a c e t i c a c i d 2 ^ , 1$ Lynen has p r o v i d e d f u r t h e r i n f o r m a t i o n f o r the - 97 -F i g u r e 3 = ~ 98 -mechanism o f b i o s y n t h e s i s o f 6 - m e t h y l s a l i e y l i c a c i d by p r e p a r i n g c e l l f r e e e x t r a c t s f r o m t h e mycelium o f P e n i c i l l i u m p a t u l u m and i n c u b a t i n g i t i n t h e p r e s e n c e o f ( l - ^ G ) a c e t y l coenzyme A and m a l o n y l coenzyme A, I t was found t h a t t h e p r e s e n c e o f m a l o n y l coenzyme A was e s s e n t i a l f o r 6 - m e t h y l s a l i e y l i c a c i d p r o d u c t i o n and t h a t ; i t s p r o d u c t i o n was a l s o TPNH dependent , When TPNH was e x c l u d e d f r o m the* medium, n e i t h e r 6 = m e t h y l s a l l c y l i c j > n or o r s e l l i n i c a c i d were produced by t h i s c e l l f r e e e x t r a c t . T h i s was e x p l a i n e d by t h e s u g g e s t i o n t h a t o n l y ( 1 5 ) a c t e d a s a p r e c u r s o r i n t h e f o r m a t i o n o f a r o m a t i c compounds i n t h i s p a r t i c u l a r mould. F i g u r e 3 i l l u s t r a t e s t h e f o r m a t i o n o f 6 - m e t h y l s a l i c y l i c a c i d by t h i s enzyme p r e p a r a t i o n , Lynen a l s o f o u n d ! t h a t i o d o a c e t a m i d e (a s u l f h y d r y l b l o c k i n g a g e n t ) p r e v e n t e d t h e f o r m a t i o n o f 6 - m e t h y l s a l i c y l i c a c i d , i n d i c a t i n g s u l f h y d r y l g roups of the enzyme system were i n v o l v e d i n t h e b i o s y n t h e s i s . On t h e b a s i s o f t h e above f i n d i n g s Lynen proposed t h a t t h e b u i l d i n g up o f 6 - m e t h y l s a l i c y l i c a c i d t a k e s p l a c e on t h e s u r f a c e o f one enzyme complex and t h a t t h e i n t e r m e d -i a t e p o l y k e t o a c i d s a r e bound t o t h e s u l f h y d r y l g r o u p s o f t h e enzyme, 1 99 -DISCUSSION The e x p e r i m e n t a l evidence so f a r a v a i l a b l e (v ide supra) s t r o n g l y i n d i c a t e s the v a l i d i t y of the p o s t u l a t e d p o l y k e t o -a c i d i n t e r m e d i a t e S o The a c t u a l involvment of the l a t t e r , however, has not as y e t been showno Thus , i t seems d e s i r a b l e t o embark on experiments des igned t o i n v e s t i g a t e the nature o f the i n t e r m e d i a r y s teps i n the a c e t a t e pathway to p h e n o l i c compoundSo A p o s s i b l e i n t e r m e d i a t e suggested i t s e l f i n the course o f work r e c e n t l y c a r r i e d out i n our own l a b o r a t o r i e s on \" p o l y p y r o n e s \" , such as b i s - a n d t r i s p y r o n e (16) and (17) r e s p e c t i v e l y o I t was found these compounds on t reatment w i t h base , r e s u l t e d i n such n a t u r a l l y o c c u r r i n g compounds as o r s e l l i n i c a c i d (13) and 4 - m e t h y l - 6 , 3 - d i h y d r o x y i s o c o u m a r i n (19) r e s p e c -26 t i v e l y o I n s p e c t i o n of the s t r u c t u r e s o f p o l y p y r o n e s (20) r e v e a l s t h a t they are b a s i c a l l y formed f rom a molecule o f t r i a c e t i c a c i d l a c t o n e ( r i n g A) w i t h s u c c e s s i v e u n i t s of malonic a c i d ( r i n g B , C , D , e t c 0 ) 0 Base h y d r o l y s i s of the l a c t o n e r i n g s l e a d s t o B - p o l y k e t o a c i d c h a i n s which spontan-e o u s l y e y c l i s e t o aromat ic s u s t r a t e s as shown i n f i g u r e 4\u00C2\u00BB The ease o f the t r a n s f o r m a t i o n of these pyrones to aromat ic compounds, and the s t a b i l i t y t h a t these l a c t o n e 1Q0 r i n g s p r o v i d e f o r otherwise h i g h l y l a b i l e polyketo-raethylene c h a i n s , suggested a p o s s i b i l i t y t h a t pyrones, or a t l e a s t the monopyrone t r i a c e t i c a c i d l a c t o n e (21), which i s b u i l t up of t h r e e a c e t i c a c i d u n i t s , are a l s o i n v o l v e d i n n a t u r a l processes., The coenzyme e s t e r of the open c h a i n form of (21) can be e n v i s i o n e d to have been c o n s t r u c t e d i n v i v o from a s t a r t e r a c e t y l coenzyme A and s u c c e s s i v e a d d i t i o n s of two u n i t s of malonyl coenzyme A, f o l l o w e d by d e c a r b o x y l a t i o n , a s shown i n f i g u r e 5\u00C2\u00B0 (17) ( 1 9 ) The ease of h y d r o l y s i s o f the l a c t o n e r i n g s i n v i t r o suggested t h a t an e q u i l i b r i u m c o u l d e x i s t between the open c h a i n a c i d and t h e l a c t o n e . In f a c t , i t has been r e p o r t e d t h a t i n the case o f s t y r y l t r i a c e t i c a c i d l a c t o n e (22), base - 101 O H b xco,H (21) ^0 (20) C ^ Q Aromatic s u b s t r a t e s F i g u r e 4; CH3-COC0A 2 >COSCoA CO, L H 3 Cok H ( 2 1 ) F i g u r e 5, - 1 0 2 -treatment f o l l o w e d by c a r e f u l a c i d i f i c a t i o n r e s u l t e d i n 27 the i s o l a t i o n o f the f r e e a c i d ( 2 3 ) <, T r i a c e t i c a c i d l a c t o n e , t h e r e f o r e , seemed t o p r o v i d e a s t a b l e u n i t , r e q u i r i n g the a d d i t i o n o f o n l y one more u n i t o f malonyl coenzyme A to form simple p h e n o l i c compounds. O (22). ( 2 3 ) The occurrence of monopyrone s t r u c t u r e s i n nature s t r e n g t h e n s the p o s s i b i l i t y of such compounds as intermed-i a t e s , i n the a c e t a t e pathway t o n a t u r a l l y o c c u r r i n g p h e n o l i c compounds, Tanenbaum e t , a l , haye found 3 - m e t h y l - 2 , 4 - d i k e t o -3 , 4 dihydro-6-methylpyrone ( 2 4 ) i n P 0 s t i p i t a t u m which c l e a r l y i s a c e t a t e d e r i v e d . C l o s e l y r e l a t e d i s o p u n t i o l ( 2 5 ) , found i n Qpuntia e l a t o r , R a d i c i n i n (26), i s o l a t e d from Stemphy- 1 ium 1 r ad i c i n ium-^ \u00C2\u00AE 3 has a s t r u c t u r e r e sembling b i s p y r one, a l t h o u g h bispyrone i s probably not i t s p r e c u r s o r . A l s o , such compounds as methoxy p a r a c o t o i n ( 2 7 ) and the s t y r y l p y r o n e s 32 (2B) are examples o f the cases where i t i s assumed t h a t enzyme a c t i v i t y has stopped short o f the necessary numbers of C 2 u n i t s t o form aromatic substances. I t would be i n t e r -- 103 -e s t i n g t o t e s t the p r e c u r s o r a c t i v i t i e s of the l a t t e r two compounds by f e e d i n g s u i t a b l y l a b e l l e d (27) or (28) t o enzyme systems capable of producing s t y r y l aromatic compounds l i k e the f l a v a n o i d s . o OH (25) (26) R R-R (27) (28) Durin g a search f o r r e p o r t e d t e s t s f o r p r e c u r s o r a c t i v -i t y by t r i a c e t i c a c i d l a c t o n e , i t was d i s c o v e r e d t h a t 33 Ehrensvard , while s t u d y i n g the e f f e c t s of v a r i o u s s u b s t r a t e s oh the p r o d u c t i o n of aromatic substances by P n patulum f l had found t h a t a d d i t i o n o f 0=2-5=0 mg of t r i a c e t i c a c i d l a c t o n e (21) enhanced the accumulation o f g e n t i s y l a l c o h o l (29), which i s an i n t e r m e d i a r y m e t a b o l i t e i n the p r o d u c t i o n of p a t u l i n (11) i n the same mould. On t h e other hand, the - 104 a c e t y l d e r i v a t i v e ; d e h y d r o a c e t i c a c i d (30), i n h i b i t e d the f o r m a t i o n o f . g e n t i s y l a l c o h o l a Although i t was subsequently found t h a t the a s s a y used i n h i s work i s perhaps at f a u l t , the r e p o r t r e p r e s e n t e d an important c l u e to the p o s s i b l e involvement of t h i s l a c t o n e i n the p r o d u c t i o n o f a'cetate d e r i v e d p h e n o l i c compoundso A l s o i n t e r e s t i n g i s Tanenbaum's o b s e r v a t i o n , d u r i n g r the i s o l a t i o n of m e t h y l t r i a c e t i c a c i d l a c t o n e ( 2 4 ) , t h a t while t h i s compound c o u l d be i s o l a t e d i n small q u a n t i t i e s (ca, 6 m g / l i t e r ) from 4 day o l d c u l t u r e s of P 0 s t i p i t a t u m , s t i p i t a t i c a c i d ( 3 D could not be d e t e c t e d u n t i l the seventh day. (29) (30) ( 3 D In view of the f a c t t h a t no i n t e r m e d i a t e s i n the postu-l a t e d b i o g e n e s i s of the a c e t a t e pathway have been r e c o g n i z e d , and t h a t no \" p r i m i t i v e \" p o l y k e t o compounds ( I t was i s o l a t e d by chromato-graphy on s i l i c a gel -Go I t was a l i q u i d h a v i n g an u l t r a -v i o l e t spectrum a t 275 mu w i t h a 13 mu r e d s h i f t on a d d i t i o n \u00C2\u00A9f base ( suggest ive of p h e n o l s ) . I t s n.oior, showed a m u l t i -p l e t a t 2o7-3?3 T ( 5 H\u00C2\u00BBs) and a s i n g l e t a t 7\u00C2\u00AB75 T(3 H ' s ) . On a d d i t i o n o f deuter ium oxide t o the n . m . r . s o l u t i o n , the aromat ic r e g i o n showed the disappearance of one p r o t o n . I t s i n f r a r e d spectrum showed i n t e r m o l e c u l a r hydrogen bonding at 3300 cm\"*** and bands c h a r a c t e r i s t i c of aromat ic systems. Comparison o f t h i s m a t e r i a l w i t h o r t h o - , rneta-, and p a r a -c r e s o l showed i t had i d e n t i c a l p h y s i c a l c h a r a c t e r i s t i c s ( I R , NMR and Rf ) t o t h a t of m e t a - c r e s o l (36). (35) (36) A t h i r d compound was i s o l a t e d once i n s m a l l q u a n t i t i e s f rom mould grown on t r i a c e t i c a c i d l a c t p n e - c o n t a i n i n g medium. C r y s t a l l i s e d f r o m e t h a n o l , i t melted a t 286-2 .87\u00C2\u00B0 . In the u l t r a v i o l e t i t showed a b s o r p t i o n s of 325 mu ( \u00C2\u00A3 = 18.500) and 240 mu (16,000) w i t h a 5 myi b l u e s h i f t on a d d i t i o n of ba se . I t showed a non-hydrogen bonded h y d r o x y l band at 3360 c m \" 1 - 1 1 2 -and two c a r b o n y l f u n c t i o n s a t 1690 and I665 cm i n the , i n f r a r e d . The mass spectrum i n d i c a t e d a m o l e c u l a r weight of 336. I t showed,as the o n l y c h a r a c t e r ! s a b l e fragment \u00E2\u0080\u009E the l o s s o f a h y d r o x y l group from the m o l e c u l a r i o n . As t h i s compound was i s o l a t e d i n s m a l l amounts and o n l y once, i t s f o r m a t i o n i s not n e c e s s a r i l y due t o t r i a c e t i c a c i d l a c t o n e . No f u r t h e r i n f o r m a t i o n on t h i s compound i s a v a i l a b l e o The presence of m - c r e s o l , a l t h o u g h p r e v i o u s l y not r e p o r t e d , i s not s u r p r i s i n g , as i t i s most p r o b a b l y formed from 6 - m e t h y l s a l i c y l i c a c i d by d e c a r b o x y l a t i o n . R e c e n t l y , Gaucher-^ 8 has been a b l e t o i s o l a t e , from P. patulum. m-hydroxy-b e n z y l a l c o h o l (37) which i s simply a h y d r o x y l a t e d m - c r e s o l . A l l attempts to d e t e c t m-cresol i n t r i a c e t i c a c i d l a c t o n e -f r e e b r o t h r e v e a l e d no t r a c e of t h i s substance. The r o l e of t r i a c e t i c a c i d l a c t o n e i n i t s p r o d u c t i o n i s not o b v i o u s . The f o r m a t i o n of methylene-bis ( 3 , 3 ' - t r i a c e t i c a c i d l a c t o n e ) i s s i m i l a r t o t h a t of dicoumarol (38)^, p h l o r o -pyrone (39)^\"\u00C2\u00B0* and phlorospyrone (40)^, as w e l l as a l a r g e OH (37) - 113 -number o f o ther f e r n p r o d u c t s of which F l a v a s p i d i c a c i d ( 4 1 i s a t y p i c a l example. (39) (41) I t was i n t e r e s t i n g to n o t e , however, t h a t when t r i -a c e t i c a c i d l a c t o n e was added to P . patulum B a i n i e r 1MI ?98Q9 (C-554)\u00C2\u00BB a s t r a i n r e p o r t e d t o produce m a i n l y g r i s e o -f u l v i n ^ , none of t h i s formaldehyde condensat ion product nor any o t h e r new m e t a b o l i t e c o u l d be d e t e c t e d i n the f e r -menta t ion p r o d u c t s . T h i s i s p r o b a b l y due t o the absence of a p a r t i c u l a r - d o n a t i n g agent i n t h i s p a r t i c u l a r s t r a i n o f P . p a t u l u m . The r e s u l t of the r a d i o a c t i v i t y d i l u t i o n e x p e r i m e n t s , and the i s o l a t i o n of these new m e t a b o l i t e s , were not too encouraging as i t was l i k e l y t h a t the p r e v i o u s chromato-114 -g r a p h i c assay methods had g i v e n m i s l e a d i n g i n f o r m a t i o n ~ about the e x t e n t , i f any, of enhancement of 6 - m e t h y l s a l i -c y l i c a c i d and t o l u q u i n o l f o r m a t i o n due to t r i a c e t i c a c i d lactone\u00E2\u0080\u009E T a k i n g i n t o account the o b s e r v a t i o n of Tanenbaum with r e g a r d t o the f o r m a t i o n of m e t h y l - t r i a c e t i c a c i d l a c -tone (24) i n Po s t i p i t a t u m (vide supra) and our own obser-v a t i o n of the pyrones t e s t e d , namely t h a t t r i a c e t i c a c i d l a c t o n e was the o n l y one showing no n e g a t i v e e f f e c t on m e t a b o l i t e growth, we f e l t j u s t i f i e d i n c o n t i n u i n g the i n v e s t i g a t i o n of the s i n g u l a r r o l e of t h i s l a c t o n e u s i n g r a d i o a c t i v e t r a c e r t e c h n i q u e s with l a b e l l e d t r i a c e t i c a c i d l a c t o n e , T r i a c e t i c a c i d l a c t o n e (3*5-\"'\"^ 'C) was prepared from s i m i l a r l y l a b e l l e d d e h y d r o a c e t i c a c i d ^ which had been obt a i n e d by s e l f - c o n d e n s a t i o n of ( 2 - ^ C ) e t h y l a c e t o a c e t a t e OEt O 1 9 2 V et (42) (30) ( 2 1 ) - 115 -T h i s l a b e l l e d l a c t o n e (40 mg, 1.5 x 10 c/m) was i n t r o d u c e d i n t o a 7 day o l d r e p l a c e d c u l t u r e of P\u00C2\u00BB\u00E2\u0080\u00A2 patulum (C-945)\u00C2\u00BB A f t e r i n c u b a t i o n f o r 72 h o u r s , the b r o t h was c o l l e c t e d and e x t r a c t e d w i t h e t h e r , f o l l o w e d by the separ-a t i o n of the b i c a r b o n a t e - s o l u b l e So From the n e u t r a l e ther e x t r a c t a crop of g r i s e o f u l v i n (1.5 mg) and of p a t u l i n (11,0 mg) was o b t a i n e d . They showed, r e s p e c t i v e l y , s p e c i -f i c a c t i v i t i e s of 160 c/ra/mg and 145 c/m/mg (ca 0 .1$ i n c o r -p o r a t i o n i n t o p a t u l i n ) . T h i s i s a very low i n c o r p o r a t i o n . Tanenbaum\"**' had r e p o r t e d a 29$ i n c o r p o r a t i o n of r a d i o -a c t i v i t y d u r i n g the c o n v e r s i o n of l a b e l l e d 6 - m e t h y l s a l i c y l i c a c i d t o p a t u l i n by P . p a t u l u m . Comparison of t h i s h i g h degree of c o n v e r s i o n w i t h t h a t o f 1$ a c e t i c a c i d 1 - C ^ 4 i n c o r p o r a t i o n i n t o g r i s e o f u l v i n by P . patulum t and 0 .2$ i n t o f l a v a p i n (43 A s p e r g i l l u s f l a v i p e s , i n d i c a t e s t h a t i n c o r p o r a t i o n of u n i t s r e q u i r e d f o r p o l y k e t o a c i d c h a i n f o r -mat ion i s c o n s i d e r a b l y l e s s than t h a t f o r aromat ic s u b s t r a t e s a l r e a d y f o r m e d . O H (43) ~ 116 -To d i f f e r e n t i a t e between low i n c o r p o r a t i o n of l a b e l l e d t r i a c e t i c a c i d l a c t o n e , or breakdown of the l a c t o n e and random i n c o r p o r a t i o n i n t o aromatic m e t a b o l i t e s , i t would be necessary t o degrade the molecule o f e i t h e r p a t u l i n or 6 - m e t h y l s a l i c y l i c a c i d and l o c a t e the p o s i t i o n s o f the l a b e l . T h i s procedure would be extremely d i f f i c u l t on the s m a l l amounts of these m e t a b o l i t e s a v a i l a b l e . However, on i n s p e c t i o n o f the s t r u c t u r e of g r i s e o f u l v i n ( 9 ) , i t became apparent t h a t , should t r i a c e t i c a c i d l a c t o n e have been i n c o r p o r a t e d d i r e c t l y i n t o t h i s molecule, on l y r i n g A would be l a b e l l e d ( f i g u r e 6,, heavy l i n e s ) , w h i le r i n g B should remain u n l a b e l l e d . Thus, the o n l y d e g r a d a t i o n r e q u i r e d would be cleavage o f r i n g s A and B by f u s i o n w i t h sodium hydroxide, ? a known p r o c e d u r e 0 . CI CH 3 F i g u r e 6, For t h i s purpose a g r i s e o f u l v i n - p r o d u c i n g mould was used. T h i s mould was a l s o u s e f u l f o r another reason. T r i -a c e t i c a c i d l a c t o n e , a l t h o u g h a u n i t , might n e v e r t h e l e s s p r e s e n t o b s t a c l e s f o r the C-4 r e d u c t i o n i m p l i e d by Lynen\"s 117 -w o r k 1 8 a t the stage of the b i o s y n t h e s i s of 6 - m e t h y l s a l i -c y l i c a c i d (vide s u p r a ) , T h i s u n c e r t a i n t y about the exact sequence of the r e d u c t i o n s c h a i n e x t e n s i o n steps i s a v oided i n the b i o s y n t h e s i s of g r i s e o f u l v i n , s i n c e i n t h i s molecule the C-4 h y d r o x y l i s r e t a i n e d , Po patulum B a i n i e r (G-554* a g r i s e o f u l v i n - p r o d u c i n g s t r a i n - ^ ) was grown on Czapek-Dox, and t h i s medium was r e p l a c e d a f t e r t e n days of growth, A t o t a l of 80 tag (3\u00C2\u00BB0 x 10^ c/m) of l a b e l l e d t r i a c e t i c a c i d l a c t o n e was added. A f t e r 7 days of i n c u b a t i o n * 15 mg o f g r i s e o f u l v i n was i s o l a t e d from the b r o t h and m y c e l i a l e x t r a c t . T h i s sample showed a s p e c i f i c a c t i v i t y o f 421 c/m/mg (0,2% i n c o r p o r a t i o n ) . U s i n g a m o d i f i -8 c a t i o n o f the method developed by B i r c h e t 0 a l , , g r i s e o -f u l v i n was fused w i t h potassium hydroxide a t 225\u00C2\u00B0 t o g i v e 3,5 mg of o r c i n o l (44* 78%) and 3,0 mg of 3-chloro - 2-hydroxy-4 * 6-dimethoxybenzoic a c i d (45* 35%)* w i t h s p e c i f i c a c t i v i -t i e s o f 385 c/m/mg and 402 c/m/mg r e s p e c t i v e l y . The o r c i n o l sample, a f t e r s u b l i m a t i o n , showed an a c t i v i t y of 375 c/m/mg. E s s e n t i a l l y a l l the r a d i o a c t i v i t y of g r i s e o f u l v i n i s accoun-t e d f o r i n the two fragments. I f a s i g n i f i c a n t q u a n t i t y o f r a d i o a c t i v e l a c t o n e had been i n c o r p o r a t e d i n t o g r i s e o f u l v i n , one would have expected a g r e a t e r f r a c t i o n o f the r a d i o a c t i -v i t y t o appear i n the o r c i n o l fragment. The r e s u l t s o b t a i n e d c l e a r l y show t h a t the l a c t o n e , a s a u n i t , had not been i n c o r -p o rated i n t o g r i s e o f u l v i n , but t h a t the r a d i o a c t i v i t y i n t h i s - 1 1 8 -molecule p o s s i b l y r e s u l t s from breakdown of, t h e l a c t o n e and i n c o r p o r a t i o n of the l a b e l l e d fragments,. T h i s seems t o support the p r e v i o u s o b s e r v a t i o n 2 ^ o f the f a i l u r e of the enzyme system t o u t i l i z e p r e c u r s o r s other than the small a c e t i c - a n d malonic a c i d m o lecules i n the c h a i n b u i l d i n g process,. The l a c k of i n c o r p o r a t i o n of t r i a c e t i c a c i d l a c t o n e i n t o g r i s e o f u l v i n i s not n e c e s s a r i l y due t o the enzyme-bound cora-1 8 p l e x t h a t has been proposed by Lynen ; t h i s would precl u d e acceptance by the enzyme s u r f a c e o f any i n t e r m e d i a t e p o l y k e t o -methylene chains,, The observed e f f e c t may a l t e r n a t i v e l y be e x p l a i n e d i n the f o l l o w i n g ways As d i s c u s s e d i n the o r i g i n a l p o s t u l a t e about the p o s s i b l e involvement of t h i s l a c t o n e i n the a c e t a t e pathway (vide s u p r a ) a the a c t u a l p r e c u r s o r e n v i s i o n e d was not t o be the l a c t o n e a s such, but the l a c t o n e d e r i v e d t r i a c e t i c a c i d as i t s coenzyme d e r i v a t i v e , . Thus, any d i f f i c u l t y encoun-t e r e d by the enzyme system t o e i t h e r open the l a c t o n e , \u00C2\u00A9r form t h e coenzyme e s t e r of the r e s u l t a n t a c i d , c o u l d l e a d t o the above r e s u l t s . Having e s t a b l i s h e d t h a t t r i a c e t i c a c i d l a c t o n e does not a c t a s a p r e c u r s o r i n the f o r m a t i o n o f g r i s e o f u l v i n and pre-sumably of other aromatic n a t u r a l p r o d u c t s , i t was decided - 1 1 9 -t o r e i n v e s t i g a t e the enhancement e f f e c t s t h a t t h i s l a c t o n e might have on m e t a b o l i t e f o r m a t i o n i n the o r i g i n a l mould, For t h i s p u r p o s e , the known a n t i b i o t i c a c t i v i t y of p a t u -l i n a g a i n s t S taphylococcus a u r e o u s ^ was t o be u t i l i s e d , Seven day o l d r e p l a c e d c u l t u r e s of P . patulum (554) were t e s t e d d a i l y by the \u00E2\u0080\u00A2papeE - d i s e * m e t h o d ^ f o r r e l a t i v e a n t i -b i o s i s , , The r e s u l t s are summarized by f i g u r e 7, The graphs show t h a t , i n i t i a l l y , t r i a c e t i c a c i d l a c t o n e has no marked e f f e c t on the a n t i b i o t i c a c t i v i t y o f the sam-p l e s . However, 4 days a f t e r t h e a d d i t i o n o f the l a c t o n e , there i s a s i g n i f i c a n t i n c r e a s e i n the d i a m e t e r s of the zones o f i n h i b i t i o n . T h i s e f f e c t i s exhausted a f t e r about 7 d a y s . A f t e r t h i s p e r i o d a n t i b i o s i s of the l a c t o n e c o n t a i n i n g samples d e c r e a s e s , w h i l e t h e r e i s a sharp i n c r e a s e f o r the c o n t r o l e x p e r i m e n t s . At t h i s stage the b r o t h o f the r e s p e c -t i v e samples was e x t r a c t e d w i t h e q u a l volumes o f e t h e r , and the d r y w e i g h t s of the t o t a l m e t a b o l i t e s produced were d e t e r -mined. These agreed w i t h the r e s u l t s o b t a i n e d by b i b - a s s a y . I n p r e v i o u s exper iments i t had been observed t h a t Czapek-Dox s o l u t i o n s prepared w i t h one l o t of reagent grade magnesium s u l f a t e t u r n e d t o a y e l l o w - b r o w n c o l o u r a f t e r s t e r i l i z a t i o n i n the a u t o c l a v e . I t was a l s o observed t h a t a medium prepared w i t h t h i s l o t o f magnesium s u l f a t e d i d not - 120 -1 2 3 4 5 6 7 8 9 10 11 Days a f t e r t r i a c e t i c a c i d lac tone had been added F i g u r e 7 . (Czapek-Dox s o l u t i o n 1*) C o n t r o l \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 Lactone added a \u00C2\u00AE \u00C2\u00AE Days a f t e r t r i a c e t i c acid lactone had been added F i g u r e 8, (Czapek-Dox s o l u t i o n 2 . ) - 1 2 1 -support mould growth as w e l l as the u s u a l reagent* When a c c i d e n t a l l y , the above b i o - a s s a y experiments were repeated on c u l t u r e s r e p l a c e d w i t h the l a t t e r medium, the l a c t o n e showed the s t r i k i n g e f f e c t shown i n f i g u r e 8, I t i s seen t h a t w h i l e samples c o n t a i n i n g t r i a c e t i c a c i d l a c t o n e , i n g e n e r a l have a constant a n t i b i o s i s , i n the absence of the l a c t o n e there i s a d e f i n i t e decrease i n a c t i v i t y . As above, the dry weights of the t o t a l meta-b o l i t e s produced were determined, and were i n good agree-ment w i t h the b i o - a s s a y r e s u l t s . These r e s u l t s are p u z z l i n g s i n c e , a s mentioned e a r l i e r , no t r i a c e t i c a c i d l a c t o n e c o u l d be d e t e c t e d i n the fermen-t a t i o n b r o t h 4 days a f t e r i t s a d d i t i o n . T h i s would i n d i -cate t h a t the observed enhancement i s not due to continued presence o f the l a c t o n e . R e t u r n i n g t o the be g i n n i n g of our experiments and t o Ehrensvard's r e s u l t s , i t i s i n t e r e s t i n g to note t h a t , while i n our work r e p l a c e d c u l t u r e s were used, Ehrensvard had added the l a c t o n e t o the growing mould and had observed the enhancement a t an e a r l i e r stage a f t e r t he a d d i t i o n . From t h i s one can speculate t h a t t r i a c e t i c a c i d l a c t o n e a c t s i n some way.-on the enzyme system of the growing mould. When the already-grown mould i s being used the e f f e c t i s not 122 -obvious u n t i l new mycelium b e g i n s t o form. T h i s e f f e c t becomes even more apparent when a medium i s used which seems not t o support mycelium and m e t a b o l i t e p r o d u c t i o n a f t e r a c e r t a i n p e r i o d . More experiments are necessary t o determine the r o l e of t r i a c e t i c a c i d on the p h y s i o l o g y of t h i s mould and a l s o the r e a s o n s f o r the i n t e r e s t i n g r e s u l t s o b t a i n e d w i t h the s p e c i a l l o t of magnesium s u l f a t e . Such'experiments are i n p r o g r e s s a t t h i s time. EXPERIMENTAL M e l t i n g p o i n t s were taken on a K o f l e r b l o c k , u l t r a -v i o l e t a b s o r p t i o n s p e c t r a were measured on a Cary 11 spectro photometer f o r s o l u t i o n s i n e t h a n o l , u n l e s s otherwise mentioned; i n f r a r e d s p e c t r a were measured with a P e r k i n Elmer 21 spectrophotometer and n,m,r, s p e c t r a were taken on a V a r i a n A 60 model f o r s o l u t i o n s i n deuterochloroform<, A n a l y s e s were c a r r i e d out by A, Bernhardt i n Germany, A l l t h i n l a y e r chromatograms were r u n u s i n g Woeim S i l i c a - g e l - G , For r a d i o a c t i v i t y measurements an end window Geiger-M u e l l e r counter (Nuclear Chicago) was used, E t h y l a c e t a t e s o l u t i o n s of the samples (0,1-0,2 mg) were p l a t e d i n t r i p l i -c a t e on p l a n e h e t s . The counts per minute were determined by r e c o r d i n g a t l e a s t 1000 count\u00C2\u00AE. Hence the s t a t i s t i c a l c o u n t i n g e r r o r i s no g r e a t e r than 10$; the counting r a t e s were c o r r e c t e d f o r background, S u b c u l t u r e s o f P e n i c i l l i u m patulum B a i n e r IMI-34909 (G945) and P e n i c i l l i u m patulum B a i n i e r IMI-39SQ9 (C554) Thorn 46402455 were k i n d l y s u p p l i e d by Glaxo L a b o r a t o r i e s , B r i s t o l , England, These were grown on Gsapek-Dox n u t r i e n t medium ( g l u c o s e , 40gm; sodium n i t r a t e , 3 gm; d i p o t a s s i u m phosphate, 1 gm; magnesium s u l f a t e heptahydrate, 0=5 gm; Potassium c h l o r i d e , 0,5 gm; f e r r o u s s u l f a t e , 0,01 gm; water, 1 l i t e r ) as s u r f a c e c u l t u r e s . The medium was d i s t r i b u t e d i n 1 l i t e r erlenmeyer f l a s k s (200 m l / f l a s k ) which had been - 124 -s t e r i l i z e d i n an \"American 1 8 s t e r i l i z e r a t 118\u00C2\u00B0 f o r 15 minutes 0 A f t e r i n o c u l a t i o n they were incubated at room temperature (23-25\u00C2\u00B0)\u00C2\u00B0 The mycelium u s u a l l y covered the s u r f a c e 2-3 -days a f t e r i n o c u l a t i o n . Unless-otherwise mentioned P, patulum G-945 was used i n these experiments. I s o l a t i o n of P a t u l i n and 6 - M e t h y l s a l i c y l i c A c i d In a p r e l i m i n a r y survey 4 l i t e r s o f the f e r m e n t a t i o n b r o t h o f a 10 day o l d c u l t u r e was separated from the mycelium by d e c a n t a t i o n and a c i d i f i e d t o pH 2 w i t h concen-t r a t e d s u l f u r i c a c i d . I t was then e x t r a c t e d w i t h e t h e r (3 x 1,5 l i t e r s ) , The e t h e r e x t r a c t c o n c e n t r a t e d t o 1/20 of i t s volume and e x t r a c t e d w i t h satiarated aqueous sodium b i c a r b o n a t e (5 x 20 m l ) . The b i c a r b o n a t e e x t r a c t s were a c i d i f i e d w i t h 6 N h y d r o c h l o r i c a c i d and the rest<ant p r e c i p i t a t e c o l l e c t e d (1,1 gm) and r e c r y s t a l l i s e d from hot water, mop, 167 - 1 6 8 \u00C2\u00B0 , ' T h i s compound moved as a s i n g l e spot on TLC Rf 0,7 u s i n g 20% a c e t i c a o i d i n c h l o r o f o r m a s s o l v e n t s and had the u l t r a v i o l e t spectrum d e s c r i b e d \" f o r 6-methyl-s a l i c y l i c a c i d ; The n e u t r a l e t h e r e x t r a c t was d r i e d over anhydrous sodium sulfate,,and c o n c e n t r a t e d i n volume..to, 1/3 and allowed t o evaporate slowly^ I t d e p o s i t e d 1,3 gm o f p r i s m shaped c r y s t a l s map, 110-112\u00C2\u00B0, A f t e r r e t r y s t a l l ! s a t i o n from ch l o r o f o r m 125 i t moved as a s i n g l e spot (Rf 0,4) using the above s o l v e n t system. E f f e c t of T r i a c e t i c A c i d Lactone ( 2 1 ) and Dehydroacetic A c i d (30.) on .Pen i c i l l imm patulum. The mould was grown f o r 7 days a f t e r which the fermen-t a t i o n b r o t h o f 9 f l a s k s ( s e l e c t e d f o r apparent equal growth of mycelium) were decanted t a k i n g care not t o break up the mycelium pads, The l a t t e r was washed w i t h s t e r i l e water (3 x 100 ml) and f r e s h Czapek-Dox s o l u t i o n was added. The f l a s k s were d i v i d e d i n groups of t h r e e . To one group was added t r i a c e t i c a e i d - l a e t o n e (3 x 100 mg i n 25 ml of w a t e r ) . To the second was added d e h y d r o a c e t i c a c i d (3 x 50 mg i n 25 ml o f w a t e r ) , The t h i r d group was made up t o e q u a l volume w i t h the o t h e r s by the a d d i t i o n \u00C2\u00A9f 25 ml \u00C2\u00A9f water t o each f l a s k and regarded a s b l a n k . A l i q u o t s (5 ml) were withdrawn with a s t e r i l e p i p e t t e , a t f i r s t every how, t h e n at every 5 hour i n t e r v a l f o r a t o t a l o f 72 hours. These a l i q i a e t s were e x t r a o t e d w i t h e t h e r ( 3 x 3 m l ) , d r i e d over anhydrous sodium s u l f a t e (1,0 gm), f i l t e r e d and the s o l v e n t removed by e v a p o r a t i o n . The r e s i d u e d i s s o l v e d i n 10 drops o f chl\u00C2\u00A9rof\u00C2\u00A9rm=methanol (1.1), 20 X of t h i s s o l u t i o n was t r a n s f e r r e d t\u00C2\u00A9 t h i n l a y e r p l a t e s (20 x 20 cm). The p l a t e s were developed w i t h c h l o r o f o r m - a c e t i c a c i d - 126 -( 4 s l ) and sprayed w i t h i o d i n e or eerie s u l f a t e reagent , D u p l i c a t e t e s t s were done. Feeding o f U - ^ C ) Sodium Malonate Seven day o l d m y c e l i a l pads (4 flasks) were washed wi t h s t e r i l e water (3 x 100 ml) and replaced with Gzapek-Dox medium, ( 2 - ^ 0 ) Sodium malonate (0,04 mc) was d i s t r i b u t e d among the f l a s k s . To two f l a s k s was added t r i a c e t i c a c i d l a c t o n e (40 mg). A f t e r 3 days the f e r m e n t a t i o n b r o t h was sep a r a t e d s The my c e l i a were washed w i t h water and the washings combined w i t h t h e f e r m e n t a t i o n b r o t h . The t r i a c e t i c a c i d l a c t o n e - c o n t a i n i n g f l a s k s and those without the l a c t o n e were combined i n two sets.(A) and (B) r e s p e c t i v e l y . Each set was e x t r a c t e d w i t h e t h e r (4 x 150 ml). An a l i q u o t (10 ml) o f each set was removed and d r i e d over sodium s u l f a t e . S p e c i f i c a c t i v i t i e s o f the respective samples were found, (A): 1,01 x 10^ c/m/mg; (B)s 2,08 x 10^ c/m/mg. The remaining e t h e r extracts were extracted w i t h s a t u r a t e d aqueous sodium bicarbonate, solution (4 x 15 ml), The b i c a r b o n a t e e x t r a c t s combined and a c i d i f i e d with 6N h y d r o c h l o r i c a c i d . The a c i d i f i e d solation was re-extracted w i t h e t h e r (3 x 50 ml). Both the a e i d i c and the n e u t r a l ' e x t r a c t s were d r i e d over sodium.sulfate, - 127 (A) ,., n e u t r a l e x t r a c t '{103 mg). s p e c i f i c a c t i v i t y , 1,8 x 10 3 e/m/mg a c i d i c extract-(63 mg) s p e c i f i c a c t i v i t y 4\u00C2\u00B05 x 10-* c/m/mg (B) n e u t r a l e x t r a c t (50 mg) s p e c i f i c a c t i v i t y 2o9 x 10 3 c/m/mg a c i d i c extract (33 mg) s p e c i f i c a c t i v i t y 5o3 x 1\u00C2\u00A93 e/m/mg The crude n e u t r a l e x t r a c t s were dissolved i n ether (3 ml) and' slowl y evaporated to y i e l d 15=0 and 15 \u00C2\u00B0 5 mg, .for 1(A) and f o r (B) r e s p e c t i v e l y a, \u00C2\u00A9f s\u00C2\u00AElld patraliac, The s p e c i f i c a c t i v i t i e s f o r these- samples were -6,0$ x. I \u00C2\u00A9 3 c/m/mg arid 6 , 0 2 d 1 0 ^ Q/ss/mg, ..'respectively. A f t e r twice rec-rystall'\u00C2\u00B1sati0n from chloroform, both samples showed one spot, R f 0 , 4 \u00C2\u00A9a T L G (etham\u00C2\u00A9l-chl\u00C2\u00A9r@form 5$); s p e c i f i c a c t i v i t y 6,03 x 1 0 3 and 5 , 9 5 x 1Q3 c/m/mg r e s p e c t i v e l y . The a c i d i c extracts were chroraat\u00C2\u00A9graphed \u00C2\u00A9a t h i c k layer s i l i c a - g e l 6 plates- (acetic a@id-c.hl\u00C2\u00A9.r\u00C2\u00A9f@rm,. 1 , 5 ) o The spots c o r r e s p o n d i n g t\u00C2\u00A9 6 - m e t h y l s a l i c y l i c acid were elated from, the s i l i c a t o y i e l d 16 and'14 mg for sets ( A ) and ( B ) respectively, The s p e c i f i c . a c t i v i t i e s found f\u00C2\u00A9r these samples were 4 0 8 0 e/m/mg and 3270 c/m/mg .respectively, . Careful t r i t u r a t i o n of set (A)', w i t h etfoylacetate afforded a small amount' \u00C2\u00A9f s o l i d . T h i s s o l i d , m 0p 0 252-254\u00C2\u00B0p shewed a g p e e i f i e a c t i v i t y of 380 c/m/mgo The mother' liqm@r of the above t r i t u r a t i o n a s w e l l a s the e l u t e d m a t e r i a l from set-B were d i l a t e d w i t h u n l a b l e d 6 - m e t h y i s a l i c y l i c a e i d (31 mg) aad r e c r y s t a l l i s e d from water t o constant radiosLetlYityo S p e c i f i c a c t i v i t y a f t e r the t h i r d r e c r y s t a l l i s a t i o n was 395\u00C2\u00A9 c/m/mg and 3980 f o r set (A) and (B) respectively\u00E2\u0080\u009E I s o l a t i o n o f New Metab\u00C2\u00A9li tes , The f e r m e n t a t i o n broth of 50 f l a s k s (10 liters) p t o which t r i a c e t i c a c i d l a c t o n e (50 x 40 mg) had been added i n the usual'mannerp was a c i d i f i e d t\u00C2\u00A9 p H 20 arad e x t r a c t e d w i t h e t h e r ( 3 x 4 l i t e r s ) o The e t h e r e x t r a c t was co n c e n t r a t e d t\u00C2\u00A9 1/30 \u00C2\u00A9f i t s voliame and e x t r a c t e d w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e ( 5 x 5 \u00C2\u00A9 m i ) 0 On a c i d i f i c a t i o n \u00C2\u00A9f the a l k a l i n e e x t r a c t w i t h esE&cenfcrated 'hydr\u00C2\u00A7> TLC showed one spot at Rf Oo75<> Later f r a c t i o n s from the column were analysed by TLG and shewed only traces \u00C2\u00A9f other new metabolites when compared with mould products without added t r i a e e t i c - a c i d lactone,, -The neutral ether layer was dried over anhydrous sodium sulfate and concentrated by slow evaporation to give successive cr\u00C2\u00A9ps of g r i s e o f u l v i n followed by patulira 0 The remaining solution upon evaporation gave 2 1 0 mg of a brown \u00C2\u00A9il which showed a new spot at Rf 0\u00C2\u00BB5 (solvents chloroform-acetic acid 9 s l K This o i l was chr\u00C2\u00A9mat\u00C2\u00A9graphed on a.column \u00C2\u00A9f s i l i c a - g e l G (30 gm) using ethanol-chloroform (.12 20) as eluento 20 ml f r a c t i o n s were collectedo F r a c t i o n 4 (18 mg)...gave mainly 'one spot on TLC Rf 0o5 (selventg a c e t i c acid-chloroform ls9)\u00C2\u00B0 It was designated as metabolite B Q \"Metabolite A8\" This compound was r e c r y s t a l l i s e d from ethanol t\u00C2\u00A9 give white prisms m 0p 0 254-255\u00C2\u00B0\u00C2\u00B0 It, showed ^m&x 28$ mp. ('X5j>0\u00C2\u00AE0)| - 1 3 0 X max (0.1N NaOH/EtOH)s 290 mu (16,000), IR(KBr). 3250, 2760, 2660, 1680, 1615, 1580, 1355* 1140, 980, 860, 825, 785, 765p 755 cm\" 1 NMR(at 60\u00C2\u00AE), 3.90 ( s i n g l e t ) ; 6.45 ( s i n g l e t ) ; 7\u00C2\u00B085 ( s i n g l e t ) T (Areas l s l s 3 ) (poor spectrum), MolcWt, by mass spectrum? 264 a,m.u, A n a l o c a l c , f o r C13Hx206= 0,59.09; H,4.58. Founds C,59.39 H,4\u00C2\u00BB63, M e t h y l a t i o n o f \" M e t a b o l i t e A\" \" M e t a b o l i t e A ' R ( 3 2 mg) was suspended i n 1 ml of methanol, ...... . . . . i and 2 ml o f e t h e r e a l diaaomethahe (prepared from p - t o l u s u l f o n -amide-N-methyl-N-hitroso) was added, A c l e a r s o l u t i o n was obta i n e d a f t e r a short time. T h i s s o l u t i o n was allowed t o stand a t room temperature f o r 2 hours. The solvent was then evaporated- t o g i v e a colourless semisolid which was t r i t u r a t e d w i t h a l i t t l e e t h e r and the' s o l i d f i l t e r e d and washed with small amounts of'; e t h e r . Y i e l d , 22 mg, m,p, 135=160\u00C2\u00B0. TLC showed one spot R f . 0.5 :(solvents acetic acid-chloroform l s 9 ) ( M e t a b o l i t e A., Rf 0,7) \u00C2\u00B0 Using ethanol-chloroform' (ls9) t h e r e were t h r e e s p o t s a t R f s 0 , 2 , 0,5 and 0,7, The Rf 0.7 compound was i s o l a t e d on a t h i c k - l a y e r ( L O m) chromatography plate t o g i v e a white s o l i d which c r y s t a l l i s e d from ether, m,p, 211-212\u00C2\u00AE, A max''303 mp (21,000); no base s h i f t , - 131 -I R (KBr)s 3120, 2940, 1705* 1645* 1555* 1350* 1250* 1135* 1025, 855* 320, 780 c n T 1 NMR8 4<>0 ( s i n g l e t ) ; 6 0 18 ( s i n g l e t ) ; 6<>42 ( s i n g l e t ) 7\u00C2\u00B080 ( s i n g l e t ) T (Area) 1 : 3 : 1 : 3 ) \u00C2\u00BB A n a l o c a l c o f o r C-j^ Q^s G,6l\u00E2\u0080\u009E64; H,5o52; Found C,62.03, H,5o69o P r e p a r a t i o n of Methylene-BIs (3 To an ethai a o l i c s o l u t i o n of t r i a c e t i c a c i d l a c t o n e form-aldehyde, was added and allowed t o stand a t room temperature \u00E2\u0080\u009E C o l o u r l e s s n e e d l e s separated a f t e r s e v e r a l hours. These c r y s t a l l i s e d from e t h a n o l m.p0 254-255\u00C2\u00B0, mixed w i t h \" m e t a b o l i t e Aro, m 0p 0 254-255\u00C2\u00B0\u00C2\u00B0 I t s i n f r a r e d spectrum was superimposable on t h a t o f A o \" M e t a b o l i t e B m T h i s compound was f u r t h e r p u r i f i e d on TLC to. g i v e a l i q u i d (12 mg)o I t showed Xmax;275 mm (1 ,900) ; Xmax ( O o l N NaOH/EtOH)g 288 mu (2,400)\u00C2\u00B0 N M R g 2.7-33 ( m u l t i p l e t ) ; 7\u00C2\u00B075 ( s i n g l e t ) . % ( A r e a s 5 \u00C2\u00B0 3 h Added deuterium oxides 2o7=3\u00C2\u00B03 ( m u l t i p l e t ) ; ; 7o75 ( s i n g l e t ) T (Areas 4 s 3 ) \u00C2\u00B0 I R ( l i q u i d f i l m ) : 3300 * 2900, 1600, 1490, 1260, 1120,, 920, 770 cnf^-o 132 -I s o l a t i o n of \" M e t a b o l i t e C\" The s o l i d , o b t a i n e d from the a c i d i f i c a t i o n of the b i c a r b o n a t e e x t r a c t from one u s u a l set o f 25 f l a s k s of mould growth t o which t r i a c e t i c a c i d l a c t o n e had been added, was washed w i t h e t h a n o l to remove 6 - m e t h y l s a l i c y l i c aeido The e t h a n o l i n s o l u b l e w h i t e s o l i d (16 mg was d i s s o l v e d i n 80 ml o f b o i l i n g ethanolo Upon c o o l i n g a crop of c o l o u r l e s s needles (6 mg) was o b t a i n e d , m.p. 286-287\u00C2\u00B0. T h i s compound was d e s i g n a t e d as \" m e t a b o l i t e C \" . The mother l i q u o r upon c o n c e n t r a t i o n y i e l d e d \" m e t a b o l i t e A \" (9 rag). \" M e t a b o l i t e G\" had A max 325, 24\u00C2\u00A9 mf* (18,000 and 16,500) Xmax (OolN NaOM/EtOH) 320, 223 mu (18,500 and 19,200) I R ( K B r k 3 3 6 0 , 3 0 5 0 ^ 2 8 8 0 , 1 6 9 0 , 1 6 6 5 , 1 6 1 0 , 1 5 3 5 * 1420, 1320, 900, 800, 765, 755* 705, cm\" 3 - . MoloWto by mass spectrum? 336 a o B t o i o P r e p a r a t i o n o f (3o5\u00C2\u00B0\u00C2\u00B01^G) Dehydroacet i c A c i d (30)^5 (2-l^KJ) Ethylacet\u00C2\u00A9acetate (181.5 mg; 0.5 me) was d i l u t e d t o 10.0 gm w i t h f r e s h l y v a c u u m - d i s t i l l e d a c e t o a c e t l c a c i d e s t e r . Of t h i s e s t e r 9.0 gm was t r a n s f e r r e d t o the r e a c t i o n f l a s k and heated f o r 9 hours i n an o i l b a t h a t 1 9 5 \u00C2\u00B0 . The temperature i n s i d e the r e a c t i o n f l a s k reached 1 9 2 \u00C2\u00B0 . The product was d i s t i l l e d t o remove unreac ted s t a r t i n g m a t e r i a l (2.8 gm). The product was then subl imed t o g i v e 0 .43 gm of - 1 3 3 -a white s o l i c L T h i s s o l i d was r e c r y s t a l U s e d from 1,2 ml o f e t h a n o l . Y i e l d , 380 mg (10%) m,p , 1 2 6 - 1 2 8 \u00C2\u00B0 showed one spot on TLG ( a c e t i c a c i d - c h l o r o f o r m 1:9) R f . 0 , 8 , S p e c i f i c a c t i v i t y 37*300 c/m/mg, P r e p a r a t i o n of ( 3 C 5 - 1 ^ G ) T r i a c e t i c A c i d Lactone (2.1)44 To 1#0 mg of l a b e l l e d d eh yd r o ace t i c a c i d was added 0,6 ml of 90% s u l f u r i c a c i d under n i t r o g e n . The r e a c t i o n tube was immersed i n an o i l b a t h a t 136\u00C2\u00B0 f o r 5 m i n u t e s . I t was then r a p i d l y cooled i n i c e and about 2 gm of i c e added. The product was kept a t 0 \u00C2\u00B0 f o r 4 hours then f i l t e r e d and washed w i t h i c e water (2 m l ) . Y i e l d 120 mg (88%), m,p , 1 8 8 - 1 8 9 \u00C2\u00B0 , S p e c i f i c a c t i v i t y a f t e r two r e c r y s t a l l i s a t i o n s from e t h a n o l , 37OO0 c/m/mg. Feeding of ( 3 , 5 - 1 2 f C ) T r i a c e t i c A c i d Lactone t o P e n i c i l l i u m patulum B a i n i e r (C 945) Seven day o l d m y c e l i a l pad (grown i n a Roux f l a s k ) was washed w i t h s t e r i l e water (3 x 100 ml) and r e p l a c e d w i t h Caapek-Dox medium, ( 3 * 5 - ^ 0 ) T r i a c e t i c a c i d l a c t o n e (40 mg) was added and the mould incubated f o r 4 d a y s . The fermen-t a t i o n b r o t h was s e p a r a t e d , the mycelium washed w i t h water and the washings combined w i t h the f e r m e n t a t i o n b r o t h . The b r o t h was e x t r a c t e d w i t h e ther (5 x 70 m l ) . The e ther e x t r a c t s 134 had a s p e c i f i c a c t i v i t y of 2.34 x 10-* c/m/mg\u00C2\u00BB The e t h e r e x t r a c t was f u r t h e r e x t r a c t e d w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e (4 x 15 m l ) . The combined b i c a r b o n a t e e x t r a c t s were a c i d i f i e d w i t h 6N h y d r o c h l o r i c a c i d and e x t r a c t e d w i t h e ther (4 x 20 m l ) . Both the a c i d i c and n e u t r a l e ther e x t r a c t s were d r i e d over sodium s u l f a t e and the e ther s t r i p p e d . n e u t r a l e x t r a c t (60 mg) s p e c i f i c a c t i v i t y : I860 c/m/mg, a c i d i c e x t r a c t (34 mg) s p e c i f i c a c t i v i t y : 2860 c/m/mg. The n e u t r a l f r a c t i o n was d i s s o l v e d i n e ther (1 ml) and a l l o w e d t o evaporate s l o w l y . A crop of c o l o u r l e s s p r i s m s separated (1.5 mg). m . p . 2 1 9 - 2 2 0 \u00C2\u00B0 , mixed w i t h a u t h e n t i c g r i s e o f u l v i n m . p . 2 1 9 - 2 2 0 \u00C2\u00B0 , s p e c i f i c a c t i v i t y 160 c/m/mg. The e ther s o l u t i o n upon f u r t h e r e v a p o r a t i o n y i e l d e d a second crop of pr ism-shaped c r y s t a l s , m . p . 1 1 0 - 1 1 2 \u00C2\u00B0 (11 mg). Re-c r y s t a l l i s e d from c h l o r o f o r m m.p . ; 1 1 1 - 1 1 2 \u00C2\u00B0 , s p e c i f i c a c t i v i t y 145 c/m/mg, showing (0.1$) i n c o r p o r a t i o n i n t o p a t u l i n . Feeding of ( 3 . 5 - ^ 0 ) T r i a c e t i c A c i d Lactone t o P e n i c i l i u m Patulum B a i n i e r (C-554) P e n i c i l l i u m Patulum B a i n i e r (G-554) was grown i n a Roux f l a s k under s i m i l a r c o n d i t i o n s as used f o r the growth of (G-945)\u00C2\u00BB P r e l i m i n a r y experiments had shown t h a t l i t t l e g r i s e o f u l v i n was produced u n t i l a f t e r about 15 d a y s , - 13$ -p r o d u c t i o n then i n c r e a s i n g c o n s i d e r a b l y * A c c o r d i n g l y , a 3 week o l d m y c e l i a l pad was washed w i t h s t e r i l e water (3 x 100 ml) and r e p l a c e d w i t h f r e s h Czapek-Dox medium (3\u00C2\u00AB 5 - l i f C ) T r i a c e t i c a c i d l a c t o n e (80 mg) was added. A f t e r a f u r t h e r 7 days growth the mycelium was d r i e d a t 50\u00C2\u00B0 under vacuum, f i n e l y powdered i n a Waring b l e n d e r and e x t r a c t e d i n a soxhle t apparatus f o r 3 days w i t h l i g h t pet ro leum e ther ( b . p . 30-60\u00C2\u00B0). The e x t r a c t conta ined l i t t l e g r i s e o -f u l v i n and was d i s c a r d e d . The mycelium was then f u r t h e r e x t r a c t e d w i t h c h l o r o f o r m f o r 2 d a y s . The c h l o r o f o r m was s t r i p p e d t o g i v e 150 mg o f an o i l . T h i s o i l was e x t r a c t e d w i t h b o i l i n g benzene (15 m l ) . ' The benzene s o l u t i o n on c o o l i n g d e p o s i t e d a c r y s t a l l i n e s o l i d (70 mg), m . p . 168-170\u00C2\u00B0 o 6 (mycelianamlde m . p . 168-170 ) . The mother l i q u o r on slow e v a p o r a t i o n y i e l d e d success ive c rops o f g r i s e o f u l v i n (12 mg), m . p . 219-220\u00C2\u00B0, s p e c i f i c a c t i v i t y 421 c/m/mg\u00E2\u0080\u009E. A f u r t h e r crop of 3 mg of g r i s e o f u l v i n was o b t a i n e d by e x t r a c t i o n of the f e r m e n t a t i o n b r o t h w i t h e ther and slow e v a p o r a t i o n of the e t h e r \u00E2\u0080\u009E A f t e r r e c r y s t a l l i s a t i o n from e t h a n o l i t showed s p e c i f i c a c t i v i t y of 418 c/m/mgo A l k a l i F i s s i o n o f G r i s e o f u l v i n ^ -G r i s e o f u l v i n (13 mg), potass ium hydrox ide \u00E2\u0080\u009E(1 p e l l e t ) and water 2 drops were heated a t 225\u00C2\u00B0 f o r 2 h o u r s . The 136 r e s i d u e on c o o l i n g was d i s s o l v e d i n water (0.5 ml) and a c i d i f i e d w i t h d i l u t e h y d r o c h l o r i c a c i d and e x t r a c t e d w i t h e ther (4 x 5 m l ) . The e ther e x t r a c t was d r i e d over sodium s u l f a t e and the e ther e v a p o r a t e d . The r e s i d u e (9 mg) was chromotographed on a t h i c k - l a y e r s i l i c a g e l - Q p l a t e (Ool x 20 x 20 cm). O r c i n o l (3 .5 rag, R f 0.4) m . p . 1 0 5 - 1 0 7 \u00C2\u00B0 and 3-chloro-2-hydroxy-4\u00C2\u00BB6-demethoxy benzoic a c i d (3 mg R f 0.7) m*,p. 2 2 0 - 2 2 1 \u00C2\u00B0 ( s o l v e n t , e t h a n o l - c h l o r o f o r m 1:?0) were o b t a i n e d by e l u t i o n from the s i l i c a , showing s p e c i f i c a c t i v i t i e s o f 385 c/m/mg and 402 c/m/mg r e s p e c t i v e l y . The o r c i n o l sample-was s u b l i m e d ; i t showed a s p e c i f i c a c t i v i t y of 375 c/m/mg, 49 P r e p a r a t i o n of 4 - C h l o r o - 4 - D e o x y t r i a c e t i c A c i d Lactone (34)-In a 50 ml round bottomed f l a s k w i t h an a t t a c h e d r e f l u x condenser and c a l c i u m c h l o r i d e tube was d i s s o l v e d 2 .0 gm of t r i a c e t i c a c i d l a c t o n e i n 15 ml of p h o s p h o r y l c h l o r i d e . The s o l u t i o n was l e f t o v e r n i g h t a t room temperature . I t was then warmed on steam b a t h f o r 3h hours c o o l e d and 100 gm of i c e was added. I t was then e x t r a c t e d w i t h e ther (7 x 50 m l ) . The e ther e x t r a c t s were washed w i t h s a t u r a t e d aqueous sodium b i c a r b o n a t e (5 x 50 m l ) , d r i e d over sodium s u l f a t e and the e ther s t r i p p e d . The r e s i d u e (1.2 gm) was subl imed a t atmos-p h e r i c pressure on a steam b a t h t o g i v e whi te p l a t e s , m.p . - 137 -8 4 - 8 5 \u00C2\u00B0 (0 .8 gm, 4 0 $ ) 0 4 9 P r e p a r a t i o n o f 4 - D e o x y t r i a c e t i c A c i d Lactone (28) The c h l o r o l a c t o n e (30, 0 .7 gm) was d i s s o l v e d i n 15 ml of e t h a n o l , and 3 gm of z i n & powder t o g e t h e r w i t h 7 ml of concent ra ted h y d r o c h l o r i c a c i d were addedo The mixture was r e f l u x e d f o r 2 h o u r s , c o o l e d , and then the p r e c i p i t a t e was washed w i t h hot e t h a n o l . The e t h a n o l washings were combined w i t h the main body of the s o l u t i o n and evaporated t o g i v e a y e l l o w o i l . I t showed on TLC 3 s p o t s , one a t R f 0 .4 ( c h l o r o -form) i n major amounts. T h i s o i l was p u r i f i e d by.VPC u s i n g a s i l i c o n e column at 1 2 0 \u00C2\u00B0 . The m a t e r i a l a t 10 minutes r e t e n t i o n t ime was c o l l e c t e d . NMR 2 .8 ( q u a r t e t ) ; 4 . 0 (doublet ) ;-s 4 .17 ( d o u b l e t ) ; 7\u00C2\u00BB8 ( s i n g l e t ) T ( A r e a , 1 : 1 : 1 : 3 ) . E f f e c t of Feeding of 4 - D e o x y t r i a c e t i c A c i d Lactone (32) . 4 - C h l o r Q - 4 - D e o x y t r i a o e t i c A c i d Lactone (34)\u00E2\u0080\u009E and Methoxy- t r i a c e t i c Afeld Lactone (33 ) . The mould was grown a s u s u a l . To the r e p l a c e d medium i n 6 f l a s k s , i n groups o f 2 , were added (2 x 40 mg) o f each 4 - d e o x y t r i a c e t i c a c i d l a c t o n e , 4 - c h l \u00C2\u00A9 r o - - 4 - d e o x y t r i a c e > i c a c i d l a c t o n e , and m e t h o x y t r i a c e t i c a c i d l a c t o n e . The f e r m e n t a t i o n b r o t h a f t e r 4 days of i n c u b a t i o n was e x t r a c t e d by u s u a l procedure and t e s t e d by t h i n l a y e r chromatography comparing w i t h a p p r o p r i a t e b l a n k s . - 1 3 8 -E f f e c t of T r i a c e t i c A c i d Lactone on P . patulum (as measured by b i o - a s s a y ) 0 The mould was grown as usual\u00E2\u0080\u009E To the r e p l a c e d medium i n 10 f l a s k s was added t r i a c e t i c a c i d l a c t o n e (10 x 40 mg), These were t e s t e d d a i l y f o r a n t i b i o t i c a c t i v i t y a g a i n s t S t a p h y l o c o c c u s a u r e o u s 4 7 by the p a p e r - d i s c m e t h o d 4 8 as f o l l o w s : Twenty ml o f s t e r i l e assay agar were added t o P e t r i disheSo A f t e r the agar had hardened 4 ml o f c o o l e d agar seeded w i t h S a aureous were d i s t r i b u t e d evenly over the s u r f a c e 0 F i l t e r paper d i s c s ( S c l e i c h e r and S c h n e l l , 740E, \u00C2\u00A3 inch) were p l a c e d f l a t s ide down on the seeded a g a r . An 0o080 ml sample of the s o l u t i o n was p i p e t t e d immediate ly ( w i t h i n 5 s e c ) onto each d i s c as i t was p l a c e d on the agar\u00C2\u00BB The paper d i s c was pressed t o the agar w i t h the t i p of the pipette<, The d i s h e s were incubated at 37\u00C2\u00B0 f o r about 24 h o u r s 0 The d iameters of the zones of i n h i b i t i o n were then measured t o the neares t one q u a r t e r mm. - 139 -REFERENCES l o L . R u z i c k a , E x p e r i e n t i a % 357 (1953) <> 2 . W.Bo O l l i s ( e d . ) , \"The Chemistry of N a t u r a l P h e n o l i c Compounds\", Pergamon, New York (1961) . 3. J 0 N 0 C o l l i e , Jo Chemo Soc. 21, 1806 (1907) . 4 . A 0 J 0 B i r c h and F 0 N 0 Donovan, ' A u s t r a l . J . Chem. 6 , 360 (1953)o 5. A 0 J 0 B i r c h . F o r t s c h r . Org. N a t u r s t o f f e , 1^ , 186 (1957) . 6 . R. Robinson, \"The S t r u c t u r a l R e l a t i o n s of N a t u r a l P r o ducts* Claredon P r e s s , Oxford (1955). 7. A . J . B i r c h , R.A. Mas'sy-Westropp and C 0 J 0 Moye, A u s t r a l . J \u00E2\u0080\u009E Chem. 8, 539 (1955\"). 8. A . J o B i r c h , RoA. Massy-Westropp, R.W. R i c k a r d s and H . Smith, J . Chem. Spc. 1958 , 360. 9 . Ro Thomas, Proc. Chem. Soc. 1959. 88 . 10o A 0 J 0 B i r c h , J o F i S n e l l and P . L . Thompson, J . Chem. Soc. 1962. 425= 1 1 . S.Wo Tanenbaum and E..W. B a s s e t t , J . B i o l . Chem. 234. 1861 (1959) . 1 2 . K 0 Mosbach, Acta Chem. Scand. 1/j., 457 (1960) o 1 3 . L . D o F e r r i t t i , J . H o R i c h a r d s Proc. N a t l . Acad. S e i . (U.SoAo) i t6 , 14 ( I 9 6 0 ) . 14 . A . J o B i r c h , Proc. Chem. S o c 0 1962 n 3 . 1 5 . A . J . B i r c h and H . Smith, Chem 0 Soc. Spec. P u b l . 195$\u00C2\u00BB 160 S. Gatenbeck and K. Mosbach, A c t a . Chem. Scand. 13, I56I (1959). 17. E.W. B a s s e t t and S.Wo Tanenbaum, Biochem. Biophys. A c t a . , itOp 535 (I960). 180 F . Lynen and M. Tada, Angew. Ghem. 21a 513 (196l) c \u00C2\u00BB 140 \u00C2\u00AB= 1 9\u00C2\u00BB F 0 Lynen, J . C e l l , Gompo Physiol. J ^ , Sup, 1 , 3 3 ( 1 9 5 9 ) . 2 0 . S o J o Wakil, J , Am. Chem. Soc* | 0 , 6 4 6 5 ( 1 9 5 8 ) 0 2 1 . R.O. Brady, P r o c N a t l . Acad, S c i 0 ( U . S . A . ) , 9 9 3 ( 1 9 5 8 ) . 2 2 o Re Bently and J.G. K e i l * Proc, Chem, S o c , 1 9 6 1 , 1 1 1 , 2 3 . J e D 0 Bu\u00C2\u00BBLock and H.M. Smalley, i b i d , 1 9 6 1 . \" 2 0 9 \u00C2\u00B0 2 4 \u00C2\u00BB M, Waite and S.-J. Wakil, J , B i o l o Chem, 2 3 8 . 81 - ( 1 9 6 3 ) \u00E2\u0080\u00A2 2 5 . J . H . Birkenshaw and A* Gowlland, J a Biochem. 3 4 , 3 4 2 ( 1 9 6 2 ) . 2 6 o T\u00C2\u00BB Money, A.I. Scott, I\u00C2\u00BB Qurashi and GoB.R, Webster 3 J o Am. Chem, Soc. ^ 2 , 3 0 0 4 ( 1 9 6 5 ) \u00C2\u00AB 2 7 . R.L. Edwards, D.G. Lewis and D.V. Wilson, J\u00C2\u00BB Chem, S o c * 1 2 6 1 9 4 9 9 5 . 2 d . S.W, Tanenbaumj T.R. Acker and P\u00C2\u00ABE, Brenneisen, J , Am, Chem., Soc. 6 6 , 1 2 6 5 ( 1 9 6 4 ) \u00C2\u00AB 2 9 o A.K. Ganguly, T.R. Govindachari and P . A . Mohamed, Tetrahydron Zl9 9 3 ( 1 9 6 5 ) \u00C2\u00BB 3 0 . J.E. Grove, J . Chem. S o c , ( 1 9 6 4 ) \u00C2\u00BB 3 2 3 4o 3 1 o G. Ciamacian and P. S i l b e r , Ber. 2 \u00C2\u00A3 , 8 4 1 * ( 1 8 9 4 ) . 3 2 . P.M. Dean, \"Naturally Occurring Oxygen Ring Compounds'*, Butterworths, London, 1 9 6 3 , p . 1 0 0 . 3 3 e Go Ehrensvard, Exp. C o l l . Res. Suppl. 2, 1 0 2 ( 1 9 5 5 ) \u00E2\u0080\u00A2 3 4 . A\u00C2\u00BB Rhodes, Private Communication. , 3 5 . R.S. Sigg i a , nThe Determination of Organic Compounds0, Wiley and Co., New York, N.Y., 1949; P\u00C2\u00AB 8 0 . 3 6 . S\u00E2\u0080\u009EW\u00C2\u00AB Tanenbaum and E.W. Bassett, Biochem, Biophys. Acta., 2 d , 2 1 ( 1 9 5 8 ) . 3 7 \u00C2\u00BB Diekmann and F. Breest, Ber. 2Z, 3 3 9 1 ( 1 9 0 4 ) . 3 0 , G.ML. Gaucher* Private 1 Goramunicat ioni . - 141 -39. M . A . Stahmann, C F . Huebner, a n d . K . P . L i n k , J\u00C2\u00BB B i ' o l . Chem. , 138, 513 (1941). .... >\u00E2\u0080\u00A2\u00E2\u0080\u009E,.... . s, .=. \u00E2\u0080\u00A2.. i \u00E2\u0080\u00A2 4 0 4 A . P e n t i l l a e and J * Sundman, A c t a . Chem. S c a n d , , 12, 1 8 8 8 X 1 9 6 3 ) . 41 o A . P e n t i l l a e and Jo Sundman, i b i d , 1 \u00C2\u00A3 , 839 (.1961). 42. A , McGookin, A . Robertson and T . H . Simpson, J . Chem. S o c , 1953, 1828. 43. J . N * C o l l i e , i b i d , 607 (.1891). 44\u00C2\u00B0 E , 8 C, H o r n i n g , Organic S y n t h e s i s , C o l l e c t i v e V o l , I I I , John W i l e y & Sons l n c , New Y o r k , N . Y . , 1955, p . 231. 45. S6W\u00E2\u0080\u009E Tanenbaum and Ei.W. B a s s e t t , J . B i o l \u00C2\u00BB Chem*, 234s 1861 (1959). 46, G . P e t t e r s s o n , A c t a . Chem. S c a n d . , 12, 35 (1965)= 47= M . I , T i m o n i n , S c i e n t i f i c A g r i c u l t u r e , 26;8, 358 (1946). 48, L , F c Johnson, EL.A. C u r l , J . H . Bond and H . A . F r i b o u r g , \"Methods f o r s t u d y i n g S o i l M i c r o f l o u r a - P l a n t Disease R e l a t i o n s ' * , .Burgess P u b l i s h i n g C o . , M i n n e a p o l i s , M i n n , , I960, p. 71. 49. M.J.D. Van Dam and F. K o g l , Rec. Trav. Cndem., 83, 46 (1964). A P P E N D I X Crystallizes from the Stobbe reaction product Fig. 1.\u00E2\u0080\u0094Synthesis of (\u00C2\u00B1)-dedimethylamino-6,12a-dideoxy-decarboxamido-7-chlorotetracycline (101, 102, 1(32) Reagents: (1), CICOOEt/N-methylmorpholine/benzene; (2), (EtOOC\u00E2\u0080\u0094CH\u00E2\u0080\u0094COOEt) M g + + (OEt); (3), aq. H,S(VAcOH/heat ; (4), diethyl succinate/NaH; (5), H 2 / N i ; (6), 1 equiv. C l j / C C U ; (7), polyphosphoric acid; (8), ethylene glycol; (9), L i A l H 4 ; (10), methanesulfonyl chloride/pyridine; (11), KCN/diniethylformamide/water; (12), LiAl(OEt)jH; (13), diethyl malonate/AcOH/piperidine; (14), Na + (CH 3 CO\u00E2\u0080\u0094CH\u00E2\u0080\u0094COOEt)/ether/reflux; (15), aq. H C l ; (16), NaH/anisole. H. M u x f e l d t , B e r . 92, 3122 (1959). M e O 0 M e O 0 (from Stage 7, F ig . l ) M e O O H M e O O M e CI C H 3 CI C H 3 C H , O H M e O O M e CI C H 3 C O O H M e O O M e O M e O O M e 0 CI C H 3 IS C O O B u 1 C O O B u 1 JL, C O O E t C O O B u 1 C O O B u 1 8,9 M e O O M e O CI ? H 3 C O O H 16 M e O O M e O H 0 |J 17,18,19 ^ C O N H , CI C H 3 H riVn M e O O H 0 O (and 12a-epimer) to C O N H 2 : i C H 3 O H O H 0 0 O H C O N H 2 Fig. 2.\u00E2\u0080\u0094Synthesis of ( \u00C2\u00B1 ) - d e d i m e t h y l a m i n o - 1 2 a - d e o x y - 5 a , 6 - a n h y d r o - 7 - c h l o r o t e t r a c y c l i n e (101) and its conversion into ( \u00C2\u00B1 ) - d e d i m e t h y I a m i n o - 5 a , 6 - a n h y d r o - 7 - c h I o r o t e t r a c y c I i n e (103, 162). Reagents: (I), Br 2/eth_er/500-wattlarnp; (2), N a O H / M e O H ; (3), C H 2 N 2 ; (4), LiAlH\u00C2\u00AB; (5), P B r , ; (6), N a + ( E t O O C \u00E2\u0080\u0094 C H j \u00E2\u0080\u0094 C ( C O O B u ' ) j ) ; (7), polyphosphoric acid; (8), dil . aq. N a O H ; (9), diethyl phthalate/170\"; _(10)t P C U or oxalyl chloride; (11), C H 2 N 2 ; (12), benzyl alcohol/180\"; (13), P C U ; (14) M g + + ( E t O O C \u00E2\u0080\u0094 C H \u00E2\u0080\u0094 C O O E t ) , ; (15), NaH/anisole ; (16) N H , / N a O M e \u00E2\u0080\u0094 M e O H ; (17), H C l / A c O H ; (18), C H 2 N , ; (19). P h C O , H / C H C l , ; (20), H C l / A c O H . H. M u x f e l d t j B e r . 92, 3122 (1959). CHaBr CI CI A ^ Y C O O E T K^J COOEt ~* OMe CN CN \u00E2\u0080\u00A2 \u00C2\u00AB.r JN ^V^ 0 0 0 \" f ^ V ^ * V ^ y ^ C O N H a MeO 0 COOH it,is, COOMe ts.ie COOMe \" BzO O \"OH (B^benzyl) CI *0 17,18, t 9>*\u00C2\u00B0 BzO COOEt COOEt MeO \u00C2\u00AB, \u00C2\u00AB. <4, OBz OMe O H O O H O H O H 0 ll Fig. 3.\u00E2\u0080\u0094Synthesis of (\u00C2\u00B1)-dedimethylamino-6-demethyl-12a-deoxy-5a,6-anhydro-tetracycline (104, 105, 135,151). Reagents: (1), N-bromosucctnimide/peroxide; (2), N a + ( E t O O C \u00E2\u0080\u0094 C H \u00E2\u0080\u0094 C O O E t ) ; (3), LiAlH\u00C2\u00AB; (4), methane sulfonyl chloride; (5), C N - ; (6), O H ~ ; (7), polyphosphoric acid; (8), oxalyl chloride; (9), Rosenmund reduction (5% Pd-BaSO\u00C2\u00AB-); (10), cyanoacetamide/piperidine; (11), coned. H C l / A c O H ; (12), benzyl chloride/boiling alkali; (13), MeOH/H 2SO<; (24), NaH/toluene; (25), Br , /NaOAc; (26), collidine (dehydrobromination); (27), Me 2 S04/K 2 C0 3 ; (18), mild alkaline hydrolysis; (29), ethyl chloro-formate/NEt,; (20), M g + + (OEt) (EtOOC\u00E2\u0080\u0094CH\u00E2\u0080\u0094COOEt) ; (21), NaH/toluene; (22), H,/10% P d - C ; (23), H C O O - N H . V 1 4 0 \u00C2\u00B0 ; (24), boiling coned. HCI/AcOH, ' J . H. Boothe, A. S. Kende, T. L. F i e l d s and R. G. W i l k i n s o n , J . Am. Chem. Soc. 81, 1006 (1959) . CI CI H H r g y C O O H _ ^ ^f^J^y^OOMe C O O H y ] f X O O H OBz O OBz O H H + (from Stage 12,Fig.3) C 1 H H OOH + O B z O O H (syn) O O H OOMe <5 O B z O H H OBz 0 O H 8yn-acid (separated by crystallization from ethanol) 4,5 C O O E t O B z O O H ^ Q E t 7,8,9 O B z O O H 0 O H O O H O Fig. 4.\u00E2\u0080\u0094Synthesis of (\u00C2\u00B1)-dedimethylamino-6-demethyl-6,12a-dideoxy-7-chlorotetracycline (106, .135). Reagents: (I), Ac 2 0/reflux/l hr.; (2), N a O M e / M e O H ; (3), NaH/toluene; (4), C I C O O E t / N E t , ; (5) M g + + ( E t O O C \u00E2\u0080\u0094 C H \u00E2\u0080\u0094 C O O E t 2 ) ; (6), NaH/toluene; (7), H s /10% P d - C ; ( "Thesis/Dissertation"@en . "10.14288/1.0062191"@en . "eng"@en . "Chemistry"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Some aspects of natural products chemistry"@en . "Text"@en . "http://hdl.handle.net/2429/37686"@en .