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UBC Theses and Dissertations

Some aspects of natural products chemistry Yalpani, Mohamed 1965

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The U n i v e r s i t y  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  o f Washington, 1961  WEDNESDAY, SEPTEMBER 8, 1965, a t 10:00 A.M. IN ROOM 261, CHEMISTRY BUILDING  COMMITTEE IN CHARGE Chairman: I . McT, Cowan L. Bo Hayward J . P. Kutney F. McCapra  External  T. Money R. E, P i n c o c k R. Stewart G. H. N. Towers  Examiner:  P r o f e s s o r W. A. Ayer  Department  of Chemistry  University  of A l b e r t a  Edmonton, A l b e r t a  SOME ASPECTS OF NATURAL PRODUCTS CHEMISTRY  ABSTRACT  In p a r t I of t h i s t h e s i s a r e d e s c r i b e d some s t u d i e s toward Attempts  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 .  t o t r a n s f o r m the key aromatic  t e r r a r u b e i n and 6-methylpretetramid  compounds  into actual  or  hypothetical biosynthetic  intermediates, f a i l e d  to  y i e l d u s e f u l non-aromatic  p r o d u c t s . However, s e v e r a l  of the t r a n s f o r m a t i o n s f u r t h e r along the r o u t e have been a c h i e v e d .  Thus, the c o n v e r s i o n of 12a-deoxy-5a,  6-anhydrotetracycline into c y c l i n e was  7-chloro-5a,6-anhydrotetra  successfully carried  In a d i f f e r e n t  approach  out.  i t was  attempted  v e r t the 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 dedimethylamino-5a,6-anhydrotetracycline 5a,6-anhydrotetracycline v i a a series amination experiments.  t o con-  7-chloro-4  to 7-chloro-  of b r o m i n a t i o n -  Chromatographic  evidence i s  presented f o r the f o r m a t i o n , i n t r a c e amounts, of 7chloro-5a,6-anhydro-4-epi-tetracycline. P a r t IT i s concerned w i t h the study of the possible precursor a c t i v i t y  of t r i a c e t i c a c i d l a c t o n e  a p o t e n t i a l polyketomethylene  chain intermediate i n  the b i o s y n t h e s i s of aromatic compounds. Triacetic  a c i d l a c t o n e was  l a b e l l e d g r i s e o f u l v i n was was  fed t o P.patulum  and  i s o l a t e d and degraded.  found t h a t r a d i o a c t i v i t y  It  i s incorporated into  g r i s e o f u l v i n a n o n - s p e c i f i c way. mould two new  (3,5-^C)  In one  s t r a i n of t h  m e t a b o l i t e s were found as a r e s u l t  of  the a d d i t i o n of t r i a c e t i c triacetic  acid lactone.  A d d i t i o n of  a c i d l a c t o n e t o the mould a l s o causes  an  e x p l a i n e d enhancement of m e t a b o l i t e f o r m a t i o n .  GRADUATE STUDIES  Field  of Study:  Organic  Topics i n Physical  Chemistry  Chemistry  A. Bree R. Coope  J . A. Seminar i n Chemistry Topics i n Inorganic  Chemical  Reaction  P, Kutney  N. B a r t l e t t W. R. C u l l e n D. G, L. James E. A. O g r y z l o  Chemistry  Stereochemistry  P h y s i c a l Organic Organic  Chemistry  Kinetics  T o p i c s i n Organic  Organic  J.  J . P. Kutney D. E. McGreer R. E. Pincock L. D, Hayward  Chemistry Mechanisms  Recent S y n t h e t i c Methods i n Organic Chemistry  R. A. G. G. A.  Stewart I. S c o t t  S. Dutton Rosenthal  un-  PUBLICATION  A. Rosenthal  and M. Y a l p a n i .  R e a c t i o n of A r a l k y l Ketone Oximes w i t h Carbon Monoxide and Hydrogen t o Y i a l d Formamides and Secondary Amines. Can. J . Chem. 43  (1965)  SOME ASPECTS OF NATURAL PRODUCTS CHEMISTRY  by Mohamed B.Sc., U n i v e r s i t y  Yalpani" o f W a s h i n g t o n , 1961  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  t h e Department of Chemistry  We  accept  required  this  thesis  as c o n f o r m i n g  to the  standard  DEPARTMENT OF CHEMISTRY THE  UNIVERSITY OF B R I T I S H COLUMBIA A u g u s t , 1965  In p r e s e n t i n g the  this  thesis  in partial  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f  British  Columbia,  I agree that  the Library  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 . mission  f o r extensive  representatives„  cation  of this  thesis  w i t h o u t my w r i t t e n  Department o f  I t i s understood for financial  thesis  that  gain  permission.  ^-fr***?*?  <f  make  i t freely per-  f o r scholarly  by t h e Head o f my D e p a r t m e n t o r by  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8 , Canada Date  shall  I f u r t h e r agree that  copying o f t h i s  p u r p o s e s may be g r a n t e d his  fulfilment of  Columbia  /  copying o r p u b l i -  shall  n o t be a l l o w e d  iii Abstract In p a r t I of t h i s t h e s i s are d e s c r i b e d some s t u d i e s toward 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 . Attempts t o t r a n s f o r m the key aromatic t e r r a r u b e i n and 6-methylpretetramid  compounds,  i n t o a c t u a l or hypo-  t h e t i c a l biosynthetic intermediateSj f a i l e d to y i e l d non-aromatic  products,.  useful  However^ s e v e r a l of the t r a n s -  f o r m a t i o n s f u r t h e r a l o n g the r o u t e have been a c h i e v e d . the c o n v e r s i o n of 1 2 a - d e o x y - 5 a 6 - a n h y d r o t e t r a c y c l i n e p  7 - c h l o r o - 5 a j , 6 - a n h y d r o t e t r a c y c l i n e was  successfully  Thus  p  . into carried  out. In a d i f f e r e n t approach i t was synthetic tetracycline derivative  attempted  t o convert the  7-chloro-4-dedimethylamino-  5ag6-anhydrotetracycline to 7-chloro-5a 6-anhydrotetracycline s  v i a - a s e r i e s of bromination-aminatipn Chromatographic evidence  experiments.  i s presented f o r the formation™  i n t r a c e amounts, o f - 7 - c h l o r o - 5 a , 6 - a n h y d r o - 4 - e p i - t e t r a c y c l i n e . Part I I i s concerned  w i t h the study of the p o s s i b l e  p r e c u r s o r a c t i v i t y of t r i a c e t i c a c i d l a c t o n e , a p o t e n t i a l polyketomethylene  c h a i n i n t e r m e d i a t e i n the b i o s y n t h e s i s of 14 .  aromatic compounds,,  (3s>5-  C) T r i a c e t i c a c i d l a c t o n e was  t o P p a t u l u m and l a b e l l e d g r i s e o f u l v i n was  isolated  degradedo  i s incorporated  3  I t was  found t h a t r a d i o a c t i v i t y  i n t o g r i s e o f u l v i n a n o n - s p e c i f i c way. mould used two  new  In one  fed  and  s t r a i n of the  m e t a b o l i t e s were found as a r e s u l t of the  iv a d d i t i o n of t r i a c e t i c acid l a c t o n e  0  A d d i 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 t o the mould a l s o causes an unexplained of m e t a b o l i t e f o r m a t i o n  0  enhancement  V  CONTENTS PART I : Approaches t o the T o t a l S y n t h e s i s o f Introduction — Synthetic  — — ~  A t t e m p t s — — —-  The B i o g e n e s i s  Tetracycline  »- — — — — — 1 — — — — — — 5  of T e t r a c y c l i n e  — — ~  ~  —  10  Discussion I„ ' ^ B i o g e n e t i c (a)  Type * Approach 1  — — —-.— — >— 17  Transformation of Terrarubein  •—  (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 II  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  Tetracycline  — — — —  Experimental  —  — — ~  References  —- —  — — —  PART I l s S t u d i e s i n R e l a t i o n t o t h e A c e t a t e A r o m a t i c Compounds I n t r o d u c t i o n «=- — « Discussion  —  —  Synthetic  ~- —  —  42  — —  54  — —  84  Pathway t o — — — — —' 89  — —  Experimental — — — — — — — References  —35  of  •— — — —• —  — 2 1  ^-  Approaches t o t h e T e t r a c y c l i n e s  =-  — —  99  — — —  123  —  139  — — — appendix  vi  L I S T OF FIGURES AND TABLES  PART Is  Figure  1  —  —  —  3  —  — —  4 „  —  —  5  —  —  —  - „ — io  —  „  .  14 20  —  —  7  .  Table  1 ~  - —  Figure  1 ~  —  ~  —  —  —  2 —  —  —  —.—  3  „  4  4  m  ... —  6  PART I I  —  37 —  29  —  -- —  91  —  —  —  — 94  —  „  .„ —  97  —  __ —  —  —  22  —  — 101  5  „  „  —  —. „  6 —  —  —  —  —  —  —  — 116  7 —  —  —  —  -- .-- —  —  ~ 120  8  —  —  —  —  —  — . 120  —  —  —  —. — 101  —  viz.  ACKNOWLEDGEMENTS  I w i s h t o e x p r e s s my t h a n k s t o P r o f e s s o r Dr  e  To M o n e y  working with during  p  and D r them  t h e course  9  0  Scott  n  F» McCapra f o r t h e p r i v i l e d g e o f  and f o r t h e i r o f my r e s e a r c h  I w i s h a l s o t o t h a n k Mr» P and  Ado  0  constant  help  and p a t i e n c e  0  Salisbury for his  expert  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 t h e i n v e s t i g a t i o n o f mould  metaboliteSo T h a n k s a r e a l s o due t o t h e L e d e r l e gift  o f t h e many s a m p l e s o f t e t r a c y c l i n e  also t o Dr  0  of Popatulum  Laboratories f o r the derivatives  9  and  A o R h o d e s o f t h e G l a x o L a b o r a t o r i e s f o r the cultureSo  gift  PART Io APPROACHES TO T H E TOTAL SYNTHESIS OF TETRACYCLINE  INTRODUCTION  A n t i b i o t i c s are metabolic  products  of microorganisms  which possess the capacity^ i n low concentration^ ing  t h e growth o f  are  elaborated  although  s  mainly  by f u n g i , b a c t e r i a and  materials exhibiting antibiotic 9  i n s e c t s and h i g h e r  terramycin  p  important  penicillin,  plants,,  Certain  c a t e g o r i e s have  Antibiotics,  and e r y t h r o m y c i n  product f i e l d  c h e m i s t f o r i n no o t h e r a r e a h a s he c o n f r o n t e d  are  i n a d d i t i o n t o being  f o rreasons of public health, are o f great  the organic  success  streptomycin;, b a c l t e r a c i n j  aureomycin, Chloromycetin  w e l l - k n o w n examples,,  to  actinomycetesg  d u r i n g t h e past two decades, c o n s i d e r a b l e  as t h e r a p e u t i c agentsj  They  properties are also  substances derived from the f i r s t three ?  inhibit-  o r destroying other microorganisms.  p r o d u c e d by l i c h e n s  attained  of  interest  of the natural  such n o v e l t y and  complexity  of structureSo The  developments i n t h e a n t i b i o t i c  field  t r a c e d b a c k t o 1929 p when F l e m i n g n o t e d t h a t a p r o d u c e d 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 bacteriostatic properties.  a  are o r d i n a r i l y principle  possessed  potent  This fortunate observation  was  c a p i t a l i z e d u p o n d u r i n g t h e e a r l y p a r t o f 1940s when commercial  p r o d u c t i o n by f e r m e n t a t i o n  commenced,,  Ghemical  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 culminated  investi~  substance  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  years  has s h i f t e d t o the  a n t i b i o t i c m a t e r i a l s produced by the actinomycetes group of microorganisms,  As a consequence  o f these e f f o r t s t h e  knowledge o f n a t u r a l product c h e m i s t r y has been e n r i c h e d correspondingly»  ISOLATION AND STRUCTURE OF THE TETRACYCLINES The t e t r a c y c l i n e s a r e a f a m i l y o f broadspectrum a n t i b i o t i c s produced i n the course o f t h e metabolism o f v a r i o u s Streptomyces s p e c i e s , o r by simple c h e m i c a l m o d i f i c a t i o n s o f n a t u r a l l y o c c u r i n g members o f t h e c l a s s . The f i r s t aureomycin  o f these y e l l o w - c r y s t a l l i n e compounds/  ( 1 , R ^ G l j R2 =H) r  iJ  Streptomyces aureofacienSo  was i s o l a t e d by Duggar  1  from  The d i s c o v e r y o f i t s powerful  a c t i v i t y a g a i n s t a broad spectrum o f pathogenic microorganisms was f o l l o w e d i n 1 9 5 0 by t h e I s o l a t i o n o f t h e second member o f the s e r i e s , t e r r a m y c i n ^ < l R|=Hj R2=0H).„ a m e t a b o l i t e p  of Streptomyces r i m o s u s  0  (1)  T h i s stimulated  an i n t e n s e e f f o r t toward t h e s t r u c t u r a l  e l u c i d a t i o n o f these complex n a t u r a l products,,  The p r e l i m i -  nary s t u d i e s e a r l y i n d i c a t e d two c l o s e l y r e l a t e d compounds^; as w e l l a s a f f o r d i n g s i m i l a r d e g r a d a t i o n ultraviolet  s p e c t r a were n e a r l y  their  superimposable* and t h e i r  orange-yellow c r y s t a l l i n e h y d r o c h l o r i d e phous o  products3  s a l t s were isomor-  The p u b l i c a t i o n ^ of the s t r u c t u r e o f aureomycin and  terramycin  i n 1953 confirmed t h e p r e d i c t i o n a s t o t h e i r  s i m i l a r i t i e s and t h e name t e t r a c y l i n e was proposed f o r the s t r u c t u r e o f ( i * R^=R =H), ~ 5  1  0  2  T h i s p r o t o t y p e t e t r a c y c l i n e was obtained hydrogenolysis discovered  by c a t a l y t i c  o f 7 - c h l o r o t e t r a c y c l i n e H j , and was l a t e r  i n a metabolite  o f S o aureofaciens- - ; 1  showed h i g h a n t i b i o t i c activity»  2  i t too  3  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 o f other t e t r a c y c l i n e s were found some o f which w i l l be d i s c u s s e d  below i n t h e s e c t i o n on the  biosynthesis of the t e t r a c y l i n e s * The  problem o f t h e t o t a l  s y n t h e s i s i s rendered  formi-  dable by t h e complex a r r a y o f f u n c t i o n a l groups l o c a t e d upon the c h a r a c t e r i s t i c hydroriaphthacene r i n g system.  Imposed  upon t h e t e t r a c y c l i c framework i s a v a r i e t y o f s u b s t i t u e n t s which r e s u l t  i n a minimum o f f i v e asymmetric c a r b o n atom  w i t h i n the molecule challenge  p  thereby imposing a major  t o the t o t a l  synthesls  0  stereochemical  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 t h e  relative  c o n f i g u r a t i o n o f t h e s i x asymmetric c a r b o n atoms o f mycin*  (see  terra-  f i g u r e 1)  F i g u r e 1» I t a p p e a r s e s t a b l i s h e d t h a t the presence amino group a t G-4 i n t h e c o r r e c t  o f the d i m e t h y 1 -  natural configuration 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 w h i c h 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 t h e 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 tetracyclines^'*^»  On t h e o t h e r hand r e m o v a l o f the G-6  h y d r o x y l and C-6 m e t h y l has no e f f e c t upon t h e o f t h e s e compounds„  activities  Thus t h e compound ^ - d e o x y - S - d e m e t h y l -  7-chlorotetraeycline'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-demethyi-7-chlor6tetracycline  by s e l e c t i v e r e d u c t i o n o f  C - 6 h y d r o x y l group e x h i b i t s t h e p o w e r f u l a n t i b a c t e r i a l v i t y associated with 7-chlorotetracycline^-?  but a l s o  the  actihas  the d e s i r a b l e feature  f o r s y n t h e s i s of h a v i n g o n l y f o u r  asymmetric c e n t e r s  Removal o f t h e 12a h y d r o x y l produces  x o  0  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 o f t h e  2-carboxamido  substituent to 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 t h e c h e m i c a l p r o p e r t i e s o f the t e t r a c y c l i n e B  0  c o v e r i n g t h e 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 B a n g e r t  °  Another i n c l u d e s  a l u c i d summary o f t h e v a r i o u s approaches w h i c h have been made t o t h e 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 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 t h e c h e m i s t r y o r 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 , t h e t e t r a c y c l i n e So  2 2 <  ~ ^ 2  Q  £  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 t h e c h e m i s t r y and b i o s y n t h e s i s o f t h e  tetracyclines  0  SYNTHETIC ATTEMPTS Attempts towards the t o t a l s y n t h e s i s of the  tetracy-  c l i n e s ^ , 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 o f d i f f e r e n t k i n d s . , g a i n e d impetus i n 1957 w i t h t h e announcement by s e v e r a l r e s e a r c h g r o u p s o f  their  projected routes^ together with t h e i r preliminary experimental resuitSo  - 6 -  W h i l e a number o f w o r k e r s have i n d e e d been a b l e synthesize t e t r a c y c l i n e  to  d e r i v a t i v e s , no s y n t h e s i s of a  naturally occurring tetracycline  has a s y e t been r e p o r t e d , ,  Two r e s e a r c h g r o u p s , t h o s e of F i e l d s , Kende and Boothe ^ 2  i n t h e U n i t e d S t a t e s and M u x f e l d t ^ i n Germany, 2  announced i n 1959 the o b t e n t i d ' n o f ( i ) 12a-deoxy-7-chlorotetracycline  (3)  dedimethylamino-6,  and (zt) d e d i m e t h y l a m i n o -  12a-deoxy-5a,6-anhydro-7-chlorotetracycline  (4)  respectively.  R e c e n t l y each group has been a b l e t o i n t r o d u c e the h y d r o x y l groups  12a  M u x f e l d t b y * t h e a c t i o n of m o l e c u l a r oxygen  i n the p r e s e n c e 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 t h e p r e s e n c e of sodium n i t r i t e  (3)  «  (4)  E m p l o y i n g a m o d i f i e d a p p r o a c h i n w h i c h the  introduction  of the d i m e t h y l a m i n o group was a c c o m p l i s h e d p r i o r t o the s t r u c t i o n o f t h e r i n g . A , Woodward e t a l were a b l e 0  con-  i n 1962  t o announce t h e 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 , (±) 6-demethyl-6-deoxytetracycline(5) A d e t a i l e d o u t l i n e of t h e s e and o t h e r a t t e m p t s toward the t o t a l  - 7 synthesis of the t e t r a c y c l i n e s i s given  More r e c e n t l y  (February,  announced the t o t a l tetracycline(2)  on a l a r g e  s y n t h e s i s was  (6).  (7)°  These  (9)»  compound  by N - f o r m y l a t i o n  s t e p s a r e shown The i m p o r t a n c e  sising,  scale»  (3),  with  i n t o the G-4  starting  i n four (7)  methyl-Nepimeric  Removal o f t h e p r o t e c t i n g  groups  and 1 2 a - h y d r o x y l a t i o n produced 2  i s w e l l w o r t h empha-  i t has solved the problem of the s y n t h e s i s o f tetracycline•<>.  a  (2)o  0  o f t h i s new method  laboratory non-biogenetic  The  of the aldehyde  by c o n d e n s a t i o n  in figure  since i n p r i n c i p l e  active tetracycline  was t r a n s f o r m e d  Reaction  tertiarybutyl-3-oxoglutaramate  which„  l-chloro^2-bromomethyl-4-  T h i s compound  hippuric acid followed  followed  b y a method  0  steps t o the aldehyde  tetracyclic  and R o g a l s k i ^  o f w h i c h had p r e v i o u s l y  time, allows b i o l o g i c a l l y  material for this  with  analog  by Woodward e t a l o ^ ° )  d e r i v a t i v e s t o be p r e p a r e d  methoxybenzene  Muxfeldt  synthesis of 6-deoxy-6-demethyl-7-chloro-  (the d e s c h l o r o  been synthesized for the f i r s t  1965)  i n a p p e n d i x !„  There  -  8  Figure  -  2  - 9 -  seems t o be no o b s t a c l e i n t h e way o f 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 t h e n  be b r o m i n a t e d 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 « 6 - a n h y d r o t e t r a c y c l i n e (11) has a l r e a d y been t r a n s f o r m e d t o  (laR-pGlgR^H) by S c o t t and  7-chlorotetracycline  Bedford32  0  (1)  (11)  The e x a c t d e t a i l s o f t h i s l a t t e r w i l l be d i s c u s s e d l a t e r synthetic tetracycline c o n s t r u c t e d and i t  which  0  It  sequence o f  s h o u l d be s t r e s s e d  reactions  that  no  p o s s e s s i n g a C ° 6 h y d r o x y l has been  seems t h a t any f u t u r e  tetracycline  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»  10 -  THE BIOGENESIS OF THE TETRACYCLINES In r e c e n t y e a r s t h e b i o s y n t h e s i s of t h e have been the s u b j e c t  tetracyclines  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  experimental investigation,,  I n s p e c t i o n o f the f o r m u l a e o f  t h e 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=HgR =0H), r e v e a l s 2  a t y p i c a l o x y g e n a t i o n p a t t e r n f o r a compound d e r i v e d f r o m acetate*.  Tracer s t u d i e s ^ using  2- *G ±l  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 t h e m o l e c u l e c o n t a i n s C - m e t h y l and N - i a e t h y l groups  d e r i v e d f r o m the l a t t e r  s o u r c e , and  t h a t t h e 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 t h e 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 was a t t h e t i m e suggested t h a t c a r b o n atoms 2 3s>kok 0  0  &  It a n d  the  carboxamido c a r b o n were d e r i v e d f r o m g l u t a m a t e ( b r o k e n l i n e s f i g u r e 3)„  F i g u r e 3c  -  Later  studies revealed  i n a n o n - s p e c i f i c way34  11 -  t h a t glutamate was  incorporated  i n a d d i t i o n ^ ' - f e e d i n g experiments  e  w i t h "^G-sodium b i c a r b o n a t e l e d t o i n c o r p o r a t i o n o f t h e t o t a l a c t i v i t y i n t o the carboxamido group o f terramycin-^,, F e e d i n g t h e organism c a r b o x y l - l a b e l l e d malonic a c i d showed t h a t the r a d i o a c t i v i t y o f the carboxamido group was approximately  10-20$ o f t h e t o t a l r a d i o a c t i v i t y o f t e r r a m y c i n  0  T h i s suggests t h a t t h e carboxylatloni r e a c t i o n i n v o l v e s t h e wintermediate f o r m a t i o n of malonate a n d  s  furthermore  that  9  malonate c o n s t i t u t e s t h e condensing p r i n c i p l e f o r t h e f o r m a t i o n o f t h e whole carbon s k e l e t o n o f t e r r a m y c i n . Consequently  s  the p r i n c i p l e of b i o s y n t h e s i s  of the t e t r a -  cyclines i s r e l a t e d t o f a t t y acid biosynt hesis35 !  a  n  Q  t o the  formation o f 6 - m e t h y l s a l i e y l i c a c i d and o r s e l l i n i c ac i  id36 which have been shown t o i n v o l v e malonate.  I n those cases  condensation i s thought t o s t a r t w i t h acetyl-coenzyme  A  p  r e a c t i n g w i t h a malonyl-coenzyme A t o form a B - p o l y k e t o methylene c h a i n o f t e n C  2  u n i t s which t h e n c y c l i z e  0  In the  t e t r a c y c l i n e s the! s t a r t i n g u n i t c o u l d be malonyl-coenzyme A (see S  0  T h i s i s supported by co-occurrence^, i n  aureofaciens37  (12) Ao  figure 4 K  g  o f 2-aeetyl~2-decarboxamid©-terramycin37  i n which the primer u n i t i s presumably a c e t y l Coenzyme Although unusual$ maXpnyl—coenzyme A has been demonstrated  t o be a c h a i n s t a r i e r u n i t i n the p o l y a c e t a t e hexamid ( 1 3 ) ^ ,  '  derived  cyclo-  -  12  These r e s u l t s l e d t o the compounds of, t h e  type  b i o s y n t h e s i s of the prepared  and  tetramid  (14p  (14)  suggestion that  c o u l d be  R  R-^CH^Rg—H), and  it  i n the  6-methyltetraeyclineSo a p p e a r s t o be  seem t©  a reasonable  other  the  =  v  0  R-^H^J  a u r e o f a e i e n s and  were  6-methylpretetramid  b i o s y n t h e s i s o f 6=demethyl  i n t e r m e d i a t e , d i d not Experiments,  t© be  show discussed  -As a r e . s u l t o f t h e s e - e x p e r i m e n t s  g e n e r a t i o n of t h e  s t e p s , namely the  moles of water the  *2 *^ 6-methylpre-  etoal<A® were a b l e t o s u g g e s t t h a t w h i l e G - m e t h y l ~  a t i o n occurs before all  o  i n d i c a t e t h a t N=>methylati@n ©csiars l a t e r ^ I n  t h e biosynthe't.ic sequence, MeCormlck  et<,ar 39-4Q  However, t e r r a r u b e i n , a l t h o u g h  s i g n i f i c a n t ' p r e c u r s o r activity© later,  = i  d e m o n s t r a t e t h a t p r e t e t r a m i d and  were n o r m a l i n t e r m e d i a t e s and  McGormick  t e r r a r u b e i n (14D  R2=NMe2) t o s u i t a b l e m u t a n t s o f S a b l e t©  i n t e r m e d i a t e s i n the  tetracyclines,,  f e d p r e t e t r a m i d •';(-14p i  naphthacenic  (at  naphthacene  1  a d d i t i o n of the  '4&,I2a a n d a t 5a,6)  c h l o r i n e f o r h y d r o g e n a t C-7  and  and the  moiety,  elements of substitution  elements of  a  two of  - 13 -  d i m e t h y l a m i n o g r o u p f o r a n atom o f h y d r o g e n place  a f t e r the nucleus  noted that  a l lfive  i s assembled.,  o f t h e asymmetric  at  take  I t s h o u l d a l s o be centers of the t e t r a -  c y c l i n e molecule a r e generated i n the course of these transformations o  (16)  (15) (a)  Rj=Rj=H:  R =CH ;  (b)  Rj=C.l; R = R - H ;  (c)  R ^ R =R=H;  (d)  R^Rj=H;  2  2  R^=OH  3  "R^=OH  3  R.fCH 3  McGormick e t o a l o ^  (b) R^=C1;  R =CH\j; R ^ OH 2  R =R =H; 2  (d) R^-H;  R =CH 2  R =CH ; 2  a l s o demonstrated that  3  3  R.=R^OH  suitably  stituted  5aj,6r-anhydr©tetracyclines were b i o l o g i c a l l y  formed.  Thus feeding  (15papbpCp  R^=OH  3  (c)- R = R = R =H;  3  R =CH ; R^OH 2  ( a ) R-j-R^H;  sub-  trans-  a n d d) r e s u l t e d i n ( I 6 a , b j , c 0  - 14 -  F i g u r e 4<>  - 15 and d) r e s p e c t i v e l y .  These r e s u l t s showed t h a t the t e r m i n a l  step o f the b i o s y n t h e s i s may  be r e p r e s e n t e d as i n t h e  s i o n o f (20) t o (1) i n f i g u r e 4° r e d u c t i o n step was 5a  s  The  conver-  e x i s t e n c e of the  i n d i c a t e d by the i s o l a t i o n of 7-chloro-  11a d e h y d r o t e t r a c y c l i n e from a mutant o f S„  aureofaciens  9  17 b l o c k e d f o r the r e d u c t i o n o f (21)  ,  I t was  f u r t h e r found  i n these and a d d i t i o n a l experiments t h a t 4-dedimethylamino5a 6-anhydrotetracycline s  (19)  (18) and  4-epianhydrotetracyclines  f a i l t o e x h i b i t any p r e c u r s o r a c t i v i t y and t h a t when-  ever 7 - c h l o r i n a t e d t e t r a c y c l i n e s were used i n a t e r r a m y c i n producing mutant and  n  S, rimosus,, no i n c o r p o r a t i o n was  s t a r t i n g m a t e r i a l was  isolated.  observed  T h i s e x p l a i n s why  no  c h l o r i n a t e d t e r r a m y c i n d e r i v a t i v e s have ever'been i s o l a t e d ^ - .  (18) Although  • (19)'^  i t seems t h a t the presence  of the 12a  hydroxyl  group in. the anhydro p r e c u r s o r s i s not necessary f o r hydroxyl a t i o n at C-6  P  i t i s n e v e r t h e l e s s a f a c t t h a t the 12a  cannot be i n t r o d u c e d a t a l a t e r  stage^^  hydroxyl  Furthermore;, since  r e c e n t l y the i s o l a t i o n and p r e c u r s o r a c t i v i t y of a s e r i e s of N-dedimethyl-5a,6-anhydrotetracyclines  (22) have been r e p o r -  t e d ^ , i t would seem t h a t 1 2 a - h y d r o x y l a t i o n precedes 2  the  - 16  -  development of the N-dimethylamino n e c e s s a r i l y t h e amino group) a t  group C-4°  More r e c e n t l y M c C o r m i c k e t . a l o ^ methyl-pretetramid  (17)  and  i n the b i o s y n t h e s i s of the The  (although not  4-hydroxy-  isolated  demonstrated  i t s intermedlacy  tetracyclines.  evidence presented  so f a r l i m i t s t h e number o f  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)  c a n be  formed t o N ~ d e d i m e t h y l - $ a 6 - a n h y d r o t e t r a c y c l i n e s  are o u t l i n e d sectiono  i n f i g u r e 4»  and  will  (22).  be d i s c u s s e d i n a  transThese later  - 17  -  DISCUSSION (I) " B i o g e n e t i c Type** Approach, In r e c e n t y e a r s the t o t a l n a t u r a l l y o c c u r r i n g organic  s y n t h e s i s o f many complex  substances has been accomplished  u s i n g what has been c a l l e d the b i o g e n e t i c type The  synthesis^.  term " b i o g e n e t i c t y p e " has been s e l e c t e d t o d e s c r i b e  organic  an  s y n t h e s i s which has; been desighed t o f o l l o w , a t  l e a s t i n i t s major aspects,  1  the proven or p o s t u l a t e d  bio-  s y n t h e s i s of the p a r t i c u l a r n a t u r a l p r o d u c t , A r e c e n t 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  s e t s out  c e r t a i n g e n e r a l guide l i n e s by  which these syntheses have been accomplished. Generally,  i n the l a b o r a t o r y d u p l i c a t i o n of the  l o g i c a l s t e p s , any  c o n d i t i o n s or r e a g e n t s ,  completion o f the r e a c t i o n , may r e q u i r e d t o simulate  the  be used.  bio-  necessary f o r the Thus i t i s not  s p e c i f i c c o n d i t i o n s which supposedly cello  approximate the environment i n the l i v i n g  T h i s type of approach i s i n g e n e r a l more a e s t h e t i c a l l y p l e a s i n g and n e a t e r , and  s a t i s f y i n g , a s i t o f t e n l e a d s to a more e f f i c i e n t  shorter,  s y n t h e s i s , than those i n which no  a t t e n t i o n i s p a i d t o the n a t u r a l p r o c e s s e s .  The  synthesis  of d , l chimonathine (23), completed e a r l i e r i n our l a b o r a t o r i e s ^ by the d r a m a t i c a l l y simple n a t u r a l product d i p t e r i n  own  d i m e r i s a t i o n of  (N-methyltryptamine), serves to  the  -  18  -  i l l u s t r a t e the u s e f u l n e s s o f t h i s approach, and we the d e s i g n of a s y n t h e s i s i n the t e t r a c y c l i n e f i e l d  f e e l that i s also  p o s s i b l e u s i n g i n f o r m a t i o n from the known b i o s y n t h e s i s of t h i s molecule.  (23) However, i t i s r e c o g n i s e d f i e l d the u s u a l one-or two and  t h a t i n the  tetracycline  step c o n s t r u c t i o n o f the l a r g e  complex m o l e c u l e s , i s not f e a s i b l e , n e i t h e r i s the a c t u a l  n a t u r a l pathway t o t e t r a c y c l i n e i n the  introduction.  a short one  , a s described  Thus the d e s i g n f o r the present  i s t o f o l l o w , i n g e n e r a l , a stepwise b u i l d u p o f the f u n c t i o n a l groups o f the t e t r a c y c l i n e moleculeo  synthesis  various  _  19  -  I n 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 polyacetate  condensation p r o d u c t , the aromatic  6-methylpretetramid (14a)o  the  substance  I f we a r e 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 t h e l a b o r a t o r y , t h i s compound t o i t s 4 - h y d r o x y a n a l o g (17),  and f r o m t h e r e v i a e i t h e r o f t h e s t e p s shown i n  figure 4 to N-demethyl-5a,6-anhydrotetraeycline 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  (22).  tetracycline  has been a c h i e v e d e l s e w h e r e ^ * 32  o  When the p r e s e n t 1  i n v e s t i g a t i o n was s t a r t e d , t h e 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 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) tetracycline  (20) „  6-methyIpretetramid  S i n c e t h e n the (17)  t e t r a c y c l i n e have been A s o l u t i o n t o the  tetra-  and 5 a , 6 - a n h y d r o -  intermediacy of 4-hydroxy-  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 demonstrated,4 f.43 2  s y n t h e t i c problem thus appears to  i n v o l v e t h e 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)  to  t e r r a r u b e i n (14b) j> r e d u c t i o n o f t h e 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 (see  f i g u r e 5p p a t h a)«,  attempt  F o r the p r e s e n t  tetracycline  i n v e s t i g a t i o n s no  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 o r t e r r a -  rubein from smaller u n i t S p neither i s the synthesis t e r r a r u b e i n from 6-methylpretetramid considered.  of  Figure  5°  - 21 B o t h t e r r a r u b e i n and 6 - m e t h y l p r e t e t r a m i d  ( s e e f i g u r e 6)•  from degradations o f t e t r a c y c l i n e  b o t h have b e e n o b t a i n e d f r o m two s o u r c e s sent e x p e r i m e n t s and  p  are available  1 0  A s showh  *^'47^  t e r r a r u b e i n p r e p a r e d by f o r m a t e  r  n  p  the pre  pyrrolysis,  6 - m e t h y l p r e t e t r a m i d b y d e h y d r o b r o m i n a t i o n method a r e  usede  (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  Examination of the,terrarubein  (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 t h e keto form, r i n g A i s i s o l a t e d substituted  as a  resorcinolo  (14b) The 1660  cm"  infrared 1  spectrum  a n d one a t 1620  of terrarubein cm"  1 D  T h e 1620  shows a band a t cm"  1  band i s  a s s i g n e d t o t h e s t r o n g l y h y d r o g e n - b o n d e d amide a t C-2 and t o t h e h y d r o g e n - b o n d e d c a r b o n y l a t C-12 band  0  The weaker 1660  em  - 1  i s a s s i g n e d t o t h e n o n - h y d r o g e n b o n d e d c a r b o n y l a t G-12.-  T h i s i s a n a l o g o u s t o t h e 1665 t o t h e naphthacene  cm""-*- band  ( 2 6 ) ^ which  i n anthrone  (25) and  i s r e p o r t e d t o have a I6l3cm~  22  Figure  6  a  - 23 -  and a 1675 cm  b a n d , 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, violet  A l s o , the  ultra-  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  s e r i e s o f a r o m a t i c t e t r a c y c l i n e s a l l show  this  characteristic  a c y l n a p h t h a l e n e a b s o r p t i o n bands (the u l t r a v i o l e t spectrum o f the a c y l n a p h t h a l e n e  (27), i s r e p o r t e d t o be 270, 450 mu^  H  (27) The p r e s e n c e o f the k e t o form i n t e r r a r u b e i n would expose t h e d o u b l e bond a t 4,4a t o h y d r o g e n a t i o n .  Several  model compounds w h i c h 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  t h e r e s o r c i n o l d e r i v a t i v e s (20) have been c o n v e r t e d t o d i h y d r o compounds (29)„  In a d d i t i o n  9  Thus the  r e d u c t i o n of a number  - 24 -  of 4a„12a-dehydrotetracyclines  have been d e s c r i b e d ^  p r o v i d e a v e r y c l o s e a n a l o g y f o r t h e case a t h a n d example, t h e r e d u c t i o n of (30) y i e l d s  a  f  and  For  12a-deoxytetracy-  c l i n e (31)«  COlMH,  CONH  Rl~H;  R =NH  Rj=H;  R =NMe|  2  z  5 1 2 2  2  R j f M e ; R "=H 3 5  2  M(CH3),  OH s-  )H  O  O  (30) F a r t h e r a l o n g t h e proposed s y n t h e t i c  O H  O H  (3D pathway i t would  be n e c e s s a r y t o i n t r o d u c e t h e h y d r o x y l g r o u p s a t C - 6 and C-12a and t o a f f e c t t h e r e d u c t i o n o f t h e r e s u l t i n g double bond.  5a,lla  The i n t r o d u c t i o n o f t h e h y d r o x y l a t G~12a has 30 28 been a c h i e v e d by Woodward e t » a l and by M u x f e l d t on t h e i r synthetic  tetracyclines<  - 25 T h u s , Woodwards f i n a l p r o d u c t  (5) was o b t a i n e d by the  a c t i o n o f oxygen i n t h e p r e s e n c e o f cesium c h l o r i d e „ f e l d t completed h i s s y n t h e s i s oxygenation  of  Mux-  (4) by u s i n g p l a t i n u m c a t a l y z e d  Other r e a g e n t s w h i c h have been found u s e f u l  for  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 benzoic  acid ^ "^ 2  0  On t h e o t h e r h a n d , t h e 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 . a l o - ^ , i n c l o s e a n a l o g y t o the b i o s y n t h e t i c by p a s s i n g oxygen t h r o u g h an i r r a d i a t e d s o l u t i o n of 5a,6-anhydrotetracycllne  (11) t o g i v e  steps,  7-chloro-  7-chloro-6-hydroperoxy-  - 26  5a,lla-anhydrotetracycline  (32), w h i c h s u b s e q u e n t l y  absorbed  two moles o f hydrogen t o f o r m 7 - c h l o r o t e t r a c y c l i n e This reaction  was r e p o r t e d t o be s p e c i f i c f o r t h e  n a t e d anhydro compound ( 1 1 ) « present  synthetic  (20)  7-chlori-  D  On t h i s  To Money of t h i s  seemed, t r a n s f o r m e d t e r r a r u b e i n v i a the to t e t r a c y c l i n e .  the  chlorinate  i n t h e Cr-7 p o s i t i o n  b a s i s a p r e l i m i n a r y experiment by D r  shown on f i g u r e 5* p a t h (b)  0  Thus f o r the p u r p o s e s o f  sequence i t became n e c e s s a r y t o  5a,6-anhydrotetracycline  ment h a d , i t  (1)  depart-  steps  Thus, a small  sample o f t e r r a r u b e i n ^ w h i c h had been s u b j e c t e d t o 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 a s c a t a l y s t , a t e d 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  was oxygen-  l i g h t s to a  compound w h i c h 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 to that of 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  This  sample,  w h i c h 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  quantities  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 w a v e - l e n g t h and i n t e n s i t y that of t e t r a c y c l i n e o  When, however, r e p e a t  to  experiments  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 n o t be r e p r o d u c e d , Thus i t became n e c e s s a r y t o p r o c e e d 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 intermediateSo  individual  A c c o r d i n g l y , t h e i n t r o d u c t i o n o f the  h y d r o x y l I n t o d i h y d r o t e r r a r u b e i n (24)  12a-  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 o f 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 t h r o u g h an i r r a d i a t e d benzene  solution  o f a s p e c i a l l y p u r i f i e d sample o f d i h y d r o t e r r a r u b e i n f r e e base ( p r e p a r e d by t h e method o f Boothe et oai,* - }, t h e 5  violet  spectrum o f 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 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  dihydroterrarubein The change  (20),  was a l s o f o l l o w e d by paper chromatography, m a t e r i a l c o u l d be d e t e c t e d .  u n t i l no  starting  The r e a c t i o n p r o d u c t 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 l 2 a ~ 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 spot f r o m t h e papergram.  ultra-  5  (probably anhydro  The i n f r a r e d spectrum of  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  this  anhydrotetra-  c y c l i n e (20), G h l o r i n a t i o n a t t h e C-7 p o s i t i o n was a c h i e v e d by the a c t i o n o f 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 o f cycline  (Id)  in glacial acetic acid.  anhydrotetra-  The p r o d u c t was  i d e n t i f i e d as 7-chlor©anhydrotetracycline  (11)  o f i t s i n f r a r e d and NMR s p e c t r a w i t h t h o s e o f material.  by  comparison  authentic  P r e v i o u s h a l o g e n a t i o n a t t e m p t s on the  tetracy-  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 t h e 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 positions using dihydroterrarubein r e m a i n e d , a s an e s s e n t i a l  (24) as s u b s t r a t e ,  s t e p , t o prepare  t o c a r r y out t h i s key s t e p i n t h e the r e d u c t i o n o f t e r r a r u b e i n t o  required  synthetic  (24 )  t  route,  it  A l l attempts namely,  i t s dihydro d e r i v a t i v e ,  have  so f a r r e m a i n e d u n s u c c e s s f u l , used  (see t a b l e  platinum  1.),  Of  these,  c a t a l y s t a l t e r e d the  a p p e a r a n c e o f a 350  mu  were  only hydrogenation using  a  t e r r a r u b e i n chromophore.  band i n p l a c e  band suggested t h a t the disrupted.  A v a r i e t y of c o n d i t i o n s  of the  acylnaphthalene  T h i s r e a c t i o n p r o d u c t was  The  u s u a l 450  mu  c h r o m o p h o r e had  not  further  been  investi-  gated,, This d i f f i c u l t y d o u b t on t h e  exact  i n the  formation  spectrum, but was  the  showed a n  change i n the  5a,  spectrum  I t was  l a t i o n a t the the  formation  (255-370 mu)  aromatic  possible biogenetic  of t h e  on e x p o s u r e  thus suggested that the  s p e c t r u m o f t e r r a r u b e i n had  a l s o be  ultraviolet  r e s u l t e d i n the  and  a t C-12a  compound i s e x p e c t e d t o h a v e t h e should  the  This  t a n e o u s o x y g e n a t i o n a t C-6  It  i n that  u n c h a n g e d t e r r a r u b e i n spectrum,,  lla-dehydrotetracycline oxygen.  resulted i n  characteristic dihydroterrarubein  s a m p l e t h a t had  l i g h t and  not  casts  terrarubein  experiments, e s p e c i a l l y since  o f t e r r a r u b e i n had  of the  of t e r r a r u b e i n  sequence o f the p r e l i m i n a r y  reduction-oxygenation case, reduction  reduction  to  observed  b e e n due  to y i e l d  to  simul-  (33)°  This  observed u l t r a v i o l e t  spectrum.  m e n t i o n e d a t t h i s p o i n t t h a t 12a  hydroxy-  stage has  of  routes  f o r t h i s type of b i o l o g i c a l  been p o s t u l a t e d  as  to tetracycline -* „  conversion  2  c a n be  one An  seen i n  analogy the  m e t a b o l i s m , by r a t l i v e r m i c r o s o m e s , o f 6 - h y d r o x y t e t r a l i n  (34)  -  29  -  TABLE'I Selected Reductions of T e r r a r u b e i n  C o n d i t i o n s used  Palladium  Product  on  charcoal  No r e a c t i o n  on  charcoal  No r e a c t i o n  Ethanol Palladium Ethanol/ Palladium Ethanol/ Palladium  sodium on  bicarbonate  charcoal  sodium on  No r e a c t i o n  hydroxide  charcoal  No r e a c t i o n  Ethanol/ trlethylamine Platinum/  ethanol  Over r e d u c t i o n o f the  moleculeo  P a l l a d i u m on c h a r c o a l P r e s s u r e s 40 p s i o Ethanol  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 P r e s s u r e s 600 p s , i 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  e  e  e  0  -  30  -  to i t s corresponding p-quinole (35). °  OH  (34)  (35)  I n t h e 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 s h o u l d be even more f a c i l e due t o t h e p r e s e n c e of two a c t i v a t i n g h y d r o x y l g r o u p s on r i n g A a t C-2 and C-4» A t t e m p t s 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, o r 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 s p e c t r u m , a s had been o b s e r v e d i n t h e p r e l i m i n a r y experiment« 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) covertible to  was  l,4j»6,ll-tetrahydro-3,6,10,12-tetrahydroxy-6-  -31  -  methyl-l ,4sll-trioxonaphthacene-2-carboxamide  {36)  i  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%  in  magnesium a c e t a t e  tetra-  57  hydrate of the was  when e x p o s e d t o a i r  , prompted an i n v e s t i g a t i o n  r e a c t i o n of t e r r a r u b e i n i n t h i s  hoped t h a t the p o w e r f u l  solvent  system.  c h e l a t i n g a c t i o n of the  magnesium a c e t a t e w o u l d f a v o u r  the  It  added  h y d r o x y l a t i o n at the  12a  position.  (17)  (36)  Terrarubein dissolved readily i n t h i s solvent r a p i d l y a b s o r b e d one  m o l e o f oxygeno  t h i s compound showed t h a t one i n the  molecule.  unchanged.  material having  an u l t r a v i o l e t  F u r t h e r exposure of t h i s i n the a b s o r p t i o n  ultraviolet  s p e c t r u m had  present  remained  sodium h y d r o s u l f i t e t o g i v e  of 4-hydroxymethylpretetramid  resulted  Nitrogen a n a l y s i s of  n i t r o g e n a t o m o n l y was  Its ultraviolet  I t reacted with  and  spectrum i d e n t i c a l w i t h  a that  (17)0 »oxyterrarubein* t o  oxygen  o f a n o t h e r m o l e o f o x y g e n and  spectrum i d e n t i c a l t o that of  (36).  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  This  an  compound  converted  to  - 32 ~  4-hydroxymethylpretetramide; to afford  sodium h y d r o s u l f i t e f a i l e d  (1?)o  On t h e 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 c o n v e r t e d t o the r i n g A quinone  (37)  f o l l o w e d by o x y g e n a t i o n at C„6 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 1 2 a h y d r o x y l a t i o n can be based on t h e use o f common p h e n o l 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 r e a s o n a b l e  f i r s t c h o i c e , and  others  such a s 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 usedc  O x i d a t i o n of p - e r e s o l  (3&) t o p - t o l u q u i n o l (39)  C a r o * s a c i d ^ , and o f o e s t e r o n e (40) 0  ether (4l)  using  t o the p - q u i n o l - m e t h y 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 b o r o n t r i f l u o r i d e ^ , a r e t y p i c a l examples o f a n g u l a r oxygenation,of  a l k y l - s u b s t i t u t e d phenols.  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 t o  oxygenations  i n v o l v i n g t h e C-12a and the C-6 p o s i t i o n of the dines,  '  o  been  tetracy-  F r e m y ' 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  giving  m u l t i p l e products (vide i n f r a ) .  experiments  For the present  t h e c h o i c e f e l l on p e r a c i d s a s oxidants<>  Of t h e s e , m - c h l o r o -  perbenzoic a c i d i s commercially a v a i l a b l e .  (3«)  (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 d i o x a n e 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 s p o t s on paper chromatograms.  As an a u t h e n t i c  sample of t h e d e s i r e d p r o d u c t (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 r e a c t i o n m i x t u r e was a f o r m i d a b l e o n e , 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  is  of  34  i t s extreme  insolubility  i n common o r g a n i c  general  i t dissolves i n solvents t o give a  ranging  f r o m 1-2 mg/10  ml.  solvents.  In  concentration  Dimethylformamide and d i m e t h y l -  s u l f o x i d e were t h e 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 the  substance t o any p r a c t i c a l A second c o m p l i c a t i n g  extent.  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. Lederle  Co. normally  The sample  showed a y e l l o w  ponding t o d i h y d r o t e r r a r u b e i n  (24).  (fading)  s u p p l i e d by spot c o r r e s -  A p u r e 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 chromatographic s o l v e n t s u s e d t o move i t f r o m t h e o r i g i n .  S u c h a sample  was  prepared by r e p e a t e d washings w i t h dimethylformamide and acetone« F u r t h e r , i t was f o u n d t h a t e x t r e m e l y oxygen l a b i l e . under a n atmosphere  solutions of terrarubein are  Thus" w h i l e  i t remained  unchanged  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 the presence of metal c a t a l y s t s , r a p i d l y changed mu.  i t sultraviolet  This ultraviolet  s p e c t r u m f r o m 440,270 mu t o 3#0,262  spectrum, although s i m i l a r t o t h a t o f  5a,lla-dehydrotetracycline hydrogenation.  (32), r e m a i n e d u n a f f e c t e d b y  I n c o n t r a s t , (32) c a n be s m o o t h l y 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 hydrogenation-^ . 2  35 -  (b) T r a n s f o r m a t i o n s  of  6-Methylpretetramid  \  Since the 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 h a d met w i t h l i t t l e focussed  s u c c e s s , a t t e n t i o n was  o n a s e c o n d r o u t e w h i c h was c o n c e i v e d  as a result  of the 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-methylpretetramid i n f i g u r e 4)  (17)  and N-deraethyl-5a,6-anhydrotetracycline  °  T h i s new r o u t e was d e s i g n e d  (22,  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 ( s e e f i g u r e 4) «>  I t required, for i t s i n i t i a l  step, the  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 4h y d r o x y d e r i v a t i v e (17)» transformed  T h i s compound c o u l d c o n c e i v a b l y  i n t o N-demethyl-5a,6-anhydrotetracycline  a sequence o f r e a c t i o n s c l o s e l y i n the b i o s y n t h e t i c process mainly of  d i r e c t e d t o the route  (17)  ( s e e f i g u r e 4)«  25  .  by  envisioned  A t t e n t i o n was  i n v o l v i n g t h e 12a  a s t h i s was c o n s i d e r e d  l o g i c a l pathway  similar to that  (22)  be  hydroxylation  t o be t h e more l i k e l y b i o -  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 5a,6-anhydrotetracycline  have a l r e a d y been r e p o r t e d  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 required f o r the completion methylpretetramid drotetracycline  (17),  of t h i s route:  „  were  4-hydroxy-6-  4-keto-4-desdimethylamino-5a,6-anhy-  ( 4 2 , f i g u r e 7), a n d 4 - o x i m i n o - 4 - d e s d i m e t h y l -  amino-5a 6-anhydrotetracycline p  prepared  '  by d e g r a d a t i v e  (43)figure  7).  These  were  procedures a t the outset of 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  facile  i d e n t i f i c a t i o n of successful reaction products  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„ 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 k e t o n e and  t h e oxime  wood e t o a l . ^ preparation the by  (43) u s i n g m e t h o d s r e c e n t l y r e p o r t e d a n d b y E s s e et al.—^»  This  0  by B l a c k -  involved the  o f t h e 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 b y  action of N-chlorosuccinimide  i n a q u o u s medium, f o l l o w e d  l i q u i d hydrogen f l u o r i d e dehydration  pound  (42)  t o t h e 4 - k e t o com-  ( s e e f i g u r e 7)o The  t e t r a c y c l o x i d e (44) w a s p r e p a r e d a s  described^.  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 h y d r o g e n r i d e f a i l e d t o produce the d e s i r e d product. 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 d i f f i c u l t i e s i n repeating t h i s reaction An a l t e r n a t i v e m e t h o d was d e v i s e d the d e s i r e d ketone d i r e c t l y .  encountered  ,  i n order  to yield  (20) r e s u l t e d i n a  a l l the predicted c h a r a c t e r i s t i c s (infrared,  u l t r a v i o l e t and a n a l y t i c a l data) o f t h e r e q u i r e d compound.  later  Thus, r e a c t i o n o f N-chlorosuc-  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 compound h a v i n g  66  We were  fluo-  4-keto  - 37 -  (43)  (51) Figure  7*  -  OH  38  -  OH (20 ) The  (42)  preparation of 4-hydroxy-6-methylpretetramid  from degradation ( v i d e supra)„"  of terrarubein  has a l r e a d y been d e s c r i b e d  (A m e t h o d r e c e n t l y d e v e l o p e d i s more c o n v e n -  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 e m p l o y s t h e 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 o p e n t o p a r a q u i n o l formation  i n 6-methylpretetramid  ( 1 4 a ) ( C - 4 , C-$« C-6,  and  C-12a);j, r i n g A was c o n s i d e r e d  ted  s i t e s a C-4 a n d C-12*a w i t h p o s i t i o n C-4 t h e l e a s t  hindered  a n d more l i k e l y  site  0  C-7  t o o f f e r t h e most a c t i v a -  - 39 -  O x y g e n a t i o n o f 6 - m e t h y l p r e t e t r a m i d was attempted u s i n g c o n d i t i o n s s i m i l a r t o t h a t used f o r t e r r a r u b e i n a t C - 4 and C-6  0  oxygenation  I t was hoped t o o b t a i n (36) a s i n the  rubein oxygenation  terra-  A c c o r d i n g l y , 6 - m e t h y l p r e t e t r a m i d was  d i s s o l v e d i n d i m e t h y l s u l f o x i d e c o n t a i n i n g 1$ 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 f r o m amber t o  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  absorption  changed d u r i n g t h i s p e r i o d f r o m 480 mu t o 520 mus e v e r a l weeks o f exposure b o t h the c o l o u r o f t h e  After 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 a t 520 mu.  Iso-  l a t i o n of the r e a c t i o n p r o d u c t a f t e r 2 weeks and r e d u c t i o n o f 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 p h e n o l 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-  tetramid o Had the i n i t i a l o x y g e n a t i o n o c c u r r e d at G-4* the i n g spectrum s h o u l d have been i d e n t i c a l t o t h a t of f i r s t o x y g e n a t i o n p r o d u c t o:f t e r r a r u b e i n ; and  result-  (37)» the  subsequently,  the s o l u t i o n s h o u l d have t u r n e d deep p u r p l e i n c o l o u r and s h o u l d have shown a 550 mu v i s i b l e spectrum ved f o r  (36),  a s was o b s e r -  On the o t h e r h a n d , o x y g e n a t i o n a t G-12a should  have r e s u l t e d i n a breakup o f t h e naphthacene chromophore, w i t h a blue s h i f t i n the v i s i b l e r e g i o n . therefore,  not a u s e f u l  T h i s compound was,  intermediate.  O x y g e n a t i o n 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 o f  the  - 40  spectrum salt  and  t h i s r e a c t i o n was  0 2 °3^2 - J K  S  N 0  a  d i m e t n  y  ^  l s  abandoned.  f o x i d e  s o l u t i o n of 6-methylpretetramid hours of s t i r r i n g  in a i r ,  a deep b l u e  was  solid  v i s i b l e .spectrum s u l f o x i d e the of  On  obtained having  spectrum  was  shifted  to  (36)  The  was  acetate  in this  mu In  band  7  solution, i n the  dimethyl-  mu.  Oxygenation  solvent leads to  c h a r a c t e r i s t i c s a s d e s c r i b e d above f o r t h e  subjected to t h i s  above r e s u l t s  The  indicated  same s p e c t r a were product  .stopping t h e r e a c t i o n a t an  of  terra-  reaction,,  t h a t Fremy's s a l t  beyond t h e d e s i r e d s t a g e ,  .6-methylpretetramide at  500  o b t a i n e d when t h e f i n a l o x y g e n a t i o n  rubein  fl  of the  a 550  solvent ,  r e a c t i o n with 6-methylpretetramide, also  n e s i u m  acidification  with ethanol as  same s p e c t r a l  m  Fremy s  became deep b l u e a f t e r  4-hydroxy-6-methylpretetramid  the  / ^6  Using  earlier  converted  A l l attempts  stage f a i l e d  to  pro-  d u c e t h e d e s i r e d compound, Simultaneously ment s the  a  an attempt  other  -attempts  with the  was  a b o v e C-4  made t o a c h i e v e  intermediates i n t h i s at the oxygenation  oxygenation  the t r a n s f o r m a t i o n of  sequence,  i n the  12a  experi-  Thus  s  sevef*al„  p o s i t i o n of  6-methyl p r e t e t r a m i d u s i n g m - c h l o r o p e r b e n z o i c  acid  4-hydroxyas  oxidant  it resulted  i n a multi-component  r e a c t i o n product*  None o f  the  r e q u i r e d 4-keto-4-dedimethylamino-5a 6-anhydrotetracyqline s  c o u l d be  detected.  (42  - 41 -  Similarly  p  several attempts  t h e 4 - k e t o compound,(43)9  failed  a n a l y s i n g f o r the expected molecule  9  cycloxide The  and  t o produce  a derivative  o f n i t r o g e n atoms i n t h e  a l t h o u g h i t was r e a d i l y f o r m e d  from the t e t r a -  (44)<> p r e s e n t i n v e s t i g a t i o n on t h e b i o g e n t i e - t y p e s y n -  t h e s i s of the t e t r a c y c l i n e stageo  number  t o p r e p a r e t h e oxime o f  It i s felt  that  molecule  was t e r m i n a t e d a t t h i s  a l t h o u g h a l a r g e number  of reactions  c o n d i t i o n s were t r i e d , , t h e number o f p o s s i b i l i t i e s  not n e c e s s a r i l y  exhausted  Q  s o l u t i o n c a n be s u g g e s t e d , r o u t e s , which terrarubein^ lation  still  remain  oxygenation  However^ no s t r a i g h t f o r w a r d The k e y s t e p s i n t h e s e v a r i o u s unsolved^, a r e h y d r o g e n a t i o n o f  o f t e r r a r u b e i n a t C-12a  of 6-methylpretetramid  latter. completed  A l l remaining  were  s  and 12a  9  4-hydroxy-  h y d r o x y l a t i o n of the  s t e p s were e i t h e r  successfully  o r do n o t seem t o p r e s e n t a m a j o r  synthetic  problem.  -  II,  Non  Biogenetic  In a l l the t i o n of was  at  overcome i n t h e  cycline the  (5)  chloro  these cule  Type T o t a l  total  nitrogen  G-4  has  b e f o r e the  and  of  (5)  nitrogen  final  closure  Although i t appears that of  (5)  can  p o t e n t i a l l y be  introduction),  the  Tetracycline introduc-  p r e s e n t e d a major problem.  synthesis  derivative  Synthesis of  s y n t h e s e s so f a r a t t e m p t e d ,  by Woodward  syntheses the  42  of  6-deoxy-6-demethyltetra-  recently by  i n the  Muxfeldt" ,  was  3 1  of r i n g  the  synthesis In both  incorporated  C-6  converted  synthesis  This  of the  of  of  i n t o the  mole-  A,  methylated  derivative  to t e t r a c y c l i n e l a t t e r has  not  (see yet  been  reported.  (4)  An  (11)  e x a m i n a t i o n of the  derivatives  showed t h a t  drotetracycline  (4)  available  synthetic  tetracycline  7-chloro-4-dedimethylamino-5a 6-anhy-  possessed a p o t e n t i a l l y a c t i v a t e d  9  methylene  -  group a t C-4,  43  Functional!sation of t h i s position  could  p r o v i d e an e n t r y i n t o t h e dimethylamirio-5ag6-anhydr©tetrac y c l i n e s s u c h a s ( 1 1 ) w h i c h c a n be c o n v e r t e d t o t e t r a p  cycline. During the present study the 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 (a)  attempteds  Bromination amine  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  dimethy1-  0  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  (b)  either  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 tion (c)  Q  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 o r 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  ketones or 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\  are w e l l reviewed 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 mentioning here.  Thus Arakawa and I r i e '  reacted  t u t e d l 3-cyclohexanedione monoenolates (45) ; ?  w i t h N-bromo-  s u c c i n i m i d e a n d o b t a i n e d C - 2 b r o m i n a t e d compounds o r kr  ol)  R =H) 2  o  was b r o m i n a t e d latter amine s,  0  When t h e d i a l k y l a t e d d e r i v a t i v e t h e b r o m i n e e n t e r e d t h e G-6  ( 4 6 "R.-^-'K^"^^) a  w  a  s  found t o react  substi-  (45&-"-Ri=H  C45p  R^R^On^)  position.  smoothly  5  with  The  -  44  -  (46) In the wider realized problem  that  application  of these r e a c t i o n s i t  the t e t r a c y c l i n e s o f f e r e d  than the  above mentioned  examples c i t e d , reviews,,  In the  amino-5aj,6-anhydrotetracycline possible  reactive  Foremost  p o s i t i o n s are  i s the C-2  c a r b o n y l groups„  for the t e t r a c y c l i n e s , producto  Other  C-5p  and t h e  C-Me  halogenation of  had  or those found case of the  p  4-dedimethylof  numerous,,  i s f l a n k e d by  l e d t o G-2  reactive  two  halogenated  c e n t e r s i n (4)  aromatic p o s i t i o n s at C-6 been r e p o r t e d a t G-6  5a 6-anhydrotetracyclines resulted s  i n the  i n a l l halogenations reported  none had  potentially  complex  f r a m e w o r k ' t h e number  p o s i t i o n which  However  a more  was  and  although i n the  0-9=  are No  oxygenation  introduction  45 o f a h y d r o x y l group a t t h i s  position  c  Bromination of 4-dedimethylamino-5a06~anhydrotetra-.. cycline (47)o  (18)  i s r e p o r t e d t o y i e l d the 9~bromo  derivative^  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 position  (49)  (48)  i n the  11a  f o l l o w e d by rearrangement of bromine t o  C - 9 p o s i t i o n i n the p r e s e n c e of hydrogen bromide^?„ the p o s i t i o n o f the bromine i n (47)  the However,  has not been u n e q u i v o c a l l y  established.  (13)  (47)  F o r t h e b r o m i n a t i o n - a m i n a t i o n experiments^ chosen a s t h e f i r s t cycline  synthesis^  of the t h r e e  suggested r o u t e s f o r  i t was n e c e s s a r y a t the  the p o s i t i o n of t h e bromine  w h i c h were  outset to  tetraestablish  i n the above bromo compound  0  I t was ring  D  hoped t h a t  the  46  -  presence of the c h a r a c t e r i s t i c  a r o m a t i c p r o t o n ( s ) i n the  presence^  o r a b s e n c e o f b r o m i n e i n the  Reaction  of  (4)p  chloro-tetracycline w i t h one  n,m,r, w o u l d i n d i c a t e  molar e q u i v a l e n t  the  sample was was  acetylated  freely at  incorporation  rather  soluble  2ol T  and  bromination of  could  of the now  be  I t was  G-9  sample o f  bromine  nitrogen  i t s solubility.  in  analysis 0  As  solvents The  this it  s  acetate  was  I t s n,m,ro showed a s i n g l e t  0  The  acetate  doublets.at  hydrogens.  b r o m i n e a t C-9° r e j e c t e d f o r the  hoped t o a v o i d  ( 4 ) was  of the  2,1  and  I t was  non-brominated 2,9.T* f o r t h e  thus established  T h i s method o f  AB that  C-9  using  of  sake o f c l a r i t y  one  as w e l l as  ionic  The  atom o f bromine  one  The  C-6  and  CThus  molar e q u i v a l e n t  product analysed i n the  i n subsequent d i s c u s s i o n s A),  C-5,  experiments.  reaction conditions.  brominated using  n a t e d a s bromo compound  bromination  purposes of these  in glacial acetic acid.  incorporation  Co,)  (4) with N-bromosuccinimide r e s u l t s i n s u b s t i -  m e t h y l b r o m i n a t i o n by a  Lederle  i n common nomor0  i n chloroform  ( 4 ) showed two  system o f C-8  tution  increase  f o r 1 hydrogeno  compound  the  o f 1 mole o f b r o m i n e  insoluble  to  by  of N-bromosuccinimide r e s u l t e d  a compound whose c a r b o n , h y d r o g e n and indicated  position<>  prepared from 4-dedimethylamino-7(kindly supplied  g  s u g g e s t e d C-9  the  molecule  this  of  for  the  (for  the  sample i s d e s i g -  i n f r a r e d spectrum of  this  - 47 bromo compound was d i f f e r e n t  f r o m t h e C-9 b r o m i n a t e d  When i t was t r e a t e d w i t h a b o u t  one m o l a r  compound  equivalent of dimethyl-  amine  i n methanol f o r 4 days and s u b s e q u e n t l y a n a l y s e d by  paper  chromatography u s i n g a butanol-phosphate  yellow  s p o t was o b s e r v e d  a t R f 0,3.  chloro-5a,6-anhydrotetracycline gave a n o r a n g e - r e d tetracycline  (11), t h e e x p e c t e d o  figure  4-epi-5a,6-anhydro-  (20) h a d a y e l l o w s p o t a t R f 0,45°  (53)  et,al,^  and Blackwood e t a l o ^ ^ 0  reported the iso-  isomer  i n the r e d u c t i o n of the hydrazone  7), t h e oxime  (52, s e e f i g u r e  7) and f r o m  amination o f the tetracycloxide  (44) •>  t h a t t h e 4-epi-isomer  more e a s i l y , i n  tions i  product,  of N-demethyl-4-epi-5a,6-anhydrotetracycline  the major  7-  0  (50)  lation  a green-  (19) showed a y e l l o w s p o t a t R f 0 2S a n d t h e 5a,  6-anhydrotetracycline  Esse  system,  I n t h e same s y s t e m  s p o t a t R f 0 $, w h i l e  0  i s formed  Thus t h e p o s s i b i l i t y  (50) a s (51? see  reductive-  I t t h e r e f o r e appears these  reduc-  was r a i s e d t h a t t h e a b o v e  green  yellow  s p o t a t R f 0=3 was a l s o due t o t h e f o r m a t i o n o f t h e  epimer  o f the expected  product  (53)°  - 48 In another  bromination attempt,  which  simulated the  above c o n d i t i o n s , a p r o d u c t was o b t a i n e d w h i c h h a d a n red  spectrum  pound  indicative  of a mixture  bromo  showed i n f r a r e d  to react  (47)*> w h i l e i n some e x p e r i m e n t s ,  compound  It  i t was n o t e d  f o r longer than  t o the C-9  spectra identical  reaction times,  on (4)  brominations  b r o m i n a t i o n s t h a t were l e f t  stopped  hours,  at intermediate bromo  (A) c o u l d be d e t e c t e d „  i s believed  that t h i s observation i s p a r a l l e l facile  bromine rearrangement  brorao-4-dedimethylaminotetracycline the i n i t i a l  (49)  *  I t i s suggested  bromination takes place at a d i f f e r e n t  at C-4,  attempts  t o r e p e a t t h e p r e p a r a t i o n o f bromo compound  to either  C-9  f o l l o w e d by r e a r r a n g e m e n t  o r incomplete  When some  of these  reacted with l i q u i d was p r o d u c e d  incomplete  dimethylamine  which d i s s o l v e d  5°5°  extract  t o C-9°  site,  A l l (A) l e d  brominationSo  w h i c h c o u l d be e x t r a c t e d w i t h a pH o f  to that  i n 11a-  possibly  form  8  that  bromo-compound  some o f t h e b a n d s a s s o c i a t e d w i t h  of the aforementioned  at  com-  (A) o  In a l l subsequent  that  o f t h e C-9  infra-  b r o m i n a t i o n p r o d u c t s were f o r a s h o r t t i m e , a compound  i n dilute  h y d r o c h l o r i c a c i d and  c h l o r o f o r m from t h e a c i d  Paper chromatographic  showed a g r e e n - y e l l o w  solution  a n a l y s i s of the chloro-  s p o t a t R f 0*3  as i n the  -49first  experimento  solvent  system,  In b u t a n o l - a c e t i c acid-water  an R f  0.5,  o f 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  t h e above g r e e n - y e l l o w  i d e n t i c a l to that  i t s d e s c h l o r o and4-epi d e s c h l o r o  s p o t had  derivatives.  4 - e p i a n h y d r o t e t r a c y c l i n e and t h e  Both  r e a c t i o n product polyamide  showed a n R f a t 0 . 9  powder a s a d s o r b e n t  (12s5;3) as  solvent.  t e t r a c y c l i n e and respectively.  t o g r a m was  plates using  and b u t a n o l - a c e t i c a c i d - w a t e r  I n t h e same s y s t e m  They a l s o  and  on TLC  dimethylamine-  7-chloro  anhydro-  a n h y d r o t e t r a c y c l i n e had R f S o f 0 . 4  corresponding, both tetracycline  (12s5S3)  i n Rf, to 4 - p l  s o l v e n t system  (2s2sl).  anhydro-  e  reaction product.  o b t a i n e d when t h e  ethanol-acetone-water  A similar was  These r e s u l t s  indicated  analysed R f 0.9  The  dehydrated  thin layer  green-yellow  by and  spot at  plates.  bromination-amination experiments  had b e e n r e p o r o d u e e d  (supplied  t o t h e a n h y d r o compound  chromat©graphically g i v i n g a  on p o l y a m i d e  7-  when an a u t h e n t i c sample o f  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 C o ' o ) i t was  the  7-chlorotetracycline.  S e v e r a l months l a t e r  Lederle  chroma-  changed t o  s t r o n g l y t h a t t h e b r o m i n a t i o n - a m i n a t i o n p r o d u c t was 4-epimer of the d e s i r e d  0.9  showed t r a c e s o f s p o t s a t R f  i n q o l o u r and  t o the  0.2  and  which  originally  t w i c e , c o u l d n o t be r e p e a t e d when  an  /  - 50 authentic  sample o f  (53) became  available.  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 r e p r o d u c e the the  original results  i t was thought t h a t  possibly  s u c c e s s e s had been due t o the presence o f t r a c e  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 .  It  amounts 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  in aliphatic  n a t i o n w h i l e i n t h e absence of hydrogen bromide bromination occurs e x c l u s i v e l y ^ .  It  aromatic  seemed p r o b a b l e  s i m i l a r c o n d i t i o n s would be of v a l u e i n the p r e s e n t B r o m i n a t i o n of (4)  bromi-  that  case.  i n the p r e s e n c e of added t r a c e s o f  hydrogen bromide gas gave a produce w h i c h produced o n l y t r a c e s o f 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 i n e . 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 facile  i t was d e c i d e d t o p r e p a r e 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 .  C-9 bromine was t o be s u b s e q u e n t l y  removed by  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 .  The  hydrogenolysis  A l l attempts at  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 o f 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 .  Bromination  t h e C-9 bromo compound w i t h N - b r o m o s u c c i n i m i d e r e s u l t e d  of in  - 51 the  d i s r u p t i o n of the c h a r a c t e r i s t i c acylnaphthalene  phore o  The new u l t r a v i o l e t  5ajlla-dehydrotetracycline  chromo-  s p e c t r u m was s i m i l a r t o t h a t o f (21).  B r o m i n a t i o n a t C-6 was  suggested, Following  the f a i l u r e  o f t h e a b o v e a t t e m p t s , i t was  decided t o brominate an a c e t y l a t e d been r e p o r t e d ^ had  A s a m p l e o f (4)  had  a s i t had  t h a t bromination o f p-acetoxyacetophenone  resulted i n aliphatic  acetate  sample o f (4)  bromination.  was t h u s a c e t y l a t e d *  p e a k s i n t h e n.m.r.  I t showed 3  I t i s believed  resulted i n the formation  o f C-10, C - l l ,  that  acetylation  C-3 t r i a c e t a t e  (54).  (54) This acetate but  reacted  failed  t o react with  N-bromo-succinimide*  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  compound s h o w i n g t h e C-8 a n d 9 a r o m a t i c p r o t o n s ,  a  i n the  n.m.r,,, a s i n t h e s t a r t i n g m a t e r i a l , b u t seemed t o have lost  one o f t h e a c e t a t e  groups,  A deacetylated  sample a n a -  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 a t o m o f b r o m i n e  -  in  the  m o l e c u l e and  infrared  suggesting  carbonylo starting sis  showed a the  showed, t h e  parent  -  1720  strong  formation  Its ultraviolet non-acetylated  52  compound.  instance  of halogenation  the  bonded  unchanged f r o m  Mass s p e c t r o s c o p i c  495-6  (poor  above r e s u l t s s u g g e s t b r o m i n a t i o n a t first  in  of a non-hydrogen  s p e c t r u m was  peak a t  cm"**' b a n d  at C-2  the analy-  resolution).  C-2j(55)»  This  is  The the  of the t e t r a c y c l i n e s .  (55) Simultaneous with several  attempts to  the  above b r o m i n a t i o n  oxygenate  (4)  potassium tertiary-butoxide-oxygen change using  selenium d i o x i d e ,  failed  t o b r i n g about  i n (4)»  In a d d i t i o n attempts t o  introduce  catalytic  procedures'(e.g. f i n e l y  divided  were a l s o  n i t r o s a t i o n reactions with  p-nitroso-dimethylaniline  analysing  oxygen  platinum)  unsuccessful,  Similarlyj and  using  experiments,  f o r two  nitrogen  failed  atoms.  n-butylnitrite  to yield  derivatives  or any  - 53 The  above r e s u l t s s t r o n g l y  p o s i t i o n o f (4) believed  that  C-4  indicate that the  i s highly unreactive.  Nevertheless,  s u c c e s s had b e e n a c h i e v e d  i ti s  i n several of the  o r i g i n a l bromination-amination reactions.  The f a c t  that i n  those  material  could  e x p e r i m e n t s an a c i d s o l u b l e  be e x t r a c t e d  from a chloroform  d u c t and t h a t acid an  t h i s material  s o l u t i o n of the r e a c t i o n  could  s o l u t i o n a t a p a r t i c u l a r pH  amine  coloured  be r e - e x t r a c t e d  p r o d u c t and t h a t  were p e r f o r m e d  with the exception  spectral data,  identical  on them.  i n the separate  It i s believed  of the f i r s t  this  a l l other  of the d e s i r e d  failure  behaviour o f the  of 4-epianhydrotetracycline  bromination  w h i c h h a d r e s u l t e d i n a bromo-compound  traces  from the  a  i n . c o l o u r a n d R f were that  pro-  supports the presence of  Furthermore, the chromatographic reaction  ,  experiments  therefore,  :  compound.  that  experiments  with c h a r a c t e r i s t i c  b r o m i n a t i o n s had y i e l d e d  bromo  both  only  No e x p l a n a t i o n  in reproducibility i s available.  of  -  54  -  EXPERIMENTAL Because o f the 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 nature the m e l t i n g or decomposition t i v e s , none w e r e  p o i n t s of t e t r a c y c l i n e d e r i v a -  recorded.  Ultraviolet  11  s p e c t r a were measured w i t h a C a r y  spectrophotometer, f o r s o l u t i o n s i n ethanol unless mentioned.  Where no  peak r a t i o s are Infrared  q u a n t i t a t i v e s p e c t r a were m e a s u r e d , t h e  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 Nuclear  60,  p a p e r c h r o m a t o g r a p h y Whatman n o .  1 p a p e r , and  t h i n l a y e r c h r o m a t o g r a p h y Woelm p o l y a m i d e p o w d e r , was The  f o l l o w i n g s o l v e n t s w e r e commonly a p p l i e d :  (1)  Butanol  saturated with 0.1  (pH 3 )  (paper s t r i p s were i m p r e g n a t e d w i t h t h e  phase b e f o r e  Butanol-acetic acid-water  (3)  Ethanol-acetone-water  used.  M sodium dihydrogenphosphate aqueous  (12s3s5)  (2s2sl)  i r r a d i a t i o n experiments,  d i r e c t l y t o the  for  using)•  (2)  In t h e  21  m a g n e t i c r e s o n a n c e s p e c t r a were  w i t h a V a r i a n model A For  otherwise  given.  spectrophotometer. taken  of  the  s a m p l e s were e x p o s e d  l i g h t of f o u r f l o u r e s c e n t lamps.  t e t r a c y c l i n e , and  Samples of  some o f i t s d e r i v a t i v e s , w e r e k i n d l y  sup-  - 55 -  p l i e d by the L e d e r l e Co., P e a r l R i v e r , N.J., U.S.A.  Attempted T r a n s f o r m a t i o n s (a)  of Terrarubein  Oxidation of Terrarubein with Terrarubein and  Platinum  (8 mg), was d i s s o l v e d i n 100 ml o f benzene  20 mg o f pre-reduced p l a t i n u m  added. days.  oxide c a t a l y s t was  Oxygen was passed through t h e s o l u t i o n f o r 3 The u l t r a v i o l e t  f e a t u r e s a t \max 450,  spectrum o f the product  showed  272. .250 mu. (1:4:5) (sh)  Paper chromatography, u s i n g s o l v e n t 1, d i d not move the compound from the o r i g i n . (b)  Attempted O x i d a t i o n of T e r r a r u b e i n w i t h Terrarubein  Irradiation  (8 mg) was d i s s o l v e d i n 100 ml of benzene.  Oxygen was passed through while being d i r e c t l y a t e d f o r 24 hours. residue  irradi-  The benzene was e v a p o r a t e d .  The  showed f e a t u r e s a t Amax: 450(&), 380, 270 mu  (1:2:5) Papergram ( s o l v e n t 2) • (c)  R f 0.5 (yellow,  fading)  O x i d a t i o n o f T e r r a r u b e i n w i t h Pd/C and I r r a d i a t i o n A benzene s o l u t i o n o f t e r r a r u b e i n (8 mg), t o which 10 mg of p a l l a d i u m on c h a r c o a l (5%) had been added, was i r r a d i a t e d and oxygen was passed through the s o l u t i o n  - 56 for of  12 hourSo solvento  The p r o d u c t was c o l l e c t e d by r e m o v a l 450 ^ ) , 3 8 0 ,  I t showed /max?  g h  262 mu  (1:2:5). P a p e r g r a m ( s o l v e n t 2)?  Rf 0 9 O  ( p i n k , under U o V .  light)o  Attempted  Hydrogenation  o f Oxydized T e r r a r u b e i n (c)  The b e n z e n e 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 hydrogenated  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  in the ultraviolet  Xmax 4 5 0 | ^ » , 3 8 0 ,  at  The p r o d u c t s  absorbed 26?  mu  (1:2:5)o Papergram  ( s o l v e n t 2)s  R f 0«9  d i h y d r o t e r r a r u b e i n R f 0«5  Attempted on  (yellow)o  Hydrogenation o f Terrarubein with  Palladium  Charcoal  Terrarubein and  (yellow, fading)  (30 mg) was d i s s o l v e d i n 200 m l o f e t h a n o l  hydrogenated  charcoal violet  f o r & h o u r s u s i n g 5$ p a l l a d i u m o n  c a t a l y s t (20  spectrum a t  mg) o  The p r o d u c t h a d a n u l t r a -  Xmax 450» 295. .272 mu (sh)  P a p e r g r a m ( s o l v e n t 2) R f 0»9  (yellow-brown)  (Is4s5)  57 -  (polyamide,  T . L . G ,  s o l v e n t 2) R f 0,9  d i h y d r o t e r r a r u b e i n R f 0,7  (f)  (orange)  (yellow,fading)  0  Attempted Hydrogenatlon of Terrarubein Medium  (Sodium  Terrarubein  i na Basic  bicarbonate)  (40 mg) w a s s u s p e n d e d i n 50 m l 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 bicarbonateo  P a l l a d i u m on c h a r c o a l  added and hydrogenated f o r 8 hours.  (3Q#p 60 mg) w a 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 extracted with chloroform. of t h e product  The u l t r a v i o l e t  spectrum  showed f e a t u r e s a t ^ m a x 435p 290, 265 mu  (ls4§5) T o L o G ,  (polyamide  Papergrams  (g)  s o l v e n t 2)  ( s o l v e n t 2)  R f 0,4  R f 0,5  (yellow,  fading)  (yellow-brown)  Attempted Hydrogenatlon o f T e r r a r u b e i n  i na Basic  Medium  (Triethylamine) Terrarubein (lsi,  (40 mg) w a s d i s s o l v e d i n w a t e r - g l y m e  mixture  20 m l ) c o n t a i n i n g a b o u t 2% t r i e t h y l a m i n e a n d was  hydrogenated w i t h p a l l a d i u m on c h a r c o a l r o o m t e m p e r a t u r e f o r 20 h o u r s .  (30#  p  50 mg) a t  The s o l u t i o n w a s 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  — 58 —  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 h a d a b s o r p t i o n b a n d s a t Xmax 435? 9 5 ( j p 2  s n  270,  (Is4s5)  260,  .mu, l  shJ  P a p e r g r a m ( s o l v e n t 2) T,L,C, (polyamide The  R f 0o5  s o l v e n t 2)  (yellow, fading) R f 0,5  a b o v e r e a c t i o n was r e p e a t e d  charcoal  (yellow, fading)  u s i n g r u t h e n i u m on  (2%) a s c a t a l y s t w i t h s i m i l a r  Attempted Hydrogenation o f Terrarubein (Sodium  i n B a s i c Medium  hydroxide)  Terrarubein ethanol  results.  (10 mg) i n 100 m l o f  (1?1)  6N s o d i u m  hydroxide-  a n d p a l l a d i u m o n c h a r c o a l 5% (10 mg) was  h y d r o g e 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 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  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  A max?  R f 0,8  (yellows  of Terrarubein with  Platinum  Terrarubein and  chloroform  6N  a  450, 390, 273 mu (ls2,5)  P a p e r g r a m ( s o l v e n t 2)  Reduction  pressure  fading)  (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  prereduced  platinum  (10 mg) w a s a d d e d a n d h y d r o g e n -  - 59 -  ated f o r 4 hours.  A l i q u o t s were t a k e n every § hour  and t h e i r u l t r a v i o l e t spectrum examined. the g r a d u a l d i s a p p e a r a n c e  o f t h e 450 mu band and the  c o n s o r t e d appearance of a 350 mu b a n d . it  showed  Pressure  Xmaxs 350, 271 mpL  (40  p,s,i,)  (10 mg) i n 100 ml o f 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 o f 40 p , s , i ,  A f t e r 4 hours  (1?3)  H y d r o g e n a t l o n of T e r r a r u b e i n  Terrarubein  T h i s showed  (10$)  were shaken a t a  of hydrogen f o r 5 h o u r s ,  pressure  ^maxi 450,  270,  260 mu (Is5s5) Papergram?  (solvent  2) R f 0,4  (yellow,  P r e s s u r e H y d r o g e n a t l o n of T e r r a r u b e i n Terrarubein  fading)  (600  ($0 mg) i n 250 m l o f s a t u r a t e d  sodium b i c a r b o n a t e  was hydrogenated at 600  hydrogen p r e s s u r e and room temperature  p , S o i , )  ethanolic p,s,i,  f o r 14 h o u r s .  The s o l u t i o n was a c i d i f i e d w i t h d i l u t e h y d r o c h l o r i c a c i d , d i l u t e d w i t h w a t e r and e x t r a c t e d The u l t r a v i o l e t  spectrum o f t h e p r o d u c t remained  unchanged f r o m s t a r t i n g  material.  Oxygenation of T e r r a r u b e i n Terrarubein  with chloroform.  (Dimethyl S u l f o x i d e )  (50 mg) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e  - 60 -  (50 ml) and f r e s h l y r e d u c e d p l a t i n u m was a d d e d .  catalyst  (50 mg)  The s o l u t i o n was exposed t o s o f t l i g h t and  oxygen was passed t h r o u g h t h e Fresh catalyst  was added at  s o l u t i o n f o r 3 days.  suitable  intervals.  The  u l t r a v i o l e t spectrum of t h e p r o d u c t ^ i s o l a t e d by d i s t i l l a t i o n o f the f e a t u r e s at  s o l v e n t under reduced p r e s s u r e ,  Xmaxs  382,;  2 7 5 W°  showed  (2s5)  Attempted R e d u c t i o n of O x i d i z e d T e r r a r u b e i n (1) The o x i d i z e d t e r r a r u b e i n dioxane  ( 1 , 10 mg) was d i s s o l v e d i n  (25 m l , p a l l a d i u m on c h a r c o a l ,  5$)  was added and hydrogenated f o r 5 h o u r s , t h e u l t r a v i o l e t spectrum c o u l d be  O x y g e n a t i o n of T e r r a r u b e i n  catalyst  No change i n  detected.  (Dimethyl Sulfoxide/Magnes-  ium A c e t a t e ) Terrarubein  ( 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  tetrahydrate,  The s o l u t i o n was oxygenated i n a h y d r o g e n a t l o n After (1/2  1 mole-equivalent hour) an a l i q u o t  apparatus.  of oxygen had been absorbed  (20 ml) was w i t h d r a w n .  This  ali-  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 d a r k brown s o l i d c o l l e c t e d by c e n t r i f u gation.  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), ane)  450, 272 mu (7 100s  It  showed  Amax ( d i o x -  Hp500)  9  A n a l , C a l c , f o r C ^ I i ^ O . ^ N , 3 » 7 ; found 3»3» IR(KBr):  3300, 1700, '  g  \ 3)  740 cm"  1640,  1600  P  1220,  1100,  860,  The remainder o f the o x y g e n a t i o n m i x t u r e was f u r t h e r oxygenated f o r I S h o u r s . mole-equivalent above,  D u r i n g t h a t time a n o t h e r  o f oxygen was a b s o r b e d ,  Worked up a s  i t p r o v i d e d 250 mg o f a brown s o l i d .  It  showed  X max(DMS0/MgAc ) 2  IR(KBr):  similar to  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  hydrosulfite), The mono-oxygenated, t e r r a r u b e i n ved i n d i m e t h y l s u l f o x i d e  (37*  50 mg) was d i s s o l -  (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 quinone had changed t o o r a n g e f i e d and the fugation.  0  the  The s o l u t i o n was a c i d i -  s o l i d t h a t was f o r m e d , s e p a r a t e d  by  centri-  I t was washed s e v e r a l t i m e s w i t h w a t e r  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 o f a brown solid  Amax ( 9 7 ^ 2 3 0 ^ 1% ^ B ^ O y ) ; 520, 470, 317,  280 mu (76,000s 15 OOOs 42,000s 2 2 , 3 0 0 ) , 5  - 62 R e d u c t i o n of the Dioxygenated T e r r a r u b e i n  (sodium  (36)  hydrosulfite), 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 t h e c o l o u r o f t h e quinone had changed t o o r a n g e . i s o l a t e d as i n (o),  A f t e r a c i d i f i c a t i o n the It  showed A max.(DM50) .400, 285 mu  (4-hydroxy m e t h y l p r e t e t r a m i d had  (1:3)  s o l i d was  Xmax (DMSO):  451* 3 6 3 , 305, 266 mu ( 2 : 1 : 1 : 5 ) ) R e d u c t i o n of t h e D i o x y g e n a t e d T e r r a r u b e i n (36) Phenol  in Hi/  0  Dioxygenated t e r r a r u b e i n p h e n o l (4 m l ) ,  (20 mg) was d i s s o l v e d i n  To i t was added h y d r o i o d i c a c i d (3 ml)  and the m i x t u r e kept a t 120°  f o r 4-5 m i n u t e s  s  It  was  t h e n added t o i c e w a t e r c o n t a i n i n g sodium hydrogen sulfite.  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 d a r k - b r o w n solid,  It  showed  Xmax (97$  HgSO^-  156  Na B^0 ): 2  7  520, 470, 317> 280 mu ( 1 . 1 : 3 : 2 )  A c t i o n o f m - C h l o r o p e r b e n z o i c 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 - c h l o r o p e r b e n z o i c 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 s t a n d a t room  temperature.  A l i q u o t s were removed d a i l y .  d a y s a papergram ( s o l v e n t 3) R f s 0,2  (blue-fluorescent);  and  (brown).  0,9  (ls4)  It  After 2  showed s e v e r a l s p o t s 0,4  (brown); 0 , 6  at  (yellow)  showed Amax(Dioxane)s 4 0 2 ,  273 mu  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 Anhydrotetracycline  (20)  (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 acid  (25 m l ) .  The s o l u t i o n was l e f t a t room t e m p e r a t u r e  t h e d a r k f o r 12 h o u r s *  in  The a c e t i c a c i d was removed under  r e d u c e d p r e s s u r e and t h e 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 in  w a t e r and t h e pH a d j u s t e d t o 4 ° 0 w i t h 6N sodium h y d r o x -  ide,;  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 t h e benzene e x t r a c t d e p o s -  i t e d a r e d amorphous s o l i d (52 mg), ^(KBr) 1120, I R  NMR  l 6 k Q  1090,  (KBr)  1375,  1  o : C  (a)»  *  l62G  1 5 7  °  9  1 4 3 5  »  1 3 8  °»  1 2 6 5  »  >  1215  820 c m " . 1  A  nnydrotetracycline s  1335, (CDC1 )s  1320, 2,9  1245* 1225, (doublet),  1645,  1175 3.7  9  1620 <> 1570,  1110,  (doublet)  754 X  1445;>  cm" , 1  (Area l s i ) .  -  64 -  Preparation of 12a-Deoxytetracycline A mixture of t e t r a c y c l i n e zinc dust  (10)  h y d r o c h l o r i d e (2 gm) and  (4 gm) i n 15% aqueous ammonia (50 ml) was s t i r r e d  f o r 2 h o u r s i n a stoppered f l a s k o f i l t e r e d and t h e f i l t r a t e  The r e a c t i o n m i x t u r e was  neutralized with  concentrated  h y d r o c h l o r i c a c i d , c a r e b e i n g t a k e n t o keep t h e t e m p e r a t u r e below 2 0 ° .  A t pH 7 , 0 t h e 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 m l o f water by a c i d i f y i n g t o pH o f lo& w i t h 6N h y d r o c h l o r i c a c l d o  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 t h e p r e c i p i t a t i o n o f a f l o c u l e n t y e l l o w s o l i d w h i c h 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 hours.  i n a c o n s t a n t e t h e r e x t r a c t o r f o r 50  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  re c r y s t a l U s e d from 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) 12a-Deoxytetracycline  ( 1 0 , 42 mg) was heated w i t h 3 , 5  ml o f g l a c i a l a c e t i c a c i d and 1 ml of $1% hydrobromic i n g l a c i a l a c e t i c a c i d added,  acid  The s o l u t i o n was heated f o r  a f u r t h e r 15 m i n u t e s 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 o f a brown s o l i d . 328,  273 mu ( 3 : 2 : 2 : 4 )  I t showed  Xmax: 440j>  380,  - 65 The hydrobromide s a l t  ( 4 0 mg) was suspended i n 1 0 0 ml  o f water and t h e pH a d j u s t e d t o 7 » 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 c a r e not t o i n c r e a s e E x t r a c t i o n i n a constant  the pH above 3 . 0 .  chloroform extractor  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 . It  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 ) (solvent  1 2 & - 0 x y g e n a t i o n of  2)  Rf 0 . 1  (yellow)  Rf 0 . 5 (yellow)  Dihydroterrarubein  (21)  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 p r e s e n c e o f f r e s h l y r e d u c e d 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 solution.  Additional catalyst  suitable intervals.  ( 2 x 2 5 mg) was added a t  U l t r a v i o l e t s p e c t r a were  obtained  p e r i o d i c a l l y u n t i l t h e 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 s a p p e a r e d and the t o that of a n h y d r o t e t r a c y c l i n e .  spectrum had become i d e n t i c a l Paper chromatograms  at  t h i s stage showed no t r a c e o f 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 ( y e l l o w ) the o t h e r a t R f 0.3 (yellow-brown)  (solvent  1).  The R f 0 . 5 spot was e l u t e d f r o m the papergram and showed t h e same i n f r a r e d spectrum a s  anhydrotetracycline.  66 -  P r e p a r a t i o n o f 6-Methylpretetramid (14a) Terrarubein hot phenol  (0,15 gm] was s u s p e n d e d i n a m i x t u r e o f  (12 ml) and h y d r o i o d i c a c i d  t u r e was h e a t e d  (10 m l ) .  i n a n o i l b a t h a t 120° f o r 4 h o u r s .  s o l u t i o n was a d d e d t o i c e w a t e r t o w h i c h s u l f i t e had been added. ether.  The mixThe  sodium hydrogen  I t w a s r e p e a t e d l y washed w i t h  The a q u e o u s m i x t u r e was t h e n c e n t r i f u g e d a n d 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 a c e t o n e a n d ether t o yield  4 4 8 , 355,  0,12 gm o f a b r o w n s o l i d .  I t showed  Xmax  3 0 0 , 272 mu ( i s i , 3 , 5 )  Oxidation of 6-Methylpretetramid with Carols Acid 6-Methylpretetramid of p y r i d i n e  ( 3 1 mg) w a s d i s s o l v e d i n a m i x t u r e  (2 m l ) a n d p o t a s s i u m h y d r o x i d e  o r a n g e s o l u t i o n was c o o l e d t o 0  G  (5%* 3 ml).  and p o t a s s i u m  The  persulfate  (40 mg, i n 2 m l o f w a t e r ) w a s a d d e d d r o p w i s e d u r i n g 1 h o u r . I t w a s k e p t a t 0° f o r 5 h o u r s . produced  a black solid  acetone a n d e t h e r ,  Acidification  (22 m g ) ,  I t showed  This solid  \ma.x  t o pH 3  was w a s h e d w i t h  5 0 0 , 3 7 0 , 2 4 0 mu (.1 s5S7)  On p a p e r g r a m s i t d i d n o t move f r o m t h e o r i g i n .  Oxidation of 6-Methylpretetramid sium  ( D i m e t h y l s u l f o x i d e / Magne-  acetate), 6-Methylpretetramid  (20 mg) w a s d i s s o l v e d  i n dimethyl  - 67 -  sulfoxide  (15 m l ) c o n t a i n i n g 1% m a g n e s i u m a c e t a t e  hydrate.  The s o l u t i o n c h a n g e d f r o m o r a n g e t o r e d when  o x y g e n was p a s s e d t h r o u g h i t f o r 2 d a y s . 278 mu  520,  (DMS0/MgAc ) ; 2  (6-Methylpretetramid  tetra-  Xmax  I t showed  (2s5)  Xmax(DJ60/MgAc ) s'.. 485, 2  280  mu  (1;3)  After  s e v e r a l more d a y s o f e x p o s u r e t h e s p e c t r u m 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 b y d i l u t i o n  hydrochloric acid  269* 250 mu  Reduction The phenol the  Xmax! 452  I t showed  9  of Oxidised  6-Methylpretetramid  (1 m l ) .  (4 mg) w a s d i s s o l v e d i n  To i t was a d d e d h y d r o i o d i c a c i d  h e a t e d a t 120°  mixture  above  f o r 4-5 m i n u t e s .  (1 m l ) a n d  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 .  2 4~ ^ S 0  The  was e x t r a c t e d w i t h e t h e r and t h e a q u e o u s l a y e r c e n -  trifuged t o give H  380,  (2:ls6:5)  brown s o l i d o b t a i n e d  mixture  0,1N  I t was washed w i t h w a t e r a n d a c e t o n e t o  0  5 mg o f a b r o w n s o l i d o  yield  with  N a  2 4 7* B  0  a  dark solid 5 5 0 p  ** °*  A c t i o n o f Fremy's S a l t on 6-Methylpretetramid  9  (1 mg). 3 G  °  3  2 8 0  I t showed W  (8,5  ( l s l s 2 s / f )  6-Methylpretetramid (5 mg) w a s 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% m a g n e s i u m a c e t a t e Fremy*s s a l t  Xmax (97$  tetrahydrate  (3 m l ) ,  mg) was a d d e d a n d t h e s o l u t i o n s t i r r e d i n  -  a i r f o r two d a y s . to blue. The  68  I t s c o l o u r changed  I t showed  f r o m orange t o g r e e n  A max(DMS0/MgAc ):  6 5 0 , 6 0 0 . 280  2  mu.  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  acid  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:  A c t i o n o f Fremy's S a l t  550, 280 mu ( 1 : 4 )  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% m a g n e s i u m a c e t a t e t e t r a hydrate  (10 m l ) .  E x c e s s Fremy's s a l t  (50 mg) was  added.  T h e m i x t u r e was l e f t a t r o o m t e m p e r a t u r e f o r 2 h o u r s . s o l u t i o n became d a r k b l u e . 6 5 0 , 6 0 0 , 280 mu  The  Xmax(DMS0/MgAc2):  I t showed  (1:1:9)  A c t i o n of m-Chloroperbenzoic Acid  on  4-hydroxy-6-methyl-  pretetramid 4-hydroxy-6-methylpretetraraid tetrahydrofuran  (60 mg) was d i s s o l v e d i 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  a d d e d a n d t h e s o l u t i o n k e p t a t 0° f o r 72 h o u r s .  (25 mg) The  was  tetra-  h y d r o f u r a n was r e m o v e d u n d e r r e d u c e d p r e s s u r e a n d 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 . ation ing  showed a n u l t r a v i o l e t  The r e s i d u e o f t h i s  spectrum i d e n t i c a l t o the  m a t e r i a l , while the ethanol s o l u t i o n  showed  Amax(dioxane):  tritur-  (orange-red)  4 4 0 ( j , 3 8 0 , 279 mu s  (1:3:5)°  start-  -  Papergram  (solvent  3)  69 -  showed a y e l l o w  i n g m a t e r i a l ) and a l o n g  s p o t R f 0,7  streak from o r i g i n  (start-  ( p i n k ) <>  Brominations of Dedimethylaminoanhydro-7-chlorotetracycline (a)  (4)° To a s o l u t i o n acetic  o f 46 mg o f (4) i n 40 m l o f g l a c i a l  a c i d , b r o m i n e (1.2 m o l a r e q u i v a l e n t s ) i n  glacial  acetic  acid  (2 m l ) were a d d e d .  The  solution  a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 9 h o u r s . D i l u t i o n w i t h w a t e r f o l l o w e d by e x t r a c t i o n chloroform yielded  Xmax: 1 1 1  (KBr)  451* 350* 272 mu l  6  6  0  Anal. Calc. Found  40 mg  p  l  6  3  0  p  1  5  5  °  s  o f a brown s o l i d .  solid):  I t showed  (2,1,5). ^ 5 0 / 1 2 1 5 * 1105*  1050  cm""  1  C, 48,38, H, 3.03 =  f o r CgQH-^NOyBrGl  ( f o r crude  with  C, 47=29; H.3»80„  47 •(b)  A s o l u t i o n o f 1,08  gm o f (4) i n 20 m l o f g l a c i a l  acetic  a c i d was c o o l e d a n d s t i r r e d j u s t above i t s f r e e z i n g point. acetic  A solution  o f 0,4  o f bromine i n 1 ml o f  a c i d was a d d e d d r o p w i s e  p r e c i p i t a t i o n commenced. was f i l t e r e d  showed  s  After  w i t h i n a few m i n u t e s 2 hours the  a n d t h e p r e c i p i t a t e washed  W i t h e t h e r and d r i e d It  gm  Xmax:  to give  0,85  gm  445* 335* 275 mu  solution  thoroughly  o f a brown  (2:1:6).  solid.  - 70 -  IR(  K B r  1660, 1630, 1575* 1420, 1215, 1060, 1035*  y:  1000  cm" . 1  A n a l , C a l c , f o r C g g H ^ N Q y B r C l s . C, .48.32; H, 3=03, Found C, 47o90» H, 3»25,  in •(c)  A mixture  o f 38 mg o f (4) was  s u s p e n d e d i n 15 ml o f (18  c h l o r o f o r m and N - b r o m o s u c c i n i m i d e to  s t a n d a t room t e m p e r a t u r e  orange  solution  Precipitation an  was o b t a i n e d  reaction  8  A  S  l  n  (  b  "(d)  acetic bromine to  commenced  upon c o m p l e t i o n  and washed w i t h  ether.  o f 100  mg  o f (4)  Yield  C  s  48.22; H  3 = 03,  p  i n 50 mg  of g l a c i a l  slowly 1 molar e q u i v a l e n t of  i n 2 ml o f a c e t i c  w a t e r and e x t r a c t e d w i t h  acid.  The s o l u t i o n  chloroform.  c h l o r o f o r m was removed t o y i e l d 80 mg p  o f the  H, 3,53»  a c i d was a d d e d  440  after  (2;ls6),  s t a n d a t room t e m p e r a t u r e f o r 8 h o u r s  A maxs  clear  )  C, 48,38;  To a s o l u t i o n  solid  solid  Anal, calc, f o r C^H^NOyBrCls Founds  A  allowed  a f t e r a few m i n u t e s .  440* 335* 275 mu  /\maxi  35 mg, "(KBr)  was f i l t e r e d  The  was  f o r 3 hours,  of a yellow-brown  hour o f s t a n d i n g .  mg)  332* 273 mu  (2sls6),  a  was  left  diluted  with  After  drying, the  o f a brown s o l i d ,  - 71 -  IR/™ IKBrJ  s i m i l a r to  (b)  A n a l , CalCo f o r C^oH^NOyBrCls Found ( f o r crude s o l i d ) ?  (e)  4S°38§  C, 50.01;  H , 3,03 o  3<>92,  Hydrogen bromide gas was passed t h r o u g h a of 150 mg of  solution  (4) i n 80 ml o f g l a c i a l a c e t i c  f o r a few m i n u t e s  s  t h e bromine  (1,2  i n 2 ml o f a c e t i c a c i d was a d d e d ,  molar  acid equivalents)  A f t e r 5 hours  the  r e a c t i o n m i x t u r e 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 P 2  272  mu ( 2 : 1 : 6 ) •^(KBr)  5  S  i  m  i  l  a  r  *o  Co)  Anal,Calc, for C^H^NOyBrCls Found ( f o r crude s o l i d ) :  C  g  C, 48,38; H 50,9; H  &  s  3=03,  4,2,  R e a c t i o n s o f the Monobrominated Compounds ( a  g  b  9  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 o f d i m e t h y l a m i n e .  5 days s t a n d i n g a t room t e m p e r a t u r e spot  (Rf 0,3)  Starting  a greenish-yellow  d e v e l o p e d on a papergram  7-Chloroanhydrotetracyeline m a t e r i a l R f 0,9  Rf 0,5  (brown).  After  (solvent  (yellow).  1),  72  (b)  Monobromo  compound  (b  p  10  i n 2 ml o f  mg)  d i m e t h y l a m i n e was h e a t e d g e n t l y u n t i l  liquid  a l l t h e amine h a d  e v a p o r a t e d , c h l o r o f o r m added and t h e s o l u t i o n for  a few m i n u t e s .  addedo  (c)  compound  ( d , 20  dimethylamine a s above, a yellow  compound.  with dilute  chloroform. reduced  (5  ml)  No c o l o u r was e x t r a c t e d i n t o t h e a c i d  Monobromo  pH 5«5  10%  Hydrochloric acid  refluxed  mg)  The a c i d  i n b u l k and examined  liquid  extracted  s o l u t i o n was a d j u s t e d t o  sodium h y d r o x i d e  The p a l e y e l l o w  layer,  was t r e a t e d w i t h  Hydrochloric acid  was  and e x t r a c t e d  chloroform extract  with  was  chromatographically.  Papergrams; (1)  Solvent  (1)  R f 0,3  (green-yellow)  7-chloroanhydrotetracycline Anhydrotetracycline  R f 0,4#  4-Epianhydrotetracycline (2)  Solvent  (2)  R f 0,5  (reddish-yellow)  (yellow)  R f 0,28  (yellow)  (green-yellow)  7-Chloro-anhydrotetracycline Anhydrotetracycline  R f 0,5  R f 0,5  4-Epianhydrotetracycline  R f 0,5  (reddish-yellow)  (yellow)  R f 0,5  (yellow)  - 73  Polyamide T L C : 0  (1)  Solvent  0  0  (2)i  Rf 0 8  (yellow-brown)  o  7-ChloroanhydrotetracyGline trace  Rf,0 8  Anhydrotetracycline R f Qo8  Rf 0 4  Solvent  (3)*.  (yellow-brown)  R f 0.9  R f 0c8  Rf 0 9  Anhydrotetracycline Rf 0 9  R f 0=4  ultraviolet  a t 428  9  340  9  (yellow)  R f 0,8  (yellow-brown)  spectrum of the product  270 mu  Monobromo compound weak s p o t  (yellow)  (yellow-brown)  0  4-Epianhydrotetracycline The  Rf 0,3  (yellow-brown)  0  trace  (yellow-brown)  (yellow-brown)  7-Chloroanhydrotetracycline trace  (yellow)  3  4-Epianhydrotetracycline  (2)  (yellow)  0  (yellow-brown)  o  trace  Rf 0 2  at Rf 0 3 o  showed  features  0  (e) was t r e a t e d (solvent  as above  1) d e v e l o p e d  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  R f 0o3  A  0  very  0  (greenish-  yellow)  B r o m i n a t i o n s of 9-Bromo-dedimethylaminoanhydroaureomycin  (a)  T o a s o l u t i o n o f 48 mg  o f (47)  i n 5 ml o f warm  (47)  chloroform  - 74 -  was  a d d e d 20  carbon  mg  of N-bromosuccinimide,  tetrachloride  p i t a t e washed w i t h  was  added and  carbon  After  the  hours  resulting  tetrachloride  30 mg o f a brown s o l i d  ether to y i e l d  24  and  preci-  with  ethyl  Xmax:  390,  1135  cm" .  254  mu (1:2), IR  ( K B r )  1775* 1710, 1635* 1575* 1410,  s  Anal, calc, for C Founds.  (b)  A  at  H  1 / f  N0 ClBr : 7  C,  2  41 = 7;  o f 50  treated  mg  of  (47)  i n 25  ml  of a c e t i c  At the  end  o f t h i s time  m e t r i c t i t r a t i o n gave n e g a t i v e r e s u l t s o l u t i o n was  chloroform.  s  i  m  H  A max, a  r  to  2o41; B r , 27,2, C I ,  Found:  (a)  Dibromo compound  N,  Compound  ( b , 10  50  (2:1:6),  C,  41,7;  H  9  2,41;  6,09,  C, 43,33; H, 3,13;  R e a c t i o n s of Dibrominate  extracted with  material.  Anal, Calc, f o r C^H-^NOyBr^ls N,  bromine.  chloroform yielded  444p 338, 273 mu starting  hours  iodo-  f o r free  d i l u t e d w i t h w a t e r and  E v a p o r a t i o n of the  mg o f a s o l i d (KBr)  acid  w i t h 1 e q u i v a l e n t o f b r o m i n e f o r 43  room t e m p e r a t u r e .  The  2,44*  H,  42,5p H, 3c09<>  C,  solution  was  2 0  1  mg)  3-27;  B r , 19=11; C I ,  (b) w i t h  was  8,31,  Dimethylamine  dissolved  i n dimethyl  „ .75 -  amine  (2 ml)  and  treated as before.  oped i n the a c i d  (b)  layer during  No  colour devel-  extraction,  S e a l e d Tube R e a c t i o n  The  d i b r o m o compound  amine  (2 ml)  in a  (10  mg)  was  sealed tube.  The  s o l u t i o n became b l a c k a f t e r  was  stopped  before.  and  No  dissolved The  tube  1 hour.  i n dimethyl-  was  heated  The  reaction  t r e a t e d w i t h c h l o r o f o r m and  c o l o u r appeared  i n the a c i d  acid,  at  as  layer,  A c e t y l a t i o n of 7-chloro-dedimethylaminoanhydrotetracyeline A ml  of a c e t i c  ture  f o r 20  about  500  peared of  solution  o f 0,5  anhydride hours.  ml  of  gm  of  was  The  (4)  i n 5 ml  allowed to  stirred  w a t e r and  the  solid.  Amax:  400,  yellow  spots  was  poured  yellOw  f i l t e r e d , washed w i t h w a t e r and  a pale yellow  o f p y r i d i n e and  s t a n d a t room  r e a c t i o n mixture  dried  342,  solid  2,5  temperainto that  ap-  t o g i v e 0,2  330,  272  (4)  mu  gm (2:3:  4:8) T,L.C, N.M.R,:  (solvent 2,2  two  -f (doublet);  3 A c e t a t e and  Bromination  3)s  1 methyl  of Acetate  Brominations  2,9  a t 7-8  Rf  T(doublet) X  0,6  and  0,8  ( A r e a 1:1);  also  °  Mixture  w i t h N-bromosuccinimide  50°,  i n carbontetra-  - 76 -  c h l o r i d e f a i l e d t o change the compound, o f ( 4 ) ( 1 0 0 mg) was d i s s o l v e d in. 1 ml  Acetate mixture  a c e t i c a c i d and bromine 1 0 0 mg i n 1 , 5 ml o f  of g l a c i a l  a c e t i c a c i d was added,  The s o l u t i o n was l e f t  7 days a t room temperature i n the dark.  t o stand f o r  The a c e t i c  acid  was removed i n vacuum and r e p l a c e d w i t h 5 ml o f c h l o r o f o r m and 4 0 ml o f petroleum  ether  30-60°) was added slowly  (b.p  c a u s i n g t h e p r e c i p i t a t i o n of 80 mg of an orange y e l l o w  solid  ToL'.Co ( s o l v e n t ' 3 ) ; R f , 0 , 7 ( y e l l o w ) , NoM,R,  Showed l o s s o f one a c e t a t e  p  otherwise  no change  from  starting material, IoR,  Showed a c e t a t e s p r e s e n t .  H y d r o l y s i s of Brominated  Acetate  Brominated a c e t a t e (40 mg) was suspended i n 10 ml o f e t h a n o l and f i l t e r e d , 1 0 ml of 6 N h y d r o c h l o r i c a c i d was added t o the f i l t r a t e  and the mixture  was l e f t  on steam  b a t h f o r 5 hours a l l o w i n g t h e e t h a n o l t o evaporate, A brown s o l i d mu I R  ( 3 0 mg) separated,  (8 300p 3*400 9  :  1735p 1 6 5 5 , 1 6 1 5 , 1 5 8 0 , 1 4 1 0 , 1 2 2 0 / 8 2 5  Anal, c a l c , f o r C, 4 8 , 3 8 ;  439» 3 3 0 ^ , 2 6 4  26p300)„  ?  (KBr)  Xmax?  H  s  cm"" . 1  C QH 5N0yBrCl: 2  1  3 , 0 3 ; . N, 2 . 8 4 ; C I , 7=14; B r , 1 6 . 1 0 ,  0 , 22.47 =  - 77 -  N , 2.80;  C, 50.10;.H, 3=22;  Found:  C I , 7»25;  B r , 16.57;  0, 23.94. Molo w t o ( f o u n d by mass s p e c t r o m e t r i c a n a l y s i s ) :  Acetylation  o f 9-Bromo-dedimethylaminoanhydroaureomycine  A solution 1 ml o f a e e t i c The  and  o f 200 mg o f (47) i n 2 m l o f p y r i d i n e anhydride  was a l l o w e d  s o l u t i o n was t h e n p o u r e d  sulting  to yield  was r e p r e c i p i t a t e d ating  with  into  t o stand  stirred  130 mg o f a p a l e y e l l o w by d i s s o l v i n g  petroleum  and  w a t e r and t h e r e w i t h water  solid.  This  solid  i t i n c h l o r o f o r m and t r i t u r -  ( b . p . 30-60°)  ether  (47)  overnight.  f i l t e r e d , washed t h o r o u g h l y  precipitate  dried  495-6.  Xmax:  390, 333s  321, 273 mu (2:3:4:8) T.L.C.:  (solvent  N.MoR.:  s i n g l e t 2.1 T .  Attempted Selenium  3)  Rf  0 6, o  The t e t r a c y c l i n e acid  (50 m l ) .  and t h e m i x t u r e  (yellow)  d i o x i d e O x i d a t i o n o f 7-Chloro-5a,6  4-dedimethylamino t e t r a c y c l i n e  acetic  0.8  (4)  (4* 50 mg) was d i s s o l v e d Selenium  refluxed  anhydro-  dioxide  f o r 24 h o u r s .  in glacial  (100 mg) was added The r e a c t i o n  was c o o l e d , 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  product  chloroform.  - 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  shown t o be u n r e a c t e d s t a r t i n g  was  material,.  T h i s e x p e r i m e n t was r e p e a t e d u s i n g 70%  acetic  acid,  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 .  Attempted  Oxygenation of 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 .  Potassium metal butyl alcohol.  (200  mg)  was a d d e d t o 10 m l o f d r y t -  To t h i s s o l u t i o n was a d d e d  dimethyl formamide  (4*  80 mg)  and  (10 m l ) . Oxygen was p a s s e d t h r o u g h t h e  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 v a c u o , w a t e r added and e x t r a c ted  with ether.  I t showed a n u l t r a v i o l e t s p e c t r u m  and  chromatographic behaviour i d e n t i c a l to the s t a r t i n g  Attempted  Nitrosation  amino t e t r a c y c l i n e (a)  of  material.  7-Chloro-5ag6-anhydro-4-dedimethyl-.  (4),  The t e t r a c y c l i n e  (4* 0,5  gm)  ml) and p y r i d i n e  (8 m l ) .  dissolved  i n ethanol  Sodium carbonate  (10%,  was a d d e d f o l l o w e d b y p - n i t r o s o d i m e t h y l a n i l i n e chloride  (1.0  gm),  The  s o l u t i o n was r e f l u x e d  10  a g a i n f o r 1/2  extracted with ether.  for 6 acid,  hour, d i l u t e d w i t h water  and  The e t h e r was  The  stripped,  ml)  hydro-  hours, cooled, a c i d i f i e d with d i l u t e hydrochloric refluxed  (40  - 79 brown r e s i d u e  showed  Xmax;  showed no i n c o r p o r a t i o n of  (b)  402,  267  (ls3)o  Analysis  nitrogen.  7-Chloro-4-dedimethylamino-5a,6-anhydrotetracycline  (4*  100 mg) was d i s s o l v e d i n 15 ml o f d r y d i g l y m e i n a t h r e e necked f l a s k *  A slow stream o f 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 o f 0.03  stirred solution.  A solution  ml of n - b u t y l n i t r i t e i n 3 ml o f d i g l y m e was  added i n s m a l l p o r t i o n s . darkened p r o g r e s s i v e l y .  The c o l o u r o f the  solution  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 c o n t i n u e d f o r an h o u r . t h i s the  After  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  was e v a p o r a t e d t o y i e l d a d a r k brown s o l i d .  solvent  T h i s was  r e p r e c i p i t a t e d from i t s dioxan  s o l u t i o n by a d d i t i o n o f  petroleum ether ( b . p . 30-60$).  I n f r a r e d and u l t r a -  v i o l e t spectrum o f t h i s starting  sample showed no change f r o m  material.  P r e p a r a t i o n of 4 O x o - 4 - d e d i m e t h y l amino hemiketal  tetracycline-4a6-  (44)  Tetracycline  h y d r o c h l o r i d e (5 gm) was d i s s o l v e d i n 250  ml o f water c o n t a i n i n g c o n c e n t r a t e d h y d r o c h l o r i c (2 ml) To t h e  s o l u t i o n , s t i r r i n g a t room t e m p e r a t u r e  gm of powdered N - c h l o r o s u c c i n i m i d e .  9  acid.  was added 3 c 5  The crude p r o d u c t began  80  to precipitate  w i t h i n a few m i n u t e s .  A f t e r 30 m i n u t e s  was f i l t e r e d and washed r e p e a t e d l y w i t h w a t e r .  The  solid  was d i s s o l v e d i n e t h e r (200 ml) and washed w i t h water the aqueous l a y e r remained c o l o u r l e s s .  IR  ( K B r )  Amax(Me0H - 0.01NHG1): :  1775,  1710,  until  The e t h e r phase was  s t r i p p e d t o d r y n e s s t o y i e l d 3*5 gm (60$) solid,  it  of a l i g h t yellow  346, 268 mu (1:5).  1650, 1450, 1180,  1020, 920 c m . - 1  P r e p a r a t i o n of 4 - h y d r o x 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 4-0xo-4-dedimethyl  amino t e t r a c y c l i n e  2 gm) was d i s s o l v e d i n 10 ml o f m e t h a n o l .  4,6 h e m i k e t a l  gm) was  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  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 d i l u t e h y d r o c h l o r i c acid*, with ether  (4 x 100 m l ) .  (44,  Potassium b i c a r -  bonate (5 gm) 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 (1.0 added.  (52)  for  a c i d i f i e d t o pH2 w i t h  The a c i d s o l u t i o n was  extracted  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 t h e r was washed w i t h c o l d benzene and the  crude  p r o d u c t was c r y s t a l l i s e d f r o m t o l u e n e - i s o p r o p y l a l c o h o l mixture It I R  (3:1),  showed (KBr)  :  The y i e l d o f the p r o d u c t was 0.5 gm ( 4 0 $ ) .  A max (MeOH 0,01 1 6 6 0  »  lo  3°>  NHC1)  359, 308, 276 mu ( 3 : 4 : 2 ) .  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-dedimethylaminotetracycline  (43)°  4-hydroxyimino-4-dedimethylaminotetracycline  (52, 200 mg)  - 31  was d i s s o l v e d i n 2 m l o f 4$ m e t h a n o l i c h y d r o g e n The  chloride.  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$)  was r e c o v e r e d b y f i l t r a t i o n a n d w a s h e d w i t h m e t h a n o l . showed IR  Amax(MeOH - 0,01 NHC1): :  ( K B r )  It  421, 303, 264 my (1:2:4),  1650, 1620, 1570, 1440, 1240 c m " . 1  P r e p a r a t i o n o f 4-keto-4-dedimethylamino-5a 6-anhydro p  tetra-  c y c l i n e (42) 5a,6-anhydrotetracycline water  (500 mg) ..was d i s s o l v e d i n  (350 m l ) a n d c o n c e n t r a t e d h y d r o c h l o r i c a c i d  N-Chlorosuccinimide  (150 mg) w a s a d d e d  10 m i n u t e s t o t h e w e l l  stirred  (10 m l ) .  i n portions  solution.  during  The s t i r r i n g was  c o n t i n u e d f o r 40 m i n u t e s a t r o o m t e m p e r a t u r e .  I t was t h e n  e x t r a c t e d w i t h w a t e r u n t i l t h e w a s h i n g s were n e u t r a l t o litmus. (240  The e t h e r w a s s t r i p p e d t o y i e l d a b r o w n  mg, 45$) w h i c h w a s r e p r e c i p i t a t e d f r o m  f u r a n - p e t r o l e u m e t h e r (30-60°). mu (17,000; 75*000).  I t showed  Found: IR  ( K B r )  C, 64.51? :  Xmax:  437, 273  (3:1:1),  A n a l , G a l e , f o r C 0 H O H : C, 64,75; 1 5  tetrahydro  I t showed one s p o t a t R f 0.3 o n p a p e r  using Methanol-Acetone-water 2  solid  7  H, 4=25;  H, 4«04; 0, 30,18,  0, 30.49=  3400, 1721, 1655, 1625, 1525* 1450, 1240, 980 c m " . 1  - 82 -  A t t e m p t e d F o r m a t i o n o f Oxime o f (a)  (42)  4-keto-4-dedimethylamino-5a,6-anhydro (42,  20 mg)  tetracycline  was d i s s o l v e d i n m e t h a n o l (2 m l ) .  sium b i c a r b o n a t e  (30 mg)  chloride  was a d d e d .  (20 mg)  f o r 20 m i n u t e s . d i f i e d t o pH2  Potas-  and h y d r o x y l a m l n e h y d r o The  s o l u t i o n was  stirred  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 -  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 o f at least (b)  5  compounds.  4-keto-4-dedimethylamino-5a,6-anhydro (42,  20 mg)  was d i s s o l v e d i n 1 m l o f a c e t i c a c i d  hydroxylaminehydrochloride  was a d d e d .  The Water  was a d d e d a n d e x t r a c t e d w i t h e t h e r .  was  i a l was  amination  T o a s o l u t i o n o f (42, aqueous amonia 40 mg)  1 hour.  o f a brown  The e t h e r solid.  shown t o be i d e n t i c a l t o s t a r t i n g  Attempted Reductive  s  (20 mg)  and  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 m i n u t e So  s t r i p p e d t o y i e l d 15 mg  (10%  tetracycline  (28%,  T h i s matercompound  c  o f (42)  24 mg)  i n monoglyme (20 m l ) a n d  2 m l ) was a d d e d p a l l a d i u m o n c h a r c o a l  and s h a k e n u n d e r h y d r o g e n g a s a t 36 p . s . i . f o r  The p r o d u c t was f i l t e r e d a n d a c i d i f i e d w i t h  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  dilute  (3 x 20  ml)  and i t s pH adjusted to 5.5» 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(H 0): 273, 430 mu (1:6)} 2  - 84 REFERENCES BoM  0  A.C. J.B. I.A. III,  D u g g a r , A n n , N , Y . A c a d . S c i . , j j l , 177 ( 1 9 4 8 ) ,  F i n e l y , G , L , Hobby, S . Y . P»an, P . P , Regna, Routien, D.B, Seeley, G,M, S h u l l , B . A , Sobin, Solomons, J . W , V i n s o n and J , H . Kane, S c i e n c e 85 (1950),  G,R. Stephens, L , H , Conover, F , A , H o c h s t e i n , P . P . ' Regna, F „ J , P i l g r i m , K , J . B r u n i n g s , R , B , Woodward, J , Am, Chem, S o c , 2Jks> 4976 (1952) = L . H . Gonover, W . T , M o r e l a n d , A , R , E n g l i s h , C , R , S t e p h e n s , F . J , P i l g r i m , i b i d 2£s>. 4622 (1953) = R, P a s t e r n a c k , P , P . - R e g n a , R , L . Wagen, A , B a v l e y , F , A , H o c h s t e i n , PoN, G o r d o n , K , J , B r u n i n g s , i b i d , 21, 2400 (1951)= PoP, Regna, I , A , Solomons, K . M u r a i , A«E, Timreck^ K . J . B r u n i n g s and W . A . L a z i e r , i b i d , , %}> 4 2 1 1 ( 1 9 5 1 ) . F . A , H o c h s t e i n , P . P . Regna, K , J , B r u n i n g s , R . B , Woodward i b i d , 2^/ 3706 (1952). F , A , H o c h s t e i n , C , R , S t e p h e n s , P , N , Gordon, P , P , Regna, F , J , P i l g r i m , K . J . B r u n i n g s , R . B . Woodward, i b i d , 74» 3707(1952), F . A . H o c h s t e i n , C . R . S t e p h e n s , L . H , C o n o v e r , P , P . Regna, R . P a s t e r n a c k , K . J . B r u n i n g s , R . B . Woodward, i b i d , 74» 3708 (1952). F . A . H o c h s t e i n j C_-R Stephens^ L . H , ConoVer, P . P . Regna, R. P a s t e r n a c k , P N G o r d o n , F , J v P i l g r i m , K . J . B r u n i n g s and R . B , Woodward, i b i d , 2£» 5455 (1953h ;  5  0  L . H . Gonover, W , T , M o r e l a n d , A , R , E n g l i s h , C . R . Stephens and F . J , P i l g r i m , i b i d , 2Jb 4622 (1953)• P . P . M i n e r i , M . G . F i r m a n , A . G , M i s t r e t t a , A . Abbey,, C . E . B r i c k e r , H . E , R i g l e r and H . S o k o l , " A n t i b i o t i c s A n n u a l , 1953-1954" e d . by W e l c h , M e d i c a l E n c y c l o p e d i a I n c . New Y o r k , N . Y . p . 8 1 . Mo Schach v . W i t t e n a u , R . B , B l a c k w o o d , L . H . C o n o v e r , R . H , G l a u e r t and R . B . Woodward, J . Am. Chem. S o c , 87, 134 (1965).  _ 85 14«  So H i r o k a w a Y. Okaya, F,M, L o v e l l and R. Pep i n s k y , A b s t r S o Am Cryst, Assoc, Meeting, C o r n e l l U n i v e r s i t y , I t h a c a , N.Y,, J u l y 1959* p° 44= p  0  15,  16,  J . Donohue, J,D, D u n i t z , K.N. T r u e b l o o d a n d M.S,  J . Am, Chem. S o c , 8£, 851 (1963) = A,P. D o e r s c h u k , B,A, B i t l e r  ZL,  Webster,  and J,R,D, McGormick,  ibid,  4687 (1955)o  17o  J,R,D, M c C o r m i c k , P.A. M i l l e r , J . A , G r o w i c h , N,0, S j o l a n d e r and A.P, D o e r s c h u k , i b i d p 80, 5572 (1958).  18,  CR.  S t e p h e n s , K, M u r a l , H.H, R e n n h a r d , L.H. C o n o v e r  and K, J . B r u n i n g s , i b i d , 80, 5324 19.  (1958),  J.R.D. M c C o r m i c k , E.R, J e n s e n , P,A, M i l l e r D o e r s h u c k , i b i d 82, 3381 (1950).  a n d A,P.  ;  20,  H. M u x f e l d t  a n d R, B a n g e r t p F o r t s c h r ,  Org. N a t u r s t o f f e ,  21, 80 (1963), 21.  G.G, B a r r e t , J , Pharm, S o e . p J 2 >  22.  E , E , van Tamelen, F o r t s c h r ,  309  (1963h  Org, N a t u r s t o f f e ,  16, 90  (1958) . 23,  P.P. Regna i n A n t i b i o t i c s : T h e i r C h e m i s t r y and M e d i c a l U s e s , e d . G o l d b e r g , v a n Nor s t r a n d Co., P r i n c e t o n , N.,'J.  (1959) p.77. 24°  J.H, Boothe, A n t i m i c r o b i a l  A g e n t s a n d Chemotherapy  1962  a  213, 25.  A.I, Scott  26.  J,H, B o o t h e , A,S, Kende, T . L , F i e l d s a n d R.G, J . Am, Chem6 S o c , S i , 1006 (1959) °  27,  H, M u x f e l d t , B e r . , 22,  28.  H. M u x f e l d t ,  29,  C.E. Holmlund,. W.W.  213  Soc, 30,  a n d T, Money, i n P r e s s ,  (1962).  3122  Wilkinson,  (1959).  G. B u h r , and R. B a n g e r t , Angew.Chem.,  81, 4749  A n d e r s and A . J , S h a y , J , Am.  74",  Chem,  (1959).  L o H , C o n o v e r , K. B u t l e r , J.D. J o h n s t o n , J , J . K o r s t , a n d R.B, Woodward, i b i d , 3222 (1952).  „ 86 -  31.  H. M u x f e l d t a n d W. R o g a l s k i ,  32.  A . l . Scott  33.  ibid  a n d C. B e d f o r d , i b i d ,  82, 933 (1965)». 8/fc, 2271 (1962).  A , J . B i r c h , J . F . S n e l l a n d P.L. Thompson, J . Chem.  Soc,  1962„ 425.  34.  So G a t e n b e c k , B i o c h e m .  35.  S . J . W a k i l , J . Am. Chem. S o c , 80, 6465 (1958).  36.  E, L y n e n , Angew. Chem., 22*  37.  F.A. H o c h s t e i n , M. S c h a c h v o n W i t t e n a u , F,W. T a n n e r , J r . , a n d K, M u r a i , J . Am.•Chem. S o c , 82, 5934 (I960).  38.  So K h a r a t y a n , M. P u z a , J . S p i z e c k , L . D o l e z i l o v a , Z, V a n e k , M. V o n d r a c e k a n d R.W. R i c k a r d s , Chem, and  (1961).  B i o p h y s . R e s . Comm., 6, 422  513 (1961).  I n d . , 1963p IO380  39. 40.  J.R0D0 M c C o r m i c k , S. J o h n s o n a n d N.O. S j o l a n d e r ,  J  c  Am, Chem. S o c , 8£, 1692 (1963),  JoRoD. M c C o r m i c k , J . R e i c h e n t h a l , S. J o h n s o n a n d N,0.'Sjolander, i b i d ,  8£, 1694 (1963).  41.  J.R.D. M c C o r m i c k , P.A. M i l l e r , S, J o h n s o n , N. A r n o l d , and N.O. S j o l a n d e r , i b i d , J ^ * 3623 (1962).  42.  P,A, M i l l e r , A, S a t u r n e l l i , J . H , M a r t i n , L . A . M i t s c h e r , No B o h o n o s , B i o c h e m . B i o p h y s , R e s . Comm., 1 6 , 28'5' (1964),  43.  J,D,R, M c C o r m i c k , U.H. J o a c h i m , E.R, J e n s e n , S. J o h n s o n and N.O, S j o l a n d e r , J , Am, Chem. S o c 82, 1793 (1965).  44.  E.E. v a n T a m e l e n ,  45»  A . I . S c o t t , F . McCapra and E S  (1961).  F o r t s c h r , Org. N a t u r s t o f f e , rg,  0  o  Hall,  242,  J . Am. Chem. S o c ,  86, 302 (1964)o 46,  R,K. B l a c k w o o d ,  H,H. R e n n h a r d  82, 5194 (I960) ,  and CR,  Stephens,  47°  A. G r e e n , R,G, W i l k i n s o n a n d JoH, B o o t h e ,  48,  H.Y, T s e n g , T e t r a h e d r o n , 11, 5 2 (I960),  (I960).  ibid,  ibid,  82, 3946  - 87 49.  C.R.  Stephens J r p 0  U S 0  0  2,841,596.  Patent  Chem.Abstr.,  J52, 21086 (1958)B u l l o T o k y o , 6, 320 (1958)0  50.  Kc  51.  Co U k i t a , K  52o  L o H o G o n o v e r , i n "Symposium on A n t i b i o t i c s and M o u l d M e t a b o l i t e s ' p Chem. S o c S p e c i a l P u b l i c a t i o n N o 5,  Taminop  Chem 0  0  Pharra  Arakawa,  0  ibid,  535  (1957)»  8  0  London, E n g . , 1956, p.48o  53o  M M o Shemyakin,'A0S0 K h o k l o v , M o N K o l o s o v , L D o B e r g e l s o n and V . K . A n t o n o v , i n " C h e m i s t r y o f A n t i b i o t i c s ' " , U o S o S o R o Academy o f S c i e n c e s , V o l o 1, Moscow, 0  c  0  1961, p . I8O0  54o  CoRo Stephens J r . , U S Patent (pending) Information on t h i s p a t e n t was k i n d l y s u p p l i e d t o u s by D r . J . D . R . McCormick ( L e d e r l e C o a ) .  55»  Ao  560  E • H e c k e r and & M u e l l e r , J . B i o l .  57o  J0R0D0  580  H0J0 T e u b e r , B e r . , 88, 329 (1955).  59o  London,  60.  E  610  E.  62.  H. M u x f e l d t and A. K r e u t z e r , B e r . , 2^,  63.  C o B e d f o r d , Ph.D.  64°  R o K . B l a c k w o o d , C R . S t e p h e n s , J . Am.  65.  R 0 C 0 E s s e , J.A. Lowery, i b i d p 3875 (1964)o  66.  R . K o Blackwood, P r i v a t e  67o  JoJ  0  0  G r e e n and J.H. B o o t h e , J . Am  3950 (I960),  McCormick,  EoRo  0  Chem, S o c ,  Chem., 2^2,  J e n s e n , J . Am.  82,  991  Chem. S o c ,  1794 (1965)o  0  P r o g r e s s i n O r g a n i c Chem, e d  Bamberger,  87  ( C o o k ) , j>, 46  9  (1961)  B e r . , ^ 6 , 2028 (1903)  H e c k e r and ft. L a t t r e l l ,  thesis,  Ann,,  662,  Glasgow,  C R 0  o  48  (1963)0  892  (1961)c  (1963).  2736 (1964).  0  (19-58)»  Chem. S o c ,  T a m o r r i a and G.M.  86,  ~~.  Sieger,  Communication.  H l a r k a p P. B i t h a and J . H . B o o t h e , J . Am.  8Zp 1795 (1965)o  "  Chem,  Soc,  - 88 -  68  0  Hart* i n " S t e r o i d R e a c t i o n s An O u t l i n e f o r O r g a n i c C h e m i s t s " , edo C D j e r a s s i , Holden-Day I r i C o , S a n F r a n c i s c o , 1963* p 1 7 9 ° PoAo  0  0  69o  0 T o u s t e r , i n "Organic R e a c t i o n s " Volo V I I , John W i l e y & S o n s l n c ; , New Y o r k , 1953* P<> 327,  70  No R a b j o n i n O r g a n i c R e a c t i o n s , V o l o V, John & S o n s I n c o , New Y o r k , 1949* p> 331=  o  71,  a  5  K Arakawa a n d M (1957)o 0  0  Irie,  PharnioBull (Tokyo),  Wiley  5, 531*  PART I I o STUDIES I N RELATION TO THE ACETATE PATHWAY TO AROMATIC COMPOUNDS  89  INTRODUCTION D u r i n g t h e 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 t h e b i o s y n t h e s i s n a t u r a l products,,  of  As a r e s u l t of t h e s e 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 o r g a n i c compounds have been shown t o be d e r i v e d f r o m simple s t r u c t u r a l u n i t s  T h i s has l e d t o  0  t h e d e r i v a t i o n of c e r t a i n r u l e s w h i c h have c o n s i d e r a b l y s i m p l i f i e d t h e t a s k o f 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 chemist  0  The f i r s t of t h e s e ,  t h e " i s o p r e n e r u l e " was proposed by R u z i c k a a s a r e s u l t 1  of 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  biogenetic  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 m e v a l o n i c acid lactone  0  acid units  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  a  w h i c h i s formed f r o m c o n d e n s a t i o n of 3 a c e t i c  The c a r b o h y d r a t e  9  or the  shikimic a c i d pathway , 2  0  leads  t o a n o t h e r 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 a s a r o m a t i c amino a c i d s coumarins, s t l l b e n e s  p  p  indole a l k a l o i d s , flavanoids,  l i g n a n s and l i g n i n s  0  d i s t i n g u i s h e d f r o m a r o m a t i c r i n g s a r i s i n g by c v c l i z a t i o n , (vide i n f r a ) characteristic rings o • .  n  They a r e r e a d i l y polyacetate  since, t h e ' l a t t e r - p o s s e s s the  r e s o ' r c i n o l p a t t e r n of o x y g e n a t i o n on t h e  - 90 -  The  involvement o f a c e t i c a c i d , another  building  b l o c k o f n a t u r a l p h e n o l i c compounds, was f i r s t by C o l l l e 3  On the b a s i s of some i n t e r e s t i n g  0  proposed transfor-  mations t h a t he had observed w i t h p o l y a e e t y l compounds and the s i m i l a r i t y  of t h e p r o d u c t s t o n a t u r a l p h e n o l i c  and n a p h t h a l e n i e compounds. C o l l i e p o s t u l a t e d t h a t compounds might be formed  such  i n nature from c o n d e n s a t i o n o f  s e v e r a l "head t o t a i l " l i n k e d a c e t i c a c i d u n i t s and t h e i r subsequent  cycllssationo  F o r example, i t was found t h a t i n ,  weak a l k a l i d i a c e t y l a c e t o n e (1) c o n d e n s a t i o n s t o t h e naphthalene dehydroacetic a c i d orcinol  (4K  (3)  dimerized v i a a l d o l derivative  similarly treated  (2) and  produced  - 91 .-  ^CHf0 H 2  combustion  0,04 c/min/mg BaCOo F i g u r e !<>  - 92 .  Many y e a r s later„ a f t e r examination  of the s t r u c t u r e  of n a t u r a l l y o c c u r r i n g phenols, pyronesp q u i n o n e s B i r c h ^ ^ ^ and Robinson^,  p  flavones,  independently of C o l l i e , l a i d down  most o f the modern view o f the a c e t a t e h y p o t h e s i s and p o i n t e d out i t s broad The  scope and u s e f u l n e s s .  key s t e p i n the a c e t a t e pathway i s t h e formation,  o f a l i n e a r B - p o l y k e t o a c i d c h a i n , which c a n c y c l l z e t o i :  e i t h e r of the; two f a m i l i e s of aromatic, compounds, the a c y l p h l o r p g l u c i n o l s (•£) and t h e o r s e l l i n i c a c i d (6) C  2  as shown below  p  derivatives  or i f l e s s than the r e q u i r e d number o f  u n i t s are present  0  by  M  0  M  c y c l i z a t i o h t o pyrones  (7JU  - 93 -  The  actual  involvement of a c e t i c a c i d  i n aromatic  n a t u r a l p r o d u c t s was f i r s t demonstrated by B i r c h and h i s eolleagues7  0  They f e d l a b e l l e d a c e t a t e t o micro-organisms,  i s o l a t e d the n a t u r a l  product o f i n t e r e s t and l o c a t e d the  p o s i t i o n of t h e l a b e l by knowndegradatlve procedures,, Thus t h e y demonstrated t h e i n c o r p o r a t i o n acid into 6-methylsalicylie  fulvW?  acid  14  (8) by P e n i c i l l i u m  g i v i n g the d i s t r i b u t i o n p a t t e r n  Subsequently t r a c e r methods u s i n g have been a p p l i e d  of ( 1 - 0 ) a c e t i c  1 4  grlseo-  shown i n f i g u r e '1, G  labelled acetate,  t o the study o f the b i o s y n t h e s i s  o f many  n a t u r a l p r o d u c t s , such a s 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)°% the t e t r a c y c l i n e s  (ll)  1 0  and such aromatic  compounds a s p a t u l l n ( 1 2 ) , p e n i c i l l i c a c i d 1 1  the  tropoionic  acids  (14) <> 13  derived  (13)  1 2  and  (see f i g u r e 2)  While o f g r e a t i n t e r e s t t h e s e t r a c e r s t u d i e s  alone  d i d not throw much l i g h t on the mechanism of t h e I n i t i a l stages o f the f o r m a t i o n o f t h e B-polyketomethylene It was e a r l y r e c o g n i z e d that  chainSo  any a c i d found i n nature might  c o n c e i v a b l y i n i t i a t e a c h a i n by adding t o the methyl end of a C  2  unit.  of varying  Among such a c i d s which do so are f a t t y  c h a i n l e n g t h ( C , 03,0^, 05, Gg, G  branched c h a i n a c i d s  2  l o  acids  , G^),  such a s i s o b u t y r i c , cx - m e t h y I b u t y r i c ,  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 :  •(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 of the f i r s t by f e e d i n g  acetic acid unit incorporated  a c e t i c a c i d showed a n a c t i v i t y  than the other units '* „ 1  b u i l d i n g process  as d i s t i n c t from chain  Gatenbeck and M o s b a c h ^ i n c o r p o r a t e d 1  acetic acid into orsellinic acid. 0 content  each phenolic  8%  higher  T h i s was l a t e r e x p l a i n e d b y t h e  involvement o f malonate r a t h e r t h a n a c e t a t e  that  into the chain  1 8  0  i n the chain  initiation,, from ( l - ^ G p  ^O)  T h e y w e r e a b l e t o show  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  hydroxyl  0  This i s the r e s u l t expected i f  id the the  0 m a r k e r a s a c e t y l coenzyme A g o e s d i r e c t l y synthesis without  orsellinic  random d i s t r i b u t i o n ^  coenzyme A ( v i d e i n f r a ) s  ending i n  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 water d i l u t e s the t o t a l x o  0  p e r oxygen by h a l f o  through  carboxyl  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 four acetates  i n l e n g t h and t h e r e b y  are directly  of  incorporated  16  into orsellinic acid  0  F i n a l l y B a s s e t t and Tanenbaum ? and L y n e n 1  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 a g e n t a n d i s Incorporated  into 6 - m e t h y l s a l i c y l i c a c i d without  reverting t o free  first  acetate*  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 ^ W a k i l 1  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 a s a b i o s y n t h e t i e p r e c u r s o r of f a t t y a c i d s , B e n t l e y and K e i l colleagues ^, 2  2 2  the  and Bu^Lock and h i s  a l m o s t s i m u l t a n e o u s l y r e p o r t e d the  incor-  p o r a t i o n o f m a l o n i c 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-methylsalicyllc acid.  I t was suggested t h a t a s i n t h e  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 chain building„  Further, lack of s i g n i f i c a n t  o f l a b e l i n t h e m e t h y l group o f p e n i c i l l i c a c i d t r a t e d t h a t malonate appreciable It  amounts 2 2  demons-  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  e x t e n t and so I s a v a l i d d i r e c t  intermediate,  i s assumed t h a t a s I n t h e 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 o f 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 steps, No a t t e m p t s 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 c a r b o n p r e c u r s o r s such a s butyrate or acet©acetate.  Thus B i r k e n s h a w ^ f o u n d t h a t 2  ( 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 t h e 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 b e f o r e i n c o r p o r ation.  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  chain.  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 -  Figure  3=  ~ 98 -  mechanism o f b i o s y n t h e s i s preparing c e l l free  of 6-methylsalieylic  extracts  from 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 (l-^G)  i t i n the presence o f  a c e t y l c o e n z y m e A a n d m a l o n y l coenzyme A,  found that  I t was  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  for  6-methylsalieylic  was  a l s o TPNH d e p e n d e n t ,  medium, n e i t h e r  a c i d p r o d u c t i o n and t h a t ; i t s  6=methylsallcylicj>  the suggestion that  production  When TPNH was e x c l u d e d f r o m the*  were p r o d u c e d by t h i s c e l l f r e e by  a c i d by  only  nor o r s e l l i n i c  extract.  acid  T h i s was  explained  (15) acted a s a precursor 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 m o u l d . Figure 3 i l l u s t r a t e s the formation of 6-methylsalicylic a c i d b y t h i s enzyme  preparation,  Lynen a l s o f o u n d ! t h a t blocking  iodoacetamide (a s u l f h y d r y l  agent) prevented t h e formation 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 r o u p s o f t h e enzyme were i n v o l v e d  i n the biosynthesis.  On t h e b a s i s that  system  o f t h e above f i n d i n g s Lynen  proposed  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 c o m p l e x a n d t h a t t h e i n t e r m e d iate polyketo acids enzyme,  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 1  99 DISCUSSION The e x p e r i m e n t a l e v i d e n c e  so f a r a v a i l a b l e  (vide  supra)  s t r o n g l y i n d i c a t e s t h e 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 acid intermediateSo  The a c t u a l i n v o l v m e n t of the  however, has not a s y e t been showno  Thus, i t  seems d e s i r a b l e  t o embark on e x p e r i m e n t s d e s i g n e d t o i n v e s t i g a t e o f the  latter,  the  nature  i n t e r m e d i a r y s t e p s i n t h e a c e t a t e pathway t o  phenolic  compoundSo  A possible  intermediate  o f work r e c e n t l y "polypyrones",  suggested  itself  i n the  c a r r i e d out i n our own l a b o r a t o r i e s  such a s b i s - a n d t r i s p y r o n e  (16)  and  course on (17)  respectivelyo I t was f o u n d t h e s e compounds on t r e a t m e n t w i t h 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 a s acid  (13) 26  tively  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 o  Inspection  of the  structures  base,  orsellinic  (19)  respec-  of polypyrones  r e v e a l s t h a t t h e y are b a s i c a l l y formed f r o m a m o l e c u l e triacetic  a c i d lactone  malonic a c i d lactone  ( r i n g A) w i t h s u c c e s s i v e  (ring B, C, D, e t c ) 0  0  of  u n i t s of  Base h y d r o l y s i s of  r i n g s leads to B - p o l y k e t o a c i d chains which  eously e y c l i s e to aromatic  (20)  the  spontan-  s u s t r a t e s a s shown i n f i g u r e 4»  The ease o f the t r a n s f o r m a t i o n of t h e s e p y r o n e s t o aromatic  compounds, and the  s t a b i l i t y that these  lactone  1Q0  r i n g s provide chains, the up  f o r otherwise h i g h l y l a b i l e  suggested a p o s s i b i l i t y  monopyrone t r i a c e t i c of three  processes., c a n be  The  coenzyme e s t e r  also involved  of t h e  a c e t y l coenzyme A and  shown i n f i g u r e  c h a i n a c i d and that  i n the  a d d i t i o n s of  by  a  two  decarboxylation,  (19)  ease o f h y d r o l y s i s o f t h e  suggested t h a t an  i n v i v o from  (21)  5°  (17) The  in natural  open c h a i n f o r m of  successive followed  least  (21), which i s b u i l t  t o have b e e n c o n s t r u c t e d  u n i t s o f m a l o n y l coenzyme A, as  pyrones, or at  acid lactone  a c e t i c a c i d u n i t s , are  envisioned  starter  that  polyketo-raethylene  lactone  equilibrium could exist  the  case o f  lactone.  In f a c t ,  styryltriacetic  rings in  vitro  between the  i t has  been  acid lactone  open  reported (22),  base  - 101  OH  ^0 x  b  co,H (20)  (21)  C ^ Q Aromatic  substrates  Figure  4;  H  CH3-COC0A  CO,  2 >COSCoA  L H  3  Cok (21)  Figure  5,  -  treatment  102  -  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  resulted i n  27 the  isolation  o f the f r e e a c i d  Triacetic a  stable  unit  acid  <,  lactone, therefore,  unit, requiring  o f malonyl  (23)  seemed t o p r o v i d e  t h e a d d i t i o n o f o n l y one more  coenzyme A t o f o r m  simple p h e n o l i c  compounds.  O  (23)  (22). The  o c c u r r e n c e o f monopyrone  strengthens the p o s s i b i l i t y  structures i n nature  o f such  compounds a s  intermed-  i a t e s , i n t h e a c e t a t e pathway t o n a t u r a l l y  occurring phenolic  compounds,  3-methyl-2,4-diketo-  3,4  Tanenbaum  et,al,  dihydro-6-methylpyrone  i s acetate derived. i n Qpuntia  e l a t o r ,  1 ium r ad i c i n ium-^ ® 1  3  haye f o u n d  (24)  Closely  in P  related  Radicinin  s t i p i t a t u m which  0  i s opuntiol  (26), isolated  clearly  (25),  from  found  Stemphy-  h a s a s t r u c t u r e r e s e m b l i n g b i s p y r one,  although bispyrone i s probably not i t s p r e c u r s o r . compounds a s methoxy p a r a c o t o i n ( 2 7 )  Also,  such  and t h e s t y r y l p y r o n e s  32 (2B)  a r e e x a m p l e s o f t h e c a s e s where  enzyme a c t i v i t y of C  2  has stopped  u n i t s t o form aromatic  i t i s assumed  short o f the necessary substances.  that  numbers  I t w o u l d be  inter-  - 103 -  esting  to test  the precursor a c t i v i t i e s  compounds by f e e d i n g enzyme like  suitably labelled  systems capable  of producing  o f the l a t t e r (27)  two  o r (28) t o  s t y r y l aromatic  compounds  the f l a v a n o i d s .  o  OH (25)  (26)  R  RR (27) During ity  (28)  a search f o r reported t e s t s f o r precursor  by t r i a c e t i c  acid  activ-  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  studying the e f f e c t s of v a r i o u s  oh t h e p r o d u c t i o n o f a r o m a t i c  s u b s t a n c e s by P  n  f o u n d t h a t a d d i t i o n o f 0=2-5=0 mg o f t r i a c e t i c (21) which  enhanced t h e a c c u m u l a t i o n  (11) i n t h e same m o u l d .  patulum acid  fl  had  lactone  of gentisyl alcohol (29),  i s an intermediary metabolite  patulin  substrates  i n the production of  On t h e o t h e r  hand, t h e  -  104  acetyl derivative; dehydroaceticacid formation  of.gentisyl alcohol  found t h a t the a s s a y used the  report  represented  involvement  Also  A l t h o u g h i t was  a  an  important i n the  c l u e to the  production  the  subsequently  i n h i s work i s p e r h a p s a t  of t h i s lactone  derived phenolic  (30), i n h i b i t e d  of  fault,  possible a'cetate  compoundso  interesting  i s Tanenbaum's  observation,  during r  the  (24),  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 lactone  while  t h i s compound c o u l d be  (ca, 6 mg/liter) stipitatic  f r o m 4 day (3D  acid  could  isolated  i n small q u a n t i t i e s  o l d c u l t u r e s of P not  be  that  detected  0  stipitatum,  u n t i l the  seventh  day.  (29) In view of the lated and be  biogenesis  that  no  regarded  (3D  (30) fact  of the  that  no  acetate  " p r i m i t i v e " polyketo as precursors,of  been i s o l a t e d ,  intermediates  pathway have b e e n  reported a c t i v i t y  acetic  a c i d lactone„  postu-  recognized,  compounds (<LCg) t h a t  Cg a r o m a t i c  i t seemed a d v i s a b l e  o f the  i n the  to  compounds have  i n v e s t i g a t e the  ( p r e c u r s o r , or  might  catalytic)  of  ever  nature tri-  -  Initially  a  105  e x p e r i m e n t s were d e s i g n e d t o  Ehrensvard s observations, r  reproduce  P e n i c i l i u m p a t u l u m B a i n i e r 1ICL-  34909-(C 9 4 5 ) » a s t r a i n r e p o r t e d t o produce m a i n l y p a t u l i n and g e n t i s y l a l c o h o l  3 4  r  was grown on Czapek-Dox a s a  surface  culture,  48 h o u r s a f t e r i n o c u l a t i o n , v a r i o u s q u a n t i t i e s  triacetic  acid lactone  and d e h y d r o a c e t i c a c i d were a d d e d .  A l i q u o t s f r o m the b r o t h were t e s t e d on s u c c e s s i v e g e n t i s y l a l c o h o l content  of  by s p e c t r o s c o p i c  days, f o r  means.  E h r e n s v a r d had used t h e Liebermann n i t r o s o t e s t phenols ** f o r h i s assays. 3  D u r i n g the p r e s e n t  however, t h i s method o f measurement  for  investigations,  p r o v e d i n c o n c l u s i v e . (A  p o s i t i v e L i e b e r m a n n * s t e s t would not n e c e s s a r i l y mean the presence of g e n t i s y l a l c o h o l , as i t i s i n g e n e r a l t o any p h e n o l w i t h a f r e e p a r a p o s i t i o n ) ,  applicable  I t was t h u s d e c i d e d  t o d e t e c t any changes by c h r o m a t o g r a p h i c means.  A thin layer  c h r o m a t o g r a p h i c t e c h n i q u e was d e v e l o p e d w h i c h s e p a r a t e d  the  e t h e r - e x t r a c t a b l e mould p r o d u c t s i n t o i n d i v i d u a l s p o t s  corres-  ponding t o 6 - m e t h y l s a l i c y l i c a c i d , t o l u q u i n o l , p a t u l i n and g e n t i s y l a l c o h o l ( R f ' s 0,7*  0.5,  0,4 and 0 , 2  respectively).  I t was f o u n d t h a t a d d i t i o n o f d e h y d r o a c e t i c  acid  caused v a r y i n g d e g r e e s o f i n h i b i t i o n of m y c e l i a l g r o w t h , depending on c o n c e n t r a t i o n  (10 mg was a s u f f i c i e n t  t o p r e v e n t t h e f o r m a t i o n of any m y c e l i u m ) . acetic acid lactone  quantity  E f f e c t s of  a d d i t i o n were not so v i s i b l e .  tri-  Also,  it  - 106  -  was observed t h a t , d u r i n g t h e normal growth of Po p a t u l u m the growth r a t e v a r i e d f r o m one f l a s k to a n o t h e r . l a r i t i e s could, therefore,  0  Irregu-  be i n t r o d u c e d i n amounts of  metabolite formed. To e l i m i n a t e the f a c t o r s g o v e r n i n g m y c e l i a l growth which were not under s t u d y , the mould was grown t o m a t u r i t y (7-8 days) and the f e r m e n t a t i o n b r o t h o f the f l a s k s  (selec-  t e d on t h e b a s i s of e q u a l m y c e l i a l growth) were r e p l a c e d w i t h f r e s h medium and t h e  substrates  added.  T e s t s were  performed p e r i o d i c a l l y on t h i s new medium. I n a l l e x p e r i m e n t s performed i n t h i s way, a d d i t i o n o f 40 mg of d e h y d r o a c e t i c a c i d i n h i b i t e d c o m p l e t e l y any metab o l i t e f o r m a t i o n , and unchanged d e h y d r o a c e t i c a c i d was the only detectable after  substance  i n t h e f e r m e n t a t i o n b r o t h even  s e v e r a l weeks of i n c u b a t i o n .  Amounts up t o 5,0 mg  d e c r e a s e d the q u a n t i t y o f accumulated m e t a b o l i t e s ably.  consider-  T h i s seemed t o c o n f i r m t h e f i n d i n g of B h r e n s v a r d , On the o t h e r h a n d , t r i a c e t i c a c i d l a c t o n e  appeared t o  s t i m u l a t e t h e p r o d u c t i o n of some o f the m e t a b o l i t e s . 4 hours a f t e r  Thus  the a d d i t i o n of the l a c t o n e , a spot d e v e l o p e d  h a v i n g an R f i d e n t i c a l t o t h a t control'experiment  of t o l u q u i n o l , w h i l e i n a  (no added t r i a c e t i c a c i d l a c t o n e )  same spot appeared a f t e r  35-40 h o u r s .  this  S i m i l a r l y , there  -107  a p p e a r e d t o be  enhancement o f 6 - m e t h y l s a l i c y l i c a c i d  an  some 35-50 h o u r s  p r o d u c t i o n by  No  0  drawn a b o u t p a t u l i n p r o d u c t i o n , c a l t o t h a t o f added t r i a c e t i c  not  regarded  that there exists a  as  steady  be  s i n c e i t shows a n R f acid  g e n t i s y l a l c o h o l p r o d u c t i o n was h o w e v e r , was  conclusion could  lactone»  not  The  effect  so p r o n o u n c e d »  significant  as  idention  This,  i t i s possible  state concentration of  this  alcoholo T h e s e r e s u l t s were o b s e r v e d and  seemed t o c o n f i r m t h e  lactone  involvement  l i t e s was  thought  t h o u g h i t was  and  t o be  observed  not  experiments  of t r i a c e t i c  i n mould m e t a b o l i s m a s r e p o r t e d by  Quantitative determination  cause  i n repeated  o f the  Ehrensvard,  individual  instructive  at t h i s  that a d d i t i o n of the  i n c r e a s e d y i e l d s of t o t a l m e t a b o l i t e s  Feeding  (34)9  lactone  triacetic  (33)a  resulted  I t was  and  acid  accumulated, sodium  bicar-  l a c t o n e d e r i v a t i v e s such  lactone  (32),  considered  inhibition  acid  acid  of metabolite  significant  only t r i a c e t i c  as  4-methoxy-triacetic  4-chloro-4-deoxytriacetic  i n complete  thus  able to accept  stage,  compoundSo  4-deoxytriacetic acid acid  metabo-  lactone did  a l s o h i g h e r y i e l d s o f n e u t r a l e x t r a c t and  bonate-soluble  acid  that the  l a c t o n e and  lactone  production.  mould  was  none o f t h e  -  structurally ing  similar derivatives.  a s t h e mould  none c o u l d  108  seemed t o u t i l i s e  be d e t e c t e d  T h i s was  e v e n more  strik  the added l a c t o n e ,  since  4 days o f  i n the b r o t h a f t e r  incuba-  tion 6  (32) At  this  necessary.  (33) stage  I t was  c o u l d be g a i n e d malonate to  (2-  C)  preferential  Sodium equally was  1 4  hoped t h a t ,  initially,  some  by f e e d i n g t h e more r e a d i l y and l o o k i n g  for reduction  incorporation of t r i a c e t i c  two  added t h e u s u a l  After  r a d i o a c t i v e t r a c e r s t u d i e s were deemed  malonate  amongst  (34)  (2-  1 4  C)  (0,04  mc)  was  sets of fermentation amount o f t r i a c e t i c  a v a i l a b l e sodium of activity acid  showed, a f t e r  r a d i o a c t i v i t y , that had t a k e n p l a c e activity  no d i l u t i o n  6,03  x 10  T o one s e t  of the malonate of t h i s  c/m/mg).  (40  b r o t h was w o r k e d ,  r e c r y s t a l l i sat i o n to  i n the production  f o u n d was  lactone.  acid lactone  b y t h e method d e s c r i b e d b y Tanenbaum and. B a s s e t t isolated  due  distributed  flasks.  4 days o f growth, the f e r m e n t a t i o n  patulin  information  mg), up  The constant  precursor  substance  (specific  - 109  The  of: 6 - m e t h y l s a l i c y l i c  isolation  a c i d was a c h i e v e d by  chromatography on t h i c k l a y e r p l a t e s and e l u t i o n o f t h e spots corresponding t o authentic yield  i n the  c a s e where no t r i a c e t i c  14 mg, and i n t h e  16 mgo 10-*  These  c a s e where t r i a c e t i c  10-^  amount o f w h i t e  specific  activity  solid  o f 380 c/m/mg  acid-free  methylsalicylic lised two  When t h e  0  low-acti-  This  solid  showed a  above t r i t u r a t i o n a n d t h e 6-  sample were d i l u t e d w i t h u n l a b e l l e d  activity.  handling  The s p e c i f i c  and r e c r y s t a l -  activities of the  s a m p l e s were f o u n d t o be 1330 a n d 1345 c/m/mg  f o r t h e c a s e where t r i a c e t i c  a  (not r e c r y s t a l l i sed) <, T h e  acid t o f a c i l i t a t e  t o constant  a c t i v i t i e s o f 4»08 x  with ethyl acetate,  remained.  r e m a i n i n g mother l i q u o r f r o m t h e triacetic  a c i d had b e e n a d d e d ,  c/m/mg r e s p e c t i v e l y  sample was c a r e f u l l y t r i t u r a t e d  small  The  a c i d h a d b e e n added was  s a m p l e s showed s p e c i f i c  a n d 3o27 x  vity  6-methylsalicylic acid.  acid lactone  respectively  had been added and  f o r t h e c a s e where none was a d d e d , s h o w i n g no s i g n i f i c a n t dilution  o f r a d i o a c t i v i t y due t o i n c o r p o r a t i o n  triacetic The m,p, o f The  acid  lactone.  ethyl acetate-insoluble  252-254°  relatively  indicated  that  of unlabelled  s o l i d was f o u n d t o have  and a n u l t r a v i o l e t  absorption  l o w r a d i o a c t i v i t y o f t h i s new i t was n o t m a l o n a t e  In a separate experiment  a t 288 mu. metabolite  derived.  i t was f o u n d t h a t , u s i n g  a  -  different spot  solvent  previously  resolved  1.10  -  system f o r t h i n l a y e r  assigned  i n t o two  spots  6-methylsalicylic acid  to  0,7  at Rf's  10$  several l i t r e s hydroxyls at  of broth,  3250 cm*"  infrared  (triacetic  slightly  soluble  derivative a t . 4o.O "T, 6,42  n,m,r, a  a methylene f l a n k e d  by  r , a m e t h o x y l g r o u p a t 6,18  This  information,  data, and  acid lactone  showed i n t h e  T attached  to  e i t h e r an together  suggested that  was  acetic  This postulated  1710  and  of t r i a c e t i c  Analysis  cm" ).  when t h e  to  from  singlet olefinic unsaturated fand  neutral  very  systems at  a methyl group  had  the  at  olefin.  mass s p e c t r a l  structure  m o l e c u l e s of  shown t o be  system, r e v e a l e d  C-^  pool.  c o r r e c t when the  TLC  using  another metabolite  grown on  (35)  tri-  reaction  formaldehyde-*?.  e x t r a c t , by  the  proton  methylene u n i t from the  with  in  1  I t s methylated  m a t e r i a l , p r e p a r e d by  mould was  cm"  I t was  1  w i t h a n a l y t i c a l and  one  acid lactone  changed s o l v e n t  1680  at  aromatic r i n g or an  s t r u c t u r e was  of the  accumulated  solvents.  t h i s metabolite  compared w i t h a u t h e n t i c  present  a c i d , was  f o r m e d f r o m c o n d e n s a t i o n o f two acid lactone  former  i n q u a n t i t i e s o f up  f o r carbonyl  i n most o r g a n i c  7,80  was  I t showed b a n d s f o r h y d r o g e n bonded  and  1  the  the  acid.  which appeared  of 6 - m e t h y l s a l i c y l i c  of t h a t  0,75,  and  corresponding to 6-methylsalicylie  This metabolite,  chromatography,  glucose  only.  the not This  - Ill -  compound showed a n R f i d e n t i c a l t o t h a t o f t o l u q u i n o l i n the o r i g i n a l s o l v e n t system<> graphy on s i l i c a g e l - G o violet  I t was i s o l a t e d by chromato-  I t was a l i q u i d h a v i n g a n u l t r a -  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  ©f base ( s u g g e s t i v e  of phenols).  I t s n.oior, showed a m u l t i -  p l e t a t 2o7-3?3 T ( 5 H » s ) and a s i n g l e t a t 7«75 T ( 3 H ' s ) . On a d d i t i o n of deuterium oxide t o the n . m . r . s o l u t i o n , the a r o m a t i c r e g i o n showed t h e d i s a p p e a r a n c e  o f one p r o t o n .  Its  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 b o n d i n g a t 3300 cm"*** and bands c h a r a c t e r i s t i c  of aromatic  Comparison o f t h i s m a t e r i a l w i t h o r t h o - , 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 (IR,  rneta-,  systems. and p a r a -  characteristics  NMR and R f ) t o t h a t o f m e t a - c r e s o l ( 3 6 ) .  (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 r o m 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 from e t h a n o l , i t melted at 286-2.87°. ultraviolet  I n the  i t showed a b s o r p t i o n s o f 325 mu ( £ = 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 b a s e . It  showed a n o n - h y d r o g e n bonded h y d r o x y l band a t 3360 c m "  1  -112  and  two  carbonyl  infrared. o f 336. the  The  f u n c t i o n s a t 1690  l o s s of a hydroxyl compound was  once, i t s f o r m a t i o n acid  lactone.  and  I665 cm  i n the ,  mass s p e c t r u m i n d i c a t e d a m o l e c u l a r  I t showed,as t h e  As t h i s  -  No  only  c h a r a c t e r ! s a b l e fragment „  group from the m o l e c u l a r  isolated i s not  weight  ion.  i n s m a l l a m o u n t s and  n e c e s s a r i l y due  to  only  triacetic  f u r t h e r i n f o r m a t i o n on t h i s compound  is  availableo  OH The reported,  (37)  presence  of m-cresol,  i s not  s u r p r i s i n g , as  f r o m 6 - m e t h y l s a l i c y l i c a c i d by Gaucher-^ benzyl All  8  has  (37)  free broth revealed  The lactone) pyrone  decarboxylation.  which i s simply  attempts to detect  acid  formation  no  m-cresol  f r o m P.  of methylene-bis  formed  Recently,  p a t u l u m . m-hydroxy-  acid  substance.  i n i t s production  m-cresol. lactone-  The  i s not  role obvious.  (3,3'-triacetic acid  i s s i m i l a r to that of dicoumarol  (39)^"°* and p h l o r o s p y r o n e  not  a hydroxylated  in triacetic  trace of t h i s  lactone  previously  i t i s most p r o b a b l y  been a b l e t o i s o l a t e ,  alcohol  of t r i a c e t i c  although  (38)^,  phloro-  (40)^, as w e l l as a l a r g e  - 113  -  number o f o t h e r f e r n p r o d u c t s of w h i c h 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 t o 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 t o P . p a t u l u m B a i n i e r 1MI ?98Q9 (C-554)» fulvin^,  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 -  none o f t h i s formaldehyde c o n d e n s a t i o n p r o d u c t  nor any o t h e r new m e t a b o l i t e mentation p r o d u c t s . a particular  c o u l d be d e t e c t e d  i n the  fer-  T h i s i s p r o b a b l y due t o the absence o f  - 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  strain  of P. patulum. 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 and the i s o l a t i o n of t h e s e new m e t a b o l i t e s ,  experiments,  were not  too  e n c o u r a g i n g a s i t was l i k e l y t h a t the p r e v i o u s chromato-  -  114  graphic assay about the cylic  m e t h o d s had  extent,  acid  and  given misleading  i f any,  information  o f enhancement o f  t o l u q u i n o l f o r m a t i o n due  ~  6-methylsali-  to t r i a c e t i c  acid  lactone„  Taking  i n t o account  with regard tone  (24)  vation  the  o b s e r v a t i o n o f Tanenbaum  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  i n Po  s t i p i t a t u m ( v i d e s u p r a ) and  our  own  o f t h e p y r o n e s t e s t e d , namely t h a t t r i a c e t i c  lactone  was  metabolite  the  o n l y one  g r o w t h , we  i n v e s t i g a t i o n o f the radioactive  tracer  showing no  felt  negative  justified  singular role  techniques  with  effect  lac-  obseracid on  i n c o n t i n u i n g the  of t h i s l a c t o n e labelled  using  triacetic  acid  lactone,  Triacetic similarly obtained  acid  labelled by  OEt  lactone  (3*5-"'"^'C) was  dehydroacetic  s e l f - c o n d e n s a t i o n of  acid^  prepared  w h i c h had  (2-^C) ethyl  O  et  (30)  been  acetoacetate  192V  (42)  from  (21)  - 115 -  T h i s l a b e l l e d lactone  (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»• patulum (C-945)»  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 t h e n e u t r a l e t h e r  e x t r a c t a c r o p 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 ,  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 $ poration into p a t u l i n ) .  speciincor-  T h i s i s a v e r y 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 o f 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 o f l a b e l l e d a c i d t o p a t u l i n by P . p a t u l u m .  6-methylsalicylic  Comparison of t h i s h i g h  degree o f 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 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 i n t o f l a v a p i n (43  t  1-C^ and  4  0.2$  Aspergillus flavipes, indicates  that  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 chain f o r mation i s considerably l e s s than that f o r aromatic already formed.  O H  (43)  substrates  ~ 116 -  To  d i f f e r e n t i a t e b e t w e e n low  triacetic  acid lactone,  incorporation  and  w o u l d be  molecule  l o c a t e the  p o s i t i o n s o f the  i n s p e c t i o n o f the  i t became a p p a r e n t  have b e e n i n c o r p o r a t e d r i n g A w o u l d be B  should  required  label.  the  random  necessary  6-methylsalicylic This  procedure these  that,  s t r u c t u r e of g r i s e o f u l v i n  should  directly  triacetic  remain u n l a b e l l e d .  w o u l d be  acid  heavy l i n e s ) ,  Thus, the  c l e a v a g e o f r i n g s A and  only  B by  only while  degradation  fusion  with  a known p r o c e d u r e . 0  ?  Figure  CH  3  6,  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  used.  T h i s m o u l d was  acetic  acid lactone, although a obstacles  lactone  into t h i s molecule,  CI  present  and  s m a l l amounts o f  l a b e l l e d ( f i g u r e 6,,  sodium h y d r o x i d e ,  For  lactone  available.  However, on  ring  on  of l a b e l l e d  i t w o u l d be  o f e i t h e r p a t u l i n or  extremely d i f f i c u l t  metabolites  (9),  o r breakdown o f the  into aromatic metabolites,  t o degrade the acid  incorporation  mould  a l s o u s e f u l f o r another reason.  f o r the  C-4  u n i t , might  reduction  implied  was Tri-  nevertheless by  Lynen"s  117  work  a t the  1 8  cylic  acid  (vide  supra),  chain extension  biosynthesis of griseofulvin,  Po  ten  in this  molecule  (G-554* a g r i s e o f u l v i n - p r o d u c i n g  grown o n C z a p e k - D o x , a n d t h i s medium was r e p l a c e d  days o f growth,  of  labelled  of  incubation*  triacetic  acid  A total  o f 80 tag (3»0 x 10^ c/m)  l a c t o n e was a d d e d .  15 mg o f g r i s e o f u l v i n  b r o t h and m y c e l i a l activity  since  steps i s avoided  i s retained,  patulum B a i n i e r  s t r a i n - ^ ) was after  T h i s u n c e r t a i n t y about t h e e x a c t  reductions  C-4 h y d r o x y l  the  biosynthesis of 6-methylsali-  stage o f the  sequence o f t h e i n the  -  extract.  This  was i s o l a t e d  sample  7 days  After  from  the  showed a s p e c i f i c  o f 421 c/m/mg (0,2% i n c o r p o r a t i o n ) .  Using a modifi8  cation  o f the  method d e v e l o p e d by B i r c h  et al,  , griseo-  0  f u l v i n was f u s e d w i t h p o t a s s i u m h y d r o x i d e a t 2 2 5 ° t o g i v e 3,5 mg o f o r c i n o l  (44*  78%) a n d 3,0 mg o f 3 - c h l o r o - 2 - h y d r o x y -  4*6-dimethoxybenzoic a c i d ties  o f 385 c/m/mg a n d 402 c/m/mg r e s p e c t i v e l y .  sample, a f t e r Essentially ted  for  vity  sublimation,  showed a n a c t i v i t y  a l l theradioactivity  i n the  radioactive one  (45* 35%)* w i t h s p e c i f i c  two f r a g m e n t s .  lactone  The o r c i n o l  o f 375 c/m/mg.  of griseofulvin  I fa significant  had b e e n i n c o r p o r a t e d  into  activi-  i s accoun-  quantity of griseofulvin,  w o u l d have e x p e c t e d a g r e a t e r f r a c t i o n o f t h e r a d i o a c t i t o appear  i n the  orcinol  fragment.  clearly  show t h a t t h e l a c t o n e , a s a u n i t ,  porated  into  griseofulvin,  The r e s u l t s  obtained  had n o t been  butthat the radioactivity  incorin  this  -  118  -  m o l e c u l e p o s s i b l y r e s u l t s f r o m breakdown o f , t h e i n c o r p o r a t i o n of the  This failure the  seems t o  of the  labelled  support  enzyme  small acetic-and  building  lactone  and  fragments,.  the  previous  system t o u t i l i z e  observation ^  of  2  precursors  the  other  malonic a c i d molecules i n the  than  chain  process,.  The  l a c k of  griseofulvin  i n c o r p o r a t i o n of t r i a c e t i c  i s not  n e c e s s a r i l y due  t o the  acid lactone  into  enzyme-bound cora-  18  plex that  has  a c c e p t a n c e by methylene The the  the  enzyme s u r f a c e  observed  effect  f o l l o w i n g ways  pathway t o be acid tered  As  may  (vide  discussed  form the  o f any  would  preclude  intermediate  supra)  lactone as  a  the  the  enzyme  polyketo-  such, but  the  T h u s , any  in  postulate  i n the  acetate  e n v i s i o n e d was  lactone derived  not  triacetic  difficulty  s y s t e m t o e i t h e r open t h e  lactone,  encoun©r  resultant a c i d , could lead  to  results.  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 act as a precursor sumably o f  explained  original  actual precursor  coenzyme e s t e r o f the  above  i n the  be  of t h i s lactone  i t s coenzyme d e r i v a t i v e , .  by  ; this  alternatively  p o s s i b l e involvement  the as  Lynen  chains,,  about the  the  b e e n p r o p o s e d by  other  i n the f o r m a t i o n  aromatic  acid lactone  does  o f g r i s e o f u l v i n and  natural products,  i t was  not pre-  decided  - 119 -  t o r e i n v e s t i g a t e the enhancement  e f f e c t s that t h i s  lactone  might have on m e t a b o l i t e f o r m a t i o n i n t h e o r i g i n a l m o u l d , F o r 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 t a p h y l o c o c c u s a u r e o u s ^ was t o be  utilised,  Seven day o l d r e p l a c e d c u l t u r e s o f P . p a t u l u m (554)  were  t e s t e d d a i l y by t h e • p a p e E - 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 biosis,,  The r e s u l t s a r e  summarized by f i g u r e  The g r a p h s show t h a t ,  7,  i n i t i a l l y , t r i a c e t i c acid lactone  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 samples.  However, 4 d a y s a f t e r t h e a d d i t i o n o f the  there  i s a s i g n i f i c a n t increase  of i n h i b i t i o n .  lactone,  i n the d i a m e t e r s o f t h e  T h i s e f f e c t i s exhausted a f t e r  zones  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 ,  while there  control experiments.  i s a sharp i n c r e a s e f o r  A t t h i s stage t h e b r o t h o f the  the  respec-  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 t h e d r y w e i g h t s o f 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 a g r e e d w i t h t h e 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 e x p e r i m e n t s i t had been o b s e r v e d t h a t Czapek-Dox s o l u t i o n s p r e p a r e d w i t h one l o t of r e a g e n t 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 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 .  grade  after  I t was a l s o observed t h a t  a medium p r e p a r e d 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 Days a f t e r t r i a c e t i c  -  5 6 7 a c i d lactone  F i g u r e 7.(Czapek-Dox  8 9 10 had been added  solution  1*)  Control • • L a c t o n e added a ®  Days a f t e r t r i a c e t i c a c i d  l a c t o n e had been added  F i g u r e 8, (Czapek-Dox s o l u t i o n  11  2.)  ®  -  s u p p o r t mould g r o w t h  121  -  as well as the usual reagent*  When a c c i d e n t a l l y , t h e above b i o - a s s a y e x p e r i m e n t s were r e p e a t e d on c u l t u r e s r e p l a c e d the lactone It  showed t h e s t r i k i n g  i s seen t h a t w h i l e samples  lactone, absence  with the l a t t e r  effect  shown i n f i g u r e  containing t r i a c e t i c  i n g e n e r a l have a c o n s t a n t a n t i b i o s i s , of the lactone  activity.  A s above,  there  medium,  i sa definite  8,  acid  i n the  decrease i n  the dry weights of the t o t a l  meta-  b o l i t e s p r o d u c e d were d e t e r m i n e d , a n d were i n g o o d  agree-  ment w i t h t h e b i o - a s s a y r e s u l t s .  These  results are puzzling  no t r i a c e t i c tation  acid  lactone  b r o t h 4 days a f t e r  cate that  since,  a s mentioned  c o u l d be d e t e c t e d i t s addition.  t h e o b s e r v e d enhancement  earlier,  i n the fermen-  T h i s would  indi-  i s n o t due t o c o n t i n u e d  presence o f the l a c t o n e .  R e t u r n i n g t o t h e b e g i n n i n g o f o u r experiments and t o Ehrensvard's r e s u l t s , i n o u r work r e p l a c e d added  i ti s interesting  c u l t u r e s were u s e d , E h r e n s v a r d h a d  the l a c t o n e t o t h e growing  enhancement a t a n e a r l i e r this  t o note t h a t , while  mould a n d h a d o b s e r v e d t h e  stage a f t e r t h e a d d i t i o n .  one c a n s p e c u l a t e t h a t t r i a c e t i c  some way.-on t h e enzyme  acid lactone  system o f t h e growing  acts i n  mould.  the already-grown mould i s b e i n g used t h e e f f e c t  From  When  i s not  122 -  obvious u n t i l  new m y c e l i u m  begins to form.  becomes e v e n more a p p a r e n t when a medium not  t o support mycelium  certain  This  i s used w h i c h  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  the  triacetic  acid  on the p h y s i o l o g y  reasons f o r the i n t e r e s t i n g  special  seems a  period.  More e x p e r i m e n t s a r e n e c e s s a r y t o d e t e r m i n e of  effect  l o t o f magnesium  the r o l e  o f t h i s mould a n d a l s o  results  obtained w i t h the  sulfate.  Such'experiments are i n progress a t t h i s  time.  EXPERIMENTAL  M e l t i n g p o i n t s were t a k e n on a K o f l e r b l o c k , violet  absorption  s p e c t r a were m e a s u r e d o n a C a r y 11  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 mentioned; E l m e r 21  ultra-  infrared  spectro  otherwise  s p e c t r a were m e a s u r e d w i t h  a  Perkin  s p e c t r o p h o t o m e t e r a n d n,m,r, s p e c t r a were  taken  on a V a r i a n A 60 m o d e l f o r s o l u t i o n s i n d e u t e r o c h l o r o f o r m < , Analyses  were c a r r i e d  o u t by A, B e r n h a r d t  i n Germany,  t h i n l a y e r c h r o m a t o g r a m s were r u n u s i n g Woeim  For Mueller  radioactivity counter  C h i c a g o ) was u s e d ,  by  on p l a n e h e t s .  counting  Ethylacetate  The c o u n t s p e r m i n u t e were  recording at least  Geiger-  (0,1-0,2 mg) were p l a t e d i n t r i p l i -  s o l u t i o n s o f t h e samples cate  Silica-gel-G,  m e a s u r e m e n t s a n end window  (Nuclear  A l l  1000  count®.  Hence t h e  e r r o r i s no g r e a t e r t h a n 10$;  determined statistical  the counting  rates  were c o r r e c t e d f o r b a c k g r o u n d ,  Subcultures  o f P e n i c i l l i u m patulum Bainer  IMI-34909  (G945) and P e n i c i l l i u m p a t u l u m 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 G l a x o Bristol, medium  England,  T h e s e were grown on Gsapek-Dox n u t r i e n t  ( g l u c o s e , 40gm; s o d i u m n i t r a t e ,  p h o s p h a t e , 1 gm;  magnesium  P o t a s s i u m c h l o r i d e , 0,5 1 liter) 1 liter  Laboratories,  as surface  dipotassium  s u l f a t e h e p t a h y d r a t e , 0=5  gm;  f e r r o u s s u l f a t e , 0,01  cultures.  erlenmeyer f l a s k s  3 gm;  (200  gm;  gm; water,  The medium was d i s t r i b u t e d i n ml/flask)  w h i c h had been  - 124 sterilized minutes  i n an "American After  0  sterilizer  18  (23-25°)°  s u r f a c e 2-3  -days a f t e r  The  P,  Isolation  o f P a t u l i n and  p a t u l u m G-945 was  trated (3 x 1,5 of  acid.  liters),  i t s volume and  bicarbonate acidified  167-168°,'  and  Rf  had  0,7  the  salicylic The sodium to  of the  separated  then  t o pH  fermentation from  the  2 with  extracted with  The  bicarbonate  (1,1  gm)  and  and  ultraviolet  acetic  to  aoid  1/20  sodium  e x t r a c t s were  the rest&ltant  recrystallised  T h i s compound moved a s a  u s i n g 20%  concenether  satiarated aqueous  with 6N hydrochloric acid  w a t e r , mop,  the  experiments.  ether extract concentrated  ml).  collected  i n these  acidified  extracted with  (5 x 20  room  Unless-otherwise  4 liters  I t was  The  precipitate  on TLC  used  o l d c u l t u r e was  d e c a n t a t i o n and  sulfuric  at  6-Methylsalicylic Acid  In a p r e l i m i n a r y survey day  15  mycelium u s u a l l y covered  inoculation.  mentioned  m y c e l i u m by  for  i n o c u l a t i o n t h e y were i n c u b a t e d  temperature  b r o t h o f a 10  a t 118°  from  hot  single  i n chloroform as  spot  solvents  6-methyl-  spectrum d e s c r i b e d " f o r  acid; n e u t r a l e t h e r e x t r a c t was  sulfate,,and c o n c e n t r a t e d  evaporate  c r y s t a l s m p, a  slowly^  over  anhydrous  i n volume..to, 1/3  It deposited  110-112°,  dried  1,3  gm  and  of prism  allowed  shaped  A f t e r r e t r y s t a l l ! s a t i o n from  chloroform  125  it  moved a s a s i n g l e  spot  ( R f 0,4)  u s i n g t h e above  solvent  system.  Effect Acid  of T r i a c e t i c  (30.)  Acid  on .Pen i c i l l imm  The mould  was  The  ml) and  triacetic  To the  added  The t h i r d  washed w i t h  (3 x 100  sterile  mg  g r o u p was  water  added.  T o one  The  group  was  i n 25 ml o f w a t e r ) .  dehydroacetic acid  w i t h t h e o t h e r s by t h e a d d i t i o n  growth  c a r e n o t t o b r e a k up t h e  i n groups of t h r e e .  aeid-laetone  s e c o n d was  ml o f w a t e r ) ,  l a t t e r was  f o r apparent equal  f r e s h C z a p e k - D o x s o l u t i o n was  f l a s k s were d i v i d e d added  (selected  were d e c a n t e d t a k i n g  mycelium pads, (3 x 100  patulum.  grown f o r 7 d a y s a f t e r w h i c h t h e f e r m e n -  tation broth of 9 flasks o f mycelium)  L a c t o n e ( 2 1 ) and D e h y d r o a c e t i c  (3 x  50 mg  made up t o e q u a l  i n 25  volume  ©f 25 ml ©f w a t e r t o e a c h  f l a s k and r e g a r d e d a s b l a n k . Aliquots at  first  total (3x3  hours.  ml), dried  dissolved  cm).  every how,  o f 72  filtered  this  (5 ml) were w i t h d r a w n with a s t e r i l e  and t h e i n 10  t h e n at  every 5 hour i n t e r v a l  for a  T h e s e a l i q i a e t s were e x t r a o t e d over anhydrous  solvent  removed  sodium by  sulfate  evaporation.  d r o p s o f chl©rof©rm=methanol  s o l u t i o n was  pipette,  transferred  with  (1,0  gm),  The  (1.1),  t© t h i n l a y e r p l a t e s  ether  residue 20 X  (20 x  The p l a t e s were d e v e l o p e d w i t h c h l o r o f o r m - a c e t i c  of 20  acid  - 126 (4sl) and sprayed Duplicate  Feeding  with  i o d i n e o r eerie  s u l f a t e reagent ,  t e s t s were d o n e .  o f U - ^ C ) Sodium  Malonate  S e v e n d a y o l d m y c e l i a l pads (4 f l a s k s ) sterile  water  medium,  ( 2 - ^ 0 ) S o d i u m malonate  (40 mg).  separated  After  3 days t h e f e r m e n t a t i o n b r o t h was  fermentation broth.  was e x t r a c t e d w i t h e t h e r  ml)  o f each  (4 x 150 m l ) .  s e t was removed a n d d r i e d  activities  o f the respective  1,01 x 10^ c/m/mg; The  the lactone  i n two s e t s . ( A ) a n d (B) r e s p e c t i v e l y .  set  remaining  over  Each  An a l i q u o t sodium  (10  sulfate.  s a m p l e s were  found,  ( B ) s 2,08 x 10^ c/m/mg.  ether extracts  were e x t r a c t e d  s a t u r a t e d aqueous sodium b i c a r b o n a t e , s o l u t i o n The  The t r i a c e t i c  l a c t o n e - c o n t a i n i n g f l a s k s a n d those w i t h o u t  Specific  acid  T h e m y c e l i a were washed w i t h w a t e r and t h e  s  were combined  (A):  (0,04 mc) was d i s t r i b u t e d  T o two f l a s k s was a d d e d t r i a c e t i c  w a s h i n g s combined w i t h t h e acid  with  (3 x 100 m l ) and r e p l a c e d w i t h Gzapek-Dox  among t h e f l a s k s . lactone  were washed  with  (4 x 15 ml),  b i c a r b o n a t e e x t r a c t s c o m b i n e d a n d a c i d i f i e d w i t h 6N  hydrochloric with ether  acid.  The a c i d i f i e d  (3 x 50 ml).  ' e x t r a c t s were d r i e d  over  s o l a t i o n was  re-extracted  Both t h e a e i d i c and the n e u t r a l 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  1,8 x 1 0 acidic 4°5  (B)  extract-(63  neutral  mg)  specific activity  (50 mg)  extract  specific  3  5o3  e x t r a c t (33 mg)  specific  activity  x 1©3 e/m/mg  crude n e u t r a l  e x t r a c t s were  evaporated t o y i e l d  dissolved i n ether 1  respectively  for  these- s a m p l e s were -6,0$ x. I ©  ..'respectively.  a, ©f s®lld patraliac,  After  The  and f o r  specific a c t i v i t i e s  c/m/mg arid 6 , 0 2 d 1 0 ^  3  Q/ss/mg,  twice rec-rystall'±sati0n f r o m chloroform,  b o t h s a m p l e s showed one spot, R f 0 , 4 ©a T L G specific  (3 ml) and'  15=0 a n d 15 ° 5 mg, .for ( A )  (B)  5$);  activity  x 1 0 c/m/mg  acidic  slowly  e/m/mg  x 10-* c/m/mg  2o9  The  3  activity ,  activity  6,03  x  1 0  3  and  5,95  (etham©l-chl©r@form  x 1Q3 c/m/mg  respectively. The  acidic  silica-gel  e x t r a c t s were chroraat©graphed © a t h i c k l a y e r  6 p l a t e s - ( a c e t i c a@id-c.hl©.r©f@rm,.  c o r r e s p o n d i n g t© 6 - m e t h y l s a l i c y l i c silica The  t oyield  specific  The spots  o  a c i d were e l a t e d from, the  16 and'14 mg f o r sets  (A)  and  (B)  respectively,  . a c t i v i t i e s found f © r these samples were  e/m/mg a n d 3270 set  1,5)  4080  c/m/mg . r e s p e c t i v e l y , . C a r e f u l t r i t u r a t i o n o f  (A)', w i t h e t f o y l a c e t a t e  afforded a s m a l l amount' ©f s o l i d .  This  solid, m p 0  252-254°p  0  shewed a g p e e i f i e  activity of  380 c/m/mgo  The the  mother' l i q m @ r o f t h e above t r i t u r a t i o n  e l u t e d m a t e r i a l f r o m s e t - B were d i l a t e d  6-methyisalicylic  aeid  set  with unlabled  (31 mg) a a d r e c r y s t a l l i s e d  w a t e r t o c o n s t a n t radiosLetlYityo the t h i r d  as well as  Specific  from  activity  after  r e c r y s t a l l i s a t i o n was 395© c/m/mg a n d 3980 f o r  (A) a n d (B) r e s p e c t i v e l y „  I s o l a t i o n o f New M e t a b © l i t e s , The  f e r m e n t a t i o n broth o f 50 f l a s k s  (10 l i t e r s ) t o p  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) h a d b e e n a d d e d i n the  usual'manner was a c i d i f i e d t © p H 2 p  ether  ( 3 x 4 liters)o  The e t h e r e x t r a c t  1/30 © f i t s voliame a n d e x t r a c t e d w i t h sodium b i c a r b o n a t e alkaline  0  (5x5© mi)  0  arad e x t r a c t e d w i t h was c o n c e n t r a t e d t ©  saturated  aqueous  On a c i d i f i c a t i o n  ©f t h e  e x t r a c t w i t h esE&cenfcrated 'hydr§><s!al©ric & ® i d  a  a  selid  was o b t a i n e d whieh was washed w i t h e t h a n e l t o reme-v© 6 - m e t h y l salicylic m„po  acido  T h e w h i t e ins@Xp.fole m a t e r i a l (10 mg) h a d  252-253° a n d R f 0„75 ( s o l v e n t s  c&loroform-aceti©  9sl)o  T h i s m a t e r i a l was d e s i g n a t e d  mother  l i q u o r was e x t r a c t e d w i t h e t h e r  dried  a s m e t a b o l i t e Ao  The  (3 x 50 m l ) , t h e e t h e r  over' a n h y d r o u s s o d i u m s u l f a t e a n d e v a p o r a t e d  a n o i l (Oo80 gm)o  aeid  T h i s o i l was t r a n s f e r r e d  t© y i e l d  t© a s i l i c a - g e l 6  -  129  chromatography column and e l u t e d w i t h acid  (9slj  200ml)  0  The f i r s t  chl©r©form-aeetic  80 ml o f e l u t e d m a t e r i a l  ( O 2 5 gm) was shown by TLG t o c o n s i s t mainly 0  s a l i c y l i c a c i d and m e t a b o l i t e A  0  The mixture  w i t h e t h a n o l t o a f f o r d 26 mg ©f a white TLC  showed one spot a t R f Oo75<>  o f 6-methylwas washed  s o l i d m po 2 5 1 - 2 5 3 ° <> 0  L a t e r f r a c t i o n s from the  column were a n a l y s e d by TLG and shewed o n l y t r a c e s ©f other new m e t a b o l i t e s when compared w i t h mould p r o d u c t s  without  added t r i a e e t i c - a c i d lactone,, -The n e u t r a l e t h e r l a y e r was d r i e d over anhydrous sodium s u l f a t e and c o n c e n t r a t e d by slow e v a p o r a t i o n t o g i v e s u c c e s s i v e cr©ps o f g r i s e o f u l v i n f o l l o w e d by p a t u l i r a  0  The remaining  s o l u t i o n upon  evaporation  gave 2 1 0 mg o f a brown ©il which showed a new spot a t R f 0»5 (solvents  chloroform-acetic acid 9 s l K  T h i s o i l was  chr©mat©graphed on a.column ©f s i l i c a - g e l G (30 gm) u s i n g ethanol-chloroform collectedo  (.12 20) a s eluento  20 ml f r a c t i o n s were  F r a c t i o n 4 (18 mg)...gave mainly 'one spot on TLC  Rf 0o5 ( s e l v e n t g a c e t i c a c i d - c h l o r o f o r m l s 9 ) ° designated as metabolite B  I t was  Q  " M e t a b o l i t e A" 8  T h i s compound was r e c r y s t a l l i s e d from e t h a n o l t© g i v e white p r i s m s m p 0  0  254-255°°  I t , showed  ^m&x 28$ mp. ('X5j>0®0)|  - 130  X max (0.1N NaOH/EtOH)s  290 mu (16,000),  3250, 2760, 2660, 1680,  IR(KBr).  1580, 1355* 1140,  1615,  980, 860, 825, 785, 765p 755 cm" NMR(at  60®),  1  3.90 ( s i n g l e t ) ; 6.45 ( s i n g l e t ) ; 7°85 ( s i n g l e t ) T lsls3)  (Areas  M o l c W t , by mass s p e c t r u m ?  C 3Hx206=  Analocalc, for  1  (poor  spectrum),  2 6 4 a,m.u,  0,59.09; H,4.58. Founds C,59.39 H,4»63,  Methylation  ...... and  o f " M e t a b o l i t e A" A ' ( 3 2 mg) was s u s p e n d e d i  "Metabolite . . . .  2 ml o f e t h e r e a l d i a a o m e t h a h e  (prepared  a m i d e - N - m e t h y l - N - h i t r o s o ) was a d d e d , obtained  a f t e r a short time.  This  t o give a colourless  with a l i t t l e  s e m i s o l i d which was t r i t u r a t e d  showed one s p o t R f . 0 . 5 ( s o l v e n t s :  ( M e t a b o l i t e A., R f 0,7)  was  mp  a n d washed w i t h  acetic  135=160°. T L C  acid-chloroform l s 9 )  U s i n g e t h a n o l - c h l o r o f o r m ' (ls9) t h e r e  on a t h i c k - l a y e r  t o give a white  A max''303  °  s p o t s a t R f s 0 , 2 , 0,5  isolated  T h e s o l v e n t was then  Y i e l d , 2 2 mg, m,p,  ;  s o l u t i o n was  s o l u t i o n was a l l o w e d t o  e t h e r and the' s o l i d f i l t e r e d  s m a l l a m o u n t s of' e t h e r .  were t h r e e  from p - t o l u s u l f o n -  A clear  s t a n d a t room t e m p e r a t u r e f o r 2 h o u r s . evaporated-  i n 1 ml o f methanol,  R  a n d 0,7,  ( L O m)  chromatography  s o l i d which c r y s t a l l i s e d  (21,000); no b a s e  shift,  T h e R f 0.7  compound plate  from e t h e r , m,p, 211-212®,  - 131 -  IR  (KBr)s  3 1 2 0 , 2940, 1705* 1645* 1555* 1350* 1250* 1135* 1025, 855* 3 2 0 , 780 c n T  1  4<>0 ( s i n g l e t ) ; 6 1 8 ( s i n g l e t ) ; 6<>42 ( s i n g l e t )  NMR8  0  7°80 ( s i n g l e t ) T (Area) 1 : 3 : 1 : 3 ) » Analocalco  f o r C-j^  Q^s  G,6l„64;  H,5o52; F o u n d C , 6 2 . 0 3 ,  H,5o69o  (3  Preparation of Methylene-BIs  To  an ethaiaolic  solution  of t r i a c e t i c acid  lactone  form-  a l d e h y d e , was a d d e d a n d a l l o w e d t o s t a n d a t r o o m t e m p e r a t u r e „ C o l o u r l e s s needles separated a f t e r crystallised A, m p ro  0  0  on t h a t  f r o m e t h a n o l m.p  254-255°°  hours.  These  254-255°, m i x e d w i t h " m e t a b o l i t e  0  I t s infrared  s p e c t r u m was  superimposable  o f Ao  "Metabolite B  m  T h i s compound  liquid  (12 mg)o  (OolN  NaOH/EtOH)g  NMRg  several  was f u r t h e r  I t showed  purified  o n T L C to. g i v e  Xmax;275 mm ( 1 , 9 0 0 ) ;  a  Xmax  288 mu (2,400)°  2.7-33 ( m u l t i p l e t ) ; 7°75 ( s i n g l e t ) . % ( A r e a s 5 ° 3 h Added d e u t e r i u m  7o75 ( s i n g l e t ) IR ( l i q u i d 770  film): cnf^-o  oxides T (Areas  2o7=3°3  (multiplet);;  4s3)°  3300 * 2900, 1 6 0 0 , 1 4 9 0 , 1 2 6 0 , 1120,, 9 2 0 ,  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 f r o m t h e a c i d i f i c a t i o n o f the bicarbonate  e x t r a c t from one u s u a l s e t o f 25 f l a s k s o f mould  growth t o w h i c h t r i a c e t i c  a c i d lactone  had been a d d e d , 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 ethanol i n s o l u b l e white s o l i d ml o f b o i l i n g e t h a n o l o needles  (16 mg was d i s s o l v e d i n 80  Upon c o o l i n g a c r o p o f  (6 mg) was o b t a i n e d , m.p.  was d e s i g n a t e d a s " m e t a b o l i t e  The  286-287°.  C".  concentration yielded "metabolite  colourless  T h i s compound  The mother l i q u o r upon A " (9 rag). " M e t a b o l i t e G "  had A max 325, 24© mf* (18,000 and 16,500)  Xmax  (OolN NaOM/EtOH)  IR(KBrk  320, 223 mu (18,500 and 19,200)  3360,3050^2880,1690,1665,1610,1535* 1420,  1 3 2 0 , 900, 800, 765, 755* 7 0 5 , cm" -. 3  MoloWto by mass spectrum?  336  aoBtoio  P r e p a r a t i o n o f (3o5°° ^G) D e h y d r o a c e t i c A c i d 1  (2-l^KJ) Ethylacet©acetate  (30)^5  (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 ester.  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 h o u r s i n an o i l b a t h a t 1 9 5 ° . The temperature  i n s i d e t h e r e a c t i o n f l a s k r e a c h e d 1 9 2 ° . The  p r o d u c t was d i s t i l l e d t o remove u n r e a c t e d s t a r t i n g (2.8 g m ) .  material  The p r o d u c t was t h e n s u b l i m e d t o g i v e 0 . 4 3 gm o f  -  a white s o l i c L of e t h a n o l .  -  1 3 3  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  Y i e l d , 380 mg (10%)  m,p, 126-128°  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)  showed one  Rf .0,8,  Specific  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 To 1#0  (3 5- ^G) T r i a c e t i c C  1  A c i d Lactone  mg o f l a b e l l e d d e h y d r o a c e t i c  ml of 90% s u l f u r i c a c i d under n i t r o g e n .  (2.1)44  a c i d was added The r e a c t i o n  was immersed i n an o i l b a t h a t 1 3 6 ° f o r 5 m i n u t e s .  0,6  tube  It  was  t h e n r a p i d l y c o o l e d i n i c e and about 2 gm o f i c e a d d e d .  The  p r o d u c t was kept a t 0 ° f o r 4 h o u r s t h e n f i l t e r e d and washed w i t h i c e water Specific  (2 m l ) .  Y i e l d 120 mg (88%), m , p ,  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  188-189°,  from e t h a n o l ,  37OO0 c/m/mg.  Feeding of  (3,5-  patulum B a i n i e r  12f  C) Triacetic  (C  A c i d Lactone  to P e n i c i l l i u m  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 ) 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  Caapek-Dox medium,  (3*5-^0) Triacetic  acid lactone  was added and the mould i n c u b a t e d f o r 4 d a y s . t a t i o n b r o t h was s e p a r a t e d ,  The  was  with (40 mg) fermen-  the mycelium 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 . b r o t h was e x t r a c t e d w i t h e t h e r (5 x 70 m l ) .  The  The e t h e r 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 e x t r a c t was f u r t h e r e x t r a c t e d bicarbonate  (4 x 15 m l ) .  x 10-* c/m/mg»  The e t h e r  w i t h s a t u r a t e d aqueous sodium  The combined b i c a r b o n a t e  extracts  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 e t h e r (4 x 20 m l ) .  with  B o t h the a c i d i c and n e u t r a l e t h e r e x t r a c t s  were d r i e d over sodium s u l f a t e and t h e e t h e r s t r i p p e d . neutral extract acidic extract  (60 mg) s p e c i f i c a c t i v i t y : (34 mg) s p e c i f i c a c t i v i t y :  I860 c/m/mg, 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 t h e r (1 ml) and a l l o w e d t o evaporate separated  slowly.  A c r o p of c o l o u r l e s s  (1.5 mg). m . p . 2 1 9 - 2 2 0 ° ,  g r i s e o f u l v i n m.p. 219-220°,  mixed w i t h  authentic  s p e c i f i c a c t i v i t y 160  c/m/mg.  The e t h e r 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 c r o p of p r i s m - s h a p e d c r y s t a l s ,  m.p. 110-112°  c r y s t a l l i s e d from chloroform m.p. 1 1 1 - 1 1 2 ° , ;  prisms  second  (11 mg). specific  Reactivity  145 c/m/mg, showing (0.1$) i n c o r p o r a t i o n i n t o p a t u l i n .  Feeding of  (3.5-^0) Triacetic  Patulum B a i n i e r  A c i d Lactone to P e n i c i l i u m  (C-554)  P e n i c i l l i u m Patulum B a i n i e r  (G-554) was grown i n a Roux  f l a s k under s i m i l a r c o n d i t i o n s a s used f o r the growth o f (G-945)»  P r e l i m i n a r y e x p e r i m e n t s had shown t h a t  little  g r i s e o f u l v i n was produced u n t i l a f t e r about 15 d a y s ,  - 13$ -  p r o d u c t i o n then i n c r e a s i n g considerably*  Accordingly, 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«5- C) T r i a c e t i c lif  acid lactone  (80 mg) was a d d e d . 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 5 0 ° under vacuum, f i n e l y powdered i n a Waring b l e n d e r and  extracted  i n a s o x h l e t a p p a r a t u s f o r 3 days w i t h l i g h t p e t r o l e u m e t h e r ( b . p . 30-60°).  The e x t r a c t c o n t a i n e d l i t t l e  f u l v i n and was d i s c a r d e d . extracted  The mycelium was t h e n  with chloroform f o r 2 days.  s t r i p p e d t o g i v e 150 mg o f an o i l .  griseo-  further  The c h l o r o f o r m was  T h i s o i l was  extracted  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 cooling deposited a c r y s t a l l i n e o 6  s o l i d (70 mg), m . p .  168-170°  (mycelianamlde m . p . 168-170 )  .  The mother l i q u o r on slow  evaporation y i e l d e d successive  c r o p s o f g r i s e o f u l v i n (12 mg),  m . p . 219-220°, s p e c i f i c a c t i v i t y 421 c/m/mg„.  A further  c r o p of 3 mg o f 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 the f e r m e n t a t i o n b r o t h w i t h e t h e r and slow e v a p o r a t i o n the ether„  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  of of  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 of G r i s e o f u l v i n ^ Griseofulvin  (13 mg), p o t a s s i u m h y d r o x i d e „(1 p e l l e t )  and water 2 d r o p s were heated a t 225° 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 e t h e r (4 x 5 m l ) .  The e t h e r e x t r a c t  s u l f a t e and t h e e t h e r e v a p o r a t e d .  with  was d r i e d over sodium  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)  and 3 - c h l o r o - 2 - h y d r o x y - 4 » 6 - d e m e t h o x y 0.7)  m*,p. 2 2 0 - 2 2 1 °  m.p. 105-107°  b e n z o i c a c i d (3 mg R f  ( 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 f r o m t h e 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 . o r c i n o l sample-was s u b l i m e d ;  it  The  showed a s p e c i f i c a c t i v i t y  o f 375 c/m/mg,  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  49 (34)-  I n a 50 ml round bottomed f l a s k w i t h an a t t a c h e d  reflux  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 acid lactone s o l u t i o n was l e f t  i n 15 ml of p h o s p h o r y l c h l o r i d e .  o v e r n i g h t a t room t e m p e r a t u r e .  I t was t h e n  warmed on steam b a t h f o r 3h h o u r s c o o l e d and 100 gm o f was a d d e d .  The  ice  I t was t h e n e x t r a c t e d w i t h e t h e r (7 x 50 m l ) .  The e t h e r 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 bicarbonate  (5 x 50 m l ) , d r i e d o v e r sodium s u l f a t e and the  ether s t r i p p e d .  The r e s i d u e  (1.2 gm) was s u b l i m e d a t  atmos-  p h e r i c p r e s s u r e on a steam b a t h t o g i v e w h i t e p l a t e s , m . p .  - 137 -  84-85°  ( 0 . 8 gm, 4 0 $ )  0  P r e p a r a t i o n of 4 - D e o x y t r i a c e t i c A c i d Lactone The c h l o r o l a c t o n e  (28)  49  ( 3 0 , 0 . 7 gm) was d i s s o l v e d i n 15 ml  o f e t h a n o l , and 3 gm o f z i n & powder t o g e t h e r w i t h 7 ml o f 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 were addedo  The m i x t u r e 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 t h e n t h e 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 w a s h i n g s were combined  w i t h t h e main body o f the s o l u t i o n and e v a p o r a t e d t o g i v e a yellow o i l .  I t showed on TLC 3 s p o t s , one a t R f 0 . 4  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 a t 1 2 0 ° .  The m a t e r i a l a t 10 m i n u t e s  r e t e n t i o n t i m e was c o l l e c t e d .  NMR 2 . 8 ( q u a r t e t ) ;  ( d o u b l e t ) ;- 4 . 1 7 ( d o u b l e t ) ; 7 » 8 ( s i n g l e t ) T ( A r e a , s  Effect  of Feeding of 4 - D e o x y t r i a c e t i c A c i d Lactone  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 t r i a c e t i c Afeld Lactone  (chloro-  4.0 1:1:1:3).  (32).  ( 3 4 ) „ and Methoxy-  (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 g r o u p s o f 2 , were added (2 x 40 mg) o f each 4-deoxytriacetic  acid l a c t o n e , 4-chl©ro--4-deoxytriace>ic  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 .  acid  The f e r m e n t a t i o n  b r o t h a f t e r 4 d a y s o f 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 with appropriate blanks.  -  1 3 8  -  E f f e c t of T r i a c e t i c A c i d L a c t o n e on P . patulum (as measured by b i o - a s s a y )  0  The mould was grown a s usual„  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 Staphylococcus aureous  47  by the p a p e r - d i s c m e t h o d  48  as  f o l l o w s : Twenty ml o f s t e r i l e a s s a y agar were added t o P e t r i A f t e r the agar had hardened 4 m l o f c o o l e d a g a r  disheSo  seeded w i t h S surface  0  a  aureous were d i s t r i b u t e d e v e n l y o v e r t h e  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,  £ i n c h ) were p l a c e d f l a t 0o080 m l sample of the (within 5 s e c )  s i d e down on t h e seeded a g a r .  An  s o l u t i o n was p i p e t t e d i m m e d i a t e l y  onto each d i s c a s i t was p l a c e d on the  agar»  The paper d i s c was p r e s s e d t o the agar w i t h the t i p of the pipette<, hours  0  The d i s h e s were i n c u b a t e d a t 37° f o r about 24 The d i a m e t e r s o f the zones o f i n h i b i t i o n were t h e n  measured t o t h e n e a r e s t one q u a r t e r mm.  -  139  -  REFERENCES  lo  L . Ruzicka, Experientia  2.  W.Bo O l l i s ( e d . ) , "The C h e m i s t r y o f N a t u r a l Compounds", Pergamon, New Y o r k ( 1 9 6 1 ) .  3.  J0N0  4.  A 0 J 0 B i r c h and F N 360 (1953)o  357  5.  A0J0  6.  R. R o b i n s o n , "The S t r u c t u r a l R e l a t i o n s C l a r e d o n P r e s s , O x f o r d (1955).  7.  A . J . B i r c h , R.A. Mas'sy-Westropp a n d C 0 J 0  8.  A . J o B i r c h , R o A . M a s s y - W e s t r o p p , R.W. R i c k a r d s a n d H . S m i t h , J . Chem. S p c . 1958 , 3 6 0 .  9.  Ro Thomas, P r o c . Chem. S o c . 1 9 5 9 .  C o l l i e , Jo Chemo S o c . 21, 0  J„  %  0  (1953) <>  1806  (1907).  Donovan, ' A u s t r a l . J .  Birch. Fortschr.  Chem. 6 , 1^,  Org. Naturstoffe,  186  (1957).  of Natural Moye,  Chem. 8, 539 (1955").  Products*  Austral.  88.  10o  A0J0 Birch, 1 9 6 2 . 425=  11.  S.Wo Tanenbaum a n d E..W. B a s s e t t , (1959).  12.  K  13.  L.Do F e r r i t t i , ( U . S o A o ) i t 6 , 14  14.  A.Jo B i r c h ,  15.  A . J . B i r c h a n d H . S m i t h , Chem  160  S. G a t e n b e c k a n d K. M o s b a c h , A c t a . Chem. S c a n d . 1 3 ,  0  JoFi  Phenolic  Mosbach, A c t a  S n e l l a n d P . L . Thompson, J . Chem. S o c . J. Biol.  Chem. S c a n d . 1/j., 457  Chem. 2 3 4 .  (1960)  1861  o  J.Ho Richards Proc. N a t l . Acad. S e i . (I960).  P r o c . Chem. S o c  0  1962 0  n  3.  S o c . S p e c . P u b l . 195$»  I56I  (1959).  17.  E.W. B a s s e t t  a n d S.Wo Tanenbaum, B i o c h e m . B i o p h y s .  itOp 535 (I960). 180  F . L y n e n a n d M. T a d a , Angew. Ghem. 21a 513  (196l)  c  Acta.,  » 140 «= 19»  F  20.  SoJo  21.  R.O. Brady, P r o c N a t l . Acad, S c i  0  Lynen,  C e l l , Gompo P h y s i o l . J ^ , Sup,  J .  Wakil,  J ,  Am. Chem. Soc*  |0,  6465  1,  33  (1958)0  (U.S.A.),  0  (1959).  993  (1958).  22o  Re B e n t l y and J . G . K e i l * P r o c , Chem, S o c , 1 9 6 1 , 1 1 1 ,  23.  JeD  24»  M, Waite and S.-J. W a k i l ,  25.  J . H . Birkenshaw and A* Gowlland, J  Bu»Lock and H.M. Smalley, i b i d ,  0  J ,  1961."  B i o l o Chem, a  209°  238.  81 - ( 1 9 6 3 )  •  Biochem. 3 4 , 3 4 2  (1962).  26o  T» Money, A . I . S c o t t , I» Q u r a s h i and GoB.R, Webster J o Am. Chem, Soc. ^ 2 , 3 0 0 4 (1965)«  27.  R.L. Edwards, D.G. Lewis and D.V. W i l s o n , J» Chem, S o c * 1261  9  3  4995.  2d.  S.W, Tanenbaumj T.R. Acker and P«E, B r e n n e i s e n , J , Am, Chem., Soc. 6 6 , 1 2 6 5 ( 1 9 6 4 ) «  29o  A.K. Ganguly, T.R. G o v i n d a c h a r i and P . A . Mohamed, T e t r a h y d r o n Zl9 9 3 ( 1 9 6 5 ) »  30.  J . E . Grove, J .  31o  G. Ciamacian and P. S i l b e r , B e r .  32.  P.M. Dean, " N a t u r a l l y O c c u r r i n g Oxygen Ring Compounds'*, B u t t e r w o r t h s , London, 1 9 6 3 , p . 1 0 0 .  33e  Go E h r e n s v a r d , E x p . C o l l . Res. S u p p l .  34.  A» Rhodes, P r i v a t e  ,35. 36.  30,  (1964)»  3234o  2 £ , 841*  (1894).  2, 1 0 2  (1955)•  Communication.  R.S. S i g g i a , T h e D e t e r m i n a t i o n o f Organic Compounds , Wiley and Co., New York, N.Y., 1949; P« 8 0 . n  0  S„W« Tanenbaum and E.W. B a s s e t t , Biochem, B i o p h y s . A c t a . , 2d,  37»  Chem. S o c ,  21  (1958).  Diekmann and F . B r e e s t , B e r .  2Z, 3 3 9 1  G.ML. Gaucher* P r i v a t e G o r a m u n i c a t i o n i . 1  (1904).  - 141 39.  M . A . Stahmann, C F . Huebner, 138, 513 (1941).  40  A . P e n t i l l a e and J * Sundman, A c t a . Chem. S c a n d , , 1 2 , 1888X1963).  ....  4  >•„,.... . s, .=.  a n d . K . P . L i n k , J » B i ' o l . Chem. ,  •.. i •  41 o  A . Pentillae  and J o Sundman,  i b i d , 1 £ , 839 (.1961).  42.  A , McGookin, A . R o b e r t s o n and T . H . Simpson, S o c , 1953, 1828.  43.  J.N* C o l l i e , i b i d ,  44°  E , 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 ,  45.  S W„ Tanenbaum and Ei.W. B a s s e t t , J . B i o l » 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 . ,  47=  M . I , Timonin, S c i e n t i f i c  48,  L , F c J o h n s o n , 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 D i s e a s e 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 a n d F. K o g l ,  J . Chem.  607 (.1891).  8  J o h n W i l e y & S o n s l n c , New Y o r k , N . Y . , 1955,  p . 231.  6  12, 35 (1965)=  A g r i c u l t u r e , 26;8, 358 (1946).  Rec. T r a v .  Cndem., 83, 46  (1964).  A P P E N D I X  Crystallizes from the Stobbe reaction product  Fig. 1.—Synthesis of (±)-dedimethylamino-6,12a-dideoxy-decarboxamido-7-chlorotetracycline (101, 102, 1(32) Reagents: (1), CICOOEt/N-methylmorpholine/benzene; (2), ( E t O O C — C H — C O O E t ) Mg (OEt); (3), aq. H , S ( V A c O H / h e a t ; (4), diethyl succinate/NaH; (5), H / N i ; (6), 1 equiv. C l j / C C U ; (7), polyphosphoric acid; (8), ethylene glycol; (9), L i A l H ; (10), methanesulfonyl chloride/pyridine; (11), KCN/diniethylformamide/water; (12), LiAl(OEt)jH; (13), diethyl malonate/AcOH/piperidine; (14), Na (CH CO—CH—COOEt)/ether/reflux; (15), aq. H C l ; (16), NaH/anisole. + +  2  4  +  3  H. M u x f e l d t ,  B e r . 9 2 , 3122  (1959).  MeO  0  0  MeO  MeO  MeO  OH  OMe  (from Stage 7, F i g . l ) CI  CH  CI  3  CH  3  CH,OH  COOBu  1  COOBu  1  JL,  COOEt MeO CI  MeO  CH  MeO  OMe  COOH  MeO CH  COOBu  1  8,9  ?H  3  COOH  OMe O CI  1  OMeO  CI  3  COOBu  OMe  0  3  IS  16  17,18,19  |J ^CONH,  MeO  OMe O H CI  CH  0  3  :i  H  CH  3  OH  to CONH  riVn MeO  OH  0  CONH  2  OH  O  OH  0  2  0  (and 12a-epimer) F i g . 2.—Synthesis of ( ± ) - 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 ( ± ) - 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 /eth_er/500-wattlarnp; (2), N a O H / M e O H ; (3), C H N ; (4), L i A l H « ; (5), P B r , ; (6), N a ( E t O O C — CHj—C(COOBu')j); (7), polyphosphoric acid; (8), dil. aq. N a O H ; (9), diethyl phthalate/170"; _(10) 2  2  +  2  t  P C U or oxalyl chloride; (11), C H N ; (12), benzyl alcohol/180"; (13), P C U ; (14) M g ( E t O O C — C H — COOEt),; (15), N a H / a n i s o l e ; (16) N H , / N a O M e — M e O H ; (17), H C l / A c O H ; (18), C H N , ; (19). P h C O , H / C H C l , ; (20), H C l / A c O H . 2  2  + +  2  H. M u x f e l d t j B e r . 92, 3122 ( 1 9 5 9 ) .  CI CHaBr  CI  A^Y K^J  C O O E T  COOEt  ~*  OMe CN CN •  «.r  JN^V^  0 0 0  "  f^V^*V^y^CONH  ll  0  MeO  a  COOH  COOMe  ts.ie  it,is,  BzO O "OH  "  (B^benzyl)  CI *0  COOMe 17,18, t9  >*°  COOEt COOEt  BzO MeO  «, « . <4, OH O H OH 0  OBz OMe O H O  Fig. 3.—Synthesis of (±)-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 — C H — C O O E t ) ; (3), LiAlH«; (4), methane sulfonyl chloride; (5), C N ; (6), O H ~ ; (7), polyphosphoric acid; (8), oxalyl chloride; (9), Rosenmund reduction (5% Pd-BaSO«-); (10), cyanoacetamide/piperidine; (11), coned. H C l / A c O H ; (12), benzyl chloride/boiling alkali; (13), MeOH/H SO<; (24), NaH/toluene; (25), B r , / N a O A c ; (26), collidine (dehydrobromination); (27), M e S 0 4 / K C 0 ; (18), mild alkaline hydrolysis; (29), ethyl chloroformate/NEt,; (20), M g (OEt) ( E t O O C — C H — C O O E t ) ; (21), NaH/toluene; (22), H , / 1 0 % P d - C ; (23), H C O O - N H . V 1 4 0 ° ; (24), boiling coned. H C I / A c O H , ' +  -  2  2  2  3  + +  J . H. B o o t h e , A. S. K e n d e , T. L. F i e l d s S o c . 8 1 , 1006 ( 1 9 5 9 ) .  a n d R. G. W i l k i n s o n , J . Am. Chem.  CI  H  CI  H  r  g  ^f^J^y^OOMe  COOH _ ^ COOH  XOOH  y]f OBz O  OBz O (from Stage 12,Fig.3)  C 1  H  8yn-acid (separated by crystallization from ethanol)  O O H <5  +  H  H  OH  H OOMe  OBzO  OOH OBzO  H  y  (syn)  H  + OBz 0  OH  4,5  OBzO  COOEt OH ^QEt  7,8,9  OBzO  OH O  OH 0  OH O  Fig. 4.—Synthesis of (±)-dedimethylamino-6-demethyl-6,12a-dideoxy-7-chlorotetracycline (106, .135). Reagents: (I), Ac 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) Mg ( E t O O C — C H — C O O E t ) ; (6), NaH/toluene; (7), H / 1 0 % P d - C ; (<?), H C O O ^ N H « / 1 4 0 ° ; (9), A q . H C l . 2  + +  2  s  T. L . F i e l d s , A. S. Kende a n d J . H. B o o t h e , J . Am.-Chem... S o c . 8 3 , 4 3 9 ( 1 9 6 1 )  +  Fig. 5.—Synthesis of (±)-6-demethyl-6-deoxytetracycline (97). Reagents: (7), dimethyl succinate/NaH/dimethyIfonnamide; (2), methyl acrylate/Triton B ; (3), hot aq. H , S 0 / A c O H ; (4). H i / P d - C / AcOH/200p.s.i.; (5). C l , / A c O H / 1 5 ° ; ( t 5 ) , H F / 1 5 ° ; (7), esterify; (8), dimethyl oxalate/1 equiv. M e O H / NaH/dimethylformamide; (9), hot aq. H C l / A c O H ; (10), M g (OMe),/«-butyl glyoxalate/toluene; (11), Me»NH/ — 1 0 ° ; (12), NaBH /diglyme/low temp.; (73), toluene sulfonic acid/toluene; (14), Z n / H C O O H ; (75), H , / P d - C / E t O H / N E t , ; (70), CICOOPr'; (77), M g (EtOOC—CH—CONHBu'),: (18). NaH/dirn«*thylforniarnide/120 ; (19), hot 48% uq. H B r ; (20), CeClj/Oj/dimethylfornium.de/M e O H / p H 6. 4  + +  4  + +  o  L. H. C o n o v e r , K. B u t l e r , J . ' D . J o h n s t o n , J . J . K o r s t , a n d R. B. Woodward, J . Am. Chem. S o c . 8 4 , 3222 ( 1 9 6 2 ) . -  

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