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Studies on the synthesis of estrogen glucosiduronates Roy, Joan Elaine 1964

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STUDIES  ON T H E S Y N T H E S I S  OP E S T R O G E N  GLUCOSIDURONATES  by  Joan B.Sc,  The U n i v e r s i t y  A T H E S I S SUBMITTED  Elaine  Roy  of B r i t i s h Columbia,  1962  I N P A R T I A L F U L F I L M E N T OF THE R E Q U I R E M E N T S FOR THE DEGREE OF Master  of  in  Department  the  Science  of Biochemistry  We a c c e p t  this  thesis  as  conforming  to  THE U N I V E R S I T Y OF B R I T I S H May,  1964  the  required  COLUMBIA  standard  In the  presenting  r e q u i r e m e n t s f o r an a d v a n c e d  British  Columbia, I agree  available, for reference mission  f o r extensive  representatives.  that  the L i b r a r y  and study..  Department  of  permission.  Biochemistry  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 D a  -te  May  6  f  1964  agree  •-,  Columbia,  that  per-  f o r scholarly  by t h e Head o f my Department  I t i s understood  of •  s h a l l make i t f r e e l y  I, f u r t h e r  o r by •  t h a t ; c o p y i n g or p u b l i -  c a t i o n .of t h i s t h e s i s . f o r f i n a n c i a l g a i n w i t h o u t my w r i t t e n  f u l f i l m e n t of  degree a t the U n i v e r s i t y  c o p y i n g of. t h i s t h e s i s  p u r p o s e s may be g r a n t e d his  this thesis i n partial  shall  n o t be a l l o w e d  i ABSTRACT An i n v e s t i g a t i o n of the chemical to  synthesis  establish  led  of  estrogen  synthesis  T h e a- a n d ( 3 - a n o m e r s  O - a c e t y l - D - g l u c u r o n a t e were p r e p a r e d base of  and a c i d  either  compounds y i e l d e d  -1-bromo-l-deoxy-a-D-glucuronate. series  of reactions  -triene. ful,  this  reason  be  prepared.  Estradiol  in  c r y s t a l l i n e form conjugated  gave  a solid  the  undertaken  doubly  label-  1,2,3,4-ietra-  glucuronolactone  respectively. methyl  by  Bromination  2,3,4-tri-0-acetyl  E s t r o n e was  of  the  17-keto  converted  g r o u p was  estriol-178-glucosiduronate glucosiduronates  could not  in significant yields. with  deoxy-a-D-glucuronate.  in  by  a  3,16a-diacetoxy-17-keto-estra-l,3,5(10)  Attempted reduction  and f o r  cessfully  to  was  of the  of methyl from  catalyzed acetylations  of these  involved  glucosiduronates  conditions for'the  conjugates.  reactions  be  unsuccess-  could  not  obtained  E s t r o n e was  suc-  methyl 2 , 3 , 4 - t r i - 0 - a c e t y l - l - b r o m o - l -  D e a c e t y l a t i o n under  compound presumed  to  be  estrone  alkaline  conditions  glucosiduronate.  ACKNOWLEDGEMENT S I nell  for  would- l i k e  his I  structive  advice  -  would also  c r i t i c i s m and The  Council  and  is  financial  gratefully  to  express  direction like  to  my t h a n k s throughout  to  Dr. V. J .  this  t h a n k my h u s b a n d  O'Don-  work.  for  his  con-  inspiration. assistance acknowledged.  of  the  National  Research  i i T A B L E OF CONTENTS Page Introduction Materials  1  and Methods  Experimental  16  and R e s u l t s  20  Preparation of Methyl T e t r a a c e t y l by A c i d C a t a l y z e d A c e t y l a t i o n  Glucopyranuronate 20  Preparation of Methyl Tetra-O-acetyl-cc-D-glucuronate by Base C a t a l y z e d A c e t y l a t i o n and I s o m e r i z a t i o n . .  23  Preparation of Methyl osyl Bromide)-uronate  25  (tri-O-acetyl-ci-D-glucopyran-  14 Preparation Conversion  of C  - l a b e l l e d Sugar  of Glucuronic A c i d  to  Derivatives  . . .  Glucuronolactone  28 .  29  Attempted  Synthesis  of E s t r i o l - 1 7 p - g l u c o s i d u r o n a t e  Attempted  Synthesis  of E s t r a d i o l Glucosiduronates  .  35  Attempted  Synthesis  of Estrone  .  37  Glucosiduronate  .  29  Discussion  45  Summary  54  Bibliography  55  1 INTRODUCTION Relatively function of  this  of  use  estrogen  thesis  synthesis  little  of  were  known o f the  conjugates to  for  i n the  establish  estrogens  t h e s e methods  is  the  body.  conditions  conjugated  metabolism The for  objectives  the  with glucuronic  preparation  of  such  and  efficient  a c i d and  compounds  to  doubly  labelled. Concurrent in  1929-1930,  in  a water  it  was  w i t h the  discovery of  reported  (l)  soluble  form.  a water  soluble  lated  such  Their  chemical studies  one  glucuronic  showed the recent  phenolic  nancy By  their  urine  to  of  the  uronate  b o t h were  occured  two  steric  C-13  of  present,  group  times  of  the  during  was  estriol (3,4).  it  the  of  16a-and  occured  structures  the  urinary that  more  attachment. and  Kellie  i n human  preg-  i n ring D.  compounds,  of  they  estriol-16a-glucosid-  quantity  glucosiduronate at  to  and' c o m p a r i s o n  of  estriol-178-  d i s t r i b u t i o n was  (6)  urine.  they  for  178-positions  178-hydroxyl group  Neeman a n d H a s h i m o t o  While  remained  points  estriol  but  iso-  conjugated  p r o b l e m by C a r p e n t e r  isolated  This unequal  hindrance  methyl  the  excreted  f r o m human p r e g n a n c y  free,  isomeric  i n three  glucosiduronate. to  at  estriol  Cohen and M a r r i a n (2)  it  determine  and  e s t r o g e n s were  molecule  discovery that  these w i t h derivatives that  per  of this  conjugated  synthesis  found  indicated  h y d r o x y l was  investigations  l e d to  1936,  estrogen  acid residue  Examination (5)  In  that  estrone  the  attributed  by the  (5)  angular  formation. same t i m e  isolated  2 e s t r i o l - l 6 a - g l u c o s i d u r o n a t e f r o m human p r e g n a n c y u r i n e , confirmed t h i s structure the  conjugate  by a c i d h y d r o l y s i s o f d e r i v a t i v e s o f  and by n u c l e a r m a g n e t i c r e s o n a n c e  F e l g e r and K a t z m a n (7) siduronate  from the  and  spectroscopy.  i s o l a t e d an e s t r i o l  same s o u r c e as w e l l as a  b u t t h e p o s i t i o n s c o n j u g a t e d were n o t  digluco-  mono-conjugate  determined.  While q u a n t i t a t i v e l y e s t r i o l glucosiduronates t h e most i m p o r t a n t other  conjugated  filtration to  e x c r e t e d i n pregnancy  e s t r o g e n s do o c c u r .  W i t h the  and c o n c e n t r a t e  of g e l  ( 8 ) was  u r i n a r y conjugated  able  estro-  I n a d d i t i o n t o t h a t i s o l a t e d b y Cohen and M a r r i a n ,  identified  e s t r o n e and e s t r i o l - 3 - g l u c o s i d u r o n a t e ,  - 3 , 1 7 - d i g l u c o s i d u r o n a t e and e s t r i o l - and the  urine,  advent  and c h r o m a t o g r a p h y on S e p h a d e x , B e l i n g  isolate, purify  gens..  conjugates  are  l a s t two o n l y  he  estradiol!  estrone-3-sulfate,  tentatively.  E x t e n s i v e w o r k b y D i c z f a l u s y w i t h human f e t u s e s shown t h a t most o f t h e i r trogens. of  the  organs  T h i s can occur at  are  capable  of conjugating  are  mixed s u l f a t e s  es-  any o f t h e h y d r o x y l a t e d p o s i t i o n s  e s t r o g e n m o l e c u l e , and w h i l e m o n o g l u c o s i d u r o n a t e s  monosulfates  has  and  formed p r e d o m i n a n t l y , d i g l u c o s i d u r o n a t e s  and g l u c o s i d u r o n a t e s have been r e p o r t e d  C o n j u g a t i o n i s a common r e a c t i o n o c c u r i n g i n  and  (9). the  b o d y and n e a r l y t h i r t y y e a r s  o f r e s e a r c h have i n d i c a t e d t h a t  the  affected:  majority of steroids  progestins,  are  a d r e n o . l c o r t i c o i d s , and t h e i r  The b i o c h e m i c a l p r o c e s s  androgens,  estrogens,  metabolites.  of c o n j u g a t i o n i n v o l v e s the  3  combination onic  acid,  of  a suitable  sulfuric  acid,  amino  acids.  This results  water  soluble  than  acid  conjugates  C-l  of  ate; the  and the C - l is  .through The  other  are  may be  the to  form a  an e s t e r  acylal  form a  bond to  al-icyclic,  H  H-C r  glucuronic i n which of  the an  glucosiduronbond  the  where  aglycone glucuronide.  aromatic  or  hetero-  /H CI H-C-OH  0  HO-CH  H-0  5  GLUCOSIDURONATE-  curonic  the  acid,  isolated (10).  uridine  from the was  diphosphate  soluble  fraction  dehydrogenase.  fraction  of  the  GLUCURONIDE  biochemical process  uridine  It  cell  —  COOH  6  In  0  H-COH  C OOH  linked  more  R-C-0  H-C„-OH I2 HO-C„-H I3 H-C.-OH  the  compounds  type  of  and  •' RO  donor  glucur-  acid  one  c a r b o x y l group  aliphatic,  as  h y d r o x y l group  cyclic.  was  of  two k i n d s :  g l y c o s i d i c bond,  stable  such  Examination of the  j o i n e d to  l i n k e d w i t h the  aglycone  production  which possesses  a much l e s s  with acids  acid, mercapturic  i n the  there  acid is  by a s t a b l e  acetic  parent.  reveals  glucuronic  aglycone  the  acceptor  diphosphate liver,  later  was  of the  shown t o  ( U D P G ) was cell  be  acid the  with  glucuronyl  o x i d i z e d to presence  occurs  influence  i n the  glu-  (UDPGA) w h i c h  by S t r o m i n g e r  i n the  The t r a n s f e r the  conjugation  glucuronic  demonstrated  glucose  under  of  (ll)  UDPGA  of  that in  a n NAD  microsomal  of u r i d i n e  diphosphate  4 (UDP) in  glucuronyl transferase  the  liver  (13),  kidney  (11,12).  (13),  This  conjugation  i n t e s t i n a l mucosa  •> als  (15),  sequence  a n d human f e t a l of reactions  organs  (16,17,18,19).  (14),  occurs adren-  . i• The  following  dbs.> i n v o l v e d .  UDPG pyrophosphorylase g l u c o s e - l - P 0 + UTP , ^ UDPG + P P UDPG dehydrogenase UDPG + 2NAD - — ^ UDPGA + 2NADH + 2 H 4  +  —  ' i i '  -  UDP g l u c u r o n y l transferase UDPGA- + a g l y e o n e _ • ' glucosiduronate 1 1  Sulfate mechanism i s  1  conjugation  how w e l l  + ATP  APS + ATP  APS p h o s p h o k i n a s e — ^ Mg  , , ROH + 3 - p A d - 5 p s u l f a t e  crosome-free The has  been  reviews is  and  these  sugar  ft , , R-0-S O~ + 3 -pAd-5 - P 0 r  occurs v i a t h i s  the  following  from  the  The f o r m a t i o n o f condensation  derivatives.  syntheses.  the  pathway  4  in mi-  (21).  primarily  (22,27,28).  body and  (l).  sulf©kinase -  d e s c r i p t i o n of  condensed  i n the  , adenosine-5 -phospho( s u l f a t e + PP APS , , ADP + 3 - p h o s p h o a d e n o s i n e - 5 -phos, , (phosulfate 3 -pAd-5 p sulfate  estrogens  supernates  a c h i e y e d by the  sugars for  the  occurs  + UDP  ATP s u l f u r y l a s e •—Mg"*"**  sulfate  of  also  established  inorganic  Sulfurylation  1  of  following  syntheses  comprehensive  conjugates,  chemically,  a l c o h o l s and p h e n o l s  There  The f i r s t ,  chemical  are  three  w h i c h has  general  with methods  been v e r y u s e f u l  in  5 the  preparation  Knorr ides of  of  reaction. which  combine w i t h  acceptor"  lation the  of the  most  glucosiduronates,  This i n v o l v e s the  a heavy'metal  "acid  steroid  or to  alcohols  organic speed  product.  common  use  It  is  reaction  Silver  oxide  the  Koenigs-  of O-acylglycosyl  and phenols  base.  the  is  customary  and to and  i n the  presence to  add  prevent  silver  hal-  an  deacety-  carbonate  are  acceptors.  \  6 cT H-C-Br  I  HC-OAc  H-C-oAc 2 ROH + A g C 0 . + 2  2  3  A c 0  _c_  0  H  *  2  A  g  B  r  +  C00CH  ried  out  i n the  C - l is  presence  phenols Walden to  a)  presence  anomer  both  anomers  produced, conjugated  Koenigs-Knorr  and  isomers  reaction  nonhalogenated thesis.  sugars  I n the the  yields  6-glycosides  of  is  formation  is  presence  of  salts  obtained  with  A  c 1  O  C  H  3  car-  with  acetone  with  organic  base  with  by the  completely  s u c h as  a-anomer  with  either Spe-  use  of  the  conditions.  phenolic  of a c a t a l y s t  obtained  performed  conditions.  appropriate  confined to  of the  i n reactions  i n aqueous  on e x p e r i m e n t a l  aglycones  are  also  mercuric  c a n o n l y be  of  presence  chloride,  i n the  and the  Condensation  o  0  ion.  of a l k a l i  c) w i t h  depending  invariable  reaction  i n v e r s i o n o c c u r i n g b)  yield  cific  i n the  - ? ' H-C H  i  silver  The K o e n i g s - K n o r r  K  3  almost  of  2 :Ac01-:CH  H,0 + CO, ^ ^  H-C  i n v e r s i o n at  C  r  H-C-oAc  Walden  2| °3 *  H  is  acetylated,  glycoside  syn-  anhydrous  zinc  favoured while  p-toluenesulfonic  good  Conjugation sence, of  a hydrogen  a mixture  of  are  also  glycoside into  of  lyst  substitution  teration  done  sugars  i n the  Fischer reaction,  one  anomer  of the  into of  sugar  i n the  by a)  another  one  pre-  gives  (e.g.  the  for  the  steps—acetylation,  one  concata-  and c)  al-  glycosiduronic  common s u g a r  conjugation  or  appropriate  another  oxidation to  of  by t r a n s f o r m i n g  anomerization  with  aglycone  Koenigs-Knorr  i n three  synthesized  another  The p r e p a r a t i o n ployed  the  free  anomers.  version b)  alcohols with  i o n , known as  Glycosides preformed  of  acids).  derivatives  reaction  em-  is  generally  a n o m e r i z a t i o n and  halogena-  tion. Formation all  cases,  acetic  determines and  the  sulfuric  yields These  of p o l y - O - a c e t y l a t e d sugars  anhydride  anomer  acid  and a c a t a l y s t ,  obtained.  mainly y i e l d  P-acetyl derivatives reactions  generally  Zinc  a-anomers,  nate,  the  project,  group  at  acetylation  performed  reactions  Methyl  first  of  methyl  methylation  and t h e n  one  of  of  acid  acetate both.  compounds. glucosiduroof the  the  carboxyl  preceding  applied.  glucuronate  ways.  G o e b e l and B a b e r s  iodide  (23)  or  which  w h i l e sodium  in crystalline  tetra-O-acetyl  C-6 i s  choice  and p y r i d i n e a m i x t u r e  result  in this  in  chloride, perchloric  To make a c o m p o u n d s u c h a s one u s e d  the  involves,  c a n be  reacted  prepared  silver  refluxed glucuronolactone  in a variety  glucuronate  with  w i t h methanol  of methyl  (24)  to  7 obtain  the  curonic dation  methyl ester.  acid  The a c i d  or glucuronolactone  w i t h methanol  and l a c t o n i z a t i o n , as w e l l  curonate.  The b e s t  ification  preparative  w i t h methanol  i n the  catalyzed reaction  as  of  l e d to  glu-  glycosi-  formation of methyl  m e t h o d was presence  found to  of base  be  glu-  ester-  catalysts  (25,  26). To c o n v e r t fic  species  tion,  i n order  to  an a n o m e r i z a t i o n  a mixture is  a mixture  of a c e t i c  generally  allow is  anhydride,  than  the  stable  ride  but  less  less  stable  the  reactive  than  the  and  sugars,  sulfuric  a hydroxyl  group  a different most u s e f u l .  of the  than  the  ways  acid  the  halogen  at  halides,  the  reducing C - l (28). of the  liquid  or  the  than  Since the  latter  stable more  c o n f i g u r a t i o n of  more r e a c t i v e  iodide.  are  carbohydrate  u s u a l l y more  is(more  chlois  halide  in-  replacement exchange  method  is  C - l a c e t o x y l group  is  gaseous hydrogen  titanium tetrachloride,  the  commonly u s e d .  carbon atom and The f i r s t  the  iodide  a glycosyl  C - l acetoxyl group,  Replacement  a c h i e v e d by treatment w i t h  anomeric is  halides  generally yield  of preparing  of the  at  of g l y c o s y l  i n synthetic  methods  bromide,  replacement  phosphorous  speci-  on r e c r y s t a l l i z a For acyl  acid  a-anomer i s  The b r o m i d e  The numerous  with  the  irrespective  material.  of  acetic  intermediates  B-, a n d p r e p a r a t i v e  form,  the  (27).  derivatives  With halides,  starting  clude  yields  a  used.  most u s e f u l  chemistry.  better  performed  The p o l y - O - a c y l among t h e  o f a-and 8-anomers t o  halides,  or hydrogen  hal-  8 ide  in glacial  glycosyl  bromide  thod w i l l  volving silver  Closer  examination  the  ing  is  of  the  and  so  competitive molecules  of  the  the  inversion is departure  of  side  (due  open f o r  complete. the  from the  identical with  required  the  naturally  may b e r e m o v e d u n d e r the  ester  type. the  reaction  g l y c o s i d i c bond i s It  was  theoretical is  amount  i n e f f i c i e n c y and  reagents  include  mildly  sodium,  of  that  catalytic  unless  potassium  a  by  a  alcohol  of  a  conjugate  cis  pre-  Acetyl  conditions,  to  the  of  bond  i n anhydrous  is  a n d now  of base  Effective  the  medialless  required  quantities  convenience.  aceto-  respectively.  obtain  was  react-  carbonyl  formation  alkaline  alkali  hin-  by  trans  occuring one.  stable,  discovered  achieved with  to  attack  polarizable  acid-labile orthoacetate,  group.  steric  by the  i n the  is  acetyl  rear  resulting  ef-  glycosyl  followed  group,  e.g.  "pull"  is  C - 2 acetoxy an  a  to  With  halogen  in-  by f a c i l i t a t i n g  neighbouring  the  or  reaction  creating  G-hl c a t i o n  me-  group.  acceptor,  catalyst  oxygen of  which  gain  an a c i d  by the  sumably  a  as  opposite  Deacetylation  than  that  for  by any  acetoxyl  conjugation  also  glycoside  groups  second  bond and  nucleophilic attack  and  most w i d e l y u s e d  i o n from 1 , 2 - c i s - 0 - a c e t y l  protects the  a  role  C - l halide halide  leaves  sugars,  the  same s i d e  group  molecules,  the  the  of  reveals  assumes  on the  acetyl  halogeno  of  halides  is  Prolonged treatment of  Loss of  drance)  last  i n replacement  glycosyl  halides  The  preparation.  carbonate,  fect.  acid.  result  dissociation  This  acetic  to  this give  deacetylating  and b a r i u m h y d r o x i d e  as  9 well  as  milder agents  s u c h as  sodium, potassium  o r b a r i u m me-  thoxide. The c h e m i c a l l y s y n t h e s i z e d an  important  serving from  as  other  a p p l i c a t i o n to  standards  for  sources.  As the  metabolic  methods  those  used  tissues, are  they w i l l  a p p l i c a b l e to  discovery  that  be  of  considered here.  steroid  estrogens  conjugates occur  was f o l l o w e d  was  p l a c e d on d i r e c t l y e s t i m a t i n g  pure  w a s made u n t i l standard  physical tify  constants  which  l e d to  are  solvent  graphic phasized thods gates  partition,  steroid  that" none  w h i c h may b e  acid  these  fluids  emphasis  conjugates.  Little  availability  can a l s o  the  conjugates  fact,  sensitive  or  methods  Because  m a i n l y as  methods  be u s e d  pro-  of  methods.  conjugates. several  for  the  The  to  the  iden-  estimation  Those methods steps.  Among  and e n z y m a t i c h y d r o l y s i s ,  and c o l o r i m e t r i c t e s t s . of  similar  conjugates.  generally involve  techniques  more  no good g e n e r a l  i d e n t i f i c a t i o n of been used  the  detection  these  in general.  explain this  accumulated  chemically synthesized  have are  to  of  as  obtained  are  biological  r e c e n t l y when g r e a t e r  conjugates  There and  by attempts  i n urine  have  well  i n the  Most  i n urine  (2)  gress  other  as  conjugates  involved  conjugates  in investigations  conjugates  experiments  i d e n t i f i c a t i o n of  and i d e n t i f i c a t i o n of estrogen to  estrogen  are  It  these chromato-  s h o u l d be  completely general  successfully applied vary with  which  em-  and the the  me-  conju-  studied. Solvent  p a r t i t i o n has  been used  to  separate  free  es-  10 trogens  from  their  conjugates.  be  applied to  is  differential hydrolysis,  This but  not  only  releases  sulfates  are  uronates. such  as  tative drastic  the  the  can separate free  steroids  hydrolyzed  Extraction  chloroform, solvolysis  estrogens  either the  estrogens  ful  for  sulfates.  sulfates  growth old  the  use  are  the  acid.  solvents a  quanti-  However,  o f up t o  o f no  This  is  which  a c t i o n of  more acid  15-20$  samples  of  at  the  (29)  estrogen  pregnancy u r i n e . beef  loss  unstable  to  acid  glucosiduronates enzymes  such  as  respectively.  ^ - g l u c u r o n i d a s e was  when he  observed that  conjugates  room t e m p e r a t u r e . isolation  significant  p a r t i c u l a r l y use-  are  specific  sulfatase  action of  i n 1933  now o b t a i n e d f r o m  molluscs.  advantage  estrogens  and phenol  and c l e a v a g e  from  effects  glucosid-  Glucosiduronates require  of enzymes.  newer  through  enzyme f o l l o w e d nate  solution with  Chemically,  (20).  by M a r r i a n  urine  enzymatic.  conditions than  i s , heating with  The h y d r o l y t i c noticed  then  A common o n e  estimated.  Enzyme h y d r o l y s i s d i f f e r e n t i a t e s  B-glucuronidase  may  glucosiduronates  accompanied by d e s t r u c t i o n  many o f t h e  hydrolysis. from  is  c a n be  and  ethyl acetate  A method h a v i n g the of  sulfates  which  or  fraction.  chemical or  o f an aqueous  that  may b e  conjugated  by m i l d e r  ether  of  treatment,  hydrolysis of  identify  Several procedures  first bacterial  o c c u r e d i n two  day  I d e n t i f i c a t i o n of  the  of e s t r i o l - 1 6 (17)-glucosiduro-  Preparations liver,  of  bacterial  ^-glucuronidase extracts  and  marine  11 It the  phenol  This  was  1949 b e f o r e  sulfatase,  M y l a s e P,  enzyme a c t i v i t y was a l s o Indications  tic  Cohen and B a t e s  h y d r o l y s i s does  have  not  would  found  been  occur  of these  ilar  incompleteness  been  In  these  cases  different  metabolites  of not The  tion,  destroying  rather  Schneider solvent  than  and L e w b a r t  partition  have  has  been  (39,40) Gelite  i n urine  a  of  the  might  be  steroids  or  drawback of  spethe  present.  o b t a i n e d by s o l v e n t  the  and Bush which  are  (35,36).  i o n exchange  Separation  of  products  (34)  have  parti-  established  attempts  of u r i n a r y  resins  sulfates  paper fait'  or  prelim-  conjugates  and a d s o r p t i o n and  separa-  of h y d r o l y s i s .  applicable to  Isolated  extracts  has  (38)  on a n i o n exchange  employed the  columns to  urine.  This  use  extract  may b e c o m e  resins.  of a l i q u i d  and f r a c t i o n a t e a useful  sim-  acid hydrolysis.  column  glucosiduronates  a c h i e v e d b y C r e p y on a l u m i n a columns and by Bush  and Hahnel  in  (33)  p u r i f i c a t i o n of bulky  chromatography.  known  i d e n t i f i e d by chromatographic  systems  b e e n made w i t h  enzyma-  of h y d r o l y s i s has  estrogens  determination  column chromatography inary  method  i s o l a t e d conjugates,  may b e t e n t a t i v e l y  exist  it.is  oxygenated  Despite  sulfates.  complete  soluble material  highly  (l).  enzyme i n h i b i t o r s , t h i s  advantage  tion  conjugates,  that  molluscs.  and w h i l e  obtained with  remaining water  estrogen  non-steroidal cific  the  that  enzymes  found  estrogen  i n marine  (31,32)  i n h i b i t o r s of both has  cleave  obtained  that  (30)  Lately  (37)  Kushinsky  anion exchanger conjugated  and  estrogens  method of i d e n t i f i c a t i o n .  12 Beling  (8)  obtained  filtration  on  similar  now b e e n  Beling.  gen  thin  layer  Beling  by a p p l y i n g g e l Steroid  onates are  (33)  has  and  (42).  not  also  Modified  Kober  established applied of  quire,:,  the  the the  this  i n 1936, case  isolation  the  be  first  theory  of  of the  conjugates other  of  of  roles.  (41)  of  estro-  c a n be  test  for  of the  of  free  test  for  glucosidursteroid  acid  radicals.  fluorescence, estrogens,  procedures  both  have  and  two m e t h o d s , mentioned,  re-  was  demonstra-  c o n j u g a t i o n was d e t o x i c a t i o n ,  steroids  suggested  Quick  tion  mechanism f a c i l i t a t e d the  effect.  suggested  e x c r e t i o n of  in  Consequent  w i t h no b i o l o g i c a l (68)  as  comparison.  glucosiduronate  estrogenic  been  behaviour  by e l e c t r o p h o r e s i s  for  deter-  parent  conjugating  conjugates  vity  has  Sephadex.  However, these  steroid  by  numerous  rhodi-zonic a c i d  obtained  standard  a limiting  for  and s u l f u r i c a c i d  distribution.  of  on  determination  <other c h r o m a t o g r a p h i c use  Diczfalusy  V a l u a b l e i n f o r m a t i o n on the  can a l s o  When t h e ted  the  presence  conjugates  and a l s o  and g l u c o s i d u r o n a t e s  determination  successfully.  countercurrent as  by the  reactions  for  conjugates  well  gel  p r o v i d e d some p h y s i c a l c o n -  naphthoresorcinol  Methods f o r  affected  individual  values  chromatography  sulfates  and the  of  chromatography,  mined by c o l o r i m e t r i c methods; sulfates  a p p l i c a t i o n of  m a i n l y by D i c z f a l u s y  colour reactions  conjugates.  stants  separation  established,  Using  established  by the  Sephadex.  Chromatographic has  results  the  acticonjuga-  compounds by  the  13 kidneys, has  as  t h e y -would be more w a t e r  been g e n e r a l l y Over  oid  for  some d i d p o s s e s s trone free the  sulfate  biological  activity  jugates  B u t as  A number  was  results  indicate  of recent  that  biosynthetic  these  that  secreted  by an e n d o c r i n e  and  it  that  that  may b e  sulfate  stration  A recent  ported  the  drosterone  (44)  i n vivo  fate.  conjugates reports  or  equal  l a y dormant  can serve  as  g l a n d s u c h as  may h a v e  l e d to  was  supported  pregnenolone  precursor  involved  mones.  most b i o s y n t h e t i c steroids. efficient also  exists  i n the  biosynthesis  intermediates  Sulfates  serve  substrates than that  the  t h e y may be  has  sulre-  dehydroisoan-  of are  may s e r v e  that  demon-  can  a l . (64)  suggested  (70)  than  of dehydroisoandrosterone  and has  is  suggestion  by the  sulfate  c o m m u n i c a t i o n b y R o b e r t s _et  i n vivo  sulfate  the  that  The p o s s i b i l i t y  con-  human a d r e n a l  sulfate  more  until  substrates  some f u n c t i o n o t h e r  This  that  (43)  the  to  exception of  to  employing l a b e l l e d  dehydroisoandrosterone  are  and perhaps  the  than  v a r i e d and remained u n c o n f i r m e d  intermediates  the  greater  conversion of c h o l e s t e r o l sulfate  Also,  while es-  compounds  excretion.  of C a l v i n  a direct  and f o u n d t h a t  ster-  g e n e r a l l y lowC"). H o w e v e r ,  cleaved i n vivo  conjugates  of.facilitating  of  processes.  The f i n d i n g  as  it  activity  i d e a o f f u n c t i o n a l hormone  recently.  in  ago F i s h m a n e x a m i n e d a number  exhibited activity  hormone.  idea  accepted.  a decade  conjugates  s o l u b l e , and h i s  as free  sulfated steroid ionic,  horwith  niohic.: hormones.  a reservoir  for  14 unconjugated being  intermediates  an i m p o r t a n t  thesis  of  consider  step  with  i n the  s t e r o i d hormones. that  sulfates  the  s u l f u r y l a t i o n and  r e g u l a t i o n of the  On t h e  may b e  the  other  active  hand, form  rate one  of  cleavage of  syn-  should  steroid  hor-  metabolism of  glu-  mones . A l t h o u g h no r e p o r t cosiduronates  has  in  transport  the  that  active  the  yet  synthesized  been made,  in situ.  conjugate  the  serosa  dicate  i n the  where  the  travels  have  been  role  of  the  Use of  considerable  insight  tive  various  d e s i r a b i l i t y of  conjugates.  vivo.  Measurements  jugate  (45)  s u g g e s t e d (7Z)  target  and be  organ  These  the  free  testosterone  converted  to  glucosiduronate transferred  in vitro  to  act  compounds.  exists  at  present  steroid  to  studies  in a  s u c h compounds into of  conjugates  experimenting with  the  renal  i n determining whether  these  involved  in-  transport  - - ---i  biological the  the  shown t o  glucosiduronates  The u n c e r t a i n t y w h i c h  vious  to  and a n d r o s t e n e d i o l  accumulated.  of  are  Fishman has  i n t e s t i n a l mucosa and t h e n  they  ability  function.  compounds  glucuronyl radical deposits  (46)  glucosiduronate  respectively  these  Radioactive estradiol  and androstenedione estrone  direct  of hormones.  whereupon removal of the hormone  on t h e  fate  m i g h t be of  A high rate,of  would probably i n d i c a t e  the  makes  ob-  expected  rates  e x c r e t i o n was  to  doubly l a b e l l e d  conjugated  clearance  as  the  to  steroids  w o u l d be  its  in  informa-  primary fate  e x c r e t i o n of unchanged  that  give  i n vivo  function  of  conwas  15 that  of  an e x c r e t o r y  w o u l d be ling  product.  Hydrolysis  e a s i l y r e c o g n i z e d by the  ratios.  Also,  the  compounds  than  of  undiscovered metabolic  as  yet  those  excretion  The p r e s e n t tions  leading to  estrogen classical and  that  the  administered  estrogens, i n the  resultant  might  the  state.  label-  radioactive  indicate  the  a study  existence  of the  of doubly  semimicro l e v e l .  o n l y e s t r o n e was  unlabelled  in  pathways.  chemical synthesis at  conjugates  change  of different  i n v e s t i g a t i o n was  glucosiduronates  of the  successfully  reac-  labelled Of  the  conjugated  16 M A T E R I A L S AND METHODS All before  use.  mentioned  alcohols, bonate  Preparations  -  shaken w i t h  washed w i t h  and d i s t i l l e d . i n the  Absolute hours,  then  alcohol  -  doubly  Dry  ethyl acetate  concentrated  a s o l u t i o n of  benzene  refluxed with  r e f l u x e d over  zinc  and  -  - d r i e d  distilled  from  calcium  slightly  acidic  ferrous  '-sulfate,  tilled.  A n h y d r o u s c o n d i t i o n s c a n be  calcium  to  remove  sodium b i c a r chloride,  stored  in a  f i l -  tight  for  two  and p o t a s s i u m h y d r o x i d e  for  24  hours,  -  hydride.  shaken w i t h  a s o l u t i o n of  washed w i t h water o b t a i n e d by  and  dis-  distilling  hydride.  ether  -  l a y e r was with  shaken w i t h colourless.  a s o l u t i o n of  concentrated  sulfuric  acid  The e t h e r was t h e n w a s h e d sodium bicarbonate  and  until to  water  distilled.  Glacial  are  distilled.  ether  neutrality  calcium  acid  over potassium carbonate  diethyl  for  solvents  magnesium t u r n i n g s  free  Petroleum  sulfuric  T h e d i s t i l l a t e was  -  and  distilled  distilled.  filtered  latter  purified  saturated  d r i e d over  Peroxide  over  and  distilled.  and  Dry  grade  dark.  methanol -  Absolute  reagent:  o f more h i g h l y  and then.with w a t e r ,  bottle  then  used were  below.  Chloroform  tered  solvents  acetic  one h o u r  acid  and  -  refluxed with  distilled.  potassium  permanganate  17 Acetic  anhydride  move a n y Acetone  -  r e f l u x e d over  contaminating -  acetic  refluxed with  fused  acid.  potassium  sodium a c e t a t e  to  re-  Distilled.  permanganate  and d o u b l y  dis-  tilled. Carbon t e t r a c h l o r i d e chloride, Pyridine  filtered -  and  refluxed  distilled.  -  Stored  washed w i t h  i n acetic  dried  over barium oxide under over  acid  over  calcium  distilled.  potassium  B r o m i n a t i o n s were acid  water,  dry  hydroxide  performed  (Eastman Kodak  with  conditions  and  pellets. 30-35% h y d r o b r o m i c  #1161).  14 D-glucuronolactone-6-C Trac.  was  obtained  from the  Chem.  Corporation, Cambridge, Mass. Radioactivity  was d e t e r m i n e d  w i n d o w l e s s Gas P l o w C o u n t e r Samples were  plated  operating  i n duplicate  or  i n the i n the  Nuclear Chicago Geiger  triplicate  region.  onto  copper  planchets. Melting previously  dried  unless  otherwise  except  for  lary  one  •  i n vacuo  redness  flame  was no  the  solid  per  halide  or  over  indicated.  in  . Beilstein  ed to  determinations  i n the  test  for  on  samples  pentoxide  at  80°C.  m e l t i n g b l o c k was  w h i c h w a s made  halogens  oxidizing coloured.  liquid  sample  the  phosphorous  made  i n a sealed  used  capil-  paraffin.  longer  gives  were  The K o f l e r  determination  tube heated  "  point  flame  flame The  and t h e n a green  -  A copper  w i r e was  of  a burner  until  c o o l e d w i r e was reheated. colour.  heat-  the  touched  A volatile  to cop-  18 Optical (Bellingham  rotations  beam r e c o r d i n g  spectra  were  done  spectrophotometer  Spectrophotometer Materials Silica  for  and the  adsorption  gel Davison  Aluminum  Stepwise  a c t i v i t y Grade  sample not  solvent  a c h i e v e d by d i s s o l v i n g  s u c h as  of the  the  their  conjugated  Polin  -  Ciocalteu to  chloride together  adsorbed  linear  soluble the  to  in  the in  amount  evaporate.  s a m p l e was  solvent  sample  adding a small  solvent  use.  The  an  of dried  carefully applied  to  column.  Colour  subjected  acetone, the  1  80° immediately before  o f an o i l y  gel with  mixed  at  100-200  Application  silica  Ferric  spectrophotometer.  experiments.  was  condensor.  on a B e c k m a n DU  b e i n g e m p l o y e d i n some  eluant  accessories  a n d a beam obtained  double  chromatography:  dried overnight  g e l and a l l o w i n g  then  on a Beckman I R - 5  11 r e c o r d i n g  neutral,  silica  top  Cary  polarimeter  England).  e l u t i o n was u s e d p r e d o m i n a n t l y , w i t h  gradients  organic  were  923 Mesh s i z e  o x i d e WOELM -  These were  starting  spectra  on a  equipped with  m i c r o - p o t a s s i u m bromide p e l l e t s Ultraviolet  the  performed  and S t a n l e y L t d . , London,  Infrared  for  were  tests  used  for  the  detection  of  estrogens  and  derivatives: reagent  -  an ammonia  chromatographs  was  sprayed  and  atmosphere.  F e r r i c y a n i d e Reagent  i n water,  were  -  a p p l i e d as  Zimmermanrareaction - A p o r t i o n of  the  a Vfo s o l u t i o n o f a  each,  spray.  paper  chromatogram  was  19 drawn through  a 2% s o l u t i o n o f m - d i n i t r o b e n z e n e  absolute  ethanol,  solution  o f p o t a s s i u m h y d r o x i d e i n 95% e t h a n o l .  Preparation  blotted  of Perbenzoic  Perbenzoic  benzene  No p e r b e n z o i c tion.  ketone-free  drawn through  a 14%  Acid  (50,51),  o f p e r a c i d p r e s e n t was s o l u t i o n was  subsequently  a c i d was p r e p a r e d  and d i b e n z o y l p e r o x i d e amount  and  in  stored  from  and s t o r e d  sodium methoxide i n benzene.  analyzed iodometrically.  i n the  a c i d was u s e d more  cold  than  i n the  two weeks  absence after  The The of  light.  prepara-  20 E X P E R I M E N T A L AND R E S U L T S Conjugation steroid  moiety w i t h the  pyranosyl of  i  the  involves sugar,  bromide)-uronate.  following  I CHOH I H-C-OH I 0 C-H |  series  the  methyl  The  of  compound  is  the  product  methyl  CHoAc I H^C-OAc  acetylation  AcO-C-H I H-C-oAc I H-C  Q  isomerization  H-CI COOCH,  glucuronolactone  appropriate  I  H-C-OH  J  the  reactions.  TT  0 H-C I H-C-OH I — C = 0  of  (tri-O-acetyl-a-D-gluco-  latter  I CHOH I H-C-OH i HO-C-H  methylation >  reaction  glucuronate  H-C-Br  COOCH, methyl 1 , 2 , 3 , 4 - t e t r a 0-acetyl-a-(and B)-Dglucuronate  H-C-oAc 0  AcO-C-H"  bromination  H-C-oAc I H-C C00CH methyl  3  2,3,4-tri-O-acetyl-l-bromo-l-deoxy-a-D-glucuronate  Preparation  of methyl  tetraacetyl  glucopyranuronate  ,  by_ A c i d  Catalyzed Acetylation The g e n e r a l and Babers mole)  was  added  methoxide stirred  in  at  dissolved. ture  the  40 C t o  (23)  were to  procedures followed.  a solution  After  methanol yield  the  another was  of  for  half  methyl  .0O3M d r y  The  hour  (17.6  mixture  until  standing  under  and  commercial  resulting  45 m i n u t e s  evaporated  syrup,  (26)  Glucuronolactone  100 m l m e t h a n o l .  room t e m p e r a t u r e  of Bollenback  at  reduced  glucuronate.  Goebel g,  .1  sodium was  the  lactone  room  tempera-  pressure As t h i s  is  at  21 not  c r y s t a l l i z a b l e the  acetylation  followed  directly.  Addi-  ~f tion  of  acetic  anhydride  lowed by dropwise of  perchloric  that  the  tion  mixture  reaction  ated  two d a y s  for  collected ether  to  temperature  .1 ml p e r c h l o r i c at  from hot GHG1 ).  + 7.4  (02,  the  onto  The c r u d e  ethanol  CHC1 ).  *  An e f f o r t  to  3  original  crushed  solid  the  ice  g)  of  h a d a mp o f  obtain  filtrate  of the  mp  a-anomer  also  -dried rup. hot, of  over  with  anhydrous  T h i s was  subsequently  crystals  (33%,  from benzene-hexane  These  methyl  and  were  diethyl  tetra-0-  set  aside  [a]p  for  several +  3  6.4°  176.5°-178°;  proceeded  a large  by  pourproduct  quantity  chloroform.  c h l o r o f o r m and the  sodium s u l f a t e  isopropyl alcohol.  crude  tions  extracted  reac-  U n d i s s o l v e d sodium b i c a r b o n a t e  removed by f i l t r a t i o n and washed w i t h was  rate  refriger-  crystallized reaction with  ml  After  peroxide-free  176.5°-178°;  and n e u t r a l i z i n g i t  sodium b i c a r b o n a t e .  (.3  The  recrystallized  (26):  the  40°C.  l i q u o r was  fol-  such a  appeared.  crude  8-anomer,  values  at  s o l u t i o n was  crystals  The m o t h e r  Literature  3  (7.15  s y r u p was  room t e m p e r a t u r e .  acid,  which time  g i v e a 19% y i e l d  (G2,  ing  at  exceeded  by f i l t r a t i o n and washed w i t h  crystallization.  yellow  catalyzing solution  never  overnight  acetyl-8-B-glucuronate.  times  the  i n 10 m l a c e t i c , a n h y d r i d e )  remained  of  to  a d d i t i o n of the  acid  addition  (68 m l )  The  combined  was  filtrate  extracts  concentrated  dissolved i n a minimal  of  to  a  amount  syof  R e f r i g e r a t i o n y i e l d e d a c r o p : •xv:.. > 1 2 . 4 g)  which,  after o h a d a mp 1 9 0 - 1 9 2 ,  two c r y s t a l l i z a 22 [ajr. + 61.2 (C2,  22 CHCl^).  L i t e r a t u r e values (23) of the a-anomer:  (C2, CHCl^), mp 111°-112°.  [ ] p + 98° a  4  Upon r e c r y s t a l l i z a t i o n of these  c r y s t a l s from hot, absolute a l c o h o l , a compound was obtained w i t h a mp (177°) i d e n t i c a l to t h a t of the 8-anomer i n d i c a t i n g a mixture  of anomers may have been .present.  The o v e r a l l y i e l d  from t h i s methylation and a c e t y l a t i o n was 52% from nolactone.  D-glucuro-  An i n v e s t i g a t i o n i n t o the i d e n t i t y of the high  m e l t i n g compound f o l l o w e d . Glucuronolactone acetylglucuronolactone  i s r e a d i l y a c e t y l a t e d to form  tri-  (47)', the B-form of which melts a t 194°-  195° with an o p t i c a l r o t a t i o n o f +84.1" i n c h l o r o f orm.  In  order to check on the completeness of methyl glucuronate  for-  mation ( f a i l u r e of which might l e a d t o formation of t r i a c e t y l glucuronolactone) the methylation and a c e t y l a t i o n of g l u c u r o nolactone was repeated. as p r e v i o u s .  C o n d i t i o n s were e s s e n t i a l l y the same  The a l k a l i n i t y of the i n i t i a l  glucuronolactone-  sodium methoxide mixture was checked with the use of u n i v e r s a l pH paper i n case any contaminating present i n the commercial p r e p a r a t i o n .  g l u c u r o n i c a c i d was Before a d d i t i o n of the  a c e t y l a t i n g s o l v e n t s , the syrup was d i s s o l v e d i n absolute a l cohol and r e f r i g e r a t e d so t h a t any unreacted  glucuronolactone  would c r y s t a l l i z e out. Some s o l i d was removed which was i d e n t i f i e d as glucuronolactone but the experiment y i e l d e d the same r e s u l t s as b e f o r e , with a s i m i l a r y i e l d . In a f u r t h e r attempt to i d e n t i f y the h i g h melting compound, carbon-hydrogen analyses were performed on both  this  23 and the in  B-anomer.  Germany were  The r e s u l t s  from the  Bernhardt  laboratory  inconclusive. TABLE  1 Percentage C  0  C a l c u l a t e d f o r a and 8 m e t h y l t e t r a O-acetyl glucopyranuronate  47.87  46.81  5.32  Pound  48.5 48.10  46.67 45.63  4.88 5.56  47.20  47.20  5.60  a-anomer 6-anomer  Calculated actone  for  triacetylglucuronol-  The r e s u l t s values  within  of  h i g h m e l t i n g compound was  the  the  f o u n d agree w i t h none  accepted  bon-hydrogen  analysis.  5.32% a g r e e d  with  acetyl  compound had been mer  of  .3.  Switzerland for  for  methyl  The c o n c l u s i o n was d r a w n t h a t it  sample car-  C-47.64%, 0-47.04%,  calculated values  synthesized but  calculated  An i d e n t i c a l  sent to  results,  of the  was n o t  H-  tetra-0— the  desired  known w h i c h  ano-  predominated. As  tylated change the  These  the  glucuronate.  value  -H  several  compounds w a s made  preparation  tives.  and no  of  the  Preparation  (48)  performed  procedure  acetylated  was  Catalyzed- Acetylation  r  on t h e s e  obtained,  previously described methyl glucuronate  and i s o m e r i z a t i o n by the  of Methyl  The s y r u p ,  were  crystalline material  from the  Acetylation  ing~and Timell  brominations  method  ace-  a for  derivaof Bower-  undertaken.  tetra-Q-acetyl-a-D-glucuronate and  by Base  Isomerization  methyl glucuronate,  was p r e p a r e d  in  the  24 same m a n n e r  as  before  dium hydroxide tion  (.002  with  sodium methoxide  c a t a l y z e d by p y r i d i n e  instead  Solution  of methyl glucuronate  ( 6 gm)  to  anhydride  and anhydrous  -17°C resulted  ture  i n a dark  was r e f r i g e r a t e d  room t e m p e r a t u r e w i t h seventy-two  hours.  and  allowed to  tic  anhydride  (3 x e q u a l tract  was w a s h e d  chloric  acid,  four and  form  over  and  evaporated  anomers  zing and  of methyl  temperature of  o i l separated.  three washed of  times  with  times  with  anhydrous yield  allowed to an  onto  i n order  to  extracted  volume). three  ace-  times  remain  convert  ace-  chloroform  ex-  withI10% hydro-  drying the it  at  chloroform  s o l u t i o n of  sodium s u l f a t e ,  mix-  ice-water  with  This  After  syrup  of  additional  poured  a saturated  a dark  was  sodium  chlorofiltered  containing a mixture  of  tetraacetylglucuronate. B-anomers were  acetic  acid the  3 x one-half  a-and  s o l u t i o n of sulfuric  then  acid.  The r e a c t i o n  shaking for  once w i t h w a t e r .  to  These  acid,  acetyla-  i n a 50% m i x t u r e  and then  hours  So-  (66 m l ) p r e v i o u s l y c o o l e d  T h i s s o l u t i o n was five  The  of p e r c h l o r i c  solution.  once w i t h w a t e r ,  bicarbonate, extract  red  occasional  acetic  volume,  pyridine  overnight  remain for to  catalyst.  mole) worked e q u a l l y w e l l .  was  tic  as  (1.2  anhydride ml).  (46 m l ) ,  After  equal  volumes  once w i t h w a t e r ,  sodium bi c a r bona t e  6 hours  four  and  onto  the  of  times  acetic  twenty-four  s o l u t i o n was p o u r e d After  d i s s o l v e d ^ i n an  isomeri-  acid  hours  ice water,  m i x t u r e was  at  After  a  layer  extracted  a saturated  once w i t h w a t e r .  room  and  c h l o r o f o r m , and the with  (20 ml)  extract  solution  drying  over  25 sodium was of  sulfate  and removal o f c h l o r o f o r m , the r e s i d u a l  dissolved i n ethanol methyl  in  t h e manner  were  be u s e d  tions  this  observed  ted  i n vacuo  the  reaction.  from  the  isomerized  made.  alkalinity  obtained over  any t r a p p e d  appeared  quantities  D r y methanol  precau-  was u s e d  Also  during  sodium methoxide  to reduce  upon e v a p o r a t i o n  of  The f o l l o w i n g  of methyl glucuronate.  the o v e r a l l  of solvents  calcium c h l o r i d e before  were  further  at yield.  dessica-  reaction  to  solvent.  The o v e r a l l y i e l d actone  effectively  t h e p H was- c o n t r o l l e d w i t h  pH 8 a s e x c e s s syrups  obtained  a c e t y l a t i o n and i s o m e r i z a t i o n r e -  and changes  the preparation  All  also  that  (48).  s c a l e d down t o t h e s e m i m i c r o l e v e l  reaction  remove  methylation,  i n the conjugation  were  during  mp 1 1 2 - 1 1 3 ° ,  described.  These  to  o f a-and 6-anomers  c a t a l y z e d a c e t y l a t i o n were  actions  crystals,  1,2,3,4-tetra-O-acetyl-a-D-glucuronate The m i x t u r e  acid  to give  syrup  of these  reactions  from g l u c u r o n o l -  to methyl tetra-O-acetyl-a-D-glucuronate  use of semimicro q u a n t i t i e s  to give  increased  an o p t i m a l y i e l d  60-65% pure  methyl 1,2,3,4-tetra-O-acetyl-a-D  Preparation  of Methyl  with of  glucuronate.  ( t r i - O - a c e t y l - a - D - g l u c o p y r a n o s y l Bromide)  -uronate a-and 6 - a c e t y l a t e d able  to form the d e r i v a t i v e The m e t h o d  Methyl  methyl glucuronates brominated  are  equally  at C - l .  o f B o l l e n b a c k ( 2 6 ) was f i r s t  tetra-0-acetyl-8-D-glucopyranuronate  attempted.  ( l g ) was d i s s o l v e d  26 in  a minimal  perature. tion and  of  the  solvent  under  s o l u t i o n of the  was w a s h e d w i t h  over  Norit,  eration  lowing  which  hours  at  v a r y i n g the  iniacetic  saturated  acid  attempted.  were  also  cooling  dried  over  to  tallization  of  the  syrup  colour. Silica  and the  Refriggel  chro-  i m p u r i t y and  Repetitions  al-  occured of  this  and q u a n t i t i e s  of  of B o w e r i n g and T i m e l l shaken  acid.  s o l u t i o n was  sodium s u l f a t e  a  treated  a black tar  times  by d i s s o l v i n g the  0°, this  and  yielded  unsuccessful.  The B - a n o m e r was  was f o l l o w e d  After  ethanol  room t e m p e r a t u r e .  room t e m p e r a t u r e w i t h HBr i n a c e t i c solvent  solvent  in:, r e m o v i n g any  refrigeration  solution  subsequently  formation.  The b r o m i n a t i o n p r o c e d u r e was n e x t  aqueous  and  the  Decomposition to  used,  less  evapora-  The  Removal of  effective  by  tem-  i n 20 m l c h l o r o f o r m .  by water,  in crystal  crystallization.  experiment, HBr  result  room  40°C  o n l y p a r t i a l l y removed the  was n o t  twenty-four  at  at  than  dissolved i n absolute  d i d not  matography  pressure  syrup  followed  acid  was f o l l o w e d  a cold,  sodium s u l f a t e .  s y r u p w h i c h was with  reduced  residual  sodium b i c a r b o n a t e ,  dried  in  of.30-32% HBr i n a c e t i c  Overnight refrigeration  chloroform of  amount  2 l / 2 hours  in  extracted  ethanol  the  chloroform. with  chloroform removed.  from absolute  at  E v a p o r a t i o n of  syrup  (48)  yielded  ice  water,  Crysstarting  material. At  this  stage  the  nate  formed by the  tion  of methyl glucuronate  tetra-O-acetyl-a-D-glucopyranuro-  base-catalyzed was  acetylation  obtained  and  isomeriza-  and b r o m i n a t e d  accord-  27 ing  to  the  method  was  processed  of Bollenback (26).  by the  method  dition  of  of  HBr and a c e t i c  the  chloroform to  Removal ved  of  of P e l z e r  the  reaction  acid  extract syrup  was  and  (49).  This included  mixture  from the  before  evaporated  twice with  at  30°C  i n vacuo  to over  almost  colorless  calcium chloride.  The  sulted  with  (C.5, 198°  CHC1 ).  Literature  3  (C.5, CHC1 ). 3  sealed  tions  gave  on t h e rest  of the  less  yields for  the  over be  the  Kofler  Reactions or  A mp o f  c a p i l l a r y tube.  Beilstein. test.  mp 1 0 4 ° - 1 0 5 ° ,  componnd a l s o  brominations  low m e l t i n g brominated melting block,  out  of methyl  at  of  the  compound  0°C.  different  upon v a r y i n g r e a c t i o n  under  lots  a  as  a  determined  cases.  glucuronate)  gave  Optimal  d e r i v a t i v e were  in  of HBr i n a c e t i c  The  obtained.  ( w i t h one  Reproducible results  conditions.  in  positive  1 0 4 ° - 1 0 5 ° was  (80-85%).  +  4  identical condi-  50% o f t h e  semimicro l e v e l  brominated  calcium c h l o r i d e at with  i n about  tetra-O-acetyl-a-D  brominated  storage  the  gave  re-  M^  obtained  compound,  a compound m e l t i n g a t  carried  obtained  (26)  dried  [ajjp + 195°  a mp 8 2 ° - 8 3 ° ,  1 0 3 . 5 ° - 1 0 4 . 5 ° was  This  Repeated  time  of the  values  so-  chloroform  a l c o h o l and r e f r i g e r a t i o n  of c r y s t a l s  achie-  a s o l u t i o n of  The d r i e d  a thick,  was  s y r u p was d i s s o l v e d i n a b s o l u t e i n formation  ad-  removal  chloroform extract  once w i t h w a t e r .  and d e s s i c a t e d  mixture  acid.  by w a s h i n g t w i c e w i t h w a t e r ,  dium b i c a r b o n a t e  The r e a c t i o n  gram the  best  conditions vacuo could  acid,  not  even  28 Preparation  of C  14  - l a b e l l e d Sugar  Derivatives  14 D-glucurondlactone-6-C ted  with  radioinert  methanol  carrier  s o l u t i o n and the  (.1  mc i n 6 mg) w a s  glucuronolactone  ( 3 8 5 mg)  r a d i o a c t i v i t y determined  diluin  as  a  115.2  6 x  10  cpm.  counting covery tion oil  crystals  of  of  separated  a significant  the  was  solvent  11.5  nolactone thylated  crystals  solid  of  described.  the  without  yield  was  of  was  the  36.2%.  were  not  pure  enough  to  rely  Upon r e f r i g e r a t i o n  rereducthe  radioactiv-  carrier  glucuro-  directly  or  following  memanner  i n 44%  yield  i n the  r a d i o a c t i v i t y of  ex-  the  results.  Specific 8.45 7.38  No p r o o f  methyl tetra-O-acetyl-a-D-glucuronate  tals  more  good c r y s t a l s  Activity 6 3 . 1 6 x 10 cpm 6 1 . 0 5 8 x 10 cpm  The r a d i o c h e m i c a l y i e l d  after  original  obtained  Total Crystals Mother Liquor  times,  c r y s t a l l i z a t i o n i n the  Crude s o l i d  l i q u o r gave  for  Therefore  ( 3 9 0 mg) w a s  Determination of the  and mother  material  diluted with  This mixture  and a c e t y l a t e d  amount.  of  one-tenth  c r y s t a l l i z a t i o n d i d not  pected  o f t h i s \ sample  from s o l u t i o n s e v e r a l  amount  x 10^ cpm,was  (351 mg).  previously  preparation  v o l u m e was u n s u c c e s s f u l .  r e d i s s o l v e d and  ity, i . e .  but  A l t h o u g h i n the  o n a mp  was  of  Activity 3 x 10 cpm/mg 3 x 10 cpm/mg the  formation  obtained  as  crys-  determination.  and d e s s i c a t i o n  of  the  original  14 oily  C  formed. a  -glucuronolactone Similarly  with  long period of time  for  the  s i x months,  reacted  i n ethanol  crystals  product,  resulted  gradually  refrigeration  i n an i n c r e a s e  in  for  29 crystal  f o r m a t i o n when t r a n s f e r r e d  Conversion  of Glucuronic acid A t t e m p t s were  glucuronic onic  acid  Detailed  acid is  more  easily  meagre,  although  cessful  m e t h o d was  aqueous agents acid  strong  acid  ed by p a s s i n g  acid  have  starting  water.  curonic  is  the  water  pH 1 w i t h  lowed by c o n c e n t r a t i o n  to  a syrup  of  the  and b o i l i n g reported  run  with  as  suc-  was an  successful  of  glucuronic  conditions  of  these  methods  or  return  sodium glucuronate salt  exchange  was  30°C  column i n  alcohol of  free  an aqeuous  (52).  to  achiev-  h y d r o c h l o r i c a c i d was  removed w i t h water  o i l from e t h a n o l  are  a mixture  Acidificationcqf  at  lactone.  and a  radioactive  i n crystal formation  to  lization  the  from  glucur-  the  to,  and ' a d d i t i o n o f  acid  a c i d was  referred  contaminating  free  hydrochloric  than  i n an o i l f o r m a t i o n  (66).  of t h i s  radioactive  a Dowex 50 c a t i o n  resulted  another.  such a conversion  often  (mp 1 6 5 ° )  tion  acid  as  Conversion of  E v a p o r a t i o n of the  the "remaining syrup  for  Varying  resulted  through  to  glucuronolactone  cheaper  a l l been  material  and r e m o v a l o f it  is  and  mineral acid  starting-material.  free  method  only after  sodium glucuronate  the  the  found  s o l u t i o n of  prepare  procedures reaction  tube  Glucuronolactone  obtained  and g l u c u r o n o l a c t o n e .  using of  the  Heat,  when t h e  to  by an e f f i c i e n t  experimental  performed.  made  to  f r o m one  The  washings.  to glusolufol-  residual Crystal-  yielded glucuronolactone  (mp  177°-178°). Attempted  Synthesis  of E s t r i o l - 1 7 B - g l u c o s i d u r o n a t e  The s y n t h e s i s  of e s t r i o l - 1 7 - g l u c o s i d u r o n a t e  was  at-  30 tempted under but  by the  following  conditions  sequence  s i m i l a r to  modified for  of  those  semimicro  reactions,  of Leeds  -diacetoxy-17-keto-estra-l,3,5(10)-triene  yield  the  17-keto  a compound w h i c h  (53)  quantities. (I)  diacetoxy-16a,17a-epoxy-estra-l,3,5(l0)-triene  of  out*  and G a l l a g h e r  _3,17B-diacetoxy-estra-l,3,5(lO) ,16-tetraene  Reduction  carried  group  could  of  III  react  acetyl-l-bromo-l-deoxy-a-Dr-glucuronate  (II)  3,176—s*  3,16a  (III).  to  with  —a>  a hydroxyl methyl  would  2,3,4-tri-O-  t o .form a c e t y l a t e d  es-  triol-178-glucosiduronate. Epoxidation to  of  3 , 1 7 B - d i a c e t o x y - e s t r a - l , 3,5 (10) , 1 6 - t e t r a e n e  (l)  f o r m 3 , 1 7 B - d i a c e t o x y - 1 6 a , 1 7 a - e p o x y - e s t r a - l , 3,-5 ( 1 0 ) - t r i e n e  (II) Initially (mp 1 5 3 ° - 1 5 4 ° ,  l i t .  freshly  prepared  steroid  were  solution ture,  at  after  dilution  over  rotary  evaporator  which  (53)  with  for  difficulty  but  obtained  corresponded  yield  1.6  in  mg)  of  the  .18 h o u r s . ' ,  T h e r e a c t i o n mix-^  was w a s h e d w i t h with water.  benzene  was o i l .  ( 2 3 6 mg)  of  from acetone-petroleum i n mp d e t e r m i n a t i o n s  17oc-epoxy-estra-l,3,5(l0)-triene  (565  acid  a colorless  a 40% y i e l d  supplied  epoxidized with  m-mdles  and then  the  was  (I)  perbenzoic  benzene,  sodium s u l f a t e ,  diacetate  149°-150°)  8mM o f  IN sodium hydroxide  to  enol  acid,  room t e m p e r a t u r e  drying  was  value  treated with  of  terial  estrone  perbenzoic  lution  with  the  to  removed  benzene  a cold  so-  After on  the  This c r y s t a l l i z e d crystalline  ma-  ether-methanol 3,17B-diacetoxy-16cc,  (mp 1 5 5 ° - 1 5 6 °  -  L i t .  values  31 (53)  150 -152  Silica  gel  ).  The-starting  chromatography  mixture.resulted Rearrangement -triene  (II)  i n no  of to  material  of the  also  remainder'  melted  of the  4° high.  reaction  significant purification.  3,17B-diacetoxy-16a,17a-epoxy-estra-l,3.5(10) 3 ,16a-diacetoxy-17-ke to-estra^l,3,5  (lO)-triene  :  (III) A n a t t e m p t w a s made t o  rearrange  17a-epoxy-estra-l,3,5(lO)-triene  to  3,17P-diacetoxy-16a,  3,16a-diacetoxy-17-keto-  estra-l,3,5(lO)-triene  by chromatography  rivative  was d i s s o l v e d a n d a p p l i e d t o  gel  ("*84 m - m o l e s )  column i n d r y benzene  ed w i t h  increasing  ether.  Solid  same m a n n e r plied  sample,  the so  and  concentrations  material  as  (9 m l )  was  eluted  epoxide  it  was  (53).  stepwise  of dry, which  The epoxy a  silica  elution  perform-  peroxide-free  diethyl  crystallized in  a n d h a d a mp i d e n t i c a l w i t h  concluded the  rearrangement  the  procedure  de-  the the  was  ap-  unsuc-  cessful . At and K e l l i e  this  (5)  point  for  m e t h o x y e s t r o n e was lated teen  by the  to  attempted.  a boiling  ( 5 6 m l ) a n d a 10% a q u e o u s After  methyl  Estrone  (.012  (19 m l )  ml)  was f o l l o w e d  was  then  Carpenter  s o l u t i o n of  estrone  was  in  further  c o o l e d and d i l u t e d w i t h w a t e r .  fif-  methanol  addition of  (112 di-  sodium h y d r o x i d e  r e f l u x i n g , and the  3methy-  over  sodium hydroxide  a n d 10% a q u e o u s  b y one m o r e h o u r  from  mole)  (19 m l ) ,  s o l u t i o n of the  refluxing 1 l / 2 hours,  sulfate  of  forming e s t r i o l glucosiduronates  a d d i t i o n of dimethyl sulfate  minutes,  ml).  synthetic  (38  solution  Extraction with  ether  32 and  evaporation  estrone  silica  yielded ene  the  solvent  (mp 1 7 0 . 5 - 1 7 1 . 5 ,  Mnolization by  of  and  gel  chromatography  to  acid  l i t . value  acid  gave  zoyl  purifications  peroxide  time  the  method  to  bath,  ;  mixture with  rather  than  water  at  epoxidations  perbenzoic  acid.prepared  in  a two f o l d  increase  on h a n d  followed  (55)  consisted  acetic acid  alumina  acid  1  to  the the  at  of  at  at-  than  of  twice  perbenzoic the  origi-  or  Also  less  the  diben-  in a  sodium benzoyl  methanol-chloroform no h i g h e r  than  reac3 -4°C. 0  enol diacetate,  i m p r o v e d manner of  dry  epoxide.  derivative  use  resulted  It  h a d no  ef-  however.  3-methoxy-17-acetoxy-l,3,5(10),163,17 diacetoxy r  a d d i t i o n of  and r e f l u x i n g f o r  by  columns.  estrone  3-methoxy  s o l u t i o n of the  (41 m l ) ,  increased  -30°C  0°C.  yield  b y a c e t y l a t i o n was of  178-acetoxy-16a,  of preparation  in this  i n the  corresponding  To c o n v e r t  of  a temperature  of  tion  product ;,  Epoxidation with  0  i n greater  sodium methoxide  a l l subsequent  tetraene  114 -115°).  f o r m e d was r e m o v e d f r o m t h e  on the  followed  T h i s was a c h i e v e d b y a d d i t i o n o f  In  fect  169.5°-170°).  reaction  T h i s was n o t  on s e v e r a l  obtained y i e l d .  ice-ethanol  tion  this  peroxide  ipurify the  o n l y 16-24% y i e l d s  was m o d i f i e d , r e s u l t i n g  nally  (5)  3-methoxy-  isopropenyl acetate,  (5)  17a-epoxy-3-methoxy-estrone.  At  value  of  3-methoxy-17-acetoxy-estra-l,3,5(10),16-tetra-  (mp 1 1 3 ° - 1 1 4 ° ,  tempted  a 91% y i e l d  literature  acetylation with  69% o f  perbenzoic  gave  5 minutes.  derivative,  demethyla-  performed.  Demethylation  steroid  m-moles)  an equal A cold  (3.79  in  volume of hydriodic 5% a q u e o u s  solution  33. of  sodium hydrogen  solid  f o r m e d was  washed with  to  water.  After with  acetic for  d i l u t e d with water,  only of  acid  crystals  lesser  quantity  covered gel  anhydride  acetylated  ether  then and  immediately  and p y r i d i n e  This reaction  with  from t h i s  was  (8 m l )  s o l u t i o n of  i n an o i l form w h i c h  practical  from e s t r o n e  described  and As  acetylated  (11  mixture  ml)  was  and washed w i t h  IN  sodium b i c a r b o n a t e .  a c e t y l a t i o n were  The  a 5% y i e l d  115°-120°,  and an  The r e m a i n d e r  was  could not  be p u r i f i e d b y  synthesis  of e s t r i o l  the  a l l of the  yield  of  reactions  subsequent  diacetate  from e s t r o n e .  enolization. m-moles,  alytic  even re-  silica  the  of  to  C - 3 p o s i t i o n as  and p u r i f i c a t i o n s the  preparation  This latter performed  sulfuric and  acid  (1.4  previously  considered.  of fresh  step  was  involved,  low  dur-  they  were  estrone  involves  enol  acetylation  by d i s s o l v i n g d r y  i n isopropenyl acetate  acetate  glucosiduro-  crystallizable material  T h i s was  1 gm)  amounts  Ji s k > p r o p e n y l  at  i l l u s t r a t e d was a g a i n  repeated,  (3.7  T h i s was  chromatography.  nates  and  solid  3-methoxyestrone.  The more  ing  the  -  compound m e l t i n g a t  of  white  a s o l u t i o n of.sodium.: b i c a r b o n a t e  extracted  obtained  and the  diethyl ether.  26 h o u r s .  and a  an u n i d e n t i f i e d  with  with  drying,  room t e m p e r a t u r e  hydrochloric  ( 8 3 0 m l ) was a d d e d  extracted  neutrality  by treatment at  sulfite  estrone  (35 m l ) , a d d i n g  ml of  a s o l u t i o n of  .1 ml c o n c e n t r a t e d  sulfuric  cat5 ml  acid)  and  i  slowly  distilling  reaction  mixture,  for  eight  hours.  peroxide-free  After  refrigeration  d i e t h y l e t h e r was  added  of and  the  34 the  m i x t u r e washed w i t h  bonate  and w a t e r  of  the  solvent  in  hexane-benzene  a saturated  and d r i e d  over  the  same s o l v e n t  white  was  separated  solid  ponents  and  responded  obtained  to  with  estrone e n d  freshly prepared  yield  of  the  in this  An e a s i l y c r y s t a l l i z e d c o l o r e d comIts  mp  cor-  (153.5°-155°) .  compound as  perbenzoic  dissolved  column and  form i n 66.5% y i e l d .  of t h i s  product,  gel  way f r o m t h e  diacetate  bicar-  Evaporation  w h i c h was  a silica  system.  i n pure  Epoxidation  residue  a p p l i e d to  eluted with  sodium  sodium s u l f a t e .  y i e l d e d a dark red (l:l),  s o l u t i o n of  previously  a c i d - u s u a l l y gave  described  an  80-85%  3,178-diacetoxy-l6a,17ct-epoxy-estra-l,  3,5(lO)-triene. Rearrangement yielded  o f the  a maximum r e c o v e r y  estra-l,3,5(lO)-triene. tween  5-10% d i e t h y l  ferent  experiments  to  173.5-175°.  epoxide of  and the  a decrease  of  50$ o f  Elution  ether  i n the  values  rearranged  product  i n mp w i t h  of the  solution cooled  to  of  similar the  2 ° and  to  sample  that  (.02  (53)  with g in  occured  from  d i s p l a y e d the  dif-  Both  the  peculiarity  recrystallizations.  the  of  3,16cc-diacetoxy-  1 7 - h y d r o x y l was and K e l l i e  i n d r y methanol a cold  be-  177-178.5°  mp 1 7 9 ° - 1 8 0 ° .  of Carpenter  ( 4 0 mg)  treated  sodium borohydride  to  column  T h e mp v a r i e d w i t h  17 k e t o - g r o u p  17-keto-estra-l,3,5(lO)-triene a method  product  same m a n n e r  successive  gel  3,16a-diacetoxy-17-keto-  i n benzene.  performed  on a s i l i c a  of the  Literature  Reduction  by  epoxide  attempted  (5).  (6 m l )  was  methanolicsuspension  .66 ml methanol).  After  A  of  15 m i n -  35 utes' at tic  2°C the  acid,  reaction  was  d i l u t e d w i t h water  dium h y d r o x i d e .  stopped (175  w i t h a few d r o p s  ml)  and n e u t r a l i z e d  Extraction with ether,  with  water  and  evaporation  of  the  fied  solid  which melted below 1 0 0 ° .  washing  solvent  gave  an  o i l .  acetates is  attempted  can  believed  thereby  It  Literature  occur that  cifically  at  the  has  been  compound  tiz  et  in sufficient  2.4  was  (352 mg,  m-moles)  A solution  werd  of  to  to  3,16a-  158°-160° impure  (67).  product  hydrolysis  of  reductions.  enol It  in this  way,  glucosiduronate  made t o  prepare  allow a radioactive (57)  this  and  as  spe-  silver  d r y benzene  carbonate  (25 m l )  and  this  synthesis.  reported  conjugation.  by Wo-  Dried  es-  (660 mg, refluxed.  methyl 2 , 3 , 4 - t r i - O - a c e t y l - l - b r o m o - l - d e o x y - a - D -  glucuronate  (702 mg,  quantity  of  benzene  (3 m l )  and added  dropwise  tion  was  distilled.  After  reduction  of  half  its  o r i g i n a l volume,  the  unidenti-  Glucosiduronates  and M i e s c h e r  m-moles)  added  extract  estradiol-17B-glucosiduronate  followed for  1.29  so-  impossible.  of  yield  the  mp o f  removed  estriol  a n a t t e m p t was  of Meystre  (56)  of  of E s t r a d i o l  (56),  that  g r o u p was  synthesis  synthesis  reported  Eil.  tradiol  3-acetoxy  the  (52)  sodium borohydride  C-17 p o s i t i o n  the  The~ p r o c e d u r e  reported  during  Attempted Synthesis As  been  the  rendering  is  r e c r y s t a l l i z a t i o n of the  has  of  ace-  with  y i e l d e d an  diacetoxy-17-hydroxy-estra-l,3,5(l0)-triene Subsequent  of  supernatant  1.77  removed  m-moles)  the  was  reaction  from the  dissolved i n a minimal  the flask  w h i l e the  solvent was  s i l v e r bromide  to  cooled salts.  soluoneand The  36 benzene anol.  was  evaporated  and the  Unreacted e s t r a d i o l  recovered  and removed.  and the  only  tradiol  conjugated  ature  solid  values  (56)  (50$ of the  Further  at  the  with v a r y i n g times  no  i n the  1$ o f what  Additional of heating  were  added  slower  dropwise  rate  on  silica  zene. ner  gar  An impure as  gel  as  curonate  this  as  and i t s  this  would  i n a solvent  many a s  also  system appear  acetate  dicated  an e f f e c t i v e  nothing  could  was  be  but  of  the  the  sugar at  previous was  (T.L.C.)  acetate  i n .ben-  identified in this be  to  detected  be  some  man-  from  form  positive  Any conjugates  spots.  column from  crystallized.  case  of  a su-  1,2,3,4-tetra-O-acetyl-a-D-glu-  separation  equally unsuccessful  were  i s o l a t e d which  could  sprays.  distinct gel  liter-  chromatography  thought  and a p p l i c a t i o n of  be  was  b r o m i n a t e d d e r i v a t i v e gave and p h e n o l  es-  and d i s t i l l a t i o n  of 40$ e t h y l  spots  both methyl  o i l on a s i l i c a  ethyl  eight  be  r e a c t i o n mixture at  layer  system  These were  as  to  conjugations  concentration  product  e s t r a d i o l after t h i n  application.  derivative  with  the  solid  was  difficult  appeared  and i n t h i s  distilling  The r e m a i n i n g o i l c o u l d n o t  because  given  the  and i n o n e - t e n t h  experiment. identified  to  amount)  eth-  obtained.  The r e a c t i o n was r e p e a t e d was  starting  1 7 8 - p o s i t i o n (mp 1 2 1 ° - 1 2 4 ° ;  122°-124°).  results  o i l dissolved in  c r y s t a l l i z a t i o n was  o b t a i n e d was  attempted change  residual  Gradient  100$ benzene  some  of these  to  of  solid  100$  on T . L . C . spots  Another conjugation  i n terms  formed  e l u t i o n of  random samples of  results  product  in-  but  reaction obtained  ^  37 with  the  melting  exception at  duronate  o f an  212°C.  i n s i g n i f i c a n t amount  This corresponds  to  the  of  estradiol,  mp 2 1 5 ° - 2 1 6 . ' 5 ° C ,  ed by W o t i z  et_al_.(56)  to  Meystre  have  was f o l l o w e d .  Estrone  carbonate  (2.6  Calcium hydride sicant.  The  method  of Shapiro  (1.34 m-mole),  with  The  black.  by  centrifugation  its  silver  to  and the  remove  dryness  salts  different  yzed  zations  of  on t h e  and i t  at  to  shaken  at  room  rotary  alcohol.  at  occured.  suspected the  free  the  40°C  room t e m p e r a t u r e were  red  removed through was  and the of  that  re-  crystals is  two  The c r y s t a l s  ap-  estrone  b y mp—  r e c r y s t a l l i z a t i o n s of of e s t r o n e were  conjugate  steroid.  des-  dark  passed  A good y i e l d  crops  benzene.  The s o l u t i o n  evaporator  sil-  temperature to  and washings  H o w e v e r , on s u b s e q u e n t  yield  and d r y  an i n t e r n a l  on r e c r y s t a l l i z a t i o n a m i x t u r e ,  was  estrone  60 m l o f d r y  particles.  remaining s o l u t i o n additional  by heat  to  were, removed and i d e n t i f i e d as  determinations.  for  m-moles)  i n c l u d e d as  crystalline structures,  first  (58)  and c a l c i u m h y d r i d e were  any f i n e  dissolved i n absolute but  (2.4  supernate  sidue  obtained  method  c o l o u r changing from green  to  the  report-  methyl-2,3,4-tri-0-ace-  added  m i x t u r e was  taken  obtained  were  (3 m - m o l e s ) was  to  c o t t o n wool  m-moles)  resultant  20 h o u r s  pearing  by the  been  Glucosiduronate  t y l - l - b r omo-l-deoxy-oc-D-glucuronate  was  3,178-diglucosi-  w h i c h has  zero y i e l d  of Estrone  The c o n j u g a t i o n  for  crystals  and M i e s c h e r .  Attempted Synthesis  ver  of  was b e i n g  Attempted  unsuccessful.  The  always  hydrol-  crystallisolid  ob-  38 tained 235°)  from which  the  r e m a i n i n g s o l u t i o n gave  could  not  a  a sample  silica  acetate of  layer  i n benzene  and t h e  glycol  Zimmermannreaction lower  than  could not sence ing  be  al.  reported  This  repeated.  et  (38).  In this  at  60°C  room t e m p e r a t u r e  the  residue  with  acetic  i n water acid,  conjugation the  solid  ether  could not  on p a p e r  and the was  product. fraction.  to  e s t a b l i s h the  bath.  methanol  to  The w a t e r  The s o l u t i o n was evaporated.  ether  to  could not  be  pre-  for cooled  Solution  remove  free  any unchanged,  starting  it  accord-  by n e u t r a l i z a t i o n t o  remove  90% o f t h e  a  ( 1 5 mg) w a s d i s s o l v e d  material  pH  of 6.6  estrone, acetylated  remained  f r a c t i o n c o n t a i n e d 5 0 0 mg o f  m a t e r i a l w h i c h was p r o b a b l y s o d i u m a c e t a t e .  glucosiduronate  ordinary  deacetylation  The s o l i d  followed  benzene  be  in a tol-  sodium methoxide and heated  i n a water  sim-  p r o v e d i n c o n c l u s i v e as  included  (17).  procedure  applied  group  sample  attempts  extraction with  extraction with  are  means  i n d i c a t e d a compound w i t h  result  product al.  on  d e v e l o p e d i n 40% e t h y l  by t h i s  same  Further  2 ml of f r e s h l y prepared  2 l / 4 hours  in  et  i n two s t a g e s  estrone.  Diczfalusy  spotted  system and a p p l i c a t i o n o f the  of a conjugated  to  was  was  Zimmerman r e a c t i o n a p p l i e d by  Development of the  uene-propylene  and  (mp 2 0 0 ° - 2 3 5 ° )  chromatoplate,  The r e s u l t s  duplicated.  to  solid  p o t a s s i u m h y d r o x i d e and m - d i n i t r o b e n z e n e  ultaneously.  (200°-  estrone-3-glucosiduronate  a spray d e s c r i b e d by L i s b o a  the  in  whether  of the  gel thin  melt  be i m p r o v e d .  To d e t e r m i n e present  a.^'broad"  detected  on a t h i n  Estrone  layer  chromat-  39 lOgram d e v e l o p e d paper  i n a system  neider  olive  estrone  with grey  (59)  m-moles  ing  of  gave  range  for  spots  occured  the  of the  carbonate  this  which  1  Sch-  140.  (60)  Both  and no  presence  reaction  ( l m-mole  pos-  of  with  a  an  estrone,  five 5.05  time  a report  some u n k n o w n r e a s o n reactions.  i n reactions  was p r e p a r e d  followed  by f i l t r a t i o n of  exposure  to  nitrate  to  the  The p r o d u c t  (22)  which  dessicants  of  this  21% a q u e o u s  formed  was y e l l o w  s l o w down silver  nature. use  sodium  ( a n 8fo a q u e o u s solid  indicated  prepared  immediately before  sodium b i c a r b o n a t e  water.  found  only freshly  of  with  was  melt-  unidentified.  internal  Also,  s h o u l d be u s e d  carbonate  a compound h a v i n g a b r o a d  remained  Addition  Thus by  carbonate.  solution)  and r e p e a t e d  but  adding  turned  was wash-  green  on  air.  The without  conjugation  complete  reaction  calcium hydride  The m i x t u r e was sure  acid  i n d i c a t i n g the  identical results,  silver  time  by  on  acetate,60:toluene,  acid,- 60:water,  sugar  40$ aqueous  ings  ethel  conjugation  brominated  (205°-230°)  conjugation  silver  25:acetic  described  or  methyl 2 , 3 , 4 - t r i - O - a c e t y l - l - b r o m o - l - d e o x y - a - D - g l u c u r -  At that  c o n s i s t i n g of  system  conjugate.  excess  onate)  n-butanol  anisaldehyde-sulphuric  Repetition fold  saturated  simple glucuronides  40: t-butanol,  sprayed  itive  for  and L e w b a r t  75:hexane, were  i n a water  shaken  reaction.  for  was  and w i t h  44 h o u r s  at  again fresh  performed, silver  carbonate.  room t e m p e r a t u r e  A compound h e h a v i n g  as  this  before  to  in-  was  ob-  40 tained.  A water-ether  because  the  soluble.  partition  compound  (the  w o u l d be  quite  (pH 1 0 . 5 )  and b e n z e n e .  The b a s i c  extracted  with  remove  remained  217.5°, nate  i n the  literature  (56)  as  estrone  estrone  remained  carried  out  benzene  values  212.5°-215°  identified  or  between 98% o f  The y i e l d  of t h i s  i n organic  extract  estrone.  fraction  zene  was  3-methoxyestrone hydroxyl  a  solution  estrone  and the  The  glucosiduro-  ether  fraction  To c h e c k i f  a similar  i n the  was any  partition  was  sodium hydroxide  toluene  layer.  w h i c h was p r o v e n b y mp, U V , I R  and  (l7-oxo-estra-l,3,5(lO),-trien-38  and p a r t i t i o n  causes  after  between  has  peak  to of  the  ultraviolet  a significant  mu.,  a split  of the  indicated that  estrone-280  sample  solvents,  O v e r 95% o f  and a s o l u t i o n o f remained  glu-  initial  alkali  and  ben-  15%. Comparison  similar  estrone  a n d h a d a mp o f 2 1 5 ° -  acetylated  phase  in-  was n e u t r a l i z e d  melting point.  be m e t h y l  attempts  unsuccessful  was w a t e r  acetylated  yl-2,3,4-tri-0-acetyl-8-D-glucosid)-uronate, crystallization  was  sodium hydroxide  any  benzene  solid  solid,  any  225.5°-228°.  toluene  the  h y d r o l y s i s to  for  by i t s  i n the  (pHU2V2).  acid  to  a cold  solid  conjugate)  soluble  w a s made  solid  between  that  partition  ether  the  suspected  Assuming therefore  cosiduronate  of  substitution  change  i n the  A max 3 - m e t h o x y - e s t r o n e and a hypsochromic  latter  acetylated  were  obtained  estrone  spectra  estrone  the  1  spectrum.  .  both  glucosiduronate  A max The  occured. the  and  phenolic  278,287 mu.  shift with  of  of  latter Spectra  standard  from Wotiz  and  the  prepared  A max 2 8 2 m(i, 2 7 4 - 2 7 5 m | i .  pared  slightly less  S t d . A max 2 8 2 m | i , 2 7 5 - 2 7 7 m | i .  compound.  impure  as  it  had h i g h e r  obvious hypsochromic The i n f r a r e d  acetylated  estrone Further  acetylated ysis  hours. rated by i t s  of the  estrone  (52).  An o r c i n o l  Free method  reagent  was  were  added  to  obtained  ed  solutions  to  standard  Therefore conjugated  compound,  Deacetylation u r o n a t e was p e r f o r m e d acetylated  the hydrol-  for  and at  1  l/4  benzene  Estrone  (2 m l ) ,  was  c o n s i s t i n g of  sepa-  detected  sample  orcinol  and the  TV S p e c t r a were  taken  occuring at  of t h i s  estrone  methyl glycosides  (22).  similar  to  heated  of the  cool-  identical  present  spectral  i n ia manner  acid  6 6 8 . 5 - 6 6 9 mp..  i n d i c a t e d by these acetylated  (.1  mixtures  h y d r o l y z e d compound was peaks  color-  Charlalampous  hydrochloric  and g l u c u r o n i c a c i d were  as  of  c l e a v e d by h e a t i n g  o f Mejbaum i m p r o v e d by  of the  estrone  ( P i g . I.)  g l u c u r o n i c a c i d was d e t e r m i n e d  glucuronic acid,  both  a  the'prepared  glucuronic acid  reaction, mixture with  water.  and t h a t  be  and  by a c i d  hydrochloric acid  1 ml of the  30 m i n i n b o i l i n g  and  preparation  a n d F e C l ^ ^ R ^ O ( 2 2 mg) i n c o n c e n t r a t e d  ( 2 0 m l ) , was  to  peaks  identical.  and g l u c u r o n i c a c i d .  UV s p e c t r u m . by the  were  liberated  and sugar  E x t r a c t i o n of the  standard  successful  15% v / v c o n c e n t r a t e d  imetrically  for  of the  glucosiduronate  The s t e r o i d  the  minima, broader  glucosiduronate  estrone  100°C w i t h  gm)  spectra  proof  appeared  shift.  and i d e n t i f i c a t i o n o f t h e  estrone.  The s t a n d a r d  Pre-  that  The c o n j u g a t e  in  the  results. glucosid-  used (16.5  for mg)  42  Figure 1.  Comparison of infrared spectra of:  A. Standard sample of methyl (17-oxo-estra-l,3,5(10)trien-3-yl-2,3,4,-tri-0-acetyl-8-D-gluc6sid)-uronate , supplied by H. H . Wotiz. B. As above, prepared as described i n Experimental.  43 was d i s s o l v e d i n a b s o l u t e catalytic  amount  dissolved  i n methanol)  This  of  methanol  sodium methoxide was a d d e d t o  r e a c t i o n m i x t u r e was h e a t e d  bath,  c o o l e d , the  basic  s o l u t i o n was n e u t r a l i z e d  tracted  with  methanol  ether  was r e m o v e d .  estrogen  conjugates  the  deacetylated  217.5°)  with  from  material  the  a substituted  the  test  for  esters  to  (6l)  split  esters  are  to the  exof  extract pH o f  to y i e l d  conjugate  with  97$ o f  A max 2 7 3 ,  was  (215°-  comparison.  characteristic  of c a r b o x y l i c  amount  so  available for  peak  and  T h e mp ( 1 4 0 ° - 1 4 2 . 5 ° )  of a c e t y l groups  sodium s a l t s  acid  water  The  2 and e x t r a c t e d  crystallized  expected.  hydroxyl  The a b s e n c e spot  acetic  corresponding acetylated  the  added.'  been r e p o r t e d  lowered to  solid  in a boiling  and w a t e r  solutions  A  .002M s o l u t i o n .  An i n s i g n i f i c a n t  was  b u t n o k n o w n mp d a t a  UV s p e c t r u m h a d  commercial  (pH6) w i t h  acidic  The d r i e d e x t r a c t  distinct  Its  evaporated  n e u t r a l i z e d s o l u t i o n was  butanol.  (the make a  N - b u t a n o l has  from  ml) by h e a t i n g .  two m i n u t e s  and benzene.  material  the  (7.5  of  estrone  280-281 mu. was d e t e r m i n e d b y  acids  (62).  Conversion  of hydroxamic acids  m e n t w i t h h y d r o x y l amine' h y d r o c h l o r i d e i n t h e  a  occurs  presence  on of  of  treatsodium  hydroxide. RCOOR* + N H 0 H + NaOH — > R C O ( N H O N a ) + .R'OH + H 0 . 2  The  free  tified  2  hydroxamic a c i d  formed by a c i d i f i c a t i o n  by a c o l o u r r e a c t i o n w i t h  ferric  c a n be  chloride. H  NHOH R -  C = 0 + 1/3  N Fe  + 3  —> R -  O  x  C = 0""  F  e  /  3  + H  +  iden-  44 A p o s i t i v e r e a c t i o n gave a red to purple c o l o u r . was  The  performed on e t h y l acetate and a c e t y l a t e d estrone  test gluco-  siduronate as standards and on the d e a c e t y l a t e d m a t e r i a l . TABLE 2 Compound  Colour  e t h y l acetate  intensespurple  a c e t y l a t e d estrone glucosiduronate  purple  d e a c e t y l a t e d estrone glucosiduronate  negative  45 DISCUSSION The m e t h o d s for  the  synthesis  -a-D-glucuronate this  of methyl  of silver  reports  and-Babers  carbonate)  made o n a l l t h e  his  comprise  and  reactions  Bollenback  the  the  reactions  isomerization  followed  (26)  and the  reported  conditions bromination  according  to  sults—an  overall yield  Pelzer  (49).  first  has  in this  into  This  the  yield  of  60-65% on t h e  reactions  and  methylation,  bromination  i n obtaining  the  in crystalline  in  for  surprising.  tallized  counting  readily  recrystallization sibilities  for  from methanol, had  this  may h a v e b e e n f o r m e d tion 80%  of the acid  lactone  i n about  was  posed  unusual  absolute  no d i f f i c u l t y behaviour  two months  best  methyl 50%; and  a  re2,3,4-  general  isomeriza-  o f u n l a b e l l e d and solution  Glucuronolactone and  95% e t h a n o l  before.  exist.  an e q u i l i b r i u m of at  were  processed  form a f t e r  from g l u c u r o n o l a c t o n e . reaches  to  and  reactions.  mixture  glucuronolactone  procedures  (48)  gave the  of  of  acetylation  were  acetylation  radioactive methanol  The  mixtures  from g l u c u r o n o l a c t o n e  80-85% on the  Difficulty  synthesis  methylation  thesis.  combination  pre-  comprehensive  o f B o w e r i n g and T i m e l l reaction  of  ( i n the  improved these  tri-O-acetyl-l-bromo-l-deoxy-a-D-glucuronate  tion  conjugation  i n v o l v e d i n the  methods have been i n c o r p o r a t e d  bromination  (1933-35)  2,3,4-tri-0-acetyl-l-bromo-l-deoxy  from g l u c u r o n o l a c t o n e ,  glucosiduronates.  and  by G o e b e l  compound w i t h a h y d r o x y l - c o n t a i n i n g r e a c t a n t  sence  and  proposed  and  Several  Glucuronic  solu-  20% l a c t o n e while  pos-  acid  An aqueous  room t e m p e r a t u r e ,  .crys-  at  and 100°C  46 an  e q u i l i b r i u m o f 60% l a c t o n e  two hours water  In this  experiment  i n 15 m l o f m e t h a n o l  minutes in  (71).  64°C.  at  amounts  and the  Therefore  sufficient  a n d 40% f r e e  to  the  acid  is  reached  t h e r e were,.,fpur  drops  s o l u t i o n was h e a t e d  formation  i n h i b i t c r y s t a l l i z a t i o n seems  extensively polymerized degradation  occuring  is  glucuronolactone solutions. days  is  never duct  Refluxing  active  yield  formation  basic  The l o w r e c o v e r y nate;.after  probably the  original  result  of  gave  higher  fact  the  has  is  as  is  that  method  of Bollenback should y i e l d  acetylated  sugar,  the  liquior  mother  the  crude  i n that  removed from the  radio-  was  most  portion  solution  of  used. (48)  of B o w e r i n g and T i m e l l  the  Despite  isomerization isomer  acid  obtained, 8-anomer,  so  the  was  step, one  acetyla-  a-form  after  the  only  catalyzed  predominantly  material  pro-  radioactive  experiments  an added  While  has  tetra-O-acetyl-a-D-glucuro-  o f p r o d u c i n g o n l y one  required.  several  catalyst.  o f B o l l e n b a c k (26).  and has  for  o n l y i f the  a  glucuronolactone  longer  advantage,  crystallization tion  and a c t i n g  by the  y i e l d s than  former  the  occur  an i m p u r i t y p r e s e n t  radioactive  Acetylation  it  case would  compared w i t h n o n - r a d i o a c t i v e  the  of  aqueous  a desirable  m e t h y l a t i o n and a c e t y l a t i o n d u r i n g the  preparation  unlikely.  a compound w h i c h  T h i s w o u l d be  of methyl  acid  preparations  w i t h methanol  methyl glucuronate,  in this  m a t e r i a l were  as  5  products  involve boiling  glucuronolactone  c r y s t a l l i z e d (26).  been  improbable  from g l u c u r o n i c a c i d  known t o  but  e v e n more  of  for  of g l u c u r o n i c  The p o s s i b i l i t y o f from heat  in  of  the  processing  difficult  to  47 crystallize  and appeared  to  be  a mixture  tetra-O-acetyl-a-D-glucuronate cult  to  isolate  product from  by the  obtained  after  method  has  the  synthesis  minated  i s o m e r i z a t i o n was  cule  to  be  diffi-  However,  the  easily crystallized  to  bromination.  glucuronolactone.  be  the  ing" t o  mixture  i n the  by washing the formed.  has  is  difficult  hampered  step  by the  e s p e c i a l l y at  the  the  bro-  room  mole-  reaction mixture  acid  after  observed  that  Timell  by e v a p o r a t i o n  reaction  to  tem-  such a r e a c t i v e  c h l o r o f o r m s h o u l d be  and then washed  been  were  of  of HBr i n - a c e t i c  the  (49)  It  most  o f B o l l e n b a c k and B o w e r i n g and  Pelzer  solvents  syrup  treatment  chloroform to  possible.  trapped  It  i n obtaining  addition of  reaction  tion  step  The methods  i n v o l v e removal  the  the  of methyl 2 , 3 , 4 - t r i - 0 - a c e t y l - l - b r o m o - l - d e o x y -  The c r i t i c a l  appears  both  was  compound b e i n g q u i t e u u n s t a b l e ,  perature.  the  reported  Methyl  alcohol.  glucuronate,from  to  acetates.  of Bollenback (26).  The b r o m i n a t i o n r e a c t i o n in  been  of  mixture.  added  remove  i n those  as  prior  Accord-  directly much a c i d  cases where  syrup  chloroform than  As a r e s u l t  and o n ; r e m o v a l  and was  i f no p r i o r  hydrobromic acid of the  more  difficult  to  evaporation  as reac-  r e m o v e d d i r e c t l y some h y d r o b r o m i c a c i d  residual  to  became  remove was  remained d i s s o l v e d i n  c h l o r o f o r m seemed  to  hinder  of the  sugar  perthe  crystal-  lization. Once t h e they in  must  the  be k e p t  crystals anhydrous.  same m a n n e r ,  brominated  Drying  by d e s s i c a t i o n  and  over  storage  are  are  obtained  effected  calcium chloride  at  0°C.  48 Decomposition Purification composed  at  any  undegraded  sample  was  achieved  The  success  quite  variable  ences  i n the  and  lots  acetic  o i l obtained  appeared  i n the  the  be  directly  hours.  acid.  de-  compound  solution  bromination  in  and  water  of the  chlo-  reaction  was  related These  quantities  to  differ-  differences  of HBr d i s s o l v e d  Therefore  the  depending  on w h i c h a c i d was Also  material  a c i d and n o t  presence  the  conversion  crystalline  glucuronic  heat,  the  on r e m o v a l  the  acid-catalyzed  when f r e e  ing  of  of  of HBr i n a c e t i c  glucuronolactone,  zation.  to  24  in  acid. In  to  the  than  from a p a r t i a l l y  by s o l u t i o n  and y i e l d  may p o s s i b l y h a v e b e e n the  material  i n less  washing w i t h a sodium b i c a r b o n a t e  c r y s t a l l i z a t i o n of  roform.  room t e m p e r a t u r e  of  chloroform, and  occurs  of  the  successful  was  obtained  sodium s a l t  sodium a c e t a t e used,  of g l u c u r o n i c  or  was  so  polymerization products  It  was  the  used.  chloride,  may h a v e h i n d e r e d  method  only  was  sodium  acid  crystalli-  o n l y one  not  may h a v e p r e v e n t e d  involvcrystal  formation.  thoxide  observed  m u s t be u s e d  temperatures  less  nolactone  the  or  sodium hydroxide  in catalytic  than  40°C  formation  A two f o l d  that  amounts  during of  increase  cured  (.002  i n the  methylation  yield and  of  its  sodium  mole)  sodium glucuronate  16cc,17cx-epoxy-estra-l,3,5(lO)-triene rangement  the  and  and  of  will  the  second  attempt  to  synthesize  at  glucurooccur.  3,178-diacetoxysuccessful  rear-  to_ 3 , 1 6 a - d i a c e t o x y - 1 7 - k e t o - e s t r a - l , 3 , 5 ( 1 0 ) - t r i e n e  during  me-  oc-  estriol-178-glu-  49 cosiduronate. ferences trone zoic  The r e a s o n  i n the  used.  less  than  doubled. highly  and the  -30°C  These  is  were  acid  different  samples  i n the  of  difof  es-  perben-  second  epoxi-  (in a dry ice-ethanol  of an i c e b a t h )  observations  concentrated  The o n l y  used  i n s u c h a manner instead  unclear.  d i f f e r e n t .preparations  The p e r b e n z o i c  d a t i o n was p r e p a r e d at  this  two p r e p a r a t i o n s  enol diacetate acid  for  suggest  that  the  a requirement  s o l u t i o n of perbenzoic  acid  yield for  for  bath was  a more  efficient  epoxidation. It  has  been  e m p h a s i z e d b y L e e d s ejt a l .  arrangement  of the  epoxide  only  sample  is  the  i f the  m a t e r i a l used  occurs  pure.  by s i l i c a  While  gel  (53)  yield  was more r e a d i l y c r y s t a l l i z e d  different  appearance  from  have  i n a purer  state  been  the  first  lot.  which allowed  reaction the  which  176-to  the  migration with w o u l d be group the  Without  16a-position. inversion  forms  stable  have  rearrangement  occured,  first)  is  the  to  the  obtained  in  distinctly  former  may  to  3,16a  occur.  been proposed f o r  of the  this  a c e t o x y l group proposed that  retention  of  u n f a v o u r e d and would  a c e t o x y l m i g r a t i o n would  from i f  configuration  1 6 8 - p o s i t i o n of the  1 6 a - c o n f i g u r a t i o n v i a an e n o l  inversion  3,  rearrangement  Woodward (53)  e x p l a i n e d by assuming the  (which  more  involve  mechanisms  the  and had a Thus t h e  -diaeetpxy-17-keto-estra-l,3,5(lO)-triene Two d i f f e r e n t  indicated  of equal p u r i t y ,  1 7 f 3 - d i a c e t o x y - 1 6 a , 1 7 a - e p o x y - e s t r a - l , 3 ,5 ( l O ) - t r i e n e higher  re-  chromatography  mp d e t e r m i n a t i o n s  i n b o t h a t t e m p t s was  that  acetoxyl  rearrange  to  intermediate. be d i r e c t l y  to  50 the  16a-position.  are  bisectional  equally  and  out  hydrogen  the  the  of  group  estriol  However, to  the  the  bonds  at  reaction  at  C-16 nearly  was  5°C v i s -  carried  sodiumborotetramethoxide hydrolysis  of the  phenolic  the  at  the  ring  formation.  hydroxyl.  s p e c i f i c a l l y at  the  of  their  the  formed  C-3  posi-  "activating"  The  For this  latter  is  reason  the  C-17 p o s i t i o n would  has  acetylated  been  m e t h o d s w o u l d be  synthesis of  be  from  i o n i z a t i o n of  conjugation,  brominated  position.  methyl group  to  cleave  obtaining  remove the  was  of the  group  bond.  material  to  the  formation  or h i g h e r  which hinder  methyl  of  from  2,3,4-tri-0-acetyl-l-  acid released  dissacharides  this  glycosidic  attributed  Because  C-3  crystalline  with methyl  carbonic  sugar  dif-  i n the  present.  unwanted  only with  the  bromo-l-deoxy-a-D-glucuronate products  applied  and  because  estradiol  reaction  by Carpenter  conjugates  sufficient  of  estriol-16a-  of unsubstituted  difficulty.in  conjugation  3-methoxy  reported  which would q u a n t i t a t i v e l y  The  the  as  hemiacetal  yield  synthesis  would probably  from  the  l 6 B - p o s i t i o n s s h o u l d be  Acetate  toward  extreme- s t a b i l i t y  the  as  feasible.  Conditions  the  (66).  17B-glucosiduronates  ficulty  likely  w i t h sodium borohydride  occured because  The  Kellie.  and  medium and  hydrolyzed to  synthesis  and  reductions  hydrogen  acetoxyl  be  16a-  evolution occured  probably  readily  not  the  i n a methanolic  releasing tion  seems more  favoured. During  ible  This  variety of  of  water  d u r i n g the  actual  sugars  formed  are  c r y s t a l l i z a t i o n of  the  5  products. either with  In  addition,  or both  the  of  starting  made f r a c t i o n a l of  the  is  a c i d on a  a more  in  no m e n t i o n  present  obtained larger  scale  is  times  the  made o f  a  The m i n u t e  the  method  no y i e l d length  is  of  probably  time  due  and under  nide  spray  groups.  Examination  estradiol  that  conjugation  the.C-17  hydroxyl  than  phenolic  the  is  It  spray  indicates  the  at  the  corresponding  obtained  in this  c o n t i n u i n g the  different the  and  surprising  is  to  and can  for  a  greater  conditions.  sugars  due  non-specificity  acetylated  reports  by the  chloride-potassium o n l y be  and  experiment  reaction  reaction  acetylated  ferric  formed  3-position  and M i e s c h e r i n w h i c h W o t i z to  with glu-  3,17-diglucosiduronate  of m a t e r i a l  The d e t e c t i o n . o f C i o c a l t e u Phenol  along  difficult.  'conjugation • product  of Meystre  at  quantities  y i e l d of "the  amount  occur  i n semimicro  indicates  position for  can  products,  from c o n j u g a t i n g  estradiol—3,17-diglucosiduronate by  estradiol  Acetylated estradiol-178-glucosiduronate  twenty-five  only.  of  two h y d r o x y l s a n d t h e  material,  favourable  hydroxyl.  conjugation  c r y s t a l l i z a t i o n very  data Wotiz  curonic  the  1  to  the  of these  Folin-  ferricyaacetyl  colour  re-  agents . The estradiol Shapiro tively of  conjugation  and had  (58) easier,  is to  several  one  e s t r o n e was  points  much m i l d e r t h a n perform.  products.hindering  only  of  Also  in its that the  for  successful  favour.  of Meystre  removed  conjugation.  than  The method (57)  p o s s i b i l i t y of  crystallization is  hydroxyl available  more  as  a  and  of  rela-  mixture estrone  As a r e p o r t  by  has  52 Conchie should slow the  (22)  indicated that  be u s e d  for  c o n j u g a t i o n and t h a t  dowri'the r e a c t i o n ,  no d r y i n g  r e a c t i o n m i x t u r e and f r e s h  parison these  of the  yield  precautions  separate  estrone  crystallization of  the  that  of  was r e n d e r e d from  of  conjugate  and benzene  estrone the  arid t h e  acetyl  or  mu, of  uted  reaction its  (std.  prepared  to  the  later  latter.  Observation  however,  indicated  t a k i n g the  discovered that  could  be  separated  toluene.  The a l k a l i  of the  the  from  aforeaceestrone  s o d i u m h y d r o x i d e (pH extract  drastic  product  acetylated  IR s p e c t r a  The s h a r p e r  (Fig. l)  only  enough t o  removed cleave  and more  was t h e  as  in a split  that  standard.  conjugate of  peak,  whereas  was  made  prepared  clearly the  A max 2 8 2  defined  standard  peaks  was  An i n d i c a t i o n  attrib-  that  the  w a s made b y c o m p a r i s o n o f  estrone.  determined with  conjugate  a n d U V s p e c t r u m w i i h l t h a t of  2 7 5 - 2 7 7 mu:  compound compared w i t h  some i m p u r i t y i n t h e  hydroxyl,  flected  by  s o l u t i o n of  ^ max 2 8 2 m\x,  UV s p e c t r u m w i t h  nolic  estrone-3-glucosiduronate  groups.  2 7 4 - 2 7 5 mu)". the  observing to  c o n d i t i o n s were n o t  comparison of the standard  Com-  inability  a cold  Identification  a  was  into  was u s e d .  and a f t e r  d i d occur while  glucosiduronate  by a p a r t i t i o n between 10.5)  It  dessicants  i m p o s s i b l e by the  acetylated  carbonate  incorporated  carbonate  crystalline structures,  an i n c r e a s e  estrone  was  before  w i t h o u t breakdown of  different  tylated  silver  silver  internal  agent  conjugate  mentioned precautions.  by  freshly prepared  S u b s t i t u t i o n at  3-methoxy e s t r o n e ,  the  free  estrone  or  the is  phere-  a salt  of  53 estrone trone  has  a s i n g l e peak.  glucosiduronate  8-glycosidic the  link,  steroid  - acetyl trone  the  Treatment  the  acetyl  h y d r o l y s i s of  glycosidic  groups. added  showed the  peaks  groups  the  bond but  D e t e c t i o n of the  further  of the  evidence  acetylated  that  of Wotiz  estrone  to  for  indicated that  the  ing  groups.  for  and  and es-  present. with  than  by  the  The  a brief similar  deacetylated from  way s e p a r a t e d  an  acid-  from  other  c a r b o x y l i c esters by  their  subsequent  chloride proved to presence  1  detection be  an  a  efficient  of a c e t y l  d e a c e t y l a t i o h r e a c t i o n h a d be.en  by  groups. successful  2).  the  ducts  test  determining the  Development for  of  only  and  followed  effective  and i n t h i s  hydroxamic acids ferric  was  successfully extracted  The s p o t  reaction with  (Table  the  not  methyl  glucosiduronate  sodium methoxide  was  n-butanol  conjugate  conjugate  removal of a c e t y l  products.  microtechnique It  for  solution with  colour  water  glucosiduronate  conversion  es-  ring  glucuronic acid  the  p r o v e d more  in boiling  reaction  of  aromatic  removed the  free  heating  ic  and the  also  of  estrone  acetylated  characteristic  acetylated  known c a t a l y t i c q u a n t i t y  method  of  (63).  Acid cleaved  The I R s p e c t r u m  separation  of  and p u r i f i c a t i o n o f  w o u l d p r o b a b l y be  syntheses.  paration  chromatographic  With  required  for  establishment  and p a r t i t i o n  intermediates  improvement  of  of doubly l a b e l l e d conjugates  and  of the  these/systems, w o u l d be m o r e  systems  the  proexistpre-  feasible.  54 SUMMARY -1. M e t h y l  1,2,3,4-tetra-O-acetyl-a-D-glucuronate  60-65$ y i e l d tion  from  glucuronolactone  and an i s o m e r i z a t i o n r e a c t i o n .  acetyl-B-D-glucuronate acid 2.  by a base  catalyzed  Methyl  was p r e p a r e d  Methyl  was p r e p a r e d  c a t a l y z e d acetyl-a-,. 1,2,3,4,-tetra-O-  i n lower y i e l d s from  an  acetylation.  2,3,4-tri-»0-acetyl-l-bromo-l-deoxy-a-D-glucuronate  prepared  in  i n 80-85$ y i e l d  from  was  methyl tetra-O-acetyl-ct-and B-D-  glucuronate. 3.  Estrone Its  enol diacetate  c o n v e r s i o n in t h e  85$ y i e l d , umn g a v e  "the  a 50$ r e c o v e r y o f  ible 4.  synthesis  by t h i s  of  of  the  be  15$ y i e l d .  estrone  yield.  on a s i l i c a  80-  gel  col-  compound,  specifically  s o d i u m b o r o h y d r i d e , was  reduced  unsuccessful.  e s t r i o l - 1 7 B - g l u c o s i d u r o n a t e was n o t  feas-  method.  identification. to  i n 66$  accomplished i n  latter  Acetylated estrone-3-glucosiduronate mately  estrone  3,16a-diacet;pxy-17-keto-estra-l,3,5  I s o l a t i o n of t h i s  17-hydroxyl with  Thus the  from  e p o x y d e r i v a t i v e was  and r e a r r a n g e m e n t  (lO)-triene. to  was p r e p a r e d  A small  was p r e p a r e d  p o r t i o n of  D e a c e t y l a t i o n gave glucosiduronate.  this  a solid  in approxi-  was p u r i f i e d compound  for  presumed  55 BIBLIOGRAPHY (1)  Breuer, H .  Vitamins  (2)  Cohen,  S.  and  Marrian,  G. F .  Biochem.  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