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Partial synthesis of porphyrin S411 from protoporphyrin IX DME Sivasothy, Ramani 1978

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PARTIAL SYNTHESIS OF PORPHYRIN SA11 FROM PROTOPORPHYRIN I X DME  by  RAMANI SIVASOTHY B.Sc.(Hons•).  U n i v e r s i t y o f London, (1976)  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES i n t h e Department of Chemistry  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required  standard  2A THE  U N I V E R S I T Y OF B R I T I S H COLUMBIA  fa  Raman! Sivasothy, Oct. 1978  p ^  ^  i (  ' -  „  p/ruw'&o  c  In p r e s e n t i n g t h i s  thesis  an advanced degree at the I  Library shall  f u r t h e r agree  for  f u l f i l m e n t o f the requirements f o r  the U n i v e r s i t y of B r i t i s h  make i t f r e e l y a v a i l a b l e  that permission  for  Columbia,  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s of  in p a r t i a l  this  representatives. thesis  It  i s understood that copying o r p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  written permission.  Department of The  University of B r i t i s h  Columbia  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1WS  Date  \\*  Qc^Vx^  VH^T  not be allowed without my  -ii-  ABSTRACT  This study describes the partial synthesis of porphyrin SA11 (la) from protoporphyrin IX DME (32).  Conversion of 2-(2-hydroxyethyl)-A-  (2-methoxycarbonylvinyl)-deuteroporphyrin IX DME (Alb) into harderoporphyrin (3Aa) i s also outlined. Photo-oxidation of (32) gives the isomeric photoprotoporphyrins (36a) and (36b);  these isomers can be separated by column chromatography.  Compounds (36a) and (36b) are transformed into the mono-formyl-mono-vinyldeuteroporphyrins (33a) and (33b) by reduction with borohydride followed by the cleavage of the resulting glycols with periodate. Treatment of (33a) and (33b) with 2 equivalents of thallium(III) nitrate in methanol affords the mono-acetal-mono-formyl derivatives which, on condensation with malonic acid i n the presence of piperidine gives the mono-acetal-mono-acrylie aciddeuteroporphyrins.  The mono-acetal-mono-acrylic  acid derivatives are  converted into the corresponding mono-acrylic acid mono-(2-hydroxyethyl)deuteroporphyrins (Ala) and (Alb) by hydrolysis and reduction with borohydride.  Treatment of (Ala) with CBr^/Ph-jP affords the 2-bromoethyl  derivative (A2a) which, is converted into the corresponding 2-cyanoethyl derivative (A3) by cyanide in 1-methylpyrrolidone.  Compound (A3) i s  transformed into porphyrin SA11 (la) by methanolysis. Hydrogenation of the acrylic acid side-chain of compound (Alb) followed by the dehydration of the 2-hydroxyethyl side-chain effect's the conversion of compound (A5) into harderoporphyrin (3Aa).  -iii-  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF SCHEMES  iv  L I S T OF ABBREVIATIONS  v  ACKNOWLEDGEMENTS  vi  I.  INTRODUCTION  1  1.1  S t r u c t u r e and Nomenclature  2  1.2  B i o s y n t h e s i s o f Heme  5  1.3  Synthesis of Porphyrins  11  A. ^  One-Step P r o c e s s  11  i)  from Dipyrromethanes  11  ii)  from Dipyrromethenes  14  B.  Two-Step P r o c e s s  15  i)  from b - b i l e n e s  15  ii)  from a - o x o b i l a n e s  16  iii)  from b - o x o b i l a n e s  16  II.  SYNTHESIS OF PORPHYRIN  S411  22  A p p l i c a t i o n o f the P a r t i a l S y n t h e t i c I n t e r m e d i a t e s  36  III.  EXPERIMENTAL  41  3.1  G e n e r a l Methods  41  3.2  Chemicals and M a t e r i a l s  42  3.3  Preparations  42  BIBLIOGRAPHY  $1  -iv-  LIST OF SCHEMES  1.1  B i o s y n t h e s i s o f Coproporphyrinogen  1.2  A P o s s i b l e Mechanism f o r the Formation  1.3  B i o s y n t h e s i s of Heme from Coproporphyrinogen  1.4  S y n t h e s i s of D i p y r r o l i c  1.5  S y n t h e s i s of P o r p h y r i n s v i a Dipyrromethanes  1.6  S y n t h e s i s of P o r p h y r i n s v i a Dipyrromethenes  1.8  P o r p h y r i n s from b - b i l e n e s  1.9  P o r p h y r i n s from  a-oxobilanes  1.10/  Porphyrins  b-oxobilan.es  2.1  O x i d a t i o n of P r o t o p o r p h y r i n IX DME  2.2  Condensation  of Mono-formyl-mono-vinyl-deuteroporphyrin  with Malonic  acid  2.3  from  via  A P o s s i b l e S y n t h e s i s of P o r p h y r i n S411  of Uroporphyrinogen  III  III  Photoprotoporphyrins  from M o n o - a c r y l i c  IX  DME  acid-mono-  DME  A R e a c t i o n Sequence f o r the C o n v e r s i o n p o r p h y r i n IX DME  III  Intermediates  v i n y l - d e u t e r o p o r p h y r i n IX 2.4  I and  into Porphyrin  of  2-formyl-4-vinyl-deutero-  S411  2.5  R e a c t i o n Mechanism of TTN  i n Methanol w i t h a Double Bond  2.6  Mechanism of B r o m i n a t i o n  2.7  S y n t h e s i s of P o r p h y r i n S411  2.8  S y n t h e t i c Scheme f o r the T r a n s f o r m a t i o n  of an A l c o h o l w i t h  CBr./Ph„P 4 3  from P r o t o p o r p h y r i n IX  4 - a c r y l i c a c i d - d e u t e r o p o r p h y r i n IX DME  of into  DME  2-(2-hydroxyethyl)Harderoporphyrin  -v-  ABBREVIATIONS  Ac  acetyl  Acr.  -  acrylic  acid  Bu*  tertiary  DME  dimethylester  DTBE  ditertiary  d  doublet  dd  doublet of doublets  log.  logarithm  max. p  /  -  Me  Acr  butyl  butylester  maximum methoxycarbonyl e t h y l  -  methoxycarbonyl  vinyl  Me  methyl  DMF  N,N-dimethyl formamide  Ph  phenyl  P  propionic  py  pyridine  q  quartet  s  -  singlet  t  triplet  V  vinyl  acid  ACKNOWLEDGEMENTS  I w i s h t o thank t h e many p e o p l e who have a i d e d me d u r i n g the course o f t h i s s t u d y .  I would l i k e t o p a r t i c u l a r l y  f o r h i s s u p e r v i s i o n and u n d e r s t a n d i n g .  thank D a v i d D o l p h i n  My s p e c i a l thanks a r e extended  to Bob C a r l s o n f o r h i s c o n t i n u a l c o u n s e l and encouragement a l l s t a g e s o f t h i s work.  Thanks a r e a l s o due t o R o b e r t D i N e l l o f o r h i s  i n s t r u c t i o n s i n the experimental aspects of t h i s study; f o r t a k i n g time t o r e a d t h i s m a n u s c r i p t comments and s u g g e s t i o n s ;  throughout  Carl Alleyne  and f o r g i v i n g h i s h e l p f u l  members o f t h e D o l p h i n r e s e a r c h group f o r  t h e i r / a s s i s t a n c e ; Dr.A.H.Jackson f o r p r o v i s i o n o f p o r p h y r i n S411. F i n a l l y , 1 w i s h t o thank D e v i f o r h e r h e l p and c o - o p e r a t i o n i n t y p i n g this  manuscript.  INTRODUCTION  (la)  -CH=CH-COOH  -CH CH COOH  (lb)  -CH CH COOH  -CH=CH-COOH  2  2  2  2  Meconium i s an accumulated end-product of the development and metabolism of the foetus and i s r i c h i n b i l e pigments and porphyrins.  1  During an i n v e s t i g a t i o n of the ether-soluble porphyrins obtained from meconium using counter-current techniques a porphyrin having a Soret absorption band maximum, i n 5% w/v hydrochloric acid, at 410-411nm was detected.  2  This i s o l a t e was c a l l e d porphyrin S411. On the basis of i t s  behaviour on a l u t i d i n e paper chromatographic system, spectral absorption, molecular weight and c a t a l y t i c hydrogenation  the porphyrin S411  was i d e n t i f i e d as a mono-acrylate, tri-propionate tetramethyl  porphin,  3  -2and  assigned  s t r u c t u r e ( l a ) or  and  ( l b ) through t h e b - o x o b i l a n e  has been accomplished. * 1  (lb).  The  s y n t h e s i s of both  r o u t e - r e f e r e n c e to s e c t i o n 1 . 3 . B ( i i i )  Mixed m e l t i n g p o i n t and  counter-current  d i s t r i b u t i o n comparison o f the s y n t h e t i c isomers w i t h has  isomers ( l a )  d e f i n e d the s t r u c t u r e of p o r p h y r i n S411  as  ( l a ) B a s e d  models and n.m.r. and v i s i b l e s p e c t r a l e v i d e n c e the a c r y l i c a c i d group on p o r p h y r i n S411  has  the n a t u r a l m a t e r i a l upon  i t has been shown  molecular 11  that  a t r a n s - c o n f i g u r a t i o n . . As  a  p o s s i b l e i n t e r m e d i a t e i n the b i o s y n t h e s i s of heme i t assumed some importance f o r a  time.  /  I.I.  STRUCTURE AND  Porphin  NOMENCLATURE  (2) i s the p a r e n t  compound of the p o r p h y r i n s .  d e r i v a t i v e s of p o r p h i n a r e c o l l e c t i v e l y  known as  examples of n a t u r a l l y o c c u r r i n g p o r p h y r i n s The  'porphyrins'.  are l i s t e d i n T a b l e  I.U.P.A.C. system of nomenclature when a p p l i e d  p o r p h y r i n m a c r o c y c l e r e s u l t s i n a p r o l i x naming system. the c o n v e n t i o n a l name f o r p o r p h y r i n S411  porphin.  consequence^ the c l a s s i c a l system used by Hans F i s c h e r those working w i t h p o r p h y r i n n a t u r a l p r o d u c t s .  nomenclature i s i l l u s t r a t e d  Some 1.  to.the example,  (la) i s 2-(2-carboxyvinyl)-  4,6,7-tris-(2-carboxyethyl)-l,3,5,8-tetramethyl  by  For  All  using porphin  5  As  a  i s preferred  T h i s system o f  (2).  I f a l l the B - p o s i t i o n s of the p y r r o l e r i n g s a r e s u b s t i t u t e d , and  these  s u b s t i t u e n t s are of two  possible. an examplei.  This i s i l l u s t r a t e d Although  kinds  only, four p o s i t i o n a l isomers  i n F i g u r e 1.1,  members o f — a l l  using coproporphyrin  f o u r isomer s e r i e s can  be  as  are  -3-  'type isomer' Figure  1.1  I,  (3);  Type i s o m e r s  II,  (4);  III,  of c o p r o p o r p h y r i a  (5);  IV,  (6);  -4-  TABLE 1.'  TRIVIAL NAMES AND STRUCTURES OF THE PORPHYRINS OF HEME BIOSYNTHESIS  S u b s t i t u e n t s on p o s i t i o n Porphyrin  1  2  3  4  5  6  7  UROPORPHYRIN I  A  P  A  P  A  P  A  P  UROPORPHYRIN I I I  A  P  A  P  A  P  P  A  HEPTACARBOXYLIC ACID PORPHYRIN I I I  A  P  A  P  A  P  P  M  HEXACARBOXYLIC ACID PORPHYRIN I I I  M  PENTACARBOXYLIC ACID PORPHYRIN I I I  M  COPROPORPHYRIN I  M  P  M  P  M  P  M  P  COPROPORPHYRIN I I I  M  P  M  P  M  P  P  M  HARDEROPORPHYRIN  M  V  M  P  M  P  P  M  PORPHYRIN S411 o r DEHYDROCOPROPORPHYRIN  M  R  M  P  P  P  M  BIS-(HYDROXYPROPIONATE) COPROPORPHYRIN I I I  M  M  PROTOPORPHYRIN I X  M  M  Side-chain  M  A, -CH COOH;  abbreviations;  2  P, -CH CH C00H; 2  2  M  M  M  M  •M V  M  M  R, -CH=CHCOOH;  V, -CH=CH , 2  R  b  M, - C H , 3  -CH(0H)CH C00H; 2  -5-  synthetically prepared, only members of the isomer series I and III are found in nature.  Similarly, i f the substituents are of three kinds only  fifteen isomers are possible. The naturally occurring porphyrins of this type possess the IX-arrangement of substituents (7).  Porphyrins  found in hemoproteins may also be described as 2 and/or 4 substituted derivatives of deuteroporphyrin IX (7).  e.g. porphyrin S411 may be  referred to as 2-(2-carboxyvinyl)-4-(2-carboxyethyl)-deuteroporphyrin IX.  1.2.  BIOSYNTHESIS OF HEME  5  Although the general pathway of the biosynthesis of heme has been known for some twenty years,  7  the detailed description of the  sequence of steps between intermediates has only been recently established. ' 8  9  The biosynthetic precursors of porphyrins are the hexahydro derivatives of porphin (2), commonly known as the porphyrinogens (8). The porphyrins encountered  in the biosynthesis of heme and their  substituition patterns are listed in Table 1.  ,(8)  The f i r s t step i n the biosynthesis of heme i s the formation of 5-aminolaevulinic acid (ALA) (11) (Scheme 1.1).  I t i s formed by the  condensation of succinyl CoA (10), derived v i a the Krebs cycle and a-oxoglutarate, with glycine (9) i n the presence of the enzyme aminol a e v u l i n i c acid synthetase (ALA-S) (Scheme 1.1). are condensed by the enzyme ALA-dehydratase porphobilinogen (PBG) (12).  Two molecules of ALA  to give the mono-pyrrole  Under the influence of the enzyme uro-  porphyrinogen synthetase, four molecules of PBG are condensed  to give  uroporphyrinogen I. When uroporphyrinogen synthetase acts i n the presence of uroporphyrinogen co-synthetase, uroporphyrinogen I I I , i n which one of the constituent PBG units i s reversed, i s formed.  The formation of  uroporphyrinogen I I I could be considered as a stepwise process i n which four PBG molecules are combined i n a h e a d - t o - t a i l manner to give the symmetrical bilane (13) (Scheme  Formation of the unsymmetrical  1.2).^  uroporphyrinogen I I I isomer could then proceed v i a a 'spiro' intermediate (14).  Recent studies by Battersby and co-workers  l a b e l l e d intermediates have p r o v e d * 1 0  1 1  using i s o t o p i c a l l y  that the unsymmetrical uro-  porphyrinogen I I I i s formed from the symmetrical uroporphyrinogen I isomer by an intramolecular process.  Sequential decarboxylation of the  -7-  HOCCH CH COSCoA 2  HJNCHJCOOH  2  H (12)  UROPORPHYRINOGEN I 4-  UROPORPHYRINOGEN I I I  HEPTACARBOXYLIC ACID PORPHYRINOGEN I  HEPTACARBOXYLIC ACID PORPHYRINOGEN I I I  4HEXACARBOXYLIC ACID PORPHYRINOGEN I  4-  4- ' HEXACARBOXYLIC ACID PORPHYRINOGEN I I I 4-  PENTACARBOXYLIC ACID PORPHYRINOGRN I 4COPROPORPHYRINOGEN I  SCHEME 1.1  PENTACARBOXYLIC ACID PORPHYRINOGEN I I I 4COPROPORPHYRINOGEN I I I  B i o s y n t h e s i s o f c o p r o p o r p h y r i n o g e n I and I I I  -8-  Uro-  SCHEME 1.2  T.J.I  A P o s s i b l e Mechanism f o r the F o r m a t i o n  (Vt)  of Uroporphyrin I I I  -9-  four a c e t i c a c i d s i d e - c h a i n s of uroporphyrinogen  a f f o r d s the hepta-,  hexa-, p e n t a - c a r b o x y l i c a c i d p o r p h y r i n o g e n s and f i n a l l y t h e c o p r o p o r p h y r i n ogen I and I I I (Scheme 1.1).  Jackson e t . a l .  9  have demonstrated  t h e above  s e q u e n t i a l d e c a r b o x y l a t i o n t o be c l o c k - w i s e , s t a r t i n g w i t h t h e a c e t i c  acid  s i d e - c h a i n on t h e D - r i n g o f u r o p o r p h y r i n o g e n and e n d i n g w i t h t h a t on t h e C-ring.  Coproporphyrinogen  I i s not f u r t h e r metabolised.  The next s t a g e i n t h e b i o s y n t h e s i s o f heme i s t h e c o n v e r s i o n o f c o p r o p o r p h y r i n o g e n I I I t o p r o t o p o r p h y r i n o g e n I X . T h i s i n v o l v e s an o x i d a t i v e d e c a r b o x y l a t i o n r e a c t i o n which converts the propionate s i d e c h a i n s on r i n g s A and B o f c o p r o p o r p h y r i n o g e n I I I i n t o v i n y l groups. enzyme i n v o l v e d i n t h i s p r o c e s s i s c o p r o p o r p h y r i n o g e n a s e  (Scheme 1.3).  /  COPROPORPHYRINOGEN I I I  J J I 1  HARDEROPORPHYRINOGEN  PROTOPORPHYRINOGEN I X  PROTOPORPHYRIN IX  PROTOHEME  SCHEME 1.3  The  B i o s y n t h e s i s o f Heme from C o p r o p o r p h y r i n o g e n I I I  -10-  Sano d e m o n s t r a t e d  1 2  the p a r t i a l i n c o r p o r a t i o n o f t h e  b i s - ( h y d r o x y p r o p i o n a t e ) d e r i v a t i v e of c o p r o p o r p h y r i n I I I ( T a b l e 1) i n t o protoporphyrin IX.  Subsequently,  from the r a t h a r d e r i a n g l a n d , meconium,  were i s o l a t e d .  2  harderoporphyrin  1 3  1 3  a v i n y l t r i c a r b o x y l i c acid  and a d e h y d r o c o p r o p o r p h y r i n  The former  porphyrin from  p o r p h y r i n has been i d e n t i f i e d as  and t h e l a t t e r p o r p h y r i n as p o r p h y r i n S A l l  4  (Table 1 ) .  On the b a s i s o f t h e i r s t r u c t u r e s they were c o n s i d e r e d as l i k e l y i n t e r m e d i a t e s i n t h e c o n v e r s i o n of c o p r o p o r p h y r i n o g e n  I I I to  protoporphy-  rinogen IX. Newman p r o j e c t i o n f o r m u l a e d e r i v a t i v e of coproporphyrinogen  o f the b i s - ( h y d r o x y p r o p i o n a t e )  I I I have i n d i c a t e d  o f v i ^ y l and a c r y l i c a c i d s i d e - c h a i n s may o c c u r .  1 4  The  how t h e f o r m a t i o n sterically  f a v o u r e d c o n f o r m e r (15b) may undergo t r a n s - e l i m i n a t i o n o f w a t e r t o f o r m an a c r y l i c a c i d group, whereas t h e l e s s s t e r i c a l l y f a v o u r e d c o n f o r m e r (15a) may undergo t r a n s - d e c a r b o x y l a t i o n t o a v i n y l g r o u p . s t u d i e s have shown t h a t h a r d e r o p o r p h y r i n o g e n metabolism,  1 5  w h i l e porphyrinogen  biosynthetic pathway.  1 6  Isotopic labelling  i s an i n t e r m e d i a t e i n n o r m a l  S411 i s an o f f s h o o t o f t h e main  On t h e b a s i s o f t h i s e v i d e n c e ,  that the s t e r i c a l l y unfavourable  i t h a s been  suggested  t r a n s - d e c a r b o x y l a t i o n may o c c u r when t h e  s u b s t r a t e i s a t t a c h e d t o t h e enzyme s u r f a c e , whereas t h e t r a n s - e l i m i n a t i o n o f w a t e r may o c c u r s p o n t a n e o u s l y detached  from t h e e n z y m e .  (15a)  w i t h i n t e r m e d i a t e s t h a t have become  12  (15b)  -11-  Th e f i n a l stages i n the biosynthesis of heme involve the oxidation of protoporphyrinogen  IX to protoporphyrin IX and the incorporation  of iron by the enzyme ferrochelatase to give protoheme.  1.3  SYNTHESIS OF PORPHYRINS A detailed discussion of the synthesis of porphyrins i s beyond  the scope of t h i s introduction. The interested reader i s advised to consult recent reviews.17,18 Synthetic intermediates of the unsymmetrically substituted porphyrins are d i p y r r o l i c compounds.  The three b a s i c types of d i p y r r o l i c  compounds used, are the dipyrromethanes (16), dipyrromethenes (17) and the dipyrroketones (18).  Scheme 1.4 outlines the general synthetic routes  for their preparations. The methods for the r a t i o n a l synthesis of the substituted porphyrins are based upon 2+2  type synthesis, i n which two  d i p y r r o l i c components are coupled together to give the The coupling may  unsymmetrically  macrocycle.  be effected through either a one-step process or a  two-step process.  A.  One-step process In the one-step process two d i p y r r o l i c components are joined at  both ends to give a c y c l i c intermediate which on jln s i t u  oxidation gives  the porphyrin. i)  From dipyrromethanes The synthetic route to porphyrins from dipyrromethanes was  developed by MacDonald ^ and u t i l i s e s the mild acid-catalysed condensation 1  of a 5,5'-diformylpyrromethane (19) with a 5,5'-di-unsubstituted pyrromethane (20) (Scheme 1.5).  The c y c l i c intermediate i s ^ o x i d i s e d i n s i t u to the  Dipyrromethanes:  N  (18) SCHEME 1.4  S y n t h e s i s of D i p y r r o l i c  Intermediates  (20)  SCHEME 1.5  Synthesis of Porphyrins v i a Dipyrromethanes  -14-  required porphyrin. ii)  From dipyrromethenes U n t i l recently the most widely employed  synthetic route to  unsymmetrically substituted porphyrins was v i a dipyrromethenes.  It  involves the condensation of 5,5'-dibromopyrromethene s a l t s (21) with 5,5'-dimethyl- or 5,5'-di(bromomethyl)pyrromethene acid melts at temperatures up to 200°  SCHEME 1.6  s a l t s (22) i n organic  (Scheme 1.6).  2 0  Synthesis of porphyrins v i a dipyrromethenes  The high temperatures required by t h i s procedure often resulted i n low y i e l d s .  Improvements on t h i s procedure have been reported by  Paine, Chang and D o l p h i n .  21  S i g n i f i c a n t v a r i a t i o n s and extensions of this  route by Johnson and h i s c o l l e a g u e s ' ' ' have further increased i t s 2 2  applicability.  2 3  2 1  -15-  Th e above m e n t i o n e d -one-step  processess  r e q u i r e one o f t h e  d i p y r r o l i c components t o be s y m m e t r i c a l about t h e i n t e r - p y r r o l i c atom, i n o r d e r t o p r e v e n t  carbon  the f o r m a t i o n of a mixture of p r o d u c t s .  •^single "product i s o n l y o b t a i n e d when a s y m m e t r i c a l d i p y r r o l i c i s s e l f - c o n d e n s e d . "~TJse o f -two d i f f e r e n t  A  compound  d i p y r r o l i c compounds r e s u l t s i n  the f o r m a t i o n o f t h r e e p o r p h y r i n s .  B.  Two-step  process  •.}.-.-_ >••-- -The-two—step-.; process- involves t h e f o r m a t i o n o f an open-chain  t e t r a p y . r r o l i c i n t e r m e d i a t e , e.g., b i l a n e ( 2 3 ) , by  isolable joining  -the d i p y r r o l i c components a t one end, f o l l o w e d by c y c l i s a t i o n . i s u s e f u l i n the c o n s t r u c t i o n of unsymmetrical w i t h the symmetry r e q u i r e m e n t s . a,c-biladienes,  i)  2 2  a-oxobilanes  From b - b i l e n e s  T h i s method  p o r p h y r i n s , as i t d i s p e n s e s  I t i n c l u d e s routes through b - b i l e n e s , 2 6  and b - o x o b i l a n e s .  2 5  2 7  Z 5  The a p p r o a c h t o p o r p h y r i n s t h r o u g h b - b i l e n e s (24) i s o u t l i n e d i n Scheme  1.8.  -16-  /  R= Bu*"; SCHEME 1.8  Porphyrins from b-bilenes  Electron-withdrawing  substituents on the internal rings (B and C )  of the b-bilene (24) lead to complications. often used.  Cyclisation of (24) using trichloroacetic acid and  trimethyl orthoformate  ii)  As a result this route i s not  gives the porphyrin.  From a-oxobilanes  (25)  25  26  An application of this route i s shown i n the synthesis of mesoporphyrin IX DME ( 2 6 ) iii)  Frnm h-nxoh-tlanes  2e  (Scheme 1.9).  (29)  27  An example of porphyrin synthesis through b-oxobilanes i s given in Scheme 1.10 using porphyrin S411 (29) as an example.  4  -17-  (26)  SCHEME 1.9  Synthesis of Mesoporphyrin  v i a the a-oxobilane  route  (21) SCHEME 1.10  Synthesis of Porphyrin  S411 v i a the b - o x o b i l a n e  route  -19-  (29) Acr., -CH=CHCOOH,  P, -CH CH COOH 2  2  The d i r e c t synthesis of a porphyrin with an a c r y l i c a c i d side-chain i s not f e a s i b l e due to the nature of the reactions involved i n the construction of the porphyrin macrocycle  (Scheme 1.10).  One  route by which the synthesis of such a porphyrin may be accomplished involves the construction of the corresponding 6-free porphyrin (28), the 6-free p o s i t i o n being protected with a bromine atom i n the early stages of the synthesis.  The substituent i s then introduced i n t o the macrocycle,  For example, formylation of the i r o n ( I I I ) complex of (28) with d i c h l o r o methyl ether i n the presence of t i n ( I V ) c h l o r i d e (Scheme 1.10), followed by condensation with methyl hydrogen malonate would introduce an a c r y l i c acid group i n t o the g-free p o s i t i o n of (28). The a- and b-oxobilane routes have been used f o r the synthesis of unsymmetric porphyrins, but t h e i r main disadvantage of the r e a c t i o n sequence.  i s the complexity  Furthermore, i s o t o p i c l a b e l l i n g of the  side-chains - e s p e c i a l l y for biochemical s t u d i e s , i s a major undertaking as there are many steps from mono-pyrrolic precursors.  These routes  also s u f f e r from the lack of a common intermediate, n e c e s s i t a t i n g the synthesis of appropriate d i p y r r o l i c - p r e c u r s o r s f o r each porphyrin.  -20-  An a t t r a c t i v e and l e s s l a b o r i o u s a l t e r n a t i v e i s t h e p a r t i a l s y n t h e s i s o f t h e d e s i r e d p o r p h y r i n from a r e a d i l y a v a i l a b l e n a t u r a l l y occurring porphyrin.  As a consequence, t h e problem i n v o l v e d i n the  construction of the unsymmetrically  s u b s t i t u t e d p o r p h y r i n macrocycle i s  circumvented.  The most r e a d i l y a c c e s s i b l e n a t u r a l p o r p h y r i n i s p r o t o p o r p h y r i n IX (30) and may be o b t a i n e d chloride (31),  from e i t h e r h e m i n  2 8  o r h e m a t o p o r p h y r i n IX d i h y d r o -  both of which a r e commercially  2 9  V, -CH=CH  2  (30)  R, -CH CH C0 H  (32)  R, - C H C H C 0 C H  2  2  2  available.  (31) R, -CH(0H)CH  2  2  2  3  3  I n t h i s study we r e p o r t t h e p a r t i a l s y n t h e s i s o f p o r p h y r i n S411 and h a r d e r o p o r p h y r i n (30) was p r e p a r e d  from p r o t o p o r p h y r i n IX DME  (32) .  P r o t o p o r p h y r i n IX  by t h e t h e r m a l d e h y d r a t i o n o f h e m a t o p o r p h y r i n IX  d i h y d r o c h l o r i d e (31) i n r e f l u x i n g DMF.^°  E s t e r i f i c a t i o n o f (30) was  e f f e c t e d w i t h 5% s u l p h u r i c a c i d i n m e t h a n o l .  2 8  P r o t o p o r p h y r i n IX (30) has one p a i r o f v i n y l groups s u b s t i t u t e d a t p o s i t i o n s 2 and A o f t h e p o r p h y r i n m a c r o c y c l e .  P a r t i a l synthesis  i n v o l v i n g d e r i v a t i s a t i o n o f one o f t h e p a i r o f v i n y l groups o f  -21-  protoporphyrin i s commonly accomplished through p a r t i a l  derivatisation.  P a r t i a l d e r i v a t i s a t i o n involves a reaction i n which only one equivalent of the reagent i s used.  Although i t has been r e p o r t e d  group i s more reactive than the A-vinyl, subsequent found p r e f e r e n t i a l product f o r m a t i o n .  3 2 - 3 5  31  that the 2-vinyl  studies have not  As a consequence,  partial  d e r i v a t i s a t i o n would result i n a mixture of porphyrins consisting of the two isomeric (2-or A-derivatised protoporphyrin) mono-derivatives and the di-derivative.  U t i l i s a t i o n of this method not only necessitates the  separation of the r e s u l t i n g porphyrin mixture, but also gives•low y i e l d s for the mono-derivatives due to the formation of the undesired d i derivative.  Furthermore, i t i s not easy to assign an absolute struc-fcure  / to each of the two isomeric mono-derivatives.  T o t a l synthesis, however,  has the advantage of being able to unequivocally distinguish between isomers.  -22-  II.  SYNTHESIS OF PORPHYRIN S411  The synthesis of porphyrin S411 (la), from protoporphyrin IX DME (32) involves the transformation of the vinyl groups at positions 2 and 4 of the latter porphyrin into an acrylic acid group and a propionic group respectively.  acid  The major problem encountered i n syntheses involving  differently substituted deuteroporphyrins i s selective derivatisation of one of the pair of vinyl groups.  As no marked difference i n reactivity  between the 2 and 4 vinyl groups was f o u n d is not possible.  32_35  s e l e c t i v e derivatisation  An alternative method i s p a r t i a l derivatisation which  would result in a mixture consisting of the two isomeric mono-derivatised porphyrins.  Therefore, i f this method i s to be synthetically attractive  an efficient method for the separation of the porphyrin mixture i s essential.  -23-  Reports on the partial synthesis of spirographis porphyrins, (mono-formyl-mono-vinyl-deuteroporphyrins) (33a) and (33b) ,33-37 harderoporphyrins  (34a) and (34b) 34  and pemptoporphyrins (35a) and (35b) 34  from protoporphyrin IX DME (32) have appeared i n the literature.  The most  outstanding of these is the synthesis of the isomeric spirographis porphyrins (33a) and (33b) by Inhoffen.37  The mono-derivatisation of proto-  porphyrin IX DME (32) was effected via the photoprotoporphyrins (36b).  Exposure of protoporphyrin IX to sunlight gave the isomeric  photoprotoporphyrins  in a yield of 75%.  (a) Rj  (b) R  (33) ; -CHO. (34)  (36a) and  Ri  2  -CH=CH ; 2  -CH=CH , 2  (35) ; -CH=CH , 2  R2  -CH=CH , 2  -CHO;  -CH CH C0 H;  -CH CH C0 H, .  -CH=CH ;  -H;  -H,  -CH=CH :  2  2  2  2  2  2  2  2  -24The reaction involves the 1,4-addition of singlet oxygen to either of the vinyl substituted pyrrolenine rings (A or B) followed by decomposition of the peroxide (Scheme 2.1).  Mono-derivatisation is  achieved through the inhibitory effect exerted by the electrophilic formyl ethylidene group on the oxidation of the second vinyl group. This procedure, after chromatography of the isomeric photoprotoporphyrins, gave a 35% yield for each isomer.  The greater mobility compound on  s i l i c a gel using dichloromethane as eluent was identified with the 4-photoprotoporphyrin (36a).  Borohydride reduction of the photoprotoporphyrins,  followed by rearrangement and cleavage of the resulting glycols with periodate gave the corresponding formyl-vinyl derivatives in an 80% yield (Scheme 2.1). The efficiency of the synthesis i s due to mono-derivatisation, which is effected by the formation of the photoproto derivatives, and the facile separation of the two photoproto isomers on s i l i c a gel. The usual mode in which a trans-acrylic acid group i s introduced into the porphyrin macrocycle is via the condensation of a formyl group with malonic acid in the presence of a strong base. Therefore, the mono-formyl-mono-vinyl-deuteroporphyrins (33a) and (33b) are potentially useful intermediates in the synthesis of porphyrin S411. The high yields reported by Inhoffen  for the synthesis of the  isomeric formyl-vinyl derivatives (33a) and (33b) could not be reproduced using the cited reaction c o n d i t i o n s . ' 38  39  Clezy  38  was able to obtain  a yield of 25% from the photoprotoporphyrin II DME by changing the solvent from dichloromethane-benzene to dioxane. Hamilton, * 1  0  A 60% yield was reported by  who used a modification of Clezy's method.  In the present  study, a procedure similar to that used by Hamilton was employed. An average yield of 75% was  achieved.  N  1  i  SCHEME 2.1 Photo=oxidation  of Protoporphyrin IX  DME  -26-  Th e condensation of the  mono-formyl-mono-vinyl-deuteroporphyrins  with malonic acid was achieved according to the method of Hamilton * 1  (Scheme 2.2).  0  The a c r y l i c acid substituent was e s t e r i f i e d with  5% sulphuric acid i n methanol.  Hydration of the a c r y l i c acid double  bond was not observed. Conversion of the v i n y l group of mono-acrylic acid-mono-vinyldeuteroporphyrin IX DMEs (37a) and (37b) into a propionic acid group would transform the l a t t e r porphyrins into isomeric S411 porphyrins.  A sequence  of reactions to e f f e c t this conversion i s outlined i n Scheme 2.3.  This  synthetic scheme r e l i e s on the p r e f e r e n t i a l photo-oxidation of the v i n y l group.  I f the photo-oxidation i s a Diels-Alder type reaction, as has been  suggested,  07  the a c r y l i c acid diene component should be de-activated  by the electronegative carboxyl group.  Consequently, the p r e f e r e n t i a l  photo-oxidation of the v i n y l group was  expected, but at a slower rate  due to the effect of the a c r y l i c acid  group.  31  Separation of the isomeric photoprotoporphyrins by column chromatography gives three f r a c t i o n s :  the 4-photoprotoporphyrin  eluted f i r s t , the mixed isomers and the 2-photoprotoporphyrin The mixed fractions may be rechromatographed  isomer  isomer.  to obtain the pure isomers.  Accumulation of adequate supplies of the pure isomers for exploratory studies was  limited due to the tedious and costly nature of the chroma-  tographic separation technique.  The mixed isomers were, therefore, used  for preliminary studies to conserve supplies of the pure isomers. The mono-acrylic acid-mono-vinyl-deuteroporphyrins i n d i c h l o r o methane containing 10% pyridine were exposed to d i r e c t sunlight. three days a colour change from red to green was observed.  The  After thin-layer  chromatogram of the green solution revealed there were four (two pairs)  SCHEME 2.3  -29and not, as expected, two (one pair) products of the reaction.  This  observation was not shared by Hamilton* who reported the presence of 40  only two products.  The observed four products indicated that both the  vinyl and the acrylic acid diene components were susceptible towards photo-oxidation.  The fact that the reaction had occurred at the acrylic  acid diene component further indicated that the expected de-activating effect at this reaction site was removed.  Ionisation of the carboxyl  group in the presence of pyridine could, however, account for the absence of this de-activating effect.  Spectral studies of the reaction mixture  were not possible, because separation of the products by TLC could not be accomplished.  Reduction of the acrylic acid group prior to photo-  oxidation would overcome the above drawback but would require the protection of the vinyl group. Protection of the vinyl group may be achieved by either Markownikoff- * or anti-Markownikoff hydration. * 1  1  1  2  The Markownikoff  procedure was preferred to the latter, because of the facile dehydration of the mono-(l-hydroxyethyl)-derivative  (38).  was that reported by Clezy and Barrett. * 1  1  30  The method employed  However, the yield was poor.  Short reaction periods increased the yield to 40%, but further attempts to improve the efficiency of the reaction were not successful.  (38)  -30Kenner, McCorabie and Smith, * made use of the 1  2  anti-Markownikoff  hydration as an intermediate step i n the conversion of protoporphyrin IX to coproporphyrin I I I . The 2-hydroxyethyl derivative was  converted to  the 2-cyanoethyl derivative v i a the 2-bromoethyl d e r i v a t i v e .  Hydrolysis  of the 2-cyanoethyl derivative gave the required propionic acid group. Using a s i m i l a r approach the v i n y l group of mono-formyl-monovinyl-deuteroporphyrin could be converted to a propionic acid group with the condensation of the formyl group with malonic acid being effected at an appropriate stage i n the conversion. the sequence outlined i n Scheme 2.3 was Scheme 2.4  In view of this d i r e c t route abandoned.  i l l u s t r a t e s the sequence of reactions leading to  the synthesis of porphyrin S411  from 2-formyl-4-vinyl-deuteroporphyrin  IX  DME. Treatment of (33a) with two equivalents of thallium (III) n i t r a t e (TTN)  i n methanol gave the 2,2-dimethoxyethyl d e r i v a t i v e (39a) . » 3 4  S t r i c t adherence to the l i t e r a t u r e procedure,  resulted i n a 70% y i e l d  34  whereas a modified procedure gave a y i e l d of 90%.  The f i r s t equivalent  of TTN i s consumed i n the formation of the thallium complex of (Scheme 2.5).  1 + 2  (33a)  Demetallation of the thallium complex was achieved by  passing sulphur dioxide through the s o l u t i o n (T1(III)->T1(I)) and p r e c i p i t a t i n g the T1(I) with acid.  Condensation  of (39a) with malonic acid  i n pyridine with a trace of p i p e r i d i n e , as mentioned  e a r l i e r , gave (40a). *  Acid-hydrolysis of (40a) i n tetrahydrofuran followed by reduction and r e - e s t e r i f i c a t i o n gave (41a) . *» * 3t  Conversion of (41a) to (42a) may  1  1  borohydride  2  be effected with either thionyl  bromide i n dichloromethane-DMF * or carbon tetrabromide-triphenyl 1  phosphine.  34  2  The former method was  found unsatisfactory i n the presence  -32-  MeO-  -H  H  SCHEME 2.5  -33of a v i n y l group, ** owing to the formation of by-products a r i s i n g from 3  Markownikoff addition of hydrogen bromide to the v i n y l group.  The  l a t t e r method was chosen i n consideration of the unsaturated a c r y l i c acid substituent on porphyrin (41a).  The mechanism * of the reaction 1  3  i s generally accepted to proceed v i a an oxophosphonium intermediate as portrayed in Scheme 2.6. The corresponding c h l o r i n a t i o n , however, i s postulated to proceed v i a a d i f f e r e n t mechanism, i n the reaction mixture.  1+33  as no chloroform was d e t e c t e d * 1  The porphyrin (41a) i n dichloromethane  33  was treated with  with a large excess of carbon tetrabromide and triphenyl phosphine and refluxed.  The bromo derivative was obtained i n a 72% y i e l d .  The y i e l d  could probably be improved as no attempts were made to determine optimum conditions.  The r e a c t i o n was also c a r r i e d out with thionyl bromide i n a  y i e l d of 55%.  R  Me  Ri  R  .(40a);  -CH=CHCOOH  (40b);  -CH CH(0CH )  (41a);  2  "  -CH CH(OCH ) 2  3  2  3  -CH=CHC00H  -CH=CHC00H  -CH CH 0H  (41b);  -CH CH 0H  -CH=CHC00H  (42a);  -CH=CHC00H  -CH CH Br  (42b);  -CH CH Br  -CH=CHC00H  (43) ;  -CH=CHC00H  -CH CH CN  2  2  2  2  2  2  2  2  2  2  2  -34-  i RBr  SCHEME  +  Ph P = O 3  2.6.  As mentioned e a r l i e r , the p r e l i m i n a r y r e a c t i o n s were c a r r i e d out on the mixed isomers.  A separation of the isomeric mono-methylacrylate-  mono-(2-bromoethyl)-deuteroporphyrins grams.  were observed on t h i n - l a y e r chromato-  A preparative s c a l e separation was e f f e c t e d , but the r e s o l u t i o n  obtained was not as good as f o r the two photoproto isomers. 15% f o r each isomer was obtained.  A y i e l d of  The 2-methylacrylate-A-(2-bromoethyl)-  isomer was found to be the greater m o b i l i t y compound on s i l i c a g e l when dichloromethane-ether was used as eluent. Treatment of (42a) w i t h cyanide i n 1-methylpyrrolidone gave the corresponding cyano-derivative (43) i n a y i e l d of 70%. of (43) gave the porphyrin S411.  Methanolysis  CHCHO  (32)  (36a)  M*  CHO  T1(IIT)N0  3  -CH CH(OCH ) 2  (33a)  2  3  (39a)  CH (C0 H^ 2  2  2  Py/piperldlne  I I  SCHEME 2.7  S y n t h e s i s of P o r p h y r i n S411  from P r o t o p o r p h y r i n IX  DME  "36Dr.A.H.Jackson made a v a i l a b l e to us a sample of porphyrin obtained by the b-oxobilane route for comparative studies. point behaviour, spectral c h a r a c t e r i s t i c s - NMR, spectroscopy,  4  The  S411  melting  v i s i b l e and mass  and TLC mobility of the two specimens were i n good agreement.  In addition, no s i g n i f i c a n t melting point depression was  observed upon  admixture. APPLICATIONS OF THE PARTIAL SYNTHETIC INTERMEDIATES An a t t r a c t i v e feature of p a r t i a l synthesis i s that the synthetic intermediates, l i k e the natural porphyrins  derived from hemoproteins, are  2 and/or 4 derivatives of deuteroporphyrin  IX.  As a consequence, some  of the synthetic intermediates are p o t e n t i a l l y useful i n the synthesis of b i o s y n t h e t i c a l l y i n t e r e s t i n g porphyrins. One  of the many b i o s y n t h e t i c a l l y i n t e r e s t i n g porphyrins i s  harderoporphyrin. gland of the r a t .  Harderoporphyrin was 1 3  f i r s t i s o l a t e d from the  The harderian gland i s located at the rear of the  eye and i s found i n animals possessing a 'membrana n i c t i t a n s ' or eyelid'.  Preliminary studies on t h i s i s o l a t e by Kennedy  i d e n t i f i e d harderoporphyrin Its structure was  41  13  'third  have  as a mono-vinyl-tricarboxylic acid porphyrin.  formulated  of i t s trimethyl ester. * was  Harderian  as (34a) and proved by the t o t a l synthesis  Subsequently, the corresponding  established as an intermediate  porphyrinogen  i n the biosynthesis of heme between  coproporphyrinogen III and protoporphyrinogen IX.. A method for the generation of a v i n y l group involves an elimination reaction of a 2-hydroxyethyl s u b s t i t u e n t .  29  Conversion of  the 2-hydroxyethyl d e r i v a t i v e into the 2-chloroethyl d e r i v a t i v e , followed  -37by the dehydrochlorination of the porphyrin z i n c chelate w i t h potassium t-butoxide generates the v i n y l group.  More r e c e n t l y , Clezy has generated  the v i n y l group from e i t h e r the 2-chloroethyl or methane d e r i v a t i v e using sodium hydroxide i n aqueous p y r i d i n e . * 1  sulphonate  5  The above reactions h i g h l i g h t the s u i t a b i l i t y of 2-(2-hydroxyethyl)-4-methylacrylate-deuteroporphyrin IX DME (41b) as a p o t e n t i a l precursor f o r the synthesis of harderoporphyrin  (34a).  Hydrogenation of the  methylacrylate s i d e - c h a i n , followed by the dehydration of the hydroxye t h y l side-chain would r e s u l t i n harderoporphyrin  (34a) (Scheme 2.7).  C a t a l y t i c hydrogenation was c a r r i e d out on the acetate d e r i v a t i v e (44) of porphyrin (41b) . In so doing p o s s i b l e hydrogenolysis of side-chains were eliminated.  The porphyrin  deuteroporphyrin  2-(2-acetoxyethyl)-4-(2-methoxycarbonylethyl)-  IX DME (45) has been a key intermediate i n the t o t a l  synthesis of harderoporphyrin. *'*»*^  As such, the conversion of porphyrin (4  i n t o porphyrin (45) would accomplish  the synthesis of harderoporphyrin.  1  1  P r e l i m i n a r y studies proved the v a l i d i t y of t h i s s y n t h e t i c sequence. The transformation of  2-methylacrylate-4-(2-hydroxyethyl)-deutero-  porphyrin IX and 2-(2-hydroxyethyl)-4-methylacrylate-deuteroporphyrin i n t o porphyrin S411 and harderoporphyrin  IX  r e s p e c t i v e l y , i l l u s t r a t e s the  e f f i c i e n t u t i l i s a t i o n of the two isomeric mono-(2-hydroxyethyl)-monomethylacrylate-deuteroporphyrins. The exact mechanism by which coproporphyrinogen I I I i s transformed i n t o protoporphyrinogen e s t a b l i s h e d to be s e q u e n t i a l ,  IX i s yet unclear. 1 3  The transformation i s  w i t h harderoporphyrinogen as an intermediate.  The d e c a r b o x y l a t i o n - e l i m i n a t i o n of the propionate side-chain i s postulated to proceed v i a the hydroxypropionate  d e r i v a t i v e -reference to s e c t i o n 1.2,  although no evidence f o r the existence i n nature f o r the hydroxy propionate d e r i v a t i v e i s a v a i l a b l e . Further i n v e s t i g a t i o n i n t o the mechanism of the  N  (34a)  SCHEME 2.8  \ Route f o r the S y n t h e s i s o f H a r d e r o p o r p h y r i n  decarboxylation-elitnination may r e q u i r e the synthesis of the hydroxy propionate d e r i v a t i v e and could be r e a d i l y obtained by the hydration of porphyrin S411 w i t h hydrobromic a c i d - a c e t i c a c i d .  3 8  Digressing from the main theme of t h i s study, diagnosis of the porphyrias - a group of diseases a r i s i n g from disorders of heme metabolism, requires the separation and q u a n t i t a t i v e determination of the uro- to protoporphyrin precursors of heme i n blood, u r i n e and faeces.  The ready  achievement of complex separations by high pressure l i q u i d chromatography (HPLC)  has found favour as an e f f i c i e n t a n a l y t i c a l technique i n the c l i n i c a l .  i n v e s t i g a t i o n of the porphyrias. * 4  7  U t i l i s a t i o n of HPLC i n such a capacity  requires the presence of an i n t e r n a l standard f o r a c o n t i n u a l check on column and i n t e g r a t o r e f f i c i e n c y . The i n t e r n a l standard should be s t a b l e and r e a d i l y a v a i l a b l e . More i m p o r t a n t l y , i t should be completly resolved from a l l unknowns and yet e l u t e near the peak of i n t e r e s t . f i r s t eluted isomer of  The above requirements were met by the  mono-hydroxymethyl-mono-vinyl-deuteroporphyrin.  The isomeric mono-hydroxymethyl-mono-vinyl-deuteroporphyrins were prepared by the borohydride reduction of the  mono-formyl-mono-vinyl-derivatives.  The two isomeric mono-hydroxymethyl-mono-vinyl d e r i v a t i v e s (46a) and (46b) were separated by column chromatography using dichloromethane-ether  as eluent.  The greater m o b i l i t y isomer was i d e n t i f i e d w i t h the 2-vinyl-4-hydroxymethyldeuteroporphyrin IX DME.  Due to the s o l u b i l i t y p r o p e r t i e s of compounds  (46a) and (46b), these compounds were converted t o t h e i r acetates (47a) and (47b) f o r c h a r a c t e r i s a t i o n .  -An-  Ill.  EXPERIMENTAL  3.1.  GENERAL METHODS  Electronic  Spectroscopy V i s i b l e s p e c t r a were o b t a i n e d on a Cary 17  Dichloromethane  - s p e c t r o grade was  spectrophotometer.  used as s o l v e n t , u n l e s s o t h e r w i s e  specified.  Nuclear Magnetic  Resonance  Nuclear magnetic a t e i t h e r 100 MHz spectrometer.  resonance  o r 270 MHz  F o u r i e r - t r a n s f o r m s p e c t r a , were t a k e n  w i t h a V a r i a n XL-100 o r N i c o l e t M o d e l NIC-80  D e u t e r i o c h l o r o f o r m was  t h e s o l v e n t used. \ Resonances a r e  quoted on the S - s c a l e r e l a t i v e t o t e t r a m e t h y l s i l a n e (6=0).  M^ss  Spectroscopy Mass s p e c t r a were r e c o r d e d i n an A t l a s CH-4  s p e c t r o m e t e r o r an  A . E . I . MS-902 s p e c t r o m e t e r .  Melting Point Determination M e l t i n g p o i n t s were measured w i t h a Thomas-Hoover c a p i l l a r y m e l t i n g p o i n t a p p a r a t u s and a r e u n c o r r e c t e d .  Analysis Elemental a n a l y s i s f o r carbon, hydrogen,  n i t r o g e n and  bromine  were determined by Mr.P.Borda of t h e " M i c r o a n a l y t i c a l L a b o r a t o r y , U.B.C.  -42-  Chromatography Column chromatography was performed using s i l i c a g e l (WoelmA c t i v i t y I ) purchased from ICN Pharmaceuticals.  The s i l i c a g e l was de-  a c t i v a t e d to A c t i v i t y IV before use. T h i n - l a y e r chromatography  (TLC) was performed using S i l i c a Gel  GF precoated p l a t e s (Analtech-Uniplate, 250u) .  Precoated 2000u t h i c k  p l a t e s were used f o r preparative s c a l e TLC.  3.2  CHEMICALS AND MATERIALS A l l chemicals were reagent grade unless otherwise s p e c i f i e d .  Hematoporphyrin IX dihydrochloride was obtained from Sigma Chemical Company.  Methanol Dry. methanol was obtained by d i s t i l l a t i o n f TOTH- magnesium  methoxide.  Dichloromethane Dry dichloromethane was obtained by d i s t i l l a t i o n from calcium  hydride.  3.3  PREPARATIONS  PROTOPORPHYRIN IX (30) Protoporphyrin IX (30) was prepared from hematoporphyrin IX d i h y d r o c h l o r i d e (31) according to the method of D i N e l l o quantitative yields.  ---  3 0  i n almost ~-  -43-  Absorption  Spectrum  A max  409  506  542  576  632  Pyridine:  Lit.  A max  409  506  542  576  63]  2 8  NMR  3.62 (16H,m) ( r i n g m e t h y l s , p r o p i o n i c 4.66  acid  (3H,t) ( p r o p i o n i c a c i d a C H ) , 6.3  2  y  (4H,dd)  2  ( v i n y l C H ) , 8.4 (2H,m) ( v i n y l CH), 10.19, 2  10.68  BCH )  10.34,  (4H, a l l s) (meso-H).  PROTOPORPHYRIN IX DME (32) Pfotoporphyin methanol / Melting  2 8  Absorption  u s i n g 5% s u l p h u r i c a c i d i n  i n a y i e l d o f 75%.  Point Lit.  IX (30) was e s t e r i f i e d  221* - from  CHC1 -methanol 3  231* - from C H C l - m e t h a n o l  2 8  3  Spectrum  A max  407  505  541  575  630  Lit.  A max  407  505  541  575  630  2 8  NMR  3.20 (4H,t) ( p r o p i o n a t e  gCH ), 2  3.48 (12H,t) ( r i n g  m e t h y l s ) , 3.66 (6H,s) ( m e t h y l e s t e r CH ) , 4.28 ( 4 H t ) 3  (propionate  s  a C H ) , 6.16 (4H,m) ( v i n y l C H ) , 8.10 2  2  (2H,m) ( v i n y l CH), 9.72, 9.78, 9.82, 9.88 (each l H , s ) (meso-H).  Mass Spectrum (%)  590 (100) ( p a r e n t ) , 517  531 (55) ( l o s s o f - C 0 C H ) , 2  3  (82) ( l o s s o f - C H C 0 C H ) , 444 (66) ( l o s s o f 2  -CH C0 CH 2  2  3  twice).  2  3  -44-  PHOTOPROTOPORPHYRIN DMEs (36a) and (36b)  Porphyrins (36a) and (36b) were prepared from (32) according to the method of D i N e l l o  3 0  i n a combined y i e l d of 72%.  The average y i e l d  of each pure isomer, a f t e r chromatography, was 22%. Isomer I (36b)  Melting Point Lit.  220-222* - from CH Cl -methanol 2  222-223* - from CHC1  3 7  Absorption Spectrum / '  Lit.  2  3 7  NMR  A max A max  388  3  -methanol  436  568  613  671  436  565  613  671  1.26 (3H,s) ( a l i p h a t i c CH ), 3.26 (4H,m) (propionate 3  6CH ), 3.36, 3.42, 3.62 (each 3H, s) ( r i n g methyls), 2  3.71 (6H,d) (methyl ester CH ), 4.28 (4H,m) 3  (propionate aCH ), 6.14 (2H,m) ( v i n y l CH ) , 6.54 2  2  (lH,d) ( o l e f i n i c CH), 7.60 (lH,m) ( v i n y l CH), 7.60, 8.42, 9.66, 9.76 (each IH, s) (meso-H), 9.97 (lH,s) (aldehyde H).  Mass Spectrum (%)  622 (100) (parent), 579 (48) (loss -CH CH0), 549 (19) 2  (loss of -CH C0 CH ). 2  2  3  Isomer I I (36a)  Melting Point L i t . 37 3 7  241-243 244  T  from CH Cl -methanol 2  - from CHC1  2  3  -methanol  Absorption  Spectrum Lit.  3 7  NMR  610  668  608  668  1.38 (3H,s) ( a l i p h a t i c C H ) , 2.72, 3.27, 3.39 ( e a c h 3H,s) 3  ( r i n g m e t h y l s ) , 3.27 (4H,m) ( p r o p i o n a t e B C H ) , 2  3.73  (6H,s) ( m e t h y l e s t e r C H ) , A.24 (4H,m) 3  (4H,m) ( p r o p i o n a t e a C H ) , 5.91 ( l H , d ) ( o l e f i n i c CH), 2  6.17 (2H,m) ( v i n y l C H ) , 7.93 (lH,m) ( v i n y l CH), 2  7.49,  8.19, 9.59, 9.62 (each l H , s ) (meso-H), 10.06  (lH,s) (aldehyde H).  Mass Spectrum  (%)  622 (100) ( p a r e n t ) , 579 (44) ( l o s s o f -CH CH0), 2  /  549  (15) ( l o s s o f - C H C 0 C H ) . 2  2  3  2 - f o r m y l - 4 - v i n y l - d e u t e r o p o r p h y r i n I X DME (33a) 2 - v i n y l - 4 - f o r m y l - d e u t e r o p o r p h y r i n I X DME (33b) The p r o c e d u r e used was a m o d i f i c a t i o n o f H a m i l t o n ' s method. * 1  0  To a s o l u t i o n o f (36a) o r (36b) (250mg) i n d r y d i c h l o r o m e t h a n e (125ml) was added sodium b o r o h y d r i d e (250mg) i n d r y m e t h a n o l  (10ml).  r e a c t i o n m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r one h o u r .  The  TLC a t t h e  end o f t h i s r e a c t i o n p e r i o d - m e t h y l a c e t a t e - h e p t a n e 1:1 v / v , R f , s t a r t i n g material-0.46, product-0.25, i n d i c a t e d the completion of the r e a c t i o n . E x c e s s sodium b o r o h y d r i d e was d e s t r o y e d by t h e d r o p w i s e a d d i t i o n o f acetic acid.  The r e a c t i o n m i x t u r e was washed w i t h w a t e r  (200ml x 2 ) ,  d r i e d o v e r sodium s u l p h a t e and t h e s o l v e n t removed i n v a c u o . The d a r k brown r e s i d u e was d i s s o l v e d i n d i o x a n e (125ml) and sodium p e r i o d a t e (400mg) i n h o t w a t e r (0.75ml) was added f o l l o w e d by concentrated sulphuric acid  (0.5ml).  The r e a c t i o n was m o n i t o r e d by TLC-  m e t h y l a c e t a t e - h e p t a n e 1:1 v / v , R f - s t a r t i n g m a t e r i a l - 0 . 2 5 , p r o d u c t - 0 . 7 5 .  -46-  Th e reaction was complete in one hour.  The reaction mixture was worked  up and the product purified according to the method of Hamilton. yield of the reaction was  The  75%.  Isomer (33a)  Melting Point  L i t . 37 Absorption Spectrum  275-278  2  2  278-279* - from CHC1 -methanol 3  A  L i t 37  NMR  - from CH Cl -petr6lei.m ether (30-60)  max max  3.26  418  518  558  583  642  420  518  559  584  642  (4H,t) (propionate BCH ), 3.52, 3.58, 3.70,3.76 2  (12H,s) (ring methyls), 3.66 4.35  CH ) 3  (4H,m) (propionate aCH ), 6.32 (2H,m) 2  (vinyl CH ), 8.21  (lH.dd) (vinyl CH), 9.78,  2  9.92,  (6H,s) (methyl ester  10.66  9.86,  (each lll,s) (meso-H), 11.32 (lH,s)  (aldehyde H). Mass Spectrum (%)  592 (100) (parent), 446 (18) (loss of CH C0 CH 2  519 (81) (loss of CH C0 CH ). 2  2  3  Isomer (33b)  Melting Point L i t 37 Absorption Spectrum L i t 37  223  - from CH Cl -pet.ether 2  2  225* - from CHC1 -methanol 3  A  max max  420  518  558  583  642  418  518  559  584  642  2  3  twice)  -47-  NMR  3.20 (4H,m) (propionate 6CH ), 3.30, 3.42, 3.48, 2  3.58  (each 3H,s) (ring methyls), 3.69 (6H,d) (methyl  ester CH ), 3  4.26 (4H,m) (propionate aCH ), 6.12 2  (2H,dd) ( v i n y l CH ), 7.84 (lH,q) ( v i n y l CH), 9.19, 2  9.52,  9.60, 10.28 (each lH,s) (meso-H), 11.06 (lH.s)  (aldehyde H). Mass Spectrum (%)  592 (100)(parent), 519 (57) (loss of CH C0 CH ), 2  2  3  446 (18) (loss of CH C0 CH twice). 2  2  3  Monp-acrylic acid-mono-vinyl-deuteroporphyrin IX DMEs (37) A solution of the mixed isomers of porphyrin 33 (65mg) was dissolved /  i n pyridine (50ml) and malonic acid (2g) i n pyridine (10ml) was added. The mixture was heated to 50* , with s t i r r i n g , and piperidine (0.75ml) added. The temperature  of the reaction mixture was then raised to 80* and  maintained there for f i v e hours. A f t e r cooling the reaction mixture to room temperature poured into d i l u t e hydrochloric a c i d (100ml, 5% v/v).  f  i t was  The porphyrin  was extracted into chloroform and the organic layer washed with water, dried over sodium sulphate and the solvent removed i n vacuo.  The residue was  chromatographed on a s i l i c a gel preparative layer plate with chloroformmethanol 20:1 v/v as eluent.  The slow moving band was removed from the  s i l i c a gel with a chloroform-methanol in vacuo.  solution  The residue was c r y s t a l l i s e d from  and the solvent removed dichloromethane-methanol  i n a y i e l d of 72%. Absorption Spectrum r  r  Lit. * 1  0  A max  418  512  552  581  637  X max  419  512  552  580  637  -48-  3.26 (4H,m) ( p r o p i o n a t e g C H ) , 3.52-3.66 (18H,m), 2  ( r i n g m e t h y l s , m e t h y l e s t e r C H ) , 4.38 (4H,m) ( p r o p i o n a t e 3  a C H ) , 6.24 (2H,m) ( v i n y l C H ) , 6.88 (lH.m) 2  2  (acrylic  a c i d BCH), 8.16 (lH,m) ( v i n y l CH), 9.18 (lH.m)  (acrylic  a c i d aCH), 9.84 (4H) (meso-H).  P h o t o - o x i d a t i o n o f m o n o - a c r y l i c a c i d - m o n o - v i n y l - d e u t e r o p o r p h y r i n I X DME (37) A s o l u t i o n o f p o r p h y r i n (37) (20mg) i n d i c h l o r o m e t h a n e c o n t a i n i n g 10% p y r i d i n e i n a 100ml m e a s u r i n g sunlight.  c y l i n d e r was exposed  to direct  A c o l o u r change from r e d t o green was o b s e r v e d a f t e r a day.  The r e a c t i o n was m o n i t o r e d by TLC u s i n g d i c h l o r o m e t h a n e - e t h e r 20:1 v / v . The c o m p l e t i o n o f the r e a c t i o n was gauged by t h e d i s a p p e a r a n c e o f t h e s t a r t i n g m a t e r i a l on TLC.  The r e a c t i o n m i x t u r e was s u c c e s s i v e l y washed  w i t h IN h y d r o c h l o r i c a c i d , 5% sodium b i c a r b o n a t e s o l u t i o n and f i n a l l y with water.  The o r g a n i c phase was d r i e d o v e r sodium s u l p h a t e and t h e  s o l v e n t removed i n vacuo. The t h i n - l a y e r chromatogram o f t h e r e a c t i o n m i x t u r e r e v e a l e d two p a i r s o f bands which c o u l d n o t be s e p a r a t e d by TLC.  Mono-formyl-mono-(l-hydroxyethyl)-deuteroporphyrin  IX DME  (38)  The p o r p h y r i n (38) was p r e p a r e d from (33) a c c o r d i n g t o t h e method o f Clezy.* 40%.  41  The r e a c t i o n time was f o u r h o u r s .  The y i e l d o f t h e r e a c t i o n was  -49-  Absorption Spectrum X_ max peak ratios  Mass Spectrum (%)  415  516  556  583  642  13.7  4.9  6.4  4.0  1.0  610 (11) (parent), 592 (100) (loss of H 0), 2  519 (55) (loss of H 0,-CH2C0 CH ). 2  2  3  Mono-formyl-mono-(2,2-dimethoxyethyl)-deuteroporphyrins  (39a) and (39b)  The porphyrins (39a) and (39b) were prepared from (33a) and (33b) respectively, according to the method of Kenner et.al.*  42  with slight  modifications. The modifications are; i)  The reaction mixture was stirred at room temperature for 45 minutes,  ii)  Porphyrins (39a) and (39b) were purified by column chromatography using dichloromethane-ether 20:1 v/v as eluent and crystallised from dichloromethane-pet.ether, in an average yield of 90%.  2-formyl-4-(2,2-dimethoxyethyl)-deuteroporphyrin IX DME (39a)  Melting Point  167-169  Analysis  Absorption Spectrum  C  N  calculated  67.87  6.47  8.56  found  67.76  6.27  8.34  Xmax loge  NMR  H  413  515  555  578  640  5.40  4.04  4.28  4.07  3.31  3.24 (4H,t) (propionate BCH ), 3.42 (6H,s) (acetal 2  CH ), 3.48,3.53,3.56, 3.59 (each 3H,s) (ring methyls), 3  3.66 (6H,d) (methyl ester CH ), 4.24 3  (4H,m)(propionate  -50-  aCH ),4.38 (2H,t) (acetal CH ), 5.05 (lH.t) (acetal 2  2  CH), 9.62, 9.85, 10.59 (4H,s) (meso-H), 11.22 (lH,s) (aldehyde H). Mass Spectrum (%)  654 (44)  (parent), 622 (100) (loss of CH 0H), 3  549 (44) (loss of CH3OH, -CH C0 CH ). 2  2  3  2-(2,2-dimethoxyethyl)-4-formyl-deuteroporphyrin IX DME (39b) Melting Point  174-176  Analysis C37Hi4 N 0 2  4  7  Absorption Spectrum  C  H  N  calc.  67.87  6.47  8.56  found  68.17  6.00  8.14  max loge  NMR  413  515  555  578  639  5.27  3.93  4.14  3.94  3.15  3.22 (4H,t) (propionate BCH ), 3.36 (6H,s) (acetal 2  CH ), 3.48, 3.52, 3.58, 3.61 (each 3H,s) (ring methyls), 2  3.64, 3.68 (each 3H,s) (methyl ester CH ), 4.06 3  (2H,d) (acetal CH ), 4.24, 4.32 (each 2H,two t) 2  (propionate ctCH ), 4.9 (lH,t) (acetal CH ), 9.60 2  (lH,s) 9.74 (2H,s) 10.6 (lH,s) (meso-H), 11.24 (lH,s) (aldehyde H) .  Mass Spectrum (%)  654 (15) (parent), 622 (100) (loss of CH 0H), 3  549 (44) (loss CH 0H, -CH C0 CH ). 3  2  2  3  -51-  Mono-methylacrylate-mono-(2,2-diroethoxyethyl)-deuteroporphyrin  IX DMEs  (40a) and (40b) The method used was based on the procedure of Hamilton.**  0  The porphyrins (40a) and (40b) were prepared from (39a) and (39b) respectively. Porphyrin (40b) was not characterised, but converted into porphyrin (41b). A solution of (39a) or (39b) (lOOmg) and malonic acid (3g) in pyridine (100ml) was heated to 50 . To this solution piperidine (0.2ml) was added and the temperature raised to 80 . The progress of the reaction was monitored by TLC using dichloromethane-methanol product 0.12.  20:1 v/v, Rf-starting material 0.88,  After five hours the reaction mixture was cooled and poured  into IN hydrochloric acid (400ml) and extracted into chloroform (200ml). The cnloroform layer was washed with 5% sodium bicarbonate solution and repeatedly with water.  The organic phase was dried over sodium sulphate  and the solvent evaporated under reduced pressure. The porphyrin residue was treated with 5% sulphuric acid in methanol (100ml) overnight in the cold (refrigerator).  The porphyrin  ester was extracted into chloroform and the organic phase washed f i r s t with 5% sodium bicarbonate solution and then with water;  i t was dried  over sodium sulphate and the solvent removed in vacuo. The residue was chromatographed on lOOg s i l i c a gel using chloroform-acetone 30:1 v/v as eluent. The desired porphyrin was eluted second.  The eluate collected was evaporated and the residue crystallised  from dichloromethane-pet.ether  (30-60) in a yield of 78mg or 72%.  2-methylacrylate-4-(2,2-dimethoxyethyl)-deuteroporphyrin Melting Point  192-194*  Analysis Ci oHi Ni,'08 t  l6  IX DME (40a)  C  H  N  calc.  67.59  6.52  7.88  found  67.26  6.25  7.75  -52-  Absorption Spectrum r  A  max  loge NMR  3.26  412  509  549  574  635  5.34  4.16  4.32  4.09  3.55  (4H,t) (propionate gCH ), 3.43  (6H,s) (acetal  2  CH ),  3.56  3  3.68  (6H,s) (methyl ester CH ), 3  (12H,s) ( r i n g methyls), 4.08  ester CH ), 3  5.06  4.34  3.56,3.63,  (3H,s) ( a c r y l i c  (6H,m) (propionate C H , acetal CH), a  (lH,t) (acetal BCH), 6.99  BCH),  9.22  (lH,d,J=17Hz),  9.94,  9.98  (4H,s) (meso-H).  2  a  (lH,d) ( a c r y l i c acid  ( a c r y l i c acid CH),  9.87,  a  710 (44) (parent), 678 (100) (loss of CH3OH),  Mass Spectrum (%)  605  (44) (loss of CH 0H, -Cr^CC^CHg). 3  2-methylacrylate-4-(2-hydroxyethyl)-deuteroporphyrin IX DME  (41a)  2-(2-hydroxyethyl)-4-methylacrylate-deuteroporphyrin IX DME  (41b)  The porphyrins (41a) and (41b) were prepared from (40a) and  (40b)  respectively. A solution of (40a) or (40b) (53mg) i n tetrahydrofuran (30ml) containing water (0.6ml) was refluxed with cone, hydrochloric acid (0.2ml) f o r f i v e minutes.  The reaction mixture was cooled and the porphyrin extracted  into dichloromethane  (30ml) containing pyridine (10ml).  The organic phase  was washed with water, dried over sodium sulphate and the solvent removed i n vacuo.  The residue i n dichloromethane  (30ml) at 0  was  treated with  a cold (0 ), solution of borohydride (250mg) i n methanol (10ml).  While  s t i r r i n g , the reaction mixture was allowed to a t t a i n room temperature. excess sodium borohydride was acetic acid.  The  then destroyed by the dropwise addition of  The organic phase was washed with water, dried over  sodium sulphate and the solvent evaporated under reduced pressure.  The  residue was re-esterified_with 5%_.sulphuric acid i n methanol and chromatographed usingdichloromethane-ether 20:1 v/v as eluent.  The major band was  collected,  -53-  the s o l v e n t e v a p o r a t e d and the r e s i d u e c r y s t a l l i s e d  from d i c h l o r o m e t h a n e  methanol i n a y i e l d o f 70%. 2 - m e t h y l a c r y l a t e - A - ( 2 - h y d r o x y e t h y l ) - d e u t e r o p o r p h y r i n I X DME ( A l a ) Analysis C  C  38 42 4°7 H  N  A b s o r p t i o n Spectrum  NMR  H  N  c.alc.  68.A5  6.35  8.AO  found  68.08  6.72  8.16  A max loge  A12  508  5A8  57A  636  5.AA  A.30  A.A2  A.22  3.70  3.28 (6H,m) ( p r o p i o n a t e BCH , h y d r o x y e t h y l 6 C H ) , 2  .  /  2  3.50, 3.95, A.60 (18H,m) ( r i n g m e t h y l s , p r o p i o n a t e e s t e r C H ) , A.08 (3H,s) ( a c r y l i c e s t e r C H ) , A.A2 3  3  (6H,m) ( p r o p i o n a t e a C H , h y d r o x y e t h y l a C H ) , 7.07, 2  2  (lH,d,J=17Hz) ( a c r y l i c gCH), 9.35 (lH.d,J=17Hz), ( a c r y l i c aCH), 10.OA (2H,s) 10.10 ( l H , s ) 10.14 ( I H (meso-H).  Mass  Spectrum (%)  666 (100) ( p a r e n t ) , 635 (26) ( l o s s o f - 0 C H ) , 3  607  (10) ( l o s s o f - C 0 C H ) , 593 (21) ( l o s s o f 2  3  -CH C0 CH ). 2  2  3  2 - ( 2 - h y d r o x y e t h y l ) - A - m e t h y l a c r y l a t e - d e u t e r o p o r p h y r i n I X DME ( A l b ) Analysis c  C  3 8 4 2 t4°7" 2 2 H  N  l  H  0  c a l c  -  found  6 7  - 3 5  67.66  H 6.A2 6.83  N 8.29 7.79  Absorption Spectrum  \ max log  e  410  507  548  573  635  4.86  3.79  3.88  3.71  3.22  2-methylacrylate-4-(2-bromoethyl)-deuteroporphyrin IX DME (42a) Porphyrin (42a) was prepared from (41a) by two methods. Method 1 This compound (42a) was prepared from (41a) according to the method of Kenner e t . a l .  3 9  The reaction was carried out at room temperature  the y i e l d of the reaction was  and  55%.  Method 2 ^  A solution of carbon tetrabromide (lOOmg) and triphenyl phosphine  (80mg) i n dry dichloromethane  (2ml) was added to a s t i r r e d s o l u t i o n of (41a)  (30mg) i n dry dichloromethane  (10ml) and refluxed for twenty  minutes.  The cooled reaction mixture was d i l u t e d with dichloromethane (20ml) and washed with water.  The organic phase was dried over sodium sulphate and  the solvent removed i n vacuo. me thane-ether 20:1 v/v. taken to dryness.  The residue was chromatographed using d i c h l o r o -  The major band eluted f i r s t was c o l l e c t e d and  The residue was c r y s t a l l i s e d  from dichloromethane-  pet.ether to give compound (42a) i n a y i e l d of 72%. The two Isomers of (42) (42a and 42b) were also separated on a preparative scale using dichloromethane-ether 30:1 v/v as eluent.  The isomer  eluted f i r s t was i d e n t i f i e d with 2-methylacrylate-4-(2-bromoethyl)-deuteroporphyrin IX DME. Melting Point  The y i e l d for each isomer was 196-200*  Analysis C38 41 4°6 H  N  C B r  15%.  c a l c  -  found  62.55 62.50  H  N  Br  5.66  7.68  10.95  5.54  7.44  10.68  -55Absorption Spectrum  \ max log E  NMR  413  508  548  576  635  4.67  3.52  3.62  3.40  2.96  3.24 (4H,m) (propionate 6CH ), 3.50 (12H,m) ( r i n g 2  methyls), 3.64 (3H,s) 3.66 (3H,s) (propionate ester CH ), 3  4.08 (5H,m) ( a c r y l i c e s t e r CH , bromoethyl 3  6CH2),  4.34 (6H,m) (propionate otCH» bromoethyl <*CH) , 2  2  6.93 (lH,d,J=17Hz) ( a c r y l i c BCH), 9.12 (lH,d,J=17Hz) ( a c r y l i c aCH), 9.74 (2H,s) 9.80 (lH,s) 9.92 (lH.s) (meso-H).  Mass -Spectrum (%)  728/730 (50/50) (parent, 655/657 (21/21) ( l o s s of -CH C0 CH ), 648 (46) ( l o s s of HBr), 675 (37) 2  2  3  ( l o s s of HBr, -CH C0 CH ). 2  2  3  2-methylacrylate-4-(2-cyanoethyl)-deuteroporphyrin IX DME (43) To a s o l u t i o n of (42) (lOmg) i n N-methylpyrrolidone  (10ml)  was added , w i t h s t i r r i n g , a s o l u t i o n of sodium cyanide (lOmg) i n N-methylpyrrolidone temperature  (5ml).  The r e a c t i o n mixture was s t i r r e d at room  and the progress of the r e a c t i o n was monitored by TLC using  dichloromethane-ether  20:1 v/v; R f - s t a r t i n g m a t e r i a l 0.58, product 0.29,  On completion of the r e a c t i o n , the r e a c t i o n mixture was d i l u t e d w i t h dichloromethane.  The organic phase was repeatedly washed with water, dried  over sodium sulphate and taken to dryness. on s i l i c a g e l using dichloromethane-ether  The residue was chromatographed 20:1 v/v as eluent.  The major  band was c o l l e c t e d , the solvent evaporated and the residue c r y s t a l l i s e d from dichloromethane-pet.ether  i n a y i e l d of 70%.  -56Melting Point  242-244'  A b s o r p t i o n Spectrum  NMR  A max loge  412  507  547  576  634  4.59  3.52  3.57  3.38  2.97  3.26  (6H,m) ( p r o p i o n a t e BCH , c y a n o e t h y l  3.60  (9H) 3.67 (9H) ( r i n g m e t h y l s ,  6CH ),  2  2  propionate  ester  C H ) , 4.09 (3H,s) ( a c r y l i c e s t e r C H ) , 4.36 3  3  (6H,m) ( p r o p i o n a t e aCH, c y a n o e t h y l a C H ) , 7.04 2  (lH,d,J=17Hz) ( a c r y l i c a c i d BCH), 9.28 (lH,d,J=17Hz) ( a c r y l i c e s t e r aCH), 9.81 ( I H ) 9.98 (IH) 10.02 (2H) (meso-H).  Mass Spectrum (%)  675 (100) ( p a r e n t ) , 602 (30) ( l o s s o f C H C 0 C H ) , 2  2  3  635 (50) ( l o s s o f CH CN) . 2  Porphyrin  S4ll Compound (43) (lOmg) was d i s s o l v e d i n methanol (10ml)  s a t u r a t e d w i t h hydrogen c h l o r i d e and l e f t o v e r n i g h t i n t h e r e f r i g e r a t o r . The p o r p h y r i n was e x t r a c t e d i n t o d i c h l o r o m e t h a n e  and t h e o r g a n i c l a y e r  washed w i t h 5% sodium b i c a r b o n a t e s o l u t i o n and w a t e r . was removed  A f t e r the solvent  i n vacuo t h e r e s i d u e was chromatographed w i t h  e t h e r 20:1 v / v as e l u e n t .  The f i r s t e l u t e d compound was c o l l e c t e d , t h e  s o l v e n t removed i n vacuo and t h e r e s i d u e c r y s t a l l i s e d from pet.ether, i  n  Melting Point Lit.  4  dichloromethane-  dichloromethane  almost q u a n t i t a t i v e y i e l d s .  237-239 236-238*  - from  dichloromethane-pet.ether  -57A b s o r p t i o n Spectrum Lit.*  max  4  max  NMR  413  509  549  576  635  413  510  548  577  635  3.26 (6H,m) ( p r o p i o n a t e g C H ) , 3.48 (9H) 3.60 (3H) 2  3.64 (3H) 3.68 (6H) 4.08 (3H) ( r i n g m e t h y l s ,  propionate  e s t e r C H ) , 4.38 (6H,m) ( p r o p i o n a t e ctCH ), 7.04 3  2  (lH,d,J=17Hz) ( a c r y l i c BCH), 9.30 (lH,d,J=17Hz) ( a c r y l i c aCH), 9.96 (IH) 10.00 (2H) 10.04 (IH) (meso-H) Mass Spectrum (%)  708 (100) ( p a r e n t ) , 677 (7) ( l o s s o f - 0 C H ) , 649 3  (14) Melting Point of  (loss of -C0 CH ), 2  (of a d m i x t u r e )  3  635 (34) ( l o s s o f - C H C 0 C H ) . 2  2  3  235-240*  2-(2-acetoxyethyl)-4-methylacrylate-deuteroporphyrin  I X DME (44)  Compound (41b)(8mg) was d i s s o l v e d i n p y r i d i n e (10ml) c o n t a i n i n g a c e t i c anhydride  (1ml) and l e f t o v e r n i g h t .  The r e a c t i o n m i x t u r e was  poured i n t o 1% h y d r o c h l o r i c a c i d (50ml) and t h e p o r p h y r i n e x t r a c t e d i n t o ether.  The o r g a n i c laye.r was washed w i t h 5% sodium b i c a r b o n a t e  f o l l o w e d by w a t e r . i n vacuo.  solution  A f t e r d r y i n g over sodium s u l p h a t e t h e s o l v e n t was removed  The r e s i d u e was p u r i f i e d by chromatography u s i n g  dichloromethane-  e t h e r 20:1 v / v . The major band was c o l l e c t e d , t h e s o l v e n t removed and the r e s i d u e c r y s t a l l i s e d from d i c h l o r o m e t h a n e - p e t . e t h e r . A b s o r p t i o n Spectrum peak  NMR  A  max  ratios  i n a y i e l d o f 72%.  411  508  548  575  635  47.4  3.4  4.8  3.02  1.0  2.10 (3H,s) ( a c e t o x y e t h y l C H ) , 3.16 (4H,m) ( p r o p i o n a t e 3  6 C H ) , 3.56, 3.62. 3.64 (18H) ( r i n g m e t h y l s , 2  propionate  C H ) , 4.90 (2H,m) ( a c e t o x y e t h y l g C H ) , 4.02 (3H,s) 3  2  ( a c r y l i c e s t e r C H ) , 4.34 (6H,m) ( p r o p i o n a t e 3  aCH , 2  -58-  acetoxyethyl aCH ), 7.02 (lH.d,J=17Hz), ( a c r y l i c 2  BCH) , 9.A3 (lH,d,J=17Hz) , ( a c r y l i c ctCH) , 10.12 (2H) , 10.20  (1H),10.2A (IH) (meso-H).  2-(2-acetoxyethyl)-A-(2-methoxycarbonylethyl)-deuteroporphyrin Compound (AA)  (5mg) i n tetrahydrofuran  IX DME(A5)  (5ml) was hydrogenated  over 10% palladium-charcoal at room temperature u n t i l the solution turned colourless.  The catalyst was f i l t e r e d o f f and while s t i r r i n g a i r was  bubbled through the solution u n t i l the v i s i b l e spectrum indicated the complete oxidation of the porphyrinogen.  The solvent was removed i n vacuo  and the residue chromatographed using dichloromethane-ether eluent.  The major band was c o l l e c t e d , the solvent evaporated  pressure and the residue c r y s t a l l i s e d give compound  under reduced  from dichloromethane-pet.ether  to  (45) i n a y i e l d of 65%.  Absorption Spectrum Lit.  NMR  20:1 v/v as  4  X wax X max  399  500  531  567  621  A00  502  532  567  621  2.08 (3H,s) (acetoxyethyl CH ), 3.20 (6H,m) 3  (propionate BCH ), 3.67, 3.69 (21H,d) ( r i n g methyls, 2  methyl ester CH ), 3  A.2A (8H,m) (propionate aCH , 2  acetoxyethyl aCH ), A.88 (2H,t) (acetoxyethyl BCH ), 2  10.10, 10.12 (AH,d) (meso-H),  2  -592-hydroxymethyl-4-vinyl-deuteroporphyrin IX DME  (46a)  2-vinyl-4-hydroxymethyl-deuteroporphyrin IX DME  (46b)  A solution of sodium borohydride (50mg) in dry methanol (2ml) was added to a stirred solution of the mixed isomers of porphyrin (33) (50mg) in dry dichloromethane (25ml).  The solution was stirred for one hour.  The  reaction was monitored by TLC using dichloromethane-ether 20:1 v/v, Rf-starting material 0.68, product 0.05.  On completion of the reaction excess borohydride  was destroyed by the dropwise addition of acetic acid.  The organic phase  was washed with water, dried over sodium sulphate and the solvent removed in vacuo.  The residue, after chromatographic purification using  dichloromethane-ether 20:1 v/v, was crystallised from dichloromethanepet.e^her in a yield of 88%. The mixed isomers of (46) were separated by chromatography using dichloromethane-ether 10:1 v/v as eluent. The isomer eluted f i r s t was identified with 2-vinyl-4-hydroxymethyl-deuteroporphyrin IX DME yield for each pure isomer was  (46b) .  The  30%.  Compound (46a)  Analysis C  35 38 4°5 H  N  Absorption Spectrum  C  H  N  calc.  70.69  6.44  9.42  found  70.78  6.55  9.10  max log  NMR  e  404  502  537  573  628  5.14  3.95  3.82  3.63  3.45  3.24 (4H,t) (propionate gCH ), 3.67 (6H,s) (methyl 2  ester CH ), 3.53 (12H,t) (ring methyls), 4.32 3  (propionate CH ), &  2  6.24  (2H,m) (vinyl CH )» 2  (2H,s)(hydroxymethyl CH ), 8.19 2  (4H,t)  5.86  (lH,dd) (vinyl CH) ,  -60-  9.95  Compound  (4H,s) (meso-H)  (46b)  Analysis C 37^0^06  Absorption  Spectrum  C  H  N  calc.  70.69  6.44  9.10  found  71.04  6.27  9.66  A  max  loge NMR  3.24  403  502  536  572  628  5.19  4.12  4.00  3.83  3.65  (4H,t) ( p r o p i o n a t e  m e t h y l s ) , 3.66 (propionate  g C H ) , 3.50 2  (6H,s) ( e s t e r CH ) , 4.30  BCH ), 5.80  (2H,s) (hydroxymethyl  2  (2H,m) ( v i n y l C H ) ,  9.92  (4H,q) (meso-H).  8.19  2  (lH.dd) ( v i n y l  2-acetoxymethyl-4-vinyl-deuteroporphyrin  IX DME  (47a)  2-vinyl-4-acetoxymethyl-deuteroporphyrin  IX DME  (47b)  and  (47b) were prepared  from (46a)  r e s p e c t i v e l y , a c c o r d i n g to the method g i v e n i n r e f e r e n c e 28. of  the r e a c t i o n was  Compound  The  2  CH),  (46b) yield  75%.  205-207  Analysis  C  N  3  and  CH ),  (47a)  Melting Point  C 7Hi40 4°  (ring  (4H,t),  3  6.24  Compounds (47a)  (9H,s)  6  H  N  calc.  69.79  6.33  8.80  found  69.27  6.21  8.49  -61-  Absorption  Spectrum  NMR  \ max  404  502  537  573  627  loge  5.29  4.17  4.02  3.85  3.68  3.26  (4H,t) ( p r o p i o n a t e  gCH ), 3.56 (3H,s) ( a c e t a t e C H ) ,  3.60  (6H,s) 3.67 (12H,s) ( r i n g m e t h y l s , m e t h y l e s t e r  2  2  C H ) , 4.38 (4H,m) ( p r o p i o n a t e 3  a  C H ) , 6.18 (2H,m) 2  ( v i n y l C H ) , 6.48 (2H,s) (acetoxymethyl C H ) , 8.20 2  2  (lH.dd) ( v i n y l CH ) , 9.98, 10.05,  10.08 (4H,s)  (meso-H).  Mass Spectrum (%)  636  (100) ( p a r e n t  )  Comp-ound (47b)  Melting  Point  165-168  Analysis C  3 7  H  4 0  N  C  H ° 6  Absorption  Spectrum  H  N  calc.  69.79  6.33  8.80  found  69.85  6.37  8.66  A  max  405  503  537  573  628  5.34  4.42  4.26  4.10  3.91  loge NMR  3.26  (4H,t) (propionate C H ) , 3.58 (9H,m) 3.68 (12 H,s) a  ( r i n g methyls.methyl  2  ester CH ,acetate CH ), 4.36 3  3  (4H,t) (propionate cxCH ), 6.28(2H,m) ( v i n y l CH ), 2  6.44 (2H,s) (acetoxymethyl  2  CH ), 8.25 (lH,q) 2  ( v i n y l CH), 10.00 (4H,q) (meso-H). Mass Spectrum (°A  636  (100)  (parent).  -62-  BIBLIOGRAPHY 1.  J .WALDENSTROM, Z.Physio.Chem.,  I I I , (1936).  2.  J.M.FRENCH and E.THONGER, C l i n . S c i . , J31,  3.  J.M.FRENCH, D.C.NICHOLSON and C.RIMMINGTON, Biochem.J.,  3 3 7  »  (1966). 120,393,  (1970).  4.  P.W.COUCH,D.E.GAMES and A.H.JACKSON, J.Chem.Soc.Perkin I , 2492,  5.  (1976).  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