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Syntheses of affinity ligands and monoazaporphyrins Singh, Jai Prakash 1987

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SYNTHESES OF AFFINITY LIGANDS AND MONOAZAPORPHYRINS by JAI PRAKASH SINGH A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA MARCH 1987 ® J a i Prakash Singh, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of CHEMISTRY The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date APRIL 20, 1988 i i ABSTRACT Heme and heme proteins are ubiquitous i n nature and play many v a r i e d and important b i o l o g i c a l functions. The b i o l o g i c a l p r o p e r t i e s of the heme pro t e i n s are dependent on the i n t e r a c t i o n between the metal and the porphyrin where each i s dependent on the other to carry out s p e c i f i c f u n c t i o n s . This t h e s i s describes the syntheses of a f f i n i t y ligands and mono-azaporphyrins. The synthesis of the l i g a n d was based on a protoporphy-r i n - I X d e r i v a t i v e s u b s t i t u t e d on e i t h e r the 2- or 4 - v i n y l group. The two isomeric a f f i n i t y l i g a n d s , 73 and 74 (2- or 4-substituted) were synthesized from protoporphyrin-IX d i - t e r t - b u t y l ester 106b by conver-s i o n of the v i n y l groups to monoformyl d e r i v a t i v e s 122b (or 123b) v i a the intermediacy of photoprotoporphyrin-IX d e r i v a t i v e s . The spacer chain C02R3 C02R3 105 R1=R2=-CH=CH2, R3=H 106b R1=R2=-CH=CH2, R 3=tBu 122b R—CH-CH 2, R2=-CH0, R ^ t B u 123b R =-CH0, R2=-Ctt=CH2, R 3=tBu 132 R ~CH-CH 2, R2=-CH=CHC02H, R 3=tBu 133 R—CH«CHC0 2H, R2=-CH=CH2, R 3=tBu 73 Rx--CH=CH2, R2=-CH=CHC0NH(CH2)3NH2, R 3=tBu 74 Rj^—CH=CHCONH(CH2)3NH2, R2=-CH=CH2> R 3=tBu i i i using a formyl group was extended by Knoevenagel condensation with malonic acid to give monoacrylic acid derivatives 132 (or 133). This chain was further extended by l i n k i n g an a l i p h a t i c diamine (1,3-diamino-propane) to the monoacrylic acid derivatives 132 (or 133) through an acid chloride mediated amide linkage. The Fe(III) and Co(II) complexes of the a f f i n i t y ligand (73 + 74) were prepared and the t e r t i a r y butyl groups were removed by treatment with t r i f l u o r o a c e t i c acid at room temperature to give 71 and 72. An improved synthesis of monoazaporphyrins (5-aza) (see p.iv) i s described i n this work (section 3.4). Our approach consists of constructing 1,19-dibromo-l,19-dideoxybiladiene-ac dihydrobromides which were subsequently cyclized, to give corresponding monoazaporphyrins i n high y i e l d s , using dibenzo-18-crown-6 as a phase transfer agent for the transfer of an azide ion. i v 7 8 R„ " V " R 6 ~ R 7 ~ " C 2 H 5 - R 3 - R 5 - R 8 — C H 3 8 8 R..>-R 0=R 0»R /=R,-=R 0=-CH, 1 Z J 4 J o C 2 1 6 R , 2 1 7 R„ 1 0 0 2 1 8 R„ R, R-, •V- C2H5 -+ R„=-CH„ "V"C2H5 = R 3 = R 5 ^ R 8 = - C H 3 = R 7 = - C H 2 C H 2 C 0 2 M e - R 8 - C 2 H 5 = R 3 = R 5 = R y = - C H 3 = R , = - C H o C H . 0 C 0 P h = R 8 = - C H 2 C H 2 C 0 2 M e 8 2 R , = ; R 3 = - C 2 H 5 R,= :R. ^ R — R ~ — CH ~ 4 5 8 3 R , = : R = - C H 2 C H 2 C 0 2 M e 2 1 9 R, - R 3 - R 5 - R Y — C 2 H 5 R o = R 4 = R 6 = R 8 = " C H 3 V TABLE OF CONTENTS Page ABSTRACT 1 1 TABLE OF CONTENTS v LIST OF FIGURES v i i i LIST OF TABLES x i i LIST OF ABBREVIATIONS x i i i ACKNOWLEDGEMENTS xv DEDICATION xvi 1. INTRODUCTION 1 1.1 PORPHYRIN MACROCYCLE - A GENERAL SURVEY 2 1.2 ELECTRONIC SPECTRA 4 1.2.1 Etio-type Spectra 5 1.2.2 Rhodo-type Spectra 6 1.2.3 Oxorhodo-type Spectra 7 1.2.4 Phyllo-type Spectra 7 1.3 NOMENCLATURE ' 7 1.3.1 Pyrroles 9 1.3.2 Dipyrroles 11 1.3.3 Tetrapyrroles 12 1.3.4 Porphyrins 14 v i 1.4 OCCURRENCE IN BIOLOGICAL SYSTEMS 15 1.5 HEME OXYGENASE AND HEME CATABOLISM 19 1.6 PURIFICATION OF HEME OXYGENASE 37 1.7 NATURE AND AIM OF THE PRESENT WORK 38 2. RESULTS AND DISCUSSION 50 PART 1: SYNTHESIS OF AN AFFINITY LIGAND 51 2.1 AFFINITY CHROMATOGRAPHY AND AFFINITY LIGAND . . . . 52 2.2 SYNTHETIC APPROACH 53 2.3 REACTIONS AT THE PORPHYRIN PERIPHERY FOR INTRODUCTION OF A SPACER ARM 57 2.4 SYNTHESIS OF FORMYL PORPHYRINS 58 2.4.1 Vilsmeier Formylation Reaction 58 2.4.2 Reactions at the Vinyl Groups of Protoporphyrin 61 2.5 EXTENSION OF THE SPACER ARM 68 2.6 FURTHER EXTENSION OF THE SPACER ARM 69 2.7 METALLATION OF THE AFFINITY LIGAND 73 2.8 REMOVAL OF THE TERTIARY BUTYL GROUPS 74 PART 2: SYNTHESIS OF AZAPORPHYRINS 76 3.1 PYRROLES 77 3.1.1 Synthesis of Monopyrroles 79 3.1.2. Preparation of Synthetically Useful Pyrroles v i a Transformations of /9-Substituents . . '. 82 3.1.3 Preparation of Synthetically Useful Pyrroles v i a Transformations of a-Substituents 84 v i i 3.2 SYNTHESIS OF DIPYRROMETHANES AND BILADIENES-ac . . . 92 3.2.1 Bromination of 1,19-dideoxybiladiene-ac dihydrobromides 98 3.3 SYNTHESIS OF DIPYRROMETHENES 100 3.3.1 Bromination of dipyrromethenes 103 3.3.2 Synthesis of 1,19-dibromo-l,19-dideoxy-biladienes-ac from dipyrromethenes . . . . 104 3.4 SYNTHESIS OF MONOAZAPORPHYRINS FROM 1,19-DIBROMO-1,19-DIDEOXYBILADIENE-ac SALTS 104 3.5 MECHANISM FOR THE CYCLIZATION OF A 1,19-DIBROMO-1,19-DIDEOXYBILADIENE-ac DIHYDROBROMIDE TO AZAPORPHYRIN 110 3. SUMMARY AND CONCLUSIONS 112 4. EXPERIMENTAL 115 4.1 GENERAL METHODS 116 4.2 SYNTHESIS OF AFFINITY LIGANDS 119 4.3 SYNTHESIS OF MONOPYRROLES 143 4.4 SYNTHESIS OF DIPYRROMETHANES 176 4.5 SYNTHESIS OF DIPYRROMETHENES 183 4.6 SYNTHESIS OF BILADIENES-ac 191 4.7 SYNTHESIS OF MONOAZAPORPHYRINS 209 5. SPECTRAL DATA 223 5.1 1H NMR SPECTRA 224 5.2 ELECTRONIC ABSORPTION SPECTRA OF PORPHYRINS . . . . 242 REFERENCES 268 v i i i LIST OF FIGURES Figure Page 1 Typical absorption spectra (Soret omitted) of porphyrins i n chloroform 6 2 -A schematic of the Etioporphyrin series 8 3 Structures of the p r i n c i p a l t e t r a p y r r o l i c pigments i n heme catabolism 20 4 Possible chemical sequence of heme degradation catalyzed by the heme oxygenase system 25 5 Coupled oxidation of hemin to the four b i l i v e r d i n isomers of the IX series 27 6 Formation of b i l i v e r d i n from verdohemochrome. The side chains are omitted for c l a r i t y 29 7 Formation of pyridine verdohemochrome-IXa from a-oxyprotohemin-IX 34 8 Formation of bil i v e r d i n - I X a from reconstituted apomyoglobin-a-oxyprotohemin-IX complex 36 9 The p r i n c i p l e of a f f i n i t y chromatography 52 10 Synthesis of Knorr's pyrrole 78 11 Synthesis of monopyrrole 142 by v a r i a t i o n of the Knorr reaction 80 12 Synthesis of monopyrrole 143 81 13 Synthesis of synthetically useful monopyrroles 169 and 177 v i a transforma-tions of a-substituents 86 14 Synthesis of the synthetically useful pyrrole 181 v i a transformation of a-substituents 90 15 Synthesis of acetoxymethylpyrroles 91 16 Synthesis of symmetrical dipyrromethanes . . . . 93 ix 17 The synthesis of dipyrromethanes from a-acetoxymethylpyrroles 94 18 Synthesis of 5,5'-dicarboxydipyrromethanes . . . 95 19 Synthesis of 1,19-dideoxybiladiene-ac dihydrobromides 191 and 192 96 20 Synthesis of 1,19-dideoxybiladiene-ac dihydrobromides 193, 194 and 196 97 21 Brominaton of 1,19-dideoxybiladiene-ac dihydrobromides 99 22 Synthesis of unsymmetrical dipyrromethenes . . . 102 23 Bromination of dipyrromethenes 208 and 210 . . . 103 24 Synthesis of unsymmetrical 1,19-dibromo-dideoxybiladiene-ac dihydrobromide s a l t s 81 and 215 105 25 Synthesis of monoazaporphyrins from 1,19-dibromo-l,19-dideoxybiladiene-ac dihydrobromide 109 26 Mechanism of porphyrin c y c l i z a t i o n I l l 27 1H NMR Spectrum (400 MHz) of 106b i n CDC13 . . 224 28 1H NMR Spectrum (400 MHz) of 110b i n CDC13 . . 225 29 1H NMR Spectrum (400 MHz) of 126b i n CDC13 . . 226 30 XH NMR Spectrum (400 MHz) of 127b i n CDC13 . . 227 31 1H NMR Spectrum (400 MHz) of 122b i n CDC13 . . 228 32 1H NMR Spectrum (400 MHz) of 123b i n CDC13 . . 229 33 1H NMR Spectrum (400 MHz) of 134 i n CDC13 . . 230 34 •'"H NMR Spectrum (400 MHz) of 135 i n CDC13 . . 231 35 1H NMR Spectrum (400 MHz) of 73 i n CDC13 . . 232 36 1H NMR Spectrum (400 MHz) of 74 i n CDC1 . . 233 X 37 lU NMR Spectrum (400 MHz) of 78 in CDC13 . . 234 38 1H NMR Spectrum (400 MHz) of 88 in CDC13/TFA . . 235 39 XH NMR Spectrum (400 MHz) of 216 in CDC13/TFA . . 236 40 1H NMR Spectrum (400 MHz) of 100 in CDC13 . . 237 41 1H NMR Spectrum (400 MHz) of 219 in CDC13 . . 238 42 1H NMR Spectrum (400 MHz) of 217 in CDC13 . . 239 43 1H NMR Spectrum (400 MHz) of 82 in CDC13 . . 240 44 1H NMR Spectrum (400 MHz) of 218 in CDC13 . . 241 45 Electronic Absorption Spectrum of 106b . . 242 46 Electronic Absorption Spectrum of 110b . . 243 47 Electronic Absorption Spectrum of 126b . . 244 48 Electronic Absorption Spectrum of 127b . . 245 49 Electronic Absorption Spectrum of 122b . . 246 50 Electronic Absorption Spectrum of 123b . . 247 51 Electronic Absorption Spectrum of 132 . . 248 52 Electronic Absorption Spectrum of 133 . . 249 53 Electronic Absorption Spectrum of 134 . . 250 54 Electronic Absorption Spectrum of 135 . . 251 55 Electronic Absorption Spectrum of 73 . . 252 56 Electronic Absorption Spectrum of 74 . . 253 57 Electronic Absorption Spectrum of 138 . . 254 58 Electronic Absorption Spectrum of 139 . . 255 59 Electronic Absorption Spectrum (Pyridine Hemochrome) of 140 . . 256 60 Electronic Absorption Spectrum of 141 . . 257 x i 61 Electronic Absorption Spectrum (Pyridine Hemochrome) of 71 . . 258 62 Electronic Absorption Spectrum of 72 . . 259 63 Electronic Absorption Spectrum of 78 . . 260 64 Electronic Absorption Spectrum of 88 . . 261 65 Electronic Absorption Spectrum of 216 . . 262 66 Electronic Absorption Spectrum of 100 . . 263 67 Electronic Absorption Spectrum of 219 . . 264 68 Electronic Absorption Spectrum of 217 . . 265 69 Electronic Absorption Spectrum of 82 . . 266 70 Electronic Absorption Spectrum of 218 . . 267 x i i LIST OF TABLES Table Page 1 Some T r i v i a l Names i n the Fischer Nomenclature 10 2 Various Reaction Conditions Attempted for the Cyclization of 199 to monoaza-porphyrin 78 107 x i i i LIST OF ABBREVIATIONS "*"H NMR = proton nuclear magnetic resonance TMS — tetramethylsilane MHz = mega hertz tic = thin layer chromatography FAB - fast atom bombardment DMF = dimethylformamide DMSO = dimethylsulfoxide TFA = trifluoroacetic acid THF = tetrahydrofuran Et^N = triethylamine Aq. = aqueous MP — melting point Pyr = pyrrole Py = pyridine Lit. = literature tBu = tertiary butyl v/v = volume/volume mmole = millimole mg = milligram g = gram mL = m i l l i l i t e r cm — centimeter mm = millimeter nm = nanometer xiv Ph = phenyl CE = crown ether Me — methyl Et - ethyl Abbreviations i n NMR Assignments s = singlet m = multiplet d = doublet bs = broad singlet t = t r i p l e t bd = broad doublet q = quartet ppm = parts per m i l l i o n X V ACKNOWLEDGEMENT I wish to express my sincere thanks to Professor David Dolphin for his guidance, suggestions, encouragement and patience throughout the course of my research and the preparation of this thesis. I am indebted to Professor B.R. James and Professor L.G. Harrison for their valuable suggestions in the completion of this thesis. I also wish to thank Dr. Tilak Wijesekera and Mr. Nimal Rajapakse for reading the manuscript and many helpful discussions. I would like to take this opportunity to thank a l l the members of Professor Dolphin's research group, past and present, for making my stay at U.B.C. a pleasant one. I also wish to thank sincerely Drs. Ashok Singh and Mahatam Singh for taking the time and sharing their experi-ences and knowledge in synthetic chemistry. Thanks should also be extended to the staff of the Mass Spectro-metry laboratory, and particularly the staff of the U.B.C. NMR Center who provided valuable assistance in recording some of the spectra. Financial support in the form of a teaching assistantship is gratefully acknowledged. x v i To my wife A n j a l i and my parents for their love, understanding and encouragement - 1 -CHAPTER 1 INTRODUCTION - 2 -1.1 PORPHYRIN MACROCYCLE - A GENERAL SURVEY The unsubstituted t e t r a p y r r o l i c nucleus joined by four methene bridges i s known as "porphin" 1. Porphyrins are formally derived from porphin 1 by substitution of some or a l l of the peripheral positions with a variety of side-chains. The high s t a b i l i t y of the porphyrin macrocycle i s endowed by the presence of eleven double bonds giving 22 7 r-electrons on the carbon skeleton. Out of eleven double bonds, nine are i n dir e c t conjugation 1 giving an 18 electron, aromatically delocalized 7r-system. The remaining two double bonds are cross conjugated and can be reduced as i n the chlorins 2 or bacteriochlorins 3 without the loss of aromaticity.^ 1 2 3 PORPHIN CHLORIN BACTERIOCHLORIN - 3 -The aromatic character of porphyrin compounds has been confirmed by 2 the thermal s t a b i l i t y of the macrocycle. A number of other physical methods are currently being used to measure the extent of 7 r-electron d e r e a l i z a t i o n , and the aromatic character. These include nuclear magnetic resonance spectroscopy, UV-visible spectroscopy and X-ray crystallography. As an aromatic system, porphyrins and p a r t i c u l a r l y metalloporphy-3 4 rins undergo a wide variety of e l e c t r o p h i l i c substitution reactions. ' The aromaticity of the porphyrin macrocycle has been extensively inves-tigated using nuclear magnetic resonance spectroscopy. The shielded internal NH protons i n porphyrins appear at r e l a t i v e l y high f i e l d (5 ~ -2 to -5) whereas the meso protons (outer), being deshielded by the aromatic r i n g current, appear at about 6 ~ 10 ppm. Perhaps the most important feature of the porphyrin macrocycle i s the r e l a t i v e ease with which metal complexes are formed with most metals 1 3 5 i n the periodic table. ' ' During the course of a metal complex formation, the metal ion not only coordinates with the two lone pairs of electrons on the pyrrolenine (-N=) nitrogen atoms but also replaces the two hydrogen atoms on the pyrrole nitrogen atoms; thus, two more lone pairs are donated from the porphinato-ligand to the valence s h e l l of the metal ion which occupies the center of the porphyrin hole. This causes a major change i n the electronic properties of the central metal ion.'' However, the e f f e c t i s synergistic and the electronegativity of the metal w i l l exert an influence on the electron density of the porphyrin JT-system and on the r e a c t i v i t y of the ring periphery. The combination - 4 -of these two major factors results i n important oxidation/reduction properties of metalloporphyrins where changes i n the central metal or external substituents cause changes i n the oxidation and reduction potentials of the ring, this property gives r i s e to the use of metallo-porphyrins i n b i o l o g i c a l and non-biological systems as electron transfer 6 agents. 1.2 ELECTRONIC SPECTRA Due to a variety of colours possessed by the porphyrin macrocycle and related compounds, i t i s easy to study their reaction chemistry i n the laboratory. This gives a qualitative measure of the progress of the reaction, i . e . , the electronic absorption spectrum varies with the change i n the nature of the chromophoric system and with the various substituents upon i t . The major feature of these spectra i s the presence of a very intense absorption band i n the region of 400 nm, the so-called "Soret" 7 8 band, f i r s t discovered by Soret i n hemoglobin and observed by Gamgee in porphyrins. The "Soret" i s the most intense band i n the o p t i c a l spectra of porphyrins and their derivatives, and possesses molar extinc-tion c o e f f i c i e n t s sometimes as large as 400,000. The intense "Soret" band i s c h a r a c t e r i s t i c of the 18 7r-electron delocalized pathway present i n the porphyrin nucleus. The rupture of the macrocycle results i n the disappearance of the "Soret" band. - 5 -The intensity of the Soret band i s weaker i n chlorins and metallo-chlorins. Besides the "Soret" band, there are four additional bands i n the 500 to 700 run region of the v i s i b l e spectrum. The r e l a t i v e intensi-ties of these bands vary with variations i n the peripheral substituents. The complexation of porphyrins with metal ions usually results i n an increase i n intensity of the Soret band, and s i m p l i f i c a t i o n of the 3 other four v i s i b l e bands to two v i s i b l e bands, namely a and /9 bands. These can vary i n intensity and wavelengths depending upon the metal ion involved. Similarly, the two band spectrum i s also obtained by protona-tion of the porphyrin to form a dication, and also by removal of two protons to form the porphyrin dianion. The position and the r e l a t i v e i n t e n s i t i e s of the four v i s i b l e absorption bands are f a i r l y sensitive to the nature and the changes of the peripheral groups, and give r i s e to four main spectral c l a s s i f i c a -tions (Fig. l a to l c ) . 1.2.1 Etio-type Spectra Etio-type spectra (Fig la) are found i n porphyrins substituted by ethyl, methyl, v i n y l , acetic acid or propionic acid side chains at the peripheral positions. The i n t e n s i t i e s of the four v i s i b l e bands are found to be i n the order of IV > III > II > I. - 6 -F i g . 1: T y p i c a l absorption spectra (Soret omitted) of porphyrins i n chloroform. A. Etio-type; B. Rhodo-type; C. Oxorhodo-type; D. Phyllo-type. 1.2.2 Rhodo-type Spectra The presence of one strong electron-withdrawing group, e.g., carbonyl (aldehyde, ketone), a car b o x y l i c a c i d , an ester or an a c r y l i c a c i d side chain,, at a ^ - p o s i t i o n r e s u l t s i n a rhodo-type spectrum where band I I I becomes more intense ( F i g . l b , ITI > IV > II > I ) . However, the presence of two e l e c t r o n withdrawing groups (rhodofying groups) on neighbouring p y r r o l e rings gives an etio-type spectrum. - 7 -1.2.3 Oxorhodo-type Spectra When two rhodofying side chains are on diagonally opposite pyrroles of the porphyrin ring, a further change to oxorhodo-type spectrum occurs (Fig. Ic, III > II > IV > I). 1.2.4 Phyllo-type Spectra Phyllo-type spectra (Fig. Id, IV > II > III > I) are characteristic of substitution at a single meso-carbon, e.g. in chlorophyll deriva-tives, phylloporphyrin-XV, and various meso-acetoxy, meso-benzoyloxy-, 9 and meso-alkoxy-porphyrins. 1.3 NOMENCLATURE The widespread use of t r i v i a l names adopted by Fischer^' ^ a ' ^ a n ( j others has led to a haphazard nomenclature in porphyrin chemistry. The adoption of a systematic nomenclature for porphyrins will require a system flexible enough to embrace the Fischer system yet informative enough to differentiate a vast number of natural and synthetic analogues. The Fischer nomenclature is based on the numbers and letters shown in 4. In this numbering scheme, the peripheral positions are numbered from 1 to 8 and "interpyrrolic" methene positions, termed as "meso", are designated as a, /3, 7, and S. The Fischer nomenclature also involves a very large number of t r i v i a l names based on the type of peripheral substituents and an isomer numbering system based on their peripheral arrangements. For example, "Etioporphyrin" has two different substitu-ents, methyl (A) and ethyl (B) in the y9 positions, and thus four isomers exist (Fig. 2 ) . Type I Type II Type III Fig. 2: A schematic of the Etioporphyrin series Type IV - 9 -The compounds derived from the common, naturally occurring porphyrins form a complex series of structures and i n order to name them, Fischer used too many t r i v i a l names. Moreover, the names do not convey clear structural information and do not form an adequate basis for naming new compounds. Although the more systematic nomenclature for b i o l o g i c a l l y impor-12 tant porphyrins and related compounds i s outlined by Bonnett, most porphyrin chemists s t i l l prefer to use Fischer's t r i v i a l names for the most important naturally occurring porphyrins and their derivatives (Table 1). The complete numbering system for porphyrins, based on the 13 I.U.P.A.C. system, i s shown i n 5. According to this system, carbon atoms i n the porphyrin macrocycle are numbered from 1 to 20 with the four nitrogen atoms being 21 to 24. Based on this numbering scheme, a 14 j o i n t commission on biochemical nomenclature (IUPAC-IUB) has proposed a systematic nomenclature for a l l porphyrins and their derivatives. Application of the Joint Commission's recommendations permits these substances to be named more systematically using fewer t r i v i a l names than are currently used i n the l i t e r a t u r e . The nomenclature used i n this work i s based on the following guidelines. 1.3.1 Pyrroles The parent monocyclic system, pyrrole, i s numbered as shown i n 6 . Greek l e t t e r s are sometimes used to distinguish the two types of carbon positions. - 10 -Table 1: Some T r i v i a l Names In the Fischer Nomenclature Substituents at positions Porphyrins 8 Meso Protoporphyrin IX Me V Me V Me pH pH Me H Mesoporphyrin IX Me Et Me Et Me pH pH Me H Etioporphyrin I Me Et Me Et Me Et Me Et H Deuteroporphyrin IX Me H Me H Me pH pH Me H Coproporphyrin III Me pH Me pH Me pH pH Me H Uroporphyrin I A H PH Me pH Me pH Me pH H Phylloporphyrin XV Me Et Me Et Me H Me pH H Pyrroporphyrin XV Me Et Me Et pH H PH Me H Abbrev.: Me — A H --CH3; Et --CH2C02H -C 2H 5; V - -CH=CH2; pH - -CH2-CH2C02H; - 11 1.3.2 DIpyrroles Structures containing two directly linked pyrrole rings are known as dipyrroles, shown i n 7. The two pyrrole rings linked at an a-positions to a single carbon function are known as 2,2'-dipyrromethanes 8, 2,2'-dipyrroketones 9, and 2,2'-dipyrromethenes 10. - 12 -1.3.3 Tetrapyrroles The nomenclature of linear tetrapyrroles is based on that of the bile pigments suggested by Lemberg and Legge.-'--' The numbering system in line with the IUPAC recommendations for the nomenclature of porphy-ri n s . J This system takes the dioxygenated reduced system, bilane 11 the parent. 2_3 7_8 12 13 17—18 0 HaHb HcH B i l a n e system 11 - 13 -Meso-positions are designated by lower case l e t t e r s . The removal of terminal oxygen functions are ind i c a t e d by the p r e f i x , 1,19-dideoxy, and the unsaturation between the rings i s shown by the use of l e t t e r s as given below. 1,19-Dideoxybilane 12 1,19-Dideoxybilene-b 13 H Q b H c 1,19-Dideoxybiladiene-ac H - 14 m H a 1,19-Dideoxybilatriene-abc 15 The most useful linear tetrapyrrole, 1,19-dibromo-l,19-dideoxybiladiene-ac dihydrobromide 16, was used i n the synthesis of monoazaporphyrins. Br 2 Br" H a H b H H Br 1,19-Dibromo-l,19-dideoxybiladiene-ac dihydrobromide 16 1.3.4 Porphyrins Azaporphyrin nomenclature i s based on the parent porphyrin and i s numbered as i n 17. The rings are lettered A to D and side chain positions can be numbered as shown when necessary. The natural porphy-rins used i n this work s t i l l r e t a i n the Fischer nomenclature i n order to avoid the complexity and confusion with the nomenclature used i n the l i t e r a t u r e . - 15 -17 1.4 OCCURRENCE IN BIOLOGICAL SYSTEMS Heme and heme proteins are ubiquitous i n nature and carry out a large number of well d i v e r s i f i e d b i o l o g i c a l functions such as oxygen transport and storage (hemoglobin and myoglobin), electron transport (cytochrome-b, c), oxygen reduction (cytochrome oxidase), hydrogen peroxide u t i l i z a t i o n and destruction (peroxidases and catalases), and substrate oxidations (cytochrome P-450). In most of the heme proteins the active s i t e s contain an iron complex of protoporphyrin-IX as the prosthetic group 18 (myoglobin, hemoglobin, cytochrome-b); a peripher-a l l y modified form i s found i n cytochrome-a (formyl side chain), cyto-chromes-c (covalent linkage between heme and protein) and cytochrome-d (chlorin as prosthetic group). The b i o l o g i c a l properties of heme proteins are dependent on the interaction between the metal and the porphyrin i n which each i s depen 16 -dent on the other for the generation of the functionally correct electronic state. In hemoglobin, the ligand f i e l d of the porphyrin nitrogens and protein h i s t i d i n e s creates the unusual yet e n t a t i c a l l y s t a b i l i z e d equilibrium between five coordinate, high spin (S = 2) iron (II) and oxygenated, six coordinate, low spin (S = 0) iron, which allows 16 for the rapid binding and release of oxygen. In the cytochromes, the pri n c i p a l b i o l o g i c a l function of the heme protein i s electron and/or hydrogen transport by virtue of a reversible valency change of the heme 17,18 iron. Although a l l these properties are present i n simple heme complexes, the interaction of protein and i t s conformational arrangement i n biolog-i c a l systems play a very important role i n carrying out sp e c i f i c func-tions, e.g., i n hemoglobin, the globin consists of four polypeptide chains, and each protoheme group i s enfolded i n one of the four chains that create a strongly non-polar hydrophobic environment. The four chains of globin consist of two identical pairs named as a-chains and 19 /3-chains. The structure of the /9-subunits as deduced from x-ray data 17 indicates that the heme prosthetic group l i e s i n a crevice, and i s held i n place by non-bonding interactions with the protein. The single covalent attachment of the heme to the protein occurs by coordination of heme iron to the imidazole nitrogen of the so-called "proximal" h i s t i d i n e residue. It appears that, i n heme proteins, iron i s the foca l point for the attachment of protein residues, e.g., h i s t i d i n e imidazole side chain i s responsible for this complexation i n hemoglobin, myoglobin and cytochromes-b5, whereas i n cytochromes-c, one of the a x i a l p o s ition is taken by sulfur of a methionine residue. Various evidence also suggests that i n cytochrome P-450, a monooxygenase, one of the coordi-20-26 nated ligand i s su l f u r of a cysteine t h i o l . Metal-protein binding i s invariably accompanied by other inter-actions between the protein and the periphery of the porphyrin. The most common of these i s the e l e c t r o s t a t i c interaction of the two free propionic acid groups with basic amino acids, which occurs i n almost a l l heme proteins. In cytochrome-c, the prosthetic group i s linked v i a two thioether linkages between two cysteine residues and the v i n y l groups of the protoheme. A most important interaction of the prosthetic heme group 18 with protein i n the b i o l o g i c a l system i s that i n which the prosthetic group of hemoglobin and myoglobin Is degraded to give i n i t i a l l y biliverdin-IXa (amphibians and bi r d s ) , and then bilirubin-IXa (characteristic of mammals, r e p t i l e s and some f i s h ) . In mammals, b i l e pigments are simply waste material that are excreted (approximately 0.4 g of b i l i r u b i n per day i s produced by a normal adult human), and they do not possess any - 18 Me H Me Phycocyanobilin 1 9 y Me H M e / V 0 ^ ^ •NH HN V fc-NH N=< Me< 1 X z>Me Phycoerythrobilin 20 P=-CH 2CH 2C00H , Me =-CH3 V--CH=CH 2 . Et =- C 2 H 5 - 19 -b i o l o g i c a l functions. On the other hand, plant b i l e pigments (so-called because of t h e i r close structural relationship to the mammalian b i l e pigments) are probably the most abundant i n the biosphere and serve 27 important functions i n photosynthesis and photomorphogenesis. In certain lower plants, including the red algae (Rhodophyta) and the blue-green algae (Cyanobacteria), the primary photosynthetic pigments are proteins with covalently linked chromophores closely related i n structure to b i l i v e r d i n . These phycobiliproteins, including phycocyanin and allophycocyanin, are associated with the b i l e pigment phycocyano-b i l i n 19, and phycoerythrin associated with phycoerythrobilin 20. In higher plants, the photoactive pigment, phytochrome, i s also a b i l e 28 pigment protein complex 21. I t i s highly s i g n i f i c a n t that, l i k e mammalian b i l e pigments, those found i n plants and cyanobacteria are also IXa isomers. In addition, biliverdin-IX7 22, the only natural b i l e pigment not to have the IXa isomer substituent orientation, occurs i n the integumental c e l l s of the c a t e r p i l l a r of the cabbage white 29 b u t t e r f l y . 1.5 HEME OXYGENASE AND HEME CATABOLISM The biochemical mechanism of heme catabolism, leading to the forma-tion of b i l i v e r d i n , remained controversial u n t i l as l a t e as 1968 when 30 Tenhunen et a l . f i r s t described a microsomal enzyme (subsequently named, heme oxygenase), capable of converting one mole of heme to one mole of b i l i v e r d i n 23 and one mole of carbon monoxide (Fig. 3). - 20 -Heme 18 B i l i v e r d i n 23 B i l i r u b i n 24 Me - -CH3, V - -CH-CH2, P - -CH2CH2C02H Fig. 3: Structures of the p r i n c i p a l t e t r a p y r r o l i c pigments i n heme catabolism. In addition to carbon monoxide and b i l i v e r d i n , iron i s also a product of heme catabolism. The iron i s retained as f e r r i t i n and r e u t i l i z e d , and the b i l i v e r d i n 23 Is reduced enzymatically to the yellow b i l i r u b i n 24. 30-35 The enzyme, heme oxygenase, described by Tenhunen et a l . i s loc a l i z e d i n various tissues such as spleen, bone marrow, l i v e r , brain and kidney. Heme oxygenase a c t i v i t y has also been found i n rat perito-36 37 neal and rabbit alveolar macrophages, chick embryo heart, and rat 38 small i n t e s t i n a l mucosa. Enzyme a c t i v i t y has also been demonstrated in tissues obtained from man, pig, cow, rabbit and rat. The microsomal 30 heme oxygenase system requires molecular oxygen (an oxidant) and NADPH (a reductant). The a c t i v i t y and substrate s p e c i f i c i t y of heme oxygenase were 31 39-44 observed by various workers ' using synthetic and natural hemins. - 21 -The synthetic hemins such as XIII 25, hemin III 26 and iron (^3-hydroxy-ethyl) porphyrin 27 were better substrates than the natural hemin-IX 18a The replacement of vi n y l s by methyl groups (as i n 28) resulted i n hemins which were good substrates for heme oxygenase. The heme oxygenase a c t i v i t y was decreased i n hemins where one of the v i n y l group was either replaced by a propionic acid chain (harderoporphyrin 30) or by a formyl group, i n 31 and 32. The isomeric protohemins 33, 34, and 35, where the 18a, R l " R3 • -CH3; R2 = R. - -CH=CH0 4 2 25, R l = R4 = -CH=CH 2' R 2 = R 3 - -CH3 26, R l " R4 -CH3; R2 - R 3 - -CH=CH2 27, R l " R4 _ -CH2CH 20H; R 2 - R 3 - -CH3 28, R l ~ R 3 -CH3; R2 - R. = -CH. 4 3 29, R 1 - R 3 - R 4 = " CH3; R 2 - -CH=CH2 30, R l ~ R 3 - -CH3; R2 = -CH=CH2; R 4 - -CH2CH2C02H 31, R l = R3 - -CH3; R4 = -CH=CH2; R 2 = -CHO 32, R l = R 3 - -CH3; R2 -= -CH=CH2; R 4 - -CHO - 22 -array of substituents i n ring A and B were the same as i n protohemin 18a, but the substituents positions i n ring C and D were different from those of protohemin 18a, were examined as substrates for heme oxygenase but none of these showed heme oxygenase substrate a c t i v i t y . This shows that the presence of a v i c i n a l propionic acid side chain at positions 6 and 7 A- 3 is an essential requirement for the substrate a c t i v i t y . The higher homologue of protohemin-IX 18a, where the v i c i n a l propionic acid chains at positions 6 and 7 were replaced by butyric acid residues ( i n 3 6 ) , when incubated i n microsomal heme oxygenase system, showed a decrease i n oxidation rate. The shortening of the chain length to acetic acid 37 e n t i r e l y suppressed the enzyme a c t i v i t y . The i n i t i a l findings suggested that heme oxygenase required cytochrome P-450 as a terminal oxidase, ^  32,34,35 ] - ) e c a u s e h e n i e oxygenase reaction had many s i m i l a r i t i e s to those of other hydroxyla-tion reactions catalyzed by the microsomal cytochrome P-450 system. 45 46 Yoshida and coworkers ' indicated that the p u r i f i e d heme oxygenase and cytochrome P-450 were d i s t i n c t proteins. A similar observation was i ^ u w - n 47,48 also made by Mames et a l . 49 50 Yoshida and Kikuchi ' have shown that p u r i f i e d heme oxygenase, from pig spleen and rat l i v e r , i s not a heme protein by nature, but i t 49 binds heme to form a 1:1 complex. The absorption spectrum of the complex of f e r r i c heme and heme oxygenase i s very similar to that of 49 metmyoglobin and methemoglobin. The absorption spectrum of the reduced form, and the CO complex of the reduced form of this complex, resembles those of the corresponding forms of myoglobin and hemoglobin. These workers have also shown that the heme bound to heme oxygenase - 23 -C 0 2 H C02H 37 - 24 -coordinates with azide and cyanide. This suggests that the heme i s linked to an amino acid residue of the heme oxygenase protein through the f i f t h coordination position of the iron atom, similar to the other f e r r i c heme proteins such as methemoglobin and metmyoglobin. The spectral properties suggest that heme oxygenase may have a heme binding crevice s i m i l a r to those of myoglobin and hemoglobin. Two d i f f e r e n t concepts have been proposed for the mechanism by which hemoglobin and other heme compounds can be converted to b i l e pigments i n the b i o l o g i c a l medium. The f i r s t mechanism describes where the cleavage of the heme ring i s enzymatically catalyzed (Fig. 4), whereas the second mechanism postulates that hemoproteins undergo a non-enzymatic coupled oxidation followed by hydrolysis of the cleaved tetrapyrrole (Fig. 5). Coupled oxidation of heme or hemeproteins with reducing agents (ascorbate or phenylhydrazine) i s thought to involve the same chemical reactions as i n the physiological conversion of heme to b i l i v e r d i n . T h e two processes d i f f e r from each other i n the sp e c i f i c -i t y of the cleavage s i t e on the porphyrin ring of heme. Cleavage controlled by heme oxygenase during physiological degradation i s spe-44 52 c i f i c for the a-meso position and produces only biliverdin-IXa. ' Coupled oxidation, on the other hand, results i n random cleavage at a l l four meso-positions i n hemin and denatured hemo-proteins5"*'' 5 3 5 5 (Fig. 5) In contrast, coupled oxidation of heme proteins i s s p e c i f i c and does 53-57 not lead to a random mixture of isomers. For example, hemoglobin gives predominantly biliverdin-IXa 2 3 together with variable amounts of the IX/3 i s o m e r 5 3 " 5 5 3 8 , while myoglobin gives exclusively the IXa c- Q 58 isomer 2 3 . Recently, Hirota et a l . studied the formation and - 25 -P=CH2CH2COOH Fig. 4: Possible chemical sequence of heme degradation catalyzed by the heme oxygenase system. - 26 isomeric composition of b i l i v e r d i n isomers i n urine after intravenous injections of various hemoproteins and hemins into rabbits. The forma-tion of the 1X5-isomer i n addition to high levels of IXa and IXjfl-isomers from hemoglobin, and IX/9-isomer with IXa isomer from myoglobin, was observed. The Increased excretion of IXa- and IX/9-isomers over the physiological l e v e l from hemoglobin, and formation of the IXa isomer from myoglobin, suggest that a part of the heme from injected hemo-proteins i s degraded by a non-enzymatic chemical reaction (coupled oxidation). The possible chemical sequence of heme degradation catalyzed by the heme oxygenase system i s shown in Fig. 4. It i s now generally assumed that the f i r s t step i n heme catabolism involves mesohydroxylation as shown i n Fig. 4.5^ 6 ^ Though mesohydroxyheme 41 has never been isolated 6 3 from any b i o l o g i c a l system, Kikuchi and Yoshida have observed spectral changes i n their studies with p u r i f i e d heme oxygenase. They concluded that these changes may be due to the formation of mesohydroxyheme. The 64 intermediacy of a-hydroxyheme i n vivo was f i r s t shown by Kondo et a l . , 3 who demonstrated that, i n rats, injected [ H]a-hydroxymesoheme i s readily converted to mesobilirubin-IXa. This view was further confirmed 65 by Yoshida and coworkers when mesoheme and hydroxymesoheme were converted to b i l i v e r d i n s by the heme oxygenase system. In nonbiological systems for heme catabolism, the evidence for the involvement of a mesohydroxylated intermediate i s stronger. Lemberg and c o w o r k e r s 1 5 , 6 6 and Foulkes et a l . 6 7 have suggested that the i n i t i a l products of coupled oxidation were mesohydroxyhemes which were readily converted to b i l i v e r d i n s . These workers also found that the meso carbon Fig. 5: Coupled oxidation of hemin to the four b i l i v e r d i n isomers of the IX series - 28 -44 45 of the hydroxyheme i s expelled as CO and replaced by an oxygen-bridge to form verdoheme 44. The pyridine compl ex of the verdoheme 44 was termed verdohemochrome. Verdohemochrome i s hydrolyzed i n v i t r o to the iron complex of b i l i v e r d i n which upon a c i d i f i c a t i o n releases iron to give b i l i v e r d i n . The intermediacy of this oxygen-bridged complex has been 68-78 observed by several other workers. 79 Anan and Mason showed that the b i l i v e r d i n formed from the coupled 18 oxidation of hemoglobin under 0^ with ascorbate contained only one terminal oxygen from molecular oxygen, the other oxygen coming from 34 water. On the other hand, Tenhunen et a l . showed that the formation 18 18 of b i l e pigment by the heme oxygenase system i n the presence of ' 0^ resulted i n the incorporation of both terminal oxygen from molecular oxygen and none from water. Further studies based on oxygen l a b e l l i n g experiments have shown that the two oxygen atoms of b i l i r u b i n were derived from d i f f e r e n t oxygen molecules (the two-molecule mecha-n i s m ) . 8 0 " 8 4 The incorporation of the two oxygen atoms from different oxygen molecules was also observed i n the coupled oxidation of myoglo-82 70 84 bin and octaethylhemin. ' These experimental results, coupled with - 29 -the assumption that the conversion of verdohemochrome or verdohemin to b i l i v e r d i n requires a hydrolytic step, have excluded the oxygen-bridged compound as a possible intermediate i n heme degradation^^' 8 0 8 4. In order to account for the v a l i d i t y of an oxygen-bridged intermediate as 72 the precursor i n heme degradation, verdohemochrome-IXa and octaethyl-73 verdohemochrome were prepared by coupled oxidation of myoglobin with ascorbate, and octaethylhemin with phenylhydrazine, respectively. The conversion of these verdohemochromes to biliverdins was carried out i n 18 18 the presence of ' 0^ and phenylhydrazine-HC1 (Fig. 6). The product Fig. 6: Formation of b i l i v e r d i n from verdohemochrome. The side chains are omitted for c l a r i t y . - 30 -18 contained one atom of 0. These workers also proposed that the incorporation of an oxygen atom into b i l i v e r d i n comes from oxygen v i a 18 18 H 2 0 2 formation from the reaction of 0 2 and phenylhydrazine (or 18 ascorbate). The 0^ produced i n the reaction reacts with verdohemochrome to give an intermediate 47 which i s converted to b i l i v e r d i n 23 by further reduction with reductant (ascorbate or phenylhydrazine) (Fig. 6). This cleavage process suggests that the hydrolysis i s not an essential step for the conversion of verdohemo-72 73 chrome intermediates to b i l i v e r d i n , ' and hence such an intermediate cannot be excluded as a precursor i n the heme catabolic process. This process also accounts for the two-molecule mechanism because one oxygen atom i s derived from 0 2 which forms the oxygen bridge of verdohemo-18 chrome, while the other oxygen atom Is derived from the ^ P r°duced 18 by reaction of 0 2 with phenylhydrazine. This was further confirmed by Itano and H i r o t a 7 ^ who prepared octaethyl [^0]verdohemochrome and 18 octaethyl [ 0]verdohemochrome from the coupled oxidation of octaethyl-16 16 18 18 hemin and phenylhydrazine hydrochloride with ' 0 2 and ' 0 2, respectively. A further reaction of these products with ^'^0 i n the presence of phenylhydrazine hydrochloride produced octaethyl [ 1 60, 16 16 18 0 ] b i l i v e r d i n and octaethyl [ 0, OJbiliverdin. A similar reaction th _ _ , ~ - 0 2 yielded octaethyl f 1 6 ~ 1 8 - ^ - - i - • r 1 8 18 wi 1 8 ' 1 8 [ 1 60, 1 8 0 ] b i l i v e r d i n and octaethyl [ 0, 0 ] b i l i v e r d i n . This suggests that the successive incorporation of the two oxygen atoms of b i l i v e r d i n occurs i n separate steps, i . e . , the formation of verdohemochrome and the conversion of verdohemochrome into b i l i v e r d i n . Thus this process also accounts for the two-molecule mechanism i n which verdoheme i s an intermediate i n the formation of - 31 -b i l i v e r d i n . Another approach to the formation of the verdoheme interme-diate arose when zinc octaethyl-5-oxaporphyrin 8 5 49 and zinc 5-oxameso-porphyrin-IXa dimethyl e s t e r ^ 51 were obtained by c y c l i z a t i o n of the corresponding zinc b i l i v e r d i n s 48 and 50 with acetic anhydride. 48 49 Similarly, zinc 5-oxaprotoporphyrin-IXa dimethyl ester was also prepared by the c y c l i z a t i o n of the zinc b i l i v e r d i n complex. In a recent study, Saito and Itano^S have also shown that the Fe(II)octaethyl verdohemo-chrome 53 can be obtained by c y c l i z a t i o n of octaethylbiliverdin 52 i n the presence of FeSO^ and pyridine. The compound 53 was converted to 54 by reaction with tosylmethylisocyanide (TsCH^NC) and NaBF^. The complexes 53 and 54 showed a structural resemblance to the Fe(II)octa-- 32 -L X 52 L = -C5H5N 53 C 5H 5N Cl 54 TsCH2NC BF 4 ethylverdohemochrome obtained from the coupled oxidation of octaethyl-hemin and ascorbic acid with 0 2. On the other hand, the structures of the protoverdohemochrome-IXQ dimethyl esters 57 and 58 obtained by c y c l i z a t i o n of respective b i l i v e r d i n s were found to be different from those of the protoverdohemochrome-IXa dimethyl esters 55 and 56 obtained by coupled oxidation of the respective protohemin-IX a dimethyl esters. Compounds 57 and 58 were at a higher oxidation state ( F e 1 1 1 ) than the corresponding complexes 55 and 56 (products of coupled oxidation). The Fe(III) oxidation state was further confirmed by the preparation of compounds 59 and 60 by coupled oxidation of mesohemin-IX dimethyl ester, as well as by c y c l i z a t i o n of mesobiliverdin-IX dimethyl ester. The reduction of compound 57 with sodium dithionite gave compound 55, which shows that the Fe(II) protoverdohemochrome-IXa was an intermediate i n the degradation of heme to b i l i v e r d i n - I X a by coupled oxidation of 79 myoglobin i n the presence of ascorbic acid and pyridine. A Saito and Itano^5 also found that the spectrum of the pyridine complex of Fe(III) - 33 -protoverdohemochrome-IXQ was similar to that of an intermediate isolated i n pyridine from an enzymic system,^2 a n d thus they suggested that this intermediate may have been the pyridine complex of Fe(III) protoverdohe-mochrome-IXa. Recently Sano et a l . ^ 8 synthesized a-oxyprotohemin-IX (an inter-mediate i n heme catabolism) and showed that the hexacoordinated, b i s -pyridine complex of a-oxyprotohemin-IX 61, underwent autoreduction (intramolecular transfer of one electron from the enolate anion to the iron) and gave an Fe(II) a-oxyprotoporphyrin 7 r-neutral r a d i c a l b i s -pyridine complex 62. This complex upon reaction with one equivalent of oxygen gave verdohemochrome-IXa 65 v i a an intermediate 64 (a dioxygen adduct which rearranges v i a an unknown mechanism to give 65) (Fig. 7). They also found that the Fe(II) ?r-neutral ra d i c a l 62 was i n - 34 -P=CH 2CH 2C00H Fig. 7: Formation of pyridine verdohemochrome-IXa from a-oxyprotohemin-IX. - 35 equilibrium with 63. A similar equilibrium has also been observed between a Ni(III) porphyrin and the corresponding Ni(II) porphyrin A- 87,88 7r-radical. The reconstituted, pentacoordinated, apomyoglobin a-oxyprotohe-min-IX complex 66 reacted with an equimolar amount of 0^ to form an Fe(II ) - 7 r neutral r a d i c a l which goes to a dioxygen adduct 67 (see Fig. 8). This dioxygen adduct rearranges to give an apomyoglobin-verdoheme-jr-radical complex 68 or 69, which further reacted with another molecule of C>2 i n the presence of ascorbate to give Fe(III) biliverdin-IXa 70. In this process i t was suggested that the oxygenation step (up to 68) does not require any reductant. On the other hand, two molecules of oxygen were used i n the whole process, which also accounts for the two-molecule mechanism i n the formation of b i l i v e r d i n . Based on these results, the heme catabolism i n vivo was considered to occur through a verdohemochrome intermediate as suggested by other w o r k e r s , ^ ' ^ ^ as i n the following sequence: °2 Protoheme-IX * a-oxyprotohemin-IX CO o 2 Fe(III) b i l i v e r d i n - •* verdohemochrome IX a- ^ - r a d i c a l IX. a Fe B i l i v e r d i n - I X a - 36 -P=-CH 2 CH 2C00H , L = H i s t i d i ne Fig. 8: Formation of b i l i v e r d i n - I X a from reconstituted apomyoglobin-a-oxyprotohemin-IX complex. L is a h i s t i d i n e residue of apomyo-globin. - 37 -The presence of iron i s an important factor i n the heme degradation catalyzed by heme oxygenase. Cobalt(II) was considered as a replacement for iron because of the oxygen-binding c a p a b i l i t i e s of Co(II) substi-89 tuted myoglobin and hemoglobin. Several groups have studied the p o s s i b i l i t y of cobalt protoporphyrin-IX as a substrate for heme oxyge-90 83 91 92 nase. However, with one exception, a l l of the studies ' '" have concluded that the cobalt protoporphyrin-IX i s not converted to b i l e pigment by heme oxygenase. Similar to the iron protoheme-heme oxygenase complex, cobalt protoheme also binds the heme oxygenase protein to give an enzyme-substrate complex which resembles those of cobalt myoglobin and hemoglobin. However, the cobalt heme bound to heme oxygenase does not react with azide, cyanide and carbon monoxide, and i s only slowly reduced by d i t h i o n i t e . Other synthetic metalloporphyrins such as 93-95 96 Sn-protoporphyrin-IX, Cr-protoporphyrin-IX, Zn-proto-porphyrin-IX,97~101 a n c j Mn-protoporphyrin-IX 1 0^ were found to i n h i b i t the heme oxygenase a c t i v i t y i n a b i o l o g i c a l medium. 1.6 PURIFICATION OF HEME OXYGENASE Heme oxygenase i s a membrane bound enzyme and therefore i t s p u r i f i -cation to homogeneity has been d i f f i c u l t . P a r t i a l or complete p u r i f i c a -tion of heme oxygenase has been reported by Maines et a l . (from rat l i v e r ) , ' 1^ > 3 Yoshinaga and coworkers (from bovine s p l e e n ) a n d Yoshida et a l . (from pig s p l e e n ^ 5 a n d rat l i v e r ) T h e molecular weight of a heme oxygenase preparation was approximately 200,000 as - 38 -estimated by gel f i l t r a t i o n on a column of Sephadex G-200.45 However, the enzyme, p u r i f i e d to electrophoretic homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a molecular weight of 49 50 32,000. ' This molecular weight i s i n agreement with the value reported by Yoshinaga and coworkers (molecular weight <* 3 1 , 0 0 0 ) , b u t is less than one-half of that reported by Maines et a l . (molecular 102 weight - 68,000). I t i s evident from the p u r i f i c a t i o n procedures described above that the development of a new p u r i f i c a t i o n procedure i s necessary and would be helpful i n understanding the nature of the enzyme (heme oxygenase) as well as i t s role i n heme degradation i n b i o l o g i c a l system. 1.7 NATURE AND AIM OF THE PRESENT WORK It i s clear from the previous discussions that heme oxygenase i s a non-heme enzyme but binds iron and cobalt protoheme to give a heme-enzyme complex. In the iron protoheme-heme oxygenase complex, heme acts as a substrate as well as coenzyme i n which heme i s degraded exclusively to give IXa b i l i v e r d i n . P u r i f i e d heme oxygenase requires addition of NADPH-cytochrome-c reductase for a c t i v i t y , i.e., the heme-heme oxygenase complex i s unreactive towards oxygen unless electrons are supplied. This presumably occurs i n vivo v i a addition of NADPH and reductase, although i t has been shown that addition of ascorbate w i l l also promote heme breakdown. - 39 -The main aims of this investigation were: 1. The synthesis of a heme derivative with a functionalized spacer arm, which can be linked to a s o l i d support and can be used as an a f f i n i t y ligand i n an a f f i n i t y chromatography column. The main objec-tive of an a f f i n i t y ligand having a heme derivative would be to bind an enzyme to give a substrate-enzyme complex, and separate other impurities from the o r i g i n a l mixture. Experimentation with the solvent system, should allow for the enzyme to be eluted i n a much p u r i f i e d form from the column. A f f i n i t y chromatography has been shown to be a powerful tool for 105 106 protein p u r i f i c a t i o n . ' The a f f i n i t y chromatographic technique, p a r t i c u l a r l y with the heme binding protein, hemopexin, has been reported in the literature 1*"* 7 1 1 ^ but these a f f i n i t y chromatographic columns had 113 low p u r i f i c a t i o n capacity. Olsen has used hematoporphyrin as an a f f i n i t y ligand, attached on Sepharose-4B through 1,6-diaminohexane as a spacer arm. The p u r i f i c a t i o n of hemopexin was found to be better than the methods described e a r l i e r . 1 ^ * 7 Ferrochelatase, an enzyme which incorporates iron into protoporphyrin i n the f i n a l step of heme biosyn-114 thesis, was p u r i f i e d by Mailer et a l . using a protoporphyrin derivative as an a f f i n i t y ligand. The a f f i n i t y ligand used i n this study was a mixture of two isomers. However, the synthesis of two isomeric a f f i n i t y ligands (73, 74) may be useful and prove to be better in enzyme p u r i f i c a t i o n . Because heme oxygenase exhibits high binding capacity with iron and cobalt protoheme, i t was decided to synthesize the iron and cobalt protoporphyrin derivatives, 71 and 72 respectively, - 40 -41 which can be attached to a s o l i d support for future attempts at the p u r i f i c a t i o n of heme oxygenase. 2. It i s clear from the above discussion that heme degradation cata-lyzed by heme oxygenase i s sp e c i f i c for the a-meso position (Fig. 4), and produces only biliverdin-IXa which is reduced by b i l i v e r d i n reductase to b i l i r u b i n - I X . The mechanism of heme degradation i n vivo a and i n v i t r o has been studied by various workers (Section 1.5) and the intermediates involved i n the catabolic process have been suggested. To understand further the nature of this enzyme and i t s a - s e l e c t i v i t y we decided to synthesize porphyrin analogues where the a-methene carbon i s replaced by nitrogen (mono-azaporphyrIns). Fischer and Friedrich"'"''"5 obtained traces of monoazaporphyrins and diazoporphyrins by heating the 5,5'-dibromodipyrromethenes 75 or 76 in pyridine with aqueous a l k a l i for 8 days at 130-135°C i n a sealed tube. Endermann and Fischer prepared monoazaporphyrin 78 in low y i e l d (5%) by treatment of the urethane derivative 77 with bromine i n acetic acid. A better y i e l d of monoazaporphyrin 80 was obtained as a by-product i n - 42 the formation of a corrole by i r r a d i a t i o n of the 1,19-dideoxybila-diene-ac 79 i n the presence of ammonia.-'-''-^  Harris et al.^-^ and Grigg and coworkers-'--^ synthesized 5-azamesoporphyrin-IX 82 i n 4 5 % y i e l d by c y c l i z a t i o n of 1,19-dibromo-1,19-dideoxybiladiene-ac dihydrobromide 81 79 80 - 43 -83 R = -CH3 84 R - -CH2C02CH3 A M e - -CH2C02CH3, P M e - -CH2CH2C02CH3 85 R - -CH3 86 R - -CH2C02CH3 - 44 -with sodium azide i n refluxing methanol. Use of reaction conditions similar to those c i t e d by Harris et a l . , 1 1 8 led to the preparation of monoazaporphyrins 85 and 86 from the 1,19-diiodo-l,19-dideoxy-biladiene-ac s a l t s 83 and 8 4 . 1 2 0 Recently Abeysekera and coworkers 1 2 1 obtained the octaalkyl monoazaporphyrin 88 in 66% y i e l d by reacting compound 87 with sodium azide i n anhydrous methanol under n i t r o g e n . An interesting route to monoazaporphyrin 91 synthesis was f i r s t demonstrated by Lemberg.^ This scheme involved the treatment of verdohemins 90 (an intermediate obtained from iron complexes of porphyrin by coupled oxidation with 0 2 i n pyridine i n the presence of reducing agent, e.g., ascorbic acid or hydrazine) with ammonia. This - 45 -89 90 91 a, R =H b , R = OH observation was further confirmed by Jackson, D y when octaethylverdoheme 44 was converted to octaethylmonoazahemin 92 by reaction with ammonia. - 46 -82 R = -C 2H 5 95 R - -C 2H 5 96 R - -CH=CH2 - 47 -A similar approach was also used by Fuhrhop and his c o l l e a g u e s . 1 2 2 In t h e i r reaction the zinc 5-oxamesoporphyrin-IX dimethyl ester 51 and zinc 5-oxaprotoporphyrin-IX dimethyl ester 94 (obtained from the c y c l i z a t i o n of the i r respective b i l i v e r d i n s 50 and 93) were converted to the respective Zn-monoazaporphyrins 95 and 96 by treatment with NH4OH. The 5-azamesoporphyrin-IX dimethyl ester 82 was obtained i n 45% y i e l d by removal of zinc from 95 by treatment with t r i f l u o r o a c e t i c acid i n chloroform. In this study the monoazaporphyrins 95 and 96 were also prepared by c y c l i z a t i o n of zinc complexes of the corresponding 1-substi-tuted b i l i v e r d i n dimethyl esters 97 and 98 i n DMF. N N=< •R Zn N' N ff DMF C 02M e C02Me C02Me C02Me Y i e l d % 97 R - -C 2H 5 98 R - -CH=CH2 95 R - -C 2H 5 96 P - -CH-CH2 41 35 - 48 -Recently Saito and I t a n o 7 5 prepared Fe(II)-monoazaporphyrins 99 and 101 by treating the corresponding iron(II) oxaporphyrins 53 and 55 with ammonia. The removal of iron from compound 99 gave octaethylmonoaza-porphyrin 100 i n 63% y i e l d . 100 - 49 -The present work describes an improved synthesis of monoazaporphy-rins over the existing methods available i n the literature. In the present work 1,19-dibromo-1,19-dideoxybiladiene-ac dihydrobromides were prepared and cyclized to give high yields of the corresponding monoaza-porphyrins using dibenzo-18-crown-6 as a phase transfer agent (for the transfer of an azide ion) i n methylene chloride/pyridine solution at room temperature (for details see section 3.4). - 50 CHAPTER 2 RESULTS AND DISCUSSION 5 1 -PART 1 SYNTHESIS OF AN AFFINITY LIGAND - 52 -2.1 AFFINITY CHROMATOGRAPHY AND AFFINITY LIGAND The technique of a f f i n i t y chromatography exploits the unique b i o l o g i c a l s p e c i f i c i t y inherent i n ligand-macromolecule interactions to give a ligand-macromolecule complex. The b i o l o g i c a l functions of proteins are based on their a b i l i t y to bind the ligand s p e c i f i c a l l y and reversibly. Thus the concept of a f f i n i t y chromatography i s realized by covalently attaching the ligand to an insoluble support through a spacer arm (Fig. 9). In pr i n c i p l e , an a f f i n i t y ligand acts as an a r t i f i c i a l / / / / • Spacer Arm h o Bioligand Adsorption O A / / / / / -Solid Support Regeneration Elution A s /> • -• A Wash Fig. 9: The pri n c i p l e of a f f i n i t y chromatography. - 53 substrate and binds only the protein which displays an appreciable a f f i n i t y for the ligand, thus slowing the progress of the protein down the column; other proteins which show no a f f i n i t y for the ligand w i l l pass through the column bed unretarded. To effect the macromolecule-ligand interaction, the ligand should possess the following properties: (a) The ligand should possess a close structural resemblance to the natural substrate. (b) To provide good a c c e s s i b i l i t y between the ligand and the macromole-cule, the ligand should be separated from the s o l i d matrix through a spacer arm containing 3 to 6 methylene (-C^-) groups and a terminal functional group which can provide a point of attachment to the s o l i d matrix. (c) The ligand must have a functional group which can be eas i l y modi-f i e d for the purpose of spacer arm synthesis ( r e l a t i v e l y easy synthesis with few steps). (d) The ligand should be stable under the experimental conditions. 2.2 SYNTHETIC APPROACH The majority of porphyrin syntheses found i n the l i t e r a t u r e are 123 125 either based on the fusion of two p y r r o l i c intermediates (2+2 synthesis) or stepwise construction of an open chain (or "linear") l o o "I OA 1 OA tetrapyrrole. ' ' The porphyrin syntheses based on d i p y r r o l i c intermediates have symmetry re s t r i c t i o n s ; thus, this route i s limited to the synthesis of porphyrins which are centrosymmetrically substituted - 54 -or porphyrins which possess symmetry i n one or both halves of the molecule. On the other hand, the stepwise synthesis i s more f l e x i b l e because any desirable groups can be introduced at the porphyrin periphery. However, the process of stepwise construction of a l i n e a r tetrapyrrole i s lengthy, time consuming, complicated and therefore limited for an a f f i n i t y ligand synthesis. Because of these limitations i n the porphyrin synthesis v i a tetra-p y r r o l i c intermediates, i t was advantageous to use one of the most commonly occurring derivative of a natural porphyrin, hematoporphyrin-IX 102 as a synthetic s t a r t i n g material, which i s readily available from blood by treatment with s u l f u r i c acid. This treatment involves the removal of the central iron from the protohemin and the hydration of the 127 2- and 4 - v i n y l groups. Commercial hematoporphyrin (obtained from - 55 Sigma and I.C.N.) contains substantial amounts of the monohydroxyethyl monovinyl derivatives 103 and 104, and small amounts of protoporphy-rin-IX 105 (the d i v i n y l derivative). These impurities were unimportant in the present work because the f i r s t step i n the synthesis was the conversion of hematoporphyrin 102 to protoporphyrin-IX 105. This transformation (102 -»• 105) has been carried out e a r l i e r by heating 102 128 129 In ortho-dichlorobenzene containing toluene p-sulfonic acid. ' 130 Dinello and Chang prepared protoporphyrin-IX 105 i n this laboratory by simply adding hematoporphyrin dihydrochloride to b o i l i n g dimethyl-formamide under an atmosphere of nitrogen. After 30 seconds, the hematoporphyrin dissolves and dehydrates, and the hot reaction mixture is cooled to room temperature. This process results i n an 85 to 90% y i e l d of protoporphyrin-IX 105. Due to the presence of polar dicarboxylic acid groups, the chroma-tographic p u r i f i c a t i o n of protoporphyrin-IX 105 i s d i f f i c u l t . To overcome this problem i t was important to convert the dicarboxylic acid derivative to either the dimethyl or d i t e r t i a r y butyl ester derivatives. The dimethyl ester derivative 106a can e a s i l y be obtained either by 127 reaction with diazomethane, or by treatment with 5% s u l f u r i c acid i n 127 methanol. In the present work, the d i t e r t i a r y butyl ester derivative of protoporphyrin-IX was preferred over the dimethyl ester, because the regeneration of the dicarboxylic acid at the end of the synthetic scheme would be easier. The hydrolysis of the methyl ester under acidic condition can cause the hydration of the sensitive v i n y l groups, whereas the d i t e r t i a r y butyl group can be easily cleaved by dry HC1 i n anhydrous methylene chloride or by s t i r r i n g with t r i f l u o r o a c e t i c acid at room - 56 -a, R = Me b, R = tBu temperature; under these conditions, the v i n y l groups of protoporphy-r i n - IX are unaffected. 1 3- 1-The protoporphyrin-IX di-tert-butyl ester 106b was prepared by the method used by Dinello and D o l p h i n . I n this method, the dicarbox_,lie acid of protoporphyrin-IX was converted to the diacid chloride by treatment with oxalyl chloride i n anhydrous methylene chloride. The diacid chloride was subsequently refluxed with t e r t i a r y butyl alcohol i n - 57 methylene chloride i n the dark to give protoporphyrin-IX d i - t e r t - b u t y l ester 106b. The column chromatographic p u r i f i c a t i o n of the crude product over s i l i c a gel (Brockmann grade-IV) resulted i n a 78-80% y i e l d of the pure 106b, which i n methylene chloride exhibited an etio-type v i s i b l e spectrum. 2.3 REACTIONS AT THE PORPHYRIN PERIPHERY FOR INTRODUCTION OF A SPACER ARM The a- and /3-pyrrolic positions, methene bridges (meso-positions) and v i n y l groups constitute the reactive sites of the protoporphyrin 133 13 A-periphery. Molecular o r b i t a l calculations and experimental results both indicate that the methene bridges of the porphyrin macro-cycle are susceptible to e l e c t r o p h i l i c , nucleophilic and rad i c a l addi-tion reactions. The important reactions carried out at the periphery of the porphyrin macrocycle include Vilsmeier f o r m y l a t i o n , b r o m i n a -„. 134,142-145 . 144,146-152 , , _ <_. 141,153-158 _ tton, n i t r a t i o n , and deuteration. The central metal ion also plays an important role i n the r e a c t i v i t y and 134 meso-selectivity of the porphyrin ring. E l e c t r o p h i l i c substitution Is greatly enhanced with complexes of metals such as Mg(II), Zn(II), Cu(II), N i ( I I ) , Co(II), and Pd(II). This may be due to d e r e a l i z a t i o n of the negative charge into the porphyrin periphery by the metals. Because of the r e a c t i v i t y of the porphyrin periphery, the introduction of a spacer arm would be f a c i l i t a t e d by u t i l i z i n g these reactive centres (meso-positions, a- and /9-pyrrolic positions and v i n y l groups) to generate some functional groups on the porphyrin macrocycle. - 58 -2.4 SYNTHESIS OF FORMYL PORPHYRINS 2.4.1 Vilsmeier Formylation Reaction The synthesis of formyl porphyrins by Vilsmeier formylation reac-135 1 ^41 tion has been attempted by several groups. Inhoffen and 135 coworkers carried out formylation of Cu(II) deuteroporphyrin-IX dimethyl ester 107. The products obtained from this reaction were found to be complex mixtures of meso (a- and /?-) 111-113 and pyrrole (2, and 4-/9-positions) substituted formyl porphyrins 108-110. The same reaction under c a r e f u l l y controlled conditions gave a 25% y i e l d of the mixed 2-and 4-monoformyldeuteroporphyrins 108 and 109 along with a 20% y i e l d of the a~ and 0-meso-monoformyldeuteroporphyrins 111 and 112. These 137 results are i n contrast with those of Nichol who found that the similar Vilsmeier formylation of deuterohemin yielded only /3-meso-formyldeuteroporphyrin dimethyl ester and 2,/9-diformyldeuteroporphyrin 141 dimethyl ester as c r y s t a l l i n e products. Smith and Langry obtained 2-and 4-mono-formyldeuteroporphyrins 108 and 109 i n 56% y i e l d by Vilsmeier formylation of Cu(II) deuteroporphyrin-IX dimethyl ester 107 using 135 N,N-di-isobutylformamide i n place of dimethylformamide. The products also included the a- or ^-meso-monoformyldeuteroporphyrin-IX dimethyl esters 111 and 112 i n small y i e l d (18%). - 59 -1 0 8 Rj —CHO, R2-H 1 0 9 R^H, R 2—CHO 1 1 0 R J ^ - R J , — C H O 1 1 1 R^-CHO, R2=R3=H 1 1 2 R1-R3-H, R 2—CHO 1 1 3 R1-H, R 2-R 3—CHO The Vilsmeier formylation of Cu(II) octaethylporphyrin at the meso-position has been reported by Inhoffen and his coworkers.-^ 8 A similar observation was also made by Johnson and Oldf ield-^9 i n a reaction of Ni(II) etioporphyrin 1 1 6 with Vilsmeier reagent. Grigg et al.^36 studied the r e a c t i v i t y of a number of metal derivatives (M - Zn 1 1, Cu 1 1, N i 1 1 , Co 1 1, Mn 1 1 1, or F e 1 1 1 ) of etioporphyrin 1 1 4 toward the Vilsmeier reagent under the ide n t i c a l reaction conditions used by Johnson and O l d f i e l d . I n this study they found that the tri v a l e n t metal complexes (M = Mn 1 1 1 or F e 1 1 1 ) of etioporphyrin 1 1 4 did not show any formylation, whereas the Z n 1 1 porphyrin was demetallated. - 60 -However, both C u 1 1 and N i 1 1 complexes of the etioporphyrins 115 and 116 yielded the meso-monoformyl products 117 and 118, respectively. The II Co etioporphyrin was found to be very reactive and gave mixed meso-diformyl derivatives. While Vilsmeier formylation could be useful in the synthesis of formyl derivatives of porphyrins, the use of metals such as Cu(II) and Ni(II) subsequently requires forcing conditions (concentrated s u l f u r i c acid) to remove the metals after formylation, at which time the sensitive v i n y l and t e r t i a r y butyl groups w i l l be affected. Besides these factors, the d i f f i c u l t separation of various mixed monoformyl derivatives imposes another r e s t r i c t i o n i n the use of t h i s method for the synthesis of monoformyl derivatives. 115 M - Cu, R - H 116 M - Ni, R - H 117 M - Cu, R - -CHO 118 M - Ni, R - -CHO - 61 -2.4.2 Reactions at the Vinyl Groups of Protoporphyrin The v i n y l groups of protoporphyrin-IX are reactive and the conver-sion of one of them to a formyl group could be useful i n the a f f i n i t y ligand synthesis. The use of the v i n y l group also leaves two propionic acid side-chains free which may play an important role i n enzyme binding and also help i n the water s o l u b i l i t y of the f i n a l a f f i n i t y ligand. The conversion of v i n y l to formyl groups, however, introduces the problem of isomer separation, but after separation, two isomeric a f f i n i t y ligands can be synthesized. 159 Sparatore and Mauzerall oxidized the v i n y l groups of protopor-phyrin- IX dimethyl ester 106a with osmium tetroxide to give the bis-1,2 glycol 119 which upon cleavage with sodium periodate gave the diformyl-deuteroporphyrin-IX dimethyl ester 110 i n high y i e l d . The dialdehyde - 62 -110 on condensation with malonic acid under Knoevenagel conditions gave the 2,4-diacrylic acid deuteroporphyrin-IX dimethyl ester 120. The d i r e c t conversion of a v i n y l group to a formyl group has been examined by a number of workers . • -^0 ,161 Caughey et a l . ^ ^ oxidized the v i n y l groups of 106a with aqueous potassium permanganate i n refluxing acetone. The products obtained after p u r i f i c a t i o n from an alumina column were the monoformyl-monovinyldeuteroporphyrin-IX dimethyl ester i n 15 to 21% y i e l d and the diformyldeuteroporphyrin-IX dimethyl ester i n 17 to 22% y i e l d . H a m i l t o n ^ 2 repeated Caughey's procedure with protoporphyrin-IX di - t e r t - b u t y l ester 106b. The products were separated with d i f f i c u l t y by s i l i c a gel column chromatography, and the yields were lower than those reported by Caughey (monoformyl 14%, diformyl 11%). Jackson and coworkers-^-*- synthesized equimolar mixtures of monoformylmonovinyl-- 63 -deuteroporphyrin-IX dimethyl ester 122a and 123a, in quantitative y i e l d , by refluxing protoporphyrin-IX dimethyl ester 106a with p i c r y l azide 121 in chloroform. They reported that the p i c r y l azide 121 reacts with 106a by a 1,3-dipolar addition mechanism at the v i n y l group to give a p i c r y l Schiff's base, which undergoes spontaneous hydrolysis to give 2- (and 4)-formyl-4-(and 2)-vinyldeuteroporphyrin-IX 122a and 123a, and 2,4-diformyldeuteroporphyrin-IX dimethyl ester 110a. a , R - Me b , R = tBu Because of the synthetic u t i l i t y of this reaction we examined the p i c r y l azide reaction with protoporphyrin-IX di-tert-butyl ester 106b. This was refluxed with one equivalent of p i c r y l azide 121 i n 1,2-dichloroethane and the progress of the reaction was followed by t i c . The t i c , after h a l f an hour, showed very l i t t l e conversion of the starting material. However, further reflux for another ha l f an hour - 64 -showed complete conversion, giving two spots on t i c . The reaction mixture was cooled and passed through an alumina column ( a c t i v i t y I ) . The f i r s t red/pink band eluted showed a "rhodo" type v i s i b l e spectrum and was found to be a mixture of the 2-forayl-4-vinyl 123b and 2-vinyl-4-formyl isomers 122b, i n 31% y i e l d . A second dark red band, having an "etio" type v i s i b l e spectrum, was eluted and shown to be diformyl-deuteroporphyrin-IX di - t e r t - b u t y l ester 110b, isolated i n 42% y i e l d . To further improve the y i e l d of the monoformyl-monovinyl deuteroporphyrin-IX- d i - t e r t - b u t y l esters 122b and 123b, the reaction conditions were changed, e.g., solvent (chloroform) and porphyrin-azide ratios, but no improvements i n yields were observed. The purity of the mixed isomers of monoformylmonovinyldeuteroporphyrins 122b and 123b was s u f f i c i e n t to carry out further extension of the spacer chain on the porphyrin periph-ery. However, our aim was to prepare two isomeric a f f i n i t y ligands, and therefore i t was necessary to separate and purify the two isomers before the spacer chain was extended. The chromatographic separation on s i l i c a or alumina column was unsuccessful. The separation of these isomers on s i l i c a gel preparative plates showed some success but the procedure was limited to very small scale separations. The repeated developments (16 times) of 20 x 20 cm, 1 mm thick s i l i c a gel preparative plate i n chloro-form -methanol (100:0.5 v/v) gave less than 10 mg of each isomer. A 163 similar observation was also made by Sono and Asakura. The problem of isomer separation was overcome using the procedure developed by Inhoffen et al. 1 6° Their procedure made use of the f a c i l e 1,4 addition of singlet oxygen to the diene centre, formed by a v i n y l group and one of the two exocyclic double bonds i n protoporphyrin-IX, to 65 -give bright green products, named photoprotoporphyrins 126a and 127a. Inhoffen and coworkers 1 6 0 separated and characterized both isomers. The compound with a higher mobility on s i l i c a gel was i d e n t i f i e d as 4-photoprotoporphyrin (ring B isomer) isomer 126a and the slower moving band as the 2-photoprotoporphyrin isomer 127a (ring A isomer). Each isomer was converted to i t s respective formyl derivative 122a and 123a, by borohydride reduction of the terminal aldehyde of 126a and 127a to an a l l y l i c alcohol, followed by acid catalyzed rearrangement to the porphy-r i n diols 130a and 131a and f i n a l l y the periodate cleavage of the resulting d i o l . An 80% y i e l d was reported for the formyl-vinyl deriva-tives 122a and 123a, obtained from this reaction. The high yields reported for the synthesis of the isomeric, formyl-vinyl porphyrins 122a 164 and 123a could not be reproduced by Clezy and Fookes, using the c i t e d 164 reaction conditions. Clezy and Fookes obtained a y i e l d of 25% from the photoprotoporphyrin II diethyl ester by changing the solvent from methylene chloride-benzene to dioxane. However, Dolphin and Siva-sothy^ 6 5 used a modified procedure of Clezy's method and obtained a 75% y i e l d of the product. In the present study, the protoporphyrin-IX di-t e r t - b u t y l ester 106b was photolyzed i n an open beaker, with s t i r r i n g , i n methylene chloride containing 10% pyridine, on the roof of the chemistry building i n bright Vancouver sunlight. The methylene chloride loss, due to evaporation was replenished. The progress of the reaction was monitored by t i c . The reaction mixture was s t i r r e d for two to three days u n t i l most of the starting material was converted to photoprotoporphyrin. The green product obtained from the above reaction was separated on a 66 -a , R - Me b , R - tBu 67 -s i l i c a gel column e q u i l i b r a t e d with 3% d i e t h y l ether i n methylene ch l o r i d e . The f i r s t band to be eluted was unreacted 106b. The f i r s t green band eluted was the r i n g B photoprotoporphyrin 126b, the second band was a mixture of r i n g A and r i n g B photoprotoporphyrin, and f i n a l l y the t h i r d band eluted was r i n g A photoprotoporphyrin 127b. Compound 127b was always contaminated with small amounts of 126b, buc the l a t t e r was l o s t during c r y s t a l l i z a t i o n from chloroform-methanol. The column chromatographic separation was very slow, time consuming and l i m i t e d to only small scale separations. A better p u r i f i c a t i o n was obtained on 20 x 20 cm, 1 mm t h i c k s i l i c a preparative plates by repeated develop-ments i n methylene c h l o r i d e - d i e t h y l e t h e r (100:3 v/v) as solvent. The y i e l d s obtained from t h i s procedure were 23% for the r i n g A isomer 127b, 25% f o r the r i n g B 126b isomer, and about 30% f o r the mixed isomers. The conversion of each isomer of photoprotoporphyrin d i - t e r t - b u t y l e s t e r ( r i n g A, 127b; r i n g B, 126b) to i t s corresponding monoformylmono-v i n y l d e r i v a t i v e i n high y i e l d was c a r r i e d out using a mo d i f i c a t i o n of 164 Inhoffen's procedure used by Clezy and Fookes. In t h i s procedure, a s o l u t i o n of photoprotoporphyrin d i - t e r t - b u t y l ester (either r i n g A or r i n g B isomer) i n methylene chloride (anhydrous) was treated with sodium borohydride i n anhydrous methanol. A colour change from green to red-brown was observed. The i s o l a t e d red-brown product was further treated by sodium periodate i n a c i d i f i e d dioxane s o l u t i o n at room temperature. The monoformyl-monovinyl der i v a t i v e s were e a s i l y separated on s i l i c a g e l i n 65-70% y i e l d s . Several t e s t reactions were c a r r i e d out and y i e l d s were consistent. Even though the syntheses of the monoformyl-monovinyl isomers ( r i n g A or r i n g B) were lengthy and time consuming by t h i s synthetic scheme, 68 their consistency and high yields proved to be better than other meth-ods, when either the single isomer or mixed isomers of monoformyl-monovinyl derivatives was required. 2.5 EXTENSION OF THE SPACER ARM Synthesis of the spacer arm using a formyl group was carried out by two methods, i . e . , the Wittig and Knoevenagel reactions. C a l l o t ^ " ^ synthesized the monoacrylic acid derivative using the Ni(II) tetra-phenylporphyrin containing a formyl group at the /9-pyrrolic position. The products obtained were mixtures of the c i s - and trans-acrylic acid in 71% y i e l d . Sparatore and Mauzerall^"^ and Battersby and his colleagues'*"*^ used a more straightforward method. They condensed the diformylporphy-i i n 110 with malonic acid under Knoevenagel conditions, i n the presence of a base catalyst, to give the d i a c r y l i c acid derivative of porphyrin 120. The product obtained under this condition showed that the o l e f i n moieties formed were of trans-configuration. With this strategy, the monoformyl-monovinyldeuteroporphyrin-IX di-ter t - b u t y l esters 122b and 123b were converted to the monoacrylic acid derivatives 132 and 133, respectively. Because of the low s o l u b i l i t y of malonic acid i n pyridine and i t s easy decarboxylation, the temperature of the reaction was slowly raised over a one hour period and then maintained at 80°C for an additional - 69 -four hours. The reaction was followed by t i c . At the end of fiv e hours of reflux, the a c r y l i c acid derivative was evident as a slow moving polar band. The product was separated by s i l i c a gel column chromatogra-phy using chloroform-methanol (100:5 v/v) as an eluent. A slow moving band eluted giving a 50% y i e l d of the monoacrylic acid derivative. The presence of an a c r y l i c acid side chain conjugated to the porphyrin w system was observed by v i s i b l e spectroscopy. The solution of the monoacrylic acid i n methylene chloride exhibited a "rhodo" type spectrum which changed to an etio-type spectrum on addition of a base (pyridine). Ttie mass spectrum of the a c r y l i c acid exhibited a molecular ion peak at m/e - 718. The porphyrin a c r y l i c acids were further characterized as their methyl esters by e s t e r i f i c a t i o n with diazomethane. The nmr spectra of the methyl ester derivatives 134 and 135 showed a trans-configuration (J = 16 Hz) of the a c r y l i c acid groups. 2.6 FURTHER EXTENSION OF THE SPACER ARM The next step i n the a f f i n i t y ligand synthesis was to extend the spacer arm outward from the a c r y l i c acid group. Ideally, the spacer arm must be long enough to allow free access of an enzyme to the ligand without a detrimental interaction with the s o l i d support. If too long an arm i s used between the s o l i d support and the ligand, the p o s s i b i l i t y of non-biospecific adsorption of contaminant proteins onto the hydro-phobic side chain may increase and reduce the e f f i c i e n c y of the enzyme pu r i f i c a t i o n . This synthetic route involved the formation of an amide - 70 -C02tBu C02tBu 134 R x - -CH-CH2, R 2 = -CH=CHC02Me 135 R x = -CH=CHC02Me, R 2 = -CH=CH2 bond with one amine group of an aliphatic diamine, leaving the other amine free for bonding to the s o l i d matrix. For this purpose 1,3-di-aminopropane was chosen and was coupled at the porphyrin periphery. The - 71 -linkage of 1,3-diaminopropane was carried out by f i r s t converting the a c r y l i c acid 132 or 133 to the acid chloride 136 or 137 by treatment with oxalyl chloride i n anhydrous methylene chloride at room tempera-ture. After the removal of excess oxalyl chloride, the resulting a c r y l i c acid chloride was dissolved i n a minimum of anhydrous methylene chloride and mixed with a large excess of the 1,3-diamine solution i n the same solvent and allowed to react for two hours. An excess of diamine was used to prevent the formation of a porphyrin dimer and to also neutralize the acid produced i n the reaction. Under these condi-tions, the 2-(or 4)-vinyl-4(or 2)-[3'-(N-3"-aminopropyl)]acrylamido-deuteroporphyrin-IX d i - t e r t - b u t y l ester 73 or 74 was prepared i n a 30% yi e l d . 136 R-L - -CH-CH 2 73 R X - -CH«CH 2 R 2 » -CH-CH-COC1 R2 - -CH=CHCONH(CH 2) 3NH 2 137 R L - -CH-CH-COC1 74 R L - -CH=CHC0NH(CH 2) 3NH 2 R 2 - -CH-CH 2 R2 ~ -CH-CH 2 - 72 -Variation i n the reaction conditions improved the y i e l d of the product to 50%. In this reaction, the a c r y l i c acid chloride was reacted with excess diamine at 0°C under a nitrogen atmosphere. The reaction mixture was then warmed up to room temperature and refluxed for two hours. The diamine-linked a f f i n i t y ligand showed a higher R f on s i l i c a gel in comparison to the starting a c r y l i c acid. Because of the presence of the free amino group, compounds 73 and 74 on a s i l i c a plate showed streaking which was eliminated by the addition of a drop of t r i e t h y l a -nilne Compounds 73 and 74 were p u r i f i e d by column chromatography ( s i l i c a gel) using chloroform-methanol (100:1 v/v) containing a drop of triethylamine as eluent. A solution of 73 or 74 in methylene chloride showed a v i s i b l e spectrum intermediate between an etio- and rhodo-type spectrum. This may be due to the weaker electron withdrawing effect of the amide group, as compared to those of the monoformyl and a c r y l i c acid derivatives. The nmr spectra showed the presence of the a l k y l side chain i n the 6 = 3 to 4 region. The mass spectra of these compounds were d i f f i c u l t to obtain. The rapid insertion of these compounds at high temperature (280°C) gave the molecular ion peak at m/e = 774. Confirmation of the structure suggested by the mass spectrum came when compound 73 (or 74) was photolyzed to the photoprotoporphyrin derivative 138 (or 139). The compound 138 (or 139) exhibited a peak at m/e - 806. I t appears that the formation of the "chlorin" chromophore had a s u f f i c i e n t d i s t o r t i n g effect on the macrocycle to increase i t s v o l a t i l i t y . - 73 -2.7 METALLATION OF THE AFFINITY LIGAND* The main aim of the a f f i n i t y ligand synthesis was to mimic the heme prosthetic group, i n order to use i t for the p u r i f i c a t i o n of heme oxygenase. Because heme oxygenase binds both cobalt and iron protopor-phyrin- IX, but metabolises only the l a t t e r , i t was important to make both Iron and cobalt derivatives of the a f f i n i t y ligand. The iron(III) complexes of the 2-(and 4)-vinyl-4-(and 2)-[3'-(N-3"-aminopropyl)]acryl-amidodeuteroporphyrin-IX di-tert-butyl esters (73 + 74) were obtained by 168 169 refluxing with anhydrous ferrous chloride i n dimethylformamide. ' The solution was then s t i r r e d i n a i r to give the Fe(III) complex 140. 74 -The pyridine hemochrome spectrum of the a f f i n i t y ligand 140 showed a-and /?-bands at 570 and 528 nm, respectively. T h e cobalt complex of the a f f i n i t y ligand was prepared by refluxing 127 i t w i t h cobalt acetate i n chloroform under a nitrogen atmosphere. 2.6 REMOVAL OF THE TERTIARY BUTYL GROUPS* The l a s t step i n the synthesis involved the removal of the ter t i a r y b u t y l ester groups and the regeneration of the carboxylic acid deriva-tives. The removal of ter t i a r y butyl ester groups i s most commonly carried out by treatment with t r i f l u o r o a c e t i c acid at room tempera-131 ture or by passing dry HC1 gas into methylene chloride solution at - 75 -0 ° C . The dicarboxylic acid derivatives of compounds 140 and 141 were prepared by treating them in anhydrous methylene chloride with t r i f l u o r -oaceti c acid at room temperature. After 10 minutes, when the reaction was complete, t r i f l u o r o a c e t i c acid was removed under reduced pressure. The d i a c i d prepared from this reaction was found to be soluble i n an aqueous sodium carbonate solution (0.2 M), and the v i s i b l e spectrum of the pyridine hemochrome of compound 71 showed an a- and ^-bands at 568 and 528 nm. 140 M - F e 1 1 1 71 M - F e 1 1 1 141 M - Co 1 1 72 M - C o 1 1 1 Metal insertion was observed by v i s i b l e spectrum. Further characterization for the identity and nature of these metal complexes and the ligands around them i s desirable. - 76 -PART 2 SYNTHESIS OF AZAPORPHYRINS - 77 -3.1 PYRROLES The synthesis of azaporphyrins i n this work u t i l i z e d b a s i c a l l y four pyrroles 142, 143, 144, and 145. Pyrroles 142 and 143 were synthesized i n large quantities by a modified Knorr synthesis. In the c l a s s i c a l Knorr r e a c t i o n " ^ 0 (Fig. 10), ethyl acetoacetate 146 i s f i r s t reacted with 0.5 mole of aqueous sodium n i t r i t e to give an equimolar mixture of ethyl acetoacetate and ethyl oximinoacetoacetate 147. The l a t t e r i s then reduced i n s i t u by zinc and acetic acid to give the a-amino ketone 148 which i n turn reacts with 146 to form the Schiff's base 149. The Schiff's base further cyclizes to form the pyrrole 151 on loss of water. During this reaction, the a-amino ketone unfortunately s e l f condenses to - 78 -Fig. 10: Synthesis of Knorr's pyrrole. - 79 -give a mixture of S c h i f f s bases and these lead to the formation of undesirable by-products, eventually lowering the yield of the desired pyrrole. In order to minimize the formation of by-products, modification of the Knorr synthesis is possible by variations in both the oxime and the 171 172 /j diketone. ' This can be easily carried out by isolating the oxime and then slowly adding this to a mixture of reducing agent and 171 ft-dike tone. A further improvement over Kleinspehn's procedure was 172 obtained by Paine and Dolphin, where the oxime was prereduced c a t a l y t i c a l l y to an amine in the absence of diketone. The amine obtained through this procedure then reacted with the diketone to give the desired pyrrole in high yield. The presence of an ester functionality in both pyrroles 142 and 143 serves as an important blocking and protecting group, which can be e a s i l y removed or modified to another functionality. 1-1,1 Synthesis of Monopyrroles The pyrroles 142 and 143 were synthesized by variations of the Knorr reaction. In the synthesis of the pyrrole 142, ethyl oximino-acetoacetate 147 was reduced to the corresponding aminoketone in the presence of pentane-2, 4-dione 152. The reaction proceeds in a manner similar to the Knorr reaction (Fig. 11). The pyrrole 143 was prepared by reaction of diethyl aminomalonate 157 (obtained from the catalytic reduction of diethyl oximinomalonate - 80 -Fig. 11: Synthesis of monopyrrole 142 by variation of the Knorr reaction - 81 -Fig. 12: Synthesis of monopyrrole 143 - 82 -172 173 156) with ethyl 5-methyl-4,6-dioxoheptanoate 159 i n b o i l i n g acetic 172 acid (Fig. 12). The other two pyrroles 144 and 145 were obtained from Dr. P. Roussi and Dr. T.P. Wijesekera. The necessary modifications at the ft- and a-positions of these pyrroles are discussed i n the following sections, 3.1.2 and 3.1.3. 3.1.2 Preparation of Synthetically Useful Pyrroles v i a Transformations of Substituents The synthesis of porphyrins v i a d i p y r r o l i c and t e t r a p y r r o l i c intermediates involves the manipulation of a- and /9-substituents of the intermediate pyrroles. Due to the greater r e a c t i v i t y of the a- over the /3- substituent, i t was important to carry out modifications at the /3-position while the a-position was completely protected. The s y n t h e t i c a l l y useful pyrrole 162 was prepared from 142 by diborane reduction of the acetyl group, as reported by Whitlock and 174 Hanauer. The reduction was carried out by dropwise addition of boron t r i f l u o r i d e etherate, to a cooled s t i r r e d suspension of 142 and sodium borohydride i n tetrahydrofuran, under an inert atmosphere. In order to preserve the ethyl ester side chain of the pyrrole, the reduction was carried out i n the presence of ethyl acetate i n the reaction mixture. The progress of the reaction was observed by t i c . On completion of the reaction, the reaction mixture was quenched with acetic acid followed by water. The pyrrole 162 was further modified (Section 3.1.3) and used i n the synthesis of porphyrins where ethyl and methyl groups were required. - 83 -For a porphyrin where a methyl ester propionic side-chain was required, the ethyl ester analogue 143 was f i r s t converted to the corresponding benzyl ester analogue 163. This t r a n s e s t e r i f i c a t i o n was 176 -17 8 effected by a high temperature modification of the procedure developed by Kenner and c o w o r k e r s , u s i n g benzyl alcohol and sodium benzyloxide. The benzyl alcohol was d i s t i l l e d beforehand from anhydrous potassium carbonate i n order to remove most of the water and any benzoic acid present. The ethyl ester 143 was heated to reflux i n benzyl alcohol and a solution of sodium i n benzyl alcohol was added i n 1 mL portions. A vigorous evolution of ethanol vapour was observed during 84 -the reaction, with concomitant lowering of the reflux temperature. The product 163 was isolated by adding the hot reaction mixture to methanol containing s u f f i c i e n t acetic acid to quench the catalyst, followed by d i l u t i o n with water. The compound 164 was then obtained by retromethylation of the side-chain benzyl ester of 163 i n dry methanol (Mg-dried) at room temperature, using sodium methoxide ( i n anhydrous methanol) as the 175 catalyst. The s o l u b i l i t y of pyrrole 163 was enhanced by the addition of tetrahydrofuran to the reaction mixture. The progress of the reac-t i o n was followed by t i c . After the completion of the reaction, the product was c r y s t a l l i z e d by addition of water, giving an 80-90% y i e l d of the pure product 164. This transformation was necessary for the porphyrins bearing propionate side-chains. The presence of the benzyl ester at the or-position has advantage over the ethyl ester analogue because i t can be cleanly and conveniently removed by c a t a l y t i c hydroge-nation without affe c t i n g the methyl ester side chain. I t i s also important to note that the transformations at the a-position can be carried out with ease without affecting the benzyl ester. 3.1.3 Preparation of Synthetically Useful Pyrroles Via Transformations of cr-Substituents To produce synthetically useful pyrroles for the synthesis of dipyrromethanes, dipyrromethenes and biladienes-ac, the a- positions of the above pyrroles were modified. Depending on the reaction conditions 85 -and the reagents used, the a- methyl groups were converted to various f u n c t i o n a l i t i e s (e.g. acid, aldehyde or acetoxy). The different trans-formations are shown i n Figs. 13, 14 and 15. The three key intermediates required for the synthesis of dipyrro-methenes were compounds 169, 177 and 181. The conversion of the a-methylpyrrole 162 to the synthetically useful a-bromo-a'-carboxy-pyrrole 169 was carried out v i a the sequence shown i n Fig. 13. The f i r s t step i n this transformation involved t r i c h l o r i n a t i o n of the a-methyl group of 162 with three equivalents of s u l f u r y l chloride, and subsequent hydrolysis to give the a-carboxypyrrole 165. a-Methylpyrro-125 le-a'-monoesters are usually t r i c h l o r i n a t e d i n ether solution. Ether unfortunately reacts with s u l f u r y l chloride resulting i n variable yields of the product. To overcome this problem, chlorocarbon solvents (e.g. 179 180 carbon tetrachloride, methylene chloride ) have been used, but i t was found that either the t r i c h l o r i n a t i o n reaction did not go to comple-tio n or the product was damaged by the hydrogen chloride evolved. The t r i c h l o r i n a t i o n of the a-methyl group was eventually carried out according to the method reported by Battersby and coworkers1''6 using methylene chloride as solvent and ether as co-solvent. In this reac-tion, the a-methylpyrrole 162 was f i r s t dissolved i n dry methylene chloride, and anhydrous ether was added just before the addition of the oxidant ( s u l f u r y l chloride). Sulfuryl chloride was diluted with methy-lene chloride and added rapidly to the a-methylpyrrole 162 with care. The reaction was performed i n a large flask to insure that the vigorous effervescence did not make the contents overflow. The reaction mixture was s t i r r e d for h a l f an hour, the solvent removed, and the resulting - 86 -13: Synthesis of synthetically useful monopyrroles 169 and 177 via transformations of a-substituents. - 87 -viscous red o i l was hydrolyzed i n refluxing aqueous acetone i n the presence of anhydrous sodium acetate. The product, a-carboxypyrrole 165 was obtained i n greater than 80% y i e l d after p u r i f i c a t i o n . The transformation of 165 to 167 was carried out v i a decarboxyla-tive iodination. The reaction, 165 to 166, was carried out i n a two-phase system of dichloroethane and water, using sodium bicarbonate to s o l u b i l i z e the carboxypyrrole 165 i n the aqueous phase. The iodination was car r i e d out using excess iodine i n potassium iodide and the reaction mixture refluxed. In the two-phase system, the iodopyrrole 166 once formed moves into the organic phase from which i t can be easil y isolated after destroying excess iodine with sodium b i s u l f i t e . The r e c r y s t a l l i z a t i o n of the crude product gave approximately 90% of pure iodopyrrole 166. The dehalogenation of compound 166 was carried out i n a mixture of potassium iodide and hydrochloric acid, and the iodine liberated was destroyed by hypophosphorous acid, giving the a-unsubstituted pyrrole 167. The product 167 was p u r i f i e d by column chromatography to give a 90% y i e l d . Further transformation of 167 to 169 was carried out by f i r s t preparing the a-unsubstituted-a'-carboxypyrrole 168 by saponification of the ester i n strong alkaline medium (sodium hydroxide). The bromination of the re s u l t i n g a-unsubstituted-a'-carboxypyrrole 168 was carried out by s t i r r i n g i t s suspension i n g l a c i a l acetic acid with one equivalent of bromine i n acetic acid at 10 to 15°C over a period of 30 to 60 seconds. The ether extract of the bromopyrrole 169 was shaken with an aqueous sodium hydroxide solution. The neutralization of the aqueous solution 88 -resulted i n a-bromo-a'-carboxypyrrole 169 in 63% y i e l d . A direct approach to synthesize this pyrrole from 165 v i a 170 f a i l e d to give any product at the saponification step (170 -» 169). The compound 177 was synthesized through the reaction sequence given i n Fig. 13. The ethyl ester functionality of compound 162 was converted to the benzyl ester 171 by t r a n s e s t e r i f i c a t i o n as described e a r l i e r (Section 3.1.2). The a-formylpyrrole 172 was prepared from the a-methylpyrrole 171 by reaction with two equivalents of s u l f u r y l chloride followed by the hydrolysis of the chlorinated product. The chlorination of 171 was carried out i n ice-cold methylene chloride. Because of high s o l u b i l i t y and poor c r y s t a l l i z a b i l i t y of the free aldehyde, the product was often obtained i n low yields and i n impure form. To overcome this problem the free aldehyde was protected by the cyanoacrylate group by treatment with methyl cyanoacetate i n methanol in 181 the presence of base as a catalyst. The product obtained i n high y i e l d through this sequence was pure and c r y s t a l l i n e . This i s an excellent p u r i f i c a t i o n method for the formylpyrrole as the impurities are l e f t , i n the methanol solution. A further advantage of the cyanoacrylate group i s that such derivatives are very resistant to reduction and especially oxidations, therefore further modification at a'-position can be carried out with ease. The carboxypyrrole 174 was prepared by cleaving the benzyl ester group by hydrogenation using palladium/charcoal as catalyst. The carboxypyrrole thus obtained, was converted to an a-unsubstituted pyrrole v i a the iodopyrrole 175. The conversion of a-carboxypyrrole 174 to iodopyrrole 175 was effected by dropwise addition of iodine monochlo-89 -ride to 174 suspended i n g l a c i a l acetic acid i n the presence of excess sodium acetate as buffer. Excess iodine was removed by hypophosphorous acid and the product was precipitated by the addition of water. The iodopyrrole 175 was r e c r y s t a l l i z e d from methanol-water as bright yellow needles i n 83% y i e l d . Dehalogenation of 175 was effected by c a t a l y t i c hydrogenolysis with 10% palladium on charcoal or platinum oxide i n the dark. The dehaloge-nation reaction was slow and the product was often contaminated by bipyrrole which was produced by condensation of 175 with the a-unsubsti-125 181 tuted pyrrole 176 when exposed to l i g h t . ' In order to avoid these problems, the reduction of 175 was performed by heating a suspension of pyrrole and zinc dust i n g l a c i a l acetic acid on a s t i r - h o t plate for 45 minutes to one hour. The zinc dust was removed by f i l t r a t i o n and c r y s t a l l i z a t i o n of the product was induced by the addition of water. The a-unsubstituted pyrrole 176 was r e c r y s t a l l i z e d from methanol-water to give pure product In greater than 90% y i e l d . The deprotection of the formyl group was carried out by refluxing the a-unsubstituted pyrrole 176 i n strong alkaline solution (5-6 N sodium hydroxide) i n an inert atmosphere for 2-3 hours. Upon cooling the reaction mixture, the pale brown s o l i d 177 was collected by f i l t r a t i o n and washed with water. The yields of the product varied between 80-85%. This v a r i a t i o n may result from product loss by sublima-tion, which was evident by the presence of white needles In the con-denser. Compound 177 was used for the synthesis of dipyrromethenes. The sample of 177 for analysis was p u r i f i e d by r e c r y s t a l l i z a t i o n from methanol-water. - 90 -The conversion of compound 164 to 181 was carried out by the sequence shown i n Fig. 14. The f i r s t step involved the oxidation of a-methyl group to a formyl group using reaction conditions similar to those used i n the transformation of 171 to 172. In this sequence the a-formyl group was not protected because the deprotection step at the end of the synthetic scheme would require alkaline condition under which the /3-side chain ester would be saponified and subsequent i s o l a t i o n of the product would be d i f f i c u l t . To eliminate this problem, an unpro-tected reaction sequence was used. The benzyl ester 178 was cleaved by Fig. 14: Synthesis of the synthetically useful pyrrole 181 v i a transformation of a-substituents. - 91 -hydrogenolysis with palladium/charcoal i n tetrahydrofuran containing a drop of triethylamine to give the a-formyl-a'-carboxypyrrole 179. The synthesis of the iodopyrrole 180 was carried out by decarboxylative iodination of 179 i n a two-phase system (chloroform-water) using one equivalent of iodine. The organic layer was separated and taken to dryness. As i t i s known that crude iodo-aldehyde decomposes on stand-i n g , 1 2 ^ the removal of iodine was immediately carried out by hydrogena-ti o n using 10% palladium on charcoal as catalyst i n methanol containing anhydrous sodium acetate. ^ / C H 3 Pb(0Ac) 4 C K R C H 3 C 0 2 H R 2 A 0 0 162 RJ-R2—C 2H 5 182 R 1-R 2--C 2H 5 145 Rj_—CH 3, R 2=-C 2H 5 183 Rj —CH 3, R 2--C 2H 5 144 R1=-CH2CH2C02Me 184 R x—CH 2CH 2C0 2Me R 2— CH 2-C 6H 5 R2=-CH2-C6H5 Fig. 15: Synthesis of acetoxymethylpyrroles - 92 Upon completion of the hydrogenolysis, the crude a-formyl-a'-unsubsti-tuted pyrrole 181 was isolated by water-methylene chloride p a r t i t i o n , and the organic phase was taken to dryness. Compound 181 was extracted into b o i l i n g water and kept i n the refrigerator overnight to give f l a t , needle shaped, colourless crystals i n 60% y i e l d . The acetoxymethylpyrroles 182, 183, and 184 were prepared from the i r respective a-methylpyrroles 162, 145, and 144 by heating with 182 lead tetraacetate i n g l a c i a l acetic acid (Fig. 15). The acetoxy-methylpyrroles were the most useful precursors for synthesis of the symmetrical dipyrromethanes used i n this work. 3.2 SYNTHESIS OF DIPYRROMETHANES AND BILADIENES-ac The syntheses of the symmetrical dipyrromethanes 185, 186, and 187 183 were carried out by the procedure of Clezy and Liepa. In this reaction, the acetoxymethylpyrroles 182, 183, and 184 (source of pyrryl-carbinyl cation) were refluxed with 20% aqueous acetic acid to give the dipyrromethanes 185, 186, and 187 respectively (Fig. 16). The reaction by which dipyrromethane formation proceeds i s through the formation of an a-pyrrylcarbinyl cation, which s e l f condenses to give the symmetrical dipyrromethane with the elimination of formaldehyde (Fig. 17). The formation of symmetrical dipyrromethanes was confirmed by nmr, where methylene (bridge) protons appeared at the S = 3.8-4.5 ppm region as a sharp singlet. - 93 -182 R1=R2=-C2H5 183 Rj--CH 3, R 2=-C 2H 5 184 R1=-CH2CH2C02Me R2=-CH2C6H5 Fig. 16: Synthesis of symmetri 185 R1=R2=-C2H5 186 R1=-CH3, R2=-C2H5 187 R1=-CH2CH2C02Me R2=-CH2C6H5 1 dipyrromethanes After the synthesis of dipyrromethanes i n s u f f i c i e n t amount, i t was necessary to synthesize the 1,19-dideoxybiladiene-ac dihydrobromides. To obtain these i n good y i e l d , the 5,5'-substituted diesters (dipyrro-methanes) were f i r s t converted to 5,5'-dicarboxylic acids, either by saponification ( i f ethyl esters were used) or by hydrogenolysis ( i f benzyl ester was used). The saponification of compound 185 was carried out by dissolution i n ethanol and refluxing with strong a l k a l i solution (10 equivalents of NaOH i n water) for 3 to 4 hours. The diacid was precipitated by careful neutralization of the base with acetic acid i n an ice-bath. The diacid thus obtained was a white powder but slowly - 94 -Fig. 17; The synthesis of dipyrromethanes from a-acetoxymethylpyrroles changed to a l i g h t pink colour. Under similar reaction conditions of saponification (as stated above), compound 186 did not dissolve and was recovered as diester. However, the saponification conditions were changed and the diester 186 was refluxed i n strong aqueous a l k a l i solution using 2-propanol as solvent. After refluxing the solution for 12 hours, the diester was saponified i n 80% yield. I t appears that the higher reflux temperature was necessary to enhance the s o l u b i l i t y of the diester 186. The benzyl ester derivative 187 was easily converted to i t s d i a c i d derivative 190 by c a t a l y t i c hydrogenolysis over palladium on charcoal i n tetrahydrofuran. During this reaction, p r e c i p i t a t i o n of 190 as a white powder was observed. The diacid 190 was recovered from the reaction mixture by treating i t with aqueous sodium bicarbonate. The catalyst was then removed by f i l t r a t i o n and the aqueous f i l t r a t e was ca r e f u l l y neutralized by acetic acid i n an ice-bath to give the 5,5'-dicarboxy-dipyrromethane 190 as a white powder (Fig. 18). 185 R1=R2=-C2H5 186 R]_—CH3, R 2=-C 2H 5 187 R x—CH 2CH 2C0 2Me R 2=-CH 2C 6H 5 188 R } - -C 2H 5 189 R-L—CH3 190 R-L=-CH2CH2C02Me Fig. 18: Synthesis of 5,5'-dicarboxydipyrromethanes - 96 -The 1,19-dideoxybiladienes-ac compounds were prepared by the acid catalyzed condensation of two equivalents of the a-formyl-a'-unsubstituted pyrroles with the dipyrromethane-5,5'-dicarboxylic a c l d . 1 8 ^ Compounds 191 and 192 were prepared by s t i r r i n g a suspension of the 5,5'-di-carboxydipyrromethane 188 with two equivalents of 4-ethyl-5-formyl-3-methylpyrrole 177 and 2-formyl-3,4-dimethylpyrrole, respectively, i n methylene chloride, i n the presence of 48% hydrobromic acid (Fig. 19). After 10 to 15 minutes of s t i r r i n g , the solvent was concentrated under reduced pressure and the solution cooled i n an ice-bath to give the 1,19-dideoxybiladienes-ac, 191 and 192, i n greater than 90% y i e l d . Use 192 Fig. 19: Synthesis of 1,19-dideoxybiladiene-ac dihydrobromides 191 and 192 - 97 -of s i m i l a r reaction conditions gave the other 1,19-dideoxybiladienes-ac 193, 194 and 196 (Fig. 20). Compound 197 was obtained from Dr. P. Roussi and converted to 198 by f i r s t cleaving the butyl ester of 197 by TFA, followed by acid catalyzed condensation of the a-formyl-a'-unsubsti-C02Me CC^ Me 194 Fig. 20: Synthesis of 1,19-dideoxybiladiene-ac dihydrobromides 193, 194 and 196. - 98 -tuted pyrrole i n methylene chloride. The solvent was removed under reduced pressure and diethyl ether was added to the solution. The 1,19-dideoxybiladiene-ac 198 was obtained i n 90% y i e l d . 198 The formation of 1,19-dideoxybiladiene-ac dihydrobromides was confirmed by th e i r nmr spectra, which showed a singlet for the -CH2- bridge protons i n the region of 5 = 4.5 4.9 ppm and a doublet for the a-unsubstituted protons at 5 = 7.5-7.8 ppm (J = 4 Hz). 3.2.1 Bromination of 1,19-dideoxybiladiene-ac dihydrobromides Bromination of the 1,19-dideoxybiladiene-ac dihydrobromides 191, 192, 193, 194, 196, and 198, was carried out using reaction conditions similar to those used for the bromination of dipyrromethenes (section 3.3.1). In this reaction, the 1,19-dideoxybiladiene-ac salts were taken i n methylene chloride, t r i f l u o r o a c e t i c acid added, followed by the addition of a three to four f o l d excess of bromine solution i n methylene chloride (Fig. 21). The reaction mixture was kept at room temperature - 99 -R 7p.- C H 2 ^ 2 / T F A 191 R1=R4=R6=R7=-C2H5 R 2- R3- R5- R8— C H3 192 R1=R2=R3=R4=R5=R8—CH3 R 6=R 7=.C 2H 5 193 Ri -+ R8=-CH3 194 R 1=R 4—C 2H 5 R2=R3=R5=R3=-CH3 R6=R7=-CH2CH2C02Me 196 R x ->• R 8=-C 2H 5 198 R 2 - R 3 - R 5 - R 7 — CH 3 R4=R6=-CH2CH2OCOPh R1=R8=-CH2CH2C02Me 199 R1=R4=R6=R7=-C2H5 R2= R3= R5= R8=- C H3 87 R1=R2=R3=R4=R5=R8=-CH3 R 6=R 7 =.C 2H 5 200 Ri - Rg—CH 3 201 R1=R4=-C2H5 R2=R3=R5=R8=-CH3 R6=R7=-CH2CH2C02Me 202 R x -+ R 8=-C 2H 5 203 R2=R3=R5=R7=-CH3 R4=R6=-CH2CH2OCOPh R1=R8=-CH2CH2C02Me Fig. 21: Bromination 1,19-dideoxybiladiene-ac dihydrobromides. - 100 -for 10 to 15 minutes and the excess bromine was removed under reduced pressure and traces of bromine f i n a l l y removed under vacuum. The s o l i d residue was redissolved i n a minimum of methylene chloride and cooled i n an ice-bath. The c r y s t a l l i z e d product was f i l t e r e d and washed with ice cooled methylene chloride to give 1,19-dibromo-1,19-dideoxybiladienes-ac i n good y i e l d (>80%). This transformation of 1,19-dideoxybiladienes-ac to 1,19-dibromo-l,19-dideoxybiladienes-ac was observed by the disappear-ance of a doublet i n S = 7.5-7.8 ppm region and the appearance of the singlet due to methylene (bridge-CH^) protons at 5 = 4.5-4.9 ppm. The mass spectra of these compounds showed a peak at M+ - (2HBr + 2 Br), i.e., the 1,19-dibromo-l,19-dideoxybiladiene-ac cyclizes i n the probe to give a corrole. 3.3 SYNTHESIS OF DIPYRROMETHENES The dipyrromethenes produced i n this work were synthesized by acid catalyzed condensation of an a-carboxypyrrole with an a-formylpyrrole (Fig. 22) . This Is a minor modification of the c l a s s i c a l method of dipyrromethene synthesis i n which the a-formylpyrrole 204 condenses with the a-unsubstituted pyrrole 205 i n the presence of strong acid to give the dipyrromethenium s a l t 206. In the present work the a-unsubstituted pyrrole i s generated i n s i t u by acid catalyzed decarboxylation upon addition of hydrobromic acid. The free base dipyrromethenes are unst-able and therefore they are stored and used as the hydrobromide s a l t s . The four dipyrromethenes 208, 210, 211, and 212 synthesized i n this work were used for the synthesis of two biladienes-ac bearing unsymmet-r i c a l substitution patterns at their periphery. The a-carboxypyrroles 207 and 209 were obtained by c a t a l y t i c hydrogenolysis of their benzyl ester derivatives 144 and 171 over palladium/charcoal i n tetrahydro-furan. These were then reacted with the a-formylpyrrole 177 i n the presence of aqueous hydrobromic acid (48%), using tetrahydrofuran as the solvent, to give the dipyrromerhenium salts 208 and 210, respectively, in 90% y i e l d . The solvent was concentrated under reduced pressure, methanol added and the reaction mixtures were cooled i n an Ice-bath. The c r y s t a l l i z e d products 208 and 210, were f i l t e r e d and washed with ether. Using similar reaction conditions (as above), pyrrole 169 was coupled with the a-formyl-a'-unsubstituted pyrroles 177 and 181 to give the respective dipyrromethenes 211 and 212 i n less than 40% y i e l d . The yields of the products were improved when the pyrroles were dissolved i n methylene chloride and condensed using 30% HBr In acetic acid. 102 Fig. 22: Synthesis of unsymmetrical dipyrromethenes. - 103 -3.3.1 Bromination of dipyrromethenes The bromination of dipyrromethenes to bromo-bromomethylmethenes was carried out by the procedure developed by Hiom and coworkers. 1 8 5 In this procedure, the bromination of dipyrromethenes was carried out i n methy-lene chloride (or 1,2-dichloroethane)-trifluoroacetic acid (3:1) with excess bromine (usually 4 to 5 equivalents) at room temperature over 4-5 days. Using t h i s procedure, the dipyrromethenes 208 and 210 were brominated to give the 5-bromo-5'-bromomethylmethenes 213 and 214 (Fig. 23). After completion of the reaction, the excess bromine was removed under vacuum overnight. The s o l i d residue was redissolved i n methylene chloride and then treated with cyclohexene. The solution was concen-trated under reduced pressure and the products were c r y s t a l l i z e d by the addition of diethyl ether to the ice-cold solution. The 5-bromo-5'-bromomethylmethenes 213 and 214 were obtained i n greater than 80% y i e l d H Br 208 R—CH2CH2C02Me 213 R=-CH2CH2C02Me 210 R=-C2H5 214 R=-C2H5 Fig. 23: Bromination of dipyrromethenes 208 and 210. 104 -and found to be pure by analysis. The nmr spectra of these methenes showed a sharp sin g l e t for the bromomethyl proton i n the S - 4.8 to 4.9 region. 3.3.2 Synthesis of l,19-dibromo-l,19-dideoxybiladienes-ac from dipyrromethenes Bromination of the 5-unsubstituted-5'-methyldipyrromethene hydro-bromides 208 and 210 to give 5-bromo-5'-bromomethyldipyrromethene hydro-bromides 213 and 214 (Fig. 23) preceded the synthesis of the 1,19-di-bromo-1,19-dideoxybiladiene-ac dihydrobromides 81 and 215 by condensa-tion of 213 with 212, and 214 with 211, respectively, i n the presence of 126 SnCl^ at room temperature. The t i n complexes were not isolated, but converted to the corresponding dihydrobromide salts 81 and 215 i n 70% yi e l d by treatment with methanolic hydrobromic acid (Fig. 24). 3.4 SYNTHESIS OF MONOAZAPORPHYRINS FROM 1,19-DIBR0M0-1,19-DIDE0XY-BILADIENE-ac SALTS The synthesis of monoazaporphyrins, using 1,19-dibromo-l,19-di-118 121 deoxybiladiene-ac salts and 1,19-diiodo-1,19-dideoxybiladiene-ac 120 salts has been attempted (section 1.7). In this reported procedure, these salts were reacted with sodium azide i n refluxing methanol under an inert atmosphere and the product was obtained i n variable yields. - 105 -Fig. 24: Synthesis of unsymmetrical 1 (19-dibromodldeoxyblladlene-ac dihydrobromlde salts 81 and 215. 106 With the aim of finding a general procedure for the c y c l i z a t i o n of 1,19-dibromo-l,19-dideoxybiladiene-ac salts to monoazaporphyrins i n quantitative y i e l d s , the reaction conditions were varied using compound 199 as a model. The reaction conditions, reactants, and the y i e l d of the product are l i s t e d i n Table 2. The use of hydroxylamine hydrochloride and hydrazine hydrate i n pyridine produced very discouraging results. The reaction of sodium azide with 1,19-dibromo-l, 19~-dideoxybiladiene-ac dihydrobromide 199 under extremely dry conditions i n anhydrous methanol gave the product i n 50% y i e l d , whereas the change of solvent from methanol to DMSO under similar reaction conditions gave a poor product y i e l d . Due to the low y i e l d of monoazaporphyrin obtained from the above reactions, i t was necessary to search for an alternative procedure where sodium azide would be dissolved i n an organic phase, or where i t could be extracted as an ion pair, to then react with 199 in the organic phase to give monoazaporphyrin i n good y i e l d . The a b i l i t y of crown ethers to 186-190 solvate cations i n both polar and nonpolar aprotic solvents prompted us to carry out the reaction i n two phases ( s o l i d - l i q u i d phase) . I n i t i a l l y , the crown ether-sodium azide complex was prepared by dissolving both components i n methanol, followed by displacement of methanol by benzene. The complex was found to be insoluble i n benzene. To keep i t i n solution, a small quantity of methanol was used and as such reacted with compound 199 i n refluxing methylene chloride. The yi e l d of the product obtained through this procedure ranged from 80-81%. A similar observation was also made when tetrabutylammonium azide was - 107 -Table 2: Various Reaction Conditions Attempted for the Cyclization of 199 to Monoazaporphyrin 78 Reaction Solvent Reactant Reaction conditions % Y i e l d Temp Time Atmos. S t i r 1 DMSO NaN3 70°C 12 h N 2 Yes 20 2 Methanol NaN3 reflux 12-24 h N 2 - 50 3 Pyridine H2N0H.HC1 ambient 12 h N 2 - 14 4 Pyridine H2NNH2.H20 reflux 3 h N 2 - 20 5 C 6H 6 CE Dibenzo-18 crown-6-NaN3 complex reflux 3.5 h 81 6 C 6H 6 18-Crown-6-NaN3 complex reflux 3.5 h N 2 - 80 7 CH 2C1 2 18-Crown-6-NaN3 complex reflux 2 h - - 80 8 CH 2C1 2 Tetrabutyl ammonium azide reflux 12 h - - 81 9 CH 2C1 2 Dibenzo-18-crown-6-NaN3 ambient 12-15 h - vigorous 95 10 CH 2C1 2-pyridine Tetrabutyl ammonium-bromide -NaN3 ambient 12-15 h vigorous 94 - 108 -refluxed with 199 i n methylene chloride. However, a modification of this procedure gave a quantitative y i e l d of the model monoazaporphyrin 78. In this reaction, crown ether (dibenzo-18-crown-6) and tetrabuty-lammonium bromide were used as phase transfer agents. 1,19-Dibromo-1,19-dideoxybiladiene-ac dihydrobromide 199 was dissolved i n methylene chloride; crown ether or tetrabutylammonium bromide (25% of the sub-strate concentration) and s o l i d sodium azide (10 f o l d excess) were added followed by the addition of pyridine. The entire reaction mixture was s t i r r e d for 12-15 hours at room temperature. The monoazaporphyrin 78 was obtained i n greater than 90% y i e l d through this procedure. The monoazaporphyrins 88, 216, 100, and 219 were prepared i n greater than 90% y i e l d from the 1,19-dibromo-l,19-dideoxybiladiene-ac dihydro-bromides 87, 200, 202, and 215, using dibenzo-18-crown-6 as a phase transfer agent (Fig. 25). The biladiene-ac dihydrobromides bearing propionate methyl ester 0-side chains 201, 203 and 81 gave the corresponding monoazaporphyrin i n 80-81% y i e l d . Under the reaction conditions used, the /9-propionate methyl esters were saponified giving two spots on t i c . After the work up of the reaction, the methyl esters were regenerated by treatment with diazomethane. The products were purified by s i l i c a gel column chromatography using methylene chloride-methanol (100:0.5 v/v) as eluent. The monoazaporphyrins obtained showed macrocyclic aromatic conjugation, and exhibited Soret bands between the 370 and 375 nm region. The nmr spectra of these porphyrins exhibited the presence of three meso-protons (-CH=) between 9 to 11 ppm. The mass spectra of the azaporphyrins exhibited molecular ion peaks (M+) of 100% intensity. - 109 -199 87 200 201 202 203 81 215 R1=R4=R6=R7=-C2H5 R2= R3= R5 = R8=- C H3 R 1 = R 2 = R 3 = R 4 = R 5 = R 8 = - C H 3 R 6 = R 7 = . C 2 H 5 R l ~* R8 =' C H3 R1=R4=-C2H5 R2=R3=R5=R8=-CH3 R6=R7=-CH2CH2C02Me R l ^ R8 = _ C2 H5 R2=R3=R5=R7=-CH3 R4=R6=-CH2CH2OCOPh R1=R8=-CH2CH2C02Me R 1 = R 3 = - C 2 H 5 R2=R4=R5=R8=-CH3 R6=R7=-CH2 CH 2 C02Me R1=R3=R5=R7=-C2H5 R2=R4=R6=R8=-CH3 78 R1=R4=R6=R7=-C2H5 R2-R3=R5=R8=-CH3 88 R1=R2=R3=R4=R5=R8=-CH R 6-.R 7~C 2H 5 216 Rx R8=-CH3 217 R^R^-C^s R2=R3=R5=Rg=-CH3 R6=R7=-CH2CH2C02Me 100 Ri -<• R 8=-C 2H 5 218 R2=R3=R5=R7=-CH3 R4=R6=-CH2CH20C0Ph R]_=R8=- CH2CH2C02Me 82 Rr=R3=-C2H5 R2=R4=R5=R8=-CH3 R6-R7--CH2CH2C02Me 219 R 1 - R 3 - R 5 - R 7 — C 2 H 5 R2=R4=R6=R8=-CH3 25: Synthesis of monoazaporphyrins from l,19-dibromo-l,19-dideo Xy biladiene-ac dihydrobromides. - 110 -3.5 MECHANISM FOR THE CYCLIZATION OF A 1,19-DIBROMO-l,19-DIDEOXY-BILADIENE-ac DIHYDROBROMIDE TO AZAPORPHYRIN The dimension of the crown ether (dibenzo-18-crown-6) i s such that i t can e f f e c t i v e l y coordinate with sodium ion to give a crown ether-sodium ion complex. Because the complex has a hydrophobic exterior, i t is readily s o l u b i l i z e d by non-polar or dipolar aprotic solvents. These do not re a d i l y solvate the anion and, therefore the anion i s present i n the medium either as an ion pair or a free anion and as such acts as an 190 effective nucleophile, termed a "naked anion". The f i r s t step i n the formation of an azaporphyrin from 1,19-di-bromo-l ,19-dideoxybiladiene-ac salts involves the displacement of a bromine by an azide ion (Fig. 26) to give a "vinyl azide". I t appears that the v i n y l azide decomposes at room temperature to give a v i n y l nitrene intermediate which cyclizes intramolecularly to give monoaza-Hydrophob ic Ex t e r i o r Weak A n i o n - S o l v e n t Interact ion porphyrin i n high y i e l d . - I l l -Fig. 26: Mechanism of porphyrin cyclization. - 112 -CHAPTER 3 SUMMARY AND CONCLUSIONS - 113 -SUMMARY AND CONCLUSIONS The two isomeric a f f i n i t y ligands, 73 and 74 were synthesized i n 50% y i e l d from protoporphyrin-IX di-tert-butyl ester 106b. The f i r s t step i n this synthesis involved the conversion of two v i n y l groups of 106b to two isomeric monoformyl-monovinyl derivatives, 122b and 123b, through photoprotoporphyrin-IX intermediates, 126b and 127b. 73 R - tBu 73a R - H 74 R - tBu 74a R = H - 114 -The compounds, 122b and 123b were then converted to monoacrylic acid derivatives, 132 and 133 by Knoevenagel condensation with malonic acid i n pyridine. These derivatives were further linked by a diamine (containing three methylene groups i n the chain) v i a an acid chloride intermediate to give compounds, 73 and 74. The removal of the t e r t i a r y butyl groups of 73 and 74 (which was not attempted i n the present work) can provide the two isomeric ligands 73a and 74a, respectively, which can subsequently be attached to a s o l i d support for the development of an e f f i c i e n t a f f i n i t y p u r i f i c a t i o n method for a number of heme and porphyrin binding enzymes i n the future. The iron(III) and cobalt(IH) complexes, 71 and 72, prepared i n this work, may f a c i l i t a t e future attempts at developing a p u r i f i c a t i o n procedure for heme oxygenase. Besides these complexes, the preparation of iron(III) and cobalt(II) complexes of 73a and 74a may offer an e f f i c i e n t method for the p u r i f i c a t i o n of heme oxygenase i n the future. The t e t r a p y r r o l i c intermediates (1,19-dibromo-l,19-dideoxy-biladiene-ac dihydrobromides) were synthesized and then cyclized to monoazaporphyrins ( i n high y i e l d s ) , using dibenzo-18-crown-6 as phase transfer agent (transfer of an azide ion). The synthesis of monoazapor-phyrins w i l l be of use i n finding and understanding the nature of the heme oxygenase and the intermediates involved i n the heme catabolism i n vivo and i n v i t r o i n the future. 115 -CHAPTER 4 EXPERIMENTAL 116 -4.1 GENERAL METHODS Melting Point Determinations Melting points of pyrroles, dipyrromethanes, dipyrromethenes and porphyrins were obtained using a 6548-J17 microscope equipped wich a Thomas model 40 micro hot stage melting point apparatus. Elemental Analysis Elemental analyses were performed by Mr. P. Borda of the Micro-analytical Laboratory, U.B.C. Nuclear Magnetic Resonance Spectroscopy The proton nmr spectra of a l l the monopyrroles were obtained at 8 0 MHz with a Varian XL-100 spectrometer under Fourier-transform condi-tions. The spectra of dipyrromethanes, dipyrromethenes and porphyrins were either recorded at 270 MHz with a U.B.C. NMR Centre modified Nicolet-Oxford H-270 spectrometer or 400 MHz on a Bruker WH400 spec-trometer. The chemical s h i f t s were recorded i n the 5 (ppm) scale with tetramethylsilane (TMS) (S - 0) as an internal standard and deuterochlo roform as solvent. 117 -Mass Spectrometry Mass spectra were recorded on a Varian MAT CH4-B spectrometer or a Kratos/AEl MS-902 spectrometer. High resolution measurements were obtained on a Kratos/AEI MS-50 spectrometer. Infrared Spectroscopy The infrared spectra were obtained from KBr discs using a Perkin-Elmer model 457 grating spectrometer covering the frequency range 4000-250 cm'\ The spectra were calibrated with polystyrene f i l m at 1601.4 cm"1. Electronic Spectroscopy A Cary (Model 17) spectrophotometer was used to record UV and v i s i b l e spectra. Chromatography Column chromatography on s i l i c a gel was performed using BDH s i l i c a gel (60-120 mesh), Merck Kieselgel 60H, or Merck Kieselgel 60 (70-230 mesh). In certain instances Brockman aluminum oxide (neutral, a c t i v i t y - 118 -I) was used for the p u r i f i c a t i o n of pyrroles and porphyrins. Thin layer chromatography ( t i c ) was performed using precoated s i l i c a gel GF plates (Analtech-Uniplate, 250 microns) and the compounds were detected by UV l i g h t (254 nm and 366 nm) and/or exposure to iodine. The preparative layer plates were obtained from Analtech Inc. and were s i l i c a gel GF (20x 20 cm, 1000 or 2000 microns). The eluents (solvent mixtures) used i n chromatography are expressed as volume:volume percentages. Starting Materials As none of the pyrroles required for this work were commercially available at a reasonable price, a l l of the p y r r o l i c starting materials had to be synthesized. Although some of these compounds have been reported previously i n the l i t e r a t u r e , their syntheses have been included here for completeness, and the convenience of any who might wish to make use of the information contained herein. In some instance useful modifications have been made. Reagents and Solvents A l l chemicals and solvents were reagent grade unless otherwise indicated. The p u r i f i c a t i o n of solvents was performed using appropriate literature procedure. - 119 -4.2 SYNTHESIS OF AFFINITY LIGANDS Protoporphyrin-IX 105 An Erlenmeyer f l a s k (4 l i t r e capacity) containing dimethylformamide (2 l i t e r , reagent grade) was heated with s t i r r i n g , under an atmosphere of nitrogen, on a hot plate-magnetic s t i r r e r . As soon as the dimethyl-formamide started b o i l i n g , hematoporphyrin-IX dihydrochloride 102 (25.0 g, 42 mmole), i n a small beaker, was added by lowering this into the flask with the help of a metal tongue and the mixture s t i r r e d vigorously for 30 seconds. The r e s u l t i n g dark red solution was protected from l i g h t and cooled to room temperature i n an ice-bath with s t i r r i n g . The dark solution was transferred to a round bottom flask and the solvent (dimethylformamide) removed using a rotary evaporator attached to a vacuum pump (water bath temperature 40-50°C). The red s o l i d obtained was dissolved i n a minimum of formic acid (90%, analytical reagent) and then precipitated with diethyl ether. The precipitate was f i l t e r e d under vacuum and washed - 120 -several times with ether and a i r dried. The dry protoporphyrin was then thoroughly washed with water and a i r dried. After a i r drying for two hours, the s o l i d was further dried under vacuum in a desiccator for 48 hours to give 22.6 g (95.6% yield) of the product. Protoporphyrin-IX Ditert-butyl Ester 106b C 0 2 t B u C 0 2 t B u 1 0 6 b Protoporphyrin free acid 105 (obtained from the above reaction) (7.55 g, 13.4 mmole) was suspended i n dry methylene chloride (450 mL) and the solvent was brought to reflux. Oxalyl chloride (17.5 mL) was carefully added to this solution and the green solution was again refluxed for 15 minutes. After 15 minutes of reflux, the solution was cooled and excess oxalyl chloride was removed under reduced pressure. The residue was redissolved i n anhydrous methylene chloride (450 mL) and the solvent was again removed under vacuum. the residue was then redissolved i n dry methylene chloride (450 mL) and the green solution was brought to reflux under a condenser f i t t e d with a drying tube. To - 121 -this refluxlng solution dry tert-butyl alcohol (55 mL) was added and the solution refluxed for 45 minutes, when an additional amount of tert-butyl alcohol (55 mL) was added and reflux continued for an additional 45 minutes. The organic solvent and excess tert-butyl alcohol was then removed under reduced pressure. The residue was dissolved i n methylene chloride (300 mL) and extracted with water. The organic phase was collected and s t i r r e d i n a saturated solution of sodium bicarbonate u n t i l the acid was completely neutralized. This solution was again extracted and washed with water. The organic phase was collected, and dried over anhydrous sodium sulfate and f i l t e r e d and organic solvent removed under reduced pressure. The crude protoporphyrin-IX d i t e r t -butyl ester was dissolved i n a minimum of chloroform and p u r i f i e d on a s i l i c a gel (grade IV, 800 g) column using chloroform:diethyl ether (100:1 v/v) as the eluent. The fractions were collected, checked by t i c and the fractions containing the products were collected and taken to dryness. The product was c r y s t a l l i z e d from benzene (60 mL) to give 6.5 g (71.8%) of protoporphyrin-IX-ditert-butyl ester. MP: 228-2-29-. 5°C (dec.) L i t . : 228-229°C ( d e c ) . LH NMR: (6, CDC1 3): 1.39 (s, 18H, t-Bu), 3.16 (t, J - 8 Hz, 4H, /S-propionyl CH 2), 3.53 (s, 3H, ring-CH 3), 3.55 (s, 6H, ring Cfl 3), 3.57 (s, 3H, ring CH 3), 4.30 (t, J - 8 Hz, 4H, a-propionyl CH 2), 6.13 (dd, J± - 12 Hz, J 2 = 2 Hz, 2H, -CH=CH2), 6.29 (dd, J± = 12 Hz, J £ - 2 Hz, 2H, -CH»CH 2), 8.15 (dd, J1 - 12 Hz, J g - 2 Hz, 2H, -CH=CH2), 9.85 (s, IH, -CH.-), 9.95 (s, 2H, -CH.-), 9.99 (s, IH, -CH=), -4.03 (bs, 2H, NH). Anal.: Calcd. for C^H^N.O.: C, 74.75; H, 7.47; N, 8.30. Found: C, 42 50 4 4 74.76; H, 7.59; N, 8.09%. - 122 -Mass Spectrum: (m/e, r e l a t i v e intensity): 674 (M+, 100), 618 (20), 562 (26). Mol. wt.: Calcd. f o r C42 H50 N4°4 : 674.3816. Found by high resolution mass spectrometry: 674.3792. Vi s i b l e spectrum (CHgCip: A (nm) 405 503 538 573 628 max log e 5.72 4.63 4.57 4.37 4.22 2 (or 4) Monoformyl-4 (or 2) Monovinyldeuteroporphyrin-IX-ditert-butyl ester 123b and 122b by P i c r y l Azide reaction 0HC CHO + C0 2 tBu C0 2tBu ^ t B u \ 0 2 t B u 122 b 123 b Protoporphyrin-IX-ditert-butyl ester 106b (0.999 g, 1.48 mmole) was dissolved i n methylene chloride (100 mL), p i c r y l azide 121 (0.379 g, 1.49 mmole)was added and the solution refluxed for one hour i n the dark. The progress of the reaction was followed by t i c using Cl^C^-MeOH (100:2 v/v) as the developing solvent. At the end of one hour, the reaction mixture showed complete conversion of protoporphyrin-IX-ditert-- 123 -butyl ester into two components. The reaction mixture was cooled and was passed through an alumina column (grade I, 50 g), and the products then eluted with CH2Cl2-MeOH (100:2 v/v). The f i r s t pink-red band eluted was found to be 2 (or 4)-monoformyl 4-(or 2) monovinyldeutero-porphyrin-IX-ditert-butyl ester ( y i e l d - 0.321 g; 33.2%) and the second band (red-brown) as 2,4-diformyldeuteroporphyrin-IX d i t e r t - b u t y l ester ( y i e l d = 0.402 g, 40%). The samples for analysis were c r y s t a l l i z e d from CH2Cl2-MeOH to give maroon crystals of 2 (or 4)-monoformyl 4-(or 2)-monovinyldeuteroporphyrin IX-ditert-butyl ester 122b and 123b and red/brown crystals of 110b. 2 (or 4) Monoformvl-4 (or 2) monovinyldeuteroporphyrin-IX d i t e r t - b u t y l ester 122b and 123b. MP: 228-229°C ( d e c ) . XH NMR (5, CDC1 3): 1.41 (d, 18H, t-Bu), 3.16 (m, 4H, /9-propionyl CH 2), 3.49, 3.55, 3.60, 3.68 (4s, 12H, -CH ), 4.23, 4.34 (two t, J = 8 Hz, 4H, a-propionyl-CH 2), 6.31 (m, 2H, -CH=CH2), 8.10 (m, 1H, -CH=CH2), 9.63, 9.83, 10.49 (s, 4H, -CH.-) , 10.23 (s, 1H, -CHO), -4.39 (bs, 2H, NH) . Anal.: Calcd. for C ^ H ^ N ^ : C, 72.74; H, 7.15; N, 8.28. Found: C, 72.73; H, 7.21; N, 7.98%. Mass spectrum (m/e, re l a t i v e i n t e n s i t y ) : 676 (M+, 62), 620 (40), 564 (100), 547 (13), 519 (15), 505 (45). Mol. wt.: Calcd. for C ^ H ^ N ^ : 676.3613. Found by high resolution mass spectrometry: 676.3658. - 124 -2.4-Diformvldeuteroporphvrin-IX-ditert-butvl ester 110b MP: 201-203°C (dec.). LH NMR (5, CDC1 3): 1.28 (s, 18H, t-Bu), 2.97 (m, 4H, /9-propionyl- CH2>, 3.36, 3.58, 3.65, ( a l l s, 12H, ring-CH 3), 4.09 (m, 4H, a-propionyl CH ), 9.81 9.88, 10.57 ( a l l s, 4H,-CH=), 11.36, 11.40 (s, 2H, -CHO), -3.65 (bs, 2H, NH). Anal.: Calcd. for C,nH.,N,0,: C, 70.76; H, 6.83; N, 8.26. Found: C, 40 46 4 6 71.00; H, 6.90; N, 8.24%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 678 (M+, 37), 622 (20), 566 (57), 549 (9), 507 (30). Mol. wt.: Calcd. for C.AH.,N,0 • 678.3406. Found by high resolution 40 46 4 6 J ° mass spectrometry: 678.3390. Visible spectrum (CH 2C1 2): A (run) 431 520 557 588 645 max log e 5.36 4.39 4.23 4.15 3.87 - 125 -Protoporphyrin-IX-ditert-butyl ester 106b (5 g, 7.4 mmole) was dissolved i n methylene chloride (360 mL) and pyridine (40 mL) and s t i r r e d i n a beaker i n bright sunlight for three days. The progress of the reaction was checked by t i c (CH 2C1 2-diethyl ether, 100:3 v/v). After being s t i r r e d for three days, the green solution was washed three times with water to remove pyridine and then the traces of pyridine were removed under vacuum. The r e s u l t i n g dark green s o l i d was dissolved i n a minimum of CH 2CT 2 and chromatographed on s i l i c a gel (60-270 mesh, 1000 g) using CH 2Cl 2:Et 20 (100:3 v/v) as eluent. The fast moving red band that eluted f i r s t , was unreacted protoporphyrin-IX-ditert-butyl ester (0.522 g, 10%). This band was followed by the f i r s t green band as 4-photoproto-porphyrin (1.315 g, 25%), the second dark green band eluted was a mixture of both isomers (1.563 g, 29.8%) and l a s t band eluted was 2-photoprotoporphyrin-IX- d i t e r t - b u t y l ester (1.214 g, 23%). The large scale separation of each isomer (ring A and ring B) was carried out on 20 x 20 cm, 1 mm thick preparative plates using CH 2C1 2:diethyl ether (100:3 v/v) as the developing solvents. To obtain a better separation, the plates were developed for 5 to six times. A n a l y t i c a l samples were c r y s t a l l i z e d from methylene chloride-methanol. 2-Photoprotoporphyrin-IX-ditert-butyl ester 127b MP: 239-240° C. NMR (S, CDC1 3): 1.38 (s, 3H, aliphatic-CH 3), 1.47 (d, 18H, t-Bu), 2.57, 3.30, 3.36 ( a l l s, 9H, ring-CH 3), 3.16 (m, 4H, £-propionyl CH 2), 126 -4.22 (m, 4H, a-propionyl-CH 2), 5.71 (d, IH, olefinic-CH), 6.13 (dd, 2h, -CH=CH2), 7.28, 8.25, 9.50, 9.69 ( a l l s, 4H, -CH=), 7.84 (dd, IH, -CH=CH2), 10.02 (s, IH, -CHO), -3.69, -4.21 (bs, 2H, -NH). Anal.: Calcd. for C.0HC.N.0^.^Ho0: C, 70.45; H, 7.18; N, 7.82. Found: 42 50 4 6 2 2 C, 70.54; H, 7.29; H, 7.73%. Mass spectrum: (m/e, r e l a t i v e i n t e n s i t y ) : 706 (M+, 100), 690 (86), 650 (96), 634 (48), 595 (75). Mol. wt.: Calcd. for C^H^H^Og: 706.3714. Found by high resolution mass spectrometry: 706.3719. V i s i b l e spectrum (CH 2C1 2): A (nm) 390 420 500 567 608 667 max log e 5.11 5.07 4.04 4.31 4.20 4.75 ° max 4-Photoprotoporphvrin-IX-ditert-butvl ester 126b MP: 240-241° C. 1H NMR (5, CDC1 3): 1.28 (s, 3H, aliphatic-CH 3), 1.45 (d, 18H, t-Bu), 3.14, (m, 4H, /S-propionyl-CH 2), 3.39-3.58 (s, 9H, ring-CH 3), 4.22 (m, 4H, a-propionyl-CH 2), 6.07 (dd, 2H, -CH=CH2), 6.70 (d, IH, olefinic-CH), 7.58 (dd, IH, -CH=CH2), 7.74, 8.55, 9.61, 9.83 ( a l l s, 4H, -CH-), 10.04 (d, IH, -CHO). Anal.: Calcd. for C.„H C„N.0,: C, 71.35; H, 7.13; N, 7.93. Found: C, 42 50 4 6 71.11; H, 7.27; N, 7.86%. Mass spectrum: (m/e, r e l a t i v e intensity): 706 (M+, 100), 690 (80), 650 (60). - 127 -Mol. wt.: Calcd. for C 4 2 H 5 0 N 4 ° 6 : 7 0 6 - 3 7 1 4 - Found by high resolution mass spectrometry: 706.3719. V i s i b l e spectrum (CH 2C1 2): A (nm) 382 435 500 567 612 670 max log e 5.41 5.43 4.39 4.63 4.51 5.06 2 (or 4)-Monoformyl-4 (or 2)-Monovinyldeuteroporphyrin-IX-Ditert-butyl esters 123b and 122b 123 b 122 b To a solution of photoprotoporphyrin-IX d i t e r t - b u t y l ester (0.435 g, 0.62 mmole of either ring A isomer 127b or ring B isomer 126b or both mixed isomers) i n dry methylene chloride (150 mL), sodium borohydride (0.435 g) solution i n anhydrous methanol (15 mL) was added and the reaction mixture s t i r r e d for one hour at room temperature. The reaction mixture was checked by t i c (methylene chloride-ether, 10:1 v/v). This revealed that the green aldehyde was completely converted to a less mobile grey alcohol band. At the end of one hour sodium borohydride was - 128 -c a r e f u l l y destroyed by acetic acid. The reaction mixture was then extracted with methylene chloride-water p a r t i t i o n , washed with water (twice), dried over anhydrous sodium sulfate, and taken to dryness. The dark brown residue (obtained from the above reaction) was dissolved i n dioxane (150 mL), sodium periodate (0.803 g) i n b o i l i n g water (5 mL) was added, followed by concentrated s u l f u r i c acid (0.5 mL) and the solution was s t i r r e d for two hours at room temperature. The purple solution was poured into water (400 mL), saturated sodium chloride solution (50 mL) added and the product was extracted with methylene chloride. The organic phase was washed with 10% potassium bicarbonate solution and water, dried over anhydrous sodium sulfate, f i l t e r e d , and evaporated to dryness. The residue was dissolved i n a minimum volume of chloroform and chromatographed on s i l i c a gel (300 g) using chloroform-methanol (100:1 v/v) as eluent. The porphyrins were c r y s t a l l i z e d from chloroform-petroleum ether (bp 30-60°C) to give the desired product (0.293 g, 69.8% y i e l d ) . 2-Formvl-4-vinyldeuteroporphvrin-IX Di-tert-butyl ester 123b MP. : 227-229°C ( d e c ) , L i t . 1 3 2 : 228-229 C ( d e c ) . LH NMR (6, CDC1 3): 1.42 (d, 18H, t-Bu), 3.09 (m, 4H, £-propionyl-CH 2), 3.15, 3.23, 3.27, 3.37 ( a l l s, 12H, ring-CH 3), 4.15, 4.21 (two t, J = 8 Hz, 4H, a-propionyl-CH 2), 6.17 (dd, 2H, -CH=CH2), 7.91 (dd, 1H, -CH=CH2), 8.86, 9.42, 9.52, 9.67 ( a l l s, 4H, -CH=), 10.75 (s, 1H, -CHO). Anal.: Calcd. for C..H..N.0 • C, 72.74; H, 7.15; N, 8.28. Found: C, 41 48 4 5 72.32; H, 7.09; N, 8.19%. - 129 -Mass spectrum: (m/e, r e l a t i v e i n t e n s i t y ) : 676 (M+, 28), 620 (19), 564 (50), 505 (14). Mol. wt.: Calcd. for C^H^N^O: 676.3613. Found by high resolution mass spectrometry: 676.3600. V i s i b l e spectrum (CH 2C1 2): A (nm) 416 515 555 581 640 max log e 5.33 4.14 4.27 4.06 3.46 max 4-Formyl-2-vinvldeuteroporphyrin-IX Di-tert-butyl ester 122b MP. : 228-229°C ( d e c ) , L i t . 1 3 2 : 228-229°C ( d e c ) . LH N M R (5, CDC1 3): 1.42 (d, 18H, t-Bu), 3.14 (m, 4H, £-propionyl- CH2) , 3.27, 3.43, 3.45, 3.56 ( a l l s, 12H, ring-CH 3), 4.19, 4.30 (two t, J = 8 Hz, 4H, a-propionyl-CH 2), 6.09 (dd, 2H, -CH=CH2), 7.71 (dd, IH, -CH=CH2), 9.13, 9.47, 9.65, 10.30 ( a l l s, 4H, -CH=), 11.05 (s, IH, -CHO). Anal.: Calcd. for C.-H.-N.O • C, 72.74; H, 7.15; N, 8.28. Found: C, 41 48 4 5 72.73; H, 7.14; N, 7.98%. Mass spectrum: (m/e, re l a t i v e i n t e n s i t y ) : 676 (M+, 28), 620 (21), 564 (52), 505 (14). Mol. wt.: Calcd. for C..,H.oN.0c: 676.3613. Found by high resolution 41 48 4 5 mass spectrometry: 676.3666. V i s i b l e spectrum (CH 2C1 2): A (nm) 416 515 555 581 640 max log e 5.31 4.11 4.24 4.03 3.41 ° max - 130 -2 (or 4)-Monoacrylic acid-4 (or 2)-Monovinyldeuteroporphyrin-IX-d i t e r t - b u t y l ester 133 or 132 Monoformyl monovinyldeuteroporphyrin-IX d i t e r t - b u t y l ester (0.25 g, 0.37 mmole, either ring A isomer 123b or ring B isomer 122b or a mixture of r i n g A and r i n g B isomers) was dissolved i n pyridine (100 mL), malonic acid (4 g) was added and the reaction mixture heated to 40°C with s t i r r i n g . At 40°C, piperidine (1.5 mL) was added to the reaction mixture. The temperature was slowly raised to 80° and maintained there for four hours. The reaction mixture was then cooled and poured into d i l u t e hydrochloric acid (300 mL, 3% v/v). The porphyrin was extracted with chloroform, washed with water, dried over anhydrous sodium sulfate, f i l t e r e d and evaporated to dryness. The traces of pyridine were removed under vacuum. The residue was dissolved i n chloroform and p u r i f i e d on a s i l i c a gel preparative plate (200 micron thickness) using chloroform-methanol (20:1 v/v) as the developing solvent. The slow moving band was scraped from the s i l i c a plate and suspended i n chloroform-methanol solution, f i l t e r e d and dried under reduced pressure to give monoacrylic 131 -acid monovinyl deuteroporphyrin-IX d i t e r t - b u t y l esters (0.132 g, 49.7% y i e l d ) . 2-Acrvlic acid-4-vinyldeuteroporphvrin-IX-di-tert-butvl ester 133 MP: 225-227 °C ( d e c ) . Anal.: Calcd. for C / oH C AN.0 • C, 71.84; H, 7.01; N, 7.80. Found: C, 71.73; H,7.00; N,7.81%. + + Mass spectrum: (m/e, r e l a t i v e i n t e n s i t y ) : 718 (M , 8), 674 (M -CO^, 523), 618 (38), 606 (13), 562 (100). Mol. wt.: Calcd. for C,.HCAN.0-: 718.3718. Found by high resolution 43 50 4 6 J O mass spectrometry: 718.3749. V i s i b l e spectrum (CHgCl^ : A (nm) 418 512 550 578 636 max log e 5.15 4.11 4.17 3.98 3.71 ° max 4-Acrylic acid-2-vinyldeuteroporphvrin-IX-di-tert-butyl ester 132 MP: 225-227° C ( d e c ) . Anal.: Calcd. for C.,HcrtN.0-: C, 71.84; H, 7.01; N, 7.79. Found: C, 43 oO 4 6 71.28; H, 7.10; N, 7.41%. + + Mass spectrum: (m/e, re l a t i v e intensity): 718 (M , 10), 674 (M -CCv,, 54), 618 (39), 562 (100). Mol. wt.: Calcd. for C/oHc.N.0 • 718.3718. Found by high resolution 43 50 4 6 mass spectrometry: 718.3785. - 132 -V i s i b l e spectrum (CH2C12>: Amax ( n m ) 4 1 8 5 1 1 5 5 0 5 7 7 6 3 6 l o S e 5.15 4.08 4.14 3.98 3.58 2 (or 4)-Monomethyl acrylate-4 (or 2)-Monovinyl Deuteroporphyrin-IX-d i - t e r t - b u t y l ester 135 or 134 Monoacrylic acid monovinyldeuteroporphyrin-IX d i - t e r t - b u t y l ester (15.2 mg, 0.021 mmole, either ring A 133 or rin g B 132 isomers) was dissolved i n methylene chloride (20 mL) and kept i n an ice-bath. To th i s , an ethereal solution of diazomethane (10 mL) was added and the solution s t i r r e d for 10 minutes. The solution was then evaporated to dryness and chromatographed on s i l i c a gel column using CH 2C1 2: methanol (100:1 v/v) as an eluent. The f i r s t moving band was collected and found to be monomethylacrylate monovinyl deuteroporphyrin-IX di - t e r t - b u t y l esters (14.1 mg, 97% y i e l d ) . 133 -2-Moriomethvl acrylate-4-vlnvldeuteroporphyrln-IX-di-tert-butvl ester 135 MP: 241-243 °C ( d e c ) . XH NMR (6 CDC1 3): 1.40 (d, 18H, t-Bu), 3.16 (t, J - 8 Hz, 4H, £-propionyl-CH 2), 3.48, 3.53 (two s, 12H, ring-CH 3), 4.12 (s, 3H, -OCH3), 4.30 (m, 4H, a-propionyl-CHp , 6.27 (dd, J = 18 Hz, J 2 - 12 Hz, 2H, -CH=CH2), 6.92 (d, J = 16 Hz, 1H, ^-acrylate-CH), 8.16 (m, 1H, -CH=CH2), 9.09 (d, J - 16 Hz, 1H, a-acrylate-CH), 9.67, 9.70, 9.90, 9.92 (4s, 4H, -CH=), -4.27 (bs, 2H, NH). Anal.: Calcd. for C..HCON.O • C, 72.09; H, 7.16; N, 7.65. Found: C, 72.10; H, 7.23; N, 7.35%. Mass spectrum: (m/e, r e l a t i v e i n t e n s i t y ) : 732 (M+, 45), 676 (28), 620 (81), 575 (17), 561 (43). Mol. wt.: Calcd. for ^ ^ ^ 0 ^ 732.3874. Found by high resolution mass spectrometry: 732.3860. V i s i b l e spectrum (CH 2C1 2): A (nm) 417 510 549 578 634 max log e 5.26 4.18 4.22 4.02 3.75 ° max 4-Monomethvl acrylate-2-vinyldeuteroporphvrin-IX-di-tert-butvl ester 134 MP: 241-242°C ( d e c ) . 1H NMR (6, CDC1 3): 1.41 (d, 18H, t-Bu), 3.16 (t, J - 8 Hz, 4H, £-propionyl-CH 2), 3.33, 3.50, 3.55 (3s, 12H, ring -CH3), 4.12 (s, 3H, -0CH3), 4.30 (m, 4H, a-propionyl-CH 2), 6.18 (dd, J± = 18 Hz, J 2 = 12 Hz, 2H, -CH=CH2), 6.91 (d, J = 16 Hz, 1H, /3-acrylate-CH), 7.99 (dd, 1H, - 134 --CH-CH2), 9.11 (d, J - 16 Hz, 1H, a-acrylate-CH), 9.60, 9.73, 9.87, (s, 4H, -CH=), -4.37 (bs, 2H, NH). Anal.: Calcd. for C ^ H ^ N ^ : C, 72.09; H, 7.16; N, 7,65. Found: C, 71.51; H,6.96; N, 7.53%. Mass spectrum: (m/e, re l a t i v e i n t e n s i t y ) : 732 (M+, 35), 676 (18), 620 (54), 575 (14), 561 (31). Mol. wt.: Calcd. for C ^ H ^ N ^ : 732.3874. Found by high resolution mass spectrometry: 732.3888. V i s i b l e spectrum ( C H ^ i p : A (nm) max log e max 417 510 549 578 634 5.23 4.14 4.19 3.96 3.68 2 (or 4)-Vinyl-4 (or 2)-[(3'-(N-3"-aminopropyl)]acrylamidodeutero-porphyrin-IX-di-tert-butyl ester 73 or 74 H H 2 3 < C0 2tBu C0 2tBu 73 O - C N f C H ^ N H , CCK tBu Monoacrylic acid monovinyldeuteroporphyrin IX di - t e r t - b u t y l ester (0.099 g, 0.138 mmole, ring A 133 or ring B 132 isomer) was dissolved i n 135 -dry methylene c h l o r i d e (20 mL) and f r e s h l y d i s t i l l e d o x a l y l c h l o r i d e (3 mL) was added. The s o l u t i o n was s t i r r e d f o r one minute and evaporated to dryness under reduced pressure. The residue was d i s s o l v e d i n dry methylene c h l o r i d e (20 mL) and cooled i n an i c e bath under nitrogen. To t h i s s o l u t i o n a large excess of 1,3-diaminopropane (10 mL) was added and the s o l u t i o n was s t i r r e d f o r h a l f an hour. A colour change from green to red was observed. The r e a c t i o n mixture was then r e f l u x e d f o r two hours, cooled to room temperature and then was washed with water (three times). The organic phase was c o l l e c t e d , d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d and evaporated to dryness. The red s o l i d was d i s s o l v e d i n a minimum of chloroform and chromatographed on s i l i c a gel (CHClj:methanol, 100:1 v/v). The f a s t moving band was removed with chloroform-methanol s o l u t i o n to y i e l d the 2 (or 4 ) - v i n y l - 4 (or 2)-[3'-(N-3"-aminopropyl)]-acrylamidodeuteroporphyrin-IX-di-tert-butyl esters (0.054 g, 50%). 4-Vinyl-2-f3'-(N-3"-aminopropvl)1acrvlamidodeuteroporphyrin-IX-ditert-b u t y l ester 74 MP: >250°C ( d e c ) . XH NMR (5, CDC1 3): 1.34 (s, 18H, t-Bu and a l k y l - C H 2 ) , 1.39 (s, 9H, t-Bu), 2.25 (bs ; 2H, -NH^), 2.83 (s, 3H, ring-CH 3), 2.95 (m, 2H, -CH 2-), 3.09 ( t , 4H, J = 8 Hz, yS-propionyl-CH^), 3.21, 3.32, 3.37 (3s, 9H, 3 x ring-CH 3), 3.95,(q, 2H, J - 6 Hz, -CH 2), 4.09, 4.25 (2t, 4H, J = 6 Hz, a-propionyl-CH 2), 6.00 (dd, J± - 18 Hz, J 2 - 12 Hz, 2H, -CH=CH2), 7.11 (d, IH, J = 16 Hz, 0-aerylate-CH), 7.25 (bs, IH, -C0NH-), 7.71 (dd, IH, 136 -CH-CH2), 8.62 (s, IH, -CH.-), 9.14 (d, IH, J = 16 Hz, a-acrylate -CH) , 9.25, 9.42, 9.47 (3s, 3H, 3 x -CH-), -5.74 (bs, 2H, NH). Anal.: Calcd. for C.JHLoN,0 • C, 71,27; H, 7.55; N, 10.85;. Found: C, H-D J O O 5 71.40; H, 7.51; N, 10.90%. V i s i b l e spectrum (CH 2C1 2): A (nm) 402 512 549 581 636 max log e 5.13 4.04 4.03 3.88 3.59 ° max 2-Vinyl-4-f 3'-CN-3"-aminopropyl)1acrvlamidodeuteroporphvrin-IX-ditert-butyl ester 73 MP: >250°C (dec.). 1H NMR (S, CDC13): 1.35, (t, 2H, J = 8 Hz, alkyl-CH 2), 1.42 (d, 18H, t-Bu), 2.35 (bs, 2H, -NH2), 2.74 (s, 3H, ring-CH 3), 3.03 (q, 2H, J = 7.5 Hz, -CH2-), 3.12 (t, 4H, J - 8 Hz, £-propionyl-CH 2), 3.29, 3.52, 3.55 (3s, 9H, 3 x ring-CH 3), 4.02 (q, 2H, J = 6 Hz, -CH^-), 4.14, 4.21, (2t, 4H, J = 6 Hz, a-propionyl-CH 2) , 5.59 (m, 2H, -CH=CH.2) , 6.85 (dd, IH, -CH=CH2), 7.08 (d, IH, J = 16 Hz, £-acrylate-CH), 7.41 (bs, IH, -C0NH-), 7.96 (s, IH, -CH=), 9.07 (d, IH, J = 16 Hz, a-acrylate-CH), 9.32, 9.46, 9.53 (3s, 3H, 3 x -CH=), -5.88 (bs, 2H, NH). Anal.: Calcd. for C.,HCON,0C: C, 71.27; H, 7.55; N, 10.85. Found: C, 46 58 6 5 71.30; H, 7.61; N, 10.80%. V i s i b l e Spectrum (CH 2C1 2): A (nm) 402 512 551 583 638 max log e 5.06 3.97 3.97 3.82 3.55 ° max - 137 -Photoprotoporphyrin Derivative of 2(or 4)-Vinyl-4 (or 2)-[3'- (N-3"-aminopropyl)]acrylamidodeuteroporphyrin-IX-di-tert-butyl ester 138 or 139 2 (or 4)-Vinyl-4 (or 2)- L3'-(N-3"-aminopropyl)]-acrylamidodeutero-porphyrin-IX-di- tert-butyl ester 73 or 74 (5 mg) was dissolved i n methylene choride (10 mL) and the solution s t i r r e d i n the l i g h t for three days. The reaction was checked by t i c and the formation of the green chromophore was monitored by i t s v i s i b l e spectrum. The products were chromatographed on s i l i c a gel (CI^C^:MeOH, 20:1 v/v). The green photoprotoporphyrin derivative ran s l i g h t l y slower than the parent amino porphyrin and was removed from the s i l i c a gel with a metylene chloride-methanol mixture. - 138 -2-Photoprotoporphyrin Derivative of 2-Vinvl-4-[3' - (N-3"-amino-propyl) 1 -acrylamidodeuteroporphyrin-IX-di-tert-butyl ester 138 Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 808 (25), 807 (33), 806 (M+, 100), 750 (33), 749 (50), 748 (100), 747 (75). V i s i b l e spectrum (CH 2C1 2): A (nm) 400 510 553 578 635 680 max peak r a t i o 11.03 0.75 0.71 0.68 0.31 1.00 4-Photoprotoporphyrin Derivative of 4-Vinyl-2-T3'-CN-3"-aminopropyl)1 -acrylamidodeuteroporphvrin-IX-di-tert-butyl ester 139 Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 808 (26), 807 (30), 806 (M+, 100), 750 (30), 749 (49), 748 (100), 747 (75). V i s i b l e spectrum (CH 2C1 2): A (nm) 406 510 550 577 633 690 max peak r a t i o 7.31 0.63 0.51 0.49 0.31 1.00 - 139 -Iron (III)-2 (and 4)-vinyl-4 (and 2)-[3'-(N 3"-aminopropyl)]acrylamido-deuteroporphyrin-IX-di-tert-butyl ester 140 0 CN(CH 2) 3NH 2 C02tBu C02tBu 140 2 (and 4)-Vinyl-4 (and 2)-[3'-(N-3"-aminopropyl)]acryl amido-deuteroporphyrin-IX-di-tert-butyl ester 73 and 74 (10 mg, 0.013 mmole) was dissolved i n dimethylformamide (10 mL) and the solution was brought to reflux. To the refluxing solution a saturated solution of ferrous chloride i n methanol (2 mL) was added and the reflux continued for 30 minutes. The solution was then cooled and s t i r r e d i n the a i r . The solution was poured into water and extracted with chloroform. The organic phase was again washed with water, dried over anhydrous sodium sulfate, f i l t e r e d and dried to give (8 mg, 71% y i e l d ) . A pyridine hemochrome spectrum was measured using the following 127 conditions. Pyridine (1.0 mL), potassium hydroxide solution (1.0 mL of 0.2M solution ), water (2.0 mL), dithionite (2-3 mg) and Fe(III) porphyrin solution i n pyridine. V i s i b l e spectrum: A (ran) 428 528 570 max peak r a t i o 3.73 0.76 1.00 See footnote on page 75 140 Co(II)-2 (and 4)-vlnyl-4 (and 2)-[3'-(N-3"-aminopropyl)] acrylamido-deuteroporphyrin-IX-di-tert-butyl ester 141 2 (and 4)-Vinyl-4 (or 2)-[(3'-(N-(3"-aminopropyl)]-acrylamido-deuteroporphyrin-IX-di-tert-butyl ester 73 and 74 (11 mg, 0.014 mmole) was dissolved i n chloroform (10 mL) and the solution was brought to reflux under nitrogen. To this refluxing solution, a saturated solution of cobalt (II) acetate i n methanol (2 mL) was added and the solution was further refluxed for one hour. At the end of one hour, the reaction mixture was poured into water and extracted with chloroform, dried over anhydrous Na^SO^, f i l t e r e d and evaporated to dryness (8.2 mg, 61% y i e l d ) . The v i s i b l e spectrum of the complex showed the complete metallation giving two bands i n the v i s i b l e spectrum. Visible spectrum: A (in) 400 525 574 max peak r a t i o 9.87 0.73 1.00 See footnote on page 75 141 Iron(III)-2 (and 4)-vinyl-4 (and 2)-[3'-(N-3"-aminopropyl)] acrylamido-deuteroporphyrin-IX 71 Iron (III)-2 (and 4)-vinyl-4 (and 2)-[3'-(N-3"-aminopropyl)] acryl-amidodeuteroporphyrin-IX-di-tert-butyl ester 140 (12 mg, 0.014 mmole) was dissolved i n dry methylene chloride (10 mL), t r i f l u o r o a c e t i c acid (1 mL) added and the solution was s t i r r e d for 5 minutes at room temperature under nitrogen atmosphere. The solution was evaporated and dried under vacuum to give 6.2 mg, 59 % y i e l d . The pyridine hemochrome spectrum was recorded using the ide n t i c a l conditions used for the compound 140 V i s i b l e spectrum: C0 2H C02H 71 A (nm) 428 528 568 max peak r a t i o 5.21 0.60 1.00 * See footnote on page 75 142 Co(III)-2 (and 4)-vinyl-4 (and 2)-[3'-(N-3"-aminopropyl)] acrylamido-deuteroporphyrin 72 C02H C02H 72 Co(II)-2 (and 4)-vinyl-4 (or 2)-[3'-(N-3"-aminopropyl)]-acrylamido-deuteroporphyrin-IX-di-tert-butyl ester 141 (12 mg, 0.013 mmole) was dissolved i n dry methylene (5 mL), t r i f l u o r o a c e t i c acid (1 mL) was added and the solution was s t i r r e d i n the dark for 5 minutes under nitrogen atmosphere. The solvent was evaporated under reduced pressure and the residue was dried under vacuum. The dried residue was taken i n methylene chloride and the s o l i d was collected by f i l t r a t i o n (6.8 mg, 62 % y i e l d ) . The product was soluble i n sodium carbonate solution indicating the presence of a diacid. V i s i b l e spectrum: (0.2 M Na 2C0 3 solution): X (nm) 423 538 574 max peak r a t i o 5.43 0.94 1.00 See footnote on page 75 - 143 -4.3 SYNTHESIS OF MONOPYRROLES 4-Acetyl-2-ethoxycarbonyl-3,5-dimethylpyrrole 142 A saturated solution of sodium n i t r i t e (560 g, 8.1 mole) i n water was added dropwise rapidly to an ice-cooled, magnetically s t i r r e d mixture of ethyl acetoacetate 146 (1040 g, 8.0 mole) and acetic acid i n a 5 l i t e r flask. A viscous product, ethyl oximinoacetoacetate 147, was obtained which was suitable for immediate use. The ethyl oximinoacetoacetate was added dropwise along with zinc dust to a s t i r r e d mixture of anhydrous sodium carbonate (900 g), acetic acid (4 l i t e r s ) , and pentane-2,4-dione (850 g, 8.5 mole) i n a 12 l i t e r three neck flask. The reaction temperature was increased and was maintained between 75-80°C. The s t i r r i n g of the reaction mixture continued for an additional 30 minutes after the complete addition of ethyloximinoaceto-acetate. The reaction mixture was poured into water and the cr y s t a l -l i z e d product was collected by f i l t r a t i o n . The s o l i d was dissolved i n chloroform by warming on a steam bath and dried with anhydrous sodium sulfate, and the organic phase was - 144 -f i l t e r e d . The organic phase was evaporated under reduced pressure afte adding methanol. 4-Acetyl-2-ethoxycarbonyl-3,5-dimethylpyrrole was c r y s t a l l i z e d as white needles and was collected by f i l t r a t i o n to give 910.2 g (54.4%). The mother liquors were concentrated for the second and t h i r d crops of 324.4 g (19.4%). MP: 142.5-143.5°C; L i t . 1 9 1 143-144°C. 1H NMR (5, CDC1 3): 1.37 (t, 3H, J - 7 Hz, -0-CH2CH3), 2.42 (s, 3H, -C0CH3), 2.52 (s, 3H, -CH3>, 2.57 (s, 3H, -CH 3), 4.33 (q, 2H, J - 7 Hz, -0CH2CH3), 9.62 (bs, IH, NH). Mass spectrum: (m/e, r e l a t i v e i n t e n s i t y ) : 209 (M+, 27), 194 (28), 164 (10), 163 (6), 162 (9), 148 (100). Mol. wt.: Calcd. for N03: 209.1048. Found by high resolution mass spectrometry: 209.1053. 2-Ethoxycarbonyl-4-ethyl-3,5-dimethylpyrrole 162 162 - 145 A s t i r r e d mixture of 4-acetyl-2-ethoxycarbonyl-3,5-dimethylpyrrole 142 (836.7 g, 4.0 mole), sodium borohydride (160.1 g, 4.2 mole) and ethyl acetate (1400 mL) i n tetrahydrofuran (4 l i t e r s ) under nitrogen was treated dropwise with boron t r i f l u o r i d e etherate (700 mL, 807.8 g, 5.7 mole). The temperature of the reaction mixture was maintained between 15-20°C. At the end of complete addition, an aliquot of the reaction mixture was removed, quenched with water and checked by thin layer chromatogra-phy for any unreacted st a r t i n g material. The excess diborane was cautiously destroyed by acetic acid (300 mL) and water (2 l i t e r s ) . Sodium chloride (250 g) was s t i r r e d into the reaction mixture followed by ethyl acetate (1 l i t e r ) and water (1 l i t e r ) causing the reaction mixture to separate into an upper o i l y solution of the product and a lower aqueous solution of sodium fluoroborate and some boric acid. The upper organic phase was isolated. The aqueous phase was further extracted with ethyl acetate and combined with the previous organic phase. The combined organic phase was evaporated to dryness under reduced pressure. The crude product was dissolved i n ethanol (95%) on a steam bath and the product was c r y s t a l l i z e d by the addition of water. The solution was cooled In an ice-bath and the product was collected by f i l t r a t i o n , y i e l d 438.8 g (56.2%). The mother liquor was concentrated to obtain the second crop of 108.7 g (13.9%). MP: 90-91.5°C; L i t . 90-91°C 1 7 1, 95-96°C 1 1 8. XH NMR (5, CDC1 3): 1.02 (t, 3H, J ' 7.5 Hz, -CH2CH3), 1.30 (s, 3H, J = 7 Hz, -OCH2GH3), 2.16 (s, 3H, -CH3), 2.24 (s, 3H, -CE^), 2.36 (q, 2H, J - 146 -- 7.5 Hz, -CH2CH3), 4.26 (q, 2H, J - 7 Hz, -0-CH2-CH3), 8.83 (bs, IH, NH) . Mass spectrum: (m/e, r e l a t i v e i n t e n s i t y ) : 195 (M+, 34), 180 (44), 166 (4), 150 (15.6), 148 (12.5), 134 (100). 2-Ethoxycarbonyl-4-ethyl-3-methylpyrrole-5-carboxylic acid 165 2-Ethoxycarbonyl-4-ethyl-3,5-dimethylpyrrole 162 (58.5 g, 0.30 mole) was dissolved i n dry methylene chloride (300 mL) i n a 5 l i t e r round bottom flask. While the above solution was magnetically s t i r r e d , ether (500 mL) was added. Following th i s , a solution of s u l f u r y l chloride (128.36 g, 0.95 mole) i n methylene chloride (200 mL) was rapidly added (dropwise) and the reaction mixture was s t i r r e d for 20 minutes; and the solvent was then evaporated o f f under reduced pressure. The residual red o i l was poured into a hot solution of 20% water-acetone mixture and the solution mixture was heated on a steam bath for 10-15 minutes. The pale yellow s o l i d c r y s t a l l i z e d out. The s o l i d was collected by f i l t r a t i o n , washed several times with water-acetone mixture (1:1 by volume) and then with water. - 147 -The crude product was taken i n one l i t e r beaker and dissolved i n methanol (200 mL) and treated with saturated solution of sodium bicarbo-nate on a steam bath. The mixture was extracted with ether and the aqueous phase was cautiously a c i d i f i e d with concentrated hydrochloric acid i n an ice-bath. The precipitated white s o l i d was f i l t e r e d , washed several times with water and dried i n the a i r . Y i e l d 54.1 g (80.2%). MP: 210-211°C; L i t . 1 9 2 : 211°C. XH NMR (5, DMS0-d6): 1.06 (t, 3H, J - 7.5 Hz, -CH2-CH3), 1.33 (t, 3H, J - 7 Hz, -'0-CH2CH3), 2.24 (s, 3H, -CH ) , 2.72 (q, 2H, J - 7.5 Hz, -CH2-CH3), 4.29 (q, 2H, J — 7 Hz, -0-CH2-CH3), 11.18 (bs, 1H, NH), 12.62 (bs, 1H, -COOH). Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 225 (M+, 100), 210 (74), 196 (58), 180 (15), 178 (77), 164 (94). Mol. wt.: Calcd. for C..HncN0.: 225.0996. Found by high resolution 11 15 4 J ° mass spectrometry: 225.1005. 2-Ethoxycarbonyl-4-ethyl-5 -iodo-3-methylpyrrole 166 - 148 A mixture of 2-Ethoxycarbonyl-4-ethyl-3-methylpyrrole-5-carboxylic acid 165 (66 g, 0.29 mole) and sodium bicarbonate (99 g, 1.18 mole) was suspended i n water (400 mL) and dichloroethane (250 mL) i n a two l i t e r Erlenmeyer flask. The above mixture was heated slowly and s t i r r e d , the sta r t i n g material dissolved with effervescence. While the reaction mixture was s t i r r e d at low temperature, a solution of iodine (87.0 g, 0.34 mole) and potassium iodide (135.3 g, 0.82 mole) i n water (450 mL) was added dropwise with the aid of a dropping funnel. As soon as the addition was completed (within 10-15 minutes), the solution was refluxed on a hot plate for 30 minutes. The heating was then stopped and the excess iodine was destroyed by careful addition of sodium b i s u l f i t e . A pale yellow organic layer was obtained while the aqueous layer remained colourless. The entire reaction mixture was cooled to room temperature, methylene chloride (400 mL) was added and the organic layer was separated by separatory funnel. The organic layer was washed with water and f i l t e r e d . The solvent was evaporated to dryness under reduced pressure. The resu l t i n g off-white crude product was redissolved i n 95% ethanol (500 mL) and the solution was cooled. To the cooled solution, d i s t i l l e d water was added slowly and flask was shaken. The product c r y s t a l l i z e d as needle shaped fine crystals. The product was f i l t e r e d and washed by a 20% ethanol-water mixture followed by water to give 76.9 g (85.4%). The mother liquor was concentrated for a second crop, 13.2 g (14.4%). MP: 114.0-115.5°C; Lit. 1 9 3114-115°C. XH NMR ( 5 , CDC1 3): 1.03 (t, 3H, J = 7.5 Hz, -CH2~CH3), 1.33 (t, 3H, J -7 Hz, -0-CH2CH3), 2.27 (s, 3H, - % ) , 2.37 (q, 2H, J - 7.5 Hz, -CH^CH^, - 149 -4.31 (q, 2H, J - 7 Hz, -0-CH2CH3), 9.02 (bs, IH, NH). Anal.: Calcd. for C^H^M^I: C, 39.11; H, 4.59; N, 4.56; I, 41.32. Found: C, 39.05; H, 4.71; N, 4.46; I, 41.19%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 307 (M+, 95), 292 (46), 262 (17), 261 (28), 260 (12), 246 (100). Mol. wt.: Calcd. for C^U^NO^: 307.0064. Found by high resolution mass spectrometry: 307.0072. 2-Ethoxycarbony1-4-ethyl-3-methylpyrrole 167 167 2-Ethoxycarbonyl-4-ethyl-5-iodo-3-methylpyrrole 166 (35.59 g, 0.12 mole) was dissolved i n 95% ethanol (250 mL). The solution was heated on a steam bath, and a solution of potassium iodide (33.3 g, 0.20 mole) i n water (50 mL) was added. Following this concentrated hydrochloric acid (50 mL) was added, and liberated iodine was dischaged by addition of 50% aqueous hypophosphorus acid (25 mL). An additional amount of hypophos-phorus acid (25 mL) was added and the red-brown solution was further - 150 -heated, on a steam bath for 30 minutes. To the cooled solution methylene chloride (250 mL) and water (300 mL) were added. With the aid of a separatory funnel,the organic layer was separated and dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator leaving dark red o i l . The crude product was dissolved i n methylene chloride and p u r i f i e d by column chromatography on s i l i c a gel (85 g, a c t i v i t y I) using methylene chloride as an eluent. The a-unsubstituted pyrrole eluted out as a clean pale yellow l i q u i d leaving a l l coloured impurities at the beginning of the column. The solvent was evaporated o f f and the viscous o i l transferred into a weighed flask. The traces of solvent were removed under vacuum at room temperature, giving 17.2 g (94.9%) of a-unsubstituted pyrrole. MP: 22.0-23°C; L i t . 1 9 4 :25°C. -^H NMR (6\ CDC1 3): 1.14 (t, 3H, J - 7.5 Hz, -CH2CH3), 1.32 (t, 3H, J -7.2 Hz, -0-CH2CH3), 2.24 (s, 3H, -CH3), 2.40 (q, 2H, J - 7.5 Hz, -CH2-CH3), 4.27 (q, 2H, J - 7.2 Hz, -0-CH2CH3), 6.61 (d, IH, J - 2.8 Hz, 5-H), 8.88 (bs, IH, NH). Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 181 (M+, 38), 166 (35), 152 (13), 136 (18), 135 (9), 134 (22), 120 (100). Mol. wt.: Calcd. for C^H^NO^. 181.1098. Found by high resolution mass spectrometry: 181.1100. - 151 -4-Ethyl-3-methylpyrrole-2-carboxylic acid 168 2-Ethoxycarbonyl-4-ethyl-2-methylpyrrole 167 (61.04 g, 0.34 mole) was dissolved i n 95% ethanol (150 mL) i n a one l i t e r Erlenmeyer flask. To this solution, sodium hydroxide (26.97 g, 0.67 mole) i n water (300 mL) was added and the solution refluxed for 3 hours. At the end of three hours, reflux was stopped and the reaction mixture was cooled and suction f i l t e r e d . The clear aqueous solution was c a r e f u l l y a c i d i f i e d by acetic acid. The resu l t i n g white precipitate was f i l t e r e d off, and washed several times with water, and a i r dried to give 48 g (93.1%). The white colour of the pyrrole changed to a s l i g h t pink colour on standing. MP: 164-165 °C ( d e c ) . XH NMR (5, DMSO-d6): 1.11 (t, 3H, J - 7.8 Hz, -CH2CH3), 2.20 (s, 3H, -CH 3), 2.35 (q, 2H, J - 7 Hz, -CH2-CH3), 6.65 (d, IH, J - 2.8 Hz, pyr-H), 8.81 (bs, IH, NH), 12.15 (bs, IH, -COOH). Mass spectrum (m/e, r e l a t i v e intensity): 153 (M+, 32), 138 (26), 134 (5), 120 (100), 109 (10), 94 (22). Mol. wt.: Calcd. for C..H....N0 • 153.0786. Found by high resolution mass o 11 L spectrometry: 153.0796. 152 5-Bromo-4-ethyl-3-methylpyrrole-2-carboxylic acid 169 4-Ethyl-3-methylpyrrole-2-carboxylic acid 168 (10.0 g, 0.07 mole) was suspended i n g l a c i a l acetic acid (100 mL) in a 500 mL Erlenmeyer flask. The suspension was s t i r r e d in a water bath and the temperature maintained at 14-16°C. To the cooled solution, bromine (10.44 g, 0.07 mole) i n acetic acid (50 mL) was added dropwise and during the addition (15 minutes), the temperature was maintained between 14-16°C. The dark purple solution was further s t i r r e d for 10 minutes and water (350 mL)was added. The dark coloured precipitated s o l i d was collected by suction f i l t r a t i o n and washed several times with water t i l l the f i l t r a t e was colourless. The black s o l i d was dissolved in diethyl ether (200 mL) and was extracted with sodium hydroxide solutin (16.0 g) in water (250 mL) i n two to three portions. A l l aqueous extracts were combined and again extracted with an additional amount of ether (250 mL). The aqueous extract was suction f i l t e r e d three times giving a pale yellow f i l t r a t e . The aqueous extract was transferred to a one l i t e r beaker, cooled and a c i d i f i e d by acetic acid (150 mL) solution in water (100 mL). The white precipitate so obtained, changed to a s l i g h t purple colour. The precipitate was collected by suction f i l t r a t i o n and dried in the a i r for - 153 -one hour. The residual water was removed under vacuum at room temperature. Y i e l d 9.5 g (62.5%). MP: 125-126°C (dec). XH NMR (5, CDC1 3): 1.08 (t, 3H, J - 7 Hz, -CH2-CH3), 2.35 (s, 3H, - C H 3 ) , 2.42 (q, 2H, J - 7 Hz, -CH2-CH3), 8.91 (bs, 1H, -NH), 11.13 (bs, 1H, -C0 2H). Anal.: Calcd. for CgH^NC^Br: C, 41.40; H, 4.34; N, 6.04; Br, 34.43. Found: C, 41.53; H, 4.37; N, 6.15; Br, 34.34%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 232 (M+2, 25), 231 (M+l, 29), 216, 218 (30, 25), 198, 200 (100, 98), 187 (18), 172 (52). Mol. wt.: Calcd. for C^E^m^r: 232.9868. Found by high resolution mass spectrometry: 232.9877. 2-Benzyloxycarbonyl-4-ethyl-3,5-dimethylpyrrole 171 2-Ethoxycarbonyl-4-ethyl-3,5-dimethylpyrrole 162 (20.0 g, 0.10 mole) was taken i n a 500 mL Erlenmeyer flask f i t t e d with a Claisen head. R e d i s t i l l e d benzyl alcohol (250 mL) was added and the solution was - 154 -brought to reflux at 209°C under nitrogen. At this temperature, a concentrated solution of sodium i n benzyl alcohol was added in 1 mL portions; this resulted i n the vigorous evolution of ethanol vapours and caused the reflux temperature to drop. An additional amount of this solution (2 mL) was added and the mixture was heaated u n t i l i t maintained i t s reflux temperature at 209°C. The hot reaction mixture was quenched i n a s t i r r e d mixture of acetic acid (100 mL) and methanol (400 mL). Water was added to this mixture and the solution turned turbid and white precipitate was rapidly obtained. The precipitate was f i l t e r e d , washed with 50% methanol- water mixture, then with water, and a i r dried. The s o l i d was redissolved i n hot methanol and c r y s t a l l i z e d by adding water. White c r y s t a l l i n e pyrrole was obtained i n a y i e l d of 22.6 g (87.9%). MP: 102-103°C; Lit. 1 7 5:104-105°C. 1H NMR (5, CDC1 3): 1.05 (t, 3H, J = 8 Hz, -CH2-CH3), 2.20 (s, 3H, -CH 3), 2.30 (s, 3H, -CH 3), 2.39 (q, 2H, J = 8 Hz, CH 2CH 3), 5.30 (s, 2H, -CH 2C 6H 5), 7.38 (m, 5H, -CH^CglL.) , 8.55 (bs, 1H, NH) . Mass spectrxim (m/e, r e l a t i v e intensity): 257 (M+,29), 242 (12), 166 (10), 150 (10), 134 (7), 123 (15), 91 (100). Mol. wt.: Calcd. for C 1 6 H 1 9 N 0 2 : 257.1410. Found by high resolution mass spectrometry: 257.1422. - 155 -2-Benzyloxycarbonyl-4-ethyl-5-formyl-3-methylpyrrole 172 OHC H 0 72 2-Benzyloxycarbonyl-4-ethyl-3,5-dimethylpyrrole 171 (117.3 g, 0.46 mole) was dissolved i n methylene chloride (2 l i t e r ) and the solution was cooled i n an ice-bath for an hour. While the ice-cooled solution was s t i r r e d under nitrogen, s u l f u r y l chloride (77 mL, 0.96 mole) was added dropwise with the aid of a dropping funnel. At the end of the complete addition, the reaction mixture was further s t i r r e d for an additional two hours, and following th i s , d i s t i l l e d water (1 l i t e r ) was added. The reaction mixture ( l i g h t yellow) was further s t i r r e d overnight. The organic phase was separated, washed with water and evaporated to dry-ness. The o i l thus obtained was dissolved i n a minimum of methylene chloride and c r y s t a l l i z e d by the addition of hexane, giving 104.9 g (84.9%) tanned white powder. 195 MP: 86-87°C; L i t . : 86-87°C. Anal.: Calcd. for C^H^NCy C, 70.83; H, 6.32; N, 5.16. Found: C, 70.88; H, 6.45; N, 5.11%. H NMR (5, CDC1 3): 1.20 (t, 3H, J - 8 Hz, -CH2-CH3), 2.31 (s, 3H, -CH 3), 2.75 (q, 2H, J - 7.6 Hz, -Cfi 2-CH 3K 5.34 (s, 2H, -CH^H^.), 7.39 (m, 5H, -CH 0-CJL), 9.50 (bs, IH, NH), 9.78 (s, IH, -CHO). 2. o D - 156 -Mass spectrum (m/e, r e l a t i v e intensity): 271 (M +, 51), 180 (33), 164 (12), 162 (26), 91 (100). Mol. wt.: Calcd. for C^H^NO^ 271.1203. Found by high resolution mass spectrometry: 271.1216. 2-Benzyloxycarbony1-5 -(2-cyano-2-methoxycarbonyIviny1)-4-ethyl- 3-methyl-pyrrole 173 2-Benzyloxycarbonyl-4-ethyl-5-formyl-3-methylpyrrole 172 (107.4 g, 0.40 mole) was dissolved i n methanol (150 mL) on a steam bath. To this hot solution,methylcyanoacetate (75 g, 0.76 mole) and methylamine (5 mL) were added. The solution was boiled for 15 minutes thereafter and cooled i n an ice-bath. A yellow, prism shaped product c r y s t a l l i z e d out. The product was collected by suction f i l t r a t i o n and washed by cold methanol and a i r dried. Y i e l d 138.0 g (98.9%). - 157 -MP: 124-126°C. LH NMR (5, CDC1 3): 1.12 (t, 3H, J - 8 Hz, -CH -CH3) , 2.31 (s, 3H, -CH 3), 2.62 (q, 2H, J - 8 Hz, -CH2CH3) , 3.91 (s, 3H, -CC^CHy, 5.39 (s, 2H, -CH 2-C 6H 5), 7.39 (m, 5H, - C ^ ) , 8.04 (s, IH, -CH=), 10.26 (bs, IH, NH) . Anal.: Calcd. for C ^ H ^ N ^ : C, 68.17; H, 5.72; N, 7.95. Found: C, 67.97; H, 5.93; N, 7.93%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 352 (M+, 25), 334 (10), 320 (9), 305 (2), 261 (3), 245 (3), 145 (5), 130 (4), 91 (100). Mol. wt.: Calcd. for c2o H20 N2°4 : 3 5 2 • 1 4 1 6 • Found by high resolution mass spectrometry: 352.1422. 5 -(2-Cyano-2-methoxycarbonylviny1)-4-e thyl-3-methylpyrrole-2-carboxy1ic acid 174 2 -Benzyloxycarbonyl- 5 -(2 -cyano- 2-methoxycarbonylviny1)-4- ethyl- 3 -methylpyrrole 173 (60 g, 0.17 mole) was suspended along with 10% palladium on charcoal (5 g) and triethylamine (10 drops) i n tetrahydro-furan (200 mL). The s t i r r e d solution was hydrogenated at one atmosphere - 158 -and room temperature and the reaction was followed by t i c . At the end of the reaction, t i c showed no starting material. The catalyst was removed by f i l t r a t i o n and the solvent removed under reduced pressure. Following t h i s , methylene chloride was added, and the acid precipitated out as l i g h t yellow powder. The product (43.9 g, 98.6%) was collected by f i l t r a t i o n , washed with methylene chloride, and dried i n the a i r . MP: 200°C (dec). 1H NMR (5, DMSO-dr): 1.06 (t, 3H, J - 8 Hz, -CH 0-CH„), 2.25 (s, 3H, -CH 3), 2.61 (q, 2H, J = 8 Hz, -CH2-CH3), 3.84 (s, 3H, -C0 2CH 3), 8.02 (s, 1H, -CH-), 10.7 (bs, 1H, NH), 13.35 (bs, 1H, -CC^H). Anal.: Calcd. for C ^ H ^ N ^ . l / ^ O : C, 58.53; H, 5.48; N, 10.50. Found: C, 58.47; H, 5.32; N, 10.29%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 262 (M+, 100), 247 (17), 244 (14), 229 (56), 218 (10), 216 (11), 213 (22), 203 (27), 185 (40). Mol. wt.: Calcd. for C ^ H ^ N ^ : 262.0948. Found by high resolution mass spectrometry: 262.0949. 5-(2-Cyano-2-methoxycarbonylvinyl)-4-ethyl-2-iodo-3-methylpyrrole 175 0 175 159 -A suspension of 5-(2-cyano-2-methoxycarbonylvinyl)- 4-ethyl-3-methyl-pyrrole-2-carboxylic acid 174 (27.6 g, 0.11 mole), sodium acetate (32 g, 0.39 mole), acetic anhydride (21 mL, 0.22 mole), i n g l a c i a l acetic acid (400 mL) was heated i n a one l i t e r Erlenmeyer flask with s t i r r i n g . The pyrrole dissolved slowly as the temperature increased to 80°C. At this temperature a solution of iodine monochloride (20 g, 0.12 mole) i n g l a c i a l acetic acid (50 mL) was added dropwise and the temperature maintained at 80°C. At the end of the complete addition, the flask was removed from hot plate, cooled and water (600 mL) was added which gave a thick precipitate. The excess iodine was destroyed by hypophosphorus acid and the s o l i d collected through suction f i l t r a t i o n , washed with water and a i r dried. R e c r y s t a l l i z a t i o n from methanol-water gave a yellow needle shaped product (30 g, 83.1%). MP: 163-164°C. 1 H NMR (5, CDC1 3): 1.14 (t, 3H, J = 7.8 Hz, -CH2-CH3), 2.05 (s, 3H, -CH 3), 2.65 (q, 2H, J = 7.2 Hz, -CH2-CH3) , 3.91 (s, 3H, -CO^Hy , 7.85 (s, IH, -CH=), 9.64 (bs, IH, NH). Anal.: Calcd. f o r C ^ H ^ ^ O ^ : C, 41.88; H, 3.81; N, 8.14; I, 36.87. Found: C, 42.20; H, 3.96; N, 8.16; I, 36.75%. Mass spectrum (m/e, re l a t i v e intensity): 344 (M+, 100), 329 (17), 312 (35), 297 (30), 285 (39), 270 (12), 244 (24), 185 (36). Mol. wt.: Calcd. for C ^ H ^ N ^ ^ : 344.0016. Found by high resolution mass spectrometry: 344.0018. 160 -5-(2-Cyano-2-metb.oxycarbonylvinyl)-4-ethyl-3-methylpyrrole 176 5 -(2 -Cyano- 2-methoxycarbonylvinyl)-4- ethyl- 2 -iodo- 3-methylpyrrole 175 (27.5 g, 79.9 mmole) and zinc dust were suspended i n g l a c i a l acetic acid (250 mL) i n a one l i t e r Erlenmeyer flask, and the mixture was heated with s t i r r i n g on a hot plate. The pyrrole dissolved i n solution, which was further heated for one hour. The hot solution was then f i l t e r e d through a sintered funnel to remove zinc dust which was washed several times with methanol u n t i l the f i l t r a t e was colourless. The hot f i l t r a t e was diluted with water (400 mL) to give fine yellow needles. The product was collected through f i l t r a t i o n and washed with 20% methanol i n water and then with water to give 17.0 g (97.6%). The sample for analysis was c r y s t a l l i z e d from methanol-water. MP: 144°C. XH NMR (5, CDC1 3): 1.14 (t, 3H, J - 8 Hz, -CH2-CH3), 2.07 (s, 3H, -CH 3), 2.63 (q, 2H, J - 8 Hz, -CH2-CH3), 3.87 (s, 3H, -C0 2CH 3), 6.99 (d, 1H, J - 4 Hz, pyr-H), 7.99 (s, 1H, -CH.-), 9.67 (bs, 1H, NH). Anal.: Calcd. for C ^ H ^ O ^ C, 66.04; H, 6.47; N, 12.84. Found: C, 66.18; H, 6.35; N, 12.82%. - 161 -Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 218 (M+, 100), 203 (51), 187 (25), 186 (34), 185 (15), 171 (47), 159 (86), 158 (33), 144 (40), 132 (20), 118 (85). Mol. wt.: Calcd. for c 1 2 H i 4 N 2 ° 2 : 2 1 8 - 1 0 5 - Found by high resolution mass spectrometry: 218.1056. 4-Ethyl-5-formyl-3-methylpyrrole 177 5- (2-Cyano-2-methoxycarbonylvinyl)-4-ethyl-3-methylpyrrole 176 (6.3 g, 29 mmole) was refluxed with potassium hydroxide (6.7 g, 0.12 mole) solution i n water (50 mL) for three hours, under nitrogen. The pyrrole slowly dissolved and a brown o i l rose to the surface of the water. The mixture was cooled and kept i n the refrigerator overnight. Light tan needles along with a brown s o l i d were f i l t e r e d , and washed with water, and a i r dried to give 3.5 g (88.1%) of the product. The sample for analysis was obtained by r e c r y s t a l l i z a t i o n from methanol and water. - 162 -MP: 74-75°C. XH N M R (cS, CDC1 3): 1.22 (t, 3H, J - 8 Hz, -CH2-CH3> , 2.05 (s, 3H, -CH 3), 2.75 (q, 2H, J - 8 Hz), -CH2-CH3), 6.89 (d, 1H, J - 3.4 Hz, pyr-H), 9.62 (s, 1H, -CHO), 10.09 (bs, 1H, NH). Anal.: Calcd. f o r CgH-^ NO:" C, 70.04; H, 8.08; N, 10.21. Found: C, 69.82; H, 8.15; N , 10.14%. Mass spectrum: (m/e, r e l a t i v e i n t e n s i t y ) : 137 (M+, 100), 122 (92), 120 (31), 118 (5), 108 (32). Mol. wt.: Calcd. for CgH^NO: 137.0837. Found by high resolution mass spectrometry: 137.0839. I R (u , KBr): 3474 (N-H), 3281 (N-H), 1643 (C-0) cm"1, max 2-Ethoxycarbonyl- 3-(2-ethoxycarbonylethyl)-4,5-dimethylpyrrole 143 U 3 A mixture of ethyl 5-methyl-4,6-dioxoheptanoate 159 (100 g, 0.5 mole) and diethyl aminomalonate (113.75 g, 0.65 mole) was added to a gently b o i l i n g g l a c i a l acetic acid (400 mL) i n a 3 l i t e r Erlenmeyer - 163 -fl a s k heated on a magnetic s t i r hot plate. A vigorous reaction (evolu-ti o n of CC>2 and ethanol) sets in, which gradually subsides. The gentle b o i l i n g of acetic acid was continued for another hour i n the fume hood. The hot solution was then diluted with water and ice, and the s o l i d i f i e d product was f i l t e r e d and washed with water. The precipitate was dissolved i n ethanol (200 mL) by heating on a steam bath and the solution was f i l t e r e d . The f i l t r a t e was diluted with water and c h i l l e d i n an ice-bath, when the pyrrole c r y s t a l l i z e d as a white c r y s t a l l i n e s o l i d . The product was f i l t e r e d and washed with a 20% ethanol-water mixture followed by water, and a i r dried. Y i e l d 82 g (61%). MP: 90°C; L i t . 1 7 5 : 88-90°C. X H NMR (5, CDC1 3): 1.25 (t, 3H, J - 8 Hz, -C0 2CH 2-CH 3), 1.34 (t, 3H, J - 8 Hz, -C0 2CH 2CH 3), 1.97 (s, 3H, -CH3), 2.19 (s, 3H, -CH 3), 2.52 (t, 2H, J - 8 Hz, -CH2-CH2-), 3.02 (t, 2H, J - 8 Hz, -CH2-CH2-), 4.13 (q, 2H, J - 8 Hz, -C02-CH2-CH3), 4.31 (q, 2H, J -> 8 Hz, -C02-CH2-CH3), 8.80 (bs, IH, NH). Anal.: Calcd. for C^H^NO^ C, 62.90; H, 7.93; N, 5.24. Found: C, 62.63; H, 7.89; N, 5.29%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 267 (M+, 52), 253 (3), 222 (20), 194 (49), 193 (100), 180 (60), 164 (11), 152 (31), 147 (46), 134 (52). Mol. wt.: Calcd. for C^H^NO^: 267.1464. Found by high resolution mass spectrometry: 267.1468. - 164 -2-Benzyloxycarbonyl -3-<2-benzyloxycarbonyl€thyl)-4,5-dimethylpyrrole 163 2-Ethoxycarbonyl-3-(2-ethoxycarbonylethyl)-4,5 - dimethylpyrrole 143 (65.8 g, 0.25 mole) was heated i n dry d i s t i l l e d (dried over anhydrous potassium carbonate) benzyl alcohol (400 mL) i n a 2 l i t e r Erlenmeyer flask, under argon. The solution was brought to reflux temperature (209°C) to ensure the complete removal of moisture. To this hot solution, a concentrated solution of sodium i n benzyl alcohol (2 mL) was slowly added i n portions. A vigorous reaction sets i n with lowering of the temperature along with the removal of ethanol vapour. An additional amount of catalyst (2 mL) was added and the reaction mixture was heated to reflux (209"C) f o r 10 to 15 minutes. The hot reaction mixture was quenched by pouring i n a s t i r r e d mixture of acetic acid (150 mL) i n methanol (500 mL), and on addidng water (800 mL), a thick white precipitate obtained. The precipitate was s t i r r e d with a glass xod, f i l t e r e d and washed with 50% methanol-water mixture and then with water and dried i n the a i r to give 77.0 g (80%). The sample for analysis was c r y s t a l l i z e d from CH„Cl„-methanol. 163 - 165 -MP: 131-132°C; L i t . 1 7 5 : 130°C. XH NMR (5, CDCL 3): 1.93 (s, 3H, -CH3>, 2.16 (s, 3H, -CH3), 2.55 (m, 2H, -CH2-CH2-), 3.03 (m, 2H, -CH2-CH2) , 5.07 (s, 2H, -^-CgH ), 5.27 (s, 2H, -CH^CgH ), 7.35 (s, 10H, -CgH ), 8.6 (bs, 1H, NH). Anal.: Calcd. for C^H^NO : C, 73.64; H, 6.43; N, 3.58. Found: C, 73.40; H, 6.27; N, 3.50%. Mass spectrum (m/e, re l a t i v e i n t e n s i t y ) : 391 (M+, 47), 300 (35), 282 (17), 256 (30), 238 (28), 91 (100). Mol. wt.: Calcd. for C^H^NC^: 391.1776. Found by high resolution mass spectrometry: 391.1788. 2-Benzyloxycarbonyl-3-(2-methoxycarbonylethyl)-4,5-dimethylpyrrole 164 2-Benzyloxycarbonyl-3-(2-benzyloxycarbonylethyl)-4,5-dimethyl-pyrrole 163 (76.7 g, 0.20 mole) was suspended i n dry d i s t i l l e d methanol (800 mL) i n a 2 l i t e r Erlenmeyer flask. The suspension was s t i r r e d - 166 under argon, dry d i s t i l l e d (over calcium hydride) tetrahydrofuran (300 mL) was added to enhance the s o l u b i l i t y of the pyrrole. To this solution a concentrated solution of sodium (0.96 g) i n benzyl alcohol (15 mL) was slowly added and the mixture s t i r r e d at room temperature. The pyrrole slowly dissolved, giving a pale yellow coloured solution. The reaction was monitored by thin layer chromatography using 3% ether i n methylene chloride as developing solvent. When a l l the starting material had changed to methyl ester, the solution was concentrated at reduced pressure, and on addition of water, tanned white fibrous needles obtained. The s o l i d was f i l t e r e d and washed with 20% methanol-water mixture and f i n a l l y with water. The s o l i d was dried i n the a i r and then under vacuum to y i e l d 60.4 g, (97.8%) of the product. 175 MP: 90-91°C; L i t . : 93-95°C. NMR (5, CDC1 3): 1.95 (s, 3H, - C H y , 2.17 (s, 3H, -CH3), 2.50 (m, 2H, -CH2CH2), 3.03 (m, 2H, -CH^CH^ , 3.63 (s, 3H, -CO ^ H y , 5.27 (s, 2H, -CH 0-C,H„), 7.38 (s, 5H, -CH„-C,H C), 8.65 (bs, IH, NH). z o o / o 5 Anal.: Calcd. for C 1 oH o 1N0.: C, 68.55; H, 6.71; N, 4.44. Found: C, l o / l 4 68.30; H, 6.60; N, 4.41%. Mass spectrum (m/e, re l a t i v e intensity): 315 (M+, 27), 224 (25), 206 (7), 198 (15), 192 (52), 91 (100). Mol. wt.: Calcd. for c 1 8 H 2 i N ° 4 : 315.1469. Found by high resolution mass spectrometry: 315.1469. - 167 -2-Benzyloxycarbonyl-5-formyl-3-(2-methoxycarbonylethyl)-4-methyl-pyrrole 178 2-Benzyloxycarbonyl-3-(2-methoxycarbonylethyl)-4,5-dimethylpyrrole 164 (16.0 g, 51 mmole) was s t i r r e d i n methylene chloride (1 l i t e r ) and the mixture was cooled i n an ice-bath, under nitrogen. To t h i s , a mixture of s u l f u r y l chloride (8.6 mL, 14.45 g, 0.11 mole) i n methylene chloride (100 mL) was added dropwise. At the end of the complete addition, the solution was further s t i r r e d for one hour, and water (500 mL) was added and the s t i r r i n g continued overnight. The organic layer was separated, washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness. The product was chromatographed over s i l i c a (60-230 mesh, 100 g) and eluted by 3% ether i n methylene chloride. The product was c r y s t a l l i z e d from methylene chloride and n-hexane to give 14.9 g (88.8%) as an off-white powder. MP: 92-93°C. OHC 178 H NMR (5, CDC1 3): 2.33 (s, 3H, -CHp, 2.52 (m, 2H, -CH 2-CH 2-)» 3.02 (m, 2H, -CH2CH2), 3.60 (s, 3H, -C0 2-CH 3), 5.32 (s, 2H, -CHj-CgHL.), 7.37 - 168 -(s, 5H, -CH 2-C 6H 5), 9.55 (bs, 1H, NH), 9.77 (s, 1H, -CHO). Anal.: Calcd. for C l gH 1 9N0 5: C, 65.64; H, 5.83; N, 4.25. Found: C, 65.37; H, 5.78; N, 4.33%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 329 (M+, 30), 311 (7), 301 (4), 285 (9), 269 (7), 255 (26), 238 (11), 206 (39), 91 (100). Mol. wt.: Calcd. for C l gH i gN0 5: 329.1257. Found by high resolution mass spectrometry: 329.1269. 3-(2-Methoxycarbonylethyl)-5-formyl-4-methylpyrrole-2-carboxylic acid 179 0 179 A solution of 2-benzyloxycarbonyl- 5 -formyl- 3 -(2-methoxycarbonyl-ethyl)-4-methylpyrrole 178 (11.5 g, 28 mmole) i n tetrahydrofuran (100 mL), triethylamine (5 drops) was hydrogenated over 10% Pd on carbon catalyst (0.5 g) at one atmosphere. The reaction was checked by thin layer chromatography u n t i l the complete disappearance of starting material, and hydrogen uptake had ceased. The solution was f i l t e r e d to - 169 -remove the catalyst and the f i l t r a t e was concentrated. Upon the addition of methylene chloride, a white s o l i d powder precipitated out. The powder was f i l t e r e d , washed with methylene chloride, and dried to give 3.5 g (52.3%). The mother liquor was concentrated and reprecipitated by the addition of methylene chloride to give 3.04 g (45.4% y i e l d ) . MP: 143°C. XH NMR (5, DMSO-D,): 2.25 (s, 3H, -CH„), 2 . 5 (t, 2H, J = 7 . 5 Hz , fc> j -CH2-CH2-), 2.95 (t, 2H, J - 7.5 Hz, -CH^CH^, 3.58 (s, 3H, -C0 2-CH 3), 9.98 (s, IH, -CHO), 12.25 (bs, IH, NH), 12.94 (bs, IH, -CO H). Anal.: Calcd. for C-^H^NO^ C, 55.23; H, 5.48; N, 5.86. Found: C, 54.98; H, 5.48; N, 5.70%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 239 (M+, 33), 207 (19), 195 (30), 179 (100), 166 (52), 161 (50), 148 (78). Mol. wt.: Calcd. for C 1 1 H 1 3 N 0 5 : 239.0789. Found by high resolution mass spectrometry: 239.0797. 3-(2-Methoxycarbonylether)-5-formyl-2-iodo-4-methylpyrrole 180 - 170 -3-(2-Methoxycarbonylethyl)-5-formyl-4-methylpyrrole-2-carboxylic acid 179 (12.1 g, 51 mmole) was suspended i n chloroform (200 mL), and sodium bicarbonate (8.53 g, 0.10 mole) i n water (200 mL) was added with s t i r r i n g . The pyrrole dissolved with effervescence and was then treated with a mixture of sodium iodide (22.9 g, 0.15 mole) and iodine (12.9 g, 50 mmole) i n water (100 mL). The reaction mixture was refluxed for one hour and upon cooling to room temperature extracted with methylene chloride (2 x 100 mL). The solvent was evaporated to dryness and used for hydrogenation to prepare a-free pyrrole. A small amount of iodopyrrole was isolated for characterization. The crude mixture was dissolved i n methanol, water was added and cooled i n an ice-bath. Flesh coloured needle shaped iodopyrrole was obtained. The pyrrole was further r e c r y s t a l l i z e d from methanol-water (three times) to give pure pyrrole. MP: 147-149°C. XH NMR (6, CDC1 3): 2.34 (s, 3H, -Cfl-j) , 2.48 (t, 2H, J - 8 Hz, -CH2-CH2-), 2.73 (t, 2H, J = 8 Hz, -CH 2Cfl 2), 3.69 (s, 3H, -C0 2-CH 3), 9.23 (bs, 1H, NH), 9.41 (bs, 1H, -CHO). Anal.: Calcd. for C^H^NO^: C, 37.40; H, 3.77; N, 4.36; I, 39.52. Found: C, 37.44; H, 3.79; N, 4.37; I, 39.40%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 321 (M+, 11), 290 (2), 248 (51), 194 (100). Mol. wt.: Calcd. for C^H^NO,^: 320.9857. Found by high resolution mass spectrometry: 320.9865. - 171 -3-(2-Me thoxycarbonylethy1)- 5-formyl-4-me thylpyrrole 181 OHC OMe The crude mixture obtained from the iodination reaction was dissolved i n methanol (50 mL) and hydrogenated over 10% palladium on carbon (1.0 g) i n the presence of sodium acetate (5.0 g) at one atmosphere and room temperature. The completion of the reaction was observed by thin layer chromatography. After completion of the reaction, the solution was poured into water and extracted by methylene chloride. The organic phase was then evaporated to dryness. The crude product was p u r i f i e d by extraction with hot water, and c r y s t a l l i z e d on cooling i n the refrigerator. White f l a t needles were obtained (6 g, 60% y i e l d based on compound 179). MP: 74-76°C. XH NMR (5, CDC1 3): 2.31 (s, 3H, - C H y , 2.56 (t, 2H, J = 8 Hz, -CH2-CH2-), 2.76 (t, 2H, J - 8 Hz, -C^CH^), 3.68 (s, 3H, -C0 2-CH 3), 6.87 (d, IH, J - 4 Hz, pyr-H), 9.15 (bs, IH, NH), 9.60 (s, IH, -CHO). Anal.: Calcd. for C 1 ( )H 1 3N0 3: C, 61.51; H, 6.72; N, 7.18. Found: C, 61.78; H, 6.61; N, 7.24%. - 172 -Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 195 ( M + , 31), 136 (12), 123 (9), 122 (100), 120 (9). Mol. wt.: Calcd. for C^H^NO^ 195.0891. Found by high resolution mass spectrometry: 195.0901. 5-Acetoxymethyl-2-ethoxycarbonyl-4-ethyl-3-methylpyrrole 182 182 2-Ethoxycarbonyl-4-ethyl-3,5-dimethylpyrrole 162 (3.86 g, 19.8 mmole) was suspended i n g l a c i a l acetic acid (25 mL) i n a 250 mL Erlenmeyer flask. The solution was warmed on a steam bath and lead tetraacetate (9.3 g, 21 mmole) was added i n one portion, and further heated, u n t i l the pyrrole dissolved. The flask was removed from the steam bath and ethylene glycol (10 mL) was added to remove excess lead. The solution was cooled to room temperature, and the pyrrole was precipitated by the addi t i i o n of water. The precipitate was f i l t e r e d , washed several times with water and dried. Y i e l d 3.68 g (73.4%). - 173 -MP: 128°C; L i t . 1 7 5 : 127-128°C. XH NMR (5, CDC1 3): 1.12 (t, 3H, J - 7.5 Hz, -CH2-CH ) , 1.37 (t, 2H, J 7 Hz, -0-CH2-CH2-), 2.11 (s, 3H, -CHy, 2.35 (s, 3H, O-COCHy , 2.5 (q, 2H, J - 7.5 Hz, -CH2-CH3), 4.35 (q, 2H, J - 7 Hz, -0-CH.2-CH3), 5.09 (s, 2H, -CH2-0-), 9.05 (bs, IH, NH). Anal.: Calcd. for C^H^NO^ C, 61.65; H, 7.56; N, 5.53. Found: C, 61.72; H, 7.57; N, 5.38%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 253 (M+, 47), 240 (25), 224 (6), 209 (19), 194 (43), 180 (40), 166 (21), 148 (58), 134 (100). Mol. wt.: Calcd. for C 1 3H i gN0 4: 253.1293. Found by high resolution mass spectrometry: 253.1321. 5-Acetoxymethyl-2-ethoxycarbonyl-3,4-dimethylpyrrole 183 183 2-Ethoxycarbonyl-3,4,5-trimethylpyrrole 145 (4.5 g, 25 mmole) was suspended i n g l a c i a l acetic acid (25 mL) i n an Erlenmeyer flask and the solution heated on a steam bath. To this warm solution lead tetra-- 174 -acetate (11.53 g, 26 mmole) was added and the solution was warmed for an additional 10 minutes. The fla s k was removed from the steam bath, and ethylene g l y c o l (20 mL) was added to remove excess lead. The above solution was cooled to room temperature and water was added, when the acetoxymethylpyrrole precipitated as a white s o l i d . The s o l i d was f i l t e r e d , washed several times and dried to give 4.34 g ( y i e l d 72.6%). MP: 120°C. XH NMR (5, CDC1 3): 1.30 (t, 3H, J - 7.5 Hz, -0-CH2-CH3), 2.02 (s, 3H, -CH 3), 2.05 (s, 3H, -CH 3), 2.23 (s, 3H, -CH 3), 2.35 (s, 2H, -CH2-), 4-26 (q, 2H, J - 7 Hz, -0-CH2-CH3), 9.30 (bs, IH, NH). Anal.: Calcd. for C^H^NO^ C, 60.24; H, 7.16; N, 5.85. Found: C, 60.14; H, 7.17; N, 5.98%. 5-Acetoxymethyl-2-benzyloxycarbonyl-4-(2-methoxycarbonylethyl)- 3-methyl-pyrrole 184 184 - 175 -2-Benzyloxycarbonyl-4-(2-methoxycarbonylethyl)-3,5-dimethylpyrrole 144 (15 g, 48 mmole) was suspended in g l a c i a l acetic acid (100 mL) in a 500 mL Erlenmeyer flask. The solution was warmed on a steam bath and lead tetraacetate (22.17 g, 50 mmole) was added i n one portion, and the solution was heated further to complete the reaction. The fla s k was removed from the steam bath, and ethylene glycol (20 mL) added Co remove excess lead. The above solution was cooled to room temperature, and the pyrrole was pre c i p i t a t e d by the addition of water. The precipitate was f i l t e r e d , washed several times with water and dried to y i e l d 16.94 g (94.6%). MP: 109-110°C; L i t . 1 9 6 : 111-112°C. 1H NMR (5, CDC1 3): 2.06 (s, 3H, -CH3), 2.28 (s, 3H, 0-CH3), 2.45 (t, 2H, J - 7.6 Hz, -CH2CH2-), 2.79 (t, 2H, J - 7.6 Hz, -CH^CH^), 3.65 (s, 3H, -C0 2CH 3), 5.03 (s, 2H, -CH2-0-), 5.29 (s, 2H, CH^-C^), 7.37 (m, 5H, C.H C), 8.81 (bs, 1H, NH). D D Anal.: Calcd. for C--H_oN0,: C, 64.32; H, 6.21; N, 3.75. Found: C, Z U ZJ t> 64.90 H, 6.22; N, 3.79%. Mass spectrum (m/e, r e l a t i v e intensity): 373 (M+, 2), 315 (8), 313 (3), 242 (13), 134 (7), 108 (5), 91 (100). 176 -4.4 SYNTHESIS OF DIPYRROMETHANES 5-Acetoxymethyl-2-ethoxycarbonyl-4-ethyl-3-methylpyrrole 182 (5 g, 19.8 mmole) was refluxed with 80% g l a c i a l acetic acid (25 mL) for two and a h a l f hours. The hot solution was cooled to room temperature and extracted with methylene chloride. The organic phase was washed with water, dried over anhydrous sodium sulfate, f i l t e r e d , and evaporated to dryness.. The product was obtained as shiny white crystals by c r y s t a l l i z a t i o n from methanol (2.8 g, 75.6% y i e l d ) . MP: 126°C. LH NMR (5, CDC1 3): 1.04 (two t r i p l e t s , 6H, J - 8 Hz, -CH2-CH3), 1.32 (two t r i p l e t s , 6H, J - 8 Hz, -0-CH2-CH3), 2.28 (s, 6H, -CH.3), 2.41 (q, 4H, J - 8 Hz, -CH2-CH3), 3.87 (s, 2H, bridge-CH 2), 4.26 (q, 4H, J - 8 Hz, -0-CH2-CH3), 8.67 (bs, 2H, NH). Anal.: Calcd. for C 2 1H 3 QN 20 4: C, 67.35; H, 8.08; N, 7.48. Found: C, 67.05 H, 8.02; N, 7.65%. - 177 -Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 374 (M+, 21), 345 (2), 329 (5), 299 (11), 283 (10), 255 (14), 239 (6), 226 (7), 193 (100), 180 (22), 166 (13), 147 (43). Mol. wt.: Calcd. for C 2 1 H 3 0 N 2 ° 4 : 374.2196. Found by high resolution mass spectrometry: 374.2205. IR O , KBr): 3400 (N-H), 3280 (N-H), 1665 (C-0) cm"1 3,3'-Diethyl-4,4'-dimethyl-2,2'-dipyrromethane-5,5'-dicarboxylic acid 188 5,5'-Diethoxycarbonyl-3,3'-diethyl-4,4'-dimethyl-2,2'-dipyrromethane 185 (5.0 g, 13.4 mmole) was suspended with potassium hydroxide (3.5 g, 63 mmole) i n water (100 mL), 40 mL of ethyl alcohol was added and the solu-t i o n was refluxed for 5 hours. The hot solution was cooled to room temperature and the solution was carefully neutralized with g l a c i a l acetic acid. The white precipitate which slowly turned l i g h t purple i n colour was f i l t e r e d , washed with water and dried to give 3.8 g (89.2%). - 178 -MP: 142°C. ^ NMR (6, DMSO-d6): 1.0 (two t r i p l e t s , 6H, J - 7.5 Hz, -CH^CHy, 2.10 (s, 6H, -CH 3), 2.25 (q, 4H, J - 7 Hz, -CH2-CH3), 3.75 (s, 2H, bridge-CH 2), 11.10 (bs, 2H, -NH), 12.0 (bs, 2H, -C0 2H). Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 274 (M +-C0 2 > 20), 230 (52), 201 (15), 184 (9), 123 (76), 121 (100), 119 (34), 109 (30), 94 (55). Mol. wt.: Calcd. for C..H o oN„0 o, (C. ^ H o oN o0.-C0 o): 274.1674. Found by l o LL 2 / 1/ 2.2. AH- / high resolution mass spectrometry: 274.1686. IR (i/ , KBr): 3265 (broad, N-H), 1670 (broad, C=0) cm"1, max 5,5'-Diethoxycarbonyl-3,3'-4,4'-tetramethyl-2,2'-dipyrromethane 186 186 5-Acetoxymethyl-2-ethoxycarbonyl-3,4-dimethylpyrrole 183 ( 5 g, 21 mmole) was suspended i n 80% acetic acid solution (100 mL) and refluxed for 2.5 hours on a steam bath. The solution was cooled to room tempera-ture and the product was precipitated by the addition of water. The precipitate was f i l t e r e d , washed with water and dried to give 2.6 g (71.6%) of the dipyrromethane. - 179 -MP: 204°C; L i t . 1 9 6 : 196-198°C. "4 NMR (S, CDC1 3): 1.30 (t, 6H, J - 7.5 Hz, 2 x -0-CH2-CH3), 1.95 (s, 6H, 2 x -CH 3), 2.25 (s, 6H, 2 x -CH ), 3.86 (s, 2H, -CH 2-), 4.26 (q, 4H, 2 x -0-CH2-CH3), 9.3 (bs, 2H, NH). Anal.: Calcd. for C 1 9 H 2 6 N 2 ° 4 : C> 6 5 - 8 7 ; H> 7.56; N, 8.08. Found: C, 65.90; H, 7.52; N, 8.05%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 346 (M+, 83), 331 (5), 300 (24), 285 (16), 273 (18), 255 (27), 227 (85), 179 (100). Mol. wt.: Calcd. for C ^ H ^ N ^ : 346.1886. Found by high resolution mass spectrometry: 346.1895. IR (v , KBr): 3320 (N-H), 1685 (C=0) cm"1, max 3,3',4,4'-Tetramethyl-2,2'-dipyrromethane-5,5'-dicarboxylic acid 189 5,5'-Diethoxycarbonyl-3,3',4,4'-tetramethyl-2,2'-dipyrromethane 186 (1.5 g, 4.34 mmole) was suspended i n water (50 mL) with potassium hydroxide (0.971 g, 17.34 mmole), n-propanol (20 mL) was added, and the - 180 -solution refluxed for 12 hours. The solution was then cooled to room temperature and c a r e f u l l y neutralized with g l a c i a l acetic acid. The precipitated product was f i l t e r e d , washed with water and dried to afford 0.93 g (74.6%). MP: >180°C ( d e c ) . 1H NMR (5, DMS0-d6): 1.95 (s, 6H, 2 x -CH 3), 2.30 (s, 6H, 2 x -CH3>, 4.1 (s, 2H, 2 x -CH 2), 10.0 (bs, 2H, -NH), 12.0 (bs, 2H, CC^H). Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 246 (M +-C0 2 > 3), 202 (M +-2C0 2 > 66), 187 (18), 108 (100). Mol. wt.: Calcd. for C 1 4 H 1 8 N 2 0 2 (M+-CC>2) and C 1 3H l gN 2 (M+-2CC>2) are 246.1364 and 202.1466 respectively. Mol. wt.: Found by high resolution mass spectrometry for (M+-CC>2) and (M+-2C02) are: 246.1377 and 202.1467. IR ( i / , KBr): 3310 (broad, N-H), 1665 (C-0) cm"1, max i / i / 5,5'-dIbenzyloxycarbonyl-3,3'-(2-dimethoxycarbonylethyl)-4,4'-dimethyl-2,2'-dipyrromethane 187 187 - 181 -5 -Acetoxymethyl- 2-benzyloxycarbonyl-4-(2-methoxycarbonylethyl) - 3 -methylpyrrole 184 (16.94 g, 45.4 mmole) was refluxed with 80% g l a c i a l acetic acid (150 mL) for two and a h a l f hour. The hot solution was then cooled to room temperature and extracted with methylene chloride. The organic phase was washed with water, dried over anhydrous sodium sulfate, f i l t e r e d and evaporated to dryness. The viscous o i l obtained was dissolved i n a minimum of methanol and cooled. An o f f white c r y s t a l l i n e granular s o l i d was obtained. The product from three crops gave 11.10 g ( y i e l d 79.7%). MP: 94-96°C; L i t . 1 9 6 : 96-97°C. XH NMR (5, CDC1 3): 2.29 (t, 6H, 2 x -CHy, 2.51 (t, 4H, J = 7.3 Hz, 2 x -Cfl 2-CH 2), 2.76 (t, 4H, J = 7.3 Hz, 2 x -CH^CHy, 3.58 (s, 6H, 2 x -C0.CH-), 3.97 (s, 2H, -CH 0), 5.25 (s, 4H, 2 x -CH0-C\HC), 7.34 (m, / J Z Z O O 10H, 2 x -CH.-C,HC), 9.12 (bs, 2H, NH). Z D J Anal.: Calcd. for C, cH o oN„0 • C, 68.39; H, 6.23; N, 4.56. Found: C, J D J o Z o 68.68; H, 6.35; N, 4.53%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 614 (M+, 3), 524 (8), 523 (26), 506 (4), 479 (5), 415 (12), 389 (7), 91 (100). Mol. wt.: Calcd. for C ^ H ^ N ^ : 614.2616. Found by high resolution mass spectrometry: 614.2612. IR (v , KBr): 3380 (N-H), 3310 (broad, N-H), 1685 (broad, C=0) cm"1. H13.2C - 182 -3,3'-(2-Dime thoxycarbonyle thy1)-4,4'-dIme thy1-2,2'-dlpyrromethane-5,5'-dicarboxylic acid 190 190 5,5'-Dibenzyloxycarbonyl-3,3'-(2-dimethoxycarbonylethyl)-4,4'- dimethyl-2,2'-dipyrromethane 187 (6.0 g, 9.77 mmole) was suspended with 10% palladium-carbon (0.3 g) i n THF (100 mL) and triethylamine (5 drops). The mixture was shaken under one atmosphere of hydrogen at room temperature t i l l hydrogen uptake ceased (530 mL hydrogen). The reaction mixture was removed from the hydrogenator and the organic solvent was evaporated under reduced pressure. The resulting s l i d was dissolved i n methanol and treated with saturated sodium bicarbonate (50 mL). The aqueous solution was f i l t e r e d to remove the catalyst, and the f i l t r a t e was neutralized with acetic acid. A pink-white precipitate was obtained. The precipitate was f i l t e r e d , washed with water and dried i n the a i r to y i e l d 3.42 g (80.7%). MP: 160-165°C ( d e c ) . XH NMR (6\ CDC1 3): 2.30 (s, 6H, 2 x -Cfl 3), 2.65 (t, 4H, J = 7.3 Hz, - 183 -2 x -CH2-CH2), 2.76 (t, 4H, J - 7.3 Hz, 2 x -CH2-CH2>, 3.75 (s, 6H, 2 x -C0 2CH 3), 4.52 (s, 2H, 2 x -Cfl^), 10.5 (bs, 2H, NH). Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 390 (M +-C0 2, 0.8), 346 (M+-2C02, 40), 259 (100), 199 (17), 197 (24), 180 (46). Mol. wt.: Calcd. for M+-CC>2 ( c 2o H26 N2°6 a n d f ° r M + " 2 C ° 2 ^ W ^ ^ l 0 ^ respectively: 390.1782 and 346.1884. Found by high resolution mass spectrometry: 390.1787 and 346.1893, respectively. IR (u , KBr): 3320 (broad, N-H), 1655 (broad, C=0) cm"1, max 4.5 SYNTHESIS OF DIPYRROMETHENES 3'-Ethyl-4-(2-methoxycarbonylethyl)-3,4',5-trimethyl-2,2'-dlpyrro-methene hydrobromide 208 4-Ethyl-5-formyl-3-methylpyrrole 177 (1.59 g, 11.6 mmole) and 4-(2-methoxycarbonylethyl)-3,5-dimethylpyrrole-2-carboxylic acid 207 184 -(2.506 g, 11.2 mmole) were suspended i n tetrahydrofuran (100 mL, CaH^, d i s t i l l e d ) , and the mixture was f i l t e r e d to give a clear solution. To this 48% aqueous HBr (4 mL) added. The resulting dark solution was s t i r r e d for 5 minutes, the solvent was slowly removed under reduced pressure (rotary evaporator) and methanol was added. The re s u l t i n g solution was cooled i n an ice-bath. The c r y s t a l l i n e product (purple) was f i l t e r e d , washed with cold methanol followed by ether, and then a i r dried to give 3.43 g (80.7%). The mother liquor was concentrated to give and a second crop 0.50 g (11.8%). The nmr spectra of both crops were very clean. MP: 158°C (dec). NMR (5, CDC1 3): 1.20 (t, 3H, J-=7.5 Hz, -CH2-CH3), 2.09 (s, 3H, -CH 3), 2.34 (s, 3H, -CH 3), 2.49 (t, 2H, J = 7.2 Hz, -CH2-CH2), 2.70 (q, 2H, overlap, -CH2-CH3), 2.73 (s, 3H, overlap, -CH 3), 2.77 (t, 2H, J -7.2 Hz, -CH2-CH2), 7.16 (s, 1H, -CH=), 7.54 (d, 1H, J = 4 Hz, Pyr-H), 13.07 (bs, 1H, NH), 13.21 (bs, 1H, NH). Anal.: Calcd. for C ^ H ^ N ^ B r : C, 56.69; H, 6.61; N, 7.35; Br, 20.96. Found: C, 56.87; H, 6.49; N, 7.42; Br, 20.91%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 300 (M+-HBr, 85), 285 (94), 271 (31), 227 (37), 211 (83), 198 (54), 197 (100). Mol. wt.: Calcd. for C ^ H ^ N ^ (C^H^N^Br-HBr) : 300.1830. Found by high resolution mass spectrometry: 300.1834. - 185 -5-Bromo-4-ethyl-4'-(2-methoxycarbonyl ethyl)-3,3'-dimethyl-2,2'-dipyrro-methene hydrobromide 212 5-Bromo-4-ethyl-3-methylpyrrole-2-carboxylic acid 169 (0.35 g, 1.51 mmole) and 3-(2-methoxycarbonylethyl)-5-formyl-4-methylpyrrole 181 (0.31 g, 1.6 mmole) were suspended i n methylene chloride (20 mL), 30-32% HBr i n acetic acid (1.0 mL) was added. After s t i r r i n g the solution for one hour, methylene chloride was removed and ether was added to give an orange-brown s o l i d . The s o l i d was f i l t e r e d , washed with ether and dried to give 0.48 g. ( 71.7%). MP: 163-165°C XH NMR (6, CDC1 3): 1.10 (t, 3H, J = 8 Hz, -CH2-CH3), 2.34 (s, 3H, -CH 3), 2.37 (s, 3H, -CH.3), 2.49 (q, 2H, J - 8 Hz, -CH2-CH2) , 2.57 (t, 2H, J - 7.6 Hz, -CH2-CH2), 2.78 (t, 2H, J - 7.6 Hz, -CH^CHy, 3.68 (s, 3H, -C0 2-CH 3), 7.22 (s, IH, -CH-), 7.71 (d, IH, J = 4 Hz, Pyr-H), 13.66 (bs, IH, NH), 13.74 (bs, IH, NH). Anal.: Calcd. for C l yH 2 2N 20 2Br 2: C, 45.76; H, 4.97; N, 6.28; Br, 35.82. Found: C, 45.65; H, 4.98; N, 6.30; Br, 35.85%. - 186 -Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 364 (M+l-HBr, 7.0), 366 (M+2-HBr, 6.7), 349, 351 (7.0, 6.7), 291, 293 (5.4, 4.7), 270 (33.8). 5'-Bromo-5-bromomethyl-3'-ethyl-4-(2-methoxycarbonylethyl)3-4'-di-me thyl-2,2'-dipyrromethene hydrobromide 213 3 -Ethyl-4'-(2-methoxycarbonylethyl)-3'-4,5'-trimethyl-2,2'-dipyrro-methene hydrobromide 208 (3.0 g, 7.9 mmole) was suspended i n methylene chloride (50 mL) i n a round bottom flask, t r i f l u o r o a c e t i c acid (10 mL) was added and the mixture was protected i n the fume hood from moisture. To this dark solution a weighed amount of bromine (6.293 g, 40 mmole) i n methylene chloride was added. The solution was protected from moisture for 5 days, and occasionally shaken and internal pressure was released. After 5 days, the excess bromine and organic solvent were removed under reduced pressure. The dark residue was dissolved i n methylene chloride (three times) and evaporated to dryness. The resulting s o l i d was further dried overnight under vacuum, dissolved i n a minimum of methylene chloride, treated with cyclohexene (1.0 mL) and c r y s t a l l i z e d C02Me Br 213 187 -by the addition of ether. An orange-red fine c r y s t a l l i n e s o l i d was obtained. The s o l i d was collected by f i l t r a t i o n , washed with ether (3 times) and a i r dried. Y i e l d 3.92 g (92.2%). MP: 182°C ( d e c ) . ^ NMR (6~, CDC1 3): 1.21 (t, 3H, J - 8 Hz, -CH2-CH3), 2.08 (s, 3H, -CH 3), 2.32 (s, 3H, -CH 3), 2.60 (t, 2H, J - 7.6 Hz, -CH2-CH2), 2.73 (q, 2H, J - 8 Hz, -CH2-CH3), 2.86 (t, 2H, J -= 7.6 Hz, -CH2-CH2), 3.68 (s, 3H, -C0 2-CH 3), 4.96 (s, 2H, -CH^Br), 7.13 (s, IH, -CH-), 13.85 (bs, IH, NH), 13.93 (bs, IH, NH). Anal.: Calcd. for C- oH o oN o0_Br o: C, 40.10; H, 4.30; N, 5.20; Br, lo 25 2 2 5 44.46. Found: C, 40.29; H, 4.18; N, 5.11; Br, 44.50%. 3',4-DIethyl-3,4',5-trimethyl-2,2'-dipyrromethene hydrobromide 210 210 A suspension of 2-benzyloxycarbonyl-4-ethyl-3,5-dimethylpyrrole 171 (5.12 g, 19.9 mmole), 10% palladium on charcoal (0.2 g) i n tetrahydro-furan (100 mL) was hydrogenated i n the presence of triethylamine (5 188 -drops) t i l l the hydrogen uptake ceased to take (500 mL). The reaction vessel was removed from the hydrogenator and 4-ethyl-5-formyl-3-methyl-pyrrole 177 (2.6 g, 19.0 mmole) was added, the solution f i l t e r e d and to the clear f i l t r a t e 48% hydrobromic acid (5 mL) was added. The solution was concentrated under reduced pressure and cooled. The product (5.0 g, 77.8%) was obtained as red c r y s t a l s . The remaining solution was placed i n the r e f r i g e r a t o r overnight and the product (1.1 g, 17.1%) obtained as deep purple c r y s t a l s . The two were shown to be i d e n t i c a l by nmr. MP: 179°C XH NMR (6, CDCT 3): 1.10 (t, 3H, J = 8 Hz, -CH2-CH3), 1.20 (t, 3H, J = 8 Hz, -CH2-CH3), 2.09 (s, 3H, -CH ), 2.32 (s, 3H, -CHy, 2.46 (q, 2H, J = 7.6 Hz, -CH2-CH3), 2.70 (q, overlap, 2H, -CH2-CH2), 2.72 (s, overlap, 3H, -CH 3), 7.14 (s, IH, -CH=), 7.50 (d, IH, J = 4 Hz, pyr-H), 12.83 (bs, IH, -NH), 13.02 (bs, IH, NH). Anal.: Calcd. for O. ,H 0.N„Br: C, 59.44; H, 7.18; N, 8.67; Br, 24.71. l b 25 2 Found: C, 59.31; H, 7.12; N, 8.65; Br, 24.65%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 242 (M+-HBr, 77), 227 (100), 213 (65), 198 (77), 184 (45). Mol. wt.: Calcd. forM +-HBr: C-,H 0„N • 242.1778. Found by high 16 22 2 J ° resolution mass spectrometry: 242.1788. - 189 -5-Bromo-3',4-diethyl-3,4'-dimethyl-2,2'-dipyrromethene hydrobromide 211 A mixture of 4-ethyl-5-formyl-3-methylpyrrole 177 (2.07 g, 15.11 mmole) and 5-bromo-4-ethyl-3-methylpyrrole-2-carboxylic acid 169 (2.41 g, 10.39 mmole) were s t i r r e d i n anhydrous methylene chloride (CaH 2 dis-t i l l e d ) (30 mL), and 20-30% hydrobromic acid i n acetic acid (1.5 mL) was added and the s t i r r i n g continued for 15-20 minutes. The solvent was concentrated and ether added slowly with swirling. The dark brown (rusty colour) fine c r y s t a l l i n e product, 3.5 g (59.8%) was obtained. MP: > 180°C ( d e c ) . ^ NMR (5, CDC1 3): 1.20 (two t r i p l e t s , overlap, 6H, 2 x -CH2-CH3), 2.10 (s, 6H, 2 x -CH 3), 2.72 (q, overlap, 4H, 2 x -CH2-CH3), 7.17 (s, 1H, -CH-), 7.68 (d, 1H, J - 4 Hz, pyr-H), 13.63 (bs, 1H, NH), 13.71 (bs, 1H, NH). 211 Anal.: Calcd. for C 1 5H 2 QN 2Br 2: C, 46.42; H, 5.19; N, 7.22; Br, 41.17. Found: C, 46.48; H, 5.20; N, 7.30; Br, 41.20%. 190 -Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 306 (M+l-HBr, 25.6), 308 (M+2-HBr, 25.16), 291, 293 (20.6, 17.8), 277, 279 (18.3, 13.04), 212 (85.4), 198 (100). 5'-Bromo-5-bromomethyl-3'-4-diethyl-3,4'-dimethyl-2,2'-dipyrromethene hydrobromide 214 3',4-Diethyl-3,4',5-trimethyl-2,2'-dipyrromethene hydrobromide 210 (2.0 g, 6.2 mmole) was dissolved i n dichloroethane (50 mL) and t r i -f luoroacetic acid (10 mL), bromine (4.95 g, 31 mmole) were added and the mixture was protected from moisture for 5 days. The solvent and excess bromine was removed under reduced pressure. The traces of solvent were removed under vacuum (overnight). The resulting s o l i d was dissolved i n methylene chloride (20 mL), cyclohexene (5 mL) was added, the solution cooled i n an ice-bath, and the product was c r y s t a l l i z e d by the addition of diethyl ether. The dark red/orange shiny crystals were f i l t e r e d and dried to give the product, 2.5 g (83.9%). - 191 -MP: >177°C ( d e c ) . 1H NMR (S, CDCT 3): 1.21 (two t r i p l e t s , overlap, 6H, 2 x -CH2-CH3), 2.08 (s, 3H, -CH 3), 2.32 (s, 3H, -CH 3), 2.54 (q, 2H, J - 7 Hz, -CH2-CH3), 2.74 (q, 2H, J - 7 Hz, -CH2-CH3), 4.89 (s, 2H, -Cfl 2-Br), 7. (s, 1H -CH.-), 13.78 (bs, 1H, -NH), 13.83 (bs, 1H, NH). Anal.: Calcd. for c 1 6 H 2 1 N 2 B r 3 : C> 39.95; H, 4.39; N, 5.82; Br, 49.83. Found: C, 39.97; H, 4.45; N, 5.60; Br, 49.61%. Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 400 (M+-HBr, 1.4). 4.6 SYNTHESIS OF BILADIENES-AC 1,19-DIdeoxy-3,8,12,17-tetraethyl-2,7,13,18-tetramethylbiladiene-ac dihydrobromide 191 191 3,3'-Diethyl-4,4'-dimethyl-2,2'-dipyrromethane-5,5'-dicarboxylic acid 188 (3.5 g, 11 mmole) was suspended with 4-ethyl-5-formyl-3-methylpyrrole 177 (3.09 g, 22.6 mmole) i n tetrahydrofuran (100 mL) and - 192 -the solution was f i l t e r e d . To the clear solution, 48% aq. HBr (5 mL) was added with s t i r r i n g . The solution was concentrated under reduced pressure, methanol added, and the solution was cooled i n an ice-bath. The re s u l t i n g thick precipitate was f i l t e r e d , washed with ether and dried to give red-powder, 6 g (86.6%). MP: 206°C ( d e c ) . XH NMR (5, GDC13/TFA): 0.92 (t, 6H, J - 8 Hz, 2 x -CH^CHy , 1.22 (two t r i p l e t s , 6H, J - 8 Hz, -CH^CHy, 2.13 (s, 6H, 2 x -CHy, 2.33 (s, 6H, 2 x -CH 3), 2.53 (q, 4H, J - 7.6 Hz, 2 x -CH.2-CH3) , 2.74 (q, 4H, J -7.6 Hz, 2 x -CH2-CH3), 4.79 (s, 2H, -CH 2-), 7.31 (s, 2H, -CH-), 7.69 (d, 2H, J - 4 Hz, pyr-H), 12.33 (bs, 2H, -NH), 12.39 (bs, 2H, -NH). Anal.: Calcd. for C 3 1H 4 2N 4Br 2: C, 59.05; H, 6.71; N, 8.89; Br, 25.35. Found: C, 59.43; H, 6.69; N, 8.74; Br, 25.16%. 1,19-Dibromo-3,8,12,17-tetraethyl-2,7,13,18-tetramethyl-l,19-dideoxy-biladiene-ac dihydrobromide 199 199 - 193 -l,19-Dideoxy-3,8,12,17-tetraethyl-2,7,13,18-tetramethylbiladiene-ac dihydrobromide 191 (5.0 g, 7.9 mmole) was dissolved i n methylene chloride (50 mL) i n a 100 mL round-bottom flask, t r i f l u o r o a c e t i c acid (10 mL) was added and the mixture was protected from moisture. To the result i n g mixture, bromine (6.31 g, 39.5 mmole) i n methylene chloride (20 mL) was added and s t i r r e d for 15 minutes. The solvent was removed by rotary evaporator and the traces were removed under vacuum. The s o l i d was dissolved i n a minimum of methylene chloride (20 mL), cyclo-hexene (5 mL) was added and the mixture cooled i n an Ice-bath. The precipitated s o l i d was f i l t e r e d , washed with ether and dried to give an orange-red shiny powder, 6.0 g (95.9%). The sample for analysis was obtained by c r y s t a l l i z a t i o n from methylene chloride and ether. MP: 210°C ( d e c ) . XH NMR (5, CDC13/TFA): 1.04 (t, 6H, J = 8 Hz, 2 x -CH2-CH3), 1.22 (t, 6H, J - 8 Hz, -CH2-CH3), 2.09 (s, 3H, 2 x -CH 3), 2.33 (s, 3H, 2 x -CH 3), 2.56 (q, 4H, J - 7.6 Hz, 2 x -CH2-CH3), 2.76 (q, 4H, J = 7.6 Hz, 2 x -CH2-CH3), 4.59 (s, 2H, -CH2-), 7.24 (s, 2H, -CH=), 12.51 (bs, 2H, NH), 12.53 (bs, 2H, NH). Anal.: Calcd. for C 3 1H 4 QN 4Br 4: C, 47.23; H, 5.11; N, 7.11; Br, 40.54. Found: C, 47.53; H, 4.97; N, 7.35; Br, 40.23%. Mass spectrum (m/e): 466 [M+-(2HBr + 2Br)], 451, 437. FAB (m/e): 788 (M +), 626 (M+-2HBr). - 194 -1,19-Dideoxy-8,12-diethyl-2,3,7,13,17,18-hexamethyl-biladiene-ac dihydrobromlde 192 3,3'-Diethyl-4,4'-dimethyl-2,2'-dipyrromethane-5,5'-dicarboxylic acid 188 (2.0 g, 6.3 mmole) and 5-formyl-3,4-dimethylpyrrole (1.59 g, 12.92 mmole) were suspended i n methanol (50 mL). The result i n g mixture was heated and brought to reflux under nitrogen and then 48% hydrobromic acid (10 mL) was added. The dark solution was removed and s t i r r e d for 10 to 15 minutes i n an ice-bath. The precipitated product was f i l t e r e d and washed with ether. The resulting dark brown product was dried under vacuum to give 3.7 g ( y i e l d 97.6%). 117 MP: 204-210°C ( d e c ) ; L i t . : >300°C 1H NMR (6, CDC13/TFA): 1.02 (t, 6H, J - „8 Hz, 2 x -CH2-CH3), 2.12 (s, 6H, 2 x -CH 3), 2.38 (s, overlap, 12H, 4 x -CHy, 2.55 (q, overlap, 4H, 2 x -CH2-CH3), 4.6 (s, 2H, -CH2-), 7.4 (s, 2H, -CH-), 7.71 (d, 2H, J - 3 Hz, pyr-H), 11.94 (bs, 4H, -NH). - 195 Anal.: Calcd. for C ^ H ^ N ^ r ^ C, 57.82; H, 6.36; N, 9.29; Br, 26.53. Found: C, 57.59; H, 6.37; N, 9.30; Br, 26.59%. Mass spectrum (m/e): 440 (M+-2HBr), 425, 411. 1,19-Dibromo-8,12-diethyl-2,3,7,13,17,18-hexamethyl 1,19-dideoxy-biladiene-ac dihydrobromide 87 1,19-DIdeoxy-8,12-diethyl-2,3,7,13,17,18-hexamethylbiladiene-ac dihydrobromide 192 (0.516 g, 0.86 mmole) was suspended i n methylene chloride (10 mL), t r i f l u o r o a c e t i c acid was (1.0 mL) added. To the dark solution, bromine (0.548 g, 3.44 mmole) i n methylene chloride (5 mL) was added. The solution was s t i r r e d and protected from moisture for 15 minutes. The solvent was removed under reduced pressure and the traces were removed under vacuum. The product was dissolved i n a minimum of methylene chloride (10 mL) and cyclohexene (1.0 mL) added. The solution was shaken and the product was c r y s t a l l i z e d by the addition of ether. The precipitated product was f i l t e r e d , washed with a methylene - 196 -chloride-ether mixture and dried to give 0.634 g (97.1%) of red-orange shiny f l a t flakes. 121 MP: >250°C ( d e c ) ; L i t . : >300°C. NMR (6, CDC13/TFA) : 1.10 (t, 6H, J - 7.5 Hz, 2 x -CH^CHy, 2.1 (s, 6H, 2 x -CH 3), 2.35 (s, 6H, 2 x -CH 3), 2.38 (s, 6H, 2 x -CHy, 2.59 (q, 4H, J •= 7 Hz, 2 x -CH2-CH3), 4.50 (s, 2H, -CH2-), 7.29 (s, 2H, -CH=), 12.36 (bd, 4H, -NH). Anal.: Calcd. for C^H^N^Br^ C, 45.81; H, 4.77; N, 7.36; Br, 42.04. Found: C, 45.86; H, 4.76; N, 7.03; Br, 42.14%. Mass spectrum (m/e): 438 [M+-(2HBr + 2Br)], 423, 409. FAB (m/e): 760 (M +), 731, 598. 1,19-DIdeoxy-2,3,7,8,12,13,17,18-octamethylbiladiene-ac dihydrobromide 193 193 3,3',4,4'-Tetramethyl-2,2'-dipyrromethane-5,5'-dicarboxylic acid 189 (3.0 g, 13 mmole) and 5-formyl-3,4-dimethylpyrrole (3.2 g, 26.1 - 197 -mole) were suspended i n methanol and the solution brought to reflux. To this solution 48% hydrobromic acid (10 mL) was added and the solution s t i r r e d further for ha l f an hour. The solvent was concentrated, the re s u l t i n g precipitate was f i l t e r e d , and washed with methanolic hydrobromic acid, and the product dried under vacuum to give a black powder, 6.0 g (80.4%). MP: 190-195°C ( d e c ) . XH NMR (£, CDCI3/TFA): 2.1 (s, 12H, 4 x -CH3), 2.34 (s, 12H, 4 x -CH 3), 4.6 (s, 2H, bridge-CH 2), 7.38 (s, 2H, -CH-), 7.69 (d, 2H, J - 3.4 Hz, pyr-H), 12.08 (bs, 4H, -NH). Anal.: Calcd. for C^H.,. N. Br • C, 56.46; H, 5.97; N, 9.75; Br, 27 34 4 2 27.82. Found: C, 56.56; H, 6.00; N, 9.65; Br, 27.94%. l,19-Dibromo-2,3,7,8,12,13,17,18-octamethyl-l,19-dideoxybiladiene-ac dihydrobromide 200 200 1,19-Dideoxy-2,3,7,8,12,13,17,18-octamethylbiladiene-ac dihydrobro-mide 193 (1.098 g, 1.9 mmole) was suspended i n methylene chloride (20 - 198 -mL), t r i f l u o r o a c e t i c acid (5 mL) was added followed by the addition of bromine (1.22 g, 7.6 mmole) i n methylene chloride (10 mL). The solution was protected from moisture for 15 to 20 minutes and the mixture was taken to dryness. Traces of bromine were removed under vacuum and the s o l i d dissolved i n a minimum of methylene chloride (20 mL), treated with cyclohexene (2 mL) and diethyl ether was then added with s t i r r i n g . The resul t i n g dark precipitate was f i l t e r e d and washed several times with ether-CH 2Cl 2 (1:1 mixture) and dried to give 1.15 g (82.7%). MP: >230°C ( d e c ) . •^H NMR 400 MHz (6\ CDC13/TFA) : 2.07, 2.11, 2.31, 2.38 (4 x s, 24H, 8 x -CH 3), 4.57 (s, 2H, bridge-CH 2), 7.28 (s, overlap, 2H, -CH=), 12.41 (bs, 2H, -NH), 12.52 (bs, 2H, NH). Anal.: Calcd. for C^H^N^Br^: C, 44.29; H, 4.41; N, 7.65; Br, 43.65. Found: C, 44.03; H, .4.67; N, 7.44; Br, 43.75%. Mass spectrum (m/e): 410 [M+-(2HBr + 2Br)], 395. FAB (m/e): 732 (M +). 1,19-Dideoxy-2, 3,7,8,12,13,17,18-octaethylbiladiene-ac dihydrobromide 196 196 - 199 -5,5'-Benzyloxycarbonyl-3,3',4,4'-tetraethyl- 2,2'-dipyrromethane (2.63 g, 5 mmole) was suspended i n 40 mL of THF and hydrogenated over 10% Pd-C at one atmosphere at room temperature u n t i l the hydrogen uptake ceased (200 mL H^). The solution was removed from the hydrogenation apparatus and, 5-formyl-3,4-diethylpyrrole (1.51 g, 10 mmole) was added and f i l t e r e d through c e l i t e to remove the catalyst. To the clear solution, 48% hydrobromic acid (10 mL) was added and the solution s t i r r e d for h a l f an hour. The solution was concentrated under reduced pressure and cooled. The product was f i l t e r e d , washed with ethyl acetate to give greenish-brown glistening crystals. Y i e l d 3.084 g (91%). MP: >250°C (dec.) 1H N M R ' ( 5 , CDC13/TFA): 1.20 (t, overlap, 24H, 8 x -CH^CHy , 2.65 (m, overlap, 16H, 8 x -CH2-CH3), 4.58 (s, 2H, -CH2-), 7.35 (s, 2H, -CH=), 7.75 (d, 2H, J - 4 Hz, pyr-H), 12.08 (bs, 4H, -NH). Anal.: Calcd. for C o cH C AN.Br • C, 61.20; H, 7.34; N, 8.16; Br, 35 50 4 / 23.29. Found: C, 60.96; H, 7.44; N, 8.04; Br, 23.15%. - 200 -1,19-Dibromo-2,3,7,8,12,13,17,18-octaethyl-1,19-dideoxybiladiene-ac dlhydrobromide 202 202 1,19-Dideoxy-2,3,7,8,12,13,17,18-octaethylbiladiene-ac dlhydrobro-mide 196 (0.686 g, 1 mmole) was suspended i n methylene chloride (30 mL), t r i f l u o r o a c e t i c acid (1.0 mL) added, followed by the addition of bromine (0.80 g, 5 mmole) solution i n methylene chloride (10 mL). The resulting dark solution was protected from moisture for 15 minutes and then taken down to dryness. The crude product was dissolved i n a minimum of methylene chloride (10 mL), cyclohexene (2 mL) was added and the solution was s t i r r e d for 10 minutes. The product was c r y s t a l l i z e d by adding diethyl ether and after f i l t r a t i o n , the product was washed with a methylene chloride-ether mixture and dried to give bright red shiny crystals, 0.763 g ( y i e l d 90.4%). MP: >205°C ( d e c ) . XH NMR (5, CDC13/TFA): 1.18 (m, overlap, 24H, 8 x CH 2-CH 3), 2.63 (m, overlap, 16H, 8 x CH2-CH3), 4.54 (s, 2H, - % ) , 7.23 (s, 2H, -CH.-), 12.43 (bs, 4H, NH). - 201 -Anal.: Calcd. for C ^ H ^ N ^ r ^ C, 49.79; H, 5.73; N, 6.63; Br, 37.85. Found: C, 50.15; H, 5.93; N, 6.59; Br, 37.75%. Mass spectrum (m/e): 522 [M+-(2HBr + 2Br)], 493. FAB (m/e): 844 (M +), 682 [M +-(2HBr)]. 1,19-DIbromo-3,8,13,18-tetraethyl-2,7,12,17-tetramethyl-1,19-dideoxy-biladiene-ac dihydrobromide 215 A mixture of 5'-bromo-5-bromomethyl-3',4-diethyl- 3,4'-dimethyl-2,2'-dipyrromethene hydrobromide 214 (0.246 g, 0.51 mmole) and 5'-bromo-3,4'- diethyl-3',4-dimethyl-2,2'-dipyrromethene hydrobromide 211 (0.201 g, 0.52 mmole) were suspended i n dry methylene chloride (30 mL) and anhydrous stannic chloride (1 mL) was added. The resulting solution was protected from moisture and s t i r r e d at room temperature for two hours. The excess solvent was removed and methanolic hydrobromic acid (5 mL HBr i n 25 mL methanol) was added. The red coloured biladiene-ac was - 202 -separated, f i l t e r e d and washed with methanol containing few drops of hydrobromic acid, then with ether to give 0.292 g (72.5% y i e l d ) . MP: >170°C ( d e c ) . XH NMR (6, CDC13/TFA): 1.20 (m, 12H, 4 x -CH2-CH3), 2.07, 2.08 (s, 6H, 2 x -CH 3), 2.32, 2.38 (s, 6H, 2 x CH. - ) , 2.53 (m, 4H, 2 x -CH2-CH3), 2.73 (m, 4H, 2 x -CH2-CH3), 4.90 (s, 2H, -CH_2), 7.17 (s, 2H, -CH=), 13.17 (bs, 2H, -NH), 13.33 (bs, 2H, -NH). Anal.: Calcd. for C 3 ]H 4 0N 4Br 4: C, 47.23; H, 5.11; N, 7.11; Br, 40.55. Found: C, 47.16; H, 5.21; N, 7.05; Br, 40.60%. Mass spectrum (m/e): 466 [M+-(2HBr + 2Br)], 451, 437. FAB (m/e): 788 (M+). 1,19-Dideoxy-3,17-diethyl-8,12 -(2 -dimethoxycarbonylethyl)-2,7,13,18-tetramethylbiladiene-ac dihydrobromide 194 C0 2 Me C0 2Me 194 203 -3,3'-(2-Dimethoxycarbonylethyl)-4,4'-dimethyl-2,2'- dipyrromethane -5,5'-dicarboxylic acid 190 (3.042 g, 7.01 mmole), and 4-ethyl-5-formyl-3- methylpyrrole 177 (1.978 g, 14.44 mmole) were suspended i n methylene chloride (20 mL) i n a 100 mL round bottom flask. While the suspension was being s t i r r e d at room temperature, t r i f l u o r o a c e t i c acid (20 mL) followed by 48% HBr (10 mL) were added. The resulting mixture was further s t i r r e d for h a l f an hour and concentrated under reduced pressure and then diethyl ether was added, orange-red shiny c r y s t a l l i n e biladiene-ac dihydrobromide separated out. The s o l i d was collected by f i l t r a t i o n , washed with cold methylene chloride and then with ether to give 4.759 g (91%) of the t i t l e compound. MP: 195°C ( d e c ) . 1 H NMR (6, CDC13/TFA): 1.19 (t, 6H, J = 8 Hz, 2 x -CH^CH^), 2.12 (s, 6H, 2 x -CH 3), 2.32 (s, 6H, 2 x -CH3), 2.47 (t, 4H, J = 7.6 Hz, 2 x -CH„-CH 0), 2.72 (q, 4H, J = 7.2 Hz, 2 x -CH0-CH_), 2.85 (t, 4H, J = 7.6 Hz, 2 x -CH2-CH2), 3.70 (s, 6H, -CO^Hy , 4.72 (s, 2H, bridge-CHy , 7.36 (s, 2H, -CH=), 7.73 (d, 2H, J - 4 Hz, Pyr-H), 11.89 (bs, 2H, -NH), 11.96 (bs, 2H, NH). Anal.: Calcd. for C0CH.,N.0.Br0: C, 56.31; H, 6.21; N, 7.50; Br, 35 46 4 4 I 21.41. Found: C, 56.59; H, 6.25; N, 7.43; Br, 21.46%. Mass spectrum (m/e): 582 [M+-(2HBr + 2H)], 536, 521, 509. FAB (m/e): 584 (M+-2HBr), 569. - 204 -1,19-Dideoxy-3,17-diethyl-8,12-(2-dimethoxycarbonylethyl)-2,7,13,18-tetramethylbiladlene-ac dihydrobromide 194 (3.499 g, 4.69 mmole) was suspended i n methylene chloride (40 mL), t r i f l u o r o a c e t i c acid (10 mL) and bromine (3.749 g, 23.46 mmole) were added. The resulting dark solution was protected from moisture and l e f t for 10 to 15 minutes. The excess bromine and solvent were removed under reduced pressure. The traces of bromine and solvent were removed under vaccum (overnight). The s o l i d was dissolved i n a nimimum of methylene chloride, cyclohexene (5.0 mL) added and c r y s t a l l i z e d by the addition of diethyl ether. The shiny orange-red crystals were f i l t e r e d , .washed with cold methylene chloride, then with ether, and dried under vacuum to give 4.1 g (96.7%). MP: 160-165°C ( d e c ) . LH NMR (8, CDC13/TFA): 1.22 (t, 6H, J - 7.6 Hz, 2 x -CH2-CH3), 205 2.09 (s, 6H, 2 x -CH 3), 2.33 (s, 6H, 2 x -CH3-), 2.5 (t, 4H, J - 7.2 Hz, 2 x -CH2-CH2), 2.77 (q, 4H, J - 7 Hz, 2 x -CH2-CH3), 2.88 (t, 4H, J -7.2 Hz, 2 x -CH2-CH2), 3.70 (s, 6H, -C0 2CH 3), 4.80 (s, 2H, bridge-CH 2), 7.25 (s, 2H, -CH-), 12.59 (bs, 2H, NH), 12.61 (bs, 2H, NH). Anal.: Calcd. for C ^ H ^ N ^ B r ^ : C, 46.48; H, 4.90; N, 6.20; Br, 35.34. Found: C, 45.94; H, 4.76; N, 6.16; Br, 35.54%. Mass spectrum (m/e): 582 [M+-(2HBr + 2H)], 568, 553, 509. FAB (m/e): 904 (M +), 874, 847, 742. l,19-Dibromo-3,18-diethyl-8,12-(2-dimethoxycarbonylethyl)-2,7,13,17-tetrame 1,19-dideoxybiladiene-ac dihydrobromide 81 >2Br" C0 2 Me C0 2 Me 81 5-Bromo-5'-bromomethyl-3-ethyl-4'-(2-methoxycarbonylethyl)-3',4-dimethy1-2,2'-dipyrromethene hydrobromide 213 (0,244 g, 0.45 mmole) and 5-bromo-4-ethyl-4'-(2-methoxycarbonylethyl)-3,3'-dimethyl-2,2'-dipyrro-206 -methene hydrobromide 212 (0.207 g, 0.46 mmole) were suspended i n dry methylene chloride (50 mL) and condensed together by the addition of anhydrous stannic chloride (1 mL). The solution was protected from moisture and s t i r r e d for two hours. The excess solvent was removed under reduced pressure and the s o l i d residue was treated with methanolic hydrobromic acid (10 mL of 48% aq. HBr i n 50 mL of methanol). After allowing the solution to stand for 10 to 15 minutes i n an ice-bath, the biladiene-ac dihydrobromide was separated as an orange-red s o l i d . The s o l i d was f i l t e r e d , washed with methanol containing few drops of hydrobromic acid, then with ether to give 0.325 g (77.6% y i e l d ) . MP: >250°C (dec). LH NMR (5, CDC13/TFA): 1.23 (t, 6H, J = 8 Hz, 2 x -C^CHy , 2.1 (s, 6H, 2 x -CH 3), 2.36 (s, 6H, 2 x -CHy, 2.53 (t, 4H, J - 7.2 Hz, 2 x -CH2-CH2), 2.78 (q, 4H, J = 7 Hz, 2 x -CH2-CH3), 2.91 (t, 4H, J - 7.2 Hz, 2 x -CH2-CH2-), 3.72 (s, 6H, 2 x -CO^Hy , 4.79 (s, 2H, bridge - CH_2) , 7.27 (s, 2H, -CH=), 12.57 (bs, 4H, NH). Anal.: Calcd. for C.CH..N.Br.0.: C, 46.48; H, 4.90; N, 6.20; Br, 35 44 4 4 4 35.34. Found: C, 46.30; H, 4.87; N, 6.03; Br, 35.16%. Mass spectrum (m/e): 582 [M+-(2HBr + 2Br)], 567, 553, 524. FAB (m/e): 904 (M +), 742 (M+-2HBr). - 207 -12,17-(2-Dibenzoxyethyl)-1,19-dideoxy-3,7-(2-dimethoxycarbonylethyl)-2,8,13,18-tetramethylbiladiene-ac dihydrobromlde 198 C02Me C02Me 198 The t r i p y r r i n hydrobromide 197 (2.4 g, 3.13 mmole) was suspended i n methylene chloride (50 mL), t r i f l u o r o a c e t i c acid (10 mL), 5-formyl-4-(2-benzoxyethyl)-3-methylpyrrole (0.816 g, 3.18 mmole) was added immedi-ately followed by 48% hydrobromic acid (6 mL). The resulting dark solution was s t i r r e d and diethyl ether (100 mL) was added with swirling and the solution was cooled. The precipitated dark-red product was collected through f i l t r a t i o n , washed with ether and dried to give 3.07 g ( y i e l d 98%). MP: 187°C LH NMR (S, CDC13/TFA): 2.14 (2s, 6H, 2 x -CH3), 2.23, 2.34 (2s, 6H, 2 x CH 3), 2.70 (m, 4H, -CH^CH,^), 3.10 (t, 4H, J = 7 Hz, -CH2CH20-), 3.72 (s, 6H, 2 x -C02CH3) , 4.25 (m, 4H, -CH^H^-), 4.55 (t, 4H, J - 7 Hz, -CH2CH20-), 4.70 (s, 2H, -CH^-), 7.36 (s, 2H, -CH-), 7.4-8.0 (m, 12H, 2 x -C0C^Hc, pyr-H), 11.67 (bs, 4H, NH). 6 5 - 208 -Anal.: Calcd. for C ^ H ^ N ^ r 0 g: C, 59.62; H, 5.52; N, 5.68; Br, 16.20. Found: C, 59.56; H, 5.51; N, 5.70; Br, 16.30%. 12,17 -(2-Dibenzoxyethyl)-1,19-dibromo-3,7-(2-dimethoxycarbonylethyl)-2,8,13,18-tetramethyl-l,19-dideoxybiladiene-ac dihydrobromide 203 C02Me C02Me 203 12,17-(2-Dibenzoxyethyl)-1,19-dideoxy-3,7-(2-dimethoxycarbonyl-ethyl) -2 , 8 , 13 , 18-tetramethylbiladiene-ac dihydrobromide 198 (0.99 g, 1.0 mmole) was suspended i n methylene chloride (30 mL), t r i f l u o r o a c e t i c acid (2 mL), and bromine (0.80 g, 5.0 mmole) solution i n methylene chloride (5 mL) were added. The solution was protected from moisture and kept i n the hood for 15 minutes. The solvent was removed under reduced pressure, cyclohexene (5 mL) added and ethylacetate added slowly with swirling. The dark red product was f i l t e r e d and dried under vacuum to give, 0.842 g ( y i e l d 74%). MP: 155-160°C ( d e c ) . 1 H N M R (5, CDC13/TFA): 2.10 (s, 3H, - % ) , 2.20 (s, 6H, 2 x - % ) , 2.35 - 209 -(s, 3H, -CH 3), 2.71 (m, 4H, -CH^CH^ , 3.10 (t, 4H, J - 7 Hz, -CH^CH^-), 3.73 (s, 6H, 2 x -CO^Hy , 4.25 (m, 4H, -CH^CH^-), 4.55 (t, 4H, J - 7 Hz, -CH2CH20-), 4.70 (s, 2H, -CH2-), 7.40 (s, 2H, -CH=), 7.41-8.0 (m, 10H, 2 x -COC,Hc), 12.30 (bs, 4H, NH). O 5 Anal. Calcd. for C. H c oN.0 oBr.: C, 51.39; H, 4.58; N, 4.89; Br, 27.94. 49 52 4 8 4 Found: C, 51.40; H, 4.60; N,4.80; Br, 27.84%. 4.7 SYNTHESIS OF MONOAZAPORPHYRINS 5-Aza-2,8,13,17-tetraethyl-3,7,12,18-tetramethylporphyrin 78 To a solution of 1,19-dibromo-3,8,12,17-tetraethyl-2,7,13,18-tetra-methyl-1,19-dideoxybiladiene-ac dihydrobremide 199 (0.022 g, 0.028 mmole) i n methylene chloride (20 mL) and pyridine (1 mL), sodium azide (0.018 g, 0.274 mmole) and dibenzo-18-crown-6 (4.4 mg, 0.012 mmole) were added and the solution was s t i r r e d for 15 hours at room temperature. - 210 -The reaction mixture was then poured into water and extracted with CH^C^. The organic phase was dried and the solvent removed under reduced pressure. The product was p u r i f i e d over s i l i c a gel (60-230 mesh, 10 g) column chromatography using methylene chloride as an eluent. The fractions were collected and solvent evaporated to give monoazaporphyrin, 0.013 g (97.5 % y i e l d ) . The sample for analysis was c r y s t a l l i z e d from methylene chloride and methanol. MP: >307°C ( d e c ) . *H NMR (5, CDC1 3): 1.84 (4t, overlap, 12H, 4 x -CH2-CH3), 3.52 (s, 6H, 2 x CH 3), 3.59 (s, 6H, 2 x -CH3), 3.99 (4q, overlap, 8H, 4 x -CH2), 9 . 8 0 (s, 2H, meso-CH=), 9.98 (s, 1H, meso -CH=), -3.20 (bs, 2H, NH). Anal.: Calcd. for C n H 3 7 N 5 : C, 77.63; H, 7.78; N, 14.59. Found: C, 77.64; H, 7.80; N, 14.60%. + Mass spectrum (m/e, re l a t i v e i n t e n s i t y ) : 479 (M , 100), 464 (51), 450 (13), 434 (12), 240 (10). Mol. wt.: Calcd. for C^H^N^ 479.3036. Found by high resolution mass spectrometry: 479.3082. V i s i b l e spectrum (CH 2C1 2): A (nm) 373 501 533 557 609 max log e 5.39 4.15 4.65 4.16 4.67 max - 211 -5-Aza-13,17-diethyl-2,3,7,8,12,18-hexamethylporphyrin 88 88 1,19-Dibromo-8,12-diethyl-2,3,7,13,17,18-hexamethyl-1,19-dideoxy-biladiene-ac dihydrobromide 87 (0.020 g, 0.027 mmole), sodium azide (0.028 g, 0.432 mmole), and dibenzo-18-crown-6 (3.2 mg, 0.009 mmole) were suspended i n methylene chloride (20 mL), and pyridine (2.0 mL) added and then the entire solution was s t i r r e d for 15 hours at room temperature. The solution was extracted with a methylene chloride-water mixture.. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The porphyrin was c r y s t a l l i z e d from methylene chloride-methanol to give purple prisms, 11.2 mg (92.8%). 121 MP: >320°C ( d e c ) ; L i t . : >300°C. XH NMR (5, CDC13/TFA): 1.65 (t, 6H, J - 8 Hz, 2 x CH 2-CH 3), 3.37 (s, 6H, 2 x CH 3), 3.45 (s, 6H, 2 x -CH 3), 3.48 (s, 6H, 2 x -CH 3), 3.98 (q, 4H, J - 7.3 Hz, 2 x -CH^GU^), 10.18 (s, 2H, 2 x -meso-CH=), 10.43 (s, 1H, -meso-CH=), -1.91 (bs, 2H, NH). Anal.: Calcd. for C 2 9 H 3 3 N 5 : c> 7 7 - 1 3 ; H, 7.36; N, 15.50. Found: C, 76.72; H, 7.42; N, 15.52%. - 212 -Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 451 (M+, 100), 436 (28), 420 (5), 40 (3), 278 (5), 225 (12). Mol. wt.: Calcd. for C 2 9 H 3 3 N 5 : 451.2724. Found by high resolution mass spectrometry: 451.2734. V i s i b l e spectrum (CH 2C1 2): (0.2 mL TFA/CH 2C1 2) A (nm) max 396 545 593 log € max 5.43 4.23 4.57 A (nm) max 373 501 532 557 log € max 5.17 4.12 4.46 4.01 (0.2 mL Et N/CH CI ) (nm) 557 609 Z. z, BQ3.X 4.46 5-Aza-2,3,7,8,12,13,17,18-octamethylporphyrln 216 216 1,19-Dibromo-2,3,7,8,12,13,17,18-octamethyl-l,19-dideoxybiladiene-ac dihydrobromide 200 (0.046 g, 0.062 mmole) sodium azide, (0.047 g, 0.73 mmole), and dibenzo-18-crown-6 (5.5 mg, 0.015 mmole) were suspended - 213 in methylene chloride-pyridine (20 mL-2 mL) and the solution s t i r r e d vigorously for 15 hours at room temperature. At the end of the reac-tion, methanol was added to the reaction mixture. The precipitated porphyrin was collected by f i l t e r a t i o n , and washed several times with methanol, and a i r dried to give purple powder (0.024 g, 91.3% y i e l d ) . MP: >310°C ( d e c ) . NMR (S, CDC13/TFA): 3.38 (s, 6H, 2 x CH 3), 3.47, 3.51 (2 x s, 18H, 6 x -CH 3), 10.21 (s, 2H, -meso-CH=), 10.48 (s, IH, meso-CH=), -2.03 (bs, 4H, NH). Anal.: Calcd. for C^H^N^. C, 76.55; H, 6.91; N, 16.54. Found: C, 76.30; H, 6.79; N, 16.69%. Mass spectrum (m/e, re l a t i v e i n t e n s i t y ) : 423 (M+, 100), 408 (8), 278 (17), 262 (7), 211 (13). Mol. wt.: Calcd. for C0-,H00NI.: 423.2412. Found by high resolution mass 21 z y 5 spectrometry: 423.2429. V i s i b l e spectrum: (0.2 mL TFA/CH2C12) A ^ (nm) log £ ° max (0.2 mL Et„N/CH„Cl 0) A (nm) 3 2 2 max log « 395 544 592 5.16 4.26 4.65 372 500 532 557 610 5.37 4.36 4.67 4.29 4.68 max - 214 -5-Aza-2,3,7,8,12,13,17,18-octaethylporphyrIn 100 100 Method A: Methanol-Sodium Azide 1,19-Dibromo-2,3,7,8,12,13,17,18-octaethyl-1,19-dideoxybiladiene-ac dihydrobromide 202 (0.211 g, 0.250 mmole) was suspended in.dry methanol under nitrogen, sodium azide (0.321 g, 4.94 mmole) was added and the solution refluxed for 24 hours. After cooling the reaction mixture, methylene chloride (40 mL) was added and the mixture was washed with water. The purple coloured organic layer was evaporated to dryness and the product was chromatographed on s i l i c a gel (60-230 mesh) using methylene chloride as eluent. A l l fractions were combined and evaporated to give the t i t l e compound, 0.081 g, (60.6% y i e l d ) . 215 Method B: Dlbenzo-18-crown-6-NaN 3/CH 2Cl 2-pyridine 1,19-Dibromo-2,3,7,8,12,13,17,18 -octaethyl-1,19-dideoxybiladiene-ac dihydrobromide 202 (0.020 g, 0.023 mmole), dibenzo-18-crown-6 (2.8 mg 0.008 mmole), sodium azide (14 mg, 0.209 mmole) were suspended i n methylene chloride (20.0 mL), and pyridine (1 mL) was added. The res u l t i n g mixture was s t i r r e d vigorously for 15 hours, poured into water and extracted with methylene chloride. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. Column chromatography on s i l i c a gel (60-230 mesh) using CH 2C1 2 as an eluent gave the t i t l e porphyrin, 0.0124 g (99.6%). Method C: Tetrabutyl Ammonium bromide-NaN^/CI^C^-pyridine 1,19-Dibromo-2,3,7,8,12,13,17,18-octaethyl-1,19-dideoxybiladiene-ac dihydrobromide 202 (0.041 g, 0.048 mmole), tetrabutylammonium bromide (10.4 mg, 0.032 mmole), and sodium azide (0.047 g, 0.73 mmole) were suspended i n methylene chloride-pyridine (30 mL-1 mL) and the reaction mixture was s t i r r e d for 15 hours. The reaction mixture was then poured into water and extracted with methylene chloride and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure and chromatographed on a s i l i c a gel column. Various fractions were collected and evaporated to dryness to give monoazaporphyrin, 0.025 g (96.7% y i e l d ) . - 216 -MP: 301-305°C (dec.) X H NMR (5, CDC1 3): 1.90 (8t, overlap, 24H, 8 x CH 2CH 3), 4.04 (8q, overlap, 16H, 8 x -CH2-CH3), 9.91 (s, 2H, 2 x -meso-CH=), 10.05 (s, 1H, meso-Ctt=), -2.66 (bs, 2H, NH) . Anal.: Calcd. for C o c H . J • C, 78.45; H, 8.47; N, 13.08. Found: 35 45 5 C, 77.93; H, 8.53; N, 12.99%. Mass spectrum (m/e, rel a t i v e i n t e n s i t y ) : 535 (M+, 100), 520 (50), 506 (11), 490 (17), 476 (7), 268 (14). Mol. wt.: Calcd. for C H^Ng: 535.3660. Found by high resolution mass spectrometry: 535.3670. V i s i b l e spectrum (CH 2C1 2): A (run) 374 503 534 557 609 max log e 5.28 4.09 4.58 4.11 4.59 ° max 5-Aza-2,7,12,17-tetraethyl-3,8,13,18-tetramethylporphyrin 219. 219 217 -1,19-Dlbromo-3,8,13,18-tetraethyl-2,7,12,17-tetramethyl-1,19-di-deoxybiladiene-ac dihydrobromide 215 (0.012 g, 0.13 mmole), dibenzo-18-crown-6 (9.5 mg, 0.026 mmole), and sodium azide (0.091 g, 1.41 mmole) were suspended together i n methylene chloride-pyridine (30 mL-0.4 mL) and the solution was s t i r r e d vigorously for 12 hours at room temperature. The reaction mixture was then poured into water and extracted with methylene chloride. The organic layer was dried and evaporated to dryness. The product was p u r i f i e d by column chromatography ( s i l i c a gel, 60-230 mesh, 10 g), and the product was eluted" with methylene chloride. The fractions were collected and c r y s t a l l i z e d by CI^C^-CH^OH to give a maroon powder, 0.0591 g (yield 94.8%). MP: 309-313°C ( d e c ) . XH NMR (S, CDC1 3): 1.85 (4t, overlap, 12H, 4 x -CH2CH3), 3.52 (s, 6H, 2 x -CH 3), 3.54, 3.56 (2s, 6H, 2 x -Cflj), 3.99 (3q, overlap, 6H, 3 x -CH2-CH3), 4.08 (q, 2H, J - 7.5 Hz, CH 2-CH 3), 9.86, 9.87 (2s, 2H, meso-CH=), 10.01 (s, IH, meso-CH=), -2.72 (bs, 2H, NH). Anal.: Calcd. for C ^ ^ N ^ C, 77.63; H, 7.78; N, 14.59. Found: C, 77.70; H, 7.75; N, 14.62%. Mass spectrum (m/e, re l a t i v e intensity): 479 (M+, 100), 464 (75), 450 (14), 434 (10), 420 (10), 240 (25). Mol. wt.: Calcd. for C31H3-,N5: 479.3036. Found by high resolution mass spectrometry: 479.3073. V i s i b l e spectrum: (CH 2C1 2): A (nm) 373 501 533 557 609 max loe e 5.57 4.37 4.85 4.39 4.86 ° max - 218 -5-Aza-2,8-diethyl-13,17-(2-dimethoxycarbonylethyl)-3,7,12,18-tetra-methylporphyrin 217 C02Me C02Me 217 To a solution of 1,19-dibromo-3,17-diethyl-8,12-(2-dimethoxy-carbonylethyl)-2,7,13,18-tetramethyl-1,19-dideoxybiladiene-ac dihydro-bromide 201 (0.035 g, 0.038 mmole) i n methylene chloride (25 mL), sodium azide (0.028 g, 0.43 mmole), dibenzo-18crown-6 (4.3 mg, 0.012 mmole) and pyridine (1.0 mL) were added and the solution was s t i r r e d vigorously for 12 hours at room temperature. At the end of the reaction, the solution was poured into water and extracted with methylene chloride. The organic phase was dried over anhydrous sodium sulfate and the solvent removed under reduced pressure. The product was dissolved i n a minimum of methylene chloride and treated with diazomethane. The crude product was p u r i f i e d by column chromatography ( s i l i c a gel, 60-230 mesh, 10 g) using methylene chloride-methanol (98:2 v/v) as eluent. Fractions were collected and the solvent evaporated under reduced pressure. The product was c r y s t a l l i z e d from methylene chloride-methanol to give 0.0192 g (83%) as needle shaped, maroon crystals of the porphyrin. 219 -MP: 185°C. XH NMR (5, CDC1 3): 1.83 (t, 6H, J = 7.5 Hz, 2 x -CH2-CH3), 3.22 (t, 4H, J = 7.3 Hz, 2 x -CH2-CH2), 3.54 (s, 6H, 2 x -CH3>, 3.57 (s, 6H, 2 x -CH 3), 3.65 (s, 6H, 2 x -C0 2CH 3), 3.97 (q, 4H, J - 7 Hz, 2 x CH 2-CH 3), 4.32 (t, 4H, J - 7.3 Hz, 2 x -CH2-CH2-), 9.84 (s, 2H, meso-CH=), 10.03 (s, 1H, meso-CH=), -3.28 (bs, 2H, NH). Anal.: Calcd. for C o cH. 1N c0 • C, 70.56; H, 6.94; N, 11.76. 35 41 5 4 Found: C, 70.56; H, 6.84; N, 11.76%. Mass spectrum (m/e, re l a t i v e i n t e n s i t y ) : 595 (M+, 100), 580 (9), 522 (47), 508 (7), 448 (9), 434 (12). Mol. wt.: Calcd. for C o cH / 1N c0.: 595.3144. Found by high resolution 35 41 5 4 J ° mass spectrometry: 595.3148. V i s i b l e spectrum: (CH 2C1 2): A (ran) 373 500 532 557 609 max log e 5.33 4.15 4.60 4.16 4.62 ° max 5-Aza-2,7-diethyyl-13,17-(2-dimethoxycarbonylethyl)-3,8,12,18-terra-methylporphyrin 82 - 220 -1,19 -Dibromo-3,18-diethyl- 8,12 -(2 - dimethoxycarbonylethyl)-2,7,13,17-tetramethyl-l,19-dideoxybiladiene-ac dihydrobromide 81 (0.104 g, 0.115 mmole), dibenzo-18-crown-6 (6.4 mg, 0.018 mmole) and sodium azide (0.075 g, 1.15 mmole) were suspended i n methylene chloride- pyridine (30 mL-1 mL) solution. The suspension was s t i r r e d vigorously for 15 hours, poured into water, and then extracted with methylene chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The s o l i d residue was dissolved i n a minimum of methylene chloride (10 mL) and treated with diazomethane i n an ice-bath. The solvent was removed and the product was p u r i f i e d by column chroma-tography over s i l i c a gel (60 to 230 mesh) using methylene chloride-methanol (99.5:0.5 v/v) as an eluent. The product was c r y s t a l l i z e d from methylene chloride-methanol to give fine needles, 0.056 g (81.5%). MP: 190-192°C. 1H NMR (5, CDC1 3): 1.85 (2t, overlap, 6H, 3 x -CH^CHy , 3.23 (t, 4H, J = 7.3 Hz, 2 x -CH2-CH2), 3.51 (s, 3H, -CHy, 3.54 (s, 6H, 2 x -CH3), 3.56 (s, 3H, -CH 3), 3.65 (s, 6H, 2 x -C0 2CH 3), 3.97 (q, 2H, J = 7 Hz, -CH2-CH3>, 4.06 (q, 2H, J - 7 Hz, -CH2-CH3), 4.33 (t, 4H, J = 7.3 Hz, 2 x -CH2-CH2), 9.84, 9.86 (2s, 2H, meso-CH-), 10.04 (s, IH, meso-CH=), -2.67 (bs, 2H, NH). Anal.: Calcd. for C o cH / 1N c0,: C, 70.56; H, 6.94; N, 11.75. 35 41 5 4 Found: C, 70.75; H, 6.86; N, 11.65%. Mass spectrum (m/e, r e l a t i v e intensity): 595 (M+, 100), 580 (6.9), 564 (2.7), 537 (20), 522 (25.9). Mol. wt.: Calcd. for C ^ H ^ N ^ : 595.3144. Found by high resolution mass spectrometry: 595.3159. - 221 -V i s i b l e spectrum (CH2C12>: A (run) 373 501 532 557 609 max log e 5.31 4.09 4.57 4.19 4.59 max 20-Aza-3,8-(2-dibenzoxyethyl)-13,17-(2-dimethoxycarbonylethyl)-2,7,12,18-tetramethylporphyrin 218 12,17-(2-Dibenzoxyethyl)-1,19-dibromo-3,7-(2-dimethoxycarbonyl-ethyl) -2,8,13,18-tetramethyl-1,19-dideoxybiladiene-ac dihydrobromide 203-(0.031 g, 0.027 mmole) was dissolved i n methylene chloride (20 mL), sodium azide (0.030 g, 0.465 mmole), dibenzo-18-crown-6 (5.4 mg, 0.015 mmole) and pyridine (1 mL) were added and the reaction mixture was s t i r r e d vigorously for 15 hours. The reaction mixture was poured into water (50 mL) and extracted with methylene chloride. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The 222 -crude product was dissolved i n methylene chloride (10 mL) and treated with diazomethane. The solvent was removed and the product was p u r i f i e d by column chromatography ( s i l i c a gel, 60-230 mesh, 15 g) using 0.5% methanol i n methylene chloride as eluent. The fractions were collected and evaporated to dryness to give 0.0203 g (88.6% yield) of the azaporphyrin. MP: 207°C. 1H NMR (5, CDC1 3): 3.22 (m, 4H, /9-propionyl-CH 2), 3.41, 3.44, 3.56, 3.58 ( a l l s, 12H, 4 x ring-CH 3), 3.65 (d, 6H, 2 x -C0 2CH 3), 4.29 (m, 8H, a-propionyl-CH 2 and -CH2-CH20-), 5.03 (m, 4H, -CH^CH^O-), 7.25 (m, 10H, 2 x -0C,Hc), 7.45 (m, 4H, 2 x -C0C,Hc), 7.94 (m, 6H, 2 x -C0C,Hc), 9.85 6 5 6 5 o 5 (d, 2H, meso-CH=), 10.02 (s, IH, meso-CH=), -2.95 (bs, 2H, NH). Anal.: Calcd. for C H 4 gN 0 g: C, 70.39; H, 5.91; N, 8.38. Found: C, 70.40; H, 5.97; N,8.40%. Mass spectrum (m/e, re l a t i v e i n t e n s i t y ) : 835 (M+, 100), 804 (2.6), 777 (3.3), 762 (6.9), 713 (28), 700 (10.5). Mol. wt.: Calcd. for C. nH. nN c0 o: 835.3569. Found by high resolution 49 49 5 8 J ° mass spectrometry: 835.3603. Visible spectrum (CH 2C1 2): X (nm) 374 503 534 558 610 max log e 5.16 4.01 4.48 4.02 4.49 ° max - 223 -CHAPTER 5 SPECTRAL DATA - 224 -Fig. 28r *H NMR Spectrum (400 MHz) of 110b i n CDC13 10 C02tBu C02tBu 126 b A 8 7 ~r 6 T 5 LX_ 3 8 Fig. 29: H NMR Spectrum (400 MHz) of 126b in GDC1 OHC-CH - 228 -OHC 1 1 1 1 1 1 1 1 T 8 7 6 5 4 3 2 1 0 S Fig. 32: *H NMR Spectrum (400 MHz) of 123b in CDC1 Fig. 35: H N M R Spectrum (400 MHz) of 73 in CDC1 233 -- 235 -r loo" 350 400 450 500 WAVELENGTH ~ I — 550 600 650 700 nm Fig. 45 Electronic Absorption Spectrum of 106b 1.0 CHO 350 So 450 500 550 600 650 700 nm WAVE LENGTH Electronic Absorption Spectrum of 1 1 0 b Electronic Absorption Spectrum of 126b OHC-CH 750 nm WAVE L E N G T H E l e c t r o n i c Absorption Spectrum of 127b Fig. 49: Electronic Absorption Spectrum of 122b WAVE LENGTH Electronic Absorption Spectrum of 123b WAVELENGTH Fig. 51: Electronic Absorption Spectrum of 132 W A V E L E N G T H Fig. 52: Electronic Absorption Spectrum of 133 Electronic Absorption Spectrum of 134 WAVE LENGTH 'Electronic Absorption Spectrum of 135 WAVELENGTH Electronic Absorption Spectrum of WAVELENGTH Electronic Absorption Spectrum of 74 WAVE L E N G T H Electronic Absorption Spectrum of 138 WAVE LENGTH Electronic Absorption Spectrum of 139 —1 i 1 1-— 1 1 , 1 400 450 500 550 600 650 700 750 nm W A V E L E N G T H Fig. 59: Electronic Absorption Spectrum (Pyridine Hemochrome) of 140 350 400 450 500 550 600 650 700 750 nm W A V E L E N G T H Fig. 60: Electronic Absorption Spectrum of 141 Ul o z < CD CC O W CD < 0.6 0.4 0.2 C0 2H C0 2H • I r o n ( I I l ) p o r p h y r i n P y r i d i n e Hemochrome o f 71 Ul oo 350 400 450 500 550 600 650 700 7 50 nm W A V E L E N G T H Fig. 61: Electronic Absorption Spectrum (Pyridine Hemochrome) of 71 WAVE L E N G T H Electronic Absorption Spectrum of 72 i.on WAVELENGTH Electronic Absorption Spectrum of 78 WAVELENGTH Electronic Absorption Spectrum of 88 WAVELENGTH Electronic Absorption Spectrum of 216 WAVE LENGTH Fig. 66: Electronic Absorption Spectrum of 100 Fig. 67: Electronic Absorption Spectrum of 219 Fig. 68: Electronic Absorption Spectrum of 217 F i g . 69: E l e c t r o n i c Absorption Spectrum of 82 Fig. 70: Electronic Absorption Spectrum of 218 268 -REFERENCES 269 REFERENCES 1. 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