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Chemistry at the porphyrin periphery MacAlpine, Jill 1999

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Chemistry at the Porphyrin Periphery by Jill MacAlp ine B.Sc. (Hons), Queen's University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1999 ©Jil l MacAlpine, 1999 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 \ShCrtl(str<. The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract The objective of this work was to synthesize novel aromatic compounds based on tetraphenylporphyrins. These compounds are potential photosensitizers for use in photodynamic therapy. Several approaches were employed towards this goal. Numerous tetraphenylporphyrins possessing a variety of substituents were synthesized, characterized and used as starting materials to prepare the analogous diol chlorins via the patented osmium tetroxide oxidation. Compounds possessing hydroxy, methoxy, alkyl and halogen substituents were prepared. Studies involving the syntheses of these variously substituted meso-tetraphenyl-v/c-2,3-dihydroxy-2,3-chlorins and analysis of their in vitro biological test results are presented and discussed. A number of these compounds appear very promising, particularly diphenyl-2,3-dihydroxy-2,3-chlorin (125) which had an LD50 value of 0.0024 uJVI - 450 times as potent as the commercially available standard Photofrin. Attempts to improve the oxidation reaction via the reversible modification of starting materials and the use of other substrates as starting materials are also discussed. The use of electronically activated Af-alkylated tetraphenylporphyrins as starting materials was attempted. This reaction was successful for TV-methyl TPP (131) but failed when /V-phenyl TPP (132) was employed. Additionally, the osmium tetroxide mediated oxidation of octaethyltetraphenylporphyrin (137) was attempted. (me^o-Tetraphenyl-2,3-dihydroxy-2,3-chlorinato)nickel (106) can be oxidized to form (meso-tetraphenyl-2,3-dialdehyde-2,3-secochlorinato)nickel (145). An investigation of the reactivity of this novel compound (145) is presented. A wide variety of reactions were performed including reactions with acids, bases, alcohols, amines and reducing agents. Over 30 interesting and novel compounds (125) iii were synthesized from nickel bisaldehyde secochlorin (145) such as the doubly cyclized diketo product (182) from reaction of (145) with TFA followed by base, and the unsubstituted secochlorin (191) from the decarbonylation of (145) with Wilkinson's catalyst. (182) ( 1 9 1 ) Finally, a relatively unexplored field of porphyrin research was investigated. Studies probed the reactivity of both tetra- and diphenylporphyrins in a number of different 1,3-dipolar cycloaddition reactions. Although this initial investigation was met with limited success overall, reactions with mesitaldehyde oxide, tetracyanoethylene oxide (TCNEO) and diazomethane were fruitful, forming products (226), (213) and (223) respectively. Ph Ph (216 ) (213) (223) Table of Contents I V Abstract ii Table Of Contents iv List of Figures ix List of Schemes xii List of Tables xvii Nomenclature xviii List of Abbreviations xx Acknowledgments xxii Chapter One Introduction 1 1.1 Tetrapyrrolic Macrocycles 2 1.1.1 Introduction „ 2 1.1.2 Structural Characteristics 5 1.1.3 Optical Absorption Spectra 7 1.1.3.1 Optical Absorption Spectra of Porphyrins 7 1.1.3.2 Optical Absorption Spectra of Chlorins, Bacterio- and Isobacterio-chlorins 9 1.1.3.3 Theory of Optical Absorption Spectra 11 1.1.4 Preparation of Porphyrins 12 1.1.4.1 Naturally Occurring Porphyrins 12 1.1.4.2 Synthetic Porphyrins 14 1.1.4.2.1 Porphyrins from Monopyrrolic Precursors 14 1.1.4.2.2 Porphyrins from Dipyrrolic Precursors 16 1.1.4.2.3 Porphyrins from Tetrapyrrolic Precursors 19 1.1.5 Preparation of Chlorins 23 1.1.5.1 Naturally Occurring Chlorins 23 1.1.5.2 Synthetic Chlorins 24 V 1.1.5.2.1 Total Synthesis of Chlorins 24 1.1.5.2.2 Reduction of Porphyrins into Chlorins 24 1.1.5.2.3 Synthetic Conversions of Porphyrins into Chlorins 26 1.1.5.2.3.1 Diels-Alder Reactions of Natural Porphyrins 26 1.1.5.2.3.2 Reactions of Carbenes with Porphyrins 27 1.1.5.2.3.3 Chlorins with Unsaturated Exocyclic Rings 29 1.1.6 Properties of Porphyrins 30 1.1.6.1 Reactivity of Porphyrins 30 1.2 Photodynamic Therapy 31 1.2.1 History of PDT 31 1.2.2 Mechanism of Photosensitization 34 1.2.2.1 Type I Reactions 36 1.2.2.2 Type II Reactions 37 1.2.3 Desirable Properties of a PDT Drug 39 1.2.3.1 Preparation of the Drug 39 1.2.3.2 Chemically-Modifiable Characteristics of the Drug 39 1.2.3.3 Biological Characteristics of the Drug 40 1.2.3.4 Properties Required for Industrial Production/Scale-Up of the Drug 40 1.2.4 Major Second Generation Photosensitizers 41 1.2.4.1 BPDMA 41 1.2.4.2 M A C E 43 1.2.4.3 Tin Etiopurpurin 44 1.2.4.4 mes0-Tetra(ra-hydroxyphenyl)chlorin 45 1.3 Osmium Tetroxide Oxidations 46 1.3.1 History 46 1.3.2 Mechanism 47 1.3.3 Osmium Tetroxide Oxidation of Aromatic Systems 49 1.3.4 Osmium Tetroxide Oxidation of Porphyrins 50 vi 1.4 p-Hydroxychlorins from Other Methods 52 1.4.1 P-Oxochlorins 52 1.4.2 Epoxychlorins 57 1.5 P-Dihydroxychlorins from Other Methods 58 1.5.1 Photoprotoporphyrin 59 1.5.2 Secochlorins 59 1.6 Porphyrins Incorporating Six-Membered or Larger Rings 60 1.7 Research Objective and Thesis Preview 62 Chapter Two Synthesis and Testing of Potential PDT Drugs Based on TPP 63 2.1 Introduction 64 2.1.1 Background 66 2.2 Results 73 2.2.1 Porphyrins 73 2.2.2 Chlorins 75 2.2.3 Bacteriochlorins 78 2.2.4 Preliminary Biological Testing Results 80 2.3 Discussion 100 2.4 Singlet Oxygen Testing 105 2.5 Conclusions 105 2.6 Attempts to Improve the Osmium Tetroxide Reaction 105 2.6.1 Decreasing the Reaction Time 105 2.6.1.1 N-Alkylated TPPs 106 2.6.1.2 Octaethyltetraphenylporphyrin 108 2.6.1.3 Porphyrazine 110 2.6.2 Other Oxidation Systems 110 2.7 Summary 111 Chapter Three Reactivity of /M<?so-Tetraphenyl-2,3-dialdehyde-2,3-secochlorin 114 3.1 Introduction 115 3.1.1 (me^o-Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II).. 116 vii 3.1.2 Oxidations of zinc(II) and free base tetraphenyl-2,3-dihydroxychlorins with lead tetraacetate 117 3.2 Reaction with Alcohols 118 3.2.1 Acetal Formation 118 3.2.2 Reaction with Ethylene Glycol 121 3.3 Reaction with Base 121 3.3.1 Reaction of the (2-oxo-3-oxa-tetraphenylchlorinato)nickel(II) (155).. 127 3.4 Reaction with Amines 129 3.4.1 N-Methylamine 129 3.4.2 1,2-Phenylenediamine 131 3.4.3 Ammonia 133 3.5 Reaction with Phenylhydrazine 137 3.6 Reaction with HMPT 140 3.7 Reaction with Acid 143 3.7.1 Trifluoroacetic Acid (TFA) 143 3.7.1.1 Excess TFA 150 3.7.1.2 Reaction of the Doubly-Cyclized Product (172) 152 3.7.2 Sulfuric Acid 156 3.7.2.1 Sulfuric Acid/Methylene Chloride 159 3.7.3 Variety of Acids 159 3.8 Reaction with Ylides: Wittig Reaction 163 3.9 Reaction with Wilkinson's Catalyst: Decarbonylation 164 3.10 Vilsmeier-Haack Reaction of the Secochlorin (191) 170 3.11 Reaction with Hydroxylamine Hydrochloride 174 3.12 Reaction with Reducing Agents 178 3.12.1 Lithium Aluminum Hydride 178 3.12.2 Sodium Borohydride 179 3.13 Reaction with Osmium Tetroxide 180 3.14 Summary 182 viii Chapter 4 1,3-Dipolar Cycloadditions 185 4.1 Introduction to 1,3-Dipolar Cycloadditions 186 4.2 Carbonyl Ylides 191 4.3 Nitrile Oxides 199 4.4 Diazomethane 202 4.5 Azides 206 4.6 Azomethine Ylides 207 4.7 Nitrile Ylides 211 4.8 Nitrones 213 4.9 Azomethine Imines 214 4.10 Conclusion 215 Chapter 5 Experimental 216 Chapter 6 References 275 IX List Of Figures Figure 1.1 Tetrapyrrolic macrocycles 2 Figure 1.2 Hematoporphyrin(l), protoporphyrin IX (2) and prosthetic group of hemoproteins (3) 3 Figure 1.3 Expanded porphyrin analogs 5 Figure 1.4 The 1 8-TC electron pathway 6 Figure 1.5 The four types of porphyrin spectra 7 Figure 1.6 The absorption spectra of a porphyrin and a metalloporphyrin 9 Figure 1.7 The absorption spectra of chlorins, metallochlorins, bacteriochlorins and metallobacteriochlorins 10 Figure 1.8 Energy level diagram for HOMOs and LUMOs of the four macrocycles 12 Figure 1.9 The bile pigments 20 Figure 1.10 Chlorophyll a (18) and b (19) 23 Figure 1.12 Modified Jablonski diagram for a typical photosensitizer 36 Figure 1.13 The wavelength-dependent penetration of light through tissue 40 Figure 1.14 18 rc-electron derealization in porphyrins, chlorins and metallochlorins 51 Figure 2.1 raeso-Tetra(m-hydroxyphenyl)porphyrin, chlorin and bacteriochlorin 67 Figure 2.2 The optical absorption spectrum of the free base tetraphenyl-2,3-dihydroxychlorin 68 Figure 2.3 The optical absorption spectrum of the Zn complex of tetraphenyl-2,3-dihydroxychlorin 69 Figure 2.4 Diphenyl-2,3-dihydroxychlorin (125) 76 Figure 2.5 'H-NMR spectrum of mei,o-tetraphenyl-2,3-dihydroxychlorins 77 Figure 2.6 meso-5-(p-Substituted phenyl)-10,15,20-triphenyl-2,3-dihydroxychlorins 78 Figure 2.7. raesx>-Tetraphenyl-2,3-12,13-tetrahydroxybacteriochlorin (68) 79 Figure 2.8 Graph showing the cytotoxicity of compounds with the lowest LD50 values .... 82 Figure 2.9 Graph showing the cytotoxicity of hydroxy substituted compounds 83 X Figure 2.10 Graph showing the cytotoxicity of methoxy-substituted compounds 87 Figure 2.11 Graph showing cytotoxicity of hydroxy and methoxy substituted compounds .. 88 Figure 2.12 Graph showing the cytotoxicity of alkylated compounds 89 Figure 2.13 Graph showing the cytotoxicity of halogenated compounds 90 Figure 2.14 Graph showing the cytotoxicity of diphenyl chlorin, tetraphenyl diol chlorin and tetraol bacteriochlorin 92 Figure 2.15 Graph showing the cytotoxicity of the trimethoxy diol chlorin and tetraol bacteriochlorin versus those of the unsubstituted diol chlorin and tetraol bacteriochlorin 93 Figure 2.16 Graph showing the cytotoxicity of diphenyl diol chlorin 94 Figure 2.17 Graph showing the cytotoxicity of the most promosing compounds both in the presence and absence of fetal calf serum 96 Figure 2.18 Graph showing the cytotoxicity of variously substituted diol chlorins 97 Figure 2.19 Tetraphenyl-based porphyrins used 109 Figure 2.20 Porphyrazine (142) 110 Figure 3.1 The optical absorption spectrum of (meso-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) 116 Figure 3.2 The 'H-NMR spectrum of (meso-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(U) (145) 116 Figure 3.3 Optical spectrum of (155) and the free base (156) 122 Figure 3.4 Proton NMR of (2-oxo-3-oxa-tetraphenylchlorinato)nickel(II) (155) 123 Figure 3.5 UV-Vis spectrum of (160) -the product from reaction of methylamine and bisaldehyde 129 Figure 3.6 Proton NMR of mew-tetraphenyl-3-N-methylimino-2-carboxaldehyde-2,3-secochlorin (160) 130 Figure 3.7 Proton NMR spectrum of the phenylenediamine product (161) 133 Figure 3.8 Proton NMR spectrum of (meso-tetraphenyl-2,3-dioxochlorinato) nickel(H) (165) 134 Figure 3.9 UV-Visible spectrum of the polar product (166) 136 xi Figure 3.10 Proton NMR spectrum of (166) 136 Figure 3.11 Structure of (168) 138 Figure 3.12 Compound (171) 141 Figure 3.13 UV-Vis absorption spectrum of the doubly cyclized product (172) 149 Figure 3.14 Proton NMR spectrum of the doubly cyclized product (172) 149 Figure 3.15 UV-Vis absorption spectrum of the lactone chlorin product (155) 151 Figure 3.16 UV-Vis spectrum of nonpolar product (182) of reaction of TFA with (172).... 152 Figure 3.17 Proton NMR spectrum of (182) the doubly cyclized diketo compound 153 Figure 3.18 UV-Vis spectrum of the polar product (183) 154 Figure 3.19 Aromatic portion of 'H-NMR of the doubly cyclized product (183) 155 Figure 3.20 UV-Vis spectrum of the product (185) of HCI reaction with (145) 160 Figure 3.21 Proton NMR of the singly cyclized keto-containing product (185) 161 Figure 3.22 UV-Vis absorption spectrum of the unsubstituted secochlorin (191) 166 Figure 3.23 UV-Vis absorption spectrum of the monodecarbonylated secochlorin (192).... 168 Figure 3.24 UV-Visible absorption spectrum of the blue product (193) from the decarbonylation of nickel bisaldehyde (145) 168 Figure 3.25 UV-Vis spectrum of the blue product (194) 171 Figure 3.26 Proton NMR spectrum of the blue product (194) formed during the formylation of the fully unsubstituted secochlorin (191) 172 Figure 3.27 UV-Visible absorption spectrum of the polar product (196) from the reaction of the bisaldehyde secochlorin (145) with hydroxylamine hydrochloride 175 Figure 3.28 Proton NMR spectrum of the polar product (196) from the hydroxylamine reaction 176 Figure 3.29 The hemiacetal (203) formed during the osmylation of (145) 182 Figure 4.1 Allyl anion type resonance structures 187 Figure 4.2 The propargyl/allenyl anion type of 1,3-dipole 187 Figure 4.3 An azomethine ylide 207 Figure 4.4 Resonance structures of a nitrile ylide 211 Figure 4.5 Resonance structures of an azomethine imine 214 xii List of Schemes Scheme 1.1 Natural porphyrin derivatives 13 Scheme 1.2 Synthesis of octaethylporphyrin (8) 14 Scheme 1.3 Rothemund-type synthesis of meso-tetraphenylporphyrins 16 Scheme 1.4 The MacDonald synthesis of porphyrins 17 Scheme 1.5 Synthesis of porphyrins from 5,5'-diformyldipyrroketones 18 Scheme 1.6 Porphyrin synthesis from 5-bromo-5'-methyldipyrromethenes 19 Scheme 1.7 The b-oxobilane route to porphyrins 21 Scheme 1.8 The a,c-biladiene route to porphyrins 22 Scheme 1.9 Raney nickel reduction of tetraphenylporphyrin 25 Scheme 1.10 Diimide reduction of porphyrins 26 Scheme 1.11 Reaction of D M A D with protoporphyrin IX (2) 27 Scheme 1.12 The cyclopropanation of tetraphenylporphyrin 28 Scheme 1.13 Synthesis of a cyclopropanochlorin (27) 28 Scheme 1.14 Synthesis of octaethylpurpurin (28) and octaethylbenzochlorin (29) 29 Scheme 1.15 Preparation of hematoporphyrin derivative (30) 33 Scheme 1.16 Reactions of singlet oxygen with biological substrates 38 Scheme 1.17 Synthesis of BPDMA (34) from protoporphyrin IX 42 Scheme 1.18 Synthesis of M A C E (36) 44 Scheme 1.19 Synthesis of Tin Etiopurpurin (38) 45 Scheme 1.20 Synthesis of mTPC (39) 46 Scheme 1.21 Osmium tetroxide dihydroxylation of alkenes 47 Scheme 1.22 The possible intermediates in the formation of the Os0 4 glycol ester 48 Scheme 1.23 The osmium tetroxide oxidation of anthracene (40) 50 Scheme 1.24 The reaction of a benzoporphyrin (34) with osmium tetroxide 52 xiii Scheme 1.25 The hydrogen peroxide-sulfuric acid oxidation of OEP (8) 54 Scheme 1.26 Synthesis of 2-diazo-3-oxo-tetraphenylchlorin (43) 55 Scheme 1.27 Syntheses of a tetraphenylporphyrin-a-dione (45)from 2-hydroxyTPP (44) 56 Scheme 1.28 Monohydroxylation of a hexaalkylchlorin (46) via C-H bond activation by alumina 56 Scheme 1.29 Epoxidation of OEP (8) 57 Scheme 1.30 The synthesis of 10,22-dihydrochlorin-e6-trimethyl ester (50) 58 Scheme 1.31 Synthesis of furochlorophin (52) 59 Scheme 1.32 Synthesis of octaethyl-2,3-secochlorin-2,3-dione (54) 60 Scheme 1.33 Synthesis of oxipuriporphyrin (55) and oxibenziporphyrin (56) 60 Scheme 1.34 Oxidation of meso-tetraphenyl-2,3-dioxo-chlorin (57) 61 Scheme 1.35 The internal aldol condensation of mejo-tetraphenyl-2,3-secochlorin-2,3-dione (54) 61 Scheme 2.1 The osmium tetroxide mediated oxidation of tetraphenylporphyrins 68 Scheme 2.2 Formation of meso-tetraphenyl-2,3-dihydroxy-12,13-dihydrobacteriochlorins ... 69 Scheme 2.3 Osmium tetroxide oxidation of mei,o-tetraphenyl-2,3-dihydroxychlorin 70 Scheme 2.4 Formation of tetraphenyl-2,3,7,8-tetrahydroxyisobacteriochlorinato zinc(II) 71 Scheme 2.5 Proposed formation of a para-quinone from oxidation of o,m'-dihydroxyphenyl group of T(o,m'-OH)PP 85 Scheme 2.6 Synthesis of /V-phenyltetraphenylporphyrin (132) 107 Scheme 2.7 Reaction of /V-PhenylTPP (132) with tosylhydrazine 108 Scheme 2.8 The hydrogen peroxide-acid oxidation of TPP (10) I l l Scheme 3.1 Synthesis of octaethyl-2,3-secochlorin-2,3-dione (54) 115 Scheme 3.2 Synthesis of (me5,o-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) 115 Scheme 3.3 Oxidation products of the zinc diol chlorin (146) 117 Scheme 3.4 Acetal formation from nickel bisaldehyde secochlorin (145) 119 Scheme 3.5 Formation of acetals (150), (151) and (152) with various alcohols 120 Scheme 3.6 Reaction of nickel bisaldehyde secochlorin (145) with ethylene glycol 121 x i v Scheme 3.7 Expected Cannizaro reaction between nickel bisaldehyde (145) and base ... 122 Scheme 3.8 Observed reaction of base with nickel bisaldehyde secochlorin (145) 123 Scheme 3.9 Synthesis of lactone (156) using MCPBA 124 Scheme 3.10 Previously proposed autooxidation mechanism for the formation of (147) from zinc(II) dihydroxy tetraphenylchlorin (146) 125 Scheme 3.11 Radical autooxidation mechanism of aldehydes 125 Scheme 3.12 Example of base-catalyzed aldehyde oxidation without a chain mechanism.. 126 Scheme 3.13 Proposed base-catalyzed autooxidation mechanism 126 Scheme 3.14 Products formed during the reaction of DIBALH with (155) 128 Scheme 3.15 Formation of a mono-imino linkage with A -^methylamine 131 Scheme 3.16 Reaction of nickel bisaldehyde (145) and phenylene diamine 131 Scheme 3.17 Synthesis of molecular wires 133 Scheme 3.18 Syntheses of meso-tetraphenyl-2,3-dioxochlorin (167) 135 Scheme 3.19 Reaction of the nickel bisaldehydesecochlorin (145) with ammonia 137 Scheme 3.20 Proposed mechanism for the synthesis of pigment (168) 139 Scheme 3.21 The reaction mechanism of hexamethylphosphorous triamide with aldehydes. 140 Scheme 3.22 Reaction of nickel bisaldehyde secochlorin (145) with HMPT 142 Scheme 3.23 Reaction of nickel bisaldehyde secochlorin (145) with HMPT to form (170) via the epoxide 142 Scheme 3.24 Cyclization of (2-formyl-5,10,15,20-tetraphenylporphyrinato)copper(II) (173) with TFA 144 Scheme 3.25 Acidification of (2-formyl-tetraphenylporphyrinato)nickel(U) (175) 145 Scheme 3.26 Proposed mechanism for the formation of the monocyclization products 146 Scheme 3.27 Synthesis of the doubly-cyclized products (180) and (181) 147 Scheme 3.28 Synthesis of a doubly-cyclized product (172) 148 Scheme 3.29 Proposed mechanism for the formation of the doubly-cyclized product (172).. 150 Scheme 3.30 Reaction of nickel bisaldehyde secochlorin (145) with excess TFA 151 Scheme 3.31 Reaction of the doubly cyclized product (172) with base 154 Scheme 3.32 Proposed mechanism for the formation of the doubly cyclized products XV (182) and (183) 156 Scheme 3.33 Formation of the free base analog of the doubly cyclized product (184) 157 Scheme 3.34 Reaction of nickel bisaldehyde (145) in CH 2C1 2 with sulfuric acid 158 Scheme 3.35 Reaction of nickel bisaldehyde (145) with concentrated hydrochloric acid... 161 Scheme 3.36 Proposed mechanism of formation of the singly cyclized product (185) 162 Scheme 3.37 Proposed reaction of nickel bisaldehyde (145) secochlorin 164 Scheme 3.38 Reaction of Wilkinson's catalyst with an aldehyde 165 Scheme 3.39 Synthesis of the unsubstituted secochlorin (191) from bisaldehyde (145) 167 Scheme 3.40 Synthesis of the monodecarbonylated secochlorin (192) from (145) 167 Scheme 3.41 Synthesis of (193) from nickel bisaldehyde (145) 169 Scheme 3.42 Proposed mechanism of formation of compound (193) 170 Scheme 3.43 Proposed mechanism for the formation of (194) from the reaction of the unsubstituted secochlorin (191) 173 Scheme 3.44 The synthesis of (meso-tetraphenyl-2-cyanosecochlorin)nickel(II) (196) 176 Scheme 3.45 Proposed mechanism for the formation of (196) 177 Scheme 3.46 Products from the reduction of nickel bisaldehyde (145) with lithium aluminum hydride 179 Scheme 3.47 Proposed mechanism of the NaBH 4 mediated reduction of (145) 180 Scheme 3.48 Proposed osmium tetroxide oxidation of nickel bisaldehyde (145) 181 Scheme 4.1 1,3-Dipolar Cycloaddition Reaction 186 Scheme 4.2 Reaction of P-vinyl-meso-tetraphenylporphyrin (204) with T C N E 188 Scheme 4.3 Reaction of tetraphenylporphyrin with o-benzoquinodimethane (208) 188 Scheme 4.4 Resonance structures of TCNEO (212) 191 Scheme 4.5 Reaction of benzene with TCNEO (212) 191 Scheme 4.6 Reaction of tetraphenylporphyrin with TCNEO (212) 192 Scheme 4.7 Reaction of pyridine with TCNEO (212) 194 Scheme 4.8 Barton-Zard reaction of 2-nitro-5,10,15,20-tetraphenylporphyrin (26) 194 Scheme 4.9 Formation of the red product (215) from the reaction of DPP with TCNEO .. 195 Scheme 4.10 Purple product (216) from reaction of DPP with TCNEO 196 xvi Scheme 4.11 Mechanism of formation of products (215) and (216) 196 Scheme 4.12 Reaction of ZnDPP with TCNEO 197 Scheme 4.13 Reaction of ZnTPP with TCNEO (212) 198 Scheme 4.14 Resonance structures for nitrile oxides 199 Scheme 4.15 Reaction of a nitrile oxide with an alkene 199 Scheme 4.16 Synthesis and reaction of 2,4,6-trimethylbenzonitrile oxide (222) with DPP.... 200 Scheme 4.17 Products of the dimerization of benzonitrile oxide (225) 202 Scheme 4.18 Reaction of ZnTPP with diazo compounds in the presence of CuCl 203 Scheme 4.19 Reaction of diol chlorin of etioporphyrin (229) with zinc acetate in 2,4-pentadione 204 Scheme 4.20 Reaction of zinc(II) tetraphenyl diol chlorin (146) with Zn(OAc) 2 and 2,4-pentadione 205 Scheme 4.21 The reaction of an azide with an alkene 206 Scheme 4.22 Proposed reaction of azidotrimethylsilane (236) with TPP 207 Scheme 4.23 Reaction of L D A with N-benzylidene benzylamine (237) 208 Scheme 4.24 Reaction of A^-lithio-2,3-cw-diphenylaziridine (238) 208 Scheme 4.25 Generation and proposed reaction of an azomethine ylide (238) 209 Scheme 4.26 Formation of an azomethine ylide (241) and proposed reaction with TPP 210 Scheme 4.27 Formation of an azomethine ylide (244) and attempted reaction with TPP .... 210 Scheme 4.28 Proposed products (245) and (246) from reaction of porphyrins with (244).... 211 Scheme 4.29 Reaction of benzonitriliophenylmethanide (248) with acenaphthylene (249).... 212 Scheme 4.30 Formation and attempted reaction of C-benzoyl-A^-phenylnitrone (253) 213 Scheme 4.31 Synthesis of N-benzyl azomethine ylide (255) from (254) 214 List of Tables X V I I Table 2.1 Comparison of tumor photonecrosis for meta-hydroxy substituted analogs 66 Table 2.2 Tetraphenylporphyrins synthesized 73 Table 2.3 Tetraphenyl-2,3-dihydroxychlorins synthesized 75 Table 2.4 Tetraphenyl-2,3,12,13-tetrahydroxybacteriochlorins synthesized 78 Table 2.5 List of LD50 values for compounds tested in vitro in order of decreasing cytotoxicity 81 Table 2.6 List of compounds in order of decreasing dark toxicity 99 Table 2.7 List of LD50 values for compounds tested in order of decreasing cytotoxicity 100 Table 4.1 Classes of 1,3-dipoles 190 x v i n Nomenclature Monopyrrolic Systems T h e pyrrolic skeleton is numbered as depicted, with positions 2 and 5 commonly referred to as "a" and positions 3 and 4 as "P" positions. Dipyrrolic Systems Dipyrromethanes are numbered as shown below. Positions 1 and 9 are referred to as "a" positions whilst positions 2, 3, 7 and 8 are called "P" positions. Carbon 5 is referred to as the "meso "-position. XIX Porphyrins and Related Systems The numbering is shown below. The "a" positions are those at carbons 1,4,6,9,11,14,16 and 19, the "P" positions are those at carbons 2,3,7,8,12,13,17 and 18 and positions 5,10,15 and 20 are referred to as the "mew "-positions. meso ,5 " • m e S 0 P Many naturally occuring porphyrins are known by their trivial names. Those names relevant to this thesis are listed below. Trivial Name Deuteroporphyrin IX Deuteroporphyrin HI Coproporphyrin U Etioporphyrin Hematoporphyri n Protoporphyrin LX Substituent Position 12 13 17 Me H Me H Me P Me Et Me E A Me V Me H Me H Me Me Me Me Me Et Me Et Me E A Me Me V Me Me Me Me Me Me Me E A = CH(OH)CH 3 (ethyl alcohol) P = C H 2 C H 2 C 0 2 H (propionic acid) V = CH=CH 2 (vinyl) 18 P P P Et P P List of Abbreviations aq. aqueous BPD benzoporphyrin derivative BPDMA benzoporphyrin derivative monoester ring A br broad Bu butyl d doublet DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DDQ 2,3-dichloro-5,6-dicyano-l,4-benzoquinone DMAD dimethylacetylene dicarboxylate DME dimethyl ether DMF N,N-dimethylformamide DMSO dimethylsulfoxide DPBF diphenyl isobenzofuran DPP 5,15-diphenylporphyrin EI electron impact eq. equivalent FAB fast atom bombardment FCS fetal calf serum HMPT hexamethylphosphorus triamide HOMO highest occupied molecular orbital HpD hematoporphyrin derivative HR high resolution hr hour Hz hertz ISC intersystem crossing LD50 lethal dose required to kill 50% of cells LDA lithium diisopropylamide LiAlH 4 lithium aluminum hydride LR low resolution LUMO lowest unoccupied molecular orbital m multiplet MACE monoaspartyl chlorin e6 MCPBA m-chloroperoxybenzoic acid Me methyl min minute MS mass spectrometry NBS /V-bromosuccinimide NCS /V-chlorosuccinimide NMR nuclear magnetic resonance OEP 2,3,7,8,12,13,17,18-octaethylporphyrin PDT photodynamic therapy py pyridine q quartet QLT Quadra Logic Technologies Ltd s singlet Sens sensitizer t triplet TCNE tetracyanoethylene TCNEO tetracyanoethylene oxide TCQ tetracyanoquinone TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TPC meso-tetraphenylchlorin TPBC meso-tetraphenylbacteriochlorin TpiBC meso-tetraphenylisobacteriochlori TPP tetraphenylporphyrin UV-Vis ultra-violet and visible xxn Acknowledgments I would like to thank Dr. Dolphin for his advice, guidance and for allowing me the freedom to investigate porphyrin chemistry as I pleased. This freedom has allowed me to fully appreciate this field of chemistry and the many idiosyncracies of porphyrins. I would also like to thank Dr. Ethan Sternberg who, over the years, has been an extremely valuable source of information, ideas and support. Members of the Dolphin group, both past and present, are also deserving of my thanks. I greatly appreciate the assistance, advice and friendship of Dr. Al ison Thompson, Angela Desjardins and Dr. Claire Johnson. Services provided by staff in N M R , Mass Spectrometry, Elemental Analyses, and Glassblowing as well as administrative staff are deeply appreciated. Special thanks go to Rani, Marietta, Liane and Marshall. Many special thanks go to my parents (who now believe I am a professional student) for their encouragement and support, both emotional and financial, over the years. I would also like to thank my grandparents very much for their loving support. Finally, to my husband Ian, who has been my ally and my best friend for 12 years: words cannot express how grateful I am for all that you've done and I can merely say thanks for everything. I couldn't have done it without you. Chapter 1 Introduction 2 Introduction 1.1 Tetrapyrrolic Macrocycles 1.1.1 Introduction Tetrapyrrolic macrocycles form the structural backbones of some of the most important biological systems.1 Porphyrin Corrin Chlorin Bacteriochlorin Isobacteriochlorin Figure 1.1 Tetrapyrrolic macrocycles In nature, these molecules play important roles in a wide variety of biological processes. Tetrapyrrolic macrocycles bind metal atoms thereby allowing them to act as centers for critical biological reactions. The iron(U) complex of protoporphyrin LX (3) forms the prosthetic group of myoglobin and hemoglobin, whilst the iron(IJI) complex forms the prosthetic group of catalases and peroxidases.2 Hemoglobin and myoglobin both reversibly bind oxygen in the body. 3 Introduction Hemoglobin transports the oxygen throughout the body and myoglobin serves to store oxygen in muscle tissue. (3) M=Fe Figure 1.2 (1) Hematoporphyrin IX (2) Protoporphyrin IX (3) Prosthetic group of Hemoproteins Peroxidases, catalases and dismutases protect the body from intermediates such as superoxide (02") and peroxide (022~) that are strong and potentially dangerous oxidizing agents formed during the conversion of oxygen to water. Chlorophylls and bacteriochlorophylls, both pigments containing magnesium, are responsible for the photosynthetic process in plants or bacteria as these capture photons of light.3 Vitamin B l 2 , containing a cobalt metallated corrin, is the prosthetic group for a number of enzymes which carry out various rearrangement reactions and frans-methylations.4 In short, nature depends on tetrapyrrolic macrocycles for the control and modification of both the coordination and the redox properties of various metals. 4 Introduction Cyclic tetrapyrrolic pigments are ubiquitous in nature and their wide variety of functions account for the continued and growing interest of scientists into the isolation, total synthesis and structural determination of such molecules. Studies in this area over 50 years ago resulted in the discovery of metalloporphyrin derivatives in petroleum and oil shale.5 These petroporphyrins created a new field of investigation - organic geochemistry.6 Today, research into metalloporphyrins has led to their application in many fields of study such as medicinal chemistry, where these macrocycles act as potential chelators for radioactive metals in diagnostic medicine.7 Other useful properties of these molecules are currently being investigated for use in optical recording materials, as dyes in paints, as molecular conductors,8"" as synthetic models of biological enzymes,11"13 as photochemical systems for solar energy conversion and as pesticides. Non-metallated, or free base tetrapyrrolic macrocycles are also the subject of current research. Photodynamic therapy (PDT) is a relatively new and highly active field of cancer therapy. A variety of free base porphyrins and chlorins, as well as some Zn(U), Al(HI) and Sn(IV) complexes, have been found to be potential photosensitizers for singlet oxygen production. Their ability to absorb light of a particular wavelength in a selected area of the body makes these macrocycles excellent candidates for the photodegradation of cancerous tumors.14 Investigation into these molecules has also led to theoretically interesting macrocycles. Expanded porphyrin analogs, such as sapphyrin,15 porphycene,16 pentaphyrin,17 and texaphyrin,18 and novel macrocycles, such as N-confused porphyrins19 and benzoporphyrins, are testing the boundaries of modern synthetic methodology and knowledge. Investigation of the aromaticity and the ring currents of these macrocycles serves to expand our chemical knowledge. 5 Introduction Pentaphyrin Porphycene Sapphyrin Texaphyrin Figure 1.3 Expanded porphyrin analogs. 1.1.2 Structural Features The macrocyclic tetrapyrrole structure was first suggested in 1912 by Kuster, but was refuted by Fischer (and others) with claims that it was too large to be stable.20 Alas, upon the total synthesis of heme in 1929 by Fischer, the proposed structure was confirmed.21 Porphyrins are aromatic tetrapyrrolic macrocycles consisting of four pyrrole units joined by methine groups. These compounds possess 22 rc-electrons, 18 of which participate in a cyclic delocalized conjugation pathway (Figure 1.4). The vast majority of porphyrins are planar and thus fulfill the requirements for aromaticity according to Huckel's rules (4n+2; n = 4).22 This aromaticity is reflected by their physical data - bond lengths, heats of combustion and large ring current. The large diamagnetic ring current observed in porphyrins is expressed by their 'H-NMR spectra wherein the inner NH protons are highly shielded (6 = -1 to -5 ppm), and the meso-protons on the bridging carbons are strongly deshielded (6= 8 to 10 ppm).2'' The ring current which deshields protons from the external magnetic field on the outside of the macrocycle simultaneously shields 6 Introduction protons on the inside of the macrocycle. Figure 1.4 The 18-TT electron pathway Reduction of one or both of the two cross-conjugated double bonds at the P positions does not affect the aromaticity of the molecule. Chlorins are formed by the reduction of one of these bonds, whilst further reduction leads to the bacteriochlorin, which has the two opposite pyrrole rings reduced, or the isobacteriochlorin, which has adjacent P-P' bonds reduced. Porphyrins have four inner nitrogens and display four stepwise acid-base reactions. Values for pKi and pK2are approximately 16 and 16, respectively, whilst pK3 is estimated to be 5 and pK4 to be 2. The porphyrin dication can therefore be formed in strong acid via protonation of the central imino nitrogen atoms and treatment of the porphyrin with strong base forms the dianion. Porphyrins can complex with a variety of metals with the concomitant loss of the inner NH protons. The donation of o electrons from the four nitrogens to the metal ion is the main bonding force. The ability to metallate the porphyrin depends on the metal being used, for example metallation with Zn(OAc) 2 occurs under mild conditions in a relatively short time period whilst metallation with Ni(OAc) 2 requires more harsh conditions and longer reaction time. There are few metals that have not been shown to complex with porphyrins.24 7 Introduction 1.1.3 Optical Absorption Spectra 1.1.3.1 Optical Absorption Spectra of Porphyrins The absorption spectra of tetrapyrrolic macrocycles correlate and reflect many of their physicochemical properties. The absorption spectra of these aromatic tetrapyrrolic macrocycles display an intense band in the region of 400 nm. This absorption is called the Soret or P band and has a strong absorption with an extinction coefficient of 100000 - 400000 M'cm" 1 . 2 5 Porphyrins also display four accompanying bands, commonly referred to as the Q-bands, of lower intensity between 450-650 nm. These four bands denoted I to IV in order of decreasing wavelength serve to classify a porphyrin into one of four categories: etio-, rhodo-, oxorhodo- and phyllo-type.26 The four types of spectra are named for the porphyrins that were first found to exhibit the respective spectra (Figure 1.5) and give insight into the substitution pattern of the porphyrin and indicate whether it is metallated or the free base. 5 0 0 5 5 0 6 0 0 6 5 0 5 0 0 5 5 0 6 0 0 6 5 0 Figure 1.5 The four types of porphyrin spectra. 8 Introduction Etio-type absorption spectra, with relative intensities of the Q bands such that IV > HI > I I > I, are representative of all naturally occurring porphyrins and are distinguished by the presence of six or more alkyl groups on the P-positions of the macrocycle. The rhodo-type spectra are observed in porphyrins with a single electron-withdrawing group, such as an aldehyde or a vinyl group having TC-electrons that extend the conjugation of the macrocycle, at one of the P-positions. These type of spectra display Q bands with relative intensities such that HI > IV > I I > I. The electron-withdrawing group causes a bathochromic shift of all of the Q bands. Whilst conjugation-extending groups on adjacent pyrrole rings in a porphyrin cancel each other's "rhodofying" or "reddening" effect and an etio-type spectrum results, these groups on opposite pyrrole rings lead to the oxorhodo-type spectra wherein the spectrum is shifted to even longer wavelengths. The relative intensities of the Q bands are HJ > I I > IV > I. This type of spectrum is considered to be due to the additive enhancement of the rhodofying effects of two electron-withdrawing groups. The phyllo-type spectrum is observed when there are four or more unsubstituted P-positions or when one mew-position is substituted by an alkyl group. This type of spectrum has Q bands of relative intensities such that I V > n > I J I > I . A variety of factors converge to influence the shape of the absorption spectra of porphyrins. Upon metallation of the porphyrin, the four Q bands collapse to two - denoted a (longer wavelength) and p.27 The formation of the dication or dianion of the porphyrin also results in the reduction of the number of Q bands. This simplification of the spectrum is a result of the increase in symmetry as the system approaches square planar symmetry, from D 2 h point group of the free base porphyrin to D 4 h . The relative intensities of these two bands, as with the four Q bands of free base porphyrins, give powerful insight into the nature of the porphyrin. The stability of the coordination complex 9 Introduction is displayed by a spectrum wherein a> p.28 This relates to a stable square planar complex with the macrocycle and is commonly observed for metals such as Ni(II), Co(U), Cu(U) and Pd(II). Less stable coordination, as in the case of Cd(H), Zn(TJ), Mn(U) and Mg(II), is reflected in the absorption spectrum by the relative intensities of the two bands such that P > a. Figure 1.6 Absorption spectra of (i) a porphyrin (TPP) and (ii) metalloporphyrin (ZnTPP). 1.1.3.2 Optical Absorption Spectra of Chlorins, Bacterio- and Isobacteriochlorins The changes in the absorption spectra of a porphyrin upon reduction to a chlorin are significant. The lowest energy band I is bathochromically-shifted by 20-30 nm, to approximately 650 nm, and its extinction coefficient increases by a factor of approximately 10.29 The Soret is also generally red-shifted, but its intensity remains unchanged as are the intensities and positions of the other Q bands. Metallation of the chlorin does not simplify the absorption spectrum as the symmetry is permanently disturbed by the presence of the reduced bond. 10 Introduction Figure 1.7 The absorption spectra of (i) chlorins and metallochlorins and (ii) bacteriochlorins and metallobacteriochlorins. The absorption spectra of bacteriochlorins and isobacteriochlorins are quite similar to that of the analogous chlorin with the exception of band I of the bacteriochlorin spectrum is bathochromically-shifted relative to that of the chlorin system to approximately 750 nm. 3 0 This 11 Introduction band has a very high intensity with an extension coefficient almost as large as that of the Soret band. Upon metallation, the intensity of the band I shows a small increase whilst that of the Soret band shows a small decrease. 1.1.3.3 Theory of the Optical Absorption Spectra of Tetrapyrrolic Macrocycles 3 1 The theoretical explanation of the absorption spectroscopy of porphyrins is based on the relative energies of the HOMO and L U M O of the macrocycle as the longest wavelength absorption can be assigned to a H O M O - L U M O transition (TI - • TI*). The energies of the HOMOs are progressively raised as a porphyrin is increasingly saturated. The LUMOs of the porphyrin, chlorin and bacteriochlorin systems are isoenergetic, but those of the isobacteriochlorins are higher in energy. The H O M O - L U M O gap, therefore, increases on progression from a bacteriochlorinogenic to a porphyrinogenic system, and as this energy gap corresponds to the energy required for the TI ->• re* transition, the longest absorption wavelengths decrease in this order. 12 Introduction Figure 1.8 Energy level diagram for HOMOs and LUMOs of the four metallbporphyrin classes 3 2 1.1.4 Preparation of porphyrins 1.1.4.1 Naturally occurring porphyrins Protoporphyr in L X (2) and hematoporphyrin L X (1) are the on ly naturally occurr ing porphyrins that can be readily isolated in large quant i t ies . 3 3 ' 3 4 These can be isolated as their d i a c i d or diester derivatives f rom b lood in bu lk quantities, and can be converted into other na tura l ly -occur r ing porphyr ins by c h e m i c a l manipula t ion o f their substituents. 3 5 1 3 Introduction BLOOD Coproporphyrin III Mesoporphyrin (7) tetramethyl ester (6) 14 Introduction Scheme 1.1 Natural porphyrin derivatives and their readily synthesized products. 1.1.4.2 Synthetic Porphyrins 1.1.4.2.1 Porphyrins from Monopyrrolic Precursors There are two main classes of synthetic porphyrins: porphyrins with fully alkylated P-positions, such as octaethylporphyrin (OEP) (8), and those with fully arylated meso-positions, such as tetraphenylporphyrin (TPP) (10). Both OEP (8) and TPP (10) are fully symmetrical and can thus be prepared from one pyrrolic unit in a one-step, one-pot condensation reaction. The pyrrolic units may or may not include the linking carbon. OEP (8) may be synthesized via a number of routes which are based on the condensation of 3,4-diethylpyrrole with the a-position of the pyrrole which may or may not be further substituted in the a-position.36"38 A simple and commonly employed method involves the reduction and cyclization of 2-ethoxycarbonyl-3,4-diethylpyrrole.39'40 Octaethylporphyrin (8) Scheme 1.2 Synthesis of octaethylporphyrin (OEP) (8). 15 Introduction meso-Tetraarylporphyrins were originally synthesized by Rothemund via a 4 x l-type method that involved heating benzaldehyde and pyrrole in pyridine at 150°C in a sealed bomb for 24 hours. 4 1 4 2 The low yields and severe conditions that few substituents on benzaldehydes could survive pressed researchers for other methods. Porphyrin syntheses were later improved upon by Adler and Longo who modified the synthesis to allow milder conditions wherein benzaldehyde and pyrrole were refluxed for 30 mins in propionic acid (141°C) open to the air.43 More recently, Lindsey modified porphyrin syntheses allowing for even milder conditions, cleaner reactions for better purification and increased batch-to-batch reproducibility of the reaction.44 In this modified synthesis, acid-catalyzed condensation of pyrrole and benzaldehyde forms the porphyrinogen at which point an oxidant, typically DDQ or p-chloranil, is added to form the porphyrin. This procedure allows only small quantities to be produced as high dilution is required, but nonetheless yields of 30 - 40% have been reported using sensitive aldehydes. Rothemund-type syntheses have been applied to a variety of both alkyl and aryl aldehydes, with average yields of 30% for fully symmetrical porphyrins. Porphyrins that are unsymmetrically substituted may be synthesized via this type of synthesis with varying amounts of the differently substituted benzaldehydes, but the yields are drastically reduced due to the statistical formation of products (mono-, di-, tri- and tetrasubstituted TPPs). The mechanism of the synthesis is known to involve acid-catalyzed condensation of benzaldehyde and pyrrole to form an arylpyrrolyl carbinol.45 This intermediate loses a hydroxyl moiety to form a resonance-stabilized cation which reacts with another pyrrole producing a meso-substituted dipyrromethane. The dipyrromethane then adds to another benzaldehyde molecule. The chain-building reactions repeat, ultimately yielding a linear tetrapyrrolic moiety. Ring closure follows to form a porphyrinogen (9). The porphyrinogen (9) is then spontaneously 16 Introduction oxidized in air to the porphyrin, and/or phlorin which tautomerizes to a chlorin and slowly oxidizes to a porphyrin. Scheme 1.3 Rothemund-type synthesis of meso-tetraphenylporphyrins (TPP) (10). 1.1.4.2.2 Porphyrins from Dipyrrolic Precursors Porphyrins may also be synthesized from dipyrrolic precursors such as dipyrromethanes, dipyrromethenes and dipyrroketones. All naturally-occurring porphyrins can be prepared from dipyrrolic starting materials as they are symmetrically substituted, but some difficulties can arise when synthesizing unsymmetrical porphyrins. The MacDonald reaction involves a mild acid-17 Introduction catalyzed condensation of 5,5'-diformyldipyrromethane with a 5,5'-di-unsubstituted dipyrromethane or its dicarboxylic analog with hydroiodic acid or toluene sulfonic acid to give the porphyrin.46 It was this method that R.B. Woodward used for the classical total synthesis of chlorophyll.47"9 The main disadvantage of this method is the requirement that one of the two dipyrromethanes be symmetrical for the production of a single porphyrin. The advantage of this method is its use of relatively mild conditions, which allows more labile substituents to be present on the resulting porphyrin. Re R 6 R e R e Scheme 1.4 The MacDonald synthesis of porphyrins. Centrosymmetrically substituted porphyrins can be prepared via a self-condensation of a 5-formyl-5'-unsubstituted dipyrromethane under mildly acidic conditions. A variation of the MacDonald route is the oxidation of readily available 5,5'-diformyldipyrromethane, resulting in 5,5'-diformyldipyrroketone which can condense with 5,5'-di-unsubstituted dipyrromethanes. The analogous oxophlorin is produced and can be converted to raew-acetoxyporphyrin, which upon hydrogenation and reoxidation, yields the meso-unsubstituted porphyrin.50,51 18 Introduction Scheme 1.5 Synthesis of porphyrins from 5,5'-diformyldipyrroketones. Dipyrrornethen.es are also used for the synthesis of porphyrins. The self-condensation of 5-bromo-5'-methyl dipyrromethenes in acid at 180°C results in a porphyrin. 5 2 This method can also be used for the condensation of 5,5'-dimethyldipyrromethene with 5,5'-dibromodipyrromethene. 19 Introduction Scheme 1.6 Porphyrin synthesis from 5-bromo-5'-methyldipyrromethenes. 1.1.4.2.3 Porphyrins from Open Chain Tetrapyrrolic Precursors Bile pigments, such as bilanes (11), bilenes (12), biladienes (13) and oxobilanes (14), are open-chain tetrapyrrolic molecules that result from the natural catabolism of porphyrins. Obviously, the substitution pattern of the tetrapyrrolic precursors will determine that of the resultant porphyrin and therefore is easily controlled. The tetrapyrrolic chain can be constructed by the condensation of two dipyrrolic units or by the reaction of two monopyrroles and dipyrromethane. These pigments increase in sensitivity towards acid as the saturation level increases (e.g. bilanes from bilenes). Lability towards acid leads to fragmentation of the tetrapyrroles into mono-, di-, and tri-pyrrolic pigments and, as a result, many porphyrins will result from the mixture. Conditions must therefore be extremely mild or the presence of electron-withdrawing groups is required to stabilize the tetrapyrrole. Oxobilanes (14) and biladienes (13) are more stable precursors for porphyrin synthesis in this respect. 20 Introduction Figure 1.9 The bile pigments. Oxobilanes (14) as starting materials provide two possible routes: a-oxobilane and b-oxobilane. The a-oxobilane route refers to the use of oxobilanes having the ketone function on one of the terminal bridging carbons whilst b-oxobilane (15) route employs those having the ketone on the middle bridging carbon. Although the two routes are quite similar, the b-oxobilane (15) route is the more convenient of the two.53 This route involves the reaction of 5-N,N-dimethylamido-pyrromethanes with 5-unsubstituted pyrromethanes to give the dibenzyl ester of the b-oxobilane. This can be cyclized to an oxophlorin (16) without first removing the oxo group, as in the a-oxobilane route. The oxophlorin is then reduced to the porphyrin. 21 Introduction Bz0 2C Bz0 2 CONMe 9 Vilsmeier-Haack H + i. H 2 /Pd/C • ii. DDQ i. Hydrolysis ii. Catalytic hydrogenation R R b-Oxobilane (15) Scheme 1.7 The b-oxobilane route to porphyrins. The a,c-biladiene route has been favored in the past. There are several routes to a,c-biladienes. One method involves the condensation of two dipyrromethenes, one of which is unsubstituted in the 5-position. Symmetrically substituted a,c-biladienes can be prepared from 22 Introduction the condensation of dipyrromethane 5,5'-dicarboxylic acid with two equivalents of 2-formyl-5-pyrrole to give the l,19-dimethyl-a,c-biladiene (17).54 Cyclization methods vary - copper has been used to cause the biladiene to encircle it and sulfuric acid used to catalyze ring closure followed by demetallation.55 Scheme 1.8 The a,c-biladiene route to porphyrins. 23 Introduction 1.1.5 Preparation of Chlorins 1.1.5.1 Naturally Occurring Chlorins There are two types of chlorophyll found in nature, denoted a and b, which vary in the nature of one P substituent on the ring. Chlorophyll a (18) has a methyl group whilst this substituent is a formyl moiety in chlorophyll b (19). O-Phytyl R= C H 3 ; Chlorophyll a (18) R= CHO; Chlorophyll b (19) Figure 1.10 Chlorophyll a and b. Both chlorophylls have been used as starting materials for a variety of porphyrins and chlorins. These can be extracted from leaves and algae by boiling them in methanol or acetone. The chlorophylls, along with a number of other photosynthetic pigments, are then separated by column chromatography. The only type of porphyrins and chlorins that can be derived from chlorophyll have the same substitution pattern of P substituents as the chlorophyll itself. The isolation and chemistry of chlorophylls have been reviewed extensively.56"58 24 Introduction 1.1.5.2 Synthetic Chlorins 1.1.5.2.1 Total Synthesis of Chlorins The total synthesis of chlorophyll a (18), performed by Woodward et al, is the archetypal total synthesis of a chlorin.47"49 The route followed closely resembles that of the total synthesis of porphyrins but additional steps are required to introduce a reduced pyrrolic unit into the molecule. These steps change the nature of the procedure from that of an extremely simple porphyrin synthesis to an extremely tedious and intricate process. For this reason, many researchers have been discouraged to attempt such a monumental task and have opted for simpler methods such as porphyrin manipulation. 1.1.5.2.2 The Conversion of Porphyrins into Chlorins by Reduction The past decade has seen an explosion of research directed at reducing porphyrins due to the discovery of novel chlorins and bacteriochlorins coupled with their biological roles in nature and/or their potential uses in a variety of fields. Based on chemical knowledge of reducing agents and their uses, one would theorize that the reduction of a single porphyrin double bond would be an easy undertaking. In practice, this is not so. Whilst some traditional reducing agents are effective in accomplishing their task of reducing one (or more) double bonds, controlling the number and location of reduced bonds within the porphyrin has proven to be more of a challenge. Of the traditional reducing agents, only borane has proven effective for the reduction of porphyrins. Treatment of octaethylporphyrin (OEP)(8) with BFL, results in a 5:1 mixture of cis-and frans-octaethylchlorin.59 The reaction of OEP (8) with sodium metal in alcohol results in a mixture of chlorins, isobacteriochlorins and other products whilst the same reaction of 25 Introduction (octaethylporphyrinato)Fe Cl results solely in the trans-\somer. Raney nickel reduction of TPP (10) in ether has been shown to produce tetraphenylbacteriochlorin (20), producing tetraphenylisobacteriochlorin (21) for the same reaction in dioxane.61 Tetraphenyl isobacter iochlor in (21) Scheme 1.9 Raney nickel reduction of tetraphenylporphyrin (10). Photoreduction of porphyrins and metalloporphyrins in the presence of hydrogen atom donors such as ascorbic acid has also been shown to produce chlorins and other products of reduction.62 For example, photoreduction of tin porphyrins in the presence of stannous chloride yields the analogous chlorin and isobacteriochlorin. 26 Introduction Diimide is the most commonly used reagent for the reduction of porphyrins to chlorins. Refluxing p-toluenesulfonylhydrazide in pyridine generates the diimide.64 A number of problems are associated with this reaction too. Both the bacteriochlorin and the chlorin analogs are formed when TPP is used as the starting material, requiring tedious separation procedures.65'66 I. ii Scheme 1.10 Diimide reduction of porphyrins. Reaction conditions: (i) K2C03,/?-toluenesulfonylhydrazide, A (ii) /j-chloranil 1.1.5.2.3 Synthetic Conversion of Porphyrins into Chlorins 1.1.5.2.3.1 Diels-Alder Reaction of Natural Porphyrins The availability of natural porphyrins such as protoporphyrin LX (2) has led to a number of synthetic methods using these as starting materials. Protoporphyrin LX (2) has two P-vinyl groups conjugated to the P~P double bonds. This conjugated diene system reacts with singlet oxygen in the presence of light in a [4+2] cycloaddition reaction to produce a mixture of 27 Introduction regioisomers of photoprotoporphyrin.67,68 The reaction occurs on either ring A or B but not both. In this reaction the porphyrin acts as the starting material and as the sensitizer responsible for generating the singlet oxygen. Other activated dienophiles such as dimethylacetylene dicarboxylate (DMAD), nitrosobenzene and tetracyanoethylene (TCNE) have been investigated for reaction with this conjugated diene system.69"72 These have been shown to yield [4 + 2] addition product chlorins in fair yields. Scheme 1.11 Reaction of DMAD with protoporphyrin IX (2). Reaction conditions: (i) DMAD/hu. 1.1.5.2.3.2 Reactions of Carbenes with Porphyrins Diazomethane and diazoesters both form cyclopropane rings at the P~P double bonds of porphyrins. The cyclopropanation yields both endo- and exo-isomers of the analogous chlorin and bacteriochlorin. This reaction has been performed using TPP (10) as starting material, yielding endo- (5%) and exo-(20%) isomers of the cyclopropanochlorin (24) and bacteriochlorins 28 Introduction (10) (24) n (25) Scheme 1.12 The cyclopropanation of tetraphenylporphyrin (10). Reaction conditions: (i) diazoethylacetate/CuCl In a recent paper147, Smith et al. described the synthesis of a cyclopropanochlorin via the Barton-Zard reaction of zinc(U) P-nitro-tetraphenylporphyrin (26) with an a-isocyanoacetic ester in THF/isopropanol. The major product was identified as the cyclopropanochlorin (27), a single stereoisomer with an exo configuration of the isopropyl ester group. The simple proton NMR spectrum displayed peaks at 8.54, 8.41, 8.32 (each a doublet), and a single peak at 4.80 representing two protons. The UV-visible spectrum was typical of chlorins : 422 (e 240 000), 520 (8200), 560 (8700), 584 (11 700), 608 (34 900) nm. Scheme 1.13. Synthesis of a cyclopropanochlorin (27). Reaction Conditions: i. CNCH2C02Et/THF/iPrOH 29 Introduction 1.1.5.2.3.3 Chlorins with Unsaturated Exocyclic Rings T h e two major classes o f chlor ins that possess unsaturated e x o c y c l i c rings fused to the skeleton between a m e w - p o s i t i o n and the adjacent P-position are the purpurins and the benzoch lo r ins . 7 4 Purpurins contain f ive-membered exocyc l i c rings whereas benzochlor ins possess s ix -membered rings. B o t h classes are synthesized f rom the intramolecular cyc l iza t ion o f mew-subs t i tu ted porphyrins typ ica l ly via consecutive V i l s m e i e r - H a a c k and W i t t i g reactions. Octaethylbenzochlorin (29) Scheme 1.14 Synthesis of octaethylpurpurin (28) and octaethylbenzochlorin (29). Reaction Conditions: (i) DMF/POCl3, Ph3PCH2C02Et; (ii) Me2NCH=CHCHO, POCl3; (iii) TT 30 Introduction 1.1.6 Properties of Porphyrins 1.1.6.1 Reactivity of porphyrins Because porphyrins are aromatic macrocycles, they react similarly to benzene in many cases, but the field of porphyrin chemistry is filled with examples of the macrocycle's idiosyncratic chemical behavior. Porphyrins readily undergo many of the electrophilic substitution reactions that are so well known for benzene such as halogenation, sulfonation, formylation and acylation. There are two different sites where these substitutions can occur on the macrocycle - the mew position and the P position. The reactivity of each of the sites can be controlled by manipulating the electronegativity of the porphyrin itself. The presence or absence of other substituents on the macrocycle and/or the nature of the metal coordinated within can be used to modify the electronegativity of these sites. mew-Carbons are known to be more electron deficient than P carbons and hence are inherently less prone to electrophilic attack. Divalent metal atoms such as Zn, Ni, and Cu produce electronegative ligands, and therefore the electrophilic substitution will occur at the mew-positions. Introduction of metals in electrophilic oxidation states such as Sn(IV) or the absence of a coordinating metal deactivate the mew-carbons towards electrophilic substitution, and reaction will occur at the P carbons. However, there are exceptions to this theory. For example nitration only occurs at the mew-carbons if this position is free. Porphyrins also react with nitrenes and carbenes. Nitrenes react with the mew-carbons of porphyrins, opening the macrocyclic ring, to produce homoazaporphyrins. Porphyrins can also be oxidized to give mew-hydroxyporphyrins which are in equilibrium with oxophlorins. 31 Introduction 1.2 Photodynamic Therapy • Photodynamic therapy is a medical treatment that employs light and a drug to create cytotoxic forms of oxygen, and other reactive species, to effect the destruction of cancerous or hyperproliferative tissue. The drug, or 'photosensitizer', is administered and preferentially accumulates in the tumour or hyperproliferating tissue. The drug is irradiated, and thereby activated, with light of an appropriate wavelength. The activated drug interacts with molecular oxygen in the body to generate reactive oxygen species that kill the diseased cells. 1.2.1 History of PDT The use of the combination of tetrapyrrolic macrocycles and light to bring about biological photoreactions dates back many millennia. Ancient Egyptians were known to treat vitiligo by the oral ingestion of various plants that contained psoralens and the use of sunlight 4000 years ago.77 In the late nineteenth century, Finsen employed ultraviolet light and similar plants to treat psoriasis, a treatment very similar to that used today. In 1900, Raab discovered that the combination of light and acridine dyes killed paramecia.78 Skin cancer was treated in 1903 by topical eosin and the administration of light.79 The first porphyrin was used in this capacity in 1913, when Meyer-Betz injected himself with 200 mg of hematoporphyrin.80 No ill effects were observed until he was exposed to sunlight, at which time he experienced extreme swelling and photosensitivity that remained for months. Policard observed that porphyrins preferentially accumulated in tumors in 1925,81 and similar 32 Introduction results were later obtained from research performed by Auler and Banzer. For the next few decades, relatively little investigation into these compounds or their observed effects was performed. In the early 1950s, Schwartz returned to Meyer-Betz's self-experiment.83 He proved that the hematoporphyrin was quickly cleared from the body, and hence, it could not have caused the prolonged photosensitivity. This led to further investigation into the synthesis and purification of Hp which determined that the preparation of Hp had produced an oligomeric fraction from the reaction of Hp with 5% sulfuric acid in acetic acid followed by alkaline hydrolysis.84"86 This fraction was called 'hematoporphyrin derivative' (HpD) and was prepared in greater concentration. It was found to be a mixture of monomeric, dimeric and polymeric porphyrins with ether, 8 7 - 8 9 ester 9 0 , 9 1 and carbon-carbon 8 9 9 2 linkages. A coworker, Lipson, later observed the preferential accumulation of the multi-component photosensitizer in cancerous tissues in animals.93'94 Impressive properties of HpD as a photosensitizer were observed, but it still suffered from the major drawback of being a mixture of compounds. Many efforts have been made to identify the active component(s) but to no avail. The only progress in this respect was in proving that neither the monomeric nor the dimeric compounds were the most OS active species. 33 Introduction Hematoporphyrin Derivative (HpD) (30) Scheme 1.15 Preparation of hematoporphyrin derivative (HpD) (30). 34 Introduction In the early 1970s, Dougherty began clinical trials using various HpD preparations. HpD (30), refined via filtration, was named "Photofrin II" and was to be the first generation photodynamic therapy drug." Further purification and lyophilization in the late 1980s at QLT Phototherapeutics and American Cyanamid resulted in "Photofrin", which has received approval from regulatory agencies throughout the world for the treatment of certain types of cancer.100 Photofrin has several drawbacks, the first of which is its chemical composition. It remains a complex mixture of oligomers of hematoporphyrin derivative. The pharmacokinetics of the drug are also troublesome because the accumulation of the drug in skin may last up to six weeks, thereby causing the patient to be photosensitive to sunlight."" Additionally, the longest wavelength at which the drug can be photoactivated, 630 nm, is in a less than ideal region of the optical spectrum because at 630 nm light penetration through skin is limited and, therefore, effective irradiation cannot occur.102 These problems have led to the investigation and discovery of new photosensitizing drugs, termed 'second' or 'third-generation' photosensitizers. 1.2.2 Mechanism of Photosensitization The principles of photosensitization are illustrated by a modified Jablonski diagram shown in Figure 1.12. The initial step of photosensitization is the promotion of an electron in the drug's outer shell from the ground state S 0 to an excited state S n due to the absorption of a photon from light. This excited state molecule can lose energy via internal conversion until it reaches the first excited state S,. The first excited state of most molecules has a very short lifetime, usually of the order of 1-100 ns. This first excited state has a number of possible routes to return to the ground state. It may undergo internal conversion (S, - S 0 + heat) or fluorescence (S, - S 0 + hv). Intersystem crossing (ISC), a spin-forbidden process, may occur giving rise to the first 35 Introduction excited triplet state (S, - T , + heat). The efficiency with which this process occurs is quantifiable, and essentially determines how good a photosensitizer is. The triplet quantum yield is the ratio of the amount of the triplet sensitizer formed to the amount of singlet sensitizer initially formed. The excited triplet state may return to the ground state via two routes, both involving a second spin-forbidden inversion. It is for this reason that the lifetime of the first triplet excited state is longer than that of the singlet excited states, typically of the order of micro-to milliseconds. One of the possible routes the first triplet state may take to the ground state is the loss of energy via phosphorescence (T, - S 0 + hv). The second route involves the non-radiative interaction of the triplet excited state with an easily oxidized or reduced substrate ("Type I" reactions) or spin-exchange with ground state triplet oxygen. The interaction of the excited state photosensitizer with triplet state oxygen leads to the production of the very reactive singlet state oxygen and is termed a "Type II" reaction. Both "Type I" and "Type II" reactions lead to the cytotoxicity of the drug, and the prevalence of one or both of these processes is determined by a variety of factors such as oxygen and substrate concentration in the system. 36 Introduction PHOTOSENSITIZER Figure 1.12 Modified Jablonski diagram for a typical photosensitizer 1.2.2.1 Type I Reactions 1 0 3 1 0 4 Type I reactions involve the interaction of the triplet state photosensitizer with other molecules in its vicinity. The triplet state is more reactive than the singlet state sensitizer as the lone unpaired electron in the higher orbital makes the triplet state more readily oxidizable and the unpaired electron left by the promoted electron makes it more readily reducible. The triplet state photosensitizer can abstract an electron or a hydrogen atom from a substrate as shown : 3sens + substrate - sens + substrate(ox) 3sens + substrateH - sensH* + substrate* The substrate is oxidized and the photosensitizer anion is produced upon electron abstraction by the photosensitizer. The anionic sensitizer can then react with molecular oxygen to generate the 37 Introduction superoxide anion. sens" + 0 2 - sens + 02" The superoxide anion can later generate the very reactive hydroxyl radical which can interact with other substrate molecules to initiate a free radical chain reaction. Hydrogen atom transfer from a substrate to the triplet state photosensitizer yields free radical products which are very reactive and can react in a variety of ways. Reaction of these radicals with molecular oxygen produces various oxidized products, often including peroxides which can, in turn, initiate free radical chain reactions. 3sens + RH - sens-H + R* R- + 0 2 - R0 2 -RCy + RH - ROOH + R-1.2.2.2 Type II Reactions ' Type II reactions involve the reaction of the first excited triplet state photosensitizer with triplet ground state oxygen, and because this is a spin-allowed process, it is very efficient. Oxygen exists in two singlet states: a high-energy (37.5 kcal/mol), short-lived (<10"" s) species and a longer-lived (4 ps in water - 25-100 u.s in nonpolar organic media), lower energy (22.5 kcal/mol) species. The difference in lifetimes of the two singlet oxygen states suggests that the longer-lived species is that involved in reactions with photosensitizers. 38 Introduction Singlet oxygen can react with biological substrates in a variety of ways: A) addition to heterocyclic systems (yields peroxides) B) hydrogen abstraction and addition ("ene" reactions with compounds with allylic hydrogen . atoms) C) [2 + 2] additions to double bonds followed by the cleavage of that bond D) oxidation of sulfides to sulfoxides o o Scheme 1.16 Reactions of singlet oxygen with biological substrates These reactions essentially result in the destruction of biological substrates such as membranes, enzymes, proteins and nucleic acids and thereby effect cell death. 39 Introduction 1.2.3 Desirable Properties of a PDT Drug 1.2.3.1 Preparation of the Drug An ideal photosensitizer should be able to be synthesized and purified via reproducible, cost-effective methods. This is, in essence, a simple synthesis employing readily-available relatively inexpensive starting materials requiring very little and inexpensive purification. These processes must produce a single stable, enantiomerically and diastereomerically pure compound. 1.2.3.2 Chemically-Modifiable Characteristics of the Drug Ideally, the photosensitizer should inherently possess the ability to be easily derivatized. It should contain a functionality or a moiety that allows easy chemical derivatization or modification to optimize the properties of the drug for different purposes. Light scattering and the presence of endogenous chromophores such as hemoglobin result in very poor penetration of tissue by light at wavelengths below 650 nm. This means that the Q bands must be used as a means of photosensitizer activation. Light penetration of tissue doubles between 630 nm and 750 nm, as shown below in Figure 1.13. Compounds absorbing energy above 800 nm do not have a large enough energy gap between the triplet and ground states to be able to generate singlet oxygen. An ideal PDT drug should, therefore, be photoactivated at wavelengths greater than 650 nm and less than 800 nm to ensure better absorption of tissue-penetrating red light and so sensitization by an external light source. Additionally, the drug must be preferably water-soluble or at minimum, easily formulated, in order for it to be soluble in the body's tissue fluids. This is necessary for the required method of injection, whereupon the drug is carried around the body to the tumor site. 40 Introduction T3 a o u a a> a, (U o 4) <U .> 550 630 700 800 nm Figure 1.13 The wavelength-dependent penetration of light through tissue 1.2.3.3 Biological Characteristics of the Drug Most importantly, the drug must have the ability to sensitize oxygen efficiently. The quantum yield of the triplet state formation must be high with triplet energy greater than 94 kcal/mol, and this energy must be efficiently transferred to triplet oxygen. The first triplet excited state must, therefore, be long-lived. An ideal photosensitizer must localize specifically in tumors. The drug should be non-toxic in the absence of light and be rapidly cleared from the body after treatment, either by excretion or metabolism so that no toxic metabolites are formed. 1.2.3.4 Properties Required for Industrial Production/Scale-up of the Drug Many of the requirements for the efficient industrial production of a drug were mentioned in the above categories, such as efficient synthesis and purification. Additional requirements include: long shelf life of the formulated drug (i.e. stability), simple preparation for 41 Introduction administration (dissolved in sodium chloride solution for injection), a wavelength of activation suitable for the use of light emitting diodes (to reduce treatment costs), and a strong proprietary position on the drug. 1.2.4 Major Second-Generation Photosensitizers The efficacy of a porphyrin-based drug depends on its ability to absorb light in the red or near infrared region of the optical spectrum. The nature of the optical spectrum is primarily determined by the level of saturation of the tetrapyrrolic macrocycle, and consequently the research toward new photosensitizers has been focused on chlorins and bacteriochlorins. 1.2.4.1 BPDMA 1 0 5' 1 0 6 In 1984, Morgan et al.105,106 synthesized a new class of chlorins, named 'benzoporphyrin derivatives' (BPD) due to their structural similarity to benzoporphyrins. Protoporphyrin LX dimethyl ester reacts with dimethylacetylene dicarboxylate (DMAD) in [4 + 2] cycloaddition to produce the 1,4 diene chlorin adduct. This product is then isomerized in base to give a chlorin with a conjugated 1,3 diene. This results in a bathochromic shift of the absorption from 666 nm in the 1,4 diene to 688 nm in the 1,3 diene system. Acid hydrolysis of one of the methyl esters to form one free propionic acid moiety produces a compound known as benzoporphyrin derivative monoacid ring A (BPDMA) (34). 42 Introduction B P D M A (34) Scheme 1.17 Synthesis of BPDMA (34) from protoporphyrin IX dimethyl ester (31). 43 Introduction Curiously, extensive in vitro and in vivo testing showed that the analogous compound possessing two propionic methyl esters was inactive, that the diacid analog was 10-70 times more cytotoxic than hematoporphyrin and that the monoacid was five times more cytotoxic to cell lines than the diacid. There are many promising features of BPDMA (34) as a photosensitizer. The absorption maximum at 688 nm indicates very effective tissue penetration by light, approximately twice as much as Photofrin. The drug clears the body within 72 hours of administration to the patient, and has a high quantum yield (0.78 in homogeneous solution, 0.46 in vivo). This drug also shows extensive clinical uses such as treatment of basal cell carcinoma, macular degeneration and bone marrow purging. This drug is currently in phase m clinical trials. Despite its attributes, there are many non-ideal characteristics involved with the drug and its synthesis. Adduct formation occurs at both possible sites on the ring, and therefore, two products are formed in equal amounts and must be separated as only one of these is used for drug preparation. Hydrolysis of one of the propionic moieties leads to substantial amounts of the diacid as well as remaining diester, both of which must be separated. Finally, BPDMA (34) results as a mixture of regioisomers, with one or the other of the two propionic esters hydrolyzed, and each consists of a pair of enantiomers. 1.2.4.2 M A C E Mono-L-aspartyl chlorin e6 (36) is a derivative of chlorin e6 (35) which is synthesized by treatment of chlorophyll a (18) with strong base.' 0 7 ' 0 8 DCC coupling of chlorin e6 (35) with di-tert-buty\ aspartate and subsequent removal of the r-butyl protecting groups of the monoaspartyl derivative with TFA produces M A C E (36). Mono-L-aspartyl chlorin e6 (36), a water soluble 44 Introduction chlorin, is currently in clinical trials. The maximum absorption occurs at 654 nm and the dru£ has a quantum yield of singlet oxygen production of 0.70.109 ° \ M e 0 2 C 0 O-Phytyl Chlorophyl l a (18) i. N a O H / H 2 0 ii. D C C / N H 2 C H ( C 0 2 R, ) C H 2 C 0 2 R ' (R'= C M e 3 ) O-R C 0 2 R ' C 0 2 R ' Chlor in e 6 : R= O H (35) M A C E : R= N H C H ( C 0 2 H ) C H 2 C 0 2 H (36) Scheme 1.18 Synthesis of M A C E (36). 1.2.4.3 Tin Etiopurpurin A purpurin is a chlorin with a five-membered exocyclic ring. Tin etiopurpurin (38) is formed by the cyclization of meso-[P-(methoxycarbonyl-2-carboxyl)vinyl]octaethyl porphyrin followed by metallation with tin(IV) chloride."0 This lipophilic drug has a maximum absorption at 640 nm at a singlet oxygen quantum yield of 0.82. This drug is currently in phase ITJ clinical trials.1" 45 Introduction 1.2.4.4 /Meso-Tetra(/M-hydroxyphenyl)chlorin' l 2"'1 5 Bonnett et al. screened a series of variously substituted porphyrins, chlorins and bacteriochlorins as potential photosensitizers and it was found that tetra(m-hydroxyphenyl)chlorin (39) was the most active photosensitizer. The simple synthesis consists of 3 steps from pyrrole and hydroxybenzaldehyde, using diimide as the porphyrin reducing agent. This drug, having an absorption maximum at 650 nm, shows great promise as a PDT agent and is currently undergoing extensive investigation. 46 Introduction Tetra(m-hydroxyphenyl)chlorin (39) Scheme 1.20 Synthesis of niTHPC (39). 1.3 Osmium Tetroxide Oxidations 1.3.1 History In 1908 the first paper describing the reduction of osmium tetroxide by unsaturated compounds was published."6 In 1912, Hofmann showed that osmium tetroxide could be used catalytically in the presence of sodium or potassium chlorate for the hydroxylation of double bonds."7 This work was continued by Milas, who reported the osmium tetroxide catalyzed oxidation of alkenes by hydrogen peroxide."8 Crigee showed that osmium tetroxide could be used in stoichiometric amounts without secondary oxidants to effectively dihydroxylate olefins in 1936, and afterward determined much about the reaction and its intermediates."9 47 Introduction 1.3.2. Mechanism120 The ds-dihydroxylation of alkenes by osmium tetroxide is known to proceed via the formation of an osmium(IV) intermediate which upon oxidative or reductive hydrolysis yields the ds-diol analogs. Reductive cleavage using lithium aluminum hydride, hydrogen sulfide, mannitol or sodium bisulfite forms an insoluble osmium salt which is removed by filtration. Oxidative hydrolysis with terf-butyl hydroperoxide, morpholino ,/V-oxide, or sodium hypochlorite regenerates the osmium tetroxide. Addition of pyridine to the reaction leads to a marked increase in the rate of formation of the intermediate ester which forms as a te-amine complex. In the absence of an amine ligand, the intermediate exists either as a dimer consisting of two five-coordinate pyramidal osmium(IV) atoms each with cyclic ester rings or the intermediate reacts with a second alkene moiety to form a monomeric diester complex. Scheme 1.21 Osmium tetroxide dihydroxylation of alkenes. Although there has been extensive research into the osmium tetroxide oxidation of double bonds, there still remain a few important unanswered questions regarding the mechanism of action. Today, there are two possible mechanisms of formation of the glycol ester under debate. One mechanism that has been postulated is the concerted [3 + 2] cycloaddition of the osmium tetroxide to the double bond, whilst the other possibility involves the indirect attack of alkenes at 48 Introduction the electropositive osmium center in a [2 + 2] cycloaddition, thereby initially forming an organometallic intermediate. O I  * °=f<o R [3 + 2] cycloaddition [2 + 2] cycloaddition , ' \ V " / / R O O O I I / 0 v 0 = j O s > — R O R / >''/R J Scheme 1 . 2 2 The two possible intermediates in the formation of the osmium tetroxide glycol ester. Presently, although no direct evidence is available for the existence of an organometallic intermediate, there is much indirect, but nonetheless supporting, evidence for such an intermediate. A kinetic study performed recently120 monitored the influence of temperature on the enantioselectivity of this reaction. At least two rate-determining steps were indicated by the data, which precludes the one-step concerted [3 +2] mechanism. Additionally, Sharpless and co-workers have proposed the similarity of the osmium tetroxide reactions and carbonyl reactions.120 Nucleophilic attack on carbonyl functional groups occurs exclusively at the carbon center. They propose that a carbon-carbon double bond, a weak nucleophile, would therefore be expected to attack at the more electropositive osmium center as opposed to the oxygen.120 Osmium tetroxide has also shown differing reactivity towards alkenes as compared to the permanganate ion which is thought to proceed via a direct oxygen attack. It has been shown that osmium tetroxide reacts 49 Introduction more slowly with alkenes bearing electron-withdrawing groups, and in this context can be seen to be due to the decreased nucleophilicity of the alkene. The opposite trend has been observed for reactions with the permanganate ion. Additionally, aromatic hydrocarbons are oxidized at the site of greatest electron density. Alkylimidoosmium(VHI) complexes are formed upon reaction of certain primary amines with osmium tetroxide which is consistent with the reaction of primary amines with carbonyl moieties. The spectacular rate increase in the formation of the osmium ester in the presence of pyridine can be rationalized by the explanation that electron donation from pyridine to the osmium center may encourage the osmium-carbon bond cleavage. The forementioned evidence points directly at the [2 + 2] cycloaddition, step-wise mechanism and it is this mechanism that will most likely be accepted in the future. 1.3.3 The Osmium Tetroxide Oxidation of Aromatic Systems Osmium tetroxide-catalyzed dihydroxylation of olefins is highly selective and can be performed in the presence of aromatic systems. In 1942 the first aromatic system was reportedly dihydroxylated by osmium tetroxide.121 Osmium tetroxide preferentially attacks the site(s) of greatest electron density and regioselectively at the site(s) where the addition to the double bond causes the least reorganization of electrons.120 This 'principle of least motion or minimal electron reorganization' is well known and applies to many reactions.122123 An example of the preferences of osmium tetroxide can be seen in the reaction of anthracene which is initially oxidized at the 1,2 positions, maintaining aromaticity in two rings, and then at the 3,4 positions, which are conjugated but non-aromatic whilst the meso positions remain unreactive.121124 Alkenes are oxidized at a much faster rate than aromatic systems, and it follows that oxidation of 50 Introduction alkenes using osmium tetroxide requires significantly less time than the oxidation of aromatic systems. Scheme 1.23 The osmium tetroxide oxidation of anthracene (40). Reaction Conditions: (i) 1.1 eq. Os04/py/benzene; mannitol/KOH 1.3.4 Osmium Tetroxide Oxidation of Porphyrins In 1940, Fischer reported the osmium tetroxide dihydroxylation of deuteroporphyrin methylester.125 This octaalkylporphyrin was dihydroxylated at the site of greatest electron density, the (3,p' bond. This reaction produced many v/c-dihydroxychlorins. The starting material, being unsymmetrical, was oxidized at each of the four possible p,p' bonds, producing four regioisomers, each of which being a pair of stereoisomers due to the availability of two possible routes of attack by the reagent - above and below the porphyrin plane. 1 2 6 1 2 7 Octaethylporphyrin (8) was also reacted with osmium tetroxide in pyridine, yielding a sole product, the vz'c-dihydroxychlorin analog.128 A second equivalent of osmium tetroxide formed the tetrahydroxybacteriochlorin as a mixture of stereoisomers.129 Dihydroxylation of the analogous metallochlorin yields an isobacteriochlorin.127 This directing effect by a metal is well known in many reactions such as the diimide reduction of metallated tetraphenylchlorin,130 and the Raney nickel-catalyzed reduction of nickel 51 Introduction pheophorbides.131 The pathway of the delocalized 7T-electrons in a chlorin segregates the opposite cross-conjugated pyrrolic double bond and, therefore, the attack of this isolated bond is favored as this results in both minimal electron reorganization and retention of aromaticity.127,130 Insertion of a metal into the chlorin system induces a change in the preferred pathway of the delocalized re-electrons, isolating the adjacent cross-conjugated pyrrolic double bond. Porphyr in Chlor in Metal lochlor in Figure 1.14. 18 7i-electron derealization pathway in porphyrins, chlorins and metallochlorins. The osmium tetroxide-catalyzed oxidation of benzoporphyrin derivative (34) has also been performed yielding the theoretically-expected products.132 Dihydroxylation occurs, first at the non-aromatic, electron-rich double bond of the exocyclic ring, then at the remaining non-aromatic bond of the exocyclic ring and lastly at the oppositely-situated pyrrolic double bond of the chlorin system forming a bacteriochlorin (37). 52 Introduction Scheme 1.24 The reaction of a benzoporphyrin (34) with osmium tetroxide. 1.4. P-Hydroxychlorins Not Derived from An Osmium Tetroxide Oxidation 1.4.1 P-Oxochlorins Acid-catalyzed rearrangement of P,P'-dihydroxyoctalkylchlorins is the simplest and therefore the preferred method of P-oxochlorin formation. This product can also be dihydroxylated further with the selective formation of the analogous dihydroxylated 53 Introduction bacteriochlorin. The p-oxometallochlorin reacts in a typical fashion, yielding the metalloisobacteriochlorin isomers, which can be acidified to form the corresponding P,P'-dioxometalloisobacteriochlorins.133 The acidic hydrogen peroxide oxidation of porphyrins to form the P-oxochlorins was discovered by Fischer et a / . 1 3 4 1 3 5 Octalkylporphyrin was dissolved in concentrated sulfuric acid and hydrogen peroxide added to the mixture. The resultant green-colored product was mistakenly identified as the P,p'-epoxide chlorin. Thirty-nine years later, this reaction was re-investigated and the correct identification of the product as the P-oxochlorin originating from the pinacol rearrangement of a P,P'-dihydroxychlorin intermediate was published.'36 Chang et al. have performed this reaction on numerous porphyrin systems.137 Using OEP (8), they have reported the formation of the oxochlorin (37) (19%), three dioxoisobacteriochlorins (38-40) (11.3%) and two dioxobacteriochlorins (41)(42) (9%) as the major products. 1 3 8 1 3 9 54 Introduction Scheme 1.25 The hydrogen peroxide-sulfuric acid oxidation of O E P (8). This group also performed this reaction on mesoporphyrin yielding nine different compounds. 55 Introduction Cavaleiro et al. have synthesized 2-diazo-3-oxo-tetraphenylchlorins (43) by reaction of P-amino-tetraphenylporphyrin (42) with NaN0 2 and sulphuric acid in peroxide-containing THF. The diazaketone (43) was formed in 25-54% yield.141 (42) (43) Scheme 1.26 Synthesis of 2-diazo-3-oxo-tetraphenylchlorin (19). Reaction Conditions: (i) 1. NaN02/H2S04 2. THF/peroxide Crossley et al. have reported two syntheses of porphyrin-[3-diones (45): Rose Bengal-mediated photolysis of an oxygenated methylene chloride solution of copper(II) 2-hydroxy-5,10,15,20-tetraphenylporphyrin (44) (50% yield), and via oxidation of the same in refluxing dioxane with selenium dioxide (58% yield). 1 4 2 , 1 4 3 56 Introduction Recently, a communication detailing the mono-hydroxylation of a (3-carbon of an unsubstituted chlorin (46) via C-H bond activation by alumina in 70-80% yields has emerged.144 The chlorin was placed on a column of alumina gel and allowed to stand for 2 hrs after which the chlorin was eluted off the column. No dihydroxylation took place and very little oxidation of the chlorin core occurred. Chlorins employed were in both the free base and zinc metallated forms, and were all hexaalkylated. Scheme 1.28 Hydroxylation of a hexaalkylchlorin (46) via C - H bond activation by alumina. 57 Introduction 1.4.2 Epoxychlorins Only one claim of a synthetic epoxychlorin in the literature has been substantiated. 1 4 5 1 4 6 A n epoxychlorin was formed via a modified Mitsunobu reaction during the attempted formation of oxygen analogs of sulfchlorins from naturally occurring episulfides of protoporphyrin L X (2).147 This reaction was later performed on O E P (8) and etioporphyrin(II). The porphyrins were treated with osmium tetroxide to afford the vz'c-dihydroxychlorin analogs which, upon acid treatment, yielded the corresponding vinyl-substituted porphyrins. Photoxidation, borohydration and dehydration generated both the P-oxochlorin (48) and its isomer, the epoxide (49), as shown. Scheme 1.29 Epoxidation of OEP (8). 58 Introduction 1.5 P-Dihydroxychlorins From Other Methods One report of the electrochemical reduction of a porphyrin into a chlorin involving an epoxide intermediate exists in the literature wherein Inhoffen produced the 10,22-dihydrochlorin-e 6-trimethyl ester via the electrochemical reduction of chlorin-e 6-trimethyl ester.1 4 8 Photoxidation forms the hydroperoxide which dehydrates to yield the epoxide intermediate. The epoxide, in the presence of methanol, opens to give the product (50). 1 4 9 , 1 5 0 N H C H ( C 0 2 H ) C H 2 C 0 2 M e N H C H ( C 0 2 H ) C H 2 C 0 2 M e 02/fm M e O H t Scheme 1.30 The synthesis of 10,22-dihydrochlorin-e6-trimethyl ester (50). 59 Introduction 1.5.1 Photoprotoporphryin As previously mentioned, protoporphyrin IX (2) reacts with dioxygen to form the chlorin, photoprotoporphyrin. The aldehyde moiety can then be reduced to an alcohol with the use of sodium borohydride, yielding the allyldiol analog. 1.5.2 Secochlorins Secochlorins are porphyrins in which one P,P'-pyrrolic double bond is fully cleaved. In 1992, Chang et al. reported the serendipitous synthesis of the first secochlorin.146 (1,19-Dimethyloctadehydrocorrinato)nickel(II) (51) was heated to 150°C in dichlorobenzene for 20 minutes and produced the green secochlorin (52) as the major product (34%). Scheme 1.31 Synthesis of furochlorophin (52). Later, Bonnett et al. reported the secochlorin bisketone analog of octaethylporphyrin (54) from the lead tetraacetate oxidation of the dihydroxyoctaethylporphyrin (53).151 60 Introduction (53) (54) Scheme 1.32 Synthesis of octaethyl-2,3-secochlorin-2,3-dione (54). 1.6 Porphyrins Incorporating a Six-Membered or Larger Ring Oxipyriporphyrin (55) (X=N) was synthesized via a 3 + 1 approach, and is shown below. The analogous oxibenziporphyrin (56), X=CH, was synthesized in the same manner using the corresponding isophthalaldehyde.152 Scheme 1.33 Synthesis of oxipyriporphyrin (X=N) (55) and oxibenziporphyrin (X=CH)(56). Reaction Conditions: i. l.TFA 2. Et3N, DDQ. 61 Introduction Other porphyrins incorporating a six- or larger membered ring are synthesized by manipulation of synthetic porphyrins. Compound (58) was synthesized by the oxidation of the corresponding 2,3-dione system (57) whilst pigment (59) results from the base-catalyzed aldol condensation of the bisketone secochlorin of octaethylporphyrin (54).153'151 Scheme 1.35 The internal aldol condensation of «!£S0-tetraphenyl-2,3-secochlorin-2,3-dione (54). Reaction conditions: i. KOH (aq)/THF. 62 Introduction 1.7 Research Objective and Thesis Preview The objective of this work was to synthesize novel aromatic compounds based on mew-substituted porphyrins and porphyrin-based macrocycles. These compounds are potential photosensitizers for use in photodynamic therapy. Several approaches were employed towards this goal. Numerous tetraphenylporphyrins possessing a variety of substituents were synthesized, characterized and used as starting materials to prepare the analogous diol chlorins via a patented osmium tetroxide oxidation. The osmium tetroxide reaction allows the conversion of porphyrins into long wavelength absorbing compounds which are, in theory, ideal for PDT. In chapter 2, compounds possessing hydroxy, methoxy, alkyl and halogen substituents were prepared and in vitro biological tests were performed. These results are presented and discussed. (mew-Tetraphenyl-2,3-dihydroxy-2,3-chlorinato)nickel can be oxidized to form (meso-tetraphenyl-2,3-dialdehyde-2,3-secochlorinato)nickel(II). In chapter 3, an investigation of the reactivity of this novel compound is presented. A wide variety of reactions are examined including reactions with acids, bases, alcohols, amines and reducing agents in order to form novel compounds which may be regarded as either potential photosensitizers or starting materials thereof. Finally, in chapter 4, a relatively unexplored field of porphyrin research was investigated. Studies probed the reactivity of both tetra- and diphenylporphyrins in a number of different 1,3-dipolar cycloaddition reactions to form the analogous chlorin compounds possessing a new 5-member heterocyclic ring. We believed that realization of this type of reaction with porphyrins to form chlorins for potential use as PDT agents would present a novel method of chlorin synthesis with a huge potential for variety. 6 3 Chapter 2 Synthesis and Testing of Potential PDT Drugs 64 Chapter 2 Results 2.1 Introduction Photodynamic therapy is the damage of living tissue through the use of a photosensitizer, light and oxygen. The photosensitizing drug, which displays very little toxicity in the absence of light, is injected into a subject and accumulates preferentially in hyperproliferative cells. Once the drug has reached the maximum ratio of accumulation in the cancerous versus healthy cells, the tumor is irradiated with light using a laser. As the photosensitizer absorbs the light, it becomes activated through energy transfer to the triplet state and singlet oxygen is generated within the tumor. This initiates destruction of the diseased cells. The damaged cells then become necrotic and are rejected or absorbed by the body. The search for effective photosensitizers requires a two-pronged approach. The optimization of photophysical properties is key to any promising drug as the compounds must absorb at long wavelengths. The development of higher wavelength photosensitizers requires a synthetic method which can generate a number of analogs with ease as in vivo biological proficiency is known to increase on going from porphyrins to chlorins to bacteriochlorins. Compounds must also display good biodistribution properties in order to be effective. The correlation between the biodistribution of photosensitizers and the structure of the drug is complex. This complexity is increased with hydrophobic molecules which must be formulated into a suitable transport system such as liposomes, emulsions or nanoparticles. The delivery systems of these drugs are crucial and have been a key obstacle in PDT. These systems are complicated in that their nature drastically affects both the rate and the amount of drug taken up by the cells. A very large percentage of porphyrin-based photosensitizers are transported via protein binding. For example, at least 95% of hematoporphyrin (Hp) (60) at the normal dose used for PDT 65 Chapter 2 Results (3-5 mg/kg body weight) is complexed by serum proteins.'54 Human serum consists of three protein fractions: lipoproteins (high density (HDL), low density (LDL) and very low density (VLDL)), globulins and albumin. The distribution of photosensitizers in the serum is strongly dependent upon their chemical structure. Hydrophilic, polar photosensitizers are bound preferentially by albumin and globulins whilst hydrophobic molecules are bound by lipoproteins.155 Albumin and globulins are known to possess a distinct number of binding sites.156 The binding of photosensitizer molecules to albumin and globulin is governed by a chemical equilibrium between the bound and unbound photosensitizer.156 In contrast, the binding of hydrophobic dyes to lipoproteins reflects a partition of the photosensitizer between the lipid and the aqueous phase and therefore many photosensitizer molecules can bind to each lipoprotein. The relative binding of tetrapyrroles to lipoproteins has been shown to increase with decreasing polarity.157 The partitioning of hydrophobic photosensitizers is significant as these dyes tend to aggregate in aqueous systems. The extent of aggregation is dependent upon the polarity of the substituents on the porphyrin skeleton.158 Only monomeric nonaggregated molecules are photoactive and therefore any aggregation will decrease the observed cytotoxicity of the drug.'59 Hydrophobic photosensitizers must, therefore, be properly formulated in order to counteract their natural tendency to aggregate in aqueous systems. An advantage of hydrophobic drugs is their preferential binding to lipoproteins as tumor cells express a much larger number of receptors for low density lipoproteins (LDL) than do most normal cells.160 These receptors specifically recognize L D L and promote their internalization by cells via the formation of coated pits. Photosensitizers that bind to L D L are endocytosed by the neoplastic cells along with the lipoprotein.161 Once inside the cell, the photosensitizer is released into the cytoplasm and binds to apolar endocellular matrices such as mitochondria, lysosomes and plasma membranes. A photosensitizer will be most effective if it 66 Chapter 2 Results displays an affinity for tumor cells versus normal cells because low cytotoxicity of such a drug can be overcome by increasing the dose. 2.1.2 Background In the early 1980s, ortho-, meta- and para-isomers of meso-tetraChydroxyphenyOporphyrin were investigated for use as photosensitizers as mentioned in Section 1.2.4.4.162 In order to increase the absorption in the red region, the analogous chlorins and the meto-hydroxy substituted bacteriochlorin were synthesized.163 The stepwise increase in the reduction of the porphyrin skeleton led to increased absorption in the red region. Band I in methanol for meso-totra(meta-hydroxyphenyl)porphyrin (mTHPP) (61) was at 646 nm (e = 3300), meso-tetra(meta-hydroxyphenyl)chlorin (mTHPC) (62) was at 650 nm (e = 22 400) whilst that for meso-tetva(meta-hydroxyphenyl)bacteriochlorin (mTHPB) (63) was at 735 nm (e = 91 000).163 In vivo testing showed that both phototoxicity (reflected by the decreased dose) and tissue penetration (reflected by the increased depth of necrosis) increased as did the level of reduction of the porphyrin.165 As discussed in Section 1.2.3.2, light penetration of tissue doubles between 630 and 750 nm. Table 2.1 Comparison of tumor photonecrosis for meta-hydroxy substituted analogs. Photosensitizer Band I Dose Depth of tumor A m a x (nm) (umol/kg) necrosis (mm) m-THPP(61) 646 6.25 4.6 m-THPC(62) 650 0.75 5.4 m-THPB(63) 735 0.39 5.1 67 Chapter 2 Results H H H H H H H •H H-H R R R m - T H P P (61) m - T H P C (62) O H m - T H P B (63) R = Figure 2.1 meso-Tetra(/«-hydroxyphenyl)porphyrin (61), chlorin (62), bacteriochlorin (63). The bacteriochlorin (63) was found to be relatively unstable despite attempts at stabilization with peripheral substituents and, therefore, testing of this compound was discontinued.166 Tetra(m-hydroxyphenyl)chlorin (62) was chosen as the most suitable for clinical trials and was found to be 25-30 times more effective than HpD (30) in destroying tumors as observed by in vivo bioassays with LD50 = 3 mg/kg.1 6 7 This chlorin (62) showed 90% tumor necrosis with only 10% recurrence, but side effects such as extended skin sensitivity, severe chest pains and loss of appetite were also observed. Our group has reported and patented the p,P'-dihydroxylation of meso-tetraphenylporphyrins and meso-tetraphenyl chlorins via osmium tetroxide mediated oxidation. 1 6 8 , 6 9 Oxidation of meso-tetraphenylporphyrin (10) or its metallated complex occurred in a solution of chloroform or methylene chloride with a stoichiometric amount of Os0 4 in the presence of pyridine. After stirring at room temperature for 5 days in the dark, reduction of the osmate complex with gaseous H 2S yielded the previously unknown 2,3-vz'c-dihydroxy-meso-tetraphenylchlorin (64) or its metallated analog in -50% yield with -40% starting material recovery. The reaction was reasoned to be much 68 Chapter 2 Results slower than the analogous osmium tetroxide mediated oxidation of OEP (8), which requires 2 days and yields 66% diol due to steric hindrance created by the bulky phenyl groups. The resultant mew-substituted vzc-diols displayed unexpected stability with dehydration and rearrangement occurring only under harsh conditions. Tet rapheny lporphyr in Tet rapheny ld ihydroxych lor in M = H p , Zn , Ni M = H 2 ( Z N N I (10) (64) Scheme 2.1 The osmium tetroxide mediated oxidation of tetraphenylporphyrins. As noted above, these diols display chlorin or metallochlorin optical spectra and the spectra of the free base and the zinc-metallated diol chlorins are shown below. 1.01 o.o^ •  ; ^ - • • — — 400 500 600 700 wavelength (nm) Figure 2.2 The absorption spectra of the free base tetraphenyl-2,3-dihydroxychlorin (64). 69 Chapter 2 Results < 1.0" 0.54 0.0" A / i x 5 / | / \ / i ! \ 400 500 600 wavelength (nm) 700 Figure 2.3 The optical absorption spectra of the Zn complex of tetraphenyl-2,3-dihydroxy-chlorin. The meso-tetraphenylchlorins (65) and metallochlorins have also successfully been oxidized using this reaction, producing novel stable P,P'-cw-diol substituted meio-tetraphenyl-2,3-dihydroxy-12,13-dihydro-bacteriochlorins (66) and-isobacteriochlorins. H H H Ph \ Q W (65) (66) (64) Scheme 2.2 Formation of /neso-tetraphenyl-2,3-dihydroxy-12,13-dihydrobacteriochlorin(66). Conditions: i. 1.1 eq. Os04, pyridine, CHC13. H2S ( g ) ii. Reflux pyridine,K2C03, p-toluenesulfonylhydrazine Further dihydroxylation of the 2,3-vz'c-dihydroxy-meso-tetraphenylchlorin (64) with one equivalent of osmium tetroxide forms two isomers of 2,3,12,13-to-(Wc-dihydroxy)bacteriochlorins (67 and 68) in a 1:1 ratio, which are separable by chromatography. 70 Chapter 2 Results (64) (67) (68) Scheme 2.3 Osmium tetroxide oxidation of /Meso-tetraphenyl-2,3-dihydroxychlorin (64). Conditions: i. 1.1 eq. Os04 in 2.5% pyridine/CHCl3 As discussed in section 1.3.4, metallation of the chlorin drastically changes the outcome of the reaction. The osmylation of 2,3-dihydroxy-meso-tetraphenylchlorin (64) produces the two possible isomers of 2,3,12,13-tetrahydroxy-mes,o-tetraphenylbacteriochlorin (67 and 68) whilst the osmylation of the analogous zinc diol (69) forms the two possible isomers of 2,3,7,8-tetrahydroxy-meso-tetraphenylisobacteriochlorin (70) and (71). These four products can also be synthesized via treatment of their respective starting material porphyrins with two or more equivalents of osmium tetroxide. This procedure does produce lower yields of the compounds, but is efficient in terms of being a one-pot reaction. 71 Chapter 2 Results r n Ph Ph (69) (70) (71) Scheme 2.4 Formation of (/Meso-tetraphenyl-2,3,7,8-tetrahydroxyisobacteriochlorinato)zinc(II) (70) and (71). Reaction Conditions: i. 1 eq Os04in 2.5% pyridine/CHCl3 The osmium tetroxide reaction presents many potential benefits. It is a one-pot, two step synthesis with high yields and starting material recovery. Reduction of non-symmetric porphyrins generally results in the formation of all fours regioisomeric chlorins, but in this reaction only one isomer of the analogous dihydroxychlorin is formed and additional oxidation yields only two separable diastereoisomers of tetrahydroxybacteriochlorin. This allows for very high yields which is of critical economic importance when developing a drug. The use of tetraphenylporphyrins as starting materials has great advantages as these are the most accessible synthetic porphyrins. The number and nature of substituents on the phenyl groups can easily be varied and, therefore, the pharmacokinetics of potential pharmaceuticals can be adjusted to meet the requirements of different, specific physiological situations. Using this reaction, an entire library of compounds was created. As the primary requirement of a good photosensitizer for use in PDT is the absorption of light at long wavelengths, the resultant dihydroxychlorins and tetrahydroxybacteriochlorins were used as templates. These are known to absorb in the desired range (650-750 nm). The second requirement, which is of critical importance, 72 Chapter 2 Results is the selectivity of the drugs for tumor tissue over healthy cells. This was systematically addressed because the biodistribution of the drugs is drastically affected by their amphiphilicity. The rationale for the project was that an analysis of a series of related photosensitizers would lead to a better understanding of the factors involved in photosensitizing ability. By keeping the basic chromophore constant and varying the substituents on the phenyl rings, a pattern of structure versus cytotoxicity was expected to emerge. 73 Chapter 2 Results 2.2 Results 2.2.1 Porphyrins A wide variety of tetraphenylporphyrins were synthesized for use as starting materials for the osmium tetroxide-mediated oxidation reaction. Tetraphenylporphyrins were synthesized via the condensation pyrrole with aromatic aldehydes. Refluxing a solution of the appropriately substituted benzaldehyde with pyrrole in propionic acid, Adler's method, produced the desired porphyrin in yields of 5 - 40%.170 In certain cases, Lindsey's method was used.171 This method involved the formation of the porphyrinogen under acidic conditions followed by oxidation to produce the desired porphyrin. mew-Tetraphenylporphyrins synthesized are listed below: Positions R 2 and R 6 are ortho, and can be represented by o and o' respectively. Similarly, R 3 and R 5 are meta (m and m') whilst R 4 is the para position and can be represented by p. Table 2.2 Tetraphenylporphyrins synthesized. C o m p o u n d N u m b e r R 2 R 3 R 4 R 5 R 6 T(p-N0 2 )PP 72 H H N0 2 H H T(m-N0 2 )PP 73 H N0 2 H H H T(o-N0 2 )PP 74 N02 H H H H T(p-Br)PP 75 H H Br H H T(m-Br)PP 76 H Br H H H Chapter 2 Results Compound Number R 2 R 3 R 4 R 5 R 6 T(p-F)PP 77 H H F H H T(m-F)PP 78 H F H H H T(o-F)PP 79 F H H H H T F 5 P P 80 F F F F F T(o,o'-CI)PP 81 Cl H H H Cl T(p-OH)PP 82 H H OH H H T(m-OH)PP 83 H OH H H H T(m,m' -OH)PP 84 H OH H OH H T ( p - C 0 2 M e ) P P 85 H H C 0 2 M e H H T ( p - C 0 2 H ) P P 86 H H C 0 2 H H H T(p-OCOEt)PP 87 H H OCOEt H H T(p -OCH 3 )PP 88 H H O C H 3 H H T (m-OCH 3 )PP 89 H O C H 3 H H H T (m,m ' -OCH 3 )PP 90 H O C H 3 H O C H 3 H T(m-OCH 3 , p -OH)PP 91 H O C H 3 OH H H T(m,p ,m ' -OCH 3 )PP 92 H O C H 3 O C H 3 O C H 3 H T(o ,m,p-OCH 3 )PP 93 O C H 3 O C H 3 O C H 3 H H T(o ,m,m' -OCH 3 )PP 94 O C H 3 O C H 3 H O C H 3 H T(o,p ,o ' -OCH 3 )PP 95 O C H 3 H O C H 3 H O C H 3 T(m,m' -OCH 3 ,p -OH)PP 96 H O C H 3 OH O C H 3 H T(p-CH 3 )PP 97 H H C H 3 H H T(o,p,o ' -CH 3 )PP 98 C H 3 H C H 3 H C H 3 T(p-NH 2 )PP 99 H H N H 2 H H T ( p - S 0 3 H ) P P 100 H H S 0 3 H H H Tp-CN)PP 101 H H CN H H T(p-t -Bu)PP 102 H H t-Bu H H 75 Chapter 2 Results 2.2.2 Chlorins The tetraphenylporphyrins listed were used as starting materials for the osmium tetroxide oxidation. In a typical experiment, 1.1 equivalents of osmium tetroxide dissolved in pyridine are added to a chloroform solution of the tetraphenylporphyrin. The mixture is stirred at room temperature, protected from direct light, for 2 to 96 hours. Upon completion, hydrogen sulfide gas is passed through the solution and the diol purified by chromatography. Table 2.3 Dihydroxychlorins synthesized. C o m p o u n d N u m b e r R 2 R 3 R 4 R 5 R 6 H 2 T P C ( O H ) 2 103 H H H H H T ( m - N 0 2 ) P C ( O H ) 2 104 H N 0 2 H H H T(p-Br )PC(OH) 2 105 H H Br H H T(m-Br )PC(OH) 2 106 H Br H H H T(m-F)PC(OH) 2 107 H F H H H T(o-F)PC(OH) 2 108 F H H H H T F 5 P C ( O H ) 2 109 F F F F F T (p -OH)PC(OH) 2 110 H H OH H H T (m-OH)PC(OH) 2 111 H OH H H H 76 Chapter 2 Results C o m p o u n d N u m b e r R 2 R 3 R 4 R 5 R 6 T ( p - C 0 2 M e ) P C ( O H ) 2 1 1 2 H H C 0 2 M e H H T(p -OCOEt )PC(OH) 2 1 1 3 H H OCOEt H H T (p -OCH 3 )PC(OH) 2 1 1 4 H H O C H 3 H H T ( m - O C H 3 ) P C ( O H ) 2 1 1 5 H O C H 3 H H H T (m,m ' -OCH 3 )PC(OH) 2 1 1 6 H O C H 3 H O C H 3 H T(m-OCH 3 , p -OH)PC(OH) 2 1 1 7 H O C H 3 OH H H T(m,p ,m ' -OCH 3 )PC(OH) 2 1 1 8 H O C H 3 O C H 3 O C H 3 H T(o ,m,m ' -OCH 3 )PC(OH) 2 1 1 9 O C H 3 O C H 3 H O C H 3 H T(o ,p ,o ' -OCH 3 )PC(OH) 2 1 2 0 O C H 3 H O C H 3 H O C H 3 T(p -CH 3 )PC(OH) 2 1 2 1 H H C H 3 H H T(o ,p ,o ' -CH 3 )PC(OH) 2 1 2 2 C H 3 H C H 3 H C H 3 T ( p - S 0 3 H ) P C ( O H ) 2 1 2 3 H H S 0 3 H H H T(p- t -Bu)PC(OH) 2 1 2 4 H H t-Bu H H Additionally, diphenylporphyrin was oxidized with osmium tetroxide to produce the analogous diphenyl-2,3-dihydroxy-2,3-chlorin (H2DPC(OH)2) in the same manner as the tetraphenylporphyrins with the exception that this reaction only requires 3 hours, presumably due to the lack of steric hindrance as compared to that observed in the tetraphenylporphyrin reaction. ( 1 2 5 ) Figure 2.4. Diphenyl-2,3-dihydroxychlorin (H 2 DPC(OH) 2 ) (125). 77 Chapter 2 Results The UV-visible spectra of the diols are typical for chlorins with A m a x (log e) 408 (5.27), 518 (4.19), 548 (4.19), 592 (3.85), 644 (4.38) nm in methylene chloride. Typical chlorin 'H-NMR spectra were obtained for the diol chlorins. 9:-;0 1 " " 8 7 , 0 s'.B S,0 4*.0- • 3^0 • P P M . . ; Figure 2.5. Example of 'H-NMR spectrum of a metallated dihydroxychlorin (M = Zn). 78 Chapter 2 Results Additionally, mew-5-(p-te^ (mono /?-Br TPC(OH)2), mew-5-(/?-hydroxyphenyl)-10,15,20-triphenyl-2,3-dihydroxychlorin (mono /?-OH TPC(OH)2) (127) andmew-5-(/?-nitrophenyl)-10,15,20-triphenyl-2,3-dihydroxychlorin (mono/?-NO, TPC(OH)2) (128) were synthesized. Figure 2.6. /neso-5-(/j-Substituted phenyl)-10,15,20-triphenyl-2,3-dihydroxychlorins (mono p-R TPC(OH)2). 2.2.3 Bacteriochlorins Although the previously mentioned tetrahydrobacteriochlorin (63) was found to be unstable in vivo and although two isomers are formed in the synthesis, we felt it would be interesting to synthesize and test the cytotoxicity of one isomer of the tetraphenyl-2,3,12,13-tetrahydroxybacterio-chlorin (68). Table 2.4 Tetraphenyl-2,3,12,13-tetrahydroxybacteriochlorins synthesized. C o m p o u n d N u m b e r R 2 R 3 R 4 R 5 R 6 H 2TPB(OH) 4 129 H H H H H T(o,p,o'-OCH 3)PB(OH) 4 130 OCH 3 H O C H 3 H O C H 3 79 Chapter 2 Results Figure 2.7 ffi^so-Tetraphenyl-2,3,12,13-tetrahydroxybacteriochlorin (68). In the exper imental section, the condi t ions o f the reactions are specif ied a long wi th yields. It must be noted that the yields o f the d io l chlor ins and the tetraol bacter iochlorins are extremely dependent upon the amount o f o s m i u m tetroxide used and the length o f t ime the reaction was a l lowed to proceed. In many cases, the reaction was on ly a l l o wed to proceed unt i l the ratio of d io l ch lo r in to starting material was at a subjective m a x i m u m in order to obtain the m a x i m u m y ie ld o f the d i o l ch lo r in versus tetraol bacter iochlor in for b io log ica l testing. 80 Chapter 2 Results 2.2.4 Preliminary Biological Test Results Compounds were synthesized at U.B.C. and tested at QLT as part of an ongoing collaboration. All compounds were dissolved in DMSO with the exception of T(/?-S03H)PC(OH)2 (123) which was dissolved in water. The solubility of the compounds was tested by placing the drug (1 mg) in 1 mL of DMSO and then spinning at 10000 rpm for 10 minutes with checking for pellet formation. The concentration of the compounds that formed a pellet was decreased from 1 mg/mL to 0.5 mg/mL DMSO and retested to show an absence of pellet. Phototoxicity was determined towards L1210 cells in the presence of the compound as follows: the L1210 cells were exposed to varying concentrations of the compounds in 96-well microtiter plates for one hour at 37°C and 5% C0 2 . No fetal calf serum (FCS) was added at this time. The plate was then illuminated for one hour after which a 10% aqueous FCS solution was added to the wells. The plates were then returned to the C 0 2 incubator overnight. After incubation, the cells were assayed for viability using the MTT assay.172 Dark cytotoxicity is defined as the effect of the photosensitizer on the cells without light exposure. This dark cytotoxicity was determined while the plates were wrapped in aluminum foil while under the light source. Twenty four compounds which were tested for cytotoxicity are listed below in order of increasing LD50 (ng/mL) (i.e. in order of decreasing photosensitizer efficacy). 81 Chapter 2 Results Table 2.5 List of LD50 values for compounds tested in vitro in order of decreasing cytotoxicity. Compound LD50 (ng/mL) LD50dark (ng/mL) LD50 (uM) DPC(OH) 2 (125) 1.2 5000 2.4 x 10'3 mono p-OH T P C ( O H ) 2 (127) 14 9000 2.1 x 10"2 T(m-OH)PC(OH) 2 (111) 15 12500 2.1 x 10"2 T(m,p ,m ' -OCH 3 )PB(OH) 4 (130) 18 18000 1.7 x 10"2 T(m,p ,m ' -OCH 3 )PC(OH) 2 (118) 20 20000 2.0 x 10"2 T(m-OCH 3 , p -OH)PC(OH) 2 (117) 30 4500 3.6 x 10'2 T ( p - C 0 2 M e ) P C ( O H ) 2 (112) 120 15000 1.4 x 10"1 H 2 T P B ( O H ) 4 (114) 700 7500 1.0 T ( m - O C H 3 ) P C ( O H ) 2 (115) 1000 20000 1.3 T ( p - S 0 3 H ) P C ( O H ) 2 (123) 1000 » 2 0 0 0 0 1.1 H 2 T P C ( O H ) 2 (103) 1800 19000 2.8 T F 5 P C ( O H ) 2 (109) 1800 12000 1.8 mono p - N 0 2 T P C ( O H ) 2 (128) 2000 18000 2.4 T (m,m ' -OCH 3 )PC(OH) 2 (116) 2000 12000 2.3 T(o-F)PC(OH) 2 (108) 3000 15000 4.2 T (p -OH)PC(OH) 2 (110) 3500 15000 4.9 T(m-Br )PC(OH) 2 (106) 4000 4000 4.2 T ( m - N 0 2 ) P C ( O H ) 2 (104) 4500 15000 5.4 T (m-F)PC(OH) 2 (107) 5000 6000 6.9 T (p -OCH 3 )PC(OH) 2 (114) 6000 >20000 7.8 T(p-Br )PC(OH) 2 (105) 6000 8000 6.2 T(p- t -Bu)PC(OH) 2 (124) 6000 7000 6.9 T (p -CH 3 )PC(OH) 2 (121) 7000 15000 9.9 T(o ,p ,o ' -CH 3 )PC(OH) 2 (122) 10000 18000 12 82 Chapter 2 Results Concentrat ion (ng/mL) Figure 2.8 Cytotoxicity of the compounds with the lowest LD50 values in L1210 cells. No trends were apparent to us from this ordered list and therefore we chose to look more closely at the compounds which possessed the same or similar substituents. 83 Chapter 2 Results Concent ra t ion (ng/mL) Log Scale Figure 2.9 Cytotoxicity of the hydroxy-substituted compounds to L1210 cells. As a great deal of information has been gathered concerning tetra(hydroxyphenyl)-2,3-dihydrochlorins and their photosensitizing ability, the first group of compounds we chose to analyze was the hydroxy-substituted tetraphenyl diol chlorins as the forementioned are closely related to these compounds. Several observations can be made based on this series. First, tetra(m-hydroxyphenyl)-2,3-dihydroxychlorin (111) appears to be much a more effective photosensitizer than tetra(p-hydroxyphenyl)-2,3-dihydroxychlorin (110) with LD50s of 15 and 6000 ng/mL respectively. This is not unexpected as tetra(/?-hydroxyphenyl)-2,3-dihydrochlorin has been reported to be 5-6 times as potent as HpD in sensitizing tumors, whereas tetra(m-hydroxyphenyl)-2,3-dihydrochlorin (122) 84 Chapter 2 Results was 25-30 times as potent.167 Interestingly, the ortho-hydwxy isomer was reported to be 12-16 times as potent as HpD but displayed unacceptably high levels of skin sensitization in clinical trials.167 We have excluded this isomer from our study due to the fact that ortho isomers are a mixture of atropisomers caused by the hindered rotation about the phenyl-porphyrin bond. Not only is the tetra(p-hydroxyphenyl)dihydroxychlorin (110) less cytotoxic than the meta-isomer (111) but it is also less cytotoxic than the completely unsubstituted tetraphenyl-2,3-dihydroxychlorin (103) which has an LD50 of 1800 ng/mL, indicating that a hydroxyl group in the para-position is detrimental. Also of interest is the fact that the mono para-hydroxy (127), that is the diol of 5-meto-hydroxyphenyl-10,15,20-triphenylporphyrin (70), is equally as potent as T(m-OH)PC(OH) 2 (111) with an LD50 of 14 ng/mL. We also observed that T(m-OMe, p-OH)PC(OH) 2 (117) is much more cytotoxic than T(p-OH)PC(OH)2(110). Many structure-activity studies have been performed on hydroxy-substituted tetraphenylporphyrins. In 1994, Peng et al. reported testing a series of mono- and di-hydroxy substituted tetraphenylporphyrins for toxicity in HeLa cells and human melanoma lines.173 The mono-hydroxy porphyrins were all found to be toxic in the absence of light in the 1-10 uM range. Cytotoxicity increased up to 100-fold in the presence of light. The dihydroxy poiphyrins were found to be an order of magnitude less cytotoxic than the mono series. o,m-, o,p-, o,m'-, and m,p-Dihydroxy-substituted tetraphenylporphyrins were studied. Cytotoxicity of the compounds to HeLa, MM96E and MM418 cells was determined and found to decrease in the following order: T(m-OH)PP > T(o-OH)PP > T(/?-OH)PP > T(o,/?-OH)PP > T(m,/?-OH)PP > T(m,m'-OH)PP > T(o,m-OH)PP > T(o,m'-OH)PP The o,m'-isomer was found to be the least toxic and exhibited poor response to light. The difference in fates of the generated singlet oxygen was proposed to be the cause of the difference in cytotoxicity 85 Chapter 2 Results between the mono and dihydroxy series. As singlet oxygen reacts with the nearest substrate due to its short lifetime and diffusion distance, it was proposed that singlet oxygen generated by a dihydroxy TPP would react with the hydroxyl groups of the porphyrin itself. This was based on the previously observed oxidation of catechol and its derivatives into o-semiquinones by singlet oxygen. In keeping with this theory, the difference in cytotoxicity between the dihydroxy isomers was proposed to be due to differences in the accessibility of the hydroxyl groups for oxidation by the singlet oxygen due to the geometry of the hydroxyl groups and the extent of aggregation of the compounds. The o,m '-dihydroxy TPP, the least toxic of the dihydroxy series, is different from the others in that it is the only isomer which has four hydroxyl moieties on each side of the porphyrin plane in all atropisomers. This compound can be oxidized by singlet oxygen to the stable para-quinone which has been proven not to fluoresce. The o,m- and m,/?-dihydroxy tetraphenylporphyrins can also form quinones, but these are the less stable orf/zo-quinones. It was noted that the m,p-dihydroxy TPP was found to be one of the most cytotoxic of the dihydroxy compounds which somewhat discredits the proposed quinone-formation theory. Scheme 2.5 Proposed formation of a para-quinone from oxidation of o,/n'-dihydroxyphenyl group of T(o,m'-OH)PP. An observed increase in solubility of the mono hydroxy-substituted TPPs in 5% FCS led to a study of the absorption spectra in the presence and absence of F C S . 1 7 3 In the presence of serum, the monohydroxylated TPPs displayed an extremely broadened Soret band whereas the 86 Chapter 2 Results dihydroxylated TPPs displayed variable behavior. Soret broadening was observed to decrease from T(m-OH)PP > Tf>OH)PP > T(o-OH)PP > T(ra,/?-OH)PP > T(m,m'-OH)PP > T(o,m-OH)PP > T(o,/?-OH)PP > T(o,m'-OH)PP This series follows closely with that of the observed cytotoxicity with increased broadening correlating to increased toxicity. The broadening of Soret bands is known to be caused by extensive porphyrin aggregation possibly involving protein binding as opposed to simple face-to-face dimerization or of specific protein-porphyrin aggregation. The number and placement of hydroxyl groups on the phenyl rings therefore affects the binding of the porphyrin to proteins or other porphyrins. One study has focused on the photophysical properties of tetra(hydroxyphenyl)porphyrin and found that all three isomers had similar fluorescence yields (~0.1) and fluorescence lifetimes (~ 10 ns), high triplet yields (~ 0.7) and singlet oxygen quantum yields (~ 0.6).174 This investigation concluded that the differences in phototoxicity previously observed in these compounds are due to factors other than their photophysical properties. 87 Chapter 2 Results Concentrat ion (ng/mL) Log Scale Figure 2.10 Cytotoxicity of the methoxy-substituted compounds in L1210 cells. The methoxy-substituted series presented some very interesting data. Again, we observed that the meta isomer (115) is much more cytotoxic than the para isomer (114) with LD50s of 1000 ng/mL and 6000 ng/mL respectively and that the para isomer (114) is much less potent than the unsubstituted diol chlorin (103). The m,/?,m'-trimethoxy diol chlorin (118) is a very cytotoxic compound with LD50 = 20 ng/mL. Interestingly, the m,m'-dimethoxy analog (116) with LD50 = 2000 ng/mL is much less potent than the m,p,m'-trimethoxy diol chlorin (118) (LD50 = 20 ng/mL), the m-methoxy, /^-hydroxy diol chlorin (117) (LD50 = 30 ng/mL) and the m-methoxy diol chlorin (115). A methoxy group in the meta position of tetraphenylporphyrins has been reported to increase the rate constants of both singlet and triplet state quenching by molecular oxygen and increase the quantum yield of singlet oxygen formation.175 88 Chapter 2 Results Concentration (ng/mL) Log Scale Figure 2.11 Cytotoxicity of the hydroxy and methoxy substituted compounds in L1210 cells. Comparison of the methoxy versus the hydroxy substituted tetraphenyl-2,3-dihydroxychlorin series provided more information about the structure-activity relationship of these compounds. First, as already noted, the meta isomers of both the hydroxy and methoxy diol chlorins are more cytotoxic than the analogous para isomers which are both less toxic than the unsubstituted diol chlorin. Secondly, the hydroxy series is more cytotoxic than the methoxy series for all isomers investigated. Also interesting to note was the fact that T(m-OMe, p-OH)PC(OH)2 (117) was found to be much more cytotoxic than either T(ra-OMe)PC(OH)2 (115) or T(p-OH)PC(OH)2 (110). 89 Figure 2.12 Cytotoxicity of the alkylated compounds in L1210 cells. Investigation of the alkylated diol chlorins reveals that alkyl groups on the phenyl rings are detrimental to the observed cytotoxicity of the compounds as all have LD50s much lower than the fully unsubstituted diol chlorin. The para-tert-buty\ diol chlorin (124), having LD50 = 6000 ng/mL, is the most active of this series followed closely by para-methy\ diol chlorin (121), LD50 = 7000 ng/mL, and then by o,p,o'-trimethyl diol chlorin (122) with LD50 = 10000 ng/mL. 90 Chapter 2 Results 10 1000 Concentrat ion (ng/mL) Log Scale Figure 2.13 Cytotoxicity of the halogenated compounds in L1210 cells. In this series, we observed that all of the halogenated diol chlorins are less cytotoxic to L1210 cells than the unsubstituted TPC(OH) 2 (103). Within the fluorinated series, the ort/zo-substituted compound (108) is more toxic (LD50 = 3000 ng/mL) than the raeta-fluoro diol (107) (LD50 = 5000 ng/mL), but both are considerably less toxic than either H 2TPC(OH) 2 (103) or the pentafluorinated diol chlorin (109) which both have LD50 = 1800 ng/mL. Within the brominated series, the meta-isomer (106) is more cytotoxic (LD50 = 4000 ng/mL) than the para-isomer (105) (LD50 = 6000 ng/mL). Focusing solely on the halogenated diol chlorins, our findings are in agreement with previously published data from studies of halogenated tetraphenylporphyrins.176"179 In these studies, 91 Chapter 2 Results it was concluded that both the nature and the position of the halogen on the phenyl rings of the TPPs affect the photophysical properties and the singlet oxygen yield of the potential photosensitizer.176 The quantum yield of singlet oxygen formation by halogenated tetraphenylporphyrins is documented to have increased in the following order : T(p-F)PP (0.65) < T(p-Cl)PP < T(p-Br)PP < T(p-I)PP (0.97) The increase in quantum yield of singlet oxygen formation was rationalized by the strengthening of the spin-orbital interaction in the molecule, which is in agreement with the heavy atom perturbation theory. Classically, the generation of singlet oxygen is maximized by two methods: by increasing the quantum yield of the triplet state - which must be done without shortening its lifetime - and by invoking the internal heavy atom effect with halogens. Direct incorporation of halogens into a chromophore of predominantly T C , T C * character promotes intersystem crossing by increasing the level of spin-orbit coupling between reactant and product states.177 Additionally, this study showed that the position of the halogen atom affected the quantum yield of singlet oxygen such that: para (0.71) < meta (0.82) < ortho (0.84) for chlorinated TPPs. It was found that the rate constant of the triplet state quenching by molecular oxygen followed this order. This was proposed to be due to the increase in oxidation potential of the molecule and the decrease in the contribution of the donor-acceptor interactions in the quenching 1 78 process. Our systems appear to follow the trends noted in the studies of halogenated tetraphenylporphyrins, with the brominated diol chlorins being more cytotoxic than their fluorinated analogs and within one series, the orf/ioisomers being more toxic than the meta which, in turn, are more toxic than the /wa-isomers. Despite our observation of these trends, which have been 92 Chapter 2 Results attributed to differences in the quantum yield of singlet oxygen, all of our halogenated diol chlorins are less cytotoxic than the unsubstituted diol chlorin (103) (with the exception of the pentafluorochlorin (109)). This indicated that the quantum yield of singlet oxygen is not the critical factor determining cytotoxicity in our systems. H2TPB (129) H2TPC (103) H2DPC (125) Concentrat ion (ng/mL) Log Scale Figure 2.14 Cytotoxicity of the diphenyl diol chlorin, the tetraphenyl diol chlorin and tetraol bacteriochlorin in L1210 cells. Comparison of these compounds reveals that the diphenyl-2,3-diolchlorin (125) is much more cytotoxic than either the tetraphenyl-2,3-diolchlorin (103) or the tetraphenyl-2,3,12,13-tetraolbacteriochlorin (129) with LD50 values of 1.2 ng/mL, 1800 ng/mL and 700 ng/mL respectively. As there is a large difference in the molecular mass of these compounds, conversion to units of uM (2.4 x 10"3 pM, 2.8 p,M and 1.0 p.M respectively), revealed that, in any terms, the 93 Chapter 2 Results diphenyl diol chlorin (125) (H2DPC) is remarkably active. As observed in Bonnett's study, the reduction of a second bond of the porphyrin skeleton results in increased cytotoxicity as observed by the increase in toxicity in H 2 TPB (129), the bacteriochlorin, as compared to H 2 TPC (103).167 T(m,p,m'-Ome)PC (118) T(m,p,m'-Ome)PB (130) H2TPC (103) H2TPB (129) 10 1000 Concentration (ng/mL) Log Scale Figure 2.15 Cytotoxicity of the trimethoxy diol chlorin and tetraol bacteriochlorin versus those of the unsubstituted diol chlorin and tetraol bacteriochlorin in L1210 cells. Similar results are observed in the comparison of the cytotoxicity of the tetraphenyl-2,3-dihydroxychlorin versus tetraphenyl-2,3,12,13-tetrahydroxybacteriochlorin for both the unsubstituted ((103) and (129)) and the ra,/?,m'-trirnethoxy compounds ((118) and (130)). In both cases, the bacteriochlorin is more active. The m,p,m'-trimethoxy diol chlorin (118) was found to have LD50 94 Chapter 2 Results = 20 ng/mL or 2 x 10"2 pM and the analogously substituted tetraol bacteriochlorin (130) had LD50 = 18 ng/mL or 1.7 x 10"2 u,M. As previously discussed the LD50 for the unsubstituted diol chlorin (103) was found to be 1800 ng/mL (2.8 pM) and that for the tetraol (129) was 700 ng/mL (1.0 pM). 30 20 10 0 . 1 0 1 2 3 4 5 6 7 8 9 10 Concent ra t ion (ng/mL) Log Scale Figure 2.16 Cytotoxicity of diphenyl diol chlorin in L1210 cells. The cytotoxicity of the diphenyl diol chlorin (125) was extremely high, and therefore, this and other promising compounds were further tested to investigate the phototoxicity in the presence and absence of fetal calf serum. For this, cells in both the presence and absence of FCS (10% solution) were exposed to the compounds for one hour at 37°C and 5% CO,. The compounds and 95 Chapter 2 Results FCS were then removed by washing the cells in D M E , and the cells distributed into plates for light exposure with FCS provided for overnight incubation. Cell viability was assessed the following day using the M T T assay.172 The first test of cytotoxicity of the diphenyl diol chlorin (125) was performed on a scale similar to other compounds which began at 10 ng/mL. At this concentration, over 90% of the cells were killed which prompted further testing at lower concentrations. The second test (DPCremoved, DPCremoved2, and DPCmeanrem) involved fetal calf serum. Here, "DPCremoved2" is the observed cytotoxicity data for H 2DPC(OH) 2 (125) in the absence of FCS, with the compound removed from the cells, where LD50 = 1.2 ng/mL and "DPCremoved" is that in the presence of FCS, compound removed, where LD50 = 1.5 ng/mL. The latter value is very unusual because LD50s usually increase by approximately factor of 10 when FCS is added. This phenomenon has been attributed to the competition for compound between the cells and the serum, thereby increasing the apparent LD50 value as the concentration of compound actually absorbed by the cells is decreased. 96 Chapter 2 Results Concentration (ng/mL) Log Scale Figure 2.17 Cytotoxicity of the most promising compounds both in the presence and absence of fetal calf serum (FCS) in L1210 cells. As previously discussed, the most promising compounds were investigated further. Testing for cytotoxicity both in the presence and absence of FCS gave the expected results - that is the LD50 of each compound, with the exception of H 2DPC(OH) 2 (125), increased by a factor of at least 10 in the presence of FCS. The LD50s for m,/?,m'-trimethoxy tetraol bacteriochlorin (130) were observed to be 180 ng/mL (1.7 x 10"1 uM) in the presence of FCS, and 18 ng/mL (1.7 x 10"2 uM) in its absence whilst the analogous diol chlorin (120) had LD50 = 250 ng/mL (2.5 x 10"' uM) with FCS added and in its absence, LD50 = 20 ng/mL (2.0 x 10"2 uM). Tetra(/?-carbomethoxyphenyl)-2,3-dihydroxychlorin (112) was found to have LD50 = 750 ng/mL (8.5 x 10"1 uM) in the presence of FCS and 70 ng/mL (8 x 10'2 uM) without FCS. 97 Chapter 2 Results Concentration (ng/mL) Log Scale Figure 2.18 Cytotoxicity of variously substituted diol chlorins in L1210 cells. The remaining compounds were analyzed as a series. 5-(/?-nitrophenyl)-10,15,20-triphenyl-2,3-dihydroxychlorin (128) had an observed LD50 of 2000 ng/mL (2.4 pM), which is more cytotoxic than tetra(m-nitrophenyl)-2,3-dihydroxychlorin (104) (LD50 = 4500 ng/mL or 5.4 u,M). Both were less toxic than H 2TPC(OH) 2 (103). This is in keeping with the mono p-OK (127) versus tetra(/?-hydroxyphenyl)diol chlorin (110) observation. Investigation into the effect of the introduction of a nitro group into the meso position of tetraphenylporphyrins concluded that intramolecular charge-transfer levels appear thereby providing another method of fluorescence quenching.176 The competition between fluorescence quenching and population of the triplet state decreases the probability of intersystem crossing and reduces the singlet oxygen quantum yields. 98 Chapter 2 Results We believed that T0-SO 3H)PC(OH) 2 (123) would be an interesting if not effective photosensitizer because it is water soluble and the analogous tetraphenylporphyrin (100) has been shown to be effective. This study led to LD50 for this compound of 1000 ng/mL (1.1 pM) in water. It must be noted that this compound was dissolved in water as opposed to DMSO used for the other compounds. Water soluble compounds such as this are largely transported by albumin. These compounds are known to localize within the interstitial space and the vascular stroma of tumor tissues.180 Hydrophobic compounds damage tumor cells by direct interactions whereas water soluble compounds act indirectly by damaging blood vessels and disrupting the supply of oxygen and other nutrients. The observed LD50 value of 1000 ng/mL may reflect this difference in solvent and/or cell localization. Tetra(/?-carbomethoxyphenyl)-2,3-dihydroxychlorin (112) was found to be quite an efficient photosensitizer with LD50 = 70 ng/mL (8 x 10"2 pM). 99 Chapter 2 Results Table 2.6 List of compounds in order of decreasing dark toxicity in L1210 cells. Compound LD50dark LD20dark LD50 (ng/mL) (ng/mL) (ng/mL) T(p-S0 3H)PC(OH) 2 (123) » 2 0 0 0 0 >20000 1000 T(p-OMe)PC(OH)2 (114) >20000 9000 6000 T(m-OMe)PC(OH)2 (113) 20000 8000 1000 T(m,p,m'-OMe)PC(OH)2 (118) 20000 7500 20 H 2TPC(OH) 2 (103) 19000 7000 1800 T(o,p,o'-Me)PC(OH)2 (122) 18000 7500 10000 mono p-N0 2 (128) 18000 2000 2000 T(m,p,m'-OMe)PB(OH)4 (130) 18000 1200 18 T(o-F)PC(OH)2 (108) 15000 5000 3000 T(p-OH)PC(OH)2 (110) 15000 5000 3500 T(p-Me)PC(OH)2 (121) 15000 5000 7000 T(m-N0 2)PC(OH) 2 (104) 15000 4500 4500 T(p-C0 2Me)PC(OH) 2 (112) 15000 3000 120 T(m-OH)PC(OH)2 (111) 12500 10000 15 TF 5PC(OH) 2 (109) 12000 5000 100 T(m,m'-OMe)PC(OH)2 (116) 12000 5000 2000 mono p-OH (127) 9000 2000 14 T(p-Br)PC(OH)2 (105) 8000 1900 6000 H 2TPB(OH) 4 (129) 7500 2500 700 T(p-t-Bu)PC(OH)2 (124) 7000 2000 6000 T(m-F)PC(OH)2 (107) 6000 1500 5000 H 2DPC(OH) 2 (125) 5000 1500 1.2 T(m-OMe,p-OH)PC(OH)2 (117) 4500 500 30 T(m-Br)PC(OH)2 (106) 4000 1500 4000 Dark toxicity refers to the toxicity of the drug to cells in the absence of light. This toxicity is, therefore, not due to singlet oxygen mediated cellular damage. Very little investigation into the mechanism or mechanisms of dark toxicity has been performed. We propose that dark toxicity might be due to the incorporation of the drug into the cell or its localization to a critical area on the cell surface, thereby hampering regular cell functions. The values might also reflect the tendency 100 Chapter 2 Results towards aggregation with those most aggregated not reaching the cells at all. These LD50 values would then reflect the drug's localization ability but no real conclusions can be drawn. While it is critical that potential photosensitizing drugs have high LD50 values in the dark so that photosensitivity after treatment is minimal, our results in this area merely serve to show that all of the compounds tested have acceptably low dark toxicity levels. 2.3 Discussion Table 2.7 List of LD50 values in order of decreasing cytotoxicity in L1210 cells. Compound Rank LD50 Molecular LD50 Ran (pM) Weight (ng/mL) (g/mol) DPC(OH) 2 (125) 1 2.4 x 10"3 496 1.2 1 T(m,p,m'-OCH3)PB(OH)4 (130) 2 1.7 x 10'2 1042 18 4 T(m,p,m'-OCH3)PC(OH)2 (118) 3 2 x 10"2 1008 20 5 mono p-OH TPC(OH) 2 (127) 4 2.1 x 10'2 664 14 2 T(m-OH)PC(OH)2 (111) 4 2.1 x 10"2 712 15 3 BPDMA (standard) 2.6 x 10'2 718 19 T(m-OCH 3,p-OH)PC(OH) 2 (117) 5 3.6 x 10"2 832 30 6 T(p-C02Me)PC(OH)2 (112) 6 1.4 x 10"' 880 120 7 H 2TPB(OH) 4 (129) 7 1.0 682 700 8 T(p-S03H)PC(OH)2 (123) 8 1.1 904 1000 9 T(m-OCH 3)PC(OH) 2 (115) 9 1.3 768 1000 9 TF 5PC(OH) 2 (109) 10 1.8 1008 1800 10 T(m,m'-OCH 3)PC(OH) 2 (116) 11 2.3 888 2000 11 mono p-N02 TPC(OH), (128) 12 2.4 828 2000 11 H 2TPC(OH) 2 (103) 13 2.8 648 1800 10 T(m-Br)PC(OH)2 (106) 14 4.1 964 4000 14 101 Chapter 2 Results Compound Rank LD50 Molecular LD50 Ran (pM) Weight (ng/mL) (g/mol) T(o-F)PC(OH)2 (108) 15 4.2 720 3000 12 T(p-OH)PC(OH)2 (110) 16 4.9 712 3500 13 T(m-N0 2)PC(OH) 2 (104) 17 5.4 828 4500 15 T(p-Br)PC(OH)2 (105) 18 6.2 964 6000 17 T(m-F)PC(OH)2 (107) 19 6.9 720 5000 16 T(p-t-Bu)PC(OH)2 (124) 20 6.9 872 6000 17 T(p-OCH 3)PC(OH) 2 (114) 21 7.8 768 6000 17 T(p-CH3)PC(OH)2 (121) 22 9.9 704 7000 18 T(o,p,o'-CH3)PC(OH)2 (122) 23 12 816 10000 19 It is standard practice to report LD50 values in terms of ng/mL but we felt that as our compounds span a wide range of molecular weights, LD50 values would be more accurate when presented in units of pM. Although overall the differences in order were minimal, certain compounds, such as the trimethoxy substituted compounds (118) and (130), were found to be more cytotoxic than the LD50 values presented in units of ng/mL would have led us to believe. The LD50 values in terms of \xM also allowed us to compare the cytotoxicity of our compounds with that of other second and third generation photosensitizers. BPDMA (34) was discussed in section 1.2.4.1. This compound has an LD50 value of 19 ng/mL, with a molecular mass of 718 g/mol, and therefore the LD50 is 2.6 x 10"2 pM which is 70 times more potent than HpD (30) and 45 times more potent than Photofrin in sensitizing tumors.181 With this in mind, we feel that some of our compounds are extremely good candidates for further testing. Five of our compounds 102 Chapter 2 Results are more cytotoxic than BPDMA (34) based on the observed LD50 values. The most cytotoxic compound, diphenyl diol chlorin (125) is extremely cytotoxic: 10 times more potent than BPDMA (34), 450 times more potent than Photofrin and 700 times more potent than HpD (30). The singlet oxygen quantum yield, as exemplified by the halogenated diol chlorin series, appears to have only a minor influence on the observed cytotoxicity of our compounds, and therefore the difference in toxicities could be reasoned to primarily reflect the cellular uptake of the drugs. A variety of molecular properties have been proposed to be responsible for cellular uptake such as hydrophobicity, amphiphilicity, self-aggregation and the ability to bind to serum protein. An increase in lipophilicity of a photosensitizer has been found to correlate with an increase in cellular uptake of the drug due to an increase in the degree of binding to L D L . 1 8 3 As well, a study investigating water-soluble tetraphenylporphyrins has found a very strong correlation between the hydrophobicity of a porphyrin (as measured by its distribution coefficient between octanol-1 and water) and its uptake in cultured cells.'84 No such correlation could be found for self aggregation or serum albumin binding with cellular uptake in this study. Amphiphilicity has been cited as a highly desirable property of photosensitizers because these have shown improved selectivity.'82 Biodistribution properties of tetrapyrrolic photosensitizers have been found to be influenced by amphiphilicity in the molecule. In particular, the selectivity for tumor tissue over healthy tissue and the rapid excretion of the photosensitizer have been attributed to amphiphilicity, reflected by the compound's water solubility and the partition coefficient between lipid and water phases. As previously discussed, self-aggregation is dependent upon the polarity and the structure of the photosensitizer and is significant in that aggregated molecules are not photoactive.'58159 The ability of the photosensitizer to bind to serum proteins is also significant and was thoroughly discussed in section 1.2. 103 Chapter 2 Results Without further investigation into these compounds, it was a difficult and daunting task to attribute causes for the observed data, but an attempt was made. Certain trends in the cytotoxicity of the diol chlorins can be attributed to each of the forementioned factors. The unsubstituted tetraphenyl diol chlorin (103) is not a remarkably cytotoxic compound, with LD50 = 2.8 pM and we can conclude that any increase or decrease in cytotoxicity is due to the presence of the substituents on the phenyl rings. We have observed that meta isomers are more cytotoxic than the analogous para isomers for all cases studied. This phenomenon has previously been observed.'67,173'178 In many cases in our study, a single substituent in all four of the para positions is actually detrimental to the observed cytotoxicity of the compound (eg. T(/?-OH)PC(OH)2 (110)). One study proposed that the optimum distance between the hydrophilic functions on the hydrophobic porphyrin skeleton is 12 A - the approximate distance between meta substituents on the phenyl rings of TPPs- based on the fact that tubulin, which might be a principal site for photoinactivation of cells, is present in appreciable amounts during mitosis and has binding sites 12 A apart.185 We have also presented the study which showed that, for hydroxy-substituted tetraphenylporphyrins, meta- substituted TPPs bind to proteins of FCS better than the /?ara-substituted TPPs and that this appears to be reflected by the greater cytotoxicity of the meta-substituted compounds.173 It has been suggested that hydrophilic groups in the para position allow self-aggregation via TC,TC stacking and that these molecules are held together by hydrophobic forces so that a hydrophilic surface is exposed to the environment. Hindered rotation about the phenyl-chlorin bond in chlorins with meta substituents would disfavor perpendicular phenyl groups, instead favoring a tilted phenyl ring. This would prevent the tight aggregation of these chlorin molecules, especially those with bulky substituents, and hence the monomers would be free to bind to the cell thereby greatly increasing the cell uptake of these drugs. In keeping with this theory, T(m,/?,m'-OMe)PC(OH)2 (118) and T(m,/?,m'-OMe)PC(OH)4 (130), 104 Chapter 2 Results which have three bulky methoxy groups on each phenyl group, were observed to be very cytotoxic (LD50 = 2 x 10"2 and 1.7 x 10"2 pM respectively). The alkylated diol chlorins all possess /?ara-alkyl substituents and were found to show very little cytotoxicity. This may be due to self-aggregation which would form a highly hydrophobic cluster. Based on our results, the degree of hydrophobicity and amphiphilicity also appear to be important factors in the cytotoxicity of our compounds. Whereas the porphyrin skeleton is essentially hydrophobic, the incorporation of the diol into the skeleton confers a degree of amphiphilicity to our compounds. The highly cytotoxic diphenyl diol chlorin (125) differs from tetraphenyl diol chlorin (103) in that it has two fewer phenyl groups. Phenyl groups are hydrophobic, and their removal alters the degree of hydrophobicity of the molecule and at the same time increases the amphiphilicity. Additionally, the loss of the phenyl group somewhat streamlines the molecule, perhaps improving its cellular uptake. We surmise that the increased toxicity of the 5-(p-nitrophenyl)-10,15,20-triphenyl diol chlorin (128) and the 5-(/?-hydroxyphenyl)-10,15,20-triphenyl diol chlorin (127) relative to their tetra-substituted analogs is due to the increased amphiphilicity and polarity that a single hydrophilic substituent would confer. Amphiphilicity may again be reasoned to be the cause for the improved cytotoxicity of T(ra-OMe, p-OH)PC(OH)2 (117) compared to T(m-OMe)PC(OH)2 (115) and T(>OH)PC(OH) 2 (110) as a number of atropisomers have polar hydroxyl groups around the perimeter of the molecule whilst the more hydrophobic methoxy moieties are all or mostly on one face of the planar chlorin. 105 Chapter 2 Results 2.4 Singlet Oxygen Testing In light of the encouraging results obtained with these compounds, we felt it necessary to confirm that our compounds were indeed singlet oxygen sensitizers. 1,3-Diphenylisobenzofuran (DPBF) has been used as a chemical quencher to determine the singlet oxygen quantum yields of various potential PDT agents.186 Monitoring the absorption decay of the absorption band at 415 nm -that of DPBF in D M F using UV-Visible spectrophotometry whilst in the presence of our compounds and whilst being irradiated with visible light confirmed the production of singlet oxygen. 2.5 Conclusions We believe that this study has been very useful. Not only has it determined which compounds were the most phototoxic, but has also allowed us to compare the effect of different substituents at various positions on the phenyl rings. We have also confirmed the increase in cytotoxicity on going from chlorins to bacteriochlorins in a given system. Much further study is necessary in order to confirm or deny our theories about the factors involved in the cytotoxicity of these compounds and the results are greatly anticipated. Section 2.6 Attempts to Improve the Osmium Tetroxide Reaction 2.6.1 Decreasing the Reaction Time Although this reaction is very useful and promising, there are two major drawbacks to its use for drug production on an industrial scale. First and most importantly, the osmium tetroxide reagent which is used on an equimolar scale is expensive ($50/g) and relatively toxic. Any measures to decrease the amount of osmium tetroxide required would greatly improve the chances that this reaction might be used on a large scale. Investigations into solving this disadvantage focused on 106 Chapter 2 Results possible catalytic systems and the recycling of the osmium tetroxide reagent. In order to make this reaction catalytic, the reaction time would need to be decreased. This 3-5 day reaction period is the second drawback of the osmium tetroxide oxidation of porphyrins. Efforts in this area were focused on both the reversible modification of the starting materials and also on the use of other substrates as starting materials. 2.6.1.1 ^-Alkylated TPPs Increasing the distortion of the porphyrin core is known to electronically activate the P,p' bond(s) of the molecule.187 Additionally, it is known that /V-alkylated porphyrins are highly distorted, with the most highly distorted porphyrins having the largest alkyl groups bound to one of the inner nitrogen atoms of the porphyrin core.188 We synthesized an ^/-alkylated porphyrin for use as a starting material in the osmium tetroxide oxidation reaction: ./V-methyl tetraphenylporphyrin (131). Previous studies had indicated that the osmium tetroxide mediated oxidation of Af-methyl TPP (131) appeared to be faster than unsubstituted TPP (10), and our investigations confirmed this. On average, TV-methyl TPP (131) required just 6-12 hours to afford the analogous diol chlorin. With this in mind, and knowing that /V-o-tolyl TPP is much more distorted from planarity than is ./V-methyl TPP (131), we synthesized N-phenyl TPP (132).189190 N-Phenyl TPP (132) is a known molecule and is relatively simple to synthesize via CoClTPP (133). Not only is this compound easily synthesized, but the iV-phenyl group is also conveniently removed with palladium(ll).'9! 107 We believed we could ^ -alkylate our various TPPs, perform the osmium tetroxide reaction in shorter reaction times - perhaps even catalytically - and then remove the phenyl group. To our extreme disappointment, no TV-phenyl diolchlorin was ever produced despite many attempts. We propose this lack of reaction to be due to the previously observed deactivation of the P carbons. At about the same time that we realized this lack of reactivity of /V-phenyl TPP (132), a paper was published detailing the lack of reactivity of this molecule to traditional reduction methods such as the classic tosylhydrazine.192,193 Callot et al. reported that, in their hands, this compound formed the analogous phlorin (136) when reacted with a variety of reagents. We did not see any phlorin product in our reaction, but these findings indicate a lack of reactivity at the P-positions and an increase in reactivity at the meso position. This was attributed to a resultant release in steric strain that occurs when the phlorin (136) is formed. These researchers did not perform these reactions with other ^ -alkylated TPPs and, due to time constraints, nor have we investigated any other ^ -alkylated TPPs such as iV-ethyl or /V-butyl for increased reactivity but the results of such investigations would, no doubt, be very interesting. 108 Chapter 2 Results 5,21-Dihydro-pentaphenylporphyr in ( 1 3 6 ) Scheme 2.7 Reaction of TV-phenylTPP (132) with tosylhydrazine. 2.6.1.2 Octaethyltetraphenylporphyrin We also investigated the osmium tetroxide oxidation with mew-tetraphenyl-2,3,7,8,12,13,17,18-octaethylporphyrin (137). This molecule is also known to be distorted from planarity due the steric hinderance between the eight P ethyl moities and the meso phenyl groups. Again, we observed no reaction except for one instance when a very polar single green band was isolated from a preparative T L C plate and was immediately sent for mass spectral analysis. The compound had the molecular mass of the analogous diol chlorin. No further characterization of this compound was obtained because it appeared that it had spontaneously returned to the starting material porphyrin. Again, at about the same time that we were investigating this compound, a 109 Chapter 2 Results report concerning the diimide reduction of nonplanar porphyrins was published. Senge et al. prepared a series of tetraphenylporphyrins with a varying number of (3 ethyl groups as shown below.194 These were found to increase in the degree of nonplanarity from left to right, with the most conformationally distorted being the octaethyltetraphenylporphyrin (137) that we have investigated. Figure 2.19 Tetraphenyl-based porphyrins used. The diimide reduction of the di- and tetra-substituted compounds (138), (139) and (140) proved fruitful although an increase in the degree of instability with respect to reoxidation was noted on progression from left to right for these three compounds (i.e. with increasing conformational distortion). Neither the hexaethyl (141) nor the octaethyl (137) compounds were successfully reduced. These results of the diimide study are in agreement with our lack of results. 110 Chapter 2 Results 2.6.1.3 Porphyrazine Another class of compounds that we thought would be interesting to subject to our osmium tetroxide oxidation are the porphyrazines. Porphyrazines are composed of four pyrrolic units which are fused with nitrogen bridges (as opposed to carbons in porphyrins). These have an absorption maximum of 600-750 nm depending on the P substituents and show potential as PDT agents.195 We chose to investigate 2,7,12,17-tetra-ter?-butyl-5,10,15,20-tetraazaporphine (142) as it is commercially available. Reaction with osmium tetroxide produced many compounds, only one of which was formed in isolable quantities. Characterization of this product determined that it was not the diol chlorin but perhaps its product of rearrangement and, therefore the identification of this product was set aside for future studies. Figure 2.20 Porphyrazine (142). 2.6.2 Other oxidation systems Potential oxidation systems other than osmium tetroxide were also investigated. None of the classic reagents such as potassium permanganate resulted in any products and only one system appeared promising at all. Fischer's classic reaction of octalkylporphyrin with acidic hydrogen peroxide to form the analogous P-oxochlorin was discussed in section 1.4.1. This reaction has been CMe. (142) CMe 3 Ill Chapter 2 Results performed on numerous systems, but to our knowledge it had not been performed with TPPs. We, therefore, performed this reaction with concentrated sulfuric acid and hydrogen peroxide. Only one product was obtained which was determined to be the expected (3-oxochlorin, resulting, presumably, from the proposed rearrangement of a diol chlorin. Although this product has been synthesized before, and by many different methods, we found this to be quite exciting because either a diol chlorin or an epoxide must be formed at some point in the reaction. We set upon altering the reaction conditions in order to isolate either the diol chlorin or the epoxide before the acid-catalyzed rearrangement occurred but to no avail. Nonetheless, this appears to be promising start to the search for alternative oxidation systems leading to tetraphenyl diol chlorins. P h P h (10) (143) Scheme 2.8 The hydrogen peroxide-acid oxidation of TPP (10). 2.7 Summary This chapter describes the synthesis of a number of variously substituted tetraphenylporphyrins and the subsequent formation of the analogous dihydroxy chlorins via the patented osmium tetroxide oxidation reaction. Additionally, a dihydroxy diphenylchlorin (125), two mono-substituted tetraphenylchlorins and two tetraphenyltetrahydroxybacteriochlorins were 112 Chapter 2 Results synthesized. These compounds were then tested in vitro for potential as photosensitizers for photodynamic therapy. The results of these tests were analyzed and compiled in an effort to identify the factors influencing the cytotoxicity of the drug. The unsubstituted, free base tetraphenyl dihydroxy chlorin (103) was not found to be very cytotoxic compared with some of the substituted tetraphenyl dihydroxy chlorins tested. Tetra(m,/?,m'-methoxyphenyl)dihydroxychlorin (118), 5-(p-hydroxyphenyl)-10,15,20-phenyldi-hydroxy chlorin (127) and tetra(m-hydroxyphenyl)dihydroxy chlorin (111) were the three most cytotoxic tetraphenyldihydroxychlorins. Diphenyl dihydroxy chlorin (125) was found to be an extremely effective photosensitizer in vitro, with an LD50 value of 1.2 ng/mL. This compound (125) is, therefore, 450 times more potent than the commercially available photosensitizer Photofrin. Compound (125) bears no substituents on the phenyl rings and any substituents on these phenyl would affect its cytotoxicity. Further investigation into the in vitro cytotoxicity of substituted diphenyl diol chlorins is currently underway. Several trends were observed in this study. Compounds substituted at the meta (m) position were consistently found to be more cytotoxic than the analogous compound substituted at the para ip) position. This trend might be attributed to the self-aggregation of para-substituted compounds which is hindered by the presence of substituents in the meta position. We believe that hydrophobicity, aggregation and amphiphilicity also affect the observed in vitro cytotoxicity of our compounds. Efforts to improve the osmium tetroxide reaction in terms of decreasing the reaction time to allow catalysis were investigated. The use of both N-methyl (131) and /V-phenyl TPPs (132) as well as octaethyltetraphenylporphyrin (137) as a starting materials was researched as these molecules are known to be distorted from planarity, and thereby activated towards certain reactions. The osmium 113 Chapter 2 Results tetroxide oxidation of TV-methyl TPP (131) was successful as the reaction required only 6-12 hours for completion. Neither A/-phenyl TPP (132) nor octaethyl TPP (137) reacted in the presence of osmium tetroxide despite several attempts under a variety of conditions. Finally, other oxidizing reagents were tested for use in the synthesis of the diol chlorins and/or the bisaldehyde secochlorin. None of the reagents tested proved to be successful. Chapter 3 Reactions of meso-Tetraphenyl-2,3-dialdehyde-2,3-secochlorin 115 Chapter 3 Results 3.1 Introduction 3.1.1 (meso-Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) As discussed in section 1.5.2, Bonnett et al. have reported the synthesis of octaethyl-2,3-secochlorin-2,3-dione (54) from the oxidation of the analogous dihydroxy octaethylchlorin (53).151 Scheme 3.1 Synthesis of octaethyl-2,3-secochlorin-2,3-dione (54). The cleavage of 1,2-glycols by lead tetraacetate to form two carbonyl compounds is a well known reaction and was also found to be applicable to the nickel(II) tetraphenyl-2,3-dihydroxychlorin (144) compound.169 A stoichiometric amount of lead tetraacetate in THF produced (mew-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato nickel(H) (145) in 85% yield. (54) (53) Ph H O H Ph C H O C H O Ph Pb(0Ac) 4 ^ P h < x Ph T H F , 1 0 min Ph (144) Ph (145) Scheme 3.2 Synthesis of (tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) 116 Chapter 3 Results The optical spectrum of this golden brown coloured compound (145) is unique for this system as it has a split Soret band at 414 and 464 nm along with a broad Q band at 688 nm. The electron-withdrawing carbonyl groups in the molecule are conjugated with the secochlorin core and are most likely responsible for the observed spectrum. l .OH 400 500 600 700 X (nm) Figure 3.1 The optical absorption spectrum of (mes0-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145). The 'H-NMR spectrum reveals the presence of two aldehydic protons with a conspicuous singlet at 1 0 . O 9 . o a . O 7 . o Figure 3.2 'H-NMR of (tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145). 117 Chapter 3 Results 3.1.2 Oxidations of zinc(II) and free base tetraphenyl-2,3-dihydroxychlorins with lead tetraacetate. The successful oxidation of the nickel diol chlorin (144) to form the nickel bisaldehyde secochlorin (145) led us to explore the same reaction with both the zinc metallated (146) and the free base diol chlorins (103). Unfortunately, the zinc diol chlorin (146) reaction resulted in a number of products (typically 4-6), none of which could be identified as the desired bisaldehyde secochlorin. Isolation and purification of the two major products from this reaction resulted in the identification of the previously known lactone secochlorin (147) and cyclic anhydride secochlorin (58). Scheme 3.3 Oxidation products of the zinc diol chlorin (146). Crossley et al. have reported both of these compounds.153 The cyclic anhydride product (58) being synthesized from the oxidation of the analogous 2,3-dione porphyrin (57) with 3-chloroperoxybenzoic acid whilst the lactone secochlorin (147) was formed from the rapid base catalyzed hydrolysis of (58). We propose that the zinc analog of (58) is formed in our reaction from autooxidation of the zinc bisaldehyde or diol chlorin. Autooxidation reactions are well documented196 and will be discussed later. The lactone secochlorin (147) could either be a product from the hydrolysis of this compound or be due to the autooxidation of the diol chlorin (146). 118 Chapter 3 Results The analogous reaction using the free base diol chlorin (103) resulted in a large number of products, none of which were isolated because the yield of each was so low. The same reaction performed under a nitrogen atmosphere resulted in a single brown product (148). This product had an absorption spectrum very similar to that of the nickel bisaldehyde secochlorin with a split Soret band at 408 and 466 nm, a broad shoulder at 528 nm and two broad bands at 596 and 642 nm. Although this product was very unstable in air, collapsing to a polar product which we could not isolate, we believe that this brown product is the free base bisaldehyde secochlorin (148). The novelty of the meso-tetraphenylsecochlorin framework and the ease with which its properties can be altered via the presence (or absence) and the nature of substituents on the phenyl rings, led us to investigate the use of (meso-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato) nickel(II) (145) as a starting material for other potential photosensitizers. A number of reactions are known to occur at aldehydic functional groups and a literature survey indicated a number of possible reactions to explore. 3.2 Reaction with Alcohols 3.2.1 Acetal Formation The reaction of aldehydes with alcohols in the presence of acid is well known to produce acetals. Serendipitously, the (me50-tetraphenyl-2,3-dicarboxaIdehyde-2,3-secochlorinato)nickel(II) (145) was previously found to react with alcohols (a mixture of methanol and ethanol) in the presence of hydrochloric acid to form the cyclic acetals (149) and (150).169 The dark green compounds displayed optical spectra with the Soret band at 430 nm and a broad band at 640 nm. This reaction was proposed to proceed via a hemiacetal intermediate which would react very quickly with the second aldehyde moiety. 119 Chapter 3 Results M e O M e O Ph Ph " ' O M e Ph Ph Ph (145) (149) (150) Scheme 3.4 Acetal formation from nickel bisaldehyde secochlorin (145). Reaction Conditions: i. MeOH/HCl fumes, CHC13. Further investigation of this reaction led us to perform the reaction purposefully and with other alcohols. In a series of simultaneous reactions, three flasks were charged with 10 mg of nickel bisaldehyde secochlorin and 3 drops of methylene chloride. To each flask was added 3 drops of one type of alcohol: methanol, ethanol or isopropanol. Upon the addition of fumes of hydrochloric acid, the mixtures turned green. The reaction with methanol was instantaneous, as observed by the dramatic change in colour from golden brown to dark forest green, whilst that with ethanol required a few seconds longer and the reaction with the bulky isopropanol required a further few seconds in order for the colour to change. All products were less polar than the nickel bisaldehyde secochlorin (145) starting material, and each exhibited only one spot on the T L C plate. The products were characterized and found to be the double acetals of the respective alcohols. 120 Chapter 3 Results Ph R O Ph C H O C H O O Ph HCI f u m e s R O H Ph ' "OR Ph Ph (145) R= Me (150) R= Et (151) R= iPr (152) Scheme 3.5 Formation of acetals (150), (151) and (152) with various alcohols. We were also curious to investigate the effect of excess hydrochloric acid on the reaction as well as the product(s) we would obtain in the absence of any alcohol. We, therefore, performed the same reaction using 10 mg nickel bisaldehyde secochlorin (145), again in 3 drops of methylene chloride and 3 drops of isopropanol, but rather than fumes of hydrochloric acid we added 1 drop of the acid to the flask and allowed the reaction to stir at room temperature. The nonpolar green double acetal (152) was observed whilst a new polar green product (172) appeared. This same compound was formed quantitatively upon the addition of 1 drop of hydrochloric acid to a solution of 10 mg of nickel bisaldehyde secochlorin (145) in 3 drops of methylene chloride in the absence of any alcohol. The absorption spectrum of this compound (172) had a broad Soret at 452 nm and a broad band at 638 nm. A seemingly complex proton NMR spectrum prematurely stopped our investigation into the identity of this compound but its reappearance as the sole product of the reaction of TFA with the nickel bisaldehyde secochlorin finally led to its elucidation many years later. This compound (172) will be thoroughly discussed in section 3.7.1. 121 Chapter 3 Results 3.2.2 Reaction with Ethylene Glycol In accordance with the previous results, we expected the nickel bisaldehyde secochlorin (145) to react with ethylene glycol to form the corresponding acetal, which would have comprised two seven membered rings. The reaction of 10 mg nickel bisaldehyde secochlorin (145) in 2 mL of methylene chloride with excess ethylene glycol and fumes of hydrochloric acid produced a bright green product (153). The absorption spectrum looked quite similar to those of the above mentioned acetals with the Soret band at 418 nm, a second band at 450 nm, several small Q bands and an intense broad band at 638 nm. Mass spectrometry confirmed the presence of the desired acetal product (153) with the parent ion peak at 746 representing a compound with the molecular formula C 4 6 H 3 2 N 4 0 : , N i 5 8 . H 2 (145) (153) Scheme 3.6 Reaction of nickel bisaldehyde secochlorin (145) with ethylene glycol. 3.3 Reaction with base Classically, aromatic aldehydes undergo the Cannizzaro reaction upon treatment with sodium hydroxide or other strong bases. In this reaction, one molecule of aldehyde oxidizes the other to an acid whilst being reduced to an alcohol. A bisaldehyde would therefore be expected to 122 Chapter 3 Results yield a hydroxy acid. This product, the 2-hydroxy tetraphenylsecochlorin carboxylic acid (154), would be anticipated to be a very interesting compound. (145) (154) Scheme 3.7 Expected Cannizaro reaction between nickel bisaldehyde (145) and strong base. Addition of base (Af-tetrabutylammonium hydroxide, or triethylamine) to a solution of ( m « o -tetraphenyl-2,3-bisaldehyde-2,3-secochlorinato)nickel(II) (145) in THF at room temperature immediately resulted in a colour change from golden brown to a blue green. 1.0 - i c o 0.5 -o < 0.0 J , , , , 4 0 0 500 600 X ( n m ) Figure 3.3 Optical spectrum of (155) (...) and the free base (156) ( ). The simple UV-vis spectrum and the very clean NMR spectrum both indicated a highly symmetrical porphyrinic molecule, thereby revealing that the expected Cannizzaro reaction had not occurred. The sole product was promptly recognized as the nickel(II) analog (155) of the previously 123 Chapter 3 Results identified (2-oxa-3-oxo-chlorinato)zinc(II) (147) which was confirmed by high-resolution mass spectrometry. This product was easily demetallated with acid to form the free base (156). Figure 3.4 Proton NMR spectrum of (2-oxo-3-oxa-tetraphenylchlorinato)nickel(II) (155). 124 Chapter 3 Results Past studies had identified this compound (155) as a byproduct from the osmium tetroxide-catalyzed dihydroxylation of metallated tetraphenylporphyrins (144).197 Although consistently formed in less than 2% yield, it was noted that the yield of the product increased upon increasing reaction times. Crossley et al. have claimed to have synthesized the analogous free base lactone (156) using two different methods. In a brief communication, photooxidation of 2-amino-tetraphenylporphyrin (42) followed by M C P B A oxidation of the resultant product reportedly led to the lactone secochlorin product (156) as did the direct oxidation of the 2-amino-tetraphenylporphyrin (42) with M C P B A . 1 4 2 1 4 3 Scheme 3.9 Synthesis of lactone (156) using MCPBA. Reaction Conditions: i. 02/ho; ii. MCPBA. Traditionally, photooxidation products of porphyrin systems result in varying amounts of ring cleavage. Specifically, extensive investigation into the photooxidation of meso-tetraphenylporphyrins (10) has determined the products to be the analogous ring-opened bilinones. 1 9 8 As the lactone product (155) has been observed not only as a byproduct from the diol chlorin (144) but also as a distinct product of reaction between the bisaldehyde secochlorin (145) and bases, it 125 Chapter 3 Results would follow that the lactone formation is due to autooxidation that is somehow catalyzed by certain reagents, such as bases, rather than photooxidation. Previous work 1 9 7 in Dolphin's lab put forth the following proposed mechanism based on the autooxidation of the diol (146): Zn Vh - H 2 ° Zri Vh - H 2 C O Zn Vh •^L "^C, (146) (147) Scheme 3.10 Previously proposed autooxidation mechanism for the formation of (147) from zinc(II) dihydroxy tetraphenylchlorin (146). The fact that aldehydes are among the most readily autooxidizable substances is well known . 1 9 9 ' 2 0 0 Aldehydes rapidly absorb oxygen from the atmosphere to produce peroxy acids. Thermal, photochemical and base-catalyzed autooxidation mechanisms have been observed for the benzaldehyde-benzoic acid conversion. 1 9 9 In the case of aldehyde autooxidation, both the thermal and photochemical mechanisms are radical mechanisms with the standard radical mechanism shown be low. 2 0 0 ? R C H O + 0 2 — • R O + H02* ? ? R O + 0 2 — • R C O O » ? ? ff R C O O + R C H O — • R C O O H + R C » ? R C O O H + R C H O — • 2 R C O o H Scheme 3.11 Radical autooxidation mechanism of aldehydes. 126 Chapter 3 Results Base-catalyzed oxidations typically require polar solvents and strong bases and are typical for unsaturated oc-aldehydes.199 These reactions do not have radical chain mechanisms but rather ionic intermediates ultimately leading to the corresponding acids. Unfortunately, very little investigation into this type of autooxidation has been performed and we, therefore, cannot either propose this type of mechanism nor rule it out. We do believe that the mechanism for our nickel lactone chlorin (155) synthesis involves autooxidation and that this reaction requires both the presence of base and oxygen. RCH=CH-CHO RCH=CH-COOH Scheme 3.12 Example of base-catalyzed aldehyde oxidation without a chain mechanism. Ph Ph s J \ / C O O H v J v s ^ C O O -\ \\ C O O H \ \\ C O O -Scheme 3.13 Proposed base-catalyzed autooxidation mechanism. In order to test our hypothesis, we performed the same reaction with /V-tetrabutylammonium hydroxide after a solution of nickel bisaldehyde secochlorin (145) had been purged with nitrogen gas for 25 minutes, and the reaction stirred under nitrogen. We observed the formation of five products: one nonpolar green product (with the same UV-vis spectrum as the nickel lactone product), two pale 127 Chapter 3 Results green compounds slightly more polar than the lactone, a brown compound and finally a dark green, very polar compound. The difference in reaction products under nitrogen as opposed to an air atmosphere supports our contention of autooxidation. 3.3.1 Reaction of the (2-oxo-3-oxa-tetraphenylchlorinato)nickel(II) (155) The lactone secochlorin (155) is demetallated in the presence of strong acid, but no other reaction is observed. No reaction apart from demetallation was observed for the addition of TFA, HCI or concentrated sulfuric acid. Neither the free base (156) nor the nickel(H) adduct (155) of (2-oxo-3-oxa-tetraphenylchlorin) react upon further addition of base such as N H 3 ( a q ) . This compound (155) was also resistant to methanol/acid catalyzed hydrolysis. DIBALH, diisobutylaluminum hydride, typically reduces lactones to the analogous lactols.201 In our experiment, 15 mg of the nickel lactone chlorin (155) in 5 mL of dry THF under nitrogen at -20 to -25°C was reacted with 5 mL of 1M DIBALH in hexane and the mixture stirred for 40 minutes. After stirring, 0.2 mL of concentrated sulfuric acid was added and stirring continued for a further 15 mins at 0°C. Following the work up procedure, the crude mixture was chromatographed. Three burgundy bands were observed along with a very polar brownish green band. The three burgundy bands were removed and the products recrystallized yielding 1 mg of the least polar compound (157), 2 mg of the compound of moderate polarity (158) and 4 mg of the polar product (159). All three of the burgundy products displayed the exact same absorption spectrum: Soret band at 418 nm and four Q bands at 516, 550, 592 and 646 nm. Although the relative intensities of the bands differ, the bands were in exactly the same positions as both the free base lactone (156) and free base diol chlorins (103). Apparently the products were demetallated during the work up. The 128 Chapter 3 Results aromatic regions of the proton NMR spectra were also remarkably similar. These spectra not only resembled one another's spectra but also very closely resembled the proton spectrum of the free base lactone chlorin (156). This indicated that the products had retained the oxygen-incorporating five membered ring in their skeletons which is in agreement with the expected lactol products. The higher field region of the spectrum indicated that the two least polar products (157) and (158) contained moieties which were most likely alkoxy groups as singlet peaks were observed at approximately 3.2 ppm. Further characterization such as mass spectrometry revealed that we had indeed formed the corresponding free base lactol chlorin (159), the analogous compound with a methoxy linkage (158), and the isobutoxy derivative of the lactol (157). Scheme 3.14 Products formed during reaction of DIBALH with nickel lactone chlorin (155). 129 Chapter 3 Results 3.4 Reaction with Amines 3.4.1 N-Methylamine Aldehydes traditionally react with primary amines to form imines. Aromatic imines are usually stable enough for isolation and thus were of interest to us. The first reagent chosen was methylamine due to its simplicity. Reaction of (me5,o-tetraphenyl-2,3-bisaldehyde-2,3-secochlorinato)nickel(II) (145) in slightly acidic THF (1% HCI) with two equivalents of N-methylamine produced one major product - a dark green, polar compound (160). The UV-visible spectrum displayed a broad Soret band at 430 nm, a shoulder at 460 nm and the most intense Q-band at 638 nm. 400 500 600 700 wavelength (nm) Figure 3.5 UV-vis spectrum of the product (160) from the reaction of methylamine and bisaldehyde. The proton NMR spectrum indicated that the desired bis-imine had not been produced as two singlets, one at 8.6 ppm and the other at 9.1 ppm, were present as opposed to the single peak anticipated for the doubly substituted, symmetric product. The singlet at 9.1 ppm, being in the region of aldehydic protons on the bisaldehyde secochlorin (145), hinted that perhaps only one imine linkage had been created whilst the separation of the peaks representative of the six P protons on the 130 Chapter 3 Results chlorin ring indicated a lack of symmetry in the molecule, thereby reinforcing the theory of a singly-substituted product. A large singlet peak at 4.4 ppm integrated for 3 protons and was assigned to represent three methyl protons of the methyl imino group. High resolution mass spectrometry confirmed the identity of the product as the mono-imino compound (160) with a molecular formula of C 4 5 H 3 1 N 5 NiO (EI, m/e 715.18551,100%). PPM Fig 3.6 H-NMR spectrum of tetraphenyl-S-N-methylimino-l-carboxaldehyde secochlorin (160) 131 Chapter 3 Results P h (160) Scheme 3.15 Reaction of iV-methylamine with nickel bisaldehyde secochlorin (145). 3.4.2 1,2-Phenylenediamine raes0-(Tetraphenyl-2,3-bisaldehyde-2,3-secochlorinato)nickel(H) (145) was reacted with one equivalent of 1,2-phenylenediamine in the same manner as the reaction of /V-methylamine. The resultant bisimine (161) was produced quantitatively, resulting in an additional nine-membered ring bound to the secochlorin core. Scheme 3.16 Reaction of nickel bisaldehyde (145) and phenylene diamine. 132 Chapter 3 Results The UV-Vis spectrum reflects the extended conjugation of the new ring in the shifted Soret at 436 nm and the most intense Q-band at 648 nm whilst the relatively simple proton NMR reflects the symmetry of the molecule. I'.H (l .' 1 [31:1 i l l l[iil + 2 C H = N / ! 12Hm+p 21-IO + 21-I 211o GDCI , Figure 3.7 Proton NMR spectrum of the phenylene diamine product (161). Similar reactions have been performed by Crossley et al. wherein the free base tetraphenyl-7,8-dione (162) was reacted with phenylenetetramine to produce the corresponding porphyrinoquinoxaline (163) in 98% yield.1" These molecules were synthesized to produce linearly conjugated polyporphyrin systems (164) for potential use as molecular wires. 133 Chapter 3 Results (164) Scheme 3.17 Synthesis of molecular wires. Reaction Condit ions: i. 1 eq. (162) in deoxygenated toluene, heat / N 2 , 72 hr. 3.4.3 Ammon ia The addition of aqueous ammonia to a solution of (meio-tetraphenyl-2,3-bisaldehyde-2,3-secochlorinato)nickel(n) (145) in THF at room temperature yields two green products (165) and (166). The simple NMR spectrum of the nonpolar product (165) indicated a high degree of symmetry and the absence of any non-aromatic protons. The UV-visible spectrum displayed a Soret band at 408 nm and a Q-band at 606 nm. The compound was recognized as the nickel(II) complex (165) of the previously mentioned (meso-tetraphenyl-2,3-dioxochlorinato) copper(II) (45). Figure 3.8 Proton NMR spectrum of (meso-tetraphenyl-2,3-dioxochlorinato)nickel(II) (165). At this point, we are uncertain of the mechanism of this reaction. We surmise that base catalyzed autooxidation may be involved but further investigation is required. Three different syntheses of the free base analog (167) of this diketone have been reported. In one instance 2-amino-meso-tetraphenyl porphyrin (42) was oxidized by air in the presence of light \ and subsequently acidified, forming the diketone (167).143 A second synthesis employed 2-oxo-mew-tetraphenylporphyrin (44) as the starting material. The 2-oxo-TPP (44) was oxidized with four 135 Chapter 3 Results equivalents of selenium dioxide in refluxing dioxan to give the desired product (167).142 The third reported synthesis involved the DDQ-mediated oxidation of (mew-tetraphenyl-2,3-dihydroxychlorinato)zinc(II) (146) in benzene.197 Ph ( 1 4 6 ) Scheme 3.18 Syntheses of meso-tetraphenyl-2,3-dioxochlorin (167). Conditions: i. 02/hu; H7H20; ii. 4 eq Se02/dioxane, A; iii. 2 eq DDQ/benzene /H+ High resolution mass spectrometry of the second polar compound (166) formed in our reaction of the bisaldehyde (145) with ammonia determined the atomic composition for the parent ion peak found at m/e 685 to correspond to C 4 4 H 2 9 N 5 N i . The absorption spectrum reflects an extension of conjugation as the Soret is shifted to 436 nm, whilst the Q-bands lie at 526, 570 and 620 nm. 136 Chapter 3 Results .0. , .Q o 0.5 JO < "53 0.0 400 500 600 700 wavelength (nm) 800 Figure 3.9 UV-visible spectrum of the polar product (166) of the reaction with ammonia. The proton NMR spectrum suggested the structure of the compound to contain a six-membered ring formed with the nitrogen of ammonia as there are two characteristic peaks present: a peak at 9.1 ppm integrating for one proton, and a singlet at 6.61 ppm which represents two protons. 2PH. 1pH 1 i CH=N 1PH 1PH I H O / i \ 1 H o 1Ho CH,-N f. 3 G. 7 Figure 3.10 Proton NMR spectrum of (166). Investigation into the mechanism of this reaction is required. 137 Chapter 3 Results Ph Ph (145) (166) Scheme 3.19 Reaction of the nickel bisaldehyde secochlorin (145) with ammonia. 3.5 Reaction with Phenylhydrazine Hydrazones are formed from the condensation of hydrazines and aldehydes. Phenylhydrazine is commonly used to form the corresponding hydrazone as this is known to react predictably with many aldehydes. Neat phenylhydrazine (1.5 mL) was added to 38 mg of nickel(II) bisaldehyde secochlorin (145) and the reaction left to stir for 20 minutes at room temperature. Three compounds were obtained: a nonpolar golden brown product, a nonpolar burgundy product and a polar dark green compound. Each was purified and characterized. The burgundy product was immediately recognized as the nickel lactone chlorin (155). The nonpolar brown product (168) and the polar dark green compound (169) were also isolated and characterized. The UV-visible spectrum of the brown product (168) had a split Soret at 432 and 446 nm, a broad band at 548 nm and a broad Q band at 602 nm. Mass spectroscopy revealed the parent ion at m/e 114 corresponded to the atomic composition C 5 0 H 3 2 N 6 N i . Evidently, both of the aldehyde 138 Chapter 3 Results moieties had reacted. The proton NMR spectrum had a prominent singlet at 8.8 ppm and the P proton peaks were between 8.6 and 8.7 ppm, whilst the protons of the phenyl group of the reagent were at 8.0 ppm. Based on the characterization, we propose that this brown compound is the secochlorin shown below. (168) Figure 3.11 Proposed structure of (168). Such a reaction is proposed to begin with the reaction of one of the aldehyde moieties to form the mono-hydrazone. This is could then react with the second aldehyde moiety to form an addition ring which would collapse upon deprotonation to form the product (168) with one nitrile group and an imino linkage. 1 3 9 Chapter 3 Results (168) Scheme 3.20 Mechanism for the proposed synthesis of pigment (168). This compound (168) is golden brown and has a split Soret band as does the nickel bisaldehyde secochlorin (145). The second product, compound (169), is green in colour which more closely resembles a metallated chlorin. The UV-visible absorption spectrum was also indicative of a closed ring system with a Soret band at 420, and a broad Q band at 610 nm. The proton NMR spectrum reflected a degree of symmetry as there was a doublet at 8.4 ppm, a singlet at 8.25 ppm and two overlapping doublets at 8.05 ppm all of which represent the six P protons. High resolution mass spectrometry found the parent ion at m/e 116 to have a molecular formula of C S 0 H 3 4 N 6 Ni: (168) plus two hydrogen atoms. The identity of this compound is currently under investigation. 3.6 Reaction with HMPT: Epoxidation 140 Chapter 3 Results Hexamethylphosphorus triamide has long been known for its use as an epoxidizing agent of bisaldehydes.202,203 Aromatic aldehydes yield epoxides in a highly exothermic and almost instantaneous reaction. As many attempts to synthesize epoxy chlorins have failed (see section 1.4.2), and as this reaction is well known and reliable, we attempted to make the first tetraphenyl epoxy chlorin. ^ . H © H ((CH,) 2 N)IP: + C=0 • ( ( C H , ) 2 N ) , P - C - 0 + C=0 R R R t R H ( ( C H , ) 2 N ) 1 P O + I A R H Scheme 3.21 The reaction mechanism of hexamethylphosphorus triamide with aldehydes. Reaction of one equivalent of hexamethylphosphorus triamide with (me50-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) produced a green product (170) in good yields. 141 Chapter 3 Results This product has a Soret band at 430 nm and a broad Q-band at 638 nm. The NMR spectrum reflected the lack of symmetry in the molecule by the positions of the peaks representing P protons and also displayed two doublets: one at 5.6 ppm and the other at 4.4 ppm both with a coupling constant of 12 Hz. This compound (170) was obviously not the expected epoxide molecule. Whilst searching through the literature, we observed a compound (171) which reportedly had very similar characterization data as our compound (170).206 The UV-visible spectrum of this molecule (171), shown in Figure 3.12, has a Soret band at 432 nm and a broad peak at 616 nm whilst the proton NMR spectrum has two peaks of interest: a doublet at 5.64 ppm (J= 10.7 Hz, CHOU) and a doublet at 4.33 ppm (7= 16.2 Hz, CH 2 ). OMe MeO-v V - < \ Ni /)—(' v — O M e Figure 3.12 Compound (171). An examination of our data revealed that one P carbon of our product was fused to an adjacent phenyl ring. 142 Chapter 3 Results (170) Scheme 3.22 Reaction of nickel bisaldehyde secochlorin (145) with HMPT. W e believe that the epoxide is formed but e lectrophi l ic attack at one o f the epoxide carbons occurs subsequently, caus ing the epoxide r ing to open, as shown. Scheme 3.23 Reaction of nickel bisaldehyde secochlorin (145) with HMPT to form (170) via the epoxide. 143 Chapter 3 Results 3.7 Reactions with acid 3.7.1 Trifluoroacetic Acid (TFA) As previously discussed, reactions of (raeso-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) with alcohols in the presence of acid result in the formation of the analogous green acetals (150) - (152), but when the bisaldehyde secochlorin (145) is dissolved in THF and fumes of T F A are added to the reaction flask a polar, dark green compound (172) is quantitatively produced. Classically, treatment of an aromatic molecule which contains an aldehyde (or a ketone) moiety undergoes cyclodehydration upon acid treatment if the functional group is in a position which allows the formation of a six- or more rarely a five-membered ring. In this respect, addition of acid to (me5o-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) would be expected to initiate ring closure, but dehydration could not occur due to the lack of a-hydrogens. A survey of the literature revealed that such reactions had been observed in the past.204"206 Researchers reported the monocyclization of 2-formyl-5,10,15,20-meso-tetraphenylporphyrinato copper(II) (173) in 1980.204 The acid-catalyzed condensation of the aldehyde moiety with the ortho position of an adjacent phenyl group formed a triphenylporphyrin (174) with a fused benzocyclohexanone ring. In this report, a very small amount (~0.1 %) of the cyclized product (174) was formed upon addition of TFA to the copper porphyrin (173). As expected, X-ray crystallographic data revealed that the fused phenyl ring lay in the plane of the porphyrin core. 144 Chapter 3 Results Ph Ph Ph Ph A, 1 hr T F A • Ph Ph Ph (173) (174) Scheme 3.24 Cyclization of (2-formyl-5,10,15,20-tetraphenylporphyrinato)copper(II) (173) In 1990, Callot et al. repeated the same reaction with 2-formyl-5,10,15,20-meso-tetraphenylporphyrinatocopper(II) (173) but using very small amounts of TFA and obtained the corresponding naphthoporphyrin derivative (174) in 38% yield.205 In order to study the NMR spectra of the product, 2-formyl-5,10,15,20-me5,o-tetraphenylporphyrinatonickel(II) (175) was acidified. Three products were obtained from this reaction. The expected ketone (173) was produced (36% yield) as were two additional products: the reduced monocyclized naphthoporphyrin derivative (176) and another reduced monocyclized derivative (177) of undetermined structure (3 possibilities are shown). with TFA. 145 Chapter 3 Results 43% (177) Scheme 3.25 Acidification of (2-formyl-tetraphenylporphyrinato)nickel(II) (175) with TFA. The intramolecular cyclization was proposed to begin with an electrophilic attack of the protonated carbonyl moiety on the ortho position of an adjacent phenyl group, resulting in an intermediate carbocation. A proton is lost to regain aromaticity of the phenyl ring, thereby producing an adjoining six-membered ring with an alcohol moiety. This product is then postulated to undergo an intermolecular hydride shift with the hydride migrating to a carbocation produced presumably from the loss of water from the alcoholic intermediate. Both the ketone (174) and the reduced species (176) and (177) are produced. 146 Chapter 3 Results (176) Scheme 3.26 Proposed mechanism for the formation of the monocyclization products. In 1994, another report of such reactions was published. Dolphin et al. communicated the double cyclization of (2-formyl-me50-tetrakis(3-methoxy)phenylporphyiinato)nickel(II) (178) and copper(H) (179) using strong acid (15% H 2 S0 4 in TFA, 1 hr, room temp).206 Reaction of 2-formyl-147 Chapter 3 Results me^o-tetrakis(3-methoxy)phenylpoiphyrinatonickel(IT) (178) and copper(II) (179) with 15% H 2 S 0 4 in TFA resulted in two fractions - the most polar being isomers of the double cyclization product incorporating a keto group (180), and a nonpolar fraction consisting of isomers of methylene-bridged doubly cyclized product (181). Isomers of the cyclized products formed were dependent upon the position of the methoxy groups on the phenyls which were fused to the macrocycle. Scheme 3.27 Synthesis of the doubly-cyclized products (180) and (181). This reaction was shown to occur only when the electron-releasing methoxy groups were present in the meta position thereby activating the ortho positions of the phenyl groups. The reaction was attempted using the analogous /?ara-methoxy substituted TPP nickel(II) and copper(II) but merely produced the previously observed monocyclized keto products. Additionally, reaction of the meta-methoxy substituted copper complex (179) in 2.5% TFA in dichloromethane resulted in the previously observed monocyclized keto-bridged (174) and methylene-bridged (176) products, indicating the necessity of strong acid (15% H 2 S 0 4 in TFA) in order for the double cyclization to occur. The green product (172) formed in our work by the acid-catalyzed condensation of the bisaldehyde secochlorin (145) was characterized by a number of methods and the data concluded that the compound was a product of double intramolecular cyclization reactions. A product (172) 148 Chapter 3 Results possessing two additional five-membered rings - one containing a keto group, the other containing a methylene bridge - had been formed. Ph Ph (145) (172) Scheme 3.28 Synthesis of a doubly-cyclized product (172) from bisaldehyde (145) and TFA. Experimental data from the doubly cyclized product (172) indicated an extreme deformation caused by the necessary planarity of the two fused phenyl rings with the porphyrin skeleton. This was reflected by the highly atypical absorption spectra. The optical spectrum no longer showed the split Soret band that is characteristic of the bisaldehyde secochlorin (145), rather displaying a bathochromically shifted single Soret band at 452 nm and a broad intense band at 638 nm. High resolution mass spectrometry found m/e 684.14600, corresponding to C 4 4 H 2 6 N 4 N i O , whilst the 'H-NMR spectrum included a conspicuous singlet integrating for two protons at 5.58 ppm. IR spectroscopy also indicated the presence of a carbonyl group by a band at 1682 cm"1 representing C=0 stretching. Interestingly, in contrast to the reported findings of the Dolphin group paper, the NMR spectrum does not reflect much, if any, loss of aromaticity of the macrocycle because the peaks for the phenyl protons are in the expected range (7.4-7.9 ppm).2 0 6 149 Chapter 3 Results 400 500 600 wavelength (nm) 700 800 Figure 3.13 UV-Vis absorption spectrum of the doubly cyclized product (172). CH., "* i 1 1 1 * i 1 • 1 1 i * 1 1 ' " i • i i i i i • i t ' • • i 9. % 8"- 5 S .0 7 .5 7 .8 5 ,5 B.l Figure 3.14 Proton NMR spectrum of the doubly cyclized product (172). The first step of the double cyclization to this novel product can be seen as an electrophilic attack of the protonated carbonyl group on the adjacent phenyl moiety at the ortho position. Two cyclic alcohols would then result, whereupon the loss of a hydroxyl group would occur and the other 150 Chapter 3 Results hydroxyl group would form a carbonyl. An intramolecular hydride shift to the electropositive carbon of the adjacent five-membered ring would ensue. Ph Ph Ph Ph (145) (172) Scheme 3.29 Mechanism of formation of the doubly cyclized product (172). This product was also formed during the previously mentioned acetal formation reaction when HCI was added without any alcohol in the system (section 3.2.1). 3.7.1.1 Excess TFA The addition of drops of TFA (as opposed to fumes) to the (me.yo-tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(U) (172) in THF at room temperature yielded two products. The nonpolar product (172) was the expected doubly cyclized keto and methylene-bridged system, while the more polar green product was identified as the (me5,o-tetraphenyl-2-oxa-3-oxo-chlorinato)nickel(II) (155). This, therefore, represents yet another reaction where the lactone product is formed. The yield for this molecule (155) was approximately 20% but was found to be dependent on the amount of TFA added to the system. The product was characterized and confirmed by IR spectroscopy (C=0 stretch at 1745 cm"1), UV-visible absorption spectroscopy ( Am a x= 410, 500, 568, 151 Chapter 3 Results 608 nm), 'H-NMR, and mass spectral analysis (EI - C 4 3 H 2 6 N 4 Ni0 2 , M + 100%). 400 500 600 700 800 wavelength (nm) Figure 3.15 UV-vis absorption spectrum of the lactone chlorin product (155). (145) (155) (172) Scheme 3.30 Reaction of nickel bisaldehyde secochlorin (145) with excess TFA. This product was also observed as the sole product of the reaction with base. The reaction products in previous studies of involving this type of cyclization have been found to be dependent on the nature of the acid and its concentration. We believe that this lactone-incorporating product (155) is the result of a reaction other than the cyclization reaction, and that there are, therefore, two different and competing reactions occurring when there is a higher concentration of acid in the system. 152 Chapter 3 Results 3.7.1.2 Reaction of the doubly cyclized product (172): Further addition of acid to the doubly cyclized product (172) had no effect. This is in agreement with the results of the previous investigations of this type of cyclization reaction. In contrast, the addition of a variety of bases (triethylamine, /V-tetrabutylammonium hydroxide, or potassium hydroxide) or the heating of (172) formed two new red products (182) and (183). While the total yield was always 100%, the yield of each of the two products was again found to be dependent on the nature of the reagent base and its concentration. The nonpolar product (182) had a very clean proton NMR spectrum that lacked any non-aromatic protons. The UV-visible absorption spectrum was highly atypical with an intense band at 344 nm, a sharp Soret-like band at 416 nm, two overlapping bands at 496 and 536 nm of equal intensity as the two previous bands, and a broad weaker band at 668 nm. 1.0 -£ 0.5 c < Pi 400 50cT 600 700 800 wavelength (nm) Figure 3.16 UV-vis spectrum of nonpolar product (182) from the reaction of TFA with (172). Mass spectrometry found the mass to correspond to a formula of C 4 4 H 2 4 N 4 N i 0 2 which confirmed that the compound was a product of the double cyclization with both fused rings incorporating keto groups. 153 Chapter 3 Results 8.8 6.6 3. '1 8 .2 Figure 3.17 Proton NMR spectrum of the doubly cyclized diketo compound (182). This oxidation is not unexpected as both Callot and Dolphin found that their respective products containing methylene bridges (176) and (181) were oxidized in solution to the analogous ketones (174) and (180).205,206 Again we note that this reaction is apparently catalyzed by base. 154 Chapter 3 Results (172) (182) (183) Scheme 3.31 Reaction of the doubly cyclized product (172) with base. The polar product (183) produced in this reaction again only possessed aromatic protons as observed by proton NMR and also displayed an atypical absorption spectrum. The UV-visible spectrum had broad bands at 304 and 376 nm, a very intense, sharp Soret-like band at 416 nm and two less intense, broad bands at 500 and 716 nm. wavelength (nm) Figure 3.18 UV-vis spectrum of the polar product (183). Further characterization led to the conclusion that this compound (183) was the doubly cyclized product with a five-membered keto-containing ring and a six-membered cyclic lactone with the formula C 4 4 H 2 4 N 4 N i 0 3 . The observed intensity of the Soret-like band at 416 nm reflects the 155 Chapter 3 Results increased aromaticity, presumably due to the larger ring size of the lactone ring which would allow the fused phenyl group to remain more out of the plane of the porphyrin skeleton. Figure 3.19 Aromatic portion of 'H-NMR spectrum of the doubly cyclized product (183). The reaction must involve the autooxidation of the methylene bridge of (172) to a ketone to form the nonpolar diketo doubly-cyclized product (182). Whether this moiety is further oxidized to the lactone-incorporating polar product (183) or the keto group of the other ring is simultaneously oxidized to the lactone group is unknown. Upon standing in solution, the nonpolar diketo product (182) has been observed to oxidize to the polar monoketo lactone-incorporating product (183). Clearly, these products are susceptible to autooxidation. 156 Chapter 3 Results AND OR [Q] / Base N. O 0 II (172) (182) (183) Scheme 3.32 Proposed scheme for the formation of the doubly cyclized products (182) and Addition of 2 drops of concentrated sulfuric acid (neat) to 10 mg of nickel bisaldehyde (145) resulted in an immediate colour change from the dark crystals of nickel bisaldehyde secochlorin (145) to a green colour. The solution was washed with basic aqueous solution whereupon the solution turned red. T L C and UV-visible spectroscopy showed that the doubly-cyclized product (182) had been formed as the major product. In an attempt to demetallate the resulting product (182), 4 drops of concentrated sulfuric acid were added. While no colour change was observed, TLC showed the presence of a new less polar brown compound (184) above and in addition to the red spot of the doubly cyclized diketo product (182), formed in about 60% yield. A sample of the brown spot (184) was removed from the T L C plate, filtered and submitted for mass spectrometry. The compound (184) was determined to be the free base analog of the doubly cyclized keto product (182). No further characterization was performed. The formation of the diketo product (182) as opposed to (172) formed with TFA is most likely due to the basic work up whereupon a colour (183). 3.7.2 Sulfuric Acid (H2S04): 157 Chapter 3 Results change from green (the colour of (172)) to red (the colour of (182)) was observed. Ph Ph Ph (145) (182) (184) Scheme 3.33 Formation of the free base analog of the doubly cyclized diketo product (184). 3.6.2.1 Sulfuric Acid/Methylene Chloride (H2S04/CH2C12): The reaction with sulfuric acid was repeated using a methylene chloride solution of the nickel bisaldehyde secochlorin (145), as opposed to the addition of neat H 2 S0 4 , and the work up not involving base. One drop of concentrated sulfuric acid was added to a solution of 35 mg of nickel bisaldehyde (145) in 5 mL of methylene chloride. The mixture was stirred at room temperature for 5 minutes at which time no visible reaction had occurred, therefore another drop of concentrated sulfuric acid was added. The reaction was allowed to proceed at room temperature for 5 minutes after which the solution was washed with water, dried with sodium sulfate and evaporated to dryness. Preparative T L C was performed. Three bands were observed and each was removed from the plate, filtered, the filtrate evaporated to dryness and characterized by mass spectrometry. The least polar band was formed in approximately 15% yield and determined to be the previously observed lactone chlorin (155). The second least polar band, formed in 60% yield, was found to be the doubly cyclized diketo product (182) whilst the most polar product, formed in 15% yield, was 158 Chapter 3 Results found to be (183) the doubly cyclized product containing one keto and one lactone group. Scheme 3.34 Reaction of nickel bisaldehyde (145) in CH2C12 with sulfuric acid. We surmise that (182) and (183) were formed despite the lack of a basic work up procedure, due to the preparative T L C performed. Previous studies involving this type of cyclization product have shown that oxidation occurs either in the presence of air while in solution or whilst the product is adsorbed on silica or alumina gel. 159 Chapter 3 Results 3.7.3 Variety of acids: In order to determine the generality of the reaction between the nickel bisaldehyde (145) and acids, four very different acids were chosen and tested as reagents. The acids comprised TFA, sulfuric acid, glacial acetic acid and hydrochloric acid. Four flasks, each containing 10 mg of nickel bisaldehyde (145) in 3 mL of methylene chloride, were set on magnetic stirring plates at room temperature. To each of the four reaction flasks, four drops of one acid was added. Each reaction was monitored by T L C over the course of 30 minutes. Of the four reactions, only the two that contained the concentrated sulfuric acid and the concentrated hydrochloric acid had reacted. Neither the reaction between the nickel bisaldehyde (145) and the 4 drops of glacial acetic acid nor the reaction with TFA showed any visible change, therefore to each an additional 1 mL of the respective acid was added and the reaction left to stir at room temperature for a further 30 minutes. Work up of all four reactions consisted of washing the mixture with water, drying with sodium sulfate, evaporating to dryness and performing preparative T L C with a 1:1 hexane:methylene chloride solution. The sulfuric acid reaction produced two bands of varying red-burgundy colour. The least polar band was determined to be the doubly cyclized diketo compound (182), whilst the most polar band was found to be (183) as expected. The reaction with concentrated hydrochloric acid produced a single brown-burgundy, product (185) in 55% yield, with a molecular mass of 700 g/mol and a very unique UV-visible spectrum. The UV-vis spectrum displayed a broad, but intense, band at 414 nm with two less intense broad bands at 456 and 504 nm and a relatively strong band at 716 nm. 160 Chapter 3 Results 1.0 wavelength (nm) Figure 3.20 UV-vis spectrum of the product (185) of HCI reaction with (145). The proton NMR spectrum was quite similar to that of the doubly cyclized products with the exception of a peak at 9.5 ppm. Upon assignment of the P-hydrogen peaks and determination of the integration scale, it was obvious that the peak at 9.5 ppm (in the aromatic aldehydic proton region) accounted for one proton. This indicated a monocyclization product (185), which was later confirmed by high resolution mass spectrometry with the atomic composition of C^H^NtNiC^ being determined for the parent ion at m/e 700. 161 Chapter 3 Results ~ i — r — r — 7 1 — r — i — ] — T — r — I — r 10 ..$ 9 . c ; -j 1 r j ; 1 ) i i i , — i . i . . i ! ! ! 1—j ! T—\—l j — T 9 . d 8..,.,5 • 8 . 3 , 7 . 5 7 , 0 6 , Figure 3.21 Proton NMR spectrum of the singly cyclized keto-containing product (185). HCI 5 5 % yield (145) (185) Scheme 3.35 Reaction of nickel bisaldehyde (145) with concentrated hydrochloric acid. 162 Chapter 3 Results Ph Ph (185) Scheme 3.36 Proposed mechanism of formation of the singly cyclized keto product (185). It is interesting to note that when a higher concentration of HCI was added to the nickel bisaldehyde secochlorin (145) during our acetal forming reactions in the absence of any alcohol, (172) was formed. Obviously, the nature of the product(s) is also highly dependent upon the concentration of the acid. The reaction with TFA produced two compounds: a nonpolar green compound which was found to be the doubly cyclized product (172) in 41 % yield, and the second least polar product which was formed in 20% yield and was determined to be the doubly cyclized product (182). No reaction with glacial acetic acid was observed. 163 Chapter 3 Results 3.8 Reaction with Ylides: Wittig Reaction Upon realizing the nature of the doubly cyclized products, we were intrigued to see if we could synthesize a nickel bisaldehyde secochlorin but with one additional methylene group between the porphyrin core and the carbonyl carbon. We were curious to investigate the product of the acid catalysis of such a system. If, as we hoped, the system reacted similarly to the nickel bisaldehyde secochlorin (145) and electrophilic reaction with the phenyl group occurred, two six-membered rings would be formed with the phenyl group, as shown below. Dehydration could ensue to form a fully aromatic system (186). In order to lengthen the secochlorin core - carbonyl bond, we would need to perform a Wittig reaction with an ylide possessing an a-alkoxy group. The resultant enol ether could then be hydrolyzed to an aldehyde (187). 164 Chapter 3 Results Ph (186) Scheme 3.37 Proposed reaction of nickel bisaldehyde secochlorin (145). Reaction Conditions: i. Ph3PCH2OCH3Br / BuLi; FT, H20. Many Wittig reactions were performed to test the reactivity of (145). Reagents included methoxymethyl triphenylphosphonium bromide, methyl triphenylphosphonium bromide, and bromomethyl triphenylphosphonium bromide. The reactions performed using the methoxymethyl-and methyl-triphenylphosphonium bromide each produced three or four products: nickel lactone chlorin (155), nickel acetal chlorin (150) and one or two products of rearrangement possibly from the monoadduct. The reactions performed using the bromomethyl triphenylphosphonium bromide met with limited success. One major nonpolar product (189) was formed, isolated and characterized. The proton NMR spectrum was extremely messy and indicated a mixture of inseparable products as did high resolution mass spectrometry. High resolution mass spectrometry revealed that 2 165 Chapter 3 Results inseparable products had been formed (189) and (190): C 4 6 H 2 8 N 4 Br 2 Ni and C 4 5 H 2 8 N 4 OBr 2 Ni. Based on the first molecular formula, the Wittig reaction had occurred at both carbonyl groups but subsequent reaction(s) had occurred. The products are, therefore, probably quite unstable which would explain the additional products when methoxymethyl triphenylphosphonium bromide was used as a reagent. The structures of the products obtained were not determined. 3.9 Reaction with Wilkinson's Catalyst: Decarbonylation Aldehydes, both aromatic and aliphatic, are decarbonylated by heating in the presence of tris(triphenylphosphine)rhodium, (Ph3P)3RhCl or Wilkinson's catalyst. The reaction between this catalyst and aldehydes occurs as follows: ~ H CO CO ? I / P P h 3 R \ I / H I R-C-Rh ^ Rn ^ RH + Ph^P- Rh-PPfh Scheme 3.38 Reaction of Wilkinson's catalyst with an aldehyde. The unsubstituted secochlorin has been a long sought product of many failed reactions. Based on past experiments, we believed that the reaction of Wilkinson's catalyst and our nickel bisaldehyde (145) compound would produce this elusive secochlorin as the aldehyde moieties of the nickel bisaldehyde (145) for the most part behaved like tradition aldehyde functional groups. A solution of the meso-tetraphenylsecochlorinato nickel(II) (145) in very dry benzonitrile was treated with 2 equivalents of Wilkinson's catalyst and refluxed under nitrogen. The reaction produced a nonpolar, bright green compound (191), an olive-coloured compound (192) of moderate 166 Chapter 3 Results polarity and a relatively polar blue compound (193). The UV-visible spectrum of the nonpolar compound (191) was quite simple with a Soret band at 422 nm, a shoulder at 576 nm and a broad but intense band at 612 nm. The proton NMR spectrum was also very simple with all peaks in the aromatic region of the spectrum. The presence of 2 doublets each integrating for 2 protons and a singlet integrating for 2 protons indicated a high degree of symmetry in the product. A singlet at 9.85 ppm, representing 2 protons, was of interest because it was quite low field. wavelength (nm) Figure 3.22 UV-vis absorption spectrum of the unsubstituted secochlorin (191). Mass spectrometry found that the mass of this bright green compound (191) corresponded to an atomic composition of C 4 2 H 2 8 N 4 Ni - that of the desired elusive secochlorin. The synthetic procedure was later modified to enable higher yields of this molecule. It was found that longer refluxing times and a larger excess of Wilkinson's catalyst led to yields of up to 60%. 167 Chapter 3 Results Ph Ph (145) (191) Scheme 3.39 Synthesis of the unsubstituted secochlorin (191) from nickel bisaldehyde (145). The olive coloured product (192) that was formed in approximately 10% ran just below the starting material, nickel bisaldehyde (145), but much further below the fully unsubstituted secochlorin (191) on silica gel T L C plates. This suggested that perhaps only one of the aldehydic moieties had been removed. Experimental data proved this theory to be true. The IR spectrum revealed the presence of a carbonyl group with a band at 1657 cm"1. Scheme 3.40 Synthesis of the monodecarbonylated secochlorin (192) from (145). The stepwise loss of the carbonyl groups which extended the chromophore is reflected in the shift in the Soret band which is shifted to 448 nm, a shoulder at 613 nm and a broad band at 678 nm. 168 Chapter 3 Results i.o -, 0.0 400 500 600 700 800 wavelength (nm) Figure 3.23 UV-visible absorption spectrum of the monodecarbonylated secochlorin (192). The proton NMR spectrum of this compound (192) was quite similar to that of the fully unsubstituted secochlorin (191) except for the separation of PH peaks due to lack of complete symmetry and the presence of a singlet peak at 9.8 ppm due to the remaining aldehydic proton. High resolution mass spectrometry confirmed that the olive coloured compound (192) was the monodecarbonylated species corresponding to the molecular formula C 4 3 H 2 8 N 4 NiO (EI, m/e 61 A, M + The third product was a bright blue compound (193) and appeared to be quite polar. The UV-visible spectrum displayed a Soret 410 nm and an intense band at 608 nm with shoulders at 548 nm and 574 nm. This spectrum was quite similar to that of the lactone product (155). 100%). o.o 500 600 wavelength (nm) 400 700 Figure 3.24 UV-visible absorption spectrum of the blue product (193). 169 Chapter 3 Results Proton NMR spectrum showed that this compound (193) was quite symmetrical. There were 2 interesting peaks in the NMR - one at 6.08 ppm integrating for 1 proton and another peak at 3.0 ppm also integrating for one proton. Due to the high degree of symmetry in the (3H peaks it was believed that these two groups must be bound to a single carbon atom. Mass spectrometry was used to deduce the formula as being C 4 3 H 2 8 N 4 N i O - the same as that of the monodecarbonylated product. As this clearly was not the monodecarbonylated product, we focused our efforts on the possible rearrangements of the remaining aldehyde moiety. Taking into account all of the experimental data, it was determined that the observed blue product (193) was a secochlorin with a four-membered ring to which was attached a single proton and a hydroxyl group. Scheme 3.41 Synthesis of (193) from nickel bisaldehyde (145). This very unique product could be formed by the involvement of the secochlorin core in the stabilization of the single aldehyde moiety, as shown below. P h C N , A 170 Chapter 3 Results Ph (193) Scheme 3.42 Proposed mechanism of formation of compound (193). 3.10 Vilsmeier-Haack Reaction of the Secochlorin (191): As the decarbonylation of the nickel bisaldehyde (145) proved to be successful, we felt it would be interesting to attempt the reverse reaction: the formylation of the fully unsubstituted secochlorin (191). Dimethylformamide and phosphorus oxychloride (2 equivalents of each) were stirred at room temperature for one hour at which time one equivalent of the secochlorin (191) in methylene chloride was added to the solution and the reaction left to stir at room temperature for an additional hour. After the work-up, TLC analysis showed the presence of three new compounds (in order of increasing polarity): an olive coloured compound, a blue product and a green compound. 171 Chapter 3 Results The olive coloured product was the previously identified monocarbonylated secochlorin (192) and was formed in 55% yield. The blue compound (194), formed in 31% yield, displayed a UV-visible spectrum almost identical to that of the blue secochlorin (193) incorporating a four-membered ring from the decarbonylation of nickel bisaldehyde (145) with the Soret at 408 nm and the most intense Q-band at 602 nm with shoulders at 544 and 572 nm. Figure 3.25 UV-vis spectrum of the blue product (194). The proton NMR spectrum was very simple with two overlapping doublets representing the 4 P hydrogens, a singlet representing the other two P hydrogens, two multiplets denoting the protons of the four phenyl groups and two very conspicuous singlet peaks at 6.1 ppm and 1.9 ppm. The singlet at 6.1 ppm integrated for one proton whilst that at 1.9 ppm represented six protons. 172 Chapter 3 Results Figure 3.26 Proton NMR spectrum of the blue product (194) formed during the formylation of the fully unsubstituted secochlorin (191). Based on the similarity of the observed UV-vis spectrum with that of the secochlorin incorporating a four-membered ring (193), the high degree of symmetry reflected in both the proton NMR and the mass spectra with m/e = 701, it was concluded that this molecule (194) also was a secochlorin that incorporated a four-membered ring. The substituents on the four-membered ring were determined from the atomic composition report from high resolution mass spectrometry which concluded that the parent peak at m/e = 701 represented a molecule with a formula C 4 5 H 3 3 N 5 N i . Attached to the four-membered ring, therefore, was a dimethylamino group. This group would account for the observed singlet at 1.9 ppm which integrated for 6 protons and is consistent with the fragmentation observed in the EI mass spectrometry: m/e 686 (30%,-CFL,), m/e 657 (70%, -N(CH3)2). 173 Chapter 3 Results (194) (192) Scheme 3.43 Proposed mechanism for the formation of (194) from the reaction of the unsubstituted secochlorin (191). The UV-vis spectrum of the fourth and most polar product (195) had a broad Soret band at 444 nm, and a very broad band at 614 nm with a shoulder at 638 nm. The proton NMR spectrum of the green compound appeared quite messy with several peaks and multiplets in the aromatic region. On closer inspection, it was noted that almost every peak had a small shadowing peak most likely was due to an impurity. We believe that there are at least two products in this mixture but we could not separate them despite numerous attempts and, therefore, we could not identify the components of (195). 174 Chapter 3 Results 3.11 Reaction with Hydroxylamine Hydrochloride (NH 2OH-HCl) The treatment of aldehydes with hydroxylamine forms the analogous oximes. This reaction is well-known and has been studied in depth. The reaction of the bisaldehyde secochlorin (145) with 2.5 equivalents of hydroxylamine hydrochloride in THF at room temperature showed very little progress after 1.5 hours as observed by TLC and UV-vis spectroscopy. Additional reagent was added and stirring continued for 16 hours at room temperature. The reaction was filtered to remove any unreacted, undissolved hydroxylamine hydrochloride and the solvent was evaporated to dryness. At least four very minor products were formed along with a significant amount of a dark green, very polar compound (196). As the yield of the other products was extremely low, only this compound was isolated by preparative TLC and characterized. The UV-visible spectrum showed a considerable bathochromic shift of the Soret band, which appeared at 444 nm with a shoulder at 422 nm. The Q-bands were observed at 540 nm, 580 nm with the most intense band at 632 nm in typical fashion for metallated chlorins and secochlorins. Based on the shift in the Soret band, there was extended conjugation to one or more of the substituents on the secochlorin skeleton. 175 Chapter 3 Results wavelength (nm) Figure 3.27 UV-visible absorption spectrum of the polar product (196) from the reaction of the bisaldehyde secochlorin (145) with hydroxylamine hydrochloride. Mass spectrometry determined the parent peak to correspond to m/e = 671 which is much lower than the expected m/e = 732 for the dialdoxime or even the monoaldoxime which would have m/e = 717. As the molecule (196) definitely had retained the nickel atom, as observed by the UV-visible spectrum, we searched for compounds that had perhaps undergone dehydration. The proton NMR spectrum proved to be the key in the structural determination of this polar compound. A singlet peak at a remarkable 10.1 ppm which integrated for one proton hinted that perhaps one of the aldehyde moieties had been replaced by a hydrogen atom as this is the range for the protons found on the fully unsubstituted secochlorin (9.85 ppm). Two multiplets at 8.73 ppm and 8.58 ppm were determined to represent the 6 P hydrogens whilst multiplets at 7.7 ppm and 7.9 - 8.0 ppm represented the phenyl ring protons. Apart from these peaks, no others were present and therefore the substituents on the secochlorin (196) were: a single hydrogen atom, and a substituent without any protons. 176 Chapter 3 Results -r-, , , ,—r-j-T-,-T—r-r-,-,-,--f-V-^ -.~,-—r-^ -,~r- , | ,—r-i—i—p-i—r~r-r-|-r-.-r-i— |-r-1—•—r-p-r 1 3 . S 1 0 . 0 g . S 3 - B 0 . 5 B . B 7 . 5 7 . 6 B . f> 8 . 0 Figure 3.28Proton NMR spectrum of the polar product (196) from the hydroxylamine reaction. A monosubstituted secochlorin core has the molecular formula C 4 2 H 2 7 N 4 Ni , which shows m/e = 645, which is 26 units less than our parent ion. High resolution confirmed our suspicion with the atomic composition analyzed for C 4 3 H 2 7 N 5 N i - a nitrile group was bonded to the secochlorin. Ph Ph (145) (196) Scheme 3.44 The synthesis of (mes0-tetraphenyl-2-cyano-2,3-secochlorinato)nickel(II) (196). 177 Chapter 3 Results Aldehydes are c o m m o n l y converted to nitriles by reaction wi th hydroxylamine hydrochloride i n the presence o f fo rmic ac id , concentrated hydrochlor ic acid , se lenium d iox ide or pyr id ine-toluene. 2 0 7 The reaction can seen as a combina t ion o f three separate reactions: the condensation o f the hydroxylamine wi th the aldehyde to form the ox ime fo l lowed by the dehydration of the a ldoxime to the ni tr i le w i t h concurrent deformylat ion or decarboxylat ion o f the molecule . W h i l s t the acid-catalyzed deformylation o f aromatic aldehydes is k n o w n , we have prev ious ly observed reactions in wh ich we believe autooxidation to the analogous acid and subsequent decarboxylat ion occurs and, therefore, further invest igat ion into the loss o f the aldehyde group is required. Scheme 3.45 Proposed mechanism for the formation of (196). 178 Chapter 3 Results 3.12 Reaction with Reducing Agents 3.12.1 Lithium Aluminum Hydride (LiAlH4) Lithium aluminum hydride is known to reduce aldehydes to primary alcohols. Because this reagent does not reduce carbon-carbon double bonds and as the reaction is quite general, we thought it would be an appropriate reducing agent. The reaction of nickel bisaldehyde secochlorin (145) with lithium aluminum hydride in refluxing THF produced three products of varying degrees of polarity: one dark green, nonpolar product (197), one yellow-green product of moderate polarity (198), and one dark green extremely polar compound (199). Each of the three products was isolated and characterized. The nonpolar product (197) was initially suspected to be the (me5,o-tetraphenyl-2,3-dimethyl-2,3-secochlorinato)nickel(II) and the experimental data later confirmed that this dark green product was indeed the fully reduced species. The UV-vis spectrum displayed a bisaldehyde secochlorin-like spectrum, with a split Soret band at 418 and 438 nm but possessed double Q bands at 620 and 666 nm, whilst the NMR spectrum showed 6 methyl protons at 6= 2.55 ppm but no peaks beyond 8.5 ppm, as observed for the starting material (145). The yellow-green product (198) of mid-polarity was characterized by proton NMR, and mass spectrometry. The NMR spectrum was very similar to that of the nonpolar dimethyl product (197) except that the singlet at 2.55 ppm integrated for only 3 protons and there were two new peaks present: a singlet at 3.65 ppm representing one proton, and a singlet at 5.88 ppm integrating for two protons. These additional peaks led to the identification of the product as the secochlorin possessing one alcohol moiety and one methyl group, (mes0-tetraphenyl-2-methyl-3-hydroxymethyl-2,3-secochlorinato)nickel(II) (198). 179 Chapter 3 Results The major polar product (199) was identified as the expected (meio-tetraphenyl-2,3-hydroxymethyl-2,3-secochlorinato)nickel(II). The diol displays an interesting absorption spectrum, with a Soret band at 418 nm, three distinct Q-bands at 588, 608, and 632 nm, and a broad absorption band at 770 nm. Ph Ph Ph Ph (145) (197) (198) (199) Scheme 3.46 Products from the reduction of nickel bisaldehyde secochlorin (145) with lithium aluminum hydride. 3.12.2 Sodium Borohydride (NaBH4) We also performed a reaction using NaBH 4 as the reducing agent. We expected to form the tetraphenyl-2,3-hydroxymethyl-2,3-secochlorin (199) that had been observed as the polar product using LiAlH 4 . From this reaction, we obtained one blue-green product (201) of moderate polarity. The absorption spectrum was very different from that of the expected product (199) with a Soret band at 422 nm and two overlapping Q bands at 610 and 636 nm. The 'H-NMR spectrum was very clean with a moderate degree of symmetry reflected by the peaks in the aromatic region which corresponded to the P and phenyl ring protons. Additionally, there were an unusually large number of peaks between 6.5 and 2 ppm- two singlets and two doublets, each representing one proton. Mass spectrometry revealed that the parent ion (m/e 704) had the molecular formula C 4 4 H 3 0 O 2 N 4 N i 5 8 . 180 Chapter 3 Results Taking all of the data into account, we believed that this had to be a cyclic acetal (201) which formed from a reduced product (200). An acetal chlorin with one unsubstituted methylene linkage and a hydroxyl group on the other methylene bridge could be proposed to be formed from the reduction of a single aldehyde group and its subsequent reaction with the second aldehyde moiety. (145) (200) (201) Scheme 3.47 Mechanism proposed for the NaBH4 mediated reduction of the nickel bisaldehyde secochlorin (145). Reaction Conditions: i. 1 eq. NaBH4 THF. 3.13 Reaction with Osmium Tetroxide We thought that it would be very interesting if the nickel bisaldehyde secochlorin (145) could be oxidized with osmium tetroxide to form the corresponding tetraphenyl-2,3-dicarboxaldehyde-7,8-dihydroxy-2,3-secobacteriochlorin (202). 181 Chapter 3 Results Ph C H O CHO O s 0 4 ) Py CHO Ph l{ X N i ' \ Ph Vh Ph Ph (145) (202) Scheme 3.48 Proposed osmium tetroxide oxidation of nickel bisaldehyde secochlorin (145). Although it was extremely disappointing to learn that this product was not formed, we did observe other interesting products. This reaction was performed many times, in hopes that the alteration of reaction conditions would yield the desired product but to no avail. The nickel lactone chlorin (155) was one of the major products that was consistently formed. Usually two additional products were formed in this reaction, both in low yields and both being green in colour. The absorption spectrum of the nonpolar green product was identical to that of the previously observed nickel acetals and further characterization confirmed that this was indeed the nickel acetal (150) presumably derived from reaction with methanol in the solvent system. The more polar of the two green coloured compounds was found to be the hemiacetal (203) corresponding to the above-mentioned acetal. Whilst hemiacetals are usually not stable enough to be isolated, we believe that this hemiacetal is. This compound (203), upon standing in solution, yields the nickel bisaldehyde (145) and acetals (149) and (150). The proton NMR spectrum, the UV-visible spectrum and the fragmentation observed in mass spectrometry are all very different from those of the known acetal (149) with the same molecular formula and are consistent with the assigned hemiacetal structure. Stable hemiacetals of quarternary heterocyclic carbaldehydes are known to exist as well as those of certain aromatic aldehydes possessing electron-withdrawing groups. 3.14 Summary In this chapter many novel compounds have been discussed. Classic reactions with aldehydes were used to guide our investigation of the reactivity of nickel bisaldehyde secochlorin (145) and led to both expected and unexpected products. All of the products can be viewed as either potential photosensitizers or as starting materials for such compounds. Free base bisaldehyde secochlorin (148) was elusive and its synthesis will be the goal of further research. Listed below are the compounds isolated in this study. 183 Chapter 3 Results CHO ,CH=NMe (172) (182) (183) 184 Chapter 3 Results (201) (203) Chapter 4 Dipolar Cycloadditions 186 : Chapter 4 Results 4.1 Introduction to 1,3-Dipolar Cycloaddition Reactions In the 1950s and 1960s, Huisgen and coworkers performed monumental work that led to the general concept of the 1,3-dipolar cycloaddition.208"210 These types of reactions have become prominent in organic chemistry due to the vast number of bonds that undergo transformations.211"213 A "1,3-dipole" is a species that is represented by a zwitterionic octet structure and undergoes 1,3-cycloadditions with a multiple-bond system, the "dipolarophile". The 1,3-dipolar cycloaddition is a [3 + 2 -> 5] cycloaddition which forms a five-membered heterocyclic ring. The ring closure is effected by cyclic electron shifts which form two new o bonds at the expense of re bonds. Over 18 different types of 1,3-dipoles have been employed in such reactions, presenting numerous possibilities for variation over and above the variety due to the wide diversity of the nature of the dienophile. © Scheme 4.1 1,3-Dipolar Cycloaddition Reaction All 1,3-dipoles incorporate an onium center whose positive charge neutralizes the negative charge on one of the terminal atoms to form a heteroallyl anion which bears no net charge. Two of the four allylic re electrons can be delocalized at the center atom. The terminal centers of the dipoles can be either nucleophilic or electrophilic - the key to the reactivity of all 1,3-dipoles. Formally, there are two types of 1,3-dipoles: those in which the central atom is sp-hybridized and those whose central atom is 5/?2-hybridized. The latter group have allyl anion type TI systems with four electrons in three parallel atomic Tt orbitals perpendicular to the plane of the 187 Chapter 4 Results dipole. This type of 1,3-dipole is bent and the central atom can be oxygen, nitrogen or sulfur. © © Figure 4.1 Allyl anion type resonance structures 1,3-Dipoles having an ^ -hybridized central atom are referred to as propargyl or allenyl types. These are linear and the central atom is confined to nitrogen. _ © Q 0 © a=b—c ~" *~ a= b = c Figure 4.2 The propargyl/allenyl anion type of 1,3-dipole. To our knowledge, 1,3-dipolar cycloadditions with tetraphenylporphyrins have only been reported with diazoacetate and diazomethane.214 Cavaleiro et al. have performed a number of Diels-Alder reactions with tetraphenylporphyrins.2'5'216 One example of such a reaction is that of tetracyanoethylene (TCNE) with (mono-P-vinyl-me50-tetraphenylporphyrinato)nickel(U) (204).21S This reaction produced both [4 + 2] (205) and [2 + 2] (206) adducts as shown below. Rearrangement of the [4 + 2] product (205) to the thermodynamically stable [2 +2] product (206) was observed. 188 Chapter 4 Results Ph Ph Ph (204) (205) (206) Scheme 4.2 Reaction of (P-vinyl-//feso-tetraphenylporphyrinato)nickel(II) (204) with T C N E . Cavale i ro et al. also reported the dienophile- l ike nature o f raeso-tetraphenylporphyrins.216 React ion of tetraphenylporphyrin wi th o-benzoquinodimethane (207) (generated in situ by heating sulfone (208)) p roduced ch lo r in (209), naphthoporphyrin (210) and porphyr in (211). Ph P h P h P h (209) (210) (211) Scheme 4.3 Reaction of tetraphenylporphyrin with o-benzoquinodimethane (208). 189 Chapter 4 Results A s on ly the 1,3-dipolar cycloaddi t ions between diazomethane and diazoacetate have been p rev ious ly performed, and because this f ie ld presents a huge potential as a source for novel tetraphenylchlorin-based photosensitizers, we felt compel led to investigate the reactivity o f tetra- and diphenylporphyr ins w i th selected 1,3-dipoles. A survey o f the literature was performed in order to determine wh ich 1,3-dipoles were either par t icular ly reactive and/or reactive towards aromatic systems. E x a m p l e s from eight different classes o f 1,3-dipoles were selected and tested. These classes were: carbonyl yl ides , nitr i le oxides, d iazoalkanes , azides, azomethine yl ides , ni tr i le yl ides , nitrones and azomethine imines. These reagents used and reactions performed w i l l be discussed according to these classif icat ions. Table 4.1 Classes of 1,3-dipoles. 190 Chapter 4 Results Propargyl-Allenyl Type Nitri l ium Betaines — C s N - C C + r. — C = N - 0 — C = N - N \ •C=N=CC .-. + -C=N=0 -C=N=N Nitrile Ylides Nitrile Oxides Nitrile Imines Diazonium Betaines N E N - C C - • N = N - N . N = N - 0 N=N=CC N=N=ISk N = N = 0 Diazoalkanes Azides Nitrous Oxide Allyl Type Nitrogen Center ^ C = N - C : / C = N - N N i / C = N - 0 l \ + r. N = N - N > \ + •"• N = N - 0 I 0 = N - 0 l Oxygen Center c^=o-c: ^ C = 0 - N , ^ C = 0 - 0 N = 0 - N , N=0 -6 + r. 0 = 0 - 0 ;'C-N=CC ; C - N = N ^ ; C - N - - O I S N - N = N S S N - N = 0 I 0 - N - - 0 I :c-o=cc : C - O = N -:c-6=o N^-6=NS ^N-6=0 .-. + 0 - 0 = 0 Azomethine Yl ides Azomethine Imines Nitrones Azimines Azoxy Compounds Nitro Compounds Carbonyl Ylides Carbonyl Imines Carbonyl Oxides Nitrosimines Nitrosoxides Ozone 191 Chapter 4 Results: 4.2 Carbonyl Ylides In 1965, Linn and Benson reported the reaction of tetracyanoethylene oxide (TCNEO) (212) with olefins, acetylenes and benzene at high temperatures.217"219 The products were those formed from [3 + 2] cycloadditions with the carbonyl ylide. A first-order electrocyclic ring opening via cleavage of the carbon-carbon bond of the epoxide to the 1,3-dipole occurs.219 Scheme 4.4 Resonances structures of TCNEO (212). The dipole (212) then adds cleanly to a variety of olefins, including aromatic substrates. More than thirty substituted tetracyanotetrahydrofuran products were prepared in the original report. Experimental conditions involved refluxing equimolar amounts of TCNEO (212) and the dipolarophile in a suitable solvent to allow temperatures above 100°C to be maintained for 4 to 18 hours. Aromatic dipolarophiles included benzene, naphthalene, toluene, 1,3-cyclohexadiene and furan. These reagents produced the corresponding 1:1 adducts in 18-73% yields.218 © © NC \ ^ Reflux in toluene 18 hr Scheme 4.5 Reaction of benzene with TCNEO (212). 192 Chapter 4 Results The reaction of naphthalene with TCNEO (212) was of particular interest to us as the osmium tetroxide-mediated oxidation of naphthalene requires similar reaction times and conditions as does meso-tetraphenylporphyrin. Naphthalene reacts with one equivalent of TCNEO (212) in 1,2-dibromoethane to give a single monoadduct at the 1,2-position in 73% yield after 4.75 hours. Polyaddition products were not produced even in the presence of excess TCNEO (212).218 After one hour of refluxing, the reaction of TCNEO (212) with tetraphenylporphyrin in 1,2-dibromoethane produced two purple compounds. Starting material recovery was quite high (48%) but the effect of longer reaction times is unknown at the present time. The least polar burgundy product was found to be the free base lactone chlorin (156) whilst the most polar compound (213) was more interesting. The absorption spectrum was chlorin-like with a Soret band at 416 nm and four Q bands at 518, 548, 586 and 642 nm with the Q band at 642 nm being the most intense. A parent ion with m/e = 678 was observed by mass spectrometry. This is 64 units less than expected for a TCNEO (212) adduct. The proton NMR of this product indicated a high degree of symmetry with the P proton peaks at 8.7 ppm (d), 8.5 ppm (s) and 8.35 ppm (d). Additionally, a singlet integrating for 2 protons was observed at 6.85 ppm representing the pyrrolidine protons of the chlorin. High resolution mass spectrometry revealed the molecular formula of this compound to be C 4 7 H 3 0 N 6 , and we believe that this compound (213) contains a cyclopropyl moiety. Maximum yield of this compound (213) was 40% but we believe that this yield can be improved with further fine-tuning of the reaction conditions. A survey of the literature revealed the tendency of TCNEO (212) to fragment upon reaction with pyridine and other tertiary nitrogen bases.217 The product of the reaction with pyridine is pyridinium dicyanomethylide (214) and the other product which has not been isolated has been presumed to be carbonyl cyanide. Similar reactions have been reported.217 N 4> NC N ^ / C N ,C C N NC C N T H F , -20°C 2 hr O H 1 + 11 IL ^ C(CN) 2 © N ec(CN) 2 (214) Scheme 4.7 Reaction of pyridine with T C N E O (212). In 1996, a communication was published detailing the synthesis of a cyclopropanochlorin (27) from the Barton-Zard reaction of zinc(II) 2-nitro-5,10,15,20-tetraphenylporphyrin (26).147,220 In 1998, the same authors published a communication which employed the dicyanocyclopropanochlorin (213) as a starting material. No synthetic or experimental details were 194 Chapter 4 Results Scheme 4.8 Barton-Zard reaction of 2-nitro-5,10,15,20-tetraphenylporphyrin (26). As 5,15-diphenylporphyrin is known to be more reactive than tetraphenylporphyrins towards P addition reactions, such as osmium tetroxide mediated oxidation, we chose to investigate the reaction between DPP and TCNEO (212). Reaction of 1.5 eq. T C N E O (212) with DPP after refluxing in toluene for 3 hours produced two compounds: a red nonpolar compound (215) and a purple compound (216). The absorbance spectrum of this product was very unusual with a split Soret at 376 and 416 nm and two intense overlapping Q bands at 510 and 534 nm. The proton NMR spectrum of this compound (215) was quite different from that of the starting material. Two doublets at 6.5 ppm and 6.6 ppm, both integrating for 2 protons each, and a triplet at 7.1 ppm which integrated for 4 protons hinted that the molecule had lost its aromaticity but retained some sort of conjugation. Additionally, there were no peaks below 0 ppm to represent the NH protons. Instead there was a sharp singlet peak at 13.9 ppm which integrated for two protons. Protons on the nitrogen atoms of dipyrromethenes have previously been observed in this range. The protons of the two phenyl groups reported. 195 Chapter 4 Results were observed at 1 A-l.5 as a large multiplet. Mass spectrometry of the red compound (215) revealed a compound with m/e = 540, and a molecular formula of C 3 5 H 2 0 N 6 O. This corresponded to DPP plus C 3 ON 2 and a loss of two protons. Taking all of the experimental data into account, we determined that we had formed the product shown below. (215) Scheme 4.9 Formation of the red product (215) from the reaction of DPP with TCNEO. The purple product (216) again displayed an unusual absorbance spectrum with one broad Soret band at 400 nm and one intense band at 562 nm. The proton NMR spectrum of the purple product (216) had a doublet representing four protons at 6.65 ppm, a doublet at 7.2 ppm also representing four protons, a multiplet representing the phenyl protons at 7.4-7.55 ppm and a singlet at 13.75 ppm representing two protons. The peaks at 6.65 and 7.2 ppm (both doublets with 7=4.5 Hz) and the lack of splitting of the Soret and Q-band both indicated a higher degree of symmetry as compared to the red product (215). High resolution mass spectrometry determined the molecular formula of the parent ion at m/e 588 to correspond to a molecular formula of C 3 g H 2 0 N 8 . The mechanism of formation of this product (216) presumably involves electrophilic aromatic substitution at the meso positions. Product (215) could be formed by hydrolysis of (216). 196 Chapter 4 Results N C ^ ^ C N C (216) 0^ Scheme 4.11 Proposed formation of product (216). 197 Chapter 4 Results Another reaction involved the use of 3.6 equivalents of TCNEO (212) and ZnDPP. After one hour of refluxing in toluene, no starting material remained therefore the reaction was worked up and preparative TLC performed. The absorption spectrum of the product (217) was once again very unusual with a broad Soret band at 458 nm and an equally intense Q band at 638 nm. The proton NMR spectrum was similar to that observed for the purple product (216) formed in the reaction of TCNEO with free base DPP in that there were 2 doublets of equal integration at 6.5 and 7.1 ppm. High resolution mass spectrometry confirmed that this compound (217) was indeed the zinc metallated analog of the purple product (216) as the parent ion at m/e 650 corresponded to the molecular formula C 3 8 H 1 8 N 8 Zn. N C ^ / C N C N C " C " C N (217) Scheme 4.12 Reaction of ZnDPP with T C N E O . We were curious as to the outcome of this reaction when ZnTPP was used as the starting material. This reaction was performed a number of times and we consistently observed the formation of a green (218) product. The product (218) displayed an absorption spectrum with a shoulder at 428 nm, a sharp Soret band at 442 and two Q bands at 584 and 638 nm. Low resolution mass spectrometry revealed a parent ion peak at m/e 738. High resolution mass spectrometry 198 Chapter 4 Results: indicated this corresponded to a molecular formula of C 4 7 H 2 6 N 6 Z n 6 4 which is one C ( C N ) 2 fragment more and, once again, 2 protons fewer than the starting material. The proton N M R spectrum was very similar to that of the previously discussed mono-cyclized compounds derived from acid treatment of formylated TPPs. The characterization data led us to investigate the plausibility of the cyclization of the dicyano moiety. This compound is currently under further investigation. (218) Scheme 4.13 Possible reaction of ZnTPP with T C N E O (212). 199 Chapter 4 Re stilts: 4.3 Nitrile Oxides Nitrile oxides are commonly generated in situ from the reaction of triethylamine and the hydroximoyl or hydrazonoyl halides as these dipoles are unstable.221 Nitrile oxides are usually represented by the nitrilium betaine formula R-CN +-Z" but there are many important resonance structures as shown below: NEt3 © O © © © 0 © © — C = N - O H u '1 * — C = N - 0 ^ - « — » • — C = N = 0 - « — C = N - 0 ^ - « — - — C = N - 0 H " H 2 0 Scheme 4.14 Resonance structures for nitrile oxides. The reaction of nitrile oxides with alkenes produces 2-isoxazolines. Nitrile oxides have been shown to react with a variety of olefinic dipolarophiles. Benzene and naphthalene do not react with nitrile oxides presumably due to the inherent loss of resonance energy although the reaction of phenanthrene with mesitonitrile oxide (219) does yield a cycloadduct.223 .c-to© + — ^ % Scheme 4.15 Reaction of a nitrile oxide with an alkene. Despite the lack of reactivity of aromatic dipolarophiles with nitrile oxides we were optimistic about the reaction of porphyrins with nitrile oxides as the loss of resonance energy incurred during the reaction would be much less than that with benzene. Mesitaldehyde oxime (220) was prepared by reacting hydroxylamine hydrochloride with mesitaldehyde (219) according to a procedure from the literature.224 Treatment of the mesitaldehyde oxime (220) with NCS produced the corresponding mesitohydroximinoyl chloride (221) 200 Chapter 4 Results quantitatively. The reaction of the 2,4,6-trimethylbenzonitrile oxide (222) (prepared in situ via reaction of the mesitohydroximinoyl chloride (225) with triethylamine) with diphenylporphyrin was performed at room temperature. After 2 days of stirring, a second, less polar compound (223) was observed by TLC. The reaction was worked up and the new compound purified and characterized. The product had an absorption spectrum that was very similar to chlorins with a Soret band at 404 nm and Q bands at 502, 530, 580 and the strongest band at 632 nm. The proton NMR also indicated the formation of the chlorin as there were two doublets at 6.6 ppm. Mass spectrometry confirmed the identity of our product (223) as being the chlorin formed from the 1,3-dipolar cycloaddition of 2,4,6-trimethylbenzonitrile oxide (222) to diphenylporphyrin. (223) Scheme 4.16 Synthesis and reaction of 2,4,6-trimethylbenzonitrile oxide (222) with DPP. 201 Chapter 4 Remits. This reaction was also performed using ZnTPP. Although the reaction of D P P with the trimethylbenzonitrile oxide (222) was performed using 4.5 equivalents of the reagent, the reaction using zinc tetraphenylporphyrin was performed with 15 equivalents of the nitrile oxide (222) as this porphyrin is known to be less reactive than D P P with respect to (3,P'-addition reactions. This reaction was allowed to proceed at room temperature for one week at which time no reaction had occurred. We therefore repeated the same experiment but employed 25 equivalents of the 1,3-dipole. After 2 days a new green polar compound (224) was visible and the reaction left to proceed. After one week, the reaction was worked up and preparative T L C performed. Much to our dismay, the green product could not be isolated in sufficient quantities for any significant characterization. We believe that this green product was the product of the 1,3-dipolar cycloaddition but due to time constraints, this reaction was left for further investigation. We were also curious to investigate the reactivity of the tetraphenyl diol chlorin towards 1,3-dipolar cycloadditions. One equivalent of the free base tetraphenyl diol chlorin was reacted with 25 equivalents of the trimethylbenzonitrile oxide (222) and the reaction left to stir. Unfortunately, no reaction was observed but this would be an interesting area of future studies. This reaction was also attempted using the commercially available benzonitrile oxide (225). These reactions did not produce any adducts. We believe this may be due to the highly recognized polymerization reaction of this nitrile oxide (225). Due to the length of time and large excess of reagent our reaction requires, the polymerization reaction predominates. 202 Chapter 4 Results Scheme 4.17 Products of the dimerization of benzonitrile oxide (225). 4.4 Diazoalkanes Diazo compounds have been known to undergo cycloadditions with carbon-carbon multiple bonds for over 100 years.225'226 Pyrazolines are the usual product resulting from the cycloaddition of diazo compounds with alkenes. The initial five-membered rings are useful intermediates in the syntheses of cyclopropane rings and olefins due to their propensity for substituent shifts. Whereas ethylene and diazomethane react to form the corresponding pyrazoline nearly quantitatively, much lower yields are observed for the same reaction with both cyclic and acyclic dienophiles. Nonetheless, reaction does occur. Cyclopentadiene reacts with diazomethane to produce the bicyclic pyrazoline in 45% yield with subsequent pyrolysis forming the exo- and endo-bicyclo[3.1.0]hexenes. Monoadducts have been observed upon the reaction of 1,3,5-trinitrobenzene with diazomethane. In 1972, Callot reported the reactions of tetraphenylporphyrin with diazomethane, diazoacetate and diazodiacetate in the presence of copper chloride.227 2 2 8 Yields of the cyclopropanochlorins were 28%, 20% and 30% respectively. 203 Chapter 4 Results (226) R A = R B = H (227) R A = C 0 2 C H 3 ; R B = H (228) R A = R B = C 0 2 C H 3 Scheme 4.18 Reaction of ZnTPP with diazo compounds in the presence of CuCl . Prior to the discovery of this paper,227 we believed that only diazoacetate had been employed as a reagent in reactions with tetraphenylporphyrins and we, therefore, attempted to form the unsubstituted cyclopropanochlorin (226) with diazomethane. In contrast to the reactions performed by Callot, we did not use copper chloride as a catalyst. Base catalyzed decomposition of 1 gram of /V-methyl-/V-nitroso-/?-toluenesulfonamide (Diazald®) yielded the reactive 1,3-dipole diazomethane. This was added dropwise to a solution of free base tetraphenylporphyrin (0.11 g) in THF. Once the addition was complete, the reaction vessel was sealed and left to sit in the dark for 7 days. Monitoring with T L C showed the appearance of a polar green compound. This compound was isolated via preparative T L C and characterized. The yield of the reaction very low (3%). The absorbance spectrum of the new polar compound indicated the chlorin-like nature of the product with a Soret band at 418 nm, and four Q bands at 514, 548, 604 and the most intense band at 654 nm. Callot reported the same spectrum for the free base, unsubstituted cyclopropanochlorin (226). Mass spectrometry confirmed the identity of our product as being the cyclopropanochlorin (226). 204 Chapter 4 Rpxnhs Although the yield of our reaction was so low as to prevent confirmation using NMR spectroscopy we believe that the product formed is the desired cyclopropanochlorin (226). It is apparent from the difference in yields (28% versus 3%) that this reaction is much more efficient and viable when performed in the presence of CuCl. Whilst we were searching the literature for cyclopropane ring formation with poiphyrins, we discovered a Russian paper which detailed the reaction of the 2,3-diol chlorin of etioporphyrin (229) with zinc(II) acetate and 2,4-pentadione.229 This reaction reportedly produced three major products: diacetylcyclopropylchlorin (230), a b/s-chlorin dimer (231) and the (3-oxo analog (232) in good yields (18%, 14% and 19% respectively). Zn(OAc) 2 2,4-pentadione/ reflux (221) C O C H 3 COCH3 (222) (223) (224) Scheme 4.19 Reaction of diol chlorin of etioporphyrin (229) with zinc acetate in 2,4-pentadione 205 Chapter 4 Results We were intrigued and attempted this reaction employing our tetraphenyl-2,3-diolchlorin (146) using the same reaction conditions. Zinc(II) tetraphenyl-2,3-diolchlorin (146) (60 mg, 0.08 mmol) was refluxed in 2,4-pentadione (20 mL) containing excess zinc acetate for 2 days. After 24 hours, no starting material remained - a nonpolar red compound (minor product) and polar green compound (major product) were observed by T L C . As the reaction proceeded, the yield of the red compound increased. After 2 days, the reaction was worked up and preparative T L C performed to yield three compounds: a bright red compound, a blue-green compound and a green product (in order of increasing polarity). The least polar red compound was characterized and determined to be the corresponding P-oxo analog (233) as per the literature.229 The blue-green product was determined to be the zinc lactone chlorin (235) whilst the polar green compound was determined to be the p\P'-dioxo analog (234). We had not formed any cyclopropylchlorin nor were there any dimers. Ph Ph Ph Ph (146) (233) (234) (235) Scheme 4.20 Reaction of zinc(II) diol chlorin (146) with Zn(OAc)2 and 2,4-pentadione. Reaction Conditions: (i) Excess Zn(OAc)2, 2,4-pentadione, reflux 2 days. The P-oxochlorin (233) has been synthesized by a number of methods. In particular, zinc tetraphenyl diol chlorin (146) when refluxed with either traces of strong acids or in the presence of Lewis acid zinc chloride was found to produce the P-oxochlorin (233).197 Similarly, the P,P'-dioxo analog (235) has been formed from the DDQ-mediated oxidation of zinc tetraphenyl chlorin (146).197 206 Chapter 4 Results 4.5 Azides Formation of 1,2,3-triazoles from the cycloaddition reaction of multiple bonds and azides has been known, and used, for many decades.230 Classically, ring strain is measured by the ease of reaction with phenyl azide as the rate of formation of 1,2,3-triazoles is greatly increased as is ring strain. Scheme 4.21 The reaction of an azide with an alkene. In general, electron-rich dipolarophiles react fastest with electron-poor azides, and electron-poor dipolarophiles react most rapidly with electron-rich azides.232 The reaction is stereospecific and this fact has been suggested to reflect the concertedness of the reaction. All evidence points to a biplanar transition state, wherein the azide lies in one plane directly above or below the second parallel plane of the dipolarophile.233 Unactivated, unstrained carbon-carbon double bonds react very slowly with azides at room temperature, with reactions requiring well over a week. For example, /?-chlorophenyl azide reacts with 1-hexene to form the corresponding triazoline product in 89% yield but required 5.5 months at room temperature.234 Electron-withdrawing groups on the azide have been shown to accelerate the reactions, but also increase the lability of the resultant triazoline product.232 Reactions with conjugated diolefins have been reported and have been observed to react more rapidly than mono-olefins. Cycloaddition of p-bromophenylazide to 1,3-cyclohexadiene produced the corresponding triazoline (a mono-adduct) in 73% yield after 8 days at room temperature.235 Commercially available azidotrimethylsilane (236) (2 eq.) and tetraphenylporphyrin (50 mg, N © © = N = N + = N 207 Chapter 4 Results 0.08 mmol) were dissolved in dry THF (10 mL) and the reaction mixture stirred at room temperature for 24 hours. No reaction had occurred as observed by T L C and UV-Visible spectroscopy, therefore the reaction mixture was set to reflux for 36 hours. Unfortunately, no reaction was observed. M e 3 S i x Ph H / N ^ N .N Me 3 SiN 3 / / r M ^ • THF, r.t. or reflux Scheme 4.22 Proposed reaction of azidotrimethylsilane (236) with TPP. 4.6 Azomethine Ylides This type of 1,3-dipole is classified as an azomethinium betaine displaying internal octet stabilization but lacking a double bond in the sextet structure. These allyl-type dipoles are bent in the ground state. \ / \ \©^L 0 / c ^ . . \ c / \ Figure 4.3 An azomethine ylide A number of methods exist to form azomethine ylides." Thermolysis and photolysis of readily accessible aziridines and the dehydrohalogenation of immonium salts are some of the most popular techniques available. Aziridines can be heated to between 100°C and 120°C, breaking the 208 Chapter 4 Results carbon-carbon bond thereby yielding in situ the analogous azomethine ylide." Thermolysis of aziridines leads to the conrotatory ring-opening. Alternatively, photolysis has been employed for the disrotatory electrocyclic ring-opening of aziridines.237 The 4TC aziridine amidic anion is isoelectronic with the 4TX dipolar azomethine ylide, and therefore should theoretically undergo similar electrocyclic ring-opening to form a linear amidic anion capable of addition to multiple bonds. Experiments generating such amidic anions were performed by Kauffmann and coworkers using AMithio-2,3-cis-diphenylaziridine (238).238-240 1,3-Diphenyl-2-azaallyllithium (238) was prepared by reaction of N-benzylidenebenzylamine (237) with lithium diisopropylamide (LDA) at -60°C. The anion was then allowed to react with an olefin (trans-stilbene, c/s-stilbene and acenaphthalyne) in THF under nitrogen to produce the corresponding monoadduct (83%, 21% and 80% respectively).238 Li H H (237) (238) Scheme 4.23 Reaction of LDA with A -^benzylidene benzylamine (237). H N (238) Scheme 4.24 Reaction of 7V-lithio-2,3-c/s-diphenylaziridine (238). 209 Chapter 4 Results As this reagent appeared to be quite reactive, we attempted the same reaction with porphyrins. Free base tetraphenylporphyrin, the corresponding zinc(II) porphyrin and diphenylporphyrin were employed as starting materials. In a typical experiment, excess TV-lithio-2,3-cw-diphenylaziridine (238) was prepared in situ at -60°C and the porphyrin dissolved in methylene chloride or chloroform added dropwise. A new slightly polar spot was observed to have formed in the reactions with both diphenylporphyrin and ZnTPP. No reaction occurred with the free base tetraphenylporphyrin. These products could not be isolated but high resolution mass spectrometric analysis of the product from the reaction with DPP consistently determined that a benzyl ion had been added to the porphyrin. Although we do not have data to confirm or deny any proposal, we suggest, merely as a starting point for future studies, the structure shown below. Scheme 4.25 Proposed reaction of DPP with an azomethine ylide (238). An alternate method of generating azomethine ylides is the "decarboxylation" method. Following a literature procedure,241 sarcosine (/V-methylglycine) (239) and paraformaldehyde (240) were refluxed in toluene in the presence of tetraphenylporphyrin. Whereas this reaction has been reported241 to give good yields of the iV-methylpyrrolidine analog with other olefins and C 6 0 , no reaction was observed with porphyrins. 210 Chapter 4 Results: C H 3 - N H - C H 2 C 0 2 H + C H 2 Q -- C O P (239) (240) Scheme 4.26 Generation and attempted reaction of an azomethine ylide (241). A similar set of reactions were performed using glycine (242) and either paraformaldehyde (240) or benzaldehyde (243).242 The same conditions were applied and both zinc TPP and diphenylporphyrin were used as starting materials. H 2 N - C H 2 C 0 2 H + RCHO A -co2 (236) R=H (237) R=Ph (238) H A© RHC C H 2 (239) -7 toluene, A Scheme 4.27 Formation of an azomethine ylide (244) and attempted reaction with TPP. One new product from reaction of each starting material (DPP and ZnTPP) with benzaldehyde (243) and (242) was observed by mass spectrometry (EI - 5-20% yield) but could not be isolated. These products corresponded to an increase in mass of 105 g/mol. As the yields were so low and the products unstable, we discontinued our investigation of this reaction. Based on past observations, we suggest the possibility of the fragmentation of the 1,3-dipole. A mass of 105 g/mol corresponds to the mass of the PhCH=NH- fragment, which is the 1,3-dipole less a methylene group. This Chapter 4 Results reaction was left for further investigation, and a possible product is shown below. 211 H l 0 to luene, A TPP N — H " N - — \ (244) M = H 2 (245) ^ M = Z n (246) Scheme 4.28 Suggested products (245) and (246) from reaction of porphyrins with (244). 4.7 Nitrile Ylides Nitrile ylides are 1,3-dipoles which possess a C-N-C moiety with two a bonds and six electrons in both TC and n orbitals. Figure 4.4 Resonance structures of a nitrile ylide. Treatment of the analogous imidoyl chloride derivatives with triethylamine at room temperature leads to the reactive nitrile ylide via elimination of hydrogen chloride.226 Reaction of triethylamine with TV-benzyl /?-nitrobenzimidoyl chloride (247) quantitatively yields p-nitrobenzo-nitrilio phenylmethanide (248).232 Huisgen investigated the reaction of nitrile ylides with both C = N 0 © -C=N= 212 Chapter 4 Results: strained and simple conjugated olefins. Of interest, acenaphthylene (249) was found to undergo a 1,3-dipolar cycloaddition with p-nitrobenzonitrilio phenylmethanide (248) in 48% yield. 2 4 3 , 2 4 4 p - 0 2 N - C 6 H 4 — C N-CH 2-Ph \ CI (247) Et 3 N - HCI © 0 p - 0 2 N - C 6 H 4 — C = N - C H - P h (248) p - 0 2 N - C 6 H 4 (250) (48%) Scheme 4.29 Reaction of /j-nitrobenzonitriliophenylmethanide (248) with (249). This reaction was performed using zinc(II) TPP numerous times. The vast majority of these reactions did not yield any isolable products despite changes in solvent, molar ratios and temperature. Thin layer chromatography of almost all of the crude reaction mixtures did show the presence of a more polar compound but this product disappeared upon work-up procedures. One 213 Chapter 4 Results: reaction did produce a peak representing the monoadduct in mass spectrometry in approximately 1% yield. This particular reaction involved a large excess of the 1,3-dipole and was left to reflux in methylene chloride for 96 hours. Although slightly promising, reactions with nitrile ylides were left for future investigations. 4.8 Nitrones Nitrones can be formed conveniently via the condensation of /V-alkyl- or N-arylhydroxylamines with aldehydes or ketones.245 Nitrones with C-aryl substituents are most stable, those lacking such a moiety are easily dimerized or trimerized. The reaction of /V-phenylhydroxylamine (251) with phenylglyoxal hydrate (252) produces the 1,3-dipole C-benzoyl-/V-phenylnitrone (253). Tetraphenylporphyrin in methylene chloride was added to the nitrone (253) at ~0°C. After 6 hours no reaction was observed, and the mixture was slowly warmed to room temperature and later set to reflux. The reaction was monitored by TLC whilst refluxing for two days, but no reaction occurred. Ph PhNHOH + PhCOCHO (251) (252) Scheme 4.30 Formation and attempted reaction of C-benzoyl-yV-phenylnitrone (253). 214 Chapter 4 Results: 4.9 Azomethine Imines Azomethine imines have an iminium center and allyl anion stabilization.232 The resonance structures are shown below: © 0 : C = N - N 0 © X ~ N = N -I Figure 4.5 Resonance structures of azomethine imines. Foote et al.246'241 have reported the 1,3-dipolar cycloaddition of TV-benzyl azomethine ylide (255) to C 6 0 . TV-Benzyl azomethine ylide was prepared in situ from the trifluoroacetic acid catalyzed desilylation of A^-benzyl-TV-(methoxymethyl)-TV-[(trimethylsilyl)methyl]amine (254).246,247 After 4 hours at room temperature, the corresponding monoadduct was isolated in 50% yield. Me 3 Si- •OMe T F A N C H 2 P h (254) © N © C H 2 P h (255) Scheme 4.31 Synthesis of TV-benzyl azomethine ylide (255) from TV-benzyl-TV-(methoxymethyl)-TV-[(trimethylsilyl)methyl]amine (254). This reaction was performed with both DPP and ZnTPP and although 2 new products were observed and isolated, characterization was inconclusive. Due to time constraints the reaction was not repeated but appears promising. 215 Chapter 4 Results 4.10 Conclusion Although our attempts at 1,3-dipolar cycloadditions with both di- and tetraphenylporphyrins met with limited success, we believe that we have uncovered a relatively unexplored field of porphyrin research. With the vast number of 1,3-dipoles and their extreme variety, we feel this area of porphyrin chemistry will be increasingly important in the coming years. 216 Chapter 5 Experimental 217 Experimental EXPERIMENTAL INSTRUMENTATION AND GENERAL MATERIALS The infrared spectra were measured with a Perkins-Elmer Model 834 FT-ER. instrument. The 'H-NMR were measured on a Bruker AC-200 spectrometer (200 MHz) or a Bruker WH-400 (400 MHz). 1 3 C-NMR were measured on a Varian XL-300 (75 MHz) spectrometer. The NMR are expressed on the 6 scale and are referenced to residual solvent peaks and TMS. The low and high resolution FAB and EI mass spectra were obtained on a AEI MS902 and a Kratos MS50. The UV-visible spectra were measured on a Hewlett-Packard HP 8452A photodiiode array spectrophotometer and the data were processed on a microcomputer (CA Kricket Graph III software). Chromatography was performed on silica gel 60, 70-230 mesh, supplied by E. Merck Co. Preparative thin layer chromatography was prepared on pre-coated 10cm x 10cm, 0.5 mm thick Merck silica gel plates. Elemental analyses were performed by Mr. P. Borda on a Fisons CHN/O Analyzer, Model 1108. Typically, porphyrins do not combust well. Combustion analyses of the majority of the compounds synthesized herein were found to be irreproducible and are, therefore, not reported. 218 Experimental Tetraarvlporphvrin Synthesis Adler's Method According to a literature procedure,248 the appropriately substituted benzaldehyde and pyrrole were mixed in propionic acid (500 mL) and the solution refluxed for 75 minutes. The solution was cooled and concentrated to 100 mL. The reaction mixture was then filtered through silica gel (~ 50 mL) to remove any insoluble polymeric byproducts. The filtrate was concentrated (-50 mL)and chromatographed (silica gel, eluent methylene chloride to methylene:methanol 1:1) to yield the desired porphyrin. Lindsey's Method (porphyrins synthesized by this method are indicated by means of * in table below) According to a literature procedure,249 samples of T C Q (para-chloranil) (5 mol%) and FePc (iron phthalocyanin) (5 mol%) were placed in a three-necked round bottom flask. Methylene chloride (reagent grade) (-300 mL) was added and the mixture stirred. The reaction was purged with nitrogen for 15 minutes. The appropriate aldehyde (0.1 M solution) and pyrrole (1 eq., 0.1 M solution) were added and the mixture stirred. Dry BF 3 -OEt 2 (boron trifluoride etherate) (1 eq., 0.1 M solution) was added and the reaction purged with nitrogen and stirred for 30 minutes. The mixture was then purged with air for 90 minutes with stirring. The mixture was reduced via evaporation of the solvent in vacuo to approximately 100 mL at which time 20 mL of silica gel was added to the flask. The mixture was concentrated to a dry solid which was added to a chromatography column (silica gel, eluent 10% hexane/methylene chloride). The eluent was concentrated again and re-chromatographed (silica gel, eluent gradient methylene chloride to 5% methanol/methylene chloride). 219 F.xperimpntnl Tetraphenylporphyrin Syntheses Aldehyde Mass/Volume (mL/g, moi) Pyrrole (mL, moi) Porphyrin Obtained Yield (%) para-Nitrobenzaldehyde 11 (0.073) 5 (0.073) 72 25 meta-Nitrobenzaldehyde 7 (0.046) 3 (0.046) 73 14 2,6-Dinitrobenzaldehyde 1.85 (0.007) 0.5 (0.007) 0 para-Bromobenzaldehyde 3 (0.0162) 1.12 (0.016) 75 24 meta-Bromobenzaldehyde 11.1 (0.06) 4.2 (0.06) 76 2 para-Fluorobenzaldehyde 10(0.08) 5.6 (0.08) 77 12 Pentafluorobenzaldehyde 10 (0.051) 3.54 (0.051) 80 7.5 Pentachlorobenzaldehyde258 1 (0.004) 0.25 (0.004) 5 2-Chloro-6-nitrobenzaldehyde 3.64 (0.021) 1.5 (0.021) 0 2,6-Dichlorobenzaldehyde 10.5 (0.06) 4.2 (0.06) 81 2 /?ara-Hydroxybenzal dehyde 10(0.08) 5.4 (0.08) 82 15 meta-Hydroxybenzaldehyde 10(0.08) 5.4 (0.08) 83 12 o/t/zo-Hydroxybenzaldehyde 14.64 (0.12) 8.4 (0.12) 5 3,5-Dihydroxybenzaldehyde 1 (0.007) 0.5 (0.007) 84 7 3,4-Dihydroxybenzal dehyde 6 (0.043) 3 (0.043) 5 2,4-Dihydroxybenzaldehyde 8.28 (0.06) 4.2 (0.06) 12 para-Carboxybenzaldehyde 9 (0.06) 4.2 (0.06) 0(see below) para-Carbomethoxybenzaldehyde* 5 (0.03) 2.1 (0.03) 85 31 para-Methoxybenzaldehyde 10(0.07) 4.85 (0.07) 88 21 meta-Methoxybenzaldehyde 10(0.07) 4.85 (0.07) 89 25 3,5 -Dimethoxybenzal dehyde 10 (0.06) 4.2 (0.06) 90 10 3-Methoxy-4-hydroxybenzaldehyde 5 10(0.06) 4.2 (0.06) 91 15 3,4,5-Trimethoxybenzaldehyde 5 (0.024) 1.6 (0.024) 92 30 2,4,6-Trimethoxybenzaldehyde 1.65 (0.008) 0.75 (0.008) 93 20 3,4-Dimethoxybenzal dehyde 10(0.06) 4.2 (0.06) 5 3,5-DiOMe-4-OHbenzaldehyde 10.4 (0.06) 4.2 (0.06) 96 14 para-Methylbenzaldehyde 7.2 (0.06) 4.2 (0.06) 97 26 2,4,6-Trimethylbenzaldehyde 8.9 (0.06) 4.2 (0.06) 98 24 /?ara-terf-Butylbenzaldehyde 1.2 (0.007) 0.5 (0.007) 102 17 /?ara-Aminobenzaldehyde 5 (0.043) 2.8 (0.043) 99 See below para-Dimethylaminobenzaldehyde 9 (0.06) 4.2 (0.06) 7 para-Cyanobenzaldehyde 7.9 (0.06) 4.2 (0.06) 101 15 220 Frperirnentnl Nitro porphyrins (72), (73), (74) According to a literature method,250 the appropriate nitrobenzaldehyde (1 eq.) and acetic anhydride (2 eq.) were added to propionic acid with stirring. The resulting solution was brought to reflux. Pyrrole (1 eq.) in a small volume of propionic acid (-10 mL) was added and the mixture reluxed for 30 minutes. The tarry solution was allowed to cool at - 10"C for 3 hours. The mixture was filtered with water and methanol. The black precipitate was refluxed in pyridine (~120°C) for one hour. The reaction was allowed to cool to room temperature and set in the refrigerator. The mixture was filtered through a glass frit filter and washed with methanol and acetone to give a purple solid. See below for characterization. Hydrolysis of T(/;-C02Me) (85)- Synthesis of T(/>-COOH)PP (86):251 Tetra(/?ara-carboxymethylphenyl)porphyrin (28) (15 mg, 0.018 mmol) in 2 mL of THF was added to a 10% K O H solution (0.5 mL) and the mixture refluxed for 24 hours. The solvent was removed by means of a rotary evaporator and the solution acidified to pH 1-2 with IN HCI. The resulting solution was centrifuged four times to free from traces of mineral acid. The green precipitate was transferred to a separatory funnel with methylene chloride and subsequently washed three times with 10 mL of a saturated sodium carbonate solution. The organic layer was removed and the mixture evaporated to dryness to yield tetra(/?ara-carboxyphenyl)porphyrin (86)(14 mg, 0.002 mmol, 100 % yield). See below for characterization. Reduction of Tetra(para-nitrophenyl)porphyrin (72) - Synthesis of T(/>NH2)PP (99):251 A solution of tetra(/?ara-nitrophenyl)porphyrin (3.0 g, 4.7 mmol) in 100 mL of concentrated hydrochloric acid was purged with nitrogen for one hour at which time a solution of stannous chloride, SnCl 2-2H 20 (9 g, 40 mmol) in concentrated hydrochloric acid (14 mL), also previously purged with nitrogen for 30 minutes, was added to the porphyrin solution. The mixture was stirred with heating (75-80°C) for 30 minutes. The hot water bath was carefully replaced by a cold-water bath and then an ice bath. The reaction was neutralized under nitrogen 221 F.xpp.ri.m.p.nt.nl by slow addition of 125 mL of concentrated ammonium hydroxide solution. The resultant solution was filtered and the green solid stirred with 200 mL of a 5% sodium hydroxide solution. The solid was then filtered, washed with water and chromatographed (silica gel, eluant methylene chloride) to yield the desired T(p-NH2)PP (99) (1.3 g, 1.9 mmol, 40 % yield). See below for characterization. Porphyrins T(/>-N02)PP (72)252 Rf 0.77 (Silica- CH2C12); UV-vis (CH2C12) A m a x 422, 518, 554, 592, 648 nm; 'H-NMR (400 MHz, CDC13) 6= 8.59 (d, J= 6.84 Hz, 8H, Hm), 8.68 (d, J = 6.86 Hz, 8H, H0), 8.75 (s, 8H,PH); MS (EI) m/e 792 (M +, 100%). T(» i -N0 2 )PP (73)2S2 Rf 0.76 (Silica- CH2C12); UV-vis (CH 2Cl 2)Am a x 422, 516, 552, 588, 650 nm; 'H-NMR (400 MHz, CDC13) 6= 7.99 (d, J = 8.14 Hz, 4H, Hp), 8.55 (m, 4H, H J , 8.68 (d, J = 8.16 Hz, 8H, H0), 8.80 (s, 8H,pH); MS (EI) m/e 794 (M +, 100%). T(<?-N02)PP (74) 2 5 2 2 5 3 Rf 0.74 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418, 518, 552, 594, 652 nm; 'H-NMR (200 MHz, CDC13) 6= 7.95 (d, 8H, H J , 8.30 (m, 4H, Hp), 8.44 (m, 4H, H0), 8.63 (s, 8H, pH); MS (EI) m/e 794 (M +, 100%). T(/;-Br)PP (75)252R f 0.82 (Silica- CH2C12); UV-vis (CH2C12)A,11;1X 420, 516, 550, 588, 644 nm 'H-NMR (200 MHz, CDC13) 8= 7.86 (d, 8H, H J , 8.10 (d, 8H, H0), 8.80 (s, 8H, pH); MS (EI) m/e 930 (M +, 100%), 984 (CuM +, 10%), 993 (M +, 20%). T(m-Br)PP (76)252 R f 0.82 (Silica- CH2C12); UV-vis (CH2C12) kmm 422, 512, 548, 584, 642 nm H-NMR (200 MHz, CDC13) 8= 7.62 (t, 4H, H J , 7.86 (d, 4H, Hp), 8.06 (d, 4H, 222 F.Yperimpntnl H0), 8.40 (d, 4H, H0,), 8.92 (s, 8H, pH); MS (EI) m/e 930 (M + , 100%), 984 (CuM +, 30%), 993 (M +, 20%). T(/>-F)PP (77)2 5 2R f 0.72(Silica-CH2Cl2); UV-vis(CH 2Cl 2) kmm 416, 516, 548, 588, 648 nm; 'H-NMR (400 MHz, CDC1,) 8= 7.48 (2d, 8H, H J , 8.18 (2d, 8H, H0), 8.82 (s, 8H, pH); MS (EI) m/e 686 (M +, 10%), 740 (CuM +, 100%). Tim-F)PP (78)253 R f 0.71 (Silica- CH2C12); UV-vis (CH2C12) kmax 418,516, 548, 588, 646 nm; 'H-NMR (200 MHz, CDC13) 8= 7.42 (m, 4H, Hp), 7.62 (m, 4H, Hm), 7.95 (m, 8H, H0), 8.90 (s, 8H, pH); MS (EI) m/e 686 (M +, 80%), 740 (CuM +, 100%). T>-F)PP (79)253 R f 0.64(Silica-CH2Cl2); UV-vis(CH2Cl2)characterized as zinc chelate Am a x422, 518, 554, 594 nm 'H-NMR (200 MHz, CDC13) 8= 7.58 (m, 8H, H J , 7.80 (m, 4H, Hp), 8.10 (d, 8H, H J , 8.94 (s, 8H, PH); MS (EI) m/e 748 (M +, 100%). TF 5PP (80)253 Rf 0.7 (Silica- CH2C12); UV-vis (CH2C12) characterized as zinc chelate A m a x 412, 518, 552, 582 nm; 'H-NMR (200 MHz, CDC13) 8= 8.50 (s, 8H, 0H); MS (EI) m/e 1036 (M + , 100%). T(o,o'-Cl)PP(81)2 4 9 R f 0.71 (Silica-CH2C12); UV-vis (CH2CI2) A m a x 420, 548, 588, 646 nm. 'H-NMR (200 MHz, CDC13) 8= 7.20 (t, 4H, Hp), 7.40 (d, 8H, H J , 8.95 (s, 8H, PH); MS (EI) m/e 890 (M +, 10%), 943 (CuM +, 20 %). T(p-OH)PP (82)254 R f 0.16 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418,516, 552, 592, 648 nm 'H-NMR (200 MHz, CDC13) 8= 7.19 (d, 8H, H J , 7.95 (d, 8H, H0), 8.72 (s, 8H, PH); MS (EI) m/e 678 (M +, 100%). 223 Experimental T ( /M-OH )PP (83)254 R f 0.23 (Silica- CH2C12); UV-vis (CH2CI2) A m a x 414, 516, 550, 588, 644 nm; H-NMR (200 MHz, CDC13) 8= 7.20 (d, 4H, H ), 7.80 (t, 4H, H J , 7.66 (m, 8H, H J , 8.96 (s, 8H, pH); MS (EI) m/e 678 (M +, 100%), 732 (CuM +, 40%). T(m,m'-OH)PP (84)255 R t 0.05 (Silica- CH2C12); UV-vis (CH2C12) A m a x 410, 514, 548, 586, 648 nm; 'H-NMR (200 MHz, CDC13) 8= 7.32 (s, 4H, Hp), 7.84 (s, 8H, H0), 9.0 (s, 8H,pH); MS (EI) m/e 742 (M +, 60%), 796 (CuM +, 100%), 805 (ZnM+, 40%). T(p-C0 2 Me )PP (85)256 R f 0.53 (Silica- CH2C12); UV-vis (CH2C12) A m a x 420, 514, 550, 590, 644 nm; 'H-NMR (200 MHz, CDC13) 8= 4.03 (s,12H, OMe), 8.26 (d,8H, H J , 8.42 (s, 8H, H0), 8.82 (s, 8H, pH); MS (EI) m/e 846 (M + , 30%), 900 (CuM +, 60 %). T0>CO 2 H )PP (86)257 Rf 0.61 (Silica- 5% MeOH:CH 2Cl 2); UV-vis (CH2C12) Xmax 416, 516, 548, 588, 644 nm; 'H-NMR (200 MHz, CDC1-,) 8= 8.18 (d, 8H, H J , 8.38 (s, 8H, H0), 8.78 (s, 8H,pH); MS (EI) m/e 790 (M +, 100%) T( / ; -OCOEt )PP (87)256 R f 0.66 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418, 518, 558, 594, 652 nm; 'H-NMR (200 MHz, CDC13) 8= 1.40 (t,12H,CH3), 2.75 (q, 8H, CH 2), 7.40 (d, 8H, H J , 8.18 (d, 8H, H0), 8.95 (s, 8H, PH); MS (EI) m/e 908 (M + , 10%). T(p-OMe)PP (88)258 R f 0.25 (Silica- 5% MeOH: CH2C12); UV-vis (CH2C12) A m a x 422, 18, 556, 592, 652 nm; 'H-NMR (200 MHz, CDC13) 8= 4.10 (s, 12H, CH 3), 7.24 (d, 8H, H J , 8.16 (d, 8H, H0), 8.82 (s, 8H, pH); MS (EI) m/e 796 (ZnM+, 100%). 224 Experimental T(/M-OMe)PP (89)259 R f 0.44 (Silica- 5 % MeOH:CH 2Cl 2), 0.79 (CH2C12); UV-vis (CH2C12) A m a x 418, 516, 550, 588, 646 nm; 'H-NMR (200 MHz, CDC13) 5= 3.90 (s, 12H, CH 3), 7.30 (d, 4H, Hp), 7.60 (t, 4H, Hm), 7.74 (m, 8H, H0), 8.82 (s, 8H, pH); MS (EI) m/e 734 (M +, 100%), 788 (CuM +, 90%), 796 (ZnM+, 80%). T(m,m'-OMe)PP (90)260 R f 0.4 (Silica- CH2C12); UV-vis (CH2C12) X M M 422, 516, 548, 588, 646 nm; 'H-NMR (200 MHz, CDC13) 8= 4.0 (s, 12H, CH 3), 7.28 (s, 4H, Hp), 7.42 (d, 8H, H J , 8.84 (s, 8H, PH); MS (EI) m/e 793 (M +, 40%), 854 (ZnM +, 90%). T(m-OMe,/>-OH)PP (91)259 R f 0.50 (Silica- CH2C12); UV-vis (CH2C12) A m a x 424, 460 (sh), 520, 558, 594, 650 nm; 'H-NMR (200 MHz, CDC13) 8= 4.0 (s, 12H, CH 3), 5.95 (br s, 4H, OH), 7.30 (d, 4H, H J , 7.72 (d, 8H, H0), 8.90 (s, 8H, PH); MS (EI) m/e 798 (M +, 100%). T(/n,/>,/n'-OMe)PP (92)260 R, 0.8 (Silica- CH2C12); UV-vis (CH2C12) A m a x 420, 514, 548, 590, 644 nm; 'H-NMR (200 MHz, CDC13) 8= 4.1 (s, 36H, Ome), 7.7 (s, 8H, H0), 8.84(s, 8H, pH); MS (EI) m/e 914 (M +, 50%), 1028 (CuM +, 80%), 1036 (ZnM +, 90%). T(o,m,p-OMe)PP (93)260 R f 0.8 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418, 516, 548, 592, 650 nm; 'H-NMR (200 MHz, CDC13) 8= 1.58 (s, 12H, />OCH3), 3.24 (s, 12H, ffl-OMe), 4.12 (s, 12H, o-OMe), 7.04 (d, 4H, H J , 7.72 (m, 4H, H0), 8.82 (s, 8H, PH); MS (EI) m/e 914 (M +, 70%), 1028 (CuM +, 100%), 1036 (ZnM +, 30%). 225 Experimental T>,m,«i'-OMe)PP (94)260 R f 0.84 (Silica- CH2C12); UV-vis (CH2C12) A m a x 420, 516, 546, 590, 646 nm; 'H-NMR (400 MHz, CDC1J 8= 3.9 (s, 36H, OMe), 6.95 (s, 4H, H J , 7.5 (s, 4H, H J , 8.80 (s, 8H, pH); MS (EI) m/e 974 (M +, 100%), 1028 (CuM +, 20%), 1036 (ZnM +, 90%). T(o,/;,fff'-OMe)PP (95)260 R f 0.82 (Silica- CH2C12); UV-vis (CH2C12) A m a x 422, 516, 548, 590, 648 nm; 'H-NMR (200 MHz, CDC13) 8= 4.0 (overlapping s, 36H, OMe), 7.05 (s, 8H, HJ,8.83 (s, 8H, PH); MS (EI) m/e 91A (M +, 100%), 1028 (CuM +, 70%), 1035 (M +, 20%). T(/n,/n'-OMe,/)-OH)PP (96)26' R f 0.36 (Silica- CH2C12); UV-vis (CH2C12) A m a x 420, 518, 556, 592, 650 nm; 'H-NMR (200 MHz, CDC1J 8= 4.00 (s, 24H, C H 3 ) , 7.44 (d, 8H, H J , 8.98 (s, 8H, pH); MS (EI) m/e 918 (M +, 100%). T0>Me)PP (97)252 R f 0.90 (Silica-5% MeOH:CH 2Cl 2); UV-vis (CH2C12) A m a x 416, 486 (sh), 516, 554, 592, 648 nm; 'H-NMR (200 MHz, CDC1J 8= 2.68 (s, 12H, CH 3), 7.58 (d, 8H, H J , 8.10 (d, 8H, H J , 8.84 (s, 8H, pH); MS (EI) m/e 670 (M +, 100%). T(o,/),o'-Me)PP (98)253'26' R f 0.94 (Silica- 5% MeOH: CH2C12); UV-vis (CH2C12) A m a x 414, 514, 546, 588, 646 nm; 'H-NMR (400 MHz, CDC1J 8=1.85 (s, 36H, CH 3), 7.28 (d, 8H, H J , 8.62 (s, 8H, pH); MS (EI) m/e 782 (M +, 10%), 844(ZnM +, 100%). T(p-NH2)PP (99)263 R f 0.61 (Silica- CH2C12); UV-vis (CH2C12) A m a x 428, 522, 562, 594, 656 nm; 226 Rvpprinipntnl 'H-NMR (200 MHz, CDC13) 8= 4.0 ( br s, 2H, NH), 7.02 (d, 8H, H J , 7.95 (d, 8H, H0),8.95 (s, 8H, pH); MS (EI) m/e 674 (M +, 20%), 728 (CuM +, 100%). T(/)-S03H)PP (100)254 R f 0.83 (Silica- 5%CHCl 3:MeOH); UV-vis (CH2C12) A m a x 414, 514, 546, 588, 644 nm; MS (EI) m/e 934 (M + , 100%). T(/7-CN)PP (101)256 R f 0.93 (SiIica-5%CHCl3:MeOH); UV-vis (CH 2 Cl 2 )A m a x 420, 514, 550, 588, 648 nm; 'H-NMR (200 MHz, GDC13) 8= 8.10 (d, 8H, H J , 8.30 (d, 8H, H0), 8.80 (s, 8H, pH); MS (EI) m/e 714 (M +, 20%), 768 (CuM +, 80%). T(/>-r-Bu)PP (102)252 R f 0.92 (Silica- 5% MeOH: CH2C12); UV-vis (CH2C12) A m a x 404 (sh), 416, 518, 554, 592, 648 nm; 'H-NMR (200 MHz, CDC13) 8= 1.58 (s, 36H, CMe 3), 7.66 (d, 8H, H J , 8.06 (d, 8H, H J , 8.78 (s, 8H, pH); MS (EI) m/e 838 (M + , 30%), 892 (CuM +, 90%), 900 (ZnM +, 100%). 5(/;-Bromophenyl),10,15,20-triphenylporphyrin (mono/>-Br TPP) (69) R, 0.7 (Silica-CH2C12); UV-vis (CH2C12) A m a x 416, 520, 554, 594, 644 nm; 'H-NMR (200 MHz, CDC13) 8= 7.7 (m, 9H), 8.0 (d, 2H, H J , 8.1 (m, 6H), 8.8 (m, pH); MS (EI) m/e 693 (M +, 100%). 5(p-Nitrophenyl),10,15,20-triphenylporphyrin (mono/?-N02 TPP) (70) R r 0.5 (CHC13); UV-Vis (CH2C12) A m a x 418, 518, 552, 592, 646 nm; 'H-NMR (200 MHz, CDC1J 8= 7.8 (m, 8H), 7.9 (m, 2H), 8.05 (m, 6H), 8.2 (d, 2H), 8.7 (m, 8H); MS (EI) m/e 660 (M + , 100%). 227 Experimental 5(p-Hydroxyphenyl),10,15,20-triphenylporphyrin (mono/>-OH TPP) (71) R f 0.14 (Silica-CH2C12); UV-vis (CH2C12) A m a x 420, 516, 550, 590, 646 nm; 'H-NMR (200 MHz, CDC13) 6= 7.6 (t, 2H), 7.8 (m, 8H), 8.0 (d, 2H), 8.2 (dd, 6H), 8.9 (m, 8H); MS (EI) m/e 630 (M +, 100%). CHLORINS General Procedure Tetraphenylporphyrin (1 g, 1.63 mmol) was dissolved in a solution of 2-10% pyridine in reagent grade chloroform. The volume of solvent used was the minimum amount required to dissolve the particular porphyrin being used and ranged from 0.25 to 1 mL/mg. Osmium tetroxide (450 mg, 1.1 eq) was added to the solution and reaction stirred at room temperature in the dark. The reaction progress was monitored by TLC or UV-visible spectroscopy until no further reaction was observed (3-5 days). The reaction was then purged with hydrogen sulfide gas for 10 minutes, and then purged with air until the solvent had evaporated. The solid was then dissolved in a 10% MeOH:CHCl 3 solution and filtered. The filtrate was evaporated to dryness and chromatographed (silica, 5 % MeOH:CHCl 3) to yield the analogous diol chlorin in 40-60 % yield. H2TPC(OH)2 (103)'68 R f 0.7(Silica- CH2C12); UV-vis (CH2C12) A m a x (rel. intensity) 416(1.0), 520 (0.12), 546 (0.11), 594 (0.06), 644 (0.14) nm; 'H-NMR (200 MHz, CDC13) 6= -1.80 (br s, 2H), 3.12 (s, 2H), 6.36 (s, 2H), 7.72-7.82 (m, 12H), 7.80 (d, 2H), 8.10 (s, 4H), 8.15 (d, 2H), 8.35 (d, 2H), 8.44 (s, 2H), 8.64 (d, 2H), MS (EI, 320°C) m/e 648 (M +, 100%). T(m-N02)PC(OH)2 (104) R f 0.76 (Silica-2% MeOH:CHCl 3); UV-vis (CH2C12) X m a x 412, 518, 548, 548, 594, 644 nm; 'H-NMR (200 MHz, CDC13) 6= -1.80 (br s, 228 Experimental 2H), 6.78 (d, 2H, Hp), 7.42 (t, 2H, H J , 7.67 (br m, 2H, H J , 7.86 (2d, 2H,HJ, 7.95 (m, 4H, H m and H J , 8.14 (d, 2H, H J , 8.27 (br m, 2H, H J , 8.38 (d, 2H, pH), 8.48 (s, 2H, PH), 8.58 (d, 2H, PH); MS (EI, 320°C) m/e 828 (M +, 30%), 810 (M + - H 2 0, 100%). T>-Br)PC(OH)2 (105) R f 0.65 (Silica- 2% MeOH:CHClJ; UV-vis (CH2C12) A m a x 412, 486 (sh), 516, 544, 592, 648 nm; 'H-NMR (200 MHz, CDC1J 6=-1.80 (br s, 2H), 6.36 (s, 2H), 8.0 (br s, 2H, H J , 8.2 (br s, 6H, H J , 8.3 (d, J = 4.76 Hz, 2H, PH), 8.4(d, J = 7.55 Hz, 8H, H J , 8.45 (s, 2H, pH), 8.60 (d, J = 4.72 Hz, pH); MS (EI, 320°C) m/e 964 (M +, 10%), 946 (M + - H 2 0, 100%). T(»i-Br)PC(OH) 2 (106) R f0.74 (Silica-2% MeOH:CHCl 3); UV-vis (CH2C12) A m a x 418, 514, 548, 586, 644 nm; 'H-NMR (400 MHz, CDC1J 8= -1.85 (br s, 2H), 6.28 (s, 2H), 7.39 (t, 2H, H J , 7.57 (br m, 2H, H J , 7.74 (2d, 2H, H J , 7.83 (m, 4H, H m and H J , 8.04 (d, 2H, H J , 8.07 (br m, 2H, H J , 8.32 (d, 2H, PH), 8.46 (s, 2H, PH), 8.63 (d, 2H, 0H); MS (EI, 320°C) m/e 964 (M +, 15%), 946 (M + - H 2 0, 100%). T(m-F)PC(OH)2 (107) Rf 0.64 (Silica-2%MeOH:CHClJ; UV-vis (CH2C12) A m a x 414, 518, 542, 596, 650 nm; 'H-NMR (200 MHz, CDC1J 8= 6.28 (s, 2H), 7.0 (dd, 2H), 7.2 (d, 2H), 7.3 (br m, 2H), 7.4-7.6 (m, 12H), 8.4 (s, 2H), 8.6 (d, 2H); MS (EI, 320°C) m/e 720 (M +, 100%), 704 (M + - O, 30%). T(o-F)PC(OH)2 (108) Rf 0.62 (Silica-2%MeOH:CHClJ; UV-vis (CH2C12) A m a x 416, 518, 544, 592, 648 nm; 'H-NMR (400 MHz, CDC13) 8= -1.80 (br s, 2H), 6.30 (s, 2H), 7.46 (m, 6H, H m and H J , 7.70 (m, 6H, H m and H J , 8.05 (m, 2H, H J , 229 Experimental 8.30 (d, 2H, H0), 8.35 (d, J = 4.70 Hz, 2H, PH), 8.45 (s, 2H, pH), 8.62 (d, 7 = 4.31 Hz, 2H, PH); MS (EI, 320°C) m/e 702 (M + - H 2 0, 60%). TF 5PC(OH)2 (109) Rf 0.60 (Silica- CH2C12); UV-vis (CH2C12) A m a x 410, 506, 596, 650 nm; 'H-NMR (200 MHz, CDC1J 8= -1.72 (s, 2H), 6.0 (s, 2H), 8.19 (d, 2H, pH), 8.39 (d,2H, PH); MS (EI, 320°C) m/e 1008 (M + , 10%), 990 (M + - H 2 0, 20%); 974 (M + - 2 HO, 100%). T(/7-OH)PC(OH)2 (110) R f 0.52 (Silica-2% MeOH:CHCl 3); UV-vis (CH2C12) A m a x 422, 520, 558, 596, 652 nm; 'H-NMR (200 MHz, CDC1J 6= -1.8 (br s, 2H), 6.3 (s, 2H), 7.3 (m, 8H), 7.8 (d, 2H), 8.0 (br m, 2H), 8.2 (m, 4H), 8.3 (d, 2H), 8.5 (s, 2H), 8.7 (d, 2H); MS (EI, 320°C) m/e 712 (M +, 15%), 694 (M + - H 2 0, 60%). T(»i-OH)PC(OH)2 (111) R f 0.52(Silica- CH 2Cl 2);UV-vis (CH2C12) A m a x 416, 514, 548, 592, 646 nm; 'H-NMR (200 MHz, CDC13) 6= 5.17 (br s, 2H), 6.18 (s, 2H), 7.07 (dd, 2H), 7.18 (dd, 2H), 7.30 (m, 2H), 7.39-7.58 (m, 10H), 8.36 (br s, 2H), 8.43 (s, 2H), 8.69 (d, 2H), 9.75 (br s, 4H); MS (EI, 320°C) m/e 712 (M +, 5%), 694 (M + - H 2 0, 100%). T>-C0 2Me)PC(0H) 2 (112) R f 0.71 (Silica- 2% MeOH:CHCl 3); UV-vis (CH2C12) X m a x 422, 518, 558, 600, 646 nm; 'H-NMR (200 MHz, CDC13) 6= -1.80 (br s, 2H), 8.0 (br s, 2H, H0), 8.2 (br s, 6H, H J , 8.28 (d, / = 4.76 Hz, 2H, PH), 8.38 (d, J = 7.55 Hz, 8H, H J , 8.43 (s, 2H, pH), 8.59 (d, J = 4.72 Hz, PH); MS (EI, 320°C) m/e 880 (M + , 65%), 862 (M + -H 2 0, 100%). 230 Experimental T(p-OCOEt)PC(OH)2 (113) R f 0.66 (Silica-CH2C12); UV-vis (CH2C12) A m n x 416, 514, 548, 592, 644 nm; 'H-NMR (200 MHz, CDC1J 8= 7.30 (br s, 2H, H0), 7.5 (br s, 6H, H J , 8.0 (m, 8H, H J , 8.2 (d, 2H, pH), 8.4 (s, 2H, pH), 8.55 (d, pH). T(/>-OMe)PC(OH)2 (114) R f 0.67 (Silica-2% MeOH:CHCl J ; UV-vis (CH2C12) A m a x 420, 520, 556, 596, 648 nm; 'H-NMR (400 MHz, CDC1J 6= -1.8 (br s, 2H), 4.1 (2s, 12H), 6.4 (s, 2H), 7.3 (m, 12H), 7.7 (d, 2H), 8.1 (m, 6H), 8.3 (d, 2H), 8.5 (s, 2H), 8.7 (d, 2H); MS (EI, 320°C) m/e 814 (ZnM+-H 2 0, 15%). T(m-OMe)PC(OH)2 (115) R f 0.6 (Silica- CH2C12); UV-vis (CH2C12) A m a x 416, 516, 546, 584, 646 nm; 'H-NMR (200 MHz, CDC13) 8= 4.0 (2s, 12H), 6.28 (s, 2H), 7.0 (d, 2H), 7.1 (d, 2H), 7.30 (m, 2H), 7.5 (m, 4H), 8.3 (d, 2H), 8.4 (s, 2H), 8.5 (d, 2H); MS (EI, 320°C) m/e 768 (M +, 10%), 750 (M + - H 2 0, 100%). T(/«,m'-OMe)PC(OH)2 (116) R f 0.43 (Silica- CH2C12); UV-vis (CH2C12) A m a x 416, 520, 548, 592, 646 nm; 'H-NMR (400 MHz, CDC1J 8= -1.85 (s, 2H), 3.9 (m, 12H), 6.4 (s, 2H), 6.8 (s, 2H), 6.85 (s, 2H), 7.1 (s, 2H), 7.25 (s, 2H), 7.3 (s, 2H), 7.4 (s, 2H), 8.4 (d, 2H), 8.6 (s, 2H), 8.75 (d, 2H) T(»i-OMe, p-OH)PC(OH)2 (117) R f 0.50 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418, 484 (sh), 518, 544, 596, 646 nm; 'H-NMR (200 MHz, CDC1J 8= -1.95 (s, 2H), 3.88 (s, 12H), 5.96 (s, 2H), 7.25 (d, 4H), 231 Experimental 7.68 (d, 8H), 8.94 (s, 8H); MS (EL 320°C) m/e 832 (M +, 10%), 814 (M + -H 2 0 , 100%). T(/«,/),m'-OMe)PC(OH)2 (118) R f0.65 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418, 518, 548, 592, 644 nm; 'H-NMR (400 MHz, CDC13) 6= -1.98 (s, 2H), 3.81(s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 3.95 (s, 3H), 5.30 (d, 2H), 6.39 (d, 2H), 7.15 (s, 2H), 7.35 (s, 2H), 7.4l(s, 2H), 7.43 (s, 2H), 8.46 (d, 2H), 8.59 (d, 2H), 8.77 (d, 2H); MS (EI, 320°C) m/e 1008 (M +, 30%), 990 (M + - H 2 0, 100%). T>,m,»f'-OMe)PC(OH)2 (119) R f 0.60 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418, 518, 548, 592, 644 nm; 'H-NMR (200 MHz, CDC1J 6= -1.8 (br s, 2H), 4.15 (m, 36 H), 6.05 (s, 2H), 6.8 (m, 4H), 7.5 (m, 4H), 8.2 (d, 2H), 8.4 (s, 2H), 8.6 (d, 2H); MS (EI, 320°C) m/e 1008 (M +, 15%), 990 (M + - H 2 0, 100%). T>,/7,o'-OMe)PC(OH)2 (120) R f 0.70 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418,518, 544, 594, 646 nm; 'H-NMR (200 MHz, CDC1J 6= -1.7 (br s, 2H), 4.0 (m, 36 H), 6.05 (s, 2H), 6.5 (m, 8H), 8.2 (d, 2H), 8.4 (s, 2H), 8.6 (d, 2H); MS (EI, 320°C) m/e 1008 (M +, 10%), 990 (M + - H 2 0, 100%). T>-Me)PC(0H)2 (121) R f 0.90 (Silica- 5% MeOH: CHC1-,); UV-vis (CH2C12) A m a x 416, 520, 546, 594, 644 nm; 'H-NMR (200 MHz, CDC13) 6= -1.78 (br s, 2H), 2.45 (2s, 12H, Me), 6.30 (s, 2H), 7.2 (m, 8H, H J , 7.7 (m, 4H, 232 Experimental HQ), 8.0 (m, 4H, H0), 8.30 (d, 2H, pH), 8.40 (s, 2H, PH), 8.56 (d, 2H, pH); MS (EI, 320°C) m/e 704 (M +, 5%), 686 (M + - H 2 0, 10%), 670 (M + , 100%). T(o,/7,o'-Me)PC(OH)2 (122) R f 0.94 (Silica- 5% MeOH:CHCl 3); UV-vis (CH2C12) A m a x 418, 480 (sh), 516, 542, 592, 646 nm; 'H-NMR (200 MHz, CDC1J 6= -1.78 (br s, 2H), 6.0 (s, 2H), 7.25 (m, 8H, H J , 8.15 (d, 2H, pH), 8.28 (s, 2H, PH), 8.45 (d, 2H, pH); MS (EI, 320°C) m/e 816 (M +, 30%), 798 (M + - H 2 0, 100%). T>-S0 3 H)PC(OH) 2 (123) R, 0.1 (Silica- CH2C12); UV-vis (CH2CI2) A m a x 420, 520, 548, 592, 648 nm. T(/>-r-Bu)PC(OH)2 (124) R f 0.75 (Silica- CH2C12); UV-vis (CH2C12) A m a x 418, 522, 548, 594, 644 nm; 'H-NMR (200 MHz, CDC1J 6= 3.45 (2s, 36H, Me), 6.2 (s, 2H), 7.0 (m, 8H, H J , 7.5 (m, 4H, H„), 7.8 (m, 2H, H J , 8.0 (br m, 2H, H J , 8.3 (d, 2H, PH), 8.4 (s, 2H, pH), 8.6 (d, 2H, pH); MS (EI, 320°C) m/e 935 (M +, 10%), 916 (M + - H 2 0, 60%). H 2 DPC(OH) 2 (125) R f 0.3 (Silica- CH2C12); UV-vis (CH2C12) A m a x 402, 504, 530, 584, 638 nm; 'H-NMR (200 MHz, CDC1J 6= -2.1 (br s, 1H), -1.9 (br s, 1H), 6.05 (d, J= 6.3 Hz, IH), 6.35 (d, /= 6.4 Hz, IH), 7.65 (m, 6H), 7.9 (d, IH), 8.10 (d, J= 4.4 Hz, 2H), 8.25 (d, IH), 8.45 (d, J= 4.5 Hz, IH), 8.65 (d, J= 4.5 Hz, IH), 8.80 (d, J= 4.5Hz, IH), 8.95 (d, J= 4.5 Hz, IH), 9.0 (d, 7=4.4 Hz, IH), 9.15 (d, J= 4.3 Hz, IH), 9.90 (s, IH), 9.34 (s, IH); MS (EI) m/e calc'd for C 3 2 H 2 4 N 4 0 2 Ni : 496.18945, found 496.18923 (M +, 100%). 233 Experimental mono p-Br PC(OH)2 (126) R f 0.4 (Silica- CH2C12); UV-vis (CH2C12) kmm 412, 508, 528, 596, 644 nm; 'H-NMR (200 MHz,CDCl 3) 8= 6.2 (d, 2H), 7.0 (d, 2H), 7.5 (m, 12H), 7.8 (2d, 1H), 8.0 (m, 4H), 8.2 (d, 1H), 8.3 (d, 1H), 8.35 (s, 2H), 8.5 (d, 1H); MS (EI, 320°C) m/e 726 (M +, 20%), 709 ( M + - H 2 0 , 100%). monop-Oll PC(OH)2 (127) R f 0.2 (Silica- CH2C12); UV-vis (CH2C12) A m ; i x 412, 520, 548, 594, 644 nm; 'H-NMR (200 MHz,CDCl 3) 5= 6.4 (d, 2H), 7.1 (d, 2H), 7.5 (m, 12H), 7.85 (2d, 1H), 8.0 (m,4H), 8.1 (d, 1H), 8.25 (d, 1H), 8.35 (s, 2H), 8.7 (d, 1H); MS (EI, 320°C) m/e 662 (M +, 5%), 990 (M + - H 2 0, 646%). monop-N02 PC(OH)2 (128) R f 0.4 (Silica- CH2C12); UV-vis (CH2C12) A , m x 418,516, 546, 596, 646 nm; 'H-NMR (200 MHz,CDCl 3) 8= 7.2 (d, 2H), 7.65 (m, 12H), 7.8 (d, 2H), 8.10 (m, 4H), 8.2 (d, 1H), 8.3 (s, 2H), 8.4 (d, 1H). Osmium Tetroxide Oxidations of Tetraphenylporphyrins: TetraphenylporDhyrin Mass (e) OsO, Yield (%) Mass (eVEquiv. Diol Tetraol T(/?-N02)PP 0.100 0.06/ 1.9 0 0 T(m-N02)PP 0.100 0.08/2.5 15 0 T(o-N02~)PP 0.112 0.045 / 1.1 0 0 T(p-Br)PP 0.097 0.098 / 3.7 40 6.7 T(m-Br)PP 0.125 0.079/ 1.0 1.1 0 T (p -F )PP 0.050 0.019/ 1.0 23 10 T(m-F)PP 0.012 0.005 / 1.1 12 15 T(o-F)PP 0.057 0.021 / 1 1 1 3 TF 5PP 0.100 0.070/2.7 12.5 49 T(o,o'-Cl)PP 0.0985 0.076 / 2.7 0 0 234 Experimental T0-OH)PP 0.160 T(m-OH)PP 0.050 T(m,m'-OH)PP 0.0985 T(/?-C02Me)PP 0.118 T(>-COOH)PP 0.100 T(p-OCOEt)PP 0.114 T(/?-OMe)PP 0.180 T(m-OMe)PP 0.115 T(m,m'-OMe)PP 0.113 T(m-OMe,p-OH)PP 0.085 T(3,4,5-OMe)PP 0.100 T(2,3,4-OMe)TPP 0.040 T(2,4,5-OMe)PP 0.040 T(2,4,6-OMe)PP 0.020 T(m,m'-OMe,p-OH)PP 0.100 T(/?-Me)PP 0.110 T(2,4,6-Me)PP 0.100 T(/?-NH2)PP 0.137 Tf>-SO,H)PP 0.100 T(p-CN)PP 0.060 T(/?-f-Bu)PP 0.095 0.090/ 1.5 18.3 10 0.054 / 2.9 46 3 0.085 /2.5 0 0 0.036/ 1.0 10 0 0.034/ 1.0 0 0 0.070 / 2.3 17 0 0.12/ 1.9 42 0 0.050/ 1.3 22.5 0 0.051 / 1.1 31 0 0.030/ 1.1 21 0 0.029 / 1.1 45 3 0.035 / 3.4 0 0 0.010/ 1.0 25 0 0.010/ 1.9 12 2 0.029/ 1.1 0 0 0.036/0.9 45 12 0.036/0.9 38 20 0.148/2.9 0 0 0.028/ 1.0 7.2 0 0.025/ 1.2 0 0 0.024 / 0.8 23 6 A^-Methyl-5,10,15-20-tetraphenylporphyrin (131):187 Tetraphenylporphyrin (50 mg, 0.08 mmol) was dissolved in glacial acetic acid (5 mL), and m-xylene (85 mL). Methyl iodide (10 mL) was added to the solution. The mixture was refluxed for 30 hours \—N H N - - , Ph<f Me ? P h after which the solvent was removed in vacuo. The remaining solids Jf~~^ were dissolved in benzene (25 mL) and dimethylsulfate (0.5 mL) was added. The mixture was refluxed for 45 minutes, cooled to room temperature and neutralized with solid sodium carbonate. The reaction was filtered, and the solvent evaporated to dryness. 235 Experimental Column chromatography (acidic alumina; 5% MeOH:CH 2Cl 2) gave (131) in 5% yield. R F 0.1 (silica - 5% MeOH:CH 2Cl 2); 'H-NMR (200 MHz, CDC13) 6= -4.0 (br s, IH), 7.42 (s, 2H), 7.70-7.85 (m, 12H), 8.15-8.40 (m, 8H), 8.46 (d, 2H), 8.64 (d, 2H), 8.82 (s, 2H), 8.4 (d, 2H). UV-vis (CH2C12) Am a x(rel. intensity) 440 (1.0), 532 (0.13), 578 (0.21), 618 (0.20), 670 (0.17) nm; MS (LSIMS) m/e 629 (M + , 100%). Ph /V-Phenyl-5,10,15,20-tetraphenylporphyrin (132):'8 9 1 9 0 Trifluoroacetic acid (0.5 mL) was added to a solution of (5,10,15,20-tetraphenylporphyrinato)(phenyl)cobalt(HI) (134) (20 mg, 0.03 mmol) in dichloromethane (10 mL) at room temperature and was stirred for 30 minutes. The solvent was evaporated and the residue washed with water, filtered and dissolved in dichloromethane. The resultant solution was dried and chromatographed (silica; chloroform) to yield the desired product (70%). R F 0.2 (silica - CH2C12); 'H-NMR (400 MHz, CDC13) 6 = 0.3 (s, 2H, NH), 2.4 (d, 2H, o-H Ph), 5.28 (t, /= 8.0 Hz, 2H, H J , 5.7 (t, J= 7.47 Hz, IH, p-K Ph), 7.70-7.90 (m, 12H), 8.15-8.30 (m, 8H), 8.39 (s, 2H), 8.6 (d, J= 4.62 Hz, 2H), 8.68 (d, J= 4.63 Hz, 2H), 8.87 (s, 2H); UV-vis (CH2C12) A n i a x (rel. intensity) 438 (1.0), 548 (0.13), 594 (0.23), 638 (0.15), 702 (0.11) nm; MS (EI) m/e 692 (M ++ 2, 100%), 690 (M +, 100%). 236 Experimental Chloro(5,10,15,20-tetraphenylporphyrinato)cobalt(III) (133):189190 Ph Co(II)TPP (0. l g , 0.15 mmol) was stirred at room temperature in a solution of methanol (100 mL) containing concentrated HCI (1 mL). After 6 hours, the solution was filtered and the solvent was Ph removed. The remaining solid was washed with water and dried. R F 0.8 (silica - CH 2 C1 2 ) ; ' H - N M R (400 M H z , CDC1 3 ) 6= 7.8 (m, 12H), 8.0 (m, 8H), 8.4 (s, 8H); U V - v i s (CH 2 C1 2 ) A m a x (rel. intensity) 426 (1.0), 506 (0.09), 540 (0.17), 572 (0.1) nm; M S (EI) m/e 705 ( M + , 30%), 671 (Co(II)TPP, 100%). (5,10,15,20-tetraphenylporphyrinato)(phenyl)cobalt(III) ( 1 3 4 ) : , 8 9 . , 9 0 Chloro(5,10,15,20-tetraphenylporphyrinato)cobalt(III), (133), \ W p h \^ (100 mg, 0.14 mmol) was dissolved in toluene (25 mL). \ _ _ / \ t~H\ ^ /V-Phenyllithium (1 mL) was added dropwise to the solution under /fr nitrogen. After 10 minutes, methanol (10 mL) was added and the solution allowed to stir at room temperature for 30 minutes. The solution was washed with water, dried and the solvent evaporated. The residue was chromatographed (silica, chloroform) to yield the desired product (80%). R F 0.9 (silica - CH 2 C1 2 ) ; U V - v i s (CH 2 C1 2 ) kmm (rel. intensity) 410 (1.0), 528 (0.22) nm; M S (EI) m/e 748 ( M + , 80%). 237 Experimental /n£S0-Tetraphenyl-2-oxoporphyrin (143): Free base tetraphenylporphryin (10) (50 mg, 0.08mmol) was dissolved in a solution of T H F (1 mL) and dichloromethane (1 mL). Concentrated sulfuric acid (1 drop) and hydrogen peroxide (40%, 5 mL) Ph were added to the reaction mixture. The reaction was left to stir at room temperature. The solvent was removed and the residue dissolved in dichloromethane. The solution was washed with water, dried and chromatographed (silica; dichloromethane) to yield (143) (10% yield) and starting material. R F 0.7 (silica - C H C l 3 ) ; U V - v i s (CH 2 C1 2 ) A m a x (rel. intensity) 418 (1.0), 486 (0.02), 514 (0.25), 548 (0.13), 588 (0.10), 608(0.07), 644 (0.09) nm; M S (EI) m/e 630 ( M + , 100%). Ph | > O H Chapter 3 Compounds (5,10,15,20-Tetraphenyl-v/c-2,3-dihydroxy-2,3-chlorinato)nickel(H) p h < (144): / A solution of 5,10,15,20-tetraphenyl-v/c-2,3-dihydroxy-2,3-chlorin (103) (0.05 g, 0.77 mmol) in chloroform with excess nickel acetate (0.2 g, 1.4 mmol) was refluxed for 10-18 hours, quantitatively producing (144). The solution was washed with water, dried, filtered and the solvent removed in vacuo. R F 0.5 (silica - CH 2 C1 2 ) ; ' H - N M R (400 M H z , CDC1 3 ) 8= 5.8 (s, 2H), 7.60 (m, 4H), 7.8 238 F.Y.pp.ri.m.p.nt.nl (m, 4H), 8.15 (d, 2H), 8.25 (s, 2H), 8.4 (d, 2H). UV-vis (CH2C12) A m a x (rel. intensity) 416 (1.0), 612 (0.36) nm; MS (EI) m/e calc'd for Q ^ N A N i : 704.1815, found 704.1806 (M +, 100%). (5,10,15,20-TetraphenyI-2,3-secochlorinato-2,3-dialdehyde)nickel(II) (145): Lead tetraacetate (175 mg, 1.1 eq.) was added to a solution of (144) (150 mg, 0.213 mmol) dissolved in dry THF (25 mL). The reaction mixture was stirred at room temperature and monitored by TLC. Within 20 minutes, the reaction was complete and the solution was evaporated to dryness. Preparative TLC (chloroform) was performed to yield the golden brown product (145) in 80-90% yield. R F 0.85 (silica - CH2C12); IR 1684 cm"' (C=0 stretch); 'H-NMR (400 MHz, CDC13) 6= 7.60 (m, 12H), 7.75 (m, 8H), 7.8 (d, 2H), 8.0 (s, 2pH), 8.2 (d, 2bTi), 9.7 (s, 2pH). UV-vis (CH2C12) A m a x (rel. intensity) 414 (0.75), 464 (1.0), 688 (0.72) nm; MS (EI) m/e calc'd for C 4 4 H 2 8 N 4 0 2 Ni: 704.1182, found 704.1193 (M +, 100%). (5,10,15,20-Tetraphenyl-v/c-2,3-dihydroxy-2,3-chlorinato)zinc(II) (146): A solution of 5,10,15,20-tetraphenyl-v/c-2,3-dihydroxy-2,3-chlorin (0.05 g, 0.77 mmol) in chloroform and excess zinc acetate (0.2 g, 1.4 mmol) in toluene were refluxed for 1-3 hours, quantitatively producing (146). The solution was washed with water, dried, filtered and the 239 Experimental solvent removed in vacuo. Preparative TLC (silica - chloroform) was performed. R F 0.6 (silica - CH2C12); 'H-NMR (200 MHz, CDC13) 8= 6.1 (s, 2H), 7.60 (m, 12H), 7.8 (d, 2H), 7.95 (d, 2H), 8.0 (m, 6H), 8.4 (s, 2H), 8.5 (d, 2H). UV-vis (CH2C12) kmm (rel. intensity) 418 (1.0), 524 (0.08), 564 (0.13), 616 (0.34) nm; MS (EI) m/e calc'd for C 4 4 H 3 0 N 4 O Zn: 710.16612, found 704.16581 (M + , 100%). Free base (156) and (mes0-5,lO,15,2O-tetraphenyl-2-oxa-3-oxo-2,3-chlorinato)zinc(II) (147): (mei,o-Tetraphenyl-2-oxa-3-oxo-2,3-chlorinato)nickel(II) (155) (0.02 g, 0.03 mmol) was dissolved in methylene chloride (3 mL). Upon addition of HCI or sulfuric acid (1 drop), the colour changed from green-blue to a burgundy colour. The solution was washed with aqueous base, dried and evaporated to dryness quantitatively yielding (156) upon purification via preparative TLC (silica - chloroform). Refluxing (156) (0.02 g, 0.03 mmol) in chloroform with excess zinc acetate (0.2 g, 1.3 mmol) for 1-3 hours gave (147) quantitatively. Compound (147) was washed with water, dried, filtered and the solvent evaporated. Preparative TLC (silica - chloroform) isolated the compound. (156): R F 0.8 (silica - CH2C12); 1 H-NMR (400 MHz, CDC13) 6= -1.9 (s, 1H), -1.8 (s, 1H), 7.65 (m, 12H, H m and Hp), 7.95 (m, 2H, H0), 8.1 (m, 6H, H0), 8.50 (d, J= 4.73 Hz, 1H, p-H), 8.59 (d, 7=4.63 Hz, 1H, p-H), 8.64 (d, J= 4.75 Hz, 1H, p-H), 8.70 (d, 7=4.76 Hz, 1H, p-H), 8.82 (d, 240 Krpprimpntnl 7=4.70 Hz, 1 H, B-H). UV-vis (CH2C12) A m a x (rel. intensity) 420 (1.0), 522 (0.21), 558 (0.20), 588 (0.17), 640 (0.15) nm; MS (EI) m/e calc'd for C 4 3 H 2 6 N 4 0 2 Ni : 632.4357, found 632.1238 (M +, 100%). (147): R F 0.8 (silica - C H ^ l ); 1 H-NMR (200 MHz, CDC1 ), 8 = 7.65 (m, 12H, H nand H \ 7.9 (m, 2H), 8.1 (dd, 4H), 8.5 (d, IH), 8.6 (d, IH), 8.65 (m, IH), 8.7 (d, IH). UV-vis (CH2C12) A m a x (rel. intensity) 402 (0.08), 422 (1.0), 520 (0.06), 558 (0.11), 602 (0.21) nm; MS (EI) m/e calc'd for C 4 3 H 2 8 N 4 0 2 Zn: 694.1310, found 694.1303 (M +, 100%). Ph 0 mes0-Tetraphenyl-2,3-dioxo-3a-oxachlorin (58): To a solution of zinc(II) diol chlorin (146) (30 mg, 0.042 mmol) p in THF (25 mL), lead tetraacetate (38 mg, 1 eq) was added. The reaction mixture was stirred at room temperature and monitored by TLC. Within 30 minutes, the reaction was complete and the solution was evaporated to dryness. Preparative TLC (chloroform) was performed to yield numerous green products including (58). Characterized as the analogous free base compound. R F 0.4 (silica - CH2C12); 1 H-NMR (200 MHz, CDC13) 8= -1.8 (br s, 2H), 7.70 (m, 12H, H m and Hp), 8.0 (m, 4H), 8.1 (m, 4H), 8.4 (d, 2H), 8.6 (s, 2H), 8.7 (d, 2H). UV-vis (CH2C12) A m a x 428, 548, 588, 612, 670 nm; MS (EI) m/e calc'd for C 4 4 H 2 6 N 4 0 3 Zn: 722.12964, found 722.12937 241 Experimental (M +, 83%). 3.2 Reaction with Alcohols Acetals (149) and (150) Nickel bisaldehyde secochlorin (145) (10 mg, 0.014 mmol) was dissolved in chloroform and 2 drops of methanol were added to this solution at room temperature. Fumes from the head space of an aqueous concentrated hydrochloric acid bottle were then added to the mixture via a pipette. The colour of the solution immediately changed from a golden brown to a dark green. The dark green compounds were isolated and purified with preparative TLC (silica, chloroform). (149): H3CO 7.85 (d, 1H), 7.95 (d, 1H), 8.0 (d, 1H), 8.2 (dd, 2H). UV-vis (CH2C12) 1H), 3.2 (s, 3H), 6.1 (s, 1H), 6.6 (d, 1H), 7.60 (2 m, 20H), 7.8 (d, 1H), R F 0.5 (silica - CH2C12); 'H-NMR (200 MHz, CDC13) 8= 2.5 (s, f Ph Ph A 'max (rel. intensity) 430 (1.0), 640 (0.38) nm; MS (EI) m/e calc'd for C 4 5 H 3 2 N 4 0 3 Ni: 734.18762, found 734.18781 (M +, 100%). (150): 6H), 6.1 (s, 2H), 7.60 (2m, 20H), 7.8 (d, 2pH), 8.0 (s, 2PH), 8.2 (d, 2pH). R F 0.8 (silica - CH2C12); 'H-NMR (200 MHz, CDC13) 5= 3.0 (s, Ph O Ph """OCH3 Ph 242 Experimental UV-vis (CH2C12) A m a x (rel. intensity) 430 (1.0), 640 (0.30) nm; MS (EI) m/e calc'd for C 4 6 H 3 4 N 4 0 3 N i : 748.19985, found 734.19878 (M + , 100%). Acetal (151) Ethanol The double acetal (151) was synthesized in an analogous manner as was acetal (150) with the exception of replacing methanol with ethanol. R F 0.8 (silica - CH 2C1 2); 'H-NMR (200 MHz, CDC13) 8= 2.2 (m, 6H), 3.3 (m, 4H), 6.1 (s, 2H), 7.70 (m, 20H), 7.7 (d, 2PH), 7.8 (s, 2pH), 8.0 (d, 2pH). UV-vis (CH2C12) A m a x (rel. intensity) 428 (1.0), 640 (0.20) nm; MS (EI) m/e calc'd for C 4 8 H 3 8 N 4 0 3 N i : 776.17865, found 776.16988 (M + , 100%). 0CHMe2 Ph Acetal (152) Isopropanol The double acetal (152) was synthesized in an analogous manner V as were acetals (150) and (151) with the use of isopropanol as the alcohol reagent. R F 0.75 (silica - C H ^ l ^ ; 'H-NMR (200 MHz, CDC\^ 8= 3.1 (s, 12H), 4.5 (s, 2H), 6.2 (s, 2H), 7.60 (2m, 20H), 7.6 (d, 2PH), 7.9 (s, 2pH), 8.0 (d, 2pH). UV-vis (CH2C12) A m a x (rel. intensity) 430 (1.0), 642 (0.15) nm; MS (EI) m/e calc'd for C 5 0 H 4 2 N 4 O 3 N i : 804.19748, found 804.19879 (M + , 100%). 243 Experimental Ethylene Glycol Adduct (153) Nickel bisaldehyde secochlorin (145) (10 mg, 0.014 mmol) was dissolved in ethylene glycol (5 mL) at room temperature. Fumes from the head space of a concentrated hydrochloric acid bottle were then added Ph to the mixture via a pipette. The colour of the solution immediately changed from a golden brown to a dark green. The dark green compound formed quantitatively was isolated and purified by preparative T L C (silica, chloroform). R F 0.85 (silica - CH 2 C1 2 ) ; ' H - N M R (200 M H z , CDC1 3 ) 8= 4.5 (m, 4H), 6.2 (s, 2H), 7.70 (m, 20H), 7.9 (d, 2H), 8.0 (s, 2H), 8.2 (d, 2H). U V - v i s (CH 2 C1 2 ) A m a x (rel. intensity) 418 (1.0), 450 (0.68), 516 (0.19), 540 (0.15), 592 (0.13), 638 (0.23) nm; M S (EI) m/e calc'd for C 4 6 H 3 2 N 4 0 3 N i : 746.5633, found 746.5782 ( M + , 100%). 3.3 Reaction with base: (i«eso-5,10,15,20-tetraphenyl-2-oxa-3-oxo-2,3-chlorinato)nickel(II) (155): m e s 0 - T e t r a p h e n y l - 2 , 3 - d i c a r b o x a l d e h y d e - 2 , 3 -secochlorinato)nickel(II) (145) (18 mg, 0.026 mmol) was dissolved in dry T H F (2 mL) . Upon addition of fumes or of drops of A^-tetrabutylammonium hydroxide, the reaction mixture immediately turned from a golden brown to a green-blue colour. The solvent 244 Experimental was removed and the residue purified by preparative TLC (silica, eluent dichloromethane) to give the product (155) (18 mg, 100% yield). R F 0.75 (silica - CH2C12); IR 1745 cm 1 ; 1 H-NMR (400 MHz, CDC13) 8= 7.65 (m, 12H, H m and Hp), 7.83 (m, 8H, H0), 7.98 (d, J= 4.82 Hz, 1H, p-H), 8.11 (d, 7=5.01 Hz, 1H, p-H), 8.18 (d, 7=4.74 Hz, 1H, p-H), 8.25 (d, 7=4.78 Hz, 1H, p-H), 8.33 (dd, 7=4.66 Hz, 2H, P-H). UV-vis (CH2C12) A m a x (rel. intensity) 410 (1.0), 500 (0.10), 568 (0.16), 608 (0.24) nm; MS (EI) m/e calc'd for C 4 3 H 2 6 N 4 0 2 Ni : 684.14107, found 684.14093 (M +, 100%). Analysis calc'd for C 4 3 H 3 8 N 4 0 8 Ni: C 64.76, H 4.80, N 7.00; found C 64.71, H 5.15, N 7.29. 3.3.1 Reaction of the (2-oxo-3-oxa-tetraphenylchlorinato)nickel(II) (155): To a stirred solution of nickel bisaldehyde secochlorin (145) (15 mg, 0.021 mmol) in dry THF (5 mL) at -20°C, DIBALH (5 mL of 1M solution in hexane) was added. The mixture was stirred whilst in an ice bath for 40 minutes at which time cone. H 2 S0 4 (0.2 mL) was added dropwise. The reaction was allowed to warm to 0°C and stir for a further 15 mins. The solution was added to ice water (-25 mL) and extracted with methylene chloride. The extract was washed with aq. NaHC0 3 and aq. Et 3N, dried with sodium sulfate, filtered and evaporated to dryness. The three burgundy products (157), (158) and (159) were separated by preparative TLC (1% MeOHxhloroform). Yields were 7%, 14% and 33% respectively. 245 F.rpprimpntnl (»ie*o-Tetraphenyl-2-hydroxy-isobutyl-3-oxo-2,3-chlorinato)nickel(II) (157): R F 0.85 (silica - CH2C12); 1 H-NMR (400 MHz, CDC13) 8= -1.1 (s, 1H), -0.75 (s, IH), 0.7 (m, 3H), 7.55 (s, IH), 7.70 (m, 12H), 7.85 (t, IH), 8.05 (d, 7= 6.02 Hz, 2H), 8.12 (t, IH), 8.17 (d, 7=4.68 Hz, IH, p-H), 8.20 (d, 7= 6.01 Hz, 2H), 8.30 (d, 7= 4.46 Hz, P-H), 8.38 (d, /= 4.46 Hz, IH, p-H), 8.42 (d, 7= 4.99 Hz, IH, P-H), 8.47 (d, 7= 4.64 Hz, IH, P-H), 8.56 (d, 7= 5.0 Hz, IH p-H). UV-vis (CH2C12) Xmm (rel. intensity) 414 (1.0), 516 (0.08), 550 (0.09), 592 (0.06), 644 (0.12) nm; MS (EI) m/e calc'd for C 4 7 H 3 8 N 4 0 2 Ni: 690.29950, found 690.29910 (M +, 100%). («ig50-Tetraphenyl-2-methoxy-3-oxo-2,3-chlorinato)nickel(II) (158): R F 0.55 (silica - CH2C12); 1 H-NMR (400 MHz, CDC1-,) 8= -1.1 P h (s, IH), -0.75 (s, IH), 3.2 (s, 3H), 7.55 (s, IH), 7.70 (m, 12H), 7.85 (t, IH), 7.90 (d, 2H), 8.00 (t, IH), 8.10 (m, 2H), 8.20 (d, 7= 4.34 Hz, IH), P h 8.30 (d, 7= 4.86 Hz, IH, P-H), 8.37 (d, 7= 4.35 Hz, IH, p-H), 8.42 (d, 7= 4.90 Hz, 2H, P-H), 8.54 (d, 7= 4.88 Hz, IH, p-H), 8.59 (d, IH, 7= 4.98 Hz, p-H). UV-vis (CH2C12) A m a x (rel. intensity) 416 (1.0), 514 (0.09), 550 (0.10), 592 (0.06), 644 (0.14) nm; MS (EI) m/e calc'd for C 4 4 H 3 2 N 4 0 2 Ni: 648.25250, found 648.25223 (M +, 100%). (/M^50-Tetraphenyl-2-hydroxy-3-oxo-2,3-chlorinato)nickel(H) (159): P h Rh- 0.25 (silica - CH2C12); IR (cm1) 970 (m, C-H arom.), 1593 (m, O-H), 3051 (w, C-H arom.), 3340 (m, O-H); 1 H-NMR (400 MHz, 246 Experimental C D C I 3 ) 6= -1.1 (s, 1H), -0.75 (s, 1H), 3.5 (br s, 1H), 7.55 (s, 1H), 7.70 (m, 12H), 7.85 (d, 2H), 8.10 (m, 2H), 8.08 (m, 2H), 8.15 (d, J= 4.12 Hz, 1H, p-H), 8.32 (d, J= 4.43 Hz, 1H, p-H), 8.40 (2d, J= 4.42 Hz, 2H, P-H), 8.49 (d, /= 3.75 Hz, 1H, p-H), 8.57 (d, 1H, J= 4.10 Hz, 1H, P-H). UV-vis (CH2C12) A m a x (rel. intensity) 418 (1.0), 516 (0.09), 552 (0.10), 592 (0.05), 646 (0.16) nm; MS (EI) m/e calc'd for C 4 3 H 3 0 N 4 O 2 Ni: 634.23688, found 634.23620 (M + , 100%). 3.4 Reaction with Amines: 3.4.1 TV-Methylamine (mes0-TetraphenyI-2-carboxaldehyde-3-methylimino-2,3-secochlorinato)nickel(II) (160) m < ? s o - ( T e t r a p h e n y l - 2 , 3 - d i c a r b o x a l d e h y d e - 2 , 3 -secochlorinato)nickel(II) (145) (25 mg, 0.0355 mmol) was dissolved in 1%HC1:THF (2 mL). N-Methylamine (0.3 mL, 0.4 mmol) was added to the reaction flask, and the reaction was set to stir at room temperature. The reaction was monitored by TLC (1% MeOH:CH 2Cl 2). Upon completion after 2 hours, the reaction was washed with water, dried, and the solvent was removed and the residue purified by preparative TLC (silica, 1 % MeOH:CH 2Cl 2) to give a green product (160) (20 mg, 0.0279 mmol, 78.57 % yield). R F 0.2 (silica- 1% MeOH:CH 2Cl 2); IR (cm"') 3455 (m, C=N), 2988 and 2795 (m, CHO arom.), 1636 (s, C=0); 'H-NMR (400 MHz, CDC13) 6= 4.45 (s, 3H, CH 3), 6.9 (br s, 1H, H 0 nearest CH=NMe3), 7.02 (br d, 1H, H 0 near CHO), 7.65 (m, 12H), 7.85 (m, 6H), 8.0 (d, 1H, J= 247 Experimental 4.86 Hz, H 1 7), 8.18 (d, 1H, J= 4.80 Hz, H,8), 8.28 (d, 1H, 7= 4.93 Hz, H l 3), 8.33 (d, 1H, J= 4.89 Hz, H7), 8.6 (d, 1H, 7= 4.83 Hz, H„), 8.40 (d, 1H, J= 4.96 Hz, H1 2), 8.62 (br s, 1H, CH=N), 9.1 (s, 1H, CHO). UV-vis (CH2C12) A m a x (rel. intensity) 430 (1.0), 460(sh), 584(sh), 638 (0.23) nm; LRMS (EI) m/e 715 (100%), 699 (80%, -CH 3), 686 (70%, -CHO), 671 (40%, -CH, - CHO); HRMS (EI) m/e calc'd for C 4 5 H 3 1 N 5 ONi: 715.18551, found 715.18823 (M +, 100%). 3.4.2 Reaction with Phenylene Diamine me.so-Tetraphenyl -2 ,3 -d icarboxaldehyde-2 ,3 -secochlorinato)nickel(II) (145) (15 mg, 0.021 mmol) was dissolved in 1%HC1:THF (1 mL). 1,2-Phenylene diamine (0.27g, 24.9 mmol) was added to the reaction flask, and the reaction was set to reflux. After 30 minutes, the TLC (5 % MeOH:CH 2Cl 2) showed no starting material remaining and the reaction was washed, dried and the solvent removed to yield a green solid. The residue was purified by preparative TLC (silica, eluent dichloromethane) to give the green product (161) (12 mg, 73 % yield). R F 0.85 (silica - CH2C12); 'H-NMR (400 MHz, CDC13) 8= 7.11 (br, 2H), 7.22 (t, 2H, J = 7.3 Hz, Hp), 7.35 (t, J = 3.5 Hz, 2H, H2.), 7.39 (t, J = 7.4 Hz, 2H, Hp), 7.4 (d, J = 8 Hz, 2H, H0,), 7.57 (d, / = 8.2 Hz, 2H, H0,), 7.62 (m, 4H, H J , 7.78 (d, / = 4.92 Hz, 2H, H,.), 7.82 (m, 4H, H J , 7.9 (m, 2H, H0), 8.16 (m, 4H), 8.19 (d, J = 4.93 Hz, 2H, p-H), 8.34 (d, 7 = 4.82 Hz, 2H, P-H). 248 Frperimpntnl UV-vis (CH2C12) A . m a x (rel. intensity) 436 (1.0), 516 (sh), 566 (sh), 648 (0.16) nm; MS (EI) m/e calc'd for C 5 0rL, 2N 6Ni: 774.20321, found 774.20416 (M +, 100%). 3.4.3 Reaction with Ammonia me5o-Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) (26 mg, 0.037 mmol) was dissolved in 1% HC1:THF (3 mL). Aqueous ammonia solution (4 mL) was added to the reaction mixture and the whole set to stir at room temperature for one hour. The reaction was monitored by TLC (1% MeOH:CH 2Cl 2) until no starting material was visible. The reaction was washed with water, dried (MgS04) and the solvent removed under vacuum. The residue was purified by preparative TLC (silica, 5 % MeOH:CH 2Cl 2) to give the products (166) (24.5 mg, 0.0358 mmol, 97% yield) and (165) (1.5 mg, 3% yield). (ffi^o-Tetraphenyl-2,3-dioxo-2,3-chlorinato)nickel(II) (165): R F 0.48 (silica - 1% MeOH:CH 2Cl 2); IR 1726 cm"1 (C=0); 'H-P NMR (400 MHz, CDC13) 8= 7.5-7.7 (m, 12H, H m + Hp), 7.82 (dd, J = 6.05 Hz, 4H, H 0 far), 7.91 (dd, J= 6.52 Hz, 4H, H 0 near), 8.11 (d, J = 4.96 Hz, P-H), 8.2 (s, 2H, P-H), 8.33 (d, J= 5.06 Hz, 2H, p-H); UV-vis (CH2C12) A m a x (rel. intensity) 408 (1.0), 480 (0.34), 606 (0.22), broad band 688 (0.10) nm; MS (EI) m/e calc'd for C ^ N A N i : 700.14522, found 700.1493 (M +, 26.42%). 249 Experimental Pigment (166): R F 0.6 (silica - 1% MeOH:CH 2 Cl 2 ); IR 1599, 1361, 974 cm 1 ; 'H-NMR (400 MHz, CDC13) 5= 6.61 (s, 2H, CH 2), 6.92 (d, J = 7.13 Hz, 2H, H 0), 7.37 (t, J = 7.43 Hz, 2H, H ), 7.59 (m, 8H, H 7.7 (t, J = 1 A3 Hz, 2H, Hp), 7.83 (br m, 2H, H 0), 7.93 (d, J = 4.9 Hz, IH, P-H), 8.0 (d, / = 4.9 Hz, 2H, p-H), 8.02 (m, IH, H 0), 8.2 (d, / = 4.6 Hz, IH, H 0), 8.28 (d, J = 4.86 Hz, IH, pH), 8.32 (d, J = 4.86 Hz, IH, p-H), 8.35 (d, J = 4.93 Hz, IH, p-H), 8.40 (d, / = 4.73 Hz, IH, p-H), 8.42 (m, IH, H 0), 8.59 (br m, IH, H 0), 9.1 (s, IH, CH); 1 3 C-NMR (75 MHz, CDC13) 5= 86.266, 113. 875, 122.542, 123.019, 125.503, 126.466, 126.934, 127.075, 127.160, 127.221, 127.266, 127.334, 127.496, 127.737, 128.010, 129.863, 130.917, 132.721, 132.861, 133.031, 133.142, 133.456, 135.555, 135.709, 137.447, 138.507, 139.103, 139.324, 139.407, 141.089, 142.009, 142.165, 145.191, 171.150; UV-vis (CH2C12) A m a x (rel. intensity) 436 (1.0), 526 (0.05), 570 (0.13), 620 (0.17) nm; MS (EI) m/e calc'd for C 4 4 H 2 9 N 5 N i : 685.17541, found 685.17767 (M + , 100%). 3.5 Reaction With Phenylhydrazine Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) (24 mg, 0.034 mmol) was dissolved in phenylhydrazine (-1.5 mL). After 15 mins., no starting material was visible on the T L C plate therefore column chromatography (silica, 1:1 hexane: methylene chloride) was performed . Three products were isolated: a brown product (168) (15% yield), a 250 Experimental burgundy product (155) (20% yield) and a polar green compound (169) (26% yield). Compound (168) R F 0.8 (silica -CH2C12); IR (cm1) 1090 (s, C-N), 1518 (s, C=N), 2336 (s, nitrile); 'H-NMR (400 MHz, CDC13) 8= 7.4 (m, 4H), 7.5 (t, 2H), 7.7 (m, 12H), 7.9 (d, 2H), 8.0 (m, 5H), 8.65 (m, 6H), 8.8 (s, 1H). UV-vis (CH2C12) A m a x (rel. intensity) 390 (0.47), 432 (0.93), 446 (1.0), 548 (0.24), 602 (0.35) nm; MS (EI) m/e calc'd for C 5 0 H 3 2 N 6 Ni: 774.20416, found 774.20139 (M +, 100%). Compound (169) R F 0.5 (silica - CH2C12); 'H-NMR (400 MHz, CDC13) 8= 3.4 (s, 1H, NH), 5.94 (s, 1H, CH), 6.25 (s, 1H, CH), 7.3 (d, 4H, H0), 7.5-7.7 (m, 16H, Hpheny,), 8.05 (2 overlapping d, 2H, PH), 8.25 (s, 2H, pH), 8.38 (d, 2H, pH). UV-vis (CH2C12) A m a x (rel. intensity) 420 (1.0), 574(sh), 610 (0.25) nm; MS (EI) m/e calc'd for C 5 0 H 3 4 N 6 Ni: 776.21985, found 776.21987 (M +, 100%). 3.6 Reaction with HMPT ( m e 5 0 - T e t r a p h e n y l - 2 , 3 - d i c a r b o x a l d e h y d e - 2 , 3 -secochlorinato)nickel(II) (22 mg, 0.03 mmol) was refluxed under nitrogen in CH 2C1 2 (2 mL) . Hexamethylphosphoramide (0.5 mL) was added. The solution immediately turned green from a golden brown colour. The reaction was 251 Expprim.pnt.nl. monitored by TLC (1% MeOH/CH 2Cl 2) whilst refluxing. Upon completion, the solvent was removed and the residue purified by preparative TLC (silica, eluent dichloromethane) to give the green product (170) in 40% yield. R F 0.3 (silica - CH2C12); 'H-NMR (400 MHz, CDC13) 8= 4.41 (d, J= 12.76 Hz, IH, CH), 5.6 (d, J= 12.81 Hz, IH, CH), 7.35 (d, J= 7.72 Hz, 2H, H0), 7.5-7.65 (m, 10H), 7.68 (d, J= 7.46 Hz, 1HJ, 7.74 (d, J= 7.4 Hz, 2H, H J , 7.83 (br m, IH, H0), 7.90 (d, J= 4.70 Hz, IH, pH), 8.0 (br m, IH, H J , 8.1 (d, 2H, BH), 8.15 (d, J= 7.63 Hz, IH, pH), 8.25 (d, J= 4.79 Hz, IH, PH), 8.47 (br m, IH, H J , 8.55 (d, J= 4.89 Hz, IH, pH), 8.90 (d, J= 4.90 Hz, IH, pH) + one singlet integrating for 0.5 H at 8.72 ppm which has not been identified; UV-vis (CH2C12) A m a x (rel. intensity) 430 (1.0), 638 (0.47) nm; LRMS (EI) m/e 686 (M +, 100%), 668 (M +- H 2 0, 90%); HRMS (EI) m/e calc'd for C ^ H ^ O N i : 686.16168, found 686.15940 (M +, 63%). 3.6 Reaction with Acid: 3.6.1 T F A + NBA: Pigment (172): meso-Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) (25 mg, 0.036 mmol) was dissolved in dry THF (2 mL). Upon addition of fumes of TFA, the reaction mixture immediately turned from a golden brown to a green colour. The solvent was removed and the residue purified by preparative TLC (silica, eluent dichloromethane) to give the product (24 mg, 100% yield). 252 F.xpprimpntnl R F 0.8 (silica - CHjClj); IR (cm"1) 1682 (s); 'H-NMR (400 MHz, CDC1 I 8= 5.58 (s, 2H, CH 2), 7.38 (t, 7=7.4 Hz, 2H, Hp'), 7.53 (t, 7=7.6 Hz, 2H, Hm'), 7.64 (m, 10H, 10- and 15-phenyl H), 7.83 (d, 7=7.39 Hz, 2H, Hm"), 8.09 (s, 2H, H l 2 and H l3), 8.25 (d, 7=7.7 Hz, 2H, H ),, 8.52 (d, 7=4.86, 2H, H 8 and H l 7), 8.95 (d, 7=4.88 Hz, 2H, H 7 and H I 8); UV-vis (CH2C12) A m a x (rel. intensity) 452 (1.0), 638 (0.20) nm; MS (EI) m/e calc'd for C 4 4 H 2 6 N 4 ONi: 684.14581, found 684.14600 (M + , 100%). 3.6.2 Excess T F A - Pigment (155) me5,o-Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato) nickel(II) (25 mg, 0.036 mmol) was dissolved in dry THF (2 mL). Addition of 2 drops of TFA immediately turned the reaction mixture from a golden brown to a dark brown-red colour. The solvent was removed and the residue purified by preparative TLC (silica, eluent dichloromethane) to give two products (172) (19 mg, 80% yield) and green 2-oxa-3-oxo-chlorin (155) (5 mg, 20% yield). R F 0.6 (silica - CH2C12); IR 1745 cm'; 'H-NMR (400 MHz, CDC1,) 8= 7.65 (m, 12H, H m and Hp), 7.83 (m, 8H, H0), 7.98 (d, 7= 4.82 Hz, 1H, p-H), 8.11 (d, 7=5.01 Hz, 1H, p-H), 8.18 (d, 7=4.74 Hz, 1H, p-H), 8.25 (d, 7=4.78 Hz, 1H, P-H), 8.33 (dd, 7=4.66 Hz, 2 p-H). UV-vis (CH2C12) A m a x (rel. intensity) 410 (1.0), 500 (0.08), 568 (0.16), 608 (0.26) nm; MS (EI) m/e calc'd for C 4 3 H 2 6 N 4 0 2 Ni : 688.14107, found 688.14093 (M +, 100%). 253 Experimental 3.6.1.2 Reaction of the Doublv-Cvclized Product (172): To the doubly-cyclized product (172) (15 mg, 0.021 mmol) in THF (5 mL), 3 drops of base (/V-tetrabutylammonium hydroxide) were added and the solution stirred at room temperature for 5 minutes. The solution was washed with water, dried with sodium sulfate, filtered and the solvent evaporated to dryness. Preparative TLC (1:1 hexane:methylene chloride) allowed the separation of the two products (182) and (183) (combined yield 100%). Pigment (182): R F 0.8 (CH2C12); UV-vis (CH2C12) A m ; l x (rel. intensity) 344 (0.97), 416 (0.95), 496 (0.99), 536 (1.0), 668 (0.32) nm; IR (cm"1) 3412 (br s), 1710 (m), 1642 (m), 1106 (m), 790 (m); 'H- NMR (400 MHz, CDC13) 8= 7.20 (t, 7=7.40 Hz, 2H, H4.), 7.5 l(t, 7=7.55, 2H, H5.), 7.62 (m, 6H, H m and Hp), 7.70 (d, 7=7.23, 2H, H T), 7.81 (m, 4H, H0), 7.89(d, 7=7.52 Hz, 2H, H6.), 8.07 (s, 2H, H l 2 and H 1 3), 8.44 (d, 7=4.92, 2H, H l 7 and H^, 8.84 (d, 7=4.97 Hz, 2H, H 1 8and H7). "C-NMR (75 MHz, CDC13) 8= 115.990, 123.686, 125.066, 127.340, 128.027, 128.203, 131.785, 132.547, 134.095, 135.316, 135.639, 138.889, 140.595, 141.045, 144.648, 145.254, 188.829. MS (EI) m/e calc'd for C 4 4 H 2 4 N 4 0 2 Ni: 698.12482, found 698.12524 (M +, 100%). 254 Frrpprimentnl Pigment (183): R F 0.6 (CHjClj); UV-vis (CH.Cl,) A m a x (rel. intensity) 304 (0.59), 376 (0.65), 416 (1.0), 500 (0.46), 710 (0.16) nm; IR (cm1) 1 106 (s, C-O ester str.), 1643 (m, C=0 aryl str.), 1705 (m, C=0 ester str.), 2876 (m), 2920 (m); 'H- NMR (400 MHz, CDC13) 8= 7.10 (t, 7=7.28 Hz, IH, Hp„), 7.17 (t, 7=7.7, IH, Hm„), 7.30 (t, 7= 7.60 Hz, IH, Hp,.), 7.39 (d, 7=7.57, IH, H in,), 7.44 (t, 7= 7.62 Hz, IH, Hm,), 7.46 (d, 7=7.37 Hz, IH, Hm,), 7.58 (m, 10H, H p h e n y l), 7.73 (d, 7= 7.43, IH, H0,), 7.78 (m, IH, H0), 7.79 (d, 7= 4.88 Hz, IH, pH), 8.17 (2d, 7= 4.89 Hz, 2H, PH), 8.48 (2d, 7=4.78 Hz, 2H, H 1 2 andH 1 3); 1 3 C-NMR (75 MHz, CDC1-,) 8= 120.760, 122.917, 123.190, 123.428, 124.073, 124.294, 124.335, 125.115, 125.773, 127.322, 127.449, 127.948, 128.034, 128.218, 129.039, 129.194, 129.734, 130.019, 131.075, 131.184, 131.574, 132.501, 132.652, 132.993, 133.145, 133.234, 133.571, 134.655, 135.336, 135.654, 138.545, 138.651, 139.243, 139.482, 139.841, 141.631, 144.618, 145.838, 148.838, 148.400, 150.001, 153.071, 182.098, 192.053 MS (EI) m/e calc'd for C 4 4 H 2 4 N 4 0 3 Ni : 714.12093, found 714.12378 (M + , 100%); Analysis calc'd for C 4 4 H 2 4 N 4 0 3 Ni - lTHF: C 73.20, H 4.09, N 7.12; found C 73.40, H 4.05, N 7.12. 255 F.xpprimpntnl 3.6.3 Reaction with HCI Pigment (185): m e s o - T e t r a p h e n y l - 2 , 3 - d i c a r b o x a l d e h y d e - 2 , 3 -secochlorinato)nickel(II) (145) (10 mg, 0.014 mmol) was dissolved in methylene chloride (3 mL). Upon addition of 4 drops of concentrated HCI, the reaction mixture immediately turned from a golden brown to a darker brown colour. The solvent was removed and the residue purified by preparative TLC (silica, toluene/dichloromethane) to give the product (185) (8 mg, 80% yield). R F 0.7 (silica - CHjClj); IR (cm"1) 945 (s), 1234 (m), 1288 (m), 1487 (m), 1590 (m), 1713 (m); 'H-NMR (400 MHz, CDC13) 6= 7.19 (m, 4H, H p andH 6 ) , 7.50 (m, 2H, H 4 ' and H 5), 7.61 (m, 10H), 7.72 (d, 2H, 7= 7.15 Hz, H0), 7.80 (m, 4H, H m and Hm'), 7.82 (d, 1H, J= 7.6 Hz, H 3), 7.95 (d, /= 4.86 Hz, 1H, H8), 8.06 (2d, J= 4.85, 2H, H 8 and H I 7), 8.24 (d, J= 4.87 Hz, 1H, H7), 8.42 (d, /= 4.93 Hz, 1H, H1 7), 8.79 (d, J= 5.00 Hz, 1H, H1 8), 8.90 (d, J= 7.24 Hz, 1H, H0), 9.48 (s, 1H, CHO); 1 3 C-NMR (75 MHz, CDC13) 8= 108.057, 109.522, 1 15.388, 116.819, 121.263, 123.650, 125.710, 126.143, 127.332, 127.387, 127.559, 127.832, 128.011, 128.078, 128.119, 128.247, 128.457, 129.849, 130.965, 131.313, 131. 906, 132.163, 132.414, 132.841, 132.887, 134.746, 134.971, 135.374, 135.974, 136.889, 138.734, 138.813, 140.938, 141.200, 141.495, 143.985, 145.448, 145.863, 146.684, 187.286, 189.948; UV-vis (CH2C12) A m M (rel. intensity) 416 (1.0), 458 (0.83), 504 (0.71), 718 (0.29) nm; MS (EI) m/e calc'd for C 4 4 H 2 6 N 4 0 2 Ni : 700.14014, found 700.14093 (M +, 100%). 256 . F.Ypprimpntnl 3.7 Reaction with Ylides: Wittig Reaction Pigment (189) Nickel bisaldehyde secochlorin (145) (20 mg, 0.028 mmol) was dissolved in THF (3 mL) and chloroform (0.5 mL). Bromomethyl triphenylphosphonium bromide (80 mg, 0.20 mmol) was added to this solution and allowed to stir. Butyllithium (2 mL of 1M hexane solution) was added dropwise to the reaction flask under nitrogen. The reaction was left to stir at room temperature under nitrogen for 5 hours. The solution was removed in vacuo and preparative TLC (chloroform) was performed. Four compounds were isolated with the major product being the most polar (4 mg, 18% yield) R F 0.1 (silica - CH2C12); UV-vis (CH2C12) X M A X 422, 534, 574, 612, 668 nm; MS (EI) m/e calc'd for C 4 6 H 2 9 N 4 BrNi: 776.09088, found 776.08879 (M + , 100%); calc'd for C 4 5 H 2 g N 4 OBr 2 Ni: 853.99902, found 853.99981 (M + , 84%). 3.8 Reaction with Wilkinson's Catalyst: Decarbonylation A solution of (meso-tetraphenylsecochlorinato)nickel(II) (145) (15 mg, 0.02 mmol) in benzonitrile (5 mL- note: reaction only occurred with very dry benzonitrile) was treated with 2 equivalents of (Ph3P)3RhCl (40 mg, 0.04 mmol). The reaction mixture was refluxed for 75 minutes producing the monodecarbonylated meso-tetraphenylchlorin (192) in 10% yield, (meso-tetraphenylchlorophinato)nickel(II) (191) in 60% yield and (193) in 5% yield. The solution was evaporated to dryness in vacuo and the resulting residue purified by preparative thin layer 257 F.xpprimpntnl chromatography (silica - toluene). (meso-Tetraphenyl-2,3-secochlorinato-2-carboxaIdehyde)nickel(II) ((192): R f 0.75 (silica - toluene); IR (cm"1) 1657 (s, C=0 stretch); 'H-NMR (400 MHz, CDC13) 8= 7.42 (t, 1 Hz, 1H), 7.6 (m, 16H), 7.78 (d, 4.8 Hz, 2H), 7.95 (d, 7.1 Hz, 3H), 8.05 (d, 4.8 Hz, 1H), 8.07 (d, 4.8 Hz, 1H), 8.15 (d, 4.7 Hz, 1H), 8.22 (d, 4.9 Hz, 1H), 8.28 (d, 5.0 Hz, 1H), 9.80 (s, 1H), 9.85 (s, 1H); UV-vis (CH2C12 (log e)) A m a x 448 (4.06), 630 (3.15), 678 (3.34); LR-MS (EI, 280°C) m/e 674 (100, M +), 646 (85%, -CO), 597 (55%), 568 (72%), 492 (35%); HR-MS (EI, 280°C) m/e calc'd for C 4 3 H 2 8 N 4 NiO: 674.16168, found: 674.16024. (meso-Tetraphenyl-2,3-secochlorinato)nickel(II) ((191): R f 0.86 (silica - toluene); IR (cm"1) 3400 (br); 1570 (w); 1050 (w); 800 (s); 'H-NMR (400 MHz, CDC13) 8= 7.55 (t, 2H), 7.62 (m, 10H), 7.79 (d, 2H), 7.85 (m, 6H), 8.18 (s, 2H), 8.22 (d, 2H), 8.42 (d, 2H), 9.85 (s, 2H); 1 3 C-NMR (75 MHz, CDC13) 8=112.362, 123.750, 126.038, 126.958, 127.349, 128.877, 129.241, 129.476, 130.509, 131.841, 132.341, 133.236, 134.030, 134.659, 138.169, 138.934, 139.837, 140.295, 141.159, 141.560, 145.565; UV-vis (CH2C12 (log e)) A m a x 422 (4.33), 576 (3.28), 612 (3.58); LR-MS (EI, 280°C) m/e 646 (100, M +); HR-MS (EI, 258 F.xpprimpntnl 280°C) m/e calc'd for C 4 2 H 2 8 N 4 5 8 Ni: 646.16675, found: m/e 646.16675. Analysis calc'd for C 4 2 H 2 8 N 4 Ni-H 2 0: C 75.89, H 4.55, N 8.30; found C 75.81, H 4.54, N 8.42. Pigment (193): R f 0.4 (silica - toluene); 'H-NMR (400 MHz, CDC13) 8= 3.0 (s, 1H, OH), 6.08 (s, 1H, CH), 7.65 (m, 12H, 4H p and 8 H J , 8.0 (2d, 8H„), 8.3 (s, 2H, H 1 2 and H 1 3), 8.60 (2d, 4H, H 1 7 , H 1 8 , H 7 and H8); UV-vis (CH2C12) A m a x (rel. intensity) 410 (1.0), 496 (0.03), 548 (0.06), 574 (0.08), 608 (0.19); LR-MS (EI, 280°C) m/e 674 (100, M +); HR-MS (EI, 280°C) m/e calc'd for C 4 3 H 2 8 N 4 NiO: 646.13454, found 646.13567. 3.9 Vilsmeier-Haack Reaction of Secochlorin (191): To a stirred solution of dimethylformamide (3 mL, 2.83 g, 0.039 moi) at room temperature was added with stirring, phosphorus oxychloride (3.5 mL, 5.76 g, 0.038 moi). After one hour, the solution had turned pink and nickel bisaldehyde secochlorin (145) (12 mg, 0.019 mmol) in 2 mL dichloromethane was added. The reaction was stirred at room temperature for 2 hours whereupon monitoring by TLC (dichloromethane) showed no starting material remaining. The solvent was removed and the residue purified by preparative TLC (20% hexane:CH2Cl2) to yield three products: a nonpolar, olive colored product which was monodecarbonylated 259 F.Ypprimentnl secochlorin (192), a blue product of medium polarity (194) and a polar green product (195). Pigment (194) R F 0.5 (silica - CH2C12); 'H-NMR (400 MHz, CDC13) 8= 1.91 (s, 6H, CH 3), 6.08 (s, IH, CH), 7.62 (m, 12H, 4H p and 8 H J , 7.94 (2d, J= 6.83 Hz and J= 6.82 Hz, 8H0), 8.23 (s, 2H, H 1 2 and H1 3), 8.40 (2 overlapping d, 4H, J= 4.87 Hz for both, H 1 7 , H 1 8 , H v and H8); UV-vis (CH2C12) A m a x (rel. intensity) 408 (1.0), 496 (0.03), 544 (0.05), 572 (0.08), 602 (0.20) nm; MS (EI) m/e calc'd for C 4 5 H 3 3 N 5 Ni: 701.21011, found 701.20892 (M +, 100%). Pigments (195): R F 0.3 (silica - CH2C12); UV-vis (CH2C12) A m a x (rel. intensity) 446 (1.0), 572 (sh), 618 (0.20), 640 (sh) nm; MS (EI) m/e calc'd for C 4 5 H 3 3 N,Ni: 701.21011, found 701.20892 (M +, 100%). 3.10 Reaction with Hydroxylamine Hydrochloride (meso-Tetraphenyl-2,3-secochIorinato-2-nitrile)nickel(II) (196): m e 5 0 - T e t r a p h e n y l - 2 , 3 - d i c a r b o x a l d e h y d e - 2 , 3 -secochlorinato)nickel(II) (145) (22 mg, 0.031 mmol) was dissolved in THF (5 mL). Hydroxylamine hydrochloride (12 mg, 0.171 mmol) was added to the reaction flask, and the reaction was set to stir at room temperature. The reaction was monitored by TLC (1% MeOH/CH 2Cl 2). Upon completion, the solvent was removed and the residue purified by 260 F.xpprimpntnl preparative TLC (silica, eluent dichloromethane) to give many green products with the major dark green product (196) (40% yield) remaining on baseline. R F 0.1 (silica - 10% MeOH/CH 2Cl 2); IR (cm1) 3407 (br s), 1634 (m), 1096 (m) ; 'H-NMR (400 MHz, CDC13) 6= 7.7 (br m, 12H), 7.92 (d, 2H, J= 7.51 Hz, H0), 7.96 (d, 2H, J= 6.83 Hz, H0), 8.0 (2d, 2H, J= 7.48 Hz, H0), 8.58 (2 overlapping dd, 4H, 7=5.59 Hz and 7=5.16 Hz, p-H), 8.72 (2 overlapping d, 2H, 7= 4.22 Hz for both, H l 2 and H1 3), 10.5 (s, 1H). UV -v is (CH2C12) A m a x (rel. intensity) 422(sh), 444 (1.0), 540 (sh), 580 (sh), 632 (0.31) nm; LRMS (EI) m/e 671 (100%, M +), 646 (2%, -CN); HRMS (EI) m/e calc'd for C 4 3 H 2 7 N 5 Ni: 671.16050, found 671.16199 (M +, 100%). 3.11 Reaction with Reducing Agents: 3.11.1 Lithium Aluminum Hydride: me5,o-Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) (22 mg, 0.03 mmol) was dissolved in dry THF (2 mL). Lithium aluminum hydride (1 mg, 0.03 mmol) was added to the reaction and set to reflux. The reaction mixture changed from a golden brown colour to dark green. The reaction was quenched with the addition of water, washed with water and separated. The solvent of the organic layer was removed and the residue purified by preparative TLC (silica, eluent dichloromethane) to give three green products: (197) - dark forest green, nonpolar (10 mg, 47 % yield), (198) - a bright yellow-green product of moderate polarity 261 Experimental (5 mg, 25% yield) and (199) - a dark green, polar compound (2.5 mg, 10% yield). (meso-Tetraphenyl-2,3-dimethyl-2,3-secochlorinato)nickel(II) (197): R F 0.88 (silica - C H CI \ IR 1120, 1040, 980 cm 1 ; 'H-NMR (400 MHz, CDC1J 8= 2.75 (s, 6H, C H J , 6.9 (br m, 2H, H 0 nearest CH3), 7.32 (m, 4H, H J , 7.43 (t, 7= 1 Al Hz, 2H, Hp), 7.50 (t, 2H, 7= 7.72, Hp), 7.56 (m, 4H, H J , 7.58 (d, 2H, 7= 4.86 Hz, H 7 and H l g), 7.82 (br m, 4H, HH), 7.93 (s, 2H, p-H), 8.11 (d, 2H, 7=4.85 Hz, p-H), 8.34 (br m, 2H, H J . UV-vis (CH2C12) A m a x (rel. intensity) 418 (1.0), 438 (0.85), 620 (0.20), 666 (0.20) nm; LRMS (EI) m/e 61A (M +, 100%), 659 (M + -CH 3 , 12%); HRMS (EI) m/e calc'd for C 4 4 H 3 2 N 4 Ni: 674.1970, found 674.19806 (M +, 100%). (meso-Tetraphenyl-2-hydroxymethyl-3-rnethyl-2,3-secochlorinato)nickel(II) (198): R F 0.67 (silica - CH2C12); IR (cm'1) 3215 (s, O-H), 2357 (m), 1676 (m), 1584 (m), 1543 (m), 1284(s); 'H-NMR (400 MHz, CDC1J 8= 2.55 (s, 3H, C H J , 3.65 (s,lH, OH), 5.88 (s, 2H, CH 2), 7.28 (m, 4H, H J , 7.38 (t, 2H, Hp), 7.45 (t, 2H, Hp), 7.62 (br m, 8H, 4H m and 4HJ, 7.83 (br m, 4H, H J , 8.0 (d, IH, 7= 4.86 Hz, H,J, 8.05 (d, IH, 7= 4.90 Hz, H1 2), 8.19 (d, IH, 7= 2.74 Hz, H1 7), 8.21 (d, IH, 7= 2.65 Hz, H J , 8.32 (d, IH, 7= 2.53 Hz, H7), 8.34 (d,lH, 7= 2.74 Hz, H,J. UV-vis (CH2C12) A m ; i x (rel. intensity) 414 (1.0), 262 Experimental 500 (0.06), 574 (0.08), 606 (0.20), 662 (0.05), 758 (0.04) nm; LRMS (EI) m/e 690 (M +, 100%), 674 (M + -CH 3 , 90%), 660 (M + -CH 2 OH, 30%), 647 (M + -CH 3 -CH 2 OH, 50%); HRMS (EI) m/e calc'd for C ^ H ^ N i O : 690.19206, found 690.19293 (M +, 100%). ( r a e s o - T e t r a p h e n y l - 2 , 3 - d i h y d r o x y r a e t h y l - 2 , 3 -secochlorinato)nickel(II) (199): R F 0.56 (silica - CH2C12); IR 3053 (br s, O-H), 1559 (s, O-H), 1489 (s, C-H) cm 1 ; 'H-NMR (400 MHz, CDC13) 8= 3.6 (s, 2H, OH), 6.46 (s, 4H, CH 2), 7.35 (m, 9H), 7.45 (t, 2H, Hp), 7.76 (m, 6H), 7.75 (d, 1H), 7.8 (m, 4H), 7.9 (d, 1H), 8.05 (d, 1H), 8.18 (d, 2H). UV-vis (CH2C12) A m a x (rel. intensity) 418 (1.0), 544 (sh), 588 (0.14), 608 (0.13), 632 (0.12), 770 (0.10) nm; LRMS (EI) m/e 706 (M +, 50%), 675 (M + -CH 2 OH, 100%), 647 (M +-2(CH 2OH), 20%); HRMS (EI) m/e calc'd for..C 4 4H 3 2N 4Ni0 2: 706.18629, found 706.18787 (M + , 12%); calc'd for C 4 3 H 2 8 N 4 NiO: 674.16360, found 674.16168 (M + - C H O H , 100%). 3.11.2 Sodium Borohydride meso -Tetraphenyl-3-hydroxy-3a-oxa-chlorin (201): m e s 0 - T e t r a p h e n y l - 2 , 3 - d i c a r b o x a l d e h y d e - 2 , 3 -secochlorinato)nickel(II) (145) (10 mg, 0.014 mmol) was dissolved in dry THF (5 mL). Sodium borohydride (2 mg) was added to the reaction and set to reflux. The 263 F.xpprimpntnl reaction mixture changed from a golden brown colour to green. The reaction was quenched with the addition of water, washed with water and separated. The solvent of the organic layer was removed and the residue purified by preparative TLC (silica, eluent chloroform) to give one blue-green product (201) (7 mg, 70% yield). R F 0.4 (silica - CH2C12); IR (cm"1) 3446 (br s, O-H), 2819 (br s, C-H arom.), 1583 (s, O-H bend), 1360 (s, C-O ether); 'H-NMR (400 MHz, CDC13) 8= 2.3 (s, 1H), 4.3 (d, J= 10.97 Hz, 1H), 5.5 (d, J= 11.03 Hz, 1H), 6.5 (d, 7= 5.67 Hz, 1H), 6.9 (br m, 2H0), 7.4 (br m, 2H„), 7.55 (t, 7= 7.04 Hz, 2Hp), 7.6 (m, 8H p h e n y l m + p), 7.85 (m, 2Hm), 7.9 (d, 7= 4.73 Hz, 2H, PH), 8.1 (s, 2H, pH), 8.3 (d, J= 4.87 Hz, 2H, PH); UV-vis (CH2C12) A m a x (rel. intensity) 422 (1.0), 610 (0.20), 636 (0.19) nm; LRMS (EI) m/e 704 (M +, 35%), 688 (M +- OH, 30%); HRMS (EI) m/e calc'd for C 4 4 H 3 0 N 4 NiO 2 : 704.17224, found 704.17287 (M +, 100%). 3.12 Reaction with Osmium Tetroxide mei,o-Tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II) (145) (24 mg, 0.034 mmol) was dissolved in methylene chloride (10 mL) and 2 drops of pyridine were added. Osmium tetroxide (14 mg, 0.055 mmol, 1.5 eq.) was added to the reaction, stirred at room temperature in the dark. The reaction progress was monitored by TLC or UV-visible spectroscopy until no further reaction was observed (5 days). The reaction was then purged with hydrogen sulfide gas for 10 minutes, and then purged with air until the solvent had evaporated. 264 Experimental The solid was then dissolved in a 10% MeOH:CHCl 3 solution and filtered. The filtrate was evaporated to dryness and chromatographed (silica, 5 % MeOH:CHCl 3) to yield 3 compounds: (150), (155) and (203) (7 mg, 28% yield). Hemiacetal (203) R F 0.3 (silica - CH2C12); 'H-NMR (400 MHz, CDC13) 6= 2.4 (s, OH), 3.3 (s, 3H, CH 3), 7.35 (s, IH, CH), 7.6 (m, 12H), 7.8 (m, 8H), 7.95 (d, 2H, pH), 8.13 (s, 2H, pH), 8.3 (dd, 2H, PH), 9.7 (s, IH, CHO). U V -vis (CH2C12) A m a x (rel. intensity) 408 (1.0), 498 (0.10), 568 (0.12), 608 (0.28), 640 (0.11) nm; LRMS (FAB) m/e 734 (M + , 55%), 717 (M +- OH, 55%), 703 (M +- OMe, 40%), 689 (M +- OH -CHO, 60%), 675 (M +- OMe - CHO, 75%), 657 (M +- OH - OMe - CHO, 55%); HRMS (LSIMS) m/e calc'd for C 4 5 H 3 2 N 4 Ni0 3 : found 734.118016, dev. from calc'd -3.52 ppm (M +, 100%). Chapter 4 Compounds 5,10,15,20-Tetraphenyl-2,3-(3'-dicyano)cyclopropano-2,3-chlorin (213): A solution of 5,10,15,20-tetraphenylporphyrin (50 mg, 0.08 mmol) and tetracyanoethylene oxide (TCNEO) (50 mg, 0.34 mmol) in 1,2-dibromoethane (2 mL) was refluxed for 1 hour. The solvent was evaporated in vacuo, and preparative TLC performed (silica; 1:1 hexane:chloroform). 265 Experimental R F 0.7 (silica - 1% MeOH:CHCl 3); 'H-NMR (400 MHz, CDC13) 8 = -2.05 (s, 2H, NH), 7.80 (m, 8H), 7.87 (m, 4H), 7.99 (d, J = 7.47 Hz, 2H), 8.09 (d, 7 = 6.72 Hz, 2H), 8.20 (m, 2H), 8.35 (d, 7 = 7.16 Hz, 2H), 8.38 (d, 7 = 4.99 Hz, 2(3H), 8.51 (s, 2f3H), 8.70 (d, 7 = 4.84 Hz, 20H). UV-vis (CH2C12) A m a x (rel. intensity) 416 (1.0), 514 (0.14), 554 (0.18), 586 (0.10), 642 (0.22) nm; MS (EI) m/e calc'd for C 4 7 H 3 0 N 6 : 678.25317, found 678.25190 (M +, 100%). Reaction of DPP with TCNEO -Pigments (215) and (216): A solution of DPP (16 mg, 0.035 mmol) and tetracyanoethylene oxide (TCNEO) (7.5 mg, 0.05 mmol) in 1,2-dibromoethane (5 mL) was refluxed for 3 hours. The solvent was evaporated in vacuo, and preparative TLC performed (silica; 1:1 toluene:hexane) to yield (215) and (216). Pigment (215): N C V / C N R F 0.8 (silica - CHC13); IR 2210(s), 1729.8 (m), 1571.7 (s), C 1509 (m), 1384 (m), 1182 (s) cm"1 'H-NMR (400 MHz, CDC13) 8= 6.5 (d, 7 = 4.41 Hz, 2H, 0H), 6.6 (d, 7= 4.29 Hz, 2H, pH), 7.14 (t, 7= 3.95 Hz, 4H, pH), 7.4-7.55 (m, 10H), 13.9 (s, 2H); UV-vis (CH2C19) kmax (rel. O intensity) 376 (0.94), 416 (1.0), 510 (0.58), 534 (0.62) nm; MS (EI) m/e calc'd for C 3 5 H 2 0 ON 6 : 540.16986, found 540.17000 (M +, 100%); yield 36%. 266 F.xperi.m.entnl Pigment (216): N G ^ C N R F 0.6 (silica - CHC13); IR 2215 (s), 1650 (m), 1567 (m) cm"1 'H-NMR ^ / / T ^ \—N HN-—^ (400 MHz, CDC13) 8= 6.65 (d, J = 4.63 Hz, 4H, 0H), 7.19 (d, /= 4.47 Ph<^ )>Ph Hz, 4H, PH), 7.4-7.58 (m, 10H), 13.75 (s, 2H);UV-vis (CH2C12) A m a x (rel. intensity) 400 (1.0), 562 (0.63) nm; LRMS (EI) m/e 588 (M +, N C / C " C N 100%), 563 ( M + - CN, 40%); HRMS (EI) m/e calc'd for C 3 8 H 2 0 N S : 588.18109, found 588.18193 (M +, 100%); yield 45%. Reaction of ZnDPP with T C N E O Pigment (217): A solution of ZnDPP (50 mg, 0.07 mmol) and tetracyanoethylene oxide (TCNEO) (19 mg, 0.13 mmol) in 1,2-dibromoethane (5 mL) was refluxed for 2 hours. The solvent was evaporated in vacuo, and preparative TLC performed (silica; chloroform). R F 0.6 (silica - 1% MeOH:CHCl 3); IR 2213.8(s), 1563.9 (m), 1492 (s) cm"' 'H-NMR (400 MHz, CDCI3) 8= 6.45 (d, J= 4.40 Hz, 4H, pH), 7.03 (d, J= 4.41 Hz, 4H, pH);UV-vis (CH2C12) A m a x (rel. intensity) 420(sh), 458 (1.0), 638 (0.98) nm; LRMS (EI) m/e 650 (M +, 100%), 625 (M +- CN, 80%), 602 (60%); HRMS (EI) m/e calc'd for C 3 8 H 1 8 N 8 Zn: 650.09460, found 650.09565 (M + , 100%). 267 Experimental Reaction of ZnTPP with T C N E O Pigment (218): A solution of ZnTPP (91 mg, 0.13 mmol) and tetracyanoethylene oxide (TCNEO) (73 mg, 0.51 mmol) in 1,2-dibromoethane (2 mL) was refluxed for 24 hours. The solvent was evaporated in vacuo, and preparative TLC performed (silica; chloroform). R F 0.5 (silica - 1% MeOH:CHCl 3); IR (cm"') 2227 (m), 1743 (s), 1592 (m), 1437 (s), 1271 (s); UV-vis (CH2C12) A m a x (rel. intensity) 428(sh), 442 (1.0), 548 (0.10), 638 (0.16) nm; 'H-NMR (400 MHz, CDC13) 8= 7.6 (t, 2H), 7.7-7.8 (m, 10H), 7.86 (t, 2H), 8.10-8.20 (m, 6H), 8.38 (d, J= 7.24 Hz, 1H), 8.55 (d, 7= 7.36 Hz, 1H), 8.80 (m, 4H, PH), 8.95 (d, 7= 4.64 Hz, 1H, pH), 9.2 (s, 1H, p'H), 9.65 (d, 7= 4.72 Hz, 1H, pH); MS (EI) m/e calc'd for C 4 7 H 2 6 N 6 Zn: 740.16669, found 740.16489 (M +, 71%). To a mixture of mesitaldehyde (219) (10 mL, 0.68 moi) in water || I H 3 C ^ ^ C H 3 (17 mL), ethanol (17 mL) and ice (29 mL) was added hydroxylamine hydrochloride (5.21 g, 0.075 moi). Sodium hydroxide (50% soln, 50 mL) was added and the reaction mixutre stirred for 1 hour. The mixture was extracted with ether, acidifed to pH 6 with HCI and extracted with methylene chloride. The solvent was removed in vacuo to leave a white solid. The solid was washed with water, redissolved in methylene chloride and recrystallized from water. The yield of the mesitaldehyde oxime (220) was 82%. Synthesis of 2,4,6-trimethylbenzonitrile oxide (222): 268 F.rpprinipntal A solution of mesitaldehyde oxime (220) (0.22 g, 1.35 mmol) in DMF (120 mL) was stirred at room temperature. A^-Chlorosuccinimide (NCS) (0.180 g) was added slowly to maintain a temperature of 25-30°C. The solution was stirred for 12 hours and was then poured into 400 mL of ice water. Long white needles of the 2,4,6-methylbenzohydroximinoyl chloride were obtained (90%). 'H-NMR (400 MHz, CDC13) 6= 2.35 (s, 3H), 3.4 (s, 6H), 6.8 (s, 2H), 8.4 (s, 1H). Addition of base (Et3N) to the hydroximinoyl chloride generated the desired mesitaldehyde oxide (222). Reaction of mesitaldehyde oxide (222) with DPP - Chlorin (223): A solution of DPP (4.5 mg, 0.01 mmol) in methylene chloride was slowly added to a solution of 2,4,6-trimethylbenzohydroximinoyl chloride (220) (55 mg, 0.28 mmol) in triethylamine (1 mL) at room temperature. The reaction was allowed to proceed at room temperature P h for 2 days. The mixture was washed with water, dried and the solvent removed in vacuo. Preparative TLC was performed (1:1 hexane:chloroform). R F 0.7 (silica - CHC13); 'H-NMR (400 MHz, CDC13) 5= -2.5 (br s, 1H), -2.05 (br s, 1H), 2.51 (s, 6H), 2.72 (s, 3H), 6.8 (s, 1H), 7.35 (s, 1H), 7.7 (2d, J= 3.03 Hz, 2H), 7.8 (m, 6H), 7.86 (t, J= 7.44 Hz, 2H), 7.95 (d, 7= 7.13 Hz, 2H), 8.15 (br m, 2H), 8.22 (br m, 2H), 8.42 (d, 7= 7.52 Hz, 2H), 8.63 (dd, 7= 6.52 Hz, 4H), 8.74 (dd, 4H), 8.88 (dd, 2H), 9.1 (d, 7= 4.38 Hz, 2H), 269 F.xpprimpntnl 9.3 (d, 7= 4.59 Hz, 2H), 10.2 (s, lH).UV-vis (CH2C12) A m a x (rel. intensity) 406 (1.0), 474 (sh), 502 (0.2), 534 (0.05), 576 (0.06), 632 (0.16) nm; HRMS (LSIMS) m/e found C 4 2 H 3 4 N 5 0: found 624.27501, dev. from calc'd: -2.13 ppm. Reaction with diazomethane- Chlorin (226) Potassium hydroxide (1 g) was dissolved in ethanol (2 mL) and water (1 mL). To this solution was added, dropwise, a solution of Diazald (1.02 g) in diethyl ether (10 mL). The diazomethane was allowed to condense on a cold finger and the liquid added to a solution of tetraphenylporphyrin (0.11 g) in THF (10 mL). Once the addition was complete, a rubber septum was added to the reaction flask and the mixture allowed to sit at room temperature. The reaction was monitored by TLC and when no further reaction was visible, 2 drops of glacial acetic acid were added to neutralize any remaining diazomethane. Acetonitrile (10 mL) was added and the reaction mixture was evaporated to dryness. Preparative TLC was performed (methylene chloride) and the green product isolated and characterized. For complete characterization see references 227 and 228. R F 0.3 (silica - CHCl 3);UV-vis (CH2C12) A m a x 418, 514, 548, 604 nm; LRMS (EI) m/e 628 (M +, 40%). 270 Experimental Reaction of (5,10,15,20-tetraphenyl-vic-2,3-dihydroxy-2,3-chlorinato)zinc(II) (146) with zinc acetate and 2,4-pentadione: A solution of (5,10,15,20-tetraphenyl-v/c-2,3-dihydroxy-2,3-chlorinato)zinc(II) (146) (60 mg, 0.08 mmol) in chloroform (1 mL) was added to a solution of excess zinc acetate (189 mg, 0.8 mmol) in 2,4-pentadione (2 mL). The reaction mixture was refluxed for 48 hours. The crude reaction mixture was washed with water, the solvent evaporated in vacuo, and preparative TLC performed (silica; chloroform). Three products were isolated: (233), (234) and (235). (5,10,15,20-tetraphenyl-2-oxo-porphyrinato)zinc(II) (233): R F 0.8 (silica - CHC13); 'H-NMR (400 MHz, CDC13) 8= 7.70-7.80 (m, 9H), 7.85 (m, 3H), 8.09 (s, IH), 8.20 (m, 8H), 8.63 (d, 7 = 4.71 Hz, 2H), 8.86-8.95 (3d, J = 4.66 Hz, 6H).UV-vis (CH2C12) A m a x (rel. intensity) 422 (1.0), 516(sh), 550 (0.15), 592 (sh) nm; MS (EI) m/e calc'd for C 4 4 H 2 8 N 4 OZn: 692.15546, found 692.15525 (M +, 100%). (5,10,15,20-tetraphenyl-2-oxo-3-oxa-2,3-chlorinato)zinc(II) (235): R F 0.60 (silica - CHC13); 1 H-NMR (400 MHz, CDC13) 8= 7.70 (m, 12H), 7.90 (d, J = 6.2 Hz, 2H), 8.03 (d, J = 6.92 Hz, 2H), 8.08 (d, J= 6.68 Hz, 2H), 8.46 (d, /= 4.39 Hz, IH, p-H), 8.55 (d, J= 4.35 Hz, 1H,P-H), 8.62 (d, /= 4.40 Hz, 271 F.xpprimpntnl 2H, p-H), 8.65 (d, J= 4.73 Hz, 1H, P-H), 8.69 (d, J= 4.57 Hz, 1H, P-H). UV-vis (CH2C12) A m i i x (rel. intensity) 426 (1.0), 524 (0.05), 562 (0.12), 608 (0.21) nm; MS (EI) m/e calc'd for C 4 3 H 2 6 N 4 0 2 Zn: 694.13470, found 694.13573 (M +, 100%). (5,10,15,20-Tetraphenyl-2,3-dioxochlorinato)zinc(II) (234): R F 0.40 (silica - CHC13); 1 H-NMR (400 MHz, CDC13) 8= 7.60 (t, 2H), 7.68 (m, 12H), 7.78 (d, 7= 6.88 Hz, 2H), 8.02 (dd, J= 6.45 Hz, 4H), 8.28 (d, 7= 4.77 Hz, 2H, p-H), 8.42 (s, 2H,P-H), 8.51 (d, J= 4.78, 2H, p-H). UV-vis (CH2C12) A m a x (rel. intensity) 436 (1.0), 580 (0.12), 626 (0.14) nm; MS (EI) m/e calc'd for C 4 4 H 2 6 N 4 0 2 Zn: 708.15039, found 708.14967 (M +, 100%). Reaction of l,3-Diphenyl-2-azaallyllithium (238) with DPP: Lithium diisopropylamine (LDA) (0.31 mL) was slowly added to N-benzylidene benzylamine (237) (0.58 mL, 0.31 mmol) in THF (5 mL) at -60°C and under nitrogen. This solution turned red within minutes at which time a ZnTPP (15 mg, 0.02 mmol) solution in methylene chloride (2 mL) was added dropwise. The reaction was allowed to warm to room temperature under nitrogen and was monitored by TLC. After 24 hours, the solvent was removed and preparative TLC performed (silica- CHC13). MS (EI) m/e calc'd for C 3 9 H 2 8 N 4 : 552.23138, found 552.23029 (M +, 100%). 272 F.Y.pp.ri.m.p.nt.al Formation and Reaction of (241): C H 3 N © Sarcosine (239) (1 g, 0.0112 moi), paraformaldehyde (240) (0.84 g, '/ STL H p U U r l o © 0.028 moi) and DPP (10 mg, 0.022 mmol) were dissolved in toluene (20 mL) and methylene chloride (5 mL). The solution was refluxed for 48 hours and monitored by TLC. No reaction was observed. Formation and Reaction of (244): I Glycine (242) (1 g, 0.013 moi), benzaldehyde (243) (3.35 mL) and R (~f Q H 0 diphenylporphyrin (10 mg, 0.022 mmol) were dissolved in toluene (20 mL). The solution was refluxed for 48 hours and monitored by T L C . No stable products could be isolated. (245) : MS (LSIMS): m/e 569 (20%), 463 (75%). (246) : MS (EI): m/e 780 (6%), 676 (94%). N-CH2-Ph p - 0 2 N - C 6 H „ — c / Synthesis of N-benzyl /;-nitrobenzimidoyl chloride (247): \ : i Benzylamine (2.18 mL, 0.02 moi) was added dropwise to a chilled flask containing 4-nitrobenzoyl chloride (3.71 g, 0.02 moi). The reaction was stirred at 0HC for 2 hours at which time the reaction was washed with basic aqueous solution, dried and the solvent removed. This amide was then dissolved in thionyl chloride (3 mL) at room temperature and refluxed for 1.5 hours. The excess thionyl chloride was removed via distillation and the product washed, dried and recrystallized. The yield of (247) was 65%. 273 F.xpprimpntnl Synthesis and Reaction of p-nitrobenzonitriliophenylmethanide (248): To TV-benzyl p-nitrobenzimidoyl chloride (247) (0.2 g) was added excess triethylamine and the temperature maintained between 0 and 10°C to yield (248). In a typical reaction, this solution was added dropwise to a solution of porphyrin in toluene. This mixture was allowed to stir at room temperature for 30 mins. and was then set to reflux for 4 days. No reaction was observed. o© I P h N = C H C O P h Synthesis and Reaction of C-benzoyl-N-phenylnitrone (253): 0 Ammonium chloride (5 g), water (160 mL) and nitrobenzene (8.36 mL) were added to a 500 mL beaker and stirred at room temperature. Zinc dust (12.4 g) was slowly added to the reaction mixture over a period of 15 - 20 minutes. The stirring was continued for an additional 15 minutes. The solution was filtered and the filtrate poured into a 1L Erlenmyer flask containing excess saturated sodium chloride solution. The flask was set in ice for 1 hour. Suction filtration isolated the pale yellow needles of /V-phenylhydroxylamine (251) (5.3 g, 60%). A solution of N-phenylhydroxylamine (251) (0.325 g, 3 mmol) in methylene chloride (10 mL) was stirred at 0°C. Phenyl glyoxal (252) (0.4 g, 3 mmol) and triethylamine (0.5 mL) were added slowly with stirring to produce C-benzoyl-./V-phenyln krone (253). In a typical experiment, a concentrated solution of porphyrin (0.1 g TPP in 5 mL CHC13) was added dropwise to the C-benzoyl-/V-phenylnitrone (253) solution in ice. The reaction 274 F.xpprimpntnl mixture was allowed to warm to room temperature and was then refluxed whilst being monitored by TLC. No products were observed. (Chloromethyl)trimethylsilane (2 mL) and benzylamine (4 mL) were refluxed at 200°C for 3.5 hours. The reaction was cooled to room temperature and sodium hydroxide (50 mL of 0.1 N soln) was added. The mixture was extracted with diethyl ether (3 x 50 mL), dried with sodium sulfate, filtered and the solvent evaporated in vacuo to yield a yellow-orange viscous liquid. To this product, chloromethyl methyl ether (15 mL) was added and the mixture stirred at room temperature for 1 hour. The reaction mixture was washed with 10% HCI solution (2 x 100 mL), dried, filtered and the solvent evaporated to yield N-benzyl-A^methoxymethyl)-/V-[(trimethylsilyl)methyl]amine (254) as a yellow viscous liquid. In a typical experiment, A^-benzyl-A^-(methoxymethyl)-A^-[(trimethylsilyl)methyl]amine (254) (1 mL), trifluoroacetic acid (TFA) (0.5 mL), porphyrin (0.015 mmol) and toluene (5 mL) were stirred at room temperature for 2.5 hours. The solution was neutralized with aqueous basic solution, dried, filtered and preparative TLC performed. e ,CH 2 Synthesis and Reaction of /V-benzyl azomethine ylide (255): CH 2 Ph Chapter 6 References 276 Chapter 6 References 1. Krautler, B. Chimie 1985,2,19. 2. Dinello, R.K.; Chang, C.K. In The Porphyrins; Dolphin, D., Ed; Academic Press: New York, 1978; Vol 1, Chapter 7, p. 291. 3. The Science of Photobiology, 2n d Ed.; Smith, K.C., Ed.; Plenum: New York, 1989, p. 350. 4. Dolphin, D.; Abeles, R.H. Acc. Chem. Res. 1976, 9, 114. 5. Treibs, A. Angew. 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