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Chlorin-like photosensitizers for photodynamic therapy Liu, Xin 2005

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CHLORIN-LIKE PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY  by  XIN LIU B. Sc., NanKai University, 1995 M . Sc., NanKai University, 1998  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY In THE F A C U L T Y OF G R A D U A T E STUDIES CHEMISTRY  THE UNIVERSITY OF BRITISH C O L U M B I A  January 2005  © X i n Liu, 2005  11  ABSTRACT The goal of the project was to generate new photosensitizers  for use in  photodynamic therapy (PDT) by modifying the amphiphilicity of the tetrapyrrolic macrocycles. This aim was achieved by developing the chemistry of the conjugated vinyl group on tetrapyrrolic macrocycles. In  the  first  part,  the  non-diastereomeric  primary  ether  derivatives  of  pyropheophorbide a, 60, and ether derivatives of ring B-BPD-l,3-diene dimethyl ester, 62, were synthesized successfully. New synthetic methods for the intermediates and target compounds were developed and proven to be effective, convenient and safe. Preliminary PDT cytotoxicity assays showed that some of the primary ether derivatives exhibited promising PDT efficacy.  60  62  Secondly, cross-metathesis (CM) was applied successfully to vinylchlorin and vinylporphyrin substrates by employing the imidazolylidene ruthenium benzylidene catalyst 96. The optimized reaction conditions and the reactivity of different substrates towards C M were investigated.  Ill  A variety of chlorins and porphyrins with the substituted-vinyl groups, such as 105, 113, 123, 124, 135 and 125, were synthesized via cross-metathesis in good yields with excellent ^-stereoselectivity. This method has proven to be a generalizable strategy for modifying the amphiphilicity of tetrapyrrolic macrocycles starting with the conjugated vinyl group.  124  135  125  IV  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES  viii  LIST OF SCHEMES  xi  LIST OF ABBREVIATIONS  xiv  ACKNOWLEDGEMENTS  xvi  CHAPTER ONE  INTRODUCTION  1.1 Tetrapyrrolic Macrocycles  2  1.1.1 Overview.....  2  ;  1.1.2 Structural Feature  5  1.1.3 Nomenclature of Porphinoids  9  1.1.4 Optical Absorption Spectra.  12  1.2 Photodynamic Therapy 1.2.1 Concept  17  1.2.2 History and the First Generation PDT Drugs  18  1.2.3 PDT Photosensitizer Development  20  1.2.3.1 Electrocyclic Diels-Alder Reaction  21  1.2.3.2 Intramolecular Cyclization  25  1.2.3.3 Chlorophyll a Derivatives  27  1.2.3.4 Dihydroxylation of P, P-double bond  31  1.2.3.5 Target-Specific Photosensitizers  33  1.3 Research Objective REFERENCES  17  36 41  V  CHAPTER TWO  Primary Ether Derivatives of Chlorin Photosensitizers  2.1 Synthetic Approach  49  2.2 Synthesis of Target Compounds  51  2.2.1 Starting Material: Methyl Pyropheophorbide a  51  2.2.2 Preparation of 3-(2-hydroxyethyl) Derivatives  55  2.2.2.1 Dihydroxylation of the Vinyl Group  58  2.2.2.2 Pinacol-Pinacolone Rearrangement  62  2.2.3 Bromination of 3-(2-Hydroxyethyl) Derivatives  66  2.2.4 Synthesis of the Primary Ethers  68  2.3 Structure Characterization 2.3.1 Structure Determination of Compound 85  71 71  2.3.2 Structure and Spectra Characterization of Primary Ether Derivatives  76  2.4 Preliminary PDT Cytotoxicity Assessment  82  2.5 Summary  83  REFERENCES  94  CHAPTER THREE Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins 3.1 Olefin Metathesis and Cross-Metathesis  97  3.1.1 The Development of Olefin Metathesis Catalysts  98  3.1.2 Cross-Metathesis  105  3.1.3 A General Model for Selectivity in C M  107  3.1.3.1 Non-selective C M  110  3.1.3.2 Selective C M  Ill  3.1.3.3 Bridge Type Olefins  113  3.1.3.3 Multi-Component C M  114  3.1.4 Intermolecular Enyne metathesis  116  3.15 Application of C M  117  vi 3.2 Results and Discussions  118  3.2.1 Reaction Conditions  119  3.2.2 Reactivity Difference of Tetrapyrrolic Macrocycle Substrates  123  3.2.3 Cross-Metathsis with a Variety of Olefin Partners  125  3.2.4 CM Studies of Vinylchlorins Based on the Empirical Model  132  3.2.5 Enyne Metathesis of Vinylchlorin  138  3.2.6 Stereoselectivity  139  3.3 Structure Characterization of CM Products  '.  141  3.3.1 Characterization via H NMR Spectra  141  3.3.2 UV-Vis Spectroscopy of CM products  143  ]  3.4 Preliminary PDT Cytotoxicity Assessment  145  3.5 Further Modification of CM Products and Future Work  146  3.6 Summary  148  REFERENCES  159  CHAPTER FOUR EXPERIMENTAL 4.1 Nomenclature and Numbering System Used for the Synthesized Compounds 4.2 General Methods and Materials  165 166  4.3 Preparation of Methyl Pyropheophorbide a 39 from Magnesium Chlorophyllin  167  4.4 Synthesis of Primary Ether Derivatives  171  4.5 Synthesis of Cross-Metathesis Products  207  4.6 Determination of the Crystal Structure of 6 7 f by X-ray Crystallography REFERENCES  234 236  vii LIST OF FIGURES Figure 1.1  Examples of Tetrapyrrolic Macrocycles  2  Figure 1.2  Tetrapyrrolic Macrocycles DerivedfromUroporphyrinogen III  4  Figure 1.3  Delocalized Electron Pathways of Tetrapyrrolic Macrocycles  6  Figure 1.4  Structure Modifications of Porphyrin  7  Figure 1.5  Nomenclature Systems of Porphyrin  9  Figure 1.6  Name of Compound 1fromDifferent Nomenclature Systems  10  Figure 1.7  Nomenclature of Chlorophyll Related Chlorins  11  Fi ure 1 8 Typical Absorption Spectra of Porphyrins (Soret band omitted): a) ' etio-type; b) rhodo-type; c) oxorhodo-type; d) phyllo-type 13 8  Figure 1.9  Typical Absorption Spectrum of Metalloporphyrin  14  Figure 1.10  Typical Absorption Spectra of Chlorins and Metallochlorins  15  Figure 1.11  Typical Absorption Spectra of Bacteriochlorins  16  Figure 1.12 PDT Treatment Process „,  18  Figure 2.1  Low-Field Region of the 'H NMR Spectra (CDC1 , 300 MHz) of 73 J O cc and 8 55  Figure 2.2  'H NMR Spectrum (CDC1 , 300 MHz) of 63  61  Figure 2.3  'H NMR Spectra (CDC1 400 MHz) of 85 and 71  72  &  3  3  3)  Fig. 2.4 1D-NOESY Spectra (CDC1 , 400 MHz) of Compound 85. A) upon irradiation at 7.79 ppm; B) upon irradiation at 5.06 ppm; C) upon irradiation at 9.78 ppm; D) upon irridation at 7.72 ppm; E) upon irradiation at 10.44 ppm; F) *H NMR Spectrum of 85 74 UV-Vis Spectra of Methyl Pyropheophorbide a Derivatives 39, 63, 66 3  '  l g u r e  „.  _  Figure 2.5 7  a n d  6 ? f  ? ?  Figure 2.6  UV-vis Spectra of ring B-BPD derivatives 61, 68, 71 and 62g  78  Figure 2.7  'H NMR Spectrum (CDC1 , 300 MHz) of Compound 67a  79  3  Vlll  Figure 2.8  *H NMR Spectrum (CDC1 , 300 MHz) of Compound 62a  Fi ure 2 9 ' Fi ure 2 10 "  ORTEP Drawing of 67f with Major Fragment (top view, H atoms have been omitted for clarity) 81 ^RTEP Drawing of 67f with Major Fragment (side view, H atoms have been omitted for clarity) 81  8  B  3  79  Figure 2.11 *H NMR Spectrum (CDC1 , 400 MHz) of Compound 66  85  *H NMR Spectrum (CDCI3, 300 MHz) of Compound 67c  86  3  Figure 2.12  Figure 2.13 H NMR Spectrum (DMSO-J , 400 MHz) of Compound 60c.  87  „ , . Part of the H- C HSQC Spectrum (DMSO-flfc, 400 MHz) of „ , ^ Compound 60c  88  l  6  ]  13  Figure 2.14  "  Figure 2.15  !  H NMR Spectrum (CDCI3, 300 MHz) of Compound 67h  89  Figure 2.16  ]  H NMR Spectrum (CDCI3, 300 MHz) of Compound 68  90  Figure 2.17 'H NMR Spectrum (CDC1 , 300 MHz) of Compound 70  91  3  Figure 2.18  ]  H NMR Spectrum (CDCI3, 300 MHz) of Compound 62c  92  Figure 2.19  ]  H NMR Spectrum (CDC1 , 300 MHz) of Compound 86  93  Fi ure 3 1 % ' • S '  Time Line of Milestones in the Development of Olefin Metathesis Catalysts 99 Low-Field Region of the *H NMR Spectrum (CDCI3, 300 MHz) of Compound 108 142 Low-Field Region of the *H NMR Spectrum (CDCI3, 300 MHz) of Product 115 Mixed with 61 143  l  F l  ure  u r e 3  gu  2  .3  3  Figure 3.4  UV-Vis Spectra of Compounds 61,105,123 and 129  144  Figure 3.5  UV-Vis Spectra of CM Product 135  145  Figure 3.6  'H NMR Spectrum (CDC1 , 400 MHz) of Compound 102  149  Figure 3.7  *H NMR Spectrum (CDCI3, 300 MHz) of Compound 113  150  3  ix  Figure 3.8  ' H N M R Spectrum (CDC1 , 300 MHz) of Compound 119  151  Figure 3.9  *H N M R Spectrum (CDC1 , 400 MHz) of Compound 120  152  Figure 3.10 *H N M R Spectrum (CDC1 , 300 MHz) of Compound 124  153  Figure 3.11  !  H N M R Spectrum (CDC1 , 300 MHz) of Compound 129  154  Figure 3.12  ]  H N M R Spectrum (CDC1 , 400 MHz) of Compound 135  155  Figure 3.13  *H N M R Spectrum (CDCI3, 400 MHz) of Compound 136  156  Figure 3.14  *H N M R Spectrum (CDCI3, 300 MHz) of Compound 138  157  H N M R Spectrum (CDCI3, 300 MHz) of Compound 125  158  3  3  3  Figure 3.15  3  3  ]  LIST OF TABLES T KI ~> 1 1 able 2.1  The pK Values of Different Acids and the Results of Their Reactions a  w  i  m  6  3  6  5  Table 2.2  *H NMR Spectra Data of meso-R of BPD Derivatives  76  Table 2.3  PDT Cytotoxicity of Primary Ether Derivatives  83  ,•  -.  Table 3.1  Table 3.2 a b  3  Functional Group Tolerance of Transition Metal Olefin , . . „ ^. , Metathesis Catalysts r  A  i U  Olefin Categories for Selective Cross-Metathesis  „„„  102 110  Selective CM between Quaternary Allylic Olefins and Terminal Olefins Using Catalyst 96 112  Table 3.4  Selective CM between Type II and Type III Olefins.  113  Table 3.5  Cross-Metathesis of Substituted-Styrenes Catalyzed by 96  114  Table 3.6  CM of Ring B-BPD 61 Under Different Conditions  122  Table 3.7  CM of Methyl Pyropheophorbide a with 1-Alkene  123  Table 3.8  CM of Protoporphyrin IX Derivatives with 1-Octene  124  Table 3.9  CM of 112 with Different Terminal Olefin Partners  126  Table 3.10  CM between Vinylchlorin and Allyl-Substituted Olefins  129  Table 3.11  The CM of 108 with Type II and Type III Olefins  136  Table3.12  Cross-Metathesis Reactions between 137 and cc-Carbonyl Containing _ Olefins  140  Table 4.1  Crystal Data and Details of the Structures Determination for 67f  205  ,, „  ...  xi LIST OF SCHEMES Scheme 1  Biosynthesis of Uroporphyrinogen III  Scheme 2  1  3  Preparation of One of the Oligomers of Hematoporphyrin Derivative Q  1  9  ocneme J  Diels-Alder Reaction of Protoporphyrin IX Dimethyl Ester with .^,. ,. DMAD 23  Scheme 4  Double Diels-Alder Reaction on Divinylporphyrin  24  Scheme 5  Diels-Alder Reaction on Chlorin  25  Scheme 6  Intramolecular Cyclization  25  Scheme 7  Benzoporphyrins with Various R Substituents via Intramolecular Cyclization 26  Scheme 8  Synthesis of SnEt2 via Cyclization  27  Scheme 9  Synthesis of Npe<j  28  Scheme 10 Alkyl Ether Derivatives of Pyropheophorbide a  29  Scheme 11 Modification of the Isocyclic Ring of Pyropheophorbide a  30  „„ Dihydroxylation and Pinacol-Pinacolone Type Rearrangement of . 32 Porphyrin Dihydroxylation and Pinacol-Pinacolone Rearrangement of Chlorin  Scheme 12 _ \ Scheme 13  c  ,\  3  J y  to  .  Substrate  0 O  32  Scheme 14 Synthesis of Galactose-Conjugated Purpurinimide  35  Scheme 15 Synthesis of Ring B-BPD-l,3-diene Dimethyl Ester 61  38  Scheme 16 Cross-Metathesis Reaction between Terminal Olefins  40  Scheme 17 CM of the Vinyl Group Conjugated with Tetrapyrrolic Macrocycle  40  Scheme 18 Synthetic Approach Towards 60  50  Scheme 19 Synthetic Approach Towards 62  51  xii  c  1 f t  Scheme 20  Preparation of Methyl Pyropheophorbide a from MagnesiumC  h  l  o  r  o  p  h  y  l  l  i  n  5  3  Converting the 3-Vinyl Group to the 3-(2-Hydroxyethyl) Group by T1(N0 ) 56 3 3  Scheme 22  Preparation of 65 by Anti-Markovnikov Hydration  57  Scheme 23  Formation of the Osmium (VI) Ester Intermediate  59  Scheme 24  Preparation of Bis-Glycol 83  59  Scheme 25  Examples of Olefin Metathesis Reactions  98  Scheme 26  Olefin Metathesis Mechanism Proposed by Chauvin  100  Scheme 27  Synthesis of the Ruthenium Metathesis Active Complexes.  103  Scheme 28  Proposed Mechanism for Catalyst Cl (PCy ) Ru=CHPh 91  104  Scheme 29  Mechanism for CM of Two Terminal Olefins.  106  Scheme 30  Homodimerization and Secondary CM  108  Scheme 31  Olefin Categorization and Rules for CM Selectivity  109  Scheme 32  CM between Type II Olefins  Ill  Scheme 33  Three-Component Selective CM  115  Scheme 34  Intermolecular Enyne Metathesis  116  Scheme 35  Selective Intermolecular Enyne Metathesis  117  Scheme 36  Enyne Reaction of Purpurinimide  117  Scheme 37  Reaction of Ring B-BPD 61 and 1 -Hexene Using Catalyst 91  119  Scheme 38  Ring-Closing Metathesis of 101 Catalyzed by 91  120  2  3 2  xiii  Scheme 39 CM between 103 and Allyltrimethylsilane Catalyzed by 91  120  Scheme 40 CM between 126 and Terminal or Internal Allylic-Substituted Olefins.. 130 Scheme 41 Catalytic Species in CM of Terminal or Internal Olefins  131  Scheme 42 Reaction of Catalyst 91 with Terminal and Internal Olefins.  131  Scheme 43 Reaction of Zn(II) BPD 112 with Ruthenium Catalyst 96  133  Scheme 44 Pathways for Reaction between Catalyst 96 and Terminal Olefins  134  Scheme 45 Steric Effect in the Formation of CM Intermediate  140  Scheme 46 Hydrogenation of the CM Product 105  146  Scheme 47 Proposed Diels-Alder Reaction of the CM Product  147  XIV  LIST OF ABBREVIATIONS  AMD  age-related macular degeneration  aq  aqueous  BPD  benzoporphyrin derivative  BPDMA  benzoporphyrin derivative monoacid A ring  br  broad  CM  cross metathesis  Cy  cyclohexyl  d  doublet  DBU  l,8-diazabicyclo[5.4.0]undec-7-ene  DCC  dicyclohexylcarbodiimide  DMAD  dimethylacetylenedicarboxylate  DMF  A'.A'-dimethylformamide  DMSO  dimethylsulfoxide  EI  electron impact  equiv(eq)  equivalent  ESI  electrospray ionization  FDA  Food and Drug Administration  h  hour  Hp  hematoporphyrin  HpD  hematoporphyrin Derivatives  HPLC  high pressure liquid chromatography  HPPH  2-(l-hexyloxyethyl)-2-devinyl pyropheophorbide a  HSQC  heteronuclear single quantum coherence  IUPAC  International Union of Pure and Applied Chemistry  ruB  International Union of Biochemistry  LD  lethal dose required to kill 50 % of cells  50  LSIMS  liquid secondary ion mass spectrometry  XV  m  multiplet  min  minute  MS  mass spectrometry  MTS  3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt  m/z  mass/charge  NMO  N-methylmorpholine N-oxide  NMR  nuclear magnetic resonance  NOE  nuclear Overhauser effect  NOESY  nuclear Overhauser effect spectroscopy  ORTEP  Oak Ridge thermal ellipsoid plot  PDT  photodynamic therapy  PpIX  protoporphyrin IX  ppm  part per million  QSAR  quantitative structure-activity relationship  RCM  ring-closing metathesis  s  singlet  sec  second  t  triplet  TBAF  tetrabutylammonium fluoride  TBS  tert-butyl  dimethyl silane  TFA  trifluoroacetic acid  THF  tetrahydrofuran  TLC  thin layer chromatography  TMS  trimethylsilane  TNFa  tumor necrosis factor-alpha  TPP  tetraphenylporphyrin  /7-TsOH  ;?-toluenesulfonic acid  UV-Vis  ultraviolet-visible  xvi  ACKNOWLEDGEMENTS First, and foremost, an expression of sincere gratitude goes to my supervisor, Prof. David Dolphin. His constructive guidance, encouragement, kindness and patience have provided invaluable support during the course of this work. I would also like to thank all of the members of Dolphin's group, past and present, who offer me valuable help and make my life in U B C productive and enjoyable. I greatly appreciate the help from Dr. Alison Thompson and Dr. Ethan Sternberg for their great advice, critique and creative discussion. I also owe appreciation to Mr. Andrew Tovey, Ms. Gosia Modzelewska, Dr. Jiyoung Shin and Dr. Olivia New for their kindness and time for proof-reading. Special thanks give to Mr. Andrew Tovey, who offered me the great help on both chemistry and language without which the work can not be completed. I also thank Dr. Lingyun Zhang deeply for her concern and help all along the time. Thanks go as well to Dr. Raymond J. Andersen, for his time for reading the thesis. Thanks also go to Dr. Ron Boch and biological group in Q L T Inc. for performing the bioassay tests; to Dr. Brian Patrick for the X-ray crystal structure; to Dr. Nick Burlinson, Ms Marietta Austria and Ms Liane Darge for help on N M R analyses. Special thankfulness is owed to Dr. Yun Ling and the analysis service group for the effective services provided. Finally, a deep sense of gratitude is directed to my family. To my parents, Mr. Zengning Liu and Ms. Yifang Dong, for their love, support and concern. To my husband Jianyu, for his consistent understanding, encouragement and valuable help. And to my sweet one-year old angel, my daughter Michelle, for all the happiness she brought to me and the chance she gives me to enjoy the achievement of being a mom.  1 Chapter 1 Introduction  Chapter 1 Introduction  .2  Chapter 1 Introduction  1.1 Tetrapyrrolic Macrocycles 1.1.1 Overview Tetrapyrrolic macrocycles (Fig. 1.1), e.g. porphyrins, chlorins, bacteriochlorins, isobacteriochlorins, corphins and corrins, form the macrocyclic skeletons of important natural prosthetic groups that support life on this planet. " Each of these porphinoids 1  4  (porphyrins and their structural variants) consists of four pyrrole-type rings linked together through methine bridges or by a direct carbon-carbon bond. A striking feature of all these cofactors is that each of their biosynthetic pathways shares the same initial steps through to uroporphyringon III. Uroporphyrinogen III is biosynthesized from 5aminolevulinic acid by way of porphobilinogen and preuroporphyrinogen " (Scheme 1). 5  Porphyrin  Isobacteriochlorin  Chlorin  Corphin  7  Bacteriochlorin  Corrin  Fig. 1.1 Examples of Tetrapyrrolic Macrocycles  3 Chapter 1 Introduction  uroporphyrinogen III  Scheme  1  Biosynthesis o f Uroporphyrinogen III  Fine-tuning o f these porphinoid ligands for optimal biological function is performed in nature by the appropriate choice o f the oxidation level o f the macrocycle, the nature o f the peripheral substituents, and the coordinated metals " (Fig. 1.2). For example, heme, a 8  9  ferrous complex o f protoporphyrin, is the prosthetic group o f hemoglobin and myoglobin, which are responsible for oxygen transfer and storage i n living tissues.  10  Cytochromes are  also heme-containing proteins which serve as one-electron carriers i n the electron transport chain. Heme can also be found i n the enzyme peroxidase, which catalyzes the 11  oxidation o f substrates with hydrogen peroxide.  12  Chlorophyll a, a magnesium (II)  complex o f a dihydroporphyrin (i.e. a chlorin) is responsible for the light-harvesting and trapping activities i n plants and algae.  13  A magnesium containing tetrahydroporphyrin,  bacteriochlorophyll a, is the main component o f the photosynthetic apparatus o f purple and green bacteria. The outstanding work o f Deisenhofer, Huber and M i c h e l towards the bacterial photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, which illustrates the possible evolution sequence with which nature manipulates  4  Chapter 1 Introduction  uroporphyrinogen into an efficient and precise photochemical device, was awarded the Nobel Prize in 1988. ' The constitutional isomers of bacteriochlorins with two adjacent 14 15  Fig. 1.2 Tetrapyrrolic Macrocycles Derived from Uroporphyrinogen III  5 Chapter 1 Introduction  saturated pyrrolic rings are called isobacteriochlorins. They are widely distributed in bacteria and plants. One typical example is siroheme, the iron-coordinated sirohydrochlorin which is the prosthetic group of a number of sulfite and nitrite reductases.  16  Further reduction of the tetrapyrrolic macrocycle leads to the corphin ring (Fig. 1.2). Coenzyme  F430,  the nickel complex of hexahydrocorphin, exists in the active site of  methyl coenzyme M reductase that catalyzes the reductive cleavage of S-methyl conenzyme M [2-(methylthio)ethanesulfonate] to methane. Another example, and the 17  structurally most complicated member of tetrapyrrolic macrocycles, is vitamin B , the i 2  cobalt complex of a corrin. Vitamin B12 is an essential vitamin for human health, the deficiency of which will lead to pernicious anemia. It is also the prosthetic group of 18  numerous enzymes which carry out rearrangement reactions and trans-methylations. This unusual organic ligand surrounding the cobalt displays many stereo centers along its periphery while carrying reactive functional groups. The complete pathway of its biosynthesis from 5-aminolevulinic acid was uncovered in 1995 after 25 years of research.  18  1.1.2 Structural Features Porphinoids, e.g. porphyrins (fully-unsaturated), chlorins (dihydro-porphyrins) and bacteriochlorins (tetrahydro-porphyrins) are tetrapyrrolic macrocycles containing four pyrrole nuclei linked by methine bridges. They can be classified as polyene chromogens, as they contain no donor or acceptor groups, and as they are structurally similar to the  6  Chapter 1 Introduction  annulenes. Each of these species maintains aromaticity in the macrocycle through an 18 19  atom, 18 7t-electron system, that is the 18-diazaannulene, inner-outer-inner-outer derealization pathway ( F i g . 1.3) in accordance with Hiickel's 4n+2 rule. The macrocycle generally maintains the planarity demanded by the delocalized 7t-system although exceptions have been reported.  Porphyrin  Chlorin  Bacteriochlorin  F i g . 1.3 Delocalized Electron Pathways of Tetrapyrrolic Macrocycles  *H NMR spectroscopy shows clearly that this ring system is anisotropic and the shielded inner NH protons appear at relatively high field (5= 0 ~ -5 ppm). The signals of the outer methine protons appear at 8 to 10 ppm due to the deshielding effect of the aromatic ring current. The remaining two double bonds in porphyrins are crossconjugated and can be reduced to the corresponding chlorins and bacteriochlorins without markedly affecting the aromatic 7t-electron system. Many structural modifications of the porphinoids are possible. The most important modifications can be classified as follows (Fig. 1.4). 1) Peripherally modified porphyrins: modification occurs on the periphery, at the fJand weso-positions (see 1.1.3 for nomenclature) of the parent tetrapyrrolic macrocycle. Heme is a typical example. Azaporphyrins, which are obtained by replacing the carbon  7 Chapter 1 Introduction atoms o f the methine bridges by nitrogen, and the benzoporphyrins, which possess benzene rings fused to the (3, (3 'positions also belong to this category.  heme  phathalocyanine  Fig. 1.4 Structure Modifications of Porphyrin  2) Contracted porphyrins: corroles are the tetrapyrroles resulting from the removal 21 of one of the methine bridges.  8  Chapter 1 Introduction  3) Core-modified porphyrins: where one or more nitrogen atoms of the pyrrole units are substituted with chalcogen atoms.  22  4) Isomeric porphyrins: resultingfromthe scrambling of the four pyrrole units and four methine bridges.  23  5) Inverted porphyrins: porphyrin isomers which associate with the inversion of one or more of the core nitrogens; they are commonly called N-confiised porphyrins.  22  6) Expanded porphyrins: result from the expansion of electron conjugation by increasing the number of pyrrole rings.  24  The porphyrin macrocycle can be regarded as an ampholyte with the two central nitrogen atoms that can accept protons from acids such as TFA, to give the porphinoid dication, and in addition, the two NH groups can be deprotonated in strongly alkaline conditions to give the porphinoid dianion. The porphinoids that contain two hydrogen atoms at the center of the macrocycle are called thefree-baseporphinoids. Various metal ions can replace the central hydrogen atoms of free-bases, when coordination to all four nitrogen atoms is possible, as exemplified by the Mg ion in the chlorophylls. Usually, 2+  the porphinoid nucleus is a thermally-stable macrocyclic system. It is also stable to concentrated sulfuric acid and neat TFA, both of which may be used to remove coordinated metals in metalloporphyrins. The solutions of porphinoids are relatively unstable to light, which may result in photooxidation and/or photodegradation of the peripheral substituents and/or the macrocyclic ring depending on the substrates and *  12  reaction conditions.  9 Chapter 1 Introduction  1 . 1 . 3 Nomenclature of Porphinoids  Two principal nomenclatures, the Fischer and IUPAC-IUB nomenclature, are currently used in porphyrin and chlorophyll chemistry. The Fischer system, which is based on a large number of trivial names that continue to appear in the literature and a numeration scheme as shown in Fig.1.5, is simple but not comprehensive. In the IUPACIUB nomenclature, the fundamental macrocycle tetrapyrrolic ring is named porphyrin to replace the Fisher's trivial name "porphin" and the 1-24 numbering scheme is used (Fig. 1.5). The 2, 3, 7, 8, 12, 13, 17 and 18 positions are commonly referred to as the "P25  positions" (i.e. of the pyrrole rings). Similarly, positions at 1, 4, 6, 9, 11, 14, 16 and 19 are referred to generically as the "a-positions", while positions 5, 10, 15 and 20 are referred to as the "meso-positions" (a, p, y and 5 in the Fisher system). The four nitrogen atoms are numbered 21 through 24.  Porphin/Fisher  Porphyrin/IUPAC-IUB  Fig. 1.5 Nomenclature Systems of Porphyrin  While the 1-24 numbering scheme is used for the unsubstituted porphyrin nucleus, in substituted porphyrins the beginning and direction of numbering is determined by the rules for systematic substitutive nomenclature. Lowest locants are assigned according to  10 Chapter 1 Introduction  the criteria of principal group named as suffix, substituents first cited, etc. Therefore, the systematic names of substituted porphyrin derivatives according to the rules are less ambiguous, however, become very long and impractical for most purposes. A comparison of the systematic and trivial name for compound 1 is presented in Fig. 1.6.  Fisher:  Chlorin e monoethyl ester  IUPAC-IUB:  (2S, 3S)-20-(Carboxymethyl)-18-(ethoxycarbonyl)-13-ethyl3,7,12,17-tetramethyl-8-vinylchlorin-2-propionic acid  6  Fig. 1.6 Name of Compound 1fromDifferent Nomenclature Systems  Some of the trivial names in the Fisher's system are so well established that they remain in the IUPAC-IUB nomenclature system. For examples chlorin, bacteriochlorin, isobacteriochlorin and porphyrinogen, the trivial names for some parent compounds of reduced porphyrins, are still retained. For reduced porphyrins that are close relatives of the chlorophylls, some specific rules related to names and substituents locants are applied. Compounds that can be derived via demetallation of the corresponding naturally occurring chlorophylls are called pheophytins (if the ester of the 17-propionic acid group is phytyl) or pheofarnesins (if the ester of the 17-propionic acid group is farnesyl). In  11  Chapter 1 Introduction  naming such compounds, the word "pheophytin" (or "pheofarnesin") replaces "chlorophyll" in the original name. Thus demetallation of bacteriochlorophyll a gives bacteriopheophytin a. A compound that is demetallated and also possesses afreeacid at the 17-position is called a pheophorbide. The methyl ester can be named either as methyl pheophorbide or pheophorbide methyl ester. The illustration for the rule is given in Fig. 1 . 7 . For these compounds, the substituent positions are assigned according to the numeration for the unsubstituted porphyrin macrocycle. 2. Chlorophyll a R = C H , R =Phytyl, M=Mg ; 1  2  2+  3  3. Chlorophyll b R =CHO, R =Phytyl, M=Mg ; 1  2  2+  4. Pheophytin a R = C H , R =Phytyl, M=2H; 1  2  3  5. Pheophytin b R =CHO, R =Phytyl, M=2H; 1  2  6. Pheophorbide a R = C H , R =H, M=2H; 1  2  3  7. Pheophorbide b R =CHO, R =H, M=2H; 1  2  8. Methyl pheophorbide a R = C H , R = C H , M=2H. 1  C0 R  2  3  3  2  phytyl:  / ^ ^ ^ / x / x ^ x / x / C ^ rCuH.  hf  n 3  (  -CnH , nH oCnH , 3  3  L^  Fig. 1 . 7 Nomenclature of Chlorophyll Related Chlorins  In order to take advantage of both nomenclature systems, the trivial names combined with the IUPAC-IUB numeration system will be used in this work. Substituents are expressed by prefixes or suffixes as in organic nomenclature. The prefix "de" followed by the name of a substituent is used to denote the removal of that substituentfromthe parent structure. In the trivial names of porphinoids, italic letters and subscript numbers are often employed. The latter indicate the number of oxygen atoms in the molecule. For example,  12 Chapter 1 Introduction  chlorin e$ has six oxygen atoms and is a chlorin from the degradation products of chlorophyll a. The italic letters a and e are interchangeable, and are used in the chlorophyll a series only; while g is used for the chlorophyll b derivatives (e.g. in rhodin g ). Many trivial names of chlorophyll-related compounds are difficult to interpret. 7  HOOC  rhodin g  7  1.1.4 Optical Absorption Spectra Porphinoids exhibit characteristic absorptions in the visible region which make them useful as photosensitizers. The metal-free and metallated porphinoids have an intense absorption at around 400 nm (e ~10 ), known as the Soret band. This band is 5  26  the most intense absorption band in all fully conjugated tetrapyrrolic macrocycles and can therefore be regarded as characteristic of porphinoids. The intensity of the Soret band is weaker in chlorins and metallochlorins and, as might be expected, it is totally absent in the non-conjugated tetrapyrroles such as porphyrinogens. The Soret band is also present in vitamin Bi and in the metal complexes of the bile pigments. Both of these types of 2  compounds have interrupted conjugation in the ligand, but the pathway is maintained 97  through the metal atom.  13 Chapter 1 Introduction Besides the Soret band, porphyrins also possess four satellite bands in the region of 450 ~ 650 nm numbered I to IV as shown in Fig. 1.8. These bands are less intense than the Soret band and are normally referred as the "Q bands". The relative intensities of "Q bands" have been used to classify porphyrin spectra as four basic types: etio-type, rhodotype, oxorhodo-type and phyllo-type. A l l naturally occurring porphyrins have the etiotype spectrum, in which the peak intensities are IV>III>II>I. The presence of a single carbonyl, carboxylic acid (or ester) or an acrylic acid causes band III to become more intense than band IV, affording a rhodo-spectrum (III>IV>II>I). When two rhodofying groups exist on diagonally opposite rings, the effect is enhanced and results in an oxorhodo-type spectrum (III>II>IV>I). Less symmetric porphyrins, such as those with a single /neso-substituent, always exhibit phyllo-type spectra (IV>II>III>I).  i—,  500  ,  550  i  600  ,—•  650  •  -,—  500  ,  550  1 600  l  650 '  Fig. 1.8 Typical Absorption Spectra of Porphyrins (Soret band omitted): a) etio-type; b) rhodo-type; c) oxorhodo-type; d) phyllo-type.  14 Chapter 1 Introduction  Simple square-planar chelations of porphyrins with divalent metal ions change the four Q bands into two bands, usually designated as a and P, between 500 and 600 nm with the Soret band retained (Fig. 1.9). This is due to the increased symmetry of the conjugated ring. The intensities and the relative intensities of the a- and (3- bands depend both on the coordinated metal and the nature of the porphyrin ligand.  1  300  i  1  1  400  500  600  —  1 •  700  nm  Fig. 1.9 Typical Absorption Spectrum of Metalloporphyrin  The intensity and the exact peak position of the optical spectra of porphyrins are dependent on the solvent as well as the concentration of solution. More importantly, correlations have been shown to exist between the nature of the porphyrin side chains and the relative intensities and positions of the absorption bands. The effects of the peripheral substitution on optical spectra are not pronounced while changes in the electronic structure and conformation of the molecule affect the porphinoid spectra significantly. The reduction of one or two endocyclic double bonds affords dihydroporphyrins (chlorins) and tetrahydroporphyrins (bacteriochlorins) respectively. This does not affect the aromaticity of the molecule apparently, but produces visible spectra characterized by red  15 Chapter 1 Introduction  shifted Q bands for the chlorins (650-680 nm, Fig. 1.10) and bacteriochlorins (750-780 nm, Fig. 1.11).  metailochlorin chlorin  o c  o \_  o  CO  <  400  500  600  700  800 nm  Wavelength  Fig. 1.10 Typical Absorption Spectra of Chlorins and Metallochlorins  In the chlorins, band I in the visible region is very prominent and is bathochromic shift about 25 nm comparing with that in a porphyrin. The ratio of the Soret band to Q band I is only about 5 versus that of around 50 in porphyrin. The extinction coefficients of band IV and the Soret band, in neutral solvents, are comparable with those of the related bands in analogous porphyrins. Chlorin mono- and di-cations have spectra similar to those of the neutral compounds. The band I and the Soret band being moved to shorter wavelength in the dications (and in metallochlorins). For some chlorins, such as  16 Chapter 1 Introduction  chlorophyll a, there are two so-called r\ bands on the shoulder of the Soret band in the near ultraviolet region (Fig. 1.10).  12  Fig. 1.11 Typical Absorption Spectra of Bacteriochlorins  17  Chapter 1 Introduction  1.2 Photodynamic Therapy As discussed before, porphyrins are the most widespread prosthetic groups found in nature. They play such diverse and important roles in life systems ranging from oxygen and electron transfer to photosynthesis and catalytic oxidation. In addition to these critical biological functions, the practical applications of porphyrins in industry and medicine have also attracted great attentions over the past few decades. Due to the abilities of porphyrins to effectively generate singlet oxygen, they have been used successfully as photosensitizers in photodynamic therapy. Research and development in this field have been very rapid and new anti-cancer and other therapies have been brought to the market place in recent years.  1.2.1 Concept , Photodynamic Therapy (PDT) is a medical treatment that involves the combination of light and photosensitizer (light-sensitive drug) to treat a range of diseases characterized by rapidly growing tissues.  28  The standard treatment protocol for PDT consists of two  steps (Fig. 1.12). The first step involves intravenous administration of the drug, i.e. the photosensitizer. The drug localizes preferentially in diseased tissues, usually rapidly dividing cells, with some degree of selectivity. The second step involves activation of the drug using a specific dose of light at a particular wavelength. This effects the conversion of triplet oxygen ( O2) found in tissue to highly energized singlet oxygen ( O2). The generation of 0 x  is the main requirement for PDT cytotoxicity, as Oi disrupts normal x  2  29  cellular function and causes destruction of cancerous or unwanted tissues.  The PDT  treatment derives great promise from the dual selectivity that is produced by both a  18 Chapter 1  Introduction  preferential uptake of the drug by the diseased tissue and the restriction of carefully regulated light shone onto the specific sites. Step I  Step II  Injection  Localization  Light activation; • 0 generation !  2  Target cell destruction  Fig. 1.12 PDT Treatment Process  1.2.2 History and the First Generation PDT Drugs The earliest application of PDT in the treatment of disease can be traced back to 4000 years ago, when ancient Egyptians used the combination of orally ingested plants (containing light-activated psoralens) and sunlight to treat vitilago. Contemporary PDT 30  began in the late 19th century when Finsen discovered that the skin disease Lupus vulgaris could be treated with UV light. In 1903, Trappeiner treated a skin cancer with 31  eosin and light. In 1913 Meyer-Betz injected himself with 200 mg of hematoporphyrin 32  (Hp, 9) (Scheme 2) and found no ill effects until he exposed himself to sunlight, whereupon he suffered extreme swelling and photosensitivity which lasted for several months. In 1925, Policard demonstrated the phototoxic effect of porphyrins to certain 33  malignant tumors.  34  Schwartz,  35  in the early 1950's, found that it was not the monomeric  hematoporphyrin (9) but an oligomeric mixture in the material derived by isolation of Hp from blood that initiated the long-term phototoxic effect in Meyer-Betz's experiment,  19 Chapter 1 Introduction  since Hp clears quickly from the body. Schwartz enriched this oligomeric fraction by treating Hp with sulfuric acid in acetic acid followed by treatment with strong alkali. The 35  mixture thus obtained was called hematoporphyrin derivative (HpD, 10). In the late 1960's, the first combination of HpD and selective light irradiation for cancer treatment  36  marked the beginning of PDT as a cancer therapy.  HO  Hematoporphyrin Derivative (HpD, 10)  Scheme 2 Preparation of One of the Oligomers of Hematoporphyrin Derivative 10  Clinical trials utilizing various HpD preparations were begun in the early 1970's by Dougherty et al.  Dougherty refined the preparation of HpD by ultrafiltration to give a TO  material designated as Photofrin II.  Further purification during the late 1980's at QLT  Inc. and American Cyanamid along with the use of lyophilization method yielded the drug Photofrin® as the first generation PDT drug.  39  20 Chapter 1 Introduction  Photofrin® has been used in the treatment of a variety of diseases, such as lung and bladder cancers and certain ocular diseases. However, because it is a complex mixture of oligomers, it suffers some drawbacks that make it not ideal as a PDT drug. First, characterization of the oligomer mixture is very difficult. Analysis by various methods has indicated that the main components of Photofrin® are likely dimer, trimer or other 40  41  oligomers. The majority of the bonds between the porphyrin monomers are ether 42  linkages but the existence of ester, carbon-carbon bond and linkage through meso43  positions have also been proposed. Secondly, Photofrin® has limitations in its 44  pharmacokinetic profile. Its accumulation in skin may last up to six weeks before clearance to acceptable levels. Thus the patient remains photosensitive, especially to 45  strong sunlight, during this period. Another drawback is that the longest absorption wavelength for Photofrin® is 630 nm, with a low extinction coefficient of 1170 cm"'M . _1  The light at this wavelength can't penetrate the skin most effectively due to endogenous chromophore absorption, mainly hemoglobin, and light scattering.  46  The lack of  efficiency limits its application for treatment of large tumours or tumours that are deeply seated within the body.  1.2.3 PDT Photosensitizer Development Applying the aforementioned principles of PDT and the general considerations for the synthesis and marketing of any drug, a profile for the ideal PDT drug can be established to direct the development of new photo sensitizers for PDT: •  Strong absorption at high wavelengths of the visible spectrum region (>650 nm);  21 Chapter 1 Introduction  •  High quantum yield of the triplet formation, with a triplet energy greater than 94 kJ/mol, which is the energy required for the conversion of 0 to '0 ; 3  2  •  High singlet oxygen quantum yield;  •  Low dark toxicity and rapid clearancefromthe body;  •  Selectivity for the tumorous tissue versus healthy tissue;  •  Facile synthesis from readily available starting materials;  •  Strong proprietary position  2  Some aspects of the profile, such as the long wavelength absorption spectrum are relatively easy to attain, whereas others, such as the pharmacokinetic profile, are much harder to fulfill. These criteria have led to the search for new compounds with improved properties that are often referred to as 2 or even 3 generation PDT drugs. As mentioned before, nd  rd  the chlorins and bacteriochlorins exhibit absorption in the long wavelength region (650800 nm) that is ideal for PDT and most research has been focused on these macrocycles. The development of PDT photosensitizers from tetrapyrrolic macrocycles is summarized as follows according to the synthetic strategies that are applied.  1.2.3.1 Electrocyclic Diels-Alder Reaction In the porphyrin macrocycle, the vinyl group in conjunction with the ring (3,P'double bond mimics a diene system that can undergo the [4+2] Diels-Alder reaction. This reaction has been investigated extensively by different research groups to generate  22 Chapter 1 Introduction  various chlorins and bacteriochlorins. The auto-sensitized photooxidation by singlet oxygen of protoporphyrin was observed in the 1966, but it was not until 1973 that 47  Callot et al. first studied the cycloaddition reaction of protoporphyrin IX (PpIX) dimethyl ester (11) with various dienophiles. A few years later, Dolphin and co-workers 48  reinvestigated  the  reaction  of  49  PpIX  dimethyl  ester  (11)  with  dimethylacetylenedicarboxylate (DMAD) and established that the products were the chlorin-type structures (Scheme 3), which were called benzoporphyrin derivatives (BPD). The structures were misinterpreted in Callot's study. The regioisomers, resultingfromthe reaction of the A and B ring vinyl groups separately, showed a significant red shift of the Q-band (666 nm) with high extinction coefficient at -10000 cm "'M" . It was then 1  discovered that triethylamine isomerized the 1,4-diene system 12 to the 1,3-diene isomer 14 with the methoxycarbonyl moiety arranged cis to the methyl group. With the sterically hindered and stronger base DBU, the thermodynamically more stable product 15, in which the methoxycarbonyl group and methyl group are in trans positions, was produced. This resulted in a further red shift of Q-band to 690 nm.  50  23 Chapter 1 Introduction  16a  16b  1  14  5  BPDMA (16, mixture of 16a and 16b)  Scheme 3 Diels-Alder Reaction of Protoporphyrin IX Dimethyl Ester with DMAD  The selective acid hydrolysis of the propionic esters over those on the exocyclic ring was established. The partial hydrolysis product, known as benzoporphyrin 50  derivative monoacid A ring (BPDMA, 16), has been marketed for the treatment of agerelated macular degeneration (AMD) with the trade name of Visudyne®. AMD is the major cause of vision loss in people over the age of 55. Visudyne is the only drug approved for the treatment of wet AMD and it has brought benefit to more than 250,000 patients around the world. Beside this indication, BPDMA has been examined for the treatment of cancers and other diseases. The success of BPDMA demonstrates the practicality of PDT drugs and has encouraged researchers to seek out further drugs that can be applied for various clinical purposes. The cycloaddition methodology was further extended by Dolphin and co-workers  51  to the two-step double Diels-Alder reaction on divinylporphyrin (17) for the synthesis of  24 Chapter 1 Introduction  novel bacteriochlorins that exhibit long wavelength absorption at about 800 nm (Scheme  Scheme 4 Double Diels-Alder Reaction on Divinylporphyrin  Applying the Diels-Alder reaction into the chlorin substrate 22 afforded the stable bacteriochlorin 23 which exhibits absorption near 800 nm (Scheme 5). The preliminary 53  in vitro photosensitizing results obtained from this series of compounds are very  promising. The 8-vinyl group in 22 was derived from the 8-ethyl group by dihydroxylation followed by dehydration. The presence of the 7V-alkyl imide ring improved the stability of the compounds compared to their anhydride counterparts.  21  22  Scheme 5 Diels-Alder Reaction on Chlorin  23  25 Chapter 1 Introduction  1.2.3.2 Intramolecular Cyclization Extension of the chromophore presents a good way to shift the absorption spectrum to longer wavelength. Several synthetic methods based on the modification of the  meso-  position of a (3-alkylporphyrins with cyclization of the appended group at a porphyrin (3position have been developed  ( S c h e m e 6 ) . The  vinylogous version of the Vilsmeyer  reaction by which the vinylformyl is introduced into the weso-position has shown great success for this purpose. The acid-catalyzed cyclization of the vinylformyl group (in 26 54  and 27) into the porphyrin ring allowed the access to the benzoporphyrin 28, which has the longest-wavelength absorption at 672 nm ( S c h e m e  24. M = C u 25. M = N i  2 +  2 +  6).  26. M = C u  28  27. M=Ni S c h e m e 6 Intramolecular  Cyclization  Li et al. developed the method and approached the synthesis of series of analogues in which the structures can be modified by introducing different  R  groups  (Scheme 7).  5 5  The author introduced various fluorinated and nonfluorinated alkyl groups into the structure and found that the fluorinated analogues showed better PDT  in vitro  efficacy  than the corresponding nonfluorinated derivatives. When the R group was acetylene, the acid-catalyzed cyclization process yielded the benzoporphyrin 31 with ketone substituent. Further modification of 31 afforded the ether derivatives 32.  26 Chapter 1 Introduction  Seheme 7 Benzoporphyrins with Various R Substituents via Intramolecular Cyclization  Purpurins have been known for quite long time as degeneration products of chlorophyll. The first synthesis of this class of compounds was reported by Woodward in 1960's, which was achieved by intramolecular cyclization of a weso-acrylate to the p— 56  pyrrolic position, during the synthesis of chlorophyll a. This methodology was extended by Morgan et al.  57  to synthesize a series of substituted porphyrin-based purpurin  analogues. The Sn ethyl etiopurpurin (SnEt2, 35, Scheme 8), with the longest-wavelength absorption at 650 nm, shows the most effective in vivo PDT efficacy among the purpurins that have been evaluated as PDT agents. FDA just issued an approvable letter for this productfromMiravant Medical Technologies in Sep. 30, 2004 for the treatment of AMD. In the acid-promoted cyclization of the unsymmetric etioporphyrin 34, the reaction proceeds selectively toward the carbon carrying the ethyl group versus the carbon carrying the methyl group. This selectivity has not yet been adequately explained. Selman recently reported the utility of liposome encapsulated 58  of canine prostate cancer.  SnEt2  in photodynamic treatment  27 Chapter 1 Introduction  Scheme 8 Synthesis of SnEt2 via Cyclization  1.2.3.3 Chlorophyll a Derivatives Chlorophyll a (2), the green photosynthetic pigment, is one of the prototypes of the chlorin class of natural product. With its highest wavelength absorption at -660 nm, it is a readily available source for chlorin-type starting material. A large amount of work has been done to obtain chlorin-type photosensitizers derived from this source. The research can generally be divided into two categories: the modification of the side chain and the modification of the five-member isocyclic ring. The carboxylic acid (or ester) group of the chlorophyll-related starting materials can be transesterified, converted to thioester or amide by treatment with other nucleophiles together with activating agents such as DCC. These provide the fastest way to modify the structure and have proved to be very rewarding. One of the successful examples is the mono-L-aspartyl derivative of chlorin ec, known as Npe<j (38), It was formed via a DCC 59  coupling between the chlorin e<$ (36) and ^-butylaspartate followed by removal of the t-  28  Chapter 1 Introduction  butyl protecting group. Npe^ was found to exhibit good tumor response, rapid clearance and high water solubility. It is now in human clinical trials for treatment of endobronchial lung cancer in Japan. The structure of Npe^ has been confirmed to be 38, as shown in Scheme 9, by recent extensive NMR studies by Gomi. The amide is linked at position60  15 instead of position-17 as reported in previous publications.  59  Scheme 9 Synthesis of Npe^  To understand the effect of various substituents on photosensitizing efficacy, Dougherty et al. synthesized and evaluated a series of pyropheophorbide a analogues with alkyl ether functional groups. The reaction of methyl pyropheophorbide a (39) with HBr/AcOH and then the appropriate alcohol produced the corresponding ether analogues. In the final step, the methyl ester was hydrolyzed to the corresponding 61  carboxylic acid (40, Scheme 10).  29 Chanter 1 Introduction OR  1. 30 % HBr/AcOH  ff R: alkyl groups  2. ROH 3. LiOH/THF  MeO'  HPPH (41), R= n-hexyl  HO'  O  O  40  39  Scheme 10 Alkyl Ether Derivatives of Pyropheophorbide a  These analogues exhibit longest-wavelength absorption at ~665 nm and show excellent singlet-oxygen production efficiency of 45 %. The results obtained from the in vivo studies on mice demonstrated that the PDT efficacies of these photosensitizers are associated with the length of the carbon chain at the ether site. The activity increased as the length of the carbon chain was increased, reaching a maximum in the «-hexyl ether (known as HPPH, 41, the photosensitizer with the highest efficacy and optimal lipophilicity in the series of analogues). Interestingly, the PDT efficacy decreased when 61  the length of the alkylether was extended past hexyl. HPPH (41) was found to be fivefold more potent than the n-dodecyl derivative (more lipophilic) and threefold more potent than the w-pentyl analogue (less lipophilic). The data indicates that the overall amphiphilicity (i.e. a proper balance between hydrophilicity and lipophilicity) of the molecule influences the biodistribution and localization of the photosensitizer in tissue,  62  thus plays an important role in drug efficacy. However, the introduction of the rc-hexyl ether side chain at other positions, such as the 8- or 20- positions, significantly reduced the in vivo efficacy.  It has been suggested that, besides the lipophilicity, the presence  and position of the substituent are also important factors. HPPH is currently at phase I/II  30 Chapter 1 Introduction  human clinical trials for the treatment of a variety of cancers and the pharmacokinetic 64  studies of HPPH are also underway.  65  The history of the modification on the isocyclic ring of pyropheophorbide a can be traced back to the work of Fisher. Thisringis readily converted to the anhydride upon 66  treatment with base and oxygen, and in this way the methyl pheophorbide a (8) was transformed to purpurin-18 (42). A series of purpurin-18-7V-alkyl imides (44, Scheme 11) were obtained when the purpurin-18 was treated with alkyl amine followed by treatment with base. The vinyl group at position-3 was then functionalized by a variety of alkyl ethers to allow modification of the lipophilicity of the molecule. This class of purpurinimides exhibit maximum wavelength absorption around 700 nm and were found to be quite effective in animal studies. As in the aforementioned pyropheophorbide a series, the lipophilicity, presence and position of the alkyl groups in the molecule play critical roles in tumour uptake, tumour selectivity and in vivo PDT efficacy of this series of compounds.  R , R : alkyl groups 1  8  42  2  43  44  Scheme 11 Modification of the Isocyclic Ring of Pyropheophorbide a  Similar modifications have been carried out on bacteriochlorophyll a derivatives in an effort to synthesize stable bacteriochlorins 45 as photosensitizers.  68  These  31 Chapter 1 Introduction  chromophores exhibit longest-wavelength absorptions near 790 nm and show high tumour uptakes. .OR  2  45  1.2.3.4 Dihydroxylation of P, P-double bond  Osmium tetraoxide oxidation has been very frequently used for the conversion of porphyrins to the corresponding vz'c-dihydroxy chlorins, such as 47, and tetrahydroxy (iso)bacteriochlorins like 48 (Scheme 12). Chang and co-workers can be credited for full elucidation and revival of this reaction. The ketochlorin analogs are formed from the 69  diol via an acid-catalyzed pinacol-pinacolone type rearrangement. The overall lipophilicity of these molecules can be altered by such transformations. However, the outcome of the rearrangement is not straightforward but depends on both the nature of the migratory group and the electronic and steric factors on the porphyrin nucleus. Thus the rearrangement result is hard to predict.  70  32 Chapter 1 Introduction  Scheme 12 Dihydroxylation and Pinacol-Pinacolone Type Rearrangement of Porphyrin  Pandey's research group extended this methodology to chlorins and synthesized a series of v/c-hydroxybacteriochlorins and ketobacteriochlorins (Scheme 13). The 71  ketobacteriochlorin 53 obtained from 20-formyl 9-deoxypyropheophorbide a methyl ester 51 showed a longest-wavelength absorption at 768 nm. In preliminary in vivo screening, the ketochlorins 53 was found to be more photodynamically active than the corresponding vic-dihydroxy analogues 52.  Scheme 13 Dihydroxylation and Pinacol-Pinacolone Rearrangement of Chlorin Substrate  33 Chapter 1 Introduction  1.2.3.5 Target-Specific Photosensitizers Since the introduction of the first PDT drug Photofrin® there has not been much success in improving the photosensitizer's tumour selectivity and specificity because tumour cells in general have nonspecific affinity to porphyrins. Some attempts have 72  been made to direct photosensitizers to known cellular targets by creating a photosensitizer conjugate, where the other molecule is a ligand specific to the target. For 73  example, cholesterol and antibody-conjugated porphyrins have been prepared to direct photosensitizer to specific tumor antigens. Certain chemotherapeutic agents have also 74  been attached to porphyrin chromophores to increase the effectiveness of the PDT treatment.  75  Oligosaccharides play essential roles in various cellular activities as antigens, growth signals, targets of bacterial and viral infection, and glues in cell adhesion and metastasis. The saccharide-receptor interactions are usually specific and multivalent. This specificity suggests a potential utility of synthetic saccharide derivatives as carriers in directed PDT agent delivery. Recently, Aoyama et al. reported the synthesis of certain TPP-based saccharide-functionalized porphyrins and demonstrated the importance of hydrophobicity masking for the saccharide-directed cell recognition. The amide-linked 77  octa(galactose) and octa(glucose) derivatives of TPP 54 and 55 were obtained by the reaction of lactonolactone or maltonolactone with TPP-amine. These saccharide conjugates are highly hydrophilic, where no hydrophobic force for incorporation into the cells works effectively. Thus the identity of the saccharide moieties plays a crucial role in cell recognition. It has been demonstrated that the cluster 54 with the galactoside moiety is captured by the cell because the galactoside undergoes specific saccharide-receptor  34  Chapter 1 Introduction  interaction with cell, while the other one 55 is completely rejected by the cells since the glucoside has no affinity with the cell. In this way, the included porphyrins can either be delivered to the target cells or kept in solution awayfromthe cells. As the saccharidereceptor interactions exist everywhere, the well-defined and well-designed synthetic saccharide clusters may serve as a new tool in glycotechnology. xcx  A  .ox  OX  54: X = - C H C H H N - C 2  2  JjO u  r  i  O II 5 5 : X= - C H C H H N - C 2  2  The Roswell Park group has reported the synthesis and biological significance of certain p-galactose-conjugated purpurinimide 59 via enyne metathesis (Scheme 14). Molecular-modeling studies indicate that when the P-galactose moiety is placed at the appropriate position the photosensitizer does not interfere with galectin-carbohydrate recognition. Intracellular studies with known cell-surface counterstains confirmed the cell-surface recognition of the conjugate. The galactose-conjugated photosensitizer 59 showed a considerable increase in both in vitro and in vivo PDT efficacy when compared with thefreepurpurinimide analogues. This result indicates the possibility for developing a new class of tumour-specific photosensitizers for PDT based on recognition of a cellular receptor.  Scheme 14 Synthesis of Galactose-Conjugated Purpurinimide  36 Chapter 1 Introduction  1.3 Research Objective The objective of this thesis was to generate new photosensitizers for use in PDT by modifying the amphiphilicity of tetrapyrrolic macrocycles via developing the chemistry of the conjugated vinyl group. This was accomplished by modification of the vinyl group of two chlorin photosensitizers to furnish non-diastereomeric primary ether derivatives (Chapter Two) and by introducing functionalities on the vinyl group via cross-metathesis (Chapter Three). Initially, the objective for this work derived from our interest in the chlorin photosensitizer, HPPH (41). In the studies of methyl pyropheophorbide a derivatives, Dougherty (the inventor of Photofrin II®) and co-workers found that HPPH, which has a secondary n-hexyl ether functionality at position-3, exhibits excellent PDT efficacy. A n 61  in vivo quantitative structure-activity relationship (QSAR) study was carried out on the congeneric series of pyropheophorbide a photosensitizers with alkyl ether groups of different length, shape and position. The study indicated a correlation between the PDT 63  efficacy and drug amphiphilicity, which was related to the alkyl ether chain length mostly for this series, as defined by log P, the logarithm of the octanol to water partition coefficient of the compound. HPPH, with the highest efficacy, showed a log P of 5.6. Interestingly, analogues with log P < 5 just showed minimal activity and as the log P increased above 6.5, activities declined gradually as well. It was suggested from the study that the overall amphiphilicity of the photosensitizer was highly predictive for PDT efficacy. Therefore, modifying the amphiphilicity of the molecule provides the opportunity to improve the PDT efficacy of photosensitizers.  37 Chapter 1 Introduction  HPPH, however, has a major drawback in that it is prepared as a pair of diastereomers. The starting material methyl pyropheophorbide a (39) is optically active with chiral centers at C-17 and C-18. In HPPH, the vinyl group at C-3 is converted to the secondary ether, and a new chiral centre C-3 is introduced. As the configuration of C-3 1  1  can be either R or S, HPPH consists of a pair of diastereomers. The separation of these diastereomers was problematic as was control of the ratio of diastereomers during synthesis. Because stereoisomers may possibly have different bio-properties, such a mixture is generally unsuitable as a drug candidate. The synthesis and characterization of non-diastereomeric ether derivatives of methyl pyropheophorbide a were, therefore, of great interest. Introduction of a primary centre at C-3 would enable such ether derivatives to be prepared. One of the aims of the project was to synthesize and examine the PDT properties of the primary ether derivatives of pyropheophorbide a 60, where the R group was varied to modify the lipophilicity of the molecule.  Ring B-BPD-l,3-diene dimethyl ester (61, Scheme 15) was another chlorin with a modifiable vinyl group. With a benzene-like ring fused to the (3, P'-positions, 61 showed  38  Chapter 1 Introduction  the longest-wavelength absorption at 688 nm, which is ideal for a good photosensitizer. As discussed in the synthesis of BPDMA (Visudyne®, Scheme 3), both ring A and ring B isomers, 12 and 13, were obtained equally in the reaction of protoporphyrin IX dimethyl ester (11) with DMAD. By treatment with DBU, 13 was converted to ring B-BPD-1,3diene dimethyl ester (61), in 90 % yield (Scheme 15) efficacy as good as BPDMA,  79  4 9  Although 61 exhibited PDT  it was not selected as a drug candidate because of  difficulties in formulation. Therefore, as the side-product during the manufacture of Visudyne®, ring B-BPD-l,3-diene dimethyl ester (61) was readily available to us and represented a valuable source for studies of new photosensitizers.  13  61  Scheme 15 Synthesis of Ring B-BPD-l,3-diene Dimethyl Ester 61  Much chemistry has been carried out on this chlorin chromophore in our group. However, relatively little work has been done on the vinyl group largely due to the generation of poorly separable diastereomers via Markovnikov addition across the double bond. Still, it has been reported that replacement of the vinyl group on 61 with a secondary ether group resulted in a remarkable improvement in photosensitizing efficiency, even though the similar problem of diastereomers existed. With what we 80  39 Chapter 1 Introduction  have known on Doughtery's work on ether derivatives of methyl pyropheophorbide a, we anticipated that it would be advantageous to create primary ether derivatives via the vinyl group of ring B-BPD-l,3-diene dimethyl ester (61). The gains from this would be twofold: not only are new, non-diastereomeric derivatives of 61 generated for photosensitizing and structure-activity studies, but the knowledge gained in their manipulation is valuable as a model in the study of 39, which is more difficult to isolate and much more expensive to purchase. The primary ether derivatives 62, with varied R groups, were thus our synthetic targets for vinyl group modification of ring B-BPD.  In our group, we seek to establish generalizable strategies for modifying the amphiphilicity of tetropyrrolic macrocycles. With this in mind, cross-metathesis of the vinyl group caught our attention. Cross-metathesis (CM), an intermolecular olefin metathesis for the formation of carbon-carbon double bonds (Scheme 16), has gained more utilities in organic synthesis with the rapid development of highly-active metathesis 81  catalysts.  40 Chapter 1 Introduction  -J  R  1  +  ==/  R  2  CM  •  R\  \=/  .R  2  +  Scheme 16 Cross-Metathesis Reaction between Terminal Olefins  Nevertheless, olefin metathesis and C M have seldom been applied to chlorin and porphyrin substrates. One of the few applications was that enyne metathesis was applied to  purpurinimide  for the synthesis  of (3-galactose-conjugated  photosensitizers  78  (Scheme 14).  We anticipated that C M reactions would provide new approaches for  introducing various functionalities onto the vinyl group, and this would be a direct way to change the amphiphilicity of the chlorin and porphyrin photosensitizers (Scheme 17). Several tetrapyrrolic macrocycles with the conjugated vinyl groups, include ring B BPD-1,3 diene dimethyl ester (61), methyl pyropheophorbide a (39) and protoporphyrin IX dimethyl ester (11) were chosen as substrates for C M in Scheme 17. To study the reaction scope, various terminal or internal olefins with different functionalities, such as alkyl, hydroxyl, halide, aldehyde, ester and silane would be employed as C M partners. With the combination of tetrapyrrolic macrocycles and C M partner olefins, a variety of new photosensistizers with different substituted-vinyl functionalities could be reached conveniently. Furthermore, the chemo- and stereoselectivity of the C M of our substrates would be worthwhile to investigate, since it was the first time that C M had been applied to tetrapyrrolic macrocycles.  +  Scheme 17 C M of the Vinyl Group Conjugated with Tetrapyrrolic Macrocycle  41 ^_  Chapter 1 Introduction  REFERENCES •1. Montforts, F.; Gerlach, B.; Hoper, F. Chem. Rev. 1994, 94, 327 and reference therein. 2. Scott, A. I. Angew. Chem. Int. Ed. Engl. 1993, 32, 1223. 3. Eschenmoser, A. Angew. Chem. Int. Ed. Engl. 1988, 27, 6. 4. Brautler, B. Chimin 1987, 41, 277. 5. Jordan, P. M . Biosynthesis of Tetrapyrroles; Jordan, P. M . Ed.; Elsevier: Amsterdam, 1991, 1. 6. Battersby, A. R. Acc. Chem. Res. 1993, 20, 15 7. Rudiger, W.; Schoch, S. Plant Pigment; Goodmin, T. W. Ed.; Academic Press: London, 1988, Chapter 1. 8. Fajer, J. Chemistry and Industry 1991, 869. 9. Stolzenberg, A. M . ; Stershic, M . T. J. Am. Chem. Soc. 1988, 110, 6391. 10. Tait, G. H . Heme and Hemoproteins; Matteis^ F. 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Engl. 2003, 42, 1900.  48 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  49 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  The synthesis, structure characterization and PDT efficacy studies of the chlorin photosensitizers with primary ether functional groups, 60 and 62, will be discussed in this section.  2.1 Synthetic Approach To synthesize the target compounds 60 and 62, synthetic routes presented in Scheme 18 and Scheme 19 were designed and investigated. In general, the vinyl group that is connected with the tetrapyrrolic macrocycle was converted into the 2-hydroxyethyl group by vz'c-dihydroxylation using osmium tetroxide followed by a pinacol-pinacolone rearrangement. Treatment of the terminal alcohol with the appropriate bromination reagent generated the corresponding bromide. Nucleophilic substitution of the bromide with various alcohols in the presence of HgO-2HBF4 afforded the desired primary ether derivatives in good yields.  50 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  60 Scheme 18 Synthetic Approach Towards 60  51 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  77 % over 2 steps  62a, R = methyl; 2  62e, R = n-pentyl; 2  62b, R = ethyl; 2  62c, R = n-propyl;  62f, R = n-hexyl; 2  62d, R = n-butyl;  2  2  62g, R = n-heptyl 2  Scheme 19 Synthetic Approach Towards 62  52 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  2.2 Synthesis of Target Compounds 2.2.1 Starting Material: Methyl Pyropheophorbide a Methyl pyropheophorbide a (39) is an important starting material for our project. As it is closely related to chlorophyll a, it can usually be obtained from algae. Smith et al. reported the isolation of pheophytin a (4) from Spirulina 1  maxima, a type of algae  that  contains no chlorophyll b. Methyl pyropheophorbide a can then be obtained from pheophytin a by transesterification with methanol/sulfuric acid followed by decarboxylation. Ma improved the procedure by demetallating the extracts prior to chromatography isolaton to minimize the decomposition reaction, since the magnesiumfree derivatives are less sensitive. However, the extraction procedures for both methods 2  are tedious and require exhaustive chromatography. Roughly 3 g and 4.2 g of methyl pyropheophorbide  a  was obtained from 1 kg of Spirulina  maxima  according to the two  reports, respectively. Methyl pyropheophorbide a (39) is also commercially available from Porphyrin Products, but is rather expensive at the price of US$ 250/g. Therefore, it was worthwhile to search for a convenient way to obtain 39 from an inexpensive source. Efforts have been made in this work and a feasible route has been developed. Magnesium-chlorophyllin, a mixture of a variety of water-soluble magnesiumchlorins, proved to be the appropriate starting material through our investigations. It is commercially available from Pfannenschmidt GmbH, a German company, for US$ 300/kg. The mixture was first dissolved in brine and washed with diethyl ether to remove the yellow pigments. The aqueous phase was then treated with 1 M aqueous HC1, which 3  served to demetallate as well as hydrolyze the esters in the mixture. The mixture was  53 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  extracted with diethyl ether, and the organic layer was treated with excess diazomethane, generating the methyl esters. Chlorin e^, trimethyl ester (72) was isolatedfromthe ester mixture via column chromatography in a 13 % yield (based on the starting magnesiumchlorophyllin material). This trimethyl ester then underwent cyclization upon treatment with ?-BuOK under rigorously Ch-free conditions. Treatment with diazomethane 4  afforded the methyl pheophorbide a (8) in 85 % yield as a diastereomeric mixture. Refluxing of 8 in collidine for 2 h produced methyl pyropheophorbide a (39) in 90 % yield (Scheme 20).  1  1) 1 M HCI Magnesium-chlorophyllin (mixture)  2) ether extraction 3) C H N 4) column chromatography 2  2  72 1) f-BuOK, 0 -free 2  2) C H N 2  2  Scheme 20 Preparation of Methyl Pyropheophorbide afromMagnesium-Chlorophyllin  54 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  This developed method is much simpler than previous methods, requiring only one column chromatography step, as well as providing an improved yield. Once the key intermediate chlorin  trimethyl ester (72) was isolated, the cyclization and  decarboxylation steps proceeded in high yields. This method has been shown to be an effective alternative method for obtaining methyl pyropheophorbide a.  It was interesting to observe that in the cyclization reaction of 72, if the reaction system was not strictly oxygen-free, no desired product 8 was obtained. Instead, the diastereomeric mixture of methyl 13 -hydroxy pheophorbide a, 73, was isolated as the 2  major product. It has been reported that purpurin-18 (42) is formed if oxygen is present during the cyclization; however, this was not consistent with our observations. Methyl 4  pheophorbide a (8) has also been reported to undergo aerial oxidation to 73 during silica column chromatography, which was indeed found to be true in our lab.  5  Fig. 2.1 shows part of the 'H NMR spectra of 73 and 8. The major difference for the two spectra is that the OH proton in 73 appears at 5.40 ppm as a singlet, while the peak of H-13 proton in 8 is at 6.24 ppm. 2  55 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  Si's  Q.2  li.B  1  SA  '  8.0  '  7.6  '  7.2  6.8  8.4  6.0  5.6  E  (PPm)  Fig. 2.1 Low-Field Region of the *H NMR Spectra (CDC1 , 300 MHz) of 73 and 8 3  2.2.2 Preparation of 3-(2-Hydroxyethyl) Derivatives The 3-(2-hydroxyethyl) derivatives 65 and 70 (Scheme 18 and Scheme 19) are important intermediates in our synthetic approach. The widely applied method for transforming the conjugated vinyl group into the 3-(2-hydroxyethyl) group is using thallium(III) trinitrate followed by acidic hydrolysis (Scheme 21). For example, methyl 6  pyropheophorbide a was treated with two equivalents of thallium(III) trinitrate in methanol or methylene chloride, whereby the dimethyl acetal 74 was obtained. The acetal was hydrolyzed in aqueous acid to give the 3-acetaldehyde derivative 64, which was then  56 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  converted to the 3-devinyl 3-(2-hydroxyethyl) derivative 65 via reduction with sodium borohydride. This method is reasonably efficient but requires the use of two equivalents of thallium(III) trinitrate (one equiv. of which is required to chelate the chlorin nucleus), which is highly toxic.  39  74  64  65  Scheme 21 Converting the 3-Vinyl Group to the 3-(2-Hydroxyethyl) Group by T1(N03)3  The Tamiaki research group reported another method for this transformation by direct anti-Markovnikov hydration of the vinyl group (Scheme 22). Atfirst,the reactive 7  13'-keto group in the isocyclic ring of 39 was protected as its ethylene ketal. The corresponding chlorin 75 was carefully treated with BH3-THF, followed by reaction with hydroperoxide and sodium hydroxide. After the ketal-protection was removed using aqueous HC1, the regioisomeric mixture of 3-(2-hydroxyethyl) pyropheophorbide a 65 and 3-(l-hydroxyethyl) pyropheophorbide a 76 was obtained in 51 % yield. The ratio of 65:76 was 33:1 by H NMR analysis, indicating that the hydroboration proceeded !  regioselectively at the vinyl group in the expected anti-Markovnikov manner. The regioisomers were separated by HPLC to give the pure 3-(2-hydroxyethyl) pyropheophorbide 65.  57 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  Scheme 22 Preparation of 65 by Anti-Markovnikov Hydration  The direct hydroxylation at the 3 -position by applying the conventional anti2  Markovnikov hydroboration-oxidation was employed in this method. However, a regioisomeric mixture of both Markovnikov and anti-Markovnikov products 65 and 76 were still obtained from this reaction, even though 65 was the major isomer. The properties of these two isomers are so similar that the isolation of the pure regioisomer 65 has to be carried out by preparative HPLC, which makes it inconvenient to prepare the desired product in large scale. Furthermore, the 13'-keto group needed to be protected and deprotected before and after the hydration. Not only are these extra steps required but the undesired side-products 77, 78 and 79 (Scheme 22) were formed in this procedure. These 13'-(2-hydroxyethoxy) derivatives were obtained via the acid-promoted reductive  58 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  cleavage of the ketal during the deprotection step. It was observed that the application of excess BH3-THF gave a larger amount of these undesired cleavage compounds. Therefore, due to the disadvantages of these methods, we decided to seek out a new method for making the 3-(2-hydroxyethyl) derivatives, which is efficient and safe. The synthetic methods shown in Schemes 18 and 19 were designed based on the pinacolpinacolone rearrangement of a diol to give the 3-acetaldehyde derivative which is then reduced to the 3-(2-hydroxyethyl) derivative. The investigations of this new method are discussed as follows.  2.2.2.1 Dihydroxylation of the Vinyl Group In our method, the first step was to generate the dihydroxy derivatives from the vinylchlorins.  Osmium tetroxide is one of the most reliable reagents available for the cis hydroxylation of alkenes to give the corresponding cis-diol. This method has been widely applied for two reasons: first, osmium tetroxide reacts with virtually all olefins; second, it reacts slowly, i f at all, with other common functional groups. The cis hydroxylation is 8  well established to take place via the formation of an osmium (VI) ester intermediate 82 (Scheme 23), which on reductive or oxidative hydrolysis yields the corresponding cisdiol.  59 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  ><°  +  82 Scheme 23 Formation of the Osmium (VI) Ester Intermediate  Reductive hydrolysis is generally carried out using sodium or potassium sulfite or bisulfite, lithium aluminum hydride, or hydrogen sulfide. Upon treatment, the osmium (VI) ester complex is hydrolyzed to an insoluble osmium salt that can be removed by filtration. A stoichiometric amount of osmium tetroxide is required for this process. This 9  method has been applied widely to differently functionalized olefins, including porphyrin substrates. As discussed in Chapter One (Scheme 12), treatment with osmium tetroxide followed by hydrogen sulfide is a general method for m-dihydroxylation of the two double bonds that are cross-conjugated to the 18 rc-electron system, from which the corresponding vz'c-dihydroxy chlorins and tetrahydroxy bacteriochlorins are obtained. Sparatore reported the conversion of the two vinyl groups in protoporphyrin IX dimethyl ester (11) into the bis-glycol 83 using the same method (Scheme 24).  11  83 Scheme 24 Preparation of Bis-Glycol 83  10  60 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  When the osmium (VI) ester intermediate is oxidatively hydrolyzed, such as by metal chlorates, JV-methylmorpholine TV-oxide (NMO), hydrogen peroxide and tert-b\xXy\ hydroperoxide, the osmium tetroxide is regenerated and can further react, rendering this process catalytic. Among these co-oxidants, NMO has proven to be effective, leading to high yields without over-oxidation. This procedure, dihydroxylation of olefins with catalytic osmium tetroxide together with NMO in acetone and water, is called the "Upjohn process". After published in 11  1976 by research chemists at Upjohn, this method soon became one of the most used organic processes because of its efficiency, compatibility and convenience. Stephen and co-workers' found that the transformation in Scheme 24 could be attained by employing 0.1 equivalent of osmium tetroxide together with NMO.  12  In our studies to apply the "Upjohn process" to our substrates 39 and 61, that is, employing catalytic amounts of osmium tetroxide (usually 0.04 equiv.) and excess NMO, very good results were obtained. A THF solution of methyl pyropheophorbide-a (39) or 13  ring B-BPD-l,3-diene dimethyl ester (61) was treated with 0.04 equivalent of osmium tetroxide, which was dissolved in toluene-acetone-water (1.5:10:1, v/v/v) mixture, and 1.5 equivalents of Af-methylmorpholine TV-oxide (50 % aqueous solution) at room temperature in the dark. Analytical TLC showed that the starting material disappeared gradually while a new more polar spot appeared and enriched over time. After 24 h, no starting material remained and sodium metabisulfite was added to the reaction mixture to reduce the osmium tetroxide to the insoluble salt. After filtration, methylene chloride was added to dilute the filtrate. The organic layer was separated and then washed with aq. sodium acetate and water. The diol derivative 63 or 68 was afforded in 75 % yield after  61 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  column chromatography. Since the diols were quite stable when kept in the fridge in the dark, they were prepared on large scale as useful intermediates for this work. As the configuration of C-3 can be either R or S, the diol derivatives 63 and 68 1  exist as diastereomeric mixtures. This is clearly reflected in the H NMR spectrum of 63 !  (Fig. 2.2), where two sets of signals for the H-20, H-3 , 17 -C0 Me, H-12 and H-2 1  2  1  1  2  appear due to the existence of two diastereomers. No efforts were made to separate the 14  diastereomers because the chiral centre will be destroyed in the pinacol-pinacolone rearrangement in the following step.  10  9  8  7  6  5  4  3  2  1  0  Rpm  Fig. 2.2 'H NMR Spectrum (CDC1 , 300 MHz) of 63 3  -1  -2  -3  62 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  The application of catalytic osmium tetroxide with cooxidant NMO thus proved to be successful for dihydroxylation of the conjugated vinyl group in our chlorins. The reaction can be carried out under mild conditions without inert gas protection and it proceeds very smoothly with no appreciable side reactions being detected.  2.2.2.2  Pinacol-Pinacolone Rearrangement  After the diol derivatives were synthesized, attention was focused on the pinacolpinacolone rearrangement of these diols (Scheme 18 and Scheme 19). The pinacol-pinacolone rearrangement is a classical reaction that has been extensively investigated and widely used in organic synthesis. For tetrapyrrolic macrocycle substrates, this reaction has also been applied successfully to the rearrangement of v/c-dihydroxy chlorins to afford the keto-chlorins (Scheme 12 and Scheme 13 in Chapter One). However, to our knowledge, this rearrangement has never been applied to a diol functionality existing at the peripheral position of tetrapyrrolic macrocycles. Our investigation started with the 3-devinyl-3-(l,2-dihydroxyethyl) ring B-BPD1,3-diene dimethyl ester (68). The BPD diol derivative 68 was dissolved in a mixture of concentrated sulfuric acid and fuming sulfuric acid without any other solvents.  15  Treatment with the mixed acids for 0.5 h at room temperature led to the completion of the rearrangement. After pouring the reaction mixture into ice-cold water, the 3-acetaldehyde ring B-BPD derivative 69 was extracted with methylene chloride. This aldehyde, which was found to be unstable in solution and on silica gel column, was then reduced to the alcohol derivative 70 using sodium borohydride without purification. The isolated yield  63  Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  of 70 after the two steps reached 77 %, indicating the high efficiency of this method. Another observation was that when only concentrated sulfuric acid was employed, the 16  rearrangement product could still be obtained, but with a much lower overall yield of 50 % for two steps. In addition, some of the starting material always remained unchanged as shownfromanalytical TLC. With these successes with the ring B-BPD derivatives, we thought that the rearrangement for methyl pyropheophorbide a derivative 63 would be straightforward. Unfortunately, this procedure was found to be troublesome. Upon treatment with the concentrated and fuming sulfuric acid mixture, the diol derivative 63 was converted to a blue coloured water-soluble material, which could not be extracted with neat methylene chloride. By using 10 % methanol/methylene chloride, this material was partially extracted into the organic phase. After removing the organic solvent, the dark blue residue was treated with an excess of diazomethane. Some of the starting material 63 was recovered after this treatment together with other materials that were hard to identify. It was suggested from this result that under such conditions, some of the diol 63 was hydrolyzed to the carboxylic acid 84, while no desired rearrangement occurred. Therefore, searching for the appropriate acid to catalyze the pinacol-pinacolone rearrangement of 63 became a crucial task. It would determine whether the whole synthetic strategy for methyl pyropheophorbide a derivatives would be successful or not. HO  ^OH  (f  NH N  /= N  A HN \  "OH  84  64 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  Several other acids that are normally used in pinacol-pinacolone rearrangements, such as concentrated sulfuric acid, p-TsOH, trifluoroacetic acid and perchloric acid 15  17  were tried, but no positive results were obtained. Either the water-soluble materials were observed, in the case of concentrated sulfuric acid; or the unchanged starting material was recovered, with the use of other acids. However, the use of a mixture of sulfuric acid and perchloric acid gave us some promising results. Trace amount of a new product was observed on TLC, which was proved to be the desired alcohol 65 after sodium borohydride reduction. It was anticipated that, based on this result, an extremely strong acid was required for the rearrangement. At this point, our attention was turned to trifluoromethanesulfonic acid (triflic acid, CF3SO3H).  Triflic acid is one of the strongest commercially available acids with pK of a  -14, and is soluble in organic solvents, such as methylene chloride. The utilization of 18  triflic acid provided us the results we were looking for. A methylene chloride solution of 63 was treated with excess triflic acid at room temperature for 3 h in the dark. The rearrangement product 64 was observed as the major product from analytical TLC with no starting material 63 remaining. The reaction mixture was then poured into ice-water and the acetaldehyde 64 was extracted with methylene chloride. The acetaldehyde derivative 64 was unstable on silica gel column chromatography like 69, and was directly reduced to the methyl 3-(2-hydroxyethyl) pyropheophorbide a 65 by treatment with sodium borohydride at 0°C for 1 min. The overall yield for the two steps reached as high as 85 %. Triflic acid was thus proved to be the most effective acid for the pinacolpinacolone rearrangement of the methyl pyropheophorbide a diol derivative 63.  65 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  The different performances of different acids for this reaction still can not be rationalized completely, but some explanations will be attempted here. From the above results, it can be concluded that the strength of the acid is a key point: the stronger the acid, the better the result. The p/<a values for different acids are listed in Table 2.1.  18  Additionally, triflic acid is miscible with methylene chloride as an organic acid. The miscibility allowed the reaction proceeded homogeneously with methylene chloride as solvent. These advantages were thought to make triflic acid an appropriate acid for the rearrangement.  Table 2.1 The p7^a Values of Different Acids and the Results of Their Reactions with 63 pKa  Reaction Results  4  -3.0  Hydrolysis, decompose  HCIO4  -10  No desired reaction  Acid H S0 2  H S0 and HC10 2  4  4  —  rearrangement product (trace amount)  p-TsOR  -6.0  No reaction  TFA  -0.25  No reaction  Triflic acid  -14  rearrangement product (> 85 % yield)  Furthermore, in the investigation of the reaction with triflic acid, it was found that low temperature is not suitable for the reaction. During the addition of the triflic acid, the temperature of the reaction mixture increased. To avoid undesired side-reactions due to the increased temperature, we tried to carry out the reaction at 0°C instead of room temperature. Surprisingly, none of the desired rearrangement product was obtained for  66 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  this procedure. It was found that the best way for controlling the reaction is to add the triflic acid dropwise to a methylene chloride solution of 63, which maintained a temperature between 20°C ~ 25°C.  In summary, in the method described here, the 3-devinyl-3-(2-hydroxyethyl) derivatives were synthesized by dihydroxylation followed by pinacol-pinacolone rearrangement and reduction, in which only catalytic amount of osmium tetroxide and other low toxicity chemicals were utilized. This route to 3-(2-hydroxyethyl) derivatives 65 and 70 is safer, less expensive and more environmentally friendly than previous methods. Furthermore, this method has been proved to be very effective from which high yields are achieved. Therefore, it provides a new route for the conversion of the vinyl group to the terminal alcohol on tetrapyrrolic macrocycle substrates.  2.2.3 Bromination of 3-(2-hydroxyethyl) Derivatives Generally, two reagents have been used in the literatures for the bromination of peripheral 2-hydroxyethyl groups on tetrapyrrolic macrocycles: thionyl bromide and 19  triphenyl phosphine/carbon tetrabromide complex. By employing thionyl bromide as 20  bromination reagent, the 3,8-di-(2-hydroxyethyl) derivative of protoporphyrin IX in DMF was treated with excess  SOBr2  together with potassium carbonate to give the  corresponding 3,8-di-(2-bromoethyl) derivative in 76 % yield. However, when the same 20  reagents were applied to 3-(2-hydroxyethyl) ring B-BPD derivative 70 in our studies, no desired bromide was obtained. Instead, another less polar product was afforded in 70 % yield. The structure of this unexpected product has been determined to be dibromide 85  '  67  Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  via H NMR and MS analyses (structural characterization of 85 is discussed in section ]  2.3.1), in which the fused benzene ring is also brominated. Further literature search  showed that thionyl bromide has been reported to be able to brominate aromatic rings, such as phenol, to give 2,4-dibromo phenol. Therefore, 21  SOBr2  was determined to be  unsuitable for our substrate.  85  The other method, in which triphenyl phosphine and carbon tetrabromide were employed, proved to be suitable to our purposes. Treatment of the 3-(2-hydroxyethyl) derivative 65 or 70 with the mixture of 10 equivalents of triphenyl phosphine and 11 equivalents of carbon tetrabromide at room temperature for 30 min gave the corresponding bromide 66 or 71. Both of the bromides were obtained in high yields (90 % and 96 % respectively) after the reaction mixture was filtered through a short column of neutral alumina (activity III). The reaction proceeded so smoothly that 30 min led to complete conversion. Another key point was that the reaction needed to be carried out in freshly distilled dry methylene chloride under nitrogen. If any water was present in the reaction system, the yield was reduced remarkably and in some cases no product was obtained.  68 Chapter 2 Primary Ether Derivatives of Chlorin  Photosensitizers  2.2 A Synthesis of the Primary Ethers Much effort was made in our studies to work out the last step in the synthetic route, from which the desired ether could be reached via nucleophilic substitution of the bromide. At first, the conventional Williamson reaction for ether synthesis was applied to 22  our substrates, but was not successful. Strong base, such as alkoxide or hydride, is usually employed in the Williamson reaction to deprotonate the nucleophile precursor. For our substrates, no desired product was obtained for the reaction between bromide 71 and sodium methoxide under anhydrous condition, even after the reaction was continued for 12 h. Indeed, more polar materials were observed on analytic TLC over time, the identity of which was not pursued. They are likely the products of the hydrolysis of the methyl ester under strong basic condition; or the decomposition product of the chlorin dianion which is from the deprotonation of the two pyrrolic NHs. It was suggested from this 23  result that the chlorin macrocycle can not tolerate the strong alkali conditions. Therefore, a milder set of conditions were sought for the nucleophilic substitution of the chlorin substrates. Unfortunately, the chlorin bromide 66 and 71 are not highly reactive electrophiles under neutral conditions. Even though alkyl bromides have been widely used in alkylation reactions, they are not the most reactive leaving groups when compared to tosylate, triflate, etc. For this reason, nucleophile bearing a negative charge at the heteroatom site is usually employed, for instance, in the classical Williamson ether synthesis. Therefore, the bromides needed to be activated to make them reactive towards nucleophiles under neutral conditions  69 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers It is well known that the presence of heavy metal salts enhances the reactivity of alkyl halides. Among various metal salts, the mercury(II) oxide/tetrafluoroboric acid 24  complex has been proven to be effective in this regard. Upon activation with mercury(II) oxide/tetrafluoroboric acid, alkyl bromides are converted into powerful alkylation agents and can react with aliphatic alcohols under neutral conditions to afford the corresponding ethers  effectively.  25  Our  investigations  demonstrate  that  mercury(II)  oxide/  tetrafluoroboric acid complex sufficiently activate our substrates, the chlorin bromides, yielding satisfactory results. The bromide 66 or 71 was treated with excess alcohol in the presence of 0.5 equivalent of mercury (II) oxide/tetrafluoroboric acid in dry methylene chloride under nitrogen. The product ethers were obtained in moderate yields after the reaction mixture was stirred overnight and purified by column or preparative thin layer chromatography. Methylene chloride was the solvent for the reaction, thus a sufficient homogeneous system was maintained to ensure the best result. The tetrapyrrolic ring remained unchanged under the mild reaction condition. Small amounts of the 3-(2-hydroxyethyl) derivatives were obtained i f the reaction system was not completely dry, which arose from the hydrolyses of the bromides. If unsaturated alcohol was used, such as 5-hexenel-ol, the reaction needed to be carried out in refluxing methylene chloride, since very small amounts of product obtained at room temperature. The HgO-2HBF salt was 4  prepared by mixing the yellow mercury(II) oxide with 2 equivalents of tetrafluoroboric 26  acid (50 % aqueous solution) for 30 min followed by drying under vacuum.  This freshly  prepared dry HgO-2HBF salt was required to ensure the best yield of the reaction. The 4  70 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  ether dimer 86 was also obtained via this method between BPD bromide 71 and 3-(2-  The method for preparing ether derivatives of chlorin by Hg(II) salt-assisted nucleophilic substitution of the bromides was very effective. The reactions proceed smoothly under the mild neutral conditions. This is the first time that mercury(II) oxide/tetrafluoroboric acid salt have been successfully applied to the nucleophilic substitution of tetrapyrrolic macrocycles.  71 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  2.3 Structure Characterization  2.3.1 Structure Determination of Compound 85 As discussed in section 2.2.3, the less polar compound was obtained when thionyl bromide was used as bromination reagent to react with the 3-(2-hydroxyethyl) ring B BPD derivative 70. Analytical data ('H N M R and MS) showed that this compound was not the desired bromide derivative 71. Detailed analyses were carried out to determine the structure of the unexpected product. The low resolution electrospray ionization mass spectrometry (ESJJVIS) of the compound showed the protonated molecular ion peak (m+H) at 893. In addition, the +  ratio of the intensities of the isotopic peaks at 891, 893 and 895 is of 1: 2: 1, which is the characteristic pattern for a dibromide. It was hypothesized, based on this result, that this compound is a ring B-BPD dibromide derivative. This hypothesis was then confirmed by the high resolution ESJJVIS result. With the observed (m+H) peak of 891.1601 from high +  resolution ESEV1S, the formula of the compound was decided to be C42H44Br2N408, which is a dibromide with the calculated (m+H) peak of 890.1604. +  At the same time, the ' H N M R (Fig. 2.3) spectrum of the compound was recorded to determine the structure of the dibromide. B y comparing the spectrum of this compound to that of the 3-(2-bromoethyl) derivative 71 (Fig. 2.3), it was found that the significant differences only occurred in the low field region between 10.5 to 7.0 ppm. The other parts of the two spectra are very similar. Therefore, it was believed that one bromination did occur on the 3-(2-hydroxyethyl) group to lead to 3-(2-bromoethyl) in the dibromide.  MeO  0  O  OMe  85  9 § -!  U/VJLJ  §  Pi  b 2.  a-, ft  5  2. a  |TrrrpTiT|TnT|m^^ 1 1 . 0  1 0 . 0  9 . 0  8 . 0  7 . 0  6 . 0  5 . 0  4 . 0  3 . 0  2 . 0  1.0  0 . 0  Fig. 2.3 ' H N M R Spectra (CDC1 , 400 MHz) of Compound 85 and 71 3  - 1 . 0  - 2 . 0  - 3 . 0  ft & OS  ft <>5  (0  73 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  Additionally, the characteristic signals of H-7 and H-7 on the fused benzene ring in 3  4  BPD, which always exhibit as two doublets at 7.8 and 7.4 ppm, were lost in the *H NMR spectra of the dibromide. Instead, a peak appeared at 7.79 ppm as a singlet (Fig. 2.3). Furthermore, the positions of the meso-protons exhibited pronounced shifts in the dibromide when compared with that of 71. It was speculated from these results that another bromination took place on the fused benzene ring. However, whether the bromination happened on C-7 or C-7 could not be decided only based on the present 3  4  results. The selective nuclear Overhauser effect (NOE) experiments were performed to elucidate the position of the bromination. NOE experiment is a NMR technique that allows the determination of connectivities through space rather than through bands. In NOE spectroscopy, irradiation of the peak for one proton generates a small change in the intensity of the signals for other protons that are in close proximity to the irradiated proton. This change in peak intensity can be reliably detected by the difference spectroscopy which is obtained by subtracting the irradiated spectrum from the original spectrum. Therefore, the positive NOE, which is shown by a difference peak, indicates that the proton is sterically close to the irradiated proton. The selective 1D-NOESY spectra for the unexpected dibromide are shown in Fig. 2.4. When the peak at 7.79 ppm was irradiated, only the positive NOE signal at 3.99 ppm was observed (A in Fig. 2.4), which is known as the signal of 7 -C02Me. 2  Therefore the peak at 8: 7.79 ppm is for H-7 , which is proximate to 7 -C0 Me. Thus the 3  2  2  bromination position is determined to be on C-7 , and the structure of the dibromide is 85. 4  74 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  B  D  Fig. 2.4 1D-N0ESY Spectra (CDC1 , 400 MHz) of Compound 85. A) upon irradiation at 7.79 ppm; B) upon irradiation at 5.06 ppm; C) upon irradiation at 9.78 ppm; D) upon irridation at 7.72 ppm; E) upon irradiation at 10.44 ppm; F) H N M R Spectrum of 85. 3  J  75 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  In addition, the peaks of meso-protons of compound 85 were also assigned as follows based on the results of the selective NOE. 1) Irradiated peak at 5.06 ppm, which is known for H-7 ; positive NOE signal at 8.87 1  ppm. So peak at 8.87 ppm is for H-5 (B in Fig. 2.4);  2) Irradiated peak at 9.78 ppm; positive NOE signal at 4.32 and 4.18 ppm, which are known for H-17 and H-13 respectively. So peak at 9.78 ppm is for H-15 (C in Fig. 2  2  2.4);  3) Irradiated peak at 9.72 ppm; positive NOE signal at 3.57 and 3.41 ppm, which are for two peripheral methyl groups. So peak at 9.72 ppm is for H-20 (D in Fig. 2.4); 4) From 1), 2) and 3) it was concluded that peak at 10.44 ppm is for H-10. This was confirmed by the NOE spectrum E in Fig. 2.4. When the peak at 10.44 ppm was irradiated; positive NOE signal at 3.52 ppm was observed, which is for one of the peripheral methyl groups. So peak at 10.44 ppm is for H-10. By comparing the chemical shifts of meso-protons of dibromide 85 with bromide 71 (Table  2.2),  it was found that the meso H-10 exhibited significant down-field shift to  10.4 ppm. This was might caused mainly by the steric compression deshielding effect of the bulky Br. The inductive effect probably also has an impact, but won't be the major  76 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  one because the Br and H-10 are 5-bond away. Additionally, the Br induced a pronounced up-field shift of meso H-5 to 8.87 ppm. The interpretation of this change required further investigations. The comparison for the chemical shifts ofraeso-protonsin compound 85 and other ring B-BPD derivatives are summarized in Table 2.2. It was observed that the transformation of the 3-vinyl group to the substituted-ethyl groups, such as in 68, 70, 71 and 62, does not cause significant shifts of me^o-protons as in 85.  Table 2.2 H NMR Spectra Data for meso-R of BPD Derivatives !  2.3.2  Compound  H-20  H-15  H-5  H-10  61  9.77  9.68  9.38  9.15  68  9.72  9.67  9.45  9.35  70  9.73  9.73  9.38  9.00  71  9.75  9.75  9.38  8.92  62f  9.70  9.70  9.37  8.99  85  9.72  9.78  8.87  10.44  Structure and Spectra Characterization  of Primary Ether  Derivatives All of the synthesized compounds were subjected to various spectral analyses, including UV-Vis absorption spectroscopy, H and C NMR spectroscopy, and mass j  13  spectrometry. The transformation of the 3-vinyl group of vinylchlorin 39 or 61 to the substituted ethyl functionalities (ethyl alcohol, ethyl bromide or primary ethyl ether) does not  77 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  significantly affect the overall electron conjugation of the chlorin macrocycle. The UVVis spectra of the 3-devinyl-3-(2-substitutedethyl) derivatives maintain the similar patterns to that of the vinylchlorins. However, the conjugation is decreased by transferring the vinyl to the saturated ethyl group. This is reflected by the 5—10 nm blue shift of the Q band in the absorption spectra of the corresponding derivatives. As shown in Fig. 2.5, methyl pyropheophorbide a 39 has Q band at 670nm, but the Q band of the diol derivative 63 appears at 665 nm and both ethyl bromide 66 and hexyl ether derivative 67f show Q band at 660 nm. The similar blue shifts of Q band were also observed in the UV-Vis spectra of ring B-BPD derivatives as shown in Fig. 2.6.  300  400  500  600  700  800  nm  Fig. 2.5 UV-Vis Spectra of Methyl Pyropheophorbide a Derivatives 39, 63, 66 and 67f  78 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  A  0  1  300  400  1  500  1  nm  1  600  700  800  Fig. 2.6 UV-Vis Spectra of ring B-BPD derivatives 61, 68, 71 and 62g  In the H NMR spectra of the pyropheophorbide a primary ether derivatives 67, H!  3 and H-3 appear at around 4.05 to 4.08 ppm (Fig. 2.7) instead of the characteristic 1  2  peaks of 3-vinyl in the starting material 39 at 7.98, 6.25 and 6.14 ppm. The protons of the ether group -OCH2- have the signal at -3.5 ppm. In ring B-BPD primary ether derivatives 62, H-3 and H-3 show peaks in the range between 4.1 to 4.2 ppm, while the signals for 1  2  ether group -OCH - also exhibit at -3.5 ppm (Fig. 2.8). The H NMR spectra of the ether ]  2  derivatives 67a and 62a are shown in Fig. 2.7 and Fig. 2.8 respectively.  79 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  Vo  ' ' $J ' T  Vo  '  \'.0  50  4.0  3.0  '  2.0  U)  00  -1.0  '  Fig. 2.7 *H N M R Spectrum (CDC1 , 300 MHz) of Compound 67a 3  (ppm)  Fig. 2.8 H N M R Spectrum (CDC1 , 300 MHz) of Compound 62a !  3  -2.0  80 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  Detailed structural assignments and spectral characterization of the primary ether derivatives and other intermediates are presented in the experimental section (Chapter Four). Selected spectra for some representative compounds are shown at the end of this chapter, which include the H NMR spectra of compounds 66, 67c, 60c, 67h, 68, 70, 62c, l  86 and the H- C HSQC spectrum of compound 60c. 1  13  The crystal structure of compound 67f, methyl 3-devinyl-3-(2-hexyloxyethyl) pyropheophorbide a, was determined by the X-ray crystallography (Fig. 2.9). The chlorin macrocycle remains flat in the ether derivative (Fig.  2.10).  It was interesting to find that  the hexyl chain showed evidence of disorder, the C-3 to C-6 within that group were modeled in two orientations, with relative populations of 0.57 and 0.43 for the major and minor fragments respectively. The major fragment was shown in Fig. 2.9 and Fig. 2.10. The crystal data and details of the structure determination for 67f are included in Chapter Four.  81 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  Fig. 2.9 ORTEP Drawing of 67f with Major Fragment (top view, H atoms have been omitted for clarity)  Fig. 2.10 ORTEP Drawing of 67f with Major Fragment (side view, H atoms have been omitted for clarity)  82 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  2.4 Preliminary PDT Cytotoxicity Assessment The PDT cytotoxicity assay of the synthesized primary ether derivatives was carried out at QLT Inc. as follows: 7600 human microvascular endothelial cells, in the presence of 200 pL Tumor Necrosis Factor-alpha (TNF , 80 U/mL, Upstate Biotech), were seeded per well into 96a  well Costar plates (Corning, Whitby, ON) for approximately 21 h. Cells were approximately 80 % confluent for experiments. Photosensitizer dilutions from 3200 n M to 25 n M were prepared in endothelial basal medium (EBM, Biowhitaker) and 100 uL media containing different amounts of drug were added to each well. The plates were protected from light, placed back in incubator for 60 min at 37 °C with 5 % CO2. Light was delivered using a white light source with an intensity of 36 mW/cm and a dose of 5 2  J/cm . After irradiation, the drug was removed and the wells were washed twice with 200 2  uL of complete media. 200 pL of complete media with T N F was added to each well and a  the cells were incubated for 24 h. After that, the cell viability was assessed using the MTS colorimetric assay. 30 uL of MTS (Sigma, Oakville, ON) solution was added to each well. Cells were incubated for 3 h in the dark at 37 °C with 5 % CO2. Color development was measured with an automated plate reader (Dynatech, Hamilton, V A ) using a 490 nm filter. The values of L D  5 0  (nM), the lethal dose required to kill 50 % of  cells, for the photosensitizers were determined accordingly. B P D M A (16) and HPPH (41), which exhibit the L D  5 0  of 73 n M and 330 n M  respectively, were used as references. The primary ether derivatives obtained in the work were found to exhibit higher L D  5 0  values than that of B P D M A but some of the  compounds have a value lower than or similar to that of HPPH (Table 2.3).  83 Chapter 2 Primary Ether Derivatives of Chlorin-like Photosensitizers  Table 2.3 PDT Cytotoxicity of Primary Ether Derivatives  Compound  60a  60b  60c  60d  67a  R  1  methyl  ethyl  1-propyl  1-butyl  methyl  R  2  H  H  H  H  methyl  215  188  185  313  332  LD (nM) 50  The lower the LD , the higher the cytotoxicity of the photosensitizer. The results in 50  Table 2.3 suggested that the primary ether derivatives exhibit PDT cytotoxicity not as potent as BPDMA, but some of them are comparable to that of HPPH. Compound 60c shows the highest cytotoxicity in the series, which is higher than HPPH. It is inferred from these preliminary results that the introduction of the primary ether functionality onto the chlorin macrocycles maintains the good PDT efficacy, even though no significant improvement was observed over existing photosensitizers. No correlations between these preliminary in vitro results and the structures of the compounds can be inferred at this time. Further analyses will be continued at QLT Inc.  Chapter 2 Primary Ether Derivatives of Chlorin-like  84 Photosensitizers  2.5 Summary The syntheses of non-diastereomeric primary ether derivatives of pyropheophorbide a, 60, and derivatives of ring B-BPD-l,3-diene dimethyl ester, 62, were accomplished. The preliminary PDT cytotoxicity assays showed that some of these derivatives exhibit in vitro PDT efficacy higher than HPPH. Efficient synthetic routes towards the target compounds were achieved. The transformation of the conjugated 3-vinyl group on chlorin macrocycles to 3-(2hydroxyethyl) group was investigated in detail. A new method for this transformation was developed and proved to be efficient and safe. Bromination of the 3-(2-hydroxyethyl) chlorins with different bromination reagents was studied. The unexpected product was isolated and characterized. A Hg(II) salt was employed to assist in the nucleophilic substitution of 3-(2-bromoethyl) chlorins and these exhibit high efficiency in the synthesis of the desired ether derivatives.  5 SF Si  I ft -S  b ft 5. Si  a-.  9 LJ I I I I I I I I I  10.0  i ' i  9.0  8.0  7.0  6.0  1  1  1  1  i i  5.0  LJ 3.0  4.0  2.0  i  i i i i i i i i ' ' i ' ' ' ' i i i  1.0  0.0  I I I I I I I I I I I I I I  -1.0  -2.0  (ppm)  Fig. 2.11 H N M R Spectrum (CDC1 , 400 MHz) of Compound 66 !  3  ft  •t 00  05  t^n  0-n-C H 3  7  88 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  Fig. 2.16 H N M R Spectrum (CDCI3, 300 MHz) of Compound 68 !  94 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  REFERENCES 1  Smith, K . M . ; Goff, D. A.; Simpson, D. J. J. Am. Chem. Soc. 1985,107, 4946.  2  Ma, L.-F. Ph. D Degree Thesis, the University of British Columbia, 1995, 58.  3  Sato, M . ; Fujimoto, I.; Sakai, T.; Aimoto, T.; Kimura, R.; Murata, T. Chem. Pharm. Bull. 1986, 34, 2428.  4  Smith, K . M . ; Bisset, G. F.; Bushell, M . J. / . Org. Chem. 1980, 45, 2218.  5  Senge, M . ; Struck, A.; Dornemann, D.; Scheer, H.; Senger, H. Z. Naturforsh 1988, 43c, 515.  6  Smith, K. M . ; Bisset, G. M . F.; Bushell, M . J. J. Am. Chem. Soc. 1985, 107, 4946.  7  Yagai, S.; Tamiaki, H. J. Chem. Soc, Perkin Trans. 1 2001, 3135.  8  Dupau, P.; Epple, R.; Thomas, A . A.; Fokin, V . V.; Sharpless, K . B . Adv. Synth. Catal. 2002, 3+4, 344.  9  Schroder, M . Chem. Rev. 1980, 80, 187.  10 Sparatore, F.; Mauzerall, D. J. Org. Chem. 1960, 25, 1073. 11 VanRheenen, V.; Kelly, R. C ; Cha. D. Y . Tetrahedron Lett. 1976, 23, 1973. 12 Kahl, S. B.; Schaeck, J. J.; Koo, M.-S. J. Org. Chem. 1997, 62, 1875. 13 Wai, J. S. M . ; Istvan, M . ; Svendsen, J. S. J. Am. Chem. Soc. 1989, 111, 1123. 14 Smith, K. M . ; Bisset, G. F.; Bushell, M . J. J. Org. Chem. 1980, 45, 2218. 15 Adam, K. R.; Berenbaum, M . C ; Bonnett, R.; Nizhnik, A. N . ; Salgado, A.; Valles, M . A. J. Chem. Soc. Perkin Trans. 1 1992, 1465. 16 Pandey, R. K . ; Issac, M . ; MacDonald, I.; Medforth, C. J.; Senge, M . O.; Dougherty, T. J.; Smith, K . M . J. Org. Chem. 1997, 62, 1463. 17 Chang, C. K ; Sotiriou, C. J. Org. Chem. 1985, 50, 4989.  95 Chapter 2 Primary Ether Derivatives of Chlorin Photosensitizers  18 Advanced Org. Chem.; March, J., 3 Ed., John Wiley & Sons: New York, 1985, rd  220. 19 Kenner, G. W.; McCombie, S. W.; Smith, K . M . J. Chem. Soc, Chem. Commun.  1972, 1347. 20 Kenner, G. W.; Quirke, J. M . E.; Smith, K . M . Tetrahedron 1976, 32, 2753. 21 Saraf, S. D. Can. J. Chem. 1969, 47, 2803. 22 Feuer, H . ; Hooz, J. The chemistry of the ether linkage; Patai, S. Ed., Interscience Publishers: New York, 1967, 445. 23 Porphyrins  and metalloporphyrins;  Smith K . M . Ed., Elsevier Scientific  Publishing Company: New York, 1975, 12. 24 Mckillop, A.; Ford, M . E. Tetrahedron 1974, 30, 2467. 25 a) Barluenga, J.; Alonso-Cires, L.; Campos, P. J.; Asensio, G. Synthesis 1983, 1, 53. b) Barluenga, J.; Alonso-Cires, L.; Asensio, G. Synthesis 1979,12, 962.  96 Chapter 3 Cross-Metathesis  Reactions of Vinylchlorins  and  Vinylporphyrins  Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  97 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  3.1 Olefin Metathesis and Cross-Metathesis The reactions that can reliably and efficiently form carbon-carbon bonds are the foundation of organic synthesis. Among the many types of transition-metal-catalyzed carbon-carbon bond forming reactions, olefin metathesis has now been widely considered as one of the most powerful synthetic tools owing to the wide range of transformations that are possible with commercially available and easily handled catalysts. Although double-bond scrambling reactions were initially reported in the mid 1950's, it was not until several years later that Calderon and co-workers recognized that 1  both ring-opening polymerization and the disproportionation of acyclic olefins were the same reactions. They introduced the term "olefin metathesis" in 1967, which we 2  currently understand as the metal-catalyzed redistribution of carbon-carbon double bonds. This transformation has a variety of applications (Scheme 25), including ring-opening metathesis polymerization (ROMP),  3  acyclic diene metathesis polymerization  (ADMET), ring-closing metathesis (RCM), ring-opening metathesis (ROM) and cross3  4  5  metathesis (CM). Through these reactions, olefin metathesis provides a route to 5  unsaturated molecules that are often challenging or impossible to prepare by other means.  98 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  O  ROMP —  / / * \ \  -  U  u  n  Scheme 25 Examples of Olefin Metathesis Reactions  3.1.1 The Development of Olefin Metathesis Catalysts The progress of olefin metathesis was outlined in Fig. 3.1 according to the chronology of the development of metathesis catalysts. It was only through major 6  advances in catalyst design that the tremendously expanded applications have become possible. Until the early 1980's, all olefin metathesis were accomplished with ill-defined, multicomponent homogeneous and heterogeneous catalyst systems. Some of the classic systems include WCl /Bu Sn, WOCl /EtAlCl , Mo0 /Si0 and 6  4  4  2  3  2  RU2O7/AI2O3.  7  The  application of these catalysts was limited because very little of the active species was formed in the catalyst mixture, making the reactions difficult to initiate and control. In addition, the required strong Lewis acid along the harsh conditions made the catalyst systems incompatible with most functional groups.  99 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins The desire to improve the catalyst performance motivated the extensive work to better understand the mechanism of the olefin metathesis. 4 1950  discovery of olefin metathesis  4  1960  RuCI (hydrate) perform ROMP 3  Chauvin proposed metal alkylidene— based mechanism  4 1970  evidence for Chauvin's mechanism found  4  1980  4  1990  single-component catalyst developed  synthesis of Mo-alkylidene catalyst 87synthesis of Ru-alkylidene catalyst 88discovery of (PCy ) CI Ru=CHPh 91 3  2  2  mechanism of 91 investigate  4 2000  N-heterocyclic carbene catalyst developed phosphine-free catalyst  Fig. 3.1 Time Line of Milestones in the Development of Olefin Metathesis Catalysts  Among the different mechanisms that were proposed, the one that was developed by Chauvin in 1971 (Scheme 26) was finally found to be most consistent with the 8  100 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  experimental evidence. It currently remains the generally accepted mechanism. Chauvin 9  proposed that olefin metathesis involved the interconversion of an olefin and a metal alkylidene. This process occurred via a metallacyclobutane intermediate by alternating [2+2] cycloadditions and cycloreversions.  R  Scheme 26 Olefin Metathesis Mechanism Proposed by Chauvin  This mechanism provided both a design rationale to a specific catalyst and a way to understand its activity. It also influenced the work on further catalyst development. Efforts to synthesize alkylidene and metallacyclobutane complexes led to the discovery of the first single-component homogeneous catalysts during the late 1970's and early 1980's.  These  new catalysts  included (CO) W=CPh , 5  11  titanocyclobutanes,  2  19  tris(aryloxide) tantalacyclobutane,  10  bis(cyclopentadienyl) •  and various dihaloalkoxide-  alkylidene complexes of tungsten. These well-defined catalysts exhibit much better 13  initiation behavior and higher activity under milder conditions than the earlier ill-defined catalysts. The molybdenum and tungsten alkylidenes of the general formula (NAr)(OR') M=CHR were the first of these catalysts to be widely used, particularly the 2  molybdenum complex 87, where Ar = 2,6-zPr -C H , R = CMe Ph, and R'=C(Me)(CF ) , 2  which is named "Schrock's catalyst".  14  6  3  2  3 2  101 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  i-Pr'  i-Pr  (CF ) MeCO 3  2  Me  (CF ) MeCO^ 3  2  Me  87  The high activity is the most impressive feature of 87. It is able to react with terminal and internal olefins, to catalyze ROMP of low-strain monomers and to ringclose sterically demanding substrates. However, the high oxophilicity of the metal center is the limitation of molybdenum and other early transition metals based catalysts. This feature renders them extremely sensitive to oxygen and moisture and incompatible with most functional groups. Therefore, continued research was spurred by the prospect of solving these problems. The advent of single-component catalysts enabled researchers to study structureactivity relationships in more detail. The trends of reaction preference of different metal complexes with different functional groups were investigated. These catalysts were observed to react more selectively with olefins as the metals were varied from left to right and from bottom to top on the periodic table. It is shown clearly in 15  T a b l e 3.1  that  ruthenium reacts preferentially with carbon-carbon double bonds over most other functional groups. This preference makes ruthenium unusually stable toward alcohols, amides, carboxylic acids, aldehydes and water. Because of this fact, the development of catalysts with better functional group tolerance focused on ruthenium and the later transition metal elements.  102 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  Table 3.1 Functional Group Tolerance of Transition Metal Olefin Metathesis Catalysts Titanium  Tungsten  Molybdenum  Ruthenium  Acids  Acids  Acids  Olefins  Alcohols, water  Alcohols, water  Alcohols, water  Acids  Aldehydes  Aldehydes  Aldehydes  Alcohols, water  Ketones  Ketones  Olefins  Aldehydes  Esters, Amides  Olefins  Ketones  Ketones  Olefins  Esters, Amides  Esters, Amides  Esters, Amides  ' Increasing Reactivity  A breakthrough in the development of ruthenium metathesis catalyst was the synthesis of catalyst 88 in the early 1990's by Grubbs and coworkers, which is a welldefined ruthenium alkylidene complex (Scheme 27). The compound 3,3-diphenyl 16  cyclopropene was used as a carbene precursor to react with  RuCl2(PPh3)3  to give this  complex. Complex 88 exhibits good initiation behaviour and functional group tolerance. However, its activity is limited to catalyze ROMP of high-strain monomers and it is not active towards acyclic olefins. By modifying the ligand environment systematically, PCy (Cy = cyclohexyl) was utilized and led to a fortuitous discovery — the larger and more basic the phosphine ligand, the higher the metathesis activity. The  PCy3  derivative 89  catalyzes the ROMP of lower strained monomers, such as cyclopentene, and it is also the first ruthenium alkylidene complex that is active towards acyclic olefins.  3  103 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins  90  and  Vinvlporphvrins  91  92  Scheme 27 Synthesis of the Ruthenium Metathesis Active Complexes  The reaction of RuCl2(PPh )3 with alkyl- and aryl-diazoalkanes provided an 3  alternative method to obtain the substituted alkylidenes in good yields. The availability of these complexes, which had been limited by the difficulty of synthesizing the 3,3disubstituted cyclopropene, made it possible to screen for the catalyst with the best activity. The benzylidene complex 91 was found to be most reactive in the series. 91 17  showed the highest initiation rate (undergoing metathesis with ethylene within minutes at room temperature to form methylidene 92 quantitatively) and the best ROMP behavior (low catalyst loading required even for low-strain monomers and a narrow molecular weight distribution of product). This catalyst, known as the "Grubbs' catalyst", became commercially available in 1996 and has been widely applied in both polymer and small molecule syntheses. Although catalyst 91 has been demonstrated to be much more active and functional group tolerant compared with the early stage catalysts, the reaction scope of this complex is still limited. By investigating the mechanism of the catalytic process extensively, a number of critical factors that contribute to its activity were identified. The key insight  104 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  was that the catalyst  Cl2(PCy3)2Ru=CHPh  (91) formed a highly active mono-phosphine  intermediate 94 during the catalytic cycle (Scheme 28). Consequently, this intermediate 18  became a starting point for the development of improved catalysts.  PCy  3  Cl/  cr I  PCy Ph  +  C  " = PCy  PCy  + PCy  PCy Cl  3  3  3  Cl  Ru=\^  /=/=  3  •Cl—RuVv~  C H  ?~  C H F  E  product  Ph  Ph  3  R  93  3  91  PCy  3  | C l Ph - p y C l — R u ^ _  94  Scheme 28 Proposed Mechanism for Catalyst Cl (PCy ) Ru=CHPh 91 2  3 2  After exploring a variety of ligands, it was found that the mesityl-substituted Nheterocyclic carbene worked well. The complex 95, containing an unsaturated backbone, was discovered almost simultaneously by different research groups. Soon thereafter, the 19  catalyst 96 containing a ^/-heterocyclic carbene with a saturated backbone, 1,3-dimesityl4,5-dihydroimidazol-2-ylidene, was reported to be even more active. This catalyst was 20  found to catalyze efficiently reactions of previously metathesis-inactive substrates when treated with 91, including a,(3-unsaturated ketone, ester, amide, 21  21  22  trisubstituted  olefins and vinyl phosphonates. The 2-isopropoxystyrene derivatization of 96 gave the 23  24  stable complex 97 with the oxygen chelated to the ruthenium center. This phosphmefree catalyst also proved to be active in metathesis reactions and even exhibits higher activity than complex 96 toward some challenging substrates, such as acrylonitrile,  26  fluorinated molecules and vinyl sulfone. 27  6  105 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  95  96  97  This "second generation" of catalysts is most significant because they combine the best features of early and late metal centers into a single species. They display performance that was previously possible only with the most active early transition metal systems (such as molybdenum catalyst 87) while retaining the impressive functional group compatibility.  3.1.2 Cross-Metathesis Cross-metathesis (CM) can be formally described as the intermolecular mutual exchange of alkylidene fragments between two olefins promoted by metal-carbene complexes. The simplified CM between two terminal olefins is presented in Scheme 29. In fact, the CM reactions are now available between terminal, internal, di- and even trisubstituted olefins. The reaction proceeds between catalyst and olefin CFI^CHR to produce transition 1  metal carbene A. The [2+2] cycloaddition occurs between A and olefin CH2=CHR to give a metallacyclobutane B. The latter undergoes a subsequent cycloreversion to produce a CM product R CH=CHR and a new metal carbene C . Through reaction 1  2  between C and CH =CHR , metal carbene A is regenerated, which completes the 1  2  catalytic cycle.  106 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  Catalyst 91 or 96  Scheme 29 Mechanism for CM of Two Terminal Olefins  As an acyclic carbon-carbon bond forming method, cross-metathesis has advantages that are typical of modern olefin metathesis: 1) The process is catalytic. 2) High yields can be obtained under mild conditions with relatively short reaction time. 3) A wide variety of functional groups are tolerated. 4) The reaction is reversible, and ethylene is usually the only by-product, which is ideal in industrial applications. 5) The olefinic products are suitable for further structural modification, e.g. hydrogenation, halogenation, cycloaddition. 6) High levels of chemo-, regio-, and stereoselectivity can be obtained under appropriate circumstances. Until recently, CM has been relatively neglected and underutilized despite its potential due to several reasons: 1) Low product selectivity, i.e. the competition between  107 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  heterodimerization (cross-metathesis) and homodimerization (self-metathesis). 2) Low stereoselectivity (E and Z) in the CM product. 3) No enthalpic driving force, such as ring strain release in ROMP. 4) No entropic driving force advantage, such as for RCM. The last two disadvantages have been compensated for to a great extent with the discovery of catalysts with higher activity, such as 96 and 97. The selectivity for both reaction and stereochemistry continues to remain the biggest concern for CM.  3.1.3 A General Model for Selectivity in C M One of the critical goals of continuing efforts in CM is to achieve high yields of the desired cross-metathesis products while minimizing self-metathesis. Lack of prediction of product selectivity was the major obstacle that limited the application of CM as a powerful synthetic technique. By investigating the selectivity trendsfromCM reactions with a variety of olefins substrates, Grubbs and co-workers developed a general empirical model that can be used for the prediction of product selectivity in CM by using different catalysts. It has been discovered that by properly matching different types of olefins 28  with the appropriate choice of metathesis catalyst, CM with high selectivity can be achieved. This model is based on the categorization of olefins by their relative abilities to undergo homodimerization, i.e. self-metathesis,  and the susceptibility of their  homodimers towards the secondary metathesis reactions (Scheme 30).  108 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  +  homodimerization +  +  R  1  secondary metathesis  Scheme 30 Homodimerization and Secondary CM  As a general ranking of olefin reactivity in CM, the olefins can be classified as four distinct types (Scheme 31). Type I olefins are categorized as those able to undergo a 28  rapid homodimerization and whose homodimers can participate in CM as well as their terminal olefin counterparts. Type II olefins are those that homodimerize slowly, and unlike Type I olefins, their homodimers can only be sparingly consumed in subsequent metathesis reactions. Type III olefins are essentially unable to be homodimerized by the catalyst but are still able to undergo CM with type I and type II olefins. Type IV olefins are not able to participate in CM with a particular catalyst but do not inhibit catalyst activity toward other olefins. Outside these categories are olefins that deactivate the catalysts and will not be included in the discussion. In general, a reactivity gradient exists from the most active type (Type I olefin) to the least active type (Type TV). The sterically unhindered, electron-rich olefins categorized as Type I and increasingly sterically hindered and/or electron-deficient olefins falling into Types II through TV.  109 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  Type I: Rapid homodimerization, homodimers consumable Type II: Slow homodimerization, homodimers sparingly consumable Type III: No homodimerization Type TV: Olefins inert to CM, but do not deactive catalyst Reaction between two olefins of Type I: Statistical CM  A  Reaction between two olefins of same type (non-type I): Non-selective CM Reaction between olefins of two different types: Selective CM  o  Scheme 31 Olefin Categorization and Rules for CM Selectivity  The author summarized the categories of some reported olefins with different 28  metathesis catalysts 87, 91 and 96 as shown in Table 3.2. It is important to note that the category of olefin is closely related to the choice of catalyst, that is, by employing a catalyst with differing activity, the same olefin can be ranked in a different activity type. For example, the un-substituted styrene belongs to Type II with catalyst 91, while it is a Type I olefin with catalyst 96 (Table 3.2).  110 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  Table 3.2 Olefin Categories for Selective Cross-Metathesis  Olefin type  ci^T=/  ph  R  ,..Ru—  cr*" |  PCy  Type I (fast homodimerization) Type II (slow homodimerizaiton) Type III  PCy  P  h  i-Pr 3  91 3  v i n y l epoxides, 2° allylic alcohols, perfluorinated alkane olefins  (no  1,1-disubstituted olefins, nonb u l k y trisubstituted olefins, v i n y l phosphonates, phenyl  homodimerization)  v i n y l sulfones, 4 ° allylic carbons (all alkyl substits.), protected 3° allylic alcohol  Type IV v i n y l nitro olefins  (inert)  i-Pr  (CF ) MeCO,„..l| V^ 3  2  o ;  (CF ) MeC0^  96  styrenes (large o-substits.), acrylates, acrylamides, a c r y l i c acid, acrolein, v i n y l ketone, unprotected 3° allylic a l c o h o l ,  J  cr*" |  PCy  terminal olefins, 1° allylic alcohols, esters, allylboronate esters, a l l y l halides, styrene (no large o-substits.), a l l y l phoshonate, a l l y l silanes, a l l y l phosphine oxides, allyl sulfides, protected a l l y l amines  A  3  C l ?OR u= /  3  2  87 terminal olefins, a l l y l silanes, 1° a l l y l i c alcohols, ethers, esters, a l l y l boronate esters, a l l y l halide  terminal olefins, a l l y l silanes  styrene, 2° a l l y l i c alcohols, styrene, v i n y l dioxolanes, v i n y l allyl stannanes boron ates  3° a l l y l i c amines, v i n y l siloxanes acrylonitrile  1,1 -disubstituted olefins, disubstituted a,(3-unsaturated carbonyls, 4 ° a l l y l i c carboncontaining olefins, perfluorinated alkane olefin, 3° a l l y l i c amines (protected)  M e  Me  1,1 -disubstituted olefins  As illustrated in Scheme 31, selective CM can be achieved by combining olefins from various types and employing a catalyst with the appropriate activity. Examples will be provided as follows for CM reactions of different types of olefins.  3.1.3.1 Non-selective C M  When two Type I olefins are used in a CM reaction, the rates of homodimerizations are high and the reactivity of these homodimers and cross products towards secondary  Ill Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  metathesis are also high. In these reactions, the desired cross product will be equilibrated with the various homodimers through secondary metathesis. This will result in a statistical product mixture. More generally, when two olefins of the same type are combined, non-selective product mixtures are usually attained. For example, alkyl acrylate and vinyl ketone (both are Type II) reacting in the presence of catalyst 96 gave non-selective CM products 98, albeit the yield was low due to the lower overall olefin reactivity (Scheme 32).  29  O .R  o  O  1  O  5 mol% 96 R  R^ 1 equiv.  R*  1  O  98 R =Me, R =Me, isolated yield: 41 %  2 equiv.  1  2  R =C(CH ) , R =Et, isolated yield: 41 % 1  2  3  3  Scheme 32 CM between Type II Olefins  3.1.3.2 Selective C M By using two different types of olefins, whose rates of dimerization are significantly different and/or slower than CM product formation, selective CM can be achieved. The first approach involves the reaction of a Type I olefin with less reactive Type II or Type III olefins. In this reaction, although Type I olefin may initially homodimerize, the homodimer readily undergoes secondary metathesis with Type II/III olefin to give the desired cross product. The reactions between olefins with fully substituted allylic carbons (Type III) and terminal olefins (Type I), providing high yields and exclusive /f-selectivity, are good illustrations for this profile. It was found during early studies that quaternary 28  30 *  allylic olefin substrates are inert towards metathesis when catalyst 91 was employed, i.e.  112 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  Type IV substrates with catalyst 91. However, these species become Type III substrates when the more reactive catalyst 96 is employed and are then useful in selective C M reactions as illustrated in Table 3.3.  28  Table 3.3 Selective C M between Quaternary Allylic Olefins and Terminal Olefins Using Catalyst 96 Entry  4° allylic olefin (Type III)  CM partner  OAc 2  *OBz  3  OAc /—\ C- . 0  4° allylic olefin: partner  Product"  Isolated Yield %  2 :1 excess 1 :1  2 :1  a Only E isomers observed by 'H NMR.  The second approach to selective C M , shown in Table 3.4, is the reaction between Type II and Type III olefins which both dimerize at much slower rates than the formation of productive C M products. Given the low reactivity of some Type III olefins, reduced C M yields may be obtained. Also, since the undesired dimers of Type II olefins are essentially inactive for secondary metathesis, stoichiometric excess of the less reactive Type III olefin may be required to produce high yields of cross products, such as in the reaction that was carried out in neat 3,3-dimethyl-l-butene to drive the C M with various Type II olefins (Table 3.4)  28  113 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  Table 3.4 Selective CM between Type II and Type III Olefins Entry  Type II  Type III (neat)  0  i  Product"  Yieki^/  73 73  o-c^>  75  a Only E isomers observed by 'H NMR.  3.1.3.3 Bridge Type Olefins As previously mentioned, styrenes are typical examples that bridge the type categories previously outlined. With catalyst 91 employed, the dimerization of styrene to stilbene was reported to be slow. Styrene reacts as a Type II olefin to give good selectivity in CM with Type I olefins. However, with 2.5 mol% of catalyst. 96, the dimerization of styrene to £-stilbene was achieved in 94 % yield. Moreover, £-stilbene can undergo efficient CM with one equivalent of allylic acetate in the presence of catalyst 96 to produce a statistical ratio of CM products (entry 2, Table 3.5). Consequently, the CM reaction of styrene with a terminal olefin employing catalyst 96 (entry 1) produces a statistical product distribution and is different from the results using catalyst 91. These results clearly indicate that styrene is a Type I olefin when using the more active catalyst 96. While matching the olefin with an appropriate catalyst changes the category of the olefin and consequently may allow for selective CM, alterations in the styrenes structures also provide chances for selective CM since the behavior of styrenes strongly depends on  114 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  the substitution patterns. Large orr/zo-substitutents often change styrene from a Type I olefin to a Type II olefin with catalyst 96, while a jt?ara-substituents normally have no effect. For example, the use of 2-bromostyrene as the C M partner leads to selective formation of the C M product with Type I olefins. B y simply using 3 equivalents of the 2bromostyrene, quantitative conversion can be achieved with hexenyl acetate (entry 3 in Table 3.5). In this case, both the steric bulk of the bromine atom and its electronwithdrawing character contribute to make the 2-bromostyrene a Type II olefin. 4nitrostyrene acts as the Type I olefin in the reaction with acrylate (Type II) to give the selective product in high yield (entry 4). The nitro substituent is far from the vinyl group and has no steric effect on the C M reactivity. The electron-withdrawing property of the nitro group was also not observed to change the reactivity type.  Table 3.5 Cross-Metathesis of Substituted-Styrenes Catalyzed by 96 Entry  Styrenes  Cross-partner  1 AcO^  2  ^OAc  .Br  3  4  CO ^ O C H ,  Styrene: partner  Product  Isolated Yield %  1: 1 4: 1  47 71  1 : 1.2  51  1: 1 1:3  80 98  ^ ^ ^ ^ ^ ^ ^ ^ O A c  89  1 : 1.5 o  3.1.3.4 Multi-Component C M As illustrated above, the general model provides a starting point for the design of potentially selective C M reactions. Furthermore, it also can be applied to the  115 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  development of new reactions. Grubbs and co-workers investigated the multicomponent selective C M process based on the concepts of the general model.  Although the three-  component reaction has always been theoretically possible, the large mixture of compounds that would form via nonselective pathway has made this a challenging method to develop. However, with the current model it can be predicted that by using two olefins that do not cross-metathesis with each other or do so only very slowly, then a third diene-containing olefin can be functionalized at both olefmic sites to provide an unsymmetrical product. In fact, this strategy has proven to furnish the multifunctionalized olefin products as shown in Scheme 33.  T  y 1 eq. p e  89% isolated yield, All E isomer  11  R= CH , O C H 3  2  5  Scheme 33 Three-Component Selective C M  1,5-Hexadiene (Type I) was treated with vinyl ketone (Type II) and isobutylene (Type III) simultaneously to give the difunctionalized olefin. Because the Type II and Type III olefins react at a much slower rate with each other than their respective reactions with the Type I olefin, olefins of three different types may be converted predominantly into one product as a single stereoisomer. A variety of asymmetrically substituted dienes have been prepared by applying this method. Theoretically, any combination of a Type 28  I diene with a Type II and a Type III olefin would provide a three-component product.  116 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  The success of this process demonstrated the utility of the general model to new reaction development and would add a new level of complexity to olefin metathesis reactions.  3.1.4 Intermolecular Enyne metathesis Intermolecular enyne metatheses are unique and interesting transformations involving the reaction of an alkene and an alkyne (Scheme  34).  32  The products from  these processes are synthetically useful butadiene derivatives, which are appropriate substrates for structure elaboration by Diels-Alder reactions and other cycloaddition procedure. +  Scheme 34  catalyst Intermolecular Enyne Metathesis  The breakthrough of the selective intermolecular enyne cross metathesis was achieved by Blechert's group in 1997. Before that, the intermolecular enyne metathesis was underutilized as it was regarded as less favored than the intramolecular enyne metathesis which is entropic favored. In Blechert's study, 2-3 equivalents of alkene reacted with a variety of propargylic derivatives in the presence of catalyst 91 to give enyne metathesis products 1,3-dienes as mixtures of E/Z isomers (Scheme 35). It was interesting to note that the formation of alkene dimers occurred only to a limited extent of substrates under the reaction conditions. Blechert's study at that time also pointed out that internal alkynes and internal alkenes failed to undergo CM reactions. However, recent studies employing more active catalyst 96 have indicated that both internal alkynes and 34  alkenes can participate in enyne cross metathesis. 35  117 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  R—s  + ^^SiMe  3  5-7 mol% catalyst 91 • CH CI , rt, 18-24 h 2  [I ... n A ^ v S i M e 0  3  2  R= C H O T H P (81 %), C H O A C (90 % ) , C H ( C H ) O A c (63 %), C H O C 0 M e (89 %). 2  2  3  2  2  Scheme 35 Selective Intermolecular Enyne Metathesis  Recently, Pandey et al. reported the enyne metathesis of the purpurin-7Vpropargylimide. Via the enyne metathesis with (9-allylgalactopyranoside, the pgalactose-conjugated purpurinimide derivatives 58 and 59 were obtained which are useful for the development of galectin-specific photosensitizers for PDT (Scheme 14, Chapter One). The corresponding chlorin-diene system obtained from the enyne metathesis is 36  also a good building block for Diels-Alder reaction. The chlorin-fullerene dyad 100 was produced by enyne metathesis followed with the Diels-Alder reaction with C6o (Scheme  i: catalyst 91, CH =CH , CH CI , 48h; Ii: C , toluene, reflux, 2h 2  2  2  2  60  Scheme 36 Enyne Reaction of Purpurinimide  3.1.5 The Application of C M With the development of more active catalysts, cross-metathesis has become a flexible and powerful methodology in organic synthesis. It is finding increasingly wide  118 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins  application in the synthesis of biologically important molecules and natural products. 6  38  In addition, the discovery of enantioselective cross-metathesis , cross-metathesis in 39  water , and the metathesis in solid-phase organic synthesis has also further widened the 40  41  scope of this versatile reaction. Meanwhile, CM has been applied widely in industry. In the well-known Shell Higher Olefin Process (SHOP), a large-scale industrial process 42  for producing linear higher olefinsfromethene, CM is the key step in the third stage for producing internal Cn-Cu linear alkenes. In the U.S. and England, Shell Chemicals can produce up to 1.2 million tons of linear higher olefins per year through the SHOP units  43  Despite its steadily gaining prominence, CM has seldom been applied to chlorin and porphyrin substrates. With the purpose of establishing generalizable strategies for modifying the amphiphilicity of tetrapyrrolic macrocycles, CM is applied to vinvlchlorins and vinylporphyrins substrates in this work.  3.2 Results and Discussions CM of the conjugated vinyl group of vinylchlorins and vinylporphyrins with functionally diverse olefins are studied in this section of the thesis. We expected that, as a terminal aromatic olefin, the conjugated vinyl group might undergo CM as do other terminal aromatic olefins. However, the large size of the chlorin or porphyrinringswas 44  anticipated to affect the reactivity. Furthermore, resonance effects might also dramatically reduce the reactivity of these systems.  119 Chanter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  3.2.1 Reaction Conditions Our studies started with the Grubbs' catalyst 91, because of its ready availability. A mixture of the ring B-BPD-l,3-diene dimethyl ester (61) and 1-hexene was refluxed in dry THF in the presence of 10 mol% of the catalyst. However, no reaction occurred even after refluxing was continued overnight and the majority of the starting material was recovered (Scheme 37).  No reaction  Scheme 37 Reaction of Ring B-BPD 61 and 1-Hexene by Using Catalyst 91  This result may be explained in two ways: the catalyst is not active enough for substrate 61; or the catalyst is altered due to the chelation between the ruthenium complex and the chlorin macrocycle. Since the starting material could be recovered from the reaction mixture in around 80 % yield and no other BPD derivatives were observed on the analytical TLC, the possibility for chelation was unlikely. Therefore, a possible reason is that the vinyl group in this macrocycle is a fairly poor substrate in CM reaction that catalyst 91 is not active enough for it. We carried out further experiments to confirm this postulate (Scheme 38 and Scheme 39).  120 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  102  Scheme 38  In  Ring-Closing Metathesis of 101 Catalyzed by 91  Scheme 38,ring-closingmetathesis  (RCM) occurred smoothly on the terminal  vinyl groups that are far away from the BPD nucleus in the presence of catalyst 91. A 23membered ring product was obtained as the final product. It was surprising that such a large ring was formed via RCM rather than the intermolecular CM product. This has been noted before and is an advantage for RCM that such system tends to form the medium to the large sized ring. Under similar conditions, the CM product was obtained by the 4  reaction between pyropheophorbide a derivative 103 and allyltrimethylsilane in 65 %  121 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins yield. For both cases, metathesis occurred readily on the ester-bound vinyl groups with catalyst 91, while the conjugated vinyl group remained unchanged. These results indicate that the chlorin macrocycle does not apparently deactivate the catalyst 91. The reason that C M does not occur in Scheme 37 is that the catalyst is not active enough to overcome the low reactivity of the conjugated vinyl group towards metathesis. Based on these conclusions, attention was focused on the highly-active 7Vheterocyclic carbene catalyst 96 in the next stage of our investigations. As discussed before, this catalyst is so active that it can efficiently catalyze reactions of some previously metathesis-inactive substrates. Ring B-BPD-l,3-diene dimethyl ester (61) and the simple olefin 1-hexene were chosen as substrates for the initial investigations because of their availability (Table 3.6). It was very exciting to observe that by employing catalyst 96, a C M product 105 was obtained between 61 and 5 equivalents of 1-hexene upon refluxing in THF for 1 h (entry 1). The product 105 was observed as a slightly less polar spot on analytical T L C as compared to the starting material. H N M R spectrum confirmed that C M did occur on the ]  conjugated vinyl group as hoped. Further studies showed that the concentration of 61 in the reaction mixture has to be high enough to ensure the reaction proceeding. It was also determined that i f the amount of 1-hexene was increased to 20 equivalents, the C M product 105 was obtained in quantitative yield based on ' H N M R analysis (entry 2). Another simple olefin, 1-octene, was found to afford the same result as 1-hexene, with a 100 % conversion to the expected C M product 106 based on the *H N M R analysis (entry 3).  122 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins Table 3.6 C M of Ring B-BPD 61 Under Different Conditions .MeO,C, ? * 0  R MeO,Q  M e  1-alkene  MeO  O O  61 : 1alkene  M e  catalyst 96  OMe  O O  MeO  OMe  105, R = n-C H ; 106, R = n - C H  61  Entry  ?°2  4  9  6  Catalyst 96  1-alkene  Product  Yield  13  E:Z  a  C  1  1: 5  0.25 eq.  1-hexene  105  15 %  2  1 : 20  0.25 eq.  1-hexene  105  100%  65 : 1  3  1 : 20  0.25 eq.  1-octene  106  100 %  50 : 1  4  1 : 20  0.15 eq.  1-hexene  105  70%  NoZ  b  —  " Yields were calculated based on H NMR spectra of crude the products. Isolation yield. E:Z ratio was 0  determined based on 'H NMR spectra.  After a number of trials, the optimized reaction conditions were determined to be as follows: a solution of 0.25 equivalent of catalyst 96 (which was weighed in a Schlenk tube) in 1 mL freshly distilled THF was added via a syringe to the flask containing the vinylchlorin (0.04 mmol) and 20 equivalents of C M partner olefin. The mixture was refluxed under Argon in the dark for 1 h. After removing the solvent, the crude residue was purified by silica gel column chromatography or preparative T L C to give the C M product. Despite the relative stability of the catalyst 96 in the presence of moisture and air,  20  in our hands the best results were obtained when the catalyst was stored and  weighed in the glove box. The relatively high loading of the catalyst was required to ensure a high yield because of the low C M reactivity of the conjugated vinyl group. The yield dropped from 100 % to 70 % (based on *H N M R integration ratio) when 0.15  123  Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  equivalent of the catalyst was used instead of 0.25 equivalent (entry 2 and 4, Table 3.6). It was also found that extending the reaction time did not improve the yields. This observation differs from other reports where cross-metathesis products continued to be produced after 8 h. This procedure served as the general procedure for the studies that 21  follow.  3.2.2 Reactivity Differences of Tetrapyrrolic Macrocycle Substrates With these promising results in hand, CM of the conjugated vinyl group of other tetrapyrrolic macrocycle systems were studied. The studies were extended to methyl pyropheophorbide a (39) first. It was found that 39 reacted with both 1-hexene and 1-octene to afford the CM products 107 and 108 in 100 % conversions based on 'H NMR analysis (Table 3.7). Therefore, 39 exhibits similar reactivity towards CM as 61 does.  Table 3.7 CM of Methyl Pyropheophorbide a with 1-Alkene R  107, R = n-C H , 108, R = n - C H  M  4  9  6  13  39  Entry  1-alkene  A : 1-alkene  Yield"  Product  1  1-hexene  1 : 20  100%  107  NoZ  2  1 -octene  1 : 20  100 %  108  15 : 1  E:Z  b  " Yields were calculated based on 'H NMR spectra of the crude products. b* c-7 E:Z ratio wasAdetermined based ITT  on 'H NMR spectra.  ,n  TT-,  124 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and  Vinylporphyrins  Interestingly, further studies suggested that protoporphyrin IX dimethyl ester (11), a porphyrin substrate, exhibited different reactivity towards cross-metathesis. Under the general conditions, the reaction between protoporphyrin IX dimethyl ester (11) and 1-octene afforded the CM product 109 in only 58 % yield from 'H NMR analysis (entry 1, Table 3.8). This low yield, however, could be compensated for by using the Zn(II) complex of protoporphyrin IX. Under the same condition, Zn(II) protoporphyrin IX dimethyl ester (110) reacted with 1-octene to give 100 % conversion to the CM product 111 according to ' i l NMR analysis (entry 2, Table 3.8). Table 3.8 CM of Protoporphyrin IX Derivatives with 1-Octene n-C H 6  11, M=2H; 110, M=Zn  13  109, M=2H; 111, M=Zn  Entry  A  A:B  Yield "  Product  1  11  1 :40  58%  109  40 : 1  2  110  1 : 40  100 %  111  50 : 1  E:Z  b  " Yields were calculated based on 'H NMR spectra of the crude products. * E:Z ratio was determined based on *H NMR spectra.  Two conclusions can be made from these results. First, Zn-complexes are more active towards CM than the correspondingfreebases. Second, protoporphyrin IX (11) is less active thanringB-BPD (61). More generally, the vinylporphyrin is less reactive than vinylchlorin to cross-metathesis. This is demonstrated from the reactions of 11 and 61  125 Chanter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinvlporphvrins with 1-octene (entry 3 in Table 3.6 versus entry 1 in Table 3.8), 58 % yield was observed for 11 while 61 afforded 100 % conversion. The first conclusion can be rationalized by the electron density change on the tetrapyrrolic macrocycle due to Zn(II) metallation. When Zn(II) inserted, the macrocycle can be regarded as a dianion ionically bound to the metal ion.  45  Therefore, the ring is  more electron-rich than the corresponding free-base and the electron-density on the conjugated vinyl group increases as well. Since the electron-rich substrates are more favorable towards metathesis, the Zn(II) complexes exhibit higher reactivity. 46  The reactivity difference between chlorins and porphyrins is also considered as the result of electronic effects. The reactivity difference of the vinyl group in vinylporphyrin and vinylchlorin has been reported in the Vilsmeier formylation reaction of Fe(III) vinylporphyrin and Fe(III) vinychlorins. The formylation of the vinyl group of Fe(III) protoporphyrin IX with D M F / P O C l required 1 h to completion, while only 90 sec for the 3  Fe(III) chlorin e .  A1  6  It was indicated that the vinyl group in vinylchlorin is more reactive  towards electrophile than that in vinylporphyrin, which is the favorable property that makes it more reactive in metathesis reaction.  3.2.3 Cross-Metathsis with a Variety of Olefin Partners Having found out that the Zn-complexes are more reactive towards C M than the corresponding free-bases, the Zn-complexes were used for further investigations in our research. In the next stage, C M of the Zn(II) ring B-BPD- 1,3-diene dimethyl ester (112) with a variety of olefins were studied. For most reactions, the final C M products were  126 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and  Vinylporphyrins  obtained as thefreebases after the Zn(II) was removed by TFA treatment. Some of these results are summarized in Table 3.9. Besides the simple alkenes discussed above, olefins with other functional groups were introduced, such as 6-bromo- 1-hexene. CM between 6-bromo- 1-hexene with the Zn(II) complex 112 afforded 100 % conversion to the CM product 113 (entry 1).  Table 3.9 CM of 108 with Different Terminal Olefin Partners Me0 C,  R  C0 Me  C0 Me 2  2  2  25 mol% catalyst 96  B MeO'-O  6- ~  0  M  MeO^O  e  112  0  " - °  M  e  113, 114, 115, 116, 117, 119, M=2H; 118, 120, M=Zn  Entry  Yield  Product  B  a  E:Z'  1  113  100 %  NoZ  2  114  68 %  c  NoZ  3  115  72 %  c  No Z  4  116  5%  No Z  5  117  50%  NoZ  118  55 %  No Z  119  50%  NoZ  120  10%  No Z  6  NHBoc  7 8  C0 Me 2  Si(OMe)  ;  " Isolation yield. * E:Z ratio was determined based on 'H NMR spectra. Yields were calculated based on c  'H NMR spectra of the crude products.  127 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  With the bromine in closer proximity to the double bond, the conversion yield to CM product decreased. As shown in entry 2 and 3, the conversion yields to the CM product 114 and 115 were 68 % and 72 % with 5-bromo-l-pentene and 4-bromo-lbutene substrate respectively. It was suggested from this result that when the functionality is close to the reactive olefin, it will impact the reaction in an unfavorable way. Crowe and co-workers reported the dramatic yield decrease in CM between 48  styrene and 4-bromo-l-butene (50 %) when compared with the reaction between styrene and 5-bromo-l-pentene (90 %) using Schrock's catalyst 87. The difference was rationalized due to the inductive effect of the bromine. This was not believed to be applicable to our studies because of the fact that 5-bromo-l-pentene and 4-bromo-lbutene provided similar results. CM products 114 and 115 were not isolated because both polarities are so close to that of the starting material that they can not be separated by routine column chromatography or preparative TLC. In addition, it was found that when 5-hexen-l-ol was used, the reaction was retarded to a great extent and only a 5 % yield of the CM product 116 was obtained (entry 4, Table 3.9). Protection of the hydroxyl group as the acetate, afforded a much better result and the product 117 was achieved in 50 % yield (entry 5). 5-hexen-l-yl N-Bocglycinate was found to be a good CM partner and afforded the product 118 in a 49  moderate yield of 55 % (entry 6). Thus, CM provides a method for introducing a-amino acid parts directly into the vinyl group. Olefins with more than one additional functionalities, such as 1 -methoxycarbonyl- 1,6-heptdiene provided good result towards 50  cross-metathesis. CM product 119 was obtained with 50 % yield (entry 7). Multiple functionalities could therefore be directly introduced via CM. For the CM between 112  128 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  and vinyl trimethoxysilane, the reaction occurred, even though the yield was quite low with 10 % for product 120 (entry 8). However, the siloxane product from this reaction is a useful building block for further transformations, such as Suzuki-type aryl halide crosscoupling. It can be seen from the results in Table 3.9 that different functional groups, 51  such as halides, esters, and ct-amino acid could be readily incorporated into the vinylchlorin by employing this CM method. When the functionalities are further away from the vinyl group, they have little impact on the reaction; when they are in closer proximity, the yields generally become lower. In the studies of CM between the Zn(II) ring B-BPD 112 and allyl-substituted terminal olefins, such as allyl trimethylsilane, allyloxytrimethylsilane and allyl acetate, it was surprised to find that no CM products could be obtained (entries 1, 2 and 3 in Table 3.10). The starting material remained unchanged during the reaction. This result was unexpected because that allyl-substituted terminal olefins are generally reactive CM partners. Other unexpected results were obtained when we carried out the CM of Zn(II) 28  ring B-BPD 112 with symmetric internal allyl-substituted olefins, such as cw-1,4bis((trimethylsilane)oxy)-2-butene and cz's-l,4-diacetoxy-2-butene. CM products 123 and 124, the allylic-substituted vinylchlorins, were obtained in moderate yields for these reactions (entries 4 and 5). Product 123 is an allyl-hydroxy derivative which was obtained after TBAF treatment to deprotect TMS. Furthermore, CM between Zn(II) methyl pyropheophorbide a (122) and c/s-l,4-diacetoxy-2-butene afforded a much better result where the product 125 was obtained in 80 % yield (entry 6). Therefore, CM with symmetric internal allylic olefins provides a valuable method for preparing allylic functionalized chlorins.  129 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins Table 3.10 C M between Vinylchlorin and Allyl-Substituted Olefins  1)25 mol% 96, reflux in THF S  R  2)TFA  2  B R =H or R A: 112, Zn(ll) ring B-BPD; 122, Zn(ll) methyl pyropheophorbide a 2  Entry  1  Product  B  Yield "  Z/E'  1  112  No reaction  2  112  No reaction  3  112  No reaction  4  112  123 , R =CH OH  43 %  No Z  5  112  124, R =CH OAc  54 %  No Z  6  122  125, R =CH OAc  80 %  No Z  c  1  2  1  2  1  2  " Isolation yield. * E:Z ratio was determined based on 'H NMR spectra. Product 123 was obtained after c  TBAF treatment to deprotect TMS.  The reactivity difference between terminal olefins and symmetric internal olefins in C M with ruthenium catalyst 91 was studied systematically by Blackwell and coworkers. Their study indicated that, in certain cases, higher cross-metathesis yields with 30  better ?ra«s-selectivity could be achieved by employing symmetric disubstituted internal olefins instead of their monosubstituted terminal counterparts. As shown in Scheme 40, the cross-metathesis product 127 was generated in 80 % yield when the internal bisOTBS substrate was used, while allyl OTBS afforded the product in 70 % yield.  130 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins C0 Me L 2  =\  > » 6  5mol%91  2  -  2 e  q  C0 Me uu Me 2 2  L / v 126 e q u i v  + +  "  /=\ TBSO^  u i v  X  2  -  ' 127:70 %,E/Z: 7.8:1 x  5 mol% 91 5mol%91 reflux in CH CI , 12 h ^OTBS ^ 2  2 equiv.  r o f h iv in  r;H„r.U  M e  >T^ OTBS  reflux in CH CI , 12 h  1 2  1 e q u i v  1  ^  \-OTBS  +  2  c  C0 2Me Me  9°  2  w  ?  0  T  B  S  k/v^V^  2  19 h  7  ^ 7 127:80 %, E/Z: 8.8:1  Scheme 40 CM between 126 and Terminal or Internal Allyl-Substituted Olefins  This result was rationalized, as shown in Scheme 41, as the result of preferential formation of the ruthenium alkylidene species (Ru=CHR ) when disubstituted internal 2  olefin was used over the formation of the less stable ruthenium methylidene species (RU-CH2),  which is generated when employing the terminal olefin. Because the  ruthenium methylidene carbene Ru=CH2 undergoes unproductive decomposition considerably faster than the ruthenium alkylidene species Ru=CHR , minimizing the 2 52  amount of the former one will extend the metathesis activity of the catalyst. As illustrated in Scheme 41, it is possible to have a CM catalytic cycle that does not involve a methylidene intermediate when one reactant is an internal olefin.  131 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins  and  Vinylporphyrins  H Ru (H)R  ruthenium alkylidene: Ru=CHR  2  •(H)  ruthenium methylidene: Ru=CH  2  2  Scheme 41 Catalytic Species in CM of Terminal or Internal Olefins  Additionally, their studies showed that the initiation rate of the terminal allylic olefin is much higher than its internal counterpart. The reactions in Scheme 42, in which the catalytic species ruthenium alkylidene 128 was formed between catalyst 91 and either allyl OTBS or bis-OTBS substrate were examined quantitatively by H NMR spectra. It !  took 5 min for allyl OTBS to provide 90 % of ruthenium alkylidene 128, while approximately 90 min was required for the bis-OTBS substrate to make the same conversion. Therefore, the initiation rate for allyl OTBS is about 18 times faster than internal bis-OTBS olefin.  OTBS  1 equiv. 91 CD CI , 22 °C 2  2  128 OTBS  128  Scheme 42 Reaction of Catalyst 91 with Terminal and Internal Olefins  132 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  These studies provided some very useful hints to explain our results. Even though these conclusions were reached based on the catalyst 91, the reactivity trend for allylic terminal olefins and internal olefins was believed to be similar with catalyst 96, which was employed in our studies. The observation that no CM product could be obtained for the reaction between vinylchlorin 112 and a terminal allylic-substituted olefin is most likely the result of the domination of self-metathesis of the terminal olefin with the ruthenium catalyst. This process dominates the catalytic cycle but is unproductive for heterodimerization. Even though the corresponding homodimer from self-metathesis, i.e the disubstituted internal olefin, can undergo secondary CM to give the desired crossmetathesis product, the reaction rate for homodimer toward catalytic species is much slower than the terminal olefin, as discussed above, and is therefore not competitive enough to take part in the catalytic cycle. Instead, when the disubstituted internal olefins were employed in the reaction, their relatively slow reaction rate towards the catalyst is an advantage to the vinylchlorin substrate, which is also slow towards CM. Therefore, the vinylchlorin gets a chance to participate in the catalytic cycle and to reach the desired cross-metathesis product.  3.2.4 C M Studies of Vinylchlorin Based on the Empirical Model To shed further light on the reactivity of our vinylchlorins towards'CM, the reaction of the Zn(II) ring B-BPD-1,3 diene dimethyl ester (112) with the ruthenium catalyst 96 was carried out. A mixture of 96 (0.01 mmol) and 112 (0.04 mmol) in 1 mL dry THF was refluxed under Ar for 1 h. The reaction progress was monitored by analytic TLC every 5 min. After 20 min, a new green spot was observed on TLC which indicated the  133 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  appearance of a chlorin product. When no other new changes were observed on TLC the reaction was stopped after 1 h. After TFA treatment, the new material was isolated by silica column chromatography and its structure was shown to be 129 (only the E isomer observed) (Scheme 43) by NMR and MS analyses. The result indicated that the vinylchlorin 112 did not undergo homodimerization by ruthenium catalyst 96, but instead reacted with 96 to generate the 3-devinyl 3-(2-phenyl-l-ethenyl) derivative 129.  112  129  Scheme 43 Reaction of Zn(II) BPD 112 with Ruthenium Catalyst 96  It has previously been reported that when the ruthenium benzylidene catalyst reacted with olefins, the reaction could proceed via two pathways (Scheme 44). Through path A, the olefin binds with the ruthenium benzylidene carbene so that the alkyl substituent is adjacent to the metal. The transition state with structure 130 is formed through this orientation and the alkylidene complex 131 is then generated. In path B, the transition state 132 has the configuration in which the alkyl group oriented further away from the metal center, and the methylidene carbene 133 and R=CHPh are then generated from the reaction. It was pointed out in the studies that when the benzylidene catalyst reacted with sterically unhindered terminal olefins, the alkylidene 131 was the initial  134 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  carbene product observed. When the steric bulk of the olefin was gradually increased, there was a decrease in the reaction rate. For even bulkier terminal olefins, metathesis led directly to the formation of the methylidene 133, i.e. the reaction went via pathway B. Therefore, the alkylidene 131 is the kinetically favored product through path A; however, when steric effect of the bulky substituent becomes predominant, the pathway is shifted toB.  132  Scheme 44 Pathways for Reaction between Catalyst 96 and Terminal Olefins Path B is clearly the route taken by our substrate. Our results indicate that the reaction between 112 and catalyst 96 goes through transition state 132, rather than 130 (Scheme 44). This is believed primarily due to the steric hinderance of the chlorin ring. According to the selective CM empirical model that was established by Grubbs and co-workers, olefins can be categorized as one of four types based on their reactivity with a specific catalyst (Scheme 31, section 3.1). Based on the principles of the model and our studies, the conjugated vinyl groups of vinylchlorins can be categorized as Type III olefins with the catalyst 96 since they are unable to homodimerize but are able to undergo CM with other olefins. Interestingly, our results also showed the category change of a  135 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  substrate when employing different catalysts. As discussed before, when catalyst 91 is used, no CM occurs on the conjugated vinyl group of vinylchlorin while it does not inhibit catalyst activity towards other olefins (Schemes 37 and 38). Thus the conjugated vinyl group belongs to Type IV for catalyst 91. By using the highly-active catalyst 96, the category changes to Type III. To this point, most of the cross-metathesis reactions that we have carried out, according to the empirical model, are reactions between vinylchlorins (Type III) and Type I olefins (except the vinyl siloxanes, entry 8, Table 3.9). For the "isolated" terminal olefins, i.e. olefins that have functionality further away from the vinyl, the selective CM proceeds smoothly and can be regarded as good examples for CM between Type III and I olefins. When allyl-substituted olefins are employed, the model can not provide a clear explanation to our results. Both allylic mono-substituted terminal olefins and internal disubstituted olefins belong to Type I according to the empirical model (Table 3.2), but only the internal di-substituted olefins react with our vinylchlorin substrates to give CM products. Based on the empirical model, as a Type III olefin, the vinylchlorin should be able to undergo selective CM with Type II olefins. In the next stage of our studies, it was worthwhile to explore the reactions of vinylchlorin 112 with Type II olefins in order to broaden the reaction scope and to further examine the applicability of the model to our substrates. As illustrated in Table 3.2 (section 3.1), a,P-unsaturated carbonyl compounds are typical Type II substrates with catalyst 96, and they will provide an opportunity to build chlorins with ct,P-unsaturated carbonyl functionalities via CM. The commercially  136 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins  and  Vinylporphyrins  available acrolein diethyl acetal and methyl acrylate were chosen as Type II CM partners in our studies (Table 3.11). Table 3.11 The CM of 112 with Type II and Type III Olefins MeO,C,  C0 Me 2  (  25 mol% catalyst 96  OMe  MeO  M ^  O  0'  OMe  134, M:Zn, R: - C H  .OEt N  "OEt O 135, M: 2H, R: — C H O  TFA  II  136, M: Zn, R: — C - O M e  B  Entry  OEt  1  ^ ^ O E t  0  2  ^  3  OCH  Product  Yield  135  40%  No Z  136  20%  NoZ  129  —  —  a  Z/E  b  3  " Isolated yield. E:Z ratio was determined based on H NMR spectra. b  In the reaction of acrolein diethyl acetal with 112 following our general procedure, a brown-colored new spot was observed on analytic TLC. After the reaction, the mixture was treated with TFA at room temperature for 30 min. After the work-up, the product was obtained in 40 % yield via silica gel column chromatography, and its structure was shown to be the conjugated aldehyde 135 (entry 1). The TFA treatment served both to hydrolyze the acetal and to demetallate the Zn(II). Although the acid-sensitive diethyl acetal CM product 134 could likely be isolated with Et^N-treated silica gel column  137 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  chromatography, it is more convenient to obtain the final aldehyde product 135 after TFA treatment. No further attempts were made to isolate compound 134. The conjugated aldehyde product 135 is found to be quite stable both in solution and as a solid at room temperature. When methyl acrylate was employed as a substrate in our CM reaction, positive result was also obtained (entry 2). Product 136, the vinylchlorin with a conjugated methoxycarbonyl functionality at the position-3 , was obtained. The yield was not as high 2  as that with the acrolein diethyl acetal, suggesting that the reactivity of methyl acrylate is lower than the diethyl acetal. The preparation of a,(3-unsaturated carbonyl derivatives is always of great interest in organic synthesis. The synthesis of a, P-unsaturated aldehydes has been accomplished previously by Wittig reaction of the aldehyde with the aldehyde ylide, Ph3P=CHCHO, or with the acetal  54  53  or imine protected two-carbon ylides. Addition-elimination 55  methods have also been used to generate a, P-unsaturated ketones and esters. The 56  F  success with CM in our studies here provides another direct approach for making chlorins with a,P-unsaturated carbonyl functionalities. In addition, the CM between vinylchlorin 112 and 2-methyl-1-hexene, a Type III 1,1-disubstituted olefin was studied (entry 3, Table 3.11). Interestingly, none of the expected CM product was generated. Instead, compound 129, which is formed via the reaction between 112 and the catalyst 96, was obtained after the reaction mixture was treated with TFA. It is suggested from this result that the reactivity of 112 is so low that it can not undergo CM with other Type III olefins under these conditions. The result for this reaction is also different from the reactions with other active olefins, such as allyl acetate,  138 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  in which compound 112 was recovered unreacted. This is because that the reactivity for 2-methyl- 1-hexene is also quite low that it does not prevent the reaction between vinylchlorin 112 and the catalyst. The model for selective cross-metathesis that was developed by Grubbs and coworkers provides an important reference for prediction of the outcome of crossmetathesis reaction with vinylchlorins. However, because of the generally poor and unique reactivities of the vinylchlorins, the model does not apply to all situations.  3.2.5 Enyne Metathesis of Vinylchlorin The enyne metathesis of the vinylchlorin 112 with 1-hexyne and 1,4-diacetoxy-2butyne were attempted. Unfortunately, no products were generated and the starting material was recovered.  112  139 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  3.2.6 Stereoselectivity  One of the critical issues that prevent the wide application of cross-metathesis is the lack of stereoselectivity for CM products. In most of the CM reactions in the literatures, CM products were obtained as the mixtures of E and Z isomers with E isomers in a higher ratio because of their thermodynamic stability. However, it was observed in our investigations that the cross-metathesis reactions with the vinylchlorins and vinylporphyrins all proceeded with excellent Estereoselectivity, which made them synthetically practical. As shown in Table 3.6 to Table 3.11, most of the reactions in our studies provided complete /^-stereoselectivity. For the reactions in which the CM products were obtained as mixtures of E- and Zisomers, the E:Z ratios were in the range of 15:1 to 60:1, with the ^-isomers as predominant products (entries 2 and 3 in Table 3.6, entry 2 in Table 3.7, Table 3.8). The lowest stereoselectivity was observed for the CM between methyl pyropheophorbide a (39) and 1-octene, in which the product 108 was obtained as a stereoisomeric mixture with the E:Z ratio of 15:1 (entry 2, Table 3.7). However, the reaction between 39 and 1hexene proceeded with absolute /^-stereoselectivity (entry 1, Table 3.7). This difference is difficult to explain. Although different factors control the stereoselectivity of the ultimate CM products, the steric arguments provide the first level of analysis for our remarkable Estereoselectivity. In CM reactions, the steric hindrance and the close proximity of the bulky substituent to the reacting olefin have been reasonably assumed as the primary reason for the selective formation of the /^-isomers. For example, in the CM reaction between terminal olefin 137 and various ct-carbonyl containing olefins (Table 3.12) , 21a  140 Chanter 3 Cross-Metathesis  Reactions of Vinvlchlorins  and  Vinylporphyrins  the lowest ^-selectivity was obtained for acrolein substrate (entry 1). Adding a geminal methyl group (entry 2) or replacing the aldehyde with a methyl ketone group (entry 3) all evidently amplified the favorable formation of the /^-isomers.  Table 3.12 Cross-Metathesis Reactions between 137 and a-Carbonyl Containing Olefins Entry  Styrenes  1  A c O ^ ^  2  Ac(/"W>  3  A c O ^ B ^  137  137  Cross-partner  Product  O H C ^  O H C ^  0 0  137  Isolated Yield %  E:Z  62  1.1:1  92  >20:1  95  >20:1  a  " E:Z ratio based on NMR spectra.  In our substrates, the bulky tetrapyrrolic macrocycle is connected directly with the vinyl group, thus has an important impact on the stereoselectivity observed. In the intermediate that lead to the formation of the CM products (Scheme 45), the steric effect makes the formation of the less hindered intermediate A more favorable over intermediate B, from which the £-isomer is produced.  A  B  favorable  unfavorable  ( p ) : chlorin or porphyrin  Scheme 45 Steric Effect in the Formation of CM Intermediate  141 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  3.3 Structure Characterization of the C M Products The structures of all of the CM products obtained were characterized by 'H NMR spectra, UV-Vis spectra and mass spectrometry.  3.3.1 Characterization via H NMR Spectra !  ]  H NMR spectra provided a very efficient and practical method for determining the  configurations of the CM products. As shown in the 'H NMR spectrum of compound 108 (Fig. 3.2), E and Z isomers exhibit characteristic peak splitting patterns at corresponding chemical shifts. For the triplet-doublet peak at 6.70-6.63 ppm, the coupling constant of the doublet is 16.0 Hz, indicates that it is the signal for H-3 of the is-isomer. The triplet 2  2  3  •  arises from the coupling between H-3 and the CH at position-3 with the coupling 2  constant of 7.0 Hz. Similarly, the peaks that are further upfield at 6.50-6.44 ppm can be assigned as signals of H-3 for the Z-isomer since the coupling constant of the doublet is 2  11.3 Hz, which is characteristic of the Z-isomer. The same conclusion can also be reached by comparing the doublet at 7.51 ppm (J = 16.0 Hz), which is the peak for H-3  1  of £-isomer, with the doublet at 7.32 ppm (J= 11.3 Hz) for H-3 in Z-isomer. The 1  integration values of these characteristic peaks accurately represent the relative yields of the corresponding stereoisomers. Thus, the E/Z ratio of compound 108 was determined to be 15:1 (Fig. 3.2).  142 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  MeO  O  108  E:Z=1.0/0.065 =15:1  H-3 (£) 1  H-3f(E)  H-3  ^H-3 (Z) 2  J  104  96  100  18  92  84  80  t> P m)  M M  76  72  68  64  Fig. 3.2 Low-Field Region of H NMR Spectrum (CDC1 , 300 MHz) of Compound 108 ]  3  Besides providing the method for determining the E/Z ratio of CM product, *H NMR spectrum also serves as a useful method for determining the reaction yields. This is particularly useful for the reactions in which the CM product has a i?/value very close to that of the starting material, and is thus difficult to be isolate by column chromatography. Such is the case for the reaction between the Zn(II) ring B-BPD-l,3-diene dimethyl ester 112 and 4-bromo-l-butene. Fig. 3.3 shows part of the *H NMR spectrum of the crude product after TFA treatment, which is the mixture of 61 and the CM product 115. The peaks at 8.12, 6.37 and 6.17 ppm are characteristic signals of the vinyl group in 61, while the H-3 of the 3-(4-bromo-l-butenyl) substituent in 115 shows at 6.75 ppm as a 2  143 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  triplet-doublet. The ratio of the integration values of the corresponding peaks thus represents the conversion yield in this CM reaction.  Me0 C,  C0 Me  C0 Me  2  2  2  o" ~  MeO"  O M e  MeC^O  cT~  0 M e  115  61  yield=1.0/(1.0+0.47) ' =68 %  H-3 of 115 2  11 L i l  H-3 of 61  Jo.47  1  10 0  \  8  I  96  9 2  18  8 4  . , H-3 of61 2  1  — i  1  1  80  1  1  7 6  1  1  '  1  72  1  1  1 —  68  8  CO  i  O  64  o 60  fpm)  Fig. 3.3 Low-Field Region of the H NMR Spectrum (CDC1 , 300 MHz) of Product ]  3  115 Mixed with 61  3.3.2 UV-Vis Spectroscopy of C M products When alkyl substituents were introduced to the position-3 by cross-metathesis, the 2  overall u-electron conjugation of the tetrapyrrolic macrocycle was not affected. The electronic spectra remained the same type as the non-substituted vinylchlorins and no shift was observed for the Q band. This was shown from the UV-Vis spectra of compound 105 and 123 when compared with 61 in Fig. 3.4. In compound 129, a benzene  144 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  ring was introduced to the position-3 and the conjugation of the whole molecule was 2  extended. Thus the Q band exhibits a red shift of 5 nm to 695 nm (Fig. 3.4).  >  400  500  600  700  800  wavelength (nm) Fig. 3.4 UV-Vis Spectra of Compounds 61,105,123 and 129  The most pronounced change in the electronic absorption spectra was observed for the CM product 135, in which an aldehyde group was introduced to position-3 on the ring B-BPD macrocycle (Fig. 3.5). The conjugated aldehyde functionality in 135 causes a dramatic shift for both the Q band and the Soret band compared with 61. The Q band is at  145 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  705 nm and the Soret band at 455 nm, exhibit red shift of 15 nm and 25 nm respectively (Fig. 3.5). Evidently, the color of compound 135 is brown instead of the green color of 61.  0.1 -I  300  , 400  , 500  , 600  700  r ^ = ^ 800  Wabelength (nm)  Fig. 3.5 UV-Vis Spectra of CM Product 135  The complete characterizations of all of the compounds are presented in the experimental section (Chapter Four). The 'H NMR spectra of some of the representative CM products, including 102, 113, 119, 120, 124, 129, 135, 136, 138 and 125 were attached at the end of this chapter.  3.4 Preliminary PDT Cytotoxicity Assessment  The preliminary cytotoxicity assays for some of the CM products were performed in QLT. Inc following the same method described in section 2.4. The assay showed that  146 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  compound 123, 3-devinyl-3-(3-hydroxy-l-pentenyl) ring B-BPD 1,3-diene dimethyl ester, exhibits good in vitro PDT efficacy with the LD50 of 188 nM. It was indicated form this result that introducing the hydrophilic functional group to BPD dimethyl ester increased the hydrophilicity of the molecule and resulted in the PDT efficacy improvement.  3.5 Further Modification of C M Products and Future Work We expected that the C M products, the chlorins and porphyrins with substitutedvinyl functionalities, would be useful substrates for further functionalization, such as hydrogenation and cyclization. Preliminary attempts towards such research have been carried out in this work. Upon Pd/C catalyzed hydrogenation,  compound  105 was hydrogenated  to  compound 138 which possesses the saturated hexyl chain at position-3 (Scheme 46). This reaction proceeded smoothly at room temperature with quantitative conversion being achieved after 4 h. Thus, the 3-alkyl substituted chlorins were synthesized readily via the C M reaction followed by catalyzed hydrogenation.  105  138  Scheme 46 Hydrogenation of the C M Product 105  147 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  Further modifications will be worthwhile towards any future work with the vinylsubstituted chlorins as useful building blocks, for example, the intramolecular DielsAlder reaction shown in Scheme 47. By introducing the appropriate substituents to the vinyl via CM, the benzoporphyrin derivative with a fusedfive-memberedring might be obtained by the Diels-Alder cyclization.  R , R : H or alkyl 1  2  Scheme 47 Proposed Diels-Alder Reaction of the CM Product  It will thus be interesting to investigate the further applications of the CM products in synthesis. Tetrapyrrolic macrocycles with novel structures can be constructed in this manner.  148 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  3.6 Summary  Cross-metathesis (CM) has been applied successfully to vinylchlorin and vinylporphyrin substrates by employing the imidazolylidene ruthenium benzylidene complex 96. The reactions were optimized and the reactivity of different substrates in CM was studied. The Zn(II) complexes of tetrapyrrolic macrocycles were found to be more reactive than the corresponding free bases, and the chlorins exhibit higher reactivity than the porphyrins. Olefin partners with a variety of substituents were compatible with the reaction and internal olefins are more beneficial than terminal olefin in some situations. All of the CM products were achieved with high ^-stereoselectivity. The cross-metathesis reaction thus proved to be an effective way of producing chlorins and porphyrins with substituted-vinyl groups with excellent control of Estereoselectivity. This method provided a generalizable strategy for modifying the amphiphilicity of the tetrapyrrolic macrocycles.  149 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  150 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  151 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  120  L I UL  J LL_J 10.0  9.0  8.0  7.0  uu  V 6.0  5.0  4.0  3.0  2.0  1.0  (ppm)  Fig. 3.9  N M R Spectrum (CDC1 , 400 MHz) of Compound 120 3  153 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  159 Chapter 3 Cross-Metathesis Reactions of Vinvlchlorins and Vinylporphyrins  REFERENCES 1. a) Eleuterio, H . Chemtech 1991, 92. b) Anderson, A . W.; Merckling, N . G. US 2,721,189,1956. 2. Calderon, N . ; Chen, H . Y . ; Scott, K. W. 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F.; Choi, T.; Grubbs, R. H . Adv. Synth. Catal. 2002, 344(6+7), 634. b) Kawai, T.; Komaki, M . ; Iyoda, T. J. Mol. Catal. A: Chem. 2002, 190, 45. c) Yasuda, T.; Abe, J.; Yoshida, FL; Iyoda, T.; Kawai, T. Adv. Synth. Catal. 2002, 344(6+7), 705. 45. Vicente, M . G. H . The porphyrin handbook; Kadish, K . M . ; Smith, K. M . ; Guilard, R. Ed.; Academic Press: New York, 2000, 4, 153. 46. Grubbs, R. H . Tetrahedron 2004, 60, 7117. 47. Nichol, A. W. J. Chem. Soc. C. 1970, 9903. 48. Crowe, W. E.; Zhang, Z. J.  Am. Chem. Soc. 1993,115, 10998.  49. Hayashida, O.; Sebo, L.; Jr Rebek, J. J. Org. Chem. 2002, 67, 8291. 50. Gastaminza, A . E.; Ferracutti, N . N . ; Rodriguez, N . M . / . Org. Chem. 1984, 49, 3859. 51. Mowery, M . E.; DeShong, P. J. Org. Chem. 1999, 64, 1684.  163 Chapter 3 Cross-Metathesis Reactions of Vinylchlorins and Vinylporphyrins  52. Ulman, M . ; Grubbs, R. H . Organometallics 1998,17, 2484. 53. Bestmann, H . J.; Vostrowsky, O.; Paulus, H . ; Billman, W.; Stransky, W. Tetrahedron Lett. 1977, 121. 54. Daubresse, N . ; Francesch, C.; Rolando, C. Tetrahedron 1998, 54, 10761. 55. Meyer, A. I.; Tomioka, K.; Fleming, M . P. J. Org. Chem. 1978, 43, 3788. 56. a) Wollenberg, R. H.; Albizati, K . F.; Peries, R. J. Am. Chem. Soc. 1977, 99, 7365. b) Tamiai, H.; Kouraba, M . Tetrahedron 1997, 53, 10677.  164 Chapter 4 Experimental  Chapter 4 Experimental  165 Chapter 4 Experimental  4.1 Nomenclature and Numbering System Used for the Synthesized Compounds For chlorophyll a related compounds, the trivial names with IUPAC-IUB numbering system are used in this work. Ring B-BPD-1,3-diene dimethyl ester was used to refer to the compound 61 in this work for the sake of brevity. The names and numbering systems are illustrated as below. The prefix "de" followed by the name of a substituent is used to denote the removal of that substituent from the parent structure.  Methyl pyropheophorbide a (39)  V \\ / 8 N--H(g  ^10  HN-  X.  14"  )l3  17' 13 17  1  2  \13  3  4  MeO' Ring B-BPD-1,3-diene dimetyl ester (61) IUPAC: 23H, 25H-Benzo[b]porphyine-9,13-dipropanoic acid,19-ethenyl-1,22a-dihydro1,2-bis(methoxycarbonyl)-8,14,18,22a-tetramethyl-dimethyl ester,(1 S, 22aR)  166 Chapter 4 Experimental  4.2 General Methods and Materials Methylene chloride was dried by refluxing over C a H under nitrogen for 4 h before 2  use. THF was refluxed over Na/acetophenone under Argon until the color changed to blue. The ruthenium catalyst 91 was bought from Acros Organic and was handled under Ar. The ruthenium catalyst 96 was bought from Sigma-Aldrich Inc. and was handled in A r protected glove box. Ring B-BPD-l,4-diene dimethyl ester (13) was obtained from QLT Inc. as the undesired side product during the manufacture of Visudyne®. Magnesium-chlorophyllin  was  bought  from  Pfannenschmidt  GmbH  (www.pfannenschmidt.de), Germany. The other commercially available reagents were used as purchased without further purification unless stated otherwise.  Silica gel (BDH, 230-400 mesh) or neutral Brockman alumina (usually Grade III, i.e. deactivated with 6 % water) was used for (flush) column chromatography. Analytic silica gel thin layer chromatography (TLC) was performed on ALUGRAM® plates (Macherey-Nagel GmbH, silical gel 60, 0.25 mm), preparative T L C was performed on glass-backed plates (Merck KGaA, silica gel 60 without indicator, 0.5 mm).  ' H N M R spectra were recorded on Bruker AV-300 M H z or AV-400 M H z instrument using deuteriochloroform (CDCI3) or deuteriodimethyl sulfoxide (DMSO-c/g) as solvent, and the residual CHC1 (5= 7.24 ppm) or the residual D M S O (5= 2.50 ppm) 3  were used as references.  1 3  C N M R spectra were recorded on the same instruments using  the same solvents and with residual CHCI3 (5= 77.0 ppm) or the residual D M S O (6= 39.5 ppm) as references. Selective N O E experiments were performed on the same instruments.  167 Chapter 4 Experimental  U V - V i s spectra were recorded on a Varian Cary-50 spectrophotometer. A l l of the mass spectra were obtained at the Department of Chemistry, U B C . Low and High resolution electron impact (EI) mass spectrometry were recorded on Kratos/AEI MS-902 spectrometer. Low and high resolution electron spray ionization (ESI) mass spectrometry were recorded on Micromass L C T spectrometer. Low and high resolution liquid secondary ionization mass spectrometry (LSIMS) were recorded on Kratos Concept IIHQ spectrometer. Elemental analyses were carried out in the analytical lab at U B C on CarloErba elemental analyzer 1108. X-ray crystallographic data were collected on a Rigaku/ADSC CCD.  4.3 Preparation of Methyl Pyropheophorbide a 39 from Magnesium Chlorophyllin I.  Extraction of Chlorin e trimethyl ester (72) from magnesium-chlorophyllin 6  Magnesium-chlorophyllin (1.0 g) was dissolved completely in 1 % sodium chloride solution (400 mL). The aqueous solution was extracted with ether (2x100 mL) to remove the ether-soluble components. The aqueous layer was then treated with 5 % HC1 (20 mL) for 0.5 h. After that, the aqueous mixture was extracted with ether (3x120 mL). The  168 Chapter 4 Experimental  combined ether layer was washed with water (2x150 mL), dried over sodium sulfate and then concentrated to 10 mL in vacuo. The concentrated ether solution was treated with excess diazomethane for 0.5 h. Chlorin e<j trimethyl ester 72 (Rf =0.5, eluted with methylene chloride/methanol (100:1, v/v)) was separated from the mixture via column chromatography (methylene chloride/methanol, 100:0.2, v/v) as a black solid (130 mg, 13 %, based on magnesium-chlorophyllin). The analyses data were consistent with the literature values.  1  ' H N M R (400 M H z , CDC1 ) £9.67, 9.53, 8.73 (3s, 3H, H-meso), 8.06 (dd, J= 17.7 Hz 3  and 11.5 Hz, 1H, H-3 ), 6.32 (d,J= 17.7 Hz, 1H, H-3 (£)), 6.11 (d,J= 11.5 Hz, 1H, H 1  2  3 (Z)), 5.28 (q, 2H, H-15 ), 4.47-4.37 (m, 2H, H-18, H-17), 4.25 (s, 3H, 13'-C0 Me), 2  1  2  3.76 (q, J= 7.6 Hz, 2H, H-8 ), 3.76 (s, 3H, 15 -C0 Me), 3.62 (s, 3H, 17 -C0 Me), 3.57 1  1  2  2  2  (s, 3H, H-12 ), 3.45 (s, 3H, H-2 ), 3.27 (s, 3H, H-7 ), 2.58-2.50 (m, 2H, H-17 ), 1  1  1  1  2.18-2.16 (m, 2H, H-17 ), 1.76 (d, J= 7.3 Hz, 3H, H-18 ), 1.70 (t, J= 7.6 Hz, 3H, H-8 ), 2  1  -1.31,-1.47 (2br, 2H, 2NH) LREIMS (m/z): 638 (M ) +  II. Cyclization of Chlorin e trimethyl ester to methyl pheophorbide a (8) 6  8  2  169 Chapter 4 Experimental  Chlorin  trimethyl ester 72 (90 mg, 0.14 mmol) was dissolved in 5 mL dry  pyridine and carefully degassed with nitrogen at 50 °C for 30 min. A nitrogen degassed solution of /-butoxide in /-butyl alcohol (1.5 mL, from a mixture of 110 mg potassium metal in 5.5 mL /-butyl alcohol) was added. The initial bright green color turned orange after stir for 2 min at 50 °C under nitrogen. The reaction was quenched with 1.5 mL nitrogen degassed acetic acid after 15 min. The mixture was poured into water and extracted with methylene chloride (3x50 mL). The organic phase was then washed with water, 2 M hydrochloric acid, dried over sodium sulfate and then concentrated in vacuo. The residue was treated with excess ether diazomethane for 0.5 h. Methyl pheophorbide a (8) was obtained as a black solid after purification by column chromatography (74 mg, 87 %). !  2  H N M R (300 MHz, CDC1 ) 89.45 (s, 1H, H-10), 9.31 (s, 1H, H-5), 8.54 (s, 1H, H-20), 3  7.96 (dd, J = 17.4 Hz and 11.4 Hz, 1H, H-3 ), 6.26 (d, J = 17 .4 Hz, 1H, H-3 (£)), 6.16 1  2  (d, J= 11.4 Hz, 1H, H - 3 (Z)), 6.24 (s, 1H, H-13 ), 4.45-4.40 (m, 1H, H-18), 4.19-4.17 2  2  (m, 1H, H-17), 3.85 (s, 3H, 13 -C0 Me), 3.68 (q, J = 7.6 Hz, 2H, H-8 ), 3.67 (s, 3H, 17 2  1  2  2  C 0 M e ) , 3.55 (s, 3H, H-12 ), 3.39 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 2.61-2.55 (m, 1H, 1  1  1  2  Ha-17 ), 2.52-2.45 (m, 1H, Hb'-17 ), 2.31-2.21 (m, 2H, Ha'-17', Hb-17 ), 1.79 (d, 1  2  2  J= 7.3 Hz, 3H, H-18 ), 1.67 (t, J= 7.6 Hz, 3H, H-8 ), 0.52 (br, 1H, NH), -1.64 (br, 1H, 1  2  NH) LREIMS (m/z): 606 (M ) +  170 Chapter 4 Experimental  III. Decarboxylation of methyl pheophorbide a to methyl pyropheophorbide a (39)  39 Methyl pheophorbide a 8 (130 mg, 0.021 mmol) was refluxed in 20 mL collidine for 1.5 h under nitrogen in the dark. The solvent was evaporated out under high vacuum, and the residue was purified on silica column chromatography. Methyl pyropheophorbide a (39) was obtained as a black solid which was crystallized from methylene chloride/hexane to give a fine powder (100 mg, 85 %). The spectra data were consistent with the literature values.  2  ' H N M R (300MHz, CDC1 ) £9.45, 9.35, 8.53 (3s, 3H, H-meso), 7.97 (dd, J = 17.4 Hz 3  and 11.4 Hz, 1H, H-3 ), 6.25 (d, J= 17.4 Hz, 1H, H-3 (£)), 6.14, (d, J= 11.4 Hz, 1H, H 1  2  3 (Z)), 5.17 (q, 2H, H-13 ), 4.50-4.45 (m, 1H, H-18), 4.29-4.24 (m, 1H, H-17), 3.66 (q, 2  2  J= 7.6 Hz, 2H, H-8 ), 3.64 (s, 3H, H-12 ), 3.59, (s, 3H, 1 7 - C 0 M e ) , 3.38 (s, 3H, H-2 ), 1  1  2  2  1  3.20 (s, 3H, H-7 ), 2.68-2.66 (m, 1H, Ha-17 ), 2.55-2.51 (m, 1H, Hb'-17 ), 2.31-2.27 1  1  2  (m, 2H, Ha'-17\ Hb-17 ), 1.79 (d, J= 7.3 Hz, 3H, H-18 ), 1.65 (t, J= 7.6 Hz, 3H, H-8 ), 2  1  0.45 (br, 1H, NH), -1.7 (br, 1H, NH). LREfMS (m/z): 548 (M ) +  2  171 Chapter 4 Experimental  4.4 Synthesis of Primary Ether Derivatives Methyl 3-devinyl-3-(l,2-dihydroxyethyl) pyropheophorbide a (63) OH ,.OH  y  NH  N •\  N-  7  HN"" i  *0  MeC" ^0  63 To a well-stirred solution of methyl pyropheophorbide a 39 (556 mg, 1.0 mmol) in 120 mL tetrahydrofuran, 50 % aqueous N-methylmorpholine TV-oxide solution (0.31 mL, 176 mg, 1.5 mmol) was added. Then a solution of osmium tetroxide (10 mg, 0.04 mmol) in 0.20 mL toluene and 10 mL acetone-water mixture (10:1, v/v) was added to the reaction. Analytical T L C showed that no starting material left after the reaction mixture was stirred at room temperature for 24 h. 1.5 g sodium metabisulfite was added, the mixture was stirred for another 5 min and the solid was removed by filtration. The filtrate was diluted with 300 mL methylene chloride, washed with 2 % sodium acetate solution and water. After dried over sodium sulfate, the solvent was removed in vacuo. The residue was purified by flash column chromatography eluted with methanol/methylene chloride (2:100, v/v). Methyl 3-devinyl-3-(l,2-dihydroxyethyl) pyropheophorbide a 63 was  obtained  as  a black needle crystal  chloride/methanol (440 mg, 75 %).  after  crystallization  from  methylene  172 Chapter 4 Experimental  ' H N M R (300MHz, CDC1 ) £9.28 (s, 1H, H-10), 8.96 (s, 1H, H-5), 8.40, 8.39 (2s, 1H, 3  H-20), 5.86-5.84 (m, 1H, H-3 ) 4.90 (q, 2H, H-13 ), 4.29-4.26 (m, 2H, Ha-3 , H-18), 1  2  2  4.02-4.00 (m, 2H, Ha'-3 , H-17), 3.88-3.86 (m, 2H, H-8 ), 3.64, 3.62 (2s, 3H, 17 2  1  2  C 0 M e ) , 3.30, 3.29 (2s, 3H, H-12 ), 3.22, 3.21 (2s, 3H, H-2 ), 2.99 (s, 3H, H-7 ), 1  1  1  2  2.44-2.41 (m, 2H, Ha-17 , Hb'-17 ), 2.18-2.16 (m, 1H, Ha'-H ), 1.96-1.94 (m, 1H, Hb1  2  1  17 ), 1.68-1.66 (m, 3H, H-18 ), 1.52-1.48 (m, 3H, H-8 ), -0.58 (br, 1H, NH), -2.33 (br, 2  1  2  1H, NH); LREIMS (m/z): 582 (M ) +  HREHMS m/z: calcd. for  C34H38N4O5  (M ): 582.2842 +  found: 582.2848 UV/Vis. (X /Abs., CH2CI2): 320.0 (0.202), 410.0 (0.876), 505.0 (0.089), 535.0 (0.086), max  605.0 (0.178), and 665.0 (0.387)  Methyl 3-devinyl-3-(2-hydroxyethyl) pyropheophorbide a (65)  Methyl 3-devinyl-3-(l,2-dihydroxyethyl) pyropheophorbide a 63 (400 mg, 0.69 mmol) was dissolved in 6 mL dry methylene chloride. The solution was degassed by nitrogen for 0.5 h. Trifluoromethanesulfonic acid (8 mL, 13.6 g, 0.09 mol) was added slowly to the well-stirred degassed solution by syringe. The color of the mixture changed  173 Chapter 4 Experimental  to blue-green. The mixture was stirred at room temperature in dark under nitrogen for 3 h. Then the reaction mixture was poured into 100 m L ice water very slowly with stirring at the same time. The aqueous phase was extracted by methylene chloride (4x75mL). The organic layer was washed with water and dried over sodium acetate. After the solvent was removed in vacuo, the residue was redissolved in 30 ml dry methylene chloride and kept at 0°C. A solution of sodium borohydride (40 mg, 1 mmol) in 5 mL dry methanol was added to the reaction. After stirring at 0°C for 1 min, 1.5 m L acetic acid was added to quench the reaction. The reaction mixture was poured into 120 mL ice water and the aqueous layer was extracted with methylene chloride. The organic phase was washed with water, 2 % sodium bicarbonate, and water and dried over sodium sulfate. After removing the solvent, the residue was purified by silica gel column chromatography, eluted with methanol/methylene chloride (1:100, v/v). Methyl 3devinyl-3-(2-hydroxyethyl) pyropheophorbide a 65 was obtained as a black solid (330 mg, 85 %). The spectra data were consistent with literature values. ' H N M R (300MHz, CDC1 ) ^ 9.28, 9.20, 8.45 (3s, 3H, H-meso), 4.94 (q, 2H, H-13 ), 2  3  4.36-4.34 (m, 3H, H-3 , H-18), 4.15-4.12 (m, 1H, H-17), 4.05 (t, / = 6.5 Hz, 2H, H-3 ), 1  2  3.62 (q, J= 7.5 Hz, 2H, H-8 ), 3.60 (s, 3H, 17 -C0 Me), 3.45, (s, 3H, H-12 ), 3.31 (s, 1  2  1  2  3H, H-2 ), 3.21 (s, 3H, H-7 ), 2.49-2.44 (m, 2H, Ha-17 , HV-17 ), 2.04-2.21, (m, 3H, 1  1  1  2  H a ' - H , Hb-17 , OH), 1.74 (d, J = 7.2 Hz, 3H, H-18 ), 1.64 (t, J= 7.6 Hz, 3H, H-8 ), 1  2  1  0.23 (br, 1H, NH), -1.79 (br, 1H, NH); LREIMS (m/z): 566 (M ) +  2  174 Chapter 4 Experimental  Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a (66) Br  ~~V  NH  O  L  MeCTX)  66 Methyl 3-devinyl-3-(2-hydroxyethyl) pyropheophorbide a 65 (150 mg, 0.27 mmol) was dissolved in 30 mL dry methylene chloride. The solution was treated with a solution of carbon tetrabromide (986 mg, 2.9 mmol) and triphenylphosphine (707 mg, 2.7 mmol) in 6 mL dry methylene chloride. The mixture was stirred at room temperature under nitrogen for 0.5 h. Analytical T L C showed that no starting material left. After the solvent was removed in vacuo, the residue was chromatographyed over a short neutral alumina (Activity III)  column, eluted  with methylene  chloride. Methyl  3-devinyl-3-(2-  bromoethyl) pyropheophorbide a 66 was got as a black solid (153 mg, 90 %). ' H N M R (400 M H z , CDC1 ) £9.44, 9.10, 8.50 (3s, 3H, H-meso), 5.15 (q, 2H, H-13 ), 2  3  4.46 (q, J= 7.3 Hz, 1H, H-18), 4.32-4.26 (m, 3H, H-17, H-3 ), 4.00 (t, J= 5.7 Hz, 2H, 2  H-3 ), 3.64 (q, J= 7.6 Hz, 2H, H-8 ), 3.62 (s, 3H, 17 -C0 Me), 3.60 (s, 3H, H-12 ), 3.30 1  1  2  1  2  (s, 3H, H-2 ), 3.21 (s, 3H, H-7 ), 2.67- 2.53 (m, 2H, Ha-17 , Hb'-17 ), 2.30-2.12 (m, 2H, 1  1  1  2  H a ' - H , Hb-17 ), 1.80 (d, J = 7.3 Hz, 3H, H-18 ), 1.67 (t, J= 7.6 Hz, 3H, H-8 ), 0.35 (br, 1  2  1  1H, NH),-1.79(br, 1H, NH); LREIMS (m/z): 628 (M ) +  HREIMS m/z: calcd. for C s ^ y B r N ^ (M ): 628.2049 +  found: 628.2051  2  175 Chapter 4 Experimental  UV/Vis. (Twc/Abs., CH C1 ): 415.1 (1.669), 505.0 (0.287), 535.0 (0.271), 600.0 (0.258), 2  2  and 660.0 (0.514)  3-Devinyl-3-(l,2-dihydroxyethyl) ring B-BPD-l,3-diene dimethyl ester (68)  To a well-stirred solution of ring B-BPD-1, 3-diene dimethyl ester 61 (586 mg, 0.8 mmol) in 120 mL THF, 50 % aqueous /V-methylmorpholine TV-oxide solution (0.24 mL, 140.6 mg, 1.2 mmol) was added. Then a solution of osmium tetroxide (8 mg, 0.032 mmol) in 0.12 mL toluene and 8 mL acetone-water mixture (10:1, v/v) was added to the reaction. Analytical TLC showed that no starting material left after the reaction mixture was stirred at room temperature for 48 h. 1.5 g sodium metabisulfite was added, the mixture was stirred for another 5 min and the solid was removed by filtration. The filtrate was diluted with 300 mL methylene chloride, washed with 2 % sodium acetate solution and then water. After dried over sodium sulfate, the solvent was removed in vacuo. The residue was purified by flash column chromatography eluted with methanol/methylene chloride (2:100, v/v). 3-Devinyl-3-(l,2-dihydroxyethyl) ring B-BPD-l,3-diene dimethyl ester 68 was got as a black solid, which was crystallized from methylene chloride/methanol to give a black needle crystal (510 mg, 75 %).  176 Chapter 4 Experimental j  H N M R (300MHz, CDC1 ) £9.72, 9.67, 9.45, 9.35 (4s, 4H, H-meso), 7.81 (d, J= 5.7 3  Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 6.44-6.42 (m, 1H, H-3 ), 5.13 (s, 1H, H 3  4  1  7 ), 4.78-4.73 (m, 2H, H-3 ), 4.29 (t, J= 7.6 Hz, 2H, H-17 ), 4.20 (br, 1H, OH), 4.14 (t, 1  2  1  J= 7.8 Hz, 2H, H-13 ), 3.98 (s, 3H, 7 -C0 Me), 3.64, 3.62, 3.60, 3.47, 3.38 (5s, 5x3H, 1  2  2  H-18 , H-2 , H-12 , 17 -C0 Me, 13 -C0 Me), 3.13-3.22 (m, 4H, H-17 , H-13 ), 2.83 (s, 1  1  1  2  2  2  2  2  2  3H, 7'-C0 Me), 1.78 (s, 3H, 7-CH ), -2.39 (br, 2H, 2NH) 2  3  LREIMS m/z: 766 HRETMS m/z: calcd. for C H46N 0,o (M ): 766.3214 +  42  4  found: 766.3128 U V / V i s . (X /Abs., CH C1 ): 355.0 (1.301), 425.0 (1.986), 575.0 (0.433), 624.9 (0.213), max  2  2  and 685.0(0.925)  3-Devinyl-3-(2-hydroxyethyl) ring B-BPD-1, 3-diene dimethyl ester (70)  3-Devinyl-3-(l,2-dihydroxyethyl) ring B-BPD-1, 3-diene dimethyl ester 68 (500 mg, 0.65 mmol) was dissolved completely in 20 mL concentrated sulfuric acid. 3.5 mL fuming sulfuric acid was added to the solution by a syringe, and the mixture was stirred at room temperature for another 15 min. The reaction mixture was poured into 200 mL ice water slowly with stirring. The aqueous phase was extracted by methylene chloride  177 Chapter 4 Experimental  (4x75mL). The organic layer was washed with water then dried over sodium sulfate. After the solvent was removed in vacuo, the residue was redissolved in 30 ml dry methylene chloride at 0°C. A solution of sodium borohydride (40 mg, 1 mmol) in 5 mL dry methanol was added to the reaction. After the mixture was stirred at 0°C for 30 min under nitrogen, 1.5 mL acetic acid was added to quench the reaction. The reaction mixture was poured into 150 mL ice water, and the aqueous layer was extracted with methylene chloride (3x50mL). The organic phase was washed with water, 2 % sodium bicarbonate and water and dried over sodium sulfate. After the solvent was removed in vacuo, the residue was purified by flash column chromatography  eluted with  methanol/methylene chloride (1:100, v/v). 3-Devinyl-3-(2-hydroxyethyl) ring B-BPD-1, 3-diene dimethyl ester 70 was obtained as a black solid (375 mg, 77 %). ' H N M R (300MHz, CDC1 ) £9.73 (s, 2H, H-meso), 9.38, 9.00 (2s, 2H, H-meso), 7.81 (d, 3  J = 5.7 Hz, 1H, H-7 ), 7.43 (d,J = 5.7 Hz, 1H, H-7 ), 5.06 (s, 1H, H-7 ), 4.38-4.35 (m, 3  4  1  2H, H-3 ), 4.30 (q, J = 7.6 Hz, 2H, H-17 ), 4.19-4.15 (m, 4H, H-13 , H-3 ), 3.97 (s, 3H, 1  1  1  2  7 -C0 Me), 3.63, 3.62, 3.55, 3.47, 3.40 (5s, 5x3H, H-18 , H-2 , H-12 , 17 -C0 Me, 13 2  1  1  1  2  2  2  2  C 0 M e ) , 3.20-3.12 (m, 4H, H-17 , H-13 ), 2.85 (s, 3H, 7 - C 0 M e ) , 1.79 (br, 1H, OH), 2  2  2  1  2  1.74 (s, 3H, 7-CH ), -2.39 (br, 2H, 2NH) 3  LREIMS (m/z): 750 (M ) +  HREIMS m/z: calcd. for C 2H46N 09(M ): 750.3265 +  4  4  found: 750.3266  178 Chapter 4 Experimental  3-Devinyl-3-(2-bromoethyI) ring B-BPD-1, 3-diene dimethyl ester (71)  3-Devinyl-3-(2-hydroxyethyl) ring B-BPD-1, 3-diene dimethyl ester 70 (400 mg, 0.53 mmol) was dissolved in 50 mL dry methylene chloride. The solution was treated with a solution of carbon tetrabromide (1.94 g, 5.8 mmol) and triphenylphosphine (1.40 g, 5.3 mmol) in 10 mL dry methylene chloride. The mixture was stirred at room temperature under nitrogen for 1 h. T L C showed that no starting material left. The solvent was removed in vacuo, and the residue was purified by a neutral alumina (Activity III) column chromatography, eluted with methylene chloride. 3-Devinyl-3-(2bromoethyl) ring B-BPD-1,3-diene dimethyl ester 71 was got as a black solid (420 mg, 96 %). !  H N M R (400MHz, CDC1 ) £9.75 (s, 2H, H-meso), 9.38, 8.92 (2s, 2H, H-meso), 7.82 (d, 3  J= 5.7 Hz, 1H, H-7 ), 7.45 (d, J = 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.49-4.45 (m, 3  4  1  2H, H-3 ), 4.31 (t, J= 7.6 Hz, 2H, H-17 ), 4.20-4.13 (m, 4H, H-13 , H-3 ), 3.98 (s, 3H, 1  1  1  2  7 -C0 Me), 3.63, 3.62, 3.57, 3.47, 3.41 (5s, 5x3H, H-18 , H-2 , H-12 , 17 -C0 Me, 13 2  1  1  2  1  2  2  2  C 0 M e ) , 3.22-3,12 (m, 4H, H-17 , H-13 ), 2.88 (s, 3H, 7'-C0 Me), 1.80 (s, 3H, 7-CH ), 2  2  2  2  -2.39 (br, 2H, 2NH); LREIMS (m/z): 812 (M ) +  3  179 Chapter 4 Experimental  H R E M S m/z: calcd. for C 2H4 BrN 08 (M ): 812.2421 +  4  5  4  found: 812.2420 UV/Vis. (Xmax/Abs., CH C1 ): 350.0 (0.954), 430.0 (1.388), 579.9.0 (0.406), 625.0 2  2  (0.266), and 685.1 (0.703)  3-Devinyl-3-(2-bromoethyl)-7 -bromo ring B-BPD-1, 3-diene dimethyl 4  ester (85)  3-Devinyl-3-(2-hydroxyethyl) ring B-BPD-1,3-diene dimethyl ester 70 (17 mg, 0.021 mmol) was dissolved in 5 mL dry methylene chloride and 0.5 mL dry D M F in the presence of potassium carbonate (0.3 g, 2.1 mmol). Thionyl bromide (0.11 mL, 1.2 mmol) was then injected into the reaction. The mixture was stirred at room temperature in the dark under N 2 for 5 h and then was diluted with methylene chloride. The organic layer was washed with water, diluted sodium bicarbonate solution and dried over sodium sulfate. After the solvent was removed in vacuo, the residue was purified by neutral alumnia (Grade III) column chromatography to give product 85 (13 mg, 70 %). ' H N M R (400MHz, CDCI3) S 10.45, 9.78, 9.73, 8.88 (4s, 4H, H-meso), 7.79 (s, 1H, H 7 ), 5.06 (s, 1H, 7 - C H 3 ) , 4.51-4.42 (m, 2H, H-3 ), 4.34 (t, J= 7.6 Hz, 2H, H-13 ), 4.18 (t, 3  1  1  J= 7.6 Hz, 2H, H-17 ), 4.18-4.08 (m, 2H, H-3 ), 4.00 (s, 3H, 7 -C0 Me), 3.63, 3.62, 1  2  2  2  180 Chapter 4 Experimental  3.57, 3.52, 3.40 (5s, 5x3H, H-18 , H-2 , H-12 , 17 -C0 Me, 13 -C0 Me), 3.22-3.13 (m, 1  1  1  2  2  2  2  4H, H-17 , H-13 ), 2.85 (s, 3H, 7'-C0 Me), 1.82 (s, 3H, H-7 ), -2.51, 2.64 (2br, 2NH) 2  2  1  2  ESIMS (m/z): 893 ([M+H] ) +  HRESIMS m/z: calcd. for C H44Br N 0 ([M+H] ): 890.1604 +  42  2  4  8  ' found: 890.1601 U V / V i s . (X /Abs., CH C1 ): 355.0 (1.162), 440.0 (1.390), 590.0 (0.403), 624.9 (0.337), max  2  2  and 685.0 (0.751)  Mercury(II) oxide/tetrafluoroboric acid (HgO-2HBF ) 4  The mixture of yellow mercury(II) oxide (0.43 g, 2.0 mmol) and 0.52 m L 48 % aqueous solution of tetrafluoroboric acid (0.73 g, 4.0 mmol) was stirred at room temperature for 30 min. The resultant solution was evaporated in vacuo to yield a white hygroscopic solid HgO-2HBF , which was directly used for the preparation of the ethers. 4  General Procedure for the Synthesis of Primary Ether Derivatives of Methyl Pyropheophorbide a (67) and Primary Ether Derivatives of Ring B-BPD 1,3-diene Simethyl Ester (62): Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a (66) or 3-devinyl-3-(2bromoethyl) ring B-BPD-1, 3-diene dimethyl ester (71) was mixed together with 0.5 equivalent of dry mercury(II) oxide/tetrafluoroboric acid in round bottom flask. The solid mixture was dissolved in 5 mL dry methylene chloride. Three to five equivalents of the corresponding alcohol was injected into the solution. The mixture was stirred at room  181 Chapter 4 Experimental  temperature in the dark under nitrogen overnight. Methylene chloride (10 mL) and water (10 mL) were added to the reaction mixture. The organic layer was washed with 2 % sodium bicarbonate and water and dried over sodium sulfate. The solvent and the excess alcohol were removed in vacuo. The residue was purified by flush column chromatography or preparative TLC plate. The product ethers were obtained as black fine powders after crystallizationfrommethylene chloride/hexane.  General Procedure for Hydrolysis of Methyl esters (67) to Carboxylic Acids (60): Methyl ester 67 was dissolved in the mixture of tetrahydrofuran and methanol. Lithium hydroxide (50 equivalents) was dissolved in distilled water and the solution was added to the reaction. The reaction mixture was stirred at room temperature under nitrogen in the dark overnight. Methylene chloride was added to dilute the reaction mixture. The organic layer was washed with water, 2 % acetic acid and water and dried over sodium sulfate. After removing the solvent in vacuo, the residue was purified by preparative TLC using methanol/methylene chloride (1:10, v/v) as eluent. The product carboxylic acid was separated and then was crystallizedfrommethylene chloride/hexane to give a black powder.  182 Chapter 4 Experimental  Methyl 3-devinyl-3-(2-methoxyethyl) pyropheophorbide a (67a)  67a Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 66 (98 mg, 0.156 mmol), dry mercury (II) oxide/tetrafluoroboric acid (31 mg, 0.08 mmol) and 0.03 mL methanol (0.77 mmol) were utilized by following the general procedure for primary ether synthesis. Product 67a was obtained as a black powder after flush column chromatography (46 mg, 51 %). ' H N M R (300 M H z , CDC1 ) £9.43, 9.20, 8.47 (3s, 3H, H-meso), 5.19 (q, 2H, H-13 ), 2  3  (m,  4.47-4.44  m , H-18), 4.27-4.24 (m, 1H, H-17), 4.05-4.04 (m, 4H, H-3 , H-3 ), 3.65 1  2  (q, J = 7.6 Hz, 2H, H-8 ), 3.63 (s, 3H, 17 - C 0 M e ) , 3.60 (s, 3H, H-12 ), 3.47 (s, 3H, 3 1  2  1  2  2  OCH3), 3.38 (s, 3H, H-2 ), 3.22 (s, 3H, H-7 ), 2.71-2.49 (m, 2H, H-17 ), 2.33-2.20 (m, 1  1  1  2H, H-17 ), 1.79 (d, J= 7.3 Hz, 3H, H-18 ), 1.67 (t, J= 7.6 Hz, 3H, H-8 ), 0.49 (br, 1H, 2  1  2  NH),-1.68(br, 1H, NH) LSIMS (m/z): 581 ([M+H] ) +  Anal, calcd. for C35H40N4O4, C: 73.39; H : 6.94; N : 9.65 found, C: 72.27; H : 6.93; N : 9.54 UV/Vis. ( W A b s . , CH C1 ): 315.0 (0.297), 410.1 (1.556), 505.0 (0.134), 534.9 (0.131), 2  2  600.0 (0.115), and 660.0 (0.636)  183 Chapter 4 Experimental  3-Devinyl-3-(2-methoxyethyl) pyropheophorbide a (60a) OCH  3  HO  2  °  O  60a Methyl 3-devinyl-3-(2-methoxyethyl) pyropheophorbide a 67a (28 mg, 0.05 mmol) was dissolved in 3 mL tetrahydrofuran and 4 mL methanol, then was treated with a solution of lithium hydroxide (59 mg, 2.5 mmol) in 3 mL distilled water by following the general procedure. 3-Devinyl-3-(2-methoxyethyl) pyropheophorbide a 60a was obtained as a black powder after preparative T L C (20 mg, 71 %). j  H N M R (400 MHz, CDC1 ) £9.46, 9.21, 8.46 (3s, 3H, H-meso), 5.16 (q, 2H, H-13 ), 2  3  4.47-4.42 (m, 1H, H-18), 4.29-4.27 (m, 1H, H-17), 4.09-4.01 (m, 4H, H-3 , H-3 ), 3.66 1  2  (q, J= 7.6 Hz, 2H, H-8 ), 3.63 (s, 3H, H-12 ), 3.46 (s, 3H, OCH ), 3.29 (s, 3H, H-2 ), 1  1  1  3  3.22 (s, 3H, H-7 ), 2.68-2.57 (m, 2H, H-17 ), 2.35-2.25 (m, 2H, H-17 ), 1.78 (d, 1  1  2  J= 7.3 Hz 3H, H-18 ), 1.67 (t, J= 7.6 Hz, 3H, H-8 ), 0.40 (br, 1H, NH), -1.65 (br, 1H, 1  2  NH) LREEVIS (m/z): 566 (M ) +  UV/Vis. (X /Abs., CH2CI2): 320.0 (0.155), 409.9 (0.770), 505.0 (0.068), 534.9 (0.067), max  600.0 (0.058), and 660.0 (0.314)  184 Chanter 4 Experimental  Methyl 3-devinyl-3-(2-ethoxyethyl) pyropheophorbide a (67b)  67b  Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 66 (110 mg, 0.17 mmol), dry mercury (II) oxide/tetrafluoroboric acid (33 mg, 0.085 mmol) and 0.03 mL ethanol (0.5 mmol) were utilized by following the general procedure for primary ether synthesis. Product 67b was obtained as a black powder after flush column chromatography (51 mg, 51 %). !  H N M R (300 M H z , CDC1 ) £9.48, 9.25, 8.46 (3s, 3H, H-meso), 5.16 (q, 2H, H-13 ), 2  3  4.45-4.41 ( , 1H, H-18), 4.27-4.23 (m, 1H, H-17), 4.09-4.07 (m, 4H, H-3 , H-3 ), 1  2  m  3.69-3.62 (m, 2H, H-8 ), 3.62-3.60 (q, / = 7.0 Hz, 2H, O C H C H ) , 3.65 (s, 3H, 17 1  2  2  3  C 0 M e ) , 3.59 (s, 3H, H-12 ), 3.31 (s, 3H, H-2 ), 3.24 (s, 3H, H-7 ), 2.55-2.50 (m, 2H, 1  1  1  2  H-17 ), 2.30-2.29 (m, 2H, H-17 ), 1.78 (d, J= 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 7.6 Hz, 1  2  1  3H, H-8 ), 1.25 (t, J= 7.0 Hz, 3H, O C H C H ) , 0.53 (br, 1H, NH), -1.66 (br, 1H, NH) 2  2  3  U V / V i s . (> /Abs., CH C1 ): 320.0 (0.216), 409.0 (1.130), 505.0 (0.099), 535.1 (0.096), max  2  2  600.0 (0.084), and 660.0 (0.465) LREIMS (m/z): 594 (M ) +  HREIMS m/z: calcd. for C H4 N 04 (M ): 594.32061 +  36  2  4  found: 594.32066  185 Chapter 4 Experimental  3-Devinyl-3-(2-ethoxyethyl) pyropheophorbide a (60b) OC H 2  5  VNH N~Y  o  1  HO  O  60b Methyl 3-devinyl-3-(2-ethoxyethyl) pyropheophorbide a 67b (35 mg, 0.059 mmol) was dissolved in 3 mL tetrahydrofuran and 4 m L methanol, then was treated with a solution of lithium hydroxide (70 mg, 2.9 mmol) in 3 mL distilled water by following the general procedure. 3-Devinyl-3-(2-ethoxyethyl) pyropheophorbide a 60b was obtained as a black powder after preparative T L C (18 mg, 53 %). ]  H N M R (400 M H z , D M S O - 4 ) 8 12.04 (br, 1H, COOH), 9.70, 9.36, 8.77 (3s, 3H, H -  meso), 5.16 (q, 2H, H-13 ), 4.58-4.53 (m, 1H, H-18), 4.30-4.28 (m, 1H, H-17), 2  4.10-4.03 (m, 4H, H-3 , H-3 ), 3.70 (q, J= 7.6 Hz, 2H, H-8 ), 3.60 (s, 3H, H-12 ), 3.56 1  2  1  1  (q, J= 7.0 Hz, 2H, O C H C H ) , 3.32 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 2.66-2.52 (m, 2H, 1  2  1  3  H-17 ), 2.29-2.05 (m, 2H, H-17 ), 1.75 (d, J= 7.2 Hz, 3H, H-18 ), 1.62 (t, J = 7.6 Hz, 1  2  1  3H, H-8 ), 1.11 (t, ./= 7.0 Hz, 3H, O C H C H ) , 0.36 (br, 1H, NH), -1.92 (br, 1H, NH) 2  2  3  LREIMS (m/z): 580 (M ) +  HREIMS m/z: calcd. for C 5H4oN 04(M ): 580.30496 +  3  4  found: 580.30492 UV/Vis. (Xmax/Abs., CH C1 ): 320.0 (0.288), 409.9 (1.450), 505.0 (0.130), 535.1 (0.127), 2  2  600.0 (0.110), and 660.0 (0.591)  186 Chapter 4 Experimental  Methyl 3-devinyl-3-(2-propyloxyethyl) pyropheophorbide a (67c)  67c Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 66 (130 mg, 0.207 mmol), dry mercury (II) oxide/tetrafluoroboric acid (40 mg, 0.1 mmol) and 0.05 mL 1-propanol (0.8 mmol) were utilized by following the general procedure for primary ether synthesis. Product 67c was obtained as a black powder after flush column chromatography (89 mg, 54 %). ' H N M R (400 M H z , CDC1 ) £9.48, 9.25, 8.46 (3s, 3H, H-meso), 5.16 (q, 2H, H-13 ), 2  3  4.47-4.42 (m, 1H, H-18), 4.27-4.25 (m, 1H, H-17), 4.08 (s, 4H, H-3 , H-3 ), 3.46 (q, 1  2  J= 7.6 Hz, 2H, H-8 ), 3.65 (s, 3H, 17 -C0 Me), 3.59 (s, 3H, H-12 ), 3.45 (t, J= 6.7 Hz, 1  2  1  2  2H, OCH CH CH ), 3.30 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 2.69-2.49 (m, 1H, H-17 ), 1  2  2  1  1  3  2.32-2.22 (m, 2H, H-17 ), 1.78 (d, J= 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 7.6 Hz, 3H, H-8 ), 2  1  2  1.66-1.58 (m, 2H, O C H C H C H ) , 0.92 (t, J= 7.4 Hz, 3H, OCH CH CH ), 0.53 (br, 1H, 2  2  3  2  2  3  NH),-1.65 (br, 1H, NH) LSIMS (m/z): 609 (M ) +  HRESIMS m/z: calcd. for C H45N 04([M+H] ): 609.3441 +  37  4  found: 609.3442 UV/Vis. ( W A b s . , CH C1 ): 320.0 (0.337), 410.1 (1.710), 505.0 (0.154), 535.1 (0.148), 2  2  600.0 (0.131), and 660.0 (0.702)  187 Chapter 4 Experimental  3-Devinyl-3-(2-propyloxyethyl) pyropheophorbide a (60c)  60c Methyl 3-devinyl-3-(2-propyloxyethyl) pyropheophorbide a 67c (68 mg, 0.11 mmol) was dissolved in 4 mL tetrahydrofuran and 6 mL methanol, then was treated with a solution of lithium hydroxide (134 mg, 5.6 mmol) in 4 mL distilled water by following the general procedure. 3-Devinyl-3-(2-propyloxyethyl) pyropheophorbide a 60c was obtained as a black powder after preparative TLC (49 mg, 75 %). ]  H N M R (400 MHz, DMSO-J ) 8 12.08 (br, 1H, COOH), 9.60, 9.26, 8.74. (3s, 3H, H 6  meso), 5.10 (q, 2H, H-13 ), 4.54-4.52 (m, 1H, H-18), 4.28-4.26 (m, 1H, H-17), 4.01 (s, 2  4H, H-3 , H-3 ), 3.64 (q, J= 7,6 Hz, 2H, H-8 ), 3.55 (s, 3H, H-12 ), 3.42 (t, J = 6.7 Hz, 1  2  1  1  2H, OCH2CH2CH3), 3.28 (s, 3H, H-2 ), 3.17 (s, 3H, H-7 ), 2.60-2.52 (m, 2H, H-17 ), 1  1  1  2.32-2.04 (m, 2H, H-17 ), 1.74 (d, J= 7.3 Hz, 3H, H-18 ), 1.59 (t, J= 7.6 Hz, 3H, H-8 ), 2  1  2  1.51-1.44 (m, 2H, O C H C H C H ) , 0.78 (t, J= 7.3 Hz, 3H, O C H C H C H ) , 0.30 (br, 1H, 2  2  3  2  2  3  NH),-1.97(br, 1H, NH) , 3  C N M R (100 M H z , DMSO-cfe) 5 195.17 (C-13 ), 174.17 (C-17 ), 172.45 (C-l9), 1  3  161.01 (C-16), 154.24 (C-6), 149.72 (C-9), 147.78 (C-8), 141.15 (C-l), 137.04 ( C - l l ) , 136.77 (C-3), 136.56 (C-4), 135.73 (C-7), 133.11 (C-2), 129.81 (C-12), 127.49 (C-13), 105.74 (C-15), 104.20 (C-10), 96.30 (C-5), 93.16 (C-20), 71.79 ( O C H C H C H ) , 71.02 2  2  3  (C-3 ), 50.97 (C-17), 49.29 (C-18), 47.42 (C-13 ), 30.79 (C-17 ), 29.38 (C-17 ), 26.16 2  2  1  2  188  Chapter 4 Experimental (C-3 ), 22.81 ( O C H C H C H ) , 22.5 (C-18 ), 18.57 (C-8 ), 17.40 (C-8 ), 11.55 (C-12 ), 1  1  2  2  1  2  1  3  10.81 ( C - 2 , C-7 ), 10.47 ( O C H C H C H ) 1  1  2  2  3  L S i M S ( m / z ) : 595 ([M+H] ) +  H R E S I M S m/z: calcd. for Q e H ^ C U t t M + H ] " ) : 595.3284 4  found: 595.3282 UV/Vis. (X  max  / A b s . , C H C 1 ) : 319.9 (0.349), 410.1 (1.633), 505.0 (0.147), 535.1 (0.145), 2  2  600.0 (0.127), and 660.1 (0.658)  Methyl 3-devinyl-3-(2-butyloxyethyl) pyropheophorbide a (67d)  67d Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 66 (62 mg, 0.1 mmol), dry mercury(II) oxide/tetrafluoroboric acid (19 mg, 0.05 mmol) and 0.03 m L 1-butanol (0.3 mmol) were utilized by following the general procedure for primary ether synthesis. Product 67d was obtained as a black powder after flush column chromatography (39 mg, 64 %). !  H N M R (400 M H z , CDC1 ) £ 9 . 4 5 , 9.23, 8.46 (3s, 3 H , H-meso), 5.15 (q, 2 H , H-13 ), 2  3  4.47-4.42 (m, 1H, H-18), 4.27-4.21 (m, 1H, H-17), 4.07 (s, 4 H , H - 3 , H - 3 ) , 3.66 (q, 1  2  J= 7.6 H z , 2 H , H-8 ), 3.64 (s, 3 H , 1 7 - C 0 M e ) , 3.59 (s, 3 H , H-12 ), 3.54 (t, J= 6.6 H z , 1  2  1  2  2 H , O C H C H C H C H ) , 3.20 (s, 3 H , H - 2 ) , 3.22 (s, 3 H , H - 7 ) , 2.68-2.49 (m, 2 H , H 1  2  2  2  3  1  189 Chanter 4 Experimental 17 ), 2.30-2.23 (m, 2H, H-17 ), 1.79, 1.78 (d, J= 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 7.6 Hz, 1  3H,  2  H-8 ), 2  1.62-1.57  (m,  1  2H,  OCH2CH2CH2CH3),  1.40-1.24  (m, 2H,  OCH2CH2CH2CH3), 0.88 (t, 3H, J =7.3 Hz, OCH2CH2CH2CH3), 0.52 (br, 1H, NH),  -1.60 (br, 1H, NH) LREEVIS (m/z): 622 (M ) +  HREHVIS m/z: calcd. for C s s ^ N ^ M * ) : 622.35191 found: 622.35183 UV/Vis. (^ /Abs., CH C1 ): 314.9 (0.213), 410.1 (1.137), 505.0 (0.096), 535.0 (0.096), max  2  2  600.0 (0.084), and 660.0 (0.457)  3-Devinyl-3-(2-butyloxyethyl) pyropheophorbide a (60d)  60d Methyl 3-devinyl-3-(2-butyloxyethyl) pyropheophorbide a 61A (12 mg, 0.019 mmol) was dissolved in 2 mL tetrahydrofuran and 3 mL methanol, then was treated with a solution of lithium hydroxide (40 mg, 1.6 mmol) in 2 mL distilled water by following the general procedure. 3-Devinyl-3-(2-butyloxyethyl) pyropheophorbide a 60d was obtained as a black powder after preparative T L C (9 mg, 77 %). j  H N M R (300 M H z , CDC1 ) £9.46, 9.23, 8.46 (3s, 3H, H-meso), 5.20 (q, 2H, H-13 ), 2  3  4.49-4.46 (m, 1H, H-18), 4.34-4.30 (m, 1H, H-17), 4.08-4.05 (m, 4H, H-3 , H-3 ), 3.65 1  2  190 Chapter 4 Experimental (q, J = 7.6 Hz, 2H, H-8 ), 3.63 (s, 3H, H-12 ), 3.54 (t, J = 6.7 Hz, 2H, 1  1  O C H C H C H C H ) , 3.29 (s, 3H, H-2 ), 3.22 (s, 3H, H - 7 ) , 2.80-2.50 (m, 2H, H-17 ), 1  2  2  2  1  1  3  2.38-2.18 (m, 2H, H-17 ), 1.79 (d, J= 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 7.6 Hz, 3H, H-8 ), 2  1  2  1.65-1.57 (m, 2H, O C H C H C H C H ) , 1.38-1.24 (m, 2H, O C H C H C H C H ) , 0.88 (t, 2  2  2  3  2  2  2  3  J= 7.3 Hz, 3H, O C H C H C H C H ) , 0.52 (br, 1H, NH), -2.38 (br, 1H, NH) 2  LREJJMS  2  2  3  (m/z): 608 ( M ) +  U V / V i s . (X /Abs., CH C1 ): 320.0 (0.276), 410.1 (1.425), 505.0 (0.128), 535.0 (0.125), max  2  2  600.0 (0.108), and 660.0 (0.578) HREIMS  m/z: calcd. for C37H44N4O4 ( M ) : 608.33626 +  found: 608.33648  Methyl 3-devinyl-3-(2-pentyloxyethyl) pyropheophorbide a (67e)  67e Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 66 (120 mg, 0.19 mmol), dry mercury(II) oxide/tetrafluoroboric acid (37 mg, 0.09 mmol) and 0.05 mL 1-pentanol (0.57mmol) were utilized by following the general procedure for primary ether synthesis. Product 67e was obtained as a black powder after flush column chromatography (60 mg, 50 %).  191 Chapter 4 Experimental *H N M R (400 MHz, CDC1 ) £9.47, 9.24, 8.46 (3s, 3H, H-meso), 5.16 (q, 2H, H-13 ), 2  3  4.47-4.42 (m, 1H, H-18), 4.27-4.25 (m, 1H, H-17), 4.07 (s, 4H, H-3 , H-3 ), 3.67 (q, 1  2  J= 7.6 Hz, 2H, H-8 ), 3.65 (s, 3H, 17 -C0 Me), 3.59 (s, 3H, H-12 ), 3.53 (t, J= 6.7 Hz, 1  2  1  2  2H, OCH2CH2CH2CH2CH3), 3.30 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 2.68-2.49 (m, 2H, 1  1  H-17 ), 2.31-2.23 (m, 2H, H-17 ), 1.78 (d, J= 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 7.6 Hz, 1  3H,  2  H-8 ), 2  1.65-1.58  (m,  1  2H,  OCH CH CH2CH CH3), 2  2  2  1.32-1.25  (m, 4H,  OCH2CH2CH2CH2CH3), 0.81 (t, J = 7.1 Hz, 3H, OCH2CH2CH2CH2CH3), 0.53 (br, 1H, NH), -2.05 (br, 1H, NH) LREIMS (m/z): 636 (M ) +  HREIMS m/z: calcd. for  C39H48N4O4  (M ): 636.36756 +  found: 636.36700 UV/Vis. fl  max  / A b s . , CH2CI2): 315.0 (0.166), 410.0 (0.836), 505.0 (0.076), 535.1 (0.076),  600.0 (0.065), and 660.0 (0.336)  3-Devinyl-3-(2-pentyloxyethyl) pyropheophorbide a (60e)  60e Methyl 3-devinyl-3-(2-pentyloxyethyl) pyropheophorbide a 67e (40 mg, 0.063 mmol) was dissolved in 3 mL tetrahydrofuran and 4 mL methanol, then was treated with a solution of lithium hydroxide (75 mg, 3.3 mmol) in 3 mL distilled water by following  192 Chapter 4 Experimental the general procedure. 3-Devinyl-3-(2-pentyloxyethyl) pyropheophorbide a 60e was obtained as a black powder after preparative TLC (22 mg, 56 %). ' H N M R (400 MHz, DMSO) 511.97 (br, 1H, COOH), 9.73, 9.37, 8.77 (3s, 3H, H-meso), 5.23-5.07 (m, 2H, H-13 ), 4.58-4.53 (m, 1H, H-18), 4.32-4.30 (m, 1H, H-17), 4.09-4.06 2  (m, 4H, H-3 , H-3 ), 3.73 (q, J= 7.6 Hz, 2H, H-8 ), 3.61 (s, 3H, H-12 ), 3.44 (t, J= 6.7 1  2  1  1  Hz, 2H, OCH2CH2CH2CH2CH3), 3.31 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 2.66-2.26 (m, 1  1  2H, H-17 ), 2.11-2.08 (m, 2H, Ha-17 ), 1.75 (d,J= 7.3 Hz, 3H, H-18 ), 1.62 (t, J = 7.6 1  Hz,  2  1  1.47-1.41 (m, 2H, OCH2CH2CH2CH2CH3),  3H, H-8 ), 2  1.16-1.07 (m, 4H,  O C H C H C H C H C H 3 ) 0.60 (t, J= 6.9 Hz, 3H, OCH2CH2CH2CH2CH3), 0.39 (br, 1H, 2  2  2  2  NH), -1.90 (br, 1H, NH) Anal, calcd. for C 3 8 H 4 6 N 4 O 4 , C: 73.28; H: 7.44; N : 9.00 Found, C: 72.67; H: 7.45; N : 9.07 LSIMS (m/z): 623 ([M+H] ) +  UV/Vis. ( W A b s . , CH2CI2): 315.0 (0.166), 410.1 (0.836), 505.0 (0.077), 535.1 (0.076), 600.0 (0.065), and 660.0 (0.336)  Methyl 3-devinyl-3-(2-hexyloxyethyl) pyropheophorbide a (67f) 0-n-C H 6  13  — f Y * ~ Y \ V-NH rW  L MeO  0  O  67f  /  N  193 Chapter 4 Experimental Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 6 6 (85 mg, 0.135 mmol), dry mercury (II) oxide/tetrafluoroboric acid (26 mg, 0.065 mmol) and 0.06 mL 1-hexanol (0.4 mmol) were utilized by following the general procedure for primary ether synthesis. Product 6 7 f was obtained as a black powder after flush column chromatography (45 mg, 51 %). *H N M R (300 MHz, CDC1 ) £9.46, 9.23, 8.46 (3s, 3H, H-meso), 5.15 (q, 2H, H-13 ), 2  3  (m, I H , H-18), 4.27-4.24 (m, 1H, H-17), 4.07 (s, 4H, H-3 , H-3 ), 3.67 (q, 1  4.45-4.43  2  7=7.6 Hz, 2H, H-8 ), 3.64 (s, 3H, 17 -C0 Me), 3.59 (s, 3H, H-12 ), 3.53 (t, J= 6.1 Hz, 1  2  1  2  2H, O C H C H 2 C H C H 2 C H C H 3 ) , 3.30 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 2.67-2.50 (m, 1  2  2  1  2  2H, H-17 ), 2.30-2.22 (m, 2H, H-17 ), 1.78 (d, J = 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 1.6 1  2  1  Hz, 3H, H-8 ), 1.62-1.58 (m, 2H, O C H C H C H 2 C H 2 C H C H 3 ) ,  1.32-1.19 (m, 6H,  2  2  2  2  O C H C H 2 C H C H C H 2 C H 3 ) , 0.78 (t, J= 7.1 Hz, 3H, O C H C H 2 C H C H C H 3 ) , 0.54 (br, 2  2  2  2  2  2  1H, NH), -1.66 (br, 1H, NH) LREEVIS (m/z): 650 (M ) +  HREIMS m/z: calcd. for C40H50N4O4 (M ): 650.38321 +  found: 650.38311 Anal, calcd. for C oH oN 04, C: 73.82; H : 7.74; N : 8.61 4  5  4  Found, C: 72.18; H : 7.84; N : 8.81 U V / V i s . ( W A b s . , CH2CI2): 320.0 (0.416), 410.1 (2.116), 505.1 (0.185), 535.0 (0.179), 600.0 (0.156), and 660.0 (0.865)  194 Chapter 4 Experimental  3-DevinyI-3-(2-hexyloxyethyl) pyropheophorbide a (60f)  Methyl 3-devinyl-3-(2-hexyloxyethyl) pyropheophorbide a 67f (40 mg, 0.063 mmol) was dissolved in 3 mL tetrahydrofuran and 4 mL methanol, then was treated with a solution of lithium hydroxide (76 mg, 3.2 mmol) in 3 mL distilled water by following the general procedure. 3-Devinyl-3-(2-hexyloxyethyl) pyropheophorbide a 60f was obtained as a black powder after preparative T L C (36 mg, 90 %). ]  H N M R (300 M H z , DMSO-J ) $ 12.04 (br, 1H, COOH), 9.68, 9.33, 8.76 (3s, 3H, H 6  meso), 5.14 (q, 2H, H-13 ), 4.55-4.53 (m, 1H, H-18), 4.30-4.27 (m, 1H, H-17), 4.04 (m, 2  4H, H-3 , H-3 ), 3.69 (q, J= 7.6 Hz, 2H, H-8 ), 3.59 (s, 3H, H-12 ), 3.43 (t, J= 6.7 Hz, 1  2  1  1  2H, OCH CH2CH2CH CH CH3), 3.30 (s, 3H, H-2 ), 3.20 (s, 3H, H-7 ), 2.58-2.50 (m, 1  2  2  1  2  2H, H-17 ), 2.32-2.06 (m, 2H, H-17 ), 1.75 (d,J= 7.3 Hz, 3H, H-18 ), 1.61 (t, J= 7.6 1  2  1  Hz, 3H, H-8 ), 1.43-1.38 (m, 2H, OCH CH CH CH2CH2CH3), 1.10-0.93 (m, 6H, 2  2  2  2  O C H C H C H C H C H C H ) , 0.54 (t, J= 6.9 Hz, 3H, O C H C H C H C H C H C H ) , 0.36 2  2  2  2  2  3  2  2  2  2  2  3  (br, 1H, NH), -1.92 (br, 1H, NH) LREIMS (m/z): 636 (M ) +  U V / V i s . (Awe., CH C1 ): 320.0 (19460), 410.1 (99500), 505.1 (8876), 535.0 (8876), 2  2  600.0 (7682), and 660.0 (40877)  195 Chapter 4  Experimental  Methyl 3-devinyl-3-(2-heptyloxyethyl) pyropheophorbide a (67g)  67g Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 66 (64 mg, 0.102 mmol), dry mercury (II) oxide/tetrafluoroboric acid (19 mg, 0.05 mmol) and 0.05 mL 1-heptanol (0.3 mmol) were utilized by following the general procedure for primary ether synthesis. Product 67g was obtained as a black powder after flush column chromatography (39 mg, 58 %). *H N M R (300 M H z , CDC1 ) £9.46, 9.23, 8.46 (3s, 3H, H-meso), 5.15 (q, 2H, H-13 ), 2  3  4.48-4.41 (m, 1H, H-18), 4.27-4.25 (m, 1H, H-17), 4.07 (s, 4H, H-3 , H-3 ), 3.77 (q, 1  2  J = 7.6 Hz, 2H, H-8 ), 3.64 (s, 3H, 17 - C 0 M e ) , 3.59 (s, 3H, H-12 ), 3.53 (t, / = 6.7 Hz, 1  2  1  2  2H, OCH CH2CH2CH CH CH CH3), 3.30 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 2.67-2.50 1  2  2  2  1  2  (m, 2H, H-17 ), 2.29-2.22 (m, 2H, H-17 ), 1.79 (d, / = 7.3 Hz, 3H, H-18 ), 1.68 (t, 1  2  1  J= 7.6 Hz, 3H, H-8 ), 1.63-1.58 (m, 2H, OCH CH CH CH CH CH2CH3), 1.31-1.16 2  2  2  2  2  2  (m, 8H, O C H C H C H C H C H C H C H ) , 0.79 (t, / = 7.0 Hz, 3H, O C H C H C H 2  2  2  2  2  2  3  C H C H C H C H ) , 0.52 (br, 1H, NH), -1.66 (br, 1H, NH) 2  2  2  3  LREIMS (m/z): 664 (M ) +  Anal, calcd. for C4iH N 04, C: 74.06; H : 7.88; N : 8.43 52  4  found, C: 74.04; H : 8.03; N : 8.28  2  2  2  196 Chapter 4 Experimental  UV/Vis. ( W / A b s . , CH C1 ): 320.0 (0.447), 410.1 (2.230), 505.0 (0.202), 534.9 (0.198), 2  2  600.0 (0.176), and 660.0 (0.921)  3-devinyl-3-(2-heptyloxyethyl) pyropheophorbide a (60g)  Methyl 3-devinyl-3-(2-methoxyethyl) pyropheophorbide a 6 7 g (38 mg, 0.05 mmol) was dissolved in 3 mL tetrahydrofuran and 4 m L methanol, then was treated with a solution of lithium hydroxide (68 mg, 2.8 mmol) in 3 mL distilled water by following the general procedure. 3-Devinyl-3-(2-heptyloxyethyl) pyropheophorbide a 60g was obtained as a black powder after preparative T L C (28 mg, 76 %). ' H N M R (400 M H z , D M S O - 4 ) S 12.06 (br, 1H, COOH), 9.65, 9.31, 8.75 (3s, 3H, H meso), 5.14 (q, 2H, H-13 ), 4.56-4.51 (m, 1H, H-18), 4.29-4.27 (m, 1H, H-17), 4.02 (s, 2  4H, H-3 , H-3 ), 3.67 (q, J = 7.6 Hz, 2H, H-8 ), 3.58 (s, 3H, H-12 ), 3.41 (t, J= 6.7 Hz, 1  2  1  1  2H, OCH CH CH2CH CH2CH2CH3), 3.29 (s, 3H, H-2 ), 3.19 (s, 3H, H-7 ), 2.57-2.52 1  2  2  1  2  (m, 2H, H-17 ), 2.32-2.06 (m, 2H, H-17 ), 1.74 (d, J = 7.3 Hz, 3H, H-18 ), 1.61 (t, J = 1  2  1  7.6 Hz, 3H, H-8 ), 1.46-1.35 (m, 2H, O C H C H C H C H C H C H C H ) , 1.08-0.84 (m, 2  2  2  2  2  2  2  3  8H, O C H C H C H C H C H C H C H ) , 0.52 (t, J = 6.9 Hz, 3H, O C H C H C H C H C H 2  2  2  2  2  2  3  CH CH ), 0.35 (br, 1H, NH), -1.93 (br, 1H, NH) 2  3  LSIMS (m/z): 651 ([M+H] ) +  2  2  2  2  2  197 Chapter 4 Experimental  . HRESIMS m/z: calcd. for C oH iN 04([M+H] ): 651.3910 +  4  5  4  found: 651.3909 UV/Vis. (?i /Abs., CH C1 ): 410.1 (1.328), 505.1 (0.211), 535.0 (0.210), 600.0 (0.205), max  2  2  and 660.0 (0.587)  Methyl 3-devinyl-3-(2-(5-hexenyloxy)ethyl) pyropheophorbide a (67h)  67h Methyl 3-devinyl-3-(2-bromoethyl) pyropheophorbide a 66 (80 mg, 0.127 mmol), dry mercury (II) oxide/tetrafluoroboric acid (23 mg, 0.06 mmol) and 0.05 m L 5-hexenel-ol (0.3 mmol) were employed. The reaction mixture was refluxed in dry methylene chloride under nitrogen for 12 h. B y following the general procedure for work-up, product 67h was obtained after flush column chromatography (49 mg, 60 %). *H N M R (300 M H z , CDC1 ) S 9.49, 9.26, 8.47 (3s, 3H, H-meso), 5.74-5.70 (m, 1H, 3  OCH CH2CH CH2CH=CH2) 2  2  5.15  (q,  2H,  H-13 ), 2  4.94-4.84  (m,  2H,  OCH CH2CH2CH CH=CH2,), 4.48-4.41 (m, 1H, H-18), 4.27-4.25 (m, 1H, H-17), 4.08 2  2  (s, 4H, H-3 , H-3 ), 3.68 (q, J= 7.6 Hz, 2H, H-8 ), 3.66 (s, 3H, 17 -C0 Me), 3.59 (s, 3H, 1  2  1  2  2  H-12 ), 3.53 (t, J = 6.7 Hz, 2H, O C H C H C H 2 C H C H = C H ) , 3.30 (s, 3H, H-2 ), 3.24 (s, 1  1  2  2  2  2  3H, H-7 ), 2.67-2.50 (m, 2H, H-17 ), 2.29-2.22 (m, 2H, H-17 ), 2.01-1.98 (m, 2H, 1  1  2  OCH CH CH2CH CH=CH2), 1.78 (d, J= 7.2 Hz, 3H, H-18 ), 1.68 (t, J= 7.6 Hz, 3H, H 1  2  2  2  198 Chapter 4 Experimental 8 ), 1.61-1.57 (m, 2H, OCH2CH CH2CH CH=CH2), 1.45-1.41 (m, 2H, O C H C H 2  2  2  2  2  C H C H C H = C H ) , 0.53 (br, 1H, NH), -1.67 (br, 1H, NH) 2  2  2  LREDVIS (m/z): 648 (M ) +  3-Devinyl-3-(2-methoxyethyl) ring B-BPDrl,3-diene dimethyl ester (62a)  62a 3-Devinyl-3-(2-bromoethyl) ring B-BPD-1,3-diene dimethyl ester 71 (60 mg, 0.074 mmol), dry mercury(II) oxide/tetrafluoroboric acid (14 mg, 0.037 mmol) and 0.01 m L methanol (0.25mmol) were utilized by following the general procedure for primary ether synthesis. Product 62a was obtained after flush column chromatography as a black powder (30 mg, 53 %). ' H N M R (300 M H z , CDC1 ) £9.70, 9.69 9.37, 8.98 (4s, 4H, H-meso), 7.80 (d, J= 5.7 3  Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.31 (t,J= 7.8 Hz, 2H, 3  4  1  H-17 ), 4.19-4.05 (m, 6H, H-13 , H-3 , H-3 ), 3.97 (s, 3H, 7 -C0 Me), 3.64, 3.62, 3.54, 1  1  1  2  2  2  3.49, 3.48, 3.40 (6s, 6x3H, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , 3 - O C H , H-12 ), 2  2  1  2  1  2  1  2  3  3.21-3.12 (m, 4H, H-17 , H-13 ), 2.88 (s, 3H, 7 -C0 Me), 1.80 (s, 3H, 7-CH ), -2.41 (br, 2  2  1  2  2H, 2NH) LREIMS (m/z): 764 (M ) +  3  199 Chapter 4 Experimental UV/Vis. (^ /Abs., CH C1 ): 355.0 (0.895), 430.0 (1.472), 580.0 (0.325), 620.0 (0.168), max  2  2  and 680.0 (0.615) Anal, calcd. for C43H48N4O9, C: 67.52; H : 6.33; N : 7.33 found, C: 67.62; H : 6,53; N : 6.72  3-Devinyl-3-(2-ethoxyethyl) ring B-BPD-1,3-diene dimethyl ester (62b)  62b 3-Devinyl-3-(2-bromoethyl) ring B-BPD-1,3-diene dimethyl ester 71 (65 mg, 0.08 mmol), dry mercury(II) oxide/tetrafluoroboric acid (15 mg, 0.04 mmol) and 0.015 m L ethanol (0.25mmol) were utilized by following the general procedure for primary ether synthesis. Product 62b was obtained as a black powder after  flush column  chromatography (34 mg, 55 %). ' H N M R (400 MHz, CDCI3) £9.70, 9.69, 9.37, 8.99 (4s, 4H, H-meso), 7.81 (d, J= 5.7 Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.32 (t, J= 7.8 Hz, 2H, 3  4  1  H-17 ), 4.22-4.08 (m, 6H, H-13 , H-3 , H-3 ), 3.98 (s, 3H, 7 -C0 Me), 3.63, 3.61, 3.54, 1  1  1  2  2  2  3.48, 3.40 (5s, 5x3H, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 ), 3.62 (q, / = 7.6 Hz, 2  2  2  1  1  1  2  2H, O C H C H ) , 3.21-3.13 (m, 4H, H-17 , H-13 ), 2.88 (s, 3H, 7'-C0 Me), 1.80 (s, 3H, 2  2  3  2  2  7-CH3), 1.22 (t, J= 7.6 Hz, 3H, O C H C H ) , -2.47, -2.52 (2br, 2H, 2NH) 2  LREIMS (m/z): 778 (M ) +  3  200 Chapter 4 Experimental  HRETJVIS m/z: calcd. for C 4H5oN 09(M ): 778.3578 +  4  4  found: 778.3580  3-Devinyl-3-(2-propyloxyethyl) ring B-BPD-1,3-diene dimethyl ester (62c)  62c 3-Devinyl-3-(2-bromoethyl)  ring B-BPD-l,3-diene dimethyl ester 71 (103 mg,  0.123 mmol), dry mercury(II) oxide/tetrafluoroboric acid (24 mg, 0.06 mmol) and 0.03 mL 1-propanol (0.4 mmol) were utilized by following the general procedure for primary ether synthesis. Product 62c was obtained as a black powder after flush column chromatography (68 mg, 70 %). ' H N M R (300 M H z , CDC1 ) £ 9 . 7 1 , 9.70, 9.37, 8.99 (4s, 4H, H-meso), 7.81 (d, J = 5.7 3  Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.31 (t, J = 7.8 Hz, 2H, 3  4  1  H-17 ), 4.21-4.08 (m, 6H, H-13 , H-3 , H-3 ), 3.97 (s, 3H, 7 -C0 Me), 3.64, 3.62, 3.54, 1  1  1  2  2  2  3.47, 3.41 (5s, 5x3H, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 ), 3.48 (t, J = 6.7 Hz, 2  2  1  2  1  1  2  2H, OCH CH CH3), 3.21-3.12 (m, 4H, H-17 , H-13 ), 2.88 (s, 3H, 7 -C0 Me), 1.80 (s, 2  2  2  2  1  2  3H, 7-CH3), 1.70-1.59 (m, 2H, OCH CH CH ), 0.90 (t, J= 6.9 Hz, 3H, OCH CH CH ), 2  -2.41, -2.47 (2br,2H, 2NH)  2  3  2  2  3  201 Chapter 4 Experimental  LREIMS (m/z): 792 ([M+H] ) +  HREIMS m/z: calcd. for C 5H N 09 (M ): 792.3734 +  4  52  4  found: 792.3729 UV/Vis. (> /Abs., CH C1 ): 355.0 (0.816), 425.0 (1.248), ,580.0 (0.295), 620.0 (0.157), max  2  2  and 680.0 (0.566)  3-Devinyl-3-(2-butyloxyethyl) ring B-BPD-1,3-diene dimethyl ester (62d)  62d 3-Devinyl-3-(2-bromoethyl) ring B-BPD-1,3-diene dimethyl ester 71 (60 mg, 0.076 mmol), dry mercury(II) oxide/tetrafluoroboric acid (14 mg, 0.038 mmol) and 0.03 m L 1-butanol (0.3mmol) were utilized by following the general procedure for primary ether synthesis. Product 62d was obtained as a black powder after  flush column  chromatography (15 mg, 25 %). ]  H N M R (300 M H z , CDC1 ) £9.70, 9.69, 9.37, 8.98 (4s, 4H, H-meso), 7.81 (d, J= 5.7 3  Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.32 (t, J= 7.5 Hz, 2H, 3  4  1  H-17 ), 4.17-4.05 (m, 6H, H-13 , H-3 , H-3 ), 3.97 (s, 3H, 7 -C0 Me), 3.64, 3.62, 3.54, 1  1  1  2  2  2  3.47, 3.40 (5s, 5x3H, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 ), 3.53-5.55 (m, 2H, 2  2  2  1  1  1  2  O C H C H C H C H ) , 3.21-3.12 (m, 4H, H-17 , H-13 ), 2.87 (s, 3H, 7'-C0 Me), 1.80 (s, 2  2  2  2  3  2  2  202 Chanter 4 Experimental  7-CH3),  3H,  1.63-1.58  (m, 2H, OCH CH CH CH3), 2  2  1.41-1.33  2  (m, 2H,  O C H C H C H C H ) , 0.87 (t, J= 6.9 Hz, 3H, O C H C H C H C H ) , -2.41 (br, 2H, 2NH) 2  2  2  3  2  2  2  3  LREEVIS (m/z): 806 (M ) +  HREIMS m/z: calcd. for C 6H54N 09(M ): 806.3891 +  4  4  found: 806.3889 UV/Vis. (X /Abs., CH C1 ): 355.0 (0.214), 425.1 (0.332), 580.0 (0.077), and 680.0 max  2  2  (0.150)  3-Devinyl-3-(2-pentyloxyethyl) ring B-BPD-l,3-diene dimethyl ester (62e)  62e 3-Devinyl-3-(2-bromoethyl) ring B-BPD-1,3-diene dimethyl ester 71 (100 mg, 0.12 mmol), dry mercury(II) oxide/tetrafluoroboric acid (24 mg, 0.06 mmol) and 0.04 mL 1pentanol (0.36mmol) were utilized by following the general procedure for primary ether synthesis. Product 62e was obtained as a black powder after  flush column  chromatography (49 mg, 50 %). !  H N M R (400 M H z , CDC1 ) £ 9 . 7 1 , 9.70, 9.37, 8.99 (4s, 4H, H-meso), 7.82 (d, J= 5.7 3  Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 5.06 (s, 1H, H-7 ), 4.31 (t, J = 7.5 Hz, 2H, 3  4  1  Chapter 4  203 Experimental  H-17 ), 4.21-4.08 (m, 6H, H-13 , H-3 , H-3 ), 3.98 (s, 3H, 7 -C0 Me), 3.64, 3.63, 3.54, 1  1  1  2  2  2  3.47, 3.41 (5s, 5x3H, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 ), 3.54 (t, J= 6.7 Hz, 2  2  1  2  1  1  2  2H, O C H C H C H C H C H ) , 3.21-3.13 (m, 4H, H-17 , H-13 ), 2.89 (s, 3H, 7 -C0 Me), 2  2  2  2  2  2  1  3  2  1.81 (s, 3H, 7-CH ), 1.63-1.62 (m, 2H, O C H C H C H C H C H ) , 2  3  2  2  2  1.35-1.24 (m, 4H,  3  O C H C H C H C H C H ) , 0.85 (t, J = 6.9 Hz, 3H, O C H C H C H C H C H ) , -2.41, -2.48 2  2  2  2  3  2  2  2  2  3  (2br, 2H, 2NH) LREIMS (m/z): 820 ( M ) +  HREIMS m/z: calcd. for C47H56N4O9 (M ): 820.4047 +  found: 820.4045 UV/Vis. (X /Abs., CH C1 ): 355.0 (0.535), 425.1 (0.832), 580.0 (0.193), and 680.0 max  2  2  (0.370)  3-Devinyl-3-(2-hexyloxyethyl) ring B-BPD-1,3-diene dimethyl ester (62f)  3-Devinyl-3-(2-bromoethyl) ring B-BPD-1,3-diene dimethyl ester 71 (104 mg, 0.128 mmol), dry mercury(II) oxide/tetrafluoroboric acid (25 mg, 0.06 mmol) and 0.05 mL 1-hexanol (0.38mmol) were utilized by following the general procedure for primary ether synthesis. Product 62f was obtained as a black powder after flush column chromatography (60 mg, 56 %).  204 Chapter 4 Experimental J  H N M R (300 M H z , CDC1 ) £9.70, 9.37, 8.98 (3s, 4H, H-meso), 7.81 (d, J= 5.7 Hz, 1H, 3  H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.31 (t, / = 7.8 Hz, 2H, H-17 ), 3  4  1  1  4.17-4.10 (m, 6H, H-13 , H-3 , H-3 ), 3.97 (s, 3H, 7 -C0 Me), 3.63, 3.62, 3.54, 3.48, 1  1  2  2  2  3.40 (5s, 5x3H, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 ), 3.53 (t, J= 6.7 Hz, 2H, 2  2  2  1  1  1  2  O C H C H C H C H C H C H ) , 3.21-3.12 (m, 4H, H-17 , H-13 ), 2.87 (s, 3H, 7'-C0 Me), 2  2  2  2  2  2  2  3  2  1.80 (s, 3H, 7-CH ), 1.64-1.59 (m, 2H, O C H C H C H C H C H C H ) , 1.33-1.24 (m, 6H, 3  2  2  2  2  2  3  O C H C H C H C H C H C H ) , 0.80 (t, J= 6.9 Hz, 3H, O C H C H C H C H C H C H ) , -2.48 2  2  2  2  2  3  2  2  2  2  2  3  (br, 2H, 2NH) LREIMS (m/z): 834 (M ) +  Anal, calcd. for C 8H N 09, C: 69.04; H : 7,00; N : 6.76 4  58  4  Found, C: 68.91; H : 7.08; N : 6.76  3-Devinyl-3-(2-heptyloxyethyl) ring B-BPD-1,3-diene dimethyl ester (62g)  3-Devinyl-3-(2-bromoethyl) ring B-BPD-1,3-diene dimethyl ester 71 (90 mg, 0.11 mmol), dry mercury(II) oxide/tetrafluoroboric acid (21 mg, 0.05 mmol) and 0.05 mL 1heptanol (0.35mmol) were utilized by following the general procedure for primary ether  205 Chapter 4 Experimental  synthesis. Product 62g was obtained as a black powder after  flush column  chromatography (49 mg, 53 %). *H N M R (300 M H z , CDC1 ) £9.70, 9.69, 9.37, 8.98 (4s, 4H, H-meso), 7.81 (d, J= 5.7 3  Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.31 (t,J= 7.8 Hz, 2H, 3  4  1  H-17 ), 4.19-4.10 (m, 6H, H-13 , H-3 , H-3 ), 3.97 (s, 3H, 7 -C0 Me), 3.64, 3.62, 3.54, 1  1  1  2  2  2  3.48, 3.40 (5s, 5x3H, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 ), 3.54 (t, J= 6.7 Hz, 2  2  1  2  1  1  2  2H, O C H C H C H C H C H C H C H ) , 3.21-3.12 (m, 4H, H-17 , H-13 ), 2.87 (s, 3H, 7 2  2  2  2  2  2  2  2  1  3  C 0 M e ) , 1.80 (s, 3H, 7-CH ), 1.64-1.59 (m, 2H, O C H C H C H C H C H C H C H ) , 2  3  2  2  2  2  2  2  3  1.33-1.24 (m, 8H, O C H C H C H C H C H C H C H ) , 0.79 (t, J = 6.9 Hz, 3H, 2  2  2  2  2  2  3  O C H C H C H C H C H C H C H ) , -2.41 (br, 2H, 2NH) 2  2  2  2  2  2  3  LREIMS (m/z): 848 (M ) +  Anal, calcd. for C49H60N4O9, C: 69.32; H : 7.12; N : 6.60 Found, C: 69.42; H : 7.21; N : 6.43 UV/Vis. ( W A b s . , CH C1 ): 355.0 (0.480), 430.0 (0.774), 580.0 (0.172), 620.0 (0.093), 2  2  and 680.0 (0.326)  Ring B-BPD-1,3-diene dimethyl ester ether dimer (86)  206 Chapter 4 Experimental  3-Devinyl-3-(2-bromoethyl) ring B-BPD-1,3-diene dimethyl ester 70 (18 mg, 0.024 mmol), dry mercury(II) oxide/tetrafluoroboric acid (5.0 mg, 0.012 mmol) and 3-devinyl3-(2-hydroxyethyl) ring B-BPD-1,3-diene dimethyl ester 69 (21 mg, 0.026 mmol) were utilized by following the general procedure. Product 86 was obtained after flush column chromatography (5.0 mg, 15 %).  J  H N M R (300MHz, CDC1 ) 59.10 (s, 2H, 2H-meso), 9.58, 9.57 (2s, 2H, 2H-meso), 9.37, 3  9.03 (2s, 4H, 4H-meso), 7.79 (d, J= 5.7 Hz, 2H, H-7 , H-7 '), 7.41 (d, J= 5.7 Hz, 2H, H 3  3  7 , H-7 '), 5.07, 5.06 (2s, 1H, H-7 , H-7 '), 4.31-4.16 (m, 14H, H-17 , H-17 ', H-3 , H 4  4  1  1  1  1  1  3 ', H-3 , H-3 ', H-13 , H-13 '), 3.93, 3.91 (2s, 2Me, 7 - C 0 M e , 7 '-C0 Me), 3.64, 3.63, 1  2  2  1  1  2  2  2  2  3.48, 3.41 (4s, 8Me, 17 -C0 Me, 17 '-C0 Me, 13 -C0 Me, 13 '-C0 Me, H-18 , H-18 ', 2  2  4  2  2  4  2  1  1  2  H-2 , H-2 '), 3.31, 3.30 (2s, H-12 , H-12 '), 3.20-3.10 (m, 8H, H-17 , H-17 ', H-13 , H 1  1  1  1  2  2  2  13 '), 2.84 (s, 2Me, 7 - C 0 M e , 7 - C 0 M e ) , 1.79, 1.78 (2s, 2Me, 7-CH , 7'-CH ), -2.34, 2  1  r  2  2  3  2.36 (2br, 4H, 4NH) E S M S (m/z): 1483.1 ([M+H] ) +  HRESIMS m/z: calcd. for C g ^ o N g O n (M ): 1483.6424 +  found: 1483.6428  3  207 Chapter 4 Experimental  4.5 Cross-Metathesis Products Ring B-BPD-1, 3-diene di(6-hexenyl) ester (101)  Ring B-BPD-1,3-diene dimethyl ester (61, 1.0 g, 1.37 mmol) was treated with 25 % HC1 (20 mL) overnight in the fridge. The methyl ester was hydrolyzed to the dicarboxylic acid mixed with some mono acids. The diacid was separated by column chromatography (methylene chloride/methanol, 100:5) as a black solid (780 mg, 80 %). The diacid (100 mg, 0.14 mmol) was refluxed in CH2CI2 for 10 min under nitrogen. Oxalyl chloride (0.5 mL, 5.7 mmol) was added and the reflux was continued for another 45 min. The reaction mixture was cooled and the solvent was removed in vacuo. The acid chloride so obtained was redissolved in dry CH2CI2 (8 mL) and 5-hexene-l-ol (0.17 mL, 1.4 mmol) was added. The mixture was stirred at room temperature under nitrogen overnight. It was then diluted with CH C1 (50 mL), washed with water, 10 % N a H C 0 and water. The solvent and the 2  2  3  excess 5-hexene-l-ol were removed in vacuo. The residue was purified by column chromatography (CH Cl /MeOH, 100:0.4) to give ring B-BPD-1, 3-diene di(6-hexenyl) 2  2  ester 101 as a black solid (85 mg, 70 %). ' H N M R (300MHz, CDC1 ) £9.74, 9.68, 9.35, 9.13 (4s, 4H, H-meso), 8.11 (dd, J= 18.0 3  Hz and 12.0 Hz, 1H, H-3 ), 7.81 (d, J = 5.7 Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 1  3  4  208 Chapter 4 Experimental  6.35 (d, J= 18.0 Hz, 1H, H-3 (£)), 6.15 (d, J= 12.0 Hz, 1H, H-3 (Z)), 5.50-5.46 (m, 2H, 2  2  2C02(CH )4CH=CH ), 5.04 (s, 1H, H-7 ), 4.79-4.70 (m, 4H, 2C0 (CH )4CH=CH ), 1  2  2  2  2  2  4.30 (t, J = 7.8 Hz, 2H, H-17 ), 4.16 (t, J= 1.1 Hz, 2H, H-13 ), 4.05-3.99 (m, 4H, 1  1  2C0 CH CH CH CH CH=CH ), 3.97 (s, 3H, 7 -C0 Me), 3.62, 3.47, 3.41 (3s, 3x3H, H2  2  2  2  2  2  2  2  18 , H-2 , H-12 ), 3.20-3.10 (m, 4H, H-17 , H-13 ), 2.93 (s, 3H, 7 -C0 Me), 1.79 (m, 1  1  1  2  2  1  2  4H,  2C0 (CH ) CH CH=CH ), 2  2  3  2  1.76  2  (s,  3H, 7-CH ), 3  1.46-1.16  (m, 8H,  2C0 CH CH CH CH CH=CH ), -2.31 (br, 2H, 2NH); 2  2  2  2  2  2  LREIMS (m/z): 868 (M ) +  HRESIMS m/z: calcd. for C H iN 08 ([M+H] ): 869.4489 +  52  6  4  found: 869.4485  Ring B-BPD ring-closing metathesis derivative (102)  102  The mixture of ring B-BPD-1,3-diene di(5-hexenyl) ester 101 (10 mg, 0.011 mmol) and catalyst 91 (1 mg, 0.001 mmol) in 3 mL freshly distilled dry THF was refluxed for 3 h. The solvent was removed in vacuo and the residue was purified by flush column chromatograph to give product 102 as a black solid (7 mg, 78 %).  209 Chanter 4 Experimental  H N M R (400MHz, CDC1 ) £9.74, 9.66, 9.34, 9.13 (4s, 4H, H-meso), 8.11 (dd, J= 18.0  l  3  Hz and 12.0 Hz, 1H, H-3 ), 7.81 (d,J= 5.7 Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 1  3  4  6.35 (d, J= 18.0 Hz, 1H, H-3 (£)), 6.15 (d, J= 12.0 Hz, 1H, H-3 (Z)), 5.05-5.02 (m, 3H, 2  2  H-7',CH=CH), 4.30-4.28 (m, 2H, H-17 ), 4.16-4.14 (m, 2H, H-13 ), 4.10-3.99 (m, 4H, 1  1  2CO2CH2 in the tail ring), 3.97 (s, 3H, 7 -C0 Me), 3.61, 3.47, 3.41 (3s, 3Me, H-18 , H 2  1  2  2 , H-12 ), 3.22-3.10 (m, 4H, H-17 , H-13 ), 2.92 (s, 3H, 7 -C0 Me), 1.76 (t, 4H, 1  1  2  2  1  2  CH CH=CHCH ),1.76 (s, 3H, 7-CH ), 1.47-1.11 (m, 8H, CH CH CH CH=CH 2  2  3  2  2  2  C H C H CH ), -2.31 (s, 2H, 2NH) 2  2  2  ESIMS (m/z): 841.4 ([M+H] ) +  HREIMS m/z: calcd. for C oH N 08 (M ): 840.4098 +  5  56  4  found: 840.4100  6-Hexenyl pyropheophorbide a ester (103)  103 Methyl pyropheophorbide a 39 (20 mg, 0.036 mmol) in 2 mL THF was hydrolyzed to the corresponding carboxylic acid by treatment with aqueous L i O H (1 M, 2 mL). The crude acid was reflux ed with CH2CI2 for 10 min under nitrogen. Oxalyl chloride (0.1 mL, 1.15 mmol) was added and the stirring was continued for another 45 min. The reaction mixture was cooled and the solvent was removed in vacuo. The acid chloride so obtained  210 Chapter 4 Experimental was redissolved in dry CH2CI2 (3 mL) and 5-hexene-l-ol (0.05 mL 0.4 mmol) was added. The mixture was stirred at room temperature under nitrogen overnight. It was then diluted with CH2CI2 (10 mL), washed with water, 10 % NaHC03 and water. The solvent and the excess 5-hexene-l-ol were removed in vacuo. The residue was purified on preparative T L C ( C H C l / M e O H , 100:0.2) to give product 103 as a black solid (4 mg, 18 %). 2  ]  2  H N M R (400 M H z , CDCI3) £9.47, 9.37, 8.54 (3s, 3H, H-meso), 7.99 (dd,' J= 17.8 Hz  and 11.5 Hz, 1H, H-3 ), 6.27 (d, J= 17.8 Hz, 1H, H-3 (E)), 6.16 (d, J= 11.5 Hz, 1H, H 1  2  3 (Z)), 5.70-5.62 (m, 1H, OCH CH CH2CH CH=CH2), 5.17 (q, 2H, H-13 ), 4.93-4.86 2  2  2  2  2  (m, 2H, O C H 2 C H C H C H C H = C H 2 ) , 4.48-4.45 (m, 1H, H-18), 4.29-4.27 (m, 1H, H 2  2  2  17), 4.01-3.89 (m, 2H, O C H C H C H C H C H = C H ) , 3.67 (q, J= 7.6 Hz, 2H, H-8 ), 3.65 1  2  2  2  2  2  (s, 3H, H-12 ), 3.39 (s, 3H, H-2 ), 3.22 (s, 3H, H-7 ), 2.67-2.53 (m, 2H, H-17 ), 1  1  1  1  2.35-2.24 (m, 2H, H-17 ), 1.94 (q, 2H, O C H C H C H C H C H = C H ) , 1.79 (d, J= 7.3 Hz, 2  2  3H,  H-18 ), 1  1.68  (t,  J  =  7.6  Hz,  2  2  2  3H,  2  H-8 ),  1.48-1.43  2  (m,  2H,  OCH CH2CH2CH CH=CH2) 1.31-1.26 (m, 2H, O C H C H C H C H C H = C H ) 0.45 (br, 2  2  2  1H, NH),-1.69(br, 1H, NH) LREIMS (m/z): 616 ( M ) +  2  2  2  2  211 Chapter 4 Experimental  Pyropheophorbide a (7-trimethylsilanyl)-5-heptenyl ester (104)  104 An oven-dried flask was charged with 5-hexenyl pyropheophorbide a ester 103 (4 mg, 0.0067mmol), allyl trimethylsilane (8.0 mg, 0.01 mL, 0.07 mmol) and condenser. A solution of catalyst 91 (1.0 mg, 0.0012 mmol) dissolved in freshly distilled dry THF (1 mL) was added via syringe to the flask and the mixture was refluxed mildly under Argon for 3 h. The solvent was removed in vacuo and the residue was purified by preparative T L C plate (CH Cl /MeOH, 100:0.8) to give product 104 as a black solid (3 mg, 65 %). 2  2  ' H N M R (400 M H z , CDC1 ) £9.50, 9.38, 8.54 (3s, 3H, H-meso), 7.98 (dd, J= 17.8 Hz 3  and 11.5 Hz, 1H, H-3 ), 6.29 (d, ./= 17.8 Hz, 1H, H-3 (£)), 6.17 (d, J= 11.5 Hz, 1H, H 1  2  3 (Z)), 5.32-5.30 (m, 1H, O C H C H C H C H C H = C H C H T M S ) , 5.17 (q, 2H, H-13 ), 2  2  2  2  2  2  2  5.11-5.09 (m, 1H, O C H C H C H C H C H = C H C H T M S ) , 4.49-4.45 (m, 1H, H-18), 2  2  2  2  2  4.30-4.26 (m, 1H, H-17), 4.01-3.90 (m, 2H, O C H C H C H C H C H = C H C H T M S ) , 3.64 2  2  2  2  2  (q, 7=7.6 Hz, 2H, H-8 ), 3.66 (s, 3H..H-12 ), 3.39 (s, 3H, H-2 ), 3.23 (s, 3H, H-7 ), 1  2.64-2.50  (m,  1  1H, H-17 ), 1  2.27-2.20  1  (m,  1  2H, H-17 ),  1.94  2  (q, 2H,  O C H C H C H C H C H = C H C H T M S ) , 1.79 (d, J= 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 7.6 Hz, 1  2  2  2  2  2  3H, H-8 ), 1.52-1.44 (m, 2H, O C H C H C H C H C H = C H C H T M S ) , 1.31-1.29 (m, 2H, 2  2  2  2  2  2  O C H C H C H C H C H = C H C H T M S ) , 1.31-1.26 (m, 2H, O C H C H C H C H C H = C H 2  2  2  2  2  2  2  C H T M S ) , 0.45 (br, 1H, NH), -0.1 l(s, 9H, Si(CH ) ), -1.69 (br, 1H, NH); 2  3  3  2  2  212 Chapter 4 Experimental  LREIMS (m/z): 702 (M ) +  3-Devinyl 3-(l-hexenyl) ring B-BPD-1,3-diene dimethyl ester (105)  105 A n oven-dried flask was charged with Zn(II) ring B-BPD-1,3-diene dimethyl ester 112 (32 mg, 0.04mmol), 1-hexene (67 mg, 0.1 mL, 0.8 mmol) and condenser. A solution of catalyst 96 (9 mg, 0.01 mmol) dissolved in freshly distilled dry THF (1 mL) was added via a syringe to the flask. The reaction mixture was refluxed mildly under Argon for 1 h. The solvent was removed in vacuo and the residue was purified by flush column chromatography (CH Cl /MeOH, 100:0.5). The Zn(II) complex of the C M product thus 2  2  obtained (32 mg, 95 %) was redissolved in CH C1 2  2  (10 mL) and treated with  trifluoroacetic acid (0.5 mL) for 30 min at room temperature. The mixture was diluted with CH C1 (20 mL) and washed by water, 5 % sodium bicarbonate and water. After 2  2  dried over sodium sulfate, the solvent was removed in vacuo. The residue was purified by flush column chromatography to give the product 3-devinyl 3-(l-hexenyl) ring B-BPD-1, 3-diene dimethyl ester 105 as a black solid (29 mg, 98 %). ]  H N M R (300 M H z , CDC1 ) £9.72, 9.67, 9.36, 9.20 (4s, 4H, H-meso), 7.81 (d, J = 5.7 3  Hz, 1H, H-7 ), 7.65 (d, J= 15.9 Hz, 1H, H-3 (^-isomer)), 7.44 (d, J= 5.7 Hz, 1H, H-7 ), 3  1  4  213 Chapter 4 Experimental  6.64 (td, 7 = 15.9 and 7.0 Hz, 1H, H-3 ), 5.05 (s, 1H, H-7 ), 4.29 (t, J= 7.7 Hz, 2H, H 2  1  17 ), 4.17 (t, J= 7.7 Hz, 2H, H-13 ), 3.97 (s, 3H, 7 -C0 Me), 3.65, 3.63, 3.58, 3.46, 3.41, 1  1  2  2  2.95 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 , 7 -C0 Me), 3.20-3.13 (m, 2  2  2  1  1  1  1  2  2  4H, H-17 , H-13 ), 2.75 (q, J= 7.0 Hz, 2H, H-3 ), 1.91-1.68 (m, 4H, H-3 , H-3 ), 1.78 (s, 2  2  3  4  5  3H, 7-CH ), 1.15 (t, J= 7.3 Hz, 3H, H-3 ), -2.34 (br, 2H, 2NH); 6  3  1 3  C N M R (75 M H z , CDCI3) 8 173.7, 173.3, 170.6, 167.6, 165.5, 156.6, 152.0 151.0,  140.1, 139.3, 138.2, 137.5, 137.3, 137.2, 136.0, 134.6, 133.8, 132.6, 131.9, 130.9, 122.0, 121.8, 112.4, 99.3, 93.4, 91.9, 52.7, 52.2, 51.7, 51.6, 51.5, 47.9, 37.0, 36.6, 34.3, 32.1, 27.5, 23.1, 22.6, 21.8, 21.5, 14.1, 12.5, 11.6, 11.2 LREIMS (m/z): 788 (M ) +  Anal, calcd. for C48H58N4O9, C: 69.75; H : 6.73; N : 7.77. found, C: 70.03; H : 6.44; N : 7.10. UV/Vis. (> /Abs., CH C1 ): 355.0 (0.401), 435.1 (0.766), 590.0 (0.138), and 689.9 max  2  2  (0.244)  3-Devinyl 3-(l-octenyl) ring B-BPD-1,3-diene dimethyl ester (106)  106  214 Chapter 4 Experimental  A n oven-dried flask was charged with ring B-BPD-1,3-diene dimethyl ester 61 (30 mg, 0.04mmol), 1-octene (90 mg, 0.12 mL, 0.8 mmol) and condenser. A solution of catalyst 96 (9 mg, 0.01 mmol) dissolved in freshly distilled dry THF (1 mL) was added via a syringe to the flask. The reaction mixture was refluxed mildly under Argon for 1 h. The solvent was removed in vacuo and the residue was purified by flush column chromatography (CH Cl /MeOH, 100:0.5) to give 3-devinyl 3-(l-octenyl) ring B-BPD2  2  1,3-diene dimethyl ester 106 as a black solid (32 mg, 96 %). ]  H N M R (300MHz, CDC1 ) £9.72, 9.67, 9.36, 9.11 (4s, 4H, H-meso), 7.83 (d, J= 5.7 Hz, 3  1H, H-7 ), 7.76 (d, J = 16.1 Hz, 0.98H, H-3 (^-isomer)), 7.51 (d, J= 11.0 Hz, 0.02H, H 3  1  3 (Z-isomer)), 7.45 (d,J= 5.7 Hz, 1H, H-7 ), 6.81 (td, J= 16.0 Hz and 7.0 Hz, 0.98H, 1  4  H-3 (^-isomer)), 6.52 (dt, J = 11.0 Hz and 7.0 Hz, 0.02H, H-3 (Z-isomer)), 5.07 (s, 1H, 2  2  H-7 ), 4.28 (t, J= 7.5 Hz, 2H, H-17 ), 4.18 (t, J= 7.5 Hz, 2H, H-13 ), 3.99 (s, 3H, 7 1  1  1  2  C 0 M e ) , 3.66, 3.63, 3.59, 3.44, 3.41, 2.96 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H-18 , H 2  2  2  2  1  2  2 , H-12 , 7 -C0 Me), 3.20-3.13 (m, 4H, H-17 , H-13 ), 2.76 (q, J= 6.9 Hz, 2H, H-3 ), 1  1  1  2  2  3  2  1.89-1.82 (m 2H, H-3 ), 1.74-1.64 (m, 2H, H-3 ), 1.53-1.44 (m, 4H, H-3 , H-3 ), 1.79 (s, 4  5  6  7  3H, 7-CH ), 1.00 (t, J= 6.9 Hz, 3H, H-3 ), -2.36 (br, 2H, 2NH); 8  3  1 3  C N M R (75 M H z , CDCI3) 5: 173.7, 173.3, 170.6, 167.7, 165.5, 156.6, 152.0, 151.0,  140.1, 139.3, 138.2, 137.5, 137.3, 137.2, 136.0, 134.6, 133.8, 132.6, 131.8, 130.9, 121.9, 121.8, 112.4, 99.3, 98.5, 93.4, 91.9, 52.7, 52.2, 51.7, 51.6, 51.5, 37.0, 36.6, 34.7, 31.9, 29.9, 29.2, 27.5,22.8,21.8,21.5, 14.2, 12.5, 11.6, 11.2 LREIMS(m/z): 816 ( M ) +  HREIMS m/z: calcd. for C 8H N 08 (M ): 816.4098 +  4  56  4  found: 816.4100  215 Chapter 4 Experimental  U V / V i s . (?t /Abs., CH2CI2): 355.0 (1.075), 430.1 (1.972), 590.0 (0.362), and 689.9 max  (0.644)  Methyl 3-devinyl 3-(l-hexenyl)-pyropheophorbide a (107)  107 A n oven-dried flask was charged with methyl pyropheophorbide a 39 (27 mg, 0.049mmol), 1-hexene (80 mg, 0.12 mL, 0.96 mmol) and condenser. Solution of catalyst 96 (11 mg, 0.012 mmol) dissolved in freshly distilled dry THF (1 mL) was added via a syringe to the flask. The reaction mixture was refluxed mildly under Argon for 1 h. The solvent was removed in vacuo and the residue solid was purified by flush column chromatography  (CH Cl /MeOH, 2  2  100:0.3,  v/v).  Methyl  3-devinyl-3-(l-hexenyl)  pyropheophorbide a 107 was obtained as a black solid (28 mg, 96 %). ' H N M R (300 M H z , CDCI3) £9.36, 9.23, 8.47 (3s, 3H, H-meso), 7.50 (d, J= 16.1 Hz, 1H, H-3 (^-isomer)), 6.68 (td, J= 16.0 Hz and 7.0 Hz, 1H, H - 3 (.E-isomer)), 5.15 (q, 1  2  2H, H-13 ), 4.48-4.41 (m, 1H, H-18), 4.26-4.24 (m, 1H, H-17), 3.63-3.56 (q, J = 7.6 Hz, 2  2H, H-8 ), 3.61 (s, 3H, 17 -C0 Me), 3.60 (s, 3H, H-12 ), 3.31 (s, 3H, H-2 ), 3.16 (s, 3H, 1  2  1  1  2  H-7 ), 2.68-2.55 (m, 3H, H-17 , Ha-3 ), 2.33-2.24 (m, 3H, H-17 , Ha'-3 ), 1.79 (d, 1  1  3  2  3  216 Chapter 4 Experimental J= 7.3 Hz, 3 H , H-18 ), 1.65 (t, J= 7.6 Hz, 3 H , H-8 ), 1.86-1.59 (m, 4 H , H - 3 , H-3 ), 1  2  4  5  I. 11 (t, .7=7.3 Hz, 3 H , H-3 ), 0.48 (br, 1H, NH),-1.70 (br, 1H, NH); 6  13  C N M R (75 M H z , CDC1 ) 8 196.1, 173.4, 171.4, 160.0, 155.2, 150.5, 149.0, 144.8, 3  141.9, 137.6, 136.6, 135.8, 134.7, 130.5, 127.9, 122.9, 121.6, 105.7, 103.9, 97.1, 97.0, 92.6, 51.6, 50.0, 48.0, 36.5, 34.1, 31.8, 30.9, 29.8, 23.0, 22.4, 19.4, 17.3, 14.1, 12.0, 11.9, II. 1 LREIMS (m/z): 604 (M ) +  HREIMS m/z: calcd. for C a s H ^ C ^ M * ) : 604.34134 found: 604.34146 U V / V i s . (X JAbs., m  CH2CI2): 319.9 (0.395), 415.0 (1.887), 510.1 (0.170), 540.1 (0.156),  605.0 (0.139), and 665.0 (0.661)  Methyl 3-devinyl 3-(l-octenyl)-pyropheophorbide a (108)  108 Followed the same procedure for the synthesis of 107. Methyl pyropheophorbide a 39 (31 mg, 0.057mmol), catalyst 96 (12 mg, 0.014 mmol) and 1-octene (127mg, 0.16mL, 1.14 mmol) were utilized. The reaction mixture was purified by flush column  217 Chapter 4 Experimental  chromatograph to give methyl 3-devinyl 3-(l-octenyl)-pyropheophorbide a 108 as a black solid (32 mg, 90 %). !  H N M R (400 MHz, CDC1 ) £9.37, 9.24, 8.47 (3s, 3H, H-meso), 7.51 (d, J= 16.0 Hz, 3  0.94H, H-3 (^-isomer)), 7.32 (d, J= 11.3 Hz, 0.06H, H-3 (Z-isomer)), 6.66 (td, J= 16.0 1  1  Hz and 7.0 Hz, 0.94H, H-3 (^-isomer)), 6.47 (dt, J = 11.3 Hz and 7.3 Hz, 0.06H, H - 3 2  2  (Z-isomer)), 5.15 (q, 2H, H-13 ), 4.47-4.42 (m, 1H, H-18), 4.26-4.24 (m, 1H, H-17), 2  3.64-3.58 (q, J= 7.6 Hz, 2H, H-8 ), 3.61 (s, 3H, 17 -C0 Me), 3.60 (s, 3H, H-12 ), 3.32 1  2  1  2  (s, 3H, H-2 ), 3.17 (s, 3H, H-7 ), 2.68-2.24 (m, 3H, H-17 , H-3 ), 2.29 (m, 2H, H-17 ), 1  1  1  3  2  1.79 (d,J= 7.3 Hz, 3H, H-18 ), 1.65 (t, J= 7.6 Hz, 3H, H-8 ), 1.8-1.46 (m, 8H, H-3 ~H1  2  4  3 ), 0.99 (t, J= 7.0 Hz, 3H, H-3 ), 0.50 (br, 1H, NH), -1.70 (br, 1H, NH); 7  1 3  8  C N M R (100 M H z , CDC1 ) 5: 196.3, 173.5, 171.5, 160.1, 155.3, 150.5, 149.0, 144.9, 3  141.9, 140.7, 137.6, 136.6, 136.4, 135.9, 130.6, 130.2, 128.0, 121.6, 105.7, 104.0, 97.2, 92.7, 51.7, 51.6, 49.9, 47.9, 34.5, 31.9, 30.9, 29.8, 29.7, 23.1, 22.8, 19.4, 17.4, 14.2, 12.1, 12.0, 11.2 E S M S (m/z): 633 ([M+H] ) +  Anal, calcd. for .C40H46N4O3, C: 75.92; H : 7.64; N : 9.03. found: C: 76.22; H : 7.51; N : 8.85. UV/Vis. (X /Abs., CH C1 ): 319.9 (0.846), 410.0 (3.330), 510.1 (0.400), 540.1 (0.365), max  2  2  610.0 (0.330), and 665.0 (1.695)  218 Chapter 4 Experimental  3,8-Didevinyl 3,8-di(l-octenyl) protoporphyrin IX dimethyl ester (109)  109 Following the same procedure for the synthesis of 105. Zn(II) protoporphyrin IX dimethyl ester 110 (45 mg, 0.07 mmol), catalyst 96 (15 mg, 0.018 mmol) and 1-octene (318 mg, 0.45 mL, 2.8 mmol) were employed. After T F A treatment, the reaction mixture was purified by flush column chromatography to give 3,8-Didevinyl 3,8-di(l-octenyl) protoporphyrin IX dimethyl ester 109 as a red-black solid (48 mg, 92 %). j  H N M R (400 MHz, CDC1 ) £10.07, 9.95 (2s, 4H, H-meso), 7.85-7.78 (m, 2H, H-3 , H 1  3  8 ), 6.81-6.74 (m, 2H, H-3 , H-8 ), 4.37-4.33 (m, 4H, H-13 , H-17 ), 3.65, 3.60, 3.58, 1  2  2  1  1  3.57 (4s, 6Me, 13 -C0 Me, 17 -C0 Me, 2-Me, 7-Me, 12-Me, 18-Me), 3.27-3.23 (m, 4H, 2  2  2  2  H-13 , H-17 ), 2.78-2.72 (m, 4H, H-3 , H-8 ), 1.87-1.43 (m, 16H, H-3 ~H-3 , H-8 ~H2  2  3  3  4  7  4  8 ), 1.10 (t, J= 6.9 Hz, 6H, H-3 , H-8 ), -3.84 (br, 2H, 2NH) 7  8  8  LREIMS (m/z): 758 (M ) +  HRESIMS m/z: calcd. for C 8H 3N 04([M+H] ): 759.4849 +  4  6  4  found: 759.4847 UV/Vis. (X /Abs., CH C1 ): 405.0 (1.051), 505.1 (0.142), 540.1 (0.132), 575.0 (0.106), max  and 630.0 (0.096)  2  2  219 Chapter 4 Experimental  3-Devinyl 3-(6-bromo-l-hexenyI) ring B-BPD-1,3-diene dimethyl ester (113)  113 Following the same procedure for the synthesis of 105. Zn-ring B-BPD-1,3-diene dimethyl ester 112 (32 mg, 0.04 mmol), catalyst 96 (9 mg, 0.01 mmol) and 6-bromo-1hexene (163 mg, 0.1 mL, 0.8 mmol) were employed. The reaction mixture was treated with T F A to remove Zn(II). 3-devinyl 3-(6-bromo-1-hexenyl) ring B-BPD-1,3-diene dimethyl ester 113 was obtained as a black solid after flush column chromatography (31 mg, 90%). !  H N M R (300 M H z , CDC1 ) £9.72, 9.67, 9.36, 9.09 (4s, 4H, H-meso), 7.83 (d, J= 5.7 3  Hz, 1H, H-7 ), 7.77 (d, J = 16.1 Hz, 1H, H-3 (£-isomer)), 7.45 (d,J= 5.7 Hz, 1H, H-7 ), 3  1  4  6.78 (dt, J= 16.0 Hz and 7.0 Hz, 1H, H-3 (^-isomer)), 5.06 (s, 1H, H-7 ), 4.28 (t,J= 7.6 2  1  Hz, 2H, H-17 ), 4.17 (t, J= 7.6 Hz, 2H, H-13 ), 3.99 (s, 3H, 7 -C0 Me), 3.68 (t, J = 6.7 1  1  2  2  Hz, 2H, H-3 ), 3.66, 3.63, 3.59, 3.44, 3.41, 2.94 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H 6  2  2  2  2  18 , H-2 , H-12 , 7'-C0 Me), 3.18, 3.15 (2t, J= 7.6 Hz, 4H, H-17 , H-13 ), 2.80 (q, 1  1  1  2  2  2  J = 7.0 Hz, 2H, H-3 ), 2.33-2.24 (m, 2H, H-3 ), 2.08-2.01 (m, 2H, H-3 ), 1.78 (s, 3H, 73  CH ), -2.35 (br, 2H, 2NH); 3  4  5  220 Chapter 4 Experimental 13  C N M R (75 M H z , CDC1 ) 8 173.7, 173.3, 170.6, 165.5, 156.6, 152.1, 152.0, 151.0, 3  140.1, 138.6, 138.3, 138.0, 137.4, 137.1, 136.1, 134.4, 133.9, 132.7, 131.5, 131.0, 123.5, 122.8, 121.8, 112.5, 99.5, 98.6, 93.4, 91.9, 52.6, 52.2, 51.7, 51.6, 51.5, 48.0, 37.0, 36.6, 33.9, 33.7, 32.6, 28.4, 27.8, 21.8, 21.5, 12.5, 11.6, 11.2 ESHMS (m/z): 868 ([M+H] ) +  HRESIMS m/z: calcd. for C 6H BrN 08 ([M+H] ): 867.2969 +  4  52  4  found: 867.2963 UV/Vis. (knax/Abs., CH2CI2): 355.0 (0.331), 435.1 (0.709), 590.0 (0.125), and 689.9 (0.174)  3-Devinyl 3-(6-hydroxy-l-hexenyl) ring B-BPD-1,3-diene dimethyl ester (116)  116 Following the same procedure for the synthesis of 105. Zn(II) ring B-BPD-1,3diene dimethyl ester 112 (32 mg, 0.04 mmol), catalyst 96 (9 mg, 0.01 mmol) and 1hydroxy-5-hexene (82 mg, 0.1 mL, 0.8 mmol) were employed. After T F A treatment, the reaction mixture was purified by preparative T L C to give 3-devinyl 3-(6-hydroxy-lhexenyl) ring B-BPD-1,3-diene dimethyl ester 116 as a black solid (2 mg, 5 %).  221 Chapter 4 Experimental !  H N M R (400 M H z , CDC1 ) 59.12, 9.68, 9:36, 9.10 (4s, 4H, H-meso), 7.80 (d, J= 5.6 3  Hz, 1H, H-7 ), 7.69 (d, J = 16.0 Hz, 1H, H-3 (^-isomer)), 7.42 (d, J= 5.6 Hz, 1H, H-7 ), 3  1  4  6.80 (td, J= 16.0 Hz and 7.0 Hz, 1H, H-3 (^-isomer)), 5.03 (s, 1H, H-7 ), 4.31 (t, J= 7.5 2  1  Hz, 2H, H-17 ), 4.17 (t, J = 7.5 Hz, 2H, H-13 ), 3.97 (s, 3H, 7 -C0 Me), 3.93 (t, J = 6.2 1  1  2  2  Hz, 2H, H-3 ), 3.64, 3.62, 3.56, 3.47, 3.40, 2.87 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H 6  2  2  2  2  18 , H-2 , H-12 , 7'-C0 Me), 3.18, 3.16 (2t, J= 7.5 Hz, 4H, H-17 , H-13 ), 2.79 (q, 1  1  1  2  2  2  J= 7.0 Hz, 2H, H-3 ), 1.9-2.1 (m, 4H, H-3 , H-3 ), 1.75 (s, 3H, 7-CH ), -2.35 (br, 2H, 3  4  5  3  2NH). LREIMS (m/z): 804 (M ) +  HRESIMS m/z: calcd. for C46H N 09(M ): 804.3734 +  52  4  found: 804.3732  3-Devinyl 3-(5-acetoxy-l-pentenyl) ring B-BPD-1,3-diene dimethyl ester (117)  117 Following the same procedure for the synthesis of 105. Zn(II) ring B-BPD-1,3diene dimethyl ester 112 (35 mg, 0.044 mmol), catalyst 96 (10 mg, 0.011 mmol) and 1acetoxy-4-pentene (115 mg, 0.13 mL, 0.9 mmol) were utilized. After T F A treatment, the  222 Chapter 4 Experimental  reaction mixture was purified by flush column chromatograph to give 3-(5-acetoxy-lpentenyl) ring B-BPD-1,3-diene dimethyl ester 117 as a black powder (18 mg, 50 %). ' H N M R (300 M H z , CDC1 ) £9.73, 9.67, 9.36, 9.09 (4s, 4H, H-meso), 7.81 (d,J= 5.6 3  Hz, 1H, H-7 ), 7.80 (d, J= 16.2 Hz, 1H, H-3 (^-isomer)), 7.44 (d, J= 5.6 Hz, 1H, H-7 ), 3  1  4  6.77 (td, J= 16.0 Hz and 7.3 Hz, 1H, H-3 , (^-isomer)), 5.08 (s, 1H, H-7 ), 4.44 (t, J = 2  1  6.5 Hz, 2H, H-3 ), 4.29 (t, J= 7.6 Hz, 2H, H-17 ), 4.17 (t, J= 7.6 Hz, 2H, H-13 ), 3.97 (s, 5  1  1  3H, 7 -C0 Me), 3.65, 3.63, 3.59, 3.45, 3.41, 2.92 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H 2  2  2  2  2  2  18 , H-2 , H-12 , 7 -C0 Me), 3.18, 3.15 (2t, J = 7.6 Hz, 4H, H-17 , H-13 ), 2.84 (q, 1  1  1  1  2  2  2  J= 7.3 Hz, 2H, H-3 ), 2.25-2.19 (m, 2H, H-3 ), 2.19 (s, 3H, OCOCH3), 1.78 (s, 3H, 73  4  CH ), -2.35 (br, 2H, 2NH); 3  ESIMS (m/z): 833 ([M+H] ) +  Anal, calcd. for C47H N Oio, C: 67.77; H : 6.29; N : 6.73. 52  4  found, C: 67.96; H : 6.31; N : 6.58. U V / V i s . (X JAbs., m  CH C1 ): 355.0 (0.535), 435.1 (0.976), 590.0 (0.175), and 689.9 2  2  (0.325)  3-Devinyl  3-((6-/V-Boc-glycinate)-l-hexenyl)  dimethyl ester (118)  ring  B-BPD-1,3-diene  223 Chapter 4 Experimental BocHN  118 The mixture of Zn-ring B-BPD-1,3-diene dimethyl ester 112 (33 mg, 0.041 mmol), catalyst 96 (8 mg, 0.011 mmol) and 5-hexen-l-yl /V-Boc-glycinate (206 mg, 0.8 mmol) in 1 mL freshly distilled dry THF was reflux ed under Argon for l h . After the solvent was removed in vacuo, the residue was purified by flush column chromatograph to give product 118 as a black solid (23 mg, 55 %). !  H N M R (400 M H z , CDC1 ) £9.56, 9.48, 9.15, 8.80 (4s, 4H, H-meso), 7.75 (d, J= 5.7 3  Hz, 1H, H-7 ), 7.67 (d, J= 16.0 Hz, 1H, H-3 ), 7.36 (d, J= 5.7 Hz, 1H, H-7 ), 6.60 (dt, 3  1  4  J= 16.1 Hz and 7.3 Hz, 1H, H-3 ), 4.99 (s, 1H, H-7 ), 4.36 (t, 2H, H-3 ), 4.15-4.11 (m, 2  1  6  4H, H-17 , H-13 ), 3.96 (s, 3H, 7 -C0 Me), 3.64, 3.63, 3.47, 3.38, 3.31, 3.04 (6s, 6Me, 1  1  2  2  17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 , 7 -C0 Me), 3.12-3.09 (m, 4H, H-17 , H 2  2  2  1  1  1  1  2  2  2  13 ), 2.96 (d, .7=5.4 Hz, 2H, N H C H C 0 ) , 2.78-2.71 (m, 2H, H-3 ), 2.05-1.85 (m, 4H, 2  3  2  2  H-3 , H-3 ), 1.77 (s, 3H, 7-CH ), 1.47 (s, 9H, (CH ) OCO); 5  4  3  3  3  ESIMS (m/z): 1025 ([M+H] ) +  HRESIMS m/z: calcd. for C H N O i Z n ( [ M + H ] ) : 1024.3608 +  53  62  5  2  found: 1024.3672  224 Chapter 4 Experimental  3-Devinyl 3-(7-methoxycarbonyl-l,6-heptdienyl) ring B-BPD-1,3-diene dimethyl ester (119)  119 Following the same procedure for the synthesis of 105. The mixture of Zn(II) ring B-BPD-l,3-diene dimethyl ester 112 (10 mg, 0.014mmol), catalyst 96 (3 mg, 0.003 mmol) and l-methoxycarbonyl-l,6-hept-diene (50 mg, 0.05 mL, 0.32 mmol) in 1 ml freshly distilled THF were refluxed under Ar for 1 h. After T F A treatment, the mixture was purified by preparative T L C to give product 119 as a black solid (6 mg, 50 %). ' H N M R (300 M H z , CDC1 ) £9.72, 9.67, 9.35, 9.07 (4s, 4H, H-meso), 7.82 (d, J = 5.7 3  Hz, 1H, H-7 ), 7.76 (d, J= 16.8 Hz, 1H, H-3 (£-isomer)), 7.43 (d, J = 5.7 Hz, 1H, H-7 ), 3  1  4  7.20-7.14 (m, 1H, H-3 ), 6.79 (td, J = 16.8 Hz and 7.2 Hz, 1H, H-3 (£-isomer)), 6.04 (d, 6  2  J= 15.6 Hz, 1H, H-3 (£-isomer)), 5.04 (s, 1H, H-7 ), 4.29, 4.17 (2t, J= 7.5 Hz, 4H, H 7  1  17 , H-13 ), 3.97 (s, 3H, 7 -C0 Me), 3.76, 3.64, 3.62, 3.58, 3.47, 3.40, 2.91 (6s, 17 1  1  2  2  2  C 0 M e , 13 -C0 Me, 3 - C 0 M e , H-18 , H-2 , H-12 , ^-COzMe), 3.18, 3.15 (2t, / = 7.5 2  7  2  1  2  1  1  2  Hz, 4H, H-13 , H-17 ), 2.82-2.75 (m, 2H, H-3 ), 2.62-2.56 (m, 2H, H-3 ), 2.06-2.00 (m, 2  2  3  2H, H-3 ), 1.77(s, 3H, 7-CH ),-2.36 (br, 2H, 2NH) 4  3  ESIMS (m/z): 859 ([M+H] ) +  5  225 Chapter 4 Experimental  HRESIMS m/z: calcd. for C 9H55N Oio([M+H] ): 859.3918 +  4  4  found: 859.3926  Zn(II) 3-devinyl 3-(2-trimethoxysilane)-l-ethenyl ring B-BPD-1,3-diene dimethyl ester (120)  120 The mixture of Zn-ring B-BPD-1,3-diene dimethyl ester 112 (37 mg, 0.046 mmol), catalyst 96 (10 mg, 0.011 mmol) and vinyl trimethoxysilane (136 mg, 0.14 mL, 0.92 mmol) in 1 mL freshly distilled dry THF was refluxed under Argon for l h . After the solvent was removed in vacuo, the residue was purified by flush column chromatograph to give Zn(II) 3-devinyl 3-(2-trimethoxysilane)-1-ethenyl ring B-BPD-1,3-diene dimethyl ester 120 as a black solid (3 mg, 10 %). !  H N M R (400 M H z , CDC1 ) £9.62, 9.49, 9.14, 8.87 (4s, 4H, H-meso), 8.55 (d,J= 19.5 3  Hz, 1H, H-3 ), 7.75 (d, J= 5.7 Hz, 1H, H-7 ), 7.34 ( d , J= 5.7 Hz, 1H, H-7 ), 6.60 (d, 1  3  4  J= 19.5 Hz, 1H, H-3 ), 4.95 (s, 1H, H-7 ), 4.15 (t, J= 7.5 Hz, 4H, H-17 , H-13 ), 3.95 (s, 2  1  1  1  3H, 7 -C0 Me), 3.90 (s, 9H, Si(OMe) ), 3.64, 3.63, 3.54, 3.38, 3.31, 3.09 (6s, 6Me, 17 2  2  2  3  C 0 M e , 13 -C0 Me, H-18 , H-2 , H-12 , 7 -C0 Me), 3.13-3.09 (m, 4H, H-17 , H-13 ), 2  2  1  2  1.77 (s, 3H, 7-CH3);  1  1  1  2  2  2  226 Chapter 4 Experimental  UV/Vis. (A, /Abs., CH C1 ): 350.0 (0.625), 445.0 (0.894), 625.0 (0.309) and 674.9 max  2  2  (0.531) HRESIMS m/z: calcd. for C 5H ,N OioSiZn([M+H] ): 915.2540 +  4  5  4  found: 915.2545  3-Devinyl 3-(3-hydroxy-l-propenyl) ring B-BPD-1, 3-diene dimethyl ester (123)  123 Following the same procedure for the synthesis of 105. Zn(II) ring B-BPD-1,3diene dimethyl ester 112 (32 mg, 0.04 mmol), catalyst 96 (10 mg, 0.011 mmol) and 1,4bis((trimethylsilan)oxy)-2-butene (186 mg, 0.8 mmol) were employed. After refluxing, the reaction mixture was treated with T F A to remove Zn(II) and then treated with T B A F to remove TMS. The mixture was purified by flush column chromatograph to give 3-(3hydroxy-l-propenyl) ring B-BPD-1,3-diene dimethyl ester 123 as a black solid (13 mg, 43 %). !  H N M R (400 M H z , CDC1 ) £9.74, 9.67, 9.35, 9.08 (4s, 4H, H-meso), 8.01 (d, J= 16.1 3  Hz, 1H, H-3 (£-isomer)), 7.80 (d, / = 5.7 Hz, 1H, H-7 ), 7.43 (d, J= 5.7 Hz, 1H, H-7 ), 1  3  4  6.96 (td, / = 16.1 Hz and 6.4 Hz, 1H, H-3 (^-isomer)), 5.05 (s, 1H, H-7 ), 4.83 (d, 2  1  227 Chapter 4 Experimental  J= 6.4 Hz, 2H, H-3 ), 4.30 (t, / = 7.8 Hz, 2H, H-17 ), 4.16 (t, J= 7.8 Hz, 2H, H-13 ), 3  1  1  3.97 (s, 3H, 7 -C0 Me), 3.64, 3.62, 3.61, 3.46, 3.40, (5s, 5Me, 17 -C0 Me, 13 -C0 Me, 2  2  2  2  2  2  H-18 , H-2 , H-12 ), 3.18, 3.15 (2t, J = 7.6 Hz, 4H, H-17 , H-13 ), 2.89 (7 -C0 Me), 1  1  1  2  2  1  2  1.76 (s, 3H, 7-CH ), -2.23 (br, 2H, 2NH); 3  ESIMS (m/z): 763 ([M+H] ) +  HRESIMS m/z: calcd. for C43H N 0 ([M+H] ): 763.3343 +  47  4  9  found: 763.3345 UV/Vis. (X JAbs., m  CH C1 ): 355.0 (0.726), 435.1 (1.196), 585.0 (0.306), and 690.0 2  2  (0.467)  3-Devinyl 3-(3-acetoxy-l-propenyl) ring B-BPD-1, 3-diene dimethyl ester (124)  124 Following the same procedure for the synthesis of 105. Zn(II) ring B-BPD-1,3diene dimethyl ester 112 (32 mg, 0.04mmol), catalyst 96 (10 mg, 0.011 mmol) and cis-1, 4-diacetyloxy-2-butene (137mg, 0.12mL, 0.8 mmol) were employed. After T F A treatment, the reaction mixture was purified by flush column chromatograph to give 3-(3-  228 Chapter 4 Experimental  acetyloxy-l-propenyl) ring B-BPD-1, 3-diene dimethyl ester 124 as a black solid (17-mg, 54 %). R N M R (300 M H z , CDC1 ) £9.74, 9.66, 9.34, 9.08 (4s, 4H, H-meso), 7.82 (d, J= 5.7  l  3  Hz, 1H, H-7 ), 8.07 (d, J= 16.1 Hz, 1H, H-3 (^-isomer)), 7.45 (d, J = 5.7 Hz, 1H, H-7 ), 3  1  4  6.90 (td, J = 16.1 Hz and 6.4 Hz, 1H, H-3 (^-isomer)), 5.25 (d, J= 6.4 Hz, 2H, H-3 ), 2  3  5.06 (s, 1H, H-7 ), 4.27 (t, J= 7.8 Hz, 2H, H-17 ), 4.16 (t, J= 7.8 Hz, 2H, H-13 ), 3.98 (s, 1  1  1  3H, 7 - C 0 M e ) , 3.65 3.63, 3.62, 3.44, 3.40, 2.96 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H 2  2  2  2  2  2  18 , H-2 , H-12 , 7'-C0 Me), 3.18, 3.15 (2t, J= 7.6 Hz, 4H, H-17 , H-13 ), 2.32 (s, 3H, 1  1  1  2  2  2  OCOCH ), 1.78 (s, 3H, 7-CH3), -2.27 (br, 2H, 2NH); 3  ESIMS (m/z): 805 ([M+H] ) +  HRESIMS m/z: calcd. for C 5H49N Oio([M+H] ): 805.3449 +  4  4  found: 805.3447  Methyl 3-devinyl 3-(3-acetoxy-l-propenyl) pyropheophorbide a (125)  125 Following the  same procedure for  the  synthesis of  105.  Zn(II)  methyl  pyropheophorbide a 122 (26 mg, 0.042mmol), catalyst 96 (10 mg, 0.011 mmol) and cisl,4-diacetoxy-2-butene  (144mg, 0.13mL, 0.84 mmol) were employed. After T F A  229 Chapter 4 Experimental  treatment, the reaction mixture was purified by preparative TLC plate to give methyl 3devinyl 3-(3-acetoxy-l-propene) pyropheophorbide a 125 as a black solid (16 mg, 60 %). ' H N M R (300 MHz, CDC1 ) £ 9 . 5 1 , 9.34, 8.55 (3s, 3H, H-meso), 7.93 (d, J = 16.1 Hz, 3  1H, H-3 (^-isomer)), 6.82 (td, 7 = 16.0 and 6.2 Hz, 1H, H - 3 (^-isomer)), 5.17 (q, 2H, 1  2  H-13 ), 5.17 (d,J= 6.2 Hz, 2H, H-3 ), 4.51-4.44 (m, 1H, H-18), 4.30-4.27 (m, 1H, H 2  3  17), 3.70 (q, J= 7.7 Hz, 2H, H-8 ), 3.66 (s, 3H, 17 -C0 Me), 3.59 (s, 3H, H-12 ), 3.40 (s, 1  2  1  2  3H, H-2 ), 3.24 (s, 3H, H-7 ), 2.68-2.51 (m, 2H, H-17 ), 2.33-2.23 (m, 2H, H-17 ), 2.27 1  1  1  2  (s, 3H, OCOCH3), 1.79 (d, J= 7.3 Hz, 3H, H-18 ), 1.68 (t, J= 7.5 Hz, 3H, H-8 ), 0.40 (br, 1  2  1H, NH),-1.75 (br, 1H, NH); 13  C N M R (75 MHz, CDCI3) 8 197.6, 174.9, 172.7, 161.8, 156.5, 152.3, 150.4, 146.5,  142.7, 139.4, 137.6, 137.4, 135.6, 133.2, 133.2, 132.1, 130.0, 127.4, 107.6, 105.6, 98.5, 94.5, 67.1, 53.2, 53.1, 51.4, 49.5, 32.3, 31.3, 24.6, 22.5, 20.9, 18.8, 13.7, 13.5, 12.6 ESIMS (m/z): 621.4 ([M+H] ) +  HRESIMS m/z: calcd. for C 7H4,N 0 ([M+H] ): 621.3066 +  3  4  5  found: 621.3067 UV/Vis. ( W A b s . , CH C1 ): 324.9 (0.533), 415.0 (2.782), 510.1 (0.278), 540.1 (0.246), 2  2  610.0 (0.210), and 669.9 (1.185)  230 Chapter 4 Experimental  3-devinyl 3-(2-phenyl-l-ethenyl) ring B-BPD-1, 3-diene dimethyl ester (129)  129 The mixture of Zn(II) ring B-BPD-1,3-diene dimethyl ester 112 (35 mg, 0.044mmol) and catalyst 96 (10 mg, 0.011 mmol) in 1 mL freshly distilled dry THF was refluxed for lh. After T F A treatment, the reaction  mixture was purified by flush column  chromatograph to give 3-devinyl 3-(2-phenylethene) ring B-BPD-1, 3-diene dimethyl ester 129 as a black solid (7 mg, 20 %). J  H N M R (300 M H z , CDC1 ) £9.76, 9.67, 9.35, 9.19 (4s, 4H, H-meso), 8.49 (d, J = 16.6 3  Hz, 1H, H-3 (^-isomer)), 7.97 (d, J= 1A Hz, 2H, 2Ha), 7.82 (d, J= 5.7 Hz, 1H, H-7 ), 1  3  7.77 (d, J= 16.6 Hz, 1H, H-3 (^-isomer)), 7.59 (t, J= 7.5 Hz, 2H, 2Hb), 7.44-7.42 (m, 2  2H, H-7 , He), 5.06 (s, 1H, H-7 ), 4.28, 4.20 (2t, J= 7.5 Hz, 4H, H-17 , H-13 ), 3.97 (s, 4  1  1  1  3H, 7 -C0 Me), 3.69, 3.65, 3.63, 3.47, 3.41, 2.97 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H 2  2  2  2  2  2  18 , H-2 , H-12 , 7 -C0 Me), 3.19 (t, J= 7.5 Hz, 2H, H-13 ), 3.15 (t,J = 7.5 Hz, 2H, H 1  1  1  1  2  2  17 ), 1.77 (s, 3H, 7-CH3), -2.23 (br, 2H, 2NH) 2  ESIMS (m/z): 809 ([M+H] ) +  HRESIMS m/z: calcd. for  C48H49N4O8  ([M+H] ): 809.3550 +  found: 809.3557  •  231 Chapter 4 Experimental  3-DevinyI 3-(3-oxy-l-propenyI) ring B-BPD-l,3-diene dimethyl ester (135)  135 Following the same procedure for the synthesis of 105. Zn-ring B-BPD-1,3-diene dimethyl ester 112 (36 mg, 0.045mmol), catalyst 96 (10 mg, 0.011 mmol) and acrolein diethyl acetal (118 mg, 0.14 mL, 0.9 mmol) were utilized. After T F A treatment, the mixture was purified by flush column chromatograph to give 3-devinyl 3-(3-oxy-lpropenyl) ring B-BPD-1,3-diene dimethyl ester 135 as a black powder (13 mg, 40 %). ' H N M R (400 M H z , CDC1 ) 8 10.24, 10.22 (d, J= 7.6 Hz, 1H, CHO), 9.77, 9.57, 9.24, 3  9.04 (4s, 4H, H-meso), 9.04 (d, J= 16.1 Hz, 1H, H-3 ), 7.78 (d, / = 5.7 Hz, 1H, H-7 ), 1  3  7.44 (d,J= 5.7 Hz, 1H, H-7 ), 7.41 (dd, J= 16.1 Hz and 7.6 Hz, 1H, H-3 ), 5.07 (s, 1H, 4  2  H-7 ), 4.25 (t, J= 7.8 Hz, 2H, H-17 ), 4.12 (t, J= 7.8 Hz, 2H, H-13 ), 3.98 (s, 3H, 7 1  1  1  2  C 0 M e ) , 3.71, 3.63, 3.62, 3.42, 3.37 (5s, 5Me, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H 2  2  2  1  2  1  2  12 ), 3.18, 3.14 (2t, J= 7.6 Hz, 4H, H-17 , H-13 ), 2.97 (s, 3H, 7 -C0 Me), 1.80 (s, 3H, 1  2  2  1  2  7-CH ), -1.78 (br, 2H, 2NH); 3  ESJJvlS (m+l/z): 761 ([M+H] ) +  HRESIMS m/z: calcd. for C43H45N4O9 ([M+H] ): 761.3187 +  found: 761.3182  232 Chapter 4 Experimental U V / V i s . (7i /Abs., CH C1 ): 350.0 (0.485), 455.0 (0.736), 579.9 (0.199), 650.0 (0.176), max  2  2  and 799.9 (0.363)  Zn(II) 3-devinyl 3-(2-methoxycarbonyl-l-ethenyl) ring B-BPD-l,3-diene dimethyl ester (136)  136 The mixture of Zn-ring B-BPD-1,3-diene dimethyl ester 112 (37 mg, 0.046 mmol), catalyst 96 (10 mg, 0.011 mmol) and methyl acrylate (80 mg, 0.08 mL, 0.93 mmol) in 1 mL freshly distilled dry THF was reflux ed under Argon for lh. After the solvent was removed in vacuo, the mixture was purified by flush silica column chromatograph to give Zn(II) 3-devinyl 3-(2-methoxycarbonyl-l-ethenyl) ring B-BPD-1, 3-diene dimethyl ester 136 as a black powder (8 mg, 20 %). !  H N M R (400 M H z , CDC1 ) £9.58, 9.32, 9.07, 8.80 (4s, 4H, H-meso), 9.04 (d, J= 16.0 3  Hz, 1H, H-3 ), 7.70 (d, J = 5.7 Hz, 1H, H-7 ), 7.33 (d, J= 5.7 Hz, 1H, H-7 ), 6.97 (d, 1  3  4  J = 16.0 Hz, 1H, H-3 ), 4.99 (s, 1H, H-7 ), 4.05-4.01 (m, 4H, H-17 , H-13 ), 3.98 (s, 3H, 2  1  1  1  7 -C0 Me), 3.96 (s, 3H, 3 -C0 Me), 3.61, 3.59, 3.56, 3.31, 3.26, 3.07 (6s, 6Me, 17 2  2  2  2  2  C 0 M e , 13 -C0 Me, H-18 , H-2 , H-12 , 7 -C0 Me), 3.05-3.00 (m, 4H, H-17 , H-13 ), 2  2  1  2  1  1  1  2  2  1.79 (s, 3H, 7-CH ) 3  2  233 Chapter 4 Experimental  ESEVIS (m/z): 853 ([M+H] ) +  HRESIMS m/z: calcd. for C44H45N4O10Z11 ([M+H] ): 853.2427 +  found: 853.2429 U V / V i s . (X /Abs., CH2CI2): 350.0 (0.464), 445.0 (0.675), 614.9 (0.293) and 685.0 max  (0.423)  3-Devinyl 3-hexyl ring B-BPD-1,3-diene dimethyl ester 138  138 3-Devinyl 3-(l-hexenyl) ring B-BPD-1,3-diene dimethyl ester 105 (10 mg, 0.013 mmol) was dissolved in 5 mL methylene chloride. 10 wt % Pd/C catalyst (5 mg) was added and the mixture was stirred at room temperature for 5 h with a hydrogen balloon on. The catalyst was filtered out and the solvent was removed in vacuo. The residue was purified on preparative T L C to give the hydrogenation product 138 as a black solid (10 mg, 97 %). ' H N M R (300 MHz, CDCI3) £9.70, 9.69, 9.38, 8.97 (4s, 4H, H-meso), 7.82 (d, J= 5.7 Hz, 1H, H-7 ), 7.43 (d, J = 5.7 Hz, 1H, H-7 ), 5.05 (s, 1H, H-7 ), 4.31 (t, / = 7.6 Hz, 2H, 3  4  1  H-17 ), 4.18 (t, J= 7.7 Hz, 2H, H-13 ), 3.97 (s, 3H, 7 -C0 Me), 3.64, 3.62, 3.51, 3.47, 1  1  2  2  3.41, 2.90 (6s, 6Me, 17 -C0 Me, 13 -C0 Me, H-18 , H-2 , H-12 , 7 -C0 Me), 3.89 (t, J 2  2  2  1  2  1  1  1  2  234 Chanter 4 Experimental  = 7.5 Hz, 2H, H-3 ), 3.19, 3.15 (2t, J = 7.6 Hz, 4H, H-17 , H-13 ), 2.18-1.20 (m, 8H, H 1  2  2  3 ~H-3 ), 1.79 (s, 3H, 7-CH ),0.89 (t, J= 7.1 Hz, 3H, H-3 ), -2.37, -2.50 (2br, 2H, 2NH) 2  5  6  3  1 3  C N M R (75 M H z , CDC1 ) 5 173.7, 173.3, 170.6, 167.7, 165.5, 156.7, 152.1, 151.7, 3  150.6, 140.2, 138.4, 137.9, 137.3, 136.9, 135.9, 135.7, 134.9, 133.9, 133.4, 130.7, 121.6, 112.3, 98.8, 92.6, 91.9, 52.7, 52.2, 51.7, 51.6, 51.5, 47.8, 37.1, 36.7, 32.7, 32.6, 32.0, 31.9, 29.5,27.6, 26.0, 22.7,21.9,21.6, 14.1, 11.6, 11.4, 11.2 ESTMS (m/z): 791.9 ([M+H] ) +  HRESIMS m/z: calcd. for C 6H55N 0 ([M+H] ): 791.4020 +  4  4  8  found: 791.4019 UV/Vis. (X /Abs., CH C1 ): 355.0 (0.431), 430.1 (0.805), 590.0 (0.164), and 680.0 max  2  2  (0.247)  4.6  Determination  of the  Crystal  Structure of 67f  by X-ray  Crystallography Table 4.1 Crystal Data and Details of the Structure Determination for 67f Empirical Formula  C40H50N4O4  Formula Weight  650.86  Crystal Color, Habit  red, block  Crystal Dimensions  0.35 x 0.2 x 0.2 mm  Crystal System  orthorhombic  Lattice Type.  primitive  Space Group  P2i2i2,(#19)  235 Chapter 4 Experimental  Lattice Parameters  a= 7.3302(4) A b= 14.3475(8) A c= 33.139(2) A V = 3485.3(3) A  Z Value  4  D calc  1.240 g/cm  F ooo  1400.00  ju (MoKct)  0.8 cm"  Temperature  3  1  -100± 1 °C  Difiractometer  Rigaku/ A D S C C C D  Radiation  MoKot( = 0.71069 A) graphite monochromated  Detector Aperture  94 mm x 94 mm  Data Images  460 exposures @ 47.0 seconds  (j) oscillation Range (=-90.0)  0.0-190.0°  co oscillation Range (=-90.0)  -17.0-23.0°  Detector Position  37.98 mm  Detector Swing Angle  -5.60°  20 x  55.8°  No. of Reflections Measured  Total: 30532  ma  Unique: 7978 (R,„ = 0.084; Friedels) (  Corrections  Lorentz-polarization Absorption/ scaling/ decay (corr. factors: 0.7731-1.0000)  236 Chapter 4 Experimental  REFERENCES 1. Pandey, R. K . ; Bellnier, D. A . ; Smith, K . M . ; Dougherty, T. J. Photochem. Photobiol. 1991, 53, 65. 2. Smith, K . M . ; Goff, D. A.; Simpson, D. J. J. Am. Chem. Soc. 1985, 707, 4946. 3. Smith, K . M . ; Bisset, G. M . F.; Bushell, M . J. J. Org. Chem. 1980, 45, 2218.  

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