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Pyrrolic pigments Brückner, Christian. 1996

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PYRROLIC PIGMENTS by CHRISTIAN BRUCKNER Dipl.Chem., Rheinisch-Westfalisch Technische Hochschule Aachen, Germany, 1991  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER March 1996 © Christian Bruckner, 1996  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  II  ABSTRACT  Part 1  presents studies aimed at the synthesis of novel aromatic tetra- and  pentapyrrolic pigments with meso-phenyl substituents and with long wavelengths of absorption. Such compounds are potential photosensitizers for use in photodynamic therapy (PDT). Several approaches are described: 1.  The osmium tetroxide mediated dihydroxylation of variously substituted meso-  tetraphenylporphyrins (TPP) to provide novel stable chlorins such as 129 and bacteriochlorins such as 141 and 146 is reported. The directing effect of the central metal zinc in the stereo- and regiochemical outcome of the dihydroxylation of meso-tetraphenylchlorins is outlined. The resulting 8,6'-dihydroxylated amphiphilic chlorins are characterized by spectroscopic and analytical techniques. Preliminary in vitro biological results of their potency as drugs in PDT have been encouraging. One side-product of the osmylation reaction, the 2-oxa-3-oxochlorin  163, was structurally characterized by X-ray  crystallography.  TPP  Some unique physical (observable rotation of the phenyl groups) and chemical properties of the 6,6'-diolchlorins are reported. For instance, me,«?-tetraphenyl-2,3-vjcdihydroxy-2,3-chlorinato)nickel(II) can be converted oxidatively into the corresponding 2,3secochlorin-2,3-dialdehyde 167. This compound can ring-close to the novel double ketal  Pyrrolic Pigments  175.  Abstract  iii  This severely distorted pigment has been structurally characterized by X-ray  crystallography and is without precedent. Ph  163  2.  P  h  n  167  Ph  OR  R = Me, Et, 175  A directed synthesis of N-confused porphyrin 104 was developed. The key step in  the synthesis was a 2+2-type condensation of dipyrromethane 197 with the novel a,6-linked dipyrromethane 190. Although the goal of producing 104 in high yields could not be met, this study provided valuable insight into the chemistry of dipyrromethanes and provided through fortuitous circumstance tripyrrane 180.  3.  Tripyrrane 180 is the key reagent in the directed high-yield synthesis of meso-phenyl-  sapphyrins such as 200. As inferred from spectroscopic properties, the protonation dependent conformation of 200 is described.  Pyrrolic Pigments  4.  Abstract  iv  Studies towards the elucidation of the mechanism of formation of porphycyanines via  the lithium aluminum hydride (LAH) reduction pathway of 2-cyanodipyrromethanes are presented. To this end, variously substituted 2-cyanopyrroles were prepared and reduced by LAH. It was found that imine linked dimers such as 204 are directly formed during this reduction. A mechanistic proposal for this outcome is presented. A unique reaction of 204 with nickel(II) to form the tripyrrolic complex 236 is described.  The complex was  structurally characterized by X-ray crystallography and its mode of formation has been rationalized.  236  Part 2 describes  the synthesis of 5-phenyldipyrromethane (20) and the conversion  into the correspondingraeso-phenyldipyrrins(11). Their ability to complex to transition metals is described and the optical spectra of the resulting dipyrrinato complexes of nickel(II), copper(II), and zinc(II) are discussed in depth, compared to known alkyldipyrrinato complexes and correlated to their structure. The special steric properties of the novel me so -phenyl dipyrrins are highlighted by the structural characterization of two unusual complexes, namely the square planar diamagnetic nickel(II) complex 27 and octahedral copper(III) complex 48.  Pyrrolic Pigments  Part 3  Abstract  v  describes the improved preparation of dipyrromethane 2 by direct  hydrodesulfurization of thione 7 with Raney-nickel/H2-  It was also found that  2-pyrrolylthiones chelate in an N,S-bidentate fashion to a variety of transition metals. The preparation of, for instance, the square planar nickel(II) (29), octahedral copper(III) (33, two isomers) and tetrahedral mercury(II) (32) chelates of 7 are described and their spectroscopic properties are correlated to their structures. The steric requirements for the dipyrrolylthione ligand were determined. These complexes as well as the free base 7 were structurally characterized by X-ray crystallography. Furthermore, the synthesis of the 2-thioacetylpyrrole and 2-thiobenzoylpyrrole and their nickel(II) and cobalt(III) complexes is described. This is the first report of the complexing ability of the 2-pyrrolyl moiety.  2  n  n = 2; M = Ni, 29 n = 2; M = Hg, 32 n = 3; M = C o ( l l l ) , 33 (two i s o m e r s )  vi  TABLE OF CONTENTS  ABSTRACT  ii.  TABLE OF CONTENTS  vi  LIST OF TABLES  xii  LIST OF FIGURES  xiv  LIST OF SCHEMES  xvii  LIST OF ABBREVIATIONS  xxi  NOMENCLATURE  xxii  References ACKNOWLEDGMENTS  GENERAL INTRODUCTION TO PYRROLIC PIGMENTS Part 1 :  xxviii xxix  l  SYNTHESIS A N D STUDY O F PYRROLIC PIGMENTS FOR U S E IN PHOTODYNAMIC T H E R A P Y (PDT)  1.  INTRODUCTION AND LITERATURE REVIEW  5  1.1  Photodynamic Therapy (PDT)  5  1.1.1  History and General Introduction  5  1.1.2  Photophysical and Photochemical Basis of PDT  9  1.1.3  The Profile of the Ideal PDT Drug  13  1.2  Research Objective  15  1.3  Review of Synthesis and Properties of Pyrrolic Pigments  1.3.1  Potentially Useful in PDT  16  Porphyrins  16  Pyrrolic Pigments  Table of Contents  vii  1.3.1.1  Naturally Occurring Porphyrins  16  1.3.1.2  Synthetic Porphyrins  17  1.3.2  Chlorins, Bacterio- and Isobacteriochlorins  21  1.3.2.1  The Optical Properties of Chlorins  21  1.3.2.2  Naturally Occurring Chlorins  24  1.3.2.3  The Total Synthesis of Chlorins  24  1.3.2.4  The Conversion of Porphyrins into Chlorins by Reduction  1.3.2.5  Non-Reversible Conversions of Porphyrins into Chlorins  1.3.3  27  Synthesis of 6-Hydroxychlorins 1.3.3.1  The Osmium Tetroxide Oxidation in General  1.3.3.2  The Mechanism of the Osmium Tetroxide Addition to  31 38 38  Double bonds  41  1.3.3.3  The Osmium Tetroxide Oxidation of Aromatic Systems  43  1.3.3.4  The Osmium Tetroxide Oxidation of Porphyrins  46  1.3.3.5  Reactivity of Wc-Diol Chlorins  51  1.3.3.6  6-Hydroxychlorins not Derived from an Osmium Tetroxide Oxidation  1.3.3.7  8-Oxochlorins and Related Pigments  52 55  1.3.4  Porphyrin Isomers  62  1.3.5  Expanded Porphyrins  67  R E S U L T S A N D DISCUSSION  2.1  74  The Osmium Tetroxide-Mediated Dihydroxylation of  2.1.1  meso-Phenylporphyrins and -chlorins  74  The Osmylation of TPP - The Principal Reaction  75  Pyrrolic Pigments  Table of Contents  2.1.2  The Osmylation of meso-Tetraphenylchlorins  2.1.2  The Osmylation of mes0-Tetraphenyl-2,3-dihydroxy-2,3chlorins and -metallochlorins  2.1.3  Comments on the Optical Spectra of the B,B'-Diolchlorins  2.1.4  Preliminary Results of the Biological Activity of Diolchlorin 137  2.1.5  85  88 91  92  Rotation of Phenyl Rings in mes0-Tetraphenyl-B,B'v/c-diolchlorins and -metallochlorins  2.1.6  The Reactivity of the meso-Tetraphenyl-v/c-diolchlorins  95 101  2.1.6.1  Formation of Isopropylidene Ketal 150  101  2.1.6.2  Formation of B-Oxo-Derivatized Pigments  102  2.1.6.3  Formation of 2-Oxa-3-oxochlorin 163  108  2.1.6.4  Formation of a Secochlorin and Subsequent RingClosure Reactions  2.2  115  Studies Towards the Directed Synthesis of N-confused TPP  124  2.2.1  Retrosynthetic Analysis of N-Confused Porphyrin  124  2.2.2  The Directed Synthesis of N-Confused Porphyrin  126  2.3  The Directed Synthesis of meso-Phenylsapphyrins  134  2.4  The Reductive Coupling of 2-Cyanopyrroles  141  2.5  The Unexpected Formation of a Tripyrrolic Tetradentate Nickel Chelate (236)...  3.  viii  149  EXPERIMENTAL  156  3.1  Instrumentation and Materials  156  3.1  mew-Aryl-vzc-diolchlorins, -bacteriochlorins and -isobacteriochlorins  3.2  Reactions of the meso-Tetraphenyl-v/c-diol-chlorins  159 169  Pyrrolic Pigments  4.  Table of Contents  ix  3.3  The Directed Synthesis of N-Confused TPP  174  3.4  The Directed Synthesis of Sapphyrins  180  3.5  The Preparation of Cyanopyrroles  184  3.6  The Reductive Coupling of 2-Cyanopyrroles  188  3.7  The Formation of Complex 236  191  3.8  Crystal Structure Analyses of 163-pyridine, 175 and 236  192  REFERENCES  Part 2:  202  meso-PHENYLDIPYRRINS - SYNTHESIS AND METAL COMPLEX FORMATION  1.  2.  INTRODUCTION  223  1.1  Alkyldipyrrins and Their Metal Complexes  223  1.2  meso -Substituted 4,6-Dipyrrins  226  1.3  4,7-Dipyrrins  228  1.4  Tripyrrins and Related Tripyrrolic Pigments  229  R E S U L T S A N D DISCUSSION  230  2.1  Synthesis of 5-Phenyldipyrromethanes  230  2.2  Preparation and Characterization of meso-Phenyldipyrrins  231  2.3  Formation and Characterization of Transition Metal Chelates of meso -Phenyldipyrrins  234  2.3.1  The Complexes of Ni(II), Cu(II), and Zn(II)  234  2.3.2  Crystal Structure Analysis of bis(me5o-Phenyl,  dipyrrinato)Ni(II) (27) 2.3.3  The Complexes of Cobalt(II) and Cobalt(III)  2.3.4  Crystal Structure Analysis of tris(meso -Phenyl-  244 248  Pyrrolic Pigments  Table of Contents  dipyrrinato)cobalt(III) (48) 2.4  The DDQ-Oxidation ofraew-Diphenyltripyrrane(49)  x  251 253  3.  CONCLUSIONS  257  4.  EXPERIMENTAL SECTION  258  4.1  Dipyrromethanes, Dipyrrins and their Metal Complexes  259  4.2  Crystal Structure Analysis of 27 and 48  265  5.  LIST O F R E F E R E N C E S  Part 3: 1.  2-PYRROLYLTHIONES AS N,S-BIDENTATE CHELATORS  INTRODUCTION  1.1  275  2-Pyrrolylthiones as Common Precursors Used in a Porphyrin Synthesis  2.  270  275  1.2  Thiocarbonyl Compounds  278  1.3  Sulfur Donor-Metal Complexes in Biological Systems  280  1.4  Sulfur-Nitrogen Chelating Agents and their Metal Complexes  281  R E S U L T S A N D DISCUSSION  283  2.1  Synthesis of di-2-Pyrrolylthiones  283  2.2  Hydrodesulfurization of di-2-Pyrrolylthiones  284  2.3  Reaction of di-2-Pyrrolylthione (7) with Nickel(II)  286  2.4.  Solution and Solid State Conformation of di-2-Pyrrolylthione (7) and-ketone (6)  288  2.5  Spectroscopic Properties of Complex 29  291  2.6  Structural Characterization of Complex 29  293  Pyrrolic Pigments  Table of Contents  2.7  Transformations of Complex 29  2.8  The Reaction of di-2-Pyrrolylthione (7) with Various Metal Ions,  xi  297  and Structural Characterization of Selected Complexes  299  2.8.1  Reaction of Thione 7 with Mercury(II)  299  2.8.2  Reaction of Thione 7 with Cobalt(II) and Cobalt(III)  302  2.8.3  Reaction of Thione 7 with Various Metal Ions  306  2.9  Establishment of the Steric Requirements of the 2-Pyrrolylthionatomoiety  2.10  307  Synthesis of 2-Thioacetyl- (37) and 2-Thiobenzoylpyrrole (38), and Formation of the Nickel(II) and Cobalt(III) Complexes thereof  2.11  310  Preliminary Reports on the Formation of Tetradendate Ligands Containing the 2-pyrrolylthione Motif, and their Nickel(II) complexes  313  3.  CONCLUSION AND FUTURE W O R K  316  4.  EXPERIMENTAL  317  3.1  Instrumentation and Materials  317  3.2  2-Pyrrolylketones  319  3.3  2-Pyrrolylthiones  320  3.4  2-Pyrrolylthionatometal Complexes  324  3.5  Hydrodesulfurization of 2-Pyrrolylthiones  332  3.6  Crystal Structure Analyses of 6, 7, 29, 33-1, and 32  334  5.  LIST O F R E F E R E N C E S  343  xii  LIST OF TABLES  Table 0-1  Fischer nomenclature for selected naturally occurring porphyrins  Table 1 -1  xxvi  Optical spectra of the novel diol chlorins in comparison to their non-dihydroxylated analogs  92  Table 1-2  Crystallographic data for 163-pyridine, 175, and 236  193  Table 1-7  Atomic coordinates and B q_ [A ] for 163-pyridine  194  Table 1 -4  Selected bond lengths [A] in 163-pyridine with estimated  2  e  standard deviations in parentheses  196  Table 1-7  Atomic coordinates and B . [A ] for 175  197  Table 1-6  Selected bond lengths [A] in 175 with estimated standard  2  e<?  deviations in parentheses  199  Table 1-7  Atomic coordinates and B . [A ] for 236  200  Table 1 -8  Bond lengths in 236 [A]  201  Table 2-1  UV-visible data and dihedral angles of literature known and  2  e<?  novel dipyrrinato-complexes  238  Table 2-2  Selected bond lengths in 27  245  Table 2-3  Nickel-nitrogen bond lengths in selected tetrapyrrolic pigments  246  Table 2-4  Crystallographic data of compounds 27 and 48  267  Table 2-5  Atomic coordinates and B . [A ] for 27  268  Table 2-6  Atomic coordinates and B . [A ] for 48  268  Table 3-1  Crystallographic data for 6 and 7  335  Table 3-2  Crystallographic data for 29, 33-1, and 32  336  Table 3-3  Atomic coordinates and B q_ [A ] for 7  337  2  e<?  2  e(?  2  e  Pyrrolic Pigments  List of Tables  xiii  Table 3-4  Atomic coordinates and B q [A ] for 6  337  Table 3-5  Atomic coordinates and B q_[A ] for 29  338  Table 3-6  Atomic coordinates and B .[A ] for 32  339  Table 3-7  Atomic coordinates and B .[A ] for 33-1  340  Table 3-8  Selected bond lengths in 7 with estimated standard deviations in  2  e  %  2  e  2  g(?  2  e(?  parentheses Table 3-9  Selected bond lengths in 6 with estimated standard deviations in parentheses  Table 3-10  341  342  Selected bond lengths in 29 with estimated standard deviations in parentheses  342  xiv  LIST OF FIGURES  Figure 1-1  Structures of selected second generation photosensitizers  9  Figure 1-2  Modified Jablonski diagram for a typical photosensitizer  10  Figure 1 -3  Type I photoprocesses  12  Figure 1-4  Type II photoprocesses  12  Figure 1-5  Optical  Figure 1-6  The different concepts of porphyrin synthesis  17  Figure 1-7  UV-visible spectrum of chlorin 1 and bacteriochlorin TPBC  21  Figure 1 -8  Energy level diagram for the frontier orbitals of the four generic  (CHCI3) spectrum of protoporphyrin IX dimethyl ester  metalloporphyrin classes Figure 1-9  16  22  X-Ray crystal structure of Cgo-buckminsterfullerene osmate ester (from Hawkin e t a l .  I86b  )  45  Figure 1-10  X-Ray crystal structure of 53 (from Barkingia etal. )  Figure 1-11  18 7t-Electron derealization pathway in porphyrins, chlorins and  24lb  48  metallochlorins  49  Figure 1-12  Porphine - isoporphine tautomerism  66  Figure 1-13  !  H-NMR spectra of 125 (bottom trace) and NOE  difference  spectrum of 125 (top trace)  76  Figure 1 -14  Optical spectrum (CH Cl /0.5% MeOH) of 129 and 130  80  Figure 1-15  iH-NMR of 134 (bottom trace) and of 135 (top trace)  83  Figure 1-16  Measured (A) iH-NMR signal of the pyrroline protons in 141  2  2  and coupling constants determined by simulation (B)  86  Figure 1 -17  Optical spectrum (CH Cl /0.5%MeOH) of 141 and 144  86  Figure 1-18  Optical spectrum (CH Cl /0.5%MeOH) of 146 and 149  88  2  2  2  2  Pyrrolic Pigments  Figure 1-19  List of Figures  xv  Schematic representation of the face differentiation in mesotetraphenylmetalloporphyrins with additional ligands (A) and in 6,6'-vic-diolchlorins (B)  96  Figure 1-20  Temperature dependent iH-NMR spectrum of 129  99  Figure 1 -21  Temperature dependent iH-NMR spectrum of 130  100  Figure 1-22  Normalized optical spectra (CH2CI2) of 151 and 152  102  Figure 1-23  Optical spectra (CH C1 ) of 163 and 164  Figure 1 -24  iH-NMR spectrum of 163 and 164  Figure 1 -25  ORTEP representation (33% probability level) and side view of  2  2  163-pyridine  109 110  112  Figure 1 -26  Normalized optical spectra (CH2CI2) of 167 and 168  116  Figure 1 -27  Optical spectrum (CH C1 ) of 173  118  Figure 1 -28  ORTEP representation (33% probability level) of 175  121  Figure 1-29  Side view and stereo view of the crystal structure of 175  122  Figure 1 -30  Optical spectrum (CH C1 ) of 200 and 200-2HC1  136  2  2  2  2  Figure 1-31  !  Figure 1-32  Conformation of diphenylsapphyrin 200 as free base and as  H-NMR of 200 and 200-2HC1  138  diprotonated species  139  Figure 1-33  Optical spectrum (CHC13) of 236  149  Figure 1-34  !H NMR spectrum of 236  150  Figure 2-1  meso-Methyldipyrrin - meso-ylidenedipyrrane equilibrium  226  Figure 2-2  UV-visible spectrum of 11 in MeOH, and in MeOH/trace HC1, and 23 in MeOH/trace HC1  231  Figure 2-3  UV-visible spectra (CH C1 ) of 27 and 26  Figure 2-4  UV-visible spectra of 25 and 24 in CH2CI2, and of 24 in  2  235  CH Cl /20%pyridine  236  iH-NMR spectrum of 26  242  2  Figure 2-5  2  2  Pyrrolic Pigments  List of Figures  xvi  Figure 2-6  ORTEP representation (33% probability level) of 27  245  Figure 2-7  Limiting resonance forms of the dipyrrinato ligands  247  Figure 2-8  Optical spectrum (CHC1 ) of 46 and 48 in  248  Figure 2-9  ORTEP representation (33% probability level) of 48  251  Figure 2-10  Optical spectrum of equal concentrations of 50  253  Figure 2-11  *H-NMR spectrum of 50  254  Figure 3-1  Normalized optical spectra (MeOH) of 2-pyrrolylketone (6) and  3  2-pyrrolylthione (7)  279  Figure 3-2  Optical (CH Cl ) spectrum of 29  286  Figure 3-3  ORTEP representation (33% probability level) and unit cell of 6  290  Figure 3-4  ORTEP representation (33% probability level) and unit cell of 7  290  Figure 3-5  CH-COSY (500, 125 MHz, acetone-d6) of 29  292  Figure 3-6  ORTEP representation (33% probability level) and side view of 29 ..294  Figure 3-7  Bond lengths and angle changes of the ligand 7 upon metallation  295  Figure 3-8  Intermolecular interaction as seen in the crystal of 29  295  Figure 3-9  Optical spectrum (CH C1 ) of mercury complex 32  230  Figure 3-10  Stereoscopic view of the mercury complex 32  301  Figure 3-11  Optical spectra (CH C1 ) of the cobalt complexes 33-1  302  Figure 3-12  !H-NMR spectrum of the two isomeric complexes 40-1 and 40-11 ....303  Figure 3-13  ORTEP representation (33% probability level) of 33-1 (as its  2  2  2  2  2  2  acetone solvate) Figure 3-14  305  Computer generated model of a hypothetical nickel(II) complex with an a-methylthionato ligand  308  Figure 3-15  Optical spectrum (CH C1 ) of 41 and 42  311  Figure 3-16  Normalized optical spectra (CH Cl ) of 44-1, 44-II and 43  312  Figure 3-17  Optical spectrum (CH C1 ) of 47  314  2  2  2  2  2  2  xvii  LIST OF SCHEMES  Scheme 1-1  Rothemund synthesis of meso-tetraphenylporphyrin (6)  19  Scheme 1-2  Rothemund-type synthesis of meso-tetraalkylmetallochlorins 13  25  Scheme 1-3  MacDonald 2+2 synthesis of metallochlorin 17  26  Scheme 1 -4  Diimide reduction of TPP (6)  28  Scheme 1 -5  Reduction of 2-nitro-TPP (25)  30  Scheme 1 -6  Diels-Alder reactions of protoporphyrin IX dimethyl ester (27)  32  Scheme 1 -7  Preparation of BPD-MA  33  Scheme 1 -8  Cyclopropanation of TPP with a carbene  34  Scheme 1-9  Formation of octaethylpurpurin (36) and octaethylbenzochlorin 36  (37)  Scheme 1-10  Claisen-type rearrangement of a hydroxyethyl-substituted porphyrin  37  Scheme 1-11  Osmium tetroxide mediated dihydroxylation of alkenes  39  Scheme 1-12  Mechanistic alternatives for the osmylation of olefins  41  Scheme 1-13 Osmium tetroxide mediated dihydroxylation of polycyclic aromatics Scheme 1-14  Osmylation of Cgo-buckminsterfullerene  Scheme 1-15  Osmium tetroxide mediated dihydroxylation of deutero-  :  43 44  porphyrin dimethylester (48)  46  Scheme 1-16  Osmium tetroxide mediated dihydroxylation of OEP  47  Scheme 1-17  Stepwise reaction of benzoporphyrin derivative (56) with osmium tetroxide  50  Pyrrolic Pigments  Scheme 1-18  List of Schemes  Pinacol-pinacolone-type rearrangement of the vzc-diolalkylchlorins  Scheme 1-19  xviii  51  Dehydration and rearrangement of vic-dihydroxyoctaethylchlorin 53  51  Scheme 1 -20  Photochemical synthesis of bacteriodiol-monomethyl ether 67  53  Scheme 1-21  Formation of photoprotoporphyrin (73)  54  Scheme 1 -22  Hydroxylation and rearrangement of B-oxochlorins and 6-oxometallochlorins  56  Scheme 1-23  Preparation of Bonnett's chlorins with graded amphiphilicity  57  Scheme 1-24  Hydroxylation of keto-chlorin 86 and its reduction to triol 87  58  Scheme 1-25  Formation of meso -hydroxyoctaethylporphyrin  60  Scheme 1-26  Formation of octaethylporphycene (100)  63  Scheme 1-27  Formation of sapphyrin (105), pentaphyrin (106), and hexaphyrin (107)  69  Scheme 1 -28  Formation of porphocyanine (115)  69  Scheme 1-29  Formation of meso-phenyl substituted porphocyanines  73  Scheme 1 -30  Osmylation of TPP to form the corresponding osmate ester chlorins  Scheme 1 -31  78  Reduction of the osmate ester chlorins to form the corresponding diol chlorins and the one-step conversion of porphyrins into diol chlorins  79  Scheme 1 -32  Preparation of diolbacteriochlorin 141  85  Scheme 1 -33  Preparation of the diolisobacteriochlorins 143 and 144  87  Scheme 1 -34  Preparation of the tetraolbacterio- and tetraolisobacteriochlorins  Scheme 1-35  145-149  89  Formation of acetonide 150  101  Pyrrolic Pigments  Scheme 1 -36  List of Schemes  xix  Dehydration of the B,8'-diolchlorins to give the corresponding ketochlorins 151  103  Scheme 1 -37  Formation of 154  104  Scheme 1 -38  Formation of 8,6'-dione chlorin 156 and 157  105  Scheme 1 -39  Proposed formation of dioxobacterio and -isobacteriochlorins  107  Scheme 1-40  Proposed autooxidation mechanism for the formation of 163  114  Scheme 1-41  Crossley's synthesis of lactone 164  114  Scheme 1-42  Formation of meso -phenylsecochlorin bisaldehydes 167 and 168  115  Scheme 1-43  Formation of furochlorophin (70)  117  Scheme 1 -44  Formation of octaethyl-2,3-secochlorin-2,3-dione (171)  117  Scheme 1 -45  Formation of 172 and 173  119  Scheme 1-46  Formation of aldol condensation product 176..... .  123  Scheme 1-47  Retrosynthetic analysis of N-confused porphyrin (104)  125  Scheme 1 -48  Directed synthesis of N-confused TPP (104)  128  Scheme 1 -49  Rationalization of the findings of the benzoylation experiments  t  of 179  129  Scheme 1 -50  Proposed mechanism of formation of N-confused porphyrin  132  Scheme 1-51  Alternative retrosynthetic analysis of N-confused porphyrin  133  Scheme 1-52  Synthesis of the meso-phenylsapphyrins 114,199, and 200  135  Scheme 1 -52  Proposed alternative synthetic pathway for the formation of me.ro-phenylporphocyanines  141  Scheme 1 -53  Formation of imine-linked dipyrrolic compound 204  142  Scheme 1-54  Syntheses of the cyanopyrroles  144  Scheme 1 -55  Outcome of the L1AIH4 reduction of various cyanopyrroles  145  Scheme 1 -56  Mechanism of the LAH coupling of benzaldoxime  147  Scheme 1 -57  Hypothetical mechanistic scheme of formation of 204 through the L1AIH4 reduction of cyanopyrrole 207  148  List of Schemes  Pyrrolic Pigments  xx  Scheme 1-58  Proposed mechanism of formation and structure of 236  151  Scheme 1-59  Fragmentation of 236 in the EI mass spectrometer  155  Scheme 2-1  Synthetic pathways towards dipyrrins  224  Scheme 2-2  Formation of  227  Scheme 2-3  Synthesis of meso-phenyldipyrranes and raeso-phenyldipyrrins 230  Scheme 2-4  Formation of dipyrrinato complexes from dipyrrins  234  Scheme 2-5  Formation of cobalt(II) and cobalt(III) dipyrrinato complexes  250  Scheme 2-6  DDQ oxidation of weso-diphenyltripyrrane (49)  255  Scheme 2-7  Classic method for the synthesis of tripyrrinones  256  Scheme 3-1  Principal pathways for the formation of 5,10-me.w-diphenyl-  raeso-phenyldipyrrins  porphyrin (x)  276  Scheme 3-2  Synthesis of di-2-dipyrrolylmethane  276  Scheme 3-3  Formation of dipyrrolylthiones and pyrrolylthione esters  283  Scheme 3-4  Hydrodesulfurization of 7  284  Scheme 3-5  Possible conformers and tautomers of di-2-pyrrolylcarbonyl compounds  288  Scheme 3-6  N-methylation of complex 29  297  Scheme 3-7  Attempted formation of complex 35 and formation of 34  307  Scheme 3-8  Formation of nickel(II) complex 36  309  Scheme 3-9  Synthesis of 2-pyrrolylthiones 37 and 38, and their nickel(II) and cobalt(III) complex formation  Scheme 3-10  310  Formation of tetradentate 2-pyrrolylthione and -ketone ligands, and their nickel(II) complexes  313  xxi  LIST OF ABBREVIATIONS BPD-MA  benzoporphyrin derivative monoacid  CSI  chlorosulfonylisocyanate  DDQ  2,3-dichloro-5,6-dicyano-l,4-benzoquinone  DMAD  dimethylacetylene dicarboxylate  FGI  functional group interconversion  HMPA  hexamethylphosphoramide  HR-MS  high-resolution mass spectroscopy  LAH  lithium aluminum hydride  LR-MS  low-resolution mass spectroscopy  MCPBA  raeta-chloroperbenzoic acid  MO  molecular orbital  MRI  magnetic resonance imaging  NOE  nuclear Overhauser effect  OEP  2,3,7,8,12,13,17,18-octaethylporphyrin  PDT  photodynamic therapy  p-Ts  para -toluenesulfonyl-  TFA  trifluoroacetic acid  TPC  meso- tetraphenylchlorin  TPBC.  meso- tetraphenylbacteriochlorin  TPiBC  meso-tetraphenylisobacteriochlorin  TPP  meso- tetraphenylporphyrin  All other abbreviations were used as defined in the "Standard List of Abbreviations" J. Org. Chem. 1995,60(1), 12 A.  xxii  NOMENCLATURE  The nomenclature of pyrrolic compounds (pigments and non-pigments) has undergone much revision over the past several decades.  1  recommendations are adopted here. 2  The most current IUPAC  The formal nomenclature is, where appropriate,  supplemented with IUPAC accepted trivial names, which continue to be widely used because of their brevity and historic significance. For example, the Fischer system for naming the 3  naturally occurring tetrapyrrols and their isomers (Table 1). The abbreviations used for porphyrins are defined in this section.  MONOPYRROLIC S Y S T E M S  4  The parent monocyclic system, pyrrole (1), is numbered as shown at right. The Greek letters a and p are used to distinguish between the two types of carbon positions in all pyrrolic pigments. The pyrrole moiety is named '-pyrrolyl'. The isopyrrole structure 2 is the base of the formal nomenclature of dipyrrins and related pigments.  DIPYRROLIC SYSTEMS The most simple structure, 3, containing two directlylinked pyrrole rings is named 2,2'-bipyrrolyl. It is commonly referred to as bipyrrole.  2/+Pyrrole  Pyrrolic Pigments  Nomenclature  xxiii  The most important dipyrrolic molecules for this work are those comprised of two pyrrole moieties joined by a single carbon bridge, the so called mew-carbon.  4,6-Dipyrromethane  4,7-Dipyrromethane  meso-Phenyldipyrromethane  Dipyrrolylketone  Their numbering scheme and nomenclature is shown above. The parent compound 4 is named 4,6-dipyrromethane. Compound 5 is an isomer of 4. In this work, discussion of dipyrromethanes refer to the 4,6-isomer, unless otherwise specified.  Ketone 7 will be  referred to as dipyrrolylketone. The, formally, dehydro-4,6-dipyrromethane 8 is named dipyrrin. This pigment is known in the older literature as dipyrromethene.  4,6-Dipyrrin 2-(2-H-Pyrrol-2-ylidenemethyl)pyrrole  5-Phenyl-4,6-dipyrrin 2-(2-/-/-Pyrrol-2-ylidene-methyl-phenyl)pyrrole  In the case of a symmetrically substituted systems, the fast tautomeric exchange of the NHproton renders the two pyrrolic rings in dipyrrins equivalent. In this work, the 4,6-dipyrrins will simply be referred to as dipyrrins. The 5-position of dipyrrins is also referred to as the meso -position.  Pyrrolic Pigments  TRIPYRROLIC SYSTEMS  Nomenclature  xxiv  5  Tripyrrolic systems with directly-linked pyrroles are called terpyrroles.  6  Most  important to this work are tripyrrolic systems with a bridging single carbon unit. The parent compound tripyrrin (10) is the most unsaturated system. The reduced tripyrrins are named as dihydro, tetrahydro etc. tripyrrins, with numbers used to denote the positions of saturation. The fully reduced form 11 is named tripyrrane. Positions 5 and 10 are the mesopositions.  7  8  O Tripyrrin  5,10,15,17-Tetrahydrotripyrrin Tripyrrane  1  5  H  - 17H-Tnpyrnn-1-one  PORPHYRINS AND RELATED TETRAPYRROLIC SYSTEMS The  fundamental  system is the fully  unsaturated, cyclic tetrapyrrolic pigment porphyrin (13).  Positions 2,3,7,8,12,13,17, and 18 are referred  to as the 6-positions; positions 5,10,15, and 20 as the mew-positions; positions 1,4,6,9,11,14,16, and 19 are the a-positions.  The structure is tautomeric with  respect to the location of the two inner hydrogens, which may be associated with any two of the four nitrogens. All substituted porphyrins can be named systematically with this system, but for convenience, the naturally occurring porphyrins are named according to the trivial Fischer nomenclature.  A selection is presented in Table 1.  Trivial names for other  porphyrins that have not been sanctioned by the IUPAC will be defined in the text.  Nomenclature  Pyrrolic Pigments  xxv  Two synthetic porphyrins, octaethylporphyrin (14) and mesotetraphenylporphyrin (15) are simply named as OEP and TPP, respectively. Metal complexes of these compounds are abbreviated as TPPZn or OEPFe(III)Cl, for example.  Table 0-1  F i s c h e r nomenclature for selected naturally occurring porphyrins  Trivial Name  Substituents and Locants** a  2  3  7  8  12  13  17  H  Me  P  H  PH  Me  P  H  P  H  Deuteroporphyrin  Me  H  Me  H  Me  Hematoporphyrin  Me  Et(OH)  Me  Et(OH)  Me  Mesoporphyrin  Me  Et  Me  Et  Me  P  H  Me  Me  Vn  Me  Vn  Me  P  H  Me  H  AH  H  AH  P  H  AH  Protoporphyrin I X Uroporphyrin  a  c  AH  P  P  E t ( O H ) = -CH(OH)CH3, V n = -CHCH2, A = -CH2COOH, H  P  P  H  = -CH2CH2COOH;  18  PH P  b  H  see  n u m b e r i n g s c h e m e a s o u t l i n e d in F i g u r e 7; t h e r o m a n n u m e r a l s r e f e r t o F i s c h e r ' s t y p e n o m e n c l a t u r e for n a m i n g all t h e p o s s i b l e r e g i o i s o m e r s of o n e s e t of s u b s t i t u e n t s . c  2,3,7,8,12,13,17,18-Octaethylporphyrin OEP  5,10,15,20-Tetraphenylporphyrin meso-Tetraphenylporphyrin TPP  Nomenclature  Pyrrolic Pigments  xxvi  REDUCED PORPHYRINS Reduced porphyrins are named systematically as hydroporphyrins, or they can be named trivially. Both naming possibilities are shown. The parent reduced porphyrin is the 2,3-dihydroporphyrin (16), also named chlorin. The tetrahydro-system with two opposite reduced pyrrolic units is called a bacteriochlorin (17), whilst the regioisomer with two adjacent reduced pyrrolic units is called isobacteriochlorin (18).  All other reduced  porphyrins are, with the exception of porphyrinogen (19) named systematically. It has been pointed out that the name 2,3-dihydroporphyrin (16) for the chlorin 7  chromophore implies that the inner hydrogens are at the nitrogen atoms at positions 21 and 23. However, it has been found in solution state NMR investigations that the actual 7  structure of chlorins is not consistent with this nomenclature. Therefore, chlorins should be named 2,3-dihydro-22/7-,247/-porphyrins.  Only in cases where this nomenclature will  contribute to any clarification of the issues to be discussed in this thesis it will be used.  5,10,15,20,22,24-Hexahydroporphyrin  (Porphyrinogen)  5,15-Dihydroporphyrin  5,22-Dihydroporphyrin (  f o r m e rn  a  m  e  :  P  n l o r i n  )  Pyrrolic Pigments  Nomenclature  xxvii  PORPHYRIN ISOMERS One porphyrin isomer is of relevance: The porphyrin with an inverted pyrrolic unit (22) is, following a suggestion of the discoverers of this pigment, referred to as 'N-confused' porphyrin.  8  'N-confused' p o r p h y r i n 2-aza-21 -carba-porphyrin  METAL COMPLEXES OF THE PYRROLIC SYSTEMS The IUPAC recommended "Rules for Coordination Compounds" are followed. The 9  name of the ligand precedes the name of the metal. The organic ligand(s) is (are) placed in brackets, listed in alphabetically order and the (anionic) ligand name takes the ending '-ato'.  N=/ N  The  name of the metal is followed by its oxidation number  (\ )-N  //  ^^C^/j N-  (Roman numerals in parenthesis). Coordination of the 23 four central nitrogens is the common structural pattern  [P  orphyrinato](pyridine)iron(ll)  of porphyrinic compounds, and need not be specifically designated. In more ambiguous cases the kappa (K) notation is used, demonstrated at structure 24. Tris(dipyrrinato-K -N ,N )cobalt(IH) 2  10  11  'EXPANDED' PORPHYRINS 'Expanded' porphyrins are porphyrinic compounds containing either more than four pyrrolic units or more than one single carbon linking unit between these pyrrols, or both.  11  Their nomenclature is not homogenous, and the use of trivial names widespread. Here, only 'expanded' systems with relevance to this work will be defined as shown below.  Nomenclature  Pyrrolic Pigments  xxviii  REFERENCES (1)  Bonnett, R. In The Porphyrins; D. Dolphin, Ed.; Academic Press: New York, 1978; Vol. 1; Chapter 1.  (2)  IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), Pure Appl. Chem. 1987, 59, 779.  (3)  Fischer, H.; Orth, H. Die Chemie des Pyrrols; Akademische Verlagsgesellschaft: Leipzig, 1937; Vol. II, p 269.  (4)  (a)Pyrroles: Part 1: The Synthesis and the Physical and Chemical Properties of the Pyrrole Ring; Jones, A., Ed.; John Wiley & Sons: New York, 1990; Vol. 48. (b)Pyrroles: Part 2: The Synthesis, Reactivity, and Physical Properties of Substituted Pyrroles; Jones, A., Ed.; John Wiley & Sons: New York, 1992; Vol. 48.  (5)  Falk, H. The Chemistry of Linear Oligopyrroles and Bile Pigments; Springer Verlag:  Wien, New York, 1989, Chapter 2. (6)  Sessler, J. L.; Weghorn, S. J.; Hiseada, Y.; Lynch, V. Eur. J. Chem. 1995,1, 56.  (7)  (a) Crossley, M. J.; King, L. G. /. Org. Chem. 1993, 58, 4370. (b) Crossley, M. J.; Harding, M. M.; Sternhell, S. J. Org. Chem. 1988, 53, 1132.  (8)  Furuta, H.; Asano, T.; Ogawa, T. J. Am. Chem. Soc. 1994,116, 767.  (9)  (a) Commission on the Nomenclature of Inorganic Compounds (CNIC), Pure Appl. Chem. 1971, 28, 39. (b) Sloan, T.E. In Comprehensive Coordination Chemistry, Wilkinson, G., Gillard, R.D., McCleverty, J.A., Eds., Pergamon:Oxford, 1987, Vol.1, Chapter 3.  (10)  Sessler, J. L. Top. Curr. Chem. 1991, 161, 111.  xxix  ACKNOWLEDGMENTS I wish to express my sincere thanks to Prof. David Dolphin for his guidance and the academic freedom he offered. I can only hope that I have made most out of the opportunities he provided. Special tribute goes to my parents for their continued support and encouragement to develop my exploratory nature. It is my pleasure to thank the past and present colleagues of my own and other research groups for their help and entertainment over the years. Mr. Andrew Tovey and Ms. Claire Johnson diligently proof-read the manuscript. I would like to thank especially Dr. Ethan Sternberg. He introduced me to Vancouver and the Pacific North-West and he was an invaluable consultant in chemical issues. I credit thanks to Dr. Ross Boyle and Ms. Huguette Savoie for not only having performed the biological tests on some of the compounds prepared in this thesis but also for having shared their excellent home-cooked foods. This work would not have been possible with the help of the many people in the Department: my teachers, my advisory committee, the secretarial staff, the technicians in the machine shop and the crew in Chem-Stores, to list only a few. Sincere thanks go to the staff of the NMR facility for the crucial help, training and service provided, and the crew in the Mass Spectroscopy Department. Mr. Peter Boda incinerated some of my finest samples and I thank him for having done that. Special thanks go to Dr. Steve Rettig for the X-ray crystal structures appearing in this thesis. It was always a pleasure to share the excitement when presenting my perfect crystals, and it was never too harshly put when their perfection crumbled upon closer investigation. Financial support in the form of a year-round research assistantship from Prof. David Dolphin (September 1991 to present) is greatly appreciated. And, hey, thank you, Mary-Lynn, for your unfaltering faith and support.  This is another specimen in the ongoing collection of stamps, stones, bones and feathers.  1  PYRROLIC PIGMENTS  GENERAL INTRODUCTION  pigment (pig'mant) n. 1. Any of a class of finely powdered, insoluble coloring matters suitable for making paints, enamels, oil colors, etc. 2. Any substance that imparts color to animal or vegetable tissues such as melanin and chlorophyll. 3. Any substance used for coloring. [L < pigmentum < pingere to paint] {Funk & Wagnalls Canadian College Dictionary, Fitzhenry & Whiteside, Toronto: 1989)  Pyrrolic pigments contain one or more, typically conjugated, pyrrolic units. Cyclic tetrapyrrolic pigments are ubiquitous in nature and are found, for example, as chlorophylls, hemes and corroles. Due to their fundamental importance to life they have been dubbed "the pigments of life".  Naturally occurring linear pyrrolic pigments are, for instance, the  photochromes whilst others are the source of bioluminescence, metabolic diseases or antimicrobial activities. Recent advances, in medicinal chemistry have introduced pyrrolic pigments as photosensitizers in tumor therapy and as chelators for metals used in diagnostic medicine. In addition, they are utilized as ligands for metals used in transition metal catalysis, as paints, in dyes for optica] recording media, as well as insecticides. They are also players in a host of basic research areas.  General Introduction  Pyrrolic Pigments  2  Thus it becomes apparent that this thesis can focus only on partial aspects of the wide field of pyrrolic pigments. However, despite this natural limitation, the number of different subjects touched and the structural variety of the pigments displayed herein will, perhaps, give some reflection of the breadth of the field. The various topics are presented in three independent and unequally weighted parts, each containing their own introductory, results and experimental sections as well as references: These parts are defined as follows:  Part 1 Synthesis and Study of Pyrrolic Pigments for Use in Photodynamic Therapy (PDT) This part discusses the synthetic aspects of cyclic, aromatic pigments such as mesophenyl substituted porphyrins, chlorins and sapphyrins. The aim of the research was to develop rational syntheses for potential drugs for PDT.  This, in turn, has led to the  investigation of the physical, chemical and biological properties of the novel compounds prepared. Truly unique (and unexpected) pigments were synthesized in due course of these studies.  The opening chapter of Part 1 presents an introduction to the principles of PDT and a review of selected compounds which are potentially interesting compounds for use as photosensitizers. Special focus is placed on the synthesis of hydroxylated porphyrins and chlorins in general, and the osmium tetroxide mediated dihydroxylation of porphyrins and other aromatic systems in particular.  General Introduction  Pyrrolic Pigments  3  Part 2 meso-Phenyldipyrrins - Synthesis and Metal Complex Formation This part describes the oxidation of bi- and tripyrrolic non-colored matter to their corresponding pigments and the metal chelating properties of these pigments. This mainly curiosity driven project was made possible by the availability of large quantities of mesophenyldipyrromethane and -tripyrrane. This stemmed from the result of one of the rational synthetic approaches described in Part 1.  Part 3 2-Pyrrolylthiones as Bidentate N,S Chelators This part introduces mainly the use of monopyrrolic pigments containing the 2-pyrrolylthione moiety as transition metal chelators.  This part describes work which  emerged by serendipity while attempting (eventually successfully) to find an improved synthesis forraeso-diphenylporphyrinused as starting material in Part 1.  PART 1  SYNTHESIS AND STUDY OF PYRROLIC PIGMENTS FOR USE IN PHOTODYNAMIC THERAPY (PDT)  5  1.  INTRODUCTION AND LITERATURE REVIEW  1.1  PHOTODYNAMIC THERAPY (PDT)  1.1.1  HISTORY AND GENERAL INTRODUCTION  1  3  Photodynamic therapy is a medical treatment which employs the combination of light and drug to bring about a (lethal) cytotoxic effect to cancerous or otherwise unwanted tissue. It derives great promise from an idealized mode of action: A drug (photosensitizer) of negligible dark toxicity is introduced into a body and accumulates preferentially in cancerous cells. When the drug reaches its highest ratio of accumulation in diseased versus healthy tissue, a carefully regulated light dose is shone onto the diseased tissue. Light activates the drug and elicits the toxic action. The amount of light is large enough to cause a lethal response in the tissue with high levels of photosensitizer, but small enough to spare the surrounding healthy tissue from extensive damage. Shortly after the treatment, the lethally damaged cells become necrotic, and are rejected or absorbed by the body.  The  photosensitizer clears rapidly out of the body after light treatment. It is worth pointing out that PDT is ideally curative, whereas many traditional cancer treatments aim merely to prevent further growth or the spread of cancer. PDT can, due to fiber optic technology, be applied at almost any location in the body, either with or without accompanying surgical treatment. In addition, extracorporal treatments of blood for virus deactivation are under investigation.  4  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  6  While the term PDT is relatively modern, this binary modality of treating diseases can be traced far back in history. The ancient Egyptians used the combination of orally ingested plants (containing light-activated psoralens) and sunlight to successfully treat vitilago over 4000 years ago.  5  Contemporary PDT began when Raab described in 1900 the  toxic action of acridine dyes and light on Paramecia,  and he also showed that these  unicellular organisms could be effectively killed with this combination.  Jesionek and  6  Trappeiner treated, in 1903, a skin cancer with topically applied eosin and light.  7  Policard examined the ability of porphyrins to produce a phototoxic effect.  8  In 1925  One unusual  experiment highlighted the potency of drug-induced photosensitivity. Mayer-Betz injected himself in 1913 with 200 milligrams hematoporphyrin and registered no ill effects until he exposed himself to sun-light, whereupon he suffered extreme swelling and photosensitivity remained for several months. '  9 10  The modern age of PDT began when a mixture, called 'hematoporphyrin derivative' (HpD) was used in 1960 by Lipson and Baldes.  11  This multi-component photosensitizer  was, and currently is, prepared by the treatment of hematoporphyrin with sulfuric acid in acetic acid followed by an alkaline hydrolysis. A number of monomeric, dimeric and polymeric porphyrins containing ether, ester and carbon-carbon linkages are thus formed.  3  HpD was originally tried as a fluorescent tumor imaging agent and the preferential accumulation of HpD in certain cancerous tissues (in animal models) was found.  12  The  potential to use HpD as an anti-cancer drug was investigated by Dougherty et al., and this led to the discovery of HpD as the first generation PDT drug. " 13  15  Several attempts to define  the active compound(s) in this mixture failed, but these investigations did determine that neither the monomers nor the dimers of hematoporphyrin are the most active species.  16  A  somewhat purified, synthetically reproducible version of HpD, known under the trade name Photofrin®, has, in the past few years, received regulatory approval for the treatment of selected cancers in several countries.  1  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  7  In spite of this approval, Photofrin® exhibits several drawbacks. First and foremost, it is a complex mixture of compounds. This complexity makes characterization, as well as physical and biological studies very complicated, and regulatory approval for drugs consisting of mixtures is becoming increasingly more difficult. These obstacles may explain the fact that it has taken over twenty years from initial biological testing to final approval of Photofrin®.  Secondly, the longest wavelength of absorption of Photofrin®, that is the  longest wavelength at which the drug can be photoactivated, is at 630 nm. At this wavelength, effective light penetration through tissue due to endogenous chromophores, mainly hemoglobin, and light scattering is quite low (~4 mm).  17  Thirdly, the clearance rate  for Photofrin® is slow. As a consequence, a patient must remain in subdued light for several weeks post-injection of the drug to prevent skin damage.  18  The above drawbacks provided the benchmarks for the development of the so called second-generation photodynamic drugs. These components are generally single substances with longer wavelengths of absorption. These longer wavelength are desired because tissue has a 'spectral window' between around 650 nm and 800 nm. In this range, light penetration is deepest and, hence, deeper-seated tumors can be treated with these drugs.  19  Also, the  pharmacokinetic patterns of the second-generation drugs are better adjusted for their uses. Several of these second-generation drugs are currently in clinical trials. However, many of 3  these are difficult to synthesize or have other drawbacks. Selected second-generation drugs will be discussed in more detail in the following chapters. The third-generation of drugs are currently in pre-clinical stages of investigation and they aim at eliminating the disadvantages of the preceding generations. They also will be discussed in more detail in the following chapters.  The development of novel PDT drugs is complicated by the nature of the mode of action of PDT. The drugs used are non-specific in the sense that they do not target a specific  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  8  enzyme or DNA sequence or even organ. Their mechanism of accumulation is believed to be an interaction with various lipoproteins found in plasma, in cell membranes and elsewhere. This non-specificity makes the development of structure-function relationships, 1  according to which a development of an improved drug could be directed, very difficult.  The achievement of the desired photophysical properties seems not to be an impediment to the development of new drugs as the plethora of long-wavelength absorbing dyes to be discussed below will show. On the other hand, the delivery system of a PDT drug, or of any drug, has a great influence on the efficiency of the drug, implying that a good drug might give a bad biological response due to delivery problems. Consequently, a rigorous and time-consuming (biological) testing program has to accompany any PDT drug development.  20  As more drugs are being developed and tested, certain trends common to promising PDT drugs can be recognized. Most prominent of these trends is that all have pronounced amphiphilicity. A selection of the second-generation photosensitizers is shown below (Figure 1-1).  The diversity of these second-generation drugs illustrates this lack of a  generally valid baseline structure. The development of BPD-MA (1) illustrates also that fortuitous circumstances are often involved in finding the 'optimal' drug, as neither the diacid nor the diester derivative of this drug show the high biological activity of the monoacid monoester (1).  1  Although this review will be restricted to tetrapyrrolic  (porphyrinic) pigments, it must be stressed that the effect of photosensitization is not restricted to these pigments (vide supra), and other substance classes might be suitable candidates for PDT.  9  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  9  C0 H 2  Figure 1-1  Structures of selected second generation photosensitizers: 1 benzoporphyrin derivative monoacid (BPD-MA) , 2 tin etiopurpurin ; 3 mono-L-aspartyl chlorin eg*; 4 meso-tetra(m-hydroxy)phenylchlorin ; 5 aluminum phthalocyanine tetrasulfonate 21  22  23  24  1.1.2  PHOTOPHYSICAL AND PHOTOCHEMICAL BASIS OF PDT  PDT is largely dependent on the presence of molecular oxygen. molecular oxygen-free systems was found to be ineffective.  27  26  2 5  PDT performed in  This suggests that singlet  oxygen generated by the photosensitization of molecular triplet oxygen (or other reactive species) is the principal toxic species formed during PDT, although the extent to which this species is responsible for the photodynamic effect is under debate.  Nonetheless, the  generation of singlet oxygen is extremely crucial to the success of PDT, and one of the first  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  10  tests performed on new PDT drugs is to probe their capability of singlet oxygen generation.  28  The principal photophysics of photosensitization are illustrated in a modified Jablonski diagram, shown in Figure 1-2. The sensitizing compound in its singlet ground state (So) absorbs a photon to place it in an exited singlet state (Si-S ). Depending on the n  energy of the exciting photon, a higher or lower state may be reached (1). In a porphyrin, this transition corresponds to a 7t*<— n transition. A compound in a higher S-state may lose the energy radiatively through fluorescence (2) or non-radiatively (thermally) through internal conversion (3) and, thereby, returns to its ground state. Both transitions are spinallowed processes and, therefore, the lifetime of the excited singlet states is very short, typically in the nanosecond range.  1. 2. 3. 4. 5. 6.  A b s o r p t i o n of Light Fluorescence Internal C o n v e r s i o n Inter-system C r o s s i n g Phosphorescence Singlet O x y g e n Production ( S e n s i t i z a t i o n , T y p e II P h o t o p r o c e s s ) 7. H y d r o g e n or e l e c t r o n t r a n s f e r (Type I Photoprocess)  o  3  2  *- 0 1  Figure 1-2  2  Modified Jablonski diagram for a typical Photosensitizer  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  11  Of particular importance with regard to PDT is that the excited species can undergo the non-radiative process of inter-system crossing (ISC) (4). This process is a so called spinforbidden process as it requires spin inversion, thereby converting the photosensitizer to a triplet state (Ti). Any 'forbidden' pathway is less likely than an 'allowed' process, but a good photosensitizer undergoes the 'forbidden' ISC pathway with very high efficiency. The molecule can relax from the triplet state via two pathways: radiatively by phosphorescence (5), and non-radiatively by spin exchange with another triplet state molecule. Phosphorescence involves a spin-inversion and thus is also spin-forbidden, imposing a relatively long lifetime on the triplet state, typically measured in microseconds, thus allowing the interaction with molecules in the vicinity of the sensitizer. Spin exchange (also corresponding to energy transfer) with triplet oxygen generates the highly reactive species singlet oxygen (6). This process is called a Type II photoprocess. Another type of interaction with other molecules can bring about electron or hydrogen transfers, converting the sensitizer into a novel chemical species (7). These latter processes are called Type I photoprocesses.  The prevalence of these processes is dictated by many  factors such as substrate and oxygen concentration, substrate types and many physical parameters of the solution in which these processes are taking place. As shown below, both processes have the potential to cause detrimental damage to a cell by altering the chemical structure of its vital components.  Type I Photoprocesses  A sensitizer (SENS) in its triplet state (^SENS) is often a strong oxidant and, as is shown in Figure 1-3, capable of abstracting electrons or hydrogens from a particular substrate (SUB). The reduced forms of the sensitizer can react in many ways, such as with ground state oxygen to form the cytotoxic superoxide radical (Pathway A) or its protonated  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  12  form (Pathway B). In either case, the sensitizer relaxes to its ground state and is available for another photosensitization cycle. SUB-H  (SUB-H)  SENS  (SENS)"  3  3  0  SUB-H  +  2  1  SENS  3  SENS  (0 )-  +  (SENS-H)-  1SENS  0  (H0 )'  3  2  Pathway A Figure 1-3  (SUB)  2  2  Pathway B  Type I photoprocesses  Type II Photoprocesses  The singlet oxygen generated by the sensitizing action is a very reactive species with a lifetime in water of roughly four microseconds. It undergoes several reactions with biological substrates such as oxidations and cycloadditions, as shown in Figure 1-4.  Hydrogen Abstraction and Oxygen Addition (Ene Reaction)  Cycloaddition  Oxygenation Figure 1-4  Type II photoprocesses  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.1.3  Introduction  13  THE PROFILE OF THE IDEAL PDT DRUG  Using the aforementioned principles of PDT and general considerations for the synthesis and marketing of any drug, the profile for the ideal PDT drug can be constructed:  29  (The order of points does not necessarily reflect a ranking of their importance.) •  The photosensitizer should have strong absorption in the red part of the visible spectrum (< 650 nm), where light penetration into tissue is the greatest.  •  For type I photosensitizers, the photophysical properties should be such that the quantum yield of triplet formation is high, with a triplet energy greater than 94 kJmoH, and that the triplet energy is effectively transferred to the triplet oxygen.  •  The photosensitizer should have a negligible or very low toxicity in the dark.  •  The drug must exhibit a large selectivity for enrichment in tumorous tissue vs. healthy tissue, particularly skin.  General skin sensitization must be avoided.  However, certain treatment modalities call for skin sensitization, e.g. the treatment of psoriasis. In that case, rapid and selective sensitization and desensitization of the skin should be achieved. •  The solution state properties such as solubility, partition coefficients, aggregation behavior and ionic charges of the drug should allow easy formulation (in solutions, creams, etc.), and should confer the above mentioned selectivity.  •  The drug should be excreted or metabolized quickly post-treatment (within hours or maximum days) in a way that does not generate toxic metabolites of any kind.  •  The drug should be a single, enantiomerically and diastereomerically pure substance.  •  The drug should contain a functionality or moiety such as the phenyl group which allows easy derivatization or variation in order to optimize various properties of the drug.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  14  The compound should be synthetically easily attainable from readily available starting materials. The process to yield the drug must bear the potential for upscaling to industrial (multi-kilogram) scales. This severely limits, for example, the number of separations by chromatography. The necessarily Simple process of production will, to a large degree, determine the price of the drug.  This is a point of  consideration in a highly competitive market place with increasing pressure on health costs. The overall costs for a PDT treatment are also reduced by the use of light emitting diodes (LEDs) as light source. Hence, a wavelength of activation suitable for the use of LEDs would be advantageous. The formulated drug should be stable, i.e. possess a long shelf life. The preparation of the administered form of the drug should be simple, e.g. by dissolution in physiological sodium chloride solution for any injectable form of the drug. Last, but not least, the development of any drug with the enormous investment required is only worthwhile, i.e. profitable, for the developer if the patent rights for the drug and their use are exclusively in the hands of the developer.  The  development of drugs solely for the improvement of humankind is, apart from notable exceptions, an illusion.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.2  Introduction  15  RESEARCH OBJECTIVE The aim of this work is the rational development of drugs intended for PDT, focusing  primarily on mew-phenyl substituted pigments. The rationale for this focus is as follows: 1.  Certain meso-phenylporphyrins and -chlorins have shown some promise as  photosensitizers for use in PDT, although only a few meso-phenyl substituted photosensitizers have been tested for PDT. Consequently, this area has the potential for development. 2.  The chemical nature of these pigments will allow facile derivatization (to hone  pharmacokinetic or other properties of the drug) should a promising lead drug be found. 3.  Many pigment classes such as the sapphyrins had not been synthesized or  characterized with mew-phenyl substituents at the time these studies were initiated. 4.  The mew-phenylporphyrins are favorable starting compounds for development of  PDT drugs as they are available via single step syntheses in large quantities. 5.  Concurrent with this investigation a novel class of mew-phenyl substituted pigment  (N-confused porphyrin) with promising photophysical properties appeared in the literature, however, its synthesis was not suited to generate larger quantities of this pigment and, hence, a novel synthetic pathway to access this pigment was desirable.  The work focuses on the elucidation of synthetic and other chemical aspects of the potential drugs rather than on their biological properties, albeit some preliminary biological results will be reported. The chemical aspects are three-fold: one aspect is the synthesis of novel compound classes, and study of their chemical and physical properties; the second and third aspects are the elucidation of the mechanism of synthesis and the synthesis via novel pathways of known compounds, respectively.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.3  Introduction  16  REVIEW OF SYNTHESIS AND PROPERTIES OF PYRROLIC PIGMENTS POTENTIALLY USEFUL IN PDT  1.3.1  PORPHYRINS  NATURALLY OCCURRING PORPHYRINS  1.3.1.1  Porphyrins occur naturally as prosthetic groups in enzymes , as dyes in various egg 30  shells  31  or feathers , and are found in crude oils 32  33  and in the urine of humans or cattle  suffering from congenital porphyria . The only readily available source of porphyrins is 34  blood from which, depending on the procedure of isolation, protoporphyrin IX and hematoporphyrin IX as their diacids or diesters can be isolated in bulk quantities. '  The  UV-visible spectrum of protoporphyrin dimethyl ester is shown in Figure 1-5.  This  35 36  spectrum exhibiting a very strong (log e = 5-5.5) absorption around 400 nm, known as Soretband, and four low intensity bands, known as Q-bands, is characteristic of porphyrins. The weak absorption in the long wavelength region indicates that porphyrins are not ideal PDT drugs, although, as mentioned, they may cause strong photosensitization.  350  450  550  X  Figure 1-5  650  [nm]  Optical (CHCI3) spectrum of protoporphyrin IX dimethyl ester  750  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Part 1:  Introduction  The chemistry of naturally occurring porphyrins has been reviewed in depth.  17 31,36-38  Their importance within the realm of this review does not go beyond their use as starting materials in the synthesis of certain PDT drugs, as mentioned above and detailed below.  1.3.1.2  SYNTHETIC PORPHYRINS  Synthetic porphyrins fall primarily into two classes. There are those with all 6positions alkylated, the archetype of which is octaethylporphyrin (OEP), and those with all mew-positions arylated, the archetype of which is mew-tetraphenylporphyrin (TPP). As in nature, synthetic porphyrins are built up from pyrrolic precursors. OEP and TPP can, due to their symmetric structures, be synthesized from one monopyrrolic precursor in a one-step 4 x 1-type condensation reaction (Figure 1-6), although this type of reaction is much more common for the TPP-type porphyrins than it is for the OEP. The pyrrolic units may (as in most OEP syntheses) or may not (as in most TPP syntheses) carry the linking carbon.  +  4x1  I' o N +  1 +1  Q Figure 1-6  The different concepts of porphyrin synthesis  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  18  All other porphyrin syntheses have to be accomplished step-by-step. Conceptually, many approaches are possible, all of which have precedent in the literature. A 2 + 2 approach joins two bipyrrolic precursors. The two bipyrrolic precursors are generated by a 39  1 + 1 addition. They may be, depending on the particular porphyrin desired, the same or different.  They can be set up to condense in only one orientation, or in both orientations.  They can be joined in one step to form the cyclic tetrapyrrolic pigment, or first joined into a linear tetrapyrrolic species which is cyclized in a second, independent, step. A rarely performed 3 + 1 approach inserts the fourth pyrrolic unit into a tripyrrolic precursor. The particular modes are chosen depending on the porphyrin required and the starting materials available. The various methods of synthesis have been reviewed  and only those of  30  particular importance for the understanding of this work will be briefly outlined.  The Synthesis of TPP The classic preparation of meso-tetraarylporphyrins is via a 4 x 1-type methodology as originally developed by Rothemund ' and improved by Adler et al. , Gonsalves , and 40 41  then Lindsey et al. ' 44  46  43  42  The mechanism of the TPP formation has been elucidated. ' 47  48  Benzaldehyde reacts under acid catalysis with pyrrole to form an intermediate arylpyrrolylcarbinol (7).  49  This carbinol is set up to form a resonance stabilized cation (8) which reacts  with another equivalent of pyrrole to yield the dipyrromethane 9. To this is added another benzaldehyde moiety and repetition of this reaction sequence occurs. Ultimately a ringclosure condensation forms the porphyrinogen (10), which is oxidized in situ to the corresponding fully unsaturated porphyrin (6). The Rothemund-type TPP synthesis has been applied to a wide variety of aryl ' " and alkyl aldehydes. Several solid ' and, more 41  50  52  53  54  55  traditionally, liquid acid catalysts are suitable to catalyze the reaction. Under a standard set of conditions, yields of over 30% for this condensation are regularly observed. Until 1995 it was believed that TPP was the only macrocyclic pyrrolic pigment formed in this reaction. Chapter 1.3.4 and 1.3.5.1 discuss findings which led to the correction of this view.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  S c h e m e 1-1  Introduction  19  Rothemund synthesis of meso-tetraphenylporphyrins 6 Reaction conditions: (i) 1. catalytic H ; 2. oxidant (ii) catalytic H+ (iii) oxidant +  meso-Tetraarylporphyrins which are less symmetric than TPP have to be synthesized in a step-wise manner, either along a 2 + 2 or a fully stepwise approach.  56,57  This step-wise  approach results in a drastic reduction in overall yield of the final porphyrin.  Porphyrins with Extended TC-Systems  The optical properties of regular porphyrins are, as mentioned, not ideal for their application in PDT.  However, certain porphyrins with extended 7T-systems have  significantly bathochromically  shifted  spectra.  Robinson  and  Morgan prepared  porphyrins bearing thiobarbituric and barbituric acid functionalities at the meso -position, for instance compound 10.  58  Somewhat unexpectedly, its electronic spectrum exhibits one  broad and strong band at ~ 710 nm. The NMR of 10 indicates a certain loss of aromaticity of the porphyrin chromophore. Without any details, the researchers remarked that biological  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  20  studies with these compounds looked promising with respect to their further refinement for their use in PDT.  58  Lash and co-workers introduced recently a series of porphyrins in which one or more pyrrolic units were replaced by pyrroles fused to larger aromatic systems, for instance phenanthrene system l l . " 59  61  Their UV-Vis spectra are reportedly bathochromically shifted  as compared to regular porphyrins, however, the scarce data available as of now indicate that they are not shifted to an extent that they are suited as PDT agents.  11  This finding is matched by evaluations for similar systems such as monobenzoporphyrins ' , dibenzoporphyrins , or tetrabenzoporphyrins . 62 63  64  6 5  Part 1:  3.2  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  21  CHLORINS, BACTERIO- AND ISOBACTERIOCHLORINS  1.3.2.1  THE OPTICAL PROPERTIES OF CHLORINS  Chlorins are distinguished from their parent porphyrin by the presence of one reduced peripheral double bond, and this leads to an altered optical spectrum of these compounds. Figure 1-7 shows the spectrum of BPD-MA (1) which exhibits the general features of chlorin spectra. Most importantly in the context of PDT, they have a strong absorption in the long wavelength portion of the visible spectrum, and this feature makes them very attractive as photosensitizers.  1  400  1  1  500  600  1  700  1  800  Column 3  Figure 1-7  UV-visible spectrum of chlorin 1 (  ) and bacteriochlorin TPBC (  )  Also shown in Figure 1-7 is the spectrum of a bacteriochlorin (meso-tetraphenylbacteriochlorin, TPBC). Its optical spectrum is even further bathochromically shifted, and its long-wavelength absorption is within the 'ideal' region for PDT. Isobacteriochlorins 19  have spectra very similar to chlorins and the synthetic effort to establish a second reduced  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  22  site is not rewarded with an improved spectrum and, consequently, research toward novel photosensitizers has been focused on the preparation of chlorins and bacteriochlorins.  TC TC TC  TC  t l  l  TC  TC  1u.  1u  TC a  2u •  a  TC a  Figure 1-8  a  2u  a  2u  2u  1u-  Energy level diagram for the frontier orbitals of the four generic metalloporphyrin classes (adapted from Fajer ) 66  Gouterman laid the theoretical foundation of porphyrin optical spectroscopy.  67  Figure 1-8 presents a scheme based on extended Hiickel calculations of the frontier orbitals of the four generic metalloporphyrin* classes.  The longest wavelength absorption in  * Metalloporphyrins and chlorins are chosen here as example for reasons o f simplicity. A n equivalent trend is valid for the free bases of the pigments.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  23  porphyrins and chlorins can be assigned to a HOMO-LUMO transition. This is shown here as a n*<r-n transition, indicated by a dark arrow. With increasing saturation of the porphyrin macrocycle, the energy of the so called a i orbitals, the HOMOs, are progressively raised. u  The LUMOs of porphyrin, chlorin and bacteriochlorin remain largely isoenergetic, whereas those of the isobacteriochlorin are raised. The size of the HOMO-LUMO gap corresponds to the energy for this transition: the larger the gap, the higher the energy needed for the transition, and vice versa. The trends seen in the electronic spectra of porphyrins and the reduced species reflect this clearly: the higher the energy, the shorter the wavelength of the corresponding absorption band, the lower the energy, the longer the wavelength of the absorption band. The theoretical picture corresponds even further with the chemistry of these pigments.  Since (electrochemical) oxidation corresponds to an abstraction of an  electron from the HOMO, and reduction to the filling of a LUMO, it can be expected that it becomes increasingly harder to perform an oxidation the more saturated the system is, and that it easier to reduce a chlorin and a bacteriochlorin than it is to reduce a porphyrin or an isobacteriochlorin. This trend can, indeed, be confirmed experimentally. '  68 69  In the remainder of this introduction, the synthesis and properties of various chlorins and related long wavelength absorbing dyes will be discussed.  Introduction  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  24  NATURALLY OCCURRING CHLORINS: C H L O R O P H Y L L S  1.3.2.2  Chlorophylls a and b (12) are the pigments of photosynthesis, and, consequently, they are ubiquitous. Demetallated chlorophylls have a strong absorption at -660 nm and for that reason are suitable for PDT. However, their isolation and purification is non-trivial and they are chemically very unstable substances. Nonetheless, Me0 C  their use as PDT agents has been proposed and  2  O  1  2  O-Phytyl  investigated by numerous workers.  70  This review  R = -CH ; Chlorophyl a R = -CHO; C h l o r o p h y l 3  is focused on the chemistry of synthetic chlorins.  The chemistry of chlorophylls, their isolation and modification have been reviewed extensively. ' " 31 71  1.3.2.3  73  THE TOTAL SYNTHESIS O F CHLORINS  The 'Classical' Total Synthesis of Chlorins The total synthesis of chlorins follows conceptually the same routes as the total synthesis of porphyrins, but includes additional steps to introduce a hydropyrrolic unit. This necessitates synthetic strategies unknown in porphyrin chemistry and often complicates the synthesis enormously. In fact, the crucial steps in some chlorin total syntheses are more reminiscent of corrin syntheses as they are adapted from synthetic strategies devised by Eschenmoser, Woodward, and Battersby for the total synthesis of corrins. synthesis of chlorophyll a by Woodward et a l .  15,76  74  The total  is the prime example of the total synthesis  of a chlorin. This chlorophyll synthesis was at its time (and still is) of such monumental  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  25  Introduction  scale that it was cited as one of the reasons why Woodward was awarded in 1965 the Nobel Prize. It is, therefore, obvious that a chlorin total synthesis can be excluded as a viable way to produce a commercial product such as a PDT drug. Total syntheses have been performed solely for the purpose of structure elucidation and verification or the demonstration of the power of a novel synthetic strategy, as illustrated with the total synthesis of bonnelin  77,78  and  sirohydrochlorin . A review published in 1994, entitled "Discovery and Synthesis of Less 79  Common Natural Hydroporphyrins", cites more examples.  80  Chlorins through a Rothemund-type condensation  In 1946, Calvin et al. identified the pigment generally present as a contaminant in TPP when produced by a Rothemund-type synthesis, asraeso-tetraphenylchlorin(TPC).  81  Separation of these traces of TPC from the bulk material TPP is possible by chromatographic means however, this is a highly inefficient method to prepare this chlorin. fractional crystallization techniques are not much more efficient.  83  82  Extraction or  A Rothemund  condensation designed to generate meso-tetramethylporphyrin reportedly contained large amounts of the chlorin contaminant (13), ' ' 40  84  85  presumably due to a higher oxidation  potential of this chlorin versus TPC. Ulman, Ibers and co-workers found conditions under which this contamination could be, albeit in low total yields (2-4 %), formed as the exclusive product (Scheme 1-2).  86  Demetallation of these metallochlorins under strict exclusion of  oxygen gave the free base chlorins.  H H R  R  R = Me, Et, P r o p y l M = Ni, C u S c h e m e 1-2  R  13  Rothemund-type synthesis of meso-tetraalkylmetallochlorins 13 Reaction conditions: (i) 1. AcOH, 2 % A c 2 0 , M(OAc)2, A  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  26  Chlorins via 2 + 2 Synthesis  Burns et al. succeeded recently in synthesizing in an astounding yield of 24-27 % a chlorin via a 2 + 2 MacDonald-type condensation in a two-step one-pot procedure. The 87  researchers based their synthesis on the previous findings of Closs , Buchler , and 88  Whitlock and their co-workers.  90  89  They described the tautomeric equilibrium between the  anion of a metallo-5,23-dihydroporphyrin and the corresponding metallated chlorin anion. The phlorin anion seemed to be the equivalent of the monoanion of a 5,15-dihydroporphyrin. Phlorins, such as 14, can be prepared by a MacDonald 2 + 2 condensation of a dipyrromethane bisaldehyde 15 with a dipyrromethane 16, if care is taken to exclude oxygen (Scheme 1-3). Upon metallation with zinc, 14 tautomerized, to the surprise of Burns et al, over the course of several hours quantitatively to metallochlorin 17, without the need to form the anion first. For reasons not entirely clear, zinc ions seem to be exceptional in stabilizing chlorins and/or in promoting the rearrangement ofraeso-hydrogenatedporphyrins into chlorins.  72  Even more surprising, one chlorin isomer is almost exclusively formed. The  mechanism and regioselectivity of this interesting chlorin synthesis are, according to Burns et al., currently under investigation and thus no evaluation of this reaction for the production of PDT agents can be made.  Reaction conditions: (i) 1. N2, p-TsOH, CHCI3; 2. aqueous NaHC03 wash; 2. Zn(OAc)2, MeOH  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.3.2.4  Introduction  27  T H E C O N V E R S I O N O F P O R P H Y R I N S INTO C H L O R I N S B Y REDUCTION  The importance of chlorins as model systems for certain biological systems and for their use in PDT, coupled with the lack of simple or general methods for their total synthesis, resulted in profuse efforts to convert porphyrins into their reduced forms. Theoretically, this corresponds to the mere addition of one (or two) pairs of (molecular) hydrogen to the 8-positions of porphyrins. In practice, this turns out to be non-trivial. All eleven double bonds of a porphyrin are potentially reducible, and the conversion of porphyrins into chlorins is, therefore, a matter of delicate chemical selectivity. In practice, however, only a few of the double bonds will react with a given reagent. Calculations have shown that the mesoand the B-carbons are, in comparison to the others, the most reactive, a finding which is confirmed by the reduction reactions.  Phlorins are often formed as primary (kinetic)  products which subsequently rearrange into chlorins (thermodynamic products). Once the non-aromatic phlorin is formed, it is prone to further reduction. Metallation of a porphyrin often has profound consequences for the outcome of the reduction. Reactions which transfer dihydrogen are advantageous as this transfer can occur for steric reasons much more easily at the periphery of the porphyrin, i.e. at the B-positions than at any other position, leading directly to the formation of chlorins.  The common hydride containing reducing agents are generally inactive with the noticeable exception of diborane. Hydroboration of OEP results in the formation of a 5:1 mixture of the cis- and zrarcs-isomers of the octaethylchlorin (OEC), possibly due to two competing pathways. '  72 91  Pure trans-OEC  is best prepared by the sodium reduction of  OEPFe(III)Cl in isoamyl alcohol. Catalytic reductions such as hydrogen over palladium or  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  28  platinum/charcoal form the c/s-isomer but tend to over-reduce the porphyrin to the corresponding leuco-form, i.e. the porphyrinogen.  The most common, in fact the only reagent known to generally convert porphyrins into chlorins, is diimide (Scheme 1-4).  92  This reduction method was introduced in 1969 by  Whitlock et al. and is performed today essentially as described in the original report. The 83  diimide is generated in situ from /?-toluenesulfonylhydrazide in refluxing pyridine. This reduction of a porphyrin, however, produces a mixture of reduced products. For instance in the diimide reduction of TPP are starting material 6, chlorin 18 and bacteriochlorin 19 isolated by extraction techniques utilizing the different basicity of the compounds, and then these are finally purified by chromatography.  The chromatography steps have severe  practical disadvantages. The Rf-values of these chlorins, bacteriochlorins and porphyrins are only marginally different and require chromatography columns with extremely large compound to solid phase ratios or preparative HPLC or TLC techniques.  This practical  disadvantage can be mitigated somewhat by the possibility of selectively oxidizing the bacteriochlorin to the chlorin and thereby simplifying the mixture.  Ph  Ph  H  93,94  H  Ph  H  H H •H  Ph  Ph  Ph Ph = = = =  Ph  phenyl, 6 m - h y d r o x y p h e n y l , 21 o h y d r o x y p h e n y l , 22 p - h y d r o x y p h e n y l , 23  Scheme 1-4  P h = H, 18 = m-hydroxyphenyl, 20  H  H  Ph  P h = H, 19 = m - h y d r o x y p h e n y l , 24  Diimide reduction of TPP (6) Reaction conditions: (i) K2CO3, p-toluenesulfonylhydrazide, A (ii) p-chloranil  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  The diimide reduction exhibits an interesting selectivity.  29  Metallochlorins are  converted selectively into metalloisobacteriochlorins, whereas chlorins are converted selectively into bacteriochlorins. The basis for this and related selectivities is discussed in section 1.3.3.4.  Regardless of the practical difficulties, Bonnett hails the use of meso-tetra(m-hydroxyphenyl)chlorin 20, prepared by the diimide reduction of the corresponding porphyrin, as the next generation PDT drug.  3  Following the discovery that TPPs with  hydroxy substitution (21-23) are good in vivo photosensitizers, Berenbaum, Bonnett and coworkers tested the analogous chlorins and bacteriochlorins. ' 23  95,96  And, as expected, their in  vivo activity as measured by dose of sensitizer required to cause photonecrosis reaching 5 mm deep into a rat tumor model increases in the sequence porphyrin 21 « chlorin 20 < bacteriochlorin 24.  23  Since the bacteriochlorin is labile, the research efforts have been  focused on the chlorin 2 0 .  23,96  Also, a cost-benefit analysis, i.e. a comparative analysis of  the benefit of desired tumor photonecrosis versus the cost of photodamage of healthy tissue under the same irradiation conditions, has singled out chlorin 20 as the most promising drug.  97  It possesses the required photophysical properties (^  max  (MeOH) = 650 nm,  log £ = 4.52), is a single compound accessible in a few steps from simple starting materials, is non-mutagenic and has minimal dark toxicity. ' 3  optimized.  99  98  Its therapeutic index has been  As a result of these favorable characteristics, clinical studies with this drug  have been conducted or are underway and their initial results have been promising.  3,100,101  The reduction of non-symmetric porphyrins results generally in the formation of all four regioisomeric chlorins. Only in exceptional cases has a selective reduction been observed and steric ' ' 72  76  102,103  as well as electronic  104  reasons have been cited for the  observed selectivity. Another case is the specific hydride reduction of 2-nitro-TPP (25) (Scheme 1-5).  105  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  30  Reduction of 2-nitro-TPP(25) Reaction conditions: (i)1. NaBhU; 2. H2O (ii) Bu3SnH/AIBN, benzene, A  Scheme 1-5  105  2-Nitro-TPP (25) can be reduced with sodium borohydride to the nitro-chlorin 2 6 . " 106  108 jpp it if j i se  s  ner  t under the very same conditions. Apparently the presence of the nitro  group activates the porphyrin towards a nucleophilic attack of the hydride ion such that the nitrated B,6'-bond resembles in its activity an isolated nitroene system.  1091110  Radical  denitration yields the desired chlorin (18).  Photochemical, electrochemical and other special reduction methods have also been utilized.  72  The limited selection of effective reducing reagents and the reversible nature and  general non-selectivity of the reductions, have led to the development of many nonreversible chlorin syntheses. ' 111  112  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.3.2.5  Introduction  31  N O N - R E V E R S I B L E C O N V E R S I O N O F P O R P H Y R I N S INTO CHLORINS  Diels-Alder Reactions of B-Vinylporphyrins  The 8-vinyl group of a porphyrin, for instance protoporphyrin IX (27), in cooperation with a 6,6'-double bond, can act as a diene in a [4+2]-Diels-Alder cycloaddition with activated dienophiles. ' 113  114  Dimethylacetylene dicarboxylate " 115  118  (DMAD)  (28),  tetracyano-ethylene (29) , B-(phenylsulfonyl)propiolic acid , and nitrosobenzene (30) 119  116  120  have been used successfully as dienophiles (cf. to the [4+2]-cycloaddition of singlet oxygen with protoporphyrin IX as described in section 1.3.3.5). In the course of the reaction, the 8,B'-double bond of the porphyrin is lost and a stable chlorin (31-33) is generated (Scheme 1-6).  This reaction has the great advantage of being capable of utilizing a naturallyoccurring vinyl substituted porphyrin which also is available in large amounts, namely protoporphyrin IX (27).  121  This advantage has a flip-side: protoporphyrin IX is not  symmetric, i.e. the two vinyl groups are non-equivalent and any Diels-Alder reaction occurs in a roughly 1:1 ratio at both vinyl groups, generating a mixture of so called ring A and ring B isomers, or it may even react with both vinyl groups to generate an isobacteriochlorin.  119  The generated diastereomers are separable by chromatography. Moreover, the attack of the dienophile can proceed from above the plane of the porphyrin or from below the plane, resulting in a mixture of enantiomers which are much more difficult to separate.  The primary Diels-Alder adduct of 27 when treated with DMAD, the 1,4 diene (31), is prone to undergo base catalyzed rearrangement to the corresponding 1,3-diene (1) (Scheme 1-7). This rearrangement yields a chlorin chromophore with an extension of the  Part 1:  Introduction  Synthesis and Study of Pyrrolic Pigments for Use in PDT  32  7i-system. This leads to a bathochromic shift of the longest wavelength absorption (from ^max (CHCI3) = 666 nm for 31 to X  max  (MeOH) = 686 nm, log e = 4.53 for 1).  122  This  absorption is within the 'ideal' region for PDT. Other photophysical parameters of 1 such as singlet oxygen quantum yield etc. are also adequate for use as a PDT drug.  C0 R  C0 R  2  2  31  Scheme  1-6  R0 C  C0 R  2  2  32  122  R0 C  C0 R  2  2  33  D i e l s - A l d e r r e a c t i o n s of protoporphyrin IX dimethyl e s t e r (27). For clarity o n l y o n e of t h e t w o p o s s i b l e r e g i o i s o m e r s (ring A a n d ring B i s o m e r s ) i s s h o w n . T h e d i e n e m o i e t y is s h o w n in b o l d .  Acid-catalyzed hydrolysis of the propionic acid esters to the free acids generates a highly cytophototoxic compound (Scheme 1-7).  21  It was, however, the discovery that the  partially hydrolyzed regioisomeric monoester monoacid (1), now known as BPD-MA (benzoporphyrin derivative ring A mono acid), exhibited an even larger cytotoxicity when  Part 1:  Introduction  Synthesis and Study of Pyrrolic Pigments for Use in PDT  33  tested against a variety of normal and malignant cell lines which led to its evolution into a promising PDT drug.  123  31 (ring A i s o m e r )  Me0 C 2  Scheme 1-7  ^  C0 H 2  The preparation of BPD-MA. Only one possible enantiomer of the regioisomeric monoesters is shown. Reaction conditions: (i)1. DBU; 2. HCI  While 1 does not show specific affinity for tumors, it exhibits significantly higher concentration in tumors than in the surrounding healthy tissue.  124  BPD-MA demonstrated a  lower skin photosensitivity than that given by Photofrin®.  BPD-MA also clears, post  administration, rapidly from the system, which is another advantage. The presence of stereoand regioisomers in the drug impels laborious experimentation in order to meet the regulatory requirements for the approval of new drugs. To this end, clinical trials are now well under way and this drug has been approved by some national regulatory agencies for its clinical use in PDT.  1  Further derivatization of BPD for its use in, for instance, topical  applications, is still in progress.  125  The development of BPD-MA as a PDT drug has  recently been reviewed in a (printed) lecture.  1  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  34  Reactions of T P P with Carbenes  The exocyclic double bonds in porphyrins have been shown to be susceptible to cyclopropanation with carbenes from sources such as diazomethane or diazoesters. TPP, for example, combines with diazoesters in the presence of copper(I) iodide to form the chlorin 32 (X, ax m  (CH2CI2) = 650 nm,  log £ = 4.25) as a separable mixture of endo- (5 %) and exo-  isomers (20 %) and bacteriochlorins 33 (A-max (CH2CI2) = 720 nm, log e = 3.6). Although Callot reports the isolation of the two possible exo/exo isomers of 33, it is reasonable to assume the presence of other isomers.  126  TPP  Scheme 1-8  Cyclopropanation of TPP with a carbene Reaction condition: (i) diazoethylacetate/CuCI  N-Alkylated products are the sole products of the equivalent reaction with TPPZn. Callot's report has been the only one of this kind, and with a respectable yield of 30 % for the chlorin resulting from the reaction with diazoethylmalonate, this is surprising. The cyclopropane entity is apparently stable, introduces amphiphilicity, could be cleaved to the free acid and the reaction is theoretically compatible with a variety of possible phenyl substituents.  The reaction of OEP with carbenes generates lower yields and more side  products, but the cyclopropane chlorin of OEP is known.  115  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Benzochlorins and Purpurins  Introduction  35  127  Both benzochlorins and purpurins are products of an intramolecular cyclization of meso-alkene substituted porphyrins, such as 34 or 35 (Scheme 1-9). They are, typically derived from consecutive Vilsmeyer-Haak and Wittig reactions.  Scheme 1-9  Formation of octaethylpurpurin (36) and octaethylbenzochlorin (37) Reaction conditions: (i) Me2NCHCHR, POCI3; (ii) strong H+  Acid catalyzed cyclization of acrylate substituted nickel porphyrin 34 yields purpurin 3 6 . compound  exhibits  the  typical  1 2 8 , 1 2 9  This  spectroscopic  characteristics of a chlorin. A number of purpurins have shown photodynamic activity.  127  The dichloro tin(IV)  complex of an etiopurpurin, 2, has turned out to be an  CQ Et 2  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  36  attractive candidate as an PDT agent. Of special note is the fact that administration of this compound elicits only minimal skin sensitization in a rat model. treatment of cutaneous carcinoma are under way.  131  130  Clinical trials for  Purpurins with various meso-phenyl  substituents have been prepared, however, no biological data are available.  132  Metallobenzochlorins, like 37, are available through the acid-catalyzed cyclization of a mew-acrolein substituted  133  metalloporphyrin such as 35 (Scheme 1-9). > 127  Removal of  128  the metal from these chlorins creates biologically active photosensitizers.  134  'improved' (metallo)benzochlorins with eitherraeso-phenylsubstituents  135  iminium salt moiety  136  Several  or a meso-  have been prepared. The copper complex of the latter was shown to  be a photosensitizer acting via a Type I mechanism, whereas its free base showed, due a prolonged triplet life time, Type II characteristics.  137  Compounds integrating the characteristics of purpurins and benzochlorins are known but their synthesis is much more involved. It is doubtful that they will become any option for larger scale syntheses.  131  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  37  Chlorins by Claisen Rearrangement  Montforts and Zimmermann introduced in 1986 an interesting conversion of a hydroxyethyl substituted porphyrin (38) as shown in Scheme 1-10. ' 138  139  Compound 38  undergoes a Claisen-type rearrangement if treated with N,N-dimethylacetamide dimethylacetal at elevated temperatures. The resulting chlorin 39 can be reduced to the closely related chlorin 40.  Both chlorins have satisfactory photophysical properties (for 39:  A,max (CHC1 ) = 660 nm, log e = 4.62; for 40: W 3  (CHCI3) = 645 nm, log e = 4.67) , but 140  the procedure introduces two chiral centers into the porphyrin and generates a mixture of stereoisomers. In addition to this conversion, some variations of this rearrangement pathway have been published. 141  Scheme 1-10  142  Claisen-type rearrangement of a hydroxyethyl substituted porphyrin with N,N-dimethylacetamide dimethylacetal. Only one set of possible stereoisomers shown. Reaction conditions: (i) CH3C(OCH.3)2N(CH3)2, o-xylene, 160°, molsieves 3 A (ii) H2, Pd/C, methanol  With hematoporphyrin as the substrate, an isobacteriochlorin is produced and the regio- and stereochemistry of this compound are complex.  143  The researchers have,  therefore, settled for the development of PDT drugs from the rearrangement of monosubstituted deuteroporphyrins, which cuts down on the final number of isomers but introduces several non-trivial steps to the total synthetic pathway as these mono-substituted deuteroporphyrins have to be synthesized, typically from hemato- or protoporphyrin.  139  Part 1:  1.3.3  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  38  SYNTHESIS O F B-HYDROXY CHLORINS  6-Hydroxychlorins are a central topic of this thesis and, hence, will be discussed in some detail. As will be shown, most B-hydroxychlorins are synthesized via the osmium tetroxide mediated oxidation of a peripheral double bond in porphyrins. For a better understanding of these reactions, some aspects of the osmium tetroxide oxidation will be discussed first.  1.3.3.1  T H E O S M I U M T E T R O X I D E OXIDATION IN G E N E R A L  It has been stated in a review  144  that the first research paper describing the reduction  of osmium tetroxide by unsaturated species was published in 1908 by Makowka, however, in an historical account  146  145  it was noted that Butlerov mentioned, in his 1851  Masters Thesis, the oxidation of organic compounds by osmic acid. Hofmann showed in 1912 that osmium tetroxide could, in the presence of a co-oxidant, be used catalytically to hydroxylate double bonds.  147  In 1936, Criegee  148  developed the dihydroxylation of alkenes  with stoichiometric amounts of osmium tetroxide and recognized the intermediate formation of an osmate ester.  "The stoichiometric osmylation of olefins as perfected by Criegee ... is generally regarded as the most reliable synthetic transformation available to organic chemists. The reasons are simple: OSO4 reacts with all olefins, and it reacts only with olefins. Admittedly, the "all" and "only" in this latter statement are used with some poetic license; however, no other known organic reaction comes close to achieving such enormous scope coupled with such great selectivity."  149  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  39  This strong statement about the osmium tetroxide oxidation of olefins is a quote from a recent review by Kolb, Van Nieuwenhze and Sharpless. This chapter will describe the general mechanism of the osmylation and will detail the "poetic license" of this reaction.  Figure 1-11 illustrates in a schematic way the fundamental steps of the catalytic and stoichiometric osmium tetroxide dihydroxylation.  41  Scheme 1-11  42  43  44  Osmium tetroxide mediated dihydroxylation of alkenes  Osmium tetroxide (41) adds to a double bond (42) to form the stable osmate ester (43). This osmate ester can be cleaved reductively (with e.g. hydrogen sulfide, lithium aluminum hydride, mannitol or sodium bisulfite) to form an insoluble osmium salt, or can be oxidatively cleaved (e.g. with tert-buty\ hydroperoxide, morpholino N-oxide, sodium hypochlorite) to regenerate the osmium tetroxide thus opening up a pathway in which the osmium tetroxide can be used in catalytic amounts. In either case, the cis-diol (44) is formed in good yields. The structure of species 43 is only a formal representation. In the absence of any amine ligand it exists either as a dimer containing five-coordinate square-based pyramidal osmium(VI) with cyclic ester rings  150  or it reacts with a second equivalent of  alkene to form a monomeric diester complex in which the osmium is coordinated in an analogous fashion.  151  In the presence of an amine ligand it exists as a bis-amine complex.  Generally, isolated and conjugated double and triple bonds are susceptible to the dihydroxylation and aromatic systems are inert, with the notable exceptions discussed below.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  40  The osmylation reaction has found a wide field of application, ranging from its use in electron microscopy as a staining and fixative agent for biological tissue  152  (unsaturated  lipids are proposed to be osmylated, thus crosslinked and permeated with the electron-rich metal) to the preparation of fine chemicals . Crystal structures of the intermediate osmate 144  esters have been determined ' , their rates of formation and hydrolysis have been 153  154  studied ' , electron-poor substrates such as partially and fully fluorinated olefins have 144  155  been osmylated ' ' , and heterogeneous osmium tetroxide oxidation catalysts have been 154  prepared.  158  156  157  The rate of the osmium tetroxide-catalyzed dihydroxylation is considerably  accelerated by the additions of amine ligands ' 159  160  and this metal-ligand interaction has  been utilized to impose enantioselectivity on the dihydroxylation through the use of chiral amine ligands. 161  1 6 2  '  1 6 3  Reviews pertaining to the various aspects of the osmium tetroxide  hydroxylations have been published. > ' 144  164  165  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.3.3.2  Introduction  41  T H E M E C H A N I S M O F T H E O S M I U M T E T R O X I D E ADDITION T O DOUBLE BONDS  In light of the aforementioned, it seems astonishing that the mechanism of the osmylation is still under debate. The alternatives are shown below: A  A one step concerted mechanism in the form of a [3+2]-cycloaddition of the osmium tetroxide to the olefin to form the glycol ester 43.  B  A two step mechanism involving the metallaoxetane formation (45) in the form of a [2+2]-cycloaddition and subsequent rearrangement into the glycolate complex 43.  [3 + 2]-cycloaddition  °-7/  O  0  o  o  o  II o  o /  o ' •Os o 43  [2 + 2]-cycloaddition  B Scheme 1-12  45  Mechanistic alternatives for the osmylation of olefins. Possible ligation by amine ligands at any stage has been omitted for clarity.  In an attempt to clarify the situation, Gobel and Sharpless published in 1993 a kinetic study on the influence of the reaction temperature on the enantioselectivity of the asymmetric hydroxylation of olefins with chirally modified alkaloid-osmium tetroxide complexes.  166  They evaluated the modified Eyring-plots according to the isoinversion  principle, a model of chemical selectivity.  167  Clear indications of a multi-step mechanism  with at least two rate-determining diastereoselective steps were thus provided. This finding rules out the one step [3+2]-addition mechanism. Although this is strong evidence for the two step mechanism shown above, it is not proof. The findings are consistent with any two-  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  42  step mechanism. However, several other indications support the [2+2]-pathway which was initially proposed as a result of the comparison of the reactivity of alkenes with the permanganate ion and osmium tetroxide:  168  •  A C=C bond, albeit only a weak nucleophile, would be expected to attack at the more electropositive end of the Os=0 bond, thus forming 45.  169  In accord with this,  osmium tetroxide reacts with the stronger nucleophiletert-butylamineat the metal centre. •  170  It has been repeatedly observed that electron-withdrawing groups on the alkene retard its reactivity toward osmium tetroxide presumably because of the lowering of the nucleophilicity of the double bond. , 144  •  Aromatic substrates (vide infra) react with osmium tetroxide at their site of greatest electron density , i.e. the bond with the highest degree of bond localization and, 171  therefore, greatest nucleophilicity. The following section will present an extended discourse of this reaction type. •  The dramatic increase in the rate of formation of the osmate ester 43 on addition of amines like pyridine might be explained by an increase of the rate of the rearrangement due to the electron-donating effect of the amine ligand.  •  144  Theoretical approaches calculate a lower transition state energy for the [2+2]pathway as compared to the [3+2]-pathway.  163  Collectively, these indications are highly supportive of the [2+2]-addition with subsequent rearrangement of the metallacycle and, consequently, this can be considered the most likely mechanism.  Introduction  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.3.3.3  43  T H E O S M I U M T E T R O X I D E OXIDATION O F A R O M A T I C S Y S T E M S  The osmium tetroxide-catalyzed dihydroxylation of olefins is considered a mild and highly selective oxidation and is routinely performed in the presence of aromatic systems.  165  However, osmium tetroxide oxidations of aromatic systems were described as early as 1942  172  and have been studied in detail since.  173-180  This apparent paradox finds its  explanation in the rate of the oxidation of alkenes as compared to that of aromatic systems. Whereas a reaction time of some hours is sufficient to achieve quantitative turnover for the former, the latter requires days, weeks, and in extreme cases months for reasonably high yields. As will later be shown with the osmium tetroxide oxidation of a benzoporphyrin derivative (Scheme 1-17), in substrates containing both double bonds and aromatic systems, any available double bond will be oxidized first before the oxidation of the aromatic system becomes appreciable. This kinetic differentiation explains the high degree of selectivity of the osmylation reaction. HO  Scheme 1-13  OH  Osmium tetroxide mediated dihydroxylation of polycyclic aromatics Reaction conditions: (i) 1.1 equiv. Os04/3%pyridine/benzene, several days to one week; 2. mannitol/KOH  Aromatic systems undergo ring cleavage only in extreme cases.  178  Generally,  osmium tetroxide attacks polycyclic aromatics regioselectively at the site where the addition to the double bonds results in minimal electron reorganization.  181,182  Part 1:  Introduction  Synthesis and Study of Pyrrolic Pigments for Use in PDT  44  For example, phenanthrene (46) reacts at the 9- and 10-position, maintaining the aromaticity of two phenyl rings ' , and anthracene (47) adds the first equivalent to the 172  180  1,2-bond, maintaining a fully conjugated naphthalene system, and a second equivalent adds to the conjugated, yet non-aromatic 3,4-bond (Scheme 1-13). ' 176  177  The site of addition in  the phenanthrene case correlates with the site of highest electron density; in the anthracene case it does not, however, in both cases the attack occurs at the bond with the lowest localization energy Ui), defined as  Equation 1 -1  L(0 = E (S) - E (S - i) K  n  where E (S) is the 7t-electron energy computed, for instance, by HMO calculations, for the n  aromatic substrate S, and E (S - i) is the computed 7t-electron energy of aromatic substrate n  without the bond at which the addition reaction took place.  181  L(i) is then an index which  quantifies the extent to which the bond is delocalized. As a result, the smaller the value of L(i), the greater the probability of the addition reaction occurring at the bond i. In other words, the bond with the highest (localized) double bond character will be the site of reaction. This sums up the interpretation of the "poetic license" in the statement regarding the selectivity of osmium tetroxide oxidations of alkenes.  The above bond localization energy argument as put forward by Dewar and Dougherty  183  has been formulated over the years as 'the principle of least motion or  minimum electron reorganization'  182  and is known for many reaction types, being  exemplified in a formidable way by the osmylation of C60-buckminsterfullerene. Buckminsterfullerene has a spherical C60-frame work which resembles the seams of a soccer ball, i.e. it is composed of five- and six-membered rings. All vertices of the five-membered rings are connected to six-membered rings, and every second vertex of the six-membered rings is connected to another six-membered ring. This renders all carbons equivalent but  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  45  results in two bond types, namely those between two six-membered rings and those between six- and five-membered rings. Buckminsterfullerene regioselectively adds osmium tetroxide to the bonds connecting two six-membered rings  184-186  (Scheme 1-14), a finding which has  been confirmed by the X-ray crystal structure analysis of the buckminsterfullerene monoosmate ester bispyridine adduct (Figure l-8)  Scheme 1-14  The osmylation of buckminsterfullerene  186b  .  Figure 1-9 X-ray crystal structure of the buckminsterfullerene-osmium tetroxide adduct (from Hawkins et a/. ) 186b  Wudl has rationalized this on the basis of defining buckminsterfullerene as a cluster of pyracycles, i.e. cyclic conjugated 4n-7t-systems, shown in Scheme 1-14 in bold.  187  These  cyclic systems 'freeze' the inner double bond in place. Consistent with Wudl's picture, this bond is also calculated to have a lower bond localization energy than the alternative bond  181  and extended Hiickel calculations also assign it a higher electron density . In light of this, 185  it is expected that osmium tetroxide adds to this 'frozen' double bond.  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Part 1:  1.3.3.4  Introduction  46  T H E O S M I U M T E T R O X I D E OXIDATION O F P O R P H Y R I N S  Another large aromatic polycyclic system which has been successfully osmylated is the octaalkylporphyrin nucleus. To anticipate the conclusions, it can be stated that, as expected, the osmium tetroxide attacks the bond of highest double bond character, namely the B,B'-bond.  188  These bonds have also be described as "cryptoolefinic" . 111  Fischer et al. reported in 1940  the oxidation of naturally occurring  octaalkylporphyrins with osmium tetroxide to yield the corresponding vie- dihydroxychlorins.  189,190  It was noticed  191  that the reaction with naturally occurring non-symmetrical  substituted porphyrins did not yield a single product, but the chlorin-type spectrum of the products revealed formation of the diol at the B,B'-positions.  49  Scheme  1-15  50  51  52  Osmium tetroxide mediated dihydroxylation of deuteroporphyrin dimethylester (48). pMe = methyl propionate side chain Reaction condition: (i) 1.1 equiv. Os04, pyridine, 2. H2S  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  47  Porphyrins provide four such positions and this may give rise to the observed formation of regioisomers. Scheme 1-15 shows the four possible regioisomers (49-52) of the osmium tertroxide-mediated dihydroxylation of deuteroporphyrin dimethylester (48).  192  The separation of the regioisomers is cumbersome and, moreover, each regioisomer is formed as a pair of stereoisomers as a result of attack from above and below the plane of the porphyrin. The products are formed in a non-statistical manner. Steric as well as electronic influences are responsible for this. Oxidation takes place preferentially at the subunit in the quadrant opposite to a electronegative group.  193  MO calculations on this type  of porphyrins have not been published, so any explanations following the principles of minimal electron reorganization or the like would be presumptuous. The osmylation of highly symmetric porphyrin, such as synthetic etioporphyrin I or OEP, provides only one isomer (Scheme 1-16).  194  The structure of 53 has been proven by X-ray crystallography  (Figure 1-10). The diol functionality introduces some amphiphilicity into the molecule and generates a stable chlorin chromophore (53) (A,  max  (CHCI3) = 643 nm, log e = 4.62) fulfilling  some of the photophysical criteria listed in section 1.1.3. Therefore, it is not surprising that these compounds have been suggested as third generation PDT agents. ' 3  29,195  "  197  The  skeleton of OEP allows no simple functionalization, so fine-tuning of the solubility and other physical properties is limited.  OEP  Scheme 1-16  Osmium tetroxide mediated dihydroxylation of OEP Reaction condition: (i) 1. OsOVpyridine, 2. Reduction  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  X-ray structure of 5 3 (from Barkigia et a/. bonded dimer in the solid state.  Figure 1-10  Introduction  241b  ).  53  48  forms a hydrogen  A second equivalent of osmium tetroxide oxidizes the diol chlorin selectively to the tetraol bacteriochlorin 54 (k  max  stereoisomers.  195  (CHCI3) = 715 nm, log e = 4.72) as a mixture of  This selectivity is general for all types of chlorins. ' 198  199  Insertion of a metal into the chlorin changes the outcome of the reaction drastically: Dihydroxylation of a metallochlorin results selectively in the formation of an isobacteriochlorin.  192  The pronounced directing effect of the central metal has been  observed before and appears to be general, e.g. in the diimide reduction of T P C , in the 83  Raney nickel catalyzed reduction of nickel(II)pheophorbides  200  oxidation of pheophorbides  193  and in the osmium tetroxide  and meso-azachlorins . It has been suggested 201  83,192  that the  reduced double bond in a chlorin induces a pathway for the delocalized 7t-electrons that 'isolates' the diametrically opposed cross-conjugated pyrrolic double bond such that attack of this bond is favored over the attack of the double bond in the adjacent pyrrolic unit, resulting in a minimal loss of Tt-energy. Introduction of a metal (or protonation of the chlorin) changes the preferredrc-delocalizingpattern, making the double bond in an adjacent  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  49  pyrrolic unit more reactive. Figure 1-10 shows the preferred 7U-electron derealization pathway (bold) in chlorins and metallochlorins as inferred from the experimental findings.  porphyrin  Figure 1-11  chlorin  metallochlorin  18 rc-Electron derealization pathway in porphyrins, chlorins and metallochlorins  Fischer reported in a publication from 1940 that multiple additions of osmium tetroxide form pigments with a red-shifted spectrum , but he was unable to isolate and 189  characterize these pigments. Without realizing it, he surely was the first to observe this directing effect in chlorins.  With this and the above mentioned substituent directing effect and the relative rates of reaction of aromatic and non-aromatic double bonds in mind, the outcome of an osmium tetroxide mediated dihydroxylation on any porphyrin is predictable. The example of the dihydroxylation of the benzoporphyrin derivative 56 in Scheme 1-17 illustrates this. The non-aromatic double bonds will, upon exposure to osmium tetroxide, react first to yield the pigments 57 and 58. At this stage a bacteriochlorin derivative 59 is formed. The direct formation of any bacteriochlorin 60 was not observed.  202  Despite the favorable  photophysical properties and possible amphiphilic properties of 59, any large scale use for this potential PDT drug can be ruled out on the basis of the mixture of stereoisomers formed and the many synthetic steps required.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  50  OH  pMe  Scheme 1-17  Mep OH  gg  Stepwise reaction of benzoporphyrin derivative (56) with osmium tetroxide. Stereochemistry was not specified. Reaction condition: (i)1. Os04, 2. reduction 202  It should also be noted that the osmium tetroxide oxidation has been applied to mesoazaporphyrins to yield chlorins and bacteriochlorins.  201  again precludes their practical use as drugs in PDT.  Their complex synthesis, however,  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  51  REACTIVITY O F WC-DIOL C H L O R I N S  1.3.3.5  The chlorin diols undergo an acid-catalyzed pinacol-pinacolone-type rearrangement to yield 6-oxochlorins.  For non-symmetric substitution patterns on the porphyrin, the  outcome of the rearrangement carries a regiochemical bias, as shown in Scheme 1-18. The diol 61 may rearrange into the two different oxo-chlorins 62 or 63, depending on the migratory aptitude of the substituents. The migratory aptitude of the substituents has been established.  203  The gem-dialkyl oxo-chlorins are, due to the lack of enolizable hydrogens,  true chlorins.  61  62  63  Pinacol-pinacolone-type rearrangement of the w'c-diolalkylchlorins Reaction condition: (i) benzene, trace HCIO4/A.  Scheme 1-18  Under carefully controlled conditions, the diol does not undergo the pinacolic rearrangement but dehydration with subsequent rearrangement, as illustrated in Scheme  53  Scheme 1-19  64  65  66  Dehydration and rearrangement of v/'c-dihydroxyoctaethylchlorin 53 Reaction Conditions: (i) 1. OSO4, 2. H2S; (ii) dioxane/water/HCI conc./A; (iii) benzene/trace HCI conc./A  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  52  Diol 53 presumably dehydrates to the unstable ethenylhydroxychlorin 64, which, with reconstitution of the fully-conjugated porphyrin system, rearranges to 65. This hydroxyethylporphyrin can further be dehydrated to the heptaethylvinylporphyrin (66). This reaction sequence is of importance as it is otherwise very difficult to derivatize the ethyl side chains in OEP.  1.3.3.6  B - H Y D R O X Y C H L O R I N S NOT D E R I V E D F R O M A N O S M I U M T E T R O X I D E OXIDATION  Inhoffen's G,B'-Diol Monomethylether Bacteriochlorin  The osmium tetraoxide oxidation of porphyrins is possibly the most versatile but not the only way to access 6,8'-diol-type chlorins. The synthesis of a 8,B'-diol monomethyl ether 67 by an intriguing photochemical pathway, shown in Scheme 1-20, was detailed by Inhoffen and co-workers. ' 205  206  Electrochemical reduction of chlorin-e6-trimethyl ester (68) resulted in the generation of the 10,22-dihydrochlorin-e6-trimethyl ester (69). Irradiation with light in the presence of oxygen and methanol led selectively to the formation of the diol monomethyl ether bacteriochlorin 67. This finding can be rationalized by the mechanism of Scheme 1-20. Photooxidation of 68 leads to the formation of hydroperoxide 70. Its dehydration leads to epoxide 71, which opens in the presence of methanol to give the final product 67. The configuration at C3 and C4 has not been determined, but according to the proposed mechanism, the diol ought to be a trans-d\o\. This pathway appears to be an attractive method to reduce a chlorin to a bacteriochlorin with concomitant selective introduction of,  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  53  from the view point of designing PDT agents, desirable substituents. Unfortunately, this pathway cannot be considered general for variously substituted chlorins.  Scheme 1-20  Photochemical synthesis of bacteriodiol-monomethylether 67. Configuration at C3 and C4 was not determined. Reaction conditions: (i) electrolytic reduction; (ii) 1. 02/hv, 2. MeOH  Photoprotoporphyrin  Protoporphyrin (27) exposed to light and oxygen forms, most likely via an intermediate endoperoxide (72), the so called photoprotoporphyrin (73) 1-21).13,207-209 1  j j n  s  n  a  m  e  j  sa  (Scheme  misnomer in the sense that photoprotoporphyrin is a chlorin.  Only one of the two available vinyl groups of the protoporphyrin reacts, thus forming two regioisomers, and owing to the chiral centre at C2/C7, each as pairs of stereoisomers. Upon reduction, it forms the allyl-diol 74.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  OH  HO  pMe  54  pMe  72  27 Scheme 1-21  73  74  75  Formation of photoprotoporphyrin 73 Reaction conditions: (i) 02/hv; (ii) NaBH4; (iii) trace H+/A  Assessing the viability of chlorin 73 and 74 as photosensitizers for PDT reveals that 73 has the disadvantage of necessitating a large scale photo-reaction, a less common modus for the industrial preparation of chemicals. Of greater practical disadvantage, however, is the formation of a mixture of stereo- and regioisomers, which require costly and cumbersome separation. The conjugated aldehyde functionality might also prove to be too reactive under physiological conditions.  Its reduction to 74 may solve this, but 74 is acid labile. It  rearranges readily to porphyrin 75, obliviating the spectral advantages of the chlorin. In short, photoprotoporphyrin strikes one, for practical and reactivity reasons, as a less than ideal photosensitizer.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  1.3.3.7  Introduction  55  G-OXOCHLORINS AND RELATED PIGMENTS  B-Oxochlorins  As mentioned in a previous section, 6,6'-dihydroxyoctaalkylchlorins can be rearranged into 8-oxochlorins, such as 63. > 203  210  The B-oxochlorin 55 (derived from the  rearrangement of diol 53, Scheme 1-23) shows the typical selectivity of chlorins upon further dihydroxylation, i.e. the selective formation of a bacteriochlorin chromophore 76, and in a corresponding fashion, the 6-oxometallochlorin 77 forms the metalloisobacteriochlorins 78 (major) and 79 (minor) (Scheme 1-22). The rearrangement into 80 and 81 is characterized by a distinct selectivity in favor of the former. None of the third isomer 82 could be detected. The rearrangement of 76 has not been reported.  A second, much older route to these B-oxochlorins is the hydrogen peroxide-sulfuric acid oxidation of porphyrins as developed by Fischer et al. (Scheme 1-23).  211,212  He  mistakenly assigned these pigments a B,B'-epoxide structure but this misinterpretation was later corrected by Stephenson and co-workers.  210  The generation of B-oxoporphyrins via the  hydrogen peroxide oxidation is somewhat in contrast to the mild, fairly selective and high yield synthesis via the osmylation-rearrangement pathway. It is extremely harsh, nonselective and it necessitates excruciating column chromatographic separations of the product mixtures. Inhoffen and Nolte reported the oxidation of 10.0 g OEP to yield monoketone 55 in 18.5% yield, five isomeric diketones in yields between 2.2 and 0.3%, and two triketones in 1.3 and 0.9% y i e l d  213,214  , which are respectable yields compared to the results reported  elsewhere using less symmetric porphyrins  215,216  . As some of these pigments serve as heme  dl models, they have been, regardless of their cumbersome synthesis, studied in depth. " 217  221  Due to the low synthetic yield and the excessive efforts to separate the products, they are not  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  56  a practical option for drugs in PDT although some pigments meet certain of the desired criteria.  Scheme 1-22  Hydroxylation and rearrangement of B-oxochlorins and G-oxometallochlorins Reaction conditions: (i) 1. OsO^pyridine 2. H2S (ii) H2SO4  The elegant access to oxochlorins such as 55 via the acid-catalyzed pinacolpinacolone type rearrangement of the corresponding dihydroxychlorin 53 avoids the practical obstacle of separating the complex mixtures which are derived from the direct sulfuric acidhydrogen peroxide oxidation of OEP. The oxochlorin can be reduced to the corresponding  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  57  alcohol 83 which can be further converted to the bromide 84. Nucleophilic exchange of this benzylic bromide with a variety of alcohols generates a host of chlorins (85) with graded amphiphilic character.  29,195  Some of these stable chlorins have been tested in an in vivo  tumor assay and have shown considerable activity.  Scheme 1-23  195  The preparation of Bonnett's chlorins with graded amphiphilicity^ Reaction conditions: (i) HCIO4/CH2CI2 (ii) 1. NaBH^EtOH; 2. H2O (iii) HBr/HOAc (vi) HOR (vi) 1. H2O2/H2SO4; 2. chromatography 9  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Part 1:  Introduction  58  With respect to their use as practical PDT drugs, the assessment of compounds 85 is unfavorable.  While they meet the photophysical criteria and their amphiphilicity and  solubility can be fine-tuned to a wide degree, and they exhibit appreciable in vivo activity,  3,197  they have been shown to exhibit poor tumor selectivity. When tested compound  85, R=glucose, was shown to be an effective tumor photosensitizer but also a very effective skin and muscle photosensitizer, causing severe edema in the test animals upon radiation of the healthy tissue.  29  This lack of sensitivity lead Bonnett and co-workers to the conclusion  that this substance cannot be regarded as promising drug for PDT. Certain other hydroxyfunctionalized chain derivatives, seem to give better tumor to healthy tissue discrimination. Further downsides are the multistep syntheses and the introduction of stereocenters.  The carbonyl group in 55 reacts readily with Wittig reagents and magnesium or lithium alkyls. ' 222  223  None of these reactions, however, furnished products that could be  ideal PDT agents. A different set of manipulations of 55 was more promising. Bonnett and co-workers have taken the oxochlorin 55 and hydroxylated it to yield the bacteriochlorin 86 (^max (CHCI3) = 693 nm, log 8 = 4.71) (Scheme 1-24).  29  Reduction of the ketone  functionality in 86 gave the corresponding triol derivative 87, which proved, however, to be very labile.  This predicated a poor shelf-life time and further work was, therefore,  discontinued.  OH  55  86 Scheme 1-24  87  The hydroxylation of keto-chlorin 86 and its reduction to triol 87 Reaction conditions: (i) 1. O S O A . 2. HpS: (ii) NaBhtyEtOH  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  59  Alcohol 83 gave erratic biological results, presumably due to a lack of solubility in aqueous solvents. Octaethyldihydroxychlorin 53, 86, as well as the 2,3,7,8,12,13,17,18octaethyl-7,8,17,18-tetrahydroxybacteriochlorin (54) (undetermined stereochemistry) (A,  max  (CHCI3) = 715 nm, log 8 = 4.72) were tested in vivo and these compounds were potent photosensitizers, in fact, based on the dose given, more effective than Photofrin®. However, these compounds displayed poor selectivity with respect to photodamage.  Epoxychlorins  The, albeit fleeting, occurrence of an epoxychlorin during the hydrogen peroxidesulfuric acid oxidation of OEP seems reasonable, though it has not been directly observed. The two reports in the literature claiming the existence of epoxychlorins were both proven wrong. The first were compounds reported by Fischer and co-workers ' , which were, as 211  already mentioned, later proven to be oxo-chlorins.  210  212  The second example, claimed by the  Johnson group ' , proved to be a pigment belonging to the secochlorin class. 224  225  and other secochlorins, will be discussed in section 2.1.7.4.  There is, however, one synthetic procedure to form chlorin epoxides in the literature. With the goal of forming oxygen analogies of sulfchlorins ' , 227  228  naturally occurring episulfides of protoporphyrin IX, Iakovides and Smith published the synthesis of chlorin epoxides via a modified Mitsunobu reaction.  229  Its non-trivial multi-step synthesis cannot  be generalized.  226  This,  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Oxophlorins  230  Introduction  60  and meso -Hydroxyporphyrins  A second site to introduce hydroxy-substituents onto the porphyrin periphery is the meso-position. Meso-hydroxy substituted porphyrins, such as 89A, have been known for sometime. ' 72  231  Scheme 1-25 shows two out of several syntheses of these systems, one  being the oxidation of porphyrins with peracids  232  (OEP to 90, followed by hydrolysis) and  the other being the MacDonald-type condensation of a dipyrromethane diacid (91) with a dipyrroketone dialdehyde (92) ' . Meso-hydroxyporphyrin 89A is in equilibrium with its 233  234  tautomer 89B, a so called oxophlorin. In fact, the equilibrium is, in the case of an octaalkyl substitution pattern, on the side of the oxophlorin form. The name oxophlorin expresses the similarity of its optical spectrum with that of 5,22-dihydroporphyrins, and, consequently, make its use as sensitizers for PDT uninteresting.  89B  Scheme 1-25  89A  Formation of meso-hydroxy-octaethylporphyrin Reaction conditions: (i) H+; (ii) MCPBA; (iii) H2O  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  61  B-Hydroxy- and B-oxochlorins  Some naturally occurring B-hydroxy- and oxochlorins have been characterized. Among them are: Heme d (93), the prosthetic group of bacterial terminal oxidases ' , 235  236  and formally a [5,6-fran5-dihydroxychlorinato]Fe(III). It is not known whether the ,  y-spirolactone or the corresponding open structure is that of the native pigment. (94) is the prosthetic group of bacterial dissimilatory nitrite reductase dioxoisobacteriochlorinato]Fe(III). ' " 217 237  241  207,238  Heme dl  236  ' , a [7,12239  Tolyporphine (95) is a pigment with unknown  function isolated from a blue-green algae , and formally a 8,18-dioxobacteriochlorin. 242  Recently, eight more tolyporphyin-like porphinoids have been described. HQ C 2  O  H3COCO  CQ H 2  243  C0 H 2  C0 H 2  "  V=  94  These naturally occurring pigments are uninteresting for large scale use as drugs for PDT as their isolation from the natural source is much too tedious. Consider, for example, the case of the pigment with the most ideal optical spectroscopic properties, i.e. the bacteriochlorin 95. The organic extract of the cultured algae was fractionated by several consecutive reverse phase and silica gel columns to give 0.03 % yield of tolyporphin (95). Total and partial syntheses of the free bases of 9 3  2 4 4  and 94 - 04,239 j^yg ^ 142  2  e e n  242  published,  but again, they are, due to their length and associated low yields {e.g. ~ 1-2 % from protoporphyrin dimethylester for the non-stereoselective synthesis of 9 3  245  ) as well as their  complexity, only of academic value. The syntheses of naturally occurring hydroporphyrins have been elaborated in a recently published review by Montforts et al.  80  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  62  1.3.4 PORPHYRIN ISOMERS  For the longest time, no structural isomers of porphyrins were known. This has changed recently with the appearance of several such isomers, opening up a new direction in porphyrin-related research.  246  Many research papers have appeared in quick succession  describing these fascinating compounds, and although many resemble in their activity the parent porphyrin, they differ significantly in other aspects and, hence, constitute a compound class in their own right.  247  As some of these isomers show strong absorption in the red part  of the visible spectrum and have also been shown to be capable of photo-generating singlet oxygen, they have become viewed as potential drugs for PDT. Here the syntheses, visible spectra and potential as PDT drugs as well other interesting aspects of the selected porphyrin isomers will be briefly reviewed.  Porphycene and Related Pigments  Porphyrin is a cyclic, aromatic tetrapyrrolic pigment with 18 7t-electrons in a closed derealization pathway in which the pyrrole units are linked by methine bridges. A reshuffling of the pyrrolic units and the bridges generates theoretically a host of porphyrin isomers as illustrated below, and all of them should be 18 7t-electron aromatic compounds. A theoretical analysis of all conceivable isomers containing an N4 coordination site and [18]-annulene conjugation performed by Waluk and Michl gave credence to the supposition that isomers other than 96 could be porphyrin-like.  248  And indeed, porphycene (97) ,  synthesized in 1986 by Vogel et al, hemiporphycene (98)  249  250  and corrphycene  251,252  (99)  synthesized by jointly by Sessler and Vogel, have since been investigated and proven to be porphyrin-like, and they fulfill Michl's predictions satisfactorily. The remaining isomers are to this point unknown. Here, further discussion will be restricted to the porphycenes.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  96  97  Introduction  98  Porphycene, its alkyl substituted analogs such as 1 0 0  63  99  253  '  254  , and the 2,7,12,17-tetra-  phenylporphycene (103) are synthesized via the McMurry coupling of two bipyrrole dialdehydes (101), as illustrated in Scheme 1-26. The dialdehydes were prepared through the Vilsmeyer-Haak formylation of the corresponding oc-free bipyrroles, which themselves are the product of an Ullmann coupling of two a-iodopyrroles (102).  Scheme 1-26  Formation of octaethylporphycene Reaction conditions: (i) 1. SO2CI2, diethyl ether; 2. H20/NaOAc; 3. I2/KI; 4. Cu/DMF, A 5. H2, Pd/C; 6. A; 7. POCI3/DMF; 4. H20/NaOAc; (ii) 1. Zn/CuCI/TiCU, THF; 2. O2 253  The greatest drawback of the porphycene synthesis is the low yield of the McMurry coupling, which is, in the case of the synthesis of 97, only 4 %. Porphycene itself is, in strong analogy to porphin, a very nonpolar compound with a porphyrin-like nature (A-max  (benzene) = 630 nm, log e = 4.71).  249  The longest wavelength absorption band of the  octaethyl derivative 100 is, as compared to 96, 35 nm bathochromically shifted  Part 1:  (>Wiax  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  64  (CHCI3) = 665 nm, log £ = 4.48) . Porphycenes, N,N'-bridged porphycenes 253  metalloporphycenes ' ' 253  254  256,257  255  have been studied in depth. " 258  265  and  Porphycene 100  generates singlet oxygen but, mainly due to its solubility and relatively short longest wavelength absorption, does not fulfill the basic criteria for PDT agents. Porphycene can be reduced to 2,3-dihydroporphycerie, nominally a chlorin isomer.  266  Its UV-Vis spectrum has  a similar band pattern as the parent unsaturated compound - this is analogous to chlorins but it does not exhibit the pronounced increase of the longest wavelength band (^  max  (benzene) = 595 nm, log £ = 4.48) - this is in sharp contrast to chlorins .  Nonell et al. have prepared the TPP isomer 103 along an analogous synthetic pathway to that described for 1 0 0 .  267  The photophysics of 103 are consistent with its  proposed use as PDT agent (kmax (toluene) = 659 nm, log £ = 4.70) and it photoproduces singlet oxygen with a quantum yield of roughly 0.25. This ability translates into a high phototoxicity in cells and the compound has low dark toxicity. Details of the biological activity of this compound were not given, so any comparison of the efficacy or other properties with other photosensitizers cannot be made.  The phenyl substituents can be  functionalized to optimize the activity index of this compound. These pigments have the potential to be useful as novel therapeutic agents for PDT, however, further research to significantly improve the McMurry coupling reaction and a short-cut synthetic pathway would be the minimal improvement mandatory if these compounds are to be amenable to larger scale preparations.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  65  N-Confused Porphyrins  Porphyrin isomers could also be prepared, conceptually, by changing the orientation of the pyrrole moieties within the porphyrin framework. This simple concept found its experimental verification recently in two independent and serendipitous discoveries. Furuta et al.  26S  reported the preparation and crystal structure of meso-tetraphenyl-2-aza-21 -  carbaporphyrin (104), which they dubbed "N-confused porphyrin", and Latos-Grazynski p  .  and co-workers  269  reported independently the synthesis  and structure of an analogous isomer of tetra-/?tolylporphyrin. Both porphyrinoids are prepared in low yields (4-8%) via variations of classic pyrrole and aldehyde condensations, followed by oxidation, as side products of the regular me«?-tetraarylporphyrin.  N-Confused porphyrins are intrinsically interesting as potential PDT agents: They are accessible in a one step reaction, the UV-Vis spectrum of the non-protonated species displays strong absorption in the red region (^ (^max  (CH2CI2) = 730 nm, log e = 3.98)  269  (CH C1 ) = 725 nm, log 8 = 4.04) , 268  max  2  2  . The phenyl substituents can, presumably, be  widely varied, and the (basic) nitrogen at the periphery of the compound introduces some amphiphilicity into the molecule. At this time, the low yield and non-directed synthesis and the demanding chromatographic purification of the compound are at odds with any large scale preparation. To overcome this, reaction conditions for the formation of N-confused porphyrin could be optimized, or a more directed approach towards its synthesis could be developed. Studies towards the directed synthesis of N-confused meso-phenylporphyrins will be described and discussed in section 2.2.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  66  N-Confused porphyrins have also aroused interest through their unusual way of forming metal complexes. They form nickel(II) complexes in which the inner carbon has lost a proton, forming a carbon-metal bond. stabilized Arduengo-type, " 270  274  269  They have, therefore, the character of metal  "bottleable" carbenes.  275  The synthesis and step-wise  protonation of a second derivative of 2, namely the outer N-methylated derivative 2-methyl2-aza-meso-tetraphenyl-21-carbaporphyrin, has been recently reported.  276  Isoporphyrins  Woodward predicted in 1962 the existence of the isoporphyrin (96B) tautomer of porphine (96A).  277  The [18]-annulene aromatic system is interrupted in this pigment owing  to the presence of a saturated meso-carbon. The optical spectrum is characterized by a broad absorption in the long wavelength range.  96A Figure  1-12  96B  Porphine - isoporphine tautomerism  The first isoporphyrin was synthesized by Dolphin et a l r  1 %  by way of electro-  chemical oxidation, and since then a series of isoporphyrins has been prepared. " 279  288  However, they all suffer from severe instability and tend to revert to the parent porphyrin. Consequently, they will find no use in applications such as PDT.  Introduction  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  67  EXPANDED PORPHYRINS  1.3.5  Any system larger than a porphyrin, i.e. containing more than one atom between the pyrrolic units, or containing more than four pyrrolic units, is called an 'expanded porphyrin'. By virtue of containing a larger number of 7i-electrons than porphyrins, their electronic spectra are considerably bathochromically shifted as compared to porphyrins and even bacteriochlorins. The long wavelength bands and their high extinction coefficient make these expanded porphyrins appealing candidates for application as photosensitizers in PDT. Expanded porphyrins have received tremendous attention in the last few years as they also offer a larger central binding core and additional coordinating heteroatoms than their congener porphyrins. This opens up the opportunity to bind very large metals, gadolinium, for instance, which is of biomedical interest as an imaging agent in magnetic resonance imaging (MRI).  289-291  Some radiopharmaceuticals also rely on the complexation of large  metals, such as technetium. expanding field. ' 292  293  Recent reviews have given an overview of the rapidly  Here, only selected classes of expanded porphyrins with particular  relevance to this thesis will be discussed.  Sapphyrins, Pentaphyrins and Hexaphyrins  The first expanded porphyrin to be reported was a sapphyrin (105), a pentapyrrolic [22]-annulene with a direct pyrrole-pyrrole linkage.  294  First report by Woodward in 1966,  these compounds have attracted the most attention of all expanded porphyrins, partially, 292  perhaps, because their synthesis has been optimized  295  since the original preparation.  296-298  They were shown to be active in PDT and, as their gadolinium(III) complexes, as MRI agents.  292  They also show unexpected properties such as neutral substrate  recognition properties  299  300-302  as well as interactions with nucleic acids.  303  and anion  Sapphyrins feature  strong (log e = 4.32 and 4.17) long wavelength absorptions at A ax (CH C1 ) = 668 and m  2  2  Introduction  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  711 nm, respectively.  295  68  Their solution state properties have been modified through  variation of the alkyl side chains. Heterosapphyrins  295,297  ' ' ' 298  304  305  have been known for  several years and very recently, the synthesis of a sapphyrin isomer  306  has been  communicated.  The syntheses of pentaphyrin (107), ' ' 308  309  313  307-312  (106), and its next higher homologue hexaphyrin  a hexapyrrolic [26]-annulene with six methine bridges, have been reported,  but, compared to the aforementioned sapphyrin syntheses, they are still in their infancy. Pentaphyrins have strong long wavelength absorption bands,  312  but lack of data on these  types of compounds, especially with respect to photosensitization ability, stability or toxicity rules out any evaluation of their potential as drugs for PDT. Hexaphyrin has only a weak long wavelength absorption (A,  max  (CH C1 ) = 789 nm, log e = 3.60), but again, the lack of 2  2  any more data prohibits any reasoned evaluation.  The key intermediate in the synthesis of all three macrocycles is the tripyrrane 108 (Scheme 1 -27). Its synthesis involves the acid-catalyzed condensation of one equivalent of dialkylpyrrole (109) with two equivalents of (acetoxymethyl)pyrrole (110). Debenzylation of the resulting tripyrrane diester is followed by decarboxylation 108.  295  Acid catalyzed  condensation of 108 with an a,a'-diformyl dipyrrane (111), dipyrromethane (112), or tripyrrane (113), followed by oxidation of the initially formed macrocycle to the fully aromatic compound, completes the synthesis of sapphyrins (105), pentaphyrins (106), or hexaphyrins (107), respectively.  Typically, a sapphyrin synthesis is a seventeen step  procedure starting from a single pyrrole precursor.  298  A similar length synthesis is required  for the pentaphyrins, while the hexaphyrins, due to being composed of two identical halves, require fewer steps. An improved synthesis for the precursors has been published, ' but 295  314  nevertheless, more than ten steps, some requiring expensive reagents, are necessary to access sapphyrins. This might be the strongest impediment for their biomedical use.  Scheme 1-27  Formation of sapphyrin (105), pentaphyrin (106), and hexaphyrin (107) Reaction conditions: (i) 1. HOAc, &, 2. H2, Pd/C; 3. &; (ii) catalytic H+  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  70  meso-Tetraphenyl substituted sapphyrin 114 has been isolated as a minor side product of the Rothemund-type TPP synthesis.  315  This was the first report showing the  formation of a direct pyrrole-pyrrole linkage under the original Rothemund ' 316  317  conditions  or variations of i t ' , and the first report of meso- substituted sapphyrins in general. An 42  44  alternative synthesis of 114 and related meso-phenylsapphyrins will be discussed in section 2.3.  114  It is noteworthy that all but the meso-tetraphenylsapphyrin of the above mentioned expanded macrocycles have one common key intermediate: the tripyrrane 108. This tripyrrane is also a crucial intermediate in the synthesis of other aromatic aromatic macrocycles. " 319  321  318  and non-  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  71  Porphocyanine  Dolphin et al. introduced, in 1993, a novel class of =expanded porphyrins , the porphocyanines.  322  Scheme 1-28  Their structure and synthesis is shown in Scheme 1-28.  Formation of porphocyanine 115 Reaction conditions: (i) 1. NH2OH, NaOAc, EtOH, &; 2. AC2O, &; (ii) 1. UAIH4, THF; 2. H2O; 3. O2 (iii) NH3, EtOH, &, O2  Two different approaches towards this expanded system are available: The lithium aluminum hydride reduction of a 1,9-dicyanodipyrromethane (116) to, possibly, the corresponding methylamino derivative 117. This labile compound was not isolated but immediately self-condensed and oxidized by air to give porphocyanine 115 (^max (CHCI3) = 797 nm, log e = 4.43).  322  The yield of this reduction and condensation  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Introduction  72  reaction is 24 %, a very respectable yield for the formation of macrocycles through a condensation reaction. The condensation of an 1,9-diformyldipyrromethane (118) with ammonia to form, presumably, the intermediate diimine 119. This diimine, presumably, condenses to a reduced, imine linked macrocycle which is then air oxidized to the final product in 26 % yield.  323  There are precedents in the literature for such a condensation.  324  Studies related to the elucidation of the mechanism of formation of sapphyrins via the reduction of cyanodipyrromethanes will be presented in section 2.4.  The long wavelength absorption bands of porphocyanine make these compounds interesting as potential PDT agents.  An extension of the work to meso-substituted  porphocyanines was reported shortly after the initial publication (Scheme 1-29).  325  Phenyl-l,9-dicyanodipyrromethanes  meso-  (120) can be reduced and condensed to the  diphenylporphocyanines (121) (Amax (CHCI3) = 814 nm), and a mixed condensation of the meso -phenyldipyrromethane 120 and the tetraalkyldipyrromethane 122 produces a mixture of 115, 121, and the desired mono-meso-phenyltetraalkylporphocyanine 123 (Amax (CHCI3) = 804 nm). This mixture had to be separated by preparative HPLC. 123 comes closest to the ideal of a strongly amphiphilic drug, particularly when polar substituents are attached to the phenyl ring.  The meso-phenyl substituents allow a facile interchange of functional groups on the long wavelength absorbing core. Their in vitro ability to generate singlet oxygen when irradiated with light of wavelengths longer than 600 nm has been shown. No biological data are presently known and thus, it is too early to evaluate these compounds as PDT agents. Their fairly long synthetic pathway and the need to separate compound mixtures when synthesizing the compounds of type 123 make these compounds initially appear to be less than ideal as practical PDT agents.  Scheme 1-29  Formation of meso-phenyl substituted porphocyanines Reaction conditions: (i) 1. UAIH4, THF; 2. H2O; 3. DDQ  74  2.  2.1  RESULTS AND DISCUSSION  THE OSMIUM TETROXIDE-MEDIATED DIHYDROXYLATION OF meso-PHENYLPORPHYRINS AND -CHLORINS Some meso-tetraphenylchlorins have shown promising activities in the field of  PDT.  2 3 , 9 6 , 1 9 6  It appeared to us that efficient syntheses of these and related meso-phenyl  substituted long wavelength photosensitizers must be developed if these compounds were ever to be produced in large quantities. Inspection of the literature showed that syntheses of the porphyrin precursors had been optimized to a great extent but the yields to the corresponding chlorins were quite poor, due primarily to the laborious separations required. Development of an efficient method to convert the porphyrin to chlorin was seen as extremely crucial to the further advancement of research on this promising class of compounds. To this end, we decided to develop and exploit an alternative route to the largescale production of stable meso-phenyl chlorins. Emphasis was placed on the route that would produce oxidation stable compounds and which would also allow introduction of potentially useful substituents to the chlorin periphery.  Study of the literature singled out the osmium tetroxide-mediated dihydroxylation as the most promising direction of research. The introductory chapter reviewed the conversion of OEP into octaethyl-2,3-diol-2,3-chlorins by means of the osmium tetroxide mediated  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  75  dihydroxylation. However, it was not obvious whether this method of preparing chlorins could be applied to TPP. In other words, it was not immediately apparent whether the resulting G,B'-diol chlorins produced from TPPs would be stable towards, for instance, spontaneous dehydration or if they would form at all. Steric as well as electronic factors may prevent the envisioned osmylation.  This portion of research shows that the conversion of TPPs to the corresponding dihydroxychlorins is indeed possible with the use of osmium tetroxide. The chlorins thus produced are stable, have the desired photophysical properties, can be widely derivatized and, hence, they are promising drugs for PDT. The resulting diol functionality confers unique chemical, biological and physical properties onto these chlorins. The following chapter details the studies of these meso-tetraphenyl-2,3-dihydroxy-2,3-chlorins.  2.1.1  THE OSMYLATION OF TPP - THE PRINCIPAL REACTION  A chloroform solution of TPPZn or its free base TPP containing pyridine and one equivalent of osmium tetroxide produces in the course of three to five days one major polar product 125 or 124, respectively, which can be isolated from the relatively non-polar starting materials in satisfying yields by column chromatography. While considerable amounts of recyclable starting material are still present after this time, little decomposition or byproducts are observed. The optical spectrum of compound 125 exhibits a more intense and 24 nm bathochromically shifted longest wavelength absorption whereas 124 shows a more intense but moderately shifted longest wavelength absorption when compared to the starting compounds. This is typical for the formation of a (metallo)chlorin and, thus, was the first indication of the successful conversion of TPPZn and TPP into the corresponding chlorin osmate esters 125 and 124, respectively. The iH-NMR of 125 is shown in Figure 1-13.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  77  Osmylation experiments performed with deuterated pyridine-ds and H,H-COSY experiments led to the assignment of the signals at 7.25, 7.35 (v br), and 8.6 (v br) to the coordinated pyridines. Their presence is in accord with previous knowledge about osmate esters.  144  Thie remaining signals allow several conclusions. Firstly, the four-fold symmetry  of the starting material (8.90, s, 8H (6-H); 8.25, m, 8H (o-H); 7.75, m, 12H (ra+p-H)) has been lost but an aromatic porphyrinic system is still maintained. Secondly, the signals for the o-protons have split into three sets with 4:2:2 intensity and, together with the number and splitting pattern of the signals attributable to the 6-protons (2d, 1 s, 2H each) clearly identify the 8-positions as the location of addition. According to the nature of the addition of osmium tetroxide to a double bond, the formed osmate ester differentiates the porphyrin into one face bearing this group, and one face opposite of this group, thus differentiating the oprotons on the phenyl group into two non-equivalent sets. Whereas this differentiation is large enough to separate the signals for the two ortho-protons next to the osmate ester, it is too minor to be seen for the signals for the o-protons on the far side of the osmate ester. The singlet at 6.85 ppm can be attributed to the protons attached to the reduced pyrrolic unit. Irradiation of this signal causes as weak NOE-effect for both neighboring o- protons (Figure 1-13). This may show the slow rotation of the phenyl group. This rotation is subject to investigations to be described in section 2.1.6. The remaining multiplets integrating for 12H are due to the meta- and para-hydrogens of the phenyl groups. The presence of a high-field signal at -1.72 ppm in 124, attributed to the inner NH-protons, shows that the osmium, as expected,  326  did not insert into the porphyrin core of the free base porphyrin.  Scheme 1-30 shows the reaction scheme and the likely conformation of the resulting osmate ester. The frans-orientation of the oxo-substituents in the osmyl-moiety is typical for osmium(VI) complexes.  154  This enables the coordination of two pyridines at two equivalent  positions, a finding supported by their equivalent NMR-signals. Their location above the porphyrin ring is based on steric considerations. The finding that the pyridine proton signals  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  78  are not drastically shifted by the porphyrin ring current effect when compared to those of free pyridine may be explained by distance from the ring or by their location in the 'neutral shift region' of the ring. The broadness of the signals attributed to the o- and m -pyridine hydrogens may indicate rotation of the pyridines along the Os-N axis.  S c h e m e 1-30  Osmylation of TPP to form the corresponding osmate ester chlorin Reaction conditions: (i) 1. 1.1 equiv. OSO4, CHCl3/10% pyridine, several days, r.t., 2. column chromatography  These findings prove that osmylation of a peripheral double bond in TPP is a viable way to convert this porphyrin into a chlorin. The occurrence of monomeric osmium diesters or dimeric diesters  144  could not, presumably for steric reasons, be observed. The reaction  was, depending on the substituents present at the phenyl groups {vide infra), performed in mixtures of ethyl acetate, water, acetone, THF, chloroform (for obvious reasons, pentene stabilized chloroform must not be used), or benzene with pyridine. The addition of pyridine to the reaction mixtures serves two purposes. acceleration effect:  172  The osmylation is subject to a ligand  that is, the osmylation proceeds much faster when a suitable ligand  for the stabilization of the osmate ester is present. Several amine-type ligands are suitable for this acceleration. ' ' 144  161  327  In addition, the rate of the reaction is also directly dependent  on the concentrations of the reactants in solution. The solubility of meso-phenylporphyrins, particularly of TPP, is, even in the best of solvents, low when compared to the solubility of  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  other organic substrates.  Results  79  This alone predicts a slow reaction rate for the osmylation.  Experimentation resulted in the choice of a mixture of 10% pyridine in chloroform, a solvent mixture which not only exploited the ligand acceleration effect but dramatically increased the solubility of the TPP to increase the overall reaction rate.  Osmate esters of type 124 can be reduced by a wide variety of reductants to the corresponding diols.  144  In fact, osmate esters produced by osmium tetroxide-mediated  dihydroxylation procedures are generally not isolated, but are directly reductively (or oxidatively) cleaved.  We found that hydrogen sulfide gas as reductant is the most  convenient method of reduction, although sodium borohydride, sodium bisulfite or LAH are suitable for this transformation. This leads to a one-pot procedure for the production of the chlorin diols 129 to 133 (Scheme 1-31). After no further change in the osmium tetroxide reaction mixture can be detected by TLC or optical spectroscopy, hydrogen sulfide is bubbled through the mixture. The precipitated black osmium sulfides are filtered off, and the mixture is purified by chromatography.  124 or 125  TPP; M TPPZn; 126 M 127 M 128 M  Scheme 1-31  = 2H M = Zn = Ni = Fe(lll)CI =Cu  129; 130; 131; 132; 133;  M M M M M  = = = = =  2H-«— Zn Ni Fe(lll)CI Cu  R e d u c t i o n oft h e o s m a t e ester chlorins to f o r m t h e c o r r e s p o n d i n g diol c h l o r i n s a n d o n e - s t e p c o n v e r s i o n of p o r p h y r i n s into diol c h l o r i n s R e a c t i o n c o n d i t i o n s : (i) 1. 1.1 e q u i v . OSO4, CHCl3/10% p y r i d i n e , 3-5d, r.t.; 2. H2S b u b b l e ; 3. c h r o m a t o g r a p h y ; (ii) H2S (iii) Z n - a c e t a t e / M e O H /  CHCI3/A; (iv) N i ( l l ) a c e t a t e / M e O H / C H C l 3 / A (v) 5 % TFA/CHCI3.  The diols have, like their osmate esters, pronounced chlorin or metallochlorincharacter. The optical spectra of the brown-purple 129 and the green 130 are shown in  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  80  Figure 1-14. The trends seen in optical spectra of several 8,B'-diol chlorins as compared to their non-hydroxylated analogs will be discussed in section 2.1.4.  400  450  500  550  600  650  X [nm]  Figure 1-14  Optical spectrum (CH2Cl2/0.5% MeOH) of 129 (  ) and 130 (  )  The !H-NMR spectra of the diols are very similar to those of the osmate esters, without, of course, the signals attributed to the coordinated pyridines and with an additional signal for the hydroxy-hydrogens. The dynamic behavior of the proton spectra will be discussed in section 2.1.6. Metallochlorin 130 can also be prepared as its pyridine adduct. Such adducts are common for zinc(II) porphyrins and chlorins. The axial coordinated pyridine groups exhibit additional signals in the *H-NMR spectrum at 3.08 (br s, 2H), 5.95 (t, 2H), and 6.7 (t, 1H). Their drastically altered chemical shifts, compared to uncoordinated pyridine, are explained by the ring current effect of the porphyrin. Neither the shifts nor the coupling patterns conclusively indicate that the pyridines coordinate selectively on one side of the porphyrins or the other.  The diols are stable compounds in the solid state. No decomposition or re-oxidation to a porphyrin chromophore could be detected after storage at room temperature and protection from light for several years. Solutions of these chlorins are also stable under mild  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  81  conditions. Although they were generally handled under subdued light, they do not appear to be extremely light-sensitive. However, silica gel seems to promote the light-sensitivity of these compounds. Their dehydration and other reactions will be described in section 2.1.7. Also, the diol chlorins dehydrate under the conditions of electron ionization (EI) mass spectrometry and, as a result of that, no or a very little signal corresponding to the expected m/e can be seen. However, the milder conditions prevalent in fast atom bombardment (FAB) mass spectrometry allows the detection of the expected molecular ion peak. The first fragmentation pattern visible results, in all cases studied, from the dehydration.  The osmylation/dihydroxylation reaction can be performed on various metalloporphyrins, such as TPPZn, and 126 to 128. However, the solubility of the zinc chelate is higher in the solvents used than that of, for instance, the nickel chelate, and on this basis the zinc chlorin is produced at a much faster rate than the corresponding nickel chlorin. This higher solubility reflects the higher tendency for zinc porphyrins to coordinate to an additional ligand to form a penta-coordinated complex than, for instance, the nickel porphyrins.  328  Acid-catalyzed demetallation of the zinc chlorin 130 forms the free base 129.  We found that various mild techniques used to insert central metals such as copper and nickel are suitable to produce the corresponding metal complexes of the diol chlorin 129.  328  The route via the intermediate zinc-complexes was found to be more efficient and convenient to get to, for example, the nickel complex or, in fact, the free base chlorin 129.  As a method of synthesizing a 'library' of differently substituted meso-tetraphenylbased diolchlorins to be tested as photosensitizers in PDT, the dihydroxylation reaction was found to be successful with those compounds with hydroxy- and methoxy-substituents present on the phenyl group (134 to 138). Due to the known biological activity of hydroxysubstituted porphyrins, we focused on this type of compounds. 96  In addition to the  compounds listed above, spectral evidence for the successful formation of diol chlorins was  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  82  gathered for compounds with phenyl substituents as diverse as 2,5-dichlorophenyl, 4nitrophenyl, 4-cyanophenyl, 4-carbomethoxyphenyl, 4-sulfonatophenyl, 4-pyridyl, 3-, and 4propioyloxy, and pentafluorophenyl.  Hence, the osmium tetroxide oxidation can be  considered to be general for meso-arylprorphyrins and their metal complexes.  R  2  R1  R2  R3  R4  R5  134  -OH  -H  -H  -H  -H  135  -H  -H  -H  -H  -OH  136  -OH  -OH  -H  -H  -OH  137  -H  -H  -OH  -H  -H  138  -OMe  -OMe  -OMe  -OMe  -OMe  Figure 1-15 shows the ^H-NMR spectra of the two isomeric compounds 10,15,20triphenyl-5-(4-hydroxyphenyl)-2,3-dihydroxy-2,3-chlorin (134) and 5,15,20-triphenyl-10-(4hydroxyphenyl)-2,3-dihydroxy-2,3-chlorin (135). They were prepared by osmylation of the corresponding 10,15,20-triphenyl-5-(4-hydroxyphenyl)-porphyrin , and the two isomers 329  formed were separated by preparative TLC. The two spectra illustrate the side- and facedifferentiation of the proton signals due to the 6,8'-diol substituents, and the effects substituents located on the phenyl groups have on, for example, the 8-protons in relation to the distance that separates them.  Figure 1-15 134 (top trace)  !H-NMR (400 MHz, CDC1 /10% acetone-d ) of 135 (bottom trace) and of 3  6  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  84  Compared to the corresponding OEP oxidation (reaction time 2 days, 66% yield diol, 17% OEP recovery)  118  the oxidation of TPP is slower (up to one week reaction time, -50%  diol, 40% TPP recovery). Steric hindrance of the attack of the bulky osmium tetroxidepyridine complex at the 8-positions by the phenyl groups, as well as electronic reasons, are responsible for this. The oxidation of the less hindered yet electronically approximately equivalent 5,15-diphenyl-porphyrin to give 139 proceeds under similar conditions within hours in high yields.raeso-Tetraphenyl-N-methylporphyrin reacts equally fast, producing 330  140.  We assume that N-methylporphyrin is, by virtue of its distortion, electronically  activated. The latter reaction is under study by other members of the research group of Prof. Dolphin, and will not be discussed further. The sluggish reaction rate of the stoichiometric osmylation of TPP is responsible for our inability to perform the reaction with catalytic amounts of osmium tetroxide. ' 144  160  It remains to be seen, however, whether the more  reactive porphyrins like the two examples cited above, make a catalytic osmium tetroxidemediated dihydroxylation possible. Such process improvement would be, due to the high costs and toxicity of osmium tetroxide, of great economical and practical interest.  The first experiment to evaluate a prospective photosensitizer for use in PDT is to test whether it generates singlet oxygen upon radiation with visible light. Compounds 129 and 146 (page 89), as assayed by their light-mediated aerial oxidation of cholesterol to 5oc-hydro-peroxycholesterol, were found to be efficient photosensitizers in organic solvents  28  when irradiated with light of > 600 nm.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  2.1.2  Results  85  THE OSMYLATION OF meso-TETRAPHENYLCHLORINS  Reaction ofraesc-phenylchlorin(18)  83  with a stoichiometric amount of osmium  tetroxide under the conditions described above, gives selectively the pink 2,3-v/c-dihydroxymeso-tetraphenylbacteriochlorin 141, in good yield (Scheme 1-32). The optical spectrum of 141 is shown in Figure 1-17. Its spectrum unequivocally proves its bacteriochlorin structure. Alternatively, diol chlorin 129 can be reduced with diimide to also give 141, albeit in poor yields.  129  Scheme 1-32  Preparation of diolbacteriochlorin 141 Reaction conditions: (i) 1. 1.1 equiv. OSO4, CHCl3/10% pyridine, 2d, r.t.; 2. H2S bubble; 3. chromatography; (ii) pyridine, K2C03-anhydrous, excess p-toluenesulfonylhydrazine, A.  The bacteriochlorin structure is also clearly visible in the JH-NMR. The two sets of non-equivalent 8-protons show as doublets of doublets with J = 5 Hz, a typical value for 3  such 8,B'-couplings, and J = 1.5 Hz, a typical value for couplings with the inner NH-proton. 4  Inspection of the possible tautomers for bacteriochlorin chromophores reveals that the NHprotons are fixed to non-reduced pyrrolic units, and this shows their coupling with the 8protons. All other protons are detected at their expected chemical shifts.  One particularly beautiful and illustrative feature of the spectrum of bacteriochlorin 141 is shown in Figure 1-16. It is the expansion of the signal for the four protons attached to  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  86  the reduced pyrrolic unit opposite the diol moiety. The chemical shift is typical for this kind of proton. By virtue of their coupling pattern they show the face-differentiation of the diol moiety. The two hydrogens located on the same face as the diols are non-equivalent to those on the opposite side. This leads to a vicinal and a geminal coupling of each proton with the protons of the opposing side. As the chemical shift difference of the two proton sets is in the same range as their coupling constants, the complex non-first order spectrum is observed. This pattern was reproduced by a computer simulation  331  with the values as indicated in ( B ) .  These values are typical for vie- as well as gem-coupling constants.  i Figure 1-16  1  1  1  1  4.1  4.0  3.9  3.8  Measured H-NMR (400 MHz) signal of the pyrrolirie protons in 141(A) and coupling constants determined by simulation (B) 1  Insertion of zinc into the chlorin completely changes the outcome of the reaction. Thus, hydroxylation of (meso-phenylchlorinato)zinc 142 gave only the (2,3-v/c-dihydroxy-  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  87  raeso-tetraphenylisobacteriochlorinato)zinc 143. The basis for this astonishing selectivity was discussed in Section 1.3.3.4.  Demetallation afforded the 2,3-vz'c-dihydroxy-  isobacteriochlorin 144. The identities of the isobacteriochlorins were determined by their mass, optical (Figure 1-17) and ^H-NMR spectra. The symmetry of these pigments (point group Q) infers the existence of two enantiomeric forms. Due to the lack of any possible chiral induction, they are formed as a racemic mixtures.  S c h e m e 1-33  Preparation of the diolisobacteriochlorins 143 and 144 Reaction conditions: (i) 1. 1.1 equiv. OSO4, CHCl3/10% pyridine, 2-3 d, r.t.; 2. chromatography; 3. H2S .; 4. chromatography; (ii) pyridine, K2CO3, p-toluenesulfonylhydrazine, A .  The optical spectra of 1 4 1 and 1 4 4 again illustrate the greater potential of bacteriochlorin chromophores for use in PDT as compared to the corresponding isobacteriochlorin chromophores. 1E+05  E 5E+04 o  OE+00 350  F i g u r e 1-17  450  550 X [nm]  Optical spectrum (CH2Cl2/0.5%MeOH) of  650  141  (  750  ) and 144  (  )  Part 1:  2.1.2  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  88  THE OSMYLATION OF meso-TETRAPHENYL-2,3-DIHYDROXY-2,3CHLORINS AND -METALLOCHLORINS  The dihydroxylation of the diol chlorin 129 and diol metallochlorin 130 follow the same selectivities as the above pigments. 129 forms a mejo-phenyl-2,3,12,13-tetrahydroxy,  2,3,12,13-tetraolbacteriochlorin, and 130 forms a (meso-phenyl-2,3,7,8-tetrahydroxy-2,3,7,8isobacteriochlorinato)zinc chromophore (Scheme 1-34). The same products can, albeit with lowered yields, be produced by the treatment of TPP or TPPZn with (at least) two equivalents of osmium tetroxide. The optical spectra of the tetraolbacteriochlorin 145 and the tetraolisobacteriochlorin 149 are shown in Figure 1-18.  2E+05-  350  450  550  X  Figure 1-18  650  750  [nm]  Optical spectrum (CH2Cl2/0.5%MeOH) of 146 (  ) and 149 (  )  The introduction of a second diol functionality into these pigments has several implications. Firstly, they impose such hydrophilicity on the pigments that their solubility in chloroform is not sufficient as to allow H-NMR spectroscopic investigations. The more ]  hydrophilic solvents methanol, methanol/chloroform mixtures or DMSO have to be used for that purpose. Secondly, their arrangement with respect to the already present diol moiety  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  89  can be one of two ways. Either the second diol group is attached on the same face as the first group, or on the opposite face. The formation of both isomers is observed in different ratios. The two pink bacteriochlorin isomers 145 and 146 form as a -0.9:1 mixture, whereas the two blue-green isobacteriometallochlorins 147 and 148 form in favor of the brans'-isomer 148 (as a racemic mixture), in a -1:6 mixture. Demetallation of 148 afforded the 2,3-vicdihydroxy-isobacteriochlorins 149 (as a racemic mixture). The difference of the two ratios in which these pigments are formed might be explained by steric effects. The incoming bulky osmate esters might show a preference to be attached on different faces of the molecule. This preference would be the more pronounced the closer the reactive sites are. Hence, the strong face preference in the isobacteriochlorins and the weak effect in the bacteriochlorins.  Scheme 1-34  Preparation of the tetraolbacterio- and tetraolisobacteriochlorins 145-149 Reaction conditions: (i) 1. 1.1 equiv. OSO4, CHCl3/10% pyridine, ~2 d, r.t.; 2. chromatography; 3. H2S; 4. chromatography; (ii) 10% TFA/CHCI3.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  90  All isomers can be separated by preparative TLC and can be identified by their proton NMR spectra. These assignments rely on the recognition of the subtle differences in the region for the ortho-protons. For instance, the symmetry elements of structure 145 (point group C2v). in which the diol moieties are on the same side of the molecule, result in a splitting of the orf/io-protons into two sets, the o- o'-Hs (Scheme 1-34). The faces of this molecule are non-equivalent but the two sides ('north- and south-side') are equivalent. The symmetry elements in 146 (point group C2J) have a similar effect in that they differentiate between o- and o'-Hs. The two faces of the molecule are equivalent but the two sides are not. The o- and o'-Hs in 145 are subject to the double face differentiation by two sets of diols and, thus, their chemical shift difference is more pronounced than that for the analogous protons in 146, which experience merely a side differentiation. In other words, the ortho-protons in 145 experience the effect of four hydroxy groups situated either on the same (o-H) or the opposite face (o'-H), respectively, whereas all ortho-protons in 146 experience the effect of two hydroxy groups located on their respective sides. The slight difference between the o- and o'-Hs results because the hydroxy-groups on the same side as the respective orf/io-protons are in one case closer (o-H), and in the other case further away (o'-H). This all translates into the observation of two separate signals of equal intensity (for 4H) for the o- and the o'-H signals in 145 as opposed to the lone broadened signal (for 8H) for those in 146.  In further support of this assignment, 145 is much more polar as seen by its lower  Rre-  value (Rf = 0.31 (silica/5% MeOH/CH Cl )) than 146 (Rf = 0.51 (silica/5% MeOH/ 2  CH C1 )). 2  2  2  This is expected for this type of rigid, flat molecule as 145 can provide  simultaneously two sets of diols for the interaction with the silica gel surface, whereas 146 can provide only one set at a time. The steric protection evidently does not mask this effect.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  91  A similar string of arguments can be used for the assignment of the structures of the isobacteriochlorins 147 and 148 and the demetallated complex 149, although their NMR spectra are, due to their lower symmetries, more complex than those of the bacteriochlorins. Also, only a small amount of 147 was available and, thus, its structural assignment is more ambiguous than for the bacteriochlorins.  No spontaneous oxidation or dehydration to a chlorin or porphyrin is observed during handling or storage of the tetraol-bacteriochlorins. This is a significant improvement as compared to the very labile TPBC chromophore.  2.1.3  C O M M E N T S ON T H E OPTICAL S P E C T R A O F T H E B,B'DIOLCHLORINS  The optical spectra of the novel B,6'-diol derivatized compounds 129,141, 144,145, 146,  and 149 clearly demonstrate their respective chlorin, bacteriochlorin or  isobacteriochlorin character.  67  Table 1-1 lists their spectral data in comparison with the  spectral data of their non-dihydroxylated analogs.  The substitution of hydrogens by a set of vie-diols causes, in the bacteriochlorin series, a ~ 20 nm hypsochromic shift of the longest wavelength absorption, alters the extinction coefficients of all bands, and, in the bacteriochlorin series, sharpens the Soretband as compared to the parent TPBC. The two isomeric bacteriochlorins 145 and 146 show identical electronic spectra. Both electronic and conformational effects cause variations in the optical spectra of porphyrinic molecules.  332  Molecular modeling of 129,145 and 146  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  92  146 does not suggest an appreciable distortion of the porphyrin core due to introduction of pairs of hydroxy groups.  333  Hence, the observed hypsochromic shifts seem to be associated  with electronic interactions of the substituents with the 7t-system of the pigments. The nature of the spectra of the tetrahydroxy isomers 145 and 146 seems to support this prediction. A similar effect, though not as pronounced as in the bacteriochlorin series, can be seen in the isobacteriochlorin series (144 and 149 as compared to TPiBC). Table 1-1  Optical spectra of the novel diol chlorins in comparison to those of their non-dihydroxylated analogs  Compound  ^maxfnm] (log e)  Solvent^  Reference 41  TPP 6  4 2 0 (5.68), 5 1 5 (4.30), 5 4 9 (3.92), 5 9 5 (3.92), 6 5 3 (3.78)  A  T P C 18  4 1 9 (5.28), 5 1 7 (4.20), 5 4 2 (4.08), 5 9 8 (3.79), 6 5 2 (4.62)  A  129  4 0 8 (5.27), 5 1 8 (4.19), 5 4 4 (4.19), 5 9 2 (3.85), 6 4 4 (4.38)  B  this work  83  3 5 6 (5.11), 3 7 8 (5.20),  5 2 0 (4.78),  7 4 2 (5.07)  A  83  141  378(4.96),  524(4.49),  724(4.71)  B  this work  145/146  3 7 6 (5.42),  5 2 8 (5.08),  7 0 8 (4.89)  B  this w o r k  TPBC  83  5 1 6 (4.00), 5 5 2 (4.28), 5 1 6 (4.08)  A  144  3 9 4 (5.02), 4 0 8 ( s h ) , 389(sh), 5 1 4 (4.03), 5 4 6 (4.19), 5 9 0 (4.20)  B  this work  149  3 9 8 (5.17), 4 0 8 ( s h ) , 480(sh), 5 1 4 (4.12), 5 4 8 (4.29), 5 8 6 (4.20)  B  this work  TPiBC  a  3 9 0 (5.00), 4 0 8 (4.90),  T h e o p t i c a l s p e c t r a of t h e n o v e l c o m p o u n d s d o not s h o w to a l a r g e e x t e n t (< 2 nm) s o l v e n t d e p e n d e n t shifts; A = b e n z e n e ; B = C H 2 C l 2 / 0 . 5 % M e O H  Part 1:  2.1.4  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  93  PRELIMINARY RESULTS OF THE BIOLOGICAL ACTIVITY OF Wc-DIOL CHLORIN 137  The first level of testing revealed that diol chlorins have the ability to generate singlet oxygen (Section 2.1.1). The next level of testing is in vitro experiments in which defined carcinoma cell lines are irradiated with a controlled dose of light after they are allowed to soak in solutions containing with various concentrations of photosensitizers.  The cell  survival after certain time intervals is monitored. The better the photosensitizer, the lower the concentration of photosensitizer at which a high and fast cell killing rate is observed. There is, however, an ongoing debate about how indicative these type of in vitro tests are for the evaluation of a photosensitizer. For instance, Bonnett's chlorin 4 is reportedly very active when tested in vivo,  23  while our in vitro tests do not indicate such high activity.  334  Several compounds were tested in-house by Mrs. Savoie of our group. The testing work is still in progress and it is too early to derive any final conclusions. meso-Tetra(3hydroxyphenyl)-2,3-v/c-dihydroxy-2,3-chlorin 137 has been tested the most thoroughly as the results derived from this compound could be set against the results published for the analogous chlorin 4 lacking the diol functionality. 137 proved in tests against the P-815 mouse mastocytoma cell lines to be-not as active as BPD-MA (1) under the same conditions.  To gain further insight into the activity and possibly the mechanism of action of the diol chlorins versus the mechanism of action of their undihydroxylated counterparts and other photosensitizers, their photodynamic potential was tested by Dr. Ross Boyle of our laboratory in a collaboration with Prof. Matthews from the Pharmacology Department, University of Cambridge, England. employed.  Two sophisticated in vitro test techniques were  The first technique employed was cell assays against, firstly, pancreatic acini  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  94  freshly isolated from male Sprague-Dawley rats, secondly, AR4-2J cells derived from an azaserine-induced carcinoma in rat pancreas, and, thirdly, MIA cells derived from a human pancreatic adeno-carcinoma.  335-338  These in vitro assays were performed to ascertain the efficacy of the drugs particularly in photoinactivation of pancreatic carcinoma. Pancreatic carcinoma is the fourth most common cause of death by cancer, and the five year survival rate is only 3%.  339  The  screening of healthy rat acini allows the therapeutic window for PDT with this drug to be determined. This is especially important for pancreatic cancer which, being diffuse, requires irradiation of the tumor and peritumoral tissue simultaneously. Preliminary results with 137 as photosensitizer indicate that the drug is more potent than the standard employed, BPDMA (1), but because experiments are still in progress, it is too early to speculate on the magnitude of the therapeutic window.  The second technique employed was the measurement of the photodynamically mediated response (contraction upon irradiation) of freshly isolated superfused Guinea pig smooth muscle (Taenia Caecum) in the presence of the photosensitizers. The smooth muscle experiments can give insight into the mechanism by which the activated photosensitizer exerts its effect on cells.  340  Compared with 1, 137 seems to operate by a different, and  distinct, mechanism, which causes contraction of the smooth muscle tissue, but in a much more reversible manner. This drug is also more sparing of the cell membrane suggesting a different pattern of intracellular localization.  Whether these effects are specifically attributable to the presence of the diol moiety remains to be elucidated. However, the initial results of the photodynamic activity of the diol chlorins are very encouraging.  Part 1:  2.1.5  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  95  ROTATION OF PHENYL RINGS IN #r?eso-TETRAPHENYL-B,B'DIHYDROXY-CHLORINS  Restricted rotation of phenyl rings resulting from steric interactions with neighboring groups has been the subject of considerable investigation since the early work on optically active biphenyls. meso-Tetraphenylpprphyrins are in that respect an interesting case study. Although maximum derealization between the porphyrin ring and the mesophenyl substituent in TPP would be achieved through co-planarity, steric interactions between the 8-hydrogens and the ortho-hydrogens on the phenyl rings prevent this conformation and force the phenyl plane to be placed approximately perpendicular to the porphyrin mean plane. Crystal structures have determined the dihedral angle to range from 90°  341  to as small as 6 9 °  342  for most free base and metalloporphyrins.  The steric interaction of the ortho- substituents with the 8-protons allows the isolation of atropisomers of ortho-phenyl substituted TPPs. " 343  345  However, rotation of the phenyl  group is, albeit at a low rate, possible for TPPs with relatively small orr/io-substituents. '  343 346  As can be readily shown with molecular models of TPP, the plane of the porphyrin has to undergo some distortion to allow any phenyl rotation to occur, even if the o/t/io-substituent is as small as hydrogen. The rigidity of a metalloporphyrin is controlled by the type of metal. Consequently, the rate of rotation in metallo-TPPs is a function of the size of the ortho- substituent, the type of metal present and possibly the presence of other substituents on the phenyl ring. All the factors mentioned have, mainly by work of Eaton and Eaton, been studied in fair detail. " 347  350  These studies depended, with no exception, upon the use of penta-coordinated metalloporphyrins. Given the right choice of a ligand inert towards substitution, this ligand  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  96  introduces a face differentiation to the metalloporphyrin such that the ortho- and ortho'protons become non-equivalent and, consequently, separate signals in the iH-NMR spectra for these protons can be observed (A in Figure 1-19). The analysis of the temperature dependency of the NMR spectra reveals the activation parameters for variously substituted metallo-TPPs. LIGAND  A Figure 1-19  B  Schematic representation of the face differentiation in tetraphenyl metallochlorins with additional ligands (A) and in w'c-f3,B'-diolchlorins (B).  The use of metalloporphyrins is intrinsic to this methodology, and therefore, no activation parameters have been described for free base porphyrins (or chlorins). The vicdiol group in diol chlorin 129 is situated on one side of the chlorin plane and, as discussed earlier, brings about a face (and side) differentiation (B in Figure 1-19). This fact prompted the investigation of the temperature dependent NMR spectra of 129, 130 and 138 as we reasoned that this face differentiation should enable the measurement of the rotational barriers of the phenyl substituents in these free base chlorins.  Figure 1-20 shows the low-field portion of the iH-NMR (400 MHz, DMSO-d ) of 6  129 in the temperature range from room temperature to 140°C. Whereas the signals assigned to the B-protons remain unchanged, those associated with the phenyl protons undergo temperature dependent changes. The broad peaks at 8.08, 8.05, and 7.88, integrating for 4, 2, and 2 protons, respectively are assigned to the ortho-protons as indicated. The separation of the signals for the two non-equivalent orzTio-protons attached to the phenyls next to the dihydroxylated pyrrolic unit (o- and o'-H near side) indicates that the phenyl groups rotate at  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  97  room temperature slowly on the NMR time scale. The broad feature may indicate rotation to some extent, and indeed, cooling of a sample of 129 in C H C I 3 to -20°C sharpens the peaks up a little (no shown), but this broadening could also be due to non-resolved couplings by all other hydrogens in the phenyl ring.  Heating the sample to 50°C causes complete  coalescence of the o- and o '-peaks, indicative of the rotation of the phenyl groups at a rate comparable to the NMR time scale. Further heating accelerates the rate of rotation and the two ortho-protons become indistinguishable. Finally, at 140°C (the technical limit for the spectrometer used), face differentiation due to the hydroxy substituents can no longer be seen. Merely their side-differentiation is visible by virtue of the two signals corresponding to the two sets (5-, 20-phenyl and 10-, 15-phenyl) of equivalent phenyl groups. The higher temperature of the coalescence of the peaks attributed to the o- and o'- near side as compared to that for the o- and o'- far side is the basis for the conclusion that the phenyl groups situated next to the diol moieties have a higher barrier of rotation than those phenyls on the far side of the diol moiety.  The analogous spectrum for the zinc diolchlorin 130 essentially shows the same effects as seen for the spectrum of the free base chlorin 129, albeit the peaks for the orthoprotons are not as well resolved due to some overlap with peaks for the 6-protons. There is one important difference, however. Even at 140°C, the peaks for the o and o'- near side are not fully recovered, suggesting that the phenyl groups (5-, 20-phenyl) still rotate at a slow rate. We attribute this effect to the stiffening effect of the central metal, in other words, the metallochlorin moiety has a higher barrier of distortion so as to allow a rotation of the phenyl groups as the free base chlorin has.  The shift of the peak for the OH-protons in 130 is  attributable to the a change of water concentration (present as minor impurity in the DMSOd6) during the experiment. The sample was heated for several hours in excess of 100°C and this led to the evaporation of some of the moist in the solvent.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  98  Not shown are the results of the temperature dependent NMR spectra for the mesotetra(3,4,5-trimethoxyphenyl)-2,3-dihydroxy-2,3-chlorin (138). Its spectrum is, due to reduction of the number of intra-phenyl H,H-couplings, much simpler than that of the previous examples. Presumably due to the bulk of the methoxy-substituents, coalescence of the o- o'-H far side is observed at ~110°C, and coalescence of the near side-protons is observed at ~140°C. Consequently, no spectrum for the fast rotation limit could be obtained.  The single run experiments, the inaccuracy of the temperature measurements, and the uncertainty associated with the necessary estimate of the signal positions for the non-rotation limiting case due to the impossibility of measuring low temperature NMR spectra in the same solvent (DMSO-dg) as in the high temperature runs, precluded the calculation of the activation parameters associated with the phenyl rotations. However, the observed trends fit well into what is known from previous studies about this kind of dynamic process. " 348  350  The significance of the experiments lay in showing that the unique face-differentiating nature of the diol functionality can serve as a tool for the measurement of parameters previously not measured due to the lack of suitable compounds.  Figure 1-20  Temperature dependent H-NMR (400 MHz, DMSO-d6, 38mM) of 129 1  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Figure 1-21  Results  100  Temperature dependent H-NMR (400 MHz, DMSO-d6, 42mM) of 130 1  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  101  2.1.6 THE REACTIVITY OF THE meso-TETRAPHENYL-Wc-DIOLCHLORINS  2.1.6.1  F O R M A T I O N O F I S O P R O P Y L I D E N E K E T A L 150  The formation of isopropylidene ketals from acetone and 1,2- and 1,3-diols is a common reaction to protect diols in, for example, sugars.  351  The reaction is generally acid-  catalyzed. Diol 130 is, as shown below, sensitive to strong acids and, consequently, dry zinc(II) chloride was chosen as the relatively mild Lewis acid to catalyze the acetonide formation, shown in Scheme 1-35. Reaction of 130 in dry acetone containing freshly fused zinc(II) chloride produced a less polar product 150 as compared to the starting material, but, 150 displays virtually the same optical spectrum as the starting compound.  Ph Scheme 1-35  150  Formation of acetonide 150 Reaction condition: (i) freshly fused ZnCl2/acetone/A;  The iH-NMR of 150 readily allows for confirmation of the expected acetonide structure. The low-field signals of the starting material are essentially maintained. The signals for the diol hydrogens have vanished, no coupling is observed for the pyrrolidine hydrogens and two new signals at 8 0.61 (for -CH.3 ) and 1.35 ppm (for -CH.3 ) appeared, a  D  with the relative intensities corresponding to three hydrogens each. These signals can be assigned to the methyl-groups of the isopropylidene moiety.  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  2.1.6.2  102  F O R M A T I O N O F G-OXO-DERIVATIZED P I G M E N T S  B-Oxochlorins The above mentioned stability of the diol chlorins towards dehydration has limits. Reflux of these chlorins with traces of strong acids such as perchloric or sulfuric acid rapidly and cleanly produces one purple pigment of low polarity and the expected mass spectral and analytical data for a compound corresponding to the starting compound minus one equivalent of water. It appears unimportant whether the zinc metallated or the free base is used for this dehydration as the conditions are harsh enough to induce demetallation, thus forming in both cases the metal-free compound 151 (Scheme 1-36). If, however, the diol chlorins are treated for several hours at reflux temperatures with the Lewis-acid zinc chloride in chloroform/methanol or tetrahydrofuran, the formation of the metallated analog 152 can be observed. The metallated and demetallated species can also be interconverted by standard methodologies.  Their optical spectra are shown in Figure 1-22.  Both spectra are  surprisingly not chlorin- but porphyrin- or metalloporphyrin-like.  i 350 Figure 1-22  1  1  1  1  450  550 X [nm]  650  750  Normalized optical spectra (CH2CI2) of 151 • (••••) and 152 ( — )  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  151A  Ph  129  or  Scheme 1-36  Ph  Results  151B  103  151C  Ph  130  Dehydration of diol 129 or 130 to give ketochlorins 151 and 152 Reaction conditions: (i) catalytic amount HCI04/benzene/A; ZnCl2/THF/A; (iii) Zn(ll)acetate/MeOH/CHCl3/A; (iv) TFA  (ii)  The iH-NMR spectrum of 151 is surprisingly complex. This is due to the occurrence of three different tautomers (151A - 151C). The tautomeric equilibria for 151 and 152 have been studied in detail by Crossley et al.?  52  The iH-NMR spectrum for 152 is much simpler  than that for 151. It indicates the occurrence of 152B as the main species in solution.  Crossley and co-workers have synthesized 151 and 152 previously.  352 3 5 4  One of  two possible synthetic routes is shown in Scheme 1-37. It is the optimal route known to date, namely the nucleophilic displacement of the nitro group in 153 by the anion of benzaldoxime with subsequent aqueous work-up.  354  This method suffers from the  disadvantage that it requires the use of the copper(II), nickel(II) or palladium(II) derivatives of TPP. To produce the metal-free 2-oxochlorin 151, for example, a harsh demetallation step (two-phase system of cone, sulfuric acid and methylene chloride) has to be included in the synthesis. The synthesis of 151 via the dehydration of diol 129, on the other hand, is a  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  104  convenient, mild, and high yield two-step process (starting from TPP) and can be performed on the metal-free systems. Thus, it represents an excellent alternative synthetic pathway for the formation of 151 and related pigments.  Ph  TPPCu(128) TPPNi  N0  Ph  2  OH  _Jj)  (126)  Scheme 1-37  Formation of 154 Reaction conditions: (i)N02, 2. aqueous work-up 354  -108  (ii)  354  1. Na-salt of benzaldoxime,  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  105  B,B'-I)ioxochlorins  The 2-oxo-TPP 151 is not only an interesting compound in its own right, but has also been used as starting material for the synthesis of G,B'-dioxo compounds such as 156 (Scheme 1-38). Two different syntheses ' 355  356  of this diketone have been published and  interesting porphyrin dimers and oligomers have been formed from them  357  or from  tetraoxobacterio- and isobacteriochlorins derived from 156. 358  130 iii  Scheme 1-38  Formation of 3,f3'-dione chlorins 156 and 157 Reaction conditions: (i) 1. light/C-2, 2. H+/H20; (ii) dioxan, A; (iii) 2.2 equivalents DDQ/benzene 355  356  4 equiv. SeC-2/  We found that oxidation of 130 with 2 equivalents of DDQ in dry benzene produces a non-polar yellow-brown pigment in excellent yields with spectroscopic and analytical properties identifying it as 157. In particular, the symmetry inherent to this compound was clearly seen in the iH-NMR. The face-differentiation seen in the starting compound 130 had vanished and no signal other than those for three sets (two doublets, one singlet) of BT and one set for the phenyl protons could be detected. Due to time constraints, reactions were not further investigated. In the 198 4  3 5 5  and 199 1  3 5 6  157 and its  communications by  Crossley and co-workers initially reported 157, no analytical data were given, nor did the authors publish a full paper detailing their findings. Hence, any comparison of their data for the diones and those found by us is unfortunately not possible.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  106  fi-Dioxobacterio- and Isobacteriochlorins  The dehydration of diol 129 to give the oxochlorin 151 points to a synthetic path to produce dioxobacteriochlorins and -isobacteriochlorins by double dehydration of the corresponding tetraolbacteriochlorins and -isobacteriochlorins. The relative stereochemistry of the tetraols used should have no effect on the outcome of the dehydration products but each dehydration may produce several regioisomers of the resulting dioxo-compounds. These possible regioisomers are shown in Scheme 1-39.  No directing effect as seen, for instance, in the pinacol-pinacolone-type rearrangements of 6,8'-diol-6-alkylchlorins (cf. Section 1.3.3.5), is expected to govern these dehydrations. This is basically what is found by experiment. Acid treatment of the tetraols 146 or 149 produces a mixture of three and two products, respectively.  Mass spectral  analysis of the crude mixture resulting from the dehydration of isobacteriochlorin 146 showed the presence of the expected mass (m/e (EI) = 647 for M H ) , however, the +  compounds proved in our hands to be impossible to separate by preparative TLC. Furthermore, this dehydration was associated with extensive decomposition, and thus, this reaction was not further investigated.  The dehydration of bacteriochlorin 149 is not corrupted by extensive decomposition, but produces two compounds relatively cleanly with the expected mass. Again, we did not succeed in separating these regioisomers in amounts which would have allowed their individual characterization.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Scheme 1-39  Results  Proposed formation of dioxobacterio and -isobacteriochlorins Reaction condition: (i) trace HCIOVbenzene, A  107  Part 1:  2.1.6.3  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  108  F O R M A T I O N O F 2 - O X A - 3 - O X O - C H L O R I N 163  As noted above, the dihydroxylation of TPP derivatives is a 'clean' reaction, in other words, only few undesired by-products form during this reaction. Nonetheless, one byproduct which formed in very small (< 2%) amounts during the dihydroxylation of TPPZn is a non-polar (Rf between the diol and TPPZn) green pigment (163) which in dilute methylene chloride solution displays a remarkable blue-green color with a strong red fluorescence. This pigment was readily separated from the desired diol and the starting material during the column chromatography step. It was furthermore observed that more of this pigment formed the longer the TPPZn was allowed to react with the osmium tetroxide, but in no case was the yield higher than a few percent. Repeated execution of the dihydroxylation reaction led to the accumulation of enough material such that full characterization could be attained.  The pigment can be demetallated with 10%TFA in methylene chloride, and reaction of the demetallated product with zinc(II) under typical metallation conditions reconstituted the native pigment, thus proving that the pigment did not decompose during the acid treatment. The optical spectra of the zinccontaining (163) and metal-free pigment (164) are shown in Figure 1-23.  T  163,M = Zn 164,M = H 2  The Soret-bands and the well defined Q-bands of this spectrum clearly attest to the porphyrinic nature of this compound. The mass spectral analysis specified the composition of C43H26N402Zn and C 4 3 H 2 8 N 4 O 2 for 163 and 164, respectively. This highly unusual composition indicated the loss of one carbon and the uptake of two oxygen atoms as  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  compared to TPPZn (C44H2sN4Zn).  109  As the loss of a carbon from the phenyl moieties is  highly unlikely, this loss had to have occurred in the porphine framework. Certain bile pigments can be derived from porphyrins by excision of a meso-proton and the introduction of ketone-functionalities at the 'exposed' oc-carbons, but this implies in the case of mesotetraphenyl pigments also a loss of a phenyl group which, based on the mass, is not observed, nor does the optical spectrum support the presence of an open-chain pigment. The loss of an oc-carbon with retention of the macrocycle also seems very unlikely. That leaves only the loss of a 6-carbon as the viable explanation for the observed data. Several positions for the oxygen atoms are conceivable and the !H-NMR spectrum of the compound is expected to give clues to where the oxygens are situated. This, indeed, is the case. The H-NMR for ]  163 and 164 are shown in Figure 1-24. 4E+05-,  350  450  550  650  X [nm]  Figure 1-23  Optical spectra (CH2CI2) of 163 (  ) and 164 (  )  Four clusters of peaks, each with unique patterns characteristic for certain hydrogens in TPP-derived compounds can be distinguished. The signals at -2.04 and -1.69 ppm with the relative integration for two hydrogens are in the typical region for the inner NH-protons. The occurrence of two signals is, given a slow tautomeric exchange on the NMR time scale, indicative of a porphyrin without any rotational symmetry. One example for this pattern is the above mentioned 2-oxo-TPP tautomer of compound 151. The complex peak cluster  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  110  between 7.6 and 7.8 ppm can, by comparison with the spectra of related compounds and the total integration of twelve hydrogens, be attributed to the meta- and para -protons of the phenyl groups, and the peaks centered at 7.9 and 8.7 ppm to the corresponding orthoprotons. Most illustrative are the peaks above 8.5 ppm, the region for the B-hydrogens. Their total integration for six protons, the recognition of the typical AB-patterns with J = 4.5 Hz for adjacent non-equivalent B-hydrogens and the presence of a coupling of 1.5  3  Hz, typical for the J coupling of B-hydrogens with an NH-hydrogens, lead to the assignment 4  indicated in Figure 1-24. Further plausibility of this is gained from the H-NMR spectrum of 1  the metallated pigment 163, the low-field region of which is also shown in Figure 1-24. It shows essentially the same pattern as the spectrum for 164, only simplified by the absence of the J coupling (and the signals for-the NH protons). 4  These data indicate the presence of the structural unit shown in Figure 1-24. The wedge in the structural drawing indicates the non-symmetric moiety which contains both oxygens and one more carbon atom. The structure 163 (and 164)  incorporates these  elements in a plausible fashion. Formally, a B,B'-link in TPPZn has been replaced with a lactone functionality. The C-NMR spectrum of 163 shows a signal at 173 ppm, 13  and the IR  spectrum (film) a strong peak at 1740 cm . Both facts support the presence of a lactone -1  group.  The ultimate proof of the structural assignment of 163 as (meso-tetraphenyl-2-oxa-3oxo-chlorinato)zinc(II) was provided by its X-ray crystal structure.  The ORTEP  representation of 163 (as its pyridine complex) is shown in Figure 1-25, the final atom coordinates, selected bond lengths and experimental details are listed in Tables 1-2, 1-3, and 1-4, respectively.  12H-m + p  •2 "3 (0  Q  a. 0)  3 42 c 5)  .eEn a. + two additional signals at -2.05 and -1.82 ppm (br s, 1H, for NH protons)  0  CO T3 C to .<«  q> •c  1  i i l i i i •i I i i i i I i I - R - r - r - T - T - r - r r r r r r p - r n r r i 8. 4 •8. 6 8. 8'  i i | -i i i i I i i i r | 8. 0 8. 2  i i i [i i i i i i i i i | i i i i i i i i M 7. 4 7. 6 7. 8  Figure 1-24 H-NMR (300 MHz, CDCI3) of 163 and 164 * impurity Wedge symbolizes any non-symmetric moiety 1  1  Figure 1-25  O R T E P representation (33% probability level) and side view of 163-pyridine  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  113  The crystal was disordered in respect to the rotational orientation of the individual porphyrins along the axis passing through the pyridine nitrogen and the central zinc atom. This shows that the crystal packing is more determined by the bulky phenyl groups, the pyridine coordinated to the zinc and the planarity of the porphyrin than the comparatively small B-substituents. However, 45% of the molecules are oriented in a unique way. The formal replacement of the pyrrole unit by a five-membered lactone-containing nitrogen heterocycle in 163 does not lead to any appreciable distortion of the mean plane of the macrocycle. The zinc atom is a little out of the porphyrin plane, a finding typical for zinc pyridine porphyrins.  359  All bond lengths testify to the presence of a highly delocalized  system. As expected for metallochlorins, the B,B'-bonds in the pyrrolic units adjacent to the 'reduced' pyrrolic unit are slightly shorter (C6-C7 = 1.352(3) and C16-C17 = 1.354(3) A) than those in the opposite unit (C11-C12 = 1.378(3) A). Perhaps unusual is the fairly short bond length between the carbonyl carbon and the connecting a -carbon (C2C3 = 1.427(3) A), the ring oxygen and the a-carbon (CI-01 = 1.397(3) A) and the C=0 o bond itself (C2-02 = 1.148 A). This may indicate some contribution of hyperconjugated structures to the overall structure. The details of its formation remain unclear. It can be shown that the lactone is not a photooxidation product. Earlier reports of the osmium tetroxide oxidation of aromatic systems have shown that this oxidation may result in some ring cleavage.  179  Treatment of  TPPZn with a large excess of osmium tetroxide leads to extensive decomposition of the porphyrin and the formation of 163 cannot be observed. It is reasonable to assume an autooxidation mechanism as shown in Scheme 1 -40, but it is not clear whether the osmate ester or the diol is the more activated starting material for such an oxidation or whether the expelled carbon is formally in the oxidation state of formaldehyde or, following another oxidation, of carbon dioxide.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  S c h e m e 1-40  Results  Proposed autooxidation mechanism for the formation of  114  163  It must be noted that 164 was reported in 1984 in a communication.  355  The authors  claimed the preparation of this pigment either by photo-oxidation of 2-amino-TPP (165) and subsequent MCPBA oxidation of the photooxidation product 166, or by direct MCPBA oxidation of 165 (Scheme 1-41). The mechanism for this transformation also remains speculative. The authors reported only the optical spectrum and the mass, as determined by mass spectroscopy, for 164 and, to the best of our knowledge, they failed to substantiate their claims in a full paper. However, we find that their reported ? i (A X  m a x  max  and log 8 deviate only little  = 4 nm; A log 8 = 0.1) from our findings.  I  (ii)  Ph  Ph  O  164  S c h e m e 1-41  Crossley's synthesis of lactone 1 6 4 Reaction conditions: (i) 02/hv: (ii) MCPBA 3 5 5  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  2.1.6.4  Results  FORMATION O F A SECOCHLORIN A N D SUBSEQUENT CLOSURE  115  RING-  REACTIONS  Formation of a meso-Tetraphenylsecochlorin  The cleavage of a 1,2-glycol into two carbonyl compounds is performed classically either by periodate or lead tetraacetate.  360  While the attempted oxidation of nickel diol 131  with periodate led to a complex mixture, the oxidation with a stoichiometric amount of lead tetraacetate gave cleanly the beige-brown pigment 167 (Scheme 1-42). Its optical spectrum is shown in Figure 1-26.  or  131  Scheme 1-42  129  Formation of meso-phenylsecochlorin bisaldehydes 167 and 168 Reaction condition: (i) 1. 1 equiv. Pb(IV)acetate, THF, r.t.  This optical spectrum featuring the split and bathochromically shifted Soret-band as compared to the starting material and the broad Q-bands does not allow an immediate conclusion about the type of chromophore present, but any conventional chlorin or porphyrin can be excluded. High-resolution mass spectroscopy (found m/e 703.16394, corresponding to C44H2902N4Ni), H-and C-NMR (singlet at 9.7 ppm and 188.7 ppm, respectively), and !  13  IR spectroscopy (C=0 stretch at 1684 cm ) readily identify this product as the (meso-1  tetraphenyl-2,3-dicarboxaldehyde-2,3-secochlorinato)nickel(II)  (167). The analogous  conversion of the metal-free chlorin 129 produces a pigment with spectroscopic properties which allow its preliminary characterization as the metal-free analog 168 of 167, but this  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  116  product is much less stable than 167 and largely converts in C H C I 3 within several hours into a pigment of unknown structure. The investigation of 168 and its reactions was reserved for future studies. The observed optical spectrum is attributed to the presence of the strongly electron-withdrawing and conjugated aldehyde groups and the greater flexibility of the porphyrin ring.  350  450  550  650  750  X [nm]  Figure 1-26  Normalized optical spectra (CH2CI2) of 167 (  ) and 168 (  The formation of secochlorins is rare. Chang, Peng and co-workers  226  )  reproduced in  1992 the alleged formation of an epoxychlorin from (dimethyloctadehydrocorrinato)nickel(II) 169 as reported in 1969 by Johnson and co-workers ' 224  225  (Scheme 1-43). They  were able to confirm the spectral properties of the pigment formed, but also realized that some chemical properties were inconsistent with the formulation of the pigment as an epoxychlorin. Single crystal X-ray crystallography finally revealed the unusual structure of pigment 170 they dubbed furochlorophin. Its structure implies the series of rearrangements which apparently had taken place upon heating corrin 169. The corrin frame has expanded and one angular methyl group has become a methine link. One pyrrole oc-B-bond was broken and the vinylic appendage of the broken pyrrole turned 180° to reattach itself to the macrocyclic frame through an ether linkage. Molecular oxygen must be assumed to be the  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  117  source of the ether-oxygen. Several other porphyrins are also formed during the reaction. The free base of 170 shows a chlorin-type optical spectrum ( X  max  = 699 nm, log e = 4.11).  From the highly specific mode of formation of 170 it is evident that any use of the furochlorophins as PDT agents can be excluded, but the optical spectra make more general methods to prepare secochlorins an attractive field of study.  Scheme 1-43  Formation of furochlorophin Reaction conditions: o-dichlorobenzene, 160°C, O2  Apart from the first example, only two more examples of secochlorins are known to us. One is derived from the serendipitous singlet oxygen-mediated ring scission of an octakis(dimethylamino)porphyrazine.  This secochlorin was communicated in 1994 by  Barrett and Hoffman and co-workers.  361  The second, and closely related example, is the  lead tetraacetate cleavage of the OEP derived diol chlorin 54 to give the octaethyl-2,3secochlorin-2,3-dione 171, reported in 1993 by Adams et al. (Scheme 1-44).  362  Scheme 1-44  Formation of octaethyl-2,3-secochlorin-2,3-dione 1 7 1 ^ Reaction condition: (i) 1. Pb(IV)acetate, 2. chromatography  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  118  Formation of the Novel Pigments 173 and 175  During recrystallizations of 167 from chloroform and methylene chloride solutions containing methanol, the slow formation of two green pigments, 172 and 173, was observed. The halogenated solvents used are prone to the formation of small amounts of hydrochloric acid and, hence, we assumed the observed pigment formation to be an acid-catalyzed reaction. And indeed, treatment of a methanol/chloroform solution of 167 with gaseous hydrochloric acid resulted in complete and rapid disappearance of this pigment and the appearance of the two compounds 172 and 173. Further treatment with acid finally resulted, at the expense of 172, in the formation of 173 as the sole product (Scheme 1-45). The optical spectrum of 173 is shown in Figure 1-27. The optical spectrum of 172 and that found for 173 are almost identical. The spectra are reminiscent of metallochlorins but they are considerably broadened and bathochromically shifted.  7.5E+04-,  400  500  600  700  X [nm]  Figure 1-27  Optical spectrum ( C H 2 C I 2 ) of 173  The formation of 172 is, as seen in the mass spectrum, associated with an increase in mass corresponding to the uptake of methanol in the starting compound, and 173 gained another 15 mu, corresponding to the additional uptake of a methyl group. These changes  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  119  are, under the present reaction conditions, best explained by the uptake of two equivalents of methanol with a concomitant loss of water. Hence, these mass spectral results suggest already the formation of an acetal. Inspection of the structure of dialdehyde 167 reveals that it is ideally set up for an intramolecular double acetal formation (Scheme 1-45).  Acid-  catalyzed reaction of one aldehyde group with methanol forms hemiacetal 174. The alcohol functionality of this hemiacetal reacts immediately with the second aldehyde functionality to form the isolable intermediate 172, which upon further acid-treatment converts into the double acetal 173. The iH-NMR spectra of 172 and 173 support this scheme strongly. The expected overall symmetry of the compounds 172 (no mirror plane) and 173 (containing a mirror plane) can be deduced from the coupling patterns of the 8-protons. Also the presence of one or two methoxygroups, respectively, and of hydrogens attached to a non-aromatic part of the molecule are visible. 168  ii  175; R =  Scheme 1-45  Et, M e  Formation of 172 and 173. Reaction conditions: (i) MeOH, catalytic amount of HCI (g); (ii) mixture of MeOH/EtOH, catalytic amount of HCI (g)  In one experiment, 168 was dissolved in chloroform, a drop of methanol was added, and the solution was exhaustively treated with acid as described above. The resulting  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  120  pigment 175 was dissolved in chloroform and petroleum ether 40-60 was allowed to diffuse into the solution. Beautiful dark green crystals suitable for X-ray crystal structure analysis were deposited. The optical spectrum of the crystalline material was identical to that for 173. Surprisingly, though, mass spectral analysis of the crystals showed that in addition to the expected peak (m/e = 748), two more peaks with m/e = 762 and 776 were present, corresponding to compounds containing one or two more methylene groups. The iH-NMR was also unexpectedly complex, and indicated the presence of ethyl groups. Reinvestigation of the experiment revealed that chloroform stabilized with ethanol was used. As a result, the formation of a mixture of ethyl and methyl double acetals (methyl-methyl, methyl-ethyl, as well as ethyl-ethyl) was possible, thus explaining the spectroscopic findings for 175.  Despite the presence of three species in the crystalline material, it was possible to solve the crystal structure. All three compounds crystallized in the same lattice and largely in the same orientation. The presence of the methoxy-bearing compounds was noticeable only by a partial occupancy for the terminal carbon of the ethoxy side chains. This conservation of one lattice for TPP-based structures is not unusual.  363  An ORTEP  representation of 175 is shown in Figure 1-28, a side view and a stereoview of this compound are shown in Figure 1-29. Experimental details of the crystal structure analysis are listed in Table 1-2, final atomic coordinates in Table 1-5, and selected bond lengths in Table 1-6. The crystal structure brings about the final proof for the structure of 175 (and 173). The presence of the six-membered ring in 175 results a spectacular distortion of the porphyrin 'plane'. This distortion is best described by a roughly 45° twist along the axis passing through Nl-Ni-N3. Despite this, the central nickel atom is still coordinated in a square planar fashion and the median Ni-N bond length of 1.895 A is almost exactly as long as that found in (porphyrinato)Ni(II) complexes.  41  The alkoxy groups of the double acetal  functionality are exclusively arranged transoid to each other. This can be understood using steric and stereoelectronic arguments.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  121  C47 C46  Figure 1-28  ORTEP representation (33% probability level) of 175. The partially occupied positions have been assigned by the use of non-shaded spheres.  The molecular structures and the ground and excited state dynamics of non-planar porphyrins have been reviewed in 1995.  364  The host of structures discussed there are  distorted through steric interactions of side chains and groups attached to the outer or inner periphery of the porphyrin macrocycle, resulting in various ruffling modes.  365  None of the  structures derived their ruffling through the replacement of a pyrrolic unit by, as in 175, a six-membered or any other sized ring. In fact, we believe that 175 is the first structurally characterized aromatic porphyrinic macrocycle with this intriguing feature. Berlin and Breitmeyer synthesized in 1994 two non-aromatic porphyrinoids in which one pyrrole unit  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  was replaced by benzene and pyridine, respectively. '  319 320  122  Bonnett and co-workers reported  in 1993 the synthesis of an aromatic pigment in which one pyrrole was formally replaced by a six-membered ring, however, it was not structurally characterized.  362  This pigment will be  discussed below in more detail. The distortion has, as demonstrated, a pronounced effect on the optical spectrum, and it remains to be elucidated what effect this distortion has on. for example, the electrochemical characteristics of this compound. C29  Figure 1-29  Side view and stereoview of the crystal structure of 175  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  123  In contrast to dialdehyde 168, octaethyldiketone 171 contains enolizable hydrogens, and when treated with base, undergoes an intramolecular aldol condensation to form 176 (Scheme 1-46). No crystal structure has been reported for this compound, and no statement about its conformation can be made. However, the presence of an a,6-unsaturated ketone within the six-membered ring suggests a less severe distortion from planarity as found for 175.  Scheme 1-46  Formation of aldol condensation product 176 Reaction conditions: (i) base  The examples of the reactivity of the diol moiety of the meso-tetraphenylchlorins mentioned in the foregoing chapters highlight the use of the osmium tetroxide mediated dihydroxylation not only to confer specific spectroscopic and solubility properties on a potential drug for PDT, but also to introduce functionalities to modify the porphyrin skeleton so as to create unique pigments. The chemistry of these compounds has only started to be explored and it can be safely predicted that more novel pigments will be made by chemical modification of the meso-tetraphenyl-2,3-v/c-dihydroxychlorins and their metal complexes. In particular, the formations of secobacteriochlorins and secoisobacteriochlorins from the tetraols 146 and 149 or their metal complexes seem attractive goals for further research. The oxidation of dialdehyde 167 to the corresponding diacid and its decarboxylation may lead to the formation of the parent secochlorin, an, as yet, elusive compound.  Part 1:  2.2  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  124  STUDIES TOWARDS THE DIRECTED SYNTHESIS OF NCONFUSED TPP Any in-depth study of N-confused porphyrins requires the preparation of significant  amounts of this material. Given the roughly 5% yield of the original, 'random' Rothemundtype syntheses  268,269  and the laborious multiple chromatography steps involved, a better  synthesis of N-confused porphyrin would be desirable. To that end, a directed step-by-step synthesis of N-confused porphyrin was sought. Such directed synthesis may also enable the synthesis of derivatives of N-confused porphyrins such as meso- diphenyl-N-confused porphyrin or meso-phenyl-di.-N-confused porphyrins, which are likely not accessible by the Rothemund-type condensation.  2.2.1  RETROSYNTHETIC ANALYSIS OF N-CONFUSED PORPHYRIN  Retrosynthetic analysis  366  of N-confused porphyrin (104) suggests, based on the  recognition of the availability of a 2 + 2-type approach for its synthesis, a series of structurally simplifying transformations, shown in Scheme 1-46. The first transformation is the conversion of porphyrin 104 into its reduced form, the N-confused porphyrinogen 177. Compound 177 transforms by cleavage of two C -C a  meso  bonds into two bipyrrolic synthons,  178 and 179. The typical nucleophilic reactivity pattern of the pyrrole a-positions in synthon 179 requires in the corresponding synthetic step an electrophilic counterpart in 178. The typical reagent of such reactivity is the corresponding hydroxy-compound 180, available by reduction of the bisbenzoyl-raeso-phenyldipyrromethane (181). This compound should be available by direct benzoylation compound.  56  of the meso-phenyldipyrromethane , a known 367  Alternative pathways are also available. " 368  370  The bipyrrolic unit 179  containing the a-to-6-link transforms into pyrrole, with its usual reactivity at the a-position,  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  125  and 184, which is ultimately derived from 6-benzoylpyrrole 185, a readily available material. From this synthetic plan it follows that the a ,8-dipyrromethane is the key intermediate as it has, as a result of the a-to-6 pyrrole linkage, the 'N-confused' unit already locked in place. Most importantly, 179 is set up for a 2 + 2 condensation and a regular reactivity pattern is expected for this a,6-dipyrromethane, i.e. the positions of highest nucleophilicity are both depositions. Ph  Ph  Ph  Scheme 1-47  Ph  Retrosynthetic analysis of N-confused porphyrin 104  Part 1:  2.2.2  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  126  THE DIRECTED SYNTHESIS OF N-CONFUSED PORPHYRIN  The synthesis of N-confused porphyrin and its precursors proceeded along the pathways suggested by the retrosynthetic analysis; however, several unforeseen events occurred. On one hand, the synthesis became less straightforward, and on the other hand, it gave serendipitous access to a novel compound (180) with a highly interesting structure (Scheme 1-48).  Synthesis of meso-Phenyldipyrromethane (179) and meso -diphenyltripyrrane (180) <i  Benzaldehyde and pyrrole were condensed in pyrrole as solvent, catalyzed by trifluoroacetic acid, to produce meso-phenyldipyrromethane (179) according to a procedure of Lee and Lindsey  367  or, alternatively, by a method briefly mentioned in the footnotes of a  paper by Rebek and co-workers.  371  This former procedure calls for a purification step by  column chromatography with an unusually large compound to stationary phase ratio. As this makes the synthesis of multi-gram scales of 179 time-consuming, inconvenient and costly, we tested several other methods to separate the main product 179, the dimeric product from this polycondensation reaction, from other oligomers produced during the reaction. Recrystallization techniques only afforded oils enriched in 179. When these oils were transferred into a sublimation apparatus and heated (130°C at 0.1 torr), 179 sublimed, to our delight, and crystallized at the cooling finger as colorless or white crystals of analytical purity. Later a protocol was established which enabled us to synthesize more than 10 grams of 179 per day. For another use of 179, see Part 3 of this thesis.  In runs in which the above oil was prepurified by short column chromatography and treated with the sublimation protocol, the residue left in the bottom of the sublimation apparatus hardened, upon cooling, into a red-orange glass. Analysis of this glass proved that  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  127  it contained -95% meso-diphenyltripyrrane (180). In Lindsey's paper describing the synthesis of 179, the occurrence of a small amount of an unstable tailing component was mentioned and, based on H-NMR spectroscopy provisionally assigned structure 180. This 1  tripyrrane is, even when ground into a powder, stable in the solid form but could not, in our hands, be further purified by chromatography or crystallization without retrieving it as an unstable oil. Preparative TLC of the crude condensation mixture and mass spectral analysis of the bands tailing the tripyrrane confirmed the presence of higher oligomers such as mesotriphenyltetrapyrrane (m/e =531)  and meso-tetraphenylpentapyrrane (m/e =786).  Macroscopic quantities of these compounds could, however, not be isolated and characterized. One successful synthetic application of the tripyrrane is in the synthesis of raeso-phenylsapphyrins, shown in Part 1, section 2.3, and another is shown in Part 3.  Synthesis of l-Benzoyl-5-phenyldipyrromethane (181), l,10-dibenzoyl-5-phenyldipyrromethane (182), and l,9-Dibenzoyl-5-phenyldipyrromethane (183)  Wallace et al.  56  described in 1993 the synthesis of meso-tetraarylporphyrins via a  2+2 approach. Much like in the present case, a l,9-di(phenylhydroxymethyl)-5-aryldipyrromethane, produced from the reduction of the corresponding dibenzoyl-compound, was condensed with a meso-phenyldipyrromethane.  The dibenzoyl-compound was  synthesized in 39% yield by a Vilsmeyer-Haak-type synthesis from dipyrromethane and required a purification step by preparative TLC. In our hands, this reaction could only be reproduced with yields under 20%. The main impediment of the reaction was thought to be the acid-catalyzed decomposition of the dipyrromethane and, consequently, we reasoned that basic reaction conditions would be more appropriate to accomplish the benzoylation.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Scheme 1-48  Results  128  Directed synthesis of N-confused TPP (104) Reaction conditions: (i) pyrrole, cat. TFA; (ii) 1. 2.1 equiv. EtMgBr, 2. 2 equiv. BzOCI; (iii) 1. large excess EtMgBr, 2. BzOCI; (iv) 1. NaBH4, 2. AcOH; (v) 1. NaH, 2. p-TsCI; (vi) BZOCI/AICI3; (vii) KOH/dioxane (viii)1. UAIH4, 2. H2O (ix) 1 .cat. p-TsOH/toluene/A; (x) AcOH/A; (xi) 1. POCI/ BzOCI, 2. AcNa/H20; (xii) AICI3/BZOCI in 1,2-dichloroethane  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  129  We hoped that reaction of dipyrromethane 179, activated as the Grignard compound with benzoylchloride would, in analogy to the chemistry of pyrrole , provide the 372  dibenzoyldipyrromethane 183, and we were surprised to find that l-benzoyl-5-phenyldipyrromethane (181) remained the main product and only traces of the dibenzoylated product 183 were formed. Harsher conditions such as performance of the reaction at higher temperatures or the use of a ten-fold excess of ethyl-Grignard resulted in extensive decomposition. These findings are rationalized as shown in Scheme 1-49.  183 Scheme 1-49  Rationalization of the findings of the benzoylation experiments of 179  Dipyrromethane 179, treated with two equivalents of ethyl-Grignard, deprotonates to give the bis-Grignard 185. Reaction with one equivalent of benzoylchloride benzoylates the a-position and generates, formally, the mono-Grignard 186.* The proton attached to the  * F o r the subtleties o f the reactivity o f the pyrrolyl ambident anion, see W a n g and Anderson.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  130  nitrogen of the benzoylated pyrrole moiety is now more acidic than that attached to the other pyrrole. Consequently, a proton exchange to form, at least formally, structure 187 takes place. This compound cannot react in the desired way to form the dibenzoyl compound 183. The addition of large excess of Grignard reagent to deprotonate 187 in situ is pointless as this excess of reagent would be quenched quickly by the benzoylchloride added. For that reason, a two-step approach had be taken: first synthesis of mono-benzoylated 187 and then in a separate second step, addition of two equivalents of ethyl-Grignard to a solution of 187 to generate 188, and Subsequent reaction with benzoylchloride to form the desired dibenzoylated product 183. A large excess of Grignard reagent has to be avoided during this step as this results in the formation of a multitude of undesired compounds. We explain this with the finding that the mew-proton in 187 is acidic enough to be deprotonated by ethylGrignard, forming compounds of the general structure 189, which leads upon reaction with benzoylchloride to the observed by-products. This acidity of the meso -proton can be readily tested by the treatment of 187 (and even 179) with several equivalents of ethyl-Grignard and subsequent quenching with D 0 . The recovered materials shows reduced proton signals for 2  the mew-hydrogen in the iH-NMR.  In a further attempt to overcome the difficulties of synthesis of 183, 179 was treated with aluminum trichloride/benzoylchloride.  Under rigorously dry conditions, little  decomposition is observed and 181 forms in good yields but no 182 forms at all. Instead, the C,N-dibenzoylated product 182 appears in the reaction mixture.  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  131  Synthesis of N-Confused meso-phenyldipyrromethane 190  The synthesis of the key intermediate, the a-to-6 linked meso-phenyldipyrromethane 190 proceeded in a straightforward manner (Scheme 1-48). Synthesis of 3-benzoylpyrrole (191) via 1 -tosylpyrrole (192) is well described. ' 368  373  375  Reduction of either the tosylated  3-benzoylpyrrole 193 or its deprotected form generated the desired alcohols 194 or 195, respectively.  Both alcohols are relatively unstable and apart from NMR spectroscopic  analyses of the crude mixtures, no further characterization was attempted. Acid-catalyzed condensation of these alcohols with pyrrole produced in good yield the N-confused dipyrromethanes 196 or 190, respectively. Base induced detosylation converted 196 into 190. The route with the early detosylation (193—>191—> 195—»190) was given preference over the alternate route with the late detosylation (193—>194—>196—>190).  Although  oc,6-linked dipyrromethanes have been described before, they have been meso-unsubstituted and a- and 6-alkylated.  376  Unlikeraeso-phenyldipyrromethane179, 190 was always  produced as an oil and attempts to sublime it were unsuccessful.  The final 2 + 2 condensation  The dibenzoyldipyrromethane 183 was reduced to the corresponding alcohol 197, mixed in several solvents with an equimolar amount of the N-confused dipyrromethane 190 and, after the addition of catalytic amounts of various acids, the reaction mixtures turned dark. After the starting materials had, based on TLC control, disappeared, an oxidant such as DDQ or chloranil was added. To our disappointment, the yield of the desired N-confused porphyrin 104 was invariably low (< 5%, in some cases 104 was not detectable at all). Thus, the synthetic effort required to make the preformed units was not balanced with high yield of the desired product.  It is interesting to note that the yields of analogous regular  tetraarylporphyrin syntheses are relatively low as well, around 12%.  56  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Chmielewski et al.  269  Results  132  suggested the following for the mechanism of the formation of  N-confused porphyrin via the Rothemund-type condensation (Scheme 1-50): Two helical conformations of the intermediate tetrapyrromethane 198, varying only in the orientation of one terminal pyrrolic unit and interchangeable simply by rotation around an a-meso-carbon bond, are present in the reaction mixture (198A and 198B). Nucleophilic attack of the pyrrole oc-position leading to ring closure, gives, after oxidation of the initially formed porphyrinogen, regular TPP, while attack of the 6-positions gives, after oxidation, the Nconfused porphyrin. While the a-position is more reactive towards electrophiles than the 8-position,  377  there are ample examples in the literature exemplifying the reactivity of the  8-positions and the helical conformation of the linear tetrapyrrolic intermediates under similar reaction conditions.  44,378  "  383  H Ar  H Ar  H Ar  198A  198B  oxidation  TPP S c h e m e 1-50  N - c o n f u s e d T P P 104  P r o p o s e d m e c h a n i s m of f o r m a t i o n o f N - c o n f u s e d  porphyrin  2 6 9  In light of this, it is even more surprising that our attempted synthesis did not generate the desired results as the proposed intermediate 189 is very similar to the likely intermediate in the condensation reaction of 179 and 190. We have no conclusive explanation for this finding.  However, the formation of porphyrins using  2 + 2-condensations frequently requires the use of special and carefully controlled conditions unique to a particular system. Furuta and co-workers propose a pronounced anion template  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  effect directing the formation of the N-confused porphyrin.  268  133  Consequently, a series of  experiments with varying acids catalyzing the final condensation may eventually discover the right conditions for the formation of high yields of N-confused porphyrins via this attractive 2+2 approach.  Future Work  Future work towards the directed synthesis of N-confused porphyrin 104 may succeed following alternative approaches. One alternative retrosynthetic analysis of 104 is shown in Scheme 1-51. This analysis employs a 3+1-approach utilizing the tripyrrane 190 as starting material. Such a synthesis seems particularly attractive as it offers a very short synthetic route.  Ph Scheme 1-51  Ph Alternative retrosynthetic analysis of N-confused porphyrin  We have developed efficient strategies to synthesize the acyclic precursors necessary for the directed synthesis of N-confused porphyrin and we anticipate further developments towards high-yielding macrocyclization procedures in the near future.  Part 1:  2.3  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  134  THE DIRECTED SYNTHESIS OF meso-PHENYLSAPPHYRINS Sapphyrins are generally prepared by condensation of a tripyrrane with a bipyrrole  dialdehyde (see Section 1.3.5). With the novel meso-phenyltripyrrane (180) in hand, we had the opportunity to prepare in an analogous fashion meso-diphenyl substituted sapphyrins 199, and 200, and in a four-component Rothemund-type condensation meso-tetraphenylsapphyrin 114 (Scheme 1-52). At the time this work was started, no meso-phenyl substituted sapphyrins were known, but in the fall of 1995, Sessler, Kodadek and co-workers published the synthesis and structural characterization of 114 via the condensation of dipyrrane dialdehyde, benzaldehyde and pyrrole under Lindsey-type conditions  384  and, almost at the  same time, Latos-Grazynski and co-workers published the isolation of tetraphenylsapphyrin 199 as a side-product from the Rothemund synthesis of T P P .  315  Both procedures, however,  produce the particular sapphyrins in low yields (-10% and 1.1%, respectively) and both require extensive chromatographic work-up (3-4 consecutive column and preparative plate runs). The syntheses to be presented here are short, produce up to 39% yield in the final sapphyrin condensation (for 114) and, due to the absence of any other porphyrinic byproducts, require only minimal chromatographic work-up. While Latos-Grazynski's onestep synthesis is undeniably the shortest possible, our approach is a little longer (a total of 5 steps from pyrrole and benzaldehyde) but still much shorter than any traditional sapphyrin synthesis.  295  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Scheme 1-52  Results  135  Synthesis of the meso-phenylsapphyrins 114,199, and 200  The Synthesis of 5,10-Diphenylsapphyrin (200)  Bipyrrole dialdehyde (201) and tripyrrane 180 were dissolved in ethanol and several equivalents of p-toluenesulfonic acid were added. The mixture turned royal blue within minutes (broad bands at A  m a x  360 and 580 nm), indicating the formation of, possibly, the  partially conjugated pigment 202. This situation did not change over the course of hours, whether molecular oxygen was bubbled through the solution or not. When oxidants such as DDQ were added, the mixture largely decomposed and, as judged by optical spectroscopy, only traces of sapphyrin-type chromophores could be detected. Evaporation of the solution to dryness over a period of several days produced a black, brittle and insoluble film. Most surprising, tritruration of this film with chloroform gave a forest-green solution composed  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  136  almost entirely of the dication of 5,10-diphenylsapphyrin 200. This assignment is based on a series of analytical and spectroscopic techniques, some of which are detailed below. Sapphyrins are basic pigments and are generally isolated and chromatographed as their dications. The present sapphyrin could also be chromatographed on silica gel as its free base. This might suggest a lower basicity for this pigment in comparison to alkylsapphyrins. Sessler and co-workers reported a similar observation for 199.  384  Washing of a chloroform  solution with either a dilute hydrochloric acid or a dilute ammonia solution converts 200 into its diprotonated or free base form, respectively.  The interest in alkylsapphyrins as potential photosensitizers to be used in PDT stems from their strong absorptions in the long wavelength region. This strong absorption in the region above 700 nm can also be observed for the me so--phenyl substituted variety. The optical spectrum of 200 in its free base and protonated form are shown in Figure 1-30. As for decamethylsapphyrin , the Soret-band of the free base is split, and singular in the 298  protonated form. The Q-bands are, in both the protonated and non-protonated case, slightly bathochromically shifted as compared to decamethylsapphyrin.  3E+05-I  x5  ^2E+052 E w 1E+05-  0E+00 400  500  600  700  800  k [nm]  Figure 1-30  Optical spectrum (CH2CI2) of 200 ( — ) and 200-2HCI (  )  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  137  Sapphyrin 200 is dibasic. Full protonation of this mirror-symmetric molecule is seen in the iH-NMR by the presence of three non-equivalent protons in the ratios of 1:2:2 in the high-field region. One complex multiplet centered at 8.0 ppm accounts for the meta- and para -protons of the phenyl-protons, and the multiplet at 8.65 for the ortho-protons. Signals for five non-equivalent B-hydrogens, each integrating for two protons, four of which are doublets and one a singlet are expected, and found. The singlet furthest down-field can be assigned to the meso-protons.  Their extreme shift is in accord with that observed in  decaalkylsapphyrins.  295  The iH-NMR of 200 as its free base and of its dihydrochloride 200-2HC1 are shown in Figure 1-31. The iH-NMR spectrum of, for instance, TPP, changes upon protonation in that it results in doubling of the integration of the signals in the high-field region, assigned to the NH-protons, and thus clearly showing that protonation occurs at the inner nitrogens. Only small shifts are observed for the remaining signals, all of which can be explained by the presence of the dicationic charge. Comparison of the two spectra of 200 and 200-2HC1 reveal that they cannot be rationalized by such a simple protonation-deprotonation reaction. While the spectrum of the diprotonated species is readily assigned, that of the free base is not. The !H-NMR spectrum of the free base 200, on the other hand, displays in the moderate high-field region one very broad and one sharp signal, each integrating for about two hydrogens. In addition, only four typical B-hydrogen signals can be detected and one lowfield signal corresponding to one proton and exchangeable with D 0 is present. Integration, 2  peak width and number are inconsistent with the formulation of 200 existing in conformation 200A, shown in Figure 1-32. All these facts indicate that diphenylsapphyrin folds as shown in Structure 200B.  Parti:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  138  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  200A  200B  Figure 1-32  200-2H  139  +  Conformation of diphenylsapphyrin 200 as free base and as diprotonated species  Inversion of the pyrrolic unit C directs the B-protons towards the middle of the still aromatic macrocycle, and, hence, shields them effectively and directs the NH proton of ring C outwards, thus deshielding it. This explains the iH-NMR spectrum, in particular the finding of the sharp signal at -1.5 and the broad, exchangeable signal at 11.8 ppm, respectively.  Latos-Grazynski and co-workers observed and described very recently an analogous sapphyrin inversion for 5,10,15,20-tetraphenylsapphyrin (114).  315  Another indication for the  validity of this assumption can be found in the literature. N-Confused TPP 104 features in a similar way a B-proton in the centre of the macrocycle and an NH proton at the outer periphery. The observed chemical shifts for these protons in 104 are comparable to those found here. ' 268  269  Synthesis of 5,10,15,20-Tetraphenylsapphyrin (114)  This sapphyrin was synthesized under Lindsey-type conditions.  Benzaldehyde,  pyrrole and bipyrrole (203) were dissolved in degassed methylene chloride and treated with catalytic amounts of trifluoroborane etherate. Chloranil was added to this reaction mixture. This mixture of newly formed TPP and sapphyrin 114 was separated by chromatography.  Part  1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  140  The spectroscopic properties of this sapphyrin were, as expected, similar to those of 200 and identical with those reported by Chmielewski et a/.  315  The overall yield of 3.7% is more  than three times that reported by the Polish group and we think that this higher yield is due to the use of the preformed building block (dipyrrin), as compared to the synthesis from pyrrole and benzaldehyde, though any findings based on the encountered minimal absolute yields are, at best, inconclusive.  Synthesis of 3,22-Diethyl-2,23-dimethyl-5,10-diphenylsapphyf in (199) This sapphyrin was prepared along the lines described in the benchmark publication by Bauer et al. for decaalkylsapphyrins. Dipyrrin dialdehyde 112 and tripyrrane 180 were 298  dissolved in absolute alcohol and as molecular oxygen was bubbled through the solution, some p-toluenesulfonic acid was added. The mixture turned dark green and the sapphyrin was isolated by standard chromatography techniques.  Only traces of TPP ( « 1%) were  formed as by-products. The distinct optical and NMR spectra of this compound readily identify it as the desired sapphyrin. All properties proved identical with those reported by Sessler, Kodadek and co-workers for this compound.  384  In addition, these authors solved  the X-ray crystal structure of the dihydrochloride salt. This largely planar molecule, like its B-alkyl analogs, can act as chloride anion receptor in the solid state.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  2.4  Results  141  THE REDUCTIVE COUPLING OF 2-CYANOPYRROLES As mentioned in the introductory Chapter (section 1.3.5), the mechanism of the facile  formation of porphocyanine via the L A H reduction of a,a'-dicyanodipyrromethanes is not entirely clear. Given the favorable photophysical properties of this compound class and its promise for use as a third generation PDT agent, the study of its mechanism of formation with the goal to, perhaps, optimize its preparation seemed worthwhile. Also, an alternative synthesis of this compound class was sought.  Scheme 1-52 depicts one alternative  possibility, namely the fusion of two imine-linked halves, followed by aromatization of the formed macrocycle. This type of coupling is under investigation by other members of Prof. Dolphin's research group.  Scheme 1-52  Proposed alternative synthetic pathway for the formation of phenylporphocyanines  Imine 204 is a known compound.  385  meso-  Its synthesis is shown in Scheme 1-53.  2-(Aminomethyl)pyrrole (205) is reacted with 2-pyrrolealdehyde (206) in aqueous solution to form 204. 205 was reportedly synthesized by the LAH reduction of 2-cyanopyrrole (207). When we repeated these procedures, we replaced the dilute sulfuric acid quenching step of the L A H reduction reaction mixture  386  by the addition of Glauber's salt (Na2SO4-10 H2O).  It was found that imine 204 was formed as the sole product. This outcome is exclusive to the use of aluminum hydrides as reductant. Compound 207 is inert to sodium borohydride with  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  142  or without the addition of transition metals, but diisobutylaluminum hydride (DIBAL) produces, albeit not as cleanly, 204. The formation of 204 is also surprising considering that its imine-link is susceptible to reduction by L A H , giving the amine 208. This provides the first clue that the imine functionality must have formed during the quenching step.  Scheme 1-53  Formation of imine-linked dipyrrolic compound 204 Reaction conditions: (i) UAIH4, then dil. H2SO4; (ii) H2O; (iii) UAIH4, then Glauber's salt (Na2SO4-10H2O)  This finding prompts several possible explanations. Partial reduction of the cyano group forms, after hydrolysis of the initially formed imine, an aldehyde functionality which then reacts, according to the above scheme, with an aminomethylpyrrole (205), stemming from the full reduction of 207. A second, and more likely, possibility is, that the initially formed imines condense to form the imine-linkage. There are precedents for such a condensation in the literature. Hydrobenzamide (ArCH(-N=CHAr)2 can be formed from an aromatic aldehyde and ammonia via the imine (ArCH=NH).  324  One drawback of these  explanations is, however, that while partial L A H reductions of cyanopyrroles to form pyrrolealdehydes via hydrolysis of the imines are known, they require the use of very deactivated 2,4-dicyanopyrroles. '  386 387  This is clearly not the case in the reduction of 207.  This makes proposed mechanisms based on partial reductions of the cyano group unlikely. In turn, this makes in situ reductive couplings seem to be more likely. To investigate this  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  143  possibility in more detail, several cyanopyrroles were synthesized and their behavior towards the LAH reduction conditions were studied.  Synthesis of Cyanopyrroles  Several possibilities to synthesize cyanopyrroles are available. " 388  392  Possibly the  most versatile and convenient strategy is the direct introduction of cyano groups by reaction of a pyrrole with chlorosulfonyl isocyanate (CSI) intermediate chlorosulfonylamide with D M F .  386  '  394  "  398  393  followed by solvolysis of the  This methodology was applied to  pyrrole ' , 3,4-diethylpyrrole (209), 3-ethyl-2,4-dimethylpyrrole (210), and 2,5386  397  dimethylpyrrole (211), to produce the monocyanopyrroles 207, 212, 213, and 214, respectively. 2-Cyanopyrrole (207) was converted to the known l-methyl-2-cyanopyrrole  399  by methylation of the pyrrolic nitrogen. In cases in which two pyrrolic hydrogens could be substituted for cyano groups this was also observed, producing 216 and 217. By using excess CSI, the doubly cyanated pyrroles could be made the major products of the cyanation reaction. A similar dicyanation has been described for pyrrole.  397  The starting pyrroles were either commercially available (pyrrole and 211), or were synthesized in-house according to standard procedures. '  39 400  One such pyrrole substituent  manipulation is exemplified for the synthesis of 3,4-diethylpyrrole (209). The methyl group in 218 was oxidized to the corresponding acid function. This group of the resulting 219 was replaced by iodine, which subsequently was removed reductively. Ester hydrolysis of the resultant product 221 then produced the acid 222 which is prone to thermal decarboxylation to a-free pyrrole 209. This desired pyrrole was isolated by high vacuum distillation and immediately cyanated with either one or two equivalents of CSI, producing the final products 212 or 216, respectively.  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Scheme 1-54  144  Syntheses of the cyanopyrroles Reaction conditions: (i) 1 (or 2) equivalents of CSI/DMF/CH3CN, respectively, then aqueous workup; (ii) 1. NaH, 2. TosCI; (iii) 1. S02Cl2/Et20, 2. H2O; (iv) I2/KI; (v) H2 over PtC-2; (vi) KOH/H2O; (vii) Ethyleneglycol/A  With the mono-cyanopyrroles in hand, a series of reductions was performed. Each reduction followed the same protocol. A defined small amount of the cyanopyrrole was dissolved in dry THF and the solution was cooled in an ice-bath and kept under a nitrogen atmosphere. Four equivalents of L A H were added in portions and, after addition was completed, the mixture was stirred for one hour. Glauber's salt was then added until gas evolution ceased, and the resulting slurry was filtered through Celite®. The filtrate was evaporated in vacuo, and the residue analyzed by mass, H - and C - N M R spectroscopy. ]  The results are presented in Scheme 1-55.  13  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  145  + v e r y little of the imine linked d i m e r 226  CH  3  223  225  v a r y i n g ratios  e x c l u s i v e product  Scheme 1-55  227  228  Outcome of the IJAIH4 reduction of various cyanopyrroles Reaction condition: (i) IJAIH4/THF at 0°C, then Na2SO4-10H2O  Reduction of cyanopyrrole 215 resulted almost exclusively in the formation of the corresponding aminomethyl compounds 2 2 3 (product ratio 13:1 in favor of 223). B-Cyanopyrrole 214 resisted reduction under the conditions employed and was fully recovered.  This unusual stability of B-cyano groups towards nucleophilic attack has  precedent in the literature.  386  Reduction of the compounds 212 and 213 gave the imine  dimers 227 and 228. The imine was the sole product in the latter case whereas in the former the aminomethyl compound 225 was observed as well. The particular ratio of the two compounds 225 and 227 varied from run to run from 1:2 to 3:1.  Reduction of 207 with lithium aluminum deuteride and subsequent quenching with deuterated water (in the form of deuterated Glauber's salt made by diffusion of deuterated water onto dry sodium sulfate) produced 208-ds in which all but the CH-protons of the  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  146  methylene-linked ring are deuterated. Quenching with water produced non-deuterated 208, suggesting that quenching elicits exchange of the protons. This can be understood by the strongly basic nature of LAH and the strongly basic conditions of the quenching procedure.  The major difference in the starting materials is the ability of their pyrrole-nitrogens to participate in, for example, a coordinating action with the reductant. The N-methylated pyrrole 215 cannot form readily chelate-type interactions with aluminum. In order to trace possible aluminum complexes formed by (partially reduced) 207 and LAH, A1-NMR 27  spectra ' 401  402  of solutions of 207 in THF/benzene-d6 (19:1) containing various amounts of  LAH were measured. With a ratio of 207:LAH of -1:3, a strong signal for the LAH and a broad, featureless signal 60-80 ppm upfield of this signal is seen. Increase in the relative amount of 207 strengthens the broad signal at the expense of the LAH signal. At about a 1:1 ratio the L A H signal has entirely vanished. The failure in measuring well defined signals for the presumed aluminum complex is not entirely unexpected. Due to the quadrupole moment of the Al-nucleus, only highly symmetric (tetrahedral) complexes give sharp signals. 27  Study of the literature reveals two closely related publications. ' 403  404  The more  recent contribution of Wang and Sukeniki describes the reduction of oximes with LAH in HMPA-containing solvents. One particular example, the reduction of benzaldoxime (229) resulted in the formation of the imine-linked dimer 230 and the authors rationalize this finding by the mechanism shown in Scheme 1-56. They propose a series of deprotonation and reduction steps. An LAH reduction in the absence of HMPA leads to the formation of benzylamine. Their rationalization for changing of the outcome of the reaction by the presence of HMPA hinges upon the promotion of the basicity of L A H by strong complexation of the lithium ions by this co-solvent.  404  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  147  N  230  HoO  LAH N-OAIH3 H  N-0AIH3U  R—<^—A H  N(AIH Li) R—|— N - C H R H AIH3U 3  LAH J  2  2  R-CN  LAH  LAH  N-H  LAH  NAIH3U  H  H N(AIH Li) R—|—N=CHR H  N"  3  H  Scheme 1-56  2  Mechanism of the LAH coupling of benzaldoxime Reaction condition: (i) 1. LAH/THF/HMPA, 2. H 2 O  403  In the case of the reductive coupling of 207, the co-solvent HMPA is not present and yet a similar reaction can be observed. We propose the sequence shown in Scheme 1-57 as one possible mechanism of the coupling, although it is important to note that we have only just begun to collect evidence for the details, and the work is still in progress. However, this proposal bears some resemblance to known literature mechanisms.  According to this mechanism, the first step would be a combined deprotonation/ reduction of 207 to form a chelate of type 231. Similarly constructed chelates of copper(II) are known for 2-pyrrolealdehyde.  420  This chelate may dimerize to give 232. Its double  bond may be reduced by another equivalent of LAH. Hydrolysis would give the labile  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  148  species 234 which stabilizes itself by the abstraction of ammonia to produce the final product 204. And, in fact, the smell of ammonia can be detected during the hydrolysis step. The proposed mechanism may not be the only mechanism occurring as, for instance, the presence of the dimer 227 and the aminomethyl pyrrole 225 indicates. H  Scheme 1-57  Hypothetical mechanistic scheme of formation of 204 through the UAIH4 reduction of cyanopyrrole 207  It is interesting to note that although we may have been the first group to recognize this coupling, there are indications in the literature that this coupling has been observed before, but went unrecognized. Barnett et al. described in 1980 the L A H reduction of 2,4dicyanopyrroles. Following a workup employing 2.5 M sulfuric acid, they found one product to be 4-cyano-2-pyrrolecarboxaldehyde. They noted "Over half the starting material was not accounted for."  386  In light of our findings this is understandable. The strong acid  cleaved the initially formed imine into a stable pyrrolealdehyde fraction and into a very unstable aminomethyl fraction. The latter decomposed and, hence, could not be accounted for.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  149  2.5 THE UNEXPECTED FORMATION OF A TRIPYRROLIC TETRADENTATE NICKEL CHELATE (236) Our interest in dipyrrins, tripyrrins and other potential metal chelating pyrrolic pigments (see Parts 2 and 3), prompted investigations into whether the unknown imine linked, fully conjugated pigment is accessible through the oxidation of 204. Many attempts to produce an isolable compound failed, but one VNH  N  235  ^  interesting observation was made.  In some  experiments the oxidation was tried in the presence  of metal ions. With the addition of nickel(II) as its acetate, the formation of tiny amounts of a brilliantly orange pigment of very low polarity (Rf = 0.9 CHCl3/silica) was noticed. It later could be shown that under certain conditions this orange pigment could be made the major product.  This enabled the synthesis of several hundred milligrams and full  characterization of this pigment (236). Its analytical and spectroscopic properties were inconsistent with it having structure 235 or being a nickel complex of 235.  3E+04-,  0E+00 300  400  500 X [nm]  Figure 1-33  Optical spectrum(CHCl3) of 236  600  700  Results  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  150  The UV-visible spectrum of the compound, shown in Figure 1-33, exhibits some characteristics of the (dipyrrinato)nickel chromophore (compare to Part 2, section 2.3). The JH-NMR shows ten non-equivalent protons in the aromatic region and the signal for the methylene group of the starting material is still present (Figure 1-34). No proton signals indicative of NH protons could be traced, suggesting the formation of a (nickel) complex.  1  1  8.0  7. 5  Figure 1-34  ••'» •'••|;!i•I 7.0 1  £.S  6.6  -  I 6.  I  '  ST  5.0  H NMR spectrum (200 MHz, acetone-d6) of 236  The HR-MS of the compound (vide supra) corroborated the presence of a nickel atom, and because the compound, by virtue of its sharp NMR signals, proved to be diamagnetic, a square planar metal complex of nickel(II) had to be assumed.  405  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  151  Based on the potential lability of the imine linkage towards hydrolysis and the reactivity pattern of any pyrrole aldehyde equivalent formed in such a cleavage, the reaction sequence shown in Scheme 1-58, and the structure depicted for the pigment 236 was proposed: Lewis acid catalyzed hydrolysis of 204 gives pyrrole aldehyde 206 and, formally, the unstable aminomethyl pyrrole 237. The aldehyde 206 reacts with a second equivalent of 204 at its most nucleophilic position (the a-position of the pyrrolic unit which is not deactivated by the imine nitrogen) to form a dipyrrin unit. This molecule would be poised to 'wrap around' the nickel atom and to coordinate it in a square planar fashion to finally give 236. This structure would account for all spectroscopic and analytical data.  Scheme 1-58  Proposed mechanism of formation and structure of 236  A single crystal X-ray crystal structure of 236 subsequently confirmed the structural assignment, and a mechanistic investigation revealed interesting aspects of the reaction mechanism.  The Molecular Structure of 236  Slow evaporation of a THF solution of 236 provided coarse red-orange crystals suitable for X-ray crystallography. An ORTEP representation and side view of the structure are shown in Figure 1-35. Experimental details of the structure determination are listed in Table 1-2, the final atomic coordinates are listed in Table 1-7, and selected bond lengths in Table 1-8.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  152  Most obvious and intriguing in this structure is the pronounced flatness of the openchain ligand-metal complex. As a result, nickel is coordinated in a perfectly planar fashion by the four nitrogens with bond distances varying from 1.845(2) to 1.893(2) A. Three types of nitrogens are present in the molecule, two of which are part of a dipyrrin moiety, one is an imine nitrogen and one is a pyrrole nitrogen. The pyrrole and imine nitrogens are in conjugation, and so are the nitrogens in the dipyrrin moiety but the two pairs are isolated from each other by a methylene group. Neither the differences in the metal-nitrogen nor the intra-dipyrrin bond lengths can be, in analogy to the dipyrrinato complexes to be described in Part 2, rationalized with a simple resonance description.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Results  153  The Influence of Various Reaction Conditions in the Formation of 236  Pigment 236 forms in moderate yields at slightly elevated temperatures (~50°C) upon reacting 204 with nickel(II) in (wet) methanolic or ethanolic solution. Pyrrole aldehyde 206 can be detected by TLC from the onset of the reaction. This suggests that the first step of the reaction is, indeed, the cleavage of the imine moiety in 204 . The hydrolysis is catalyzed by the Lewis acidity of nickel(II) as the compound is perfectly stable under these conditions in the absence of the metal. Other Lewis acids such as zinc(II) as its acetate and Br0nsted acids (HC1, HBr, TFA) were shown to catalyze the hydrolysis of the imine linkage under the above described conditions. The highly unstable 237 could not be seen, nor could possible reaction products such as dipyrromethanes from a self-condensation of 204. Only the occurrence of highly polar and colorful materials in the course of the reaction indicate its polymerization and/or reaction with any other pyrrolic compounds in the reaction mixture. The acidcatalyzed hydrolysis of imines and the instability of any aminomethyl pyrroles is well documented in the literature.  419  The second step of the reaction requires the formation of the final ligand and, possibly concomitant, the formation of complex 236. An equimolar alcoholic solution of independently synthesized pyrrole aldehyde (206) and 204 form in the presence of nickel(II) complex 236 in yields almost double those obtained in the absence of additional aldehyde. This corroborates the assumption that pyrrole aldehyde is the reactive species in the ligand formation derived from cleavage of the imine moiety. The formation of 236 is of particular interest as it is susceptible to a profound metal template effect. Only nickel(II) is capable of forming a complex of type 236. Some metals (zinc) catalyze only the hydrolysis of the imine step, others (cobalt, iron, copper) form highly colored precipitates with 204. They are, however, known complexes of the type ( 2 0 4 ) M . 2  3 8 5  '  4 0 6  A metal-free mixture of  2-pyrrolealdehyde (206) and imine 204 does not show any reaction. This mixture treated  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  154  with Br0nsted acids such as HBr exhibits at first the typical optical spectrum of a dipyrrin hydrobromide (^max~500 nm, compare to Part 2), but rapid decomposition sets in and no defined product can be isolated.  Neutralization and reaction with nickel(II) yields no  isolable products, indicating no formation of, for instance, the free base of 236. On the other hand, an alcoholic solution of pyrrole and pyrrole aldehyde (206) does not form the corresponding dipyrrin in the presence of nickel(II), indicating that the presence of ligand 204 is required for the reaction to proceed.  The Mass Spectrum of Complex 236 The electron impact LR-mass spectrum ( 1 8 0 ° C ) of 236 (A) together with the calculated isotope pattern for Ci5H)2N4Ni ( B ) is shown in Figure 1-36.  305  C H N Ni  (B)  15  12  4  (A) JL  306 308 310 312  226 •l r l ' l I'r 220  2 30  >39 M 240  139  278 T T  i j' 7 i rn- i ii,i11i1 i i i' 1 1  120 Figure 1-36  1 1  140  h 11  I | I II' I II I I I I I II I I I I | II 260 280 300  53  161  i |Ti 160  175  I'I  i rl'i i |'i 180  1193 ,201 'I I'l i'l' I I ' l ' .  1  2Z3  1  1  320  214 1 I'm 220  Measured (El, 180°C) (A) and expected (B) mass spectrum of 236  111 M 111 340  Part 1:  Results  Synthesis and Study of Pyrrolic Pigments for Use in PDT  155  There are clear discrepancies between the measured and the expected spectrum, both in the value of the molecular peak as well as in the isotope pattern. Instead of the expected molecular ion peak of m/e = 306 (or 307 for the often observed M H ) one molecular peak +  with m/e = 305 is observed. The high resolution mass of 305.03370 mu revealed its composition to be C i 5 H n N 4 N i (expected: 305.03372 mu). This fragmentation pattern, corresponding to a hydride abstraction of complex 236, finds its explanation in the highly resonance stabilized structure of the resulting cation (238) (Scheme 1-59). The computed overlay of a -1:2 ratio of 239 (resonance stabilized radical cation, m/e - 306) and 238 yields the observed spectral pattern. No indications for the formation of a cyclized product such as 240 were found in the mass spectrum. Attempts to form macroscopic amounts of 238 under solution phase conditions failed.  236  Ci5H  1 2  N Ni/306 4  239 S c h e m e 1-59  C H-|-| N N i / 3 0 5 15  4  238  F r a g m e n t a t i o n o f 2 3 6 i n t h e El m a s s s p e c t r o m e t e r  C  1 5  H N N i / 303 9  4  240  156  3.  3.1  EXPERIMENTAL  INSTRUMENTATION AND GENERAL MATERIALS Melting points were determined on a Thomas Model 40 Micro Hot Stage and are  uncorrected. The infrared spectra were measured with a Perkin-Elmer Model 834 FT-IR instrument or an a Perkin Elmer 710D. The !H-NMR spectra were measured on a Bruker AC-200 spectrometer (200 MHz) or a Bruker WH-400 with data processing on a Bruker data station, or a Varian XL-300 (300 MHz).  13  C-NMR spectra were measured on a Bruker AC-  200 Fourier-transform spectrometer (50 MHz), a Varian XL-300 (75 MHz) or a Bruker AMX 500 (125 MHz). The NMR spectra are expressed on the 5 scale and are referenced to residual solvent peaks. The low and high resolution FAB and EI mass spectra were obtained on a AEI MS902 and a Kratos MS50. The UV-visible spectra were measured on a Hewlett Packard HP 8452A photodiode array spectrophotometer and the data were processed on a microcomputer (CA Kricket Graph III software). Elemental analyses were performed on a Fisons CHN/O Analyzer, Model 1108.  For experimental details of the X-ray crystal  structure determination, see Table 1-2 (p. 193).  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  157  Materials  The silica gel used in the flash chromatographies was Merck Silica Gel 60, 230-400 mesh. Rf-values were measured on Merck silica TLC aluminum sheets (silica gel 60 F254). whilst preparative TLC was performed on pre-coated 20x20 cm, 0.5 or 1.0 mm thickness, Whatman or Merck silica gel plates (with or without fluorescence indicator).  TPP and its zinc(II), nickel(II) (126), iron(III)Cl (127), and copper(II) (128) complexes and the variously substituted meso-tetraarylporphyrins were synthesized along standard Rothemund-type procedures ' ' with subsequent metallations. 41  44  45  328  TPC (18) and  TPCZn (142) were synthesized from TPP following the procedure described by Whitlock et a/.  83  DPP (139)was produced according to a procedure outlined by Manka et al.^  1  3-Benzoylpyrrole (191)  374  has. been synthesized from 1-tosylpyrrole (192)  407  following procedures described.  2,2'-Bipyrrole (203) was prepared from pyrrole and 2-pyrrolidinone/POCl3, followed by dehydrogenation of the resulting l-pyrrolinyl-2-pyrrole.  408  Violent pyrrole  polymerization was observed in several runs upon the addition of POCI3 to the reaction mixture. Thus, it was later added slowly (over several hours) with the help of a syringe pump. The dehydrogenation was, in a second variation to the original procedure, performed in trimethyleneglycol dimethylester.  An alternative synthesis of 203 by palladium(II)  catalyzed coupling of 1-aroylpyrrole, and subsequent hydrolysis of the l,l'-dibenzoyl409  2,2'-bipyrrole proved to be by far not as cost efficient and convenient as the latter synthesis. 2,2'-Bipyrrole dialdehyde (112) was prepared from 203 following a standard VilsmeyerHaak formylation procedure as first described by Bauer et al.  29%  3,3'-dimethyl-2,2'-bipyrrole  410  4,4'-Diethyl-5,5'-diformyl-  (114) was prepared by hydrolysis of the corresponding  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  dicyanovinylgroup  411  Experimental  158  protected compound. This compound was available from stocks of  the research group.  2-Pyrrolealdehyde (206) was prepared by Vilsmeyer-Haak formylation of pyrrole.  412  3,4-Dimethyl-4-ethylpyrrole (210), and 3,4-diethylpyrrole (209) were prepared by standard procedures  39,413  from in-house pyrrolic starting materials.  2-Cyanopyrrole (207) was  prepared from pyrrole and chlorosulfonyl isocyanate according to a procedure detailed by Barnett, Anderson, and Loaner.  386  All other reagents and solvents were commercially available and of reagent grade or higher, and were, unless specified, used as received.  NOTE OF CAUTION  Some chemicals used throughout this work deserve special attention. H 2 S , O S O 4 , chlorofulfonyl isocyanate (CSI), and B r 2 are volatile, corrosive and severely toxic chemicals. They must be handled in a well-ventilated fumehood and proper personal protective equipment must be worn. Also, familiarization with the recommendations, warnings, first aid measures and disposal procedures for these chemicals as outlined in the Material Safety Data Sheets (MSDS) should be mandatory before working with these compounds.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  3.1  meso-ARYL-  Experimental  159  WC45IOLCHLORINS, -BACTERIOCHLORINS  AND -ISO-BACTERIOCHLORINS mesoTetraphenyl-2,3-osmate ester-2,3-chlorin bispyridine adduct (124) A solution of TPP (160 mg, 0.26 mmol) and O S O 4 (65 mg, 1 equivalent) in pyridine (10 mL) was stirred at room temperature in a tightly stoppered flask for 72 h. The mixture was then evaporated in vacuo at temperatures not exeeding 50°C. The resulting solid was loaded onto column (silica gel, 20x3 cm) and eluted with CH^Ch/MeOH (gradient ranging from 100% C H C H toCH2CH2/10% MeOH). The first fraction was starting material (50 2  2  mg, 31% recovery), and the second main purple fraction the desired compound. Slow evaporation from CH2CH2/10% MeOH with the exclusion of light produces 124 as purple microcrystalline material (85 mg, 32% yield). MW = 1027.15 (bis-pyridine adduct); mp = d > 120°C; Rf = 0.49 (silica-CH Cl /5% 2  MeOH);  2  iH-NMR (400 MHz, CDCI3) - 1.72 (br s, exchangeable with D 2 O , 2H), 6.99 (s,  2H), 7.30 (m, 6H), 7.51 (tr, J = 4.8 Hz, 2H), 7.57 (tr, J = 4.5 Hz, 2H), 7.61-7.72 ( m, 8H), 7.97 (d, J = 4.3 Hz, 2H), 8.05 (d, 7 = 4.3 Hz, 2H), 8.09 (d, J = 4 Hz, 4H), 8.26 (d, J = 3 Hz, 2H), 8.43 (s, 2H), 8.57 (m, 6H); ^C-NMR (75 MHz, CDCI3) 96.59, 114.06, 121.97, 124.34, 124.71, 126.16, 126.55, 126.88, 127.04, 127.43, 127.47, 132.13, 132.24, 133.86, 134.11, 135.41, 139.82, 140.91, 142.07, 142.22, 149.74, 152.67, 162.95; UV-Visible (CHCI3) X  m a x  (rel. intensity) 420 (1.0), 520 (0.12), 548 (0.11), 594 (0.05), 646 (0.13) nm; LR-MS (EI, 3-NBA) m/e 1159 (<1, MH+ + 2 NBA), 1106 (<1, C44H30N4O4OS), 631 (10, C44H31N4O), 615 (21, C 4 4 H 3 1 N 4 ) ; HR-MS (EI, 3-NBA) m/e expected for C H 4 3 N 0 9 0 s (= 58  C44H oN 030s-2 matrix-H+): 1159.27121, found: 1159.27200. 3  4  6  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  160  (meso-Tetraphenyl-2,3-osmate ester-2,3-chlorinato)zinc(ll) bispyridine adduct (125) Prepared from TPPZn in 58% yield according to a procedure analogous to the one described for the preparation of 124. Column chromatography (silica-Et20/CH2Cl2 7:3). MW = 1091.51; mp = d > 150°C; R = 0.41 (3:2-Et 0:CH Cl2); H-NMR (400 MHz, 1  f  2  2  CDC1 ) 8 6.86 (s, 2H), 7.23 (m, 2H), 7.38 (br s, 4H), 7.45 (tr, J = 7Hz, 2H), 7.53 (tr, J = 3  7Hz, 2H), 7.58-7.64 (m, 8H), 7.90-7.96 (m, 4H), 8.0-8.08 (overlapping m and d J = 4.5 Hz, 6H), 8.37 (s, 2H), 8.41 (d, J = 4.5, 2H), 8.64 (br s, 4H); UV-Visible (CHCI3) k  max  (rel.  intensity) 424 (1.0), 570 (sh), 618 (0.18) nm; LR-MS (EI, 3-NBA) m/e 932 (< 1, M+-2 pyridine), 692, (5, C4 H28N OZn), 976 (9, C 4H 8N Zn); HR-MS (EI, 3-NBA) m/e 4  expected for C H 8 N 0 4 4  2  4  4  6 4 4  Zn  4  1 9 2  2  4  O s : 932.09911, found 932.09752; Analysis calc'd for  C H4oN60 OsZn (125H 0): C, 58.51; H, 3.64; N, 7.58; found: C, 58.97; H, 3.68; N, 7.38. 54  5  2  (meso-Tetraphenyl-2,3-osmate ester-2,3-chlorinato)zinc(ll) bispyridine-ds adduct Prepared analogous to 124 in 1:9 pyridine-d5:CHCl3 as solvent mixture. 1  H-NMR (400 MHz, CDCI3) 5-1.73 (s, 2H), 6.99 (s, 2H), 7.33, (br tr, J = 7 Hz, 2H), 7.53,  (tr, / = 7 Hz, 2H), 1.65-1.1 A (m, 8H), 7.99 (d, J = 4.5 Hz, 2H), 8.43 (s, 2H), 8.57 (d, / = 4.5 Hz, 2H).  meso-Tetraphenyl-3,4-w'c-dihydroxy-3,4-chlorin (129) General procedure for the OsQ4 mediated dihydroxylation of meso -arylporphyrins  TPP (1.0 g, 1.63 x 10~ mol) was dissolved/suspended in freshly distilled, ethanol3  stabilized CHCl3/10% pyridine (200 mL, in general, the least amount of solvent possible) and was treated with O S O 4 (540 mg, 1.3 equivalents). The reaction flask was stoppered and stirred at ambient temperature in the dark until no change could be detected by optical spectroscopy or TLC control (ca. 4 days). The reaction was then quenched by purging with gaseous H2S for 5 min. Following the addition of MeOH (-20 mL), the precipitated black OsS was filtered off through Celite®. The filtrate was evaporated to dryness by a stream of  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  161  air or N 2 . (FUMEHOOD !!; the use of a rotary evaporator connected to an aspirator is not recommended for the evaporation step as this carries residual H2S into the waste-water streams, resulting in a pungent stench from the drains. Washings of the organic reaction mixture performed with dilute Na2CG*3 solutions to extract the H2S forms, depending on the phenyl substituents, very stable emulsions.) The resulting residue was loaded onto a silica gel column (200 g, 280 - 400 mesh) and eluted with CH2CI2. The first fraction is starting material (400 mg, 40 % recovery). 1.5 % MeOH/CH^Ch eluted then 125. Slow evaporation from a MeOH/CH Cl mixture gave 125 (520 mg, 8.02 x 10" mol, 49 % yield) as bright 4  2  2  purple crystalline material. Further elution of the column with 5% MeOH/CH2Cl2 gave a crude mixture of 145 and 146 (ca. 40 mg, 3.5 %). MW = 648.76; m.p. > 300°; R = 0.68 (silica-CH Cl2/1.5% MeOH); iH-NMR (400 MHz, 2  f  CDCI3) 5-1.78 (br s, 2H), 3.14 (s, exchangeable with D 0 , 2H), 6.36 (s, 2H), 7.68 - 7.80 2  (m, 12H), 7.92 (d, 7=8.5 Hz, 2H), 8.09 (br s, 4H), 8.15 (d, J = 8.5 Hz, 2H), 8.33 (d, J = 4.5 Hz, 2H), 8.48 (s, 2H), 8.63 (d, 7 = 4.5 Hz, 2H); C-NMR (125 MHz, CDCI3) 8 73.9, 113.2, 13  123.1, 124.2, 126.7, 127.5, 127.7, 127.9, 128.1, 132.2, 132.7, 133.9, 134.1, 135.5, 140.6, 141.2, 141.8, 153.2, 161.4; UV-Visible (CHCI3) X  m a x  (log e) 408 (5.27), 518 (4.19), 544  (4.19), 592 (3.85), 644 (4.38) nm; fluorescence (CH C1 ) at 649 nm (excitation 512 nm); 2  2  LR-MS (EI, 300°C) m/e 648 (0.5, M+), 646 (0.9, M+ - 2H), 630 (100, M+ - H 0), 614 2  (42.7); HR-MS (EI, 300°C) m/e calc'd for C44H32N4O2: 648.2525, found 648.2525; Analysis calc'd for C44H32N4O2 • 1/2 H 0 : C, 80.34; H, 5.06; N, 8.52; found: C, 80.26; H, 2  4.93; N, 8.46.  (5,10,15,20-Tetraphenyl-Wc-2,3-dihydroxy-2,3-chlorinato)zinc(ll) (130) Prepared in 72% yield from TPPZn following the general procedure. Washing with dilute acetic acid removes coordinated pyridine. Slow evaporation of a CHCI3 /MeOH solution containing a drop of pyridine produces blue-green crystals of 125 as its pyridine  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  162  adduct. Alternatively, refluxing of a solution of 129 in CHCl3/MeOH 9:1 containing excess zinc acetate converts the free base quantitatively into its metallated form 130 within 2 h. MW = 712.12; mp (d) > 300°; Rf = 0.62 (silica gel-1.5 % MeOH/CH Cl ); *H-NMR (400 2  2  MHz, CDC1 ) 8 5.30 (s, 2H, exchangeable with D 0 ) , 6.12 (s, 2H), 7.55 - 7.72 (m, 12H), 3  2  7.81 (d, J= 7Hz, 2H), 7.94 (d, J = 7Hz, 2H), 7.96- 8.06 (m, 6H), 8.37 (s, 2H), 8.46 (d, J = 4.5 Hz, 2H) (pyridine adduct: additional signals at, 3.8 (m, 2H), 5.9 (br tr, 2H), 6.69 (tr, J = 2.5 Hz, 1H); C - N M R (75 MHz, CDCI3) 6 50.63, 126.48, 126.59, 126.63, 127.23, 127.35, 13  127.48, 127.68, 127.77, 127.82, 129.31, 132.11, 132.52, 133.63, 133.68, 133.79, 141.73, 142.57, 146.52, 148.04, 154.22, 156.28; UV-Vis (CH Cl /0.5%MeOH) ? i 2  2  m a x  (log e) 418  (5.41), 524, 564, 596 (sh), 614 (4.71) nm; LR-MS (FAB, 3-NBA) m/e 710 (29.2, M+), 693 (7.0, M+-OH), 676 (3.7, M+-20H); HR-MS  (FAB, 3-NBA)  m/e calc'd for  C 4H oN40 Zn: 710.16602, found 710.16595; Analysis calc'd for C 4 H o N 0 Z n 64  4  3  2  4  3  4  2  •C5H5N: C, 74.38; H, 4.46; N, 8.85; found: C, 73.50; H, 4.25; N, 7.87.  (5,10,15,20-Tetraphenyl-Wc-2,3-dihydroxy-2,3-chlorinato)nickel(ll) (131) Prepared in 23% yield from TPPNi following the general procedure. Alternatively, refluxing under nitrogen a solution of 129 (CHCb/MeOH 9:1) containing excess nickel acetate converts the free base quantitatively into its metallated form 130 within 24 h. Crystallization by slow solvent exchange from CHCI3 to MeOH gives bright green crystals of 131. MW = 705.40; mp (d) > 250°; Rf = 0.48 (silica-CH Cl /0.5% MeOH); !H-NMR (400 MHz, 2  2  CDC1 ) 5 2.85 (s, 2H, exchangeable with D 0 ) , 5.81 (s, 2H), 7.55 - 7.65 (m, 12H), 7.70 (m, 3  2  4H), 7.82 (m, 4H), 8.22 (overlapping s and d = J, 4H), 8.38 (d, J = 4.5 Hz, 2H);  1 3  C NMR  (50 MHz, DMSO-d ) 8 76.0, 111.4, 123.2, 127.3, 127.5, 128.2, 128.5, 132.2, 132.6, 137.5, 6  139.1, 139.4, 140.4, 145.8, 148.6; UV/Vis (CH Cl /0.5%MeOH): X 2  2  m a x  (log e) 416 (5.10),  516 (3.66), 552 (sh), 572 (sh), 612 nm (4.30); LR-MS (FAB, thioglycerol) m/e 705 (25.5, MH+), 704 (20.2, M+), 693 (18.0, M+-OH); HR-MS (FAB, thiogycerol) m/e calc'd for  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  163  C 4 H 3 i N 0 2 N i : 705.18005, found 705.17945; Analysis calc'd for C 4H3oN Ni02 • 1/3 58  4  4  4  4  H 0 : C, 74.29; H, 4.34; N, 7.88; found: C, 74.33; H, 4.06; N, 7.59. 2  5-(4-hydroxyphenyl)-10,15,20-triphenyl-2,3-vic-dihydroxy-2,3-chlorin (134) and 10(4-hydroxyphenyl)-5,15,20-triphenyl-2,3-vic-dihydroxy-2,3-chlorin (135) Prepared from 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin  (260 mg, 4.04 x  10~ mol) according to the general procedure for the dihydroxylation of meso-arylporphyrins. 4  The two isomeric products 134 (22% yield) and 135 (16% yield) were separated by preparative TLC (silica-CH.2Cl2/1.5% MeOH, multiple developments). (134) :  MW = 664.76; R = 0.31 (CH Cl /2.5% MeOH/silica); iH-NMR (400 MHz, CDC1 /10% f  2  2  3  acetone-d ) 5 -1.96 (br s, 2H), 3.63 (s, 2H), 6.19 (d, J = 5.1 Hz, 1H), 6.25, d, J = 5.1 Hz, 6  1H), 7.02 (dd, J = 7, 23 Hz, 2H), 7.45-7.60 (m, 12), 7.78 (dd, J = 7, 23 Hz, 2H), 7.9-8.0 (m, 4H), 8.18 (d, J = 4.5 Hz, 1H), 8.23 (d, J = 4.5 Hz, 1H), 8.30 (s, 2H), 8.47 (dd, J = 4.5, 1.2 Hz, 1H); UV-visible (CHCl /10%MeOH) X 3  m a x  410, 508, 526, 596, 642; LR-MS (EI,  thioglycerol) m/e 665 (80, MH+), 647 (8, MH+-H 0); HR-MS (EI, thioglycerol) m/e 2  expected for C44H33N4O3: 665.25527, found 665.25569. (135) :  MW = 664.76; Rf = 0.55 (CH C12/2.5% MeOH/silica); iH-NMR (400 MHz, CDC1 /10% 2  3  acetone-d ) 8 -1.85 (br s, 2H), 3.50 (s, 2H), 7.08 (d, J = 8.5 Hz, 2H), 7.52-7.65 (m, 9H), 7.83 6  (br s, 4H), 7.95-8.08 (m, 4H), 8.20 (d, J = 4.5 Hz, 2H), 8.36 (d, J = 4.5 Hz, 1H), 8.42 (d, J = 4.5 Hz, 1H), 8.51 (d, J = 4.5 Hz, 1H), 8.59 (d, J = 4.5 Hz, 1H); UV-visible (CHCI3/ 10%MeOH) ? i  m a x  410, 508, 526, 596, 642; LR-MS (EI, thioglycerol) m/e 665 (75, MH+),  647 (4, MH+-H 0); HR-MS (EI, thioglycerol) m/e expected: 665.25527; found 665.25681. 2  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  meso-Tetra-(3-hydroxyphenyl)-2,3-vic-dihydroxy-2,3-chlorin  164  (137)  Prepared in 3 3 % yield from [mera-tetra-(3-hydroxyphenyl)porphyrinato]zinc according to the general procedure. The crude reaction mixture was demetallated with 5%TFA/CH Cl2 and then purified by chromatography (silica-5%MeOH/CH Cl ). 2  2  2  MW = 712.76; R = 0.37 (silica- 10%MeOH/CH Cl ); ipI-NMR (400 MHz, DMSO-d ) 8 f  2  2  6  - 1.98 (br s, 2H), 5.17 (br s , 2H), 6.18 (s, 2H), 7.07 (dd, J = 2.2, 7 Hz, 2H), 7.18 (dd, / = 2.2, 7 Hz, 2H), 7.30 (m, 2H), 7.39-7.58 (m, 10H), 8.36 (br s, 2H), 8.43 (s, 2H), 8.69 (d, J = 4.6 Hz, 2H), 9.75 (br s, 4H); UV-visible (MeOH) X  (rel. intensities) 414 (1.0), 516 (0.11),  max  542 (0.10), 592 (0.07), 644 (0.14); LR-MS (EI, thioglycerol/MeOH) m/e 713 (16, MH+); HR-MS (EI, 3-NBA/CHCI3) m/e calc'd for C 4 H 3 N 0 : 713.24001, found: 713.23925. 4  2  4  6  /77eso-Tetra-(3,4,5-trimethoxyphenyl)-2,3-vic-dihydroxy-2,3-chlorin (138) 138 was prepared in 66% yield from meso-tetra(3,4,5-trimethoxyphenyl)porphyrin according to the standard procedure. Chromatography silica-CH Cl /2.5%MeOH. 2  2  MW = 1009.08; mp < 300°C; R = 0.72 (silica-CH Cl /5.0% MeOH); !H-NMR (400 MHz, f  2  2  DMSO-d ) 8-1.98 (s, 2H), 3.81 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 3.90 (s, 3H), 3.92 (s, 6  3H), 3.95 (s, 3H), 5.31 (d, 4.8, 2H), 6.19 (d,7. = 4.8 Hz, 2H), 7.15 (s, 2H), 7.37 (s, 2H), 7.41 (s, 2H), 7.43 (s, 2H), 8.46 (d, J = 4.8 Hz, 2H), 8.49 (s, 2H), 8.77 (d, J = 4.8 Hz, 2H);  1 3  C-  NMR (75 MHz, CDCI3) 8 56.35, 61.26, 74.50, 110.15, 111.7, 112.1, 112.26, 113.20, 123.00, 124.33, 128.19, 132.69, 135.62, 136.15, 137.23, 138.10, 141.00, 141.09, 151.50, 152.34, 152.55, 153.20, 161.80; UV-visible (CH Cl /0.5% MeOH) ? i 2  2  max  420, 520, 548, 594, 646;  LR-MS (FAB, 3-NBA) m/e 1009 (60, M+), 991 (10, M+ - H 0), 975 (5, M+- 20H); HR2  MS (FAB, 3-NBA) m/e calc'd for C56H N Oi4: 1008.37922, found 1008.38109. 56  4  5,10-Diphenyl-2,3-vic-dihydroxy-2,3-chlorin (139) 139 was prepared in 45% yield from 5,10-diphenylporphyrin (49 mg in 10 mL solvent) according to the general procedure. However, the reaction time was considerably  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  165  shortened (5h) and the mixture was separated on a preparative T L C (silica1.5%MeOH/CH Cl , double development). 2  2  MW = 496.57; R = 0.28 (silica-CH Cl /1.5%MeOH); H-NMR (400 MHz, CDC1 / 1  f  2  2  3  10%acetone-d ) 8 -2.23 (br s, 1H), -1.88 (br s, 1H), 5.99 (d, 7= 6.5 Hz, 1H), 6.34 (d, J = 6.5 6  Hz, 1H), 7.62-7.75 (m, 6H), 7.91 (d, J = 5.2 Hz, 1H), 8.09 (m, 2H), 8.18 (m, 1H), 8.42 (d, J = 4.5 Hz, 1H), 8.64 (d, J = 4.5 Hz, 1H), 8.83 (d, / = 4.5 Hz, 1H), 8.92 (d, / = 4.5 Hz, 1H), 8.97 (d, J = 4.5 Hz, 1H), 9.11 (d, J = 4.5 Hz, 1H), 9.90 (s, 1H), 9.34 (s, 1H); UV-visible (CHCl /5%MeOH) X 3  m a x  (rel. intensities) 398 (1.0), 502 (0.10), 530 (0.06), 584 (0.04), 636  (0.19); LR-MS (EI, 3-NBA) m/e 496 (79, M+), 478 (100, M+-H 0); HR-MS (EI, 3-NBA) 2  m/e calc'd for C H N 0 : 496.18993, found 496.18979. 3 2  2 4  4  2  meso-Tetraphenyl-2,3-w'c-dihydroxy-2,3,12,13-bacteriochlorin (141) Two different methods are available for the synthesis of 141. Method 1: The compound was prepared in 53% yield fromraeso-tetraphenylchlorin(50 mg, 8.1 x 10" mol) according to the general osmium tetroxide mediated dihydroxylation 5  procedure. Method 2: meso-Tetraphenylchlorin (100 mg, 1.62 x 10~ mol) was dissolved in a 4  suspension of 200 mg dry Na C03 in dry pyridine (20 mL). The mixture was kept under N 2  2  and refluxed. p-Toluenesulfonylhydrazide (150 mg, 5 equivalents) were added in portions over a period of 4 h. Reflux was continued for another 2 h. The mixture was cooled, filtered, the filtrate evaporated to dryness and the residue was seperated on a preparative TLC plate.  The pink band of 141 separating from the brown band of 129 (silica,  1.5%MeOH/CH Cl ) was isolated to give, after recrystallization from CH C1 /CC1 , 141 in 2  2  2  2  4  12 % yield. MW = 650.78; R = 0.78 (silica gel-2.5% MeOH/CH Cl ); f  2  2  1  H-NMR (400 MHz, CDCI3)  8 -1.58 (s, 2H), 3.00 (s, 2H), 3.94-4.21 (m, 4H), 6.13 (s, 2H), 7.58-7.73 (m, 12H), 7.79 (br tr, 7 = 6.8 Hz, 4H), 7.86 (br d, 7 = 4.4 Hz, 2H), 7.97 (dd, J = 4.8, 2 Hz, 2H), 8.13 (2 overlapping  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  166  d, J = 4.5Hz Hz, 4H); UV-Vis (CH C1 ) X x (log e): 378 (4.96), 524 (4.49), 724 (4.71) nm; 2  2  ma  LR-MS (+ FAB, 3-NBA) m/e 650 (100, M+), 633 (19.2, M+-OH); HR-MS (+ FAB, 3-NBA) m/e calc'd for C 4H34N 0 : 650.26818, found 650.27118; 4  4  2  (mes<>Tetraphenyl-2,3-wc-dihydroxy-2,3,7,8-isobacteriochlorinato)zinc(ll) (143) and /Deso-Tetraphenyl-2,3-w'c-dihydroxy-2,3,7,8-isobacteriochlorin (144) Prepared in invariably low yields (< 10%) from 142 following either of the two methods described for 141. The main product appeared to be 130. In no case, however, was the formation of a bacteriochlorin or metallobacteriochlorin chromophore, respectively, observed. The green pigment 143 {HR-MS (FAB, 3-NBA) calc'd m/e for C H 3 N 0 Z n : 44  2  4  2  712.18167; found 712.17630} was directly demetallated by 5% TFA/CH C1 to produce the 2  2  fuchsia-colored 144. 144:  MW = 650.78; R = 0.28 (silica-CH Cl /0.5% MeOH); !H-NMR (400 MHz, CDCl /4% f  2  2  3  MeOH-d ) 5 3.2-3.5 (m, 4H), 5.28 (d, / = 6.5 Hz, 1H), 5.34 (d, J = 6.5 Hz), 7.38 (d, J = 4.5 4  Hz), 7.4-7.6 (m, 18H), 7.6-7.75 (m, 2H), 7.7-7.8 (m, 2H); UV-Vis (CH C1 ) ? i 2  2  max  (log e):  394 (5.02), 480 (sh), 514 (4.03), 546 (4.19), 590 (4.20) nm; LR-MS (+FAB, thioglycerol/ CHC1 ) m/e 651 (38, MH+), 633 (MH+-H 0); HR-MS (+FAB, thioglycerol/CHCl ) m/e 3  2  3  calc'd for C 4H34N 0 : 650.26818, found 650.26675. 4  4  2  meso-Tetraphenyl-2,3-12,13-di- Wc-dihydroxy-2,3,12,13-bacteriochlorin ( 1 4 5 ) and meso-Tetraphenyl-2,3-12,13-frans-di-v'/c-dihydroxy-2,3,12,13-bacteriochlorin (146) 129 (100 mg, 1.54 x 10" mol) was dissolved in a minimal amount of CHC1 4  3  containing 10 % pyridine (ca. 4 mL). O S O 4 (51 mg, 1.3 equivalents) was added and the stoppered solution was stirred at r.t. until the chlorin peak at 644 nm was largely replaced by the bacteriochlorin peak at 708 nm (16 h). The reaction was quenched by bubbling gaseous H S through it. After filtering of the solution (Celite®) and evaporation of the filtrate, the 2  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  mixture was separated on a preparative TLC plate (silica gel, 2 mm, 5 % MeOH in  167  CH2CI2  as eluent, two developments). Yield 19 % and 20% for 145 and 146, respectively. (145) : MW = 682.78; mp = d > 150°C; R = 0.51 (silica gel-CH Cl /5.0 % MeOH); iH-NMR (300 f  2  2  MHz, DMSO-d ) 8 = -1.65 (s, 2H), 4.99. d (J = 4.9 Hz, 4H), 5.87 (d, J = 4.9 Hz Hz, 4H), 7.6 6  (br m, 12H), 7.86 (br s, 4H), 7.96 (overlapping s and broad s, 8H); C - N M R (75 MHz, 13  DMSO-d ) 8 = 73.11, 115.63, 122.88, 127.10, 131.54, 133.85, 136.22, 141.22, 160.07; UV6  Vis (CH C1 ) X 2  2  m a x  = 376 (5.42), 528 (5.08), 708 (4.89) nm; LR-MS (+ FAB, 3-NBA) m/e  682 (100, M+), 665 (31.1, M+ - OH), 648 (5.8, M+ - 20H), 613 (6.4, M+ - 40H - H); HRMS (+ FAB, 3-NBA) m/e calc'd for C44H34N4O4: 682.25801, found 682.25470. (146) : MW = 682.78; mp = d > 150°C; R = 0.30 (silica gel-CH Cl /5.0 % MeOH); !H-NMR (400 f  2  2  MHz, DMSO-d ) 8 -1.75 (s, 2H), 5.05 (br s, 4H), 5.95 (s, 4H), 7.65 (br s, 12H), 7.93 (br s, 6  8H), 8.09 (s, 4H); UV-Vis (CH C1 ) X 2  2  max  (log e): 376 (5.40), 528 (5.01), 708 (4.86) nm;  LR-MS (+ FAB, 3-NBA) m/e 682 (19.4, M+), 665 (7.4, M+ - OH), 649 (9.4), 648 (7.5, M+ 20H), 613 (1.5, M+ - 40H - H).; HR-MS (+FAB, 3-NBA) m/e calc'd for C44H34N4O4: 682.25797, found 682.25518.  (meso-Tetraphenyl-2,3,7,8-c/'s-di-w'c-dihydroxy-2,3,7,8-isobacteriochlorinato)zinc(ll) (147)  and  (/77eso-Tetraphenyl-2,3,7,8-frans-di-v/c-dihydroxy-2,3,7,8-isobacterio-  chlorinato)zinc(ll) (148) Prepared in low yields from 130 following the general dihydroxylation procedure. TPPZn treated with 3-4 equivalents of O S O 4 also produces these compounds and they are found, albeit in very small amounts, in the polar material left on the chromatography column used in the preparation of 130. Their purification was always performed by preparative TLC plate chromatography.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  168  147: MW = 746.12; R = 0.21 (silica-CH2Cl2/10%MeOH); LR-MS (+ FAB, 3-NBA/CHCl ) m/e f  3  744 (40, M+), 726 (11, M+ - H 0 ) ; HR-MS (+ FAB, 3-NBA) m/e calc'd for 2  C 4 H 3 4 N 0 4 Z n : 744.17150, found 744.17210. 64  4  4  148: MW = 746.12; R = 0.33 (silica-CH2Cl2/10%MeOH); strongly fluorescent purple-green f  color in solution; !H-NMR (400 MHz, CDCl /20%acetone-d ) 8 = -2.75 (brs, 4H), 5.68 d (7 3  6  = 8,8 Hz, 4H), 7.37 (d, J = 4.5 Hz, 2H), 7.48-7.57 (m, 12H), 7.76 (m, 4H), 7.82 (d, J = 4.5 Hz, 2H), 7.83-7.89 (m, 4H); UV-Vis (CH Cl /0.5%MeOH) ?i ax (rel. intensities) 394 (sh), 2  2  m  418 (1.0), 512 (0.07), 558 (0.12), 596 (0.27), 638 (0.04) nm; LR-MS (+ FAB, 3-NBA/CHCI3) m/e 744 (45, M+), 727 (13, M+ - OH); HR-MS (+ FAB, 3-NBA) m/e calc'd f o r C H 3 4 N 0 4 Z n : 744.17150, found 744.17176. 64  44  4  mesc^Tetraphenyl-2,3,7,8-frans-di-w'c-dihydroxy-2,3,7,8-isobacteriochlorin (149) Prepared by demetallation of 148 in 5%TFA/CH2C12 MW = 682.78; R = 0.71 (silica-CH Cl /5%Me0H); fuchsia color in solution; H-NMR !  f  2  2  (400 MHz, CDCI3) 8 = 2.55 (br s, 2H), 2.70 (br s, 2H), 4.04 (br s, 2H), 5.49 (d J = 1 Hz, 2H), 5.58 (d, J= 1 Hz, 2H), 7.22 (d, 7 = 4.5 Hz, 2H), 7.50-7.60 (m, 14H), 7.68-7.78 (m, 6H), 7.82 (dd, J = 4.5,2 Hz, 2H); UV-Vis (CH Cl /0.5%MeOH) X 2  2  max  (log e) 398 (5.17), 514  (4.12), 548 (4.29), 586 (4.20), 638 (3.56) nm; LR-MS (+ FAB, thioglycerol/CHCl ) m/e 683 3  (70, MH+), 665 (12, M+ - H 0); HR-MS (+ FAB, 3-NBA) m/e calc'd for C44H35N4O4 2  683.26583, found 683.26604.  Part 1:  3.2  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  169  REACTIONS OF THE MESO-TETRAPHENYL- WODIOLCHLORINS  (A7?eso-Tetraphenyl-Wc-3,4-di-0-isopropylidene-3,4-chlorinato)zinc(ll) (150) 130 (20 mg, 2.81 x 10~ mol) dissolved in 20 mL dry acetone containing freshly 5  fused ZnCl (100 mg) was refluxed for 20 min under anhydrous conditions. The reaction 2  mixture was evaporated to dryness. The resulting residue was separated on a preparative TLC plate (silica gel-CH2Cl2/20%CCl ). The main bright green band was isolated after one 4  development, to give 150 in 60% yield. A small amount (-10% yield) of 152 is also formed during this reaction. MW = 752.19; Rf = 0.31(silica gel-CH Cl /20%CCl ); !H-NMR (300 MHz, CDCI3) 5 0.61 2  2  4  (s, 3H), 1.37 (s, 3H), 6.46 (s, 2H), 7.55-7.76 (m, 12H), 8.05 (dd, J = 8.0, 2.1 Hz, 4H), 8.12 (m, 4H), 8.16 (d, / = 4.5 Hz, 4H), 8.41 (s, 2H), 8.53 (d, 7 = 4.5, 2H); UV-visible (CH C1 ) 2  2  418, 520, 564, 594 (sh), 612; LR-MS (+ FAB, 3-NBA) m/e 750 (11, M+), 693 (23, MH+ C H 0 ) ; HR-MS (+ FAB, 3-NBA) m/e expected for C 7H34N 02 Zn: 750.19732, found 64  3  6  4  4  750.19422.  meso-Tetraphenyl-2-hydroxy-porphyrin (151) and (meso-tetraphenyl-2-hydroxyporphyrinato)zinc(ll) (152) 129 or 130 (0.154 mmol) was dissolved in benzene (10 mL) and refluxed. Three drops of HC10 (70% aqueous solution) were added and the reflux was continued for 3 min. 4  The bright green solution was then washed with dilute aqueous ammonia, dried over anhydrous Na2C03 and evaporated to dryness. The resulting solid was chromatographed on a short column (CHCl3/silica gel). The main purple fraction was precipitated by solvent exchange with hexanes. Drying (50°C/high vacuum) of the microcrystalline material produces in 85% yield analytically pure 151. The material was identical to that described by  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  170  152 is produced from 151 in quantitative yields by standard metallation  Crossley et al.  352  with Zn(II)acetate in hot CHCl /MeOH. 3  (/77eso-Tetraphenyl-2,3-dioxochlorinato)Zn(ll) (157) A benzene solution of DDQ (66 mg, 2.94 x 1(H mol in 5 mL) was added to 130 (100 mg, 1.40 x 10~ mol) dissolved in benzene (10 mL). Stirring at r.t. converted the green 4  starting material quantitatively into the yellow product 157. Short column chromatography (silica, benzene) separated the excess reagent from the product. Evaporation of the solvent produces 157 as a brown-blue solid in high yield. MW = 708.10; R = 0.8 (silica-CH Cl /CCl 1:1); *H-NMR (300 MHz, CDC1 ) d 7.6-7.75 f  2  2  4  3  (m, 12H), 7.77 (dd, 7=8,2 Hz, 4H), 8.25 (dd, 7 = 8,2 Hz, 4H), 8.32 (d, J = 4.5Hz, 2H), 8.45 (s, 2H), 8.53 (d, J = 4.5 Hz, 2H); UV-visible (CH C1 ) ? i 2  2  max  (rel. intensities) 416 (1.0), 496  (0.13), very broad, featureless band centred -650 nm; LR-MS (EI, 350°C) m/e 708 (85, M+), 692(100,M+-O).  meso-Tetraphenyl-2-oxa-3-oxo-chlorinato)zinc(ll) ( 1 6 3 ) and mesotetraphenyl-2oxa-3-oxo-chlorin (164) 163 is isolated during chromatography of the crude reaction mixture resulting from the treatment of TPPZn with OsOVpyridine, followed by reduction with H S. It is the band 2  following the separation of the starting material. Slow diffusion of petroleum ether 40-60 into a CHCI3 solution (green) containing a trace of pyridine of 164 results in the formation of purple X-ray quality crystals. 164 is prepared by acid treatment (10% TFA/CH C1 ) of 163. 2  2  Washing of the acidic solution with dilute aqueous NH3, drying of the isolated organic phase over anhydrous Na CC>3 and recrystallization by slow solvent exchange to hexane produces 2  164 as dark purple powder.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  171  163: MW = 696.09; mp = d > 200°C; R = 0.82 (silica-ethyl acetate/hexane 3:1); ipI-NMR (300 f  MHz, CDCI3) 5 7.65-7.77 (m, 12H), 7.93 (dd, J = 7.8, 2 Hz, 2H), 8.05 (dd, J = 7.8, 2 Hz, 2H), 8.10 (dd, J = 7.8, 2 Hz, 4H), 8.51 (d, J = 4.5 Hz, 1H), 8.58 (d, J = 4.5 Hz, 1H), 8.65, 8.67, 8.69 (three overlapping d, J = 4.5 Hz, 1H), 8.75 (d, J = 4.5 Hz, 1H); C - N M R (75 13  MHz, CDCI3) 5 173.5, 154.1, 143.4, 142.1, 142.0, 139.0, 138.0, 134.3, 134.0, 133.8, 132.4, 132.3, 132.2, 132.0, 130.9, 130.7, 130.6, 129.5, 128.8, 127.9, 127.8, 127.7, 127.6, 127.5, 126.7, 126.6; UV-visible (CH C1 ) X 2  2  m a x  (log e) 402 (sh), 422 (5.53), 520 (3.54), 558 (4.07),  602 (4.44); LR-MS (EI, 250°C) m/e 694 (30.9, M+), 638 (7.3), 561 (17.4), 483 (3.5), 28 (100); HR-MS (EI, 200°C) m/e calc'd for C 4 3 H N 0 2 6  4  6 4 2  Z n : 694.1347, found 695.1355.  See below for the X-ray crystal structure data. 164: MW = 632.72; R = 0.50 (silica-CCl /CH Cl 1:1); *H-NMR (300 MHz, CDCI3) 6 = -2.05 f  4  2  2  (s, 1H), -1.82 (s, 1H), 7.66-7.78 (m, 12), 7.97 (m, 2H), 8.00-8.16 (m, 6H), 8.52 (d, 7 = 4.5 Hz, 1H), 8.57 (dd, J = 4.5, 15 Hz, 1H) overlapping with 8.59 (d, J = 4.5 Hz, 1H), 8.69 (d, J = 4.5, 1.5 Hz, 1H), 8.78 (two overlapping dd, J = 4.5, 1.5 Hz, 2H); UV-visible (CHCI3) ? w c (log e) 420 (5.56), 522 (4.15), 558 (4.16), 588 (3.95), 640 (3.66); UV-visible (TFA/CHCI3) V a x 430, 588 (sh), 614; LR-MS (FAB, 3-NBA) m/e 633; HR-MS (FAB, 3-NBA) m/e calc'd for C 3 H N 0 : 632.2212, found 632.2168. 4  2 8  4  2  (/77eso-Tetraphenyl-2,3-secochlorinato-2,3-dialdehyde)Ni(ll) (167) 131 (170 mg, 9.94 x 10" mol) dissolved in dry THF (10 mL) were treated with 5  Pb(IV)(acetate)4 (120 mg, moist). Within 10 min at r.t. the solution turned from green to dark yellow. After no starting material was detectable by TLC (20 min), the solution was then evaporated to dryness in vacuo and the resulting residue was purified by flash chromatography (silica, 3x7 cm, CHCI3).  The first yellow fraction was collected and  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  evaporated to dryness, dissolved in  CH2CI2  172  and solvent exchange to hexane gave 167 in  80% yield as a dark purple-brown powder of analytical purity. MW = 703.61; mp > 250°C; Rf = 0.88 (silica-CHCl , yellow-brown spot which turns forest3  green within a few minutes); iff-NMR (400 MHz, DMSO-d ) 8 7.55-7.60 (m, 12H), 7.706  7.75 (m, 8H), 7.85 (d, J = 4.8 Hz), 7.98 (s, 2H), 8.18 (d, J = 4.8 Hz), 9.50 (s, 2H); the lHNMR in C D C I 3 exhibit additional broad signals at 7.1 (s, 2H), 7.8 (s, 2-4H) and 8.8 (s, 1-2H) which have not found any explanation yet;  13  C - N M R (50 MHz, C D C I 3 ) 8 120.0, 126.7,  127.4, 127.8, 128.2, 128.6, 130.8, 131.4, 132.9, 133.4, 134.0, 134.6, 135.7, 138.8, 140.8, 144.1, 146.9, 188.7; IR (film): v = 1684 cm" (C=0); UV/Vis (CH C1 ) X 1  2  2  max  (log e) 312  (4.55), 344 (sh), 414 (4.63), 466 (4.78), 686 nm (4.04); LR-MS (EI, 280°C) m/e 702 (4.0, M+), 700 (3.5, M+-2H), 686 (33.6, M+-O), 684 (8.1, M+-H 0), 673 (100, M+-CHO); HR2  MS (FAB, 3-NBA/CHCI3) m/e calc'd for C 4 H N 4 N i 0 : 703.16440, found 703.16394; 58  4  29  2  Analysis calc'd for C H N 4 N i 0 : C, 75.13; H, 4.01; N, 7.96; found: C, 75.04; H, 4.34; N, 44  28  2  7.34.  Pigment 172 and pigment 173 167 (50 mg, 7.11 x 10  -5  mol) dissolved in C H C I 3 (2 mL, pentene stabilized)  containing 2% MeOH were fumed carefully with some head space from a cone. HCI bottle applied through a pipette. 172 and 173 appear immediately. The mixture was separated on a preparative TLC plate (silica, CHCI3).  Depending on the intensity of the acid treatment,  variable yields of the two green products were produced. Other than the two pigments 172 and 173, no other pigments or decomposition can be observed. 172 is labile. 172:  MW = 733.45; R = 0.45 (silica-CHCl ); M.p. > 150 °C (d); H-NMR (400 MHz, CDCI3) 1  f  3  8 2.45 (d, 7 = 7.6 Hz, 1 H), 3.12 (s, 3H), 6.12 (s, 1 H), 6.47 (d, / = 7.6 Hz, 1 H), 7.50-7.65 3  3  (m, 20 H), 7.78 (d, J = 4.6 Hz, 1 H), 7.84 (d, 7 = 4.6 Hz, 1 H), 8.06 (two overlapping d, 3  3  second order, 2 H), 8.22 (dd, J = 4.6, 1.2 Hz, 2 H); C - N M R (125 MHz, DMSO-d ) 3  13  6  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  173  5 54.1, 89.2, 96.7, 110.5, 111.1, 125.3, 125.4, 127.4, 127.7, 127.8, 127.9, 128.0, 128.1, 128.8, 128.8, 129.0, 132.4, 133.1, 137.7, 137.8, 137.9, 138.5, 138.7, 140.8, 141.3, 141.4, 145.5, 145.6; UV/Vis (CH C1 ) A, ax (log e) 430 (4.82), 640 (4.10); LR-MS (EI, 32  2  m  NBA/CHCI3) m/e 734 (20, MH+); HR-MS (EI, 3-NBA/CHCl ) m/e calc'd for 3  C45H 2N4 Ni0 : 734.18279; found 734.18314. 58  3  3  173:  MW = 749.49; R = 0.80 (silica-CHCl ); M.p. > 200 (d); 'H-NMR (400 MHz, CDC1 ): f  3  3  5 3.03 (s, 6 H), 6.08 (s, 2 H), 7.5-7.65 (m, 20 H), 7.81 (d, J = 4.5 Hz, 2 H), 8.06 (s, 2 H), 3  8.22 (d, J = 4.5 Hz, 2 H); C-NMR (50 MHz, CDC1 ): 6 54.6,97.4, 111.9, 122.6, 124.4, 3  13  3  125.9, 126.0, 126.2, 127.1, 127.6, 127.7, 127.8, 128.9, 129.2, 132.8, 132.9, 133.3, 13.8.7, 138.8, 139.0, 139.7, 142.4, 146.5; UV/Vis (CH C1 ) l 2  2  max  (log e) 430 (4.81), 640 (4.09); MS  (EI, 3-NBA/CHCl ) m/e 748 (18, MH+), 717 (8, M+-OCH ); HR-MS (EI, 3-NBA/CHCl ) 3  m/z calc'd for C  3  4 6  H  3 4  N  5 8 4  3  N i 0 : 748.19844; found 748.19938; Analysis calc'd for 3  C H N N i 0 : C, 73.73; H, 4.57; N, 7.48; found: C, 73.81; H, 4.63; N, 7.52. 4 6  3 4  4  3  Pigment 175 Prepared analogous to 173 from 167 in CHCl /MeOH/EtOH.  Its UV-visible  3  spectrum is identical to that of 173. The three components present cannot be separated by TLC. Their presence is indicated by the presence of three strong molecular ion peaks at m/e 748 (corresponding to C 6 H N 4  (corresponding to C H N 4 8  3 8  34  5 8 4  58 4  N i 0 ) , 762 (corresponding to C H 6 N 3  47  3  58 4  N i 0 ) , and 776 3  N i 0 ) . The iH-NMR (400 MHz, CDCI3) and C-NMR (75 13  3  MHz, CDC1 ) have a complex fine structure. Compared to the spectrum of 173, additional 3  signals at 5 0.8 (two overlaying t) and at 3.5 (m) and at 14.8 and 62.4 are attributed to the presence of the ethyl groups. determination.  See below for details of the X-ray crystal structure  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  174  THE DIRECTED SYNTHESIS OF N-CONFUSED TPP  3.3  5-Phenyldipyrrane (179) and 5,10-diphenyltripyrrane (180) Method A (adaption from Lindsey and co-worker)  367  Benzaldehyde (6.0 mL, 59 mmol) was mixed with pyrrole (150 mL, 2.16 mol) and the mixture was deoxygenated by bubbling dry N2 through it for 15 min. With the mixture still under nitrogen, TFA (0.45 mL, 5.8 mmol) was added and the mixture was stirred for 15 min at ambient temperature. After this time, the mixture was evaporated under vacuum (5 torr) and slight heating on a rotary evaporator yielded a dark oil. The oil was taken up in a minimal amount of CH2CI2, and loaded onto a flash chromatography column (silica gel, 5.5 x 30 cm, CH2CI2). The colorless, dipyrrane 3 and tripyrrane 4, and some minute amounts of unidentified material (< 1 %) containing fractions (TLC control, upon Br2-fuming of the TLC spots, 179 turns bright orange, 180 turns beige) were collected and evaporated on a rotary evaporator to yield a tan oil. This oil was transferred into a sublimation apparatus and subjected to high vacuum (0.1 torr). A slow heating rate (~ 0.75°C/min) was maintained until visible sublimation set in at 130°C. After further sublimation had ceased, the white crystalline sublimate, consisting of crystalline 179 (7.20 g, 55 % yield), and the orange, glassy sublimation residue (2.45 g, 11 % yield) consisting mainly (> 95 %, based on H [  NMR and analysis of the residue) of 180 were collected.  Method B (adaptation from Carell  371  ):  This method is, in principal, useful for the preparation of tripyrranes which are not made from unsubstituted pyrroles (as the pyrroles are here not needed as solvent). Benzaldehyde (12 mL, 0.118 mol) and pyrrole (52 mL, 0.745 mol) are dissolved in toluene (750 mL). A catalytic amount of /?-toluenesulfonic acid monohydrate (100 mg, 4.9 x 10" mol) was added and the mixture was refluxed under N 2 for 1 h. After this time, the 4  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  175  mixture was evaporated on a rotary evaporator to produce a brown oil. This oil was subjected to the same treatment (chromatography, sublimation) as described in Method A to give 179 (44 % yield) and 180 (9.8 %). 179:  Analytical and spectroscopic properties identical to those described in the literature.  367  R = 0.78 (silica-CH Cl /CCl f  2  2  4  1:1).  180:  MW = 377.49; mp = 75-80°C, Rf = 0.63 (silica CH C1 :CC1 1:1); !H-NMR (200 MHz, 2  2  4  CD C1 ) 5 5.35 (s, 2H), 57.8 (d, J = 4 Hz, 2H), 5.89 (s, 2H), 6.14 (m, 2H), 6.66 (m, 2H), 2  2  7.15-7.38 (m, 10H), 7.75 (br s, 1H), 7.88 (br s, 2H); H-NMR (50 MHz, CDC1 ) 5 44.1, 13  3  107.2, 107.4, 108.4, 117.2, 127.0, 128.4, 128.6, 132.3, 132.5, 142.1; LR-MS (EI, 150°C) m/e 311 (45, M+), 221 (18), 156 (14), 145 (23), 80 (20), 67 (100); HR-MS (EI, 200°C), expected f o r C H N : 377.1892, found 377.1881; Analysis calc'd for C H N : C, 82.73; H, 6.14; 26  23  3  2 6  2 3  3  N, 11.13; found 82.16, 6.03, 10.72.  1 -Phenylcarbonyl-5-phenyldipyrromethane (181) Sublimed 179 (533 mg; 2.40 mmol) was dissolved under anhydrous conditions in dry THF (20 mL). Ethylmagnesium bromide (1.80 mL 3M solution in Et 0; 2.2 equiv.) was 2  syringed in, and the mixture was stirred for 15 min at r.t., then refluxed for 10 min and finally cooled to 0°C. The reaction mixture was taken up by a syringe and freshly distilled benzoylchloride (330 u,l; 1.15 equiv.) in dry THF (10 mL) was added to the (now empty) flask. The dipyrromethane-Grignard solution was, under ice cooling, slowly syringed back into the flask. The mixture was stirred at r.t. for lh and refluxing for an additional 20 min., TLC analysis indicated the disappearance of the starting material. The reaction mixture was quenched with MeOH and subsequently diluted with water (50 mL). The products were extracted with CHC1 (3x50 mL) and the combined organic layers were dried over MgS0 3  4  and evaporated under vacuum to give a yellow oil. Flash column chromatography (silica, 20  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  x 3 cm,  CH2CI2) separated  176  the title compound from traces of starting material and higher  polarity materials (containing also traces of 183). Evaporation of the solvent in vacuo afforded 181 as slightly yellow oil which solidified (540 mg, 69 %). The procedure is amenable to a 5-fold scale-up. For an alternative synthesis, see preparation for 182. MW = 326.40; mp = 72-77°C; Rf = 0.65 (silica-CH Cl /5.0% MeOH); !H-NMR (400 MHz, 2  2  CDCI3) 8 5.65 (s, 1H), 6.03 (s, 1H), 6.17 (s, 2H), 6.61 (m, 1H), 6.86 (m, 1H), 7.18-7.33 (m, 5H), 7.44-7.64 (m, 3H), 7.82 (d, 2H), 8.75 (s, 1H), 10.81 (s, 1H); C - N M R (75 MHz, 13  CDCI3) 8 44.17, 107.78, 108.26, 110.88, 117.90, 121.66, 127.14, 128.33, 128.38, 128.64, 129.12, 130.78, 131.23, 131.80, 138.50, 141.06, 142.68, 184.99; LR-MS (EI, 200°C) m/e 326 (100, M+), 249 (25.4), 221 (71.1), 156 (21.3), 105 (76.2); HR-MS (EI, 150°C) m/e calc'd for C22H18N2O: 326.14191, found 326.14194.  1,9-Diphenylcarbonyl-5-phenyldipyrromethane  (183)  181 (880 mg, 2.69 mmol) dissolved under anhydrous conditions in dry THF (20 ml) was treated with ethylmagnesium bromide (2.25 mL 3M solution in Et20; 2.5 equiv.). The rate of addition was such that a slow reflux was maintained. The mixture was refluxed for an additional 40 min after the completion of the addition. The solution turned red during this time. Benzoylchloride (0.93 mL, 3 equiv.) was added to the cooled solution by syringe at a slow rate. The mixture was then refluxed for an additional 1.5 h. The tan solution was then poured into aqueous ammonium acetate and stirred for 1 h. Extraction with CHCI3 (4x50 mL), drying of the organic phase over Na2C03 and evaporation of the solvent in vacuo provided crude 183. Purification by flash chromatography (silica, 20x3.5, CH2Cl2/0.5% MeOH) and evaporation of the appropriate fractions provided a tan oil which solidified to produce 0.92 g (79% yield) of 182. MW = 430.29; no sharp mp; Rf = 0.33 (silica-CH2Cl2/5.0% MeOH, spot stains yellow with Br ); !H-NMR (400 MHz, CDCI3) 5 6.05 (m, 2H), 6.40 (m, 2H), 7.30-7.55 (m, 11H), 7.82 2  (dd, J = 7.5, 2 Hz, 4H), 11.2 (br s, 2H); C-NMR (75 MHz, CDCI3) 8 44.9, 11.2, 120.7, 13  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  177  127.6, 128.1, 128.7, 129.0, 129.4, 131.0, 131.7, 138.2, 140.0, 140.5, 184.5; LR-MS (EI, 250°C) m/e 430 (57.0, M+), 325 (48.9, M+-PhCO), 105 (100, PhCO+); HR-MS (EI, 200°C) m/e calc'd for C29H22N2O2: 430.1861, found 430.1674; Analysis calc'd for C29H22N2O2: C, 80.91; H, 5.15; N, 6.51; found: C, 80.80; H, 5.13; N, 6.23.  1,10-Diphenylcarbonyl-5-phenyldipyrromethane (183) 179 (100 mg, 5.41 x 10~ mol) dissolved in 1,2-dichloroethane (5 mL) was added 4  dropwise under anhydrous conditions into a freshly prepared mixture of AICI3 (133 mg, 2.2 equiv.) and benzoyl chloride (0.11 mL, 9.0 x 10~ mol) in 10 mL of 1,2-dichloroethane. 4  After being stirred for 15 h at r.t., the mixture was quenched by pouring it into aqueous Naacetate. Extraction with CHCI3 (3 x 25 mL), drying over Na2C03 and evaporation of the solvent, produced a solid which was chromatographed (silica preparative TLC, 1.5%MeOH/ CH C1 ) to give 181 (75 mg, 42 % yield) and 183 (46 mg, 19 % yield). 2  2  MW = 430.29; Rf = 0.39 (silica, 2.5%MeOH/CH Cl ); !H-NMR (200 MHz, CDCI3) 8 5.42 2  2  (s, 1H), 5.91 (m, 1H), 6.13 (q, J = 10Hz), 6.43 (m, 1H), 7.15-7.35 (m, 7H), 7.35-7.50 (m, 4H), 7.75 (dd, 7= 13, 3 Hz, 2H), 8.10 (dd, 7= 13, 3 Hz, 2H), 8.59 (br s, 1H); C-NMR (50 13  MHz, CDC1 ) 5 43.8, 108.5, 108.9, 117.8, 124.8, 125.6, 127.3, 128.2, 128.4, 128.9, 130.2, 3  131.4, 133.7, 134.7, 139.8, 141.0, 172.0, 192.3; LR-MS (EI, 180°C) m/e 430 (<1, M+), 326 (82, M+-PhCO), 249 (24), 221 (M+-2PhCO), 105 (100, PhCO+).  Phenyl-2-pyrrolyl-3-pyrrolymethane (190) Method A (detosylation of 196): To a solution of 196 (500 mg, 1.33 mmol) in dioxane (20 mL) was added dropwise concentrated (viscous) aqueous KOH until the resulting mixture started to separate into two layers. The solution was then stirred for 12h at r.t. The reaction was then diluted with H2O (100 mL) and extracted with Et20 (4x20 mL). Evaporation of the ether produced 190 (60 mg, 87 % yield) as an oil, which, as judged by NMR, was pure.  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  178  Method B (condensation of 195 with pyrrole): 191 (620 mg, 3.62 mmol) dissolved in dry THF (5 mL) was, under anhydrous conditions and cooled by an ice-bath, dropped into a suspension of L A H (150 mg, 1 equiv.) in dry THF (30 mL). After the addition is completed, Glauber's salt was (0.5 g) slowly added to the mixture. The resulting sludge was filtered through Celite® and the filter cake thoroughly extracted with THF. The combined filtrates were evaporated to dryness in vacuo and immediately used in the next step. TLC analysis (silica, 5%MeOH/CH2Cl2) of the crude mixture exhibits 195 as a spot with low Rf (Rf = 0.1), which stains brown with Br2 fumes, possibly some starting material (Rf = 0.3) and possibly some low polarity material (Rf = 0.95) which stains bright red with Br2. This proved to be 3-benzylpyrrole which resulted from an over-reduction of 191 and which is hard to separate from the final product 190. 195:  MW = 173.21; iH-NMR (200 MHz, CDC1 ) 8 2.45 (br s, 1H), 5.81 (s, 1H), 6.16 (m, 1H), 3  6.55 (m, 1H), 6.68 (m, 1H), 7.20-7.55 (m, 5H), 8.4 (br s, 1H); C-NMR (50 MHz, CDCI3) 5 13  71.0, 107.2, 116.1, 118.5, 126.4, 127.2, 128.2, 128.3, 144.6. 195 (crude mixture from the reduction of 620 mg, 3.62 mmol of 191) was dissolved in toluene (25 mL) containing 1 drop of acetic anhydride, pyrrole (0.5 mL) and a catalytic amount of p-toluenesulfonic acid-4 H2O. The mixture was refluxed under N2 for 10 min. No starting material could be detected (TLC) after this time. The mixture was evaporated in vacuo to dryness (the facilitate the chromatographic purification, it is important to remove all of the pyrrole) and the resulting oil was purified by flash chromatography (silica, CHCI3). The first main fraction was collected and evaporated to produce 190 in 52% yield. 190:  MW = 222.29; Rf = 0.76 (silica-CH2Cl2), stains brown upon treatment with Br ; *H-NMR 2  (200 MHz, CDCI3) 8 5.38 (s, 1H), 5.91 (m, 1H), 6.15 (q, J = 7 Hz, 1H), 6.43 (m, 1H), 6.66 (m, 1H), 6.77 (q, J = 6 Hz, 1H), 7.22-7.35 (m, 5H), 8.00 (br s, 2H); C - N M R (50 MHz, 13  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  179  CDCI3) 8 43.5, 106.7, 108.3, 108.8, 116.7, 118.3, 125.6, 126.5, 128.5, 128.6, 128.9, 135.2, 144.7; LR-MS (EI, 180°C) m/e 222 (100, M+), 156 (20.1, M+-pyrrolyl), 154 (20.1, M+pyrrolyl-2H), 145 (65.4, M+-phenyl); HR-MS (EI, 150°C) m/e calc'd for C15H14N2: 222.11569; found 222.11571.  3-(phenylhydroxymethyl)-1-tosyl-pyrrole (194) 193 (100 mg, 3.08 x 10" mol) dissolved in dry THF (10 mL) was treated under 4  anhydrous conditions with L A H (58 mg, 5 equiv.). No starting material was detectable {TLC, Rf = 0.8 (silica, CH Cl /0.5 % MeOH)} after lh at r.t. The reaction was carefully 2  2  quenched by the addition of Glauber's salt and then some H2O. The mixture was partitioned between H2O and CHCI3 (50 mL each), the organic phase was isolated, dried over Na2C03 and evaporated in vacuo to give a colorless oil (90 mg, 90 % yield) which showed partial crystallization upon standing. As judged by NMR, the material thus produced is pure. Although 195 seemed to be stable, it was generally directly used in the next step. MW = 328.41; R = 0.65 (silica-CH Cl /0.5 % MeOH); iH-NMR (200 MHz, CDCI3) 8 2.25 f  2  2  (s, 3H), 2.75 (br, 1H), 5.05 (s, 1H), 6.06 (overlapping d, 1H), 6.94 (m, 2H), 7.10-7.23 (m, 7H), 7.59 (d, J = 8 Hz, 2H); C - N M R (50 MHz, CDCI3) 8 21.60, 70.50, 112.76, 117.91, 13  121.38, 126.89, 127.11, 127.75, 128.26, 130.05, 132.67, 135.95, 143.16, 145.11; LR-MS (EI, 180°C) m/e 327 (31), 325 (18), 310 (M+-H 0, 8), 248 (22), 155 (52), 105 82), 91 (100); 2  HR-MS (EI, 150°C) m/e calc'd for C i H N S 0 : 328.1007; found: 328.0972. 8  18  3  Phenyl-2-pyrrolyl-(1 -tosyl-3-pyrrolyl)-methane (196) 195 (1.22 g, 3.72 mmol) dissolved in glacial acetic acid (50 mL) containing five drops of acetic anhydride and pyrrole (1.2 mL, 5 eq) was refluxed under anhydrous conditions for lh. The mixture was evaporated to dryness under high vacuum (it is important to remove all excess pyrrole as it proves hard to separate large amounts of pyrrole from the desired compound by chromatography), and the resulting oil was purified by flash  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  180  chromatography (silica-CHCl3). The fraction containing the least polar compound was isolated. 900 mg (91 % yield) of 196 as an oil were thus produced. MW = 376.47; Rf = 0.75 (silica-CH Cl ); H-NMR (200 MHz, CDC1 ) 8 2.42 (s, 3H), 5.25 1  2  2  3  (s, 1H), 5.83 (m, 1H), 6.14 (m, 1H), 6.17 (q, J = 1.7 Hz, 1H), 6.65 (m, 1H), 6.86 (m, 1H), 7.10-7.22 (m, 3H), 7.25-7.35 (m, 5H), 7.72 (d, J= 8 Hz, 2H), 7.86 (br s, 1H); C-NMR (50 13  MHz, CDCI3) 8 21.66, 43.19, 107.13, 108.23, 114.70, 117.16, 118.93, 121.34, 126.85, 128.43 (br), 128.59, 130.03, 131.45, 133.10, 136.10, 142.57, 145.00.  N-Confused porphyrin (104) 183 (125 mg, 2.9 x 10" mol) dissolved in MeOH/CH Cl (4 mL of a 1:1 mixture) 4  2  2  and NaBH4 (100 mg, 2.6 mmol) was added in portions over 10 min. After 10 more min. of stirring, brine was added to the solution, the organic phase was separated, the aqueous phase extracted with CH C1 (3 x 10 mL) and the combined and the dried (Na C0 ) extracts were 2  2  2  3  evaporated to dryness. The presence of the dihydroxy compound 197 was seen by TLC {Rf = 0.2 (silica, CH Cl /5%MeOH), stains bright red with Br }. The freshly prepared 197 2  2  2  was dissolved in CH C1 (25 mL) and, an equimolar amount of 190 (65 mg) was added. A 2  2  catalytic amount of p-toluenesulfonic acid-H 0 was added and the slowly darkening mixture 2  was stirred at r.t. for 2h. After this time, DDQ (65 mg, 2 equiv.) was added and the mixture was stirred for 2 d. After this time, the optical spectrum of the crude mixture exhibited a Soret-type band at 434 nm and a weaker band at 740 nm. Both bands are indicative for the presence of 104, however, preparative TLC separation of the mixture did not allow isolation of 104 in larger amounts than 6-9 mg, corresponding to 3-5% yield. No TPP was formed during the reaction.  Part 1:  3.4  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  181  THE DIRECTED SYNTHESIS OF SAPPHYRINS  10,15-Diphenylsapphyrin (200) Bipyrrole dialdehyde 201 (94 mg, 5.0 x 10" mol) and tripyrrane 180 (188 mg, 1 4  equivalent) were dissolved, with the help of a heat gun, in a beaker containing dry EtOH (450 mL). To facilitate the dissolution of 201, it has to be either finely powdered or dissolved in DMSO (0.5-1 mL) first, and this solution is then mixed with the EtOH. pToluenesulfonic acid monohydrate (380 mg, 2 mmol) dissolved in 1 mL EtOH was slowly added to the vigorously stirred solution. The mixture turned royal blue (broad bands at 360, 580 nm) over the course of 15 minutes. The mixture was allowed to evaporate completely over several days. The resulting dark crust is collected and broken up (and dried under high vacuum in cases where DMSO had been used to dissolve the dialdehyde), and thoroughly triturated with CHCI3 (5 x 20 ml). The combined brilliant green extracts were evaporated in vacuo and the resulting crude 200 was chromatographed on alumina preparative TLC plates with CH2Cl2/0.1% E t N as eluent. The main band was isolated, 3  extracted with  CH2CI2 and  the product was precipitated by solvent exchange with  cyclohexane to give 22 mg (8 %) of 200. Washing of a C H C I 3 solution of 200 with aqueous HC1 or HIO3 converts the green-yellow free base solution into a bright yellow solution of the corresponding protonated form. MW = 539.58; Rf = 0.18 (silica-5%MeOH/CH Cl ); H-NMR (400 MHz, CDCI3) 5 -1.52 J  2  2  (s, 2H), -0.1-0 (br s, 2H), 7.15-7.20 (m, 1H); 7.25-7.30 (m, 1H), 7.60 (tr, *7 = 7 Hz, 2H), 7.85 (tr, ^/ = 8 Hz, 4H), 8.49 (br s, 4H), 9.24 (d, J = 4.5 Hz, 2H), 9.43 (d, J = 4.5 Hz, 2H), 3  3  9.60 (d, J = 4.5 Hz, 2H), 10.20 (d, J = 5.0 Hz, 2H), 10.27 (s, 2H); UV-Vis (CH Cl /trace 3  3  2  2  Et N) X x (log e) 478 (4.86 ), 506 (4.71 ), 626 (3.67), 686 (3.97), 708 (sh), 786 (3.62) nm; 3  ma  LR-MS (EI, 200°) m/e 527 (100, M+), 450 (14.6, M+-phenyl), 264 (M++, 27.9); HR-MS (EI, 180°) m/e calc'd for C36H25N5: 527.21100, found 527.21015.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  182  200-2HC1:  !H-NMR (400 MHz, CDC1 ) 6 -4.69 (s, 2H), -4.48 (s, 2H), -3.87 (s, 1H), 7.26 (d, J= 8 Hz, 3  3  1H), 7.80 (d, J = 8 Hz, 1H); 8.65 (d, ^7=5 Hz, 4H), 9.33 (d, J = 0.8 Hz, 2H), 9.63 (dd, J 3  4  3  = 5 H z , / = 0.8 Hz, 2H), 10.25 (dd, ^/ = 5 H z , / = 0.8 Hz, 2H), 10.46 (dd, J = 5 H z , / = 0.8 4  4  3  4  Hz, 2H), 10.78 (dd, J=5 Hz, J = 0.8 Hz, 2H), 11.92 (s, 2H); "C-NMR (75 MHz, CDCI3) 3  4  8 145.69, 140.61, 136.45, 131.68, 130.56, 130.28, 129.91, 129.83, 129.79, 128.83, 128.56, 126.75, 124.90, 123.15, 104.22, 102.71; UV-Vis (CH Cl /trace HC1) X 2  2  max  (log e) 482  (5.47), 656 (4.13), 682 (4.13), 724 (sh), 758 (4.70) nm;  3,22-Diethyl-2,23-dimethyl-5,10-diphenylsapphyrin (199) Tripyrrane 180 (22.6 mg, 6 x 10"^ mol) and bipyrrole dialdehyde 112 (16.3 mg, 1 equivalent) were dissolved under anhydrous conditions in absolute EtOH (60 mL). Molecular oxygen was bubbled at r.t. through the solution. p-Toluenesulfonic acid monohydrate (45.6 mg, 4 equivalents) was added to this solution, and a slow oxygen bubble was maintained for 12 h. The solution turned from an initial red through aquamarine to green. The dark green solution was evaporated to dryness in vacuo and the resulting dark solid was subjected to column chromatography (alumina neutral, activity 1-2.5% MeOH/CH2Cl2). The first (purple) fraction (minute amount of TPP) was discarded and the second (green) main fraction was collected. Precipitation from hexane gave 14.3 mg (39% yield) of 199 as dark green powder. It had identical spectroscopic properties to those reported by Sessler, Kodadek and co-workers.  384  5,10,15,20-Tetraphenylsapphyrin (114) Bipyrrole (203) (26.4 mg, 2 x 10"^ mol), tripyrrane 180 (75 mg, 1 equivalent), and benzaldehyde (42 pL, 2 equivalents) were dissolved under anhydrous conditions in dry CH2CI2 (20 mL), and the solution was degassed by bubbling nitrogen through it for 20 min. One drop of BF3-etherate was added and the mixture was stirred for 1 h. /?-Chloranil  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  183  (100 mg, 4 x 1 0 mol) was added and the mixture was refluxed for 1 h. After this time, the 4  mixture was evaporated in vacuo to dryness and the residue was subjected to flash chromatography (alumina, neutral, activity II-CH2CI2), separating the by-product TPP and a yellow-green fraction containing the sapphyrin. The latter fraction was evaporated to dryness and chromatographed on a preparative plate (alumina-CH2Cl2/CCl4l:l ). The main band was extracted with CH Cl2 and slow evaporation of this solution provided 3.6 mg 2  (3.7%) of dark green sparkly crystals of 114. tetraphenylsapphyrin described by Chmielewski  etal.  315  It proved identical with the  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  3.5  184  THE PREPARATION OF CYANOPYRROLES  2-Cyano-1 -methylpyrrole (215) This product has been described before by Barnett et a/.  386  These authors prepared it  from in 67% from 1-methyl pyrrole and chlorosulfonyl isocyanate. The synthesis to be described here details an alternative synthesis, provided by Anderson.  414  and complements the analytical data  399  2-Cyanopyrrole (207) (1.1 g, 11.58 mmol) dissolved in C H C N (5 mL) was added 3  dropwise under N  2  into a suspension of KH (~ 700 mg, 1.5 equiv., from a mineral oil  suspension, washed with dry C H 3 C N ) in C H 3 C N (25 mL). The suspension was refluxed for 1 h, during which time most of the KH went into solution. The reaction mixture was cooled and methyl iodide (865 mL, 1.2 equivalent) was syringed into the ice-cold solution. The mixture was then refluxed for an additional 1.5 h. The fine white precipitate of KI was then filtered (glass frit-M) from the cooled solution, and the filtrate evaporated in vacuo to give a pale yellow oil. This oil was subjected to flash column chromatography (silica gel, 25 x 3 cm, CHCI3). The first major fraction was collected, to give, after evaporation of the solvent, 745 mg (60.7 %) of a colourless liquid, which, based on the iH-NMR, was pure. Due to its low boiling point (72-75°C/9 torr) , it is likely that some product was lost during the work386  up. MW = 106.13; R = 0.71 (silica-CHCl ); H-NMR (200 MHz, ) 5 3.72 (s, 3H), 6.10 (dd, J  f  3  7 = 4.0, 4.0 Hz, 1H), 6.78 (dd, 7 = 4.0, 2.0 Hz, 1H), 6.71 (m, 1H); C - N M R (50 MHz, 13  CDC1 ) 5 35.2, 104.3, 109.4, 113.8, 119.8, 127.6; MS (EI, 150°C) m/e 106 (100, M+), 105 3  (82.0, M+-H), 78 (31.0), 64 (12.3), 52 (14.0); HR-MS (EI, 150°C) m/e calc'd for C H N : 6  106.05310, found 106.05302.  6  2  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  185  3-Cyano-2,5-dimethylpyrrole (214) General Procedure for the preparation of cyanopyrroles from CSI and the corresponding pyrroles  2,5-Dimethylpyrrole (211) (1.0 g, 10.5 mmol) was dissolved under anhydrous conditions in a mixture of DMF (2 mL) and C H 3 C N (10 mL) in a flask equipped with a septum, and was cooled in an icebath to -5°C. Chlorosulfonylisocyanate (CSI) (1 equivalent, 911 mL, 1.48 g) dissolved in dry C H 3 C N (5 mL) was syringed into the stirred solution. No starting material was detectable by TLC after 15 min. The orange solution was then quenched by pouring it into aqueous Na2C03 (100 mL, 5 % w/w). After the solution was stirred for 1 h at room temperature, it was extracted with CHCI3 (3 x 50 mL). The combined organic layers were thouroughly washed with water, dried over Na2CC>3 and evaporated to dryness. The partially solidified oil was loaded onto a flash chromatography column (silica gel, 25 x 3 cm-CHCl3). The first major fraction gave after vacuum evaporation of the solvent and drying (20°C/0.2 torr; the compound sublimes easily) the desired compound in 70.5 % (1.25 g) yield as fine, colorless crystals. An analytical sample was sublimed at 70°C/0.2 torr. A second, minor (~ 5%) fraction proved to be 3,4-cyano-2,5-dimethylpyrrole (217): MW = 120.15; m.p. = 85.0°C; R = 0.63 (silica-CH Cl ); iH-NMR (200 MHz, CDCI3) 8 f  2  2  2.18 (s, 3H), 2.35 (s, 3H), 5.95 (d, 1.5 Hz, 1H), 8.40 (br s, 1H); C-NMR (50 MHz, CDC1 ) 13  3  5 12.0, 12.5, 90.0, 107.8, 118.0, 127.7, 136.7; LR-MS (EI, 150°C) 120 (52.5, M+), 119 (100), 105 (13.9), 92 (12.4), 78 (9.3), 65 (8.9); HR-MS (EI, 150°C) m/e calc'd for C H N : 7  8  2  120.06875, found 120.06822; Analysis calc'd for C H N : C, 69.97; H, 6.71; N, 23.31,; 7  found: C, 70.15; H, 6.44; N, 23.18.  8  2  Part 1:  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  186  3,4-Dicyano-2,5-dimethylpyrrole (217) Either isolated as side (~ 5%) product of the preparation of 214, or 62 % yield after chromatography from 2,5-dimethylpyrrole following the general procedure, albeit with 2.5 equivalents of CSI: MW = 145.16; m.p. 242-244° (dried at 80°C/ 0.2 torr, 15 h); R = 0.27 (silica-CH Cl ); f  2  2  iH-NMR (200 MHz, acetone-d ) 5 2.35 (s, 6H), 11.2 (v br s, 1H); C - N M R (50 MHz, 13  6  acetone-d ) 8 11.7, 93.00, 114.5, 138.9; LR-MS (EI, 150°C) 145 (46.8, M+), 144 (100, M+6  H), 5.0 (11.7, C H N +); HR-MS (EI, 150°C) m/e calc'd for C H N : 145.06400, found 7  7  2  8  7  3  145.06332.  2-Cyano-3,5-dimethyl-4-ethylpyrrole (213) Prepared in 69 %yield from 3,4-dimethyl-4-ethylpyrrole (210) and 1.2 equivalents of CSI according to the general procedure. The crude product can be filtered off after the hydrolysis step (48 h, 5°C). Either column chromatography (silica-CH Cl ) or sublimation 2  2  (110°C/760 torr) gives analytical pure material as a white solid or long, colorless needles, respectively. MW = 148.21; m.p. 133-134° (sublimed material); R = 0.31 (silica-CH Cl /0.5% MeOH); f  2  2  !H-NMR (200 MHz, CDC1 ) 5 1.04 (tr, 3.6 Hz, 3H), 2.12 (s, 3H), 2.18 (s, 3H), 2.35 (q, 3  3.6 Hz, 2H), 8.40 (br s, 1H); C - N M R (50 MHz, CDCI3) 8 10.0, 11.4, 15.0, 96.7, 102.8, 13  115.5, 122.6, 130.4, 131.1; LR-MS (EI, 150°C) m/e c 148 (25.3, M+), 133 (100, M+-CH ), 3  32 (63.8); HR-MS (EI, 150°C) m/e calc'd for C H i N : 148.0005, found 148.0001; Analysis 9  2  2  calc'd for C H i N : C, 72.94; H, 8.16; N, 18.90; found: C, 73.17; H, 8.23; N, 19.00. 9  2  2  2-Cyano-3,4-diethylpyrrole (212) Prepared from 3,4-diethylpyrrole (1.165 g, 9.46 mmol) in 76 % yield according the general procedure. Chromatographic (CHCl3/silica, 12x3 cm) workup and evaporation of  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  187  the solid in vacuo produces 212 as a slightly pink, coarse crystalline solid. An analytical sample was sublimed at 70°C/0.2 torr. Chromatography also yielded a minor amount of 216. MW = 148.20; mp = 133-134°C (sublimed material); Rf = 0.39 (silica-CH Cl ); !H-NMR 2  2  (300 MHz, CDCI3) 5 1.98 (two overlaying (separated by 1 Hz) tr, J = 7.5 Hz, 6H), 2.41 (d of q, 7= 0.8, 7.5 Hz, 2H), 2.57 (q, 7.5 Hz, 2H), 6.65 (d, 2.5 Hz, 1H), 8.95 (br s, 1H); C-NMR 13  (50 MHz, CDCI3) 5 14.6, 15.0, 18.0, 18.2, 98.6, 115.2, 120.8, 125.8, 136.5; LR-MS (EI, 150°C) 148 (29.8, M+), 133 (100, M+-CH ), 118 (20.7, M+-C H ), 106 (10.4), 91 (9.9), 77 3  2  6  (16.5); HR-MS (EI, 150°C) m/e calc'd for C H i N : 148.10005, found 148.10045; Analysis 9  2  2  calc'd for C H i N : C, 72.94; H, 8.16; N, 18.90; found: C, 73.00; H, 8.27; N, 19.02. 9  2  2  2,5-Dicyano-3,4-diethylpyrrole (216): Isolated in small amounts as side-product in the preparation of 212 or prepared from 3,4-diethylpyrrole (209) (0.5 g, 4.06 mmol) and excess CSI in 47 % yield according to the general procedure. After the addition of the CSI (0.833 mL, 2.5 equ.), the mixture was refluxed for 1 h. Hydrolysis was accomplished over an extended period of time (12 h) on the steam bath. The crude mixture was purified by flash chromatography (silica, 10x3 cm, CH C1 ). 2  2  MW = 173.22; R = 0.4 (silica-CH Cl /2.5% MeOH); 'H-NMR (200 MHz, acetone-d ) f  2  2  6  5 1.18 (tr, 7.5 Hz, 6H), 2.56 (q, 7.5 Hz, 4H), 10.9 (br s, 1H); C-NMR (50 MHz, acetone13  d ) 5 11.7, 93.0, 116.5, 139.8; LR-MS (EI, 150°C) 173 (25.5, M+), 158 (100, M+-CH ); 6  3  HR-MS (EI, 150°C) m/e calc'd for C i H n N : 173.09529, found 173.09512; Analysis calc'd 0  3  for C10H11N3: C, 69.34; H, 6.40; N, 24.26; found: C, 69.62; H, 6.38; N, 24.30.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  3.6  Experimental  188  THE REDUCTIVE COUPLING OF 2-CYANOPYRROLES  (2-Pyrrolylmethene)-(2-pyrrolylmethyl)imine  (204)  General Procedure for the reductive coupling of 2-cyanopyrroles  2-Cyanopyrrole (207)(2.0 g, 21.67 mmol) dissolved in dry THF (10 mL) was carefully added dropwise under anhydrous conditions into a cooled (0°C) and vigorously stirred suspension of L A H (960 mg, 24.2 mmol) in THF (30 mL) over 20 min. After completion of the addition, the reaction mixture was stirred for one additional hour at 0°C. To quench the reaction, Glauber's salt (Na2SC"4 • 10 H2O, -2.0 g) was slowly added until gas evolution ceased. The resulting colorless thick slurry was filtered through a pad of Celite® 545 and the filter cake thoroughly rinsed with CHCI3 (~ 50 mL). The combined filtrates were evaporated to dryness on a rotary evaporator to give the desired imine (1.61 g, 85.5 %) as an off-white solid. Recrystallization from EtOH/H 0 gives 204, after drying at high 2  vacuum at 50°C, as analytically pure shiny plates. If fumed with Br2, the colorless spot of this compound on a silica gel TLC plate turns slowly and characteristically into a yellow spot with purple edges. MW = 173.22; mp = 155-157°C ; R = 0.16 (silica-CH Cl /5% MeOH); !H-NMR (200 385  f  2  2  MHz, acetone-d ) 5 4.59 (s, 2H), 5.93 (s, 1H), 5.98 (t, / = 2.0 Hz, 1H), 6.14 (dd, / = 2.0, 2.0 6  Hz, 1H), 6.42 (dd, 7 = 1.9, 2.0 Hz, 1H), 6.67 (narrow m, 1H), 6.91 (narrow m, 1H), 9.85 (br s, 1H), 10.65 (br s, 1H); C NMR (50 MHz, CDCI3) 6 = 56.7, 106.2, 108.5, 110.0, 115.3, 1 3  117.6, 122.5, 129.3, 129.8, 152.9; LR-MS (EI, 150°C) m/e 173 (63.6, M+), 96 (86.1), 92 (89.2), 80 (100, C H6N+), 68 (50.5); HR-MS (EI, 150°C) m/e calc'd for C10H11N3: 5  173.09529, found 173.09535.  '  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  189  Bis(2-pyrrolylmethyl)amine (208) 204 (0.1 g, 5.8 x 10"4 mol) dissolved in THF (10 mL) was added under anhydrous conditions and at 0°C to a suspension of L A H (44 mg, 2 equivalents) in dry THF (5 mL). The reaction mixture was quenched with Glauber's salt ( N a 2 S 0 • 10 H 2 O , 0.2 g) and the 4  resulting slurry filtered through a pad of Celite®. The filtrate was evaportated on a rotary evaporator to give the desired amine as a colourless and odorless oil. It was, based on Kl  NMR, > 95% pure. The amine is not visible under a UV lamp if spotted on a TLC plate (with fluorescence indicator), however, Br2 fumes make it visible.: MW = 175.26; R = 0.1 (silica-CH Cl /7% MeOH); !H-NMR (200 MHz, CDCI3) 8 2.65 (br f  2  2  s, 1H), 6.08 (s, 2H), 6.15 (s, 2H), 6.68 (s, 2H), 8.70 (br s, 2H); C - N M R (50 MHz, CDCI3) 13  8 45.6, 106.9, 108.2, 117.6, 129.9; LR-MS (EI, 180°C) m/e 175 (30.1, M+), 158 (37.8), 108 (57.9, M+-pyrrole), 95 (93.8, C5H7N+), 80 (100, C H6N+), 68 (63.1, C H6N+); HR-MS (EI, 5  4  180°C) 77z/e calc'd for C10H13N3: 175.11095, found 175.11107.  The Reduction Experiments of Cyanopyrroles 212, 213, and 215  Following the general procedure, ca. 100 mg of the respective cyanopyrroles were reduced. The crude reaction mixtures were evaporated to dryness and dried under high vacuum at ambient temperature. The resulting oils or solids were taken up in CDCI3 and the success of the experiments was judged, in addition to the evaluation of the LR-MS of the crude mixtures, by the resulting H - and C-NMRs. The product ratios are determined by J  13  the integration of the H-NMR. 1  Reduction of 2-cyano-3,4-diethylpyrrole (212) This reduction produces 2-(aminomethyl)-3,4-diethylpyrrole (225) and the imine linked dimer 227 in variable ratios between 3:2 and 2:1. H-NMR (200 MHz, CDCI3) 8 1.31  1.4 (m, -CH2CH3 of 225 and 227), 2.25-2.65 (m, - C H C H of 225 and 227), 3.2 (br s, - N H 2  3  2  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  190  of 225), 3.82 (s, - C H N H of 225), 4.78 (s, =N-CH - of 227), 6.5-6.6 (pyrrole-H of 225 and 2  2  2  227), 8.30 (s, -C =N of 227), 8.80 (br s, pyrrolylmethyl-NH of 227, the corresponding signal H  for the pyrrolylmethene-NH is observed at shifts >12 ppm), 9.1 (br s, NH of 225).  Reduction of 2-cyano-4-ethyl-3,5-dimethylpyrrole 213 This reduction leads to the exclusive formation of the imine linked dimer 228. Hl  NMR (200 MHz, CDC1 ) 5 1.05-1.2 (m, 6H), 2.03 (s, 3H), 2.07 (s, 3H), 2.10 (s, 3H), 2.19 (s, 3  3H), 2.35-2.47 (m, 4H), 4.62 (s, 2H), 8.20 (s, 1H); C-NMR (50 MHz, CDCI3) 6 8.8, 9.1, 13  10.7, 11.1, 15.5, 15.8, 17.3, 17.7, 55.4, 113.2, 120.7, 121.8, 122.7, 123.0, 124.1, 126.7, 127.9, 150.6; LR-MS (EI, 200°C) m/e 283 (23, M+2H), 179 (20), 163 (82), 136 (100).  Reduction of 2-cyano-1-methylpyrrole (215) This reduction gives 2-(aminomethyl)-l-methylpyrrole (223) and a second product (imine linked dimer (?) !H-NMR (200 MHz, CDCI3) 5 1.2, 3.8, 4.6, 6.5, 6.6, 8.2; m/e = 201) in the ratio of 1:13 as judged by !H-NMR). However, this second product could not be produced in a large enough quantity to undoubtedly determine its structure. 223: !H-NMR (200 MHz, CDCI3) 5 3.61 (s, 3H), 3.82 (s, 2H), 5.95-6.1 (m, 2H), 6.58 (m, 1H); 13  C-NMR (50 MHz, CDCI3) 8 33.6, 38.1, 106.3, 106.6, 122.2, 134.2; LR-MS (EI, 200°C)  m/e 110 (53, M+), 94 (100, M+-NH2).  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  3.7  191  THE FORMATION OF COMPLEX 236  [SP-4]-[(2-dipyrrinato-K N, N'^ 2  (236) Imine 204 (200 mg, 1.16 mmol), 2-pyrrolealdehyde (206) (110 mg, 1.16 mmol) and nickel(II) acetate tetrahydrate (500 mg, 2.0 mmol), dissolved in a 2:1 mixture of EtOH and CHCI3 (15 mL), were heated at reflux temperature for lh. The initially pale green color of the solution turned time dark orange during that time. Evaporation in vacuo of the reaction mixture gave a solid which was triturated with CHCI3 (4x5 mL). The volume of the dark orange solution was reduced and chromatographed (silica gel, 2x12 cm, CHCI3). The first major orange band was collected and evaporated to dryness. The resulting solid was dissolved in CH2CI2, and slow solvent exchange with cyclohexane resulted in the formation of analytically pure 236 as shiny metallic green dichroic short needles (125 mg, 35%). For the details of the X-ray crystal structure determination see below. MW = 306.98; m.p. = 184 °(drying conditions 50°C at 0.1 torr); R = 0.90 (silica-CHCl ); f  3  !H-NMR (400 MHz, acetone-d ) 5 = 4.60 (s, 2H), 6.01 (dd, 7= 2.0 Hz, 3.8H, 1H), 6.29 (d, / 6  = 4.2 Hz, 1H), 6.41 (dd, 7 = 4.2, 1.7 Hz, 1H), 6.57 (dd, 7 = 3.7, 0.8 Hz, 1H), 7.02 (d, 7 = 4.2 Hz, 1H), 7.09 (dd, 7 = 4.1, 1.1 Hz, 1H), 7 (25, s, 1H), 7.53 (s, 1H), 7.69 (s, 1H); C-NMR 13  (50 MHz, CDC1 /10% acetone- 6) 6 54.0 (-C-), 111.3 (=C-), 112.8 (=C-), 117.0 (=C-), 117.8 3  d  (=€-), 129.7 (=C-), 129.9 (=C-), 131.6 (=C-), 133.5 (=C=), 136.9 (=C-), 139.7 (=C=), 146.2 (=C=), 151.1 (=C-), 158.9 (=C-), 162.9 (=C=); UV-Vis (CH C1 ) X 2  2  (log e) 314 (4.08),  m a x  354 (4.11), 394 (4.12), 508 (4.40) nm; LR-MS (EI, 150°C) m/e : 311 (1.1), 310 (4.5), 309 (10.5), 308 (34.2), 307 (50.0), 306 (83.9), 305 (100.0); HR-MS (EI, 150°C) m/e calc'd for Ci Hi N 5  2  5 8 4  N i : 306.04153, found 306.04013; Analysis calc'd for C i H i N N i : C, 58.69; H,  3.94; N, 18.25; found: C, 58.51; H, 3.74; N, 18.00.  5  2  4  Part 1:  3.8  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  192  CRYSTAL STRUCTURE ANALYSES OF 163-PYRIDINE, 175 AND The  236 crystals of 163 (175 and 236) were mounted on a glass fibre and all  measurements were made at 21±1°C on a Rigaku AFC6S diffractometer. Selected crystallographic data appear in Table 1-2. The final unit-cell parameters were obtained by leastsquares on the setting angles for 25 reflections in the range 93.9 < 28 < 104.9° (80.6 < 20 < 97.5, and 34.1 < 20 < 38.8). Of the 9000 (7834 and 5135) reflections which were collected, 8602 (7492 and 5014) were unique (Ri = 0.020, 0.017 and 0.032). The intensities of three nt  standard reflections, measured every 200 reflections throughout the data collections, decreased by 1.2% in 236. A linear correction factor was applied to the data to account for this phenomenon. No decay correction was applied for 163 and 175. The data were processed and corrected for Lorentz and polarization effects. All structures were solved by the heavy atom Patterson method and and expanded using Fourier techniques. Hydrogens were refined isotropically. Neutral atom scattering factors for all atoms and anomalous dispersion corrections for the non-hydrogen atoms were taken from the International Tables for X-Ray Crystallography. ^ 4  Final atomic coordinates and equivalent isotropic thermal  parameters of and selected bond lengths in 163'pyridine, 175 and 236 appear in Tables 1-3 to 1-8.  There is one molecule of pyridine in 163'pyridine in the asymmetric unit. The lactone group in 163-pyridine was modeled as disordered over five states. The site occupancies of the carbonyl oxygen atoms were constrained to total of 1.0 and were adjusted as the refinement progressed to yield approximately equal equivalent isotropic thermal parameters. The associated ring atoms were treated as part C and part O, based on the appropriate carbonyl oxygen populations.  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  193  The crystal of 175 is a solid solution of four different compounds differing only in the alkoxy groups OR and OR': R = R' = Me; R = Me, R' = Et; R = Et, R' = Me; R = R' = Et. Table 1-2  Crystallographic data for 163-pyridine, 175, and 236 compound  163 ^pyridine  175  236  crystal  purple p r i s m  green prism  red p r i s m  dimensions, mm  0.17x0.28x0.35  0.20x0.25x0.30  0.35x0.35x0.50  empirical formula  C53H36N602Zn  C47H36N4ND3  Ci Hi2N Ni  MW  854.29  763.53  306.99  crystal s y s t e m  triclinic  triclinic  monoclinic  space group  P T(#2)  P T(#2)  C2/c (#15)  a, A  13.2954(8)  13.447(2)  21.461(1)  b, A  14.899(2)  13.950(1)  5.362(1)  11.226(2)  11.015 (2)  22.0841 (9)  93.46(1)  102.180(9)  104.728(8)  112.768(9)  Y. d e g  99.707(7)  94.074(9)  v, A  2107.4(4)  1835.4(4)  2538.4(4)  2  2  8  1.346  1.381  1.606  CuK  CuK  MoKa  c,  A  a, d e g  P,  deg  3  z Dcalc.g/cm  3  radiation  x, A  a  a  5  4  92.748(4)  1.54178  1.54178  0.71069  u, c m " Transmission factors  12.08  11.57  15.22  R; Rco  0.036, 0.038  0.044; 0.042  0.032; 0.024  gof  2.46  1.13  1.68  1  0.921-1.00  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Table 1-3  Experimental  Atomic coordinates and B q . [ A ] for 163-pyridine e  2  a  Atom*  x  y  z  B .  Zn(1)  0.16039(2)  0.21047(2)  0.42785(3)  3.724(7)  0.2056(2)  -0.0285(1)  0.2341 (2)  5.04(5)  0(2)  0.0581(4)  -0.1160(3)  0.1476(4)  7.1(1)  0.45  0(2a)  0.2265(7)  -0.0872(7)  0.1718(9)  7.3(3)  0.22  0(2b)  0.1002(7)  0.4675(6)  0.7549(10)  7.2(3)  0.21  0(2c)  -0.242(3)  0.157(2)  0.486(3)  8.1(8)  0.07  0(2d)  0.270(3)  0.497(3)  0.794(4)  6.4(9)  0.05  N(1)  0.1583(1)  0.0813(1)  0.3458(2)  3.78(4)  N(2)  0.0080(1)  0.1624(1)  0.4393(2)  3.79(4)  N(3)  0.1721(1)  0.3150(1)  0.5631 (2)  3.70(4)  N(4)  0.3232(1)  0.2314(1)  0.4723(2)  3.71 (4)  N(5)  0.1165(2)  0.2750(1)  0.2615(2)  4.30(5)  N(6)  0.652(2)  0.2261(8)  -0.0199(7)  18.2(4)  C(1)  0.2405(2)  0.0527(1)  0.3139(2)  3.77(5)  0.0946(2)  -0.0510(1)  0.2153(2)  4.86(5)  C(3)  0.0685(2)  0.0191(1)  0.2879(2)  3.86(5)  C(4)  -0.0332(2)  0.0205(1)  0.2967(2)  3.76(5)  C(5)  -0.0597(2)  0.0853(2)  0.3737(2)  3.97(5)  -0.1623(2)  0.0816(2)  0.3923(2)  5.02(6)  C(7)  -0.1579(2)  0.1567(2)  0.4688(2)  4.92(6)  C(8)  -0.0509(2)  0.2081(2)  0.4968(2)  4.05(5)  C(9)  -0.0143(2)  0.2927(2)  0.5697(2)  3.95(5)  C(10)  0.0911(2)  0.3399(1)  0.6026(2)  3.85(5)  0(11)^  0.1310(2)  0.4199(2)  0.6912(2)  4.78(6)  C(12)  0.2386(2)  0.4406(1)  0.7046(2)  4.74(5)  C(13)  0.2625(2)  0.3751(1)  0.6255(2)  3.84(5)  C(14)  0.3651(2)  0.3717(1)  0.6194(2)  3.77(5)  C(15)  0.3925(2)  0.3041(1)  0.5486(2)  3.77(5)  C(16)  0.4987(2)  0.2983(2)  0.5481 (2)  4.38(5)  C(17)  0.4944(2)  0.2229(2)  0.4719(2)  4.45(6)  C(18)  0.3837(2)  0.1806(2)  0.4258(2)  3.83(5)  C(19)  0.3470(2)  0.0968(1)  0.3493(2)  3.77(5)  C(20)  -0.1223(2)  -0.0503(1)  0.2162(2)  3.73(5)  5  0(1 )  a  C{2)°  C(6)  c  e  eq  Occupancy  194  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  C(21)  -0.1672(2)  -0.0347(2)  0.0962(2)  5.08(6)  C(22)  -0.2513(2)  -0.0969(2)  0.0207(2)  5.58(7)  C(23)  -0.2904(2)  -0.1757(2)  0.0626(2)  5.10(6)  C(24)  -0.2454(2)  -0.1927(2)  0.1799(3)  5.85(7)  C(25)  -0.1609(2)  -0.1303(2)  0.2564(2)  5.31(6)  C(26)  -0.0923(2)  0.3350(2)  0.6171(2)  4.16(5)  C(27)  -0.1235(2)  0.3080(2)  0.7195(3)  5.81(7)  C(28)  -0.1960(3)  0.3489(3)  0.7614(3)  7.24(10)  C(29)  -0.2372(3)  0.4175(3)  0.7016(4)  7.6(1)  C(30)  -0.2089(3)  0.4433(2)  0.5992(4)  7.24(10)  C(31)  -0.1363(2)  0.4031(2)  0.5575(3)  5.57(7)  C(32)  0.4534(2)  0.4424(2)  0.7016(2)  4.09(5)  C(33)  0.5025(2)  0.4258(2)  0.8190(2)  6.11(7)  C(34)  0.5838(2)  0.4912(2)  0.8956(3)  6.93(8)  C(35)  0.6169(2)  0.5720(2)  0.8565(3)  6.46(8)  C(36)  0.5691 (3)  0.5892(2)  0.7401 (4)  7.58(9)  C(37)  0.4875(2)  0.5252(2)  0.6628(3)  6.34(7)  C(38)  0.4245(2)  0.0527(2)  0.3037(2)  3.96(5)  C(39)  0.4781 (2)  0.0950(2)  0.2255(2)  4.97(6)  C(40)  0.5486(2)  0.0530(2)  0.1811(3)  6.16(8)  C(41)  0.5672(2)  -0.0309(2)  0.2151(3)  6.40(8)  C(42)  0.5160(3)  -0.0722(2)  0.2947(3)  6.07(8)  C(43)  0.4445(2)  -0.0318(2)  0.3385(2)  4.89(6)  C(44)  0.1257(3)  0.2419(2)  0.1538(3)  6.16(8)  C(45)  0.0940(3)  0.2801(3)  0.0457(3)  7.06(9)  C(46)  0.0500(3)  0.3549(3)  0.0471 (3)  7.12(9)  C(47)  0.0376(3)  0.3896(2)  0.1556(3)  8.2(1)  C(48)  0.0724(3)  0.3476(2)  0.2611 (3)  6.64(8)  C(49)  0.6010(7)  0.275(1)  0.042(2)  15.7(4)  C(50)  0.642(2)  0.3018(6)  0.146(2)  15.5(5)  C(51)  0.733(2)  0.2885(8)  0.2044(7)  16.2(4)  C(52)  0.7850(7)  0.240(1)  0.153(2)  16.0(4)  C(53)  0.748(2)  0.2107(7)  0.038(2)  15.7(5)  a  b  B . = (8/3)K IZUjja*ia*j{araj). Superscripts refer to C/O ratios: eq  2  a  55/45,  b  78/22,  c  93/7,  d  95/5,  e  79/21  195  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Table 1-4.  Experimental  196  Selected bond lengths [A] in 163-pyridine with estimated standard deviations in parentheses Bond  Distance [A]  Bond  Distance [A]  Zn(1)-N(1)  2.075(2)  Zn(1)-N(2)  2.071(2)  Zn(1)-N(3)  2.066(2)  Zn(1)-N(4)  2.058(2)  Zn(1)-N(5)  2.146(2)  0(1)-C(1)  1.397(3)  0(1)-C(2)  1.415(2)  0(2)-C(2)  1.148(4)  0(2a)-0(1)  1.198(9)  0(2c)-C(7)  1.18(4)  0(2d)-C(12)  1.21(4)  N(1)-C(1)  1.357(3)  N(1)-C(3)  1.366(3)  N(2)-C(5)  1.373(3)  N(2)-C(8)  1.370(3)  N(3)-C(10)  1.360(3)  N(3)-C(13)  1.368(3)  N(4)-C(15)  1.381(2)  N(4)-C(18)  1.365(3)  N(5)-C(44)  1.320(3)  C(1)-C(19)  1.402(3)  C(2)-C(3)  1.427(3)  C(3)-C(4)  1.384(3)  C(4)-C(5)  1.404(3)  C(4)-C(20)  1.501(3)  C(5)-C(6)  1.425(2)  C(6)-C(7)  1.352(3)  C(7)-C(8)  1.444(3)  C(8)-C(9)  1.400(3)  C(9)-C(10)  1.403(3)  C(9)-C(26)  1.490(3)  C(10)-C(11)  1.437(2)  C(11)-C(12)  1.378(3)  C(12)-C(13)  1.410(3)  C(13)-C(14)  1.392(3)  C(14)-C(15)  1.397(3)  C(14)-C(32)  1.501 (3)  C(15)-C(16)  1.431 (3)  C(16)-C(17)  1.354(3)  C(17)-C(18)  1.447(3)  C(18)-C(19)  1.409(3)  Experimental  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Table 1-5 Atom  197  Atomic coordinates and B g . [A ] for 175 e  2  a  Occupancy  X  y  z  Beg.  Ni(1)  0.19083(3)  0.28677(4)  0.30436(5)  3.926(10)  0(1)  -0.1932(2)  0.2892(2)  0.0787(2)  6.37(6)  0(2)  -0.1273(2)  0.3209(2)  0.3132(2)  6.37(6)  0(3)  -0.1145(2)  0.2233(2)  -0.0649(3)  7.81(8)  N(1)  0.0380(2)  0.2880(2)  0.2233(2)  4.08(5)  N(2)  0.1644(2)  0.1698(2)  0.3562(2)  4.07(5)  N(3)  0.3433(2)  0.2837(2)  0.3794(2)  4.08(5)  N(4)  0.2171(2)  0.4065(2)  0.2593(2)  4.22(5)  C(1)  -0.0077(2)  0.3725(2)  0.2232(3)  4.30(6)  C(2)  -0.1255(2)  0.3566(2)  0.2030(3)  5.15(7)  C(3)  -0.1427(2)  0.2102(3)  0.0428(3)  5.37(8)  C(4)  -0.0393(2)  0.2025(2)  0.1608(3)  4.42(6)  C(5)  -0.0278(2)  0.1157(2)  0.1979(3)  4.15(6)  C(6)  0.0675(2)  0.1064(2)  0.3060(3)  4.37(6)  C(7)  0.0767(2)  0.0348(3)  0.3837(4)  5.85(8)  C(8)  0.1788(2)  0.0560(3)  0.4844(3)  5.63(8)  C(9)  0.2362(2)  0.1376(2)  0.4631 (3)  4.39(6)  C(10)  0.3483(2)  0.1701(2)  0.5233(3)  4.34(6)  C(11)  0.3980(2)  0.2336(2)  0.4734(3)  4.36(6)  C(12)  0.5122(2)  0.2498(2)  0.5026(4)  5.34(8)  C(13)  0.5252(2)  0.3065(2)  0.4222(4)  5.36(8)  C(14)  0.4211(2)  0.3304(2)  0.3487(3)  4.41(6)  C(15)  0.4030(2)  0.4014(2)  0.2741 (3)  4.40(6)  C(16)  0.3083(2)  0.4418(2)  0.2401 (3)  4.56(7)  C(17)  0.2940(3)  0.5333(2)  0.2029(4)  5.55(8)  C(18)  0.1995(3)  0.5566(2)  0.2072(4)  5.75(8)  C(19)  0.1500(2)  0.4758(2)  0.2381(3)  4.55(7)  C(20)  0.0448(2)  0.4646(2)  0.2376(3)  4.43(7)  C(21)  -0.2313(3)  0.3056(4)  0.3207(6)  10.0(2)  C(22)  -0.224(1)  0.246(1)  0.422(2)  12.7(6)  0.38  C(23)  -0.2025(7)  0.1775(8)  -0.2001(9)  9.1(3)  0.65  Part 1: Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  C(23a)  -0.132(2)  0.159(1)  -0.171(2)  9.2(5)  C(24)  -0.177(1)  0.184(1)  -0.305(2)  10.0(5)  C(24a)  -0.161(4)  0.051(4)  -0.238(5)  10.4(9)  C(25)  -0.1214(2)  0.0298(2)  0.1344(3)  4.41 (6)  C(26)  -0.1933(3)  0.0208(3)  0.1930(3)  6.29(9)  C(27)  -0.2813(3)  -0.0568(3)  0.1358(4)  7.3(1)  C(28)  -0.2971(3)  -0.1246(3)  0.0213(5)  6.78(10)  C(29)  -0.2291(3)  -0.1163(3)  -0.0378(5)  8.9(1)  C(30)  -0.1400(3)  -0.0398(3)  0.0180(4)  7.9(1)  C(31)  0.4203(2)  0.1324(2)  0.6387(3)  4.68(7)  C(32)  0.4363(3)  0.0343(3)  0.6211(4)  6.07(9)  C(33)  0.5059(3)  0.0026(3)  0.7299(5)  7.3(1)  C(34)  0.5594(3)  0.0679(3)  0.8551(4)  7.2(1)  C(35)  0.5447(3)  0.1649(3)  0.8750(4)  6.65(9)  C(36)  0.4760(2)  0.1975(2)  0.7667(3)  5.51(8)  C(37)  0.4941(2)  0.4428(2)  0.2442(3)  4.59(7)  C(38)  0.5017(3)  0.4030(3)  0.1239(4)  6.13(9)  C(39)  0.5859(3)  0.4417(3)  0.0958(4)  7.1(1)  C(40)  0.6626(3)  0.5204(3)  0.1880(5)  6.36(10)  C(41)  0.6566(3)  0.5591 (3)  0.3072(5)  7.5(1)  C(42)  0.5734(3)  0.5206(3)  0.3364(4)  7.3(1)  C(43)  -0.0096(2)  0.5546(2)  0.2446(3)  4.49(7)  C(44)  -0.0167(3)  0.6001(3)  0.3642(3)  5.82(9)  C(45)  -0.0637(3)  0.6859(3)  0.3754(4)  7.0(1)  C(46)  -0.1055(3)  0.7242(3)  0.2654(5)  7.1(1)  C(47)  -0.1008(3)  0.6794(3)  0.1452(5)  7.3(1)  C(48)  -0.0530(3)  0.5947(3)  0.1349(4)  6.19(9)  a  B. eq  = (8/3)K IZUija*ia*j{araj). 2  198  Part 1:  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Table 1-6.  Experimental  199  Selected bond lengths [A] in 175 with estimated standard deviations in parentheses  Bond  Distance [A]  Bond  Distance [A]  Ni(1)-N(1)  1.900(2)  Ni(1)-N(2)  1.892(2)  Ni(1)-N(3)  1.898(2)  Ni(1)-N(4)  1.887(2)  0(1)-C(2)  1.399(4)  0(1)-C(3)  1.401 (4)  0(2)-C(2)  1.414(4)  0(2)-C(21)  1.436(4)  0(3)-C(3)  1.419(4)  0(3)-C(23)  1.468(9)  0(3)-C(23a)  1.24(1)  N(1)-C(1)  1.368(4)  N(1)-C(4)  1.379(3)  N(2)-C(6)  1.363(3)  N(2)-C(9)  1.388(4)  N(3)-C(11)  1.375(4)  N(3)-C(14)  1.380(3)  N(4)-C(16)  1.398(3)  N(4)-C(19)  1.363(4)  C(1)-C(2)  1.505(4)  C(1)-C(20)  1.368(4)  C(3)-C(4)  1.521(4)  C(4)-C(5)  1.358(4)  C(5)-C(6)  1.409(4)  C(5)-C(25)  1.503(4)  C(6)-C(7)  1.429(4)  C(7)-C(8)  1.352(4)  C(8)-C(9)  1.440(4)  C(9)-C(10)  1.386(4)  C(10)-C(11)  1.393(4)  C(10)-C(31)  1.494(4)  C(11)-C(12)  1.431 (4)  C(12)-C(13)  1.355(4)  C(13)-C(14)  1.424(4)  C(14)-C(15)  1.392(4)  C(15)-C(16)  1.379(4)  C(15)-C(37)  1.495(4)  C(16)-C(17)  1.424(4)  C(17)-C(18)  1.350(4)  C(18)-C(19)  1.435(4)  C(19)-C(20)  1.411(4)  C(20)-C(43)  1.499(4)  C(21)-C(22)  1.51(2)  C(23)-C(24)  1.35(2)  C(23a)-C(24a)  1.47(5)  C(25)-C(26)  1.366(4)  Part 1:  Table 1-7  a  Experimental  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Atom Coordinates and B g . [A ] for 236 e  e . eQ  2  Atom  X  y  z  Beg.  Ni(1)  0.13527(1)  0.17533(6)  0.34572(1)  3.298(7)  N(1)  0.07988(8)  -0.0955(4)  0.35457(9)  3.63(5)  N(2)  0.1437.1(9)  0.2216(4)  0.42848(8)  3.61(5)  N(3)  0.19076(8)  0.4435(4)  0.34637(9)  3.57(5)  N(4)  0.13797(9)  0.2095(4)  0.25883(8)  3.60(5)  C(1)  0.0550(1)  -0.2484(5)  0.3124(1)  4.46(7)  C(2)  0.0153(1)  -0.4260(6)  0.3368(2)  5.29(8)  C(3)  0.0159(1)  -0.3822(6)  0.3967(2)  5.17(8)  C(4)  0.0562(1)  -0.1788(5)  0.4095(1)  4.30(6)  C(5)  0.0728(1)  -0.0775(6)  0.4654(1)  4.78(7)  C(6)  0.1140(1)  0.1151(5)  0.4763(1)  4.14(6)  C(7)  0.1348(2)  0.2438(6)  0.5297(1)  5.16(8)  C(8)  0.1755(1)  0.4221(6)  0.5139(1)  4.88(7)  C(9)  0.1800(1)  0.4056(5)  0.4505(1)  3.89(6)  C(10)  0.2137(1)  0.5477(6)  0.4049(1)  4.30(7)  C(11)  0.2012(1)  0.5403(5)  0.2943(1)  4.22(7)  C(12)  0.1725(1)  0.4173(5)  0.2443(1)  3.83(6)  C(13)  0.1698(1)  0.4561 (6)  0.1820(1)  4.57(7)  C(14)  0.1334(1)  0.2685(6)  0.1573(1)  4.63(7)  C(15)  0.1146(1)  0.1229(5)  0.2049(1)  4.39(7)  = (8/3)7i 2£l/,ya*/a*y(a/-ay). 2  200  Part 1:  Table 1-8  Synthesis and Study of Pyrrolic Pigments for Use in PDT  Experimental  Bond lengths in 236 [A] Bond  Distance [A]  Bond  Distance [A]  Ni(1)-N(1)  1.893(2)  Ni(1)-N(2)  1.845(2)  Ni(1)-N(3)  1.867(2)  Ni(1)-N(4)  1.931(2)  N(1)-C(1)  1.332(3)  N(1)-C(4)  1.409(3)  N(2)-C(6)  1.384(3)  N(2)-C(9)  1.334(3)  N(3)-C(10)  1.470(3)  N(3)-C(11)  1.291(3)  N(4)-C(12)  1.384(3)  N(4)-C(15)  1.352(3)  C(1)-C(2)  1.402(4)  C(2)-C(3)  1.343(4)  C(3)-C(4)  1.412(4)  C(4)-C(5)  1.381(4)  C(5)-C(6)  1.373(4)  C(6)-C(7)  1.419(4)  C(7)-C(8)  1.352(4)  C(8)-C(9)  1.411(3)  C(9)-C(10)  1.480(4)  C(11)-C(12)  1.405(4)  C(12)-C(13)  1.390(3)  C(13)-C(14)  1.371 (4)  C(14)-C(15)  1.387(4)  201  202  4.  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Chem., Int. Ed. Engl. 1992,57, 891.  (416)  Cheng, R.-J.; Chen, Y.-R.; Chuang, C.-E. Heterocycles 1992, 34, 1.  (417) Manka, J.S.; Lawrence, D.S. Tetrahedron Lett. 1989 30(50) 6989. (418)  (a)  International  Tables for X-Ray Crystallography;  Kynoch Press: Birmingham,  UK, 1974; Vol. IV, pp 99-102, 149-150. (b) International Tables for Crystallography; Kluwer Academic Publishers: Boston, 1992; Vol. C, pp 219-222. (419) (420)  Kim, J. B.; Adler, A. D.; Longo, F. R. In The Porphyrins; D. Dolphin, Ed.; Academic Press: New York, 1978; Vol. 1; pp 85-100. Adams, H.; Bailey, N.A.; Fenton, D.E.; Moss, S.; Rodriguez de Barbarin, CO.; Jones, G. 7. Chem. Soc, Dalton Trans., 1986, 693.  PART 2  meso-PHENYLDIPYRRINS SYNTHESIS AND METAL COMPLEX FORMATION  223  1  As  INTRODUCTION  described in Part 1, the synthesis of large quantities of meso-phenyl-  dipyrromethane and meso-diphenyltripyrrane became feasible. Aside from their main use as starting material for the directed synthesis of 'N-confused' and meso-diphenyl porphyrins etc., their oxidation to the corresponding unsaturated novel raeso-phenyl substituted dipyrrins and tripyrrins, and a study of their metal-complexing properties appeared to be an interesting goal.  1.1  ALKYLDIPYRRINS AND THEIR METAL COMPLEXES Dipyrrins (1) are basic, brightly colored, fully conjugated and flat bipyrrolic  pigments. Their propensity to strongly chelate transition metals has long been recognized.  1  Scheme 2-1 outlines 1  the principal pathways for the synthesis of alkyldipyrrins (2) and their chelate type mode of metal complex formation. Five main synthetic pathways 2  can be distinguished: A  The 'classic' and most versatile reaction is the acid catalyzed condensation of an a,B-  alkyl-a'-free pyrrole (2) with a trialkylpyrrole-a-aldehyde (3)  1-3  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  B  Introduction  224  The reaction of an a,6-alkyl-a'-ethyloxycarbonylpyrrole (5) in concentrated formic  acid induces the cleavage of the ester, spontaneous decarboxylation to the a-unsubstituted pyrrole, and subsequent fusion by formic acid of two units to form the desired dipyrrin C  4  The halogenation of an a-unsubstituted a'-methylpyrrole (6) induces a head-to-tail  condensation of two molecules of the initially formed a-(halomethyl)-a-unsubstituted pyrrole to give 2 (R = B r , C H 2 B r )  R  u  8 R  M 9, M = C a  2 +  10, M = C o , Ni , C u , Z n 2 +  Scheme 2-1  2+  2 +  ;  Synthetic pathways towards dipyrrins Reaction conditions: (i) HBr/EtOH; (ii) HCOOH cone; (iii) Br2/AcOH; (iv) oxidation by a variety of oxidants; (v) Br2; (vi) I.CaO, 2. M(ll)X ; (vii) M(II)X2 n  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  D  Introduction  225  The oxidation of hexaalkyldipyrromethanes (7) by a wide variety of oxidants, e.g.  ferric chloride , or DDQ 1  5  The dipyrrins prepared by methods A - D are generally isolated as their stable and well crystallizable hydrobromide or hydrochloride salts. E  The meso-bromination of a dipyrromethane to yield the meso-bromo-dipyrromethane  8, and subsequent reaction with calcium oxide to form the calcium complex 9, yield directly a metal chelate.  This calcium chelate can easily be transmetallated with a variety of  transition metals.  6-8  In all other cases, reaction of dipyrrin 2 with a divalent transition metal salt yields the corresponding dipyrrinato complexes 10. None of the methods A , B , C or E have the potential to give access to meso-substituted, a,B-free dipyrrins such as, for instance, 11. Only route D offers access to the title compounds by oxidation of a meso-phenyldipyrromethane . As will be 9  outlined later in detail, this route was, indeed, successful in providing the title compounds. 11  Hexaalkyldipyrrins, such as 2, have been of interest because of their relation to the biological important porphyrins and bile pigments and, consequently, their use as intermediates in the synthesis of them. ' 10  11  In fact, dipyrrins were the obligatory  intermediates in Fischer's porphyrin synthesis, historically the first discovered method for preparing these macrocycles.  12  It was also Fischer who pointed out the strong sternutatory  (causing sneezing) effect of these compounds, which were particularly strong of 6-free dipyrrin hydrobromides.  2,13,14  Luckily, this effect could not be detected in the 8,8'-  unsubstituted meso-phenyldipyrrins or its salts.  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  1.2  Introduction  226  meso-SUBSTITUTED 4,6-DIPYRRINS meso-Alkyl substituted dipyrrins are less common. ' 1  15,16  This can, perhaps, be  explained by the fact that most naturally occurring porphyrins are meso-unsubstituted. In the case of meso-substituents containing an oc-hydrogen atom the dipyrrin salt (12) will tautomerize, upon deprotonation, to yield the correspondingraeso-ylidenedipyrrans(13) (Figure 2-1). > 10  Figure 2-1  17  18  One such example of this class was recently described by Xie et al.  19  meso-Methyldipyrrin - meso-ylidenedipyrrane equilibrium  Reports ofraeso-phenylsubstituted dipyrrins are even rarer. In fact, the author is aware of only three previous syntheses, two of which are shown in Scheme 2-2. Rogers confirmed in 1943 a finding from 1908 , which described the formation of 14 by reaction of 20  2,4-diphenylpyrrole (15) with in situ generated benzoylchloride. '  21 22  In a similar approach,  Treibs and co-worker reacted pyrrole 16 with benzoylchloride to yield the hexasubstituted raeso-phenyldipyrrin hydrochloride 17. It is noteworthy that in both instances the pyrroles 4  were substituted, particularly at one a - and at least one B-position.  This prevents  polymerization of the pyrroles during the harsh reaction conditions. Consequently, these methods are not options to synthesize me so-phenyl, a-unsubstituted dipyrrins.  A  disadvantage of the protecting a-phenyl moieties is that they introduce severe steric interligand interactions upon metal complex formations. Moreover, meso-phenyl substitution concomitant with B-substituents introduces intra-ligand steric interactions which can, for  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Introduction  227  instance in the case of 14, lead to deviations from planarity and even chemical instability. No report was given on the metal complexation properties of either 14 or 17.  16  Scheme 2-2  17  Formation of meso-phenyldipyrrins Reaction conditions: (i) P O C I 3 / A ; (ii) A  In 1985 an X-ray structure of  bis[l-(2,6-dichlorobenzyl)-5-(2,6-dichlorophenyl)-  dipyrrinato]-zinc(II) was reported. This meso-phenyl and a-substituted dipyrrin complex 24  was the kinetic product in the Rothemund-type condensation of the sterically hindered 2,6dichlorobenzaldehyde with pyrrole, and its isolation was unexpected and fortuitous. Thus, this synthetic pathway towards meso -phenyl dipyrrins and their metal complexes cannot be generalized.  Recently some reports appeared in the literature in which me so-phenyl  substituted dipyrrin moieties were integral parts of larger molecules. The BF2-complex of an a-methyl-mes'o-phenyl-dipyrrin unit was the input unit of a molecular photonic wire and an 25  a-thiophenyl and 6-alkyl substituted meso-phenyldipyrrin was synthesized in the course of research towards polyheterocyclic ligands ; however, neither the complexing properties nor 26  the conformation of this compound was reported.  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Introduction  228  The stereochemistry of these ligands around the metal is dependent on the bulkiness of the substituents in the a - and a'-position, and has found interest since Porter called 27  28  attention to this phenomenon. However, only limited structural data are available. ' ' 27  29  30  Few a-unsubstituted dipyrrinato complexes have been prepared " and in no case has a 31  34  crystal structure been described. Therefore, it was interesting to investigate the complex geometry of the a,a'-unsubstituted meso-phenyldipyrrin ligands of type 2 and to contrast these findings with published data. This and the general interest for novel ligand classes for use in transition metal catalysis, photometric metal detection or biomedical purposes 35  36  37  prompted the investigation of the synthesis and the metal complexing properties of a , B unsubstituted meso-phenyldipyrrins.  Other more distantly related meso-substituted pigments have been described. For instance, Wagner et al.  38  reported the crystal structure of a lO-aryl-bilatriene-a&c.  Structurally related to dipyrrins are 1,9-dioxodipyrrins (propentdyopent). For their synthesis see Bonnett and co-workers , and for the structure of a bis(l,9-dioxodipyrrinato)Cu(II) 39  complex see Balch and co-workers . 40  1.3  4,7-DIPYRRINS Reports on a-B-linked 4,7-dipyrrins (as well as on B,B'-linked dipyrrins) are scarce. 41  They are reportedly fairly unstable and this might be the reason that they are not well 42  characterized.  As the a-6-linked isomer to 20, meso-phenyl-2,3'-dipyrromethane, was  synthesized in due course of the studies described in Part 1, section 2.2.2, attempts to oxidize this compound to the corresponding 4,7-meso-phenyldipyrrin suggested itself. However, all experiments led to no avail.  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  1.4  Introduction  229  TRIPYRRINS AND RELATED TRIPYRROLIC PIGMENTS Although the biosynthetic pathway for all tetrapyrrolic pigments involves the  porphobilinogen deaminase catalyzed tetramerization of porphobilinogen to a porphyrinogen via, undoubtedly, a tripyrrane intermediate, these tripyrrolic compounds have received only little attention, as have their dehydro species. " 43  45  Also in comparison to the wealth of  knowledge on linear tetrapyrrolic compounds such as bilanes, biladienes, bilines and bilones, the knowledge about the tripyrrolic congeners is minimal.  To the best of our knowledge,  10  only one tripyrrinone has been structurally characterized.  46  Coordination compounds of  tripyrrinones have been investigated and used in intriguing carrier mediated proton driven 47  countertransport chains in which metal ions are transported across a liquid membrane against a concentration gradient.  48  Apparently the only tripyrrolic pigments of wider interest are those related to prodigiosene (18), the first isolated member of a class of naturally occurring pigments with strong antimicrobial and cytotoxic properties, all possessing the 2-pyrrolyldipyrrin skeleton. '  49 50  OCH  3  As outlined in Part 1, section 2.2.2, no meso-phenyl substituted tripyrrane was previously known. It was, therefore, interesting to study the oxidation of this tripyrrane in order to see whether the corresponding meso-phenyltripyrrin, similarly not described before, could be derived.  Section 2.4 of this Part describes the results of these studies.  230  2.  2.1  RESULTS AND DISCUSSION  SYNTHESIS OF 5-PHENYLDIPYRROMETHANES The meso-phenyldipyrrins 19 and 20 were synthesized by the acid catalyzed  condensation of benzaldehyde (21) or p-nitrobenzaldehyde (22), with pyrrole.* Pyrrole was also used as solvent according to a procedure of Lee and Lindsey (see Scheme 2-3). The 9  synthesis of 19 offers the great practical advantage over the synthesis of 20 or other dipyrromethanes described by Lee and Lindsey, in avoiding any chromatography during the workup or purification of the compound, thus it is amenable to large scale (> 10.0 g product per experiment) preparations. The higher electrophilicity of /?-nitro benzaldehyde compared to benzaldehyde likely results in a faster reaction rate and a stabilization of the resulting dipyrromethane towards acid catalyzed decomposition. Both these aspects in combination with the simple workup explain the high overall yield of 82 % for 19 vs. the reported 49 %  9  for 20. R  22, R = N 0 21, R = H  Scheme 2-3  2  19, R = N 0 20, R = H  R  2  23, R = N 0 11, R = H  2  Synthesis of meso-phenyldipyrranes and meso-phenyldipyrrins Reaction conditions: (i) r.t., TFA; (ii) benzene, DDQ; (iii) NaBH4/MeOH  * F o r an optimized procedure for the synthesis of 20, see Part 1, section 3.3.  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  2.2  Results  PREPARATION AND CHARACTERIZATION OF  231  meso-  PHENYLDIPYRRINS Dehydrogenations with DDQ have found wide application in the synthesis of pyrrolic pigments.  In particular, DDQ is useful in the conversion of any type of reduced  51  porphyrins (e.g. porphyrinogens or chlorins) to the corresponding fully unsaturated porphyrins.  52  Porphyrinogens are intermediates in the TPP synthesis according to the  methods of Adler  53  or Lindsey , i.e. the acid catalyzed cyclization of pyrrole and 54  benzaldehyde. Hence, it was not unexpected that the reaction of raeso-phenyl-a,a'-dipyrrins 19 or 20 with one equivalent of DDQ smoothly formed the desired dipyrrins 23 and 11, respectively (Scheme 2-3). p- and o-Chloranil are equally well suited to perform the conversion. Reduction of 11 or 23 with NaBHi in MeOH regenerates the leuko form 20 or 19.  In dilute solution, the oxidation products are bright yellow in color. The optical  spectrum of 20 under acidic and basic conditions is shown in Figure 2-2.  6E+04-I i > 4E+04-  •  E —  2E+04-  0E+00 250  350  450  550  650  X [nm]  Figure 2-2  UV-visible spectrum of 11 in MeOH/trace N H 3 ( HCI ( — ) , and 23 in MeOH/trace HCI (----)  ), and in MeOH/trace  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Results  232  The two-band pattern of the protonated species is analogous to that of 3,3',4,4',5,5'tetramethyldipyrrin , but -14 nm hypsochromically shifted, with slightly lower extinction 15  coefficients.  The bands have been assigned to 7U*<—n transitions and are indicative of the  marked planarity of these fully conjugated aromatic systems. Addition of acid protonates the basic imine-type nitrogen of the 2H-pyrrole unit and this removal of non-degeneracy of the linear resonator in combination with the presence of a positive charge induces a bathochromic shift of 42 nm and a doubling of the extinction coefficient.  55  For steric  reasons, it can be inferred that the phenyl moiety is approximately perpendicular to the plane of the dipyrrin. Consequently, the phenyl group is not in full conjugation with the pyrrolic system and substituents on the phenyl group minimally influence the 7t-cloud of the dipyrrin. This explains the close similarity of the optical spectrum of 11 and its /?-nitroderivative 23; a similar situation is also found in variously phenyl substituted TPPs.  56  The  optical spectrum of 11 is more similar to that of the hexamethyldipyrrin than to the spectrum of 14 which is about 110 nm bathochromically shifted , possibly reflecting the extended 23  conjugation (and distortion) of the system by the a-phenyl groups. The jc-nitro compound 23 and its metal complexes exhibit an additional band at -258 nm which can be attributed to the p-nitrophenyl moiety.  The observed numbers of signals in the !H and C-NMR of the meso -phenyl-a,a 13  dipyrrins indicate a plane of symmetry. This is consistent with formulating the dipyrrins as adopting a planar conformation and a rapid tautomeric exchange of the NH-proton between the two nitrogens. As a result of this fast exchange, the proton is lodged between the two nitrogens, indicated by the low field resonance of 12.5 ppm for this strongly hydrogenbonded proton.* It can be concluded that meso-phenyldipyrrins have the above shown Z  * F o r the energetics of the tautomeric exchange, see F a l k and Hofer interchanges see Falk and M i i l l e r  .  ; for the energetics o f the conformational  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Results  233  configuration (based on the exo-cyclic double bond) and syn conformation (based on the exo-cyclic single bond). The iH-NMR spectrum shifts for the B-protons of 11 of 6.39 and 10  6.47 ppm and for the a-protons of 7.78 ppm attest to the aromatic character of these compounds.  Alkyldipyrrins of type 2 are, owing to their basicity, generally isolated and purified as their hydrobromide or hydrochloride salts.  1  Although conditions for thin-layer and  column chromatography of dipyrrin hydrobromides have been described it is not a common practice. Their free bases are also reportedly less stable. Hence, it was surprising to find that in the case of theraeso-phenyldipyrrins,column chromatography (CH2Cl2/silica gel) of their free bases posed no difficulty.  It should also be noted that, as assessed by UV-visible spectroscopy, the oxidation of several a- and B-substituted meso-Phenyldipyrromethanes such as 2,2'-dicarbonylethoxy-5phenyl-3,3',4,4'-tetramethyldipyrromethane, with DDQ, to their corresponding dipyrrins is possible.  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  2.3  Results  234  FORMATION AND CHARACTERIZATION OF TRANSITION METAL CHELATES OF roeso-PHENYLDIPYRRINS  2.3.1  THE COMPLEXES OF Ni(ll), Cu(ll), AND Zn(ll)  General Data and Synthesis  A concentrated MeOH-solution of the meso-phenyldipyrrins 11 or 23 when mixed with a methanolic solution of the divalent metal ions C o , N i , C u , and Z n 2+  2 +  2 +  2 +  as their  acetates, yields the corresponding highly colored metal complexes (Scheme 2-4). The nickel, copper, and zinc complexes are stable and do not require any special handling. The cobalt complexes are characterized by some special features, and are discussed in a separate chapter (Section 2.3.3).  Analyses confirmed the stoichiometry of the precipitates as  M(Ligand)2- The metal complexes formed X-ray quality dichroic (metallic dark green/red) crystals.  M (11)  Scheme 2-4  24, 25, 26, 27,  R R R R  = = = =  N0 N0 N0 H, M  Formation of dipyrrinato complexes from dipyrrins Reaction conditions: (i) M(ll)(acetate)2/MeOH/(base)  2  2  2  M M M =  = Zn = Cu = Ni Ni  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Results  235  IR Spectra  The vibrational spectrum of the metal complexes are similar to those of their ligands. This is not unexpected as conjugation is already attained in the planar ligand moiety before coordination to a metal. Thus, intraligand vibrations will undergo only minor shifts upon metal chelation. This has been rationalized before for the metal complexes of hexaalkyldipyrrins.  59  Optical Spectra  The optical properties of the metal compounds are strongly dependent on the central metal and, as expected, the spectra of the two nickel chelates 26 and 27 are very similar. (Figure 2-3 and 2-4). In solution, they are brilliant orange in color.  6E+04-1  250  350  450  550  650  X [nm]  Figure 2-3  UV-visible spectra of 27(  ) and 26 (  This similarity, particularly with respect to X  max  ) in CH2CI2  and log e, is indicative of a very  similar stereochemistry of these two compounds. The optical spectrum of the zinc chelate 24 (bright yellow color) resembles that of the protonated ligand suggesting the absence of any metal->metal transitions (Figure 2-4). However, charge transfer ligand—>metal transitions  Results  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  are generally observed in this energy region and they cannot be excluded.  27  236  However, other  authors have assigned this band exclusively to intraligand n*<—n transitions and the bands 60  in the 320-350 nm region to charge transfer transitions. The electronic spectra of the nickel and copper chelates 26, 27, and 25 follow the same pattern as that of 24. The optical spectra of the metal chelates are nearly indistinguishable in non- or weakly coordinating solvents such as benzene, methanol, methylene chloride or chloroform but show changes in pyridine, most noticeable for the zinc chelate 24, as also shown in Figure 2-4. Table 2-1 lists selected optical data of some known dipyrrinato-metal complexes (shown below) and of the novel compounds 24, 25 and 26.  6E+04-,  250  350  450  550  650  X [nm]  Figure 2-4  UV-visible spectra of 25( CH.2Cl2/20%pyridine  ) and 24 (  ) in CH2CI2, and of 24 (  ) in  When comparing the longest wavelength absorption of the zinc chelate 24 at 486 nm against the equivalent transitions of the alkyl substituted analogies 27-34, it is remarkable that the meso-phenyldipyrrin chromophore is distinguished by the highest energy transition. An equivalent trend can be seen in the nickel (26 vs. 35-38) and copper (25 vs. 39-42) chelate series.  Hyperconjugation effects have been suggested for the progressive  bathochromic shift with increasing methyl-substitution.  32  Extendedft-conjugationcan be  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Results  237  evoked for the bathochromically shifted optical spectrum in case of dipyrrins 30 and 34. The absence of both effects in the zinc, nickel and copper chelates 24-27 rationalizes their relatively high transition energies. The introduction of araeso-methylsubstituent in chelates 31 and 32 leads, when compared to their meso-unsubstituted analogies 29 and 30, to a relatively small change in the energy of the longest wavelength transition, however, their extinction coefficients significantly decrease. This, based on theoretical considerations, may be taken as a sign of distortion from planarity.  23  /  \  R"  R  R"  \  /  R  M  \  12 p/neso  R  R'  R"  19  -H  -H  -H  -Ph  25  -H  -Me  -Me  -H  Zn  26  -Me  -H  -Me  -H  Zn  27  -Me  -Me  -Me  -H  Zn  28  -Me  -C02Et  -Me  -H  Zn  29  -Me  -C0 Et  -Me  -Me  Zn  30  -Me  -Me  -Me  -Me  Zn  31  -C02Et  -CI  -CI  -H  Zn  32  -Ph  -H  -H  -H  Zn  22  -H  -H  -H  -Ph  Ni  33  -Me  -H  -Me  -H  Ni  34  -Me  -Me  -Me  -H  Ni  35  -Me  -C02Et  -Me  -H  Ni  36  -Ph  -H  -H  -H  21  -H  -H  -H  -Ph  37  -H  -Me  -Me  -H  Cu  38  -Me  -Me  -Me  -H  Cu  39  -Me  -C02Et  -Me  -H  Cu  40  -Ph  -H  -H  -H  Cu  2  c  M  2 +  Zn  Ni c  Cu  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Results  238  Based on the foregoing, the high extinction coefficients of the metal chelates of the meso -phenyldipyrrins seem to indicate that the ligands are flat. In the absence of any 8substituent and hence any intraligand steric crowding, and in analogy to the conformation of TPPs , this appears to be a reasonable assumption. As will be detailed later, the single 61  crystal X-ray structure of 27 (and of 48) shows that the assumption of planarity is, in fact, valid. Table 2-1  UV-visible data and dihedral angles of literature known and novel dipyrrinato-complexes  ^max (log  e)  a  909  19  486 (4.97)  25  500 (5.08) , 483 (4.97)  909  thisvok 27  26  488 (4.22) , 469, sh  909  27  27  505 (4.93) , 487 (5.07)  909  27  28  490 (5.33) , 446, sh  909  27  d  e  e  e  e  15  29  501 (5.05)  30  505 (4.06)  31  537 (5.13)  32  552 (4.93) , 510 (5.06)  909  27  22  484 (4.63)  d  42^, 38.5'  33  512 (4.85)  e  th'swoik 29,59  34  531 (4.70) , 462 (4.57)  59  35  495 (4.99)  e  60  36  540 (4.85)  e  21  474 (4.72)  e  37  471 (4.85) , 409 (4.61)  38  525 (4.65) , 471 (4.76)  39  495 (5.05)  40  564 (4.93) , 509 (4.70)  f  e  15  f  8,62  e  e  76.3'  e  £60/, 66  h  e  e  e  in  n*<r-n  CHCI3;  Motekaitis a n d Martell  8  31 thiSWDlk 31  e  o f longest wavelength  d\n CH2CI2;  reference  b  [nm]  a  dihedral angle [°]  50 ,63i 68/  59  66'  30,60  90^,73/  31  h  transition, ^ b e t w e e n p l a n e s formed by the ligands,  'solvent not s p e c i f i e d ;  9assumed  angle;  c  p-N02-phenyl;  ^ c a l c u l a t e d a c c o r d i n g to  'from X-ray c r y s t a l structure; / b a s e d o n ligand field t r a n s i t i o n a n a l y s i s  3 1  Part 2: meso-Phenyldipyrrins - Synthesis and Metal Complex Formation  Results  239  The stereochemistry of the ligands around the central metal is strongly dependent on the metal type. The preference of zinc(II) for a tetrahedral and of nickel(II), and, even more so, of copper(II) for a square planar coordination sphere is well documented.  63  Regardless  of the a-substituents present in the dipyrrin ligands, the realization of a tetrahedral coordination sphere poses no inter-ligand steric interactions. Consequently, and in analogy to the stereochemistry of the zinc chelates of alkyldipyrrins, compound 24 can be assigned a tetrahedral structure. ' ' ' 8  27  32  60  The picture is more complex for the nickel and copper  chelates. It has been found in previous studies that a-substituents prevent square planar coordination due to inter-ligand crowding. This forces the complex into a distorted tetrahedral structure in which the two approximately planar a-methyldipyrrinato ligands are inclined (as determined by X-ray crystal structure analysis) at an angle (referred to as dihedral angle) of 76.3° for the nickel chelate 3 5  29  and 66° for the copper chelate 4 1 . With 30  hydrogen as the sole cc-substituents no a priori  statement can be made about the  stereochemistry around the metal. It has been suggested that some electronic interaction ex