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Synthesis of porphyrins, chlorins and bacteriochlorins by chemical modifications of chlorophyll a Ma, Lifu 1995

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SYNTHESIS OF PORPHYRINS, CHLORINS AND BACTERIOCHLORINSBY CHEMICAL MODIFICATIONS OF CHLOROPHYLL aBYLIFU MAB. Sc., Wuhan University, 1985M. Sc., Wuhan University, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHThOSOPHYinTHE FACULTY OF GRADUATh STUDIESDEPARTMENT OF CHEMISTRYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJuly 1995© Lifu Ma, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department ofDateCke,vvWt°The University of British ColumbiaVancouver, CanadaDE-6 (2/88)11ABSTRACTThe goal of this project was to synthesize new porphyrins, chlorins andbacteriochlorins via chemical modifications of chlorophyll a so that new chemistry andapplications of chlorophyll a and its related derivatives could be developed. Amagnesium-free yet stable derivative of chlorophyll a, 7, isolated from the blue algaSpirulina maxima, was employed as the common intermediate in this work.As the first objective, new methods of asymmetric hydroxylation andregioselective oxidation of chlorophyll derivatives were successfully developed.Stereoselective synthesis of natural antioxidative chlorins 81, 82(S), 82(R), 83(R), 84 and102, isolated from marine metabolites, was performed in a short and effective way. Thisbiomimetic synthetic approach helped to elucidate the possible biogenetic evolution ofthese antioxidative chlorins from chlorophyll a.The second objective of this work was to design a method to convert chiorin 112into porphyrin 114. A novel and effective acid-catalyzed tautomerization reaction wasdiscovered and optimized, which has provided a new view on the migration of hydrogensin the saturated ring IV to the exocyclic ring V. The porphyrins so produced were usedas intermediates for the further preparation of chlorophyll related petroporphyrins andregiochemically-pure benzoporphyrin derivatives (BPD5).Making use of the aforementioned tetrapyrrolic materials, the third objective ofthis work was to develop new photosensitizers for photodynamic therapy of tumors. Newmonovinylporphyrins and an [A,C]-divinylporphyrin 147 were synthesized. Diels-Alderreaction of these (di)vinylporphyrins with dimethyl acetylenedicarboxylate (DMAD)afforded new regiochemically-pure BPDs 125 and 141 and dibenzoporphyrin derivative165. These new sensitizers have characteristics that meet or exceed the promisingchemical features of benzoporphyrin derivative monoacid ring A (BPDMA), a secondgeneration sensitizer in Phase-TI clinical trials.The final objective of this work was to exploit the nucleophilic behaviour of thebicyclic amidines, l,8-diazabicyclo[5.4.O]undec-7-ene (DBU) and 1,5-diazabicyclo-111(I7jNHN\83(R)(,N HNC02R114MeOOC N HN’NH 000Me‘I COOMeC02R3165jNHN\82(R)C02R OH112HN’NHNC’CO2Me147[4.3.O]non-5-ene (DBN). Two reactions were examined. Firstly, DBU acting as adifunctional nucleophile quantitatively reacted with DMAD to afford a fused tricyclicderivative 176. Secondly, 7, a weak electrophile which alone does not electrophilicallyreact with DBU or DBN, has reacted, through catalytic activation by Lewis acids, withnucleophilic DBU and DBN to form chlorin e6 amides 185 and 186. These results havebrought about further understanding of the nucleophilicity as well as the basicity of thesecommon organic bases.82(S)t!O2MeMeOOC /—MeOOC ,NH N\—N HN\C02R3125NH N(N HNJ102MeOOC y..NH N.ç<N HN(’--)OeMeO2R3141OMeH176i-NH NI(_A ç’ ONHNCO2MeCO2Ne n185. n=3186. n=1ivTABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTS ivLIST OF TABLES viiiLIST OF FIGURES ixLIST OF SCHEMES xiiiLIST OF ABBREVIATIONS xviACKNOWLEDGEMENTS xixCHAPTER ONE INTRODUCTION 11.1 Overview 21.2 Structural Features 91.3 Nomenclature of Porphinoids Related to Chlorophylls 111.4 Optical Absorption Spectra 141.5 Overview of Chlorophyll Chemistry 181.5.1 Structure and Occurrence of Chiorophylls 191.5.2 Chemical Modifications of Chlorophyll a Reaction of the Isocyclic Ring Epimerization and Enolization Ring Opening Pigments from Standard ChlorophyllDegradation Chemistry 311.5.2.2 Peripheral Reactions 331. Electrophilic Substitution 331. Oxidation 351.6 Synthetic Aspects of Natural Chlorins 381.6.1 Partial Synthesis of Dimethyl Tunichlorin fromChlorophyll a 39V1.6.2 Partial Synthesis of Methyl Bacteriopheophorbide c[Et,Me] from Methyl Pyropheophorbide a 401.6.3 Partial Synthesis of Heme d 431.6.4 Total Synthesis of Bonellin Dimethyl Ester 45CHAPTER TWO STEREOSELECTIVE SYNTHESIS OF NATURALANTIOXIDATIVE CHLORINS 472.1 Synthetic Targets and Research Objective 482.2 Synthetic Approach 532.3 Starting Materials : Natural Pheophorbides 552.4 Hydroxylation Studies Leading to AsymmetricHydroxylation 592.5 Model Studies for Hydroxylactonization 682.6 Regioselective Oxidation 712.7 Structure and Spectral Characterization 752.8 Biogenetic Rationalization 832.9 Summary 87CHAPTER THREE PHYTOPORPHYRINS: NOVEL SYNTHESIS ANDAPPLICATIONS 1083.1 Research Objective 1093.2 Synthesis of Divinylchlorin and its Photooxygenation 1103.3 Novel Synthesis of Phytoporphyrins 1173.4 Tautomerization Mechanism 1223.5 Applications 1253.6 Summary 127CHAPTER FOUR THE THIRD GENERATION PHOTOSENSITIZERS 1294.1 Background and Research Objective 130vi4.2 Syntheses of Regiochemically-pure BenzoporphyrinDerivatives 1364.2.1 Rationale 1364.2.2 Via 3-Vinylrhodoporphyrin XV Dimethyl Ester 1374.2.3 Via 3-Vinylphytoporphyrin 114c 1444.3 Synthesis of [A,C]-Dibenzoporphyrin Derivative(Bacteriochiorin) 1474.3.1 Rationale 1474.3.2 Synthesis of [A,C]-Divinylporphyrin 1494.3.2.1 Via 3-Vinylpurpurin 113 1494.3.2.2 Via 3-Vinylrhodoporphyrin XV DimethylEster (23) 1594.3.3 Synthesis of [A,Cj-Divinylporphyrin Derivative 1664.4 Monoacid Analogues of New Regiochemically-pureBPDs and [A,C]-Dibenzoporphyrin Derivative 1684.5 Summary 169CHAPTER FIVE DBU AND DBN: NUCLEOPHThICITY vs. BASICITY 1715.1 Background and Research Objective 1725.2 DBU as a Difunctional Nucleophile 1765.3 Nucleophilic Reaction with Pheophorbide a Methyl Ester 1825.3.1 Rationale 1825.3.2 The Generation and Fate of Nucleophilic DBUandDBN 1845.3.3 Structural Elaboration of the Products 1875.3.4 Reaction Mechanism 1945.4 Summary 196CHAPTER SIX EXPERIMENTAL 1976.1 General Methods 198vii6.2 Stereoselective Synthesis of Natural AntioxidativeChlorins 2036.2.1 Starting Materials: Natural Pheophorbides 2036.2.2 Asymmetric Hydroxylation 2086.2.3 Model Studies for Hydroxylactonization 2236.2.4 Regioselective Oxidation 2306.3 Synthesis of Phytoporphyrins 2406.4 Synthesis of Benzo- and Dibenzo-porphyrin Derivatives 2606.4.1 Synthesis of Regiochemically-pureBenzoporphyrin Derivatives 2606.4.2 Synthesis of [A,C]-Divinylporphyrin viaPurpurin 113 2696.4.3 Synthesis of [A,Cj-Divinylporphyrin viaPorphyrin 23 2766.4.4 Synthesis of [A,C]-DibenzoporphyrinDerivative 165 2846.5 Nucleophilic Reactions of DBU and DBN 287REFERENCES 294viiiLIST OF TABLESTable 2.1 Asymmetric Hydroxylation of 5, 7 and 81 with Oxaziridines91, 93 and 94 62Table 2.2 Selected 1H NMR Spectral Data (CDC13,400 MHz) 65Table 2.3 Selected 1H NMR Spectral Data (CDC13,400 MHz) 79Table 2.4 Selected1H- Coupling Constants J (Hz, CDC13) 81Table 2.5 Selected 13C NMR Spectral Data (CDC13) 83Table 3.1 Comparison of Proton Chemical Shifts (CDC13,400 MHz) 112Table 5.1 1H NMR Spectral Data (CDC13,400 MHz) 191Table 5.2 13C NMR Spectral Data (CDC13,400 MHz) 193ixLIST OF FIGURESFig. 1.1 Important Tetrapyrrolic Macrocycles 2Fig. 1.2 The Cofactors Derived from Uroporphyrinogen III 3Fig. 1.3 Biosynthesis of Uroporphyrinogen III 4Fig. 1.4 Examples of Expanded Porphyrins 8Fig. 1.5 Delocalized Electron Pathways of Tetrapyrrolic Macrocycles 9Fig. 1.6 Numbering Systems of Chlorins 11Fig. 1.7 The Four Types of Porphyrin Spectra 14Fig. 1.8 Typical Visible Spectrum of Metallated Porphyrins 16Fig. 1.9 Typical Spectra of Chlorins and Metallochlorins 17Fig. 1.10 Typical Spectrum of Bacteriochlorins 18Fig. 1.11 Chlorophyll a (1) and Chlorophyll b (2) 21Fig. 1.12 Chlorophylls of the c-type (3). Chl c : R1=Me,R2=Et;Chl c2 : R1=Me,R2=CH3;Chl C3 :R1=COOMe,R2=CH3;and Bacteriochlorophyll a (4) 22Fig. 1.13 Esterifying Alcohols of Chlorophylls and Bacteriochlorophylls.From top to bottom: geranylgeraniol (A2,6,1O,14); geranylgeraniol (A2,4,1O,14); phytol; farnesol 23Fig. 1.14 Epimerization at C-132 of Chlorophylls 26Fig. 1.15 Electronic Spectra of 1 (- - -) and the Silylated Enol Ether13( ) 27Fig. 1.16 Cleavage of the Exocyclic Ring by Nucleophiles 28Fig. 1.17 Structures of Bacteriochlorophylls c 40Fig. 2.1 HPLC Chromatography 64Fig. 2.2 Structures and UVIVis Spectra (CDC13)of 7, 82(5), 82(R),83(R) and 87 76Fig. 2.3 Structures and UV/Vis Spectra (CDC13)of 81, 102, 84 and 16 78xFig. 2.4 1H NMR Spectral Comparison (the Low Field Region) of NaturalHydroxychiorins 82(5), 82(R) and 83(R) (CDC13,400 MHz) 80Fig. 2.5 1H NMR Spectrum (CDC13)of Pheophorbide a Methyl Ester (7). 89Fig. 2.6 13C NMR Spectrum (CDCI3)of Pheophorbide a Methyl Ester (7). 90Fig. 2.7 1H NMR Spectrum (CDC13)of132,173-Cyclopheophorbide aEnol (81) 91Fig. 2.8 13C NMR Spectrum (CDC13)of13,17-Cyclopheophorbide aEnol (81) 92Fig. 2.9 1H NMR Spectrum (CDCI3)of132S-Hydroxychlorophyllone a[82(S)] 93Fig. 2.10 13C NMR Spectrum (CDC13)of135-Hydroxychlorophyllone a[82(S)] 94Fig. 2.11 1H NMR Spectrum (CDC13)of132R-Hydroxychlorophyllone a[82(R)] 95Fig. 2.12 1H NMR Spectrum (CDC13)of13R-Hydroxychlorophyllone-lactone a [83(R)] 96Fig. 2.13 1H- COSY Spectrum (CDC13)of132R-Hydroxychlorophyllone-lactone a [83(R)] 97Fig. 2.14 1H NMR Spectrum (CDC13)of Chiorophyllonic Acid a MethylEster (84) 98Fig. 2.15 13C NMR Spectrum (CDC13)of Chlorophyllonic Acid a MethylEster (84) 99Fig. 2.16 1H NMR Spectrum (CDC13)of Purpurin-18 Methyl Ester (87).... 100Fig. 2.17 1H NMR Spectrum (CDC13)of Chlorin P6 Trimethyl Ester (92).. 101Fig. 2.18 1H NMR Spectrum (CDC13)of132R-Hydroxypheophorbide aMethyl Ester [95(R)] 102Fig. 2.19 13C NMR Spectrum (CDC13)of13R-Hydroxypheophorbide aMethyl Ester [95(R)] 103xiFig. 2.20 1H NMR Spectrum (CDC13)of132S-Hydroxypheophorbide aMethyl Ester [95(S)] 104Fig. 2.21 1H NMR Spectrum (CDC13)of132R-Hydroxypheophytin a[96(R)] 105Fig. 2.22 13C NMR Spectrum (CDC13)of132R-Hydroxypheophytin a[96(R)] 106Fig. 2.23 1H NMR Spectrum (CDC13)of132-Oxopyropheophorbide aMethyl Ester (102) 107Fig. 3.1 1H NMR Spectrum (CDC13)of13-Deoxo-13,1d hydro-pheophorbide a Methyl Ester (112a) 113Fig. 3.2 Structures and Comparison of UV/Vis Spectra (CH2C1)ofllOaand7 114Fig. 3.3 1H NMR (CDC13)and UV/Vis (CH2C1)Spectra ofPurpurin 113 115Fig. 3.4 1H- COSY Spectrum (CDC13)of Phytoporphyrin 114a 118Fig. 3.5 Structure and UV/Vis Spectrum (CH2C1)ofPhytoporphyrin 114a 119Fig. 3.6 Structures and Comparison of UVIVis Spectra (CH2C1)of27and116 122Fig. 4.1 Structure and UVIVis Spectrum (CH2C1)of BPD 141 141Fig. 4.2 The Difference nOe Spectra on Regiochemically-pure BPD 141 142Fig. 4.3 Structure and UVIVis Spectrum (CH2C1)of BPD 141 145Fig. 4.4 1H NMR Spectrum (CDC13)of BPD 125 146Fig. 4.5 Structure and UVIVis Spectrum (CH2C1)of Purpurin 150 154Fig. 4.6 1H NMR Spectrum (CDC13)of [A,C]-Divinylporphyrin 125 158Fig. 4.7 Structure and UVIVis Spectrum (CH2C1)of[A,C]-Divinylporphyrin 147 159Fig. 4.8 1H NMR Spectrum (CF3COOD) of Porphyrin Monoacid 157 161Fig. 4.9 1H NMR Spectrum (CDC13)of Imidazoylporphyrin 158 163xiiFig. 4.10 1H NMR Spectrum (CDC13)of Acetylporphyrin 161 165Fig. 4.11 UV/Vis Spectrum (CH2C1)of [A,C]-DibenzoporphyrinDerivative 165 167Fig. 4.12 Synthesis of Monoacid Analogues of BPDs and[A,C]-Dibenzoprphyrin Derivative 169Fig. 5.1 Comparison of 1H NMR Spectra (CDC13)of DBU and TricyclicDerivative 176 177Fig. 5.2 1H- COSY Spectrum (CDCI3)of Tricyclic Derivative 176 178Fig. 5.3 13C NMR and APT Spectrum (CDC13)of TricyclicDerivative 176 179Fig. 5.4 X-Ray Structure of 176 180Fig. 5.5 1H-’H COSY Spectrum (CDC13)of Chiorin e Amide 185 189Fig. 5.6 1H-’3CHETCOR Spectrum (CDC13)of Chiorin e Amide 185 190xiiiLIST OF SCHEMESScheme 1 Syntheses of Silyl Enol Ethers 12 and 13 27Scheme 3 Allomerization and its Proposed Mechanism 30Scheme 4 Electrophilic Substitution Reactions of Chlorins 34Scheme 5 Peripheral Oxidation of Chiorins by 0s04 35Scheme 6 Photooxygenation of 3-Vinylporphinoids 36Scheme 7 Photooxygenation of Bacteriochiorophylls c and e 37Scheme 8 Partial Synthesis of Dimethyl Tunichiorin 39Scheme 9 Partial Synthesis of Bacteriopheophorbide c 42Scheme 10 Partial Synthesis of (±) Porphyrin d 44Scheme 11 Total Synthesis of (±) Bonellin Dimethyl Ester 46Scheme 12 Natural Antioxidants and Their Origins 50Scheme 13 General Strategy for the Synthesis of the Antioxidative Chiorins 54Scheme 14 Synthesis of Enol 81 59Scheme 15 Oxidative Cleavage of Exocyclic Rings 61Scheme 16 Asymmetric Hydroxylation of Chlorophyll Derivatives 63Scheme 17 Model Studies for Periodate Oxidation 70Scheme 18 Regioselective Periodate Oxidation of Hydroxychlorin 82 72Scheme 19 Chlorin 84 and Its Unfavored Non-Esterified Isomers 74Scheme 20 “HI Isomerization” of Chlorophyll Derivatives 109Scheme 21 Synthesis of Divinylchlorin and Its Photoreaction 111Scheme 22 A Possible Mechanism for the Photooxygenation 116Scheme 23 Direct Conversion of Chlorins to Phytoporphyrins 120Scheme 24 Reaction of 3-Vinylchlorin 25 Using Benzoyl Chloride 121Scheme 25 Proposed Tautomerization Mechanism of Chlorins toPhytoporphyrins 123Scheme 26 Structures of Hydroxychlorin 95 and Polyhydroxychiorins119 and 120 124xivScheme 27 Synthesis of Petroporphyrin 121 126Scheme 28 Synthesis of Petroporphyrin 123 127Scheme 29 Synthesis of Benzoporphyrin Derivative 125 131Scheme 31 Various Components of Hematoporphyrin Derivative 133Scheme 32 Major Second Generation Photosensitizers 134Scheme 33 Synthesis of the Biological Active BPD Monoacids (BPDMA) 134Scheme 34 Synthesis of 3-Vinylporphyrin 23 via Basic Aeration ofPheophytin a 137Scheme 35 Purpurin- 18 Methyl Ester (87) and Purpurin-7 TrimethylEster (16) 138Scheme 36 Synthesis of 3-Vinylporphyrin 23 via Hydroxychlorin 95 139Scheme 37 Synthesis of Regiochemically-pure BPD 141 140Scheme 38 Regiochemically-pure BPDs 142 and 143 143Scheme 39 Synthesis of Regiochemically-pure BPD 125 144Scheme 40 Dolphin’s Synthesis of [A,C]-Dibenzoporphyrin Derivative 148Scheme 41 Retrosynthetic Analysis of [A,C]-Divinylporphyrin 147 150Scheme 42 Synthesis of Purpurin 113 via Photooxidation 151Scheme 43 Synthesis of Purpurin 113 via Periodate Oxidation ofDihydroxychlorin 119 152Scheme 44 Protection of Purpurin 113 via the Formation of EthyleneAcetal 150 153Scheme 45 Unsuccessful Transformation of Zn(II) Chiorin 152 156Scheme 46 Synthesis of [A,C]-Divinylporphyrin 147 via Porphyrin 148 157Scheme 47 Synthesis of [A,CJ-Divinylporphyrin 147 via Porphyrin 23 160Scheme 48 Synthesis of [A,C]-Dibenzoporphyrin Derivative 165 166Scheme 49 Resonance Structures of DBU and DBN 173Scheme 50 Examples of DBU and DBN Acting as Nucleophiles 175Scheme 51 Michael-type Addition of DBU to DMAD 181Scheme 52 Nucleophilic Reaction of EtNH2with Pheophorbide aMethyl Ester (7) 183Scheme 53 Nucleophilic Reaction of DBU and DBN with Pheophorbide aMethyl Ester (7) 186Scheme 54 Observed nOe Enhancement for Chlorin Amide e 185 192Scheme 55 Proposed Mechanism for the Formation of Chiorin Amide e185 and 186 195xvxviLIST OF ABBREVIATIONSAc acetylAnal. microanalysesAPT attached proton testBCh1 bacteriochiorophyllBPD benzoporphyrin derivativeBPDMA benzoporphyrin derivative monoacid (ring A modified)br broadBu butylcalcd calculatedCD circular dichroismChl ChlorophyllCOSY two dimensional proton homonuclear correlation spectroscopyd doubletd.e. diastereomeric excess(decomp.) decomposesDBN 1 ,5-diazabicyclo[4.3 .O]non-5-eneDBU 1 ,8-diazabicyclo[5.4.O]undec-7-eneDDQ 2,3-dichloro-5,6-dicyano- 1 ,4-benzoquinoneDMAD dimethyl acetylenedicarboxylateDMF N,N-dimethylformamideEl electron impactEt ethylether diethyl etherFAB fast atom barbardmentHETCOR two dimensional proton heteronuclear correlation spectroscopyHOAc acetic acidHpD hematoporphyrin derivativeHPLC high performance liquid chromatographyxviiHREIMS high resolution electron impact spectroscopyHRFABMS high resolution fast atom bombardment spectroscopyHz Hertzisc intersystem crossingIUPAC International Union of Pure and Applied ChemistryJ coupling constantL. lipid radicalLDA lithium diisopropylamideLiBHT lithium salt of 2,6-t-butyl-4-methylphenolLREIMS low resolution electron impact mass spectroscopyLRFABMS low resolution fast atom bombardment mass spectroscopym multiplet (in NMR)m mass (in mass spectroscopy)mCPBA m-chloroperbenzoic acidmeq milli-equivalentMe methylm.p. melting pointNMR nuclear magnetic resonancenOe nuclear Overhauser effectobsd observedP phytyl (in NMR assignments)PDT photodynamic therapyPh phenylPhCOC1 benzoyl chloridePOV peroxide valueppm parts per millionPr propylpy pyridineq quartetxviiiR R configuration at C-132 or C-151 or C-131 position whereappropriatere face in which the groups in this sequence are clockwiset tripletS S configuration at C-132 or C-151 or C-13’ position whereappropriatesh shouldersi face in which the groups in this sequence are anti-clockwiseTBD 1 ,5,7-triazabicyclo[4.4.O]dec-5-eneTBDMSOTf tert-butyldimethylsilyl triflateTEA triethylamineTFA trifluoroacetic acidTHF tetrahydrofuranTLC thin layer chromatographyTMS trimethylsilylTMSOTf trimethylsilyl triflatetR retention timeUV ultravioletUVIVis ultraviolet and visiblez electron charge (in mass spectroscopy)xixACKNOWLEDGEMENTSFirst, and foremost, an expression of sincere gratitude goes to my supervisor, Prof.David Dolphin, who introduced me to the “colourful world of porphinoids” and whokindly taught me how to keep research “green”.Without the respect and desire for the search for knowledge instilled in me by myparents, Mr. Changquan Ma and Mrs. Shuizhen Song, such an undertaking would noteven have been contemplated. Their constant support and concern have been invaluableand are deeply appreciated. By a happy coincidence, the completion of this thesiscoincided with the fourth birthday of my son Zhifang, who has been a “fatherless child”living with his grandparents now for more than 3 years, a circumstance he has enduredwith good humor and understanding. Had it not been so, this work would still beuncompleted.My intellectual life of the past three years or so has been greatly enriched throughthe friendship of Dr. Ethan Sternberg, one of the most creative of individuals. With him,I have shared the disappointment of failure, the stimulation of discussion and the joy ofsuccess.My efforts towards the preparation of this thesis would have been far less fruitfulwithout the efficient and cheerful assistance and invaluable critique of Dr. VeranjaKarunaratne, Mr. Andrew Tovey and Dr. Ross Boyle, who have been kind enough to readportions of the manuscript.I also owe gratitude to my group, past and present members, too numerous tomention by name. Their collaborations with me have always been stimulating,productive, and above all, enjoyable. A special debt of gratitude goes to Mr. Jack Chowwho provided me technical assistance during the preparation of the manuscript. Thanksgo as well to Drs. Martin Tanner and Tom Money, members of my advisory committee,for suggestions made during the course of the work.1Chapter 1Introduction21.1 OverviewTetrapyrrolic macrocycles (Fig. 1.1), e.g. porphyrins, chiorins, bacteriochiorins,isobacteriochiorins, corphins and corrins, form the macrocyclic skeletons of importantnatural prosthetic groups of living systems on this planet.14 Each of these porphinoidsconsists of four pyrrole-type rings linked together directly or more commonly throughmethine bridges. A striking feature of all these cofactors is that the substitution aroundeach macrocycle is based on the same pattern derived from uroporphyrinogen Ill (Fig.1.2). This common intermediate is biosynthesized from 5-aminolevulinic acid by way ofporphobilinogen and preuroporphyrinogen57(Fig. 1.3).isobacteriochiorin corphin corrinporphyrin chiorin bacteriochiorinFig. 1.1 Important Tetrapyrrolic Macrocycles3CH3H3C‘N N—Fe‘N’ “NH3C \‘ — / CH3HO2C CO2Hhemep AAp“NH HN’A,NH HNApp \uroporphyrinogen III—4’ H3C. 1H3CH3C ..LNNHL/CH3co2%OPhytylbacteriochlorophyll aFine tuning of these porphinoid ligands for optimal biological function isperformed in nature by the appropriate choice of the oxidation levels of the macrocycle,the nature of the peripheral substituents and the coordinated metals8-9(Fig. 1.2). Forexample, porphyrins, the tetrapyrrolic macrocyles at the highest oxidation level,functionCH3H3CH3C.5N’NHL/CH3co2%OPhytylchlorophyll aI /CH3)fl<p /( ‘N<ASLAP PsirohemeA — Corboxymethyl —Cl-I2CO2HP = Carboxyethyl—CH2CHCO2 H-NH2coenzyme F430OHvitamin B12 coenzymeFig. 1.2 The Cofactors Derived from Uroporphyrinogen III4as their ferrous complexes in the binding of dioxygen and electron transport (hemoglobin,myoglobin, cytochromes and cytochrome P-450) and ferric complexes for numerousoxidations utilizing hydrogen peroxide (catalases and peroxidases).1° Chlorophyll a, amagnesium (II) complex of a dihydroporphyrin (a chlorin) is responsible for the light-harvesting and trapping activities in plants and algae.1’ A magnesium containingtetrahydroporphyrin, bacteriochlorophyll a, is the main component of the photosyntheticapparatus of purple and green bacteria. Recent advances in the study of the bacterialphotosynthetic reaction center from the purple bacterium Rhodopseudomonas viridisillustrate the fundamental atomic structure of the basic machinery and the possibleevolutionary sequence with which nature precisely manipulated the basicuroporphyrinogen molecule into an efficient photochemical device (for whichDeisenhofer, Huber and Michel won the Nobel Prize in Chemistry in 1988). 2-13CoASy.%.)l.. OHsuccinyl CoA HN OHH N 5—aminolevulinic acid H H H H H2 II porphobilinogen0 A preuroporphyrinogenglycine THO’.-r_J’OHH2Nglutamic acid AA —p“NH HN’A = Carboxymethyl —CH2CO2 H / NH HNA— —AP = Carboxyethyl—CH2CHCO2 H Puroporphyrinogen IllFig. 1.3 Biosynthesis of Uroporphyrinogen ifi5The constitutional isomers of bacteriochiorins with two adjacent saturated pyrrolicrings are called isobacteriochiorins. They are widely distributed in bacteria and plants.One typical example is siroheme, the iron-coordinated sirohydrochlorin which is theprosthetic group of a number of sulfite and nitrite reductases.14 These enzymes catalyzethe six-electron reduction of sulfite to sulfide and nitrite to ammonia, respectively.’5Further reduction of the tetrapyrrolic ring leads to the nickel hexahydrocorphin ringsystem of coenzyme F4309 (Fig. 1.2). Coenzyme F430 is a prosthetic group of methylcoenzyme M reductase, an enzyme which catalyzes the reductive cleavage of S-methylcoenzyme M [2-(methylthio)ethanesulfonate] to methane.16-17 A final example, thestructurally most complicated member of such macrocycles is vitamin 12. the cobaltcomplex of a corrin. Vitamin 12 is an essential vitamin for human health, a deficiencyof which leads to pernicious anemia.’8 It is also the prosthetic group of numerousenzymes which carry out various rearrangement reactions and trans-methylations.18 Thisunusual organic ligand surrounding the metal cobalt displays many stereogenic centersalong its periphery carrying reactive functional groups. The saga around the completepathway of its biosynthesis from 5-aminolevulinic acid has been successfully uncoveredin 1995 after 25 years of research.18The above examples illustrate the wide diversity of biological functionsperformed by the metallated tetrapyrroles in nature and account for the continued interestof researchers in the isolation, structural and biosynthetic elucidation, and total synthesisof such molecules. Interest in studies aimed at the isolation of new tetrapyrroles led toessentially two phases of development. The first phase, shortly after the discovery of6metalloporphyrin derivatives in petroleums and sendiments in the 1930’s, was initiatedby Treibs’s hypothesis linking these fossil pigments with two classes of biologicalmolecules, chlorophylls and hemes.19 The development in this area has given birth to thefield known today as “organic geochemistry” and these sendimental porphyrin pigmentsare appropriately named “petroporphyrins”. Up to the time of writing this thesis, morethan 80 petroporphyrins have been characterized, each of them either isolated as metal-free or complexed by 4 different metals: nickel, vanadium, copper and gallium.20 Thesecond phase, begun a half century later, resulted from the investigation of vitamin 12biosynthesis and consisted of the search for the new porphinoid structures in marinemetabolites, which has actually brought about the discovery of previously unknownporphinoid macrocycles (some of these new macrocyles even have unusual biologicalfunctions).1 In both cases, isolation, identification, and structural elucidation of the novelstructures, which often occur in trace amounts, had only just become possible due to thesimultaneous development of new separation techniques and Fourier transform NMRspectroscopy.Following the isolation and elucidation of these novel structures there are twoadditional fields of endeavor: total and (sometimes) partial chemical synthesis todemonstrate “the synthetic art of the chemical architect” and last, but not least, deeperunderstanding of the origin of these macrocyclic molecules. Usually the researchbetween these two fields is closely related. The former research not only develops thenew synthetic methodology (e.g. the outstanding synthesis of chlorophyll a21) and thusthe ability to (in most cases) produce sufficient amounts of material for subsequentinvestigations, but also provides conceptual reasons and insights on the latter7(biosynthetic pathways). A good example of this is the synthetic approach of vitaminB12, which uncovered the “dark” variant of the biosynthetic AID-decocorrin —> coffincycloisomerization.3A new practical application of the chemistry of porphinoid compounds is theburgeoning interest in photodynamic therapy that takes advantage of the tetrapyrrolephotosensitized generation of singlet oxygen to attack tumours.22 Porphyrin derivativesincorporating boron clusters are also successfully used in boron neutron capture therapyto deliver radiation to tumors in situ.23 Originally, hematoporphyrin derivatives wereshown to have necrotic activity.24 More recently, attention has shifted to chlorins andbacteriochlorins with their red-shifted absorption spectra since red light penetrates deeperinto tissues than blue light.25 Besides the medicinal application, there is a group ofchemists fascinated by the possibilities of their practical application in a wide diversity offields, such as solar energy conversion26,catalysis27,and the possibilities offered by therapidly expanding area of materials with novel electrical and optical properties.28In addition to medical and industrial applications, tetrapyrrolic macrocycles serveas important model compounds for the study of theoretical concepts such as aromaticity,electron transfer, quantum chemistry and diamagnetic ring currents. Interest in thesystematic exploration of potentially aromatic porphinoid chromophores has led to thesynthesis and study of the larger aromatic pyrrole-containing systems, the so-called“expanded porphyrins”.29 Some of examples are shown in Fig. 1.4. Such systems, byvirtue of containing a greater number of it-electrons, a greater number of donating (e.g.pyrrohc) groups, or a larger central binding core have properties which differ8substantially from the far better studied porphyrin analogues. It has been demonstratedthat the (4n+2) rule of Hückel is valid in these large systems as long as sufficientstablization of planar conformations is provided.30sapphyrin penta;hyrLn rubyrtnJJN Hr[22] poiphycene ( 1.4 Examples of Expanded PorphyrinsThe continuing exploitation and further understanding of the seeminglyinexhaustible treasury of porphinoids bear witness to the enigmatic inventiveness withwhich nature is able to manipulate molecular architecture. It is not unreasonablyoptimistic to believe that an effort with contributions from chemistry, physics, geneticsand biochemistry will make progress toward the ultimate understanding of chemistry ofthese important molecules and, in turn, benefit mankind.[26] porphyrin ( Structural FeaturesPorphinoids, e.g. porphyrins (fully-unsaturated porphinoids), chiorins (dihydroporphyrins) and bacteriochiorins (tetrahydroporphyrins), are tetrapyrrolic macrocyclescontaining four pyrrole units linked by methine bridges. They can be classed as polyenechromogens, as they contains no donor or acceptor groups, and as they are structurallysimilar to the annulenes.31 Each of these species maintains aromaticity in the macrocyclethrough an 18 atom, 18 it-electron system, i.e. 1 8-diazaannulene, inner-outer-inner-outerdelocalization pathway (Fig. 1.5) in accordance with Hückel’s 4n+2 rule (n=4). Themacrocycle generally maintains the planarity demanded by the delocalized It-systemalthough exceptions have been synthesized.32 1H NMR spectroscopy shows clearly thatthis ring system is diatropic and the shielded inner NH protons appear at relatively highfield (ö= 0 to -5 ppm). The outer methine protons, deshielded by the aromatic ringcurrent, appear at 8 to 10 ppm. The remaining two double bonds in porphyrins are cross-conjugated and can be reduced to the corresponding chlorins and bacteriochlorins withoutmarkedly affecting the aromatic it-electron system despite the apparently large decreasein conjugation.NH N.$)N HN(porphyrin chiorin bacteriochiorinFig. 1.5 Delocalized Electron Pathways of Tetrapyrrolic Macrocycles10Many structural modifications of the porphinoids are possible. Azaporphyrins areobtained by replacing the carbon atoms of the methine bridges [i.e. the ci. to positions ofporphyrin in Fig. 1.5) by nitrogen, and the benzoporphyrins possess benzene rings fusedto the 2-3, 7-8, 12-13, or 17-18 positions of macrocycle. Phthalocyanine is acommercially valuable blue pigment which embodies both modifications, and can bedescribed as tetrazatetrabenzoporphyrin. The porphinoids that contain two hydrogenatoms at the center of the molecule, are called the free-base porphinoids. The centralnitrogen atoms are basic, and can accept two more protons, to give the porphinoiddications, and in addition, the two N-H hydrogen atoms are acidic and can be removed bystrong bases to give the porphinoid dianions. Various metal ions can replace the centralhydrogen atoms of the free-bases, when coordination to all four nitrogen atoms ispossible, as exemplified by the Mg2 ion in the chlorophylls.Usually the porphinoid nucleus is a highly thermal-stable macrocyclic system. Itis even stable towards concentrated sulfuric acid and trifluoroacetic acid, both of whichare often used to remove coordinated metals. Conversely, the non-fully conjugatedporphyrinogens such as uroporphyrinogen III are considerably less stable and arerandomized in acids. Solutions of porphinoids are relatively unstable to light, which mayphthalocyanine11result in photooxidation and/or photodegradation of the peripheral substituents and/orporphinoid macrocycles depending on the substrate and reaction conditions.101.3 Nomenclature of Porphinoids Related to ChiorophyllsTwo systems of tetrapyrrole nomenclature are currently in wide use. The Fischersystem for chlorophyll derivatives is shown in Fig. 1.6, and features eight peripheralpositions, numbered 1-8, two additional positions (9,10) associated with the isocyclic ringE, and four interpyrrolic (or meso) positions, designated a, B, y and & Other carbons arenumbered with primes (‘) inward, or with alphabetical letters outward from the centralchelating core. The second nomenclature system, as shown in Fig. 1.6 is recommendedby the JUPAC-ITJB and is based on the corrin (1-20) system of nomenclature. The fourmethine positions are numbered 5, 10, 15, 20, and the nitrogen atoms are numbered 21through 24. Further, the HJPAC-IUB system provides a less ambiguous and more widelyapplicable way to number the extra exocyclic ring or rings fused to the porphinoidnucleus. Systematic names of substituted porphinoids are formed by the application of2a(827b[_0S_&7cCO2PhytyIFischer IUPACFig. 1.6 Numbering System of Chlorins12the rules of systematic organic nomenclature. Despite the advantages of the IUPAC-IUBsystem, the systematic names of chlorins related to chlorophyll a become too long andimpractical. Therefore, the Fischer system is widely used today and has the merit that itenables contemporary and historical work to be integrated and allows continued use of alarge number of classical and indispensable trivial names.Throughout this work, in order to take advantage of both nomenclature systems,the trivial names with the IUPAC-]IJB numbering system, which are also widely adoptedin the literature, will be used. For the chlorophyll derivatives, the chlorin (17,18-dihydroporphyrin) derived from direct demetallation of chlorophyll a is calledpheophytin a. With the further loss of a phytyl group, the compound possessing a freepropionic acid residue at position 17 is called pheophorbide a; its ester can be named intwo alternate ways, e.g., methyl pheophorbide a or pheophorbide a methyl ester.Fischer: Chlorin eIUPAC: (2S, 3S)- 1 8-Carboxy-20-(carboxymethyl)- 1 3-ethyl-3 ,7, 12,17-tetramethyl-8-vinylchlorin-2-propionic acidTrivial nomenclature of chlorophylls often uses italic letters and subscriptnumbers; the latter indicate the number of oxygen atoms in the molecule, therefore,chlorin e has six oxygen atoms and is a chlorin from the chlorophyll a series of13degradation products. Letters a and e are interchangeable, and are used in the chlorophylla series only (e.g. in chlorin e6), while g is used for chlorophyll b derivatives (e.g. inrhodin g7). Much of the remaining chlorophyll nomenclature is difficult to interpret.1.4 Optical Absorption SpectraPorphinoids exhibit characteristic absorption and fluorescence properties in thevisible region which make them useful as photosensitizers. The metal-free andmetallated porphinoids have an intense absorption (E —1O) around 400 nm, known as theSoret band.33 This band is by far the most intense absorption found in all fullyconjugated tetrapyrroles and can therefore be regarded as characteristic of thismacrocyclic conjugation. The intensity is weaker in chlorins and metallochiorins and, asmight be expected, it is totally absent in the non-conjugated tetrapyrroles such asporphyrinogens. The Soret band is also present in vitamin 12 and in the metalcomplexes of bile pigments; both of these types of compounds have interruptedconjugation in the ligand, but the pathway is maintained through the metal atom.34Besides the Soret band, porphyrins also possess four accompanying less intenseabsorptions usually referred as “Q bands” which appear in the 450-650 nm region. Therelative intensities of these four satellite bands, numbered I to IV as shown (Fig. 1.7),have been used to identify porphyrin spectra as four basic types: etio-type, rhodo-type,oxorhodo-type and phyllo-type.14a)C)00)-a)C)C00)650Fig. 1.7 The Four Types of Porphyrin Spectraa) Etio-type spectrum:The etio-type spectrum (Fig. 1.7) is characterized by a IV>IIbll>I order of theband intensities and found in all naturally occurring porphyrins in which six or moreperipheral positions carry side-chains such as methyl, ethyl, acetic, or propionic acidgroups, the remaining positions being unsubstituted. Naturally occurring porphyrins suchas copro-, hemato-, uro-, meso- and deutero-porphyrins all exhibit this type of spectrum.500 550 600 650 500 550 60015b) Rhodo-type spectrum:Porphyrins which display this type of spectra are those which have one stronglyelectron-withdrawing group (e.g. formyl, acetyl or carboxyl) conjugated with theporphyrin ring. This situation causes band III to be more intense than band IV resultingin the rhodo type spectrum (Ill>IV>IbI) as shown in Fig. 1.7 (named afterrhodoporphyrin, a degradation product of chlorophyll a). The extended conjugationresulting from the strongly electron-withdrawing group (“rhodofying” effect) produces abathochromic shift of all the bands, which distinguishs this type of spectrum from theetio-type. Rhodo-type spectra are also exhibited by porphyrins which have a benzenering fused to one pyrrolic ring (benzoporphyrin) and by those with vinyl groupsubstituents. An interesting feature to note is that two rhodofying groups on adjacent“pyrrole” subunits cancel out each other’s rhodofying effects and an etio-type spectrumresults (e.g. 2,4-diformyl- and 3,8-diacetyl-deuteroporphyrins IX). However, thebathochromic shift of the absorption bands, being additive, still take place.c) Oxorhodo-type spectrum:Two rhodofying groups on diagonally opposite rings enhance each other’s effectand the oxorhodo spectrum results (Fig. 1.7). This is characterized by a band intensityratio of Ill>IbIV>I. The oxorhodo spectrum is considered to be the result of a furtherenhancement of the rhodofying effect.d) Phyllo-type spectrum:This spectral pattern (IV>IbIIb.I, Fig. 1.7), named after a chlorophyll adegradation product phylloporphyrin, is distinguished from the etio-type by less intense16bands Ill and I. Two substitution patterns on the periphery produce this spectrum: (i) asingle meso-alkyl substitution and (ii) four or more unsubstituted 13-positions.Metallation of the porphyrin (the dianion formed by the removal of the NHprotons) which acts as a tetradentate ligand often changes the four band spectrum to onewith two bands, designated a and B, between 500 and 600 nm while retaining the “Soret”band absorption around 400 nm (Fig. 1.8). This is due to the increasing symmetry of theconjugated ring. The relative intensities and the absorption maxima of the a and the Bbands depend on the coordinated metal as well as on the nature of the porphyrin ligand.C.,C0-D0Co________________________-D300 500 600 700 nmWavelengthFig. 1.8 Typical Visible Spectrum of Metallated PorphyrinsThe intensity and the exact peak positions are dependent on the solvent as well asthe concentration. More importantly, correlations have been shown to exist between thenature of the porphyrin side chains and the positions and the relative intensities of theabsorption bands. Actually, the effects of the external substitution on optical spectra arenot pronounced, and changes in the electronic structure and conformation of themolecules affect the porphinoid spectra significantly. The reduction of one and/or twoa500 550 600 650 70040017endocyclic double bonds respectively to dihydroporphyrins (chlorins) andtetrahydroporphyrins (bacteriochlorins), although not affecting the aromaticity of themolecule, produces visible spectra characterized by absorptions at 650-680 nm (chlorins)(Fig. 1.9) and 750-780 nm (bacteriochlorins) (Fig. 1.10).metallochiorinci)0C0Cl)-oFig. 1.9 Typical Spectra of Chlorins and MetallochlorinsnmIn chiorins, band I in the visible region is very prominent (Fig. 1.9), and is about25 nm longer wavelength than in porphyrins. The Soret to band I ratio is only about 5(versus about 50 in porphyrins). The extinction coefficients of band IV and the Soretband, in neutral solvents, are comparable with those of the related bands in analogousporphyrins. Chlorin mono- and di-cations have spectra similar to those of the neutralchiorinIV IIIX5Wavelength18compounds, the band I and the Soret absorption being moved to shorter wavelengths inthe dications (and in metallochiorins). The Soret band in chlorins has a tendency to besplit, this being more noticeable where there is distortion of the resonance pathway, as inthe chiorins described in Chapter 2 and Chapter 3.500 600 700 800WAVELENGTH (nm)Fig. 1.10 Typical Spectrum of Bacteriochiorins1.5 Overview of Chlorophyll ChemistryChiorophylls are the pigments of photosynthesis, and photosynthesis is theenergetic basis of life on earth. The green colour of leaves has fascinated poets andscientists alike since the beginning of human civilization. The pioneering work byWillstätter and Stoll at the beginning of this century marked the establishment of methodsfor the isolation of the plant chlorophylls and some of their basic chemistry, which werehighlighted in the first scientific book in this area.3540019As the field was established, many famous names became associated with thechemistry of these fascinating compounds. The chemistry and structures of the mostnaturally occurring chlorophylls a and b were first elucidated by the efforts of HansFischer at a time when science contributed more to destruction than to the support of life.The wealth of these works, which was highlighted in one volume of his book “DieChemie Des Pyrroles”, has become as a classic in chlorophyll chemistry.36 Woodwardand his team achieved the enormous task of the total synthesis of chlorophyll a inl960;21,3738 the final question of stereochemistry at the carbons 17 and 18 was settled byFleming in l967.The past 30 years also marked an enormous progress in chlorophyll research.Along with improvements in the separation, analytical techniques, coupled with betterunderstanding of their chemistry, the structures of more than 50 chlorophylls and relatedderivatives from photosynthetic organisms have been established, and the number is stillgrowing.11 The biosynthesis has been elucidated in considerable detail; there is abeginning in understanding their biological breakdown; and the fossil record nowprovides good evidence for their fate in geological timescales.1.5.1 Structure and Occurrence of ChiorophyllsChlorophylls, named originally for Chl a and Chl b, now actually represent twolarge families, chlorophylls and bacteriochlorophylls. Bacteriochlorophylls are namedafter photosynthetic pigments present in bacteria and are different from chlorophyllspresent in plants and algae. They are a group of tetrapyrrolic pigments with common20structural elements and photosynthetic functions. In chemical terms, they aremagnesium-metallated tetrapyrroles characteristic of a fifth, isocyclic ring that isbiosynthetically derived from the C- 13 propionic acid side chain of protoporphyrin.Under this definition, the cores of chlorophyll macrocycles can be any porphinoids. Onlymagnesium complexes of porphyrins, chiorins, or bacteriochiorins possess photosyntheticactivity and have been found in nature. Conventionally, the magnesium-free structuralanalogs are excluded and are called chlorophyll related derivatives, although some ofthem do function in photosynthesis. Chiorophylls do not occur solitarily within the plant,alga and bacterium world. They are associated with one or two carotenes and severalxanthophylls, and sometimes are accompanied by blue or red proteinaceous pigments.Although more than 15 different chlorophylls and bacteriochlorophylls have been isolatedso far, only the following are widely distributed in nature.Chlorophyll aChlorophyll a (Chl a) (1) (Fig. 1.11) is present in all organisms capable ofoxygenic photosynthesis, where it occurs in both reaction centers (photosynthetic systemsI and II) and in all light-harvesting complexes. It is the most abundant and mostimportant chlorophyll. The molecular structure of Chl a has been established by totalsynthesis of the tetrapyrrole moiety and the 20 carbon terpenoid alcohol, phytol.21 Thestereochemistry at carbons 17 and 18 has been determined by relation to (—)ct-santoninand dimethylpentane39,that at C-132 and of phytol by a combination of synthetic andspectroscopic techniques.21Chi a has been used as a reference compound in the structural elucidation of manyother chiorophylls and related pigments. Originally chlorophyll a was isolated fromgreen vegetable spinach and alfalfa.4° Now it can be readily available fromcyanobacteria (blue-green algae) which do not contain ChI b.4’ Chl a provides a chiraland substituent pooi from which a variety of reactions can produce modified structuresand allows extensive modifications and correlations among the chiorophylls.cooFig. 1.11 Chlorophyll a (1) and Chlorophyll b (2)Chlorophyll bChlorophyll b (Chl b) (2) (Fig. 1.11) is distinguished from Chi a by a 7-formylinstead of the 7-methyl-substituent. Its structure has been established by chemicalcorrelation with Chi a; the stereochemistry and esterifying alcohol (phytol) of bothpigments are identical. Due to the electron-withdrawing effects of the carbonyl at C-7,the basicity of the central nitrogen is decreased42,and the spectroscopic properties aremarkedly changed. In its electronic absorption spectrum, Chi b has a wider absorption ofvisible light since the Q band of Chi b is shifted to shorter wavelengths and the Soretband appears at longer wavelength than the corresponding absorptions of Chi a.1COOPhytyl222Chi b accompanies Chi a in the “green” series of oxygenic photosyntheticorganisms and is generally present as a light-harvesting pigment in about a 1:3 ratio. Ithas also recently been identified together with Chlorophyll c in a few chromophytes, e.g.Mantionella squamata.Chlorophyll cChlorophyll c (Chl c) (3) (Fig. 1.12) or chlorophyllide c is the common name forwhat were originally considered two, and now at least three chlorophylls, which arewidely distributed and abundant in the chromophyte algae.43 These pigments differ fromall other chlorophylls in being fully unsaturated porphyrin macrocycles rather thanchiorin derivatives. They generally do not carry a long-chain esterifying alcohol at the C-17 acrylic acid side chain and the stereochemistry at the only asymmetric C-i 32 positionis unexplored. Three Chl c structures have been currently established and they all havean acrylic side chain at C- 17. Chl c and Chl c2 differ by the respective presence of an 8-ethyl- and 8-vinyl-substituent44’5and Chi C3 has a methoxycarbonyl group at the C-7position.46R2Fig. 1.12 Chiorophylls of the c-type (3).Chl c:R1=Me,R2=Et; Chi c2: R1=Me,R=CH3;Chl c3:R1=COOMe,R2=CH;and Bacteriochlorophyll a (4)03COOR423Bacteriochiorophyll aBacteriochiorophyll a (BCh1 a) (4) (Fig. 1.12) is the most widely distributedbacteriochlorin pigment.47 It occurs in most photosynthetic bacteria, and is the onlybacteriochiorophyll in most Rhodospirillales. The stereochemistry at the reduced ring IVand the isocyclic ring V is identical to that of Chi a (17S, 18S, 132R).48 The commonasymmetric C-7 and C-8 at ring II are both R-configured. The crystal structure of a BCh1a derivative and several BCh1 a proteins has confirmed this stereochemistry.49Phytol is the most common esterfying alcohol of chiorophylls. Conversely,bacteriochlorophylls carrying alcohol other than phytol are most frequent and theesterifying alcohol varies in different bacteria. For example, BCh1 a fromRhodospirillum rubrum and Rb sphaeroides respectively, contains z2,6,10,l4- and2,4, 10, 14-(geranylgeraniol).50 The most popular esterifying alcohol inbacteriochiorophylls is farnesol which is present in most bacteria containing BCh1 c, d,e.51 The above mentioned specific esterification of bacteriochlorophylls pointsundoubtedly to the biological significance of these bacteria, but this is still poorlyinvestigated on the molecular scale.HOCH2f(HOCH2HOCH2HOCH2Fig. 1.13 Esterifying Alcohols of Chiorophylls and Bacteriochlorophylls. From top tobottom: geranylgeraniol (z2,6,10,l4); geranylgeraniol (2,4,10,14); phytol; farnesol241.5.2 Chemical Modifications of Chlorophyll aThe isolation of intact chiorophylls from natural sources is known to be a difficulttask because of their extreme susceptibility to various modification reactions, such asenolization, epimerization, allomerization, demethoxycarbonylation, solvolysis,demetallation, dephytylation, and photooxidation. Mild acid treatment of thechiorophylls affords the metal-free pheophytins and this is usually the form in which thepigments are stored prior to further degradation. The mixture of pheophytin a (5) andpheophytin b (6) is conveniently separated on a large scale by making use of the reactionof the formyl group in the ‘b’ series with Girard’s reagent T, followed bychromatographic separation.52 Methanolysis of pheophytin a and b produces theconesponding methyl pheophorbides a (7) and b (8). It is possible to transesterify thephytyl residue without removal of the magnesium atom; with methanol, the methylchiorophyllides (9) and (10) result.COOR2 COOR25. R1=Me, R2=Phytyl 9. R1=R2Me6. R1CHO, R2=Phytyl 10. R1=CHO, R2=Me7. =R2Me8. R1CHO, R2=Me251.5.2.1 Reaction of the Isocyclic RingThe most prominent group in chiorophylls is probably the enolizable B-ketoestergroup at the isocyclic ring V. which (together with the central Mg) is responsible for thestrong and specific self-aggregation of chlorophylls.53 In addition, the C- 132 is in aposition comparable to a benzylic position in aromatic compounds. It is therefore subjectto epimerization, enolization and other reactions under basic conditions. Epimerization and EnolizationThe extent to which ring V exists in the enol form has been much exploited in thepast; it is ordinarily almost entirely in the keto form.54 Closure of ring V introducesstrain into the chlorin ring and any further strain that would be introduced by a doublebond between C-131 and C-132 probably inhibits the presence of appreciable amounts ofenol under nonbasic conditions. Due to the activation by the two carbonyl functions, theC-132 hydrogen atom is highly acidic (no pKa values have been reported) and doesexchange with protons of solvent methanol, even in neutral solution, and the rateconstants have been measured by a magnetic resonance technique. Exchange is muchaccelerated in the presence of base, and in pyridine, even the C-132 hydrogens ofpyrochlorophyll (demethoxycarbonylation product of chlorophyll) are exchanged at anappreciable rate.The Chl enolate ions occur as intermediates in the formation of Chl C-i 32epimers (11) (Fig. 1.14), which were named Chi a’ and Chl b’ by their discoverers, Strainand Manning, in 1942. These chlorophylls and their closely related Mg-free derivatives26have recently been studied thoroughly by several investigators.56 The epimerization ratewas found to increase according to the polarity (Lewis basicity) of the organic solventused. Thus, in pyridine or triethylamine the reaction occurred rapidly, whereas inbenzene, it was very slow.132R H=K e 1325CH300C CH300C 0 CH300CFig. 1.14 Epimerization at C-132 of ChlorophyllsThe higher thermodynamic instability of the 132 (S)-stereoisomers compared tothe 132 (R)-isomers has been accounted for by the increase of steric crowding and strainin the periphery of the molecule when the epimerization occurs. The ratio of 132 (R)..isomers to 132 (S)-stereoisomers is about 83:17 in CDC13 at 25°C.The existence of Chi a’ in nature has been hypothesized, and it is thought tofunction as a kind of chain breaker in the chlorophyll aggregation in vivo or/and in thereaction center(s) of photosynthesis.57 So far, no experimental evidence for Chl a’ hasbeen found.Recently, the potassium enolate of Chl a has been prepared in adequate purity togive a well-defined 1H NMR spectrum.58 Methyl pheophorbide a trimethylsilyl enolether59 (12) (Scheme 1) and chlorophyll a t-butyldimethylsilyl enol ether60 (13) (Scheme1) have been obtained, respectively, by tetrapropylammonium fluoride and the lithiumsalt of 2,6-t-butyl-4-methylphenol (LiBHT) combining with the conventional silylatingagents. The creation of a new double bond into ring V in the aforementionedpheophorbide and chlorophyll enol derivatives has a profound effect on the delocalized27system. A considerable perturbation in the it-system can be experimentally observed ( 1H NMR). The comparison of their absorption spectra is shown in Fig. 1.15.Pr4NeFeMe3SiC1/Et3N—78°C/‘j [ 1) t—BuMe3SiC1/Py( Ag2) BHT/BuLi(CoCO2Phytyl1Scheme 1 Syntheses of Silyl Enol Ethers 12 and 137 12C02i13AFig. 1.15 Electronic Spectra of 7 (- - -) and the silylated enol ether 12 ( ) Ring OpeningThe isocyclic ring can be opened by hydrolysis, methanolysis, or aminolysiswithout simultaneous oxidation at C-132. The formed products usually are chiorin e andits derivatives depending on the attacking nucleophiles. Alkaline hydrolysis(saponification) of Chi a or b under oxygen-free conditions yields a mixture of thesodium salts, which are water-soluble compounds and consist of more than 9 differentchlorophyll degradation products with the major component being chiorin ej trisodiumsalt. By treatment with Cu(ll)-acetate, they are converted to the corresponding Cu(ll)complexes, called “Cu chlorophyllin” in industry. Thousands of tons of Cu chiorophyllinare produced each year and it is widely used in the food industry, i.e., in oral hygienesuch as chewing gums, toothpastes and in medicinal industry as a treatment for anemiaand hypertension, etc. •61CH2COORHOR7 I7 COOMe7/VMeOOC THNRR’CH2ONRR’COOMeFig. 1.16 The Cleavage of the Exocyclic Ring by Nucleophiles29The reaction mechanism of ring cleavage was first proposed by us and will bediscussed in Chapter 5.Alkaline hydrolysis in the presence of oxygen leads to a variety of potentiallyuseful degradation products by way of the “allomerization” reaction. The term“allomerization” was introduced to chlorophyll chemistry by Willstätter35,who used it todescribe the then unknown modification reactions of chlorophylls occurring on standingin alcohol solution in contact with air. Thus, allomerization is synonymous with the term“autooxidation”, which implies oxidation by triplet oxygen.The allomerization reaction is the main difficulty in the isolation of chlorophyllsfrom natural sources and in the handling of chlorophylls to yield Chl-related chlorin andporphyrin derivatives, not easily accessible by total synthesis. When a pure sample ofChi a is permitted to stand in methanol for several days in the dark, a great number ofallomers and other products can be separated from the mixture. Besides allomerization,solvolysis of the isocyclic ring, demetallation, dephytylation, and photooxidation, if lightis present, may occur as side reaction. These reactions appear to be complex, and theprecise nature of the products depends upon the conditions. Many of the allomers consistof two 132 or 15-stereoisomers, some of which have been separated bychromatography.54Among the allomers (Scheme 3) that have been identified are 132 (R,S)-hydroxy(14) and 132 (R,S)-methoxy-chlorophylls (15), the Mg-complexes of purpurin-7 trimethylester (16), 15 (R,S)-hydroxypurpurin-7-lactone dimethyl ester (17), 15 (R,S)-methoxy-30purpurin-7-lactone dimethyl ester (18), and purpurin- 18 methyl ester (19) (instead of the174-methyl, there can be a phytyl in these derivatives).62MeQOC MeQOC 0 MeQOC•0—0’,c\.o—o. c;3\ R’O•R’>MeQOC Q MeQOC 0 MeOOC 0RH or R’OHr 15.R• or R’O.Hoo:oH r R45O20 16. R’=H 18. R’=H17. R’Me 19. R’MeScheme 3 Allomerization and Its Proposed MechanismAllomerization has been attributed by Fischer and Pfeiffer to the formation of a132-hydroperoxide6 (20). The 132-hydroperoxide intermediate 20 subsequentlyundergoes a nucleophilic attack by hydroxide or methoxide ion with simultaneousheterolytic cleavage of the C- 131—C- 132 bond and the peroxide bond to result in anumber of allomers (Scheme 3). The plausibility of this free-radical mechanism is that it31can account for the formation of most allomers, though no intermediates have beendetected. Pigments from Standard Chlorophyll Degradation ChemistryUsually pheophytin a (5) and pheophorbide a methyl ester (7) are the stagingpoints for literally dozens of subsequent degradation products. Thus, depending on theprecise conditions, “unstable chlorin” (21) and a number of purpurins can be obtainedalong with rhodo-, phyllo-, and pyro-porphyrins, etc.. In view of the fact that chlorophylldegradation is a complicated process and usually yields many by-products, only thosedegradations which furnish chlorins and porphyrins in reasonable and useful quantitieswill be discussed.Aerial oxidation of pheophytin a (5) in alkaline solution cleaves the C- 131 to C-132 bond with concomitant loss of phytol, giving the ‘unstable chlorin’36 (21).Evaporation of the solution produces purpurin-18 (22), whereas esterification withdiazomethane furnishes purpurin-7 trimethyl ester (16).52 If the latter compound isheated in collidine, a 76% yield of 3-vinylrhodoporphyrin XV dimethyl ester (23) isobtained.52 In an analogous fashion, the meso (dihydro, with the 3-vinyl reduced toethyl) compound affords an 81% yield of rhodoporphyrin XV dimethyl ester (24).If methyl pheophorbide a (7) is heated in collidine the corresponding methylpyropheophorbide a (25) is produced in virtually quantitative yield;41 heating in pyridineaffords a lower yield. Similar reactions take place with the meso-series of compounds.3223 R1=C2H,R2=COOMe, R3=H24 R1=Et, =COOMe, R3=H29 R1=Et, R2=H, R3=Me30 R1=Et, R2=R3H‘ _r’ u‘‘27 R1=EtTreatment of chiorins with high-potential quinones, such as 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) gives the corresponding porphyrins. The oxidation yieldsvary widely depending on the substrates. For example, methyl pheophorbide a (7) gives3-vinylpheophorphyrin a dimethyl ester (26) in 28% yield and methylmesopyropheophorbide a (27) yields phylloerythrin methyl ester (28) in 35% yield.3421 2226 2833Various alkaline treatments of chlorophyll, followed by methanolysis, affords, inlow yields, phylloporphyrin XV methyl ester (29) and pyroporphyrin XV methyl ester(30); pyroporphyrin XV(31) is also obtained from phylloporphyrin XV (32) by treatmentwith alkali.361.5.2.2 Peripheral ReactionsThe chlorin macrocylic system, as compared to that of porphyrins, shows twodistinct differences in reactivity. The methine positions next to the pyrroline ring have ahigher electron density and are, thus, susceptible to electrophilic attack; and chiorins aremore easily oxidized both by one- and two-electron oxidants. Both features have beenlinked to the reactivity of chlorophylls, which are, with the exception of chlorophyll c,either chiorins or bacteriochlorins. The easy proton exchange of the ö-H in chlorophyll a(1) is a direct result of the increased electron density at this position, and especiallyphotooxidation to the cation radical is of eminent biological importance as the genuinelight conversion step in photosystem J• Electrophilic SubstitutionThe higher susceptibility of the 15 and 20-positions to electrophiles in simplechiorins (20 position in pheophorbides) was qualitatively predicted and experimentallyverified by Woodward.64 These positions can therefore be selectively deuterated,halogenated, acylated, or nitrated by deuterium, halonium, acylium, or nitronium34electrophiles. In substituted chiorins, the reactivities of the free meso and 8-pyrrolicpositions in electrophilic substitutions depend on the number and nature of substituents inoccupied positions. In general, any substituent increasing the electron density (e.g., alkyl,amino) in the aromatic it-system will activate the meso positions and deactivate the B-7. R=COOMe25. R1=HPod3/DMF33. R =COOMe34. R1=HcECOOMeH20/HCtCICH2 COOMeCOOMe35COOMe36BrCN/AICL3NC37 38Scheme 4 Electrophilic Substitution Reactions of Chiorins35pyrrolic positions to electrophilic attack. In contrast, an electron-withdrawing substituent(e.g. formyl, acetyl) will deactivate the meso and activate the 8-pyrrolic positions in thisrespect. Also the central metal atom should be looked at as an electron-donating (e.g.Mg2,Cu2,Ni2)or electron-withdrawing (e.g. Sn4) substituent. Further, steric factorsare also of importance. Bulky substituents exert steric hindrance toward electrophilicsubstitution at positions close to these substituents.Some examples of electrophilic substitutions are showed in Scheme OxidationChlorins and bacteriochlorins can be dehydrogenated by several oxidants, such asoxygen, quinones, FeCl3 or Fe(CN)63. 3-Vinyl chiorins are dehydrogenated by 02 orFe(CN)63 in alkaline solution to 3-vinyl porphyrins.65 These oxidation reactions arerarely controllable and usually lead to overoxidation and produce large amounts of byproducts, and, as such, are not useful from a preparative viewpoint.0EIsEI4/Py No.2S03COOMe39 40Scheme 5 Peripheral Oxidation of Chiorins by 0s0436Chiorins can also form adducts (39) (Scheme 5) with osmium tetraoxide, whichare hydrolyzed in sodium sulfite solution to dihydroxychiorins (40).66 The 3-vinylgroup, if present, is oxidized by 0504 to the 3-glycol group.Chlorophylls can be photooxygenated by singlet oxygen to n-cation radicals. Thisis a “self-destruction” pathway of chiorophylls due to its photosensitizing capability.Photooxygenation of chlorins and bacteriochlorins is responsible for the cleavage of themacrocyclic ring system and subsequent degradation to smaller carbon- and/or nitrogen-containing fragments. This breakdown probably represents the fate of 1O tons of annualchlorophyll destruction occurring in nature.0HO42 43Scheme 6 Photooxygenation of 3-VinylporphinoidsIf a 3-vinyl group is present, as in chlorophylls, photoxidation happens viaaddition of singlet oxygen in a 1,4-fashion with ring I and 3-vinyl group to form a cyclicN in41HN N4437peroxide 41 (Scheme 6). This compound can be transformed to the possible strucutures42, 43, or 44. The presence of oxygen and EtOH or pyridine was found necessary for thereaction.67 In addition, the reaction required a complexed Mg2+ or Zn2+, but not anintact cyclopentanone ring. However, no detailed structural analysis of thephotooxygenation products was presented to corroborate the proposed mechanism.The photooxygenations of the non-vinyl chlorins such as bacteriochiorophylls c(45a) and e (45b) and their Mg-free derivatives have been studied by Troxler and coworkers68 and Risch et al. 69 The mechanism of these photooxygenations involves the[2+2] cycloaddition of singlet oxygen to positions 1 and 20 to yield the 1, 20-cycloperoxide intermediate (46) (Scheme 7), which then undergoes cleavage to afford abilitriene (47). Because the reaction is typical of all Chl derivatives bearing a C-20substituent, it may provide a route for the degradation of chlorophylls in vivo. Unlike thephotooxygenation of Chl a , these photooxygenations do not require the presence ofMg2 in the molecule.hva R =Me45 b. R’=CHOHO HOCO2Farnesyl47Scheme 7 Photooxygenation of Bacteriochlorophylls c and e381.6 Synthetic Aspects of Natural ChiorinsAlthough myriad synthetic routes to tetrapyrrolic macrocycles have beendeveloped in the past century, most of them have been applied to the synthesis ofporphyrins, which are planar with no stereogenic centers at the periphery. The progressin the synthesis of chlorins, bacteriochlorins and isobacteriochiorins has been very slowand in most cases these compounds have been proved to be elusive targets. The mainreason for this lack of activity was probably the challenge posed by the presence ofstereogenic centers in the molecule and the trans geometry in the reduced ring IV ofnatural chiorins and bacteriochiorins.With the exceptions of Woodward’s unique approach to the synthesis ofchlorophyll a, up to the present time no synthetic methods have been devised in whichthe trans geometry of chlorin (17,18-dihydroporphyrin) ring systems is built-in in arational stepwise fashion, although various “reduced” systems bearing geminallysubstituted (nonoxidizable) moieties have been synthesized.7°Methods for preparationof cis-reduced chlorins71 (e.g. catalytic hydrogenation, di-imide reduction, Raney nickelreduction, photoreduction) and cis-oxidized chlorins76 (e.g. bishydroxylation withosmium tetraoxide) from porphyrins have also been developed. However, these methodsare generally complicated by the possibility of stereoisomers, and, when unsymmetricallysubstituted porphyrins are to be modified, of structural isomers.The following sections review the most recently synthetic work in the field ofnatural chlorins, most of which has been carried out by chemical modifications ofpheophorbide a, the more stable and more readily accessible derivative of chlorophyll a.391.6.1. Partial Synthesis of Dimethyl Tunichiorin From Chlorophyll aThe nickel-containing tunichlorin (49) was discovered by Rinehart et al.72 in thetunicate Trididemnum solidum which occurs in the Caribbean Sea. Structural elucidationwas canied out by UV-Vis, CD, MS and 1H NMR spectroscopy on dimethyltunichlorin(50) (Scheme 8), which was prepared by etherification of the 3-hydroxymethyl group andesterification of 17-propionic acid side chain. Dimethyltunichiorin synthesized fromtunichlorin (49) is in all respects identical to dimethyltunichlorin prepared from49 50Collidine1 7251) 0s04 ,Na1042) NaBH43) Ni(OAc)24) TsOH/MeOHCO 2H CO2MeScheme 8 Partial Synthesis of Dimethyl Tunichiorin40chlorophyll a (1) by partial synthesis72 (Scheme 8). However, the biological function oftunichlorin (49) is still unknown. Its substitution pattern suggests biogenesis fromchlorophyll a as being the most likely.1.6.2. Partial Synthesis of Methyl Bacteriopheophorbide c[Et,MeJ from Methyl Pyropheophorbide aBacteriochlorophylls c (45) (Bchls c) are found in bacterial strains such asProsthecochioris aestuarii, and in the gliding filamentous bacterium Chioroflexusaurantiacus. The latter produces only one homologue (8-Et, 12-Me) of the Bchl c whilethe former occurs as a mixture of homologues (45 a-f)73 (Fig.1.17). Early structuralwork by Holt and coworkers74 led to the derivation of the gross structures for thehomologous mixtures of the Bchl c. The absolute stereochemistry in ring IV is the sameas for Chl a and the chirality of the 3-(l-hydroxyethyl) was established on the basis ofR8 R1 R2 Chiralityat 31 positiona Et Me RR12 b Et Et Rc n-Pr Et Rd n-Pr Et Se i-Bu Et Rf i-Bu Et SHO.45Fig. 1.17 Structures of the Bacteriochiorophylls c41HPLC, NMR and X-ray crystallography.73 The major structural difference between theBchl c [Et, Me] and Chi a is in the -meso-methyl substituent found in the former.The Bchls are most correctly designated as “pyro” compounds, because they aredevoid of the 132-methoxycarbonyl group present in Chl a. Esterifying alcohols on the17-side are mainly farnesol (instead of the phytol found in Chl a).In 1985, Smith et al.41 accomplished a partial synthesis of Bchl c [Et, Me] frompheophorbide a methyl ester (5) (Scheme 9). Treatment of 5 with hot collidine affordedmethyl pyropheophorbide a (25). This compound when treated with two moleequivalents of thallium(III) nitrate in methanol gave the dimethoxyacetal 53 after removalof chelated thallium(Ill). Aqueous acid treatment followed by immediate reduction withsodium borohydride of compound 53 yielded the 3(2-hydroxyethyl) derivative 54, whichunderwent halogenation with benzoyl chloride in dimethylformamide, to give therequired vinyl-protected compound 55 in 72% yield. Insertion of copper(ll) intocompound 55, followed by treatment with titanium tetrachloride and chloromethyl methylsulfite provided the 20-methylthiomethyl derivative 57 in 69% yield. With Raney nickelin acetone, the thiomethyl derivative 57 gave the required 20-methylchlorin 58 in 59%yield, which was demetalated to give compound 59. Vinylation, using KOH in pyridine,gave the 3-vinyl compound 60, in 93% yield. The final step in the partial synthesisrequired Markovnikov hydration of the 3-vinyl group and this was accomplished withHBrIHOAc, in 49% yield, to give a mixture of diastereomers 61 and 62 in 3:2 ratio.Reversed-phase HPLC separated the diastereomers, and the 31 -(R) configuration wasshown to be identical with that of 8-ethyl-12-methyl natural product.42MeO OH..OMe/ (3) SO2 • /N HN/ (2) N HN / (2) NoBH4 HNMeOH‘(1) TI(N03 (1) HCI ‘ NH N—(4) HCI 53%o 0 0CO2Me CO2Me CO2Me25 53 iPhCOCI/DMFICI CI 72%__________Cu(OAcHNM“NMe—s/ (1) Raney (2) TiCI4HN(2) HCI j \ / MeSCHCIo 31% ( 0 69%CO2Me CO2Me CO2MeKOH59 57 5593%HHO HO-HHBr/HOAcMe Me_(HN+ MeCO2Me CO2Me CO2Me60 61 62Scheme 9 Partial Synthesis of Bacteriopheophorbide c431.6.3. Partial Synthesis of Heme dHeme d (63) is the prosthetic group of the terminal oxidase cytochrome d, one ofthe two terminal oxidases found in many bacteria. Heme d (63) is bishydroxylated in theperiphery of ring III, which gives it the characteristic geminally disubstituted structureand the typical chlorin chromophore. The absolute and relative configurations of heme d(63) have still not been determined.75 The correct configurational formula of heme dmay therefore be a stereoisomer of formula 63. In addition, there is no certainty as towhether the spirocyclic lactone structure is the natural structural element or whether it isgenerated during isolation.Porphyrin d, the metal-free ligand system of heme d, was synthesized fromprotoporphyrin IX (64, Scheme 10) by Sotiriou and Chang.76 Before bishydroxylation ofring III of compound 64 is carried out, the vinyl groups have to be protected. This isachieved by converting them into chloroethyl residues. Subsequent bishydroxylationwith osmium tetraoxide yields the four possible constitutional isomers as expected. Thedesired isomer rac-66 is present in the mixture in 22% yield along with 26% ring IVisomer, 6.8% ring I isomer and 6.8% ring II isomer. In the presence of sodium acetatethe bis-hydroxyporphyrin rac-66 forms the spirolactone structure rac-67, which can betransformed into either of the stereoisomeric porphyrins rac-68 or rac-69 depending onthe reaction conditions chosen. Since the stereochemistry of natural heme d is unknown,both stereoisomers formed may be of interest.44Cl Cl—NH N( “(1) Tl(N03 \ NH N—\ /4.( )N HN) (2) NaBH4 N HN—cc?---- — /0H(3) PhCOCI/DMFNOHCOOMe COOMe COOMe COOMe COOMe COOMe64 65 rcw—66NaOAcClMeOHClYOHOMep OH (1) KOHCOOMe (1) KOH(2) HCI(2763rac—67(3) silica gelsilica ge‘.-NH N.J ‘-NH/\H) N HN)OMepOHOMeOHrac—68 rac—69Scheme 10 Partial Synthesis of (±) Porphyrin d451.6.4. Total Synthesis of Bonellin Dimethyl EsterBonellin (72) is the green sex-differentiating pigment of Bonellia viridis, a marineanimal found throughout the Mediterranean and belonging to the Echiuroida class ofanimals. Bonellia viridis possesses a remarkable sex dimorphism, which is induced bybonellin. Any of the initially asexual larvae that come into contact with the body wall ofthe female, which contains the green bonellin, develop into males of about 1-3 mm insize. After contact with bonellin the males live inside the body cavity of the largerfemale (15 cm). The female of the species develop from those larvae which have had nocontact with bonellin.Although pure crystalline bonellin (72) was first isolated by Lederer et al.77 in1939, it was not until 1976 that Pelter et al.78 determined the constitutional formula ofbonellin by modern spectroscopic methods. The ring IV degradation product of bonellin(72) was found to be constitutionally and configurationally identical to the alreadyfamiliar ring Ill degradation product of vitamin B 12.The photochemical cyclization opened the way to the synthesis of bonellindimethyl ester.79’8° The bonellin macrocycle was divided along the north-south linegiving rise to a western and an eastern block. The western half (74, Scheme 11) with areduced pyrrolic ring (IV) was prepared from the nitropyrrole 73. The eastern half (adipyrromethene, 75) was prepared separately from readily available pyrroles.Condensation of the western block 74 with the eastern block 75 under acidic conditionsgenerated a seco-system 76 which gave the recemate mixture of bonellin dimethyl ester77 after methanolysis of the nitrile with methanolic sulphuric acid.46H}NHCNH“—\ HNH N— (1)1w\ /HN/(2) H2S04MeOH20%COOMe COOMeNH4F79%4PCN+H H H- HOAc CH3NO2NH _.NHNaBH OHCNO2 57 0N73TFA71%OHC.74 75COOMe7672 rcLc—77Scheme 11 Synthesis of (±) Bonellin Dimethyl Ester47Chapter 2Stereoselective Synthesis of Natural AntioxidativeChlorins482.1 Synthetic Targets and Research ObjectiveThe metallated complexes of tetrapyrrolic macrocycles constitute the basicmacrocyclic pigments of living systems on this planet (Chapter 1, Fig. 1.2). Thefunctions of these cofactors are determined by the incorporation of different metal ionsinto the centers of their tetrapyrrolic macrocycles. As mentioned in the introduction,complexation with a central metal ion by tetrapyrroles allows fine tuning of its electronicand redox properties. In this way, these coordination compounds have developed uniquereactivity and biological interaction with their various molecular environments in cells.However, other than the metal-containing cofactors, metal-free naturaltetrapyrroles having special biological functions may be not the exception rather than therule, since the number of related compounds isolated in metal-free form from marinemetabolites is still increasing. In addition to the well-known pigment bonellin78 (Chapter1), which controls the sex of the larvae of the mediterranean sea worm, Bonnellia viridis,another new class of chlorins (called “new chlorophyll a related chlorins”) with strongantioxidative activity has also been recently discovered.The term “antioxidative activity” can be defined as a function which inhibitsoxygen-mediated oxidations. In nature, antioxidative activity is inherent in a class ofsimple compounds and/or complex bio-macromolecules which have evolved in manyorganisms and microorganisms as a defense against the detrimental effects of oxygen.81Oxidative by-products (most commonly in the form of highly reactive and potentiallyharmful free-radicals such as HO., 02•, L.)82 of normal cellular metabolism causeextensive damage to DNA, proteins, and lipids.83 This damage (the same as that49produced by radiation) appears to be a major contributor to aging and to degenerativediseases of aging such as cancer, cardiovascular disease, cataracts, immune systemdecline, and brain dysfunction.84 Antioxidative defenses against this damage include twotypes of natural antioxidants that are highly effective in neutralizing free-radicals or/andin inhibiting the bio-reactions responsible for free radical production.85 The first type islow-molecular-weight compounds such as vitamin C (ascorbic acid), vitamin E (atocopherol), glutathione, and uric acid. These hydroxylated compounds can donatehydrogen atoms to other free radicals, resulting in the formation of relatively stable, oftenoxygen-centered antioxidative radicals. The long life-spans (usually seconds forantioxidative radicals in contrast to 106 seconds for most lipid radicals)86 of theseantioxidative radicals allow them to slow down the progression of the radical-chainpropagations and to finally react with other free-radicals to terminate the radical chains.Therefore, these compounds are free radical scavengers and are thus able to inhibit lipidperoxidation (lipid damage). The second type of natural antioxidants is antioxidativeenzymes, e.g. copper-, zinc- or manganese-containing superoxide dismutases, iron-containing catalase and selenium-containing glutathione peroxidase, and metal-bindingproteins (such as transferrin, ferritin, and ceruloplasmin).85 Instead of scavenging freeradicals, these bio-macromolecules inactivate reactive electrophilic mutagens and inhibitthe reaction systems responsible for the radical production.85The discovery of antioxidative activity for the new chlorophyll a related chiorinsdates back to 1986, when Karuso et al.87 reported the structure of a marine chlorin, 132,l73-cyclopheophorbide a enol (81) (Scheme 12). 132,17-Cyclopheophorbide a enol, as50(‘I132,17—Cyclopheophorblde132S—Hydroxychlorophyllone a[ 82(s)] SpongeShort—necked clam, Scallop, OysterAttached diatoms mixture(I,N HNa enol(81)132R—Hydroxychlorophyllone a[ 82(R)]Scallop(‘ICOORPurpurln— 18(22 ,R=H)Purpurln—1 8 methyl ester(87 ,RMe)Short—necked clamNrevealed by single-crystal X-ray crystallography, exists only in the enolic form and is theonly tetrapyrrole isolated from the sponge Darwinella oxeata. Its biological function(‘Ii-NH N(I)Jrçvy13i71\Vl423172173 OH//5.-NH N( N!tI,L3l117 \ /OH eo2c%o O2PhyI151 R—Hydroxychloro—phyllonelactone a[83(R)] Chlorophyll a( i)Short—necked clam, Scallop, Oysterco2Me17 0 pyropheophorblde a(86)Chlorophyllonic acid a methyl ester(84) 132_i-—aShort—necked clam, Scallop, Oysterpheophorbide a(85 ,R=H)a methyl ester(102 ,RMe)Short—necked clamScheme 12 Natural Antioxidants and Their Origins51remained unknown until the closely related chiorin, 132S-hydroxychlorophyllone a[82(S)] (“S” or “R” denotes the absolute configuration at C-132 or C-15’ position whereappropriate) was found as an antioxidant in the short-necked clam, Ruditapesphilippinarum in 1990.88 The antioxidative biological activity of these chiorins wasdiscovered as a result of studies on the peroxide value (POV, which is an index of freeradical-mediated oxidative damage in lipids) and mutagenicity among the extracts ofvarious marine species that contain high content of highly unsaturated fatty acids (themost readily oxidized lipids), e.g. various kinds of marine fish, bivalves, and attached andwafting diatoms. However, instead of expected high POV (larger than 100 meq/per kgextracts) found in their extracts, much lower POV (less than 30 meq/per kg extracts) andmutagenicity were identified, suggesting the existence of strong antioxidants in theseorganisms.89 Further screening of these extracts has revealed the presence of new strongantioxidative chiorins, 132S-hydroxychlorophyllone a [82(S)], 132R-hydroxy-chiorophyllone a [82(R)], 15’R-chlorophyllonelactone a [83(R)], chiorophyllonic acid amethyl ester (84)°,13-oxopyropheophorbide a (85) as well as the known chiorins,pyropheophorbide a (86), purpurin-18 (22) and purpurin-18 methyl ester (87).’ Adetailed description of their structures and their sources is shown in Scheme 12.The finding of antioxidative activity in these marine metabolites not only suggestsa new biogenetic pathway but also provides new insights into the evolution of marinechiorophylls. These novel antioxidative chiorins share a similar structural framework andmolecular substitution pattern to chlorophyll a (1). For example, the structural differencebetween 132,17-cyclopheophorbide a enol (81) and chlorophyll a is in the presence of52an additional exocyclic ring VI in 81. It is, therefore, most likely that such naturalantioxidants are evolved from chlorophyll a. In nature, antioxidants have evolved inmany organisms and microorganisms as a defense against the detrimental effects ofoxygen since the time photosynthetic organisms began releasing oxygen into the primitiveatmosphere about 3.5 billion years ago.92 It is interesting to note that instead ofemploying the common antioxidants, hydroxylated and polyhydroxylated aromatic andheterocyclic compounds such as vitamin A (retinol), vitamin C (ascorbic acid) andvitamin E (a-tocopherol),93’4nature chose to modify the very molecule (chlorophyll) thatwas producing oxygen to protect the unwanted oxidation processes. As can be deducedfrom the occurrence in animals, these pigments have no photosynthetic activity.The rich stereostructural diversity of these antioxidants has been a challenge to thesynthetic chemist and will continue to be so as more skeletal types are found. The usefulbiological properties, coupled with their interesting and challenging structures, makethem targets for extensive synthetic studies. The objective of our research has been twofold: The first was to develop new and efficient synthetic routes to these unusualcompounds and thus be able to provide sufficient amounts of materials for subsequentantioxidative and biomedical investigations. Antioxidants as therapeutic agents haveshown potential in the treatment of inflammation, cancer, aging and neurodegenerativediseases.95 Secondly, we have striven to design synthetic routes that are both simple andbiomimetic. This requires the synthetic routes to be as close as possible to their possiblyenzymatic pathways.53The following sections describe our synthetic strategy and detail the syntheticroutes that are required to achieve the transformations of these unusual structures.2.2 Synthetic ApproachThe exocyclic rings of these unusual chlorins, as a class of naturally occurringantioxidants, display different oxidation states. Based on the number of the oxygen atomsin these natural antioxidative molecules, purpurin-18 (22) and its methyl ester (87) bothpossess 5 oxygen atoms and are in the highest oxidation state. The latter can be producedfrom the former upon treatment with diazomethane. Purpurin-18 is the fully oxidizedproduct from the four-oxygen compounds,15R-hydroxychlorophyllonelactone a [83(R)]and/or132-oxopyropheophorbide a (85). Chiorophyllonic acid a methyl ester (84) is themethylation product of1R-hydroxy-chlorophyllonelactone a [83(R)].The regioselective mono-oxidation of the seven- and five-membered exocyclicrings in the three-oxygen compounds, 132S- and/or132R-hydroxychlorophyllone a, 82(S)and/or 82(R), should bring about the generation of the four-oxygen compounds 85 and83(R), respectively (Scheme 13). The exact way in which the regioselective oxidationmight be performed was not known at the start of the research. However, the naturallyoccurring hydroxylactone 83(R) has an a 132-OH moiety, a stereochemistry resultingfrom the cisoid geometry between positions 151 and 17, which minimises the exocyclicring strain of the peripheral substituents associated with the chiral centers (C- 151, C- 17).Thus, it could be envisioned that the thermodynamically-favoured epimer 83(R) shouldbe obtainable under equilibrating conditions. Further, the diketone 85, is also present in54the thermodynamically-favoured form. Its corresponding hydroxylactone isomer 88,which should possess an eight-membered exocyclic ring, is thermodynamicallyunfavoured and has not been found (Scheme 13). Consequently, the goal was simplified/NH-NH N(N HN) N HN N HNLrO H0COOH 2288 87E /-NH N1 “N‘N HN)j17 115110CO2MeOHCOOH8584 83(R) x/N HN)132LHO•OHO82(R)82(S)/ \llh/X) NH HNN HN)eO2bCO2Ph’t)I CO2H OH1 86 81Scheme 13 General Strategy for the Synthesis of the Antioxidative Chlorins.55to devise a new regioselective oxidation to the seven- and five-membered exocyclic rings,which should ensure high yields and avoid the appreciable formation of the di-oxidizedproduct, purpurin-18 (22).Development of a successful asymmetric hydroxylation of 132, 17-cyclopheo-phorbide a enol (81) to produce compound 82(R) or/and 82(S) was thus thought to becentral to the entire effort. These compounds could then be used in the oxidativeformation of hydroxylactone 83(R) and diketone 85.As was described in Chapter 1, the partial synthesis of compound 81 frompyropheophorbide a (86), a degradation product of chlorophyll a, had been completed byEschenmoser and coworkers96’7some years before it was found in nature. The strategyfor the cyclization between the two peripheral substituents to form the additionalexocyclic ring VI involved a Claisen-type intramolecular condensation.Based on molecular modelling studies (Insightll, Biosym), formation of the enolicexocyclic ring in compound 81 should result in steric compression on its chlorinmacrocycle. Further oxidation to form 132S- and132R-hydroxychlorophyllone, 82(5)and 82(R), should relieve the strain to a great extent. The cisoid (C- 17, C- 132) geometricisomer 82(5) should be more stable than the transoid epimer 82(R) if one considers thering-strain of the exocyclic rings discussed above. A retrosynthetic analysis linking allthe antioxidative chiorins to chlorophyll a (1) is summarized in the antithetic format ofScheme 13. Once effective routes to these antioxidative chlorins were devised, next thestarting material was required to achieve these chemical transformations.2.3 Starting Materials : Natural Pheophorbides56Because chiorophylls are extremely light-sensitive and labile pigments, it isvirtually impossible to extract these plant materials without the formation of variousalteration products (even when samples are handled and chromatographed in the dark).The nature of these undesirable transformations and the yield of the secondary productsvary with the plant material, its treatment, and the conditions to which the extracts areexposed.98The usual chlorophyll extraction consists of the following steps: a) primaryextraction, b) separation of contaminating substances, c) conversion to stablederivatives.99 The last two steps should immediately follow the first, as a variety ofartifacts have been reported from stored samples.’°°Chiorophylls are highly insoluble in water and are always accompanied by otherlipophilic compounds within living cells. Therefore, isolation of chlorophylls requires,firstly, their detachment from the chlorophyll-binding proteins. This is achieved byextraction from photosynthetic tissues into polar organic solvents after mechanicaldisruption of cells. The solvents most suitable for the extraction of the chlorophylls fromfresh plant material are those miscible with water, such as acetone and methanol.’°’The isolated coloured matter of plants and algae is a mixture, with yellowpigments tending to dominate the extracts, coupled with a very small amount of the greenpigments. The ratio of the total green to total yellow pigments is approximately 1:3 inhigher plants. In blue algae, the ratio of the green to yellow pigments can be as low as1:300.102 Purification and separation of the mixture of pigments leads to the two principalgreen pigments (chiorophylls a and b), the orange to red carotenoids and the yellow to red57xanthophylls. The ratio of chlorophyll a to chlorophyll b obtained is usually 3:1; that ofcarotene to xanthophyll, 1:2.As mentioned in the introduction (Chapter 1), the blue alga Spirulina maxima iscommercially available and contains no chlorophyll b. This advantage makes it a goodsource of chlorophyll a and its closely-related derivatives. Thus, we have employed theblue alga Spirulina maxima as the source of chlorophyll a in our work. In 1985, Smith etal.4’ reported an isolation procedure for pheophytin a (5) from Spirulina maxima usingacetone as the extracting solvent, followed by chromatography on alumina. Nochlorophyll a could be obtained directly from this procedure due to repeatedchromatography on alumina. However, the demetallated derivative of chlorophyll a,pheophytin a was obtained as the major product. According to their report,approximately 2.4-3.6 g pheophorbide a methyl ester (7) was obtained from 1 kg ofSpirulina maxima alga after the final methanol/sulfuric acid step was used to transesterifythe phytyl ester to give the methyl ester.’°3However, a lower isolated yield(-j-1.0-1.5 g of compound 7 from 1 kg of alga),was observed (by other researchers’°4and also in our hands), along with a relatively largeamount of alteration products when this procedure was repeated. The main problemassociated with this method is the presence of large quantity of carotenoids andxanthophylls in the original acetone extract. Separation of these yellow-red pigmentsfrom the acetone extracts to afford the desired green chlorophyll required exhaustivechromatography which led to oxidation and alteration of the material. To overcome thisproblem, an improved method was developed in this work by introducing a two-phase58extraction in the purification step (to remove large amounts of yellow-red pigments)before the extracts were subjected to chromatography. Furthermore, pheophytin a (5), themagnesium-free derivative of chlorophyll a, is less sensitive, can better be handled inchromatographic systems, and is also the initial intermediate for chemical synthesis of theantioxidative chlorins in the present work. Therefore, demetallation of chlorophyll beforechromatography was performed in our isolation in order to minimize the alterationreaction in the chromatographical purification.In our method, the acetone extract was dissolved in petroleum ether. Successivewashing of the petroleum ether solution with 30% methanol in water removed most of theyellow-red pigments while the chlorophyll remained in the petroleum ether layer. Whenthe aqueous phase became colorless, the organic layer was collected, dried over sodiumsulphate, filtered and evaporated in vacuo. The residue obtained was redissolved indiethyl ether and conc. HC1 was added to remove the magnesium ion in chlorophyll. Theether layer was immediately washed with water, dried over sodium sulphate, filtered andevaporated in vacuo, affording crude pheophytin a (5). The remaining yellow-pigmentsin the crude pheophytin a were readily removed by flash chromatography on neutralalumina (Brockman Grade ifi). About 6.5 g of pheophytin a (5) was obtained fromapproximately 1 kg of Spirulina alga. Treatment of pheophytin a (5) with 5% sulfuricacid in methanol, followed by recrystallization from methylene chloride/methanol, gave4.2 g of pheophorbide a methyl ester (7).The conversion of pheophorbide a methyl ester into132,173-cyclopheophorbide aenol (81) was based on the modification of a method reported by Eschenmoser and co59Collidine (1) (TMS)2NNOReflux,9Omin (2) phosphate buffer(98%)(85%) 171VI3$17 17 OH7 25 81Scheme 14 Synthesis of Enol 81workers96 (Scheme 14). Thus, decarbomethoxylation of pheophorbide a methyl ester (7)in collidine afforded a 98% yield of pyropheophorbide a methyl ester (25) which was thentreated with seven equivalents of (TMS)2NNa in TFIF for 3 mm to bring about theClaisen-type intramolecular condensation to produce, after flash chromatography ondeactivated silica,132,173-cyclopheophorbide a enol (81) in 85% yield. Enol 81 is thecommon intermediate to new chlorophyll a related chlorins and exists only in the enolicform in solution and in the crystalline state87.After the key intermediate 132, 17-cyclopheophorbide a enol (81) was madeavailable, attention was then focused on its asymmetric hydroxylation.2.4 Hydroxylation Studies Leading to Asymmetric HydroxylationTo bring about the conversion of enol 81 to hydroxychlorophyllone a 82, anumber of methods were investigated, which included aerial and iodine oxidation. Noneof them were successful since decomposition and overoxidation readily occurred duringthese procedures. It is important to note that the aerial oxidation of pheophorbide amethyl ester (7) occurs during chromatography on silica, especially on a TLC plate, to60give a diastereomeric mixture of132-hydroxypheophorbide a methyl ester (95) (in —20%yield).’°5 Unfortunately, enol 81 is so unstable that it decomposes when applied to silicagel plates. Attempts at aerial oxidation of enol 81 in an alcoholic solution of zinc acetatealso failed in our hands. Although this method of aerial oxidation was used in thepreparation of 132-hydroxychlorin 95 from pheophorbide a methyl ester (7) in 30%yield’°6,it did not work in the case of compound 81, which gave back neither 82 nor zincmetallated 81, but an overoxidized green mixture which was not identified.Hydroxylation of enol 81 using molecular iodine’07”8,a mild oxidizing agent, in thepresence of sodium acetate in aqueous THF solution was only partially successful, givinga mixture (19% yield) of 132-hydroxychlorophyllone a (82) and l32acetoxychiorophyllone a (90). However, in both cases (82 and 90) no epimericenrichment was noted and no attempts have been made to separate the optically-pure 82.c//IMeO2c1eO’0ocH3Due to the unacceptably low yield and stereoselectivity of the above method,alternative reactions for introduction of a 132-hydroxy group were sought. Thus, weturned our attention to the oxidation of enolates with N-sulfonyloxaziridines, a methodintroduced by Davis and coworkers.’°9”°Following the standard procedure’ 10, treatmentof enol 81 with (TMS)2NNa or LDA followed by oxidation with 1-phenyl-N-8261(phenylsulfonyl)oxaziridine” (91) at —78°C for 15 mm, failed to give the desired product82 after the standard workup, but resulted in a very polar yellowish-green mixture. Thevisible spectrum (2max 672 nm, 404 nm) of this mixture indicated cleavage of bothexocyclic rings. Treatment of this mixture with diazomethane gave chlorin P6 trimethylester (92) (24% yield from 81) (Scheme 15). The amount of base and/or oxaziridine aswell as reaction conditions were varied in order to avoid the cleavage of the exocyclicrings, but all were largely unsuccessful. These results suggested that the two exocyclicrings of enol 81 could not withstand strong ionic bases such as (TMS)2NNa and LDA.(1) (TMS)2NNa,—78°CH? (2) (±)91. —78°C0JH\!J I THai.4o (3) CH2N ( CO2MOH CO2MeHb (24%)81 92Scheme 15 Oxidative Cleavage of Exocyclic RingsTherefore, the matter of choosing a suitable base which would not ring-open enol81 had to be attended to before attempting the asymmetric hydroxylation. Towards thisend, l,8-diazabicyclo[5.4.Ojundec-7-ene (DBU), a strong organic base, was used as thebase which was found to be very efficient at promoting this reaction. Upon addition ofDBU (0.4 mL) to a THF solution (10 mL) of enol 81 (20 mg) at 0°C under N2, the greensolution changed instantly to the reddish-brown enolate (a typical color of the Molishphase-test intermediate112) After 10 minutes, 1.2 equivalents of (±)- 1 -phenyl-N-(phenylsulfonyl)oxaziridine (91) was injected and allowed to react for 2 hours at 0°C (the62reaction was monitored by UVIVis and TLC). Following a standard workup and flashchromatography on deactivated silica, a dark green solid was collected (entry 1 in Table2.1). The 1H NMR of the material obtained showed that it was a diastereomeric mixture.The most significant changes were that the 173-OH peak ( 13.24 ppm) of 81 disappearedand two new broad OH peaks at ö 4.56 and 4.14 ppm (exchangeable with CD3O )appeared. The absorption spectrum Q band showed a blue shift of 20 nm to 670 nm.A FABMS analysis showed a molecular mass increase of 17 units to 533 (M+l)and reversed-phase HPLC analysis confirmed a 68:32 mixture of diastereomers (close tothe intensity ratio shown in the 1H NMR). The two diastereomers were separated bypreparative HPLC. The more mobile band (tR = 15.37 mm) (Fig. 2.1) was 132S-hydroxychiorophyllone a [82(S)] and the less mobile band (tR = 17.14 mm) was 132R-Table 2.1 Asymmetric Hydroxylation of 5, 7 and 81 with Oxaziridines 91, 93 and 94entry oxa- sub- reaction temp. product d.e.%a 132R: 132S isolated yield(%)ziridine strate and time [hi1 (±)-91 81 0°C [2] 82 24 38 : 62 752 (±)-91 81—25°C [12] 82 78 11:89 983 (—)-93 81 0°C [2] 82 90 5 : 95 934 (—)-93 81—25°C [12] 82 90 95 : 5 945 (+)-94 81 0°C [2] 82 14 43 : 57 776 (+)-94 81—25°C [12] 82 36 68 : 32 887 (±)-91 7 0°C [2] 95 38 69: 31 858 (—)-93 7 0°C [2] 95 88 94 : 6 889 (—)-93 7 —25°C [12] 95 90 95 : 5 9210 (+)-94 7 0°C [2] 95 14 57 : 43 4911 (+)-94 7 —25°C [12] 95 16 42:58 8212 (±)-91 5 —25°C [12] 96 60b 80: 20 5513 (—)-93 5 —25°C [12] 96 100b 100 : 0 9114 (+)-94 5 —25°C [12] 96 32b 66: 34 87a: The % d.e.’s determined by reverse-phase HPLC. b: The % d.e.’s determined by 1H NMR.63hydroxychlorophyllone a [82(R)]. The newly introduced 132-OH group causeddownfield shifts in the 1H NMR of nearby protons [H-17, Ha’-171Hb’-172,in 82(S); H18, Ha471Hb-172,in 82(R)] comparison to those in the starting material 81 (Table 2.2).The synthetic chlorins 82(S) and 82(R) exhibit the same spectral data as their naturalcounterparts.89(1) DBU,—25°C >NH N((2) (+)94.—25°C HN(88%) H0 HH01 / 0Hi HbOH82(R) (36% de)(+) 94:00(1S)—(+)(1O—CamphorsuLfonyDoxzIrIdIne(I_NH N( ‘ (1) DBU,—25°C(2) (+)94.—25°c) H0....( (82%)02J2Me095(S) (16% de)(INH N(N HNRC0Oic: 82(s) (90% de)C—) 93:(1R)—(—)(lO—CamphorsuLfonyfloxazlrldlne(‘I(1) DBU.—25°C NH N— ‘\ /(2) (—)93,—25°C HN(94%)OH 0081(IN HN...wYM6o7(1) DBU,—25°C NHHN(2) (—)93.—25°c132(92%)Me0295(R) (90% de)(1) DBU—25°C(2) (+)94,—25°C(87%)(1) DBu,—25°C(2) (—)93.—25°c) (91%)RO2Me02 OH0 ROJMeO2 OH0596(R) (32% de) R: 96(R) (90% de)Scheme 16 Asymmetric Hydroxylation of Chlorophyll Derivatives64BL.10.00 15.00 20.00 25.00 mmI I I I I I I10.00 15.00 20.OCFig. 2.1 HPLC Chromatography: (A) The epimeric mixture of 95% 82(S) and 5%[82(R)]. (B) The epimeric mixture of 32% 82(S) and 68% 82(R). (C)Epimerically-pure 82(S) from the semipreparative HPLC separation of(A). (D) Epimerically-pure 82(R) from the semipreparative HPLC separationof (B). HPLC conditions: Waters 600E HPLC system using a Waters C18 4p.60 A (3.9 mmxl5 cm) column; solvent system was 25% (0.1% TFA in H20)and 75% (0.1 %TFA in CH3N) with flow rate 1 mL/min and - 1300 psi backpressure.AC D65Table 2.2 Selected1H-NMR Spectral Data (CDC13,400 MHz)protonCompound 132OHIH H47 H18 Hp471 Hp’471 Hb472 Hb’-1727a 6.24(s) 4.22(ddd) 4.52(dq) 2.60(dddd) 2.58(dddd) 2.20(ddd) 2.18(ddd)81a 13.24(s) 2.58(m) 2.93(q) 1.71(m) 2.58(m) 2.45(t) 2.45(t)82(S) 4.56(br s) 4.90(ddd) 4.33(dq) 2.23(dddd) 2.88(dddd) 2.78(ddd) 4.31(ddd)82(R)b 4.14(br s) 3.82(ddd) 4.75(dq) 3.71(dddd) 2.65(dddd) 3.83(ddd) 2.95(ddd)95(R)b 5.32(s) 4.69(dd) 4.49(q) 2.13(m) 2.46(m) 2.09(m) 2.29(m)95(S) 5.43(s) 4.15(dd) 4.49(q) 2.92(m) 2.28(m) 2.55(m) 2.26(m)96(R) 5.32(s) 4.68(d) 4.47(dq) 2.09(m) 2.42(m) 1.99(m) 2.24(m)96(S)c 5.49(s) 4.15(d) 4.55(dq) 2.91(m) 2.25(m) 2.53(m) 2.25(m)a: Concentration: 1.5 mg / 0.6 mL, b: 1.0 mg / 0.6 mL, c: 2.0 mg / 0.6 mL 38%d.e., d: overlap peaksTo achieve direct diastereoselective hydroxylation, two commerically-availableenantiomerically-pure oxidants have been used. Reaction of 81 with (—)-(1R)-(10-camphorsulfonyl)oxaziridine 93 (98%, Aldrich) using the above procedure, gave a 93%yield of 82(S) (90% d.e.) (entry 3 in Table 2.1). Higher selectivity was obtained at lowertemperature where reaction of 81 with (±)-91 and (—)-93 at —25°C for 12 hours (thereaction was monitored by UVIVis and TLC analyses) respectively, gave a 98% (78%d.e.) and 94% yield (90% d.e.) of 82(S) (Scheme 16). Reaction with (+)-(1S)-(10-camphorsulfonyl)oxaziridine [(+)-94] (98%, Aldrich), the enantiomer of (—)-93, gaveslightly lower yields and diastereomeric selectivity (entry 5 and 6 in Table 2.1). Theseobservations can be rationalized when one considers the steric bulk of the 17-propionicgroup which hinders the approach from the re face of the chlorin enolate.In a similar fashion, treatment of pheophorbide a methyl ester (7) and pheophytina (5) with the oxaziridines gave excellent yields and diastereoselectivity. As describedabove, the 1 7-propionic group affects the stereoselectivity. With the increased size of the17-propionic ester chain from methyl ester 7 to phytyl ester 5, the d.e.% of the products66after reaction with (±)-91 and (—)-93 increased (entry 9 and 13 in Table 2.1), in the lattercase, a 100% d.e. was observed with re face recognition of the nucleophilic oxaziridine93. This is in contrast to a 90% d.e. in the reaction with the smaller ester where only a32% d.e. was observed in the reaction of 5 with (+)-94 (entry 14 in Table 2.1) along withreversed stereoselectivity such that 96(R) and 96(S) were produced in a ratio of 66:34(entry 14 in Table 2.1) (Scheme 16). The bulky phytyl ester (methyl ester) and 132methoxycarbonyl moieties are on opposite sides of the molecular plane in 96(S) [95(5)]while they are at the same side in 96(R) [95(R)]. 132S configuration (which gave lowerd.e. and yields) is the thermodynamically more stable form than the 132R configurationshowing that these reactions are under kinetic control. Diastereomerically-pure 95(R) and95(S) were prepared by HPLC separation of the products from entries 9 and 11 in Table2.1. Attempts to purify132S-hydroxypheophytin a [96(5)] from entry 14 (in Table 2.1)were partially successful. A sample of 32% d.e. ‘S 96(S) was obtained after preparativenormal-phase HPLC separation. Further purification by HPLC (reversed and normalphases) and preparative TLC was attempted and was largely unsuccessful due to thedecomposition of the material.96(S)67In these hydroxychiorins, the downfield shifts (of the nearby protons of 132-OHmoiety) in the 1H NMR were less pronounced due to the greater distance between the 17-alkyl protons and the newly introduced OH group (Table 2.2).Additional hydroxylation methods were also tested to better understand thedifferent reactivities between ionic bases [(TMS)2NNa and LDA] and the organic base(DBU). For example, reaction of the enolate from pheophorbide a methyl ester (7)formed by DBU, with mCPBA at room temperature for 10 hours, gave less than 5%hydroxylation product. Bubbling molecular oxygen into the enolate of 7 in the presenceof DBU and triethyl phosphite’13 at room temperature for five hours gave no reaction andthe starting material was recovered. Furthermore, reaction the enolate of 7, formed byionic bases, with mCPBA’14 (30 mm at room temperature) or 02 (5 mm at 0°C) led toexocyclic ring cleavage.To our knowledge, this method is the first to use DBU as a base for ohydroxylation and the first successful diastereoselective oxidation of chlorophyllderivatives. The mild conditions, high yields and d.e.% make it an efficient method tointroduce a hydroxy group into B-dicarbonyl chiorins and other similar systems. Thesehydroxychlorins are not available from other methods and will serve as models for anunderstanding of chlorophyll allomerization mechanisms.132S-hydroxypheophytin a [96(S)], recently isolated from Silkworm excreta, isreported to possess a high quantum efficiency (50%) for the photosensitized production ofsinglet oxygen’15 suggesting that the hydroxychlorins produced here have a potential asdrugs in photodynamic therapy.682.5 Model Studies for HydroxylactonizationAs mentioned earlier, 15’R-hydroxychlorophyllone a [83(R)] and diketone 85 arethe mono-oxidized products at different exocyclic rings of 12-hydroxychlorophyllone a(82). The purpurin-18 (22) results from their further oxidation (di-oxidized product from82). Therefore, with sufficient amounts of hydroxychlorin 82 having been produced, ournext objective was to generate these further oxidized products. Initially, because 132hydroxychlorophyllone a (82) possesses two similar carbonyl groups which are bothreactive (though the carbonyl at the 173-position appeared to be less stericallyencumbered and less conjugated), the monoketone132-hydroxypheophorbide a methylester (95) was used as a model for the hydroxylactonization.The first hydroxylactonechlorin 21, a diastereomeric mixture of hydroxylactone21 (S) and 21(R), was prepared by Fischer”6 via aerial oxidation of pheophorbide amethyl ester (7) in the presence of pyridine and alkali in the 1930s. This mixture, called“unstable chlorin” due to its extremely instability, is the precursor to many chlorophyll adegradative products (detailed discussion appears in Chapter 1). The “unstable chlorin”2 1 C02R21(S). R12H 21(R). R12H 99. R=COOMe97(S). R1=Me, R2=H 97(R). R1=Me R2—H 100. R=H101 (5). =R2Me 101 CR). =R2Me69monomethylester 97 is more stable and was obtained by Fischer and Kahr from KMnO4oxidation of pheophorbide a followed by acid fractionation.117Treatment of the (95%R, 5%S)132-hydroxypheophorbide a methyl ester (95) withmethanolic alkali at room temperature for 12 h under N2, resulted in the hydrolyticcleavage of the ring V generating “unstable chiorin” 21, a result similar to the alkalineaerial oxidation of pheophorbide a methyl ester (7). Attempts to purify “unstablechiorin” 21 were unsuccessful due to its ready decomposition. However, purpurin-7trimethyl ester (16) (total 45% yield from 82) was obtained after treating the “unsta1lechlorin” (21) with diazomethane. Attempts to prepare “unstable chlorin” dimethyl ester(101) by partial esterification (via Et3N/MeOH) of 21 were also unsuccessful sincemethylation of the hydroxylactone is fast and readily proceeds without selectivedifferentiation from the esterification of the 173-carboxylic acid. Rather than expendfurther efforts on optimizing this process, it was decided to establish whether this routewas suitable to the oxidation of132S-hydroxychlorophyllone a [82(S)]. Treatment of the(95%S, 5%R)132-hydroxychlorophyllone a (82) with KOHIMeOH under N2 for 10 hoursled to the cleavage of both the exocyclic rings and gave chiorin p trimethyl ester (92)after methylation with diazomethane.Chemically, the transformation of hydroxyketones 82 to hydroxylactone 83(R) isoxygen atom insertion and Sakata et al.89 have suggested that this conversion isbiogenetically via a Baeyer-Villiger type oxidation. With this in mind, we alsoinvestigated oxidation reaction of chiorins with peroxycarboxylic acids. Unfortunately,reaction of 95 (95%R, 5%S) or 82 (95%S, 5%R) with mCPBA or CF3000H in the70dark resulted in overoxidation and no products could be identified. Conversely, treatmentof the non-hydroxylated chiorins, pheophorbide a methyl ester (7) and pyropheophorbidea methyl ester (25), with the above oxidants gave no reactions if electrophiles were notpresent in solution. Instead of oxidation, electrophilic substitution (detailed descriptionof electrophilic substitution reaction appears in Chapter 1) at the H-20 position of 7 and25 occurred when only a trace amount of electrophile was present. For example, the traceamount of HC1 present in solvent chloroform brought about the formation of 20-chloropheophobide a methyl ester (99) and 20-chloropyropheophobide a methyl ester(100).The only method which worked in our hands was found to be periodateoxidation”8,which occurred only in acidic conditions. Periodic acid (0.1 N) in aqueous0.1 N H5106(79%) + ..hA JHO115 [TIMeO2cIMeO2C0 ‘) OMe O2COHCO2Me CO2Me95 101(S) 101(R)84% : 16%CH2NNH NN HN’CO2MeO2MeCO2Me16Scheme 17 Model Study for Periodate Oxidation71dioxane was found to be the most satisfactory and 15’-hydroxypurpurin-7-lactonedimethyl ester (101) was isolated in 79% yield (the reaction was monitored by UVIVisspectroscopy and completed in 16 h at room temperature). Under these conditions 101was obtained as a diastereomeric mixture of 84% 101(S) and 16% 101(R) (from signalintegration of the 400 MHz 1H NMR spectrum). Both epimers of 95 were found to givethe same diastereomeric mixture of 101 under the above oxidation conditions. Thereason for this is that hydroxylactone 101 is very sensitive to both acid and base whichgive rise to epimerization via the reversible opening of the hydroxylactone ring. Theequilibration of epimerization under acidic conditions predominantly favours formationof the thermodynamically more stable epimer 101(5) in which the methoxycarbonylmoiety at position 151 is on the opposite side to that of the bulky 17-propionic group.Therefore, in subsequent reactions, we used the (95%S, 5%R) 82 instead of using theepimerically-pure compounds. Reaction of hydroxylactone 101 with an excess of etherealdiazomethane caused almost quantitative conversion to purpurin-7 trimethyl ester (16).2.6 Regioselective OxidationWhen the initial experiment utilizing the above periodate oxidation was used tomake hydroxylactone 83 and 1 ,2-diketone 85 from hydroxychlorin 82, only a low yield(total <10%) of the desired products were obtained, the major product (>50%) being thedi-oxidized product, purpurin-18 (22). This result indicated that the mono-oxidizedproducts 83 and 85 were more reactive than the starting material 82. This was further72confirmed by following oxidations of 83 and 85, with periodic acid, where both morerapidly gave purpurin-18 (22).Subsequent work showed that regioselective oxidation of one of the two carbonylgroups can be modestly achieved by using different reaction media. Addition of pyridine(15 mL) to the periodic acid solution (40 mL) resulted in the formation of l32oxopyropheophorbide a (85) (49%) and two minor products, an 11% yield of purpurin-184 I / /7\NH N NH N NH N—J151 13 13 131“N HN/ N HN / N HN): 0OH OH R0 0o 0 COO COOR22 R=H 87 R=Me83(R) 83(S) 85 R=H 102 R=MeA: 0.08 N H5106/pyridine(26%) (2%)B: 0.06 N H5106/MeOH(55%) (3%)82B(49%)(2%)(11%)(2%)(5%) (7%)Scheme 18 Regioselective Periodate Oxidation of Hydroxychlorin 8273(22) and a 28% yield of 15’-hydroxychlorophyllonelactone a (83) after preparative TLCseparation on deactivated silica (Scheme 18, Condition A). Structural assignments arebased on visible spectra and ‘H and ‘3C NMR spectroscopy (see the following spectraldiscussion). This procedure which preferably oxidizes the seven-membered ring(exocyclic ring VI) allowed for the preparation of132-oxopyropheophorbide a (85) in 4steps from pheophorbide a methyl ester (7) in —40% overall yield.Another procedure which oxidizes the exocyclic ring V over ring VI results fromreplacement of pyridine with methanol (45 mL) as a component in the reaction medium(144 mL) (with periodic acid). Under these conditions, the (95%S, 5%R) 82 waspredominantly converted into 15’-hydroxychlorophyllonelactone a (83) (57%), togetherwith four minor products, purpurin-18 methyl ester (87) (7.2%), purpurin-18 (22) (2.7%),132-oxopyropheophorbide a methyl ester (102) (5.4%) and13-oxopyropheophorbide a(85) (1.8%) (Scheme 18, Condition B). Separation was achieved by preparative TLC ondeactivated silica gel. The two purple-red bands, 22 (the second least mobile band) andits methyl ester 87 (most mobile band), could be readily identified from their different Rfvalues and their identical visible spectra (700 nm, 404 nm) while the two yellow bands,85 (the least mobile band) and its methyl ester 102 (the second mobile band) displayedthe same visible spectra (678 nm, 420 nm, 390 nm). The gray-green major band (83) wasanalyzed by 400 MHz ‘H NMR to be a (R,S) diastereomeric mixture of 94% 15’R-hydroxychlorophyllonelactone a [83(R)] and 6% 15’S-hydroxychlorophyllonelactone a[83(5)]. Hydroxylactone 83 was found, like the model hydroxylactone 101, to be verysensitive to both acid and base which give rise to epimerization via the reversible opening74of the hydroxylactone ring. Nevertheless, the principal epimer 83(R) has an cX(downward) 15’-OH group rather than a p-OH (upward) as in the model compound101(S). The reversed orientation of 15’-OH group in 83(R) is due to the conformation atC-15’ position which reduces the major steric congestion with the carbonyl group at C17g. Optically-pure 83(R) was obtained by subjecting the above diastereomeric mixtureto preparative TLC separation on deactivated silica gel. The synthetic 83(R) exhibits aspectrum identical to that of the natural product (see the following spectral discussion).This procedure provided an efficient synthesis of 15’R-hydroxychlorophyllonelactone a[83(R)] in 4 steps from pheophorbide a methyl ester (7) in —45% overall yield.As expected, direct reaction of the (94%R, 6%S) 15’-hydroxychlorophyllone-lactone a (83) with an excess of ethereal diazomethane gave a good yield (82%) ofchlorophyllonic acid a methyl ester (84) (Scheme 19). Attempts to isolate 88, thelactonized isomer of 132-oxopyropheophorbide a (85) by using milder oxidationconditions failed. In addition, attempts to obtain a pure sample of chiorophyllonic acid a(104), a non-lactonized isomer of 15’-hydroxychlorophyllonelactone a (17), were alsounsuccessful. These observations, and a consideration of the peripheral overcrowding,84 88 104Scheme 19 Chlorin 84 and Its Unfavored Non-Esterified Isomers75indicate that the formation of the six-membered hydroxylactone ring (as in chiorins 22,83, and 101) is predominant and cyclization to the eight-membered hydroxylactone ring(as in 88) is unfavoured under our reaction conditions. Although chiorins 84 and 102 are1 ,2-diketones, their oxidation by periodic acid was found to be even faster than the ohydroxy- 1 ,3-diketone 82, a difference perhaps mainly attributed to the ring strain of the1 ,2-diketone twisted conformation in 84 and 102 and the obviously more encumberedconformation of 82.2.7 Structure and Spectral CharacterizationAll the synthetic chiorins were subjected to various spectral analyses, includingmass, UV-Vis absorption, ‘H and ‘3C NMR spectroscopies. All compounds werehomogeneous and diastereomerically-pure materials, as ascertained by reversed-phaseHPLC analyses (except 81 and 83(R) due to their instability). Further, their ‘H and ‘3Cspectral assignments were carefully compared with the reported spectral data89 of thecorresponding natural compounds including their ‘H-’H coupling constants.The conformational changes of the chiorin macrocycles resulting from variationsof the peripheral substituents and the resulting shift on the frontier orbitals of thesechiorins are reflected in their electronic absorption spectra. We have divided thesechiorins into 4 different types as a result of their optical absorption spectra. Each typerepresents special exocyclic structures and the conjugation effects of the exocyclic ringswith the chiorin framework.76Type I (Fig. 2.2), including hydroxychlorins 82(R), 82(S), 95(R) and 95(S), ischaracterized by a strong Soret band, in chloroform, at —412 nm and a relatively strong Qa)C.)0WavelengthjNHN\82(R)(I[eO2c?oCO2Me783(R)(I,COOle87Fig. 2.2 Structures and UV/Vis Spectra (dC!3)of Pheophorbide a Methyl Ester(7), 1325-Hydroxychlorophyllone a [82(S)], 13R-Hydroxychlorophyllonea [82(R)], 15’R-Hydroxychlorophyllonelactone a [83(R)] and Purpurin-18Methyl Ester(87)82(R)82(S) 82(S)78783(R) —82(R)jNHN\82(5)82(5)Type IType II400 500 600 700 800nm77band at —670 nm. They exhibit electronic spectra similar to that of pheophorbide amethyl ester (7). The only difference among them is that conformational twisting of thering VI in 82(R), due to the trans-orientation of 17-17’ and l32173 bond, make its Q bandshift 4 nm to the red.Type II (Fig. 2.2), including chlorins 22, 83(R), 87, 92 and 101, is another type ofchlorin spectra. These chlorins (except 92) contain the exocyclic rings V in the form ofsix-membered lactone. With the insertion of oxygen into the five-membered exocyclicring V of type I, the exocyclic twisting is relieved which results in blue-shifted (—8 nm)Soret band and disappearance of the Soret shoulders. The pronounced red shifts (—30nm) of the Q bands in purpurin-18 (22) and its ester 87 result from conjugation with the151 carbonyl group which markedly extends the conjugation of the aromatic macrocycle.When there are two conjugated sp2 group (two carbonyl or one carbonyl and oneC=C double bond) directly connected in the five-membered exocyclic ring V, theconformational changes of the chlorin macrocycles resulting from the effects of thetwisted and electron-withdrawing exocyclic rings become so significant that the Soretbands of Type ifi, enol 81, chlorins 85 and 102, are split, as shown in Fig. 2.3. Inaddition to the split Soret bands, their Q bands are also significantly red-shifted 20 nm(81) and 8 nm (85 and 102) in contrast to that of chiorin 7.The Q bands of Type IV, including chlorins 16 and 84, are somewhat resolved andslightly split into two peaks as shown in Fig. 2.3. Two electron-withdrawing groups(conjugated sp2 groups) at C-15’ and C-iS2 significantly extend the conjugation of thechlorin macrocycle which results in more than 10 nm bathochromic shifts of the Q bands78although no shift of their Soret bands was observed. It should be noted that the splittingof these Soret bands is solvent-dependent and has only been observed in chlorinatedCOOMe102(‘I084(‘I,1N HN— /22MeCO2Me16WavelengthFig. 2.3 Structures and UV/Vis Spectra (CHC13)of132,173-Cyclopheophorbidea Enol(81), 32-Oxopyropheophorbide a Methyl Ester(102), Chlorophyllonic Acid a Methyl Ester(84) and Purpurin-7 Trimethyl Ester(16)8181Type IIIType IV400 600 BOOnm79solvents such as methylene chloride and chloroform. In non-chlorinated solvents the twopeaks of the split Q band become a peak with a shoulder on the long-wavelength side.Consequently, the formation of ring VI has less effects on the macrocyclicconformation if the electron-withdrawing carbonyl (C-173) is not conjugated with thechlorin ring. The sp2 conjugate groups (electron-withdrawing) of the exocyclic ringsdeform the conformations of exocyclic rings and thereby the macrocycles, which in turnaffects the absorption spectra.The meso hydrogens in the chlorins of Type I and Type II do not show any shiftsin their ‘H NMR spectra since the macrocyclic conformation remains almost identical.The hydroxy groups at C-132 [82(R), 82(S), 95(R) and 95(S)] and C-15’ [83(R) andTable 2.3 Selected 1H NMR Spectral Data (CDC13,400 MHz)CompoundProton 7 81 82(S) 82(R)b 95(R)b 95(S) 1O1(S) 83(R)b 102b 84bH-b 9.52 8.64 9.40 9.47 9.53 9.62 9.77 9.68 9.90 9.70H-5 9.39 8.43 9.35 9.35 9.47 9.48 9.55 9.50 9.86 9.49H-20 8.57 7.38 8.70 8.52 8.61 8.63 8.80 8.78 9.00 8.60H-18 4.52 2.93 4.33 4.75 4.49 4.49 4.43 4.38 4.68 4.40H-17 4.22 2.58 4.90 3.82 4.69 4.15 4.05 4.42 5.16 4.53Ha471 2.60 1.71 2.23 3.71 2.13 2.92 2.46 2.19 2.78 2.38Ha’471 2.58 2.58 2.88 2.65 2.46 2.28 2.18 2.85 2.36 2.90Hb-172 2.20 2.45 2.78 3.83 2.09 2.55 2.45 3.49 2.67 3.83Hb’-172 2.18 2.45 4.31 2.95 2.29 2.26 1.80 3.01 2.32 3.05132-OH/H 6.24 4.56 4.14 5.32 5.4315’/173-OH 13.24 6.05 5.86aConcentration 1.5 mgIO.6 mL bConcentration 1.0 mgIO.6 mL(‘I(I82(R)(‘I82(S)Fig. 2.4 ‘H NMR Spectral Comparison (the Low Field Region) of NaturalHydroxychlorins 82(S), 82(R) and 83(R) (CDC13,400 MHz)83(R)80CH31se81101(S)] cause pronounced anisotropical effects on their nearby protons, which results indownfield shifts for H17, Ha’17’ and Hb’-172 in 82(S), 95(R), 83(R) and for H-18, Ha17’ and Hb-172 in 82(R), 95(S), 101(S) (Table 2.3).For chiorins with Type ifi and Type IV absorption spectra, the effects of the itelectrons at C-15’ and/or exocyclic rings on their chemical shifts in 1H NMRspectroscopy are less pronounced than on their electron absorption spectra. However,two peak shifts are observed for chiorins 81 and 102, aromatic rings with split Soretbands (Type ifi). For enol 81, the zS.ö for H-b resonance was —0.88 ppm, for H-5 —0.96ppm, and for H-20 —1.19 ppm together with general up-fielded shifts more than 0.40 ppmin comparison with the corresponding protons in a normal chiorin such as 7. These shiftssuggest an apparently increased local aromatic ring current in 81 due to the strain on theTable 2.4 Selected 1H-1H Coupling Constants J (Hz, CDC13)_____CompoundProtons 7 81 82(S) 82(R) 95(R) 95(S) 101(S) 83(R) 102 8431,32(E) 17.1 18.0 18.1 18.1 18.2 18.3 17.6 17.6 18.5 17.431,32(Z) 11.6 11.6 11.4 11.6 11.9 11.5 11.2 12.4 12.1(E),3Z) 1.6 1.2 1.0 1.0 0.8 1.2 1.0 1.3 1.081,82 7.8 7.9 8.1 7.2 7.7 7.6 8.0 7.9 7.7 7.7181,18 7.1 7.2 7.4 7.0 7.0 7.3 6.8 7.5 7.6 7.318,17 1.7 3.8 8.3 1.6 1.3 1.717,17a 3.1 13.3 11.0 1.7 2.2 2.4 11.5 2.9 12.317,17a 9.3 3.5 1.6 8.5 10.2 10.4 5.3 8.8 6.617’a, 17a’ 13.3 12.4 13.1 12.8 12.317a, 172b 7.1 2.1 6.2 8.3 10.017a, 172b’ 6.2 14.0 12.8 2.5 1.4171a, 172b 9.3 4.4 1.5 9.0 10.017a’, 172b’ 5.3 3.5 5.2 9.6 8.3172b, 172b’ 15.1 11.7 15.0 11.5 12.782chiorin nucleus resulting from the conjugated enol conformation. This conformation isenergetically-favoured through formation of an intramolecular hydrogen bond betweenthe 173-OH (enol) and the 132-carbonyl group. Compound 102, another chlorin of Typeifi, has showed pronounced down-fielded shifts [Aö for H-l0 (0.38 ppm), H-5 (0.47 ppm)and H-20 (0.43 ppm)] in contrast to the corresponding hydrogens in 7.The large coupling constants between H-17 and Hal7’ in 82(S) (J 13.3 Hz),82(R) (J = 11.0 Hz), and 83(R) (J = 11.5 Hz) indicate their 1,2-trans diaxial relationships(Table 2.4). The relative stereochemistry around rings IV, V and VI was deduced by a2D COSY spectrum (Fig. 2.13) and decoupling experiments. These assignments shownin Table 2.3 and 2.4 are consistent with their assigned structures.The ‘3C NMR spectra of epimeric 82(R) and 82(S) showed upper field shifts of C-17’ (iS.6 = —15.28 ppm) and C-18’ (A6 = —5.38 ppm) in 82(R) from 82(S), which resultfrom the deformation of the seven-membered ring VI in 82(R) where the 17-17’ and l3217 bonds are trans-oriented (Table 2.5). This deformation does not exist in the absenceof exocyclic ring VI, such as in epimers 95(R) and 95(S). The ‘3C NMR spectra ofdiketonic isomers 84 and 102, shows a different effect of exocyclic rings VI and V on themacrocyclic conformations. The downfield shift of the carbonyl group bonded to C-15position, C-15’ (ö = 192.38 ppm) of 84 (with the exocyclic ring VI) from that of the C132 ( = 185.19 ppm) in 102 (with the exocyclic ring V) is attributed to a change inhybridization modified by the more twisted ring V (five-membered ring) than ring VI(seven-membered ring).83Table 2.5 Selected 13C NMR Spectral Data (CDC13)_______CompoundCarbon 7 81 82(S) 82(R) 95(R) 95(S) 83(R) 102 84C-173 173.36 167.35 208.00 206.21 173.46 172.83 203.62 174.87 196.90C-131 189.63 191.78 195.44 193.39 191.93 192.00 162.57 192.79 166.90C-132 64.71 116.83 93.43 92.66 89.09 88.94 185.19 52.31C-17 52.88 52.47 51.91 53.71 50.75 51.75 49.84 52.67 50.16C-18 51.70 49.33 51.51 50.31 50.16 50.29 51.18 51.63 51.20C-172 29.86 34.00 40.12 43.17 30.99 31.40 34.17 29.70 36.25C-171 31.06 25.03 37.99 22.71 30.18 31.11 31.37 31.50 29.47C-151 104.68 192.38C-181 23.10 19.06 22.37 16.99 22.69 22.65 23.56 23.85 23.62Detailed structural assignments of these antioxidative chlorins appear in theexperimental section (section 6.2). Selected spectra for these unusual chlorins are shownat the end of this chapter (from pp89 to pp107). These spectra are: ‘H NMR spectra: 7(Fig. 2.5); 81 (Fig. 2.7); 82(5) (Fig. 2.9); 82(R) (Fig. 2.11); 83(R) (Fig. 2.12); 84 (Fig.2.14); 87 (Fig. 2.16); 92 (Fig. 2.17); 95(R) (Fig. 2.18); 95(5) (Fig. 2.20); 96(R)(Fig.2.21); 102 (Fig. 2.23). ‘3C NMR spectra: 7 (Fig. 2.6); 81 (Fig. 2.8); 82(5) (Fig. 2.10);84 (Fig. 2.15); 95(R) (Fig. 2.19); 96(S) (Fig. 2.22). ‘H-’H COSY spectrum: 83(R) (Fig.2.13).2.8 Biogenetic RationalizationThe concern about the origin of these antioxidative chlorins takes the form of twobasic questions: “Where are they from ?“ and “How are they formed?”. The first questionis not difficult to answer. Although these compounds have been identified in diversemarine organisms such as sponge, fish, bivalves, attached and wafting diatoms, thesespecies are all plankton feeders.”9 These marine animals themselves do not contain any84chiorophylls, but the food chain of these herbivorous species provides them a dietarysource of chlorophyll a.The second question should be answered with discussion. The key point is how todetermine with certainty whether these chlorins were created from chlorophyll a bydigestion of these herbivorous species or whether they were biosynthesized by thesemarine species to serve a special biological function. The former (degradation) viewappears to suggest that these antioxidative chlorins are yet another example of chlorophyllbreakdown, which happens everyday on this planet. Many degradation products ofchlorophyll a have been reported, which includes simple oxidation and complicatedbreakdown to colourless products.12°For instance, the acidic environment of the stomachalone would bring about the loss of Mg to produce pheophytin.7”2’The removal of thephytol group from chlorophyll to form pheophorbide and the oxidation (ring 1V) anddecarboxylation (methoxycarbonyl moiety in ring V) steps to form phytoporphyrin(porphyrin derived from chlorophylls) have been discovered to be produced in the gut ofmicroflora.’22However, the degradation view of these compounds does not explain theirbiological (antioxidative) function since degradation usually occurs randomly orstochastically without any biotic evolution and no chlorophyll degradation products havebeen reported to have any biological function. Further, it also can not explain the fact thatchiorins 81, 82(S), 83(R), 84 and 82(R), 22, 87, 85 have been isolated and that manycomplicated chemical-specific steps (based on our synthetic research) are involved intheir pathways from chlorophyll a. The epimers at C-i 32, hydroxychlorin 82(S) (found in85short-necked clam and diatoms)89 and hydroxychiorin 82(R) (found in the scallop), occurin different marine species, strongly suggesting that both 82(S) and 82(R) areenzymatically produced in each bivalve or diatom and that new biosynthetic pathways ofchlorophyll a to these antioxidative chlorins are present. Compound 82(S) was alsoisolated from the mixture of attached diatoms91,indicating that 82(S) was produced bythe attached diatoms themselves. Thus, these antioxidative chlorins are not serendipitouschlorophyll degradation products. Rather, it appears reasonable that they arebiosynthesized from chlorophyll a. They are a class of compounds having a specificmetabolic function (antioxidative activity). The biogenetic evolution may result frombiotic regulation of these marine organisms in order to develop antioxidative function as adefence of the detrimental effects of oxygen. It is not clear at this stage why these specieschoose to modify chlorophyll from their food chain rather than other simpler molecules.Many enzymes, which mimic the synthetic transformations of these antioxidativechlorins from chlorophyll a, have been found in nature. Enzymatic transformation ofchlorophyll a to pyropheophorbide a has been confirmed in a mutant strain of themicroalga, Chiorella fusca.’23 The assumed biosynthetic origin of 132, 17-cyclopheophorbide a enol (81), via an intramolecular Claisen-type condensation ofpyropheophorbide a methyl ester (25), is firmly supported by the facile chemicaltransformation. Actually, only pyropheophorbide a and its esters (not their metallatedcompounds) can participate in this cyclization reaction. Our attempts to extend thisreaction to chlorophyll a and its closely related derivatives, pheophytin a (5) andpheophorbide a methyl ester (7) were unsuccessful.86Enzymatic hydroxylation of chlorophyll a to132-hydroxychlorophyll and 132S-hydroxypheophytin a has been found in alga’24 and in silkworm”5,respectively. Thechemical possibility [oxidation of13,173-cyclopheophorbide a enol (81) to give 82(S)and 82(R)] has been confirmed by our asymmetric hydroxylation. Periodate oxidation ofeither epimer of 82, 82(R) or 82(S), was found to give the similar products, i.e., a mixtureof mono- and di-oxidized products 22, 83 and 85, which coincidently parallels all theantioxidative chlorins isolated from the short-necked clam, Ruditapes philippinarum.This observation suggests that 22, 83(R) and 85 were probably biosynthesized by“periodate type” oxidation of 82(S). In fact, flavin-dependent monooxygenases havebeen identified which catalyse oxidation of acyclic and alicyclic hydroxyketones tohydroxylactones in soil bacteria.’25 However, the natural oxidation of hydroxychlorin82(R) has found to be highly regioselective in the scallop, Pactinopecten yessoensis,which only metabolizes the mono-oxidized products of the exocyclic ring V,hydroxylactone 83(R) and its methyl ester 84.Esterification is a common reaction in marine metabolism. Chlorophyllonic acida methyl ester (84) and purpurin- 18 methyl ester (87) presumably arise from thecorresponding methylation of hydroxylactone 83(R) by ring V opening and directmethylation of purpurin-18 (22). Thus, the biosynthetic pathway to these compoundsfrom chlorophyll a has been predicted to be parallel to their chemical synthetic routes,which has met our initial objective that the chemical synthetic routes to these unusualstructures should be developed in a simple and biomimetic way.872.9 SummaryShort and efficient stereoselective synthesis of new chlorophyll a related chlorinsfrom chlorophyll a has been accomplished in a way that parallels their probablebiogenesis. The key stereochemical issues were addressed via DBU-promotedasymmetric hydroxylation and via the anticipation that the rigid exocyclic ring VI (inhydroxylactone 83) would provide an exploitable diastereofacial bias for ensuinghydroxylactonization to the desired epimer 83(R).Effective improvements on chlorophyll extraction from the blue alga Spirulinamaxima have been achieved. The improved method affords isolation of pheophytin aeasily and in high yield. DBU as a base for promoting the hydroxylation reactions iscertain to be applicable to other 1,3-dicarbonyl systems, particularly those sensitive toionic bases. Model studies for hydroxylactonization have shown that periodate oxidationof hydroxyketone 95 stereoselectively and predominantly forms hydroxylactone 101(5).Periodate oxidation of oc-hydroxy-l,3-diketone 82(S) and/or 82(R) to furnish hydroxylactone 83(R) and diketone 102 was found out to be regioselective and the site of reactiondepended on the appropriate choice of reaction media.The effects of the formation of an additional exocyclic ring (ring VI) on themacrocyclic conformation and electronic absorption spectra were also discussed. Thebiogenesis of these antioxidative chlorins has been rationalized and a new biosynthetic88route has been proposed. These new chlorophyll a related chlorins will also providestrong structural evidence to support the hypothesis that the antioxidative chlorinssynthesized in this work are the precursors to the so-called disturbing petroporphyrinscharacterized by exocyclic rings, the molecular fossils from chlorophyll a derivatives inthe marine sediment.I2Me2O‘1tf.OfO.O9.‘HNMRSpectrum(CDCI3)of PheophorbideaMethyl Ester(7)‘-NHN(NHAWc7—I.._.—LPPNCFig.2.6Structureand13CNMRSpectrum(CDC13)ofPheophorbideaMethyl Ester(7)II141286Fig.2.7Structureand‘HNMRSpectrum(CDC13)of132,173-Cyclopheophorbideaenol(81)HNIIIIIIIIIIIIIIIIIIIIIIIIIIIIII420—2—4t’38124018012080Fig.2.8Structureand‘3CNMRSpectrum(CDC13)of132,173-Cyclopheophorbideaenol (81)82(S)!‘.06.0S.0a.PPH3.0—:0-1.0-2.—4.0czFig.2.9Structureand1HNMRSpectrum(CDCI3)ofI32S-Hydroxychlorophyllonea[82(S)]If)0Ifr-I—IDI—r-r-eincvcva.—r-In(001u,mr’,,inminFin‘(o(0cvn‘IfQ(‘‘cva.øIDIL)0)0)r(‘3r-CDIf)If)‘If‘IfininininincuCucvcvIbID00)1”-m—‘3mIfl’’j000)0)0)IV——(11010)IL)—0incvcv—0——‘.0I’-.(‘30)c-cvcv..;‘3c71NH\82(S),IIIIIIIppmI.I20018014012010080Fig.2.10Structureand‘3CNMRSpectrum(CDC13)of132S-Hydroxychlorophyllonea[82(S)](‘INHN1NHNHO 082(R)Fig.2.11Structureand‘HNMRSpectrum(CDCI3)of132R-Hydroxychlorophyllonea[82(R)](‘Ii83(R)AIL_LiVjj_________1l‘i.i9.117.1.1t4.43.112.0).Ill211Fig.2.12Structureand‘HNMRSpectrum(CDCI3)of15’R-HydroxychlorophyllonelactOflea[83(R)]Fig. 2.13 Structure and 1H-’H COSY Spectrum (CDC13)of 15’R-Hydroxychiorophyllonelactone a [83(R)] (88% d.e.)83(S)6%973.6.083(R)94%;..PP”Fig.2.14Structureand‘HNMRSpectrum(CDCI3)ofChiorophyllonicAcidaMethylEster(84)-1JH-.,-i.a--4.1(‘IJwNHN((\)84)NHW\co2u..111I1I11111(111111111111111IIIIIIPIIlPIIIIIIIIIPIIliii,.,,1111111111i.,..i...iiii,ii,i..i20018016014012010080604020PPMFig.2.15Structureand‘3CNMRSpectrum(CDC13)ofChiorophyllonicAcidaMethylEster(84)876.aIIi.eFig.2.16Structureand‘HNMRSpectrum(CDCI3)of Purpurin-18Methyl Ester(87)Ci-NH•—t’‘HN92O2MeCO2MeIt’..Ij.IIr’9.11.1•Ø6.‘HNMRSpectrum(CDCI3)ofChlorinp6TrimethylEster(92)(I NHN95(R)Hi_tI.Ii’..e2•Ø:,‘.:2’:.::.:4’.Fig.2.18Structureand‘HNMRSpectrum(CDCI3)of132R-HydroxypheophorbideaMethylEster[95(R)]C cz(I‘$LNHN?95(R)1.111 HIIII11111111IIIIIIliiII11111111I11111III111111111111111111111111111I1III!11111111III•IIJIlIllIllilIIIIliii22020018016014012010080604020PPM0Fig.2.19Structureand‘3CNMRSpectrum(CDCI3)of132R-HydroxypheophorbideaMethylEster[95(R)]Fig.2.20Structureand‘HNMRSpectrum(CDCI3)of1325-HydroxypheophorbideaMethylEster[95(S)]i-NHNf95(S))HO0jo2MeO96(R)‘HNMRSpectrum(CDCI3)of132R-Hydroxypheophytina196(R)]LQ__J/RIiLLLLL1LL4)4JIIIIIIhiIIIiIIIIIppm2202001801601401201008060.io2JC96(R)I iII hiIhi[Fig.2.22Structureand‘3CNMRSpectrum(CDCI3)ofI32R-Hydroxypheophytina[96(R)](‘I102Fig.2.23Structureand‘HNMRSpectrum(CDC13)of132-OxopyropheophorbideaMethylEster(102)108Chapter 3Phytoporphyrins: Novel Synthesis and Applications1093.1 Research ObjectivePhytoporphyrins are porphyrins directly derived from chiorophylls and theirclosely related derivatives.65 Chemically, they are defined as porphyrins characterized bya five-membered exocyclic ring that is typical of chlorophylls. Research onphytoporphyrins can be dated back to the 1930’s, when Fischer and Bäumler’26 usedhydrogen iodide in glacial acetic acid to convert 3-vinylchlorins into 3-ethylporphyrin forthe structural elucidation of chlorophylls (Scheme 20). Based on this transformation,chlorophyll and its derivatives were believed to be vinyldihydroporphyrins(vinylchlorins). This ready transformation from vinylchlorins to simple porphyrins waseasily understood as a process simply involving the transference of two hydrogens fromthe dihydroporphyrin nucleus to the peripheral vinyl group, which is thus converted intoethyl. This reaction, later termed as “HI reduction” or “HI isomerization”, was a usefulmethod for the preparation of chlorophyll related porphyrins (i.e. phytoporphyrins)although a large amount of by-products was always observed and the major productseparated from the complex mixture was usually obtained in less than 20% yield.65However, the reaction mechanism, involving isomerization and migration of hydrogens atNH N HI NH NCH3COOH+ OthersR1 =COOMe,H; R2=Me,PhytylScheme 20 “HI Isomerization” of Chlorophyll Derivatives110positions 17, 18 to the exocyclic 3-vinyl group, remained obscure. Although acarbocation rearrangement mechanism has been recently suggested by Hynninen’27,theharsh acidic condition and complex product distribution have made mechanistic researchdifficult.The objective of our research was to develop a new method for the conversion ofchlorins into phytoporphyrins which may help to elucidate this reaction mechanism,especially the acid-catalysed migration of H atoms at postions 17, 18. With this in mind,we focused our attention on the active position (131 carbonyl group in the exocyclic ringV) of chlorophyll, where most chemical reactions related to chlorophylls are known. Itwas assumed that if another vinyl group is introduced into chlorophyll derivatives,especially into the exocyclic ring V, the acid-catalysed migration of protons at H- 17, 18may compete for the two peripheral vinyl groups. Comparison of hydrogen migrationcapabilities to these two groups should provide some new insight on the reactionmechanism. In an effort to verify this assumption, synthesis of a chlorin with two vinylgroups was envisioned.The following discussions detail the synthetic studies toward132-deoxo-13’,13dehydropheophorbide a methyl ester (llOa) (a chlorin with two exocyclic vinyl groups)and its various reactivities. During the course of our studies, we discovered a novelmethod for the isomerization of chlorins to phytoporphyrins which is addressed in thefollowing sections.3.2 Synthesis of Divinyichiorin and its Photooxygenation111The reaction sequence used for the synthesis of 132-deoxo- 131,132-dehydro-pheophytin a (111)128, a chiorin with two vinyl groups, was previously elaborated instudies related to the enolic tautomer of the 13-keto ester in ring V of chlorophyll a. Thisenolic tautomer was once considered to be a possible active intermediate in thephotosystem I reaction center (P700).’29 Thus, following the procedure128 previouslydescribed for the transformation of pheophytin a (5) into 132-deoxo- 131,1 32dehydropheophytin a, 13’-deoxo-13’,13-dehy ropheophorbide a methyl ester (110a) wasprepared (Scheme 21). In this synthetic approach, the first step was NaBH4reduction’3°of pheophorbide a methyl ester (7) to give a diastereomeric mixture of 131 -deoxo- 131hydroxypheophorbide a methyl ester (112a). Conversion of alcohol 112a into atrifluoroacetyl ester, followed by elimination of a molecule of trifluoroacetic acid (viaproton sponge), provided the desired compound llOa in 80 % overall yield.NH N( NaBH4/1(86%)i ci5 7 12aCO2Me(1) trifluoroocetylimidazole, 0°C(2) proton sponge(94%)(I,‘-NH Nq ‘ sunlight/airI) (100%)O=j HO) CO2MeO2Me 113 llOa.R=Me111. R=PhytylScheme 21 Synthesis of Divinylchlorin and Its Photoreaction112The proton chemical shifts in the NMR spectrum (Fig. 3.1) of llOa differsubstantially from those of pheophorbide a methyl ester (7) (Table 3.1). For example,the H-5, H-b and H-20 methine protons of pheophorbide a methyl ester (7) exhibit largedownfield shifts characteristic of an effective 1 8ic electron macrocycle. However, theresonance of the corresponding protons in llOa are shifted upfield by nearly 1 ppmrelative to those in 7. Therefore, compound llOa is somewhat less diatropic than chlorin7. This indicates that the formation of a 131132 double bond in the exocyclic ring ofllOa significantly perturbs the it electronic structure of the chiorin macrocycle.Table 3.1 Comparison of Proton Chemical Shifts (CDC13,400 MHZ)aProtonH-b H-5 H-20 H-3’ H-32(E) H-32(Z) H-13’ H-17 H-187 9.52(s) 9.39(s) 8.57(s) 8.O1(dd) 6.30(dd) 6.19(dd) — 4.20(dd) 4.47(g)llOa 8.41(s) 8.33(s) 7.42(s) 7.42(dd) 6.O1(dd) 5.94(dd) 7.19(s) 4.77(d) 3.77(g)aConcentration 1.5 mgIO.6 mLThis view is further supported by a comparison between the electronic spectra ofllOa and pheophorbide a methyl ester (7) (Fig. 3.2). The Soret band of llOa is split intotwo distinct maxima at 434 nm and 354 nm. This is quite different from the single broadSoret band of 7 at 416 nm. The 630 nm absorption band of llOa is substantially blueshifted from the corresponding 670 nm band in 7. In addition, the strength of this band isonly 10% that of the band in chiorin 7. This loss in strength is consistent with a decreasein the dipole moment of llOa along the ring I -4 ring ifi axis due to the absence of the13’-keto group. The most interesting feature in the spectrum of compound llOa (Fig.3.2) is the broad, low energy absorption centered at 800 nm.(I’ NHNi1100RC0e_________________________I.—.—...—......———.—‘HNMRSpectrum(CDC13)of DivinyichiorinllOa114ci)0C0I.0(I)Fig. 3.2 Structures and Comparison of UV/Vis Spectra (CH2C1)of llOa and 7Although llOa (m.p.>300°C) has high thermal stability, we found that it isextremely photosensitive, and reaction (in dichloromethane solution) in air withVancouver sunlight (20 mm) oxidatively cleaved the 131132 double bond toquantitatively give vinylpurpurin 113 (Scheme 21), while the 3-vinyl group remainedunchanged.The ‘H NMR and UVIVis spectra of purpurin 113 appear in Fig. 3.3. The maindifferences in the NMR spectrum of purpurin 113 from divinylchlorin llOa are the loss ofthe 13’ vinyl hydrogen located at 7.19 ppm and the addition of the -CHO proton at 10.93ppm. An interesting feature is the downfield shift of the all proton resonances in purpurin113 relative to those in compound llOa. For example, the H-5 and H-b meso protons of113 are appeared at 9.50 and 9.20 ppm, with the H-20 meso proton next to the reduced$-NH N/ $-.NH NilHN)(ACO2Me CO2Me7BOOnmWavelengthI2.I11.1I.aj...J: Pr”...-I.e-2.113Fig.3.3‘HNMR(CDC13)andUV/Vis(CH2CI)SpectraofPurpurin113116ring, at higher field (8.45 ppm) suggesting the expected reappearance of the common 18 itelectronic ring current. With a formyl group at C-13, compound 113 has thecharacteristic color (dark brown) and visible spectrum of a purpurin. In addition, the Qband of purpurin 113 had shifted to longer wavelength (686 nm), an observation similarto what was reported by Woodward and coworkers during the corresponding oxidation ofpurpurin21 in the synthesis of chlorophyll a.The photoreaction of compound llOa is regarded as a photooxygenation in whichsinglet oxygen is generated from ground state oxygen by triplet purpurin21,as follows:Purpurin (llOa) S0 liv > Purpurin (llOa) SiiscPurpurin (llOa) Si ) Purpurin (llOa) T1Purpurin (llOa) T1 + ) Purpurin (llOa) So + 102Purpurin (llOa) So + ‘ Purpurin (113)The detailed mechanism is not known, but a dioxetane intermediate as shown inScheme 22 is regarded as plausible. That the reaction should have left the 3-vinyl groupuntouched is remarkable but not exceptional.2’1e MeOOC Hiioa 113Scheme 22 A Possible Mechanism for the Photooxygenation1173.3 Novel Synthesis of PhytoporphyrinsThe high reactivity of the isocyclic double bond in the ring V reflects strongperturbation in the chiorin chromophore of llOa. An acid-catalysed isomerization of thisdouble bond should, we felt, bring about the conversion of llOa into the more stableporphyrin product 114a.Benzoyl chloride (PhCOC1) in DMF, which significantly reduces side-reactions,was found to be a much milder reagent than HI in acetic acid (which gave less than 65%yield for the same conversion) to achieve this transformation. The reaction of llOa with1 equiv benzoyl chloride in DMF at 100°C, under an atmosphere of nitrogen for 20 mm(reaction was monitored by TLC and UVIVis spectroscopy), gave phytoporphyrin 114a(Scheme 23) in quantitative yield after recrystallization from dichloromethane/hexane.This compound was characterized by ‘H NMR, high resolution mass and UV/Visspectroscopies. Its ‘H-’H COSY spectrum is shown in Fig. 3.4. The main differences inthe NMR spectrum of phytoporphyrin 114a from divinylchlorin llOa are the loss of thesignals: H-13’ (s, 7.19 ppm), H-17 (d, 4.77 ppm), H-18 (q, 3.77 ppm) and the appearanceof a new signal at 6.69 ppm (1H, dd, Jtrans=17.5 andJ1=7.7 Hz) denoting the H-132. Thecharacteristic signals of 3-vinyl protons remained largely unchanged and located at 8.27,6.27 and 6.12 ppm. The peaks at 3-4.5 ppm region are well-resolved and were somewhatdifficult to assign to the corresponding hydrogens because of their complex multicouplings. Thus, an COSY spectrum was performed, which revealed theirconnectivity and enabled assignments of all the hydrogens in the molecule. Theinteresting features of the ‘H-’H COSY spectrum are that six hydrogens of H-17’, H-172118(IO2M2MeIi114o1 I III•izo.—._____I /—__:9 0- -— 8:*——c— ....... .... .1.18.8 9.0 8.0 7.0 6.0 5.0 .0 3.0 2.0 1.0PPMFig. 3.4 1H-’H COSY Spectrum (CDC13)of Phytoporphyrin 114a119and H-13’ (2H each) are well-resolved and are assigned as H-17’ [4.42 ppm (ddd), 4.31(ddd)]; H-172 [(3.12 (dt), 3.00 (dt)] and H-13’ [(4.52 (dd), 4.18 (dd)]. The fine resolutionof four protons at 171 and 172 was exceptional and not observed in other porphyrins. Theelectronic spectrum of 114a as shown in Fig. 3.5 is characteristic of a phyllo-typeporphyrin (IV>ll>ffl>I, max(CH2Cl) = 404 nm) suggesting a single meso alkylsubstitution in the molecule. Therefore, the tautomerization of the two protons atpositions 17, 18 is regioselective with respect to the exocyclic double bond in ring Vwithout generating any migration to the 3-vinyl group.a)C)0Fig. 3.5 Structure and UVIVis Spectrum (CH2C1)of Phytoporphyrin 114aAdditionally, it was also found that a weaker acid such as acetic acid is not strongenough to promote the tautomerization of the exocyclic double bond. For instance,(IS’LN HN— /CO2MeCO2Me114aWavelength120treatment of llOa with glacial acetic acid in DMF at 100°C for 2 h gave no products andthe starting material was recovered.Encouraged by this exciting finding, we decided to expand the same reactionconditions to hydroxychiorin 112a; after reaction under the above conditions for 45 mm,it gave the same product 114a in 87% yield. Similarly, the conversion of another twohydroxychlorins 112b and 112c gave 114b and 114c in 81% and 73% yield, respectively(Scheme 23).NOBH4(83—86%)(-47. R= COOMe, R2= Me llOa5. R= COOMe, R2= Phytyl25. R1 = H, R2 = Me PhCOC(/DUF100°C(100%)a. R1= COOMe, R2= Meb. R1= COOMe, R2= Phytylc. R1= H, R2= Me114Scheme 23 Direct Conversion of Chiorins to PhytoporphyrinsWhen the above procedure was applied directly to monovinylchlorin (3-vinylchlorin) pyropheophorbide a methyl ester 25, a mixture (Scheme 24) ofphytoporphyrin methyl ester 27 (19% yield) and 3-vinylphytoporphyrin methyl ester 116(15% yield) was obtained after the preparative TLC separation, along with recovery of112(I,‘s-NH N1N HN\‘ — /C02R121the starting material 25 (40% yield) (the reaction was monitored by UVIVis and TLCanalyses). The formation of the non-isomerized (oxidized) product 116 is probably dueto direct autooxidation under the acidic reaction conditions.PhCOCI/DMF NH N NH N—100°C “N HN/ +N HN/CO2Me CO2Me27 116(19%) (15%)Scheme 24 Reaction of 3-Vinyichiorin (25) Using Benzoyl ChlorideThe products 27 and 116 were characterized by ‘H NMR, UV/Vis, and highresolution mass spectroscopies. The most important difference between ‘H NMR spectraof the two products is the presence of 3-vinyl protons in phytoporphyrin 116. Furtherstructural confirmation was derived from the 2 unit difference of the molecular ions (mlz)in their mass spectra. Their UVIVis spectra are shown in Fig. 3.6. Compound 116 has anoxorhodo-type spectrum (ffl>ll>IV>I) characteristic of two rhodofying groups (3-vinyland 13’-carbonyl) on diagonally opposite rings. Compound 27 has a rhodo-type spectrum(ffl>IV>ll>I) suggesting only one electron-withdrawing group (13’-carbonyl) in themolecule. The Soret of the former compound (420 nm) is 4 nm to the red in contrast tothat of the latter (416 nm) (Fig. 3.6).25122Q)C)0700nriFig. 3.6 Structures and Comparison of UV/Vis Spectra (CH2C1)of 27 and 1163.4 Tautomerization MechanismA possible tautomerization mechanism is presented in Scheme 25. The formationof phytoporphyrin 114 from hydroxychiorin 112 can be regarded as acid-catalyzed doubleisomerization of the intermediate 110, which is formed by elimination of a molecule ofN HNCO2Me27116(I’N HN?CO2Me116116400 500 600Wavelength123benzoic acid from the initial intermediate 117. Migration of the proton at position 17,with the relief of the steric strain in the intermediate 110, gives the intermediate 118which subsequently tautomerizes, with the loss of a hydrogen at position 18, to give thefully conjugated product 114.- wPhCOCVDMF H%rH4\ —PhCOOH(I \)-HCI (181lN HN C02R OOCPh C02R... IA 117 I 110112 7/C02R OH I‘NH N—a. R1= COOMe, R2= MeHN HNb. R1= COOMe, R2= Phytylc. R1= H, R2= MeR? ( R+C02R 02R1 H114 118Scheme 25 Proposed Tautomerization Mechanism of Chlorins to PhytoporphyrinsAlthough there is no direct evidence implicating the formation of the intermediate118, support for the formation of intermediate 110 and the fate of stereogenic center (C132) involved in the reaction have been found experimentally. For instance,phytoporphyrin 114a, directly derived from hydroxychlorin 112a (132R), was found to bea racemic mixture (at asymmetric c-i 32 position) by ‘H NMR determination using a shiftreagent, tris-[3-(trifluoromethylhydroxymethylene)-(+)-camphorato]praseodymium (ifi)derivative [Pr(tfc)3]. Furthermore, the yield (87%, 114a) of the PhcOcl-inducedisomerization of 112a was greater than that (73%, 114c) of 112c. This observation can be124related to the electron-withdrawing methoxycarbonyl group at C- 132, which increasesacidity of the H- 132 and thereby facilitates the elimination of a molecule of benzoic acidfrom the exocyclic ring V to furnish the intermediate divinylpurpurin 110 as shown inScheme 25. Several pathways can be written for the further transformation of 110 to thefinal products and the most straightforward appears the one via the intermediate 118 asshown in Scheme 25.Water present in the reaction medium will quench the carbocation intermediatesand generate side products or cause decomposition. Similar results were also observed inthe transformation of polyhydroxychlorins. For example, reaction of benzoyl chloride (2equiv) with the diastereomeric diol 119 (Scheme 26), obtained (89% yield) from theNaBH4 reduction of132R-hydroxypheophorbide a methyl ester (95) (90% d.e., seeChapter 2), gave 2% yield pheophorbide a methyl ester (7) and an unidentified mixture ofporphyrins. Similarly, the diastereomeric triol 120 (Scheme 26), prepared (72% yield)from the LiA1H4 reduction of pheophorbide a methyl ester (7), also afforded acomplicated porphyrin mixture.MeOOC oH MeOOC OH OH 119CH2O 120Scheme 26 Structures of Hydroxychlorin 95 and Polyhydroxychlorins 119 and 1201253.5 ApplicationsMechanistic research aside, the synthesis of these phytoporphyrins has opened theway to a variety of porphyrins and their reduced derivatives, especially petroporphyrins’9.For example, we have employed phytoporphyrins as intermediates to synthesizedeoxophylloerythroetioporphyrin’31(12 ),one of the most abundant porphyrin derivativeson earth. Compound 121, occurring largely in oil shales and related deposits, is the majorpigment in most samples of petroporphyrins. Its isolation from natural sources is madedifficult by the complexity of the porphyrin mixtures, this fact explaining why the totalsynthesis’3’ of 121 has been carried out. Although it has been long believed thatcompound 121 is derived by degradation of chlorophyll over the course of time, its partialsynthesis’32 from pheophytin a resulted in very low yield. However, we sought out todevelop an efficient way to obtain this unique reference compound for identification andchemical reactivity studies.Making use the ready availability of phytoporphyrins 114a, 114b and 114c fromour method, petroporphyrin 121 was synthesized from chlorophyll a in a short andefficient way (Scheme 27). Thus, alkaline (10% KOHIMeOH) or acidic (25% HC1)hydrolysis of either of the phytoporphyrins, 114a, 114b or 114c, gave the correspondingporphyrin diacid or monoacid, which could be decarboxylated and reduced to givedeoxophylloerythroetioporphyrin (121) (Scheme 27). It appeared likely that both thesesteps could be carried out by a one-pot procedure described by Kampfen andEschenmoser’33,where protoporphyrin was transformed into etio-porphyrin ifi (reductionof vinyl to ethyl groups and decarboxylation of side-chains) in one step by heating in1261,5 ,7-triazabicyclo[4.4.0]dec-5-ene (TBD). When the phytoporphyrin mono- or di-acidsubjected to these conditions (large excess TBD; tube sealed under vacuum; 200°C; 4 h) areaction mixture largely containing compound 121 was obtained. Purification of productby flash chromatography (elution with dichloromethane) gave petroporphyrin 121(Scheme 27) in 67-75% yield after recrystallization from dichloromethanelhexane.KOH/MeOH TBDor 25%h1C02R C02R 121114a. R1= COOMe, R2= Me R1 COOH, H; R2= Hb. R1= COOMe, R2= Phytylc. R1= H, R2= MeScheme 27 Synthesis of Petroporphyrin 121Another petroporphyrin acid’34 123 (Scheme 28), which is the major portion inMessel oil shale, can also be prepared from catalytic hydrogenation of phytoporphyrin114c (prepared according to our method) followed by saponification with KOH in EtOH.Pt/c. H2 KOH/EtOHCO2Me CO2Me CO2H114c 123Scheme 28 Synthesis of Petroporphyrin 123127Another application of phytoporphyrins was to develop new photosensitizers forphotodynamic therapy of tumors. For example, Diels-Alder reaction ofmonovinylporphyrin 114c with dimethyl acetylenedicarboxylate, followed by DBUpromoted rearrangement of the cycloadduct, gave 36% yield of 125 (Scheme 29), a newclass of regiochemically-pure benzoporphyrin derivative, which can act as photosensitizerin the photodynamic therapy. The work on this topic had brought about the research ofanother new field (“the third generation photosensitizers”), which is described in Chapter4./ MeOOC MeOOC /‘NH N- DMAD MeOOC/NH DBU MeOOC / NHHN 110°C, 30h —N HN (36%) —N HNCO2Me CO2Me CO2Me114c 125Scheme 29 Synthesis of Benzoporphyrin Derivative 125(Only One Enantiomer of 125 Is Shown)3.6 SummaryChlorophyll derivatives 110, 112a, 112b and 112c have been readily converted totheir corresponding phytoporphyrins 114a, 114b and 114c in excellent yields usingbenzoyl chloride in DMF. This new approach has added more scope to the acid-catalysedmigration of hydrogens at the saturated ring IV into the exocyclic ring V. This methodalso affords a novel and efficient route to prepare monovinyl porphyrins which can be128used as the important intermediates for the further preparation of chlorophyll relatedpetroporphyrins and regiochemically-pure benzoporphyrin derivatives for use in thephotodynamic therapy.129Chapter 4The Third Generation Photosensitizers1304.1 Background and Research ObjectivePhotodynamic therapy (PDT), known also as photochemotherapy of cancer, is amedical treatment which employs a combination of light and drug to create cytotoxic(‘cell-lethal’) forms of oxygen (singlet-oxygen and superoxide radical), as well as otherreactive species, to bring about the destruction of cancerous or unwanted tissue.135The present treatment schedules which are used in the clinic are based on theretention of a photosensitizer such as PhotofrinTM in tumor tissue in concentrations whichare higher than in surrounding nonmalignant tissues. Subsequent photoactivation(usually by a laser source) of the sensitizer evokes tumor destruction. The exactmechanisms of action for effective PDT in cancer treatment are unclear. While studies invitro have indicated that generation of singlet oxygen is the main mechanism for PDTcytotoxicity,’36”7studies in vivo with animal models have suggested that photodynamicdamage to the blood vessels of the tumor may be the major cause of tumordestruction. 138,139Contemporary PDT began in the late 19th century when Finsen140 discovered thatthe skin condition Lupus vulgaris could be treated using UV light. Alexandra, the futurequeen of Edward VII, brought the discovery back to the London Hospital.141 In 1913,Meyer-Betz142 demonstrated the phototoxicity of porphyrins by injecting himself with200 mg of hematoporphyrin, and in 1924 Policard143 discovered that the certainmalignant tumors were selective accumulators of porphyrins.Although the potential for photochemotherapy with porphyrins was exposed bythese pioneers, it was not until 1972, when the first sustained series of tests on animals131H3 C)_—N HN.4’ OH<)NH NH3 C-)__LCHHO2C CO2H\HO2C(CH2)2 CH3 CH3 CHOHHO2C(CH2)2cH3H3C ci-—H NH H— 2)2,.N.?HOHè!.o_C (CHH3CCH3 Q )( CR3— IHO—CH CH3 (cH2) CH3CH3 CO2Hand humans were begun by Dougherty et al.144 and others using hematoporphyrinderivative (HpD), that the field of clinical PDT truly began. By 1975, there was ampleproof that HpD and light could be used to destroy cancers in animals,145 and in 1976 thefirst successful trials in humans were initiated. 146CR3H3H3H3 C.H3 CHO2C CO2HHO2C(CH2)2 CH3H02 C(CH2H3HO2C CO2HHO—CH3 (CH2)2C02HH3C (CH2)2CH0—CH3H3C HCOHCH3CR3Scheme 31 Various Components of Hematoporphyrin Derivative132HpD (the first generation photosensitizer), together with its commercial variantsPhotofrinTM, Photosan, Photogem, and Photocarcinorin, has been used to treat a variety oflung and bladder cancers, breast cancer, and certain occular cancers. However, because itis a mixture of compounds (the major components of HpD are listed in Scheme 31), someof which are PDT-inactive, HpD is not the ideal sensitizer. It also localises in healthy aswell as cancerous tissues, where it persists, leading to generalised photosensitivity. HpDalso locates in the liver and brain, and its clearance rate from human body requires 4-6weeks, post-injection, for systemic concentrations to fall to acceptable levels.147 Duringthis time the patient must remain in subdued light to prevent skin photosensitivity.Another disadvantage is that the excitation wavelength (630 nm, e 1 170) is not themost efficient for producing photodamage or for penetrating the skin (less than 4 mmdepth for HpD) from an external light source. This lack of efficiency, resulting from poorlight absorption (E 1170) of HpD, coupled with light loss from optical absorption byendogenous tissue chromophores (mainly hemoglobin) and light scattering, disables thetreatment of large tumors or tumors which are deeply seated within the body.In an effort to improve on the first generation drug, new “second generationphotosensitizers” 148 were sought out. During the last few years, a number of dyes whichstrongly absorb in the range of 650-700 nm, such as a benzoporphyrin derivative (BPD),tin etiopurpurin, zinc (II) phthalocyanine, m-THPC [5,10,15 ,20-tetrakis(m-hydroxyphenyl)-chlorin], and monoaspartyl chiorin e (‘MACE’) (Scheme 32) have beenpatented as potential clinical photosensitizers.133In the chiorin series, benzoporphyrin derivative monoacid ring A (BPDMA),developed in our laboratory, has generated interest due to low skin phototoxicitycompared with the first generation drug PhotofrinTM, the industry standard used in theclinic. As a result, BPDMA is one of the long wavelength photosensitizers (?max = 690nm, E = 33 000) which are currently in Phase-TI clinical trials. To synthesize BPDMA149,protoporphyrin IX dimethyl ester (131) is reacted with dimethyl acetylenecarboxylateCI COCH/0CH3OCH3Oc0Benzoporphyrinderivative mono—acid (BPDMA)Tin EtiopurpurinOHZinc Phthalocyanine meso —Tetra—(m—hydroxy—phenyl)chlorinScheme 32 Major Second Generation PhotosensitizersMono—aspartylchiorin—e6134(DMAD) in refluxing toluene (Scheme 33). The ring A Diels-Alder adduct (i.e. 1,4-cyclohexadiene derivative) is then separated by crystallization from the ring B adduct andis subsequently rearranged with base (DBU). The rearranged ring “A” 1,3-cyclohexadiene derivative is then partially hydrolyzed to afford, after purification, thetwo regioisomers, BPDMAad (132) and BPDMAaC (133), which are used for biologicalstudies.150 From the biological data reported so far, photodynamic activity is maximised/MeO2CCCCOe MeO2CCO2MeM:2cCO2Me CO2Me131/BU/,MeOCO2Me CO2MeMeO2C...f\MeO2CH —N HN’NH N\ —. I,CO2H CO2Me CO2Me CO2H132 133Scheme 33 Synthesis of the Biologically Active BPD Monoacids (BPDMA)(Only One Enantiomer of Each Molecule is Shown)MeO2CMeO2C.#\N HN-.’CO2H CO2HHCI135for BPD when: (a) “ring A” of the porphyrin nucleus is modified by the Diels-Aldercycloaddition/base rearrangement procedure, (b) only one of the two methyl propionategroups is hydrolysed to the free acid.Due to the troublesome separation involved with BPDMA, porphyrins with onemonovinyl group (in ring A) and only one propionic side chain in the moleculesimmediately stand out as target molecules. Further, various chlorophyll derivatives, suchas pheophorbide a, pyropheophorbide a and chlorin e have been reported asphotosensitizers for the photodynamic therapy of tumors.151 Preliminary in vivo resultsled us to conclude that the five-membered exocyclic ring plays an important role in thephotosensitizing ability of these compounds. To our knowledge, further research, aimedat comparing the structure/activity relationships among chlorins, pheophorbides andBPDs and at understanding the basic requirements for an effective long wavelengthphotosensitizer, has not been performed.The objectives of this section of research (the third generation photosensitizers)were two-fold. The first was to eliminate the problem of isomer formation in the DielsAlder reaction of protoporphyrin and to synthesize regiochemically-pure benzoporphyrinderivatives (BPDs) characteristic of the successful and promising chemical features ofBPDMA. These regiochemically-pure BPDs should be more readily characterized andpurified and would subsequently lead to less ambiguity at the biological screening stage.Synthesis of porphyrins with a single exocyclic vinyl group and one propionic side chain,therefore, became the initial goal of this project.Secondly, because depth of penetration through tissue varies with the wavelengthof light, the ideal photosensitizer for treatment of more deep-seated tumors should absorb136at wavelengths >700 nm.’5° There is therefore a need to synthesize new macrocycleswhich will absorb in the far red or the near infrared region of the electromagneticspectrum. It was reasoned that since natural bacteriochiorins (tetrahydroporphyrins withtwo opposite reduced rings) absorb at 750-800 nm, they could be good candidates forphotodynamic therapy.With the goals of isomeric purity and long wavelength absorption in mind,therefore, the syntheses of regiochemically-pure benzoporphyrin derivatives (BPDs) and[A,C]-dibenzoporphyrin derivatives (bacteriochiorins) were attempted. The followingsections describe the detailed syntheses and characterization of these compounds.4.2 Syntheses of Regiochemically-pure Benzoporphyrin Derivatives4.2.1 RationaleThe brief introduction to photodynamic therapy (PDT) and benzoporphyrinderivatives (BPD5) above described the relative cytotoxicities of the ring A and ring BBPD compounds. It was shown that the monoacid derivatives of ring A had the bestinitial biological results. However, in the industrial production of BPDMA, an equalamount of the ring B material is produced as an “unwanted” by-product. A solution tothis problem would be to use monovinylporphyrins to prepare regiochemically-purebenzoporphyrin derivatives (BPD5). As a continuation of our research into naturalantioxidative chlorins and phytoporphrins, two series of photosensitizers related to BPDwere synthesized by using 3-vinylrhodoporphyrin XV (23) and 3-vinylphytoporphyrin137(114c) as starting materials. These are ideal substrates for the synthesis of compounds tobe used in probing structure/activity relationships of BPDs because both systems have avinyl group at position 3 (i.e. in ring “A”, and thus will produce only ring “A” chiorinafter the Diels-Alder reaction), and also have only one propionic ester side chain, whichcan be hydrolyzed to the corresponding monocarboxylic acid at the final step of thesynthesis.4.2.2 Via 3-Vinylrhodoporphrin XV Dimethyl EsterAs described in the Introduction (Chapter 1), 3-vinylrhodoporphyrin XV could beprepared from the stardard degradation of chlorophyll a.52 Thus, pheophytin a (5) (fromthe blue alga Spirulina maxima) was transformed into purpurin-7 trimethyl ester (16) by(1) Py, KOH/PrOHHN’ (2)°2HN’(3) H2S04PhytyO2 MeO2c 0 CO2HCO2H 21collidineNH N/ \_____...NHN 170°C, 2hO2MeO2Me (76%)23CH2N(58%),2Me16Scheme 34 Synthesis of 3-Vinylporphyrin 23 via Basic Aeration of Pheophytin a (5)138dissolution in pyridine, dilution with ether and addition of KOH in n-propanol. Afteraeration for 30 mm Fischer’s ‘unstable chiorin’ was obtained.52 Esterification of‘unstable chlorin’ (21) with diazomethane gave purpurin-7 trimethyl ester (16). Theoverall yield from pheophytin a was 58%. Deglyoxylation, and concomitant oxidation ofpurpurin-7 trimethyl ester in collidine, furnished a 76% yield (44% overall yield frompheophytin a) of 3-vinylrhodoporphyrin XV dimethyl ester (23) (Scheme 34).One important characteristic of the above method is that 21 was somewhatunstable and had a tendency to oxidize further in the aerial oxidation step. Theesterification with diazomethane therefore produced both purpurin- 18 methyl ester (87) inaddition to the desired purpurin-7 trimethyl ester (16) (Scheme 35). Separation ofpurpurin- 18 methyl ester from purpurin-7 trimethyl ester by chromatography is difficultdue to their very close structures and polarities.CO2H 21CH2N(100%)(‘I,2Me16 CO2Me 87Scheme 35 Purpurin- 18 Methyl Ester (87) and Purpurin-7 Trimethyl Ester (16)CH2N(100%)22139Based on our synthetic methods developed in the research of antioxidativechiorins (Chapter 2), an alternate route to synthesize purpurin-7 trimethyl ester (16) wasvia 15’-hydroxypurpurin-7-lactone methyl ester (101). As described in Chapter 2,asymmetric hydroxylation of pheophorbide a methyl ester (7) using (—)-(1R)-(10-camphorsulfonyl)oxaziridine [(-)93] and DBU gave 132R-hydroxypheophorbide a methylester (95) (90% d.e.) in 94% yield. Periodate oxidation of13R-hydroxypheophorbide amethyl ester followed by esterification with diazomethane gave purpurin-7 trimethyl ester(16) in 79% yield (73% overall yield from pheophytin a). Final deglyoxylation andconcomitant oxidation of purpurin-7 trimethyl ester gave 3-vinyirhodoporphyrin XVmethyl ester (23) in 76% yield (55% overall yield from pheophytin a) (Scheme 36). This(IiiN HNMe02 MeO2 07H5106(79%)(I,HNCO2Me 101CH2N(100%)(IDBU(—)93 ‘ NH N—/—25°C,12h I I-IN(92%)Me02Me02 OH 095(R)(90% d.e.)çNH N‘2Me23collidine170°C, 2h(76%)16Scheme 36 Synthesis of 3-Vinylporphyrin 23 via Hydroxychlorin 95140route has the advantages of higher yield and facile separations although it necessitatestwo extra steps compared with the original aerial oxidation of pheophytin a. Bothmethods have been used in the preparation of 3-vinylrhodoporphyrin XV (23) frompheophytin a (5) in this work.MeOOC/NH tMe000-CEc-CUeMeOOC/NH-NHN\110°C, 28h -NHN\O2MeO2Ue (50%)O2MeO2Me23 DBU/ 140MeOOC / (90%)141Scheme 37 Synthesis of Regiochemically-pure BPD 141(Only One Enantiomer of 141 is Shown)For the preparation of new regiochemically-pure benzoporphyrin derivatives, theporphyrin 23 was heated in degassed toluene solution at 110°C with a 50 fold molarexcess of dimethyl acetylenedicarboxylate (DMAD). The desired Diels-Alder reactionwas complete in 28 hours. Isolation and purification of the product by chromatographyafforded the cycloadduct 140 in 50% yield (Scheme 37). The ring A 1,4-cyclohexadienederivative 140 was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene in dichloromethane toprovide the chiorin-like BPD 1,3-cyclohexadiene dimethyl ester 141 in 90% yield afterchromatography (45% overall yield from 23). The reaction progress was monitored by141UV/Vis spectroscopy which showed a new absorbance at 672 nm (Fig. 4.1). Thisrearrangement gave the thermodynamically more stable product, i.e., the carbomethoxysubstituent attached at 21 in relation to the angular methyl group located at C-2 is in atransoid orientation. This transoid orientation was confirmed by a positive nOe effectobserved for the C-21 proton (6 5.04 ppm) and the methyl group at C-2 (6 1.79 ppm)(Fig. 4.2).C)CC0(1)500 600 700 800nmWavelengthFig. 4.1 Structure and UV/Vis Spectrum (CH2C1)of BPD 141(Only One Enantiomer of 141 is Shown)The absorption spectrum of BPD 141 is shown in Fig. 4.1. It is characteristic of achlorin-like chromophore but it has an unusually broad Soret band and the lowest energyabsorption (Q band) at 672 nm ( 14 600) is blue-shifted some 18 nm in comparison withthat (690 nm) of the BPDMA. The difference is due to the presence of an 8-vinyl groupin BPDMA, which extends the conjugation of the molecule.We have also used olefinic dienophiles to synthesize photosensitizers related tothe BPD system, which absorb at shorter wavelengths than the parent BPD 141, but wereprepared in much higher yields due to the higher reactivity of these dienophiles. The400142IIIMeoocH\MeOOC—N HN\02M2UepPNi •. —i.e —2.IRRRfl1T€D WT 5.P4PPJMeOOC%QMeOOC,NH N.O2MeéO2M.141IRRD!TD 3.7SPPN6.8 7.8 8.8 8.8Fig. 4.2 The Difference nOe Spectra on Regiochemically-pure BPD 141143Diels-Alder adduct 142 (77% yield) obtained from 3-vinyirhodoporphyrin XV (23) withtetracyanoethylene (TCNE) exhibits an absorption maximum at 642 nm (E 20 500).Using a similar approach, reaction of the powerful heteroatomic dienophile, 4-phenyl-1,2,4-triazoline-3,5-dione, with 3-vinylrhodoporphyrin XV (23) gave an adduct 143 withmaximum absorption at 640 nm (E 19 500) in 90% yield (Scheme 38). The blue shift inthe absorption maximum of adducts 142 and 143 is explained by lack of conjugation inthese Diels-Alder adducts when compared with the Diels-Alder product 141.3 \70°C,lh N\ 0°C,4h2MeO2Me(77 O2Me (90%) O2Me142 23 143Scheme 38 Regiochemically-pure BPDs 142 and 143In addition, Pandey et al.152 have very recently published a communication inwhich they exploited chemistry similar to that which we have described above, andobtained a similar Diels-Alder adduct 141 from 3-vinylrhodoporphyrin XV and DMAD.However, these authors reported a 23% overall yield (141 from 23) in contrast to a 45%total yield for the same reaction sequence in our hands.1444.2.3 Synthesis of Regiochemically-pure Benzoporphyrin DerivativeVia 3-Vinyl-13-Deoxophytoporphyrin Methyl Ester (114c)As has been described in Chapter 3, phytoporphyrins with a vinyl group in theposition 3 can be used to synthesize the new regiochemically-pure benzoporphyrinderivatives bearing an isocyclic ring. Thus, for the preparation of BPD 125, reaction ofphytoporphyrin 114c with dimethyl acetylenedicarboxylate in refluxing toluene undernitrogen gave the desired chiorin 134 in 42% yield. Compound 134 was then treated with1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in dichloromethane to provide the fully-conjugated chiorin 125 in 91% yield (36% overall yield) after chromatography (Scheme39). The reaction progress was monitored by UV/Vis spectroscopy which showed a newabsorbance at 686 nm ( 37 000) (Fig. 4.3) and the disappreance of the peak at 648 nm ofcompound 134./ MeOOC MeOOC /‘NH N— DMAD MeOOC/NH DBU MeOOC / NH r:2I:AeN h1::30h1(91%)114c 134 125Scheme 39 Synthesis of Regiochemically-pure BPD 125(Only One Enantiomer of 125 is Shown)The FAB mass spectrum of BPD 125 showed a molecular ion at mlz 675([MH]j. High resolution mass spectroscopy gave an accurate molecular mass at675.3176 (calculated value forC40H3N06= 675.3182). Compound 125 exhibited145almost the same electronic spectrum as that of the BPD 141. The difference is that the Qband of compound 125 shifts about 14 nm to the red in comparison with BPD 141. Thisis due to the “rhodofying effect” of the five-membered isocyclic ring. The 1H NMRspectrum (Fig. 4.4) showed the three meso protons as singlets at 8 9.59, 9.30 and 8.95ppm, each corresponding to one proton. The H-132 and H-131 appeared at 65.20 (triplet)and 3.93 ppm (multiplet) respectively corresponding to two protons. The two aromaticprotons on the exocyclic ring characteristic of the benzoporphyrins were seen as doubletsat 8 7.83 and 7.45 ppm (J = 5.8 Hz) as expected. Another hydrogen located at 21 in thisexocyclic ring was observed as singlet at 8 5.05 ppm. This observation indicated anisomerization of the double bond in the six-membered ring thus extending theconjugation in the molecule.cjC)C0Cl)-800nmFig.4.3 Structure and UV/Vis Spectrum (CH2C1)of BPD 125(Only One Enantiomer of 125 is Shown)MeOOC /400 500 600 700WavelengthMeOOCooc/MeOOC’125/F13.—1.9-2.9-3.9-4.9PPKFig.4.4Structureand1HNMRSpectrum(CDC13)ofBPD1250\1474.3 Synthesis of [A,C]-Dibenzoporphyrin Derivatives(Bacteriochiorins)4.3.1 RationaleThe penetration of light through tissue is attenuated by a number of factors butabsorption and scattering are the most important in limiting the penetration depth foreffective treatment.153 Heme proteins account for most of the absorption of light in thevisible region. Since this drops off rapidly beyond 550 nm, the effective depth ofpenetration doubles in going from 550 nm to 630 nm and doubles again in going to 700nm. The difference of light penetration is the main reason that BPDMA (a chiorin, max= 690 nm, e = 33 000) is about 70-80 times more effective than is HpD (a porphyrinmixture, 2max = 630 nm, E = 1 170) in terms of tumor photonecrosis. Biologicaleffectiveness could be further enhanced by going to the corresponding bacteriochiorinsystems. The light absorption properties of bacteriochiorins (750-800 nm) have thuscaused them to be regarded as prospective candidates as photosensitizers forphotodynamic therapy. In recent years, the search has begun for a third generationphotosensitizer which absorbs strongly in the far visible red or near infra red region,thereby conferring a therapeutic advantage of greater tissue penetration and thus enablingtreatment of large tumors. The advent of low-cost, reliable diode lasers operating at 790nm to 850 nm has spurred in the development of several new compounds havingabsorption maxima in the 750-800 nm range.Naturally-occurring bacteriochlorins, which absorb in the red region, have beenreported to show both in vitro and in vivo photosensitizing activity; however by virtue of148their tetrahydro reduction states, they can be readily oxidized back to the parent(dihydro)porphyrins with an accompanying loss of their long-wavelength absorptionbands (thus reducing the photodynamic efficiency).’50 This potential lack of stability hasled researchers to examine other ways of producing stable bacteriochlorin-likechromophores.In 1991, Dolphin et al.153 extended the Diels-Alder cycloaddition reaction ofvinylporphyrins to include several methods for synthesis of stable bacteriochlorins withabsorption maxima up to 786 nm. Their synthetic strategy was based on two successiveDiels-Alder cycloadditions on a symmetric [A,C]-divinylporphyrin (145) (Scheme 40).The [A,C]-divinylporphyrin 145 was further totally synthesized via a stepwise route frompyrrolic precursors by following the MacDonald dipyrromethane approach. In 1992,another research group led by Smith154 at UC, Davis also published their work in thisEtOOC/N rEtOOC—CC—COOEt EtOOCN145DBUEtOOCfEtOOC’ ‘s_—N HN’NH N” COOEt/ COOEtScheme 40 Dolphin’s Synthesis of [A,C]-Dibenzoporphyrin Derivative149area in which they exploited chemistry similar to that described by Dolphin et al., andobtained a similar bacteriochlorin (a dibenzoporphyrin) derivative.Preliminary biological results by the above two research groups indicated that thesynthetic dibenzoporphyrin (bacteriochiorin) derivatives were highly photocytotoxic.’55However, further biological investigation of these compounds as potential phototoxins inPDT was delayed by problems associated with the availability of the [A,C]divinylporphyrin 145. The total synthesis of [A,C]-divinylporphyrin 145 described bythese groups requires a large number of steps and therefore requires a huge amount ofeffort in order to obtain the quantities required for clinical investigations. It wasenvisioned, however, that performing a partial synthesis, from a readily-available naturaltetrapyrrole, might facilitate and greatly simplify the synthetic sequence of an [A,C]divinylporphyrin. Continuing our work on Diels-Alder reactions of vinylporphyrins andon chemical modifications and applications of chlorophyll derivatives, it was thought thatan [A,C]-divinylporphyrin could be synthesized by chemical modification of achlorophyll related 3-vinyl-porphyrin or chiorin. So the partial synthesis of an [A,CJdivinylporphyrin described below was undertaken.4.3.2 Synthesis of [A, CI-Divinylporphyrins4.3.2.1 Via 3-Vinylpurpurin 113Attention was focused then on the development of a new and efficient route toobtain an [A,C]-divinylporphyrin from a chlorophyll derivative. To keep the number of150chemical modifications involved in this program to a minimum, the porphinoidssynthesized in the study of natural antioxidative chiorins and of phytoporphyrins werescreened and 3-vinylpurpurin 113 (Chapter 3) was chosen as the required precusor to an[A,CJ-divinylporphyrin. With a 3-vinyl group at ring A and a 13-formyl group at ring C,purpurin 113 was susceptible to a Wittig olefination reaction on the 13-formyl group toform a second vinyl group at ring C and, after further decarboxylation and oxidation, togive the desired target [A,C]-divinylporphyrin 147 (Scheme 41). It was reasoned that theglyoxylic ester group at position 15 of the purpurin 113 could be lost with concomitantoxidation of the chlorin macrocycle into the porphyrin 148 in a one pot procedure as wasused in the transformation of purpurin-7 trimethyl ester (16) into 3-vinyirhodoporphyrinXV (23) in collidine at 170°C. Further, Wittig olefination of the resulting 148 withCO2Me 113 148llNH N/ \NHNc\O2Ue147149Scheme 41 Retrosynthetic Analysis of [A,C]-Divinylporphyrin 147151methylene triphenyiphosphorane would furnish the desired [A,C}-divinylporphyrin 147.Alternatively the [A,CJ-divinylporphyrin 147 may be formed firstly by a Wittigolefination of the purpurin 113, to furnish the [A,C]-divinylpurpurin 149, followed bydeglyoxylation of the glyoxylic ester and oxidation in situ in refluxing collidine. Bothpathways appeared plausible because of the known transformation procedures and theready-availability of the starting material 113 from the synthetic method developed inChapter 3.For the preparation of 3-vinylpurpurin 113, two synthetic methods wereinvestigated and the one giving the higher overall yield was subsequently used to make113 on a 400 mg scale. In the first method, as described in Chapter 3, 3-vinylpurpurin_1LLk_NH N< NOBH4 _NH N’(86%)‘ ‘ /MOOH1120(1) trifluoroacetylimidazole, 0°C(2) proton sponge(94%)(“INH N— ‘ sunlight/air\ /N HN (100%)...0O2MeO2Me 113 llOaScheme 42 Synthesis of Purpurin 113 via Photooxidation152113 was derived from the photooxidation of13-deoxo-13,12dehydrophe phorbide amethyl ester (llOa), which was prepared from pheophorbide a methyl ester (7) in 80%overall yield (Scheme 42). In the second method, 3-vinylpurpurin 113 was derived fromthe periodate oxidation (Scheme 43) of the diastereomerical mixture, 132-deoxo-131hydroxy-132R-hy roxypheophorbide a methyl ester (119), which was in turn obtainedfrom the NaBH4 reduction of132R-hydroxypheophorbide a methyl ester [95(R)] asdescribed in chapter 3. With the second method, purpurin 113 has been obtained in 67%overall yield from pheophorbide a methyl ester (7) in contrast to 80% overall yield from 7in the first method by means of photooxidation.NH N— ‘ DBU,(—)93 NH NHN’ —25°C12h HN”(94%)Me02 MeO2 0Me02Me02 OH 0 95(R)7(90% d.e.)NaBH4(89%)H 5106(80%)MeO2J20HOH113 119Scheme 43 Synthesis of Purpurin 113 via Periodate Oxidation of Dihydroxychlorin119153When purpurin 113 was heated in collidine at 170°C for60 minutes, 13-decarboxyl-13-formyl-3-vinylrhodoporphyrin XV methyl ester (148) (Scheme41) wasobtained only in 15% yield; the remainder of the starting material was decomposed oroveroxidized under these conditions. Attempts to avoid theseside-reactions by reducingreflux time and temperature (using pyridine instead of collidine) were all unsuccessful.The problem seemed to be due to the susceptibility of the 1 3-formyl group which led tooveroxidation when the purpurin-into-porphyrin conversion occurred.Protection of the 1 3-formyl function to circumvent this unwanted side reactionwas therefore considered next. The ethylene acetal 150 was readily formed in 90% yieldby heating purpurin 113 with ethylene glycol in THF with p-toluenesulfonic acid as thecatalyst (Scheme 44). In this case, the 151 carbonyl function was found to be unaffected.The difference in reactivities between the 13-formyl group and the 15-ketone group,coupled with the steric hindrance of the 151 -ketone moiety, is responsible for\ HObHHN p—touenesuIfonic ocdO2Me(90%)o:MeO2Me113 150Scheme 44 Protection of Purpurin 113 via the Formation of Ethylene Acetal 150154The salient features of the electronic spectrum (Fig. 4.5) of acetal 150 Qmax 660nm), which looks like a hydroxychiorin described in Chapter 3, are the hypsochromicshift of its Q band in comparison with that (Amax = 686 nm) of the purpurin 113 and theappearance of large absorbance band at 496 nm,which suggested that the electron-withdrawing group at C-13 was 4.5 Structure and UV/Vis Spectrum (CH2C1)of Purpurin 150Unfortunately, when the ethylene acetal 150 was refluxed in collidine even for 4hours, no reactions occurred and the starting material was recovered. Various attempts toachieve the purpurin-into-porphyrin conversion forthe ethylene acetal 150 were allunsuccessful. It appears that a electron-withdrawing group at position 13 is required forthe loss of the 15-glyoxylic ester group and concomitant oxidation of a purpurinmacrocycle to a porphyrin as observed in purpurin-7 trimethyl ester (16) and in purpurinCO2MeWavelength155113. The key reason appears to be the dramatic change in the oxidation potential of theethylene acetal 150 with the protection of the 13-formyl group. The presence of aelectron-withdrawing group at position 13 in these purpurins seems to destablize the it-system of the molecule and render the molecule easier to oxidize than the correspondingprotected derivative.The conversion of purpurin-7 trimethyl ester (16) into 3-vinylrhodoporphyrin XV(23) was originally investigated by Fischer36,who found the loss of the glyoxylic esterresidue from purpurin-7 trimethyl ester (16) occurred upon refluxing in pyridine. Later,Kenner et al.52 reported that the use of refluxing collidine in place of pyridine results inhigher yield for the purpurin-into-porphyrin conversion, because prolonged reflux inpyridine often gives rise to decomposition products. Interestingly, we found that thepresence of oxygen in the reaction medium has a beneficial effect on the conversion, butis not required, since the conversion was also observed under oxygen-free condition,suggesting the facile transformation ability of purpurin 16.Given the anticipated difficulty in the purpurin-into-porphyrin conversion for the[A,CJ-divinylpurpurin 149 (Scheme 41), a purpurin with a non-electron-withdrawinggroup (i.e. vinyl) at position 13, the planned synthetic route to [A,C]-divinylporphyrin147 via the [A,C]-divinylpurpurin 149 was abandoned. Another attempt to synthesis[A,C]-divinylporphyrin 147 was via purpurin 154 (Scheme 45), a purpurin with a 13-acetyl (electron-withdrawing) group, which should undergo the purpurin-into-porphyrinconversion. The planned synthetic route to purpurin 154 was via Grignard reaction withZn(II)132R-acetoxypheophorbide a methyl ester [152(R)] to afford 132R-acetoxy-13’-Scheme 45 Unsuccessful Transformation of Zn(II) Chlorin 152collidine170°CFinally, we came back to the direct conversion of purpurin 113 and preparedsufficient 1 3-decarboxy- 1 3-formyl-3-vinylrhodoporphyrin XV methyl ester (148) by156deoxo-13’-hydroxy-13-methylpheoph rbide a methyl ester (153), which could befurther oxidized by periodic acid to give 13’-decarboxy-13’-acetylpurpurin-7 dimethylester (154) as shown in Scheme 45. Unfortunately, the Grignard reaction at —78°C withZn(II)132R-acetoxypheophorbide a methyl ester [152(R)] {prepared from acetoxylationof 13R-hydroxypheophorbide a methyl ester [95(R)] (90% d.e.) followed by metallationwith zinc acetate } was unsuccessful and gave none of the desired product 153, and led todecomposition of the material under these conditions.MeMgBr H5106= ;= = = = = =MeO2d020 MeO26J02152(R) 153(90% d.e.)R=COMe154147= = ==155157(1) Zn(OAc)2(2) CH2=PPh31 1 0°C 1CHO (3) CF3COOH 2Me148 (71%) 147Scheme 46 Synthesis of [A,CJ-Divinylporphyrin 147 via Porphyrin 148refluxing purpurin 113 in collidine. Treatment of 13-formyl-3-vinylporphyrin 148 withzinc(II) acetate in methanol/dichioromethane gave the zinc complex 156 which wassubjected to a Wittig reaction to give, after removal of the zinc by treatment withtrifluoroacetic acid, a 71% yield of the desired [A,C}-divinylporphyrin 147 (Scheme 46).With this synthetic approach the [A,C]-divinylporphyrin 147 was obtained in 6 stepsfrom pheophorbide a methyl ester (7) in 9% overall yield. The structure of 147 wasconfirmed by its 1H NMR (Fig. 4.6) and UV/Vis spectra (Fig. 4.7). In its 1H NMR, bothvinyl groups in this non-symmetrical porphyrin were magnetically equivalent and theywere analyzed as an ABX system as before. The doublet of doublets at 6 6.15 ppm wasassigned to H-32(Z) and H-132(Z). The doublet of doublets centered at 6 6.33 ppm wasassigned to H-32(E) and H-132(E). The doublet of doublets centered at 6 8.27 ppm wasassigned to H-31 and H- 131. The coupling data found here agreed with the informationalready gained for the other vinylporphyrins. Its electronic spectrum as shown in Fig. 4.7exhibited a rhodo-type pattern (max = 404, 506, 544, 572, 630 nm, III>IV>Ib.I) and thisobservation corresponded to that reported by Dolphin et al. 15314713.112,1tI.l11.11.18.1.l6.85.84.•—2.8-3.8-4.8PPMFig.4.6Structureand‘HNMRSpectrum(CDC13)of[A,CJ-Divinylporphyrin147Go159ci)0C00C’)Fig. 4.7 Structure and UVJVis Spectrum (CH2C1)of [A,CJ-Divinylporphyrin 1474.3.2.2 Synthesis of [A,C]-Divinylporphyrin Via Porphyrin 23At this point, it was felt that the 15% yield of the formyl-rhodoporphyrin XV(148) obtained by the oxidation and deglyoxylation of purpurin 113 in collidine wasunsatisfactory and should be improved upon by pursuing an alternative approach.Previous studies led us to conclude that the 13-methoxycarbonyl group and othernon-formyl groups are advantageous in this regard. 3-Vinyirhodoporphyrin XV dimethylester (23) was chosen as the starting material since it is readily available from theoxidation of purpurin-7 trimethyl ester (16) as described before.In this approach (Scheme 47), 3-vinyirhodoporphyrin XV dimethyl ester (23) washydrolyzed to its di-acid analog and was partially remethylated on the propionic sidechain using 5% H2S04 in methanol to give the 3-vinyirhodoporphyrin XV monomethylçNH N147400 500 600 700nmWavelength160(IN HNdaMeCO2Me23(1) KOH/MeOH..NH N1cN HN)(2) HS04/MeOH(70%) O2HCO2MeN(4-N HN)c-ck2Me H3161ester (157) in 70% overall yield. The reesterification step was based on the reactivitydifference of the two carboxy groups which was found by Kenner and coworkers156. Itwas predicted that protonation of the nuclear carboxy-group was inhibited by the doublepositive charge on the macrocycle, whereas the side-chain carboxy-group retainedaliphatic character and therefore a greater disposition to esterification. A monoester wasindeed obtained without difficulty, and its structure 157 was confirmed by the lack of thelow-field methoxy-resonance (ring current effect) in the 1H NMR spectrum (Fig. 4.8) (indeuterotrifluoacetic acid) compared to the presence of the singlet signal located at ö 4.45ppm (i.e. 13-COOCH moiety) in the porphyrin dimethyl ester 23.(INH N—N HN—. ,O2Me 0157(91%)4—DMAP90°Cphosphatebuffer(pH=7)(75%)1594-NH N( NaBH44-N HN) (82%)H-0H02Me H3162 (1) PhCO”\105°C(2) KOH(60%)(70%)‘b-NH N-1SN HN&O2Me 147Synthesis of [A,Cj-Divinylporphyrin 147 via Porphyrin 23Scheme 47JNIILM.714DOTI10-6SF480.SF0.0DI5080.9!12768TO42760908920.HZIPTP88.RD0.ITOI.or.I05640TL298FO11280020.OP191P18 GB0.Cx48JCF26.ElIS.P2-S.HI/CIT179.PPM/CMSR2735.8! 10 I0 lOOP1081’199‘SI‘4 101‘II 71 45 ‘35157I12.!11.0900!7!6059i3!28II9II203049Fig.4.8Structureand1HNMRSpectrum(CF3COOD)ofPorphyrinMonoacid157162The resulting monomethyl ester (157) was added to a solution of N,N’-carbonyldi-imidazole in THF and heated to reflux for 30 mm.157 The desired imidazolide 158was obtained in 91% yield after flash chromatography on silica gel. The FAB massspectrum of 158 showed the molecular ion ([MHJ/z 601) and a fragment derived from theloss of the imidazole moiety (M/z 533). The 1H NMR spectrum (Fig. 4.9) of thecompound was in agreement with the structure of the expected porphyrin.The nucleophilic displacement’56of the imidazolide 158 with the magnesium saltof methyl hydrogen malonate (159)158 gave the expected B-keto-ester 160 in 70% yield.This step was based on a method which was introduced by Bram and Vilkas158 and laterimproved by Kenner and coworkers156. The magnesium salt of methyl hydrogenmalonate (159) was prepared by treatment of methyl hydrogen malonate with 2 equiv ofisopropylmagnesium bromide in THF. The advantage of this procudure was itsselectivity which allowed the reaction to proceed without deprotonation of the porphyrinmacrocycle to the green dianion.With the enolizable 8-keto-ester 160 in hand, we were able to attempt thedecarboxylation step. A high yield of the desired acetylporphyrin 161 was obtained bymodifying the decarboxylation method described by Taber and coworkers159. In thisreaction, the 8-keto-ester 160 was stirred in a solution of 4-(dimethylamino)pyridine (4-DMAP) in phosphate buffer (pH = 7) at 90°C for 12 hours under nitrogen to effect thedemethoxycarbonylation (75% yield). The progress of the reaction was monitored byTLC analysis and its completion was indicated by the disappearance of the less mobileenolizable 8-keto-ester 160.Fig.4.9Structureand1HNMRSpectrum(CDC13)of Imidazoyl Porphyrin158158II_ILl9. FAB mass spectrum of acetylporphyrin 161 showed a molecular ion at m/z549 ([MH]j. High resolution mass spectroscopy gave an accurate molecular mass at549.2861 (calculated value forC34H7N40 549.2865). The electronic spectrum ofthis compound was almost the same as that of the imidazoylporphyrin 158 (oxorhodotype spectrum). The 1H NMR spectrum of porphyrin 161 (Fig. 4.10) showed the fourmeso protons as singlets at 10.75, 10.10, 10.07, and 9.98 ppm, each corresponding toone proton. The H-132 (i.e. 13-COCH) appeared as singlet at 3.32 ppm. Thisobservation indicated the presence of the desired 13-COCH moiety.The acetylporphyrin 161 was reduced with sodium borohydride in THF to givethe expected hydroxyporphyrin 162. With the absence of the electron-withdrawing groupat position 13, hydroxyporphyrin 162 exhibited a rhodo-type electronic spectrum incontrast to an oxorhodo-type spectrum in the starting material 161, suggesting only onerhodofying group (i.e the 3-vinyl moiety) present in the molecule.The last step in the present synthetic sequence involves the generation of the 13-vinyl group. This transformation was achieved by subjecting the hydroxyporphyrin 162to benzoyl chloride in DMF at 105°C for 2 hours.160 Under these conditions, thehydroxyporphyrin 162 was dehydrated to give the desired [A,C]-divinylporphyrin 147 in60% yield after chromatography. This material was found identical to be thedivinylporphyrin prepared from the Wittig reaction of 13-formylporphyrin 148. With thissynthetic approach, the [A,C]-divinylporphyrin 147 was prepared in 9 steps frompheophorbide a methyl ester (7) in 12% overall yield.F161IIJiJ_th_LJ-z12.0IIIIl.Pe:piø5.’4.P3.e2.,1.0-2.0-4.1-5.1Fig.4.10Structureand111 NMRSpectrum(CDC13)ofAcetylporphyrin1611664.3.3 Synthesis of [A,C]-Dibenzoporphyrin DerivativesAs has already been mentioned, the synthetic strategy for the [A,C]-dibenzoporphyrin derivative relied on two cycloaddition reactions on an [A,C]-divinylporphyrinsystem. The first step in this program was to prepare an [A,C]-divinylporphyrin by thechemical modification of chlorophyll derivatives. Two routes were investigated and bothgave the desired [A,C]-divinylporphyrin 147 in moderate yields (described in section4.3.2). Having solved the problem of preparing the precusor, we next turned to theultimate goal of this program, the synthesis of a bacteriochlorin chromophore.MeOOC...(1DMAD MeOOC110°C _itQeCO2Me 147 164I DBU(25%)MeOOC..(1MeO\OOMeCOOMeCO2Me 1 65Scheme 48 Synthesis of [A,C]-Dibenzoporphyrin Derivative 165Refluxing the [A,CJ-divinylporphyrin 147 with 100-fold molar excess of dimethylacetylendicarboxylate in toluene for three days gave a mixture with a strong absorption at720 nm. The desired bis-adduct 164 (Scheme 48) was obtained as the major product167along with a small amount of chiorin in which oniy one of the vinyl groups (either ring Aand ring C) was transformed (identified by spectrophotometry). The best reaction timewas found to be 80 hours with a yield of —30%, while prolonged reflux resulted indecomposition and thus gave the product 164 in a lower yield. Flash chromatography ona silica gel column followed by further purification by preparative TLC plate gave thebis-adduct 164 in 25% yield. The electronic spectrum of the bis-adduct exhibited thecharacteristic features of a bacteriochiorin (Amax = 720 nm). The FAB mass spectrum ofthis adduct showed the molecular ion as the base peak at mlz 817 ([MHJj. Highresolution mass spectroscopy gave an accurate mass at 817.3455 (calculated value forC46H9N010= 817.3449).a)C)ccS0800nm400 500 600 700WavelengthFig. 4.11 UV/Vis Spectrum (CH2I)of [A,C]-Dibenzoporphyrin Derivative 165168When the above bis-adduct was stirred overnight with 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) at room temperature, a dramatic bathochromic shift was observedQrnax = 784 nm, £ = 38 000) (Fig. 4.11). This [A,CJ-dibenzoporphyrin derivative 165showed almost the same electronic spectrum as the bacteriochiorin reported by Dolphin etat. 153• The most significant features in the 1H NMR spectrum of the new adduct 165were the appearance of a signal at 6 4.89 ppm for the new sp3 center generated at C-2’and C-121 and two doublets at 6 7.30 ppm and 6 7.81 ppm for the new sp2 centers. Thisobservation indicated an isomerization of the double bond in both 1 ,4-cyclohexadienesthus extending the conjugation in the molecule as described before.4.4 Monoacid Analogues of New Regiochemically-pure BPDs and[A,C]-Dibenzoporphyrin DerivativesAs described earlier, the monocarboxylic acid analogue of benzoporphyrinderivative BPDMA shows better photosensitizing efficacy than its corresponding diester.Therefore, to parallel the structural requirements for this biologically activebenzoporphyrin derivative and to satisfy our originally planned goals, the newregiochemically-pure benzoporphyrin derivatives 141 and 125, and the [A,C]dibenzoporphyrin derivative 165 were partially hydrolyzed with 25% HC1 at thepropionic ester side chain to afford the corresponding monocarboxylic acids (i.e.propionic acid analogues) (Fig. 4.12). These monocarboxylic acids together with theircorresponding esters described herein are being evaluated for photodynamic ability.169MeOOC / MeOOC /MeOOC /NH 25% HCI MeOOC / NH.-NHN\ 0°C ...NHN\CO2Me CO2MeO2Me CO2H141 141—MAMeOOC / MeOOC /MeOOC/NH 25% HC1 MeOOC/ NH125 125—MAMeOOC-11 MeOOC-..ç725% HCI MeOO)çY”I) I)__Cji1cPOMe 0°C 5J)çpOOMeI_Ij-COOMe &j)-COOMeCO2Me CO2H165 165—MAFig. 4.12 Synthesis of Monoacid Analogues of BPDs and Dibenzoporphyrin Derivative4.5 SummaryTwo series of new regiochemically-pure benzoporphyrin derivatives 141 and 125have been synthesized from Diels-Alder reactions on 3-vinyirhodoporphyrin XV (23) and3-vinyiphytoporphyrin (114c) with dimethyl acetylenedicarboxylate. The correspondingmonocarboxylic analogues of these new regiochemically-pure BPDs have been preparedby partial hydrolysis. These new photosensitizers have characteristics which meet or170exceed the promising chemical features of BPDMA, a second generation photosensitizerin Phase-IT clinical trials.The [A,C]-divinylporphyrin 147 was synthesized via two routes and its DielsAlder reaction with dimethyl acetylenedicarboxylate was studied. The resulting bisadduct 165, being a stable bacteriochlorin derivative, absorbs strongly at 784 nm, a factwhich, according to cunent thinking, renders it eminently desirable as a photosensitizerfor treatment of large tumors or tumors which are deeply-seated within the body.In conclusion, the present work has led to the synthesis of a class of compounds,via chemical modifications of chlorophyll a, with potential as future drugs in the field ofphotodynamic therapy. All these new photosensitizers are of high purity and fullycharacterized, and thus are readily accessible for subsequently biological investigations.171Chapter 5DBU and DBN: Nucleophilicity vs. Basicity1725.1 Background and Research ObjectiveThe effectiveness of the bicyclic amidines, 1 ,8-diazabicyclo[5.4.O]undec-7-ene(DBU) and 1 ,5-diazabicyclo[4.3.0]non-5-ene (DBN) as “non-nucleophilic strong bases”in organic and inorganic chemistry has been widely demonstrated.161 These compoundsinitially became known because of their activity in dehydrohalogenations, which was firstnoted in the synthesis of vitamin A, and was rapidly exploited in other organic syntheses.The applications of DBU and DBN have rapidly increased because of their favorable“non-nucleophilic”, yet strongly basic, properties. They can, therefore, be applied to thepreparation of even relatively sensitive molecules. It was also found beneficial to usethese compounds in condensation162, substitution163, addition 164, isomerization andrearrangement 165, cyclization and cyclocondensation166, and oxidation and reduction 167reactions; and as catalysts in the syntheses of macromolecules 161The base strength of DBU has been determined by several groups.168’9 ThepKa value of the corresponding conjugate acid of DBU is in the range of 11.5-13.4depending on the determination medium in contrast to that of 10.7 for triethylamine(TEA)170. Therefore, DBU and DBN are slightly stronger bases than triethylamine. Aplausible explanation is the resonance contribution of their bicyclic amidine structures.The unshared pair of electrons at N-i position in DBU and DBN can be delocalized overtwo nitrogen atoms, as represented by the corresponding resonance structures 168 and169 (Scheme 49). Since the actual molecule is a hybrid of these resonance forms, theelectron density of the nonbridged nitrogen atoms (i.e., N-8 in DBU and N-5 in DBN) is“increased” and the unshared pair of electrons in these positions is more available for173attack by an acid (a proton) or an electrophile. In other words, DBU and DBN can act aseither base or nucleophile. It is, therefore, of great interest to be able to predict whenDBU and DBN will act as nucleophiles and when they will act as bases.cccç.DBU 168DBN 169Scheme 49 Resonance Structures of DBU and DBNBased on Lewis acid-base theory, any nucleophile is also a Lewis base. However,there is a clearly conceptual relationship between the properties called basicity andnucleophilicity. The term basicity is defined in terms of the position of an equilibriumreaction with a proton or some other acid, while nucleophilicity is generally accepted torefer to the effect of a Lewis base on the rate of a nucleophilic substitution reaction.Therefore, basicity is the so-called thermodynamic basicity and nucleophilicily is the socalled kinetic basicity. 170Nucleophilicity is used to describe trends in the kinetics of reactions. Thecompetition between nucleophilicity and basicity has been observed in many organicreactions, e.g., SN1 Substitution Reaction versus E1 Elimination; and NucleophilicAddition at a Carbonyl Carbon versus Enolate Formation. In some cases, nucleophilicity174is correlated directly with basicity, although the relative nucleophilicity as well as therelative basicity of a given species may differ from substrate to substrate. It has not beenpossible to devise an absolute scale of nucleophilicity or basicity. Steric influences oftenplay a major part in nucleophilicity. For example, NH3 (conjugate acid has pKa 9.2)170is a much weaker base than Et3N, DBU and DBN, but a much stronger nucleophilebecause its molecular size facilitates close approach to a substrate (electrophile).From the above discussion, DBU and DBN should also be capable of acting asnucleophiles under a certain set of circumstances, although the steric hindrance of themolecules usually blocks the approaching of an electrophile. In 1981, McCoy and Ma117became the first to provide chemical evidence for the nucleophilic nature of DBU. In aserendipitious discovery, they found DBU acting as a difunctional nucleophile during thereaction of DBU with methyl cyclopropene- 1 ,2-dicarboxylate derivatives 170 (Scheme50). In 1993, Bertrand et al.172 found that DBU and DBN showed remarkablenucleophilicity in their reactions with halogenated phosphanes, a reaction which providedion-pair products 171 (Scheme 50). In 1994, Lammers and his coworkers173 alsoobserved another reaction in which DBU and DBN acted as nucleophiles when reactedwith 4-halo-3,5-dimethyl-1-nitro-1H-pyrazoles to form lactam-type products 172(Scheme 50). In the products 172, one of the bicyclic rings in DBU and DBN wasopened by water.175170cc(R2N)Pc1 1eKPF6PF(64%)171n=1,_________X=CI, Br, I NO2 (21—74%) H172Scheme 50 Examples of DBU and DBN Acting as NucleophilesTherefore, DBU and DBN can act as nucleophiles (via the N-8 atom in DBU andthe N-5 atom in DBN) to form a nitrogen-phosphorus bond, a nitrogen-carbon bond andact as difunctional nucleophiles. The objective of this portion of research, which was alsoinitiated by a serendipitious discovery from a side-reaction product in the research on “thethird generation photosensitizers” (Chapter 4), was to further exploit the nucleophilicbehaviour of DBU and DBN; and thus be able to bring about further understanding of thenucleophilicity as well as the basicity of these common organic bases. In the firstreaction, DBU acting as a difunctional nucleophile quantitatively reacted with a strongelectrophile (DMAD) to afford a fused tricyclic derivative 176 through concomitantformation of new nitrogen-carbon and carbon-carbon bonds. In the second reaction,176pheophorbide a methyl ester (7), a weak electrophile which alone does notelectrophilically react with DBU or DBN, has reacted, through catalytic activation byLewis acids, with nucleophilic DBU and DBN to form chiorin e lactams 185 and 186.The nucleophilic reaction mechanisms will also be discussed.5.2 DBU as a Difunctional NucleophileIn studies related to the photosensitizers (BPD series) for the photodynamictherapy of tumors (detailed discussion appears in Chapter 4), we have used DBU as abase to tautomerize the isolated double bond formed in the Diels-Alder reaction of vinylporphyrins with dimethyl acetylenedicarboxylate (DMAD), to the fully conjugatedchromophores. A yellow material is occasionally observed in the chromatographicpurification of the rearranged benzoprophyrin derivatives (green). Since this materialshows no electronic absorptions in visible region (all porphyrins have visibleabsorptions), we considered that it was not a porphyrin related by-product. When DBUwas added to DMAD (1:1 ratio), in chloroform at room temperature, a golden yellowproduct was instantly formed with a strong exothermicity. After recrystallization(CH2C1/hexane), red crystals (mp 158°C) were obtained (96% yield) and, surprisingly,mass spectrometry (mlz 262) and microanalysis indicated this product to be an adduct ofDBU and DMAD minus a molecule of methanol. The ‘H NMR spectrum is clean anddistinct at 200 MHz (Fig. 5.1), and exhibits two singlets at 6 5.94 (1H) and 6 3.33 (3H)together with seven completely resolved methylene groups. The connectivity of these177CcDBU—il--çç)0/4-COOMeH 176• IU.O O.O g.O e.o 7.0 6.0 5.0 4.0 3.0 2.0 .0 0.0PPMFig. 5.1 1H NMR Spectra (CDC13)of DBU and Tricyclic Derivative 176Qç)0&—COOMeH 176178I I I-___.— II.— • I.7.0 6.0 5.0 4.0 3.0 2.0PPM2. PPMFig. 5.2 1H- COSY Spectrum (CDC13)of Tricyclic Derivative 176‘II‘I•II•I180160140120100806040PPMFig.5.313CNMRandAPTSpectra(CDCI3)ofTricyclicDerivative176200cc 0’—COOMeH176220200—20180methylene signals was correlated by a ‘H-’H COSY spectrum (Fig. 5.2) as two isolatedspin systems with three and four methylene groups, respectively. In combination with its13C NMR (Fig. 5.3) and attached proton experiments (APT, Fig. 5.3), all fourteencarbons and eighteen hydrogens were accounted for and structures 176 and 178 wereproposed. Since the spectral analysis was unable to discriminate between these twostructures, a single crystal suitable for X-ray analysis was prepared and we have nowshown this product to have the unusual structure 176, as shown in Fig. 5.4.Formation of 176 was initiated by Michael addition of DBU with DMAD via theN-8 nitrogen atom (Scheme 51), affording the quanternary ammonium salt’74 173.Abstraction of the H-6 proton in 173 leads to an intermediate 174 which could react withFig. 5.4. X-Ray Structure of 176. Five-membered ring is planar, six-membered ring hasan envelope-like conformation, and seven-membered ring adopts a distortedchair conformation. Important bond lengths(A): C(2)-C(12) 1.340(2), C(10)-C(11) 1.389(2).C(7)C(9)C(7)C(9)C(3)0(3)C(3)0(3)C(14) C(14)181either ester functionality to give either a five (Path A) or a six (Path B) membered ring(175 or 177). Loss of the H-6 proton from the cycloadduct (175 or 177) generates theincipient double bond conjugated with the carbonyl to produce the neutral tricyclicproduct 176 or 178. Although the formation of a six-membered ring (Path B) seems to bethermodynamically more favourable, the reaction is highly selective and produces onlyisomer 176, suggesting that this reaction is similar to typical Michael reactions and isunder kinetic control.DBUMeOOC—=—COOMeDMADC)OfMeMeOi H 0 174Path B,’ \ Path AMeOG 0OMe )OMe175—MeOHccMe 0’—C00MeH 178 H 176173I —MeOHScheme 51 Michael-type Addition of DBU to DMAD182We have also found that DBU reacts with other activated triple bonds, such as,methyl propiolate and methyl cyanoformate, but, so far, no products have beencharacterized from these complex reaction mixtures. The reaction of DBN with DMADis also strongly exothermal and gives an, as yet, intractable unidentified red mixture. Theconformational rigidity of the five membered ring undoubtedly hinders a transformationsimilar to that of 174 to 175.5.3 Nucleophilic Reaction with Pheophorbide a Methyl Ester5.3.1 RationaleAt this point, the remarkable Michael-type addition of DBU to dimethylacetylenedicarboxylate (DMAD) had been discovered, where DBU acts as a difunctionalnucleophile to form the unusual tricyclic derivative 176. Unfortunately, the nucleophilicbehaviour of DBN had been brought into question by the unidentified products from thereaction of DBN and DMAD. It was envisioned that designing a reaction, in which bothDBU and DBN should act as nucleophiles to form identifiable products, would provide abetter example to understand the nucleophilic behaviour of these bicyclic amidines.Continuing our research into the chemical modifications of chlorophyll a, it is desirablethat this reaction should make use of a chlorophyll derivative as the electrophilicsubstrate.Previous studies led us to conclude that the five-membered exocyclic ring inchlorophyll a and its non-metallated derivatives is advantageous in this regard. As183described in Chapter 1, this exocyclic ring (ring V) can be nucleophilically cleaved byprimary and secondary amines. For example, reaction of pheophorbide a methyl ester (7)with an excess amount of ethylamine at room temperature for 25 hours gave chlorin e13-(2-N-ethyl)amide-15,17-dimethyl ester (179) in 63% yield (Scheme 52). The reactionmechanism was not clear at that stage. It was reasoned that DBU and DBN are strongerbases than EtNH2 but are poorer nucleophiles for steric reasons. As was described inChapter 2, DBU is effective at promoting the asymmetric hydroxylation of pheophorbidea methyl ester (7) and its derivatives at position 132 by protonating the 8-ketoester in theexocyclic ring V without cleavage of the exocyclic ring. Therefore, DBU and DBNthemselves can not act as nucleophiles to react with the B-ketoester in the exocyclic ringV because of the poor reactivity activity of DBU and the substrate.(I, (IEtNH2N HN N HN(63%),I ...‘—4 / \ 6ONHEtCO2M.CO2MeO OeMe2M07 179Scheme 52 Nucleophilic Reaction of EtNH2with Pheophorbide a Methyl Ester (7)The facility with which an electrophile-nucleophile reaction takes place dependsof course on the strengths of the electrophile and the nucleophile. Low nucleophilicactivity of DBU and DBN presents the reaction with the weak electrophilic substratepheophorbide a methyl ester (7). Therefore, increasing either the nucleophilic strength ofDBU and DBN or the electrophilic strength of pheophorbide a methyl ester (7) was184required for bringing about the generation of the reaction. Since we were trying todemonstrate the nucleophilicity of DBU and DBN, improving the electrophilic activity ofpheophorbide a methyl ester (7) was attempted.An electrophile with a positive charge is always a more powerful electrophile thanits neutral counterpart (assuming the latter is also an electrophile). Therefore, the easiestmethod to increase the electrophilic activity of a given substrate is by increasing its “acidstrength”. However, common protic acids such as H2504,CH3OOH or ArSO3H aretotally ineffective in this special case because these acids protonate the substrate andsubsequent addition of DBU or DBN will deprotonate the substrate back to its originalstate, i.e., the procedure virtually becomes an acid-base reaction, without any effects onthe desired reaction. After a number of experiments, strong Lewis acids, the trialkylsilyltriflates, were found to be the best at promoting the nucleophilic reaction of DBU andDBN with pheophorbide a methyl ester (7). The following sections will present thisreaction, the characterization of the reaction products and the mechanistic rationalization.5.3.2 The Generation and Fate of Nucleophilic DBU and DBNTrimethylsilyl triflate (TMSOTf) and tert-butyldimethylsilyl triflate(TBDMSOTf), super-reagents bearing a highly electron-withdrawing triflate moiety, canactivate various oxygen-containing organic compounds through a one-center (not multicenter) interaction at the electron-deficient silicon atom and, in some cases, generatereactive ion-pair intermediates even in aprotic solvents.175 Many reports have shown thatthey can act as catalysts to accelerate a variety of nucleophilic reactions in aprotic185media.176 For example, reactions of enol silyl ethers with acetals or related compoundshave been reported to be catalyzed efficiently by trialkylsilyl triflates, TMSOTf andTBDMSOTf, leading to the aldol-type products in a directed manner.177 They are alsopowerful silylating agents and a wide range of active hydrogen containing compounds aresilylated in the presence of amines (including DBU and DBN).178 Their combination withhindered amidine DBU has also been used in the selective ring-opening reactions ofoxiranes.179Instead of simple protonation observed in the protic acids such as H2S04,CH3OOH or ArSO3H, trialkylsilyl triflates catalyze a substrate by converting it to arelatively stable ion in which the positive charge on the carbon is greatly increased, thusmaking it more susceptible to nucleophilic attack.180 This was found to be the case in thereaction of pheophorbide a methyl ester (7) with DBU and DBN. Upon activation for 10mm by trialkysilyl triflates, trimethylsilyl triflate or tert-butyldimethylsilyl triflate,pheophorbide a methyl ester (7) behaved as a powerful electrophile and reacted with DBUgiving a green product 185 (>50% yield). The product 185 was characterized surprisinglyas the chlorin e6 13- [1 -(3-N-propyl)-2-azacycloheptanejamide- 15,1 7-dimethyl ester,where the exocyclic ring V of pheophorbide a methyl ester and one of the bicyclic rings inDBU were opened. An analogue {found to be chlorin e6 13-[1-(3-N-propyl)-2-pyrrolidinone]amide-15,17-dimethyl ester, 186} was also obtained on the reaction ofcompound 7 with DBN. Their exhaustive characterization is fully described in thefollowing section.186The reaction can run in either dry THF or dry DMF. In THF, DBU and DBNreacted with pheophorbide a methyl ester (7) giving yields greater than 50% along with-2O% starting material 7 (no side reaction was observed). In DMF the reactionsproceeded faster, reaching completion in -3 h, but only -45% yields were obtained alongwith -2O% side- (degradative) products. The reactions promoted by TMSOTf andTBDMSOTf were both efficient, and no difference between them was observed.i. TMSOTf or TBDMSOTfii.DBU, n=3DBN, n=10 3’i’a185. R3=i’c i’ a’186. R = HN’-N1. 3’i’bidPc179. R3= HN-’i’d i’bScheme 53 Nucleophilic Reaction of DBU and DBN with Methyl Pheophorbide a (7)Therefore, the “planned nucleophilic reaction” of DBU and DBN withpheophorbide a methyl ester (7) has been successfully achieved through catalyticpromotion by trialkylsilyl triflates, TMSOTf and TBDMSOTf. This present work hasdemonstrated that DBU and DBN are not only strong bases that can deprotonate the f3-ketoester of the exocyclic ring V, but also can act as nucleophiles to cleave the ring Vj747. R1=COOMe, R2=H25. =R2H187under certain conditions. These observations could help explain the previously observedand anomalous products in the enolate-trapping reaction of chlorophyll a and relatedcompounds. 181,1825.3.3 Structural Elaboration of the ProductsUnambiguous structure assignments of 185 and 186 were carried out using massspectroscopy, 1H NMR, proton decoupling, 13C NMR, attached proton test (APT), 1Hhomonuclear correlation spectra (COSY) and 1H-3C heteronuclear correlation spectra(HETCOR). Furthermore, the structure of 185 was confirmed by nuclear Overhausereffect (nOe) while the structure of 186 was confirmed by its direct preparation throughreaction of commercially available l-(3-aminopropyl)-2-pyrrolidinone with pheophorbidea methyl ester (7). The assignments are described as follows.HRMS and elemental analysis showed 185 to be an adduct of DBU and methylpheophorbide a methyl ester (7) plus the elements of a water molecule (formula:C45H56N60). 13C NMR showed that 185 consists of 45 carbons (36 carbons in 7) andan attached proton test (APT) confirmed that eight of the additional nine carbon atoms aremethylene carbons in the high field and only one sp2 carbon at low field. The 13C signalsof the eight additional methylene carbons are matched with related chemical shifts inDBU. In its 1H- homonuclear correlation spectrum (COSY) (Fig. 5.5), chlorin 185,like the starting material 7, only has three downfield methine peaks (1 H each) suggestingthat one of the compound’s four bridging carbons (i.e. C-15) is still functionalized. Fivesharp singlets (3H each) between 3.0 and 4.0 ppm suggest three aryl-substituted methyl188groups as well as two methoxyl groups, which showed these groups were unchanged afterthe reaction. A doublet of quartets at 4.46 ppm (1H) was coupled to one proton (ddd) at4.39 ppm (1H), suggesting the former (4.46 ppm) is the H-18 and the latter is H-17. H-17is also coupled to another two multiplet hydrogens at 2.23 and 1.83 ppm, and these twoprotons were found to be coupled with the corresponding carbon at 29.66 ppm (see Table5.2 for 13C NMR data) as shown by a HETCOR spectrum (Fig. 5.6). This methylene isassigned to 17-CH2and the two hydrogens are assigned as Ha-171 (2.23 ppm) and H&17’ (1.83 ppm). Consequently, Hb-172(2.52 ppm) and Hb-172(2.12 ppm) were identifiedbased on their coupling to Ha- 171 and Ha’- 171. There are two other obvious couplingpairs at low field. (i). A triplet (1H, J = 6.0 Hz) at 7.55 ppm was exchangeable withCD3O and coupled to the multiplets at 3.68 and 3.64 ppm (1H each), suggesting apossible CONHCH2unit; (ii) an asymmetrically substituted methylene was suggested by asplit AB quartet at 5.54 and 5.27 ppm (1H each) with coupling constant J = 19.1 Hz,whose corresponding carbon is at 38.02 ppm. Assignment of this methylene unit as C-15’was based on the nOe enhancement (Scheme 54) of the H-17 (4.39 ppm). Theseassignments confirmed that the exocyclic ring V was the only part of the molecule to havereacted and also suggested that 185 has a chlorin-e6-type structure. Since it contains aCONHCH2moiety, it was believed to be a chlorin e6 amide derivative. This conclusion isfirmly supported by its absorption spectrum, a typical chlorin-e6-type spectrum with theSoret band at 404 nm (E 171 900) and Q band at 666 nm (48 700).189-NH—1185— ,II I I Al III dh2. 5.5 1H- COSY Spectrum (CDC13)of Chiorin e Amide 185(between H 0-10 ppm for Proton Resonances)190(/NH N—\ / 185N HN,CONO2MeCO2Me3 2Fig. 5.6. 1H-3C Heteronuclear Correlation Spectrum of Chiorin e Amide 185(12.0mg in 1.0 mL CDC13)between 6 0.0-6.0 ppm for protonresonances, and between 6c 0-60 ppm for the carbon resonancesQ00ooppI20•4040pp.191Table 5.1. 1H NMR Spectral Data (CDC13,400 MHz)CompoundProton 185k 186b 179b 7H-b 9.69(s) 9.70(s) 9.70(s) 9.52(s)H-5 9.62(s) 9.63(s) 9.62(s) 9.39(s)H-20 8.89(s) 8.80(s) 8.80(s) 8.57(s)H-3’ 8.07(dd) 8.08(dd) 8.05(s) 8.01(s)H-32(E) 6.32(dd) 6.34(dd) 6.38(dd) 6.30(dd)H-32(Z) 6.10(dd) 6.12(dd) 6.15(dd) 6.19(dd)H-8’ 3.78(q) 3.79(q) 3.79(q) 3.67(q)H-121 3.57(s) 3.57(s) 3.55(s) 3.68(s)H-2’ 3.47(s) 3.48(s) 3.48(s) 3.39(s)H-71 3.30(s) 3.30(s) 3.32(s) 3.21(s)H-18’ 1.71(d) 1.68(d) 1.70(d) 1.80(d)H-82 1.70(t) 1.70(t) 1.70(t) 1.68(t)H-18 4.46(dq) 4.44(q) 4.46(dq) 4.47(q)H-17 4.39(ddd) 4.36(dd) 4.35(dd) 4.20(dd)Ha-171 2.23(m) 2.21(m) 2.15(m) 2.62(dt)Ha-171 1.83(m) 1.80(m) 1.78(m) 2.31(dt)Hb-172 2.52(ddd) 2.52(ddd) 2.50(ddd) 2.50(dt)Hb’-172 2.12(ddd) 2.13(ddd) 2.15(m) 2.24(t)NH -1.60(br s) -1.60(br s) -1.87(br s) 0.53(br s)NH -1.82(br s) -1.82(br s) -1.96(br s) -1.62(br s)H-151/132 5.54(d) 5.51(d) 5.56(d) 6.27(s)5.27(d) 5.23(d) 5.25(d)H-174 3.60(s) 3.58(s) 3.59(s) 3.57(s)H-153/13 3.69(s) 3.71(s) 3.80(s) 3.88(s)H-D-l’d 7.55(t) 7.31(t) 6.40(t)H-D-l’c 3.68(m) 3.81(m) 3.80(q)3.64(m) 3.60(m)H-D-l’b 2.08(m) 2.08(m) 1.41(t)H-D-l’a 3.64(m) 3.51(m)H-D-3’ 2.48(dd) 2.37(t)H-D-4’ 1.64(m) 2.08(m)H-D-5’ 1.66(m) 3.SOdd)H-D-6’ 1.70(m)H-D-7’ 3.41(dd)______________ _____________ __________aConcentration 1.5 mgIO.6 mL bConcenti.ation 1.0 mg/0.6Reaction of ethylamine with pheophorbide a methyl ester (7) gave chiorin e6ethylamide 179 (Scheme 52), an analog of 185. Compound 179 exhibited a relativelysimple and easily-assigned NMR spectrum. Comparing the spectral data of 185 and 179,the assignment of 185 as a chiorin e6 amide with 13-substitution was confirmed.192For the 13 side-chain protons in 185, the methylene (83.68 ppm, 3.64 ppm) of theCONHCH2unit was assigned as H-D- 1 ‘c and the corresponding carbon (C-D- 1 ‘c) is at36.87 ppm. A multiplet at 2.04 ppm (2H) was believed to be H-D-l’b based on theircoupling with H-D-l’c. Two protons at 3.64 ppm (m) were assigned as H-D-l’a sincethey are coupled with H-D-l’b (2.04 ppm). A doublet of doublet (2H) at 3.41 ppm,coupled with 2 protons at 1.70 ppm, were assigned as H-D-7’ (NCH2) because itscorresponding carbon is at 49.64 ppm and the three other carbons are all in the higherfield. With the same technique, the remaining three methylenes, whose proton chemicalshifts overlap with H-181 and H-82 at 1.64-1.70 ppm, were assigned as H-D-4’, H-D-5’and H-D-6’ based on the relative cross couplings.NH N—HN’ )nOe02MeHOiOCO2MenOe185Scheme 54 Observed nOe Enhancement for Chlorin e Amide 185Further details of the NMR assignments are given in Table 5.1 and Table 5.2. Tnaddition, nOe experiments (Scheme 54) have confirmed the above assignments.Compound 185 is thus chlorin e6 1 3-[ 1 -(3-N-propyl)-2-azacycloheptane]amide- 15,17-dimethyl ester.193A similar analysis of spectral data has shown that product 186 is chiorin e6 13-[1-(3-N-propyl)-2-pyrrolidinone]amide- 15,1 7-dimethyl ester. This was further confirmed bycomparison with the product from direct nucleophilic reaction of connnercially-available1-(3-aminopropyl)2-pyrrolidinone with methyl pheophorbide a (7). The productsprepared by these two methods were identical.Table 5.2 13C NMR Spectral Data (CDC13,125 MHz)Compound CompoundCarbon 185k 186b 179C 7d Carbon 185 186b 179C 7dC-173 173.49 173.57 173.54 173.36 C-15’1132 38.02 38.01 37.80 64.71C-13’ 173.55 173.57 174.18 189.63 C-17 53.12 53.11 53.08 52.88C-152/133 169.25 169.36 169.39 169.60 C-18 49.20 49.23 49.23 51.70C-19 168.53 168.64 168.73 172.14 C-172 31.09 31.10 31.12 31.06C-16 166.65 166.67 166.62 161.16 C-171 29.66 29.66 29.65 29.86C-6 153.93 154.04 154.16 155.50 C-18’ 23.03 23.03 23.05 23.10C-9 149.07 149.08 149.08 150.85 C-8’ 19.67 19.68 19.70 19.29C-14 144.62 144.68 144.74 149.61 C-82 17.68 17.69 17.75 17.36C-8 138.65 138.76 138.83 145.05 C-121 12.11 12.15 12.20 12.09C-i 135.96 136.76 136.11 141.99 C-21 12.05 12.08 11.92 12.06C-il 135.32 135.23 134.95 137.85 C-7’ 11.30 11.31 11.37 11.08C-3 134.70 134.76 134.87 136.39 C-174 51.54 51.55 51.63 50.10C-4 134.58 134.69 134.77 136.12 C-153/13 52.06 52.08 52.13 51.10C-7 134.34 134.43 134.02 136.02 C-D-l’c 36.87 37.17 35.52C-2 130.07 130.00 130.13 131.76 C-D-l’b 27.82 17.93 14.79C-12 129.89 130.00 130.13 128.87 C-D-l’a 45.50 40.00C-31 129.54 129.53 129.49 128.94 C-D-2 176.64 175.66C-13 128.76 128.48 128.34 128.94 C-D-3 37.05 30.80C-32 121.39 121.48 121.63 122.66 C-D-4 23.36 27.16C-is 102.42 102.32 102.12 105.13 C-D-5 28.52 47.32C-b 101.34 101.39 101.36 104.28 C-D-6 29.90C-S 98.78 98.81 98.84 97.39 C-D-7 49.64C-20 93.53 93.57 93.66 93.07aConcentration 12.0mg /i.OmL bConcentration 9.0mg /1.0 mLcConcentration 18.0mg /1.OmL dConcentration 25.0mg /1.OmL1945.3.4 Reaction MechanismThis reaction requires the -ketoester system of pheophorbide a methyl ester (7).Reaction with pyropheophorbide a methyl ester (25), a decarboxylated product ofpheophorbide a methyl ester (7), failed. Because of the similarity of methyl 2-oxocyclopentanecarboxylate to the ring V in pheophorbide a methyl ester (7), we alsoinvestigated its reaction with the amidine bases but found that ring opening did not occur.The initial reaction between methyl 2-oxocyclopentanecarboxylate and DBU (DBN)resulted in the formation of a trace amount of 1:1 “adduct” which could be only identifiedby mass spectra and chromatographical analysis.Since direct mixing of TMSOTf with DBU resulted in no new products and theenolate from deprotonation of pheophorbide a methyl ester (7) by DBU (or DBN) isrelative stable in the absence of oxygen in the dark, the nucleophilic behaviour of DBUand DBN must be initiated from the activation of compound 7 by Lewis acids (TMSOTfand TBDMSOTf). In Scheme 55 a mechanism is proposed for the formation of 185 and186. The first step is coordination of the l3 carbonyl group of 7 by TMSOTf orTBDMSOTf to generate a reactive ion-pair intermediate 180. This kind of ion-pairintermediate is common and found to form easily in the TMSOTf (or TBDMSOTf)catalyzed aldol-type reaction of silyl enol ethers and acetals.177 The pentacoordinatesilicon species183 are so electron-deficient that they can react with the nonbondingelectrons in DBU and/or DBN nitrogen to give 181. The further cleavage of bond (C-13’—C- 132) of ring V, step 181 to 182, drives the reaction towards completion. Indeed, theformation of 179 from the nucleophilic reaction of ethylamine with 7 is also through this195type of bond cleavage. The intramolecular rearrangement to generate 183 is facilitatedthermodynamically by the formation of the amide (with higher bond energy). For 2-oxocyclopentanecarboxylate, it can also form an activated ion-pair of this kind, but due tothe lack of delocalization which should stabilize the charges, no ring cleavage occurred.The intramolecular rearrangement step arises from the generation of 183, no doubtfacilitated by both the adjacent nonbonding nitrogen electrons and by the breaking of theMeOOC 07TMSOTf ITfMeOOC SiMe3OTfSiMe30ççDBU, n=3DBN, n=1180.ISjMe30182 181MeO183184185. n=3186. n=1Scheme 55 Proposed Mechanism for the Formation of Chiorin e amides 185 and 186196C-N bond to relieve ring strain. Similar rearrangements have been extensively studiedthrough the hydrolysis of bicyclic imidates by Deslongchamps et al. 184 Compound 183 istrapped by water in the NH4C1 workup to release TMSOTf (or TBDMSOTf) to give themore stable amide 184. Consequently, 184 tautomerizes to afford products 185 and 186.5.4 SummaryIn the first part of this work, the remarkable Michael-type addition of DBU (as adifunctional nucleophile) to dimethyl acetylenedicarboxylate was discovered. Theunusual tricyclic structure of the reaction product 176 (kinetically-controlled product) hasbeen confirmed by X-ray diffraction. A possible mechanism was proposed.In the second part of this work, unusual nucleophilic reactions of DBU and DBNwith pheophorbide a methyl ester (7) were successfully achieved. The catalytic promotionof these reactions by Lewis acids, the trialkylsilyl triflates, as well as the reactionmechanism was discussed.In conclusion, the present work demonstrates the nucleophilic behaviour of thebicyclic amidines DBU and DBN and serves to remind researchers of possiblenucelophilicity when they choose DBU and/or DBN as a base.197Chapter 6Experimental1986.1 General MethodsThis general section covers the techniques and instruments used for the analysisand the purification of the products.Melting Point DeterminationsMelting points were performed on a 6548-J17 microscope equipped with aThomas model 40 hot stage melting apparatus; the values are uncorrected.Nuclear Magnetic Resonance SpectroscopyProton nuclear magnetic resonance (1H NMR) spectra were obtained fromsamples in deuteriochlorform (CDC13)on a Bruker AC-200 (200 MHz), a Varian XL-300(300 MHz), a Bruker WH-400 (400 MHz) or a Bruker AMX-500 (500 MHz)spectrometer. The chemical shifts are expressed in parts per million (ppm) on the scalewith residual chloroform (6 = 7.24 ppm) as internal standard. Signal multilicities,coupling constants, integration ratios and assignments appear in parentheses. Selectivedecouplings were performed on the same instruments.Carbon-13 NMR (13C NMR) spectra were obtained in CDC13 with a Varian XL300 (75 MHz) or a Bruker AMX-500 (125 MHz) spectrometer. The chemical shifts arereported on the 6 scale with residual chloroform (6 = 77.0 ppm) as internal standard.Signal assignments appear in parentheses.199Attached proton test (APT) spectra were obtained in CDC13 with a Bruker AC-200 (50 MHz) or a Varian XL-300 (75 MHz) spectrometer. The chemical shifts arereported on the 6 scale with residual chloroform (6 = 77.0 ppm) as internal standard.Two dimensional proton homonuclear correlation spectra(1H- COSY) wereperformed in deuteriochlorform (CDC13)on a Bruker WH-400 (400 MHz) spectrometerto help assign the signals.Two dimensional proton heteronuclear correlation spectra (HETCOR) wereperformed in CDC13 on a Bruker AMX-500 (500 MHz) spectrometer to help assign thesignals.Nuclear Overhauser effect difference (flOe) spectra were performed indeuteriochlorform (CDC13)on a Bruker WH-400 (400 MHz) spectrometer.Exchange test experiments of active hydrogens (OH and NH) in hydroxychiorinsand chlorin e amides were performed in a CDC13 + l0%CD3OD on a Bruker WH-400(400 MHz) spectrometer to help assign the OH and NH signals by comparison with thespectra obtained in CDC13.Elemental AnalysisMicroanalyses were carried out in the microanalytical laboratory at the Universityof British Columbia by Mr. Peter Borda using a Carlo Erba Elemental Analyzer 1106.Mass SpectroscopyLow and high resolution fast atom bombardment (FAB) mass spectra weremeasured on a Varian Mat CH 4-B spectrometer and 1-thioglycerol or 3-200nitrobenzylalcohol was used as a matrix. Low and high resolution electron impact (El)mass spectra were recorded on a Kratos/AEI MS-902 spectrometer.Electronic SpectroscopyElectronic spectra were measured in chloroform or dichloromethane or methanolusing a Hewlett Packard Model 8452A Diode Array spectrophotometer.ChromatographyFlash column chromatography was performed on silica gel 60 (70-230 mesh,supplied by E. Merck Co; usually silica III, i.e deactivated with 6% water or silica V, i.e.deactivated with 15% water) or neutral alumina (usually Brockman Grade III, i.e,deactivated with 6% water or Brockman Grade V, i.e., deactivated with 15% water).Analytical thin layer chromatography (TLC) was performed using commercially-available Merck 60 F254 silica gel (precoated sheets, 0.2 mm thick).Preparative TLC was prepared on pre-coated 20 x 20 cm 0.5, 1 or 2 cm thickWhatman or Merck silica gel plates. The deactivated silica gel plates, in some caseswhen necessary, were prepared by blank development of the commercially availableplates with 10% methanol in dichloromethane followed by air-drying before use.Analytical high performance liquid chromatography (HPLC) was obtained using aWaters Novapak C18 4t 60 A (3.9 mm x 15 cm) column with a flow rate of 1 mL min’and detection at 410 nm using a Waters 994 photodiode array detector. Semi-preparativeHPLC separations were performed on a Waters Novapak C18 lOji 125 A (7.8 mm x 30201cm) column with a flow rate of 3 mL mm-1 and detection at 410 nm using a Waters 994photodiode array detector. Solvent systems used are specified where appropriate.Extraction and Reaction ConditionsDue to the inherent light sensitivity of these compounds, in particular the isolatedchlorophyll and antioxidative chiorins, all extractions, separations and reactions wereperformed in the dark. Reactions were monitored by TLC and spectrophotometry andwere carried out under a atomsphere of nitrogen.Reagents and SolventsAll chemicals and solvents were reagent grade. When necessary, the solventswere purified according to procedures given in the literature. Spirulina maxima alga(food quality) was purchased from Sosa Texcoco S. A., Mexico. (—)-(1R)-(10-camphorsulfonyl)oxaziridine and (+)-( 1 S)-( 1 0-camphorsulfonyl)oxaziridine werepurchased from Aldrich. 1-Phenyl-N(phenylsulfonyl)oxaziridine11and monomethylmalonate158 were prepared by following the literature procedure.Nomenclature and Numbering System Used for the Synthesized CompoundsThe trivial names with IUPAC-IUB numbering system, which is exemplifiedbelow, will be used in this work. When no corresponding trivial names are available, thecompounds will be named in the JUPAC-IUB nomenclature. These compounds includethe regiochemically-pure benzoporphyrin derivatives (BPDs) and the dibenzoporphyrin202derivatives. “S” or “R” (labelled after a compound number) denotes the absoluteconfiguration at C-132 or C-15’ or C-131 position where appropriate.2331/67\: 81/34212\NH N 82 NH N21 2220\ 24 23/1018 N HNN HN H/18•.. 17’..- /181 17171 132 i121Ha H 132172 COOMeO Ha’ 17 OHOMeQOC 13 13 Hb;H 017 17Pheophorbide a methyl ester (7) 132S-Hydroxychlorophyllone a [82(S)]p10 p12 p14 p16P17 P20Numbering of Phytyl Group2036.2 Stereoselective Synthesis of Natural Antioxidative Chiorins6.2.1 Starting Materials : Natural PheophorbidesPheophorbide a methyl ester (7) from Spirulina maxima(IApproximately 1 kg of dried Spirulina maxima alga was slurried in 3 L acetone ina 5 L three-neck round-bottom flask and liquid nitrogen was added to rupture the cells.After 1 h the frozen slush was refluxed under nitrogen with continuous stirring for 2 h.The supernatant was then filtered through Whatman filter paper on a Buchner funnel andwas washed with more acetone (—2 L) (even though the solid remains dark blue, the yieldof pigment obtained from further extraction was low). The green filtrate was evaporatedand the viscous oil so obtained was dissolved in 1.5 L petroleum ether (b.p. 35-60°C).The petroleum ether layer was successively washed with water (3 times), to remove theresidual supernatant and water solubles, and 30% aqueous methanol until the aqueousphase was colorless. Petroleum ether was evaporated and the dark green residue wasfurther dissolved in diethyl ether and treated with cone. HC1 (—80 mL) for 1 mm (colorchanged from green to black), immediately washed with water (2 x 800 mL) and 20%aqueous methanol (3 x 800 mL). The organic layer was dried over anhydrous sodiumsulphate, filtered and evaporated in vacuo. The extract was purified by flash204chromatography on neutral alumina (Brockmann Grade Ill), first eluting with methanol toremove most of the yellow-red band (carotenoids), further eluting with dichioromethaneto remove the black pheophytin a band. Flash chromatography was repeated, first elutingwith hexane (or petraleum ether) to remove the residual yellow band, and further elutingwith 30% dichioromethane in tetrachioromethane to remove the pheophytin a. Removalof solvent, followed by recrystallization from dichloromethane/methanol, gavepheophytin a (6.5 g) as a black solid. The pheophytin a was treated with 5% sulphuricacid in methanol (vlv) (900 mL) (degassed by bubbling with nitrogen) for 13 h at roomtemperature in the dark, followed by dilution with dichloromethane, successively washedtwice with water, once with saturated sodium bicarbonate and finally three times withwater. The organic layer was dried over anhydrous sodium sulfate, filtered andevaporated in vacuo. Recrystallization of the product from dichloromethane/methanolgave the title compound (4.2 g) as blue prisms.M.p.: 24 1°C (lit.41 228°C, lit.36 206°C)1H NMR (400 MHz, 1.5 mg/0.6 mL CDC13) (the major epimer) 6: 9.58 (s, 1H, H-b),9.40 (s, 1H, H-5), 8.59 (s, 1H, H-20), 8.01 (dd, 1H, H-31 J= 17.1 and 11.6 Hz), 6.38 [d,1H, H-32(E), J= 17.1 Hz], 6.22 [d, 1H, H-32(Z), J= 11.6 Hz)], 6.24 (s, 1H, H-132), 4.52(dq, 1H, H-18, J= 9.3 and 1.7 Hz), 4.22 (ddd, 1H, H-17, J= 9.3, 3.1 and 1.7 Hz), 3.90 (s,3H, H-134), 3.64 (q, 2H, H-8’, J= 7.8 Hz), 3.62 (s, 3H, H-121), 3.59 (s, 3H, H-174), 3.40(s, 3H, H-21), 3.21 (s, 3H, H-71), 2.60 (dddd, 1H, Ha-171J= 13,3, 7.1, 6.2 and 3.1 Hz),2.58 (dddd, 1H, Ha’171J = 13,3, 9.3, 9.3 and 5.3 Hz), 2.20 (ddd, 1H, Hb-17’, J = 15.1,2059.3 and 7.1 Hz), 2.18 (ddd, 1H, Hb’-17’,J= 15.1, 6.2 and 5.3 Hz), 1.82 (d, 3H, H-181,J= 7.1 Hz), 1.70 (t, 3H, H-82 J= 7.8 Hz), 0.39 (hr s, 1H, NH), —1.60 (hr s, 1H, NH)l3 NMR (75 MHz, 25 mg/i.0 mL CDC13)& 189.63 (C-131), 173.36 (C-173), 172.14(C-133), 169.60 (C-19), 161.16 (C-16), 155.50 (C-6), 150.85 (C-9), 149.61 (C-14),145.05 (C-8), 141.99 (C-i), 137.85 (C-li), 136.39 (C-3), 136.12 (C-4), 136.02 (C-7),131.76 (C-2), 128.87 (C-i2), 128.94 (C-13), 128.94 (C-31), 122.66 (C-32), 105.13 (C-15), 104.28 (C-b), 97.39 (C-5), 93.07 (C-20), 62.71 (C-132), 52.88 (C-17), 51.70 (C-i8),51.10 (C-134), 50.10 (C-174), 31.06 (C-172), 29.86 (C-171), 23.10 (C-181), 19.29 (C-81),17.36 (C-82), 12.09 (C-121), 12.06 (C-21), 11.08 (C-71)UV/Vis max (CHC13)412 nm (E 104 800), 506(15 800), 536(14 000), 610(13 100), 668(45 300) [lit.41 max (CH2C1)412 nm (E 106 000), 506 (10 800), 538 (9 710), 610 (8620), 668 (44 600)]Pyropheophorbide a methyl ester (25)(IPheophorbide a methyl ester (7) (930 mg, 1.53 mmol) was dissolved in collidine(150 mL) and stirred at reflux for 1.5 h under nitrogen in the dark. Removal of solvent bydistillation at reduced pressure (2.0 mm Hg) gave a dark blue residue, which was206crystallized fromCH21/C3O to give the title compound (840 mg, 98%) as tiny blueneedles.M.p.: 233°C [lit.41 217-219°C]1H NMR (400 MHz, 1.5 mgIO.6 mL CDC13) & 9.50 (s, 1H, H-b), 9.39 (s, 1H, H-5),8.59 (s,1H, H-20), 8.00 (dd, 1H, H-3’, J= 17.4 and 11.4 Hz), 6.30 [dd, 1H, H-32(E), J=17.4 and 1.1 Hz], 6.18 [dd, 1H, H-32(Z), J 11.4 and 1.1 Hz], 5.29, 5.10 (ABq, 2H, H-132), 4.49 (dq, 1H, H-18, J = 7.3 and 2.2 Hz), 4.27 (ddd, H-17, J = 7.9, 3.0 and 2.2 Hz),3.68 (q, 1H, H-81 J 7.6 Hz), 3.66 (s, 3H, H-121), 3.60 (s, 3H, H-174), 3.39 (s, 3H, H2k), 3.22 (s, 3H, H-71), 2.68 (m, 1H, Ha17’, J = 7.9 Hz), 2.55 (m, 1H, H b’-172), 2.30(m, 1H, Ha’47’, J= 3.0 Hz), 2.28 (m, 1H, Hb-172), 1.79 (d, 1H, H-181J= 7.3 Hz), 1.68(t, 3H, H-82 J= 7.6 Hz), 0.45 (br s, 1H, NH), —1.70 (br s, 1H, NH)UV/Vis 2max (CHC13)418 nm (e 147 700), 510 (13 500), 538 (9 800), 608 (11 200), 668(53 500) [lit.41 max (CH2C1),410 (E 113 000), 508 (11 500), 538 (9 800), 610 (8 500),668 (47 100)132,17-Cyclopheophorbide a enol (81)To a solution of pyropheophorbide a methyl ester (25) (546 mg, 1 mmol) in dryTHF (60 mL) under an atmosphere of nitrogen was added, NaN[Si(CH3)12(7.0 mL, 7.0207mmol, 1.0 M in THF). The resultant yellow solution was stirred at room temperature for3 mm then poured into a deoxygenated (N2) mixture of dichloromethane (800 mL),saturated NaH2PO4(200 mL) and ice (200 g). The mixture was strongly shaken until theyellow color turned to bright-green. After separation of the aqueous phase, the organiclayer was dried over sodium sulphate, filtered and evaporated in vacuo. The residue waspurified by chromatography on silica V, eluting with dichloromethane (2 L). The productwas crystallized from methylene chloride/hexane under nitrogen, giving the title product(438 mg, 85%) as lustrous dark green needles.M.p.: > 300°C [lit.6 >300°C, lit.87 >360°C]1H NMR (400 MHz, 1.5 mg/0.6 mL CDC13) 6: 13.24 (s, 1H, OH), 8.64 (s, 1H, H-b),8.43 (s, 1H, H-5), 7.38 (s, 1H, H-20), 7.70 (dd, 1H, H-31 J= 18.0 and 11.6 Hz), 6.12 [dd,1H, H-32(E), J= 18.0 and 1.6 Hz], 6.04 [dd, 1H, H-32(Z), J= 11.6 and 1.6 Hz], 3.31 (q,2H, H-81 J= 7.9 Hz), 3.08 (s, 3H, H-121), 3.02 (s, 3H, H-21), 2.94 (s, 3H, H-71), 2.93 (q,1H, H-18, J= 7.2 Hz), 2.58 (m, 1H, H-17), 2.45 (t, 2H, H-172), 1.71 (m, 2H, H-171), 1.80(d, 3H, H-18’, J= 7.1 Hz), 1.52 (t, 3H, H-82 J= 7.9 Hz), 0.30 (br s, 1H, NH), —1.72 (brs, 1H, NH)13C NMR (75 MHz, 18.0 mg/0.6 mL CDC13)6: 191.78 (C-131), 169.63 (C-19), 167.35(C-173), 157.77 (C-16), 154.65 (C-6), 150.04 (C-9), 144.15 (C-14), 143.21 (C-8), 141.27(C-i), 136.35 (C-il), 135.95 (C-3), 135.04 (C-4), 134.99 (C-7), 130.80 (C-2), 128.93 (C12), 127.78 (C-31), 127.52 (C-13), 121.80 (C-32), 116.83 (C-132), 104.10 (C-iS), 104.02(C-b), 96.74 (C-5), 91.02 (C-20), 52.47 (C-17), 49.33 (C-18), 34.00 (C-172), 25.03 (C-20817k), 19.06 (C-18’), 17.24 (C-81), 16.69 (C-82), 11.69 (C-121), 11.58 (C-21), 10.90 (C71)UVIVis max (CHC13)364 nm (e 67 200), 430 (66 000), 456 (47 200), 592 (5 200), 630(4 800), 690 (33 400) [lit.6 max(CH2C1)361 nm (E 65 500), 429 (64 000), 455 (44400), 629 (9 000), 688 (33 000); lit.87 max 359 nm (E 63 000), 426 (63 000), 452 (50000), 626 (10 000), 686 (32 000)]EREIMS (mlz): 516 (M, 100%), 501 (M—CH3,28)HREIMS: C33H2N402(Mj: calcd 516.2525obsd 516.2534Anal. calcd forC33H2N402: C, 76.72; H, 6.24; N, 10.84 %found: C, 76.91; H, 6.19; N, 10.37 %6.2.2 Asymmetric Hydroxylation13S-Hydroxychlorophyllone a [82(S)](‘I82(S)A cold (—25°C) solution of 132,17-cyclopheophorbide a enol (81) (103 mg, 0.2mmol) in dry THF (60 mL) was blanketed with N2 and stirred vigorously while DBU (1.0mL) was injected dropwise via a syringe. The mixture was kept at this temperature for 15209mm while a solution of (1R)-(—)-(10-camphorsulfonyl)oxaziridine (50 mg, 0.22 mmol) incold (—25°C), dry THF (12 mL) was transferred into the reaction vessel via a cannula.This mixture was stirred at —25°C for 12 h and quenched with saturated NH4C1. Theaqueous phase was extracted with dichloromethane (2 x 100 mL) and the combinedorganic phases were dried over sodium sulphate, filtered and evaporated in vacuo. Theresidue was purified by flash chromatography on silica V, eluting with dichloromethane.The product was recrystallized from methanol, giving 100 mg (94%) of a blue powder,which was analyzed by reversed-phase HPLC as a diastereomeric mixture of 95% 132S-hydroxychlorophyllone a [82(S)] and 5%132R-hydroxychlorophyllone a [82(R)].M.p.: >300°CAnal. calcd forC33H2N40: C, 74.41; H, 6.06; N, 10.52%found: C, 74.65; H, 6.10; N, 10.19%After HPLC separation (12 mg) using a Waters C18 lOj.i 125A (7.8 mm x 30 cm)column with a flow rate of 3 mL min1 and detection at 410 nm [the mobile phase: 75%(0.1% TFA in CH3N)/25% (0.1% TFA in water)],132S-hydroxychlorophyllone a[82(S)] (10 mg) was obtained. After treatment with methanol, the optically-pure titlecompound (9.1 mg) was collected as a dark green solid.M.p.: >300°C1 NMR (400 MHz, 1.5 mg/0.6 mL CDC13) ö: 9.40 (s, 1H, H-b), 9.35 (s, ill, H-5),8.70 (s, 1H, H-20), 7.96 (dd, 1H, H-31 J= 18.1 and 11.4 Hz), 6.28 [dd, 1H, H-32(E), J=18.1 and 1.2 Hz], 6.18 [dd, lH, H-32(Z), J = 11.4 and 1.2 Hz], 4.90 (ddd, 1H, H-17, J =13.3, 3.8 and 3.5 Hz), 4.56 (br s, 1H, OH), 4.33 (dq, 1H, H-18, J= 7.4 and 3.8 Hz), 4.31210(ddd, 1H, Hb’-172J= 14.0, 11.7 and 3.5 Hz), 3.60 (s, 3H, H-12’), 3.59 (q, 2H, H-81 J=8.1 Hz), 3.39 (s, 3H, H-21), 3.20 (s, 3H, H71), 2.88 (dddd, 1H, Ha’47’, J 12.4,4.4, 3.5and 3.5 Hz), 2.78 (ddd, 1H, Hb-172J= 14.0, 11.7 and 2.1 Hz), 2.23 (dddd, 1H, Ha47’, J= 14.0, 13.3, 12.4 and 2.1 Hz), 2.19 (d, 3H, H-181J = 7.4 Hz), 1.64 (t, 3H, H-82 J = 8.1Hz), 0.41 (br s, 1H, NH), —2.05 (hr s, 1H, NH)l3 NMR (125 MHz, 7.0 mg/0.6 ml CDCI3)& 208.00 (C-173), 195.44 (C-13’), 172.68(C-19), 163.20 (C-16), 154.53 (C-6), 150.92 (C-9), 147.72 (C-14), 144.88 (C-8), 142.16(C-i), 138.19 (C-il), 136.38 (C-3), 136.31 (C-4), 135.75 (C-7), 131.58 (C-2), 129.13 (C-12), 129.08 (C-31), 127.77 (C-13), 122.85 (C-32), 105.36 (C-15), 104.06 (C-b), 98.10(C-5) , 93.43 (C-132), 92.87 (C-20), 51.92 (C-17), 51.51 (C-18), 40.12 (C-17), 37.99 (Ci7), 22.37 (C-i81), 19.24 (C-81), 17.29 (C-82), 12.20 (C-121), 12.08 (C-21), 11.14 (C7)UV-Vis max (CHC13)416 nm ( 111 000), 506 (12 500), 536 (10 200), 612 (9 200), 670(50 900); max (CH3OH) 408 nm (E 103 000), 504 (11 600), 534 (9 000), 608 (8 300),666 (47 600) [1it.899l max(CH30IT) 408 nm, 503, 534, 608, 665]LRFABMS (mlz): 533 ([MH], 100%)HRFABMS: C33HN40([MH]): calcd 533.2552obsd 533.2540=r‘() 1-H‘HI‘P13169‘(zH111P9L1=I‘jE-H‘HI‘PP)96L‘(oz-H‘HI‘s)‘(c-H‘HI‘s)‘(01-H‘HI‘s)LV6:(iDUD‘1”90Im01‘ZHJA0017)IISINHjaoo<:djpqosuJppsipifoSM(gui)punodmoDona1nd-I(I113idoqi‘ou1qwqiiiiuwpanij’punqosit(wc17)[(i)z1‘(JuMuvu.%ro)%clI(NDHDuV[I%1o)%L:siqdTqowqiJmu0117uoTppu1_uuijuijoiiOfjtqlTMuiunio(uio0xm’WL)yczid0181DSJ1Miusn(gui6)uonirndsXIJH1JV%W0T‘N17F9‘HEI17L‘:punoj%ZOJ‘N909‘H11717L‘D:OI7NZEHEEDjojpio0<rd,sjv%pui[(>J)zx]v-)JJ%9Joainxtwuoisinpiqol3npoJdpoqsstsApuiD]JHsiqd-psJAaiyou1qiuJqliMuon1.in1u1ioij‘m6c17)pqosnqipu1AO1?pquspsipijundsiionpodzqQowmg10‘uu)uipuizixo(ii(uojjns-Joqduw3-oJ)-(+)-(J)q’(10mm10‘uiç)(ix)iouvpiqJoqdoqdopA-LJ‘jjouoioiaitCqpi(oiduisi’(g)giojs1atnpoiduomAxoipAqurnsqj0OH/—,NHN\—NHN\[(i)zx]vITi21217.6 and 1.0 Hz], 6.18 [dd, 1H, H-32(Z), J= 11.2 and 1.0Hz], 4.75 (dq, 1H, H-18, J= 8.3and 7.0 Hz), 4.14 (br s, 1H, OH), 3.83 (ddd, 1H, Hb-172 J = 15.0, 6.2 and 1.5 Hz), 3.82(ddd, 1H, H-17, J = 11.0, 8.3 and 1.6 Hz), 3.71 (dddd, 1H, Ha471 J 13.1, 12.8, 11.0and 6.2 Hz), 3.68 (s, 3H, H-12’), 3.68 (q, 2H, H-81 J = 7.2 Hz), 3.35(s, 3H, H-21), 3.20(s, 3H, H-71), 2.95 (ddd, 1H, Hb’-172 J = 15.0, 12.8 and 5.2 Hz), 2.65 (dddd, 1H, Ha’171, J= 13.1, 5.2, 1.6 and 1.5 Hz), 2.20 (d, 3H, H-181 J= 7.0 Hz), 1.68 (t, 3H, H-82 J=7.2 Hz), 0.90 (br s, 1H, NH), —1.56 (br s, 1H, NH)13C NMR (75 MHz, 4.0 mgIO.6 mL CDC13) ö: 206.21 (C-173), 193.39 (C-131), 172.36(C-19), 162.82 (C-16), 154.68 (C-6), 150.80 (C-9), 149.43 (C-14), 144.95 (C-8), 142.44(C-i), 138.18 (C-il), 136.32 (C-3), 136.26 (C-4), 135.89 (C-7), 131.60 (C-2), 129.55 (C12), 128.93 (C-31), 127.11 (C-13), 122.83 (C-32), 105.73 (C-15), 104.73 (C-b), 98.29(C-5), 92.66 (C-132), 91.73 (C-20), 53.71 (C-17), 50.31 (C-18), 43.17 (C-172), 22.71 (C17k), 19.36 (C-81), 17.39 (C-82), 16.99 (C-181), 12.25 (C-121), 12.01 (C-21), 11.16 (C7)UV-Vis max (CHC13)416 nm (E 113 000), 508 (15 500), 538 (11 100), 616 (10 000),674 (48 300); max (CH3OH) 408 nm (E 119 000), 504 (13 500), 534 (11 000), 612 (10500), 670 (50 000) [lit.89,91max (CH3OH) 408 nm, 505, 535, 612, 670]LREIMS (mlz): 532 (M, 100%), 501 (M—OCH3,21)HREIMS: C33H2N40([M]): calcd 532.2474obsd 532.2474213Chiorin P6 trimethyl ester (92)EH2Me92CO2MeMethodA A cold (—78°C) solution of132,173-cyclopheophorbide a enol (81)(30 mg, 58 imol) in dry THF (15 mL) was blanketed with N2 and stirred vigorouslywhile a similarly cold solution of sodium hexamethyldisilazide in dry THF (0.18 mL of1.0 M, 0.18 mmol) was introduced dropwise via a syringe. The mixture was kept at thistemperature for 15 mm while a solution of 1 -phenyl-N-(phenylsulfonyl)oxaziridine”(20.8 mg, 79 pmol) in cold (—78°C), dry THF (1 mL) was transferred into the reactionvessel via a cannula. This mixture was stirred at —78°C for 30 mm before it wasquenched with saturated NH4C1. The aqueous phase was extracted withdilchloromethane (3 x 30 mL) and the combined organic phases were dried over sodiumsulphate and evaporated in vacuo. TLC analysis showed that the product did not moveon a TLC plate even with development by 5% methanol in dichloromethane. The residuewas dissolved in THF and acidified with 1M HC1. The aqueous layer was re-extractedwith dichloromethane before the organic layer was treated with an excess of etherealdiazomethane. The evaporated residue was purified by chromatography on silica gel,eluting with dichioromethane (100 mL). The product was crystallized fromdichloromethane/methanol, giving the title compound (8.8 mg, 24%) as small dark greenneedles.214Method B A solution of the foregoing (95%S, 5%R) mixture of 132Whydroxychiorophyllone a (82) (15 mg, 0.0282 mmol) in dry THF (15 mL) was blanketedwith N2 and stirred vigorously while KOH (0.5 g) in CH3O (5 mL) was added. Thismixture was stirred at room temperature in the dark for 10 h before the mixture wasacidified to pH 3 by 2N HCI and extracted with dichioromethane. The organic layer waswashed with water 3 times before being subjected to an excess of ethereal CH2N. Thematerial was purified as described in (a) to give chiorin P6 trimethyl ester (92) (8.0 mg,46%).M.p.: 237°C [lit.185 235-236°C, lit.186 236°C]1H NMR (400 MHz, 1.5 mg/0.6 ml CDC13)& 9.70 (s, 1H, H-b), 9.49 (s, 1H, H-5),8.77 (s, 1H, H-20), 8.00 (dd, 1H, H-31 J = 16.8, 12.0 Hz), 6.31 [dd, 1H, H-32(E), J =16.8 and 1.2 Hz], 6.15 [dd, 1H, H-32(Z), J = 12.0 and 1.2 Hz], 5.15 (dd, 1H, H-17, J =9.2 and 2.8 Hz), 4.38 (q, 1H, H-18, J = 7.6 Hz), 4.22 (s, 3H, H-152), 4.14 (s, 3H, H-132),3.72 (q, 2H, H-81 J 7.7 Hz), 3.63 (s, 3H, H-174), 3.52 (s, 3H, H-121), 3.40 (s, 3H, H-21), 3.23 (s, 3H, H-71), 2.38 (m, 1H, Hb’-172), 2.20 (m, 1H, Ha’171), 2.05 (m, 1H, Hb172), 1.87 (m, 1H, Ha47’), 1.84 (d, 3H, H-181 J = 7.6 Hz), 1.69 (t, 3H, H-82 J = 7.7Hz), —0.82 (br s, 1H, NH), —1.00 (br s, 1H, NH)13C NMR (75 MHz, 7.5 mg/0.6 ml CDC13)6: 173.54 (C-173), 172.84 (C-19), 170.72 (C151), 167.23 (C-131), 167.02 (C-16), 154.93 (C-6), 148.89 (C-9), 145.24 (C-14), 141.19(C-8), 137.75 (C-i), 135.98 (C-il), 135.80 (C-3), 135.71 (C-4), 135.46 (C-7), 130.87 (C2), 129.53 (C-12), 129.06 (C-31), 122.44 (C-13), 122.34 (C-32), 104.64 (C-iS), 103.08(C-b), 100.33 (C-5), 93.60 (C-20), 52.67 (C-i7), 52.56 (C-152), 52.14 (C-132), 51.48215(C-174), 49.39 (C-18), 31.43 (C-172), 31.25 (C-171), 23.56 (C-181), 19.56 (C-81), 17.64(C-82), 12.54 (C-121), 12.04 (C-2’), 11.20 (C-71)UV-Vis max (CHC13)404 nm (E 158 500), 500 (12 000), 532 (7 300), 616 (6 200), 672(44 900) [lit.185 max (CH2C1)402 nm (E 137 000), 498 (9 900), 532 (5 500), 614 (4900), 668 (40 900)]LRFABMS (mlz): 625 ([MH], 100%)HRFABMS: C36H41N06([M+H]j: calcd 625.3026obsd 625.3043Anal. calcd forC36H40N06:: C, 69.21; H, 6.45; N, 8.97 %found: C, 68.75; H, 6.38; N, 8.80 %132R-Hydroxypheophorbide a Methyl Ester [95(R)](I95(R)Meo2CM O2 OH0The same hydroxylation procedure as for 82(S) was employed by reaction ofpheophorbide a methyl ester (7) (61 mg, 0.1 mmol) with (1R)-(—)-(10-camphorsulfonyl)-oxaziridine (27 mg, 0.118 mmol). The product was purified as described forhydroxychlorin 82(S) and, after treatment with methanol, gave a blue powder (57 mg,92%) which was analyzed by reversed-phase HPLC as a diastereomeric mixture of 95%13R-hydroxypheophorbide a methyl ester [95(R)] and 5%132S-hydroxypheophorbide amethyl ester [95(S)].216M.p.: >300°CAnal. calcd forC36H8N406: C, 69.44; H, 6.15; N, 9.00 %found: C, 69.10; H, 6.16; N, 8.70 %After HPLC separation (10 mg) using a Waters C18 lOp 125A (7.8 mm x 30 cm)column with a flow rate of 3 mL min and detection at 410 nm [the mobile phase: 85%(0.1% TFA in CH3N)/15% (0.1% TFA in water)], 132R-hydroxypheophorbide amethyl ester 95(R) (7.5 mg) was obtained. After treatment with methanol, the optically-pure title compound (7.0 mg) was collected as shiny blue plates.M.p.: >300°C1H NMR (400 MHz, 1.0 mg/0.6 mL CDC13) 6: 9.53 (s, 1H, H-b), 9.47 (s, 1H, H-5),8.61 (s, 1H, H-20), 7.96 (dd, 1H, H-31 J= 18.2 and 11.9 Hz), 6.29 [dd, 1H, H-32(E), J=18.2 and 1.0 Hz], 6.17 [dd, 1H, H—32(Z), J= 11.9 and 1.0 Hz], 5.32 (s, 1H, OH), 4.69 (dd,1H, H-17, J= 8.5 and 1.7 Hz), 4.49 (q, 1H, H-18, J= 7.0 Hz), 3.70 (q, 2H, H-81 J= 7.7Hz), 3.69 (s, 3H, H-134), 3.66 (s, 3H, H-121), 3.56 (s, 3H, H-174), 3.39 (s, 3H, H-21),3.18 (s, 3H, H-71), 2.46 (m, 1H, Ha’47’, J = 8.5 Hz), 2.29 (m, 1H, Hb’-172), 2.13 (m,1H, Ha47’, J = 1.7 Hz), 2.09 (m, 1H, Hb-172), 1.68 (d, 3H, H-181 J = 7.0 Hz), 1.65 (t,3H, H-82 J = 7.7 Hz), 0.39 (br s, 1H, NH), —1.74 (br s, 1H, NH)13C NMR (75 MHz, 7.0 mg/0.6 mL CDC13) 6: 191.93 (C-131), 173.46 (C-173), 173.42(C-iS2), 172.71 (C-19), 161.80 (C-16), 155.46 (C-6), 150.88 (C-9), 150.19 (C-14),145.15 (C-8), 142.09 (C-i), 137.68 (C-il), 136.40 (C-3), 136.35 (C-4), 136.24 (C-7),131.87 (C-2), 129.56 (C-12), 128.96 (C-31), 126.22 (C-i3), 122.31 (C-32), 107.58 (C15), 104.15 (C-b), 97.79 (C-5), 93.40 (C-20), 89.09 (C-132), 53.77 (C-134), 51.33 (C-21717), 50.75 (C-17), 50.16 (C-18), 30.99 (C-172), 30.18 (C-171), 22.69 (C-181), 19.35 (C81), 17.41 (C-82), 12.27 (C-121), 12.08 (C-21), 11.15 (C-71)UV-Vis ?max (CHC13)416 nm (E 136 600), 506 (15 500), 538 (11 600), 560 (4 200), 612(11 500), 670 (62 100)LRFABMS (mlz): 623 ([MH], 100%)HRFABMS: C36H9N406([MH]j: calcd 623.2869obsd 623.2874132S-Hydroxypheophorbide a Methyl Ester [95(S)](IN HN..i95(S))HOO2M02Me0The same hydroxylation procedure as for 95(R) was employed by reaction ofpheophorbide a methyl ester (7) (61 mg, 0.1 mmol) with (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (27 mg, 0.118 mmol). The product was purified as above and, after treatmentwith methanol, gave a blue solid (51 mg, 82%) which was analyzed by reversed-phaseHPLC as a diastereomeric mixture of 58%13S-hydroxypheophorbide a methyl ester[95(S)] and 42%13R-hydroxypheophorbide a methyl ester [95(R)].M.p.: >300°CAfter HPLC separation (10 mg) using a Waters C18 lOp 125A (7.8 mm x 30 cm)column with a flow rate of 3 mL min1 and detection at 410 nm [the mobile phase: 85%218(0.1% TFA in CH3N)/15% (0.1% TFA in water)], 132S-hydroxy pheophorbide amethyl ester [95(S)] (4.1 mg) was separated and was treated with methanol, giving theoptically-pure title product (3.9 mg) as a dark blue solid.M.p.: >300°C1H NMR (400 MHz, 1.5 mgIO.6 mL CDC13) & 9.62 (s, 1H, H-b), 9.48 (s, 1H, H-5),8.63 (s, 1H, H-20), 8.01 (dd, 1H, H-31 J= 18.3 and 11.7 Hz), 6.30 [dd, 1H, H-32(E), J=18.3 and 0.8 Hz], 6.20 [dd, 1H, H-32(Z), J = 11.7 and 0.8 Hz], 5.43 (s, 1H, OH), 4.49 (q,1H, H-18, J= 7.3 Hz), 4.15 (dd, 1H, H-17, J= 2.2 and 10.2 Hz), 3.72(s, 3H, H-134), 3.69(q, 2H, H-8’, J= 7.6 Hz), 3.64 (s, 3H, H-121), 3.59(s, 3H, H-174), 3.41(s, 3H, H-21), 3.23(s, 3H, H-71), 2.92 (m, 1H, Ha17’, J= 2.2 Hz), 2.55 (m, 1H, Hb-172), 2.28 (m, 1H, Ha’171, J 10.2 Hz), 2.26 (m, 1H, Hb’-172), 1.68 (t, 3H, H-82 J = 7.6 Hz), 1.58 (d, 3H, H181, J= 7.3 Hz),0.31 (br s, 1H, NH), —1.83 (br s, 1H, NH)l3 NMR (75 MHz, 3.5 mg/0.6 mL CDC13)& 192.00 (C-13’), 173.96 (C-19), 172.83(C-173), 172.37 (C-iS1), 162.37 (C-16), 155.35 (C-6), 151.03 (C-9), 149.84 (C-14),145.22 (C-8), 142.03 (C-i), 137.80 (C-il), 136.52 (C-3), 136.29 (C-4), 136.21 (C-7),131.76 (C-2), 129.41 (C-12), 129.04 (C-31), 122.89 (C-13), 122.88 (C-32), 107.59 (C15), 104.26 (C-b), 97.97 (C-5), 93.62 (C-20), 88.94 (C-132), 53.44 (C-13), 51.78 (Cb7), 51.75 (C-17), 50.29 (C-18), 31.40 (C-172), 31.11 (C-17), 22.65 (C-181), 19.47 (C81), 17.45 (C-82), 12.30(C-12), 12.11 (C-2), 11.26 (C-71)UV-Vis“max (CHC13)414 nm (E 141 200), 506 (9 800), 536 (15 800), 612 (ii 300), 670(63 500)LREIMS (mlz): 622 (M, 56%), 563 (M—COOCH,100)219HREIMS: C36H8N406(Mj: calcd 622.2791obsd 622.2802132R-Hydroxypheophytin a [96(R)]A cold (—25°C) solution of pheophytin a (5) (50 mg, 0.0575 mmol) in dry THF(30 mL) was blanketed with N2 and was stirred vigorously while DBU (0.3 mL) wasinjected dropwise via a syringe. The mixture was kept at this temperature for 15 mmwhile a solution of (1R)-(—)-(10-camphorsulfonyl)oxaziridine (15.8 mg, 0.069 mmol) incold (—25°C), dry THF (6 mL) was transferred into the reaction vessel via a cannula.This mixture was stirred at —25°C for 12 hours before the reaction was quenched withsaturated NH4CI. The aqueous phase was extracted with CH21 and the resultingorganic layer was washed with water, dried, filtered and evaporated in vacuo. Theresidue was purified by flash chromatography on silica V. eluting with CH21. Theproduct was recrystallized from MeOH, giving 46.2 mg (91% yield) of a black solid,which was analyzed by 1H NMR to be an 100% d.e. of132R-hydroxypheophytin a[96(R)].M.p.: 199-200°C96(R)220114 NMR (400 MHz, 1.5 mg/0.6 mL CDC13) ö: 9.70 (s, 1H, H-b), 9.45 (s, 1H, H-5),8.60 (s, 1H, H-20), 8.00 (dd, 1H, H-31 J= 17.6 and 11.2 Hz), 6.31 [dd, 1H, H-32(E), J=17.6 and 1.0Hz], 6.21 [dd, 1H, H-32(Z), J= 11.2 and 1.0Hz], 5.32 (s, 1H, OH-132), 5.15(t, 1H, H-P2. J = 8.3 Hz), 4.68 (d, 1H, H-17, J = 9.6 Hz), 4.47 (dq, 1H, H-18), 4.47 (d,2H, H-P1 J= 8.0 Hz), 3.75 (q, 2H, H-81 J— 7.1 Hz), 3.74 (s, 3H, H-121), 3.61 (s, 3H, H13), 3.41 (s, 3H, H-21), 3.25 (s, 3H, H-71), 2.42 (m, 1H, H-171), 2.24 (m, 1H, H-172),2.09 (m, 1H, H-171), 1.99 (m, 1H, H-172), 1.86 (t, H-P4), 1.68 (t, 3H, H-82 J = 7.1 Hz),1.65 (d, 3H, H-181), 1.55 (s, 3H, H-P17), 1.52 (s, 3H, H-P15), 1.19 [m, 16H, H-P(CH2)8],0.82 (m, 8H, H-P20.H-P16,H-P7, and H-P11), 0.39 (hr s, 1H, NH), —1.73 (hr s, 1H, NH)l3 NMR (125 MHz, 14 mg/i .0 mL CDC13)& 191.96 (C-131), 173.45 (C-173), 173.00(C-133), 172.78 (C-19), 161.90 (C-16), 155.54 (C-6), 150.96 (C-9), 150.23 (C-14),145.25 (C-8), 142.86 (C-P3), 142.10 (C-i), 137.74 (C-li), 136.49 (C-3), 136.41 (C-4),136.32 (C-7), 131.91 (C-2), 129.62 (C-12), 129.06 (C-31), 126.29 (C-13), 122.87 (C-32),117.73 (C-P2), 107.65 (C-iS), 104.23 (C-b), 97.86 (C-5), 93.43 (C-20), 89.07 (C-i32),53.78 (C-134), 50.76 (C-17), 50.17 (C-18), 39.78 (C-P4), 39.33 (C-P14), 37.36 (C-P8),37.28 (C-P10), 37.23 (C-P12), 36.61 (C-P6), 32.74 (C-P11), 32.59 (C-P7), 31.20 (C-172),30.19 (C-i71), 27.94 (C-P15), 24.97 (C-P5), 24.75 (C-P13), 24.39 (C-P9), 22.68 (C-P16and C-P20), 22.60 (C-181), 19.70 (C-P18), 19.61 (C-P19), 19.46 (C-81), 17.44 (C-82),16.25 (C-P17), 12.29 (C-i21), 12.09 (C-21), 11.25 (C-71)UV/Vis max (CHC13)416 nm (E 101 000), 506 (ii 800), 536 (9 500), 612 (9 000), 670(45 600)LREIMS (mlz): 886 (M, 4 1%), 607 (M— Phytyl, 62%)221HREIMS: C55H74N406(M): calcd 886.5608obsd 886.5616132S-Hydroxypheophytin a [96(S)]0(A cold (—25°C) solution of pheophytin a (5) (60 mg, 0.069 mmol) in dry THF (35mL) was blanketed with N2 and was stirred vigorously while DBU (0.35 mL) wasinjected dropwise via a syringe. The mixture was kept at this temperature for 15 mmwhile a solution of (1S)-(+)-(10-camphorsulfonyl)oxaziridine (19.0 mg, 0.083 mmol) incold (—25°C), dry THF (6 mL) was transferred into the reaction vessel via a cannula.This mixture was stirred at —25°C for 12 hours before the reaction was quenched withsaturated NH4C1. The aqueous phase was extracted with CH2I and the resultingorganic layer was washed with water, dried, filtered and evaporated in vacuo. Theresidue was purified by flash chromatography on silica V. eluting with CH21. Theproduct was recrystallized from MeOH, giving 52.9 mg (87% yield) of a black solid,which was analyzed by 1H NMR to be a diastereomeric mixture of 66% 132R-hydroxypheophytin a [96(R)] and 34%132S-hydroxypheophytin a [96(S)]. After normalphase HPLC separation (30 mg), using a Rainin silica lOp. 125 A (7.8 mm x 25 cm)column with a flow rate of 4 mL min1 and detection at 414 nm using a Waters 99496(S)222photodiode array detector (the mobile phase: hexane: isopropanol : ethyl acetate = 92: 26), a sample of 38% d.e. of132S-hydroxypheophytin a [96(S)] was obtained. Furtherpurification by HPLC (normal-phase) and preparative TLC was largely unsuccessful dueto decomposition of the material.M.p.: 187°C1H NMR (400 MHz, 2.0 mgIO.6 mL CDC13) [96(S)] & 9.73 (s, 1H, H-b), 9.49 (s, 1H,H-5), 8.65 (s, 1H, H-20), 8.01 (dd, 1H, H-31 J= 17.9 and 11.9 Hz), 6.30 [d, 1H, H-32(E),J= 17.9 Hz], 6.21 [dd, 1H, H-32(Z), J= 11.9 Hz], 5.49 (s, 1H, OH-132), 5.20 (t, 1H, Hp2, J = 7.5 Hz), 4.55 (dq, 1H, H-18, J = 7.6 Hz), 4.47 (d, 2H, H-P1 J = 5.5 Hz), 4.15 (d,1H, H-17, J= 10.0 and 1.0Hz), 3.71 (s, 3H, H-121), 3.69 (q, 2H, H-81 J= 7.3 Hz), 3.60(s, 3H, H-134), 3.40 (s, 3H, H-21), 3.24 (s, 3H, H-71), 2.91 (m, 1H, H-171), 2.53 (m, 1H,H-172), 2.25 (m, 1H, H-171), 2.25 (m, 1H, H-172), 1.90 (t, H-P4), 1.71 (t, 3H, H-82 J =7.3 Hz), 1.67 (d, 3H, H-18’, J = 7.6 Hz), 1.59 (s, 3H, H-P17), 1.54 (s, 3H, H-P15), 1.21[m, 16H, H-P(CH2)81,0.84 (m, 8H, H-P20.H-P16,H-P7 and H-P11), 0.29 (br s, 1H, NH),—1.83 (br s, 1H, NH)13C NMR (125 MHz, 18 mg/1.0 mL CDC13)[96(S)] & 192.01 (C-13’), 173.58 (C-173),172.99 (C-133), 172.39 (C-19), 162.45 (C-16), 155.33 (C-6), 151.03 (C-9), 149.82 (C14), 145.22 (C-8), 142.77 (C-P3), 142.01 (C-i), 137.80 (C-li), 136.52 (C-3), 136.27 (C4), 136.20 (C-7), 131.74 (C-2), 129.41 (C-12), 129.06 (C-31), 122.95 (C-13), 122.88 (C32), 117.84 (C-P2), 107.65 (C-iS), 104.25 (C-b), 97.97 (C-5), 93.64 (C-20), 88.95 (C132), 61.54 (C-P1), 53.40 (C-134), 51.80 (C-17), 50.31 (C-18), 39.82 (C-P4), 39.33 (CP14), 37.37 (C-P8), 37.30 (C-P10), 37.24 (C-P12), 36.64 (C-P6), 32.74 (C-P11), 32.61 (C-223P7), 31.56 (C-172), 31.12 (C-171), 27.95 (C-P15), 25.00 (C-P5), 24.76 (C-P13), 24.41 (CP9), 22.70 (C-P16 and C-P20), 22.61 (C-18), 19.71 (C-P18), 19.64 (C-P19), 19.49 (C-81),17.46 (C-82), 16.32 (C-P17), 12.31 (C-12), 12.09 (C-21), 11.25 (C-71)6.2.3 Model Studies for Hydroxylactonization15-Hydroxypurpurin-7-lactone dimethyl ester (101)MeO2COH0CO2Me CO2Me101(S) 101(R)84% : 16%A solution of the foregoing (95%R, 5%S) mixture of 32-hydroxypheophorbide amethyl ester (95) (30 mg, 0.05 mmol) in dioxane (20 mL) was stirred with an aqueoussolution (20 mL) of periodic acid dihydrate (900 mg, 3.95 mmol) at room temperature for20 h before the mixture was extracted with dichioromethane (2 x 40 mL). The organiclayer was dried over sodium sulphate, filtered and evaporated in vacuo. The residue waspurified by flash chromatography on silica III, eluting with 2% methanol indichioromethane. The product was crystallized from dichioromethane/hexane, giving ablack solid (25.2 mg, 79%), which was analyzed by 1H NMR as a diastereomeric mixtureof 84% 15S-hydroxypurpurin-7-lactone dimethyl ester [101(S)] and 16% 15R-hydroxypurpurin-7-lactone dimethyl ester [101(R)].M.p.: 217°C2241H NMR (400 MHz, 1.5 mg/0.6 mL CDC13) [101(S)] 6: 9.77 (s, 1H, H-b), 9.55 (s, 1H,H-5), 8.80 (s, 1H, H-20), 8.00 (dd, 1H, H-31 J = 17.6 and 11.5 Hz), 6.34 [dd, 1H, H32(E), J = 17.6 and 1.2 Hz], 6.19 [dd, 1H, H-32(Z), J = 11.5 and 1.2 Hz], 6.05 (s, 1H,OH), 4.43 (q, 1H, H-18, J = 6.8 Hz), 4.05 (dd, 1H, H-17, J = 10.4 and 2.4 Hz), 3.89(s,3H, H-153), 3.76 (s, 3H, H-121), 3.75 (q, 2H, H-81 J = 8.0 Hz), 3.51 (s, 3H, H-174), 3.40(s, 3H, H-21), 3.26 (s, 3H, H-7’), 2.46 (m, 1H, Ha17’, J= 2.4 Hz), 2.45 (m, 1H, Hb-172),2.18 (m, 1H, Ha’47’, J= 10.4 Hz), 1.80 (m, 1H, Hb’-172), 1.70 (t, 3H, H-82 J= 8.0 Hz),1.59 (d, 3H, H-181J= 6.8 Hz),—1.10 (br s, 1H, NH), —1.41 (br s, 1H, NH)UV-Vis max (CHC13)404 flffi (8 189 000), 502 (17 300), 530 (13 600), 562 (4 200), 614(9 300), 672 (61 200)LREIMS (mlz): 638 (M, 20%), 622 (M—O, 80)HREIMS: C36H8N407(Mj: calcd 638.2740obsd 638.2745Anal. calcd forC36H8N407: C, 67.70; H, 6.00; N, 8.77 %found: C, 68.00; H, 6.14; N, 8.95 %Purpurin-7 trimethyl ester (16)(‘I16¶ co2CO2Me225A solution of the foregoing (84%S, 16%R) mixture of15-hydroxypurpurin-7-lactone dimethyl ester (101) (15 mg, 0.05 mmol) in dichloromethane (20 mL) was treatedwith an excess of ethereal diazomethane and then washed with water 3 times before theorganic layer was dried over sodium sulphate, filtered and evaporated in vacuo. Theproduct was crystallized from dichloromethane/hexane, giving the title compound(14.8mg, 97%) of a purple solid.M.p.: 232°C [lit.186 227-230°C]1H NMR (400 MHz, 1.5 mg/0.6 mL CDC13) 6: 9.56 (s, 1H, H-b), 9.27 (s, 1H, H-5),8.47 (s, 1H, H-20), 7.87 (dd, 1H, H-31 J= 17.9 and 11.7 Hz), 6.28 [dd, 1H, H-32(E), J=17.9 and 1.1 Hz], 6.11 [dd, 1H, H-32(Z), J= 11.7 and 1.1 Hz], 4.66 (d, 1H, H-17, J= 7.6Hz), 4.29 (q, 1H, H-18, J= 7.2 Hz), 4.12 (s, 3H, H-iS2), 3.86 (s, 3H, H-132), 3.63 (q, 2H,H-81, J = 7.6 Hz), 3.58 (s, 3H, H-121), 3.51 (s, 3H, H-174), 3.31 (s, 3H, H-2’), 3.14 (s,3H, H-71), 2.35 (t, 1H, Hb-172), 2.08 (m, 2H, H-171), 1.77 (d, 3H, H-18’, J = 7.2 Hz),1.75 (t, 1H, Hb’-172), 1.64 (t, 3H, H-82 J = 7.6 Hz), —0.01 (br s, 1H, NH), —0.09 (br s,1H,NH)UV-Vis2’max (CHC13)410 nm (e 99 500), 506 (7 500), 548 (10 000), 680 (25 300), 688(24 800) [lit.186 ?max 408 nm, 504, 544, 682 ( 23 900)]LREIMS (mlz): 652 (M, 11%), 565 (M—COCOOCH 100)HREIMS: C37H40N07(Mj: calcd 652.2897obsd 652.2898Anal. calcd forC37H40N07: C, 68.08; H, 6.18; N, 8.58 %found: C, 67.72; H, 5.92; N, 8.36 %22620-Chioropheophorbide a methyl ester (99)(‘ICI—CÔ2MeOCO2MeA mixture of pheophorbide a methyl ester (7) (20 mg, 0.033 mmol), metachloroperbenzoic acid (8.7 mg, 0.05 mmol), and CHC13 (15 mL) was magnetically stirredat room temperature in the dark for 12 hours. The mixture was diluted with CH21,washed once with water, once with 2N Na2SO3,twice with water, dried over Na2SO4,filtered and evaporated in vacuo. The residue was purified by flash chromatography onBrockman III, eluting with CH21, first to recycle 5.4 mg (27% yield) of the startingmaterial, pheophorbide a methyl ester (7), and further eluting to furnish a brwonish-redband. The main band was collected, evaporated and recrystalized fromCH21/MeOH togive the title compound (11.4 mg, 54% yield) as a brown-red solid.M.p.: 217°C1H NMR (400 MHz, CDC13)& 9.60 (s, 2H, H-b and H-5), 7.95 (dd, 1H, H-3’, J =18.8 and 11.8 Hz), 6.29 [dd, 1H, H-32(Z), J= 11.7 and 0.5 Hz], 6.23 (s, 1H, H-132), 6.15[s, 1H, H-32(E), J= 18.8 and 0.5 Hz], 4.78 (q, 1H, H-18, J= 8.0 Hz), 4.12 (dd, 1H, H-17,J = 8.1 and 1.0 Hz), 3.90 (s, 3H, H-21), 3.68 (s, 3H, H-174), 3.68 (q, 2H, H-81 J = 7.5Hz), 3.59 (s, 3H, H-71), 3.52 (s, 3H, H-134), 3.22 (s, 3H, H-121), 2.45 (m, 2H, H-171),2.17 (m, 2H, H-172), 1.69 (t, 3H, H-82 J = 7.5 Hz), 1.60 (d, 3H, H-181 J = 8.0 Hz), —1.87 (br s, 2H, NH)227UV/Vis ?max (CH2C1)416 nm (E 109 000), 518 (10 500), 550 (15 000), 616 (7 500),678 (45 000)LREIMS (m/z): 640 (M, 16%), 608 (M— OCH4, 12%),HREIMS: C36H7N4051(M): calcd 640.2452obsd 640.244920-Chioropyropheophorbide a methyl ester (100)(ICI—, 100CO2MeA mixture of pyropheophorbide a methyl ester (25) (20 mg, 0.036 mmol), mesochloroperbenzoic acid (8.3 mg, 0.048 mmol), and CHC13 (15 mL) was magneticallystirred at room temperature in the dark for 12 hours. The mixture was diluted withCH2I,washed once with water, once with 2N Na2SO3,twice with water, dried overNa2SO4, filtered and evaporated in vacuo. The residue was purified by flashchromatography on Brockman Ill, first eluting with CH2I, to recover 10.2 mg (50%yield) of the starting material, pyropheophorbide a methyl ester (25), and further elutingto furnish a brwonish-red band. The appropriate band was collected, evaporated andrecrystallized fromCH21/MeOH to give the title compound (8.4 mg, 40% yield) as tinydark needles.M.p.: 234°C2281H NMR (400 MHz, CDC13)& 9.56 (s, 1H, H-b), 9.50 (s, 1H, H-5), 7.92 (dd, 1H, H31, J= 18.2 and 11.7 Hz), 6.26 [dd, 1H, H-32(Z), J= 11.7 and 1.9 Hz], 6.14[s, ill, H32(E), J= 18.2 and 1.9 Hz], 5.23 (s, 1H, H-132), 5.20 (s, 1H, H-132), 4.80 (q, 1H, H-18, J= 7.3 Hz), 4.22 (dd, 1H, H-17, J = 9.3 and 2.0 Hz), 3.68(s, 6H, H-21 and H-174), 3.59 (s,3H, H-71), 3.59 (q, 2H, H-81 J= 7.8 Hz), 3.23 (s, 3H, H-121), 2.55 (m, 2H, H-171), 2.19(m, 2H, H-172), 1.62 (d, 3H, H-18’, J= 7.3 Hz), 1.68 (t, 3H, H-82 J= 7.8 Hz), —1.95 (brs, 2H, NH)UVIV1s max (CH2C1)420 nm (E 111 000), 520 (18 000), 552 (20 000), 614 (15 000),676 (39 000)LREIMS (mlz): 582 (M, 100%)HREIMS: C34H5N401(Mj: calcd 582.2398obsd 582.240615-Hydroxypurpurin-7-lactone methyl phytyl ester(‘I Io=02C0H81% 19%A solution of the foregoing 132R-hydroxypheophytin a [96(R)] (25 mg, 0.029mmol) in dioxane (35 mL) was stirred magnetically at room temperature. To this was229added an aqueous solution (20 mL) of periodic acid dihydrate (400 mg, 1.76 mmol) andthe resulting mixture was stirred in the dark for 25 hours before the mixture was extractedwith CH21. The organic layer was washed with water 3 times, dried over anhydrousNa2SO4,filtered and evaporated. The residue was purified by flash chromatography onsilica Ill, eluting with 2% MeOH in CH21. The product was recrystallized fromCH2lIhexane, giving a black solid (19.6 mg, 75% yield), which was analyzed by 1HNMR as a diastereomeric mixture of 81% 5S-hydroxypurpurin-7-lactone methyl phytylester and 19% l5’R-hydroxypurpurin-7-lactone methyl phytyl ester.M.p.: 183°C1H NMR (400 MHz, 1.1 mg/0.6 mL CDC13) ö: (the major epimer) 9.76 (s, 1H, H-b),9.55 (s, 1H, H-5), 8.71 (s, lH, H-20), 8.00 (dd, 1H, H-31 J= 18.4 and 12.1 Hz), 6.51 [d,1H, H-32(E), J= 18.4 Hz], 6.19 [d, 1H, H-32(Z), J= 12.1 Hz], 6.10 (s, 1H, OH-151),5.15(t, 1H, H-P2.J= 7.1 Hz), 4.07 (dd, 1H, H-17, J= 10.6 and 1.2 Hz), 4.43 (q, 1H, H-18, J=6.7 Hz), 4.43 (d, 2H, H-P1. J = 8.0 Hz), 3.73 (q, 2H, H-8’, J = 7.6 Hz), 3.76 (s, 3H, H12’), 3.88 (s, 3H, H-153), 3.41 (s, 3H, H-21), 3.25 (s, 3H, H-71), 2.58 (m, 1H, H-l71),2.44 (m, lH, H-172), 2.19 (m, 1H, H-171), 1.89 (m, 1H, H-172), 1.86 (t, H-P4), 1.70 (t,3H, H-82 J = 7.6 Hz), 1.59 (d, 3H, H-181), 1.58 (s, 3H, H-P17), 1.52 (s, 3H, H-P15), 1.19[m, 16H, H-P(CH2)8],0.82 (m, 8H, H-P20. H-P’6. H-P7 and H-P11), —1.10 (br s, 1H,NH), —1.43 (br s, 1H, NH)UVIV1s ?max (CHC13)404 nm (E 112 000), 500 (9 300), 532 (7 200), 564 (1 600), 614 (4800), 672 (36 300)230LRFABMS (mlz): 904 ([M+ H], 56%), 625 ([M+ H]— Phytyl, 20%), 565 ([M+ HJ—Phytyl — COOMe — H, 100%), 503 ([M+ H]— Phytyl — COOMe — COOH — H20, 87%)HRFABMS: C55H74N40([M+ 2H]): calcd 904.5714obsd 904.57146.2.4 Regioselective OxidationOxidation of132-Hydroxychlorophyllone a (82) byH5106in MethanolA solution of the foregoing (95%S, 5%R) mixture of13-hydroxychlorophyllonea (82) (53 mg, 0.1 mmol) in dioxane (54 mL) and methanol (45 mL) was mixed with anaqueous solution (45 mL) of periodic acid dihydrate (2.1 g, 9.17 mmol) and stired atroom temperature for 14 h before the mixture was extracted with dichioromethane (80mL). The organic layer was washed with water 3 times before it was dried over sodiumsulphate, filtered and evaporated in vacuo. The residue was separated by preparativeTLC on deactivated silica gel (developed twice by 5% acetone, 1% methanol indichioromethane), giving 5 distinct bands: the most mobile band (purple-red, 87), thesecond mobile band (yellow, 102), the major band (gray-green, 83), the second leastmobile band (purple-red, 22) and the least mobile band (yellow, 85). Unambiguousstructure assignments of these compounds were accomplished by electronic absorptionand 1H and 13C NMR spectroscopy.231Purpurin-18 methyl ester (87)4.2 mg (7.2% yield) of a purple red shiny small flakes, recrystallized frommethylene chloride/methanol.M.p.: 267°C [lit.185 >270°C; lit.186 >260°C (decomp.)]1H NMR (400 MHz, 1.0 mg/0.6 mL CDC13) 8: 9.60 (s, 1H, H-b), 9.39 (s, 1H, H-5),8.58 (s, 1H, H-20), 7.90 (dd, 1H, H-31 J= 18.3 and 11.3 Hz), 6.30 [dd, 1H, H-32(E), J=18.3 and 1.6 Hz], 6.20 [d, 1H, H-32(Z), J= 11.3 and 1.6 Hz], 5.20 (dd, 1H, H-17, J= 9.9and 2.5 Hz), 4.38 (dq, 1H, H-18, J= 9.9 and 7.7 Hz), 3.77 (s, 3H, H-121), 3.63 (q, 2H, H82, J= 8.3 Hz), 3.32 (s, 3H, H-21), 3.15 (s, 3H, H-71), 2.73 (m, 1H, Hb’-172), 2.45 (m,1H, Ha’471), 2.43 (m,1H, Hb-172), 1.99 (m, 1H, Ha-17’), 1.73 (d, 3H, H481 J 7.7Hz), 1.65 (t, 3H, H-82 J= 8.3 Hz), 0.25 (br s, 1H, NH), —0.08 (br s, 1H, NH)13C NMR (75 MHz, 12.0 mg/0.6 mL CDC13)6: 177.52 (C-173), 176.61 (C-13), 176.61(C-151), 173.66 (C-19), 164.20 (C-16), 156.35 (C-6), 150.17 (C-9), 146.05 (C-8), 144.15(C-i), 140.04 (C-140), 139.15 (C-il), 137.82 (C-7), 136.70 (C-3), 136.64 (C-4), 131.87(C-2), 131.84 (C-12), 131.61 (C-13), 128.42 (C-31), 123.75 (C-32), 111.56 (C-15),107.73 (C-b), 103.14 (C-5), 95.01 (C-20), 55.01 (C-17), 51.63 (C-174), 49.26 (C-18),32.55 (C-172), 31.27 (C-171), 23.85 (C-181), 19.38 (C-81), 17.43 (C-82), 12.41 (C-12),11.96 (C-21), 11.09 (C-7’)I‘LI-H‘HI‘PPP)9vc‘[ziljpu=I’‘(z) 1-H‘HI‘P1919‘[ziljpucgj1‘()zH‘HI‘PPIVE9‘(ZH1,{PUcsi1‘j-H‘HI‘PP)0V8‘(or-H‘HI‘s)006‘(c-H‘HI‘s)986‘(01-H‘HI‘s)066:(i3U3‘T11190IW0t‘ZHJAJoo)Hj30ç:dpjWOJJpzijpiscjoai‘P!I°isoI1njo(p1iX%vc)woNHN—NHN\(zor)iAtnnupqJoqdoqdoJAdoxo-çJ%0L6‘N€09‘Hc6oL‘3:punoj%896‘N‘HLcoL‘3:c0I7NI7EHVEDLcl8Lcpsqo6Zc8LcP3°(+JAI)cO’NIHt’E3AJ[3)JH(001‘H3OO3lH3ZH3÷N)16I‘(%c‘+i”i)8L:(zjui)svirmi[(0086fr)869‘(0086)‘(009t’)9j7c‘(oocL)80S‘(001c)8L17‘(0001T3)WUOJ1(I3HD)”çl!I(oocic3)OOL‘179‘6ç‘ctc‘Loc‘8Lfr‘mu1117(E13H3)981111L69‘i179‘8oc‘8L17‘LOt’‘6c(HOEHD)X1UI681111(001ic)ZOL‘(00176)17179‘(oocci)817S‘(ooiL)80ç‘(008fr)0817‘(0019i1)iTt’‘(0068173)11Wi9C(EIDHD)X1UIS!A-AfliEZ2338.8, 2.9 and 1.3 Hz), 4.68 (dq, 1H, H-18, J = 7.6 and 1.3 Hz), 3.72 (s, 3H, H-121), 3.70(q, 2H, H-82 J= 7.7 Hz), 3.56(s, 3H, H-174), 3.42 (s, 3H, H-21), 3.35 (s, 3H, H-71), 2.78(m, 1H, Ha47’, J = 2.9 Hz), 2.67 (m, 1H, Hb-172), 2.36 (m, 1H, Ha’171 J = 8.8 Hz),2.32 (m, 1H, Hb’-172), 1.87 (d, 3H, H-18’, J = 7.6 Hz), 1.75 (t, 3H, H-82 J = 7.7 Hz),0.26 (hr s, 1H, NH), —2.32 (hr s, 1H, NH)l3 NMR (75 MHz, 6.2 mg/0.6 mL CDC13)& 192.79 (C-131), 185.19 (C-132), 174.87(C-173), 173.67 (C-19), 166.86 (C-16), 153.87 (C-6), 152.58 (C-9), 151.30 (C-14),144.71 (C-8), 142.33 (C-i), 137.77 (C-il), 137.40 (C-3), 136.32 (C-4), 134.51 (C-7),131.29 (C-2), 130.34 (C-13), 128.95 (C-31), 126.42 (C-12), 123.65 (C-32), 105.05 (C-10), 104.41 (C-15), 101.50 (C-5), 95.59 (C-20), 52.67 (C-17), 52.67 (C-174), 51.63 (C-18), 31.50 (C-17’), 29.70 (C-172), 23.85 (C-181), 19.48 (C-81), 17.51 (C-82), 12.60 (Ci2), 12.22 (C-21), 11.32 (C-71)UV-Vis max (CHC13)390 nm (E 49 500), 420 (42 900), 518 (6 900), 622 (3 300), 678(29 000); max (CH3OH) 386 nm (E 40 000), 514 (7 200), 622 (4 700), 676 (20 800)[lit.89‘max (CH3OH) 386 nm, 514, 618, 676]LREIMS (mlz): 562 (M, 100%), 475 (M—CH2CHOO396)HREIMS: C34HN40(M): calcd 562.2580obsd 562.2589Anal. calcd forC34HN40: C, 72.58; H, 6.09; N, 9.96 %found: C, 72.09; H, 6.01; N, 9.80 %23415-Hydroxychlorophyllonelactone a (83)83(R) 83(s)94% 6%31 mg (57% yield) of a gray-green solid, recrystallized fromdichloromethane/hexane.M.p.: >300°CAnal. calcd forC33H2N40: C, 72.24; H, 5.88; N, 10.21 %found: C, 71.89; H, 6.01; N, 10.35 %1H NMR analysis showed that it is a diastereomeric mixture of 94% 15R-hydroxychlorophyllonelactone a [83(R)] and 6%15S-hydroxychlorophyllonelactone a[83(S)]. Further purification of this diastereomeric mixture (14 mg) by preparative TLCon deactivated silica gel (developed three times by 5%acetone, 1 %methanol indichloromethane) gave the optically-pure1R-hydroxychlorophyllonelactone a [83(R)](11.5 mg, 100% d.e. from 1H NMR) of a dark green powder after recrystallization fromdichloromethane/hexane.I83CR)235M.p.: >300°C1H NMR (400 MHz, 1.0 mg/0.6 mL CDC13) 6: 9.68 (s, 1H, H-b), 9.50 (s, 1H, H-5),8.78 (s, 1H, H-20), 8.00 (dd, 1H, H-31 J= 17.6 and 11.2 Hz), 6.32 [dd, 1H, H-32(E), J—17.6 and 1.0Hz], 6.20 [d, 1H, H-32(Z), J= 11.2 and 1.0 Hz], 5.86 (br s, 1H, OH), 4.42(ddd, 1H, H-17, J= 11.5, 5.3 and 1.6 Hz), 4.38 (dq, 1H, H-18, J= 7.5 and 1.6 Hz), 3.79(s, 3H, H-121), 3.69 (q, 2H, H-81 J= 7.9 Hz), 3.49 (ddd, 1H, Hb-172 J= 11.5, 9.0 and8.3 Hz), 3.42 (s, 3H, H-21), 3.33 (s, 3H, H-71), 3.01 (ddd, 1H, Hb’-172J= 11.5, 9.6 and2.5 Hz), 2.85 (dddd, 1H, Ha’47’, J= 12.8, 9.3, 9.0 and 5.3 Hz), 2.19 (dddd, 1H, Ha17’,J= 12.8, 11.5, 8.3 and 2.5 Hz), 1.84 (d, 3H, H-181J= 7.5 Hz), 1.69 (t, 3H, H-82 J= 7.9Hz), —0.93 (br s, 1H, NH), —1.45 (br s, 1H, NH)l3 NMR (75 MHz, 8.5 mgIO.6 mL CDC13) 6: 203.62 (C-173), 173.31 (C-19), 163.85(C-16), 162.57 (C-131), 155.84 (C-6), 150.35 (C-9), 145.68 (C-8), 141.89 (C-i), 138.89(C-12), 136.59 (C-7), 136.39 (C-3), 136.06 (C-4), 134.07 (C-14), 131.62 (C-il), 131.45(C-2), 128.96 (C-31), 123.01 (C-32), 111.69 (C-13), 104.68 (C-iS1), 104.61 (C-b),100.24 (C-iS), 99.82 (C-5), 93.44 (C-20), 51.18 (C-18), 49.84 (C-17), 34.17 (C-172),31.37 (C-171), 23.56 (C-181), 19.54 (C-81), 17.57 (C-82), 12.44 (C-121), 12.15 (C-21),11.33 (C-71)UV-Vis max (CHC13)404 nm (E 147 000), 500 (12 400), 532 (11 900), 614 (7 700), 670(50 200);‘max (CH3OH) 400 nm (E 164 000), 498 (14 200), 528 (12 500), 610 (8 200),666 (53 000) [lit.89 ‘ax (CH3OH) 400 nm, 498, 529, 610, 667]LREIMS (mlz): 549 ([MH], 50%)HREIMS: C33H2N4O([MH]): calcd 549.2502obsd 549.2506236Purpurin-18 (22)1.5 mg (2.7% yield). Treatment of this product with excess of etherealdiazomethane gave purpurin-18 methyl ester (87). After crystallization fromdichloromethane/methanol, the product was found identical to 87 as described before.132-Oxopyropheophorbide a (85)1.0 mg (1.8% yield). Treatment of this product with excess of etherealdiazomethane gave132-oxopyropheophorbide a methyl ester (102). After crystallizationfrom dichloromethanelhexane, this material was found identical to 102 as describedbefore.Oxidation of132-Hydroxychlorophyllone a (82) byH5106in PyridineA solution of the foregoing (95%S, 5%R) mixture of13-hydroxychlorophyllonea (82) (27 mg, 0.05 mmol) in dioxane (20 mL) and pyridine (15 mL) was stirred with anaqueous solution (20 mL) of periodic acid dihydrate (1 g, 4.38 mmol) at roomtemperature for 18 h before the mixture was extracted with dichloromethane (60 mL).The organic layer was washed with water 3 times before it was dried over sodiumsulphate, filtered and evaporated in vacuo. The residue was separated by preparativeTLC on deactivated silica gel (developed twice by 5% acetone, 1% methanol indichloromethane), giving 3 distinct bands: the most mobile band (gray-green, 83); thesecond mobile band (purple-red, 22) and the least mobile band (yellow, 85).23715-Hydroxychlorophyllonelactone a (83)7.7 mg (28% yield) as a gray-green solid, recrystallized from dichloromethane/hexane.M.p.: >300°CThis material was analyzed by 1H NMR as a diastereomeric mixture of 92%15 R-hydroxychlorophyllonelactone a [83(R)] and 8% 151 S-hydroxychlorophyllonelactone a [83(S)].Purpurin-18 (22)3.2 mg (11.3% yield). Treatment of this product with excess of etherealdiazomethane gave purpurin- 18 methyl ester (87). After crystallization fromdichloromethane/methanol, the product was found identical to 87 as described previously.132-Oxopyropheophorbide a (85)13.4 mg (49% yield). Treatment of this product with an excess of etherealdiazomethane, followed by recrystallization from dichloromethane/hexane gave 1 32oxopyropheophorbide a methyl ester (102) (12.7 mg). This material was found identicalto the product 102 as described previously.Chiorophyllonic acid a methyl ester (84)(‘IO2Me238A solution of the foregoing (94%R, 6%S) mixture of 15’-hydroxychlorophyllonelactone a (83) (8 mg, 0.0 146 mmol) in dichloromethane (20 mL)was treated with an excess of ethereal diazomethane and then washed with water 3 times,dried over sodium sulphate, filtered and evaporated in vacuo. The product wascrystallized from dichioromethane/hexane, giving the title compound (6.7 mg, 82%) of agray-brown solid.M.p.: 219°C1H NMR (400 MHz, 1.0 mg/0.6 mL CDC13) 8: 9.70 (s, 1H, H-b), 9.49 (s, 1H, H-5),8.60 (s, 1H, H-20), 7.96 [dd, 1H, H-3’, J = 17.4 and 1.0 Hz], 6.30 [dd, 1H, H-32(E), J =17.4 and 1.0 Hz], 6.15 (dd, 1H, H-32(Z), J = 12.1 and 1.0 Hz), 4.53 (ddd, 1H, H-17, J =12.3, 6.6 and 1.7 Hz), 4.40 (dq, 1H, H-18, J = 7.3 and 1.0 Hz), 4.03(s, 3H, H-132), 3.83(ddd, 1H, Hb-172 J = 12.7, 10.0 and 10.0 Hz), 3.70 (q, 2H, H-81 J = 7.7 Hz), 3.60 (s,3H, H-121), 3.38 (s, 3H, H-21), 3.21 (s, 3H, H-71), 3.05 (ddd, 1H, Hb’-172J = 12.7, 8.3and 1.4 Hz), 2.90 (dddd, 1H, Ha’17’, J= 12.3, 10.0, 8.3 and 6.6 Hz), 2.38 (dddd, 1H, Ha17k, J= 12.3, 12.3, 10.0 and 1.4 Hz), 1.73 (d, 3H, H-181 J= 7.3 Hz), 1.67 (t, 3H, H-82 J= 7.7 Hz), —0.68 (br s, 2H, NH)13C NMR (75 MHz, 6.5 mg/0.6 mL CDC13)6: 196.90 (C-173), 192.38 (C-151), 173.24(C-19), 166.90 (C-131), 164.07 (C-16), 155.22 (C-6), 149.61 (C-9), 145.48 (C-8), 142.09(C-i), 138.42 (C-12), 136.38 (C-3), 136.31 (C-4), 135.61 (C-7), 135.07 (C-b4), 130.82(C-2), 130.12 (C-li), 128.85 (C-31), 122.77 (C-32), 121.12 (C-13), 108.52 (C-15),105.87 (C-b), 101.48 (C-5), 93.41 (C-20), 52.31 (C-132), 51.20 (C-18), 50.16 (C-17),23936.25 (C-172), 29.47 (C-171), 23.62 (C-181), 19.49 (C-8’), 17.56 (C-82), 12.57 (C-121),12.00 (C-21), 11.17 (C-71)UV-Vis max (CHC13)408 nm ( 163 000), 504 (14 500), 544 (20 000), 628 (10 500),680 (53 000), 688 (53 000); 2max (CH3OH) 400 nm (E 170 000), 504 (15 400), 540 (20000), 628 (11 000), 678 (52 400), 686 (sh 46 300) [lit.89 max (CH3OH) 400 nm, 504,540, 610, 677]LRFABMS (mlz): 563 ([MH], 55%)HRFABMS: C34H5N40([MHJj: calcd 563.2658obsd 563.2665Anal. calcd forC34HN40 C, 72.58; H, 6.09; N, 9.96 %found: C, 72.19; H, 5.92; N, 9.85 %Oxidation of15-Hydroxychlorophyllonelactone a (83) byH51O6A solution of the foregoing (94%R, 6%S) mixture of 151hydroxychiorophyllonelactone a (83) (5.5 mg, 0.01 mmol) in dioxane (10 mL) wasstirred with an aqueous solution (10 mL) of periodic acid dihydrate (50 mg, 0.22 mmol)at room temperature for 8 h before the mixture was extracted with dichloromethane (30mL). The organic layer was washed with water 3 times before it was dried over sodiumsulphate, filtered and evaporated in vacuo. The residue was redissolved indichloromethane and treated with an excess of ethereal diazomethane. The product waspurified by chromatography on silica gel, eluting with dichloromethane. Aftercrystallized from dichloromethane/methanol, purpurin-18 methyl ester (87) (4.5 mg,24078%) was obtained as a purple-red shiny flakes, which was identical to the materialprepared from previous methods.Oxidation of132-Oxopyropheophorbide a Methyl Ester (84) byH5106A solution of the foregoing132-oxopyropheophorbide a methyl ester (102) (3.5mg, 6.2 j.imol) in dioxane (5 mL) was stirred with an aqueous solution (4 mL) of periodicacid dihydrate (25 mg, 0.11 mmol) at room temperature for 6 h before the mixture wasextracted with dichloromethane (20 mL). The organic layer was washed with water 3times before it was dried over sodium sulphate, filtered and evaporated in vacuo. Theresidue was purified by chromatography on silica gel, eluting with dichloromethane. Theproduct was crystallized from dichloromethane/methanol, giving purpurin- 18 methylester (87) (3.0 mg, 84%) of a purple-red shiny flakes, which was found identical to thematerial prepared from previous methods.6.3 Synthesis of Phytoporphyrins13-Deoxo-13hydroxypheophorbide a methyl ester (112a)(I,N HNMOOH241Pheophorbide a methyl ester (7) (100 mg, 0.16 mmol) was dissolved in 50 mL ofpyridine/methanol solution (pyridine : methanol = 1:1) and the solution stirred at roomtemperature. NaBH4 (90 mg) was added and the reaction mixture was stirred for 2 h atwhich time the UVIVis spectrum indicated no more starting material. The reactionmixture (bright green) was poured into cooled 2N HCI and the chlorin was extracted withCH21. The green organic layer was carefully washed once with water, twice withsaturated NaHCO3, three times with water, and then dried over anhydrous Na2SO4,filtered and evaporated. The residue was chromatographed on a alumina III columneluting with 1%MeOH in CH21. The main band was collected, evaporated andrecrystallized from CHCl3Ihexane to give the required compound (83.7 mg, 86% yield) asa dark-green powder.M.p.: 135-136°C1H NMR (200 MHz, CDC13)& (the major epimer) 9.84 (s, 1H, H-b), 9.60 (s, 1H, H-5), 8.88 (s, 1H, H-20), 8.20 (dd, 1H, H-31), 6.63 (dd, 1H, H-13’), 6.34 [dd, 1H, H-32(E)],6.18[s, 1H, H-32(Z)], 5.90 (s, 1H, H-132), 4.60 (m, 1H, H-18), 4.45 (m, 1H, H-17), 3.81,3.55, 3.55, 3.39, 3.22 (5s, 15H, H-21 H-7’, H-121H-134 and H-174), 3.80 (q, 2H, H-81),2-2.70 (m, 4H, H-171 and H-172), 1.90 (d, 3H, H-181), 1.75 (t, 3H, H-82), —1.39 (br s,1H, NH), —3.05 (br s, 1H, NH)UVIV1s max (CH2C1)400 nm (e 124 400), 498 (12 000), 656 (35 800)LREIMS (mlz): 608 (M, 100%)HREIMS: C36H40N05(Mj: calcd 608.2999obsd 608.2993242Anal. calcd forC36H40N05: C, 71.03; H, 6.62; N, 9.20 %found: C, 70.81; H, 6.79; N, 8.97 %13-Deoxo-13, 32-dehydropheophorbide a methyl ester (llOa)(IN HNiRC13-Deoxo-13hydroxypheophorbide a methyl ester (112a) (50 mg, 0.0822mmol) was dissolved in 20 mL of pyridine and the solution stirred at 0°C under N2.Excess trifluoroacetylimidazole (150 mg) was added via a syringe and the reactionmixture was stirred at this temperature for 5 minutes. After addition of proton sponge(1.81 mmol), the reaction mixture was kept at 0°C for 30 minutes and 25°C for 3 hoursat which time the UV/Vis spectrum indicated no more starting material. The reactionmixture (bright green) was poured into water and the chiorin was extracted with CH21.The green organic layer was washed three times with water, and then dried overanhydrous Na2SO4, filtered and evaporated. The residue was chromatographed on asilica column eluting with CH21. The main band was collected, evaporated and theresidue was recrystallized from CHC13IMeOH to give the title compound (45.6 mg, 94%yield) as a dark-green powder.M.p.: >300°C114 NMR (400 MHz, 1.5 mg/0.6 mL CDC13) ö: 8.41(s, 1H, H-b), 8.33 (s, 1H, H-5),7.42 (s, 1H, H-20), 7.42 (dd, 1H, H-31 J= 18.5 and 11.8 Hz), 7.19 (s, 1H, H-131), 6.01243[d, 1H, H-32(E), J= 18.5 Hz], 5.94 [d, 1H, H-32(Z), J= 11.8 Hz], 4.77 (d, 1H, H-17, J=8.6 Hz), 3.93 (s, 3H, H-134), 3.77 (q, 1H, H-18, J= 8.5 Hz), 3.57 (s, 3H, H-174), 3.25 (q,2H, H-81 J 7.1 Hz), 2.96 (s, 3H, H-121), 2.91 (s, 3H, H-21), 2.80 (s, 3H, H-71), 2.44(dd, 1H, H-171 J= 8.6 Hz), 2.20 (dd, 2H, H-172), 1.85 (dd, 1H, H-171), 1.69 (d, 3H, H18’, J= 8.5 Hz), 1.45 (t, 3H, H-82 J= 7.1 Hz)UV/Vis max (CHC13)354 nm ( 55 600), 434 (67 900), 586 (4 100), 630 (11 600), 760(4 100), 820 (4 100)LREIMS (mlz): 590 (M, 100%)HREIMS: C36H8N40(M): calcd 590.2893obsd 590.2895Anal. calcd forC36H8N40: C, 73.21; H, 6.48; N, 9.48 %found: C, 72.73; H, 6.53; N, 9.21 %13-Decarboxy-13-formyl-purpurin-7 dimethyl ester (113)(‘I113O2MeA solution of13-deoxo-13’,132dehydropheophorbide a methyl ester (llOa) (20mg, 0.0339 mmol) in CH21 (100 mL) was stirred in Vancouver sunlight for 20 minutes.Removal of solvent in vacuo and crystallization from CH21/hexane gave a brownpowder of the aldehyde 113 (21 mg, 100% yield).244M.p.: 216-217°C1H NMR (400 MHz, 1.5 mgIO.6 mL CDC13)8: 10.93 (s, 1H, H-131), 9.50 (s, 1H, H-b),9.20 (s, 1H, H-5), 8.45 (s, 1H, H-20), 7.90 (dd, 1H, H-31 J 18.2 and 11.9 Hz), 6.30 [d,1H, H-32(E), J= 18.2 Hz], 6.11 [d, 1H, H-32(Z), J= 11.9 Hz], 4.48 (dd, 1H, H-17, J=9.4 Hz), 4.28 (q, 1H, H-18, J = 9.0 Hz), 3.93 (s, 3H, H-121), 3.66 (s, 3H, H-174), 3.61 (q,2H, H-81 J = 7.5 Hz), 3.52 (s, 3H, H-153), 3.30 (s, 3H, H-2’), 3.13 (s, 3H, H-71), 2.35(m, 1H, H-171), 2.10 (m, 2H, H-172), 1.80 (m, 1H, H-171), 1.73 (d, 3H, H-181 J= 9.0Hz), 1.65 (t, 3H, H-82 J= 7.5 Hz), 0.2 (br s, 2H, NH)13C NMR (75 MHz, 15 mg/1.0 mL CDC13)6: 186.83 (C-131), 184.92 (C-151), 174.85(C-152), 173.30 (C-173), 168.22 (C-19), 163.28 (C-16), 157.05 (C-6), 149.12 (C-9),145.98 (C-14), 143.24 (C-8), 140.40 (C-i), 137.34 (C-il), 136.62 (C-3), 135.97 (C-4),134.20 (C-7), 131.90 (C-2), 129.56 (C-12), 124.36 (C-13), 128.57 (C-31), 122.89 (C-32),106.90 (C-5), 105.80 (C-iS), 100.45 (C-b), 93.74 (C-20), 53.27 (C-17), 52.63 (C-iS),51.53 (C-174), 49.71 (C-18), 31.44 (C-172), 30.93 (C-171), 22.78 (C-181), 19.40 (C-81),17.47 (C-82), 11.90 (C-12’), 11.39 (C-21), 11.06 (C-71)UV/Vis max (CH2C1)414 nm (e 113 200), 508 (7 400), 544 (10 600), 630 (7 000), 680(35 400), 686 (31 700)LREIMS (mlz): 622 (M, 15%), 563 (M — COOMe, 27%), 535 (M — COOMe — CO.27%)HREIMS: C36H8N406(Mj: calcd 622.2791obsd 622.2786Anal. calcd forC36H8N406: C, 69.44; H, 6.15; N, 9.00 %found: C, 69.11; H, 5.93; N, 8.92%24513-Deoxo-13hydroxypheophytin a (1 12b)112bCOHPheophytin a (5) (50 mg, 0.057 mmol) was dissolved in 30 mL ofpyridine/methanol solution (pyridine: methanol = 1:1) and the solution stirred at roomtemperature. NaBH4 (50 mg) was added and the reaction mixture was stirred for 2 h atwhich time the UV/Vis spectrum indicated no more starting material. The green reactionmixture was poured into ice-cooled saturated NH4C1 and the chlorin was extracted withCH21. The organic layer was carefully washed three times with water, and then driedover anhydrous NaSO4, filtered and evaporated. The residue was purified by flashchromatography on silica III eluting with 1 %MeOH in CH21. The main band, afterevaporation, gave the title compound (41.2 mg, 83% yield) as a black solid.M.p.: 124-126°C1H NMR (400 MHz, CDC13)6: (the major epimer) 9.89 (s, 1H, H-b), 9.68 (s, 1H, H5), 8.89 (s, 1H, H-20), 8.20 (dd, 1H, H-31), 6.70 (dd, 1H, H-131), 6.38 [dd, 1H, H-32(E)],6.20 [s, 1H, H-32(Z)], 5.95 (s, 1H, H-132), 5.20 (t, 1H, H-P2), 4.60 (m, 2H, H-18 and HP1), 4.50 (m, 1H, H-17), 3.83, 3.72, 3.65, 3.55, 3.40, 3.15 (5s, 15H, H-21 H-71, H-121,H-134 and H-174), 3.80 (q, 2H, H-81), 2-2.70 (m, 4H, H-17’ and H-172), 1.90 (d, 3H, H-24618k), 1.75 (t, 3H, H-82), 0.82-1.90 (m, 38H, H-Phytyl), —1.35 (br s, 1H, NH), —3.12 (br s,1H, NH)UV/Vis max (CH2C1)400 nm (e 156 600), 498 (16 000), 656 (46 500)LRFABMS (m/z): 874 ([M+ 2H], 100%)HRFABMS: C55H79N40([M+ 2H]): calcd 874.5972obsd 874.597213-Deoxo-13-hydroxypyropheophorbide a methyl ester (1 12c)(I,112cCO2Me OHPyropheophorbide a methyl ester (25) (150 mg, 0.274 mmol) was dissolved in100 mL of pyridine/methanol solution (pyridine:methanol = 1:1) and the solution stirredat room temperature. NaBH4 (200 mg) was added and the reaction mixture was stirredfor 2 h at which time the UVIVis spectrum indicated no more starting material. Thegreen reaction mixture was poured into cooled 2N HC1 and the chlorin was extracted withCH21. The organic layer was carefully washed once with water, twice with saturatedNaHCO3,three times with water, and then dried over anhydrous Na2SO4,filtered andevaporated. The residue was purified by flash chromatography on silica III eluting with1% MeOH in CH21. The main band was collected, evaporated and recrystallized fromCH21/hexane to give the title compound (129 mg, 86% yield) as a dark-green powder.247M.p.: 178-180°C1H NMR (200 MHz, CDC13)ö: 9.98 (s, 1H, H-b), 9.82 (s, 1H, H-5), 9.05 (s, 1H, H-20), 8.30 (dd, 1H, H-31), 6.45 [dd, 1H, H-32(E)J, 6.32 [s, 1H, H-32(Z)], 6.27 (dd, 1H, H13k), 5.30 (m, 1H, H-131), 4.48 (m, 2H, 132), 4.20 (m, 1H, H-18), 4.10 (m, 1H, H-17),3.90 (q, 2H, H-81), 3.83, 3.72, 3.65, 3.40, 3.15 (5s, 12H, H-21 H-71,H-121 and H-174),2.70 (m, 2H, H-171), 2.30 (m, 2H, H-172), 1.95 (d, 3H, H-181), 1.82 (t, 3H, H-82), —1.40(br s, 1H, NH), —3.10 (br s, 1H, NH)UV/Vis ?max (CH2C1)402 nm (E 117 600), 502 (9 100), 654 (32 900)LREIMS (mlz): 550 (M, 5%), 532 (M— H20, 100%)LRFABMS (m/z): 551 ([M+ H], 100%)HREIMS: C34H6N40([M—H2O]): calcd 532.2838obsd 532.2828HRFABMS: C34H9N40([M+ H]j: calcd 551.3021obsd 551.30193-Vinyl-13-deoxo-132-methoxycarbonyl-phytoporphyrin methyl ester(114a)(I,11402Me2Method A 13-Deoxo-13,1dehydr pheophorbide a methyl ester (llOa)(20 mg, 0.0339 mmol) was dissolved in dry DMF (15 mL) and the solution stirred at248100°C under N2. Benzoyl chloride (0.04 1 mmol) was injected via a syringe and thereaction mixture (which turned red immediately) was stirred for 20 minutes at which timethe UVIVis spectrum indicated no more starting material. The reaction mixture waspoured into ice-cooled 10% NaOH and the porphyrin was extracted with CH21. Theorganic layer was carefully washed three times with water, dried over anhydrous Na2SO4,filtered and evaporated. Recrystallization of the residue from CH2llhexane gave thetitle compound (19.2 mg, 100% yield) as violet prisms.Method B 3-Deoxo-13hydroxypheophorbide a methyl ester (112a) (50mg, 0.0822 mmol) was dissolved in dry DMF (35 mL) and the solution stirred at 100°Cunder N2. Benzoyl chloride (0.106 mmol) was added via a syringe and the reactionmixture (which turned red in 15 minutes) was stirred at this temperature for 45 minutes atwhich time the UVIVis spectrum indicated no more starting material. The reactionmixture was poured into ice-cooled 10% NaOH and the porphyrin was extracted withCH21. The organic layer was washed three times with water, and then dried overanhydrous Na2SO4, filtered and evaporated. The residue was purified by flashchromatograph on silica eluting with CH21. The main band was collected, evaporatedand the residue was recrystallized from CH2llhexane to give the title compound (42.1mg, 87% yield) as violet prisms.M.p.: 250°C1H NMR (400 MHz, CDC13)& 10.19 (s, 1H, H-b), 10.15 (s, 1H, H-5), 10.00 (s, lH,H-20), 8.27 (dd, 1H, H-31 J= 17.5 and 11.8 Hz), 6.69 (dd, 1H, H-132 J= 7.7 Hz), 6.27[dd, 1H, H-32(E), J = 17.5 and 1.5 Hz], 6.12 [s, 1H, H-32(Z), J = 11.8 and 1.5 Hz], 4.54249[dd, 1H, H-131(E), J = 17.5 and 7.7 Hz], 4.18 [dd, 1H, H-131(Z), J = 17.5], 4.42 (ddd,1H, H-171), 4.31 (ddd, 1H, H-17’), 4.10 (q, 2H, H-8’, J = 7.7 Hz), 3.72 (s, 3H, H-121),3.69 (s, 3H, H-134), 3.66 (s, 3H, H-174), 3.64 (s, 3H, H-71), 3.63 (s, 3H, H-181), 3.56 (s,3H, H-21), 3.12 (dt, 1H, H-172), 3.00 (dt, 1H, H-172), 1.87 (t, 3H, H-82 J = 7.7 Hz), —3.00 (s, 1H, NH), —3.73 (s, 1H, NH)UV/Vis 2’max (CH2C1)404 nm (E 179 800), 504 (13 600), 540 (6 900), 568 (7 300), 618(4 800)LREIMS (mlz): 590 (M, 100%), 531 (M— COOMe, 88%)HREIMS: C36H8N40(Mj: calcd 590.2893obsd 590.2886Anal. calcd forC36H8N40: C, 73.20; H, 6.48; N, 9.48 %found: C, 73.51; H, 6.62; N, 9.68 %3-Vinyl-13-deoxo-132-methoxycarbonylphytoporphyrin phytyl ester(114b)(I114 bCO2Me13-Deoxo-13hydroxypheophytin a (112b) (35 mg, 0.040 mmol) was dissolvedin dry DMF (20 mL) and the solution stirred at 100°C under N2. Benzoyl chloride (0.052mmol) was added via a syringe and the reaction mixture (which turned red in 15 minutes)250was stirred at this temperature for 60 minutes at which time the UVIVis spectrumindicated no more starting material. The reaction mixture was poured into ice-cooledsaturated NaHCO3and the porphyrin was extracted with CH2I. The organic layer waswashed three times with water, and then dried over anhydrous Na2SO4, filtered andevaporated. The residue was purified by flash chromatograph on silica eluting withCH21. The main band was collected, evaporated and the residue was recrystallizedfrom CH21/hexane to give the title compound (27.5 mg, 81% yield) as a black-purplesolid.M.p.: 137-138°C1H NMR (400 MHz, CDC13)& 9.58 (s, 1H, H-b), 9.50 (s, 1H, H-5), 9.40 (s, 1H, H-20),8.14 (dd, 1H, H-3’, J= 18.2 and 12.8 Hz), 6.35 (d, 1H, H-132J= 7.3 Hz), 6.25 [dd, 1H,H-32(E), J= 18.2 and 1.5 Hz], 6.10 [s, 1H, H-32(Z), J= 12.8 and 1.5 Hz], 5.40 (t, 1H, Hp2, J = 7.3 Hz), 4.74 (d, 2H, H-P1 J = 7.3 Hz), 4.47 [dd, 1H, H-131(E), J = 17.8 and 8.4Hz], 4.10 [dd, 1H, H-131(Z), J 17.8 and 7.3 Hz], 3.98 (m, 2H, H-171), 3.82 (q, 2H, H81, J = 7.8 Hz), 3.67 (s, 3H, H-121), 3.64 (s, 3H, H-134), 3.52 (s, 3H, H-71), 3.46 (s, 3H,H-181), 3.34 (s, 3H, H-21), 2.96 (m, 1H, H-172), 2.73 (m, 1H, H-b72), 1.72 (t, 3H, H-82J = 7.8 Hz), 0.8-2.0 (m, 33H, H-phytyl), —3.20 (br s, 1H, NH), —3.95 (br s, 1H, NH)UV/Vis max (CH2C1)408 nm (E 165 500), 506 (9 400), 538 (14 000), 568 (12 600),580 (11 200)LRFABMS (mlz): 856 ([M+ 2H], 100%), 577 ([M+ 2H]— phytyl, 69%)HRFABMS: C55H77N40(Mj: calcd 856.5866obsd 856.58502513-Vinyl-131-deoxo-phytoporphyrin methyl ester (114c)(IN HN114c131-Deoxo- 131 -hydroxypyropheophorbide a methyl ester (112c) (105 mg, 0.191mmol) was dissolved in dry DMF (50 mL) and the solution stirred at 100°C under N2.Benzoyl chloride (0.250 mmol) was added via a syringe and the reaction mixture (whichturned red in 40 minutes) was stirred at this temperature for 120 minutes at which timethe UV/Vis spectrum indicated no more starting material. The reaction mixture waspoured into ice-cooled 10% NaOH and the porphyrin was extracted with CH2I. Theorganic layer was washed three times with water, and then dried over anhydrous Na2SO4,filtered and evaporated. The residue was purified by flash chromatograph on silicaeluting with CH21. The main band was collected, evaporated and the residue wasrecrystallized from CH2lIhexane to give the title compound (74.0 mg, 73% yield) as apurple solid.M.p.: > 300°C1H NMR (400 MHz, CDC13)6: 10.10 (s, 1H, H-b), 9.99 (s, 1H, H-5), 9.93 (s, 1H, H20), 8.26 (dd, 1H, H-31 J= 17.9 and 11.6 Hz), 6.27 [dd, 1H, H-32(E), J= 17.9 and 1.7Hz], 6.11 [dd, 1H, H-32(Z), J= 11.6 and 1.7 Hz], 5.20 (br s, 2H, H-132), 4.25 (t, 2H, H171, J= 8.3 Hz), 4.10 (q, 2H, H-81 J= 7.1 Hz), 3.96 (br s, 2H, H-131), 3.78 (s, 3H, H121), 3.65 (s, 6H, H-174 and H-71), 3.59 (s, 3H, H-181), 3.52 (s, 3H, H-21), 3.03 (t, 2H,252H-172,J= 8.3 Hz), 1.86 (t, 3H, H-82 J= 7.1 Hz), —3.02 (hr s, 1H, NH), —3.89 (hr s, 1H,NH)UV/Vis Amax (CH2C1)404 nm (E 161 300), 504 (15 900), 540 (7 800), 566 (8 300), 620(5 800)LREIMS (mlz): 532 (M, 100%), 459 (M—CH2OOMe, 47%)HREIMS: C34H6N402(M): calcd 532.2838obsd 532.2835Anal. calcd forC34H6N402: C, 76.66; H, 6.81; N, 10.52 %found: C, 76.50; H, 6.85; N, 10.58 %Phytoporphyrin methyl ester (27) and 3-vinyiphytoporphyrin methylester (116)Pyropheophorbide a methyl ester (25) (45 mg, 0.082 1 mmol) was dissolved in dryDMF (20 mL) and the solution stirred at 100°C under N2. Benzoyl chloride (0.107mmol) was added via a syringe and the reaction mixture was stirred at this temperaturefor 120 minutes after which time TLC analyses indicated no more formation of theproducts. The reaction mixture was poured into ice-cooled 10% NaOH and the porphyrinwas extracted with CH21. The organic layer was washed three times with water, andthen dried over anhydrous Na2SO4,filtered and evaporated. The residue was purified byflash chromatograph on silica eluting with CH21 to give the unreacted starting material25 (20.0 mg, 44% yield), further eluting with 1% MeOH in CH21 to give the porphyrinband. The porphyrin hand was then Subjected to preparative TLC separation on silica253(0.5 cm thick) eluting with 2% MeOHICHC1to give two bands. The separated bandscontaining the porphyrins were carefully dissolved in THF, filtered, evaporated and theresidue was recrystallized from CH2lIhexane to give 8.5 mg (19% yield of a blacksolid) of the phytoporphyrin 27 as the first product, followed by 6.7 mg (15% yield of ablack solid) of the phytoporphyrin 116 as the other product.Phytoporphyrin methyl ester (27)‘NH N—M.p.: 234-236°C1H NMR (200MHz, CDC13)& 9.79 (s, 1H, H-b), 9.71 (s, 1H, H-5), 9.40 (s, 1H, H-20),5.10 (br s, 2H, H-132), 3.68 (s, 3H, H-121), 3.64 (s, 3H, H-174), 3.60 (s, 3H, H-71), 3.58(m, 2H, H-171), 3.50 (s, 3H, H-181), 3.40 (q, 4H, H-31 and H-81, J = 8 Hz), 3.25 (s, 3H,H-21), 2.78 (t, 2H, H-172), 1.90 (t, 6H, H-32 and H-82,J= 8 Hz), —3.35 (br s, 1H, NH), —4.21 (br s, 1H, NH)UV/Vis 2’max (CH2C1)416 nm (E 179 300), 524 (15 300), 564 (17 800), 586 (12 800),636 (3 600)LREIMS (mlz): 548 (M, 100%), 475 (M—CH2OOMe, 26%)HREIMS: C34H6N40(M): calcd 548.2787obsd 548.27962543-Vinyiphytoporphyrin methyl ester (116)(IN HN’116M.p.: 206-208°C1H NMR (200 MHz, CDC13)& 9.80 (s, 1H, H-b), 9.60 (s, 1H, H-5), 9.30 (s, 1H, H-20), 8.18 (dd, 1H, H-31 J = 18.1 and 12.0 Hz), 6.30 [dd, 1H, H-32(E), J = 18.1 and 1.8Hz), 6.19 [dd, 1H, H-32(Z), J =12.0 and 1.8 Hz], 5.01 (br s, 2H, H-132), 3.98 (q, 2H, H8, J= 7.5 Hz), 3.70 (s, 3H, H-121), 3.64 (s, 3H, H-174), 3.59 (s, 3H, H-71), 3.59 (m, 1H,H-171), 3.50 (s, 3H, H-181), 3.22 (br s, 2H, H-172), 3.20 (s, 3H, H-21), 2.72 (s, 1H, H-171), 1.80 (t, 3H, H-82 J= 7,5 Hz), —3.70 (hr s, 1H, NH), —3.80 (br s, 1H, NH)UV/Vis max (CH2C1)420 nm (E 148 000), 526 (7 100), 568 (14 500), 590 (11 200),640 (3 000)LREIMS (mlz): 546 (M, 58%)HREIMS: C34HN40(Mj: calcd 546.2631obsd 546.262825513-Deoxo-13-hydroxy-132R-hydroxypheophorbide a methyl ester(119)NHN1NcNMeOOC OH H MeOOC OH OH119RR 119SR56% : 44%132R-Hydroxypheophorbide a methyl ester (95) (90% d.e.) (50 mg, 0.0803 mmol)was dissolved in 25 mL of pyridine/methanol solution (pyridine:methanol = 1:1) and thesolution stirred at room temperature. NaBH4 (70 mg) was added and the reaction mixturewas stirred for 3 h at which time the UVIVis spectrum indicated no more startingmaterial. The reaction mixture was poured into ice-cooled 2N HC1 and the chlorin wasextracted with CH21. The organic layer was carefully washed once with water, twicewith saturated NaHCO3,three times with water, and then dried over anhydrous Na2SO4,filtered and evaporated. The residue was purified by flash chromatography on silica ifieluting with 1% MeOH in CH2J. The main band was collected (44.5 mg, 89% yield)and was analyzed by 1H NMR to be a diastereomeric mixture of 56% 13-deoxo-13Rhydroxy-132R-hy roxypheophorbide a methyl ester (119RR) and 44% 13-deoxo-13Shydroxy-13R-hy roxypheophorbide a methyl ester (119SR). Further purification of thisdiastereomeric mixture (20 mg) by preparative TLC on silica (developed by 2% MeOH,5% acetone in dichloromethane) gave the optically-pure 13’-Deoxo-13’R-hydroxy-132R256hydroxypheophorbide a methyl ester (119RR) (100% d.e. from 1H NMR) (8.5 mg) as agreen solid after recrystallization fromCH2I/hexane.13-Deoxo-13R-hydroxy-13R-hydroxypheophorbide a methyl ester(119RR)(‘I119RRMeOOC OH HM.p.: 180°C1H NMR (400 MHz, 1.4 mg/0.6 mL CDC13)S: 9.80 (s, 1H, H-b), 9.65 (s, 1H, H-5),8.80 (s, 1H, H-20), 8.20 (dd, 1H, H-31 J 18.3 and 12.0 Hz), 6.42 (br s, 1H, OH-l32),6.33 [dd, lH, H-32(E), J=18.3 and 1.4 Hz], 6.16 {dd, 1H, H-32(Z), J= 12.0 and 1.4 Hz],5.60 (s, 1H, H-131), 4.93 (d, 1H, H-17, J= 8.2 Hz), 4.59 (q, 1H, H-18, J= 7.5 Hz), 4.48(m, 2H, 132), 3.81 (q, 2H, H-81 J 7.6 Hz), 3.57 (s, 3H, H-174), 3.55 (s, 3H, H-134),3.54 (s, 3H, H-121), 3.53 (s, 3H, H-71), 3.38 (s, 3H, H-21), 2.45 (ddd, 1H, H-l71), 2.32(ddd, lH, H-172), 2.10 (ddd, 1H, H-171), 1.96 (ddd, 1H, H-172), 1.74 (d, 3H, H-181 J=7.5 Hz), 1.71 (t, 3H, H-82 J 7.6 Hz), —1.40 (br s, 1H, NH), —3.10 (br s, 1H, NH)13C NMR (75 MHz, 12 mg/i.0 mL CDCI3)3: 176.03 (C-13), 173.81 (C-173), 167.27(C-19), 163.05 (C-16), 150.59 (C-6), 150.48 (C-9), 143.18 (C-14), 142.49 (C-8), 139.81(C-i), 138.18 (C-li), 136.49 (C-3), 135.63 (C-4), 134.48 (C-7), 132.21 (C-2), 129.88 (C3k), 128.83 (C-l2), 128.41 (C-13), 121.56 (C-32), 108.87 (C-15), 99.75 (C-b), 99.29(C-5), 95.00 (C-132), 93.46 (C-20), 82.41 (C-13’), 53.32 (C-134), 51.57 (C-174), 50.98(C-17), 49.95 (C-18), 30.66 (C-171), 29.33 (C-172), 23.54 (C-181), 19.64 (C-81), 17.71(C-82), 12.31 (C-12), 11.43 (C-21), 11.43 (C-71)257UV/Vis Amax (CH2C1)400 nm (s 114 100), 498 (10 700), 656 (33 700), 682 (6 200)LREIMS (mlz): 624 (M, 38%)HREIMS: C36H40N06(M): calcd 624.2948obsd 624.2938Anal. calcd forC36H40N06: C, 69.20; H, 6.46; N, 8.97 %found: C, 68.97; H, 6.44; N, 8.70 %13-Deoxo-13-hydroxy-132R-hydroxymethyl-17-depropionate- l7-(y---hydroxypropyl)pheophorbide a (120)(I120CH2O OHCH2OPheophorbide a methyl ester (7) (50 mg, 0.0825 mmol) was dissolved in driedTHF (50 mL) and the solution stirred at 0°C under N2. LiA1H4 (30 mg) was quicklyadded and the reaction mixture was stirred for 5h at which time the UV/Vis spectrumindicated no more starting material. The reaction was quenched by addition of ethylacetate to destroy excess LiA1H4;aqueous ammonium chloride was then introduced andthe solution was extracted with CH21. The organic layer was carefully washed oncewith 2N HC1, once with water, twice with saturated NaHCO3,three times with water, andthen dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified byflash chromatography on silica III eluting with 2%MeOH in CH21. The main band was258collected and was subjected to further purification by preparative TLC on silica(developed by 2%MeOH, 5% acetone in dichloromethane) to give the title compound(32.1 mg, 72% yield) as a green solid after recrystallization fromCH21/hexane.M.p.: >300°C1H NIvIR (200 MHz, CDC13)6: (the major epimer) 9.82 (s, 1H, H-b), 9.59 (s, 1H, H-5),8.90 (s, 1H, H-20), 8.20 (dd, 1H, H-31), 6.32 [dd, 1H, H-32(E)], 6.16 [s, 1H, H-32(Z)],5.18 (s, 1H, H-13’), 6.00 (br s, 1H, OH-132),4.58 (d, 1H, H-17), 4.38 (br s, 2H, H-173),4.10 (m, 1H, H-18), 3.20-3.60 (4s, l2H, H-81 H-121,H-71 and H-21), 2.25 (m, 1H, H17k), 2.02 (m, 1H, H-17’), 1.82 (d, 3H, H-181), 1.79 (m, 5H, H-172 and H-82), —3.35 (brs, 2H, NH)UV/Vis 2max (CH2C1)402 nm (E 163 000), 500 (17 400), 654 (41 700)LRFABMS (mlz): 553 ([M+ H], 100%)HRFABMS: C34H41N0(Mj: calcd 553.3178obsd 553.3165Deoxophylloerythroetioporphyrin (121)N—1213-Vinyl-13’-deoxo-phytoporphyrin methyl ester (114c) (10 mg, 0.0188 mmol) or3-vinyl-13-deoxo2methoxycarbonyl-phytoporphyrin phytyl ester (114b) (16 mg,0.0188 mmol) or 3-vinyl-13-deoxomethoxycarbonyl-phytoporphyrin methyl ester259(114a) (11 mg, 0.0188 mmol) was saponified in the following conditions: 114c, 10%KOH/MeOH (10 mL) at room temperature for 10 hours, 114a or 114b, 25% HC1 (10 mL)at room temperature for 100 minutes. After that time TLC analyses showed goodconversion to the desired mono-acid andlor diacids. The porphyrin acid (the productfrom the basic saponification was acidified with HC1 to pH 3 before it was transferred)was transferred to a separatory funnel and diluted to 100 mL with ice. An equal portionof CHC13 was added to extract the aqueous layer. After the extraction was repeated 6times, the combined organic layers were pooled, washed with water (100 mL). Theorganic layer was quickly dried with Na2SO4, filtered and evaporated in vacuo. Theresidue was dissolved in THF and tranferred to a small vial. The vial was kept at roomtemperature until the solvent had evaporated. The vial was then sealed with a smallrubber septum and was dried (via a needle) under high vacuum (0.5 mm Hg) at 120°C for8 hours. After that, 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) (0.6 g) was quicklysqueezed into the vial and dried under high vacuum at 120°C for another 16 hours. Thevial was then flame-sealed under high vacuum, was heated at 200°C for 4 hours, and,after cooling down, the vial was broken and the porphyrin mixture was immediatelypurified by flash chromatography on silica eluting with 2% MeOH in CH2I. The mainband was collected, evaporated and recrystallized from CH21IMeOH to give the titlecompound (5.9-6.7 mg, 67-75% yield) as a red solid.M.p.: >300°C (decomp.)1H NMR (200 MHz, CDC13)8 9.96 (s, 1H, H-b), 9.90 (s, 1H, H-5), 9.89 (s, 1H, H-20),5.32 (br s, 2H, H-132), 4.05 (br s, 211, H-13), 4.10, 4.08, 4.00 (3q, 6H, H-31 H-81 and260H-171), 3.50-3.70 (4s, 12H, H-181H-121,H-71 and H-21), 1.70-1.90 (3t, 9H, H-32 H-82and H-172),—3.00 (br s, 1H, NH), —3.90 (br s, 1H, NH)UV/Vis max (CH2C1)402 nm (e 195 000), 500 (15 000), 536 (3 000), 566 (5 200), 620(6 100)LREIMS (m/z): 476 (M, 100%)HRFABMS: C32H6N4(Mj: calcd 476.2940obsd 476.29516.4 Synthesis of Benzo- and Dibenzo-porphyrin Derivatives6.4.1 Synthesis of Regiochemically-pure Benzoporphyrin DerivativesPurpurin-7 trimethyl ester (16)(I,16¶CO2MeMethod A Described in section 6.2.3.Method B Pheophytin a (5) (1.52 g, 1.75 mmol) was dissolved in warmpyridine (25 mL) and the solution was diluted with ether (700 mL). The solution wasstirred with a stream of air passing through it, and a solution of potassium hydroxide(11.5 g) in n-propanol (40 mL) was added. The bright green mixture (containingprecipitated KOH) was stirred and aerated for 30 mm and then extracted with water (2 x261300 mL). The ethereal solution was discarded; the aqueous extracts were combined,acidified with concentrated H2S04 (11.5 mL) in water (60 mL), and then extracted withmethylene chloride (2 x 350 mL). The extracts were washed with water (350 mL) andimmediately treated with an excess of ethereal diazomethane. The brownish-purplesoluion was left at room temperature for 10 mm, and then evaporated in vacuo.Chromatography (alumina V) of the residue (elution with methylene chloride) gave amajor band which was collected, evaporated and subjected to preparative TLC separationon deactivated silica plates (elution with 0.5% MeOH in CH21) to give the desiredproduct (660 mg, 58% yield) after crystallization fromCH2lIhexane, along with 35 mgof a unseparated mixture of purpurin- 18 methyl ester (87) and purpurin-7 trimethyl ester(16) as the other products. The corresponding physical data of this compound appear insection XV dimethyl ester (23)NH N23Purpurin-7 trimethyl ester (16) (450 mg, 0.69 mmol) was heated in an oil-bath at180-185°C in 2,4,6-collidine (60 mL) during 2 h. After cooling, the solvent wasevaporated off at 0.5 mm Hg and the residue was recrystallized from CHC13/CHO ,giving the title porphyrin (295 mg, 76%) as purple needles.M.p.: 266-267°C (lit.52 271-272.5°C)2621H NMR (400 MHz, CDCI3)& 10.81, 9.89, 9.82, 9.68 (4s, 4H, H-5, H-10, H-15 and H-20), 8.12 (dd, 1H, H-3’, J = 18.4 and 11.8 Hz), 6.23 [dd, 1H, H-32(E), J = 18.4 and 1.6Hz), 6.10 [dd, 1H, H-32(Z), J= 11.8 and 1.6 Hz], 4.42 (s, 3H, H-132), 4.30 (t, 2H, H-171J = 8.0 Hz), 3.93 (q, 2H, H-8, J = 8.0 Hz), 3.84, 3.67, 3.52, 3.50, 3.48 (5s, 15H, H-21 H71, H-121 H-181 and H-174), 3.23 (t, 2H, H-172 J = 8.0 Hz), 1.79 (t, 3H, H-82 J = 8.0Hz), —4.50 (br s, 2H, NH)UVIV1s 2max (CH2C1)408 nm (E 187 000), 514 (10 000), 552 (20 000), 576 (11 000),636 (1 600)LREIMS (mlz): 564 (M, 100%)Anal. calcd forC34H6N40: C, 72.32; H, 6.43; N, 9.92 %found: C, 71.98; H, 6.45; N, 9.65 %21,2-Bis(methoxycarbonyl)-8-ethyl-13-methoxycarbonyl-17-methoxy-carbonylethyl-2,7,12,18-tetramethyl-2-hydrobenzo[bjpo phyrin (140)MeOOC1403-Vinyirhodoporphyrin XV dimethyl ester (23) (57 mg, 0.1 mmol) and dimethylacetylenedicarboxylate (710 mg, 5 mmol) were suspended in degassed toluene (N2) (20mL) and the mixture heated at 110°C for 28 hours. After removal of the toluene in vacuo,the residue was chromatographed on silica (methylene chloride eluant). After elution ofthe excess dienophile, the solvent polarity was increased to 2% methanol in methylene263chloride to elute the major reaction product. After evaporation to dryness, the residuewas recrystallized fromCH2J/hexane to afford the 1 ,4-cyclohexadiene adduct 140 as agreen solid (35 mg, 50% yield).M.p.: 244-245°C1H NMR (400 MHz, CDC13)& 10.75, 9.80, 9.25, 9.01 (4s, 4H, H-5, H-b, H-15 and H-20), 7.40 (dd, 1H, H-31 J = 7.4 and 2.1 Hz), 4.31 (t, 2H, H-171 J = 8.3 Hz), 3.95 (dd,1H, H-23 J 3.0 and 0.5), 3.94 (q, 2H, H-81 J = 7.5 Hz), 3.62 (s, 1H, H-23 J =3.3 and0.5 Hz), 3.25 (t, 2H, H-172 J= 8.3 Hz), 4.33, 4.02, 3.89, 3.75, 3.69, 3.45, 3.41 (7s, 21H,H-7’, H-121H-181,H-174,2-COOCH3and2-COOCH3),2.07 (s, 3H, 2-CH3), 1.78 (t,3H, H-82 J = 7.5 Hz), —2.26 (br s, 1H, NH), —2.30 (br s, 1H, NH)UVIVis max (CH3COCH)410 nm (peak ratio, 1.40), 516 (0.119), 646 (0.138)LREIMS (mlz): 706 (M, 100%), 690 (M — CH4, 60%)21,2-Bis(methoxycarbonyl)-8-ethyl-13-methoxycarbonyl-17-methoxy-carbonylethyl-2,7,12,18-tetramethyl-2-hydrobenzo[b]porphyrin (141)MOOC MeOOC141The adduct 140 (32 mg, 0.045 mmol) was dissolved in methylene chloride and afew drops of DBU were added. The reaction mixture was stirred in the dark andmonitored by visible spectroscopy (complete in 4 hours). The mixture was poured into1M HC1 and extracted with methylene chloride. The organic layer was washed twice264with brine, once with water and dried over anhydrous magnesium sulfate. The solventwas evaporated in vacuo and the compound was chromatographed on silica gel (1%MeOH in CH21 eluent). The pure 1 ,3-cyclohexadiene fraction was evaporated in vacuoyielding 29 mg (90% yield) of a dark green tiny needles after recrystallization fromCH2lIhexane.M.p.: 248-249°C1H NMR (400 MHz, CDC13)6: 10.70, 9.80, 9.30, 8.90 (4s, 4H, H-5, H-b, H-15 and H-20), 7.80 (d, 1H, H-24 J = 6.0 Hz), 7.42 (d, 1H, H-23 J = 6.0 Hz), 5.40 (s, 1H, H-21),4.29 (t, 2H, H-17’, J= 7.7 Hz), 3.92 (q, 2H, H-81 J= 7.9 Hz), 3.25 (t, 2H, H-172J= 7.7Hz), 4.34, 3.98, 3.75, 3.70, 3.43, 3.40, 2.98 (7s, 21H, H-71 H-121,H-181,H-174, 2-COOCH3 and2-COOCH3), 1.79 (s, 3H, 2-CH3), 1.76 (t, 3H, H-82 J = 7.9 Hz), —1.95(br s, 1H, NH), —2.00 (br s, 1H, NH)UV/Vis A.max (CH2C1)356 nm (E, 27 000), 440 (70 400), 592 (19 900), 672 (14 600)LREIMS (mlz): 706 (M, 100%)HREIMS: C40H2N08(M): calcd 706.3002obsd 706.3010Anal. calcd forC40H2N08: C, 67.97; H, 5.99; N, 7.93 %found: C, 68.03; H, 5.96; N, 7.92 %26521,2-Tetracyano-ethyl-13-methoxycarbonyl-17-methoxycarbonyl-ethyl-2,7,12,18-tetramethyl-23-hydrobenzo[b]porphyrin (142)NCNCNcH2Me142TCNE (50 mg) was added to 3-vinyirhodoporphyrin XV dimethyl ester (23) (57mg, 0.1 mmol) dissolved in dry chloroform (20 mL) and the reaction mixture wasrefluxed for 1 hours (monitored by spectrophotometry). The solvent was evaporated andthe residue was purified on an alumina column (Brockman Grade III; elution withmethylene chloride). The eluates were collected and evaporated to give a residue whichwas crystallized fromCH2lIhexane to afford the title compound as a green powder (53mg, 77% yield).M.p.: 252-253°C1H NMR (400 MHz, CDC13)ö: 10.90, 9.90, 9.28, 9.19 (4s, 4H, H-5, H-10, H-15 and H-20), 7.03 (dd, 1H, H-24 J = 8.0 and 3.6 Hz), 4.34 (s, 3H, H-132), 4.32 (t, 2H, H-171 J =7.6 Hz), 4.02 (d, 1H, H-23 J= 8.0 Hz), 3.98 (q, 2H, H-81 J= 7.3 Hz), 3.95 (d, 1H, H-23J = 3.6 Hz), 3.29 (t, 2H, H-172 J = 7.6 Hz), 3.76, 3.69, 3.50, 3.42 (4s, 12H, H-71, H12k, H-181 and H-174), 2.37 (s, 3H, 2-CH3), 1.80 (t, 3H, H-82 J = 7.3 Hz), —2.26 (br ,1H, NH), —2.30 (br s, 1H, NH)UV/Vis ?max (CH2C1)414 nm (E, 172 000), 518 (8 500), 552 (15 700), 588 (5 900), 642(20 500)LRFABMS (m/z): 692 (M, 13%)266HRFABMS: C40H36N08(M): calcd 692.2859obsd 692.2853Anal. calcd forC40H36N08: C, 69.35; H, 5.24; N, 16.17 %found: C, 68.67; H, 5.44; N, 15.83 %21,2-Diazo(N-phenylmaleimide)-ethyl-13-methoxycarbonyl-17-methoxycarbonylethyl-2,7,12,18-tetramethyl-2-hydrobenzo[b]-porphyrin (143)0—1434-Phenyl- 1 ,2,4-triazoline-3 ,5-dione (25 mg) was added to 3-vinyirhodoporphyrinXV dimethyl ester (23) (29 mg, 0.05 mmol) dissolved in dry methylene chloride (10 mL)and the reaction mixture was stirred at 0°C for 4 hours (monitored by spectrophotometry).The solvent was evaporated and the residue was purified on an alumina column(Brockman Grade V; elution with methylene chloride). The eluates were collected andevaporated to give a residue which was crystallized from CH2lIhexane to afford thetitle compound as a green powder (37 mg, 90% yield).M.p.: 242°C1H NMR (400 MHz, CDC13)& 10.90, 10.80, 9.80, 9.10 (4s, 4H, H-5, H-b, H-15 and H20), 7.80 (d, 2H, Ph-H, J= 7.2 Hz), 7.62 (dd, 2H, Ph-H, J= 8.1 and 7.2 Hz), 7.50 (t, 1H,J =8.1 Hz), 6.93 (t, 1H, H-24 J = 3.6 Hz), 5.10 (dd, 1H, H-23 J = 17 and 3.6 Hz), 4.59267(dd, 1H, H-23 J = 17 and 3.6 Hz), 4.33 (s, 3H, H-132), 4.30 (t, 2H, H-171 J = 7.3 Hz),3.95 (q, 2H, H-8’, J= 7.0 Hz), 3.22 (t, 2H, H-172J= 7.3 Hz), 3.73, 3.69, 3.48, 3.41 (4s,12H, H-71, H-121,H-181 and H-174), 2.08 (s, 3H, 2-CH3), 1.79 (t, 3H, H-82 J = 7.0Hz), —2.57 (br s, 1H, NH), —2.65 (br s, 1H, NH)UV/Vis max (CH2C1)414 nm (E, 180 000), 518 (8 400), 552 (13 900), 584 (6 700), 640(19 500)LRFABMS (mlz): 739 (M, 10%)HRFABMS: C42H1N706(Mj: calcd 739.3118obsd 739.3105Anal. calcd forC40H36N08: C, 68.19; H, 5.59; N, 13.25 %found: C, 67.84; H, 5.52; N, 13.52 %21,2-Bis(methoxycarbonyl)-13,15-ethano-8-ethyl-17-methoxycarbonyl-ethyl-2,7,12,18-tetramethyl-2-hydrobenzo[b]porphyrin (125)MeOOC / MeOOCMeOOC’125MeOOCPhytoporphyrin 114c (27 mg, 0.05 mmol) and dimethyl acetylenedicarboxylate(360 mg, 25 mmol) were suspended in degassed toluene (N2) (20 mL) and the mixtureheated at 110°C for 30 hours. After removal of the toluene in vacuo, the residue waschromatographed on silica (methylene chloride eluant). After elution of the excess268dienophile, the solvent polarity was increased to 2% methanol in methylene chloride toelute the major reaction product. After evaporation to dryness, 14 mg (42% yield) of the1 ,4-cyclohexadiene adduct 134(2max 404, 550, 650 nm) was obtained.The adduct 134 (14 mg, 0.02 mmol) was dissolved in methylene chloride and afew drops of DBU were added. The reaction mixture was stirred in the dark andmonitored by visible spectroscopy (completed in 6 hours). The mixture was poured into1M HC1 and extracted with methylene chloride. The organic layer was washed twicewith brine, once with water and dried over anhydrous magnesium sulfate. The solventwas evaporated in vacuo and the compound was chromatographed on silica gel (1%MeOH in CH21 eluent). The pure 1 ,3-cyclohexadiene fraction was evaporated in vacuoyielding 12.7 mg (91% yield) of a dark green tiny needles after recrystallization fromCH21/hexane.M.p.: 258°C1H NMR (400 MHz, CDC13)6: 9.59 (s, 1H, H-b), 9.30 (s, 1H, H-5), 8.95 (s, 1H, H-20),7.83 (d, 1H, H-24 J = 5.8 Hz), 7.45 (d, 1H, H-23 J = 5.8 Hz), 5.20 (t, 2H, H-132), 5.05(s, 1H, H-21), 4.30 (m, 2H, H-171), 3.93 (m, 2H, H-131), 3.93 (q, 2H, H-8’, J = 7.9 Hz),3.10 (t, 2H, H-172), 3.97, 3.78, 3.46, 3.42, 3.39, 2.92 (6s, 18H, H-71 H-121,H-181,H17,21-COOCH3and2-COOCH3),1.78 (t, 3H, H-82 J = 7.9 Hz), 1.77 (s, 3H, 2-Cl3),—1.42 (br s, 1H, NH), —2.22 (br s, 1H, NH)UV/Vis 2max (CH2C1)354 nm (E, 86 000), 438 (120 000), 580 (18 000), 620 (14 000),680 (47 000), 686 (37 000)LRFABMS (mlz): 675 ([M+H], 100%)269HRFABMS: C40H3N06([M+H]j: calcd 675.3182obsd 675.31766.4.2 Synthesis of [A,C]-Divinylporphyrin 147 via Purpurin 11313-Decarboxy-13-formyl-purpurin-7 dimethyl ester (113)(I11302MeMethodA A solution of13-Deoxo-13,1dehydr pheophorbide a methylester (llOa) (20 mg, 0.0339 mmol) in CH21 (100 mL) was stirred in Vancouversunlight for 20 minutes. Removal of solvent in vacuo and crystallization fromCH2llhexane gave a brown powder of the aldehyde 113 (21 mg, 100% yield).Method B A solution of 132-deoxo- 131 -hydroxy- 1 R-hydroxypheophorbidea methyl ester (119) (31 mg, 0.05 mmol) in dioxane (20 mL) was stirred with an aqueoussolution (20 mL) of periodic acid dihydrate (900 mg, 3.95 mmol) at room temperature for20 h before the mixture was extracted with dichloromethane (2 x 40 mL). The organiclayer was dried over sodium sulphate, filtered and evaporated in vacuo. The residue waspurified by flash chromatography on silica III, eluting with dichioromethane.Recrystallization from dichloromethane/hexane gave the title compound as a brownpowder (24.8 mg, 80%).270M.p.: 216-217°C1H NMR (400 MHz, 1.5 mg/0.6 mL CDC13)8: 10.93 (s, 1H, H-131), 9.50 (s, 1H, H-b),9.20 (s, 1H, H-5), 8.45 (s, 1H, H-20), 7.90 (dd, 1H, H-31 J= 18.2 and 11.9 Hz), 6.30 [d,1H, H-32(E), J = 18.2 Hz], 6.11 [d, 1H, H-32(Z), J = 11.9 Hz], 4.48 (dd, 1H, H-17, J =9.4 Hz), 4.28 (q, 1H, H-18, J = 9.0 Hz), 3.93 (s, 3H, H-121), 3.66 (s, 3H, H-174), 3.61 (q,2H, H-81 J = 7.5 Hz), 3.52 (s, 3H, H-153), 3.30 (s, 3H, H-2’), 3.13 (s, 3H, H-71), 2.35(m, 1H, H-171), 2.10 (m, 2H, H-172), 1.80 (m, 1H, H-171), 1.73 (d, 3H, H-181 J 9.0Hz), 1.65 (t, 3H, H-82 J= 7.5 Hz), 0.2 (br s, 2H, NH)l3 NMR (75 MHz, 15 mg/1.0 mL CDC13)6: 186.83 (C-131), 184.92 (C-151), 174.85(C-152), 173.30 (C-173), 168.22 (C-19), 163.28 (C-16), 157.05 (C-6), 149.12 (C-9),145.98 (C-14), 143.24 (C-8), 140.40 (C-i), 137.34 (C-li), 136.62 (C-3), 135.97 (C-4),134.20 (C-7), 131.90 (C-2), 129.56 (C-12), 124.36 (C-13), 128.57 (C-31), 122.89 (C-32),106.90 (C-5), 105.80 (C-15), 100.45 (C-b), 93.74 (C-20), 53.27 (C-17), 52.63 (C-iS),51.53 (C-174), 49.71 (C-18), 31.44 (C-172), 30.93 (C-171), 22.78 (C-181), 19.40 (C-81),17.47 (C-82), 11.90 (C-121), 11.39 (C-21), 11.06 (C-71)UV/Vis max (CH2C1)414 nm (E 113 200), 508 (7 400), 544 (10 600), 630 (7 000), 680(35 400), 686 (31 700)LREIMS (mlz): 622 (M, 15%), 563 (M — COOMe, 27%), 535 (M— COOMe — CO,27%)HREIMS: C36H8N406(Mj: calcd 622.2791obsd 622.2786Anal. calcd forC36H8N406: C, 69.44; H, 6.15; N, 9.00 %found: C, 69.11; H, 5.93; N, 8.92 %27113-Decarboxyl-13-formyl-3-vinylrhodoporphyrin XV methyl ester (148)..NH N148Purpurin 113 (150 mg, 0.24 mmol) was heated in an oil-bath at 180-185°C in2,4,6-collidine (30 mL) during 60 minutes. After cooling, the solvent was evaporated offat 0.5 mm Hg and the residue was recrystallized from CHCI3ICHOH, giving the titleporphyrin (19.3 mg, 15% yield) as purple needles.M.p.: 245-246°C1H NMR (400 MHz, CDC13)ö: 11.40 (s, 1H, 13-CHO), 10.70, 9.92, 9.88, 9.76 (4s, 4H,H-5, H-b, H-iS and H-20), 8.15 (dd, 1H, H-31 J= 17.5 and 11.0 Hz), 6.29 [dd, 1H, H32(E), J= 17.5 and 1.0 Hz], 6.15 [dd, 1H, H-32(Z), J= 11.0 and 1.0 Hz], 4.37 (t, 2H, H17k, J = 7.5 Hz), 4.00 (q, 2H, H-81 J = 8.3 Hz), 3.80, 3.69, 3.58, 3.58, 3.58 (5s, 15H, H2, H-71H-121,H-181 and H-174), 3.23 (t, 2H, H-172 J= 7.5 Hz), 1.80 (t, 3H, H-82 J=8.3 Hz), —4.50 (br s, 2H, NH)UV/Vis max (CH2C1)414 nm ( 189 000), 518 (9 000), 560 (21 400), 582 (15 300),636 (2 500)LREIMS (mlz): 534 (M, 100%)HREIMS: C33H4N40(Mj: calcd 534.2631obsd 534.263427213-Decarboxyl-13-formyl-15-methoxyglyoxylic-3-vinylrhodoporphyrinXV methyl ester—NH NçHOO2Me2A mixture of15-hydroxypurpurin-7-lactone methyl ester (101) (60 mg, 0.094mmol) and N,N’-carbonyldi-imidazole was refluxed in THF (30 mL) for 35 minutes. Thesolution was evaporated and the residue was chromatographed on alumina V (elutionwith methylene chloride). The appropriate band was collected and the elute wasevaporated. The residue was recrystallized from CHCl3Ihexane, giving the titleporphyrin (4.6 mg, 7.9%) as a red solid.M.p.: >300°C1H NMR (400 MHz, CDC13)6 10.30 (s, 1H, 13-CHO), 10.19, 10.15, 8.40 (3s, 3H, H-5,H-b and H-20), 8.19 (dd, 1H, H-31 J = 18.2 and 11.5 Hz), 6.30 [dd, 1H, H-32(E), J =18.2 and 1.0 Hz], 6.15 [dd, 1H, H-32(Z), J= 11.5 and 1.0 Hz], 4.81 (m, 1H, H-171), 4.48(m, 1H, H-171), 4.15 (q, 2H, H-81, J = 8.0 Hz), 4.10, 3.78, 3.78, 3.65, 3.65, 3.30 (6s,18H, H-21H-71,H-121,H-181,H-174 and15-COOCH3),3.39 (m, 1H, H-172), 3.23 (m,1H, H-172), 1.90 (t, 3H, H-82 J= 8.3 Hz), —3.18 (br s, 1H, NH), —3.40 (br s, 1H, NH)UV/Vis Amax (CH2C1)414 nm (E 142 000), 522 (6 400), 564 (11 300), 582 (9 300), 634(2 600)LR1?ABMS (m/z): 621 ([M+H], 73%)HREIMS: C36HN406(M): calcd 621.2713obsd 621.271427313-Decarboxy- 13- (ethyleneacetal)-purpurin-7 dimethyl ester (150)(I,150CO2MeThe foregoing purpurin 113 (20 mg, 0.032 mmol) in dried THF (50 mL) undernitrogen was stirred in a 3-necked round-bottom flask equiped with a Soxhiet apparatusand 4A molecular sieve inside. Ethylene glycol (0.9 mL) and p-toluenesulfonic acid (2mg) were added to the solution and the reaction mixture was brought to reflux. Progressof the reaction was followed by spectrophotometry, using the change in absorptionspectrum from Amax = 686 nm to max = 660 nm; the reaction was complete after 1 hour.The mixture was diluted with methylene chloride (50 mL), washed with water (3 x 50mL), dried over Na2SO4,and evaporated to dryness. The residue was chromatographedon alumina V (elution with methylene chloride) to give the principal green band.Removal of solvent in vacuo and recrystallization fromCH2llhexane gave a dark greenpowder of the title acetal 150 (19.2 mg, 90% yield).M.p.: 265°C1H NMR (400 MHz, 1.5 mg/0.6 mL CDC13)& 10.93 (s, 1H, H-131), 9.75 (s, 1H, H-b),9.68 (s, 1H, H-5), 8.80 (s, 1H, H-20), 8.12 (dd, 1H, H-31 J= 18.3 and 11.7 Hz), 6.33 [d,1H, H-32(E), J = 18.3 Hz), 6.15 [d, 1H, H-32(Z), J = 11.7 Hz), 4.70 (m, lH, l3-OCH2),4.40 (q, 1H, H-18, J = 7.3 Hz), 4.18 (dd, lH, H-l7, J = 13.3 and 3.6 Hz),4.06 (s, 3H, 15-COOCH3),4.01 (m, 3H, 13-OCH2O),3.79 (q, 2H, H-81, J =2747.56Hz), 3.59 (s, 3H, H-174), 3.50 (s, 3H, H-121), 3.44 (s, 3H, H-21), 3.33 (s, 3H, H-71),2.38(m, 1H,H-17),2.20(m, 1H,H-172), 1.89(m, 1H,H-17’), 1.82(d,3H,H-18,J=7.3 Hz), 1.80(m, 1H,H-172), 1.73 (t, 3H,H-8,J=7.6Hz),—1. 5 (brs, 1H,NH),—2.03(br s, 1H, NH)UV/Vis ?max (CH2I)400 nm (E 149 000), 496 (13 000), 606 (5 000), 660 (39 000)LREIMS (m/z): 666 (M, 100%)HREIMS: C38H42N07(M): calcd 666.3053obsd 666.306113-Decarboxyl-3,13-divinylrhodoporphyrin XV methyl ester (147)NH N14713-Decarboxy-13-formyl-3-vinylrhodoporphyrin XV methyl ester (148) (18 mg,0.0337 mmol) was dissolved in methylene chloride (10 mL), and a saturated solution ofZn(OAc)2 in methanol (0.5 mL) was added. The resulting solution was stirred at roomtemperature for 3 hours at which time TLC and UV/Vis spectroscopy showed fullconversion of the desired compound. The solvent was removed by evaporation in vacuoand the product was redissolved in methylene chloride, filtered through a short cake ofalumina V, and evaporated to dryness. The product [i.e. Zn(II) porphyrin 148] wasfurther dried by an oil pump at 0.5 mm Hg for 4 hours.275Dry methyltriphenylphosphonium bromide (30 mg, 0.084 mmol) in dry THF (25mL) and diisopropylamine (0.5 mL) were treated with n-butyllithium in n-hexane (50 j.LL,1.6 M). The resulting ylide was stirred at 0°C under nitrogen gas and a THF solution (10mL) of the above Zn(II) porphyrin 148 was added via a cannule. After 30 mm thesolution was evaporated to dryness to give a residue which was taken into methylenechloride (30 mL), filtered through anhydrous sodium sulfate, and then evaporated againto dryness. The residue was dissolved in trifluoroacetic acid (3 mL) and washed twicewith water (30 mL). The organic phase was evaporated to dryness and the residue waschromatographed on amulina III (elution with methylene chloride). The red eluates wereevaporated to dryness to furnish a residue which was recrystallized from CHC13/hexaneto give the title porphyrin (12.6 mg, 71%) as a purple-red powder.M.p.: 227°C1H NMR (400 MHz, CDC13)6: 10.16, 10.15, 10.08, 10.07 (4s, 4H, H-5, H-b, H-15 andH-20), 8.27 (dd, 2H, H-31 and H-131), 6.33 [dd, 2H, H-32(E) and H-132(E)], 6.15 [dd,2H, H-32(Z) and H-132(Z)], 4.42 (t, 2H, H-171 J = 7.6 Hz), 4.10 (q, 2H, H-81 J = 7.5Hz), 3.69, 3.67, 3.66, 3.65, 3.63 (5s, 15H, H-21 H-71,H-121,H-181 and H-174), 3.26 (t,2H, H-172J= 7.6 Hz), 1.86 (t, 3H, H-82 J= 7.5 Hz), —3.75 (br s, 2H, NH)UV/Vis ‘max (CH2C1)404 nm (E 108 000), 506 (7 500), 544 (9 800), 572 (5 600), 630(1 500)LRFABMS (m/z): 533 (M, 100%)HRFABMS: C34H7N402(Mj: calcd 533.2916obsd 533.29162766.4.3 Synthesis of [A,C]-Divinylporphyrin 147 via Porphyrin 233-Vinyirhodoporphyrin XV methyl ester (157)1561573-Vinyirhodoporphyrin XV dimethyl ester (23) (850 mg) in warm pyridine (175mL) was refluxed during 4 hours with a solution of potassium hydroxide (20 g) in water(25 mL) and methanol (150 mL). The solution was cooled, diluted with iced water (1 L),acidified with concentrated sulphuric acid (15 mL) in water (100 mL), and then stirred for10 mm. The precipitated porphyrin diacid was filtered off on Celite, and washed withwarm water followed by dry methanol. A mixture of dry methanol (650 mL) andconcentrated sulphuric acid (20 mL) was passed slowly through the bed of Celite, therebydissolving the porphyrin, and the resultant solution was kept at room temperature in thedark for 16 hours before addtion to water (1.5 L). The porphyrin was extracted withmethylene chloride (3 x 500 mL) and the extracts were washed with water (1 L), driedover anhydrous MgSO4, and evaporated. The residue was recrystallized fromTHFfbenzene to give the title porphyrin monocarboxylic acid (605 mg, 70%) as a darkred powder.M.p.: >300°C [lit.156 >300°C]277.1H NMR (400 MHz, CF3OOD) ö: 11.90, 11.20, 11.00, 10.95 (4s, 4H, H-5, H-10, H-15and H-20), 8.28 (dd, 1H, H-31 J = 18.3 and 11.0 Hz), 6.65 [d, 1H, H-32(Z), J = 11.0Hz], 6.40 [d, 1H, H-32(E), J = 18.3 Hz], 4.62 (t, 2H, H-171), 4.25 (q, 2H, H-81), 4.19,3.80, 3.78, 3.76, 3.72 (5s, 15H, H-2’, H-71 H-121, H-181 and H-174), 3.40 (t, 2H, H-172), 1.82 (t, 3H, H-82)UV/Vis ‘umax (CH2C1)408 nm (E 154 000), 514 (7 900), 554 (16 300), 578 (9 500)[lit.156 ?max (pyridine) 409 nm (E 189 000), 511(10 000), 552 (17 200), 579 (9 200);?max (CHC13-HC1) 427 nm (E 206 000), 569 (12 000), 620 (12 900); max (0.1 MNaOMe-MeOH) 399 nm ( 146 000), 503 (10 500), 550 (12 000), 572 (6 700), 624 (1900)]LREIMS (mlz): 550 (M, 100%), 506 (M-CO2,30%)HREIMS: C33H4N40(M): calcd 550.2580obsd 550.2580Anal. calcd forC33H4N40: C, 71.98; H, 6.22; N, 10.17 %found: C,71.93;H,6.16;N, 9.86%13-Imidazoyl-3-vinylrhodoporphyrin XV methyl ester (158)158N HN— /O2Me278A mixture of 3-vinyirhodoporphyrin XV methyl ester (157) (600 mg, 1.09 mmol)and N,N’-carbonyldi-imidazole (600 mg) was refluxed during 30 mm in THF (125 mL).The solution was evaporated to about 60 mL and then applied to a short alumina column(Brockman Grade V; elution with methylene chloride). The appropriate eluates wereevaporated and the residue was recrystallized from CH2lIbenzene to give the titleporphyrin (595 mg, 91%) as a dark red powder.M.p.: 239°C1H NMR (400 MHz, CDC13)6: 10.01, 9.97, 9.88, 9.70 (4s, 4H, H-5, H-b, H-15 and H-20), 8.38 (s, 1H, H-imidazole), 8.12 (dd, 1H, H-3’, J = 17.5 and 12.0 Hz), 7.95 (s, 1H, Himidazole), 7.35 (s, 1H, H-imidazole), 6.29 [dd, lH, H-32(E), J = 17.5 and 1.5 Hz], 6.15[dd, 1H, H-32(Z), J= 17.5 and 1.5 Hz], 4.25 (t, 2H, H-171J=8.1 Hz), 4.00 (q, 2H, H-81J =8.0 Hz), 3.67, 3.65, 3.55, 3.53, 3.51 (5s, 15H, H-21 H-71, H-12’, H-181 and H-174),3.18 (t, 2H, H-172J= 8.1 Hz), 1.80 (t, 3H, H-82 J= 8.0 Hz), —4.30 (br s, 2H, NH)UV/Vis max (CH2C1)408 nm (E 160 000), 514 (6 900), 554 (15 300), 574 (9 900)LRFABMS (m/z): 601 ([M+H], 73%), 533 (M-CHN2,100%)LREIMS (m/z): 533 (M-CHN2,100%)HRFABMS: C36H7N604([M+H]j: calcd 601.2927obsd 601.2929HREIMS: C33HN40(M-C3HN2):calcd 601.2927obsd 601.292927913-Decarboxy-13-methoxycarbonylacetyl-3-vinylrhodoporphyrin XVmethyl ester (160)160A solution of 2-PrMgBr was prepared by refluxing under dry nitrogen a mixtureof Mg turnings (1.2 g) and 2-PrBr (4 g) in freshly distilled THF (150 mL). When themetal had dissolved the solution was cooled to 0°C and redistilled methyl hydrogenmalonate158 (3 g) in dry THF (30 mL) was added via a cannula. The mixture (i.e. 159)was warmed to 65°C and stirred for 10 mm before introduction of a solution of 13-imidazoyl-3-vinylrhodoporphyrin XV methyl ester (158) (400 mg) in dry THF (200 mL).Stirring was maintained for 2.5 hours while heating under reflux, and glacial AcOH (15mL) was added. Heating and stirring were continued for a further 15 mill after which themixture was diluted with chloroform (1400 mL), washed with 0.1 M HC1 (1100 mL) andwater (2 x 800 mL), and then dried over anhydrous Na2SO4 and evaporated to dryness.The residue was chromatographed on alumina V (elution with 5% acetone in methylenechloride) and after evaporation of the appropriate eluates the product was recrystallizedfromCH2llhexane to give the required porphyrin-B-ketoester 160 (280 mg, 70%) as adark red solid.M.p.: 270-274°C [lit.156 250-254°C]02801H NMR (400 MHz, CDC13) 6: (the enol tautomer) 13.30 (s, 1H, OH), 10.62, 10.62,10.42, 9.75 (4s, 4H, 11-5, H-10, H-15 and H-20), 6.10 (s, 1H, H-132); 6: (the ketonetautomer) 9.98, 9.97, 9.86, 9.85 (4s, 4H, H-5, H-b, H-15 and H-20), 4.70 (s, 2H, H-132);6: (their mixture) 8.10 (m, 111, H-31), 6.25 [m, 1H, H-32(E)], 6.12 [m, 1H, H-32(Z)], 4.41(t, 2H, H-171), 3.98 (q, 2H, H-8’), 4.02-3.45 (6s, 18H, H-21 H-71, H-12’, H-181 H-174and132-COOCH3),3.25 (t, 2H, H-172), 1.83 (t, 3H, H-82), —4.26 (br s, 1H, NH), —4.30(br s, 1H, NH)UV/Vis max (CH2C1)410 nm (E 140 000), 512 (7 400), 552 (14 000), 576 (9 200)[lit.156 max (CH2I)409 nm (E 176 000), 512 (7 200), 553 (15 400), 574 (9 900), 635(1 300); max (CHC1- F3OOH) 412 nm (E 267 000), 559 (12 200), 608 (8 600);max (0.1 M-NaOMe-MeOH) 401 nm ( 154 000), 504 (10 500), 542 (12 400), 572 (6900), 625 (2200)]LRFABMS (m/z): 607 ([M+H], 16%)HRFABMS: C36H9N405([M+H]): calcd 607.2920obsd 607.291928113-Acetyl-13-decarboxy-3-vinylrhodoporphyrin XV methyl ester (161)(I161N HN— /O2Me IA mixture of porphyrin-B-ketoester 160 (180 mg, 0.297 mmol), 4-(dimethylamino)pyridine (36 mg, 2.92 mmol), 1.0 M phosphate buffer (pH = 7) (40 mL),and toluene (150 mL) was stirred at 90°C for 12 hours at which time TLC analysisshowed the full disapperance of the porphyrin-B-ketoester band and apperance of a moremobile red band. The reaction mixture was extracted with ethyl acetate (250 mL) and theorganic layer was washed once with saturated NH4CI, twice with water (200 mL), driedover MgSO4, filtered and evaporated. The residue was chromatographed on silica Ill(elution with methylene chloride) to give a red major band. The appropriate eluates wereevapoarted and the residue was recrystallized from CH2llhexane to give the titleacetylporphyrin (122 mg, 75%) as purple-red small leaves.M.p.: 284°C1H NMR (400 MHz, CDC13)ö: 10.75, 10.10, 10.07, 9.98 (4s, 4H, H-S. H-b, H-15 andH-20), 8.20 (dd, 1H, H-3’, J = 17.4 and 12.5 Hz), 6.30 [d, 1H, H-32(E), J = 17.4 Hz],6.12 [d, 1H, H-32(Z), J= 12.5], 4.43 (t, 2H, H-171 J= 7.5 Hz), 4.09 (q, 2H, H-81 J=8.8Hz), 3.88, 3.67, 3.65, 3.61, 3.61 (5s, 15H, H-21 H-71,H-121,H-181 and H-174), 3.33 (t,2H, H-172 J= 7.5 Hz), 3.32 (s, 3H, H-132), 1.85 (t, 3H, H-82 J= 8.8 Hz), —3.80 (br s,2H, NH)282UV/Vis Amax (CH2C1)410 nm (E 147 000), 514 (7 400), 554 (13 800), 576 (8 600), 608(1 300)LRFABMS (mlz): 549 ([M+H], 100%)HRFABMS: C34H6N40([M+Hjj: calcd 549.2865obsd 549.286113-Decarboxy-13-(o-hydroxy)ethyl--3-vinylrhodoporphyrin XV methylester (162)162To a mixture of the foregoing porphyrin 161 (100 mg, 0.182 mmol) in methylenechloride (80 mL) and methanol (15 mL) was added sodium borohydride (80 mg). Themixture was stirred for 90 mm at room temperature, after which time TLC monitoringindicated reduction of the acetyl group to be complete. The reaction was quenched byslow addition of ice-cooled 1 M HC1. The organic layer was separated, washed twicewith water, dried over MgSO4, filtered and evaporated. The residue waschromatographed on silica V (elution with 0.5% methanol in methylene chloride) to givea red major band. The appropriate eluates were evaporated and the residue wasrecrystallized fromCH21/hexane to give the title hydroxyporphyrin (82 mg, 82%) as apurple solid.283M.p.: 2 19°C1H NMR (400 MHz, CDC13)6: 10.18, 10.00, 9.88, 9.88 (4s, 4H, H-5, H-10, H-15 and H-20), 8.20 (dd, 1H, H-31 J = 17.1 and 11.4 Hz), 6.31 [dd, 1H, H-32(E), J= 17.1 and 1.9Hz], 6.15 [dd, 1H, H-32(Z), J= 12.5 and 1.9 Hz], 4.27 (t, 2H, H-171J= 8.5 Hz), 4.00 (q,2H, H-81 J=8.2 Hz), 3.58, 3.58, 3.54, 3.51, 3.49 (5s, 15H, H-21H-71,H-121,H-181 andH-174), 3.45 (q, 1H, H-131 J 7.0 Hz), 3.16 (t, 2H, H-172 J = 8.5 Hz), 2.72 (br s, 1H,OH-131), 2.14 (d, 3H, H-132 J= 7.0 Hz), 1.81 (t, 3H, H-82 J= 8.2 Hz), —4.30 (hr s, 2H,NH)UVN1s max (CH2C1)404 nm (e 178 000), 502 (10 600), 540 (12 000), 572 (9 300),624 (2700)LRFABMS (mlz): 551 ([M+H], 100%)HRFABMS: C34H8N40([M+H]): calcd 551.3022obsd 551.301113-Decarboxy-3,13-divinylrhodoporphyrin XV methyl ester (147)—147The foregoing hydroxyporphyrin 162 (55 mg, 0.1 mmol) was dissolved in dryDMF (20 mL) and stirred at 105°C under nitrogen. Benzoyl chloride (1 mL) was addedto this solution via a syringe and the mixture was kept at this temperature for 2 hours.284The solution was then diluted with methylene chloride (100 mL), and successivelywashed with 2M sodium hydroxide (75 mL) and water (2 x 100 mL). The organic layerwas dried over MgSO4, filtered and evaporated in vacuo. The residue waschromatographed on silica III (elution with methylene chloride) to give a major red band.The appropriate fractions were collected, evaporated and recrystallized fromCH21/hexane to give the title divinylporphyrin 147 (31.9 mg, 60%) as a purple-redsolid. This material was found identical to the compound prepared from the Wittigreaction of 13-formylporphyrin 148. The corresponding physical data of this compoundare shown in section Synthesis of [A,C]-Dibenzoporphyrin Derivative 16521,212-Tetrakis(methoxycarbonyl)-8-ethyl-17-methoxycarbony-lethyl-2,7,12,18-tetramethyl-2312-dihydrobenzo[b,1]porphyrin (164)MeOOC....ç(M.OOC164NH N COOMeO2MeCOOMeThe foregoing 3,13-divinylporphyrin 147 (30 mg, 0.0564 mmol) and dimethylacetylenedicarboxylate (800 mg, 5.6 mmol) were suspended in degassed toluene (N2) (20mL) and the mixture heated at 110°C for 80 hours. After removal of the toluene in vacuo,the residue was chromatographed on silica (methylene chloride eluant). After elution ofthe excess dienophile, the solvent polarity was increased to 2% methanol in methylene285chloride to elute the major reaction product. After evaporation to dryness, the resultingresidue was subjected to preparative TLC on silica (1 mm thick plate; elution with 2%methanol in methylene chloride). The more mobile band was found to be the monoadduct (showing a band at 656 nm) in negligible amount; the major band was found to bethe bis-adduct which was recrystallized fromCH21/hexane to afford the bis-adduct 164as a green solid (10.8 mg, 25% yield).M.p.: 198°CUVIVis 2’max (CH3COCH)406 nm (peak ratio, 1.10), 484 (0.21), 538 (0.15), 666 (0.13),720 (0.338)LREIMS (mlz): 817 (M, 100%)1,212-Tetrakis(methoxycarbonyl)-8-ethyl-17-methoxycarbonyl-ethyl-2,7,12,18-tetramethyl-2-dihydrobenzo[b,1]porp rin (165)MeOOC.1-,MeOOC’ “N HN..( ‘/) 165OOMeCOOkIeCO2MeThe bis-adduct 164 (9 mg, 0.011 mmol) was dissolved in methylene chloride anda few drops of DBU were added. The reaction mixture was stirred in the dark andmonitored by visible spectroscopy (completed in 16 hours). The mixture was poured into1M HC1 and extracted with methylene chloride. The organic layer was washed twicewith brine, once with water and dried over anhydrous magnesium sulfate. The solvent286was evaporated in vacuo and the compound was chromatographed on silica gel (1%MeOH in CH21 eluent). The pure 1 ,3-cyclohexadiene fraction was evaporated in vacuoyielding 8.0 mg (90% yield) of a dark green solid after recrystallization fromCH2Cl2lhexane.M.p.: 289-291°C1H NMR (400 MHz, CDC13)6: 9.15, 9.15, 8.74, 8.74 (4s, 4H, H-5, H-b, H-15 and H-20), 7.82 (d, 2H, H-24 and H-124,J 7.8 Hz), 7.35 (d, 2H, H-23 and H-123,J = 7.8 Hz),4.95 (s, 2H, H-21 and H-12’), 4.10 (t, 2H, H-171 J= 8.1 Hz), 3.98 (q, 2H, H-81 J= 7.7Hz), 3.25 (t, 2H, H-172J= 7.7 Hz), 3.90, 3.86, 3.63, 3.60, 3.32, 3.01 (6s, 18H, H-71Hb8, H-174 2’-COOCH3,2-COOCH3,12’-COOCH3and 122-COOCH3),1.81 (s, 6H,2-CH3), 1.74 (t, 3H, H-82 J= 7.7 Hz), —1.90 (br s, 2H, NH)UV/Vis max (CH2C1)468 nm (E, 81 000), 620 (20 900), 700 (4700), 784 (38 000)LRFABMS (mlz): 817 ([M+H], 100%)HRFABMS: C40H2N08(M): calcd 817.3455obsd 817.34492876.5 Nucleophilic Reactions of DBU and DBN3,4,5,6,7,8,9-Heptahydro-1-oxo-2a,5a-diazacyclohepta[cd]indan-2[2(Z)-methoxycarbonyl]-methylene (176)176/1COOMTo a solution of dimethyl acetylenedicarboxylate (1.42 g, 0.01 mol) in chloroform(10 mL) at room temperature was added, a solution of DBU (1.52 g, 0.01 mol) inchloroform (10 mL). The golden-red solution was stirred at room temperature for 5 mm.Removal of solvent, followed by recrystallization from CH21/hexane, gave the titlecompound (2.51 g, 96% yield) as red prisms.M.p.: 158°C1H NMR (200 MHz, CDC13) & 5.94 (s, 1H, H-21), 3.98 (t, 2H, H-3), 3.33 (s, 3H,OCH3), 3.30 (t, 2H, H-5), 3.23 (dd, 2H, H-6), 2.22 (dd, 2H, H-9), 2.05 (m, 2H, H-4), 1.95(m, 2H, H-7), 1.86 (m, 2H, H-8)13C NMR (50 MHz, CDCI3)6: 179.21, 166.84, 165.64, 145.25, 93.57, 93.31, 54.39,51.44,48.96, 42.32, 28.06, 25.52, 22.32, 20.47LRFABMS (mlz): 263 ( [MHJ, 100%)LREIMS (mlz): 262(M, 100%)288HREIMS: C14H8N203([M]): calcd 262.1317obsd 262.1309Anal. calcd forC14H8N203: C, 64.09; H, 6.92; N, 10.50 %found: C, 64.04; H, 6.86; N, 10.50 %A single crystal suitable for X-ray diffraction was obtained by slow diffusion of nhexane into a concentrated CHC13 solution of compound 176.Crystal data for 176: Red prism,C14H8N203M = 262.31, triclinic, space groupP1 (No. 2), a = 12.066(1), b = 12.946(2), c = 9.0900(8) A, a = 103.695(9), 13 =103.828(8), ‘y = 101.68(1)°, V = 1288.1(3) A3, Z = 4 (two molecules in the asymmetricunit), Dc 1.190 g cm3. The final unit-cell parameters were obtained by least-squaresanalysis on the setting angles for 25 reflections with 20 = 110.5-114.6°. The intensitiesof three standard reflections were measured every 200 reflections throughout the datacollection: no decay correction was necessary. Data were corrected for Lp and absorption(azimuthal scans for three reflections, relative transmission factors 0.86-1.00). Thestructure was solved by direct methods. Non-hydrogen atoms were refined withanisotropic thermal parameters and hydrogen atoms were refined with isotropic thermalparameters. An isotropic Zachariasen type I secondary extinction correction was applied,the final value of the extinction coefficient being 5.58(6) X iO. The refinementconverged at R = 0.042 and R = 0.047 for 4303 independent reflections with 1 3a(1).Calculations were performed using the teXsan structure analysis package (MolecularStructure Corporation, 1985 & 1992).289Typical Procedure of Nucleophilic Reaction of DBU and DBN withPheophorbide a Methyl Ester (7)To a solution of pheophorbide a methyl ester (7) (60.7 mg, 0.1 mmol) andimidazole (10 mg) in dry THF (15 mL) under nitrogen, TMSOTf or TBDMSOTf (0.3mmol) was injected via a syringe. After 15 mm at room temperature, DBU (—1.Oml, 6.7mmol) or DBN (—0.83ml, 6.7 mmol) was added slowly to this blue-gray solution. Thebrown-red mixture was then stirred in the dark for 5 h and was poured into an ice-cooledmixture of saturated NH4C1 (30 mL) and dichloromethane (50 mL). The organic layerwas washed with water (2 x 30 mL), saturated aqueous sodium bicarbonate (30 mL) andwater (2 x 30 mL), dried over anhydrous sodium sulfate, filtered and evaporated in vacuo.The product was purified by flash chromatography on silica III, first eluting withdichloromethane to give unreacted starting material 7 (20-25% yield) and further elutingwith 1.5% methanol in dichioromethane to give the products. The products wererecrystallized from dichloromethanelhexane.Chlorin e6 13- [1 -(3-N-propyl)2-azacycloheptanone]amide-15,17-dimethyl ester (185)NH NN HN’185O2MeDCO2Me45.3 mg (58% yield) as a green powder.290M.p.: 124°C1H NMR see Table 5.1.l3 NMR see Table 5.2.UV-Vis max(CHC13)404 nm (E 171 900), 502 (15 100), 528 (5 000), 610 (5 700), 666(48 700)LRFABMS (mlz): 777 ( [MH], 100%), 579 ([MHJ—C10H8N2O76)LREIMS (mlz): 776 (M, 73%), 744 (M—CH3OH, 20), 717 (M—COOCH3,37),606 (M—C9H18N20, 51)HRFABMS: 45H576O([MH]): calcd 777.4339obsd 777.4365HREIMS: C45H56N6O(M): calcd 776.4261obsd 776.4267Anal. calcd forC45H56N60: C, 69.55; H, 7.27; N, 10.82 %found: C, 69.77; H, 7.60; N, 10.52 %Chiorin e6 13- [1-(3-N-propyl)2-pyrrolidinone]amide-15,17-dimethylester (186)(Ii,N HN186CO2Me38.7 mg (52% yield) as a green powder.291M.p.: 127°C1H NMR see Table 5.1.l3 NMR see Table 5.2.UV-Vis max(Q13)404 flffi (E 162 000), 502 (12 500), 528 (2 800), 610 (3 600), 666(45 000)LRFABMS (mlz): 749([MH], 100%), 579([MH]—C8H14N2O43)HR1?ABMS: C43H5N60([MH]j: calcd 749.4026obsd 749.4017Anal. calcd forC43H52N60: C, 68.95; H, 7.00; N, 11.23 %found: C, 68.80; H, 7.11; N, 11.04%Chiorin e 13-(2-N-ethyl)amide-15,17-dimethyl ester (179)(IN HNi179.wçCO2MeCO2MeTo a solution of pheophorbide a methyl ester (7) (30 mg, 0.05 mmol) in THF (10mL) at room temperature under nitrogen, ethylamine (20 mL) was added and the mixturewas allowed to react in the dark for 25 h before the solvents were evaporated in vacuo.The product was purified by preparative TLC on silica gel (developed by 2% methanol indichloromethane). Recrystallization of the product from dichlomethane/hexane gave thetitle compound (20.5 mg, 63%) as a green solid.292M.p.: 134°C1H NMR see Table 5.1.l3 NMR see Table 5.2.UV-Vis max(C112C1)402 nm (E 194 000), 500 (16 900), 528 (5 000), 608 (5 600), 664(52 000)LRFABMS (mlz): 652( [MH],100%), 580([MH]—CONHEt, 19)HRFABMS: C38H46N50([MH]j: calcd 652.3499obsd 652.3475Anal. calcd forC38H45N50 C, 69.90; H, 7.11; N, 10.73 %found: C, 69.55; H, 6.85; N, 10.76 %Nucleophilic reaction of 1-(3-aminopropyl)2-pyrrolidinone withpheophorbide a methyl ester (7)To a solution of pheophorbide a methyl ester (7) (15 mg, 0.025 mmol) in THF (5mL) at room temperature under nitrogen was added, l-(3-aminopropyl)2-pyrrolidinone(tech., Aldrich) (2 g) in THF (10 mL). The mixture was allowed to react in the dark for30 h and poured into a mixture of saturated sodium chloride and dichloromethane. Theorganic layer was washed with water (3 x 30 mL), dried over anhydrous sodium sulphate,filtered and evaporated. 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