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Investigations towards the synthesis of vinblastine and vinblastine analogs Pedersen, Ove 1992

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INVESTIGATIONS TOWARDS THE SYNTHESIS OF VINBLASTINE AND VINBLASTINE ANALOGS by OVE PEDERSEN M.Sc.,  University of Copenhagen,  1984.  A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January 1992 ©Ove Pedersen,  1992  In presenting this thesis in  partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Chemistry  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  March 5.  1992  ii  ABSTRACT  This dissertation describes research towards a feasible synthesis  of  vinbiastine  1,  coupling  by  catharanthine analog with vindoline 4. reinvestigation undertaken  in  of  order  the  modified gain  to  a  suitable  In this connection a  Polonovski  better  a  of  reaction  understanding  of  was the  factors affecting the yield of the reaction. The Diels-Alder reaction of the methyl a-chloroacrylate 31 with the dihydropyridine 30 afforded the isomeric Diels Alder adducts 32 and 33.  The two adducts were separated and  transformed into the indole-amides 40 and 51 as well as the corresponding  thioamides.  Photochemical  cyclization  of  the  amides and the thioamides gave lactam 55 and thiolactam 61. However,  with  a best  overall  yield  of  7%  of  lactam  55  the  investigated strategy did not lead to a practical synthesis of vinbiastine. The  low  catharanthine  yields derivatives  reinvestigation using  obtained  fractional  of  the  with  in  vindoline  modified  factorial  the 4  Polonovski  design  as  coupling necessitated reaction.  experimental  of a In  strategy  and exocatharanthine 89 as a suitable catharanthine analog, a new intermediate was  discovered and  the yield of dimeric  alkaloid was improved from 35% to 70%. The  better  influencing  the  understanding yield  of  the  obtained  modified  the  factors  Polonovski  reaction  of  iii  should now make it possible to couple catharanthine analogs with vindoline 4 in good yields.  H .C H 3 3 CH 3 CH  3 CH  1  3 CH  C  4  h H0CH3  30  31  iv  0 Ph  P hON  OIl  0  c’  CH  CH 2 CO 3  1 32  33  C  3 H  Cl  c 2 c0  H 3 3 H  51  40  0  H  H  CH 3  3 H 55  89  V  TABLE OF CONTENTS Title Page  i  Abstract  ii  Table of Content  V  List of Figures  Viii  List of Schemes  ix  List of Tables  X  List of Abriviations  Xiii  Acknowledgements 1.  2.  Introduction 1.1. Background 1.2. Biosynthetic Considerations 1.3. Synthetic Considerations 1.4. Synthetic Strategy Chosen for a Feasible Synthesis of Vinbiastine 1 Results 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. 2.8. 2.9. 2.10.  2.11.  3.  XV1  1 1 3 5 9  and Discussion 14 Synthesis of the Isoquinuclidine Skeleton 14 Transformation of the Endo Isomer 32 18 Transformation of the Exo Isomer 33 27 Photochemical Cyclization of the Amides 40 and 51, and of the Thioamides 41 and 52 37 Evaluation of the Chosen Strategy 57 The Modified Polonovski Reaction 58 Synthesis and Characterization of Exocatharanthine 89 65 Investigation of the Formation of Catharanthine N-oxide 10 and Exocatharanthine N-oxide 90 . . .84 Reinvestigation of the Modified Polonovski Reaction 91 Identification and Characterization of the Second Intermediate 99 in the Modified Polonovski Reaction 111 Preliminary Investigation of the Reactivity of the 19’, 20’-Double Bond in 19’, 20’Anhydrovinblastine 91 126  Experimental 3.1. General Experimental Conditions 3.2. Catharanthine N-oxide 10 3.3. N-Benzyloxycarbonyl-l, 2-dihydropyridine 30. 3.4. a-Chloro methyl acrylate 31  .  .  135 135 137 .138 140  vi 3.5.  3. 6.  3.7.  3.8.  3.9.  3.10.  3.11.  3.12.  3.13.  N-Benzyloxycarbonyl-endo-7-methoxycarbonyl7-chloro-2-azabicyclo [2,2,2] octan-5-ene 32 and N-Benzyloxycarbonyl-exo-7 —methoxycarbonyl 7-chloro-2-azabicyclo [2,2,2] octan-5-ene 33. .142 N-Benzyloxycarbonyl-encZo-7-methoxycarbonyl-7-chloro-2-azabicyclo [2,2,2] octan-endo-6-ol 34 and N-Benzyloxycarbonyl-endo- 7-methoxycarbonyl-7-chloro-2-azabicyclo [2,2,2] octan endo-5-ol 35 146 N-Benzyloxycarbonyl-endo-7--methoxycarbonyl-7-chloro-2-azabicyclo [2,2,2] octan-6-one 36 and N-Benzyloxycarbonyl-endo-7-methoxycarbonyl-7-chloro-2-azabicyclo [2,2,2] octan 5-one 37 150 N-Benzyloxycarbonyl-endo-7-methoxycarbonyl-7chloro-2-azabicyclo [2,2,2] octan-6-one 2’, 2’ -dimethyl-1’ , 3’ -propanediyl acetal 38.. . .154 N-(3’ ‘-Indolylmethylenecarbonyl)-endo-7methoxy-carbonyl -7 -chloro-2 -azabicyclo [2,2,2] octan-6-one 2’,2’-dimethyl-1’,3’propanediyl acetal 40 156 N-(3’ ‘-Indolylmethylenethiocarbonyl )-endo-7methoxy-carbonyl-7-chloro-2-azabicyclo [2,2,2] octan-6-one 2’, 2’ -dimethyl-1’ .3,propanediyl acetal 41 158 N-Benzyloxycarbonyl-exo-7-methoxycarbonyl-7chloro-2-azabicyclo [2, 2,2] octan-endo-6-ol 44 and N-Benzyloxycarbonyl-exo-7-methoxycarbonyl7-chloro--2-azabicyclo [2,2,2] octan endo-5-ol 45 and N-Benzyloxycarbonyl 7-methoxycarbonyl-2-azatricyclo [2,2,2,06,7] octane 46 160 N-Benzyloxycarbonyl-exo--7-methoxycarbonyl-7chloro-2--azabicyclo [2,2,2] octan-6-one 47 and N-Benzyloxycarbonyl-exo-7-methoxycarbonyl—7-chloro-2-azabicyclo [2,2,2] octan 5-one 48 and N-Benzy1oxycarbony--methoxycarbonyl-2-azatricyclo [2,2,2,0 ‘ ] octane 46 165 N-Benzyloxycarbonyl-exo-7-methoxycarbonyl-7chloro—2--azabicyclo [2,2,2] octan-6-one 2’, 2’ -diinethyl-1’ , 3’ -propanediyl acetal 49.. .168 N-(3’ ‘-Indolylmethylenecarbonyl)-exo-7methoxycarbonyl -7 -chioro- 2- azabicyclo [2,2,2] octan-6-one 2’ ,2’-dimethyl-l’ 13’propanediyl acetal 51 171 N-(3’ ‘-Indolylmethylenethiocarbonyl)-exo-7methoxycarbonyl-7-chloro-2-azabicyclo [2,2,2] octan—6-one 2’, 2’ -dimethyl-1’,3’propanediyl acetal 52 174 20-Desethyl-15,20-dihydro-5-oxo-catharanthin20-one 2’ ,2’ -dimethyl-1’ ,3’- propanediyl acetal 55 and the Isomeric By-product 56 177 .  3.14.  3.15.  3.16.  vii 3.17.  20-Desethyl-15, 20-dihydro-5-thioxo-catharanthin20-one 2’ ,2’-dimethyl-l’ propanediyl acetal 61 and the Isomeric By-product 62 180 3.18. Exocatharanthine 89 183 3.19. Exocatharanthine N-oxide 90 185 3.20. 19’,20’-Anhydrovinblastine 91 and Epi 19’ , 20’ -anhydrovinbiastine 94 187 3.21. 7-Hydroxy cathraranthine N-oxide 92 193 3.22. 7-Hydroxy exocatharanthine N-oxide 93 195 3.23. 4’-Benzyl-19’ ,20’-anhydrovinblastine 108 197 3.24. 19’-Hydroxy vinbiastine 109 and 19’-Hydroxy leurosidine 110 198 3.25. Bis-indolic mesylate 112 from the Mesylation of 19’-Hydroxy vinbiastine 109 203 3.26. General Experimental Conditions for the Optimization of the Photochemical Cyclization of the Amides 40 and 41 and the Thioamides 51 and 52 205 3.27. General Experimental Conditions Used in the Optimization of the Yield of Exocatharanthine 89 206 3.28. General Experimental Conditions Used in the Investigation of the Formation of Catharanthine N-oxide 10 and Exocatharanthine N-oxide 90 .207 3.29. General Experimental Procedure Used in the Systematic Investigation of the Modified Polonovski Reaction 208 3.30. Preparation of the Second Intermediate 99 212 13’-  4.  References  214  5.  Appendix 5.1. Factorial Design 5.2. Fractional Factorial Design  220 220 224  viii  List of Figures  Figure 2-1. Figure Figure Figure Figure  2-2. 2-3. 2-4. 2-5.  Figure 2-6. Figure 2-7.  Figure 2-8.  Figure 2-9. Figure 2-10. Figure 2-11.  Figure 2-12. Figure 2-13.  Figure 2-14. Figure 2-15.  Figure 2-16.  Figure 3-1.  ‘H NMR spectra of the Diels-Alder adduct 33 in DMSO-d 6 17 X-ray diffraction analysis of ketone 36 20 X-ray diffraction analysis of thioamide 41.. .26 X-ray diffraction analysis of alcohol 45 29 X-ray diffraction analysis the tricyclic by product 46 30 The conformers 52A and 52B of thioamide 52.. .37 A graphic representation of the results of the fractional factorial designs in table 2-6 and 2-7 45 A graphic representation of the results of the fractional factorial design in table 2-9 52 The overall yields of cyclized products 55 and 61 from the Diels-Alder adducts 32 and 33. . .58 The structure of the isolated carbomethoxy dihydrocleavamines 84 The CD spectrum of 19’,20’-anhydrovinblastine 91 and epi 19’,20’-anhydrovinbiastine 94 93 Dehydration of vinblastine 1 94 Monitoring of the Polonovski reaction by HPLC 101 Possible structures of the second intermediate 99 112 -H NMR spectrum of the second intermediate 99 114 The structure of the mesylate decomposition product 112 determined by X-ray diffraction analysis 133 Apparatus used in the photolysis 205  ix  List of Schemes  Scheme 1-1. Scheme 1-2. Scheme 1-3. Scheme 1-4.  Scheme 1-5. Scheme 2-1. Scheme 2-2. Scheme 2-3. Scheme 2-4. Scheme 2-5. Scheme 2-6. Scheme 2-7. Scheme 2-8.  Scheme 2-9. Scheme 2-10. Scheme 2-11. Scheme 2-12. Scheme 2-13. Scheme 2-14. Scheme 2-15. Scheme 2-16. Scheme 5-1. Scheme 5-2. Scheme 5-3. Scheme 5-4.  Possible pathways for the biosynthesis of vinbiastine 1 4 Synthesis of 15’,20’-anhydrovinblastine 5 6 Hypothetical formation of vinblastine 1 by introduction of an ethyl group into a suitable ketone 9 Strategies used in the synthesis of catharanthine 3 and desethyl catharanthine 17 11 Synthetic strategy chosen for a feasible synthesis of vinbiastine 1 13 Synthesis of the diene 30 and the dienophile 31 14 Synthesis of the Diels-Alder adducts 32 and 33 15 Transformation of the Diels-Alder adduct 32.. 19 Synthesis of the amide 40 and the thioamide 41 22 Transformation of the Diels-Alder adduct 33 .28 Synthesis of the amide 51 and the thioamide 52 32 Photochemical cyclization of the amides 40 and 51, and the thioamides 41 and 52 38 Possible path ways in the photochemical cyclization reaction of aromatic a-chloro acetates 56 The mechanism of the Polonovski reaction 59 The pathways in the modified Polonovski reaction 60 The coupling of catharanthine 3 with vindoline 4 61 Possible mechanisms for the coupling of catharanthine 3 with vindoline 4 63 Coupling of exocatharanthine 89 with vindoline 4 92 The reaction pathways in the modified Polonovski reaction 100 Transformation of 15’20’-double bond in 15’,20’-anhydrovinblastine 5 126 Mesylation of the major diol 109 followed by decomposition to 112 134 The factors and their levels used in the factorial design in scheme 5-2 221 The factorial design 222 A three factor two level factorial design. .226 Fractional factorial design 227  x  List of Tables  Table 2-1. Table 2-2.  Table 2-3. Table 2-4.  Table 2-5.  Table 2-6.  Table 2-7.  Table 2-8.  Table 2-9.  Table 2-10. Table 2-li. Table 2-12. Table 2-13. Table 2-14. Table 2-15. Table 2-16. Table 2-17. Table 2-18. Table 2-19. Table 2-20.  Chemical shifts observed in the ‘ C APT 3 NMR spectrum of thioamide 41 24 Chemical shift correlations obtained in the HETCOR spectrum of thioamide 41 in the region 0-100 ppm 25 Chemical shifts observed in the C APT NMR spectrum of thioamides 52A and 52B 34 Chemical shift correlations obtained in the HETCOR spectrum of thioamide 52A in the region 0-100 ppm 36 Investigation of the effect of the nature of the solvent, the wave length and the temperature on the photochemical cyclization of amide 40 40 Investigation of the photochemical cycliza tion of amide 40 using fractional factorial design with four factors on three levels 41 Investigation of the photochemical cycliza tion of amide 51 using fractional factorial design with four factors on three levels 43 Investigation of the photochemical cycliza— tion of thioamide 41 using fractional factorial design with seven factors on two levels 48 Investigation of the photochemical cycliza tion of thioamide 41 using fractional factorial design with four factors on three levels 50 The photochemical cyclization of thioamide 52 compared to thioamide 41 53 Coupling of catharanthine derivatives with vindoline 4 64 Chemical shifts observed in the H NMR spectrum of exocatharanthine 89.4 68 Chemical shifts observed in the C APT NMR spectrum of exocatharanthine 89 69 Chemical shift correlations obtained in the HETCOR spectrum of exocatharanthine 89 70 Chemical shift correlations obtained in the COSY spectrum of exocatharanthine 89 71 Observed enhancements in SINEPT experiments on exocatharanthine 89 72 Observed enhancements in NOE experiments on exocatharanthin 89 72 Assignment of the -‘C chemical shifts of 3 catharanthine 3 77 Assignment of the H and chemical shifts of exocatharanthine 89 78 Conditions investigated for the metal catalyzed isomerization of catharanthine 3 to exocatharanthine 89 80  xi Table 2-21. Table 2-22.  Table 2-23. Table 2-24. Table 2-25.  Table 2-26. Table 2-27.  Table 2-28. Table 2-29.  Table 2-30.  Table 2-31.  Table 2-32. Table 2-33. Table 2-34. Table 2-35.  Table 2-36.  Optimization of the isomerization of catharanthine 3 to exocatharanthine 89 82 The factors and their levels used in the fractional factorial design in table 2-23 88 Investigation of the formation of 7-hydroxy catharanthine N-oxide 92 89 The influence of acid on the formation of 7-hydroxy catharanthine N-oxide 92 90 Investigation of the effect of the reaction temperature, the purity of the m chloroperbenzoic acid and the addition rate of TFAA on the yield of 19’,20’-anhydrovinblastine 91 96 The Polonovski reaction carried out at low temperature 97 Investigation of the effect of the amount of vindoline 4, the nature of solvent and the amount of TFAA on the yield of l9’,20’anhydrovinblastine 91 98 Addition of TFAA to exocatharanthine N-oxide 90 prior to the addition of vindoline 4 103 Addition of vindoline 4 prior to the addition of TFAA. The importance of concentrating the reaction mixture after the reaction has gone to “completion” at -65°C 105 The effect of concentrating (versus not concentrating) the reaction mixture after the reaction has gone to “completion” at -65°C on the yield and product distribution 107 The type of carbon signals expected for each of the five possible structures in figure 2-14, together with th actual types of carbons found in the APT spectrum for the second intermediate 99 113 Chemical shifts observed in the ‘H NMR spectrum of the second intermediate 99 115 Chemical shift correlations obtained in the COSY spectrum of the second interediate 99. .116 Chemical shifts observed in the C APT NMR spectrum of the second intermediate 99 117 Chemical shift correlations obtained in the HETCOR spectrum of the second intermediate 99 118 Observed enhancements in the SINEPT spectrum on the second intermediate 99. Iradiation at 7.39.ppm and observation in the C chemical shift region of 0 90 ppm 120 Observed enhancements in the SINEPT experiment of the second intermediate 99. Irradiation at 457 ppm and 4.09 ppm and observation in the chemical shift region of 80 175 ppm.. . .122 -  Table 2-37.  -  xii Table 2-38. Observed enhancements in the SINEPT experiment of the second intermediate 99. Irradiation at 7.73 ppm (H-9) and 746 ppm (H-12) and observation in the C chemical shift region of 83 175 ppm. 123 Table 2-3g. Assignment of the ‘H and ‘C chemical shifts to the second interediate 99 125 Table 2-40. Assignment of the C chemical shifts in vinblstine 1 and leurosidine 113 130 Table 2-41. The chemical shifts of the aliphatic methylenes and quaternary carbons groups in vinblastine 1, leurosidine 113 and the two diols 109 and 110 132 Table 5-1. Confounding of main effect and two-factor interactions in a fractional factorial design of three factors each on two levels. .227 -  .  xiii  List of Abbreviations:  Ac  Acetate  Addn  Addition  APT  Attached proton test  Ar  Aryl  Ar—H  Aromatic proton  t-BuOH  tert—Butanol  Caic  Calculated  CD  Circular Dichroism  COSY  Correlated spectroscopy  d  Doublet  2-D  Two-dimensional  DCC  1, 3-Dicyclohexylcarbodiimide  dd  Doublet of doublets  DMSO  Dimethyl sulfoxide  dt  Doublet of triplets  Et  Ethyl  Ether  Diethyl ether  N 3 Et  Triethyl amine  eq  Equivalent  evac  Evacuation  exo  Exocatharan-thine  exp  Experiment  G.  Don  Genus Don  HETCOR  Hetereonuclear correlation  HPLC  High pressure liquid chromatography  xiv  hrs  Hours  i  Iso  ipr N 2 H  Iso-propylamine  IR  Infared  m  Multiplet  m/z  Mass to charge ratio  Me-morph  N-Methylmorpholine  MeOH  Methanol  MHz  Mega hertz  zn-Cl PBA  zneta-Chloroperbenzoic acid  mp  Melting point  MS  Mass spectroscopy  nm  Nanometer  NMR  Nuclear magnetic resonance  NOE  Nuclear Overhauser effect  Nu  Nucleophile  PCC  Pyridinium chlorochromate  Ph  Phenyl  PLC  Preparative thin layer chromatography  ppm  Parts per million  p-TsOH  para-Toluene sulfonic acid  RT  Room temperature  5  Singlet  sec  Seconds  SINEPT  Selective insensitive nuclei enhancement by polarization transfer  solv  Solvent  xv  t  Tert  temp  Temperature  TFAA  Trifluoroacetic acid  THF  Tetrahydrofurane  TLC  Thin layer chromatography  UV  Ultraviolet  vac  Vacuum  VCR  Vincristine  VLB  Vinblastine  xvi  ACKNOWLEDGEMENTS  I wish to express my appreciation to Professor James P. Kutney for the opportunity to pursue this project and for his advice both during the progress of this research and in the preparation of this thesis. wish I to express my appreciation to the members of Dr. Kutney’s research group for their advice, enlightening discussions and friendship and in this connection I would especially like to thank Dr. Katalin Honty for her invaluable advice. I would also like to express my appreciation to colleagues my at Niels Clauson-Kaas Chemical Research Laboratory for their support throughout the preparation of this thesis. Finally, I wish to thank Dr. Mike McHugh for proof reading and criticism of this thesis and to thank Dr. Trotter, J. Department of Chemistry, University of British Columbia, for performing the X-ray diffraction analyses.  1  INTRODUCTION  1.  1.1.  BACKGROUND The  bisindole  Catharanthus  (vincaleukoblastine or VLB) or  VCR)  2  are  important  alkaloids  vinbiastine  1 and vincristine (leurocristine  anticancer  which  agents  are  used  routinely in the treatment of a number of human cancers.  CH 3 Ac  3 CF-I CH 2 0 3  Vinbiastine vincristine 2 the  leaves  =  3 CH  2 R  =  CHO  first  was  three years  of  Catharanthus  1  1 R  the  later in  periwinkle  roseus  isolated  although  19581,2  in  and  l961 by extraction from  Catharanthus native  to  roseus  Madagascar  G. has  Don. now  spread throughout the tropics . 4  Attention had been drawn to  this  reported use  an  plant oral  on the  basis  hypoglycemic  of  its  . 5 agent  The  in  reported  folklore as hypoglycemic  2  properties the  could  not,  investigations  oncologic  led  activity  culminating  in  isolated  to  of  the  21,2.  vincristine  however, the  some  Some  70  verified  clinically  discovery of  isolation  the of  5 roseus  of  plant  vincristine  only  100  2,  being  700 mg/kg  -  minor  dried  for  and  have  been  the  4  and  Vinbiastine 1  constituents,  leaves  1  vindoline  catharanthine 3 being the major constituents. and  extracts’,  alkaloids with  but  significant  vinbiastine  different  Catharanthus  from  be  found  are  former  and  in 6  -  30 mg/kg dried leaves for the latter . 6 The  major  treatment of used  use  Hodgkin’s disease  against  Hodgkin’s  clinical  childhood  . 79 lymphomas  of  vinbiastine  1  is  and vincristine 2  leukemia,  Wilm’s  The mechanism of  in  the  is mainly  tumor  and  action of  1  nonand  2  is not known but the activity is probably connected to the fact that they both are mitotic inhibitors causing metaphase arrest  in  dividing  . 0 1 cells  A  major  problem  with  both  alkaloids is their relatively high 7 toxicity ° . Vinbiastine 1  shows  mainly  hematologic  toxicity  and  vincristine  2  neurotoxicity. The structure of vincristine 2 was established in 1965 by  an  X-ray  vincristine  diffraction  . 1 methiodide-  study The  of  a  structure  single of  crystal  vinbiastine  followed from its known relationship to vinblastine 212.  of 1  3  1.2. BIOSYNTHETIC CONSIDERATIONS Since  catharanthine  alkaloidal proposed  3  and  4  vindoline  are  the  major  components of Catharanthus roseus Atta-Ur-Rahman  that  vinblastine  1  they and  might  the  be  biological  213.  vincristine  Feeding  precursors  of  experiments  by  Scott et al. with carbon-14 labelled vindoline 4 and tritium labelled  catharanthine  using  3  whole  roseus  Catharanthus  plants gave incorporation of labelled material into 15’,20’anhydrovinbiastine feeding  5  experiments  well  as  using  as  14a• 1  vinbiastine  apical  of  cuttings  Similar  Catharanthus  roseus plants gave incorporation of labelled catharanthine 3 and cell  vindoline free  labelled  4  into  extracts  vinbiastine  have  catharanthine  anhydrovinbiastine  also and  3  5151)  as  14b• 1  shown  Experiments  incorporation  the  vindoline  well  as  the  using  4  into  of  15’,20’-  transformation of  labelled 15’,20’-anhydrovinblastine 5 to vinbiastine Kutney  et  al  have  with vindoline 4  shown  using  that  cell  coupling  free  of  extract  catharanthine  3  initially results  7 7a (scheme 1in the formation of the iminium intermediate 1 1).  The  same  intermediate  anhydrovinbiastine 5  is  also  formed  when  15’,20’-  b. 7 is incubated in cell free extractsl  Furthermore it has been shown that the iminium intermediate 7 can be reduced to the enamine 12 which subsequently can be oxidized  to  vinblastine  17c,27,28 1  Whether  the  actual  biosynthesis of vinbiastine 1 in Catharanthus roseus plants proceeds via 15’,20’-anhydrovinblastine 5 or via the enamine 12 is still to be established.  4  3 H H  3 +  H  3 H  7 4 3 CH  H  H CH 2 3  INDOLINE  INDOLINE  5 1  I H  if  INDOLINE  12  Scheme 1-1. Possible vinbiastine 1.  pathways  for  the  biosynthesis  of  5  1.3.  SYNTHETIC CONSIDERATIONS The  as  the  first approach to these bisindole alkaloids, “chloroindolenine  approach”,  consisted  l613-carbomethoxy-2O3-dihydrocleavamine methoxycleavamine the  corresponding  6b  with  of  or  6a  7-chioroindolenine  8.  treating  1613-carbo-  hypochlorite  tert-butyl  known  to  Treatment  give of  8  H  Cl  6a t—BuOC 1 3 H  8a Sb: H  15,2O’  3 CH /HC1’MQH,  6b  /  3 H 3 CH  9a  9b:  15’,2O’  6  with 13,18  vindoline  under  acidic  conditions  gave  X-ray diffraction analysis revealed,  possessed the unnatural stereochemistry (R)  the  dimer  however,  that 9  at C-16’ . 19  CO H 0  •C H 3  10  3  1)  vindoline  2)  CO) 3 (CF 0 2  H  C 2 CH ’ —EO 1 °C Pc 3 CH  NaB H 4 7 3 H  3 CH CH 2 CO 3  5 Scheme 1-2.  Synthesis of 15’,20’-anhydrovinblas-tine 5.  7  The and by oxide  simultaneous discovery in  Potier et 10  , 21 al.  that  treatment of  with trifluoroacetic  vindoline  4  gave  by Kutney et  1975  anhydride  catharanthine N-  in the presence of  15’,20’-anhydrovinblastine  natural stereochemistry at C-16’,  5  However,  with  the  opened for the first time  a viable route to the synthesis of vinbiastine 1 2).  ° 2 al  despite considerable effort,  (scheme 1-  transformation of  the 15’,20’-double bond in 5 to either vinblastine 1 or it’s 20’  epimer leurosidine were unsuccessful . 2 ’ 22 3 The first reported synthesis of vinblastine 1 consisted  of coupling  the N-oxide of  the catharanthine derivative  11  with vindoline 4 and subsequent removal of the acetyl group at  the  20’  . 24 position  The  structure  11,  of  formed  in  a  11  modified however, study  Prévost  the  from  catharanthine  been questioned after it was found,  of  the  chemistry  catharanthine 3, of  reaction  of  the  C-15  and  325,  has,  in an extensive  C-20  positions  in  that nucleophilic approach from the a-side  quinuclidine  system  was  , 26 impossible  thus  drawing  the correctness of structure 11 into question. Vinbiastine 1  8  has  also been synthesized  either  with  chloride  from the enamine  thallium(III)  and  28 oxygen  27 acetate  followed  by  or  12,  treating it  with  reduction  iron(III)  with  sodium  borohydride. The later reaction has been reported to give an over-all  yield  of  40%  of  vinbiastine  1  based  on vindoline  428  Ti (OPc >3 1)  or 2 ’ 3 FeC1 0  H 2)  CH 3 3 CH  12  3 CH  H 3 C 2 CO H Pc  CH 3 CH  1  CH 2 0 3  4 NaBH  9  1.4.  SYNTHETIC STRATEGY CHOSEN FOR A FEASIBLE SYNTHESIS OF VINBLASTINE 1 In the investigation of the reactivity of the  double bond in 15’,20’-anhydrovinblastine 5  it was observed  that electrophiles always attacked the double bond 3-f ace of the piperidine ring system . 2 ’ 22 3  15’,20’-  from the  Assuming that the  3 H  E  H  5 observed  approach  bond in 5  of  electrophiles  to  the  15’,20’-double  is due to steric hindrance of the piperidine ring  system then addition of  an ethyl  group  to  the ketone  13  0 E  H  H  tNDOLINE  INOOLINE  13  1  Scheme 1-3. Hypothetical formation of vinbiastine 1 by introduction of an ethyl group into a suitable ketone.  in  10  scheme  1-3  support  should  for  lead  this  vinblastine  to  view  found  is  1.  the  in  Experimental synthesis  of  velbanamine 15 by Büchi et OH  3 H  LiEt  H  H  14  We ethyl  15  therefore  group  adopted  last  as  the  outlined  strategy in  of  scheme  introducing  1-3.  This  in  the turn  requires the synthesis of the catharanthine analog 16 shown below.  16  Several  syntheses  catharanthine  17  have  of been  catharanthine reported  in  3  the  and  desethyl  literature.  In  scheme 1-4 are summarized the different strategies employed in  the  formation  catharanthine formation strategies  of  3  and  the  have  of the  the final  isoquinuclidine ring  catharanthine been  applied  closure  skeleton. in  the  part  leading Two  formation  to  of the  different of  the  11  Bu 2 ) 3 (CH OSi t  CH 2 CO 3  CH 2 CO 3  Ph 2 0 21  19  /fl\ 1  H  CH 2 ;0 3  H3®  20  19  R=Et R=H  3 17  0 C H  CH 2 CO 3 22  24a 25a  H  R1 3 0CH  0 2 R R H 1 S 2 Et R 1 R  26  0 P C Ph 2 0 23  24b 25b  =PhCH R O 2 CO R = 1 H =CH 2 R O CO Et 3 1 R  27 28  Scheme 1-4. Strategies in used the catharanthine 3 and desethylcatharanthine 17.  R R  =  PhCH 2 OCO 2 PhCH  synthesis  of  12  isoquinuclidine strategies  part  of  231,  i O 3 ,  and  the 332  catharanthine  skeleton.  In  isoquinuclidine system  the  is  formed by an intermolecular or intramolecular reaction of a dienophile  attached  dihydropyridine. skeleton  is  to  with  a  suitable  1,2-  The final cyclization to the catharanthine  achieved  photochemical  indole  either  cyclization  through as  an  intramolecular  strategy  in  an  1,  intramolecular nucleophilic substitution as in strategy 2 or an  intramolecular  addition  reaction  as  in  strategy  3.  In  strategy 433,34 the isoquinuclidine unit was first built and then  attached  to  the  indole  tramolecular photochemical skeleton. part  of  system,  followed  by  an  in  cyclization to the catharanthine  In strategy 529,35 the carbomethoxy group is not a  the isoquinuclidine  system but  is  introduced after  the acid catalyzed cyclization to the ibogamine skeleton. Based on the background outlined above a retrosynthetic analysis steric 522  carried  was  requirements  and  velbanamine  out  as  observed 1529  outlined for  vindoline 4.  scheme  1-5.  The  15’,20’-anhydrovinblastine  dictated  as the catharanthine analog 16,  in  our  immediate  sub-target  which was to be coupled to  The resulting bisindole 14 was subsequently to  be transformed into vinbiastine 1. group could be  introduced  into  Alternatively,  the ethyl  the catharanthine analog  16  prior to the coupling with vindoline 4 as outlined in scheme 1-5. to was  The strategy chosen for the formation of 16 was similar  route  4  formed  in  scheme  first,  1-4  where  transformed,  the  isoquinuclidine  coupled  to  system  indole  and  13  finally  cyclized  to  give  the  desired  catharanthine  analog  16  H 3 C 2 CO H  INDOLINE  1  0  3 H H  OH CH 2 CO 3  NDOLINE  29  14  16  Ph  C0 CH 2 3 32/33  Scheme 1-5. Synthetic strategy chosen for a feasible synthesis of vinbiastine 1.  14  2.  2.1.  RESULTS AND DISCUSSION  SYNTHESIS OF THE ISOQUINUCLIDINE SKELETON  The necessary diene and dienophile for the formation of the  desired  isoquinuclidine  outlined in scheme 2-1.  skeleton  were  synthesized  as  Addition of benzyl chloroformate in  h  I  4 NaBH  OCOC1 2 PhCH  +  I  OH/—?8°C 3 CH  -  30 (867.)  3 H0CH +  HCHO  OH 3 CH  +  4 S 2 H / i00°C 0  (357.)  Scheme 2-1.  anhydrous  Synthesis of the diene 30 and the dienophile 31.  ether  borohydride . 36 yield present  in  The as  6.71,  suspension  a  anhydrous NMR  a mixture  The chemical at  to  spectrum of  shifts of  5.83,  5.53  methanol  and  two  of  pyridine  of at  -78°C  30  revealed  conformers  in  the olefinic protons 5.10 ppm  (for  and 30  in  86%  that  it  was  gave  a  sodium  ratio in 30  of  4:6.  appeared  the major conformer)  15  unambiguously showing 30 to be a 1,2-dihydropyridine. NMR of  30  also  indicated the presence of  1,4—dihydropyridine integral  of  multiplet  isomer  the  at  H—4  as  a  2.79 ppm , 37  the corresponding  contaminant.  protons, the  an  The ‘H  Based  the  recognizable  easily  content  on  of  the  1,4-  dihydropyridine isomer was calculated to be about 7%. The  dienophile  31  prepared  was  by  treatment  of  trichioroethylene with aqueous  formaldehyde and methanol in  concentrated  . 3 , 38 1000C 9  sulfuric  acid  at  The  structure  of  the dienophile 31 was established by a coupling constant of 1.5 Hz for the olefinic protons.  3 )CH  + .c1  30  31 benzene r e f’ 1 ux  0  3 C 2 ‘0 H 32  (357.)  Scheme 2-2.  +  33 (267.)  Synthesis of the Diels-Alder adducts 32 and 33.  16  The  Diels-Alder  benzene yield  gave  of  only  70%  reaction  two  (scheme  of  products, 2-2).  32  The  with  30  and  yield  31  in  33,  of  in  32  refluxing combined  a  and  after  33  separation by repeated column chromatography was 35% and 26% respectively.  The  ratio of  1.3:1  reflect the real  does not  ratio in which the two adducts were formed since compound 33 showed  some  conditions.  decomposition The  ‘H  NMR  under  spectra  the  of  chromatographic  the  two  Diels-Alder  adducts are relatively complicated due to splitting of most of  the  signals,  as  illustrated  compound 33 in figure 2-1.  by  ‘H  the  NMR  spectrum  of  Heating either compound to 120°C  .  in  6 DMSO—d  causes  establishing  that  the  this  splitting  not to the presence of isomers. the  ratio  of  the  two  dependent.  For example,  conformers  of compound 32  is  52:48  but  57:43  same  in CDC1 . 3  conformer  due  to  conformers  and  For each Diels-Alder adduct  conformers  is  62:38, the  33  However,  which  is  collapse,  to  somewhat  is  solvent  in DMSO-d 6 the ratio between the two  For compound  .  signals  split  in CDC1 3  but  ratio  is  71:29  in DMSO-d , 6  for both compounds,  predominates  in  both  the ratio  is the  it  solvents.  The  most noticeable difference in the 1 H NMR spectra (in CDC1 ) 3 of  the  two  Diels-Alder  adducts  is  the  methoxy signal,  which in one case is 4 Hz  case  The cause  is  64  Hz.  of  this  splitting  of  the  and in the other  splitting can only  from different conformations of the carbamate group,  arise  and the  spectrum showing the larger splitting is therefore assigned to  structure  32,  in which  the methoxy  group  is  closest  to  17  a)  ‘H NMR spectrum of 33 in DMSO-d 6 at 25°C.  7  b)  6  5  4.  3  2  -H NMR spectrum of 33 in DMSO-d 5 at 120°C.  Figure 2-1. DMSO-d . 6  ‘H NMR spectra of the Diels-Alder adduct 33  in  18  the carbamate group.  In the  NMR spectrum of 33 in DMSO-d 6  at 120°C the doublet at 5.19 ppm (J H-i.  This  olefinic  assignment proton  at  is  verified  6.44 ppm  4.0 Hz)  =  5.19 ppm to collapse to a singlet.  irradiation  by  which  is assigned to  causes  the  of  doublet  the at  The -H NMR spectrum of 32  in DMSO-d 6 at 120°C revealed the H-i proton to be a doublet (J  =  4.0  Hz)  too.  Since the  H-i  protons  in bothcompounds  are doublets the structure of the Diels—Alder Alder adducts must be as shown in scheme 2-2.  2.2. TRANSFORMATION OF THE ENDO ISOMER 3240. Treatment of 32 with borane dimethyl sulfide complex at 26  -  28°C in THF and subsequent oxidation with 30% hydrogen  peroxide gave 80  85% of 34 and 7  -  10% of 35  -  (scheme 2-3).  Both alcohols gave the expected molecular ion at 353/355 m/z in the low resolution mass spectrum and the presence of the hydroxy group was established through cm  and  at  3460  cm  for  separation of the alcohols was  found  mixture  to  to  more  be  the  34  and  IR absorption at 3445 35  respectively.  34 and 35 was difficult,  convenient  corresponding  to  ketones  oxidize which  separated by fractional crystallization. hydroxy group the  in 34  corresponding  and  The it  the  alcohol  were  readily  The position of the  and 35 was established by oxidation to ketones  36  and  37.  The  observed  regioselectivity must be due to a directional effect of the carbamate  nitrogen  hydroboration step.  through  complexation  Furthermore,  to  borane  in  the  complexation of the borane  19  0  0 S(CH 3 BH 2 1) ) THF/26°C  P O 3 CH  ci  2 2) NaQH’30X HD  1  THF/MeOH/26°C  35  34 (80—857.)  32  (7—lOX)  /cc C 2 CH / Ref1LLx 1  0 P +  3 CH  C  1 37  36  (777.)  H  3 H  HO  OH, p—TsOH  benzene/reflUX  0  CH 3 o’JJ CH 3 3 CH 38 (997.)  Scheme 2-3.  Transformation of the Diels-Alder adduct 32.  20  molecule to group in 34,  the carbamate  nitrogen would  to be endo.  and perhaps also in 35,  Oxidation  of  chlorochromate  in  the  alcohol  refluxing  34  and  an  carbonyl  of  showed  36  increased at  1740  lack  of  absorption cm,  due  the  pyridinium  the  the  gave  Compared to 34 the IR  absorption  at  3445  position  of  the  the  at to  with  dichioromethane  corresponding ketone 36 in 77% yield. spectrum  cause the hydroxy  keto  group.  The  cm’ ester  ‘H  NMR  spectrum in CDC1 3 revealed that the ketone 36 exists as two conformers 4.85 ppm  in  a  ratio  40:60.  of  (major conformer)  H-i  proton  is a sharp singlet,  the keto group to be in position 6. ray diffraction analysis  The  in  36  at  establishing  This was confirmed by X  (figure 2-2),  which also confirmed  the position as well as the stereochemistry of the chlorine  c1  C14 C13  02  •03  Figure 2-2. X-ray diffraction analysis of ketone 36.  21  The mass spectrum of the isomeric ketone 37 showed the  atom.  expected  molecular  ion  at  353/351  In  m/z.  solution  the  isomeric ketone 37 also exists as two conformers in a ratio of  39:61.  The  H-i  proton  5.01 ppm is a dd with J  in  the  -H 1  NMR  3.5 Hz and J’  =  spectrum of 2 Hz,  =  37  at  establishing  the keto group to be in position 5. The  ketal  38  ketone  36  benzene  overnight  (scheme  2-3).  was  with  methyl  groups  99%  yield  by  2,2-dimethyl-i,3-propanol using p-toluenesulfonic  The  mass  spectrum  molecular ion but only M+ chlorine was,  in  formed  however, of  -  of  the  ref lux  in  at acid  did  38  heating  Cl at 402 m/z.  as  not  catalyst show  the  The presence of  confirmed by elemental analysis.  the  group  ketal  in  appear  the  The  H 1  NMR  spectrum at 1.22 and 0.81 ppm (major isomer). The  benzyloxycarbonyl  hydrogenolysis  in  THF  group  at  room  was  was  used  without  work-up  in  the  in  54% yield  yield  of  chloride  40  amine  in  triethylamine.  (scheme 2-4). 39  was  the  The crude amine  much  mass m/z  lower  spectrum followed  yield of  40  by  a  than gave peak  in a at  coupling  giving the amide  treated  with  in  crude  3-indole  the  product  the weak 424  DCC  promoted  molecular m/z  due  of  in  this  resulting in reaction.  peak to  acetyl  presence obtained  way turned out to be very difficult to purify, a  10%  In an attempt to improve the  dichloromethane However,  using  subsequent  reaction with DCC and 3-indole acetic acid, 40  by  temperature  palladium on activated carbon as catalyst. 39  removed  at  the  The  460/462 loss  of  22  hydrogen chloride.  The presence of chlorine was established  0 0 Ph  H Pd/C , 1) 2 THFI25°C  N  ) 2  O 3 CH  H’  COOR  3 C 0 H  ci  C1 25°C 2 CH ,  38  •CH 3 3 CH  CH 0  3 CH  40 (547.)  PPQo CR3  C H3O—  Toluene, 75°C  3 CH 41 (537.)  Scheme 2-4. Synthesis of the amide 40 and the thioamide 41.  by  elemental  analysis.  The  IR  characteristic N-H stretch at 3250 cm a typical  spectrum  showed  a  and the UV spectrum  indole absorption pattern at 272,  279  and 289 nm.  The most remarkable difference in the 1 H NMR spectrum of 40 compared  to  that  of  38  (besides  the  replacement  benzyloxycarbonyl group with 3-indole acetyl group)  of  the  is that  different conformers are no longer observed and the position of the H-i proton has shifted down field from 5.38 ppm in 38 (major conformer) to 5.88 ppm in 40.  23  Treatment of 40 with Lawesson’s reagent 76 in toluene at 75°C for 10 hours gave the corresponding thioamide 41 in 53% yield of  (scheme  Lawesson’s  complicated expected the H-i order  Using  reagent  the  phosphorous  gave  work-up.  molecular  ion  at  the  same  spectrum  of  about mass  The  476/478  pentasulfide  In  m/z.  ‘H  to  verify  this  (table  Correlation) expected, contained  5  attached,  carbons  at  C 3  APT  and  a  region  with 22.0,  at  22.0  and  an  of  41  the  spectrum  6.88 ppm. Proton  In  Test)  (Heteronuclear  were  the  odd  23.1, 23.1  of  but  gave  NMR  (Attached HETCOR  2-2)  (table  aliphatic  namely  signals  2-1)  spectrum  the  a  instead  yield 41  proton was assigned to the singlet at  spectrum  The  2-4).  obtained.  number  27.4,  53.3  correspond  to  spectrum  APT  C 13  As  of  hydrogens  and  53.4 ppm.  the  two  methyl  groups of the ketal and the signal at 27.4 ppm is attributed to  C-4.  Judged  by  their  relative  intensities  the  smaller  signal at 53.4 ppm was assigned to C-i and the larger signal at  53.3  ppm  confirmed  this  to  the  methoxy  group.  The  HETCOR  interpretation by correlating  the  spectrum signal  at  53.3 ppm with the three proton singlet at 3.73 ppm in the NMR spectrum and the signal  at  53.4 ppm with the proton at  6.88 ppm.The downfield shift observed for H-i in 41 compared to the H-i  proton in the corresponding amide 40,  the introduction of  sulfur,  caused by  must either be due to a change  in equilibrium conformation or to the larger size of sulfur which results in an increased deshielding of H-i.  24  2-1. Chemical shifts 41 spectrum of thioamide a  Table  5 (ppm)  +/_b Interpretation  observed  S (ppm)  in  the  C 3 ‘  APT  NMR  _b Interpretation 1  22.0  -  -C 3 CH  109.5  +  Indole-C  23.1  -  -C 3 CH  111.2  -  Indole-CH  27.4  -  C-4  118.3  -  Indole-CH  29.1  +  >C<  119.5  -  Indole-CH  37.1  +  2 -CH  122.1  -  Indole-CH  41.2  +  2 CH  122.3  -  Indole-CH  42.6  +  2 CH  126.8  +  Indole-C  49.9  +  2 CH  136.0  +  Indole-C  53.3  -  O3 CH  170.3  +  C=S  53.4  -  C-i  202.9  +  C=0  62.2  +  C-7  71.5  +  O2 -CH  73.2  +  O2 -CH  98.1  +  c-6  a Solvent: CDC1 3 b The +/- sign indicate whether the signal s positive (>C<, -) or negative ( >CH-, CH 2 -CH -) in the C APT spectrum. 3  25 Table 2-2. Chemical shift correlations obtained HETCOR spectrum of thioamide 41 in the region 0-100  shift (ppm)  the  of  the  ‘H correlation  22.0  0.80  23.1  1.20  27.4  2.12  29.1  no correlation  37.1  3.09 and 1.88  41.2  4.24 and 4.10  42.6  1.88 and 1.71  49.9  3.40  53.3  3.73  53.4  6.88  62.2  no correlation  71.5  3.35 and 4.01  73.2  3.35 and 4.42  98.1  no correlation  a Solvent:  in  . 3 CDC1  Raucher  et  ’ 4 al.  have  reported  that  reaction  compound 42 with Lawesson’s reagent 76 in 1,2-dimethoxyethane at 65°C caused the chlorine to epimerize:  26  ‘S  1,2—dietoxyethariel65°C  H’ O 3 CH  C  3 CH  ci.  43  42  In order to establish if this had happened in our case, 41  was  submitted  for  X-ray  crystallography.  diffraction analysis of 41  (figure 2-3)  the  in  chlorine  work-up that  the  of  had  41  no  remained other  remaining  the  isomer  material  was  observed,  lost  X-ray  clearly showed that  exo position.  was  The  due  to  During  and  it  seems  decomposition  under the reaction conditions.  LB  Figure 2-3.  the  X-ray diffraction analysis of thioamide 41.  27  2.3. TRANSFORMATION OF THE EXO ISOMER 3340 Treatment dimethyl  of  sulfide  the  Diels-Alder  complex  in  THF  adduct room  at  with  33  borane  temperature  subsequent oxidation with basic hydrogen peroxide at 0 gave the alcohols 44  and 45  -  and 4°C  in a combined yield of 73% and  the by-product 46 in 6% yield  (scheme 2-5).  Upon monitoring  the reaction by TLC it was found that the by-product 46 was formed during  the oxidation stage.  was carried out at  room temperature leading to a 30% yield  of the two alcohols product  46.  (44 and 45)  However,  oxidation to 0  -  Initially the oxidation  lowering  and a 47% yield of the by of  the  temperature  of  4°C reduced the amount of by-product to 6%  and increased the combined yield of the alcohols to 73%. temperatures mass  below  spectra of  at 353/355 m/z.  44  0°C  the  and 45  hydroxy  oxidation  became  too  slow.  group.  and 3400 cm respectively,  Both  alcohols  are  seen  in  the  NMR spectrum of 44 the H-i proton is a doublet (J  group proton  to  (major conformer).  be  on  C-6.  In  a  dd  (J  4  is  The  gave the expected molecular ions  due to H 1  spectrum to exist as two conformers in a 46:54 ratio.  at 4.67 ppm  At  The IR spectra of 44 and 45 showed a strong  absorption at 3440 cm the  the  =  the Hz,  In the =  2 Hz)  This establishes the hydroxy  -H NMR J’  NMR  =  1  spectrum of  45  Hz)  ppm  at  4.65  the H-i (major  conformer),  thereby establishing the hyciroxy group to be in  position 5.  The directional effect of the carbamate nitrogen  makes it reasonable to assume that the hydroxy groups in 44 and 45 are endo as  drawn in scheme 2-5.  This assumption was  28  0 1) BHS(CH THF”25°C 2 ) 3 1  Cl.  P) NaUH’307. H ,THF/MEQH/O—4°C 0 2  33 0 P ho  0 F C  i-L-4.o I H  P H  Cl  +  0  H CH 2 0 3  3 C 2 CO H 44  45  +  C  46 (6Z)  (737.) PCC CH 2C 2 1 / R ef lux  0  0  P +  P hoN1 1O 1 Cl_ 3 C 2 CO H 48 (207.)  47 (527.)  C CH 3 H 3 HO  OH,  p—T sO H  benzeneireflux  0 Pho_Lj  3 3 C 2 CO H CH 49 (737.) Scheme 2-5.  Transformation of the Diels-Alder adduct 33.  29  C14  ot  CA  03  Figure 2-4.  X-ray diffraction analysis of alcohol 45.  shown  correct  to  be  analysis also  of  which  confirmed  for  is  alcohol in  shown  the  45,  X-ray  an  figure  The X-ray of  2-4.  regiochemistry  diffraction  well  as  45 the  as  stereochemistry of the chlorine atom to be as predicted for the Diels-Adler adduct 33, The  mass  spectrum  of  molecular ion at 301 m/z of  chlorine.  correct 301.  The  elemental  The  hydroxy indicated  an  of  analysis of  but ester  the  the  46  did not  broad  and/or  on  a  showed  a  indicate the presence  chlorine  based  46  by-product  and did not  absence  IR spectrum group  based on its 1 H NMR spectrum.  a  was  confirmed  molecular  show  the  absorption  carbamate  by  weight  of  presence of at  group.  1680 The  H 1  a  a  1 cm NMR  spectrum confirmed the presence of both a benzyloxycarbonyl and  a methoxycarbonyl  group.  A tricyclic  structure such as  30  46  seemed  data  to  and  be  was  in  best  finally  agreement  confirmed  with by  spectroscopic  the X-ray  diffraction  analysis (figure 2-5).  Ct3  C13  clz  c1z  C14  cli  cii  cis  01  02  02  04  04  Figure 2-5. product 46.  The out  to  X-ray diffraction analysis the tricyclic by  separation be  iterative  very  of  difficult  chromatography  corresponding ketones, by  column  the  two  alcohols  and  could  on  0.5 mm  44  only  and be  silica  45  turned  achieved plates.  by The  on the other hand could be separated  chromatography,  and  the  mixture  of  the  two  alcohols was therefore oxidized to the corresponding ketones  31  47 and 48 in a yield of 52% and 20% respectively (scheme 25).  Alternatively,  crude  reaction  the  oxidation  mixture  obtained  carried  was  the  after  without removing by-product 46 first.  Diels-Alder  adduct  32  the  In this way 34% of 47,  interaction periplanar  between chlorine  lone pairs),  the bond  Compared to  regioselectivity  hydroboration step of 33 is reduced.  the  in  This is probably due to  nitrogen (and/or  the  hydroxylation  14% of 48 and 11% of compound 46 were obtained. the  on  out  lone  pair  the  and  interaction with  chlorine  thereby reducing the capability of the nitrogen  lone pair to interact with borane in the hydroboration step, thus leading to lower regioselectivity. 47  The mass spectra of  and 48 gave the expected molecular ions at 351/353 m/z.  In the -H NMR spectrum 47 was found to be a 43:57 mixture of two  conformers.  conformer), The  ketone  conformers. J’  =  H-i  is  a  sharp  singlet  at  4.72 ppm  (major  establishing the keto group to be in position 6. 48  was  found  be  to  The H-i proton at  a  48:52  mixture  5.06 ppm is a dd  (J  of =  two  4 Hz,  2 Hz) showing the keto group to be in position 5. Treatment  of  the  ketone  47  with  2,2-dimethyl-1,3-  propanediol and p-toluene sulfonic acid in refluxing benzene gave  the  mass  spectrum of  desired  ketal 49  formula  as  well  in  did not  m/z but an ion at 402 correct elemental  49  73%  yield  (scheme  show the molecular  corresponding to  2-5).  The  ion at  437  loss of chlorine.  A  analysis confirmed the expected molecular as  the  presence  of  chlorine.  The  NMR  spectrum showed 49 to be a 32:68 mixture of two conformers.  32  The reactivity of the carbamate group in 49 turned out to be quite different from that of the isomeric ketal 38 in that  the  the  conditions  carbamate  palladium  on  group  used  in  for  carbon  in  49 38  could  not  be  removed under  (hydrogenation  THF  at  1  using  48  psi  was  also  iodotrimethylsilane starting material anticipated  amine  Treatment  unsuccessful. (formed  in  ) 42 situ  to disappear on TLC but was  unsuccessful.  Attempted  atm).  hydrogenation of 49 in a Parr hydrogenator at 30  The  -  of  did  10%  35°C and 49  with the  cause  isolation of the removal  of  the  carbamate group was finally achieved by passing dry hydrogen bromide through an anhydrous solution of 49 in benzene.  P  1 )H8r benzene Cod Cl 2)cJ—( N 3 ,Et H çC 3 5 o. H CH 2 CO 3 3 CH CN, 25°C 3 CH 49  /  H 3 H C 51 (71Z)  CH 3  C  H’ CH 3 Toluene, 60°C  52 (757.)  Scheme 2-6. Synthesis of the amide 51 and the thioamide 52.  33  The amine hydrogen bromide was treated, purification, 41 chloride  with  in  triethylamine  acetonitrile  give  to  71% yield based on the ketal 49 Treatment 5376  reagent desired  of  or  the  5443  thioamide  in  52  51  desired  the  with  in  75  3-indole  and  acetyl  amide  in  51  (scheme 2-6).  amide  reagent  without further  toluene  at  Lawesson’s  60°c  yield.  76%  -  either  the  gave  However,  HPLC  analysis of this compound revealed it to be a mixture of two components.  Reinvestigation of  observation,  and  the  subsequently isolated eluting 52A,  the  and  52B  conclusively  that  -H  ion NMR  differences, H-i. The  at  52B  and  were  41  under  neither  52A  TLC comparison of  the nor  same  52B  The low resolution mass  are virtually  molecular The  52A  by preparative TLC on alumina plates,  thioamide  to the thioamide 41. and  components  iteratively with dichioromethane.  52B  showed  two  52 by TLC confirmed the HPLC  identical,  476/478  spectra  for  m/z  of  both  52A  the  was  desired 52B  identical  spectra of  showing  and  conditions  the  52A  expected  thioamide  showed  52.  several  the most noticeable being the chemical shift of  In 52A H-i appeared at 7.05 ppm and in 52B at 5.05 ppm. results  listed  in  of  the  table  C APT 13  2-3.  NMR  spectra  Comparison  of  the  of C 13  52A  and  APT  52B  are  of  52A  NMR  and 52B strongly suggests that both the carbon skeletons and the  patterns  verify  that  of the  substitution signal  HETCOR experiment table 2-4.  was  at  are  7.05 ppm  carried  out.  identical. in The  52A  is  result  In due is  order to  H-i  listed  to a in  34 a 3 _ Table 2 Chemical shifts observed in the 13 C APT NMR 2Bb. spectrum of thioamides 52A and 5  52A 6  (ppm)  52B 1 _ c  41  8(ppm)  6(ppm)  22.0  21.8  22.0  22.5  22.0  23.1  26.9  27.1  27.4  +!_C  29.3  +  30.1  +  29.1  +  37.7  +  34.6  +  37.1  +  40.3  +  36.3  +  41.2  +  41.2  +  39.7  +  42.6  +  50.3  +  52.6  49.9  +  53.2  53.3  56.3  62.5  +  53.3 53.4  66.7  +  65.5  +  62.2  +  71.6  +  70.1  +  71.5  +  72.5  +  70.3  +  73.2  +  98.3  +  98.6  +  98.1  +  a The table is continued on the following page. b Solvent: CDC1 . 3 C See note at the end of the table on the next page.  35  Table 2-3 continued.  52A  52B  8 (ppm)  1 _ b  41  ö(ppm)  109.2  +  109.8  111.3  —  111.2  118.0  -  —  121.6  —  122.5  —  119.1  —  122.1  +  118.4  -  119.3  6(ppm)  —  123.8  -  -  109.5  +  111.2  —  118.3  -  119.5  —  122.1  —  122.3  -  126.8  +  127.1  +  126.8  +  135.9  +  135.8  +  136.0  +  168.8  +  168.9  +  170.3  +  203.8  +  204.1  +  202.9  +  b The +1- sign indicate whether the signal s positive (>C<, -) or negative ( >CH-, CH 2 -CH -) in the 1 c APT spectrum. 3  This  experiment  clearly  demonstrates  that  the  singlet  7.05 ppm is due to H-i.  The mass spectral data and the  APT  52B  spectra  compounds  of  are  52A  and  structurally  strongly identical.  suggests The  that  only  the  at  two  conclusion  possible on basis of this physical data is that 52A and 52B are unusually stable conformers of the same molecule 52.  36  Table 2-4. Chemical shift correlations obtained in the HETCOR spectrum of thioamide 52A in the region 0-100 ppma.  shift  (ppm)  correlation  26.9  2.15  29.3  no correlation  37.7  1.91 and 3.06  40.3  1.76  41.2  4.41  50.3  3.53 and 3.64  53.2  3.87  56.3  7.05  66.7  no correlation  71.6  3.29 and 3.73  72.5  3.38 and 4.26  a Solvent:  . 3 CDC1  The only region of the molecule that might give rise to restricted rotation is around the C-N bond in the thioamide group.  Conformer  52A  containing  the  most  deshielded  H-i  proton must then be the conformer with sulfur syn to the H-l proton this 52B  as  depicted  in  interpretation to  100°C  in  figure  is the toluene  2-6.  fact  the  evidence  that heating  results  formation of a mixture of the two comparison of  Supporting  in  both  for  either 52A or cases  “compounds”.  in  the  Furthermore,  H NMR spectra of 52A and 52B 1  6 in DMSO-d  37  at  room  temperature  spectra  of  the  and  two  120°C  show  are  “compounds”  while  that  the  different  ‘H  at  NMR room  temperature then they are identical at 12000.  .c1  I—  3 C 2 CO H  CH 2 0 3  C. 3 H  Cl  3 H 52B  Figure 2-6.  24.  The conformers 52A and 52B of thioamide 52.  PHOTOCHEMICAL CYCLIZATION OF THE AMIDES  40 AND 51,  AND  OF THE THIOAMIDES 41 AND 52 Irradiation of the amide 40 in aqueous methanol using a Pyrex filter gave 4% of the desired lactam 55 and 15% of the undesired expected  isomer  56  molecular  (scheme ion  at  2-7).  424  Both  m/z.  compounds  The  two  gave  the  compounds  are  differentiated by the chemical shifts of the indole protons. In the  ‘H NMR spectrum of 55 the indole N-H is a singlet at  9.04 ppm.  The protons  7.54 ppm  (J  respectively.  =  The  8.0  at 0-9  Hz)  protons  and 0-12 appear as doublets at  and at  triplets at 7.07 and 7.12 ppm.  7.28 ppm 0-10  and  (J C-il  =  8.0 appear  Hz) as  This assignment was confirmed  38  I-1  / 3 CH  3 H =  40 R 41 R  =  0 S  CH 3  hu 3 CH R  +  CH 3  3 C 2 CO H 55 R 61 R  = =  0 S  CH 3 12  56 R 62 R  =  =  0 S  hu  H’ C1 3 H 51 R 52 R  =  0  =  S  Scheme 2-7. Photochemical cyclization of the amides 40 and 51, and the thioamides 41 and 52.  39  by irradiation of the triplets at 7.07 and 7.12 ppm,  whereby  the doublets at 7.54 ppm and 7.28 ppm collapse to singlets. In the  ‘H NMR spectrum of  at 8.72 ppm as a singlet.  56 the indole N-H proton appears The four indole C-H protons appear  as a doublet at 7.25 ppm (J (J  Hz),  8.0  =  6.84 ppm  (J  7.25  at  or  a  singlet  8.0 Hz).  =  6.84 ppm  collapse to a doublet. therefore  assigned  as  =  8.0 Hz),  a triplet at 7.09 ppm  6.94 ppm  at  and  doublet  a  at  Irradiation of either the doublet at the  causes  triplet  7.09 ppm  at  to  The doublets at 7.25 and 6.84 ppm are C-b  and  7.09 ppm to the proton at C-li.  C-12  and  triplet  the  Irradiation of the  at  singlet  at 6.94 ppm results in a sharpening of the indole N-H signal at  8.95 ppm  establishing  the  singlet  as  the  cyclization  the  6.94 ppm  at  proton at C-2. In order influence  of  examined.  The  2-5  indicated  to  improve  various  the photochemical  factors  preliminary that polar  the photochemical  on  the  yield  investigation protic  cyclization  to  55  summed up  solvents take  of  in  was table  are necessary  for  Comparison of  place.  entry 20 with 21 suggests that the cyclization is favored by an increase in reaction temperature. -  In methanol  (Entries 18  20) the cyclization is about three times more efficient at  wavelengths  above  However,  this  methanol  where  300 nm  observation a  cut  off  than a cut off at 220 nm, marked  decrease  in  the  compared does at  shorter  to  not  200 nm  hold is  wavelengths.  true  only  in  aqueous  slightly worse  whereas longer wavelengths show a yield.  On  the  basis  of  these  40 a• 5 _ Table 2 Investigation of the effect of the nature of the solvent, the wave length and the temperature on the b• photochemical cyclization of amide 40  exp  solvent  nm  1  THF  200  ref lux  0.0  1.0  2  THF  220  reflux  0.0  1.5  3  THF  300  reflux  1.0  0.5  4  0 2 THF/H  300  ref lux  0.0  3.5  5  EtOAc  200  ref lux  0.0  0.0  6  EtOAc  220  reflux  0.0  0.0  7  EtOAc  300  ref lux  0.0  0.0  8  EtOAc  300  23  0.0  0.0  9  N 3 EtOAc/Et  300  23  0.0  0.0  10  EtOAc/HOAc  300  23  0.5  0.0  11  Benzene  200  reflux  1.5  0.0  12  Benzene  220  ref lux  1.0  0.0  13  Benzene  300  ref lux  1.0  0.0  14  CH C 3 N  200  ref lux  0.0  0.0  15  CH C 3 N  220  reflux  0.0  1.5  16  CH C 3 N  300  ref lux  0.0  0.5  17  2 C 3 CH 0 N/H  300  reflux  0.0  2.5  temp (°C)  % 55  a Table 2-5 is continued on the following page.  % 56  41  Table 2-5 continued.  exp  solvent  nm  18  QH 3 CH  200  ref lux  0.0  4.0  19  OH 3 CH  220  reflux  0.5  4.5  20  QH 3 CH  300  reflux  1.0  15.0  21  OH 3 CH  300  23  1.0  7.0  22  2 O 3 CH 0 H/H  200  ref lux  0.0  13.5  23  2 O 3 CH 0 H/H  220  reflux  0.5  15.0  24  2 Q 3 CH 0 H/H  300  ref lux  0.0  5.5  25  OH/H 3 CH 0 2 N /Et  300  ref lux  0.0  2.0  26  O 3 CH 0 2 /NaHCO H/H  300  23  0.0  4.5  temp (°C)  % 55  % 56  b The yields were determined by HPLC (conditions as described in section 3.26) using pure 55 and 56 as external standards.  a• Investigation of the photochemical cyclization 6 _ Table 2 of amide 40 using fractional factorial design with four factors on three levels.  Factor temp:  level 1 temperature (°C)  base:  nm:  none  wavelength (nm)  solv:  0  solvent  level 2  level 3  25 N-methyl morpholine  65 triethyl amine  220  300  260  MeOH  THF  CN 3 CH  a The table is continued on the following page.  42 Table 2-6 continued.  55 % b  56 % b  exp  temp  base  nm  solv  1  0  none  260  THF  0  2  2  25  none  220  OH 3 CH  0  12  3  65  none  300  CN 3 CI{  0  0  4  0  Me morph  300  OH 3 CH  0  1  5  25  Me morph  260  CN 3 CH  4  14  6  65  Me morph  220  THF  0  0  7  0  N 3 Et  220  CN 3 CH  0  5  8  25  N 3 Et  300  THF  0  0  9  65  N 3 Et  260  0H 3 CI-1  4  7  1/3 El  (55)  0.0  0.0  0.0  1.3  1/3 E2  (55)  1.3  1.3  0.0  0.0  1/3 E3  (55)  1.3  1.3  2.7  1.3  1/3 El  (56)  2.7  4.7  5.7  6.7  1/3 E2  (56)  8.7  5.0  0.3  0.7  1/3 E3  (56)  2.3  4.0  7.7  6.3  b The yields were determined by HPLC (conditions as described in section 3.26) using pure 55 and 56 as external standards.  43 Table 2-7. Investigation of the photochemical cyclization of amide 51 using fractional factorial design with four factors on three levelsa.  55 b % b 56 %  exp  temp  base  nm  solv  1  0  none  260  THF  0  11  2  25  none  220  OH 3 CH  0  5  3  65  none  300  CN 3 CH  0  2  4  0  Me morph  300  OH 3 CH  0  8  5  25  Me morph  260  CN 3 CH  0  28  6  65  Me morph  220  THF  31  20  7  0  N 3 Et  220  CN 3 CH  0  17  8  25  N 3 Et  300  THF  0  0  9  65  N 3 Et  260  OH 3 CH  35  22  1/3 E 1(55)  0.0  0.0  10.3  10.7  1/3 E 2(55)  0.0  10.3  0.0  10.3  1/3 E 3(55)  22.0  11.7  11.7  0.0  1/3 E 1(56)  12.0  12.7  20.7  18.3  1/3 E 2(56)  17.7  18.7  3.3  10.3  1/3 E 3(56)  14.7  13.0  20.3  15.6  a The levels of the factors are defined in table 2-6. b The yields were determined by HPLC (conditions as described in section 3.26) using pure 55 and 56 as external standards.  44  preliminary  findings  carried out on the to  make  the  fractional  levels, and as  investigation  A  design  design  was chosen,  design  and 51.  efficient  as was  with  used  four  how  the  four  experimental on  three  for the amide 40  for the amide 51.  dictates  possible  each  factors,  was  In order  as  the  as  shown in table 2-6  as  shown in table 2-7  factorial  investigation  systematic  isomeric amides 40  two  factorial  . 44 strategy  more  a  The fractional are  factors  to  be  varied in the 9 experiments while all other factors are kept In  constant.  the  last  six  rows  in  each  table  the  average  yield of 55 and 56 are summed up for each level of the four factors. A graphic representation of the results is shown in figure  2-7.  It  of  the  analysis that no  should  be  fractional  noted  that  factorial  the  in  design  interactions exist between the  four  it  following is  assumed  factors.  As  in  the preliminary investigation of 40 all the factors examined in table  2-6  had  only a  the desired product all  the  factors  formation of 55. 2-7)  55.  very small However,  examined  effect on the yield  for the  show  some  From the results  isomeric amide influence  in table 2-7  51 the  (and figure  it is clear that the photochemical cyclization of 51 to  55 is favored by high reaction temperature, and wave  lengths below 300 nm.  different take  on  of  place  protic  solvents in  solvent  a  is  rather  non-polar but  not  in  presence of base  The observed effect of the odd.  aprotic an  That  solvent  aprotic  should  cyclization  and  polar  in  a  polar  solvent  of  comparable transparency seems unlikely. A closer look at the  45  Cyclization of 40 (o) and 51 () X YIELD  30  Z YIELD  TEMPERPTURE  /  10  0  25  X YIELD OflSE  20  to 55  30  20  20  10  °  // 65  None  N 3 Et  X YIELD  WPVE LENGTH  SOLVENT  20  Zr  2 _A 220  260  300  THF MeCN MeDH  Me—morph  Cyclization of 40 (a) and 51 (es) X YIELD  30  X YIELD  TEMPERflTURE  to 56 X YIELD  BSE  30  X YIELD  IJPVE LENGTH  SOLVENT  30  :  10  0  25  65  None  N 3 Et  220  260  300  A  THF MeCN  MeOH  rle—riiorph  Figure 2-7. A graphic representation of the results of the fractional factorial designs in table 2-6 and 2-7.  levels  of  the  acetonitrile  other  (table  factors 2-7  in  entry  3,  the 5  three  and  7)  experiments reveals  that  in in  each of these experiments at least one of the other factors is at a level where the graphic analysis in figure 2-7 shows that no reaction occurs. why no  This is the most likely the reason  reaction is observed  in acetonitrile.  The  fact that  no response is observed in a majority of the experiments in the fractional  factorial design makes the interpretation of  46  the design less reliable. 55,  the  amides  undesired under  examined. cyclization  product  most  The  of  effect  of  In contrast to the desired product  the  the  the  amides  formed  readily  different  of  two  is  56  reaction  different to  56  from  is  conditions  factors seen  both  on  the  follow  to  a  similar trend. Raucher et al. 34 reported that photochemical cyclization of  the  amide  product 59,  57  gave  only  trace  amount  of  the  desired  but that the corresponding thioamide 58 cyclized  in 41% yield to the thiolactam 60.  hv CH 3  On  the  basis  of  this  report  the  thioamides  were synthesized from the corresponding amides. of  thioamide  41  in  aqueous  methanol  using  a  41  and  52  Irradiation Vycor  filter  gave 11% of thiolactam 61 and 10% of the undesired isomer 62 (scheme 2-7). at  440  indole  mhz.  Both compounds gave the expected molecular ion The  protons  two in  thiolactams  their  ‘H  NMR  are  differentiated  spectra.  In  by  the  the NMR  spectrum of 61 the N-H proton is a singlet at 7.86 ppm.  The  H-9 and H-12 protons appear as doublets at 7.58 ppm (J  7.4  =  47  Hz) and 7.28 ppm (J  7.4 Hz).  =  triplets appearing at 7.17 ppm (J =  7.4 Hz).  triplet  7.13 ppm to  the assignments.  into  collapse  The remaining four indole protons  resonate as a doublet at 7.29 ppm (J a triplet at 7.11 ppm  6.81 ppm  confirming  a doublet,  In the 1 H NMR spectrum of 62 the N-H proton  is a singlet at 8.50 ppm.  at  and 7.13 ppm (J  7.4 Hz)  =  Irradiation of the proton at 7.58 ppm causes the  at  7.22 ppm,  and H-il protons are  The H-b  (J  7.11 ppm causes,  Hz).  7.0  =  as  7.0 Hz),  =  (J  7.0 Hz)  =  Irradiation  expected,  and a doublet  the  of  the doublets  6.81 ppm to collapse to singlets.  a singlet at  at  triplet  7.29 ppm  at and  For steric reasons C-9 on  the indole ring must be substituted,  since a molecular model  shows  system  that  the  quinuclidine  ring  cannot  stretch  across to C-12 in the indole ring. Six  factors  were  screened  for  effect  their  on  the  photochemical cyclization of 41 using a fractional factorial design  with  seven  factors  each  on  The  levels.  two  seventh  variable which the chosen design allowed was not assigned to any  factor  size  but  needed  was  for  a  instead  used  response  to  to be  some  give  idea  significant.  shows the design and the results obtained.  of  the  Table  2-8  The effect of the  different factors on the yield of the desired thiolactam 61 is  given  in  column  E(61).  Only  the  temperature  and  the  solvent have any significant effect on the yield of  61  comparison of  showed  that  the  methanol.  the  better For  two  yield  the  levels  for  these  61  was  obtained  of  undesired  product  two  62  factors at  the  and  and  in  presence  or  65°C  48  absence  of  largest  triethylamine  effect  triethylamine preferred factors yield  have  of  important  the  on  and  levels  62.  In  the  these  order  factors  two  minor  a  light  yield  wavelength  of  only  and  found  the  with  up  to  factors.  further in  the  have  the  presence  of  220 nm The  insignificant  or  to  frequency  being  the  remaining  four  effect  on  the  investigate  the  experiments  above  most a  fractional factorial design with four factors each on three levels was chosen. The of  the  design,  representation  of  is the  general plan, shown  in  results  as well as the results  table is  2-9.  given  in  A  graphical  figure  2-8.  a Investigation of 8 _ Table 2 photochemical cyclization of thioamide 41 using fractional factorial design with seven factors on two levels.  Factor  level 1  level 2  time:  time (mm)  temp:  temperature (°C)  25-30  65-85  base:  base 3 (Et N )  absence  presence  mt  :  light intensity  low  nm  : wavelength (nm)  solv:  solvent  20  40  high  >220  >300  MeOH  0 (3:2) 2 MeOH\H  a The table is continued on the following page.  CD  Ij  01  [‘a) IS.)  [-.3 01  01  Ca.)  0  01 [‘3 Ca) •  0  01 IS.) 01 •  0  IS.)  0  QJc-I-  Q’O  H [‘.3  H 0 • CD  IS.) 0  ‘.  Ca.) 0  (I)  H  H  H  H  H ‘.0  0  0  IS.)  H  [-.3  H  H  [‘.3  H  -)  H  Ca)  H  CA)  •  H  0  H  CD  •  CD  01  01  Ca)  H IS.)  H 0  IS.)  IS.)  L’3  CD  0) 0)0)  C’U) [“3  0  0  •  •  Ca)  H • 01  [“a) CD . 01  0  CD  H ‘.0 • 01  [‘.3  0  Ca)  [‘a) 01  CD  •  01  H 0  01  H [‘.3 •  IS.) •  (J1  Ca)  01  •  H C’a  CD  Ca)  IS.) [“3  •  .  .  01  Ca)  Ca)  H 01  CD  .  [‘a) C.a)  Ca)  Ca) H  H  CD  •  01  H  [‘a)  01  Ca)  01  CD  H 0  H  ) )  L’J 01  F’.)  [“3  H  H  •%)  •  (DO  (Q  H-  [‘S3  •H•  CD  Ort  U) H-CD  I-jo  Q)(D(D  jCflM  rtH  I—’(DH c1,Q Cl) Cl)  C’  --S  C’  —S  M H  C’  ctU)(D (DC)  M  C’ L%J  ts)  (DQ  M H  H 01  —J  H  [‘3  [‘a)  H  H  H  [‘a)  C’  [‘.3 i  01  Ca) CD  [‘3  IS.)  [“3  H  [“3  [‘.)  H H  [‘.3  H  H  IS.)  H  H  01  IS.) 01  [‘-.3  H  IS.)  [‘s.)  [‘3  t-’.)  IS.)  H  Ca)  IS.)  (a)  i  H  H  H  [“3  [“3  H  [“3  t’J  CD  01  H  [‘-.3  H  H  H  H  [‘a)  C’  H  C’  o\O  <1  H  C’) 0  CD  0) U)  CD  rF  CD  I-’.  r1  CD  0  C)  (D  H  0)  50  Changing the solvent from tetrahydrofuran to acetonitrile or methanol results both  products.  favored  by  in a The  polar  temperature,  significant photochemical The  solvents.  base  increase  and  type  in the yield of  cyclization  in  variations  examined  wavelength  clearly  is  have  only  a  the  small  effect on the yield of the desired thiolactam 61.  The trend  observed  indicated  in  the  for the two  temperature  level  discrepancy investigation  is  design not  to  be  in  is opposite table  clear  and  unravelled.  temperature on the formation of 62 a  significant increase  2-8.  to that The  would  cause  of  require  further of  the  is more pronounced,  with  The  effect  this  in the yield going from 25  to  10°C.  a• Investigation of the photochemical cyclization 9 _ Table 2 of thioamide 41 using fractional factorial design with four factors on three levels.  Factor  temp:  level 1  temperature (°C)  base:  nm:  N-methyl morpholine  wavelength (nm)  solv:  10  solvent  level 2  25 triethyl amine  level 3  65 diisopropyl amine  260  300  330  MeOH  THF  CH C 3 N  a The table is continued on the following page.  51 Table 2-9 continued.  exp  temp  base  nm  solv  61 % b  62 % b  1  10  Me morph  330  THF  16  14  2  25  Me morph  260  OH 3 CH  22  38  3  65  Me morph  300  CN 3 CH  20  30  4  10  N 3 Et  300  OH 3 CH  21  39  5  25  N 3 Et  330  CN 3 CH  16  28  6  65  N 3 Et  260  THF  10  5  7  10  NH 2 ipr  260  CN 3 CH  20  51  8  25  NH 2 i-pr  300  THF  11  10  9  65  NH 2 i-pr  330  OH 3 CH  16  29  1/3 El  (61)  19  19  17  20  1/3 E2  (61)  16  16  17  12  1/3 E3  (61)  15  16  16  19  1/3E1  (62)  35  27  31  35  1/3E2  (62)  25  24  26  10  1/3E3  (62)  21  30  24  36  b The yields were determined by HPLC (conditions as described in section 3.26) using pure 61 and 62 as external standards.  52  Cyclization of 41 to 61 () 7. ‘IIELO  to 62 (o)  7. IIELO  X 13ELO  TEMPERATURE  BASE  :  :  10  10  I 25  10  I 65  I Me—morph  7. YIELO  WAVE LENGTH  SOLVENT  70::  0::  10  10  I j—pr N 2 H  260  300  0  I THF MeCN  330  IleOH  N 3 Et  Figure 2-8. A graphic representation of the results of the fractional factorial design in table 2-9.  As  found  in  the  design  in  table  2-8  the  yield  drops  with  increasing wavelength.  In order to determine if yield could  be  by  increased  experiment formamide  further in  3 (E  =  table  111)  increasing 2-9  instead  was of  solvent  the  repeated  using  acetonitrile  yield of 42% of 62 and 8% of 61 was obtained. solvent  and  cyclization ratio  of  solvent as  the  polarity gave  in  experiment  two  products.  3 In  same  the  but  (E  =  methyl 36).  total  yield  changed  fractional  base,  62  was  strongly  whereas  the  desired  disfavored  in  thiolactam 61  the  of the  factorial  design in table 2-8 it was found that the formation of by-product  A  This change in  profoundly  the  polarity  absence  the of  was unaffected.  a In  order to check if the disfavoring of 62 could be translated into an increased yield of 61 experiment 2 in table 2-9 was  53  repeated  but  without  any  yield of 62 was obtained. 62  did  not  result  base. Thus,  an  in  A  yield  22%  of  61  and  14%  the decrease in formation of  increase  the  in  yield  61.  of  Lowering of the temperature to —10°C under these conditions did not have any effect on the yield of the two products. The  photolysis  examined under  of  isomeric  the  the conditions  table  2-10.  Both  the  % 61  % 62  The results are shown  conformers  pure  Table 2-10. The photochemical compared to thioamide  compound time  cyclization  % 41  % 52A  52A  of  and  52B  % 52B  * 52 (total)  15  22  14  52A  15  1  1  52B  15  2  1  52A  75  3  2  52B  75  4  2  0 —  -  -  -  were  thioamide  (mm)  41  was  found to give the best yield  of compound 61 from the thioamide 41. in  52  thioamide  -  -  -  40  29  69  33  30  63  22  17  39  17  14  31  a Conditions: MeOH, >260 nm, 25°C, no base. b The yields were determined by HPLC (conditions as described in section 3.26) using pure 61 and 62 as external standards.  52  54  used  in these  starting  studies.  material  desired product  41  61  15  After had  had  and  consumed  been  In  formed.  photolysis  mm  the  same  all  of  the  of  22%  of  period  the time  only one third of the starting material of 52A and 52B had been consumed, 61.  producing only 1  2% of the desired product  -  The remaining and initially pure 52A and 52B had now,  both cases, clear  become  from table  a  mixture of  2-10  that  52  two  conformers.  irradiation of  leads mainly to decomposition. exited  the  in  It  is  the thioamide  52  It appears that the decay of  occurs mainly through translation into rotational  energy around the thioamide bond  and  some chemical  through  process other than cyclization.  The mechanism of the photochemical cyclization of aromatic 45 detail the  a-chloroacetamides  indicate  ct-chloroacetates,  that  they  45 chloroacetam . ides aromatic  corresponding  behave  Assuming  the  results  a-chloroacetamides  holds  a-chloroacetates,  the  obtained the  to  results  the  some  been carried out on  similar  that  in  studied  been  and even though less work has  corresponding  the  has  a  obtained  for  for  the  mechanism  of  true  photochemical cylization of aromatic a-chloroacetates can be represented by the simplified model outlined in scheme 2-8. The  excited molecule  64  has  several  may decay to the ground state. cleavage  of  electron  transfer  group  and  the  2)  carbon-halogen from  subsequent  the  fates:  1)  It  It may undergo homolytic bond.  aromatic  heterolytic  possible  3)  system  cleavage  It to  of  may  undergo  the  acetate  the  carbon-  55  halogen bond. has  been  Homolytic scission of the carbon-chlorine bond  found  abstraction  generally  from  the  solvent  rather than cyclization. been  found  to  be  to  followed  be  or  from  the  proton  by  molecule  itself  The major route to cyclization has  electron  transfer  from  the  aromatic  ring  system to the acetate group followed by heterolytic cleavage of the halogen bond and subsequent ring closure of the thus formed diradical. gave  a  much  amide 40. higher  As it is seen from figure 2-7 the amide 51  higher  yields  of  cyclized  products  than  the  The higher reactivity of 51 can be explained by a  reactivity  coplanarity  with  of  the  the  lone  chlorine  pair  of  bond  the  due  carbamate  to  its  nitrogen.  That the overall yield of the cyclization for both amides 40 and  51  (figure  2-7)  increased  with  towards  mechanism  a  therefore  and  increasing  the  polarity  involving  reasonable  thioamide  to  of  the  charged  assume  41  (figure  solvent,  points  intermediates.  that  the  2-8)  It  is  photochemical  cyclization of the investigated amides and thioamides follow a  mechanistic  subsequent as  path  involving  heterolytic cleavage  outlined  in  scheme  2-8.  electron of  This  the  transfer  and  carbon-halogen  bond  reaction  mechanism  could  also explain why the thioamide 41 gave a much higher yield of cyclized products than amide 40, of  sulfur  initially  probably  makes  accepting  the  system  and  group.  Model  subsequently studies  the  in that the introduction  electron  electron  from  transferring  have  transfer  indicated  the  it  the  that  easier  aromatic  by  ring  a-chloroester the  diradical  56  0 II  1 63  jhv *  CH C 2 1J  LC H C 1] 2  C1  ®I  65  0 0  [—cH . 2 .c1]  cI._0CH2’D1  Cl.:]  63  66  67  H  [H=°1  Scheme 2-8. Possible path in ways photochemical the cysclization reaction of aromatic a-chloroacetates.  formed upon the release of the chloride ion has lifetime  that  conformer The  change  of in  cyclization the the  starting ratio  takes  place  material of  the  in  the  dominant  ground  . 48 state  from its  desired  so short a  lactam  55  to  the  57  undesired  lactam  56  cyclized  products  might be  a  starting  conformers. have  indole  ring  . 49 position  This  the  4  by  reflection  indolylamines the  as  well  the  yields  the  of  two  increasing  the  of a  in the equilibrium of the  shift  Model  shown is  in  as  reaction  studies  that  N-chloroacetyl-3-  on  cyclization  preferred  in  to  6:1  a  temperature  position 4  ratio  the  to  correlates with the general preference  position  observed  for  both  amides  the  on 2  for  and  the  thioamide 41 resulting in the undesired isomer.  2.5. EVALUATION OF THE CHOSEN STRATEGY.  the  Having  investigated  amides  40  and  51  the  photochemical  and  thioamides  51  cyclization and  52  to  of the  ibogamine skeleton the time has come to evaluate the results of  this  strategy against  the goal  of  the project,  viable synthetic route to vinbiastine 1. from  the  Diels-Alder  thiolactam  61  are  adducts shown  to in  the  The overall yields  desired  figure  namely a  lactam  2-9.  and  55  Taking  into  consideration that at least another five steps remain in the synthesis  of  formation strategy and  of  viriblastine the  unfeasible  further work  this point.  on  1  the  ibogamine as  a  this  low  overall  skeleton  synthetic  makes  route  strategy was  yield  to  the  of  the  chosen  vinbiastine  therefore  stopped  1 at  58  0  3 H C 2 0 55  P  3 C 2 CO H  0 3 CH 32  s  3  H  3 0 2 0 H 61  Figure 2-9. The overall yields of cyclized products 55 and 61 from the Diels-Alder adducts 32 and 33.  2.6.  THE MODIFIED POLONOVSKI REACTION The  tertiary  discovery amines  Max  by  can  be  and  Michael  demethylated  , 50 Polonovski  by  treatment  corresponding N-oxides with acetic anhydride, versatile tool in alkaloid synthesis . 51 reaction  is  outlined  in  scheme  of  that the  has provided a  The mechanism of the  2_952.  The  intermediate  59  species found  7O that  dramatic  the  the  nature on  is  the  most  the  the  isolated.  acylating  regiochemical  treated  with  acetic anhydride,  trifluoroacetate  intermediate follows  of  been  In the modified Polonovski  N-oxide  instead of of  have  effect  . 55 reaction amine  and  The  I i-,. —C—N——--  has  reagent course  , 56 reaction  trifluoroacetic  ion  disfavors  modified  a to  the  Polonovski  nitrogen  I-  I  is  formation  reaction  abstracted,  -HOPc  flc 0  I  —C—N  72  11+  C—O 3 CH 0Pc  0 2 flc  Oflc  71  —C--N—flc  /  —b  \ N—Pc \ iiO / / +  +  0 2 Pc  75  73  Scheme 2-9.  of  mainly  In pathway A  70  \  the  anhydride  6  /  a  when the  H  C_N  has of  t0flc  I  0 2 flc  H  \+/  been  the diminished nucleophilicity  the two pathways shown in scheme 2-10. acidic proton  It  The mechanism of the Polonovski reaction.  giving  60  the  “normal  Polonovski  fragmentation adjacent 57 rules  to  reaction the  for  C-N  intermediates. place  takes  bond  cleaved.  is  hetereoatomic  in  In  which  pathway  B  the  bond  According  fragmentation  C-C to  reactions  a  Grob’s the  C-C  bond to be cleaved must be antiperiplanar to the N-C bond.  cF C 3 00cF c 3 00  0  1+  —N—C---—C—C-—H  I  1+  r  —N—C—C——C—H  -CF C 3 OO  COO 3 CF  \+  /  /  SC—C—H  N=C  I I (0  I  I  I  I  B COQ 3 CF  CF C 3 OO  \+  N=zC  / /  I +1C—C—H  / H  I”\ CC/ /\ II  Nu—C—C—H  82  81  Scheme 2-10. reaction.  Kutney et  The  pathways  in  the  modified  Polonovski  ° and Potier et ai. 2 al. 21 were the first to  utilize the modified Polonovski reaction in the coupling of  61  catharanthine  3  vinbiastine  with the natural  (scheme  5  2-11).  with  vindoline  4  give  5  was  found  epi-15’,20’-anhydrovinblastine that  at C-16  (S) to  be  50%  by  Under optimal conditions for the formation of  15’,20’-anhydrovinblastine 5 Potier et al.  groups  15’,20’-anhydro-  stereochemistry  The best yield of  both groups.  to  the  amount  of  21•  It  also found 12% of  was  shown  by  both  epi-15’,20’-anhydrovinblastine  9  increases with increasing reaction temperature . 2 ’ 20 1  1) rn—Cl PBP 2) VINDOLINE -C H 3  3) TFPfl, —50°C  3  C  3 H  3 C 2 0 H  VINDOLINE  INDOLINE  3 C 2 CO H 9  5  Scheme 2-11. 4.  As  a  The coupling of catharanthine 3 with vindoline  reasonable  rationale  for  the  observed  Polonovski  fragmentation process two routes were suggested as outlined  62  in scheme 2-12:  1) A concerted mechanism in which the attack  by vindoline on C-16’ 21’  and the fragmentation of the C-16’,C-  bond takes place simultaneously.  2) A stepwise mechanism  in which fragmentation of the C-16’,C-21’ intermediate  which  give the dimer. reasons,  catharanthine 3,  For  the  of  formation  must  leading  configuration.  would be  this the  approach to  to  the  reason dimer  concerted process. able  reacts  with  vindoline  Molecular models suggested that,  vindoline  through a  subsequently  bond generates an  explain  the  from  it  is  with  for steric  the  a-face  with  dimer  However,  a  of  the natural  difficult  unnatural  to  to  imagine  configuration  stepwise mechanism  formation of  both  dimers.  If  the initially formed intermediate 85a remains in the “iboga” conformation,  attack by vindoline  4 would occur  from the a  face leading to the dimer with the natural conformation.  By  conformatiorial change of 85a to the conformer 85b the 3-f ace of  the  the  indole unit becomes the more accessible,  unnatural  vindoline 4.  configuration  It  is  reasonable to  reaction temperature might from  85a  unnatural  to  85b  giving  configuration.  concerted  and  a  at  lead an It  stepwise  C-16’  suppose that to  a  is  attack  in  also  mechanism  by  an increased  conformational  increase  simultaneously and that the latter is temperature.  upon  leading to  dimer  possible are  change  with that  the a  operating  favored by increasing  63 Concert.ed process -C H 3  VINDOLINE  ‘Co CF 3 CF C 3 OO  3 -C H  INDOLINE 3 C 2 0 H 5  Stepuise process 3 Co CF COO 3 CF  3 •CH 84  83  4  3 -C H  3 C 2 :0 H 85b  85a  VINDOLINE  I  I  -C H 3  H  3 -CH  INDOLINE  I NDOL INE 3  5  9 Scheme 2-12. Possible mechanisms catharanthine 3 with vindoline 4.  for  the  coupling  of  64  The  coupling  of  various  catharanthine  derivatives  of  catharanthine 3 with vindoline 4 has been examined (table 211).  It  dependent  was  found  that  the  yield  of  dimers  on the reaction 20 conditions 2 ’ 1  and  is  strongly  that none of  the examined catharanthine derivatives gave as good a yield as catharanthine 3 itself.  2-11. Table vindoline 4.  Coupling  catharanthine  of  derivatives  with  3 R •1  VINDOLINE  H  87a—87f  Compound  88a  1 R  a  2 R  =  0  INDOLINE  —  3 R  4 R  5 R  Et  H  H  50  20,21  Reference  b  H  H  Et  H  H  10  21b  b  H  H  Et  H  H  a 20  20b  H  Et  H  20  21b  H  Et  H  11  21b  C  d  =  H  H  e  -0-  Et  H  H  20  58  e  -0-  Et  H  H  6  59  Et  H  OAc  6  60  f  H  H  a +20% where R =Et, 1  =H, 2 R  =H, 3 R  =H, 4 R  =H. 5 R  65  In  the  vinbiastine  strategy 1  it  is  chosen  by  necessary  for  the  synthesis  couple  the  catharanthine  us  to  of  derivative 16 or 29 with vindoline 4 to form the appropriate dimeric  intermediate.  Based on  the  literature  it was  clear  that a moderate to significant drop in the yield of dimer by using either 16 or 29 had to be anticipated.  This,  combined  with the provisional status of the Polonovski reaction, with respect to the mechanism, the yield of  the  of  the  as well  desired dimer,  Polonovski  as  the  factors  prompted a  affecting  reinvestigation  reaction primarily in order to  determine  the factors having the most significant effect on the yield.  CH 3  16  2.7.  SYNTHESIS AND CHARACTERIZATION OF EXOCATHARANTHINE 89 Utilization  of  catharanthine  3  itself  in  the  reinvestigation of the Polonovski reaction is complicated by two due  factors. to  Firstly,  a  facile  reactio b 2 T h , n very  catharanthine  difficult.  making  [2,3] the  Secondly,  N-oxide  10  sigmatropic isolation the  of  formed  is  unstable  rearrangement  the  pure  N-oxide  15’,20’-anhydro-  66  vinbiastine  5  reacts  very  readily  with  oxygen  under  the  formation of the corresponding epoxide . 16  H  H  105  10  H  H  vindoline  indoline 88e  5  Thus  we  decided  to  look  at  exocatharanthine  89,  a  structurally closely related derivative of catharanthine 3.  6  53  8  14 4N 15  13  2  21  16  3 \\cH 19  CH 2 C0 3 22  89  23  18  67  A preliminary investigation showed that exocatharanthine Noxide 90 did not undergo the rearrangement reaction observed for catharanthine N-oxide 10 and could easily be isolated in its  pure  the  corresponding  form.  appeared  19’,20’-Anhydrovinblastine  more  Furthermore  epoxide  stable  when  exposed  91  not  form  air,  and  did the  to  15’,20’-anhydrovinblastine  than  19’,20’-anhydrovinblastine  91  5. an  up  opened  opportunity to compare the reactivity of the 19’,20’-double bond in 91 with the  15’,20’-double bond in 5,  as well as a  potential new route to vinblastine 1. Exocatharanthine thine 3, was  89,  synthetic  a  was first reported by Szantay eL a1 . 46  formed  as  hydrogenation  a  side  of  product  was  done  exocatharanthine way,  exhibited  the  at 89.  a  time  in  3  The compound catalytic  methanol,  in a yield of 20 to  indole  UV  89,  using 30%.  No  the  yield  of  formed  in  optimize  Exocathararithine  typical  catharan  the  during  catharanthine  palladium on carbon as catalyst, effort  of  isomer  spectrum  -  and  this  the  low  resolution mass spectrum showed a molecular ion at 336 m/z. A  molecular  elemental that  of  weight  analysis.  of  336  This  catharanthine  3.  was  confirmed  molecular However,  weight TLC  by is  clearly  correct  a  the  same  showed  as  that  exocatharanthine 89 is not identical to catharanthine 3,  and  this fact pointed towards that exocatharanthine 89 might be an  isomer  of  catharanthine  exocatharanthine  89  (table  3.  The 2-13)  C 13  APT  showed  spectrum the  of  same  distribution of the 21 carbons on the different “bond types”  68  as  that  found  exocatharanthine  for 89  is  catharanthine an  isomer  of  3,  confirming  catharanthine  that 3.  The  Table 2-12. Chemical shifts observed in the ‘H NMR spectrum a of exocatharanthine 89  Shift (ppm)  # H  Correlation  1.56 Cd)  3  H-18  1.81  (d)  1  H-17  2.16  (m)  1  H-14  2.32 (s)  2  H-15  2.78  1  H-17  2.90-3.05 (m)  2  H-6,  3.08-3.16  Cm)  1  H-3  3.22-3.41  Cm)  2  H-5,  3.43-3.57  (m)  1  H-5  3.70 (s)  3  H-23  4.00 (s)  1  H-21  5.36  (q)  1  H-19  7.07  (t)  1  H-10  7.16 Ct)  1  H-il  7.25  (d)  1  H-12  7.50 (d)  1  H-9  7.65  1  N-H  (dt)  (s)  a Solvent:  . 3 CDC1  H-3  H-6  69 Table 2-13. Chemical shifts observed in the ‘ C 3 spectrum of exocatharanthine 89 (solvent CDC1 ). 3  Shift (ppm)  1 _ a  12.85 21.44  Correlation  +  C-6 C-14  29.72  +  C-15  37.32  +  C-17  50.28  +  C-3  52.72  C-23  53.02  +  C-5  55.53  +  C—16  63.59  C-21  110.34  C-il +  C-7  118.22  C—9  118.36  C—19  119.36  C—10  121.83  C—12  128.72  +  C-8  135.12  +  C-13  136.98  +  C-2  137.09  +  C-20  174.47  +  C-22  a  NMR  C-lB  27.31  110.38  APT  indicate whether the signal is positive or negative.  70 2-14. Chemical shift correlations a• HETCOR spectrum of exocatharanthine 89 Table  C 13  correlation  (ppm)  C 13  obtained  ‘H correlation  (ppm)  (ppm)  (ppm)  1.56  110.34 (C—il)  7.16  21.44 (C-6)  2.95 and 3.28  110.38  27.31  (C-14)  2.16  118.22 (C-9)  7.50  29.72 (C—15)  2.32  118.36  (C-19)  5.36  37.32 (C-17)  1.81 and 2.78  119.36  (C-iC)  7.07  50.28 (C-3)  2.99 and 3.10  121.83  (C-12)  7.25  52.72  3.70  128.72 (C-8)  53.02 (C-5)  3.30 and 3.50  135.12  (C-13)  55.53  (C—16)  none  136.98  (C—2)  63.59  (C-21)  4.00  137.09  (C—20)  12.85  (C—18)  (C-23)  (C-7)  174.47 (C-22)  a Solvent:  . 3 CDC1  in  -  -  -  —  -  —  the  71 Table 2-15. Chemical shift correlations obtained in the a COSY spectrum of exocatharanthine 89  Shift  (ppm)  # H  Correlation  1.56  (d)  3  5.36,  2.32.  1.81  (d)  1  2.78,  2.32,  2.16.  2.16 (m)  1  3.10,  2.78,  2.32,  1.81.  2.32 (s)  2  5.36,  2.95,  2.16,  1.81.  2.78 (dt)  1  3.10,  2.16,  1.81.  2.90-3.05 (m)  2  3.50,  3.30.  3.10 (m)  1  2.78,  2.16.  3.22-3.41 (m)  2  3.50,  2.95  3.50  (m)  1  3.30,  2.95.  3.70  (s)  3  4.00  (s)  1  5.36 (q)  1  2.32,  1.56.  7.07 (t)  1  7.50.  7.16 (t)  1  7.25 (d)  1  7.50 (d)  1  7.65  1  (s)  a Solvent:  . 3 CDC1  7.07.  72  Table 2-16. Observed enhancements a• exocatharanthine 89  Irradiation (ppm)  63.59  4.00 (H—21)  136.98/137.09  (H—18)  a Solvent:  137.09  (100%),  Irradiation (ppm)  (100%),  50.28  (17%).  (100%).  enhancements  4.00 and 1.56.  4.00 (H—21)  5.36 and 3.50.  3.70 (H—23)  7.65,  1.56  5.36 and 2.32.  (H-18)  . 3 CDC1  in  NOE  experiments  Observed enhancement (ppm)  (H-19)  a Solvent:  29.72 (29%).  . 3 CDC1  Table 2-17. Observed a exocatharanthine 89  5.36  on  Observed enhancement (ppm)  5.36 (H-19)  1.56  experiments  in SINEPT  7.25 and 5.36.  on  73  highest field signal in ‘H NMR spectrum of exocatharanthine 89  (table  2-12)  integrating  to  is  a  three  doublet  at  protons.  the methyl group 0-18.  This  1.56  ppm  signal  (J  was  6.0  =  Hz)  assigned  The COSY spectrum (table 2-15)  to  showed  that the methyl group was coupled to the olefinic proton at 5.36  ppm,  thereby establishing exocatharanthine 89  to be  a  double bond isomer of catharanthine 3,  with the double bond  shifted  catharanthine  from  position  C-15/C-20  in  3  to  position C—15/C-19 in exocatharanthine 89.  Position 15.  18.  and 21:  19,  As mentioned above the ‘H NMR spectrum (table 2-12) and the COSY spectrum 1.56  ppm  and  (table 2-14) at  118.36  ppm,  (table 2-15)  ppm.  The  correlated  Hz  field  HETCOR  spectrum  12.85 ppm and C-19 aliphatic  signal  to be  at  63.59 was  In the HETCOR spectrum this carbon signal  to  the in  proton table  three bond  between  hydrogen. 19)  lowest  listed  detection of 7  to be at  The  with a negative amplitude in the 13 C APT spectrum,  experiments  of  respectively.  showed C-18  assigned to 0-21. was  ppm  5.36  established H-18 and H-19 at  C  at  2-16  4.00 are  couplings with  and H  by  ppm.  The  optimized a  coupling  irradiation of  the  SINEPT for  the  constant  appropriate  Irradiation of the olefinic proton at 5.36 ppm (H  resulted  in  a  maximum  enhancement  at  63.59  ppm  corresponding to 0-21 and a smaller enhancement at 29.72 ppm indicating signal  at  0-15. 29.72  The ppm  APT  C 13 was  a  spectrum  methylene  confirmed  group  and  that  the  the  HETCOR  74  spectrum established the corresponding protons to be at 2.32 ppm  (table  2-14).  The NOE experiments  listed in table  2-17  shows that irradiation at 5.36 ppm (H-19) resulted in an NOE enhancement group at  at  H-21  1.56 ppm.  (4.00  ppm)  well  as  as  the  at  methyl  Irradiation of  the methyl  group at  ppm gave a NOE enhancement at H-19  (5.36 ppm),  but not at H-  21.  Instead the irradiation of the methyl group resulted in  an enhancement at 2.32 ppm, to  1.56  the  C-15  methylene  confirming this signal to belong  group,  and  firmly  establishing  the  stereochemistry of the double bond to be E. Next  to  the  H-19  proton  observed a small quartet to 1/6 of a proton. proton was  =  the  6 Hz)  NMR  spectrum  is  at 5.26 ppm integrating  The COSY spectrum established that this  coupled to a doublet  1/6 of a methyl group. and H-18  (J  in  at  1.65 ppm integrating to  These to signals are assigned to H-19  in the corresponding Z-isomer of exocatharanthine,  and from the HETCOR spectrum C-18 and C-19  (in the z-isomer)  were found to resonate at 13.10 and 118.36 ppm respectively.  Position 14 and 23: The  remaining  hydrogens  in  52.72  and  ppm  the are  aliphatic carbons C 13  APT  assigned  spectrum to  0-14  with an odd number of resonate  at  and  respectively.  C-23  27.31  The HETCOR spectrum correlated H-14 at 2.16 ppm and 3.70 ppm.  and  H-23 at  75  Position 3,  5,  6,  16 and 17:  Of the remaining aliphatic carbons 3,  5,  16 and 17,  6,  C-16 was assigned to the signal  at  of the five carbon signals that  did not show correlation to  55.53 ppm,  the only one  any hydrogens atoms in the HETCOR experiment.  The remaining  four aliphatic carbon,  were  all  methylene groups,  the 13 A APT spectrum to resonate at 21.44, 53.02 ppm. H—14  proton  3.10,  2.78,  In the COSY experiment at  2.16  2.32,  ppm was  and  1.81  ppm in the ‘H NMR revealed, cross peaks,  in  50.28 and  listed in table 2-15 the  found  ppm.  37.32,  found  to  couple to protons  at  Decoupling of H-14 at 2.16  besides confirming the observed  a coupling to a proton in the multiplet 2.90  -  3.05 ppm. Consequently coupling from H-14 to all the vicinal protons was  were  accounted  correlated  experiment  above  listed  in  for. to  The  C-15  table  proton at  2-14  signal  29.72  at  2.32  ppm  ppm.  The  HETCOR  correlated  the  proton  signals at 3.10 and 2.99 ppm and to the carbon at 50.28 ppm and  the  proton  signals  signal at 37.32 ppm.  at  2.78  and  1.81  ppm  to  the carbon  Taking the size of the chemical shifts  into consideration the carbon signals at 50.28 and 37.31 ppm are  assigned  chemical  to  C-3  and  shifts  the  remaining  C-17  respectively. carbon  Based  signals  on  their  53.02  and  21.44 ppm are then assigned to C-5 and C-6 respectively.  The  at  HETCOR experiment established the H-5 protons to be at 3.30 and 3.50 ppm and the H-6 protons to be at 2.95 and 3.28 ppm.  76  Position 1,  10,  9,  11 and 12:  The singlet observed in the  NMR spectrum at 7.65 ppm  was readily assigned to the indolic N-H proton. of  the  protons  resulted  in  in  small  a  group  methoxy NOE  at  ppm  3.70 of  enhancement  the  Irradiation (table  N-H  2-17)  proton  as  well as the doublet at 7.25 ppm establishing this proton to be  the  indolic  doublet listed ppm  at  proton  7.50  ppm  H-12  must  it was  coupled  the  to  Thus  2-17).  H-9.  be  in table 2-15  was  (table  From  found that triplet  COSY  the  found to  resonate at  118.22,  spectrum  ppm  7.07  7.50  thereby  By way of exclusion it  followed that the triplet at 7.16 ppm must be H-li. HETCOR experiment (table 2-14) C-9,  other  the doublet at  at  establishing this signal to be H-b.  the  C-b,  119.36,  From the  C-li and C-12 were  110.34  and  121.83  ppm  respectively.  Position 2, The  7,  8,  13,  carbonyl  C-22  shift at 174.47 ppm. ppm  resulted  (table  in  2-16),  20, is  22: readily assigned  to the  chemical  Irradiation of the methyl group at 1.56  enhancement which  at  the  therefore  carbon  was  at  137.09  assigned  to  ppm  C-20.  Irradiation of H-21 at 4.00 ppm resulted in a enhancement of the  carbon  signals  at  137.09,  136.98  signal at 136.98 was assigned to C-2. carbon  spectrum  of  signals at 110.38, 7,  catharanthine  and  50.28  ppm.  The  By comparison with the  (table  2-18),  the  carbon  128.72 and 135.12 ppm were assigned to C  C-8 and C-13 respectively.  77  Assignment of the a• catharanthine 3 Table 2-18.  Position  C (ppm) 3 ‘  chemical shifts of  Position  C (ppm) 13  2  136.0  13  134.7  3  52.9  14  30.4  5  49.3  15  123.4  6  21.0  16  55.0  7  110.2  17  38.0  8  128.4  18  10.5  9  117.7  19  25.9  10  118.9  20  148.5  11  121.3  21  61.5  12  110.2  22  173.6  23  52.0  a Solvent:  The  . 3 CDC1  numbering  of  the  carbon atoms  in  exocatharanthine  89 is shown below and the assignment of the chemical shifts for 89 are summarized in table 2-19. 6  5  “013N2  21  3 C 2 CO H 22  23  20 19  H 18  78 Table 2-19. Assignment of the ‘H and 13 C chemical shifts of exocatharanthine 89 (solvent CDC1 ). 3  Position  1  H ã(ppm)  C ö(ppm)  7.65  2  136.98  3  2. 99/3. 10  50.28  5  3.30/3.50  53 .02  6  2.95/3.28  21.44  7  110.38  8  128. 72  9  7.50  118.22  10  7.07  119.36  11  7.16  110.34  12  7 .25  121.83  ‘3  135.12  14  2.16  27.31  15  2.32  29.72  16 17  55.53 1.81/2.78  37.32  18  1.56  12.85  19  5.36  118.36  20  21  137.09 4.00  22 23  63. 59 174.47  3.70  52. 72  79  In  an  attempt  catharanthine for metal  3  to  to  the  optimize  isomerization  exocatharanthine  89  isomerization of  the double bond  catalyzed  homogeneous conditions were  looked  various  that  isomerization  conditions.  The  hydrogenation  conditions  hydrogen  present  for  occur  However,  under  therefore  under  which  under  it turned  any  these  of  turned back  the  to the  isomerization was  to  present  be  take place. the  The  in  fact  isomerization  to  for  order  take  addition-elimination  Even  though  over  hydrogenation  to  be  slightly  favored  prehydrogenated catalyst was 4)  it  was  for  optimization hydrogen  practical  using  a  atmosphere.  used  However, 15,  the too  87b/89 much  to  non-prehydrogenated Change  of  catharanthine 3 in the range of 1 influence  decided  ratio  catalyst  the -  (table  the  significant  solvent  improvement  entry 4 and 6).  from of  a  1  and  continue  the  entry  in  concentration  a of  3 mg/mL does not seem to 2-21,  resulted  methanol the  when  catalyst  entry  in  20-dihydrocatharanthine 87b (table 2-21,  Changing  1,2-  isomerization  (table 2-21,  reasons  to  indicates  place  a metal hydride  reaction.  the  that hydrogen has  that the mechanism of the reaction is  seemed  are  A preliminary investigation established  has  isomerization to be  not  attention was  first discovered . 46 that  did  conditions  In table 2-20  . 47 at  listed the different conditions examined. out  of  87b/89  to  an  1  and  increase  3). in  entry 1 and 2). benzene  ratio  gave  (table  a  2-21,  80  Table 2-2O. Conditions catalyzed isomerization of a ranthine 89  EXP METAL  1  Pd  investigated catharanthine  for the metal 3 to exocatha  WORKUPb  CATALYST  SOLVENT  °C/hr  CO 3 Pd(CF 2 )  COCH CH 3  RT/4  A  then  and  60/1.5  B  2  PdC1 2 Na 4  AcOH  50/24  C  3  -AgBF 4 PdC1  CN 3 CH  RT/24  A  then  and  70/4  B  4  (PhCN) 2 PdC1  3 CHC1  61/7  C  5  (PhCN) 2 PdC1  CN 3 CH  82/3  A  6  (PhCN) 2 PdC1  benzene  79/11  B  7  (Ph 2 PdC1 P 3 )  3 CHC1  RT/24  A  then  and  61/5  B  a,b The table is continued on the following page, and b at the end of the table.  see note a  81  a 2 _ 0 continued. Table 2  EXP METAL  WORK_UPb  CATALYST  SOLVENT  °C/hr  2 . 3 RhC1 0 H  EtOH  78/4  C  9  2 . 3 RhC1 0 H  t—BuOH  108/4  C  10  2 . 3 RhC1 0 H  i—PrOH  82/4  C  11  P) 3 RhC1(Ph  benzene  RT/20  B  8  Rh  12  Ru  P) 3 RuHC1(Ph  benzene  13  Fe  5 Fe(CO)  n-octane  5 Fe(CO)  n-octane  14  then  and  79/3  C  79/5  C  127/4  70/96  B/D  C  then 127/12  a The reactions were carried out using 10 mg of exocatharanthine 89. b Method of work-up: A) Reductive (NaBH ). 4 B) Ligand exchange (aqueous KCN). C) Extractive. D) Decomposition by FeC1 /EtOH. 3  82 2-21. Optimization of the a catharanthine 3 to exocatharanthine 89  Table  exp solvent  TPb  [3]  (%)  (mg/mL)  Pd-C:3  isomerization  of  Temp MethodC 3:87b:89 (°C)  1  MeOH  1.00  1.2:1  rt  A  0:7:3  2  MeOH  1.67  2.4:1  rt  A  0:9:1  3  MeOH  3.00  1.0:1  rt  A  0:7:3  4  MeOH  0.75  1.0:1  rt  B  0:8:2  5  benzene  2.20  1.1:1  rt  B  0:6:4  6  benzene  1.10  1.1:1  rt  B  0:6:4  7  benzene  1.00  1.0:1  40  B  0:5:5  8  benzene  1.79  1.0:1  45  B  0:5:5  9  benzene  0.015  1.00  1.2:1  rt  B  2:3:5  10  benzene  0.030  1.00  1.2:1  rt  B  1:5:4  11  benzene  0.100  1.10  1.1:1  rt  B  1:4:5  12  benzene  1.000  5.10  1.0:1  rt  B  10:0:0  13  benzene  0.030  1.00  1.2:1  60  B  0:3:7  14  toluene  0.030  0.89  1.2:1  80  B  0:2:8  a The reaction were carried out using 10 50 mg of catharanthine 3. b (vol/vol) of thiophene (TP) added to the solvent. C A: prehydrogenated catalyst. B: non-prehydrogenated catalyst. For a description of method A and B see chapter 3.27 in the experimental section. -  83  Also  in  benzene  it  holds  concentration  of  product  (table  2-21,  reaction  temperature  from  further  increase  ratio  that  true  catharanthine  exocatharanthine  in 89  3  does  entry room  the  (table  an  and  5  increase  in  the  not  influence  the  6).  Increasing  the  temperature  yield 2-21,  from entry  the catalyst with thiophene gave a  40°C  to  40  to  6,7).  gave  50%  of  Poisoning  significant  of  increase in  the isomerization at room temperature but also prevented the reaction from going to completion (table 2-21, -  11).  entry 6 and 9  Too large amount of catalyst poison totally inhibited  any reaction reaction  (table 2-21,  temperature  thiophene  resulted  to in  entry 12). 60°C  in  an  However increasing the the  presence  of  0.03%  amount  of  exo  increased  catharanthine 89 and consumption of all the catharanthine 3 (table  2-21,  solvent  as  entry  well  as  10  and  13).  Changing  increasing the  to  a  less  toxic  temperature another  10°C  it was possible to obtain a further improvement of the yield of  exocatharanthine  the  temperature  yield side  of  above  (table 80°C  exocatharanthine  products.  subsequent they  89  and  of  in  the  these  and  13  resulted  89  Isolation  2-21,  a  14).  decrease  formation side  Increasing  of  products  in  the  several and  a  investigation by mass spectroscopy revealed that  were  carbomethoxydihydrocleavamines.  Comparison  of  their ‘H NMR spectra as well as their fragmentation pattern b, 63 with the data reported in the literature to  determine  the  structure  of  the  it was possible  isolated  dihydrocleavamines as shown in figure 2-10.  carbomethoxy  84  H  H  H  Figure 2-10. The structure dihydrocleavamines.  In  large  R  =  Et  2 = Et, R 1 86c R  =  H  = H, 1 86b R  86a  scale  of  the  preparations  of  isolated  carbomethoxy  exocatharanthine  89  it  turned out to be necessary to lower the reaction temperature to  70°C  to  cleavamines.  prevent In  exocatharanthine obtained  the  the 89  formation optimized  could  exocatharanthine  be 89  of  carbomethoxydihydro  large  scale  obtained was  83%  yield.  The  by  proton  MNR  shown  spectroscopy to be a mixture of the E and the Z typical ratio of 5:1.  It was not possible  procedure  isomer in a  to separate this  mixture.  2.8.  INVESTIGATION OF THE FORMATION OF CATHARANTHINE N-OXIDE 10 AND EXOCATHARANTHINE N-OXIDE 90 The  oxide  10  examination and  of  the  exocatharanthine  reinvestigation exocatharanthine  of 89  the  formation N-oxide  modified  instead  of  of 90  catharanthine was  Polonovski  prompted reaction  catharanthine  3.  N-  by  a  using  In  the  85  preliminary Kutney’s  experiments, research  following  group,  exocatharanthine  8928,  chloroperbenzoic  acid  about well  as for  N_oxi ( 2 t), Th1 de corresponding 21 vindoline 4.  In this  experience  the  excess  50% an  as  excess  generation  the  of  Dr. of  of of  the  was used in the coupling with  way the coupling reaction afforded an  average of 30% of 19’,20’-anhydrovinblastine 91.  The applied  -c H 3 1) peracid 2) vindoline 3) TFRR H  INDOLINE  4 4) NaBH  91  89  excess after  of the  starting  exocatharanthine coupling  material  procedure,  reaction  and  necessitated  3  chloroperbenzoic  with acid  catharanthine  catharanthine  a  of  an  not the  be  accounted  loss  closer  of  look  N-oxide  the  the  10 92.  formed  instead  of  and  at  In  at  the  of to  of  m  gave  50%  7-hydroxy keep  a minimum  to  catharanthine  whole  Treatment of  -70°C  order  for  valuable  amount  16%  62 m 2 Noxide ),  dissolved  kept below 10°C at all times. chloroperbenzoic  equimolar  in dichloromethane  N-oxide  rearrangement of temperature  could  starting with the N-oxide formation.  catharanthine  of  89  N-oxide  10  the the was  By using two equivalents of m one  the  yield  of  7—hydroxy  86  catharanthine Similarly,  N-oxide  could  92  exocatharanthine  exocatharanthine  N-oxide  increased  be  N-oxide were  93  and  90  prepared  in  70%.  to  7-hydroxy and  71%  68%  yield respectively.  PERPCID H  •C H I  15 A ° 2 3 ’ 19,2O  8  HO +  H  U13 I  Q L  10 t  19,O  19,2O  Catharanthine Kutney et  N-oxide  ° and 2 a1.  10  has  Potier et  been  al. T 2 h.  characterized  by  Exocatharanthine N-  oxide 90 exhibited a typical indole UV spectrum absorbing at 221,  274  (shoulder),  280 and 289 nm.  The expected molecular  ion at 352 m/z was observed as well as a peak of significant intensity  at  336  molecular ion. double  bond  showed, compared  corresponding  loss  of  oxygen  from  the  The ‘H NMR spectrum showed that the 19’,20’-  was  still  intact  as would be expected, to  to  exocatharanthine  and  the  carbons  alpha  to  N-4  a significant downfield shift 89.  The  mass  spectrum  of  7-  87  hydroxy exocatharanthine N-oxide 93 showed a small molecular ion  at  368  mhz  with  corresponding to  daughter  peaks  loss of one and  352  at  and  two oxygens  336  m/z  respectively.  The UV spectrum of 93 did not contain the characteristics of an  indole  nucleus.  showed the  Compared  absence of  to  the  90  C 13  spectrum  NMR  a quaternary olefinic carbon and the  appearance of a quaternary aliphatic carbon at 85.0 ppm.  The  presence of the 19,20-double bond was demonstrated in the NMR spectrum. A fractional factorial design 44 with seven variables at two  levels  was  carried  out  in  order  to  screen  various  factors which might influence the reaction of catharanthine 3  with m-chloroperbenzoic  acid.  The  variables  investigated  in the design are listed in table 2-22 and in table 2-23 is given the layout of the design as well as the results. Row  E(92)  in  table  2-23  shows  the  effect  of  the  different  factors on the formation of 7-hydroxy catharanthine N-oxide 92.  It  is  seen that the excess  addition  have  whereas  the  insignificant. peracid  to  the  the  largest  effect In  of  fact,  of  effect  on  the  addition  experiment  peracid and  leads  the  formation  remaining of to  the mode of  another  of  factors equivalent  conversion  of  all  92 are of the  formed catharanthine N-oxide 10 into 7-hydroxy catharanthine N-oxide 92. different 3.  With  Row E(3)  factors  in table 2-23  on the  an absolute  amount  uncertainty  of  shows  the effect of the  remaining  catharanthine  in the order of  1%  on the  yields none of the factors can be said to have a significant  88  influence on the amount of remaining catharanthine. excess  of  peracid  was  used  all  in  the  Since an  experiments  in  fractional factorial design all the catharanthine should, theory,  the in  have been used up. A prolonged reaction time did not  have any influence on either the ratio or the amount of the three compounds, to completion.  showing that  the reaction has  indeed gone  That catharanthine remains despite the excess  of peracid must be due to protonation of the catharanthine 3  Table 2-22. The factors and their levels fractional factorial design in table 2-23. Factor  level 1  solv:  solvent  conc:  concentration  100 mg/mL  temp:  temperature  -10°C  mode of addn:  light:  dichioromethane  used  in  the  level 2  acetonitrile 20 mg/mL -30°C  peracida added as a  3 added as a solid  solid to the solu  to the peracida  tion of 3.  solution.  reaction vessel not  reaction vessel is  covered with Al  covered with Al  foil.  foil.  peracid purity:  n0 ,u 0  oDo o.J 0  peracid excess:  DrO Li 0  5%  a zn-chloroperbenzoic acid.  89 Table 2_238. Investigation of the formation of 7-hydroxy catharanthine N-oxide 92.  expb  solv conc temp mode of light peracid addn  purity  92 % peracid C  C 3 %  excess  1  1  1  1  2  2  2  1  24  5  2  2  1  1  1  1  2  2  10  1  3  1  2  1  1  2  1  2  10  1  4  2  2  1  2  1  1  1  22  2  5  1  1  2  2  1  1  2  16  5  6  2  1  2  1  2  1  1  20  0  7  1  2  2  1  1  2  1  20  1  8  2  2  2  2  2  2  2  12  1  ¼  El  18  18  17  15  17  17  22  ¼  E2  16  16  17  19  17  17  12  E(92)  2  2  0  4  0  0  10  ¼E1  3  3  2  1  2  2  2  ¼E2  1  1  2  3  2  2  2  E(3)  2  2  0  2  0  0  0  a The levels of th factors are defined in table 2-22. b The reactions were performed as described in the general procedure in chapter 3.28 on a 50 mg scale. C The yield is determined by HPLC using 92 and 3 as external standards. The absolute uncertainty on the yield is ± 1%.  90  and/or that the peracid reacts faster with catharanthine Noxide 10  than it does with catharanthine 3.  The latter can  be ruled out since it is possible to minimize the amount of 7-hydroxy catharanthine N-oxide 92 by lowering the excess of peracid  used.  In  catharanthine therefore  order  slows  indirectly  determine  to  down  the  if  formation  promotes  the  protonation  of  N-oxide  of  formation  and  7-hydroxy  of  catharanthine N-oxide 92 2.3 equivalence of acetic acid were added to  the  catharanthine  3  addition  of  the  but  conditions shown  in  as  peracid,  in experiment 3  table  2-24.  This  solution  7 mm  otherwise  using  in table 2-23.  experiment  Table 2-24. The influence of acid hydroxy catharanthine N-oxide 92.  prior  the  the  same  The result is  confirmed  on  to the  that  formation  proto  of  7-  exp solvent conc temp mode of light peracid peracid % 92 % 3 addn  9  1  2  nation of and  1  1  2  catharanthine 3  slows  thereby  indirectly promotes  catharanthine N-oxide 92. 500 mg)  purity  1  down the  excess  2  27  13  the N-oxide  formation  formation of  7-hydroxy  In large scale preparation  (200  -  it was found that equimolar amounts of catharanthine  91  3  and  peracid  overall  the  gave  catharanthine N-oxide 10,  yield  best  namely 85  of  desired  the  90%.  -  For exocatharanthine 89 the use of equimolar amounts of peracid was also found to be optimal, 95%  of  exocatharanthine  N-oxide  giving a yield of 90 with  90  equal  amounts  -  of  unreacted exocatharanthine 89 and 7-hydroxy exocatharanthine N-oxide 93  accounting for the remaining 5  10% of  -  starting  material. Acetonitrile and acetone were also examined as possible solvents.  The  yield  of  N-oxide  in  solvents  these  was  found  to the same as in dichloromethane.  2.9.  REINVESTIGATION OF THE MODIFIED POLONOVSKI REACTION Coupling  conditions O 2 b  of  exocatharapthine  gave  a  yield  of  vinbiastine 91  (scheme 2-13).  molecular  of  ion  792  spectrum  contained  expected  dimeric  the  protons  6.5 Hz) figure  at  stereochemistry  the  appear  -  using  35%  of  Compound 91 and  the  In as  a  as quartet at 5.49 ppm. indicates at  that  . 66 C-16’  resolution  at  mass  of  the  of  spectrum 1.68 ppm  (J  91 =  The CD spectrum in has  91  Another  fragments  NMR  doublet  standard  gave the expected  low  the  the  19’,20’-anhydro-  characteristic  . 64 structure  C-lB’  and H-l9’ 2-ha  all  m/z  30  89  correct  the  compound  was  isolated  from the reaction mixture with the desired molecular ion at 792  m/z  and  the  expected  fragmentation  pattern.  The  ‘H NMR  spectrum showed that this compound also contained a C-19’,C20’-double  bond.  The  CD  spectrum  in  figure  2-lib  indicates  92  that  this  compound must be the C-16’  Analogs of  epimer 94.  H 3 CO  1 iJL •C H  3  3 C 2 CD H  3 C 2 CO H 90 1) VINDOLINE 2)  / /1/  4 3) NaBH  3 H  3 /_CH + H  H  ‘VINOOLINE / CH 2 CO 3  INDOLINE 3 CH  91 Scheme 2-13.  Coupling of exocatharanthine 89 with vindoline  4.  91 have been synthesized by Miller et al., vinblastine  with  desacetylated 12). the  concentrated  and  dehydrated  Comparison of dimers  found  for  indicate  96  and  H-19’ that  no  the  products  chemical  acid  95  shifts  -  9765  the  (figure  2-  H-19’  shift  of  5.49  19’,20’-anhydrovinblastine  91  strongly  with  the  isomerization  chemical  of  the  found  give  to  for  97  in  sulfuric  by dehydration of  19’,20’-double  in ppm  bond  93  has  taken place during  the coupling  of  exocatharanthine 89  with vindoline 4.  a:  CD of 91  Le  1  —1 —2 —3 -4’  250  200  300  350 nm  b: CD of 94 LE  10  200  250  300  Figure 2-11. a) The CD spectrum of vinbiastine 91. b) The CD spectrum of epi vinbiastine 94 (solvent CH CN). 3  350 nm  19’,20’-anhydro19’,20’-anhydro-  94  —5.46 ppm H  95 ppm +  H  4 S 2 H 0 VINBLflSTINE H  96 3 CH +  ppm  28 -H—--5.  97  R: 17_(desacety1)V1fld0l1te  Figure 2-12.  Using strategy influence  Dehydration of vinblastine 1.  fractional  various in  the  factorial  factors modified  were  44 design  investigated  Polonovski  as to  experimental study  reaction with  their  respect  95  to the yield of 19’,20’-anhydrovinblastine gi. of the effect of the reaction temperature,  Investigation  the purity of the  m-chloroperbenzoic acid and the addition rate of TFAA on the of  yield  91  (table  2-25)  disclosed  only  that  reaction  the  temperature had a significant influence on the yield of 91. Comparison  of  the  temperature  two  that  shows  levels  a  reaction temperature of -60°C gives a better yield than at 40°C.  -  Experiment 5 in table 2-26 confirmed this conclusion.  Lowering the temperature below -73 to -78°C did not increase the  yield  but  reaction  time.  resulted  in  a  Comparison  substantial  of  experiments  1  and  experiments 2 and 4 in table 2-25 indicates that, either  the  addition  rate  of  TFAA  strongly affects the yield whereas Fast  -60°C.  increase  of  addition 10  to  15°C  temperature  drops  temperature  within  TFAA  of  in  back two  established that this brief in  a  significant  drop  in  the  than to  minutes.  the with  3  at -40°C,  peracid  purity  is not the case at  this  in  results  less  down  or  in  increase  15  the  temperature  a  seconds actual  Further  but  the  reaction  experiments  increase in temperature results the  yield  when  the  design  in  table  reaction  is  carried out at -40°C. The  fractional  that with respect  factorial  to the amount of vindoline 4,  2-27  shows  the nature  of the solvent and the amount of TFAA both the nature of the solvent and the amount of TFAA have a significant effect on the  yield  of  19’,20’-anhydrovinblastine  solvent to acetone,  91.  Changing  the  but otherwise using the same conditions  96 Table 2-25. Investigation of the effect of the temperature, the purity of the zn-chloroperbenzoic the addition rate of on TFAA yield the of a• anhydrovinbiastine 91  Factor  level 1  level 2  -60°C  -40°C  96%  69%  Reaction temp Peracid purity Addn rate of TFAA  exp  Reaction temp  <  1 sec  20 mm  PeracidC addn rate purity  of TFAA  reaction acid and 19’,20’-  HPLCb  Isolated  * 91  % 94  34  2  1  -60°C  96%  20 mm  2  -40°C  96%  <  1 sec  17  3  3  -60°C  69%  <  1 sec  33  2  4  -40°C  69%  20 mm  27  1  ½E(9J-)  33.5  25.5  25.0  ½E(91)  22.0  30.0  30.5  E(91)  11.5  4.5  % 91  -  -  -  -  5.5  a 200 mg exocatharanthine 89 in 2 C1 used. The reactions CH were performed as described in chapter 3.29 using steps la and 7a. The reactions were not followed by HPLC. b HPLC conditions as described for exocatharanthine 89 in chapter 3.28. The yield of 94 is estimated from the height ratio of 91 and 94 assuming that 94 has the same response factor as 91, since no actual calibration curve has been established for 94. c zn—chloroperbenzoic acid.  97 26 _ 2 Table a temperature.  exp  The  reaction  carried  PeracidC Addn rate  Reaction temp  5  Polonovski  purity  -73°C  HPLCb % 91  % 94  1 sec  41  2  <  low  at  Isolated  of TFAA  96%  out  % 91  -  a See note a under table 2-25. b See note b under table 2-25. c see note c under table 2-25.  as  in  yield  experiment (isolated)  investigation  (table  7 of  of  91  2-27),  from  44  acetonitrile,  an  gave  to  After  51%.  acetone  increase  and  an  in  the  extensive  acetonitrile/  dichloromethane (1:1) as solvents for the reaction it became clear  that  the  increased  dichioromethane  to  was  the  not  due  to  coupling which evaporation  obtained  yield  switching  acetonitrile/di-chioromethane or solvent  as  such,  took place during  step  by  is  the  point  but  the  in  an  to  from  acetone  additional  evaporation  step.  The  procedure  where  the  the  reaction is “finished” and the cooling bath is removed while the  solvent  reaction  is  is  evaporated  raised  to  off.  about  The  20°C  temperature  during  the  of  the  evaporation.  After the solvent has been evaporated off methanol is added, the reaction is cooled down to -20°C and reduced with sodium borohydride. revealed but  A  that  first  closer in  when  examination  acetone, the  of  the  evaporation  step  no coupling took place at -60°C  reaction  was  heated  above  _2000.  98  Table 2_278. Investigation of the effect of the amount of vindoline 4, the nature of the solvent and the amount of 918. TFAA on the yield of 19’,20’-anhydrovinblastine  Factor  level 1  level 2  Vindoline 4  1.5 eq  1.0 eq  3 C CH / 2 C CH N 1  C1 2 CH  Solvent  (1:1)  .  TFAA  exp  4.8 eq  Vindoline  Solvent  HPLCb  TFAA  (4)  ½1  1.2 eq  %91  Isolated  %94  6  1.5 eq  3 C CH / 2 C CH N 1  1.2 eq  19  3  7  1.0 eq  3 C CH / 2 C CH N 1  4.8 eq  48  7  8  1.5 eq  C1 2 CH  4.8 eq  30  5  9  1.0 eq  C1 2 CH  1.2 eq  8  1  1  24.5  33.5  39.0  2  28.0  19.0  13.5  3.5  14.5  25.5  E(9l)  a See note a under table 2-25. b See note b under table 2-25.  %91  -  44 -  -  99  Even in dichioromethane, obtained  by  lowering  where  the  an  increased yield of  temperature  from  -60  to  91  is  —73°C,  additional product is formed upon heating the reaction above -30°C.  This apparent contradiction of the dependence of the  yield  on  the  temperature  can  only  be  explained  by  the  existence of two different intermediates of exocatharanthine 89,  each  desired  capable dimer  of  coupling  (scheme  with vindoline  2-14)  give  the  acylated  vindoline 4  Addition  .  exocatharanthine N-oxide 90 must  4  give  the  TFAA  to  to of  lead to acylation of 90 to  intermediate  98  which  reacts  then  with  at -60°C to give 19’,20’-anhydrovinblastine 91.  To account for the observed formation of dimer on heating of the reaction mixture a second intermediate must be present, structurally different from 98,  which is capable of reacting  with vindoline 4 but only at a much higher temperature than the temperature needed for the reaction of 98 with vindoline 4.  That the yield of 91  reaction  temperature  competitive  reaction  transforming 98 of  coupling  from  -60  between  into a  with  actually increases on lowering the  second  vindoline  to  -73°C  vindoline  points 4  and  intermediate 4  but  99  only  towards  reaction  a also  at  a  capable higher  a  temperature. In  order  exocatharanthine  to  substantiate  intermediates  the  capable  idea of  of  two  coupling  with  vindoline 4 the coupling reaction was followed by HPLC using the same conditions as those described for exocatharanthine 89 in  chapter 3.28.  In  figure  2-13  are  shown  the  I-IPLC  100  H 3 CO  0  LC1  CH 2 CO 3  CH 2 0 3  90  89 C003 CF 3 OCF TFflR  SECOND INTERFIEDIPTE H  99  3 H 98 VINDOLINE  >—30°C  _73o  COO 3 CF  3 H Pc 3 H 100 4 NaBH  91  Scheme 2-14. The Polonovski reaction.  reaction  pathways  in  the  modified  101  .3  A  5  B  1  6 8  E  7  1 0  6  1: 2: 3: 4: 5: 6:  1.2 mm. 2.4 mm. 3.1 mm. 3.9 mm. 9.3 mm. 11.2 mm  6: 11.2 mm. 9.7 mm. 7: 8 :11.4 mm.  1: m-Chloroperbenzoic acid. 2: 7-Hydroxy-exocatharanthine Noxide 93. 3: Exocatharanthine N-oxide 90. 4: Vindoline 4. 5: Second intermediate 99. 6: Iminium dimer 100. 7: Epi 19’ ,20’ -anhydrovinbiastine 94. 8: 19’, 20’ -Anhydrovinbiastine 91. A) Exocatharanthine N-oxide 90 and vindoline. 4 B) Acylated exocatharanthine N-oxide. C) The reaction mixture at -73°C after the reaction has stopped. D) The reaction mixture after concentration and heating. E) The reaction mixture after reduction with NaBH . 4 Figure 2-13. Monitoring of the Polonovski reaction by HPLC (same conditions as for exocatharanthine 89 in chapter 3.28).  102  chromatograms at different stages of the reaction. be  noted  intermediates itself  the  that  but  might  peaks not  attributed  represent  the  to  actual  the  rather an artifact created  It should various  intermediate  from that  particular  intermediate by reaction with methanol. In  experiment  exocatharanthine 4  vindoline  10  (table  N—oxide  90  (step  1).  After  2-28) prior  TFAA the  to  completion  of  added  to  addition  of  was  the  reaction  of  TFAA with 90 the excess of TFAA was evaporated of f prior to the addition of vindoline 4 (step 2). TFAA  is  absolutely  necessary  acylation of vindoline 4 reaction  is  heated  chromatogram  that  in  prior  added  experiment in  only  11 16  to  the  at  If,  -65°C.  the  These  from  place  TFAA,  reaction  the  HPLC  unless  the  the vindoline 4 as  is  was  78%  done  in  “complete”  experiments  two  the  present when the  however,  of  prevent  to  clear  is  takes  addition  (table 2-29), mm  It  coupling  reaction is heated above —30°C. is  order  and any dimer(s)  above —30°C. no  This removal of excess  clearly  demonstrate that exocatharanthine N-oxide 90 in the presence TFAA  of  only  is  transformed  into  an  intermediate  (99)  capable of coupling with vindoline to the desired dimer, only at was  temperatures above  subsequently confirmed  -30°C.  This NMR  by  observation by HPLC (figure  the nature of this second intermediate 99, a  fragmentation product  with  the  of  trifluoroacetate  98 ion  or  a  but  As  to  it must either be  reaction  released  2-15).  upon  trifluoroacetic anhydride with the N-oxide 90.  of  98  reaction  of  product  103 a 2 _ 8 Addition of TFAA to exocatharanthine N-oxide 90 Table 2 prior to the addition of vindoline 4. CONDITIONSb.  EXP  10  1)  90 at _6000,  HPLCC  add TFAA,  stirring for  Remaining N-oxide 90: 20% after 16 mm  2 hr.  7% after 1 hr 3 mm 3% after 1 hr 56 mm 2)  -60  -  -30°C over 30 mm  while evac.  on vac.  line,  stop evacuation and cool to  3)  -40°C,  yellow foam dissolved  in 2 ml, of , C1 2 CH  cool to  HPLC profile looks the same after as before the evaporation  4)  -55°C, 1 hr,  5)  -30°C,  add 4,  stirring for  then heating to  stirring for 2.5 hrs,  then heating to  time temp %4  100 % %99 d  0  -52  100  100  0  40  -33  98  96  0  57  -30  94  94  0  a The table is continued on the following page. b,c,d See the end of the table on the next page.  104 Table 2-28 continued. CONDITIONSb.  EXP  HPLCC  time temp %4 6)  0°C,  stirring for 4 hrs,  then workup  Yield:  100 % %99 d  94  —36  97  86  0  138  —42  88  77  0  165  -30  92  75  0  193  0  96  38  46  335  0  55  8100  9% 91 11% 94  b 200 mg of exocatharant hine 89 in 2 C1 1.00 equivalent CH , of m-chloroperbenzoic acid and 1.5 equivalent of virido line 4 used. The experiment was performed as described in chapter 3.29 (using steps la and 7b) except for the changes in addition, temperatures and reaction times described in table 2-28. c HPLC conditions as described for exocatharanthine 89 in chapter 3.28. d The time is in minutes and time 0 mm is the point when vindoline 4 was added. The amount of 4 and second intermediate 99 is the height of the peak at time x mm in percent of the peak height at time 0 mm. The amount of intermediate 100 is the peak height at time x mm in percent of the peak height at time 335 mm.  105 29 Addition of vinc7oline 4 prior to the addition _ 2 Table a• of TFAA. The of importance concentrating the reaction mixture after the reaction has gone to “completion” at  —65°C. CONDITIONSb  EXP  11  1) 90 and 4 at -65°C, of TFAA,  2)  —65  -,  HpLCc,d  addition  stirring for 3 hr.,  time %90  %4  0  100  100  0  0  16  15  65  91  78  39  5  55  93  90  96  0  55  103  103  159  0  51  100  100  end  0  3  0  185  -40°C over 20 mm  while evac.  on vac.  line to  give a red foam. Vacuum released and the foam dis solved in 1 mL CH C1 at 2  3)  -45°C over 15 mm  4)  -45°C evac. 25 mm.  on vac.  line for  Vacuum released and  heating from  a The table is continued on the following page. b,c,d See the end of the table on the next page.  %99 %100  106 Table 2-29 continued  CONDITIONSb  EXP  5)  HPLCC  -45°C to RT over 1 hr.  6) Yield: HPLC:  72% 91 12% 94  Isolated:  70% 91  b 100 mg of exocatharanthine C1 1.00 equivalent CH , 89 in 2 of m-chloroperbenzoic acid and 1.0 equivalent of vindo line 4 used. The experiment was performed as described in chapter 3.29 (using steps la and 7b) except for the changes in temperatures and reaction times described in table 2-22. c See c under table 2-28. d The time is in minutes and time 0 mm is the point when TFAA was added. The amount of exocatharanthine N-oxide 90, vindoline 4 and the second intermediate 99 is the height of the peak at time x mm in percent of the peak height at time 0 mm. The amount of intermediate 100 is the peak height at time x mm in percent of the peak height at time 159 mm. The amounts at time “end” refers to the composition of the reaction after evaporation and heating (entry 5).  107 30 The effect of concentrating (versus not concen _ 2 Table a• trating) the reaction mixture after the reaction has gone to “completion” at -65°C on the yield and product distri bution. CONDITIONSb  EXP  12  1) 90 at -60°C, TFAA,  I-IPLC°  addition of  stirring for 1.5 hr.,  Remaining 90: <1% after 22 mm  heating to 2)  -45°C,  evac.  for 20 mm,  on vac.  line  then heating to  -30°C for 5 mm  whereby a  yellow foam is formed, cooling to 3)  -50°C,  the yellow foam is  dissolved in 2 mL 2 C1 CH  12A 1)  99 at -65°C,  addition of 4,  HPLC profile looks the the same as before evap  time temp  %4  100 % %99 d  stirring for 1 hr. 2)  -65°C,  add 4.8 eq.  TFAA,  stirring for 1 hr.  0  -65  100  100  0  45  -55  92  89  0  -50  85  85  0  <  100  3)  -50°C for 30 mm  238  4)  -50°C  545  TFAA,  a b,c,d,e  +  another 4.8 eq.  0  e 0  stirring for 4.5 hr.  The table is continued on the following page. See the end of the table on the next page.  108 Table 2-30 continued.  CONDITIONSb  EXP  HPLCC  12A 5)  -30°C,  evac.  for 20 mm,  on vac.  line  thick oil,  then heating to 6)  0°C for 2.5 hr  7)  Yield  55% of 91 12% of 94  123 1) 99 at -65°C,  addition of 4,  stirring for 2 hr., 2)  -50°C,  %4  %99  then  stirring for 5 hr.,  heating to 3) 0°C and stirring for 2 hr.  4) Yield  time temp  0  -60  100  100  0  90  -50  99  92  0  203  -50  97  95  0  480  0  81  <17  100  11% of 91 7% of 94  b 100 mg of exocatharanthine 89 in 2 C1 and 1.00 equi CH valent m-chloroperbenzoic acid used. After step 3 in exp. 12 the reaction mixture was divided equally into exp. 12A and 12B. 1.5 equivalent vindoline 4 (used in exp. 12A and 12B. The experiments were performed as described in chapter 3.29 (using steps la and 7c) except for the changes in addition, temperatures and reaction times described in table 2-30. c See c under table 2-28. d See d under table 2-28. c Substantial amounts of acylated vindoline 4.  109  From the HPLC chromatogram of experiment 11 in table 229  it can be seen that the reaction stops after 1 hr 30 mm  at  —65°C  either  because  the  dimeric  all  the  N-oxide  exocatharanthine  intermediate  temperature  100.  been  intermediate  Prolonged  not  does  has  90  give  converted  reaction  any  or  99  increase  the  to  time  this  at  in  to  either  intermediates. After evaporation of the solvent at -45°C and subsequent  heating  intermediate prior  100  the  has  and  amount of the second  step  (step  2,  table  The  unnatural  experiment heating exp.  12  not  12A  stereochemistry in  only  table  2-30.  gives  compared  a  its  height  giving  an  formed at  of  the  -65°C in  concentration  as well  as on the  stereochemistry to the one  at  C-16’,  can  yield  12B)  of  91  also  but  seen  be  prior  Concentration  higher  exp  to  is  on the yield,  ratio of dimers with the natural with  to  It is clear that a considerable  importance  2-29)  dimeric  the  2-29)  (table  intermediate 99  100.  of  compared  85%  heating  isolated yield of 70% of 91.  competition with  height  increased  evaporation  to  peak  from the  to  2-30,  (table  suppresses  the  performing  the  formation of the epimer 94. In modified  conclusion, Polonovski  formation of 3)  the  reduction  reaction  N-oxide, with  traditional  2)  way  consists  of  of  three  coupling with vindoline  sodium  borohydride  at  -50°C,  work-up.  and  1)  steps;  As  a  result of this investigation into the factors affecting the yield  of  leading  the to  a  modified second  Polonovski intermediate  reaction 99  was  a  side  reaction  discovered.  This  110  intermediate coupling  has  99  with  also  vindoline  been  4,  shown  under  to  capable  be  appropriate  of  conditions.  Taking advantage of this discovery an increased yield of the desired  dimeric  traditional  alkaloid  procedure.  is  obtained  This  improved  compared  to  the  procedure  of  the  Polonovski reaction consists of five steps; N-oxide,  2)  coupling with vindoline  evaporation of almost  dry  the  solvent  residue  to  borohydride and work-up.  below  0°C,  at  73  -  -30°C,  1)  4)  formation of  to  heating  reduction  5)  78°C,  -  of  3) the  sodium  with  By monitoring the reaction by HPLC  it has been found that incorporation of these two new steps after the point where the reaction was  “complete”  at  -70°C  resulted in an increase of the peak height relating to the intermediate 100 of up to 75% 3,  85%  case  in  of  advisable  the  case  of  in the case of catharanthine  exocatharanthine  dihydrocatharan-thine to  first  second intermediate  transform 99  since 99  form both 16’S and 16’R dimers, the  coupling  formed.  -73°C  shows  Concentration  temperature modified  stages  of  of  way  it  the  the  been  yield  of  and  is,  45%  in  the  however,  N-oxide  not  into  90  the  reacts with vindoline 4 to whereas the HPLC profile of  an  only  the  was  found  that  Ob, dimer 2  16’S  dimer  role  important  coupling  reaction  has  the  that  plays  Polonovski  traditional decrease  at  It  87b. all  89  reaction.  at  is  both  When  the  performed  in  the  dilution  lead  .  In  the  to  a  improved  procedure the concentration has an extreme influence on the coupling  reaction  in  terms  of  the  yield  and  the  product  111  ratio  between  mixture  is  16’S  the  not  intermediate  16’R  and  concentrated not  99  isomers.  much  as  shows  only  If  reaction  the  as  possible  a  higher  second  the  degree  of  decomposition and thereby lower yield upon heating but also a  substantial  increase  in  formation  the  of  undesired  16’R  dimer.  2.10.  IDENTIFICATION AND CHARACTERIZATION OF THE SECOND INTERMEDIATE 99 IN THE MODIFIED POLONOVSKI REACTION As  demonstrated  exoca-tharan-thirie Polonovski  in  the  previous  intermediate  reaction which  is  99  is  also  90  was  intermediate are  listed  in  capable  all  intermediate 99.  the  the  together second  five with  possible  this  figure  2-14  In  into  for  second  this  99  group.  types  shown  of  carbons  excluding It  can  in  the be  figure found  from  signals  seen  for  that  the  possible structures for the second intermediate are 101, and  99.  These  with  summarized the different  structures  actual  intermediate  trifluoroacetate  coupling  signals that are to be expected for each  possible the  4.  structures are  modified  the  The exocatharanthine  of vindoline  In table 2-31  types of carbon-13 of  absence  of  second  a  exclusively  transformed  the  in  formed  vindoline 4 to give the desired dimer. N-oxide  chapter  possibilities  all  contain  one  2-14 the the only 103  quaternary  aliphatic carbon and one tertiary aliphatic carbon.  From the  l3 APT spectrum listed in table 2-34 the chemical shift for the  quaternary  aliphatic  carbon  was  found  to  be  at  112  85.89 ppm. shift be  for  This  a positively 200  above  excludes  ppm  (  structure  101  charged carbon  e.g.  C ) 3 (CH  ;  since  would  328 ppm  the  be  chemical  expected  and  C 3 (Ph)  to ;  211 ppm).  6  9  5  3 OCOCF  1o  15  11 12  3 C 2 CO H 22  19  ‘3  23  98  101  103  10  CF C 3 O  3 H  3 -CH  C0 CH 2 3  CH 2 0 3  3  99  Figure 99.  2-14.  Possible  structures  of the second  intermediate  113  Table 2-31. The type of carbon signals expected for each of the five possible structures in figure 2-14, together with the actual types of carbons found in the APT spectrum for the second intermediate 99 (excluding the signals from the trifluoroacetate group).  Structure  =0<  =CH-  >C<  >CH-  2 >CH  3 —CH  98  6  5  1  2  5  2  101  6  6  1  1  5  2  102  7  6  0  1  5  2  103  6  6  1  1  5  2  99  6  6  1  1  5  2  second inter mediate99  6  6  1  1  5  2  This second  leaves  103  intermediate.  spectrum  of  the  and In  second  99  as possible  figure  results  of  two  the  NMR  is  shown  intermediate  99  and  HETCOR  the  The results of  spectrum are summarized up in table 2-34. the  for the  2-15  shifts are summarized in table 2-32.  shown  structures  chemical APT  In table 2-35 are  experiments,  one  the aliphatic region the other the aromatic region.  covering  I-’  ‘.0 ‘.O  CD  CD ci F.-’. 0)  CD ‘-1  rt  H  ci  U) CD C) 0  CD  H  0  CD C) r1  (n  z  QJ.  CD(D rt  01•1  (.Q  I-I. j I-I.  5  4  h  3  2  7  1  115  Table 2-32. Chemical shifts observed in the a spectrum of the second intermediate 99  # H  Shift (ppm)  2.12  Correlation  (d)  3  H-18  2.60 (d)  1  H—15  2.67—2.82  (m)  2  H-15,  H-17  2.84-3.01  (m)  2  H-14,  H-17  3.42 (t)  1  H-6  3.67 (s)  3  H-23  3  H-3,  3.71-3.82  Cm)  4.12  (t)  1  H-5  4.59  Cd)  1  H—5  7.15  (t)  1  H_lOb  7.25  Ct)  1  H_llb  7.39  (q)  1  H-19  7.46  (d)  1  H--12  7.73  (d)  1  H-9  9.10 (s)  1  N—21  11.75(s)  1  N—H  H-6  a The assignment might have to be reversed b Solvent:  . 6 Acetone-d  NMR  116 Table 2-33. Chemical shift correlations obtained in the a• C0SY spectrum of the second intermediate 99  Signal (ppm)  Correlation (ppm)  2.60 (d)  2.77,  2.92,  3.77.  2.77 (d)  2.60,  2.92,  3.77.  2.87 (m)  3.77.  2.92 (t)  2.60,  2.77.  3.42 (t)  3.75,  4.12,  4.59.  3.75 (d)  3.42,  4.12,  4.59.  3.77 (s)  2.60,  2.77,  2.87.  4.12 (t)  3.42,  3.75,  4.59.  4.59 (d)  3.42,  3.75,  4.12.  a Solvent: Acetone-d . 6  117 Table 2-34. Chemical shifts observed in the ‘ C APT NMR 3 a• spectrum of the second intermediate 99  6(ppm)  +/_b  16.71 26.56  +  27.46  Correlation  6(ppm)  1 _ b  C-18  110.83  C-6  112.98  C-14  115  +  ÷  Correlation  C—7 C—12 quartet; 2 C 3 CF O  30.78  +  C-15  119.52  -  C-9  33.03  +  C-17  121.19  -  C_lob  54.31  +  C-3  124.55  -  C-23  128.14  +  C_2C  54.31  C_llb  60.87  +  C-5  128.34  +  C-8  85.89  +  C-16  131.14  +  C_2OD  136.73  +  C-13  157  +  quartet;  162.03  -  2 C 3 CF O  C—19  167.94  +  C—22  170.56  -  C-21  a Solvent: Acetone-d . 6 b The +/- sign indicate whether the sina1 is positive (>C<, -) negative (>CH-, CR 2 -CR -) in the ‘ 3 C APT spectrum. 3  118 Table 2-35. Chemical shift correlations obtained in the a. HETCOR spectrum of the second intermediate 99  l c 3 b  l C 3 0  ‘H correlation  (ppm)  correlation  (ppm)  16.71  (C—18)  2.12  112.98  26.56 (C-6)  3.42 and 3.75  119.52 (C-9)  27.46 (C-14)  2.84  121.19  (C_ O 1 )e  7.15  30.78 (C-15)  2.60 and 2.77  124.55  (C_ll)e  7.25  33.03 (C-17)  2.77 and 2.92  162.03  (C-19)  7.39  54.31  (C-3,C-23)  3.77 and/or 3.67 170.56 (C-21)  9.10  60.87  (C-5)  4.12 and 4.59  a b c e  -  3.01  (C-12)  7.46 7.73  Solvent: Acetone-d . 5 1.8 ppm to 4.8 ppm correlated with 10 ppm to 100 ppm 7.0 ppm to 11.4 ppm correlated with 105 ppm to 175 ppm The correlation might have to be reversed  Position 18, From 18,  22  27.46,  19,  the  and 16.71,  22,  C 13  23  23 and C-14  APT  were  spectrum easily  H 1  NMR  table  identified  2-34, as  the  167.94 and 54.31 ppm respectively.  attached to carbons 18, the  in  spectrum  carbons signals  14, at  The protons  19 and 23 were readily identified in  listed  in  chemical shifts and multiplicity.  table  2-32  based  on  their  The HETCOR spectrum listed  in table 2-35 established the position of H-14 to be in the multiplet at 2.84-3.01 ppm and the position of C-19 to be at  119  162.03 ppm.  The  shift  chemical  assignment  for  position  18  was also confirmed in the HETCOR spectrum.  Position 5 and 6 The  positive  spectrum  must  positively  be  signal one  charged  the  of  methylene N-4.  nitrogen,  in  60.87 ppm  at  the  alpha  groups  The  APT  C 13  the  to  spectrum  HETCOR  in  table 2-35 correlates the carbon signal at 60.87 ppm  to the  protons  listed  in  4.12 ppm  at  table  2-33  each other, 3.75 ppm carbon  establishes  at  in  assignment  are  turn  26.56 ppm  that  in  of  The  these  COSY  spectrum  protons  two  couple  to  and to the protons at 3.42 ppm and  as expected,  which  possible  4.59 ppm.  and  correlated  the  HETCOR  -CH 2 -CH -  this  to  the  methylene The  spectrum. fragment  is  only  that  the  protons at 4.12 ppm and 4.59 ppm and the carbon at 60.87 ppm belong  to  and  0-5,  3.42 ppm  protons  and  3.75 ppm  and  the  carbon at 26.56 ppm to 0-6.  15  Positions 3 The spectrum  next  17 and H-14 highest  appears  at  correlation of 0-3 unfortunately chemical  54.31 ppm  and  in  only  group  and was  to its protons  ambiguous  shift  methylene  that one  in  the  assigned  as  13C  APT  0-3.  The  in the HETCOR spectrum is  peak  C-23  and  0-3  is  seen  have in  the  the  same  HETCOR  spectrum which correlates to the area in the proton spectrum where  the  resonate.  methoxy  signal,  as  well  as  the  The remaining two methylene groups  H-3  protons,  in the carbon-  120  13 spectrum resonate at 30.78 ppm and 33.03 ppm.  The SINEPT  Table 2-36. Observed enhancements in the SINEPT spectrum of the second intermediate 99. Irradiation at 7.39.ppm and Observation in the chemical shift region of 0 90 ppma. -  Irradiation (ppm)  7.39  Observed enhancement (ppm)  (H—19)  a Solvent:  16.71  (100%),  30.78 (68%)  . 3 CDC1  experiment in table 2-36 is optimized for detection of three bond  couplings  and  H  by  was  at  H-19  16.71 ppm.  observed  at  at the  coupling  at In  and  constant  of  the  addition  a  This  68%  7  Hz  between C hydrogen.  in enhancement of  relative  signal  the chemical  of  appropriate  7.39 ppm resulted  30.78 ppm.  assigned as C-15 17.  a  irradiation  Irradiation of C-18  with  could  enhancement therefore  be  shift at 33.03 ppm to C—  The chemical shifts of the H-15 protons were found to be 2.60 COSY  and  2.77 ppm  spectrum  in  showed  coupled to each other.  the the  HETCOR H-15  spectrum. protons  to  As be  doublets  The HETCOR spectrum established that  the H-17 protons resonate at 2.77 ppm and 2.92 ppm. showed the 1-1-17 protons, other and that  expected,  as expected,  the proton at  The COSY  to be coupled to each  2.77 ppm is  a doublet whereas  the proton at 2.92 ppm is a triplet. Unfortunately it is not  121  possible  to  see  the coupling  between the  and  H-15  the H-17  protons with the H-14 since the COSY is too crowded region.  By  protons  aliphatic 3.77 ppm protons 2.60 as  ppm  to  a  process  a  and at  must  in  are  located  the  H-3  protons.  showed  coupling  3.77 ppm  and the H-15 triplet  elimination  of  (or H-17)  centered  at  the the  2.87  ppm.  H-15  The  at  the  COSY  2.77  proton at  two  singlet  broad  In to  remaining  the  the  in this  H-3  proton  ppm  at  as well  triplet  2.87  at  ppm was assigned to H-14.  Position 21 and the Indole NH Of the two protons at 9.10 ppm and 11.75 ppm the HETCOR spectrum correlated the proton at 170.56 ppm,  accounting  for  H-21  9.10 ppm to the carbon at  and  respectively.  C-21  The  indole NH proton must then be the signal at 11.75 ppm.  Position 9, The HETCOR  10,  protons  position  12.  resulted  in  was  and 12 7.46 ppm  at  the  to  respectively,  which  11,  carbons  and  at  NOE  of  7.73 ppm  112.98 ppm  corresponds  Irradiation an  and  either  the  enhancement  therefore  assigned  to  N-H  of be  to  correlate and  the  The  9  or  11.75 ppm  at  proton  H-12.  the  119.52 ppm  position  proton  in  at  7.46 ppm  protons  at  7.15 ppm and 7.25 ppm correlate in the HETCOR to the carbons at  121.19 ppm  and  124.55 ppm  respectively  either to position 10 or position 11.  and  corresponds  122  Position 7 In  the  SINEPT  experiment  listed  in  table  2-37  it  is  seen that irradiation of each of the H-5 protons at 4.59 ppm and 4.12 ppm gave, quaternary carbon is  therefore  concluded  in both cases, 110.83 ppm.  at  assigned  that  the  to  be  structure  maximum enhancement of the This  C-7. of  shift value  chemical  It  can  the  unambiguously  be  second  intermediate  corresponds to structure 99 since the chemical shift of the quaternary aliphatic carbon is at 85.89 ppm.  Table 2-37. Observed enhancements in the SINEPT experiment of the second intermediate 99. Irrdiation at 4.57 ppm and 4.09 ppm and observation in the chemical shift region of 80 175 ppma. -  Irradiation  Observed enhancement  (ppm)  (ppm)  4.59  (H—5)  110.83  (100%),  170.56  4.12  (H-5)  110.83  (100%),  128.14 (60%),  a Solvent:  . 6 Acetone—d  Position 8  and  2,  and 20  10,  11,  13  and the tentative  In the SINEPT experiment  (11%) 128.34 (25%)  assignment of  in table 2-38  are  position  listed the  enhancements observed in the aromatic/clef inic carbon region by irradiation of the H-9  and H-12  protons.  Irradiation at  123  7.73 ppm (H-9)  results in maximum enhancement at 136.73 ppm  and this chemical shift value is therefore assigned as C-13. Irradiation at 7.46 ppm (H-12) gave a maximum enhancement at 128.34 ppm  Table  2-38.  and  is  assigned  Observed  as  C-8.  enhancements  in  The  the  irradiation  SINEPT  at  experiment  of the second intermediate 99. Irradiation at 7.73 ppm (H-9) and 7.46 ppm (H-12) and observation in the chemical shift region of 83 175 ppma. -  Irradiation  Observed enhancement  (ppm)  7.73  (H—9)  (ppm)  136.73  (100%),  110.83 (26%),  128.34 (44%), 112.98  (18%),  124.55 121.19  (31%), (10%),  119.52 (7%). 7.46 (I-{—12)  128.34 (100%), 110.83  (26%),  b 0 • 162 3 (65%), 119.52  (18%),  b 5 • 170 6  112.98  (15%),  a Solvent: Acetone—d . 6 b The enhancements observed at 170.56 ppm (C-21) and 162.03 ppm (C-19) are due to the fact that the chemical shift of H-19 at 7.39 ppm is too close to the chemical shift of the proton irradiated at 7.46 ppm, so that some irradia tion of H-19 also occurred.  7.73 ppm also gave a 10% relative enhancement for the carbon at 121.19 ppm and 31% relative enhancement for the carbon at 124.55 ppm.  124  Again  assuming  bonds  away,  the  larger  a  signal  enhancement at  121.19 ppm  and the signal at 124.55 ppm to C-il.  for  the  was  assigned  carbon  three  as  C-10  The olefinic/aromatic  quaternary carbons at 128.14 ppm and 131.14 ppm did not show any enhancement and are therefore assigned as position 2 or 20. The  numbering  intermediate  99  is  of shown  the  carbon  below  and  atoms the  3 C 2 CO H 22  23  the  assignments  chemical shifts are summarized in table 2-39.  COO 3 CF  in  second of  the  125 Table 2-39. Assignment of the ‘H and a the second intermediate 99 Position  1  H ö(ppm)  C 6(ppm)  11.75  2 3  3.77  54.31  5  4. 12/4. 59  60.87  6  3.42/3.75  26.56  7  110.83  8  128.34  9  7.73  119.52  10  7.15  c 1 . 121 9  11  7.25  c 12455  12  7 46  112.98  .  13 14 15  136.73 2. 87 2. 60/2. 77  16 17  27.46 30.78 85.89  2.77/2.92  33.03  18  2. 12  16.71  19  7.39  162.03  9.10  170.56  20 21 22 23  167.94 3.67  54.31  a Solvent: 6 Acetone-d . b,c c5 values might have to be reversed.  chemical shifts to  126  2.11.  PRELIMINARY INVESTIGATION OF THE REACTIVITY OF THE 19’,20’-DOUBLE BOND IN 19’,2O’-ANHYDROVINBLASTINE 91. The  reactivity  of  the  15’,20’-anhydrovinblastine detail,  5  with the objective of  leurosidine  113,  the  C-20’  C—15’,C-20’-double  bond  has  in  been  studied  synthesizing vinbiastine epimer  of  vinblastine  in some 1 or The  1.  C00H ) 3 (CH H  NOOLINE  88e  )I-I  0504 H  H  5  ‘INOOLINE  CH 2 3  CH 2 , 3  8H 1) 3 /H0 0 2 2) H  106  IH INDOLINE  107 Scheme 2-15. Transformation 15’ ,20’ -anhydrovinbiastine 5  of  the  15’,20’-double  bond  in  127  results  of  these  2_1522,23,67. 106  to  investigations  are  outlined  Attempts to convert leurosine 88e or the diol  either  vinblastine  . 23 unsuccessful  1  or  leurosidine  91  different  15’,20’-double  from  that of  anhydrovinblastine 5.  air.  This  into is  exposed  contrast  in  to  quite  be in  bond  epoxide the  to  which,  5,  significant  air.  to  were  15’,20’-  The 19’,20’-double bond in 91 was not  sharp  bond  forms  the  found  was  the corresponding  in  15’,20’-double solution,  113  The reactivity of the 19’,20’-double bond in  19’,20’-anhydrovinblastine  transformed  scheme  in  both  amounts  Furthermore  the  when exposed  as  of  of  reactivity a  solid  leurosine  the  and  in  when  88e  19’,20’-double  to  bond  in  19’,20’-anhydrovinblastine 91 did not react with tert-butyl hydroperoxide Treatment  under  of  the  conditions  described  19’,20’-anhydrovinblastine  91  567•  for  with  mercuric  acetate followed by reduction with sodium borohydride 68 led neither  to  hydroxylation  double bond. due  to  nor  epoxidation  of  the  19’,20’-  Since the failure of the hydroxylation might be  the heterogeneous  nature of  the reaction conditions  or the fact that oxymercuration is potentially a reversible , 7 ’ 69 reaction 0  H NMR was employed in order to determine if 1  any addition or coordination actually took place to 19’,20’double  bond.  In  order  to  ensure  homogeneous  reaction  conditions mercuric trifluoroacetate in methanol was used . 71 The  NMR  deuterated  spectrum  methanol  revealed that  of  and  19’,20’-anhydrovinblastine in  deuterated  the position of  the  91  trifluoroacetic  olefinic proton  in  acid  (H-19’)  128  did  not  change  upon  addition  mercuric trifluoroacetate, did not coordinate add palladium(II) to the  of  an  fold  eight  excess  of  establishing that the mercury ion  to the 19’,20’-double bond. acetate or palladium(II)  Attempts to  trifluoroacetate  19’,20’-double in l9’,20’-anhydrovinblastine 91 were  also unsuccessful. Treatment of bromide  gave  an  19’,20’.-anhydrovinblastine almost  quantitative  91 with benzyl of  yield  benzylatec3  19’ ,20’ -anhydrovinblastine 108.  Br 2 Br H H  INDOLINE  INOOLINE CH 2 .0 3 108  91 1) 0504 9 2 2) H N 3 3) Et  H OH  3 H 0H  3 CR +  INDOLINE  INDOLINE  H  CH 2 :0 3 109  110  129  Reaction  108  of  tetrahydrofuran  with  and  osmium  in  tetroxide  subsequent  aqueous  debenzylation  with  triethylamine gave two dihydroxy isomers 109 and 110 in 36% and  yield  14%  respectively.  The  compounds  dihydroxy  identified by their molecular ion at 826 m/z. was  indeed  the  molecular  characteristic M of  ion  the  ion  14 and M  +  +  of  fragment  28  That this ion  confirmed  was  . 7 ’ 64 ions 3  170  were  by  the  The presence that  confirmed  m/z  dihydroxylation in the catharanthine half had taken place . 64 Comparison of the 13 C chemical shifts for vinblastine 1 and leurosidine for C-3’,  11372  C-6’,  reveal  C-19’,  that  C-20’  significant differences exist  and C-21’  (table 2-40). All the  remaining chemical shifts are seen to be identical, -  2 ppm.  within 1  If the chemical shifts of aliphatic methylenes and  quaternary carbons of vinbiastine 1 and leurosidine 113 are listed  in  descending  order  respective  carbons)  as  differences  found  in  table  spectrum of  diols  109  (without  shown  in  2-40  and 110  correlation  stand  the  out.  chemical  shifts  are  In  then  the  the  C APT 13  aliphatic methylenes  quaternary carbons are readily identified, their  2-41  table  their  to  listed  in  and  and in table 2-41 descending  order.  Comparing the lists of chemical shifts of the diols 109 and 110 with those of vinbiastine 1 that  the  the diol  diol  109  best  and leurosidine 113 reveal,  corresponds  with  vinbiastine  1  and  110 best corresponds with the list for leurosidine  113.  Based on this simple comparison the stereochemistry of  the  C-20’  hydroxy  groups  of  109 and 110 are tentatively  130 40 _ 2 Table a• Assigent of th, 13 c vinbiastine 1 and leurosidine 1132.  chemical  shifts  1 R 5’  6’ 9’  11 14 15  .C H 3 Pc  3 CH  3 C 2 0 H  Position  1:  : 1 R  113:  : 1 R  1  OH,  : 2 R  3 2 CH , CH 19’  113  3 — 2 CH CH 19’  : 2 R  18’  OH  18’ Position  1  113  2’  130.9  130.2  2  83.0  83.1  3’  47.5  43.9  3  50.2  50.2  5  55.5  b 555  5  50.2  50.2  6’  28.7  21.4  6  44.3  44.5  7’  115.9  116.8  7  52.9  53.1  8’  129.0  128.9  8  122.6  123.0  9’  118.1  117.9  9  123.1  123.4  10’  122.2  122.0  10  120.4  120.4  11’  118.8  118.6  11  157.8  157.6  12’  110.4  110.2  12  93.9  94.0  a The table is continued on the following page. b See the end of the table on the next page.  in  131 Table 2-40 continued.  Position  1  113  Position  1  113  13’  134.7  134.5  13  152.5  152.8  14’  29.2  29.8  14  124.3  124.3  15’  40.0  c 4 . 40  15  129.7  129.7  16’  55.3  55.4  16  79.3  79.5  17’  34.1  17  76.2  76.2  18’  6.7  18  8.1  8.3  19’  34.1  19  30.4  30.7  20’  68.6  71.8  20  42.6  42.6  21’  f3..1  5 q qb  21  65.2  65.5  CH O 3 CO-  52.1  52.1  H 0 3 C0-  51.8  51.9  CH O 3 QO-  174.6  173.9  CH O 3 QO-  170.6  170.7  2 C 3 H O  21.7  21.0  2 0 3 CH  171.4  171.4  CH O 3 -  55.3  55.7  CH N 3 -  38.0  38.2  7.1  b,c The chemical shifts may be reversed.  132 2-41. Table The shifts chemical of the aliphatic methylenes and quaternary carbons groups in vinbiastine 1, a leurosidine 113 and the two diols 109 and 110  1  113  109  110  28.7  21.4  28.5  18.8  30.8  30.7  30.8  30.6  34.1  35.5  34.7  35.5  40.0  40.4  37.4  38. 5  42.3  42. 6  42. 7  42. 5  44.3  43.9  44.6  43. 5  47.5  44.5  47.8  44.7  50.0  50.2  50.3  50.0  50.2  50.2  50.3  50.2  52 9  53. 1  53.3  52.2  55.3  55.4  55.7  53. 1  55. 5  55. 5  55.8  54.4  63.1  59.9  65.8  55.1  68.6  71.8  71.3  71.9  79.3  79. 5  79 7  79. 6  .  a Solvent:  correlated  to  respectively.  .  . 3 CDC1  vinbiastine  In  order  to  stereochemistry of the C-20’ an  attempt  was  made  to  1  obtain  and direct  leurosidine  113  evidence  the  for  hydroxy group in the two diols  remove  the  19’-hydroxy  group.  The  133  major did but  109 did not react with tosyl chloride in pyridine  treatment  conditions,  with  mesyl  chloride  under  instead,  gave one major product  111  on TLC.  the  same  By addition  of water to the reaction mixture the crude product could be filtered of f as a purple solid. be  One  unstable.  of  the  This product (111) proved to  major  decomposition  products  112  could however be readily obtained on column chromatography. The same material to  purify  the  formed in significant amounts on attempts  crude  mesylate  flash  by  chromatography  crystallized surprisingly easily as thin needles. spectrum of 112, showed  that  a  as well  mesylate  diffraction  analysis  presence of  a mesylate group.  of  The 1 H NMR  as the elemental analysis, group 112  still in  was  figure  present. 2-16  and  clearly  The  X-ray  confirmed  From the structure of  112  the it  3 CH :0  H Cl  mc 3 CH  2 CR SO 3  112  Figure 2-16. The structure of the mesylate decomposition product 112 determined by X-ray diffraction analysis.  134  is clear that treatment of the did  109 with mesyl chloride  resulted in mesylation of the hydroxy group at C—16 as well as  A  C-19’.  subsequent  cleavage  of  the  C-0  bond  at  C-19’  followed by a 1,2 shift then lead from the initially formed dimesylate 111 to the rearranged product 112 as outlined in scheme 2-16.  OH 3 CH H  2 S 3 CH C O 1  INIJOLINE 109 S  3 CH  CH 3  H 112  111  R: 1—mesy1vindoNne  Scheme 2-16. Mesylation decomposition to 112.  of  the  major  diol  109  followed  by  135  EXPERIMENTAL  3.  3.1.  GENERAL EXPERIMENTAL CONDITIONS  Physical data: Melting  points  (with  the  solvent  used  for  recrystallization given in brackets) were determined using a Nagle  or  a  Herschberg  uncorrected. Elmer  Infrared  157,  710B  Ultraviolet  spectra  or  spectra  melting  1710  point  were  apparatus  recorded  spectrometer  were  recorded  on  pellets.  Cary  a  are  Perkin  a  KBr  as  on  and  or  15  a  Perkiri Elmer lambda 17 spectrometer using 1 cm quartz cells. Mass or  spectra  were  KRATOS-MS-50  a  the  electron  the  probe  recorded (high  impact  is  given  on  WH—400  reported  in  ionization in  standard.  COSY  spectrometer recorded structures  on  spectra  a  were  with graphite  relative  and  C 13  method.  were  Varian  on  monochromated  a Cu  and  on  Ka  were  a  shifts  AFC6S  radiation.  internal  as  Bruker  SINEPT  are  WH-400  spectra  were X-ray  The  diffractometer The  elemental  analysis were determined using combustion analysis by Dr. Borda,  Microanalytical  Columbia. commercial  Laboratory,  of  Variari XL-300 or  spectrometer. Rigaku  employing  spectra  NMR  chemical  recorded  XL-300  recorded  H 1  all  HETCOR  resolution)  temperature  tetramethylsilane  to  APT,  The  Bruker WH-250, and  (low  spectrometer  parenthesis.  spectrometer  ppm  AEI-MS-9  resolution)  recorded on a Bruker WP-80, Bruker  an  University  of  P.  British  Thin layer chromatography (TLC) was carried out on aluminum-backed  silica plates  (Merck  art.  5554)  136  or commercial glass-backed aluminum oxide plates (Merck art. 5731).  The  a guide.  Rf  values given  Visualization  should  was  case  sulfuric acid  of  alkaloids,  with  spraying  heating,  with  1%  as  ultraviolet  ammonium molybdate in  5%  followed by brief by  considered  be  accomplished  light and/or by spraying with aqueous  only  or  ceric  10%  in the  ammonium  sulfate in phosphoric acid followed by brief heating. Solvents: Anhydrous  ether,  tetrahydrofuran,  benzene  and  toluene  were prepared by distillation from sodium in the presence of benzophenone. distillation was  prepared  pyridine  was  Anhydrous  dichloromethane  from phosphorous pentoxide. by  distillation  prepared  hydroxide.  Anhydrous  distillation  from  from  magnesium.  acetonitrile  over 4A molecular sieves.  hydride  prepared  and  by  Anhydrous methanol  distillation  by  calcium  was  from  was  Anhydrous potassium  prepared  subsequently  by  stored  Anhydrous acetone was prepared by  distillation from Drierite. Reagents: All unless  the  reagents  otherwise  purified  by  buffer  stated.  distillation  chloroperbenzoic commercial  used  acid was  of  reagent grade  Trifluoroacetic from  acid  potassium  material  anhydride  phosphorous  purified  m-chloroperbenzoic  (1.775 g  were  pentoxide.  was  m  by suspending  10.0 g of  200 mL  phosphate  in  dihydrogen  of  phosphate  and  12.23 g of disodium hydrogen phosphate dissolved to 1000 mL in distilled water)  and  sonicated  for 20 mm.  The  solution  137  filtered  was  100 mL  of  water.  The  the  and  the  buffer  dried  The  purified  over  at  4°C.  Kept  acid  three  times  two  pentoxide,  m-chloroperbenzoic  97% peracid for 2  -  by  washed  transferred  phosphorous  plastic container as 95  followed  filter cake was  and  cake  filter  to in  times  with  100 mL  vacuo, kept  in this way  of  plastic beaker  a  was  with  overnight. in  it  a  closed 74 assay  will  3 months.  -  Column chromatography: Unless  otherwise  performed  using  TLC grade  aluminum  column  was  stated  TLC grade oxide  pressurized  column  silica gel (neutral)  with  chromatography (Merck  (Merck  nitrogen  or  art.  art.  7730)  1090).  argon gas  was or The  pressure  to obtain a suitable flow rate.  3.2.  CATHARANTHINE N-OXIDE 10  0  H  3 H  3 C 2 0 H Catharanthine dichloromethane  3  (400 mg,  (70 mL)  and cooled  m-Chloroperbenzoic acid (98%) in  dichloromethane  (10 mL)  under vigorous stirring. reaction  was  stirred  1.19  mmol)  was  dissolved  in  to  -70°C under nitrogen.  (210 mg,  1.19 mmol) dissolved  was  added  dropwise  over  15  mm  After the addition was complete the  for  another  10  mm  at  —70°C  and  the  138  solvent was evaporate off in ethyl long)  The residue was dissolved  acetate and loaded on a column  made of silica gel  Elution  successively  acetate/methanol (8:2)  at 0°C.  with  (9:1)  (200 mL).  (40 g)  suspended in ethyl acetate.  ethyl  (200 mL)  The  (3 cm wide and 8 cm  (50 mL),  acetate  and  fractions  ethyl  acetate/methanol  ethyl  the  containing  desired  material was evaporated off at 0°C to give catharanthine Noxide 10  (210 mg,  (70 mg,  50%) and 7-hydroxycatharanthine N-oxide 92  16%).  The  NM?.  and  MS  data  of  10  found  was  to  in  be  Ob, iiterature . identical to the data published in the 2  3.3.  N-BENZYLOXYCARBONYL-1, 2-DIHYDROPYRIDINE 30  °r°A  dry  1  mechanical bubbler.  The  to  methanol  the  a  dropping  apparatus (95%)  -78°C (115 mL)  exothermic. and  three—necked  stirrer,  borohydride cooled  liter  was  (13.1 g, in  a was  Dry pyridine  resulting white  dry  h  flask  funnel,  purged 0.33  ice/acetone  added  slowly.  (25.8 mL, slurry was  thermometer  a  argon  with  mol)  equipped  was  was  a  and  a  sodium  introduced  and  bath.  Dry  reaction  was  cooling The  0.32 mol) cooled  and  with  to  was  then added  -78°C.  Benzyl  139  chioroformate  (45.0 mL,  diethyl ether  (35 mL)  added dropwise below The  -70°C  0.30  mci)  was  dissoived  dry  in a dropping funnel and the solution  at such a rate that the temperature was kept  (the addition took approximately  reaction  in  was  stirred  for  another  3  hr  1  hrs  at  30 mm).  -78°C.  The  cold reaction mixture was poured into 1 liter of ice and the reaction Sodium  vessel  rinsed  with  chloride  (10 g)  was  emulsion.  The  extracted  with  cold  diethyl added  solution  diethyl  in  was  ether  (5  ether  (2  x  order  to  break  the  argon  and  kept x  under  The  100 mL).  50 mL).  combined  extracts were washed successively with 1 M sodium hydroxide (50 mL),  1  M  hydrochloric  acid  (50 mL),  carbonate (100 mL) and water (2 x 100 mL). was  dried  over  magnesium  sulfate,  cake washed with diethyl ether removed on drying oil,  rotary evaporator  in  vacuo gave  30  2.5%  The organic phase  filtered  and  (2 x 50 mL).  the  86%)  as  filter  The solvent was  at 35°C under vacuum.  (56 g,  sodium  Further  slightly  a  yellow  which could be kept for a few weeks when stored below  -  30°C and under argon.  Physical data of 30: TLC (silica, NMR  (400  (broad s, (m, H,  1 H,  MHz,  CDC1 ) 3 :  5 H,  Ph-H),  C 3 H),  5.53  -CH O 2 -),  x 2 C H).  benzene/EtOAc 9:1) Rf:  6.71  Conformer (d,  (broad s,  5.10 (broad s,  0.78.  1 H,  J  =  1 H,  A  6  (ppm):  7.36  1 H,  C 6 H),  5.83  (60%)  7.0 Hz,  olefinic H),  olefinic H),  4.39  5.20 (m,  (s,  2  2 H,  2  140  Conformer B (d,  J  s,  1  7.0 Hz,  =  H,  (ppm):  7.36  (broad s,  1 H,  C 6 H),  5.83  Cm,  olefinic  olefinic H),  3.4.  6  (40%)  H),  5.20  Cm,  4.39  2 H,  (s,  2  Ph-H),  5 H, C 3 H),  1 H,  -CH O 2 -),  H,  6.79  (broad  5.45 5.20  (1  H,  -H). 2 2 x C  a-CHLORO METHYL ACRYLATE 3138139  :>=0cH3  2  A  liter  mechanical  three-necked  stirrer,  ref lux condenser. sulfuric 0.03  acid  mol)  1.98  vigorously, reaction  (400 mL)  was  and  mixed  (37%)  mol)  a dropping  basic  and  (150 g,  added  kept  between  60  mol),  carbonate  in  added  over  evolved.  The  kept  this  at  mixture added  to  was the  a  period  reaction  methanol  (80 mL,  while  stirring  The temperature of the during  65°C  steam  reaction  15  mm,  temperature  temperature  then  of  for  the  addition.  mixture  hydrochloric  was  another  distilled in  and hydroquinone was added to the  98%  (3.2 g,  The temperature was raised to 90°C and methanol (80 mL, mol)  a  Aqueous  70°C.  reaction,  to  2.01  to  mol)  with  thermometer and a  (180 mL,  over a period of 35 mm.  was  a  heated  the  equipped  copper  1.85  to  was  funnel,  Trichloroethylene  were  formaldehyde  flask  raised hour.  (paraffin order  to  receiver  acid was 100°C  to The  oil  1.98  reaction  (2 mL)  prevent  and  was  foaming  flask).  The  141  temperature in the reaction vessel was during  the  separated with  steam  from  the  were  (70 mL), filter  washed  dried  cake  crude product was mm  Hg).  with  over  washed  (2  50 mL).  x  the The  sodium  5%  magnesium with  and  combined  31  (77 g,  35%)  organic solution  filtered  dichloromethane  (2  was  x -  was  extracted  bicarbonate  sulfate,  130°C  phase  latter  fractionally distilled (57  Compound  at about  organic  The  phase  aqueous  dichioromethane  phases  64  distillation.  kept  and  the  10 mL).  The  58°C at 57  obtained  as  -  a  colorless liquid.  Physical data of 31: IR (neat on KBr) Vmax stretch),  1605  (cm):  2950  (C-H stretch),  1730  (C=0  (C=C stretch),  1280  (C-0 stretch),  1120  (C-0  stretch). NMR C 3 H),  (80 MHz,  ) 3 CDC1  6.03  J  3 C 2 CO ) H .  (d,  =  6  1.5  (ppm): Hz,  1  6.53 H,  (d,  C 3 H),  J  =  3.85  1.5 (s,  Hz, 3  1 H,  H, -  142  3.5.  N-BENZYLOXYCARBONYL-endo-7-METHOXYCARBONYL-7-CHLORO-2AZABICYCLO  [2,2,2]  OCTAN-5-ENE 32 and N-BENZYLOXY-  CARBONYL-exo---7-METHOXYCARBONYL-7-CHLORO-2-AZABICYCLO [2,2,2]  OCTAN-5-ENE 33  P C CH  A  bar,  500 mL  1  3 C 2 0 H  32  33  flask  was  equipped  with  magnetic  a  stirring  a reflux condenser and a bubbler and the apparatus was  purged  with  (53.0 g,  a  positive  0.229 mol)  dissolved  in  pressure  and compound 31  benzene  (250 mL).  heated under  ref lux  for  evaporation  on  rotary  vacuum giving  a  dark  a  of  6  days.  nitrogen.  The  reaction  orange  oil  mixture  solvent was  evaporator  (90.5 g).  was  removed by  45°C  at  30  0.374 mol) was  (45.0 g,  The  Compound  at  This  full  oil  was  divided into three portions and each portion was purified by chromatography made of The  on  a  TLC grade  crude  oil  column  silica  was  at  flow  rate  product  (54.2 g,  70%)  purified  product  crystallized  out.  gel  loaded  benzene  on  of  wide  (150 g)  on 6  was a  (7.5 cm  -  the  column  obtained.  Hexanes/ethyl  line  5.5 cm  suspended  7 mL/min.  vacuum  and  y 3  and  A  compound  acetate  in benzene. eluted  partly  drying  long)  with  purified  the  partly  33  slowly  (8.5:1.5)  (50 mL)  143  was  added  the  to  suspension  was  partly  filtered  crystalline and  the  hexanes/ethyl  acetate  (8.5:1.5)  hexanes  (2  10 mL).  The  dryness  and  time,  on  x  obtained.  partial  chromatography of isomers  (27 g,  was  9 cm  a  flow  with  12 g  -  long)  in  rate  then  evaporated  10  suspended  until  the eluent was  This  (19.5 g,  and  and  with  at  made  a of  benzene.  of  to  The  4 mL/min  was  When mixed exo and endo adduct began to elute off  the column  two  wide  (200 g)  pressurized  liquor  obtained  washed  10 mL)  x  chromatographed,  (7.5 cm  silica gel  was  (8:2).  residue  column  a  TLC grade column  the  crystals  (2  mother  The  oil.  35%)  to benzene/ethyl  crystallization  the mother  had  26%)  changed  been  and  liquor was  separated.  In  acetate  subsequent  column  repeated until  this  way  the  compound  33  was obtained as a white solid and compound 32  as a yellowish oil.  Physical data of 32: M.p.:  44  49°C (the solidified oil).  -  TLC (silica, UV  (CH C 3 N)  (2.46), IR  ‘>max  262  (KBr)  stretch),  benzene/EtOAc 9:1) Rf: (nm)  (2.36),  267  E):  207  (4.10),  250  (2.48),  257  2950  (C-H  (2.22).  (cm):  Umax 2855  (log  0.58.  3005  (C-H stretch),  (=C-H  stretch),  1730 (ester C=0 stretch),  1685  (carbamate C=0 stretch). ‘H NMR (400 MHz, 5  H,  (m,  Ph-H), 3 H,  CDC1 ) 3 :  6.40  Ph-CH O 2 -,  -  6.60 C 1 H),  Conformer A (52%) 6 (ppm): (m,  2  H,  3.70 (s,  C 5 H, 3 H,  C 6 H), 3 C 2 -CO ) , H  7.35 (m,  4.97 3.27  -  5.31 (d,  J  144  =  10 Hz,  1 H),  10 Hz,  1 H),  14 Hz,  1 H).  3.02  2 H,  (m,  C 5 H,  3.53 (s, =  14 Hz, 1 H,  s, MS  C 4 H),  2.94  1  4.97  2.94  (broad d,  J  =  C 4 H),  1.84  (broad d,  J  =  (m,  3.20 (d,  (broad d,  (31.4),  1 H),  5.31  -  (relative  215  H,  7.35  1.84 (broad d,  m/z  <0.1/0.1),  14 Hz,  =  (ppm):  3 C 2 -CO ) I-{ ,  1 H),  (120°C)  8  —H), 6 C  3 H,  J  (broad s,  2.87  Conformer B (48%)  (d,  J J  3 H,  (m,  J  10 Hz,  =  1 H), 1 H),  6.60  C 1 H),  3.02 (d, J (broad  2.87  337/335  10%):  92  (30.7),  -  1 H).  14 Hz,  =  6.40  Ph-CH O 2 -,  10 Hz,  =  intensity  170  Ph-H),  5 H,  (13.9),  (M,  (100),  91  80  (18.1). Elemental  analysis:  5.40,  10.56,  Cl: N:  10.56,  Caic.  N:  for  4.17.  C1N0 C 1 H : 4 7 8  Found:  C:  60.80,  C:  60.81,  H:  5.58,  H: Cl:  4.14.  Physical data of 33: M.p.:  97  98°C (EtOAc/hexanes 3:17).  -  TLC (silica, UV  benzene/EtOAc 9:1) Hf:  (CH C 3 N)  256(2.32), IR  ‘Xmax  263 (2.21),  (KBr)  Umax  stretch),  (nm)  (log  0.52.  E):  250(2.25),  204(4.05),  266 (2.02).  (cm):  3020  2850 (C-H stretch),  (=C-H  1745  stretch),  2920  (ester C=0 stretch),  (C-H 1685  (carbamate C=0 stretch). NMR (400 MHz, 5  Ph-H),  H,  6.44  7.2  Hz,  1  (d,  J  12 Hz,  =  H,  CDC1 ) 3 : (t,  C 6 H), 1 H,  J  Conformer A (57%) 8 =  5.30  7.2  Hz,  1  (d,  J  7.2  Ph-CH O 2 -),  =  5.17  H,  (d,  (ppm):  C 5 H), Hz, J  1 =  H,  6.36  7.36 (m, (t,  C 1 H),  12 Hz,  1 H,  J  =  5.20 Ph-  145  CH O 2 -), 1  H,  3.77  (s,  C 3 H),  3.07  (broad s,  1 H,  1.98  J  (dd,  Hz,  1  (d,  J  7.2  CH O 2 -),  =  2.73  J’  1 H,  1 J  Cdt, 2  =  H,  <0.1/<0.1),  J  Hz,  1  m/z  215  (10.4),  C 3 H), 1 H,  J  1  H,  J’  2 Hz,  =  C 3 H),  14 Hz,  =  =  Ph-H),  5 H,  Hz,  7.2  H, =  Hz,  10,  =  1 H,  2.84  C 7 H),  J  (d,  1  1  2.88  C 7 H),  H,  1  3.76  J  H, =  =  5.16 Ph— H,  3  3.07  1  s,  (dd,  J  H,  (s,  C 3 H),  (broad 1.99  Hz,  12  =  (t,  C 6 H),  H,  Ph-CH O 2 -),  2 Hz,  6.48  Cdt,  C 4 H),  14,  J’  =  5.40,  10.56, 4.20.  intensity  170  (18.8),  92  10%):  (12.8),  337/335  91  (M,  (100),  80  65(12.9).  analysis:  N:  J’  (relative  Elemental  10.42,  1  J  C — 8 H).  5.20  Hz,  H,  J  (t,  10,  =  2  =  (m,  7.36  14 Hz,  J  77(11.1),  Cl:  12  J’  1 H,  C — 1 H),  =  Cdt,  C 8 H).  (150°C)  (14.3),  (ppm): 6.31  3.50  (broad d,  2 Hz,  =  5  (d,  (broad d,  2 Hz,  MS  Hz,  3.50  10,  J’  10,  =  2.73  C — 5 H),  5.17  3 C 2 CO ) , H J  H,  J  C 4 H),  (43%)  7.2  3 C 2 CO ) , H  (dt,  14,  =  Conformer B  =  3 H,  N:  Caic. 4.17.  for  C1N0 C 1 H : 4 7 8  Found:  C:  61.00,  C: H:  60.81, 5.49,  H: Cl:  146  N-BENZYLOXYCARBONYL-endo-7-METHOXYCARBONYL-7-CHLORO-2-  3.6.  AZABICYCLO  [2,2,2]  OCTAN-endo-6-OL 34 and N-BENZYLOXY  CARBONYL-endo-7-METHOXYCARBONYL-7-CHLORO-2-AZABICYCLO [2,2,2]  OCTAN-enc7o-5-OL 35  P  H 35  34 100 mL  A  mechanical  three-necked  stirrer,  thermometer  a  equipped with a bubbler. overnight, positive mmol)  nitrogen  dimethyl  -  28°C.  additional  4  as eluent. slowly  peroxide  funnel  dry  Compound  32  tetrahydrofuran  complex  (1.70 mL,  17.2  (5.77 g, (55 mL)  and  mmol)  was  17.8  at such a rate that the temperature was kept The reaction mixture was then stirred  hrs  between (silica)  26  and  The  28°C.  added  followed (6.0 mL,  with  for an  reaction  using benzene/ethyl acetate  The heating bath was removed and methanol  hydroxide (6.0 mL, release)  in  sulfide  monitored by TLC  was  dropping  a  a  The apparatus was dried in the oven  atmosphere.  dissolved  added dropwise, at 26  and  with  fitted  was  assembled while still hot and cooled down under a  was  borane  flask  vigorous  stirring.  Then  3  was  (9:1)  (10 mL)  N  sodium  18.0 mmol) was added slowly (vigorous gas by the dropwise 53  mmol)  at  addition of such  a  30%  rate  hydrogen that  the  147  temperature  of  the  reaction was  The reaction mixture was 28°C,  then  organic  extracted  phase  hydrosulfite 5%  sodium  The  (2  under  and  with  cake  washed  the  crude  x  with  on  the  filter.  crude  phosphorus pentoxide gave 34  (5.6 g)  was  suspended  Evaporation of  the mother  yellowish  (0.42 g)  oil  benzene/ethyl  acetate  alcohols 34 and 35, 35  could  be  repeated  PLC  which, was  partly  a  1:1  0.5  mm  the  mother  silica  TLC:  -  100°C (EtOAc/hexanes 3:20).  (silica,  3 C CH / 2 O CH 1 40:1) Rf: H  in  (9:1)  cake  over  crystalline  TLC  liquor  plates  Physical data of 34: 98  to  mixture  dichioromethane/methanol (40:1).  M.p.:  acetate  of  together with other impurities. from  a  The filter  filter  according a  as  85%) as a white solid.  liquor gave  8:2),  isolated on  the  (5.15 g,  acetate  product  hexanes/ethyl of  30 mL).  a rotary evaporator  product  Drying  The  sulphate,  ethyl  in  (20 mL)  x  magnesium  cake  resuspended  -  sodium  5%  (2  (20 mL) and filtered.  then  26  20 mL).  hexanes/ethyl acetate (9:1) was  28°C.  hrs at  with  brine  solvent on the  and  negative peroxide test),  over  gave  This  a  dried  filter  vacuum  solid.  (until  was  (3  acetate  successively  (30 mL)  Evaporation of  40°C  white  30 mL)  the  ethyl  washed  phase  and  (20 mL).  x  26  between  stirred for another 2  with  bicarbonate  organic  filtered  at  was  kept  0.25.  (silica, the  two  Compound  residue  eluted  by  with  148  UV  (CH C 3 N)  (2.55),  (nm)  ‘max  260 (2.49),  (log  263  IR (KEr) Vmax (cm): 2950  (C-H  206  ):  (2.48),  3445  stretch),  (4.00),  (2.53),  251  257  267 (2.40).  (OH stretch),  3030 (=CH stretch),  (ester  C=O  Conformer A  (58%)  1735  stretch),  1670  (carbamate C=0 stretch). NMR (250 MHz, 5 H,  Ph—H),  4.20  (m,  3.08 H), H,  5.19  1 H,  15 Hz,  1 H),  1.84  J  (d,  -CH O 2 -),  3.80  -  2 H),  1.98  —  15 Hz,  =  4.60 (dd,  3.68  3.25 (m,  -  CDC1 ) 3 :  (m,  2.70 1 H),  5.03  3.5 Hz,  =  =  7.32 (d,  1 H),  (m, J  =  4.10  -  1 H),  3.65  (s,  2.90 (m,  1 H),  2.03 —2.18 (m,  Cm, -  —CH O 2 —),  1 H,  J  6(ppm):  1.62  (m,  1.80  —  3 H,  1 H),  CH O 3 -),  1.72  (s,  1 1  OH).  Conformer B  (42%)  15 Hz,  -CH O 2 -),  1 H,  (ad,  J  3.80  (m,  2.70  -  J’  =  1  3.5  =  H),  1.62  MS  (150°C)  0.3/0.8),  262  (5.6),  5.70,  10.02,  N:  N:  (m, J  H),  =  15 Hz,  1 H,  4.20  -  (m,  1.60 (s,  3.08 1 H),  1 H,  intensity  92(8.7),  3.96.  Ph-H),  CH O 3 -),  2.03 —2.18  Caic.  5 H,  4.10  3 H,  (relative  analysis:  9.90,  (d,  1 H),  Elemental Cl:  (s,  1 H),  m/z  1  Hz,  1.80 (m,  —  7.32  5.04  3.48  2.90 (m,  H),  8(ppm):  for  130  TLC (silica,  H),  1 3.25  1.84  5%):  —  J  4.55  3.68  (m,  =  -  2 H),  1.98 (m,  1  355/353  (M,  91(100). C1N0 1 C 2 H : 5 7 0  Found:  C:  57.80,  3.93.  -  -CH O 2 —),  (m, -  (d,  OH).  Physical data of 35: M.p.:  5.14  131°C (EtOAc/hexanes 2:20). 3 C CH / 2 O CH H 1 40:1) Rf:  0.20.  C: H:  57.71, 5.74,  H: Cl:  149  UV  (CH C 3 N)  (2.43),  (nm)  ‘max  260 (2.32),  262  IR (KBr) Umax (cm): 2940  (C—H  E):  205  (2.95),  (2.29),  266  (2.12).  (log  3460 (OH stretch),  stretch),  1735  (ester  250  (2.44),  256  3020 (=CH stretch), stretch),  C=O  1695  (carbamate C=O stretch). ‘H NMR (300 MHz,  CDC1 ) 3 :  5 H,  Ph-H,  12.2  Hz,  1  H,  -CH O 2 -),  3.68  (s,  3  H,  3 C 2 -CO ) , H  (m,  1 H),  1.97  (m,  5.18  2.97  12.2 Hz, -  3.33  -  1 H), 1.52 MS  H,  4.75  (m,  (m,  2  H,  (m,  1.52  7.35  5.07  3.17  (m,  -  1 H),  (d,  -  2 H),  7.35  -CH O 2 -),  5.07  (d,  -H, 1 C  4.75  (m,  2  H,  3.42  (m,  1  H),  3.17  2.28  (m,  2 H),  2.08  -  (m, J  =  -H), 5 C -  3.30  1.67  -  1 H). (m, J  3.30 1.67  H,  Ph-H,  12.2  Hz,  5 =  -H), 5 C  -H, 1 C  1 H),  m/z  309  Elemental  (relative  (6.4),  analysis:  Cl: N:  3.33  (m, —  -  1 H,  6(ppm):  3.53 (m, -  H),  1.97  1  3  (s,  1  5.21 H,  (m,  J  =  -CH O 2 -),  H,  2.97  (d,  3 C 2 —CO ) , H -  3.08  2 H),  (m,  1.40  -  1 H).  2.7/6.5),  9.85,  4.58  6(ppm):  2.28  -  (150°C)  5.70,  12.2 Hz,  =  —CH O 2 -),  (m,  3.42  2.08  1.40  (46%)  1  J  3.08  -  2 H),  Conformer B  4.58  Cd,  Conformer A (54%)  10.02,  3.86.  N:  218  intensity  (5.6),  Calc. 3.96.  for  92  5%):  (11.5),  91  C1N0 1 C 2 H : 5 7 0  Found:  C:  57.56,  355/353  (M,  (100). C: H:  57.71, 5.76,  H: Cl:  150  3.7.  N-BENZYLOXYCARBONYL-enc7o-7--METHOXYCARBONYL-7-CHLORO-2AZABICYCLO [2,2,2]  OCTAN-6-ONE 36 and N-BENZYLOXY  CARBONYL-endo-7-METHOXYCARBONYL-7-CHLORO-2-AZABICYCLO [2,2,2]  OCTAN-5-ONE 37  0 Ph  3 CH  o  1  37  36 A  250 mL  mechanical  two-necked  stirrer  and  was  flask reflux  a  calcium chloride guard-tube.  equipped  condenser  with  fitted  a  with  a  The apparatus was dried in the  oven overnight.  Pyridinium chioroformate  (3.0 g,  13.9 mmol)  was  mixture  34  35  8.48  added  to  mmol)  reaction  a  dissolved  mixture  of  in  the  dry  alcohols  dichioromethane  heated  was  reflux  to  stirring and monitored by TLC (silica, acetate  3:1).  (0.42 g,  1.95  After mmol)  3 was  hrs  more  added  under reflux for another hour. carefully  filtered  and  long).  3 cm  reaction vessel, stirring The  and  reaction  through  a  and  under  vessel  was  vigorous  dichioromethane/ethyl  the  chioroformate  reaction was  of  bed  florisil  (40 mL)  was  heated to reflux for 15 mm filtered  The  heated  The cold reaction mixture was  Dichloromethane  then  (60 mL).  pyridinium  and  (3.0 g,  through  similarly  the  added  to  wide the  under vigorous  same  treated  (4.5 cm  florisil  two  times  bed. more  151  with  dichioromethane  was  rinsed  with  (40 mL)  after  dichioromethane  necessary to thoroughly rinse  which x  (6  the  florisil  the  20 mL).  florisil  was  in order to  bed  ensure that all of the product was washed out.  It  bed  The solution  was evaporated to dryness and the solid greenish residue was dissolved was  in boiling  slowly  added,  solution was  ethyl  keeping  decanted  acetate the  (5 mL).  solution  from a green oil  at  Hexanes  (50 mL)  reflux.  The  which separated out  of the solution and this in turn was rinsed with hexanes x  5 mL).  The  combined  organics  were  hot  evaporated  to  (2  dryness  and the residue redissolved in boiling ethyl acetate (6 mL). Hexanes  (60 mL)  at reflux.  was slowly added while keeping the solution  The clear hot solution was seeded and allowed to  cool to room temperature and then at 5°C overnight. the  day  solution  crystals (10 mL)  collected and  obtained  allowed white  as  liquor gave  a  was  cooled  were to  rinsed  dry.  oil  and  0°C with  Compound  crystals.  turbid  to  cold 36  filtered. (0°C)  (2.23 g,  Evaporation  (0.64 g).  of  Compound  isolated from this oil by column chromatography. dissolved  in  a  minimum  of  The next  hexanes/ethylacetate  hexanes 77%)  the 37  The  was  mother can  be  The oil is (8:2)  and  loaded onto a column made of TLC grade silica gel suspended in  hexanes/ethylacetate  (9:1).  Elution  with  hexanes/ethyl  acetate (9:1) affords 37 (approximately 0.4 g).  Physical data of 36: M.p.:  99  -  100°C (EtOAc/hexanes 1:10).  152  TLC (silica, UV  (CH C 3 N)  hexanes/EtOAc 9:1, (nm)  ‘Xmax  (log  (2.45),  260  (2.34),  (2.19),  323  (shoulder,  262  IR (KBr) Vmax(cm’): 1740  5 H, 12  (300 MHz,  Ph-H),  Hz,  1  CH O 3 -),  5.27  H,  2.60  (broad d,  J  2.42  (broad d,  J  (4.13),  207 266  1695  256  302  (2.20),  311  (carbamate C=0 stretch).  Conformer A (60%)  (a,  12 Hz,  J  =  4.85  1 H, 1  (s,  2 H), 16 Hz,  3.10  H,  2.58  1 H),  2.16  6(ppm):  ArCH O 2 -), C 1 H),  (broad d,  1 H),  16 Hz,  =  (2.44),  2940 (C-H stretch),  CDC1 ) 3 :  =  250  (2.20),  3020 (=CH stretch),  (m,  0.07.  1.92).  PhCH O 2 -),  3.3-3.48  E):  (2.34),  (ester C=0 stretch),  H NMR 1  developed twice) Rf:  J  (a,  5.01  3.51  (s,  (broad s, (broad d,  H,  C 4 H),  15 Hz,  =  =  1 H),  1 H, J  J  3  15 Hz,  =  (m,  7.39  1  H). Conformer B 12  Hz,  4.91  1  (s,  H),  3.10  Hz,  1 H),  Hz,  1 H),  MS  H,  5.16,  N:  C 1 H),  (broad d, 2.58 2.16  316  Elemental  6(ppm):  PhCH O 2 -),  1 H,  (140°C)  /1.6),  Cl:  (40%)  m/z  J  3.93  (broad d,  analysis: Cl:  Caic.  5 H, =  12  Ph-H),  5.16  Hz,  H,  CH O 3 -),  1 3.3  -  (d,  J  PhCH O 2 -), 3.48  (m,  (broad d,  J  =  16  C 4 H),  2.42  (broad d,  J  =  16  1 H).  intensity  4%):  (24.9),  for Found:  92  353/351  (12.6),  C1N0 C 1 H : 5 7 8 C:  57.86,  H:  C:  (M,  91  0.6  (100).  58.04,  5.17,  N:  developed twice) Rf:  H:  4.00,  Physical data of 37: hexanes/EtOAc 9:1,  2  2.60  9.93.  TLC (silica,  =  H),  15 Hz,  =  (10.1),  10.08.  1  1 H,  (relative 159  3 H,  Hz,  J  J  (d,  (s,  15  (broad s,  (5.6),  3.98,  5.09  =  (m,  7.39  0.13.  153  UV 3 (CH C N) ‘>max IR  (KBr)  (nm):  (ester  1735  251,  (cm):  max 1  stretch),  207,  257,  3000  (=CH  (shoulder).  stretch),  stretch),  C=0  266  262,  (C-H  2930  (carbamate  C=0  (ppm):  (m,  1700  stretch). NMR (300 MHz, 5 H,  Ph-H),  CDC1 ) 3 :  5.16  (d,  J  12 Hz,  1 H,  PhCH O 2 -),  C 1 H),  3.68  (s,  (m,  1 H),  2.50  3 H, -  Conformer A (61%) 6  =  12 Hz,  5.01  (dd,  CH O 3 -),  2.75  (m,  1 H, J  3.4  3.5 Hz,  =  3.7 (m,  -  2 H),  PhCH O 2 -),  2.28  (d,  5.07  J’  2 Hz,  =  3 H),  (broad d,  7.35  3.02 J  -  J  1 H, 3.18  16 Hz,  =  =  1  H). Conformer B 12  Hz,  4.88  1  H,  (dd,  J  CH O 3 -), 2.75  MS  3.4  (m,  (39%)  =  -  3.5 Hz, (m,  3.7 2.28  m/z  0.3/0.91,  231  (100),  (8.0).  65  7.35  5.06 J’  3  H),  (broad d,  (16.8),  186  Calc.  (m,  (d, 2  =  (relative  High resolution MS: 351.0871.  (ppm):  PhCH O 2 -),  2 H),  (120°C)  6  J  Hz,  3.02 J  5 H,  =  12 1 H, -  5.25  Hz,  H,  16 Hz,  159  1  C 1 H),  3.18  intensity (4.6),  Ph-H),  (m,  J  =  PhCH O 2 -),  3.51  1  (d,  (s,  H),  3 H,  2.50  -  1 H). 4%):  353/351  (4.7),  92  for 5 C1N0 C 1 H : 7 8  (M,  (8.0),  351.0874,  91  Found:  154  3.8.  N-BENZYLOXYCARBONYL-endo-7-METHOXYCARBONYL-7-CHLORO-2AZABICYCLO  OCTAN-6-ONE 2’,2’-DIMETHYL-1’,3’-  [2,2,21  PROPANEDIYL ACETAL 38  P  A  250 mL  O 3 CH  ci  was  equipped  flask  CH 3 3 CH  0  with  a  magnetic  stirring  bar and a Dean-Stark trap fitted with a ref lux condenser and a  calcium  mmol),  chloride  guard-tube.  Compound  2,2-dimethyl-1,3-propanediol  (6.00 g,  p-toluene sulfonic acid monohydrate (0.60 g, added to reflux  benzene  for  (silica,  18  (100 mL) hrs.  and  The  hexanes/ethyl  57.6 mmol)  acetate  was  8:1).  The  and  3.15 mmcl) were  the mixture was  reaction  11.4  (4.00 g,  36  heated under  monitored reaction  by  TLC  mixture  was cooled to room temperature,  washed with saturated sodium  bicarbonate  x  brine  (50 mL).  sulfate, x  (30 mL),  10 mL).  The  water organic  (5  phase  50 mL) was  and  dried  over  with  magnesium  filtered and the filter cake washed with benzene (2 Evaporation  of  the  solvent  at  40°C  followed by drying in vacuo gave 38 (4.92 g, solid.  Physical data of 38: M.p.:  finally  124-126°C (hexanes).  under  99%)  vacuum  as a white  155  TLC (silica, UV  (CH C 3 N)  (2.39), IR  hexanes/EtOAc 7:3) Rf:  260  (KBr)  (nm)  ‘Xmax  (2.28),  Ph-H  5.02  1 H),  3.32 Cd, (m,  1 H),  (s,  3 H,  206  (4.05),  (2.26),  267  (2.10).  CDC1 ) 3 :  3  (d,  3.56  =  J  -  12.5 Hz,  =  (s,  10 Hz,  1.80  H,  228 92  3 H,  (8.7), (13.3),  6.45, Cl:  N:  7.95.  256  (ester  C=0  8(ppm):  (64%)  2.02  1 H,  CH O 3 -),  1 H),  3.03  (m,  PhCH O 2 -), PhCH O 2 -),  3.38  Cd, 3  3.40  -  3 H),  (140°C)  Elemental  H,  1 H,  3.03  2.02 (m, MS  1  CH O 3 -),  H),  1735  (2.38),  3.40 (m,  -  3 H),  12.5 Hz,  =  PhCH O 2 -),  3.38 Cd,  1.22  J  2.12  3 H,  3  =  1 H),  10 Hz,  =  (m, 1 H,  (d,  3.97  4 H),  (s,  7.35  2.28  -  0.81  CH C 3 ),  CH C 3 ).  Hz,  12.5 Hz,  stretch),  Conformer A  Conformer B (36%) 6(ppm): 12.5  (C-H  2940  250  -H), 5.19 Cd, J 1 ), 5.38 Cs, 1 H, C  PhCH O 2 -), 10 Hz,  E):  1700 (carbamate C=0 stretch).  NMR (300 MHz, 5 H,  262  (cm):  Umax  stretch),  (log  0.33.  (m,  91  analysis, 3.20,  Cl:  (d,  10 Hz,  4  H),  (14.6),  (100),  Cs,  =  m/z(relative 154  5.18 3.97  1.13 (s,  69  3 H,  138  —H), 1 C  -  (10.6),  Found:  1  H,  =  CH C 3 ),  for  Ph-H  1 H),  2.12  (26.7),  5 H,  3  intensity  Caic. 8.10.  (m,  7.35  65  ), 5.19 (d, J  10 Hz,  1 H),  3.32  Cd, 3  Cm,  2.28  0.68 (s, 402  5%): 129  1  (d,  3  =  (5,  3  10 Hz,  1  3.61 =  1.80  H),  3 H,  -  CH C 3 ). (M-Cl),  (7.8)  (58.0),  128  (13.0),  (10.6).  C1N0 C 2 H : 6 2 8 C:  5.10  =  60.16,  H:  C: 6.30,  60.37, N:  H:  3.18,  156  3.9.  N- (3’ ‘-INDOLYLMETHYLENECARBONYL) -endo-7-METHOXYCARBONYL-7-CHLORO-2-AZABICYCLO  [2,2,2]  OCTAN-6-ONE  2’, 2’ -DIMETHYL-1’,3’ -PROPANEDIYL ACETAL 40  0 3  CH 3 3 H To a 250 mL flask equipped with a magnetic stirring bar was added 10% palladium on charcoal  (0.50 g)  tetrahydrofuran  38  (150 mL).  Compound  followed by dry 17.4  (7.60 g,  mmol)  was added and the reaction stirred at room temperature under an  atmosphere  filtered  of  through  hydrogen a  0.5 cm  washed with tetrahydrofuran solvent crude  at amine  (4.86 g) argon, the From  35°C  was  and  reddish a  acetic  suspension  hexylcarbodiimide dichloromethane reaction with  as  dissolved  dropping  a in  hrs.  bed  of  celite  (3  15 mL).  was  dichloromethane  cooled was  to  the  vacuo The  19.1 0°C  slowly 20.4  was  celite  the  crude  amine  (50 mL)  under  mmol)  added  in  ice  an  added  mmol)  gave  and  bath.  1,3-dicyclodissolved  in  After stirring at 0°C for 5 hrs the  filtered (2  and  in  foam.  (3.35 g,  solution  Evaporation of the  drying  white  (4.21 g,  was  x  The  dichloromethane  acid  funnel  (25 mL).  mixture  24  subsequent  (4.86 g)  3-indole  for  x  and  10 mL).  the The  filter mother  cake  washed  liquor  was  157  washed successively with 1 N sodium hydroxide N  hydrochloric  acid  (15 mL).  The  magnesium  sulfate,  (2  combined  and  product  further  (8.77 g)  organic  (2 x  water  dried  cake  1  over washed  was  Evaporation at 35°C under  vacuo  a reddish  solid.  54%)  with  were  filter  the  and  in  (4.32 g,  finally  and phases  10 mL).  drying  as  methanol gave 40  15 mL)  filtered  with dichloromethane vacuum  x  (2 x 15 mL),  resulted  in  the  crude  Recrystallization from  as white crystals.  Physical data of 40: M.p.:  215  216°C 0 C (MeOH/CH / 2 Et 1 5:2:5).  -  TLC (silica,  heptane/acetone 1:1)  (neutral alumina,  /hexanes/Et 7:3:0.05) Rf: 3 CHC1 N  (neutral alumina,  C1 Rf: 2 CH )  (KBr)  (cm):  2940  (C-H  (ester C=0 stretch), NMR  MHz,  (300  (d,  1  indolic C-H),  H,  7.12 H), 2  (t, 5.88  H),  (dt,  J  J  =  =  =  (s,  3.44  0.63. 0.26.  0.17.  (log  E)  3250  (indole N-H stretch),  :221  1640  7.5 Hz,  (4.60),  (3.74),  272  279  7.19  1 H,  14 Hz,  3 J’  8(ppm):  H, =  8,15  (s,  indolic C-H), (t,  1 H,  C 1 H),  (C-H  2850  3045  (=C-H  stretch),  1730  (carbamate C=0 stretch).  1 H,  7.5 Hz,  (s,  stretch),  ) 3 CDC1  7.61  J  Rf:  (3.71).  Vm8x  stretch),  (nm)  ‘max  289  (3.77), IR  0.49.  (neutral alumina,  (CH C 3 N)  UV  3 hexanes/acet N one/Et 10:10:0.6) Rf:  J  7.5  =  (t,  CH O 3 CO-), 3 Hz,  1 H),  J  Hz,  2.19  1  H,  7.07  12 Hz,  =  3.26  H,  7.34  indolic C-H), 4.02  1  -  3.39  indolic NH),  (d,  J  7.5 Hz,  =  indolic C-H), (s,  1 H,  2 H), (m,  (broad s,  4  C , 2 -  3.66 H),  1 H,  (5,  3.18  C 4 H),  158  1.97 H),  (t,  J  1.21  M.S.  (s,  3 H),  (160°C)  0.3/0.1), (6.7),  168  (6.0),  139  Elemental 6.34,  Cl:  2 H),  0.80  m/z  426  268  (11.7),  N:  12 Hz,  =  425  (37.6),  267  (7.2),  157  N:  (dt,  =  intensity  14 Hz,  3 Hz,  J  1  5%):  460/462  (M,  (13.2),  424 (38.1),  270  (14.3),  269  (26.8),  236  (9.3),  198  (6.6),  182  (27.2),  151  (6.6),  150  (27.4),  Caic.  6.08.  J  3 H).  (23.1),  131  analysis: 7.69,  (s,  (relative  (8.9),  (6.4),  1.81  154  130  for  Found:  (100).  C1N C 2 H : 5 0 2 2 9 C:  62.53,  H:  62.54,  C: 6.27,  Cl:  H:  7.52,  6.08.  3.10.  N- (3’ ‘-INDOLYLMETHYLENETHIOCARBONYL) -endo-7-METHOXYCARBONYL-7-CHLORO-2-AZABICYCLO  [2,2,2]  OCTAN—6-ONE  2’ ,2’-DIMETHYL-l’ ,3’-PROPANEDIYL ACETAL 41  •CH 3 3 CH  O 3 CH  To  a  dry  thermometer, (17.26 g, mmol)  and  heated mixture  three-necked  dry  was  liter  flask  equipped  with  a  a bubbler and a mechanical stirrer was added 40  37.44 mmol),  to  1  toluene  75°C then  in  an  Lawesson’s (600 mL). oil  allowed  to  bath cool  76 reagent The for to  (15.80 g,  reaction 10  hrs.  room  39.06  mixture The  was  reaction  temperature,  the  159  excess  of  Lawesson’s  reagent  cake washed with toluene  (2 x 30 mL).  were evaporated to dryness, dichioromethane and 2 cm  thick)  suspended  of  three  8.5 cm  TLC grade  (600  g).  of  Compound  times  41  alumina  (9.43 g,  60/E)  This  column  a  filter  (8.5 cm wide and (200 g)  Evaporation (20 g).  on  the  dissolved in  g)  alumina (type  product  chromatographed wide)  (25  filtered through a bed  crude  and  The combined filtrates  dichioromethane. the  off  the residue  TLC grade  in  filtrate gave  filtered  was  high  and  without  binder)  obtained  was  the  material  (8.5 cm  (neutral 53%)  of  a  as  yellowish foam.  Physical data of 41: M.p.:  193  195°C (MeOH).  -  TLC (neutral alumina, (neutral alumina, UV  (CH C 3 N)  (4.20),  ‘>‘max  288  C1 2 CH )  Rf:  E):  (log  0.45.  0.40  196  (4.44),  221  (4.55),  282  (4.16).  IR (KBr) Umax stretch),  (nm)  /hexanes/Et 7:3:0.05) Rf: 3 CHC1 N  (cm ) 1 :  2910  (C-H  3350  (indolic N-H stretch),  stretch),  2845  (c-H  3040  (=C-H  stretch),  1730  (ester C=0 stretch). ‘H NMR 7.52 H,  (300 MHz,  (d,  J  =  8 Hz,  indolic C-H),  (t, 6.88  J  =  (s,  15 Hz,  8  Hz,  1 H,  1 H),  ) 3 CDC1  1  1  H, (t,  H,  indolic  (d,  4.42 J  8.19  (s,  indolic C-H),  7.18  C 1 H), 4.10  8(ppm):  =  J  =  (d,  8  Hz,  C-H), J  15 Hz,  1  7.32 H,  7.04  12 Hz, 1 H),  1  indolic N-H),  H, (d,  J  =  8  Hz,  indolic C-H), (s,  1  1  H),  4.01  Cd,  H, 4.24 J  =  1  7.10  C , 2 ,-H), (d,  J  12 Hz,  =  1  160  H), J  =  (m,  Cs,  3.73 15  Hz,  2 H),  0.80  3  3  =  1.71  OCO-), 3 CH  3.30  Hz  2.12  (dt,  1  3  H),  15 Hz,  =  -  3.48  (m,  (broad  J  =  1  s,  3Hz,  4 H,), H,  3.09  Cdt,  -H), 4 C  1.88  1.20  1 H),  Cs,  3 H),  3 H).  (s,  M.S.  3 H,  (150°C)  5.7/12.1),  m/z  443  intensity  (relative  (16.3),  442  10%):  (50.8),. 441  478/476  (M,  (18.3),  440  (45.2),  312  (13.5),  270  (45.2),  268  (29.3),  195  (19.6),  174  (20.4),  173  (58.4),  168  (10.4),  154  (11.8),  138  (15.5),  131  (17.6),  130  (100).  Elemental  analysis:  6.13,  7.43,  Cl:  Cl:  7.40,  3.11.  N:  N:  5.59,  for  Caic. 5.87,  S:  C1N C 2 H S 4 0 2 4 9 :  6.72.  Found:  C:  60.43,  C:  60.15,  H:  H:  6.08,  6.62.  S:  N-BENZYLOXYCARBONYL-exo-7-METHOXYCARBONYL-7- -CHLORO-2AZABICYCLO  [2,2,2]  OCTAN-endo-6-OL 44 and N-BENZYLOXY  CARBONYL-exo-7-METHOXYCARBONYL-7-CHLORO-2-AZABICYCLO [2,2,2]  OCTAN-enc7o--5-OL 45 and N-BENZYLOXYCARBONYL-7-  METHOXYCARBONYL-2-AZATRICYCLO [2,2,2, o6, 7  OCTANE 46.  0  0 P  PhON  OH  Cl  C1OH  3 C 2 CO H 3 C 2 CO H 44 A  100 mL  mechanical  C  45  three-necked  stirrer,  a  46  flask  thermometer,  a  was  fitted  bubbler  and  with a  a  septum.  161  The apparatus was dried in the oven overnight, and  cooled  (1.15 g,  down  3.42  (10 mL).  under  mmol)  nitrogen  a  was  assembled hot  atmosphere.  dissolved  in  33  tetrahydrofuran  dry  Borane dimethyl sulfide complex  Compound  4.2 mmol)  (0.4 mL,  was added and the reaction mixture stirred overnight at room temperature.  The  reaction  was  monitored  TLC  by  (silica,  benzene/ethyl acetate 9:1). Methanol (5 mL) was added slowly with  vigorous  stirring,  another hour. on  a  mmol)  was  followed  by  (0.8 mL,  7.8  the  bath  added  the  and  reaction  at  kept  was  N  3  slowly  dropwise  mmol)  reaction  the  The reaction mixture was  methanol/salt  5.2  and  such  a  below  The  0°C.  -5°C  (1.8 mL,  release  of gas),  hydrogen peroxide  30%  that  to  for  hydroxide  (vigorous  rate  stirred  then cooled  sodium  addition of  was  the  temperature of  reaction  mixture was  then stirred for another 2 hrs at 0°C and at 4°C for another 18 hrs. mmol)  A 5%  (w/w)  added  was  and  reduced pressure. with  ethyl  phase  and  filtered (10 mL).  organic  (5  x  successively  brine  and  the  the  solvents  evaporated  25 mL) with  (15 mL), filter  and 5%  dried cake  the  sodium over  washed  combined  x  sulphate,  magnesium with  (2  ethyl  acetate  Evaporation of the filtrate on a rotary evaporator  a clear glass.  cm).  under  organic  bicarbonate  at 40°C under vacuum gave the crude product (1.07 g,  silica  4.8  The remaining aqueous phase was extracted  acetate  washed  10 mL)  solution of sodium bisulfite (10 mL,  gel  The crude product was  (50 g)  suspended  in  93%) as  purified on TLC grade  benzene  (column  diameter  4  The crude product was loaded onto the column dissolved  162  in benzene (9:1) a  eluted  (200 mL)  flow  73%)  and  rate  and then with benzene/ethyl acetate 4 mL/min.  of  was obtained  Compound  44  initially with benzene/ethyl  and  A  as well 45  mixture  44  of  45  and  as pure compound 46  could  separated  be  by  acetate (8:2)  (878 mg,  (62 mg,  PLC  at  on  6%). mm  0.5  silica plates eluted with chloroform/methanol (20:1).  Physical data of 44: M.p.:  97  98°C (EtOAc/hexanes 1:6).  -  TLC (silica,  benzene/EtOAc 8:1) Rf:  (silica, UV  chloroform/methanol 20:1) Rf:  (CH C 3 N) ‘max  (2.39),  (nm)  260 (2.28),  (log  263  IR (KBr) Vmax (cm ): 1 2860  (C-H  0.05.  E)  :  204  (2.28),  267  (3.99),  1740  (2.36),  251  257  (2.11).  3440 (OH stretch),  stretch),  0.24.  (ester  2940 (C-H stretch),  C=0  stretch),  1690  (carbamate C=0 stretch). NMR (300 MHz, H,  Ph—H),  Hz,  1 H,  (m,  1 H),  (m,  1  H),  1.90  5.25  ) Conformer A (54%) 8(ppm): 3 CDC1  (d,  -CH O 2 -), 3.82  H),  13 Hz,  =  4.67 (d, 3 H,  (s,  2.54 —  J  (d,  J  2.08 (m,  4.58  H),  3.82  (s,  3 H,  H),  2.21  (d,  J  -  2.08  (m,  (d,  4 Hz,  =  2 H),  =  2 H),  J  2  =  1.31  1 H, -  H,  —  1.43  1  —OH),  (m,  -OH),  1.43  H,  3.47  1 H,  3.50 (s,  7.34 (m, Hz,  —CH O 2 —),  2 Hz,  1  1.31  2 C 3 CH ), O 6 Hz,  =  2 C 3 CH ), O  Conformer B (46%) ö(ppm): -CH O 2 -),  J  1 H,  2 H),  2.64  Ph-H), 3.77  2 H),  1 H).  3.77  -  -  13  J -  -  2.20  5  3.95 2.80 (m,  1  1 H).  C 1 H),  2.08  (d,  C 1 H),  2.08  (m,  5 H,  (s,  5.15  7.34 (m,  2.64  5.21 —  -  2.20 (m,  (s,  2 H, (m,  1  2.80 (m,  1  3.95  1 H),  1.90  163  MS  (160°C)  m/z  (relative intensity 5%):  (6.1),  108  (10.1),  141  (100),  80  (7.3),  Elemental 5.70, Cl:  N:  79  (6.1),  105  (28.2),  77  Caic.  10.02.  Cl:  (9.5),  (21.9),  analysis: 3.96,  107  for  353/355  C1N0 1 C 2 H : 5 7 0  Found:  C:  91  70 (12.0). 57.71,  C:  H:  58.00,  (13.2),  92  (7.9),  73  (1.5/0.5),  5.71,  N:  H:  3.93,  10.00.  Physical data of 45: M.  112  p.:  113°C (EtOAc/hexanes 4:11).  -  TLC (silica,  benzene/EtOAc 8:1) Rf:  (silica, UV  chloroform/methanol 20:1) Rf:  (CH C 3 N) ‘Xmax  (2.35), IR  262  (nm)  (2.22),  (KEr)  (log  267  2930  (C-H  5 H, Hz,  (300 MHz,  Ph-H), 1 H,  3.90  2 C 3 CH — O ), 2.05 1 H),  3.42  1.69  (m, —  J  4.65  (m,  2.20  -  1660  5.24 (d,  3.99  -  3400  CDC1 ) 3 :  CH O 2 -),  (d,  J  4.54  3.99  (m,  1 H),  3.42  (d,  J  =  (C-H  4 Hz,  3.72 11  =  J  =  -  4  3.90  -  Hz,  1  (2.30),  3000  257  (=C-H  stretch),  Cm,  J’  (m, 2.78  =  8(ppm):  CH O 2 -), J’  (m,  =  1780  H),  1  2 Hz,  1.73  1  H,  3.82 (s,  2.88  2.88  Ph-H),  5 H,  (m,  1 H,  3.82  -  1 H),  7.38  5.17 (d,  2 Hz,  2.78  (m,  1 H), -  (54%)  H),  2.04  -  7.38  3.90  1 H),  =  Hz,  1 H).  11 Hz,  2860  J  (dd,  (m,  3.72  stretch),  1 H,  1.92  (dd,  (0-H  13 Hz,  Conformer B (46%) 6(ppm): CH O 2 -),  251  (carbamate 0=0 stretch).  2 H),  1.73  (3.92),  Conformer A  =  H),  1  206  stretch),  (ester C=0 stretch), NMR  E):  0.32.  (2.02).  Vmax (cm):  stretch),  0.05.  J  (s,  H),  1.82  (m,  C 1 H),  1 H),  H,  3 1  5.20 (s,  3 H,  13  =  C 1 H),  (m, -  (m,  2 H,  3.90  -  2 C 3 CH ), O  2.05  -  2.20  164  (m,  2 H),  1.73 MS  (m,  1.92  2.04 (m,  —  m/z  1.82 (m,  -  (relative intensity 5%):  105 (5.5),  92 (11.2),  Elemental  analysis:  Cl:  1.73  1 H),  1.69  —  1 H).  (160°C)  5.70,  1 H),  N:  3.96,  Cl:  91  (100),  Caic.  10.02.  (0.7/0.4),  353/355  77 (14.6).  for  C1N0 1 C 2 H : 5 7 0  Found:  C:  57.75,  57.71,  C:  H:  5.64,  N:  H:  3.87,  9.95.  Physical data of 46: M.p.:  67  69°C 2 (EtOH/H 0 ).  -  TLC (silica, UV  benzene/EtOAc 8  (CH C 3 N) ‘max  (2.32), IR  260  (KBr)  (nm)  (2.22),  (log E)  262  204  (4.24),  (2.23),  266  (2.06).  3075  (=CH  stretch),  2975  stretch),  1680 (ester C=0 stretch).  (CH  stretch),  ‘H NMR (300 MHz,  CDC1 ) 3 :  5 H,  (d,  Ph—H),  12 Hz, 3 H, 1.78  1 H,  5.21  -CH O 2 -),  2 C 3 CH ), O —  1.99  12 Hz,  =  3.33  -  2 H),  2 C 3 CH ), O 1.99 MS (M, 166  (m,  4.12 3.12 2 H),  (150°C)  rn/z  (d, -  J  J  (m,  1.70 (m,  Conformer B (45%) 6(ppm): -CH O 2 -),  2940  7.35 Hz,  8  =  3.33 Cm,  1.70 (m,  (CH  2 H),  (2.25),  256  3020  (=CH  stretch),  (55%)  1 H, =  251  stretch),  Conformer A  4.26 (d,  3.12  (m,  J  0.35.  :  (cm):  Vmax  1) Rf:  :  2 H),  1 H,  2.08  (CH  7.35  (m,  8(ppm):  -CH O 2 -),  8 Hz,  2870  5.08  C 1 H), 2.35  -  (d,  J  (s,  3.66 (m,  3 H),  1 H). (m, 1  Ph—H),  5 H, H,  2.08  C 1 H), -  5.15 (s,  3.66  2.35 (m,  3  (s,  3 H),  2 H, H,  1.78  -  1 H).  (relative  intensity  10%):  302  (13.1),  301  68.9),  257  (50.0),  242  (14.1),  199  (17.8),  198  (10.7),  (35.2),  158  (33.2),  139  (36.4),  134  (13.7),  107  (30.0),  165  106  (11.2),  (18.5),  (10.0),  93  79(80.6),  Elemental  92  (69.7),  78(11.0),  analysis:  Caic.  91  (100),  81  (16.8),  80  C:  67.76,  H:  77(23.2).  for  C1N0 C 1 H : 4 7 9  6.36,  N:  3.12.  N-BENZYLOXYCARBONYL-exo-7-METHOXYCARBONYL-7-CHLORO-2-  4.65.  Found:  AZABICYCLO  C:  [2,2,2]  H:  67.65,  6.25,  4.60.  N:  OCTAN-6-ONE 47 and N-BENZYLOXY  CARBONYL-exo-7-METHOXyCARBONYL--7-CHLORO-2-AZABICyCLO [2,2,2]  OCTAN-5-ONE 48 and N-BENZYLOXYCARBONYL-7-  , 6 METHOXYCARBONYL-2-AZATRICYCLO [2,2,2, o  OCTANE 46  0  0 Ph  Fh  Ph  C’ CH CO 3 47  A  250 mL  mechanical calcium (4.00 g, alcohols  two-necked  stirrer  chloride 18.6  CH 3  48  and  reflux  guard-tube.  mmol)  (3.86 g)  a  flask  was  was  equipped  condenser Pyridinium  added  to  a  4  with  fitted  a  with  a  chlorochromate  solution  dissolved in dichioromethane  of  crude  (75 mL).  The  reaction mixture was heated to reflux with vigorous stirring for 5 hrs and methane/ethyl filtered  was  acetate  carefully  and 2.5 cm  monitored  long).  3:1).  through  by The  a  bed  Dichioromethane  TLC cold of  (silica, reaction  florisil  (40 mL)  was  dichioro mixture  (3.5 cm added to  was wide the  166  vessel  reaction vigorous  stirring  florisil bed. was  solution  was  vacuo gave  reflux  to  then  silica gel  suspended  in  acetate  (7:3)  a  at  made  wide)  rate  same  drying  in  crude product was (200 g)  TLC grade  of  with  and  loaded  silica gel  hexanes/ethyl  25 mL/min.  of  the  The greenish  further  This  Elution  hexanes. flow  20 mL).  400 mesh)  -  under  Then the florisil bed  and  (3.86 g).  (230  (7.5 cm  (200 g)  dryness  to  mm  through  x  (6  15  for  filtered  dichioromethane  greenish oil  column  a  and  evaporated  a  adsorbed on  heated  This was repeated twice.  with  rinsed  onto  and  The  column  chromatography had to be performed twice in order to achieve complete  separation,  as a colorless oil,  giving 48  solid and 46 (397 mg,  47  (1.412 g,  (576 mg,  based on 33)  34%  14% based on 33)  as a white  11% based on 33).  Physical data of 47: TLC (silica, (silica,  hexanes/EtOAc 7:3) Rf: C 2 CH / EtOAc 1 3:1) Rf:  UV 3 (CH C N) ‘Xmax (nm): IR  (KBr)  (cm):  Umax  stretch),  208,  1740  (ester  251,  0.70  257,  3005 C=0  0.10  262,  (=C-H  stretch),  267.  stretch),  2875  (C-H  (carbamate  1700  C=0  stretch). NMR 5 H, Hz,  ), (m,  (300 MHz,  Ph-H), 1 H,  3.45  5.24 (d,  CH O 2 -), -  1 H),  CDC1 ) 3 :  3.70 2.38  J  4.72 (m,  =  (s,  2 H),  (broad s,  Conformer A 13 Hz, 1 H, 2.78 2 H),  1 H,  CH O 2 -),  -H), 1 C -  2.90  2.15  8(ppm):  (57%)  -  3.82 (m,  5.14 (d, Cs,  1 H),  2.28(m,  3 H, 2.50  1 H).  (m,  7.38 J  =  13  2 C 3 CH O -  2.62  167  Conformer B 13  Hz,  (s,  1  H),  2.78  H,  1 H,  2 H),  MS  (150°C)  2.15  m/z  (6.1),  (9.7),  77  —  (m,  7.38  5.18  3.82  2.90  -  6(ppm):  CH O 2 -),  -H), 1 C  s,  159  (43%)  Cd, 3  (s,  (m,  1 H),  2.28  (m,  J  Ph-H),  5 H, 13  =  Hz,  2.50  -  2.62  2.38  J  =  4.84 Cm,  3.70  -  1 H),  (m,  (d,  CH O 2 -),  3.45  2  (broad  1 H).  (relative intensity 5%):  158  H,  1  2 C 3 CH ), O  H,  5.24  (18.7),  107  (5.3),  92  351/353  (1.0/0.5),  91  (9.6),  (100),  79  (6.7).  High resolution MS:  Caic.  for 5 C10 C 1 H : 7 8  351.0874.  Found:  351.0879.  Physical data of 48: M.p.:  133  135°C (EtOAc/hexanes 1:3)  -  TLC (silica, (silica, UV  hexanes/EtOAc 7:3) Rf:  dichioromethane/EtOAc 3:1) Rf:  (CH C 3 N) max  (2.32),  256  (shoulder,  (nm)  (2.38),  (log  260  C):  205  (2.28),  0.70  (4.00),  262  (2.30),  246  (2.27),  250  266  (2.16),  288  1.38).  IR (KBr) ‘umax (cm): 2875  0.15  (CH stretch),  3005 (=CH stretch),  1740  (shoulder,  2945  (CH stretch),  ester C=0 stretch),  1705  (carbamate C=0 stretch). ‘H NMR H,  Ph—H),  Hz,  1  3.86 (m, H).  (300 MHz,CDC1 ) 3  H, (s,  1  H),  5.24  (d,  O—), 2 CH 3  H, 2.60  J  Conformer A (52%) 12 Hz,  =  5.06  (dd,  2 C 3 CH ), O -  2.78  J  3.67 (m,  2  1 H, =  CH O 2 -),  2 Hz,  -  H),  o(ppm):  3.82 2.43  J  =  (m, (m,  5.18  5 Hz, 2  H), 1  H),  7.38 (d, 1 H, 3.07 2.32  Cm, J  =  5 12  —H), 1 C -  3.18 Cm,  1  168  Conformer B CH 0 2 -), H,  MS 232  4.94  2.78  -  (5.3),  231  Elemental 5.16,  N:  -  7.38  2 Hz,  =  3.82  2 H),  (m, rn/z  91  J  3.67  (120CC)  (15.4),  Cl:  (dd,  2 C 3 CH ), O  2.60  ô(ppm):  (48%)  J (m,  2.43  (relative (27.2),  (m, =  2  (m,  5 H,  Ph-H),  5 Hz,  1 H,  H),  3.07  1 H),  2.32  intensity 5%): 186  (9.0),  159  5.23  C 1 H), -  3.85  3.18  (m,  2 H,  (s,  Cm,  (s, 1  3  H),  1 H)  351/353 (8.5),  (1.2/0.4), (5.8),  92  58.04,  H:  96  (100). analysis:  3.98,  Cl:  for  Calc.  10.08.  Found:  C1N0 C 1 H : 5 7 8 C:  58.23,  H:  C:  5.15,  N:  3.89,  9.89.  Physical data of 46: As described in the synthesis of 44.  3.13.  N-BENZYLOXYCARBONYL-exo- 7-METHOXYCARBONYL--7-CHLORO-2AZABICYCLO  [2,2,2]  OCTAN-6-ONE 2’ ,2’-DIMETHYL-l’ ,3’-  PROPANEDIYL ACETAL 49  0 P C’ 3 C 2 CO H  CH 3 3 CH  A 25 mL flask was equipped with a magnetic stirring bar and  a  Dean-Stark  calcium mmol),  chloride  trap  fitted with a  guard-tube.  reflux  Compound  2,2-dimethyl--1,3-propanediol  47  (68 mg,  condenser  and  (206 mg, 0.65  mmol)  a  0.59 and  169  p-toluene sulfonic acid monohydrate  (26 mg,  added  mixture  to  reflux  benzene  for  4  (10 mL)  hrs.  The  and  the  reaction  mixture  0.14 mmol)  heated  was  was  were to  with  diluted  benzene (50 mL) and washed with saturated sodium bicarbonate (10 mL)  and water  (3  x  15 mL).  over magnesium sulfate, with benzene (25 mL).  The organic  layer was dried  filtered and the filter cake washed Evaporation of the solvent and drying  of the residue in vacuo overnight gave an oily white solid (241 mg).  This  crude  product  stirred  was  in  acetate (5:1) until a fine powder had formed. washing of after  the filter cake with hexanes (2  drying  evaporated  in vacuo, to  49  dryness  and  dichloromethane  (2 mL)  wide)  TLC grade  made  of  (146 mg).  and  the  onto gel  5 mL/min gave  of  49,  gave,  liquor was  column  (2 g)  rate  42 mg  2.5 mL)  dissolved  a  Elution with hexanes/ethyl acetate another  x  mother  hexanes. of  Filtration and  residue  loaded silica  The  hexanes/ethyl  (1.2 cm  suspended  (7:3)  in  in  at a flow  giving  a  total  yield of 188 mg (73%) of 49.  Physical data of 49: M.p.: TLC  141  -  143°C (EtOAc/hexanes 1:2).  (silica,  hexanes/EtOAc  7:3)  Rf:  0.22/0.38.  Double  spot  due to the two conformers. UV  CN) ‘“max 3 (CH  (2.51), IR  (KBr)  1745  262  (nm)  (2.46),  Umax  (log  266  (cm):  E)  (2.36,  2925  (ester C=0 stretch),  :  207  (3.86),  252  (2.46),  257  shoulder).  (CH stretch),  2860  (CH stretch),  1690 (carbamate C=0 stretch).  170  NMR 5 H,  (300 MHz,  Ph-H),  -H), 1 C  5  H),  (d,  0.85 (s,  1  H),  3 H,  Conformer B 12  Hz,  4.99  1  (s,  Hz,  1 H,  H),  2.15  s,  2 H),  M.S.  0.87  154 (5.6),  Elemental 6.45,  m/z  N:  8.00.  Cl:  7.35  3.79  H),  3 H,  1.92  C), 3 CH  1.07  Cs,  H),  1.92  H,  3  H,  3  3.50 (m,  -  1  (s,  Calc.  12  =  for  Found:  Hz,  5 H),  2.07  0.75  92  Ph-H),  (s,  (9.4),  Cm,  -  C), 3 CH  -  1 H), C 3 CH  5%):  60.35,  (d,  402  J  =  O-), 2 PhCH  3.73 (d, 3.03 1.61  J  12  =  Cm,  1  (broad  ). (M  -  Cl,  (100).  C1N0 C 2 H : 6 2 8 C:  H,  2.80  3 H,  91  5.40  1  2 C 3 CH O ),  (m, —  5 H,  intensity  (10.0),  8.10.  J  3.50  -  (relative 129  Cm,  (d,  (s,  1 H),  3 H,  analysis: 3.20,  2  3.10  (m,  2.28  —  1 H,  5,22 (s, 3.81  O-), 2 C—CH  2.15 s,  O-), 2 PhCH  O-), 2 PhCH  1 H,  H),  5.12  3.10  Cm,  Cs,  1  (broad  -H), 1 C  2.28  H,  6(ppm):  O-), 2 C-CH —  Hz,  1  O-), 2 PhCH  1 H,  1 H,  Cm,  7.35  ).  C 3 CH  (150°C)  28.6),  Cl:  H,  Cm,  1.78  (32%)  12 Hz,  12 Hz,  J  o(ppm):  Conformer A (68%)  12  3.03  —  J  J  3.58 (d,  2.80 (m,  2.07  5.26 (d,  5.18  2 C 3 CH O ),  CDC1 ) 3 :  H:  C: 6.45,  60.34, N:  H:  3.02,  171  3.14.  N- (3’’ -INDOLYLMETHYLENECARBONYL) -exo-7-METHOXYCARBONYL-7-CHLQRQ-2-AZABICYCLO [2,2,2]  OCTAN-6-ONE  2’,2’ -DIMETHYL-1’,3’ -PROPANEDIYL ACETAL  51  5  C’ •C H 3 3 CH Compound  49  benzene  (50 mL)  bubbled  through  solution was  (3.418  g,  7.81  under argon. the  was dissolved in dry  mmol)  Anhydrous hydrogen bromide was  solution  for  purged with argon.  45  mm,  after which  Dry diethyl  ether  the  (200 mL)  was slowly added to the vigorously stirred solution until a fine white precipitate had formed.  The solution was filtered  and the filter cake was stirred twice with dry diethyl ether (20 mL).  Drying in vacuo gave the crude amine salt (2.730 g,  91%) as a white powder.  Method A: The crude amine salt in  dry  acetonitrile  (210 p1, 3-indole added, for  (10 mL)  1.51 mmol) was added, acetyl  giving  4.5  (439 mg,  hrs..  a  under  Water  argon  and  was suspended triethylamine  giving a clear solution.  chloride  clear  1.14 mmol)  brownish (0.5 mL),  (290 mg, solution followed  150 which by  mmol) was  Then was  stirred  triethylamine  172  (0.2 mL) a  was added to the now clear yellow solution,  dark greenish  The  residue  solution  was  which  evaporated  was  dissolved  in  and  and 14 cm long)  packed with silica gel  onto  a  flash  column  (230  dryness.  volume  minimum  a  dichioromethane  loaded  to  of  (3 cm wide  400 mesh)  -  hexanes/ethyl acetate/triethylamine (12:8:0.6).  in  Elution with  the above solvent system at a flow rate of 5 cm/mm (377 mg,  giving  gave 51  71% based on 49) as a white powder.  Method B: The crude amine salt in  dry  acetonitrile  (290  iii,  Then  3-indole  2.08  (800 mg,  (6 mL)  mmol) was acetic  under  was  argon  triethylamine  to give  added  acid  2.08 mmol)  (365 mg,  and  a  thick  2.08  mmol)  suspended  suspension. was  added,  giving a clear brownish solution which was cooled to 0°C in an  ice  mmol) 0°C.  bath.  was The  1,3-Dicyclohexylcarbodiimide  added and the reaction was reaction mixture was  (472 mg,  stirred  filtered and  for 19  the  2.29 hrs at  filter cake  washed with dichloromethane (3 x 5 mL). After evaporation of the  solvent  the  dichioromethane. column gel  residue This  (4.5 cm wide  (230  -  400 mesh)  (14:6:0.6).  was  redissolved  solution  and  14  cm  in  was  loaded  long)  packed  minimum  a  onto with  flash  a of  of  silica  in hexanes/ethyl acetate/triethylamine  Elution,  first  acetate/triethylamine  (14:6:0.6)  hexanes/ethyl  triethylamine  acetate!  with  hexanes/ethyl  (1000 mL) (12:8:0.6)  with  then at  a  flow  173  of  rate  cm/mm  5  51  gave  (559 mg,  based  58%  49)  on  as  a  white solid.  Physical data of 51: M.p.:  174  179°C (MeOH/CH C1 6:1). 2  —  TLC (silica, UV  (CH C 3 N)  (4.58), IR  3 hexanes/acet N one/Et 10:10:0.6) Rf: (nm)  ‘max  273  (3.75),  (KBr)  stretch),  (log  279  E)  :  (3.77),  Vmax  (cm ) 1 :  1735  (ester  202  (4.40,  2.89  stretch),  stretch),  C0  shoulder)  220  ,  (4.79).  (N-H  3200  0.40.  1630  2900  (C-H  (amide  C=0  stretch). ‘H NMR (250 MHz, J  7.5  =  Hz,  H,  C — 1 H),  (d,  J  Hz,  J’’  1 H), H,  =  H,  1  indolic C-H), 3.78  C-H),  7.24  -  1 H),  (m,  Cm,  3.88  -  2 Hz,  1.75  indolic  7.07  10 Hz, =  ) ö(ppm): 3 CDC1  1 H),  (broad s,  3.59  —  2.13  -  2 H),  1 H,  N-H),  J  7.5  (d,  indolic C-H),  3.80  (s,  (m,  5 H),  2.20  1.08  (s,  7.37  3 H,  2 H),  3.16  8.06  3 H,  Hz,  5.84  (dt,  1.87  CH — 3 C),  -  (s,  1  3.66  J’  15  =  2.00  0.77  H,  1  CH O 3 CO),  3.01  1 H),  (m,  (s,  3 H,  (d,  7.59  (m,  (s,  3  CH 3 C).  M.S.  (150°C)  1.9/0.8), (10.7),  m/z  426  268  (5.9),  167  (100),  129  (19.6),  55  (relative  (19.4),  (16.1), (8.5),  236 157  (23.4), (6.5),  425  41  intensity  (11.0), (15.2),  128  424  (47.8),  (6.2),  (18.6),  198 154  5%):  460/462  (39.5),  307  (M,  (5.5)270  (5.9),  182  (11.1),  168  (20.4),  131  (14.7),  130  103  36 (5.5).  (5.6),  77  (5.7),  69  174  Elemental 6.34,  analysis:  Cl:  7.69,  N:  Calc.  for  6.08.  Found:  C1N C 2 H : 5 0 2 4 9 C2:  62.73,  C:  62.54,  H:  6.40,  H: Cl:  7.55,  N:  3.15.  N- (3’ ‘-INDOLYLMETHYLENETHIOCARBONYL) -exo-7-METHOXY-  6.10.  CARBONYL-7-CHLORO-2-AZABICYCLO  2’ ,2 ‘-DIMETHYL-l  1,3T  [2,2,2]  OCTAN-6-ONE  -PROPANEDIYL ACETAL 52.  5  H’  3 •CH 3 C 2 CO H  Dry mmol)  benzene  and  oil  argon.  (293 mg, The  51  to  dichioromethane). and  mmol)  0.717  in  reaction mixture was  bath for 3.5 hrs  vacuum  added  was  (331 mg,  0.718  2, 4-bis(thiophenyl)-1, 3-dithia-2, 4-diphosphetane-  2,4-disulfide under  (12 mL)  solvent  residue  was  dry  heated  and monitored by TLC  The  the  a  in  flask  60°C  in an  (neutral alumina,  removed  dissolved  to  25 mL  at a  35°C  under  minimum  of  dichioromethane and loaded onto a column (3 cm wide) of made TLC grade alumina G dichioromethane. of  6 mL/min  dissolved  (neutral,  type 60/E)  (30 g)  suspended in  Elution with dichioromethane at a flow rate  gave  the  crude  product  (261 mg)  which  was  in a minimum volume of dichioromethane and loaded  175  equally  onto  two  PLC  plates  (neutral  alumina,  20 cm) and eluted with dichioromethane.  1  mm,  20  x  The partly separated  products so obtained were loaded onto new alumina plates and re-eluted with dichioromethane.  In this way 52A (120 mg) and  52B (89 mg) were isolated.  Physical data of 52A: TLC (neutral alumina,  C1 Rf: CH ) 2  UV (CH CN) ‘>max (nm): 3  195,  IR  (KBr)  ‘H NMR 7.60  (cm ) 1 :  max 1  stretch),  J  ) 3 CDC1  8 Hz,  =  280,  3320  2840 (C-H stretch),  (300 MHz,  (d,  220,  288  (N-H  1735  6(ppm):  1 H,  0.27.  stretch),  8.36  7.38  indolic C-H),  7.25 Cs,  1 H,  ,,-H), 2 C  H,  indolic C-H),  7.14  J  8 Hz,  1 H,  1 H), Cd, 3.36 Hz, 2.15 (s, M.S.  -H), 1 C  3.85  J  (s,  3 H,  12 Hz,  =  (dd, J’  4.30 (s,  =  J  s,  1.11  3.72  J’  1 H,  (d,  3.06  Cdt, J  1  1.91  (broad d,  (s,  3 H),  0.81  (s,  =  1 H),  1 H), H),  J  J  441  (17.6),  J  =  8 Hz,  1  8 Hz,  1  J  11.2 Hz,  1 H),  J’  =  3.27  =  =  16 Hz,  12 Hz,  2 Hz,  (dd,  J’  =  J  3.64 1 H), 11.2  =  2 Hz, 1  7.05  1 H),  H),  1.75  3 H).  (relative intensity):  442 (11.8),  (t,  =  4.24 (d,  16 Hz,  =  J  indolic C-H),  12 Hz,  =  2 Hz,  =  (C-H  indole N-H),  Cd,  7.22  OCO-), 2 -CH  Cdt, J  3.54  11.2 Hz,  C220°C) m/z  3.1/1.1),  O-), 3 CH  1 H),  2 Hz,  (broad 2 H),  =  2 H,  1 H,  (s,  H,  (s,  2910  (ester C=0 stretch).  indolic C-H),  (t,  (shoulder).  (M,  476/478  440 (50.8),  354 (21.2),  268 (13.2),  195 (15.6),  182  (12.8),  174 (12.6),  173 (46.9),  154  (28.8),  139  (10.7),  138  (22.1),  131  130  129  (22.4),  128  (17.4),  77 (11.7),  69  (12.6),  (50.7),  68  (84.7),  (25.5),  59  176  (12.0),  57  (19.7),  43(21.8),  (16.1),  (19.3),  56  42(13.5),  45 (11.4),  (29.8),  55  44  41(70.6).  Physical data of 52B: TLC (neutral alumina,  C1 Rf: 2 CH )  UV 3 (CH C N) ‘>%max (nm):  221,  IR  (KEr)  stretch), NMR 7.75  2825  Cd,  J  ) 3 CDC1  8 Hz,  =  stretch),  8.11  indolic C—H),  7.32 (s,  1 H,  ,-H), 2 C 7  H,  indolic C-H),  7.14  J  8 Hz,  Cs,  1 H,  J  14 Hz,  =  (s,  3  C 1 H),  H,  1  1 H),  H),  (broad d, M.S.  =  3.8/1.4),  442  3.38  J  (broad s,  15.2,  =  Cd,  15.2 Hz,  (220°C) m/z  J  1 H),  3.12 (t,  2.43 J  (q,  2 Hz,  =  CH O 3 -),  11.2 Hz, Hz,  J’  4.57  3.75 J  =  1  2 H,  11.2  =  Hz,  11.2 Hz, H),  1 H),  0.87  (s,  441  (14.2),  440  Ct,  =  J  1  8 Hz,  1  =  1  H),  3 I-I),  1 H),  3.32  2.80 Cd, Cm,  5.05  3.94 (dt,  14 Hz,  =  2.08  -  8 Hz,  indolic C-H),  2 H),  1.90  J  -CH O 2 CO-), J  (relative intensity):  (13.3),  (C-H  indole N-H),  (d,  7.19  1 H,  ((d,  H,  7.37  H,  =  1  Cs,  indolic C-H),  (t,  2900  1730 (ester C=0 stretch).  o(ppm):  1 H,  (shoulder).  (N-H  3350  (C-H stretch),  (300 MHz,  279  270,  (cm):  umax  0.15.  (d, J  =  2 H),  0.73  3.46  (s,  J  15.2 1.70  3 H).  476/478 (M, (36.5),  354 (12.1),  268 (15.1),  195  (11.7),  182  (23.0),  174 (14.3),  173 (53.4),  168  (10.7),  166  (12.1),  155 (12.1),  154 (28.9),  140 (16.2),  139  (24.9),  138  (38.7),  131  130 (100),  128  (16.3),  124 (10.6),  (14.2),  80 (18.9),  77 (11.0),  (51.1),  68  (25.8),  59  (10.4),  59  (19.1),  55  (33.4),  45  (11.4),  44 (17.0),  (11.7),  41  (72.1).  (14.1),  57  129 71  (22.0),  (11.4),  (19.7),  56  43 (26.0),  42  =  69  177  3.16.  20—DESETHYL-15, 20-ANHYDRO-5-OXO-CATHARANTHIN-20-ONE 2’, 2’ -DIMETHYL-1’  ,  3’ -PROPANEDIYL ACETAL 55 AND THE  ISOMERIC BY-PRODUCT 56  CH 3  L 3 CH 0 CH 3 H  3 •CH  CO C 2 H  3 CH 55  56  Sodium hydrogen carbonate in  water  (180 mL)  was  added  dissolved in methanol. with  argon  for  The  solution  which  was  photolysed  Vycor  filter and with so heated  evaporated the  from  remaining  40 mL of ethyl order  to  (300  mm.  6  suspension  lamp  40  to  the the  resulting phase  acetate.  prevent  mg,  dissolved mmol)  0.65  slowly for  became 20  a  to  thin  using  mm  little cooling that the heat  solution  aqueous  6.4 mmol)  The mixture was degassed by bubbling  white  the  (541 mg,  a  from  The  methanol  was  clear yellowish  solution  and  extracted  times  was  reflux.  five  with  Sodium chloride had to be added in  formation  of  an  emulsion.  The  combined  organic extracts were dried over magnesium sulfate,  filtered  and the  filter cake washed with ethyl  Evaporation (258 mg)  of  the  which was  solvent dissolved  at in  acetate  40°C gave  (2  x  20 mL).  yellow-brown  dichioromethane  (2 mL)  oil and  178  loaded  equally  20 cm).  The  following amine  onto  plates  solvent  (30:60:4),  four were  2 mm  silica  eluted  systems;  once  PLC  plates  with  each  hexanes/ethyl  hexanes/ethyl  acetate  (20 of  x  the  acetate/diethyl and  (30:60)  finally  with hexanes/ethyl acetate/diethylamine 30:60:10.  The second  band from the top  as a white  solid.  The  (44 mg)  last  which  (Rf band  was  loaded  equally  20 cm)  .  on  0.7) gave 55  =  (Rf  two  in  purified  dichloromethane mm  0.5  4%)  partly  0.3) gave  dissolved to  (11 mg,  PLC  silica  56  (1 mL)  plates  and  (20  x  The plates were developed twice with hexanes/ethyl  acetate 1:3.  Compound 56  (and 41 mg,  15%)  was obtained as a  white solid. The compounds scale  55  and  56 were not  from the amide 51.  synthesized on large  That the amide 51 does  lead to 55  and 56 under the photochemical conditions was established by comparative TLC and HPLC with isolated 55 and 56.  Physical data of 55: TLC (silica,  hexanes/EtOAc 1:2) Rf:  UV (CH CN)’Xmax (nm): 3 IR  (KBr)  (indole stretch), stretch),  (cm):  Vmax N-H  192,  stretch),  2925  219,  1735  282,  3400  (ester  C=0  CDCN)  8(ppm):  290.  (indole  3050  (C-H stretch),  0.54.  (=C-H 2865  N-H  stretch),  stretch),  2945  (C-H stretch),  stretch),  1645  3280 (C-H  2850  (C-H  (lactame  C=0  stretch). NMR J  8.0  (400 MHz, Hz,  1  H,  indolic  C-H),  9.06  (s,  7.28  1 H,  (d,  J  N-H), =  8.0  7.54 Hz,  Cd, 1  H,  179  indolic C-H),  Ct,  J  =  7.12  8.0  Hz,  16 Hz,  4.27  Cd,  J  3.65  (m,  2 H),  3.32  (d,  J  2  Cm, 0.85 MS  H),  =  H,  indolic  1  (a,  =  1 H),  3.58  1.46  Cs,  3 H,  =  5.25 J  Cd,  -  14 Hz,  1  3.42  1 H),  3.07  Cm,  2 H),  H),  1.11  Cs,  3.55  1.75 3 H,  -  2 H),  (m,  3.53  -  7.07  -H), 2 C 1  H,  1  Cs,  16 Hz,  =  CH O 3 CO),  2.98  indolic C-H),  1 H,  C-H),  3.74  1 H), J  8.0 Hz,  -  1.87  CH — 3 C),  CH 3 C).  m/z  C180°C)  J  12 Hz,  =  3 H,  (s,  Ct,  (relative  intensity):  424  (M,  89.0),  296  C17.2),  267  (16.6),  255  (12.0),  241  C10.3),  223  (23.5),  214  (18.4),  209  (10.3),  207  (10.5),  195  (14.7),  194  C15.4),  182  (21.7),  181  (15.6),  180  (16.9),  169  C13.5),  168  (29.9),  168  (28.7),  129  (14.3),  128  C100),  155 127  C17.1),  154  (18.5),  (41.6),  81  C12.5),  130  (45.4),  69  C12.3),  68  C14.2),  54  (16.5) High resolution MS:  Caic.  for 2 N C 2 H 5 0 4 8  424.1998.  :  Found:  424.1998.  Physical data of 56: TLC (silica,  hexanes/EtOAc 1:2) Rf:  UV 3 (CH C N) ‘>‘max (nm): (KBr)  IR  (indole  (cm):  Vmax N-H  stretch),  198,  3400  stretch),  2865  226,  288,  296.  (indole  3040  (C-H stretch),  0.15.  (=C-H  N-H  stretch),  stretch),  2940  1732 (ester C=0 stretch),  3260 (C-H 1635  (lactame C=0 stretch). NMR  (400 MHz,  7.25  (d,  1 H,  -H), 1 C 1  J  =  ) 3 CDC1  8.0 Hz, 6.94  (s,  ö(ppm):  1 H, 1 H,  8.72  (s,  indolic C-H), C 2 H),  6.84  1 H,  7.09 Cd,  J  indolic N-H),  (t, =  J  =  8.0 Hz,  8.0 Hz,  1 H,  180  indolic C-H), H),  3.47  3.33 s,  1.06 MS  3.41  -  1  -  H),  3.60 (m,  1.93 3 H,  (s,  (180°C)  (7.7),  5.38  365  Cs,  1  5  H,  (m, 2 H),  2.04  —  -C), 3 CH  m/z  H,  4.25  (d,  J  -0), 2 C-CH  3.45  (s,  3  3.24 (m, 0.90  (relative  (6.7),  -H), 2 C  338  (d, 2  J  H),  (s,  12 Hz,  =  1.75  3 H,  J  =  OCO-), 3 CH  2.26  14  1  (broad  Hz,  1  H),  -C). 3 CH  intensity):  (12.1),  H,  1 H),  Cd,  14.0 Hz,  =  294  424  (17.6),  (M, 251  100),  392  (10.2),  223  (11.5),  208  (11.8),  207  (13.3),  196  (12.8),  195  (10.7),  194  (13.6),  182  (15.4),  181  (15.0),  180  (12.2),  169  (15.0),  168  (28.5),  167  (71.8),  155  (16.9),  154  (59.2),  149  (11.4),  141  (10.2),  130  (10.2),  129  (10.0),  128  (11.5),  127  (16.7),  115  (11.5),  70(22.3),  69(54.2).  High resolution MS:  Caic.  for 2 N C 2 H 5 0 4 8  :  424.1998.  Found:  424.2016.  3.17.  20-DESETHYL-15, 20-ANHYDRO-5-THIOXO-CATHARANTHIN-20-ONE 2’, 2’ -DIMETHYL-1’ ,3! -PROPANEDIYL ACETAL 61 AND THE ISOMERIC BY-PRODUCT 62  S  S  0 C  H  CH 2 C0  •C H 3 CH 3  61  62  181  Sodium hydrogen carbonate (146 mg, in  water  (300 mL)  was  dissolved in methanol  added  41  to  (450 mL).  filter.  for  50  mm  mmol)  0.67  and the solution was then  room  at  (319 mg,  The mixture was degassed by  bubbling with nitrogen for 10 mm photolysed  1.74 mmol) dissolved  temperature  using  Vycor  a  The methanol was evaporated from the clear yellowish  solution and the remaining aqueous phase was extracted with chloroform (5 x 50 mL). order  to  prevent  Sodium chloride had to be added  formation  of  an  emulsion.  extracts were dried over magnesium sulfate, filter cake washed with chloroform  The  in  combined  filtered and the  (2 x 20 mL).  Evaporation  of the combined filtrates at 40°C gave a brown oil (352 mg). The  crude  product  chromatography  on  was  partly  deactivated  chioroform/hexanes  (2:1)  material  so  obtained  (20 cm  x  20 cm)  as  was  neutral  eluent.  then  and  loaded  eluted  dichloromethane/methanol (200:1). and  compound  62  (28.6 mg,  purified  The on  alumina partly  0.5  three  column  by  mm  using  purified  PLC plates  times  with  Compound 61 (31.4 mg,  10%)  were  obtained  as  11%) white  solids. The large  scale  compounds from  the  61  and  62  thioamides  were 52A  not and  synthesized 52B.  That  on the  thioamides 52A and 52B do lead to 61 and 62 under the photo chemical conditions was established by comparative HPLC with isolated 61 and 62.  182  Physical data of 61: TLC  (neutral alumina,  UV (CHCN) ‘>max IR  (KBr)  (nm):  2920  stretch),  197,  (cm):  Umax  stretch),  CHC1 / 3 hexanes 2:1) Rf: 223,  3340  (ester  289  (shoulder)  (indole N-H  (C-H stretch),  1735  272,  2865  C=0  0.15.  stretch),  2945  (C-H  (C-H stretch),  2850  (C-H  (ester  C=0  1718  stretch),  stretch). H NMR 1  (300 MHz,  ) 3 CDC1  7.58  (d,  1  indolic N-H),  H,  J  7.13  Ct,  4.50  (dd,  3.62  (s,  J  J J  H),  1.85  (s,  3 H),  MS  (120°C)  (9.2),  (d,  7.17  7.4  =  16 Hz,  Hz,  2.36 J  =  J  =  -  3.65  1 H),  1 H),  7.28  7.4 Hz,  indolic N-H),  (d,  H,  J  (m  1  Cs,  5 H),  ,  J  =  H, J  J  -H), 2 C 1 13 Hz),  =  (broad d,  3.06  (broad d,  (d,  7.4 Hz,  =  indolic N-H),  (broad d,  2.15  1.56  1  5.52  4.05  H,  1  (s,  C-H),  -H), 6 C  3.35  (broad s,  Cs,  m/z  (t,  2 H,  13 Hz,  7.86  indolic C-H),  indolic  OC-), 3 CH  0.92  354  1 H,  =  3 H,  13 Hz),  =  7.4 Hz,  =  8(ppm):  =  13 Hz,  1  13 Hz,  1 H),  1.08  (M,  100),  408  (10.3),  267  3 H)  (relative  (61.1),  321  intensity):  (15.9),  295  440  (15.9),  280  (34.9). High resolution MS:  Calc.  for 2 N C 2 H S 4 0 4 8 :  440.1770.  Found:  440.1757.  Physical data of 62: TLC  (neutral alumina,  UV 3 (CH C N) ‘max NMR 7.29  (300 MHz,  (d,  J  (nm):  CHC1 / 3 hexanes 2:1) 193,  ) 3 CDC1  7.0 Hz,  228,  8(ppm):  1 H),  7.22  272, 8.50 Cs,  295 (s, 1 H,  Rf:  0.10.  (shoulder). 1  H,  indolic N-H),  ,-H), 2 C  7.11  (t,  J  183  =  7.0 Hz,  1 H,  -H), 1 C 1  6.81  C — 2 H),  4.65  (d,  14,  H,  C 6 H),  3.47  (broad s, 14 Hz,  1 H),  (s,  3 H).  MS  (150°C)  (0.6),  326  =  H,  1  Cs,  1 H),  J  m/z  J  =  (relative  Caic.  3.38 J  1 H),  4.28 (d, -  3.70  1 H),  intensity):  1.02  440  5.78 Cs,  J  (m,  1 H),  14 Hz,  =  14 Hz,  130 (20.5),  High resolution MS:  7.0 Hz,  =  C — 5 H),  2 C 3 CH — O ),  (d,  (1.0),  J  1 H,  (broad d,  2.07  1.75  (d,  =  7  14,  1 H,  H),  2.34  1.90 (d,  (s,  (M,  1  J  =  0.93  3 H),  4.7),  353  (100).  83  for 2 N C 2 H S 4 0 4 8 :  440.1770.  Found:  440.1763.  3.18. EXOCATHARANTHINE 89  H  In  a  thermometer, rubber The  1000 mL a  was  followed  a  equipped  magnetic  with  nitrogen  containing  catharanthine  3  0.05%  (1509 mg).  with  stirrer  10% palladium on carbon  flushed  (600 mL) by  flask  condenser,  septum was placed  (spectro grade) added  three-necked  ref lux  apparatus  3 -CH  and  and  a  (700 mg). toluene  thiophene The  a  was  reaction  vessel was attached to a hydrogenation apparatus through the top  of  the  reflux  hydrogen without  condenser,  stirring.  The  evacuated  and  refilled  with  reaction was heated to 70°C  184  and stirred at a moderate rate for 3 hours. was  filtered  toluene  and  the  (2 x 100 mL).  product  was  filter  washed  was  with  hot  The solvent was removed and the crude  purified  hexanes/ethyl  paper  The hot solution  flash  by  acetate/triethyl  Compound 89 (1249 mg,  chromatography  amine  10:10:0.6  using eluent.  as  83%) was obtained as a white solid.  shoul be noted that compound 89  It  a mixture of the E and  is  the Z isomer in a typical ratio of 5:1.  Physical data of 89. TLC (silica,  N 10:10:0.1) Rf: 3 hexanes/acetone/Et  UV 3 (CH C N) Xmax (nm): IR (KBr)  223  Vmax (cm):  H stretch),  2935  (4.49),  232  241  (3.89),  2845  (3.84).  ),  3370 (indole N-H stretch  (C-H stretch),  0.47.  3050 (=C  (C-H stretch),  1750  (ester C0 stretch). NMR (300 MHz, 7.50 (d,  J  H),  (t,  7.16  10 C -H), H), 3.41 -  (m,  8 Hz, J  (m,  J  3 H,  1 H,  O), 3 CH  2.32 J  7.25  1 H,  (d, 7.07  3.57  -  -  (t,  J  =  12 C -  8 Hz,  1 H,  -H), 5 C  1 H,  21 C 3.22  -H), 3 C  14 Hz,  =  1 H,  1 H,  (s,  1 H,  3.16 Cm,  2.78 (dt,  J  4.00  (m,  indole N-H),  8 Hz,  J  19 C -H),  3.08  -H), 6 C  1 H,  7.65 (s,  11 C —H),  3.43  -H), 6 C  -H, 3 C  (d,  -H), 9 C  6 Hz,  =  -H, 5 C  2 H,  1.81  1 H,  8 Hz,  =  (q,  2 H,  17 C -H),  14 C —H), 3 H,  5.36  3.70 (s,  3.05  1 H,  =  ) 8(ppm): 3 CDC1  J  =  2.90 2 Hz,  (broad s,  2 H,  15 C -H),  2.16  (broad s,  14 Hz,  1 H,  17 C —H),  1.56  (a,  =  J  =  -  1 H,  6 Hz,  18 C -H).  MS (150°C) m/z  (relative intensity 10%):  (M,  (19.3),  100),  335  321  (M  —  , 3 CH  337 (23.6),  6.2),  277  336  (12.5),  249  185  (10.4),  214  (43.3),  195  (12.9),  (12.0),  154 (22.7),  122  (46.6).  Elemental analysis: N:  8.33.  3.19.  Found:  C:  Caic. 75.17,  170  (11.6),  for 2 N C 2 H : 0 1 4 H:  7.20,  N:  168  (20.2),  74.97,  C:  167  H:  7.19,  (500 mg,  1.49  8.26.  EXOCATHARANTHINE N-OXIDE 90  0  3 CH  To mmol)  a  in  dry  argon was mmol) the  in  solution  of  exocatharanthine  dichioromethane  (5 mL)  cooled  added m-chloroperbenzoic acid one  solvent  portion. was  After  evaporated  stirring  off  at  89  to  (98%) for  10°C.  20 The  -30°C  under  (265 mg, mm  at  1.46 -30°C  residue  was  dissolved in ethyl acetate and loaded on a column (3 cm wide and 8 cm long) acetate. ethyl  made of  Elution  silica gel  successively  acetate/methanol  acetate/methanol  (8:2)  (40 g)  with  (9:1)  (200 mL).  ethyl  Compound  Physical data of 90: (vacuo):  acetate  (200 mL)  was obtained as a white solid.  M.p.  suspended in ethyl  170-171°C 2 OH/Et 1:4) 3 (CH 0  90  (50 mL),  and (370 mg,  ethyl 71%)  186  UV (CHCN)  (nm)  shoulder),  (log E):  280 (3.89), ): 1 vmax (cm  stretch),  2950 (C-H stretch),  3000  ‘H NMR (400 MHz,  CD O 3 D)  H),  8 Hz,  (d,  -H 1 C 0 or 5.69 -  H),  J  =  (m,  3.04  14 Hz,  =  -H), 1 C 1  (q,  4.02  J  3 H),  1 H),  1.68  MS (200°C) m/z  Ct,  1 H,  7.46  J  2.30  J  7.10 1  4.30  2.51  -  Cm,  J  1 H,  8 Hz,  =  9 C 1 H,  11 C -H 1 C 0 or -H),  H,  —H), 2 C 1  1 H,  (s,  3 H,  8 Hz,  =  (t,  CH O 3 CO-),  7.2 Hz,  =  Cd,  Hz,  8  =  3 H,  Cs,  J  -H), 1 C 2  —H), 1 C 9  2 H),  (a,  1740 (ester C=0 stretch).  5(ppm):  3.67  3.20 (m,  -  0 and indole N-H 2 3500 (H  -  1 H,  7.04  7.2 Hz,  274 (3.87,  (4.57),  2.89 (3.81).  IR (KBr)  7.29  221  3.30  3.45  -  3 H),  1.75  3.92 (m,  (d,  2  J  =  -H). 1 C 8  (relative intensity 10%):  352 (M,  3.0),  337  (12.3),  336  (52.9),  334  (13.3),  277  (12.9),  249  (13.6),  235  (11.9),  229  (18.4),  218  (12.2),  214  (33.4),  205  (13.1),  182  (17.6),  170  (33.5),  169  (10.3)  (17.0),  167  (19.7),  156  (10.2),  154  (30.2),  141  (11.0),  122  (65.3),  121  (10.5),  86  (24.9),  (26.6),  51  (30.3),  (100),  43  (27.7),  (21.5),  139  (64.9),  127  (10.0),  123  (11.3),  113  (11.1),  111  (38.1),  108  84  (43.1),  77  (11.7),  75  (20.3),  73  50  (16.9),  49  (78.2),  47  (12.6),  44  42 (15.2),  High resolution MS:  168  Caic.  41  for  (27.1). N C 2 H : 3 0 2 1 4  352.1787.  Found:  352.1779. Elemental 67.29,  H:  analysis: 7.01,  N:  Caic.  7.47.  for  Found:  N C 2 H , 3 0 2 1 4 C:  67.37,  H:  0: 2 1.25 H 7.15,  N:  C:  7.19.  187  3.20.  191 ,20’-ANHYDROVINBLASTINE 91 and epi-19’ ,20’ANHYDROVINBLASTINE 94  3  H Pc  3 CH  3 CH  I 3 CH  3 CH  94  3 CH  91 A 50 mL three-necked flask, and  cooled  stirrer,  down  a gas  under  argon,  bubbler,  a  dried in the oven overnight equipped  was  thermometer  with  and  a  magnetic  a glass  stopper.  To a stirred solution of exocatharanthine 89 mmol) acid 30°C  (100 mg,  0.297  in dichioromethane (1 mL) was added m-chloroperbenzoic (54 mg,  (96%) under  temperature temperature formation  argon.  was  of  kept  Et N 3 ,  1.5 mL/min).  phase  0.300 4  of at  had  , 1 C 8  in one portion,  reaction 5 -10  exocatharanthine  (reverse  vindoline  The  increase  HPLC  (137 mg,  0.297 mmol),  exothermic  was  to  10°C  to  -15°C  N-oxide  0 2 MeOI-{/H  -20 to  at  observed.  was for 90  10 was  23:77  and  mm  -  a The the  and  monitored  containing  by  0.3%  After cooling to -30 to -40°C vindoline 4  mmol)  was  dissolved  added the  and trifluoroacetic anhydride  as  a  mixture (0.2 mL,  solid.  When  all  the  was  cooled  to  -78°C  1.42  mmol)  was  added  188  in  one  portion.  The  reaction  exothermic  very  was  and  the  temperature rose 10 to 15°C within 10 to 15 sec. After 3 mm the  temperature  followed  had  by HPLC  returned  and  kept  -78°C.  to  at  -78°C  until  complete according to MPLC (that is; disappeared,  and  the  peaks  The  reaction  was  coupling was  the  the N—oxide 90 peak had  corresponding  to  acetylated  N-  oxide 99 and the iminium salt 100 respectively remained the same  (hight  apart).  wise)  in  two  successive  The reaction time was 3  -  samples  taken  30  mm  4 hrs and the color of the  reaction changed from yellowish to deep red within the first hour.  The  bubbler  thermometer  was  an  by  replaced  adapter  a  by  fitted  stopper  with  a  and  the  stopcock.  The  reaction vessel was then connected to a vacuum line and the solvent the  evaporated  cooling  for  bath  5  was  mm  at  allowed  -78°C. to  The  rise  temperature  to  -40°C  and  evaporation continued until a sticky foam had formed. pressure cooling (1 mL)  bath was  allowed until mm. any  was  to  the  restored was  lowered  added. rise  The  to  sticky  with  foam  the  solvent  evaporation  is  —60°C  and  dry  and  had  on  the reaction vessel was and  temperature  temperature  -40°C  sitting  The  to  the  kept  swirled sides.  in  of f order  the  Normal of  the  dichioromethane  cooling this  at  which  bath was  temperature  took  about  15  in order to wash down When  again connected  evaporated  necessary  was  the  of  dissolved,  The reaction vessel was material  argon.  of  all to  had  dissolved  the vacuum line  at  -40°C  to  remove  (this the  second  remaining  traces of trifluoroacetic anhydride). When a sticky foam had  189  once again  30°C  and  formed the temperature was the  additional a  dry  reaction  15 mm.  collapse  Normal  pressure  give  very  viscous  then  was  the cooling bath removed. to  temperature  this  at  -  an  for  The foam should at this point have quite  appearance.  argon and  kept  allowed to rise to  foam should  The  oil,  restored  barely  with  slowly  stirable.  1  hr  after removing the cooling bath the oil was dissolved in dry dichloromethane  (1 mL)  methanol  was added and the temperature was restored  to  (15 mL)  -25°C.  An  borohydride period of 3  tended  orange-red  (115 mg,  the  solution  The  The  The  reaction  evaporator dried  mixture 20  further  on  foam  which  was  and  vacuum  (10 mL)  yellow  the  -  dryness  to  yellow  and  35°C.  hrs gave  the  give  was  dissolved  10  to in  a  degassed  This solution was washed  (the pH of the washings fell from >10  cake  was  washed  organics  Further drying on  crude  rotary  mm  subsequently  -  on  obtained  5  the combined  vacuo at  pH  residue  for  filter  the  10°C in 5 mm.  dried over magnesium sulfate for 5  The  and  -  and  exothermic  line  with water (3 x 10 mL)  filtered.  Sodium  The reaction was kept at -20 to  (with argon) ethyl acetate (30 mL).  to 8 to 7),  was  became  evaporated  25°C  -  a  Degassed  obtained.  and was then heated up to 8  at  yellow  -25°C.  was  reaction  solution  increased to greater than 8. -25°C for 5 mm  to  the temperature being kept at -20 to  addition.  foam.  to  cooled  304 mmol) was added in portions over a  4 mm,  -  during  25°C  and  product  with  evaporated a vacuum  (255 mg)  as  a  -  10 mm  ethyl  and  acetate  to dryness in  line  for  yellowish  2  to  4  foam.  190  This  material  was  hexanes/acetone/  dissolved  (10:10:0.6)  and loaded onto  14 cm long)  packed with silica gel  with  the  above  solvent  triethylamine  a flash column (4.5 cm wide and  system  (230  at  flow  a  gave t 19’,20 anhydrovinblastine 91  400 mesh).  -  of  rate  (165 mg,  Elution mL/min  5  70%).  Physical data of 91: TLC UV  (silica, (CH C 3 N)  stretch),  8.00 H),  7.08  6.59  (s,  5.85  (dd,  Hz, 1  1 H,  H,  (s,  1  212  258  (4.69),  (4.17),  288  J  CDC1 ) 3 :  N-H), (m,  10 Hz,  ,—H), 1 C 9  1  H,  2.73  (s,  (m,  1 H),  (s,  3  H),  1.68  J  (s,  3  H,  Hz,  3  H,  J  2950  (C-H  (m,  6.5 Hz, 1 H),  2.67  (m,  2.00  6  =  2  (s,  H),  2.08  -  3 H,  0.81  (t,  1 1  H), H,  2.14 (m,  1  -H), 1 C 81 J  =  (q,  (d,  (s, 3.17 2.77  2.22  H),  -  8 Hz,  —  3 H,  J =  6.5  10 Hz,  3 C 2 CO ) , H (m,  9  2.89  (m,  2  2.39  -  =  3.52  -  (m,  1.72  1.19  J H,  3  —H), 2 C 1  -  -H), 1 C 11  —H or 9 C 5.49  OCH ) 3 ,  Hz,  -OH), 1 C 6  —H, 1 C 01  5.30  3.80  H,  ,—H or C 9 C ,1 2  1 H,  -H), 1 C 7  3 H,  H,  -H), 1 C 4  3 C 2 CO ) , H  J  1  (s,  1 H,  (s,  CH N 3 ), 2.39  -  =  15  6.13  1 H,  (s,  8 Hz,  =  4 Hz,  =  J  2 C 3 CH ) , O  1.12  Cg,-H or C ,-H, 1 2  H,  J  1  (s,  3  3.62  =  9.82  (d,  -H), 2 C  2.29  (d,  stretch),  7.50  5.46  3.83  H),  (ppm):  8  —H or C 9 C —H), 1 2  =  N-H  (ester C=0).  1 H,  (dd,  -  (indole  7.19  -  3.04  1.02  3467  H,  H),  H,  (cm) 1 :  MHz,  -H), 1 C 5  3.74  E):  1741  (400  (s,  (log  0.26.  4.07).  (KBr)Vmax  NMR  (nm)  Xmax  (shoulder, IR  N 10:10:0.6) Rf: 3 hexanes/EtOAc/Et  1  H),  1.90  1.40  -  (m,  -H). 1 C 8  2.49 2.11 (m,  4  2 H),  191  MS (260°C) m/z (relative intensity 5%): 806  (M  791  (6.5),  1.8),  14,  +  (7.9),  525  (24.3),  (6.2),  (12.8),  524  (6.6),  (7.0),  , 3 CH  -  732 469  2.2),  28,  +  792  (M,  2.4),  761  (M  (7.9),  633  (21.1),  793  (M  777  733  (9.5),  335  794  (9.4),  790  732  (5.2),  1.8),  820 (M  42.5), , 3 OCH  -  (6.8),  631  (10.9),  337  (11.7),  336  282  (20.9),  278  (5.6),  251  323  (7.1),  283  (5.0),  265  (6.1),  263  (6.1),  (17.6),  214  (14.8),  202  (8.4),  200  (5.2),  277  (5.8),  250  (5.8),  194  (5.0),  188  (8.6),  185  (5.2),  182  (5.5),  170  (7.8),  169  (5.2),  168  (8.2),  167  (5.9),  156  (6.1),  154  (8.8),  144  (12.0),  137  (8.9),  136  (14.9),  123 109  (7.7),  249  (9.0),  143  (5.0),  138  (7.7),  130  (6.6),  (55.6),  135  (54.8),  134  (9.7),  (18.0),  122  (56.0),  121  (22.6),  (6.4),  108  (7.4),  91  (6.7),  (5.7),  44  (100).  Elemental H:  7.47,  (19.4),  107  82  (5.4),  79  Caic.  for  analysis: N:  6.56.  Found:  Cab,  for 4 N 4 C 5 H , 8 0 6  6.35.  Found:  C:  (21.1),  4 S 2 2H : 0  52.97,  H:  124  120  (7.2),  110  (5.3),  106  (11.1),  93  (14.8),  92  (6.0),  55  (7.8),  77  (7.7),  N 4 C 5 H , 8 0 4 6  68.45,  C:  252  C:  6.33,  H:  N:  CH O 3 H: N:  7.28,  52.62,  H:  5.37,  67  6.79. N:  6.50,  5:  68.62,  C:  5.26,  S:  6.14.  Physical data of 94: TLC (silica,  3 hexanes/EtO N Ac/Et 10:10:0.6) Rf:  (CH C N) ‘Xmax (nm): UV 3 ‘H  NMR  (400  7.36  (d,  1  Cg,—H  H,  J  =  MHz, 8 Hz, or  214,  CDC1 ) 3 : 1 H,  -H), 1 C 21  261, 6  291.  (ppm):  9.05  —H 9 C ,-H), 1 1 or C 2 7.10  0.34.  (t,  J  =  8  Cs,  1  7.25 Hz,  1  -OH), 1 C 5  H,  8 Hz,  (d,  J  H,  ,—H 1 C 0  =  or  192  11 C ,—H), 1 H, J  6.99  (t,  10 Hz,  J  =  5.44 (s,  H),  Cs,  3.85 O), 3 CH J  Cd,  =  1 H,  3 H,  2.60  17 C -H),  H,  3  1 H,  12 C -H or -H), 9 C  1 H,  14 C -H),  CO 3 CH ) 2 ,  (s,  6.5 Hz,  1 H,  11 C 10 or ,—H), C ,—H  1 H,  6.02 (s,  4 Hz,  191 C —H),  8 Hz,  =  Cg-H or -H), 12 C  =  H,  J  5.51  (q,  5.26 (d, 3 H,  3.78 (s,  N), 3 CH  2.08  18 C ,-H),  J  0.57  J  5.88 (dd,  6.5 Hz,  1 H,  1 H,  15 C —  10 Hz,  =  CO 3 CH ) 2 ,  Cs,  Ct,  H,  3 J  6.96 (s,  Cs, 3  3.76  1.66  CO 3 CH ) 2 ,  8 Hz,  3 H,  18 C -  (M  14,  1.0),  777  (M  H). MS (260°C) m/z  (relative intensity 5%):  794 , 3 CH  (5.5),  793  (17.9),  1.4),  761  (M  -  792  (M,  , 3 OCH  33.3),  1.3),  733  790  +  (5.5), 732  (8.5),  716  (14.1),  524  (5.4),  249  (7.6),  631  (5.6),  (8.9),  337  (7.5),  336  (13.6),  282  (15.9),  (6.0),  222  (5.7),  (6.2),  202  (5.5),  200  (5.5),  188  (5.3),  187  (6.1),  156  (48.4), (8.7),  144  135  (58.9)  122  121  (40.9),  (22.8),  107  (10.0),  91  (23.4), (8.6),  170  (5.7),  168  (5.9),  157  (10.1),  138  (7.2),  137  (7.7),  136  (6.0),  124  (7.4),  123  (13.7),  109  (6.2),  108  93  (16.3),  134  ,  (7.9),  (6.0),  79  (7.7),  60  (7.3),  58  (5.7),  53  (5.2),  51  (7.8),  45  (10.5),  resolution  792.4088.  MS:  Caic  120  (25.7),  65  High  130  (32.4),  106  82  272  (6.6),  174  (6.3),  525  (7.2),  633  (11.9),  (5.2),  —  (5.3),  214  526  806  for  105  (5.1),  (13.7), 57  (5.8),  77 56  92  (13.6),  67  (7.7),  (5.2),  55  (8.2),  44 (100). 45 C 5 H : 8 0 4 N 6  792.4098.  Found:  193  3.21.  7-HYDROXYCATHARANTHINE N-OXIDE 92  0  HO  CH 2 CO 3 To a solution of catharanthine 3 dichioromethane  (5 mL)  Chloroperbenzoic  was  acid  cooled  (420 mg,  to  2.38  (400 mg, -20°C mmol)  1.19 mmol)  under was  in  m  argon.  added  in  portion and the reaction was stirred for another 20 mm  one at  -  20°C before the solvent was evaporated off at 0°C in vacuo. The  residue was  column  (3  dissolved  cm wide and 8 cm long)  suspended in ethyl acetate ethyl  in ethyl  (50 mL),  acetate.  ethyl  the  Compound 92  and  loaded on  silica gel  a  (40 g)  Elution successively with ethyl  (8:2)  desired material  (318 mg,  made of  acetate/methanol  acetate/methanol  containing  acetate  (9:1)  (200 mL). was  (200 mL)  The  evaporated  and  fractions off  at  0°C.  70%) was obtained as a white solid.  Physical data of 92. M.p. UV  (in vacuo):  (MeOH)  ‘Xmax  137 (nm)  138°C (EtOAc/MeOH/Et 0 7:1:3). 2  -  (log  E):  221  (4.27),  266  (3.75),  281  (3.73). IR  (KBr)  stretch),  Vmax 2960  (cm):  3590  (C-H stretch),  (free 1745  0-H  stretch),  3360  (ester C=0 stretch).  (0-H  194  NMR (400 MHz,  OD), 3 CD  1 H,  —H or -H), 9 C 12 C  7.38  (t,  1  10 or -H), C -H 11 C  H,  21 C -H), 3.70  J  4.39  3.02  Cm,  2 H),  (t,  3 H,  2.00  —  6.15 1  Cm,  H,  8 Hz,  =  (d, 2.99  Cm,  1  H,  1 H,  1 H),  J  7.32  3.80  (broad s, 1.75  —  J  J  Ct,  8 Hz,  =  1.95  1  Cs, Cm,  1 H), 1 H),  8 Hz,  =  5.26 3.92  -  12 Hz,  =  Cd,  Cg-H or -H), 12 C  15 C -H),  -OH), 7 C  3.65  2 H),  2.15  J  10 or -H), C -H 11 C  s,  Cm,  (d,  7.48  1  3.32 2.34  (m,  H, H),  Cm,  1  2.55  -  2 H),  1.10  18 C -H).  (200°C)  relative 3.4),  1 H,  OCO), 3 CH  3.12  -  7.41  (broad  3 H,  Cs,  H),  MS  8 Hz,  =  400 MHz) 8(ppm):  m/z  (relative  intensity  350  (M  -  intensity 10%  20%)):  0, 2 H  (M,  368  9.0),  194  (from m/z 352  37.6),  (28.8),  188  91  200  -  (M  -  (27.4),  0, 188  (32.0),  175  (22.4),  174  (32.0),  173  (25.3),  171  (24.0),  164  (20.8),  163  (21.9),  160  (37.1),  158  (22.1),  157  (32.0),  156  (51.2),  155  (20.8),  146  (28.8),  135  (73.6),  133  (48.0),  130  (41.6),  129  (25.9),  128  (21.1),  119  (32.0),  117  (25.6),  107  (43.2),  105  (29.6),  91(100).  (54.4),  Elemental analysis: H:  6.37,  N:  7.24.  103  :  (31.2),  Calc.  Found:  C:  102  (21.0),  93  for 2 N C 2 H , 4 0 1 4  0: 2 1 H  65.22,  N:  H:  6.92,  (25.6),  C:  7.06.  92  65.20,  195  3.22.  7-HYDROXY-EXOCATHARANTHINE N-OXIDE 93  0  HO  3 CH  To mmol)  solution  a  in  dry  argon was mmol) the  in  column  one  ethyl  (3  portion.  After  evaporated  dissolved  cm wide  and  in ethyl  (50 mL),  in  the  (5 mL)  the  dark.  off  long)  acetate.  ethyl  desired Compound  at  for  room  1.19  (400 mg, to  -30°C  under 2.49  (450 mg,  (96%) 20  mm  -30°C  at  temperature.  acetate  and  made  silica gel  of  loaded  The on  a  (40 g)  Elution successively with ethyl  acetate/methanol (8:2) material 93  89  cooled  stirring  ethyl  8 cm  acetate/methanol  containing in  dichioromethane  was  was  suspended acetate  exocatharanthine  added nz-chloroperbenzoic acid  solvent  residue  of  (200 mL). was  (298 mg,  (200 mL)  (9:1)  68%)  was  fractions  The  evaporated  and  off  at  obtained  10°C as  a  white solid.  Physical data of 93: M.p. UV  (vacua): (MeOH)  (4.31),  231  181 -184°C  ‘>\max  (nm)  (shoulder,  2 (EtOAc/MeO 0 H/Et 4:1:5). (log  4.20),  E): 270  218  (shoulder,  (3.67).  4.26),  223  196  IR  V  (KBr)  stretch),  (cm):  2950 (C-H stretch),  NMR (400 MHz, or C -H), 1 2 J  8 Hz,  =  1 H,  1 H,  3 H,  OCO), 3 CH  Hz,  1 H),  2.30  MS  8 Hz,  5.50 (broad s,  Cs,  Cm,  =  1 H),  -OH), 7 C 3.26  2.63  —  1.61  (200°C) m/z  1 H,  Cm,  Cd,  J  3.76 Cd, Cm,  293  (15.2),  188  (27.3),  186  (12.4),  160  (33.4),  157  (10.1),  156  145  (10.4),  136  (57.8),  132  (12.4),  130  121  (18.9),  108  (66.0),  High  =  352  (26.2),  351  1 H,  J  =  C 9 H  7.39  1 H,  1 H,  (s,  3.18  (d,  3 H,  (M  8 Hz,  11.2 Hz,  =  (d,  -H 1 C 0  -H), 2 C 1  2 H),  (d,  J  3.65 15.2  =  11.2 Hz,  1 H),  —H). 1 C 8  (relative intensity 10%):  7.1),  —  J  2 H),  7.2 Hz,  =  8 Hz,  =  5.05  J  2.00  , 3 CH  (M  (t,  -H), 1 C 9  4 H),  J  11 C -H 1 C 0 or -H),  7.33  3.35  -  (0-H  3360  (ester C=0 stretch).  7.47 (d,  1 H,  C 9 H or C -H), 1 2  (broad s,  1.85  J  stretch),  0-H  1742  ) 6(ppm): 3 CDC1  7.40 (t,  or C -H), 1 1 4.21  (free  3600  368 (M,  2.0),  353  4.4),  335  (11.2),  164  (11.6),  163  (10.3),  (11.7),  149  (11.1),  146  (15.0),  135  (20.7),  134  (13.8),  133  (32.7),  (23.5),  129  (10.1),  123  (12.7),  122  (15.8),  120  (20.8),  119  (17.5),  117  (13.7),  109  (15.2),  107  (21.2),  resolution MS:  106  Caic.  (22.2), for  OH,  —  105  (35.7).  N C 2 H : 4 0 2 1 4  368.1736.  Found:  368.1738. Elemental analysis: H:  6.77,  N:  6.92.  Calc.  Found:  for 2 N C 2 H , 4 0 1 4 C:  63.79,  H:  1.5 2 H 0 :  6.83,  N:  C:  7.09.  64.12,  197  3.23.  4’ -BENZYL-19’,20’ -ANHYDROVINBLASTINE 108  3 cl-I  Br  H  19’,20’-Anhydrovinblastine dissolved in dry benzene (2 mL, the  16.8 mmcl)  dark  at  solvent  was  added  and  homogeneous.  room  was  (10 mL)  the  at  40°C  overnight. in  resulting  vacuo.  (10 mL),  Compound 108  was  stirred in  next  Hexanes  suspension  mmcl)  Benzyl bromide  The  day  the  (10 mL)  was  stirred  The resulting precipitate was  (10 mL).  0.675  under argon.  washed successively with hexanes and hexanes  (535 mg,  added and the reaction was  temperature  removed  94  until  filtered off and  benzene (2 x 1 mL)  (513 mg,  79%) was obtained  as white solid.  Physical data of 108: UV 3 (CH C N) ‘Xmax (nm): IR  (KBr)  stretch),  Vmax 2820  (cm):  216, 3360  269,  309.  (indole  (C-H stretch),  1715  N-H  stretch),  (ester C=0).  2880  (C-H  198  NMR (250 MHz,  6 (ppm):  DMSO-d ) 6 :  173.58,  170. 13,  171.14,  157.60,  152.98,  135.71,  133.49,  131.38,  130.46,  129.03,  128.59,  128.31,  127.91,  126.30,  123.83,  122.06,  119.58,  118.90,  117.94,  70.42,  64.00,  62.34,  60.83,  57.80,  56.35,  54.83,  52.94,  52.50,  51.77,  49.86,  49.25,  44.86,  42.50,  42.21,  37.90,  37.00,  3.24.  30.86,  112.03,  20.96,  111.35,  94.31,  13.24,  19.38,  82.08,  79.47,  75. 90,  7.84.  19’-HYDROXY VINBLASTINE 109 and 19’-HYDROXY LEUROSIDINE 110  3 H H  nc  3 CH 3 CH . 3 CH  CH 2 O 3  110 109  Compound  108  (513 mg,  tetrahydrofuran/water (94 p1, (298 mg,  1.16 mmol) 1.17  aluminium foil. (20 mL)  was  (1:1)  was  mmol).  0.532  (10 mL)  added,  The  mmol)  was  under  dissolved  argon.  reaction vessel  hydrogen  Pyridine  followed by osmium tetroxide was  covered  After stirring for 1 hr and 30 mm.  added and  in  sulfide  bubbled  with  Methanol  through the  199  reaction mixture for 15 mm. at  40°C  under  residue  dried  mixture  of  (7 mL)  washed  heated  with  was  dichloromethane  and  silica  loaded  on  the  eluted  (10:10:0.6)  (1.5  1)  (20:0.3)  (500 mL)  at  (160 mg,  36%) and compound 110 (60 mg,  and a  mm.  30  black  a  The cake  filter of  the  dissolved  was  column  initially  with  and  suspended  in  trie-thylamine  Evaporation  a  black  suspended  dry  which  to  (17 g)  was  hrs  and  residue  gel  2  resultant  the  and  (10 mL).  brown  a  for  filtered  methanol  solvents left  (7 mL)  ref lux  to  and  residue  The  acetonitrile  mixture  TLC grade  pressure  vacuo.  in  dry  and  reaction  reduced  The solvent was evaporated off  (3 cm in  in of  wide)  hexanes  and  hexanes/acetone/triethylamine then  with  acetone/triethylamine  flow rate of  19 mL/min Compound 109 14%) were obtained as  white solids.  Physical data of 109:  TLC (neutral alumina, (silica, (CH C 3 N)  UV  288.6  3 C CH / 2 O CH 1 20 : H  3 acetone/hexa N nes/Et 10  ‘>max  (4.06),  (nm)  296.0 (4.05),  IR (KBr) umax (cm): 2971  (CH stretch),  ‘H NMR J  H,  =  (400 Mz,  8 Hz,  1 H,  (log  3469  2586,  ) 3 CDC1  E):  :  214.8  1) Rf: 10  :  0.6) Rf:  (4.70),  306.2 (shoulder) (OH stretch),  2472,  0.40.  261.4  0.11. (4.22),  (3.95).  3009  (=CH stretch),  1741 (ester C=O stretch).  o(ppm):  8.05  C , 9 -H or C -H), 1 27  -H 1 C 11 and 9 C , -H or C -H), 1 21  (s,  7.09 6.65  -  Cs,  1 H,  N-H),  7.20  (m,  1  H,  7.54  3 H,  Cd,  ,1 C 0  C 9 H or C 1 2  200  H),  6.11  (s,  Hz,  1 H,  —H), 1 C 4  H,  _H), 1 C 5  3.73  CH O 3 -),  H,  3.41  (q,  5 H),  3.13  Hz,  1 H),  2.80  Cs,  1 H),  2.64  J  14  =  2 C 3 CH ), O H),  1.33  Hz, 1.75  (q,  H,  ,—H), 1 C 8  MS  (260°C)  (12.0),  5.48  826  J  J  1 H,  Cs,  J  3.65  (broad d, 2.90  —  1 -  (M,  H),  2.36  1.85  (m,  J  2 H,  (relative  1  H),  3.62  3 H,  2.49  Cm,  2 H),  (m,  1.40  1  3 H,  (7.6),  H),  1.12  (s,  J  CH N 3 -), 2.29  J  (d,  1  3.40 14  =  2.67  (broad  (s, =  J  4  H,  3 -  (d,  2.11  (broad d,  =  OCO-), 3 CH  3.25  3.06  Cs,  J  10 Hz,  =  6 H,  2.71  H,  3  14 Hz,  2  6 Hz,  3  =  —H). 1 C 8  intensity  824  J  (s,  2 H),  -H), 1 C 9  6 Hz,  =  8.0),  —  7 H),  7.2 Hz,  3.81  (m,  2.22  -  10 Hz,  =  5.30 Cd,  12 Hz,  =  J  -H 1 C 91 (COSY)),  2 H),  2.16  0.82 (t, m/z  J  H),  =  3.72  1 H,  (m,  1  (s,  1 H),  —  6 Hz,  =  5.85 (dd,  -H), 1 C 7  14 Hz,  =  -H), 2 C  (m,  d,  C 9 H or —H), 12 C  3.96 (t, 1  (s,  1 H,  841  5%):  795  (7.0),  (6.6),  840  (6.2),  767  (14.5),  341  768  (7.8),  766  (5.2),  327  (5.7),  325  (6.5),  313  (5.1),  311  (7.0),  310  (5.1),  309  (5.6),  299  (5.1),  298  (5.0),  297  (8.4),  295  (5.4),  284  (6.0),  283  (6.1),  282  (18.3),  272  (6.9),  240  (5.3),  222  (9.4),  215  (5.2),  214  (10.9),  212  (5.2),  202  (7.8),  200  (8.7),  188  (12.5),  (6.3),  183  (5.0),  172  (7.1),  168  (8.7),  157  (6.3),  765  (8.1),  (10.0),  171  (16.0),  170  (16.9),  156  (10.5),  154  469  (100),  (10.5),  184 169  371  (7.7),  152  (7.6),  144  (11.3),  138  (6.4),  136  (15.2),  135  (48.9),  134  (5.1),  124  (18.9),  123  (6.4),  122  (26.7),  121  (20.5),  110  (6.5),  108  (13.9),  107  (19.4), 84  (5.1)  106  (5.7),  83  (5.5),  94 82  (5.4), (8.6),  93 81  (13.2),  92  (5.6),  91  (5.1),  (5.8),  70  (7.3),  60  (6.2),  201  58  (24.4),  (5.4), High  45  44  (8.0),  30 (6.6),  28  (40.7),  43  (27.7),  42  (12.7),  41  (9.4).  resolution MS:  Calc  for N : 4 C 5 H 1 0 4 6 8 0  826.4153.  Found:  for N , 4 C 5 H 1 0 4 6 8 0  C1 C: CH : 2  61.94,  826.4171. Elemental analysis: H:  6.64,  6.10,  N:  Cl:  Calc.  6.15,  Cl:  7.79.  Found:  61.95,  C:  H:  6.79,  N:  7.50.  Physical data of 110: PLC (neutral alumina, (silica, UV  3 C CH / 2 O CH 1 20:1) Rf: H  0.2  N 10:10:0.6) Rf: 3 acetone/hexanes/Et  CN) ‘>\max 3 (CH  (nm)  (log  E):  215.0,  263.0,  0.00  288.2,  295.6,  307.2 (shoulder). IR (KBr) Vmax (cm): OH  stretch),  2635,  1741  3010  8 Hz,  =  H,  ) 3 CDC1  1 H,  6.13 (s,  Hz,  1 H,  (s,  O-), 3 CH H),  2.15  1 H,  8(ppm):  8.08  1  H,  3.27  2.45 -  (CH  2.25  -  (s,  J  3.44 =  6.43  3360 (broad,  stretch),  2879,  (m,  (m, 1  H),  4  1 H), 2.11  7.27  (m,  1  (s,  H,  7.47  3 H,  (d,  107 C -  -H or 9 C 12 C  J  10 Hz, J  17 C —H),  5.35  (d,  J  H),  1 H),  N-H),  (dd,  3.81 (m,  3.78  —  -  1 H,  5.91  OCO-), 3 CH  3.64  18 Hz,  2.55 (m,  H,  3  -H), 2 C -  1 H,  (s,  (s,  7.13  -H or -H), 9 C 12 C 5.46  3.81  2.83 (d,  1 H),  2970  ,-H or -H), 9 C 127 C  14 C -H),  15 C -H),  3.78  01-I stretch),  stretch),  111 and -II C —H 97 or -H), C 121 C  H),  H,  (CH  (sharp,  (ester C=O stretch).  ‘H NMR (400 Mz, J  3465  3.15  Cs,  H),  3 H,  (broad d, 3  H,  3.64 J  =  N-), 3 CH J  =  2 C 3 CH O ),  4  10 Hz,  1  OCO-), 3 CH  3 H,  (broad d,  2.75 (s, 2.39  5  (s,  =  =  (s,  3  12 Hz,  H, 1  2.65 (s,  14 Hz, 1.87  1 H), -  1.98  202  (m,  1 H),  (q,  J  H),  1.11  1,72  7.2 Hz,  =  (d,  J  1.87  —  2 H, =  (m,  3 H),  19 C —H),  14 Hz,  1.54  1.16  1 H),  (d,  0.80  1.65  -  J  (t,  2 H),  6 Hz,  =  J  Cm,  3 H,  6 Hz,  =  1.34 18 C ,-  3 H,  18 C -  H). MS (260°C) m/z (relative intensity  5%):  (6.6),  766  (5.0),  607  (5.8),  393  (5.0),  353  (5.0),  351  (8.0),  343  (5.6),  327  (5.5),  325  (5.0),  323  (5.3),  313  (7.0),  312  (5.2),  311  (9.3),  310  (15.9),  309  (7.0),  308  (5.0),  299  (6.5),  298  (5.4),  297  (11.4),  295  (5.5),  284  (7.6),  283  (8.0),  282  (18.2),  265  (5.1),  240  (6.8),  226  (5.0),  224  (5.1),  (9.7),  215  214  (11.9),  212  (6.0),  210  (5.5),  202  (9.1),  201  (5.1),  200  (13.4),  198  (6.6),  197  (6.4),  196  (5.2),  194  (5.8),  188  (13.6),  186  (7.1),  185  (7.2),  184  (5.8),  174  (7.3),  (9.9),  168  (10.2),  167  (7.5),  152  (7.0),  (28.0),  135  (47.9),  122  (15.6), (9.0),  154 136  222  172  (17.1),  123  (8.1),  (12.8),  107  (22.7),  (14.8),  92  79 65  (6.2),  (9.8), 77  (5.6),  (22.9),  44  60  (10. .0),  (100),  43  768  (5.5),  182  (6.1),  180  171  (13.8),  170  (65.3),  169  (5.5),  158  (11.0),  157  (13.0),  156  144  (26.5),  83  (6.3), 58  70  121  143  (5.2),  138  (6.2),  130  (6.9),  124  (21.4),  110  (7.2),  108  94  (20.4),  (18.1),  96  93  (5.2),  82  (8.5),  81  (6.4),  (6.0),  60  (6.2),  67  (5.0),  (37.8),  (79.4),  (14.9),  134  (10.7),  (16.3), 74  183  (8.6),  0.6),  (10.4),  106  91  (6.5),  (9.7),  826 (M,  42  55  (7.3),  (17.1),  41  (5.3),  45  (10.64),  30  50  (8.6). High  resolution MS:  826.4180.  Calc  for N : 4 C 5 H 1 0 4 6 8 0  826.4153.  Found:  203  3.25.  THE BIS-INDOLIC MESYLATE 112 FROM THE MESYLATION OF 19’- HYDROXY VINBLASTINE 109  H 3 H  flc IS  Methanesulfonyl chloride to  a  solution  (2 mL).  of  109  0.194  (160 mg,  2.58 mmol)  mmcl)  in  was  added  dry pyridine  After stirring at room temperature under argon for 3  hrs water (20 mL) was added. filtered vacuo  (200 p1,  3 CH  off  and  over  (97 mg). converted  The precipitate thus formed was  washed with water  phosphorous  (5  x  10 mL).  pentoxide gave  a  Drying in  purple  solid  On standing in chloroform this material was partly 112  to  and  attempts  on  flash  chromatography  on  silica similarly gave decomposition to 112.  Physical data of 112: TLC (silica,  3 acetone/hexa N nes/Et 10:10:0.6) Rf:  UV 3 (CH C N)’Xmax (nm): IR  (KBr)  stretch),  vmax 2750  (cm ) 1 :  214, 3360  279,  296.  (indole N—H stretch),  (C-H stretch),  1700 (ester 0=0).  288,  0.54  1730  (0=0),  1750  2850  (C-H  (ester C=0),  204  NMR J  6.10  (s,  Hz,  1 H,  -H), 1 C 4  -H), 1 C 5 3  H),  3.24  H,  3.95  2.30 1.67  -  —  2.89 (m,  6 H),  1.85  0.88  (m,  Cd,  2.10 4 H),  (d,  -H), 2 C  3.88  3 H,  3.01 (m,  3 H,  2  -  3.22  H),  -  2 C 3 CH ), O 1.48  (m,  -H), 1 C 87  (s,  1.90  1H), 0.60  J  -  =  J  =  4  10 Hz,  1  1.12 (t,  3.52  (m,  2  3.10  (s,  3  CH N 3 -),  1 H),  2.13  2.00  -  J  3.77  H,  3  16 Hz,  =  (d,  ,1 C 0  10 Hz,  H),  5  2.75  3 H,  OCO—), 3 CH  3.42  (m,  J  =  3 H,  CH O 3 -),  (broad d,  3 H,  1.39  6 Hz,  =  5.18  (s,  7.50  C 9 H or C 1 2  -H), 1 C 7  2.43 Cs,  H,  1  (s,  1 H,  3.00  -  Cm,  J  H),  1 H),  (m, J  2  7.20  (dd,  (s,  3.60  N—H),  5.86  Cs,  1 H,  (S,  3.39  -  2.75  Cm,  5.71  1 H,  -  6.47  12 C C 9 H or -H),  OCO-), 3 CH  S), 3 CH O  2.70  H),  H,  1 H,  Cs,  7.05  and Cg,-H or C ,-H), 1 2  ,—H 1 C 1  (s,  8.02  Cg,-H or C ,-H), 1 2  H),  H,  5(ppm):  ) 3 CDC1  1 H,  8 Hz,  =  H,  (400 Mz,  —  =  Cm,  1.23  -  1H), Cm,  6 Hz,  1  3 H,  -H). 1 C 8 Elemental 62.35, N:  H:  5.84,  analysis: 6.91, S:  N:  3.44.  Caic. 5.82,  S:  for  S, 4 C 6 H 1 0 4 N 7 0 2  3.33.  Found:  C:  COCH CH : 3  62.31,  H:  C:  6.80,  205  3.26.  GENERAL EXPERIMENTAL CONDITIONS FOR THE OPTIMIZATION OF THE PHOTOCHEMICAL CYCLIZATION OF THE AMIDES 40 AND 41 AND THE THIOAMIDES 51 AND 52  d  ii)  e  a  I:.  b C  4cm  Figure 3-1. Apparatus used in the photolysis. I) This apparatus was used for “high” light  intensity and was wrapped in aluminum foil. For reactions room at temperature compressed air was used for cooling. II) This apparatus was used for “low” light intensity. In both setups magnetic stirring was used and a mantle used for heating the reaction. (a) Quartz immersion well. (b) Glass filter (Corex, Vycor or Pyrex). Cc) Low-pressure mercury Hanovia lamp. (d) Stainless steel needle. (e) Condenser. (f) Quartz test tube. (g) Nitrogen inlet.  A  solution  (0.5 mg/mL)  51) or thioamide (41, 5  mm  and  Aliquots  then  52A,  of  irradiated  through  the  appropriate  amide  (40,  52B) was purged with nitrogen for using  were withdrawn every  photolysis,  the  15  stainless  the mm,  appropriate  filter.  without stopping  steel  needle  by  the  using  a  206  positive nitrogen pressure as the driving force.  The samples  were diluted in methanol and analyzed by HPLC.  HPLC conditions used for the analysis: Column:  Waters Reverse Phase Radial Pak C 8  lOp,  8 mm x 10  cm. Solvent:  2 C 3 CH 0 N/H (60:40).  Flow:  1.00 mL/min.  Detection:  254 nm.  In  the  photochemical  cyclization  of  the  amides  40  and  51 the yields of 55 and 56 were determined by HPLC using the pure lactams 55 and 56 as external standards. thioamides  41,  52A  and  the  52B  pure  In case of the  thiolcatams  61  and  62  were used as external standards.  3.27.  GENERAL EXPERIMENTAL CONDITIONS USED IN THE OPTIMIZATION OF THE YIELD OF EXOCATHARANTHINE 89  Method A: Methanol (19.3  mg)  under  with  hydrogen  mm.  The  with  argon.  was  argon.  and  reaction  added  stirred  (2.0 mL)  The  to  by  the  vessel  TLC  was 3  catalyst  argon  at  argon  catalyst  Catharanthine  under  monitored  the  added to  was  atmosphere  stirred  evacuated  (9.3  mg)  suspension  room  (silica  10% palladium on carbon  in  briefly  toluene/ethyl acetate/ MeOH 10:10:2).  vigorously and  then  methanol  and  temperature.  was  the The  exposed  replaced for  10  refilled (2.1  mL)  mixture  was  reaction  was  to  ammonia,  207  Method B: Benzene (10.0  mg)  (10.0 mL)  and  reaction  and  the  temperature. briefly  was  The  exposed  argon.  under  then  and  the by  toluene/ethyl  The with  refilled  at  monitored  was  ammonia,  mg)  stirred  was  reaction to  (10.0  3  evacuated  mixture  10% palladium on carbon  added to  catharanthine  vessel  hydrogen  was  appropriate TLC  (silica  acetate/methanol  10:10:2).  3.28. GENERAL EXPERIMENTAL CONDITIONS USED IN THE INVESTIGATION  OF THE FORMATION OF CATHARANTHINE  N-OXIDE 10 AND EXOCATHARANTHINE N-OXIDE 90. A  dry  magnetic  50 mL  stirrer,  stopper.  clear,  a  of  quality which  necked  a  bubbler,  gas  catharanthine  exocatharanthine give  three  was  perbenzoic  89  was  often  the  dissolved  yellowish  to  a  -10  (107 mg,  was  or  in  or  -30°C  0.595  and  0.594  dry  with a  on  exocatharanthine under  mmcl)  argon.  was  or  (2 mL)  (depending  a  glass  mmol)  solvent  solution 3  equipped  thermometer  (200 mg,  catharanthine  cooled acid  3  flask  to the  89),  m-Chloro  added  in  one  portion  (it  is crucial  for the reproducibility that all the  peracid  is  introduced  into  quickly  as  possible).  temperature returned mm.  increased  to the  The 5  -  the  solution  reaction 10°C  temperature of  in  was less  and  dissolve  exothermic than  the cooling  30  and sec..  as the It  bath with in 2  The reaction was complete in less than 10 mm.  After 10  208  mm  sample  a  was  withdrawn  and  diluted  methanol  with  for  HPLC analysis. HPLC  conditions  for  monitoring  and  analysis  of  the  catharanthine N-oxide reaction:  Column:  Waters reverse phase Radial Pak 1 C 8 lOp,  8 mm x  10 cm. Solvent:  0 (23:77) containing 0.1% Et 2 MeOH/H N. 3  Flow:  1.5 mL/min.  Detection:  254 nm.  HPLC  conditions  for  monitoring  and  analysis  the  of  exocatharanthine N-oxide reaction:  Column:  Waters Reverse Phase Radial Pak 1 C 8  lOu,  8 mm x  10 cm. Solvent:  0 (23:77) 2 MeOH/H  Flow:  1.5 mL/min.  Detection:  254 nm.  containing 0.3% Et N. 3  TLC conditions for monitoring the formation of N-oxide: Silica  plates  using  dichioromethane/methanol  (10:1)  as  eluent.  3.29.  GENERAL  EXPERIMENTAL PROCEDURE USED  IN THE SYSTEMATIC  INVESTIGATION OF THE MODIFIED POLONOVSKI REACTION Setup: a  magnetic  A dry 50 mL three-necked flask was equipped with stirrer,  a  gas  bubbler,  a  thermometer  and  a  209  rubber  septum.  Dry  glass  syringes  were  solvents and trifluoroacetic anhydride  used  to  transfer  and the reaction was  performed under argon.  Steps in the procedure: la)  Exocatharanthine 3 (200 mg,  0.594 mmol) was dissolved  in dry solvent (1.0 mL). ib)  Exocatharanthine 89  (200 mg,  0.594 mmol) was dissolved  in dry solvent (2.0 mL). 2)  Cooling to -20 to -30°C.  3)  96% m-Chloroperbenzoic acid (112.5 mg, added in one portion.  0.626 mmol) was  The reaction was exothermic.  temperature rises 5 to 10°C less than 30 sec.  The  and then  return to the temperature of the cooling bath. 4)  The temperature was kept at -10 to -15°C for 10 mm.  5)  The N-oxide formation was monitored on HPLC or TLC using the condition described in chapter 3.28.  6)  Cooling to -30 to -40°C.  7a)  Vindoline 4 (272 mg,  0.596 mmol) was placed in a  and dissolved in dry solvent (0.8 mL) reaction.  vial,  and added to the  The vial was rinsed with dry solvent (0.2 mL)  which was also added to the reaction. Alternative,  and probably better,  methods of adding  vindoline 4 when hygroscopic solvents are used are: 7b)  Vindoline 4 (272 mg, reaction (1.0 mL).  0.596 mmol) was added to the  as a solid followed by the dry solvent  210  7c)  The N-oxide was generated in 2.0 mL of dry solvent instead of 1.0 mL and vindoline 4  (272 mg,  was added as a solid at this stage. was  0.596 mmcl)  The temperature  kept at -20 to -10°C until all the vindoline has  dissolved and a clear solution was obtained. 8)  Cooling to the desired reaction temperature (-60°C)  9)  Addition of trifluoroacetic anhydride (0.4 mL. mmcl) in one portion.  The reaction was very exothermic.  The temperature rose 10 to 15°C in 10 to 15 3 mm  2.83  sec. After  the temperature had returned to the desired  temperature (-60°C). 10)  The reaction was kept at the desired reaction temperature (-60°C) for 3 hrs.  11)  The bubbler and the septum are replaced by stoppers and the thermometer by an adapter.  The reaction vessel was  connected to a vacuum-line and evacuated for 5 mm  at  the reaction temperature (-60°C). 12)  Removal of the cooling bath and evacuation for another 15 mm.  13)  The sticky foam was dissolved in dry degassed (with argon) methanol  (5 mL) at room temperature under Ar.  The residue dissolved in 3 mm  and an orange solution  was obtained. 14)  Cooling to -25 to -30°C.  15)  The temperature was kept at -20 to -25°C during the portion-wise addition of sodium borohydride (225 mg, 595 mmol).  The reaction was exothermic and tended to  211  foam.  The addition takes 3  -  4 mm.  The reaction turned  yellow and pH rose above 8. 16)  the 17)  10°C over 5 mm.  The reaction mixture was transferred  rotary evaporator at 20  -  to a 100 mL round  evaporated off on a  25°C.  This took 15  -  20 mm.  The yellow residue was dried further on a vacuum-line for  20)  after  addition of tlxe sodium borohydride was complete.  bottom flask and the solvent  19)  to -25°C for 5 mm  The reaction was heated up by the palm of the hand to 8 -  18)  -o  The reaction was kept at  5  -  10 mm.  The yellow foam was dissolved in degassed  (with argon)  ethyl acetate (50 mL). 21)  Extraction once with water (25 mL). washing was  >  10.  Then with water (2 x 15 mL).  pH of the washing fell  22)  The pH of the The  from about 8 to 7.  Drying of the organic phase over magnesium sulfate for 5 to 10 mm.  23)  Filtration,  washing of the filter cake with ethyl  acetate (2 x 10 mL). 24)  Evaporation of the solvent on a rotary evaporator at 35°C to dryness.  25)  Further drying on a vacuum-line for 2 to 4 hrs.  26)  The crude product (520 foam,  27)  -  550 mg) was obtained as a  often yellow in color.  Isolation of 19’,20’-anhydrovinblastine 91 was performed as described in section 3.20.  212  3.30.  PREPARATION OF THE SECOND INTERMEDIATE 99. In  a  magnetic stopper  dry  50 mL  stirrer,  bubbler,  a gas  (0.8 mL)  6 acetone-d  flask  three-necked  thermometer  a  (from  freshly  a  sion  argon. was  The  obtained  propanol/dry (0.3 mL, of  the  then  cooled  bath.  cooling  to  ampule)  0.170 mmol) suspen  fine  a  glass  -65°C  in  iso—  an  anhydride  Trifluoroacetic  2.1 mmol) was added in one portion and the progress  reaction was  described the  ice  and  a  opened  stirred until  reaction was  in  stopcock.  HPLC  followed by  section  thermometer was  When  3.28).  the  replaced with  Evaporation on  a vacuum  (using  anhydride.  Normal  reaction was  pressure was  cooled  to  an adapter line  for  conditions complete  fitted with 30 mm  about  of  restored The  -65°C.  the  reaction was  —50°C removed the majority of the excess  NMR  kept  tube  under  with  and  argon  reaction was  argon.  precooled in order to prevent NMR  tube  was  then  transferred  The  the  syringe  NMR  at  the  transferred  was  introduction of moisture. to  a  trifluoroacetic  as quickly as possible with a dry syringe to a precooled 65°C)  a  with  and  (60 mg,  was added to exocatharanthine N-oxide 90 under  equipped  probe  which  (-. not The had  also been precooled to -40°C. Comments: 2 was Dichloromethane-d that  the  transformation  99  intermediate  dichioromethane.  below  -40°C  in  was Compound  both  also used as of  the  faster  99  solvents  N-oxide in  seemed but  solvent. to  acetone  to  more  be  It  the  second  than  equally  stable  seemed  in  in  stable acetone  213  above —40°C.  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L.; Schreiber, S.; Scrinivasan, P. C.; Lamb, P.; Forcier, P.; Bryan, R. F.: J. Org. Chem., 1978, 43, 4859.  1980,  S; Yonemitsu, 28, 900.  0:  Chem.  Chem. Pharm. Bull.,  50.  Polonovski, M; Polonovski, 1927, 41, 1190.  51.  a) Lounasmaa, M; Koskinen, A: Heterocycles, 1591. b) Volz, D. H: Kontalcte, 1984, 14.  52.  a) Huisgen, R.; Kolbeck, W.: Chem. Ber.., 1959, 92, 3223. b) Michelot, R: Bull. Soc. him. Fr., 1969, 4377.  53.  Gartner, H.: Acetylierte Aminoxide. Eine Untersuchung zum Mechanismus der Polonovski-Reaction. Universität Karisruhe (TH), 1981. Dissertation.  54.  Volz,  H;  55.  Cave,  A.; 669.  265,  Ruchti,  L:  Michelot,  a) Cave,  Liebigs Ann. R.:  56.  A.; Tetrahedron, Kan-Fan, C.; J. Am. Chem.  Kan-Fan,  57.  Grob,  Angew.  C.  A.:  M: Bull.  C.  R.  Soc.  Chern.,  Acad.  Sci.  Fr.,  Chim.  1984,  1972,  763,  Paris,  22,  184.  1967,  C.; Potier, P.; Le Men, J.: 1967, 23, 4681. b) Ahond, A.; Cave, A.; Husson, H. P.; De Rostolan, J Potier, P.: Soc., 1968, 90, 5622.  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G.  219  K.:  L,loydia 1964,  27,  397.  73  Biemann,  74  Vogels Textbook of Quantitative Inorganic Analysis. ed. Longman Scientific & Technical, 1978, pp 375 and p 386.  -  75  a) Kempthorne, 0.: The Design and Analysis of Experiments. New York: John Wiley & Sons, 1952. b) Davies, 0. L. Design and Analysis of Industrial Experiments. New York: Hafner Publishing Co., 1954. c) Box, G. E. P.; Hunter, W. G.; Hunter, J. S.: Statistics for Experimenters. New York: John Wiley & Sons, 1978. d) Bayne, C. K.; Rubin, I. B.: Practical Experimental Designs and Optimization Methods for Chemists. Weinheim: VCH Publishers, 1986.  76  a) Pedersen, B. S.; Scheibye, S.; Lawesson, S.-0.: Bull. Soc. Chim. Belg., 1978, 87, 223. b) Scheibye, Pedersen, B. S.; Lawesson, S.-0.: Bull. Soc. Chim. Beig., 1978, 87, 229.  4th 377  S.;  220  APPENDIX  5.  5.1.  FACTORIAL DESIGN 75 Experiments  engineers  are  carried  out  in enera1  chemists,  by  intended  determine  to  and  physicists, the  effects  of one or more factors on the yield or quality of a product, the  performance  of  a  or  machine  measuring  instrument,  the  and so on.  The  resistance of a material to chemical attack, traditional  strategy  keeping  other  all  select  another  However, to  this  be  is  factors  factor  gained  by  at  for  and  among  next  the  lacks  factors.  studying  the  and  of  strategy  then  to  experiments.  has  ability  factors  while  factor  value  set  Increased  several  one  constant  a  one-factor-at-a-time  inefficient  interactions  investigate  to  been  shown detect  to  efficiency  can  be  using  simultaneously  factorial design as experimental strategy. The sense  term  to  which  factor  denote  may  experiment. temperature, qualitative experiment,  be  any  (or  variable)  feature  of  deliberately  A  factor  may  stirring (e.  magnetic  versus  from  either  rate,  used  in  condition  quantitative  person  mechanical  general  experiment  concentration the  a  experimental  varied  be  solvent,  g.  the  is  (e.  etc.)  carrying  stirring  to g. or  out  the  etc.).  The  various values of a factor examined in a design are known as  levels.  The  chosen  observable  effect of a factor is the change observed in a (the  yield,  the  product  quality,  the  221.  purity,  the rate of reaction and so on) produced by a change  in the levels of the factor in question.  Example 1: Hydrogenation of the  influence  yield  of  of  the  an olef in  required an and  temperature  the  examination of  pressure  on  the  interaction  between  temperature and pressure. A factorial design of two  factors  product  as  FACTOR  well  the  as  LEVEL  VALUE  1  1 atm.  2  2 atm.  1  10°C  2  40°C  AC pressure)  B(temperature) ,  Scheme 5-1. The factors and factorial design in scheme 5-2.  their  each on two levels was carried out.  levels  used  in  the  Scheme 5-1 shows the two  factors and their levels and scheme 5-2 shows the factorial design.  The design consists of four experiments and for each  experiment  the  design  dictates  the  combination  and  the  222  levels of the factors, on which  factorial  according to the mathematical  design  FACTOR  EXP  A  founded.  The  four  AB  Yield  (%)  (°C)  1  1  1  2  50  2  2  1  1  82  3  1  2  1  61  4  2  2  2  92  Z1  55.5  66.0  71.5  ½E2  87.0  76.5  71.0  E  31.5  10.5  0.5  Scheme 5-2.  in scheme  The factorial design.  5-2  temperature  are performed by varying the pressure and the  as  dictated  other factors constant. be  50%.  experiments  INTERACTION  B  (atm)  to  is  theory  The  levels  by  the  design  while  keeping  all  The yield in experiment 1 was found given  in the  column representing the  223  of  calculation on  factors  between the two factors relates only to the  AB,  interaction,  the  the  of  effect  yield,  the  after  interaction  the  between  the  experiments  are  calculated  been  have  carried out. In yields level  ros  the  factor A,  for 1  and  ½E1  and  level  calculated the  ½E2  factor  2  B  and  respectively.  at  E,  is  last  For factor A the effect of  pressure from 1 atm.  row,  between  the  for each of the factors and  average yield at level 1 and 2, their interaction.  the  In  average  interaction AB  absolute difference  the  effect,  their  the  to 2 atm.  (level 1)  changing the  (level 2)  resulted  in an increase of the yield of 31.5% whereas the effect of changing factor B from 10°C (level 1) to 40°C (level 2) only in  results  of  effect  an  increase  factor A  is  in  the  larger  than  the variation in the pressure has the yield. at  level  of  yield  effect  the the  10.5%.  largest  of  Since  the  factor B,  influence on  The effect of the interaction of the two factors 1  compared  to  level  2  is  only  0.5%  and  the  influence of the interaction on the yield can be said to be insignificant.  At this point it should be stressed that the  importance  the  design, chemist.  but  of  observed  depends  effects  entirely  on  In the example above,  is  the  not implicit in the  interpretation  of  the  the effect of the interaction  between the two factors is insignificant,  but it might be a  very  to  important  result  interaction exists  for  the  between the  chemist pressure  the range examined for the two factors.  and  know  that  temperature  no in  It is also important  224  to realize that the conclusions reached on the basis of the design can only be expected to have validity as individual levels.  factors  In  the  changing  the  increase  in  the  which  pressure  the  to  it  atm.  3  On basis  possible  result  should  compared  is  above  to  the yield.  within  kept  example  it  design  are  in  ones  be  result  the  obtained  in  that  obtained  in  experiment  of  the  design.  the  lower  further  a  new  a  improvement in  and  assumed  results  construct  to  further  a  not  will of  upper  their  can  long as the  yield  This  is  accomplished by combining the best levels for each factor in a new experiment.  In the example, above that would cor;espond  to an experiment where both factors are kept at this  case  also  that happens the  gave  calculating effects  of  the  correspond  to  highest of  effects  factor  B  yield.  and  factor  the  level 2.  experiment  to  4  which  Mathematically, A,  scheme  in  AB  interaction  are  In  when  5-2,  the  cancelled  out; Effect  of  fA(level B(level  A  =  2) 2)  AB(level 2))  +  ½[tA(level B(level AB(level  +  =  1)  1)  AB(level  +  1))  A(level 1)  -  B(level  +  1) 1))  A(level 2).  +  2)  tA(level  -  AB(level  +  +  2))  -  {A(level  1)  +  B(level  2)  +  The same is true for  the effect of factor B and the interaction AB.  5.2.  FRACTIONAL FACTORIAL DESIGN In  scheme  5-3  is  shown  factors each on two levels. scheme  5-3  in  two  and  a  factorial  design  with  three  Dividing the factorial design in  assuming  that  no  interactions  exist  225  between  the  scheme  5-4  scheme  factors, is  5-4  the  obtained.  could  fractional  The  also  factorial  fractional  have  been  design  in  factorial design in  constructed  the  from  factorial design in scheme 5-2 by assigning factor C to the column containing the interaction AB. The  calculation  of  the  in  effects  the  fractional  factorial design in scheme 5-4 is performed in the same way as described in For  the  reliable, true,  example  interpretation  that  different factors. is  of  the  observed  effects  to  be  it is important that the initial assumption holds  namely  true  1.  due  different  to  interactions  exist  the  between  The necessity for this assumption to hold  the  factors  no  fact  are  that  the  the  sum of  effects the  itself and the effect of one set of  for  observed  effect  of  the  interactions.  the  factor  In table  5-1 are shown how the effects of the factors are confounded with the effect of the interactions between the factors for the  fractional  factorial design in scheme  from the table that due  to  5-4.  It  is clear  the worst mistake which might be made,  interactions  in  a  fractional  factorial  design,  is  that a factor might be attributed an importance it does not have.  Normally  such  misinterpretations  subsequent experiments. half (experiment 5 3,  -  However,  are  revealed  by carrying out  in  the other  8) of the factorial design in scheme 5-  and thereby completing the full  factorial design,  possible to resolve the factors from the interactions.  it  is  226  FACTORS  INTERACTIONS  EXP  A  B  C  AB  AC  BC  1  1  1  2  1  2  2  1  2  2  1  1  1  1  2  2  .3  1  2  1  1  2  1  2  4  2  2  2  1  1  1  1  5  1  1  2  2  1  1  2  6  2  1  1  2  2  1  1  7  1  2  1  2  1  2  1  8  2  2  2  2  2  2  2  ABC  Scheme 5-3. A three factor two level factorial design.  227  FACTOR EXP  A  B  C  1  1  1  2  2  2  1  1  3  1  2  1  4  2  2  2  Scheme 5-4.  Fractional factorial design.  two-factor and main effect Table Confounding of 5-1. three of design factorial fractional in a interactions factors each on two levels.  observed effect  +  effect  factor A  =  factor A  +  interaction BC  factor B  =  factor B  +  interaction AC  factor C  =  factor C  +  interaction AB  The design, of  effect  the  possibility into  the  parent  interactions,  preferred  of  strategy  in  expanding factorial  makes order  a  design  fractional to  obtain  in as few experiments as possible.  fractional for  factorial  investigation  factorial the most  design  the  information  

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