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Elsheikh, Mohammed Osman Abdalla

ISOLATION, IDENTIFICATION AND STRUCTURAL ELUCIDATION OF TERTIARY ALKALOIDS FROM THE ROOT OF THALICTRUM MINUS L. RACE C

The Ohio Stale University Ph.D. 1985

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University Microfilms International ISOLATION, IDENTIFICATION AND STRUCTURAL ELUCIDATION OF TERTIARY ALKALOIDS FROM THE ROOT OF THALICTRUM MINUS L. RACE C

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University

Mahammed Osman Abdalla Elsheikh B.Sc., M.Sc. *****

The Ohio State University

1985

Reading Committee: Approved by Prof. J. L. Beal Prof. R. W. Doskotch Prof. L. W. Robertson Advi so F ollege of Pharmacy ACKNOWLEDGEMENTS

I would like to express my deep and sincere appreciation and gratitude to vay adviser, Dr. Jack L. Beal, for his excellent guidance, concern, constant encouragement, understanding, extreme patience, and endless support throughout my graduate education. I would like to extend my gratitude and appreciation to Dr. Raymond W. Doskotch for his deep concern and interest in this work and for his continuous encouragement and valuable discussions. My appreciation and thanks are extended to Dr. Larry W. Robertson for his deep concern, interest and encouragement.

I would like to thank the professors, staff and personnel of the College of Pharmacy (OSU), for their encouraganent and constant help and the College of Pharmacy (OSU) for financial support as a graduate associate.

I w u l d like to thank my fellow graduate students and postdoctors for their suggestions, discussions and assistance. Special thanks are extended to my friends Shoet-Sheng Lee, A. Adejare, R. Shrivastava, J. Loper, R. McClanahan, M. Obeidat, A. Chandrasekaran, S. Balaih and Dr. M. Sheikh for many valuable discussions, concern and continuous encouraganent and assistance.

I wish to acknowledge the assistance of Mr. P. Caipbell, Mr. C. Weiseriberger, Mr. J. Fowbel and Mr. N. Felt and his assistants.

I would like to thank my country, the Sudan, and the Sudanese National Council for Research for the award of scholarship.

I would also like to thank Mrs. Rose Sknith for typing this thesis and for her extreme patience and concern.

I thank the Sudanese Ehibassy and the Sudanese ccnmunity and all my friends for deep concern and help, and I also wish to express my gratitude to my fanily in Sudan for constant concern, encouragement and love.

And last, but not least, I thank my loving wife whose constant encouragement, understanding and sacrifices have all helped to make this work a reality. My thanks are extended to her fanily for constant encouragement.

- ii - VITAE

December II, 1947 ...... Bom-Qndurman, Sudan 1971. . B.S.C. (Horticulture-Major), University of Khartoun (Sudan) 1979. . . M.S.C. (Medicinal Plants), University of Khartoun (Sudan) 1979-1984 ...... Sudanese National Council for Research 1984-1985 Graduate Research Associate College of Pharmacy The Ohio State University

PUBLICATIONS

"Studies in Sudanese Medicinal Plants III: Indigenous Hyoscyanus muticus as Possible Ccmnercial Source for Hyoscyamine. Planta Medica, 45, 116 (1982).

FIELD OF STUDY

Major Field: Pharmacognosy and Natural Products Chemistry. TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... ii VITA AND FIELDS OF STUDY ...... iii LIST OF T A B L E S ...... vi LIST OF F I G U R E S ...... is GENERAL INTRODUCTION...... EXPERIMENTAL...... 16 (A) METHODOLOGY...... 16 1. Physical Analyses ...... 21 2. Chemical A n a l y s e s ...... 21 (B) MATERIAL ...... 21 (C) EXTRACTION AND FRACTIONATION OF THE ALKALOIDS FROM THE ROOTS OF THALICIRIJM MINUS L. RACE C ...... 21 1. Extraction of total" alkaloids ...... 21 2. Fractionation of the ethanolic e x t r a c t ...... 22 (D) SEPARATION OF ALKALOIDS FRCM THE TOLUENE-ETHER-SOLUBLE NONPHENOLIC FRACTION ...... 25 1. Isolation of thalrairine (1) and N-methylcorydaldine (2) . . 31 a) Thalmirine ...... " ...... 31 b) N-Methylcorydaldine...... 32 2. Isolation of 6a , 7-dehydrothaliaciine ( A ) ...... 34 3. Gtxidation of adiantifoline (5) to 6 a , 7-dehydro thaliadine (4) 35 4. Isolation of thalfine (6 ) . 7 ...... 35 5. Chromatography of the tfialmelatidine f r a c t i o n ...... 37 6 . Isolation and identification of 7 ’ -dihydrodehydro thaliadine ( 7 ) ...... 38 7. Preparation of ccopound 2 fran 6 a , 7 -dehydrothaliadine (4) . 39 8 . Isolation and identification of coco thaliadine (8 ) . . . 40 9. Isolation and identification of 7-dihydrooxothaliadine (9) 41 10. Isolation of thalisopynine (10) ...... 7 . 42 11. Isolation and identification of 7-dihydrothaliadine (11) 43 12. Reduction of 6a, 7-dehydrothaliadine (4) to 7' -dihydrothaliadine ( 1 1 ) ...... " ...... 44 13. Isolation of thalmelatldine (12) 45 14. Isolation of adiantifoline (5)7 ...... 46 15. Isolation and identification'of 6 a ,7-dehydroadiantifoline (14)...... 47 16. Oxidation of adiantifoline (5) to 6a ,7-dehydroadiantifoline 49 17. Isolation and identificaticn'of squarosine ( 1 5 ) ...... 50 18. Isolation of thalfinine (16), O-methylthalicSerine (17) and thalicarpine (18) ...... 7*. . . 52 TABLE OF CONTENTS (continued)

a) thalfinine ...... 54 b) O-methylthalicberine...... 54 c) thalicarpine...... 56 19. Isolation of thaliracebine (19) and thalmineline (20). . . 56 a) thaliracebine ...... ~ 7 ...... "7 . . . 57 b) thalmineline...... 57 20. Isolation of obaberine (21) and thalrugosine(22) ...... 58 a) obaberine ...... “7 ...... ' 7 ...... 59 b) thalrugosine...... 59 21. Isolation of O-methylthalibrine (23) and 6 -noradiantifoline (24)...... 7"...... 60 a) O-methylthalibrine...... 61 b) 6 -noradiantifoline...... 61 22. Isolation of thalistine (25) and thalmirabine (26) .... 62 a) thalistine...... 7"...... 7“ ...... 63 b) thalmirabine...... 63 (E) SEPARATION OF ALKALOIDS FRCM THE TCOJENE-ETHER-9QLUBLE PHENOLIC FRACTION ...... 64 1. Isolation of thalisopynine (10)...... 65 2. Isolation of isoboldine (22)”7 ...... 6 6 3. Isolation of delporphine (28)...... 67 4. Isolation of (+)-laudanidine (24) and thalmineline (20). . 6 8 5. Isolation of thalistine (25) . ' 7 ...... “7 . . 69 (F) RESULTS AND DISCUSSION . . . 7"...... 71 S i m V R Y ...... 154 APPENDIX OF SPECTRA ...... 156 REFERENCES ...... 205

_ v - LIST OP T&BLBS

1. Alkaloids of Thalictrum minus C o m p l e x ...... 4

2. Results of chromatographic separation of the tertiary ncnphenolic base fraction (colunn " A " ) ...... 2 7

3. Results of chromatographic separation of the tertiary norphenolic base fraction (colunn " B " ) ...... 2 9

4. Results of colunn chromatography of the thalmelatidine fraction (20 ml/fraction)...... 36

5. Separation of thalfinine, O-methylthalicberine and thalicarpine by R L - O O C ...... 53

6. Results of chromatographic separation of the tertiary phenolic base fraction ...... 4 5

7. The M4R data of thalmirine (JJ (90 Miz, C D Cl ^) ...... 7 8

8. The tt® data of thalactamine (3) (90 ttiz, C D C l ^ ) ...... 7 9

9. tWR and NOE difference results for thalmirine (1)...... 81

10. tWR (67.925 ttiz) data of thalmirine in (270 ttiz, CDCl^) . .81

11. Sinple isoguinolone alkaloids isolated from natural sources (69) 82

12. tWR and NOE difference results for oonpound 9 ...... 92

13. The *H tWR data of oxothaliadine (8) (90 ttiz, CDCl^) ...... 9 5

14. The *H bWR data of 7'-dehydrooxothaliadine ( 9 ) ...... 9P

15. Conparative H-nmr and ir spectra of ccnpound 11 and thaliadine H-nmr spectra, (90 ttiz, CDC13) ...... 9 8

16. Si tWR and NOE difference results for 6a, 7-dehydroadiantifoline (270 ttiz, C D C l ^ ) ...... 102

17. *H tWR data for 6a, 7-dehydroadiantifoline (14) (270 ttiz, C D C l ^ ...... 104

- vi - 18. Hi tWR NOE difference results for squaresine (15)...... 106

19. Comparison of nmr data (S) of adiantifoline, 2 ’-noradiantifoline and compound 24...... 109

20. Values and intensities of m s s spectral fragments of 2'-noradiantifoline and ccnpound 2 4 ...... 1 1 2

2 1 . The Hi IWR data of oonpound 24 (6-noradiantifoline) (90 ttiz, CDC13 ) ...... 1 1 2

22. The coupling constants of C-3 and C-4 protons of thalfine , . 3 3 3

23. Hi M © and NOE difference results for thalfine (6 ) (270 ttiz, 120 24. Carbon-proton correlation via long range coupling constant (coloc) of thafine (6 ) ...... 1 2 2

25. 13C tMR (CDC13) data of thalfine (6 ) (270 Miz, CDCl-j) .... 123

26. The Hi tWR data of thalfine (j6), (270 Miz, CDCl-j) ...... 124

27. Hi tHR and NOE difference results for thalfinine (16). (270 Miz, CDC13 ) ...... "T ...... 132

28. Carbon-proton correlation via long range coupling constant (coloc) of Thalfinine ( 1 6 ) ...... 133

29. 13C M © data of thalfinine (16) (270 Miz, CDC13 ) ...... 134

30. The XH DWR data of thalfinine (16), (500 Miz, CDC13) .... 135

31. Hi MIR and NOE difference results for thalmelatidine (500 Miz, CDC13 ) ...... 138

32. Chemical shifts data of methoxyl groups on the aporphine moiety of thalmelatidine and related analogs ( £ ) ...... 138

33. The Hi tWR data for delporphine (28) (270 Miz, CDC 1 3 ) .... 1 4 3

34. 13C NKR data of delporphine (28) (270 Miz, CDC13) ...... 1 4 4

35. Hi-tMR and NOE difference results for thalrugosine (2 2 ) (270 Miz, CDC13) ...... 150

36. Carbon-proton correlation via long range coupling constant (coloc) of thalrugoeine ( 2 2 ) ...... 151

- vii - 37. IWR data of thalrugosine (22) (270 Miz, CDCl^) • ■ ■ . . 152

38. The Hi NMR data of thalrugosine {22) (270 Miz, CDC13> . . . 153

- viii - LIST OF FIGURES

1. Fractionation of the ethanolic extract of the roots of T. minus race C ...... 22

2. Mass spectral fragmentation pattern of 6 a, 7-dehydrothaliadine (Jl)...... 84

3. Mass spectral fragmentation pattern of 6 a, 7-dehydroadiantifoline (14)...... 100

4. Mass spectral fragmentation pattern of compound 24.... 110

5. Rationalization of the mass spectral fragmentation pattern of 2 '-noradiantifoline...... Ill

6 . IR spectrum (CHC13) of thalmirine U ) ...... 157

7. Hl-tMR data of delporphine (28) (270Miz, CDCl^)...... 158

8 . 13C NMR SFORD spectrum (CDC13) of thalmirine U ) ...... 159

9. ^3C (MR BB Decoupling spectrum (CDCl^) of thalmirine (1_)...... 160

10. IR spectrum (CHC13) of 6 a, 7-dehydrothaliadine (4)..... 161

1 1 . Hi (MR spectrum (CDC13 ) of 6 a, 7-dehydrothaliadine 162

12. Mass spectrum of 6 a, 7-dehydrothaliadine (4)...... 163

13. IR spectrum (CHC13) of 7 1 -dihydrodehydrothaliadine... 164

14. Hi (MR (CDC13) of 7*-dihydrodehydrothaliadine...... 165

15. Mass spectrum of 7'-dihydrodehydrothaliadine (7)...... 166

16. IR spectrum (CHC13) of oxothaliadine (8 )...... 167

17. Hi (MR spectrum (CDC1 3) of oxothaliadine (8 )...... 168

18. Mass spectrum of oxothaliadine (8 )...... 169

19. IR spectrum of (CHC13) of 7'-dihydrooxothaliadine (9)..- 170

- ix - 20. W1R spectrum (CDC1-) of 7' -dihydrodehydrooxotnal iad ine...... 171

21. Mass spectrum of 7'-dihydrooxothaliadine (_9)...... 172

22. IR spectrum -r (CHCl^) of 7'-dihydrothaliadine (11_)..... 173

23. M4R spectrum (CDClj) of 7'-dihydrothaliadine(11).... 174

24. Mass spectrum of 7'-dihydrothaliadine (11)...... 175

25. IR spectrum (CHCl^) of 6 a, 7-dehydroadiantifoline (14)-. 176

26. lH NMR spectrum (CDCU) of 6 a, 7-dehydroadiantifoline (14)...... 177

27. Mass spectrum of 6 a, 7-dehydroadiantifoline (14)...... 178

28. IR spectrum (CHCl^) of squarosine (15)...... 179

29. Si FWR spectrum (CDCl^) of squarosine (15)...... 180

30. Mass spectrum of squarosine (15)...... 181

31. 33C bWR DEPT spectrum (CDCl^) of squarosine (15).... 182

32. C-H oorrelation spectrum (CDCl^) of squarosine (15).... 183 12 ___ 33. C H ® BB Decoupling vs SFORD spectrum (CDCl^) of squarosine (15)...... 184

34. IR spectrum (OJCl^) of 6 -noradiantifoline (24)...... 185

35. »1R spectrum (CDCl^) of 6 -noradiantifoline (24).... 186

36. Mass spectrum of 6 -noradiantifoline (24)...... 187

37. iMR spectrum (CDCl^) of thalfine (j6 )...... 188

38. 13C t«R SFORD spectrum (CDCl.^) of thalfine (6 ^ ...... 189

39. 13C HMR BB Decoupling spectrum (CDCl^) of thalfine (j6 ).. 190

40. C-H oorrelation spectrum (CDC1 ) of thalfine (6) at the aliphatic region...... 191

41. C-H correlation spectrum (CDC1.) of thalfine (6 ) at the aromatic region...... TT...... 192

- x - 42. OOLOC correlation spectrum (CDCl^) of thalfine (6J at the aliphatic region...... 193

43. COLOC correlation spectrum (CDC1 ) of thalfine (6J at the arcmatic region...... 194

44. Hi (MR spectrum {CXCl^)of thalfinine (16)...... 195

45. 13C (MR SPORD spectrum (CDC13) of thalfinine (16)...... 196

46. Grated Decoupling ^3C (MR spectrum (CDC1-) of thalfinine (16)...... 197

47. *3C (MR BB Decoupling spectrum (CDC1.) of thalfinine (16)...... 198

48. IR spectrum (CMCl^) of delporphine (28)...... 199

49. Hi (MR spectrum (CDCl^) of delporphine (28)..... 200

50. 13C (MR SPORD spectrum (CDCl^) of delporphine (28)..... 201

51. ^3C (MR BB Decoupling spectrum (CDCl^) of delporphine (28)...... 2 0 2

52. Hi (MR spectrum (CDCl^) of thalrugosine (22)...... 203

53. 13C (MR SPORD spectrum (CDC13) of thalrugosine (22).... 204 13 54. C (MR BB Decoupling spectrum (CDC1.) of thalrugosine (22)...... 205 General Introduction

In the last three decades, there has been a world-wide interest in the genus ’Ihalictruin . Members of this genus have been shown to be rich sources of constituents which exert hypotensive, antimicrobial, and antitumor activities (1,2). Particular emphasis has been given to the chemistry of the Thalictrum alkaloids with the major work being done in

Japan, Bulgaria, the Soviet Union, and the United States. As a result, many research papers and a number of review articles (1,2,3) have been published in this laboratory and in other laboratories reporting on the isolation and characterization of several new classes of alkaloids.

Of great taxonomic, evolutionary, and phytochemical interest is the complex (polymorphic species) T. minus L. which is wide-spread in most of Europe, the Caucasus, Siberia, and Southwestern Asia (4). Cytologi es 1 investigations during the last fifty years have revealed cytological as well as morphological variability in T. minus (4). Accordingly, sev eral cytotypes have been found in different localities. These include diploids (2n - 14), pentaploids, hexaploids, decaploids, dodecaploids, and aneuploids (2n - 40). The hexaploids are the most abundant whereas the diploids and dodecaploids are the rarest (4). Hence, it is not unusual that a number of subspecies have been recognized though no clear correlation between the known cytotypes and the morphotypes (subspecies) has yet been found (4). Numerous alkaloids have been isolated previous ly from the members of this oonplex (table 1); several of them were new.

- 1 - 2

Since the early sixties, a great effort has been given in this labo

ratory to study plant material of different Thalictrum species from var

ious sources. As part of a continuation of work on Thai let rum, a

detailed study of the alkaloids of the roots of T. minus race C has been

undertaken in the hope of discovering new alkaloids with potential phar

macological activity.

The seeds of this plant, orginally labeled as Thalictrum saxatile,

were obtained from Poland (July, 1969). However, after the seeds were

planted in the medicinal plant garden of The Ohio State University Col

lege of Pharmacy, it was found that the plants developing from these

seeds fit the taxonomic description of T. minus L. This finding was

established by Dr. Bernard Boivin, a noted expert in the taxonomy of

Thalictrum and Botanist of Central Experimental Farm, Plant Research

Institute, Department of Agriculture, Ottawa, Ontario, Canada. Two mor phologically identical chemical races of T. minus had already been rec ognized and designated race A and B on the basis of qualitative differ ences in alkaloid content (2). The plant material used in the present

investigation differs significantly in alkaloid content and hence it can be regarded as a new chemorace, C. Table 1: Alkaloids of Thalictrum minus Complex

Type of Alkaloid Alkaloid Plant part Plant Source

Simple isoquinolines N-Methylcorydaldine Root T. minus L. Race B, USA (7)

Noroxyhydrastinine* Root T. minus L. var. adiantifolium*, USA (11, 13)

Root !• minus L. var. elatum, Bulgaria (12)

Thalactamlne Aerial parts T. minus L. Bulgaria (14)

Aerial parts T. minus, Bulgaria (15)

Thalifoline* Root T. minus var. adiantifolium*, USA (11, 13)

Numbers between brackets denote references f*Alkaloids isolated as new compounds and the source in which they were first found ,

CO Type of Alkaloid Alkaloid Plant part Plant source

Benzyltetrahydroiso (+)- Reticuline Root T. minus race B, USA (16) quinolines (16)

Takatonine Leaf T. minus var. hypoleucum (previously T. thunbergii DC.), Japan (17)

Stem and leaf T. minus var. hypoleucum, Japan (28)

Thalmeline Root T. minus var. elatum, Bulgaria (12) ((-)-Thalifend- lerine)

Pavines and isopavines Argemonine Aerial parts T. minus, Bulgaria (18)

Aerial parts T. minus, USSR (50)

N-Methylargemo- Aerial parts T. minus, USSR (50) nine

Eschscholzidine Aerial parts T. minus, Bulgaria (18) Type of alkaloid Alkaloid Plant part Plant source

Pavlnes and Isopavlnes N-Methylargemonine Aerial parts T. minus, USSR (50) (cont.) Thalipoline Aerial parts T. minus, Bulgaria (18)

Aporphines Corunnlne Root T. minus, USSR (50)

Glaucine Root T. minus, USSR (18)

Aerial parts T. minus, Bulgaria (55)

Isoboldine Root T. minus race B, USA (62)

Magnoflorine Root T. minus, USSR (6) (Thalictrine) Root T. minus var. adiantifolium, USA (13) Root T. minus var. hypoleucum, China (19) Root T. minus var. hypoleucum, Japan (28) Stem (callus T. minus var. hypoleucum, Japan (51) tissue) Rhizome and T. minus var. microphyllum, Turkey (54) Root Root T. minus var. minus, Iran (58) Root T. minus race B, USA (59) Type of alkaloid Alkaloid Plant part Plant source

Aporphines (cont.) O-Methylisoboldine* Root T. minus*, race B, USA (62)

Preocteine N-oxide* Root T. minus*, USSR (48,60)

Thalicmidine Root T. minus, USSR (5,21,22,48) (Thaliporphlne)

Thalicmidine N-oxide* Root T. minus*, USSR (48,60)

Thallcmlne Root T. minus, USSR (5,21) (Ocoteine) Root T. minus, USSR (48) Root T. minus, USSR (50) Aerial parts T. minus, Bulgaria (55)

Thalicminine* Root T. minus*, USSR (24,48)

Thallcslmldine Root T. minus, USSR (50)

(+)-Thalphenine Root T. minus race B, USA (7) Type of alkaloid Alkaloid Plant part Plant source

Penanthrenes Thalflavldlne Root T. minus. USSR (57.61)

Thalicthuberlna* Root T. minus var. hypoleucum*, Japan (25)

Root T. minus var. elaturn, Bulgaria (49)

Thaligluclne Root T. minus race B, USA (62)

Thailgluclnone Root T. minus race B, USA (16)

Root and T. minus var. mlcrophyllum, Turkey (54) Rhizome

Protoberberlnea Berberine Root T. minus. USSR (6) (Thalalne) Entire plant T. minus var. adiantifolium. USA (13)

Aerial parts T. minus, Bulgaria (15)

Aerial parts T. minus, Bulgaria (18)

Root T. minus var. adiantifolium, USA (33)

Root T. minus var. elatum, Romania (26) Type of alkaloid Alkaloid Plant part Plant source frotoberberlnes Berberlne Leaf and T. minus var. hypoleucum, Japan (28) (cont.) Stem Stem (callua T. minus var. hypoleucum, Japan (51) tissue) Root and T. minus var. microphyllum, Turkey (56) Rhizome

Aerial parts I* minus, Bulgaria (55)

Root T. minus var. minus, Iran (58)

Root T. minus race B, USA (59)

Berlamblne Root T. minus race B, USA (59)

L-Canadlne beta- Aerial parts T. minus, USSR (29) methochloride

Columbamlne Root T. minus race B, USA (7)

Stem (callus T. minus var. hypoleucum, Japan (51) tissue) Deoxythalldastlne Stem (callus T. minus var. hypoleucum, Japan (51) tissue) Jatrorrhlzlne Root T. minus race B, USA (7) Root I* minus var. hypoleucum, China (19) Stem (callus I* minus var. hypoleucum, Japan (51) tissue) Root and T. minus var. mlcrophyllus, Turkey (54) Rhizome Type of alkaloid Alkaloid Plant part Plant source

Protoberberlnea N-Methylcanadlne Aerial parte Thalictrum minus, Bulgaria (18) (cont.) Aerial parts Thalictrum mlnuB, USSR (29) Palmatine Root I* minus var. hypoleucum. China (19) Root and I* minus var. mlcrophvllus. Turkey (54) Rhizome Root T. minus race B. USA (59)

Tballdastlne Stem (callus T. minus var. hypoleucum. Japan (51) tissue)

Thallfendlne Root T. minus race B, USA (7) Entire plant I* minus var. adiantifolium, USA (13) Stem (callus T. minus var. hypoleucum, Japan (51) tissue) 8-Trlchloromethy1- Root and I* minus var. mlcrophyllum, Turkey (54) dlhyd roberbe rlne** Rhizome

8-Allocryptoplne Aerial parts T. minus, USSR (29) Protoplnea (Thailetrimlne) Entire plant T. minus, USSR (20.30) Part not I* minus, USSR (61) specified

Protopine Root T. minus var. hypoleucum, China (19) Hemandaline type Thaliadine* Root I* minus race B*, USA (31) Root T. minus var. elatum, Bulgaria (49) **It la considered to be an artifact. Type of alkaloid Alkaloid Plant part Plant source

Blsbenzyllsoqulno- Aromollne Root T. minus var. hypoleucum, Japan (32,33) lines (Thalierlne) Aerial parts T. minus, USSR (50)

Root T. minus, race B, USA (62)

Berbamlne Part not T. minus, Bulgaria (4,34) specified Root T. minus, var. hypoleucum, China (19)

Homoaromollne Root T. minus var. hypoleucum, Japan (32,33) (Homothallcrlne)

Isotetrandrine Part not T. minus, Bulgaria (4,34) (O-Methylberbamine) specified

O-Methylthallbrlne* Root T. minus race B*. USA (7) (Thallatyllne) Type of alkaloid Alkaloid Plant part Plant source

Blabenzyli aoq uIno- O-Methylthalicber- Root T. minus, Bulgaria (8) llnes (cont.) lne Root T. minus var. adlantlfollum, USA (18)

Root T. minus, Bulgaria (15)

Aerial parts T. minus, Bulgaria (18)

Stem and T. minus var. hypoleucum, Japan (27,36, Leaf 37)

Root and T. minus var. mlcrophyllum, Turkey (54) Rhizome

O-Methythalme- Aerial parts T. minus, Bulgaria (15) thine* Aerial parts T. minus, Bulgaria (16)

Aerial parts T. minus, Bulgaria* (38)

Part not T. minus, Bulgaria (43) specified Obaberlne Root T. minus race B, USA (16)

Root and T. minus var. mlcrophyllum, Turkey (54) Rhizome Thalbadenzlne Aerial parts T. minus, USSR (50) (Thalbadenslne)

Thaifine Root T. minus race B, USA (16,59) (Thalphin) Type of alkaloid Alkaloid Plant part Plant source

Bisbenzylisoquino- Thalfinine Root T. minus race B, USA (16) llnes (cont.) (Thalphlnin)

Thallcberlne Root T. minus, Bulgaria (15) Bulgaria (15)

Aerial parts T. minus, Bulgaria (18) Bulgaria (18)

Stem and T. minus var. hypoleucum, Japan (27,36, Leaf 37)

Thalictlne* Stem and T. minus var. hypoleucum*, Japan (9) Leaf Thalldasine Root T. minus, Bulgaria (8)

Root T. minus race B, USA (16)

Thaligoslne Root and T. minus var. mlcrophyllum, Turkey (54) Rhizome

Thalirabine* Root T. minus race B*, USA (16)

Thallraceblne* Root T. minus race B*, USA (16)

Thai1stine* Root T. minus race B*, USA (7) Type of alkaloid Alkaloid Plant part Plant source

Blsbenzyllsoquino~ Thalmethlne* Aerial parts T. minus, Bulgaria (15) lines (cont.)

Aerial parts T. minus, Bulgaria (18)

Aerial parts T, minus*, Bulgaria (38)

Part not T. minus, Bulgaria (43) specified Aerial parts T. minus, USSA (47)

Thalmlne Entire plant T. minus, USSR (5,39)

Thalnlrablne* Root T. minus race B*, USA (7)

Thalrugoaaminlne* Root T. minus race B*. USA (16)

Thalrugosine Root T. minus race B, USA (7) (Thallglne) Rhizome and T. minus var. mlcrophyllum, Turkey (54) Root

Aporphlne-Benzyl- Adiantlfollne* Entire plant T. minus var. adlantlfolium, USA (10,13) lsoqulnolines Root T. minus var. elatum, Bulgaria (12)

Root T. minus race B, USA (31)

Rhizome and T. minus var. mlcrophyllum, Turkey (54) Root Root T. minus race B, USA (59) Type of Alkaloid Alkaloid Plant part Plant source Aphorphine-Benzyl- (+)-Bursanine* Root and T. minus var. mlcrophyllum*, Turkey (52) isoqulnollnes (cont.) Rhizome

Dehydrothalicar- Root T. minus var. elatum*, Bulgaria (42) pine* (Thalictrucar- pine)

De hydrothal- Part not T. minus var. elatum*, Bulgaria (44) melatlne* (9) specified

O-Desraethyla- Root T. minus var. elatum, Bulgaria (12,31) dlantlfoline

(+)-Istambula- Rhizome and T. minus var. mlcrophyllum*, Turkey (52) mine* Root (+)-Iznikine* Rhizome and T. minus var. mlcrophyllum*, Turkey (52) Root Noradlantlfoline Rhizome and T. minus var. mlcrophyllum, Turkey (56) Root Thali-adanlne Root T. minus race B, USA (31) Root and T. minus var. mlcrophyllum, Turkey (54) Rhizome Thallblaatlne Part not T. minus var. elatum, Bulgaria (64) specified Thalicarplne Part not T, minus , Bulgaria (35,44) specified Aerial parts T. minus var. elatum, Bulgaria (40,41) Type of alkaloid Alkaloid Plant part Plant source

Aporphine-Benzyl- Thaiicarpine Aerial parts T. minus, Bulfiaria (48) isoquinolines (cont.) (cont.) Aerial parts T. minus, Bulgaria (55)

Root T. minus, USSR (57)

Thalipine Aerial parts I* minus, Bulgaria (55)

Thalmelatidine* Root T. minus, Bulgaria (12)

Root T. minus var. elatum*, Bulgaria (45)

Rhizome and T. minus var. microphyllum, Turkey (54) Root

Thalmelatine* Aerial parts T. minus var. elatum*, Bulgaria (40,41)

Aerial parts T. minus, Bulgaria (55)

Thalmineline* Root T. minus var. elatum*, Poland (46)

(+)-Uskuduramine* Entire plant T. minus var. microphyllum*, Turkey (53)

Other Alkaloids Elatrine ** Part not T. minus var. elatum, Japan (63) specified

**No further information has been reported about this con^ rixnce 1945 16

EXPERIMENTAL

(A) METOQDOLQGY

1. Physical analyses

i. Melting points were determined on a Fisher-Johns hot stage

or Thomas-Hoover Uni melt apparatus and were un corrected.

ii. Optical rotations were obtained on a Perk in-Elmer Model 241

polarimeter at the Na-D line (589 ran) in a 1 dm cell using

the stated solvent.

iii. Circular diciiroism spectra (CD) were measured in metha

nol using Jasco J-50GA spectrcpolarimeter, Japan Spectro

scopic Co. Ltd. and were reported in molecular ellipticity

units.

iv. Ultraviolet spectra were recorded an a Beckman 5260 ultra

violet and visible spectrophotometer using methanol as sol

vent.

v. Infrared spectra were taken in chloroform solution using a

Beckman 4230 infrared spectrophotometer in Na Cl cells. 13 1. vi. C and H fWR spectra were obtained in the stated deuterat-

ed solvent using tetramethylsilane as an internal standard

an a (a) Bruker WF-80; (b) Bruker HX-90E; (c) IBM AF-270 (d)

- 16 - Bruker VM-300; or (e) Bruker AM-500. Chemical shifts were

listed in ppm on the delta scale (5), coupling constants

reported in Hz, and multiplicities given as s, singlet; d,

doublet; t, triplet; q, quartet; m, multiplet; br, broadened

signal. vii. Mass spectra were measured on a AEI-MS-9 mass spectrome

ter and provided by Mr. C. Weisenberger, Chemistry Depart

ment, The Ohio State University, or determined on a Du Pent

Model 21-491 by Mr. D. O'Mathuna of the College of Pharmacy,

The Ohio State University.

Chemical analyses.

All solvents for chromatography and chemical transformations were distilled and reagents were of analytical purity. i. Thin Layer Chromatography.

Layers of 0.25, 0.50 or 0.75 mm were made using 20 x 20

cm, 10 x 20 cm, or 5 x 20 cm glass plates and a Shandon

Southern Unoplan spreader. After spreading, the plates were

air-dried for 15—20 minutes, even-dried for 25-30 minutes at

110-120°C, and then allowed to stand at room temperature

overnight before usage. The alkaloidal spots were detected

by spraying with Dragendorff ’s reagent, in case of suspect

ed phenolic alkaloids, phosphomolybolic acid reagent was

also used as a spray. ii. Adsorption coluun chromatography. 18

Adsorptive colunns of silica gel 60 and basic or neutral

alumina were prepared and used extensively throughout the

oourse of work. iii. Chromatographs.

a) A droplet counter-current chromatograph (Model DCC-A,

Tokyo Rikakikai Co., Ltd.) was used at times in the pre

liminary investigation of the alkaloids of this plant.

b) A rotation locular counter-cur rent chromatograph (Tokyo

Rikakikai Co., Ltd.) was used in certain cases to sepa

rate alkaloids in a complex mixture. iv. Adsorbents.

a) Silica gel 60 FF-254 (particle size (63-200 m, EM

reagents) was used for colum chromatography of crude

extracts.

b) Silica gel 60 (particle size 40-63 m, EM reagents) was

used for flash and gravity column chrcnatography.

c) Silica gel 60 G (EM reagents) was used for analytical

thin layer chromatography (tic) plates.

d) Sililca gel 60 PF-254 (EM reagents) was used for final

column chromatographic purification.

e) Aluminium oxide, basic powder, (Brockman activity grade

1) or aluminium oxide, basic powder, (particle size

63-200 m, EM reagents, activity grade 1) were used for

column chromatography. f) Aluminium oxide, neutral powder, (particle size 80-200

mesh, Fisher Scientific C o.) was also used for column

chromatography. g) Aluminium oxide 60 HF-254 (type E), basic powder (EM

reagents) was used for analytical thin layer chromatog

raphy (tic).

Reagents. a ) Valser's reagent:

This reagent was prepared by slowly adding 15 g of

mercuric iodide with stirring to a solution of potassium

iodide (10 g in 100 ml of water). The mixture was then

filtered to remove excess potassium iodide.

The presence of alkaloids was indicated by a white

or yellow precipitate formed when one or two drops of

this reagent was added to about one ml of aqueous acidic

solution of the sample. b) Dragendorff's reagent:

Stock solution: - The stock solution was prepared by

boiling 2.6 of bismuth subcarbonate and 7.0 of dry sodi

um iodide in 25 ml of glacial acetic acid for a few min

utes until the orange color disappeared. The solution

was kept at room tenperature overnight, and filtered.

The filtrate was mixed with four times its volume of

ethyl acetate. 20

Spray reagent: - The spray reagent was prepared by

mixing 10 ml of the stock solution with 50 ml of glacial

acetic acid and 120 ml of ethyl acetate. To this solu

tion, 10 ml of water was slowly added while stirring.

An orange to red color at alkaloid spots on paper or

thin layer chromatograms was shown when sprayed with

Dragendorff1 s reagent. The orange background was deco

lorized by spraying with 2% aqueous acetic acid solution

c) Ehosphcmolybdic acid reagent:

This reagent was prepared by dissolving phosphcxno-

lybdic acid (2 g) in 100 ml of acetane-water mixture

(1:1) and filtered.

The spots on paper or thin layer chromatograms were

sprayed with phosphomolybdic acid reagent and exposed to

ammonium hydroxide fumes. The spots of phenolic com

pounds showed a dark blue color. d) Gibbs' reagent and test:

A small amount (about 1 mg) of the compound to be

tested was placed in a 10 ml volumetric flask and dis

solved in 1 ml of saturated sodium bicarbonate aqueous

solution. To the solution was added a freshly prepared

suspension of Gibbs reagent (2,6-dichlorobenzoqulnone

chloroimide, 30 mg in 25 ml of water) and the mixture

was diluted to 10 ml with saturated sodium bicarbonate 21

aqueous solut ion. The appearance of a blue color

(absorption at the 500-700 run region) indicated the

presence of an unsubstituted CH para to a phenolic

group.

(B) MATERIAL

The plant material used in this study was the root of Thaiictrum

minus L. race C (Ranunculaceae). The plant was cultivated in the

medicinal plant garden of The Ohio State University, College of

Pharmacy fran seed obtained from Poland. * A herbarium sample is on

file in the herbarium of the Division of Medicinal Chemistry and

Pharmacognosy. The roots were washed with water, and then oven-

dried at 40° C. The dried roots were ground to a fine particle size

(80-100 mesh) by means of a Wiley Mill.

THALICTRUM MINUS L. RACE C

1. Extraction of total alkaloids

Powdered plant material (7.7 kg) was extracted with 95% eth

anol ( 102 liters) by percolation at room temperature. The

plant material was macerated in the solvent for about 24 hours

before percolation. The percolate was collected and concentrat

ed to a thick syrup in vacuo at 37° C. The recovered solvent

was returned to the plant material. The percolation was carried

*The source of seeds are Ogrod Farmacognostyczny, Wydzielu, Farmaceuty- cznegb, Akademii Medyczneij W Pcznaniu, Poznan, Sieroca 10, Poland. out until a residue of 50 ml of percolate showed a negligible positive result upon addition of Valser's reagent. An amount of

1.13 kg of a dark brown semi sol id material was obtained (14.7%

of the total weight of the powdered roots).

Fractionation of the ethanolic extract

The fractionation of the ethanolic extract is illustrated in

fig. 1.

To the ethanol ic extract was added a small amount of 95% ethanol to make the extract flow freely. The extract was poured

into a 3% citric acid aqueous solution in 2000-ml flasks, me<±ianically stirred and filtered. The procedure was repeated until the filtrate gave a negligible positive result to Valser's reagent. During extraction with citric acid, a yellowish material precipitated. It was collected by filtration to give

10.70 g. The citric acid solutions were combined to give a total volume of about 9.0 liters which was chilled in the cold room overnight and a yellowish precipitate was removed by fil tration (1.01 g).

The citric acid solution was extracted twice with one half volume of ethyl acetate to remove acidic and neutral substances.

The ethyl acetate solutions (9.0 liters) were washed with a small amount of distilled water and the solvent removed in vacuo at 37° C to leave a dark syrupy extract (20.50 g). The citric acid solution was made alkaline with ooncentrated

ammonium hydroxide solution to pH 8-9 and extracted successively

with toluene-ether (1:1, 27 liters) and chloroform (17.6 lit

ers). The toluene-ether solution was washed with water (3.0

liters) and evaporated to dryness to yield the toluene-ether

soluble tertiary bases (57.90 g). The residue was dissolved in

toluene-ether (1:1) to give a workable volume of about 1.5 lit

ers and extracted four times with 750 ml of 5% sodium hydroxide

solution. An excess of a saturated aqueous solution of ammonium

chloride (4.5 liters) was added to the aqueous alkaline solution

to yield a cloudy suspension which was extracted with toluene-

ether until a negative Valser's test was obtained from the

toluene-ether fraction. Then the organic layers ( 4.0 liters)

were combined, washed with water and evaporated to dryness to yield the toluene-ether soluble tertiary phenolic bases (7.9 g).

The toluene-ether solution from which the phenolic bases were

removed was washed with water (2 liters), and evaporated to dry ness to yield the toluene-ether soluble tertiary nonphenolic bases (49.9 g). The chloroform extract was also washed with water and evaporated to dryness to yield the chloroform-soluble

tertiary bases (14.6 g). Thaiictrum minus L. race C Fractionation Scheme Powdered boots (7.7 kg)

95? Ethanol (102 liters)

Ware Ethanolic Extract (1.1 kg 14.7*) (discard) Citric Add Solution (9 liters, pH - 1) 1------1 Insoluble Material Citric Acid Solution (pH ■ 3) Yellow Ppt.

Ethyl Acetate (9 liters) I------1 Ethyl Acetate Extract (20.5 g) Citric Acid Solution ( 9 liters), pH ■ 4-5 1 NH^QH (28*. 3.7 liters) (aqueous phase pH - 8-9) 2 Toluene: Ether (1:1). ri7 " T i te rs) 3 Chloroform (17.6 liters) I------1------1------1 Aaueous Phase Interphase Toluene:Ether Extract Chloroform Extract (pH « 9) p (14.6 g)

n-BuOH (22.3 liters) Partition with 5? NaOH ------p m — n-BuLHI Extract i---- 1------(117 «j Tertiary MonphenoUc 5? HaQH Laver Fracticio n (50 g) 1 Saturated cl Aqueous Phase (pH • B) Solution (4.5 1) 1 Glacial Acetic ------2 Toluene.Ether 2 Ammonium TT7T) heTneckate I Aqueous Phase Organic Phase Reineckate Salt (Tertiary Phenolic Tract on 1 HeOH: Acetone: Water (3:1:1) T7-9"g7 2 Ion Exchange Resin (Cl)

Crude Quaternary Chloride ?94gJ Figure 1: Fractionation scheme of the ethanolic extract of the roots of T. minus race C 25

The aqueous solution, after removal of the tertiary alka

loids, was extracted three times with n-butyl alcohol (22.3 lit

ers) and the n-butanoic extract was evaporated to give a residue

of 116.6 g. The mother liquor was made weakly acidic with gla

cial acetic acid to pH 5-6. An excess of freshly-prepared,

aqueous, saturated solution of ammonium reineckate (2%) was add

ed to the acidic solution to precipitate the quaternary alka

loids as reineckate salts. The alkaloid reineckate precipitate

was collected, washed with water and air dried to give 319.5 g

of residue which was triturated several times with a mixture of

methanol-acetone-water (3:1:1), (15.0 liters). The aqueous

methanol-acetone solution was magnetically stirred with 1600.0 g

of anion exchange resin (IRA 400, Cl-form) for three days and

then filtered. The filtrate was evaporated to dryness to give

93.8 g of crude quaternary chlorides.

(D) SEPARATION OF ALKALOIDS FRCM THE TOLUEME-EMER-SQLUBIJ: HOMFHEMOLIC

FRACTION:

The tertiary nonphenolic alkaloid fraction (6.6 g) from a previ

ous preliminary investigation of alkaloids of the root (2 kg) of

this plant were combined with the nonphenolic bases (50 g) to give a

total weight of 57 g. This amount of the crude nonphenolic fraction

(57 g) was divided into two parts, and each part was separately

chromatographed on a silica gel oolunn (60 PF-254, E. Merck No. 26

7747). Effluent fractions of 60 ml were collected and the alka

loids, therein, analyzed by tic.

One part, "A", (32.0 g) was chromatographed on a silica gel col-

unn (I.D, 6.4 cm x 102.3 cm 1, 1.3 kg). The eluting solvents were

chloroform (2.5 liters) and the following mixtures of methanol in

chloroform: 0.5% (1.0 liter), 1% (2.8 liters), 2% (26.0 liters), 3%

(2.0 liters), 5% (1.5 liters), 10% (3.0 liters), 20% (2.8 liters),

50% (3.0 liters), and finally cue liter of methanol. Results of the

separation are given in table 2.

The remainder of the crude nonphenolic fraction, "B", (23 g) was

chromatographed on a similar silica gel column (l.D, 6.4 cm x 94.5

cm, 1.1 kg). Elution was started with chloroform (500 ml) and fol

lowed successively by increasing amounts of methanol in chloroform

in the following order: 0.5% (1.0 liter), 1% (21.0 liters) 2% (23.3

liters), 5% (10.3 liters), 10% (2.5 liters), 20% (3.0 liters), 50%

(3.0 liters), and methanol (1.0 liter). The results of separation are recorded in table 3.

The collected residues from both colunns were divided into sub groups on the bas is of results from t lc (toluene: acetone tNH^OH,

35:15:0.5; silica gel PF 254), and the detected alkaloids (Dragen- dorff's) were numbered and categorized into majors, minors or traces. Matching residues that were eluted in the same sequence from both colunns, having the same Rf values and showing similar

fluorescence under UV light, were combined prior to further separa tion and purification. 27

Table 2: Results of chromatographic separation of the tertiary nonphenolic base fraction (coluitn "A")

Fraction Eluent Weight of Compounds Number Composition residue (mg)

1-132 CHCL 0.5-2% MeOH^CHCl3 8,360 Nonalkaloids

133-142 2% MeOH-CHCl3 510 Nonalkaloids

143-164 2% Me0H-CHCl3 150 Minor alkaloids

165-177 2% Me0H-CHCl3 90 6a, 7-Dehydro- thaliadine

178-202 2% Me0H-CHCl3 200 Minor alkaloids

203-211 2% MeOH-CHCl3 810 Thalfine

212-223 2% MeCH-CHCl3 130 Mixture

224-236 2% MeOH-CHCl3 960 Dihydrodehydro- thaliadine, Oxothalia- dine, Dihydrooxo- thaliadine, Thalisopynine, 7' -Dihydrothaliadine, Thalmelat idine

237-243 2% Me0H-CHCl3 620 Minor alkaloids

244-278 2% Me0H-CHCl3 2,600 Adiantifoline

279-302 2% Me0H-CHCl3 390 Adiantifoline, De- hydroadiant i foline

303-310 2% Me0H-CHCl3 120 Adiantifoline with mixture

311-318 2% Me0H-CHCl3 240 Adiantifoline, Squaresine

319-322 2% MeQH-CHCl3 160 Mixture

323-400 2% Me0H-CHCl3 2,240 Thalf i nine, O-Methyl-thalicber ine Thalicarpine 28

Table 2 (continued)

Fraction Eluent Weight of Compounds Number Composition residue (mg)

401-443 2% MeOH-CHCl. 600 Mixture 444-511 2% MeOH-CHCi: 640 Thaiiracebine, Thalmineline

512-571 2% MeOH-CHCl. 340 Mixture

572-601 5% MeOH-CHCl- 1,200 Qbaberine, Thalrugosine

602-619 5% MeOH-CHCl- 210 Mixture

620-671 10% MeOH-CHCl. 560 Thalrugosine, O-Methylthal ibr ine, 2-Noradiant i fol i ne

672-727 20% MeOH-CHCl. 1,220 Thalmi rabine, Thaiistine

728-796 50% MeOH-CHCl. 4,050 A mixture of alkaloidal and nonalkaloidal minor confounds

I 797-847 100% MeCH 2,900 An undifferentiated I mixture of confounds I I + ■ ------29

+------h

Table 3* Results of chromatographic separation of the tertiary I nonphenolic base fraction (column "B") I

Fraction Eluent Weight of Confounds I number composition residue (mg) I

1-114 CHC1 , 0.5-1% MeOH-CHCl* 6,090 Nonalkaloids 115-1221% MeOH-CHCl. 300 Nonalkaloids 123-175 1% MeOH-CHCl:: 200 Minor alkaloids 176-104 1% MeOH-CHCl^ 30 6a, 7-Dehydro- thaliadine 185-208 1% MeOH-CHCl* 160 Minor alkaloids 209-258 1% MeOH-CHCl* 680 Thalfine 259-288 1% MeCH-CHCl* 390 Mixture 289-560 1-2% M b 0H-CHC13 1,310 Dihydro- dehydrothal i adine, Oxothaliadi ne, 7' -Dihydr ooxotha 1 i ad Thalisopynine, in® Thalmelat idi ne 561-572 2% MeOH-CHCl 180 Minor alkaloids 573-616 2% MeOH-CHCl^ 970 Adiantifoline, Dehydroadi anti foli ne 617-681 2% MeOH-CHCl 3 2,640 Adiantifoline + Minor alkaloids 682-739 2% MeOH-CHCl3 640 Adiantifoline, Squarcsine 740-910 2-5% MeOH-CHCl3 1,380 Adiantifoline, Squarosine, Thalf inine 911-950 5-10% MeCH-CHCl3 480 Squarosine, Thalfinine, O-methylthal i carpi ne 951-968 10% MeOH-CHCl* 460 Thalicarpine 969-976 10% MeOH-CHCl^ 240 Thai i r acebine, Thalmineline 977-995 20% Me0H-CHCl3 2,310 Thaliracebine, Thalmineline, Thaliracebine, Thalmineline, Obaberine, Thalrugosine, 2-Noradiantifoline 996-1015 20% Me0H-CHCl3 430 Thalmi rabi ne, Thalistine, Mixture of minors 30

I Table 3 (continued) I I I I Fraction Eluent Weight of Ccrpounds 1 1 number composition residue (mg) I

I 1016-1055 20-50% MeOH-CHCl 1,840 A mixture of I j alkaloidal and I j nonalkaloidal minor I I compounds 1 j 1056-1077 50% MeOH-CHCl3 640 An undifferenti- I I ated mixture of I I compounds I j 1078-1098 100% MeOH 1,130 An undifferenti- I t ated mixture of I I compounds I

+------h 31 1. Isolation of thalmirine (1) and N-methy 1 corydaldine (2).

The oily brown residue (468 mg) fran the combined fractions no. 133-137 (eluted from coluim "A") and 115-119 (eluted from coluitn "B") was dissolved in chloroform, adsorbed to a snail amount of basic alumina and applied to the top of a basic alumi na colunn (65 g, activity I) packed in toluene. The colunm was eluted with toluene (300 ml) and the following mixtures of chlo roform in toluene:2% (1.3 liter), 5% (1.8 liter), 10% (1.5 lit er), 20% (1.0 liter), 50% (1.0 liter) and chloroform (1.0 lit er). Finally, the column was eluted successively with 2% (1.0 liter) and 5% methanol in chloroform (1.0 liter).

No alkaloids were obtained from the toluene eluates while the 2% chloroform-in-toluene effluents afforded 2 mg of a mix ture of alkaloids.

a) Thalmirine.

The toluene-chloroform (95:5) effluent gave 37 mg of

an amorphous solid which exhibited a faint orange spot

(Rf 0.43) on tic (silica gel, Dragendorff *s) with

toluene-aoetone-ammonium hydroxide (35:15:0.5) as the

solvent system. The crude base was rechranatographed on

a 1.5 g colunn of silica gel 60 (particle size 40-60 "m)

packed in chloroform. The column was eluted with chlo

roform (100 ml) followed by 1% (100 ml) and 2% (150 ml)

methanol in chloroform. The last two solvents eluted 24 mg of a greenish-white amorphous base which was crystal lized frocn ethanol to yield 17 mg of colorless fine nee dles (mp 173-175°) of N-methyl-5,6-methylene

-dioxy-7-methoxyisoquinolone, the new natural isoquinolone oonpound (1) which was named thalmirine.

The ir spectrum (CHCL^) of thalmirine (1) revealed ' “1 absorption bands aty 1650 and 1620 cm for a lactam max carbonyl group and a conjugated double bond, respective ly (66); and a band a tV 935 cm ^ (C-0 stretching) u d X for a methylenedioxy group (14). The maxim in the uv spectra of thalmirine were at 256 nm (log €. 3.92), 261 run (log £. 4.62), 287 nm (log £ 4.12), 318 nm (log6

3.74), 330 nm (log €. 3.91) and 343 nm (log £. 3.85). The proton nmr spectrum (CDCl^, table 4) indicated the pres ence of a N-tnethyl group, a methoxy group, a methylene dioxy peak, two one-proton doublets, and an aronvatic proton singlet. No mass spectrum was obtained for the oonpound due to its instability during electron inpact and chemical ionization processes, b) N-Methylcorydaldine.

The toluene-chloroform (90:10) eluates afforded 64 mg of an oily dark brown neterial. Thin layer chroma tography of this fraction, using silica gel as adsorbent and chloroform-methanol-ammonium hydroxide (98:2:0.5) as 33

the solvent, indicated the presence of two alkaloidal

spots (Dragendorff's), a minor (Rf 0.49) and a major (Rf

0.35). Preparative tic (silica gel 60 FF-254) with the

above solvent system resulted in the separation of two

bands with Rf values of 0.49 and 0.35. The extracted

upper band afforded 2 mg of a light brown residue while

the lower one yielded 25 mg of a yellowish-brown resi

due.

The major residue (25 mg) was dissolved in an ethyl

acetate and n-hexane mixture frcm which 17 mg of white

rosettes crystallized (up 134-136°); nmr (90 MHz, CDCl^)

3.12 (s, tWe), 3.89 (OMe), 3.90 (CMe), 6.61 and 7.60

(2s, 2 Ar H), 2.91 and 3.51 (2t, 2 aliphatic H,

system, = 6.4 Hz); ir (CHCL3 ) 1645 (lactam c=o); ms

(El) m/z 221 (M+ ,C^2 *H-L5 N°3 ), 178 (base peak). This

oonpound gave identical tic mobility, nmr, ir and ms with those of N-methylcorydaldine (2) which was previ ously isolated from Thalictruro fendleri (65), and formed by KMnO^ oxidation of certain bisbenzylisoquinoline and

aporphine-benzylisoquinoline dimers including adiantifo

line, thalicarpine and cissampareine (64).

The combined fractions of chloroform, 2% and 5% methanol in chloroform effluents gave a mixture of more

them five minor alkaloids (51 mg) neither of which was

isolated in a substantial amount for study. Isolation of 6a,7-dehydrothaliadine (4).

The crude dark brown residue (118 mg) from the combined

fractions 165-177 (eluted from column "A") and 176-184 (from

column "B") showed a complex mixture of compounds present in

trace amounts and a major component with an value of 0.45 on

silica gel 60 G with the solvent system of toluene-acetone-

ammonium hydroxide (35:15:0.5). This residue was chromato

graphed on a column of neutral alumina (grade I, 15 g) with

toluene (50 ml), toluene-chloroform (4:1, 200 ml), toluene-

chloroform (3:1, 200 ml), toluene-chloroform (2:1, 150 ml),

toluene-chloroform (1:1, 200 ml), and chloroform (250 ml) as

eluents. From the toluene-chloroform (3:1) fraction, 47 mg of

an alkaloidal residue was obtained, Which showed two spots on

tic (silica gel 60 G) at an value of 0.45 (major) and 0.34

(minor) with the above-mentioned solvent system. Preparative

tic of the residue (silica gel G, 0.25 nm layer) with the sol

vent system of chlorofonn-methanol-ammonium hydroxide (95:5:1)

separated two components having values of 0.81 (major, 30 mg)

and 0.47 (minor, 3 mg).

The major ccqponent, a yellowish-green residue, showed the

following spectral properties: uv A max (MeOH) 274 nm (log 6.

4.73), 314 nm (log £. 4.16) and 336 nm (log & 3.60); ir (CHCl^)V

max 2937, 2860, 2830, 1680, 1608, 1504, 1480, 1454, 1442, 1405,

1346 and 875 cm * ms (El) m/z 533 (M+, C^q H ^ O^N, 100%), 531 (M-2H, 75%), 516 (m-17, 74%), 502 (M-31, 7.8%), 500 (11.5),

486 (10.2), 368 (0.8); ^ nmr (CDC13, 90 Miz) S 3.02 (s, N-Me>,

3.27 (in, 4 aliphatic Hs), 3.78 (s, CMe), 3.96 (s, 2 QMe), 4.00,

4,03, 4.09 (3s, 3 OMe), 6.51 (s, 2 Ar-H), 7.05, 7.44, 9.16 (3s,

3 Ar-H, the latter assigned to C-ll H), and 10.40 (s, aldehyde

H). This alkaloid gave identical nmr, uv, and ir spectra to those of 6a,7-dehydrothaliadine (4). The tic mobilities were also identical.

Oxidation of adiantifoline (J5) to 6a, 7-dehydrothaliadine (4_).

To a solution of 100 mg of adiantifoline (5) in 10 ml of acetone, 180 mg of KMnO^ in 15 ml of acetone was added drcpwise for half an hour while stirring. After the solution was stirred for three hours, 10 ml of methanol was then added to destroy the reagent and stirring was continued for two hours. The solvent was evaporated at reduced pressure and the residue suspended in acetone and filtered. The filtrate residue (121 mg) on tic

(silica gel) showed four alkaloidal spots with chloroform- methanol-ammonium hydroxide (95:5:1) at 0.81 (6a,7- dehydru- thaliadine), o . 6 3 0.4 5 (N-methy 1 corydaldine) and 0.38 (adianti foline). Preparative tic (silica gel G 60) with the above solvent system gave 30 mg of pure 6a, 7-dehydrothaliadine, 15 mg of N-methyloorydaldine (2} and 1.3 mg of adiantifoline (5).

Isolation of thalfine (6). 36

The foamy orange residue (1.50 g) from the combined frac tions (203-211 from colunn "A" and 209-258 from column "B") was placed on a neutral alumina ooluim (70 g. activity I) packed in toluene. The eluting solvents were toluene (50 ml) and the fol lowing mixtures of chloroform in toluene: 5% (100 ml), 10% (200 ml), 15% (1.0 1), 20% (1.5 1), 25% (1.0 1), 33% (1.0 1), and 50%

(600 ml). The final column washes were made with chloroform

(200 ml) followed by 2% of methanol in chloroform (200 ml) and

5% of methanol in chloroform (200 ml).

An orange residue (174 mg) was eluted with 20% chloroform in toluene which gave 147.0 mg of yellowish-orange prisms on crys tallization from ethanol, mp 140-142° (lit. (59) 139-140°),

Ca327D + 75 (C 0.10, MeOH). The ir spectrum (CHCl^) showed a peak at 1600 cm-^ consistent with an imino function (59). Other peaks were exhibited at 1500, 1455, 1412, 1345 and 1265 cm

The uv spectrum revealed ^ 348 run (log €• 3.86) and 260 nm mnX (4.58). The nmr spectrum in deuterchloroform (270 Mlz, S ) exhibited a single N-methyl peak (2.28)? nine aliphatic protons

]4H 1.87 (m), 2.13 (m), 2.15 (m), 2.53 (m); 3.18 (m, part of an aliphatic ABX system), 3.32 (m, part of the aliphatic ABX sys tem) and 3.54 (m, part of the ABX (H^) partially obscured by the

O-methyl group at 3.59), = 15.8 Hz, J^, = 2.5 Hz, =5.0

Hz); 4.56 (d) and 4.81 (d) (an aliphatic AB system, J - 14.3 A d Hz)]; four O-methyls [3.47 (s), 3.59 (s), 3.75 (s), 3.89 (s)]; ten arcmatic protons £5 . 9 9 (d, 3.0 Hz), 6.47 (s), 6.61-6.85 (m,

4 Ar-H), 7.10-7.20 (m, 2 Ar-H), 7.45 and 8.40 (2 X d, AB system,

= 5.8 Hz); 6.15 ppm (m, a O-CI^-O function). The ms peaks were m/z 648 (100%, m+, C3SH36N2

392 (2) and 369 (8).

This confound was identified as thalfine (6) by direct com parison ([aID, nmr, ir, uv and ms) with authentic samples

(16,59). An additional amount of thalfine (139 mg) was obtained by preparative tic of a residue (292 mg, from 20% chloroform/ toluene) containing the compound in a mixture with minor compo nents. The solvent system used was chlorofonn-raethanol-airanonium hydroxide (95:5:1) that separated thalfine which appeared as a greenish-blue band under uv with an value of 0.50.

The dark green residue (515 mg) from the combined fractions

(212-223 from colunn "A" and 259-288 from colunn "B") gave a oonplex mixture of several minor alkaloids from which no pure constituent was isolated.

Chromatography of the thalmelatidine fraction.

The dark brown residue (2.34 g) from the combined fractions

(224-236 from column "A" and 289-560 from column "B") revealed a number of alkaloidal spots on tic (silica gel 60, toluene- acetone-NH^OH (35:15:0.5)). It was chromatographed on a silica gel 60 colunn (particle size 40-63/4 m, 70 g) packed in toluene 38 and eluted with toluene (50 ml) and the following mixtures of

chloroform in toluene: 2% (200 ml), 5% (500 ml), 7.5% (400 ml),

10% (3 1), 15% (2.9 1), 20% (900 ml), and 50% (200 ml). Results

of chromatography were shown in table 4.

Table 4: Results of colunn chromatography of the thalmelatidine fraction (20 ml/fraction)

Fractions % QiCl^/toluene Weight (rag) Alkaloids

1-23 0% - 7.5% 13 nonalkaloids 24-47 10% 27 alkaloid !_ 48-67 10% 53 alkaloid Q_ 68-78 10% 22 mixture 79-128 10% 53 alkaloid 9 129-252 10% - 15% 210 alkaloids IQ 253-312 15% 83 alkaloids 11 313-334 15% - 20% 235 alkaloid 12 335-380 20% - 50% 210 alkaloid 389-402 50% 93 alkaloid and nonalkaloid mixture

Hie 10% chloroform in toluene effluents gave alkaloidal com

ponents in mixtures that were separated by preparative tic with

chloroform-methanol (9:1) as the developing system in the fol

lowing order:

6. Isolation find identification of 7 *-dihydrodehydrothalia-

d i n e (7) . The early 10% chloroform in toluene effluents (1200 ml) gave a light brown residue (27 mg) that contained a relatively major component (R^ 0.67) as revealed by the (toluene-acetone-NH^OH

(35:15:0.5)). Preparative tic of this residue (silica gel 60

PF-254, CHCl^-MeOH (9:1)) gave a band of an value of 0.75 w h ic h was removed and extracted. The crude material obtained (8 mg) was further rechrcmatographed on a colunn of silica gel 60

(40-63 ~m, 1 g) packed in chloroform and eluted successively with chloroform and 1% methanol in chloroform to yield 5.3 mg of an amorphous light brown solid (7J|.

This compound (7'dihydrodehydrothaliadine) showed the fol lowing spectral properties: Hi nmr (CDC13) 5 3.00 (s, N-Me),

3.25 (m, 4 aliphatic methylene protons), 3.76, 3.93, 3.95, 3.99,

4.03 and 4.08 (6s, 6CMe), 4.68 (m, 2 methylene protons of a ben zyl alcohol), 6.48, 6.64, 6.92 and 7.02 (4s, 4 ArH), and 9.12

(s, ArCHO); ir (CHCl-j) 3610 and 3670 cm-1 (d, O ^ O H ) ; ms (Cl and

El) SS/l 535 (100%, M+ , c i2HllNC>4 rec3u^res 535533 (M~2 * 64%), m/z 520 (M-Me, 30), and m/z 504 (M-MeO, 25); uv X max (MeOH) 243 nm (243 nm (log 6. 4.54), 272 nm (log 6. 4.59), 315 nm shld (log €-4.11) and 325 nm shld (log 6 4.06).

Preparation of canpound front 6a, 7-dehydrothaliadine {4) i

To a solution of 5 mg of 6a, 7-dehydrothaliadine (4}, in methanol (5 ml), NaBH^ (2 mg) in methanol (2 ml) was added drcpwise for five minutes while stirring (room tenp.). The reaction 40

was allowed to proceed for thirty minutes then 10 ml of water

was added and the reaction mixture was then partitioned with

ether (15 ml x 4). The ether layers were combined, washed with

water (30 ml), dried over anhydrous MgSO^, and then evaporated

to dryness to yield a 5.2 mg residue which was chromatographed

on a silica gel 60 colunn (1 g). The column was eluted with

chloroform followed fcy 1% methanol in chloroform to give 5 mg of

compound T. (7' -dehydrodehydrotha 1 ladi rve).

8. Isolation and identification of oxothaliadine (8)

The 1000 ml of effluent collected after the early 10% chlo

roform in toluene effluents (1.2 1), on evaporation, afforded 53

mg of a dark brown residue. Tic of the brown residue on silica

gel 60 with toluene-acetone-NH^OH (35:15:0.5) showed a mixture

of components in trace amounts with a major spot of 0.61.

Preparat i ve tic of thi s res idue (silica gel 60 PF-254,

CHCl^-MeCH (9:1)) gave a band, 0.72, which was removed and

extracted. The residue obtained (13 mg) was purified by chroma

tography on a silica gel ooluim (40-63yHm, 2g) which was eluted

successively with chloroform and 1% methanol in chloroform to 23 o X yield 10 mg of an orange amorphous solid ([ajD = 0 ); H nmr

(CDC13, 90 MHz) 3.81, 3.96, 4.09, 4.12, 4.15 and 4.20 (6s, 60

Me), 6.51, 7.43, 8.06, and 8.88 (4s, 4 ArH), 8,20 and 8.96 (2d,

2 ArH, AB pattern, = 5.4 Hz), 10.36 (s, ArCHO); ir (CHC13 )

1675 cm 1 (aldehyde c=o) and 1660 cm-^ (ketone c»o), 2817 and 2830 cm 1 (CHO), 1580, 1545, 1505, 1460, 1395, 1300, 1352, 1275,

1251, 1197, 1175, 1137 and 957 cm"1 ; u v V (MeOH) 243 nm (log max €4.60), 276 nm (log €.4.77), 4.42 nm (log € 4.42), and 346 nm

shld (log £ 4.16); ms (Cl and El) m/z 531 (M+ C ^ H ^ N O g , 21%

requires 531), m/x 516 (M-CH^, 6.6), 500 (M-MeO, 5.3), 219 (42)

and 131 (26).

Isolation and identification of 7 ’-dihydrooxothaliadine (9)

The combined fractions of 79-120 from the 10% chloroform in

toluene effluents (table 4) afforded 52 mg of a brownish-red

residue that gave a major spot, 0.62 (tic, silica gel 60,

toluene-acetane-NH^OH (35:15:0.5)). Preparative tic of this

residue (silica gel 60 PF-254, CHCl^-MeOH (9:1)) gave a band at

0.54 which was removed and extracted. The obtained naterial

(14 mg) was further rechrcmatographed on a coluitn of silica gel

60 (40-63M m, 1.5 g) packed in chloroform and eluted successive

ly with chloroform, 1% and 2% methanol in chloroform to yield 11 n n _ mg of a red-orange amorphous solid (C»]D = 0 ). This new alka

loid showed the following spectral properties: proton nmr

(CDC13, 270 fr«z) S 3.78, 3.93, 4.10, 4.12, 4.13 and 4.19 (6s, 6

OMe), 4.66 (s, 2H, -OH, D^O exchangeable), 6.59, 7.02, 7.89 and

8.85 (4s, 4 ArH), 8.19 and 8.94 (d, 2 ArH, AB system, J 5.3 AB Hz); ir (CHCl^) 3638 and 3680 cm-1 (OH), 1650 cm-1 (ketone c=o),

1588, 1494, 1492, 1387, 1347, 1291, 1269, 1188, 1131 and 955; uv

A (MeOH) 244 nm (log € 4.52), 277 nm (log £ 4.56), 315 nm InaX 42

(shld, log 4.13), and 346 ran (shld, log 3.84); and ms (El)

m/z 533 (M+, C 2g H 2 ?N.09 , 100%), m/z 517 [M-(Me+H), 03], m/z 502

(M-OMe, 58),m/z 367 (06), 352 (91) and 330 (55).

1 0 . Isolation of thalisapynine (1 0 )

The late 10% MeOH in CHCl^ effluents and the early 15% MeOH

in CHClg effluents (table 4) gave a brown residue (210 mg) that

showed two major alkaloidal spots on tic (toluene-acetone-

ammonium hydroxide (35:15:0.2)) with values of 0.35 and 0.21.

Other minor spots were also detected. Attempts to separate the

alkaloidal conponents by chromatography on several coluims were

not successful. Preparative tic of the alkaloidal mixture (sil

ica gel 60 PF-254) using toluene-acetone—ammonium hydroxide

(35:15:0.5) as the developing solvent gave two nain bands (R^

0.35 and 0.21) which were removed and extracted. The residue

from the band with R^ 0.35 was rechraraatographed an a colunn of

silica gel (particle size 40-63 yKm) packed in chloroform and

eluted with chloroform followed by 1 % and 2 % methanol in chloro

form to give 22 mg of a homogeneous alkaloid. This compound 27 showed the following physical properties: + 42 (cO.Ol,

MeOH); uv A (MeOH) 219 (log £ 4.63), 270 (shld, log £ 4.17), max 280 (log € 4.26), 301 (log € 4.19) and 312 (log £ 4.14); CD

[e]3U - 24800, Ce]298 - 27100, te]279 - 33000 and Ce ] 2 4 5 +

214000; ir (CHC13) 3936, 3535 cm" 1 (OH); ms (El) m/z 371 (90%,

M+ , C 21H 250 5N), 370 (100, M-H), 356 ( 49, M-Me), 340 (77, M-CMe), 43

324 (39), 310 (35), 297 (33), 282 (24), 149 (28), 109 (70), 95

(82) and 43 (70); proton nmr (CDCl^, 500 MHz) ^ 2.40-2.50 (m,

2H), 2.55 (s, N-Me), 2.81-3.12 (m, 5H), 3.72 (s, CMe), 3.89,

3.92, 3.96 (3s, 3 CMe), 6.82 and 7.93 (2s, 2 ArH). This com

pound. gave a positive result with phosphamolybdic acid (presence

of phenolic OH). The physical properties were in agreement with

those reported for thaliscpynine (10) by Abduzhabfoarova et al

(75). An additional amount of thaliscpynine (52 mg) was

obtained from the phenolic fraction.

1 1 . Isolation and identification of 7'-dihydrothaliadine (1 1 ).

The residue extracted from the band with 0 . 2 1 (prepara

tive tic, silica gel 60, toluene-acetone-^IH^OH (35:15:0.5)) was

rechromatographed on a colunn of silica gel 60 (particle size

40-60>«tin) packed in chloroform and eluted with chloroform fol

lowed by 1%, 2% and 4% methanol in chloroform which on evapora

tion of the solvents yielded 35 mg of an amorphous conpound with

[cdD = + 38 (c = 0.010, MeOH). An additional amount of this

alkaloid (5 mg) was obtained from preparative tic (silica gel 60

PF-254, CHC1 ^-MeOH-NH^OH (98:2:0.5)) of the residue from the

combined fractions 253-312 (83 mg) that gave two alkaloidal

bands (R^ 0.62 for compound 11 and 0.45 for thalmelatidine

(12)). The lower band yielded 27 mg of thalmelatidine.

Compound 11 (named 7'-dihydrothaliadine) revealed the fol

lowing spectroscopic properties: proton nmr (CDCl^, 90 MHz) 3 44

2.53 (s, N-Me), 2.40-2.50 (m, 2H), 2.80-3.20 (m, 5H), 3.77,

3.79, 3.90, 3.91, 3.92 and 3.96 (6 s, 6 CMe), 4.63 and 4.66 (2d,

CH^OH, AB system = 12.5 Hz, OH is D^O exchangeable), 6.56,

6.63 and 6.98 (3s, 3 ArH), 8.04 (C-ll ArH); ir (CHC13) 3550 and

3530 cm- 1 OH), 3010, 2935, 2395, 1508, 1416, 1376, 1341, 1210

(br), 1113, 927 and 750 cm 1 (br); uv ^ (MeOH) 281 nm (log£ HlcLX 5.04), 275 nm (shld, log £ 4.58), 280 nm (log £ 4.66), 301 nm

(log £ 4.60) and 313 nm (shld, log £ 4.50); CD - 53200,

Le ]29q “ 61700, [0^279 “ 7780 and ^ 2 4 0 + 58530; 311(3 1115 m/z 502 (5.3%), 464 (4), 426 (2.7), 414 (4), 376 (5.3%), 264

(9.3) and 231 (2.7%).

12. Reduction of 6 a,7-dehydrothaliadine (4) to dihydrothaliadine

(11).

To a solution of 20 mg of 6 a, 7-dehydrothaliadine in 15 ml of

acetic acid, 2 0 mg of PtO^ were added and the mixture was

mechanically stirred under an atmosphere of hydrogen gas at room

temperature. After 7 hours, the mixture was filtered and 50 ml

of water was added to the filtrate. The solution was basified

with ammonium hydroxide (pH 9) and extracted with ether (70 ml x

3). The ether solution was washed with water, dried over anhyd

rous Mg SO^ and evaporated to dryness under reduced pressure to

afford 21 mg of a brawn residue. The residue gave one spot (R^

0.21) on tic (toluene-acetone-ammonium hydroxide (35:15:0.5))

but the nmr spectrum showed peaks for a mixture of two compounds 4b

in approximately 1:1 ratio. Preparative tic of this mixture

(silica gel 60 FF * 254) with CHCl 3 -MeOH-NH4O H (98:2:0.5)

gave two bands with 0.23 and 0.16. The two bands were sepa

rately removed and extracted. The upper band afforded compound

11a (6.7 mg) with a methyl group on C-2' (nmr spectrum) instead

of the CH^OH function, while the lower band afforded 7.0 mg of

compound lib which was identical (nmr, ir, uv) with dihydrothal

iadine (1 1 ).

13. Isolation of thalmelatidine (12).

The dark brown residue (445 mg) from the combined fractions

313-334 (table 4) was chromatographed on a basic alumina colunn

(45 g, activity I) packed in toluene. Elution was carried out

with toluene (100 ml), 1% (250 ml), 2% (250 ml), 5% (200 ml) and

10% (150 ml) methanol in toluene; 20 ml fractions were collect

ed. The 2% and 5% methanol in toluene fractions, on evaporation

of solvent, yielded 236 mg of an orange-brown residue that was

further purified by preparative tic (silica gel 60 PF-254,

CHCl3 -MeOH-NH4OH (98:2:0.5)). A band of Rf 0.45 was relieved and

extracted to give 178 mg of alkaloid 12_ which was further

rechromatographed on a 3 gm colunn of silica gel 60 (40-63 /am)

packed in CHCl^. Elution was carried out with chloroform fol

lowed by 1% and 2% methanol in chloroform to yield 173 mg of the

alkaloid which was identified as thalmelatidine on the basis of

identity of nmr, ms and uv spectral properties with those of 46

thalimelatidine isolated from T. minus var. elatum by Mollov et

al. (45) and also by comparing its physical properties (nmr, ir,

uv) with those of adiantifoline (_5) which is a related

aporphine-benzylisoquinoline alkaloid.

14. Isolation of adiantifoline (5).

The dark brown residue (2.60 g) from fractions 244-278 of

colunn "A" were chromatographed on a rotation locular counter

current chromatograph (RL-CCC) in which fractionation occurred

by liquid-liquid partition chromatography (Rikakikai Co. Ltd.,

flow rate:34.2-42.0 ml/hr; angle of inclinations 60°, rota

tion: "D"; ascending mode). The solvent used for separation was

CHC13 : MeOH: aqueous O ^ O O ^ a / a ^ C O ^ buffer (5:5:3) with the

upper aqueous layer being used as the mobile phase. The pH of

the aqueous layer was gradually decreased (5.00-2.52) by varying

the CH^X)^Ha/Oi^CO^i ratio to facilitate fractionation. Frac

tions of 16 ml were collected and the presence of alkaloids was

moni tored by tic (s i 1 i ca gel 60, toluene-acet one-ammonium

hydroxide (35:15:0.5)). Fractions 131-224, upon removal of the

organic solvents (flash evaporation), basification (NH4 0H, pH

9-10), extraction with chloroform and evaporation of the organic

solvent to dryness, afforded 1.40 g of the major alkaloid,

adiantifoline (5). Identification was based on direct compari

son of nmr, ir, ms anduv data with those of an authentic sanple

(10). Additional amounts of adiantifoline were obtained from 47

fractions 279-302 and 303-318 eluted from colunn "A" and from

the combined residues of tractions 573-681 from coluim "B".

These fractions were separately chromatographed on different

colums [basic alumina packed in toluene and eluted with toluene

followed by increasing amounts of ethyl acetate in toluene (p.

48) and silica gel (40-63 /in) colunns packed in toluene and

eluted with toluene followed by increasing amount of methanol in

toluene (p. 50)3* Also, 15 mg were obtained from the phenolic

base fraction. Residues of adiantifoline were purified by

rechromatography on colunns of silica gel 60 (40-63yx m) packed

in chloroform and eluted with chloroform and 1 - 2 % of methanol in

chloroform. On the other hand, the combined fractions 617-681

(coluim "B") afforded 1.74 g of adiantifoline on crystallization

from ethanol. The combined amounts of adiantifoline gave a

total of 3.98 g of the compound.

15. Isolation and identification of 6 a, 7-dehydroadiantifoline (14).

The dark brown residue (1.36 g) obtained from the combined

fractions 279-302 eluted from colunn "A” and 573-681 from colunn

"B" were dissolved in a little amount of toluene and chronmto-

graphed on a coluim of basic alumina (77 g, activity I) packed

in toluene. The column was eluted with toluene (500 ml) and the

following mixtures of ethyl acetate in toluene: 2 % ( 2 2 0 0 ml),

3% (6400 ml), 5% (2100 ml), 10% (600 ml), 25% (1000 ml) and 50%

(750 ml). The later effluents of the 3% EtQAc-in-toluene fractions and

the 5% EtCftc-in-toluene fractions gave a brown residue (108 mg)

that showed two main alkaloidal spots on tic (silica gel 60,

toluene-acetane-ammonium hydroxide (35:15:0.5)) with R^ 0.43 and

0.26 (adiantifoline). Preparative tic of this residue on silica gel 60 PF-254 (0.75 0m thick layer) using chloroforra-methano 1 - ammonium hydroxide (98:2:0.5) as the developing solvent gave two bands with 0.53 and 0.22. The two bands were separately removed and extracted. The band with 0.22 yielded 30 mg of a residue that was rechranetographed on a silica gel colunn (1.5 g, particle size 40-63/xm) packed in CHCl^ and was eluted with

CHC13 (50 ml) followed by 1% (150 ml) and 2% (150 ml) MeOH in

CHCl^ to yield 25 mg of andiantifoline (nmr, ir, tic). Addi tional amounts of adiantifoline (477 mg) were obtained from the combined fractions of 10%, 25% and 50% EtQftc-in-toluene effl uents by crystallization frcm ethanol.

The band with R^ 0.53 yielded 25 mg of a residue that was rechromatographed on a column of silica gel (1.5 g, particle size 40-63/4m ) packed in chloroform and was eluted with CHCl^

(50 oil) and 300 ml of 1% MeOH in CHCl^ to afford 18.3 mg of a

A ^ light brown amorphous base? Ca3Q + 58° (£ 0.01, MeOH); cd ^6 ^ 3 5 2

- 2900, Ce] 3 0 6 - 7240, iel275 + 9400, CeJ2 4 8 + 62260; ir (CHCl^ y 2950, 1600, 1490, 1438, 1383, 1240, 1184, 1121, 1078, 1000, max 855 cm uv^M (MeOH) 206 nm (log £. 4.97), 217 nm (log£ h e x 49

4.89), 252 nm {log € 4.82), 275 nm (log €. 4.81), 315 nm (shld,

log €. 4.38), 328 nm (shld, log €. 4.31); nmr (90 ffiz, CDCl^) 5

2.47, 298 (2s, 2 NMe), 2.50-2.91 (m, 4H), 3.15-3.31 (m, 6 H), (m,

H, obscured by 3 OHe at 3.76, 3.78 and 3.80, 3.35, 3.76, 3.78,

3.80, 3.94, 3.98, 4.04 and 4.09 (8 S, 8 OMe); 6.18, 6.44, 6.54,

6.67, 6.72, 6.80 (6 s, 6 ArH), 9.10 (s, C-ll H) and MS (El, FAB)

m/z 724 (M+, c 4 2 H 4 eO gN 2 100%), 609 (M-Me, 16%), 518 (M -

280, 10%), 461 (52), 368 (3), 206 (2) and 185 (22).

16. Oxidation of adiantifoline (5) to 6 a,7-dehydroadiantifoline

(14).

KMnO^ (55 mg) in 10 ml of acetone was added to a solution

of 210 mg of adiantifoline in 50 ml of acetone. After the solu

tion was stirred for 5 minutes, about 10 ml of methanol was add

ed and stirring was continued for 35 minutes to destroy the

reagent. The solution was filtered, the solvent evaporated at

reduced pressure, and the residue suspended in acetone and

refiltered. Chromatography of the filtrate residue (213.2 mg)

on a silica gel colunn packed in chloroform was not successful

in separating the different oonponents. Therefore, preparative

tic of the mixture residue on silica gel 60 PF-254 with

CHCl^-MeOH-NH^OH (98:2:0.5) as the developing solvent gave six

bands with R f values 0.83, 0.77, 0.71, 0.67

(N-methy loorydaldine), 0.59 (dehydroadiantifoline) and 0.31

(adiantifoline). The bands were separately removed and extract 50 ed to give 18 mg of N-me thylcorydaldine {2), 85 mg of adiantifo

line (5) and 17 mg of 6 a,7-dehydroadiantifoline (nmr, ir, uv,

tic).

17. Isolation and identification of squarosine (15).

The dark brown residue (240 mg) obtained from column "A"

fraction 311-318 was chromatographed on a colunn of silica gel

60 (12 g, particle size 40-63/im) packed in toluene. The column

was eluted with toluene (140 ml) and the following mixtures of

methanol in toluene - 0.5% (100 ml), 1% (150 ml); 2% (500 ml),

3% (150 ml), 4% (350 ml), 5% (400 ml), 8 % (400 ml), 107 (300 ml)

and 20% (200 ml). A volume of 60 ml was collected for each

fraction and the alkaloids analyzed by tic (silica gel 60) with

solvent system toluene-acetone-NH^OH (35:15:0.5).

The 2% MeOH in toluene gave an oily brown residue (78 mg).

Preparative tic with silica gel PF-254 (0.75 mm layer) and

CHCl^-MeOH (9:1) of this residue gave a band of 0.62 which

was removed and extracted to afford 50 mg of a li^it brown

material. On the other hand, preparative tic (CHCl^-MeOH (9:1))

of the residue (55 mg) obtained from the combined 4% and 5% MeOH

in toluene effluents gave 18 mg of this material and 14 mg of

adiantifoline. An additional amount of this material was also

obtained from flash chromatography (silica gel 70-230 /J-m,

toluene-acetane-NHjOH (35:15:0.5)) of oolunn "B” fractions

682-739; this source afforded 35 mg of the conpound and 201 mg

of adiantifoline. 51 Hie combined material (103 mg) was rechrcmatographed on a

silica gel 60 coluim (4 g, particle size 40-63/in) packed in

chloroform to afford 98 mg of a homogeneous base (15), named

squarosine, Ca^D + (s 0.01, MeOH); od £0 ^ 2 2 5 + ®^^50, C9 3285

+ 6000, Lej.1£. - 1300; ir (CHC1-) V 2942, 2843, 1650, 1615, 316 3 max 1509, 1651, 1380, 1341, 1275, 1220 (br), 1132, 1059, 955, 834

cm S uv)\ (MeOH) 211 nm (log £ 5.02), 276 nm (log €. 4.04), max 285 nm, shld (log £ 3.98); nmr (CDCl^, 90 NHz) £ 2.48, 2.53 (2s,

2 tWe), 2.42-2.52 (m, 2H), 2.63-2.85 (m, 6H), 3.05-3.28 (m, 4H),

3.62-3.72 (m, 2H, one H obscured by the two CMe groups at 3.61 and 3.64), 3.61, 3.64, 3.80 (3s, 3 CMe), 5.70 (s, C-8 ArH), 5.75

(s, C-8 ArH), 6.70 (d, ArH, part of an ABX system (Hx, = 1.8

Hz)), 6.79 (t, ArH, part of an AA’BB' system), 6.81 (t, ArH, part of the AA'BB' system), 6.84 (dd, ArH, part of the ABX sys tem (Hg, 7^g = 8.3 Hz, = 1.8 Hz)), 6.89 (d, ArH, part of the

ABX system (H^, - 8.3 Hz), 6.99 (t, ArH, part of the AA'BB* system), 7.02 (t, ArH, part of the AA'BB' system), 5.91-5.94 (m,

4H, 2 0CH20); 13C nmr (CDC13, 270 f«z) 18.7, 18.8 (C-4, C-4'),

40.3, 40.6 (Ca, Ca'), 42.4, 42.5 (N-O^, N'-CH3 ), 45.2, 45.3

(C-3, C - 3 '), 56.2, 56.3, 56.4 (C-7 O C H ^ C-7' 0CH3, C-12 0CH3 ),

64.8, 65.0 (C-l, C-l’), 101.5 (2 O-O^-O), 106.6, 106.7 (C-8,

C-8'), 112.6 (C-14), 116.8 (C-ll', C-13'), 122.4 (C-10), 125.9

(C-13), 130.8, 131.4 (C-10*, C-14'), 109.3, 109.6, 127.4, 132.9,

133.0, 133.8 (4° C-C, 6C), 141.4, 141.4, 144,7, 145.9, 145.9, 5?

149.8, 149.8, 156.4, 156.4 (4° C-0, 9C) ms (El) m/z 220 (100%,

a), 205 (a-Me, 12), 204 (8 ), 203 (5), 178 (12), 91 (9), 81 (15),

69 (15), 57 (16), 55 (17) and 41 (17).

18. Isolation of thalfinine (16) O-methylthalicberine (17.) and tha-

licarpine (18).

The light brown residue (2.23 g) from coluim fractions

323-400 of colunn "A" revealed a complex mixture of major and

minor alkaloids on tic using silica gel G-254 and toluene-

acetone-aanonium hydroxide (35:15:0.5) as the solvent system

(Dragendorff's reagent). Rotary locular countercurrent chroma

tography (RL-CCC) was enployed to separate the components of

this mixture [Rikakikai Co.; Model DCC-A: pressure 6 -8 .5 kg/cm ;

flow rate = 34.2 ml/hr: angle of inclination = 45°r ascending

mode]. The following solvent system was used for separation:

CHC1 ^-MeOH-aqueous CH^CXXNa/CH^COCH (5:5:3) with the upper aque

ous layer being used as the mobile phase. A volume of 20 ml was

collected for each fraction for analysis by tic while the pH of

the aqueous layer was gradually decreased (5.00-2.52) by varying

the CH^COCNa/CH^COOH ratio to facilitate fractionation. After

removal of the organic solvents under reduced pressure, the dif

ferent fractions were separately basified with ammonium hydrox

ide to pH 9-10 and the free bases were extracted with chloro

form. The chloroform extract was washed with water, dried over

anhydrous magnesium sulfate and evaporated to dryness to yield 53

Table 5: Separation of thalfinine, O-methylthalicberine and tha ll carpi ne by RL-COC

Fraction PH Weight of free Compounds Number base (mg)

1 - 2 0 5.00 - 4.00 Negligible Ndnalkaloidal residue 2 1 - 2 2 4.00 - 3.76 1 1 0 Nonalkaloidal residue 23 - 80 3.76 - 3.00 80 Complex mixture of trace alkaloids 81 - 157 3.76 - 3.00 2 2 0 Thalf i nine, trace alkaloids 158 - 180 3.00 150 ThaIfinine-O-methyl- thalmine, traces 181 - 207 3.00 - 2.52 380 O-Methyl thalmi ne, thalicarpine 208 - 283 2.52 190 Mixture of minor alkaloids 284 - 339 2.52 30 Mixture of trace alkaloids and nonalkaloids

the free alkaloidal residues. Table 5 shows the results of

the RL-CCC. 54 a) Thalfinine.

The crude alkaloidal residue (225 mg) obtained frotn

the combined fractions 81-157 (table 5) was chrcmoto-

graphed over a colunn of basic alumina (22.5 g, activity

I) packed in diethyl ether. The colunn was eluted with

diethyl ether ( 2 0 0 ml) followed successively by 2 % (1 . 1

1 ), 5% (1.5 1), 10% (2.5 1 ), 20% (1.0 1) and 40% (1.0 1 )

ethyl acetate in diethyl ether. Finally, the colunn was

washed with 1000 ml of chloroform. Frcxn the 10% and 20%

ethyl acetate in ether eluates, a major amorphous compo

nent (59 mg) was isolated and identified as thalfinine

(16j on the basis of identical tic* nmr, ir, and uv

properties with those of an authentic sanple (16).

An additional amount of thalfinine was obtained by

preparative tic (silica gel PF-254, 0.5 mm layers) of

the combined alkaloidal residues of fractions 158-180

(table 5) us ing toluene-aetone-ammonium hydroxide

(35:15:0.5) as the developing solvent system. Two major

bands of values 0.38 and 0.23 were removed and

extracted. The crude alkaloidal residue obtained frotn

the band with 0.38 was rechronatographed on a coluim

of silica gel 60 ( 40-63/tm) packed in chloroform and the

column was eluted with chloroform followed by 1 % and 2 %

methanol in chloroform to afford 35 mg of thalfinine. b) O-Methylthalicberine. 55

Preparative tic of the combined fractions 158-180

(table 5) that yielded 35 mg of thalfinine (extracted

from the upper band# Rj 0.38) also afforded an alkaloi

dal residue that was extracted from the lower band (R^

0.23). The compound was purified in the same manner

described for thalfinine by rechrcnBtography on a column

of silica gel packed in GHCl^ and eluted with CHCl^ fol

lowed by 1% and 2% MeOH in CHCl^ to yield 34 mg of a

homogeneous base. This alkaloid was identified as

O-methylthalicberine (17) by direct comparison of its

physical properties (nmr, uv, ir and mx) with those of a

known sanple (13) and found to be identical. An addi

tional amount of this compound was obtained from the

chromatography of the combined fractions 181-207 (376

mg, table 5) on a colunn of basic alumina (38 g, activi

ty I) packed in diethyl ether. The colunn was eluted with ether (200 ml) followed by mixtures of EtQAc in ether in the following order: 2% (1.1 1), 5% (3.0 1),

10% (2.0 1), 40% (1.0 1), then the column was washed with 1 liter of chloroform. The 5% and the early 10% ethyl acetate i n ether e f fluents gave

O-methylthalicberine as the major component in mixture with minor alkaloids. Preparative tic of the residue

(113 mg) obtained front these combined 5% and early 1 0 % EtQAc in ether effluents with toluene-acetone-NH„0H 4 (35:15:0.5) as the develqping solvent afforded

O-methylthalicberine Which was purified by rechromatog

raphy on silica gel (40-63/tm, particle size) in CHCl^-

The 1% and 2% MeOH in CHCl^ effluents afforded 73 mg of

the compound to give a total of 107 mg.

c) Thalicarpine.

The late 10% and the early 40% EtQAc in ether

afforded 162 mg of a residue that indicated the presence

of a major alkaloidal spot on tic (silica gel 60,

toluene-acetone-NH4OH (35:15:0.5)) with 0.19. Pre

parative tic of this residue using silica gel 60 and the

above solvent system gave a band (R^ 0.19) Which was

removed, extracted and purified in the same manner as

previously described for thalfinine and

O-methylthalicbrine. The alkaloid obtained (113 mg) was

shown to be identical to thalicarpine (18) by the com

parison of its ir, uv and nmr spectral data with authen

tic thalicarpine (1, 78). An additional amount of tha

licarpine (105 mg) was obtained in the same manner

(preparative tic and purification) from the residue (460

mg) collected from colunn "B" fractions 951-968 to give

a total of 218 mg of thalicarpine.

Isolation of thaliraoebine (19) and thalmineline (20). 57

The crude dark brown residue {643 mg) obtained from colunn

"A" fractions 444-511 was chromatographed on a colunn of neutral alumina (27 g, activity I) packed in toluene and was eluted with toluene (150 ml) and the following mixtures of chloroform in toluene: 10% (100 ml), 50% (400 ml), 60% (800 ml), and 75% (400 ml). The final elution was made with CHCl^ (600 ml) followed by

2% (1,0 1) and 5% (500 ml) MeOH in CHCly Effluent fractions of

50 ml were collected and analyzed by tic (silica gel 60, toluene-acetcne-NH4 0H (35:15:0.5)).

a) Thaliracebine.

The late 50% CHCl-j in toluene effluents and the ear

ly 60% CHCl^ in toluene effluents gave a brawn residue

(138 mg) that showed a major alkaloidal spot (R^ 0.28)

and a number of minor spots on tic with the above sol

vent system. Preparative tic of this residue (silica

gel 60 PF-254, CHC1 ^-MeOH-NH^OH (98:2:05)) gave a major

band (R^ 0.23) which was removed and extracted. The

residue obtained (54 mg) was rechranatographed on a col

umn of silica gel 60 (1.5 g, particle size 40-63 /um)

packed in GHCl^ and eluted with CHCl^ followed by 1% and

2% MeCH in CHCl^ to yield 39 mg of an amorphous compound

which was identified as thaliracebine (19) by direct

cotiparison (tic, uv, ir and nmr) with a known sanple

(16).

b) Thalmineline. 58

The 75% CHCl^ in toluene, CHCl^, and the 2% MeOH in

CHcl^ effluents, on evaporation of solvents, yielded 236

mg of a yellowish-green residue that showed a major

alkaloidal spot (R^ 0.14) on tic (silica gel 60) with

toluene-acetone-tJH^OH (35:15:0.5) in addition to minor

alkaloidal spots (Dragendorff's). Preparative tic of

the residue on silica gel 60 PF—254 (0.5 nm layer) and

CHCl^-MeOH-NH^OH (98:2:0.5) as the developing solvent

gave a major band (R^ 0.13) Which was removed and

extracted. The residue obtained (193 mg) was further

rechranatographed on a ooluxn of silica gel 60 (2.5 g,

particle size 40-63^m) packed in CHCl^. The column was

eluted with CHCl^ followed by 1%, 2% and 4% MeOH in

CHCl^ to yield 185 mg of an amorphous compound that gave

a positive phosphomolybdic acid test. The compound was

identified as thalmineline (2 0 ) by caoparison of its

physical data (C«]jy nmr, ir, uv) with those of thal

mineline obtained from the roots of Thaiictrum minus

var. elatum by Reisch et al. (46). An additional

amount of thalmineline (35 mg) was obtained from the

phenolic fraction.

20. Isolation of obaberine (21) and thalrugosine (22). 59

The light brown residue (1.19 g) obtained from column "A"

fractions 572-601 (5% MeOH in CHCl^ effluents) was dissolved in

CHCl^ and adsorbed to silica gel (particle size 70-230/Um). The adsorbed material was chromatographed on a oolurm of neutral alumina (60 g, activity I) packed in toluene and eluted with

toluene (150 ml) and the following mixtures of chloroform in toluenes 1 0 % ( 1 0 0 ml), 50% (300 ml), 60% (1.2 1), 75% (800 ml) and 80% (700 ml), then chloroform (500 ml) followed by 2% (500 ml) and 5% (500 ml) methanol in chloroform.

a) Obabenne.

The late 60% CHCl^ in toluene effluents, on evapora

tion of solvents gave a light brown residue (176 mg)

that showed a major spot (R^ 0.35) on tic (silica gel

60, toluene-acetone-NHjOH (35:15:0.5)). Preparative tic

of this residue (silica gel 60 PF-254, CHC1 ^-MeOH-NH^OH

(95:5; 1)) gave a band with 0.44 which was removed and

extracted to give 152 mg of a residue which was further

rechramatographed on a column of silica gel (particle

size 40-63/im, 2g) packed in CHCl^. Elution with CHCl^

followed by 1% and 2% MeOH in GHCl^ afforded 148 mg of a

pale yellow powder that was identified as bbaberine (2 1 )

from its nmr, ir and tic behavior when compared with the

data of authentic sanples (16, 79, 80).

b) Thalrugosine. 60 The late 75% CHCl^ in toluene effluents and the 80%

CHCl^ in toluene effluents afforded a light green resi

due that showed a mjor alkaloid with 0 . 1 0 on tic

(silica gel, toluene-acetone—NH^OH (35:15:0.5)). Pre

parative tic of this residue (silica gel 60 PF-254,

CHCl3 -MeOH-NH4OH (95:5:1)) afforded a band with Rf 0.42

which was removed and extracted to give 96 mg of an

amorphous residue that was rechranatographed on a silica

gel ooluim (1.5 g, particle size 40-63 /xm) packed in

CHCl^. The colunn was eluted with CHCl^ followed by 1%,

2% and 4% MeOH in CHCl^ to give 8 8 mg of a homogeneous

base that gave a pos i t i ve phosphcmolybdi c acid test.

This oonpound was identified as thalrugosine (22) from

its nmr, ir, ms and tic behavior when compared with ref

erence samples (7,81,82). An additional amount of thal-

rugoeine (71 mg) was obtained from colunn "A" fractions

620-671 (10% MeOH in OiCl3 effluents).

The combined late effluents of the alumina column

(late 80% CHCl^ in toluene, CHCl^, 2% and 5% MeOH in

CHCl^ eluates) gave a complex mixture of minor alkaloi

dal and nonalkaloidal oorpounds (460 mg) from which no

pure constituent was obtained.

21. Isolation of O-methylthalibrine (23) and 6 -noradiantifoline

(24). 61 Ihe dark brawn residue (560 mg) obtained from coluim "A" fractions 620-671 (10% MeOH in CHCI3 effluents) was dissolved in

CHCl^ and adsorbed to silica gel (particle size 70-230 /Am. The adsorbed material was chromatographed on a column of neutral alumina (28 g, activity I) packed in toluene. The column was eluted with toluene (100 ml) and the following mixtures of CHCl^ in toluene: 10% (100 ml), 50% (200 ml), 60% (500 ml), 75% (1.0

1), and 80% (500 ml). Finally, the column was eluted succes sively with CHC1.J (500 ml), 2% (100 ml) and 5% MeOH in CHCl^

( 1 0 0 ml).

a) O-Methylthalibrine.

The 60% and the early 75% CHCl^ in toluene effluents

afforded a foamy residue (156 mg) that showed a major

alkaloidal spot (0.56). Preparative tic of this residue

(silica gel 60 FF-254) with CHCl3 -MeaH-NH4OH (90:10:1)

as the developing solvent gave a band with 0 . 6 8 which

was removed and extracted to afford a yellowish-brown

residue (123 mg). The residue was further purified by

chromatography on a silica gel coluim (2.5 g, particle

size 40-63 /u m) packed in CHCl^ and eluted with CHCl^

followed successively by 1%, 2% and 4% MeOH in CHCl^ to

afford 1 1 2 mg of an amorphous base that showed physical

propert ies (t lc, nmr, i r and u v ) ident i ca 1 wi th

O-methylthalibrine (23).

b) 6 -noradiantifoline. fi? The late 75% and the 80% CHCl^ in toluene effluents

in addition to the early CHCl^ effluents gave 179 mg of

a dark brown residue. Preparative tic (silica gel 60

PF-254, CHC1 3-MeOH-NH^OH (90:10:1)) gave two bands. The

lower band (R^ 0.62) was removed and extracted to yield

a residue that was further purified (silica gel colurm,

2 g, CHC13, 1%, 2%, 5% (MeOH/CHCl3)) to afford 71 mg of

thalrugosine (22). The upper band, after removal,

extraction and purification of the residue, yielded 1 2

mg of a compound named 6 -noradiantifoline that had the 27 following properties: Ca]D + 6 6 (c 0.01, MeOH) cd

Ce]3io ” 10320, ^ 0^241 + ^-^O; Protan

nmr (CDC13, 90 PWz) S 2.44 (s, N-Me), 3.56 (s, CMe),

3.77 (s, 3 CMe), 3.82, 3.90, 3.93, 3.96 (4s, 4 OMe),

6.17, 6.47, 6.55 (3s, 3 ArH), 6.58 (s, 2 ArH), 8.06

(c-11 ArH); ir (CHC1,) V 3005, 2935, 1610, 1510, J max 1466, 1414, 1395, 1338, 1257, 1192, 1102 and 1015 cm-1;

uv h (MeOH) 220 nm, shld (log £. 4.55), 281 nm (log£ JDBlX

4.22), 203 nm, shld (log €, 4.07), 310 nm, shld (log 6 -

4.03); ms (El) m/z 506 (17%), m/k 356 (100%), 206 (70%),

150 (76%), 119 (21%) and 97 (24 %).

22. Isolation of thalistine (25) and thalmirabine (26).

The dark brown residue (1.12 g) obtained from ooluitn "A"

fractions 672-727 (20% MeOH in CHC1 3 effluents) was dissolved in 63

CHCl^ and adsorbed to silica gel (particle size 70-230yum). The adsorbed material was chromatographed on a column of neutral alumina (60 g, activity I) packed in toluene. The coluim was eluted with toluene (200 ml) and the following mixtures of CHCl^ in toluene: 10% (100 ml), 50% (200 ml), 60% (650 ml), 75% (800 ml) and 80% (500 ml). Finally, the coluim was successively eluted with CHCl^ (600 ml) followed by 5% and 10% MeOH in CHCl^

(500 ml each).

a) thalistine.

The 60% and 75% CHCl^ in toluene eluates yielded 165

mg of a light brown residue that showed two mjor alka-

loidal spots (R^ 0.70 and 0.63) and two minor spots (Rf

0.11 and 0.06) on tic with CHC13 -MeOH-NH4OH (90:10:1) as

the solvent system. Preparative tic of this residue

(silica gel 60 PF-254) with the above solvent system

gave four bands (R^ values 0.70, 0.63, 0.11 and 0.06)

which were separately removed, extracted and purified as

described before.

The band with R^ 0.70 yielded 29 mg of an alkaloid

that was shown to be identical to thalistine (24) by the

comparison of its tic behavior as well as its ir, uv and

nmr spectral data with those of authentic thalistine

(7). An additional amount of thalistine (16 mg) was

obtained from the phenolic fraction.

b) thalmirabine. 64

Hie band with R^ 0.63 afforded 40 rag of thalmirabine

(25) (identification based on identical nmr, ir and uv

with authentic thalmirabine (7)) While the two bands

with 0.11 and 0.06 yielded 4 mg and 2.0 mg, respec

tively.

The combined 80% CHCl^ in toluene eluates, the CHCl^

and the 5% and 10% MeOH in CHCl^ eluates gave a complex

mixture of polar compounds (656 mg).

(E) SEPARATION OF ALKALOIDS FRCM TOE TOLUENE-EMER-SOIJUELE PHENOLIC

FRACTION;

Hie tertiery phenolic alkaloid fraction ( 8 mg) was dissolved in

a minimum amount of chloroform and chromatographed over a deactivat

ed silica gel coluim (I.D. 5.4 cm x 67.5 cm L, 450 g, 60 PF-254, E.

Merck No. 7747, activated at 120°C for 5 hr, left to cool then shak

en well with 10% W/W for 8 hr). Hie oolunn was eluted with

CHCl^ (1 liter) and the following mixtures of MeOH in CHCl^: 1% (6.5

liters), 2% (6.5 liters), 5% (6.5 liters), 10% (4.6 liters), 20%

(4.0 liters), 80% (2.0 liters). Finally, the colunn was eluted with

MeOH (1.5 liters) followed by 1.5 liters of 10% NH^OH in MeOH.

Effluent fractions of 60 ml were collected and the alkaloids, there

in, analyzed by tic (acetone-toluene-NH^OH (21:19:0.6), silica gel

FF-254, Dragendorff's)). Hie results of separation are recorded in

table 5. The following alkaloids were isolated from the phenolic

fraction. 65 ------f 1 1 Table 6: Results of chromatographic separation of the tertiary 1 phenolic base fraction 1I 1 Fraction Eluent Weight of Compounds 1 number composition residue (mg) 1 1 1-71 CHC1 IS 1 MeOH- CHC1. 82 Nonalkaloids 72-101 1% MeOH-CHCI-, 118 Mixture 102-116 1% MeOH-CHClf 145 Thai i scpyni ne 117-138 1-2/5 Me0H-CHCl3 72 Mixture of very Minor alkaloids 139-177 2 % MeOH-CHCl 106 Minor alkaloids 178-192 2% MeOH-CHClf 52 Isbboldine 193-204 2% MeOH-CHClf 117 Delporphine 205-237 2-5/5 MeOH-CHCl, 159 238—252 5S MeOH-CHCl 04 Minor alkaloids 253-262 5% MeOH-CHClf 82 263— 304 5% MeOH-CHCl'; 585 Minor alkaloids 305-306 5% MeOH-CHCl:? 34 Mixture 307-332 5% MeOH-CHClf 515 Minor alkaloids 333-397 5-10% MeOH-CHCl. 818 Minor alkaloids 398-457 10-20% MeOH-CHCl, 640 Minor alkaloids 458-508 20-40% MeOH-CHClf 457 Polar alkaloids 1 509-543 40-80% MeOH-CHClf 682 Cooplex mixture 1 1 544-576 80 Me0H-CHCl3 2 1 1 Dark brown 1 1 material I 1 577-626 MeOH + 10% 318 Dark brown 1 1 material 1 1 NH 4 0H-Me0H 1 1 1 •1------

1. Isolation of thaliscpynine.

The dark brown residue obtained from fractions 102-116 (145

mg) showed a major alkaloidal spot (R^ 0.40) and a number of

very minor spots on tic (silica gel 60, acetone-toluene-NH^QH

(21:19:0.6)). Preparative tic of this residue (silica gel 60

PF-254) with the above solvent system gave a major band (R, 0.40) which was removed and extracted to give a crude alkaloidal

residue (61 mg). Further rechromatography of this residue on a

coluim of silica gel 60 (1.5 g, particle size 40-63/i.m) packed

in CHCl^ and successive elution with CHCl^, 1% and 2% MeOH in

CHC1.J gave 52 mg of a compound that showed the same physical

properties of thaliscpynine (1 0 ) isolated earlier from the non phenol ic base fraction (p. 42).

Isolation of isoboldine (27).

The dark brown residue (91 mg) obtained from fractions

178-192 revealed two major alkaloidal spots (R^ 0.41 and 0.32) and two minor ones (R£ 0.48 and 0.25) cm tic (silica gel G, acetone-toluene-NH^OH) 21:19:0.6)). Preparat ive tlc of the

residue (silica gel 60 PF-254) with the above-mentioned solvent

system gave four conspicuous bands with R^ values as previously mentioned. The bands with R^ 0.48 and 0.25 gave minor residues of 4 mg and 1.4 mg, respectively on removal and extraction while the bands with R^ 0.41 and 0.32, on similar treatment, gave crude alkaloidal residues of 2 1 mg and 18 mg, respectively.

The residue from the band with R^ 0.41 (21 mg) was purified by further chromatography on a coluim of silica gel 60 (1.5 g, particle size 40-63/(w.m) in CHCl^. Elution was irade with CHCl^ followed by 1% and 2% MeOH in CHCl^ to give 15 mg of adiantifoline. The identification was based on comparison of spectro scopic data with that of a known sauple. Preparat ive 11 c of the other ma jor res idue (18 m g ) us ing silica gel 60 PF-254 and CHC1 ^-MeOH-NH^OH (95:5:1) gave a band with Rj 0.23 which was removed and extracted. The residue obtained was further chromatographed on a column of silica gel

60 (1.5 g, particle size 40-63/ atb) packed in CHCly Elution with CHCly 1%, 2% and 4% MeOH in CHCl^ afforded 11 mg of a homogeneous alkaloid: Hi nmr (CDCl^/ 90 MHz) £ 2.53 (s, N-Me),

3.90, 3.92 (2s, 2QMe), 6.10 (br, s, phenolic OH), 6.54, 6.80,

8.01 (3s, 3 Ar-H, the latter C-ll H); ir (CHCl^) V max 3520 cm-^

(phenolic OH). The alkaloid was identified as isoboldine (27) by comparing its physical properties (nmr, ir, Ca]D ) with those reported in the literature (83,84). Isoboldine showed a posi tive phosphomolybdic acid test.

Isolation of delporphine.

The brown residue (117 mg) obtained from fractions 193-204 showed an alkaloidal spot (R^ 0.36) in a complex mixture of oth er very minor compounds on tic with silica gel G and acetone- toluene-NH^OH (21:19:0.6). Preparative tic of the residue (sil ica gel 60 PF-254) of the residue with the same solvent system gave a band (R^ 0.36) that was removed and extracted. A further preparative tic of the obtained crude residue (42 mg) with sili ca gel 60 PF-254 and CHCl^-MeOH (9:1) gave a band (R^ 0.23) that was scraped off and extracted to yield an alkaloidal residue (25 mg). This residue was rechrcmatographed on a column of silica 68

gel 60 (1.5 g, particle size 40-63/Um) packed in CMCl^. Succes

sive elution with CHCl^, 1%, 2% and 4% MeOH in CHCl-j afforded 17

mg of an alkaloid: C a ] ^ = 40 (s 0.01, EtOH); ir (CHCl^) V nax

3420 (phenolic OH); u v ^ (MeOH) 215 nm (log £ 4.74), 281 nm

(log €.4.40) with change in 0.01 Na OH (MeOH) to 260 nm (log€

4.53), 300 nm (log £ 4.35), 311 nm, shld (l o g £ 4.28); cd (MeOH)

C03212 - 37100, Ce3242 + 45300, Ce3282 - 8900; Hi nmr (CDC1 3,

270 ftiz) 7 aliphatic protons [2.90-2.93, m, a C-4 H; 2.45-2.58,

m, partially obscured by N-Me signal, a C-7 H, br; 2.76-2.80,

dd, a C-4 H; 2.88-2.92, m, a 0-5 H; 2.94-2.98, dd, a C-7 proton,

&; 3.00-3.03, m, C- 6 & H; 3.10-3.14, m, a C-5 H]; 2.55 (s, N-Me),

3.70, 3.91, 3.97 (3s, 3 CMe), 5.85 (br, 2 phenolic OH), 6.79 and

7.85 (2s, 2 Ar-H). The compound showed a positive phosphcmo-

lybdic acid test and it was identified as delporphine (28) by

comparing its physical properties with those reported in the

literature (85).

4. Isolation of {+)-laudanidine (29) and thalmineline (20).

The dark brown residue (159 mg) from fractions 205-237

showed two major alkaloidal spots (Rf 0.37 and 0.32) on tic

(silica gel G, acetone-toluene-NH OH (21:19:06)). Preparative

tic of this residue (silica gel 60 PF-254) with CHCl^-MeOH-NH^OH

(95:5:1) as the developing solvent system gave two major bands

with 0.40 and 0.29 which were separately removed and extract

ed. The residue obtained from the upper band (40 mg) was further purified by chromatography on a 1.5 g colunn of silica gel (par

ticle size 40-63/* m) packed in CHCl^ and eluted successively with CHC13, 1%, 2% and 4% MeOH in CHCl^ to afford 36 mg of a compound identified as thalmineline (2 0 ) by direct comparison of

its physical properties (nmr, ir, tic behavior) with those of an authentic sample isolated earlier from the nonphenolic base

fraction (p. 58).

The residue obtained from the lower band (38 mg) was also purified by chromatography on a similar silica gel colunn (1.5 27 gm) packed in CHCl^ to yield 35 mg of an alkaloid: C*3p + 65 (c

0.01, MeOH); ir (CHCl^) V nax 3525 ctn-^ (phenolic OH); nmr

(CDCl^, 90 MHz) S 2.60-3.90 (m, 7 aliphatic protons), 2.57 (s,

N-Me), 3.54, 3.84, 3.85 (3s, 3 CMe), 5.97 (s, C - 8 Ar-H,

6.45-6.57 (m, 2 Ar-H, and part of an ABX system), 6.69 (s,

0 5 Ar-H), 6.78 (d, Hj^ of the ABX system) and 6.15 (br, phenolic

OH). This compound also showed a positive phosphcmolybdic acid test. Thi s oonpound was shown to be ident i ca 1 to

(+)-laudanidine (29) by comparison of its Ca3p* nmr and ir spec tral data (86,87).

Isolation of thalistine (25).

The dark brown residue obtained form fractions 253-262 (82 mg) showed two alkaloidal spots: a major (R^ 0.16) and a minor

(R^ 0.24) on tic with acetone-toluene-NH^OH (21:19:0.6). Pre 70 parative tic of this residue {silica gel 60 PF-254) with

CHC1 ^-MeOH-NH^OH (90:10:1) as the developing solvent gave two conspicuous bands. The upper band (R^ 0.78) was removed, extracted, and further purified by rechromatography on a small coluim of silica gel 60 (particle size 40-63ydm, 0.5g) packed in

CHCl^ The coluim was successively eluted with CHCl^, 1%, 2% and

4% MeOH in CHCl^ to give 3 mg of a minor alkaloid. Likewise, the lower band (R^ 0.70) was removed, extracted, and purified by rechromatography on a similar coluim of silica gel 60 (1.0 g) which was eluted successively with CHCly 1%, 2% and 4% MeOH in

CHC1, to yield mg of thalistine (25). Its identification was J W * based on its identical spectral properties as well as it identi cal R^ value (mixed spots) with those of thalistine isolated earlier from the teritary nonpihenolic base fraction (p. 63). 71

RESULTS AND DISCUSSION

The tertiary base fraction from the roots of Thaiictruin minus race C has yielded twenty seven alkaloids of which two are sinple isoquinolones ithalmirine (JO and N-methylcorydaldine (2); one is a benzyltetrahydroisoquinoline: (+)-laudanidine (29); three are aporphines: thalisopynine (10.), isoboldine {21) and delporphine (28); three are hemandaline type alkaloids:

6a,7-dehydrothaliadine (4J, 7'-dihydrodehydrothaliadine (7) and dihydrothaliadine (11); two are oxoaporphines: oxothaliadine (8) and dihydrooxothaliadine (9J; ten are bisbisbenzyl isoquinolines of which five have one ether linkages squarosine (15),

O-methylthalicberine (1J7), thaliracebine (19),

O-methylthalibrine (23) and thalistine (25) while the other five: thalfine (6), thalfinine (16)/ obaberine (21), thalrugosine (22) and thalmirabine (26) have di-ether linkages; and six are aporphine- isoquinoline dimers: adiantifoline (5*K thalmelatidine (12), 6a,7-dehydroadiantifoline (14), thalicarpine (18), thalmineline (20) and 6-noradiantifoline.

Of these alkaloids isolated from the tertiary base fraction, three were exclusively obtained from the fractions that normally contain phenolic alkaloids; these were isoboldine (£7), delporphine and (+)-laudanidine (29). Thaliscpynine (10), thalmineline (20), thalistine (25) and a little amount of adiantifoline

(5, 15 mg) were isolated from both phenolic and nonphenolic base 72 fractions. The remainder (twenty bases) of which nine were new

naturally occurring alkaloids were isolated from the fractions

that normally contain nonphenolic alkaloids. The new alkaloids were thalmirine (1), squarosine (15), 6a, 7-dehydroadiantifoline

(4J, 71 -dihydrodehydrothaliadine (1), oxothaliadine (8),

7' -dehydrooxothaliadine (9), 7'-dihydrothaliadine (11),

6a,7-dehydro adiantifoline (14) and 6-noradantifoline (24).

The last seven alkaloids were related to adiantifoline (J5_), the major alkaloid that was obtained in a substantial amount (3.98 g)*

The isolation of thalfine (6), thalfinine (16) and thalmela- tidi ne (12) was reported in the literature (16,45,88) and the structural characterization of each alkaloid was based on cer tain evidence obtained by chemical and spectroscopic means. In this study, their structures were confirmed in terms of the basic skeleton and the stereochemistry, however, the positions of the methylenedioxy and the methoxyl groups on adjacent car bons were found to be interchanged.

The completely characterized known alkaloids were identified by cooparison of their physical properties with reported values and with authentic samples. Structural assignments of the new and structurally revised alkaloids will be discussed. The remainder of the alkaloids isolated from the tertiary base frac tion will be discussed in turn. 73 M e O M e O N M e

2 o M e O N M e 3 O

M e O

OMe

4; 6a,7-dehydro, R = CHO ?; R = CHO 7; 6a,7-dehydro, R = CH^OH 9; R - Ch2oh IX; R = CH20H 13; R = CHO

R O N M e M e N

16a; as in 6a !6b; as in £6 6a; Rj « Me, R2 * R3 “ CH,v"2 26; Rj ■ H,R2 = r^ = Me (rest as in 6a) 6b; Rj * R2 * ^ 2 '^3 ~ 74

5; Rj * Me , R 3 = H,R 2 = R 4 = R 5 = OMe

12; Rx = Me,R 2 = R3 = Ome ,R 4 = R& = 0CH20

14; 6 ,7-dehydro, rest as in 5

18; Rx = Me,R 2 = R 3 = H,R 4 = Rg = OMe

20; Rj = Me,R 2 = R 4 3 r 5 = OM e , R 3 = OH

24; Rj = R 3 = H, R 2 = r 4 = r 5 = OMe

M e O M e O N M e N M e M e O

OH OH

29 10; Rj = Ome,R 2 = Me

27; R x = R 2 = H

28; Rj * OH,R 2 = Me 75

Me Me M e 1 N M e OH

M e

Me MeO. M e N M e M e O N M e

23 76

O M e Me M e O M e N M e

M e O

M e N

19; R = H 25; R = o h

M e N

15 77 (A) THALMIRINE U J •

This new simple isoquinolone alkaloid was isolated from the non

phenol ic base fraction by colunn chromatography, preparative tic and

recrystallization. Comparison of the chemical shifts for the

N-methyl, the methoxy groups, the conjugated double bond protons and

the aromatic proton of thalmirine (table 7) with those of thalacta-

mine (3), a related isoquinoline (14), (table 8) showed only a very

small difference from the thalactamine positions (the largest dif

ference was 0.17 Hz and an average of 0.07 Hz). The absorption of

the N-methyl group of 1 (as well as in thalactamine) was shifted

downfield by the neighboring carbonyl group and the double bond rel

ative to the normal absorption of analogous N-methyl groups of sim

ple benzyltetrahydroisoquinoline alkaloids (14). The AB type quar

tet at ^6.41 and 6.95 (J = 7.4 Hz) indicated the presence of the AB protons at the C^-C^ double bond. Hie aromatic proton (C-8 H) at 5

7.60 ppm was shifted downfield because of the deshielding effect of

the carbonyl group.

Location of the methylenedioxy and the methoxy groups on the

remaining three positions of the isoquinolone skeleton was assisted

by Nuclear Overhauser Effect (NOE) experiments (table 9). When the

resonance region at S 7.60 (C-8 proton) was irradiated, a NOE

enhancement of 10 % was observed for the methoxy group at §3*99

ppm. Alternatively, irradiation at the methoxy resonance region { £

3.99) caused a NOE enhancement of 20 % for the proton at S 7.60. 78

These higher NOE enhancement values indicated the ortho relation

between the C-8 proton and the methoxy group suggesting that the

methoxy group

Table 7: The "Si NMR data of thalmirine (Jj (90 NHz, CDCl^)

Compound Structure Functional Chemical Protons group Shift, m e 3.57 3 (s) CMe 3.99 3 (s) n -O-CH.-O- 6.16 2 (s) C-4 H 6.41 (J 7.4) 1

nust be located between C-5 and C-6. That the methylenedioxy group

was located between C-5 and C-6 rather than between C-6 and C-7 was

supported by irradiation of the methylenedioxy resonance region at

6.16. No NOE enhancement was observed for the C-8 proton while the

resonance region of the C-4 proton was enhanced by 1.0%. Thus the

structure of this ocopound was established as structure 1. 79

Table 8: The NMR data of thalactamine (JJ) (90 NHz, CDCl^)

Compound Structure Functional Chemical Protons group Shift,

M e O NMe 3.54 3 (s) OME 3.90 3 (s) OME 3.93 6 (s) C-4 H 6.58 (J 7) 1 (d) N M e0 C-3 H 6.91 (J 7) 1

The car bon-13 tWR spectrum (table 10) showed 12 carbons and was

fully in accord with the proposed structure. The single frequency

off-resonance decoupled spectrum showed a carborryl singlet at 161.9

indicated the carbonyl carbon of the lactam. The two doublets at

131.0 and 99.1 counted for the conjugated double bond at C-3 and

C-4. The two singlets at 117.0 and 121.8 Which were relatively at

upfield positions in oonparison with the other three singlets were

4 assigned to the two carbon atoms at the two ring junctions. The

remaining three singlets were assigned to the three oxygen-bearing

carbons which were expected to be strongly deshielded. The fact

that 104.9 was a doublet was the key to the assignment of this value

for C-8 whereas the values of 37.3, 56.6 and 103.1 were assigned to

the carbons of N-CH^, OOJ^ and O-d^-O, respectively.

Isoquinolones can be considered to form a distinct group of

alkaloids and they probably originate in the plant by biochemical 80 oxidation of benzylisoquinolines (68). The presence of isoquinolones can be oansidered as a strong evidence for the presence of

larger and more complex molecules bearing them as moieties. The sinple isoquinolones obtained from natural sources reported so far are thirteen compounds (table 11) with substitution patterns of the

6,7-dioxygenated or 5,6,7-trioxygenated types. Conpounds of both types may exist in the same species (69).

The pharmacological effects of simple isoquinoline alkaloids in general have been the subject of a vast number of reports in the literature (69). However, none of the naturally occurring compounds appeared to possess activities interesting enough for more elaborate use as a pharmacological tool.

The dimeric isoquinolone alkaloids so far isolated from natural scurces were baluchistanamine, punjabine and revolutinone (69). 81

+— — + 1 1 1 Table 9: W4R and NOE difference results for thalmirine (1). 1 1 1t 1 (270 Miz, CDC13 ) 1 1 [l 1 Protons Irradiated Region (5)ppm Comments 1 1 1I 1 MeO 3.99 20.8% enhancement for 1 I H-8 at 7.59 1 1 H-8 7.59 10.2% enhancement for I 1 MeO 1 1 O-CH-O 6.16 1% enhancement for £ 1 1 H-4 at 6.41 1 1 H-4 6.41 7% enhancement for 1 1 H—4 at 6.95 1 1 H-3 6.95 7% enhancement for 1 1 H-4 at 6.41 I 1 N-CH- 3.57 11.7% enhancement 1 J 1 for H-3 at 6.95; 1 I 1I -L

+— — +

Table 10: IWR (67.925 MHz) data of thalmirine in (270 Hiz, CDCl3 )a

Carbon Multipl: ‘ ty ppm

C-l s 161.9 C-3 d 131.0 C-4 d 99.1 C-4a s 117.0 C-5* s 142.8 C-6* s 139.0 C-7* s 144.7 C-8 d 104.9 C-8a s 121.8 N-CHt q 37.3 O-CHT q 56.6 O-CH^-O t 103.1

* my be interchangeable a (BB, SPORD)

+ + 82

Table 11: Simple isoquinolone alkaloids isolated from natural sources (69) i Alkaloid Plant Species I I Corydaldine Enantia polycarpa Engl, St Diels I Doryanine Doryphora sassafras Endl. I Dory fornine Doryphora sassafras Endl. I N-Methyl corydaldine Papaver bracteatum Lindl. Papaver urbanium Fedde Thaiictrum fendleri Engelm. I N-Methy1-6,7- I dime thoxy-1-isoquinolone Hernandia ovigera L. Thalictrum alpinum L. I Thalictrum isopyroides C.-A. Meyer 6,7-Methylenedioxy- 1-isoquinolone Thalictrum rugosum Ait. (T. glaucum

Desf.) N-Methylthalidaldine Thalictrum fendleri Englem. Noroxyhydrast inine Thalictrum alpinum L. Thai ict rum foliolosum EC. Thalictrum minus L. var. adiantifolium Thalictrum rugosum Ait. (T. glaucum Pesf.) Oxyhydrastinine Fumaria schleicheri Scyer-Willem. Thalactamine Thalictrum minus L. Thalflavine Thalictrum flavum L. Thalifoline Thalictrum minus~~L. var. adiantifolium Si amine Cassia siamea Lam. 83

(B) 6a, 7-DEHYDRPMALIADINE (a ).

This oonpound has been obtained as a KMnO^ oxidation product of

adiantifoline (10) but it has not been previously isolated front a

natural source. Its nmr spectrum clearly showed six methoxy1 groups

at relatively lower field positions (3.78-4.09) in addition to the

N-methyl peak at the deshield&Zposition of 3.02, characteristic for

the dehydroaporphines, and the aldehyde proton at 10.40 ppm. The ir

showed an intense peak at 1680 cm ^ consistent with a conjugated or

an aromatic aldehyde function (70) in addition to the two character

istic stretching bands of the aldehyde proton at 2830 and 2860 cm~^

The mass spectrum showed significant peaks only in the high mss

region with a molecular ion peak at m/z 533 (100%). As the apor-

phine system is additionally aromatized due to the double bond

between C-6a and C-7, it is expected that the molecule is rather

stable (71). The m s s peak at m/z^ 531 (75%) could be assigned to

fragment a in figure 2 due to the loss of two hydrogen atoms result

ing in further aromatizatian of the aporphine system. Fragmentation

along the ether linkage C(b) and (c)j appeared to be insignificant

as it was exhibited in the small peak at m/z 368 (0.8%). 84

M * 0

m/z 533 a m/z 531

— OM*

OM« m/z 516 m/z 500 m/z 502 b m/z 368

Figure 2; Mass Sprectral fragmentation pattern of 6a,7-dehydrothaliadine ( 4).

MeO. NM*

U 8*

The structure of the alkaloid was further confirmed by the WtoO. 4 oxidation of adiantifoline Which gave the compound as the major

product.

M e M e O

N M e M e O

M e O

' ^ O M e O M e

Assignment of structure 1_ to this new compound with the double

bond at 6a, 7 position was made on the basis of the intensity of the

ultraviolet absorption and the characteristic downfield shifts

observed in the nmr spectrum for the C-ll proton and N-methyl sig

nals of the dehydroaporphine system (75). The signal for the C-ll

proton in compound 7 occurred at 9.12 ppm.

When the proton nmr and ir spectra of oonpound were compared

with those of 6a,7-dehydrothaliadine 04) (table 11) certain similar

ities in spectrosoapic properties were observed. Table 11 Comparative H-nmr and ir spectra of compounds and 7, H-nmr spectra, 6(80 MHz, COCK) (a) “ 6

Functional N-CH 3 och 3 och 3 och 3 0CH 3 och 3 och 3 H H H H H Group

Compound 4 3.02 3.78 3.96 3.96 4.00 4.03 4.09 6.51 6.51 7.05 7.44 9.16

Compound 7 3.01 3.77 3.94 3.95 3.99 4.04 4.09 6.48 6.63 7.02 6.94 9.12

Ir spectra (cnf*, CHC1,) ib]______L Compound 4 3010 2960 2937 2860 2830 1675 1608 1504 1442 1108 875

Compound 7 3670 3610 3018 2965 2936 1665 1605 1505 1440 1 1 0 0 870

oo CTN 87

Regions of absorption were quite similar in the two ir spectra

although peak intensities were not identical. Likewise, the six

methoxyl groups, the four methylene protons of C-3 and C-4, the

N-methyl group, and C-ll proton in both confounds had similar chemi

cal shifts in the proton nmr spectrum. The main difference between

compounds 4_ and 1_ in the nmr spectra was the relative dcwnfield

shift of the signals in case of compound 4_ in comparison with those

of compound which could probably be attributed to the presence of

the carbonyl group in compound 4_ with its inisotrophy and conjuga-

tive effects. In the ir spectra, the main difference was the band

at 1670 cm * for compound 4_ due to the absorption of the conjugated

carbonyl group and the two bands (2830 and 2860 an *) of the aldeh yde proton (absent in compound 1) and the presence of the peaks at

3610 and 3670 cm ^ (-0H) in compound 1_ (absent in compound 4).

Additional evidence for the structure of this hemandaline-type compound (7_) was obtained by the sodium borchydride redaction of oottpound 4_ (6a, 7-dehydrothaliadine) that yielded a compound having

identical nmr, ir, uv and tic properties with those of compound _7.

Hence, its relation to adiantifoline (5) and 6a,7-dehydrothaliadine

(4) was established. 68

MeOH.RT

(D) Oxothaliadine (8).

The proton nmr spectrum of this ccnpound showed six O-methyl

groups and six aromatic protons (4 singlets and two doublets). The

two doublets (at the downfield positions of $ 8.20 and 8.96 ppm

formed an AB system (J^g 5.4 Hz) characteristic for protons at C-3

and C-4 of an isoquinoline system (C-4 and C-5 of the oxoaporphine

system). The signal at 5^ 10.36 ppm was assigned to an aldehyde pro

ton. There was neither absorption for methylene or me thine protons,

nor was there any absorption for a N-methyl or a N-H group in the

nmr spectrum. The ir spectrum showed two peaks for conjugated car

bonyl absorption at 1675 (aldehyde c=o) and 1660 cm~^ (ketone c=o)

in addition to a doublet at 2817 and 2830 cm-'*' (absorption of aldeh

yde proton).

The molecular ion peak at m/z 531 (100%) in the mass spectrum

suggested a size intermediate to a monomeric bisbenzylisoquinoline

or an aporphine—benzylisoquinoline dimer. In addition, the ness

spectrum with significant peaks only in the high mass region also

suggested a highly conjugated aromatic system more stable under the 89 conditions of mass spectral fragmentation. Comparing the spec tral properties of this conpound with those of

6a, 7-dehydrothaliadine (4) indicated similarities in ahsorption both in the ir and the proton nmr spectra. Absorption bands in the ir spectra were very similar in intensity and in the region of absorp tion while the nmr spectra showed similarities in the pattern of substitution and the number of substituting functions (six O-methyls in both conpounds, four aromatic protons in this conpound and five in conpound 4). The difference of one in the number of aromatic protons could be accounted for by the presence of the second carbo nyl group in the new conpound (1660 cm ^ absorption in the ir, miss ing in case of conpound ^4). Accordingly, the new conpound could be assigned structure whose spectroscopic data was also in good agreement with spectroscopic data of a number of oxoaporphine alka loids (76). 90

(E) 7 1 -DIHYDROOXOTOALIADINE (1).

The spectroscopic properties of this conpound showed a great

similarity to those of conpound J3. The proton nmr spectrum (CDCl^,

270 !*Hz) revealed six O-methyl groups, a singlet at <5^ 4.66 (2H,

CH^OH, D^O exchangeable), and six aromatic protons (4 singlets and

two doublets at S 8.19 and 8.94 (AB system, J 5.3 Hz)). There was A d no absorption for methylene protons, me thine protons, N-methyl or

N-H groups in the nmr spectrum. The ir spectrum (CHCl^) exhibited a

peak for a conjugated carbonyl absorption at 1650 cm”1 (ketone C O )

and insignificant absorption at 3638-3680 (OH absorption).

The great similarity in the proton nmr, uv, ir and mass spectral

properties between this conpound and conpound suggested that this

conpound was the alcoholic form of conpound with the alcoholic

group located on C-2 of the assigned structure (9) for this new

oxoaporphine compound. In order to determine the exact location of

the substitution positions of the protons and other functional

groups of conpound % a double irradiation experiment was employed

(270 MHz). Results of the irradiation experiment were shown in

table 12. o h ,o h

O M e O M e £

Two nain features were taken into consideration in interpreting and correlating the results of irradiation. First, the two doublets at £ 8.19 and 8.94 were characteristic for aromatic protons at C-4 and C-5, respectively. Second, it was noted that exceptionally high

£ values were found for protons in the 11 position (C-ll position) of aporphines (or oxoaporphines) if position 1 was substituted by a methoxyl group (75, 77) and therefore the value of & 8.85 was assigned to the C-ll proton.

When the resonance region of the C-5 proton (£8.94) was irradi ated, 16 % Nuclear Overhauser Effect (NOE) enhancement was exhibit ed for the C-4 proton (£8.19). On the other hand, irradiation of the C-4 proton caused a NOE enhancement of 2% of a methoxyl group at

£ 4 .18 which was assigned to the OCH^ group on C-3 as it was the most adjacent group. As expected, irradiation of the protons of the

C-3 methoxyl group caused a NOE enhancement of 1% for the C-4 pro ton. 92

Table 12; M4R and NOE difference results for conpound J5.

(270 f*iz, c d c i 3 )

Absorption Region Effects On Other Groups Irr^iiated (S)

8.95 (H) 16% enhancement for (H,8.19) 8.19 (H) 2% enhancement for (Me0(4.18) 8.85 (H) 12% enhancement for (MeO, 4.10); 4% enhancement for (MeO, 4.12); 0.3% enhancement for (MeO,6. 59); 6.59; insignificant enhancement for (MeO#4.13) 4.10 (MeO) 2% enhancement for (H,8.85) (MeO,4.12) and (MeO,4.13) irradiated 4.12 (MeO) 6% enhancement for (H,8.85) (MeO,4.10) and (MeO,4.13) irradiated 4.13 (MeO) 6% enhancement for (H, 8.85) (MeO,4.10) and (Me0,4.12) irradiated 4.19 (MeO) 1% enhancement for (H,8.19) 7.89 (H) 3% enhancement for (H, 6.59) MeO groups (54.12, 4.13, 4.19) insignificantly affected 7.02 (H) 3% enhancement for CH-0H (54.66) 10% enhancement for (MeO, 3.93) MeO groups (4.12, 4.13, 4.19) insignificantly affected 6.59 (H) 2% enhancement for (H,7.84) 2% enhancement for (MeO, 3.78) MeO groups (4.12, 4.13, 4.19) insignificantly affected 4.66 (2H) 5% enhancement for (H,7.02) 3.78 (MeO) 13% enhancement for (H.6.59) 3.93 (MeO) 11% enhancement for (H,7.02)

Irradiation of the C-ll proton ( 5 8.85) caused a significant 93 enhancement of 11% for a methoxyl group at S 4.12 which was assigned

to C-10. An enhancement of 4 % was also observed for the methoxyl

group at % 4.12 which was assigned to the C-l position. The proton

at S 6.59 and the methoxyl at <5 4,13 were also affected by the irra diation of the C-ll proton but the effect was insignificant.

Methoxyl groups at <5 4.11, 4.12 and 4.13 seemed to be spatially

related since irradiation at the resonance region of any of these

three functional groups caused an effect on the absorption of the other two. In addition, the three methoxyl groups were spatially related to the C-ll proton ( S"8. 85) since enhancements of 17, 6 and

6% were caused by the irradiation of these methoxyl groups at the resonance regions of S 4.10, 4.12 and 4.13, respectively. This observation established the position assignments for the C-10 and

C-l methoxyls in addition to the C-2 methoxyl group which was assigned the resonance region of S 4.13.

Upon irradiation of the proton at <5 7,89, a NOE enhancement o f

3% was observed for the proton at S 6.59 while the absorption regions for the methoxyl groups at 5 3.78 and 3.93 were not a f f e c t ed. This observation established the assignment of position 8 f o r the proton at S 7.89 which was spatially related with the proton absorbing at <5 6.59.

From the previous observations, the remaining methoxyl groups

( h 3.78 and 3.93) were considered to be located on the substituted benzene moiety of the molecule. Irradiation at 5 3.93 (0CH3) caused 94

an enhancement of 13% for the proton at 5 6.59. On the other hand,

irradiation at the resonance region of <5*6.59 (H) resulted in 9% NOE enhancement for the methoxyl at <5 3.78 and 1.9% for the proton at 5

7.89 (on the oxoaporphine moiety) While the methoxyls at S 4.12,

4.13, and 4.19 were also affected but not so significantly. At the same time, no effects were observed in the ChÔH group resonance region. This observation placed this proton (<5~6.59) on an ortho position to the ether linkage. On the other hand, irradiation at the CHÔH resonance region (<5~4.66) resulted in a 5 % NOE enhance ment for the proton at 7.02. Alternately, irradiation at & 7.02 absorption region resulted in a 3% NOE enhancement for the CH2OH group at & 4.66 in addition to an enhancement of 10% for the methox yl group at S 3.93. These observations placed the CHÔH group on

C-2', the proton with the 6.59 chemical shift on C-6', and the pro ton with the £ 7.02 chemical shift on C-3'. On the other hand, methoxyl groups at <§" 3.78 and 3.93 must be placed on C-5' and C-4', respectively. This conclusion led to the assignment of the positions for all the protons and other functional groups (methoxyls and

CH^OH) of conpound and also to the related conpound, 8_ (tables 13 and 14). The position of the carbonyl group on C-7 in both com pounds was thus established. The location of the carbonyl group on

C-7 also accounted for the downfield positions of protons on C-8 in both coopounds. In addition, the substitution pattern in both com pounds related them to adiantifoline (5) and 6a, 7-dehydrothaliadine 95

Table 13: The NMR data of oxothaliadine (8) {90 Miz, CDCl^)

Functional group Chemical Shift, Protons

(C-l) MeO 4.12 3 (s) (C-2) MeO 4.15 3 (s) (C-3) MeO 4.20 3 (s) (C-4) H 8.20 (J * 5.7) 1

OM«

8;R = CH O 9;R = CH 2OH Conpounds 8 and 96

I Table 14; The Hi tMR data of 7'-dehydrooxothaliadine (9)

(90 MHz, CDC13 )

Funtional group Chemical Shift, Protons

(C-l) MeO 4.12 3 (s) (C-2) MeO 4.13 3 (s) (C-3) MeO 4.18 3 (s) (C-4) H 8.19 (J = 5.8) 1

*J = 5.8 Hz +-

(F) Dihydrothaliadine (11)

MeO.

0M« OMe it; R = CH 2OH R = CH O When the proton nmr and ir spectra of conpound 11^ and thaliadine

(13,2) were oonpared (table 15), certain similarities in spectro- 97 sccpic properties were observed. Certain regions of absorption in the ir spectra were quite similar although peak intensities were not identical. Likewise, the six methoxy groups, the N-methyl group and the aromatic protons in both compounds had similar chemical shifts in the proton nmr spectrum. The main difference between the two compounds in the nmr spectra was the resonance at 5 4.67 (CH^-CH) in conpound Id and the resonance at 5^ 10.38 CHO) in thaliadine (13).

In the ir spectra, the main difference was the band at 1680 cm-* for thaliadine (13) due to the absorption of the conjugated carbonyl group and the two bands (2850 and 2820 cm *) of the aldehyde proton

(absent in conpound Id) and the presence of the absorption of OH at

3550 and 3530 cm-1 in oonpound 11_ (absent in conpound 13).

These observations suggested that oonpound 11_ was the alcohol analog of thaliadine (13J which had been isolated from the roots of

T. minus race B in our laboratories (31). Additional evidence for the structure of this hemandaline-type conpound,

7' -dihydrothaliadine (11), was obtained by hydrogenation of

6a, 7-dehydrothaliadine (4) that yielded a conpound having identical nmr, ir, uv and tic properties with those of dihydrothaliadine (11).

The CD spectrum of dihydrothaliadine (11), with two negative mxima at 302 and 278 nm and a positive mximum at 242 nm was akin to that observed for thaliadine (31), and thus S stereochemistry was estab lished for the C-6a configuration of 7 '-dihydrothaliadine. Table 15 Comparative H-nmr and ir spectra of compound 11 and thaliadine

(a) 1 H-fWR spectra, 6 {90 MHz, C0C13)

Functional N-Me MeO MeO MeO MeO MeO MeO * H HHH

Compound 11 2.53 3.77 3.79 3.90 3.91 3.92 3.96 4.67 6.56 6.63 6.98 8.04

Thaliadine 2.50 3.79 3.81 3.91 3.91 3.93 3.97 10.38 6.46 6.77 7.40 8.08

Ir spectra (cm-*, CHCU) lbj______

Compound 11 3550 3530 - - 2395 1416 1376 1341 1280 1 2 1 0 1080 927

Thaliadine 2850 2820 2395 1680 1410 1380 1350 1280 1240 1080 940

♦CH^OH for compound 11 and CHO for thaliadine

UD CO 99 Me OMe Me MeN NMe OMe Me O

OMe MeO Me

(G) 6a, 7-DEHYDRCftDIAHTIFGUNE (&).

The mass spectrum with the molecular ion peak at m/z 724 was was

in good agreement with the assignments of the functional groups in

the proton nmr spectrum. Both spectra supported the formula

The nmr signal for one N-methyl group appeared at an

unusally low field position 2.98) while the nmr signal at 9.10

was characteristic for a downfield shift for the C-ll proton of a

dehydroaporphine. The downfield shift of the signal for an N-methyl

group adjacent to the 64-7 double bond of a dehydroaporphine had

been noted earlier by Mol lav et al. (77). Although the number of

the methoxyl groups and their relative positions in the nmr spectrum

was similar to that of adiantifoline, the ultraviolet spectrum with

its maximal absorption, as had been shown earlier, supported a high

ly conjugated chrctnophore. This latter structural feature was indi

cated also by the more intense absorption at 1600 cm * in the ir

spectrum (75). All these observations supported the assignment of

structure 14, 6a,-dehydroadiantifoline, for this ccnpound. 100 The mass spectral fragmentation pattern supported certain peaks

as expressed in figure 3.

M»l

>M* 'M*

369

Figure 3; Mass spectral fragmentation pattern of 6a,7-dehydroadiantifoline (14)

Additional evidence for the assigned structure was obtained from

the short time KMnO^ oxidation of adiantifoline (4) that yielded a

conpound having identical nmr, ir, uv and tic properties with those

of 6a, 7-dehydroadianti foline. Hence its relation to adiantifoline

was also established. 101

*MwO *0,,0n* *RT

S n

In order to determine the positions of the different protons and methoxyl groups of 6a, 7-dehydroadianti foline, an extensive nmr

Nuclear Overhauser Effect (NOE) study of the alkaloid was carried out (table 16). Certain criteria had been taken into consideration for assigning the spectral information. First, the positions of the

C-ll proton at ^ 9.10 and the N-methyl group at S' 2.98 of the dehy droaporphine moiety were previously observed and these two positions comprised very inport ant features in correlating the positions of certain protons and other substituents. In addition, the relative upfield shift of the C-8' proton at & 6.18 of the benzyl isoquinoline moiety was also an inportant criteria in correlating on C-7* posi tion and other relative positions.

As a result of the double irradiation process, the resonance positions for the protons and the methoxyl groups were assigned

(table 16). 1 ^

Table 16i WR and NOE difference results for 6a,7-dehydroadiantifoline (270 Miz, CDCl^)

Absorption Region Effects on Other Groups Irradiated (£)

C-ll H (9.10) OCR, at 4.04 enhanced by 12% ortho relation, assigned to C-10 C O T OCH3 at 3.99 enhanced by 3%; assigned to C-l 0CH3 H at 6.44 H at 6.80 enhanced by 18% NCH- at 2.98 enhanced by 11% Aliphatic protons enhanced by 0.7% C-8' H (6.18) H at 6.80 enhanced by 1% H at 6.67 irradiated OCH- at 3.53 enhanced by 13%; ortho relation, assigned to C-7'OCH-. 0CH3 at 2.98 enhanced by 4% 0CH3 at 3.53 H at 6.18 enhanced by 13% (ortho relation) Aliphatic protons H at 6.18 enhanced by 4% (<*') at 2.85-2.98 H at 6.54 enhanced by 12% (assigned to C-6" H) H at 6.80 H at 6.44 enhanced by 9% OCH. at 3.80 enhanced by 5% H at 6.57 enhanced by 1% H at 6.18 enhanced by 1% H at 9.1 enhanced by 0.2% 0CH3 at 3.78 H at 6.54 enhanced by 18% 0CH3 at 3.80 irradiated 0CH3 at 3.80 H at 6.72 enhanced by 12% (ortho relation) H at 6.54 enhanced by 7% 0CH3 at 3.78 irradiated OCH3 at 3.94 irradiated 0CH3 at 3.76 H at 6.67 enhanced by 23.1% (ortho relation) Aliphatic protons CH30 at 3.94 enhanced by 10%; at 3.21-3.28 assigned to C-3 OCH.. N-CH- at 2.98 enhanced by 9% OCH3 at 3.94 No hr enhanced; OCH- at 3.80 and 0CH3 at 3.98 irradiated OCH3 at 4.09 H at 6.18 enhanced by 6% H at 9.10 enhanced by 2% OCH3 at 4.04 irradiated 0CH3 at 3.98 H at 9.10 enhanced by 10% 103 Table (centi nued)

Absorption Region Effects on Other Groups Irradiated ($)

OCH., at 3.94 irradiated 0CM3 at 4.04 H at 9.10 enhanced by 4% OCH,, at 4.09 irradiated H at 6.54 OQi^ at 3.78 enhanced by 11% (ortho relation) OCH, at 3.76 enhanced by 3% Aliphatic protons at 2.85-298 {*’) enhanced by 3.0% H at 6.67 OCH, at 3.76 enhanced by 14% (ortho relation) H at 6.80 enhanced by 0.6% H at 6.54 irradiated

(H) SQUAROSINE (15).

Squarosine was named after T. squarrosum Steph. from which it

was first obtained in a small amount that prevented further study of

its structure.

The nmr spectrum indicated a bisbenzyltetrahydroeioqinoline

structure with fourteen protons in the me thy lene-me thine region.

This bisbenzylisoquinoline structure was also supported by a typical

absorption at 276 and 285 nm in the uv spectrum. Tie presence of

one diphenyl ether linkage joined tail to tail was evident from the

AA'BB' and ABX patterns at the two benzyl moieties. On the other

hand, the presence of two protons at the high field positions of

5.70 and 5.75, expected for the C-8 and C-8' protons, indicated the

absence of an ether linkage at either position and restricted the 104

MHz, CDC13 )

Position (ppm) Substituent and Multiplicity

C-l 3.98 CMe, s, 3H C-2 4.09 CMe, s, 3H C-3 3.94 Ome, s, 3H C-4, C-5 3.21-3.25 CH^-CH-, Conplex (m), 4H N-6 2.98 Me7 s, 3H C-7 6.44 H , S C-8 6.80 H, s C-10 4.04 OMe, s, 3H C-ll 9.10 H, s C-l* 3.78-3.82 H (buried under methoxyl groups at 3.78, 3.80 & 3.82) N - 2 ' 2.48 CMe, s, 3H C-3', C-4' 2.50-2.91 CH~-CH_, conplex (m), 4H C-5' 6.67 H, s C-6' 3.76 CMe, s, 3H C-7' 3.53 CMe, s, 3H C-8' 6.18 H, s &’ 2.86-2.93 CH_, conplex (m), 2H C-3" 6.72 H, s C-4" 3.80 CMe, s, 3H C-5" 3.78 CMe, s, 3H C-6" 6.54 H, s s = singlet; m = multiplet I H-

presence of the remaining substituents (two me thy 1 ened i oxy and two

methoxyl groups) on carbons 5,5',6,6',7 and 7'. Thus, structure Aa

or Ab could be postulated for this conpound.

Analysis of the mass spectral peaks of this conpound (figure 3)

showed the absence of any mass fragments higher than m/z, 220 (the

base peak). This base peak on accurate ness measurement gave conpo- 105 sition corresponding to the fragment ion a or b which was in good

agreement with the nature of the proposed substituents on the two

isoquinoline moieties.

NM*

A j ;N,5«,:CMi.IVM* 4 a .*, = *,= CM, / \

<5 a * i Double irradiation and NOE difference results (table 18) indi cated the clear ortho relation between the protons at positions 8 and 8' and the corresponding methoxyl groups. On irradiation of the proton at 5.75 ppm, a NOE enhancement of 16% was observed for the methoxyl group at 3.64. Likewise, irradiation of the proton at 5.70 ppm caused a NOE enhancement of 8% for the methoxyl group at 3.61 ppm. Alternatively, irradiation of the methoxyl group at 3.61 ppm caused a NOE enhancement of 11% for the proton at 5.70 ppm. These results indicated that the methoxyl groups at 3.64 and 3.61 must be located on carbons 7 and 7', respectively. On the other hand, one of the two methylenedioxy groups nust be located between C-5 and C-6 of one isoquinoline unit and the second group between C-5* and C-6' of the other isoquinoline unit. 106

This conpound was thus assigned structure Aa or 15. The two

asymmetric centers were assigned S,S-configuraticn from the circular

dichroism spectrum which had similar characteristics to that of tha-

liracebine (19) of known stereochemistry (16).

MeN NMe

Aa (is)

Table 181 "Si IsMR NOE difference results for squarosine (15).

(270 NHz, CDC13)

Protons Irradiated Region (5) ppm Comments

H-8' 5.70 8% enhancement for MeO at 3.61

H-8 5.75 16% enhancement for MeO at 3.64; H (5.70) irradiated

MeO 3.61 11% enhancement for C-8' H at 5.70

MeO 3.80 13% enhancement for Ar-H (C-13 H, part of ABX system) (6.89)

+ 107

(I) 6-N0RADIANTIF0L1ME (^4);

This alkaloid was obtained as a minor amorphous base. Its spec

tral data (nmr, ir, uv) showed a marked resemblence to those of

adiantifoline (5) indicating that it is a benzyltetrahydro-

isoquinoline-aporphine dimer. The main difference in the nmr spec

trum was the presence of only one N-Me group at

~~Generally, the benzylisoquinoline-aporphine dimers are obtained~~

~~as tertiary amines in which each of the two nitrogen atcms bears a~~

~~methyl group (88). The only two exceptions so far known are northa-~~

~~licarpine (jt0,89) and 2'-noradiantifoline (31,88) isolated from T.~~

~~revolutum D.C. and T. minus L. var micrcphy 1 lum Boiss., respective~~

~~ly. In both alkaloids, the N-methyl group is present on the apor-~~

~~phine rather than the benzyltetrahydroisoquinoline moiety. Since~~

~~2 '-noradi anti foline is directly related to adiantifoline (5), its~~

~~nmr data was compared to that of adiantifoline and compound 24~~

~~(table 19).~~

~~30; R= H 31 i R - OMe 108~~

Hie data showed clear ly that the three compounds were very much related but they were different. In particular, the mass spectra were most revealing (figures 4 and 5). The fragmentation patterns shewed different values and intensities for peaks displayed by

~~2'noradiantifoline and conpound 24 (table 20). That the N-methyl group was present on the benzy ltetrahydroisoquinoline rather than the aporphine residue was indicated by the presence of peaks at m/z~~

506 (17%), 356 (100%) and 206 ( 70%) (figure 4). Thus, conpound 24 was established as 6-noradiantifoline and its nmr data were assigned as in table 21. The absolute configuration of 6-noradi anti foline as

~~S,S was indicated by the general similarity of its CD curve with that of adiantifoline and 2'-noradiantifoline. 109 — h~~

~~Table 19; Campar i son of nmr da ta (5) of adi ant i fol i ne, 2*-noradiantifoline and compound 24.~~

~~Group Adiantifoline 2-noradiant i foline Compound~~

N-Me 2.43 2.44 N-Me 2.48 2.51 — O—Me 3.58 3.57 3.56 O-Me 3.77 3.60 3.77 O-Me 3.77 3.74 3.77 O-Me 3.79 3.78 3.77 O-Me 3.82 3.81 3.82 O-Me 3.89 3.90 3.90 O-Me 3.95 3.90 3.93 O-Me 3.96 3.96 3.96 Ar-H 6.19 6.21 6.17 Ar-H 6.50 6.47 6.47 Ar-H 6.55 6.55 6.55 Ar-H 6.61 6.56 6.58 Ar-H 6.62 6.73 6.58 Ar-H 8.05 8.05 8.06

~~■+ 110~~

~~Me OMe Me i.+ NH MeN Me MeO~~

~~OMe MeO OMe m/z 356~~

~~MeO^y^N^ NMe~~

~~m/z 206 / OMe m/z 506 OMe~~

~~m/z 356~~

~~Figure 4; Mass spectral fragmentation pattern of oonpound 24 Me°T^NlT^+~~

~~m/z 192~~

~~MeO~~

~~OMe m/z 519 OMe~~

~~m/z 369~~

Figure 5: Rationalization of the mass spectral fragmentation pat tern of 2 '-noradiantifoline Table 20 Values and intensities of mass spectral fragments of 2'-noradiantifoline and compound 24 Peak Value 712 681 520 369 192 177 2'-Noradiantifoline

~~Intensity 0.3 0 . 6 6 . 0 1 . 0 1 0 0 . 0 8 . 0~~

~~Peak Value 506 356* 206 150 119 97 Compound 24~~

~~Intensity 17.0 1 0 0 . 0 70.0 76.0 2 1 . 0 24.0~~

*This value could also account for the double-charged mass fragment 712/2z

~~Table 21 The nmr data of compound 24 (6 -noradiantifoline) (5, 90 MHz, CDC1 ^)~~

~~Function C-l C-2 C-3 C-10 C-6 ' C-7’ C-4H C-5" C - 8 C-ll C-5' C-8 1 C-3 '1 C-6 1' al Group 2'N Me OMe OMe OMe OMe OMe OMe OMe OMe H HH H H H~~

~~6 Value 2.44 3.77 3.96* 3.90 3.93* 3.82 3.56 3.77 3.77 6.55** 8.06 6.47 6.17 6.58** 6.58** * and ** may be Interchangeable 113 (J) THMJTNE (&) AND THALFIMINE (16).~~

~~Thaifine and thalfinine were first isolated from T. foetidum L.~~

~~by Abdizhabbarova et al in 1968, and their structures were predicted~~

~~and reported two years later by the same authors (88). Thalfine was~~

~~also isolated from T. minus L. race B by Geiselman et al (59).~~

~~Reduction of thalfine with Zn-HCl followed by N-methylation yielded~~

~~two compounds identified as thalfinine and its diasterecmer epithal~~

~~finine that had the opposite stereochemistry at C-l' of the mo ncme-~~

~~thylated benzyl isoquinoline unit (16). The asynmetric center of the~~

~~trimethoxylated benzylisoquinoline unit of thalfinine at C-l was~~

~~designated S as in thalfine (16) and the remaining asynmetric center~~

~~at C-l* was also deduced to be S on the basis of the biogenetic~~

~~relation of thalfinine and thalirabine (32) that had an established~~

~~S,S configuration.~~

~~^NM* OM* "H~~

~~As in several isolated bisbenzylisoquinoline alkaloids with~~

~~adjacent methoxyl and methylenedioxy groups on carbons 5, 6, and 7~~

~~of the isoquinoline unit, thalfine and thalfinine were assigned~~

~~structures 6a_ and 16a, respectively (p 73) with the methylenedioxy~~

group between C-6' and C -7’ and the methoxyl group on C-5'. In 114 order to assess the validity of this assignment and also to deter mine the exact location of the protons and other substituent groups on the bisbenzylisoquinoline skeleton of thalfine and thalfinine, an extensive study was carried out employing double irradiation and NOE experiments in addition to the techniques of carbon-proton correla tions and correlation by long range coupling (coloc). The 1^C-nmr data of both compounds supported their fully-characterized struc tures.

~~a) Thalfine.~~

~~The Hi-nmr spectrum of thalfine (CDCl^, 270 Miz,~~

~~Figure37 ) exhibited a single N-methyl peak at 2.28 ppm,~~

~~nine aliphatic protons, four methoxyl groups, a methy~~

~~lenedioxy group and ten aromatic protons.~~

~~Four of the nine aliphatic protons appeared as mul-~~

~~tiplets at regions 1.78-1.93, 1.94-2.01, 2.08-2.18 and~~

~~2.49-2.56 ppm. Spin-decoupling and NOE experiments~~

~~revealed the spectral relations between these four pro~~

~~tons. Double irradiation of the doublet of triplet pro~~

~~ton at the 2.49-2.56 region resulted in the collapse of~~

~~the two raultiplets at the resonance regions of 1.78-1.93~~

~~and 1.94-2.01 ppm to two triplets While the nultiplet at~~

~~2.08-2.18 was caused to collapse into a doublet of doub~~

~~let. On the other hand, NOE experiments (Table 25)~~

~~resulted in 14%, 6% and 8% enhancements for the protons 115~~

~~at 2.08-2.18, 1.94-2.01 and 1.78-1.93 ppm, respectively~~

~~when the proton at 2.49-2.56 (2.53 ppm) was irradiated.~~

~~It is clear from these enhancement values and from the~~

~~carbon-proton correlation spectrum (Figure 4 0 ) that the~~

~~two protons at 1.78-1.93 and 1.94-2.01 ppm were the C-4~~

~~protons while the two protons at 2.08-2.18 and 2.49-2.56~~

~~ppm were the C-3 protons. Calculations of the coupling~~

~~constants supported the suggested conformational posi~~

~~tions of the four protons as in table 22.~~

~~Table 22: The coupling constants of C-3 and C-4 protons of thal fine~~

~~Protons Resonance Region Coupling Constants (in Hz)~~

C-3 He 2.49-2.56 11.7 (gem), 4.6 (ae), 4.1 (ee) C-3 Ha 2.08-2.18 11.7 (gem), 9.2 (aa), 4.1 (ae) C-4 He 1.94-2.01 16.0 (gem), 4.1 (ae), 4.1 (ee) C-4 Ha 1.78-1.93 16.0 (gem), 9.2 (aa), 4.6 (ae) gem = geminal, a = axial, e = equatonal

~~Three of the five remaining aliphatic protons formed~~

~~an ABX system that could be related to an AMX pattern~~

~~with as a doublet of doublets (3.32 ppm, J^g = 15.8~~

~~Hz, J = 2.0 Hz), Hg as a doublet of doublets (3.18~~

~~ppm, = 5.0 Hz) and H^ as a multiplet (3.54 ppm) 116 which was partially buried by the methoxyl group at 3.59 ppm. Spin decoupling of the proton at 3.54 ppm col~~

~~lapsed the other two protons into an AB quartet (J^ =~~

~~16.5 Hz). These two protons were assigned as C-& pro tons while the partially buried proton was assigned as~~

~~C-l proton.~~

~~The two remaining aliphatic protons appeared as an~~

~~AB quartet (J^ = 14.3 Hz) at the downfield positions of~~

~~4.56 and 4.81 ppm and were assigned to C-a' on the basis of their multiplicities as well as their position on a carbon alpha to two aromatic systems.~~

In the aronatic region, one of the ten aromatic pro tons appeared as a doublet at 5.99 ppm (J = 2.0 Hz) and was shown by double irradiation and NOE experiments to be C-10 proton (H^ of an aromatic ABX system). When this proton was irradiated, one of the two overlapping aromatic protons at the 6.72-6.75 resonance region had its peaks sharpened. The NOE experiments showed enhancements of 5% for the proton at the resonance region 6.72-6.75 ppm and 3% for another proton, a doub let, at 6.79 ppm when the C-10 proton at 5.99 ppm was irradiated. Thus, the two protons at 6.72-6.75 (6.73) and 6.79 ppm were assigned to the C-14 and C-l3 protons, respectively. 117 The singlet at 6.46 ppm was assigned to the C-Q pro

~~ton. The assignment was supported by the NOE experiment~~

~~since irradiation of its resonance region enhanced the~~

~~C-l proton by 4% and the methoxyl group at 3.75 ppm by~~

~~13%; the methoxyl group was thus assigned the C-7 posi~~

~~tion as a result of this significant value {ortho rela~~

~~tion) .~~

~~The two overlapping triplets at the resonance region~~

~~7.11-7.20 ppm were assigned the C-10’ and C-14' protons~~

~~since irradiating the resonance region at 4.81 (one of~~

~~the C-&* protons) resulted in 8% enhancement for these~~

~~two protons. Alternatively, irradiation of 7.11-7.20~~

~~resonance region resulted in 2% and 3% enhancement for~~

~~the C—&' two protons. Since the location of the C-10'~~

~~and C-14' protons was already assigned, then the C-ll'~~

~~and C-l3' proton could be assigned to two separate reso~~

~~nance regions. A doublet of doublets appeared at the~~

~~resonance region of 6.65-6.69, and a partially overlap ping doublet of doublets seemed to appear at the~~

~~6.74—6.78 resonance region (part of the complex multiplet between 6.71 and 6.81 ppm integrated for three pro~~

~~tons). These two protons were thus assigned as the~~

~~C-ll* and C-13’ protons. 118~~

~~The four methoxyl groups resonances were exhibited~~

~~at 3.47, 3.54, 3.75 and 3.89 ppm. The NOE experiments~~

~~indicated the ortho relation between the C-8 proton at~~

~~6.46 ppm and the methoxyl group at 3.75 ppm as well as~~

~~the ortho relation between the C-l3 proton at 6.79 ppm~~

~~and the methoxyl group at 3.89 ppm. Irradiation at the~~

~~3.75 resonance region caused a 10% enhancement for the~~

~~C-8 proton at 6.46 while irradiating the methoxyl group~~

~~at 3.89 resulted in 12% enhancement for the C-l3 proton at 6.79 (table 23). Thus the methoxyl groups at 3.75 and 3.89 were assigned positions on C-7 and C-l2,~~

~~respectively. Irradiation of the methoxyl at 3.59~~

~~resulted in the enhancement of 1.5% and 1.6% for the~~

~~N-Me group at 2.28 and the C-8 proton at 6.46, respec tively which indicates the possible location of this methoxyl group on C-6. Additional evidence was obtained~~

~~from the coloc technique (table 24). Since the protons of the methoxyl group at 3.59 and the C-8 proton corre~~

late to the carbon having 137.8 ppm value (figure 4 2), this carbon must be C-6 and the methoxyl group at 3.59 ppm .-nmr) must be located on this carbon while the methoxyl group at 3.47 must be located on C-6'.

~~The methylenedioxy group was shown in the proton nmr spectrum as an AB quartet (J = 1.5 Hz) at 6.15 and 6.16 119 ppm. Irradiation of this resonance region resulted in~~

~~0.8% enhancement for the methoxyl group at 3.47 and 0.6%~~

~~enhancement for the C-4* proton. In addition, the pro-~~

~~ten on C-4' was shown to correlate with the carbon at 13 137.1 in the C-nmr spectrum. At the same time, the~~

~~methylenedioxy protons were shown to correlate with car~~

~~bons 137.1 and 139.6 (coloc) which suggests that the 13 singlet at 137.1 ( C-nmr) must be C-5' resonance and~~

~~that the singlet at 139.6 must be C- 6 ' resonance (table~~

~~24). The afore-mentioned observation indicates that the~~

~~methylenedioxy group is located between C-5* and C- 6 ‘~~

~~and the methoxyl group at 3.47 (^H-nmr) must be on C-7’.~~

~~Accordingly, analogous assignments must be considered~~

~~for thalfinine.~~

~~As a result, thalfine was assigned structure 6 b~~

~~(page 122) and table 26 showed its proton nmr data. The 13 . . . C-nmr spectrum (table 25, figure 3 9 ) showed thirty eight carbons and was in accord with the suggested~~

structure. Assignment of the carbons was based on the miltiplicities obtained from the singlet frequency off- resonance decoupling experiments (SPORD), the carbon- proton oorrelat1 0 ns and the long range carbon-proton coupling correlations (coloc) tediniques (Figures 38,

~~4b and 43). 120~~

~~Table 23: XH tWR and NOE difference results for thalfine (6) (270 MHz, CDC13~~

~~Absorption Region Effects on Other Groups Irradiated {5)~~

2.29 (N-GH) 6% enhancement for the C-3H at % 2.53 1% enhancement for one of C- a Hs (5 3.18-3.24) 3% enhancement for the other C-a H (S 3.29-3.35) 7% enhancement for the C-l H (obscured proton at 6 3.54) 1% and 0.9% enhancement for C-l3 & C-14 protons at 6.68 & 6.79, respectively 3% enhancement for C-14 proton

2.53 (C-3 H,m) 14% enhancement for the other C-3H (52.10-2.18); collapsed from ddd to dd 6% enhancement for one of C-4 Hs ( 51.91-1.99); collapsed from ddd to dd 8% enhancement for the other C-4 H (S l.79-1.90); collapsed from ddd to dd

~~C-a ‘ H (4.56) 13% enhancement for the other C-a ' proton (geminal) 1% enhancement for OCH^ (3.75) 12% enhancement for C-14 proton 3% enhancement for O-CHj-O~~

4.81 (C-a' H) 14% enhancement for the other C-a' proton at 4.56 2% enhancement for OCJU (3.89); 3% enhancement for C-l proton (3.54); 10% enhancement for C-10 proton (5.99) 8% enhancement for C-10' and C-14'

~~5.99 (C-10 H) 5% enhancement for C-14 H 3% enhancement for C-l 3 H 0.8% enhancement for C-8 H~~

~~6.15 (0-CH2-0) 0.8% enhancement for OCH, (3.47); 0.6% enhancement for C—4 proton~~

~~7.45 (C-4* H) 20% enhancement for C - 3 1 H (collapsed into a singlet) C-10 H ( 5.99) enhanced by 2.3% 12 Table 23 (continued)~~

~~Absorption Region Effects on Other Groups Irradiated (3)~~

~~7.11-7.20 (two 40% and 38% enhancements for overlapping dd; C-10' C-ll' H & C-l3* H (collapsed to two d) H & C-14' H) 7% enhancement for the MeO at 3.59 2% & 3% enhancements for the C—&' protons~~

~~C-8 H (6.46) 7% & 1% enhancements for C-& protons 4% enhancement for C-l proton 13% enhancement for 0CH3 (3.75);~~

~~3.75 (0CH3) OCH at 3.89 irradiated (- 17.8%) 2% enhancement for C-10 proton (5.99) 10% enhancement for C-8 proton (6.46)~~

3.59 (0CH3) 2% enhancement for N-CH. (2.28) 2% enhancement for C-10 proton (5.99) 2% enhancement for C-8 proton (6.46) 1% enhancement for C-14 proton (6.68) 6% enhancement for overlapping C-10' and C-141 protons (7.13-7.17)

~~0CH3 (3.89) 12% enhancement for the C-l3 proton; 4% enhancement for the C-14 proton; 0.9% enhancement for C-10 proton; 0.2% enhancement for C-8 proton; 2% enhancement for N-CH3 (2.28) 122~~

~~Table 24i Carbon-proton correlation via long range coupling con stant (coloc) of thafine (6)~~

~~Carbon ppm Long Range Correlated Protons~~

C-4a 119.6 C-4 H, C-8 H C-5 148.6 C-8 H C-6 137.8 C-8 H, C-6 OMe 3H C-7 151.5 C-8 H, C-7 OMe 3H C-8a 132.6 C-1H, C-a 2H C-9 130.9 C-1H, C-a 2H, C-l3 H C-ll 148.6 C-13H C-l 2 147.9 C-14 H, C-l2 OMe 3H C-l' 157.9 C-3 'H, C -a’ 2H C-4'a 118.8 C-3 'H C-5’ 137.1 C-4'H, O-CH-O 2H C-6' 139.6 O C H 9-0 2H C - 7 1 134.8 C-7’CMe 3H c-a* 142.2 — C-8a* 118.2 C-4'H C-9' 136.4 C-a* 2H, C-11'H, C-l31H C-l 2 ' 154.5 C-10'H, C-14'H

~~Thalfine (6b) 123~~

~~^------h~~

~~Table 25i 13C NMR (CDC13 ) data of thalfine (6) (270 Miz, CDC13 )~~

Carbon Mult. ppm Carbon Mult. ppm C-l d 63.1 C—4 a 1 s 118.8 C-3 t 51.2 C-5* s 137.1 C-4 t 22.8 C-6' s 139.6 C-4a s 119.6 C-7' s 134.8 C-5 s 148.6 C-8' s 142.2 C-6 s 137.8 C-8'a s 118.2 C-7 s 151.5 C-a’ t 46.7 C-8 d 105.3 C-9' s 136.4 C-8a s 132.6 C-10' d 129.4* C-a t 36.1 C-ll' d 120.8 C-9 s 130.9 C-l 2' s 154.5 C-10 d 118.4 C-l 3' d 120.8 C-ll s 148.6 C-14' d 128.8* C-l 2 s 147.9 N-Me q 43.6 C-l 3 d 111.7 C-6 CMe q 60.2 C-14 d 123.5 C-7 CMe q 55.9 C-l" s 157.9 C-l2 CMe q 55.9 C-3* d 140.5 C-7' CMe q 59.6 C-4' d 110.6 o -c h 2-o t 102.6

~~s = singlet , d = doublet, t = triplet, q - quartet singlets were assigned via coloc technique doublets, triplets and quartets were assigned via C-H correlation ♦Assignments may be interchanged~~

~~+- + Table 26 The proton nmr data for thalfine (6 ), (C0C1^* 270 MHz)~~

~~Functional Chemical shift Functional Chemical shift group (ppm) Protons group (ppm) Protons~~

~~1 C-l H 3.54 (m) a C-l 3 H 6.79 1 (d)~~

~~N-He 2.28 3 (s) C-14 H 6.73 1 (dd) C-3 H 2.08 - 2.18 1 (m) a C-3* H 8.39 1 (d)~~

~~1 C-3 H 2.49 - 2.56 (dt) e C-4' H 7.45 1 (d)~~

~~1 C-4 H 1.78 - 1.93 (m) a 0-CH--0 (6.15, 6.16) 2 (q) C-4 H 1.94 - 2.01 1 (dt) eC-7 OMe 4 3.47 3 (s)~~

~~C - 6 OMe 4 3.59 3 (s) C-a' (a-H)4 4 4.81 1 (d)~~

~~C-7 OMe 3.75 3 (s) C-a' (e-H)44 4.56 1 (d)~~

~~C - 8 H 6.46 1 (S) C-ll' 6.65 - 6.69 2 (two d, C-a (a-H) 3.18 1 (dd) C-13' 6.74 - 6.78 one over C-a (e-H) 3.32 1 (dd) lapping)~~

C-10 H 5.99 1 (d) C-10' 7.11 - 7.15 2 (two C-12 OMe 3.89 3 (s) C-14' 7.15 - 7.18 overlap- Dino dd) Apartlally burled by the OMe group at 3.59 ♦Data obtained via correlation through long range coupling (coloc) ♦♦Assignments may be Interchanged; e * equatorial, a = axial 125 b) Thalfinine.

~~The ^H-runr spectrum of thalfinine revealed peaks for~~

~~two N-methyl groups, fourteen aliphatic protons, four~~

~~methoxyl groups, a methylenedioxy group and eight aro~~

~~matic protons.~~

~~The aliphatic protons were exhibited as multiplets~~

~~of varying complexities. Spin-decoupling and NOE exper~~

~~iments performed at 500 NHz revealed spatial relations~~

~~between certain protons and led to the assignment of~~

~~most of these aliphatic protons. Irradiation of the~~

~~nultiplet at 4.46 ppm partially collapsed two complex~~

~~2-proton multiplets at 3.09-3.14 and 3.18-3.22 ppm into~~

~~an AB quartet {J = 16.3 Hz). On the other hand, irradi~~

~~ation of the partially buried proton at 3.49 ppm col~~

~~lapsed a doublet of doublets at 3.35-3.39 ppm and one of~~

~~the overlapping multiplets (a doublet of doublets) at~~

~~3.18-3.23 ppm into another AB quartet (J = 16.4 Hz).~~

~~This helped identify the six protons of two ABX systems.~~

~~To locate the positions of these protons, a series of~~

~~irradiations were carried out. First, irradiation of~~

~~the aromatic proton (a doublet characteristic for the~~

~~C-10 proton) at 5.98 ppm, beside collapsing the doublet~~

~~of doublets at 6.76 ppm (Hg of the aromatic ABX system)~~

into a doublet, it also caused the peaks of the doublet 126 of doublets at 3.35-3.39 (3.37) ppm to sharpen signifi cantly. In addition, irradiation of the singlet at 6.42 ppm (characteristic for the C - 8 proton) resulted in a

~~10% NOE enhancement for the same proton at 3.37 ppm.~~

This helped identify this proton as one of the C-a pro tons since the protons on this carbon are spatially related to both protons on C - 8 and C-10. Thus, the pro ton at 3.49 ppm was established as the C-l proton while the two protons at 3.18-3.23 (3.20) and 3.35-3.39 (3.37) ppm were assigned as the C-a protons. On the other hand, the three protons that form the other aliphatic

~~ABX system could be assigned to the C-l* proton at 4.46 ppm and to the C-a* protons at 3.09-3.14 (3.12) and~~

~~3.20-3.23 (3.22) ppm.~~

The two N-methyl groups were exhibited in the 'Hl-nmr as two singlets at 2.37 and 2.62 ppm. To locate their positions on the two benzyl isoquinoline units, both resonance regions were separately irradiated. When the signal at 2.62 was irradiated, a 7% NOE enhancement was observed for the proton at 4.46 (the C-l' proton).

~~Accordingly, the signal at 2.62 was assigned to the~~

N-methyl group at position 2 ’ on the mono-methoxy 1 ated benzylisoquincline unit. Alternatively, irradiation of the signal at 2.37 resulted in a 10% enhancement for the 127 proton at 3.49 (the C-l proton), a 2% enhancement for the proton at 5.98 (the C-10 proton) and a 12% enhance ment for a proton at 2.81 (expected for one of C-3 pro tons). Thus, the signal at 2.37 was assigned to the

~~N-methyl group at position 2 on the trimethoxylated ben zyl isoquinoline unit.~~

The complex multiplet at 2.79-2.86 integrated for three protons. One of these protons (at 2.81) had already been observed as a C-3 proton on irradiation of the N-Me signal at 2.37. Irradiation of the doublet of triplets at 2.52-2.57 (2.54) resulted in the partial collapse of the two one-proton multiplets at 2.21-2.27 and 2.30-2.34, as well as the multiplet at 2.79-2.82

(2.81). This indicated that these four protons were coupled and were located at C-3 and C-4. The carbon- proton correlation spectrum gave further evidence for the aforementioned location of the four protons. Since

~~C-4 generally resonates at a higher field coopered to~~

~~C-3 (91,92), the signal at 23.6 was assigned to C-4 and the signal at 52.6 was assigned to C-3 (figure 4 7 , table~~

~~29). The carbon-proton spectrum related the two protons at 2.21-2.27 and 2.52-2.57 to C-4, the two protons at~~

~~2.30-2.34, and 2.79-2.82 to C-3. In addition, the four remaining aliphatic protons exhibited as multiplets at 128 3.06-3.09 (one proton), at 2.83-2.91 (two protons) and~~

~~2.63-2.66 (one proton) were related to C-3 1 and C - 4 1.~~

~~Accordingly, the carbon-protcn spectrum related the two protons at 2.86-2.91 and 3.06-3.09 to C-3’ while the two protons at 2.63-2.66 and 2.83-2.85 were related to C-4'.~~

The singlet at 6.42 was assigned to the C - 8 aromatic proton as mentioned earlier. The assignment was sup ported by an NOE experiment since irradiation of its resonance region enhanced the methoxyl group at 3.74 and the C-10 proton at 5.98 by 13% and 4%, respectively.

~~Irradiation of the doublet at 5.98, assigned earlier as the C-10 proton, collapsed the doublet of doublets at~~

~~6.76 into a doublet (J =8.0 Hz) which formed an AB quartet with the doublet at 6.80. As in thalfine, this indicated that these three protons formed an ABX system with at C-10, at C-13, and Hg at C-14.~~

~~The four remaining aromatic protons on carbons 10', 11',~~

~~13' and 14' appeared as broad signals at 6.70 and 7.15~~

(at 270 and 500 f>tlz). However, the ^H-nmr at 90 MHz showed two multiplets at 6.64-6.74 and 7.13-7.21 each integrating for two protons. The carbcn-proton correla tion spectrum showed the two protons at 6.64-7.74 coupled to carbons resonating at 120.5 ppm. The signal at 120.5 was also shown by broad band decoupling inte grating for two carbons. 129

~~As in thalfine and other similar bisbenzylisoquino-~~

~~line alkaloids, C-ll* and C-13' generally resonate at~~

~~higher fields conpared to C-10 1 and C-14* and usually~~

~~appear at the same resonance region (93). Therefore,~~

~~the signal at 120.5 integrating for two carbons could~~

~~possibly be assigned to C-ll' and C-13*. The two broad~~

~~signals at 127.5 and 129.7 obtained by gated decoupling~~

~~could be assigned to C-10' and C-14'.~~

~~The four methoxyl groups were exhibited in the~~

~~Hi-nmr as four singlets at 3.40, 3.50, 3.74 and 3.88.~~

~~The NOE experiments indicated the ortho-relation between~~

~~the C - 8 proton at 6.42 and the methoxyl group at 3.74 as~~

~~well as the ortho relation between the C-13 proton at~~

~~6.80 and the methoxyl group at 3.88. Irradiation at the~~

~~3.74 resonance region resulted in 9% NOE enhancement for~~

~~the C - 8 proton at 6.42 while irradiation at 3.88 result~~

~~ed in 14% NOE enhancement for the C-13 proton at 6,80~~

~~and 3% NOE enhancement for the C-14 proton at 6.76,~~

~~Thus, the methoxyl groups at 3.74 and 3,88 were assigned positions on C-7 and C-12, respectively. Irradiation of~~

~~the methoxyl at 3.40 resulted in 2% NOE enhancement for~~

~~the methoxyl at 3.47 while irradiating the methoxyl at~~

~~3.50 casued only 0.8% NOE enhancement for the methoxyl~~

at 3.74 (table 30). This observation suggested that the 130 methoxyl at 3.40 could be assigned position 6 . The spectrum of the long range carbon-proton coupling corre lation (coloc) gave further support for this assignment.

~~Since the protons of the methoxyl group at 3.40 and the~~

~~C - 8 proton correlate to the carbon resonating at 137.3~~

~~Table 28), this carbon must be C - 6 and the methoxy group at 3.40 (Hi-nmr) must be located on this carbon.~~

As in the case of thalfine, the remaining methoxyl group at 3.50 should be assigned on C-7‘ and the methy lenedioxy group at 5.89 should be located between C-5* and C-6 1. The evidence supporting the above-mentioned assignments was obtained from the long range carbon- proton coupling spectrum (coloc). The protons of C-4' and those of the methylenedioxy group correlate to the carbon resonating at 141.0 (three-bond correlation) whereas the protons of the methoxyl group at 3.50 only correlate with the carbon resonating at 133.0 (table 28.

~~Accordingly, thalfinine was assigned structure 16b instead of 16a as has been previously reported. Table 13 30 shows its proton nmr data. The C-nmr spectrum~~

(table 29, figure 4 5 ) showed 39 carbons and was in accord with the suggested structure. Assignment of the carbons was based on the multiplicities obtained from the single frequency off-resonance decoupling experi- llil ments (SFV3RD), the carbon-proton correlations and the long range carbon-proton coupling correlations (coloc) techniques.

~~16b 132~~

~~Table 27: "Si fcWR and NOE difference results for thalfinine (16) (270 M z , CDC13 )~~

~~Absorption Region Irradiated (S) Effects on Other Groups~~

2-37 (N-Me) 12% enhancement for H at2.81; 10% enhancement for H at3.49; 2 0 % enhancement for H at 5.98 2.62 (N-Me) 7% enhancement for H at4.46 2.24 (H) 20% enhancement for Hat 2.54; 23% enhancement for Hat 2.81 4.46 (H) Hs at 3.12 and 3.22 were significantly enhanced. 6.42 (H) 13% enhancement for MeO at 3.74; 4% enhancement for H at5.98; 10% enhancement for H at3.37 5.98 (H) H at 6.76 was significantly enhanced 5.89 (O-CH.-O) 4% enhancement for MeO at 3.50 3.40 (OMe) 2% enhancement for MeO at3.74; 1% enhancement for MeO at3.88 3.50 (OMe) 1% enhancement for MeOat 3.40; 1% enhancement for MeO at 3.74 3.74 (OMe) 9% enhancement for H at6.42 3.88 (OMe) 3% enhancement for H at 6.76 14% enhancement for H at 6.80

~~+ ■+ 133~~

~~♦ — — ■ ' ' ------i i ------' ------...... — — — - ■ ■■■ \ I I Table 28; Carbon-proton correlation via long range coupling oon- | stant (coloc) of Thalfinine (16)~~

~~Carbon ppm Long Range Correlated Protons~~

C-4a 119.9 C-4 H, C-8 H C-5 147.3 C-4 H C - 6 137.3 C - 8 H, C- 6 CMe 3H C-7 151.4 C - 8 H, C-7 OMe 3H C- 8 131.7 C-l H, C-a 2H C-9 131.1 C-l H, C-a 2H, C-13H C-ll 148.7 C-13H C-l 2 147.8 CMe, C-l2 OMe 3H C-4a' 124.2 C-l* C-5' 141.0 C-4', 0-CH2-0 2H C-6 ' 136.0 0-CH„-0 2H C-7' 133.0 C-7 CMe 3H C-8 ' 142.3 C-l' C-8 a ' 109.8 C-4* C-9' 137.6 C-a 1 C—12' 153.2 —

~~+• 134~~

~~Table 29: 13C tMR data of thalfinine (16) (270 NHz, CDC1~~

Carbon Mult. ppm Carbon Mult. ppm C-l d 64.1 C-4a' s 124.2 C-3 t 52.6 C-5' s 141.0 C—4 t 23.6 C-6 * s 136.0 C-4a s 119.9 C-7* s 133.0 C-5 s 147.3 C-8 ' s 142.3 C - 6 s 137.3 C-8 a ' s 109.8 C-7 s 151.4 C-a' t 37.1 C - 8 d 104.5 C-9' s 137.6 C-8 a s 131.7 C-10' d 127.5* C-ll' d 120.5 C- a t 36.1 C-12 1 s 153.2 C-9 s 131.1 C-13* d 120.5 C-10 d 116.9 C-14' d 129.7* C-ll s 148.7 N-Me q 44.1 C-l 2 s 147.8 N-Me q 42.1 C-13 d 1 1 1 . 8 C - 6 MeO q 59.0 C-14 d 123.3 C-7 MeO q 56.5 C-l' d 59.0 C-12 MeO q 56.2 C-3' t 45.1 C-7* MeO q 59.5 C -4’ t 2 0 . 2 o -c h 2-o t 101.3

~~s = singlet , d - doublet, t - triplet, q - quartet singlets were assigned via ooloc technique doublets, triplets and quartets were assigned via C-H correlations ♦Assignments may be interchanged~~

~~+- -+ Table 30 The proton nmr data for thalfinine (!§)» (CDCl^* 500 MHz)~~

~~Functional Chemical shift Functional Chemical shift group (ppm) Protons group (ppm) Protons~~

~~C-1H 3.49 1 (m) part C-14 H 6.80 1 (dd)~~

~~ially buried C - T H 4.46 1 (d), 0 2 0 exchangeaDl* N-Me 2.37 3 (s) N-Me' 2.62 3 (s)~~

~~C-3 H 2.30 - 2.34 1 (s) C-3 1 H 2.86 - 2.91 1 (m) C-3 H 2.79 - 2.82 1 (m) C-3' H 3.06 - 3.09 1 (m)~~

~~C-4 H 2.21 - 2.27 1 (m) C-4' H 2.63 - 2.66 1 (m)~~

~~C-4 H 2.52 - 2.57 1 (dt) C-4’ H 2.83 - 2.85 1 (m) C - 6 OMe* 3.40 3 (s) 0-CH,-0 5.89 2 (dd) C-7 OMe 3.74 3 (s) C-7 OMe 3.50 3 (s)~~

~~C - 8 H 6.42 1 (s) C-a' (a-H) 3.22 1 (d) C-a (a-H) 3.20 1 (dd) C-6 ' (B-H) 3.12 1 (dd)~~

C- 6 (B-H) 3.37 1 (dd) C-ll' ** 6.64 - 6.74 , 2 (m) C-10 H 5.98 1 (d) C-13* C-12 OMe 3.88 3 fs) C-10' ** 7.13 - 7.21 2 (m) C-13 H 6.80 1 id) C-14' *Data was obtained via correlation through Tong range coupling (coloc) **Data was obtained from a 90 MHz 'H-nmr spectrum and may be Interchangeable 136

~~c) Thalmelatidine (12).~~

~~Thalmelatidine was isolated from the roots of T.~~

~~minus L. subspecies elatum by Mollov et al (45) and was~~

~~assigned structure 1 2 a based on spectroscqpic and chemi~~

~~cal evidence. However, reexamination of the proton nmr~~

~~spectrum of thalmelatidine confirmed the aporphine-~~

~~benzylisoquinoline character but provided no evidence~~

~~for the position of the methylenedioxy group and a~~

~~methoxyl group on carbons 5 ’, 6 ’ and 7’ of the benzyli-~~

~~soquinoline moiety. In order to study the spatial rela~~

~~tionship between that specific methoxyl group and the~~

~~methylenedioxy group with the proton on C- 8 ' and also~~

~~the spatial relationship between certain protons and~~

~~functional groups, a NOE experiment was carried out.~~

~~The proton at § 5.83 with its relative upfield posi~~

~~tion (being shielded considerably by the currents of~~

~~ring C) was characterized for the C- 8 ' proton of the~~

~~benzyl isoquinoline moiety. Irradiation at that reso~~

~~nance region (5.83) caused a significant NOE enhancement~~

~~for the methoxyl group at 3 3.59. This significant~~

~~effect suggested an ortho relation between the methoxyl~~

~~group (3.59) and the C- 8 ‘ proton and that this methoxyl~~

~~group should be located on C-7', the only position for 137 such an ortho relation. To sustain this observation,~~

~~irradiation of the 3.59 resonance region (OC34^) caused a significant NOE enhancement for the C-8 ' proton (Table~~

31). This dual observation strongly supported the ortho relation between the proton at § 5.83 and the methoxyl group at % 3.59. The me thy 1 enedi oxy group, previously suggested to occupy the C- 6 ' and C-7' positions (45) must therefore be located at C-5' and C- 6 ' positions.

~~On the other hand, irradiating the resonance region of the proton at S 8.04 (characteristic position for the~~

C-ll proton of the aporphine moiety) caused a signifi cant NOE enhancement for the methoxyl group resonating at % 3.94. This resonance region must therefore be assigned to the methoxyl group on position 1 0 (ortho position, C—10) of the aporphine moiety. Other methoxyl groups enhanced were at £ 3.59, 3.76, 3.79, 3.89 and

3.96. Three methoxyls of the last four groups (3.59 assigned for the C-7' OCH^) could therefore be assigned to the three methoxyl substituents of the aporphine moiety. These tentative assignments were facilitated by comparisons with structurally-related analogs of thalme latidine (12) (table 32, 79).

~~« a = R3= M e O ; R 4,R5= C H 202 « b = R3,R4 — CH 20*.R5 = M« 0~~

~~(rest as in 12 ) 138~~

-t------h 1 I Table 31* Hi tWR and NOE difference results for thalmelatidine | I ' ------(500 Mlz, CDC13 ) | 1 r 1 Absorption Region Irradiated (5) Effects on Other Groups | 1 1I 1 5.83 (H) Enhancement for MeO (3.59) | 1 Enhancement for MeO (3.89) | 1j 1 1 3.59 (MeO) Enhancement for (5.83) I I MeO (3.89) irradiated j |1 1 1 8.04 (H) Enhancement for MeO (3.94) I 1 Methojyls affected: (3.59, | 1 3.76, 3.79, 3.89, 3.96) | 1 1 1 1 + ™ " '■ ------.---- 1-

~~+- -~~

~~1 Table 32: Chemical shifts data of methoxyl groups on the apor- | 1 phine moiety of thalmelatidine and related analogs («5) 1~~

~~1 Compound C-l (&) C--2 (5) C-3 (h) C-10 (&) I~~

~~1 Bursanine 3.75 3.96 3.88 3.98 | 1 2 -Noradian- 3.60 3.90 3.90 3.96 I 1 tifoline~~

~~1 Iznikine 3.72 3.96 3.88 4.02 1~~

~~1 Huangshanine 3.75 3.97 3.88 4.00 I~~

~~I Istambu- 3.76 3.96 3.90 3.93 j 1 lamine~~

1 TTialine lati- 3.76 (3-79) 3.96 3.89 3.94 | dine*

~~•Tentative assignment~~

~~+■ + 139 d) N-methylcorydaldine (2):~~

~~This alkaloid was obtained from the early eluates of~~

~~the tertiary non-phenolic alkaloid fraction. It was~~

~~identified on the basis of identical tic, nmr, ir and uv~~

~~properties with those of an authentic sample obtained as~~

~~a KMnO^ oxidation product of adiantifoline. The alka~~

~~loid was also known as an oxidation product of a number~~

~~of bisbenzylisoquinoline and benzylisoquinoline-~~

~~aporphine alkaloids as early as 1966 (78). However, it~~

~~was not until 1971 that it was reported as a naturally~~

~~occurring compound by Shamma and Fodczasy, who isolated~~

~~this i soqu inol one alkaloid from the roots of T. fendleri~~

~~Engelm. It was then reported from other natural sourc~~

~~es, namely, Thalictrum and Papaver species. e) Adiantifoline (5j:~~

~~This alkaloid was isolated as the najor component.~~

~~The major amount (3.98 g) was obtained from the tertiary~~

~~nonphenol ic base fraction while only a minute amount (15~~

~~mg) was found in a mixture with the components of the~~

~~tertiary phenolic base fraction. f) Thalisqpynine (10):~~

~~This aporphine alkaloid was obtained as a minor com~~

~~ponent from both tertiary phenolic and nonphenol ic base~~

~~fractions. Its Sl-nmr (CDCl^, 500 f-Hz) shewed signals 140~~

~~for a single N-Me group, multiplets of aliphatic protons~~

~~(7 H), and two aromatic protons with one at a character~~

~~istic downfield position (c-11 H of the aporphine). It~~

~~gave a positive result with phosphcmolybdic acid which~~

~~indicated the presence of a phenolic CM. The ^H-nmr, uv~~

~~and ms spectral date of this alkaloid agreed with those~~

~~reported in the literature for thaliscpynine (75). The~~

~~circular dichroism curve (CD) which was not reported~~

before shewed maxima at “ 24800, £ ^ 2 9 8 ~ 2710°*

~~[tfQ27g - 33000 and C^]245 + 214000. g) O-Methylthalicberine (17) and thalicarpine (18):~~

~~O-Methylthalicberine was obtained from the tertiary~~

~~nonphenolic base fraction, and it yielded 107 mg of~~

~~material. The 1H-nmr, ms and the mobility in the thin-~~

~~layer chromatographic system were in agreement with~~

~~those reported in the literature (13,36). The CD curve~~

~~showed maxima at C^214 + ^42100, £^3252 “ 19300 and~~

~~^ 2 8 7 + 6 2 2 0 0 * Thalicarpine was obtained as a major ooopound (218~~

~~mg) from the tertiary nonphenolic base fraction and was~~

~~identified by comparison of its ir, uv and ^H-nmr spec~~

~~tral data with those of authentic thalicarpine. h) Thaliraoebine (19), O-methylthalibrine (23), thalmira-~~

~~bine (26) and thalistine (25): 141~~

~~The first three alkaloids were isolated from the~~

~~tertiary nonphenolic base fraction while the cryptophe~~

~~nol ic alkaloid, thalistine, was isolated from both phe~~

~~nolic and nonphenolic base fractions. These four alka~~

~~loids were only the second reported from nature; they~~

~~were first isolated from the roots of T. minus race B as~~

~~major aonponents (7,16). However, in this study they~~

~~were obtained as minor compounds. All were identified~~

~~by comparison of physical properties with those of the~~

~~authentic sanples. i) Thalmineline (20) and obaberine (21):~~

~~Thalmineline was obtained from both phenolic and~~

~~nonphenolic base fractions (220 mg). Its identification~~

~~was based on comparison of its physical data (nmr, ir~~

~~and uv) with those reported by Reisch et al, who isolat~~

~~ed this alkaloid from the roots of T. minus L. var. ela-~~

~~tum Koch (46).~~

~~Obababerine was obtained as a powder from the non~~

~~phenolic base fraction and was identified by comparing~~

~~its physical data (nmr, ir, and uv) with those of~~

~~authentic smaples reported in the literature (16,79,80). j) Isoboldine (27) and (+)-laudanidine (29):~~

~~Both isoboldine and (+)-laudanidine were obtained in~~

~~minute amounts with the latter obtained as colorless 142~~

~~prisms from MeOH-CHCl^ (rip 182-183°). Both alkaloids~~

~~were identified by comparison of their physical date~~

~~with those of authentic samples. k ) Delporphine (28):~~

~~Delporphine was isolated as a homogeneous amorphous~~

~~compound from the phenolic base fraction in minute~~

~~amounts. Extensive spin-decoupling and NOE difference~~

~~experiments resulted in the assignments of its protons 13 (table 33). Hie C-nmr spectrum (table 34) showed 20~~

~~carbons and was in accord with the structure of this~~

~~alkaloid. Hie assignment of the carbons was based on~~

~~the multiplicities obtained from the single frequency~~

~~off-resonance decoupling and from comparison of the data~~

~~to that of related oocpounds. This alkaloid was only~~

~~the second reported from a natural source and the first~~

~~to be isolated from a Hialictrum species. Hie spectro~~

~~scopic data (nmr, ir and uv) were in good agreement with~~

~~that reported for delporphine, which was isolated by~~

~~Salimov et al from Delphinium dictyocarpum L. (Ranuncu-~~

~~laceae) (85). 143~~

+■ ■ ■1 1 ■ ------■ ■ |— — ------—— ------■ —— — ■ Y- 1 1_ 1 I Table 33: The Ti NMR data for delporphine (28) {270 M z , CDC1,) I i ~ 3 i Functional Chemical Shift Protons group (ppm)

C-4 2.45 - 2.50 Ha (m) C-4 2.76 - 2.80 He (dd) C-5 2.88 - 2.92 Ha (dt) C-5 3.10 - 3.14 He (dd) C-6a 3.00 - 3.03 H (m) C-7 2.53 - 2.60 ct-H (m) C-7 2.94 - 2.98 g —H (dd) C-8 6.80 H (s) C-ll 7.85 H (s) N-Me 2.55 3H (s) C—1 MeO 3.70 3H (s) C—2 MeO 3.97 3H (s) C—10 MeO 3.91 3H (s) C-3, C-9 OH 5.85 2 (br)

~~a = axial, e = equatorial, m = multiplet, s = singlet, dd = doublet of doublets, dt = doublet of triplets, br = broad signal~~

~~H------Y~~

~~1) Thalrugosine (22)~~

~~This cryptophenolic alkaloid was isolated from the~~

~~tertiary nonphenolic base fraction. Its identification~~

~~was based on direct comparison of physical properties~~

~~(uv, ir, and ^H-nmr) with authentic talrugosine (7).~~

~~The ^H-nmr (CDCl^, 270 MHz) showed two N-Me groups~~

~~at 2.30 and 2.48 ppm, fourteen aliphatic protons exhib~~

~~ited as coqplex nultiplets (2.30 - 4.01) and three~~

~~methoxyl groups at 3.76, 3.93 and 3.94. Ten protons 144~~

~~— *-~~

~~Table 34; M4R data of delporphine (28) (270 NHz, CDCl^)~~

~~Carbon Mult. ppm Carbon Mult. ppm~~

C-l s 148.2 C-9 s 145.5° C-2 s 138.8 C-10 s 144.4° C-3 s 145.5 C-ll d 110.7 C-3a s 116.3 C-l la s 124.3 C-4 t 23.3 C-l a s 119.2 C-5 t 53.0 C-16 s 131.1 C-6a d 63.0 N-Me q 44.0 C-7 t 34.0 C-l MeO q 61.1* C-7a s 129.3 C-2 MeO q 60.3* C-8 d 114.2 C-10 MeO q 56.4 s - singlet, d = doublet, t = triplet, q = quartet °, ‘Assignments may be interchanged h------

~~were inhibited in the aromatic region. Of the ten aro~~

~~matic protons, three were singlets, four were part of an~~

~~AA’BB1 pattern (all are doublets of doublets), while the~~

~~other three were part of an ABX system.~~

~~In order to provide further support for the struc~~

~~ture of this alkaloid, extensive NOE difference and 13 spin-deooupling experiments were carried out. C-rnnr~~

~~values were assigned on the basis of the observed C-H~~

~~nultiplicities due to bond couplings (using single fre~~

~~quency of f-resonance decoupling (SPDRD), broad band~~

~~decoupling and DEPT techniques. The coloc technique was~~

~~enplcyed for the possible assignment of the substituted~~

~~carbon atoms. 145 When the aromatic proton (dd) at 7.30 was irradiat~~

~~ed, the doublet of doublets at 6.42 collapsed into a~~

~~doublet (J = 9 Hz) and the doublet of doublets at 7.04~~

~~also collapsed into a doublet (J = 2.2 Hz). These~~

~~observations indicated the ortho relation of the irradi~~

~~ated proton with the proton at 7.04 and its roeta rela~~

~~tion with the proton at 6.42. The doublet of doublets~~

~~at 6.84 was not affected which suggested the possibility~~

~~of its para position with relation to the irradiated~~

~~proton as shown in the partial structure below:~~

~~On the other hand, irradiation of the doublet at~~

~~6.28 resulted in the collapse of the doublet of doublets~~

~~at 6.67 to a doublet (J = 8.1) and the fometion of what~~

~~seemed to be an AB pattern by this collapsed doublet and~~

~~the doublet at 6.79. This observation suggested that~~

~~the irradiated proton could be considered as H^. in an~~

~~ABX pattern while the proton at 6.67 could be assigned as Hg and that at 6.79 as 146~~

The series of double irradiation of the complex aliphatic multiplets resulted mainly in partial collapse of certain protons within the same or other multiplets and provided little infornatian about their spatial relations. However, NOE difference experiments provided more infatuation about the spatial relations between the different substituents (table 35). On irradiation of the proton at 6.35, an NOE enhancement of 10% was observed for the MeO at 3.76. Alternatively, irradia tion of the resonance region of the MeO at 3.76 resulted in 20% NOE enhancement for the proton at 6.35. Similar results were obtained for the protons at 6.73 and 6.79 with their corresponding methoxyl groups at 3.93 and

~~3.94, respectively. This indicates the ortho relation between these protons andtheir corresponding CMe groups.~~

In order to differentiate between the relative posi tions of the various groups on each of the two benzylisoquinoline units of thalrugosine, and consequently locate the exact positions of their different substit uents, certain criteria were considered. First, the

C-8' proton with its relative high field position was assigned the value of 6.06. From the C-H correlation spectrum of this proton and its corresponding carbon 147 (C-8‘), this carbon was given the value of 121.5 ppm.

~~On the other hand, the coloc results (table 36) indicat~~

~~ed that this C-8' proton correlated with one carbon~~

~~(143.7) with which another singlet proton (at 6.73) also~~

~~correlated. It was concluded that the latter proton~~

~~must be locat ed on the same benzene molecu le. Si nee~~

~~there were only three singlet protons that correspond to~~

~~the C-5, C-5' and C-8' protons, it was concluded that~~

~~these protons at 6.06, 6.73 and 6.35 mist be assigned~~

~~the positions of C-8', C-5' and C-5, respectively.~~

~~Double irradiation (spin-decoupling) experiments~~

~~indicated that the C-5' proton (6.73) was coupled to a proton with signals at the 2.30-2.42 resonance region.~~

~~From the C-H correlation spectrum that showed character~~

~~istic relative upfield value for its corresponding car bon, it was suggested that this aliphatic proton was either C-4 or C-4' proton. Since the proton at~~

~~2.30-2.42 was coupled to the C-51 proton, it was con~~

~~cluded that this aliphatic proton oust be the C-4* pro~~

ton. Since this proton was observed to couple with two other aliphatic protons at 3.21-3.25 and 2.74-2.83, it was concluded that the latter protons must be the C-31 protons. This conclusion was supported by the absorp tion of this particular carbon (C-31) at 44.4 ppm (C-H 148

~~correlation and SFQRD spectra), a relatively lower field~~

~~position compared to C-4 and C-a resonance regions.~~

~~That the cartoon whose protons absorb at 2.30-2.42 reso~~

~~nance region is on (the right-hand side) benzyl isoquino~~

~~line unit is supported toy the ooloc spectrum. The pro~~

~~tons of this carbon were correlated with two carbons at~~

~~112.7 and 131.14 ppm that correspond to C-5' and C-8a',~~

~~respect ively.~~

~~The protons of the IWe group at 2.30 was shown to be~~

~~coupled to the aliphatic proton at 3.31-3.40 resonance~~

~~region. The C-H correlation and SFOBD spectra correlate~~

~~this particular proton to a carbon which was deduced to~~

~~be C-3. This suggested that the IWe at 2.30 should be~~

~~located on (the left-hand side) benzylisoquinoline unit.~~

~~Irradiation of this NMe group resulted in an NOE~~

~~enhancement of 11% for the aliphatic proton at 4.01, which from the C-H correlation spectra and the relative~~

~~value of its corresponding carbon, must be assigned as~~

~~the C-l proton. Hence the analogous proton at 3.59 ppm mist be assigned as the C-l' proton since this proton was shown to couple to the IMe protons at 2.48. Accord~~

~~ingly, the corresponding C-« and C-a' protons that were shown to be coupled to either C-l or C-l' protons were assigned their particular values. The results were recorded in table 37. 149~~

~~Table 36 shows the ^C-nmr values of thalrugosine which were assigned according to information deduced from the DEPT, COLOC and the C-H correlation techniques. 150~~

~~Table 35; Hi-MIR and NOE difference results for thalrugoeine (22) (270 f«z, c d c i 3 )~~

~~Absorption Region Irradiated Effect On Other Groups~~

2.48 (N-Me) 51 enhancement for part of the 4H-nultiplet at 2.80 (2.74 - 2.83) 10% enhancement for dd (H) at 3.59 (3.56 - 3.62) 2.30 (N-Me) 11% enhancement for part of the nultiplet (4H) at (2.74 - 2.83) 6% enhancement for dd (H) at 4.01 (3.98 - 4.03) 3% enhancement for H at 6.06 5% enhancement for dd (H) at 7.04 6.73 (H) 5% enhancement for part of nultiplet (3H) at 2.87 - 2.94 13% enhancement for MeO at 3.94 6.35 (H) 14% enhancement for MeO at 3.76 4% enhancement for nultiplet (H) at 2.30 - 2.42 6.28 (H) 13% enhancement for H at 4.01 5% enhancement for H (dd) at 6.84 5% enhancement for H (dd) at 7.04 6.06 (H) 11% enhancement for H (m) at 3.59 10% enhancement for H (dd) at 4.01 4.01 (H) 20% enhancement for H (d) at 6.28 I 14% enhancement for H at 6.06 | 2.59 - 2.68 (H) 5% enhancement for N-Me at 2.48 j 8% enhancement for part of nultiplet at 2.74 - 2.83 I 4% enhancement for part of nultiplet at 3.17 - 3.25 i 8% enhancement for H at 6.06 I 3.93 (OMe) 19% enhancement for H at 6.73 j 3.94 (OMe) 20% enhancement for H at 6.79 j 3.76 (OMe) 20% enhancement for H at 6.35 I 151

~~-t------~~

~~I Table 36: Carbon-proton correlation via long range coupling con- t stant (coloc) of thalrugosine (22)~~

Carbon ppm Long Range Correlated Protons C—4a 124.8 — C-6 147.2 C-6 OMe 3H C-7 136.6 C-5 H C-8 144.4 — C-8a 122.7 C-5 H C-9 133.9 C-l 3 H C-ll 150.5 C-10 H, C-l 3 H C-l 2 147.0 C-10 H, C-l4 H, C-12 CMe 3H C-4a' 131.15 C-8* H C-6' 149.4 C-8' H, C-6' Ome 3H C-7* 143.7 C-5' H, C-8' H C-8a ’ 131.1 C-4' H, C-5' H, C-8' H C-9* 135.6 C-ll* H, C-l3' H C-12' 154.8 C-10' H, C-141 H

~~+■ 152~~

~~^ ^ 1 O to Table 371 ^ C NMR data of thalrugosine (22) o c d c i 3>~~

Carbon Mult. ppm Carbon Milt. ppm C-l d 65.4 C-4* t 23.0 C-3 t 46.4° C-4a* S 131.1 C-4 t 26.0 C-5' d 112.7 C-4a s 124.8 C-6' s 149.4 C-5 d 107.9 C-7' s 143.7 C-6 s 147.2 C-8' d 121.5 C-7 5 136.6 C-Sa' s 131.1 C-8 S 144.4 C-a' t 38.3 C-8a S 122.7 C-9' s 135.6 C-a t 39.4 C-10* d 132.1* C-9 s 133.9 C-ll’ d 123.0** C-10 d 115.4 C-12' s 154.8 C-ll s 150.5 C-13' d 122.8** C-12 d 147.0 C-14* d 130.2* C-l 3 d 112.0 N-Me q 42.6 C-14 d 122.2 N-Me' q 43.3 C-l' d 60.5 C-6 CMe q 56.3 C - 3 1 t 44.4 C-6* Ome q 56.3 C-12’ One q 56.4

~~s - singlet, d * doublet, t * triplet, q = quartet *, **, Assignments may be interchanged Table 38 The proton nmr data for thalrugosine {22) (CDC1^* 270 MHz)~~

~~Functional Chemical Shift Functional Chemical shift group (ppm) Protons group (ppm) Protons~~

~~C-l H 4.01 1 (dd) N-Me' 2.48 3 (s)_ N-Me 2.30 3 ~~c C-3' H 2.74 - 2.83 1 (m) 2.74 - 2.83 1 C-3 H (m) C-3' H 3.21 - 3.25 1 (dt) 1 C-3 H 3.31 - 3.40 (dt) C-4' H 2.30 - 2.42 1 (m)~~~~

1 C-4 H 2.87 - 2.91 (m)- C-4' H 2.74 - 2.83 1 (m) C-4 H 2.87 - 2.91 1 (m)C C-5' H 6.73 1 (s) C-5 H 6.35 1 (s) C-6 ' OMe 3.93 3 (s) C - 6 OMe 3.76 3 (s) C-8 ' H 6.06 1 (s)r 1 C-a H 2.63 (d) C-a' H 2.74 - 2.83 1 (m)

C-a 1 H 2.91 - 2.94 (m) C-a' H 3.17 - 3.21 1 (m) C-10 H 6.28 1 (d) C-10’ H 6.42 1 (dd)*

C-12 OMe 3.94 3 (s) C-ll* H 6.84 1 (dd)**

1 C-13 H 6.67 (dd) C-13' H 7.04 1 (dd)**

~~C-14 H 6.79 1 (d) C-14' H 7.30 1 (dd)* C-l 1 H 3.59 1 (dd) c * proton signals exhibited as a part of a complex 4-proton multiplet * ( **Assignments may be Interchanged 154~~

~~SUMMARY~~

Extensive chromatographic separation methods of the toluene-ether- soluble tertiary base fraction from the roots of Thaiictrum minus L. race C have resulted in the isolation of twenty seven alkaloids which were identified by spectral and chemical methods. Of these compounds,

~~6 a ,7-dehydrothaliadine, 7'-dihydrodehydrothaliadine, 7'-dihydrothalia~~

~~dine, oxothaliadine, dihydrooxothaliadine, 6 a,7-dehydroadiantifoline,~~

6 -noradiantifoline, and thalmirine are new natural products. The first seven compounds are related to adiantifoline, which was isolated as the major component while thalmirine is a simple isoquinolone. A complete structure was assigned to squarosine, which was pre viously isolated as a minor component from T. squarrosum steph. In addition, the structures of thalmelatidine, thalfine and thalfinine were revised and the latter two were given full structure assignments. N-methylcorydaldine, thalisopynine, 0-methylthalicberine,

thalicarpine, thaliracebine, 0 -methylthalibrine, thalmerabine, thalistine, thalmineline,obaberine, isoboldine, (+)-laudanidine, del porphine and thalrugosine were the completely characterized known alkaloids. They were identified by comparison of their physical proper ties with reported values and with authentic samples. Additional 155

physical data were given where possible. Although T, minus race B and T. minus race C are morphologically identical and were cultivated under the same environmental conditions, this study proved that they are indeed different chemoraces. Adianti foline, as previously mentioned, was isolated from the chemorace C as the major alkaloid while it was isolated as a minor component from the chemorace B. On the other hand, thaliracebine, O-methylthalibrine, thalmirabine and thalistine were obtained from the chemorace B as major components whereas they were only isolated in small amounts from the chemorace C. 156

APPENDIX OP SPECTRA «UMMN ctr*

~~Figure 6: IR spectrum (CHC1^) of thalmirine {1) JV_ JL±~~

~~1 —f " -» r t 1~ i p » r -i -1-- i-- --- 1---- ■-- 1-- *-- »-- <-- »-- *-- 1 7.i I i Li.4 * 1 158 Figure 7 : H NMR spectrum (CDC1^) of thalmirine (1) I 9 5 1~~

~~Figure 8 C NMR SFORD spectrum (CDC1^) of thalmirine (1) I~~

~~n*. • s*.* •a.* ?».»~~

~~Figure 9 : NMR BB Decoupling spectrum (CDCl^) of thalmirine (1) 160 Figure 10: IR spectrum (CHCl^) of 6 a,7-dehydrothaliadine (4) JL JLUt JL I .... 1 1---. t.... t~~

~~Figure 11: H NMR spectrum (CDC13) of 6a,7-dehydrothaliadine (4) Figure 12: Mass spectrum of 6 a ,7-dehydrothal iadine (4) I~~

~~U U 14 l>~~

~~n i~~

~~UtlU M m IV * ' lM*i 1JUU IHM* lUUO WAV t*UMH « t m '~~

~~Figure 13: IR spectrum (CHCl^) of 7 '-dihydrodehydrothaliadine (7) 164 r T~~

Figure 14: NMR spectrum (CDCl^) of 7'-dihydrodehydrothaliadine (7) 166., 80 60] 220 40" 231 20] 0 TTTI HiVirrTTTTTTTTTTTTTTT 1 I III HfT IITM ! 11IIIIH IIH M I m l ! 11 f I M T l|» t1111' 11 Ml IIIIII H II III1111 III 220 240 260 260 300 320 340 360

~~118 31 81 1169~~

~~M’^T L H m T n hiTiiill u III? iiMi|nnrrt im iininiiiiniMHifti»n>rfiiiwrrrr rrrrrli K u e 120 140 160 180 200~~

~~Figure 16: Mass spectrum of 7'-dihydrodehydrothaliadine (7) U II ft~~

~~I ;rr in iniiiimik I I H I I i n i M M~~

~~iliBii iiifi~~

~~! !~~

~~Figure 16: IR spectrum (CHCl-j) of oxothaliadine (8 ) I~~

~~Figure 17: *H spectrum (CDCl^) of oxothaliadine (8 ) 168 2 S L~~

~~m W tMH ffiiiiH>|iniinH»MM imnmTTT»ii|m iitniiHiiiHw i»nTTii«mfiiTtnr 248 2M 280 308 320 340 368 160 480 <~~

~~rtiliifirimiMiniiiMf iiiii 40 66 W 100 120 140 160 V~~

Figure 18: Mass spectrum of oxothaliadine (8) wwwiwwi or*

~~Figure 19: IR spectrum (CHC13) of 7'-dihydrooxothaliadine (9) / / Jl D Jll I_J.JLJ.L1 1 .~~

~~Figure 20: *H NMR spectrum (CDCl^) of 7'-dihydrooxothaliadine (9) 100 533 90. 80. 517 70. 60. 50. 40. 38. 20. ie. m nj m 520~~

~~100- 90. 68. 70. 66. 307 338 58. 292 40. 30. 28. 10. nfnim rn m t n m im iiiir 280~~

~~Figure 21: Mass spectrum of 7 '-dihydrooxothaliadine (9) 172 i~~

~~I 11 * ' 1 j * \ • l | ' | 1 f |at | M~~

~~f y v~~

~~Figure 2 2 : IR spectrum (CHC1 ) of 7'-dihydrothaliadine (11) 3 173 “r~~

~~Figure 23: lW NMR spectrum (CDC13) of 7'-dihydrothaliadine {11} 106.~~

~~.502 517~~

~~80! 601 461~~

~~Figure 24: Mass spectrum of 7 ’-dihydrothaliadine (11) 175 176 IM i 5 IBM wm Hi t* mm 1 - 1 Fijure 25: Fijure 25: spectrum of IR 6a,7-dehydroadiantifoline {CHC13 ) (14) 1 I 1 NO* m l-i T~~

Figure 26: H NMR spectrum (CDCl^) of 6a,7-dehydroadiantifoline (14) 177 723 96 eo. 70. 60L 50. 40. 38. 20J 16. 656709 1 |737 7^ 0 imu mip »>nn inimMfii|iiiiiiiMiiin'iiffniiim »i|i™iTtn\TiMiini|THiniMnliwiiiM eae 620 648 690 ,80 720 740 760 730 k»

~~J08. 9 6 80. 70. 60. 5 6 j 461 40 30- 553 20. 10. a |fiiiilffinnim m innMiim nim niiMiiiinnimirliiiiniw piifn iinuimniinnHnr ^ 420 W ^ SM 520 M 360 580 600~~

~~Figure 27: Mass spectrum of 6 a,7-dehydroadiantifoline (14) 178 WCIN* iue 8 IR spectrum 28;Figure iMi~~

~~)Uua TtUO MOD~~

~~|---1 T I | T 'I T T |---1----1----I---1---|---1---ITT] ---1---I ¥ I |---1---1--T 1 | —T~ ~|-¥*--T ‘ T ■—1---1 f —T"-T T 7 5 70 6 5 AO 5* 50~~

~~Figure 29: H NMR spectrum (CDCl^) of squarosine (15) 100 as: 6 £ 40: 20: 0 :n n r i ii't iti 1 i |■ 1 i 1 i-r 1 1 "i1 i-i 1 iti 1 r i T"i l i r > 1 n i 1 r 525 550 575 666 625 650 675 760 100., 80:~~

~~6 0 : 40 20: 'r i i i m n i n i r n 1 t 1 1 1 i i 1 r n i-i > i n iti iti i i'i ri 1 275 360 325 350 375 400 425 450 475 100, ,220 80. 60. 401 203~~

~~a! ^*11^ > ^ 'r r r 1 t v i tt i 1 1 ri^ n ^ ‘pt t t t t t 50 75 \9d 125 150 \ 75 few 225 251~~

~~Figure 30: Mass spectrum of squarosine (15) 181 Li T n T~~

~~r~l— 130 HO II 0 IOC 90 BO 70 60 50 JO 20 13 Figure 31: C NMR DEPT spectrum (CDCl^) of squarosine (15) 182 ’60 ’60 140 120 100 PO 60 40 20~~

~~figure 32: C-H correlation spectrum (CDC1 squarosine (15)~~

~~CD I~~

~~"iVN*^Vl(J v < N w ^~~

~~Figure 33: *3C NttR BB Decoupling vs SFORD {CDCl^} of squarosine (15) 183 11 WAVfllNCilH~~

~~W A W t l M N t C m~~

~~Figure 34: IR spectrum (CHCl^) of 6-noradiantifoline (24) t~~

~~Figure 35: *H NMR spectrum (CDC1of 6 -noradiantifoline (24) 106 9 0 . 80. 70 . 60. 50. 40. 30. 20.~~

~~10- . I 0 fTni i i i i i ri i t i I i i i i 11 i it I t i n | i i i T'f n i r»i i~i i in tti 500Cfc 525 558 575 600 625 650 675 700 725 750~~

~~iti i jTi1 i"T|"i rrr p ti r p i-Jf 400 425 450 475 ? 0 0 186~~

~~Figure 36: Mass spectrum of 6 -noradiantifoline (24) A /~~

~~ill~~

ill11___ . 0 «• 1 *

~~Figure 37: NMR spectrum (CDCl^) of thalfine (6) 187 |~~

~~Sf" * * ■* * n» »n~~

~~■"r" — — T ” i m i iM. i m i .* m i i.’t.i n i l in.• 9i.i 91.1 71 I M l ‘jl. I FI'H~~

Figure 38: 13 C NMR SFORD spectrum (CDC13) of thalfine (6) I *

~~I I I I~~

I .. iiiki |«*f' iiiiniiiiwTwyin ii H » |» n m (q ii ihi m ^ iim iiiib|iii m m ii|iih i 11 II | II ITU 11 H I "*T ISQ.O 140.0 110.0 100.0 00.0 10.0 40.0 20.0 0.0 fPN 13 Figure 3 9 : C NMR BB Decoupling spectrum (CDCl^) of thalfine (6 ) 9 8 1 •at HI •• 0 10 0 rt i

~~Figure 40: C-H correlation spectrum (CDCl^of thalfine (6) at the aliphatic region q 7.9 ■n It It~~

~~JUU U v _ J L A L~~

~~Figure 41: C-H correlation spectrum (CDCl^) of thalfine (6) at the aromatic region 1~~

~~f t ft~~

t * : * *

~~_AJ~~

~~Figure 42: COLOC correlation spectrum (CDCl^) of thalfine (6) at the aliphatic region liti~~

~~Figure 43: COLOC correlation spectrum (CDC13) of thalfine (6) at the~~

~~aromatic region 193 T -rr~ r i ** i,1 1 §~~

Figure 44: *H NMR spectrum {CDC13 ) of thalfinir.e (16) 194 J lA jU U l . -H "'T... . ■^i I'll.I H I I m i i ;i i in.i m i la.I M l III til 'ill M l IS.* pr*

13 Figure 4 5 : C NMR SFORO spectrum (CDCl^) of thalfinine (16) 195 [ ■ . I . . ■ m* . | , , r P , |-..,.F f „ »m a m a m i i n na • i«a a M t rrm w.< r» | c«,I ,11 411 ill 13 Figure 46: Gated decoupled C NMR spectrum (CDCl^) thalfjnine (16) 196 197 1 ii'» *1 y yyni C C i i M C 9) tujet o LQ) rias iidoa g yN _ sjnfiij 3_T y^N fiuiodnoDaa gg wruioads LDQ3) ( stnuijieitt jo (91) -.1 *1/ .., ^ n in in j .i^. HJJ ■•i • cm ... ■ ■ - - ■ i m c e r i i t v v V rr.i • m I f iI I,-. m*NiM iue48: R pcrm CCj o eprhn (28) delporphine of (CHClj) spectrum IR : 8 4 Figure w»wiiii> c*r< - 1 - 1 , 1 . - - - I I

~~Figure 4 9 : NMR spectrum (CDC1-J of delporphine (28) >1 ‘f I I~~

~~Jj Liu **w i«».i m i i:• i m.a in i ** I m II I 'I.I II. I 'jf I 4 1 1 11 1 II I~~

~~13 Figure 5 0 : C NMR SFORD spectrum (CDCl^) of delporphine (28) 200 I~~

~~MWW fNMPMI~~

~~■ '-T ' I" 11 \ *p "fl • H.« M l I# 9 IM.t ►r* M l •• I »»~~

~~I ^ Figure 51: C NMR BB Decoupling spectrum (CDCl-j) of delporphine (28) 201 ,1 1~~

jJlA-i L MJ u u *

~~74 <4 34~~

~~Figure 52: H NMR spectrum (CDCl^) of thalrugosine (22) 202 iij • * i ■ " ■ *•» ■ • i - * - ’ f 4 ■ ■ « i , ’i" r j '1* j r «i j Ml J »< J~~

13 Figure 53: C NMR SFORD spectrum (CDCI^) of thalrugosine (22) 3 0 2 ', 1*

~~t i~~

~~’"r ” '' T"1 l>J 4 144.4 U 4 *14.4 H I 14.1 ;J J *J J 4 I '<■ t m * \u* i.’i-J I ‘ •~~

~~Figure 54: 13C NMR BB Decoupling spectrum (CDC13) of thalrugosine (22) 4 0 2 References~~

~~1. P. L. Schiff, Jr. and R. W. Doskotch, Lloydia, 33, 403 (1970).~~

~~2. W.-T. Liao, Ph.D. Ohio State University, Columbus, Ohio, 1976.~~

~~3. T. Tomimatsu, Yakugaku Zasshi 30 1,1 (1976).~~

~~4. Kh. B. Dutschewska and B. A. Kuzmnov, J. Nat. Prod., 45, 295 (1982).~~

~~5. S. Yu. Yunusov and N. N. Progressov, Zhur. Qbehchei Khim. 20, 1151 (1951); Chem. Abetr. 45; 1608.~~

~~6. Kh. G. Pulatova, S. Abdizhabbarova, Z. F. Ismailov, and S. Yu. Yunusov. Khim. Prir. Soedin., 4, 59 (1968); Chem. Abetr. 68:112177.~~

~~7. W.-N. Wu, W.-T. Liao, Z. F. Mahmoud, J. L. Beal and R. W. Doskotch. J. Nat. Prod., 43, 472 (1980).~~

~~8. N. M. Mo Llov, P. Panov, L. N. Thuan, and L. Panova, Dokl. Bolg. AKad. NauR. 23, 181 (1970); Chem. Abetr. 73: 32285. ------~~

~~9. T. Tomimatsu and M. Sasakawa, Chem. Fharm. Bull. 23, 2279 (1975).~~

~~10. R. W. Doskotch, P. L. Schiff, Jr. and J, L. Beal, Tetrahedron Lett., 46, 4999 (1968).~~

~~11. R. W. Doskotch, P. L. Schiff, Jr. and J. L. Beal, Tetrahedron, 25, 469 (1969).~~

~~12. N. M. Mollov, P. P. Panov, L. N. Thuan, and L. Panova, Dokl. Bolg. Akad. Nauk, 23, 1243 (1970).~~

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