New Sesquiterpene Lactones from the Genera Calea and Berlandiera (Asteraceae) and the Fragmentation Reactions of 1,3-Dihydroxyeudesmanolide Derivatives

Louisiana State University LSU Digital Commons

LSU Historical Dissertations and Theses Graduate School

1983 New Sesquiterpene Lactones From the Genera Calea and Berlandiera (Asteraceae) and the Fragmentation Reactions of 1,3-Dihydroxyeudesmanolide Derivatives. Ihl Young Lee Louisiana State University and Agricultural & Mechanical College

Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses

Recommended Citation Lee, Ihl Young, "New Sesquiterpene Lactones From the Genera Calea and Berlandiera (Asteraceae) and the Fragmentation Reactions of 1,3-Dihydroxyeudesmanolide Derivatives." (1983). LSU Historical Dissertations and Theses. 3895. https://digitalcommons.lsu.edu/gradschool_disstheses/3895

This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS

This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted.

The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction.

1.The sign or “target” for pages apparently lacking from the document photographed is “Missing Page(s)”. If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity.

2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blurred pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame.

3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of “sectioning” the material has been followed. It is customary to begin filming at the upper left hand comer of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete.

4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department.

5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed.

University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106

8400127

Lee, Ihl Young

NEW SESQUITERPENE LACTONES FROM THE GENERA CALEA AND BERLANDIERA (ASTERACEAE) AND THE FRAGMENTATION REACTIONS OF 1,3-DIHYDROXYEUDESMANOLIDE DERIVATIVES

The Louisiana State University and Agricultural and Mechanical Col. Ph.D. 1983

University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106

PLEASE NOTE:

In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V .

1. Glossy photographs or pages______

2. Colored illustrations, paper or print______

3. Photographs with dark background______

4. Illustrations are poor copy______

5. Pages with black marks, not original copy______

6. Print shows through as there is text on both sides of page______

7. Indistinct, broken or small print on several pages

8. Print exceeds margin requirements______

9. Tightly bound copy with print lost in spine______

10. Computer printout pages with indistinct print______

11. Page(s)______lacking when material received, and not available from school or author.

12. Page(s)______seem to be missing in numbering only as text follows.

13. Two pages n u mbered______. Text fol lows.

14. Curling and wrinkled pages______

15. Other______

University Microfilms International

NEW SESQUITERPENE LACTONES FROM THE GENERA CALEA AND BERLANDIERA (ASTERACEAE) AND THE FRAGMENTATION REACTIONS OF 1,3-DIHYDROXYEUDESMANOLIDE DERIVATIVES.

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Chemistry

Ihl Young Lee B.S., Kyung Puk National University, 1973 M.S., Seoul National University, 1976 August 1983 To

my family

ii ACKNOWLEDGEMENT

I would like to express my sincere appreciation to my major

professor, Nikolaus H. Fischer for his enthusiastic guidance and assistance through this research.

The collection and identification of the various plants by Dr. 0.

R. Wussow and Professor Lowell Urbatsch is greatly appreciated.

Special thanks to Mrs. Helga Fischer for her technical assistance

in the extraction of the many plant populations and to Dr. Frank

Fronczek for the X-ray data.

Also, extreme gratitude is extended to my husband and daughter, Ji-

Eun, who have been spiritual support.

Lastly the financial support received from the Coates Memorial

Foundation is gratefully acknowledged. TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... iii

LIST OF TABLES...... vii

LIST OF FIGURES...... viii

LIST OF SCHEMES...... xi

ABSTRACT...... xii

CHAPTER

1. Introduction

1.1 Systematic background in the

genus Calea ...... 2

1.2 Sesquiterpene lactones in the

genus Calea ...... 3

2. The isolation, structure elucidation, and

chemistry of sesquiterpene lactones from

Calea te rn ifo lia ...... 22

2.1 Isolation of tem ifo lin s,

zexbrevin derivatives,

atripliciolide derivatives,

and 2,3-epoxycalein A ...... 23

2.2 The structures of ternifolin

derivatives 32 and 33 ...... 29

2.3 Oxidation reactions of compounds

32 and 33 ...... 31

2.4 The structure of zexbrevin-

type furenones

2.41. 9a-Acetoxyzexbrevin ...... 40

iv Page

2.42 83-Angeloyloxy-9a-hydroxy-

calyculatolide and 9a-hydroxy-

zexbrevin ...... 41

2.5 The structure of atripliciolide

derivatives...... 47

2.51 11,13-Dihydro-11a,13-

epoxyatripliciolide-83-0-angelate

and 11,13-di hydro-11 ci-13-

epoxyatri pii ci oli de-83-0-

methacrylate...... 47

2.52 9 o-Hydroxy-11,13-dihydro-

11 a, 13-epoxyatri pi i ci ol i de 83-0-

angel ate and 9a-hydroxy-l1,13-dihydro -

11 a ,13-epoxy-atri pii ci oli de-8 3-0-

metacrylate...... 52

2.53 15-Hydroxy-l1,13-di hydro-11 a,

13-epoxyatripii ci oli de -

83-0-angelate ...... 55

2.6 The tentative structure of 2,3-epoxycalein A.. 61

2.7 Epoxidation of calein A ...... 62

2.8 Experimental ...... 68

3. The isolation and structure elucidation

of sesquiterpene lactones from Berlandiera

texana and B. ly atra ...... 76

3.1 Introduction ...... 77

3.2 Isolation of pumilin and 3a-epoxypumilin 77

v Page

3.3 The structure of pumilin ...... 78

3.4 The structure of 3a-epoxypumilin ...... 84

3.5 Experimental ...... 91

4. Preparation and fragmentations of

1,3-dihydroxyeudesmanolide derivatives...... 94

4.1 Introduction ...... 95

4.2 Isolation of costunolide ...... 104

4.3 Reductive and oxidative

modifications of costunolide ...... 107

4.4 Cyclization reactions of germacranolides 108

4.5 Preparation and chemical

transformations of eudesmanolides ...... 110

4.6 Experimental ...... 137

REFERENCES...... 152

VITA...... 158

v i LIST OF TABLES

Table Page

1-1. Sesquiterpene lactones isolated from the

genus Calea ...... 5

1-2. Common ester side chains in the genus Calea ...... 9

2-1. Sesquiterpene lactones of Calea ternifolia

varieties and populations...... 25

2-2. NMR data of compounds 32 and 33 ...... 35

2-3. H NMR parameters of compounds 36 and 89 ...... 36

2-4. H NMR parameters of compounds 36 - 38 ...... 43

2-5. 1H NMR parameters of compounds 51 - 56 ...... 56

2-6. 13C NMR parameters of compounds 47 and *51^ ...... 57

2-7. *H NMR parameters of calein A, 2,3-epoxycalein A,

and epoxide derivative ...... 64

3-1. Sesquiterpene lactones present in the genus

Berlandiera...... 78

3-2. *H NMR parameters of 3a-epoxypumilin, pumilin,

and the pyrazoline ...... 89

3-3. 13C NMR parameters of 3a-epoxypumil in ...... 90

4-1. *H NMR spectral data comparison between C-15-oxo-

elemanolides derivatives (140) and the synthetic

aldehyde (112) ...... 131

vi i LIST OF FIGURES

Figure Page

1-1 Sesquiterpene lactones known from the genus Calea. . 10

2-1 Sesquiterpene lactones isolated from Calea

tern i folia...... 26

2-2 Major mass spectral fragmentation of

ester side chains...... 28

2-3 200 MHz *H NMR spectrum of 83-angeloyloxy-9a-

[2-methylbutanoyloxy]temifol in ...... 37

2-4 200 MHz *H NMR spectrum of 83-angel oyloxy-9a-

acetoxyternifolin ...... 38

2-5 200 MHz *H WR spectrum of 83-angeloyl oxy-9a-

acetoxycalyculatolide ...... 39

2-6 Molecular structure of 9a-acetoxyzexbrevin ...... 41

2-7 200 MHz *H NMR spectrum of 9a-

acetoxyzexbrevin ...... 44

2-8 200 MHz *H WR spectrum of 83-angeloyloxy-

9a-hydroxy calyculatolide...... 45

2-9 200 MHz *H NMR spectrum of 9a-hydroxyzexbrevin.... 46

2-10 200 MHz *H NMR spectrum of 11613-di hydro-11 a,

13-epoxyatripliciolide-83-0-angelate (CDCl^).... 48

2-11 200 MHz *H NMR spectrum of 11,13-di hydro-11 a,

13-epoxyatripiici olide 83-0-angelate (CgDg) ...... 49

2-12 50 MHz 13C NMR spectrum of 11,13-di hydro-11 a,

13-epoxyatri pi iciol ide 83-0-angelate ...... 50

2-13 200 MHz *H NMR spectrum of 11,13-dihydro-

11 a-1 3-epoxyatri pi iciol ide 83-0-methacrylate 51

v iii Page

2-14. 200 MHz NMR spectrum of 9a-hydroxy-l1,13-

di hydro-11 a,13-epoxyatri pii ci oli de-8 3-0-

methacrylate...... 53

2-15. The 3-face of 9a-hydroxy 11,13-dihydro-

11 a,13-epoxyatri pii ci oli de- 83-0-[ 2-methyl-

acrylate]...... 54

2-16. 200 MHz *H NMR spectrum of 9a-acetoxy-ll,13-

di hydro-11 a ,13-epoxyatri pii di olide-8 3-0-

methacrylate...... 58

2-17. 200 MHz *H NMR spectrum of 9a-hydroxy-ll,13-

dihydro-11 a ,13-epoxyatripiiciolide-83-0-

angel ate ...... 59

2-18. 200 MHz *H WR spectrum of 15-hydroxy-ll ,13-

dihydro-11 a ,13-epoxyatripliciolide-83-0-

angel ate ...... 60

2-19. 200 MHz *H NMR spectrum of 2,3-epoxycalein A ...... 65

2-20. 200 MHz *H NMR spectrum of calein A ...... 66

2-21 . 200 MHz *H MIR spectrum of 2 ',3'-epoxycalein A 67

3-1. The structures of sesquiterpene lactones

isolated from Berlandiera species ...... 79

3-2. The CD spectrum of pumilin ...... 82

3-3. 200 MHz M^IR spectrum of pumilin ...... 83

3-4. Stereoscopic view of pumilin ...... 84

3-5. 200 MHz *H M4R spectrum of 3ot-epoxypumilin ...... 85

3-6. X-ray structures of 3a-epoxypumilin ...... 86

3-7. 50 MHz 13C NMR spectrum of 3a-epoxypumilin ...... 88

ix Page

4-1. Preparative HPLC of the costus root oil ...... 105

4-2. 200 MHz *H NMR spectrum of costunolide...... 106

4-3. 200 MHz *H NMR spectrum of dihydrocostunolide-

l,(10)-epoxide ...... 109

4-4. 200 MHz *H NMR spectrum of dihydrosantamarine Ill

4-5. 200 MHz *H NMR spectrum of dihydroreynosin ...... 112

4-6. 200 MHz *H NMR spectrum of 13-mesyloxy-

eudesmano-3-eno-l 2,6a-lactone ...... 114

4-7. 200 MHz *H NMR spectrum of cyclopropyl

derivative acid ...... 115

4-8. 200 MHz *H NMR spectrum of (11S)-1 3-tosyl-

eudesm-3-eno-l 2,6a-lactone ...... 119

4-9. 200 MHz *H NMR spectrum of (11S)-l 3-tosyl-

3-epoxyeudesmano-l 2,6a-lactone ...... 122

4-10. 200 MHz !H NMR spectrum of (11S)-13-tosyl-3-

hydroxyeudesm-4(l 5)-eno-l 2,6a-l actone ...... 124

4-11. 200 MHz *H NMR spectrum of (11S)-13-tosyl-

eudesm-4(l 5)-eno-l 2,6a-1 actone ...... 127

4-12. 200 MHz XH NMR spectrum of (11S)-1 3-tosyl-

3-hydroxyeudesmano-l 2,6o-l actone ...... 129

4-13. 200 MHz *H NMR spectrum of 15-oxo-saussurea

lactone ...... 132

4-14. 200 MHz NMR spectrum of hemiacetal...... 134

4-15. 200 MHz *H fWR spectrum of hemiacetal acetate ...... 135

4-16. Molecular structure of hemiacetal ...... 136

x LIST OF SCHEMES

Scheme Page

1-1. Biogenetic relationships of skeletal types

germacranolides and endesmanolide ...... 4

1-2. Biogenetic relationships of Calea

sesquiterpene lactones ...... 8

2-1. Sesquiterpene lactone extraction procedure ...... 24

2-2. Oxidation of 8e-angeloyloxy-9a-acetoxy-

tern ifo lin ...... 32

2-3. Acetylation of 9a-hydroxy-l1,13-dihydro-

1 lo.l 3-epoxyatripl iciol ide-80-O-methacryl ate ...... 55

2-4. Attempted acid-catalyzed rearrangement of

2,3-epoxycalein A ...... 61

2-5. Epoxidation of Calein A ...... 62

4-1. Total synthesis of costunolide ...... 98

4-2. Proposed synthetic strategy for germacranolides

from eudesmanolides ...... 99

4-3. Synthesis of dihydrosantamarine and

dihydroreynosin ...... 100

4-4. Synthesis of the germacrolide skeleton via

15-oxo-saussurea lactone ...... 101

4-5. Proposed synthesis of an elemanolide and

germacrolide skeleton ...... 102

4-6. Three possible approaches for the introduction of a

hydroxy group at C-3 of eudesmanolide-l-O-tosylate.... 117

4-7. Proposed mechanism of selenium dioxide oxidation 126

xi Abstract

Chemical analysis of Calea te m ifo lia var. calyculata (syn. Calea hypoleuka, Calea salmaefolia, and Calea albida) yielded the known sesquiterpene lactone calein A (21) and the atripliciolide derivatives, ll,13-dyhydro-lla,13-epoxyatripiiciol ide-8e-0-angelate (51), 11,13- dihydro-lla,13-epoxyatripliciolide-8e-0-methacrylate (52), and 9a- hydroxy-ll,13-dihydro-lla,13-epoxyatripliciolide-8e-angel ate (54). In addition, three new germacranolides, 8e-angeloyloxy-9a-[2- methylbutanoyloxy] temifolin (32), 8e-angeloyloxy-9- acetoxytemifolin (33) and five furanoheliangolides, 9a- acetoxyzexbrevin (36), 9a-hydroxyzexbrevin (37), 83-angeloyloxy-9a- hydroxycalyculatolide (38), 9a-hydroxy-ll,13-dihydro-lla,13- epoxyatrip!iciolide 8e-0-methacrylate (55) and 15-hydroxy-ll,13- dihydro-lla,13-epoxyatripliciolide (56) were isolated. The structures of the new compounds were elucidated by NMR and-mass spectral methods.

Single crystal X-ray diffraction of the lactones 36 and 55 established their molecular structure. Calea tem ifo lia var. tem ifo lia (syn. Calea liebmanii) provided the new sesquiterpene lactone 2,3-epoxycalein A

(81). Epoxidation of calein A (21) gave the epoxyangelate derivative (91) but not the expected 2,3-epoxycalein A (81).

Acid-mediated chromate oxidation of 83-0-angeloyloxy-9a-acetoxytemifolin (33) provided the furan-type germacranolide (89).

The isolation and structure determination of two new guaianolides, pumilin (95) and 3a-epoxypumilin (94) from Berlandiera texana and B. lyatra are described. Their structures were determined by NMR and mass spectral methods, and single crystal X-ray analyses.

xii Epoxidation of dihydrosantamarine tosylate (119) followed by treatment with aluminum isopropoxide gave in high yield (llS)-l3-tosyl-

3a-hydroxyeudesmano-4(15)-eno-12,6a-lactone (111). The latter compound was also obtained in good yield from dihydroreynosin tosylate (117) by

Se02 oxidation. Hydrogenation of lactone 111 with Adam's catalyst provide the (llS)-l3-tosyl-3-hydroxeudemano-12,6a-lactone (121).

Based-mediated fragmentation reactions of (llS)-l3-tosyl-3- hydroxyeudesmano-4(15)-eno-12,6a-lactone 111 and (llS)-l3-tosyl-3- hydroxyeudesmano-12,6a-lactone (121) using t-BuOK in t-BuOH provided 15- oxo-saussurea lactone (112) and the hemiacetal (142), respectively.

Hydroboration-oxidation of the mesylate of dihydrosantamarine (115) gave a 1,3-elimination product, the cyclopropyl-type acid (131) in low yield.

xiii Chapter 1

Introduction

1 2

1.1 Systematic background in the genus Calea.

Calea with its approximately 100 species is a fairly large genus

belonging to the family Asteraceae tribe Heliantheae. The genus is

neotropical; its centers of greatest diversity include Mexico, Central

America, northern South America, and Brazil. Most species are shrubs

with opposite leaves, but the genus is by no means uniform in these and

other features. Many species are vine-like and a few are herbaceous or

arborescent, and they vary greatly in their morphology, or have

dissimilar chromosome numbers. A few species are abundant and weedy

while many have restricted distributions and are rare.

Calea urticifolia and C. temifolia are widespread, often fairly

abundant, and largely found in Mexico and Central America. Prior to a

morphological study by Professor Urbatsch and coworkers, Department of

Botany, Louisiana State University, numerous segregate taxa had been

proposed for both species. Presently, they recognize two varieties in

_C. urticifo lia and four in C. te m ifo lia .* Based on our recent chemical

study, C. urticifolia and C. temifolia are nearly identical in their

sesquiterpene lactone constituents. These findings, along with their

largely sympatric distributions and suspected hybridization, support the

close relationship of the two species. The name _C_. tern if ol ia has

priority over all others in the complex including C. prinqlei, C.

zacatechichi, C. albida, C. hypoleuka and C. salmaefolia.* Calea

temifolia is a morphologically variable, wide-ranging species,

occurring from northeastern Mexico to northern Costa Rica. The chemical

studies of various varieties of Calea temifolia form the body of

discussion in the second chapter of this dissertation. 3

1.2 Sesquiterpene lactones in the genus Calea.

Sesquiterpenes are among the most universally distributed naturally

occurring compounds in higher plants. They possess a wide range of

structural variation and therefore have provided an almost lim itless

field of chemical investigation. The subclass of sesquiterpene lactones

are far less widely distributed, occurring predominantly in the family ? 3 Asteraceae. With nearly 1500 compounds known today, * sesquiterpene

lactones display considerable structural diversity resulting from a

variety of cyclizations, ring fissions, oxidations, reductions, etc.

Biogenetically, sesquiterpene lactones are derived from famesyl

pyrophosphate (1) which upon cyclization and subsequent biomodifications

lead to the various skeletal types. The possible biogenetic

relationships of the four subgroups of germacranolides (2 to 5) and the

eudesmanolide skeleton (6) are shown in Scheme 1-1.

To date about 21 taxa of the genus Calea, eleven species and ten

varieties, have been investigated chemically, yielding over 60

sesquiterpene lactones. Most newly isolated compounds from Calea

species represent germacrolides (2) [1(10)-trans,4-trans-

cyclodecadienolides] and mainly he!iangolides (4) [1 -(10) -trans,4-cis-

cyclodecadienolides] and their biogenetic derivatives. Table 1-1 lists

those species and varieties that have been chemically examined along with the numbers and structural types of compounds found in each taxon.

Heliangolides and their bio-derivatives are common constituents in

most Calea species. Eudesmanolides are more restricted and have been l? found to be the major constituents of C. rotundifolia from Brazil.

Scheme 1-2 shows the biogenetic relationships of the 12 structural trans,trar.s-farnesyl pyrophosphate Q)

germacranolide (2) eudesmanolide (EU) (6)

melampolide (GM) Q)

cis,cis-germacranolide (GC) (£)

Scheme 1-1. Biogenetic relationships of skeletal types of

germacranolides and eudesmanolide. 5

Table 1-1. Sesquiterpene lactones isolated from the genus Calea.

Taxon No. of compounds and structural type Ref. SG FH EU

C. temifolia var. zacatechichi 5 2

6,7 4

8 3 4

C. u rticifo lia (Muller) DC. 4 1

8 8

9 3 4

10 2

C. pilosa Baker 11 1 15

C. morii H. Rob. 11 6

C. rotundifolia (Less) Baker 12 3

C. teucrifolia (Gardn.) Baker 13 1

C. scabra 5 no lactones

C. lantanoides 14 1

C. temifolia var. calyculata 15 3

16 2

17 6

C. temifolia var. temifolia 23 3

C. hispida (DC.) Baker 18 1 4

C. hymenolepis Baker 19 2

C. oxylepis Baker 20 3

C. pinnatifida Banks 21 1 6

Table 1-1. Sesquiterpene lactones isolated from the genus Calea.

Taxon No. of compounds and structural type Ref. GG FH EU

C. augusta 22 7

C. new sp. 13 1 4

Germacranolides (GG), Furanoheliangolides (FH), Eudesmanolides (EU). 7 subgroups with increasing biogenetic complexity. These skeletal types of germacranolides which have been found in Calea species, may be derived from a common precursor, the heliangolide (7) and its modification products 18-20. Germacranolides 10, 11 may have been derived from precursor 9 by loss of water and subsequent epoxidation, respectively. Hemiketal formation of diketone 20 would lead to the furanone (13). Subsequent loss of water from 13 would produce furanone heliangolide derivatives, the atripliciolides Q4, 16, and U), and zexbrevin type ring skeleton (15).

One interconversion performed in our laboratory is presented here as an in vitro model reaction for the formation of the furanoheliangolide skeleton from the possible biogenetic precursor of structural type 19. For example, acid-mediated oxidation of 83- angel oyloxy-9a-acetoxytemifolin (33) produced the furanoheliangolide

(89) presumably via the intermediate diketone (87) and the hemiketal 88

(see Scheme 2-2, page 32).

Figure 1-1 tabulates the known sesquiterpene lactones which have been isolated from Calea species. Scheme 1-2. Biogenetic relationships of Calea sesquiterpene lactones.

9 10 11 7

OR OR OR

8 18 19 'O VO VO

'OR OR OR

12 20 13 VO

OH ♦Structural types shown in brackets have not yet been oo

isolated as natural products. 9

Table 1-2. Common ester side chains in the genus Calea.

Structure of side chain Type of Ester Abbreviation

acetate Ac H3

isobutyrate i -But

methacrylate Mac

isovalerate 1-Val

senecioate Sen

2-methylbutanoate 2-Mebut

ti glate Tigl

angel ate Ang 10

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

Calein derivatives R R* Ref

(21) Calein A OAng OAc 6, 15, 23

(22) Calein B OAc OAng 6

(23) Calein C OMac OAc 7. 8

(24) Calein D OAc OMac 7. 8, 21

(25) Caleurticolide- '

[2-methylacrylate] OMac OMac 8, 9, 10

(26) Caleurticolide-angel ate OAng OMac 8, 9

(27) Caleurticolide-i sobutylate OiBut OMac 9

(28) OMac OiBut 8

(29) OMac OAng 8

(30) OTigl OAc 8

(31) OAc OTigl 8 11

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

OR OR'

Temifolin derivatives ______R______IV______Ref.

(32) 8£-Angeloyloxy-9a-

[2-methylbutanoyloxy]-ternifolin iBut Ang 15

(33) 8B-Angeloyloxy-9a-

acetoxyternifolin Ac Ang 15, 23

Zexbrevin derivatives R R1 Ref.

(34) Zexbrevin H OMac 8

(3§) H OTigl 8

(36) 9a-Acetoxyzexbrevin OAc OMac 16

(3Z) 9a-Hydroxyzexbrevin OH OMac 17

(38) 80-An geloyloxy-9a-hydroxy

calyculatolide OH OAng 17 12

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued).

Atripliciolide derivatives R_ JiL Jil Ref

(39) Calaxin OMac H H 4, 11, 13,

(40) 83-Angeloyloxyatripliciolide OAng H H 11, 13,

14, 19, 22

(41J 83-Tiglinoyloxyatripliciolide OTigl H H 11, 22

(42) 93-Hydroxyatri pii ci oli de-8-0-

angel ate OAng OH H 11, 18, 22

(43) 9a-Hydroxyatri pii ci oli de-8-0

tig late OTigl OH H 11, 22

(44) 93-Hydroxyatripii ciolide-8-0

methacrylate OMac OH H 9, 11

(45) 83-Angeloyloxy-15-hydroxy-

atrip licio lid e OAng H OH 8, 18

(46) 83-[methacryloyloxy]-l5-hydroxy-

atripliciolide OMac H OH 8

(47) 83 -[Tiglinoyloxy]-l5-hydroxy-

atripliciolide OTigl H OH 8

(48) 9a-[Isovaleryloxy]-l5-hydroxy

atripliciolide-8-0-[methacrylate] OMac OVal OH 9 13

Figure 1-1. Sequiterpene lactones known from the genus Calea.

(Continued)

R R1 R11 Ref.

(49) 9a-[Seneci oyloxy]-l5-hydroxy-

atripliciolide-8-0-[methacrylate] OMac Osen OH 9

(50) 9a-[Angeloyloxy]-l5-hydroxy-

atripliciolide-8-0-[methacrylate] OMac OAng OH 9

Atripliciolide derivatives R R1 R" Ref.

(51) 11»13-Dihydro-11 a,13-epoxy-

at ri pii ci oli de-83-0-an gel ate OAng H H 11, 13, 17

(52) 11,13-Dihydro-11 a,l3-epoxy-

atri pii ci olide-8g-0-methacrylate OMac H H 11, 13, 17

(53) 11,13-Dihydro-ll a-13-epoxy-

atripliciolide-8e-0-tiglate OTigl H H 11

(54) 9a-Hydroxy-l1,13-dihydro-

11 a,13-epoxyatri pi icioli de-

8e-0-angelate OAng OH H 11, 17 14

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

R R1 R" Ref

(55) 9a-Hydroxy-l1,13-dihydro-

11 a,l3-epoxyatripii ci oli de

83-0-methacrylate OMac OH H 11, 16

(56) 15-Hydroxy-ll,13-dihydro-

11 a, 13-epoxyatri pii ci oli de-

83-0-angelate OAng H OH 17

(57) 9a-Hydroxy-l1,13-dihydro-

11 a,l3-epoxyatri pii ci oli de-

83-0-tiglate OTigl OH H 11

(58) 11,13-Dihydro-11 a,13-epoxy

atri pii ci ol i de-83-0-[2-methyl-

butanoate] iBut H H 22

(59) 9a-Hydroxy-ll,13-dihydro-

11 a, 13-epoxyatri pii cioli de-

83-0-[2-methylbutanoate] iBut OH H 22 15

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

R R1 R" Ref

(60) 11-Hydroxy-13-chloro-ll,13-

di hydro-at ri pii ci oli de-8-0

. an gel ate OAng H H 11

(61) 11-Hydroxy-13-chloro-ll ,13-

di hydro-atri pii ci oli de-8-0-

tig late OTigl H H 11

(62) 9a,11-Dihydroxy-13-chloro-l 1,

13-di hydroatri pii ci oli de-8-0

an gel ate OAng OH H 11 16

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

(63) 5-Myrtenyl-4,5-11,13- o c tetrahydro-11,13-

epoxyatri pii ci oli de-8-0-

angel ate 11

OH (64) 5B-Myrtenyl-9a-hydroxy-4,5- OC' di hydro-atri pii ci oli de-8-0- 1 fi angelate.

(65) 5e,9a-Dihydroxy-4,5- 'OC- di hydro at ri pi i ci ol i de-8-0-

angelate.^® 17

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

1,10-Dihyrocostunol ide derivatives R R' Ref.

(66) 33-Acetoxy-8g-angeloyloxy-

1,10-di hydro-1 a-10g-

epoxycostunolide OAc An g 11

(67) 13,1Oa-Epoxy-83-

tiglinoyloxy-1,10-dihydro-

costunolide H Tigl 12

Ref.

(68) Heliangin-3-0-acetate Tigl OAc 12

(69) Heliangin Tigl OH 12, 18,

19 18

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

OR*

Zacatechinolide derivatives _R R1 R" Ref.

(70) 1o-Acetoxyzacatechinolide H Ac Mac 5

( Z D 1a-Acetoxy-8-deacylzacatechinoli de-

2-methylbutyrate H Ac 2-Mebut 20

(72) 10-Hydroxy-8-deacylzacatechinolide-

[2-methylbutyrate] H H 2-Mebut 20

(73) 1-Epiniveuin C acetate H Ac Ang 13

RO''

Zacatechinol ide derivatives ______R______RJ______Ref.

(74) 1-Oxo-zacatechinolide H Mac 5

(75) l-0xo-8-deacylzacatechinolide-

[2-methylbutyrate] H 2-Mebut 20 19

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

OR HO

2,3-Epoxycalein derivatives R ______RJ______Ref.

(Z5) Mac Ang 8 Ang Mac 8

(77) Mac iVal 8 i Va 1 Mac 8

(78) Mac iBut 8

iBut Mac 8

(Z5) Mac Ac 8

Ac Mac 8

(85) Mac Mac 10

(§1) Ang Ac 24 20

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

1 p (82) 83-Tigl inoyloxyreynosin

1 p (83) 83-Tiglinoyloxybalchanin .

(84) 10-Hydroxy-8 0-tiglinoyloxy- Ip arbusculin . 21

Figure 1-1. Sesquiterpene lactones known from the genus Calea.

(Continued)

(85) Isoatripliciolide 19 angelate .

(86) Caleamyrcenolide^. Chapter 2.

The isolation, structure elucidation, and chemistry

of sesquiterpene lactones from Calea ternifol ia.

22 23

In this chapter, the structure elucidation and chemistry of a number of sesquiterpene lactones isolated from Calea ternifolia var. calyculata and C. ternifolia var. temifolia are described.

2.1 Isolation of tem ifo lin s (32, 33), zexbrevin derivatives (36 - 38),

atrip licio lid e derivatives (55-56) and 2,3-epoxycalein A (81) .

The dried and ground plant material was extracted according to the general procedure outlined in Scheme 2-1, The *H NMR spectrum of the crude syrup indicated the presence of vinylic protons centered at 6.3 ppm and 5.8 ppm, suggesting the presence of a -methylene lactones.

Furthermore, signals between 1 and 2 ppm were indicative of acetates and vinylic methyl groups and methyls attached to saturated carbons (1.0-1.5 ppm).

Subsequently, the crude syrup was examined by thin layer chromatography in order to determine the approximate number of components and to find a suitable solvent system for separation by colurm chromatography. Separations were carried out as described in the experimental section which resulted in the isolation of twelve compounds

(see Figure 2-1). Of these twelve lactonic constituents, four have been recently reported by Bohlmann et al.** The structure determinations of eight new compounds (32, 33, 36-38, 55-56, and 81) are described in this chapter. These twelve lactones which were isolated from the various population of the subspecies Calea ternifolia are listed in Table 2-1.

Hereafter, assignments of mass spectral fragmentation pattern notations as given in Figure 2-2 will be used in the discussion. 24

DRY PLANT MATERIAL (1 Kg)

CHCI3 (6 L)

CRUDE EXTRACT

v EtOH (2L)

PLANT FATS (PPT) + TERPENOID SOLUTION

2L-5% Pb(OAc)2

TERPENOID SOLUTION + PHENOLICS, CHLOROPHYL (PPT)

REMOVE EtOH vacuo

OIL + WATER MIXTURE

3X400 mL CHCI3

w FILTER AND EVAPORATE

CRUDE SYRUP (1-20 g)

Scheme 2-1. Sesquiterpene lactone extraction procedure. 25

Calea ternifolia var. calyculata (syn. C^. hypoleuka) , 32, 33.

(syn. C. salmaefol ia) 36, 55 .

(syn.' " _ C. _ a1bida)37, - • /V.rs. 7 38, /N.A. * 51, ft.#*.' 52, 4S./S.* 54, 56.

Calea ternifolia var. ternifolia (syn. C. 1iebmanii) 21, 33, 81.

Table 2-1. Sesquiterpene lactones of Calea ternifolia varieties and

populations. 26

21. Calein A

OC:

32. 8^-Angeloxyloxy-9a-[2- OR OR' methy 1 butan oyl oxy ]-tem i fo lin ,

R = iBut, R1 = Ang

33. 8B-angeloyloxy-9o-

acetoxyternifolin, R = Ac, R1 =

Ang.

34. Zexbrevin, R = H, R‘ = OMac

9a-Acetoxyzexbrevin, R = OAc,

R‘ = OMac

9a-Hydroxyzexbrevin, R = OH, R1

= OMac.

38. 8 3-An geloyloxy-9a-Hydroxy-

calyculatolide, R = OH, R1 =

OAng.

Figure 2-1. Sesquiterpene lactones isolated from Calea tern ifo lia. 27

Figure 2-1. Sesquiterpene lactones isolated from Calea ternifolia.

(Continued)

5U 11,13-Dihydro-11 a,13-epoxyatripliciolide-8e-0-angelate, R =

OAng, R' = H, R" = H.

52. 11,13-Dihydro-ll a,13-epoxyatripliciolide-8e-0-methacrylate, R =

OMac, R‘ = H, R" = H.

54 . 9a-Hydroxy-ll ,13-Dihydro-ll a,13-epoxyatripliciolide-8e-0-

angelate, R = OAng, R' = OH, R" = H.

55. 9a-Hydroxy-ll,13-Dihydro-ll o,13-epoxyatripliciolide-83-0-

methacrylate, R = OMac, R' = OH, R" = H.

56. 15-Hydroxy-l1,13-dihydro-11 a,13-epoxyatripiiciolide-8e-0-

angelate, R = OAng, R' = H, R' = OH.

OAc

81 2,3-Epoxycalein A 28

A' A* B B B 102 |85 157 100 155 19 !9h3 I 9 I9h3 H 0 h -C -LC-CH2CH3 H 0 *-C —rc = CH—Cl-fa I |' 1 2' 3' 4 |' i 2' 3' 4

D D D 86 69 1 41 116 [99 [r f 1 9 19H3 ,9 l9H3 ?H3 H 0 -j C—jcH=CH2 HO—|- C—|C ——-C H I I V , 1 I' 1 2' V I l ' 12' 3

Figure 2-2. Major mass spectral fragmentation of ester side chains. 29

2.2 The structures of temifolin derivatives 32 and 33.

8g-Angeloyloxy-9a-[2-methylbutanoyloxy]-ternifolin (32), ^ 258350 ^, was a colorless gum, which displayed in the 200 MHz *H NMR spectrum

(Fig. 2-3) two one-proton doublets at 6.29 (Jj ^ = 2.0 Hz, H-13a) and

5.79 ppm (Jj ^ 3k = 2.0 Hz, H-13b) and a broad mutiplet at 2.60 ppm (H-

7) that are characteristic of an a-methylene-y-lactone. An IR absorption at 1765 cm -1 corroborated the presence of a y-lactone moiety. Further IR bands at 3450 and 1710 cm"* suggested the presence of hydroxyl and ketone functions. The presence of two ester side chains in 32 was evident in the NMR spectrum; absorption typical of an gel ate

(1.77q , 3H, _J=1.5 Hz; 1.95 dq, 3H,_J=1.5, 7.5 Hz; 6.13 qq, 1H, _Jj=l .5,

7.5 Hz) and 2-methyl butanoate (0.88t, 3H; 1.16d, 3H, J_=6.5 Hz; 1.42dq,

2H; 2.38 sext, 1H) were observed. The mass spectrum of 32 verified the above NMR assignments showing strong peaks at m/z_ 85(A') and _m/z_

83(B1). This was also supported by IR bands at 1745 (angelate) and 1725 cm"* ( 2-methylbutanoate).

Irradiation of the mutiplet at 2.60 ppm (H-7) changed the doublet of doublets at 5.94 (H- 8, J_7 g = 1.5 Hz) to a doublet, simplified the threefold doublet at 4.92 (H- 6, _J7 6 = 3.5 Hz) to a doublet of doublets and collapsed the two H-13 doublets at 5.79 and 6.29 ppm to singlets.

On the basis of chemical shift arguments the absorption at 4.92 ppm was assigned a proton at a lactonic carbon whereas the signals centered at

5.94 ppm were ascribed to a proton at a carbon carrying an ester group. Double irradiation at 5.94 ppm collapsed the doublet at 5.85 (H-

9» i s ,g = 10.5 Hz) the chemical shift suggesting the attachment of the second ester moiety to C-9. 30

Irradiation at 4.92 ppm (H- 6) sharpened the H-7 multiplet at 2.60 ppm to a broad singlet and affected the one-proton multiplets centered at 1.62 (H-5b) and 2.12 ppm (H-5a) which were coupled to a multiplet at

1.75 ppm (H-4). In return, irradiation of the center of the H-4 signal affected the two H-5 absorptions, collapsed the three-proton doublet at

1.14 ppm (C-4-CH3) to a singlet and simplified the threefold doublet at

4.31 (H-3, _J_3 4 = 2.5 Hz). Saturation of the H-3 signal at 4.31 collapsed the two doublets at 2.95 ppm (H-2b, 3 = 6.5 Hz) and 3.25 ppm (H-2a, _J_2a 3 = 10 Hz) to an A,B-pattem 2b = 1® Hz). The chemical shift of H-3 suggested the attachment of a hydroxyl group to C-

3 and the absorptions near 3 ppm of the two geminally coupled protons at

C-2 indicated their positioning next to a carbonyl function.

The above NMR data accounted for all atoms in compound 32 except one of each of carbon, oxygen, hydrogen and a methyl group as indicated by a three-proton singlet at 1.28 ppm.

Since most Calea-derived sesquiterpene lactones possess a C-l

Q carbonyl and commonly carry a hydroxyl group at C-l0 , the structure of the new compound could be tentatively formulated as the ten-membered ring 32 exclusive of stereochemistry and sites of attachement of the two ester groups. Due to the great sim ilarity of the medium ring proton

*H NMR absorptions of 32 and 33 , compound 33 must possess the same ring skeleton and differ from 32 by the absence of the 2-methylbutanoate group and the presence of an acetate moiety. (Compare the *H NMR spectra shown in Figure 2-3 and Figure 2-4).

The stereochemistry at C-6, C-7, C-8 and C-l0 of compounds

32 and 33 was assigned as H- 6e, H-7a, H-8a, and C-10g-CHg by chemical OC transformation of 33 to the furenone-type compound 89 . 31

Acid-mediated chromate-oxidation of 33 provided after PLC separation compound 89 (Scheme 2-2) which exhibited medium ring proton signals nearly identical with the NMR parameters of 9a- acetoxyzexbrevin (36)1®, another C. ternifolia constituent the structure of which was established by X-ray diffraction. The large coupling constant (J_g g = 10Hz) for both, 32 and 33, indicated antiperiplanar orientation of H -8 and H-9 suggesting H-9& in both,

32 and 33/V.A. , * which is in accord with the NMR correlations of the oxidation product 89 and the X-ray established 9a-acetoxyzexbrevin

(36) stereochemistry (Table 2-3).

2.3 Oxidation reactions of compounds 32 and 33.

Oxidation with pyridinium chlorochromate (PCC) of lactones

A.N32 and 33 A.A. resisted reaction. However, acid-mediated ' chromate oxidation of 33 with Jones' reagent provided compound 89 with medium ring proton signals in the 200 MHz NMR (Figure 2-5) which were nearly identical Pd with the spectral parameters of zexbrevin (34) and its derivatives

36-38. The la tte r compounds, which were isolated from other

Calea ternifolia collections, will be discussed below.

A possible mechanism for the oxidative transformation of

33 to 89 is shown in Scheme 2-2. The C-10-hydroxyl of the in itia lly formed diketone (87) would attack the carbonyl at C-3 to give hemiacetal (8§)which after loss of water would provide 89. The stereochemistry of the C-4 methyl in (89) was established by *H NMR 15 correlation with 9a-acetoxyzexbrevin (36) (compare data in Table

2-3). 32

OAc OAc l y ) HO1 HO'

>00

89 «w 88 v o

Scheme 2-2. Oxidation of 8B-angeloy1oxy-9a-acetoxyternifo1in. 33

The deshielding of the acetate singlet at 2.26 ppm in compound

89 is similar to analogs which had been prepared by acetylation of 9a-

hydroxyfuranogermacranolides.Therefore, in compound 33 the

attachment of the acetoxy moiety is tentatively assigned to C-9 and the

angelate to C- 8. In compounds 32 and 33, the NMR chemical shifts of

the angelate proton absorptions are the same within experimental error

suggesting a similar chemical environment. Therefore, in compound 32

the attachment of the angelate group must be at C -8 and the 2- methylbutanoate moiety at C-9.

The stereochemistry at C-3 and C-4 was tentatively assigned by correlation of the dihedral angles of the medium ring protons with the experimentally observed _J_-values by application of the Karplus ?c correlation. Using stereomodels, two major conformations were considered in this treatment. One with a downward orientation of the

C-l carbonyl and the other with the C-l carbonyl being oriented upward. In both conformations the skeleton of the medium ring was fixed at C-6 and C-9 so that the proton dihedral angles were: H-9/H-8

^180°C; H-8/H-7 ^80°; and H-7/H-6 ^140°; those angles correlated very well with the experimental _J_-values of the *H NMR absorptions. The dihedral angles of H-2a, H-23, H-3, and H-4 in a conformation with an orientation of the C-l carbonyl function below the plane of the medium ring correlated poorly with the observed photon couplings in all four configurational isomers at C-3 and C-4. The four possible configurational relationships at the chiral centers C-3 and C-4 in a conformation with an upward oriented C-l carbonyl function were considered next. The dihedral angles H-4a/H-30« 110°; H-36/H-2B « 50° were derived from model considerations and correlated best with the 34

experimental J-values suggesting a 3a-0H and 4e-CH 0 in 32 and 33. ^ rs.A, 35

Table 2-2. NMR data 3 of compounds' 32 (S. and 33. .

32 33

H-2b 3.25 dd (18, 10.5) 3.25 dd (18, 10)

H-2a 2.95 dd (18, 6.5) 2.96 dd (18, 6.5)

H-3 4.31 ddd (10.5, 6.5, 2.5) 4.34 ddd (10, 6.5, 2.5)

H-4 1.75C c

H-5a 2.12 ddd (4, 6.5, 16) c

H-5b - 1. 6C c

H-6 4.92 ddd (6.5, 6.5, 4) 4.90 ddd (6.5, 6.5, 3.5)

H-7 2.60 m 2.59 br s

H-8 5.94 dd (10.5, 1.5) 5.88 dd (10, 1.5)

H-9 5.85 d (10.5) 5.54 d (10)

H-l 3a 6.29 d (2.0) 6.31 d (2.0)

H-l 3b 5.79 d (2.0) 5.80 d (2.0)

C-4-Me 1.14 d (6.5) 1.14 d (6.5)

C-10-Me 1.28 s 1.27 s

OAc b 2.06 s

OAng 6.13 qq (7.5, 1.5) 6.12 qq (7.5, 1.5)

1.95 dq (7.5, 1.5) 1.94 dq (7.5, 1.5)

1.77 q (1.5) 1.77 q (1.5)

3 Spectra were run at ambient temperature in CDCI 3 at 200 MHz; TMS- was used as internal standard. Values are in ppm(<5) and signals are designated as follows: singlets, s; doublet, d; triplet, t; quartet, q; m ultiplet, m; broad singlet, br s. Figures in parentheses are coupling constants or line separation in hertz.

b Chemical shift data of the 2-methylbutanoate group: 2.38 sext (H- 2 '); 1.42 dq (2H-31); 1.16 d (6.5 Hz, C-2'-Me); 0.88 t (3H, C-3'-Me).

c Obscured by other signals. 36

Table 2-3. *H NMR parameters3 of compounds 36 and 89.

36 IN.A. 39"

H-2 5.58 s 5.58 s

H-4 3.05 m 3.05 m

H-5a 2.69 m 2.57 m

H-5b 2.11 m 2.10 m

H-6 4.32 dd 4.45 dd (10, 4.5)

H-7 3.62 m 3.59 m

H-8 4.99 dr d (5) 5.06 dd (5.1)

H-9 5.38 dr d (5) 5.40 d (5)

H-l 3a 6.33 d ( 2. 2) 6.33 d (3)

H-13 b 5.38 d ( 2. 2) 5.50 d (3.5)

C-4-Me 1.39 d (7) 1.40 d (7)

C-10-Me 1.38 s 1.37 s

OAc 2.20 s 2.26 s

C-2'-Me 1.88 s 1.96 dq (7.5, 1.5)

C-3'-Me — 1.82 dq (1.5)

H-3'a 6.02 br s 6.16 qq (7.5, 1.5)

H-3 'b 5.66 br s

3 Spectra were run at ambient temperature in CDCI 3 at 200 MHz; TMS was used as internal standard. Values are in ppm ( 6) and signals are designated as follows: singlets, s; doublet, d; triplet, t; quartet, q; multiplet, m; broad singlet, br s; Figures in parentheses are coupling constants or line separation in hertz. Figure 2-3. 200 MHz 1H NMR spectrum of80-angeloyloxy-9a-[2-methybutanoyloxy]temifolin (32)

(CDC1)3-

trrod. H - 7 3 -CH 3 2- CH3 3-CH-

13a 13b

2b OH

T T 7 .0 G.O 3 04.0

OJ Figure 2-4. 200 MHz *H NMR spectrum of 8g-angeloyloxy-9a-acetoxytemifol in (33) (CDCI3 ).

OAc

HO' j-c^ HOT OAc

130 !3 b

OH 3 0

TO 6.0 5 .0 4-.0 3 0 2.0 1.0

u> CO Figure 2-5. 200 MHz *H NMR spectrum of 8&-angeloyoxy-9ct-acetoxyca1ycu1atolide (89) (CDC13).

OAc

OAc 2-CHa

13a 13b

7.0 5 .0 4 .0 2.0

COUD 40

2.4 The structures of zexbrevin-type furenones

2.41 9a-Acetoxyzexbrevin (36), 02^ 2403, mp 200-203°C, a colorless crystalline compound exhibited UV [X max 258 nm) and IR (1700 and 1590 I 0*7 cm" ) absorptions suggesting a furenone carbonyl chromophore (F) .

The 200 MHz NMR (Figure 2-7) of 36 was very similar to zexbrevin 24 (34) except that spin docoupling experiments on 36 indicated only one

( 0

C-9 proton with a chemical shift (5.38 ppm) that suggested an ester function. A three-proton singlet at 2.20 ppm together with a mass spectral peak at jn/z_ 43 was in agreement>with an acetate moiety in

36. Since the chemical shift of the acetate methyl was the same as in a zexbrevin-type compound obtained from a 9a-hydroxy precursor by a c ety la tio n ,^ it allowed tentative assignment of the acetate to C-9 and 2ft the methacrylate to C-8. The coupling 1 Hz suggested on 8a-H^° and Jjj 9 = 5Hz correlated well with a H-93 or a 9a-acetoxy group in 36, as derived from stereomodel consideration. The change of the C-4 stereochemistry in the transformation of 33 to 89 could have occurred during the acid-mediated oxidation of 33. Alternatively, the configuration at C-4 in 9o-acetoxyzexbrevin (36) and by analogy zexbrevin (34) required revision. Because of this ambiguity, single crystal X-ray crystallography (Figure 2-6) was performed on

9a-acetoxyzexbrevin, and the stereochemistry of the C-4-Me has thus been 2R established to be 3 as depicted in Figure 2-6. 3 41

Figure 2-6. Molecular structure of 9a-acetoxyzexbrevin (36).

2.42 8e-Angeloyloxy-9a-hydroxycalyculatolide (37) and 9a-

hydroxyzexbrevin (38) .

Compound 37 and 38 were isolated from Calea te m ifo lia var. calyculata (syn. Calea albida No. 320). These two compounds displayed

*H NMR and MS signals which were identical except for absorptions that indicated differences in the ester groups attached to the two molecules.

8g-Angelo.yloxy-9a-h.ydrox,ycalycu1atolide (37) , ^20^24^7* mP- 175°C

(dec.) exhibited *H NMR spectral signals (Figure 2-8) which were very similar to 9a-Acetoxyzexbrevin (36) a compound of known X-ray structure. The spectral parameters of the two compounds differed in the chemical shifts of the H-9 absorptions which in 36 appeared at 5.38 ppm together with an acetate methyl singlet at 2.20 ppm. In the calyculatolide 37 the acetate signal was missing and H-9 represented a doublet at 4.19 ppm (J_= 6; 4.8 Hz) suggesting the presence of a hydroxyl group at C-9; this was also supported by an IR OH absorption at 3420 cm"* as well as a mass spectral peak m/z_ 358 (M-H2O). The presence of 42 an angelate ester side chain at C -8 was indicated by diagnostic fWR and MS signals. The new compound exhibited methyl absorptions, doublets of quartets at 1.92 (C-2'-methyl) and 1.82 ppm (C-3'-methyl), respectively, and a one-proton quartet at 6.15 ppm which are typical for the angelate moiety. Furthermore MS peaks at _m/z_ 276 (M-B), 83 (B1), and 55 (B") were in accord with the above assignments.

The great similarity of the NMR parameters of 9a-acetoxyzexbrevin

(36) and calyculatolide (37) suggests the same skeletal arrangement, including all stereochemical centers of the two compounds, and therefore

8B-angeloyloxy-9a-hydroxycalyculatolide must have structure 37. The acetylation of 37 with acetic anhydride/pyridine could not be attempted because of the small amount of material.

The other minor gummy consituent, 9a-hydroxyzexbrevin (38),

^19H24®7» whic^ could not be freed from 37 , exhibited *H NMR signals

(Figure 2-9) very similar to those of the calyculatolide 37. The two compounds had nearly identical *H NMR parameters for the medium ring proton absorptions but they differed in the ester side chain at C- 8.

Compound 38 showed NMR and MS signals typical for the methacrylate moiety. A broadened three-proton singlet at 1.89 ppm and a pair of broad one-proton signals at 5.64 and 5.98 ppm together with diagnostic

MS peaks at 69 (C1) and 41 (C") were in accord with a methacrylate group at C-8. Therefore, the stereochemical representation 38 is suggested for 9a-hydroxyzexbrevin. The NMR data of compounds 36, 37, and 38 are listed in Table 2-4. 43

Table 2-4. *H NMR parameters 3 of compounds 36-38.

rs.r.36 MS37 38

H-2 5.58 s 5.56 s 5.56 s

H-4 3.05 m 3.03 m 3.03 m

H-5a 2.69 m 2.62 m 2.62 m

H-5b 2.11 m 2.10 m 2.11 m

H-6 4.32 dd 4.52 dd 4.45 dd

(9.6, 4.8) (9.6, , 4.8)

H-7 3.62 m 3.79 m 3.81 m

H-8 4.99 br d (5) 5.12 d (4.8) 5.07 d (4.8)

H-9 5.38 br d (5) 4.19 t ( 6) 4.18 t (6)

H-l 3a 6.33 d (2.2) 6.35 d (3) 6.35 d (3)

H-13 b 5.38 d (2.2) 5.78 d (3) 5.78 d (3)

C-4-Me 1.39 d (7) 1.39 d (7) 1.39 d (7)

C-10-Me 1.38 s 1.64 s 1.64 s

OAc 2.20 s ------

C-2'-Me 1.88 s 1.92 dq 1.89 s

(7.5, 1.5)

C-3'-Me ------1.82 dq (1.5) ------

H-3'a 6.02 br s 6.15 qq 5.98 br s

(7.5, 1.5)

H-3'b 5.66 br s ------5.64 br s

9-OH ------3.30 d ( 6) 3.19 (6)

a Spectra were run at ambient temperature in CDCI 3 at 200 MHz; TMS was used as internal standard. Values are in ppm ( 6) and signals are designated as follows: singlets, s; doublet, d; triplet, t; quartet, q; m ultiplet, m; broad singlet, br s. Figures in parentheses are coupling constants or line separation in hertz. Figure 2-7. 200 MHz *H MR spectrum of 9a-Acetoxyzexbrevin (36) (CDC13).

OAc

13b 13a

7.0 5.0 4.0 3.0 2.0

4=» Figure 2-8. 200 MHz NMR spectrum of 8g-angeloyloxy-9a-hydroxyca1yculatolide (37) (CDCI3 ).

OC-

*CH,

13 a 13b

7.0 6.0 50 4.0 3.0 2.0

-F» Figure 2-9. 200 MHz 1H NMR Spectrum of 9a-hydroxyzexbrevin (38) (CDC13).

OH 2-CH: OC;

13a OH

5 a

7.0 6.0 5.0 4.0 3.0 2.0 1.0

-t* cr> 47

2.5 The structure of atripliciolide derivatives

(51, 52, 54, 55 and 56). 'MV ' NAi ' IS./V. •

r______\v r

51^ OAng H H

52 OMac H H

54 OAng OH H

55 OMac OH H

56 OAng H OH

2.51. 11,13-Di hydro-11 a,13-epoxyatripliciolide-8s-0-angelate (51_)

and 11,13-Dihydro-11a,l3-epoxyatripiicioli de- 83-O-methacrylate

(52) .

Two known compounds were isolated from Calea ternifolia var. calyculata (syn. Calea albida, No. 338). Compound 51_, C2 qh2287> mp* 88"

91°C, exhibited a *H NMR spectrum (Figure 2-10, CDCI 3 and Figure 2-11,

CgDg) in which the parameters were identical with those of a gummy compound described by Bohlmann et a l ^ as a constituent of C. pilosa from Brazil. 1 ^ The C WR spectrum of 51^ is shown in Figure 2-12 and spectra 1 O data are listed in Table 2-6. The C NMR parameters were tentatively assigned on the basis of proton noise decoupling (PND) and by comparison O with reference compound 47 described by Herz et al.

Compound 52, 0^ 2(307, mp 155-158°C showed the 1H NMR (Figure 2- Figure 2-10. 200 MHz 'li NMR spectrum of 11,13-dihydro-11 a,13-epoxyatripliciolide 8e-0-angelate (51) (CDClj).

7 0 6 0 5 .0 4 . 0 3 .0 3-CH

7 8 |3ab

3 ° o 9 a 9 b Aa _

6.0 5.0 4.0 3.07.0 2.0 1.0 -P* 00 Figure 2-11. 200 MHz NMR spectrum of 11,13-dihydro-11 o,13-epoxyatripliciolide 86-0-angelate (51) ( W -

2 8> 6 2-Qi 13b 9b 3-a

7.0 60 5.0 4.0 3.0 2.0 1.0

-F» io Figure 2-12. 50 MHz 13C NMR spectrum of 11,13-dihydro-ll a,13-epoxyatripliciolide 8g-0-ange1ate (51) (CDC13)-

C -7

C-13 C-6 C -3 C-9

C -2 C-2 -CH3 C- 8 C -2 ' C - 6 C-4 C -3 C -lO C-W C-3-CHj C-3

C -ll C -l C-12

■mTpTH m n w m M iiii m im iiin mTTnm 'm fm i n m |iiiin iiT|m m M i|m m n ijim n 'in| 100 on o Figure 2-13. 200 MHz lH NMR spectrum of 1 1 , 13-dihydro-ll a, 13-epoxyatripiiciolide 8B-0-methacrylate (52) (CDC13).

2'- ch 3

9a 9b

7.0 0.0 4 .0 J o 2 .0 52

13, CDC13) spectrum which was identical with data described for compound 52 reported in the literature.

2.52. 9a-Hydroxy-l 1,13-Dihydro-11 a,l3-epoxyatripii ci o lide- 80-O-an gel ate

(54) and 9a-hydroxy-ll,13-dihydro-ll a,l3-epoxyatripliciolide 86-

O-methacrylate (55).

Compound 55, C^H^Og, a colorless crystalline compound with a melting point of 200°C (dec.) showed UV (A max 262 nm) and IR (1700 and

1590 crrf*) absorptions suggesting a furenone carbonyl chromophore p 7 (F) The presence of hydroxyl(s) and a y-lactone moiety was indicated by IR bands at 3415 and 1785 cm-*, respectively. The presence of an additional methacrylate ester function was suggested on the basis of diagnostic *H NMR signals: two broadened one-proton singlets at

5.66 and 5.94 ppm and a broad three-proton singlet at 1.91 ppm as well as MS peaks at jn/z_ 290 (M-C 4Hg02) and 69 (C‘). The NMR spectral pattern (Figure 2-14) was in agreement with a furanone-type heliangolide related to the known atripiiciolide skeleton. However, singals typical for the exocyclic methylene lactone protons at 6.3 and 5.7 ppm were missing. Instead, two one-proton signals appearing as doublets (AB pattern) were found near 3.6 ppm typical for an epoxide function between

C-ll and C-l3 . 11

A multiplet at 3.99 ppm was attributed to H-7 and verified by double irradiation. Two other signals, which must correspond to H -6 and

H-8, were affected. A broad multiplet at 5.35 ppm sharpened distinctly and the doublet of doublets centered at 5.32 ppm (1H, J_ = 5.2, 2.0) was transformed into a doublet (J_ = 5.2 Hz). Reciprocal decoupling Figure 2-14. 200 MHz NMR spectrum of 9a-hydroxy-l1,13-dihydro-lla , 13-epoxyatripliciolide 86-0- methacrylate (55)(CgD5 N).

OH >OC

30,5

6.0 5 .0 4 .0 3 .0 2.0 u> 54 experiments confirmed the assignments of H -6 and H- 8. Based on the above results the signals at 5.32 and 5.35 ppm were assigned to H -6 and

H-8, respectively. One remaining signal was assigned to H-9 on the basis of coupling with H -8 whose irradiation produced a sharpening of the H-9 triplet.

The crystal structure of 55 unambiguously confirmed the basic skeletal arrangement and relative stereochemistry of all chiral centers and established the C-l1(13)-epoxide stereochemistry to be a as shown in

Figure 2-15.

Figure 2-15. The B-face of 9a-hydroxy-l1,13-dihydro-l1 a ,l3-

epoxyatri pii ci oli de- 8B-0-[ 2-methylaery1 ate].

Acetylation of 55 with Ac 20/pyridine produced a monoacetate,

^21^22®9» which was identified as compound 90 (Scheme 2-3). The NMR spectra of 90 (Figure 2-16) indicated modification of the hydroxyl group causing a shift of H-9 from 4.47 in 55 to 5.17 ppm in 90 . 55

Scheme 2-3. Acetylation of 9a-hydroxy-l1,13-dihydro-11 a ,l3-

epoxyatripiiciolide-8g-0-methacrylate (55)

The known compound 54 , a colorless crystalline substance, mp 205-

207°C, exhibited an *H NMR spectrum (C 5D5N) which was almost identical with NMR data reported in the literature.Figure 2-17 shows the

MR spectrum (CDC13) of compound 54.

2.53 15-Hydroxy-11,13-di hydro-11 a, 13-epoxyatri pii ci oli de- 83-O-an gel ate

(56) .

Compound 56, mP 118-121°, displayed *H NMR signals

(Figure 2-18) which were similar to those of compound 55 with known X- ray structure. Detailed spin decoupling experiments of this substance led to assignments which are summarized in Table 2-5. The data indicated the lack of a hydroxyl function at C-9 and a methyl at C-4.

Instead, the presence of an OH group at C-l5 was indicated by a two- proton doublet at 4.20 ppm. The attachment of an angelate ester group at C-8 was supported by diagnostic *H NMR signals (Table 2-5) and characteristic MS peaks at m/z_ 83 (B1) and 55(B"). Table 2-5. (HR param eters of compounds 51-56^

51a 51b 52a 56a 54a 54c 55c

H-2 5.59 s 5.04 s 5.60 s 5.69 s 5.61 s 5.86 s 5.84 s

H-4 6.01 dd 5.32 dd 6.02 dd 6.30 m 6.04 m 5.96 m 5.94 m (4 .4 , 2 .0 ) (4 .4 , 2 .0 ) ( 4 .4 , 2 .0 )

H-6 5.32 5.04 m 5.24 m 5.33 m 5.23 m 5.47 m 5.35 m h e p te t (2 .0 )

H-7 3.31 dd 2 .9 9 dd 3.31 m 3.35 m 3.43 dd 3.99 dd 3.99 dd (4 .5 , 2 .2 ) (3 .6 , 2 .0 ) (4 .4 , 1 .6 ) (4 .0 , 2 .0 ) (4 .0 , 2,

H-B 5.14 4.76 m 5.06 m 5.14 m 5.03 dd 5.42 dd 5.32 dd (h e p te t) (5 .2 , 1 .6 ) (4 .8 , 2 .0 ) (4 .0 ) (2 .0 )

H-9a 2.41 dd 1.94 dd 2.44 dd 2.43 dd 4.00 dd 4.48 dd 4.47 t (14.4, 6) (14.4, 6) (14.4, 6) (14.4, 6) (7 .2 , 4 .2 ) (7 .6 , 4 .8 ) (5 .6 )

H-9b 2.19 dd 1.47 dd 2.1 9 dd 2.20 dd ------(1 4 .4 , 4) (1 4 .4 , 4) (1 4 .4 . 4) (1 4 .4 , 4)

C-4-Me 2.09 s 1.43 s 2.09 s 4.43 br 2.08 s 1.90 s 1.90 s (-CHoO) C-10-He 1.46 s 1.06 s 1.47 s 1.47 s 1.54 s 1.76 s 1.76 s

H-13a 3.35 d 3.27 d 3.35 d 3.36 d 3.37 d 3.67 d 3.64 d (4.8) (4.8) (4.0) (4.8) (4.8) (5.0) (5 .0 ) H-13b 3 .2 8 d 2.82 d 3.28 d 3.30 d 3.34 d 3.61 d 3.59 d (4.8) (4.8) (4.8) (4.8) (4.8) (5.0) (5 .0 )

C-2'-Me 1.97 dq 1.88 dq 1.88 br s 1.95 dq 1.95 dq 1.99 dq 1.91 s (7 .5 , 1.5) (7 .5 . 1 .5 ) (7 .5 , 1 .5 ) (7 .5 , 1.5) (7 .5 , 1 .5 )

C-3'-M e 1.92 dq 1.77 dq —- 1.83 dq 1.84 dq 1.95 dq (1 .5 ) (.1 5 ) (1 .5 ) (1 .5 ) (1 .5 )

H-3a 6.14 qq 5.71 qq 6.08 br s 6.17 qq 6.19 qq 6.02 qq 5.94 br (7 .5 , 1 .5 ) (7 .5 , 1.5) (7 .5 , 1.5) (7 .5 , 1.5) (7 .5 , 1.5)

H-3b 5.67 br s .... — 5.66 br

9-OH ------3.02 d (7 .2 ) ------

^Spectra were run at ambient temperature in CDC^3, CgDgb, and dg-pyridinec at 200 MHz. TMS was used as internal standard. Values are in ppm (4) and signals are desiccated as follows: singlets, s; doublet, d; triplet, t; quartet, q; multiplet, m; broad singlet, br *. Figures in parentheses coupling constants or line separations in hertz. 57

Table 2-6. NMR parameters3 of compounds 47 and 51. 1 « a . ^

47 51 Carbon 6,multiplicity 6,multipl icity

1 205.42 s 205.42 s

2 104.94 d 103.97 d

3 182.76 s 184.65 s

4 138.88 s 132.74 s

5 133.85 d 134.49 d

6 75.28 d 78.81 d

7 48.23 d 47.51 d

8 74.97 d 71.13 d

9 42.63 t 41.68 t

10 87.99 s 87.43 s

11 135.99 s 59.63 s

12 168.95 s 172.03 s

13 123.38 t 49.98 t

14 21.38 q 21.94 q

15 60.26 t 20.48 q

1' 166.44 s 166.16 s

2' 127.33 s 126.04 s

3' 139.76 d 143.17 d

2'-Me 14.62 q 19.95 q

3'-Me 11.84 q 17.14 q

3 Spectra were obtained in CDC1 3 at ambient temperature at 50.32 MHz. Chemical shifts 6 ( ) are in ppm relative to TMSi as internal standard as determined by proton noise decoupling. Peak m ultiplicity was obtained by off-resonance decoupling (3.5 ppm above TMSi). Multiplicities are designated by the following symbols: s = singlet, d = doublet, t = triplet, q = quartet. Figure 2-16. 200 MHz *H NMR spectrum of 9a-acetoxy-ll,13-dihydro-11 a,13-epoxyatripliciolide 80-0 methacrylate (90) (CDCI3 ).

QAc

7.0 e.o 5.0 4.0 3.0 20 To

cn CD :-17. 200 MHz XH NMR spectrum of 9a-hydroxy-l1,13-dihydro-11 a,13-epoxyatripiiciolide 8p-0- Figure angelate (54) (CDC13) .

* S / r

7.0 6.0 5.0 4.0 3.0 2.0 1.0

cn Figure 2-18. 200 MHz *H NMR spectrum of 15-hydroxy-l1,13-dihydro-l1 a,13-epoxyatripliciolide 8g-0- angel ate (56) .

3 - CH-

2 -C H

7 , I3 a b

9 a 9 b

—»-- T T T T T 7 .0 5 .0 3 . 0 2.0

Ch o 61

2.6 The tentative structure of 2,3-epoxycalein A (81) .

2,3-Epoxyjuanislamin (80) , which was isolated by Castillo et 1 n l _al_. , and 2,3-epoxycalein A (81J had nearly identical H NMR

parameters (spectrum in Figure 2-19, data in Table 2-7) for the medium

ring protons but they differed in the signals of the ester side chains

at C-8 and C-9. The presence of two ester groups in 81^ was supported by

the NMR spectrum with absorptions typical of angelate (1.77 q, 3H,

= 1.5 Hz, 1.95 dq, 3H, = 1.5, 7.5 Hz; 6.13 qq, 1H, ± = 1.5, 7.5 Hz)

and an acetate (2.07, s, 3H). The mass spectrum of 81^ verified the

above assignments, showing strong peaks at ni/z_ 83 (B1), 55 (B"), and 43

(Ac). This was also supported by IR bands at 1695 and 1730 cm"1.

In order to test whether the 2,3-epoxycalein skeleton might

represent a biogenetic precursor for the furenone type heliangolides, a

Lewis acid-mediated transformation of compound 81_ to skeletal type 89 by

the use of BF3*Et 20 or p-TsOH was attempted (Scheme 2-4), but without

success.

OC O...J Hi or p-T*OH

Scheme 2-4. Attempted acid-catalized rearrangement of 2,3-epoxycalein

A. 62

2.7 Epoxidation of calein A (21) .

The 2,3-epoxidation of the known calein A (21) , which was obtained from C_. ternifolia var. te m ifo lia from Michoacan, Mexico, was of interest for two reasons: Firstly, it would possibly establish by chemical correlation the sites of attachment of the two ester groups at

C-8 and C-9. Secondly, this would represent an in vitro reaction analog to the possible biogenetic transformation.

m - C P B A ,

R T . 4 8 h r CH2CI2

Scheme 2-5. Epoxidation of Calein A

The portion of the a,B-unsaturated ketone has abnormal geometry with the ketone and alkene double bond being strongly distorted from coplanarity. Therefore, one would assume that the 2,3-double bond might show the reactivity of an isolated olefinic bond and 2,3-epoxidation of calein A could be expected. However, reaction of calein A (21) with m-

CPBA provided the epoxyangel ate derivative 91_ but no trace of the expected 2,3-epoxycalein A (Scheme 2-5). The *H NMR spectrum (compare 63

Figures 2-20 and 2-21) suggested the disappearance of angelate vinyl proton and new peaks, a quartet at 2.99 ppm (H-3*) and a doublet at 1.5 ppm (C-3'-methyl) appeared. The mass spectrum did not show the molecular ion, but a peak typical for the epoxyangelate fragment at m/z_

116 (D) verified the above NMR assignments. Therefore, structure (81J for 2,3-epoxycalein remains tentative with respect to the site of attachments of the two ester groups at C -8 and C-9. 64

Table 2-7. NMR parameters3 of calein A (21), and 2,3-epoxycalein A (81^) , and epoxide derivative .

2l 81 91

H-2 6.61 d ( 12) 4.25 d (4.5) 6.62 d (11.7)

H-3 6.03 dd ( 12, 11) 3.35 dd (9, 4.5) 6.04 t (11.7)

H-4 3.13 dddq (5.5) b 3.13 dddq (5.5)

H-5a 1.84 dd b b

H-5b 1.45 dd b

H-6 4.60 dd (11.5, 5) 4.81 dd (11.5, 5) 4.59 dd (11.7, 5)

H-7 2.64 br s 2.38 br s 2.64 br s

H-8 5.69 dd ( 10, 2) 5.76 br s 5.62 br s

H-9 5.59 d ( 10) 5.76 br s 5.62 br s

H-13a 6.33 d (1.5) 6.34 br s 6.60 d ( 1)

H-l 3b 5.85 d (1.5) 5.84 br s 5.84 d ( 1)

C-4-Me 1.14 d ( 6) 1.21 d (6) 1.15 d ( 6. 8)

C-10-Me 1.35 s 1.47 s 1.36 s

OAc 2.02 s 2.07 s 2.14 s

C-2'-Me 1.93 dq (7.5, 1.5) 1.95 dq (7.5, 1.5) 1.39 s

C-3'Me 1.76 q 0.5) 1.86 q 0.5) 1.15 d (5.8) H-3' 6.19 qq (7.5, 1.5) 6.14 qq (7.5, 1.5) 2.99 q (5.8)

a Spectra were run at ambient temperature at CDCI 3 at 200 MHz. TMS was used as internal standard. Values are in ppm ( 6) and signals are designated as follows: singlets, s; doublet, d; quartet, q; broad singlet, br s. Figures in parentheses are coupling constants or line separations in hertz.

k Obscured by other signals. Figure 2-19. 200 MHz, *H NMR spectrum of 2,3-epoxycalein A (81) (CDC13).

OAc 2PCH3

OAc I3a 13b

8 . 9

h ----- T T T T T T 7 .0 5 0 4 0 3 .0 2 0 LO

cncr> Figure 2-20. 200 MHz NMR spectrum of calein A (2 1 J (CDC1 3).

OAC

3-CH3

2ktt

T T T 7.0 6.0 5.0 4.0 3.0 20 1.0

CTlO'! Figure 2-21 . 200 MHz NMR spectrum of epoxide derivative (91^) (CDCI3 ).

OAc

3 -0 % 2f-CH

OAC

13b 13a 8 ,9

r T T T T T T T 7.0 6 0 5 .0 3 .0 2J0 CTl 6 8

2.8. Experimental

The following generalization may be made about equipment and conditions with regard to data presented in this section:

JR_ spectra were obtained on a Perkin Elmer 621 Infrared

Spectrophotometer;

UV_ spectra were recorded on a Cary-14 Recording Spectrophotometer using 95% EtOH as solvent;

CD spectra were run on a Durram-Jasco J-20 Spectrophotometer using methanol as solvent; theta values for wavelengths below 220 nm bear great uncertainty due to light absorption by the solvent, although the signs of the cotton effects remain certain;

Melting points were determined on a Thomas Hoover Capillary Melting

Point Apparatus and are uncorrected;

NMR spectra were recorded on Bruker WP 200 Fourier Transform NMR

Spectrometer at 200 MHz; conditions are presented with individual data;

Mass spectra were obtained on a Hewlett Packard 5895 GCMS at 70 eV, source temperature 200°C; sample were introduced via direct inlet probe.

HRMS data were obtained on a Varian MAT 711 High Resolution Mass

Spectrometer at 70 eV.

Isolation of 83-angeloyloxy-9a-[2-methylbutanoyloxy]-ternifo1in

(32) and 8 3-angeloxyloxy-9cx-acetoxytemifolin (33) . Dried leaves

(1 kg) of Calea tern ifo lia (collected in Chiapas Co., Mexico on July 29,

1978; L. Urbatsch, No. 3333, voucher deposited at LSU, U.S.A) were extracted with CHCI3 and worked up as outlined in Scheme 2-1 (vide supra) yielding 6.2 g of crude syrup. The crude syrup was 69 chromatographed over 200 g of silica gel (EM Reagents 70-230 mesh) using petroleum ether (bp. 30-60°) and mixture of P.E.: EtOAc (10, 20, 25, 50,

75%) as eluent and taking 100 ml fractions. Fractions were monitored by

TLC.

Fractions 17-18 provided 30 mg of calein A (21J which was identical with authentic material by NMR and MS®. Fractions 26 gave

50 mg of 32 and fractions 27-28 yielded 120 mg of 33.

8g-Angel oyl oxy-9ot-[2-methylbutanoyl ]-ternifol in (32) , C25H36°9» mp 55-60°; UV, X max (EtOH), 213 nm (e, 2.55 x 104); CD (c, 2.08 x

10"4, MeOH); C©]225 - 4.88 x 103, CG^253 + x ^ 2 9 0 ”

5.76 x 102, IR, v ^ ]3 cm'1: 3450 (OH), 1765 (y-lactone), 1745 max (ester), 1725 (o,e-unsaturated ester), 1710 (ketone), 1650 (double bond); MS, m /z.(rel. in t.): 480 (M+), 462 (0.2, M-18), 444 (1 , M-36),

378 (6.2, M-A), 85 (25.5, A'), 83 (100, B'), 57 (23.9, A"), 55 (29.7,

B"). Calc, for C25H3609; 480.2359. Found: (MS) 480.2370.

8g-Ange1oyloxy-9a-acetoxyternifolin (33) , C 22H3q09, glass; UV,

X max (EtOH), 213 nm (e , 1.86 x 104); CD (c, 1.52 x 10'4, MeOH):

[ 0] 212 - 8.5 x 104, [0]25Q + 5.33 x 103, [©]2go + 3.03 x 103; IR,

v ^ ^ 3 , 3425 (OH), 1745 (y-lactone), 1730 (acetate), 1710 (ketone), max 1695 (a, 6-unsaturated ester); MS, m/z_ (rel. int.): 438 (M+), 420 (0.4,

M-18), 402 (5.1, M-36), 278 (M-A-B), 83 (100, B'), 55 (30, B"), 43

(18.9, Ac). Calc, for C 22H3009: 438.1889. Found: (MS) 438.1888.

Oxidation of compound 33 . A solution of 50 mg of 33 in acetone containing a few drops of Jones' reagent was stirred at 0°C until orange 70

color persisted. The residue was diluted with H20 and extracted with

Et20. The solvent was evaporated in vacuo and the crude product

purified by PLC (P.E.: EtOAc = 7:3). The main fraction gave 3 mg of

compound 89, C 22H2g08, gum; UV, X max (EtOH), 213 nm (e, 1.91 x 104),

259 (e, 9.73 x 103); CD (c, 4.78 x 10'4, MeOH): [e ]221 - 7.17 x 103,

[0]26Q = 2.38 x 103, [0 ]298 + 6.12 x 102; IR, v ^ ]3 1760 ( y-

lactone), 1740 (ester), 1703 (a, 3-unsaturated ester), 1690 (a, 3 -

unsaturated ketone), 1590 (enolic double bond); MS, m/z_ (re l. in t.): 418

(M+) , 277 (21.5), 125 (19.9), 83 (100, B'), 55 (30.7, B"), 43 (15,

Ac). Calc, for C22H2608; 418.1625. Found: (MS) 418.1657.

Isolation of 9a-Acetox.yzexbrevin (36) and 9a-H.ydroxy-11,13- dihydro-11 a,13-epoxyatripiiciolide- 83-0-methacrylate (55). Calea tem ifo lia var. calyculata, (syn. Calea salmaefolia) was collected in

Nuevo Leon, Mexico on October 14, 1980 (J. Wussow, No. 278, voucher deposited at the Herbarium of Louisiana State University at Baton Rouge,

Louisiana, U.S.A.).

Dried leaves (405 g) of C. ternifolia var. calyculata were extracted and worked up as outlined in Scheme 2-1 to yield 11.7 g of crude syrup. The syrup was chromatographed over 230 g silica gel taking

100 ml fractions. Petroleum ether was used as an eluent followed by petroleum ether-ethyl acetate mixtures (90:10, 80:20, 70:30, etc.).

Fractions 14 and 15 provided 100 mg of not further investigated flavonoid material, fractions 18 and 19 contained 110 mg of 36 and fractions 20-23 gave crude lactonic material which upon rechromatography yielded 48 mg of pure crystalline 55 . 71

9a-Acetoxyzexbrevin (36) , C g ^ ^ g , mp 200-203°C; UV, X max

(EtOH), 208 nm (e , 2.34 x 105), 258 (e , 1.39 x 1O5); CD (c, 4.95 x

10"4, MeOH): C©3217 ' 2,3 x ]°5’ ^ 2 5 8 + 1,3 x 1q5, ^ 3 1 0 2,5 x 103; IR, v max 1767 (y-lactone), 1759 (ester), 1715 (unsaturated ester), 1698 (ketone), 1593 (enolic double bond); MS _m/z_ (rel. in t.):

404.1470 (.5, M+), 318.1102 (1.3, M-C4H602), 259.096 (.3, M-C 6Hg04),

125.1 (45.8, C7H902), 69.1 (100, C'), 41.2 (15.2, CH). Calcd. for

C21H24°8: 404.1470. Found: (MS) 404.1470.

9a-H.ydrox.y-l 1,13-di hydro-11 a,13-epoxyatri pi i ci ol i de -8 3-0-[2- methylacrylate] (55), CigHggOg, mp 200°C (dec); UV, X max (EtOH),

197 nm (e, 2.0 x 104), 262 (e, 1.20 x 104); CD (c, 5.19 x 10"4, MeOH):

[©]2i 5 + 3.7 x 103, C®]232 + ^ x ^ (maximum), [Q ^ O + 4,2 x ^°3

(minimum), [©1267 + x ^ (maximum), [©^293 + x ^

(minimum), [©^315 + 3.4 x 1O3 (maximum); IR, v max 3415 (OH), 1785 ( y- lactone), 1705 (ester), 1698 (ketone), 1587 (enolic double bond); MS, m/z_ (rel. int.) 376 (14.7, M+), 360 (.9, M-0), 290 (2.2, M-C 4H602), 69

(100, C'). Calcd. for C 19H2008: 376.1157. Found: (MS) 376.1171.

Acetate (90) . Acetylation of 30 mg of 55 in 1 ml pyridine and 1 ml of AC2O for 2 hr followed by the usual work-up gave 17 mg of acetate

(90) ; mp 57-60°C; IR, v max (CHCI 3), 1798 (y-lactone), 1760, 1720,

(esters), 1700 (ketone), 1598 (enolic double bond); MS, m/z_ (rel. in t.)

418 (11.7, M+), 402 (.4, M-0), 69 (100, C'), 43 (18.3, Ac), 41, (15.3,

C").

Isolation of compounds 37, 38, 51, 52, 54, and 56. 72

Plant material-Dried aerial parts of Calea ternifolia var.

calyculata (syn. albida) were extracted with Ch^Clg, and worked up according to a standard procedure (Scheme 2-1) to give the crude terpenoid syrup which was then chromatographed over silica gel taking 50 ml fractions. Petroleum ether (P.E.) was used as eluent followed by

P.E.: EtOAc mixtures (90:10; 80:20; 70:30; etc.). The crude extracts of three different populations of C. temifolia var. calyculata were analyzed.

A. Collection J. Wussow and G. Landry No. 320: collected on

October 16, 1980 in Mexico: San Luis Potosi, along small paved road to

Canoas; 2.2 miles North of junction with Hwy. 70. 200 g of dried plants gave 2.77 g of crude syrup. Chromatography provided 10 mg 37 and 8 mg

of a mixture of 37M S . and 38. /V.A.

B. Collection J. Wussow and G. Landry No. 336: collected on

October 21, 1980 in Mexico: Jalisco, in oak-grass savanna region, ca. 4 miles North of north edge of Guadalajara on Hwy. 50 to Zacatecas. Dried aerial parts (400 g) gave 4.1 g of the crude syrup. Chromatography afforded 145 mg 51^ from fractions 8-9 and 20 mg 56 from fractions 19-

20.

C. Collection J. Wussow and G. Landry No. 338: collected on

October 21, 1980 in Mexico: Jalisco, along Hwy. 15 to Tepic, 11.9 miles west of west junction with bypass around Guadalajara and Hwy. 15 to

Tepio. The crudy syrup obtained from 136 g of dried aerial parts consisted of 10 mg the known Caleochromene ADD from fractions 1-3, 90 mg

51 from fractions 13-14, 15 mg 52 from fraction 16, 15 mg 54 from fractions 21-22, and 10 mg 56 from fractions 24 and 25. Vouchers deposited at LSI), U.S.A. 73

8g-Angeloyloxy-9a-hydroxycalycu1atolide (37) , C 2qH2407, mP 175°

(dec.); CD (c, 5.1 x 10"4, MeOH), lelZZ} -1.1 x 104, [e ] 265 3*8 x

103, [0]31O 1.5 x 103; IR, v max (CHC1 3) 3420 (OH), 1770 (y-lactone),

1720 (ester), 1690 (ketone), 1596 (enolic double bond); MS, _rn/z_ (rel. int.), 376 (5.3, M+), 358 (0.4 M-H 20), 276 (0.7, M-B), 83 (100, B'), 55

(11.5, B"). Calcd. for C 20H2407: 376.1521. Found: (MS) 376.1490.

11.13-Di hydro-11a,l 3-epoxyatri pii ci oli de-8g-0-angelate (51),

82082287» mP 58-91°; UV, X max (EtOH) 262 nm (e, 4.35 x 103); CD (c,

5.15 x 10"4, MeOH), [©]228 + 1*° x 1q4» to]292" 2,9 x 1()3* te]325 + 1.4 x 103; IR, v max (CHC1 3 ) 1792 (y-lactorie), 1716 (ester), 1708

(ketone), 1650, 1593 (double bond); MS, m/z_ (rel. int.), 374 (1 7.7, M+),

358 (0.5, M-16), 274 (0.9, M-B), 83 (100, B1), 55 (33.5, B"). Calcd. for C2qH2207: 374.1365. Found: (MS) 374.1364.

11.13-Di hydro-11a,l3-epoxyatri pii ci oli de- 8B-methacrylate (52),

C19H20°7» mP 155-158°; UV, X max (EtOH) 262 nm (e , 4.1 x IO 3); CD (c,

6.28 x 10“4, MeOH), [°3235+ 7.4 x 103, [©]295 " 2,1 x 1C)3’ Ce^335 +

1.1 x 103; IR, v max (CHC1 3) 1793 (y -lactone), 1718 (unsaturated ester), 1708 (ketone), 1591 (enolic double bond); MS, m/z_ (rel. in t.):

360 (24.5, M+), 274 (1.4, M-C), 69 (100, C ) , 41 (21.7, C"). Calcd. for CigH 2007: 360.1208. Found: (MS) 360.1232.

15-Hydrox.y-l 1,13-di hydro-11 a,13-epoxyatri pi i ci ol i de-8g-0-an gel ate

(56), C20H2208, mp 118-121°, UV, X max (EtOH), 262 nm (e ,7200); CD (c.

5.8 x 10-4, MeOH), [e]227 + 1.3 x 104, C©]264+ 2.8 x 103, C©3293 “3-2 x 103, [ 0]329 + 1.8 x 103; IR, v max (CHC1 3) 3440 (OH), 1762 74

(y-lactone), 1723 (ester), 1704 (ketone), 1597 (double bond): MS, _m/z_

(rel. in t.) 390 (11.3, M+), 372 (0.9, M-HgO), 290 (0.7, M-B), 138

(29.4), 83 (100, B'), 55 (31.0, B"). Calcd. for C 20H2208: 390.1313.

Found: (MS) 390.1300.

9a-Hydrox.y-l 1,13-di hydro-11 a,13-epox.yatri pi i ci ol i de-8g-0-an gel ate

(54) , C20H2208, mp 205-207°; CD (c, 4.1 x 10"4, MeOH), [ 0]229 + 2.6 x 104, [O] 260 - 951, [©]282 + 3.56 x 103, C©]315 + 6.7 x 103; IR,

v max (CHC13) 3420 (OH), 1793 (y-lactone), 1712 (ester), 1697

(unsaturated ketone), 1650, 1595 (double bond); MS, _m/z_ (re l. in t.): 390

(6.7, M+), 290 (1.6, M-B), 83 (100, B'). Calcd. for C 2qH2208: 390.1302.

Isolation of 2,3-epoxycalein A (81) . Calea tem ifo lia var. tern ifo lia (syn. C. liebmanii) was collected in Michoacan, Mexico on

October 24, 1980 (J. Wussow and G. Landry No. 343, voucher deposited at

LSU, U.S.A.).

Dried leaves (476 g) of C. temifolia var. temifolia were extracted with CHC13 and worked up as outlined in Scheme 2-1 to get 7.7 g of crude syrup. The syrup was chromatographed over 240 g silica gel taking 100 ml fractions. Petroleum ether was used as eluent followed by

P.E.: EtOAc mixture (90:10, 80:20, 70:30; 60:40; etc .). Fractions were monitored by TLC.

Fractions 11-12 provided 60 mg of calein (21) which was shown to be identical with authentical material by *H NMR and MS®. Fractions 13-14 gave 260 mg of 81 and fraction 18 yielded 20 mg of 32 .

2,3-Epoxycalein A (81), C 22H280g, gum; UV, A max.(MeOH) 216 nm (e, 75

1.74 x TO4) ; IR, v max (CHC1 3), 3470 (OH), 1765 (y-lactone), 1730

(ketone, ester), 1695 (unsaturated ester); MS, m/z_ (rel. int.): 436

(1.1, M+), 418 (14, M-H20), 394 (.4, M-42), 376 (.9, M-C 2H402), 277

(2.5, C5 H702), 83 (100, B'), 55 (37.3, B"), 43 (42.5, Ac).

Epoxide (91^) Epoxidation of 42 mg of calein A (21_) in 10 ml dichloromethane and 1.2 eq. of m-CPBA (17 mg) was allowed for 2 days at

RT. The reaction mixture was treated with 5% Na^Og, saturated NaHCOj, washed with water, and dried over Na 2S04. After filteration, evaporation and purification by PLC (P.E.: EtOAc = 6:4), 7.5 mg 21^ and

9.6 mg of 91^ were obtained. Compound 91^ , C 22H2g09, mp 223-225°C; IR, v max (CHCI 3) 3420 (OH), 1775 (epoxy ester), 1765 (y-lactone), 1745

(ester), 1695 (a,e -unsaturated ketone); MS, m/z_ (rel. int.): 436 (M+, not obs.), 394 (1.6, M-42), 376 (.3, M-C 2H402), 321 (4.6, M-(D-l)), 125

(19.1), 116 (19.1, D), 82 (57), 43 (100, Ac). Chapter 3

The isolation and structure elucidation of sesquiterpene lactones from

Berlandiera texana and B. lyatra.

76 77

3.1 Introduction

The genus Berlandiera DC, with its four species belongs to the 7 ?Q family Compositae, tribe Heliantheae. * These four species are

Berlandiera subacaulis (Nutt), _B. pumi1 a (Michx) Nutt., B^. texana DC, and JJ. lyatra Bentham. The firs t species is found in the south of the

U.S. and the other three are restricted to the state of Texas. on In 1972, Herz et_ al_. described two guaianolides, berlandin

(92) and subacaulin (93) from _B. subacaulis (Nutt.). A further guaianolide, pumilin (95) was firs t isolated in our laboratory /from _B. O 1 pumi1 a (Michx) Nutt, by Mr. Charles Leonard. _B. texana and B. lyatra also contain pumilin as well as 3a-epoxypumilin (94). Table 3-1 listed the constituents found in the four .Berlandiera species.

3.2 Isolation of pumilin and 3a-epoxypumilin.

The dried and ground plant material of J 8. texana and B_. lyatra was extracted according to a general procedure outlined previously in

Chapter 2. The crude terpenoid syrup exhibited *H NMR signals typical for vinylic protons (5.5-7.0 ppm) that must belong to an a, B-unsaturated carbonyl system, vinylic methyls (1.75-2.25 ppm) and methyl groups attached to saturated carbons (1.5 ppm).

The syrup was examined by thin layer chromatography to determine the approximate number of compounds and to find a suitable solvent for separation by coluim chromatography. The separation was carried out as described in the experimental section providing two new sesquiterpene lactones the structure determination of which is described below. 78

Species Berlandin Subacaulin Pumi1 in 3o-Epoxypumilin

B.subacaulis + + - -

(Nutt.)

B.pumila - - + -

Michx) Nutt.

B.texana DC - - + +

B.lyatra Bentham - “ + +

Table 3-1. Sesquiterpene lactones present in the genus Berlandiera.

3.3 The structure of pumilin (95)

A colorless, crystalline compound which we named pumilin was

isolated from the extracts of dried aerial parts of J3. pumi la (Michx)

Nutt., JB. texana, and B_. lyatra. Pumilin (95), C 2oH22° 7» mP 244-245°C

(dec), showed UV (A max 257 nm) and IR (1680, 1640, and 1620 cm"1)

absorptions which were characteristic of the dienone chromophore

(G) present in a number of guaianolides.^ The 1H NMR signals at 2.42

(C-4-CH3), 2.61(C-10-CH3) and 6.33 ppm (H-3) also supported a

cyclopentenone moeity in pumilin.

(S)

The presence of hydroxyl(s) and an a-methylene-y-lactone moiety was 79

OC •’"OAc

92 93 — •*

•OH

94 95 A A| A2 OC c h -c h3 >"'OH

Figure 3-1. The structures of sesquiterpene lactones isolated from

Berlandiera species. 8 0

indicated by IR bands ant 3460 and 1775 cm"*, respectively. An IR absorption at 1715 cm“* suggested an additional ester function in the molecule. The NMR and MS data corroborated the above assignments. A series of diagnostic signals, a one-proton quartet of quartet at 5.98 ppm (H-31, _Jh_3' 3'cHg = ^ ^z> _JjH-3' 3 '-CH3 = anc* two met,1yl absorptions, a broadened quartet at 1.97 ppm (C-2'-CH 3) and a doublet of a quartet at 2.02 ppm (C-31-CH3), suggested the presence of an angelate moiety in lactone 95 . This was supported by a series of strong MS peaks at _m/z_ 274 (M-A), 83 (A^), and 55 (A£).

Further assignments of the basic skeleton of pumilin were deduced from extensive double resonance experiments in pyridine-dg. Irradiation of the multiplet at 4.51 ppm (Hc) removed the large couplings of the two allylicly coupled protons at 6.46 and 6.60 ppm (Hfl and H^), respectively, collapsed the doublet at 4.23 ppm (Hd) to a singlet and simplified the broadened doublet of doublets at 4.22 ppm (Hg) to a doublet. When the signal at 4.22 ppm (Hg) was irradiated, the broadened doublet at 6.98 ppm (Hf) collapsed to a broadened singlet. On the basis of the above chemical shifts and coupling data the partial structures H and I can be formulated. It was hoped that upon acetylation of pumilin the hydrogen attached to the carbon carrying the hydroxyl group would shift downfield, allowing an unambiguous decision between partial formulae H and I. . Attempts to prepare pumilin acetate in 81

acetic anhydride/pyridine resulted in decomposition of the substrate. o n However, preparation of the pyrazoline derivative* (96) allowed

assignment of Hd and Hg in the spectrum indicating that Hd, a doublet at

4.96 ppm (J = 10.5 Hz), was shifted considerably farther downfield than

Hg at 3.78 ppm suggesting that Hd represent the lactonic proton and He

the one attached to the hydroxyl-containing carbon.

lAng OAng He He OH *>Hb

H I

This excluded partial structure I and by combining fragments G and H

allowed the formulation of a guaianolide skeleton for pumilin. The only

position for attachment of the unassigned oxygen in pumilin could be at

C-5. This led to formular 95 exclusive of the stereochemistry at C-5 to

C-9.

Assuming that H-7 be a as in all sesquiterpene lactones from higher O 1 plants, the configurations at C -6 to C-9 could be derived from the H

NMR couplings when correlated with the dihedral angles obtained from

stereomodel considerations. The large coupling between H -6 and H-7

(J^ 7 = 10.5 Hz) suggested an antiperiplanar orientation of the two nuclei, that is H-63 or the presence of a trans- 6,12-lactone in 95 .

*The pyrazoline derivative (96) was prepared by Dr. Ngo Le Van. 82

A negative band at 268 nm in the CD spectrum (see Figure 3-2) OO of 95 supported this assignment although it is known that this 34 criterion does not hold in all cases. Further large couplings near

o K

220 >50 'aoo 350 400 nm

Figure 3-2 The ,CD spectrum of pumilin.

10Hz between H-7 and H-8 as well as H-8 and H-9 also suggested antiperiplanar hydrogens at these centers from which H-83 and

H-9a configurations were derived. Further evidence for an a-oriented OH

group at C-8 was provided by the geminal coupling between the two C-13 protons together with a paramagnetic shift of H-13 below 6 ppm. The configuration of the OH group at C-5 could not be derived from NMR data of 95 . The *H NMR spectrum of 95 is shown in Figure 3-3 and NMR data of 94,A./V. 9 95, and 96 are listed in Table 3-2. The remaining structural ambiguities of pumilin were established by single crystal X-ray diffraction. Figure 3-4 shows the B-face of the skeleton of pumilin, which represents a guaianolide with a 12,6a-

1 actonized a-methylene-y-lactone and a 5a-0H group. Figure 3-3. 200 MHz 'H NMR spectrum of pumilin (95) in pyridine-dg.

4-C^ ,OC IO-CH3

7.0 6.0 5.0 4.0 3.a ZO

U)oo 84

C19 'CIS c n reo Cl 7 1CI6 'Cl B cm

CIO 05 CIO

CIS, CIS Cll C15

Figure 3-4. Stereoview of pumilin (94)

3.4 The structure of 3a-epoxypumilin (94).

3a-Epoxypumi1 in (94), C2 qh22^8» a crystalline compound, mp 178-

183°C, was isolated as a minor constituent from JJ. texana and is the major component in B. lyatra. The NMR spectrum (Figure 3-5) exhibited absorptions which were very similar to pumilin (95) except that the lowfield narrow quartet at 6.33 ppm assigned to H-3 in 95 was replaced by a sharp singlet at 3.87 ppm in the new compound.

Furthermore, instead of the C-4-methyl absorption at 2.42 ppm in 95 , compound 94 exhibited a three-proton singlet at 1.93 ppm. This strongly suggested that the new compound represented a 3,4-epoxide derivative of pumilin.

The configurations at the centers Cg to Cg in 94 were found to be the same as in pumilin (95) on the basis of the *H NMR couplings (Table

3-2).

The configurations at C-3, C-4, and C-5 of 94 were established by single crystal X-ray diffraction (Figure 3-6) and shown to be H-33, H-4e, and 5a-0H. Figure 3-5. 200 MHz 'H NMR spectrum of 3a-epoxypumilin (94)%(CDC13)

H<3 T 6 . 7 . a 8

7.0 60 5 .0 4 .0 3.0 2.0 8 6

eii

.CI7)

cib;

c j #J

C I3 u ay

Figure 3-6. X-ray structure of 3a-epoxypumilin (94).

1 3 The C NMR spectrum (Figure 3-7) was tentatively assigned on the

basis of single frequency selectively decoupled spectrum (SFSD) and by

correlation with reference compounds described in the

literature. A number of the NMR signals could be assigned on the basis of their chemical shift and the multiplicity of the signals.

Other assignments required SFSD experiments. For instance, when the C4-CH3 proton carbon mutiplet was irradiated, the C-15 quartet in

13 the noise-decoupled C spectrum collapsed into a sharp singlet at 14.36 ppm with signal enhancement at 65.14 ppm. When in the NMR spectrum of 3a-epoxypumi1 in the maximum intensity of the decoupling field was set at the resonance frequency of the C-3 1 methyl at 1.92 ppm, not only the

splitting of the directly bonded nuclei (^C-*H) of the C-3'-methyl multiplet and to a lesser extent the C-2'-methyl signal at 2.03 ppm were perturbed, but the long-range couplings (^C-C-H and ^C-C-C-H) 87

occurring in their resonances were also affected. As a direct result of this decoupling, the intensities of the nonproton-bearing carbons C-2‘ and the C -l' carbonyl carbon singlets at 125.65 and 166.06 ppm, respectively, increased in the SFSD spectrum. By the application of the

1 3 same technique described above partially designated C NMR methyl signals at 13.07, 14.36, 14.98, and 19.45 ppm as well as the methine *3C resonances at 47.24, 69.17, 73.26, 78.16, could be assigned to C-4-CH3,

C-lO-CHg, C-3'-CH3, C-2'-CH3, C-7, C-8, C-9, and C-6, resepctively.

The 13C NMR signals at 125.64, 132.27, 135.69, and 155.22 ppm corresponding to non proton-bearing olefinic carbon atoms as well as the carbonyl carbons at 166.06, 167.59, and 193.99 ppm were sorted out during the SFSD experiments by observing the enhancements in the

13 intensities of the resonances of the nonproton-bearing C nuclei. A

1 3 summary of C NMR parameters is given in Table 3-3. Figure 3-7. 20 MHz NMR spectrum of 3a-epoxypumilin (94\ (CDC1^)

OH f 15 13

0 W 13

10 Z

...... | i i " n 11 »t r r r r n i r n i r n 111111111111 n I I111 I m JTI n FI FH> m 111n n 1111 M n n 111111III |T TTMrFT m11 FIT n11 ' «rrrrri g n u fi n11 n rrTTTTrrrrrtu r i n in il HTT[H im >H 200.0 75o 100 5 0 Table 3-2. *H fWR parameters3 .of 3a-epoxypumilin (94), and pumilin (95), and pyrazoline (96)

94 95 96

H-3 3 . 8 7 [3 .78] 6.33[6.22] q (1.5) 6.15 br

H-6 4.24[3.93] (c )d {10.5) 4.13[3.93] (c)d(10.5) 4.94 d (10.5)

H-7 4.61[3.93] (c) dddd 4.51[3.93] (c) dddd 3.53 dd (10, 10.5)

( 3 .0 ; 3 .5 ; 10; 10.5) (3.0; 3.5; 10; 10.5)

H-8 [3.93] (c) 4.22(3.93] brdd (10; 10) 3.78 dd (10, 10.5)

H-9 7.11[6.25] (c)brd(10) 6.98(6.1] brd (10) 5.93 brd (10)

H-13a 6.46[6.25] (c)dd(3.5; 1.5) 6.46(6.22] (c) dd (3.0; 1.5) 2 .05 m (b )

H-13b 6 .5 6 [6 .2 5 ] (c)dd(3.0; 1.5) 6.60(6.22] (c) dd (3.0; 1.5)

C-4-Me 1 .9 3 [1 .8 ] 2.42[2.33] d (1.5) 2.14 br

C-10-Me 2 .4 6 [2 .2 2 ] br 2.61[2.30] br 2.33 br

C -3 '-H 5 .9 5 [6 .2 5 ] (c)qq(7.25; 1.5) 5.98(6.22] (c) qq (7.5; 1.5) 6.21 qq (7.0, 1.5)

C -2'-M e 1 .9 2 [2 .0 5 ] brq ( 1 .5 ) 1.97[2.02] brq (1.5) 1.90 br

C -3'-M e 2.03C2.01] dq (7.2; 1.5) 2.02(2.03) dq (7.5; 1.5) 1.98 br

C-13-CH2 4.74 dd (7.0; 8.0)

aThe spectrum of 94, and 95, and 96 were run at 200 MHz in p yrid in e -d g w ith TMS as

internal standard. The data given in brackets were obtained in deuterochloroform.

Chemical shifts (6) are in parts per million; coupling constants or line separations

are given 1n parenthesis; m ultiplicities are designated as follows: d, doublet; q, quartet;

m, multlplet with center given; br broadended signal, ^obscured by other signals.

cPart of a complex m ultlplet. 90

Table 3-3 13C NMR parameters3 for 3a-epoxypumi1 in (94).

Carbon 5,multiplicity Carbon 6,multiplicity

1 155.22 s 11 135.69 s

2 193.99 s 12 167.69 s

3 63.64 d 13 122.23 t

4 65.14 s 14 13.07 q

5 74.62 s 15 14.06 s

6 78.16 d 1' 166.06 s

7 47.24 d 2 ' 125.65 s

8 69.17 d 3' 140.97 d

9 73.26 d 4' 19.45 q

10 132.27 s 5‘ 14.98 q

aSpectra were obtained in CDC1 3 at ambient temperature at 20 MHz. Chemical shifts (6) are in ppm relative to TMSi as internal standard as determined by proton noise decoupling. Peak multiplicity was obtained by off-resonance decoupling (3.5 ppm above TMSi). Multiplicities are designated by the following symbols: s = singlet, d = doublet, tr = triplet, q = quartet. 91

3.5 Experimental

The following generalization may be made about equipment and conditions with regard to data presented in this section:

IR spectra were obtained on a Perkin Elmer 621 Infrared

Spectrophotometer;

_UV_ spectra were recorded on a Cary-14 Recording Spectrophotometer using methanol as a solvent;

Melting Point were determined on a Thomas Hoover Capillary Melting

Point Appratus and are uncorrected;

NMR spectra were recorded on a Bruker WP 200 Fourier Transform NMR

Spectrometer at 200 MHz; conditions are presented with individual data;

Mass Spectra were obtained on a Varian MAT 711 High Resolution Mass

Spectrometer at 70 eV. 92

Isolation of pumilin (95) and 3a-epoxypumi1 in (94)

A. Dried aerial part (250g) of Berlandiera texana DC. (G. Newsom, collected on July 1, 1979 in Travis Country, Texas; voucher is deposited at the Ohio State University Herbarium, Columbus, Ohio) were extracted and worked up as outlined before yielding 3.1g of crude syrup which was chromatographed over lOOg silica gel (EM Reagents 70-230 mesh) using a mixture of petroleum ether (P-E.) and ethyl acetate as eluent and taking

100 ml fractions. Progress was monitored by thin lay6r chromatography

(TLC).

Lactones were found through fractions 7-12. Fractions 7-8 provided, after preparative layer chromatography (PLC) (P.E.: ether,

1:2), 14 mg of 3a-epoxypumi1 in (94). Fraction 9 yield 200 mg of pumilin (95).

B. A collection of Berlanderia lyatra Bentham (N.H. Fischer, H.

Fischer, and A. Malcolm, Fi 132) was made on June 17, 1982, 3-3.5 miles south of Alpine, Texas. Voucher specimens are deposited at the

Louisiana State University Herbarium in Baton Rouge, Louisiana. Dried leaves (400g) were extracted and worked up as previously outlined providing 5.2 g of crude syrup which was chromatographed over silica gel using mixtures of P.E. and ethyl acetate with increasing amounts of ethyl acetate. Fractions 12-14 gave 351 mg 3a-epoxypumil in and 97 mg of pumilin from fractions 15-17. In addition, 328 mg of a mixture of the two compounds was obtained.

3ot-epox.ypumi 1 in (94). C20H22^8» mP 178-183°C (dec); UV

(methanol):X max 253 nm (e, 2400); 203 nm (e, 7,100); IR: v max 3400 (OH),

1765 (y-lactone), 1710 (ester), 1680 sh (cycl openten one) 1620-1640 broad 93

(double bonds), 1225 (epoxide); MS _m/z_ (intensity, assignments): 390

(0.2, M+), 374 (0.1, M-Q), 354 (0.3, M-2H20), 308 (0.7, M-A^, 291 (4.4,

M A-H or M-Ar 0), 277 (1.6, 261 (1.0), 244 (0.7, M-A-H20-C0), 216 (0.6,

M-A-H2-2C0), 195 (3.3), 177 (2.9), 165 (9.2), 161 (1.2), 149 (2.5), 128

(1.7), 83 (100,Aj), 69 (8.5), 55 (25.5, A2). Anal. Calcd. for CjjjHj^Og

(M-C5H702) Mr = 291.0867. Found: Mr (ms) = 291.0862.

Pumilin (95), C2 qH2207, mp 244-245°C (dec, maroon color);

UV X max (methanol) 257 nm (e = 8,400); CD [0]222 + 4»® x ^®^268

-4.7 x 104; [0]3OO 0; [e]351 + 2 x 104; IR (KBr): v max 3460 (OH), 1775

(y-1actone), 1715 (ester), 1680 (eye1 openten one), 1640 (double bond),

1620 (cisoid double bond); MS jm/z_(intensity, assignments) 374 (1.0, M+),

356 (1.8, M-H20), 338 (1.6, M-2H20), 291 (6.1, M-A^, 274 (4.6, M-A),

256 (3.6, M-A-H20), 228 (1.8, M-A-H20-C0), 200 (1.1, M-A-H20-2C0), 185

(1.0, M-A-H20-2C0-CH3), 178 (2.5), 165 (1.2), 161 (13.2), 150 (3.4), 83

(A^), 69 (5.5), 55 (15.8, A2). Chapter 4

Preparations and fragmentations of 1,3-dihydroxyeudesmanolide deri vati ves.

94 95

4.1 Introduction

The costus plant, Saussurea lappa Clark, which grows on the slopes of the Himalayas, belongs to the Compositae family. The essential oil obtained from costus roots is highly valued in perfumery as a blending a g e n t.^

The chemistry of costus oil was investigated for the first time by

Semmler and Feldstein in 1914.^° Romanuk _et__al_. had isolated costus lactone by fractional distillation in 1 9 5 8 .In 196Q, Bhattacharyya et

AO al. obtained costus oil by a solvent extraction procedure. The oil thus obtained was quite different from the oil examined by previous workers. One of the constituents of this oil was a new crystalline sesquiterpene lactone which they named costunolide (97). This lactone is rather unstable, particulary at elevated temperature.

97 VI/ This may explain why the earlier investigations failed to detect its presence in the costus root o il. The primary constituent of the costus oil seemed to be this lactone. Two communications dealt with the structure and absolute configuration of 97 .43,44 Bohlmann et al.^ isolated costunolide from the aerial part of Cosmos sulphureus Cav. and

Cosmos hybridus Klondyke by colutm chromatography. Toribio and

Geissman^® obtained costunolide from Hymenoclea monogyra by the combination technique of the solvent extraction procedure and column chromatography. 96

Over the years the cyclodecadiene ring system, including the

germacranolides, of which costunolide is the simplest member, has

received limited attention by synthetic organic chemists. The first

synthesis of a germacranolide was described by Corey and Hortman47 which

involved the photochemical transformation of the eudesmanolide

derivative (98) for construction of the 1,5-cyclodecane ring system

100 (equation 4-1).

98 99 100

In 1977, Grieco and Nishizawa reported the total synthesis of

costunolide (97) via synthetic dehydrosaussurea lactone (108) by

utilizing the Cope rearrangement for construction of the ten-membered

carbocyclic unit (Scheme 4-1). The starting point of their synthesis was keto-lactone (102) , which was prepared from santonin (101) by a two-step procedure involving hydrogenation and epimerization at C-4.^

Treatment of the keto-lactone with tosylhydrazine provided the

corresponding hydrazone which, when treated with excess lithium diisopropyl amide in dry tetrahydrofuran at 0°C, gave the olefin

(103). Ozonolysis of 103 followed by reaction with sodium borohydride

gave the diol 104 . Treatment of diol 104 with o-nitropheny-

selenocyanate in pyridine-tetrahydrofuran (1:1) containing tributyl- phosphine gave exclusively the monoselenide 105 in excellent yield. 97

Prior to oxidation of selenide 105 to its corresponding selenoxide, they attempted to convert monoselenide 105 to the bis-selenide 110 by resubmission of 105 to the reaction conditions described above, but

110

without success. Upon oxidation, monoselenide 105 was smoothly transformed into the olefinic alcohol 106 . Direct conversion of alcohol 106 to selenide 107 proceeded without difficulty. Elimination of the corresponding _o-nitrophenylselenide gave saussurea lactone

108 which in a sealed tube at 210°C under nitrogen gave a 1:1 mixture of dihydrocostunolide (100) and saussurea lactone (108).

Selenylation of saussurea lactone (108) gave exclusively the selenide at C-ll which was converted upon treatment with 30% hydrogen peroxide in tetrahydrofuran into dehydrosaussurea lactone

(109). Thermolysis of dehydrosaussurea lactone at 220°C gave a 20% yield 50 of costunolide (97). 98

H 2 . P d / C SrC O -i '

101 102

1 . t * n h n h 2 1. O j , ^ ^ P h H , B F3 E t2 0 ► 2. LDA.THF, - 70 Or - 78 -*0° C 2. N0BH4. . H -78°C-*2S C

103 104

I. 50XH202 , THF H

105

I.N 0 2 p g H 4 S » C N . B u jP , T H F SOX^gOg. THF

108

1.LDA. P h S o S « P h , 2 2 0 * C HMPA.THF.5 -7a»C --20*C 2. 30% HgOg

109 97

Scheme 4-1. Total synthesis of costunolide. , = >

OH 112

100 113

OTs

HO'

111

Scheme 4-2. Proposed synthetic strategy for germacranolides from eudesmanolides.

VO vo NaBH4 . M«OH m - C P B A ,

H2 . P d / C M«OH

Scheme 4-3. Synthesis of dihydrosantamarine (115) and dihydroreynosin (116)

O O OH OT»5

T«CI. P y S«02 .

HO*1' " 0 r t f l u x 116 117

t - B u O K , t-B u O H I h r . 5 0 * C

112 118

Scheme 4-4. Synthesis of the germacrolide skeleton via 15-oxo-saussurea lactone (112) OTs

TsCI, Py m - C P B A , 6 5 "C , 2 4 h rs>

115 119 120

A l( iP r O ) t-BuOK toluene M eO H ^ > HCT t - B uO H . HO' 3hr, 50*C

111 121

Aco

p-T * O H Aci AcO'

123 124

o Scheme 4-5. Proposed synthesis of an elemanolide and germacrolide skeleton. no 103

Scheme 4-2 outlines an alternative strategy towards the synthesis of the germacranolide skeleton which involves a fragmentation reaction of 1,3-dihydroxyeudesmanolide derivatives (111) via elemanolides ( 112).

Attempts towards the synthesis of the germacranolide skeleton via the above reaction sequence is discussed in this chapter. Scheme 4-3 describes the synthesis of the starting eudesmanolides, dihydrosantamarine (115) and dihydroreynosin (116) which can be prepared from costunolide (97) in 43% overall yield in three steps. The overall reaction plans are shown in Schemes 4-4 and 4-5. As outlined in Scheme

4-4, treatment of dihydroreynosin (116) with j>-toluenesulfonyl chloride/pyridine could provide the corresponding tosylate which, when treated with Se02 in dioxane, would give the alcolhol ( 111) .

Fragmentation reaction of alcohol (111) with t-BuOK/t-BuOH would lead the 15-oxo-saussurea lactone (112). Finally, Cope rearrangement of aldehyde (112) above 150°C could furnish the germancranolide (118).

According to Scheme 4-5, dihydrosantamarine (115) might be converted to the corresponding tosylate (119) which, when treated with excess of m-chloroperbenzoic acid in dichloromethane would give the epoxide (120). Meerwein-Ponndorf elimination of (120) followed by hydrogenation would provide the saturated alcohol ( 121) and treatment of alcohol (121) with t-BuOK/t-BuOH give aldehyde (122) by a fragmentation process. Upon reaction of aldehyde (122) with isoprenyl acetate in p- toluenesulfonic acid one could expect enol acetate (123) by an ester exchange reaction. Thermolysis of enolacetate (123) should provide n dihydrotamaulipin-B acetate (124). 104

4.2 Isolation of costunolide (97).

The oil widely used as a perfume base (Pierre Chauvet, S.A.,

France) is the steam-distilled oil containing the following constituents: costunolide (97), dehydrocostuslactone (125), and dihydrodehydrocostuslactone (126) .

125 125 97

Generally, isolation of 125 and 126 from costus roots is accomplished with organic solvents giving a 4-6% yield of a mixture of lactones, which can be crystallized directly from methanol or hexane/benzene.

Another crop of lactones can be extracted with alcoholic KOH of the residual oil. The crystalline mixture is composed of costunolide

(97) (50%) and dehydrocostuslactone (125) (50%). Separation of lactones

(97) and (125) is possible by recrystallization from methanol.

Since larger amounts of costunolide were required, separation of resinoid costus by preparative high pressure liquid chromatography

(HPLC) were performed using a mixture of petroleum ether-ether as eluant. This provided pure costunolide (97) and dehydrocostunolide

(125) as major constituents (see the HPLC trace in Figure 4-1). The *H

NMR spectrum of (97) is presented in Figure 4-2. Figure. 4-1. Preparative HPLC of the costus root oil.

125

I 20 (mln)

Coluim: PrePAK-500/sil ica

Solvent: P.E./Et 20 (8:1); Flow rate: 0.5 liter/min

o U1 Figure 4-2. 200 MHz 'H NMR spectrum of costunolide (97)

(CDC13)

I3b 13a

7 .0 6 0 5 .0 3 0 20 o CTl 107

4.3 Reductive and oxidative modifications of costunolide.

Generally, catalytic hydrogenation of sesquiterpene lactones with

Pd-C as a catalyst as well as reduction with NaBH^ in methanol proceed with ease under saturation of the lactonic exocyclic methylene group to form the 11,13-dihydroderivatives. As outlined in equation 4-2, catalytic hydrogenation of costunolide (97) provides a mixture of the

11,13-dihydroderivatives, the predominant product being the isomer with an a-oriented C-ll-methyl group. In the presence of platinum oxide at

60 atm the hexahydroderivatives is formed.^ Reduction using NaBH^ results in an a-oriented C-ll-methyl group (equation 4-2). This latter route was used for the.preparation of dihydrocostunolide ( 100) .

N0BH4

100 108

Epoxidation of 7,6-lactonic germacranolides (100) with peracids

provides preferentially 1,10-epoxides. Dihydrocostunolide (100) was

therefore converted to the acid-labile 1,10-epoxide (114) with m-

chloroperbenzoic acid in CHCI 3 in the presence of sodium acetate as a

buffer since without buffer the epoxide cyclizes under the reaction

CO conditions forming a mixture of eudesmanolides. The epoxidation occurred within 15 minutes at room temperature (equation 4-3). The *H

NMR spectrum of 114 is shown in Figure 4-3.

m- CPBA

CHCI'

4.4 Cyclization reactions of germacranolides

In the process of structure elucidation, many Lewis acid-catalyzed cyclization reactions of the cyclodecadienolides or their 1,10- and 4,5-

O CO epoxide derivatives have been performed. * In general, cyclizations of germacra-l,5-diene provide eudesmanolides. As shown in equation 84 55 4-4, costunolide (97), when treated with a cation exchange resin * or

HClO^/AcOH,^ undergoes an acid-initiated cyclization via the presumed cation intermediate (127) to give a mixture of the eudesmanolides

128 and 129. Cyclization of costunolide via C-13-amine adducts are

reported to give higher y ie ld s .^ Figure 4-3. 200 MHz 'H WR spectrum of dihydrocostunolide l,(10)-epoxide (114),

(CDC13)

6 070 20 1.0

O UD 1 1 0

-H

Q 7 127 (eq. 4-4)

Cyclization of 1,10-epoxide (114) with BF3*Et20 gives the eudesmanolides dihydrosantamarine (115) and dihydroreynosin (116)

(equation 4-5).52 The 1H NMR spectra of dihydrosantamarine (115) and dihydroreynosin (116) are shown in Figure 4-4 and Figure 4-5, respecti vely. (eq .4-5) OH

114 115 116

4.5 Preparation and chemical transformation of eudesmanolide deri vati ves.

The treatment of dihydrosantamarine (115) with methanesulfonyl chloride in pyridine at room temperature for 2 hr gave mesylate derivative 130 in 90% yield.The *H NMR spectrum of 130 (Fig 4-6) showed the typical mesylate methyl signal at 3.04 ppm. An attempt to introduce the hydroxyl group at the C.-3 position by a hydroboration- Figure 4-4. 200 MHz 'H NMR spectrum of dihydrosantamarine (115)

(cdci3)

70 60 40 30 20 Figure 4-5. 200 MHz ‘H NMR spectrum of dihydroreynosin (116),

(CDC13)

I5a.b

70 6.0 50 40 3 0 20 1.0 113 oxidation reaction was unsuccessful. Instead, hydroboration of the mesylate derivative followed by treatment with aqueous sodium hydroxide and hydrogen peroxide provided the cyclopropyl derivative (131) (equation

4-6). The stereochemistry of the cyclopropyl ring and the C- 4-CH3 group could be assigned a 3-orientation by using stereomodels which show ca.

30° for the dihedral angle between H-4ct and H-3a corresponding to the coupling constant J_ = 8 Hz which is in agreement with the NMR data for acid 131 (see Figure 4-7).

o*c 2 hr 'OH 115 13C 131

The formation of the cyclopropyl type derivative 131 from 130 can be rationalized as a 1,3-elimination of the tertiary boryl derivative 130a . Similar reactions leading to compounds analogous to the formation of 130a have been previously reported . 59 The carboxylic acid group of 131 was formed by the ring opening of the lactone under basic condition. The proposed mechanism for the transformation of mesylate 130 to the acid 131 is given in equation 4-6a. Figure 4-6 200 MHz 'H NMR spectrum of (llS)-l8-mesyloxy-eudesmano-3-eno-12,6a-1actone (130).

'Ms

— i— T 7 .0 6.0 SO 40 30 20 10 Figure 4-7. 200 MHz 'H NMR spectrum of cyclopropyl acid derivative (131).

(d5-pyridine)

c„ -ch

-COOH

7.0 5.0 4.06.0 3.0 2.0 116

MsO OH

130

hJo

COO COOH 130a 131

(eq. 4-6a)

After the initial failure in the hydroboration-oxidation reaction of the mesylate (130) , the key step in this reaction sequence was the introduction of the hydroxyl group at the C-3 position using tosylates. Three possible approaches were considered: 1) hydroboration; 2) sensitized photoxygenation of olefin; 3) epoxidation, followed by Meerwein-Ponndorf elmination/hydrogenation (Scheme 4-6).

The fir s t and the second method did not give high yields of the desired products. The highest yields were provided by the third approach which also occurred with high stereo- and regioselectivity. 117

076 OTs

I) 2 . N qO H H2°2 HO

121a

OTs OTs

119 111

OTs OTS OTs m -C P B A , 3)

119 120 121

OTs

HO'

121

Scheme 4-6. Three possible approaches for the introduction of a

hydroxyl group at C-3 of eudesmanolide-l-O-tosylates. 118

Tosylation of dihydrosantamarine (115) at 0°C and at room temperature for 24 hr failed, possibly due to steric hindrance by the angular methyl group at C-10. Therefore, the reaction was performed at

65°C with p-toluenesulfonyl-chloride in pyridine for 24 hr®** which provided tosylate (119) in 63% yield, (eq. 4-7). The *H NMR spectrum of 119 (Figure 4-8) showed the typical tosylate signals at 2.45 (tosyl methyl), 7.34 and 7.78 ppm (aromatic protons).

OTs (eq. 4-7) T s C I. Py 65*C, 24hr*

115 119

Hydroboration of lactone 119 followed by treatment with aqueous sodium hydroxide and hydrogen peroxide, yielded a mixture of unidentified products containing small amounts of alcohol 121a, but mainly starting material®* (equation 4-8). Probably the introduction of the sterically demanding tosyl group prohibits the approach of diborane to the double bond.

cm OTs

119 121a Figure 4-8. 200 MHz 'H NMR spectrum of (llS)-lp-tosyl-eudesm-3-eno-12,6a-1actone (119)

(CDC13)

OTs OTs

3 .5 2.6

8.0 7.0 6 0 5 .0 4 .0 3.0 2.0 1 2 0

The second plan for the introduction of a hydroxyl group at C-3 of 119 involved a photooxygenation reaction. It is well known that aerated solutions containing a monoolefin, diene, or polyene, when irradiated in the presence of a sensitizer, give oxygenated products whose nature depends on the structure of the substrate and the lability of the initial photoproduct produced under the given reaction conditions.®^

The photosensitized oxygenation of dihydrosantamarine tosylate (119) in the presence of tetraphenylporphyrin produced in 10.5% yield hydroperoxide (133) which contained the undesired endocyclic double bond. The overall structural change is similar to that of other singlet oxygen ene reactions involving the hydrogen at C-5 of 119. The reaction was too sluggish presumably because of the steric hindrance of the tosyl group and the angular C-10-methyl group (equation 4-9).

Therefore, the route via compound 133 was not further considered to obtain alcohol 132 .

OTs OTs

P P h

HO"'

119 133 132

Finally, hydroxylation at C-3 in 119 was attempted by epoxidation of the double bond and subsequent transformation to the ally lic alcohol (111) by based-mediated opening of the expoxide function.®^

The epoxidation of tosylate 119 with m-chloroperbenzoic acid in dry dichloromethane proceeded stereoselectively from the a-face to give 1 2 1

3a-epoxide (120) in almost quantitative yield, (equation 4-10). The

stereochemistry of 3a-epoxide function was fully supported by the *H NMR

spectrum (Figure. 4-9).®^ The dihedral angle H-20/H-3&* 30° was derived

from model considerations which correlated best with the experimental _J_-

value (J_ = 3.4 Hz) suggesting a 3a-epoxide. The a-attack can be

rationalized on the basis of steric hindrance by the angular C-10 methyl

group as shown in the favored a-transition state represented by 119a .

m - c p b a . (eq. 4-10) CH2 CI2 , 7 2 h r*

119 120

Treatment of 120 with aluminum isopropoxide in boiling dry toluene65*®® gave allyl alcohol 111 in 99% yield (equation 4-11). The high regioselectivity of this reaction is presumed to be due to the

preferred geometry of the possible intermediate complex (120a) which

involves a C-15 methyl hydrogen. This procedure has considerable preparative advantages for the following reasons:

119a 120a Figure 4-9. 200 MHz 'H NMR spectrum of (llS)-3a-epoxy-l0-tosyl-eudesman-12,6a-lactone (120).

(CDC13)

CH-

OTs

3’. 5' 2.7,88=

8.0 6.0 4*0 3.0 2^0

ro ro 123

(1) aluminum isopropoxide is easy to handle; (2) the reaction can be applied to compound sensitive to strong base; (3) this reaction generally proceeds in high yield and with high regioselectivity. The 1H

NMR spectrum at 5.15 and 4.98 ppm (Figure 4-10) showed two olefinic singlets which were assigned to the two exocyclic methylene protrons at

C-15. A trip le t near 4.3 ppm was due to the 33-H.

A I ( iP r Q )3 toluene (eq. 4-11) HOT'

120 111

Haruna and Ito^7 reported in 1981 a regio- and stereospecific oxidation of german crane-type sesquiterpene lactone using as reagents selenium dioxide and t-butylhydroperoxide. Allylie oxidation of epitulpinolide (134) with 0.5 mole eq. Se02 and 2 mole of 70% t-butyl hydroperoxide in anhydrous CH 2CI2 at room temperature for 2 hr afforded the desired alcohol 135 in 90% yield along with 5% of the melampolide type aldehyde 136 (equation 4-12). Figure 4-10. 200 MHz 'H NMR spectrum of (llS)-l6-tosyl-3-hydroxyendesm-4(15)-eno-12,6a-lactone (CDC13 )

OTs

c,,-o

1 5 a ,b 2 7 , 8 , 9 3 .5

80 7.0 6.0 4 .05.0 3.0 2.0 125

t-BuOOH

An attempted allyic oxidation of the tosylate of dihydroreynosin

(116) using the above conditions with Se02/t-Bu00H failed.

The tosylate of dihydroreynosin (116) was prepared by the same procedure as previously described for dihydrosantamarine (115). The

NMR spectrum of 117 (Figure 4-11) showed a singlet at 2.45 ppm for the tosylate methyl, two doublets at 7.34 and 7.38 ppm for the aromatic protons, and two singlets at 4.84 and 4.97 ppm for the exocyclic methylene protons. The dihydroreynosin tosylate (117) was regio- and stereoselectivily oxidized with Se0268*69 to give the desired alcohol (111) (equation 4.13). Alcohol 111 had been previously prepared from the epoxide (120) with aluminum isopropoxide. (eq. 4-13).

Scheme 4-7 outlines the proposed mechanism for the selenium dioxide oxidation of olefin 117 . After an initial ene reaction followed by a conformational rotation to intermediate 117a, sigmatropic rearrangement of the intermediate allylselenic acid 117a would lead stereoselectivity to the anticipated a-isomer 111.

HO 0H

E ne HO' reaction

H 117

117a

Scheme 4-7. Proposed mechanism of selenium dioxide oxidation. Figure 4-11. 200 MHz 'H NMR spectrum of (llS)-lg-tosyl-eudesm-4(15)-eno-12,6a-lactone (117).

(CDC13)

OTs

3 .5 2 ' 6’

8.0 7.0 6 j0 5 .0 4.0 3.0 2.0 128

Attempted catalytic hydrogenation of (111) in the presence of platinum on carbon failed. However, catalytic hydrogenation of 111 with

13% platinum oxide on activated charcoal (Adam's catalyst) underwent smoothly to give alcohol 121 as a single product in nearly quantitative yield (equation 4-14). The *H NMR spectrum of product 121 is shown in

Figure 4-12. In compound 121 , the signals typical for the exocyclic methylene at 5.15 and 4.98 ppm were missing. Instead, a three-proton doublet appeared at 1.07 ppm typical for the saturated C- 4-CH3.®6

HO" HO''

The formation of carbon-carbon double bonds by the fragmentation of the monotoluene-p-sulfonates or methane-sulfonates of suitable cyclic

1,3-diols can be accomplished by treatment with base .^0 Reaction of the tosylate 111 with t-BuOK in t-BuOH gave aldehyde 112 in 20% yield which was separated by preparative layer chromatography (equation 4-15). The

1H NMR spectrum (Figure 4-13, Table 4-1) of 112 was in accordance with the data of aldehyde 140 reported in the literature^* except for the C -8 proton resonance.

An attempt to thermally rearrange the elemolide (112) to the desired germacranolide (118) at 220°C for 5 min failed. Figure 4-12. 200 MHz 'H NMR spectrum of (llS)-l8-tosyl-3-hydroxyeudesmano-12,6ct-lactone (121)%

(CDCI3 )

OTs

HO"'

IS*

2 .5 .7 . . 809

8.0 7.0 6.0 5.0 4 :0 3.0 2 .0

UDro 130

t - B u O K . t-B u O H I h r , SO *C

118

(eq. 4-15)

Bohlmann and Zdero7* reported an interesting finding related to the

Cope rearrangement of a C-15-oxygenated germacrolide. Conversion of

germacrolides to elemanolides generally requires elevated temperatures

(140°C or above). In contrast, the C-15-aldehyde intermediate 139, which had been obtained from alcohol (137) by Mn02~oxidation,

spontaneously converted to the elemanolide 140 in 30 minutes at 60°C.

(equation 4-16). These considerably milder rearrangement conditions

suggest that spontaneous, instead of enzyme-controlled Cope

rearrangement of C-14 and C-15-oxygenated germacrolides possibly also

occur in the living plants. This would explain the biogenesis of most

C-15-oxygenated elemanolide, e.g. the anti-tumor active elemanolide

vemolepin (138).

T 38

-OC 6 0 * C 60°C

(eq. 4-16) 131

Table 4-1. *H NMR spectral data comparison between C-15-•oxoelemano-

Tides derivatives (140) and the synthetic aldehyde ( ^ 2)

112 112 140

200 MHz 80 MHz 270 MHz

(CDC13) (c6d 6) (c6d 6)

H-l 5.69 (dd, 17,11) 5.70 5.82 (dd, 17,10)

H' 2a 4.89 (dd, 17,1) 4.82 5.01 (d) H-2b 4.80 (dd, 11, 1) 4.65 4.81 (d)

5.63 5.73 H-3a 6.25 (s) (s) H-3b 6.23 (s) 5.60 5.70 (s)

H-5 3.00 (d, 10. 8) 2.88 3.03 (d, 12)

H-6 4.20 (dd, 10. 8, 3.65 3.68 ( 12, 10)

9 .8 )

H-7 obs 2.12 (m)

H-8 5.23 (ddd, 10, 10, 2. 1.63 (m) H-9 1.87 (dd,13, 2.5)

H-U 2.36 (dq,13.3,6.8) ------

C10-CH3 1.02 (s) 0.64 0.60 (s)

0.95 ------C11“CH3 1.24 (d, 6.8) H—13 ------6.31 a (d, 1) H-13b ------5.58 (d, 1)

15-CHO 9.44 (s) 9.19 9.16 (s)

COR 2.20 (tq, 7)

1.65 (m)

1.06 (d, 7) Figure 4-13. 200 MHz 'H NMR spectrum of 15-oxo-saussurea lactone (112).

(CDC13)

15 a,b

CHO 7 .8 ,9 2 a,b

10.0 9.0 8.0 7.0 6 .0 5.0 40 3.0 2.0 1.0

GJ rv> 133

The fragmentation reaction of the monotosylate 121 with t-BuOK/t-

BuOH did not give the desired aldehyde product (122) . Although the

fragmentation reaction had occurred, as indicated by vinyl proton

signals, no aldehyde proton absorption was detected in the NMR spectrum

(Figure 4-14). Cooccuring ring opening of the lactone ring by t-BuO"

could have given the intermediate 141 followed by an attack of the C-6-

alkoxyl group at the aldehyde to give a hemiacetal which under acidic

work-up condition would result in the hemiacetal 142 (equation 4-17).

(eq. 4-17)

TsoJ 122

•5 ° :oo h O t Bu •s v_ HO 1 O t Bu 142 07 O O tBu

141

For further confirmation of the structure of acid 142, acetylation

of the hemiacetal hydroxy group with acetic anhydride/pyridine was performed to give acetate derivative 143 (equation 4-18). In the NMR

spectrum (Figure 4-15) of the acetate derivative, the chemical shift for

H-3 moved downfield from 4.90 to 5.79 ppm because of the introduction of the electron withdrawing acetate group. The hemiacetal structure was fully supported by the *H NMR spectrum (Figure 4-14) and X-ray crystallography provided the stereochemistry of all chiral centers in 142 (Figure 4-16). Figure 4-14. 80 MHz 'H NMR spectrum of hemiacetal (142)

(CDC13)

:00H

6 0 5 0 4 07.0 3 0 20 10 00 134 Figure 4-15. 80 MHz 'H NMR spectrum of hemiacetal acetate (143)

(CDC13)

OAc

AcO’

2 a

8.0 70 6.0 50 4.0 3.0 20 1.0 136

An attempt to recyclize the hemiacetal acetate 143 to the enol

acetate lactone (123) with p-toluenesulfonic acid in boiling benzene was without success.

(eq. 4-18)

:o o h COOH HO AcO’ 142 143

p- T*OH I

AC'

After this work was completed, Masayoshi et_jil_. 73 published the novel fragmentation reaction of dihydrosantamarine mesylate epoxide with

aluminum isoproperoxide. The synthetic approach of the Japanese authors was very similar to the aforementioned synthetic route.

Figure 4-16. Stereoview of hemiacetal (142). 137

4-6. Experimental

General Comments

Melting points were determined on a Thomas-Hoover Uni-Melt apparatus in capillary tubes and are uncorrected. Infrared spectra were recored on either a Perkin Elmer IR-137 or an Beckmann IR-9 grating spectrophotometer. NMR spectra were recorded on either an IBM Bruker

N/R 80, Bruker WP 200 FT spectrometer. Mass spectra were obtained on a

Hewlett Packard 5895 GCMS at 70 eV, sourpe temperature 200°C. The X-ray crystallographic analyses were performed by Dr. Frank R. Fronczek on a

Enraf-Nonius CAD4 automatic diffractometer. Preparative HPLC was performed on.Waters Prep LC Systems 500.

Unless otherwise indicated in a specific experiment, all of the chemicals used were reagent grade and no purification has been done.

Benzene and toluene were distilled over sodium and stored over molecular sieves. The CH2CI2 was d istilled over P 2O5 or CaH2* Tetrahydrofuran

(THF) was d istilled from sodium benzeophenone ketyl or UAIH 4 immediately prior to use. _t-Butanol was d istilled over BaO and stored over molecular sieves. Pyridine was distilled over NaOH and stored over molecular sieves.

Isolation of costunolide (97) . The resinoid costus syrup was purchased from Pierre Chauvet S. A., France. The costus oil (228g=8 oz.) was pre-chromatographed through a short silica gel colurm to remove polar material. 10 ml injection of 5g/ml solution of the pre-treated syrup in Et 20 were loaded onto a Waters Prep-Packed colurm (PrePak-

500/Silica). Eluant was P.E.-Et 20 (8:1) at a flow rate of 0.5 L/min and 138

a column pressure of 2000 psi. The retension times were 10-12 min for

dehydrocostunolide (125) and 13-16 min for costunolide (97) . About 35g

(15.4%) of impure dehydrocostunolide (125) and 23g (10.1%) of

costunolide (97) were obtained.

Costunolide (97), C 15H2002, mp 106-107°C (Lit. 107-108°C)42 !H WR

(CDC13) 6 4.55 (dd, H- 6, ± = 10.9 Hz, 1H), 4.75 (d, H-5, J_ = 10 Hz, 1H),

4.85 (dd, H-l, J_ = 6,1 Hz, 1H), 5.33 (d, H-13a, J_ = 3.2 Hz, 1H), 6.26

(d, H-13b, = 3.2 Hz, 1H); MS (70eV) _m/z_ (relative intensity) 232

(30.2, M+), 109 (64.1), 81 (100), 79 (52.8), 53 (49.9); IR (CHC13) 2833,

1764 (6-lactone), 1666, 1443, 1376, 1323 cm-1.

No BH4

97 100

NaBH/| Reduction of costunolide (97) To a solution of costunolide

(97) (1.1 g, 4.7 mmol) in 10 ml of MeOH was added with stirring NaBH^

(278 mg, 7.05 mmol) at 0°C. Stirring was continued for 1 hr. The

solution was acidified with 5% HC1, evaporated at reduced pressure,

diluted with 10 ml of water, and extracted with chloroform. The extract was evaporated to give 1.08 g (98%) of dihydrocostunolide (100) as white crystals; mp. 76-77°C (Lit. 77-78°C).42

Catalytic hydrogenation of costunolide (97) Costunolide (5.14g, 139

0.022 mol) was reduced catalytically at atmospheric pressure using 100 ml of anhydrous methanol as solvent and 250 mg of pre-reduced 5% Pd/C as catalyst. The consumption of 567 ml (0.0231 mol) of hydrogen was allowed over a period of 1 hr. The catalyst was filtered and the solvent evaporated to give spectroscopically pure dihydrocostunolide (100) : 4.18g (82%); mp. 70-73°C (Lit. 77-78°C);42 l H

NMR (CDC13) 6 1.26 (d, Cn -CH3, J_ = 6.9 Hz, 3H) 1.43 (s, C 10-CH3, 3H),

1.5-2.8 (ob, H-2, 3,8, and 9, 9H), 4.5-5.0 (ob, H-1,5, and 6, 3H); MS

(70eV) m/z_ (relative intensity) 234 (26.4, M+), 109 (50.0), 81 (100), 79

(38.1); IR (CHC13) 2883, 1779 (y-lactone), 1667, 1450, 1383, 1340,

994 cm"**

m - CPBA ^

CHCI3 . r.t.

114 °

Epoxidation of dihydrocostunolide (100) . A mixture of dihydrocostunolide (3.8 g, 0.015 mol) and m-chloroperoxybenzoic acid

(0.35 g, 0.0192 mol) in CHC1 3 (150 ml) was allowed to stand at room temperature for 15 min. The reaction mixture was washed with 5% Na 2S03, a saturated NaHC0 3 aqueous solution, and a saturated NaCl solution, dried over Na 2S04, and concentrated in vacuo to furnish 2.95 g (72%) of 114 as white crystals, mp. 120-123°C (Lit. 124-125°C);^2 NMR

(CDC13) 6 1.14 (s, C10-CH3, 3H), 1.23 (d, Cn -CH3, ^ = 8 Hz, 3H), 1.84

(s, C4-CH3, 3H), 2.69 (dd, H-l, J_= 10.8, 2 Hz, 1H), 4.60 (dd, H- 6, = 140

10.0, 8.4 Hz, 1H), 5.19 (bp d, H-5, J_ = 10 Hz, 1H); MS (70eV) m/z_

(relative intensity) 250 (85.8, M+), 232 (65.2), 165 (58.1), 159 (48.9),

81 (100); IR (CHC13) 2935, 1764 (y-lactone), 1670 (C=C) cm'1*

b f 3 E f2 0 Benzan

Acid-catalyzed rearrangement of dihydrocostunolide-1,10- epoxide (114). To a solution of dihydrocostunolide 1,10- epoxide 100 (4.6 g, 0.018 mol) in 200 ml benzene, 4.5 ml of BF 3*Et 20 was added. The reaction mixture was stirred at room temperature for 30 min., and then ethyl acetate was added to dissolve a brownish oil. The mixture was washed with 5% aqueous NaHC03 solution, water, and dried over anhydrous ^ S O ^ The evaporation of solvent yielded a mixture of

4.6 g of two isomers, dihydrosantamarine (115) and dihydroreynosin

(116) in a 2:1 ratio. The mixture was chromatographed on silica gel by using a mixture of P.E.:Et0Ac = 6:4 as eluant to provide 1.8 g pure 115 (39%) and 920 mg 116 (20%).

Dihydrosantamarine (115). mp 117 119°C (Lit. 134-136°C)j^1 ^ NMR

(CDC13) 6 0.89 (s, C 10-CH3, 3H), 1.22 (d, Cn -CH3, J_ = 6.8 Hz, 3H), 1.81

(s, C4-CH3, 3H), 3.65 (dd, H-l, J_= 9.5, 6.5 Hz, 1H), 3.96 (dd, H- 6, J_ =

10.5, 9.0 Hz), 5.32 (br s, H-3, 1H); MS, _m/z_ (relative intensity) 250 141

(2.5, M+), 232 (100, M-18), 158 (51.6), 143 (49.5), 86 (82.3). The

above NMR parameters are identical with the data described in the

litera tu re . 51’74 lR (CHC13) 3500 (OH), 1767 (y-lactone) cm"1.

Dihydroreynosin (116). mp 110-112°C (Lit. 129°C);74 1H NMR

(CDC13) 0.83 (s, C10-CH3, 3H), 1.23 (d, Cn -CH3, = 10.5, 9 Hz, 1H),

4.97, 4.82 (2 br s, C 4 = CH2, 2H); MS, m/z_ (relative intensity) 250

(10.4, M+), 232 (100, M-18), 165 (73.8), 159 (56.9), 91 (62.1); IR

(CHC13) 3500 (OH), 1767 (y-lactone), 1672 (C=CH2), 872 cm"1.

o*c 2 hr

115 130

(llS)-lg-Mesyloxy-eudesmano-3-eno-12,6ot-lactone (130). To a solution of dihydrosantamarine 115 (250 mg, 1 mmol) in 2 ml of dry pyridine at 0°C, was added freshly distilled methanesulfonyl chloride 75

(0.2 ml). The mixture was allowed to stand at 0°C for 1 hr, poured into ice water, and extracted with CHC1 3 (2 x 25 ml). The combined extracts were washed successively with 5% HC1, a saturated NaHC0 3 solution, saturated aqueous NaCl solution, dried over anhydrous Na 2$04, and concentrated in vacuo to afford an oil (290 mg; 90%); 1H NMR (CDC13) 6

1.01 (s,C10-CH3, 3H), 3.04 (s, CH3S02-, 3H), 3.94 (dd, H- 6, J_ = 10.5, 9

Hz, 1H), 4.69 (dd, H-l, J_= 9.5, 6,5 Hz, 1H), 5.34 (br s, H-3, 1H); MS m/z_ (relative intensity) 232 (100, M-CH 3S02), 159 (60.9), 158 (71.4), IR

(CHC13) 2915, 1765 (y-lactone), 1345, 1165, 915 cm-1. 142

THF J / 2 NoOH, L p' HgOj, n H'

Hydroboration of Mesylate (130) . To a 3-necked 25 ml flask equipped with a mechanical stirre r and a thermometer, mesylate 130 (1 g, 3.05 mmol) in THF (10 ml) was added under nitrogen. After cooling to

0-5°C, 1.3 ml of the 1M borane-tetrahydrofuran (BH3*THF) complex was added. The solution was stirred at room temperature for 1 hr to complete the reaction. Excess hydride was destroyed by the careful addition of 1 ml of water. After 5 min., 4.5 ml of 3 M NaOH was added to the reaction mixture, 4.5 ml of 30% hydrogen peroxide solution was introduced into the dropping funnel and added dropwise to the stirred reaction mixture at a rate such that the temperature of the reaction mixture did not exceed approximately 40°C. When the addition was complete, the reaction mixture was heated to 50°C and maintained there for 1 hr to ensure complete oxidation. The two-phase reaction mixture was poured into separatory funnel, acidified with 5% HC1 solution and extracted with chloroform. The extract was washed with water, a saturated NaCl solution and dried over ^SO^. The evaporation of solvent provided 150 mg of cyclopropyl type acid (131) which was chromatographed on silica gel with P.E.: EtOAc (6:4) as an eluant to give pure 131, mp 166-167°C; *H NMR (dg-Pyridine) 6 0.52 (ddd, H-2a, J_ =

9, 5.6, 3.6 Hz, 1H), 0.88 (s, C10-CH3, 3H), 0.99 (ddd, J_ = 5.6, 3.6 Hz, 143

1H), 1.15 (d, C4-CH3, _J = 7.2 Hz, 3H), 1.24 (dd, H-l, J_ = 9, 3.6 Hz,

1H), 1.39 (d, C 13-CH3, ± = 7.4 Hz, 3H), 1.49 (dd, J. = 9, 3,6 Hz, 1H),

2.27 (dd, H-5, J_ = 11, 8 Hz, 1H), 3.74 (dd, H- 6, ± = 11, 9 Hz, 1H), 3.71

(m, H-ll, 1H); MS jn/z_ (relative intensity) 252 (.5, M+). 234 (6.0, M

-18), 81 (100), 55 (78.4), 41 (41.3), 29 (50.8); IR (Csl) 3350 (OH,

COOH), 1685 (carboxylic C=0) cm”*.

T»CI, Py 63*C, 24hr*

115 119

(ll,S)-lg-tosyl-eudesm-3-ene-12,6a-l actone (119). To a solution of dihydrosantamarine 115 (2 g, 8 mmol) in 50 ml of dry pyridine at 65°C, was added 2.56 g (16 nmol) of freshly recrystallized p-toluenesulfonyl chloride.7® The reaction was allowed to proceed for 24 hr at 65°C, and then poured into ice water. The reaction mixture was extracted with chloroform, washed with water, brine, and dried over anhydrous Na 2S04.

After evaporation of solvent a crude syrup was chromatographed on silica gel by eluting with P.E.:Et0Ac (6:4) to obtain 2.05 g (63%) of pure tosylate 119 as white crystals, mp 155-158°C; *H WR (CDC13) 6 0.95 (s,

C10"CH3» 3h)» *20 i (d> C11“CH3» 1 = 6*9 Hz» 1H)> 1*77 (s. C4-CH3, 3H),

2.45 (s, C4'-CH3, 3H), 3.90 (dd, H- 6, J_ = 10.6, 9.9 Hz, 1H), 4.51 (dd,

H-l, J. = 9, 4.7 Hz, 1H), 5.22 (s, H-3, 1H), 7.34 (d, H-2', 6', J_ = 8.0

Hz, 2H), 7.78 (d, H-3', 5',^= 8.2 Hz, 2H); MS m/z (relative intensity)

404 (M+, not obs), 249 (.7, M-CH 3C6H4S02- ) , 232 (100, M-CH3CgH4S03H), 91

(65.1, CH3C6H4); IR (CHC13) 2950, 1778 (y-lactone), 1353, 1180, 930 144

OH OTs

T»CI. Py „ 65*C, 24hr»

116 11Z

(llS)-lg-tosyleudesm-4(15)-eno-12,6a -lactone (117) . To a solution of dihydroreynosin 116 (270 mg, 1.08 mmol) in 7 ml of dry pyridine at 65°C, was added p-toluenesulfonyl chloride (346 mg, 2.16 mmol). The reaction was allowed to proceed for 24 hr at 65°C, and then poured into ice water. The reaction mixture was extracted with chloroform (2 x 25 ml), washed with water, brine, and dried over anhydrous Na 2S04. After evaporation of the solvent, a crude syrup was chromatographed on silica gel by eluting with P.E.:EtOAc (6:4) to get

239 mg (54.1%) of 117, mp. 113 115°C; *H NMR (CDC13) 6 0.86 (s, C10-CH3,

3H), 1.21 (d, Cn -CH3, J_ = 6.9 Hz, 3H), 2.45 (s, C 4'-CH3, 3H), 3.98 (t,

H-6, 2 = 10.4 Hz, 1H), 4.40 (dd, H-l, 5.5 Hz, 1H), 4.97 , 4.84 ( 2s,

C4=CH2, 2H), 7.34 (d, H-2', 6', J_ = 8.2 Hz, 2H), 7.78 (d, H-3', 5', J_ =

8.2 Hz, 2H); MS m/z (relative intensity) 404 (M+, not obs), 232 (100, M-

CH3C6H4S02-), 159 (55.7), 158 (56.2), 91 (85.9); IR (CHC13) 2942, 1775

(y-lactone), 1355, 1180, 930 cm"**

OTs

m - CPBA,

119 120 145

(llS)-3a-Epoxy-lg-tosy1eudesmano-12,6ot-lactone ( 120). A mixture of 119 (101 mg, 0.25 mmol) and m-chloroperoxybenzoic acid (55.8 mg, 0.32 mmol) in dry CH 2CI2 (10 ml) was allowed to stand at room temperature for

72 hr. The reaction mixture was washed with 5% aqueous ^ 6 0 3 solution, a saturated NaHC03 solution, brine, dried over anhydrous ^ S O ^ , and concentrated in vacuo to give 99 mg (94%) of 120 as white crystals which were triturated with ether, mp 122-124°C (dec); NMR (CDCI3) 6 0.99

(s, C10-CH3, 3H), 1.21 (d, Cn -CH3, J_ = 6.8 Hz, 3H), 1.43 (s, C4-CH3,

3H), 2.46 (s, C4 ’-CH3, 3H), 2.94 (d, H-3, J_ = 3.4 Hz, 1H), 3.88 (t, H- 6,

J_ = 10.3 Hz, 1H), 4.27 (dd, H-1,_J = 9.8, 6.6 Hz, 1H) 7.35 (d, H-2', 6',

J_ = 8.0 Hz, 2H), 7.77 (d, H-3‘, 5', J_ = 8.0 Hz, 2H); MS, m/z, (relative intensity) 420 (.4, M+), 248 (24.0, M-CH 3C6H4S03H), 156 (24.3), 172

(26.6, CH3C6H4S03H), 91 (100, CH3CgH5); IR (CHC13) 2920, 1775 (y- lactone), 1350, 1170, 920, 850 cm"*.

Preparation of aluminum isopropoxide^

27.5 g of clean aluminum foil was added to 0.5 g of mecuric chloride and

300 ml of anhydrous isopropyl alcohol. The mixture was refluxed on a water bath under nitrogen atmosphere. 2 ml of carbon tetrachloride was added as a catalyst when the reaction mixture was boiling. The reaction mixture turned grey and, within a few minutes, a vigorous evolution of hydrogen gas commenced. Heating was discontinued and reaction flask was allowed to be cooled in icewater. After the reaction was less vigorous the reaction mixture was refluxed until all metal was reacted ( 6-12 hr). The reaction mixture became dark because of the presence of 146

suspended particles. After cooling of the reaction mixture isopropyl

alcohol was removed under water pump pressure. The aluminum

isopropoxide passed over as a colorless viscous liquid at 140-150°C/12 mm.; the yield was 190 g. The resulting molten aluminum isopropoxide was stored in the refrigerator over a week to get the solidified product

(mp 118°C).

Her'

120 i n

(llS)-lg-tosyl-3-hydroxyeudesm-4(15)-eno-12,6ct -1 actone (111). A solution of 120 (390 mg, 0.93 mmol) in anhydrous toluene (50 ml) was

refluxed with aluminum isopropoxide (388 mg, 4.64 mmole), under nitrogen for 10 hr. After further addition of aluminum isopropoxide (400 mg,

4*78 mmole), the reflux was continued for 10 hr. The solvent was removed from the reaction mixture under reduced pressure. The residue was stirred with a mixture of ethyl acetate (20 ml) and 2M HC1 (20 ml) until the residue was dissolved. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 30 ml). The combined organic layers were washed successively with a saturated NaHC 03 solution, brine, and concentrated in vacuo to furnish pure 111 (370 mg,

95%) as a crystalline material, which was recrystallized from ether, mp

173-175°C (dec); *H NMR (CDC13) 6 0.84 (s, C10-CH3, 3H), 1.21 (d, Cn - ch3, J_ = 6.8 Hz, 3H), 2.30 (dq, H-ll, J_ = 13.0, 6.8 Hz, 1H), 2.46 (s,

C4‘-CH3, 3H), 2.68 (d, H-5, ± = 10.5 Hz, 1H),- 3.97 (t, H-6, = 10.5 Hz, 147

1H), 4.28 (brt, H-3, J_ = 2.8 Hz, 1H), 4.82 (dd, H-l, J_ = 10.5, 5.2 Hz,

1H), 4.98, 5.15 (2br s, C 4-CH2, 2H), 7.40 (d, H-2', 6', J_= 8.2 Hz, 2H),

7.80 (d, H-31, 5‘, J_ = 8.2 Hz, 2H); MS, m/z_ (relative intensity) 420

(M+, not obs), 250 (.4), 172 (49.5), 107 (37.7), 91 (100), 65 (30.1); IR

(CHC13) 3480 (OH), 2940, 1768, (y-lactone), 1358, 1183 cm’1.

M«OH HO'

(llS)-le -tosyl-3-hydroxyeudesmano-12,6a-lactone (121). A mixture

of 111 (51.1 mg, 0.12 mmol), methanol (10 ml), Pt0 2 (14 mg), and

activated charcoal powder (30 mg) was stirred under 1 atm of hydrogen

for 1 hr. The filte rate was concentrated in vacuo to give pure 121 (99% yield) as white crystals, mp 137-138.5°C; 1H NMR ( 00013) 6 0.96 (s, C^g-

CH3, 3H), 1.07 (d, C4-CH3, J_= 6.8 Hz, 3H), 1.13 (d, Cn -CH3, J_ = 6.9

Hz, 3H), 2.45 (s, C4 '-CH3, 3H), 3.77 (br s and t, H-3, 6, 2H), 4.69 (dd,

H-l, J_ = 10.5, 6.3 Hz, 1H), 7.34 (d, H-2‘, 6', = 8.2 Hz, 2H), 7.80 (d,

H-3', 5‘, J_ = 8.2 Hz, 2H); MS, mjz_ (relati ve intensity) 422 (.5, M+),

250 (61.8), 159 (54.4), 152 (33.4), 91 (100); IR (CHC13) 3490 (OH),

2920, 1772 (y-lactone), 1365, 1185, 940 cm"1.

HO>

r t f lux

117 111 148

(1 IS)-l3-tosy1-3-hydrox.yeudes-4( 15)-en0-12,6g-lactone (111). A

mixture of the tosylate of dihydroreynosin (200 mg, 0.495 mmol),

selenium dioxide (65.9 mg, 0.594 mmol), and 20 ml of dioxane was

refluxed for 3 hr, filtered hot through Celite, and the residue on the

funnel was washed with a lit tl e dioxane. After concentration of the

f iltr a te in vacuo, colurm chromatography of crude syrup on silica gel

resulted in 112 mg (54%) of pure tosyl alcohol 111. The 1H NMR data

were identical with those of compound 111 which was previously obtained

from 120 with aluminum isopropoxide.

t- B u O K . t-B uO H HO' Ihr, 50*C

15-0xo-saussurea lactone (112). Tosylate 111 (180 mg, 0.43 mmol) was dissolved in 5 ml of dry t-butyl alcohol. The solution of ca. 3

equivalent of potassium t-butoxide in t-butyl alcohol was added. A white precipitate started to form immediately. After stirring for 1 hr

at 50°C, the reaction mixture was acidified with 5% HC1 and then water was added to break the emulsion. The reaction mixture was extracted with chloroform, washed with water, brine, dried anhydrous Na 2S0^, and

concentrated in vacuo to yield crude mixture. The crude was separated

on preparative layer chromatography (PLC) to give 20 mg (22%) of pure aldehyde 112, mp 133-135°C;1H NMR (CDC13) 6 1.02 (s, C10-CH3, 3H), 1.24

(d, Cn -CH3, _J = 6.8 Hz, 3H), 1.63 (m, H-8, 9, 4H), 2.36 (dq, H-ll, J_ =

13.3, 6.8 Hz, 1H), 3.00 (d, H-5, J_ = 10.8 Hz, 1H), 4.20 (dd, H- 6, J_ - 149

10.8, 9.8 Hz, 1H), 4.89 (dd, H-2a, J_ = 11, 1 Hz, 1H), 4.80 (dd, H-2b, J_

= 17, 1.0 Hz, 1H), 5.69 (dd, H-l, J_= 17, 11 Hz, 1H), 6.23, 6.25 (2s, H-

15a,b, 2H), 9.44 (s, C^-aldehyde, 1H), MS, _m/z_ (relative intensity) 250

(.3, M+), 175 (74.5), 91 (73.7), 81 (100), 79. (72.7); IR (CHC13) 2912,

1771 (y-lactone), 1682 cm"1(a,3-unsaturated aldehyde). OTs OTs

119 121a

Hydroboration of tosylate (119). To a 3-necked 100 ml flask

equipped with s tirre r, thermometer, tosylate (305 mg, 0.75 mmol) in THF

(25 ml) was added under nitrogen. The borane-tetrahydrofuran (0.28 ml,

1M, 1.1 equiv.) complex was added and stirred at room temperature for 3

hr and the 1 ml of water was added to quench the reaction. The alkali,

0.1 ml of 3M sodium hydroxide, was added, followed by the slow dropwise

addition of 0.1 ml of 30% hydrogen peroxide at a rate that the

temperature did not rise above 50°C. The solution was acidified with 5%

HC1, extracted with chloroform (2 X 25 ml). The combined extracts were washed with water and brine. The solution was dried over anhydrous

sodium sulfate and the chloroform was removed by evaporation in vacuo to

get the mixture of two compounds, starting material 119 (120 mg) and impure desired product, alcohol 121a (17 mg), yield 5.4%.

so*c HO' 3W :o o h HO"

Hemiacetal (142). Pure tosyl alcohol 121 (145 mg, 0.344 rmiol) was 150 dissolved in t-butyl alcohol (15 ml), the solution of ca. 3 equivalent of potassium t-butoxides in t-butyl alcohol was added dropwise under nitrogen. A white precipitate formed immediately, the solution was stirred for 3 hr at 55°C, acidified with 5% HC1 and water was added to dissolve the white precipitate. The solution was extracted with chloroform, washed with water, dried over anhydrous ^ S O ^ , and evaporated in vacuo to provide hemiacetal 142 (60 mg, 70%), mp 161-

163°C; XH NMR (CDC13) 6 1.04 (s, C10-CH3, 3H), 1.08 (d, C4-CH3, J_ = 6.8

Hz, 3H), 1.18 (d, Cn -CH3,_J = 7.0 Hz, 3H), 2.30 (m, H-ll, 1H), 3.73

(dd, H-6, J_ = 10.5, 9 Hz, 1H), 4.85 (d, H-2b, J_ = 1.8 Hz, Hz, 1H), 4.96

(d, H-3, J_ = 4.0 Hz, 1H), 5.02 (d, H-2b, J_ = 4.0 Hz, 1H), 5.18 (br s,

COOH), 5.70 (dd, H-l, J_ = 10, 8 Hz, 1H); MS, jn/z_ (relative intensity)

268 (2.7, M+), 250 (4.6, M-18), 148 (100), 149 (83.2), 93 (67.2), 81

(57.0); IR (CHC13) 3270 (OH), 2910, 1710 (carboxylic C=0), 1176, 995

:o o h COOH HO AcO' 142 143

Acetylation of acid (142). To 10 mg of acid, 2 ml of AcgO and a few drop of pyridine was added and stirred at room temperature for 1 hr. after evaporation of AC 2O, pyridine gave acetate 143 as white crystals. *H NMR (80 MHZ, CDC13) 6 1.05 (s, C10-CH3, 3H), 1.09 (d, C4-

CH3, J. = 6.8 Hz, 3H), 1.16 (d, Cn -CH3, J_ = 7.0 Hz, 3H), 2.06 (s, OAc.

3H), 2.87 (m, H-ll, 1H), 3.69 (t, H-6, J_ = 10.5 Hz, 1H), 4.88 (t, H-2b, 151

J_ = 1.8 Hz, 1H), 5.06 (d, H-2a, J_ = 4.0 Hz, 1H), 5.75 (dd, H-l, J_ = 10,

8 Hz, 1H), 5.79 (d, H-3, ^ = 4.0 Hz, 1H); MS, m/z_ (relative intensity)

310 (.1, M+), 265 (1.8, M-45), 249 (46.7, M-59), 233 (100), 148 (75.5),

81 (50.1).

OTs OTs

133119

Singlet oxygen reaction of tosylate (119). A solution of 88 mg

(119, 0.219 mmol) and a few milligrams of tetraphenylporphyrin in 1 ml of chloroform was irradiated with a 650 watt lamp (Sylvania, DWY

Tungsten Halogen) in a water-cooled immersion apparatus through which oxygen was bubbled. The reaction was monitored by TLC every 2 hrs.

After 8 hr, the solvent was evaporated in vacuo and chromatographed on

PLC to obtain impure peroxide 133 as a syrup (yield: 10.5%); *H NMR

(CDC13) 1.10 (s, C10-CH3, 3H), 1.24 (d, Cn -CH3, = 7.0 Hz, 3H), 1.98

(s, C4-CH3, 3H), 2.45 (s, C4'-CH3, 3H), 4.20 (br s, H-3, 1H), 4.49 (d,

H-l, J_ = 10 Hz, 1H), 4.75 (dd, H-6, J_ = 13, 5 Hz, 1H), 7.36 (d, H-2',

6 ', J_ = 8.2 Hz, 2H), 7.80 (d, H-3', 5', d_ = 8.2 Hz, 2H); MS, m/z

(relative intensity) 436 (M+, not obs), 420 (1.4, M-0), 107 (30.4), 91

(100), 55 (28.4), 28 (33.8). REFERENCES

1. Wussow, J. P. Ph.D. Dissertation, Louisiana State University,

Baton Rouge, 1981.

2. Fischer, N. H.; Olivier, E. J.; Fischer, H. D. "Progress in the

Chemistry of Organic Natural Products"; Springer-Verlag: Vienna,

1979: Vol. _38, p. 47.

3. Seaman, F. C.; "The Botanical Review", A. Cronquist, editor; The

New York Botanical Garden, Bronx, N.Y. 1982; Vol. 48^ p. 121.

4. Ortega, A.; Romo de Vivar, A.; Diaz, E.; Romo, J. Rev. Latinoamer.

Quim. 1970. _1_, 81.

5. Bohlmann, F.; Zdero, C. Phytochemistry 1977. 16, 1065.

6. (a) Quijano, L.; Romo de Vivar, A.; Rios, T. Rev. Latinoamer. Quim.

1978. _9, 86. (b) Quijano, L.; Calderon, S.; and Rios, T. Rev.

Latinoamer. Quim. 1977, 8, 90.

7. Quijano, L.; Romo de Vivar, A.; Rios, T. Phytochemistry 1979. 18,

1745.

8. Herz, W.; Kumar, N. Phytochemistry 1980. 19, 593.

9. Bohlmann, F.; Jakupovic, J. Phytochemistry 1979, 18, 119.

10. Castillo, J.B.; Ferrero, M. T. M.; Luis, F. R.; Bueno, P. V.;

Leon or, N. G.; Arevalo, S. C. 0. Nat. Prod. 1981. 44, 348.

11. Bohlmann, F.; Fritz, U.; King, R. M.; Robinson, H. Phytochemistry

1981. 20, 743.

12. Bohlmann, F .; Gupta, R. K.; Jakupovic, J .; King, R. M.; Robinson,

H. Phytochemi stry 1981. 20, 1635.

13. Bohlmann, F.; Zdero, C.; King, R. M.; Robinson, H. Phytochemi stry

1981. 20., 1643. 153

14. Vichnewski, W.; Goulart, E. G.; Herz. W. Phytochemitry 1982. 21,

464.

15. Lee, I. Y.; Olivier, E. J.; Urbatsch, L. E.; Fischer, N. H.

Phytochemistry 1982. 21, 2313.

16. Lee, I. Y.; Fronczek, F. R.; Malcolm, A; Fischer, N.H. J. Nat.

Prod. 1982. _45, 311.

17. Lee. I. Y.; Urbatsch, L. E.; Fischer, N. H. _J_. Nat. Prod. ,

submitted.

18. Bohlmann, F.; Gupta, R. K.; Jakupovic, J.; King, R.M.; Robinson, H.

Phytochemistry 1982. 21, 2899.

19. Bohlmann, F.; Mathur, R.; Jakupovic, J.; King, R. M.; Gupta, R. K.;

Robinson, H. Phytochemistry 1982, 21, 2045.

20. Bohlmann, F.; Bapuji, M.; King, R. M.; Robinson, H. Phytochemi stry

1982. 2U 1164.

21. Ferrerira, Z. S.; Roque, N. F.; Gottlieb, 0. R.; Oliveira, F.;

Gottlieb, H.E. Phytochemistry 1980. 19, 1481.

22 Bohlmann, F .; Gupta, R. K.; King, R. M.; Robinson, H.

Phytochemistry 1980. 21, 2117.

23. Lee, I. Y.; Fischer, N. H., unpublished.

24. Romo de Vivar, A.; Guerrero, C.; Diaz, E.; Ortega, A. Tetrahedron

1970. 26, 1657.

25. Fronczek, F. R.; Lee, I. Y.; Fischer, N. H. _J. Nat. Prod. 1983. 46,

104.

26. Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. "Spectroscopic

Identification of Organic Compounds" 3rd ed.; Wiley: New York,

1974; p. 191.

27. Lequesne, P. W.; Levery, S. B.; Menachery, M. D.; Brennan, T. F.; 154

Raffauf, R .F. _J. Chem. Soc. Perkins Trans. I, 1978. 1572.

28. Baruah, N. C.; Sharma, R. P.; Madhusadana, K. P.; Thyagarajan, G.;

Herz, W.; Murari, R. _J. Org. Chern. 1979. 44, 1831.

29. Stuessy, T. F. "The Biology and Chemistry of the Compositae"

(Heywood, V. H.; Harbome, J. B.; and Turner, B. L., editors),

Academic Press, London, 1977.

30. Herz, W.; Bhat, S. V.; Srinivasan, A. J_. Org. Chem. 1972. 37, 2532.

31. Korp, J. D.; Bernal, L.; Fischer, N. H.; Leonard, C.; Lee, I. Y.;

Levan, N. J_. Heterocyclic Chem. 1982. 19, 181.

32. Herz, H.; Santhanam, H. S.; J. Org. Chem. 1967. 32, 507.

33. Stoecklin, W.; Waddell, T. G.; Geissman, T.A. Tetrahedron 1970. 26,

2397.

34. Cox, P. J.; Sim, G. A.; Herz, W. J. Chem. Soc. Perkin Trans. II.

1975. 459.

35. Herz, W.; Kumar, N. Phytochemistry 1980. 19, 2387.

36. Herz, W.; Kumar, N. Phytochemistry 1981. 20, 93.

37. Bhacca, N. S.; Wehrli, F. W.; Fischer, N. H. ^J. Org. Chem. 1973.

_38, 3618.

38. Ito, K.; Sakarkibara, Y.; Haruna, M. Phytochemistry 1982. 21, 715.

39. Guenther, E. Essential Oils 1952. _5, 446.

40. (a) Semmler, F. W.; Feldstein, Ber. Dtsch. Chem. Ges. 1914. 47,

2433, (b) Semmler, F. W.; Feldstein, Ber. Dtsch. Chem. Ges. 1914,

47, 2687.

41. Romanuk, M.; Herout, V.; Sorm, F. Col 1. Czech. Chem. Commun. 1958,

_23, 2188.

42. Rao, A. S.; Kelkar, G. R.; Bhattacharyya, S. C. Tetrahedron 1960.

9, 275. 155

43. Rao, A. S.; Kelkar, G. R.; Bhattacharyya, S. C. Chem. and Ind.

1958. 1359.

44. Bhattachrayya, S. C.; Kelar, G. R.; Rao, A. S. Chem. and Ind. 1959.

1069.

45. Bohlmann, F.; Boronowski, H.; Kohn, S. Chem Ber. 1964. 97, 2583.

46. Tori bio, F. P.; Geissman, T. A. Phytochemistry 1969, _8, 313.

47. Corey, E. J.; Hortman, A. G. _J_. Am. Chem. Soc. 1965, 87, 5736.

48. Grieco, P. A.; Nishizawa, M. J_. Org. Chem. 1977. 42, 1717.

49. Hendrickson, J. B.; Ilogard, T. L. _J_. Chem. Soc. 1962. 1678.

50. Rao, A. S.; Sadgopal, P.; Bhattacharyya, S. C. Tetrahedron 1961,

13_, 319.

51. Yoshioka, H.; Renold, W.; Fischer, N. H.; Higo, A.; Mabry, T. J.

Phytochemistry 1970, _9, 823.

52. Rodrigues, A. A. A.; Garcia, M. Rabi, J. A. Phytochemistry 1978,

_17_, 953.

53. Sutherland, J. K.; McCloskey, J. E. Tetrahedron 1974. 30, 1651.

54. Jain, T. C.; McCloskey, J. E. Tetrahedron Letters 1960, 34, 2917.

55. Jain, T. C.; McCloskey, J. E. Tetrahedron 1975, 31, 2211.

56. Kulkami, G. H.; Kelkar, G. R.; Bhattacharyya, S. C. Tetrahedron

1976. 32, 765.

57. Jain, T. C.; Banks, C. M.; McCloskey, J. E. Tetrahedron 1976, 32,

2740.

58. Kato, M.; Kurihara, H.; Yoshikoshi, A. J. _J. Chem. Soc. Perkin

Trans. I 1979. 2740.

59. Gordon, M.; Gragg, L. "Organoboranes in Organic Synthesis" Wiley:

New York, 1973.

60. Heathcock, C. H.; Badger, R. A.; Patterson, J r ., J. W. J. Am. Chem. 156

Soc. 1967. 89, 4133.

61. Brown, H. "Organic Syntheses via Borans": Wiley: New York, 1975.

p. 23.

62. Denny, R. W.; Nickon, A. "Organic Reaction"; Wiley: New York, 1973;

Vol. 20, p. 133.

63. Wilds, A. L. "Organic Reactions"; Wiley: New York, 1944; Vol. _2,

p .178.

64. Bhacca, N. S.; Williams D. H. "Application of WR Spectroscopy in

Organic Chemistry"; Holden-Day: San Fransisco, 1964; p. 100.

65. (a) Barton, D, H. R. _J. Chem. Soc. 1953. 1027. (b) Siegel, S.;

Smith, G. V. _J_. Am. Chem. Soc. 1960. 82, 6082. 6087.

66. Masayoshi, A.; Akahane, A.; Yamaoka, H.; Kahei,T. _J. Org. Chem.

1982. 47, 3909.

67. Ito, K.; Ha run a, M. J. Chem. Soc. Chem. Commun. 1981. 22, 483.

68. Ley, S. V.; Mahon, M. Tetrahedron Letters 1981, 22, 3909.

69. Rabjohn, N. "Organic Reactions"; Wiley: New York, 1976. Vol. 24, p.

261.

70. (a) Wharton, P. S.; Hiegel, G. A. _J_. Org. Chem. 1965. 30, 3254.

(b) Wharton, R. S.; J. Org. Chem. 1961. 26, 4781.

71. Bohlmann, F.; Zdero, C. Phytochemistry 1979. 18, 95.

72. Kupchan, S. M.; Hemingway, R. J .; Werner, D.; Karim, A. _J. Org.

Chem. 1979. 34, 3903.

73. Masayoshi, A.; Kiyoshi, T.; Kahei, T. Org. Chem. 1983. 48, 1210.

74. Clark, A. M.; Hufford, C. D. J. Chem. Soc. Perkin Trans. I, 1979.

3022.

75. Fieser, L. F .; Fieser, M. "Reagents for Organic Synthesis"; Wiley:

New York, 1967; Vol. _1_, p. 662. 157

76. Fieser, L. F.; Fieser, M. "Reagents for Organic Synthese"; Wiley:

New York, 1967; Vol. J_, p. 1180.

77. Vogel, A. I. "Practical Organic Chemsitry"; 3rd ed.; Longmans:

London, 1956; p. 883. VITA

I hi young Lee was bom on September 29, 1950 in Taegu, Korea, the

daugther of Jae Bok Choi and Phil Hee Lee. After receiving her high

school dipolma from Kyung Puk Girls High School in 1969 she entered the

Kyung Puk National University, Taegu, Korea. She acquired the degree of

Bachelor of Science with a major in chemistry in 1973 with honor of

Summa Cum Laude. Thereafter she entered the Graduate School of the

Seuol National University, Seoul, Korea in 1974. She obtained the

degree of Master of Science with a major in organic chemistry in 1976.

In 1978 she married Hyo Won Lee. She has a daughter, Ji-Eun. In 1978,

she entered the Graduate School at Louisiana State University in Baton

Rouge where she is currently a candiate for the degree of Doctor of

Philosophy in the Department of Chemistry. She has accepted a position of postdoctoral fellowship offered by Professor Y. Kishi at Harvard

University to begin August 15th., 1983.

158 EXAMINATION AND THESIS REPORT

Candidate: Ihl Young Lee

Major Field: Organic Chemistry

Title of Thesis: New Sesquiterpene Lactones fromthe.Genera Calea and Berlandiera (Asteraceae) and the Fragmentation Reactions of 1 ,3-Dihydroxyeudesmanolide Deri vati ves

Approved:

Major Professor and Chairman

Dean of the Graduate Schoolschjsol

EXAMINING COMMITTEE:

h .X - &

Date of Examination: