ALKALOIDAL CONSTITUENTS OF THE MARINE SPONGECLION …

ALKALOIDAL CONSTITUENTS OF THE

MARINE SPONGE CLIONA CELATA

by

RICHARD J . STONARD

B.Sc. (Honours), McMaster U n i v e r s i t y , 1977

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

(DEPARTMENT OF CHEMISTRY)

We accept t h i s t h e s i s as conforming to

the r e q u i r e d standard

THE UNIVERSITY OF BRITISH COLUMBIA

JULY 1981

(c)Richard J . Stonard

In present ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I fur ther agree that permission for extensive copying of t h i s t h e s i s for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representa t ives . It i s understood that copying or p u b l i c a t i o n of t h i s thes is for f i n a n c i a l gain s h a l l not be allowed without my wr i t ten permiss ion.

Department of

The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5

Date

D K - f i 12/19)

ABSTRACT

Methanol e x t r a c t s of the marine b u r r o w i n g sponge C l i o n a

c e l a t a Grant have y i e l d e d a complex m i x t u r e of ' i m p e r f e c t '

a l k a l o i d s . F r a c t i o n a t i o n of t h i s m i x t u r e by a r a p i d a c i d

p a r t i t i o n i n g p r o c e d u r e has f a c i l i t a t e d the i s o l a t i o n of the

major m e t a b o l i t e c l i o n a m i d e (E53) . The s t r u c t u r e of t h i s m o d i f i e d

amino a c i d was d e t e r m i n e d by i t s c o n v e r s i o n t o

t e t r a c e t y l c l i o n a m i d e • The a s s i g n e d s t r u c t u r e of 46,

p r e v i o u s l y i s o l a t e d from a c e t y l a t e d e x t r a c t s of C c e l a t a , was

c o n f i r m e d by the s y n t h e s i s of i t s u l t i m a t e h y d r o g e n a t i o n p r o d u c t

49. C l a r i f i c a t i o n of the a b s o l u t e s t e r e o c h e m i s t r y of c l i o n a m i d e

has been a c c o m p l i s h e d by h y d r o l y s i s and subsequent

h y d r o g e n o l y s i s of 46 t o g i v e ( S ) - N - a c e t y l t r y p t o p h a n a m i d e (52)

which was c h a r a c t e r i z e d by comparison w i t h an a u t h e n t i c sample.

The r e m a i n i n g 18-22 a l k a l o i d s e l a b o r a t e d by C l i o n a c e l a t a

bear c l o s e resemblance t o a l a r g e f a m i l y of p e p t i d e a l k a l o i d s

found i n h i g h e r p l a n t s . D e r i v a t i z a t i o n of the crude sponge

e x t r a c t by a c e t y l a t i o n and s u b j e c t i o n of the r e s u l t i n g r e s i d u e

t o r e p e t i t i v e s i l i c a and r e v e r s e d - p h a s e chromatography have

e f f e c t e d the i s o l a t i o n of f o u r n o v e l l i n e a r p e p t i d e a l k a l o i d s .

S t r u c t u r e s of the 6 - b r o m o t r y p t o p h a n - c o n t a i n i n g a l k a l o i d s ,

h e x a c e t y l c e l e n a m i d e A ( 5 8 ) , B (52) and p e n t a c e t y l c e l e n a m i d e C

( 5 9 ) , and the b i s d e h y d r o t r i p e p t i d e n o n a c e t y l c e l e n a m i d e D (60),

were e s t a b l i s h e d by i n t e r p r e t a t i o n of s p e c t r a l d a t a and c h e m i c a l

d e g r a d a t i o n by o z o n o l y s i s and a c i d - c a t a l y z e d h y d r o l y s i s .

D e m o n s t r a t i o n of the p r e s e n c e of the unique amino a c i d oC,J$-

d i d e h y d r o - 3 , 4 , 5 - t r i h y d r o x y p h e n y l a l a n i n e as a c o n s t i t u e n t of each

of t h e s e compounds was a i d e d by c o r r e l a t i o n w i t h s y n t h e t i c

a n a l o g u e s . A s m a l l s c a l e i s o l a t i o n e m p l o y i n g a c e t i c a n h y d r i d e - d g

has i n d i c a t e d t h a t c e l e n a m i d e s A ( 7 0 ) , B (7J_) , C (72) and D (74)

are the n a t u r a l l y o c c u r r i n g sponge m e t a b o l i t e s .

M H H 58 57 59

60 74 70

IH

IH

H 72 H 71 A c h e m i c a l comparison of specimens of C l i o n a c e l a t a from

M a s s a c h u s e t t s , Southern C a l i f o r n i a and B r i t i s h Columbia i s

r e p o r t e d .

iv

TABLE OF CONTENTS

page

A b s t r a c t i i

Table of Contents i v

L i s t of F i g u r e s v i i

L i s t of Schemes ix

L i s t of Tables x

L i s t of Appendices x i

Acknowledgments x i i

D e d i c a t i o n x i i i

A b b r e v i a t i o n s x i v

INTRODUCTION

I. N a t u r a l Products from the Sea 1

I I . Nitrogenous Secondary M e t a b o l i t e s from Sponges..4

I I I . N a t u r a l - P r o d u c t s H i s t o r y of the C l i o n i d a e 17

NATURAL-PRODUCTS CHEMISTRY OF CLIONA CELATA

I. T e t r a c e t y l c l i o n a m i d e (£6) 21

I I . The O b j e c t i v e s 28

I I I . C o n f i r m a t i o n of the S t r u c t u r e of

T e t r a c e t y l c l i o n a m i d e (46) 29

V

IV. The Absolute C o n f i g u r a t i o n of

T e t r a c e t y l c l i o n a m i d e (46) 31

V. Clionamide (53) 36

VI. The Celenamides: I s o l a t i o n 47

V I I . The Celenamides: S t r u c t u r e Determination 50

V I I I . D i s c u s s i o n 95

CHEMICAL ECOLOGY OF CLIONA CELATA

I. B i o l o g y of the C l i o n i d a e 107

I I . L i f e - C y c l e , Geographic and Taxonomic

D i s t r i b u t i o n of the C . c e l a t a A l k a l o i d s I l l

I I I . Experiments Aimed at Determining the

Involvement of the B.C. C . c e l a t a

A l k a l o i d s in the Burrowing Process 115

IV. C o n c l u s i o n * 121

SYNTHETIC STUDIES

I . R a t i o n a l e 122

I I . P r e p a r a t i o n of Simple Model Compounds 123

I I I . Geometry C o n s i d e r a t i o n s 132

IV. P r e p a r a t i o n of D e h y d r o t r i p e p t i d e s p o s s e s s i n g

N-terminal Alpha-Amino A c i d s 138

vi

EXPERIMENTAL 143

APPENDICES 177

BIBLIOGRAPHY 189

vii

L i s t of F i g u r e s

page

1. C l i o n a c e l a t a Grant 20

2. EIMS of t e t r a c e t y l c l i o n a m i d e (46) 23 3. 270 MHz XH NMR spectrum of t e t r a c e t y l c l i o n a m i d e ( 4 6 ) ..24

4. Comparison of the a r o m a t i c r e g i o n s of the 270 MHz XH NMR s p e c t r a of 47 and 48 25

5. 270 MHz JH NMR spectrum of 49 ( n a t u r a l ) 27

6. 270 MHz *H NMR spectrum of 49a ( s y n t h e t i c ) 30

7. 270 MHz JH NMR spectrum of 51 34

8. 270 MHz :H NMR spectrum of 52 35

9. RP-HPLC of the u n d e r i v a t i z e d C l i o n a m e t a b o l i t e s 39

10. 270 MHz lH NMR spectrum of t e t r a c e t y l c l i o n a m i d e

- d n (54) 41

11. IR spectrum of the u n d e r i v a t i z e d C l i o n a m e t a b o l i t e s ..42

12. 400 MHz JH NMR spectrum of c l i o n a m i d e (53) 43

13 . 270 MHz »H NMR spectrum of 55 45


15. RP-HPLC of cru d e a c e t y l a t e d methanol e x t r a c t s of

C l i o n a c e l a t a 49

16. S i l i c a HPLC of p a r t i a l l y p u r i f i e d , a c e t y l a t e d

C l i o n a m e t a b o l i t e s 48

17. IR s p e c t r a of h e x a c e t y l c e l e n a m i d e A (58) and B (5_7) ..51

18. IR s p e c t r a of p e n t a c e t y l c e l e n a m i d e C (59)

and n o n a c e t y l c e l e n a m i d e D (60) 52

19. 400 MHz lH NMR spectrum of h e x a c e t y l c e l e n a m i d e A (58).53

via

20. 270 MHz 1H NMR spectrum of h e x a c e t y l c e l e n a m i d e B (57)..54

21. EIMS of h e x a c e t y l c e l e n a m i d e A (58) 58

22. EIMS of h e x a c e t y l c e l e n a m i d e B (57) 59

23. 270 MHz »H NMR spectrum of 65 63

24. 270 MHz lH NMR spectrum of 66 64

25. EIMS of 6_5 66

26. EIMS of 66 67

27. 270 MHz XH NMR spectrum of 68 74

28. 270 MHz *H NMR spectrum of 69 75

29. 270 MHz lH NMR spectrum of p e n t a c e t y l c e l e n a m i d e C

(59) , 77

30. 270 MHz *H NMR spectrum of 65 79

31. EIMS of 65 80

32. EIMS of p e n t a c e t y l c e l e n a m i d e C (59) 81

33. 270 MHz XH NMR spectrum of n o n a c e t y l c e l e n a m i d e D (60).84

34. EIMS of n o n a c e t y l c e l e n a m i d e D (60) 85


36. EIMS of 73 88

37. A l i p h a t i c r e g i o n of the 270 MHz *H NMR spectrum

of a p a r t i a l l y p u r i f i e d sample of unknown

p e p t i d e a l k a l o i d s 92

38. 270 MHz *H NMR spec t ruin of a p a r t i a l l y p u r i f i e d ,

unknown p e p t i d e a l k a l o i d 94

39. Remains of an H . m u l t i r u g o s u s s h e l l i n u n d a t e d

by C . c e l a t a 109

40. Schematic r e p r e s e n t a t i o n of the b u r r o w i n g p r o c e s s ....110

IX

L i s t of Schemes

page 1. Mechanism of s t y r y l a c e t a m i d e h y d r o l y s i s 32

2. Summary of the d e g r a d a t i v e and s y n t h e t i c r e a c t i o n s

employed i n the d e t e r m i n a t i o n of

t e t r a c e t y l c l i o n a m i d e (4_6) 37

3. I n t e r p r e t a t i o n of the EIMS of 57 and 58 60

4. I n t e r p r e t a t i o n of the EIMS of 59 82

5. I n t e r p r e t a t i o n of the EIMS of 60 86

6. B i o s y n t h e t i c pathways to s t y r y l a m i n e s 98

7. B i o s y n t h e s i s of mescaline and L-ep i n e p h r i n e 99

8. B i o s y n t h e t i c routes to

oi, j3-didehydro-3 , 4 , 5 - t r i h y d r o x y p h e n y l a l a n i n e 105

9. Proposed route to C - t e r m i n a l s t y r y l a m i n e s 128

10. Summary of the methods of formation of dehydropeptides

i n c o r p o r a t i n g N-terminal o^-amino a c i d s 139

X

L i s t of Tables

page

1. 270 MHz * H NMR s p e c t r a l data 55

2. S e l e c t e d f e a t u r e s of the lH NMR s p e c t r a of

s y n t h e t i c dehydroamino a c i d d e r i v a t i v e s 65

3. EIMS of s y n t h e t i c model compounds 71

4. Calcium Analyses ....117

XI

L i s t of Appendices

page

1. Comparison of the s u b s t i t u t e d benzaldehydes

d e r i v e d from the o z o n o l y s i s of 46, 5_7, 58, 5_9 , 6_5

and £6 with s y n t h e t i c samples 177

2. 20 MHz 1 3 C NMR spectrum of 5_5 178

3. 20 MHz 1 3 C NMR spectrum of a p a r t i a l l y p u r i f i e d

mixture of a c e t y l a t e d celenamides 179

4. 100.6 MHz 1 3 C NMR spectrum of hexacetylcelenamide

A (58) ' 180

5. Computer s i m u l a t i o n of the aromatic region of the

400 MHz »H NMR spectrum of 57 181

6. 20 MHz 1 3 C NMR spectrum of hexacetylcelenamide

B (57) 182

7. 20 MHz 1 3 C NMR spectrum of 65 183

8. 20 MHz 1 3 C NMR spectrum of 66 184

9. IR and 100 MHz :H NMR spectrum of a mixture of

oligomers d e r i v e d from the h y d r o l y s i s of the

C-t e r m i n i of a c e t y l a t e d celenamides 185

10. 100.6 MHz 1 S C NMR spectrum of an unknown

a c e t y l a t e d p e p t i d e a l k a l o i d 186

11. C i r c u l a r d i c h r o i s m spectrum of

hexacetylcelenamide B (57) 187

12. Calcium t r a n s l o c a t i o n experiment 188

xii

Acknowledgements

Authors of d i s s e r t a t i o n s such as t h i s i n v a r i a b l y owe an

indebtedness to numerous persons who have, i n one way or

another, i n f l u e n c e d the course or outcome of t h e i r work. T h i s

t h e s i s i s no e x c e p t i o n . Two people deserve s p e c i a l mention;

Raymond Andersen and my wife Nancy, f o r q u i t e d i f f e r e n t but

e q u a l l y important reasons.

The myriad of eager s o u l s who w i l l i n g l y s u b j e c t e d

themselves to the o f t times f r i g i d waters of B r i t i s h Columbia to

a i d i n the c o l l e c t i o n of C l i o n a are warmly thanked. The r e l i a b l e

and f r i e n d l y a s s i s t a n c e , and c o n t i n u e d p a t i e n c e d i s p l a y e d by

Mike LeBlanc and the s t a f f of the departmental NMR and MS

f a c i l i t i e s are acknowledged. F i n a l l y , I humbly acknowledge the

most important c o n t r i b u t o r s , C l i o n a c e l a t a and the other marine

c r e a t u r e s whose l i v e s were taken to f u r t h e r the course of t h i s

r e s e a r c h .

G l o r i a , G l o r i a , M a u r i c e and Nancy.

XI V

L i s t of A b b r e v i a t i o n s

Ac20 = A c e t i c anhydride

CD = C i r c u l a r d i c h r o i s m

DCC = D i c y c l o h e x y l c a r b o d i i m i d e

DMAP = Dimethylamino p y r i d i n e

DMF = DimethyIformamide

DMSO = D i m e t h y l s u l f o x i d e

DCU = D i c y c l o h e x y l u r e a

DOPA = 3,4-Dihydroxyphenylalanine

EIMS = El e c t r o n - i m p a c t mass spectroscopy

EtOAc = E t h y l a c e t a t e

GC = Gas chromatography

HPLC = High p r e s s u r e l i q u i d chromatography

IR = I n f r a r e d

'H NMR = Proton n u c l e a r magnetic resonance 1 3 C NMR = Carbon-13 nuc l e a r magnetic resonance

PND = Proton-noise-decoupled

PTLC = P r e p a r a t i v e t h i c k l a y e r chromatography

Py = P y r i d i n e

RP = Reversed-phase

S = Solvent s i g n a l

THF = Tet r a h y d r o f u r a n

TLC = Thin l a y e r chromatography

U = Unknown i m p u r i t y s i g n a l

UV = U l t r a v i o l e t

W = Water s i g n a l

1

INTRODUCTION

"For i n and out, above, about, below, ' T i s nothing but a Magic Shadow-show, Played i n a Box whose Candle i s the Sun, Round which we Phantom F i g u r e s come and go."

-Omar Khayyam (1)

I. N a t u r a l Products from the Sea

S t u d i e s aimed at understanding b i o l o g i c a l systems at the

molecular l e v e l have r e s u l t e d i n the c l a s s i f i c a t i o n of carbon

compounds present i n l i v i n g organisms i n t o two groups - the

primary and secondary m e t a b o l i t e s . The former are molecules

encountered i n most, i f not a l l , b i o l o g i c a l systems and are

c o n s i d e r e d to be of fundamental importance to the e x i s t e n c e of

a l l organisms. The study of these substances has, in the past,

been p r i m a r i l y the domain of the b i o c h e m i s t . In c o n t r a s t , the

s t r u c t u r a l l y d i v e r s e secondary m e t a b o l i t e s are s p o r a d i c i n

occurrence and are not g e n e r a l l y c o n s i d e r e d to be q u i n t e s s e n t i a l

to l i f e (2). However, i t i s becoming i n c r e a s i n g l y apparent that

the f l o r a and fauna which host these compounds have unique

metabolic and s o c i a l requirements. Recent work in the areas of

i n s e c t pheromones (3), p l a n t hormones (4), p h y t o a l e x i n s (5), and

marine a l l e l o p a t h i c agents (6) may be c i t e d as examples. In

these i n s t a n c e s , the secondary substances serve important

p h y s i o l o g i c a l f u n c t i o n s . The study of the nature, occurrence,

2

o r i g i n and r o l e of these compounds c o n s t i t u t e s the realm of the

n a t u r a l - p r o d u c t s chemist. The more u t i l i t a r i a n g oal of u t i l i z i n g

secondary substances f o r mans b e n e f i t i s the r e s p o n s i b i l i t y of

pharmacologists and other pragmatic s c i e n t i s t s .

The search f o r n a t u r a l products from marine organisms i s a

recent endeavour. The proceedings of the Food-Drugs from the Sea

Conference of August, 1969 (7b) c i t e fewer than two dozen

p u r i f i e d and i d e n t i f i e d marine secondary m e t a b o l i t e s ( e x c l u d i n g

s t e r o l s , pigments and simple l i p i d s ) . T h i s s e r i e s of meetings

(7) s t i m u l a t e d i n t e r e s t i n marine n a t u r a l - p r o d u c t s r e s e a r c h and

pr o v i d e d d i r e c t i o n to a f i e l d i n i t s i n f a n c y . Advancements i n

the technology of SCUBA have enabled marine chemists to c o l l e c t

specimens which were p r e v i o u s l y u n a t t a i n a b l e . Increased ease of

c o l l e c t i o n , coupled with the development of modern s e p a r a t i o n

techniques and s p e c t r o s c o p i c methods of a n a l y s i s , have r e s u l t e d

in the examination of thousands of marine s p e c i e s . From these,

hundreds of novel substances have been i s o l a t e d and s t r u c t u r a l l y

d e f i n e d i n the past decade. Many of the f a s c i n a t i n g s t r u c t u r e s

which have been encountered are presented i n the f o l l o w i n g

subsect i o n .

Although marine organisms were o r i g i n a l l y h a i l e d as being

p o t e n t i a l sources of 'miracle e l i x i r s ' (7,8), no true

pharmaceutical of marine o r i g i n has yet been produced. The

absence of immediate pragmatic r e s u l t s has dampened the

enthusiasm of many r e s e a r c h e r s and funding a g e n c i e s . However,

Faulkner (9) r i g h t f u l l y p o i n t s out that the development of a

drug i s a time consuming and expensive p r o c e s s . Time and a

3

s u b s t a n t i a l l y enhanced e f f o r t by the i n d u s t r i a l community w i l l

be r e q u i r e d before success i s ac h i e v e d . In the meantime,

chemists i n v o l v e d i n marine n a t u r a l - p r o d u c t s r e s e a r c h w i l l

c o n t i n u e to p r o v i d e a p l e t h o r a of unique organic s t r u c t u r e s

which serve to f u r t h e r our comprehension of nature's

a r c h i t e c t u r e . Of equal importance i s the i n c r e a s e d tendency

towards i n t e r d i s c i p l i n a r y c o o p e r a t i o n . In t h i s context marine

chemists can make s u b s t a n t i a l c o n t r i b u t i o n s to problems of

taxonomy and to the enhancement of our knowledge of marine

b i o l o g i c a l phenomena. The c o o p e r a t i v e examination of marine

phytoplankton - the ocean's 'primary producers' - should prove

to be most rewarding. Research i n t o chemical communication and

r e l a t e d a l l e l o p a t h i c phenomena in marine communities i s j u s t

b e g i n n i n g .

4

I I . Nitrogenous Secondary M e t a b o l i t e s from Sponges

Members of the phylum P o r i f e r a - the aq u a t i c sponges - are

among the simplest and most p r i m i t i v e of a l l m u l t i c e l l u l a r

animals. These s e s s i l e organisms are represented by

approximately 4,500 s p e c i e s of which 150 r e s i d e i n f r e s h water

h a b i t a t s . G e n e r a l l y c l a s s i f i e d i n the subkingdom Metazoa, the

s t r u c t u r a l and developmental uniqueness of the P o r i f e r a has l e d

many b i o l o g i s t s to suggest that they are e v o l u t i o n a r y dead-ends

and, as such, should be r e l e g a t e d to the d i s t i n c t subkingdom,

Parazoa (10).

Sponges e x i s t i n most marine h a b i t a t s , over a wide range of

depths. T h e i r abundance, d i s t i n c t i v e appearance, and apparent

lack of defence coupled with a general absence of p r e d a t i o n make

them n a t u r a l c a n d i d a t e s f o r n a t u r a l - p r o d u c t s r e s e a r c h . Of the

three l a r g e c l a s s e s i n t o which the P o r i f e r a are s u b d i v i d e d , the

Demospongiae 1 have r e c e i v e d the most a t t e n t i o n . C l a s s i f i c a t i o n

of the Demospongiae i s d i f f i c u l t and repr e s e n t s a major problem

for the chemist i n the f i e l d .

The most p r o l i f i c source of unique a n t i m i c r o b i a l substances

has been s p e c i e s of the order Keratosa (eg-Verongidae) (11b).

T h i s g e n e r a l i z a t i o n may r e s u l t from the f a c t that the Keratosa

are the only demosponges which do not possess s p i c u l e s . Instead,

these organisms have s k e l e t o n s composed e n t i r e l y of p r o t e i n . The

The other two c l a s s e s are the C a l c a r e a and the H e x a c e t i n e l l i d a .

5

absence of a s k e l e t a l d e f e n s i v e mechanism may have given r i s e to

the e v o l u t i o n of b i o s y n t h e t i c pathways f o r the p r o d u c t i o n of

p r o t e c t i v e substances.

The l i s t of marine n a t u r a l products which c o n t a i n n i t r o g e n

i s s h o r t i n comparison with the abundance of such compounds from

t e r r e s t r i a l sources such as p l a n t s and f u n g i . A thorough

treatment of marine n a t u r a l - p r o d u c t s chemistry p u b l i s h e d i n 1971

(12) i n c l u d e d only seven n i t r o g e n - c o n t a i n i n g sponge-derived

compounds. As a r e s u l t of the r a p i d expansion i n the number of

compounds d e f i n e d i n recent y e a r s , a comprehensive review of

these substances would be beyond the scope of t h i s t h e s i s .

Reviews by Minale et a l . (11b), Faulkner (11c), Faulkner and

Andersen (11a), and those i n the s e r i e s of books e d i t e d by

Scheuer ( 1 l d , e , f , g ) , have pr o v i d e d thorough coverage of the

marine n a t u r a l - p r o d u c t s l i t e r a t u r e to 1978. In t h i s s u b s e c t i o n ,

an attempt has been made to b r i n g together those compounds of

sponge o r i g i n which c o n t a i n n i t r o g e n and which have been

re p o r t e d subsequent to January 1978. The i n t e n t i o n of t h i s b r i e f

survey i s to demonstrate the a r r a y of n i t r o g e n - c o n t a i n i n g

compounds which have been found i n the P o r i f e r a i n order to

p r o v i d e a framework f o r a d i s c u s s i o n of the m e t a b o l i t e s of

C l i o n a c e l a t a i n Chapter Two. Many of these compounds have no

t e r r e s t r i a l c o u n t e r p a r t s , while in a few i n s t a n c e s , c l o s e

analogy to the p l a n t a l k a l o i d s i s apparent.

The term ' a l k a l o i d ' has been used i n t h i s t h e s i s to d e f i n e

substances of n a t u r a l o r i g i n which c o n t a i n b a s i c n i t r o g e n .

I n c l u s i o n i n t h i s c l a s s has been based p r i m a r i l y on s t r u c t u r a l

6

c o n s i d e r a t i o n s . The more r e s t r i c t i v e taxonomic and

p h a r m a c o l o g i c a l a c t i v i t y requirements of P e l l e t i e r (13) and

o t h e r s have been excluded. Nakanishi et a l . (14) have d e f i n e d

m o d i f i e d amino a c i d s and p e p t i d e s as imperfect a l k a l o i d s .

The compounds d i s c u s s e d below are presented in a sequence

based upon the s t r u c t u r a l groupings i n which t h e i r c o n s t i t u e n t

n i t r o g e n atoms are found. Those molecules p o s s e s s i n g n i t r o g e n i n

a c y c l e and i n an a c y c l i c s t r u c t u r a l f e a t u r e are ordered on the

b a s i s of t h e i r c y c l i c moiety.

A. Compounds C o n t a i n i n g Nitrogen i n an Aromatic H e t e r o c y c l e .

V a r i o u s c o l l e c t i o n s of the I n d o - P a c i f i c sponge, Dysidea

herbacea , have pr o v i d e d a number of compounds c o n t a i n i n g

t r i c h l o r o m e t h y l groups. Two of these compounds were found to

possess an uncommon C-terminal a m i n o - a l k y l - t h i a z o l e r e s i d u e . The

s t r u c t u r e and a b s o l u t e c o n f i g u r a t i o n of i s o d y s i d e n i n (J_, [cxr] D +

47°) was s o l v e d by X-ray d i f f r a c t i o n a n a l y s i s of the quaternary

ammonium s a l t 2. Compound 2 was prepared from j_ by s e q u e n t i a l

treatment with diborane-THF complex and methyl i o d i d e (15).

D y s i d e n i n (3, _ 98°) a diastereomer of J_ was present i n

small amounts in the sponge sample from which j_ was o b t a i n e d .

Compound 3 had p r e v i o u s l y been i s o l a t e d from D.herbacea by

Kazlauskas et a l . (16) and i t s s t r u c t u r e c o r r e c t l y deduced on the

b a s i s of s p e c t r a l a n a l y s i s and r e d u c t i o n ( Z n - a c e t i c a c i d ) to i t s

d e c h l o r o d e r i v a t i v e 4. Employing chemical degradation by a c i d

h y d r o l y s i s , C h a r l e s et a l . (17) have e s t a b l i s h e d that d y s i d e n i n

7

d i f f e r s from i s o d y s i d e n i n o n l y i n t h e c o n f i g u r a t i o n about the

s o l e amino a c i d ct-methine carbon i n the m o l e c u l e and have t h u s ,

by c h e m i c a l c o r r e l a t i o n , succeeded i n d e t e r m i n i n g the a b s o l u t e

c o n f i g u r a t i o n of 3.

In c o n t r a s t t o the t e r r e s t r i a l s i t u a t i o n , few i n d o l i c

compounds have been i s o l a t e d from marine o r g a n i s m s . The m a j o r i t y

of the m o l e c u l e s which have been d e f i n e d a r e s i m p l e i n d o l e

d e r i v a t i v e s . P r i o r t o the i n v e s t i g a t i o n of C l i ona c e l a t a

(Chapter 2 ) , no complex m e t a b o l i t e s r e m i n i s c e n t of the p l a n t

a l k a l o i d s had been i s o l a t e d from s p o n g e s . 1

^ y n b y a t o x i n A ( 6 , 1 8 ) , h y e l l a z o l e ( 7 , 1 9 ) , c h l o r o h y e l l a z o l e ( 8 , 1 9 ) , f l u s t r a m i n e A (9) and B ( 1 0 , 2 0 7 and s u r u g a t o x i n ( 1 1 , 2 1 ) a r e among the most complex i n d o l e and i n d o l e - b a s e d a l k a l o i d s o b t a i n e d from marine o r g a n i s m s .

9

The simple bromotryptamine d e r i v a t i v e s , 12 and 12a, were

i s o l a t e d from Smenospongia echina and S. aurea , r e s p e c t i v e l y , by

Djura et a l . (22). The bromine atom i n 12a was p o s i t i o n e d at C-5

on the b a s i s of *H NMR comparison with 5-bromo- and 6-

bromoindole-3-carboxaldehyde.

Three tryptophan d e r i v a t i v e s , c l o s e l y r e l a t e d to the sponge

compound a p l y s i n o p s i n (.13,23), have r e c e n t l y been i s o l a t e d . The

s t r u c t u r e s of 2'-de-N-methylaplysinopsin (14) and 6~bromo-2'-de-

N- m e t h y l a p l y s i n o p s i n • (1_5) , o b tained from the sponge D e r c i t u s

sp., were a s s i g n e d on the b a s i s of s p e c t r a l i n t e r p r e t a t i o n s

(24). Djura and Faulkner confirmed the s t r u c t u r e s of 14_ and 15

by t o t a l s y n t h e s i s and i n the l a t t e r case by comparison with a

s y n t h e t i c sample of the 5-bromo compound. The absence of meta-

c o u p l i n g between protons at p o s i t i o n s 5 and 7 of the i n d o l e

nucleus in the 1H NMR spectrum of compound 1J5 a p p a r e n t l y l e d to

an i n c o r r e c t assignment f o r the protons at.C-4 and C-5 (24). The

s t r u c t u r e of the brominated i n d o l e 1_6 was determined (22) by

s p e c t r a l comparison with 5-bromo- and 6-bromoindole

carboxaldehyde and with a s y n t h e t i c sample of 5-

bromoaplysinopsen. Compound 1̂ 6 was o b t a i n e d from a c o l l e c t i o n of

Smenospongia aurea which, d i d not c o n t a i n e i t h e r of the bromo

i n d o l e s 12 or 12a.

12 R = B r

12a R = H

10

R R - , R 2 R3 13 Me Me N H H

14 H Me N H H

15 H Me N H B r

16 H H O B r

D e t e r m i n a t i o n of the geometry about the t r i s u b s t i t u t e d

d ouble bond i n a r o m a t i c dehydroamino a c i d d e r i v a t i v e s p r e s e n t s

s u b s t a n t i a l d i f f i c u l t i e s (see Chapter f o u r f o r f u r t h e r

d i s c u s s i o n ) . K a z l a u s k a s et a l . (23) suggested t h a t a p l y s i n o p s i n

(13) e x i s t s as a m i x t u r e of dou b l e bond isomers ( 9 : 1 ) , but d i d

not a s s i g n the major isomer. A n a l y s i s of the NMR d a t a

p r o v i d e d by D j u r a and F a u l k n e r (22,24) f o r _1_4, J_5, j_6 and

r e l a t e d compounds does not a l l o w c o n c l u s i v e s t e r e o c h e m i c a l

a s s i g n m e n t s t o be made.

The s t r u c t u r e of the n o v e l i n d o l e - c o n t a i n i n g compound 17,

o b t a i n e d from the C a r i b b e a n sponge H a l i c h o n d r i a m e l a n o d o c i a i n

minute q u a n t i t i e s , was e l u c i d a t e d by Gopichand and Schmitz (25)

s o l e l y on the b a s i s of s p e c t r a l c o n s i d e r a t i o n s .

H

17

S e v e r a l p y r r o l l i c compounds have been i s o l a t e d from sponges

i n c l u d i n g a number of b r o m o - p y r r o l l e d e r i v a t i v e s . S t i e r l e and

F a u l k n e r (26) have r e c e n t l y d e s c r i b e d a f a m i l y of 5-

11

a l k y l p y r o l l e - 2 - c a r b o x a l d e h y d e s (JJJ-Ô) i n c l u d i n g the f i r s t

s t a b l e , n a t u r a l l y o c c u r r i n g c y a n o h y d r i n 2_0. A s e r i e s of 3-

a l k y l p y r o l l e - 2 - c a r b o x a l d e h y d e s had been i s o l a t e d p r e v i o u s l y by

C imino et a l . (27) from O s c a r e l l a l o b u l a r i s . The c y a n o h y d r i n

f u n c t i o n p r e s e n t i n 20_ was i n d i c a t e d by 1H- and 1 3 C NMR

c omparisons w i t h 2 - h y d r o x y n o n y l c y a n i d e .

F u r t h e r i n v e s t i g a t i o n of the aqueous e x t r a c t of the sponge

P h a k e l l i a f l a b e l l a t a by Sharma e t a l . ( 2 8 ) a f f o r d e d the i s o l a t i o n

of the n o v e l p y r o l l e - c o n t a i n i n g m e t a b o l i t e 2J_. Sharma and

B u r k h o l d e r ' s o r i g i n a l i n v e s t i g a t i o n of t h i s sponge (29) gave

r i s e t o the s t r u c t u r e e l u c i d a t i o n of two of the e a r l i e s t

n i t r o g e n o u s secondary m e t a b o l i t e s o b t a i n e d from marine sponges-

4 - b r o m o p h a k e l l i n and d i b r o m o p h a k e l l i n . The s t e r e o c h e m i s t r y about

the e x o c y c l i c double bond i n 2J_ was a s s i g n e d on the b a s i s of a

marked d o w n f i e l d s h i f t of the a d j a c e n t methylene p r o t o n s

r e s u l t i n g from the a n i s o t r o p y of the c l o s e l y s i t u a t e d amide

c a r b o n y l .

18 n = 1 4 - 1 6 , 1 8 1 9

20

1 2

21

Few p y r i d i n e a l k a l o i d s have been i s o l a t e d from marine

organisms. Substances present i n s e v e r a l s p e c i e s of the genus

H a l i c l o n a are t o x i c to f i s h and mice. Working with H.rubens , H

v i r i d i s , and H.erina Schmitz et a l . (30) demonstrated that t h i s

a c t i v i t y was caused by a polymeric compound composed of 3-

a l k y l p y r i d i n e u n i t s as shown in 22.

The substance r e s p o n s i b l e f o r the a n t i b a c t e r i a l a c t i v i t y of

the blue sponge Reniera sp.has been i s o l a t e d by M c l n t y r e et a l .

(31). Renierone (2_3) was d e f i n e d by X-ray c r y s t a l l o g r a p h y .

23

13

B. Compounds C o n t a i n i n g N i t r o g e n i n a Nonaromatic H e t e r o c y l e .

In a d d i t i o n t o the t h i a z o l e d e r i v a t i v e s _1_ and 3 D y s i d e a

herbacea i s known t o produce two o t h e r n i t r o g e n o u s m e t a b o l i t e s

p o s s e s s i n g t r i c h l o r o m e t h y l g roups. The s t r u c t u r e of the

d i k e t o p i p e r a z i ne 2_4 ( 3 2 ) , d e r i v e d from t r i c h l o r o l e u c i n e , was

deduced by s p e c t r a l a n a l y s i s and s e q u e n t i a l r e d u c t i o n t o i t s

s a t u r a t e d d e c h l o r i n a t e d d e r i v a t i v e 2_5. The E c o n f i g u r a t i o n was

a s s i g n e d t o the dehydroamino a c i d double bond on the b a s i s of

the c h e m i c a l s h i f t of the a l l y l i c i s o p r o p y l methine p r o t o n i n 24

and 25. The p o s i t i o n of the s e resonances (S 5 . 1 8 24 and 3.96 25)

was a t t r i b u t e d t o d e s h i e l d i n g by the a d j a c e n t c a r b o n y l group. An

e a r l i e r i n v e s t i g a t i o n of t h i s sponge a f f o r d e d d y s i d i n (2_6, 33).

The d i v e r s e n a t u r e of the n a t u r a l p r o d u c t s i s o l a t e d from

s e p a r a t e c o l l e c t i o n s of D.herbacea has been a t t r i b u t e d t o the

presence of d i f f e r e n t b l u e - g r e e n a l g a l symbionts (32, 15).

24 25

Mel

26

1 4

Three n o v e l t o x i c m a c r o l i d e s , l a t r u n c u l i n A {2T), B (28)

and C p o s s e s s i n g an unique 2 - t h i a z o l i d i n o n e t e r m i n u s have been

o b t a i n e d by Kashman e t a l . (34) from the Red Sea sponge

L a t r u n c u l i a magni f i c a . S t r u c t u r a l d e t e r m i n a t i o n of t h e s e

m e t a b o l i t e s was f a c i l i t a t e d by X-ray d i f f r a c t i o n a n a l y s i s of the

methyl e t h e r 29. The a u t h o r s s p e c u l a t e t h a t t h e s e compounds are

d e r i v e d from a mixed p o l y a c e t a t e - a m i n o a c i d s o u r c e .

27 R = H

29 R = M e

In the r e p o r t d e s c r i b i n g the i n d o l i c l a c t a m _1_7' Gopichand

and Schmitz (25) d e t a i l the s t r u c t u r e e l u c i d a t i o n of a second

l a c t a m 3_0, a l s o o b t a i n e d from H. melanodoc i a . C o n f i r m a t i o n of

the s t r u c t u r e p r oposed f o r 3_0 from s p e c t r a l a n a l y s i s was

p r o v i d e d by a c e t y l a t i o n and s e l e c t i v e r e d u c t i o n of the ketone

f u n c t i o n .

30

15

. Sponges of the order Verongidae have been a r i c h source of

brominated t y r o s i n e - d e r i v e d m e t a b o l i t e s ( 1 1 b ) , many of which

c o n t a i n an i s o x a z o l e moiety fused i n a s p i r o - s e n s e to a

c a r b o c y c l i c r i n g . The s t r u c t u r e of a e r o t h i o n i n (3J_) o r i g i n a l l y

i s o l a t e d from V.aerophoba in 1970 (35) has been confirmed by X-

ray a n a l y s i s ( 3 6 ) . The r e s u l t s of the X-ray d i f f r a c t i o n

experiments in c o n j u n c t i o n with c i r c u l a r d i c h r o i s m measurements

enabled McMillan et a l . to e s t a b l i s h i t s a b s o l u t e c o n f i g u r a t i o n .

R e c e n t l y , s e v e r a l novel m e t a b o l i t e s belonging to t h i s c l a s s have

been a c q u i r e d from two Verongidae sponges. A p l y s i n a f i s t u l a r i s

p r o v i d e d f i s t u l a r i n - 1 ( 3 2 ) , -2 (33) and -3 (.34, 3 7 ) . These

compounds were e l u c i d a t e d p r i n c i p a l l y by 1H NMR. The second

Verongidae sponge r e c e n t l y i n v e s t i g a t e d produced m e t a b o l i t e s

b e a r i n g only a c y c l i c n i t r o g e n . These w i l l be presented below.

16

C. Compounds C o n t a i n i n g A c y c l i c N i t r o g e n .

A b i o a s s a y g u i d e d f r a c t i o n a t i o n p r o c e d u r e e n a b l e d C a r t e r

and R i n e h a r t (38) t o i s o l a t e the p o t e n t a n t i b i o t i c s , 35-3_7, from

the red-orange sponge Acarnus er i t h a c u s . The s t r u c t u r e

e l u c i d a t i o n of the a c a r n i d i n e s (35-3_7) was a i d e d by p r o t e c t i o n

of the g u a n i d i n e f u n c t i o n w i t h 2,4-pentanedione. The

h y d r o g e n a t i o n p r o d u c t of one of the p r o t e c t e d d e r i v a t i v e s was

s y n t h e s i z e d i n o r d e r t o c o n f i r m the homospermidine s k e l e t o n .

From an u n i d e n t i f i e d b l a c k sponge Yunker and Scheuer (39)

i s o l a t e d a s e r i e s of f a t t y a c y l d e r i v a t i v e s (3_8-4_0 of 2-

methylene-^g-alanine. The amino a c i d i t s e l f and r e l a t e d f a t t y

a c y l d e r i v a t i v e s had p r e v i o u s l y been r e p o r t e d by Kashman et a l .

(40) from the sponge F a s c i o s p o n g i a c a v e r n o s a . Holm and Scheuer

(41) s u b s e q u e n t l y d e s c r i b e d the s y n t h e s i s of t h i s t o x i c amino

a c i d and i t s amide ^9 ( n = l 3 ) .

38 R

39 R

4 0 R

41 R

Me, R,= COCH 2 (CH2)nCH 3

Me, R|= C O C O ( C H 2 ) n C H 3 , n = 11-13

Me, Ri = COCHOH(CH2>nCH 3 , n =11-13

H , R,= C O C H 2 ( C H 2 ) n C H 3

17

Methanol e x t r a c t s of the V e r o n g i d a e sponge, I a n t h e l l a b a s t a

, gave the h i g h l y m o d i f i e d p e p t i d e s , b a s t i d i n - 1 (42) and

b a s t i d i n - 2 (£3, 4 2 ) . S e v e r a l complex r e l a t e d compounds were a l s o

o b s e r v e d .

I I I . N a t u r a l - P r o d u c t s H i s t o r y of the C l i on i d a e

The sponge f a m i l y C l i o n i d a e have been l a r g e l y o v e r l o o k e d by

n a t u r a l - p r o d u c t s c h e m i s t s as a r e s u l t of the s u b s t a n t i a l

c o l l e c t i o n problems they g e n e r a l l y p r e s e n t . The v a s t m a j o r i t y of

c l i o n i d s a r e e n d o l i t h i c and i f v i s i b l e (many a r e not) must be

c h i s e l l e d or p r i e d from t h e i r s u b s t r a t e s as b l o c k s c o n s i s t i n g

m o s t l y of c o r a l or r o c k . ^ I t i s not s u r p r i s i n g , t h e r e f o r e , t h a t

i t has been p r i m a r i l y those sponges which a t t a i n the l a t e r , b e t a

or gamma, s t a g e s of growth (see Chapter t h r e e f o r e x p l a n a t i o n )

which have r e c e i v e d a t t e n t i o n . However, i n t e r e s t i n the

Br

42 R = H

43 R = B r

18

b u r r o w i n g sponges i s i n t e n s i f y i n g (43) and even the r e s i s t a n t

e n d o l i t h i c organisms w i l l soon become the s u b j e c t s of c l o s e r

s c r u t i n y .

C l i o n a c e l a t a , i d e n t i f i e d by G rant i n 1826, was f i r s t

s t u d i e d f o r i t s c h e m i c a l c o n s t i t u e n t s by Doree i n 1909 (44) as

p a r t of h i s g e n e r a l s u r v e y of c h o l e s t e r o l i n a n i m a l s . H i s

c o l l e c t i o n s a f f o r d e d an unique s t e r o l which he named

c l i o n a s t e r o l . Bergmann r e p e a t e d t h i s i s o l a t i o n i n 1941 (45) and

s u g g e s t e d a p a r t i a l s t r u c t u r e f o r t h i s s t e r o l i n 1942 ( 4 6 ) .

Bergmann a l s o demonstrated the p r e s e n c e of c l i o n a s t e r o l and

p o r i f e r s t e r o l i n C . c a r r i b o e a ( 4 7 ) . R e c e n t l y , Erdman and Thomson

( 4 8 ) , employing modern GC s e p a r a t i o n t e c h n i q u e s , have shown t h a t

Doree's ' c l i o n a s t e r o l ' was a m i x t u r e of c l i o n a s t e r o l (4_4) and

p o r i f e r a s t e r o l ( 4 5 ) .

A New Z e a l a n d specimen of C . c e l a t a has been i n v e s t i g a t e d

f o r i t s f r e e amino a c i d c o n t e n t ( 4 9 ) . T a u r i n e and h y p o t a u r i n e ,

amino s u l f o n i c a c i d s commonly e n c o u n t e r e d i n marine

i n v e r t e b r a t e s , were shown t o be r e l a t i v e l y abundant

c o n s t i t u e n t s . Taurocyamine was a l s o o b s e r v e d .

The d e m o n s t r a t i o n t h a t marine sponges c o n t a i n s u b s t a n c e s

which i n f l u e n c e the growth of b a c t e r i a was f i r s t made by

N i g r e l l i i n 1959 ( 5 0 ) . He s u b s e q u e n t l y showed t h a t a n t i m i c r o b i a l

19

a c t i v i t y was a s s o c i a t e d w i t h the o r g a n i c s o l v e n t e x t r a c t s of a

v a r i e t y of sponges i n c l u d i n g C. c e l a t a (gamma s t a g e ) ( 5 1 ) . C.

c a r t e r i and C . c o p i o s a were r e p o r t e d by B u r k h o l d e r and R u e t z l e r

(52) t o c o n t a i n c h e m i c a l s which i n h i b i t e d the growth of s e v e r a l

t e s t b a c t e r i a , a l t h o u g h C. v i r i d i s e x t r a c t s d i d n o t . The

c h e m i c a l s r e s p o n s i b l e f o r th e s e a c t i v i t i e s have not been

d e t e r m i n e d .

F i g u r e 1. C l i o n a c e l a t a ( G rant) ( y e l l o w ) .

21

NATURAL-PRODUCTS CHEMISTRY OF CLIONA CELATA

I . T e t r a c e t y l c l i o n a m i d e (46)

Andersen i n i t i a t e d our study of C l i o n a c e l a t a i n 1977 when

he observed that ethanol e x t r a c t s of sponge c o l l e c t e d i n Barkley

Sound showed marked _iri v i t r o a n t i b i o t i c a c t i v i t y a g a i n s t the

gram-positive bacterium, Staphylococcus aureus . H i s i n i t i a l

attempts at i s o l a t i n g the e t h y l a c e t a t e s o l u b l e substance

r e s p o n s i b l e f o r t h i s a c t i v i t y were u n s u c c e s s f u l owing to i t s

f a i l u r e to undergo chromatography by c o n v e n t i o n a l methods. The

presence of p h e n o l i c compounds i n the sponge e x t r a c t was

i n d i c a t e d by an i n t e n s e l y p o s i t i v e f e r r i c c h l o r i d e r e a c t i o n .

T h i s d i s c o v e r y l e d to the pr o p o s a l that d e r i v a t i z a t i o n might

pr o v i d e a v i a b l e s o l u t i o n to the i s o l a t i o n problem. A c e t y l a t i o n

employing a c e t i c anhydride-sodium a c e t a t e proved to be the most

s u c c e s s f u l method i n terms of ease of performance and y i e l d . The

d e r i v a t i z a t e d r e s i d u e was s u b j e c t e d to r o u t i n e f r a c t i o n a t i o n on

a column of s i l i c a g e l to g i v e the major a c e t y l a t e d m e t a b o l i t e ,

t e t r a c e t y l c l i o n a m i d e (46,53) .

O A c

46

22

T e t r a c e t y l c l i o n a m i d e (£6) c r y s t a l l i z e d from T H F - i s o p r o p y l

e t h e r as v e r y f i n e t h r e a d - l i k e c l u s t e r s which were not amenable

t o X-ray c r y s t a l l o g r a p h i c e x a m i n a t i o n . The m o l e c u l a r f o r m u l a

C27H26BrN3C>8 was d e t e r m i n e d by e l e m e n t a l a n a l y s i s (C,H,N) and

was c o n f i r m e d by HR a n a l y s i s of the p a r e n t i o n d o u b l e t o b s e r v e d

at m/e 599/601 (1:1) i n the LRMS ( f i g u r e 2). The base peak

ob s e r v e d a t m/e 210/208 (1:1) was a t t r i b u t e d t o the bromoindole

i o n shown below (54):

A n a l y s i s by *H NMR ( f i g u r e 3, t a b l e 1) suggested that the

molecule c o n t a i n e d a bromotryptophan r e s i d u e . T h i s was confirmed

by h y d r o l y s i s i n MeOH-H 2 SO 4 to give the methyl e s t e r £7. The

s t r u c t u r e of £7 was i d e n t i f i e d by comparison with N - a c e t y l - 5 -

bromotryptophan methyl e s t e r (£8) prepared from commercially

a v a i l a b l e (±)-5-bromotryptophan ( A l d r i c h ) . The e s t e r s £7 and £8

d i f f e r e d i n t h e i r IR and *H NMR s p e c t r a ( f i g u r e 4) and were

d i s t i n g u i s h a b l e by s i l i c a - H P L C .

The remaining f e a t u r e s of t h i s molecule were determined by

s p e c t r a l a n a l y s i s and two c l a s s i c a l d e g r a d a t i v e r e a c t i o n s .

O z o n o l y s i s of £6 a f f o r d e d 3,4,5-triacetoxybenzaldehyde which was

shown to be i d e n t i c a l to an a u t h e n t i c sample prepared from

g a l l i c a c i d (see appendix 1). T h i s r e s u l t i m p l i e d the presence

of the f o l l o w i n g p a r t i a l s t r u c t u r e :

F i g u r e 2. EIMS of t e t r a c e t y l c l i o n a m i d e (46).

F i g u r e 3 a. P a r t i a l l y exchanged 270 MHz *H NMR spectrum of t e t r a c e t y l c l i o n a m i d e (46) in acetone-dg b. Expansion (1000 Hz) of the non-exchanged aromatic region of 46.

25

F i g u r e 4. Comparison of the aromatic regions of the 270 MHz *H NMR s p e c t r a of 47 and 4 8 .

26

O A c

The 1 H NMR spectrum of 46 i n CDC1 3 (53) a l s o c o n t a i n e d

s i g n a l s a t t r i b u t a b l e to two coupled o l e f i n i c protons; a doublet

at cT 6.09 (J=14,Hz), and a doublet of doublets at 6 7.32

( J=10,14HZ). Exchange of a s i g n a l (bd) at cf 8.64 (J=10HZ) with

D zO c o l l a p s e d the s i g n a l at 6 7.32 to a doublet (J=14Hz). These

o b s e r v a t i o n s together with the r e s u l t s of double resonance

experiments suggested that the remainder of the molecule not

accounted f o r by the N - a c e t y l t r y p t o p h a n moiety, was an unusual

t r a n s ~ d i s u b s t i t u t e d enamide (N-vinylamide, N - a c y l s t y r y l a m i n e ) .

Hydrogenation of 46 i n the presence of a p a l l a d i u m c a t a l y s t

a f f o r d e d the expected dihydro-debromo d e r i v a t i v e 49 ( f i g u r e 5),

thereby c o n c l u d i n g Andersen's s t r u c t u r e e l u c i d a t i o n of

t e t r a c e t y l c l i o n a m i d e (46).

H

49

s

I I • • • • • • • • • I • • • • • • • • • I • • • • • • • • • I I I.... • —

7 6 5 4 3 2 1 p p m ( £ )

F i g u r e 5 a. 2 7 0 MHz ' H NMR spectrum of 49 ( n a t u r a l ) in acetone- * J d 6 b. V e r t i c a l expansion c. O f f s e t . Impurity s i g n a l s a r i s e from the presence of the incompletely reduced degradation product 5 0 .

28

I I . The O b j e c t i v e s

The c o n t i n u a t i o n of the n a t u r a l - p r o d u c t s i n v e s t i g a t i o n of C.

c e l a t a i n i t i a t e d by Dr. Andersen , c o n s t i t u t e s the subject

matter of t h i s t h e s i s . T h i s study was o r i g i n a l l y envisaged to be

of three p a r t s : the c o n f i r m a t i o n of the t e t r a c e t y l c l i o n a m i d e

(46) s k e l e t o n and d e t e r m i n a t i o n of i t s a b s o l u t e c o n f i g u r a t i o n ;

the i s o l a t i o n of u n d e r i v a t i z e d 46, assessment of i t s b i o l o g i c a l

a c t i v i t y , and i d e n t i f i c a t i o n of the source of the a n t i m i c r o b i a l

a c t i v i t y a t t r i b u t e d to C. c e l a t a e x t r a c t s ; and f i n a l l y , the

i s o l a t i o n and s t r u c t u r e e l u c i d a t i o n of the remaining substances

present i n the a c e t y l a t e d sponge e x t r a c t .

During the course of t h i s i n v e s t i g a t i o n these o b j e c t i v e s

were expanded to encompass s e v e r a l other f e a t u r e s of i n t e r e s t .

These i n c l u d e d : a p a r t i a l survey ' of the chemistry of North

American c l i o n i d s ; experiments aimed at e x p l o r i n g the nature of

the burrowing process of C . c e l a t a ; and s t u d i e s towards the

s y n t h e s i s of dehydropeptides to be used in a s s e s s i n g the r o l e of

dehydroamino a c i d r e s i d u e s i n metal b i n d i n g , to a i d i n the

s t r u c t u r e assignment and f o r b i o l o g i c a l t e s t i n g .

The r e s u l t s of t h i s work are d e s c r i b e d and d i s c u s s e d i n the

f o l l o w i n g s e c t i o n s of t h i s chapter and are continued i n two

subsequent c h a p t e r s .

29

I I I . C o n f i r m a t i o n of the S t r u c t u r e of T e t r a c e t y l c l i o n a m i d e (46)

In o r d e r t o v e r i f y the carbon s k e l e t o n of

t e t r a c e t y l c l i o n a m i d e (4_6) i t was deemed n e c e s s a r y t o s y n t h e s i z e

e i t h e r 4_6 i t s e l f , or i t s h y d r o g e n a t i o n p r o d u c t £9 which

c o n t a i n e d a l l of the s k e l e t a l e l ements of £6. In view of the

d i f f i c u l t i e s a s s o c i a t e d w i t h the s y n t h e s i s of N - v i n y l a m i d e s i n

s e n s i t i v e m o l e c u l e s ( t o be d i s c u s s e d ) the h y d r o g e n a t i o n p r o d u c t

49 c o n t a i n i n g the s t a b l e p h enethylamine moiety was chosen as the

t a r g e t m o l e c u l e .

N - A c e t y l t r y p t o p h a n was p r e p a r e d from t r y p t o p h a n by

a c e t y l a t i o n w i t h a c e t i c a n h y d r i d e ( 5 5 ) . C o u p l i n g of the above

w i t h 5-hydroxydopamine h y d r o c h l o r i d e i n t h e p r e s e n c e of DCC and

t r i e t h y l a m i n e a f f o r d e d , upon workup, an o i l which f a i l e d t o

r e a c t w i t h n i n h y d r i n . A c e t y l a t i o n of t h i s m a t e r i a l w i t h A c 2 O-py

gave, i n 30 p e r c e n t o v e r a l l y i e l d , a p r o d u c t (4 9a) ( f i g u r e 6)

i d e n t i c a l (TLC, UV, MS), except f o r o p t i c a l r o t a t i o n , w i t h 4_9.

H

50

F i g u r e 6 a. 270 MHz 'H NMR spectrum of 49a ( s y n t h e t i c ) i n a c e t o n e - d 6 b. V e r t i c a l expansion c. O f f s e t .

CO o

31

The dihydro-bromo d e r i v a t i v e 50 (see experimental) was

obtained d u r i n g the c o n v e r s i o n of t e t r a c e t y l c l i o n a m i d e (46) to

i t s u l t i m a t e hydrogenation product 49.

IV. The Absolute C o n f i g u r a t i o n of T e t r a c e t y l c l i o n a m i d e (46)

The d e t e r m i n a t i o n of the a b s o l u t e c o n f i g u r a t i o n about the

s o l e c h i r a l carbon atom present i n £6 r e q u i r e d the p r o d u c t i o n of

an o p t i c a l l y a c t i v e - d e b r o m i n a t e d d e g r a d a t i o n product which was

e i t h e r commercially a v a i l a b l e or r e a d i l y s y n t h e s i z e d . The methyl

e s t e r 4_7 produced by Andersen was r u l e d out owing to the very

low y i e l d of product o b t a i n e d i n the MeOH-H 2S0 4 h y d r o l y s i s

r e a c t i o n .

S y n t h e t i c N - v i n y l amides have been s t u d i e d i n connec t i o n

w i t h t h e i r a b i l i t y to p o l y m e r i z e . Kutner (56), among o t h e r s ,

noted that these monomers r e a c t e d i n a manner s i m i l a r to v i n y l

e t h e r s and e s t e r s when t r e a t e d with d i l u t e s o l u t i o n s of m i n e r a l

a c i d s . The mechanism of s t y r y l a c e t a m i d e h y d r o l y s i s has been

s t u d i e d i n d e t a i l (57,58) and one p o s s i b l e route i s o u t l i n e d i n

scheme .1.

Scheme 1. A p o s s i b l e mechanism f o r the a c i d h y d r o l y s s t y r y l a c e t a m i d e (57,58).

33

When t e t r a c e t y l c l i o n a m i d e (£6) was r e a c t e d w i t h HCl i n

a c e t o n i t r i l e (0.1M), 2 - a c e t a m i d o - 3 - ( 6 ' - b r o m o i n d o l - 3 ' - y l )

p r o p i o n a m i d e (5_1, f i g u r e 7) was o b t a i n e d . A maximum y i e l d of 50-

60 p e r c e n t c o u l d be a c h i e v e d f o l l o w i n g 48-72h a t room

t e m p e r a t u r e . The 3 , 4 , 5 - t r i a c e t o x y p h e n y l a c e t a l d e h y d e produced,

p o l y m e r i z e d under the r e a c t i o n c o n d i t i o n s employed.

H y d r o g e n o l y s i s of 51 p r o v e d t o be u n u s u a l l y d i f f i c u l t . 2-

A c e t a m i d o - 3 - ( i n d o l - 3 ' - y l ) p r o p i o n a m i d e (5_2, f i g u r e 8) was

e v e n t u a l l y o b t a i n e d f o l l o w i n g r e p e a t e d l o w - p r e s s u r e

h y d r o g e n a t i o n employing a p a l l a d i u m - o n - c h a r c o a l c a t a l y s t i n the

pre s e n c e of a c e t i c a c i d . The a b s o l u t e c o n f i g u r a t i o n of 5_2 ( [ o ; ] D

+ 15" (c 1.0, CH 3OH)) was d e t e r m i n e d t o be S by com p a r i s o n w i t h

a u t h e n t i c N - a c e t y l - L - t r y p t o p h a n a m i d e ([a]D + 17° (c 1.0, MeOH);

l i t . (59) [a]D23 + 20 ± 1 ° (c 2%, MeOH)) pu r c h a s e d from the

A l d r i c h C hemical Company. Hence, t e t r a c e t y l c l i o n a m i d e i s (2S)-N-

( ( l B E ) - 5 " , 6 " , 7 B - t r i a c e t o x y s t y r - l " - y l ) - 2 - a c e t a m i d o - 3 - ( 6 ' - b r o m o -

i n d o l - 3 ' - y l ) p r opionamide ( 6 0 ) . The d e g r a d a t i v e and s y n t h e t i c

F i g u r e 7 a. 270 MHz 'H NMR spectrum of 51 in DMSO-d_6 b. V e r t i c a l expansion c. O f f s e t .

CO

36

r e a c t i o n s employed i n the s t r u c t u r e e l u c i d a t i o n of t h i s

molecule are summarized in scheme 2.

O A c

V. Clionamide (53 , 61)

The s o l v e n t e x t r a c t s of C . c e l a t a once a c e t y l a t e d showed no

i n h i b i t o r y a c t i o n towards S. aureus or s e v e r a l fungi (see

e x p e r i m e n t a l ) . S i m i l a r l y , the l i p i d c o n t a i n i n g f r a c t i o n o b tained

by t r i t u r a t i o n of the crude EtOAc e x t r a c t with ether p r i o r to

a c e t y l a t i o n a l s o was devoid of a c t i v i t y . T h e r e f o r e , we sought to

i s o l a t e u n d e r i v a t i z e d p h e n o l i c m e t a b o l i t e s with the hope that

one of these compounds would be r e s p o n s i b l e f o r the rn v i t r o

a n t i b i o t i c a c t i v i t y . P h e n o l i c substances are f r e q u e n t l y

r e s p o n s i b l e f o r the a n t i m i c r o b i a l p r o p e r t i e s a s s o c i a t e d with

e x t r a c t s of marine organisms ( l l g ) .

P u r i f i c a t i o n attempts u t i l i z i n g chromatography on

c e l l u l o s e , s i l i c a , Sephadex LH-20, Sephadex G-10 or r e v e r s e d -

H

4 6

Scheme 2. Summary of the d e g r a d a t i v e and s y n t h e t i c r e a c t i o n s employed i n the d e t e r m i n a t i o n of t e t r a c e t y l c l i o n a m i d e ( 4 6 ) .

38

phase m a t e r i a l s i n a v a r i e t y of s o l v e n t s f a i l e d to provide

d i s c r e t e compounds. RP-HPLC on a small s c a l e was capable of

p a r t i a l l y s e p a r a t i n g the u n d e r i v a t i z e d m e t a b o l i t e s ( f i g u r e 9).

However, attempts to s c a l e up t h i s procedure l e d to s u b s t a n t i a l

l o s s of column e f f i c i e n c y because of the high percentage of the

e x t r a c t which was extremely slow to e l u t e . The r e l u c t a n c e of

these compounds to be p u r i f i e d by chromatography was a t t r i b u t e d

to t h e i r high p o l a r i t y and i n s t a b i l i t y and to the complex nature

of the mixture.

The EtOAc s o l u b l e - e t h e r t r i t u r a t e d e x t r a c t s of C. c e l a t a

e x h i b i t e d p o s i t i v e r e a c t i o n s not only towards f e r r i c c h l o r i d e

but a l s o to n i n h y d r i n . When a small sample of sponge (40 g, wet

weight) was worked up using a c e t i c anhydride-d§ as the

a c e t y l a t i n g agent, t e t r a c e t y l c 1 i o n a m i d e - d 12 ( 5 4 ) and a

d e u t e r o a c e t y l a t e d mixture of the remainder of the sponge

m e t a b o l i t e s were i s o l a t e d .

D 3 C O H N

C O C D 3

:OCD3

H

54

39

F i g u r e 9. RP-HPLC of the u n d e r i v a t i z e d C l i o n a m e t a b o l i t e s . A n a l y s i s c o n d i t i o n s ; H Bondapak-Ci8 , 4 x 250 mm; s o l v e n t C H 3 C N -H 2 O , g r a d i e n t 35-100% at 1%/min; f l o w r a t e , 1.0 mL/min; room t e m p e r a t u r e ; A 290 nm.

40

A n a l y s i s of 54 ( f i g u r e 10) and the m i x t u r e by lH NMR

i n d i c a t e d the absence of s i g n a l s a r i s i n g from a c e t a m i d e s (-

NHCOCH3) and phenol a c e t a t e s (PhOCOCH 3), t h e r e b y p r o v i n g t h a t

t h e s e were not n a t u r a l f e a t u r e s of the C l i o n a compounds (see

a l s o f i g u r e 1 1 ) . These r e s u l t s l e d t o the s u g g e s t i o n t h a t a

c l a s s i c a l a l k a l o i d e x t r a c t i o n w i t h a c i d might be u s e f u l i n

i s o l a t i n g c l i o n a m i d e (5_3) . E x e c u t i o n of t h i s p r o c e d u r e i n the

presence of n i t r o g e n a f f o r d e d a p a l e y e l l o w s o l i d r e s i d u e , which

was shown t o be p r a c t i c a l l y pure c l i o n a m i d e (—80%). Attempts t o

c r y s t a l l i z e t h i s m a t e r i a l were u n s u c c e s s f u l due t o i t s

i n s t a b i 1 i t y .

C l i o n a m i d e (5_3, f i g u r e 12) was i s o l a t e d i n pure form

f o l l o w i n g r a p i d chromatography on s i l i c a p l a t e s a l t h o u g h t h i s

l e d t o s u b s t a n t i a l l o s s e s of m a t e r i a l . C l i o n a m i d e d i d not g i v e

an i n t e r p r e t a b l e EIMS and i t s IR spectrum d i s p l a y e d l i t t l e of

OH

H

53

F i g u r e 10. P a r t i a l l y exchanged 270 MHz 'H NMR spectrum t e t r a c e t y l c l i o n a m i d e - d 12 (M> i n a c e t o n e - d 6 .

MICROMETERS (pm)

F R E Q U E N C Y (CM" 1 )

F i g u r e 11. IR spectrum (KBr) of the u n d e r i v a t i z e d C l i o n a ^ m e t a b o l i t e s . ^

44

d i a g n o s t i c value beyond a broad OH/NH a b s o r p t i o n at 3300 cm-1

and a broad amide a b s o r p t i o n at 1635 cm- 1. Attempts t o o b t a i n 'H

NMR s p e c t r a i n DMSO-d6 l e d to decomposition of the molecule.

Formation of the deuteroacetonide 55 occ u r r e d when 'H NMR

s p e c t r a ( f i g u r e 13, see a l s o appendix 2) were obtained i n

acetone-d_6. The acetonide methyl resonances were observed at <f

1.42 and 1.47 i n the non-deuterated a c e t o n i d e 56 ( f i g u r e 14). A

'H NMR spectrum of clionamide (5_3) procured i n methanol-d4

showed none of the exchangeable protons. The non-exchangeable

protons were t r i v i a l l y i d e n t i f i e d by analogy with the assig n e d

s i g n a l s f o r t e t r a c e t y l c l i o n a m i d e (£6). The i d e n t i t y of

cli o n a m i d e (5_3) was confirmed by c o n v e r s i o n to

t e t r a c e t y l c l i o n a m i d e (£6) upon a c e t y l a t i o n .

H H

55 56

F i g u r e 13 a. 270 MHz *H NMR spectrum of 55 in acetone-cU b. V e r t i c a l expansion c. O f f s e t .

F i g u r e 14 a. 270 MHz 'H NMR spectrum of 56 in acetone-d(; b. O f f s e t .

47

C l i o n a m i d e (5_3), the major m e t a b o l i t e of C . c e l a t a , showed

o n l y weak a c t i v i t y a g a i n s t S.aureus and was i n a c t i v e towards

f u n g i . T h i s unexpected r e s u l t c o m p e l l e d us t o r e - e v a l u a t e our

o r i g i n a l sponge b i o a s s a y s . When we c o l l e c t e d , worked up, and

b i o a s s a y e d C . c e l a t a i n one day a r e s u l t q u a l i t a t i v e l y s i m i l a r t o

t h a t o b t a i n e d w i t h c l i o n a m i d e was o b s e r v e d . As a r e s u l t of the

weak l e v e l of i n h i b i t i o n o b t a i n e d i n t h i s experiment we

c o n c l u d e d t h a t the s t r o n g l y p o s i t i v e b i o a s s a y s o r i g i n a l l y found

were a r t i f a c t s . D e c o m p o s i t i o n of one of the C l i o n a m e t a b o l i t e s

must g i v e r i s e t o t h i s e f f e c t but the compound r e s p o n s i b l e was

not pursued f u r t h e r . 1 The u n d e r i v a t i z e d C l i o n a m e t a b o l i t e s a r e

c u r r e n t l y b e i n g c o m m e r c i a l l y t e s t e d f o r t h e i r p h a r m a c o l o g i c a l

p r o p e r t i e s .

V I . The C e lenamides: I s o l a t i o n (62,63)

P r e l i m i n a r y c h r o m a t o g r a p h i c a n a l y s i s of crude a c e t y l a t e d C.

c e l a t a e x t r a c t s by TLC showed two i l l - d e f i n e d s p o t s w i t h Rf

v a l u e s g r e a t e r than t e t r a c e t y l c l i o n a m i d e (46) upon v i s u a l i z a t i o n

w i t h UV l i g h t or c h a r r i n g w i t h s u l p h u r i c a c i d . P a r t i a l

p u r i f i c a t i o n of t h i s e x t r a c t was a c h i e v e d by s i l i c a column

chromatography employing a s t e p w i s e CH2CI2 -EtOAc g r a d i e n t .

1 T h i s r e s u l t i s c o n s i s t e n t w i t h the o b s e r v a t i o n t h a t C. c e l a t a grows i n c l o s e a s s o c i a t i o n w i t h numerous a l g a e , t u n i c a t e s , b ryozoans and o t h e r marine organisms. The area s u r r o u n d i n g sponges which p o s s e s s p o t e n t a n t i m i c r o b i a l agents i s u s u a l l y f r e e of o t h e r forms of marine l i f e . However, we have never o b s e r v e d s i g n s of p r e d a t i o n of C . c e l a t a .

48

E x a m i n a t i o n of the column e l u a t e by RP- (eg. f i g u r e 15) and

s i l i c a - H P L C i n d i c a t e d t h a t the f r a c t i o n e l u t i n g w i t h 10-50

p e r c e n t EtOAc i n CH 2 C l 2 c o n t a i n e d a p p r o x i m a t e l y t e n compounds. XH NMR i n v e s t i g a t i o n of t h i s m a t e r i a l s u b s e q u e n t l y caused us t o

i n c r e a s e our e s t i m a t i o n of the number of a c e t y l a t e d c e l e n a m i d e s

p r e s e n t t o 18-22. However, the m a j o r i t y of th e s e a r e e x c e e d i n g l y

minor components.

C a r e f u l r e p e t i t i o n of the column p u r i f i c a t i o n s t e p

f a c i l i t a t e d the p a r t i a l f r a c t i o n a t i o n of t h i s m i x t u r e (e.g.

f i g u r e 1 6 ) . The most p o l a r c o n s t i t u e n t , h e x a c e t y l c e l e n a m i d e B

( 5 7 ) , was o b t a i n e d i n pure form f o l l o w i n g r e p e a t e d s i l i c a PTLC

i n c l u d i n g m u l t i p l e developments of each p l a t e . T h i s method

f a i l e d t o s e p a r a t e h e x a c e t y l c e l e n a m i d e A (5_8) and

p e n t a c e t y l c e l e n a m i d e C ( 5 9 ) . These m e t a b o l i t e s were i s o l a t e d by

re p e a t e d chromatography on r e v e r s e d - p h a s e p l a t e s .

N o n a c e t y l c e l e n a m i d e D (60) was o b t a i n e d i n an an a l o g o u s f a s h i o n .

The r e m a i n i n g C l i o n a a l k a l o i d s proved t o be e x t r e m e l y d i f f i c u l t

t o p u r i f y and c o n s e q u e n t l y were s t u d i e d as m i x t u r e s . CO

cc E

I—I 1 1 1 1 | I I 1 1 ' — I ' 1 ' — 1 — • — » I1 i I 1 — I r — I — I — I — i — I 1 1 1 1 — ' T 0 2 4 6 8 1 0 1 2 V4 min 0 2 4 6 8 1 0 1 2 1 4 min

F i g u r e 16. S i l i c a HPLC of p a r t i a l l y p u r i f i e d a c e t y l a t e d C l i o n a m e t a b o l i t e s a. F r a c t i o n e l u t i n g w i t h 25% EtOAc b. F r a c t i o n e l u t i n g w i t h 50% EtOAc. A n a l y s i s c o n d i t i o n s : L I C h r o s o r b S1:SO; 10 x 4 x 250 mm; s o l v e n t . CH 2C1 2-CH 3CN ( 4 : 1 ) : f l o w r a t e , 1.2 mL/m1n; room temperature; A 290 nm.

F i g u r e 15. RP-HPIC of c r u d e a c e t y l a t e d methanol e x t r a c t s of CJMqna c e l a t a a . ^ - s t a g e b.CX-stage. A n a l y s i s c o n d i t i o n s ; fl B o n d a p a k - C i o . 4 x 250 mm; s o l v e n t . CH 3CN-H 20 ( 3 : 2 ) ; flow r a t e . 1.2 mL/min; room temperature; A 290 nm. Chromatogram 'a' from 5.59-11.36 min Is I d e n t i c a l to the chromatogram d i s p l a y e d by a s i l i c a column f r a c t i o n e l u t i n g w i t h 10-50% EtOAc.

50

VII. The Celenamides: Structure Determinat ion

A l l of the p u r i f i e d acetylated celenamides were white

powders and none have c r y s t a l l i z e d to date. The homogeneity of

these compounds was established by spectral analysis ( JH NMR,

MS) and p a r t i c u l a r l y by HPLC on s i l i c a and reversed-phase

supports using several solvent combinations. These powders

decomposed on heating and on prolonged standing in solvents and

did not give acceptable elemental analyses.

The peptidyl nature of the acetylated celenamides was

evident from intense amide absorptions in th e i r infrared spectra

( v max 3300 (NH stretch), 1660 cm-1 (amide I band)) (figures 17

and 18) and peptide alpha-methine and NH signals present in the

*H NMR spectrum of each compound (figures 19, 20, 29, 33, table

1) (see also appendix 3, 4 and 6). Many of the spectral features

observed were reminiscent of those exhibited by

tetracetylclionamide (4_6) . However, the abundance of signals in

the 1H NMR spectra suggested much greater complexity. The

preliminary spectral data confirmed our suspicions that chemical

degradation would be required to simplify, and so deduce, these

structures. Hexacetylcelenamide A (58, 0.03% wet weight) and

hexacetylcelenamide B (5_7, 0.02% wet weight) were the most

abundant of t h i s new class of Cliona metabolite and therefore,

were the obvious choices for d e t a i l e d chemical examination. The

structures of pentacetylcelenamide C (59) and

nonacetylcelenamide D (6_0) were solved largely by comparison

with 57 and 5_8. The proposed structures of the minor

51

F i g u r e 17. IR s p e c t r a a. Hexacetylcelenamide A (58) b. Hexacetylcelenamide B, ( 5_7 ) .

52

F i g u r e 18. IR (KBr) s p e c t r a a. Pentacetylcelenamide C (59) b. Nonacetylcelenamide D (50).

10 9 8 7 6 5 4 3 2 1ppm(S)

F i g u r e 19 a. 400 MHz *H NMR spectrum of hexacetylcelenamide A (58) in acetone-d_6, b. V e r t i c a l expansion, c,d,e. Expansions (600 Hz) of the aromatic region of 58.

F i g u r e 20 a. 270 MHz *H NMR spectrum of hexacetylcelenamide B (57) in acetone-d_6 b. V e r t i c a l expansion c,d. Expansion (1000 H z T of the aromatic region of 5_7. See appendix 5 f o r p a r t i a l 400 MHz spectrum and computer s i m u l a t i o n .

TABLE 1. 270 MHZ 'H NMR SPECTRA 1

P r o t o n

pos 11 Ion 1

4G 58 57 59 60 65 66 73 Mul t l p l I d ty

(H2)

I n d o l e HI

I n d o l e H2

I n d o l e H4

I n d o l e H5

I n d o l e H7

Trp NH

Trp CH2

Trp CH

Leu NH

Leu CH2

Leu CH

Val NH

Val CH

1 -PrCH

1-Pr(CH3)2

10.22'

7 . 20

7 .59

7.16

7 . 57

7.41 d,8

3 . 14dd,

6, 15

3. 28dd,

7 . 15

4.77ddd,

6.7,8

10. 19

7.31

7 .62

7 . 16

7 . 57

7.91 d. 10

3 . 26dd.

11,15

3.54dd,

4 . 15

4.75ddd,

4. 10.1 1

7 .93

1 . 73

4 . 32

1 . 86

0 . 97

1 .02

10. 19

7.31

7 .65

7 . 16

7 .60

7.76d.10

3 . 48dd,

15.4

3 . 29dd.

15 . 4

3, 29dd,

15,11

7 .80d,6

4 . 46dd,

6.8.2

2 . 24

0.99

1.01

10. 21

7 . 32

7 .64

7.15

7 . 55

7.88 d,9

3. 28dd,

11,15

3.55dd,

4,15

4.77ddd,

4.9,11

7.94

1 . 75

4 . 32

1 .89

0.98

1 .03

7.91

1 .78

4 .48

1 . 86

0.95

0.98

10. 16

7 . 26

7 .64

7 . 15

7 .58

7.81 d,8

3 . 25dd,

11,15

3.51dd,

4 . 15

4.66ddd,

4,8,11

7.91

1 .67

4 . 28

1 .87

0.95

0.99

10. 24

7 . 26

7 .64

7 . 17

7 .60

7 65d,8

3.44dd,

4 . 15

3.16dd.

4 . 15

3.16dd,

10, 15

7 . 82d.6

4 . 39dd.

6.8.5

2 .02

0.97

0.99

7 . 76

1.71

4 . 38

1 .85

0.94

0.99

bs

d. 2 . 2

d.8.8

dd,2.0,8.8

d. 2 .0

d.5

d. 7

TABLE 1. CONTINUED.

P r o t o n 46

pos1t1on

58 57 59 GO 65 66 73 M u l t l p l I c i t y

_iH2J

-NHAc

-OAc

-NHCH=CH

-NHCH=CH

-NHCH=CH

Phenyl H

-CO(NHR)C=CH

-CO(NHR)C=CH

-C0NH2

1.91

2.26 6H

2.27 3H

7 .46

6.14

9.54

7.13 2H

1 .98

2.25 3H

2.27 3H

2.28 9H

7 . 50

6 . 54

9.51

7.17 2H

7 . 15d.

8 . 1H

7 . 21d.

2 , 1H

7 . 23dd,

2.8, 1H

6 . 80

9 . 44

1 .97

2 28 9H

2.25 3H

2.24 9H

7 . 49

6 . 40

9.54

7.4 1 2H

7 . 12d.

8 . 1H

7.21d,

2 . 1H

7 . 17dd.

2.8 1H

7 . 16

9 . 47

1 . 99

2.24 3H

2.28 6H

2.29 3H

7.51

6 . 56

9.46

7.23 2H

7 04d,

9. 1H

7 . 38d.

9 , 1H

6.84

9 . 46

1 .90

2.26 9H

2.27 3H

2.29 3H

2.31 9H

7.59

6.60

9.65

7.43 2H

7.26d,

2 . 1H

7 . 28dd.

2.9.1H

7.47 2H

7.06.7.59

9. 18,9 . 89

1 . 98

2.28 9H

1 .86

2 . 23 6H

7.21 2H 7.42 2H

6.88

9.33

6.51,7.21

7 . 22

9.46

6.54,7.22

1 .88

2.24 6H

2.27 3H

2.29 6H

2 . 30 3H

7.38 2H

7.44 2H

7 . 09 . 7 . 4 1

9. 10,9.54

6.53,7.30

s

dd,10,15

d. 15

d. 10

s

each 1H,s

each 1H,s

' A l l 1H NMR s p e c t r a were r e c o r d e d 1n ( C D 3 ) 2 C O . 'In ppm from I n t e r n a l tetramethy1s11ane 'Assignments were made by e x t e n s i v e

d e c o u p l i n g , D 20 exhange e x p e r i m e n t s . comparison w i t h model compounds and In s e v e r a l Instances e x a m i n a t i o n of e x p a n s i o n s

o b t a i n e d at 400 MHz and computer s i m u l a t e d s p e c t r a ( see appendix 5 ) . 'Derived from 57 or 59.

57

c o n s t i t u e n t s , not amenable to p u r i f i c a t i o n , were suggested

s o l e l y on the b a s i s of JH NMR c o n s i d e r a t i o n s .

M o l e c u l a r ions were not observed i n the EIMS (10-70 eV) of

hexacetylcelenamides A (58, f i g u r e 21) and B (57, f i g u r e 22).

The elemental compositions of C46H48BrNsO 14 and C4sH46BrN 50i4

were i n f e r r e d from JH NMR (58, 43 CH and 5 NH; 57 , 41 CH and 5

NH), 1 3 C NMR (58, 46C, appendix.4), and mass s p e c t r a [SB and 57

d i s p l a y e d the fragment ion C 1 2 H 1 3 N O 4 • f o r C-terminal

d i a c e t o x y s t r y r y l a m i n e (53,64,65, see scheme 3)] i n c o n j u n c t i o n

with the molecular formulas, C 3 i H 3 8 B r N s 0 7 and C 3 0 H 3 6BrNs07, of

t h e i r methylated h y d r o l y s i s products 61_ and 62 determined from

exact mass measurments.

M e O M e O

H H

61 62

The *H NMR spectrum of p e p t i d e 58 ( t a b l e 1) d i s p l a y e d

resonances -at 5 6.52 (d, IH, J=15Hz), 7.50 (dd, IH, J=10, 15Hz),

and 9.50 (d, IH, J=10Hz, exchangable) i n d i c a t i v e of a t r a n s -

F i g u r e 21. EIMS of hexacetylcelenamide A (58).

59

*«o

m/a 818

C 0 H 7 B r

m/a 210/208

j lô 52,74

flCr

m/a ass S£,i* 57,40

C20H22N2O8 . C 2 1 M 2 4 M 2 ° 8 m/a 418 m/a 4*2

2 •

C 4 H * > * m/a 72 m/a> 88

78 *4

*.1t

5#" § f CajH^MaOair

m/a 484/482

C e M t t , » ' C 7 » * 1 4 , | 0 m/a 114 m /a 1 2 8 ~ 38 18

HNAa

C j H f j " ^ • C8**14"°2 in/a 142 m/a 188

8 8

Scheme 3. I n t e r p r e t a t i o n of the EIMS of hexacetylcelenamide A (58) and B (57) a. R=CH 2(CH 3) 2, b. R=CH(CH 3) 2, c. Consecutive l o s s e s of C 2 H 2 O are observed from these ions, d. Exact mass measurement ob t a i n e d on daughter ions only (-C 2H 20), e. Exact mass measurement not obtained, f. Fragment ion i n t e n s i t i e s vary with instrument c o n d i t i o n s , g. Base peak m/e 43, h. Calc d formulas are w i t h i n 0.0015 amu of obsd v a l u e s .

o

61

d i s u b s t i t u t e d enamide (53) as i n £6. The o b s e r v a t i o n of i n t e n s e

fragment i o n s a t m/e 235, 193, and 151 i n the mass spectrum

( f i g u r e 21, scheme 3) s u g g e s t e d t h a t t h e enamide s u b s t i t u e n t was

a d i a c e t o x y p h e n y l group. E v i d e n c e f o r the same r e s i d u e was

ap p a r e n t i n the s p e c t r a l d a t a of p e p t i d e 5_7 ( t a b l e 1, f i g u r e 22,

scheme 3 ) . The most i n t e n s e fragments o b s e r v e d i n the EIMS of

p e p t i d e s and p e p t i d e - r e l a t e d m e t a b o l i t e s (66) a r i s e from the C-

and N - t e r m i n a l r e s i d u e s . O z o n o l y s i s of enamides 5_7 and 5J3 i n

methanol a t -78°C f o l l o w e d by r e d u c t i o n of the o z o n i d e s w i t h

d i m e t h y l s u l f i d e y i e l d e d e q u i m o l a r amounts of 3,4,5-

t r i a c e t o x y b e h z a l d e h y d e (6_3) and 3 , 4 - d i a c e t o x y b e n z a l d e h y d e (64)

p l u s p o l a r p r o d u c t s r e s u l t i n g from t h e remainder of the

m o l e c u l e . The s u b s t i t u t i o n p a t t e r n s about the benzene r i n g s of

t h e s e a l d e h y d e s j*a-s' d e t e r m i n e d by comparison w i t h a u t h e n t i c

samples (see appendix 1 ) . 3 , 4 , 5 - T r i a c e t o x y b e n z a l d e h y d e was

p r e p a r e d from g a l l i c a c i d by a c e t y l a t i o n , f o r m a t i o n of the a c i d

c h l o r i d e , and Rosenmund r e d u c t i o n ( 6 7 ) , w h i l e 3,4-

d i a c e t o x y b e n z a l d e h y d e was o b t a i n e d s i m p l y by a c e t y l a t i n g

c o m m e r c i a l l y a v a i l a b l e 3 , 4 - d i h y d r o x y b e n z a l d e h y d e ( A l d r i c h ) . The

p r o d u c t i o n of 6£ i n t h i s o z o n o l y s i s r e a c t i o n i s c o n s i s t e n t w i t h

t h e p r e s e n c e of ( E ) - l - a m i n o - 2 - ( 3 ' , 4 ' - d i a c e t o x y p h e n y l ) e t h e n e as

the C - t e r m i n a l r e s i d u e of both h e x a c e t y l c e l e n a m i d e A (58) and B

( 5 7 ) .

63 64

62

Treatment of enamides 58 and 57 with 0.1 M HC1, as

d e s c r i b e d f o r 4_6, gave the peptide amides 65 and 66. The r i c h l y

d e t a i l e d *H NMR s p e c t r a of these products ( f i g u r e s 23,24; t a b l e

1) r e v e a l e d a number of s t r u c t u r a l f e a t u r e s . Two peptide a l p h a -

methine protons were observed at 5 4.28 and 4.66 f o r 6_5, and at

S 4.39 and 4.71 f o r 66. Both compounds showed uncoupled o l e f i n i c

s i g n a l s (65, 6 6.88; 66, 5 7.22) and h i g h l y d e s h i e l d e d NH

s i n g l e t s (6_5, 5 9,33; 66, 5 9.46) c h a r a c t e r i s t i c of phenyl-

substi-tuted dehydroamino a c i d r e s i d u e s (68). A t a b l e of

comparative v a l u e s from s y n t h e t i c (see experimental)

dehydroamino a c i d d e r i v a t i v e s i s p r o v i d e d on the f o l l o w i n g page.

A l s o d i s c e r n i b l e i n the XH NMR s p e c t r a were an acetamide

resonance (6_5, 5 1.98; 6_6, <5 1.86) and two primary amide

hydrogens 65, 6 6.51, 7.21; 66, 5 6.54, 7.22), i n d i c a t i n g that

65 and 66 were t r i p e p t i d e s with the N-terminus a c e t y l a t e d and

the C-terminus p r o t e c t e d as a primary amide.

The nature of the two alpha-amino a c i d r e s i d u e s was

A c O

F i g u r e 23 a. 270 MHz 'H NMR spectrum of 65 in acetone-d6 b. Expansion (1000 Hz) of the aromatic region of 65.

F i g u r e 24 a. 270 MHz XH NMR spectrum of 66 in acetone-de b. Expansion (1000 Hz) of the aromatic region of 66.

TABLE 2. SELECTED FEATURES OF THE *H NMR1 SPECTRA OF

SYNTHETIC DEHYDROAMINO ACID DERIVATIVES 2

Compound R 2C=CHAr ArH NH NHAc Deu t e r a t

(s) (2H,s) (S) (3H,s) s o l v e n t

AcATPheNH 24 7.06 3 6.90 9.37 2.00 DMSO

AcATPheGlyOMe 7.19 6.88 8.69 2.12 Acetone

AcATPheValOH 6.99 6.89 9.46 2.01 DMSO

AcATPheLeuOH 6.99 6.87 9.38 2.00 DMSO

AcATPheATPheNEt 2 7.29 6.83 9.77 2.13 Acetone

5.94 (4H,s) 8.97

AcATPheATPheNHtBu 7.32 6.92 9.38 2.21 Acetone

7.03 6.84 8.85

AcATPheATPheOMe 7.23 7.02 9.58 2.02 DMSO

7.15 6.91 9.54

AcATPheATPhe-DMD 5 7.35 6. 94 10.06 2.11 DMSO

(400 MHz) 6.97 6.92 9.65

AcATPheATPhe-DMD 7.37 6.9(m) 9.68 2.23 Acetone

7.00 9.05

l S p e c t r a were r e c o r d e d a t 270 MHz u n l e s s o t h e r w i s e i n d i c a t e d . 2See e x p e r i m e n t a l f o r p r e p a r a t i o n p r o c e d u r e s . 3 A l l c h e m i c a l s h i f t s a r e i n ppm from i n t e r n a l t e t r a m e t h y l s i l a n e . 4ATPhe r e f e r s t o a:,/3"clidehydro- ( 3 , 4 , 5 - t r imethoxy p h e n y l a l a n i n e . SDMD r e f e r s t o O,0-dimethyldopamine.

F i g u r e 25. EIMS of 65.

68

apparent from the s p e c t r a l data of hexacetylcelenamides A (58)

and B (57). An abundant fragment ion at m/e 210/208,

c h a r a c t e r i s t i c of bromoindole d e r i v a t i v e s (54, f i g u r e s 21 and

22) was observed in the mass s p e c t r a , and comparison of UV (Xmax

289 nm) and 1H NMR s p e c t r a ( t a b l e 1) with the corresponding

s p e c t r a of t e t r a c e t y l c l i o n a m i d e (4_6; UV X max 290 nm) c l e a r l y

i n d i c a t e d that t h i s moiety was present as a 6-bromotryptophan

r e s i d u e . Strong ions at m/e 86 (58,34%) and 72 (57,78%) i n the

mass s p e c t r a and s i g n a l s at h i g h f i e l d i n the 'H NMR s p e c t r a

c orresponding to i s o p r o p y l groups (58, 6 0.97, 0.99; 57, 6 0.99,

1.01 (each d, 3H, J=7Hz)) suggested the presence of a l e u c i n e

and v a l i n e r e s i d u e i n 58 and 5_7, r e s p e c t i v e l y . H y d r o l y s i s of

p e p t i d e s 5_8 and 5_7 i n 6N HCL at 100°C f o r 18h a f f o r d e d l e u c i n e

and v a l i n e as expected. When mil d e r h y d r o l y t i c c o n d i t i o n s were

employed an amino a c i d having chromatographic ( c e l l u l o s e , and

s i l i c a 2D-TLC) and n i n h y d r i n c o l o u r i n g p r o p e r t i e s s i m i l a r to

(±)-5-bromotrytophan was a l s o observed. A l l the r e q u i r e d s i g n a l s

fo r the 6-bromotryptophan, l e u c i n e , and v a l i n e r e s i d u e s c o u l d be

found i n the *H NMR s p e c t r a of the h y d r o l y s i s products 65 and 66

( t a b l e 1, see a l s o appendix 7 and 8).

The exact nature of the t h i r d amino a c i d r e s i d u e , known to

be a p h e n y l - s u b s t i t u t e d dehydroamino a c i d from the 1H NMR data

was determined by chemical d e g r a d a t i o n . O z o n o l y s i s of the

primary amides 6_5 and 66 gave 3 , 4 , 5 -triacetoxybenzaldehyde,

i n d i c a t i n g t h at an cx:„<5-didehydro-3 , 4 , 5 - t r i h y d r o x y p h e n y l a l a n i n e

r e s i d u e must be pr e s e n t . T h i s r e s u l t excluded the p o s s i b i l i t y of

69

having an a,/3-didehydro-6-bromotryptophan r e s i d u e . 1 A d d i t i o n a l

evidence f o r the dehydroamino a c i d was p r o v i d e d by the i s o l a t i o n

of o x a l i c a c i d from an a c i d h y d r o l y s a t e of the p o l a r products

(eg. 67) o b t a i n e d from the o z o n o l y s i s of 5_7 and 58. Reduction of

a small sample of a 1:1 mixture of the t r i m e t h y l a t e d h y d r o l y s i s

products _61 and 62_ with sodium borohydride gave in high y i e l d a

mixture of the expected d i h y d r o d e r i v a t i v e s (see e x p e r i m e n t a l ) .

Sodium borohydride r e d u c t i o n s are c h a r a c t e r i s t i c r e a c t i o n s of

dehydroamino a c i d s (69c).

67

We c o u l d conclude from the above data that t r i p e p t i d e 65

c o n t a i n e d 6-bromotryptophan , a , / 3-didehydro-3 , 4 , 5-

t r i h y d r o x y p h e n y l a l a n i n e and l e u c i n e r e s i d u e s , while t r i p e p t i d e

66 c o n t a i n e d 6-bromotryptophan, a , / 3-didehydro-3 , 4 , 5 - t r i h y d r o x y -

p h e n y l a l a n i n e , and v a l i n e r e s i d u e s . Hence, the s t r u c t u r e s of

hexacetylcelenamides A (58) and B (57) must c o n s i s t of

t r i p e p t i d e s 65 and 66 b e a r i n g C-terminal (E)-l-amino-2-(3',4'-

1 a , / 3-Didehydrotryptophan r e s i d u e s are components of s e v e r a l marine (23,24) and t e r r e s t r i a l n a t u r a l products (69d). T y p i c a l UV s p e c t r a l c h a r a c t e r i s t i c s are Xmax 360 nm (e 1 0 s ) .

70

d i a c e t o x y p h e n y l ) e t h e n e r e s i d u e s i n p l a c e of the primary amides.

The m i l d a c i d h y d r o l y s i s of the t r a n s - s u b s t i t u t e d enamide i n 58

and 5_7 generates the primary amide f u n c t i o n a l i t i e s i n 65 and 66

(see a l s o appendix 9 ) .

The amino a c i d sequences of hexacetylcelenamide A (58) and

B (5_7) were deduced from t h e i r mass s p e c t r a ( f i g u r e s 21, 22 and

scheme 3 ) . As mentioned e a r l i e r , i n the mass s p e c t r a of most

p e p t i d e a l k a l o i d s the s t r o n g e s t ions o r i g i n a t e from the N-

t e r m i n a l amino a c i d (66) and inte n s e ions o r i g i n a t e from the C-

t e r m i n a l grouping. Abundant fragment ions f o r 5_8 at m/e 86 (34%)

and f o r 5_7 72 (78%) suggested, t h e r e f o r e , t h a t l e u c i n e and

v a l i n e represented the N-terminal r e s i d u e s of these compounds,

r e s p e c t i v e l y . T h i s was confirmed by the presence of d i a g n o s t i c

ions observed at m/e 128 (15%) and 156 (5%) i n the spectrum of

58 and at m/e 114 (38%) and 142 (9%) i n the spectrum of 57 ,

o r i g i n a t i n g from N - a c e t y l l e u c i n e and N - a c e t y l v a l i n e . These

i n t e r p r e t a t i o n s were s u b s t a n t i a t e d by the mass s p e c t r a of

s e v e r a l model compounds ( t a b l e 3 ) .

58 57

71

TABLE 3. EIMS OF SYNTHETIC MODEL COMPOUNDS

Compound m/e r e l % m/e r e l % m/e r e l % other m/e r e l %

AcValNH 2 72 100 114 67 142 <1 158(M+) 2

43 14

AcLeuNH 2 86 90 128 76 156 2 172(M+) 3

43 100

A c ( 3 , 4 , 5 - t r i - 72 18 114 <1 142 6 207 100

MeO)A PheValOH 1 43 76

A c ( 3 , 4 , 5 - t r i - 86 3 128 1 156 5 340 100

MeO)A PheLeuOH 2 277 50

43 25

^ - A c e t y l - Q i j / S - d i d e h y d r o - (3,4, 5-tr imethoxy ) p h e n y l a l a n y l v a l i ne . JN-Acetyl-a:,/3-didehydro- (3,4, 5-tr imethoxy ) p h e n y l a l a n y l l e u c ine .

72

The 6-bromotryptophan and c ^ ^ - d i d e h y d r o - 3 , 4, 5 - t r i h y d r o x y

p h e n y l a l a n i n e r e s i d u e s were sequenced on the b a s i s of two

d i a g n o s t i c fragment i o n s . Each of t h e s e fragment i o n s l e a d t o a

s e r i e s of daughter i o n s r e s u l t i n g from l o s s e s of C 2 H 2 0 t y p i c a l

of phenol a c e t a t e s . The mass s p e c t r a of b oth 58 and 57 d i s p l a y e d

an m/e 484/482 i o n (scheme 3) r e s u l t i n g from the e l i m i n a t i o n of

NHCOR and H which i s commonly observed i n t r y p t o p h a n - c o n t a i n i n g

m e t a b o l i t e s (53,70). The presence of t h i s c h a r a c t e r i s t i c i o n

s u g g e sted t h a t 6-bromotryptophan was l i n k e d t o the C - t e r m i n a l

s t y r y l a m i n o group. Support f o r t h i s h y p o t h e s i s comes from the

o b s e r v a t i o n of m/e 432 and 418 i o n s i n the s p e c t r a of 5_8 amd 57,

r e s p e c t i v e l y , i n d i c a t i n g t h a t the a ; , / 3-didehydro-3 , 4 , 5-

t r i h y d r o x y p h e n y l a l a n i n e r e s i d u e was l i n k e d t o the N - t e r m i n a l

l e u c i n e and v a l i n e r e s i d u e s . One o t h e r s t r u c t u r e i n which the

r e l a t i v e p o s i t i o n s of the 6-bromotryptophan and dehydroamino

a c i d r e s i d u e a r e r e v e r s e d , c o u l d be c o n s i d e r e d f o r b o t h 5_8 and

57. The i o n s e x p e c t e d from the f r a g m e n t a t i o n of these

h y p o t h e t i c a l s t r u c t u r e s a r e not o b s e r v e d i n the MS of 58 or 57.

Only the s t r u c t u r e s proposed f o r t h e s e two a l k a l o i d s are

c o n s i s t e n t w i t h the d a t a o b t a i n e d .

E l e c t r o n - i m p a c t MS of the n o n v o l a t i l e C l i o n a a l k a l o i d s a r e

i n g e n e r a l d i f f i c u l t t o o b t a i n and d i f f i c u l t t o r e p r o d u c e . The

h y d r o l y s i s p r o d u c t 65_ f a i l e d t o g i v e an i n t e r p r e t a b l e f i e l d -

73

d e s o r p t i o n mass s p e c t r u m . 1

The geometry about the dehydroamino a c i d double bond has

not been d e t e r m i n e d . N u c l e a r Overhauser e f f e c t e x p e r i m e n t s

p r o v e d t o be ambiguous and comparison w i t h s y n t h e t i c model

compounds ( t o be d i s c u s s e d ) d i d not a l l o w us t o draw d e f i n i t i v e

c o n c l u s i o n s .

The i s o l a t i o n of h e x a c e t y l c e l e n a m i d e - d i j A (6jU ( f i g u r e 27)

and h e x a c e t y l c e l e n a m i d e - d i s B (69, f i g u r e 28) from

d e u t e r o a c e t y l a t e d C . c e l a t a e x t r a c t s , as d i s c u s s e d i n c o n n e c t i o n

w i t h t e t r a c e t y l c l i o n a m i d e - d i 2 ( 5 4 ) , l e d t o the c o n c l u s i o n t h a t

c e l e n a m i d e s A (70) and B (7_1) were the n a t u r a l l y o c c u r r i n g

sponge m e t a b o l i t e s .

1 P e p t i d e amide 65 c r y s t a l l i z e d from C H C l 3 _ a c e t o n e as v e r y f i n e n e e d l e s (mp 153-156°C). E l e m e n t a l a n a l y s i s s u g g e s t e d the m o l e c u l a r f o r m u l a C34H38BrNsOio-2/3H20. The a d h e s i o n of s o l v e n t s of c r y s t a l l i z a t i o n t o t h i s m a t e r i a l i s c h a r a c t e r i s t i c of p e p t i d e a l k a l o i d s (64,65) and d e h y d r o p e p t i d e s ( 6 9 ) .

F i g u r e 27. 270 MHz XH NMR spectrum of 68 in a c e t o n e - d 6 . D i s c r e t e s i g n a l s a t t r i b u t a b l e to pentacetylcelenamide C - d i j are i n d i c a t e d by the l e t t e r c.

w

I 4 I I

F i g u r e 28. 270 MHz >H NMR spectrum of 69 in acetone-djj. S i g n a l s a r i s i n g from 6j5 are designated by the l e t t e r a.

76

The *H NMR spectrum of p e n t a c e t y l c e l e n a m i d e C (5_9, f i g u r e

29, t a b l e 1) showed remarkable s i m i l a r i t y t o the spectrum

d i s p l a y e d by h e x a c e t y l c e l e n a m i d e A (5_8). Comparison of the two

s p e c t r a i n d i c a t e d the presence of r e s o n a n c e s c o r r e s p o n d i n g t o a

1 , 4 - d i s u b s t i t u t e d and a 1 , 3 , 4 , 5 - t e t r a s u b s t i t u t e d p h e n y l r i n g i n

t h e spectrum of 59, 1 i n c o n t r a s t t o t h e p r e s e n c e of s i g n a l s

a r i s i n g from 1 , 3 , 4 - t r i s u b s t i t u t e d and 1 , 3 , 4 , 5 - t e t r a s u b s t i t u t e d

p h e n y l r i n g s i n the spectrum of 58. Hence, the s t r u c t u r e

e l u c i d a t i o n of the g r o s s s k e l e t o n of 59 r e q u i r e d o n l y the

p o s i t i o n i n g of the two p h e n o l i c s u b s t i t u e n t s i n o r d e r t o

d i f f e r e n t i a t e between the two p o s s i b l e s t r u c t u r e s shown below:

59

1 O z o n o l y s i s of a m i x t u r e of a c e t y l a t e d c e l e n a m i d e s s i m i l a r i n c o m p o s i t i o n t o t h a t shown i n f i g u r e 15 a f f o r d e d 3,4,5-t r i a c e t o x y , 3 , 4 - d i a c e t o x y and 4 - a c e t o x y b e n z a l d e h y d e as the o n l y i s o l a b l e p r o d u c t s . The l a t t e r was i d e n t i f i e d by comparison w i t h an a u t h e n t i c sample p r e p a r e d by a c e t y l a t i o n of 4-h y d r o x y b e n z a l d e h y d e (Sigma).

F i g u r e 29. 270 MHz *H NMR spectrum of pentacetylcelenamide C (59) in acetone-d6 b. V e r t i c a l expansion (1000 Hz) of the aromatic r e g i o n of 59.

78

T h i s problem was t r i v i a l l y s o l v e d by h y d r o l y s i s of 59 with HCl

i n a c e t o n i t r i l e to give the p e p t i d e amide £5 ( f i g u r e s 30 and 31)

which proved to be i d e n t i c a l ( t a b l e 1) to the p e p t i d e amide

obt a i n e d when 5_8 was t r e a t e d i n a s i m i l a r f a s h i o n . The amino

a c i d sequence of 5_9, which corresponded to that of 5_8, was

a s s i g n e d on the b a s i s of i t s EIMS ( f i g u r e 32 and scheme 4).

Pentacetylcelenamide-d15 C was not separated from

hexacetylcelenamide-d le A (68) but was c l e a r l y v i s i b l e i n the 'H

NMR spectrum of a mixture of the two compounds ( f i g u r e 27). Thus

we c o u l d conclude that celenamide C (7_2) was present i n the

u n d e r i v a t i z e d sponge e x t r a c t .

HO

H

72

I t was immediately apparent from 1H NMR s t u d i e s ( t a b l e 1)

that u n l i k e hexacetylcelenamide A (58), B (57) and

p e n t a c e t y l c e l e n a m i d e C (59), nonacetylcelenamide D (60, f i g u r e

F i g u r e 30 a. 270 MHz JH NMR spectrum of 65 in acetone-cU b. V e r t i c a l expansion c. Expansion (1000 Hz) of the aromatic region of 65.

F i g u r e 31. EIMS of 65.

81

10 w _ 7 m / « 2 1 0 / 2 0 8

XL. X L C g M ^ ^ M ^ M O

m / « 8 6 m / * ia« SO 17

Scheme 4 . I n t e r p r e t a t i o n of the EIMS of pentacetylcelenamide C (59) a. Fragment ion i n t e n s i t i e s vary with instrument c o n d i t i o n s , b. Base peak m/e 4 3 , c.. Consecutive l o s s e s of C 2 H 2 O are observed from these i o n s , d. C a l c d formulas are with 0.00015 amu of obsd v a l u e s .

83

33) d i d not possess 6-bromotryptophan and that l e u c i n e (Of CH ,

5 4.48,m) represented the s o l e alpha-amino a c i d present i n the

molecule. Intense ions at m/e 86 and 128 ( f i g u r e 34, scheme 5)

d e f i n e d N - a c e t y l l e u c i n e as the N-terminal r e s i d u e .

C h a r a c t e r i s t i c resonances at <5 6.60 (d, J=15 Hz), 7.59 (dd,

J=10, 15 Hz), and 9.65 (d,J=10 Hz) i n the JH NMR spectrum of 60

suggested the presence of a C - t e r m i n a l s t y r y l a m i n e r e s i d u e .

Abundant fragment ions at m/e 235, 193 and 151 i n the EIMS

f u r t h e r suggested that the C-terminus was an (E)-l-amino-2-

(3',4'- diacetoxypheny 1)ethene group as i n 57 and 5_8. T h i s was

confirmed by h y d r o l y s i s to the peptide amide 7_3« T n e 1 H N M R (<5

6.53 and 7.30 (each l H , s ) ; f i g u r e 35, t a b l e 1), IR (3640, 3540

cm- 1), and MS ( f i g u r e 36) of 21 i n d i c a t e d that the C-terminal

s t y r y l a m i n e r e s i d u e of 60 was absent in 7_3 and that the

A c O

O A c

60

w

' * - • • I I I .... I • ... I .... I .... I • . . . I .... I .... I . . . I I .... I . . . 1 1 . . . . I .... I 1 . . . . I . . .

1 0 9 8 7 6 5 4 3 2 1 ppm( c f )

F i g u r e 33 a. 270 MHz XH NMR spectrum of nonacetylcelenamide D (60) i n acetone-d6 b. Expansion (1000 Hz) of the aromatic region of 60 c. P a r t i a l expansion (1000 Hz) of the aromatic region of a f u r t h e r p u r i f i e d sample of 60.

F i g u r e 34. EIMS of nonacetylcelenamide D (60).

m/«ae m/jiaa m/. 166

Scheme 5. I n t e r p r e t a t i o n of the EIMS of nonacetylcelenamide D ^ (60) a. Fragment ion i n t e n s i t i e s vary with instrument c o n d i t i o n s , b. Base peak m/e 43, c. Consecutive l o s s e s of C 2H 20 are observed from these ions, d. Calcd formulas are with i n 0.00015 amu of obsd v a l u e s .

F i g u r e 35 a. 270 MHz *H NMR spectrum of JA i n acetone-d_6 b. Expansion (1000 Hz) of the aromatic region of 7_3.

89

h y d r o l y s i s r e a c t i o n had c h a r a c t e r i s t i c a l l y a f f o r d e d a C-

t e r m i n a l p r i m a r y amide.

The r e m a i n i n g s t r u c t u r a l f e a t u r e s of n o n a c e t y l c e l e n a m i d e D

(60) were e l u c i d a t e d by 1 H NMR ( t a b l e 1 ) . Two u n c o u p l e d o l e f i n i c

s i g n a l s a t 5 7.06 and 7.59, two h i g h l y d e s h i e l d e d NH s i n g l e t s a t

5 9.18 and 9.89, and two 2H a r o m a t i c s i n g l e t s a t <5 7.43 and 7.47

c l e a r l y i n d i c a t e d the p r e s e n c e of two a,/?-didehydro-3,4,5-

t r i a c e t o x y p h e n y l a l a n i n e r e s i d u e s . The o b s e r v a t i o n of fragment

i o n s a t m/e 638, 432 and 319 ( f i g u r e 34) i n the EIMS of 60

f u r n i s h e d c o n c l u s i v e e v i d e n c e f o r t h i s p r o p o s a l .

N o n a c e t y l c e l e n a m i d e - d _ 2 7 D was not i s o l a t e d . However, as

p r e v i o u s l y mentioned, *H NMR a n a l y s i s of the PTLC p u r i f i e d

d e u t e r o a c e t y l a t e d celenamide m i x t u r e d i d n-ot d i s p l a y any

resonances c o r r e s p o n d i n g t o phenol a c e t a t e s or a c e t a m i d e s . T h i s

90

r e s u l t , i n c o n j u n c t i o n w i t h absence of t h e s e s i g n a l s i n a l l of

the p a r t i a l l y p u r i f i e d d e u t e r o a c e t y l a t e d c e l e n a m i d e s (68,69) and

t e t r a c e t y l c l i o n a m i d e - d i 2 (5_4) l e d t o the a s s u m p t i o n t h a t

c e l e n a m i d e D (7£) was a n a t u r a l l y o c c u r r i n g sponge m e t a b o l i t e .

HO

74

S u b s t a n t i a l e f f o r t has been expended i n a t t e m p t i n g t o

p u r i f y the l e a s t p o l a r a c e t y l a t e d c e l e n a m i d e s . The RP-HPLC of

t h i s complex m i x t u r e (below: see f i g u r e 15 f o r g e n e r a l

c o n d i t i o n s ) c l e a r l y i n d i c a t e s the n a t u r e of the p u r i f i c a t i o n

i — ' — i — " — i — " — i — i — i — i — i — i — i i i

0 2 4 6 8 10 min

91

S e v e r a l s u c c e s s i v e PTLC s t e p s , each i n v o l v i n g m u l t i p l e s o l v e n t

development, and RP-TLC have been employed. T h i s c o m b i n a t i o n of

p r o c e d u r e s o c c a s i o n a l l y g e n e r a t e d m a t e r i a l which was

s u b s t a n t i a l l y pure by HPLC c r i t e r i a . However, X H NMR i n d i c a t e d

t h a t i n each case a m i x t u r e of 2-4 compounds remained. Enhancing

t h i s problem was the g r a d u a l d e t e r i o r a t i o n of th e s e s u b s t a n c e s

when h a n d l e d e x c e s s i v e l y , the s m a l l q u a n t i t i e s p r e s e n t i n the

sponge e x t r a c t s , and the s l i g h t v a r i a t i o n i n the c o m p o s i t i o n of

the minor c o n s t i t u e n t s of C. c e l a t a from c o l l e c t i o n t o

c o l l e c t i o n .

Owing t o our i n a b i l i t y t o p r o c u r e t h e s e compounds i n a

homogeneous s t a t e o n l y a b r i e f o v e r v i e w of t h e g e n e r a l f e a t u r e s

d i s c e r n i b l e from the lH NMR s p e c t r a of m i x t u r e s w i l l be

p r e s e n t e d .

S e v e r a l of the minor c o n s t i t u e n t s bear c l o s e resemblance t o

h e x a c e t y l c e l e n a m i d e s A (58_) and B (5_7), p e n t a c e t y l c e l e n a m i d e C

( 5 9 ) , and n o n a c e t y l c e l e n a m i d e D ( 6 0 ) . The p r i n c i p a l s i t e of

v a r i a t i o n a ppears t o be the a l i p h a t i c amino a c i d r e s i d u e . Amino

a c i d a n a l y s i s of the h y d r o l y z e d (6NHC1, r e f l u x ) a c e t y l a t e d

c e l e n a m i d e m i x t u r e d e m o n s t r a t e d the p r e s e n c e of a v a r i e t y of

common alpha-amino a c i d s , i n c l u d i n g : t h r e o n i n e , s e r i n e , g l y c i n e ,

v a l i n e , i s o l e u c i n e and l e u c i n e .

Two c l a s s e s of n o v e l m e t a b o l i t e s have been d e t e c t e d . From

one c o l l e c t i o n what i s p o s s i b l y a m o d i f i e d t e t r a p e p t i d e , s i m i l a r

t o 5_8 but c o n t a i n i n g one unique r e s i d u e , was o b t a i n e d .

U n f o r t u n a t e l y , t h i s compound decomposed d u r i n g r o u t i n e s p e c t r a l

e x a m i n a t i o n and has not been e n c o u n t e r e d i n subsequent

92

c o l l e c t i o n s . The second group of novel compounds s t u d i e d are

d i s t i n c t i n t h a t , u n l i k e 57-60, they appear to l a c k a t y p i c a l N-

t e r m i n a l acetamide grouping (ca. 2.10-1.80 ppm) (eg. f i g u r e s 37

and 38).

F i g u r e 37. A l i p h a t i c r e gion of the 270 MHz *H NMR spectrum of a p a r t i a l l y p u r i f i e d sample of unknown a c e t y l a t e d celenamides in acetone-d_6.

T h i s o b s e r v a t i o n can be e x p l a i n e d by three s t r u c t u r a l

p o s s i b i l i t i e s : an N-terminal dehydroamino a c i d (cf. t a b l e 2); a

c y c l i c molecule or a c y c l i c N-terminal r e s i d u e ; or an N-

a l k y l a t e d N-terminal r e s i d u e . S t r u c t u r a l c o n c l u s i o n s c o u l d not

be drawn from the 'H NMR s p e c t r a alone and the m a t e r i a l s

93

r e s p o n s i b l e f o r the 1H NMR spectrum shown i n f i g u r e 40 d i d not

g i v e an i n t e r p r e t a b l e EIMS. A n a l y s i s of t h i s m a t e r i a l by 1 3 C NMR

(appendix 10) d i d not a i d the s t r u c t u r e assignment due t o the

s m a l l amount of m a t e r i a l a v a i l a b l e and the h i g h m o l e c u l a r

w e i g h t . I t i s c l e a r from the i n f o r m a t i o n o b t a i n e d t h u s f a r t h a t

t h e s e m o l e c u l e s a l l p o s s e s s a 6-bromotryptophan r e s i d u e and a C-

t e r m i n a l t r _ a n s - d i s u b s t i t u t e d enamide m o i e t y . The n a t u r e of the

r e s t of the s t r u c t u r a l f e a t u r e s of t h e s e d i s t i n c t m o l e c u l e s

remains t o be d e t e r m i n e d .

1 . 1 ' 1 ' — - 1 ' -I 10 8 6 4 2 ppm ( S )

F i g u r e 38. 270 MHz 'H NMR spectrum of a p a r t i a l l y p u r i f i e d unknown pept i d e a l k a l o i d i n acetone-dg.

95

V I I I . D i s c u s s i o n

C a r l e and C h r i s t o p h e r s e n (20) r e c e n t l y s t a t e d t h a t "the

l i t e r a t u r e on marine a l k a l o i d s i s v e r y s c a r c e ; moreover, the few

b r o m o s u b s t i t u t e d members of t h i s c l a s s seem t o bear no

resemblance t o p a t t e r n s e s t a b l i s h e d from t e r r e s t r i a l s o u r c e s " .

Celenamides A (7_0) , B (7J_) and C (72) p o s s e s s bromine, and t h e i r

s t r u c t u r e s , as w e l l as t h a t of c e l e n a m i d e D (7_4) , c l o s e l y

p a r a l l e l those of the p e p t i d e a l k a l o i d s i s o l a t e d from s e v e r a l

t e r r e s t r i a l p l a n t f a m i l i e s (64,65) (eg. Rhamnaceae). The

s i m i l a r i t y i s p a r t i c u l a r l y apparent when cele n a m i d e B (7_1_) and

i n t e g e r r i n (75, 71) a r e compared. L a s i o d i n e A (7_6, 72) i s the

o n l y o t h e r example of an a c y c l i c p e p t i d e a l k a l o i d .

76

96

The b i o g e n e s i s of the p l a n t p e p t i d e a l k a l o i d s has not been

i n v e s t i g a t e d to any s i g n i f i c a n t e x t e n t . Tschesche and Kaussmann

(65a) suggest that the lone l i n e a r example, l a s i o d i n e A (76),

might a r i s e from secondary opening of a c y c l i c p r e c u r s o r .

A l t e r n a t i v e l y , the c y c l o p e p t i d e a l k a l o i d s may a r i s e v i a 1,4-

a d d i t i o n of the C-terminal p h e n o l i c r e s i d u e of a l i n e a r

p r e c u r s o r onto an a p p r o p r i a t e l y s i t u a t e d c^^-didehydroamino a c i d

(65b). The s t y r y l a m i n e r e s i d u e present i n the marine and

t e r r e s t r i a l peptide a l k a l o i d s i s unique to t h i s f a m i l y of

n a t u r a l products. L a s i o d i n e A (76) and a l l of the t e r r e s t r i a l

m e t a b o l i t e s possess t h i s grouping in a c i s o i d arrangement i n

c o n t r a s t to the t r a n s o i d c o n f i g u r a t i o n found in c l i o n a m i d e (53)

and the celenamides ( 7_0 , l_\_,l_2,ljk) . No evidence of i somer i z a t ion

to the thermodynamically l e s s s t a b l e c i s c o n f i g u r a t i o n or

subsequent c y c l i z a t i o n to the p h e n c y c l o p e p t i n e 1 nucleus has been

encountered i n the l a t t e r . The p o s s i b l i t y e x i s t s that the

n a t u r a l l y o c c u r r i n g sponge m e t a b o l i t e s possess c i s o i d

s t y r y l a m i n e r e s i d u e s and that the t r a n s o i d c o n f i g u r a t i o n s

observed are a r t i f a c t s . We f e e l that t h i s i s u n l i k e l y due to the

f a c t t h at the harsh acid-base e x t r a c t i o n procedure used to

i s o l a t e l a s i o d i n e A d i d not r e s u l t i n i s o m e r i z a t i o n and that i n

comparison, the procedure u t i l i z e d i n o b t a i n i n g the C l i o n a

a l k a l o i d s i s extremely m i l d . The o x i d a t i v e enzymes which give

r i s e to the s t y r y l a m i n e moiety i n the p l a n t s and i n the sponge

1Rapoport (73) has proposed the nomenclature "phencyclopeptines", to d e s c r i b e the 14-membered r i n g nucleus common to many peptide a l k a l o i d s .

97

may have d i f f e r e n t s t e r e o c h e m i c a l s p e c i f i c i t i e s .

I n v e s t i g a t i o n of the b i o s y n t h e t i c o r i g i n of the novel

s t r u c t u r a l f e a t u r e s of the C l i o n a m e t a b o l i t e s has not been

attempted f o r obvious l o g i s t i c a l reasons. However, recent

success by Dr. Pomponi in c u l t i v a t i n g C. c e l a t a c e l l s i n

suspension should f a c i l i t a t e the performance of b i o g e n e s i s

experiments at a l a t e r date.

C o n s i d e r a t i o n of the b i o g e n e t i c o r i g i n of a m e t a b o l i t e i s a

c h a l l e n g i n g i n t e l l e c t u a l process and can f r e q u e n t l y be an

important a i d to s t r u c t u r e d e t e r m i n a t i o n . However, in the

absence of experimental evidence over emphasis should not be

given to s p e c u l a t i v e schemes. A p l a u s i b l e b i o s y n t h e t i c route to

the C-terminal s t y r y l a m i n e r e s i d u e of p e p t i d e a l k a l o i d s i s

o u t l i n e d i n scheme 6. The pathway shown i s based upon the w e l l -

e s t a b l i s h e d b i o s y n t h e s i s of the catecholamines (74) and the

peyote a l k a l o i d s (75). These are summarized i n scheme 7. The

catecholamines occur widely throughout the animal and p l a n t

kingdoms and are a l s o known from microorganisms (74). T h e i r

mechanism of formation in i n s e c t s i s analogous to that i n

v e r t e b r a t e s (74). The p l a u s i b i l i t y of the b i o s y n t h e t i c pathway

d i s p l a y e d i n scheme 6 i s s u b s t a n t i a t e d by the n a t u r a l occurrence

of pandamine (77.' 65) and pandaminine (28, 65).

jS-Hydroxytyramine

x HN—(

Tyr

Tyramine^ .^5-Hydroxy dopamine dopamine ^ > 5-Hydroxynorepinephrine » Hi

Norepinephrine ^

1 ii

<Tyramine

Dopa ^

R=R t=H ii R=OH,R!=H

iii R=R1=OH

I

Scheme 6. P o s s i b l e b i o s y n t h e t i c r o u t e s t o C - t e r m i n a l s t y r y l a m i n e s .

A . L-Phe •> L < L-Dopa S 5-Hydroxydopamine

Dopamine < ^ ^>~* Mescaline Tyramine ^ 3-Methoxytyramine'

B. L-Tyr •» L-Dopa * Dopamine * L-Norepinephrine * L-Epinephrine

Scheme 7. A. B i o s y n t h e s i s of M e s c a l i n e (75). B. B i o s y n t h e s i s of L - e p i n e p h r i n e (74).

100

The t e r r e s t r i a l and marine p e p t i d e a l k a l o i d s have s e v e r a l

f e a t u r e s i n common i n a d d i t i o n to t h e i r C - t e r m i n a l s t y r y l a m i n e

r e s i d u e s . A l k a l o i d s from e i t h e r source e x i s t as complex

mixtures. The p r i n c i p a l s i t e of v a r i a t i o n i n these molecules i s

the nature of the s i d e chains of the a l i p h a t i c amino a c i d s . The

b a s i c i t y of p e p t i d e a l k a l o i d s i s a t t r i b u t a b l e to t h e i r N-

t e r m i n a l cxramino group. A l l of the C l i o n a a l k a l o i d s i s o l a t e d i n

a pure s t a t e possess a f r e e amino terminus, as do a number of

t e r r e s t r i a l p e p t i d e a l k a l o i d s (65). Most of the p l a n t

c y c l o p e p t i d e a l k a l o i d s are s t r o n g l y l e v o r o t a t o r y (-200° to

400°, 65). The o p t i c a l r o t a t i o n s of hexacetylcelenamide A (58, +

40°), hexacetylcelenamide B (57, +22°), and p e n t a c e t y l c e l e n a m i d e

C (5_9, +14°) are d e x t r o r o t a t o r y and of s i m i l a r magnitude to

l a s i o d i n e A (7_6, + 38°). Most s y n t h e t i c d i - and t r i p e p t i d e s

composed of L-amino a c i d s and one aromatic dehydroamino a c i d are

d e x t r o r o t a t o r y (75,76). Nonacetylcelenamide D (60, - 25°) i s

l e v o r o t a t o r y , a c h a r a c t e r i s t i c of p e p t i d e s c o n t a i n i n g L-amino

a c i d s other than p r o l i n e and two aromatic dehydroamino a c i d

r e s i d u e s (75,76). U n f o r t u n a t e l y , these g e n e r a l i z a t i o n s cannot be

used to p r e d i c t the a b s o l u t e s t e r e o c h e m i s t r y of the tV-amino

a c i d s of 5_7, 5_8, 5_9 and 6̂0 as a r e s u l t of the d e x t r o r o t a t i o n of

l a s i o d i n e A {IS). L a s i o d i n e A i s the only p e p t i d e a l k a l o i d known

to possess an amino a c i d having the D c o n f i g u r a t i o n .

The bromotryptophan r e s i d u e present i n c l i o n a m i d e (53) has

been shown to possess the L c o n f i g u r a t i o n . Since t h i s r e s i d u e i s

destroyed d u r i n g a c i d h y d r o l y s i s , the a b s o l u t e s t e r e o c h e m i s t r y

of the two c h i r a l carbon atoms present i n hexacetylcelenamide A

101

( 5 8 ) , B (57) and p e n t a c e t y l c e l e n a m i d e C (59) has not been

d e t e r m i n e d . The s y n t h e t i c s t u d i e s d e s c r i b e d i n a subsequent

c h a p t e r were d e s i g n e d t o r e s o l v e t h i s problem.

S e v e r a l n a t u r a l p r o d u c t s c o n t a i n i n g bromoindole r e s i d u e s

have been i s o l a t e d from marine p l a n t s and a n i m a l s (80-89, see

a l s o _H, V2, _1_5, J_6, p. 9,10), as have a number of c l o s e l y

r e l a t e d compounds (9-_n_, p. 8; 2_9) • T ^ e monobrominated

m e t a b o l i t e s , w i t h one e x c e p t i o n (_1_2) , p o s s e s s a halogen atom i n

the 6 - p o s i t i o n . The C l i o n a m e t a b o l i t e s 53, 70, 7J_ and 72 conform

t o t h i s g e n e r a l p a t t e r n . A s p e c i f i c h a l o p e r o x i d a s e may be a c t i v e

i n the organisms from which t h e s e compounds were o b t a i n e d .

The s p e c i f i c i t y of the enzyme systems r e s p o n s i b l e f o r the

p r o d u c t i o n of c l i o n a m i d e and the c e l e n a m i d e s would n o t , a t f i r s t

g l a n c e , appear t o be p a r t i c u l a r l y r i g i d . On c l o s e r e x a m i n a t i o n

c e r t a i n c o n s t a n c i e s are a p p a r e n t . A l l of the c e l e n a m i d e s

p o s s e s s , as p a r t of the C - t e r m i n a l t r a n s - d i s u b s t i t u e d enamide,

a p h e n y l r i n g which i s l e s s o x i d i z e d than the c o r r e s p o n d i n g

group of c l i o n a m i d e ( 5 3 ) . O x i d a t i o n of t h i s r e s i d u e t o the

t r i h y d r o x y s t a t e may i n h i b i t f u r t h e r e l o n g a t i o n of the p e p t i d e

c h a i n , or c o n v e r s e l y , o x i d a t i o n t o t h i s s t a t e may not be

p o s s i b l e i n the l o n g e r c h a i n . A l s o , w i t h the e x c e p t i o n of

c elenamide D (14), a l l of the C l i o n a m e t a b o l i t e s bear a 6-

bromotryptophan r e s i d u e i n the p e n u l t i m a t e C - t e r m i n a l p o s i t i o n .

102

"2

R R, (77) R R1 R 2 (78) yg c i H 82 H Br Me

80 Br H 8 3

81 C . B r 8 4 B r B r H

85 Br Br Me Hemichordata

Rhodophyta

II I H

R (79)

86 Me

87 H

Porifera

H R

Me

c o 2

88 ( r o )

Porifera

89 ( s o )

Mollusca

103

Ribosomal p r o t e i n and p e p t i d e s y n t h e s i s i s i n i t i a t e d a t the N-

t e r m i n a l r e s i d u e , however, i t i s i n t r i g u i n g t o note t h a t the

maximum c o n s t a n c y of the C l i o n a a l k a l o i d s o c c u r s a t , and

a d j a c e n t t o , the C-t e r m i n u s . T h i s o b s e r v a t i o n l e a d s t o the

s u g g e s t i o n t h a t the sponge a l k a l o i d s may be b i o s y n t h e s i z e d

i n d e p e n d e n t l y of the normal R N A - c o n t r o l l e d p e p t i d e s y n t h e s i s as

i s the case f o r c y c l i c p e p t i d e a n t i b i o t i c s ( 8 1 ) .

oc^- D i d e h y d r o a m i n o a c i d s have been s t u d i e d e x t e n s i v e l y

owing t o t h e i r o c c u r r e n c e as c o n s t i t u e n t s of many b i o l o g i c a l l y

a c t i v e - m i c r o b i a l p e p t i d e s , the m a j o r i t y of which a r e c y c l i c .

o/^/3-Didehydrophenylalanine has been e n c o u n t e r e d i n the c y c l i c

p e p t i d e t e n t o x i n (82) and a m o d i f i e d o ^ / J - d i d e h y d r o t y r o s i n e

r e s i d u e i s p r e s e n t i n mycelia n a m i d e ( 8 3 ) . D e r i v a t i v e s b e a r i n g

h i g h e r degrees of s u b s t i t u t i o n on the ph e n y l r i n g have not been

p r e v i o u s l y r e p o r t e d . 3 , 5 - D i s u b s t i t u t e d t y r o s i n e - d e r i v e d r e s i d u e s

a r e components of numerous marine (eg 3_3, 43) and t e r r e s t r i a l

n a t u r a l p r o d u c t s . To d a t e , we have been unable t o l o c a t e a

p u b l i s h e d a r t i c l e r e p o r t i n g the o c c u r r e n c e of 3,4,5-

t r i h y d r o x y p h e n y l a l a n i n e i n n a t u r e ( 8 4 ) . 1 An amino a c i d b e a r i n g

t h i s s u b s t i t u t i o n p a t t e r n i s not i n v o l v e d i n the b i o s y n t h e s i s of

m e s c a l i n e (75, see scheme 7 ) . A d e t a i l e d s e a r c h f o r t h i s amino

a c i d and f o r L-dopa i n the m e s c a l i n e s y n t h e s i z i n g c a c t u s ,

Lophophora w i l l i a m s i i f a i l e d t o demonstrate t h e i r p r e s e n c e ( 7 5 ) .

The b i o s y n t h e s i s of dehydroamino a c i d s and d e h y d r o p e p t i d e s

1 3 , 4 , 5 - T r i m e t h o x y p h e n y l a l a n i n e has been s y n t h e s i z e d by Acheson e t a l . ( 8 5 ) .

104

has r e c e n t l y been c o m p r e h e n s i v e l y r e v i e w e d by Schmidt e t a l .

( 8 1 ) . Three of t h e s e v e r a l h y p o t h e t i c a l r o u t e s t o the f o r m a t i o n

of a , / 3 _ d i d e h y d r o - 3 , 4 , 5 - t r i h y d r o x y p h e n y l a l a n i n e a r e g i v e n i n

scheme 8. The c o n v e r s i o n of dopa t o 5-hydroxydopa ( i n d i c a t e d by

an a s t e r i s k ) i s u n p r e c e d e n t e d .

D e h y d r o p e p t i d e s have p r e v i o u s l y been c o n s i d e r e d t o o c c u r

p r i m a r i l y i n m i c r o o r g a n i s m s , e s p e c i a l l y f u n g i . 1 Sponges

f r e q u e n t l y h o s t a v a r i e t y of m i c r o o r g a n i s m s , p a r t i c u l a r l y

b a c t e r i a , f u n g i , and m i c r o s c o p i c a l g a e . In some i n s t a n c e s t h e s e

a l g a e e x i s t i n a s y m b i o t i c a s s o c i a t i o n w i t h the sponge and a r e

r e f e r r e d t o a s z o o x a n t h e l l a e . Z o o x a n t h e l l a e have been r e p o r t e d i n

s e v e r a l c l i o n i d s (86), i n c l u d i n g C. c e l a t a c o l l e c t e d o f f the

c o a s t of B r i t i s h C o l u m b i a . 2 The o r i g i n of the C l i o n a m e t a b o l i t e s

must be c o n s i d e r e d u n c e r t a i n a t t h i s t i m e .

S o l u t i o n of the remainder of the C l i o n a a l k a l o i d s w i l l

r e q u i r e v e r y c a r e f u l and t e d i o u s l y r e p e t i t i o u s f r a c t i o n a t i o n by

RP-HPLC. The knowledge g a i n e d from the s t r u c t u r a l e l u c i d a t i o n of

the s e a p p a r e n t l y unique compounds w i l l u n d o u b t e d l y j u s t i f y the

e f f o r t r e q u i r e d t o i s o l a t e them.

D u r i n g any n a t u r a l - p r o d u c t s i n v e s t i g a t i o n q u e s t i o n s

i n v a r i a b l y a r i s e c o n c e r n i n g the o r i g i n of the s u b s t a n c e s

i s o l a t e d , t h e i r f u n c t i o n i n t h e organisms p h y s i o l o g y , and t h e i r

d i s t r i b u t i o n i n the s p e c i e s and f a m i l y b e i n g s t u d i e d .

1One e x c e p t i o n i s l a s i o d i n e A ( 7 6 , 7 2 ) . 2 S . A. Pomponi, p e r s o n a l communication.

Scheme 8. Three p o s s i b l e b i o s y n t h e t i c r o u t e s t o ctf , j8-didehydro -3 , 4 , 5 - t r i h y d r o x y p h e n y l a l a n i n e - c o n t a i n i n g p e p t i d e s . R and R' r e p r e s e n t amino a c i d r e s i d u e s , a l t h o u g h , s e v e r a l of the s t e p s shown may o c c u r a t the f r e e amino a c i d l e v e l .

1 0 6

F r e q u e n t l y , e x p e r i m e n t s aimed a t a n s w e r i n g th e s e q u e s t i o n s

p r o v i d e o n l y ambiguous r e s u l t s . However, the s e a r c h f o r the s e

answers s h o u l d be a prime c o n c e r n of a l l n a t u r a l - p r o d u c t s

c h e m i s t s as t h i s i s one of our p r i n c i p a l means of c o n t r i b u t i n g

t o man's i n c r e a s e d u n d e r s t a n d i n g of n a t u r e .

T h i s c h a p t e r has o u t l i n e d i n d e t a i l the f i r s t s t a g e

i n v o l v e d i n n a t u r a l - p r o d u c t s r e s e a r c h , namely the i s o l a t i o n and

s t r u c t u r e d e t e r m i n a t i o n of the m e t a b o l i t e s of i n t e r e s t .

The f o l l o w i n g c h a p t e r p r e s e n t s our p r e l i m i n a r y e f f o r t s a t

u n r a v e l l i n g some of the m y s t e r i e s a s s o c i a t e d w i t h the u b i q u i t o u s

b u r r o w i n g sponge C l i o n a c e l a t a G r a n t .

107

CHEMICAL ECOLOGY OF CLIONA CELATA

I . B i o l o g y of the C l i o n i d a e

The marine sponge f a m i l y C l i o n i d a e , c o n s i s t i n g of about 100

d e s c r i b e d s p e c i e s , i s c l a s s i f i e d as f o l l o w s (87):

Phylum : P o r i f e r a

C l a s s : Demospongiae

Order : Hadromerida

Family : C l i o n i d a e (Gray).

The genus C l i o n a with i t s 65 members i s the most abundant

of the 13 C l i o n i d a e genera. C l i o n i d s are p r e v a l e n t i n most of

the world's oceans but are uncommon i n a r c t i c waters. Specimens

have been encountered from Alaska and B a f f i n I s l a n d to C h i l e and

B r a z i l . C l i o n a s p e c i e s commonly occur in England, the

Mediterranean, Japan, and throughout the west c e n t r a l P a c i f i c

(88). Owing to t h e i r d i s t i n c t i v e y ellow or yellow-orange

c o l o u r a t i o n (see f i g u r e 1), s p e c i e s of the genus C l i o n a are

o f t e n r e f e r r e d to as 'sulphur sponges'. However, a few green (eg.

C. v i r i d u s ) or p u r p l e (eg. C.schmidt i i ) s p e c i e s e x i s t . The

C l i o n i d a e are i d e n t i f i e d by the nature of t h e i r s p i c u l e s and the

m o r p h o l o g i c a l f e a t u r e s of t h e i r e x c a v a t i o n s i n t o submerged

c a l c a r e o u s m a t e r i a l s .

The C l i o n i d a e have three r e c o g n i z a b l e m u l t i c e l l u l a r l i f e

stages (89): the alpha stage, i n which the organism i s almost

completely e n d o l i t h i c and i s exposed to the environment only by

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way of i n c u r r e n t and e x c u r r e n t p a p i l l a e ( e g . f i g u r e 1 ) ; the be t a

s t a g e , i n which the sponge becomes e x o l i t h i c as w e l l as

e n d o l i t h i c and the gamma or " f r e e - l i v i n g " s t a g e . The presence of

the o r i g i n a l h o s t i s not d e t e c t a b l e i n the l a t t e r s t a g e and the

sponge no l o n g e r engages i n b u r r o w i n g . E x t r e m e l y l a r g e specimens

of gamma-stage c l i o n i d s have been e n c o u n t e r e d ( e g . C . c e l a t a ).

The sponge of p r i n c i p a l i n t e r e s t i n our i n v e s t i g a t i o n ,

C l i o n a c e l a t a G r a n t 1 , i s perhaps the most w i d e s p r e a d and

c o n s p i c u o u s of the c o s m o p o l i t a n C l i o n i d a e . 2 I t i s one of the

most common marine i n v e r t e b r a t e s i n the w a t e r s of the c o a s t of

B r i t i s h Columbia and i s f r e q u e n t l y found b u r r o w i n g i n t o e i t h e r

the s h e l l s of the g i a n t b a r n a c l e , Balanus n u b i l i s , and the red

rock s c a l l o p , H i n n i t e s m u l t i r u g o s u s , or i n t o c a l c i u m c a r b o n a t e

b e a r i n g r o c k s . The be t a s t a g e i s the predominant form of C

c e l a t a t h r o u g h o u t Howe Sound a l t h o u g h gamma-stage sponge i s

o c c a s i o n a l l y e n c o u n t e r e d . B a r k l e y Sound, w i t h i t s abundance of

s h e l l f i s h , p r e d o m i n a n t l y h o s t s a l p h a - s t a g e sponge.

The b u r r o w i n g b e h a v i o u r of C l i o n a has been most w i d e l y

documented i n r e g a r d t o i t s d e s t r u c t i v e i n f l u e n c e on r e e f c o r a l s

(91) and commercial s h e l l f i s h ( 9 2 ) . The honeycombed s k e l e t o n of

a rock s c a l l o p which has been i n f e s t e d by C . c e l a t a i s shown i n

f i g u r e 39.

1 C. c e l a t a was i d e n t i f i e d on the b a s i s of morphology, c o l o u r , and m i c r o s c o p i c s p i c u l e e x a m i n a t i o n ( 9 0 ) . The s p i c u l e s a r e s l i g h t l y c u r v e d t y l o s t y l e s w i t h t e r m i n a l heads.

JTwo o t h e r s p e c i e s of C l i o n a a r e b e l i e v e d t o r e s i d e i n B.C. w a t e r s . B. A u s t i n , p e r s o n a l communication.

109

The c h i p s produced d u r i n g C l i o n a b o r i n g a r e an i m p o r t a n t

component of b i o g e n i c c a r b o n a t e sediments (91 , 9 7 ) . In some

s h a l l o w water e n v i r o n m e n t s th e s e p a r t i c l e s c o n s t i t u t e up t o 30

p e r c e n t of the t o t a l sediment ( 9 7 ) .

F i g u r e 39. Remains of an H . m u l t i r u g o s u s s h e l l i n u n d a t e d by C. c e l a t a . Arrow (upper l e f t c o r n e r ! i n d i c a t e s a sponge p a p i l l a e opening on the upper s h e l l s u r f a c e ( m a g n i f i e d 3X).

Rock and s h e l l b o r i n g by marine organisms i s a common

phenomenon ( 9 3 ) . The b i o l o g i c a l advantage o b t a i n e d as a r e s u l t

of t h i s b e h a v i o u r i s most c e r t a i n l y p r o t e c t i o n and i s r a r e l y

a s s o c i a t e d w i t h f e e d i n g . T h i s i s e s p e c i a l l y t r u e of b u r r o w i n g

sponges which a r e thought t o f e e d e x c l u s i v e l y on u n i c e l l u l a r

a l g a e , b a c t e r i a , and o r g a n i c d e t r i t u s .

C l i o n i d b u r r o w i n g has been a s u b j e c t of s c i e n t i f i c

a t t e n t i o n f o r over 100 y e a r s . The c u r r e n t l y a c c e p t e d mechanism

1 10

of s u b s t r a t e p e n e t r a t i o n i s o u t l i n e d i n f i g u r e 40. The e t c h i n g

c e l l (a s p e c i a l i z e d amoebocyte) i s b e l i e v e d t o s e c r e t e a

s u b s t a n c e which f a c i l i t a t e s C aC0 3 d i s s o l u t i o n . A c i d s (eg.

c a r b o n i c ) , enzymes (eg- c a r b o n i c a n h y d r a s e ) , and o r g a n i c

c h e l a t o r s have been v a r i o u s l y s u g g ested t o be the r e s p o n s i b l e

a g e n t s ( 9 4 ) . Copious q u a n t i t i e s of a c i d s a r e not s e c r e t e d (95)

by c l i o n i d sponges. Aqueous e x t r a c t s of B.C. C. c e l a t a a r e

n e u t r a l . The s i l i c e o u s s p i c u l e s a r e not i n v o l v e d i n the

b u r r o w i n g p r o c e s s ( 9 5 ) , however, Yonge (93) has proposed t h a t

growth p r o c e s s e s may a s s i s t . R u t z l e r and R i e g e r (96) have

e s t i m a t e d t h a t 2-3 p e r c e n t of the e x c a v a t e d CaCC>3 i s removed by

s o l u t i o n . T h i s e s t i m a t i o n has been c o n f i r m e d by the o b s e r v a t i o n

t h a t C l i o n a c e l a t a grown on c a l c a r e o u s s u b s t r a t e s i_n v i t r o does

not i n f l u e n c e the c a l c i u m i o n c o n c e n t r a t i o n of a q u a r i a water

( 9 5 ) . The f a t e of the d i s s o l v e d c a l c i u m has not been d e t e r m i n e d .

F i g u r e 40. Schematic r e p r e s e n t a t i o n of the b u r r o w i n g p r o c e s s . (A) E t c h i n g c e l l i n c o n t a c t w i t h s u b s t r a t e ( s t i p p l e d ) . (B) I n i t i a l p e n e t r a t i o n of c e l l p r o c e s s e s by c h e m i c a l e t c h i n g of s u b s t r a t e . (C,D) C o n t i n u e d d i s s o l u t i o n of s u b s t r a t e . (E) E t c h e d c h i p removed from p i t t e d s u b s t r a t e . (Reproduced by k i n d p e r m i s s i o n of Dr. S. A. Pomponi).

I l l

I I . L i f e - C y c l e , Geographic and Taxonomic D i s t r i b u t i o n of the

C . c e l a t a A l k a l o i d s

The i s o l a t i o n of c l i o n a m i d e and the c e l e n a m i d e s as the

p r i n c i p a l p h e n o l i c secondary m e t a b o l i t e s of C . c e l a t a l e d t o the

s u g g e s t i o n t h a t t h e s e a l k a l o i d s might p a r t i c i p a t e i n e i t h e r the

p r i m a r y d i s s o l u t i o n of CaC03 or i n the subsequent t r a n s p o r t of

d i s s o l v e d c a l c i u m . P r i o r t o embarking on an i n v e s t i g a t i o n of the

r o l e p l a y e d by t h e s e compounds i n the p h y s i o l o g y of C. c e l a t a ,

we sought t o a s c e r t a i n the n a t u r e of t h e i r o c c u r r e n c e w i t h i n the

C l i o n i d a e f a m i l y . The reason f o r o b t a i n i n g t h i s i n f o r m a t i o n was

t o d e t e r m i n e whether c o n c l u s i o n s c o n c e r n i n g the f u n c t i o n of the

C. c e l a t a a l k a l o i d s would be r e l e v a n t t o the C l i o n i d a e as a

whole. I t was a l s o hoped t h a t chemotaxonomic s i g n i f i c a n c e c o u l d

be a s c r i b e d t o t h e s e compounds.

Specimens of C. c e l a t a were o b t a i n e d from t h r e e p r i n c i p a l

l o c a t i o n s : Woods H o l e , M a s s a c h u s e t t s 1 ; La J o l l a , C a l i f o r n i a ;

and Howe Sound, B a r k l e y Sound and the S t r a i t s of G e o r g i a ,

B r i t i s h C o l u m b i a . C.lampa , C. c a r i b b a e a , and C. d e l i t r i x were

c o l l e c t e d o f f P l a n t a t i o n Key, F l o r i d a . Sponge samples were

e x t r a c t e d as d e s c r i b e d f o r C. c e l a t a i n the e x p e r i m e n t a l s e c t i o n ,

t o g i v e EtOAc and water s o l u b l e f r a c t i o n s . P h e n o l i c s u b s t a n c e s

were d e t e c t e d by a s t a n d a r d FeCl3 s p r a y reagent t e s t . The EtOAc

^ e thank Dr. S. A. Pomponi (Woods Hole and F l o r i d a c o l l e c t i o n s ) and Dr. D. J . F a u l k n e r (La J o l l a n c o l l e c t i o n s ) f o r t h e i r generous c o n t r i b u t i o n s of sponge samples.

1 12

f r a c t i o n s were a c e t y l a t e d i n the u s u a l f a s h i o n and the crude

r e s i d u e s o b t a i n e d were a n a l y z e d by TLC and RP-HPLC. 1

C. c e l a t a i n d i g e n o u s t o B r i t i s h Columbian (B.C.) w a t e r s

showed a c o n s t a n t p a t t e r n of a l k a l o i d s when sponges of the same

l i f e - s t a g e were compared. Beta and 7 - s t a g e sponges p o s s e s s an

e s s e n t i a l l y i d e n t i c a l a r r a y of m e t a b o l i t e s . However, e x a m i n a t i o n

of f i g u r e 15b i n d i c a t e s the absence of p e n t a c e t y l c e l e n a m i d e C

(59) i n o i - s t a g e B.C. sponge. T h i s chromatogram a l s o shows the

p r e s e n c e of a n o v e l compound h a v i n g a r e t e n t i o n time between

t h a t of 58 and 5_7. Comparison of f i g u r e 15a and 15b f u r t h e r

i n d i c a t e s t h a t fewer l e s s p o l a r c o n s t i t u e n t s are found i n the on

stage organism. These r e s u l t s suggest t h a t the b i o s y n t h e s i s of

a l k a l o i d s o c c u r s even i n the e a r l i e s t s t a g e s of the sponge's

development and t h a t s u b t l e a l t e r a t i o n s i n m e t a b o l i s m do t a k e

p l a c e as the organism matures. The absence of a l k a l o i d s i n e a r l y

o^-stage sponge would have r u l e d out the p o s s i b i l i t y of the

i n v o l v e m e n t of t h e s e m o l e c u l e s i n the b u r r o w i n g p r o c e s s . The

reason f o r the a n a b o l i c changes obse r v e d i s not c l e a r . The

c o m p o s i t i o n of p e p t i d e a l k a l o i d m i x t u r e s o b t a i n e d from

t e r r e s t r i a l p l a n t s has been r e p o r t e d t o be dependent upon the

m a t u r i t y of the p l a n t ( 7 3 ) .

S e a s o n a l v a r i a t i o n of the minor m e t a b o l i t e s has been

ob s e r v e d but i s i n g e n e r a l , i n s i g n i f i c a n t . Only i n one i n s t a n c e

'Statements r e g a r d i n g s i m i l a r i t y or d i s s i m i l a r i t y are based p r i m a r i l y on the o c c u r r e n c e of 46, 57, 58 and 59, s i n c e the l e s s p o l a r , minor m e t a b o l i t e s have n o t , f o r the most p a r t , been c h a r a c t e r i z e d .

113

has a compound f a i l e d t o reappear i n subsequent c o l l e c t i o n s .

La J o l l a n 7 - s t a g e C . c e l a t a i s q u a l i t a t i v e l y i d e n t i c a l t o

i t s B.C. c o u n t e r p a r t . T e t r a c e t y l c l i o n a m i d e (46) and

h e x a c e t y l c e l e n a m i d e A (58) and B (5_7) were i s o l a t e d from

a c e t y l a t e d e t h a n o l e x t r a c t s of t h i s sponge but the minor

components were not d e t e r m i n e d . I t i s of i n t e r e s t here t o note

t h a t B.C. C. c e l a t a i s e n c o u n t e r e d a t an average depth of 18 m

compared w i t h 33 m f o r La J o l l a n C . c e l a t a . The o c c u r r e n c e of

t h i s P a c i f i c sponge at s h a l l o w e r depths i n temperate as opposed

t o t r o p i c a l e n v i r onments i s c o n s i s t e n t w i t h l i t e r a t u r e

d e s c r i p t i o n s of C. c e l a t a r e s i d i n g i n western A t l a n t i c w aters

(91, 9 5 ) . The sponge's change of h a b i t a t w i t h l a t i t u d e may be

due t o s e v e r a l f a c t o r s i n c l u d i n g , l i g h t or t e m p e r a t u r e

r e q u i r e m e n t s .

Gamma-stage C . c e l a t a from Woods Hole does not c o n t a i n

p e p t i d e a l k a l o i d s or r e l a t e d compounds. E x a m i n a t i o n of

a c e t y l a t e d EtOAc f r a c t i o n s of t h i s sponge show o n l y pigments and

s t e r o l s . No o t h e r secondary m e t a b o l i t e s are o b s e r v e d . The

aqueous f r a c t i o n gave an orange c o l o u r a t i o n when r e a c t e d w i t h

F e C l 3 , i n c o n t r a s t t o the more t y p i c a l g r a y - b l a c k c o l o u r a t i o n

o b t a i n e d w i t h p h e n o l s . A c e t y l a t i o n of the f r e e z e d r i e d m a t e r i a l

d i d not p r o v i d e r e a d i l y i d e n t i f i a b l e , d i s c r e t e compounds. The

n a t u r e of the water s o l u b l e component r e s p o n s i b l e f o r t h i s

r e a c t i o n i s b e i n g p e r s u e d i n our l a b o r a t o r y .

The t h r e e F l o r i d i a n e n d o l i t h i c sponges a s s a y e d were d e v o i d

of a l k a l o i d s . The EtOAc and aqueous f r a c t i o n s f a i l e d t o r e a c t

w i t h F e C l 3 and d i d not i n h i b i t the growth of S.aureus .

114

Woods Hole and B.C. C. c e l a t a burrow i n t o b i o g e n i c and

g e o l o g i c s o u r c e s of CaCC"3 and both grow on I c e l a n d s p a r 1 i_n v i v o

The morphology, c o l o u r a t i o n , and s p i c u l e c h a r a c t e r of t h e s e

sponges a r e i d e n t i c a l . The o b v i o u s c h e m i c a l d i f f e r e n c e s which

e x i s t might a r i s e as a r e s u l t of one of the f o l l o w i n g f a c t o r s :

1. G e n e t i c a l l y d i s t i n c t s t r a i n s ,

2. A s s o c i a t e d s y m b i o n t s ,

3. D i e t ,

4. S o c i a l r e q u i r e m e n t s ,

5. T a x o n o m i c a l l y d i s t i n c t s p e c i e s .

The secondary m e t a b o l i t e s of many marine and t e r r e s t r i a l

organisms are thought t o be d e r i v e d from s y m b i o t i c

m i c r o o r g a n i s m s or d i e t a r y s o u r c e s ( 9 8 ) , Rapoport (73) s u g g e s t s

t h a t p e p t i d e a l k a l o i d s of the shrub f a m i l y Ceanothus a r e

produced by r o o t - a s s o c i a t e d s y m b i o t i c a c t i n o m y c e t e s . As

mentioned, s y m b i o t i c z o o x a n t h e l l a e e x i s t i n C . c e l a t a and o t h e r

c l i o n i d s .

S e v e r a l f u n g i and one y e a s t have been i s o l a t e d from B.C. C.

c e l a t a t i s s u e and i n t e r s t i t i a l f l u i d s . 2 None of t h e s e organisms

were found t o produce p e p t i d e a l k a l o i d s or r e l a t e d compounds i n

v i t r o (see e x p e r i m e n t a l f o r d e t a i l s ) . Z o o x a n t h e l l a e from

1A t r a n s p a r e n t form of c a l c i t e . 2 F u n g a l b i o a s s a y s and the c u l t u r i n g of i s o l a t e s were performed by Gary H e w i t t .

1 15

A t l a n t i c and P a c i f i c specimens of C . c e l a t a are c u r r e n t l y being

compared by Dr. Pomponi and her c o l l a b o r a t o r s . A genetic

comparison of the two sponges has a l s o been proposed. U n t i l the

g e n e t i c and p o s s i b l y the taxonomic r e l a t i o n s h i p between these

c l i o n i d s i s understood, the chemotaxonomic s i g n i f i c a n c e of the

a l k a l o i d s d i s c u s s e d i n chapter two can not be f u l l y a p p r e c i a t e d .

In l i g h t of the b i o c h e m i c a l c o n t r a d i c t i o n s observed, i t may not

be i n a p p r o p r i a t e to suggest that re-examination of the taxonomy

of these sponges i s i n order.

The i n v e s t i g a t i o n of specimens of C . c e l a t a from the e a s t e r n

A t l a n t i c and western P a c i f i c oceans would a i d i n the extension

of the r e s u l t s presented above.

I I I . Experiments Aimed at Determining the Involvement of the

B.C. C c e l a t a A l k a l o i d s i n the Burrowing Process

Rapoport has r e c e n t l y suggested ( 9 9 ) that t e r r e s t r i a l

c y c l o p e p t i d e a l k a l o i d s may f u n c t i o n as ionophores i n t h e i r host

p l a n t s . The b a s i s of t h i s suggestion was the o b s e r v a t i o n that

the i n h e r e n t l y l i p o p h i l i c n a t u r a l and s y n t h e t i c a l k a l o i d s of

t h i s c l a s s which possess a 14-membered r i n g system (eg. 75) were

capable of b i n d i n g c e r t a i n d i v a l e n t and monovalent metal

c a t i o n s . T h i s work i n c o n j u n c t i o n with the n a t u r a l p r o p e n s i t y of

C . c e l a t a to burrow i n t o c a l c a r e o u s s u b s t r a t e s s t i m u l a t e d our

c u r i o s i t y concerning the f u n c t i o n of c l i o n a m i d e (5_3) and the

celenamides (7JD, 7J_, 7 2 , in the sponge's metabolism. The

i n s t a b i l i t y of the u n d e r i v a t i z e d C l i o n a a l k a l o i d s and the

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d i f f i c u l t i e s a s s o c i a t e d with o b t a i n i n g these i n a pure s t a t e

meant t h a t , i d e a l l y , the t o t a l s y n t h e s i s , or at the very l e a s t

the s y n t h e s i s of analogues, would be necessary i n order to

perform p r e c i s e l y c o n t r o l l e d b i o l o g i c a l and p h y s i c o - c h e m i c a l

experiments. These s y n t h e t i c s t u d i e s were i n i t i a t e d as p a r t of

t h i s t h e s i s and are d e s c r i b e d i n the f o l l o w i n g c h a p t e r .

As s y n t h e t i c m a t e r i a l s were not immediately a v a i l a b l e , we

sought to answer the f o l l o w i n g p e r t i n e n t q u e s t i o n s by u t i l i z i n g

simple and i n some i n s t a n c e s r a t h e r crude experimental methods.

F i r s t , do a l l l i f e - s t a g e s possess the compounds of i n t e r e s t ?

Secondly, do h i g h c o n c e n t r a t i o n s of c a l c i u m e x i s t w i t h i n the

sponge, and i f so, where? T h i r d l y , are the a l k a l o i d s l o c a l i z e d

i n a s p e c i f i c c e l l - t y p e , and f i n a l l y , are these compounds

capable of t r a n s p o r t i n g c a l c i u m a c r o s s a hydrophobic b a r r i e r ?

The answer to the f i r s t q u e s t i o n i n t h i s s e r i e s was p o s i t i v e and

has been d e a l t with i n the f o r e g o i n g s u b s e c t i o n .

Before proceeding with t h i s d i s c u s s i o n i t should be p o i n t e d

out that the m a j o r i t y of the _in v i v o and _in v i t r o burrowing

s t u d i e s which have been performed with C . c e l a t a as s u b j e c t have

u t i l i z e d the western A t l a n t i c organism. As a r e s u l t of the

f i n d i n g s presented i n the p r e c e d i n g s u b s e c t i o n , the p o s s i b i l i t y

e x i s t s t h at the i n f o r m a t i o n p r o v i d e d by these s t u d i e s i s not

p e r t i n e n t to the e a s t e r n P a c i f i c v a r i e t y . Although i t may be

proven to have been i n a p p r o p r i a t e the c u r r e n t d i s c u s s i o n w i l l

employ the assumption that a l l of the l i t e r a t u r e p e r t a i n i n g to C

c e l a t a i s r e l e v a n t to the present study.

If Warburton's (95) i n v i t r o r e s u l t s are taken i n

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c o n j u n c t i o n w i t h t h o s e of R u t z l e r and R i e g e r (96) and

e x t r a p o l a t e d t o t h e i_n v i v o s i t u a t i o n t h e n i t can be assumed

t h a t 2-3 p e r c e n t of the t o t a l c a l c i u m removed from the s u b s t r a t e

d u r i n g a sponge's l i f e t i m e i s e i t h e r r e l e a s e d i n t o the

e n v i r o n m e n t , o r t o t a l l y or p a r t i a l l y r e t a i n e d w i t h i n the sponge.

In o r d e r t o d e t e r m i n e the p o s s i b i l i t y of e i t h e r of the l a t t e r

two s i t u a t i o n s , specimens were d i v i d e d i n t o f o u r f r a c t i o n s as

f o l l o w s . F r e s h sponge (wet weight 12.1 g) was e x t r a c t e d

t h o r o u g h l y w i t h aqueous methanol. The m e t h a n o l i c e x t r a c t was

then f u r t h e r d i v i d e d i n t o EtOAc and water s o l u b l e f r a c t i o n s . The

s o l i d s r e m a i n i n g a f t e r t r e a t m e n t w i t h methanol were d r i e d and

one h a l f was t r e a t e d w i t h h y p o c h l o r o u s a c i d t o g i v e p u r i f i e d

sponge s p i c u l e s . S u b j e c t i o n of each of t h e s e samples t o

e l e m e n t a l a n a l y s i s produced the r e s u l t s i n d i c a t e d i n T a b l e 4.

T a b l e 4. C a l c i u m A n a l y s e s 1

F r a c t i o n Weight

9 C a l c i u m — c o n c e n t r a t i o n

% mg O r g a n i c s o l v e n t s o l u b l e Water s o l u b l e D r i e d t o t a l s o l i d s S p i c u l e s N o n - s p i c u l e s o l i d components 2

Sand and d e b r i s

0.04 0.4 3.6 3.0 0.5 0.1

0.10 0.04 3.9 1.9 17

<1 <1 -140 -57 -83

l C a l c i u m a n a l y s e s were performed by Chemex L a b o r a t o r i e s , i n f e r r e d .

118

Few r e p o r t s r e g a r d i n g the m i n e r a l c o m p o s i t i o n of sponges

a r e a v a i l a b l e i n the l i t e r a t u r e . Schwab and Shone (100) examined

the s i l i c e o u s s p i c u l e s of Acarnus e r i t h a c u s and d e t e r m i n e d t h e i r

c a l c i u m c o n t e n t t o be 0.014 p e r c e n t . Bowen and S u t t o n

i n v e s t i g a t e d t h e s i l i c o n and c a l c i u m (101) c o n t e n t of s e v e r a l

whole d r i e d sponges of the genera D y s i d e a , C h o n d r i l i a , and

T e r p i o s . C a l c i u m c o n c e n t r a t i o n i n t h e s e Demosponges ranged from

0.53 t o 30.20 p e r c e n t .

The l a t t e r s tudy i n d i c a t e s t h a t the c a l c i u m c o n c e n t r a t i o n s

found i n the s t r u c t u r a l f e a t u r e s of C . c e l a t a a r e not anomalous.

However, p o t e n t i a l s i t e s of c a l c i u m s e q u e s t r a t i o n a r e i m p l i e d by

the r e s u l t s shown i n T a b l e 4 and may be i n k e e p i n g w i t h Yonge's

s u g g e s t i o n (93) t h a t growth p r o c e s s e s may a s s i s t i n the

b u r r o w i n g p r o c e s s . The n a t u r e of the c a l c i u m - r e t a i n i n g s p e c i e s

p r e s e n t i n h i g h c o n c e n t r a t i o n i n the sponge t i s s u e has not been

i n v e s t i g a t e d .

The e n d o l i t h i c t i s s u e s of b u r r o w i n g sponges a r e composed of

s e v e r a l d i s t i n c t t y p e s of c e l l s i n c l u d i n g a s p e c i a l i z e d c e l l

b e l i e v e d t o be r e s p o n s i b l e f o r the s e c r e t i o n of a c h e m i c a l

e t c h i n g agent ( 8 6 b ) . In o r d e r t o a s c e r t a i n the i n v o l v e m e n t of

the C . c e l a t a a l k a l o i d s i n the p r i m a r y d i s s o l u t i o n p r o c e s s ,

d i s s o c i a t e d c e l l s from t h i s sponge were f r a c t i o n a t e d 1 i n t o c e l l -

t y p e s by d i s c o n t i n u o u s d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (see

e x p e r i m e n t a l f o r d e t a i l s ) . F i v e c e l l s u b - p o p u l a t i o n s (—50 mg wet

wt. each) were o b t a i n e d and a n a l y z e d f o r the p r e s e n c e of

1 C e l l s e p a r a t i o n s were performed by Dr. S.A. Pomponi.

119

c l i o n a m i d e (5_3) and the celenamides. A l k a l o i d s were not d e t e c t e d

i n any of the c e l l f r a c t i o n s . S e v e r a l i n t e r p r e t a t i o n s of t h i s

s u r p r i s i n g r e s u l t have been put forward. However, u n t i l f u r t h e r

i n f o r m a t i o n i s a c q u i r e d , an a c c u r a t e c o n c l u s i o n cannot be

reached. At the time of w r i t i n g , the necessary i n f o r m a t i o n was

i n the process of being gathered.

I t i s i n t r i g u i n g that the only c e l l f r a c t i o n observed to

c o n t a i n f r e e s t e r o l s and pigments a l s o possessed the sponge's

z o o x a n t h e l l a e . S t e r o l s have been suggested as taxonomic a i d s to

sponge i d e n t i f i c a t i o n (102). I t would be important, t h e r e f o r e ,

to determine whether sponge s t e r o l s are s y n t h e s i z e d de novo or

are e x c l u s i v e l y of symbiont o r i g i n .

Rapoport (99) has been able to show that c y c l o p e p t i d e

a l k a l o i d s bind c e r t a i n monovalent and d i v a l e n t metal c a t i o n s . A

s i m i l a r r e s u l t was obtained when CD s p e c t r a of

hexacetylcelenamide B (5_7, appendix 11) were measured in the

presence and absence of C a 2 * . The c o n f o r m a t i o n a l change

i n d i c a t e d i n appendix 11 c o u l d be a t t r i b u t e d to i o n - b i n d i n g

a s s o c i a t e d with e i t h e r the a c e t y l a t e d c a t e c h o l or g a l l o l

r e s i d u e s , or the amide groupings which c o n s t i t u t e the backbone

of the p e p t i d e s t r u c t u r e .

Simple l i n e a r p e p t i d e s do not complex metal ions to a

n o t i c e a b l e degree. Examination of molecular models suggested

that celenamides A {10), B (71), and C (72) c o u l d e x i s t i n

conformations s i m i l a r i n nature to the p h e n c y c l o p e p t i n e

s k e l e t o n . The p o s s i b i l i t y e x i s t s that the s o l e dehydroamino a c i d

r e s i d u e i n these molecules i s r e s p o n s i b l e f o r the g e n e r a t i o n of

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a c o n f o r m a t i o n a l s t a t e which e x h i b i t s metal b i n d i n g p r o p e r t i e s .

Ionophores, many of which possess amino a c i d r e s i d u e s or

amide f u n c t i o n a l i t i e s , are c h a r a c t e r i z e d by t h e i r a b i l i t y to

form h i g h l y l i p o p h i l i c complexes with metal ions (103).

The C l i o n a a l k a l o i d s were t e s t e d f o r p o t e n t i a l i o n o p h o r i c

p r o p e r t i e s i n a simple U-tube system (104, see experimental fo r

d e t a i l s ) . Ion t r a n s l o c a t i o n i s d i s p l a y e d i n t h i s system by

i o n i z a b l e l i g a n d s but not by n o n i o n i z a b l e l i g a n d s . The r e s u l t of

the f i r s t experiment performed i s i l l u s t r a t e d i n appendix 12.

During the course of t h i s experiment, a s u b s t a n t i a l amount

of p r e c i p i t a t e formed at the i n t e r f a c e of the aqueous donor

phase (+Ca 2*) and the organic phase. I t was suggested,

t h e r e f o r e , that the low percentage t r a n s p o r t observed was due to

i n s o l u b i l i t y of the a l k a l o i d - m e t a l complex(es). The p o s s i b i l i t y

t h a t the C l i o n a a l k a l o i d s are n o n i o n i z a b l e l i g a n d s i s not

excluded by t h i s experiment. Complexes of metal ions and

n o n i o n i z a b l e ionophores n e c e s s a r i l y r e t a i n the net charge of the

metal i o n . Hence,the s o l u b i l i t y of the complex i s dependent upon

the nature of the s a l t and the p a r t i t i o n i n g p r o p e r t i e s of the

c o u n t e r i o n .

In a second experiment, the s o l v e n t d i e l e c t r i c c onstant was

i n c r e a s e d by the use of n itrobenzene as a component, of the

organic phase and the t r a n s p o r t d i s t a n c e was decreased by

employment of a Pressman c e l l (104). The r e s u l t o b t a i n e d was

roughly analogous to that shown i n appendix 12. T h i s confirmed

the f a c t that c l i o n a m i d e and the celenamides are not i o n i z a b l e

ionophores. F u r t h e r experimentation i s necessary to determine

121

the n o n i o n i z a b l e i o n o p h o r i c c a p a b i l i t i e s of the s e m o l e c u l e s .

IV. C o n c l u s i o n

The statement was made d u r i n g the d i s c u s s i o n of Chapter 2

t h a t e x p e r i m e n t s aimed a t e l u c i d a t i n g the b i o l o g i c a l r o l e of a

secondary m e t a b o l i t e o f t e n l e a d t o ambiguous r e s u l t s . The

i n i t i a t o r y i n q u i r y p r e s e n t e d above, i l l u s t r a t e s t h i s p o i n t .

A l t h o u g h , the m e t a b o l i c f u n c t i o n of the C . c e l a t a a l k a l o i d s has

not been c l a r i f i e d , many i n t r i g u i n g q u e s t i o n s have a r i s e n as a

r e s u l t of t h i s b r i e f i n v e s t i g a t i o n . These w i l l w i t h o u t doubt

s t i m u l a t e f u r t h e r c o l l a b o r a t i v e r e s e a r c h . The c h e m i s t r y of

C l i o n a z o o x a n t h e l l a e , the n a t u r e of sponge-symbiont s t e r o l

r e l a t i o n s h i p s , the s i t e of p r o d u c t i o n of the C l i o n a m e t a b o l i t e s ,

and the o r i g i n of the d i f f e r e n c e s of A t l a n t i c and P a c i f i c C

c e l a t a a r e s u b j e c t s worthy of f u r t h e r e x a m i n a t i o n .

122

SYNTHETIC STUDIES

I . R a t i o n a l e

The advent of p o w e r f u l s p e c t r o s c o p i c methods f o r the

e l u c i d a t i o n of o r g a n i c s t r u c t u r e s , has i n i t i a t e d a t r e n d among

n a t u r a l - p r o d u c t c h e m i s t s away from the use of c h e m i c a l

d e g r a d a t i o n s and p a r t i a l s y n t h e s e s . T h i s tendency has on

o c c a s i o n , l e d t o i n c o r r e c t s t r u c t u r a l a s s i g n m e n t s . In many

i n s t a n c e s , the a p p l i c a t i o n of r e a d i l y a v a i l a b l e s p e c t r o s c o p i c

t e c h n i q u e s does not g i v e r i s e t o the complete e l u c i d a t i o n of a

n o v e l s t r u c t u r e . The d e t e r m i n a t i o n of s t e r e o c h e m i c a l and s u b t l e

r e g i o c h e m i c a l f e a t u r e s , f r e q u e n t l y r e q u i r e s the i m p l e m e n t a t i o n

of more c l a s s i c a l e l u c i d a t i v e p r o c e d u r e s .

C o r r e c t s t r u c t u r e s f o r the c e l e n a m i d e s were not proposed

u n t i l s i m p l i f i c a t i o n of t h e s e complex m o l e c u l e s had been

a c c o m p l i s h e d by c h e m i c a l d e g r a d a t i o n . To p r o v i d e f u r t h e r

e v i d e n c e f o r our s t r u c t u r a l h ypotheses we sought t o c o r r e l a t e

the a c q u i r e d s p e c t r a l i n f o r m a t i o n w i t h d a t a r e p o r t e d f o r n a t u r a l

p r o d u c t s and s y n t h e t i c m a t e r i a l s p o s s e s s i n g s i m i l a r s t r u c t u r a l

f e a t u r e s . S p e c t r a l i n f o r m a t i o n p e r t a i n i n g t o a r o m a t i c

dehydroamino a c i d and N - a c y l s t y r y l a m i n e f u n c t i o n a l i t i e s ,

p a r t i c u l a r l y *H NMR d a t a , was found t o be g e n e r a l l y u n a v a i l a b l e .

Hence, the p r e p a r a t i o n of s e v e r a l s i m p l e model compounds was

u n d e r t a k e n i n an attempt t o f i l l t h i s v o i d .

Analogues were a l s o d e s i r e d t o a i d i n the c l a r i f i c a t i o n of

the c o n f i g u r a t i o n s of the c e l e n a m i d e dehydroamino a c i d s .

123

P e p t i d e s i n c o r p o r a t i n g s u b s t i t u t e d and u n s u b s t i t u t e d

d e h y d r o p h e n y l a l a n i n e r e s i d u e s were r e q u i r e d f o r a proposed

i n v e s t i g a t i o n r e g a r d i n g the a b i l i t y of u n s a t u r a t e d p e p t i d e s t o

b i n d m e t a l i o n s .

To a v o i d m u l t i - s t e p t o t a l s y n t h e s e s or the development of

n o v e l s y n t h e t i c methods, we e x p e r i m e n t e d w i t h s e v e r a l l i t e r a t u r e

p r o c e d u r e s f o r p r e p a r i n g d e h y d r o p e p t i d e s i n o r d e r t o a s c e r t a i n

whether a method was a v a i l a b l e which encompassed o n l y a few

w e l l - c h a r a c t e r i z e d s y n t h e t i c s t e p s .

11. P r e p a r a t i on of Model Compounds

Aro m a t i c dehydroamino a c i d s have most commonly been

p r e p a r e d v i a o x a z o l o n e s ( a z l a c t o n e s , o x a z o l i n o n e s , 105). The

l a t t e r may be o b t a i n e d by s e v e r a l r o u t e s , the most w i d e l y

u t i l i z e d b e i n g the c l a s s i c a l E r l e n m e y e r - P l b c h l s y n t h e s i s (106)

i l l u s t r a t e d below. The f o r m a t i o n and p r o p e r t i e s of o x a z o l o n e s ,

p a r t i c u l a r l y u n s a t u r a t e d o x a z o l o n e s , have been the s u b j e c t s of

much s c i e n t i f i c a t t e n t i o n d u r i n g the p a s t 100 y e a r s . Recent

r e v i e w s (105, 107, 108) a t t e s t t o the c o n t i n u e d i n t e r e s t i n t h i s

a r e a . In a d d i t i o n t o dehydroamino a c i d s , o x a z o l o n e s a r e a l s o

i m p o r t a n t p r e c u r s o r s t o c h i r a l a-amino a c i d s and cx-keto a c i d s .

124

AcGlyOH A C 2 < > ft> f N\7^P1 NaOAc ^ J±-d J

RCHO

Condensation of a c e t y l g l y c i n e (109) with 3,4,5-

trimethoxybenzaldehyde (Sigma) i n ' t h e presence of AC2O-sodium

a c e t a t e p r o v i d e d b r i g h t yellow 2 - a c e t y l - 4 - ( 3 ' , 4 ' , 5 ' -

t r i m e t h o x y b e n z y l i d e n e ) - 5 ( 4 H ) - o x a z o l o n e (9_0) i n a maximum y i e l d

of 70 p e r c e n t . T h i s p r e p a r a t i o n was repeated s e v e r a l times with

the average y i e l d being 62 p e r c e n t . Oxazolone 9_0 had p r e v i o u s l y

been prepared i n 30 percent y i e l d by Acheson et a l . ( 8 5 ) .

Attempts t o s y n t h e s i z e £0 by the m o d i f i e d Erlenmeyer r e a c t i o n

(110) employing sodium b i c a r b o n a t e i n p l a c e of sodium a c e t a t e ,

r e s u l t e d i n lower y i e l d s ( 2 0 - 3 0 % ) . Two other oxazolones, 91

(111) and !92, were made f o r comparative purposes. The phenyl

oxazolone JJ2 was i d e n t i c a l to that r e p o r t e d by Mauthner ( 1 1 2 ) .

125

T r a n s f o r m a t i o n of u n s a t u r a t e d o x a z o l o n e s i n t o dehydroamino

a c i d s i s r e a d i l y a c h i e v e d by r e a c t i o n w i t h w a t e r , a l c o h o l s o r

amines. Oxazolones d e r i v e d from a c e t y l g l y c i n e ( a c e t u r i c a c i d )

r e a c t r e a d i l y w i t h t h e s e r e a g e n t s , whereas, tho s e formed from

h i p p u r i c a c i d ( b e n z o y l g l y c i n e ) a r e more r e s i s t a n t t o r i n g

opening and f r e q u e n t l y r e q u i r e the p r e s ence of a c i d i c or b a s i c

c a t a l y s t s ( 1 1 3 ) . S e v e r a l d e r i v a t i v e s were p r e p a r e d i n ' e x c e l l e n t

y i e l d s i n t h i s manner, i n c l u d i n g compounds 93-96_. The a c i d s 94

and 95 can be q u a n t i t a t i v e l y r e c o n v e r t e d i n t o t h e i r r e s p e c t i v e

o x a z o l o n e s by t r e a t m e n t w i t h A c 2 O - d i m e t h y l a m i n o p y r i d i n e .

P e p t i d e s i n c o r p o r a t i n g an N - t e r m i n a l dehydroamino a c i d

r e s i d u e can be o b t a i n e d by r e a c t i o n of the a p p r o p r i a t e

u n s a t u r a t e d o x a z o l o n e w i t h the sodium s a l t s of amino a c i d s or

p e p t i d e s ( 1 2 3 ) . The {Z)-dehydrodipeptides 99-103 were p r e p a r e d

i n t h i s f a s h i o n .

126

101 i-Pr H 1 0 2 s -Bu H

S e v e r a l b i s d e h y d r o d i p e p t i d e s were p r e p a r e d f o r s t r u c t u r a l

a n a l o g y w i t h n o n a c e t y l c e l e n a m i d e D (6JD). A p p l i c a t i o n of the

Erlenmeyer r e a c t i o n t o the g l y c i n e c o n t a i n i n g d e h y d r o d i p e p t i d e s

99 and 103 a f f o r d e d the p e p t i d y l o x a z o l o n e s 104 and 105. T h i s

r e a c t i o n sequence has been p r e v i o u s l y employed by Doherty et a l .

(124) and a l s o by P i e r o n i e t a l . (76) i n the s y n t h e s i s of

compound 105. R e a c t i o n of the p e p t i d y l o x a z o l o n e s 104 and 105

w i t h n u c l e o p h i l e s , as d e s c r i b e d f o r the o x a z o l o n e s 90-92, gave

the b i s d e h y d r o d i p e p t i d e s 106-111. Treatment of the o x a z o l o n e 104

w i t h ammonia under a v a r i e t y of c o n d i t i o n s g e n e r a t e d the amino

a c i d 96 as the s o l e i s o l a b l e p r o d u c t .

127

D e s c r i p t i o n s of the s y n t h e s i s of N - a c y l s t y r y l a m i n e s are not

p r e v a l e n t i n the l i t e r a t u r e . Methods which have been employed

i n c l u d e the condensation of acetamide and phenylacetaldehyde,

the r e a c t i o n of styrene epoxide with amides (116) and the

d e c a r b o x y l a t i o n of od-benzoylamido cinnamic a c i d s i n the presence

of copper chromite (113) or ammonia (110a). The route envisaged

f o r the p r o d u c t i o n of these f u n c t i o n a l i t i e s i s shown in scheme

9. The avoidance of a c i d s i n the f i n a l e l i m i n a t i o n r e a c t i o n i s

c r i t i c a l due to the a c i d - s e n s i t i v e nature of the moiety being

produced.

Scheme 9. Proposed s y n t h e s i s of C-terminal s t y r y l a m i n e s .

129

A p p l i c a t i o n of Corey's epoxide s y n t h e s i s (117) u t i l i z i n g

d i m e t h y l s u l f o n i u m methylide gave the styrene epoxide 112 i n 90

percent y i e l d . The f o l l o w i n g three steps shown in scheme 9 were

performed i n immediate s u c c e s s i o n without i s o l a t i o n and

c h a r a c t e r i z a t i o n of the i n t e r m e d i a t e products 113 and 114.

Treatment of the epoxide 112 with aqueous ammonia at room

temperature f o r four days r e s u l t e d i n the formation of

predominately the d e s i r e d r e g i o i s o m e r . T h i s s e l e c t i v i t y i s

f r e q u e n t l y observed in b a s e - c a t a l y z e d n u c l e o p h i l i c ring-openings

of epoxides (118 ) .

For the purposes of t h i s e x p l o r a t o r y r e a c t i o n sequence,

v a l i n e (scheme 9, R=CH(CH3)2) was chosen as the amino a c i d

component of the d e s i r e d t a r g e t molecule. T h i s c h o i c e was made

s o l e l y on the c o n s i d e r a t i o n of s i m p l i f i c a t i o n of the end-product

*H NMR spectrum. S i m p l i f i c a t i o n of the o l e f i n i c r e g i o n of t h i s

spectrum was r e q u i r e d for d e t e c t i o n of p o s s i b l e g e o m e t r i c a l

isomerism i n the s t y r y l a m i n e 116. Condensation of the crude

amine 113 with N - a c e t y l v a l i n e , by the c a r b o d i i m i d e method

in t r o d u c e d by Sheehan et a l . (119), a f f o r d e d the m o d i f i e d amino

a c i d 114. R e t e n t i o n of o p t i c a l a c t i v i t y was not a major concern

d u r i n g t h i s f i r s t s e r i e s of r e a c t i o n s . In f u t u r e t r i a l s the

c o u p l i n g a d d i t i v e h y d r o x y b e n z o t r i a z o l e should be employed in

t h i s step and the use of t r i e t h y l a m i n e e l i m i n a t e d (120, 121).

A c e t y l a t i o n of the a l c o h o l 114 a f f o r d e d the a c e t a t e 115 i n an

o v e r a l l - y i e l d of 52 percent from the epoxide 112.

Although the syn e l i m i n a t i o n of a c e t a t e s i s a f r e q u e n t l y

employed r e a c t i o n i n organic s y n t h e s i s , t h i s procedure has not

130

a p p a r e n t l y been used i n the p r o d u c t i o n of N - a c y l s t y r y l a m i n e s . N-

V i n y l - 2 - o x a z o l i d o n e has been p r e p a r e d from N - ( 2 - a c e t o x y e t h y l ) - 2 -

o x a z o l i d o n e as i l l u s t r a t e d below ( 5 6 ) .

OAc

(56)

Attempts t o e l i m i n a t e a c e t i c a c i d from a c e t a t e 115 have so

f a r proven t o be u n s u c c e s s f u l . A v a r i e t y of ' p y r r o l y t i c

c o n d i t i o n s has been t r i e d , i n c l u d i n g m u l t i p l e passages t h r o u g h a

hot tube (200-450°) f i l l e d w i t h g l a s s beads. Rather than r e s o r t

t o h i g h e r t e m p e r a t u r e s t o f a c i l i t a t e e l i m i n a t i o n , we chose t o

persue a l t e r n a t i v e , m i l d e r methods of double bond f o r m a t i o n .

Harsher p y r o l y t i c c o n d i t i o n s were c o n s i d e r e d t o be u n d e s i r a b l e

f o r f u t u r e s y n t h e s e s i n which , /3-hydroxymescal i n e or 3,4-

dimethoxydopamine would be l i n k e d t o s e n s i t i v e amino a c i d s or

pept i d e s .

In v e r y r e c e n t work, we have s u r p r i s i n g l y been unable t o

d e h y d r a t e the a l c o h o l 114 t o g i v e the enamide 116. Methods t r i e d

i n c l u d e ; r e f l u x i n g i n DCC-EtOAc, t r e a t m e n t w i t h t h i o n y l c h l o r i d e

i n p y r i d i n e , and t o s y l a t e and m e s y l a t e f o r m i n g r e a c t i o n s . No

change was o b s e r v e d a f t e r 24 hours i n the pr e s e n c e of DCC.

De c o m p o s i t i o n of the s t a r t i n g m a t e r i a l was ob s e r v e d when t h i o n y l

131

c h l o r i d e , t o s y l c h l o r i d e or mesyl c h l o r i d e were employed.

Experiments aimed at r e s o l v i n g t h i s s i t u a t i o n are i n p r o g r e s s .

Two other methods f o r forming the a l c o h o l 1 1 4 were

attempted. R e f l u x i n g the epoxide 112 i n anhydrous DMF i n the

presence of N - a c e t y l v a l i n a m i d e (117) f o r s e v e r a l days at 180°

r e s u l t e d i n the i s o l a t i o n of s t a r t i n g m a t e r i a l s and small

amounts (<5%) of the ketone 118 and the aldehyde 119 (apparently

present as polymer). The a d d i t i o n of one e q u i v a l e n t of NaH to

t h i s r e a c t i o n e f f e c t e d the formation of the a l c o h o l 114 i n

approximately 10 percent y i e l d ( f o l l o w i n g a c e t y l a t i o n ) .

C o n d i t i o n s r e q u i r e d to improve the y i e l d of t h i s r e a c t i o n have

not been e x p l o r e d . The opening of styrene epoxide with amines,

amides, amino a c i d s and aromatic h e t e r o c y c l e s has been d e s c r i b e d

(116, 122, 123) .

N H A c

117

H

Me Me

116 119

132

I I I . Geometry C o n s i d e r a t i o n s

HPLC and *H NMR a n a l y s i s of the o x a z o l o n e s 90-92 and TLC

and *H NMR a n a l y s i s of the amino a c i d s 9_3-98 i n d i c a t e d the

p resence of one g e o m e t r i c a l isomer i n each c a s e . T h i s

o b s e r v a t i o n a g r e e s w i t h the c o n c l u s i o n a r r i v e d at by P i e r o n i et

a l . (76) t h a t , a t l e a s t f o r b e n z a l d e h y d e , the Erlenmeyer

s y n t h e s i s g i v e s o n l y one isomer h a v i n g the Z - c o n f i g u r a t i o n .

C o n d e n s a t i o n s c a r r i e d out i n s u l f u r i c a c i d or s u l f u r i c a c i d -

a c e t i c a n h y d r i d e g i v e m i x t u r e s of E and Z isomers ( 1 0 5 ) . The

l a b i l e E - o x a z o l o n e s can be opened w i t h oxygen n u c l e o p h i l e s t o

the c o r r e s p o n d i n g E-dehydroamino a c i d s . C o n t a c t w i t h amines or

p y r i d i n e cause o x a z o l o n e s p o s s e s s i n g the E - c o n f i g u r a t i o n t o

i s o m e r i z e t o the more s t a b l e 2 form ( 1 0 5 ) .

Assignment of the s t e r e o c h e m i s t r y about the d o u b l e bond i n

compounds such as 93-9_B i s u s u a l l y performed by c o mparison of

the p h y s i c a l p r o p e r t i e s [eg. UV ( X max, e ), *H NMR (6"), IR (CO,

v max)] of one isomer w i t h the o t h e r . The c h e m i c a l s h i f t of the

v i n y l p r o t o n i n t h e s e m o l e c u l e s i s measurably s e n s i t i v e t o t h e i r

geometry. Employing *H NMR s p e c t r o s c o p y M o r g e n s t e r n e t a l . (124)

e s t a b l i s h e d t h a t the s t a b l e c o n f i g u r a t i o n of a r o m a t i c

dehydroamino a c i d s i s Z, i n agreement w i t h a much e a r l i e r X-ray

assignment ( 1 2 5 ) . T h i s s t u d y c o r r e c t e d the e r r o n e o u s c o n c l u s i o n

of B r o c k l e h u r s t e t a l . ( 1 2 6 ) .

133

M o r g e n s t e r n demonstrated t h a t the o l e f i n i c hydrogen atom i s

d e s h i e l d e d i n the E, r e l a t i v e t o the 1, c o n f i g u r a t i o n .

Subsequent c o n f i r m a t i o n of thes e o b s e r v a t i o n s has been made by

Rao and F i l l e r (105).

In t he lH NMR s p e c t r a of d i k e t o p i p e r a z i n e s which posse s s an

a r o m a t i c dehydroamino a c i d r e s i d u e and the s y n t h e t i c c y c l o p e n i n

p r e c u r s o r s 120 (127, 128, 129), the v i n y l p r o t o n of isomers w i t h

the 1 c o n f i g u r a t i o n i s ob s e r v e d a t lower f i e l d than i n the

c o r r e s p o n d i n g E is o m e r s . C l o s e s p a c i a l p r o x i m i t y of the o l e f i n i c

hydrogen atom and the a d j a c e n t c o n f o r m a t i o n a l l y - f i x e d c a r b o n y l

group i n the Z isomers g i v e s r i s e t o t h i s d e s h i e l d i n g e f f e c t . In

marked c o n t r a s t , g e o m e t r i c a l isomers of u n s a t u r a t e d a r o m a t i c

o x a z o l o n e s show the o p p o s i t e v i n y l p r o t o n s h i f t s . T h i s has been

a t t r i b u t e d t o the pronounced d e s h i e l d i n g i n f l u e n c e of an -N=C-R

moiety on a c i s - o l e f i n i c hydrogen atom (105). In s i m p l e a c y c l i c

s i t u a t i o n s c o n f o r m a t i o n a l freedom would be e x p e c t e d t o g i v e r i s e

t o m o l e c u l e s which e x i s t p r e d o m i n a t e l y i n the more s t a b l e s-

t r a n s o i d c i n n a m i c a c i d c o n f o r m a t i o n . An e x p l a n a t i o n f o r the

v i n y l p r o t o n s h i f t s o b s e r v e d i n the *H NMR s p e c t r a of a r o m a t i c

dehydroamino a c i d s i s e v i d e n t i n the work of Mo r g e n s t e r n et a l .

(124) and of B r o c k l e h u r s t e t a l . (126). These a u t h o r s c l e a r l y

d emonstrate t h a t the a d d i t i o n of an o«J-acylamido group t o e i t h e r

m e t h y l cinnamate (126) or methyl a c r y l a t e (124) r e s u l t s i n

s h i e l d i n g of a t r a n s - v i n y l p r o t o n and d e s h i e l d i n g of a c i s - v i n y l

p r o t o n , w i t h the l a t t e r b e i n g the most pronounced of the two

e f f e c t s ( A 6* c i s s+1.00, AS t r a n s =-0.37). These observed

s h i f t s d i f f e r markedly from th o s e employed by P a s c u a l et a l .

The e m p i r i c a l a d d i t i v e increment r u l e s of P a s c u a l e t a l .

( 1 3 0 ) , and o t h e r s (131), do not a s s i s t i n the assignment of

dehydroamino a c i d c o n f i g u r a t i o n s i n the a c e t y l a t e d c e l e n a m i d e s

5_7-6_0. These r u l e s o r i g i n a t e d from o b s e r v a t i o n s made on s i m p l e

i s o l a t e d c h e m i c a l systems. N u c l e i i n complex m o l e c u l e s such as

p e p t i d e s may e x i s t i n unique c h e m i c a l e n v i r o n m e n t s c r e a t e d by

the t e r t i a r y s t r u c t u r e of the m o l e c u l e . In th e s e s i t u a t i o n s ,

c o m p a r i s o n s w i t h s p e c t r a l d a t a o b t a i n e d on m o l e c u l e s l a c k i n g

t h i s i n f l u e n c e a r e d i f f i c u l t . Tobey (132) i n t r o d u c e d the p a r e n t

compound method of c o r r e l a t i o n i n an attempt t o c i r c u m v e n t t h i s

p roblem. However, c h e m i c a l s h i f t s of v i n y l p r o t o n s i n (E) -

d e h y d r o p h e n y l a l a n i n e - c o n t a i n i n g p e p t i d e s a r e not a v a i l a b l e f o r

c o m p a r i s o n . 1 S i g n a l s c o r r e s p o n d i n g t o v i n y l p r o t o n s were

ob s e r v e d i n the 1H NMR s p e c t r a of compounds 96, £7, and 99-103

a t 6 6.99 t o 7.19. M e t h y l N-acety l v a l y l - o ^ -

1'H NMR d a t a f o r E and Z isomers of BocMeAlaLeu A PheGlyOMe have been r e p o r t e d but the s p e c t r a p r o v i d e d have not been a s s i g n e d (133) .

135

didehydrophenylalanate (to be d i s c u s s e d ) d i s p l a y e d the

corresponding s i g n a l at <5*7.25.

In s e v e r a l i n s t a n c e s , chemical s h i f t c o r r e l a t i o n s do

f a c i l i t a t e the assignment of aromatic dehydroamino a c i d

geometries [eg. 98, (5"7.46; 121, (Z) <5"7.44, (E) , <5"8.00 (124)].

The *H NMR s p e c t r a of p e p t i d e s 57-59, 6_5 and 66 (Table 1)

possess v i n y l s i g n a l s w i t h i n a s i m i l a r range (S 6.80 to 7.22) to

these co r r e s p o n d i n g resonances i n compounds 9_6, 9_7 and 99-103.

Although these v a l u e s are suggestive of the Z-contiguration, the

temptation to draw t h i s c o n c l u s i o n has been r e s i s t e d .

Isomerizaton of these r e s i d u e s has not been observed. Many

authors d e p i c t t h e i r dehydroamino a c i d - c o n t a i n i n g s y n t h e t i c and

n a t u r a l products as p o s s e s s i n g the Z - c o n f i g u r a t i o n s o l e l y on the

b a s i s of t h i s c o n s i d e r a t i o n . As a r e s u l t of our a n a l y s i s of many

dehydroamino a c i d s and dehydropeptides, we suggest that

c o r r e l a t i v e procedures or s t a b i l i t y assumptions alone are not

s a t i s f a c t o r y methods of determining the s t e r e o c h e m i s t r y of the

o l e f i n i c components of the l a t t e r . T h i s i s e s p e c i a l l y t rue of

marine n a t u r a l products which f r e q u e n t l y d i s p l a y uncommon

arrangements of f u n c t i o n a l groups and unexpected s t e r e o c h e m i c a l

and r e g i o c h e m i c a l f e a t u r e s . With the ex c e p t i o n of X-ray

c r y s t a l l o g r a p h y and t o t a l s y n t h e s i s , comparison of iso m e r i c

analogues i s the only d e f i n i t i v e method of assignment a v a i l a b l e

at t h i s time. U n f o r t u n a t e l y , both isomers of a n a t u r a l product

or c l o s e l y r e l a t e d analogues are seldomly r e a d i l y a v a i l a b l e .

136

The *H NMR s p e c t r a of n o n a c e t y l c e l e n a m i d e D (6_0) and i t s

h y d r o l y s i s p r o d u c t 73 each d i s p l a y s i g n a l s c o r r e s p o n d i n g t o two

dehydroamino a c i d v i n y l p r o t o n s ( T a b l e 1 ) . The d i s p a r i t y i n the

observ e d c h e m i c a l s h i f t s (60, 8 7.59, 7.06; 73, <$"7.41, 7.09)

can be e x p l a i n e d by c o n s i d e r i n g a r e c e n t X-ray c r y s t a l l o g r a p h i c

a n a l y s i s performed by P i e r o n i e t a l . ( 7 6 ) . T h e i r study i n d i c a t e d

t h a t the r e l a t i o n s h i p between the o l e f i n s and t h e i r a d j a c e n t

c a r b o n y l groups i n the two dehydroamino a c i d r e s i d u e s of a

{Z,£)-bisdehydrotripeptide (122) a r e d i f f e r e n t . One r e s i d u e

e x i s t s i n an s - t r a n s o i d c o n f o r m a t i o n , whereas the o t h e r i s s-

c i s o i d . I f , i n s o l u t i o n , compounds 72 and 60 r e s i d e d i n a

s i m i l a r c o n f o r m a t i o n a l s t a t e , then a marked d i s p a r i t y i n the

v i n y l p r o t o n c h e m i c a l s h i f t s would be e x p e c t e d .

121 122

1 37

The 1H NMR s p e c t r a of the s y n t h e t i c b i s d e h y d r o d i p e p t i d e s ,

106-111 known to possess the Z, Z - c o n f i g u r a t i o n (76), showed

v i n y l proton s i g n a l s having s i m i l a r chemical s h i f t s e p a r a t i o n s

to those observed i n the s p e c t r a of p e p t i d e s 60 and 73 (see

experimental and Table 2). Although these o b s e r v a t i o n s do not

f a c i l i t a t e assignment of the geometry of the C l i o n a p e p t i d e

a l k a l o i d s , they do provide an a l t e r n a t i v e to the e x p l a n a t i o n

that the observed chemical s h i f t d i s p a r i t i e s r e s u l t from the

presence of dehydroamino a c i d r e s i d u e s having o p p o s i t e

c o n f i g u r a t i o n s .

T e n t o x i n , a fungal m e t a b o l i t e r e c e n t l y s y n t h e s i z e d by R i c h

et a l . (133), i s p o s s i b l y the only aromatic dehydroamino a c i d -

c o n t a i n i n g p e p t i d e i n which the o l e f i n i c geometry has been

c o n c l u s i v e l y determined. Celenamide D (7_4) i s the f i r s t example

of a n a t u r a l l y o c c u r r i n g dehydropeptide p o s s e s s i n g m u l t i p l e

aromatic dehydroamino a c i d r e s i d u e s .

IV. Preparat ion of Dehydropept ides P o s s e s s i n g N-terminal

o6-Amino Ac i d s

Syntheses of dehydropeptides are few i n number and those

which p r o v i d e dehydropeptides with aromatic dehydroamino a c i d s

s i t u a t e d at other than the N-terminus are even fewer. The

s y n t h e t i c methods which have been employed, or which are

c o n c e i v a b l e , are o u t l i n e d i n a b b r e v i a t e d form i n scheme 10 f o r

the h y p o t h e t i c a l t r i p e p t i d e 123. I t i s not my i n t e n t i o n here to

d i s c u s s each of these s i x routes i n i n t i m a t e d e t a i l . A recent

138

comprehensive r e v i e w by Schmidt et a l . (81) p r o v i d e s e x c e l l e n t

coverage of t h i s s u b j e c t .

As i n d i c a t e d e a r l i e r , the purpose of our i n q u i r y was t o

de t e r m i n e the a v a i l a b i l i t y of a r a p i d method t o compounds such

as 123. I t i s ap p a r e n t from c l o s e e x a m i n a t i o n of scheme 10 t h a t

r o u t e s A, C and E encompass the l e a s t number of s y n t h e t i c s t e p s .

Routes B, D and F a r e not o n l y s u b s t a n t i a l l y l o n g e r (9-12 s t e p s )

but they a l s o have the added d i s a d v a n t a g e of e n t a i l i n g the

s y n t h e s i s and m a n i p u l a t i o n of f r e e amino a c i d s . Methods r e l a t e d

t o r o u t e E have a p p a r e n t l y not been r e p o r t e d , a l t h o u g h , two

r e l a t e d s y n t h e s e s (128, 137) l e n d c r e d a n c e t o i t s p o t e n t i a l (see

r e a c t i o n sequence b e l o w ) .

Scheme 10. Summary of employed and p o s s i b l e r o u t e s t o l i n e a r d e h y d r o p e p t i d e s p o s s e s s i n g N - t e r m i n a l cx-amino a c i d s .

1 40

The f i r s t examples of the f o r m a t i o n of d e h y d r o p e p t i d e s i n

which the dehydroamino a c i d component was not a t the N - t e r m i n a l

end can be a t t r i b u t e d t o Bergmann (115, 138). In 1932 (138)

N H 2 G l y A P h e O H 1 and i n 1943 (11 5) AcPheAPheOH were p r e p a r e d by

d e h y d r a t i o n of the c o r r e s p o n d i n g ^ - h y d r o x y a m i n o a c i d r e s i d u e s .

U n f o r t u n a t e l y , ^ - h y d r o x y a r o m a t i c amino a c i d s cannot be

r o u t i n e l y p r e p a r e d . In t h i s same 1943 r e p o r t (115) Bergmann

p r e s e n t e d the f o r m a t i o n of AcPheAPheOH by the c o n d e n s a t i o n of

AcPheGlyOH and benzaldehyde. The i n t e r m e d i a t e p e p t i d y l o x a z o l o n e

124 was i s o l a t e d i n 19 p e r c e n t y i e l d .

The a p p l i c a t i o n of t h i s s t r a i g h t f o r w a r d p r o c e d u r e has

s u b s e q u e n t l y been atte m p t e d by Konno and Stammer ( 1 3 4 a ) . They

were a b l e t o i s o l a t e the d e s i r e d o x a z o l o n e from CbzGlyGlyOH i n

l e s s than 5 p e r c e n t y i e l d .

In o r d e r t o determine whether the f a i l u r e of t h i s r e a c t i o n

was i n d e e d a g e n e r a l phenomenon we p r e p a r e d the p r o t e c t e d

1The a b b r e v i a t i o n A Phe r e f e r s t o <*,,$-didehydrophenylalan i n e .

141

d i p e p t i d e AcLeuGlyOH as d e s c r i b e d by Smart e t a l . (139) u s i n g

DCC. S e v e r a l a t t e m p t s have been made t o condense t h i s m a t e r i a l

w i t h 3 , 4 , 5 - t r i m e t h o x y b e n z a l d e h y d e by the Erlenmeyer p r o c e d u r e ,

but a l l have been u n s u c c e s s f u l . The reason f o r the s u c c e s s of

the Erlenmeyer r e a c t i o n when the N - a c y l s u b s t i t u e n t of g l y c i n e

i s Ac, Bz, -CH^Cl or a dehydroamino a c i d , i n c o n t r a s t t o i t s

f a i l u r e when t h i s group i s an oc-amino a c i d , i s not o b v i o u s .

P a r t i c u l a r l y , i n view of the apparent ease of f o r m a t i o n of

s i m i l a r s a t u r a t e d p e p t i d y l o x a z o l o n e s (134, 140).

The Bergmann-Grafe r e a c t i o n (141, r o u t e C) e n t a i l s the

c o n d e n s a t i o n of an c^-keto a c i d and an u n s u b s t i t u t e d amide.

W i e l a n d et a l . (135a) e x p l o i t e d t h i s r e a c t i o n i n the s y n t h e s i s

of s e v e r a l d e h y d r o a l a n i n e d e r i v a t i v e s i n c l u d i n g C b z G l y A A l a O H .

3,4,5-Trimethoxyphenyl p y r u v i c a c i d (142, 112) f a i l e d t o

condense w i t h the amide 117 under t h e s e c o n d i t i o n s . The use of a

v a r i e t y of a c i d i c c a t a l y s t s i n t h i s p r o c e d u r e has r e c e n t l y been

i n t r o d u c e d by S h i n e t a l . (135b) . Employment of

p h o s p h o r u s o x y c h l o r i d e i n the c o n d e n s a t i o n mentioned above l e d t o

p o l y m e r i z a t i o n of the Otf-keto a c i d , as d i d the use of p y r i d i n e .

Low y i e l d s (16%) of AcValAPheOMe (125) were o b t a i n e d when 1 17

was r e a c t e d w i t h m e t h y l p h e n y l p y r u v a t e i n the p r e s e n c e of

p h o s p h o r u s o x y c h l o r i d e . 1 Attempted c o n d e n s a t i o n of A c V a l A P h e O H

(126) w i t h e t h y l g l y c i n a t e has been u n s u c c e s s f u l . The a c i d 125

was u n r e a c t i v e towards h y d r a z i n e h y d r a t e , D C C - p - n i t r o p h e n o l ,

T h i s work was performed i n a s s o c i a t i o n w i t h K. Leavens ( C h e m i s t r y 449 s t u d e n t 1980 - 8 1 ) .

142

D C C - E t j N - h y d r o x y b e n z o t r i a z o l e and i t f a i l e d t o form an o x a z o l o n e

i n t h e presence of AcÔ-DMAP as d i d the dehydroamino a c i d s 94

and 95. These r e s u l t s a r e c o n s i s t e n t w i t h those r e p o r t e d by R i c h

e t a l . (133) .

R e g r e t t a b l y , we have been u n a b l e , t o d a t e , t o d i s c e r n a

r o u t e t o the d e s i r e d d e h y d r o t r i p e p t i d e s which f u l f i l l s the

r e q u i r e m e n t s o u t l i n e d p r e v i o u s l y .

143

EXPERIMENTAL

G e n e r a l

The JH NMR s p e c t r a were r e c o r d e d on N i c o l e t - O x f o r d 270,

Va r i a n XL-100, V a r i a n HA-100, Bruker WP-80, and Bruker WH-400

s p e c t r o m e t e r s . 1 3 C NMR s p e c t r a were r e c o r d e d on B r u k e r WH-400,

Bruker WP-80 and V a r i a n CFT-20 s p e c t r o m e t e r s . T e t r a m e t h y 1 s i l a n e

(t5"=0) was employed as an i n t e r n a l r e f e r e n c e i n a l l i n s t a n c e s .

L o w - r e s o l u t i o n and h i g h - r e s o l u t i o n EIMS were measured on A.E.I.

MS-902 and MS-50 s p e c t r o m e t e r s , r e s p e c t i v e l y . U l t r a v i o l e t

s p e c t r a l d ata was o b t a i n e d w i t h a Cary-14 u l t r a v i o l e t

s p e c t r o p h o t o m e t e r , i n f r a r e d s p e c t r a were r e c o r d e d on a P e r k i n -

Elmer model 700B s p e c t r o p h o t o m e t e r and o p t i c a l r o t a t i o n s were

measured on P e r k i n - E l m e r model 241C or model 141 p o l a r i m e t e r s . A

10 cm m i c r o c e l l was used f o r o p t i c a l r o t a t i o n measurements

u n l e s s o t h e r w i s e i n d i c a t e d . A J a s c o J-20 s p e c t r o - p o l a r i m e t e r was

employed f o r c i r c u l a r d i c h r o i s m measurements.

Gas chromatography and h i g h p r e s s u r e l i q u i d chromatography

were performed on H e w l e t t - P a c k a r d 5830A and P e r k i n - E l m e r S e r i e s

2 i n s t r u m e n t s , r e s p e c t i v e l y . A P e r k i n - E l m e r LC55 UV d e t e c t o r

system was employed f o r peak d e t e c t i o n d u r i n g HPLC. M e l t i n g

p o i n t s were d e t e r m i n e d on a F i s h e r - J o h n s a p p a r a t u s and are

u n c o r r e c t e d . Merck s i l i c a g e l 60 PF-254 was used f o r PTLC. The

HPLC s o l v e n t s were F i s h e r HPLC grade, Caledon HPLC grade or

B u r d i c k and Jac k s o n UV q u a l i t y ; water was g l a s s d i s t i l l e d ; a l l

o t h e r s o l v e n t s were reagent grade.

144

Sponge c o l l e c t ion

The sponge C l i o n a c e l a t a Grant was c o l l e c t e d by hand using

SCUBA i n Barkley Sound, Howe Sound, and the S t r a i g h t s of

Georgia, B r i t i s h Columbia, at an average depth of 18 metres'.

Specimens obtained from La J o l l a , C a l i f o r n i a were c o l l e c t e d at

33 metres' depth and those from Woods Hole, Massachusetts were

obtained at v a r y i n g depths. A l l other s p e c i e s of C l i o n a s t u d i e d

were c o l l e c t e d i n 3 metres of water at Hen and Chickens Reef,

two m i l e s o f f P l a n t a t i o n Key, F l o r i d a .

Specimens were p l a c e d i n methanol or ethanol at the dive

s i t e immediately f o l l o w i n g c o l l e c t i o n . The organisms were

homogenized i n a Waring Blendor, and allowed to stand o v e r n i g h t

at 3-5° C. The aqueous methanol e x t r a c t obtained f o l l o w i n g

f i l t r a t i o n was co n c e n t r a t e d j_n vacuo and the r e s u l t a n t

suspension p a r t i t i o n e d between b r i n e and EtOAc. The EtOAc l a y e r

was d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and

evaporated in vacuo. T r i t u r a t i o n (hexane, e t h e r , chloroform) of

the green s o l i d o b t ained a f f o r d e d a light-brown s o l i d mixture

c o n s i s t i n g of a l k a l o i d s f r e e of s t e r o l s , pigments and l i p i d s .

B r i t i s h Columbian specimens y i e l d e d , on an average, 3 g (0.3%)

of crude a l k a l o i d s from every 1 kg of wet sponge c o l l e c t e d . The

s o l e C a l i f o r n i a n c o l l e c t i o n was not q u a n t i t a t e d .

145

D e r i v a t i z a t i o n of the Crude A l k a l o i d E x t r a c t w i t h Acet i c

Anhydr ide and Acet i c Anhydr i d e - d f i

D e r i v a t i z a t i o n r e a c t i o n s were performed under n i t r o g e n i n

e i t h e r A c 20-py ( 5 : l , v / v ) or Ac20-sodium a c e t a t e ( 5 : 1 , w/w). In

e i t h e r c a s e , the s l u r r y was warmed u n t i l a c l e a r s o l u t i o n was

o b t a i n e d and then a l l o w e d t o s t i r o v e r n i g h t at room t e m p e r a t u r e .

Removal of the s o l v e n t s i_n vacuo and p a r t i t i o n of the r e s u l t a n t

gum between b r i n e and e t h y l a c e t a t e p r o v i d e d a s o l i d r e s i d u e of

a c e t y l a t e d a l k a l o i d s , i n e s s e n t i a l l y q u a n t i t a t i v e y i e l d ,

f o l l o w i n g d r y i n g (anhyd N a 2 S 0 4 ) and e v a p o r a t i o n of the EtOAc

l a y e r .

A s m a l l s c a l e i s o l a t i o n p r o c e d u r e (40 g wet weight of

sponge) employing a c e t i c anhydride-d_6 (10 ml, 99 + atom %,

A l d r i c h ) as the a c e t y l a t i n g agent y i e l d e d a m i x t u r e of

d e u t e r o a c e t y l a t e d a l k a l o i d s . The HPLC of t h i s m i x t u r e was

analogous t o t h a t shown i n f i g u r e 15a. The XH NMR (270 MHz,

acetone-de ) spectrum i n d i c a t e d the absence of s i g n a l s a r i s i n g

from n a t u r a l l y o c c u r i n g a c e t a m i d e s (-NHCOCH3 ) and phenol

a c e t a t e s (Ph-OCOCH 3).

I s o l a t i o n of C l i o n a m i d e (53)

The crude u n d e r i v a t i z e d a l k a l o i d s were d i s s o l v e d i n EtOAc

and r a p i d l y p a r t i t i o n e d w i t h 0.5-1M H 2 S O 4 . The a c i d l a y e r was

c o v e r e d w i t h f r e s h e t h y l a c e t a t e , the two phases were mixed and

146

deoxygenated by a r a p i d flow of n i t r o g e n , and sodium bicarbonate

was added u n t i l carbon d i o x i d e e v o l u t i o n ceased. A f t e r d r y i n g

over anhydrous Na^C^ and c o n c e n t r a t i o n , the e t h y l a c e t a t e

e x t r a c t was p u r i f i e d by PTLC (CHCI3-CH3OH, 5:2) to g i v e 53 as an

unstable yellow powder; Rf ( s i l i c a , C H C 1 3 -CH 3 OH, 3:1) 0.24

( F e C l 3 and n i n h y d r i n p o s i t i v e ) ; [cx] D + 32.1" (c 2.12, CH3OH,

l=lcm); UV (CH 3OH) (nm) X max 227 ( e 3.1 x 1 0 4 ) , 291 ( e 1.1 x

1 0 4 ) , 296 ( e 1.2 x 1 0 4 ) , 311 sh ( e 9.4 x 1 0 3 ) ; IR (KBr),p max

3350-3200 br, 1635, 1605, 1500 cm- 1; XH NMR (400 MHz, CD3OD,

f i g u r e 12) £ 3 . 0 1 (m, IH), 3.17 (m, IH), 3.66 ( t , IH, J=7), 5.97

(d, IH, J= 15 Hz, CH=CHNH), 6.32 (s, 2H, Ph-H), 7.08 (s, IH,

i n d o l e H-2), 7.09 (dd, IH, J=2, 8Hz, i n d o l e H-5), 7.15 (d, IH,

J=15 Hz, CH=CHNH), 7.48 (d, IH, J=2 Hz, i n d o l e H-7), 7.50 (d,

IH, J=8 Hz, i n d o l e H-4). A 5 mg sample of 53 a p p l i e d to a 0.65

cm d i s c of S c h l e i c h e r and S c h u e l l adsorbent paper gave a 1 mm

zone of i n h i b i t i o n of the growth of Staphylococcus aureus.

A c e t y l a t i o n of Clionamide (53 ) : T e t r a c e t y l c l i o n a m i d e (46)

Excess a c e t i c anhydride (lOmL) was added to a s o l u t i o n of

53 (100 mg, 0.23 mmole) i n p y r i d i n e (2 mL) and the r e a c t i o n

mixture was s t i r r e d o v e r n i g h t , at room temperature, under an

atmosphere of n i t r o g e n . E v a p o r a t i o n i_n vacuo and p u r i f i c a t i o n by

PTLC ( C H C I 3-CH 3 OH, 10:1) gave t e t r a c e t y l c l i o n a m i d e (46, 82 mg,

0.14 mmole, 61%), mp 209-211° (TH F - i s o p r o p y l e t h e r ) ; [c*0D + 45°

(c 0.7, acetone); UV (CH 3OH) X max (nm) 227 (e 5.9 * 1 0 4 ) , 290

(€ 3.7 x 1 0 4 ) ; IR (KBr) v max 3418, 3280, 1760, 1627 cm- 1; JH

147

NMR ( f i g u r e 3, t a b l e 1 ) ; 1 3 C NMR (CD 3CN) 20.4 ( I C ) , 20.9 (2C),

23.1 ( I C ) , 28.3 ( I C ) , 55.2 ( I C ) , 111.3 ( I C ) , 112.1 ( I C ) , 115.1

( I C ) , 115.5 ( I C ) , 118.3 ( 2 C ) , 121.0 ( I C ) , 122.7 (2C), 125.5

(2C) , 127.7 ( I C ) , 136.3 ( I C ) , 138.4 ( I C ) , 145.0 ( 2 C ) , 168.1

( I C ) , 168.9 ( 2 C ) , 171.0 ( I C ) , 171.9 ppm ( I C ) ; MS ( f i g u r e 2 ) ;

Exact mass c a l c d f o r C 2 7 H 2 6 * 1 B r N 3 0 8 , 601.0874; obsd, 601.0889.

A n a l c a l c d f o r C 27H 26BrN 308 C54.01, H4.36, N7.00; found, C53.89,

H4.40, N7.09.

I s o l a t i o n of T e t r a c e t y l c l i o n a m i d e di2 (54)

Workup of the crude d e u t e r o a c e t y l a t e d a l k a l o i d m i x t u r e as

d e s c r i b e d f o r the i s o l a t i o n of the a c e t y l a t e d c e l e n a m i d e s

(below) gave t e t r a c e t y l c l i o n a m i d e - d _ 6 ( 5 4 ) . S i g n a l s at (51.85

(s,3H) f o r acetamide (-NHCOCH3) and 8 2.23 (s,9H) f o r t h r e e

p h e n o l a c e t a t e s (Ph-OCOCH 3) p r e s e n t i n the spectrum of

t e t r a c e t y l c l i o n a m i d e (4_6) were absent i n the *H NMR spectrum of

54 ( f i g u r e 1 0 ) .

C l i o n a m i d e A c e t o n i d e (56) and C l i o n a m i d e Acetonide-d6 (55)

C l i o n a m i d e (53, 30 mg, 0.07 mmole) was d i s s o l v e d i n

anhydrous a c e t o n e or acetone-d§ f o r l h a t room t e m p e r a t u r e .

C o n c e n t r a t i o n and p u r i f i c a t i o n by PTLC (CHCl 3-MeOH, 5:2) gave

the a c e t o n i d e 5_6 (19 mg, 0.04 mmole, 57%) or the

d e u t e r o a c e t o n i d e 5_5, r e s p e c t i v e l y , as s t a b l e o i l s . 56; Rf

148

(CHC1 3-CH 30H, 3:1) 0.54 ( F e C l 3 and n i n h y d r i n p o s i t i v e ) ; UV

(CH 3OH) X max (nm) 229 (e 2.5 x 1 0 4 ) , 297 (e 1.7 * 1 0 4 ) , 315 sh

(€ 1.4 * 1 0 4 ) ; IR (KBr) ^max 3300 b r , 1635 b r , 1605, 1508 cm- 1; JH NMR (270 MHz, acetone-d6, f i g u r e 14) 6 1.42 ( s , 3H,

a c e t o n i d e - C H 3 ) , 1.47 ( s , 3H, a c e t o n i d e - C H 3 ) , 3.11 (dd, IH,

J=7.5, 14.5 H z ) , 3.31 (dd, IH, J=5, 14.5 H z ) , 3.86 (dd, IH, J=5,

7.5 H z ) , 6.50 ( s , 2H), 6.75 (d, IH, J=14.2 H z ) , 6.83 (d, IK,

J=14.2 H z ) , 7.14 (dd, IH, 3 = 2, 8.5 H z ) , 7.28 ( s , I H ) , 7.57

( d , l H , J=2 H z ) , 7.58 ( d , IH J=8.5 H z ) , 10.09 ( b s , IH, D 20

exchange); 1 3C NMR ( ( C D 3 ) 2 C O ) , 20 MHz, appendix 2, 5_5) , 59.1

( I C ) , 105.7 ( 2 C ) , 106.0 ( I C ) , 120.7 ( I C ) , 121.3 (2C), 122.6

( 2 C ) , 125.6 ( I C ) , 125.7 ( I C ) , 129.5 ( I C ) , 146.8 ( I C ) , 174.2 ppm

( I C ) ; MS (10-70 eV) m/e 471/473 (0.02, 1:1), 210/208 (100, 1:1).

H i g h r e s o l u t i o n mass measurement was performed on the d i a c y l

d e r i v a t i v e of 5_6 o b t a i n e d f o l l o w i n g a c e t y l a t i o n w i t h Ac20-py.

E x a c t mass c a l c d f o r C 2 6 H 2 68 1 B r N 2 0 6 , 557.0965; obsd, 557.0990.

The d e u t e r o a c e t o n i d e 5_5 was i d e n t i c a l (TLC, UV, JH NMR

( f i g u r e 1 3 ) , MS) t o 5_6 w i t h the e x c e p t i o n of the absence of JH

NMR s i g n a l s a t 6 1.42 ( s , 3H) and 8 1.47 ( s , 3H) f o r two

a c e t o n i d e m e t h y l groups and the complex n a t u r e of the s i g n a l s i n

t h e mass spectrum.

H y d r o l y s i s of T e t r a c e t y l c 1 i o n a m i d e (46) w i t h H y d r o c h l o r i c Ac i d

T e t r a c e t y l c l i o n a m i d e (46, 145 mg, 0.24 mmole) was d i s s o l v e d

i n a c e t o n i t r i l e (15 mL, 0.1% H 20) and c o n c e n t r a t e d HC1 (0.25 mL

i n 2 mL CH 3CN) was added. A f t e r 48h a t room t e m p e r a t u r e the

149

s o l v e n t was e v a p o r a t e d and the r e s i d u e p u r i f i e d by PTLC ( C H C I 3 -

CH3OH, 5:1) t o g i v e the bromo amide 5_1 (21 mg, 0.06 mmole, 2 7 % ) ;

[« ] D + 4.6° (c 3.2, CH3OH, l = l c m ) ; XH NMR (100 MHz, (CD 3 )2CO) <5"

1.87 ( s , 3H), 3.13 (dd, IH, J=8, 14 H z ) , 3.29 (dd, IH, J=6, 14

H z ) , 4.71 (dd, IH, J=6, 8 H z ) , 6.40 (bs, IH, D2O exchange, -

CONH 2), 6.93 (b s , IH, D2O exchange, - CONH 2), 7.17 (dd, IH, J=9,

2 H z ) , 7.61 ( d , IH, J=2 H z ) , 7.64 ( d , IH, J=9 H z ) , MS m/e, M*

325/323 ( 3 , 1:1), 266/264 (43, M*-C 2H 5NO, 1:1), 210/208 (100,

1:1), 129 ( 3 5 ) , 128 ( 1 2 ) . E x a c t mass c a l c d f o r C 2 3 H 1 47 ' B r N 3 0 2 ,

323.0269; obsd, 323.0251.

C a t a l y t i c H y d r o q e n a t i o n of T e t r a c e t y l c l i o n a m i d e (46)

P a l l a d i u m on c h a r c o a l c a t a l y s t ( 5 % , 15 mg) was added t o a

s o l u t i o n of 46 (60 mg, 0.10 mmole) i n methanol (20 mL). The

s o l u t i o n was s t i r r e d under an atmosphere of hydrogen o v e r n i g h t ,

f i l t e r e d t h r o u g h C e l i t e , c o n c e n t r a t e d , and p u r i f i e d by PTLC

(EtOAc) t o g i v e 5_0 (26 mg, 0.04 mmole, 43%) as a c o l o u r l e s s o i l ;

[<x]D +6.2' (c 4.37, a c e t o n e , l = l c m ) ; UV (CH 3OH) X max (nm) 224

(e 3.38 x 1 0 4 ) , 283 (e 6.02 x 1 0 3 ) ; *H NMR (100 MHz, (CD 3)2CO) (5"

1.88 ( s , 3H), 2.24 ( s , 9H), 2.67 ( t , 2H, J=7 Hz,-CH 2CH 2NH-),

3.12 (m, 2H), 3.38 (m, 2H, D 2 0 - t , J=7 Hz,-CH 2CH 2NH-), 4.63 (m,

I H ) , 6.94 ( s , 2H), 7.11 (m, I H ) , 7.24 ( s , I H ) , 7.60 (d, IH, J=2

H z ) , 7.62 ( d , IH, J=9 H z ) , 10.40 (b s , IH, D 20 exchange); MS m/e,

M* 603/601 (0.3, 1:1), 544/542 ( 3 , M*-C 2H 5NO, 1:1), 210/208

(100, 1:1). E x a c t mass c a l c d f o r C 2 7 H 2 8 * I B r N 3 0 8 , 603.1040; obsd,

603.1020.

150

H y d r o g e n o l y s i s of the Bromo Amide 50

P a l l a d i u m on c h a r c o a l c a t a l y s t ( 5 % , 10 mg) was added t o a

s o l u t i o n of 5_0 (26 mg, 0.04 mmole) i n C H 3 O H (15 mL) c o n t a i n i n g

0.2% C H 3 C O 2 H . The r e a c t i o n m i x t u r e was s t i r r e d a t room

t e m p e r a t u r e , under an atmosphere of hydrogen, f o r 72h, f i l t e r e d

t h r o u g h C e l i t e , c o n c e n t r a t e d , and p u r i f i e d by PTLC (EtOAc) t o

g i v e 4_9 (8 mg, 0.02 mmole, 38%) as a c o l o u r l e s s o i l ; IR (KBr) j /

max 3400, 1760, 1635 cm- 1; 1H NMR (270 MHz, acetone-d_6 , f i g u r e

5) S 1.81 ( s , 3H), 2.25 ( s , 9H), 2.64 ( t , 2H, J=7 H z ) , 3.10 (m,

2H), 3.32 (m, 2H, D 2 0 - t , J=7 H z ) , 4.48 (m, I H ) , 6.85 ( s , 2H),

7.00 (m, IH, i n d o l e H-5), 7.0 ( I H , D 20 exchange), 7.06 (m, IH,

i n d o l e H-6), 7.10 ( s , IH, i n d o l e H-2), 7.25 (d, IH , J=8 Hz, D 20

exchange), 7.35 ( d , IH, J=8 Hz, i n d o l e H-7), 7.64 (d, IH, H=9

Hz, i n d o l e H-4), 10.14 (b, IH, D 20 exchange, i n d o l e H - l ) ; S p i n

d e c o u p l i n g (100 MHz, ( C D 3 ) 2 C O - D 2 0 ) , x H o b s - { x H i r r } , $ 2.65 ( s ,

2H) - {3.36}, 3.12 (b, 2H) - {4.52}, 3.36 ( s , 2H) - {2.65}, 4.52

( s , IH) - {3.12}; MS m/e, M* 523 ( 0 . 3 ) , 481 (0.5, M* - C 2 H 2 0 ) ,

464 (15, M* - C 2H 5NO), 422 ( 1 5 ) , 380 ( 1 4 ) , 130 (100). E x a c t mass

c a l c d f o r C 2 7 H 2 9 N 3 0 8 , 523.1954; obsd, 523.1970.

H y d r o g e n o l y s i s of the Bromo Amide 51

The p r o c e d u r e o u t l i n e d f o r the h y d r o g e n o l y s i s of the bromo

amide 50 was f o l l o w e d ( 5 % Pd/C, 6 mg). P u r i f i c a t i o n was

performed by HPLC ( s i l i c a A, h e x a n e - i s o p r o p y l a l c o h o l g r a d i e n t )

151

t o g i v e the debromo amide 52 ( f i g u r e 8) (8.4 mg, 0.03 mmole, 34%

from 31.3 mg, 0.10 mmole of 5 1 ) ; [oc]D + 15° (c 1.0, CH 3OH) ( l i t .

[c*]£ 3 + 20 ± 1° (c 2%, CH 3OH) 50). The debromo a c i d 52 was

i d e n t i c a l t o a u t h e n t i c ( S ) - 2 - a c e t a m i d o - 3 - ( i n d o l e - 3 ' - y l ) -

p ropionamide (Sigma; [o6]D + 17° (c 1.4, CH 3OH)).

S y n t h e s i s of the U l t imate H y d r o g e n a t i o n Product (49) of T e t r a c e t y l c 1 i o n a m i d e (46)

5-Hydroxydopamine h y d r o c h l o r i d e (86.0 mg, 0.42 mmole) was

added, as a s o l i d t o a s o l u t i o n of DCC (90.7 mg, 0.44 mmole),

( S ) - N - a c e t y l t r y p t o p h a n (105.3 mg, 0.43 mmole), and t r i e t h y l a m i n e

(0.25 mL) i n THF -CH3CN (10:1, 50 mL). The r e a c t i o n m i x t u r e was

s t i r r e d a t room tem p e r a t u r e o v e r n i g h t , f i l t e r e d t h r o u g h C e l i t e ,

and e v a p o r a t e d . The crude r e s i d u e was a c e t y l a t e d (Ac£0-py,

20:1), e v a p o r a t e d i_n vacuo and p u r i f i e d t w i c e by PTLC (CHCI3-

C H 3 O H , 20:1) t o g i v e a c o l o u r l e s s o i l (66.5 mg, 0.13 mmole, 30%)

i d e n t i c a l (TLC UV, *H NMR, ( f i g u r e 6) , IR, MS) t o the u l t i m a t e

h y d r o g e n a t i o n p r o d u c t 49 of t e t r a c e t y l c l i o n a m i d e ( 4 6 ) .

I s o . l a t i o n of A c e t y l a t e d Celenamides

Chromatography of the a c e t y l a t e d r e s i d u e on a column of

s i l i c a g e l G p r o v i d e d a f r a c t i o n e l u t i n g w i t h 10-50% e t h y l

a c e t a t e i n c h l o r o f o r m c o n t a i n i n g a m i x t u r e of a c e t y l a t e d

c e l e n a m i d e s and a second f r a c t i o n e l u t i n g w i t h e t h y l a c e t a t e

152

c o n t a i n i n g t e t r a c e t y l c l i o n a m i d e (46; 0.13%, wet weight). Pure

hexacetylcelenamide A (58; 0.03%, wet weight),

hexacetylcelenamide B (57; 0.02%), p e n t a c e t y l c e l e n a m i d e C (59,

0.003%) and nonacetylcelenamide D (60; 0.002%) were obtained as

n o n c r y s t a l l i n e white s o l i d s by repeated chromatography on s i l i c a

p l a t e s with c h l o r o f o r m - m e t h a n o l - a c e t o n i t r i l e (19:1) as eluant

and i n s e v e r a l i n s t a n c e s RP chromatography on Whatman K C i 8

p l a t e s with water-methanol (3:5:2) as e l u a n t .

I s o l a t i o n of P e u t e r p a c e t y l a t e d Celenamides

D e u t e r o a c e t y l a t e d celenamides were i s o l a t e d by PTLC as

d e s c r i b e d above in the case of the a c e t y l a t e d celenamides.

Hexacetylcelenamide A - d 1 8 (68; , f i g u r e 27), B-d 1 8 (69, f i g u r e

28), and p e n t a c e t y l c e l e n a m i d e C-dis ( f i g u r e 27) were i s o l a t e d as

p a r t i a l l y p u r i f i e d mixtures. Nonacetylcelenamide D-d i 7 and the

remainder of the d e u t e r o a c e t y l a t e d celenamides were examined by 1H NMR as a crude mixture. 1H NMR a n a l y s i s of the

d e u t e r o a c e t y l a t e d celenamides i n d i c a t e d an absence of s i g n a l s

a r i s i n g from n a t u r a l l y o c c u r r i n g acetamides (-NHCOCH^) and

phenol a c e t a t e s (Ph-OCOCHj).

Hexacetylcelenamide A (58)

58: [<*]D+ 40° (c 1.1, acetone); UV (MeOH) X max (nm) 227 (V

7.2 x 10"), 289 (br, € max 5.1 x 10"); IR (CHCI3) V max 3300

153

( b r ) , 1680, 1660 ( b r ) , 1380, 1200 cm- 1; »H NMR ( f i g u r e 19, t a b l e

1 ) ; 1 J C NMR (100.6 MHz, CDC1 3, appendix 4) 20.0, 20.6 ( 2 C ) , 20.7

(2C), 21.4, 22,8, 23.1, 25.2, 26.6, 39.8, 53.2, 55.3, 111.3,

112.9, 114.5, 115.6, 120.1, 120.3, 121.8 (2C), 122.8, 123.5,

123.8, 124.1, 124.4, 125.8, 126.8, 130.2, 131.4, 135.0, 136.0,

137.2, 140.6, 142.5, 143.5 ( 2 C ) , 165.3, 167.0, 168.2 ( 4 C ) ,

169.4, 172.0, 174.1 ppm; MS ( f i g u r e 21, scheme 3 ) .

H e x a c e t y l c e l e n a m i d e B . (57)

57: [t*] D+ 22° (c 1.1, a c e t o n e ) ; UV (MeOH) X max (nm) 227 (e

8.0 x 1 0 4 ) , 289 ( b r , e max 6.0 * 1 0 4 ) ; JH NMR ( f i g u r e 20, t a b l e

1 ) ; IR (CHCI3 ) v max 3340 ( b r ) , 1780, 1660 ( b r ) , 1510 ( b r ) ,

1380, 1200 cm- 1; 1 3C NMR (20.1 MHz, CDCl3-DMSO-d_6 [ 3 : 2 ] ,

appendix 6) 170.2, 168.3, 166.2, 164.9, 164.5, 163.6, 161.7,

140.4 (2C, s ) , 1 139.0 ( s ) , 139.2 ( s ) , 132.7 ( s ) , 132.0 ( s ) ,

129.0 ( s ) , 127.4, 124.2, 123.2, 121.7, 121.3, 120.4, 120.2,

113.9 (2C), 118.5, 116.7 ( 2 C ) , 111.4, 111.2, 108.1, 107.1, 56.5

( d ) , 51.9 ( d ) , 39.1 ( t ) , * 27.2 ( d ) , 23.5, 19.5, 17.4 (q, 3C),

17.0 ( q ) , 16.4 ( q ) , 15.3 ( q ) ; MS ( F i g u r e 22, Scheme 3 ) ; CD

(CH 3CN), (appendix 1 1 ) .

M u l t i p l i c i t i e s were o b t a i n e d i n a s i n g l e f r e q u e n c y o f f - r e s o n a n c e d e c o u p l e d spectrum (100.6 MHz).

*T h i s s i g n a l was not ob s e r v e d due t o masking by the DMSO m u l t i p l e t c e n t e r e d a t 37.0 ppm. The c h e m i c a l s h i f t was o b t a i n e d a t 100.6 MHz i n CDC1 3.

154

P e n t a c e t y l c e l e n a m i d e C (59)

59: [oc]D + 14 c (c 0.30, a c e t o n e ) ; IR (CHCI 3 ) v max 3300 ( b r ) , 1780, 1660 ( b r ) cm- 1; JH NMR ( f i g u r e 29, t a b l e 1 ) ; MS (70 eV, f i g u r e 32, scheme 4 ) .

N o n a c e t y l c e l e n a m i d e D (60)

60: [ & ] D -25° (c 0.54, a c e t o n e ) ; IR (CHCI 3) v max 3300 ( b r ) , 1780, 1680 (br) cm- 1; JH NMR ( f i g u r e 33, t a b l e 1 ) ; MS (70 eV, f i g u r e 34, scheme 5 ) .

H y d r o l y s i s of A c e t y l a t e d Celenamides w i t h H y d r o c h l o r i c Ac i d

To 200 mg (0.21 mmole) of p a r t i a l l y p u r i f i e d 5_8 was added

50 mL of a c e t o n i t r i l e and 0.5 mL of h y d r o c h l o r i c a c i d . The

s o l u t i o n was s t i r r e d a t room t e m p e r a t u r e f o r 24 h. E v a p o r a t i o n

of the s o l v e n t s gave an o i l which was chromatographed on s i l i c a

p l a t e s w i t h c h l o r o f o r m - m e t h a n o l (5:1) as e l u a n t t o g i v e 21 mg

(0.03 mmole, 14%) of the p e p t i d e amide 65; mp 153-156°C (CHCl 3 -

a c e t o n e ) ; IR (CH 3CN) v max 3600, 3530, 3340, 1780, 1670 cm- 1; lH

NMR ( f i g u r e 23, t a b l e 1 ) ; 1 3 C NMR ( ( C D 3 ) 2 C O , 20 MHz, appendix 7)

175.4, 174.6, 173.3, 168.5 (2C), 167.6, 165.3, 144.6 ( 2 C ) ,

133.0-112.2 ( c o m p l e x ) , 55.7, 54.6, 40.4, 33.2-26.9 (masked by

acetone s i g n a l ) 25.5, 23.4, 22.8, 21.8, 20.6 ( 2 C ) , 20.1; MS

( f i g u r e 2 5 ) ; A n a l c a l c d f o r C 3 4 H 3 8 N 5 O 1 0 B r • 2/3H 20, C 53.13, H

155

5.16, N 9.11; found, C 5 3.12, H 5 . 0 4 , N 8 . 9 3 and C 5 3 . 1 5 , H 5 . 0 1 , N 8.98.

S i m i l a r t r e a t m e n t of 57 ( 150 mg, 0 . 1 6 mmole) gave the

p e p t i d e amide 66 (18 mg, 0 . 0 2 mmole, 16%) as a n o n c r y s t a l l i n e

w h i t e s o l i d : IR (CH 3CN) v max 3 6 0 0 , 3 5 3 0 , 3 3 4 0 , 1 7 8 0 , 1670 cm- 1; aH NMR ( f i g u r e 24, t a b l e 1 ) ; 1 3C NMR (CD 3CN, 20 MHz, appendix 8)

1 7 5.2, 1 7 3 . 6 ( 2 C ) , 169.4 ( 2 C ) , 1 6 8.2, 1 6 5.4, 144.7 ( 2 C ) , 1 3 9 . 0 -

111.3 ( c o m p l e x ) , 6 0.3, 56.4, 31.1, 27.1, 23.0, 20.9 ( 2 C ) , 2 0 . 5 ,

19.7, 1 8 . 6 ; MS ( f i g u r e 2 6 ) .

P e n t a c e t y l c e l e n a m i d e C ( 5 9 , 3 mg, 0 . 0 0 3 mmole) a f f o r d e d the

p e p t i d e amide 65 (1 mg, 0 . 0 0 1 mmole, 4 0 % ) . Compound 65 was

i d e n t i c a l (TLC, lH NMR ( f i g u r e 3 0 , t a b l e 1 ) , MS ( f i g u r e 3 D ) t o

the h y d r o l y s i s p r o d u c t i s o l a t e d f o l l o w i n g s i m i l a r t r e a t m e n t of

h e x a c e t y l c e l e n a m i d e A ( 5_8 ) .

N o n a c e t y l c e l e n a m i d e D (6_0 8 mg, 0 . 0 0 8 mmole) gave the

p e p t i d e amide 7_3 (3 mg, 0 . 0 0 4 mmole, 4 7 % ) : IR (CH 3CN) v max

3 6 4 0 , 3 5 4 0 , 3 3 5 0 , 1 7 8 0 , 1 6 8 0 ( b r ) cm- 1; JH NMR ( f i g u r e 3 5 , t a b l e

1 ) ; MS (70 eV, f i g u r e 3 6 ) .

O z o n o l y s i s of H e x a c e t y l c e l e n a m i d e A ( 5 8) and B (57)

Ozone was passed t h r o u g h a methanol s o l u t i o n ( 40 mL) of 58

(32 mg, 0 . 0 3 mmole) a t -78°C f o r 4 min. E x c e s s ozone was removed

i n a stream of oxygen, d i m e t h y l s u l f i d e (2 mL) was added, and

the m i x t u r e was a l l o w e d t o warm t o room t e m p e r a t u r e . E v a p o r a t i o n

of the s o l v e n t s and p u r i f i c a t i o n on s i l i c a p l a t e s w i t h e t h y l

a c e t a t e as e l u a n t gave 3 , 4 , 5 - t r i a c e t o x y b e n z a l d e h y d e (4 mg, 0 . 0 1

156

nunole, 43%) and 3 , 4 - d i a c e t o x y b e n z a l d e h y d e (3 mg, 0.01 mmole,

41%) which were i d e n t i f i e d by comparison (TLC and IR, *H NMR,

and MS) w i t h a u t h e n t i c samples p r e p a r e d from g a l l i c a c i d (see

p.6l) and 3, 4 - d i h y d r o x y b e n z a l d e h y d e ( A l d r i c h ) , r e s p e c t i v e l y (see

appendix l ) .

3 , 4 , 5 - T r i a c e t o x y b e n z a l d e h y d e (3 mg, 0.01 mmole, 47%) and

3 , 4 - d i a c e t o x y b e n z a l d e h y d e (2 mg, 0.01 mmole, 39%) were a l s o

o b t a i n e d by o z o n o l y s i s of 5_7 (22 mg, 0.02 mmole).

O z o n o l y s i s of the P e p t i d e Amides 65 and 66

Treatment of 65 (10 mg, 0.01 mmole) and 66 (10 mg, 0.01

mmole) as d e s c r i b e d f o r the o z o n o l y s i s of 5_7 and 5_8 gave 3,4,5-

t r i a c e t o x y b e n z a l d e h y d e (2 mg, 0.007 mmole each; 65, 54%; 66 53%;

TLC, XH NMR, MS) as the s o l e b e n z a l d e h y d i c p r o d u c t .

O z o n o l y s i s of a Crude A c e t y l a t e d Celenamide Sample

O z o n o l y s i s of a m i x t u r e of a c e t y l c e l e n a m i d e s s i m i l a r i n

c o m p o s i t i o n t o t h a t shown i n f i g u r e 15 a f f o r d e d 3,4,5-

t r i a c e t o x y - , 3 , 4 - d i a c e t o x y - , and 4- a c e t o x y b e n z a l d e h y d e as the

o n l y i s o l a b l e p r o d u c t s . The s u b s t i t u t i o n p a t t e r n of each of the

b e n z a l d e h y d i c p r o d u c t s was d e t e r m i n e d by comparison w i t h

s y n t h e t i c samples.

157

H y d r o l y s i s of H e x a c e t y l c e l e n a m i d e s A (58) and B (57) w i t h 6N

H y d r o c h l o r i c Ac i d

A g l a s s ampoule c o n t a i n i n g 14 mg (0.01 mmole) of 5_8 and 5

mL of 6 N h y d r o c h l o r i c a c i d was s e a l e d under n i t r o g e n and heated

at 100°C f o r 18 h. The ampoule was c o o l e d t o room t e m p e r a t u r e ,

opened, and the c o n t e n t s were washed out w i t h 5 mL of d i s t i l l e d

w a t e r . The s o l v e n t was e v a p o r a t e d i n vacuo, and the s o l i d

o b t a i n e d was t r i t u r a t e d w i t h 1 N a c e t i c a c i d . F i l t r a t i o n of the

m i x t u r e and e v a p o r a t i o n of the s o l v e n t i n vacuo gave 1 mg of a

l i g h t brown s o l i d . Two-dimensional TLC ( c e l l u l o s e ; f i r s t

d i m e n s i o n , 1-butanol s a t u r a t e d w i t h ammonia; second d i m e n s i o n ,

1 - b u t a n o l - a c e t i c a c i d (9:1) s a t u r a t e d w i t h water) i n d i c a t e d the

pre s e n c e of l e u c i n e . S i m i l a r t r e a t m e n t of 5_7 w i t h 6N HCl gave

v a l i n e .

O x a l i c Ac i d

The complex m i x t u r e of p o l a r p r o d u c t s (20 mg) o b t a i n e d from

the o z o n o l y s i s of 58 was d i s s o l v e d i n 3 N h y d r o c h l o r i c a c i d (10

mL), and the s o l u t i o n was r e f l u x e d f o r 1 h. The s o l v e n t was

e v a p o r a t e d i n vacuo, and the s o l i d o b t a i n e d was t r i t u r a t e d w i t h

m ethanol. The m e t h a n o l - s o l u b l e m a t e r i a l was t r e a t e d w i t h excess

diazomethane i n d i e t h y l e t h e r , and the m i x t u r e was a n a l y z e d by

gas chromatography (10% DEGS, 4 mm x 2 m column, He f l o w r a t e 30

mL/min). The s o l e peak o b s e r v e d i n the gas chromatogram had an

158

i d e n t i c a l r e t e n t i o n time (R T=4.61 min) w i t h a u t h e n t i c d i m e t h y l

o x a l a t e .

D i m e t h y l o x a l a t e was a l s o o b s e r v e d when the p o l a r m a t e r i a l

o b t a i n e d from the o z o n o l y s i s of 5_7 (14 mg) was t r e a t e d i n a

s i m i l a r f a s h i o n .

M e t h a n o l y s i s of the P e p t i d e Amides 65 and 66

The p e p t i d e amide 65 (6 mg, 0.006 mmole) was d i s s o l v e d i n

methanol (20 mL), and the s o l u t i o n was s t i r r e d at 50°C f o r 4 h

under an atmosphere of n i t r o g e n . The r e a c t i o n m i x t u r e was c o o l e d

t o room t e m p e r a t u r e , and e x c e s s diazomethane i n d i e t h y l e t h e r

was added. The s o l v e n t s were e v a p o r a t e d t o o b t a i n 61: IR (KBr) v

max 3340 ( b r ) , 2950, 1640 ( b r ) cm- 1; 1H NMR ( ( C D 3 ) 2 S O ) 6 0.92

(d, 1 H , J=7 H z ) , 0.98 (d, IH, J=7Hz), 1.7 (m, 3H), 1.98 ( s ,

3H), 3.33 (m, 2H), 3.67 (s,3H, OMe), 3.78 ( s , 6H, OMe), 4.26 (m,

I H ) , 4.61 (m, I H ) , 6.87 ( s , 2H), 7.06 ( s , I H ) , 7.14 (dd, IH,

J=2, 8Hz), 7.12 (br s, IH, e x c h ) , 7.28 ( d , IH, H=2 H z ) , 7.40 (br

s , l H e x c h ) , 7.50 (d, IH, J=2, 8 H z ) , 7.60 (d, IH, J=8 H z ) , 8.33

(m, 2H, e x c h ) , 9.96 ( s , IH, e x c h ) , 10.90 (br s, IH, e x c h ) ; MS

(70 e V ) , m/e M* 673/671 (1:1, 2.4), 266/264 (1 : 1 , 1 6 ) , 210/208

(1: 1 , 5 8 ) , 181 ( 3 0 ) , 130 ( 2 8 ) , 129 ( 5 3 ) , 128 ( 4 4 ) , 86 ( 1 0 0 ) , 43

( 9 5 ) ; E x a c t mass c a l c d f o r C 3 1 H 3 8 * 1 B r N 5 0 7 , 673.1927; obsd,

673.1934.

Treatment of 66 w i t h methanol as d e s c r i b e d f o r 65 a f f o r d e d

the t r i m e t h o x y d e r i v a t i v e 62; IR (KBr) v max 3340 ( b r ) , 2950,

1640 ( b r ) cm- 1; 'H NMR ( ( C D 3 ) 2 S O ) £ 0.86 (d, 3H, J=6.Hz), 0.91

159

(d, 3H, J=6 Hz), 1.80 (s, 3H), 1.97 (m, IH), 2.97 (dd, IH, J=10,

15Hz), 3.17 (dd, IH, J=5, 15 Hz), 3.67 (s, 3H, OMe), 3.78 (s,

6H, OMe), 4.16 (dd, IH, 3=2, 8.4 Hz), 4.61 (ddd, IH, J = 5, 8.6,

10 Hz), 6.98 (s, 2H), 7.09 (dd, IH, 3=2 Hz), 7.11 (br s, IH,

exch), 7.17 (s, IH), 7.22 (d, IH, 3=2 Hz), 7.40 (br s, IH,

exch), 7.43 (d, IH, J=8.6 Hz, exch), 7.51 (d, IH, 3=2 Hz), 7.62

(d, IH, J=8.4 Hz), 8.36 (d, IH, 3=1 Hz, exch), 10.01 (s, IH,

exch), 11.03 (br s. IH, exch); MS (70 eV), m/e M* 659/657 (1:1,

0.4), 266/264 (1:1, 32), 210/208 (1:1, 100), 181 (57), 129 (40),

114 (27), 72 (52), 43 (52); Exact mass c a l c d f o r C 3 0 H 3 6 • 1 B r N 5 0 7 ,

659.1786; obsd, 659.1778.

127 1 2 8

The t r i p h e n o l i c compounds 127 and 128 were observed i n JH

NMR experiments i n which (CD 3) 2CO s o l u t i o n s of 6_5 and £6 were

warmed (50°C, 30 min) i n the presence of water, D 20 or CD3OD.

For 127: »H NMR 6 0.91 (d, 3H, 3=1 Hz), 0.98 (d, 3H, H=7 Hz),

1.7 (m, 3H), 1.89 (s, 3H), 3.31 (m, 2H), 4.42 (m, IH), 4.72 (m,

1H), 6.44 (br s, IH, exch), 6.72 (s, 2H), 7.08 (s, IH), 7.16

(dd, IH, 3=2, 8.4 Hz), 7.21 (br s, IH, exch), 7.30 (d, IH, 3=2

Hz), 7.54 (d, IH, 3=2 Hz), 7.60 (d, IH, J=8.4 Hz), 7.79 (s, IH,

exch, ArOH), 7.83 (s, 2H, exch, ArOH), 8.12 (m, 2H, exch), 8.88

160

( s , 1H, e x c h ) , 10.21 (br s, IH, e x c h ) . For 126: *H NMR <5" 0.94

(d , 3H, J=7 H z ) , 0.97 (d, 3H, J=7 H z ) , 1.9 (m, I H ) , 1.97 ( s ,

3H), 3.3 (m, 2H), 4.35 (m, l H ) , 4.78 (m, I H ) , 6.76 ( s , 2H), 7.14

(dd, IH, J=2, 8 H z ) , 7.20 ( s , I H ) , 7.35 (br s, I H ) , 7.57 (d, IH,

3=2 H z ) , 7.64 ( d , IH, J= 8 H z ) . F e r r i c c h l o r i d e p o s i t i v e

r e a c t i o n s were g i v e n by both 127 and 128.

R e d u c t i o n of the Enamides 61 and 62 w i t h Sodium B o r o h y d r i d e

To 2 mg of a 1:1 m i x t u r e of 61 and 62 d i s s o l v e d i n 15 mL of

e t h a n o l was added 100 mg of sodium b o r o h y d r i d e . The s o l u t i o n was

s t i r r e d a t room tem p e r a t u r e f o r 20h. Excess reagent was

d e s t r o y e d w i t h 1 N h y d r o c h l o r i c a c i d , and the s o l u t i o n was

c o n c e n t r a t e d and e x t r a c t e d w i t h d i c h l o r o m e t h a n e . The

d i c h l o r o m e t h a n e l a y e r was d r i e d over anhydrous sodium s u l f a t e

and e v a p o r a t e d t o g i v e a m i x t u r e of the two e x p e c t e d d i h y d r o

d e r i v a t i v e s . *H NMR ( ( C D 3 ) 2 S O ) £ 1 . 0 (m, ( C H 3 ) 2 C H ) , 1.7 (m, Leu

JB-CH2), 1.78 ( s , NHAc), 1.9 (m, V a l ^ - C H ) , 2.73 (dd, J= 8, 16

Hz, T r p ^ 6 - C H 2 ) , 2.90 (dd, 3 = 6, 16 , T r p ^ - C H 2 ) , 3.36 ( s , OMe),

2.66 (m, ^ - C H 2 ) , 3.93 (m, Leu a-CH), 4.02 (m, V a l a-CH), 4.45

(m, cxs-CH), 4.73 (m, <*-CH), 7.09 (dd, 3=2, 9 Hz, i n d o l e H 5 ) , 7.11

161

(br s, C0NH 2), 7.13 ( d , J=3 Hz, i n d o l e H 2 ) , 7.54 ( d , J=2 Hz,

i n d o l e H 7 ) , 7.56 (d, J=9 Hz, i n d o l e H 4 ) , 7.80 (d, J=8 Hz, NH),

7.92 (br s, C0NH 2), 8.31 ( s , PhH), 10.92 (br s, i n d o l e H 2 ) .

Fungi s t a t i c A s s a y s and F ungal I s o l a t e s

Samples composed of p u r i f i e d c l i o n a m i d e (5_3, 1.0 mg),

c l i o n a m i d e and a m i x t u r e of c e l e n a m i d e s (0.5, 2.5, 10 mg), and

t e t r a c e t y l c l i o n a m i d e (4_6) and m i x t u r e of a c e t y l a t e d c e l e n a m i d e s

(20 mg) were a p p l i e d t o f i l t e r paper d i s k s , d r i e d and t e s t e d f o r

i n h i b i t o r y a c t i v i t y a g a i n s t the f o l l o w i n g f u n g i : A s p e r q i 1 l u s

n i g e r , A u r e o b a s i d i u m p u l l u l a n s , G i b e r e l l a zeae , P e n i c i Hum

p a t u l i n , P o l y p o r u s v e r s i c o l o r , T r i c h o d e r m a v i r i d e , and a p i n k

y e a s t i s o l a t e . No f u n g i s t a t i c a c t i v i t y was d e t e c t a b l e f o l l o w i n g

i n c u b a t i o n at 25" f o r s e v e r a l days. A s s a y s were performed on

i n o c u l a t e d p o t a t o d e x t r o s e agar p l a t e s i n d u p l i c a t e .

In o r d e r t o i s o l a t e the f u n g i a s s o c i a t e d w i t h C . c e l a t a ,

f r e s h sponge was c o l l e c t e d and s t o r e d i n a s m a l l volume of sea

w a t e r . I n o c u l a were o b t a i n e d from the sponge s u r f a c e , the sea

water and an i n t e r n a l p o r t i o n o b t a i n e d by s u r f a c e s t e r i l i z a t i o n

(10 min. 70% EtOH) and d i s s e c t i o n . The c o l o n i e s which a r o s e

f o l l o w i n g i n c u b a t i o n a t 22-25° f o r s e v e r a l days were s e p a r a t e d

by p i c k i n g , and i n c u b a t e d f o r a f u r t h e r 10 days on peptone

d e x t r o s e agar ( i n c l u d i n g r o s e bengal and a u r e o m y c i n ) . In t h i s

f a s h i o n , t w e l v e u n i d e n t i f i e d i s o l a t e s were o b t a i n e d which were

162

subsequently s u b j e c t e d to chemical a n a l y s i s . C o l o n i e s were

scraped from t h e i r agar p l a t e s , homogenized i n MeOH and

f i l t e r e d . The methanolic e x t r a c t was c o n c e n t r a t e d and analyzed

by TLC. Samples were a c e t y l a t e d as d e s c r i b e d above and analyzed

by TLC comparison with a sample of known C l i o n a a l k a l o i d s .

Compounds bearing resemblance to those i s o l a t e d from C . c e l a t a

were not d e t e c t e d in any of the twelve fungal e x t r a c t s .

C. c e l a t a C el1 F r a c t i o n a t i o n

F r e s h l y c o l l e c t e d sponge was p l a c e d i n s e v e r a l t e s t tubes

c o n t a i n i n g s t e r i l e c a l c i u m - f r e e seawater and immediately

submitted f o r c e l l f r a c t i o n a t i o n by Dr. S.A. Pomponi. 1 C e l l s

were d i v i d e d i n t o sub-populations by the f o l l o w i n g a b b r e v i a t e d

procedure: "The c e l l s were d i s s o c i a t e d i n calcium-and magnesium-

free seawater (CMF) and then separated in d i s c o n t i n u o u s d e n s i t y

g r a d i e n t s of 6%, 12%, 18%, 24%, and 30% F i c o l l / C M F . The bands

were c o l l e c t e d by p i p e t t i n g , c e n t r i f u g e d , F i c o l l supernatant

decanted o f f , and p e l l e t s resuspended in a b s o l u t e e t h a n o l " .

F r a c t i o n s from the 6-12%, 12-18%, 18-24% and 24-30% i n t e r f a c e s

and the p e l l e t were returned and a n a l y z e d as per the fungal

c e l l s d e s c r i b e d above. A l k a l o i d s were not d e t e c t e d i n any of the

samples. C h o l e s t e r o l and a green pigment were present in the

c e l l - p e l l e t e x t r a c t but the usual a r r a y of yellow pigments found

xHorn Point Environmental L a b o r a t o r i e s , Box 775, Cambridge, Maryland. 21613.

163

in nonfractionated C.celata tissue was not apparent in any of

the c e l l f r a c t i o n s .

Calc ium I on Transport Exper iments

In one experiment, the lower portion of a U-tube (id.0.9

cm) equipped with a magnetic s t i r r i n g bar was f i l l e d with 6.0 mL

of CHCl3-MeOH (9:1) containing 10 mg mL-1 of crude t r i t u r a t e d

Cliona a l k a l o i d s . In one upper portion of the tube was placed

1.5 mL of an acceptor-phase (0.5 M sodium c i t r a t e , pH 5.5) and

in the opposing upper portion was placed 1.5 mL of a donor-phase

(0.5 M Tris-HCl, pH 8.5 containing 1.0 M C a C l 2 ) . The apparatus

was sealed under nitrogen and s t i r r e d at room temperature for 24

h. Aliquots (100 f i D were taken by syringe for analysis (flame-

atomic absorption) at T=l,2,7 and 24 (500 fiL) h. Two control

experiments were performed, one in which the donor-phase was

replaced by 0.5 M Tris-HCl (pH 8.5) and one in which the

organic-phase was free of a l k a l o i d s . The results of these

experiments are shown in appendix 12.

A second transport experiment was performed in a Pressman

c e l l under conditions similar to those described above. CHC13-

PhN02~MeOH (60:39:1; 5.0 mL) was employed as the organic solvent

mixture and the donor and acceptor aqueous-phase volumes were

1.2 mL. The result of t h i s experiment and the control experiment

performed in the absence of a l k a l o i d s are described in the text.

164

( Z ) - 2 - A c e t y l - 4 - ( 3 ' ,4' ,5'-Tr imethoxybenzylidene)-5(4H)-Oxazolone

(90) and (Z)-2-Acety1-4-Benzylidene-5(4H)-Oxazolone (91)

A c e t y l g l y c i n e (109, mp 207-208°, 2.39 g, 0.020 mole) and

3,4,5-trimethoxybenzaldehyde (Sigma, 4.00 g, 0.020 mole) were

re a c t e d as d e s c r i b e d by Herbst and Shemin (111), using

benzaldehyde, to give the b r i g h t yellow a z l a c t o n e 90 (3.80 g,

0.014 mole, 70%) f o l l o w i n g r e c r y s t a l 1 i z a t i o n from EtOAc.

Treatment of f r e s h l y d i s t i l l e d benzaldehyde (5.8 g, 0.050 mole)

under s i m i l a r c o n d i t i o n s a f f o r d e d 91 (111, 6.68 g, 0.036 mole,

71%). 90: mp 159.5-162°; IR (KBr) v max 1790, 1765, 1660 cm- 1;

IR (CHC1 3) v max 1800, 1770, 1660 cm- 1; JH NMR (100 MHz,

acetone-d§) 8 2.44 (s, 3H), 3.85 (s, 3H), 3.91 (s, 6H), 7.10 (s,

IH), 7.65 (s, 2H); MS m/e, M* 277, 207, 192, 43; Exact mass

c a l c d f o r C i 4 H 1 5 N 0 5 , 277.0950; obsd, 277.0946. 91: mp 151.5-153

( l i t . 148-150°); IR (KBr) v max 1790 sh, 1768, 1650 cm- 1; :H NMR

(100 MHz, CDC1 3) <5" 2.41 (s, 3H), 7.17 (s, IH), 7.47 (m, 3H),

8.46 (m, 2H); MS m/e, M* 187, 158; Exact mass c a l c d f o r c l l H 9 N 0 2 ' 187.0634; obsd, 187.0634.

The a z l a c t o n e s 9_0 and 9_1 were shown to be s i n g l e

g e o m e t r i c a l isomers by lH NMR and HPLC (RP and S i l i c a - A )

a n a l y s e s .

Dehydroamino Ac i d s Derived from 90 and 91

{!)-2-Acetamino-(3',4',5'-trimethoxy)cinnamic a c i d methyl

165

e s t e r (9_3) was p r e p a r e d i n q u a n t i t a t i v e y i e l d by r e f l u x i n g the

a z l a c t o n e 9_0 i n anhydrous methanol f o r 48 h. 93: mp 220°

(decomp.); IR (KBr) v max 3300 b r , 1720, 1660 br cm- 1; XH NMR

(100 MHz, DMSO-d§) £2.03 ( s , 3H), 3.72 ( s , 3H), 3.81 ( s , 6H),

7.05 ( s , 2H), 7.22 ( s , I H ) , 9.64 ( s , I H ) ; MS, m/e, M* 309, 267,

252, 192, 43; Ex a c t mass c a l c d f o r C 1 4 H 1 9 N 0 6 , 309.1213; obsd,

309.1210.

Treatment of 9_0 w i t h r e f l u x i n g aqueous a c e t o n e (113)

a f f o r d e d an 80% y i e l d of the a c i d 9_4 a f t e r r e c r y s t a l l i z a t i o n

from methanol. 94: mp 208-210°; IR (KBr) v max 3400 b r , 3300,

2950 b r , 1710, 1630 cm- 1; XH NMR (100 MHz, DMSO-d6) £ 2.04 ( s ,

3H), 3.72 ( s , 3H), 3.81 ( s , 6H), 7.03 ( s , 2H), 7.27 ( s , I H ) ,

9.49 ( s , I H ) . (2)-2-Acetaminocinnamic a c i d (95) was o b t a i n e d i n

85% y i e l d f o l l o w i n g s i m i l a r t r e a t m e n t of 91. 9_5: mp 189-190°

( l i t . ( I l l ) mp 191-192°) IR (KBr) v max, 3280, 3050, 2950, 1724,

1600, 1540(br) cm- 1. The a c i d s 9_4 and 95 c o u l d be q u a n t i t a t i v e l y

c o n v e r t e d back t o the a z l a c t o n e s 90 and 9_1 by r e a c t i n g w i t h A c 2 0

(1 e q u i v ) and 4 - d i m e t h y l a m i n o p y r i d i n e (0.1 e q u i v ) i n E t 3 N at

40°C f o r 12 h.

Ammonolysis of the a z l a c t o n e s 9_0 and 91 i n anhydrous

methanol a t 0 eC f o r 2 h gave the p r i m a r y amides 96 and 97,

r e s p e c t i v e l y , i n q u a n t i t a t i v e y i e l d s . 9_6: mp 211-213° (decomp);

IR (KBr) r max 3200 b r , 1660 br cm- 1; JH NMR (270 MHz, DMSO-d§)

£2.00 ( s , 3H), 2.68 ( s , 3H), 3.78 ( s , 6H), 6.90 ( s , 2H), 7.06

( s , I H ) , 7.11 ( b s , I H ) , 7.41 (bs, I H ) , 9.37 ( s , I H ) ; MS m/e, M*

294, 276, 261, 252, 237, 207. 97: mp 202.5-204° (decomp); IR

(KBr) v max 3250 b r , 1650 br cm- 1; JH NMR (270 MHz, DMSO-d6) £

166

1.96 ( s , 3H), 6.98 ( s , I H ) , 7.11 (bs, IH) 7.30 (tn, 3H), 7.42 (bs, I H ) , 7.48 (m, 2H), 9.32 ( s , I H ) ; MS m/e, M* 204, 187, 162;

Exac t mass c a l c d f o r C11H12N2O2, 204.0899; obsd, 204.0897.

( Z ) - 2 - P h e n y l - 4 - ( 3 ' , 4 ' , 5 ' - T r i m e t h o x y b e n z y l i d e n e ) - 5 ( 4 H ) - O x a z o l o n e

(92) and (Z)-2-Benzamido-(3' ,4',5'-Trimethoxy) Cinnamic Ac i d

M e t h y l E s t e r (98)

Con d e n s a t i o n of h i p p u r i c a c i d (Sigma, 24.0 g, 0.13 mole)

and 3 , 4 , 5 - t r i m e t h o x y b e n z a l d e h y d e (Sigma, 24.5 g, 0.12 mole) as

d e s c r i b e d by Mauthner (112) a f f o r d e d the a z l a c t o n e 92 (27.1 g,

0.08 mole, 6 7 % ) ; mp 162.5-165° ( l i t . 165-166°, 112); IR (KBr) v

max 1790, 1655, 1580 cm- 1. The a z l a c t o n e 92 underwent

q u a n t i t a t i v e c o n v e r s i o n t o the methyl e s t e r 9_8 upon r e f l u x i n g i n

anhydrous methanol f o r 48 h. 98: mp 127-129°; IR (KBr) v max

3250 b r , 2950, 1719, 1655, 1580 cm- 1; XH NMR (100 MHz, a c e t o n e -

d § ) <5" 3.88 ( s , 9H), 7.06 ( s , 2H) , 7:46 ( s , IH) , 7.60 (m, 3H),

8.14 (m, 2H), 9.13 (bs, I H ) ; MS m/e, M* 371, 266, 234.

N - B e n z o y l - 3 - B e n z y l t h i o - ( 3 ' , 4 ' , 5 ' - T r i m e t h o x y p h e n y l a l a n i n e M e t h y l

E s t e r (129)

MeO

MeO

MeO

O

167

The t h i o e t h e r 129 was p r e p a r e d as a m i x t u r e of

d i a s t e r e o m e r s i n q u a n t i t a t i v e y i e l d a c c o r d i n g t o the p r o c e d u r e

of R i c h e t a l . ( 1 3 3 ) . The r e a c t i o n was complete w i t h i n 6 h a t

room t e m p e r a t u r e . 129: mp 132-140° (decomp); IR (KBr) v max

3325, 2950, 1740, 1640, 1590, 1510 cm- 1; lH NMR (100 MHz, CDC1 3)

$ 3.8 (m, 14H), 4.27 (d, IH, J=6Hz), 5.20 (dd, 0.6H, J=5, 6Hz),

5.33 (m, 0.4H), 6.58 ( s , 2,H), 6.8 (bm, IH) , 7.28 ( b s , 5H) , 7.50

(m, 3H), 7.78 (m, 2H), MS m/e, 371 (M*-C 7H 8S), 339, 303, 246,

181, 121, 105, 91.

S e v e r a l a t t e m p t s were made t o c o n v e r t 129 i n t o i t s

c o r r e s p o n d i n g f r e e amino a c i d . H y d r o l y s i s i n r e f l u x i n g 3N HC1,

IN NaOH, or m i x t u r e s of f o r m i c a c i d and aqueous HC1 (133) f a i l e d

t o p r o v i d e the d e s i r e d p r o d u c t .

D e h y d r o d i p e p t i d e s D e r i v e d from 90 and 91

(Z)-N-Acetyl-o <;, J6-didehydro-(3' ,4' , 5 ' - t r i m e t h o x y ) - p h e n y l -

a l a n y l g l y c i n e (99, 95%) and (Z) -N-Acetyl-c*,£ -

d i d e h y d r o p h e n y l a l a n y l g l y c i n e (103, 92%) were p r e p a r e d from 9_0

and 91, r e s p e c t i v e l y , by the method of Doherty et a l . ( 1 1 5 ) . 99:

mp 207° (decomp); IR (KBr) v max 3300 b r , 1740, 1630 br cm- 1; 'H

NMR (270 MHz, DMSO-d§) 6 2.01 ( s , 3H), 3.70 (m, H H ) , 6.88 ( s ,

2H), 7.11 ( s , I H ) , 8.20 (bs, I H ) , 9.43 ( s , I H ) , 12.39 ( b s , I H ) ;

MS m / e , M* 352, 334, 319, 290. 1_03: mp 189-191° ( l i t . 194-195°,

115); IR (KBr) v max 3380, 3230 b r , 1700, 1630 br cm- 1; *H NMR

(270 MHz, DMSO-de) 8 1.99 ( s , 3H), 3.79 ( d , 2H, J=6Hz), 7.09 ( s ,

I H ) , 7.34 (m, 3H), 7.49 (m, 2H), 8.23 ( t , IH, J=6Hz), 9.39 ( s ,

168

I H ) , 12.53 ( b s , I H ) . 99 was f u r t h e r c h a r a c t e r i z e d as i t s methyl

e s t e r 100 ( C H 2 N 2 / CH3OH); IR (KBr) v max 3400 b r , 1755, 1640 br

cm- 1; JH NMR (270 MHz, acetone-djg_) 8 2.12 ( s , 3H) , 3.67 ( s , 3H),

3.74 ( s , 3H), 3.83 ( s , 6H), 4.00 (m, 2H), 6.88 ( s , 2H), 7.19 ( s ,

I H ) , 7.73 (bs, I H ) , 8.69 ( s , I H ) ; MS m/e, M 4 366, 348, 324, 309;

Exact mass c a l c d f o r C 17H2.2N2.O7, 366.1427; obsd, 366.1426.

The a z l a c t o n e 90 (3.0 g, 0.011 mole) was d i s s o l v e d i n

acetone (25 mL) and 1.29 g (0.011 mole) of L - v a l i n e i n 20 mL of

IN NaOH was added (115). The r e a c t i o n m i x t u r e was s t i r r e d a t

room te m p e r a t u r e o v e r n i g h t and then a c i d i f i e d w i t h 2 M HCl t o

g i v e the d e h y d r o d i p e p t i d e 101 (2.76 g, 0.01 mole, 91%) as a

whit e powder. When 90 (3.38 g, 0.012 mole) was t r e a t e d i n an

analogous f a s h i o n w i t h L - l e u c i n e (1.60 g, 0.012 mole) the

d e h y d r o d i p e p t i d e 102 was i s o l a t e d (3.10 g, 0.01 mole, 8 3 % ) . 101:

mp 221-223° (decomp); IR (KBr) v max 3400 b r , 2950, 1720 b r ,

1640 br cm- 1; JH NMR (270 MHz, DMSO-d$) £0.89 ( d , 3H, J=7Hz),

0.92 (d, 3H, J=7Hz), 2.01 ( s , 3H), 2.12 (m, l H ) , 3.68 ( s , 3H),

3.77 ( s , 6H), 4.20 (dd, IH, J=7,8Hz), 6.89 ( s , 2H), 6.99 ( s ,

IH ) , 7.71 ( d , IH, J=8Hz), 9.46 ( s , I H ) . 102: mp 199-201°

(decomp); IR (KBr) v max 3400 b r , 3230 b r , 2950, 1730, 1640 br

cm- 1; JH NMR (270 MHz, DMSO-dg) 8 0.86 (d, 3H, J=7Hz), 0.90 (d,

3H, J=7Hz), 1.54 (m, I H ) , 1.67 (m, 2H), 2.00 ( s , I H ) , 3.68 ( s ,

3H), 3.78 ( s , 6H), 4.32 (m, I H ) , 6.87 ( s , 2H), 6.99 ( s , l H ) ,

8.02 ( d , IH, J=8Hz), 9.38 ( s , I H ) .

169

B i s d e h y d r o p e p t i d e s D e r i v e d from 99 and 103

The d e h y d r o p e p t i d e £9 (0.85 g, 2.43 mmole) was t r e a t e d w i t h

3 , 4 , 5 - t r imethoxybenzaldehyde (0.48 g, 2.31 mmole), e x c e s s AC2.O and anhydrous sodium a c e t a t e (0.200 g, 2.4 mmole) at 40°C f o r 24

h (115) t o g i v e the p e p t i d e a z l a c t o n e 104 (0.94 g, 1.83 mmole,

8 0 % ) . When the d e h y d r o d i p e p t i d e 103 (4.16 g, 0.016 mole) was

r e a c t e d i n an analogous f a s h i o n w i t h r e d i s t i l l e d benzaldehyde

(1.70 g, 0.016 m o l e ) , the p e p t i d e a z l a c t o n e 105 (4.46 g, 0.013

mole, 81%) was o b t a i n e d . 104: mp 239-242°; IR (KBr) v max 3400

( b r ) , 2950, 1800, 1640, 1580 cm- 1; *H NMR (270 MHz, CDC1 3) $

2.43 ( s , 3H), 3.87 ( s , 6H), 3.89 ( s , 6H), 3.91 ( s , 3H), 6.75 ( s ,

2H), 7.11 ( s , I H ) , 7.13 ( s , I H ) , 7.38 ( s , 2H), 7.64 ( s , IH, NH); JH NMR (270 MHz, DMSO-d_6) 8 2.07 ( s , 3H.) , 3.71 ( s , 3H) , 3.75 ( s ,

3H), 3.79 ( s , 6H), 3.83 ( s , 6H), 7.13 ( s , 2H), 7.23 ( s , I H ) ,

7.44 ( s , I H ) , 7.66 ( s , 2H), 9.77 ( s , IH, NH); MS m/e, M* 512,

470; E x a c t mass c a l c d f o r C 2 6 H 2 8 N 2 O 9 , 512.1794; obsd 512.1770.

105: mp 184-186.5° ( l i t . (115) mp 184-186°); IR (KBr) v max

3250, 1800 ( s h ) , 1780, 1650 ( b r ) cm- 1; JH NMR (270 MHz, a c e t o n e -

d § ) 8 2.19 ( s , 3H), 7.00 ( s , I H ) , 7.41 (m, 7H), 7.74 (m, 2H),

8.23 (m, 2H), 8.91 ( s , IH, NH); MS m/e, M* 332, 290, 244; E x a c t

mass c a l c d f o r C2oHi6N 20 3, 332.1161; obsd 332.1163.

The a z l a c t o n e s 104 and 105 were r e a c t e d w i t h a v a r i e t y of

r e a g e n t s t o g i v e the b i s d e h y d r o d i p e p t i d e s d e s c r i b e d below. 106

( a c e t o n e / H 2 0 ) : IR (KBr) vmax 3300 ( b r ) , 2950, 1660 ( b r ) , 1584

cm- 1; XH NMR (270 MHz, DMSO-de) 8 2.02 ( s , 3H), 3.67 ( s , 3H),

3.69 ( s , 3H), 3.78 ( s , 12H), 6.91 ( s , 2H), 6.99 ( s , 2H), 7.16

170

( s , 1H), 7.25 ( s , 1H), 9.41 ( s , 1H), 9.55 ( s , I H ) ; MS m/e, 512

(M-18), 468. 102 (MeOH): IR (KBr) ii max 3300 ( b r ) , 2950, 1720,

1640 ( b r ) cm- 1; >H NMR (270-MHz, DMSO-d6) 6 2.02 ( s , 3H), 3.66

( s , 3H), 3.68 ( s , 3H), 3.70 ( s , 3H), 3.78 ( s , 12H), 6.91 ( s ,

2H), 7.02 ( s , 2H), 7.15 ( s , l H ) , 7.23 ( s , I H ) , 9.54 ( s , I H ) ,

9.58 ( s , I H ) ; MS m/e, M" 544, 526, 511; Ex a c t mass c a l c d f o r

C 2 7 H 3 2 N 2 O 1 0 , 544.2057; obsd 544.2055. 1,08 (NH 2C (CH 3 ) 3 ) : *H NMR

(400 MHz, acetone-dg) S 1.42 ( s , 9H), 2.21 ( s , 3H), 3.72 ( s ,

3H), 3.77 ( s , (H), 3.87 ( s , 6H), 6.84 ( s , 2H), 6.92 ( s , 2H),

7.03 ( s , 3H), 3.77 ( s , 9H), 3.87 ( s , 6H), 6.84 ( s , 2H), 6.92 ( s ,

2H), 7.03 ( s , I H ) , 7.32 ( s , I H ) , 7.35 (b s , IH, NH), 8.85 ( b s ,

I H ) , 9.38 ( b s , I H ) . 109 ( N H ( C H 2 C H 3 ) Z ) : JH NMR (400 MHz, a c e t o n e -

d § ) £ 1.16 (m, 3H), 1.28 (m, 3H), 2.13 ( s , 3H), 3.34 (m, 2H),

3.66 (m, 2H), 3.73 ( s , 3H), 3.74 ( s , 3H), 3.79 ( s , 6H), 3.88 ( s ,

6H), 5.94 ( s , I H ) , 6.83 (s,' 4H), 7.29 ( s , IH) , 8.97 ( s , IH) ,

9.77 ( s , I H ) . I l l (NH 4OH): mp 220-223° (decomp); IR (KBr) V max

3300 ( b r ) , 1640 ( b r ) , 1490 cm- 1; »H NMR (270 MHz, DMSO-de) 8

2.07 ( s , 3H), 6.93 ( s , I H ) , 7.4 (m, 9H), 7.7 (m, 4H), 9.63 ( s ,

I H ) , 9.97 ( s , I H ) ; MS m/e, M* 349, 332; Ex a c t mass c a l c d f o r

C 2 o H 1 9 N 3 0 3 , 349.1427; obsd 349.1427. 110 (NH 2CH 2CH 2(3,4-

MeO)C 5H 2): lH NMR (400 MHz, DMSO~d 6) <?2.11 ( s , 3H) , 2.75 ( t ,

2H, J=7 H z ) , 3.40 (m, 2H), 3.68 ( s , 3H), 3.71 ( s , 3H), 3.72 ( s ,

3H), 3.75 ( s , 9H), 3.82 ( s , 6H), 6.80 ( d , IH, J=8 H z ) , 6.85 (m,

2H), 6.92 ( s , 2H), 6.94 ( s , 2H), 6.97 ( s , l H ) , 7.35 ( s , I H ) ,

7.89 ( t , IH, 3 = 1 , NH), 9,65 ( s , I H ) , 10.06 ( s , I H ) .

171

Racemic A c e t y l v a l i n a m i d e (117)

A c e t y l - L - v a l i n e (109, 8.0 g, 0.05 mole, [c*0 D + 3.4 e (c 2.0

EtOAc) ( l i t . (143) [oc]D + 4° (c 2.0, EtOH)) was converted to i t s

p-nitropheny1 e s t e r by the procedure of Hassner and Alexanian

(144). A f t e r 16 h at room temperature the r e a c t i o n mixture was

washed r e p e a t e d l y with water and 5% a c e t i c a c i d to give a pale

yellow o i l (13 g) f o l l o w i n g e v a p o r a t i o n in vacuo . T h i s m a t e r i a l

was d r i e d and subjected to ammonolysis without f u r t h e r

p u r i f i c a t i o n . Treatment with ammonia in anhydrous methanol

overnight at 0 CC a f f o r d e d the amide 117 (6.2 g, 0.039 mole, 78%

from a c e t y l - L - v a l i n e ) . 117; mp 239.5°; [oc]D + 0.1° (c 2.0,

EtOH); IR (KBr) V max 3250, 2960, 1650br cm- 1; 2H NMR (270 MHz,

DMSO-d|) & 0.82 (d, 3H, J=7 Hz), 0.86 (d, 3H, J=7 Hz), 1.86 (s,

3H), 1.93 (m, IH), 4.11 (dd, IH, J=7, 8.5 Hz), 6.97 (bs, IH),

7.35 (bs, IH), 7.77 (d, IH, J=8.5 Hz); Anal c a l c d f o r

C 7H 1 4N 2O z,C53.15, H8.92, N17.71; found, C53.25, H8.76, N17.73.

A c e t y l - L - V a l i n a m i d e (117a)

A c e t y l - L - v a l i n e d e s c r i b e d above (14.0 g, 0.088 mole) was

t r e a t e d with t h i o n y l c h l o r i d e (16.8mL, 0.117 mole) i n anhydrous

methanol (140 mL) at 0° f o r 24 h and then at room temperature

f o r 48 h (145). The s o l u t i o n was r e f l u x e d f o r 4 h, con c e n t r a t e d

in vacuo and the o i l y r e s idue obtained p a r t i t i o n e d between EtOAc

and aqueous potassium carbonate. The EtOAc l a y e r was washed with

172

water, d r i e d over anhydrous Na^SC^ and e v a p o r a t e d t o g i v e 8.2 g

(0.047 mole, 53%) of the m e t h y l e s t e r 130 as a c o l o u r l e s s o i l ;

IR (KBr) v max 3200, 2960, 1725 cm- 1. T h i s m a t e r i a l was

s u b j e c t e d t o ammonolysis w i t h o u t f u r t h e r p u r i f i c a t i o n . A c o l d

s o l u t i o n of 200 mL of anhydrous MeOH s a t u r a t e d w i t h ammonia was

added t o 130 (7.6 g, 0.044 mole) and the m i x t u r e was kept a t 0°C

f o r one week. Removal of the s o l v e n t _in vacuo and t r i t u r a t i o n of

the r e s u l t i n g o i l w i t h e t h e r a f f o r d e d the amide 117a (420 mg,

2.7 mmole, 7%). 117a was i d e n t i c a l t o racemic N - a c e t y l v a l i n a m i d e

w i t h the e x c e p t i o n of i t s o p t i c a l r o t a t i o n ; [oc]D - 17° (c 0.68,

IN H C l ) . The unchanged methyl e s t e r i s o l a t e d from t h i s r e a c t i o n

(7.2 g, 0.041 mole) was r e s u b j e c t e d t o ammonolysis at room

tem p e r a t u r e f o r 1 week t o g i v e a f u r t h e r 0.44 g (2.8 mmole, 7%)

of the d e s i r e d amide; [ c * ] D - 16° (c 1.10, IN H C l ) . The o p t i c a l

p u r i t y of t h i s m a t e r i a l has not been d e t e r m i n e d .

2 - O x o - 3 - P h e n y l p r o p i o n i c A c i d (131) and 2-Oxo-3-(3' ,4 ' , 5-

Trimethoxypheny1) P r o p i o n i c Ac i d (132)

H y d r o l y s i s of the dehydroamino a c i d 94 (7.0 g, 0.024 mole)

i n r e f l u x i n g IN HCl f o r 3h a f f o r d e d 2.7 g (0.011 mole, 45%) of

the s u b s t i t u t e d p h e n y l p y r u v i c a c i d 132. I d e n t i c a l t r e a t m e n t of

c*-acetamino c i n n a m i c a c i d (9_5, 2.0 g, 0.010 mole) gave

p h e n y l p y r u v i c a c i d (131, 1.5 g, 9.1 mmole, 9 1 % ) . 132: mp 168-

171° [ l i t . 167-168 (142, 1 1 2 ) ] ; IR (KBr) V max 3360, 3220 ( b r ) ,

1690, 1580, 1506 cm- 1; F e 3 * p o s i t i v e . 131: mp 152-154° [ l i t .

150-154° ( 1 4 6 ) ] ; IR (KBr) z/ max 3450, 3150b, 1700sh, 1650b, 1450

173

cm- 1; F e 3 4 p o s i t i v e .

3,4,5-Trimethoxyepoxystyrene (112)

The styrene epoxide 112 (2.3 g, 11.0 mmole, 90%) was

prepared from 3,4,5-trimethoxybenzaldehyde (2.4 g, 12.4 mmole)

acc o r d i n g to Corey's method of epoxide formation using

t r i m e t h y l s u l p h o n i u m methylide (117). The c o l o u r l e s s o i l ,

i s o l a t e d f o l l o w i n g repeated passage through a s i l i c a p lug with

d i e t h y l e t h e r, c r y s t a l l i z e d upon c o o l i n g : mp 5 3-54°; IR (neat) i>

max 2940, 2830, 1586, 1500, 1460, 1421, 1383, 1328, 1239, 1190,

1130, 1010, 942, 840 cm- 1; JH NMR (100 MHz, C D C I 3 ) 8 2.75 (dd,

IH, J=3, 6 Hz), 3.13 (dd, IH, J=4, 6 Hz), 3.83 (m, IH), 3.85 (s,

3 H ) , 3.86 (s, 6H), 6.55 (s, 2H); MS m/e, M* 210, 195, 181 (base

peak).

Conversion of the Epoxide 112 to Acetate 115

The styrene epoxide 112 (864 mg, 4.1 mmole) was d i s s o l v e d

in 3 mL of DMF and the s o l u t i o n cooled to 0°. Ammonium hydroxide

(2 mL) was added and the mixture s t i r r e d at room temperature

u n t i l a l l of the reagent had re a c t e d (TLC, 4 days). The s o l v e n t s

were removed jjn vacuo and the r e s u l t i n g crude n i n h y d r i n

p o s i t i v e , r e s i d u e was d i s s o l v e d i n dry C H 2 C I 2 (5 mL) c o n t a i n i n g

one e q u i v a l e n t of E t 3 N . T h i s s o l u t i o n was c o o l e d to 0° and a

mixture of 847 mg of DCC (4.1 mmole), 652 mg of N - a c e t y l v a l i n e

174

(4.1 mmole) and 5 mL of d r y C H 2 G I 2 was added. The r e a c t i o n was

s t i r r e d o v e r n i g h t , ( u n t i l n i n h y d r i n n e g a t i v e ) p r e c i p i t a t e d DCU

was removed by f i l t e r e d and the s o l v e n t s e v a p o r a t e d in vacuo t o

g i v e a p a l e y e l l o w o i l . A c e t y l a t i o n of t h i s m a t e r i a l o v e r n i g h t

( A c 2 0 - DMAP-Et3N) f o l l o w e d by s i l i c a column chromatography

(CH 2C1 2-EtOAc) a f f o r d e d the m o d i f i e d - a m i n o a c i d 115 i n 52

p e r c e n t y i e l d from 112 (874 mg, 2.1 mmole). 115; IR (ne a t ) 1/ max

3270, 2950, 1735, 1640b, 1590, 1520b cm- 1; lH NMR (100 MHz,

a c e t o n e - d 6 ) S 0.88 (d, 6H, J=7Hz), 1.95 ( s , 3H), 2.06 ( s , 3H),

2.07 (m, I H ) , 3.61 (m, I H ) , 3.72 ( s , 3H), 3.84 ( s , 6H), 3.85 (m,

I H ) , 4.25 (m, I H ) , 5.82 (dd, IH, J=5,6Hz), 6.71 ( s , 2H), 7.27

(bd, IH, J=9Hz), 7.57 (bm, I H ) ; MS m/e, m* 410, 350, 252 (base

p e a k ) , 210, 197, 142, 114, 72. The a c e t a t e 115 would be

c o n v e r t e d i n t o the a l c o h o l 114 i n 82 p e r c e n t y i e l d by o v e r n i g h t

t r e a t m e n t w i t h MeOH/K2.C03 .

Attempted Opening of the Epo x i d e 112 w i t h the Amide 113

The e p o x i d e 112 (150 mg, 0.7 mmole) was d i s s o v e d i n

anhydrous DMF c o n t a i n i n g 126 mg (0.8 mmole) of N-

a c e t y l v a l i n a m i d e ) . The r e a c t i o n m i x t u r e was r e f l u x e d f o r 72 h,

the s o l v e n t was removed _i_n vacuo and a p o r t i o n of the r e s u l t i n g

o i l y r e s i d u e p u r i f i e d by PTLC. *H NMR a n a l y s i s of the f r a c t i o n s

which d i d not c o n t a i n s t a r t i n g m a t e r i a l s i n d i c a t e d the pr e s e n c e

of s m a l l q u a n t i t i e s (<5%) of the ketone 118 and t h e al d e h y d e

119.

T h i s r e a c t i o n was r e p e a t e d w i t h the pr e s e n c e of one

175

e q u i v a l e n t (2 mg) of hexane-washed NaH. The r e s i d u e o b t a i n e d was

a c e t y l a t e d as d e s c r i b e d above t o g i v e 28 mg (0 .07 mmole, 10%) of

a compound i n d e n t i c a l (TLC, 'H NMR) t o the a c e t a t e 115 f o l l o w i n g

p u r i f i c a t i o n by PTLC.

M e t h y l N - A c e t y l v a l y l - o ^ - d i d e h y d r o p h e n y l a l a n a t e

To a 50 mL round bottom f l a s k f i t t e d w i t h a Dean-Stark t r a p

and a condenser, was added 10 mL of anhydrous benzene, 2.12 g

(0.012 mole) of the ctf-keto a c i d J_32, and 2.42 g (0.015 mole) of

the u n s u b s t i t u t e d amide 117. Phosphorus o x y c h l o r i d e (0.6 mL, .6.5

mmole) was added d r o p w i s e w i t h s t i r r i n g , and the m i x t u r e was

r e f l u x e d f o r 19 h. Removal of the s o l v e n t s j_n vacuo and

p u r i f i c a t i o n by s i l i c a column chromatography (CH2Cl2~EtOAc)

a f f o r d e d two major p r o d u c t s ; the d e h y d r o d i p e p t i d e 133 (0.6 g,

1 . 9 mmole, 16%) and the b i p r o d u c t 134 (0.41 g, 1.5 mmole, 2 2 % ) .

133; IR (KBr) y max 3250b, 1720, 1640b, 1520 cm- 1; 1H NMR (270

MHz, acetone-d_6) 6 1.00 dd, 3H, J=7Hz), 1.03 ( d , 3H, J=7Hz),

2.00 ( s , 3H), 2.20 (m, I H ) , 3.75 ( s , 3H), 4.43 (dd, 1H,

176

J=6,7Hz), 7.25 ( s , I H ) , 7.30 (bd, IH, J=6Hz), 7.37 (m, 2H), 7.69

(m, 3H), 8.87 ( b s , 1H); MS m/e, M* 318, 177 (base p e a k ) , 117,

114, 72. 133_: mp. 193.5-194.5°; IR (KBr) y max 3300b, 2800,

1650b cm- 1; XH NMR (270 MHz, a c e t o n e - d 6 ) £0.97 ( d , 3H, J=7Hz),

1.01 (d, 3H, J=7Hz), 1.13 ( d , 6H, J=7Hz), 1.98 ( s , 3H), 2.17 (m,

I H ) , 2.29 ( s , 3H), 2.73 (m, I H ) , 4.44 (dd, IH, J=b,7Hz), 7.35

(bd, IH, J=7Hz), 9.09 ( b s , I H ) ; MS m/e, M* 281, 140 (base p e a k ) ,

125, 114, 72.

177

Appendix 1. Comparison of the S u b s t i t u t e d Benzaldehydes D e r i v e d from the O z o n o l y s i s of £6, 5_7, 58, 59 1 , 65_, and 66 w i t h S y n t h e t i c Samples.

O A c

N a t u r a l S y n t h e t i c N a t u r a l 2 S y n t h e t i c N a t u r a l 3 S y n t h e t i c

IR 4 1760 1760 1776 1775 1780 1780 1700 1700 1703 1700 1704 1705 1600 1600 1606 1610 1603 1603 1505 1505 1504 1504 1500 1500 1435 1432 1440 1400 1443 1444 1375 1375 1380 1378 1378 1380

EIMS 5 164 164 222 222 280 280 122 122 180 180 280 280

138 138 196 196 154 154

*H NMR' 2.33 2.30 2.34 2.34 2.24 2.25 7.31 7 7.30 1 7.41' 7.42 7 7.61 7.62 7.95 1 7. 9 1 1 7.76* 7.76*

7.82' 7.82' 10.01 10.00 10.00 9.99 9.88 9.90

>4-Acetoxybenzaldehyde was d e r i v e d from the o z o n o l y s i s of a m i x t u r e of a c e t y l a t e d c e l e n a m i d e s . JFrom 58 and 59. 'From 46, 57, 58, 65 and 66. 4 C H C l 3 , v max, cm- 1. '1OVMHZ7 /§DC1 3 or C C U , ppm from i n t e r n a l t e t r a m e t h y l s i l a n e (5=0). 7 d , J=8 Hz. *d, J=2 Hz. 'dd, J=2, 8 Hz.

Appendix 2. 20 MHz 1 3 C NMR of 55 in acetone-de ,PND.

I . I . I J I I I 1 I I I I I . I . I . J I L

4TOO Hz

?Joo 1J00

600

J I I I I I

-i—i—i—i—r -i—I—i—I—i—T"i—I—i—r—r

1 5 0 1 0 0 5 0

-I—|—i—|—i—|—r

Oppm

Appendix 3. 20 MHz 1 3 C NMR of a mixture of celenamides i n C D 3 C N , P N D .

a c e t y l a t e d

Appendix 4. 100.6 MHz 1 3 C NMR of hexacetylcelenamide A (58) i n CDC1 3 ,PND.

181

7.6 7.4 7.2 7.0 ppm Appendix 5 a. E x p a n s i o n of the a r o m a t i c r e g i o n of the 400 MHz spectrum of 5_7, b. Computer s i m u l a t i o n of the spectrum shown i n 'a'. C o u p l i n g c o n s t a n t s and c h e m i c a l s h i f t s were o b t a i n e d from 57, 58, and 6_6 ( t a b l e 1 ) ; l i n e w i d t h s a r e 2 Hz. I n s e r t s show the s p i n systems, c h e m i c a l s h i f t s and c o u p l i n g c o n s t a n t p a t t e r n s used f o r s i m u l a t i o n .

182

- A o

o 10

E CL CL

O

O

o CD

10 . O

O o

z z

o CM

o

I CRM t ' I l I L _ J I I I I L

Hs

i • i . i , i - T - r

AcO

AcO.

AcO

NHAc

NH.

N'Ô^Br H

65

1 1 1 1 1 1 1 1 1 1 1 < 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 I 180 150 120 90 60 30 Oppm

00

Appendix 7. 20 MHz 1 3 C NMR of 65 in acetone-d 6 , P N D .

Appendix 8. 20 MHz 1 3C NMR of 66 in CD 3CN ,PND.

I I I . I L I I • I • . • . — L L

9 8 7 6 5 4 3 2 1 p p m ( £ ) ca

Appendix 9. IR (CHCI3) and 100 MHz XH NMR of a mixture of 0 1

oligomers d e r i v e d from the a c i d h y d r o l y s i s of a mixture of 57, 58 and 59.

Appendix 10. 100.6 MHz 1 3 C NMR spectrum of an unknown peptide a l k a l o i d , PND.

CD

Appendix 11. C i r c u l a r d i c h r o i s m s p e c t r a of h e x a c e t y l c e l e n a m i d e ( 5 7 ) , 1.54 x 1 0 - 4 M i n CH 3CN. No s a l t added, ; 2.31 x 10 M Ca 2(C104.)4, (e i n L m o l - 1 cm- 1).

15 18 — r —

21

2c2_j0 24

T i m e |h)

Appendix 12. T r a n s l o c a t i o n of C a J * through a CHCl 3-MeOH (9:1) l a y e r m e d i a t e d by a m i x t u r e of crude t r i t u r a t e d C l i o n a a l k a l o i d s ( ). c i and c 2 r e p r e s e n t c o n t r o l e x p e r i m e n t s , see e x p e r i m e n t a l f o r d e t a i l s .

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