Synthesis and biosynthesis of some natural products

by

R • . A. RUSSELL

A thesis submitted in p2.rtial ful f i lment of tJ1e requi rements for the degree of

DOCTOR OF PHILOSOPHY

in ~he .Australian National University

Research School 0£ c._eTd_stry, At1.stralian National Unlversi ty , C 71 _'1B.,_, r: RA 11

c·

-

1

.

l .

DECLARATION

The work described in this thesis is the candidate's own, except where otherwise stated, and h as not been submitted in support of an application fer any other degree. It was carried out at The Australian

National University (1969-72), during the tenure of a Commonwealth Postgraduate Award.

SUMMARY

Part I of this thesis is a review of those metab-olites produced by micro-organisms which contain a unit derived from tryptophan and a C-5 isoprene group.

Part IIA describes the structural elucidation of brevianamides-B,

-c,

-D and -F, which are indoloid metabolites of the mould Penici l lium brevi-compactum. A relationship is established between the known

metab-olite brevianamide-A and the minor brevianamides-B,

-c

and -D .

Part IIB describes attempts to elucidate tl1e bio-synthesis of breviaI1amide-A. Synthetic approaches to key intermediates are discussed and the biosynthetic sig·nificance of brevianamide -F is elucidated.

Part III describes a new synthesis of nidulol methyl ether and an isomeric phthalide previously

isolated from Aspergillus nidulans . A possible synthetic approach to 5-hydroxyphthalides is discussed~

Part IV describes attempts to extend the linear synthesis of rnycophenolic acid to the preparation of

2~nalogues. The structure and spectra of rnycophenolic

.

. .

l l l . ACKNOWLEDGEMENTS

I would like to express my gratitude to Professor A. J. Birch, F.R.S., for his supervision and interest

thro~ghout this work.

I would also like to thank the mycologists, technical staff and spectroscopists of the Australian National

University, and in particular, Mrs. M. Anderson, M.Sc., Mrs .. J. Rothschild, B.Sc., Dr. J. K. McLeod and

Mr! C. Aranjelovic.

I am indebted to my colleagues, Dr. J. Corrie and

Dr. J. Baldas, for many stimulating discussions throughout the duration of this work.

My thanks are also due to Mrs . J. Madden for typing t..ri.is script.

The financial assistance of a Commonwealth Postgraduate Award and an Australian National University Res·earch

Scholarship are . gratefully acknowle~ged.

TABLE OF CONTENTS

Page

Surmnary _{• • • •} • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

..

].].

Introduction .•••••..••••••.•••••••• ~ •• ~~-~~~~~ - 1 Part I

Part IIA

Part IIB

A REVIEW: MOULD METABOLITES DERIVED FROM TRYPTOPHAN AND A C-5 ISOPRENOID UNIT

THE BREVIANAMIDES . Discussion

Introduction • • • • • • • • • • • • • • • • •

(a)

(b) Brevianamide-C, the chromophore (~) Brevianamide-C, the

diketo-3

28 29

piperazine ••.•••••••••••••••• 35 ( d)

(e)

( f)

Brevianamide-D • • • • • • • • • • • • • • • Inter-relation of

-c

and -D ••• Brevianamide-B • • • • • • • • • • • • • • • •

(g) Relationship between -A, -B,

40

41

44

-C, - D • • • • • • • • • • • • • • • • • • • • • • • 4 7

(h) Brevianamide-F • • • • • • • • • • • • • • • 51 (i) The synthesis of Brevianamide-F 53

(cyclo-L-tryp.-L-pro.)

SOME ASPECTS OF THE BIOSYNTHESIS OF BREVIANAM.IDE-A

Discussion

(a)

(b)

Introduction • • • • • • • • • • • • • • • • •

The synthesis of 1-(3-methylbut--2-enyl) tryptophan . . . ..

('c) Approaches to the syn thesis of

56

57

2- (1, 1-dimethylallyl) tryp.tophan 59 (d) The involvement of brevianamide

-F •••••••••••• 0 • • • • • • • • • • • • • 70 I

(e) Cyclo-2- (1,1-dimethylallyl)

t~"P. -L-pro. • • • • • • • • • • • • • • • • : 75 Experimental • • • • • • a • • • • • • • • • • • • • • • 78

Part III

Part IV

CHEMISTRY RELATED TO THE PHTHALIDES OF ASPERGILLUS NIDULANS

(a)

(b)

Introduction

Discussion

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • E xpe rimen tal • • • • • • • • • • • • • • • • • • • • • • •

SOME CHEMISTRY RELATED TO MYCOPHENOLIC ACID

(a) (b)

Introduction • • • • • • • • • • • • • • • • • Discussion of analogues· ••••• ~. (c) Discussion of mycophenolic acid

diol lactone ~

. .

.

.

.

.

.

.

. .

. .

. .

. .

v.

142

162 167

176 Experimental •••••••••••••• ~ •••••••• ~ 181 References cited • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 197

CORRECTIONS

?114 _{l ine} 12

Pl20 l ine 1 NH n ot NY

1.

Introduction

Nature would seem to be a_ good o~ganic chemist and

i t is desirable to explain biological processes in chemical

mechanistic terms. In the quest of this goal there have

arisen several aspects of natural products chemistry, namely

structural elucidation, synthesis and biosynthesis, which

interact with one another. Consequently L~e results

obtained in one particular aspect of an investigation can

often indicate worthwhile areas of study within another

branch of the subject.

This interaction between different chemical aspects

is illustrated by the brevianamides, which pose some

inter-esting problems in biosynthesis and, in particular, the

formation of a bri~ged diketopiperazine ring system. This

interesti1:1g structural unit of these metabolites· will

undoubtedly initiate future synthetic studies, and indeed

i t seems likely that the penultimate stage of the

biosyn-thetic pathway will remain obscure until such time as

syn-thetic chemistry can provide sui ta'ble precursors . .

The unexpected structures of brevianamides-C, and

-D could not be easily explained in terms of current

biosynthetic theories and subsequently lead to an unusual

photocheroical reaction, suggesting some interesting me

chan-istic chemistry.

Nidulol has an unexpected structure, which seems to

2 •

synthesis of this type of structure is a further example of synthetic chemistry initiated by peculiarities observed in structural elucidation studies. Mycophenolic acid

represents an interesti~g biosynthetic problem with poten-tially important repercussions for chemotherapy. The

synthesis of substrates which can be metabolised by fungi to yield unnatural metabolites is of considerable pharmac-eutical importance, and illustrates a practical union of synthetic and biosynthetic studies. The selectivity and specificity of naturally occurring enzyme systems is often superior to purely chemical methods of achieving a

par-ticular transformation. The difficulty experienced in synthesising analogues of mycophenolic acid would suggest that i f large quantities of such compounds are to be

prepared then the subtle manipulation of the biosynthetic

.

pathway is of considerable importance.

PART.

MOULP .METABOLITES DERIVED FROM TRYPTOPHAN· AND A

PART 1

Mould Metabolites Derived from Tryptophan and a C'-5

Isoprenoid

c~s

Isoprenoid Unit

A review of this subject is of general interest, since apart from the ergot alkaloids no such review exists in the literature. It is also relevant to the

3.

work de.scribed on the mode of introduction of terpene units into the indoloid metabolites of Penicillium brevi-compactum1, the brevianamides.

Tryptophan can undergo electrophilic attack at the nitrogen hetero-atom or within the heterocyclic and

carbo-cyclic rings. In the latter case, electron density cal-culations2 do not accurately predict the positions most susceptible to electrophilic attack, although experimental work suggests a selectivity for the 4 and 6 positions]'. Such selectivity is not strictly observed in mould metab-olites, possibly as a consequence of enzymatic assistance

to electrophilic substitution.

Streptomyces islandicus and Streptomyces atratus

d t · f ·d ·b· · h ·1 · 4 , 5

pro uce wo series o pepti e ant1. 1ot1cs, t 1.e 1 amycins

and the rufomycins, 617 which, although reported

indepen-dently, are, in most cases, identical. I lamycin B

1 ( 8a)

contains, as part of its peptide chain, a tryptophan moeity

substituted at the pyrrole nitr?gen with a reversed

iso-prene unit, whilst ilamycin ( 8b) and ilarnycin B

2 ( 8c)

A

1

0

C

B

D

0

E

-¢o

0

5

BREVIANAM!DES THE

2

'

H 0

N'Y'~

H N

-9'

0

R' I

N H

8

I

a) R= CH:CH R = Me _2•

I

b) R= ~ R=CHO

V I I

c) R= ~ , R= Me

5 •

h as b een epoxi ise ·d· d 8 . Altho~gh no direct tracer studies

have been made to show the stage in biosynthesis at which

alkylation of the nitrogen occurs, i t seems likely that i t

does so prior to the formation of the peptide skeleton.

Cultures of S. atratus in which normal antibiotic production

has been inhibited with D.L-isoleucine, have been shown to

accumulate the amino acid (9) 9 . Such a compound could

conceivably be produced by an SN2'-substitution10 of

3,3-dimethylallyl pyrophosphate (10), although no in vitro

analogue of the reaction has been reported.

C0₂H

9

N

~~ ~

_{/~\_:_OPP}

_*o

11 R= H

Amongst the group of metabolites which show substitution

in the benzenoid ring of tryptophan, the most extensively

studied are the ergot alkaloids. These may be divided

· t 1 11 h . d f 1 . . d ( 11} f into wo c asses , t e ami es o ysergic aci o

which ergotamine (12) is an example, and the clavines

examplified by chanoclavine-1 (13), agroclavine (14) and

elymoclavine (15).

t,JHMe

14 R

=

Me

15 R =CH₂0H

7.

. 12 h . . . f

Early experiments showed that t e inJection o trypto-phan into the phloem of ergot-bearing rye plants increased

the yield of alkaloids obtained and suggested that trypto-phan may be a key biogentic precursor of these compounds.

14 Subsequently i t was shown that injection of D.L-[S- C]-tryptophan into the internodes of rye plants infected with Claviaeps purpurea yielded radioactive alkaloids which

could be degraded to active lysergic acid (11) and iso-lysergic acid (16) 13.

H

---~

+

11 16

The repetition of this experiment using replacement cultures not only confirmed the result, but indicated that

14 both D- and L-tryptophan could be metabolised by the fungus . The suggestion that these results might be attributed to

the decarboxylation of the incorporated tryptophan was

con-. .

firmed by the fact that D.L-[carboxy-14cJ-tryptophan yielded totally inactive alkaloids when fed to cultures which could yield active metabolites from D.L-[s-14cJ -tryptophan14115 . The biosynthesis of the clavines was shown to involve

8.

Similar results have been obtained with the lysergic acid

derivative, ergosine (17) 18 .

17

C

0

H

NMe

The biosynthesis of isopentenyl pyrophosphate (18)

involves decarboxylation of mevalonic acid19 , and the

isolation of inactive alkaloids from cultures containing

[1- 14c]-mevalonic acid indicated the involvement of (18)

or dimethylallyl pyrophosphate (10). Direct proof of

this hypothesis was obtained by the incorporation of

deuterated isopentenyl pyrophosphate into the clavines20 •

Despite the involvement of tryptophan and (18) in ergot

alkaloid biosynthesis, there still remained the problem

of the position of alkylation and its sequence in the

bio-synthetic pathway. As the N-methyl_ group was known to be derived from methionine21 (19), and since neither tryptamine

(20), N-(a)-methyltryptophan (21} nor N-(w)-methyltryptamine

(22) were invo ve . 1 d . in th e b. iosynt esis h . 21; 22 , i t appeare . d

that alkylation must occur directly on a tryptophan molecule

9.

A A

+

_{~ 0 - P P _ _ _ ...,_}

FLapp

18 10

A

23

\

24

15

R=OH

e ~ C O i H

....

MeS

19 NH2

1

~

C0

2H

NH₂ NHMe NHMe

20 21 22

10

The latter of these processes would involve a simultaneous

decarboxylation, which would accommodate the previous

·observation that a decarboxylation of tryptophan occurs

after substitution has taken place, Initial studies23

utilising 4-(3-methylbut-2-enyl)-[ s- 14c]-tryptophan (23)

3 .

and a-(3-methylbut-2-enyl)-[ H] tryptamine (24) showed that

both compounds were incorporated into elymoclavine (15).

Further examination of the rates of incorporation showed

that (23) was a more efficient precursor than (24), although

24 both were less efficiently incorporated than tryptophan .

This result could have been due to the_ greater permeability

of tryptophan through the cell walls in comparison with its

alkylated derivaties. The inhibition of elymoclavine

production with ethionine in an unidentified strain of ergot,

resulted in the accumulation of 4- ( 3-methylbut .... 2-enyl)

tryptophan (23), a result which further suggests tha~ (23)

· · f t th t f the clavi'nes25126 • is, in ac, e correc precursor o

Decarboxylation must therefore follow the alkylation of the

aromatic ring, a suggestion confirmed by the incorporation

of 4-(3-methylbut-2-enyl)tryptamine (25) into agroclavine

( 14) •

In the case of elymoclavine and chanoclavine, the

hydroxylation of the C-17 position has also to be considered.

The experimental results are not definitive as

4-(trans-4-hydroxy-3-methylbut-2-enyl)tryptophan (26), the corresponding

tryptarnine (27), as well as (25), were all found to be

rnetab-olised by Claviceps -Stamm Jl3. These results may be explain-ed by postulating two separate pathways for the rne.tabolisrn

11,

23 ₂₆

N~

25 27

That hydroxylation precedes the first cyclisation, at least in a Claviceps strain from Pennisetum typhoideum,

has been established by the fact that neither (2 8) nor (29) are incorporated into chanoclavine-I or the

tetra-. 28 29 cyclic ergolines ' _•

· . 3

o

dB' h 31 h h th t . th . Arigoni an ire aves own a in e iso-pentenyl pyrophosphate isomerase reaction, the methylene

group, which arises from C-2 of mevalonic acid, becomes the trans-methyl group in 3,3-dimethylallyl pyrophosphate, a process which is accompanied by the stereospecific

removal of the proton originating from the 4-S position of mevalonic acid32 • It might be expected that if [2-14 c]-mevalonic acid were incorporated into elymoclavine or chano-clavine-I, the activity would be retained at C-17. As

previously noted, this is true for elymoclavine, but recent work has revealed that chanoclavine-I (13), chanoclavine-II

(29) and isochanoclavine-I (30) retain 90% of the activity at the C-methyl group irrespective of the ste:reochemistry of the double bond33134 .

13 29 30

Furthermore, i t has been shown that mevalonic acid

contain-ing tritium in the 4R position will lead to active ergolines,

•

OPP

0

HOH₂C 0

26

A0

•

HOH

2C

13

0 A0

14

,

=

observed

0 o

=

predicted

Figure 2

cis,.,,trans

C0

2H

~

13.

I I

I

0

I

H

'

NH2

25

0 I

HOH₂C

0

i

H

NH₂

27

A ₀ OHC

31

14 ..

give rise to an active trans-methyl group in the

isopre-noid side chain34r 35 . It is, therefore, necessary to

postulate a reversal of sterecchemistry about the double

34

bond , a process which presumably occurs before the

for-mation of 4-(trans-4-hydroxy-3-methylbut-2-enyl)tryptamine

(Figure 2) .

Since the activity from [2-14c]-mevalonate is found

at C-17 in elymoclavine (15), there can exist two possible

biosynthetic pathways. The first of these would involve

chm1oclavine-I, which must undergo a second cis-trans

iso-merisation in the isoprenoid side chain, whilst the second

would involve isochanoclavine-I, in which case, no such

isomerisation is necessary.

13

15.

Tracer experiments using chanoclavine-I (13), with a 14

c

label in the hydroxymethyl. group, have shown that 96.4% of the activity occurs in the C-7 position of agroclavine. Further work has suggested the possible involvement of the

. 33 36

chanoclavine-I aldehyde (31) ' . These observations, together with the fact that isochanoclavine-I is not

incor-d . h 1 · 1 · 2 8, 33 .

porate into t e tetracyc ic ergo. ines, confirms that a second cis-trans isomerisation is involved in the bio-synthesis of elymoclavine (14) and related molecules. The overall biosynthetic pae~way is outlined in Figure 2. Such a pathway however leaves several points unexplained. It would be enlightening to invest~gate the role of 4-(ais-4-hydroxy-3-methylbut-2-enyl)tryptophan (32) to see

if

this provides a link between (23) and (26).

32

The problem of the decarboxylation of tryptophan still remains unanswered. I t has been shown that (23)

labelled with 14

c

and 3H (as shown in Figure 2) is incor-

.

porated into agroclavine (14) without any change in the

Furthermore, since both D- and L-tryptophan are metabolised,

and a decarboxylation step confirms this fact, there is the

problem of converting a racemic centre (33) containing

tritium into the fixed sterochemistry of chanoclavine-I.

This latter problem is also associated with the involvement

of (25} and (27). Since these would be derived from a

(+) mixture, i t is difficult to find a cyclisation process

that would not involve the loss of tritium.

33 ₁₃

34

The role of isoprenylated tryptophan units in the

biosynthesi.s of the ergot alkaloids remains somewhat

obscure, and the further use of optically active amino

acids should prove rewarding. There is also the problem

f 1 . . . 38 ( )

17.

not being involved in the biosynthesis of the tri cyclic

and tetracyclic ergolines. Perh aps i t should be pointed

out that much of the biosynthetic work report ed has been

conducted on a wide variety of Claviceps strains. This

raises the question as to how valid i t is to correlate

results obtained in one

.

system with those obtained from

another .

A cyclisation similar to that found in the ergot

alkaloids must also be involved in the biosynthesis of

cyclopiazonic acid (35), a metabolite from Penicillium

l . 39

cy c opi-um •

----li::-ro HN

OH 0

37

e.,... .

Me

I OH

36

(

0

The expected involvement of ace tate , rnevalonate and

tryptophan has been observed. By the use of cultures wi th

acetate l abelled bisseaodehydrocyclopiazonic acid (36),

which proved to be an efficient precursor of the

penta-cyclic system (35) 40 .

The close similarity between cyclopiazonic aci~ and

the ergolines suggests that 4-(3-methylbut-2-enyl)tryptophan

(23) may be the alkylated species involved in the biosynthesis,

but no evidence exists to rule out the possibility of an

alkylation occurring on a structure such as (37).

Neoechinulin (38), a metabolite of Aspergillus

t -,

d · 41 . . . :, . t th C 6 . .

ams e&o am~ , contains an isoprenoia uni on e - position

of the benzenoid ring of tryptophan, together with a reversed

isoprene unit at C-2. The oxidative cleav~ge of an alanine

or serine side chain attached to a 2,5 - diketopiperazine ring

which apparently occurs in the minor me t abolites (39), (40),

f P · ·-,-,· -,·k k' · 42 ld h t (41) (42)

o en1,,c1,,&&1,,um ter&1,, ow 1,,1,, , wou suggest t a or

may be possible key intermediates in the biosynthesis of (38).

©CHH

_OH

_H

0 O

39 40

The involvement of (41) would be particularly interesti ng, as

this structure is closely re.lated to that of echinul in (43),

the major 2,5-diketopiperazine of

A

.

amstelodami and

4 h . -, . 4 3, 44 .

, • ec .1,,nu&at1,,s • Recent studies , suggest that cyclo

o-19.

h . f h. 1 · 45 1 . . h

synt esis o ec inu in , a postu ate consistent wit the

previously established incorporation of tryptophan and

alanine43 r 46 .

II

44

0

H N ~ O

NH

'?'

H N ~

' NH

0

0

0

H N ) y

NH

41

/

0

H N Y y

NH

The close relationship between (38) and (43)

illus-trates the apparent ease with which tryptophan undergoes

isoprenylation in the aromatic ring. The substitution at

C-7 in (43) is particularly noteworthy, as this position is

considered least susceptible to electrophylic attack3. The

C-alkylation of relatively unactivated systems has been

. 4 7 4 8 49 postulated to proceed

via

a carbene-type mechanism ' '

and recently i t has been suggested that a similar mechanism

could be involved in the case of (38) and (43) 47 . Such a

mechanism would involve the formation of a cyclopropane

derivative (45) which could presumably undergo

re-aromatis-ation by either of two different routes to yield (46} and

( 4 7) .

46 45 47

A somewhat similar hypothesis involving an arene oxide, has

been suggested as an explanation for the biological

5-hydroxy-50 lation of tryptophan .

The reversed isoprene unit at C-2, common to bot.11.

neoechinulin (38) and echinulin (43) ₁ has been the cause of

considerable speculation. _{Whilst i t is conceivable that i t}

is derived directly from an SN2' substitution of

3,3-dimethyl-allyl pyrophosphate [ca. (10)~(9)], attempts to produce

suit-able model reactions have been unsuccessful. 3-Methylindole

3-21.

dimethylallyl bromide to yield (49). Under similar con-ditions N-(w)-acetyltryptamine (50) gave a mixture of (51) and (52) 51 .

Me Me

48 49

NHCOCH3

50 51

These products are consistent with initial substitution

taking place at the more nucleophilic C-3 position, followed

either by cycilisation (51), or a 1,2-shift (49), (52).

The possibility of a Claisen type rearrangement from C·-3

had previously been shown to be unlikely52 . _{Another possible} mechanism of substitution could involve an acid catalysed

Claisen rearrangement from the nitrogen hetero-atom, and

recent reactions of this type have been successfully

con-53

ducted . _{There is, however, in the case of echinulin a} lack of biological evidence to support any of these propo-sitions.

found in Penicillium brevi-compactum are derived from the precursor (53). Whilst attempts to detect this inter-mediate in P. brevi-compactum have been unsuccessful, i t has recently been isolated from Aspergillus ustus, where i t occurs in conjunction with austamide (56) 54 •

53 56

54 55

23.

0 0

59

53

•

The substituted 2,5-diketopiperazine (53) contains the

reversed isoprene unit at C-2, and to this extent resembles both echinulin and neoechinulin. However, the presence of only one isoprene unit in (53) should make it easier to

establish its biogenetic origins. Work in this thesis has attempted to clarify the origin of the isoprene group in brevianamide-A(l) on the assumption that the precursor (53) is involved. The amino acid (57) is clearly not involved in the biosynthesis of brevianamide-A, although a possible rearrangement of (58) has yet to be disproved.

The conversion of (53) to (1) has been the subject of considerable discussion, and the formation of the proposed intermediates (55) can be attributed to a number of

mechan-.

1.sms. Two early suggestions, . . 47 55 ' involved the displace-ment of sulphur [(61)7(62)] and the generation of a cationic centre [(63)7(64)] which would initiate addition of the

double bond to the diketopiperazine ring.

N

+

S2 .

61 _N H

---N

H

~

+ ~ " - - H

N _N

~

N N

H _H

25.

If i t can be assumed that the diketopiperazine (53) is derived from two L-amino acids, then the former mechan-ism would involve the inversion of the two asymmetric

centres whilst the latter would result with retention of stereochemistry. _{Attempts to determine} the absolute

stereochemistry of the chloroform adduct of brevianamide-A, 56

by X-ray crystallography have been unsuccessful , so that at present i t is not possible to rule out either possible mechanism. _{More recently, i t has been proposed that (55)} may be derived from an internal Diels-Alder reaction involv-ing an intermediate (65) 57 . _{Such a suggestion closely}

resembles the postulated biosynthesis of gambogic acid and the structurally simpler bronianone (66) 58 , both of which are believed to be derived by the Diels-Alder addition of an isoprene unit to a cyclohexa-2,4-dienone system, Recent studies59 have demonstra.ted. the feasibility of btiis suggest-ion.

I

R, R

=

Terpenoid

HO

HO

65

0

?

OH

I

0

- - - -

~

HO

[OJ

HO

Diels Alder

R

ofn

In the case of austarnide (56) the addition of the

isoprene unit to the 2,5-diketopiperazine ri~g is

presum-ably prevented by the presence of a double bond in an early

intermediate such as (67). _{Whilst i t is possible to}

spec-ulate about the mechanism whereby the nitrogen attacks the

double bond of the isoprenoid unit, one plausible scheme

would involve the epoxide (68). _{The intermediate (70)}

would then undergo oxidation and rearrangement similar to

1

that proposed for brevianamide A. Clearly both the

bio-synthesis of brevianamide A and austamide must remain

spec-ulative until such times as proposed intermediates can be

synthesised and fed to the appropriate mould.

67 68

2 7.

ADDENDUM

Since the text of this thesis was prepared, some

unpublished experimental data relating to the biosynthesis

f 1 . ( 73} h b . 1 bl 195 h · o eye openin . as ecome avai a e • Te conversion

of cyclopeptin (71) to cyclopenin (73) does not appear to

involve the hydroxylated intermediate (72) and is controlled

by a dehydrogenase.

Similar reactions must therefore be considered as

possible steps in the biosynthesis of neoechinulin (38) and

austamide (56).

28.

PART IIA

THE· BREVIANAMIDES

DISCUSSION

(a) Introduction

· . . . 60 f th b 1 ·

During an early investigation o e meta o ites

produced by the mould PeniciZZium brevi-compactum, i t was

observed that cultures grown on Czapek-Dox broth produced

an intensely fluorescent pigment which diffused into the

culture medium. _{A subsequent investigation 1 resulted in}

the identification of the major pigment, brevianamide-A,

together with a related metabolite, brevianamide-E. It

was noted during the same investigation that three· minor

pigments, brevianamides-B,

-

c

and -D, were also present in

the culture medium, although their structures remained

speculative.

A number of common moulds which grow on poorly stored

corn are known to be responsible for the production of

met-abolites which exhibit marked toxicity and are believed to

be partly responsible for the high incidence of liver

car-61 cenoma amongst the Bantu .

The isolation from mouldy corn of Penicillum

brevi-compactum and Penici lZium viridicatum, both known sources

f b . .d 61,62 . . . .

o revianami e-A , prompted a detailed investigation

into the chemistry of the minor neutral metabolites of these

moulds. Although the toxicity of the neutral extract of

P. brevi-compactum has been established61, the relative

toxicities of the six indoloid metabolites, now known to

29.

Because of the small quantity of pigments present

in the culture of P. brevi-compactum when grown on

Czapek Dox broth, considerable effort was directed towards

producing a strain of the mould, more suitable for the

present work, which would give high yields of the pigments.

An initial screening of a group of mono-spore cultures

enabled ·a strain to be selected which, when cultured under

optimum conditions, would yield 20 mg of pigments per litre

of culture medium. Attempts to isolate artificially

induced mutant strains of the mould which would produce

exclusively brevianamides-C and -D were unsuccessful and

subsequent work suggested that such a mutant was most

unlikely to exist.

A separation of the minor components of the pigment

mixture was achieved by preparative tl1in layer chromatography,

and a sufficient quantity of the pure compounds was obtained

to enable the measurement of their physical properties and

a restricted investigation of their chemistry. The latter

was however sufficient to enable a chemical relationship

between brevianamide-A and brevianamides-B, -C and -D to be

established.

(b) Brevianam.ide-C:

The nature of the chromophore

Brevianarnide-C was isolated as an orange glass which

showed an appreciable solubility in ch1oroform and more

...

.-....--r--r-.-.-....--r--.--.-.-....--...--...--r-,-.---.--r--r-r-.--....--r---r-,-.---,--r--r-.-.---,--r--r-.-.--....--r--r-.-.--....--r--r-.-.--....--r---r-,-.---,--,---r-,-.---,--,.---.-,-r-·....-...---r-,-.--....-....---r-,-.---,-...,...,..-,-,---,--r--r-.-.--....--r--r-r-.--.--r--r-,-.--_{-,--r--r-,-r-.---,--,--,--r-..-"T-,}

1000 I

500 400

I

250

I

100

I 50

BREVIANAMIDE C

C

10·0 9·0 3·0 7·0

300 200 100

6·0 5·0 _{PPM (S4-0} 3·0 2·0

COCI_..., )

/

I

~

l·O ·

>-H~ C -ps

30.

The mass spectrum of brevianamide-C showed a

molec-ular ion at m/e 365 of composition

c

₂₁H₂₃N₃

o

₃ , which

indicated that brevianaIPide-C was isorreric with the major

p~gment brevianarnide-A.

The ultra-violet spectrum, which had maxima at 234 nm,

259 nm, and: 450 nm, did not immediately s~g-gest the ·nature

of the chromophore. However, the ·1o~g wave le~gth

absorp-tion at: 450 nm did indicate th.at the chromophore was not

. the simple pseudo-indoxyl system found in brevianarnide-A .•

The NMR spectrum (Figure 1) showed resonances due to

four aromatic protons, and a resonance at 85.94 which was

considered to belo~g to an olefinic proton. These

obser-vations , t~gether with the colour of the compound, and a

possible relationship with brevianarnide-A, sug-gested the

chromophores (1) and (2), both of which appeared to be

consistent with the NMR da~a.

CHR 0

0 CHR

1 2

R= Alkyl

63

Previous workers had prepared 3'-oxindolidenethane (3)

by the hydr~genolysis of 1-(3'-oxindoliden)ethanol (4),

but an attempt to repeat this work yielded only colourless

products, of which 3-ethyloxindole (5) was the major

CHMe

6

- - - >

0 3

HO-.../Me

5 0 ... 45,---_ _ _ _ _ 0 4

H

The desired compound (3) was subsequently prepared by the

condensation of oxindole (6) with acetaldehyde64. The

ultraviolet and NMR spectra of (3) showed little

similar-ity to the corresponding spectra of brevianamide--C and

the chromophore (1) was excluded from further

consider-ation.

A model chromophore of the second type was ~ore

difficult to find. Whilst indoxyl undergoes base catalysed

condensation with aromatic aldehydes 65166 and ketones67 to

yield 2'-indoxylidenarenes (2 R=Ar), the reaction with

aliphatic aldehydes, with the exception of one

unsubstan-tiated report68, does not appear to have been investigated.

The condensation of 3S-hydroxy-5-androsten-17-one (7) with

o-nitrobenzaldehyde produces a yellow compound which has

been assigned the structure of

3S-hydroxy-16,17-seco-16-nor-5-androsten-15-(2'indoxylidene)-17-oic acid (8), this

being the first reported, although indirect, synthesis of

2'-indoxylidenalkane69 . The stereochemistry of the

indoxy-lidene double bond appeared to be trans with respect to

the carbonyl, as the product would cyclise to (9) when

32.

0

HO HO

7 8

0

OAc AcO

9

The ultra-violet spectrum of (8) A _max 238, 262, 455 nm was in close agreement with that observed for

brevian-amide-C and i t appeared that the natural product possessed

a chromophore of type (2) (R=alkyl) in which the alkyl

group was trans to the indoxyl carbonyl.

h f 1 th . 70

f

.

Te recent success u syn esis o abs and

trans-6-methoxyaurone (10}, (11) suggested that the photolysis

of a 2'-indoxylidenalkane might yield an analogous mixture

of isomers which would provide further evidence to confirm

the sterochemistry of the double bond. It appeared desirable to have the simplest possible model compound

for this photochemical isomerisation, and i t was

consid-ered that a suitable system might well be produced by the

condensation of 2,2~dimethylpropanal with indoxyl. The

aldehyde was chosen to prevent self condensation occurring

0

MeO MeO

10

0

H

Indoxyl generated in situ from its diacetate was found to

readily condense with the aldehyde in the presence of

sodium methoxide. The NMR spectrum of the product, a

yellow crystalline solid, showed a nine proton resonance

of a tertiary-butyl group at ol.27, an olefinic proton

resonance at 86.00 togethe~ with signals attributed to

four aromatic and one labile proton. The resonance at

o 6. 0 was considered to be consistent \·li th the proton

attached to the exocyclic doQble bond of (12) and this

structure was supported by an intense absorption of a

con-jugated double bond at 1640 cm-l in the infra-red spectrum.

A carbonyl absorption at 1695 cm-l was in agreement with

the published values 71 for a fused aromatic cyclopentenoid

ketone. Final confirmation of the structure came from

the high-resolution mass spectrum which showed a molecular

ion of composition

c

13H15

No.

The spectra of (12) showed

excellent agreement with the related parts of tlLe spectra

34.

( 12) A _max 237, 260, 444 nm, confirmed the correct choice

of chromophore.

The irradiation of a benzene solution of (12) with

310 nm lamps produced a mixture of two compounds which was

shown by thin layer chromatography to contain the yellow

starting material and a second orange compound with a

greater Rf value. Mass spectrometry showed that this new

product was isomeric with the starting material (12) and

the similarity between the fragmentation patterns of the

two compounds was consistent with the anticipated

geomet-rical isomerism. In the NMR spectrum of this orange isomer

the tertiary-butyl group resonance occurred at ol.36, and

that of the proton attached to the exocyclic double bond

at

o

5 . 7 8, _{The continued presence of the} double bond and

the indoxyl carbonyl were confirmed by the absorptions at

1632 and 1685 cm-l in the infra-red spectrum.

A detailed consideration of _{the NMR spectrum of the}

two isomers suggested that the resonance of the olefinic

proton at a lower field in the case of yellow isomer (12)

was consistent with the tertiary-butyl group being trans

to the carbonyl. _{This stereochemis try would allow the}

olefinic proton to be slightly deshielded by the carbonyl

72

group . The lowering of the methyl resonance by 9Hz

in

the case of the orange isomer (13) _{was consistent with a}

lessened deshielding influence of the carbonyl on the more

remote methyl _{protons of the tertiary-butyl group in the}

C'l,S isomer, It appeared from these experi_{ments that}

2,2'-2·0

0,0

0

©O=CHIB

,

H

200

WAVELENGTH IN NM

I I I I I I I I I I I 225 I I I I I I I , .... , ' \ ,' ' I 250

' ' I

\ \ \ \ \ 275 I I I I \ I I I I I I I I

FIGURE 2

400 450 500

---CIS ISOMER

- - TRANS ISOMER

100

300 325 350 400 450

2·0 . . . - - ~ - - - . . - - - -- - - r - - - , - - - . - - - . - - - , - - - , - - - ,

BREVIANAMIDES I I I

--,

,' \

I \ / I ,' \ / I

I \

/ I

I ' ' \ \ I I I I I I \ I \ I \ I I I X3 400

--- --- D - - - c

... -

--

... ... , ', ' _',, ' _{... _} 450 .... ---' ', ' 500 '

O·O ...__...._ _ _ _ _ __. _ _ _ _ _ _._ _ _ _ _ _ L..._ _ _ _ _ ___._ _ _ _ _ _ . . . . _ _ _ _ _ _ _ . . _ _ _ _ _ _ _ . _ _ _ _ _ ______.

100 450 200

WAVELENGTH IN NM

35.

dimethylpropanal to yield the thermodynamically more stable

isomer (12) in which the alkyl group is trans to the

car-bonyl.

The slight but characteristic differences in the

ultra-violet spectra of the cis and trans isomers (13) and

(12) (Figure 2) were sufficient to allow the assignment of

the trans stereochemistry to brevianamide-C, _{A partial}

structure for brevianamide-C (14) could now be proposed.

14

(c) The dipeptide fr·agment

By assuming a close relationship between brevianamide

-c

and brevianamide-A i t was possible to interpret certain

of the spectroscopic features.

N2

o

2 , of the remaining fragment of the pigment appeared to

be consistent with a 2,5-diketopiperazine ring and a C-5

carbon unit. _{The NMR spectrum showed a triplet at 03.48}

which could be attributed to the methylene protons (H H '

A A

in18) adjacent to the pyrrolidine nitrogen, and the multiplet

at 82.8 was consistent with the protons HDH D~ a similar

pattern having been observed in brevianamide-A. _Further

confirmation of the presence of the 2,5--diketopiperazine

structural unit was obtained from the NH band at 3351 cm-l

and the amide II band at 16 80 cm -l, in the infra-red

spec-73

trum . _{As was the case with brevianamide-A} 1 _{, the amide}

I band was missing from the infra--red spectrum and its

absence was taken as further proof of the cyclic amide

structure. The absence of resonances in the region

83.7-4.5 in the NMR spectrum suggested the absence of protons

from the 3 and 6 positions of the diketopiperazine ring 1 ,

and i t appeared that the C-5 unit must, as a consequence,

be attached to these positions.

Confirmation of this hypothesis could be obtained

from the mass spectrum which showed a major fragment ion

m/e 295 corresponding to tt,_e loss of

c

5H10 from the

molec-ular ion. _{It has been shown that in the fragmentation of}

3,6-dialkyl-2,5-diketopiperazines the alkyl groups may be

lost as radicals or olefins 74 . This fact, coupled with

the NMR data obtained from brevianamiae-c, confirmed that

a C-5 bridge linked the 3 and 6 positions of the

diketo-piperazine ring, present in the pigment. More recently,

a cleavage similar to that postulated for brevianamide-C

has been observed75 in the mass spectrum of

N,N'-diphenyl-2,5-diaza-3,6-dioxobicyclo [2.2.2.J octane (15).

©l

N

0

15

The exact nature of the C-5 bridge was determined by the

presence of an isopropyl group. The evidence for the

existence of this group came from a six proton doublet at

3 7.

remained the same in both 100MHz and 60MHz spectra,

thereby indicating that the signal was a coupled

doubled, rather than two non-equivalent singlets, The

loss of

c

3H7 from the molecular ion in the mass spectrum

confirmed the presence of an isopropyl group, and two

structures (16) and (17) could now be proposed for the

N

~~c

~r 0

--16

0

N

H/C N 0

17

From the physical evidence i t was not possible to

dis-tinguish between these two partial structures but

bio-genetic considerations and. a possible relationship to

brevianarnide-A favoured (17).

The total structure (18) for brevianarnide-C could

now be proposed, and this structure can be used to explain

the ready loss of the

c

5H10 fragment in the mass spectrum.

The cleavage of two bonds S t o a nitrogen atom and S t o a

carbonyl group, both known favourable cleavages, would

produce the stable ion (19), It is interesting to note

that the anticipated loss of CONH, normally found in the

mass spectra of 3,6-dialkyl-2,5-diketopiperazines, was

absent from the spectrum of brevianarnide~C, possibly

A detailed assignment of the NMR spectrum of brevianamide-C

in relationship to the structure (18) is given in Table 1.

0

18

0

0

+

0

19

TABLE 1

NMR Spectrum of Brevianamide-C ·(T8)

Assignment Protons Shift (cS)

Aromatic 4 7.7-6,,8

eHMe

2 6 0.84

HA 1 5. 9 3

HD HD' 2 2.8

HF HF' 2 3.48

HG HG' b 2 7. 9 8, 7.34

HB He He• HE HE' 5 2.5-1.7

a s-singlet, a-doublet, t-triplet, m-multiplet

b disappears with D 20

0

Me

Me

18

39.

a

Pattern

AA'XX'

d

s

m

t

s, s

(d) Brevianamide-D:

Brevianamide-D was obtained as a red glass and_ gave

a mass spectrum which, with the exception of some minor

intensity differences, was identical with that of

brevian-amide-C.

The infra-red spectrum showed two NH bands, one at

-1 -1

3420 cm and a broad band at 3200 cm . The carbonyl

region could not be clearly resolved even in dilute

solu--1

tion, and showed a shoulder at 1705 cm and a broad band

-1

at 1680 cm •

The ultra-violet spectrum A 233, 264, 306, 479 nm · max

closely resembled that of 2,2-dimethyl-cis-(2'-indoxylidene)

propane (13) which showed maxima at 239 nm, 269 nm, 298 nm

and 462 nm. As a consequence of this similarity, i t seemed

reasonable to suggest that brevianamide-D (20) was the

. geometrical isomer of brevianamide-C.

20

This suggestion was confirmed by the close similarity between

the NMR spectra of the two compounds . The most significant

difference between the two spectra was the appearance of

the brevianamide-D isopropyl_ group resonance as a quartet

(Figure 3) rather than a doublet. This could be explained

FIGURE 3

~

1000 I

500 400 300 200 JOO

)-H~

(1 Hz

I

250

I CDCl3

100

I

50

BREVIANAMIDE D

l

10·0 9·0 8·0 7·0 6·0 5·0 PPM(6) 4·0 3·0 2·0 J.Q _O·O

~

the indoxyl carbonyl in the· ais isomer m~ght provide

76

differing environments for the methyl protons • However,

whilst this suggestion is not unreasonable, i t should be

pointed out that the isopropyl groups in both isomers are

attached to an asymmetric carbon atom and this might be

expected to produce a quartet in both the ais and trans

. 76 77

isomers ' • Consequently little significance was

attached to the difference in pattern of the methyl

reson-ances in the NMR spectra of brevianamides-C and -D. It

was significant that the resonance positions of the

ole-finic proton attached to the exocyclic double bond of (19)

and (20) were almost identical and that without suitable

model compounds (12) and (13) i t would have been difficult

to deduce the sterochemistry of the two natural products.

(e) The inter-relation of Brevianamides-C and -D

'I'he suggestion that the brevianamides-C and -D were

geometrical isomers about the indoxylidene double bond

required some clarification. Despite the close similarity

between the spectra of the two compounds the possibility of

differences in the diketopiperazine moiety, such as a

rev-ersal of the bridge, could not be ruled out. Direct

photo-chemical interconversion seemed to provide one possible

method of relating the two isomers. However an initial

attempt to effect this conversion by irradiating a benzene

solution of brevianamide-C at 310 nm resulted in the total

rupture of the molecule. Subsequently, in a different

42.

affect the desired photolytic interconversion between

brevianamides-C and -D.

In view of the difficulties experienced with the

initial photochemical interconversion, i t appeared to be

necessary to relate the two pigments by a chemical

con-version to a common product which lacked the centre of

.

.

isomerism. Ozonolysis of the exocyclic double bond ·

presented one possible method of producing common products

from the two isomers, but the small quantity of material

available indicated that a more profitable approach would

be to try and reduce the double bond of the chromophore.

A preliminary investigation into the hydrogenation of

2,2-dimethyl-trans-(2'-indoxylidene)propane (12} showed

that this approach to the reduction of the chromophore

was unsatisfactory. Although hydrogenation proceeded

smoothly, the product rapidly oxidized when exposed to

air, regenerating the starting material. Attention was

then directed towards the reduction of (12) with sodium

borohydride. Considerable difficulty was experienced in

obtaining a homogeneous product from this reaction.

How-ever, prolonged reduction of (14) with sodium borohydride ·

in a methanolic sodium methoxide solution yieloed the

3-hydroxyindoline (22) which, when treated with acid, was

rapidly converted to the indole (23). From a study of

the ultraviolet spectrum78 of the reduction mixture i t would

appear that (12) is rapidly reduced to (21), which is

slowly converted to (22). Attempts to isolate (21) were

on exposure to the air.

.

0 OH

12 _{- . r - - - - -} 21

OH

23 22

Brevianamides-C and -D were reduced under similar

conditions and the 3-hydroxyindolines (24) converted

directly to the indoles deoxydihydrobrevianamides-C and -D

respectively. The two reduction products were identical

in all respects and showed spectroscopic properties in

agreement with structure (25). The major fragmentations

in the mass spectrum of (25) corresponded to the loss of

the C-5 bridge and cleavage at the benzylic carbon; both

expected and facile cleavages.

in Figure 4.

The NMR spectrum is shown

The successful conversion of brevianamides-C and -D

to a common reduction product, coupled with the

photo-chemical interconversion, confirmed the hypothesis that

1000

s1

I

2.50

I

100 I

.50

10·0

400

C

9-0 8·0 _7·0

FIGURE 4

300 200

DEOXYDIHYDROBREVIANAMIOE C

D₂0

6-0 5·0 PPM(6)4·0 3·0

100

CDC'3

2·0 1·0

~

>-H~

0 CPS

(}0

.:i,.

w

24

OH

f

(f) Brevianamide-B

25

Apart from a much lower solubility and different

behaviour on TLC, brevianamide-B,

c

21H23N3

o

3, was found to

closely resemble brevianamide-A. In particular ·the

ultra-violet spectrum confirmed the presence of a simple

pseudo-indoxyl chromophore78 , whilst the mass spectrum showed a

fragmentation pattern identical to that given by the major

pigment. The loss of a

c

5H₉ fragment in the mass spectrum

strongly suggested that the structure of brevianamide-B

must closely resemble that of brevianamide-A. Unfortunately,

the low solubility of brevianamide-B made i t difficult to

obtain satisfactory NMR spectra. However, certain features

common to the spectra of the two pigments could be ~bserve~

and the presence of two non equivalent methyl groups in

brevianamide-B was confirmed by resonances at o0.98 and 1.40

in an NMR spectrum determined in d

6-dimethyl sulphoxide.

One hypothesis which appeared to be consistent with the

physical data was that brevianamide-B was stereoisomeric at

45.

HN---co

Relative Stereochemistry

0

26

- - -CO

If this was the case, i t appeared reasonable to assume that

a chemical conversion of brevianamide-A to -B could be

achieved, and i t became apparent that reactions similar to

those proposed for the biosynthesis of brevianamide-A ~ght

be particularly useful. Previous work1 had established

that brevianamide-A could be reduced to the hydroxyindoline

(30), which could be rearranged to deoxybrevianamide-A (31).

0

b i

-28 29

0 0

30

H+ OH

0 0

1)

Oz

f Pt

,...

2) H2

I

Pt

This indole (31) is believed to have the inverted structure

of the biosynthetic precursor (28) of brevianamide-A, and

this now suggested that deoxybrevianamide-A could be

re-oxidised to yield a mixture of brevianamides-A and -B.

Deoxybrevianamide-A (31) obtained by the reduction

and dehydration of brevianamide-A1, was submitted to

cata-lytic re-oxidation by the procedure of Witkop79180 • The

intermediate hydroperoxyindolenine was reduced and

hydroxy-indolenine (32) rearranged with base. Only one

rearrange-ment product, brevianamide-B, was observed.

was surprising and is difficult to explain.

This result

An

examin-ation of Dreiding models of (31) or (32) does not give any

indication as to why a single product is obtained in

prefer-ence to a mixture of stereoisomers. One possible

explan-ation seemed to be that the overall asyrr~etry of

deoxy-brevianamide-A (31) resulted in a preferential approach of

the molecule to the catalytic surface on which the

oxida-tion occurred. This in turn implied a stero-specific e

A.

oxidation, the product of which could conceivably undergo

a stereospecific rearrangement to brevianamide-B. In order

to test this hypothesis, deoxybrevianamide-A was subjected ·

to aerial oxidation in the absence of a catalyst.

Su~pris-ingly, the methanolic solution turned yellow indicating a

direct conversion to a pseudo-indoxyl. Again brevianamide

-B was the only rearrangement product observed and this ·

result was in conflict with the previous hypothesis. I t

is not impossible that even in the absence of a catalyst, a

stereospecific oxidation occurs. Unfortunately this

inter-47.

mediate hydroxyindolenine (32) could not be isolated.

These results confirmed the suggestion that

brevianamide-B was in fact a stereoisomer of brevianamide-A

with the isomerisation occurring at the spirocentre. The

results of the oxidation experiments also raise an

inter-esting biosynthetic concept. The seemingly selective

oxidation and rearrangerrent of (31) in air makes i t

tempt-ing to speculate that the biosynthetic precursor (28) may

also oxidise and rearrange selectively to brevianamide-A.

Such a suggestion is not inconsistent with the natural

abundance of the two pigments -A and -B. No evidence

exists to suggest whether or not the large number of known

indoxyl alkaloids are metabolites of enzymatic origin or

simply artifacts produced by oxidation. In the case of

P. _{bre~i-compactum the culture conditions would be in}

accord with the latter possibility.

(g) _{The inter-relationship of brevianamides-A,· -B,}

_-c

and -D

An examination of the structures of brevianarnides-A

and -B, together with the structures proposed for

-c

and

-D, suggested that the latter two pigments might be derived

from the former pair by an interesting

_c-c

_{bond cleavage.}

The generation of a saturated alkyl group under relatively

mild conditions by the cleavage at an appropriately

activated quaternary carbon atom is not unknown, but is

certainly unusual. _{The thermal conversion}81 _{of (33) to}

(34), whilst not directly related to the brevianamides,

did suggest that brevianamides

_-c

_{and -D may be artifacts}

produced during the isolation procedure.

H

©X)<:

H

33

©X:}R

+

RH

H

34

Despite the relatively mild conditions of the isolation

procedure, the possible occurrence of a thermal cleavage

during large scale evaporations could not be discounted.

However attempts to convert brevianamide-A to

-c

by a

thermal process were unsuccessful and the possibility of a

photochemical conversion was considered.

The irradiation of solutions of brevianamide-A, in

mixtures of benzene and methanol at 245 and 310 nm yielded

complex mixtures which contained brevianamides-C and -D.

Methanolic solutions of -A were then _{irradiated with white}

light and a quantitative conversion to _{the isomers-C and}

-D was achieved. _{During the early stages of the photolysis}

with white light, traces of brevianarnide-B were also

49.

-B can be explained by postulating a ring-open radical

intermediate (35) in which free rotation can occur, thereby

· allowing ring closure to occur either to -A or -B.

Alter-natively, hydrogen transfer would produce brevianamide-C

or -D. Since the latter two isomers were equilibrated

under the conditions of photolysis i t was not possible to

determine any sequence of

repeated in methanol-d 1

82

production. The photolysis was

but after the exchange of labile

deuterium no further deuterium remained in the product.

Unfortunately this experiment could only point to the

non-involvement of labile protons from the solvent and as yet

i t has not been possible to prepare suitably deuterated

brevianamide-A to enable a further examination of this

reaction.

In an attempt to gain further insight into this

cleavage, the photolysis of (36) was examined, but no

reaction analogous to that observed with brevianamide-C

was observed. Dreidi~g models su9gest that the "drivi~g

force" for the conversion -A to

-c

is the removal of steric

strain. _{If this is the case, (36) must be considered a}

poor model compound and its failure to undergo a similar

cleavage would not be unexpected.

0 ₀

+

so.

0

A

hv

35

0 ₀

51.

The surprisingly facile cleavage of brevianamide-A

implied that brevianamides-B,

-c

and -D may be produced

photochemically during their isolation. In order to

test this hypothesis a culture of P. brevi-compactum was

grown in the dark and extracted under conditions of low

light intensity. Thin layer chromatography of the neutral

fraction showed that -A and -B were present but no -C or -D

were detected, thereby confirming that they are unusual

photochemical artifacts.

(h) Brevianarnide-F

An examination of the residues remaining after the

removal of brevianamides-B,

-c

and -D, was carried out to

see if any simple indole precursors, related to the

brevian-amides could be detected. The presence of a small amount

of a compound which gave a blue colour with Ehrlich's

reagent was revealed. By working up the residues from

an initial 40 litres of culture medium, i t was possible to

isolate, by thin layer chromatography, 3 mg of a white

crystalline compound which had an ultraviolet spectrum).

max

277 (inf), 283, 292 nm indicative of a simple indole78·.·

The mass spectrum showed a molecular ion at m/e 283

which had the composition

c

₁₆H

17N3

o

2 , whilst the base peak

m/e 130 corresponded to the indolenin-3-methylene ion83•

This latter -res·ult suggested that the compound was a

deriv-ative of tryptophan. A peak at m/e 154 has previously

been observed1 in the praline-containing diketopiperazines,

(40), and is possibly due to the ion (41). _{From this}

evidence i t was reasonable to suggest that (42) was the

structure of brevianamide-F.

The small amount of material available did not

per-mit ORD studies to determine the stereochemistry of the

2,5-diketopiperazine, but the predominant natural occurrence

of L-amino acids prompted a comparison of brevianamide-F

with synthetic cyclo-L-tryptophanyl-L-proline. There was

no mixed melting point depression and the ultraviolet and

mass spectra were identical.

0

0

39 ₄₀

0

0

42

m;e 130 _m/e 154