Isolation and biosynthesis of (−)-indolactam I, a new congener of indole alkaloid tumor promoter teleocidins

Teuahedron Lenas. Vo1.31.No.50,pp 7337-7340, 1990 Printedin GreatBritain

GO404039I90 53.00+ .OO Pcrgmm Rss pk

ISOLATION AND BIOSYNTHESIS

I , A NEW CONGENER OF

OF (-)-INDOLACTAM

INDOLE ALKALOID TUMOR PROMOTER TELEOCIDINS Kazuhiro

Irie,*

Shin-ichiro

Kajiyama,

Hideo Hayashia Department

Koichi Koshimizu,

and Motoo Araia

of Food Science and Technology, Faculty Kyoto University, Kyoto 606, Japan

aDepartment

of Agricultural

University

Chemistry,

of Osaka Prefecture,

of Agriculture,

College of Agriculture, Sakai 591, Japan

Abstract: (-)-Indolactam I. a new congener of indole alkaloid tumor was isolated from S treptoverticillium blastmyceticum, and was shown from N-methyl-L-isoleucyl-L-tryptophanol.

(-)-Indolactam has attracted involving

V,t.2)

an active fragment

much interest

in the area of organic

a nine-membered

biosynthetic

precursors,

suffix

Endo

V.5)

indolactam

L-tryptophan

et d.5)

with

have recently

respectively,

instead

shown to be biosynthesized blustmyceticum,@

for new

indolactam

V.

(-)-indolactam

and biosynthesis

characteristic

of its peculiar

DL-leucine, V.

structure

on the logical

which

of indolactam

provides

(*)-

DL-phenylalanine

and

Since (-)-indolactam

indolactam

the

analogues,

and L-methionine

coloration

for These

increasing

of acetone

volumes column

communication

by Ehrlich on TLC.

45hr,

dichloromethane.

of producing

of (-)-indolactam

(green)

extracts

and

in the culture

(-)-indolactam

This

of 1 was guided

fermentation

by repeated

whether

feature

valine

Purification

L-valine

teleocidins.3)

V is based

and L-valine,

a series

DL-alanine,

analogues

which has the characteristic

determination

ring

promoter

because

in (-)-indolactam

to examine

of teleocidins.

indolactam

an indole

of L-valine

thus found a new congener in

tumor

V was

in Streptoverticillium

analogues

with amino

acids

occur naturally.

We searched NA34-17.7)

chemistry

synthesized

from L-tryptophan,

it is of interest

other than L-valine

alkaloid

lactam ring. 4) The name (-)-indolactam

G, A, L, F and t-L with DL-glycine.

DL-t-leucine,

aerated

of indole

promoter teleocidins, to be biosynthesized

the

were

of S.

(-)-indolactam

blastmyceticum

V in quantity,

I (1) with L-isoleucine deals

with

the

and

in place of L-

isolation,

structure

I (1). reagent,*)

with which

S. blastmyccticum culture

broth

chromatographed

to give 40% acetone

chromatography

broth

and HPLC

eluates

(-)-indolactam

NA34-17 (100 on

was cultured

liters) silica

V showed

was gel

using

along

with

with

toluene

(2Og). which were further

to give 1 (0.8mg)

by deep

extracted

3.5g

and

purified of

(-)-

V.

Compound 1 was obtained as an amorphous powder. HR-EI-MS established its molecular fomula as Cl8H25N302 (obs. m/z 315.1963, talc. 315.1947). The presence of an indole chromophore was confirmed by UV spectrum [Xmax (MeOH) nm (E): 228 (27.600), 289 7337

7338

181

(-)-Indolactam I (1)

(-)-Indolactam V

Teleocidin B-4

Fig. 1 Structureof tekocidin-related compounds.

Table I

1H NMR (400MHz) chemical shifts of (-)-indolactam I (1) and (->indolactam V in chloroform-da

No

(-)-indolactam I (1)

(->indolactamv

7.97 (lH, br.s)

7.98 (lH, br.s)

6.89 (lH, s)

6.90 (II-I, s)

6.51 (1H. d, J=7.3Hz)

6.51 (lH, d, J=7.6Hz)

7.07 (IH, t, J=7.9Hz)

7.06 (1H. t, J=7.9Hz)

6.90 (lH, d, J=7.9Hz)

6.90 (lH, d, J=8.2Hz)

3.00 (1H. dd, J=17.4, 3.7Hz)

3.01 (lH, dd, J=17.4, 3.7I-k)

3.21 (lH, br.d, J=17.4Hz)

3.20 (lH, br.d, J=17.4Hz)

4.28 (lH, m)

4.30 (lH, m)

10

6.62 (lH, br.s)

6.56 (lH, br.s)

12

4.51 (IH, d. J=IO.IHz)

4.39 (lH, d, J=10.4Hz)

14

3.54 (lH, m)

3.54 (lH, m)

3.75 (1H. m)

3.75 (1H. m)

9

15

2.42 (1H. m)

2.61 (1H. m)

16

0.90 (3H. d, J=6.4Hz)

0.93 (3H, d, J=6.4Hz)

17

0.63 (lH, m)

0.63 (3H, d, J=6.7Hz)

18

0.59 (3H. t, J=6.7Hz)

1.37 (lH, m) 19 OY

2.93 (3H, s)

2.92 (3H, s) 2.02 (IH, m)

1.95 (lH, m)

aChemica shifts for twist conformer are expressed as ppm down field from TMS. twist : sofa = 2.5 : 1 in (-)indolactam I (1); 3 : 1 in (-)-indolactam V at O.O04M,27 K!.

7339

(7500),

300 (7500)].

pattern of 1 [m/z (%): 315 (M+, 82). 297 (12),

The EI-MS fragment

(23), 258 (21), 215 (54),

171 (loo)]

was quite similar

to that of (-)-+ndolactam

268

V [m/z (46): 301

(M+. 83), 283 (19). 268 (22). 258 (18), 215 (50), 171 (loo)]. The fragment ion m/z 258 (M+ -57) of 1 suggests the presence of a butyl moiety in 1 in place of an isopropyl group in (-)indolactam

V. of 1 in chloroform-d

The tH NMR spectrum two stable conformers2)

: sofa = 2.5 : 1).

(twist

that of (-)-indolactam

(0.004M.

V, except

for signals

27 OC) revealed

The spectrum

ascribable

that 1 existed

as

resemblance

to

bore a striking

to the substituent

at position

12.

A

comparison between the spectra of 1 and (-)-indolactam V (Table I) revealed the presence of a set-butyl group [S 0.59 (3H, t), 0.63 (lH, m), 0.90 (3H, d), 1.37 (lH, m) and 2.42 (lH, m) for twist 0,

conformer]

c=O.O0016M) indolactam

was closely

indolactam

similar

V had the same absolute

analogue

containing

In our previous

V,t)

9 and 12.

publication,@

deuterium

medium

(4mg/lOOml)

indolactam

V production

indolactam

I (cu. 40% yield) was obtained

The indolactam

NMR,

rate

(-)-

signals

established

of the isolation

of an

labeled

N-methyl-L-valyl-L-tryptophanol,

its biosynthetic

previously. of

pathway,

immediately

this

was added

cu. 15mg of (-)-

broth.

(-)-indolactam

to

to the start of (-)-

11) After 48hr of cultivation, “synthetic”

of 1 using

synthesis

prior

from lOOOm1 of the culture

which

into (-)-indolactam

N-methyl-L-isoleucyl-L-tryptophanol*o)

of this micro-organism

EI-MS)

incorporated

The spectral

I coincided

data

with

1,

of 1 unambiguously.

the structure high

was tried:

as reported

CD and

1 and

that

On the basis of these

of all proton

This is the first example

of 1 and to examine

the structure

indicating

191217. 0,

amino acid other than L-valine.

the culture

supporting

The assignments

I.

in Table I.

NA34-17

lH

at positions

at 28 OC (]0]320

-55,800,

[8122ij

configuration

S. blastmyceticum

(UV,

-19,900,

by Sakai et al.,g) was shown to be efficiently

was first isolated V. To confirm

h1245

to that of (-)-indolactam

to be (-)-indolactam

COSY are summarized

of 1 in methanol

The CD spectrum

101294 0, h1255 -24,900,

data, 1 was deduced by lH-lH

12 of 1.

at position

[81306 +9600,

of

I indicates

N-methyl-I_.-isoleucyl-L-tryptophanol that (-)-indolactam

biotransconversion

I is biosynthesized

through

into

biosynthetic

(-)-

pathway

analogous to (-)-indolactam V.6) The very low levels of naturally occurring (-)-indolactam I (ca. 10~g/1000ml) compared with (-)-indolactam V (ca. 35mg/lOOOml) can be interpreted by the hypothesis substrate ring.

that the condensation specificity

The above results

structure-activity

enzyme

also indicates

studies

is easily

that a variety obtainable

indolactam

L was similarly

synthesized

Moreover,

such indolactam

precursors

useful

for studying

indolactam

I (l),

for L-valine

and L-tryptophan

to the valine moiety than the cyclization

the cyclization rarely

Studies on the isolation progress.

and

substrate

of indolactam this

has a much

at position and teleocidin

biotransconversion.

the cyclization

and the efficiency specificity

of this

for

In

(-)-

fact,

shown).

are especially

products,

of this cyclization cyclization

analogues (not

as N-methyl-L-isoleucyl-L-tryptophanol because

higher

4 of the indole

from N-methyl-L-leucyl-L-tryptophanol

enzyme

occur naturally,

by

enzyme

for example

(-)-

was very high.

enzyme

are

now

in

7340

Acknowledgements The authors University Institute

thank

Dr. K. Shudo

for supplying for Chemical

at Kyoto

University

Research

at Kyoto

Research

for NMR measurements,

for Scientific

References

and Footnotes

Irie, K.; Hirota,

Research

M.; Hagiwara,

Endo,

3)

Fujiki,

Y.; Shudo,

K.; Itai,

Sciences

of the Faculty

and Dr. K. Soda of the Institute

from Ministry

N.; Koshimizu.

Science

K.; Hayashi,

in part by a

and Culture

H.; Murao.

of the

of Science

for Chemical

This work was supported of Education,

at Tokyo

and Ms. K. Omine

Mr. R. Imamura

for CD measurements.

Ito. Y. Agric. Biol. Chem. 1984, 2)

of Pharmaceutical

A, F and L, Dr. J. Oda

at Kyoto University,

University

Grant-in-Aid

1)

of the Faculty

(f)-indolactam

of Japan.

S.; Tokuda.

H.;

48, 1269-1274.

A.; Hasegawa,

M.; Sakai,

S.

Tetrahedron

M.; Sugimura,

T.; Moore,

1986,

42, 5905-

5924. H.; Mori. M.; Nakayasu,

Acad. Sci. USA

1981,

M.; Terada,

Endo,

Y. Yuki Gosei Kagaku Kyokaishi.

1988,

5)

Endo,

Y.; Hasegawa.

K.

Irie,

7)

Irie,

M.; Itai, A.; Shudo,

K.; Kajiyama.

Lett. 1990.31,

S.; Funaki,

A.; Koshimizu,

N.; Koshimizu,

Heacock,

R. A.; Mahon,

9)

Hitotsuyanagi,

Y.; Yamaguchi,

M.; Sugimura.

T.; Endo,

Products,

(33,700),

1990,

Tetrahedron

H.; Murao,

S.; Ito,

Y.

Agric.

Biol.

1965,

17, 338-348.

K.; Ogata. K.; Aimi, N.; Sakai, S.; Fujiki, H.; Suganuma, on the Chemistry K.; Koyama. Y. 27th Symposium 1985.

Symposium

papers,

was synthesized

5400),

281 (SSOO), 290 (5000).

0.78 (3H, d, J=7.0Hz),

(3H, s), 2.79 (1H. d, J=4.6Hz),

11) Irie,

M.

Y.; Shudo,

Hiroshima,

6.7, 0.6Hz), 3.67 (lH, 7.06 (1H. d, J=2.1Hz), (lH, (IH,

there cited.

43, 3695-3704.

H.; Arai.

p. 624-631.

from Boc-N-methyl-L-isoleucine

methyl ester by the method reported previously.@ [U]D -38.20 (c=O.50, MeOH, 26eC). UV hmaz

275 (sh.,

t, 5=7.3Hz),

K.; Hayashi,

K.; Hayashi,

M. E. J. Chromatogr.

10) N-methyl-L-isoleucyl-L-tryptophanol L-tryptophan tryptophanol:

and references

1987,

49, 845-847.

8)

of Natural

46, 344-355,

Tetrahedron

101-104.

K.; Hagiwara,

Chem. 1985,

Proc. Natl.

78, 3872-3876.

4) 6)

R. E.

0.92

2.99 (lH,

dd, J=ll.O, 7.11 (IH,

(1H.

m),

and

N-methyl-L-isoleucyl-L(MeOH) nm (E): 222

tH NMR 6 (CDC13) ppm: 0.75 (3H, 1.21 (lH,

dd, J=14.7,

m),

7.9Hz),

1.66 (1H.

3.06 (lH,

m), 2.27

ddd. J=14.7,

6.1Hz), 3.77 (1H. dd, J=ll.O, 3.5Hz), 4.26 (lH, m), dt, J=7.9, 1.2Hz), 7.19 (1H. dt, J=8.2, 1.2Hz), 7.35

dd, J=7.9, 0.9Hz), 7.49 (1H. br.d, J=7.OHz), 7.65 (1H. dd, J=8.2, 0.9Hz), br.s). HR-EI-MS m/z : 317.2101 (M+. talc. for ClgH27N302, 317.2103). K.; Kajiyama,

S.; Funaki,

46, 2773-2788.

(Received in Japan 24 August 1990)

A.; Koshimizu,

K.; Hayashi,

H.; Arai,

M.

8.23

Tetrahedron