Bioactive marine metabolites II. Halistanol sulfate, an antimicrobial novel steroid sulfate from the marine sponge halichondria cf. moorei bergquist

Tetrahedron Letters, Vo1.22, Printed in Great Britain

BIOACTIVE

pp 1985 - 1988, 1981

N0.21,

MARINE METABOLITES

STEROID

II.

of Marine

The University

A new C29 steroid

has been isolated constituent.

Marine a variety

of bioactive waters

and Gram-negative

AN ANTIMICROBIAL

Faculty

Bunkyo-ku,

NOVEL

of Agriculture,

Tokyo

(Japan)

trisodium

sponge Halichondria cf. moorei Bergquist

sulfate

as an antimicrobial

in a search for useful drugs from the sea, and 2-5 have been isolated. Our screening6 of marine invertebrates

compounds

for antimicrobial

bacteria.

is a sulfate

SULFATE,

245,25-dimethylcholestane-2S,3o,6o-triyl

sulfate,

from the Okinawan

studied

substances

sponge HaZichondria cf. moorei

Okinawan

which

Biochemistry,

of Tokyo,

sponges have been extensively

from Japanese

HALISTANOL

FROM THE MARINE SPONGE HALICHONDRIA CF. MOOREI BERGQUIST * S. Matsunaga and S. Konosu N. Fusetani,

SULFATE

Laboratory

Abstract:

1

0040-4039/81/211985-04$02.00/O 01981 Pergamon Press Ltd.

showed that the methanol

Bergquist

We have isolated

inhibited

the growth

from this sponge a water

ester of a novel polyhydroxylated

steroid,

extract

of the

of fungi, Gram-positive soluble

active

compound

24<,25-dimethylcholestane-28,3a,6a-

triol. The water collected

TSK G3000S, EtOH-H20 Besides

soluble portion

at Ishigaki

of the aqueous

silica gel and Sephadex

yielded

the antimicrobial

sulfate(i) the sulfate

activity,

extract

Recrystallization

LH-20.

300 mg of halistanol

ethanol

of the frozen sponge

Japan, was chromatographed

Island of the Ryukyus,

as colorless

of the active needles,

showed hemolytic

(500 g)

successively fraction

on from

mp 159.5-160.5";

and ichthyotoxic

[alD+17".

activity.

Compound&contained three sulfate groups, which was deduced by a strong IR absorption7 at 1250 -1 cm , sodium rhodizonate test 8 and calorimetric determination. 9 Atomic absorption analysis demonstrated

that all three sulfates

EI mass spectral

data suggested

this formula, which

supported

[(lo0 MHz, CD30D) 0.70(s,3H), presence

of a trioxygenated

Acid hydrolysis which no longer molecular combustion

a molecular

revealed

EI mass spectrum(m/z --

(Table 1).

2.00(s,3H),

of A afforded

l.O6(s,3H),

halistanol(2)

triacetate

448.3917;

m,lH),

as colorless

4.74(m,2H)]

also indicated

Cl0 side chain(e.g.,

4.72(m,2H);

plates,

calcd for 448.3914),

2 [m/z 514(M+-AcOH);

m,lH),

4.13(br

The high resolution

activity.

The mass spectrum

of a saturated

4.50(br

three oxygen-bearing

carbons

indicated

the

steroid moiety.

3 H20, as well as ring D fission peak at 211).l" corresponding

including

FD and

The l3 C NMR spectrum

of C2gH52012S3Na3.

signals

analysis,

412, 394, 392, 379, 275, 253, 229, 211) and 'H NMR spectrum

of C2gH5203(m/z

analysis.

as the presence

Combustion

as sodium salts.

formula

29 carbon

0.86(s,9H),

showed antimicrobial

formula

were present

V

[al, +37', established

which was supported

a trihydroxylated

steroid

Halistanol

was easily

converted

1740, 1240 cm-'; 'H NMR l)].

a

by

skeleton

as well

peak at -m/z 253 for loss of side chain +

13C NMR ?gzble

1985

mp 248-249";

mass spectrum

to the

(CDC13) 1.95(s,6H),

1986

28 26 29 RO

HO

RO

0

OR

Table 1. C 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

1 = 40.1 75.5 a 75.5 a 25.1 45.4 78.8 a 39.2 35.2 55.9 37.7 21.9 41.2 43.8 57.4 b 25.1 29.2 57.7 b 12.5 15.3 c 37.7 19.5 36.6 29.2 45.4 34.1 27.8 27.8 15.0 c 27.8

2 R=Ac

1, 2, 3 and 4 13C NMR data of Compounds = = = = 2 = 40.4 d 69.3 e 69.9 e 25.3 45.8 70.4 e 41.6 33.9 54.8 36.6 20.9 40.0 d 42.7 56.2 f 24.3 28.2 56.3 f 12.1 15.2 g 36.6 19.1 35.5 28.2 44.1 33.3 27.4 27.4 14.6 g 27.4

;(calcd) 40.3 71.7 70.6 25.5 45.9 70.0 41.7 34.3 54.9 36.6 20.9 40.2 42.6 56.7 24.2 28.3 56.4 12.2 14.7

4 = 47.9 72.0 211.0 n 35.2 58.7 208.1 n 46.5 37.6 53.6 42.8 21.9 39.3 43.1 56.5 24.0 28.0 o 55.9 12.1 13.9 36.3 19.0 35.3 28.1 o 44.1 33.3 27.4 27.4 14.6 27.4

21.1(3C) 169.4(2c) 170.7

OAc

k

3 = 38.0 h 69.2 i 69.7 i 23.8 j 44.1 k 72.2 i 37.7 h 33.9 54.6 36.6 20.9 39.9 42.8 56.3 24.1 j 28.5 1 56.3 12.1 14.8 m 36.5 19.1 35.5 28.1 1 44.2 k 33.3 27.4 27.4 14.7 m 27.4

The values are in ppm down field relative to TMS. CD30D(5:1); 2 and 4, CDC13

a-o Assignments may be interchanged.

The solvents: i, CD30D; 2, CDC13-

1987

13

The signals

C NMR spectrum

including

a three-carbon

MHz 'H NMR spectrum at 0.65(s)

to the C-21 methyl, methyl

isopropyl

exhibited

five methyl

This was also observed

groups at c-18 and 19 were assignable

in the 400

to the signals

The three proton doublet at 0.93(J=7Hz) was attributed 11 the 202 configuration. When recorded in pyridine-d5 the C-19

respectively.

suggesting

signal shifted

at 27.4 (Table 1).

quartet

The methyl

(CDC13).

and 1.01(s),

[25 MHz, CDC13-CD30D(5:1)]

of halistanol(2)

to 1.50, indicating

the proximity

nor ethyl groups were recognized

of a hydroxyl

in the spectrum,

Since neither

function.

a t-butyl

moiety(0.85,s,9H)

must be

The presence of a t-butyl group was also confirmed by the placed at the end of the side chain. 13 The position of the remaining C NMR spectrum(27.4,q,3C) and IR spectrum(1375,1360 cm-'). 13 C NMR chemical shifts for the methyl group(0.80,d,J=7Hz) was determined by calculating the side chain12; observed

the result was consistent

carbon

chemical

with a 24,25_dimethylated

for the side chain of halistanol(2) 13 those of 24~-methylcholestane-lS,3S,5o,68-tetrol. The position

and configuration

of three hydroxyl

NMR study of trio1 2 in pyridine-d5, carbinol

methine

Irradiation

signals:

including

3.93(ddd,L=4,11,11Hz,66-H),

of the 6B-H at 6 3.93 collapsed

and 1.39(br at 2.34(br

d,J=llHz) _

to a triplet

at 6 4.57 and 4.61 sharpened as well as the C-4 geminal

the C-l geminal

protons

proton

resonance

doublet,

m,2o-H),

protons

m,3S-H).

at 1.37(q,J=llHz)

but also the C-5a proton

and 2.90(br

the C-5a proton

4.6l(narrow

of the two carbinol

at 2.02(dd,J=3,13Hz)

at 2.42(ddd,J=3,11,13Hz)

at 6 2.90 collapsed

There were three

protons

respectively,

irradiation

the

well with

from the 400 MHz lH

experiments.

4.57(narrow

Simultaneous

coincided

groups were deduced

not only the C-7 geminal

and a sharper

t,.J=llHz) to a broad doublet. _

of the C-4 equatorial

double

Furthermore,

side chain.

shift values

and 2.16(br d,J=13Hz).

methines d,J=13Hz)

Irradiation

at 2.34 to a broad doublet

and

Thus the 2f3,3a,6a_trihydroxyl feature was 13 C NMR spectrum of compound 2 This structure was further confirmed by the fully established. 13 C NMR signals of sterols (Table 1). When the substituent effect of hydroxyl groups on the 14,15 the calculated values for this structure were was applied as reported by Djerassi -_--a> et al the C-4 axial proton

in good agreement Further

with the observed

support

gave a diketone 0.80(d,J=7Hz,3H),

at 2.42 to a double doublet.

for structure

alcohol

ones

(Table 1).

2 was provided

by Sarett oxidation

i [M+ -m/z 444; Vmax 3500,172O

0.84(s,9H),

0.93(d,J=7Hz,3H),

(Table l)], and by a 13C NMR study of compound

16

of halistanol(z),

cm -l; 1H NMR (400 MHz,CDC13)

l.O4(s,3H),

4.26(dd,L=7,12Hz,lH);

i using the lanthanide

which

0.69(s,3H), 13C NMR

shift reagent

(Yb(fod)3).

Various

steroids with unusual side chains or unconventional ring systems have been obtained 17-19 organisms. Particularly polyhydroxylated sterols have recently been isolated 12,20,21 17 from gorgonians and soft corals. However, both the side chain and the hydroxylation from marine

pattern

of halistanol(2)

possesses

the unusual

butyl moiety methylated

unknown,

at the end of the side chain.

naturally

Acknowledgement: reading

are hitherto

2S,3o,ba-trihydroxyl

occurring

intriguing

An even more unusual

Halistanol(2)

since halistanol

feature

is the first example

is the -t-

of a C-25

sterol.

We thank Professor

this manuscript.

and biosynthetically

functions.

Paul J. Scheuer

We are also grateful

of the University

to Dr. T. Hoshino

of Hawaii

for his

of the Mukaishima

Marine

1988

Biological Furihata

Station

of Hiroshima

of the Institute

400 MHz 'H NMR spectra, Pharmaceutical

analyses.

This work was partly of Education,

for the identification

supported

by a Grant-in-Aid

and Culture

Part I:

2.

D. J. Faulkner,

3.

Y. Hashimoto, Press,

N. Fusetani,

S. Matsunaga

Tetrahedron,

Marine

and Development

and FD mass spectral for Scientific

of

of

and combustion

Research

from the

of Japan.

REFERENCES

1.

to Mr. K.

for the measurement

of Research

Co., Ltd., for the high resolution

Science

of the sponge,

of this university

and to the staff of the Institute

Yamanouchi

Ministry

University,

of Applied Microbiology

AND NOTES

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