STEROLS FROM THE MARINE SPONGES ORINA ARCOFERUS A N D GEODIA MEGASTRELLA JOHSF. K I ~ G S T OEARL S , BESSOS,BRIAXGREGORY and ALEXG. FALLIS Department o j Cheinzstry, Memorial r m e r s z t y os S e q f o u n d l a n d , St. John‘s, Se7Ljoiindland, Canada,A I B 3 x 7
these were separated by glc on both the acetate and free sterol and the mass spectra and retention times compared with authentic samples. Occelasterol (3) and cis-22-dehydrocholesterol (2) cannot be distinguished by
As part of our investigation of local marine natural productc. v e have examined a number of marine invertebrates inhabiting the cold waters off Kewfoundland and Labrador (1). Warm n i t e r sponges have proved a rich source of various natural products including sesquiterpenes ( 2 ) , sesterterpenes ( 3 ) , purines (4) and steroids (5). Sponges of the Ckoristida and Haplosclerida orders have received relatively little attention (6). V e report herein the isolation and identification of the sterols isolated from the sulphur sponge Geodia megastrella (Class Desmospoiagiae, Order Choristida) collected from a depth of 400 fatlionis off the Labrador coast (Hamilton Bank) and the glass sponge Orilia arcoferus (Class Desmospoizgiae, Order Haplosclerida) from the same general area. Fractionation of the acetone extract of freeze-dried animals by high speed liquid chromatograph>- on silica gel afforded, from the chloroform eluent, a lipid fraction containing the sterol components. This mixture was acetylated and further fractionated into four bands by tlc on silver nitrate imprenated silica gel. In each case the major component was shon-n to be 24-meth~-lenecholesterylacetate (8) (’7) located in the first band (lowest Rr) and identified by direct comparison (mp. ‘H nmr, ms) with an authentic sample. The acetate n-as hydrolyzed (KOHIEtOH H 2 0 ) t o the free sterol, a sample of which was purified by glc. The second band included all the other qteryl acetates containing a As and sidechain double bond at either AZ2 or AZ4. As indicated in the table
RO
dP
I. R = H or A c ,
RI=+
2. R = H or
R,
AC,
=
3. R = H or Ac,
R, =
5
AC,
R,=
AC,
R,=
R = H or
-5. R = H
or
#
6. R = H or Ac,
RI=
7. R = H or Ac,
RI=
8. R
H or Ac,
R,=
9. R = H or Ac,
R,=
c
c
I
IO. R = H -
or Ac,
-I I.
or Ac,
R
-12 R
=
H
H or
AC,
-
%
+ \_.rl”u
y”t
fi Y‘I;
RR,= ‘= R,=
A
&
glc, and their mass spectra are the same as the t r a m AZ2 isomer (4) (8). At least two of these three sterols Jvere present in each of the sponges ex528
SEP-OCT
19791
K I S G S T O S E T AL.
: sTEROLb FROM lI.%RISE SPOSGES
4
m
Im
-
' d
m
a
6
0
Y)
1 1
t
b. 0
'*
0
10,
0
' 0
In (D
-
0
9 -
(D (D
-
In
W
-
Footnotes for Table 1 on next page.
530
JOURSAL OF NATURAL PRODUCTS
amined. I t is not clear if the cis Az2 sterol (2) iq actually a natural product as its existence has recently been questioned (9). The third band contained mainly the acetates of sterols \{-it11 a saturated side chain. The fourth band was mainly cholestanyl acetate but showed traces of the acetates of CZS and C2sqtanolq. n hich were not present in sufficient quantity for glc collection and identification. The t n o sponges contained similar mixtures of sterols but in considerably different amounts, sterols accounting for 1.1~ of7~ the dry \\eight of 0. arcoferus and only 0.09G7c of the dry weight of G. megastrella.
EXPERIlIESTAL The glass sponge (Orzna arcojerirs Class D e s i n o s p o i i g ~ a e , Order Hnplosclerida) m s collected a t a depth of -85 fathoms in the Bradore Trough on the Hamilton Bank (Labrador), and the sulfur sponge (Geodze ntegestrelln Class Desii70spongzneJ Order Chorzstzdaj n a s collected using a deep Rater trawl (400 fathoms) on the South Hamilton Bank in the North H a a k e Channel. The later sample mas elliptical in shape 92 x 66 cm, therefore, the entire investigation was conducted w t h one animal The sponges were cut in pieces and freeze dried. They were pulverized in a mortar and pestle and extracted tnice a i t h acetone The oily extracts so obtained were fractionated by high speed liquid chromatography (Waters Porasil -4,75-125 1-1, 122 cm x 7 m m 1.d.) w t h chloroform as eluent. Thus O r i m arcaferzrs (239 g freeze dried) gave 8.32 g of crude extract; further purification of 365 mg of this extract afforded 124 mg of sterols (sterol yield from freeze dried sponge, 1 . 1 8 5 ) . Similarly, Geodin niegastrelln (167 g, freeze dried) afforded 7iG mg of crude eytract, mhich provided 160 mg of sterols (0 096%).
[VOL.
42. s o . 5
The sterol fractions were acetylated (acetic anhydride, pyridine, 23", 16 h ) and separated into four major bands b y preparative tlc on silver nitrate impregnated silica gel (.igSOa:silica gel PF 254 and 366 1:5 W / R , developed tn-ice with benzene-petroleum ether, 2 : 3 ) . Portions of these bands x-ere hydrolyzed ( 8 5 a q KOH/EtOH, 4 h j to afford the free sterols. These sterols and the sterol acetate mixtures were independently analyzed and collected b y prep. glc (1.5% OT--li on 100/120 mesh Gas Chrom Q, 3 m x 6 mm i.d., 80 ml/min He, temp. 270" sterols, 282' acetates) as summarized in table 1. The mass spectra of the pure sterols and acetates collected from the glc were recorded (Hitachi Perkin Elmer RMCBE, 70 eY) and compared with those of authentic samples and,/or published spectra. A4CKKOWLEDGhlEKT We thank I B l I Canada, hlemorial University of Sen-foiindland, and the Sational Research Council of Canada for financial support of this research, R. Bow-ring for collecting the sponges, H . M. Reism-ig and E. L. Bousfield for their taxonomic identification, and E . Bullock for his encouragement. The following individuals generously provided samples of authentic sterols or their acetates: L. J . Goad, R . H . Thompson, and P. J. Scheuer.
Receiaed 12 Merch 1979. LITERATURE C I T E D 1. For previous work see J . F. Kingston,
B . Gregory and -1.G. Fallis, J . Chev7. SOC.Perki77 I , 1979, in press. 2. B. J . Burreson, P . J. Schener, J . Finer and J. Clardy, J . Ani. Ciieni. Soc., 97, 4763 (1975). 3. F. Cafieri, E. Fattorusso, C. Santacroce and L. hlinale, Tetrahedron, 28, 1579 11972'1. -, \--
E. Cullen and J. P. Devin, Can. J . Cheni., 53, 1690 (1975). 5 . F. J. Schmitz, in P. J . Scheuer, hIarine 4.
S a t u r a l Products, Chemical and Biological Perspectives Yo1 I . Academic 19i8, pp. 211Press, S e w Tork, ?i.>7. 297.
Footnotes for Table 1 (a) (b) (c) (d) (e)
The remaining percentages consist of a number of sterols present in too small a quantity to be identified. These two compounds cannot be distinguished b y glc or ms. The presence of at least tn-o of these compounds was indicated by a leading shoulder on the glc peak. Collection on the leading and trailing sides of the peak afforded compounds with identical ms. 24-a and 0 isomers cannot be distinguished by these methods. -4uthentic samples n-ere not available for these compounds. Glc and ms data were in agreement n-ith the literature values (8-10, 12). Relative retention times are reported t o accuracies of 10.02.
SEP-OCT
19i9]
KISGSTOS ET 1 L . :
STEROLS FROM J i h R I S E SPOSGES
6. 11.de Rosa, L . JIinale and G . Podano, Comp. Biochem. Physiol., 46B, 823 t 1973 I. i . T. I?. Erdman and R . H . Tliomson, T e f r z l i e d r o u , 28, 5163 119i21. S. D . R. Idler and P . \Viseman, Comp. Biochem. Physiol., 38A, 581 (19i1).
.j3 1
9. 31. Kobayashi and H. Xitauhashi, Steroids, 24,399 tl9i4). 10. D . R . Idler, P . 11.Kiseman and L. 11. Safe, S f e r o i d s , 16,451 11970). 11. I . Rubinstein and L. J . Goad, Phy!oclieiii., 13, 485 (19i4). 12. B. A. Knight and C. J . IT. Brooks, P k ~ i o c l i e m .8, , 463 11969).
