Journal of Natural Products Vol. 5 5 , No. 3 , p p . 311-320, March 1992
311
UNIQUE 3 ~-0-METHYLSTEROLSFROM THE PACIFIC SPONGE JERElCOPSlS GRAPHlDlOPHORA M.VALERIA DAURIA,LUIGI GOMEZPALOMA,LUIGI MINALE,' RAFFAELERICCIO, Dipartinento di Chimira delle Sostanze Naturali, Univerrita di Napoli F&co Via D . Montesano 49, 80131 Napoli, ltaly
11,
CECILDEBITUS, Centre ORSTOM, B . P . AS, Noumh, NouveIIe Calhhnie
and CLAUDELBVI Labmatoire de Biologie des lmwt&t5 mrim et Malesologie, Museum National GHistoire Naturelle, 5 7 , me Cuvier, 75005 Paris, France
A s s m c r . - T h e lipid fraction of the Pacific sponge]creiropsisgraphidio~~a has been investigated for its sterol composition. Although the conventional 3B-hydroxysteroidswere totally absent, the extracts contained unique 3f3-methoxysteroids. Fifteen 3f3-methoxysteroids with six different nuclei [A7*%"),A8*14,A8,A7,A5,5a-saturated] and with five usual C8X3,, side chains, have been identified. In addition, the extracts also contained 3f3-methoxysteroids with oxygenated functionalities in the nuclei: A7-9a, 1 la-epoxy,A7-9-hydroxy,A8-7-one,A814-hydroxy,Ax")-8a, 14a-epoxy. Characterization was accomplished by gc-ms, hreims, 'Hnmr, "C-nmr, Ft-ir, and uv spectroscopy.
Sponges have been found to be the most diverse source of sterols, particularly in terms of unique side chain structures and unusual functionalization (1-3). During our ongoing program to isolate novel marine natural products from New Caledonian invertebrates, we undertook an investigation of the extracts of the sponge Jereicopsis grapbidiopbora Uvi & IRvi (family Leiodermatiidae) (4). In a preceding paper (5) we described the occurrence inJ . grapbidiopbora of two very unusual 3P-methoxysecosteroids. An analysis of the lipidic fraction showed the absence of conventional 3P-hydroxysteroids, while a number of 3P-methoxysteroids were isolated. Here we report the isolation and structure determination of these compounds. RESULTS AND DISCUSSION The sponge was ground and freeze-dried, and the dried material (0.7 kg) was extracted with n-hexane. Si gel chromatography of the extract (1.4 g) using increasing concentrations of EtOAc in n-hexane provided a fraction (0.23 g) of mixed 3P-Omethylsterols and two very minor more polar fractions, one containing a mixture of compounds 16 and 17and the other a mixture of compounds 18-20. The compounds were separated by hplc. The 'H-nmr spectrum of the less polar fraction, which contained methoxyl singlets at 6 3.23-3.37 and the C-3 methine protons highfield shifted to 6 3.08-3.15 ppm, immediately indicated the presence of mixed 3P-0-methylsterols. Gc-ms analysis revealed a complex mixture with fifteen components. Argentation cc gave seven fractions, which were subjected to reversed-phase hplc on a Whatman M9 10/50 ODs-2 column with MeOH-CH,CI, (8:2) to give pure 1,2,>10,12, and 15 (Table 1)characterized by ms, 250 MHz nmr spectroscopy, and uv spectroscopic measurements. The remaining 3P-0-methylsterols in Table 1 were identified by their ms data. The configuration at C-24 of the isolated 24-methylsteroids was assigned by comparison with 1 H-nmr spectra of known compounds. The chemical shift of the C-2 1methyl is a diagnostic measurefor differentiating 24R and 2 4 s epimers of (22E)-24-methyl-A22-sterols (6,7) (compounds 5 and 6)and their 27-nor-analogues (8)(compound 1).In the case of
3 12
Journal of Natural Products TABLE1. 36-0-Met
Compound
1 . . . . .
lsterols from jenicopsiJ
aphidiopbara. Mobility
Side chain
-+
mol. 5 5 , No. 3
%
gc
hplc
398
0.79
0.81
1.06
398
0.81
0.81
0.82
2
. . . . . . .
3
. . . . . .
400
0.82
4
......
402
0.84
0.93
4 12
0.87
0.85
5 . . . . . . .
3.45 2.94
7.80
6
. . .
412
0.87
0.89
7
.......
412
0.95
0.84
4.30
8
. . . . . . .
4 14
0.96
0.93
9.75
4 12
0.97
0.81
6.93
10 . . . . . . .
4 14
1.00
1.00
32.69
11 . . . . . . .
428
1.03
12
430
1.04
13 . . . . . . .
428
1.06
3.64
426
1.07
1.16
428
1.11
9 . . . . . .
14
..
15 . . . . . . .
-l+ -l+
3.25
1.01
1.07
1.30
20.83
24-methylsterols with saturated side chains (compounds 8-10, 16-20),slight differences in chemical shift of the 26- and 27-methyls between epimers create diagnostic patterns (6). In the 24-ethylsteroid series the chemical shift differences of the Me-26
March 1992)
3 13
D'Auriaet a(. : 3P-0-Methylsterols from Sponge
I
CH30
CH30
16
18
17
19
20
and Me-27 signals between epimers are too small for differentiation; therefore we prefer to leave the stereochemistry at C-24 unassigned. Interestingly the 3P-0-methylsterols mixture also includes the rare A8,14,A8,and A7,9(11' nuclei (compounds 7, 8, and 9 in Table 1). Compound 7, probably an artifact originating from 20 during isolation, exhibited a mass spectrum with molecular ion peak at m/z 4 12 and fragments at mlz 285 (loss of the side chain) and 253 (loss of the side chain plus MeOH), indicating a di-unsaturated nucleus and a saturated C, side chain. The uv spectrum {A max (MeOH) 248 nm) (9) and the 'H-nmr signals of the angular methyls (6 0.83 and 0.89) and of the olefinic protons (6 5.37) were closely comparable with those of synthetic 5a-cholesta-8,14-dien3p-01 (10). Conclusive support for the existence of the A8*"-diene came from a comparison of the 13C-nmr spectrum of 7 (Table 2) with that of the synthetic model compound. Compound 8, with the rare A8-unsaturated nucleus, exhibited a molecular ion peak in the ms at mlz 4 14 and a fragment ion at mlz 287 (loss of the side chain), indicating a mono-unsaturated nucleus and a saturated C, side chain (11,12). The existence of A8-unsaturation was confirmed by 'H nmr, since 8 exhibited the angular methyl signals at 6 0.62 and 0.95, virtually identical to those reported for A8-sterols (14), while
3 14 TBLE 2 .
"C-nmr Data for the Compounds 7.18.19.and 20. Compound
Carbon
. . . . . . . . . . . . .
c-1 c-2 c-3 c-4 c-5 C-6 c-7 c-8
............. . . . . . . . . . . . . .
............. . . . . . . . . . . . . .
.............
............. . . . . . . . . . . . . .
c-9 c-10 c-11 c-12 C-13 C-14 C-15 C-16 C-17 c-18 C-19 c-20 c-21 c-22 C-23 C-24 C-25 C-26 C-27 C-28 -0Me
mol . 55. No . 3
Journal of Natural Products
............. ............. . . . . . . . . . . . . .
............. . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
............. . . . . . . . . . . . . .
.............
. . . . . . . . . . . . .
............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . .
. . . . .
. . . . .
. . . . .
......... . . . . . . . . . ......... . . . . . . . . . .........
............
7
18
19
20
34.7 28.3 79.6 34.3 41.1 25.6 26.7 120.0 140.4 37.0 22.0 37.1 45.2 150.7 117.3 35.5 57.4 15.8 18.3 33.8 19.0 34.3 30.4 39.0 32.5 18.3 20.2 15.5 55.6
34.9 28.0 78.7 34.7 41.3 42.6 186.6 133.4 164.7 38.7 24.9 35.9 42.6 48.5 25.3 29.4 53.8 11.6 17.1 36.3 18.9 34.0 30.7 39.1 32.5 18.3 20.2 15.5 55.5
33.2' 27.3 79.2 33.7 35.8 29.4 121.3 135.5 73.9 39.2 27.7 30.0' 43.7 51.2 23.1 27.9 56.1 11.1 15.0 36.3 19.0 34.5 30.5 38.9 32.4 18.3 20.2 15.4 55.5
34.4 28.0 79.8 33.7 41.4 24.9 26.0' 140.0 130.3 37.4 21.5 31.5 45.0 82.2 31.1 25.8' 44.8 17.9 17.6 36.0 18.6 34.4 30.4 39.1 32.5 18.3 20.2 15.5 55.5
These assignments may be reversed.
signals due to olefinic protons were absent . 11'-Marine sterols have been previously reported only from the sponge Axinella cannabina (13.14) . For compound 9. the eims gave the molecular ion peak at mlz 4 12 and intense fragments at mlz 285 and mlz 253. arising from loss of the side chain and loss of side chain plus MeOH and indicating a di-unsaturated nucleus with a saturated C, side chain (1 1.12). The uv spectrum showing absorption at 242 nm. typical of A7.% 1 1 ) sterols (15). and two olefinic signals at 6 5.38 and 5.46 indicated a conjugated system of two double bonds . The structural deduction was confirmed by comparison of 'H-nmr signals of the olefinic protons at C-7 and C- 11 and of the angular methyl protons (6 0.52 and 0.90 ppm) with.those of 24.methyl.5a.cholesta.7. 9( 11).24(28)-trien.3P.01. the first naturally occurring A7.9(1 l ) unsaturated marine sterol. isolated from the sponge Haliclona fiwcms ( 16). The nmr spectral data indicated that all of the more polar compounds 1 6 2 0 possessed a 3P-methoxy group and the same saturated (24R)-methyl side chain . For compounds 18. 19. and 20. obtained in amounts adequate to measure the 13C-nmr spectra. the 24R configuration received further support by comparison of their 13C-nmr spectra with those of known compounds (17). (24R).9a. 1la-Epoxy-3~-methoxy-24-methyl-5a-cholest-7-ene f161.obtained
March 1992)
D'Auriaetal. : 3~-O-Methylsterolsfrom Sponge
3 15
in 0.8mg quantities (from 0.7 kg dry wt), gave the molecular ion in hreims at d z 428.364 1, corresponding to a molecular formula C29H4802, and the major fragment, due to the loss of a saturated C,side chain, at d z 30 1.2 167 (C2&12902), thereby locating both oxygen functions in the nucleus. An olefinic signal at 6 5.63 dong with the high field shift of the C-18 angular methyl signal at 6 0.57 in the 'H-nmr spectrum suggested a A7-unsaturation (18).A resonance at 6 3.24 (lH, d ,J = 5.8 Hz) indicated the presence of an epoxide moiety. The signal at 6 3.24 has the same doublet appearance reported by Tori et al. (19)for the 118proton of 9a, 1la-epoxysteroids while the 1la proton of a 9 8 , 1+epoxide appears as a triplet (J= 1.5 Hz) (19). Furthermore H-[H'] decoupling experiments showed that the epoxy proton was only coupled to a double doublet, at 6 2.14 ( J = 15.0, 5.8 Hz), assigned to a H-128 and that this latter was coupled to a broad doublet at 6 1.60 attributable to H-12a. Thus the A'-9a,llaepoxy functionality was established in 16.The 9 a , 1la-epoxy functionality has been previously found in two marine polyhydroxysteroids from a sponge Dysidur sp. (20,2 1) and from the marine gastropod P f u ~ x isnkatur s (22). (24R)-8a, 14a-Epoxy-3f3-methoxy-24-methyl-5a-cholest-9( 11)-ene 1171, obtained in 0.5 mg quantities (from 0.7 kg dry wt) is isomeric with 16.Peaks observed in the hreims spectrumat dz428.3647 [m+(C29H4802)and301.2154[M-C,H19]+ indicated a conventional C9H19 side chain and a tetracyclic nucleus containing both oxygen functions and two formal unsaturations. The 'H-nmr spectrum at 400 MHz contained an olefinic signal at 6 5.14 ( l H , t , J = 6.5 H t ) coupled with two double doubletsobservedat63.02(1H, dd,J= 13.0,6.5 Hz)and62.10(1H,dd,J= 13.0, 6.5 Hz), which are coupled to each other by 13.0 Hz, thus proving the presence of a methylene group next to an olefinic proton and to a blocked position as in the partial structure >C=CH-CH,C
