Secondary metabolites from the cnidarian Cavernularia sp.: structures of the new briaranes cavernulin A and Bi"l• 2

Novel cyclizcd cernbranoids (briantnes) cavernulin A and cavernulin B along with ubiquitous wax esters (derived from satu rated fatty acids and some analogous unsaturated fatty acids), cholesterol, ceramides as well as 1-0-alkylglycerols have been isolated from the cnidal"ian Cavemularia sp., the extract of which shows some toxicity to guppy fingerlings and in brine shrimp assays. The structures of cavernulin A as (IS*,2S*,5Z,7S*,8R*,9S*,IOS*,llR*,l2R*,I7R*)-2,9-diacetoxy-12-buta noyloxy-8-hyd roxybria ra-5, 13-dien-18-one and cavern ulin B as (IS* ,2S* ,5Z, 7 S* ,8R * ,9S*, I OS* ,II R* ,12R* ,17 R* )-2,9-diacetoxy-8,12-dihydroxybl"iara-5,13-dien-18-one are established f.-om one-dimensional ) 1 11 and homodecoupling as well as 13c (NDC and DEPT-135)1 and two-dimensional (COSY-90, X-H correlation optimized for 1 Jc-H and NOESY) NMR and other· (IR and Mass) spectral and some chemical studies. The lengths of the alkyl chains in wax esters, ceramidcs and 1-0-alkylglyccrols arc established by appropriate gas chromatographic studies.

Tremendous surge of interests in marine natural product research prevailed throughout the globe during the past couple of decades 3 . As a part of our search for bioactive components from marine sources a cnidarian, Cavernularia sp., was collected from the coastal Bay of Bengal. Preliminary bioactivity studies on the organism indicated that the CH 2 ClrMeOH extract of raw crushed Cavernularia sp. had some toxicity in brine-shrimp assays (LC 50 _386 pg per ml at 24 h) and caused distress of guppy fingerlings {35-40 mm and 0.8-1.0 g) which sank and died_ in about 45-50 min. This observation further prompted the present investigators to carry out systematic chemical investigations of the aforesaid marine organism. This paper deals with the results of the said chemical investigation including the structural elaboration of interesting and novel marine metabolites cavernulin A (1) and B (2) which incidentally belong to cyclized cembranoid or briarane (3) group of diter-penoids4. Such briarane diterpenoids have earlier been re-ported3·4 from Gorgonacea (Genus : Briareum 5 , Soleno-podium6, Erythropodium 7 , Junceella 8 , Gorgonella 9 and Menella 10 ), Alcyonacea (soft coral) (Genus: Minabea 11 ), Stolonifera (Genus: Tubipora 12 ) and Pennatulacea (Genus : Ptilosarcus 13 22 ) and continue to attract the attention of investigators because of the structural complexity and wide range of biological activities, e.g. toxic 22 , cytotoxic 19 , antiinflamatory 23 , antivirai 24 , antiinsecticidai 13 , antifouling 21 , immunomodulatory 25 and antibacteriai 9 . Results and Discussion The Cavernularia species was thoroughly extracted with CH 2 CI 2 and subsequently with CH 2 CI 2 -MeOH (I : I). Chromatographic resolution (column and preparative thin layer chromatography) of the CH 2 Cl 2 extracted mass afforded wax esters (fatty esters of saturated fatty acids and of some analogous unsaturated fatty acids) ( 4-), ceram ides ( 5), cholesterol (6), 1-0-alkylglycerol (7) and also a novel cyclized cembranoid (briarane), cavernulin A (I), C 28 H 40 0 9 , Rf 0.
It has further been noted that a broad doublet signal at Again, homodecoupling and COSY spectra of 1 revealed that a broad doublet signal at c5 5.52 (1 H, 1 I 0.0 Hz) was found to couple to a doublet signal at c5 5.23 (11-1, J 10.0 Hz) while the former signal was also weakly coupled to a vinyl methyl signal at c52.02 (br s). The XHCORR experiment confirmed that the corresponding protonated carbon signals were at c5c 118.6 (d), 77.8 (d) and 28.2 (q), respectively, in conformity with the presence of fragment-Ill, requiring the presence of a quaternary olefinic carbon atom which incidentally resonated at c5c 146.9.
The I H and 13 c spectra also displayed signals assignable to fragment-TV as it was observed that a doublet signal at c51.15 (3H, 17.2 Hz) coupling with a quartet signal at c5 2.42 ( 1 1-1, J 7.2 Hz) in the 1 H spectrum of I were linked to carbon resonances at c56.6(q) and 42.3( d), respectively. The upfield carbon resonance at c5 6.6 for the methyl group was an indication that the methyl group was attached to a-carbon of a five-membered lactone moiety.
A triplet at c50.93 (31-1, 17.5 Hz) coupled with a multip-let at c5 1.62 (2H) which in turn was further coupled to a triplet at 152.24 (2H, 17.4 Hz). The signal positions indicated the presence of a butanoyl group (fragment-V) in cavernulin A (I). This was fLuiher confirmed by the 13 C NMR spectrum of the compound 1 (Table I).
Besides, the compound also exhibited additional 1 H signals for a quaternary methyl at 15 1 The aforesaid observations on cavernulin A were thus in conformity with the alternative structures I and 8. The gross structure as I and the position of three ester functions, i.e. two acetates and one butyrate group at C-2, C-9 and C-12, respectively, were, however, ascertained by accounting for the ion peaks observed in the mass spectrum of cavernulin A (I) and subsequently by its derivation from the congener diterpenoid cavernulin B (2) by the action of butyric anhydride and pyridine.
An ion peak at m!z 238 (40%) [ca. C 13 H I 8 0 4 ](ion fragment a generated by RDA collapse of six-membered ring and simultaneous cleavage ofallylic C(3)-C(4) bond) was clearly in conformity with the gross structure 1 and that the butyrate group could be present at C-2 or C-12. Thus an acetate function was present at C-9. Again an ion peak at mlz 265 (10%) [ca. C 15 H 21 0 4 ) (ion fragment b formed by a 01\c l+ b the cleavage ofC(1 )-C(2) and C(8)-C(9) bonds) further supported the gross structure I with the butyrate group present at C-9 or C-12. Consequently, the presence of butyrate group at C-12 would account for the formation of both the ion ,fragments a and b, and thus two acetate functions \\{ere :at Patra et ul. : Secondary metabolites from the en idarian Cavernularia sp.
C-2 and C-9, respectively. Similar ion fragments are not achievable from 8.
TheIR spectrum (KBr) of cavernulin B (2) indicated the presence of hydroxyl (3400-3450 cm-1 ) and five-membered lactone ( 1770 and 1225 cm-1 ) functional ities. The 1 H NMR spectrum of compound 2 clearly accounted for the various structural fragments present in the molecule. The presence of a cis-double bond as a patt of the structural unit -CH=CH-CHOH-CH(CH 3 )CH-CH(OCOR)-, similar to fragment-! in 1, was clearly discernible since a double doublet signal at r55.73 (IH, J 10.1 and 5.9 Hz) was found to couple with a doublet resonance at r5 5.42 (I J-1, J 10.1 Hz) as well as with a double-doublet appearing at r5 3.85 (I H, J 5.9 and 1.7 Hz). FUJther, a multiplet at r52.10 (1H) was coupled to three signals resonating at r53.85 (I H, dd), 0.96 (3H, d) and 2.64 (l H, dd). The latter signal was also coupled to an oxygenated methine proton signal at r5 5.27 (I H, d). Thus this oxygenated carbon was attached with a quaternary system. The spectrum of compound 2 also showed a broad doublet at r5 4.39 (I H) and four multiplets at r52.74, I .65, 2.52 and 2.02 ( 1 H each). The 1 H-1 H correlation study of compound 2 fUiiher indicated them to be for two adjacent methylene units. Further, the multiplets at r52.74 and 1.65 for a set of geminal protons were coupled to the oxygen bearing methine proton resonating at r54.39 (br d). The 13 C-1 H correlation experiment identi fled the carbon resonances associated with the fragment-II of the compound 2 at b~ 81.2 (d), 32.0 (t) and 29.0 (t), respectively.
Again, homodecoupling and COSY spectra of2 revealed that a broad doublet signal at r5 5.52 (I H, J I 0.1 Hz) was found to couple to a doublet signal at 6 5.21 (I H, J I 0.1 Hz) while the former signal was also weakly coupled to a vinyl methyl signal at r5 1.97 (br s). The XIICORR experiment confirmed that the corresponding protonated carbon signals in 2 were ate">~ 118.7 (d), 77.9 (d) and 28.4 (q), in conformity with the presence of fragment-Ill, requiring the presence of a quaternary oleti nic carbon atom for which the singlet resonance at o~ 146.3 is ascribable ..
The 1 1-1 and 13 C spectra of 2 also displayed signals as-signable to fragment-tV as it was observed that a doublet signal at c5 I . 18 (3 H, J 7.2 Hz) coup! ing with a quartet signal at r52.37 (IlL J7.2 Hz) in the 1 11 spectrum of2 were linked to carbon resonances at c5 6.7 (q) and 42.5 (d). respectively. The upficld carbon resonance at r5 6.7 for the methyl group was an indication that the methyl group was attached to a-carbon of a five-membered lactone moiety. Besides, the compound also displayed additional 1 H signals for a quaternary methyl at 15 1.02 (311, s) and two ac-   26 . To our knowledge this is the first report of the occurrence of briaranes in the genus Cavernularia belonging to the order Pennatulacea.

Experimental
Column chromatography was carried out with silica gel (60-120 mesh) and TLC was performed on silica gel G plates. IR spectra (KBr) were recorded on a Perkin-Elmer 782 spectrophotometer. PMR, CMR, 20-NMR (IH_IH COSY and XHCORR) spectra were recorded on a Bruker AM 300L supercon spectrometer equipped with ASPECT 3000 computer fitted with an array processor using programme version DISR87.1 or DISR94.1 in CDCJ 3 as solvent at 300.13 MHz for proton and at 75.47 MHz for carbon. The chemical shifts values are in J(ppm) down field from TMS. Standard procedures were used for two-dimensional NMR experiments. Optical rotations were measured in a Perkin-Elmer M241 electronic polarimeter in CHCJ 3 at 25°. Gas chromatographic experiments were done with Hewlett-Packard M5890, Series II gas chromatograph fitted with a Hewlett-Packard integrator M3394A using appropriate experimental conditions. Mass spectra were taken in a Hitachi RMU 6L spectrometer operating at 70 eV. Extraction and isolation: The raw organism (6 kg) was crushed mechanically and extracted with CH 2 Cl 2 (5 dm 3 ) and then with CH 2 Cl 2 -MeOH (I : 1; 5 dm 3 ) while the liquid expelled by the raw uncrushed organism was extracted with EtOAc (2 dm 3 ) after saturation with NaCI. Solvents were removed under reduced pressure to afford the respective extracts (~5, 4 and 1.5 g, respectively). Both CH 2 Cl 2 -MeOH and EtOAc extracts showed similar TLC behavior and were mixed together. Chromatographic resolution of the CH 2 CI 2 extract and subsequent preparative TLC of the appropriate fractions afforded a novel cyclized cembranoid (briarane ), cavernulin A ( 1) together with a few ubiquitous compounds such as wax esters (fatty esters of saturated fatty acids and also of some analogous unsaturated fatty acids) ( 4), ceramides (5), cholesterol ( 6), 1-0-alkylglycerol (7).

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Simi Jar chromatographic resolutions of the mixed CH 2 Clz-MeOH (I : 1) and EtOAc extracts afforded another new briarane diterpenoid cavernulin B (2) together with small amounts ofceramides (5) as well as 1-0-alkylglycerol (7) as the polar components. 3 mg) in pyridine (0.2 ml) was treated with butyric anhydride (0.2 ml) at about oo and the reaction mixture was kept as such for 20 h. It was then treated with methanol (I ml) and after ~2 h the solvents were removed under reduced pressure. The product was purified by chromatography to afford cavernulin A (1; 2 mg) identified by TLC and PMR spectral comparison with the natural specimen. Basic hydrolysis of wax ester fraction and extraction with ether gave the alcohol fraction which was converted into trimethylsi lyl ether and analyzed by gas chromatography in gradient mode (180-320°) over a column ofSP 2100 (1.8 m x 2 mm glass column) with an increase of temperature at the rate of I 0° per min and employing inlet temperature at 350°, outlet 380° and a flow rate of N 2 at 30 ml per min whereby the major trimethylsilylated alcohol components 16:0 (36.2), 17:0 (4.1), 18:0 (33.3) and 21: a (10.5%) eluted out successively.