New lipid metabolites from a new Sinularia species soft coral

A new monohydroxysterol, 22,23-dimethyl cholest-5-en-3fi-ol (I) has been identified in the monohydroxysterol fraction and two new ceramides (2 and 3) have been isolated from the EtOAc-solubles of a new species of Sinularia soft coral, collected from the Andaman and Nicobar Islands. Their structures have been deduced from spectral data, and in the case of I, by GLC and GC-MS analysis also.

As part of our studies on the isolation and structure elucidation of new polyhydroxysterols 1 , steroidal glycosides 2 .4, sphingosine derivatives 3 -5 , cerebroside 6 , lipid glycosides 7 • 8 and diterpenoids 9 from the soft corals of the Andaman and Nicobar Islands, we have examined a new species of Sinularia collected at Hori Island, and the results are reported here. The soft coral was extracted with ethanol and the residue from the ethyl acetate soluble portion of the ethanolic extract on extensive chromatography over silica(Si0 2 )gel with solvents of increasing polarity from n-hexane through EtOAc gave ethyl arachidonate 3 , II, 12epoxyisoneocembrene-A 3 , a monohydroxysterol mixture, a novel norditerpenoid 10 , and two new ceramides, compound-S (2) and compound-C (3).

Results and Discussion
The monohydroxysterol fraction was found to be homogeneous over silica gel thin layers. It was crystallized from methanol as colourless crystals, m.p. I 86-I 88°. It gave positive Liebermann-Borchard test, characteristic for sterols. Its IR spectrum showed a broad band at 3500 cm-1 for hydroxyl absorption. However, its 1 H NMR spectrum showed more secondary methyl signals than expected for a cholestane or ergostane type steroid, and duplicated carbon chemical shifts found in its 13 c NMR spectrum also suggested that it could be a mixture ofmonohydroxysterols and hence it was acetylated. The sterol mixture formed a monoacetate with Ac 2 0/pyridine at room temperature for 24 h and the resultant product upon crystallization from methanol gave colourless plates, m.p. I48-I50°. IR spectrum of the acetyl derivative showed the lack of hydroxyl absorption, indicating it to be the acetate ofmonohydroxysterols. The acetate showed several close running spots on   •RR 1 with respect to cholesteryl acetate whose retention time under the same experimental conditions was 3.70 min.
Compound-A, is the major constituent of the sterol mixture (35%). Its molecular formula was found to be C 31 H 52 0 2 from the ion at mlz 396 (M+ -AcOH) in its mass spectrum. The prominent peaks at mlz 255 [M+ -Ac0H-sidechain(C10H21) (70%)] and 120 (25%) were due to cholest-5-ene nucleus. The saturated 22,23-dimethylcholestane sidechain was deduced from the fragment ions at mlz 296 [M+-AcOH-C 6 Hn-CH 3 (10%)] due to the cleavage ofC-22-C-23 bond and ion at mlz 28J[M+-Ac0H-C 8 H 17 -2H(25%)] due to the cleavage ofC-20-C-22 bond. Therefore, the structure was assigned as 3,8-acetoxy-22,23-dimethylcholest-5ene (l)to compound-A. It is a new addition to the literature ofmonohydroxysterols. The monohydroxysterol was a new sterol based on its retention time different from other known sterols and the tentative structure was derived from the mass spectral fragmentation. The mass spectral fragmentation of compound-A is shown in Chart I; its relative retention time and fragment ions are shown in Table I.
Compound-B. m.p. 130-132° was analyzed for C3 4 H 65 N0 3 and its IR spectrum showed strong bands for hydroxyl (3350, 1 040) and secondary amide ( 1640, 1540), in addition to trans-double bond (970) and an aliphatic chain (2918, 2851, 1453 cm-1 ), suggesting it to be a fatty acid amide. Furthermore, compound-S was found to possess norma/ 11 types of side-chains, since the carbon atom signals due to terminal methyl groups were observed at o 14.1 (normal form) in the De NMR spectrum of compound-S (Table 2). The existence of an unbranched fatty acid and an unbranched long-chain base was also suggested by its 1 H NMR spectrum. The 1 H NMR spectrum (90 MHz, pyridine-d5, TMS standard) of compound-S (Table 2)   It showed IR bands at 3350 (OH) and 1640 (amide) in addition to trans-double bond (970 cm-1 ). eompound-e was found to possess norma/ 11 types of side-chains since the carbon signals due to terminal methyl groups were observed at 814.1 (normal form) in the 13 e NMR spectrum ofcompound-e ( Table 2). The existence of an unbranched fatty acid and an unbranched long-chain base was also suggested by its 1 H NMR spectrum. A carbonyl carbon signal at 8 175.5 (s) in the Be NMR spectrum, a downfield proton signal at 88.55 (d, 19.0 Hz) in the 1 H NMR spectrum and a strong IR band at 1640 cm-1 indicated the presence of a secondary amide group. A very strong signal at 8 1.25 in the 1 H NMR spectrum and lack ofupfield methyl signals in the 13 C NMR spectrum revealed that compound-C must be derived from a long-chain fatty acid precursor.
The 1 H NMR spectrum (90 MHz, pyridine-d 5 , TMS standard) of compound-C (Table 2)  The 13 C NMR spectrum of compound-C (Table 2) showed the presence of an amide functionality at c5 175.5 The 1 H NMR and 13 C NMR (Table 2) data of compound-C was almost identical with that of N-(2' -hydroxyicosanoyl)-1 ,3,4-trihydroxy-2~amino heptadeca-5-ene ( 4) isolated from Sinularia gravis 4 Tixier-Durivault in our laboratories. The difference lies only in the mass spectral data of these two compounds 3 and 4. In the former, the molecular ion was observed at mlz 569 in El-MS spectrum and in the latter, the [M + H] ion at mlz 612 4 in the positive FABMS. Therefore, they differ only in the lengths of the side-chains.
The acyl chain and sphingosine part of compound-C were deduced from mass spectroscopy. The prominent mass fragment ions at mlz [314(M+-255)(45%)] and [255(M+-314)(40%)] due to the cleavage ofNH-CO-bond3, 2 5 suggested that the sphingosine part contains a 18 carbon unit and the fatty acid chain posseses a 16 carbon unit. There-fore, the structure of compound-C was established as N-(2' -hydroxyhexacosanoyl)-1 ,3 ,4-trihydroxy-2-aminooctadeca-5-ene (3) and is also a new addition to the literature of ceramides. The ma5s spectral fragmentation of compound-Cis given in Chart 3. University, Visakhapatnam, with code number MF-VA/31. The specimen (dry weight 0.9 kg) was washed with fresh water, soaked in ethanol. The extraction was carried out using EtOH by percolation for every 48 h (7 times). The solvent was stripped off by distillation under reduced pressure. The dark coloured residue was extracted with ethyl acetate several times. The ethyl acetate soluble portion was washed with distilled water and dried over anhyd. MgS0 4 and concentrated under vacuum. The residue (40 g) was chromatographed over a column ofsillica gel (500 g, I 00--200 mesh; Acme) using eluants with increasing polarities of solvent mixtures starting from pet. ether, ethyl acetate to methanol (each 800 ml fractions). Pet. ether, pet. ether-ethyl acetate ( 19 : I) as eluants yielded II, 12-epoxyisoneocembrene-A (ISO mg) as an oil and ethyl arachidonate (450 mg) as a pale yellow oil; pet. ether-ethyl acetate (9: I) eluate gave a mixture of inseparable monohydroxysterols (2.0 g), containing compound-A; pet. ether-ethyl acetate ( 4 : 1) furnished a norditerpeniod; pet. ether-ethyl acetete (3 : 2) afforded compound-S (I 0 mg); and pet. ether-ethyl acetate (I : !)yielded compound-C (5 mg). The known compounds were identified by comparison of their physical and spectral data with authentic samples2 6 .
The monohydroxysterol mixture showed a single spot on TLC (Rr = 0.45, pet. ether : EtOAc; 4 : 1), which on repeated crystallization from chloroform-methanol yielded colourless shining plates of sterol mixture (2.0 g), m.p. 186-1880. Monohydroxysterol mixture (I 00 mg) was dissolved in pyridine (3.5 ml), acetic anhydride (3.5 ml) added and kept at room temperature for 24 h. The reaction product was worked-up by usual procedure and the resultant solid upon crystallization from methanol gave colourless plates ofmonohydroxysterol mixture acetate, m.p. 148-150°. The monohydroxysterol mixture acetate was subjected to GLC and GC-MS analysis.
Compound-A :The percentage composition, relative retention times and mass spectral fragmentation ions are presented in Table I