Galactocerebroside and other secondary metabolites of sea mouse Chloeia

Novel galactocerebrosides derived from 2-amino-4-alkene-1,3-diol along with fatty acid methyl esters (FAME), cholesterol, ceramides, 1-0-acylglycerols and 1-0-alkylglycerols have been isolated from a sea mouse Cllloeia parva. The structure of galactocerebrosides is established from one-dimensional [ 1 H, homodecoupling and 13c (NDC and DEPT-135)] NMR spec tral studies of the metabolite and its acetate as well as two-dimensional (COSY -90, X-II correlation optimized for I J C-ll) NMR spectral studies of the latter. The remaining components isolated have been characterized from chemical studies and det11iled NMR spectral analyses of parent materials and products derived thereof. The lengths of the alkyl and acyl chains in galactocerebrosides, FAME, ceramides, 1-0-acylglycerols and l-0-alkylglycerols are established by gas chromatographic analysis of appropriate derivatives. spectrophotometer and IR were examined in KBr disc on a Perkin Elmer-782 spectro photometer. NMR ( 1 H, 13 c, 1 H- 1 H COSY and l3c-1 H XHCORR) experiments on Bruker AM 300L supercon spectrometer equipped with ASPECT 3000 computer fitted with an array processor using programme version DISR87.1 or DISR94.1 CDCI3, unless other wise stated, as solvent at 300.13 MHz for proton and at 75.47 MHz for carbon. Multiplicity of the carbon signals was determined from DEPT-135 experiments. The chemi cal shift values are in 8 (ppm) downfield from TMS. Deuterio-solvent signal served as an internal standard in carbon spectral measurements; SrMs = 8 (CDCJ 3) + 77.0 ppm. Standard procedures were used for two-dimensional NMR experiments. Optical rotations were measured in a Perkin Elmer M241 electronic polarimeter at 25°.

Past three decades have witnessed a tremendous surge of interests in marine natural product research throughout the globe 5 . As a part of our search for bioactive components from marine sources a sea mouse, Clzloeia parva was collected from the Eastern coast of the Bay of Bengal, adjoining West Bengal. This paper deals with the results of the chemical investigations including the structural elaboration of interesting and novel marine metabolites, galactocerebrosides. Cerebrosides are important membrane components in both plant and ;,p1imal cells, and they appear to participate in cell regulatory functions and trans-membrane signaling. Such galaclocerebrosides have earlier been reported 4 from star fish (Pentaceraster regulas 6 ), sponge (Ciwndropsis sp. 7 • 8 , Haliclwndria japonica 9 , Halichondria panicea 10 , Age las longissima ll-l3, Age/as clrrrl!rodes 14 , Age/as conifera 15 , Age/as dispar 16 , Age/as sp. 17 , Ax in ella sp. 18 , Plakortis simplex 19 and Age/as mauritianus 20 · 21 ), sea star (Stellaster equestris 22 and Astropecten latespinosis 23 ), annelid (Marphysa sanquinea 24 ) and red algae (Corallina pilulifera2 5 ) and continue to attract the attention of investigators because of their structural complexity and wide range of biological activities, e.g., coronary vasodialators, antiherpertensive and histidine carboxylase inhibitors 7 • 8 , cytotoxic 17 • 23 , immunosuppressive prop~rties 19 ; antitumour and immunostimulatory 2 0· 21 , active ingredients for the treatment of AIDS and HIV -related diseases 26

Galactocerebroside :
The UV spectrum of the component 1 in aldehyde free ethanol showed no characteristics absorption for conjugated chromophore. The IR spectrum of the material displayed absorption bands for the presence of -OH and -NHCO-functions and methylene units. The detailed ID ( 1 1-l-, 13 C-, homodecoupling) and 20 (COSY and XHCORR) NMR experiments of the metabolite and its pentaacetate were in consonance with formulations 1 and 7 respectively. The 1 H spectrum of the component 1 in CDClrCD 3 0D displayed a signal at 8 5.59 (I H, d, 1 6.5 Hz, very slowly exchangeable by D 2 0) for an amide proton ( -CONH-) that was coupled to a multiplet signal at 8 3.93 (I H). The spectrum also showed few signals in the 3.3-4.2 ppm region and most of them were for the protons on oxygenated carbons as in a sugar moiety of a glycoside. Besides, the 1 H spectrum contained the signals at 8 5 bon resonances were made by using substituent chemical shift parameters applicable to olefinic and alkyl sys-tem28-30. The signals for the terminal methyl carbon and inner methylene carbons of the long aliphatic chain at 813.6 and 29.5 respectively were also observed in the 13 c spectrum of 1.
Treatment of the component 1 with Ac 2 0-pyridine yielded a pentaacetate 7, Rr0.5 in CHC1 3 . TheIR spectrum of the product showed new absorption bands at 1750 and 1240 cm-1 (for acetate function) along with reduction in intensity of the broad band at 3400-3300 cm-1 confirmed the changeover to the acetate 7. The formation of the pentaacetate upon acetylation was indicated by the generation of five acetate methyl signals at 8 2.
The CH 2 CI 2 and CH 2 CI 2 -MeOH {l : 1) extracts of the organism were subjected separately to column chromatography over silica gel (60--120 mesh, 120 g) using solvents of increasing polarity. The eluates of each silica chromatogram were tested by running micro-tic, fractions having identical spots were mixed up and the different fractions were purified further by preparative tic over silica gel G using solvents of increasing polarity. The less polar eluates of the chromatograms afforded fatty acid methyl esters (FAME), cholesterol while the relatively polar CHCI 3 -MeOH (95 : 5) eluates yielded ceramide and CHClrMeOH (90 : 10) eluates gave 1-0-alkylglycerol and 1-0-acylglycerol. Again, the relatively more polar Acetylation of galactocerebrosides 1 : Galactocerebroside 1 (25 mg) dissolved in pyridine ( 1 ml) was mixed with distilled Ac 2 0 ( 1 ml), warmed on water bath for brief period and kept at room temperature for 24 hours. Dilution of the reaction mixture with cold H 2 0 (50 m1) and subsequent acidification with aqueous HCI (IN; 20 ml) was followed by extraction with CHC1 3 (3 x 25 ml). The material obtained from organic layer was purified by column chromatography to yield an amorphous pentaacetate 7 (20 mg); Rr 0.5 in CHC1 3 ; IR Vmax 3400-3300, 2955, 2920, 2845,1750,1680,1505,1465,1370,1240,1170, 1070and 1045 cm-1 • FAME and Alc-OTMS from galactocerebroside 1 : The galactocerebroside fraction (8 mg) was retluxed with dry MeOH (2 ml) and cone. H 2 S0 4 (2 drops) for half an hour. Then the reaction mixture was cooled, diluted with water and extracted with CHCI 3 (3 x lO ml). Usual work-up of the organic layer followed by solvent removal afforded FAME fraction that was purified by column chromatography on silica gel (100-200 mesh). Finally the purified FAME (3 mg) so produced was gas chromatographed over I 0% DEGS column and found to be consisted of methyl esters of 14: 0 (4.4%), 16 The aqueous phase was treated with a bit of NaN0 2 and stirred for half an hour, then refluxed with Nal0 4 (5 mg) for an hour under N 2 atmosphere. The reaction mixture was cooled, extracted with CHCI 3 (3 x 10 ml).
Usual work-up and removal of solvent afforded a material which was as such dissolved in MeOH ( 1 ml) and stirred with KBH 4 ( -5 mg) in the cold on a magnetic stirrer for an hour, diluted with water (25 ml) and extracted with CHCI 3 (3 x 10 ml). Usual work-up of CHCI 3 layer and removal of solvent yielded fatty alcohol (3 mg) as a colourless semi-solid mass, Rr 0.5 in CHCI 3 -MeOH (99 : 1) after purification by column chromatography (silica gel, 100-200 mesh). The fatty alcohol thus produced was dissolved in 50 ,ul of dry pyridine to which 45 ,ul of hexamethyldisilizane (HMOS) and 30 ,ul of trimethylchlorosilane were added. The vial was vigorously shaken for 30 second and the reaction was allowed to complete at 80° for 20 minutes and the content was directly injected into the column (3% OV 17). GLC analysis of the product allowed determination of the carbon chain length (as c,8) of the basic sphingosine unit.