tricyclic alkaloids represented by cylindricines , lepadiformine ( 3 )   and fasicularin   are unique metabolites of ascidians . cylindricines a and b isolated from clavelina cylindrical   were the first members of the family based on the perhydropyrrolo[2,1-j]quinoline and the perhydropyrido[2,1-j]quinoline ring systems , respectively . the structures and relative stereochemistries were unambiguously established by x - ray crystallography of their corresponding picrates . moreover , these two compounds were reported to interconvert through aziridinium intermediate forming 3:2 equilibrium mixture upon standing in a solution . compound 3 exhibited cytotoxicity against several tumor cell lines   and in vivo and in vitro cardiovascular activity . more recently , fasicularin , isolated from nephteis fasicularis , was reported as a cytotoxin to vero cells and was found to act as a dna - damaging agent in the assay using a dna repair - deficient yeast strain . as a part of collaborative investigation of biologically active compounds from indonesian marine organisms , we herein report the isolation and structures of two new tricyclic alkaloids polycitorols a ( 1 ) and b ( 2 ) together with known lepadiformine ( 3 ) from a marine ascidian . specimens ( 65.0 g , wet ) of the ascidian of the family polycitoridae were collected by scuba ( 25 m ) off flores , indonesia in august 2001 and were stored in ethanol after its collection . the sample was exhaustively extracted with meoh and the resulting extract was chromatographed on a silica gel column followed by reversed - phase hplc to give polycitorol a ( 1 ) , b ( 2 ) , and lepadiformine ( 3 ) . polycitorol a ( 1 ) , colorless oil , [ ]d + 15 ( c 0.2 , chcl3 ) , was shown to have a molecular formula of c17h31no by hreims ( m / z 265.2410 , m ,  + 0.4 mmu ) . strong absorption bands at 3384 and 1671 cm in the ir spectrum suggested that 1 contain hydroxyl and amine functionalities . the h nmr spectrum contained a number of unresolved multiplets in the high field region (  1.212.24 ) , while no olefinic signals were observed . the c nmr spectrum revealed the presence of a quaternary carbon (  77.9 ) , two heteroatom bearing methines (  67.0 , 71.9 ) , one methine (  44.2 ) , one ch2oh (  65.0 ) , one methyl , and eleven methylenes . therefore , alkaloid 1 is tricyclic , and the nitrogen atom was suggested as a tertiary amine . the c nmr spectrum of 1 displayed 17 carbon signals indicating that 1 has two less carbons than 3 . a mass fragment ion at m / z 208 was formed by the cleavage of c4h9 , thus , differing in the length of the alkyl side chain from 3 . cosy correlations revealed that a methine proton at  2.97 ( c 67.0 ) correlated with two methylene protons at  1.58 and 2.05 confirming the c2/c15 linkage . further 2d nmr analysis ( table 1 ) allowed us to elucidate the planar structure of 1 . enhancement of h-2 at  2.97 and h-4ax at  1.57 upon irradiation of h-9ax at  2.15 suggested a cisfusion between rings a and b. the alkyl side chain at c-2 occupied  equatorial position on the basis of mutual noe enhancements between h-2 and h-5 . noe between one of h-12 protons and h-13 may indicate the primary hydroxyl group as shown . polycitorol b ( 2 ) , [ ]d 10.3 ( c 0.6 , chcl3 ) , was isolated as a colorless oil . the molecular formula c17h31no was determined by hreims , indicating that it was isomeric to 1 . comparison of nmr data of 1 and 2 revealed that considerable higher field shifts were observed for a methine at  58.3 and a quaternary carbon at  66.5 in the c spectrum of 2 . furthermore , additional methine signal at  4.06 ( c 60.5 ) suggested that 2 had a secondary rather than a primary alcohol . the cosy spectrum indicated that this proton coupled to two nitrogenattached methylene protons (  3.53 , 3.36 ) and two other protons (  1.78 , 2.11 ) . thus , the aforementioned data indicated that the pyrrolidine ring was rearranged to a piperidine ring . the relative stereochemistry of a / b rings was shown to be identical to that in 1 with a c-2 butyl side chain based on similar noe data , particularly a set of mutual noe enhancements between h-2 , h-4ax and h-9ax . a trans fusion between rings a and c was suggested by noe between h-13/h-2 , h-14ax / h-2 and h-5/h-2 . thus , rings a and c of 2 adopted a much more rigid trans 1-azadecalin system and the c-13 hydroxyl group occupied  equatorial position . two new tricyclic alkaloids , polycitorols a ( 1 ) and b ( 2 ) , have been isolated along with the known lepadiformine ( 3 ) from a marine ascidian . the new compounds contain a butyl side chain instead of a hexyl group observed more commonly in known related compounds . nmr spectra were taken on a jeol  500 ft nmr spectrometer and referenced to chcl3 solvent signal at  7.26 for h nmr and  77.0 for c nmr spectra in cdcl3 as a solvent and also referenced to tms signal at  0.00 for h nmr and  49.0 for c nmr spectra in cd3od as a solvent . hplc separations were carried out on a hitachi l-6000 or shimadzu lc 9a pumps equipped with waters 486 or hitachi l-4000 uv detector and waters r401 differential refractometer . columns used for hplc were reverse phase nacalai 5c18-ar ii ( 10  250 mm ) . tlc was carried out on precoated silica 60f254 plates and visualized with vanillin - etoh-1% h2so4 . the specimens ( 65.0 g , wet wt . ) of the ascidian ( family polycitoridae ) were collected by hand using scuba ( 25 m ) at misa is . , labuhanbajo , flores , indonesia in august 2001 and were stored in ethanol after its collection . adrian gittenberger , national museum of natural history naturalis , leiden , the netherlands . a voucher specimen ( 0117j78 ) is deposited at department of chemistry , biology , and marine science , university of the ryukyus . a photo of the specimen can be viewed at http://www.ascidians.com/families/polycitoridae/polycitoridae_blue/polycitoridaeblue.htm a sample of tunicate ( 65.0 g wet weight ) was cut into small pieces and soaked in acetone ( 300 ml ) for 7 hr . after decantation , fresh solvent was added , and the procedure was repeated three times . the oil was chromatographed on silica gel by eluting with step gradient of hexane / ch2cl2/etoac / meoh . fraction v was selected for further purification using hplc ( rp-18 , meoh / h2o / tfa ) to afford compounds 1 , 2 and 3 . polycitorol a ( 1 ) : colorless oil ; [ ]d + 15.3 ( c 0.2 , chcl3 ) ; ir ( film ) 3384 , 2937 , 2867 , 1671 , 1455 , 1201 , 1132 cmh and c nmr see table 1 ; eims m / z 265 ( 25 , m ) , 234 ( 100 ) , 222 ( 97 ) , 208 ( 45 ) , 194 ( 5 ) , 178 ( 5 ) ; hreims found m / z 265.2410 ( calcd for c17h31no , 265.2406 ) . polycitorol b ( 2 ) : colorless oil ; [ ]d 10.3 ( c 0.6 , chcl3 ) ; ir ( film ) 3384 , 2937 , 2869 , 1670 , 1457 , 1201 , 1132 cmh and c nmr see table 1 ; eims m / z 265 ( 6 , m ) , 222 ( 29 ) , 208 ( 100 ) , 193 ( 7 ) , 150 ( 5 ) ; hreims found m / z 265.2406 ( calcd for c17h31no , 265.2406 ) . nmr spectra were taken on a jeol  500 ft nmr spectrometer and referenced to chcl3 solvent signal at  7.26 for h nmr and  77.0 for c nmr spectra in cdcl3 as a solvent and also referenced to tms signal at  0.00 for h nmr and  49.0 for c nmr spectra in cd3od as a solvent . hplc separations were carried out on a hitachi l-6000 or shimadzu lc 9a pumps equipped with waters 486 or hitachi l-4000 uv detector and waters r401 differential refractometer . columns used for hplc were reverse phase nacalai 5c18-ar ii ( 10  250 mm ) . tlc was carried out on precoated silica 60f254 plates and visualized with vanillin - etoh-1% h2so4 . the specimens ( 65.0 g , wet wt . ) of the ascidian ( family polycitoridae ) were collected by hand using scuba ( 25 m ) at misa is . , labuhanbajo , flores , indonesia in august 2001 and were stored in ethanol after its collection . adrian gittenberger , national museum of natural history naturalis , leiden , the netherlands . a voucher specimen ( 0117j78 ) is deposited at department of chemistry , biology , and marine science , university of the ryukyus . a sample of tunicate ( 65.0 g wet weight ) was cut into small pieces and soaked in acetone ( 300 ml ) for 7 hr . after decantation , fresh solvent was added , and the procedure was repeated three times . the oil was chromatographed on silica gel by eluting with step gradient of hexane / ch2cl2/etoac / meoh . fraction v was selected for further purification using hplc ( rp-18 , meoh / h2o / tfa ) to afford compounds 1 , 2 and 3 . polycitorol a ( 1 ) : colorless oil ; [ ]d + 15.3 ( c 0.2 , chcl3 ) ; ir ( film ) 3384 , 2937 , 2867 , 1671 , 1455 , 1201 , 1132 cmh and c nmr see table 1 ; eims m / z 265 ( 25 , m ) , 234 ( 100 ) , 222 ( 97 ) , 208 ( 45 ) , 194 ( 5 ) , 178 ( 5 ) ; hreims found m / z 265.2410 ( calcd for c17h31no , 265.2406 ) . polycitorol b ( 2 ) : colorless oil ; [ ]d 10.3 ( c 0.6 , chcl3 ) ; ir ( film ) 3384 , 2937 , 2869 , 1670 , 1457 , 1201 , 1132 cmh and c nmr see table 1 ; eims m / z 265 ( 6 , m ) , 222 ( 29 ) , 208 ( 100 ) , 193 ( 7 ) , 150 ( 5 ) ; hreims found m / z 265.2406 ( calcd for c17h31no , 265.2406 ) .
two new tricyclic alkaloids , polycitorols a ( 1 ) and b ( 2 ) have been isolated along with the known lepadiformine ( 3 ) from a marine ascidian of the family polycitoridae . 
 the structures of the new compounds were elucidated by analysis of nmr data and comparison with those of 3 and other related compounds [ 15 ] . 
 compounds 1 and 2 are closely related to cylindricines a and b , lacking c-4 oxygenation found in cylindricines and having a butyl instead of a hexyl appendage . 
 noe experiments on compounds 1 and 2 suggested the a / b ring fusion to be cis .