corticium niger specimens were collected near 
 luzon , on the west side of the calumpan peninsula of the philippines 
 in july 1994 , and extracted using the standard nci protocol for marine 
 invertebrate animals . a portion ( 224 
 mg ) of the sponge organic solvent extract was initially fractionated 
 by step gradient elution through an spe cartridge containing diol 
 solid - phase media , which concentrated the cytotoxic activity into 
 fractions eluted with etoac  meoh ( 5:1 ) and 100% meoh . these 
 materials were combined and subjected to size - exclusion chromatography 
 on lh-20 ( meoh  h2o , 9:1 ) and c18 hplc 
 to yield two new plakinamines , n ( 1 ) and o ( 2 ) , along with the known plakinamines , i ( 3 ) and j ( 4 ) . the structures of the new compounds were unambiguously 
 assigned by comprehensive analysis of their 1d and 2d nmr data , in 
 conjunction with accurate mass measurements ( hresims ) , and by comparison 
 of their spectroscopic data to appropriate literature values . plakinamine n ( 1 ) was obtained as a pale yellow 
 oil , 
 and a preliminary examination of the h and c nmr spectra revealed the steroidal nature of the compound , as they 
 were very similar to those reported for plakinamine k ( 5 ) . the hresims spectrum established 
 a molecular formula of c29h48n2 , 
 requiring seven degrees of unsaturation . two of these were attributed 
 to olefinic groups assigned to  ( c 118.5 , 140.4 ) and  ( c 128.4 , 130.6 ) on the basis of their hmbc correlations ( table 1 ) . analysis of cosy and hsqc data allowed assignment 
 of the c-1 to c-7 , c-9 to c-12 , and c-14 to c-17 spin systems , which 
 were characteristic of a tetracyclic steroidal backbone with a c-3 
 amine substituent . the chemical shift of c-3 ( c 48.7 ) , 
 together with cosy correlations from h-3 ( h 3.53 ) 
 to methylene resonances assigned to h2 - 2 ( h 1.71 , 1.91 ) and h2 - 4 ( h 1.75 ) , confirmed 
 placement of the amine at this position . hmbc correlations from the 
 methyl singlet at h 0.85 ( me-19 ) to c-1 , c-5 , c-9 , 
 and c-10 helped define the composition and junction of rings a and 
 b. the fused nature of rings b , c , and d was established by hmbc correlations 
 from the olefinic methine resonance ( h-7 , h 5.22 ) 
 to the ring junction methine carbons c-5 , c-9 , and c-14 and from the 
 methyl singlet ( me-18 , h 0.63 ) to c-12 , c-13 , c-14 , 
 and c-17 . hmbc correlations from the methyl doublet ( me-21 , h 1.12 ) to c-17 , c-20 , and c-22 helped elaborate the steroidal 
 side chain . deshielded carbon and proton resonances at c-23 ( c 59.2 , h 4.40 ) and c-29 ( c 45.1 , h 3.43 and 3.32 ) , which were indicative 
 of nitrogen substitution , and characteristic h h vicinal coupling between h2 - 28 and h2 - 29 were consistent with the presence of a pyrrolidine ring system . an exocyclic tetrasubstituted olefinic group was assigned to  based on hmbc correlations from the coincident vinyl 
 methyl protons of me-26 and me-27 ( h 1.71 , 6h ) to 
 c-23 , c-24 , and c-28 . homoallylic coupling observed from me-26/27 
 to the h2 - 28 methylene resonances at h 2.61 and 2.66 supported this assignment . characteristic hmbc correlations 
 from h-29 to c-23 , c-24 , and c-28 helped confirm that compound 1 has the same substituted heterocyclic ring system that is 
 found in plakinamine k ( 5 ) . proton coupling , roesy correlations , and c comparisons 
 were used to define the relative configuration of 1 . the small vicinal couplings observed for h-3 ( h 3.53 , 
 br s ) , together with its roesy correlations ( figure 1 ) to h-2 ( h 1.91 ) and h-4 
 ( h 1.75 ) , were consistent with a -(equatorial)-orientation 
 of this proton . the chair conformation and trans - ring - fusion 
 of the a and b rings were established from 1,3-diaxial roesy correlations 
 between me-19 ( h 0.85 ) , h-2 ( h 1.91 ) , h-4 ( h 1.75 ) , and h-11 
 ( h 1.52 ) and a correlation between h-5 ( h 1.62 ) and h-9 ( h 1.84 ) . a roesy 
 cross - peak between me-18 ( h 0.63 ) and one of the 
 h-11 protons ( h 1.52 ) revealed these two substituents 
 had a 1,3-diaxial relationship , or -orientation . a roesy correlation 
 between h-14 and h-7 supported an -orientation for h-14 and , 
 thus , trans - fusion of the c and d rings . all of the 
 previously reported plakinamine steroidal alkaloids possess a trans - fused c / d ring junction , a -oriented side chain 
 at c-17 , and an -methyl group at c-20 . thus , a comparison of 
 the c nmr data recorded for 1 with the data 
 reported for other structurally related plakinamines allowed assignment 
 of the relative configuration at these centers . there are nine compounds 
 in the plakinamine class with a pyrrolidine or dehydropyrrolidine 
 ring in the side chain , and the c chemical shifts for 
 c-13 , c-14 , c-17 , and c-20 in these compounds all fall within very 
 characteristic ranges ( c-13 , c 44.3  0.7 ; 
 c-14 , c 56.8  1.7 ; c-17 , c 58.0  0.9 ; c-20 , c 35.2  0.7 ) . the c nmr data for 1 were 
 in close correspondence with these values ( c-13 , c 44.7 ; c-14 , c 56.2 ; c-17 , c 57.8 ; 
 c-20 , c 34.7 ) , which indicated it also had a  
 side chain and an  methyl group at c-20 . a roesy correlation 
 between me-18 and me-21 ( h 1.12 ) and additional 
 correlations between h-23 ( h 4.40 ) , h-20 
 ( h 1.56 ) , me-21 ( h 1.12 ) , and 
 me-26 were all consistent with these assignments , but they were not 
 sufficient to define the relative configuration at c-23 . a cursory examination 
 of the h and c nmr 
 spectroscopic data ( table 1 ) of plakinamine 
 o ( 2 ) showed that the chemical shifts from c-1 to c-19 
 were virtually identical to those recorded for compound 1 , thus establishing the presence of a closely related steroidal alkaloid . the hresims spectrum of 2 defined a molecular formula 
 of c31h50n2o2 , which differed 
 from 1 by the substitution of an oac group . the presence 
 of an acetate functionality was confirmed by an hsqc correlation from 
 a methyl proton singlet at h 2.10 to a carbon resonance 
 at c 20.8 and an hmbc correlation from this methyl 
 signal to an ester carbonyl carbon at c 171.5 . cosy 
 correlations from both h-3 ( h 3.45 , br s ) and h-5 
 ( h 1.80 ) to an oxymethine group ( h 5.00 , this was confirmed by an hmbc correlation from the acetate 
 methyl protons to c-4 . the remainder of the tetracyclic backbone was 
 defined using cosy spin systems from c-1 to c-7 , c-9 , to c-12 and 
 from c-14 to c-17 and a comprehensive set of hmbc correlations ( table 1 ) . both the carbon and proton nmr chemical shifts 
 for c-20 to c-29 in compound 2 were virtually identical 
 to those of 1 , which indicated they shared the same side 
 chain and substituted pyrrolidine moiety . hmbc correlations from h-23 
 ( h 4.40 ) to c-20 , c-22 , c-24 , c-25 , c-28 , and c-29 
 supported this assignment . the relative configuration of 2 was assigned primarily 
 from roesy cross - peaks and c nmr data that were similar 
 to those observed in 1 . roesy correlations between h-4 
 and h-5 and those between me - ac and me-19 ( h 1.07 ) 
 were used to unequivocally assign a -orientation for the c-4 
 acetate group ( figure 2 ) . the corresponding 
 -orientation for h-3 ( h 3.45 , br s ) was confirmed 
 by the very small magnitude of its vicinal couplings and roesy correlations 
 with me - ac ( h 2.10 ) and h-2 ( h 2.12 ) . all of the other roesy cross - peaks observed in 2 were analogous to those seen in 1 , and the c data were also very similar , which indicated that they 
 shared the same relative configuration throughout the tetracyclic 
 steroidal core and the pyrrolidine - containing side chain . some key roesy 
 correlations for 2 . compounds 14 were evaluated 
 for 
 cytotoxicity in the nci-60 anticancer screen , which includes a panel 
 of seven human colon carcinoma cell lines ( colo 205 , hct-15 , sw-620 , 
 hcc-2998 , ht29 , hct-116 , km12 ) . plakinamines 
 n ( 1 ) , o ( 2 ) , and j ( 4 ) showed 
 sufficient growth - inhibitory activity when tested at a single concentration 
 to warrant full dose plakinamine i ( 3 ) had only modest activity and was not 
 subjected to further testing . while compounds 1 , 2 , and 4 contain a substituted pyrrolidine ring 
 in the steroidal side chain , the pyrrolidine nitrogen in compound 3 forms an additional fused piperidine ring system , which 
 may affect the cytotoxic activity of these alkaloids . results from 
 the complete 60-cell study revealed that plakinamines o ( 2 ) and j ( 4 ) were the most potent , with mean gi50 ( 50% growth inhibition ) values of 2.4 and 1.4 m , respectively , 
 while plakinamine n ( 1 ) was less potent , with a mean 
 gi50 of 11.5 m . all three of these plakinamines 
 showed an enhanced antiproliferative effect against the entire panel 
 of colon cell lines at the mean gi50 , mean tgi ( total growth 
 inhibition ) , and mean lc50 ( 50% cell death ) levels ( supporting information , s15s17 ) . while 
 their potencies were only low micromolar , the observation of greater 
 colon cancer sensitivity at all three of the standard indices of inhibition 
 was striking , and it provided strong evidence that these plakinamines 
 are indeed colon cell line selective . while the differential cytotoxic 
 activity of the plakinamines was real and highly reproducible , the 
 degree of colon selectivity was relatively modest , as the total variation 
 about the mean for all three indices was approximately 10 to 10 . in our experience , it is extremely 
 rare to find a class of compounds that is more active against all 
 cell lines in a particular tumor panel , at all three indices of inhibition . these findings indicate that the plakinamine steroidal alkaloid skeleton 
 provides a structural scaffold that could potentially be modified 
 to generate analogues with greater potency and enhanced colon - selective 
 antiproliferative properties . optical rotations ( [ ]d ) were measured on a perkinelmer 241 polarimeter in a 100 
  2 mm cell ( units 10 deg cm g ) . uv absorption spectra were obtained using a varian 
 cary 50 bio uv  visible spectrophotometer . nmr spectra were 
 acquired on a bruker avance iii spectrometer operating at 600 mhz 
 for h and 150 mhz for c and equipped with 
 a 3 mm cryogenically cooled probe . lcms data were obtained using a hewlett - packard series 1100 msd , and 
 hrms data were acquired on an agilent 6520 accurate mass q - tof instrument 
 with internal reference masses calibrated at 121.050  87 and 
 922.009  79 , both within 5 ppm . diol spe fractionation of the 
 extract was performed on dio spe - ed spe cartridges , and subsequent 
 fractions were separated on a sephadex lh-20 column attached to a 
 model ua-6 uv detector and foxy 200 fraction collector ( teledyne isco ) . final purifications were performed using a varian prostar 218 solvent 
 delivery module hplc equipped with a varian prostar 325 uv the sponge corticium niger pulitzer - finali 1996 
 was collected using scuba at a depth of 15 
 to 20 m at luzon ( west side of calumpan peninsula ) , philippines , 
 in 1994 , and it was immediately frozen and stored at 20 c 
 until extraction . taxonomic identification of the sponge was provided 
 by michele kelly ( national institute of water and atmospheric research , 
 new zealand ) , and a voucher specimen is maintained at the smithsonian 
 institution , washington , d.c . the frozen sponge was ground 
 and processed using the standard nci method for marine samples . the sponge material ( 203 g ) was first extracted 
 with 100% h2o to give an aqueous extract ( 39.9 g ) and then 
 sequentially extracted with ch2cl2meoh 
 ( 1:1 ) followed by 100% meoh . removal of the combined organic solvents 
 under reduced pressure provided 6.1 g of organic extract ( nsc no . a portion of the organic extract ( 224 mg ) was utilized for 
 the colon cancer cell based bioassay - guided isolation of the cytotoxic 
 compounds . the extract was loaded onto two separate diol dio spe - ed 
 spe cartridges ( 112 mg each ) , and each cartridge was sequentially 
 eluted with 6.0 ml of hexane ch2cl2 ( 9:1 ) , 
 ch2cl2etoac ( 20:1 ) , 100% etoac , etoac  meoh 
 ( 5:1 ) , and 100% meoh to give five fractions ( a  e ) , which weighed 
 42.5 , 34.5 , 2.0 , 13.5 , and 98.5 mg , respectively . the colon cancer 
 cytotoxicity of fractions d and e was more potent than the other fractions , 
 so they were combined ( 92.0 mg total ) and then chromatographed on 
 a 90  2.0 cm i.d . column of sephadex lh-20 eluted with meoh  h2o ( 9:1 ) , with 275 drop fractions retained in each collection 
 tube . on the basis of their uv absorption at 220 nm , seven fractions 
 ( a  g ) were collected by combining tubes 1217 , 1820 , 
 21/22 , 23/24 , 25/26 , 27/28 , and 2945 , respectively . among 
 these , fractions d , e , and f exhibited colon cancer cytotoxicity . fraction d ( 30.4 mg ) was further purified by hplc onto a 250  
 10 mm phenomenex luna 5  c18 ( 2 ) 100  column . the detector wavelength was set to 220 nm , and the solvent flow rate 
 was 2.5 ml / min . , running isocratic for 10 min and then followed by a linear 
 gradient of 50% to 80% meoh ( 0.1% tfa ) over 
 20 min to yield compounds 3 ( 5.1 mg ; tr = 17.3 min ) and 4 ( 3.6 mg ; tr = 25.6 min ) . fraction e ( 31.4 mg ) was purified 
 using the same c18 hplc column and almost identical conditions 
 to fraction d , except 
 that the starting elution composition was 55% meoh of the five fractions ( 15 ) that were collected , 
 only fractions 2 ( 8.5 mg ; tr = 23.3 min ) 
 and 4 ( 8.5 mg ; tr = 25.6 min ) were found 
 to be active in the colon cancer cell bioassay . fraction 4 was found 
 to be solely composed of compound 4 , while fraction 2 
 appeared to be a mixture of two closely related compounds . it was 
 then subjected to further hplc purification on the same c18 column using an isocratic elution with 55% meoh  h2o ( 0.1% tfa ) to yield the two new compounds 1 ( 2.3 mg ; tr = 23.3 min ) and 2 ( 4.2 mg ; tr = 25.1 min ) . pale yellow 
 oil ; [ ]d25 + 8.8 ( c 0.17 , meoh ) ; ir ( nacl ) 3450 , 2956 , 2833 , 
 2150 , 1651 , 1630 , 1455 , 1017 cm ; h 
 nmr ( 600 mhz , cd3od ) and c nmr ( 150 mhz , cd3od ) data , see table 1 ; hresims found m / z 425.3903 [ m + h ] ( calcd 
 for c29h49n2 , 425.3890 ) . pale yellow 
 oil ; [ ]d25 + 17.5 ( c 0.40 , meoh ) ; ir ( nacl ) 3440 , 2951 , 2085 , 
 1681 , 1670 , 1651 , 1450 , 1408 , 1205 , 1143 , 1018 cm ; h nmr ( 600 mhz , cd3od ) and c nmr ( 150 mhz , cd3od ) data , see table 1 ; hresims found m / z 483.3952 
 [ m + h ] ( calcd for c31h51n2o2 , 483.3945 ) . chromatography fractions 
 were made up in dmso and assayed in an in vitro cytotoxicity 
 assay employing the colo205 and km12 colon cancer cell lines to direct 
 purification . general experimental details of the 2-day antiproliferative 
 assay for testing in a 96-well format have been published previously . in the current study , the assay was modified 
 for use in a 384-well plate . in brief , cells were harvested and plated 
 ( 45 l ) at a seeding density of 3.0  10 cells 
 per well for the colo205 cell line and 2.5  10 cells 
 per well for the km12 cell line into a 384-well  assay plate  
 and then incubated for 24 h. dmso solutions of the test materials 
 ( 8 l ) were diluted 1:25 with medium ( 192 l ) and then 
 subjected to five 2:1 serial dilutions ( 100 l each ) on a 96-well 
 plate . duplicate 40 l aliquots of each sample concentration 
 were then transferred to a 384-well  dilution plate  , 
 which could accommodate the duplicate samples from two 96-well plates . a 5 l portion of each solution on the dilution plate was transferred 
 to the cell cultures on the 384-well assay plate to give a final volume 
 of 50 l and a dmso concentration of 0.4% . cells were incubated 
 for 48 h at 37 c in the presence of the test samples and then 
 treated with the tetrazolium salt xtt ( 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2h - tetrazolium-5-carboxanilide ) . viable cells reduced the 
 xtt to a colored formazan product , and after an additional 4 h incubation 
 period the amount of formazan produced was quantified by absorption 
 at 450 nm , using a 650 nm reference . pure compounds were tested in 
 the assay described above and in the nci 60 cell assay .
bioinformatic analysis of data from 
 the nci-60 cell cytotoxicity 
 screen revealed a subset of extracts that showed selective cytotoxic 
 activity toward human colon carcinoma cell lines . 
 bioassay - guided 
 fractionation of a colon cancer selective extract from a philippines 
 collection of the marine sponge corticium niger provided 
 two new steroidal alkaloids , plakinamines n ( 1 ) and o 
 ( 2 ) , along with two known compounds of the plakinamine 
 class ( 3 , 4 ) 
 . the structures of these compounds 
 were elucidated by interpretation of combined ms and nmr spectroscopic 
 data . 
 plakinamines n ( 1 ) , o ( 2 ) , and j ( 4 ) were tested for antiproliferative activity in the nci-60 
 screen , and they showed enhanced inhibitory effects against all of 
 the colon cell lines with mean gi50 values of 11.5 , 2.4 , 
 and 1.4 m , respectively .