Patent Abstract:
investigation of the activity of extracts of the clam spisula polynyma has led to antitumour long - chain , straight - chain alkane or alkene compounds which have a 2 - amino group and a 3 - hydroxy group .

Detailed Description:
referring to fig1 in the l1210 assay some of the cells changed from being spherical ( fig1 a ) to ovoid with long points approximately 180 ° c . apart ( fig1 b ). several other forms have also been observed in assays of these extracts , including cells with points not 180 ° c . apart ( fig1 c ), cells with more than two points ( fig1 d ), cells with a bulge ( fig1 e ) and cells with a bulge replacing one of the points ( fig1 f ). however , the form with two sharp , opposing points was by far the predominant and characteristic one observed . this type of morphological change had not previously been observed during the screening of over 1000 marine extracts . for this invention , spisula polynyma were collected , at a depth of − 110 feet , from a clam bed on the eastern edge of stellwagon bank which is located off the coast of new england , stretching from near gloucester , mass ., north to maine . they were shipped live by the new england clam corporation ( formerly new dawn seafoods , inc .) and then immediately frozen . a purification scheme similar to the extraction procedure described above for the original testing of the bioactivity was employed . first 35 clams were thawed and the shells removed to give 1 . 9 kg ( wet wt ). these were allowed to stand in 3 : 1 methanol / toluene and filtered after several hours . this step was repeated followed by homogenization and extensive extraction with this same solvent to give a crude extract . to this was added a 1 m sodium chloride solution which caused the extract to separate into two layers . the lower aqueous layer was further extracted with toluene and the toluene layers combined . the resulting aqueous layer was then extracted with dichloromethane as shown in fig2 . the toluene extract was partitioned between methanol and hexane . the cytotoxicity and cellular alteration were observed almost exclusively in the methanol fraction . the methanol extract thus obtained was applied to a silica flash column , eluting with a chloroform / methanol step gradient ( 100 : 0 , 99 : 1 , 95 : 5 , 90 : 10 , 85 : 15 , 80 : 20 , 70 : 30 , 50 : 50 , 0 : 100 ). the main cytotoxic and pointed - cell - forming activity eluted off the column very late , although earlier fractions did show some cytotoxicity , but no pointed cells . this late eluting was further purified by flash silica chromatography , using 8 : 12 : 1 : 1 chloroform / 1 - butanol / acetic acid / water . fractions were neutralized with sodium bicarbonate before removing the solvent to prevent possible decomposition when they were concentrated in acid . this resulted in a series of three bioactive fractions . it had been observed in earlier attempts at isolation that the bioactivity did not wash off of a cyano solid - phase extraction ( spe ) column with methanol , but the cytotoxicity was found to elute with 3 : 1 methanol / 0 . 01 m ammonium formate ( 0 . 5 ml / min ). this was confirmed by chromatographing a small amount of a bioactive fraction on a cyano hplc column with this same solvent system and then repeating the injection under the same conditions except replacing the ammonium formate solution with water . the chromatograms appeared identical except that a peak eluting at 15 . 6 min was only observed in the first . the three bioactive fractions from the second silica column were each further purified by cyano hplc with the same conditions used above ( except 1 ml / min ) to give three series of bioactive fractions . the ammonium formate was removed by passing the sample through a c - 18 spe column , washing first with water and then eluting with methanol . the main cytotoxicity and morphology - changing activity of each series ( fractions a , b , and c ) was found in a peak comparable to that discussed above . however the activity was spread throughout most of the fractions . silica tlc ( 3 : 12 : 2 : 2 chloroform / 1 - butanol / acetic acid / water ) indicated that fraction a ( 0 . 4 mg ) contained one spot ( r f 0 . 47 ), which was pink by ninhydrin . fraction b ( 1 . 3 mg ) showed this same spot as well as one slightly lower ( r f 0 . 44 , red by ninhydrin ), while fraction c ( 0 . 2 mg ) contained both of these and a third one ( r f 0 . 34 , purple by ninhydrin ). all three showed good cytotoxicity and pointed - cell forming activity , with a exhibiting slightly more activity than b and significantly more than c . this indicated that the uppermost tlc spot must be from compound ( s ) which caused the morphological change in l1210 cells . these fractions were not purified further , but analyzed as mixtures . quantitative bioassay results are discussed below . an attempt was made to determine if a particular organ of spisula polynyma contained most or all of the bioactivity . a live clam was anesthetized with diethyl ether and then dissected into nine parts : foot , digestive system , gonads , siphon , gills , heart , mantle , adductor muscles , and the remainder of the visceral mass ( with foot , digestive system and gonads removed ). these were identified by comparison to illustrations of other clams . each organ was homogenized in 3 : 1 methanol / toluene and the resulting extract was then triturated with dichloromethane and methanol to remove salts . while all of the extracts showed cytotoxicity ( table ), only those from the gills and the gonads exhibited strong morphology - changing activity . that from the digestive system and the remainder of the visceral mass also showed weak pointed - cell forming activity , possibly due to incomplete separation from the gonads . the lack of pointed - cell - forming activity in other organs may have resulted either from a lack of 1 - 3 or from a much lower concentration . in another experiment , one foot that had been cooked for a brief period was extracted in an analogous manner . this also showed cytotoxicity , but no morphology - altering activity . however , when a larger sample of cooked material was more extensively extracted , some pointed cells were observed in the l1210 assay . silica tlc ( 3 : 12 : 2 : 2 chloroform / 1 - butanol / acetic acid / water , 100 mg ) of the extracts of the digestive system and gonads showed a weak ninhydrin - positive spot at r f 0 . 49 . 250 μg / ml 125 μg / ml 50 μg / ml organ % inhibition % pointed a % inhibition % pointed a % inhibition % pointed a foot 100 0 , ad b 100 0 , ad 93 0 , 0 digestive 96 0 , 18 62 0 , 0 0 0 , 0 system gonads nr c nr 99 56 , 100 90 32 , 100 siphon 100 ad , ad 50 0 , 0 0 o , 0 gills 100 ad , ad 100 50 , ad 98 100 , 93 heart 100 ad , ad nr 0 , 0 38 o , 0 mantle 100 0 , 0 99 0 , 0 95 o , 0 adductor 100 added 100 0 , 0 95 o , 0 muscles visceral 100 10 , ad 100 2 , ad 94 o , 0 mass cooked 100 0 100 0 97 o foot d cooked 91 21 25 0 0 o foot e several clues to the structure of the bioactive compounds could be found in the isolation procedure . the tlc spot which correlated with the activity visualized as pink or red by ninhydrin , suggesting that the compounds contained primary amines . also , they exhibited amphiphilic character . they were originally extracted into toluene from aqueous methanol , but they then partitioned into methanol versus hexane . while they are soluble in nonpolar solvents , they require a very polar solvent ( 3 : 12 : 2 : 2 chloroform / 1 - butanol / acetic acid / water ) to be eluted from silica . only fractions a and b were reasonably pure from inactive contaminants as shown by tlc . most of the structure determination studies were carried out on fraction b because of its size relative to the others . fig3 and 4 show the 1 h nmr spectra of this fraction in cdcl 3 and cd 3 od , respectively . what was immediately obvious in these spectra was a peak corresponding to a long methylene chain ( 1 . 25 ppm ) and several overlapping terminal methyl groups ( 0 . 87 ppm ). other peaks were not as well defined . no peaks corresponding to aromatic protons were observed , but several peaks appeared in the alkene proton region . several others seemed to correspond to protons attached to heteroatom - substituted carbons . the major difference between the spectra in the two different solvents was that , in cd 3 od , a methyl doublet ( 1 . 21 ppm ) downfield of the terminal methyl groups were clearly observed , while in cdcl 3 this resonance appeared only as an upfield shoulder on the methylene chain peak . an authentic sample of d - trans - erythro - sphingosine ( 4 ) was obtained from sigma for comparison with the isolated material . the 1 h nmr spectrum thereof was similar in many respects to that of fraction b . as expected , 4 exhibited a large peak due to the long methylene chain ( 1 . 25 ppm ), a terminal methyl group ( 0 . 87 ppm ) and two vinyl protons ( 5 . 75 and 5 . 46 ppm ). of particular note was the broadness of the resonances corresponding to protons on the heteroatom - substituted carbons ( 4 . 40 , 3 . 66 , 2 . 85 and 2 . 18 ppm ). also , on silica tlc ( 3 : 12 : 2 : 2 chloroform / 1 - butanol / acetic acid / water ), 4 had r f 0 . 43 and appeared red by ninhydrin , like the lower spot in fraction b and the middle spot in c . palmeta and pro { overscore ( s )} tenik have reported that 2 - amino - 3 - octadecanol and 4 exhibited very similar r f values ( 0 . 32 and 0 . 29 , respectively ) when eluted on paper impregnated with silicic acid with the solvent system di - isobutyl ketone / acetic acid / water ( 40 : 25 : 5 ). fractions a - c were also studied by several mass spectrometric methods . the largest ion in all of the spectra was m / z 286 . high resolution measurement of this peak ( m / z 286 . 3109 ) allowed the assignment of the molecular formula c 18 h 40 no ( δ0 . 1 mmu ) to spisulosine 285 ( 1 ). this compound derived its name , in part , from its molecular weight . this molecular formula indicated that the molecule is totally saturated . a strong peak corresponding to the loss of water from this m + h ion was observed at 268 . 3019 ( d − 1 . 5 mmu ). thus , 1 must contain a hydroxyl group . ions corresponding to matrix adducts of m / z 286 were observed at m / z 438 . 3078 ( c 22 h 48 no 3 s 2 , d − 0 . 2 mmu ), 590 , and 592 . one well - known primary metabolite that , like 1 , consists of an 18 carbon chain substituted with hydroxyl and amine functionalities is sphingosine ( 4 ). this compound has one more oxygen and two less hydrogens than 1 . the analogy appeared valid because high resolution measurement of m / z 300 for the spisulosines indicated that it was a doublet corresponding to the m + h of a higher homologue ( 2 ) of m / z 286 ( 300 . 3270 , c 19 h 42 no , ?− 0 . 4 mmu ), together with sphingosine ( 4 ) itself ( 300 . 2914 , c 18 h 38 no 2 , d − 1 . 1 mmu ). this also helped to explain the presence of alkene protons in the 1 h nmr spectrum . several other peaks were evident in all three spectra . the ion at m / z 314 was also a doublet corresponding to c 20 h 44 no ( 314 . 3439 , d − 1 . 6 mmu ), which was the molecular ion of another homologue of 1 , spisulosine 313 ( 3 ), and c 19 h 40 no 2 ( 314 . 3075 , d − 1 . 6 mmu ) which was a homologue of sphingosine ( 5 ). compound 4 showed matrix adducts of the m + h ion at m / z 452 . 2885 ( c 22 h 46 no 4 s 2 , d − 1 . 7 mmu ), 604 . 2831 ( co 26 h 54 no 6 s 4 , d 0 . 3 mmu ) and 606 . 2995 ( c 26 h 56 no 6 s 4 , d 3 . 6 mmu ), 5 exhibited matrix adducts of the m + h ion at m / z 464 . 2888 ( co 23 h 46 no 4 s 2 , d − 2 . 0 mmu ) and 618 . 2940 ( c 27 h 56 no 6 s 4 , d 5 . 1 mmu ). it should be noted that , while m / z 300 and 314 were doublets of nearly equal intensity in fraction b , only one peak was measurable for the matrix adducts listed here from fraction b . this suggested that these two series of compounds , although very similar in general structure , behaved differently in fabms . the spisulosine series ( saturated ) gave strong molecular ions and weaker matrix adducts , while the reverse was observed for the sphingosine series ( unsaturated ). to better establish the structures identified by the data discussed above , several derivatives were prepared . the most informative was the diacetyl derivative of spisulosine 285 ( 8 ). because fraction b was the largest , a portion of it was acetylated with acetic anhydride in pyridine . this mixture of acetyl derivatives will be referred to here as acb . by silica tlc ( 3 : 12 : 2 : 2 chloroform / 1 - butanol / acetic acid / water ), the reaction appeared quantitative , with a new spot appearing at r f 0 . 86 . for comparison , the triacetyl derivative of authentic 4 ( 9 ) was also synthesized by the same method . two series of compounds related to the spisulosines have been previously isolated . gulavita and scheuer reported that a xestospongia sp . sponge from papua - new guinea contained two epimeric 14 - carbon amino alcohols 134 and 135 . these were not isolated as the free amines , but rather the mixture was acetylated to give both the mono - ( 136 , 137 ) and diacetyl compounds ( 138 , 139 ) which were then separated . jimenez and crews have isolated several molecular ion of the underivatized 1 at m / z 286 . this m + h ion ( m / z 370 ) fragmented to give m / z 310 and 268 , presumably by losing acetic acid and then the second acetyl group , respectively . the comparable ions for the other spisulosines were small , but present : m / z 384 , 324 and 282 for the diacetyl derivative of 2 ( 144 ), and m / z 398 , 338 and 296 for the diacetyl derivative of 3 ( 145 ). the ions from the sphingosine in the sample were too small to state definitively that they were present . this again showed that the two series of compounds had very different ionization potentials . the cims spectrum showed strong m / z 370 and 310 ions , but here the m / z 268 ion was very weak . the higher homologues were again seen at m / z 384 and 324 for 144 , and m / z 398 and 338 for 145 . weak ions at m / z 426 and 366 were indicative of 133 . to confirm the structure and determine the stereochemistry of spisulosine 285 the compound was synthesized . none of the isomers of 2 - amino - 3 - octadecanol were previously known as natural products , but both the 2 s , 3 s and 2s , 3r isomers have previously been synthesized . the higher homologues are novel compounds . a modified version of the synthesis of pro { overscore ( s )} tenik and alaupovic ( scheme ix ) was used to obtain the authentic material for comparison . first , dibenzyl malonate ( 147 ) was alkylated with tetradecyl bromide ( 148 ). the resulting dibenzyl tetradecylamalonate ( 149 ) was then condensed with n - phthaloyl - l - alanyl chloride ( 150 ) to give 2 - phthalimido - 3 - octadecanone ( 151 ) after removal of the benzyl groups and decarboxylation . this ketone was treated with excess sodium borohydride , which resulted in the reduction of one of the phthalimido carbonyls in addition to the ketone , producing both 152 , which had one phthalimido carbonyl reduced to the carbinolamine , and 153 , which was further reduced . these two products could be readily separated from each other by silica flash chromatography . the reduction of 151 to 152 produced a mixture of four diastereomers because of the formation of two new chiral centers . at this point , the diastereomers were separated by cyano hplc . the protecting group was then removed from each by further reduction with sodium borohydride followed by acetic acid . as one stereocentre was removed with the protecting group , this resulted in the production of two diastereomers . since this synthesis started with l - alanine , the two products were ( 2s , 3s )- 2 - amino - 3 - octadecanol ( 154 ) and ( 2s , 3r )- 2 - amino - 3 - octadecanol ( 155 ). while the spisulosines were quite simple compounds , as illustrated in fig1 a - 1 f , they exhibited a very unusual type of bioactivity . as discussed above , the spisulosines caused a distinct morphological change in l1210 leukemia cells , in addition to cytotoxicity . this bioactivity , which was recorded as the percentage of living cells in which altered morphology was observed , could be observed sometimes as early as 13 h after the start of the assay and reached a maximum at 50 - 60 h , after which it decreased . generally 60 cells were observed to determine this number , except in assays in which less than this number of cells remained alive . the morphological effect was usually measured 30 - 35 h after the start of the assay and again about 24 h later , while the cytotoxicity was determined when the number of cells in the controls reached approximately 8000 , usually in 3 days after the assay was begun . it should be noted that the pointed cells were live cells and that they were counted as such for the cytotoxicity reading . also , assays in which 100 % cytotoxicity was recorded may still have contained live cells (& lt ; 0 . 5 %) which may or may not have been pointed . all morphologically - changed cells were counted in the pointed cell percentage . this change in morphology was always observed in fractions with fairly high cytotoxicity . generally , no significant number of pointed cells were observed in assays with less than 70 % growth inhibition . however , assays in which the cytotoxicity approached 100 % often had lower percentages of cells with altered morphology than those with 90 - 98 % growth inhibition . this suggested that the altered cells might be more easily killed . it is unknown whether the cytotoxicity and the morphology change resulted from the same mechanism of action . in one instance , pointed cells from an assay were recultured and found to revert to the normal state . this suggested that the effect was reversible after the compound had been metabolized . acetylation drastically reduces the bioactivity . to determine if the change in morphology of l1210 cells was caused by sphingosine ( 4 ) or related compounds , several authentic compounds were obtained and assayed against l1210 cells . both sphingosine and stearylamine ( 131 ) exhibited moderate cytotoxicity , but no morphological effect . sphingomyelins are well - known derivatives of 4 in which a phosphoryl choline unit has been added to the primary alcohol and the amine is acylated by a fatty acid . a mixture of sphingomyelins isolated from bovine brain ( sigma ), which consisted mainly of stearoyl and nervonoyl sphingomyelins ( 161 , 162 ), showed minimal cytotoxicity and no pointed cells . the cytotoxicity of the phosphorylcholine derivative of 4 ( 163 , sigma ) may be , at least , partially due to hydrolysis of 163 to 4 . cytotoxicity of model compounds concentration % % pointed compound ( μg / ml ) inhibition cells 128 5 100 0 2 . 5 100 0 1 75 0 0 . 5 31 0 0 . 25 13 0 0 . 1 0 0 161 + 162 50 7 0 25 0 0 10 0 0 131 5 99 0 2 . 5 96 0 1 19 0 0 . 5 0 0 0 . 25 0 0 0 . 1 0 0 163 50 88 0 25 50 0 10 38 0 sphingosine and other long - chain amines , including stearylamine , are known to be cytotoxic . this bioactivity , as measured against chinese hamster ovary ( cho ) cells , has been shown to be maximal for 18 - carbon homologues . all four stereoisomers of sphingosine were found to be almost equally active . reduction of the double bond of 4 to produce dihydrosphingosine ( 164 ) did not affect the cytotoxicity . addition of an n - methyl group to 164 also caused no significant change in the bioactivity , while acylation of the amine caused a large decrease in the cytotoxicity . no cytotoxicity was reported for the related compounds ( 134 , 135 , 140 - 142 ) which have been isolated from other marine sources , however , they may not have been tested in this type of assay . a mixture of 134 and 135 was active against c . albicans ( 8 - mm zone of inhibition for 19 mg of a mixture of the two ). xestaminol a was reported to exhibit weak activity against several gram - positive and gram - negative bacteria and fungi . it also showed antihelminthic activity against nippostrongylus brasiliensis . both 140 and 142 showed some activity against reverse transcriptase . the activity of fractions a - c , the acetyl derivative of fraction b and compounds 154 and 155 is summarized in the table . the assay results clearly confirmed the nmr analysis assigning 155 , not 154 , as the same as 125 . also , acetylation drastically reduces the bioactivity table ix bioactivity of fractions a - c , acb , and 154 and 155 concentration % % pointed cells a / b sample ( μg / ml ) inhibitiion c time 1st 2nd fraction a 2 . 5 100 35 , 59 ad ad 1 . 25 100 25 ad 0 . 5 90 42 45 0 . 25 85 45 55 0 . 125 75 8 35 0 . 05 19 0 0 fraction b 2 . 5 100 35 , 59 0 7 1 . 25 93 3 21 0 . 5 90 2 43 0 . 25 80 7 37 0 . 125 75 5 21 0 . 05 7 0 0 fraction c 2 . 5 90 55 0 1 . 25 88 0 0 . 5 63 0 acb 10 31 27 0 5 38 0 2 13 0 1 0 0 0 . 5 0 0 0 . 2 0 0 154 5 100 27 0 2 . 5 100 0 1 63 0 0 . 5 0 0 0 . 25 0 0 0 . 1 0 0 155 5 100 27 ad 2 . 5 100 22 1 100 64 0 . 5 99 56 0 . 25 96 40 0 . 1 63 33 the bioactivity of the spisulosines may be due to their similarity to sphingosine . in the nomenclature of sphingolipids , spisulosine 285 would be considered 1 - deoxysphinganine . the spisulosines may compete with sphingosine for binding sites or be incorporated into sphingolipids such as sphingomyelins , ceramides or gangliosides . in either case , the spisulosines could disrupt the cellular functions controlled by these compounds . sphingosine and its derivatives are involved in the regulation of cell growth and differentiation . sphingosine is a potent inhibitor of protein kinase c , competing with diacylglycerol for the binding site , which may explain its cytotoxicity . structure - activity studies have shown that this inhibition requires a positively charged amine and thus n - acyl derivatives were inactive . if the spisulosines act by competing with sphingosine , this would explain the relative lack of activity of the acetylated compounds ( acb ). there is growing evidence that sphingosine may act as a second messenger by regulating protein kinase c activity . it has also been shown to inhibit the differentiation of hl - 60 cells treated with phorbol 12 - myristate - 13 - acetate , a known protein kinase c activator . the spisulosines should be tested for inhibition of protein kinase c . it is unknown whether inhibition of this enzyme could cause the morphological effects observed for the spisulosines , but protein kinase c is involved in the control of cell growth and differentiation . nmr spectra were obtained on general electric gn 500 and qe 300 and varian u400 spectrometers . samples for nmr analysis were dissolved in cdcl 3 or cd 3 od . chemical shifts ( d ) are reported in ppm downfield of tetramethyl - silane ( tms ) and referenced to the residual solvent peak or tms . low and high resolution fabms spectra were recorded on either a vg zab - se or a vg 70 - se4f spectrometer , using a 3 : 1 mixture of dithiothreitol - dithioerythritol ( magic bullet ) as the matrix . fabms / ms spectra were recorded on a vg 70 - se4f with the same matrix , using helium as the collision gas . ci mass spectra were recorded on a vg vse spectrometer , operating in the alternating ci / ei mode with methane as the reagent gas . ir spectra were obtained on an ibm ir / 32 ftir spectrometer . optical rotations were measured on a jasco dip - 370 digital polarimeter . hplc was carried out using an alltech econosphere cyano column ( 4 . 6 × 250 mm , 5 mm particle size ). the hplc system used consisted of a beckman model 114m pump , a rheodyne 71 injector and either an isco v 4 or beckman 165 variable wavelength detector or a waters 990 photodiode array detector . analytical thin layer chromatography ( tlc ) was performed on a pre - coated silica gel ( merck 60 f - 254 ) and cyano bonded - phase ( em science cn f 2548s hptlc ) plates . spots were visualized by uv ( 254 nm ), ninhydrin ( 5 % in ethanol ), phosphomolybdic acid ( 5 % in ethanol ) and / or iodine . silica column chromatography was carried out on either 50 - 200 mm or 40 - 63 mm silica gel ( merck ). other column chromatography used chromatorex ods ( fuji - division 100 - 200 mesh ) and sephadex lh - 20 ( pharmacia ). high speed countercurrent chromatography ( hsccc ) was performed on an ito multi - layer coil separator - extractor ( p . c ., inc .) with a # 10 coil and a milton - roy mini - pump . solid phase extraction ( spe ) was carried out on normal phase ( silica , alltech maxi - clean ), reversed - phase ( c - 18 , waters sep - pak ), and bonded - phase ( cn , fisher prepsep ) columns . cytotoxicity assays against l1210 murine lymphocytic leukemia cells were performed by dissolving the samples in methanol and / or hexane were applied to the dry assay wells and the solvent was allowed to evaporate . cells ( 1000 ) were added in minimum essential medium ( mem , 1 ml ) and incubated 37 ° c . inhibition of growth was recorded as the estimated percentage of living cells in sample wells versus that in control wells . this was measured when the control wells reached 8000 cells , generally three days after the start of the assay . morphologically - changed cells ( fig1 a - 1 f ) were counted as living cells when determining the percent inhibition of growth . morphological changes were assessed throughout the assay period . the percentage of pointed cells was determined by counting the number of altered cells in approximately 60 living cells . this percentage varied with the length of time the assay had been running . it generally reached its maximum about 50 hours after the start of the assay , but pointed cells could be observed as early as 13 hours after the start of the assay and could usually still be seen when the percent growth inhibition was measured . the percentages of pointed cells were often counted both after about 35 and after 55 hours . the time that this measurement was made is indicated with the data . antimicrobial assays were performed using the filter disk diffusion method . paper disks ( 6 . 35 or 12 . 7 mm , schleicher & amp ; schuell ) were impregnated with samples ( 50 - 500 μg ) in solution and allowed to dry . these disks were then placed on agar seeded with either bacillus subtilis , penicillium melinii ( formerly p . atrovenetum ), micrococcus luteus ( formerly sarcina lutea ), escherichia coli or saccharomyces cerevisiae . these plates were incubated for 12 - 24 h ( 32 - 35 ° c ., except p . melinii , 25 - 27 ° c .). one clam ( spisula polynyma ) was thawed and the shell removed ( 35 . 32 g , wet wt ). this was placed in a blender with 350 ml of 3 : 1 methanol / toluene and homogenized . the yellow - brown extract was filtered and added to a 1m sodium chloride solution ( 100 ml ). the upper toluene layer was removed and the aqueous layer extracted with toluene ( 75 ml ). the two toluene layers were combined and the solvent was removed to give a brown oily residue ( 333 . 9 mg ). the aqueous layer was further extracted with dichloromethane ( 2 × 75 ml ), which gave a yellow - brown residue ( 18 . 6 mg ) after removal of the solvent . the aqueous layer was then extracted with ethyl acetate ( 75 ml ). the lower phase was the organic layer due to the presence of some dichloromethane which had remained in the aqueous phase after the last step . the upper layer was further extracted with the ethyl acetate ( 245 ml ), the upper organic layer back - extracted with water ( 100 ml ), and the two ethyl acetate extracts were combined to give a yellow residue ( 36 . 8 mg ) after removal of the solvent . the combined aqueous layers were concentrated by one - half and extracted twice with 1 - butanol ( 150 ml , 75 ml ). the combined butanol layers were back - extracted with water ( 75 ml ), resulting in a yellow residue ( 132 . 8 mg ) after removal of the butanol . the combined aqueous layers were concentrated to give an oily light yellow residue ( 946 . 1 mg ). each extract was triturated with dichloromethane and methanol to remove salts to give the toluene ( 302 . 2 mg ), dichloromethane ( 18 . 6 mg ), ethyl acetate ( 36 . 7 mg ), butanol ( 120 . 9 mg ) and aqueous ( 590 . 4 mg ) extracts which were assayed . thirty - five clams were thawed and the shells removed to give a sample of spisula polynyma ( 1 . 9 kg ) which was soaked in methanol / toluene ( 3 : 1 , 2 × 1 . 5 l ). the solids were then ground in the same solvent ( 6 × 1 . 5 l ) and the resulting extracts filtered . a 1 m solution of sodium chloride ( 3 l ) was added to this crude extract ( 12 l ) and the resulting upper toluene layer removed . the aqueous layer was further extracted with toluene ( 2 × 2 . 5 l ), followed by dichloromethane ( 4 × 2 . 5 l ) as shown in fig1 . after removal of the solvent , the toluene extract ( 21 . 55 g ) was partitioned between methanol and hexane ( 1 . 5 l each ). the methanol layer was further extracted with hexane ( 4 × 1 l ). the combined hexane layers were concentrated to about 1 . 8 l and both extracts chilled (− 10 ° c .). the two layers which resulted in each case were separated . the combined hexane layers were then extracted with methanol ( 0 . 5 l ). this process resulted in a hexane and three methanol extracts of which the first methanol extract ( 6 . 8 g ) contained the most bioactivity . this bioactive methanol fraction was separated by flash silica chromatography employing a chloroform / methanol step gradient ( 100 : 0 , 99 : 1 , 95 : 5 , 90 : 10 , 85 : 15 , 80 : 20 , 70 : 30 , 50 : 50 , 0 : 100 ) to give 12 fractions . while the third , fourth , seventh and eighth fractions possessed some cytotoxicity , they showed no pointed - cell forming activity . this activity was found in the last two fractions along with most of the cytotoxicity . these two fractions were combined ( 370 mg ) and further purified by another flash silica column , using chloroform / 1 - butanol / acetic acid / water ( 8 : 12 : 1 : 1 ). to remove the acetic acid , each of the 12 fractions thus obtained was neutralized by ( a ) adding chloroform ( one - quarter volume ), ( b ) washing with 5 % sodium bicarbonate until the ph of the aqueous layer was above 7 ( 2 - 3 × half volume ), and then ( c ) washing the organic layer with water ( half volume ). the third , and fourth and fifth fractions possessed all of the pointed cell - forming activity and essentially all of the cytotoxicity . each of these fractions was separately purified by hplc on a cyano column with 3 : 1 methanol / 0 . 01 m ammonium formate ( 1 ml / min ). six fractions , of which the most bioactive was the fifth , were collected from each silica fraction . the ammonium formate was removed from each fraction by adding water ( 2 - 8 ml ), applying the sample to an spe column ( c - 18 ), washing with water ( 5 - 10 ml ) and then eluting with methanol ( 5 ml ). this resulted in fractions a ( 0 . 4 mg , 2 × 10 − 5 % yield ), b ( 1 . 3 mg , 7 × 10 − 5 % yield ) and c ( 0 . 2 mg , 1 × 10 − 5 % yield ), from the third , fourth and fifth silica fractions , respectively , which all eluted at t r 7 . 9 min . white solid ; silica tlc ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o ) r f 0 . 47 ( ninhydrin - positive , pink ); ir ( nacl ) 2922 , 2853 , 1734 , 1593 , 1462 , 1377 , 1061 cm − 1 ; 1 h nmr ( cdcl 3 ) d 5 . 38 , 5 . 15 , 3 . 82 , 3 . 67 , 3 . 44 , 3 . 24 , 2 . 31 , 2 . 03 , 1 . 67 , 1 . 60 , 1 . 55 , 1 . 25 , 1 . 10 , 0 . 86 ; fabms m / z 606 , 604 , 592 , 590 , 466 , 452 , 438 , 314 , 300 , 286 , 268 ; cims m / z 354 , 340 , 338 , 328 , 326 , 324 , 314 , 312 , 310 , 300 , 298 , 296 , 286 , 284 , 268 , 266 , 149 , 139 , 137 , 1 , 123 , 111 , 109 , 97 , 95 , 85 , 83 , 71 , 69 , 59 , 57 , 55 . anal . calcd . for c 18 h 40 no : 286 . 3110 ( m + h ). found : 286 . 3115 ( hrfabms ). white solid ; silica tlc ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o ) r f 0 . 47 ( ninhydrin - positive , pink ), 0 . 44 ( ninhydrin - positive , red ); ir ( nacl ) 3273 , 2953 , 2918 , 2851 , 1639 , 1591 , 1510 , 1466 , 1379 , 1344 , 1059 , 970 cm − 1 ; 1 h nmr ( cdcl 3 ) d 5 . 98 , 5 . 78 , 5 . 55 , 5 . 44 , 5 . 32 , 4 . 43 , 3 . 78 , 3 . 65 , 3 . 24 , 2 . 15 , 2 . 08 , 2 . 00 , 1 . 95 , 1 . 70 , 1 . 44 , 1 . 25 , 1 . 19 , 0 . 87 ; fabms m / z 6 . 18 . 2940 , 616 , 606 . 2955 , 604 . 2831 , 592 , 590 , 480 , 466 , 464 . 2888 , 452 . 2885 , 438 , 314 . 3439 , 314 . 3075 , 300 . 3273 . 300 . 2914 , 286 , 268 ; cims m / z 354 , 352 , 342 , 340 , 338 , 328 , 326 , 324 , 314 , 312 , 310 , 300 , 298 , 296 , 286 , 284 , 282 , 280 , 268 , 266 , 219 , 193 , 179 , 165 , 149 , 137 , 123 , 111 , 109 , 97 , 95 , 85 , 83 , 71 , 69 , 59 , 57 , 55 . white solid ; silica tlc ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o ) r f 0 . 47 ( ninhydrin - positive , pink ), 0 . 44 ( ninhydrin - positive , red ), 0 . 34 ( ninhydrin - positive , purple ); ir ( nacl ) 2924 , 2853 , 1593 , 1456 , 1352 , 1063 , 972 cm − 1 ; fabms m / z 620 , 618 , 616 , 606 , 604 , 602 , 466 , 464 , 452 , 438 , 314 , 300 , 298 . 2741 , 296 , 286 , 280 , 268 ; cims m / z 354 , 352 , 340 , 338 , 336 , 328 , 326 , 324 , 322 , 314 , 312 , 310 , 308 , 300 , 298 , 296 , 294 , 292 , 286 , 284 , 282 , 280 , 278 , 268 , 179 , 165 , 149 , 137 , 135 , 1 , 123 , 121 , 111 , 109 , 97 , 95 , 85 , 83 , 81 , 7 1 , 69 , 60 , 59 , 57 , 55 . twenty - two s . polynyma claims were thawed and the shells removed to give 1 . 3 kg of the organism ( wet wt ). this was placed in waring blender with 3 : 1 methanol / toluene ( 1 . 5 l ) and ground into a thick slurry which was filtered through a layer of celite . the solid residue was further extracted ( 4 × 1 . 5 l ) and filtered in a similar manner . the remaining solids were then placed in 5 : 1 methanol / toluene ( 750 ml ) and allowed to soak for 36 h , before filtering . to the combined filtrates ( 7 . 8 l ) was added 1 m sodium chloride ( 2 l ). after removal of the upper toluene layer , the aqueous phase was extracted with toluene ( 2 × 1 . 5 l ) and dichloromethane ( 3 × 1 . 5 l ). the remaining aqueous phase was concentrated by one - half and extracted with ethyl acetate ( 2 × 1l ). the resulting aqueous layer was diluted with water ( 2 l ) and extracted twice with 1 - butanol ( 1 . 5 l , 1 l ). removal of the solvents and trituration with dichloromethane and methanol resulted in the toluene ( 14 . 1 g ), dichloromethane ( 0 . 75 g ), ethyl acetate ( 1 . 3 g ), 1 - butanol ( 0 . 2 g ) and aqueous ( 1 . 9 g ) extracts which were assayed . the toluene extract was partitioned between hexane and methanol ( 750 ml each ). the resulting methanol layer was further extracted with hexane ( 2 × 750 ml , 2 × 500 ml ). the hexane layers were combined and concentrated to about 3 l and then both extracts were chilled (− 10 ° c .) which caused each to separate into two layers . the combined methanol layers were concentrated in vacuo to give a brown residue ( methanol extract 1 , 536 g ). the hexane layers were further concentrated to about 1 l and back - extracted with methanol ( 500 ml ). the solvent was removed from each of these to give the methanol extract 2 ( 4 . 26 g ) and the hexane extract ( 4 . 52 g ). a portion of the first methanol extract ( 594 mg ) was separated by hsccc , using hexane / ethyl acetate / methanol / water ( 4 : 7 : 4 : 3 , mp = up ) at 4 ml / min . this gave 12 fractions of which the third , fourth and fifth contained most of the bioactivity . these three fractions were combined ( 158 mg ) and chromatographed on sephadex lh - 20 , eluting with methanol . this resulted in eight fractions of which the fourth ( 8 . 4 mg ) possessed the majority of the biological activity . this bioactive fraction was further purified by hplc on a cyano column with 3 : 1 methanol / 0 . 01 m ammonium formate ( 0 . 5 ml / min ). eight fractions were collected and the ammonium formate was removed from each by adding water ( 2 - 8 ml ), applying the sample to an spe column ( c - 18 ), washing with water ( 5 - 10 ml ) and then eluting with methanol ( 5 ml ). the seventh fraction ( t r . 15 . 8 min , white amorphous solid , 0 . 3 mg , 2 × 10 − 4 % yield ) proved to contain the bioactive compounds and is referred to here as fraction d . silica tlc ( 1 - buoh / acoh / h 2 o , 4 : 1 : 5 , upper layer ) showed four spots by phosphomolybdic acid visualization : r f 0 . 53 ( major ), 0 . 35 ( major ), 0 . 31 ( minor ), and 0 . 19 ( minor ). the inactive sixth fraction showed all the same spots except rf 0 . 53 . the fabms spectrum of fraction d showed intense peaks at m / z 286 . 3019 , 300 . 3270 and 268 . 3019 , and weaker peals at m / z 314 , 438 , 452 , 464 , 590 , 592 , 669 , 797 , 809 and 825 . the last three ions listed were also observed in most of the other hplc fractions and appeared to correspond to the tlc spot at r f 0 . 35 . anal . calcd . for c 18 h 40 no : 286 . 3110 ( m + h ). found : 286 . 3109 ( hrfabms ). a second portion of the first methanol extract described above ( 633 mg ) was subjected to hsccc . the solvent system employed was hexane / methanol / water ( 5 : 4 : 1 , up = mp , 5 ml / min ), which gave poor stationary phase retention . this resulted in 10 fractions with the bioactivity spread throughout most of them . the first three fractions ( 310 mg ) were combined and further purified by hsccc using hexane / ethyl acetate / methanol / water ( 4 : 7 : 4 : 3 , lp = mp , 2 ml / min ) to give 12 fractions . the second to fifth fractions ( 85 mg ), containing the majority of the bioactivity , were chromatographed on a c - 18 flash column , eluting with a methanol / water / chloroform step gradient ( 90 : 10 : 0 , 95 : 5 : 0 , 100 : 0 : 0 , 95 : 0 : 5 , 90 : 0 : 10 , 50 : 0 : 50 ). this gave 10 fractions which were all bioactive . the fourth to sixth fractions from the first hsccc run were combined with a side fraction from the sephadex lh - 20 column discussed under fraction d ( 270 mg ). this material was subjected to hsccc , using the same conditions as the second run just described except that the flow rate was 3 ml / min . this resulted in nine fractions of which the second and third contained most of the cytotoxicity and cell - altering activity . these two fractions were combined ( 42 mg ) and separated on a flash c - 18 column , using a methanol / water step gradient ( 80 : 20 , 90 : 10 , 95 : 5 , 100 : 0 ). this resulted in 12 fractions of which the eighth to eleventh showed morphology - altering activity and cytotoxicity . all but the first and fifth fractions from the first c - 18 column were combined with the eighth to eleventh fractions from the second ( 50 . 4 mg ) and separated by preparative silica tlc with chloroform / 1 - butanol / acetic acid / water ( 3 : 12 : 2 : 2 ). the plate was divided into eight fractions , which were scraped off and eluted with methanol . the residue from each fraction after removal of the solvent was triturated with dichloromethane and filtered . the fraction second from the top of the plate ( rf 0 . 80 - 0 . 42 ) contained the bioactive material and is referred to as fraction e ( 5 . 7 mg ). analytical silica tlc of fraction e , eluting with the same solvent system , showed a single spot by ninhydrin visualization ( r f 0 . 44 ), but phosphomolybdic acid spray regent showed other material which streaked throughout the middle third of the plate . the fabms spectrum of fraction b showed m / z 286 as the major peak , with lesser peaks at m / z 268 , 300 , 438 , 452 , and 592 . a third portion of the first methanol extract ( 468 mg ) was separated by flash silica chromatography , using the solvent system chloroform / 1 - butanol / acetic acid / water ( 8 : 12 : 1 : 1 ). to remove the acetic acid , each of the 10 fractions thus obtained was neutralized by ( a ) adding water ( half the volume of the fraction ) and separating the two phases , ( b ) extracting the aqueous layer with chloroform ( half volume × 2 ), ( c ) washing the combined organic layers with 5 % sodium bicarbonate until the ph of the aqueous layer was above 7 ( 2 to 3 × half volume ), and then ( d ) washing the organic layer with water ( half volume ). the third fraction ( 24 mg ), which possessed the majority of the bioactivity , was chromatographed on sephadex lh - 20 , eluting with methanol , to give eight fractions . the sixth fraction ( 2 . 3 mg ) was separated by repeated hplc , using the same conditions as for the separation of fraction a - c . the ammonium formate was removed as for fraction a - c . the fraction eluting at t r 8 . 1 min was the most biologically active and is referred to as a fraction f . it was so small that an accurate weight could not be obtained , but probably was 100 - 200 μg ( approximately 1 to 2 × 10 − 4 % yield ). the fractions eluting later than this one also showed both cytotoxic and pointed cell - forming activity , although less potent . this suggested that either the bioactive compound ( s ) did not elute as a well - defined peak or that different homologues eluted at different times , but were not well separated . silica tlc ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o ) showed one ninhydrin - positive spot at r f 0 . 44 . the later eluting fractions also showed this same spot , but less intense . the fabms spectrum of fraction f shows ( in decreasing order of intensity ) m / z 286 , 268 , 300 , 314 , 344 , 438 , 452 , 592 , 669 . a live clam was placed in a container with about 10 ml of diethyl ether and chilled ( 4 ° c .) for 20 h . it was dissected into nine organs : foot , digestive system ( including the stomach , intestines and crystalline style sac ), gonads , siphon , gills , heart , mantle , adductor muscles , and the remainder of the visceral mass . each organ was first soaked in methanol / toluene ( 3 : 1 , 10 ml / g sample ) and then homogenized in a virtis blender . the extracts were filtered and the solvent was removed . the residue was triturated with dichloromethane and methanol to give 155 mg ( foot ), 60 mg ( digestive system ), 147 mg ( gonads ), 10 l mg ( siphon ) 65 mg ( gills ), 2 . 5 mg ( heart ), 168 mg ( mantle ), 101 mg ( adductor muscles ) and 252 mg ( visceral mass ). in a separate experiment , one foot that had been cooked was extracted in an analogous fashion ( 189 mg ). a larger sample of cooked clams ( 483 g ) was more extensively extracted by first soaking in 3 : 1 methanol / toluene ( 3 × 500 ml ) and then homogenizing the sample in the same solvents ( 5 × 500 ml ). a small sample of the combined extracts was evaporated and redissolved in methanol for assaying . optical rotations were measured on a jasco dip - 370 digital polarimeter , with a 3 . 5 × 50 mm 1 ml cell . melting points were taken on a thomas hoover capillary melting point apparatus . 1 h and 13 c nmr were recorded on a varian unity - 400 or unity - 500 spectrophotometer . chemical shifts are reported in ppm relative to the solvent ( 7 . 26 , cdcl 3 and 3 . 30 , cd 3 od ). high resolution ( hrfab ) and fast atom bombardment ( fab ) mass spectra were recorded on a vg zab - se or a 70 se4f mass spectrometer . tlc was done on merck silica gel 60 thin - layer plates . chromatographic separations were done by flash chromatography using 230 - 400 mesh merck silica gel . all moisture sensitive reactions were run in oven - dried glassware under an atmosphere of n 2 . solvents were distilled prior to use : thf from benzophenone ketyl , ch 2 cl 2 from cah 2 other solvents used were reagent grade . to a 300 ml round bottom was added 20 ( 10 . 0 g , 71 . 6 mmol ), benzyl bromide ( 25 . 73 g , 150 . 4 mmol ), k 2 co 3 ( 9 . 90 g , 71 . 6 mmol ) and ch 3 cn ( 172 ml ). the mixture was stirred at 60 ° c . until the reaction was complete by tlc . the reaction was cooler to room temperature and the solid was separated by filtration . the filtrate was concentrated in vacuo to give an oil which was purified by flash chromatography on silica gel ( 9 : 1 hexane / etoac ) to give a colorless oil : [ a ] 25 d = 113 . 6 ( c 1 . 2 , chcl 3 ); 1 h nmr ( 400 mhz , cdcl 3 ) d 1 . 35 ( d , 3h , j = 7 . 1 hz ), 3 . 53 ( q , 1h , j = 7 . 0 hz ), 3 . 65 ( d , 2h , j = 1 . 38 hz ), 3 . 75 ( s , 3h ), 3 . 85 ( d , 2h , j = 13 . 8 hz ), 7 . 22 - 7 . 42 ( m , 10h ); 13 c nmr ( 100 mhz ) d 14 . 9 , 51 . 1 , 54 . 3 , 56 . 0 , 2 . 8 , 4 . 1 , 4 . 5 , 139 . 1 , 175 . 1 ; fabms m / z 284 . 1 ( m + h ), 282 . 1 ( m − h ), 224 . 2 ( m - cooch 3 ); hrfabms calcd for c 18 h 22 no 2 m r 284 . 165 . 1 ( m + h ), found m , 284 . 1650 . to a suspension of lialh 4 ( 550 mg , 14 . 5 mmol ) in thf ( 20 ml ) a solution of 30 ( 910 mg , 3 . 21 mmol ) in thf ( 2 ml ) was added dropwise . the solution was stirred for 15 minutes and then heated to 65 ° c . for 3 hours . the reaction was cooled to 0 ° c . and quenched with 0 . 1 n hcl . the reaction was filtered through celite and the celite washed with thf ( 2 × 15 ml ) and the solvent removed in vacuo . flash chromatography on silica gel ( 4 : 1 hexane / etoac , r f = 0 . 30 ) gave 750 mg ( 92 % yield ) of a colorless solid : mp 40 - 41 ° c . ( from hexane ) literature mp 40 - 41 ° c . ( from hexane ) see , stanfield et al ., j . org . chem . 1981 , 49 , 4799 - 4800 ; [ a ] 25 d =+ 86 . 6 ( c 1 , chcl 3 ) literature [ a ] 23 d =+ 88 . 2 ( c 1 , chcl 3 ); 1 h nmr ( 500 mhz cdcl 3 ) d 0 . 98 ( m , 3h ), 2 . 98 ( m , 1h ), 3 . 13 ( m , 1h ), 3 . 35 ( m , 3h ), 3 . 45 ( m , 1h ), 3 . 81 ( m , 2h ), 7 . 19 - 7 . 41 ( m . 10h ); 13 c nmr ( 1 mhz ) d 8 . 6 , 52 . 9 , 54 . 1 , 62 . 7 , 3 . 2 , 4 . 5 , 5 . 0 , 5 . 3 ; fabms m / z 256 . 2 ( m + h ), 22 4 . 2 ( m - ch 2 oh ); hrfabms calcd for c 17 h 22 no m r 256 . 1701 ( m + h ), found m r 256 . 1702 . dry dmso ( 0 . 53 ml , 7 . 43 mmol ) was added to a stirred solution of oxalyl chloride ( 0 . 31 ml , 3 . 6 mmol ) in ch 2 cl 2 ( 7 . 5 ml ) at − 78 ° c . the solution was allowed to stir 15 minutes followed by the addition of 40 ( 740 mg , 2 . 90 mmol ) in ch 2 cl 2 ( 7 . 5 ml ). after 30 minutes , et 3 n ( 1 . 0 ml , 7 . 2 mmol ) was added and allowed to warm to room temperature . the solution was extracted with saturated . nahco 3 ( 20 ml ) and the aqueous layer was extracted with ch 2 cl 2 ( 2 × 15 ml ). the organic layer was washed with saturated . nacl solution , dried with mgso 4 an concentrated in vacuo at room temperature to give 720 mg ( 98 %) yield ) of a yellow oil which became a solid when cooled to − 20 ° c . the aldehyde was used without further purification . mp 52 - 54 ° c ., literature mp 55 . 5 ° c . see , dix et al ., arch pharm ( weinheim ) 1995 , 328 , 203 - 205 . ; [ a ] 20 d =− 36 . 0 ( c 1 , chcl 3 literature [ a ] 20 d =− 35 . 1 ( c 1 , etoac ); 1 h nmr ( 400 mhz , cdcl 3 ) d 1 . 19 ( d , 2h , j = 7 . 0 hz ), 3 . 34 ( q , 1h , j = 7 . 0 hz ), 3 . 58 ( d , 2h , j = 13 . 7 hz ), 3 . 74 ( d , 2h , j = 13 . 7 hz ), 7 . 26 ( m , 2h ), 7 . 33 ( m , h ), 7 . 42 ( m , 4h ), 9 . 74 ( s , 1h ); 13 c nmr ( 100 mhz ) d 6 . 7 , 54 . 9 , 62 . 8 , 3 . 3 , 4 . 4 , 4 . 8 , 139 . 1 , 204 . 6 ; fabms m / z 408 . 2 ( m + mb ), 254 . 2 ( m + h ), 22 . 2 ( m − cho ); hrfabms calcd for c 17 h 20 no m r 254 . 1545 ( m + h ), found m r 254 . 1545 . mg ribbon ( 237 mg , 9 . 75 mmol ), dibromoethane ( 16 μl , 0 . 189 mmol ) in thf ( 160 μl ) were added to a two neck flask fitted with a reflux condenser . a ½ ml of a 1 - bromopentadecane solution ( 970 mg , 3 . 33 mmol , 3 . 25 ml thf ) was added . after the reaction had started the remainder was added dropwise . to the grayish solution , 50 ( 105 mg , 0 . 413 mmol ) in thf ( 0 . 5 ml ) was added dropwise . the reaction was allowed to stir overnight followed by the addition of h 2 o ( 5 ml ) and 0 . 1 n hcl until the solution became clear . the mixture was extracted with etoac ( 3 × 10 ml ). the organic layer was washed with 5 % nahco 3 then saturated . nacl solutions and dried with mgso 4 . the solvent was removed in vacuo to give an oil - solid mixture ( 750 mg ). the crude material was purified by flash chromatography on silica ( 8 : 1 hexane / etoac , r f = 0 . 34 ) to give 120 mg of a solid . this solid was further purified by hplc on silica ( 93 : 7 hexane / etoac ) to give a colorless waxy solid ( 94 . 3 mg , 49 % yield ): [ a ] 25 d =+ 16 . 3 ( c 1 , chcl 3 ); 1 h nmr ( 500 mhz , cdcl 3 ) d 0 . 88 ( t , 3h , j = 7 . 0 hz ), 1 . 10 ( d , 3h , j = 6 . 7 hz ), 1 . 16 - 1 . 41 ( bm , 26h ), 1 . 56 ( m , 1h ), 1 . 69 ( m , 1h ), 1 . 79 ( m , 1h ), 2 . 72 ( quin , 1h , j = 6 . 7 hz ), 3 . 47 ( d , 2h , j = 13 . 8 hz ), 3 . 60 ( m , 1h ), 3 . 76 ( d , 2h , j = 13 . 8 hz ), 7 . 22 ( m , 2h ), 7 . 30 ( m , 4h ), 7 . 34 ( m , 4h ); 13 c nmr ( 1 mhz ) d 8 . 67 , 14 . 11 , 22 . 68 , 25 . 90 , 29 . 35 , 29 . 61 , 29 . 64 , 29 . 68 , 29 . 69 , 31 . 91 , 34 . 27 , 54 . 79 , 57 . 26 , 73 . 65 , 2 . 89 , 4 . 25 , 4 . 77 , 140 . 17 ; fabms m / z 465 ( m + h ), 448 ( m − h 2 o ), 464 ( m − h ), 388 ( m − ph ), 224 ( m − c 16 h 33 o ); hrfabms calcd for c 32 h 52 no m r 466 . 4049 ( m + h ), found m r 466 . 4037 . the assignment of the 2s , 3r configuration is based on comparison of the chemical shifts of the benzyl protons in 60 to literature values for the syn and anti diastereomers of 2 -( n , n - dibenzylamino )- 3 - pentanol . the anti isomer has a chemical shift difference of 0 . 29 ppm and the syn is 0 . 52 ppm . comparison of other syn - anti pairs show the range for the syn isomer to be 0 . 44 to 0 . 54 ppm and the anti 0 . 05 to 0 . 29 ppm . the value for 60 is 0 . 29 ppm . to a 15 ml round bottom was added 60 ( 88 . 2 mg , 0 . 189 mmol ) in meoh ( 2 ml ) and 20 % pd ( oh ) 2 - c ( 11 mg ). the mixture was stirred under 1 atmosphere of hydrogen overnight . the catalyst was removed by filtration through a 25 mm syringe filter ( 0 . 2 μm nylon membrane ) and the filter was washed with 4 ml of meoh . the solvent was then removed in vacuo to give 51 . 50 mg of a white solid . the product was purified by chromatography over a 6 ml lc - si spe tube ( 90 : 10 ch 2 cl 2 / meoh followed by 100 % meoh ) to give 49 . 47 mg ( 92 % yield ) of a white solid : mp 66 - 67 ° c . ; [ a ] 26 d =+ 24 . 9 ( c 1 , chcl 3 ); 1 h nmr ( 500 mhz , cd 3 od ) d 0 . 89 ( t , 3h , j = 7 . 0 hz ), 1 . 05 ( d , 3h , j = 6 . 6 hz ), 1 . 20 - 1 . 56 ( bm , 31h ), 2 . 81 ( qd , 1h , j 1 = 6 . 6 hz , j 2 = 3 . 8 hz ), 3 . 42 ( dt , 1h , j 1 = 8 . 8 hz , j 2 = 3 . 8 hz ); 13 c nmr ( 1 mhz ) a 14 . 60 , 16 . 82 , 23 . 90 , 27 . 40 , 30 . 65 , 30 . 90 , 30 . 95 , 30 . 96 , 33 . 23 , 34 . 13 , 52 . 33 , 76 . 16 ,; fabms m / z 286 . 3 ( m + h ), 268 . 3 ( m − oh ), hrfabms calcd for c 18 h 40 no m r 286 . 3110 ( m + h ), found m r 286 . 3109 . a mixture of diastereomers of 3 - hydroxy - 2 -( 1 - methyl - 2 - 2 - hydroxy - heptadecyl )- isoindolin - 1 - one ( 152 , 22 mg ) were separated by cyano hplc with hexane / 2 - propanol ( 98 : 2 , 1 ml / min ) to give four compounds ( 152a - 152d ). the purity of each peak was determined by reinjection on hplc . anal . calcd . for c 26 h 44 no 3 : 418 . 3321 ( m + h ). found : 418 . 3321 hrfabms ). 152a : 4 . 2 mg ; t r 13 . 3 min ; 1 h nmr ( cdcl 3 ) d 7 . 77 ( 1h , d , 7 . 3 ), 7 . 58 ( 2h , m ), 7 . 50 ( 1h , m ), 5 . 91 ( 2h , s ), 4 . 51 ( 1h , m ), 3 . 78 ( 1h , m ), 1 . 58 ( 2h , m ), 1 . 40 ( 3h , d , 7 . 1 ), 1 . 24 ( 26h , m ), 0 . 87 ( 3h , t , 6 . 5 ); fabms m / z 418 , 400 ; relative ratio of diastereomers 17 : 1 : 0 : 0 ( 152a : 152b : 152c : 152d ). 152b : 13 . 7 mg ; t r 13 . 9 min ; 1 h nmr ( cdcl 3 ) d 7 . 70 ( 1h , d , 7 . 3 ), 7 . 54 ( 2h , m ), 7 . 47 ( 1h , m ), 5 . 88 ( 2h , s ), 4 . 37 ( 1h , m ), 3 . 85 ( 1h , m ), 1 . 52 ( 2h , m ), 1 . 27 ( 3h , d , 7 ), 1 . 25 ( 26h , m ), 0 . 87 ( 3h , t , 6 . 5 ); fabms m / z 41 , 400 ; relative ratio of disastereomers 1 : 6 . 8 : 0 : 0 ( 152a : 152b : 152c : 152d ). 152c : 1 . 4 mg ; t r 20 . 0 min ; 1 h nmr ( cdcl 3 ) d 7 . 78 ( 1h , d , 7 . 3 ), 7 . 59 ( 2h , m ), 7 . 51 ( 1h , m ), 5 . 93 ( 2h , s ), 4 . 12 ( 1h , m ), 3 . 99 ( 1h , m ), 1 . 58 ( 2h , m ) 1 . 37 ( 3h , d , 7 . 0 , 1 . 25 ( 26h , m ), 0 . 87 ( 3h , t , 6 . 5 ); fabms m / z 418 , 400 ; relative ratio of diastereomers 0 : 2 . 5 : 45 : 1 ( 152a : 152b : 152c : 152d ). 152d : 1 . 5 mg ; t r 21 . 7 min ; 1 h nmr ( cdcl 3 ) d 7 : 77 ( 1h , d , 7 . 3 ), 7 . 59 ( 2h , m ), 7 . 51 ( 1h , m ), 5 . 86 ( 2h , s ), 4 . 12 ( 1h , m ), 3 . 90 ( 1h , m ), 1 . 58 ( 2h , m ), 1 . 45 ( 3h , d , 6 . 6 ), 1 . 24 ( 26h , m ), 0 . 87 ( 3h , t , 6 . 5 ); fabms m / z 418 , 400 ; relative ratio of diastereomers 0 : 1 : 2 : 21 ( 152a : 152b : 152c : 152d ). each diastereomer was separately deprotected by the method of osby et al . each isomer was dissolved in 2 - propanol / water ( 6 : 1 , 0 . 1 m for 152a and 152b , 0 . 7 m for 152c and 152d ). sodium borohydride ( 5 - 10 equivalents ) was added to each solution , which was then stirred at 25 ° c . for 24 h . each solution was then adjusted to ph 4 . 5 with acetic acid and stirred at 80 ° c . for an additional 24 h . ammonium formate was added to bring the ph of each solution to above 7 and then the solvent was removed from each by a stream of nitrogen . the residue from each was applied to a silica spe column , which was first washed with hexane : 2 - propanol ( 9 : 1 ) and then the product eluted with 2 - propanol . 1 h nmr indicated that 152a and 152d produced 154 ( 1 . 15 mg , 40 %, and 0 . 48 mg , 47 % respectively ), while 152b and 152c produced 155 ( 3 . 35 mg , 42 %, and 0 . 38 mg , 40 %, respectively ). 154 : white solid ; silica tlc ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o ) r f 0 . 48 ( ninhydrin - positive , pink ), ir ( nacl ) 2919 , 2851 , 1563 , 1466 , 1406 , 758 cm − 1 ; fabms m / z 438 , 286 , 268 , 85 , 70 , 69 , 57 , 55 , 44 . anal . calcd . for c 18 h 40 no : 286 . 3110 ( m + h ). found 286 . 3115 ( hrfabms ). 155 : white solid ; silica tlc ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o ) r f 0 . 50 ( ninhydrin - positive , pink ); ir ( nacl ) 3281 , 2917 , 2849 , 1568 , 1520 , 1470 , 1412 cm − 1 ; fabms m / z 438 , 286 , 268 , 85 , 70 , 69 , 57 , 55 , 44 , anal . calcd . for c 18 h 40 no : 286 . 3110 ( m + h ). found : 286 . 3109 ( hrfabms ). a portion of fraction b ( 560 μg ) dissolved in acetic anhydride ( 200 pl ) and pyridine ( 400 μl ) and was stirred at 25 ° c . for 4 . 5 h , at which time no starting material could be observed by tlc . the solvent was removed by a stream of nitrogen to give acb : off - white solid ; silica tlc r f 0 . 86 ( 3 . 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o , phosphomolybdic acid ), 0 . 65 ( 9 : 1 chcl 3 / meoh , phosphomolybdic acid ); ir ( nacl ) 2922 , 2853 , 1741 , 1651 , 1547 , 1460 1371 , 1234 , 1022 , 970 cm − 1 ; fabms m / z 370 , 310 , 268 ; cims m / z 426 , 424 , 412 , 410 , 398 , 384 , 370 , 368 , 364 , 338 , 324 , 310 , 165 , 149 , 139 , 1 , 121 , 111 , 97 , 86 , 61 , 57 , 55 . anal . calcd . for c 22 h 44 no 3 : 370 . 3321 ( m + h ). found : 370 . 3326 ( hrfabms ). in a procedure similar to grode and cardellina d - erythro - sphingosine ( 4 , 2 mg , 6 . 7 μmol , sigma ) in acetic anhydride ( 1 ml ) and pyridine ( 2 ml ) was stirred at 25 ° c . for 4 . 5 h , at which time no starting material could be observed by tlc . the solvent was removed by a stream of nitrogen to give 133 : white solid ; silica tlc r f 0 . 86 ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o , phosphomolybdic acid ), 0 . 65 ( 9 : 1 chcl 3 / meoh , phosphomolybdic acid ); fabms m / z 580 , 426 , 366 , 306 , 264 ; cims m / z 468 , 454 , 426 , 424 , 394 , 366 , 364 , 306 , 264 , 144 , 85 , 84 , 83 , 61 . ( 2s , 3s )- 2 - amino - 3 - octadecanol ( 154 , 150 μg , 0 . 5 μmol ) in acetic anhydride ( 50 μl ) and pyridine ( 100 μl ) was stirred at 25 ° c . for 5 h , at which time no starting material could be observed by tlc . the solvent was removed by a stream of nitrogen to give 156 : white solid ; silica tlc r f 0 . 86 ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o , phosphomolybdic acid ); ir ( nacl ) 3286 , 2924 , 2853 , 1740 , 1653 , 1541 , 1456 , 1371 , 1238 cm − 1 ; fabms m / z 522 , 370 , 328 , 310 , 286 , 268 . anal . calcd . for c 22 h 22 no 3 370 . 3321 ( m + h ). found : 370 . 3326 ( hrfabms ). ( 2s , 3r )- 2 - amino - 3 - octadecanol ( 155 , 750 μg , 2 . 6 μmol ) in acetic anhydride ( 200 μl ) and pyridine ( 400 μl ) was stirred at 25 ° c . for 5h , at which time no starting material could be observed by tlc . the solvent was removed by a stream of nitrogen to give 157 : white solid ; silica tlc r f 0 . 86 ( 3 : 12 : 2 : 2 chcl 3 / 1 - buoh / acoh / h 2 o , phosphomolybdic acid ); ir ( nacl ) 3289 , 2917 , 2849 , 1728 , 1637 , 1545 , 1464 , 1369 , 1240 cm − 1 ; fabms m / z 522 , 370 , 328 , 310 , 286 , 268 . anal . calcd . for c 22 h 44 no 3 : 370 . 3321 ( m + h ). found : 370 . 3319 ( hrfabms ). a portion of fraction a ( 40 μg ) was dissolved in acetone ( 200 μl ) to which 0 . 1 n hydrochloric acid ( 20 μl ) was added . this solution was stirred at 25 ° c . for 24 h , after which the solvent was removed by a stream of nitrogen . fabms indicated that a small amount of the acetonide 146 was formed : m / z 592 , 452 , 438 , 326 . 3430 , 300 , 286 , 268 . anal . calcd . for c 21 h 44 no : 326 . 3423 ( m + h ). found 326 . 3430 ( hrfabms ). ( 2s , 3r )- 2 - amino - 3 - octadecanol ( 155 , 750 μg , 2 . 6 μmol ) was dissolved in dichloromethane ( 100 μl ) to which 1 , 1 ′- carbonyldiimidazole ( 0 . 85 mg , 5 . 3 μmol ) and triethylamine ( 0 . 4 μl , 2 . 9 μmol ) was added . the solution was stirred for 5 h and then the solvent removed by a stream of nitrogen . the crude product 158 was analyzed without purification : ir ( nacl ) 36 , 2919 , 2851 , 1742 , 1713 , 1551 , 1470 , 1395 , 1321 , 49 , 1239 , 1094 , 1061 , 1001 , 768 , 743 , 664 cm − 1 ; fabms m / z 785 , 623 , 474 , 406 , 362 , 328 , 312 , 286 , 268 . anal . calcd . for c 19 h - 38 no 2 : 312 . 2903 ( m + h ). found : 312 . 2903 ( hrfabms ). lysophosphatidic acid ( lpa ), antibodies against tubulin and phalloidin were all obtained from sigma . fluorescein - and texas red - labelled goat antimouse antibody were obtained from amersham ( u . k .). antibody raised against the rho protein was obtained from sta cruz biotechn . vero cells were grown in dulbecco &# 39 ; s modified eagle medium supplemented with 10 % foetal bovine serum . spisulosine or lpa were added to these cultures to a concentration of 0 . 2 - 1 . 0 mg and 50 - 10 mm respectively , from 4 to 24 hours . cells were counted with the drug exclusion haemocytometer procedure using a solution of 0 . 4 % trypan blue in hanks buffered saline ( celis and celis , “ general procedures for tissue culture in cell biology , a laboratory handbook ” academic press inc , vol 1 , pp . 5 - 17 .) vero cells were incubated with spisulosine 285 ( 0 . 5 mm ) for 4 hours . fig3 is a microphotograph for the results in example a . cell shaped was altered from polygonal ( untreated cells , panel a ) to a fusiform shape ( panel b ). panel c represents a higher magnification of the culture to which spisulosine was added . change in cell morphology is due to an effect on the cell microfilaments in order to identify the organisation of the microfilament and microtubule organisation in cells treated with spisulosine , cells were stained with phalloidin to detect actin polymers , and an antitubulin antibody to detect tubulin . vero cells were incubated in the presence ( panel b , d ) or absence ( a , c ) of 0 . 5 mm spisulosine for 4 hours . cells grown in coverslips were fixed with methanol at − 20 ° c . ( for tubulin antibody ) or with 4 % paraformaldehyde in phosphate buffered saline pbs ( w / v ) for phalloidin incubation . in the second case the cells were washed with 0 . 2 % triton x100 in pbs . the coverslips were washed with pbs and incubated for 1 hour at room temperature with the tubulin antibody ( diluted 1 / 1000 in pbs ) or with phalloidin ( 1 mg / ml ). after washing with pbs the coverslips incubated with the tubulin antibody were overlaid with fluorescein or texas red - labelled goat antimouse antibodies ( diluted 1 : 50 in pbs ). the coverslips were mounted with mowiol and stored in the dark at 4 ° c . until observation . [ 0160 ] fig4 is a microphotograph for the results in example b . panel ‘ a ’ represents cells stained with phalloidin ( actin stain ) and not treated with spisulosine . panel ‘ b ’ represents cells stained for phalloidin and treated with spisulosine . panel ‘ c ’ represents cells stained for tubulin and not treated with spisulosine . panel ‘ d ’ represents cells stained for phalloidin and treated with spisulosine . there is a dramatic decrease in actin in spisulosine - treated cells , in comparison with untreated cells . under the same conditions , the microtubule network remains in a polymerised form . the small gtp binding protein rho is involved in the formation of actin - myosin “ stress fibres ” ( hall , a ., science , 279 , 1998 , p 509 - 514 ). therefore , the electrophoretic mobility and cellular distribution of rho was analysed in cells treated with spisulosine . [ 0162 ] fig5 is an electrophoretogram of example c . in panel a , equivalent amounts of protein from a cell extract from untreated ( a ) or from 0 . 5 mm spisulosine treated ( 20 hour ) cells ( b ) were fractionated by gel electrophoresis and blotted onto nitrocellulose paper to analyse the amount of the rho protein . in panel b the experiment was carried out as above , except that the homogenate was fractionated into a particulate ( membrane , “ m ”) fraction and soluble (“ s ”) fraction . subcellular fractionation was carried out by placing cells in a hypotonic buffer ( 0 . 25 m sucrose , 20 mm hepes ph 7 . 4 , 2 mm edta , 1 mm pmsf , 10 mg / ml aprotinin , leupeptine and pestatine ), and lysing them with a dounce . the homogenate was first centrifuged at 750 g for 5 minutes to remove nuclei and unbroken cells , and the supernatant was further centrifuged at 30 , 000 g for 1 hour ( 4 ° c .) to isolate a pelleted particulate fraction ( putative membrane fraction ) and a supernatant . the different fractions were characterised by electrophoresis and western blotting using an antibody against the rho protein . no significant change in the amount or mobility of rho was observed on treatment of cells with spisulosine . however , a decrease in the proportion of rho associated with the particulate fraction was observed . lpa is known to increase the level of stress fibres in cells by activation of the rho protein . the effect of lpa on cells treated with spisulosine , and untreated cells was examined . vero cells were incubated in the absence ( a ) or presence ( b ) of 10 mm lpa for 2 hours , or in the presence ( c ) of 0 . 5 mm spisulosine for 20 hours , or in the presence ( d ) of first 10 mm lpa ( 2 hours ) and afterwards with 0 . 5 mm spisulosine for an additional 18 hours . [ 0168 ] fig6 is a microphotograph for the results in example d . panel b indicates the effect of lpa in increasing the level of actin . incubation of vero cells with spisulosine for 24 hours results in the appearance of rounded cells , see panel c . these cells detach from the culture dish and die . the addition of lpa prior to spisulosine prevents the morphological change promoted by spisulosine . spisulosine 285 was tested in in vivo studies against xenograft models of human - prostate cancer ( pc - 3 ) and human renal cancer ( mri - h - 121 ). these models use subcutaneously implanted solid human tumours that grow and increase in volume over time . the mean volume of tumour growth in control animals provides the basis for comparison . for active compounds the tumour growth is inhibited either completely (% t / c values & lt ; 1 %, or negative ), or partially (& gt ; 1 % t / c − 50 % t / c ). a level of activity that is less than 40 % t / c is considered statistically significant . the doses of spisulosine used were given at the maximum tolerated , non lethal dose ( mtd ), ½ mtd and ¼ mtd . delivery of the drug was by the intraperitoneal route . human prostate cancer pc - 3 total dose % compound ( mg / kg ) t / c day comments spisulosine 285 9 . 990 − 21 % 11 stasis ( complete remission ) spisulosine 285 5 . 010 − 1 % 11 stasis ( complete remission ) spisulosine 285 2 . 499 223 % 15 control 100 % 15 [ 0171 ] human mri - h - 121 renal cancer total dose compound ( mg / kg ) % t / c day comments spisulosine 285 9 . 990 28 % 11 inhibition ( partial remission ) spisulosine 285 5 . 010 35 % 11 inhibition ( partial remission ) spisulosine 285 2 . 499 43 % 15 control 100 % 15 spisulosine 285 is effective against both tumour types , significantly reducing the tumour size in the case of the human prostate cancer model pc - 3 at higher doses . spisulosine 285 reduces the growth of the human renal cancer , with effects continuing up to a few weeks after the last dose of the drug . an expanded in vitro screen was performed of spisulosine 285 against a series of different cell lines . the following data was obtained : cv - 1 therapeutic category line tumor ic50 index solid sk - hep - 1 liver 3 . 51e − 15 7863 panc - 1 pancreas 1 . 71e − 12 16 ht - 29 colon 2 . 56e − 12 11 786 - 0 renal 2 . 75e − 12 10 fadu pharnynx 4 . 99e − 12 6 hs 746t stomach 7 . 89e − 12 3 sk - ov - 3 ovary 1 . 40e − 11 2 mx - 1 mammary 3 . 89e − 11 1 ramos burkitts 4 . 82e − 11 1 p3hr1 burkitts 6 . 73e − 11 0 sw684 fibrosarcoma 1 . 05e − 09 0 lymphoma u - 937 lymphoma 1 . 96e − 11 1 h9 lymphoma 3 . 10e − 11 1 leukemia hl60 leukemia 8 . 50e − 12 3 arh77 leukemia 1 . 36e − 12 2 k562 leukemia 1 . 57e − 11 2 ccrf - sb leukemia 1 . 05e − 09 0 normal cv - 1 kidney 2 . 76e − 11 1 fibroblasts the range of ic 50 potencies against the tumour cell lines are from nanomolar , 1 . 05 e - 09 nm , to fentamolar , 3 . 51 e - 15 mm . it is exceptional to go beyond the nm and pm range to find a drug which has activity in the mm range . the activities against the solid tumours were generally 1 log more potent than against the leukemias and lymphomas . among the solid tumours , the most slow growing were the most sensitive , culminating with the very slow growing hepatoma sk - hep - 1 . the best therapeutic indices compared to the cv - 1 normal cell line were seen with the slow growing solid tumours , since the ic 50 potency ( 2 . 76 e - 11 ) was comparable to the leukemia / lymphomas . the solid tumour tis ranged from 1 - 20 units and the ti for the hepatoma was & gt ; 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