Patent Application: US-96068110-A

Abstract:
latrunculin derivatives are disclosed , as are anti - invasive and cytotoxic uses for latrunculins and latrunculin derivatives , and semisyntheses of latrunculin derivatives . the latrunculins and latrunculin derivatives are useful , for example , in treating cancers .

Description:
we prepared several semisynthetic derivatives of latrunculin a with diverse steric , electrostatic , hydrogen bond donor ( hbd ), and hydrogen bond acceptor ( hba ) properties . several c - 17 lactol hydroxyl or thiazolidinone nh - substituted latrunculin a derivatives were semisynthesized , compounds 2 - 14 . see fig1 ( scheme 1 ) and table 1 . analogs were designed to modulate binding affinity toward g - actin . examples of the reactions used in the semisynthesis were esterification , acetylation , and n - alkylation . alternative semisynthetic routes include reaction schemes proceeding via lactol hydroxy and n - substituted derivatives . the semisynthetic latrunculins have been tested for their ability to inhibit pyrene - conjugated actin polymerization , and for their antiproliferative and anti - invasive properties against mcf7 , mda - mb - 231 , and pc - 3 cells using mtt and invasion assays , respectively . the red sea sponge negombata magnifica was collected at the egyptian coast at hurghada in june to afford a high dry - weight yield of latrunculin a , ˜ 2 to 3 %. at this time of the year there are only low levels of latrunculin b ( 0 . 0 - 0 . 024 % dry weight yield ), which saved time , effort , and resources during purification , e . g ., through reverse phase chromatography or hplc chromatographic methods . collections from hurghada , safaga , and qusier in december gave high yields of latrunculin b , ˜ 2 % dry weight yield , with only low concentrations of latrunculin a ( 0 . 0027 - 0 . 0052 %). n . magnifica extract is subjected to vacuum liquid chromatography using n - hexane with increasing concentrations of ethyl acetate . latrunculin - containing fractions are identified by tlc , and subjected to mplc using isocratic 4 % meoh in chcl 3 . where necessary , hplc final purification is conducted semi - preparatively on a dionex summit iii hplc unit , with a phenomenex luna 250 × 10 mm , c18 column , and isocratic ch 3 cn — h 2 o ( 5 : 95 ) elution with uv detection at 235 nm . tlc analysis is carried out using the developing systems n - hexane - etoac ( 1 : 1 ) or chcl 3 - meoh ( 9 : 1 ). for column chromatography , si gel 60 ( particle size 63 - 200 μm ) or bakerbond octadecyl ( c 18 ), 40 μm , is used . reactions are monitored by tlc . after complete depletion of the starting material , the mixture is diluted with brine and extracted with chcl 3 . the dry extract is fractionated on si gel 60 using mplc . latrunculin a ( compound 1 ) and latrunculin b ( compound 20 ) were isolated from the red sea marine sponge negombata magnifica , and identified by detailed 1d and 2d nuclear magnetic resonance ( nmr ) studies and a comparison with literature descriptions . reaction of compound 1 with methanol or phenethyl alcohol in the presence of bf 3 - diethyl etherate ( et 2 o — bf 3 ) yielded the corresponding 17 - methoxy analog compound 2 and the phenethoxy derivative compound 3 ( scheme 1 , fig1 ). the configuration at c - 17 is maintained after acetalization of the c - 17 hydroxyl group , as confirmed by molecular modeling , nmr measurements , and circular dichroism measurements . the distance between h - 18 and the c - 17 methoxy group in derivative 2 was calculated ( using sybyl ) as 2 . 403 å , within the range for noe coupling . the h - 18 and c - 17 methoxy group showed space dipole - dipole coupling in noesy measurements . additionally , the 13 c chemical shift of c - 17 in compound 2 ( δ c 99 . 9 ) was almost identical to the reported value ( δ c 99 . 8 ). this observations indicated the preservation of the c - 17 configuration between the natural and semi - synthetic derivatives . high resolution mass spectrometry ( hrms ), 1 h nmr data , and 13 c nmr data for compound 3 indicated the presence of a 17 - o - phenethyl side chain . the downfield shift of the c - 17 carbon and h - 18 signals in compound 3 (+ 3 . 2 and 0 . 20 ppm , respectively ), compared with those for the starting material compound 1 , suggested possible etherification at c - 17 . the doublet of triplets of oxygenated methylene h 2 - 1 ′ ( δ h 3 . 81 , 3 . 51 ) showed cosy coupling with the benzylic h 2 - 2 ′ triplet ( δ h 2 . 89 ). protons h 2 - 1 ′ showed 3 j hmbc correlations with c - 17 and the aromatic quaternary carbon c - 3 ′ ( δ c 138 . 3 ). protons h 2 - 2 ′ showed 3 j hmbc correlation with the aromatic methine carbons c - 4 ′/ 8 ′ ( δ c 128 . 9 ). protons h - 4 ′/ h - 8 ′ showed cosy couplings with protons h - 5 ′/ 7 ′ and 3 j hmbc correlation with c - 6 ′ ( δ c 126 . 7 ). compound 4 was prepared by treating compound 1 with benzoic anhydride in chcl 3 in the presence of 4 - dimethylaminopyridine ( dmap ) as catalyst ( scheme 1 ). 1 h and 13 c nmr data indicated benzoylation at c - 17 . the hrms data of compound 4 suggested the molecular formula c 29 h 35 no 6 s . the aromatic double doublet h - 3 ′/ 7 ′ ( δ h 7 . 69 ) showed cosy coupling with protons h - 4 ′/ 6 ′ ( δ h 7 . 42 ) and 3 j hmbc correlation with the carbonyl carbon c - 1 ′ ( δ c 169 . 2 ). protons h - 4 ′/ 6 ′ in turn showed cosy coupling with h - 5 ′ ( δ h 7 . 53 ) and 3 j hmbc correlation with the aromatic quaternary carbon c - 2 ′ ( δ c 133 . 4 ). the downfield shift of h - 18 ( δ h 5 . 07 , & gt ;+ 1 . 00 ppm ) compared with that of the starting material compound 1 was possibly due to the anisotropic effect of the newly introduced c - 17 - o - benzoyl functionality . n - substitution of compound 2 was carried as shown in scheme 1 using nah in anhydrous thf at 0 ° c . and the corresponding alkyl and aryl halides to produce compounds 5 - 11 . the identity of compound 5 was confirmed by nmr data analysis , which showed the replacement of the thiazolidinone nh proton ( δ h 5 . 80 ) with an n - methyl singlet ( δ h 2 . 97 and δ c 36 . 7 ) and comparison with literature . similarly , the n - ethyl functionality in compound 6 was evident from the cosy coupling between the downfield nitrogenated methylene h 2 - 1 ′ ( δ h 3 . 71 and 3 . 35 ) and the methyl triplet h 3 - 2 ′ ( δ h 1 . 17 ). protons h 2 - 1 ′ also showed 3 j hmbc correlation with the carbonyl c - 20 , connecting the new ethyl group with the thiazolidinone ring . the hrms data of compound 7 suggested an additional degree of unsaturation . analysis of 1 h and 13 c data further confirmed the presence of a new n - cyclopentyl moiety . the methine quintet h - 1 ′ ( δ h 3 . 88 ) showed 3 j hmbc correlations with the thiazolidinone carbonyl carbon at δ c 171 . 5 and the methylene carbons c - 3 ′/ 4 ′ ( δ c 24 . 5 ). protons h 2 - 3 ′/ 4 ′ showed cosy coupling with both h 2 - 2 ′/ 5 ′ protons , which in turn cosy - coupled with h - 1 ′. 1 h and 13 c data for compound 8 suggested n - hydroxypropylation of the starting material 2 . the new methylene singlet h 2 - 1 ′ ( δ h 3 . 55 ) showed a 3 j hmbc correlation with c - 29 carbonyl ( δ c 170 . 2 ), connecting this moiety to the thiazolidinone ring . protons h 2 - 1 ′ also showed a 3 j hmbc correlation with the oxygenated methylene carbon c - 3 ′ ( δ c 59 . 2 ). the two chemically nonequivalent h 2 - 3 ′ at ( δ h 3 . 76 and 3 . 58 ) showed cosy coupling with h 2 - 2 ′ ( δ h 1 . 78 ), confirming the introduction of the new n - hydroxypropyl side chain . high resolution electrospray ionization mass spectrometry ( hresims ) of compound 9 suggested the molecular formula c 30 h 39 no 5 s , twelve degrees of unsaturation , and the n - benzylation of compound 2 . the downfield benzylic nitrogenate methylene protons h 2 - 1 ′ ( δ h 5 . 11 and 4 . 35 ) showed a 3 j hmbc correlation with c - 20 carbonyl ( δ c 170 . 2 ), connecting this moiety to the thiazolidinone ring . protons h 2 - 1 ′ also showed a 3 j hmbc correlation with the symmetric aromatic methine carbons c - 3 ′/ c - 7 ′ ( δ c 128 . 4 ). protons h - 3 ′/ 7 ′ showed a 3 j hmbc correlation with the aromatic methine carbon c - 5 ′ ( δ c 127 . 7 ), and cosy correlation with protons h - 4 ′/ 6 ′ ( δ h 7 . 34 ). the latter protons also showed cosy correlation with h - 5 ′ ( δ h 7 . 31 ) and a 3 j hmbc correlation with the quaternary aromatic carbon c - 2 ′ ( δ c 132 . 5 ). 1 h and 13 c nmr data for compound 10 were similar to those for compound 9 , with an n - benzoyl functionality replacing the n - benzyl of compound 9 . the aromatic methine protons h - 3 ′/ 7 ′ ( δ h 7 . 72 ) showed a 3 j hmbc correlation with the carbonyl carbon c - 1 ′ ( δ c 169 . 7 ). the benzoylation of the c - 17 lactol hydroxy in compound 4 and of the thiazolidinone nh in compound 10 resulted in a significant downfield shifting of proton h - 18 (+ 1 . 22 and + 1 . 49 ppm , respectively ) compared with that of compound 1 , possibly due to the anisotropic effect of the benzene ring and the carbonyl group . reaction of compound 2 with p - methoxy phenylacetyl chloride produced compound 11 , which was highly unstable in the reaction pot , with the amide bond rapidly degrading . we therefore shortened the reaction time to five minutes after reagent was added . the hresims data of compound 11 suggested the molecular formula c 32 h 41 no 7 s . 1 h and 13 c nmr data of compound 11 suggested there had been a successful n - p - methoxy phenylacetylation . the methoxy singlet h 3 - 9 ′ ( δ h 3 . 77 ) showed a 3 j hmbc correlation with quaternary aromatic oxygenated carbon c - 6 ′ ( δ c 159 . 8 ). the aromatic protons h - 4 ′/ 8 ′ ( δ h 7 . 21 ) showed 3 j hmbc correlations with c - 6 ′ and the benzylic methylene c - 2 ′ ( δ c 42 . 3 ). they also show cosy coupling with protons h - 5 ′/ 7 ′ ( δ h 6 . 81 ). proton singlet h 2 - 2 ′ ( δ h 3 . 79 ) showed 2 j hmbc correlations with the carbonyl carbon c - 1 ′ ( δ c 175 . 7 ) and the quaternary aromatic carbon c - 3 ′ ( δ c 126 . 3 ). proton h - 18 ( δ h 5 . 30 ) showed a 3 j hmbc correlation with c - 1 ′ carbonyl , connecting the new p - methoxy phenylacetyl moiety with the thiazolidinone ring . to explore the effect of n - substitution on the pharmacological activity of the unsubstituted c - 17 lactol group , compounds 6 and 9 were demethylated by heating with aqueous acoh to produce compounds 12 and 13 , respectively , with a free c - 17 lactol hydroxyl functionality ( scheme 1 ). compounds 12 and 13 showed 1 h and 13 c nmr spectra essentially identical to those of compounds 6 and 9 , respectively , but with the c - 17 methoxy replaced with a hydroxyl group . the d 2 o - exchangeable broad proton singlets at δ 4 . 01 and 3 . 91 were assigned as the new c - 17 hydroxy signals in compounds 12 and 13 , respectively . n - hydroxymethyl latrunculin a ( compound 14 ) was prepared as described in d . blasberger et al ., liebigs ann . chem . 1989 , 12 , 1171 - 1188 , to study the effect of extending the location of the only hbd in compound 1 ( the thiazolidinone nh ) by one carbon , and replacing it with a primary alcohol group ( see scheme 1 ). compound 1 reversibly binds cytoskeleton actin monomers , forming a 1 : 1 complex with g - actin , and disrupting its polymerization . compound 1 has shown antiangiogenic , antiproliferative , antimicrobial , and anti - migratory activities . compound 1 and the other latrunculins and derivatives described here will inhibit cancer and other diseases whose pathology is linked to the stability of the cytoskeleton actin . an actin polymerization kit was used to assess the direct actin - binding activity of each analogue at two concentrations : 100 nm and 1 μm . this kit is based on the enhanced fluorescence of pyrene - conjugated actin that occurs during polymerization . when pyrene - g - actin ( monomer ) forms pyrene - f - actin , fluorescence is enhanced . the enhanced fluorescence is used to follow polymerization over time . table 2 lists activities as the percentage of actin polymerization inhibition , compared to the negative vehicle control ( dmso ), along with calculated ic 50 . fig3 shows the time - course inhibition of actin polymerization by compounds 1 and 3 . compounds 3 and 14 were substantially more active than the parent latrunculin a ( compound 1 ) in inhibiting actin polymerization at both doses , while compounds 4 , 9 , 12 , and 13 were almost equipotent to 1 . this specific binding assay demonstrates that the antiproliferative and anti - invasive activities of latrunculin a and its derivatives are generally due to the disruption of actin polymerization , with some exceptions , such as compound 11 . the antiproliferative and anti - invasive activities of compounds 1 - 14 were quantified using mtt and cell invasion assay kits , respectively . latrunculin a ( compound 1 ) was used as a positive control in all biological assays , because it is known to be an actin polymerization inhibitor , with cytotoxic , antiproliferative , and anti - invasive activities . the 3 -[ 4 , 5 - dimethylthiazol - 2 - yl ]- 2 , 5 - diphenyl tetrazolium bromide ( mtt ) assay selectively detects living cells in a quantitative , colorimetric fashion , based on a living cell &# 39 ; s ability to reduce mtt to an insoluble , purple formazan dye . the mtt assay is routinely used to assess cytotoxicity and proliferation inhibition activities . the antiproliferative activities of compounds 1 - 14 were measured against two human mammary gland adenocarcinoma cell lines , mcf7 and mda - mb - 231 , and against human prostate cancer cells pc - 3 at five different doses ( 0 . 1 , 0 . 5 , 1 , 10 , and 50 μm ). the ic 50 values are shown in table 3 . while many of the semisynthetic latrunculins had similar activity levels , some had greater activity than compound 1 . in particular , compounds 3 and 14 were both more potent than compound 1 against both cell lines , with 4 . 8 and 4 . 0 - fold increases in the activity against mcf7 , respectively , and with noticeable cytotoxic effect at doses higher than 1 μm . although the mda - mb - 231 cells were more resistant than mcf7 against most latrunculin derivatives tested , compound 14 had potent activity against both cell lines . compounds 3 and 14 were both more potent than compound 1 against prostate cancer cells pc - 3 , with 3 . 2 and 10 . 8 - fold increases in activity , respectively . although derivative 10 was less active than compound 1 in the invasion assay , compounds 10 , 4 , and 9 were almost equipotent with the parent natural product 1 in the proliferation assay . the anti - invasive activities of compounds 1 - 14 were measured using a 96 well basement membrane extract ( bme ) cell invasion assay kit against the highly metastatic mda - mb - 231 cells . this assay employs a simplified boyden chamber with a polyethylene terephthalate membrane ( 8 μm pore size ). cell invasion was quantified using calcein - acetomethylester ( calcein - am ). cells internalize calcein - am ; intracellular esterases then cleave the acetomethylester moiety to generate free calcein . free calcein fluoresces brightly . this fluorescence may be used to quantify the number of cells that have invaded across the bme . the anti - invasive activities of compounds 1 - 14 at three different doses are shown in fig3 . to correlate the anti - invasive activities of the latrunculin a derivatives with their actin - disrupting activities , compounds 1 - 14 were each tested at non - toxic concentrations . except for compound 3 , the mtt assay showed no or insignificant toxicity at concentrations below 1 . 0 μm over a period of 72 hours . accordingly , all compounds were tested at 0 . 1 , 0 . 5 , and 1 . 0 μm . compound 3 killed 17 % of mda - mb - 231 cells at 1 . 0 μm , while the same dose of compound 3 inhibited 95 % of mda - mb - 231 cells &# 39 ; invasion . these observations showed a clear difference between the cytotoxic and anti - invasive activity levels . the anti - invasive activities of compounds 1 - 14 are attributed primarily to the inhibition of actin polymerization , not to cell death per se . derivatives 3 , 11 , 13 , and 14 were 3 . 3 , 4 . 6 , 3 . 1 , and 2 . 1 - fold more active , respectively , against cell invasion than latrunculin a ( compound 1 ) at 0 . 5 μm . 17 - o - phenylethyl - latrunculin a ( compound 3 ) was the most potent , with a 100 nm dose inhibiting mda - mb - 231 cell invasion , as compared to a 1 μm dose of compound 1 . upon the addition of compound 1 , 3 , or 14 to mda - mb - 231 cells in the upper assay chamber , rapid changes in cell morphology were seen ; actin filaments were disrupted , as characterized by remarkable cell deformity ( data not shown ). during cell migration , cytoskeletal actin dynamically reorganizes to impart the forces that cause cell migration . these observations suggest that the anti - invasive activities of compounds 1 , 3 , and 14 are mediated through direct disruption of actin cytoskeleton remodeling . while compounds 4 and 12 were equipotent to compound 1 at 1 and 0 . 5 μm doses , the others were less active . interestingly , the second most active derivative , n - p - methoxyphenylacetyl - 15 - o - methyl - latrunculin a ( compound 11 ) showed potent anti - invasive activity at a 100 nm dose ( fig2 ). this activity was nearly 4 - fold greater than that of compound 1 at the same dose , but without antiproliferative , cytotoxic , or cell shape modifying activities . although compound 11 was dramatically less active than compound 1 in the antiproliferation assay , it was nevertheless surprisingly potent in attenuating the metastasis of mda - mb - 231 cells . because compound 11 inhibits actin polymerization only weakly ( see table 2 ), compound 11 presumably has different target ( s ), target ( s ) other than the microfilament actin . n - substitution of compound 1 with ethyl and benzyl groups preserves the potency , with ic 50 values of 0 . 63 μm and 0 . 95 μm for compounds 13 and 12 , respectively , against mcf7 . mda - mb - 231 was more resistant , with ic 50 values of 2 . 72 μm for compound 13 , and 7 . 19 μm for compound 12 . latrunculin derivative 14 showed greater activity than the parent latrunculin a ( compound 1 ) by factors of 4 and 1 . 4 , respectively , against mcf7 and mda - mb - 231 cells in the proliferation assay ; and by a factor of 2 . 7 against mda - mb - 231 cells in the invasion assay ; all assessed at 1 μm . the pronounced activity of compound 14 may be due to improved direct actin binding via the more approachable hbd n - hydroxymethylene as compared to the thiazolidinone nh in compound 1 . optical rotation was measured on a rudolph research analytical autopol iii polarimeter . ir spectra were recorded on a varian 800 ft - ir spectrophotometer . 1 h and 13 c nmr spectra were recorded in cdcl 3 , using tms as an internal standard , on a jeol eclipse nmr spectrometer operating at 400 mhz for 1 h , and 100 mhz for 13 c . the hreims experiments were conducted on a micromass lct spectrometer . analytical hplc analyses were performed on a dionex ® summit ii system using a phenomenex luna 250 × 4 . 6 mm , rp - c - 18 column , isocratic elution ( 100 % meoh ), and uv detection set at 235 nm to verify the purity of each latrunculin compound . each of latrunculins 1 - 14 had a purity of & gt ; 98 %, except for analogue 9 , which had 91 % purity . tlc analysis was carried out on precoated si gel 60 f 254 500 μm tlc plates ( emd chemicals ), using the developing systems n - hexane - etoac ( 1 : 1 ) or chcl 3 - meoh ( 9 : 1 ). for column chromatography , si gel 60 ( emd chemicals , 63 - 200 μm ), fine si gel 60 ( em science , & lt ; 63 μm ), and c - 18 si gel ( bakerbond , octadecyl 40 μm ) were used . for sephadex lh - 20 column chromatography , n - hexane - chcl 3 ( 1 : 3 ), chcl 3 , and chcl 3 - meoh ( 9 : 1 ) solvent systems were used . general chemical reaction procedures . ( a ) acetalization of the c - 17 hydroxyl group of latrunculin a : to a solution of compound 1 in roh , bf 3 - diethyl etherate ( et 2 o — bf 3 ) was added . the mixture was stirred for 12 h at room temperature , and was neutralized with 10 % aq . nahco 3 solution . the solvent was evaporated , and the residue was extracted with chcl 3 and dried over mgso 4 . after solvent evaporation the residue was chromatographed on a si gel 60 column . ( b ) alkylation of the thiazolidinone nitrogen : a solution of compound 2 in dry thf was gradually added to a suspension of nah ( 60 % dispersion in mineral oil ) in dry thf at 0 ° c . the mixture was then stirred for 1 h at 0 ° c . alkyl halide was added and the mixture stirred at room temperature until compound 2 was completely depleted . ether and water were then added , the two layers were separated , and the organic layer was dried over na 2 so 4 . evaporation of the solvents left a residue , which was chromatographed on si gel 60 column . ( c ) demethylation of 17 - o - methyllatrunculin a derivatives : a solution of 17 - o - methyllatrunculin a analogue in acoh / h 2 o / thf ( 3 : 1 : 1 ) was stirred and heated to 60 ° c . the reaction was monitored by tlc until complete depletion of the starting material (˜ 0 . 5 - 1 h ). the mixture was then cooled to room temperature , neutralized with 10 % aqueous nahco 3 solution , and ether was then added . the upper organic layer was then washed with water , dried over anhydrous na 2 so 4 , and evaporated under reduced pressure . the residue was chromatographed on a si gel 60 column . 17 - o - methyllatrunculin a ( compound 2 ): compound 2 was prepared according to procedure a from compound 1 ( 200 mg , 0 . 72 mmol ), meoh ( 5 ml ), and et 2 o — bf 3 ( 0 . 22 ml , 1 . 77 mmol ). elution with n - hexane / etoac ( 8 : 2 ) afforded compound 2 ( 144 mg , 72 . 0 %). 17 - o - phenylethyllatrunculin a ( compound 3 ): compound 3 was synthesized according to the general procedure a from 1 ( 15 mg , 0 . 036 mmol ), phenethanol ( 1 ml , 7 . 131 mmol ), and et 2 o — bf 3 ( 15 μl , 0 . 117 mmol ). elution with n - hexane / etoac ( 8 : 2 ) afforded 3 ( 5 . 5 mg , 36 . 7 %): colorless oil , [ α ] d 25 + 34 . 5 ( c = 0 . 18 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . 17 - o - benzoyllatrunculin a ( compound 4 ): a solution of compound 1 ( 20 mg , 0 . 047 mmol ) in anhydrous chcl 3 ( 3 ml ) was stirred with benzoic anhydride ( 11 mg , 0 . 050 mmol ) and a catalytic amount of dmap for 12 hours at room temperature . the reaction mixture was neutralized with nahco 3 solution . the organic layer was washed with h 2 o ( 2 × 5 ml ), dried over anhydrous na 2 so 4 , and evaporated under reduced pressure . the reaction residue was chromatographed over si gel 60 using isocratic n - hexane / etoac ( 9 : 1 ) to afford compound 4 ( 4 . 7 mg , 23 . 5 %): colorless oil , [ α ] d 25 + 47 . 1 ( c = 0 . 25 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . 17 - o - methyl - n - methyllatrunculin a ( compound 5 ): compound 5 was prepared according to procedure b using compound 2 as starting material ( 10 mg , 0 . 022 mmol ), in thf ( 1 ml ), nah ( 2 mg , 0 . 041 mmol ) in thf ( 1 ml ) and mei ( 0 . 1 ml , 1 . 600 mmol ). elution with n - hexane / etoac ( 8 : 2 ) afforded compound 5 ( 7 . 1 mg , 70 . 1 %). 17 - o - methyl - n - ethyllatrunculin a ( compound 6 ): compound 6 was prepared according to procedure b using compound 2 as starting material ( 10 mg , 0 . 022 mmol ) in thf ( 1 ml ), nah ( 2 mg , 0 . 041 mmol ) in thf ( 1 ml ), and etl ( 0 . 1 ml , 1 . 147 mmol ). elution with n - hexane / etoac ( 8 : 2 ) afforded compound 6 ( 7 . 6 mg , 70 . 6 %): colorless oil , [ α ] d 25 + 33 . 7 ; ( c = 1 . 3 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . 17 - o - methyl - n - cyclopentanelatrunculin a ( compound 7 ): compound 7 was prepared according to procedure b starting with compound 2 ( 10 mg , 0 . 022 mmol ), in thf ( 1 ml ), nah ( 2 mg , 0 . 041 mmol ) in thf ( 1 ml ) and iodocyclopentane ( 0 . 1 ml , 0 . 867 mmol ), elution with n - hexane / etoac ( 8 : 2 ) afforded compound 7 ( 7 . 6 mg , 70 . 6 %): colorless oil , [ α ] d 25 + 53 . 6 ( c = 0 . 63 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . 17 - o - methyl - n -( 3 ′- hydroxypropyl ) latrunculin a ( compound 8 ): compound 8 was prepared according to procedure b from compound 2 ( 10 mg , 0 . 022 mmol ) in thf ( 1 ml ), nah ( 2 mg , 0 . 041 mmol ) in thf ( 1 ml ), and 3 - iodo - 1 - propanol ( 0 . 1 ml , 1 . 043 mmol ). elution with n - hexane / etoac ( 8 : 2 ) afforded compound 8 ( 4 . 6 mg , 40 . 6 %): colorless oil , [ α ] d 25 + 53 . 6 ( c = 0 . 83 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . 17 - o - methyl - n - benzyllatrunculin a ( compound 9 ): compound 9 was prepared according to procedure b using compound 2 as starting substrate ( 15 mg , 0 . 033 mmol ) in thf ( 1 ml ), nah ( 2 mg , 0 . 041 mmol ) in thf ( 1 ml ), and benzyl chloride ( 0 . 1 ml , 0 . 866 mmol ). elution with n - hexane / etoac ( 9 : 1 ) afforded compound 9 ( 11 . 3 mg , 75 . 3 %): colorless oil , [ α ] d 25 + 28 . 5 ( c = 0 . 48 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . 17 - o - methyl - n - benzoyllatrunculin a ( compound 10 ): compound 10 was prepared according to procedure b using compound 2 as starting substrate ( 10 mg , 0 . 022 mmol ) in thf ( 1 ml ), nah ( 2 mg , 0 . 041 mmol ) in thf ( 1 ml ), and benzoyl chloride ( 0 . 1 ml , 0 . 866 mmol ). elution with n - hexane / etoac ( 8 : 2 ) afforded compound 10 ( 5 . 2 mg , 52 . 0 %): colorless oil , [ α ] d 25 + 42 . 7 ( c = 0 . 15 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . 17 - o - methyl - n -( 7 ′- methoxyphenylacetyl ) latrunculin a ( compound 11 ): compound 11 was prepared according to procedure b from compound 2 ( 10 mg , 0 . 022 mmol ) in thf ( 1 ml ), nah ( 2 mg , 0 . 041 mmol ) in thf ( 1 ml ), and p - methoxyphenylacetyl chloride ( 0 . 1 ml , 0 . 636 mmol ). elution with n - hexane / etoac ( 8 : 2 ) afforded compound 11 ( 2 . 2 mg , 22 . 0 %): colorless oil , [ α ] d 25 + 63 . 6 ( c = 0 . 1 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . n - ethyllatrunculin a ( compound 12 ): compound 12 was prepared according to procedure c starting with compound 6 ( 5 mg , 0 . 011 mmol ) in acidic solution ( 1 ml ) for 3 h at 60 ° c . elution with n - hexane / etoac ( 9 : 1 ) afforded compound 12 ( 1 . 3 mg , 26 . 0 %): colorless oil , [ α ] d 25 + 63 . 6 ( c = 0 . 11 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . n - benzyllatrunculin a ( compound 13 ): compound 13 was prepared according to procedure c from compound 9 ( 9 mg , 0 . 0176 mmol ) in acidic solution ( 1 ml ) for 5 h at 60 ° c . elution with n - hexane / etoac ( 9 : 1 ) afforded compound 13 ( 1 . 8 mg , 20 . 0 %): colorless oil , [ α ] d 25 + 63 . 6 ( c = 0 . 17 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . n - hydroxymethyllatrunculin a ( compound 14 ): a solution of 10 mg of compound 1 in 3 ml etoh was treated with 3 ml 35 % aq . ch 2 o , and stirred for 24 hours at 60 ° c . brine ( 5 ml ) was then added , and the mixture was extracted with chcl 3 ( 2 × 5 ml ). the residue was chromatographed over si gel 60 with an n - hexane / etoac ( 8 : 2 ) solvent system to afford compound 14 ( 2 . 8 mg , 28 %): colorless oil , [ α ] d 25 + 70 ( c = 2 . 2 in chcl 3 ). nmr , ir , and ms data are presented in the 61 / 267 , 575 priority application . pyrene - actin polymerization assays . actin polymerization assays were performed in accordance with the manufacturer &# 39 ; s instructions ( cytoskeleton , denver , colo .). briefly , 5 mm final concentration of monomeric actin ( 1 : 10 pyrene labeled ) was incubated on ice for 10 min with one of various concentrations of one of the latrunculin derivatives . samples were then equilibrated 10 min in an elisa plate reader ( biotek , vt ), after which polymerization was induced by adding kcl , mgcl 2 , and atp . compounds 1 - 14 were tested at 0 . 1 , 0 . 5 , 1 . 0 , and 10 μm . the ic 50 for each compound was calculated using graphpad prism 5 . 0 . cell cultures . breast cancer cell lines mcf7 and mda - mb - 231 were purchased from atcc ( manassas , va .). the cell lines were grown at 37 ° c . under 5 % co 2 in 10 % fetal bovine serum ( fbs ) and rpmi 1640 ( gibco - invitrogen , ny ) supplemented with 2 mmol / l glutamine , 100 μg / ml penicillin g , and 100 μg / ml streptomycin . preparation of latrunculin a derivatives for cell culture assays : a latrunculin derivative was first dissolved in dmso at 50 mm ( for mtt assays ) and 1 mm ( for cultrex assays ). about 1 μl of the solution was transferred to 1 ml of serum - free medium to obtain 50 μm and 1 μm dilutions ( 0 . 001 % dmso ) for mtt and cultrex assays , respectively . serial dilutions were then conducted to produce various concentrations . mtt proliferation assay . the growth of mcf7 , mda - mb - 231 , and pc - 3 cancer cell lines was measured using an mtt kit ( tacs ™, trevigen ®, inc .). exponentially growing cells were plated in a 96 - well plate at a density of 8 × 10 3 cells per well , and allowed to attach for 24 h . complete growth medium was then replaced with 100 μl of rpmi serum - free medium containing various concentrations ( 50 , 10 , 1 , 0 . 5 , 0 . 1 μm ) of the particular test compound . culture continued at 37 ° c . under 5 % co 2 . after 72 h of culture , the cells were treated with mtt solution ( 10 μl / well ) at 37 ° c . for 4 h . the color reaction was stopped by adding solubilization / stop solution ( 100 μl / well ). incubation continued at 37 ° c . continued to completely dissolve the formazan dye product . absorbance of the samples was determined at 570 nm with an elisa plate reader ( biotek , vt ). the number of cells per well was calculated against a standard curve that had been prepared by plating various concentrations of cells , as determined by hemocytometer , at the start of each experiment . ic 50 for each compound was calculated using a nonlinear regression curve fit of log concentration versus the number of cells / well , using graphpad prism 5 . 0 ( graphpad software , la jolla , calif .). cultrex ® cell invasion assay . anti - invasive activities were measured using trevigen &# 39 ; s cultrex ® cell invasion assay as described in k . tamilarasan et al ., bmc cell biol . 2006 , 7 , 17 - 30 ; and p . borghesani et al ., development . 2002 , 6 , 1435 - 1442 . about 50 μl of basement membrane extract ( bme ) coat was added to each well . after incubation for 4 h at 37 ° c . under 5 % co 2 , 50 , 000 mda - mb - 231 cells in 50 μl of serum free rpmi medium were added to each well in the top chamber , along with the test compound at one of several concentrations ( 0 . 1 , 0 . 5 , 1 μm ). about 150 μl of rpmi medium was added to the lower chamber in 10 % fbs and penicillin / streptomycin , using fibronectin ( 1 μl / ml ) and n - formyl - met - leu - phe ( 10 nm ) as chemoattractants . cells were allowed to migrate to the lower chamber at 37 ° c . under 5 % co 2 . after 24 h , the top and bottom chambers were aspirated and washed with the washing buffer that had been supplied with the kit . about 100 μl of cell dissociation solution / calcein - am solution was added to the bottom chamber , and the cells were incubated at 37 ° c . under co 2 for 1 h . briefly , the cells internalize calcein - am , and then the intracellular esterases cleave the am moiety to generate free calcein . fluorescence of the samples was observed at 485 nm excitation , 520 nm emission , using an elisa plate reader ( biotek , vt ). the number of cells that had invaded through the bme coat was calculated using a standard curve . molecular modeling . three - dimensional structure building and modeling were performed using the sybyl program package , version 8 . 0 . the x - ray crystallographic structural of compound 1 has been reported : w . m . morton et al ., nat . cell biol . 2000 , 2 , 376 - 378 . this reported structure was used as a template to model other latrunculins . conformations for each compound were generated using confort ™ conformational analysis . energy minimizations were performed using the tripos force field with a distance - dependent dielectric and the powell conjugate gradient algorithm , with a convergence criterion of 0 . 01 kcal /( mol - å ). partial atomic charges were calculated using the semiempirical program mopac 6 . 0 and applying the am1 . see j . j . stewart , j . comput . aided mol . des . 1990 , 4 , 1 - 105 . results are presented in the 61 / 267 , 575 priority application . molecular docking . the surflex - dock program , version 2 . 0 , interfaced with sybyl 8 . 0 was used to model the docking of the compounds to the active site of actin . surflex - dock employs an idealized active site ligand ( protomol ) as a target to generate putative poses of molecules or molecular fragments . these putative poses were scored using the hammerhead scoring function . the program was used to dock the training set molecules into the active site of g - actin . the 3d structure was taken from the brookhaven protein databank ( pdb code : 1 esv ). c - 17 / c - 15 lactol acetalization products are synthesized via acetalization of the lactol group of compound 1 or 20 in a substituted or free alcohol ( ar —( ch 2 ) 1 - 3 — oh or hetar —( ch 2 ) 1 - 3 — oh ), bf 3 - diethyl etherate ( et 2 o — bf 3 ). the mixture is stirred for 12 h at room temperature and then neutralized with 10 % aq . nahco 3 . the solvent is evaporated , and the residue is extracted with chcl 3 and dried over mgso 4 . after evaporation of the solvent , the residue is subjected to mplc . hydroxymethylation / hydroxyethylation is conducted through alkylation of the thiazolidinone nitrogen . a solution of compound 1 or 20 in 35 % aqueous formaldehyde or acetaldehyde in ethanol is kept overnight at 60 ° c . reaction workup and chromatography will afford 30 - 40 % yield . compound 26 is prepared by the acetalization reaction as otherwise described above , using 2 -( 4 - aminophenyl ) ethanol after protecting the amino group with boc , followed by later de - protection . the product is hydroxymethylated with 3 % aqueous hcho . compound 27 is prepared by carbamoylation of compound 1 using benzenesulfonylmethyl isocyanate in toluene , followed by hydroxymethylation . compound 28 is prepared via oxidation of the n - hydroxymethyl group of compound 14 to an aldehyde using pyridinium chlorochromate in ch 2 cl 2 , followed by reaction with nh 2 oh . hcl , pyridine - etoh ( 1 : 3 ), and heating at 100 ° c . for 18 h . compound 29 is prepared by treating compound 3 with 35 % aqueous formaldehyde in ethanol for 12 - 24 hours in a 60 ° c . water bath . product is purified on sephadex lh20 ( 1 - 5 % meoh / chcl 3 ) or c - 18 rp ( ch 3 cn — h 2 o gradient elution ). si gel 60 is not used , to avoid chemical instability or degradation . compounds 30 and 31 are prepared by essentially the same procedure used to prepare compound 3 , but replacing phenylethyl alcohol with ethyl 5 - hydroxypentanoate or ethyl - 4 -( hydroxymethyl ) benzoate , respectively . the ethyl ester is hydrolyzed with acetic acid , hcl ( because latrunculins are generally more stable in acids than in bases ), aqueous t - buok in thf , me 3 sii , kosime 3 , cyclodextrins , or dowex - 50 . compound 32 is prepared by reacting compound 1 with methanol and et 2 o — bf 3 overnight to give compound 2 in 70 - 80 % yield . bromination of ch 3 nh 2 . hcl in ethanol under nitrogen for 5 hours at 40 ° c . gives bromomethaneamine . hcl in 90 % yield . compound 2 in dry thf is gradually treated with a suspension of nah ( 60 % dispersion in mineral oil ) in dry thf at 0 ° c . the mixture is then stirred for 1 hour at 0 ° c . brch 2 nh 2 is then added , and the mixture is stirred at room temperature until compound 2 is completely depleted . ether and water are added , the two layers are separated , and the organic layer is dried over anhydrous na 2 so4 . after evaporation the residue is subjected to mplc on sephadex lh20 ( 1 - 5 % meoh / chcl 3 ) or c - 18 rp ( ch 3 cn — h 2 o gradient elution ) to give the 17 - o - methyl analogue of compound 32 . compound 32 . alternatively , compound 2 is added to bromomethaneamine in anhydrous et 2 o , and the mixture is treated with metallic na with ice cooling under nitrogen for 3 hours , after which naoet is added to give the 17 - o - methyl analogue of compound 32 , which is then demethylated using acoh / h 2 o / thf ( 3 : 1 : 1 ) and stirring at 60 ° c . the reaction is monitored using tlc until complete depletion of the starting material (˜ 0 . 5 - 1 hours ). the mixture is cooled to room temperature , neutralized with 10 % aqueous nahco 3 , and extracted with ether . the ether layer is washed with water , dried over anhydrous na 2 so 4 , and evaporated under reduced pressure . the residue is subjected to sephadex lh20 or c18 reverse phase chromatography to give compound 32 . compound 33 . compound 2 in dry thf is gradually treated with a suspension of nah ( 60 % dispersion in mineral oil ) in dry thf at 0 ° c . the mixture is then stirred for 1 hour at 0 ° c . commercially available 4 -( bromomethyl ) imidazole ( cas # 80733 - 10 - 44 ) is then added , and the mixture is stirred at room temperature until compound 2 is completely depleted . ether and water are added , the two layers are separated , and the organic layer is dried over anhydrous na 2 so4 . after evaporation the residue is subjected to mplc on sephadex lh20 ( 1 - 5 % meoh / chcl 3 ) or c - 18 rp ( ch 3 cn — h 2 o gradient elution ) to afford the 17 - o - methyl analogue of compound 33 , which is then demethylated by using acoh / h 2 o / thf ( 3 : 1 : 1 ) as otherwise described above for compound 32 , to afford compound 33 . an alternative scheme for the semisynthesis of compound 33 proceeds by the 5 % cui - catalyzed n - methyl - imidazolation of compound 1 directly , using 4 -( bromomethyl )- imidazole in the presence of ( s )- pyrrolidinylmethylimidazole and cs 2 co 3 in dmf at 110 ° c . for 24 hours . this method allows the incorporation of diverse functional groups such as ester , nitrile , nitro , ketone , free oh , and free nh 2 on the halide . compound 34 : 17 - o - methyl - n -( 2 ′- carboxyfuroate ) latruncul in a . compound 34 is prepared according to procedure b from compound 2 , otherwise similar to the method for preparing compound 11 , using nah in thf and 2 ′- carboxyfuroylchloride and reacting at room temperature for 5 minutes . small molecule analogs . the invention may also be practiced with small molecule latrunculin analogs . examples of smaller analogs that may be used in practicing the present invention include the following : r 1 ═ h , ch 2 oh , cho , cooh , conh 2 , nh 2 , c1 to c6 hydroxyalkyl or carboxyalkyl , hydroxylaryl , hydroxyheteroaryl , r 2 ═ h , ch 2 oh , cho , c1 to c6 hydroxyalkyl or carboxyalkyl , hydroxylaryl , hydroxyheteroaryl , r 3 ═ cooh , cho , conh 2 , oh , o — c1 to c4 alkyl or hydroxyalkyl , nh 2 , nh — c1 to c4 carboxyalkyl or hydroxyalkyl , sh our molecular modeling studies suggest that these maleidimide structures will exhibit high binding affinity toward the actin - binding proteins gelsolin , cofilin , and capg , and other gelosolin - relating actin binding proteins . these compounds are expected to be bioisosteres of the latrunculin active tetrahydropyran / thiazolidinone segment . therefore they should show selective anti - invasive activity similar to that of compound 11 . these analogs may be totally synthesized through methods otherwise known in the art , and therefore should not require harvesting marine fauna to obtain semi - synthetic precursors . the in vitro antiproliferative and anti - invasive activities of various latrunculin analogues are observed and measured . some preliminary in vitro studies have been conducted , with encouraging results . effects on hela cells . in an in vitro assay , latrunculin h ( compound 14 ) strongly inhibited the growth of cervical cancer hela cells at early interphase over a dose range 0 . 04 - 1 . 25 μm , as analyzed by flow cytometry . latrunculin 11 showed 3 . 5 - 51 . 0 times greater inhibition against g2 - m - phase hela cells as compared to the control antimitotic compound nocodazole in various g2_m assays . latrunculin b ( compound 20 ) and latrunculin h ( compound 14 ) were less active than compound 11 in these assays , but still showed greater activity than did nocodazole . effects on wnt / β - catenin . latrunculin b ( compound 20 ) and compound 11 strongly inhibited wnt / β - catenin signaling , a key pathway in the proliferation of epithelial cancer cells . these two compounds affected both alkaline phosphatase and β - catenin levels in c2c12 cells . compound 11 was the more potent of the two , with an ic 50 value of 0 . 007 μm for β - catenin , and 0 . 004 μm for alkaline phosphatase . effects on angiogenesis . angiogenesis inhibition assays were conducted using endothelial colony forming cells ( ecfcs ). the microvessel density marker cd31 was quantified using confocal laser scanning microscopy and immunohistochemistry . compounds 11 , 14 , and 20 all down - regulated the microvessel density marker cd31 in the ecfcs . each of these three compounds caused 100 % inhibition of cd31 in ecfcs at concentrations of 10 μm and 2 μm . the ic 50 values for compounds 14 and 20 were 0 . 063 and 0 . 043 μm , respectively . the ic 50 value for compound 11 was not precisely quantified , but must be below the lowest tested level , 0 . 043 μm . pyrene - actin polymerization assays . actin polymerization inhibitory activity is assessed using the cytoskeleton actin polymerization kit ( cytoskeleton , denver , colo .). briefly , 5 mm ( final concentration ) of monomeric actin ( 1 : 10 pyrene labeled ) is incubated on ice for 10 min with one of various concentrations of the test latrunculin compound . samples are equilibrated 10 min in an elisa plate reader ( biotek , vt ), after which polymerization is induced by adding kcl , mgcl 2 , and atp . compounds are tested at 0 . 1 , 0 . 5 , 1 . 0 , and 10 μm . the ic 50 for each compound is calculated using graphpad prism 5 . 0 . cell culture assays . the antiproliferative activity of the latrunculins is measured in vitro using various cell culture assays . breast cancer cell lines mcf7 and mda - mb - 231 , and normal mammary epithelial cells mcf10a ( controls ) are obtained from atcc . the cell lines are grown in 10 % fetal bovine serum ( fbs ) ( gibco - invitrogen , ny ) and leibovitz &# 39 ; s l - 15 ( atcc ) or rpmi , supplemented with 2 mmol / l glutamine , 100 μg / ml penicillin g , and 100 μg / ml streptomycin , at 37 ° c . under 5 % co 2 . mtt proliferation assay . the effect of latrunculins on the growth of mcf7 and mda - mb - 231 cancer cell lines , and on mcf10a normal mammary epithelial cells is measured using an mtt kit ( tacs ™, trevigen ®, inc .). exponentially - growing cells are plated in a 96 - well plate at a density of 8 × 10 3 cells per well , and allowed to attach for 24 hours . complete growth medium is then replaced with 100 μl of rpmi serum - free medium ( gibco - invitrogen , ny ) containing one of various doses ( 50 . 0 , 10 . 0 , 1 . 0 , 0 . 5 , 0 . 1 μm ) of the test latrunculin compound . culture continues at 37 ° c . under 5 % co 2 . after 72 h of culturing , the cells are treated with mtt solution ( 10 μl / well ) at 37 ° c . for 4 hours . the color reaction is stopped by adding the solubilization / stop solution ( 100 μl / well ). incubation continues at 37 ° c . until complete dissolution of the formazan product . absorbance of the samples is determined at 570 nm with an elisa plate reader ( biotek , vt ). the number of cells per well is calculated against a standard curve prepared at the start of each experiment by plating various concentrations of cells , as determined by hemocytometer . ic 50 for each compound is calculated using non - linear regression of log concentration versus number of cells / well , implemented in graphpad prism 5 . 0 . cultrex ® cell invasion assay . anti - invasive activities are measured using trevigen &# 39 ; s cultrex ® cell invasion assay . 50 μl of basement membrane extract ( bme ) coat is added to each well . after incubation for 4 h at 37 ° c . in 5 % co 2 , mda - 50 , 000 mb - 231 cells in 50 μl of serum - free rpmi or leibovitz &# 39 ; s l - 15 medium are added to the top chamber of each well with the test compound at one of various concentrations ( 0 . 1 , 0 . 5 , 1 μm ). 150 μl of rpmi medium is added to lower chamber , which contains 10 % fbs and penicillin / streptomycin , and which uses fibronectin ( 1 μl / ml ) and n - formyl - met - leu - phe ( 10 nm ) as chemoattractants . cells are allowed to migrate to the lower chamber at 37 ° c . under co 2 . after 24 h , the top and bottom chambers are aspirated and washed with the washing buffer supplied with the kit . about 100 μl of cell dissociation solution / calcein - am solution is added to the bottom chamber and incubated at 37 ° c . under co 2 for 1 hour . the cells internalize calcein - am , and intracellular esterases cleave the acetomethylester ( am ) moiety to generate free calcein . fluorescence measurements of the samples are taken at 485 nm excitation , 520 nm emission using an elisa plate reader ( biotek , vt ). the numbers of cells that invade through the bme coat are calculated using a standard curve . wound - healing assay . mda - mb - 231 cells are plated onto sterile 24 - well plates and allowed to form a confluent cell monolayer (& gt ; 95 % confluence ) overnight . wounds are then inflicted on each cell monolayer using a sterile 200 μl pipette tip . media are removed , and cells are washed twice with pbs and once with fresh serum - free media . test latrunculin compounds at various concentrations ( 100 nm - 10 μm ) in fresh , serum - free media are added to each well and incubated for 24 h under serum - starved conditions . media are then removed , and the cells are washed , fixed and stained using diff - quick ™ staining ( dade behring diagnostics , aguada , puerto rico ). cells that have migrated across the inflicted wound are counted under the microscope in three or more randomly selected fields ( magnification : 400 ×). all experiments are conducted independently in triplicate replications . evaluation of antiproliferative and antimetastatic activities of latrunculins in balb / c athymic nude mice . we have demonstrated that the novel latrunculin derivatives significantly inhibit the growth and viability of mda - mb - 231 cells ( atcc - htb - 26 ) in vitro . the mda - mb - 231 cell line is known to be highly malignant , to be estrogen - independent , and to display anchorage - independent growth when cultured in soft agarose gels . when mda - mb - 231 cells are injected subcutaneously into the flanks of female balb / c athymic nude mice , they form rapidly - growing , anaplastic adenocarcinomas that are highly invasive , and that typically metastasize into the lungs and bones . the most active latrunculin analogues from the in vitro assays are examined for their antiproliferative and anti - invasive activity in vivo . mda - mb - 231 cells transfected with green fluorescence protein ( gfp ) gene are grown in culture , isolated with 0 . 25 % trypsin and 0 . 53 mm edta solution , washed , counted and diluted to a desired concentration in fresh , complete growth culture medium . briefly , 1 × 10 6 mda - mb - 231 - gfp cells in 100 pl of serum - free rpmi 1640 are injected subcutaneously into the flanks of nude mice using a 1 - ml syringe with a 26 - gauge , sterile needle . all surgical operations are performed under aseptic conditions . nude mice with similar tumor sizes , about 100 mm 3 ( 2 weeks post - inoculation ) are selected and randomly divided into seven groups ( n = 9 per group ): control ( dmso vehicle ); two groups for the two most antiproliferative latrunculins ; two groups for the two most anti - invasive latrunculins ; and one group for each of latrunculins a and b . animal weights and tumor growth and metastases are monitored weekly . average tumor diameter for each palpable tumor is determined as the mean of the two largest perpendicular diameters , measured by vernier calipers . four to six weeks after tumor cell implantation , tumors in all mice reach approximately 1 cm in diameter . detection and quantification of primary and metastatic tumors in anesthetized animals in each treatment group are performed with the kodak in - vivo imaging system fx pro imaging system . animals are then given intraperitoneal injections daily for one week , either with vehicle ( dmso ) or with the test latrunculin compound . a dose of the test compound equivalent to 0 . 05 mg per kg body mass is used . ( this dose was chosen based on previous reports with latrunculin a . if the dose should be too toxic , an alternative effective dose ( middle dose ) can be estimated by extrapolation from results obtained from the in vitro studies . a lower dose ( ½ middle dose ) and a high dose ( 2 × middle dose ) can then be tested . the doses for each compound can also be titrated to the maximum tolerated doses that do not produce apparent toxicity .) afterwards , primary and metastatic tumors in each animal are re - scanned with the same imaging system to determine treatment effects on primary and metastatic tumor growth . animals are examined every three days for evidence of tumors by fluoroimaging , and the rate of tumor growth is monitored by estimating tumor volume . animals are examined for secondary tumors in multiple organs by observing the presence of fluorescent cell colonies . at the end of the experimental period , animals are sacrificed . tumors are excised , weighed , and prepared for later histological examination . wet sections of organs are examined for the presence of green fluorescent protein . other tissue portions are fixed in neutral buffered formalin and embedded in paraffin . 5 . 0 μm - thick sections of tumors are processed for h & amp ; e staining . testing activity against established tumors provides a different ( and generally more stringent ) test of in vivo anti - tumor activity than the ability to inhibit the formation of new tumors . these experiments determine whether latrunculins can prevent or significantly delay tumor formation and metastasis in vivo . all animal experiments will be approved by the applicable institutional animal care and use committee . all surgical and treatment procedures will be consistent with the iacuc policies and procedures , and applicable laws and regulations . following successful animal testing , clinical trials in humans will be conducted in accordance with applicable laws and regulations . statistical analysis . all experimental treatments are run at least in triplicate . one - way anova is used to evaluate results statistically . significance is determined by the newman - keuls test . differences are considered statistically significant at p & lt ; 0 . 05 . compounds used in the present invention may be administered to a patient by any suitable means , including intravenous , parenteral , subcutaneous , intrapulmonary , and intranasal administration . parenteral infusions include intramuscular , intravenous , intraarterial , or intraperitoneal administration . the compounds may also be administered transdermally , for example in the form of a slow - release subcutaneous implant . they may also be administered by inhalation . pharmaceutically acceptable carrier preparations include sterile , aqueous or non - aqueous solutions , suspensions , and emulsions . examples of non - aqueous solvents are propylene glycol , polyethylene glycol , vegetable oils such as olive oil , and injectable organic esters such as ethyl oleate . aqueous carriers include water , alcoholic / aqueous solutions , emulsions or suspensions , including saline and buffered media . parenteral vehicles include sodium chloride solution , ringer &# 39 ; s dextrose , dextrose and sodium chloride , lactated ringer &# 39 ; s , or fixed oils . the active therapeutic ingredient may be mixed with excipients that are pharmaceutically acceptable and are compatible with the active ingredient . suitable excipients include water , saline , dextrose , glycerol and ethanol , or combinations thereof . intravenous vehicles include fluid and nutrient replenishers , electrolyte replenishers , such as those based on ringer &# 39 ; s dextrose , and the like . preservatives and other additives may also be present such as , for example , antimicrobials , anti - oxidants , chelating agents , inert gases , and the like . the form may vary depending upon the route of administration . for example , compositions for injection may be provided in the form of an ampoule , each containing a unit dose amount , or in the form of a container containing multiple doses . a method for controlling the duration of action comprises incorporating the active compound into particles of a polymeric substance such as a polyester , peptide , hydrogel , polylactide / glycolide copolymer , or ethylenevinylacetate copolymers . alternatively , an active compound may be encapsulated in nanoparticles or microcapsules by techniques otherwise known in the art including , for example , by coacervation techniques or by interfacial polymerization , for example , by the use of hydroxymethylcellulose or gelatin - microcapsules or poly ( methylmethacrylate ) microcapsules , respectively , or in a colloid drug delivery system . colloidal dispersion systems include macromolecule complexes , nanocapsules , microspheres , beads , and lipid - based systems including oil - in - water emulsions , micelles , mixed micelles , and liposomes . as used herein , an “ effective amount ” of a compound is an amount , that when administered to a patient ( whether as a single dose or as a time course of treatment ) inhibits or reduces the growth of targeted tumors to a clinically significant degree ; 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