Patent Application: US-52931901-A

Abstract:
the present invention describes the design , synthesis and therapeutic use of a variety of novel inhibitors of polyamine transport . the main feature of this class of transport inhibitors is to incorporate a linker or side chain that prevents the uptake of polyamines and helps to conjugate polyamine analogs to form dimers with high inhibitory potency against polyamine uptake . these new compounds incorporate features that are designed to maximize their chemical and metabolic stability and their ability to bind to the polyamine transporter , and to minimize their toxicity by preventing their absorption by the cells . the purpose of such inhibitors is to prevent the uptake or salvaging of circulating polyamines by rapidly proliferating cells such as tumor cells , in order to potentiate the effect of therapeutic inhibitors of polyamine biosynthesis such as alpha - difluoromethylornithene .

Description:
sym - norspermidine , ornithine dihydrochloride and other reagents for organic syntheses were purchased from aldrich ( milwaukee , wis .) and sigma ( st . louis , mo .). reversed phase silica gel liquid chromatography was performed with a lichroprep ™ rp - 18 c 18 silica gel column ( 40 - 63 μm ; bdh , st . laurent , qc ., canada ) using a gradient of ch 3 cn : meoh : h 2 o ( 25 : 35 : 40 to 50 : 30 : 20 ) as eluent . homogeneity of synthetic products was assessed by thin - layer chromatography performed on 0 . 20 mm f 254 silica gel 60 plates or 0 . 25 mm f 254 s rp - 18 reversed phase silica gel plates ( e . merck , darmstadt , germany ). fir spectra were obtained on a perkin - elmer 1600 spectrophotometer ( ftir series ) and were expressed in cm − 1 . 1 h and 13 c nmr spectra were recorded with a bruker ac / f 300 ( 300 mhz ); 13 c were recorded at 75 . 47 mhz . chemical shifts ( δ in ppm ) were referenced to cdcl 3 ( 7 . 26 ppm for 1 h and 77 . 00 ppm for 13 c ). mass spectra ( ms ) were recorded at the mass spectrometry region center ( university of montreal , montreal , qc ., canada ) by fast atomic bombardment mass spectrometry ( fabms ) or liquid secondary ion mass spectrometry ( lsmis ), using a vg autospecq ™ and a kratos ms50 tcta , respectively . [ 2 , 3 - 3 h ( n ] putrescine dihydrochloride ( 4 . 1 × 10 4 cl / mol ) and [ 1 , 8 - 3 h ( n )] spermidine trihydrochloride ( 1 . 5 × 10 4 cl / mol ) were obtained from dupont - new england nuclear ( lachine , qc ., canada ). [ 5 , 8 - 14 c ] spermine tetrahydrochloride ( 108 cl / mol )) was purchased from amersham ( arlington heights , ill .). dfmo was provided by the marion merrell dow research institute ( cincinnati , ohio ). fetal bovine serum ( fbs ) and cosmic ™ calf serum were from hyclone ( logan , utah ). the heterobifunctional reagent 1 (- p - azidosalicylamido )- 4 - iodoacetamido ) butane ( asib ) as obtained from pierce ( rockford , ill .). lucifer yellow ( oy ) iodoacetamide was purchased from molecular probes ( eugene , oreg .). putrescine dihydrochloride , spermidine trihydrochloride , spermine tetrahydrochloride , iodoacetamide , 5 , 5 ′- dithio ( 2 - nitrobenzoic acid ) and 3 , 4 - diaminobenzoic acid as well as tissue culture reagents were purchased from sigma . ortho - phthaldialdehyde was purchased from fluka ( ronkonkoma , n . y .) and other reagents for high - performance liquid chromatography ( hplc ) were from fisher scientific ( montreal , qc ., canada ) or aldrich ( milwaukee , wis .). unless otherwise indicated , reactions were performed at room temperature . compound 1 of fig1 , namely 5 - carboxyspermine , was synthesized using a known scheme ( behr , j . p . 1989 . j . chem . soc . chem . commun . 101 - 103 ). briefly to a stirred solution of 10 . 0 g ( 59 . 3 mmol ) of ornithine hydrochloride dissolved in 250 ml meoh were added 18 . 0 g ( 197 mmol ) of tetramethylammonium hydroxide . after dissolution of ornithine salt , meoh was evaporated , the mixture was then dissolved in 350 ml of dry dimethylformamide ( hplc grade ; aldrich , milwaukee , wis .) and the residual ammonium salt was filtrated , yielding ornithine as its free base . following the addition of acrylonitrile ( 2 . 2 equivalents , 130 . 9 mmol ), the mixture was stirred for 16 hours in the dark to give 10 . 6 g ( yield = 74 %) of crude n a , n b - diethylcyanide ornithine , which was subsequently used without further purification . white solid : ir ( film ) v cm − 1 3372 ( oh , acid ), 2247 ( cn ); 1 h nmr δ ( cdcl 3 300 mhz ) 1 . 48 ( m , 4h , c h 2 chcooh ), 2 . 63 ( m , 6h , 3 × c h 2 n ), 2 . 86 ( 2xt , j 1 = 5 . 9 and j 2 − 2 . 7 hz , 4h , 2 × ch 2 cn ), 3 . 07 ( t , j = 7 . 2 hz , 1h , c h cooh ). to obtain 5 - carboxyspermine koh ( 2 . 7 g , 48 . 0 mmol ) was dissolved with vigorous stirring in 8 ml of 95 % ( v / v ) etoh and 10 . 5 g ( 44 . 1 mmol ) of n a , n b - diethylcyanide ornithine were then added . the resulting mixture was placed under h 2 at 40 psi in a burgess - parr hydrogenator , using 2 . 09 g ( 24 . 4 mmol ) or raney nickel as catalyst ( behr , j . p . 1989 . j . chem . soc . chem . commun . 101 - 103 ; bergeron , r . j . and garlich , j . r . 1984 . synthesis : 782 - 784 ). after 22 hours , raney nickel was removed by filtration , and the solvent evaporated in vacuo , yielding 16 . 07 g of crude 5 - carboxyspermine potassium salt . yellow oil ; ir ( film ) v cm − 1 3363 ( oh , acid ), 2937 ( nh 2 ) no cyanide band ; 1 h nmr δ ( cdcl 3 , 300 mhz ) 1 . 53 ( m , 2h , c h 2 nh 2 ), 2 . 65 ( m , βh , c h 2 nh ), 3 . 09 ( t , j = 5 . 7 hz , 1h , c h cooh ). amine protection of 5 - carboxyspermine by tert - butyl carbonyl ( boc ) groups was performed as described ( ponnusamy , e ., fotadar , u ., spisni , a . and fiat , d . 1986 . syntheses : 48 - 49 ). to 16 . 0 g ( 65 . 0 mmol ) of crude 5 - carboxyspermine potassium salt dissolved in 1 . 5 l meoh were added 9 . 64 ml of 10 % ( v / v ) triethylamine and 54 . 3 g ( 4 . 4 equivalents , 286 mmol ) of di - tert - butyl dicarbonate . after stirring for 24 hours , solvent was evaporated , 100 - 150 ml h 2 o were added and the resulting mixture was chilled at 0 ° c . after adjusting ph at 2 . 2 with 2 n hcl , the boc - product was extracted with ethyl acetate , dried over anhydrous mgso 4 and purified by c 18 reversed phase silica gel chromatography , yielding 3 . 3 g of pure tetra - boc - 5 - carboxyspermine ( compound ii , fig1 ). light yellow solid ; ir ( film ) v cm − 1 3356 ( oh , acid ), 1682 ( c ═ o , amide ); 1 h nmr δ ( cdcl 3 , 300 mhz ) 1 . 32 ( 2 × s , 36 h , ( c h 3 ) 3 c from boc - n ), 1 . 90 - 1 . 40 ( m , 9h , c h 2 ch 2 n ), 3 . 20 - 2 . 90 ( m , 10h , c h 2 n ); m ( for c 31 h 58 o 10 n 4 )− 646 . 41 ; m / z ( lsims )= 647 . 42 [( m + 1 )*]. coupling of tetra - boc - 5 - carboxyspermine ( compound ii ) to cystamine was then performed in two steps based on the method of venkataraman ( venkataraman , k . 1979 . tetrahedr . lett . 32 , 3037 ). to a solution of 1 . 15 g ( 1 . 78 mmol ) of compound ii in 20 ml dry acetone was added 0 . 27 ml ( 1 . 1 eq , 1 . 96 mmol ) of triethylamine ( freshly distilled on koh ) and 361 mg ( 1 . 1 eq , 1 . 96 mmol ) of cyanuric chloride and the reaction mixture stirred overnight under n 2 to form the corresponding acid chloride . cystamine dihydrochloride ( 241 mg ; 1 . 07 mmol ) was then suspended in dry triethylamine and added to the acid chloride form of compound ii , with the resulting triethylamine concentration being at ≧ 4 - fold excess relative to the latter . after stirring for 12 hours , the residual triazine oxide was filtrated , acetone was evaporated and the product extracted with chcl 3 , dried over anhydrous mgso 4 and evaporated in vacuo . the crude compound was then purified by reversed - phase c 18 column chromatography , yielding 0 . 682 g of 2 , 2 ′- dithiobis [ n - ethyl -( n 1 , n4 , n c , n 12 - tetra - boc - 5 - carboxamide ( compound iv , fig1 ). ( m ) yellow oil ; ir ( film ) v cm − 1 1693 ( c ═ o , amide ); 1 h nmr δ ( cdcl 3 , 300 mhz ) 1 . 38 ( s , 36h , ( c h 3 ) 3 c ), 1 . 59 ( m , 8h , ch 2 c h 2 ch 2 ), 2 . 53 ( 6 , j = 5 . 7 hz , 1h , con h ch 2 ), 2 . 73 ( t , j = 6 . 1 hz , 2h , c h 2 s ), 3 . 11 ( m , 10h , c h 2 nh ), 3 . 51 ( m , 2h , nc h 2 ch 2 s ): m ( for c xx h 123 o 18 n 10 s 2 )= 1408 . 85 ; m / z ( fabms )= 1409 . 9 [( m + 1 )*]. compound iii ( 215 mg in meoh ) was then deprotected by addition of 1 ml of 3 n hcl , bringing the ph from 6 . 0 to = 0 . 5 . after stirring vigorously for 15 hours , the solvent was dried out in vacuo and the resulting compound purified by cation exchange chromatography with a dowex 50w - x4 column ( dry mesh ; 100 - 200 ; sigma ) pre - equilibrated with h 2 o and successively washed with h 2 o , 1 n hcl , 2 n hcl , 4 n hcl and 6n hcl . ninhydrin - positive fractions eluted with 6 n hcl were pooled and evaporated in vacuo , yielding 96 mg of pure 2 , 2 ′- dithiobis ( n - ethyl - spermine - 5 - carboxamide )- octahydrochloride ( desc , compound v , fig1 . white solid ; mp 75 - 78 ° c . ; bp 118 ° c . 1 h nmr δ ( cdcl 3 , 300 mhz ) 1 . 62 ( m , 2h , c h 2 chconh ), 1 . 97 - 1 . 80 ( m , 6h , c h 2 ch 2 ch 2 ), 2 . 74 ( t , j = 6 . 2 hz , 2h , c h 2 s ), 2 . 92 ( m , 10h , c h 2 hn ), 3 . 46 ( dt , j = 7 . 1 hz , 2h , c h 2 ch 2 s ), 3 . 84 ( t , j = 7 . 0 hz , 1h , c h conh ); m ( for c 25 h 60 o 2 n 10 s 2 )= 608 . 96 ; m / z ( fabms )= 609 . 4 ( m *). compound iv was similarly deprotected to yield n -[ 2 , 2 ′- dithio ( ethyl , 1 ′- aminoethyl )] spermine - 5 - carboxamide ( deasc , compound vi , fig1 ). yellow solid ; mp 50 - 54 ° c . ; bp 109 ° c . 1h nmr δ ( cdcl 3 , 300 mhz ) 1 . 89 ( m , 2h , c h 2 chconh ), 2 . 10 - 2 . 29 ( m , 6h , c h 2 ch 2 ch 2 ), 3 . 04 ( t - j = 6 . 0 hz , 2h , conhc h 2 ch 2 s ), 3 . 19 ( t , j = 7 . 4 hz , 2h , ssch 2 c h 2 hn 2 ), 3 . 25 ( m , 10h , c h 2 , nh ), 3 . 51 ( t , j = 6 . 5 hz , 2h , ssch 2 c h 2 hn 2 ), 3 . 78 ( m , 2h , conhc h 2 ch 2 s ), 4 . 11 ( t , j = 6 . 7 hz , 1h , c h conh ), m ( for c 15 h 41 on 5 s 2 )= 380 . 62 ; m / z ( lsims )= 381 . 24 . desc was dissolved in 50 mm sodium phosphate bugger , ph 8 . 0 , containing 250 mm dithiothreitol ( dtt ), and incubated for 30 minutes at 37 ° c . in a water bath . the mixture was then loaded on a dowex ™ 50w - x4 cation exchange column equilibrated with h 2 o , and after washing with 5 column volumes each of 1 n hcl and 2 n hcl , the free thiol was eluted with 10 volumes of 4 n hcl . amine - containing fractions , as identified by mixing 5 μl aliquots with 200 μl of an o - phthaldialdehyde solution ( 3 . 7 mm o - phthaldialdehyde ; 0 . 4 m boric acid , ph 10 . 4 ; 1 % v / v msoh ; 0 . 45 % v / v 2 - mercaptoethanol ; 0 . 03 % w / v bri ? 35 ) and hearing for 20 minutes at 37 ° c ., were then pooled . the amount of n -( 2 - mercaptoethyl )- spermine - 5 - carboxamide [ mesc ] tetrahydrochloride ( compound vii , fig1 ) thus isolated was titrated for thiol equivalents with 5 , 5 ′- dithio - bis -( 2 - nitrobenzoic acid ) ( jocelyn , p . c . 1987 . meth . enzymol . 143 , 44 - 67 ) using either cysteamine or dtt as standard . the yield of mesc using this procedure was virtually 100 %, based on the number of thiol equivalents determined with 5 , 5 ′- dithio - bis -( 2 - nitrobenzoic acid ) and the expected number of thiol equivalents expected per mass of desc . finally , mesc purity was configured by ion - pair reversed - phase hplc using post - column derivatization with o - phthaldialdehyde ( pegg , a . e ., wechter , r ., poulin , r ., woster , p . m . and coward , j . k . 1989 . biochemistry 28 : 8446 - 8453 ). 1 nmr δ ( cdcl 3 , 300 mhz ) 1 . 91 ( m , 2h , c h 2 chconh ), 2 . 08 - 2 . 24 ( m , 6h , ch 2 c h 2 ch 2 ), 2 . 82 ( t , j = 6 . 3 , 2h , con hch 2 c h 2 sh ), 3 . 22 ( m , 10h , c h 2 nh , 3 . 56 ( m , 2h , conhc h 2 ch 2 sh ), 4 . 11 ( t , j = 6 . 6 , 1h , c h conh ). to 1 ml of an extemporaneously prepared , dtt - free solution of mesc ( 20 mm in h 2 ) were added 50 μl of 50 mm tris - hcl ( ph 7 . 0 ) and 105 μl of a 40 mm solution of either iodoacetamide , ly iodoacetamide or asib in a light - protected microcentrifuge tube , and the mixture was incubated for 2 hours at 37 ° c . the extent of thiol modification was assessed by measuring the amount of thiol remaining at the end of the incubation with 5 , 5 ′- dithio - bis -( 2 - nitrobenzoic acid ) as described above , and was determined to be essentially complete . excess iodoacetamide was then inactivated by adding dtt to a final concentration of 40 mm and incubating the solution for 2 hours at 37 ° c . the resulting solutions of mesc adduct was used without further purification for [ 3 h ] spermidine uptake assays conducted as described below . the effect of the respective dtt - inactivated iodoacetamide on spermidine transport was determined in parallel by incubating cells with the same reaction mixture from which mesc was omitted . both zr - 75 - 1 human breast cancer cells and chinese hamster ovary cells ( cho - k1 ) were obtained from the american type culture collection ( rockville , md .). zr - 75 - 1 cells were maintained in phenol red - free rpmi 1640 medium supplemented with 10 % fetal bovine serum , 2 mm l - glutamine , 1 mm sodium pyruvate , 15 mm hepes , 10 nm 17β - estradiol , and antibiotics [ mezr medium ] ( huber , m . and poulin , r . 1995 . cancer res ., 55 , 934 - 943 ). cho - k1 cells were routinely grown in α - minimal essential medium supplemented with 10 % cosmic ™ calf serum in a 5 % co 2 humid atmosphere at 37 ° c . even though the present invention has been described with a certain degree of particularity , it is evident that many alternatives , modifications , and variations will be apparent to those skilled in the art in light of the following disclosure . accordingly , it is intended that all such alternatives , modifications , and variations which fall within the spirit and the scope of the invention be embraced by the defined claims . for growth studies , zr - 75 - 1 cells were cultured in mezr medium or in phenol red - free rpmi 1640 supplemented with 2 mm l - glutamine , 1 mm sodium pyruvate , 15 mm hepes , antibiotics , 1 nm 17β - estradiol , 0 . 5 μg of bovine insulin per ml and 5 % ( v / v ) charcoal - treated fetal bovine serum ( sd medium ), as indicated in the text . when polyamines or polyamine analogs were added to serum - containing media , 1 mm aminoguanidine was added to inhibit bovine serum amine oxidase ( bsao ) activity ( morgan , d . m . l . 1989 , in the physiology of polyamines ( bachrach , u ., and heimer , y . m . eds ) vol . i , pp . 203 - 229 , crc press , boca raton ). the effect of the transport inhibitors on cell growth was measured by incubating zr - 75 - 1 cells for 11 days in medium supplemented with antagonist , polyamines and / or 1 mm dfmo as indicated , followed by colorimetric determination of dna content with 3 , 4 - diaminobenzoic acid ( simard , j ., dauvois , s ., haagensen , d . e ., lėvesque c ., mėrand , y . and labriė , f . 1990 . endocrinology 126 : 3223 - 3231 ). medium was changed every other day in these experiments because of the slow reaction of the compound with an unknown component present in the imem and rpmi 1640 medium formulation . zr - 75 - 1 cells were plated in 100 mm culture dishes at 5 × 10 5 cells / dish in mezr medium and grown for 5 days with medium changes every other day . fresh mezr medium containing the indicated concentration of transport antagonist was then added , plus or minus 200 μm cycloheximide ( chx ), and cells were incubated for 1 or 6 hours . medium was then removed , cell monolayers rinsed twice with 10 ml of ice - cold ca 2 + / mg 2 + - free phosphate buffered - saline ( pbs ) ( 2 . 7 mm kcl ; 1 . 5 mm kh 2 po 4 ; 8 . 1 mm na 2 hpo 4 ; 137 mm nacl ), and harvested by centrifugation ( 2000 × g × 90 s at 4 ° c .) following a 5 - to 7 - minute - incubation with bovine trypsin / edta solution ( 0 . 05 %/ 0 . 02 %, w / v ) in hanks &# 39 ; balanced salt solution ( huber , m . and poulin , r . 1995 . cancer res . 55 ; 934 - 943 ). cell pellets were resuspended in 300 μl of 10 % ( v / v ) trichloroacetic acid or tris - dtt buffer ( 50 mm tris / hcl , 0 . 1 mm edta , 5 mm dtt , ph 7 . 5 ) and stored at − 20 ° c . until further analysis . for chromatographic analysis , samples were first quickly thawed and incubated for 15 minutes at 37 ° c . trichloroacetic acid was then added to dtt - containing samples to a final concentration of 10 % ( wt / v ). samples were dispersed for 2 minutes in a sonicating water bath , and pelleted in a microcentrifuge for 5 minutes . the trichloroacetic acid - insoluble pellet was solubilized in 300 - 500 μl of 1 n naoh and used to determine protein content using bovine serum albumin ( fraction v ) as standard . polyamine contents were then analyzed by ion pair reverse - phase hplc with fluorometric detection after postcolumn derivatization with o - phthaldialdehyde as described ( pegg , a . e ., wechter , r ., poulin , r ., waster , p . m ., and coward , j . k . 1989 . biochemistry 28 : 8446 - 8453 ; huber , m ., and poulin , r . 1996 . cancer res ., 55 : 934 - 943 ). in this system , putrescine , spermidine , spermine , mesc and deasc were resolved with retention times of 18 . 5 , 31 . 0 , 35 . 0 , 36 . 5 , 37 . 5 , and 44 . 0 minutes respectively . desc stability was tested by incubating the compound dissolved ( at 50 μm ) in pbs or in imem medium containing 10 % ( v / v ) fetal bovine serum plus or minus 1 mm aminoguanidine in a humid 5 % co 2 atmosphere at 37 ° c . and in the absence of cells . at indicated times , trichloroacetic acid was added to aliquots of this solution to a final concentration 10 % ( w / v ) and the samples directly analyzed by hplc as above . the rate of putrescine and spermidine transport was determined in zr - 75 - 1 cells incubated in serum - free rpmi 1640 medium as described ( lessard , m ., zhao , c ., singh , s . m . and poulin , r . 1995 . j . biol . chem . 270 : 1685 - 1694 ), using [ 3 h ] putrescine ( 30 ci / mol ) and [ 3 h ] spermidine ( 20 ci / mol ), respectively as substrates for a 20 minute - assay period . spermine uptake was similarly determined , using 1 μm [ 14 c ] spermine ( 32 ci / mol ) as substrate . uptake activity was expressed per amount of dna as fluorometrically determined using 3 , 4 - diaminobenzoic acid ( simard , j ., dauvois , s ., haagensen , d . e ., lėvesque , c ., mėrand , y . and labrie , f . 1990 . endocrinology , 126 : 3223 - 3231 ). for the determination of spermidine uptake activity in cho - k1 cells , 80 % confluent cell monolayers were rinsed twice with pbs and incubated for 20 minutes at 37 ° c . in 400 μl of buffer a ( 20 mm tris - hcl , ph 7 . 4 ; 0 . 42 mm cacl 2 ; 0 . 41 mm mgso 4 ; 103 mm nacl ; 5 . 7 mm kcl ; 1 . 1 mm d - glucose ) containing 5 μm [ 3 h ] spermidine ( 20 ci / mol ). cell cultures were then washed twice with 1 ml pbs containing 5 . 7 mm sym - norspermidine . cells were then lysed with 200 - μl aliquot of 1 n naoh and incubated for 30 minutes at 60 ° c . after neutralization with 200 μl of 1 n hcl , radioactivity was determined from a 250 - μl of the cell lysate by scintillation counting . uptake activity was expressed per amount of total cellular protein as determined by the method of bradford ( bradford , m . m . 1976 . anal . biochem . 72 : 248 - 254 ). non - specific binding of radioactive substrate was similarly determined in parallel for both cell lines after a 15 second - incubation with 400 μl of ice - cold uptake solution . kinetic analysis of polyamine transport was performed by determining uptake activity in the presence of a 3 μm [ 3 h ] putrescine or 1 μm [ 3 h ] spermidine plus increasing concentrations of nonradioactive substrate . k ?? k i and v max values were then estimated by lineweaver - burke analysis . for competitive inhibitors , k i values were also estimated by measuring uptake activity in the presence of logarithmically increasing concentrations of antagonist , and using the cheng - prusoff equation ( cheng , y .- c . and prusoff , w . h . 1973 . biochem . pharmacol . 22 : 3099 - 3108 ) by iterative curve fitting for a sigmoidal curve . for mixed competitive / noncompetitive inhibition , two methods were used to calculate kinetic constants . first , the equation where v , s , and i are the transport velocity , substrate concentration and inhibitor concentration respectively , was used to calculate the inhibition constants for inhibitor / carrier complex formation ( k i ) and carrier / inhibitor / substrate complex formation ( k i ′) ( dixon , m . and webb , e . c . 1976 . enzymes , 3rd ed ., academic press , san diego , calif .). alternatively , the value of k i for a mixed competitor / non - competitor was estimated from the intersect of equations v − 1 vs i at two different substrate concentrations ( dixon , m . and webb , e . c . 1976 . enzymes , 3rd ed ., academic press , san diego , calif .). the time course of intracellular accumulation of spermidine in the presence of transport antagonists was determined by incubating zr - 75 - 1 cells in 24 - well plates with desc ( 50 or 200 μm ) or mesc ( 200 μm ) in dissolved in mezr medium containing 5 μm [ 3 h ] spermidine in the presence or absence of cycloheximide ( chx , 200 μm ), and harvesting at the indicated times for the determination of intracellular radioactive contents , as described above for polyamine uptake assays . statistical significance of differences between means was assessed by unpaired student &# 39 ; s t - tests . unless otherwise indicated , results are expressed as means ± sd of determinations from triplicate cell cultures . the original rationale for synthesizing mesc ( compound vii of fig1 ) was to generate an affinity reagent with a thiol side chain that could be derivatized with fluorescent or radioactive sulfhydryl reagents to label the polyamine transporter . the precursor chosen for the synthesis , namely 5 - carboxyspermine , has been previously used to prepare lipopolyamines for efficient dna transfection ( behr , j . p . 1989 . j . chem . soc . chem . commun . 101 - 103 ; behr , j . p ., demeneix , b ., loeffler , j .- p . and perez - mutul , j . 1989 . proc . natl . acad . sci . usa 86 : 6982 - 6986 ), and more recently , as a photoaffinity reagent to label the polyamine - binding site of casein kinase 2 ( leroy , d ., schmid , m ., behr , j .- p ., filhol , o ., pares , s ., garin , j ., bourgarit , j .- j ., chambaz , e . m . and cochet , c . 1995 j . biol . chem . 270 : 17400 - 17406 ). the scheme used to prepare mesc involved the coupling of a cystamine bridge through amide bonds with two boc - protected 5 - carboxyspermine molecules to form desc after removal of the boc groups ( compound v of fig1 ), followed by reduction of the desc disulfide bridge . a small amount ( 10 - 15 %) of the mixed mesc - cysteamine disulfide ( deasc , compound vi ; fig1 ) was also generated in the coupling process . complete separation of deasc from desc on a preparative basis proved to be difficult even using ion exchange chromatography ( data not shown ). consequently , most desc preparations contained a small amount ( 1 - 2 %) of deasc after reversed - phase liquid chromatography on c ?? silica gel . desc and deasc were stable for months in aqueous solutions buffered at ph = 7 . 0 , whereas mesc solutions were supplemented with dtt to prevent oxidation . affinity of desc , deasc and mesc for the mammalian diamine and polyamine transport in order to evaluate the suitability of the spermine conjugates as prospective affinity ligands , their relative ability to inhibit putrescine and polyamine uptake was evaluated . as shown in fig2 , desc was the most potent antagonist of [ 14 c ] spermine transport in zr - 75 - 1 cells , with a k i value about 5 - fold and 16 - fold lower than that of deasc and mesc , respectively . the ability of spermine to compete against [ 3 h ] putrescine and [ 3 h ] spermidine uptake was in fact only about 7 - fold higher than that of desc ( fig3 ). desc ( fig4 a ) and mesc ( data not shown ) were pure competitive inhibitors of [ 3 h ] putrescine uptake at concentrations up to 100 and 200 μm , respectively . on the other hand , inhibition of putrescine transport by deasc belonged to a mixed competitive / non - competitive type ( fig4 b ). table i summarizes the k i values determined for desc , mesc and deasc toward putrescine , spermidine and / or spermine uptake , in relation with the mutual transport interactions between the latter substrates . notably , k i values of the three spermine conjugates with respect to putrescine uptake were 3 - fold to 5 - fold higher than for spermine uptake , unlike spermidine and spermine which both inhibited the uptake of either substrate with similar potency , and with a k i roughly equal to their k m as substrate . k i values of inhibition of diamine and polyamine transport by data annotated with a are from lessard , m ., zhao , c ., singh , s . m ., and poulin , r . 1995 . j . biol . 270 : 1685 - 1694 , b indicates data obtained with this work ; mean ± sd of triplicate determinations from 2 to 4 different experiments ; c corresponds to values of inhibition constants for carrier / inhibitor complex formation ( k i ) and for carrier / inhibitor / putrescine complex formation ( k i ′) assuming a mixed competitive / non - competitive model ; mean ± sd of triplicate determinations at 3 three inhibitor concentrations at two different substrate concentrations for a series of increasing inhibitor concentrations ( dixon , m , and webb , e . c . 1976 . enzymes , 3rd ed ., academic press , san diego , calif .). the relative potency of desc and mesc as competitive inhibitors of polyamine uptake was also evaluated in cho - k1 cells , in which they respectively exhibited k i values of 0 . 92 ± 0 . 15 and 33 . 6 ± 7 . 2 μm ( fig5 ). effect of side chain length and substituents on spermidine transport inhibition by mesc derivatives the observation that mesc was a less potent inhibitor of diamine and polyamine transport than desc and deasc suggested that the nature of the side chain strongly influences the interaction of these compounds with the carrier . the thiol side chain of mesc was thus derivatized with substituting groups of different sizes and charges through thioether linkage with three different iodoacetamides , namely ly iodoacetamide , asib and iodoacetamide itself , and the ability of the resulting complexes ( mesc - ly , mesc - asib , and mesc - acetamide , respectively ) to inhibit spermidine uptake was then evaluated . these studies were conducted using cho - k1 cells . as shown in fig5 , derivatization of the thiol group of mesc did not significantly ( p & gt ; 0 . 10 ) increase the k i toward spermidine uptake for the three conjugates studied . in the case of mesc - asib , ki values might have been underestimated by partial inactivation of the polyamine carrier at the assay temperature , although the uptake reaction was conducted under subdued lighting . thus , the results show that specific recognition of the spermine head of mesc can accommodate considerable variation in length , size , polarity or charge for the side chain without detrimental effect on its affinity for the polyamine carrier . consequently , inhibitors having different side chains , while maintaining their inhibitory activity on polyamine transport are also encompassed by the present invention . lack of permeation of desc and mesc through the polyamine transport system the ability of zr - 75 - 1 cells to accumulate desc and mesc was determined . since desc was eluted as a late , broad peak in the hplc system used , dtt was added to cell extracts to reduce desc to mesc and decrease the detection threshold . results are shown in table ii . zr - 75 - 1 cells were incubated for 1 or 8 hours in mezr medium in the presence of 50 or 200 μm desc or mesc prior to determination of polyamine contents . chx was added at 200 μm where indicated . other details are provided under “ materials and methods .” values are the mean ± sd of triplicate determinations from 2 independent experiments . a significantly different ( p & lt ; 0 . 5 ) from control value at time = 1 h . (?) as shown in table ii , only trace amounts of desc could be recovered in zr - 75 - 1 cells after a 6 - hour incubation with 200 μm desc , but not with 50 μm , desc could be detected only after reduction with dtt . these levels represent only about 1 . 5 % of the accumulation measured in zr - 75 - 1 cells under identical conditions for spermine ( lessard , m ., zhao , c ., singh , s . m . and poulin , r ., 1995 . j . biol . chem . 270 : 1685 - 1694 ). moreover , inhibition of protein synthesis by cycloheximide ( chx ), which is known to upregulate polyamine uptake by preventing the synthesis of a polyamine - induced feedback repressor of transport ( lessard , m ., zhao , c ., singh , s . m . and poulin , r . 1995 . j . biol . chem . 270 : 1685 - 1694 ; mitchell , j . l . a ., diveley , r . r ., jr . and bareyal - leyser , a . 1992 . biochem . biophys . res . commun . 186 : 81 - 88 ), did not enhance desc internalization , in marked contrast with its effect on spermidine accumulation under similar conditions ( fig6 b ) ( lessard , b ., zhao , c ., singh , s . m . and poulin , r ., 1995 . j . biol . chem . 270 : 1685 - 1694 ). likewise , mesc was accumulated to measurable levels by zr - 75 - 1 cells only when present at 200 μm ( cf . table ii ). thus , neither desc or mesc appear to be used as substrates for the polyamine transport system despite the high affinity of the former compound as an antagonist of diamine and polyamine uptake . the time course of internalization of radio - labeled spermidine was determined in zr - 75 - 1 cells incubated for up to 6 hours in the presence of the impermeant agonists . as illustrated in fig6 a , steady - state [ 3 h ] spermidine accumulation in the absence of competitor abruptly reached a near plateau after about 1 hour , which results from the induction of feedback inhibition of polyamine transport ( lessard , m ., zhao , c ., singh , s . m . and poulin , r ., 1995 . j . biol . chem . 270 : 1685 - 1694 ). mesc and desc decreased the initial rate of spermidine uptake according to their respective potency as competitive antagonists . interestingly , spermidine accumulation in the presence of either inhibitor followed a pattern similar to that of control cells , i . e . a rapid phase during the first 60 minutes , followed by a much slower rate of accumulation thereafter , which was nearly independent of antagonist concentration . this pattern suggests that even cellular levels of newly internalized spermidine as low as 20 % of those found under control conditions , e . g ., in cells treated with 200 μm desc , may induce a near maximal degree of feedback repression of polyamine transport . nevertheless , even a 40 - fold excess of the most potent antagonist ( i . e . 200 μm desc ) only decreased net spermidine accumulation by only 50 % after 6 hours . as previously observed (( lessard , m ., zhao , c ., singh , s . m . and poulin , r ., 1995 . j . biol . chem . 270 : 1685 - 1694 ), chx abolished the induction of feedback transport inhibition , resulting in a 4 - fold increase in spermidine accumulation after 4 hours ( fig6 b ). protein synthesis inhibition also enhanced spermidine accumulation in desc - treated cells , a finding consistent with the onset of substantial feedback transport repression by subthreshold levels of internalized substrate . thus , in the absence of the feedback mechanism , the highest concentration of desc tested ( 200 μm ) decreased net [ 3 h ] spermidine accumulation by 80 to 85 % after 6 hours and to a level lower than that found in control cells with a fully repressed uptake activity . due to the analogy of the novel transport antagonists with spermine , it might be surmised that they would exhibit significant cytotoxicity like the parent molecule . the marked toxicity of low (& lt ; 10 − 3 m ) spermine concentrations in biological media mostly results from catabolism by copper amine oxidases , which generates a dialdehyde , acrolein and h 2 o 2 as deleterious products and can be irreversibly inhibited by carbonyl reagents such as aminoguanidine ( morgan , d . m . l . 1989 . in the physiology of polyamines ( bachrach , u ., and heimer , y . m . eds ) vol . i , pp . 203 - 229 , crc press , boca raton ). the biocompatibility of desc , mesc , and deasc was thus evaluated during a long - term ( 11 - day ) incubation with zr - 75 - 1 cells grown in rpmi 1640 containing 10 % ( v / v ) fbs in the absence and presence of 1 mm aminoguanidine . as shown in fig7 , aminoguanidine alone had a slight inhibitory effect on zr - 75 - 1 cell growth as previously observed ( huber , m . and poulin , r . 1995 . cancer res . 55 : 934 - 943 ). although desc was only mildly growth inhibitory at 50 μm , there was an abrupt , aminoguanidine - resistant increase in toxicity at 200 μm . in contrast , spermine was acutely cytotoxic at 50 μm , an effect that was only partly prevented by aminoguanidine . mesc was considerably less toxic than its dimer , with a 35 % decrease in cell growth at 200 μm which was not blocked by aminoguanidine . on the other hand , 50 μm deasc caused a 20 % inhibition of cell proliferation which could be completely prevented by the amine oxidase inhibitor . thus , desc , and to a much lesser degree , its thiol monomer mesc , are cytotoxic toward breast cancer cells at high concentrations through a mechanism that does not involve bsao . weak growth inhibition caused by the mixed mesc - cysteamine disulfide , however , apparently involved degradation by a copper amine oxidase . effect of desc on rescue of demo - induced growth inhibition by exogenous spermidine although desc is indeed a potent antagonist of polyamine accumulation , the slow residual uptake that occurred even at a 40 - fold molar excess of inhibitor might be sufficient to counteract polyamine depletion by inhibitors of polyamine biosynthesis . this possibility was assessed by comparing the ability of desc to prevent the reversal of dfmo - induced growth inhibition by increasing concentrations of exogenous spermidine . at concentrations superior to 0 . 3 μm , spermidine inhibited zr - 75 - 1 cell proliferation by up to 20 % ( fig8 ). this effect could be due to an incomplete inhibition of bsao by aminoguanidine ( seiler , n . 1987 . in inhibition of polyamine metabolism . biological significance and basis for new therapies ( mccann , p . p ., pegg , a . e . and sjoerdsma , a . eds . ), pp . 49 - 77 , academic press , orlando ), since it was not observed in media supplemented with equine serum , which does not contain amine oxidase activity ( blaschko , h . and hawes , r . 1959 . j . physiol . 145 : 124 - 131 ), instead of fbs ( data not shown ). the approximately 50 % growth inhibition induced by 1 mm dfmo after an 11 - day incubation was completely reversed by as little as 0 . 3 μm spermidine , whereas 0 . 1 μm spermidine already restored growth of dfmo - treated cells to 78 % of control value . however , addition of 50 μm desc was unable to prevent the reversal of dfmo - induced growth inhibition by spermidine , even at a desc : spermidine ratio of 500 . essentially similar results were obtained using horse serum instead of fbs , or replacing rpmi 1640 medium , which contains 3 . 2 μm reduced glutathione that might undergo thiol / disulfide exchange with desc , with thiol - free imem ( data not shown ). the inability of desc to block the biological effect of exogenous spermidine , even when present at large molar excesses , might have been caused by its degradation in growth media . to assess this hypothesis , desc solutions ( 20 μm ) made in pbs or in sterile imem medium enriched with 10 % ( v / v ) fbs were incubated for 20 minutes or 48 hours under cell - free conditions at 37 ° c . in a humid 5 % co 2 atmosphere , and the polyamine analog was then analyzed by ion - pair reversed - phase hplc . after 48 hours , degradation of desc to two new amine - containing derivatives occurred in imem ( fig9 a , b ) but not in pbs ( fig9 c ), as evidenced by the appearance of a major ( compound 1 ) and minor ( compound 2 ) peaks of o - phthaldialdehyde - reactive material eluting earlier than desc . although aminoguanidine did not prevent desc degradation to the two unknown products , it did prevent the degradation of a trace amount of deasc ( indicated as compound 3 ) initially present in the desc preparations , thus confirming that deasc can indeed be a substrate of serum copper amine oxidase ( fig7 ). mesc could not be detected , indicating that desc does not undergo reduction to mesc under conditions used for cell culture . furthermore , the decomposition of desc in imem showed an identical pattern in the presence or absence of fbs , which thus ruled out a serum component as being responsible for the degradation . fig1 shows that desc was slowly degraded to compounds 1 and 2 . after 48 hours , i . e . the interval at which freshly made desc - containing media were added to cell cultures in growth experiments , 40 % of the desc originally present had been decomposed by imem . identical results were obtained using rpmi 1640 medium instead of imem . thus , the present inventors propose that a component present in imem and rpmi 1640 medium , but not in pbs , must be responsible for the degradation of desc . desc , a novel type of spermine derivative , is shown to be endowed with high affinity for the polyamine transport system while being highly resistant to cellular uptake . the combination of these two attributes confers unique characteristics to desc as a pure competitive antagonist of polyamine uptake . as compared with spermine , the higher ki of mesc against putrescine , spermidine and spermine uptake could owe to the presence of an amide linkage , which decreases the basicity of the neighboring secondary amino group of the spermine head ( pka = 5 . 5 in comparison with 8 . 9 - 9 . 8 for spermine ) ( tabor , c . w . and tabor , h . 1984 . ann . rev . biochem . 53 : 749 - 790 ; remy , j .- s ., kichler , a ., mordvinov , v ., schuber , f . and behr , j .- p . 1995 . proc . natl . acad . sci . usa 92 : 1744 - 1748 , and / or may cause steric hindrance for its interaction with the polyamine binding site ( bergeron , r . j . and seligsohn , h . w . 1986 . bioinorg . chem . 14 : 345 - 355 ; porter , c . w ., cavanaugh , p . f ., jr ., stolowich , n ., ganis , b ., kelly , e ., and bergeron , r . j . 1985 . cancer res . 45 : 2050 - 2057 ). despite the particular structural features of mesc as a ligand , its dimerization into desc increased by up to 20 - fold the affinity of the resulting structure for the polyamine transporter . there is no precedent for dimeric polyamine structures like desc . its overall design is reminiscent of that of 2 - n - 4 -( 1 - azi - 2 , 2 , 2 ,- tri - fluoroethyl ) benzoyl - 1 , 3 - bis ( d - mannos - 4 - yloxy )- 2 - propylamine , an impermeant ligand which binds to the exofacial domain of facilitative glucose transporters and bears two symmetrical sugar moieties linked tail to tail ( clark , a . e . and holman , g . d . 1990 . biochem . j . 269 : 615 - 622 ). at least one mammalian glucose transporter , namely glut - 1 , exists as a tetrameric complex in its native form ( hebert , d . n . and carruthers , a . 1992 . j . biol . chem . 267 : 23829 - 23838 ; gould , g . w . and holman , g . d . 1993 , biochem . j . 295 : 329 - 341 ). the stronger affinity of desc relative to mesc could reflect a dyad symmetry in the organization of the transporter complex . alternatively , dimerization of mesc into desc could impose conformational constraints ( e . g . due to electrostatic repulsion ) that would favor recognition of the polyamine binding site of the carrier by each of the symmetrical spermine moieties . mesc thioethers as diverse in size as mesc - ly , mesc - asib , or mesc - acetamide had k i values virtually identical to that of mesc , indicating that the thiol group of mesc does not specifically determine its lower affinity as a polyamine transport inhibitor as compared with desc . these data suggest that additional bulk on the side chain has little influence on the interaction of mesc with the polyamine transporter , in agreement with the observation that large substituents attached to the distal end of a spacer of sufficient length do not notably decrease the affinity of spermidine as a substrate for uptake ( holley , j . o ., mather , a ., wheelhouse , r . t ., cullis , p . m ., hartley , j . a ., bingham , j . p ., and cohen , g . m . 1992 . cancer res ., 52 : 4190 - 4195 ). unexpectedly , the mesc - cysteamine mixed disulfide ( deasc ) was found to block putrescine uptake as a mixed competitor / non - competitor , whereas mesc and desc behaved like pure competitive inhibitors of putrescine transport . since the interaction of desc or mesc with the polyamine transporter was strictly competitive , and because deasc exhibits higher affinity than mesc as an inhibitor of diamine and polyamine transport , the spermine head and the cysteamine side chain of deasc might be respectively responsible for the competitive and non - competitive components of its transport inhibition . the biochemical properties of desc clearly illustrate that the binding affinity of a compound can be dissociated from its ability to serve as a substrate for the polyamine transporter . the large size of desc cannot be the main factor preventing its internalization through the channel - like portion of the transporter since mesc was also virtually impermeant . thus , the mere attachment of an amido side chain on the spermine backbone would appear to be responsible per se for the impaired internalization of mesc and its derivatives . indeed , n 4 - alkylated spermidine derivatives are far better competitors of spermidine uptake than their n 4 - acyl counterparts in mouse leukemia cells , in support of the notion that charged secondary amino groups are important in the interaction with the polyamine carrier ( porter , c . w ., cavanaugh , p . f ., jr ., stolowich , n ., ganis , b ., kelly , e ., and bergeron , r . j . 1985 . cancer res . 45 : 2050 - 2057 ). however , the latter argument cannot account for the fact that long - chain aliphatic , α , ω - diamines with at least 6 to 7 methylene groups have an affinity comparable to that of spermidine ( lessard , m ., zhao , c ., singh , s . m . and poulin , r ., 1995 . j . biol . chem . 270 : 1685 - 1694 , bergeron , r . j . and seligson , h . w . 1986 . bioinorg . chem . 14 : 345 - 355 ; porter , c . w . and bergeron , r . j . 1983 . science 219 : 1083 - 1085 ; minchin , r . f ., martin , r . l ., summers , l . a . and hett , k . f ., 1989 . biochem . j . 262 : 391 - 395 ; gordonsmith , r . h ., brooke - taylor , s ., smith , l . l . and cohen , g . m . 1983 . biochem . pharmacol . 32 : 3701 - 3709 ). a more likely explanation for the poor affinity of polyamines bearing an acyl side chain might be the steric hindrance due to the amide group , which restricts the freedom of rotation around the adjacent carbon and nitrogen atoms . there are indications that cyclic or pseudocyclic conformations of polyamines stabilized by hydrogen bonds might be energetically favored for recognition and / or internalization of substrates of the polyamine transport system ( lessard , m ., zhao , c ., singh , s . m . and poulin , r ., 1995 . j . biol . chem . 270 : 1685 - 1694 ; bergeron , r . j . and seligsohn , h . w . 1986 . bioinorg . chem . 14 : 345 - 355 ). the formation of such folded conformers would be impaired by the presence of an amide group next to the polyamine chain . in support of this hypothesis , chlorambucil - spermidine , which bears a n - propyl chlorambucil carboxamide side chain on the central nitrogen of spermidine , is a good substrate of the polyamine transport system , with a k m averaging that of spermidine ( holley , j . l ., mather , a ., wheelhouse , r . t . cullis , p . m ., hartley , j . a ., bingham , j . p ., and cohen , g . m . 1992 . cancer res . 52 : 4190 - 4195 ). in marked contrast , a spermidine conjugate with a chlorambucil carboxamide side chain directly attached at the c5 position of the spermidine head is a very poor substrate of the polyamine uptake system ( stark , p . a ., thrall , b . d ., meadows , g . g ., and abdel - monam , m . m . 1992 . j . med . chem . 35 : 4264 - 4269 ). although a 40 - fold molar excess of desc dramatically reduced the rate of spermidine uptake in zr - 75 - 1 cells , slow but continuous spermidine accumulation was still observed in the presence of the inhibitor . the low rate of polyamine internalization observed , even in the presence of a large excess of desc , in addition to the slow decomposition of the inhibitor , may largely explain the complete inability of desc to prevent polyamine - mediated prevention of growth inhibition by dfmo . since the affinity of mesc thioethers remains virtually unaffected relative to the unconjugated polyamine , mesc - asib might serve as a photoaffinity label to detect polyamine - binding proteins , including the polyamine carrier . experiments are currently conducted with 125 i - labeled mesc - asib to assess its usefulness as a probe to identify the mammalian polyamine transporter . a recent report has described the specific labeling of discrete plasma membrane proteins using 125 i - labeled n 1 - azidosalicylamido - norspermine and n 4 - azido - salicylamidoethylspermidine as photoaffinity reagents ( felschow , d . m ., macdiarmid , j ., bardos , t ., wu , r ., woster , p . m . and porter , c . w . 1995 . j . biol . chem . 270 : 28705 - 28711 ). however , these conjugates are internalized by mammalian cells ( felschow , d . m ., macdiarmid , j ., bardos , t ., wu , r ., woster , p . m . and porter , c . w . 1995 . j . biol . chem . 270 : 28705 - 28711 ), and mesc - asib or similar derivatives could be useful as a photoactivatable probes to exclude labeling of intracellular proteins . while not intended to be limited to any particular theory , the slow degradation of desc observed in growth media , but not in pbs , was likely due to l - cystine , which is present at 100 and 200 μm in imem or rpmi 1640 medium , respectively , through the formation of mixed disulfides with desc . nevertheless , the cytotoxicity of high concentrations of desc and mesc is unlikely to be solely due to the formation of such adducts , since mesc was less toxic than desc , despite the fact that the free thiol group of the former would make it more reactive toward l - cystine ]. the present data clearly show that desc has remarkably low toxicity in comparison with its homolog spermine . thus , the basic features of this molecule , including its resistance to bsao , should be useful for the design of potent transport inhibitors with minor non - specific effects on cell viability . the inherent structural features of desc that confer its high affinity and resistance to uptake should thus provide a useful framework for the design of potent irreversible inhibitors of polyamine transport , which could incorporate an alkylating group such as that used in the design of specific suicide substrates of mammalian glucose transporters ( clark , a . e , and holman , g . d . 1990 . biochem . j . 269 : 615 - 622 ; lehmann , j ., and scheuring , m . 1995 . carbohydrate res . 276 : 57 - 74 )]. polyamine derivatives ( natural or synthetic ) comprising sulfur in the side chain have been made , because they conducted to the formation of dimers simply by forming a disulfide bridge . by - products which are not dimers have also shown an activity . however , it will be readily apparent to those skilled in the art that compounds being more stable than those containing sulfur atoms are contemplated . therefore , the side chains used for increasing the affinity of the derivatives for a polyamine transporter and / or as substrates for labeling molecules and / or as a spacer in the making of a dimer can be varied to optimize the characteristics of the derivatives of the present invention . any equivalent structures of modifications obtainable without departing from the teachings and the spirit of this invention are considered as part of the scope thereof because the invention is in no way limited to the particularly disclosed embodiments , as reflected in the appended claims . synthesis and evaluation of spermine dimers as inhibitors or polyamine transport and enhancers of eflornithine action in tumor cells and tumor - bearing animals novel spermine analogs will be synthesized and evaluated as blockers of transport in tumor cells simultaneously treated with d , l - α - difluoromethylornithine ( fmo = eflornithine ). these molecules are based on the overall design of a prototype , 2 , 2 ′- dithiobis ( n - ethyl - spermine - 5 - carboxamide ) ( desc ). desc has recently been reported to act as a competitive and potent antagonist of polyamine uptake in leukemia and breast cancer cells . desc is proposed here to potentiate the chemotherapeutic efficacy of dfmo . while not intending to be limited to any particular theory , it is proposed that such effect is provided by preventing the replenishment of dfmo - treated tumor cells with polyamines from exogenous sources . structural modifications to the molecule will improve it to a pharmacologically useful compound . these modification include : [ i ] the replacement of the disulfide bridge with a fully reduced aliphatic chain to prevent its rapid reaction with biological thiols and disulfides and [ ii ] the addition of substituents to prevent its oxidative deamination by the ubiquitous plasma enzyme , serum amine oxidase . two types of desc analogs will be synthesized , and characterized for their ability to inhibit polyamine transport and to enhance the therapeutic action of dfmo in various tumor cell types , including animal models . the first type of analogs will be simply obtained by substituting the original cystamine side chain of desc with α , ω - diamine cross - linkers of varying length . the synthesis of these analogs will help in the short term to optimize the length of the cross - linker chain , and to rapidly evaluate their relative ability to potentiate dfmo action in vitro . the second type of analogs will be made according to a new route of synthesis to introduce methyl groups at the extremities of the spermine - like backbone , and will also incorporate alkylation instead of acylation of the aliphatic , α , ω - diamine cross - linker in order to improve their affinity for the polyamine transport system , their potency as antagonists of uptake and as enhancers of dfmo therapeutic action . the pharmacological evaluation of the second - type analogs will be conducted in a standard mouse model bearing l1210 leukemia tumor cell treated with dfmo . 2 , 2 ′- dithiobis ( n - ethyl - spermine - 5 - carboxamide ) ( desc ) and its thiol monomer , n -( 2 - mercaptoethyl ) spermine 5 - carboxamide ( mesc ) ( fig1 ) have been synthesized as precursors of photoaffinity labeling probes of polyamine - binding proteins ( 21 ). characterization of the potency of desc and mesc to inhibit polyamine transport unexpectedly showed that desc has ≢ 20 - fold higher affinity than mesc for the polyamine carrier . the marked difference in transport inhibitory potency between mesc and its dimer suggested that the carrier protein might have a dyadic symmetry , and that the conjugation of two spermine molecules through a cross - linking side chain could markedly enhance the interaction with the polyamine transporter . moreover , neither desc nor mesc is significantly internalized by human breast cancer cells or mouse leukemia cells at concentrations that saturate the polyamine carrier , indicating that they are essentially membrane - impermeant ( 21 ). the combination of high affinity and lack of carrier - mediated permeation of desc provided the basis for a novel design of pure polyamine transport antagonists that could be used in combination with dfmo to enhance polyamine depletion in tumor cells exposed to physiological levels of exogenous polyamines . desc was designed for biochemical use . it was found to degraded in physiological media due to thiol - disulfide reaction with compounds such as l - cystine . desc cannot efficiently counteract the ability of exogenous spermidine to reverse dfmo - induced cytostasis in breast cancer cells as a result of this instability ( 21 ). desc is also subject to attack by serum amine oxidase ( sao ), an ubiquitous plasma enzyme which oxidatively deaminates aminopropyl groups , albeit to a much lesser degree than the parent compound spermine . modifications that further improve the design of desc analogs that are part of particular embodiments of the present invention are : ( 1 ) to use chemically inert , aliphatic diamines as cross - linkers to conjugate two spermine - like moieties ; and / or ( 2 ) to introduce methyl groups on the terminal carbons of the spermine - like backbones of the molecule . this latter modification will prevent / reduce the oxidation of spermine by sao . 8 . 2 ) synthesis and biochemical evaluation of unmethylated , stable desc analogs desc analogs are prepared with unmodified - spermine backbones but different side chain lengths as lead compounds to guide us in the design of methylated analogs described herein . this series of compounds will be synthesized in order to : ( i ) perform a structure - function study in the short - term to determine the optimal length of the cross - linker for inhibition of polyamine uptake . compounds viiia to viiid ( fig1 ) will be rapidly available in amounts sufficient for in vitro testing . a refinement to the originally propose route of synthesis will be the use of fmoc - blocked diamine precursors . one such diamine precursor is nh 2 ) ch 2 ) n nh 2 where n = 3 to 6 . instead of simultaneously coupling two spermine - like moieties to a diamine cross - linker , each amino group of the diamine cross - linker was sequentially amidated to the spermine - like precursor with the n - fmoc - diamine , and then the other amine group was deprotected for the second amidation reaction . this sequential reaction scheme improves the purification of the spermine dimer from the spermine monoamide . this was difficult to achieve with the original method . the present method will improve the yield of desired product through a better control of the reaction stoichiometry . the kinetic properties of these desc analogs ( abbreviated as bs - 3 , bs - 4 , bs - 5 and bs - 6 ; fig1 ), as inhibitors of polyamine transport will be determined by uptake assays of radiolabeled putrescine , spermidine and spermine , according to procedures in huber et al . ( 1996 ), j . biol . chem ., 271 : 27556 - 27563 , which is specifically incorporated herein by reference . these structures are shown below . ( ii ) use of analogs to potentiate the effect of dfmo in the presence of exogenous polyamines , which is the main criterion of pharmacological activity for polyamine transport inhibitors . these compounds are expected to be stable under cell culture conditions in the presence of aminoguanidine , a sao inhibitor ( 13 , 28 , 40 , 46 , 49 , 66 , 67 ). these polyamine transport inhibitors will be evaluated using zr - 75 - 1 human breast cancer cells and l1210 mouse leukemia cells . briefly , the rate of cell proliferation will be determined in zr - 75 - 1 and l1210 cells grown in the presence or absence of dfmo ( 1 and 5 mm , respectively ), and of the transport inhibitor candidate to be analyzed , in the presence of increasing concentrations of putrescine or spermidine . the ability of the transport antagonist to prevent the reversal of dfmo - induced growth inhibition by exogenous putrescine or spermidine will provide a valid measurement of the pharmacological potential of these compounds as enhancers of dfmo action in vivo . these studies will also include ( a ) dose - response experiments to evaluate the cytotoxicity of these analogs and the optimal concentration for their use as inhibitors of polyamine uptake , and ( b ) measurement of the uptake of the transport inhibitors during incubation with tumor cells by hplc , along with their effect on polyamine pools . since the latter type of inhibitors will rapidly provide the first stable desc analogs available , the thorough analysis of their biological properties with cultured tumor cells will be important to validate the concept of spermine dimers as polyamine transport blockers . moreover , the structure - function relationships of this series will help in refining the design of the methylated analogs described in the following section . 8 . 3 ) design , synthesis and evaluation of oxidation - resistant , stable methylated desc analogs while replacing the nature of the cross - linking chain is a rather straightforward modification , the second alteration required considerable changes in the preparation scheme originally used for desc synthesis . the reduction step proposed to obtain a 1 , 12 - dimethylspermine 5 - carboxyl methyl ester from a 1 , 4 - bis ( 3 - azidobutyramido ) ornithine methyl ester ( fig1 , xiii ) could not spare the ester group , resulting instead into the formation of 1 , 12 - dimethylspermine 5 - carbinol ( fig3 , xiv ). changing the nature of the ester group did not improve the synthesis since steric hindrance problems prevented amidation of the amino groups of ornithine methyl ester the most proximal to the ester group . the nature of the proposed precursor was modified , and two 1 , 12 - dimethylspermine 5 - methyl chains were conjugated to an α , ω - diamine cross - linker through alkylation rather than through amide bonds ( fig1 to 16 ). this modification represents an improvement over the original design , since direct alkylation will lead to compounds with a higher affinity for the polyamine transporter — and higher potency as transport antagonists — as compared with the more rigid acylated analogs , as previously shown for spermidine analogs ( 8 , 19 , 44 , 52 , 54 , 56 ). the proposed scheme of synthesis , for which steps ix to xiv have already been realized , is provided in fig1 to 16 . this improved scheme also includes the use of mono - fmoc - protected diamines as building blocks for cross - linking the dimethylspermine - 5 - methyl precursors , as described above for the unmethylated desc analogs . the resulting compounds are abbreviated as bms - 3 , bms - 4 , bms - 5 and bms - 6 ( fig1 ; compounds xxa to xxd ). the in vitro evaluation of this series will be conducted for the unmethylated desc analogs . the effectiveness of a combination of polyamine depletion with the selected pa transport antagonist as an antitumor strategy will then be assessed in vivo . for this purpose , an established experimental cancer model , namely l1210 mouse leukemia , will be used to evaluate the therapeutic potential of the candidate transport inhibitor . this leukemia model is an aggressive tumor type with a median host survival time of 9 days in the absence of treatment . moreover , it is completely resistant to dfmo as a single tumor agent in vivo ( albeit very sensitive in vitro ), whereas pa transport deficiency and / or reduction of exogenous pa sources confers a striking ability to dfmo to extend survival rates , with complete cure being observed in ≧ 75 % of animals ( 1 , 50 ). protocol 1 — toxicity will first be determined by single i . v . and i . p . injections of logarithmically increasing drug concentration to mice and estimating the ld 50 . blood samples will be taken at intervals to measure the plasma drug concentration by ion pairing reverse - phase hplc ( 22 , 23 ). body weight and liquid consumption will also be monitored for 10 days , at the end of which period animals will be sacrificed to evaluate the incidence of liver and kidney damage . a similar experiment will be conducted by dissolving the drug in the drinking water with free access to the animals . protocol 2 — on day 0 , mice will be injected with l1210 cells , with concomitant treatment with dfmo or vehicle , plus or minus 2 different sublethal doses of the transport antagonist on a daily schedule . oral . i . v . and i . p . routes will be compared for the transport antagonist . survival will be evaluated for up to 120 days , with regular body weight measurements and blood sampling to determine the steady - state plasma concentrations of inhibitor . l1210 cells are strongly immunogenic tumors and cured animals develop extended immunity against this leukemia ( 1 ). thus , to evaluate the curative potential of the drug combination , survivors will be rechallenged with l1210 cells in the absence of treatment and survival monitored . the present example demonstrates the utility of the present invention with the use of compounds that are analogs of spermine that include two chains connected to one another through a linker . the linker molecule that attaches the two spermine chains may be any spacer chain that is capable of bridging the polyamine chains . the two chains may attach to the linker at an internal c atom or an n group within the chain . it is also possible for one chain to be connected to the linker through one of its carbon molecules , while the second chain attaches to the linker molecule through an n group within its chain . ( 1 ) the central carbon chain of the spermine backbone can have between 3 and 7 methylene groups or carbon atoms . this is the range of central chain length that can be accommodated with good affinity by the mammalian polyamine transporter ( 81 ). ( 2 ) each methylene group of the polyamine chains can be modified by methyl groups without compromising the ability of the inhibitor to interact with the polyamine transporter . ( 3 ) the linkage between the polyamine chains and the spacer may comprise any type of linkage compatible with a k i ≦ 20 μm ( relative to spermine ) for the resulting inhibitor , such as direct alkyl substitution or ether group on the central methylene groups ( structure 1 ), or alkylation on the secondary amino ( structure 2 ) groups of the polyamine chain . wherein r 1 is h , methyl , ethyl or propyl , r 2 is h or methyl , x is greater than two and less than five ( 2 & lt ;×& lt ; 5 ), and the sum of y + z is greater than or equal to 2 and less than or equal to 6 ( 2 ≦ y + z ≦ 6 ). r 3 = ch 2 , s , c ═ o or nh ; 2 & lt ;×& lt ; 5 ; 2 ≦ y +≧≦ 6 ; l = a chemical structure ( the linker ) connecting covalently the two polyamine chians via alkyl , amide , ether or thioether bonds with a substituent group ( r 3 ) attached on a carbon atom located between the two most internal amino groups of the polyamine chain . wherein r 1 is h , methyl , ethyl or propyl , r 2 is h or methyl , x is greater than two and less than five ( 2 & lt ;×& lt ; 5 ), w is greater than 2 and less than 8 ( 2 & lt ;×& lt ; 8 ) and the sum of y + z is greater than or equal to 2 and less than or equal to 6 ( 2 ≦ y + z ≦ 6 ). the invention when a carbon of one chain is attached by a linker to the nitrogen of a second chain is represented in structure 3 . wherein r 1 is h , methyl , ethyl or propyl , r 2 is h or methyl , x is greater than two and less than five ( 2 & lt ;× 5 ), w is greater than 2 and less than 8 ( 2 & lt ;×& lt ; 8 ) and the sum of y + z is greater than or equal to 2 and less than or equal to 6 ( 2 ≦ 7 + z ≦ 6 ). r 3 = ch 2 , s , c ═ o or nh ; 2 & lt ;×& lt ; 5 ; 2 ≦ y +≧≦ 6 ; l = a chemical structure ( the linker ) connecting covalently the two polyamine chains via alkyl , amide , ether or thioether bonds with a substituent group ( r 3 ) attached on a carbon atom located between the two most internal amino groups of the polyamine chain . alkylation can be preferred over amidation because the former allows a greater flexibility to the polyamine chain to adopt the optimal conformation to interact with the polyamine transporter ( 81 ). ( 4 ) the linker ( l ) can be of any nature or chain length , as long as the total mass of the final structure does not exceed 3 , 000 . these molecules may in other embodiments be described as having a total mass of between about 50 to about 2 , 500 , or about between 500 to about 1500 or about 1 , 000 as a total mass . by way of example , such linkers may comprise alkyl , ether , a thioether , amide , phosphono , keto , amine , or sulfonyl groups or a combination thereof . the linker may comprise a carbon chain by a length of 2 to 50 carbons . in some embodiments , the carbon chain will have a length of between 5 to about 25 carbons , or between 10 and 20 carbons , or in even other embodiments , the carbon length of 2 to about 15 or 12 carbons . in another family of synthetic derivitives of original polyamines , the synthetic original polyamine is either sym - norspermidine , sym - homospermidine , or spermidine , and is cross linked to a second original polyamineby a side group on the central amino group of said polyamines . various examples of such molecules , which by no means include all embodiments made possible by this alternative design , are ( a ) spermidine dimers cross - linked by an α , α ′- dialkyl disulfide side chain via n - alkyl bonds with the central amino group of each triamine chain as depicted for type compound i . n , n ′- bis ) 3 - aminopropyl ), n , n ′- bis ( 4 - aminobutyl ) cystamine or babac ( structure i , fig2 ), ( b ) sym - norspermidine dimers [ bnspd -( n + 2 ), structure ii , fig2 ] or spermidine dimers [ bspd -( n + 2 ), structure iii . fig2 ] cross - linked by an aliphatic chain with n + 2 methylene groups ( 0 & lt ; n & lt ; 8 ) via n - alkyl bonds with the central amino group of each triamine chain . ( c ) sym - norspermidine dimers cross - linked by an aryl - containing side chain via n - alkyl bonds with the central amino group of each triamine chain as depicted for type compound iv , n , n , n ′, n - tetrakis ( 3 - aminopropyl )- p - xylylenediamine or tadax ( structure iv , fig2 ), or their sym - homospermidine and spermidine - like homologs ( not illustrated ), ( d ) sym - homospermidine , sym - norspermidine or spermidine dimers cross - linked by an alkene side chain via n - alkyl bonds with the centralamino group of each triamine chain with either a trans ( baba - trans , structure v , fig2 ) or a cis configuration around the single double bond ( baba - cis , structure vi , fig2 ) and ( e ) sym - homospermidine , sym - norspermidine or spermidine dimers cross - linked by an alkyne side chain via n - alkyl bonds with the central amino group of each triamine chain as depicted for structure vii ( baba - yne , fig2 ). the general structure of the noval compounds claimed ( structure 4 ) include the following characteristics : ( 1 ) the two polyamine backbones are either spermidine [ n -( 3 - aminopropyl )- 1 , 4 - diaminobutane ], sym - homospermidine [ n -( 4 - aminobutyl )- 1 , 4 - diaminobutane ] or sym - norspermidine [ n -( 3 - aminopropyl )- 1 , 3 - diaminopropane ]. ( 2 ) each methylene group of the polyamine chain can be modified by methyl groups without compromising the ability of the inhibitor to interact with the polyamine transporter . ( 3 ) each primary amino group of the polyamine chains can be modified by methyl , ethyl or propyl groups without compromising the ability of the inhibitor to interact with the polyamine transporter . ( 4 ) the central secondary amino groups of the two polyamine chains are connected via n - alkyl bonds by a linker ( l ) so that the resulting inhibitor has a k i ≦ 20 μm ( relative to spermine ). ( 5 ) the linker ( l ) can be of any nature or chain length , as long as the total final structure does not exceed 3 , 000 . these molecules may in other embodiments be described as having a total mass of about 50 to about 2 , 500 , or about between 500 to about 1 , 500 , or about 1 , 000 as a total mass . by way of example , such linkers may comprise alkyl , aryl , ether , thioether , disulfide , amide , phosphono , keto , amine and sulfonyl groups or a combination thereof . the general design and use of dimers of spermidine , sym - homospermidine and sym - norspermidine corresponding to general structure 4 comprise yet another aspect of the present invention . for example , in some embodiements , these synthetic derivatives comprise a structure of a first polyamine chain and a second polyamine chain according to the structure . wherein r 1 is h , methyl , ethyl or propyl , r 2 is h or methyl , x is greater than two and less than five ( 2 & lt ;×& lt ; 5 ), y is greater than two and less than five ( 2 & lt ; y & lt ; 5 ), and l is a chemical structure ( the linker ) covalently connecting said second polyamine chain to said second chain through an alkyl bond , such as a α , ω , - diamine cross - linker . in order to evaluate the effectiveness of dimeric triamines as polyamine transport inhibitors , we synthesized n , n ′- bis ( 3 - aminopropyl ), n , n ′- bis ( 4 - aminobutyl ) cystamine or babac ( structure 1 , fig2 ; fig3 ), and its thiol form n -( 3 - aminopropyl ), n -( 4 - aminobutyl ) cysteamine or aac ( fig3 ) according to scheme 4 described above ( fig3 ), using spermidine as a precursor polyamine . the ability of babac and aac to inhibit the intracellular uptake of radio - labeled putrescine and spermidine in the zr - 75 - 1 human breast cancer cell line was then determined according to standard published procedures ( lessard , et al . and poulin , et al .) briefly , zr - 75 - 1 cells were grown for four days in twenty - four well plates in rpmi 1640 medium supplemented with 10 % fetal bovine serum . 1 nm estradiol , 2 mm l - glutamine , 1 mm sodium pyruvate , 15 mm hepes and antibiotics , and specific uptake of [ 3 h ] putrescine , [ 3 h ] spermidine and [ 14 c ] spermine was measured as described ( lessard , et al . ), using 20 μm and 3 μm substrate , respectively . zr - 75 - 1 cells are a convenient system to assess the potential of polyamine transport inhibitors because they exhibit elevated polyamine uptake activity ( lessard , et al .) as shown in fig3 , babac was a potent inhibitor of putrescine , spermidine and spermidine uptake in zr - 75 - 1 cells , with calculated apparent k i values of 0 . 15 μm , 0 . 68 and 2 . 1 μm , respectively . its thiol monomer aac was a less potent transporter inhibitor ( k i = 0 . 54 , 4 . 37 and 3 . 9 μm , respectively , as expected from the potentiating effect of dimerization on polyamine transport inhibition . although the potency of babac to inhibit putrescine and spermidine uptake was only slightly lower than that of spermine ( k i of 0 . 0745 and 0 . 40 μm , respectively , for spermine ), it was clearly greater than that of the dimeric spermine - like inhibitor desc ( fig3 ), which inhibited putrescine , spermidine and spermine uptake with apparent k i values of 0 . 54 , 5 . 6 and 7 . 5 μm , respectively . these data clearly indicate that dimerization of a shorter polyamine chain ( i . e . spermidine relative to spermine ) can still generate potent polyamine transport inhibitors , and that the nature of the linker l as well as its site of covalent attachment on the polyamine backbone can strongly influence the potency of the dimer to inhibit polyamine uptake . a comparison between the structures of desc and babac suggests that a n - alkyl type of attachment leads to superior properties of polyamine transport inhibition than an amide linkage on a methylene group of the polyamine backbone , and that this effect dominates over the effect of elongating the polyamine chains . to further evaluate the validity of the design of dimeric triamines as polyamine transport inhibitors , we have assessed the ability of a sym - norspermidine dimer , namely n , n , n ′ n ′- tetrakis ( 3 - aminopropyl )- p - xylylenediamine ( tadax ) ( fig2 ), to inhibit polyamine uptake in zr - 75 - 1 cells . tadax ( fig2 ) was synthesize according to the scheme 2 described above ( fig2 ). briefly , into a solution of norspermidine ( 2 . 55 g ) and diethylamine ( 8 . 0 ml ) in chloroform ( 100 ml ) was added trityl chloride ( 10 . 87 g ) portionwise . after addition , stirring was continued for 24 h . the reaction mixture was washed with water and dried over anhydrous potassium carbonate . the solution was concentrated under reduced pressure and the residue was re - crystallized from dichloromethane - methanol to obtain n 1 , n ′- bis ( trityl ) norspermidine . 1 h nmr ( 300 mhz , chcl 3 ) δ2 . 26 ( m , 12h , aromatic h ), 7 . 12 - 7 . 30 ( m , 18h , aromatic h ), 2 . 36 ( t , 4h , j = 8 hz , 2 × ch 2 n ), 2 . 18 ( t , 4h , j = 8 hz , 2 × ch 2 n ), 1 . 66 ( m , 6h , 2 × ch 2 and 2 × nhtr ). a mixture containing n ′, n ′- bis ( trityl ) norespermidine ( 770 mg ), α , α ═- dibromo - p - xylene ( 166 mg ), anhydrous na 2 co 3 ( 573 mg ), acetonitrile ( 45 ml ) and dmf ( 3 drops ) was refluxed under nitrogen atmosphere for 3 days . then , the mixture was filtered while it was still hot and the filtered solid was washed thoroughly with water and dried to give n , n , n ′, n ′- tetrakis ( n - trityl - 3 - aminopropyl ) p - xylylene diamine . 1 h nmr ( 300 mhz , chcl 3 ) δ7 . 24 ( m , 24h , aromatic h ), 6 . 92 - 7 . 34 ( m , 36h , aromatic h ), 3 . 42 ( s , 4h , 2 × ch 2 ), 2 . 42 ( m , 8h , 4 × ch 2 n ), 2 . 08 ( m , 8h , 4 × ch 2 n ), 1 . 64 ( m , 12h , 4 × ch 2 and 4 × nhtr ). n , n , n ′, n ′- tetrakis ( n - trityl - 3 - aminopropyl ) p - xylene diamine ( 374 mg ) was suspended in a solution of hcl 6 m ( 20 ml ) and the mixture was refluxed for 24 hours . the solid was removed by filtration and the aqueous phase concentrated to 2 - 3 ml by rotary evaporation . addition of ethanol into the concentrated solution afforded the hexahydrochloride salt of n , n , n ′, n ′- tetrakis ( 3 - aminopropyl ) p - xylenediamine , or tadax ( fig2 ). the ability of tadax to inhibit [ 14 c ] spermine transport was next compared in zr - 75 - 1 human breast cancer cells using 3μm substrate ( fig3 ). in this assay , spermine has an apparent k m = 0 . 6 μm . it is clear that the p - xylylene norspermidine dimer tadax ( k i = 1 . 5 μm ) is a much more potent spermine transport inhibitor than the prototypic spermine dimer desc ( k i = 17 μm ), and was nearly as potent as the substrate spermine at interacting with the polyamine carrier in these cells . these data further demonstrate the importance of the crosslinker l in the relative ability of triamine dimers to inhibit polyamine transport . to further substantiate the usefulness of triamine dimers such as tadax as transport inhibitors , the cytotoxicity of tadax and its ability to serve a transported substrate for the polyamine carrier were then evaluated . as shown in fig3 . tadax was remarkably non - toxic to zr - 75 - 1 cells ( ic 50 & gt ; 500 μm ), as compared to sym - norspermidine or the disulfide babac , which had lethal effects on zr - 75 - 1 cells with ic 50 values of 6 . 4 and 17 μm , respectively . while the cytotoxicity of sym - norspermidine has been well documented ( bergeron and seligshon , komori and ohsugi , porter and bergeron ), a less pronounced , but yet significant cytotoxicity of desc has been found using zr - 75 - 1 breast cancer - cells . that cytotoxicity may well be related to the reactivity of the disulfide groups of desc and babac with biological thiols and disulfides found in growth media and at the cell surface . thus , despite the presence of four 3 - aminopropyl groups on the tadax backbone , and its two sym - norspermidine - derived moieties , it is remarkably inert toward biological functions . this is consistent with the fact that no detectable accumulation of the compound was found in zr - 75 - 1 cells even after a 6 - hour incubation , as determined by ion pairing - reverse phase high - pressure liquid chromatography of cell extracts performed as described ( lessard , et al .) ( data not shown ). these data suggests that the very low cytotoxicity of tadax is related to its lack of uptake by zr - 75 - 1 cells . moreover , these properties are those expected from an impermeant , pure antagonist of polyamine transport such as desc , thus supporting the potential usefulness of tadax and similar triamine dimers to preempt polyamine uptake by tumor cells . ( 1 ) unmethylated spermine analogs ( fig1 a ): n 1 , n 4 , n 8 , n 12 - tetra - boc - 5 - carboxyspermine ( iv , fig1 ) is first prepared as described ( 88 ). if the linker is going to be amidated to the polyamine chain , the carboxyl group used as an acceptor is activated with cyanuric chloride ( 88 ), and conjugated with a n - mono - fmoc diaminoalkane of the desired length to generate the corresponding n 1 , n 4 , n 8 , n 12 - tetra - boc - spermine - 5 - n -( n - fmoc - aminoalkyl ) carboxamide ( v , fig1 ). the fmoc group of the latter compound is removed with 20 % piperdine / dmf , and the resulting n 1 , n 4 , n 8 , n 12 - tetra - boc - spermine - 5 ( n - ω - aminoalkyl ) carboxamide ( vi , fig1 ) is then reacted with the acid chloride form of n 1 , n 4 , n 8 , n 12 - tetra - boc - 5 - carboxyspermine ( iv , fig1 ). the latter compound is then deprotected with hcl / ch 3 cooh to obtain the corresponding n α , n ω - bis ( spermine - 5 - oyl )- diaminoalkane , the desired transport inhibitor ( vii , fig1 ). these compounds are symbolized as bs compounds , and bs - 3 , bs - 4 , bs - 5 and bs - 6 corresponds to the forms where the diaminoalkane linker is 1 , 3 - diaminopropane , 1 , 4 - diaminobutane , 1 , 5 - diaminopentane and 1 , 6 - diaminohexane , respectively . if the spacer is going to be alkylated to the polyamine chain , the carboxyl group used as an acceptor in an amidation reaction is first reduced to an alcohol with liah 4 . after protecting the amine groups with carbobenzoxy ( cbz ) groups , the alcohol is then converted to a bromide with pbr 3 . the resulting cbx - protected spermine bromide is then reacted with a diamine spacer with a 2 : 1 stoichmetry to generate the cbz - protected spermine dimer . this dimer is finally deprotected by catalytic hydrogenation with pd / c ( 82 ) to generate the unmethylated spermine dimer ( the transport inhibitor ). if an ether linkage is desired , the alcohol obtained as above is then converted to an alkoxide with sodium metal , and then reacted with an alkyl dihalide ( e . g . 1 , 3 - diiodopropane ) with a 2 : 1 stoichiometry to generate the cbz - protected polyamine dimer , which is then deprotected as above to generate the unprotected polyamine dimer ( the transport inhibitor ). as an alternative precursor to ornithine , one may use instead 2 - hydroxyputrescine , synthesized as described ( 83 ), and proceed with carboxyethylation and catalytic hydrogenation as in the route using ornithine as precursor , to obtain 6 - hydroxyspermine . the four amino groups of the latter are protected with carbobenzoxy groups , and the alcohol is converted into an alkyl halide or alkoxide for subsequent reaction with the spacer as described above . ( 2 ) methylated spermine analogs ( fig1 a ): for example , ornithine methylester ( x , fig1 ) is synthesized as described ( 89 ) and is diamidated with two equivalents of 3 - azidobutyric acid ( xii , fig1 ) using dcc / ohb , ( 90 ) to generate n 1 , n 4 - bis ( 3 - azidobutyryl )- ornithine methylester ( xiii , fig1 ). the latter is than reduced using bh 3 / thf ( 90 ) to obtain 1 , 12 - dimethylspermine - 5 carbinol ( xiv , fig1 ). after protection of all four amino groups with boc groups ( xv , fig1 ), the carbinol group is activated with pb 3 to generate 1 , 12 - dimethyl - n 1 , n 4 , n 8 , n 12 - tetra ( boc )- 5 - bromomethyl spermine ( xvi , fig1 ), and reacted with fmoc - nh -( ch 2 ) n nh 2 ( where 3 ≦ n ≦ 6 ) to generate the corresponding 1 , 12 - dimethyl - n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine - 5 ( n α - methyl , n ω fmoc - diaminoalkane ) ( xvii , fig1 ). after removing the fmoc group with piperdine / dimethylformamide ( xviii , fig1 ), the free amino group of compound xviii is alkylated with one equivalnet of compound xvi to generate a n α , n ω bis [ 1 , 12 - dimethyl - n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine ]- 5 - methyl )- diaminoalkane ( xix , fig1 ). the latter compound is then deprotected with hcl / ch 3 cooh to finally obtain the methylated spermine analog ( bms - 3 , bms - 4 , bms - 5 and bms - 6 ), which are the desired transport inhibitors ( xxa - d , fig1 ). ( 3 ) unmethylated , n - alkylated spermine analogs ( fig1 b ): a symmetrical dimer that can be made where the linker ( l ) bridges two polyamine derivatives chains through one of the innermost , secondary nitrogens of each polyamine chain . ( a ) n - benzyl - 1 , 3 - diaminopropane ( xxi , fig1 ) is first obtained by catalytic hydrogenation of 3 -( benzylamino ) propiononitrile with raney nickel as described ( 84 ). ( b ) n - benzyl - 1 , 3 - diaminopropane is then n - alkylated with 3 - bromobutyronitrile to generate n 1 - benzyl , n 3 -( 3 - cyanopropyl )- 1 , 3 - diaminopropane ( xxii , fig1 ) ( 85 ). ( c ) n 1 - benzyl , n 3 -( 3 - cyanopropyl )- 1 , 3 - diaminopropane is protected with a boc group ( 86 ) to generate n 1 - benzyl , n 3 - boc , n 3 -( 3 - cyanopropyl )- 1 , 3 - diaminopropane ( xxiii , fig1 ) ( d ) n 1 - benzyl , n 3 - boc , n 3 -( 3 - cyanopropyl )- 1 , 3 ,- diaminopropane is reduced to n 1 - benzyl , n - boc - spermidine ( xxiv , fig1 ) by catlytic hydrogenation with raney nickel ( 84 ). ( e ) n 1 - benzyl , n 4 - boc - spermidine is then cyanoethylated with acrylonitrile to generate n 1 - benzyl , n 4 - boc , n 8 - cyanoethyl - spermidine , and reduced to n 1 - benzyl , n 4 - boc - spermine by catalytic hydrogenation with raney nickel ( 84 ) ( xxv , fig1 ). ( f ) the two free amino groups of n 1 - benzyl , n 4 - boc - spermine are protected wtih cbz groups are described ( 87 ) to generate n 1 - benzyl , n 4 - boc , n 8 , n 12 - di ( cbz )- spermine ( xxvi , fig1 ). ( g ) n 1 - benzyl , n 4 - boc , n 8 , n 12 - di ( cbz )- spermine is then deprotected to n 1 - benzyl , n 8 , n 12 - di ( cbz )- spermine with trifluoroacetic acid as described ( 87 ) ( xxvii , fig1 ). ( h ) n 1 - benzyl , n 8 , n 12 - di ( cbz )- spermine can then be cross - linked with an α , ω - dibromoalkane of the desired chain length to generate the corresponding bis ( n 1 - benzyl , n 8 , n 12 - di ( cbz )- spermine ) dimer ( xxviii , fig1 ), which is then deprotected by catalytic hydrogenation with pd / c ( 87 ) to generate the unmethylated , n - alkylated spermine dimer ( the transport inhibitor ) ( xxix , fig1 ). ( a ) the amino acid group of 3 - aminobutyric acid is protected with boc as described ( 89 ), and the resulting n - boc - 3 - aminobutyric acid ( xxx , fig2 ) is condensed with n - fmoc - 1 , 4 - diaminobutane using dcc / ohbt ( 88 ) to obtain n 1 -( n - boc - 3 - aminobutyryl ), n 4 - fmoc - 1 , 4 - diamonobutane ( xxxi , fig2 ). ( b ) n 1 -( n - boc - 3 - aminobutyryl ), n 4 - fmoc - 1 , 4diaminobutane is then reduced to n 1 - boc - n 8 - fmoc - 1 - methylspermidine with bh 3 / thf ( 88 ) ( xxxii , fig2 ). ( c ) two equivalents of n 1 - boc - n 8 - fmoc - 1 - methylspermidine are then n 4 - alkylated with one equivalent of the α , ω - diiodalkane of the desired length to obtain the corresponding n α , n ω - bis ( n -( nboc - 3 - amino , 3methylpropyl ], n -[ n - fmoc - 4 - aminobutyl ]- α , ω - diaminoalkane ( xxxiii , fig2 ). ( d ) the fmoc groups of the resulting n α , n ω - bis ( n -[ nboc - 3 - amino , 3 - methylpropyl ], n -[ n - fmoc - 4 - aminobutyl ]- α , ω - diaminoalkane are then deprotected with 20 % piperidine / dmf to yield the corresponding n α , n ω - bis ( n -[ nboc - 3 - amino , 3 - methylpropyl ], n -[ n - 4 - aminobutyl ])- α , ω - diaminoalkane ( xxxiv , fig2 ), which is then condensed with 3 - azidobutyric acid , prepared as described ( 88 ), using dcc / ohbt , to generate the corresponding n α , n ω - bis ( n -[ nboc - 3 - amino , 3 - methylpropyl ], n -[ n - 8 - amino - 5 - aza - octanoyl ]- α , ω - diaminoalkane . ( e ) the n α , n ω - bis ( n -[ n - boc - 3 - amino , 3 - methylpropyl ], n -[ n - 8 - amino - 5 - aza - octanoyl ])- α , ω - diaminoalkane is then reduced to the corresponding n α , n ω , bis -( n -[ n - boc - 3 - amino , 3 - methylpropyl ], n -[ n - 8 - amino - 5 - aza - octyl ])- α , ω - diaminoalkane with bh 3 / thf ( 88 ) ( xxxvii , fig2 ). ( f ) the boc groups of n α , n ω - bis ( n -[ n - boc - 3 - amino , 3 - methylpropyl ], n -[ n - 8 - amino - 5 - aza - octyl ])- α , ω - diaminoalkane are then removed with hcl / ch 3 cooh to generate the desired transport inhibitor , a n α , n ω - bis ( n -[ 3 - amino , 3 - methyl - propyl ], n -[ 8 - amino - 5 - aza - octyl ])- α , ω - diaminoalkane ( xxxviii , fig2 ). ( 5 ) 1 , 12 - dimethylated spermine dimers cross - linked n - alkyl / c - alkyl attachments of the linker ( fig1 c ): dimeric polyamine transport inhibitors of a different type can be generated by cross - linking one polyamine chain to a linker through a n - alkyl bond as in examples 3 and 4 above , and the other polyamine chain via a c - linked anchor lying between the two innermost secondary amino groups as in examples 1 and 2 above . such compounds ( as terminal - c - methylated spermine analogs ) can be obtained as follows : ( a ) n 1 - boc - n 8 - fmoc - 1 - methylspermidine ( xxxii , fig2 ), obtained as described above ( example 4 , steps a to b ), is n 4 - alkylated using an ω - bromoalkyphthalimide of the desired length as described ( 92 ), to generate the corresponding n 1 - boc , n 4 - alkylphthalimide , n 8 - fmoc - 1 - methylspermidine ( xxxix , fig2 ). ( b ) the phthalimide group of n 1 - boc , n 4 - alkylphthalimide , n 8 - fmoc - 1 - methylspermidine is removed with hydrazine in . etoh ( 88 , 90 ) to generate the corresponding n 1 - boc , n 4 -( ω - aminoalkyl ), n - 8 fmoc - 1 - methylspermidine ( xl , fig2 ). ( c ) the free amino group of n 1 boc , n 4 -( ω - aminoalkyl ), n 8 - fmoc - 1 - methylspermidine is then alkylated with n 1 , n 4 , n 8 , n 12 - tetra ( boc )- 1 , 12 - dimethyl - 5 - bromomethylspermine prepared as described in example 2 ( xvi , fig1 ) to obtain the corresponding n α ( n - boc - 3 - amino , 3 - methylpropyl ], n -[ n 8 - fmoc - 4 - aminobutyl ]), n ω -[ 5 -( n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine )- methyl ] α , ω - diaminoalkane ( xli , fig2 ). ( d ) the fmoc group of n α ([ n - boc - 3 - amino , 3 - methylpropyl ], n -[ n - fmoc - 4 - aminobutyl ]), n ω -[ 5 -( n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine )- methyl ] α , ω - diaminoalkane is then removed with 20 % piperidine / dmf , and the resulting n α ([ n - boc - 3 - amino , 3 - methylpropyl ], n -[ 4 - aminobutyl ]), n ω -[ 5 -( n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine )- methyl ] α , ω - diaminoalkane ( xliii , fig2 ) is condensed with 3 - azidobutyric acid ( xxx . fig2 ) as in example 4 ( step d ) above , to generate the corresponding n α ([ n - n - boc - 3 - amino , 3 - methylpropyl ], n -[ 8 - amino - 5 - aza - octanoyl ]), n ω -[ 5 -( n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine )- methyl ] α , ω - diaminoalkane ( xliii , fig2 ). ( e ) n α ( n -[ n - boc - 3 - methylpropyl ], n -[ 8 - amino - 5 - aza - octanoyl ], n ω -[ 5 -( n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine )- methyl ]- α , ω - diaminoalkane is then reduced to n α ( n -[ n - boc - 3 - amino , 3 - methylpropyl ], n -[ 8 - amino - 5 - aza - octyl ]), n ω -[ a5 -( n 1 , n 4 , n 8 , n 12 - tetra ( boc )- spermine )- methyl ]- α , ω - diaminoalkane ( xliv , fig2 ) with bh 3 / thf as in example 4 ( step e ) above , and the boc groups of the resulting compound are removed with hcl / ch3cooh to generate the corresponding n α ( n -[ n -[ 3 - amino , 3 - methyl - propyl ], n -[ 8 - amino - 5 - aza - octyl ]), n ω -[ 5 -( 1 , 12 - dimethyl - spermine )- methyl ]- α , ω - diaminoalkane ( xlv , fig2 ), which is the desired polyamine transport inhibitor . ( 6 ) scheme 1 ( fig2 ): synthesis of sym - norspermidine dimers using total cyanoethylation of aliphatic or aromatic diamines this scheme is used to prepare symmetrical dimers of sym - norspermidine by michael addition via total cyanoethylation of hydrochloride salts of aliphatic or aromatic α , ω - diamines . this route is the simplest one to generate polyamine dimers since no amine protection is neccessary . the diamin hydrochloride of the desired nature and chain length ( viii , fig2 ) to be used as a crosslinker is stirred with a 4 - fold excess of acrylonitrile in the presence of triethylamine ( et 3 n ) is aqueous solution , yielding a n , n , n ′, n ′- tetrakis ( cyanoethyl )- α , ω - diamine ( ix , fig2 ). cyanoethyl groups are then reduced with nabh 4 in the presence of methanolic cocl 2 as a reducing agent , resulting in the formation of a sym - norspermidine dimer ( x , fig2 ) with the desired linker l . the compound is obtained as its hexahydrochloride salt after alkaline extraction with chcl 3 / na 2 co 3 and solubilization in hcl . various linkers can be used according to that scheme , including aliphatic α , ω - diaminoalkanes ( preferably with a chain length greater than two and less than ten ) which may incorporate one double bond in a cis or trans configuration , or one triple bond , and aromatic diamines where the amino groups are present as aminoalkyl substituents at two different positions of the aromatic cycle , such as the para configuration shown in fig2 . ( 7 ) scheme 2 ( fig2 ): synthesis of spermidine , sym - homospermidine and sym - norspermidine dimers using direct alkylation of the central secondary amino group with a dibromoalkene or dibromoarene spermidine , sym - homospermidine and sym - norspemidine dimers can be prepared by protecting the primary amino groups of the precursor triamine with trityl groups , and directly alkylating the secondary amino group of the triamine with a dibromoalkene , a dibromoalkyne or a dibromoarene with the desired crosslinking carbon chain ( zang and sadler ). for this purpose , the primary amine groups of the triamine ( fig2 ) are reacted with trityl chloride in the presence of diethylamine in chcl 3 to obtain the n α , n ω - bis ( trityl ) polyamine chain xii ( fig2 ). two equivalents of the latter compound are then refluxed with the alkyl or aryl dibromide form of the desired linker l to generate the trityl - protoected form of the desired triamine dimer ( xiii , fig2 ). the trityl groups of this dimer are then removed by refluxing with 6 n hcl to obtain the hexahydrochloride salt of the polyamine transport inhibitor xiv ( fig2 ). dibromoalkanes tend to form heterocyclic by - products with the triamine precursor , and therefore the present scheme is chiefly used as a simply synthetic method to generate dimeric triamine - derived polyamine transport inhibitors with unsatured or aromatic linkers . ( 8 ) scheme 3 ( fig2 ): synthesis of spermidine sym - homospermidine and sym - norspermidine dimers by amidation with an acyl chloride followed by reduction of the amide groups an alternative , even more versatile approach to generate spermidine , sym - homospermidine and sym - norspermidine dimers as polyamine transport inhibitors is to crosslink the secondary amino groups of the triamine chains , protected with trityl groups as in scheme 2 ( xii , fig2 and fig2 ), with the α , ω - diacyl chloride form of the desired linker l , and then reduce the amide bonds of the resulting molecules . for this purpose , two equivalents of the trityl - protected triamine precursor xii are reacted with the diacyl chloride of choice in the presence of triethylamine / chcl 3 to obtain the corresponding diamide form of a trityl - protected triaminen dimer xv ( fig2 ). the diamide is then reduced to its n - alkyl form with lialh 4 in dry thf under nitrogen to obtain the trityl - protected form of the triamine dimer ( xiii , fig2 and fig2 ). the primary amine groups are then freed with 6 n hcl as in scheme 2 to generate the desired dimeric polyamine transport inhibitor xiv ( fig2 and fig2 ). this scheme is superior to scheme 2 for dimerizing triamine molecules with aliphatic linkers of the general structure (—( ch 2 ) n —, which tend to form heterocycles with the polyamine chain when reacted in their dihalide form . ( 9 ) scheme 4 ( fig3 ): synthesis of dimeric spermidine , sym - homospermidine and sym - norspermidine disulfides synthesis of useful dimeric polyamine transport inhibitors is not limited to those containing aliphatic or aromatic carbon chains . triamines can be dimerized via n - alkyl bonds with a linker l containing a disulfide bond , generating polyamine transport inhibitors with even higher potency than desc , which is a dimeric spermine disulfide cross - linked through amide bonds with position c5 of each spermine skeleton . the following scheme allows the synthesis of dimeric triamines cross - linked via n - alkyl bonds with a diethyl disulfide chain . the primary amino groups of the desired triamine xi ( fig2 and fig3 ) are first protected with ethyl trifluoroacetate in aqueous acetonitrile to obtain the bis - trifluoroacetyl )- protected triamine xvi ( fig3 ). the latter compound is then reacted with one equivalent with ethylene sulfide ( cohen , et al . ), 1 . 2 equivalent of diisopropylethylamine ( hunig &# 39 ; s base ) and 0 . 1 equivalent of cucl in dry acetonitrile in the presence of 2 , 2 ′- dithiobis ( benzothiazole ) to catalyze the formation of the disulfide from an intermediate thiol ( brzezinska and ternay ), to generate the trifluoroacetyl - protected triamine dimeric disulfide xvii ( fig3 ). trifluoroacetyl groups are removed from the latter compound using meoh / nh 3 ( 4 : 1 ) at ph 11 , yielding the free dimeric triamine disulfide xviii ( fig3 ). the latter disulfide can be further reduced to its free thiol xix ( fig3 ) with either dithiothreitol or tris ( carboxyethyl ) phosphine ( burns , et al .). the following references are specifically incorporated herein by reference for the purposes indicated : 1 . ask , a ., l . persson , and o . heby . increased survival of l1210 leukemic mice by prevention of the utilization of extracellular polyamines . studies using a polyamine - uptake mutant , antibiotics and a polyamine - deficient diet . cancer lett . 66 : 29 - 34 , 1992 . 2 . aziz , s . m ., m . n . gillespie , p . a . crooks , s . f . tofiq , c . p . tsuboi , j . w . olson , and m . p . gosland . the potential of a novel polyamine transport inhibitor in cancer chemotherapy . j . pharmacol . exper . ther . 278 : 185 - 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