Patent Application: US-201414256280-A

Abstract:
compounds of the general structure s - 1 , wherein x and r 1 - r 5 are defined in the specification , are capable of abrogating the deubiquitinating activity of the 19s rp dubs and are useful in methods and compositions for treating cancer , in particular , cancer tumors refractory to treatment by state - of - the - art chemotherapy .

Description:
in vitro proteasome activity assays are performed in black 96 - well microtitier plates using human 20s proteasome ( boston biochem ) in reaction buffer ( 25 mm hepes , 0 . 5 mm edta , 0 . 03 % sds ) with suc - llvy - amc , z - lle - amc or boc - lrramc used as substrates for proteasome activity . de - ubiquitinase activity assays are performed with human 19s rp ( boston biochem ) with ubiquitin - amc as substrate . for fadu xenograft studies a 100 - μl - cell suspension containing 1 × 10 6 cells is injected subcutaneously into the flank of scid . upon tumor take mice are randomized into control or treatment groups and administered with 5 mg kg − 1 compound of the invention or vehicle . in vivo levels of apoptosis and cell death are determined from the detection of caspase cleaved and total levels of cytokeratin - 18 in plasma using m30 apoptosense ® and m65 elisa ® s assays ( peviva ). the methods are described below in more detail . reagents . reagents were obtained from the following sources : 20s proteasome ( e - 360 ), 26s proteasome ( e - 365 ), 19s proteasome ( e - 366 ), suc - llvy - amc ( s - 280 ), z - lle - amc ( 5 - 230 ), boc - lrr - amc ( s - 300 ), ubiquitin - amc ( u - 550 ), tetra - ubiquitin k63 ( uc - 310 ), tetra - ubiquitin k48 ( uc - 210 ), deconjugating enzyme set ( ke10 ), ha - ubiquitin vinyl sulfone ( u - 212 ) ( boston biochem ); anti - β - actin ( ac - 15 ), odc - 1 ( hpa001536 ) ( sigma aldrich ); anti - lc - 3 ( 2775 ), anti - gapdh ( 2118 ), anti - p44 / 42 mapk ( 4695 ), anti - phospho - p44 / 42 mapk ( 9101 )( cell signaling ); n - ethylmaleimide ( 34115 ) ( emd chemicals ); anti - ubiquitin k48 ( apu2 ), anti - ubiquitin ( mab1510 ) ( millipore ); anti - p53 ( do1 ), anti - uchl5 ( h - 110 ), hdm2 ( smp14 ) ( santa cruz ); anti - parp ( c2 - 10 ), anti - p27 ( g173 - 524 ), anti - active caspase 3 ( c92 - 605 ) ( bd biosciences ); anti - usp14 ( a300 - 919a ) ( bethyl laboratories ); anti - ha ( 12ca5 ) ( roche ). bortezomib was obtained from the department of oncology , karolinska hospital , sweden . cell culture . mcf7 cells are maintained in mem / 10 % fetal calf serum . hct - 116 p53 +/+, p53 −/−, bcl - 2 +/+, puma −/− and bax −/− cells are maintained in mccoy &# 39 ; s 5a modified medium / 10 % fetal calf serum . the hct - 116 p53 +/+, p53 −/−, puma −/− and bax −/− are generated as described ( 25 ). the hct - 116 bcl - 2 +/+ cell line was generated by transfecting parental hct - 116 p53 +/+ cells with pcep4 bcl - 2 ( addgene plasmid 16461 ) ( 26 ) and isolating high expression clones . fadu and llc3 cells are maintained in dmem high glucose medium supplemented with 10 % fetal calf serum , na pyruvate , hepes and non - essential amino acids . 4t1 . 12b carcinoma cells are maintained in rpmi medium supplemented with 10 % fetal calf serum . the proteasome reporter cell line meljuso ub - yfp was generated as described ( 12 ). cells were maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium / 10 % fetal calf serum . the retinal epithelial cell line was generated as described ( 12 ). all cells are maintained in dulbecco &# 39 ; s modified eagle medium / 10 % fetal calf serum . the retinal epithelial cell line was generated as described ( 28 ). all cells are maintained at 37 ° c . in 5 % co 2 . proteasome and dub inhibition assays . in vitro proteasome activity assays using 20s cp ( 2 nm ) ( boston biochem ) are performed at 37 ° c . in 100 - μl reaction buffer ( 25 mm hepes , 0 . 5 mm edta , 0 . 03 % sds ). samples are incubated for 10 min with indicated compound followed by addition of 10 μm suc - llvy - amc , z - lle - amc or boc - lrr - amc for the detection of chymotrypsin - like , caspase - like and trypsin - like activity respectively . for dub inhibition assays 19s rp ( 5 nm ), 26s ( 5 nm ) uch - l1 ( 5 nm ), uch - l3 ( 0 . 3 nm ), usp2cd ( 5 nm ) usp7cd ( 5 nm ) usp8cd ( 5 nm ) and bap1 ( 5 nm ) are incubated with the compound of the invention followed by addition of ubiquitin - amc ( 1000 nm ). fluorescence is monitored using wallac multilabel counter or tecan infinite m1000 equipped with 360 nm excitation and 460 nm emission filters . substrate overlay assays . native gel electrophoresis is performed as described ( 29 ). in brief 4 μg of purified 26s proteasome ( boston biochem ) is mixed with 10 or 50 μm of the compound of the invention and incubated at 37 ° c . for 10 min . samples are resolved on 4 % non - denaturing page . gels are submerged in assay buffer ( 20 mm tris - hcl , 5 mm mgcl 2 , 1 mm atp , 0 . 1 mm suc - llvy - amc ) and proteasomes are visualized under uv illumination . ubiquitin - cleavage assay . the recombinant ub - gfp plasmid pet19b ub - m - gfp is generated as described ( 30 ). in brief recombinant ub - gfp is purified from bl21 e . coli cells by his affinity purification . for cleavage assays 19s rp ( 25 nm ) is incubated with 10 mm nem , 250 μm tpen or 50 μm of the compound of the invention for 10 min followed by the addition of recombinant ub - gfp ( 200 nm ). ubiquitin chain disassembly reactions are performed essentially as above except k48 - or k63 - linked ubiquitin tetramers ( 50 ng ) are substituted for ub - gfp . the level of ub - gfp cleavage or ubiquitin disassembly is determined by immunoblotting with anti ubiquitin antibodies . the ubiquitinated hdm2 substrate is generated according to the boston biochem protocol ( k - 200 ). for the cleavage assay 19s rp ( 25 nm ) is incubated with 50 μm of the compound of the invention or dmso for 10 min followed by the addition of ubiquitinated hdm2 substrate ( 100 nm ). the cleavage of ubiquitinated hdm2 substrate and ubiquitinated hdm2 is determined by immunoblotting with anti - hdm2 antibodies . proteasome isolation : hct - 116 cells are treated with bortezomib ( 100 nm ) or the compound of the invention ( 1 μm ) for 3 hours . after stimulation , the cells are lysed in 50 mm hepes ph 7 . 4 , 250 mm sucrose , 10 mm mgcl 2 , 2 mm atp , 1 mm dtt and 0 . 025 % digitonin . samples are sonicated briefly and incubated for 15 min on ice . proteasomes from these samples are isolated according to the manufacturer &# 39 ; s protocol . ubvs labelling of dubs . for labelling of dubs in cell lysates sub confluent cells are harvested by trypsinization , washed three times with pbs , and centrifuged at 1500 rpm for 5 min . cell pellets are lysed with buffer ( 50 mm hepes ph 7 . 4 , 250 mm sucrose , 10 mm mgcl 2 , 2 mm atp , 1 mm dtt ) on ice for 15 min . debris is removed by centrifugation and 25 μg of protein is labelled with 1 μm ha - ubvs for 30 min at 37 ° c . samples are resolved by sds - page and analyzed by immunoblotting with indicated antibodies . determination of cell apoptosis and viability . for determination of apoptosis parental hct - 116 p53 +/+ cells are treated with the increasing doses of the compound of the invention for 24 h . treatment doses are based on the drug concentration that resulted in maximal apoptosis over a 24 h period . hct - 116 cells are seeded in 96 - well microtiter plates at 10 , 000 cells per well and incubated overnight . cells are treated with indicated drug for 24 h . at the end of the incubation period , np40 is added to the tissue culture medium to 0 . 1 % and 25 μl of the content of each well was assayed using the m30 - apoptosense ® elisa as previously described ( 31 ). cell viability is determined by measuring acid phosphatase activity or using the fmca method ( 32 ). for the acid phosphatase activity cells are seeded at 5000 cells per well in 96 - well culture plates and incubated for 12 h at 37 ° c . compounds are added to the cells in growth media and incubated for 72 h at 37 ° c . cells are washed with 200 μl warm pbs . 100 μl of para - nitrophenyl phosphate ( pnpp , 2 mg / ml ) in na acetate buffer ph 5 ( naac 0 . 1 m , 0 . 1 % triton - x - 100 ) is added per well . cells are incubated for 2 h after which reaction was stopped by addition of 1n naoh . absorbance is measured at 405 nm . the dose - dependent cytotoxicity of a number of embodiments of the compound of the invention is illustrated in fig1 a - 1 o . for the fmca assay cells are seeded in the drug - prepared 384 - well plates using the pipetting robot precision 2000 ( bio - tek instruments inc ., winooski , vt .). the plates are incubated for 72 h and then transferred to an integrated hts saigan core system consisting of an orca robot ( beckman coulter ) with co 2 incubator ( cytomat 2c , kendro , sollentuna , sweden ), dispenser module ( multidrop 384 , titertek , huntsville , ala . ), washer module ( elx 405 , bio - tek instruments inc ), delidding station , plate hotels , barcode reader ( beckman coulter ), liquid handler ( biomek 2000 , beckman coulter ) and a multipurpose reader ( fluostar optima , bmg labtech gmbh , offenburg , germany ) for automated fmca . survival index ( si ) is defined as the fluorescence of test wells in percentage of controls with blank values subtracted . cell - cycle analysis . for determination of cell cycle hct - 116 cells are treated with the compound of the invention or dmso cells are harvested by trypsinisation , washed and fixed in 70 % ice cold etoh for 12 h . the cells are re - suspended in staining solution containing propidium iodide ( 50 μg / ml ) and rnase a ( 0 . 5 μg / ml ) in pbs . samples are run on bd facscalibur . the percentage of cells in each phase of the cell cycle is determined using modfit software . general information . all solvents used were of hplc grade or better . when anhydrous conditions were required , an excess of 3 å molecular sieves were added to the solvent at least 24 h before use to ensure dryness . 1 h nmr nuclear magnetic resonance ( nmr ) was recorded on a bruker advance dpx 400 spectrometer at 400 . 1 mhz . low resolution electrospray ionization mass spectra were obtained using an agilent mass spectrometer in positive ionization mode . flash chromatography was performed on merck silica gel 60 ( 230 - 400 mesh ). analytical lcms data were obtained with an agilent mass spectrometer ; agilent 1100 system ; a : ace c8 column ( 50 × 3 . 0 mm , 5 μm ); gradient : 10 - 97 % acetonitrile in water / 0 . 1 % tfa , in 3 min 1 . 0 ml / min , or b : xbridge c18 column ( 3 . 5 μm . 50 × 3 . 0 mm ), gradient 10 % to 97 % acetonitrile in 10 mm nh 4 hco 3 ( ph 10 ) in 3 min , 1 ml / min ). names of chemical structures were determined using marvin scech 5 . 2 . 6 , chemaxon . hexahydro - 4h - azepin - 4 - one ( 0 . 45 g , 3 . 0 mmol ), together with either benzaldehyde ( 0 . 70 g , 7 . 0 mmol ), 4 - methoxybenzaldehyde ( 0 . 90 g , 7 . 0 mmol ) or 4 - chlorobenzaldehyde ( 0 . 92 g , 7 . 0 mmol ) was dissolved in acetic acid ( 10 ml ). then sulfuric acid ( conc . 1 ml ) was added drop - wise and the reactions were stirred for 24 hours at rt . water ( 30 ml ) was added and the precipitate filtered and dried in vacuo over night . no further purification was performed . compound # 1516 was obtained with 99 % purity determined by lcms ( system a ) ms esi + m / z 290 [ m + h ] + . compound # 1517 was also obtained in 99 % purity determined by lcms ( system a ), ms esi + m / z 350 [ m + h ] + . compound # 1518 was obtained in 91 % purity ; lcms ( system a ). ms esi + m / z 358 [ m ] + , 360 [ m + 2 ] + . ( 3e , 5e )- 3 , 5 - bix ( phenylmethylidene ) azepan - 4 - one (# 1516 ) ( 50 . 0 mg , 0 . 182 mmol ) and acrylic acid ( 14 . 4 mg , 0 . 20 mmol ), hbtu ( 58 . 4 mg , 0 . 182 mmol ), triethylamine ( 36 . 7 mg , 0 . 364 mmol ) were dissolved in dmf ( 2 ml ) and stirred over night . ethyl acetate and brine were added and the products were extracted . the combined organic layers were dried and evaporated . the crude product was diluted with methanol and purified by preparative hplc . compound # 1520 was obtained in 96 % purity , ms - esi + m / z 344 [ m + h ] + . n - boc - azepanone ( 100 mg , 0 . 47 mmol ) and 4 - nitrobenzaldehyde ( 156 mg , 1 . 03 mmol ) were dissolved in acetic acid ( 10 ml ). then sulfuric acid ( conc . 1 ml ) was added dropwise and the reactions were stirred at room temperature for three days . then more aldehyde and sulfuric acid were added and the reaction stirred another 24 hours , more acid was added twice 24 hours apart . the reaction was quenched by addition of water and the precipitated crude intermediates were filtered off and washed with water . after drying the product in vacuo over night 2 × 35 mg ( 0 . 09 mmol ) of the crude intermediate was weighed into two flasks and dissolved together with monoethyl succinate ( 14 . 8 mg , 0 . 10 mmol ) in dcm / dmf ( 2 ml , 4 : 1 ). triethylamine ( 19 . 3 μl , 0 . 14 mmol ) was added and the mixture stirred for 5 min before addition of hatu ( 38 . 6 mg , 0 . 10 mmol ). after continuing stirring for 12 hours more triethylamine and hatu was added and the stirring continued for 4 hours . the solvents were evaporated and the residue purified by preparative hplc . the residue was dissolved in dichloromethane / trifluroacetic acid ( 5 ml , 4 : 1 ), stirred for 40 min and concentrated again . compound # 1505 was obtained in 93 % purity by lcms ( system a ). ms esi + m / z 477 [ m + h ] + . ( 2r )- 2 -{[( 3e , 5e )- 3 , 5 - bis [( 4 - nitrophenyl ) methylidene ]- 4 - oxoazepan - 1 - carbonyl } pyrrolidinium trifluoroacetate ( compound # 1505 ). n - boc azepan - 4 - one ( 0 . 10 g , 0 . 47 mmol ) and 4 - nitrobenzaldehyde ( 156 mg , 1 . 0 mmol ) were dissolved in acetic acid ( 10 ml ), conc . h 2 so 4 ( 1 ml ) was added drop - wise and the reaction stirred at rt over the weekend . more aldehyde ( 156 mg ) and h 2 so 4 ( 1 ml ) were added and stirring continued at rt over night . another ml conc . h 2 so 4 was added and reaction stirred over night again . conc . h 2 so 4 was added once more and the reaction stirred until complete ( for two weeks ). upon addition of water a brown precipitate was formed , filtered off , washed with water , and dried under vacuum to give 339 . 5 mg of brown solid intermediate 1 , which was used without further purification . intermediate 1 ( 35 mg , 0 . 09 mmol ) and n - boc proline ( 22 mg , 0 . 10 mmol ) were dissolved in dcm / dmf ( 4 : 1 , 2 ml ). tea ( 19 μl , 0 . 14 mmol ) was added and the mixture stirred for 5 min , then hatu ( 38 . 6 mg , 0 . 10 mmol ) was added and the reaction stirred at rt overnight . more tea ( 19 μl , 0 . 14 mmol ) and hatu ( 38 . 6 mg , 0 . 10 mmol ) was added , and the reaction stirred for another 4 h . the reaction mixture was concentrated and then purified by preparative lc ( 40 - 70 % acn in 0 . 1 % tfa ) to give the product as a yellow solid . the solid was dissolved in dcm / tfa ( 4 : 1 , 5 ml ) and the solution stirred at rt for 40 min to remove the boc protective group . the tfa salt of the product was recovered as a yellow solid of 93 % purity . lcms a : rt 1 . 94 / 1 . 99 , m / z [ m + h ] + 477 . 1 , b : rt 2 . 28 . ( 3e , 5e )- 1 -( 4 - ethoxy - 4 - oxobutanoyl )- 3 , 5 - bis [( 4 - nitrophenyl ) methylidene ]- 4 - oxoazepan - 1 - ium trifluoroacetate ( compound # 1507 ). intermediate 1 ( 35 mg , 0 . 09 mmol ) and n - boc proline ( 22 mg , 0 . 10 mmol ) were dissolved in dcm / dmf ( 4 : 1 , 2 ml ). tea ( 19 μl , 0 . 14 mmol ) was added and the mixture stirred for 5 min , then hatu ( 38 . 6 mg , 0 . 10 mmol ) was added and the reaction stirred at rt overnight . more tea ( 19 μl , 0 . 14 mmol ) and hatu ( 38 . 6 mg , 0 . 10 mmol ) were added and the reaction stirred for another 4 h . the reaction mixture was concentrated and then purified on preparative lc ( 40 - 70 % acn in 0 . 1 % tfa ) to give the tfa salt of the product as a yellow solid of 95 % purity . lcms a : rt 2 . 48 / 2 . 50 m / z [ m + h ] + 508 . 1 . b : rt 2 . 48 / 2 . 52 . ( 3e , 5e )- 3 , 5 - bis [( 4 - chlorophenyl ) methylidene ] azepan - 4 - one ( compound # 1518 ). azepan - 4 - one hydrochloride ( 0 . 45 g , 3 . 0 mmol ) and 4 - chlorobenzaldehyde ( 0 . 92 g , 6 . 6 mmol ) were dissolved in acetic acid ( 10 ml ), conc . h 2 so 4 ( 1 ml ) was added drop - wise and the reaction stirred at rt for 24 h . after addition of water ( 30 ml ) a precipitate was formed , filtered off , and dried in vacuum to give the product in 91 % purity as a yellow solid . lcms a : rt 2 . 04 m / z [ m ] + 358 . 1 . ( 3e , 5e )- 3 , 5 - bis ( phenylmethylidene )- 1 -( prop - 2 - enoyl ) azepan - 4 - one ( compound # 1520 ) . azepan - 4 - one hydrochloride ( 50 mg , 0 . 182 mmol ), acrylic acid ( 14 μl , 0 . 20 mmol ), tbtu ( 58 mg , 0 . 182 mmol ) and tea ( 37 mg , 0 . 364 mmol ) were dissolved in dmf ( 2 ml ) and stirred at rt overnight . brine and ethyl acetate were added and the phases separated . the organic phase was dried and the solvents evaporated after filtration . the crude product was dissolved in acetic acid ( 2 ml ) and h 2 so 4 ( 0 . 2 ml ). benzaldehyde ( 50 μl ) was added and the reaction stirred for 24 hours . methanol and water were added to the mixture , which was purified by preparative lc . the title compound was isolated in 96 % purity as a yellow solid . lcms a : rt 2 . 68 m / z [ m + h ] + 344 . 1 . ( 3e , 5e )- 3 , 5 - bis ( phenylmethylidene )- 1 - cyclobutanecarbonylazepan - 4 - one ( compound # 1521 ). azepan - 4 - one hydrochloride ( 50 mg , 0 . 182 mmol ), cyclobutyric acid ( 14 μl , 0 . 20 mmol ), tbtu ( 58 mg , 0 . 182 mmol ) and tea ( 37 mg , 0 . 364 mmol ) were dissolved in dmf ( 2 ml ) and stirred at rt overnight . brine and ethyl acetate were added and the phases separated . the organic phase was dried and the solvents evaporated after filtration . the crude product was dissolved in acetic acid ( 2 ml ) and h 2 so 4 ( 0 . 2 ml ). benzaldehyde ( 50 was added and the reaction stirred for 24 h . methanol and water were added to the mixture , which was purified by preparative lc . the title compound was isolated in 96 % purity as a yellow solid . lcms a : rt 2 . 68 m / z [ m + h ] + 372 . 1 . ( 3e , 5e )- 1 -( 2 - cyclopropylacetyl )- 3 , 5 - bis [( 4 - methoxyphenyl ) methylidene ] azepan - 4 - one ( compound 1526 ). azepan - 4 - one hydrochloride ( 0 . 45 g , 3 . 0 mmol ) and 4 - methoxybenzaldehyde ( 0 . 90 g , 6 . 6 mmol ) were dissolved in acetic acid ( 10 ml ), conc . h 2 so 4 ( 1 ml ) was added drop - wise , and the reaction stirred at rt for 24 h . water ( 30 ml ) was added . the precipitate was filtered off and dried in vacuum over night . the crude material ( 30 mg , 0 . 107 mmol ), cyclopropylacetic acid ( 12 mg , 0 . 12 mmol ), tbtu ( 41 mg , 0 . 13 mmol ) and tea ( 26 mg , 0 . 26 mmol ) were dissolved in dmf ( 2 ml ) and stirred at rt over night . methanol ( 1 . 5 ml ) and water ( 0 . 5 ml ) were added and the product was purified by preparative lc to yield the solid product in 95 % purity . lcms a : rt 2 . 51 m / z [ m + h ] + 432 . 2 . ( 3e , 5e )- 5 -[( 3 - nitrophenyl ) methylidene ]- 3 -( phenylmethylidene ) azepan - 4 - one ( compound # 1560 ). n - boc - azepan - 4 - one ( 0 . 10 g , 0 . 47 mmol ) and 3 - nitrobenzaldehyde ( 156 mg , 1 . 0 mmol ) were dissolved in acetic acid ( 5 ml ), concentrated h 2 so 4 ( 0 . 5 ml ) was added drop - wise and the reaction stirred at rt for 4 days . then more concentrated h 2 so 4 ( 0 . 5 ml ) and aldehyde ( 156 mg , 1 . 0 mmol ) were added and stirring continued at rt for three weeks . a mixture of the mono - and di - condensation products was obtained . the mixture was purified by column chromatography ( dcm / methanol ) to give the intermediate amine intermediate 2 as a brown oil ( 19 mg ). intermediate 2 was dissolved in acetic acid ( 1 . 5 ml ) together with benzaldehyde . conc . h 2 so 4 ( 0 . 05 ml ) was added and the reaction stirred at rt overnight . then more h 2 so 4 was added and the stirring continued for a week . more aldehyde ( 156 mg , 1 . 0 mmol ) and h 2 so 4 was added and stirring continued for an additional 4 days . the reaction mixture was concentrated and purified by preparative lc to give the tfa - salt of the product as yellow solid in 98 % purity . lcms system a : rt 1 . 78 m / z [ m + h ] + 335 . 1 , system b : rt 2 . 43 / 2 . 28 . ( 3e , 5e )- 1 - methyl - 3 , 5 - bis [( 4 - nitrophenyl ) methylidene ] azepan - 4 - one ( compound # 1563 ). n - methylazepan - 4 - one . hcl ( 50 mg , 0 . 30 mmol ) and 4 - nitrobenzaldehyde were dissolved in acetic acid ( 5 ml ) and stirred for 10 min , then conc . h 2 so 4 ( 50 μl ) was added slowly and the mixture was stirred at rt overnight . more concentrated h 2 so 4 ( 100 μl ) was added and stirring was continued at rt for 6 h . additional 500 μl of concentrated h 2 so 4 was added and the reaction stirred overnight . a further 350 μl of conc . h 2 so 4 was added and stirring continued for additional 5 h , during which period further h 2 so 4 was added in two portions ( 500 μl and 250 μl ). then water ( 3 × reaction volume ) was added and the mixture was stirred until rt was reached . the reaction mixture was extracted with ethyl acetate ( 3 × reaction volume ). the phases were separated and the organic phase concentrated to yield a dark yellow viscous oil . the crude product was purified by preparative hplc , ( xbridge column ; eluents 50 mm ammonium carbonate buffer at ph 10 and methanol ) giving the title product as a yellow solid ( 26 . 3 mg ). lcms system a : rt 1 . 87 m / z [ m + h ] + 394 . 1 , system b : rt 2 . 57 . ( 3e , 5e )- 3 , 5 - bis [( 4 - fluorophenyl ) methylidene ]- 1 - propylazepan - 4 - one ( compound # 1574 ). azepan - 4 - one hydrochloride ( 0 . 25 g , 1 . 68 mmol ) and 4 - fluorobenzaldehyde ( 0 . 416 g , 3 . 36 mmol ) were dissolved in acetic acid ( 20 ml ) and the solution stirred for 10 min , then conc . h 2 so 4 ( 200 μl ) was slowly added and the solution was stirred at rt overnight . more conc . h 2 so 4 ( 1 ml ) was added and stirring continued at rt . another ml of conc . h 2 so 4 was added after 6 h , and the reaction stirred again overnight . the next day further 800 μl of conc . h 2 so 4 was added and stirring continued for a period of five days , during which two portions of h 2 so 4 ( 1 ml and 0 . 5 ml ) were added to the reaction mixture . then water ( 3 × reaction volume ) was added and the mixture stirred until rt was reached . the reaction mixture was extracted with ethyl acetate ( 10 × reaction volume ). the organic phase was concentrated by evaporation . water was added to the residue . a precipitate was formed and filtered off . the solid was washed with water and dried in vacuum to give intermediate 3 as a yellow solid . a portion ( 15 mg , 0 . 05 mmol ) thereof was dissolved in dce - propanal ( 4 μl , 0 . 06 mmol ) was added , and the mixture stirred for 15 min at rt . then nabh ( oac ) 3 ( 15 . 7 mg , 0 . 07 mmol ) and acetic acid ( 2 . 6 μl , 0 . 05 mmol ) were added and the reaction stirred at rt over night . the reaction was concentrated and the crude product purified by preparative lc giving the product ( 7 . 2 mg ) in 90 % purity . lcms system a : rt 2 . 02 m / z [ m + h ] + 368 . 1 , system b : rt 3 . 21 . ( 3e , 5e )- 3 -[( 4 - methoxyphenyl ) methylidene ]- 5 -[( 4 - nitrophenyl ) methylidene ] azepan - 4 - one ( compound # 1575 ). azepan - 4 - one hydrochloride ( 0 . 25 g , 1 . 68 mmol ) and 4 - nitrobenzaldehyde ( 253 mg , 1 . 68 mmol ) were dissolved in acetic acid ( 20 ml ) and stirred for 10 min , then conc . h 2 so 4 ( 1 ml ) was slowly added and the mixture stirred at rt for 8 days . on days 1 - 3 one portion conc . h 2 so 4 per day was added ( 0 . 5 ml , 0 . 75 ml , and 0 . 5 ml ). water ( 2 × reaction volume ) was added and the mixture extracted with ethyl acetate ( 2 × reaction volume ). the organic phase was concentrated by evaporation and dried to yield crude intermediate 4 . a portion of intermediate 4 ( 100 mg , 0 . 41 mmol ) was dissolved in acetic acid ( 6 ml ) and stirred for 10 min , then concentrated h 2 so 4 ( 0 . 6 ml ) was added slowly and the reaction stirred at rt for 6 days . upon addition of water the product precipitated as a yellow solid . the precipitate was filtered off , washed with water and dried in vacuum to give the title compound as a yellow solid in 98 % purity . lcms system a : rt 1 . 82 m / z [ m + h ] + 365 . 1 , system b : rt 2 . 41 . 1 h - nmr ( 400 mhz , cdcl 3 ) [ ppm ]= 2 . 97 - 2 . 99 ( m , 2h ), 3 . 41 - 3 . 44 ( m , 2h ), 3 . 83 ( bs , 3h ), 4 . 28 ( s , 2h ), 7 . 06 - 7 . 08 ( d , 2h ), 7 . 47 ( s , 1h ), 7 . 59 - 7 . 62 ( d , 2h ), 7 . 76 ( s , 1h ), 7 . 78 - 7 . 80 ( d , 2h ), 8 . 27 - 8 . 29 ( d , 2h ). ( 3e , 5e )- 5 -[( 4 - fluorophenyl ) methylidene ]- 3 -[( 4 - methoxyphenyl ) methylidene ]- 1 - methylazepan - 4 - one ( compound # 1577 ). n - methylazepan - 4 - one hydrochloride ( 75 mg , 0 . 46 mmol ) and 4 - fluorobenzaldehyde were dissolved in acetic acid ( 7 ml ) and stirred for 10 min , then conc . h 2 so 4 ( 350 μl ) was added slowly and the mixture was stirred at rt for 8 days . more conc . h 2 so 4 was added during days 2 - 4 ( 0 . 175 ml , 0 . 35 ml , 0 . 25 ml respectively ). water was added and the solution extracted with ethyl acetate ( twice the volume of reaction mixture ). the organic phase was concentrated to give intermediate 5 . a portion of this intermediate ( 35 mg , 0 . 15 mmol ) and 4 - methoxybenzaldehyde ( 17 μl , 0 . 15 mmol ) were dissolved in acetic acid ( 2 . 5 ml ) and stirred for 10 min , then conc . h 2 so 4 ( 0 . 20 ml ) was added slowly and the reaction stirred for five days . water ( 2 × reaction volume ) was added and the reaction mixture extracted with ethyl acetate ( 2 × reaction volume ). the organic layer was concentrated and water was added . a precipitate was formed and filtered off to give the title product ( 11 . 2 mg ) in 91 % purity as a yellow solid . lcms system a : rt 1 . 86 m / z [ m + h ] + 352 . 1 , system b : rt 2 . 79 . ( 3e , 5e )- 1 - acetyl - 5 -[( 4 - fluorophenyl ) methylidene ]- 3 -[( 4 - ethoxyphenyl ) methylidene ] azepan - 4 - one ( compound # 1579 ). azepan - 4 - one hydrochloride ( 0 . 25 g , 1 . 68 mmol ) and 4 - fluorobenzaldehyde ( 179 μl , 1 . 68 mmol ) were dissolved in acetic acid ( 20 ml ) and stirred for 10 min , then conc . h 2 so 4 ( 1 ml ) was slowly added and the mixture was stirred at rt for 8 days with addition of conc . h 2 so 4 during the first three days ( 0 . 5 ml , 0 . 75 ml and 0 . 5 ml respectively ). water ( 2 × reaction volume ) was added and the mixture extracted with ethyl acetate ( 2 × mixture volume ). the organic phase was concentrated and dried to give the crude intermediate 6 . a portion of this intermediate ( 100 mg , 0 . 46 mmol ) was dissolved in acetic acid ( 6 ml ) and stirred for 10 min , then concentrated h 2 so 4 ( 0 . 6 ml ) was added slowly and the reaction stirred at rt for 7 days . water was added ( 1 × volume ) and the mixture was neutralized with saturated aqueous nahco 3 . the formed precipitate was filtered off , washed with water and dried in vacuum to yield intermediate 7 ( 31 . 5 mg ) as a yellow solid of 91 % purity . lcms system a : rt 1 . 85 m / z [ m + h ] + 338 . intermediate 7 ( 10 mg ) was dissolved in dcm ( 1 ml ) and tea ( 5 . 0 μl , 0 . 04 mmol ) was added . the mixture was stirred for 10 min , then acetyl chloride ( 2 . 3 μl , 0 . 03 mmol ) was added and the reaction stirred at rt for 30 min . the reaction was washed with water , saturated aqueous nahco 3 and brine . the organic phase was concentrated to give the title compound ( 6 . 4 mg ) as a yellow solid of 90 % purity . lcms system a : rt 2 . 35 m / z [ m + h ] + 380 . 1 , system b : rt 2 . 37 . 1 h - nmr ( 400 mhz , cdcl 3 ): [ ppm ]= 1 . 70 , 1 . 90 , 1 . 98 and 1 . 99 ( 4 × s , 3h , ch 3 co —, signals from the two regioisomers and their acetate rotamers ), 2 . 89 - 3 . 01 ( m , 2h ), 3 . 68 - 3 . 77 ( m , 2h ), 3 . 79 , 3 . 79 , 3 . 79 , 3 . 08 ( 4 × s , 3h , — ome , signals from the two regioisomers and their acetate rotamers ), 4 . 65 - 4 . 68 ( m , 2h ), 7 . 0 - 7 . 04 and 7 . 098 - 7 . 103 ( 2 × m , 2h ), 7 . 22 - 7 . 30 ( m , 3h ), 7 . 48 - 7 . 62 ( m , 5h ). ( 3e , 5e )- 5 -[( 4 - chlorophenyl ) methylidene ]- 3 -[( 4 - nitrophenyl ) methylidene ] azepan - 4 - one ( compound # 1583 ). n - methylazepan - 4 - one hydrochloride ( 75 mg , 0 . 46 mmol ) and 4 - chlorobenzaldehyde ( 64 mg , 0 . 46 mmol ) were dissolved in acetic acid ( 7 ml ) and stirred for 10 min , then conc . h 2 so 4 ( 350 μl ) was added slowly and the mixture was stirred at rt for 8 days . more conc . h 2 so 4 was added during days 2 - 4 ( 0 . 175 ml , 0 . 35 ml , 0 . 25 ml respectively ). water ( 2 × reaction volume ) was added and the solution extracted with ethyl acetate ( 2 × reaction volume ). the organic phase was concentrated to give intermediate 8 . a portion of the intermediate ( 35 mg , 0 . 14 mmol ) and 4 - nitrobenzaldehyde ( 69 . 5 mg , 0 . 46 mmol ) were dissolved in acetic acid ( 2 . 5 ml ) and stirred for 10 min , then conc . h 2 so 4 ( 200 μl ) was added slowly and the mixture was stirred at rt for 5 days . more conc . h 2 so 4 ( 0 . 2 ml ) was added , and stirring continued for 5 more days . water ( 2 × reaction volume ) was added and the solution extracted with ethyl acetate ( 2 × reaction volume ). the organic phase was concentrated and the residue purified by preparative lc to give the title compound ( 1 . 8 mg ) as a yellow solid of 94 % purity . lcms system a : rt 1 . 98 / 2 . 04 m / z [ m + h ] + 383 . 1 , system b : rt 2 . 82 / 2 . 98 . pharmaceutical composition a ( aqueous suspension ). the compound of the invention ( 25 mg ) is dissolved in 1 ml of dimethyl sulfoxide . the solution is added drop - wise to 10 ml of vigorously stirred saline . the formed suspension , which can be stabilized by adding 1 % by weight of pvp , can be used for intramuscular , intravenous or subcutaneous administration . pharmaceutical composition b ( tablet ). tablets for oral administration are produced by blending 2 . 0 g of the compound of the invention ( powder , & lt ; 10 mμ , 90 %) with microcrystalline cellulose ( 1 . 30 g ), corn starch ( 0 . 50 ) g , silica ( 0 . 20 ) g , mg stearate ( 0 . 12 mg ). the mixture is dry compressed to 400 mg tablets , which are sugar coated . pharmaceutical composition c ( solution ). the compound of the invention ( 10 mg ) is dissolved in 0 . 5 ml of cremophor el ( basf corp .) and absolute ethanol was added to 1 . 0 ml . the clear solution is filled into glass vials for injection . pharmaceutical composition d ( solution ). for intraperitoneal administration in animal studies an aqueous composition a stock solution was prepared by dissolving the compound of the invention to a concentration of 2 mg / ml in chremaphor el / polyethylene glycol 400 1 : 1 . ( v / v ) at room temperature or by heating to up to about 80 ° c . assisted by ultrasonication . a aliquot of the stock solution was diluted 1 : 10 with 0 . 9 % saline and used immediately for ip injection . pharmaceutical composition e ( solution ). for intraperitoneal administration a 25 % by weight kolliphor hs15 stock solution was prepared by melting an entire container of kolliphor hs15 ( sigma 42966 ) by warming to 60 ° c . and diluting with deionized water to 25 % w / w . to compound # 1570 ( 18 . 0 mg ) in a 10 ml sample tube was added 10 . 0 ml of the stock solution and the tube vortexed , treated with ultrasound at about 50 ° c . for about 2 h , and occasionally heated to about 83 ° c . the clear solution obtained was sterile filtered through a 0 . 2 μm cellulose syringe filter prior to injection . by the same procedure solutions of compounds # 1546 and # 1571 were prepared ; these compounds were however not fully dissolved . the non - dissolved residue was weighed , and the weight deducted from the starting weight of compound ( 18 mg ). it was found that the prepared solutions ( 10 ml ) contained 8 . 5 mg and 11 . 0 mg , respectively , of compounds # 1546 and # 1571 . pharmaceutical composition f ( solution ). for intraperitoneal administration a stock solution of 2 - hydroxypropyl - β - cyclodextrin ( aldrich 332593 ) was prepared by dissolving the cyclodextrin in deionized water to a concentration of 30 % w / w . to compound # 1649 ( 15 . 0 mg ) in a 10 ml sample tube was added 10 . 0 ml of the stock solution . the tube was vortexed , treated with ultrasound at about 50 ° c . for about 2 h , and occasionally heated to about 83 ° c . the solution obtained was sterile filtered through a 0 . 2 μm cellulose syringe filter prior to injection . the weight of residual compound # 1659 not dissolved was determined and used for correcting the concentration of the filtered solution to 82 . 5 % of the attempted concentration . by the same procedure a solution of compound # 1546 was prepared . the compound of the invention induces proteasome inhibition . the reporter cell line meljuso ub - yfp , which is engineered to accumulate yellow fluorescent protein ( yfp ) upon proteasome inhibition ( 12 ), was used for compound evaluation . the accumulation of yfp was measured for 48 hours in an incucyte - flr system ( essen bioscience , essen , uk ), which is an automated fluorescence microscope . numbers of positive cells per field were used as a measure of proteasome inhibition . determination of solubility of compounds of the invention in aqueous media . in the diagrams of fig2 a - 2 e solubility is expressed as log s ( mmol / ml ; software acd / labs inc .) solubility is determined in aqueous buffer at various ph values and predicted for pure water at 25 ° c . the algorithm uses a set of & gt ; 6 , 800 compounds as reference . the diagrams show that azepanones of the invention can have a substantially increased solubility , such as by a factor 2 or more , in aqueous media at physiological ph , such as at a ph from 6 to 8 , in particular of from 7 . 0 to 7 . 5 , in comparison with correspondingly substituted piperidin - 4 - ones . azepanes / azepanones of the invention exhibit higher cytotoxicity than structurally corresponding piperidines / piperidin - 4 - ones fig3 b , 3 d , 3 f are diagrams illustrating the cytotoxicity of compounds of the invention nos . 1546 , 1547 , and 1570 with a 7 - membered ring moiety in comparison to structurally corresponding compounds not comprised by the invention with a 6 - membered ring moiety . their induction of dose - dependent cytotoxicity was determined after 72 hours of continuous compound exposure to the reporter cell line hct - 116 . treated cells were compared with untreated controls . cytotoxicity is visualized as survival index ( si ) over the range of about 90 % si to about 0 % si in dependence on compound concentration . it appears from the figures that the compounds of the invention are more cytotoxic that the reference compounds since they are producing the same level of cytotoxicity at lower concentration . 1 . masdehors , p et al ., increased sensitivity of cll - derived lymphocytes to apoptotic death activation by the proteasome - specific inhibitor lactacystin . br j haematol 105 , 752 - 757 , doi : bjh1388 [ pii ] ( 1999 ). 2 . demartino , g n et al ., pa 700 , an atp - dependent activator of the 20 s proteasome , is an atpase containing multiple members of a nucleotide binding protein family . j biol chem 69 , 20878 - 20884 , http :// www . ncbi . nlm . nih . gov / entrez / query . fcgi ? 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