Patent Application: US-201514613881-A

Abstract:
the present invention concerns novel carbonic anhydrase ix inhibitors comprising a nitroimidazole moiety and their use in therapy of hypoxic conditions , in particular cancer treatment , especially chemotherapy and radiotherapy . the compounds of the invention have an increased specificity for the carbonic anhydrase ix enzyme compared to the art . the present invention relates to novel nitroimidazole derivates represented by formula , wherein r 1 , r 2 , and z are as defined herein :

Description:
reagents and conditions : ( i ) 1 equiv . of 2 - nitroimidazole , 1 equiv . of tert - butyl bromoacetate , 4 equiv . of potassium carbonate , mecn , rt , 1 night ; ( ii ) cocktail of trifluoroacetic acid / water / thioanisole 95 / 2 . 5 / 2 . 5 v / v , room temperature , 1 night ; ( iii ) 1 equiv . of 4 - dimethylaminopyridine ( dmap ), 1 equiv . of 1 -( 3 - dimethylaminopropyl )- 3 - ethylcarbodiimide hydrochloride ( edc ), n , n - dimethylacetamide ( dma ), room temperature , 2 days . reagents and conditions : ( i ) 1 equiv . of 1 -( 2 - aminoethyl )- 2 - methyl - 5 - nitroimidazole dihydrochloride monohydrate , 1 equiv . scn - ph - so 2 nh 2 , 2 equiv . of triethylamine , mecn , room temperature , 1 hour . reagents and conditions : ( i ) 1 equiv . of 1 -( 2 - aminoethyl )- 2 - methyl - 5 - nitroimidazole dihydrochloride monohydrate , 4 equiv . of triethylamine , 1 equiv . of chlorosulfonylisocyanate , 1 equiv . of tert - butanol , ch 2 cl 2 , rt , 1 hour ; ( ii ) trifluoroacetic acid ch 2 cl 2 7 / 3 , rt , 6 hours . fig4 shows the scheme of the second preferred compound of the invention . reagents and conditions : ( i ) 1 equiv . of 2 - methyl - 5 - nitro - 1 - imidazolylethanol , n , n - dimethylacetamide , 3 equiv . sulfamoyl chloride , rt , 1 night . the compounds of the present invention can be synthesised according to the following procedures . all reagents and solvents were of commercial quality and used without further purification , unless otherwise specified . all reactions were carried out under an inert atmosphere of nitrogen . tlc analyses were performed on silica gel 60 f 254 plates ( merck art . 1 . 05554 ). spots were visualized under 254 nm uv illumination , or by ninhydrin solution spraying . melting points were determined on a büchi melting point 510 and are uncorrected . 1 h and 13 c nmr spectra were recorded on bruker drx - 400 spectrometer using dmso - d 6 as solvent and tetramethylsilane as internal standard . for 1 h nmr spectra , chemical shifts are expressed in δ ( ppm ) downfield from tetramethylsilane , and coupling constants ( j ) are expressed in hertz . electron ionization mass spectra were recorded in positive or negative mode on a water micromass zq . it is referred to the attached fig1 and 2 . to a mixture of 2 - nitroimidazole 1 ( 17 . 7 mmol , 1 equiv .) and anhydrous potassium carbonate ( 70 . 74 mmol , 4 equiv .) in 20 ml of acetonitrile is added dropwise a solution of tert - butylbromoacetate ( 17 . 7 mmol , 1 equiv .) in 10 ml of acetonitrile . the mixture is stirred one night at room temperature and concentrated under vacuum . the residue is purified by chromatography on silica gel using a mixture ch2cl2 / meoh 95 / 5 as eluent to give the expected compound as white powder in 71 % yield . mp 95 - 97 ° c . ; 1 h nmr ( dmso - d6 , 400 mhz ) δ 1 . 46 ( s , 9h ), 4 . 99 ( s , 2h ), 7 . 06 ( d , 1h , j = 1 . 01 hz ), 7 . 17 ( d , 1h , j = 1 . 01 hz ). ms ( esi + / esi − ) m / z 226 . 15 [ m − h ] − , 262 . 13 [ m + cl ] − , 250 . 20 [ m + h ] + compound 2 ( 2 . 5 g ) is dissolved in 20 ml of a cocktail of tfa , water , thioanisole 95 - 2 . 5 - 2 . 5 and stirred at room temperature for one night . the mixture is then concentrated under vacuum and co - evaporated several times with diethyl ether until formation of a powder . after filtration , the precipitate is washed with dichloromethane and acetonitrile to give quantitatively the expected product . mp 143 ° c . ( decomposition ); 1 h nmr ( dmso - d 6 , 400 mhz ) δ 5 . 21 ( s , 2h ), 7 . 21 ( d , 1h , j = 1 . 01 hz ), 7 . 64 ( d , 1h , j = 1 . 01 hz ). 13 c ( dmso - d 6 , 101 mhz ) δ 50 . 65 , 127 . 69 , 128 . 44 , 168 . 56 , 168 . 57 ; ms ( esi + / esi − ) m / z 170 . 12 [ m − h ] − , 341 . 05 [ 2m − h ] − , 194 . 14 [ m + na ] + . to a solution of compound 3 ( 1 . 17 mmol , 1 equiv .) in 8 ml of n , n - dimethylacetamide was added the aminoalkylbenzene sulfonamide ( 1 . 17 mmol , 1 equiv . ), 4 - dimethylaminopyridine ( 1 . 17 mmol , 1 equiv .) and 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ( 1 . 17 mmol , 1 equiv .). the mixture was stirred two days at room temperature , then diluted with ethyl acetate and washed three times with water . the organic layer was dried over anhydrous magnesium sulfate , filtrated and concentrated under vacuum . the residue was then purified by chromatography on silica gel using methylene chloride — methanol 98 - 2 v - v as eluent . yield : 68 %; mp 163 - 165 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 5 . 36 ( s , 2h ), 7 . 24 ( d , 1h , j = 1 . 01 hz ), 7 . 28 ( s , 2h ), 7 . 67 ( d , 1h , j = 1 . 01 hz ), 7 . 70 ( d , 2h , j = 8 . 9 hz ), 7 . 77 ( d , 2h , j = 8 . 9 hz ), 10 . 7 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 52 . 20 , 118 . 70 , 126 . 81 , 127 . 57 , 128 . 84 , 138 . 76 , 141 . 18 , 144 . 79 , 165 . 02 ; ms ( esi + / esi − ) m / z 324 . 09 [ m − h ] − , 359 . 92 [ m + cl ] − , 649 . 15 [ 2m − h ] − , 685 . 01 [ 2m + cl ] − , 348 . 14 [ m + na ] + . yield : 79 %; mp 195 - 197 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 5 . 35 ( s , 2h ), 7 . 24 ( d , 1h , j = 1 . 01 hz ), 7 . 38 ( s , 2h ), 7 . 52 ( m , 2h ), 7 . 65 ( m , 1h ), 7 . 67 ( d , 1h , j = 1 . 01 hz ), 8 . 14 ( s , 1h ), 10 . 81 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 52 . 16 , 116 . 10 , 120 . 72 , 121 . 92 , 127 . 59 , 128 . 86 , 129 . 67 , 138 . 67 , 144 . 72 , 164 . 87 , 167 . 75 ; ms ( esi + / esi − ) m / z 324 . 24 [ m − h ] − , 360 . 18 [ m + cl ] − , 685 . 13 [ 2m + cl ] − , 326 . 24 [ m + h ] + , 348 . 07 [ m + na ] + , 364 . 17 [ m + k ] + , 673 . 18 [ 2m + na ] + . yield : 83 %; mp 181 - 183 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 4 . 37 ( d , 2h , j = 5 . 7 hz ), 5 . 22 ( s , 2h ), 7 . 18 ( d , 1h , j = 1 . 01 hz ), 7 . 35 ( s , 2h ), 7 . 43 ( d , 2h , j = 8 . 4 hz ), 7 . 67 ( d , 1h , j = 1 . 01 hz ), 7 . 76 ( d , 2h , j = 8 . 4 hz ), 9 . 15 ( t , 1h , j = 6 . 06 hz ); 13 c ( dmso - d 6 , 101 mhz ) δ 41 . 81 , 51 . 55 , 106 . 87 , 125 . 58 , 127 . 39 , 138 . 91 , 142 . 66 , 142 . 99 , 156 . 82 , 165 . 88 ; ms ( esi + / esi − ) m / z 338 . 15 [ m − h ] − , 374 . 22 [ m + cl ] − , 713 . 16 [ 2m + cl ] − , 340 . 15 [ m + h ] + , 362 . 17 [ m + na ] + . yield : 89 %; mp 139 - 141 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 2 . 80 ( t , 2h , j = 6 . 9 hz ), 3 . 16 ( m 2h ), 5 . 07 ( s , 2h ), 7 . 18 ( d , 1h , j = 1 . 01 hz ), 7 . 30 ( s , 2h ), 7 . 40 ( d , 2h , j = 8 . 2 hz ), 7 . 61 ( d , 1h , j = 1 . 01 hz ), 7 . 74 ( d , 2h , j = 8 . 2 hz ), 8 . 46 ( t , 1h , j = 5 . 6 hz ); 13 c ( dmso - d 6 , 101 mhz ) δ 34 . 59 , 51 . 49 , 125 . 64 , 127 . 38 , 128 . 74 , 129 . 09 , 142 . 05 , 143 . 39 , 144 . 87 , 165 . 57 ; ms ( esi + / esi − ) m / z 352 . 19 [ m − h ] − , 388 . 07 [ m + cl ] − , 354 . 12 [ m + h ] + , 376 . 09 [ m + na ] + , 729 . 21 [ 2m + na ] + . to a solution of the commercially available compound 5 ( 0 . 76 mmol , 1 equiv .) in 10 ml of acetonitrile , was added the corresponding isothiocyanate ( 0 . 76 mmol , 1 equiv .). the reaction was stirred for one hour at room temperature and then filtered . the filtrate was concentrated under vacuum , and the residue obtained purified by chromatography on silica gel using methylene chloride - methanol 95 - 5 as eluent . yield : 72 %; mp 186 - 188 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 2 . 42 ( s , 3h ), 3 . 91 ( m , 2h ), 4 . 5 ( t , 2h , j = 5 . 68 hz ), 7 . 3 ( s , 2h ), 7 . 46 ( d , 2h , j = 8 . 7 hz ), 7 . 71 ( d , 2h , j = 8 . 7 hz ), 8 . 05 ( s , 2h ), 10 . 0 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 13 . 81 , 42 . 87 , 44 . 86 , 122 . 46 , 126 . 30 , 133 . 19 , 138 . 66 , 139 . 14 , 141 . 88 , 151 . 35 , 180 . 88 ; ms ( esi + / esi − ) m / z 385 . 17 [ m + h ] + , 407 . 07 [ m + na ] + , 769 . 22 [ 2m + h ] + , 383 . 21 [ m − h ] − , 419 . 18 [ m + cl ] − , 767 . 16 . [ 2m − h ] − . yield : 75 %; mp 66 - 68 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 2 . 43 ( s , 3h ), 3 . 91 ( m , 2h ), 4 . 49 ( t , 2h , j = 5 . 68 hz ), 7 . 39 ( s , 2h ), 7 . 51 ( m , 1h ), 7 . 55 ( s , 1h ), 7 . 57 ( m , 1h ), 7 . 81 ( s , 1h , 1h ), 7 . 93 ( m , 1h ), 8 . 04 ( s , 1h ), 9 . 94 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 13 . 81 , 42 . 82 , 44 . 99 , 120 . 29 , 121 . 39 , 126 . 62 , 129 . 09 , 133 . 18 , 138 . 65 , 139 . 51 , 144 . 35 , 151 . 37 , 181 . 28 ; ms ( esi + / esi − ) m / z 385 . 23 [ m + h ] + , 406 . 94 [ m + na ] + , 791 . 19 [ 2m + na ] + , 383 . 12 [ m − h ] − , 419 . 09 [ m + cl ] − , 767 . 26 [ 2m − h ] − . yield : 82 %; mp 67 - 69 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 2 . 37 ( s , 3h ), 2 . 87 ( m , 2h ), 4 . 43 ( t , 2h , j = 5 . 18 hz ), 4 . 70 ( br s , 2h ), 7 . 32 ( s , 2h ), 7 . 34 ( d , 2h , j = 8 . 4 hz ), 7 . 68 ( s , 1h ), 7 . 75 ( d , 2h , j = 8 . 4 hz ), 8 . 03 ( s , 1h ), 8 . 15 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 13 . 84 , 30 . 64 , 42 . 70 , 45 . 44 , 125 . 52 , 127 . 23 , 133 . 17 , 138 . 61 , 142 . 48 , 151 . 41 , 181 . 44 ; ms ( esi + / esi − ) m / z 399 . 23 [ m + h ] + , 421 . 16 [ m + na ] + , 797 . 08 [ 2m + h ] + , 819 . 26 [ 2m + na ] + , 397 . 10 [ m − h ] − , 433 . 09 [ m + cl ] − , 795 . 33 [ 2m − h ] − . yield : 86 %; mp 75 - 77 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 2 . 35 ( s , 3h ), 2 . 83 ( m , 2h ), 3 . 62 ( m , 2h ), 3 . 82 ( m , 2h ), 4 . 41 ( m , 2h ), 7 . 31 ( s , 2h ), 7 . 37 ( d , 2h , j = 8 . 2 hz ), 7 . 52 ( s , 1h ), 7 . 63 ( s , 1h ), 7 . 74 ( d , 2h , j = 8 . 2 hz ), 8 . 03 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 13 . 78 , 30 . 64 , 45 . 4 , 125 . 65 , 129 . 05 , 133 . 18 , 138 . 54 , 142 . 03 , 143 . 45 , 151 . 42 , 180 . 83 ; ms ( esi + / esi − ) m / z 413 . 06 [ m + h ] + , 435 . 02 [ m + na ] + , 825 . 09 [ 2m + h ] + , 847 . 21 [ 2m + na ] + , 411 . 06 [ m − h ] − , 447 . 20 [ m + cl ] − , 822 . 99 [ 2m − h ] − , 859 . 26 [ 2m + cl ] − . to a solution of 5 ( 3 . 83 mmol , 1 equiv .) and triethylamine ( 30 . 63 mmol , 4 equiv .) in 10 ml of methylene chloride was added a solution of chlorosulfonyl isocyanate ( 4 . 59 mmol , 1 . 2 equiv ), tert - butanol ( 4 . 59 mmol , 1 . 2 equiv .) in 2 ml of methylene chloride ( prepared ab - initio ). the mixture was stirred at room temperature for one hour , then diluted with ethyl acetate and washed with water . the organic layer was dried over anhydrous sodium sulfate , filtered and concentrated under vacuum . the residue was purified by chromatography on silica gel using methylene chloride - methanol 98 - 2 as eluent . this intermediate was then diluted in a solution of trifluoroacetic acid in methylene chloride ( 30 % volume ), and stirred at room temperature for 6 hours . the mixture was then concentrated under vacuum and co - evaporated several times with diethyl ether to give the expected sulfamide as a white powder . overall yield : 70 %; mp 122 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 2 . 52 ( s , 3h ), 3 . 26 ( m , 2h ), 4 . 37 ( t , 2h , j = 5 . 81 hz ), 6 . 65 ( s , 2h ), 6 . 86 ( s , 1h ), 8 . 1 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 14 . 03 , 41 . 8 , 46 . 0 , 132 . 68 , 138 . 26 , 151 . 65 ; ms ( esi + / esi − ) m / z 250 . 19 [ m + h ] + , 272 . 34 [ m + na ] + , 499 . 32 [ 2m + h ] + , 249 . 09 [ m − h ] − , 284 . 12 [ m + cl ] − , 533 . 14 [ 2m + cl ] − . to a solution of the commercially available compound 8 ( 1 . 75 mmol , 1 equiv .) in n , n - dimethylacetamide , was added sulfamoyl chloride ( 5 . 25 mmol , 3 equiv .). the mixture was stirred at room temperature for one night , then diluted with ethyl acetate , and washed three times with water . the organic layer was dried over anhydrous magnesium sulphate , filtered and concentrated under vacuum . the residue was purified by chromatography on silica gel using methylene chloride - methanol 9 - 1 as eluent . yield : 81 %; mp 166 - 168 ° c . ; 1 h nmr ( dmso - d 6 , 400 mhz ) δ 2 . 45 ( s , 3h ), 4 . 35 ( t , 2h , j = 5 . 05 hz ), 4 . 61 ( t , 2h , j = 5 . 05 hz ), 7 . 57 ( s , 2h ), 8 . 06 ( s , 1h ); 13 c ( dmso - d 6 , 101 mhz ) δ 14 . 04 , 44 . 98 , 57 . 21 , 133 . 10 , 138 . 32 , 151 . 82 ; ms ( esi + / esi − ) m / z 250 . 3 [ m + h ] + , 272 . 32 [ m + na ] + , 521 . 30 [ 2m + na ] + , 770 . 16 [ 3m + na ] + . the compounds of the invention were tested for their effects on ca inhibition and the resulting effect on extracellular acidosis using classical chemistry and biology assays . the compounds of the invention were tested on their inhibitory activity on carbonic anhydrase in the following experiment : the inhibition constants ( k i ) the compounds for four ca isozymes , ca i , ii , ix and xii were determined . an applied photophysics ( oxford , uk ) stopped - flow instrument has been used for assaying the ca - catalyzed co 2 hydration activity ( khalifah , 1971 ). phenol red ( at a concentration of 0 . 2 mm ) has been used as indicator , working at the absorbance maximum of 557 nm , with 10 mm hepes ( ph 7 . 5 ) as buffer , 0 . 1 m na 2 so 4 ( for maintaining constant the ionic strength ), following the ca - catalyzed co 2 hydration reaction for a period of 10 - 100 s . the co 2 concentrations ranged from 1 . 7 to 17 mm for the determination of the kinetic parameters and inhibition constants . for each inhibitor at least six traces of the initial 5 - 10 % of the reaction have been used for determining the initial velocity . the uncatalyzed rates were determined in the same manner and subtracted from the total observed rates . stock solutions of inhibitor ( 1 mm ) were prepared in distilled - deionized water with 10 - 20 % ( v / v ) dmso ( which is not inhibitory at these concentrations ) and dilutions up to 0 . 1 nm were done thereafter with distilled - deionized water . inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay , in order to allow for the formation of ca ix - inhibitor complex . the inhibition constants were obtained by non - linear last - squares methods using prism 3 and represent the mean from at least three different determinations . the experiment was set up as previously described ( dubois et al , 2007 ). a colorectal ( ht - 29 ) ( fig5 a ) and a cervical ( hela ) ( fig5 b ) carcinoma cell line were tested in normoxic ( ambient oxygen concentration ) and hypoxic ( 0 . 2 % oxygen ) conditions . ht - 29 cells are known to be constitutive hypoxia inducible ca ix ( ca ix expression under normoxia and increased ca ix expression upon hypoxia ) expressing cells . therefore , compounds were added after 1 h of hypoxic exposure ( to ensure active ca ix ) and incubation was done for another 23 h ( total of 24 h hypoxia ). hela cells are hypoxia inducible ( at lower density ) ca ix expressing cells . therefore , hela were first incubated for 24 h hypoxia to ensure ca ix expression in the first place ( time point assessed in time series experiments ). afterwards , compounds were added and cells were incubated for another 24 h . experiments were performed in triplicate for each condition . the described conditions were done both for ht - 29 and hela under normoxic and hypoxic conditions . blank 1 mm or 0 . 1 mm ( no addition of compound , only dmso / pbs ) s = aebs ( 4 -( 2 - aminoethylbenzenesulfonamide ); sigma ) 1 mm or 0 . 1 mm ( known to reduce extracellular ph as described by ( svastova et al , 2004 )) cells were seeded in 6 cm dishes ( ht - 29 : 10e6 ; hela : 4 × 10 5 : to compensate for cell size ) in 5 ml of dmem supplemented with 10 % fcs . the day after , medium was replaced by 3 . 6 ml of freshly prepared dmem / fcs 10 %, from which the ph was measured = ph at incubation ), after which dishes were placed in the hypoxic chamber ( normoxic dishes remained at ambient air in the incubator : 37 ° c ., 95 % humidity , 5 % co 2 ). compounds were added ( 1 h for ht - 29 or 24 h for hela after start hypoxic exposure ) to have a final concentration of 1 mm or 0 . 1 mm , by adding 400 μl to the dishes , starting from a 10 or 1 mm stock ( dmso final concentration 0 . 1 %). blanc controls received dmso / pbs without compound . the ph of the medium was measured after 24 h ( ht - 29 ) or 48 h ( hela ) inside the hypoxic chamber after calibration of the electrode to reduced oxygen concentrations . delta ph ( ph at end hypoxic exposure − ph of replacement medium before incubation ) were calculated and shown in the graph of fig5 a and 5 b . hypoxia results into an extracellular acidification . the medium is more acid in blank conditions upon hypoxia compared with normoxia . the control compound ( s ) effectively reduced the extracellular acidification upon hypoxia at 1 mm concentration ( less at 0 . 1 mm ) for both cell lines . no effect was seen upon normoxic exposure all compounds were able to reduce the extracellular acidification . for ht - 29 cells , dh348 ( compound 7 ) and dh338 ( compound 9 ) give the best results ( 1 mm ). at 0 . 1 mm dh348 gives an additional reduction compared with the other compounds . for hela , all compounds effectively reduced the extracellular acidification . dh310 even made the medium alkaline ( 1 mm ) and also 0 . 1 mm caused a strong reduction . since this effect was not seen in ht - 29 , interpretation should be done carefully . dh348 demonstrated the best results in hela , and confirms the obtained data for ht - 29 . aim of these experiments was to assess the in vivo therapeutic effect of dh348 in combination with conventional treatment modalities such as radiotherapy and chemotherapy . we hypothesize that blocking ca ix may both decrease extracellular acidosis , and thus increase the effect of irradiation , as well as increase the uptake of the weak base doxorubicin , and therefore its therapeutic effect . furthermore , a clinically approved sulfonamide acetazolamide ( aza )— a known general carbonic anhydrase inhibitor ( meaning no preference for one ca )— was used to prove the ca ix specificity of the investigated compounds . two experimental setups were used . the first experiments were carried out on parental ht - 29 xenografts . the second were done on ht - 29 xenografts harbouring a knock - down for ca ix . a shrna construct against ca ix was introduced in the ht - 29 cells using the pretro - super vector . after selection and screening , cells with a 95 % efficient knock - down for ca ix were selected and designated kd cells . as a control , a scrambled shrna construct was used and those cells were designated ev cells . these cells still demonstrate ca ix mrna and protein expression . tumor xenografts were produced by injecting the colorectal carcinoma cells ( 1 , 5 10e6 ) subcutaneously into the lateral flank of nmri - nu mice ( 28 - 32 g ). tumor growth was monitored 3 ×/ week by measuring the tumor dimensions in 3 orthogonal directions . measurements were corrected for skin thickness (− 0 . 5 mm ) and tumor volumes were calculated using the formula a × b × c × pi / 6 , were a , b and c represent the orthogonal diameters . at an average tumor volume of 250 mm 3 , dh348 was injected intravenously ( 5 × 5 mg / kg intravenously ) using the lateral tail vein . at day 3 animals were anaesthetized using sodium pentobarbital ( nembutal , 0 . 1 ml / 100 g body weight ) and positioned in the irradiation field using a custom - built jig and subjected to irradiation with a single dose of 10 gy ( 15 mev electron beam ) using a linear accelerator ( siemens ). another group of animals started at day 3 with doxorubicin treatment ( 5 mg / kg i . v . ix / week for 3 weeks ). tumor growth and potential treatment toxicity was monitored ( 3 ×/ week ), by daily evaluation of the body weight . when tumors reached four times the treatment starting volume , animals were sacrificed and tumors excised for further histopathological evaluation . in summary the treatment groups are arranged as follows , with the results presented in the figures : 6 a : ht - 29 parental tumors ( wt ): effect of irradiation 10 gy alone compared to no treatment ( controls ). 6 b : ht - 29 parental tumors ( wt ): effect of combination treatment ( dh348 + irradiation 10gy ) compared with dh348 , irradiation or no treatment . 6 c : effect of irradiation 10 gy alone compared to no treatment on ht - 29 shscrambled ( ev ) and shca ix ( kd ) tumors . 6 d : effect of dh348 treatment compared to no treatment on ev and kd tumors 6 e : effect of aza treatment compared to no treatment on ev and kd tumors . 6 f : effect of combination treatment with dh348 and irradiation 10 gy compared to the single treatment modalities on ev tumors . 6 g : effect of combination treatment with dh348 and irradiation 10 gy compared to the single treatment modalities on kd tumors . 6 h : effect of combination treatment with aza and irradiation 10 gy compared to the single treatment modalities on ev tumors . 6 i : effect of combination treatment with aza and irradiation 10 gy compared to the single treatment modalities on kd tumors . 6 j : ht - 29 parental tumors ( wt ): effect of doxorubicin treatment compared to no treatment ( controls ). 6 k : ht - 29 parental tumors ( wt ): effect of combination treatment ( dh348 + doxorubicin ) compared with the single treatment modalities . the tumor growth curves are shown in fig6 a - 6 d . tumor volumes are normalized to start treatment = 100 mm 3 ). no significant treatment toxicity was observed as monitored by total body weight changes during treatment . treatment with the compound of this invention results in a growth delay compared with no treatment . when combined with conventional clinically available treatment schedules , it results in a sensitization for radiotherapy and to a smaller extent for chemotherapy . this is indicated by the enhanced growth delay for the combination treatment , compared with the individual treatment arms . furthermore , we have demonstrated similar results for the parental and scrambled shrna bearing tumors . more important , we have shown a specific effect of the compounds on ca ix , since ( 1 ) the compound had no therapeutic effect on the ca ix shrna ( kd ) tumors and ( 2 ) no enhanced growth delay was observed when combined with irradiation compared with irradiation alone . the specificity of the compound for ca ix was further confirmed by evaluation of the general ca inhibitor aza , which demonstrated also an effect on the kd tumors . since several other ca isozymes then ca ix are present in healthy tissues and aza has inhibitory properties against those , normal tissue toxicity in prolonged treatments can be expected , a phenomenon what will be greatly reduced using the ca ix specific compounds of this invention . the daily dose of active ingredient can be administered to a host in a single dose or it can be an effective amount divided into several smaller doses that are administered throughout the day . the pharmacotherapeutic regimen for treating the aforementioned diseases with a compound of the invention and / or with the pharmaceutical compositions of the present invention will be selected in accordance with a variety of factors , including for example age , body weight , sex and medical condition of the patient as well as severity of the disease , route of administration , pharmacological considerations and eventual concomitant therapy with other drugs . in some instances , dosage levels below or above the aforesaid range and / or more frequent may be adequate , and this logically will be within the judgment of the physician and will depend on the disease state . the compounds of the invention may be administered orally , parenterally , rectally or topically , by inhalation spray o aerosol , in dosage unit formulations containing conventional non - toxic pharmaceutically acceptable carriers , adjuvants and vehicles as desired . topical preparations can be administered as solutions , suspensions or emulsions ( dispersions ) in an acceptable vehicle . topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices . the term “ parenteral ” as used herein includes subcutaneous injections , intravenous , intramuscular , intrasternal injection or infusion techniques . injectable preparations , for example , sterile injectable aqueous or oleaginous suspensions may be formulated according to known art using suitable dispersing or wetting agents and suspending agents . the sterile injectable preparation may also be a sterile injectable solution or suspension in a non - toxic parenterally acceptable diluent o solvent . among the acceptable vehicles and solvents are water , ringer &# 39 ; s solution and isotonic sodium chloride solution . suppositories for rectal administration of the drug can be prepared by mixing the active ingredient with a suitable non - irritating excipient , such as cocoa butter and polyethylene glycols . solid dosage forms for oral administration may include capsules , tablets , pills , powders , granules and gels . in such solid dosage forms , the active compound may be admixed with at least one inert compound such as sucrose , lactose or starch . such dosage forms may also comprise , as in normal practice , additional substances , e . g . lubricating agents such as magnesium stearate . in the case of capsules , tablets and pills , the dosage forms may also comprise buffering agents . tablets and pills can additionally be prepared with enteric coatings . liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions , solutions , suspensions , syrups and elixirs containing inert diluents commonly used in the art , such as water . such compositions may also comprise adjuvants such as wetting agents , emulsifying and suspending agents , and sweetening , flavouring and the like . it is further contemplated that the compounds of the present invention can be used with other medicaments known to be useful in the treatment of carcinomas . the above examples are only to be considered as an illustration of the invention and do not limit the scope of the invention in any way . hence , obvious variations and variants of the invention will be apparent to one skilled in the art . brizel d m , schroeder t , scher r l , walenta s , clough r w , dewhirst m w , mueller - 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