Patent Application: US-5726001-A

Abstract:
the present invention relates to anti - tumor compounds , compositions and methods . in particular , the invention relates to indolocarbazole analogues of the following general formulas that inhibit topoisomerase i activity

Description:
the compounds disclosed in the present invention are useful as antitumor agents for the treatment or prevention of cancer , either alone or with a carrier . cytotoxic agents are often employed as anticancer agents to control or eradicate tumors . topo i poisons are useful cytotoxic agents , and two topo i poisons related to camptothecin , camptosar and hycamtin ( topotecan ) are currently used clinically for the treatment of tumors . indolocarbazoles are a different class of topo i poison that represent useful agents for the treatment of tumors . in particular , topo i - poisoning compounds disclosed in this invention were shown to be highly cytotoxic against human ovarian and prostate tumor cells . indolocarbazole analogues of this invention may be formulated as a solution of lyophilized powders for parenteral administration . powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use . the liquid formulation is generally a buffered , isotonic , aqueous solution . examples of suitable diluents are normal isotonic saline solution , standard 5 % dextrose in water or in buffered sodium or ammonium acetate solution . such formulation is especially suitable for parenteral administration , but may also be used for oral administration . it may be desirable to add excipients such as polyvinylpyrrolidone , gelatin , hydroxycellulose , acacia , polyethylene glycol , mannitol , sodium chloride , or sodium acetate . alternatively , the compounds of the present invention may be encapsulated , tableted , or incorporated into an emulsion ( oil - in - water or water - in - oil ) syrup for oral administration . pharmaceutically acceptable solids or liquid carriers , which are generally known in the pharmaceutical formulary arts , may be added to enhance or stabilize the composition , or to facilitate preparation of the composition . solid carriers include starch ( corn or potato ), lactose , calcium sulfate dihydrate , terra alba , croscarmellose sodium , magnesium stearate or stearic acid , talc , pectin , acacia , agar , gelatin , maltodextrins and microcrystalline cellulose , or colloidal silicon dioxide . liquid carriers include syrup , peanut oil , olive oil , corn oil , sesame oil , saline , and water . the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate , alone or with a wax . the amount of solid carrier varies but , preferably , will be between about 10 mg to about 1 g per dosage unit . the dosage ranges for administration of indolocarbazole analogues disclosed in this invention are those to produce the desired affect . the dosage will generally vary with age , body weight , extent of the disease , and contraindications , if any . the dosage will also be determined by the existence of any adverse side effects that may accompany the compounds . it is always desirable , whenever possible , to keep adverse side effects to a minimum . one skilled in the art can easily determine the appropriate dosage , scheduling , and method of administration for the exact formulation of the composition being used in order to achieve the desired effective concentration in the individual patient . however , the dosage can vary from between about 1 mg / kg / day to about 500 mg / kg / day , and preferable from between about 1 mg / kg / day to about 50 mg / kg / day . one skilled in the art will recognize that modifications may be made in the present invention without deviating from the spirit or scope of the invention . the invention is illustrated further by the following examples , which are not to be construed as limiting the invention in spirit or scope to the specific procedures or compositions described in them . the required 5 , 6 - indolodioxan and 5 , 6 - indolodioxole precursors can be prepared starting from 4 - methylcatechol , as illustrated in scheme a for 5 , 6 - ethylenedioxyindole ( 4 ). protection of the ortho - dihydroxy function was achieved using 1 , 2 - dibromoethane , dichloromethane , and acetone , respectively . nitration using fuming nitric acid followed by indole formation using the batcho - leimgruber protocol 12 afforded the desired indoles . construction of indolocarbazole analogues can be conducted as illustrated in scheme b for ia . n - benzyloxymethyl - 3 , 4 - dibromomaleimide was prepared as previously described 13 . reaction with an appropriate indole , which had been pre - treated using an organometallic , preferably but not limited to methylmagnesium halide or lithium hexamethyldisilazide , afforded the bromoindolomaleimide intermediates represented by 5 . glucosidation at the indole nitrogen was achieved with 2 , 3 , 4 , 6 - tetra - o - benzyl - d - glucose under mitsunobu conditions 14 ( 3 equivalents each of the glucose , pph 3 , and diisopropylazodicarboxylate ( diad ), followed by reversed - phase purification to afford 6 . introduction of the second indole unit was conducted under conditions similar to introduction of the first indole unit , providing the bis - indolylmaleimides represented by 7 . oxidative cyclization of the bis - indolylmaleimides was achieved using either palladium ( ii ) trifluoroacetate in dmf , or via photochemical cyclization , providing indolocarbazoles represented by 8 . others 14 reported oxidative cyclization using alternative reagents such as cucl 2 and pdcl 2 , but these failed to catalyze the reaction in our hands . hydrogenolysis of the protective groups ( palladium hydroxide , hoac ) afforded the 6 - n - hydroxymethyl derivatives represented by 9 , which were readily converted to the desired final products , represented by ia , using ammonium acetate in methanol . all compounds provided spectral and analytical characteristics ( 1 h nmr , 13 c nmr , ms and elemental analysis ) consistent with the targeted structures . the following examples are illustrative of the invention but do not serve to limit its scope . a mixture of 4 - methylcatechol ( 33 . 04 g , 266 . 1 mmol ), 1 , 2 - dibromoethane ( 100 g , 532 . 3 mmol ), k 2 co 3 ( 75 . 4 g , 545 . 5 mmol ) and sodium iodide ( 0 . 2 g , 1 . 33 mmol ) in ethylene glycol ( 500 ml ) was heated to 130 ° c . under nitrogen for five hours . the solution was allowed to cool to ambient temperature and stirred overnight . after the mixture was filtered through celite , the solution was diluted with brine ( 800 ml ) and extracted with organic solvents ( ch 2 cl 2 / hexane / etoac : 1 : 3 : 1 , 3 × 500 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated to provide a crude oil . flash silica gel chromatography eluting with hexane ( 100 %) gradient to ether / hexane ( 8 : 2 ) afforded the title intermediate as a colorless oil 20 . 0 g ( 50 %). to a solution of 1 ( 20 . 0 g , 133 . 3 mmol ) in acetic acid ( 135 ml ) was added a solution of fuming hno 3 ( 10 ml ) in acetic acid ( 50 ml ) dropwise over 30 minutes . the mixture was stirred at ambient temperature for 10 minutes , then poured into a beaker containing ice to give a crystalline precipitate , which was collected by vacuum filtration and washed with water to afford the product ( 25 . 8 g , 99 . 2 %) as an off - white solid . a solution of 2 ( 19 . 5 g , 100 mmol ), n , n - dimethylformamide dimethyl acetal ( 23 . 63 g , 198 . 3 mmol ) and pyrrolidine ( 14 . 1 g , 198 . 3 mmol ) was heated to 110 ° c . and stirred for 24 hours under nitrogen . the reaction mixture was cooled , and 250 ml absolute methanol was added . the product crystallized as a bright red solid . recovery by suction filtration afforded the product ( 24 . 3 g , 88 . 0 %). to a solution of 3 ( 24 . 0 g , 87 . 0 mmol ) in methanol and thf ( 240 ml , 1 : 1 ) was added raney nickel ( 2 . 0 ml ) and hydrazine hydrate ( 3 × 3 . 6 ml , 348 mmol ) every half hour at ambient temperature under nitrogen . then , the solution was heated at 45 ° c . for two hours . the mixture was cooled to room temperature and the catalyst is removed by filtration through a bed of celite and washed three times with methylene chloride . the filtrate was evaporated and the residue dried by evaporating with toluene ( 100 ml ) to give a crude oil . flash silica gel column purification eluting with hexane ( 100 %) gradient to ethyl acetate / hexane ( 40 / 60 %) afforded the title intermediate as an off - white solid ( 7 . 0 g , 46 . 1 %). to a solution of 5 - benzyloxyindole ( 8 . 93 g , 40 mmol ) in benzene ( 150 ml ) was added methylmagnesium iodide ( 14 . 7 ml , 44 . 0 mmol , 3 m in ether ) at 0 ° c . the solution was stirred for one hour , and then a solution of n - benzyloxymethyl - 3 , 4 - dibromomaleimide ( 15 . 0 g , 40 mmol ) in benzene ( 50 ml ) and thf ( 100 ml ) was added . the reaction mixture was stirred at 0 ° c . for 30 minutes and then warmed to room temperature and stirred for one hour . the mixture was diluted with etoac ( 350 ml ) then washed with hcl ( 150 ml , 0 . 3 n ), nahco 3 ( 200 ml ) and h 2 o ( 200 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . crystallization of the crude oil with methanol afforded the title intermediate as a yellow solid ( 12 . 50 g , 66 . 0 %). a solution of 5 ( 15 . 0 g , 29 . 0 mmol ), 2 , 3 , 4 , 5 - tetra - o - benzyl - d - glucopyranose ( 47 . 02 g , 87 . 0 mmol ) and triphenylphosphine ( 22 . 8 g , 87 . 0 mmol ) in thf ( 800 ml ) was cooled to − 78 ° c . diisopropylazodicarboxylate ( 17 . 14 ml , 87 . 0 mmol ) was added dropwise , maintaining the temperature at − 78 ° c ., and then stirred for three hours . the solution was warmed to 0 ° c . with the aid of an ice - water bath and stirring was continued for two hours . the mixture was diluted with etoac ( 1200 ml ), washed with hcl , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated . the crude mixture was applied to a reversed - phase biotage cartridge and eluted with a ch 3 cn / h 2 o ( 50 / 50 ) gradient to ch 3 cn ( 100 %) afforded the title intermediate as a yellow solid 22 . 3 g ( 74 . 0 %). to a solution of 4 ( 303 . 2 mg , 1 . 73 mmol ) in thf ( 35 ml ) was added lithium hexamethyldisilazide ( lihmds , 3 . 46 ml , 3 . 46 mmol , 1 m in thf ) at 0 ° c ., and the resulting solution stirred for 40 minutes . a solution of 6 in thf ( 20 ml ) was added slowly to the above mixture , followed by stirring for 20 minutes at 0 ° c . the mixture was diluted with etoac ( 300 ml ), washed with hcl ( 2 m ), nahco 3 , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated to give a crude mixture . flash silica gel chromatography eluting with a hexane ( 100 %) gradient to etoac / hexane ( 40 / 60 ) afforded the title intermediate as a red solid 1 . 20 g ( 73 . 2 %). to a solution of 8 ( 550 mg , 0 . 485 mmol ) in dmf ( 28 ml ) was added palladium ( ii ) trifluoroacetate ( 338 . 5 mg , 1 . 18 mmol ), and stirred at 80 ° c . for one hour . the solution was cooled to room temperature , diluted with etoac ( 280 ml ), and washed with hcl ( 1 m ), nahco 3 , brine ( 150 ml ) and h 2 o ( 3 × 120 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel chromatography eluting with etoac / hexane ( 3 : 7 ) afforded 404 mg ( 73 . 6 %) as a yellow solid . to a solution of 8 ( 140 mg , 0 . 1236 mmol ) in hoac ( 25 ml ) was added palladium hydroxide [ pd ( oh ) 2 , 140 mg ]. the reaction was shaken under a hydrogen atmosphere ( 50 psi ) at ambient temperature for 63 hours . the mixture was filtered through an acrodisc syringe filter and concentrated in vacuo to give a crude solid . flash chromatography eluting with meoh / hoac / etoac ( 12 / 1 / 87 ) afforded 42 . 0 mg ( 57 . 5 %) as a yellow solid . to a solution of 9 ( 5 . 0 mg , 0 . 00845 mmol ) in meoh ( 0 . 5 ml ) was added nh 4 oh ( 1 . 5 ml ). the mixture was stirred at ambient temperature for 3 hours , then concentrated in vacuo to give a crude solid . recrystallization with meoh / hexane / chcl 3 afforded 4 . 3 mg ( 90 . 5 %) as a yellow solid . a mixture of 4 - methylcatechol ( 26 . 0 g , 209 . 4 mmol ) and naoh ( 18 . 4 g , 461 . 0 mmol ) in ch 2 cl 2 ( 40 . 0 ml ) was heated to 100 ° c . under nitrogen for 2 hours . the solution was allowed to cool to ambient temperature and diluted with ethyl acetate ( 500 ml ). the mixture was washed with nahco 3 ( 200 ml ) and h 2 o ( 2 × 200 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated to give a crude oil . flash chromatography eluting with hexane ( 100 %) gradient to ether / hexane ( 1 : 1 ) afforded the title intermediate as a colorless oil 20 . 5 g ( 71 . 9 %). to a solution of 10 ( 19 . 0 g , 139 . 6 mmol ) in acetic acid ( 180 ml ) was added a solution of fuming hno 3 ( 10 ml ) in acetic acid ( 70 ml ) dropwise over 30 minutes . the mixture was stirred at ambient temperature for 10 minutes , then poured into a beaker containing ice to give a crystalline precipitate , which was collected by vacuum filtration and washed with water to afford the crude product . further purification via recrystallization from ch 2 cl 2 / hexane gave the pure product ( 16 . 3 g , 64 . 4 %). a solution of 11 ( 15 . 7 g , 86 . 74 mmol ), n , n - dimethylformamide dimethyl acetal ( 15 . 5 g , 130 . 1 mmol ) and pyrrolidine ( 9 . 25 g , 130 . 1 mmol ) was heated to 110 ° c . and stirred for 3 hours under nitrogen . the reaction mixture was cooled , and a mixture of absolute methanol and ethanol ( 1 : 1 ; 250 ml ) was added . the product crystallized as a bright red solid . recovery by suction filtration afforded the product ( 16 . 2 g , 71 . 4 %). to a solution of 12 ( 15 . 7 g , 59 . 92 mmol ) in methanol and thf ( 200 ml , 1 : 1 ) was added raney nickel ( 1 . 5 ml ) and hydrazine hydrate ( 3 × 2 . 56 ml , 240 mmol ) in three equal portions every half hour at ambient temperature under nitrogen . the solution was then heated at 45 ° c . for two hours . the mixture was cooled to room temperature and the catalyst was removed by filtration through a bed of celite , then washed three times with methylene chloride . the filtrate was evaporated and the residue dried by azeotroping with toluene ( 100 ml ) to provide a crude oil . flash silica gel column chromatography eluting with hexane ( 100 %) gradient to ethyl acetate / hexane ( 30 / 70 %) afforded the title intermediate as an off - white solid ( 5 . 1 g , 52 . 9 %). to a solution of 13 ( 279 . 0 mg , 1 . 73 mmol ) in thf ( 35 . 0 ml ) was added lithium hexamethyldisilazide ( lihmds , 3 . 46 ml , 3 . 46 mmol , 1 m in thf ) at 0 ° c . and stirred for 40 minutes . a solution of 6 in thf ( 20 ml ) was added slowly to above mixture , followed by stirring for 20 minutes at 0 ° c . the mixture was diluted with etoac ( 350 ml ), washed with hcl ( 1 m ), nahco 3 , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated to give a crude mixture . flash silica gel column chromatography eluting with hexane ( 100 %) gradient to ethyl acetate / hexane ( 40 / 60 %) afforded the title intermediate as a red solid 0 . 73 g ( 45 . 3 %). to a solution of 14 ( 650 mg , 0 . 58 mmol ) in dmf ( 36 ml ) was added palladium ( ii ) trifluoroacetate ( 405 mg , 1 . 22 mmol ), and the reaction was stirred at 80 ° c . for one hour . the solution was cooled to room temperature and diluted with etoac ( 350 ml ), then washed with hcl ( 1 m ), nahco 3 , brine ( 150 ml ) and h 2 o ( 3 × 150 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel column chromatography eluting with etoac / hexane ( 3 : 7 ) afforded 248 mg ( 38 . 2 %) as a yellow solid . to a solution of 15 ( 150 mg , 0 . 1341 mmol ) in hoac ( 10 ml ) was added palladium hydroxide ( 150 mg ). the reaction was shaken under an atmosphere of h 2 ( 50 psi ) at ambient temperature for 60 h . the mixture was filtered through an acrodisc syringe filter and concentrated in vacuo to give a crude solid . flash silica gel column chromatography eluting with meoh / acoh / etoac ( 12 / 1 / 87 ) afforded 56 . 2 mg ( 76 . 7 %) as a yellow solid . to a solution of 16 ( 30 . 0 mg , 0 . 052 mmol ) in meoh ( 2 . 0 ml ) was added nh 4 oh ( 4 . 0 ml ). the mixture was stirred at ambient temperature for 3 hours , then concentrated in vacuo to give a crude solid . flash silica gel column chromatography eluting with meoh / acoh / etoac ( 12 / 1 / 87 ) afforded 26 . 1 mg ( 91 . 1 %) as a yellow solid . a mixture of 4 - methylcatechol ( 75 . 0 g , 604 . 2 mmol , aldrich ), phosphorous pentoxide ( 85 . 8 g , 302 . 3 mmol ) in acetone ( 200 ml ) and toluene ( 200 ml ) was refluxed under nitrogen for 50 hours . the solution was allowed to cool to ambient temperature and diluted with ether ( 500 ml ). the mixture was washed with 2 m naoh ( 2 × 200 ml ) and h 2 o ( 2 × 200 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated to give a crude oil . flash chromatography eluting with hexane ( 100 %) gradient to ether / hexane ( 1 : 1 ) afforded the title intermediate as a colorless oil ( 82 . 0 g , 82 . 7 %). to a solution of 17 ( 65 . 0 g , 395 . 9 mmol ) in hoac ( 450 ml ) was added a solution of fuming hno 3 ( 35 ml ) in acetic acid ( 100 ml ) over 30 minutes . the mixture was stirred at ambient temperature for 10 minutes , then poured into a beaker containing ice to give a crystalline precipitate which was collected by vacuum filtration and washed with water to afford the crude product ( 77 . 1 g , 93 . 8 %). a solution of 18 ( 45 g , 215 . 1 mmol ), n , n - dimethylformnamide dimethyl acetal ( 38 . 45 g , 322 . 7 mmol ) and pyrrolidine ( 22 . 95 g , 322 . 7 mmol ) were heated to 110 ° c . and stirred for 16 hours under nitrogen . the reaction mixture was cooled , and a mixture of absolute methanol and ethanol ( 1 : 1 ; 600 ml ) was added . the product crystallized as a bright red solid . recovery by suction filtration afforded the product ( 51 . 0 g , 81 . 5 %). to a solution of 19 ( 50 . 0 g , 171 . 7 mmol ) in methanol and thf ( 400 ml , 1 : 1 ) was added raney nickel ( 4 . 0 ml ) and hydrazine hydrate ( 3 × 7 . 13 ml , 686 . 6 mmol ) in three equal portions every minutes at ambient temperature under nitrogen . the solution was heated at 45 ° c . for two hours . the mixture was cooled to room temperature and the catalyst was removed by filtration through a bed of celite , then washed three times with methylene chloride . the filtrate was evaporated and the residue dried azeotropically with toluene ( 100 ml ) to give a crude oil . flash silica gel column chromatography eluting with hexane ( 100 %) gradient to ethyl acetate / hexane ( 30 / 70 %) afforded the title intermediate as an off - white solid , which was futher purified via recrystallization with benzene / petroleum ether ( 2 : 8 ) to give the pure product ( 15 . 1 g , 46 . 2 %). to a solution of 20 ( 2 . 27 mg , 12 . 02 mmol ) in thf ( 150 ml ) was added lithium hexamethyldisilazide ( lihmds , 12 . 02 ml , 12 . 02 mmol , 1 m in thf ) at 0 ° c . and the solution was stirred for 40 minutes . a solution of 6 ( 5 . 0 g , 4 . 81 mmol ) in thf ( 50 ml ) was added slowly to the above mixture , followed by stirring for 20 minutes at 0 ° c . the mixture was diluted with etoac ( 400 ml ), then washed with hcl ( 1 m ), nahco 3 , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated to give a crude mixture . flash silica gel column chromatography eluting with hexane ( 100 %) gradient to ethyl acetate / hexane ( 40 / 60 %) afforded the title intermediate as a red solid ( 3 . 02 g , 54 . 71 %). to a solution of 21 ( 1 . 60 g , 1 . 39 mmol ) in dmf ( 85 ml ) was added palladium ( ii ) trifluoroacetate ( 657 mg , 2 . 93 mmol ), and the solution was stirred at 80 ° c . for two hours . the solution was cooled to room temperature and diluted with etoac ( 250 ml ), then washed with hcl ( 1 m ), nahco 3 , brine ( 150 ml ), and h 2 o ( 3 × 150 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel column chromatography eluting with etoac / hexane ( 3 : 7 ) afforded 1 . 02 g ( 63 . 7 %) as a yellow solid . to a solution of 22 ( 280 mg , 0 . 244 mmol ) in hoac ( 12 ml ) was added palladium hydroxide ( 100 mg ). the reaction was shaken under an atmosphere of h 2 ( 50 psi ) at ambient temperature for 60 h . the mixture was filtered through an acrodisc syringe filter and concentrated in vacuo to give a crude solid . flash silica gel column chromatography eluting with meoh / acoh / etoac ( 12 / 1 / 87 ) afforded 135 mg ( 91 . 4 %) as a yellow solid . to a solution of 23 ( 100 . 0 mg , 0 . 165 mmol ) in meoh ( 7 . 0 ml ) was added nh 4 oh ( 6 . 0 ml ). the mixture was stirred at ambient temperature for 2 hours , then concentrated in vacuo to give a crude solid . flash silica gel column chromatography eluting with meoh / hoac / etoac ( 12 / 1 / 87 ) afforded 81 . 0 mg ( 85 . 3 %) as a yellow solid . to a solution of 5 , 6 - dimethoxyindole ( 441 mg , 2 . 49 mmol ) in thf ( 50 ml ) was added lithium hexamethyldisilazide ( lihmds , 4 . 98 ml , 4 . 98 mmol , 1 m in thf ) at 0 ° c . and the solution was stirred for 40 minutes . a solution of 6 ( 2 . 25 g , 2 . 16 mmol ) in thf ( 30 ml ) was added slowly to the above mixture , followed by stirring for 20 minutes at 0 ° c . the mixture was diluted with etoac ( 250 ml ), then washed with hcl ( 1 m ), nahco 3 , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated to give a crude mixture . flash silica gel column chromatography eluting with hexane ( 100 %) gradient to ethyl acetate / hexane ( 60 / 40 %) afforded the title intermnediate as a red solid ( 2 . 79 g , 98 . 6 %). to a solution of 24 ( 1 . 50 g , 1 . 32 mmol ) in dmf ( 60 ml ) was added palladium ( ii ) trifluoroacetate ( 923 mg , 2 . 77 mmol ), and the solution was stirred at 80 ° c . for two hours . the solution was cooled to room temperature and diluted with etoac ( 250 ml ), then washed with hcl ( 1 m ), nahco 3 , brine ( 150 ml ), and h 2 o ( 3 × 150 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel column chromatography eluting with etoac / hexane ( 3 : 7 ) afforded 769 mg ( 51 . 4 %) as a yellow solid . to a solution of 25 ( 600 mg , 0 . 529 mmol ) in hoac ( 25 ml ) was added palladium hydroxide ( 600 mg ). the reaction was shaken under an atmosphere of h 2 ( 50 psi ) at ambient temperature for 60 hours in a parr shaker . the solution was filtered through an acrodisc syringe filter and concentrated in vacuo to provide a solid . the solid was dissolved in meoh ( 150 ml ) and aqueous nh 4 oh ( 50 ml ). the mixture was stirred at ambient temperature for 1 . 5 hours , then concentrated in vacuo to provide the crude product . flash silica gel chromatography eluting with meoh / hoac / etoac ( 12 / 1 / 87 ) afforded 166 mg ( 60 . 0 %) of desired product as a yellow solid . to a solution of 4 ( 2 . 1 g , 12 . 0 mmol ) in benzene ( 100 ml ) was added methylmagnesium iodide ( 4 . 4 ml , 13 . 19 mmol , 3 m in ether ) at 0 ° c . after stirring for one hour , a solution of n - benzyloxymethyl - 3 , 4 - dibromomaleimide ( 4 . 5 g , 12 . 0 mmol ) in benzene ( 30 ml ) and thf ( 50 ml ) was added slowly . the reaction mixture was stirred at 0 ° c . for 30 minutes and then warmed to room temperature for one hour . the mixture was diluted with etoac ( 250 ml ), then washed with hcl ( 100 ml , 0 . 3 m ), nahco 3 ( 100 ml ) and h 2 o ( 100 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel chromatography , eluting with a hexane gradient to etoac / hexane ( 3 : 2 ) afforded the title intermediate as a yellow solid ( 2 . 83 g , 50 . 5 %) to a solution of 26 ( 2 . 7 g , 5 . 75 mmol ), 2 , 3 , 4 , 5 - tetra - o - benzyl - d - glucopyranose ( 9 . 33 g , 17 . 26 mmol ) and triphenylphosphine ( 4 . 53 g , 17 . 26 mmol ) in thf ( 150 ml ) at − 78 ° c . was added diisopropylazodicarboxylate ( diad ) ( 3 . 4 ml , 17 . 26 mmol ) dropwise . stirring was continued at − 78 ° c . for 3 hours , then the solution was warmed to 0 ° c . with the aid of a ice - water bath and stirring continued for 2 hours . the mixture was diluted with etoac ( 300 ml ), then washed with hcl , brine and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel chromatography , eluting with a toluene gradient to toluene / etoac ( 25 : 1 ) afforded the title intermediate as a yellow solid 3 . 15 g ( 55 . 3 %). to a solution of 5 - benzyloxyindole ( 1 . 52 g , 6 . 8 mmol ) in thf ( 70 . 0 ml ) was added lithium hexamethyldisilazide ( lihmds , 6 . 8 ml , 6 . 8 mmol , 1 m in thf ) at 0 ° c ., and the solution stirred for 30 minutes . a solution of 27 in thf ( 80 ml ) was added slowly to the above mixture , followed by stirring for 30 minutes at 0 ° c . the mixture was diluted with etoac ( 300 ml ), then washed with hcl ( 1 m ), nahco 3 , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated to give the crude product . flash silica gel chromatography eluting with a hexane ( 100 %) gradient to etoac / hexane ( 2 : 3 ) afforded the title intermediate as a red solid 1 . 62 g ( 52 . 6 %). to a solution of 28 ( 1 . 0 g , 0 . 882 mmol ) in dmf ( 50 ml ) was added palladium ( ii ) trifluoroacetate ( 615 . 4 mg , 1 . 85 mmol ), and the solution was stirred at 80 ° c . for 1 hour . the solution was cooled to room temperature and diluted with etoac ( 350 ml ), then washed with hcl ( 1 m ), nahco 3 , brine ( 150 ml ) and h 2 o ( 3 × 150 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel chromatography eluting with etoac / hexane ( 3 : 7 ) afforded 471 . 0 g ( 47 . 2 %) of desired product as a yellow solid . to a solution of 29 ( 350 mg , 0 . 309 mmol ) in hoac ( 12 ml ) was added palladium hydroxide ( 350 mg ). the reaction was shaken under an atmosphere of h 2 ( 50 psi ) at ambient temperature for 62 hours in a parr shaker . the solution was filtered through an acrodisc syringe filter and concentrated in vacuo to provide a solid . the solid was dissolved in meoh ( 200 ml ) and aqueous nh 4 oh ( 10 ml ). the mixture was stirred at ambient temperature for 3 hours , then concentrated in vacuo to provide the crude product . flash silica gel chromatography eluting with meoh / hoac / etoac ( 12 / 1 / 87 ) afforded 164 mg ( 94 . 5 %) of desired product as a yellow solid . to a solution of 13 ( 2 . 7 g , 16 . 75 mmol ) in benzene ( 100 ml ) was added methylmagnesium iodide ( 6 . 14 ml , 18 . 42 mmol , 3 m in ether ) at 0 ° c . the solution was stirred for 1 hour , and then a solution of n - benzyloxymethyl - 3 , 4 - dibromomaleimide ( 6 . 28 g , 16 . 75 mmol ) in benzene ( 30 ml ) and thf ( 50 ml ) was added slowly . the reaction mixture was stirred at 0 ° c . for 30 minutes , then warmed to room temperature and stirred for 1 hour . the mixture was diluted with etoac ( 200 ml ), then washed with hcl ( 100 ml , 0 . 3 m ), nahco 3 ( 100 ml ) and h 2 o ( 100 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . crystallization of the crude oil with meoh afforded the title intermediate as a yellow solid ( 2 . 92 g , 38 . 2 %). to a solution of 30 ( 2 . 3 g , 5 . 05 mmol ), 2 , 3 , 4 , 5 - tetra - o - benzyl - d - glucopyranose ( 8 . 2 g , 15 . 2 mmol ) and triphenylphosphine ( 4 . 0 g , 15 . 2 mmol ) in thf ( 150 ml ) at − 78 ° c . was added diisopropylazodicarboxylate ( diad ) ( 2 . 98 ml , 15 . 2 mmol ) dropwise . the solution was stirred at − 78 ° c . for 3 hours , then warmed to 0 ° c . and stirred further for 2 hours . the mixture was diluted with etoac ( 300 ml ), then washed with hcl , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel chromatography eluting with a toluene gradient to toluene / etoac ( 25 : 1 ) afforded the title intermediate as a yellow solid 3 . 05 g ( 61 . 8 %). to a solution of 5 - benzyloxyindole ( 822 . 0 mg , 3 . 68 mmol ) in thf ( 35 ml ) was added lithium hexamethyldisilazide ( lihmds , 3 . 68 ml , 3 . 68 mmol , 1 m in thf ) at 0 ° c ., and the resulting solution was stirred for 40 minutes . a solution of 31 in thf ( 20 ml ) was added slowly to the above mixture , followed by stirring for 20 minutes at 0 ° c . the mixture was diluted with etoac ( 300 ml ), then washed with hcl ( 1 m ), nahco 3 , brine , and h 2 o . the organic layer was dried over na 2 so 4 , filtered and concentrated to provide the crude product . flash silica gel chromatography eluting with a hexane ( 100 %) gradient to etoac / hexane ( 2 : 3 ) afforded the title intermediate as a red solid ( 1 . 56 g , 91 . 2 %). to a solution of 32 ( 1 . 45 g , 1 . 294 mmol ) in dmf ( 75 ml ) was added palladium ( ii ) trifluoroacetate ( 904 mg , 2 . 72 mmol ), and the solution was stirred at 80 ° c . for 1 hour . the solution was cooled to room temperature and diluted with etoac ( 350 ml ), then wash with hcl ( 1 m ), nahco 3 , brine , and h 2 o ( 3 × 150 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated . flash silica gel chromatography eluting with etoac / hexane ( 3 : 7 ) afforded 1 . 05 g ( 72 . 6 %) of the desired product as a yellow solid . to a solution of 33 ( 300 mg , 0 . 268 mmol ) in hoac ( 14 ml ) was added palladium hydroxide ( 300 mg ). the reaction was shaken under an atmosphere of h 2 ( 50 psi ) at ambient temperature for 60 hours in a parr shaker . the solution was filtered through an acrodisc syringe filter and concentrated in vacuo to provide the crude product as a solid . the solid was dissolved in meoh ( 20 . 0 ml ) and aqueous nh 4 oh ( 30 . 0 ml ), stirred at ambient temperature for 2 hours , and then concentrated in vacuo to provide the crude product . flash silica gel chromatography eluting with meoh / hoac / etoac ( 12 / 1 / 87 ) afforded 92 . 0 mg ( 62 . 7 %) as a yellow solid . typical experimental procedure for the synthesis compounds with the following general formula to a solution of the anhydride ypx - 2 - 21 ( 32 mg , 0 . 05834 mmol ) in dmf ( 1 . 8 ml ) was added appropriate hydrazine or amine [ 0 . 5834 mmol ] ( see table 1 ). the reaction was placed under atmosphere of nitrogen at 95 ° c . for 2 h . the mixture was diluted with water ( 12 ml ) and stirred at 0 ° c . for two h . the precipitate was filtered and washed with water and ethyl ether to obtain the product ( see table 1 for yields ). a .) topoisomerase i assay . reaction buffer ( 10 . 3 μl h 2 o , 2 . 0 μl 10 × buffer , 1 . 5 μl 100 μm mgcl 2 , and 3 . 2 μl 500 mm kcl ) was prepared and kept on ice . 10 × buffer was prepared by mixing 2 ml 2m tris ph 7 . 5 , 15 . 3 μl 10 % dtt , 100 μl 0 . 5 m edta , 75 μl 20 mg / ml bsa , and 7 . 935 μl h 2 o . test poisons were prepared in dmso at such concentrations that the final incubation mixture was 5 % dmso . dna mix was prepared by dissolving 55 ml of phot1 dna solution ( 0 . 25 μg / μl ) with 715 μl reaction buffer . topo i mix was prepared by mixing 14 μl of topo i solution ( 2 units / μl ) with 266 μl of reaction buffer . proteinase k solution was prepared fresh as 10 mg / ml in 1 % sds . gel loading buffer was prepared by dissolving 1 mg bromophenol blue in 100 μl h 2 o , then adding 900 μl 50 % glycerol . topotecan ( camptosar ) and 3 , 9 - dihydroxy - 12 -( β - d - glucopyranosyl )- 6 , 7 , 12 , 13 - tetrahydroindolo [ 2 , 3 - a ] pyrrolo [ 3 , 4 - c ] carbazole - 5 , 7 - dione 11 ( als - 007 ) were used as positive controls . the assay was conducted as follows . dna mix ( 14 μl ) was added to sample tubes containing 1 μl of test poison solution and stored on ice . next , 5 μl of topo i mix was added , the solution was mixed with the pipettor , then incubated with gentle rocking in a 37 ° c . water bath for 30 minutes . the reactions were stopped via addition of 2 μl of proteinase k solution , and incubation was continued for another 30 minutes , then placed on ice . 2 . 2 μl of 5 m nacl and 75 μl of etoh were added to the tubes , the tubes were vortexed briefly , then placed on dry ice for 1 hour . the dna was pelleted by centrifugation at 16 , 000 × g for 10 minutes at 4 ° c . etoh was removed from each tube using a gel loading pipette tip , and the dna pellet was re - suspended in 18 μl of reaction buffer and 2 μl of gel loading buffer . the samples were vortexed briefly , then spun for 15 seconds in a micro - centrifuge to force all the liquid to the bottom of the tubes . the samples were loaded onto a 1 % agarose gel made with 1 × tbe containing 2 μg / ml chloroquine . the gels were run at 35 v for 15 hours in 1 × tbe . gels were stained with 0 . 5 μg / ml ethidium bromide in 1 × tbe for 1 hour , then destained for 30 minutes in h 2 o . gels were photographed with a digital camera , and the digitized images analyzed using nih software . ic 50 values were determined by comparing supercoiled dna band density ( negative controls containing 5 μl reaction buffer in place of topo i mix ) with the supercoiled dna bands remaining in the test samples . b .) in vitro cytotoxicity assay . 96 - well tissue culture cluster plates were seeded with 100 μl of cell suspension ( 5 × 10 3 cells / ml ), and incubated overnight for cell anchorage and acclimation . cells were propagated under sterile conditions in rpmi 1640 or dmem with 10 % fetal bovine serum , 2 mm l - glutamine , and sodium bicarbonate ( complete medium ), and incubated at 37 ° c . the test compounds were prepared in dmso and then diluted in complete media . a range of eight concentrations was used for each test drug to establish cytotoxicity , with eight replicates for each concentration . all dosing was conducted using a biomek 2000 robotic liquid handler . the plates were incubated at 37 ° c . with 5 % co 2 and 95 % relative humidity . the data were analyzed for cytotoxicity using the mts assay 3 - 5 days ( depending upon the growth rate of the cell lines ) after commencement of treatment . mts ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carboxymethoxyphenyl )- 2 -( 4 - sulfophenyl )- 2h - tetrazolium ) is bio - reduced by viable cells into a soluble formazan that absorbs at 490 nm , allowing simple spectrophotometric measurement of viable cells . topotecan ( camptosar ) and 3 , 9 - dihydroxy - 12 -( β - d - 1 - glucopyranosyl )- 6 , 7 , 12 , 13 - tetrahydroindolo [ 2 , 3 - a ] pyrrolo [ 3 , 4 - c ] carbazole - 5 , 7 - dione 11 ( als - 007 ) were used as positive controls . c .) mts assay . a solution of 40 μl of mts / pes solution ( purchased from promega corporation ) was added to each well , and incubated for one to four hours . the absorbance of formazan in each monolayer was measured at 490 nm on a coulter microplate reader . the data were processed in a spreadsheet program to provide a dose - response curve , allowing determination of the ic 50 . as illustrated by the results in table 3 , in a side - by - side comparison , the compounds diclosed in this invention provide a stronger poisoning effect against human topo i than the control compounds , topotecan ( a clinically - used topo i poison / antitumor agent ) and als - 007 , an experimental indolocarbazole previously disclosed 11 . additionally , in a side - by - side comparison , the compounds disclosed in this invention exhibit in vitro cytotoxicity profiles against a series of three human tumor cell lines similar to the two control compounds ( table 2 ). 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