N-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-oxobutanoyl]-hydroxyamino]pentyl]-N'-(5-aminopentyl)-N'-hydroxybutanediamide

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: An aliquot of 120 mg of Desferrioxamine and 233.64 mg of fluorescein isothiocyanate (FITC) are mixed in 50 ml of 0.1 M Na carvonate pH 9.5. A molar ratio of 3:1 of FITC: Desferrioxamine is used as described in Hentz et al. (1997) Anal. Chem. 69:4994-5000. The reaction mixture is wrapped in aluminum foil to avoid light exposure and allowed to react for at least 24 hours at room temperature. To separate fluorescent-Desferrioxamine from free fluorescein and free Desferrioxamine, the mixture is electrophoresed over a P-2 column (Bio-Rad) equilibrated in saline and samples are eluted in 2 ml fractions. The fluorescein-Desferrioxamine is eluted in the void volume of the column. The first peak is collected and used for preparing fluorescein-Desferrioxamine, Desferrioxamine, and rhodamine-liposomes. to react for at least 24 hours at room temperature To separate fluorescent-Desferrioxamine from free fluorescein and free Desferrioxamine are eluted in 2 ml fractions The fluorescein-Desferrioxamine is eluted in the void volume of the column The first peak is collected used for preparing fluorescein-Desferrioxamine, Desferrioxamine, and rhodamine-liposomes

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: To a solution of polyacrolein (125 mg) in dimethylsulfoxide (2.5 ml), a solution of deferoxamine (105 mg) in dimethylsulfoxide (2.5 ml) was added, and the resultant mixture was agitated at room temperature for 3 hours to produce a solution containing the polyacrolein-deferixamine condensation product, which is useful as a non-radioactive carrier. to produce a solution containing

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: The deferoxamine B base was then suspended in a mixture of ethanol (3.1 L) and water (0.46 L). Methanesulfonic acid was slowly added until the deferoxamine B base dissolved and the solution reached a pH of 3.5. Another 30 L of ethanol was added to the solution to precipitate deferoxamine B mesylate. The resulting suspension was then cooled to between −5° C. and 0° C. and maintained at reduced temperature for 24 h. The crystals were isolated by filtration, washed with ethanol and dried under vacuum to yield deferoxamine B mesylate (299 g) with a purity of 98% (w/w) as determined by HPLC analysis. dissolved to precipitate deferoxamine B mesylate The resulting suspension was then cooled to between −5° C. and 0° C. maintained for 24 h The crystals were isolated by filtration washed with ethanol dried under vacuum

反応条件: temp:-10.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: The deferoxamine free base was suspended in a 14:3 mixture of ethanol and water (42.5 L). Dilute methanesulfonic acid was slowly added to the suspension until the free base dissolved and the solution attained a pH of between 3.7 and 5. Ethanol (200 L) was added to the solution to precipitate the mesylate salt and the resulting suspension was cooled to −10° C. and maintained at −10° C. for several hours. The resulting crystals were isolated by filtration, washed with ethanol (50 L) and dried under vacuum to yield 3.4 kg of deferoxamine mesylate. HPLC chromatography showed that the mesylate contained 1.84 weight % impurities. The moisture content as determined by Karl-Fischer Analysis was 0.2 mole %. USP/NF 24/19, pp. 2003-2004 (1999). Coloration was better than Y5 as measured according to the European Pharmacopeia criteria for grading coloration. dissolved to precipitate the mesylate salt maintained at −10° C. for several hours The resulting crystals were isolated by filtration washed with ethanol (50 L) dried under vacuum

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Crude deferoxamine B hydrochloride (7.88 kg) containing 5.30 kg deferoxamine B was dissolved in water (362 L). The aqueous solution was then passed through an equal volume of Diaion® SP 207 adsorption resin (Mitsubishi Chemical Corp.) in a 10 liter chromatographic column at a flow rate of 14 L h−1. The eluent contained 5.04 kg deferoxamine. Ammonium chloride was then added in an amount of 5 g L−1 to the eluent with stirring until the ammonium chloride completely dissolved. The solution was then loaded onto a column containing Amberlite® XAD 1180 adsorption resin (132 L) (Rohm & Haas) and driven into the bed with salt water. The column was eluted with 10% acetonitrile-water and then 20% acetonitrile-water at a flow rate of 14 L h−1. The eluent was collected in fractions. The main fraction yielded deferoxamine hydrochloride solution containing 4.53 kg of deferoxamine B. The main fraction was then decolorized over activated carbon (45 g) and Duolite® C 467 (Rohm and Haas) ion exchange resin in H+ form yielding deferoxamine hydrochloride solution containing 4.03 kg of deferoxamine B. completely dissolved The column was eluted with 10% acetonitrile-water The eluent was collected in fractions

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: The deferoxamine B base was then suspended in a mixture of ethanol (3.1 L) and water (0.46 L). Methanesulfonic acid was slowly added until the deferoxamine B base dissolved and the solution reached a pH of 3.5. Another 30 L of ethanol was added to the solution to precipitate deferoxamine B mesylate. The resulting suspension was then cooled to between −5° C. and 0° C. and maintained at reduced temperature for 24 h. The crystals were isolated by filtration, washed with ethanol and dried under vacuum to yield deferoxamine B mesylate (299 g) with a purity of 98% (w/w) as determined by HPLC analysis. dissolved to precipitate deferoxamine B mesylate The resulting suspension was then cooled to between −5° C. and 0° C. maintained for 24 h The crystals were isolated by filtration washed with ethanol dried under vacuum

反応条件: temp:-10.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: The deferoxamine free base was suspended in a 14:3 mixture of ethanol and water (42.5 L). Dilute methanesulfonic acid was slowly added to the suspension until the free base dissolved and the solution attained a pH of between 3.7 and 5. Ethanol (200 L) was added to the solution to precipitate the mesylate salt and the resulting suspension was cooled to −10° C. and maintained at −10° C. for several hours. The resulting crystals were isolated by filtration, washed with ethanol (50 L) and dried under vacuum to yield 3.4 kg of deferoxamine mesylate. HPLC chromatography showed that the mesylate contained 1.84 weight % impurities. The moisture content as determined by Karl-Fischer Analysis was 0.2 mole %. USP/NF 24/19, pp. 2003-2004 (1999). Coloration was better than Y5 as measured according to the European Pharmacopeia criteria for grading coloration. dissolved to precipitate the mesylate salt maintained at −10° C. for several hours The resulting crystals were isolated by filtration washed with ethanol (50 L) dried under vacuum

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Crude deferoxamine B hydrochloride (7.88 kg) containing 5.30 kg deferoxamine B was dissolved in water (362 L). The aqueous solution was then passed through an equal volume of Diaion® SP 207 adsorption resin (Mitsubishi Chemical Corp.) in a 10 liter chromatographic column at a flow rate of 14 L h−1. The eluent contained 5.04 kg deferoxamine. Ammonium chloride was then added in an amount of 5 g L−1 to the eluent with stirring until the ammonium chloride completely dissolved. The solution was then loaded onto a column containing Amberlite® XAD 1180 adsorption resin (132 L) (Rohm & Haas) and driven into the bed with salt water. The column was eluted with 10% acetonitrile-water and then 20% acetonitrile-water at a flow rate of 14 L h−1. The eluent was collected in fractions. The main fraction yielded deferoxamine hydrochloride solution containing 4.53 kg of deferoxamine B. The main fraction was then decolorized over activated carbon (45 g) and Duolite® C 467 (Rohm and Haas) ion exchange resin in H+ form yielding deferoxamine hydrochloride solution containing 4.03 kg of deferoxamine B. completely dissolved The column was eluted with 10% acetonitrile-water The eluent was collected in fractions

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: DACB was synthesized by covalently conjugating iodoacetyl-deferoxamine and N-cysteinyl-biotin by the following Scheme II. ##STR20## Solid deferoxamine mesylate was added to methanol for a concentration of 20 mM, followed by addition of NaOH to reach a final concentration of 20 mM NaOH. The solution was heated and maintained at 65°. Solid iodoacetic acid N-hydroxysuccinimide ester (NHS-IA) was added to the deferoxamine solution to a final molarity of 50mM NHS-IA and incubated for at least one hour at 65°. The resulting iodoacetyl-deferoxamine (IA-DFO) was purified by HPLC with an EM LiChroCART reverse phase column using a linear gradient of 0-10 ml 100% Buffer A (0.025M phosphate, pH 6, 2 mM NTA) and 10-60 ml 0-100% Buffer B (0.025M phosphate, pH 6, 2 mM NTA, 75% methanol). IA-DFO, but not unconjugated DFO, has an absorbance at 260 nm and thus was purified by measuring the absorbance of the eluate and collecting the fraction with absorbance at 260 nm. The solution was heated maintained at 65° incubated for at least one hour at 65° The resulting iodoacetyl-deferoxamine (IA-DFO) was purified by HPLC with an EM LiChroCART reverse phase column thus was purified collecting the fraction with absorbance at 260 nm

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: A mixture of 500.0 g of desferrioxamine B-methanesulfonate in 2,500 ml of distilled water changes into a clear solution after having been stirred for 10 minutes; this is cooled to 10° and 80 g of sodium carbonate in 500 ml of water are added thereto within a period of 5 minutes. Inoculation with free desferrioxamine B yields a viscous milky suspension which is filtered. The residue is washed with 500 ml of water and with 500 ml of a 1:1 mixture of water and acetone, then with portions, totalling 2,000 ml, of acetone, and dried for 16 hours at 40° under a pressure of approximately 20 mm/Hg and for a further 16 hours at 40° C. under a high vacuum. this is cooled to 10° within a period of 5 minutes

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: A mixture of 500.0 g of desferrioxamine B-methanesulfonate in 2,500 ml of distilled water changes into a clear solution after having been stirred for 10 minutes; this is cooled to 10° and 80 g of sodium carbonate in 500 ml of water are added thereto within a period of 5 minutes. Inoculation with free desferrioxamine B yields a viscous milky suspension which is filtered. The residue is washed with 500 ml of water and with 500 ml of a 1:1 mixture of water and acetone, then with portions, totalling 2,000 ml, of acetone, and dried for 16 hours at 40° under a pressure of approximately 20 mm/Hg and for a further 16 hours at 40° C. under a high vacuum. this is cooled to 10° within a period of 5 minutes

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: An aliquot of 120 mg of desferrioxamine and 233.64 mg of fluorescein isothiocyanate (FITC) are mixed in 50 mL of 0.1 M sodium carbonate pH 9.5. A molar ratio of 3:1 of FITC: desferrioxamine is used as described in Hentz et al. (1997) Anal. Chem. 69:4994-5000. The reaction mixture is wrapped in aluminum foil to avoid light exposure and allowed to react for at least 24 hours at room temperature. To separate fluorescently labeled desferrioxamine from free fluorescein and free desferrioxamine, the mixture is separated using a P-2 column (Bio-Rad) equilibrated in saline and samples are eluted in 2 mL fractions. The fluorescein-desferrioxamine is eluted in the void volume of the column. The first peak is collected and used for preparing fluorescein-desferrioxamine, desferrioxamine, and rhodamine liposomes. to react for at least 24 hours at room temperature To separate fluorescently labeled desferrioxamine from free fluorescein and free desferrioxamine the mixture is separated are eluted in 2 mL fractions The fluorescein-desferrioxamine is eluted in the void volume of the column The first peak is collected used for preparing fluorescein-desferrioxamine, desferrioxamine, and rhodamine liposomes