N,N'-bis(3-aminopropyl)butane-1,4-diamine

Conditions: temp:0.00; pressure:0.00; stir:

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of spermine (1,4-bis [{(3-amino) propyl}amino]butane, 460 mg) in N,N-dimethylformamide anhydride (5 ml) is added a solution of para-nitrophenyl 2,4-dibenzyloxyphenylacetate (100 mg) in N,N-dimethylformamide anhydride (5 ml). The resulting solution is concentrated to dryness under reduced pressure and thereto are added 5 % aqueous sodium hydrogencarbonate solution (0.1 ml) and then water (30 ml). The resulting precipitate is filtered and washed thoroughly with water, followed by drying, to yield crude 2,4-dibenzyloxyphenylacetylspermine (72 mg, yield 64%). 3) To a solution of crude 2,4-dibenzyloxyphenylacetylspermine (72 mg) in acetic acid (5 ml) is added 10% palladium-carbon (50 mg) and then catalytic reduction is carried out in a hydrogen stream for one and half hours. After the reaction is over, the catalyst is removed by filtration and the filtrate is concentrated to dryness under reduced pressure. The residue is subjected to separation and purification by high-performance liquid chromatography (reversed-phase partition column TSK gel ODS-120T manufactured by Toyo Soda Mfg., CO., Ltd.; inner diameter of the column: 7.8 mm, length: 30 mm; resin diameter; 3 μm; column temperature: 40° C.; eluent: water/acetonitrile mixed solvent (98/2, V/V) to which trifluoroacetic acid has been added to final concentration of 0.05% by volume). The solvent is distilled off to yield 2,4-dihydroxyphenylacetyl-spermine trifluoroacetic acid salt (3.0 mg, yield 4.2%). The resulting solution is concentrated to dryness under reduced pressure are added 5 % aqueous sodium hydrogencarbonate solution (0.1 ml) The resulting precipitate is filtered washed thoroughly with water by drying

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of spermine (1,4-bis [{(3-amino) propyl}amino]butane, 460 mg) in N,N-dimethylformamide anhydride (5 ml) is added a solution of para-nitrophenyl 2,4-dibenzyloxyphenylacetate (100 mg) in N,N-dimethylformamide anhydride (5 ml). The resulting solution is concentrated to dryness under reduced pressure and thereto are added 5 % aqueous sodium hydrogencarbonate solution (0.1 ml) and then water (30 ml). The resulting precipitate is filtered and washed thoroughly with water, followed by drying, to yield crude 2,4-dibenzyloxyphenylacetylspermine (72 mg, yield 64%). 3) To a solution of crude 2,4-dibenzyloxyphenylacetylspermine (72 mg) in acetic acid (5 ml) is added 10% palladium-carbon (50 mg) and then catalytic reduction is carried out in a hydrogen stream for one and half hours. After the reaction is over, the catalyst is removed by filtration and the filtrate is concentrated to dryness under reduced pressure. The residue is subjected to separation and purification by high-performance liquid chromatography (reversed-phase partition column TSK gel ODS-120T manufactured by Toyo Soda Mfg., CO., Ltd.; inner diameter of the column: 7.8 mm, length: 30 mm; resin diameter; 3 μm; column temperature: 40° C.; eluent: water/acetonitrile mixed solvent (98/2, V/V) to which trifluoroacetic acid has been added to final concentration of 0.05% by volume). The solvent is distilled off to yield 2,4-dihydroxyphenylacetyl-spermine trifluoroacetic acid salt (3.0 mg, yield 4.2%). The resulting solution is concentrated to dryness under reduced pressure are added 5 % aqueous sodium hydrogencarbonate solution (0.1 ml) The resulting precipitate is filtered washed thoroughly with water by drying

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Monoquaternary spermine containing hydrophilic head-group was prepared in a similar way to compound 3. Briefly, TriBoc-Spermine was reacted with excess of 1-azido-3-iodo-propane in the presence of excess TBA. The resulting quaternarized derivative was hydrogenised under (Pd—C) and Boc protecting group were removed using gaseous HCl in dry methanol. were removed

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Monoquaternary spermine containing hydrophilic head-group was prepared in a similar way to compound 3. Briefly, TriBoc-Spermine was reacted with excess of 1-azido-3-iodo-propane in the presence of excess TBA. The resulting quaternarized derivative was hydrogenised under (Pd—C) and Boc protecting group were removed using gaseous HCl in dry methanol. were removed

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A composition was prepared by dissolving human insulin with spermine and polyvinylpyrrolidone (PVP-40), sodium dodecanoate (SD) and methylcellulose (MC-400) in DDW containing NaOH. The solution was then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil, further containing sorbitan monopalmitate (Span-40). Components and concentrations are detailed in Table 4. A composition was prepared Components and concentrations

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A composition contemplated by the instant invention was prepared by dissolving human insulin with spermine and phytic acid in double distilled water (“DDW”) containing NaOH. The solution was then lyophilized and suspended with sodium dodecanoate (SD), octanol and geraniol in a mixture of mineral oil, medium chain triglyceride (MCT) oil and castor oil. Components and concentrations are detailed in Table 1. was prepared in double distilled water (“DDW”) containing NaOH suspended with sodium dodecanoate (SD), octanol and geraniol in a mixture of mineral oil, medium chain triglyceride (MCT) oil and castor oil Components and concentrations

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 1-L round-bottom flask equipped with a magnetic stir bar and rubber septum was purged with nitrogen and charged with 600 mL of USP-grade dry ethanol, 60 mL of anhydrous THF, 4.95 grams (10.5 mmol, 1.05 equivalents) of dexamethasone-mesylate and 10 grams (19.5 mmol, 4.95 equivalents) of spermine. 2-Iminothiolane, 1.38 grams (10 mmol, 1.0 equivalents) in 3 mL of water, was added dropwise to the solution over 5 minutes with vigorous mixing. The solution changed color from clear to clear light yellow. The reaction was monitored by TLC and by analytical HPLC. After three hours at room temperature, the TLC spot at R1=0 (the charged DS) (Rt=0.7 minutes DS by analytical HPLC) was maximized, and dexamethasone-mesylate (Rt 0.47, 2.8 minutes HPLC) spot minimized. The crude reaction mixture was diluted with 15.25 mL (198 mmol, 19.8 equivalents) of trifluoroacetic acid (or alternatively formic acid), forming a white precipitate (spermine tetra TFA salt or tetra-formate salt). Solvent was removed with a rotary evaporator and 50 mL of ethanol was added, forming a white precipitate (spermine-4TFA). The pH 3 solution was filtered twice to remove spermine acid salt, and ethanol was removed with a rotary evaporator. 50 mL of water was added to the yellow viscous oil, yielding a precipitate (dexamethasone and dexamethasone mesylate), and the solution was filtered through 0.2 μM filter to yield a clear yellow liquid. Water was removed with a lyophilizer over several days to yield dexamethasone-spermine and disubstituted spermine tetra-acid salts. Total yield ˜30% for combined products). A 1-L round-bottom flask equipped with a magnetic stir bar rubber septum was purged with nitrogen The crude reaction mixture

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The 21-hydroxy group of dexamethasone is not required for anti-inflammatory activity, and therefore is an ideal choice for conjugation to a polycation (FIG. 1A) (Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics, 1459-1485, eds. Hardman and Limbird, McGraw-Hill, New York). A one-pot reaction between spermine, 2-iminothiolane (Traut's reagent) and dexamethasone mesylate yielded the dexamethasone-spermine and disubstituted conjugates, (DS and D2S, respectively) (FIG. 1B). Traut's reagent is selectively ring-opened by the primary amines (Hermanson, 1996, Bioconjugate Techniques, Academic Press) on spermine, forming a hydrolytically sensitive amidimide bond (Hermanson, 1996, Bioconjugate Techniques, Academic Press) between spermine and iminothiolane and a reactive thiolate anion that reacts with the α-keto mesylate on the 21 position of dexamethasone mesylate (Simmons et al., 1980, J. Org. Chem. 45:084-3088), yielding an α-keto thioether linkage between the dexamethasone and iminothiolane. Dexamethasone mesylate (prepared or received from Steraloids, N.H.) or dexamethasone (Steraloids, N.H.), 2-iminothiolane (Traut's reagent) (Pierce), spermine (Sigma), and ethanol or DMSO (Aldrich) were used as received. A total of 105 mg (223 μmol) of dexamethasone-mesylate, and 28.4 mg (206 μmol) of Trout's reagent were dissolved together in either DMSO or ethanol prior to addition of 31.9 μl (145 μmol of spermine at room temperature. The conjugation reaction was complete in 45 minutes by TLC. HPLC purification (60/40 0.1% TFA/acetonitrile, Hamilton PRP-1 column), evaporative solvent removal, and freeze-drying yielded both DS and D2S as white powders, 1H NMR of DS confirmed the presence of the 3-bis-enone and 11- and 17-hydroxy groups required for glucocorticoid activity (FIG. 1A) as well as 1H signals from the conjugated spermine and the 21-α-keto thioether group. Hydrolysis of DS in 1M NaOH for 20 minutes resulted in the cleavage of the amidimide linkage between spermine and iminothiolane, forming a dexamethasone-amide (DA) (FIG. 1B), which has a 21-substituted butyl thioether amide side-chain on dexamethasone. For DA, FIG. 1B: Calc: C, 63.26; H, 7.35; N, 2.84. Found: C, 63.44; H, 7.27; N, 2.83. Water solubility: DS>100,000 mg/l; DA 60 mg/l; (dexamethasone 100 mg/l).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The 21-hydroxy group of dexamethasone is not required for anti-inflammatory activity, and therefore is an ideal choice for conjugation to a polycation (FIG. 1A) (Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics, 1459-1485, eds. Hardman and Limbird, McGraw-Hill, New York). A one-pot reaction between spermine, 2-iminothiolane (Traut's reagent) and dexamethasone mesylate yielded the dexamethasone-spermine and disubstituted conjugates, (DS and D2S, respectively) (FIG. 1B). Traut's reagent is selectively ring-opened by the primary amines (Hermanson, 1996, Bioconjugate Techniques, Academic Press) on spermine, forming a hydrolytically sensitive amidimide bond (Hermanson, 1996, Bioconjugate Techniques, Academic Press) between spermine and iminothiolane and a reactive thiolate anion that reacts with the α-keto mesylate on the 21 position of dexamethasone mesylate (Simmons et al., 1980, J. Org. Chem. 45:084-3088), yielding an α-keto thioether linkage between the dexamethasone and iminothiolane. Dexamethasone mesylate (prepared or received from Steraloids, N.H.) or dexamethasone (Steraloids, N.H.), 2-iminothiolane (Traut's reagent) (Pierce), spermine (Sigma), and ethanol or DMSO (Aldrich) were used as received. A total of 105 mg (223 μmol) of dexamethasone-mesylate, and 28.4 mg (206 μmol) of Trout's reagent were dissolved together in either DMSO or ethanol prior to addition of 31.9 μl (145 μmol of spermine at room temperature. The conjugation reaction was complete in 45 minutes by TLC. HPLC purification (60/40 0.1% TFA/acetonitrile, Hamilton PRP-1 column), evaporative solvent removal, and freeze-drying yielded both DS and D2S as white powders, 1H NMR of DS confirmed the presence of the 3-bis-enone and 11- and 17-hydroxy groups required for glucocorticoid activity (FIG. 1A) as well as 1H signals from the conjugated spermine and the 21-α-keto thioether group. Hydrolysis of DS in 1M NaOH for 20 minutes resulted in the cleavage of the amidimide linkage between spermine and iminothiolane, forming a dexamethasone-amide (DA) (FIG. 1B), which has a 21-substituted butyl thioether amide side-chain on dexamethasone. For DA, FIG. 1B: Calc: C, 63.26; H, 7.35; N, 2.84. Found: C, 63.44; H, 7.27; N, 2.83. Water solubility: DS>100,000 mg/l; DA 60 mg/l; (dexamethasone 100 mg/l).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: An assay buffer solution was prepared which was 100 mM HEPES pH 8.0, 10 mM ATP, 2 mM MgCl2, 5 mM DTT, 0.5 mM PMSF. A fructosyl-spermine stock solution was prepared which was 2 mM fructosyl-spermine HCl. A spermine control solution was prepared which was 2 mM spermine HCl. An assay buffer solution was prepared which

Conditions: temp:0.00; pressure:0.00; stir: