Usage And Synthesis
Ambutyrosin is the another name of butirosin. Ambutyrosin obtained from
fermentation broths of Bacillus circulans contains a major proportion of N1-(4-
amino-2-hydroxybutyryl)-4-O-2,6-diamino-2,6-didexy-D-glucopyranosyl)-5-O-D-xelofuranosyl-2-deoxystreptamine (ambutyrosin A) and a minor proportion
(up to 10-15%) of N1-(4-amino-2-hydroxybutyryl)-4-O-2,6-diamino-2,6-
didexy-D-glucopyranosyl)-5-O-D-ribofuranosyl-2-deoxystreptamine
(ambutyrosine B). A process for preparation of ambutyrosin by semisynthetic
procedure of acylating of 5-β-D-xylofuranosylneamine or 5-β-
ribofuranosylneamine is described below.
Preparation of 5-β-D-pentofuranosyIneamine: ambutyrosin A (1.0 g) in 30 ml of 0.5 N sodium hydroxide solution was refluxed for one hour, then neutralized with 6 N hydrochloric acid and applied on a column of Amberlite CG-50 (NH4+ form, 30 ml). The column was washed with water (ca. 100 ml) until the ninhydrin test became negative, and then eluted with 0.2 N ammonium hydroxide. Biologically active fractions were collected, concentrated in vacuum to 5 ml and treated with 5 ml of methanol to induce precipitation. It was further purified by re-precipitation from aqueous methanol to yield 607 mg (65%) of deacylated ambutyrosin A hereafter referred to as DA1.
The wash water of the above CG-50 column was adjusted to pH 7.0 and applied on a column of Amberlite IR-120 (H+ form, 30 ml). The column was washed with water and then eluted with 1 N ammonium hydroxide. Ninhydrin-positive fractions were combined, treated with active carbon and neutralized with IRC-50 resin (H+ form). The solution was concentrated to a small volume, treated with ethanol, and kept in the cold overnight to yield 160 mg (75%) of γ-amino-α-hydroxybutyric acid as colorless needles which melted at 217°C-218°C. [α]D23= -30.3° (c=1.0%, H2O).
The above experiments were also carried out on 1.0 g of ambutyrosin B yielding 579 mg (62%) of deacylated ambutyrosin B, hereafter called DA2.
L-(-)-γ-benzyloxycarbonylamino-α-ydroxybutyric acid: L-(-)-γ-amino-α- hydroxybutyric acid (7.4 g, 0.062 mole) was added to 50 ml of aqueous sodium 3 hydroxide solution (5.2 g, 0.13 mole). To the solution was added dropwise 11.7 g (0.068 mole) of carbobenzoxy chloride with stirring at 0°C to 5°C for one hour. The reaction mixture was washed with 50 ml of ether, adjusted to pH 2 with dilute hydrochloric acid, and extracted with four 80-ml portions of ether. The extracts were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated in vacuum, and the residue was crystallized from benzene to give 11.6 g (74%) of 4 colorless plates. M.P: 78.5°-79.5°C.
N-Hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid: a solution of L-(-)-γ-benzyloxycarbonylamino-α- hydroxybutyric acid (10.6 s g, 0.042 mole) and N-hydroxysuccinimide (4.8 g, 0.042 mole) in 200 ml of ethyl acetate was cooled to 0°C and added to 8.6 g (0.042 mole) of dicyclohexylearbodiimide. The mixture was stirred overnight at 5°C. The dicyclohexylurea which separated was removed by filtration, and the filtrate was concentrated in vacuum. The product was obtained as colorless plates. Yield: 13.4 g (92%). M.P: 117°-118°C [α]D23= +1.5° (c= 2.0%, CHCl3).
N-(Benzyloxycarbonyloxy)succinimide: N-hydroxysuccinimide (23 g, 0.2 mole) was dissolved in 200 ml of aqueous NaOH solution (9 g, 0.22 mole). To the stirred solution was added dropwise 34 g (0.2 mole) of carbobenzoxy chloride with water-cooling. The mixture was stirred overnight at room temperature, and the precipitate which separated was collected by filtration, washed with water, and air dried. Yield: 41.1 g (82%). Recrystallization from benzene-n-hexane (10:1) gave colorless prisms melting at 78°-79°C.
6'-Carbobenzoxy-DA1 and 1,6'-dicarbobenzoxy-DA1:
To a solution of 9.1 g (20 mmoles) of above prepared DA1 in 150 ml of water and 60 ml of tetrahydrofuran (THF) was slowly added, under vigorous stirring and cooling (5°C), a solution of 5.17 g (20.8 mmoles) of N- (benzyloxycarbonyloxy)succinimide in 60 ml of THF. The mixture was stirred for 24 hours at 5°C and for an additional 16 hours at room temperature and then concentrated in vacuum to dryness. The crude product thus obtained was roughly separated into two fractions by a preparative counter-current distributor (52 tubes, 100 ml/tube) using a solvent system of n-BuOH-CHCl3·H2O (4:1:5). Tube No. 1-15 were combined and evaporated in vacuum to give 9.75 g of solid. The solid was dissolved in 20 ml of water and applied on a column of amberlite CG-50 (NH4+ form, 120 ml). The column was washed with water and then eluted with aqueous 0.1 N ammonium hydroxide collecting each 20-ml fraction. Fractions No 62-93 were combined and concentrated in vacuum to give 5.75 g (49%) of 6'-carbobenzoxy-DA1 as a white solid. NMR in D2O and elememtal analysis confirmed its structure.
Tubes No. 36-49 from the above current distribution were combined and evaporated in vacuum to give 2.91 g of solid. The solid was further purified by silica gel column chromatography developed with methanol-ethyl acetate (4:1) to yield 1.11 g (7.5%) of 1,6'-dicarbobenzoxy-DA1. TLC [silica gel plate, MeOAc-n-PrOH-28% NH4OH (45:105:60)]. Rf=0.44.
1-[L-(-)-γ-Amino-α-hydroxybutyryl]-DA1: a solution of 6'-carbobenzoxy-DA1 (588 mg, 1 mmole) in 10 ml of water and 5 ml of THF was added dropwise to a solution of N-hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α- hydroxybutyric acid (350 mg, 1 mmole) in 5 ml of THF with stirring and cooling (5°C). The mixture was stirred overnight at room temperature and then concentrated in vacuum to dryness. The residue was shaken with a mixture of n-butanol-ethyl acetate-water (4:1:5). The upper layer of the solvent mixture was separated and evaporated in vacuum to dryness. The solid thus obtained was dissolved in 30% aqueous ethanol and hydrogenated over 250 mg of 10% palladium-on-charcoal at room temperature. The reaction mixture was filtered to remove the palladium catalyst, and the filtrate was concentrated in vacuum to dryness. The residual solid was dissolved in 10 ml of water and chromatographed on a column of CG-50 (NH4+ form, 40 ml). The column was washed by water and then eluted fractionally with aqueous 0.5 N NH4OH. Biologically active fractions which showed Rf=0.20 by TLC were collected, concentrated in vacuum and lyophilized to give 94 mg (14%) of product as a white solid, which was identified with ambutyrosin A in every respect. It melts with decomposition over a wide range beginning at about 149°C. [α]D25= +26°C (1.4%, water). Ambutyrosin B was prepared in a similar manner. It melts with decomposition at about 146°C [α]D25=+33° (1.5%, water).
Preparation of 5-β-D-pentofuranosyIneamine: ambutyrosin A (1.0 g) in 30 ml of 0.5 N sodium hydroxide solution was refluxed for one hour, then neutralized with 6 N hydrochloric acid and applied on a column of Amberlite CG-50 (NH4+ form, 30 ml). The column was washed with water (ca. 100 ml) until the ninhydrin test became negative, and then eluted with 0.2 N ammonium hydroxide. Biologically active fractions were collected, concentrated in vacuum to 5 ml and treated with 5 ml of methanol to induce precipitation. It was further purified by re-precipitation from aqueous methanol to yield 607 mg (65%) of deacylated ambutyrosin A hereafter referred to as DA1.
The wash water of the above CG-50 column was adjusted to pH 7.0 and applied on a column of Amberlite IR-120 (H+ form, 30 ml). The column was washed with water and then eluted with 1 N ammonium hydroxide. Ninhydrin-positive fractions were combined, treated with active carbon and neutralized with IRC-50 resin (H+ form). The solution was concentrated to a small volume, treated with ethanol, and kept in the cold overnight to yield 160 mg (75%) of γ-amino-α-hydroxybutyric acid as colorless needles which melted at 217°C-218°C. [α]D23= -30.3° (c=1.0%, H2O).
The above experiments were also carried out on 1.0 g of ambutyrosin B yielding 579 mg (62%) of deacylated ambutyrosin B, hereafter called DA2.
L-(-)-γ-benzyloxycarbonylamino-α-ydroxybutyric acid: L-(-)-γ-amino-α- hydroxybutyric acid (7.4 g, 0.062 mole) was added to 50 ml of aqueous sodium 3 hydroxide solution (5.2 g, 0.13 mole). To the solution was added dropwise 11.7 g (0.068 mole) of carbobenzoxy chloride with stirring at 0°C to 5°C for one hour. The reaction mixture was washed with 50 ml of ether, adjusted to pH 2 with dilute hydrochloric acid, and extracted with four 80-ml portions of ether. The extracts were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated in vacuum, and the residue was crystallized from benzene to give 11.6 g (74%) of 4 colorless plates. M.P: 78.5°-79.5°C.
N-Hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid: a solution of L-(-)-γ-benzyloxycarbonylamino-α- hydroxybutyric acid (10.6 s g, 0.042 mole) and N-hydroxysuccinimide (4.8 g, 0.042 mole) in 200 ml of ethyl acetate was cooled to 0°C and added to 8.6 g (0.042 mole) of dicyclohexylearbodiimide. The mixture was stirred overnight at 5°C. The dicyclohexylurea which separated was removed by filtration, and the filtrate was concentrated in vacuum. The product was obtained as colorless plates. Yield: 13.4 g (92%). M.P: 117°-118°C [α]D23= +1.5° (c= 2.0%, CHCl3).
N-(Benzyloxycarbonyloxy)succinimide: N-hydroxysuccinimide (23 g, 0.2 mole) was dissolved in 200 ml of aqueous NaOH solution (9 g, 0.22 mole). To the stirred solution was added dropwise 34 g (0.2 mole) of carbobenzoxy chloride with water-cooling. The mixture was stirred overnight at room temperature, and the precipitate which separated was collected by filtration, washed with water, and air dried. Yield: 41.1 g (82%). Recrystallization from benzene-n-hexane (10:1) gave colorless prisms melting at 78°-79°C.
6'-Carbobenzoxy-DA1 and 1,6'-dicarbobenzoxy-DA1:
To a solution of 9.1 g (20 mmoles) of above prepared DA1 in 150 ml of water and 60 ml of tetrahydrofuran (THF) was slowly added, under vigorous stirring and cooling (5°C), a solution of 5.17 g (20.8 mmoles) of N- (benzyloxycarbonyloxy)succinimide in 60 ml of THF. The mixture was stirred for 24 hours at 5°C and for an additional 16 hours at room temperature and then concentrated in vacuum to dryness. The crude product thus obtained was roughly separated into two fractions by a preparative counter-current distributor (52 tubes, 100 ml/tube) using a solvent system of n-BuOH-CHCl3·H2O (4:1:5). Tube No. 1-15 were combined and evaporated in vacuum to give 9.75 g of solid. The solid was dissolved in 20 ml of water and applied on a column of amberlite CG-50 (NH4+ form, 120 ml). The column was washed with water and then eluted with aqueous 0.1 N ammonium hydroxide collecting each 20-ml fraction. Fractions No 62-93 were combined and concentrated in vacuum to give 5.75 g (49%) of 6'-carbobenzoxy-DA1 as a white solid. NMR in D2O and elememtal analysis confirmed its structure.
Tubes No. 36-49 from the above current distribution were combined and evaporated in vacuum to give 2.91 g of solid. The solid was further purified by silica gel column chromatography developed with methanol-ethyl acetate (4:1) to yield 1.11 g (7.5%) of 1,6'-dicarbobenzoxy-DA1. TLC [silica gel plate, MeOAc-n-PrOH-28% NH4OH (45:105:60)]. Rf=0.44.
1-[L-(-)-γ-Amino-α-hydroxybutyryl]-DA1: a solution of 6'-carbobenzoxy-DA1 (588 mg, 1 mmole) in 10 ml of water and 5 ml of THF was added dropwise to a solution of N-hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α- hydroxybutyric acid (350 mg, 1 mmole) in 5 ml of THF with stirring and cooling (5°C). The mixture was stirred overnight at room temperature and then concentrated in vacuum to dryness. The residue was shaken with a mixture of n-butanol-ethyl acetate-water (4:1:5). The upper layer of the solvent mixture was separated and evaporated in vacuum to dryness. The solid thus obtained was dissolved in 30% aqueous ethanol and hydrogenated over 250 mg of 10% palladium-on-charcoal at room temperature. The reaction mixture was filtered to remove the palladium catalyst, and the filtrate was concentrated in vacuum to dryness. The residual solid was dissolved in 10 ml of water and chromatographed on a column of CG-50 (NH4+ form, 40 ml). The column was washed by water and then eluted fractionally with aqueous 0.5 N NH4OH. Biologically active fractions which showed Rf=0.20 by TLC were collected, concentrated in vacuum and lyophilized to give 94 mg (14%) of product as a white solid, which was identified with ambutyrosin A in every respect. It melts with decomposition over a wide range beginning at about 149°C. [α]D25= +26°C (1.4%, water). Ambutyrosin B was prepared in a similar manner. It melts with decomposition at about 146°C [α]D25=+33° (1.5%, water).
Preparation Products And Raw materials
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