SU965359A3 - Process for producing 1n-(l-hydroxy-w-aminoalkanoyl)-6n-methyl-3,4-dioxykanamycin b - Google Patents

Abstract

1-N-(alpha-hydroxy-omega-aminoalkanoyl)-6'-N-methyl-3',4'-dideoxyka namycins B or pharmaceutically acceptable acid addition salts thereof are prepared by selective acylation of the 1-amino group of an amino-protected derivative of the formula <IMAGE> in which R1 is a monovalent amino protective group and R2 is hydrogen or a monovalent amino protective group, with an alpha -hydroxyomega -amino acid of the formula V or with a functional derivative thereof, in which n is 1, 2 or 3, R3 is an amino protective group and R4 is hydrogen, or R3 and R4 together are a divalent amino protective group, after which the amino protective groups are eliminated from the 1-N-acylated product obtained in this way. The prepared compounds are effective not only against Gram-negative and Gram-positive bacteria with the usual kanamycin sensitivity but also against kanamycin-resistant bacteria. <IMAGE>

Description

one

This invention relates to a process for the preparation of a new derivative of kanamycin B, which possesses valuable pharmacological properties.

The purpose of the invention is the synthesis of a new derivative of kanamycin B, which expands the arsenal of means of influencing

living organism — achieved by synthesis based on the known acylation reaction of amino sugars tl

According to the invention 1, a process for the preparation of N- (cC-hydroxy-sh-aminoalkanoyl) -b N-methyl-3, 4-dideoxycanamycin in the formula

where n is 1-3, consisting in the fact that the amino-protected derivative of b

(CH,) „TJH,

-methyl-3, 4-dideoxyanamycin B of the general formula: where R is a t-butyryloxycarbonyl group, 10 in a mixture of water and dimethoxyethane, aci-

BHir- (CHj) n-CH (OHl-coo-:

where R and n are specified values.

b Yngono,

KHN) nuCHj) H | 4.CO-Jllf where R, n have the indicated meanings, is treated with a 90% aqueous solution of trifluoroacetic acid, followed by separation of the target product by column chromatography. The novel compounds of formula 1 have the following physical, chemical and biological properties. 1 N- (OH-isoseryl) -bN-methyl-3, 4-dideoxy anamine B is a colorless crystalline powder with a decomposition point of 165-1690С, + 96 ° (s, 1.17 water). Its formula is as follows: (C, 47.81%, H 8.03%, N 15.21%). Gives one spot (positive reaction with ninhydrin), R 0.51, with thin-layer chromatography on silica gel, using as mixture: p-butanol-ethanol-chloroform-2 8% aqueous ammonia solution (4: 5: 2: 8 by volume). 1-S (L-isoseryl) -b N-methyl-3, 4-dideoxyanamycin B is a colorless crystalline powder with a decomposition point of 1b2-166 ° C, Gs (+ 80 ° (s, 1.02, water) .. Formula its following: - 47.81%, H 8.03%, N 15.21%). Gives one spot (positive reaction with ninhydrin), H 0.51 with thin-layer chromatography on silica gel. 1 I- (L-4-amino-2-hydroxybutyryl) -b N-methyl-3, 4-dideoxycanamycin -. colorless crystalline powder with a decomposition point of 158-161 ° С, +71 (s, 0.8, water). Foelloe his next:, (C 48.75% H 8.18%, N 14.83%). Gives one spot

l

R

(P

gpoluchevoe 1 N-acylated derivative of the formula

KN CHjlTHR

W is cast with a cL -oxy-w-amino acid derivative of the formula (positive reaction with ninhydrin), Rr 0.38 with thin-layer chromatography on silica gel. „1-N (L-5-amino-2-oxivaleryl N-methyl-3 , 4-dideoxycanamycin B, a colorless β-crystalline powder with a decomposition point of 152-155 ° C, dlll +79 (s, 0.71, water). Its elementary formula is C24H4eN (, (С 49.64%, H 8.33 %, N, 14.47%). Gives one spot (positive reaction with ninhydrin), R 0.39 upon thin-layer chromatography on silica gel. New compounds of the formula are not sensitive to inactivation of b-acetone iltransferazoy so as 6-amino group of the novel compound methyl "Evan, are not to inactivate chuvstvitelnmi -nukleotidiltransferaeoy 2 and W-phospho .tranferazoy since 1-amino group of the new compounds of formula 1, is acylated oxy-o1 and -aminoatsilnoy group, they they are also not sensitive to the inactivation of another type of phosphotransferase, since the 3 and 4 hydroxyl groups present in kanamycin B are removed from the new compound. Thus, the new compounds of formula 1 have significant advantages, since they possess high antibacterial activity not only against various types of gram-positive and gram-negative bacteria that are sensitive to kanamycin, but also against strains of these bacteria resistant to kanamycin, in particular against kanamycin-resistant strains of Eschericnfa coli, Pseudomonas aeruginosa.

Example 1. Synthesis of 1 N- (L-amino-2-hydroxybutyryl) -6 N-methyl-3, 4-dideoxycanamycin B (compounds corresponding to formula D, where n is 2), Preparation of starting materials:

but). To a solution of 930 mg (2 mmol) of N-methyl-3, 4-dideoxyanamycin. In a 5 ml solution of water and 960 mg (4 mmol) of t-butyl-S-4,6-dimethylpyrimide-2-ylthiocarbonate are added. 5 ml of dioxane. The mixture was shaken quickly at room temperature to react with the t-butoxycarbonyl group. The reaction mixture is concentrated by drying under reduced pressure to obtain a solid product, which is taken up in 40 ml of water and the insoluble residue is filtered off. The solution (filtrate) is passed through a 100 ml column (20x290 mm) containing Amberlite CG 50 (NH-form) cation-exchange resin for the resin to absorb the reaction products with the t-butoxycarbonyl group. The column is washed with water (500 ml) and then eluted with 0.1 aqueous ammonia solution. Those fractions of the eluate, which gave a positive reaction with ninhydrin and a positive effect on the reaction of Rydon-1IT, and those that also gave the main spot Rf 0.60 in thin layer chromatography on silicon and used in quality. the developing solvent mixture of butanol-ethanol-chloroform — 17% si 1miak (in a volume ratio of 4; 5: 2: 3), combined and concentrated by drying under reduced pressure. The result is a colorless powder containing mainly 2N, b N-di-butoxycarbonyl-b N-methyl-3, 4-dideoxyanamycin B. Yield 41%;

b). This colorless powder (100 mg, containing mainly 2 N, 6 N-di-t-bytoxycabronyl-b N-methyl-3, 4-dideoxyanamycin B (0.16 mmol) is dissolved in a mixture consisting of 1 ml of water and 1 ml of dimethoxyethane, and to the resulting solution was added a solution of 54 mg (0.17 mmol) of N-oxysuccinimide ester and L-4-t-botoxycarbonyl-amide-2 o-hydroxybutyric acid in 2 ml of dimethoxymethane. The mixture was shaken at room temperature in for 22 hours to obtain an acylation reaction of the material containing the amino-protected N-methyl-3, 4-dideoxyanamycin B. The reaction mixture is then concentrated to suturing under reduced pressure to give a solid

A product containing a mixture of N-acylated N-methyl-3 b derivatives, 4-dideoxycanamycin B with N-substituted groups;

with); The solid residue from step 15) is dissolved in 2.4 ml of a 90% aqueous solution of trifluoroacetic acid, and the solution is kept at room temperature for 1 hour to achieve the effect of removing the t-butoxy carbonyl group. The reaction mixture is concentrated by drying at. reduced pressure and the residue is dissolved in 4 ml of water. The pH of the solution was adjusted to 8 by adding a concentrated aqueous solution of ammonia, and the solution was then passed through a chromatographic column (8x400 mm) with a volume of 20 ml with Amberlite CG 50 cation-exchange resin (LND form) to absorb the resin N-acylated products B of N- methyl-3, 4-dideoksikanamitsin V .. The column is washed successively with 100 ml of water; 100ML 0.3 ammonia water solution and 0.5N aqueous ammonia solution.

5, 0.75 N aqueous ammonia solution. The resulting eluate is collected in fractions (2 ml) and each fraction is examined using a conventional method for antibacterial activity against a strain sensitive to kanamycin Bacillus subtili PCI 219 and against a kanamycin-resistant Escherichia coli strain IR66 / W 677. The fractions that have a high antibacterial, activity against these two strains, are combined (in a volume of 26 ml) and then concentrated by drying, resulting in 39 mg of a colorless powder,

0 containing mainly 1 N- (L-4-amino-2-hydroxybutyryl) -b N-methyl-3, 4-dideoxycanamycin B.

I

For further purification, this colorless powder is dissolved and 0.5 ml.

5 mixtures of methanol - chloroform - 17% aqueous solution of ammonia (in a volume ratio of 4: 1 2) and the resulting solution is passed through a chromatography column with 3 g of silica gel,

0 as an eluent using a mixture of methanol - chloroform - 10% aqueous ammonia (in a volume ratio of 4 j 1: 2). The eluate is collected in 1 ml fractions, fractions 46-78 contain only 1 N5 - (L-4-amino-2-hydroxybutyryl) -b N-methyl-3, 4-dideoxyanamycin B, give only one spot at RP 0, 38 at thin-layer chromatography on silica gel (/ ART 5721); as

0 eluate use a mixture of butazol ethanol - chloroform - 28% aqueous ammonia solution (in a volume ratio of 4: 5: 2: 8). These fractions are combined and concentrated by drying, to obtain

15 mg of purified 1 N- (L-4-amino-oxybutyryl) -6 N-methyl-3, 4-dideoxyanamycin B as a colorless powder. Decay point 158-161 °

Example 2. Synthesis of 1 N- (DL isoseryl) -6 N-methyl-3, 4-dideoxycanamycin B (compound corresponding to formula I, where n 1).

but). A colorless powder (403 mg) containing mainly 2 Nb NT-di-t-bycoxycarboneJrl-b M-methyl-3, dideoxyanamycin B (0.6 mmol) obtained in Example 1a, dissolved in a mixture 4 ml of water and 4 ml of dimethoxyethane. The resulting solution is mixed with a solution of 221 mg (0.66 mmol) of ester of y.-oxysuccinimide and N-t-butoxycarbxy-OH-isoserine in 8 ml of dimethoxyethane. The mixture was shaken at room temperature for 23.5 to achieve an acylation effect. The reaction mixture is then concentrated by drying under reduced pressure to obtain a solid residue containing mainly a mixture of N-apylated 2 N-6 N-di-b-butoxycarbonyl-N N-methyl-3 4-dideoxycanamycin B derivatives

b). The resulting solid was dissolved in 7.5 ml of an aqueous solution. (90%) - of trifluoroacetic acid, the solution was allowed to stand for 1 hour at room temperature to achieve the effect of removing the t-butoxycarbonyl group. The reaction mixture is concentrated by drying under reduced pressure, the resulting product is dissolved in 10 ml of water. The pH of the aqueous solution thus obtained is adjusted to 8 by adding a concentrated aqueous solution of ammonia, then this solution is passed through a column (10x560 mm) with a volume of 43 ml with Amberlite CG 50 (NH form) cation exchange resin to absorb the mixture of N-acylated products. The resin in the column is washed with 200 ml of water and then 400 ml of 0.3 n. ammonia and elute 0.5 n. ammonia water solution. The eluate was collected in fractions of 4 ml, each fraction was examined by a conventional method for its antibacterial activity against Bacillus Subtilus PCI 219 and Escherichia coli 3R bb / w 677. The fractions, which show high antibacterial activity against these two strains, are combined ( 100 ml) and then concentrated by drying, the result is 135 mg of a colorless powder, containing mainly 1K- (OH, isoseryl-bN-methyl-3, 4-dideoxycanamycin B). For further purification, this colorless powder is dissolved in 2 , 6 ml of a mixture of methanol - chloroform - 17%

ammonia water solution (in a volume ratio of 4: 1: 2). The resulting solution was passed through a chromatographic column on silica gel (8 g), in which methanol – chloroform 17% aqueous ammonia (4: 1 2) was used as an eluent. The eluate is collected in a fraction of 2 ml. It was found that fractions No. 20-27 contain only 10 of the target product, which gives only one spot (at Rf 0.51) on the chromatogram when performing thin-layer chromatography on silica gel, using a mixture of buto15 nol-ethanol-chloroform-28% as the eluate aqueous solution of ammonia (in a volume ratio of 4: 5: 2: 8). These fractions are combined and concentrated by drying, to give 0 38 mg of purified 1 N- (DL-iso-propyl) -b N-methyl-3, 4-dideoxycanamycin B as a colorless powder with a decomposition point of 165-1 ° C °.

Example 3. Synthesis of 1 N- (L5 -isoseryl) -6 N-methyl-3, 4-dideoxycanamycin B (a compound corresponding to formula I, in which 1).

a), t-bytol cycarbonyl-azide (465 mg, 3.2 mmol) is added to

0 solution of 500 mg (1.1 mmol). b N-methyl-3, 4-dideoxyanamycin B in a mixture of 21 ml of pyridine, 21 ml of triethylamine and 12.6 ml of water. Shake quickly at room temperature.

5 to react with a t-butoxycarbonyl group. The reaction mixture is concentrated by drying under reduced pressure, to obtain 727 mg of a colorless powder,

0 consisting mainly of a mixture of 2 N, b N-di-t-bytoxycarbolyl-b N-methyl-3, 4-dideoxyanamycin B and 6 N-t-butoxycarbonyl-b N-methyl-3, 4-dideoxycanamycin B.

5 B). The resulting colorless powder (510) ml, consisting mainly of a mixture of partially amino-substituted derivatives of bN-methyl-3, 4-dideoxycanamycin B, without purification, is mixed with 5 ml of water and 5 ml of dimethoxyethane. To the resulting solution was added a solution of 254 mg (0.84 mmol) of N-oxysuccin-Imide ester and N-t-butoxycarbonyl-o1-isoserine in 10 ml | dimethoxyethane. Shake mixture

e g for 19 hours at room temperature to achieve an acylation effect. The reaction mixture is concentrated by drying under reduced pressure, the result is

0 794 mg of solid product, consisting mainly of a mixture of N-acylated derivatives of 2 N, b N-di-t-butoxycarbonyl- and b N-t-butonescarbonyl-bIsI-methyl-3, 4 -dideoxy5 kanamycin B.

with). The solid product is treated with a 90% aqueous solution of trifluoroacetic acid to remove the t-butoxycarbonyl group, purified on chromatographic with Amberlite CG-50, and then purified on a chromatographic column on silica gel in the same manner as in Example 25. Pure lN- (L-H3oceryl) -.6 N-methyl-3, 4-dideoxyaneamycin. B is obtained as a colorless powder with a yield of 34 mg, with a decay point of 162-1bb ° C.

Example 4. Synthesis of 1 N- (L-5-amino-2-oxivaleryl) -6 N-methyl-3, 4-dideoxyanamycin in (the compound corresponding to formula 1, in which item 3).

but). Colorless powder (610 mg), consisting mainly of 2N, -di-t-bytoxycabrbonyl-b N-methyl-3, 4-dideoxyanamycin B and bN-t-butoxycarbonyl-b N-methyl-3, 4 - Dideoxycanamycin B, prepared by the same method as in Example 3, was dissolved in 5 ml of water and 5 ml of dimethoxyethane without purification. To the resulting solution was added a solution of 278 mg (0.84 mmol) of N-oxysuccinimide ester and L-5-t-botoxycarbonylamido-2-oxivaleric acid in 10 ml of dimethoxyethane. The mixture was shaken for 18 hours at room temperature to achieve an acylation effect. The reaction mixture is concentrated by drying under reduced pressure, resulting in 834 mg of a solid product consisting mainly of a mixture of N-acylated 2 N derivatives, b N-di t-butoxycarbonyl and

H, K

where, characterized in that it is protected. prod4 CHjOH

b N-t-butoxycarbonyl-b N-methyl-3, 4-dideoxyanamycin B.

b). The solid was dissolved in 8 ml of a 90% aqueous solution of trifluoroacetic acid, and the solution was allowed to stand for 1 hour at room temperature to remove the t-butoxycarbonyl group. The reaction mixture is concentrated by drying under reduced pressure.

The product obtained is dissolved in 16 ml of water. The pH of the resulting solution was adjusted to 8 by the addition of concentrated aqueous ammonia, then the solution was adsorbed onto a column (10x560 mm) with a volume of 40 ml.

5 Amberlite CG 50 resin (NH4 form). . After that, the column is washed successively with 200 ml of water, 250 ml of 0.3 n. aqueous ammonia and 240 ml of 0.5 n, aqueous solution of am-.

0 miak, the eluate is collected in fractions of 4 ml. Those fractions that exhibit high antibacterial activity against Bacillus-subtilus PCI 219 and Escheria coli 3R bb / w 677,

5 was collected together, concentrated by drying, and then purified by thin layer chromatography on silica gel in the same manner as in Example 1c. Pure 1 N0 - (L-5-amino-2-oxivaleryl) -6 N-methyl-3, 4-dideoxy 1 Micin B is obtained in the form of a colorless powder with a yield of 29 mg and a decomposition point of 152-155 C.

5 i

Claims (1)

Invention Formula The method of obtaining IN- (| A-hydroxy-sh-aminoalkanoyl) -b N-methyl-3, 4-dideoxycanamycin in formula I CHjUHCHs  water. 6N -methyl-3, 4 -dideoxyamine B of the general formula II four where R is a tert-butyroylcosicarbonyl group, in a mixture of water and dimethoxyethane is acylated and BHN- (CH2) n-fiH (OH) -COO- where R and p have the indicated meanings R, n have the indicated meanings; treated with 90% -BBM aqueous solution triperoxonane acid, followed by separation of the target product by column chromatography, o (.- hydroxy- (A) -amino acid derivative of general formula III VIA obtained 1 N-acidified derivative of the general formula IV sJ CHjIUIR Sources of information, 25 taken into account in the examination 1. USSR patent 576047, cl. C 07 N. 15A22, 01.27.72.