KR810001044B1 - Preparing process for amino-glycocides derivatives - Google Patents

Abstract

Amino-glycocide derivs. (I; R = H, lower alkyl, aralkyl, aralkoxycarbonyl; n = 1,3; R1 = aminomethyl, hydroxymethyl, 1-aminoethyl, 1-methylaminoethyl; R2,R3,R6 = H, hydroxy; R4 = hydroxy, amino group; R5 = amino, methylamino group; R7 = hydroxy, methyl; R8 = H, hydroxy methyl, carbamoyloxymethyl; dotted line means either double bond or single bond), useful as antibactrial agent, were prepd. by reacting aminoglycocide(II) with compd.(III) followed by elimination of functional group.

Description

Process for preparing novel aminoglycoside derivatives

The present invention relates to a novel aminoglycoside derivative of the formula (I) having a good antibacterial action and a process for preparing the salts thereof.

Wherein R is hydrogen, lower alkyl, aralkyl or aralkoxycarbonyl group and n is an integer of 1 or 3.

R ¹ is an aminomethyl, hydroxymethyl, 1-aminoethyl or 1-methylaminoethyl group, and R ² , R ³ and R ⁶ are each hydrogen or a hydroxy group.

R ⁴ is a hydroxy or amino group, R ⁵ is an amino or methylamino group, R ⁷ is a hydroxy or methyl group, R ⁸ is a hydrogen, hydroxymethyl, or carbamoyloxymethyl group, and the dotted line is the presence of a double bond Or absent.

In the past, amino glucoside antibiotics such as streptomycin, kanamycin, zenamycin, tobramycin, and the like have been used as a broad-spectrum antimicrobial agent against gram-positive bacteria, gram-negative bacteria and anti-acid bacteria. However, aminoglycoside antibiotics are sometimes accompanied by unwanted side effects such as kidney disease and deafness. The development of resistant bacteria to aminoglycosides is another problem to be solved. Therefore, in order to increase the antimicrobial activity of such amino glycosides and to significantly reduce the side effects, it has been attempted to improve by introducing a specific acyl group to the amino group of 1 position. For example, amikacin, an excellent anti-regulator made by acylating the amino group at position 1 of kanamycin A with 4-amino-2-hydroxybutyric acid, is effective against kanamycin A resistant bacteria and its toxicity is almost the same as that of kanamycin A [ See: Gucci River et al. J. Antibiotic. 25,695 (1972), US Pat. No. 3,78,268 (1973) and J. Antibiotc, 27,677 (1974).

The inventors of the present invention acylated the amino group at the 1-position of the known amino glycosides with 3-hydroxypiperidine-3-carboxylic acid or 3-hydroxy-azetidine-3-carboxylic acid to increase the antimicrobial range and antimicrobial activity. It was found to increase. The present invention is based on this finding.

The amino glycosides used as starting materials in the present invention include 2-deoxystrepamine residues and are represented by compounds of the following structural formula.

In the above formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and the dotted line are the same as above.

Specific examples of the known aminoglycosides represented by compounds of formula (III) and their alicyclic compounds are shown in Table 1 below.

TABLE 1

Lower alkyl in R in the compound of formula (I) is an alkyl group having 1 to 5 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary-butyl, tert-butyl, pentyl, isopentyl , Tert-pentyl, neopentyl and the like; An aralkyl group means an aralkyl group having 7 to 20 carbon atoms such as benzyl, phenethyl, phenylpropyl and the like; Alkoxycarbonyl means an alkoxycarbonyl group having 8 to 20 carbon atoms, for example benzyloxycarbonyl, phenoxycarbonyl, 1-phenylethoxycarbonyl, 3-phenylpropoxycarbonyl.

The aminoglycoside antibiotic derivatives represented by formula (I) in the present invention include free bases and salts thereof, particularly pharmaceutically toxic acid addition salts.

Acids forming acid addition salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, carbonic acid, acetic acid, fumaric acid, malic acid, tartaric acid, maleic acid, citric acid, mandelic acid, ascorbic acid, gallic acid, and the like. The same organic acid is mentioned.

Representative compounds of formula (I) are as follows.

1) 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin

2) 1-N- (3-hydroxyazetidine-3-carbonyl) canamycin A

3) 1-N- (3-hydroxyazetidine-3-carbonyl) canamycin B

4) 1-N- (1-methyl-3-hydroxyazetidine-3-carbonyl) tobramycin

5) 1-N- (1-methyl-3-hydroxyazetidine-3-carbonyl) canamycin A

6) 1-N- (1-methyl-3-hydroxyazetidine-3-carbonyl) canamycin B

7) 1-N- (1-benzyl-3-hydroxyazetidine-3-carbonyl) tobramycin

8) 1-N- (1-benzyl-3-hydroxyazetidine-3-carbonyl) canamycin A

9) 1-N- (1-benzyl-3-hydroxyazetidine-3-carbonyl) canamycin B

10) 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine-3-carbonyl) tobramycin

11) 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine-3-carbonyl) canamycin A

12) 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine-3-carbonyl) canamycin B

13) 1-N- (3-hydroxypiperidine-3-carbonyl) tobramycin

14) 1-N- (3-hydroxypiperidine-3-carbonyl) canamycin A

15) 1-N- (3-hydroxypiperidine-3-carbonyl) canamycin B

16) 1-N- (1-methyl-3-hydroxypiperidine-3-carbonyl) tobramycin

17) 1-N- (1-methyl-3-hydroxypiperidine-3-carbonyl) canamycin A

18) 1-N- (1-methyl-3-hydroxypiperidine-3-carbonyl) canamycin B

19) 1-N- (1-benzyl-3-hydroxypiperidine-3-carbonyl) tobramycin

20) 1-N- (1-benzyl-3-hydroxypiperidine-3-carbonyl) canamycin A

21) 1-N- (1-benzyl-3-hydroxypiperidine-3-carbonyl) canamycin B

22) 1-N- (1-benzyloxycarbonyl-3-hydroxypiperidine-3-carbonyl) tobramycin

23) 1-N- (1-benzyloxycarbonyl-3-hydroxypiperidine-3-carbonyl) canamycin A

24) 1-N- (1-benzyloxycarbonyl-3-hydroxypiperidine-3-carbonyl) canamycin B

The compound of formula (I) can be easily prepared by acylating the above-mentioned compounds of aminoglycosides (II) with the carboxylic acids of the following formula (III) or reactive derivatives thereof.

Wherein R and n are as defined above.

Since aminoglycosides (II) compounds as starting materials have many functional groups (for example, amino groups) in addition to the amino groups at the 1-position, it is appropriate to protect them with protecting groups arbitrarily before acylation.

As the protecting group, a protecting group that can be easily removed from the peptide chemistry after the acylation of the amino group in the 1 position is used. Such protecting groups are benzyloxycarbonyl, formyl, tert-butyloxycarbonyl tert-amyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, p-toluenesul which may be optionally substituted on the benzene nucleus Phonyl, phthaloyl, m-nitrophenylthio, triphenylmethylthio and the like.

Reactive derivatives of the aforementioned carboxylic acids used as acylating agents include those originally used in peptide synthesis such as acid halides, acid azides, acid anhydrides, mixed acid anhydrides, reactive esters, and the like. [Note: Synthesis Volume 453 (1972) and Petide Synthesis Volume 75 ~ 135 (1966) by Bodansky]

In the acylating agent it is preferred to protect the nitrogen atom with a suitable protecting group as mentioned in aminoglycoside protection when R is a hydrogen atom. Acylating agents can be easily made by conventional methods. For example, 1-benzyl-3-hydroxypiperidine-3-carboxylic acid is reacted with 1-benzyl-3-piperodon hydrochloride with potassium cyanide or hydrogen cyanide. Drin is produced and made by hydrolyzing it with concentrated hydrochloric acid.

Acylation of aminoglycosides can be accomplished by reacting the starting material aminoglycoside with an active acylating agent in a suitable solvent, where the functional group is protected other than the amino group at the 1 position. In carrying out this acylation, an equimolar amount or excess of acylating agent, preferably 1.0 to 2.0 moles, is used relative to 1 mole of aminoglycosides. This reaction is carried out at a temperature of 0 ° C to 50 ° C, preferably 20 to 25 ° C.

Examples of the solvent used include lower alcohols such as methanol, ethanol and ethylene glycol, ethers such as diethyl ether, 1,2-dimethoxyethane tetrahydrofuran and dioxane, ketones such as acetone and methylmethyltone, dimethylform Amide, dimethylacetamide, pyridine, water and the like, which may be used alone or in mixture of two or more.

After the acylation is complete, the protecting group is removed by conventional methods such as acid treatment or catalytic hydrogenation to yield the desired compound (I).

The aminoglycoside derivatives and their nontoxic salts according to the invention exhibit good antibacterial action. These compounds have hundreds to tens of times more antimicrobial activity than the corresponding unsubstituted aminoglycosides against Gram positive and Gram negative bacteria. The minimum inhibitory concentrations (MIC, μg / ml) of known unsubstituted aminoglycosides corresponding to acylated compounds according to the invention are shown in Tables 2 and 3.

TABLE 2

MIC (μg / ml)

Note: Compound (1) = 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin

TOB = Tobramycin

Compound (2) = 1-N- (3-hydroxyazetidine-3-carbonyl) canamycin A

KM-A = Kanamycin A

* Tobramycin resistant bacteria.

TABLE 3

MIC (μg / ml)

Note: Compound (3) = 1-N- (3-hydroxypiperi-3-carbonyl) tobramycin

* Tobramycin resistant bacteria.

As shown in Tables 2 and 3, the compound of formula (I) according to the present invention has a strong antibacterial action against a variety of Gram-positive bacteria and Gram-negative bacteria and is useful as a medicament and veterinary medicine. Ie these compounds are Gram-positive bacteria (e.g., Staphylococcus aureus, Staphylococcus, Epidermis) and Gram-negative bacteria (e.g., E. coli, Crabsilla pneumoniae, Proteus mirabilis, Proteus vulgaris, It can be used for the prevention and treatment of infectious diseases caused by Monas aeruginosa). The compound of formula (I) according to the present invention can also be used as a fungicide, as an additive of perishable food, as a disinfectant, or in a place or apparatus where bacteria are present.

The compound of formula (I) according to the present invention can be administered orally or parenterally, either alone or in the form of a mixture with other active substances. The pharmaceutical composition may be in a mixture of 0.01 to 99% with a pharmaceutical carrier such as a liquid which may be dissolved, dispersed or suspended in the compound of formula (I), or a solid material.

They may be in a single dosage form. Solids are solid preparations such as tablets, powders, dry syrups, troches, granules, capsules, pills, suppositories. The agent is an injection, ointment, dispersant, inhalant, suspension, liquid, emulsifier, syrup, or elixir. All diluents (eg, starch, white sugar lactose, calcium carbonate, kaolin); Weight agents (eg, lactose, sugar, salt, glycine, starch, calcium carbonate, calcium phosphate, kaolin, bentonite, talc, sorbitol); Binders (eg, starch acacia gum, gelatin, glucose, sodium alkate, tragacanth, carboxymethylcellulose, sorbitol, polyvinylpyrrolidone); Disintegrants (eg, starch, agar, carbonates, sodium lauryl sulfate); Lubricants (eg, stearic acid, talc, paraffin, boric acid, silica, sodium benzoate, polyethylene glycol, cacao oil, magnesium sulfate); Emulsifiers (eg, lecithin, sorbitan monooleate, acacia gum); Suspending agents (eg, sorbitol, methylcellulose, glucose, sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, hydrogenated fat); Solvents (eg, water, peanut oil, sesame oil, methyl oleate); Preservatives (e.g. methyl or ethyl p-hydroxybenzoate, sorbic acid), edible colorants, aromatics, solubilizers, buffers, stabilizers, dispersants, wetting agents, antioxidants, etc., are normally used as long as they do not adversely affect the compound of formula (I). It can be used in the way.

The compounds of formula (I) according to the invention, in particular their mineral salts, are readily soluble in water and can be conveniently used as intravenous, intramuscular, or subcutaneous injection solutions according to conventional methods. The compound of formula (I) is dissolved in an aqueous ampoule or an oily solvent to form an injection solution, and in order to preserve it as an injection for a long time, it is appropriate to use a vial formulation with crystals, powders, microcrystals, or lyophilized products. This vial preparation may be dissolved or suspended in the aforementioned solvent for injection just before use. This formulation may contain the preservatives described above.

Furthermore, the compounds of formula (I) according to the invention can be used as suppositories, ointments for topical or ophthalmic use, as powders for topical use and as preparations which can be formulated according to methods well known to those skilled in the art. .

The external preparation may comprise 0.01 to 99% of the compound of formula (I) according to the present invention together with the required amount of the aforementioned pharmaceutical carrier.

In addition, the compound of formula (I) may be administered in single or divided doses in a daily dosage of 0.01 to 5 g / kg for injection or 0.01 to 10 g / kg for oral administration for humans or animals, and 0.01 for local treatment. To 10 g may be used at intervals of 3 to 12 hours.

Compounds according to the invention are infectious diseases caused by bacteria, such as staphylococcal infectious dermatitis, animal parasitic disease, cystitis, pyelitis, pneumonia, alveolaritis, bronchitis, sinusitis, nasopharyngitis Iv), tonsillitis, rhinitis, dermatitis, pustules, abscess wounds and soft cell infections, otitis media osteomyelitis, sepsis, enteritis, urinary tract infections, pyelonephritis.

The following examples illustrate the invention in more detail.

Example 1

1-N- (3-hydroxyazetidine-3-carbonyl) tobramycinsulfate

(A method)

(1) 252 mg (1.0 mmol) of 1-benzyloxycarbonyl-3-hydroxyazetidine-3-carboxylic acid 116 mg (1.0 mmol) N-hydroxysuccinimide in 4 ml of dimethylformamide And 208 mg (1.0 mmol) of dicyclohexylcarbodiimide are stirred at room temperature for 3 hours. To this was added 3,2 ', 6', 3'-tetra-N-formyltobramycin (300 mg; 0.502 mmol) and the mixture was stirred at room temperature and then further stirred at 50 ° C. overnight. Precipitated dicyclohexylurea is filtered and washed with dimethylformamide. The combined filtrate and washings were mixed with 70 ml of ethyl acetate, stopped for 30 minutes and filtered. Treatment of the residue with water yields 317 mg of water-soluble material and 84 mg of water-insoluble mucus. Concentration of the combined filtrate and washings yields 0.42 g of residue.

The obtained water-soluble substance (317 mg) was dissolved in a mixture of 15 ml of water and 15 ml of methanol, and one drop of acetic acid was added, and then catalytically hydrogenated in the presence of 150 mg of 10% palladium-charcoal under hydrogen pressure. The catalyst is isolated by filtration and washed with a mixture of water and methanol. The combined filtrate and washings were concentrated under reduced pressure to give 261 mg of 1-N- (3-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3′-tetra-N-formyltobramycin. (Iv) Obtained as a product.

The obtained water-insoluble mucus material and residue (0.42 g) were dissolved in a mixture of 2 ml of N, N-dimethylformamide, 15 ml of water and 15 ml of methanol, and catalytically hydrogenated in the same manner to give 326 mg. Obtain 1-N- (3-hydroxyazetidine-3-carbonyl-3,2 ', 6', 3′-tetra-N-formyltobramycin as a crude product (total yield: 587 mg). ).

Crude 1-N- (3-hydroxyazetidine-3-carbonyl-3,2 ', 6', 3'-tetra-N-formyltobramycin (587 mg) was added to 35 ml of 10% hydrazine monohydrate. Dissolved, adjusted to pH 6 with acetic acid, refluxed for 6 hours, diluted with 700 ml of water, adsorbed onto a column of 137 ml of Amberlite CG-50 (NH ₄ ⁺ ), washed column with 1.76 l of water, and then 1.76 l. Was eluted with a mixture of 0.4% ammonium hydroxide and 2 liters of 0.8%. (1 fraction: 15 g) Collect fractions 91 to 100 and evaporate under reduced pressure to yield 127 mg of 1-N- (3-hydroxyazetidine- 3-carbonyl) tobramycin is obtained as a powder, which is dissolved in 0.9 ml of water and treated with activated carbon, adjusted to pH 5 with 2N acid and mixed with 20 ml of ethanol, followed by 2 at 0 ° C. Stop for hours and filter The residue is washed with ethanol and dried over phosphorus pentoxide under reduced pressure to give a total yield of 28% Was obtained in a 1-N- (3- hydroxy-azetidin-3-carbonyl) tobramycin 2.5H ₂ SO ₄ and 2.5H ₂ O of 120㎎.

TLC: Rf = 0.30 [Rf = 0.55 (Tobramycin)]

[Silk gel 60 F ₂₅₄ plate solvent system made by Cerck: Isopropanol-ammonium hydroxide (1: 1)]

Elemental analysis: C ₂₂ H ₄₂ N ₆ O ₁₁ ㆍ 2.5H ₂ SO ₄ ㆍ 2.5H ₂ O

Calculated (%): C, 30.84; H, 6.12; N, 9.81; S, 9.36

Found (%): C, 30.71; H, 5.95; N, 9.75; S, 9.08

(2) 6.225 g (10.4 mmol) of 3,2 ', 6', 3'-tetra-N-formyltobramycin, 3.137 g (1.2 equivalents) of 1-benzyloxycarbonyloxy-3-hydroxyase Tidin-3-carboxylic acid and a solution of 1.437 g (1.2 equivalents of N-hydroxysuccinimide in 97 ml of dimethylformamide) were stirred, and stirred at 3.095 g (1.4 equivalents) of dicyclohexylcarbodiamide at room temperature. The mixture is stirred overnight at room temperature The resulting precipitate is filtered off and washed with dimethylformamide The combined filtrate and wash are washed with 600 ml of ethyl acetate and dissolved in a mixture of methanol and water (2: 1). Concentration of this solution gave 8.1 g of 1-N- (1-benzyl oxycarbonyl-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3'-tetra-N-formyltobra Mycin is obtained as a powder (yield: 95.8%).

Crude 1-N- (1-benzeneoxycarbonyl-3-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3′-tetra-N-formyltobramycin (7.2 g; 8.86 Mmol) is dissolved in a mixture of 360 mL of water and 360 mL of methanol and catalytically hydrogenated under hydrogen pressure for 2 hours in the presence of 3.6 g of 10% palladium charcoal. After filtration through this catalyst and the filtrate was concentrated under reduced pressure, 6.9 g of crude 1-N- (3-hydroxy-azetidine-3-carbonyl) -3,2 ', 6', 3'-tetra-N- Formyltobramycin is obtained.

의 물과 3

의 1N 수산화암모늄 수용액으로 용출한다.(일획분 : 18㎖). 획분 번호 319에서 348까지를 감압하 농축시키면 2.420g의 1-N-(3-히드록시아제티딘-3-카르보닐)토브라마이신을 순수한 생성물로 수득한다.The crude product (6.9 g) is dissolved in 15 ml of water and mixed with 95.8 ml of concentrated methanolic hydrochloric acid (11 ml → 65.5 ml) solution. The resulting solution is stirred in an oil bath at 36 ° C. for 24 hours. After the reaction was completed, the reaction mixture was neutralized with 220 ml of Amberlite 1R-45, and then the resin was filtered and washed with water. Combined area and washings are evaporated under reduced pressure. The residue (5.34 g) is adsorbed onto a column of 1.2 L of Amberlite CG-50 (NH ₄ ⁺ ) and eluted by fractionation with 3 L of water and 3 L of 1N aqueous ammonium hydroxide solution (fraction: 16 mL). Concentration from fractions 296 to 345 yields 4.06 g of 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin as crude product. The crude product was chromatographed on a column of 200 g silica gel (Merck silica gel), eluted with a mixture of isopropanol, ammonium hydroxide and chloroform (2: 1: 1), and then into a mixture of isopropanol and ammonium hydroxide (1: 1). Elutes. The eluate was again adsorbed onto 2.3 L of Amberlite CG-50 (NH ₄ ⁺ ) and 3

Of water and 3

Elution with 1N aqueous ammonium hydroxide solution. (Fraction: 18 ml). Concentration under fractions 319 to 348 under reduced pressure yields 2.420 g of 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin as pure product.

2.347 g of 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin dissolved in 20 ml of water was adjusted to pH 4.6 with 6.6 ml of 10% sulfuric acid and mixed with 250 ml of ethanol. A precipitate (3.10 g) is obtained, which is collected by filtration, washed with ethanol, dissolved in 10 ml of water and mixed in 200 ml of ethanol. The precipitate is collected by filtration, washed with ethanol, dissolved in water and concentrated under reduced pressure. The residue was dried in vacuo over phosphorus pentoxide and absorbed in a desiccator containing saturated sodium bromide to absorb 2.419 g of 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin 2.5H ₂ Obtain SO ₄ 8H ₂ O. (Total yield 28.5%).

Elemental analysis: C ₂₂ H ₄₂ N ₆ V ₁₁ 2.5H ₂ SO ₄ ㆍ 8H ₂ O

Calculated (%): C, 27.64; H, 6.64; N, 8.79; S, 8.39

Found (%): C, 27.48; H, 6.35; N, 8.51; S, 8.20

[B method]

시간 동안 교반한다. 생성되는 디시클로헥실우레아(7.40g)를 여과하여 60㎖의 디메틸포름아미드로 세탁한 후 에틸아세테이트로 세척한다. 합한 여액과 세척액에 진탕하면서 2.6

의 에틸아세테이트를 서서히 가한다. 10분 후 침전된 고형물을 여과하여 수집하고 에틸아세테이트로 세척한다. 이 침전물을 600㎖의 뜨거운 메탄올로 처리하면 결정화합물이 수득된다. 이 결정화합물에 3.0

의 디에틸에테르를 가하므로서 융점(분해) 230 내지 240℃의 1-N-(1-벤질옥시카르보닐-3-히드록시아제티딘-3-카르보닐)-3,2',6',3＂-테트라-포르밀토브라마이신 28.3g을 수득한다. 상기 생성물(500㎎)의 일부분을 250㎖의 메탄올로 재결정시키면 융점 243 내지 247℃의 침상의 1-N-(1-벤질옥시카르보닐-3-히드록시아제티딘-3-카르보닐)-3,2',6',3＂-테트라-포르밀토브라마이신 435㎎을 수득한다.(1) 3,2 ', 6', 3'-tetra-N-formyltobramycin (20.0 g; 33.4 mmol) and 1-benzyloxycarbonyl-3-hydroxyazetidine in 200 ml of dimethylformamide In a solution of 3-carboxylic acid (10.1 g; 40.3 mmol, 1.20 equiv) and 1-hydroxybenzotriazole (452 mg; 3.34 mmol; 0.1 equiv), 8.3 g (40.2 mmol) in 100 ml of tetrahydrofuran (1.20 equivalents) of picyclohexylcarbodiimide was added dropwise for 2.5 hours, and the mixture was then stirred at room temperature.

Stir for hours. The resulting dicyclohexyl urea (7.40 g) was filtered, washed with 60 ml of dimethylformamide, and then washed with ethyl acetate. 2.6 while shaking in the combined filtrate and wash solution

Ethyl acetate was added slowly. After 10 minutes, the precipitated solid is collected by filtration and washed with ethyl acetate. Treatment of this precipitate with 600 ml of hot methanol yields a crystalline compound. 3.0 to this crystalline compound

Melting point (decomposition) of 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3 at 230-240 ° C by adding diethyl ether 28.3 g of VIII-tetra-formyltobramycin are obtained. Recrystallization of a portion of the product (500 mg) with 250 ml of methanol gave needle-like 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine-3-carbonyl) -3 at a melting point of 243 to 247 ° C. 435 mg of 2 ', 6', 3'-tetra-formyltobramycin is obtained.

Elemental analysis: C ₃₄ H ₄₈ N ₆ O ₁₇ ㆍ 3 / 2H ₂ O

Calculated (%): C, 48.62; H, 6.12; N, 10.01

Found (%): C, 48.66; H, 6.19; N, 10.03

+92.3±1.3° (c=1.007, 디메틸포름아미드)

+ 92.3 ± 1.3 ° (c = 1.007, dimethylformamide)

에 녹이고 14g의 10% 팔라듐/목탄 14g 존재하 수소압하에서 2시간 동안 촉매적으로 수소 첨가시킨다. 이 촉매를 여과하고 물로 세척한다. 합한 여액과 세척액을 감압하 증발시키면 24g의잔유물이 생성되며 이것을 물에 녹이고 1.0g의 활성탄으로 처리한 후 라이렉스 필터를 통해 여과한다. 이 여액을 감압하 증발시키면 22.8g의 1-N-(3-히드록시아제티딘-3-카르보닐)-3,2',6',3＂-테트라-N-포르밀토브라마이신을 수득한다.(2) A crude product (27.8 g) obtained in the above (1) was mixed with methanol and water (1: 1) 1.0.

And catalytically hydrogenated for 2 hours under hydrogen pressure in the presence of 14 g of 10% palladium / charcoal. This catalyst is filtered off and washed with water. The combined filtrate and washings are evaporated under reduced pressure to produce 24 g of residue, which is dissolved in water, treated with 1.0 g of activated carbon and filtered through a Relex filter. Evaporation of this filtrate under reduced pressure yields 22.8 g of 1-N- (3-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3'-tetra-N-formyltobramycin. .

(3) 361 ml (0.71 mol; 4.25 equiv) of methanolic hydrochloric acid solution made of hydrochloric acid (110 ml) and methanol (545 ml) were added to a solution of 22.8 g of the product in 63.5 ml of water. Stir at a temperature between 35 ° C. After 24 hours the mixture is adjusted to pH 7.15 with 48.0 mL (0.7 mol) of ammonium hydroxide at 35 ° C. or lower. The resulting solution is concentrated to about 200 ml at about 30 ° C. under reduced pressure. Water (400 mL) was added thereto, and the solution was adsorbed onto a column of 2.0 L of Amberlite CG-50 (NH ₄ ⁺ ) on a column, and water and 1N ammonium hydroxide were mixed with 5.7 L (388 mL of concentrated ammonium hydroxide and 5312). Eluted with a mixture of mL). (1 portion: 20 mL). Pure 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin (11.10 g) was obtained from 492 in fraction 430.

의 물과 2.0

의 0.5N 수산화암모늄, 68㎖의 농수산화암모늄과 1932㎖ 물의 혼합물로 획분법에 의해 다시 크로마토그라피했다(1획분 : 20㎖). 획분 195~229로부터 순수한 1-N-(3-히드록시아제티딘-3-카르보닐)토브라마이신(1.15g)이 수득되었다. 획분 230에서 255까지를 감압하 농축시키면 3.698의 잔유물이 수득된다. 이 잔유물 일부분(3.44g)을 10㎖의 물에 녹여 10㎖의 이소프로판올로 혼합한다. 생성된 용액을 150g의 실리카겔 컬럼에 흡착하고 획분 32까지 이소프로판올과 농수산화암모늄(2 : 1)의 혼합물로 용출한 후 이소프로판올과 농수산화암모늄(1 : 1)의 혼합물을 용출한다.Combine fractions 418 to 429 and fractions 493 to 418 on a column of 600 ml Amberlite CG-50 (NH ₄ ⁺ ) 2.0

With water 2.0

Was chromatographed again by fractional method with a mixture of 0.5N ammonium hydroxide, 68 mL of concentrated ammonium hydroxide, and 1932 mL of water (1 fraction: 20 mL). Pure 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin (1.15 g) was obtained from fractions 195-229. Concentration of the fractions 230 to 255 under reduced pressure yields a residue of 3.698. A portion of this residue (3.44 g) is dissolved in 10 ml of water and mixed with 10 ml of isopropanol. The resulting solution was adsorbed onto a 150 g silica gel column and eluted with a mixture of isopropanol and ammonium hydroxide (2: 1) to fraction 32, followed by elution of a mixture of isopropanol and ammonium hydroxide (1: 1).

Fractions 54 to 100 were concentrated under reduced pressure to 50 ml, treated with 300 mg of activated charcoal and filtered. The filtrate was chromatographed on a column of 120 mL Amberlite CG-50 (NH ₄ ⁺ ) and eluted with 1N ammonium hydroxide (1 portion: 20 mL). Target substance (3.19 g) was obtained in fractions 11-16.

The total yield of pure 1-N- (3-hydroxyazetidine-3-carbonyl) tobramycin was 15.44 g (81.7%).

(4) A solution of product (15.44 g; 27.3 mmol) dissolved in 180 ml of water was treated with 1.5 g of activated carbon and filtered.

The residue is washed with 70 ml of water. The combined filtrate and washings were adjusted to PH 6.0 with 111.5 ml of 1 N sulfuric acid under stirring at room temperature and filtered. Lyophilization of the filtrate yields 21.55 g of lyophilized product, which is 23.027 g of 1-N- (3-hydroxyazetidine-3-carb) when placed in a desiccator containing a saturated solution of sodium bromide until a constant volume. carbonyl) tobramycin 2.5H ₂ SO ₄ and to give the 7H ₂ O. (73.5% total yield).

Elemental analysis: C ₂₂ H ₄₂ N ₆ O ₁₁ ㆍ 5 / 2H ₂ SO ₄ ㆍ 7H ₂ O

Calculated (%): C, 28.17; H, 6.56; N, 8.96; S, 8.55

Found (%): C, 28.02; H, 6.69; N, 8.86; S, 8.38

Example 2

1-N- (3-hydroxyazetidine-3-carbonyl) canamycin A sulfate

[A method]

180 mg (0.716 mmol) in 2.6 mL of dimethylformamide was dissolved in 1-benzyloxy-3-hydroxyazetidine-3-carboxylic acid and 82.4 mg (0.716 mmol) of N-hydroxysuccinimide. MG dicyclohexylcarbodiimide is added and the mixture is stirred at room temperature for 30 minutes. The precipitated dicyclohexylurea is collected by filtration and washed with 1 ml of dimethylformamide. The combined filtrate and washes comprise the corresponding reactive esters.

To the solution of 313 mg (0.54 mmol) of 3,6 ', 3'-tri-N-formylkanamycin A dissolved in 3 ml of dimethylformamide was added the reactive ester solution, and the mixture was stirred at room temperature for 5 hours. Then mixed again with the same amount of reactive ester solution. After 1.5 hours, the precipitated dicyclohexylurea was removed by filtration and washed with a small amount of dimethylformamide. The combined filtrate and washings were mixed with 150 ml of ethyl acetate and cooled with ice. The resulting precipitate was collected by filtration, washed with ethyl acetate and dissolved in a mixture of methanol and water (2: 1). The solution was treated with activated carbon and evaporated under reduced pressure to give 405 mg of 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine-3-carbonyl) -3,6 ', 3'-tri-N. Formylkanamycin A was obtained.

This product was dissolved in 7 ml of methanol and catalytically hydrogenated in a solution in which 200 mg of 10% palladium / charcoal was suspended in 7 ml of water under hydrogen pressure. The catalyst was filtered off and the filtrate was concentrated under reduced pressure to give 307 mg of 1-N- (3-hydroxyazetidine-3-carbonyl) -3,6 ', 3'-tri-N-formylkanamycin A. Was obtained.

의 물과 1

의 1N 수산화암모늄 용액으로 획분법에 의해 용출했다(1획분 : 12㎖). 획분번호 73에서 84까지를 농축시키면 181㎎의 1-N-(3-히드록시아제티딘-3-카르보닐)카나마이신 A를 수득한다.This precipitate was dissolved in 0.64 mL of water and mixed with a concentrated methanolic hydrochloric acid solution (11.0 mL → 65.5 mL), and the resulting mixture was stirred at 35 to 37 ° C. for 23 hours. The resulting mixture was diluted with 5 mL of water and neutralized with 20 mL of Amberlite 1R-45. The resin was filtered and washed with water. The combined filtrate and washings were evaporated under reduced pressure to yield 329 mg of serum, which was dissolved in 4 ml of water and adsorbed onto a column of 153 ml of Amberlite CG-50 (NH ₄ ⁺ ) and 440. After washing the column with 1 ml water

Water and 1

Was eluted with 1 N ammonium hydroxide solution in 1 fraction (1 fraction: 12 ml). Concentration from fractions 73 to 84 affords 181 mg of 1-N- (3-hydroxyazetidine-3-carbonyl) canamycin A.

This product is taken up in 6 ml of water, adjusted to pH 4.6 with 10.85 ml of 0.0955 N sulfuric acid and concentrated to 1-2 ml under reduced pressure. This residue is collected by filtration, washed with ethanol and dissolved in water. The solution is treated with 100 mg of activated carbon and filtered through a Pyrex filter. The filtrate was lyophilized and left to absorb moisture until it was weighed to give 246 mg 1-N- (3-hydroxyazetidine-3-carbonyl) canamycin A. 2H ₂ SO ₄ .7H ₂ O. . 50% total yield, melting point 222 (colored) to 246 to 257 ° C (decomposition).

Elemental analysis: C ₂₂ H ₄₁ N ₅ O ₁₃ ㆍ 2H ₂ SO ₄ ㆍ 7H ₂ O

Calculated (%): C, 29.17; H, 6.56; N, 7.73; S, 7.08

Found (%): C, 29.35; H, 6.43; N, 7.52; S, 7.21

[B method]

시간에 걸쳐 점적하여 가한 다음 이 혼합물을 실온에서 7시간 동안 교반한다. 생성되는 디시클로헥실우레아를 여과해내고 100㎖의 디메틸포름아미드로 세척한다. 이 여액과 세척액을 감압하 약 200㎖까지 농축한 후 얼음으로 냉각시킨다. 나타나는 디시클로헥실우레아를 여과해내고 100㎖의 냉 디메틸포름아미드로 세척한다. 합한 여액과 세척액을 3.0ℓ의 에틸아세테이트로 처리하면 무색 침전이 생성되며 이것을 여과하여 수집하고 에틸아세테이트로 세척하면 27.4g의 1-N-(1-벤질옥시카르보닐-3-히드록시아제티딘-3-카르보닐)-3,6',3＂-트리-N-포르밀카나마이신 A을 수득한다.19.0 g (32.39 mmol) of 3,6 ′, 3′-triformylcanamycin A, 10.57 g (42.109 g mmol; 1.3 equivalents of 1-benzyloxycarbonyl-3- in 380 ml of dimethylformamide In a solution of 438 mg (3.239 mmol; 0.1 equivalent) of 1 hydroxybenzotriazole in hydroxyazetidine-3-carboxylic acid, 8.688 g (42.109 mmol; 1.3 equivalent) of dicyclohexyl in 160 ml of dimethylformamide. The solution of carbodiimide

It is added dropwise over time and the mixture is stirred at room temperature for 7 hours. The resulting dicyclohexylurea is filtered off and washed with 100 ml of dimethylformamide. The filtrate and washings are concentrated to about 200 ml under reduced pressure and cooled with ice. The dicyclohexyl urea shown is filtered off and washed with 100 ml of cold dimethylformamide. The combined filtrate and washings were treated with 3.0 L of ethyl acetate to give a colorless precipitate which was collected by filtration and washed with ethyl acetate and 27.4 g of 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine- 3-carbonyl) -3,6 ', 3′-tri-N-formylkanamycin A is obtained.

(2) The product (27.4 g; 32.39 mmol) was dissolved in 200 ml of a mixture of methanol and water (1: 1) and catalytically hydrogenated in the presence of 9.5 g of 10% palladium charcoal under hydrogen pressure to 24.2 g of 1-. N- (3-hydroxyazetidine-3-carbonyl) -3,6 ', 3′-tri-N-formylkanamycin A is obtained.

(3) To a solution of 24.2 g (37.39 mmol) of the above product in 57 mL of water, 283.5 mL (557 mmol, 4.3 equivalents) of methanolic hydrochloric acid as described above was added and the mixture was stirred at 35 to 36 DEG C for 22 hours. Then dilute with 50 ml of water. The mixture is adjusted to pH 8 with 47.5 mL (704 moles) of concentrated ammonium hydroxide. This residue was dissolved in 100 ml of water and the resulting solution was concentrated to give 71 mg of residue. This residue was taken up in 190 ml of water and adsorbed onto a column of 2.0 L of Amberlite CG-50 (NH ₄ ⁺ ) and eluted by fractionation with 5.7 L of water and 5.7 L of 1.5N ammonium hydroxide. (1 fraction: 20 ml) From 321 to 403, fractions were concentrated under reduced pressure, treated with 5 g of activated carbon, filtered, and the filtrate was evaporated under reduced pressure. 16.6 g of 1-N- (3-hydroxyazetidine-3- Carbonyl) canamycin A was obtained. Yield 87.8%.

(4) A solution of 250 ml of water (16.6 g; 28.44 mmol) in water was adjusted to pH 4.6 with 102.5 ml of 1N sulfuric acid, concentrated to a concentration of about 60 to 70 ml, mixed with 700 ml of ethanol, and then iced. Cool. The resulting colorless precipitate is collected by filtration, washed with ethanol and dissolved in 300 ml of water. The solution is evaporated to dryness under reduced pressure. The residue was taken up in 200 ml of water, treated with 5 gf of activated carbon and filtered through a Pyrex filter. This filtrate was concentrated under reduced pressure. The residue was dried over phosphorus pentoxide, milled, and then absorbed by a desiccator (57% humidity) with a saturated aqueous sodium bromide solution to absorb 22.85 g of 1-N- (3-hydroxyazetidine-3-carbonyl). Kanamycin A.2H ₂ SO ₄ .7H ₂ O is obtained. Yield 77.8%

Elemental analysis: C ₂₂ H ₄₁ N ₅ O ₁₃ ㆍ 2H ₂ SO ₄ ㆍ 7H ₂ O

Calculated (%): C, 29.17; H, 6.56; N, 7.73; S, 7.08

Found (%): C, 29.27; H, 6.57 N, 7.57; S, 7.02

Example 3

Preparation of 1-N- (3-hydroxyazetidine-3-carbonyl) canamycin B sulfate

163.3 mg (0.65 mmol) of a solution of 307 mL (0.5 mmol) of 3,2 ', 6', 3'-tetra-formylcanamycin B dissolved in a mixture of 1 mL of water and 6 mL of dimethylformamide. Benzyloxycarbonyl-3-hydroxyazetidine-3-carboxylic acid 75 mg (0.65 mmol) of 1-hydroxy-succinimide is added with stirring to give a uniform solution. To this is added dicyclohexylcarbodiamide (16 mg; 0.78 mmol) and the mixture is stirred at room temperature for 24 hours. The resulting dicyclohexylurea is collected by filtration and washed with 2 ml of dimethylformamide. The combined filtrate and washings were mixed with 80 ml of ethyl acetate and cooled to 0 ° C. The precipitate was collected by filtration, washed with ethyl acetate, dissolved in 10 ml of water, treated with 200 mg of activated carbon, and filtered through a Pyrex filter. The filtrate was concentrated under reduced pressure, and 45 mg of 1-N- (1-benzyloxycarbonyl-3-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3'-tetra-N- Formylcanamycin B is obtained.

This product is dissolved in 14 ml of 50% methanol and catalytically hydrogenated in the presence of 200 ml of 10% palladium / charcoal under hydrogen pressure. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. 331 mg of 1-N- (3-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3'-tetra-N-formyl Kanamycin B is obtained.

This product is dissolved in 0.8 ml of water and mixed with 5.72 ml of a mixture of concentrated hydrochloric acid (11.0 ml) and methanol (54.5 ml) and stirred at 35 to 37 ° C. for 20 hours on a water bath. The reaction mixture is diluted in 5 ml of water and neutralized with 20 ml of Amberlite 1R-45. The resin is filtered off and washed with water. The combined filtrate and washings are adsorbed onto a column of 60 ml of Amberlite CG-50 (NH ₄ ⁺ ) and eluted by fractionation with a mixture of 1 liter of water and 1 liter of aqueous ammonium hydroxide solution washed with 500 ml of water. (1 fraction; 12 ml). Collect fractions 60 to 69, treat with 100 mg of activated charcoal, and use the Pytex filter for leisure.

The filtrate is adjusted to pH 4.6 with 13.66 ml of 0.0955 N sulfuric acid and concentrated under reduced pressure to 1-2 ml. This residue is mixed with 50 ml of ethanol and cooled with ice. The precipitate is collected by filtration, washed with ethanol, dissolved in 20 ml of water and evaporated to dryness under reduced pressure. The residue is again dissolved in 10 ml of water, treated with 100 mg of activated carbon and filtered through a Pyrex filter. The filtrate was lyophilized to produce anhydride, which absorbed moisture yielding 252 mg of 1-N- (3-hydroxyazetidine-3-carbonyl) canamycin B.2.5H ₂ SO ₄ .9H ₂ O. (Total yield: 51%).

Elemental analysis: C ₂₂ H ₄₂ N ₆ O ₁₂ ㆍ 2.5H ₂ SO ₄ ㆍ 9H ₂ O

Calculated (%): C, 26.69; H, 6.26; N, 8.49; S, 8.10

Found (%): C, 26.63; H, 6.37 N, 8.28; S, 8.38

Example 4

Preparation of 1-N- (3-methylhydroxyazetidine-3-carbonyl) tobramycin sulfate

. 이것을 2㎖의 디메틸포름아미드에 용해하고 0.38㎖(10당량)의 100% 히드라진모노하이드레이트와 혼합하고 실온에서 밤새 정치한 후, 감압하 농축하고 15㎖의 앰버라이트 CG-50(NH₄ ⁺)상에 흡착시키고 물로 용출한다(1획분: 5㎖). 획분번호 11에서 50까지를 감압하 증발시키면 72㎎의 잔유물을 얻는다. 이를 메탄올, 아세톤 및 에테르의 혼합물로 처리하면 50㎎의 1-메틸-3-히드록시아제티딘-3-카르복실산히드라지드가 얻어진다. 이 생성물을 1.2㎖의 디메틸포름아미드와 0.3㎖의 디메틸설폭시드의 혼합물에 녹이고 0.24㎖의 4.25N 디옥산성 염산용액과 0.102㎖(0.756밀리몰)의 이소아밀니트리트(아질산이소아밀)과 혼합했다.To a solution of 100 mg (0.762 mmol) of 1-methyl-3-hydroxyazetidine-3-carboxylic acid in 2 ml of methanol was added 0.3 ml of concentrated sulfuric acid, and the mixture was heated for 2 hours. It was refluxed. After cooling the reaction mixture was diluted with 20 mL of water and neutralized with 25 mL of Amberlite 1R-45. The resin was filtered off and washed with water. The combined filtrate and washings were concentrated under reduced pressure to yield 138 mg of methyl-1-methyl-3-hydroxyazetidine-3carboxylate as a syrup.

. This was dissolved in 2 ml of dimethylformamide, mixed with 0.38 ml (10 equivalents) of 100% hydrazine monohydrate, left at room temperature overnight, concentrated under reduced pressure and concentrated on 15 ml of Amberlite CG-50 (NH ₄ ⁺ ). Is adsorbed and eluted with water (1 portion: 5 ml). Evaporation of fractions 11 to 50 under reduced pressure yields 72 mg of residue. Treatment with a mixture of methanol, acetone and ether yields 50 mg of 1-methyl-3-hydroxyazetidine-3-carboxylic acid hydrazide. This product was dissolved in a mixture of 1.2 ml of dimethylformamide and 0.3 ml of dimethyl sulfoxide and mixed with 0.24 ml of 4.25N dioxane hydrochloric acid solution and 0.102 ml (0.756 mmol) of isoamylnitrite (isoamyl nitrite). did.

The reaction mixture was held at −20 ° C. for 50 minutes, cooled to −60 ° C. and neutralized with 0.144 mL of triethylamine. To a mixture of 0.6 ml of dimethylformamide and 0.6 ml of dimethyl sulfoxide, 171 mg (0.287 mmol) of 3,2 ', 6', 3'-tetra-N-formyltobramycin and 0.04 ml of triethylamine were added. The dissolved solution is added to the solution. The mixture is stirred at 0 ° C. overnight. The precipitate is isolated by filtration and washed with dimethylformamide. The combined filtrate and washings are mixed with ethyl acetate. This precipitate is collected by filtration, washed with ethyl acetate, dissolved in water, treated with activated charcoal and filtered. The filtrate was concentrated under reduced pressure, and 213 mg of 1-N- (1-methyl-3-hydroxyazetidine-3-carbonyl) -3,2 ', 6', 3′-tetra-N-formyltobra Mycin was obtained as crude product.

The product was dissolved in 0.5 ml of water, mixed with 2.85 ml of a mixture of concentrated hydrochloric acid (11 ml) and methanol (54.5 ml), stirred at 35 ° C. for 24 hours and neutralized with Amberlite IR-45. This resin was filtered off and the filtrate was concentrated under reduced pressure. This residue is adsorbed onto a 20 ml column of Amberlite CG-50 (NH ₄ ⁺ ) and eluted with a mixture of 500 ml of water and 500 ml of 0.4% aqueous ammonium hydroxide solution (1 fraction: 9 ml). Evaporation of fractions 80 to 104 under reduced pressure yielded 133 mg of 1-N- (1-methyl-3-hydroxyazetidine-3-carbonyl) tobramycin. The product was taken up in 3 ml of water, adjusted to pH 4.5 with 10% sulfuric acid and 0.09 N sulfuric acid and mixed with 40 ml of ethanol. The precipitate was collected by filtration, washed with ethanol, dissolved in water, treated with activated carbon and filtered. Lyophilization of the filtrate yields anhydride, which absorbs moisture until it is weighed, and 1-N- (1-methyl-3-hydroxyazetidine-3-carbonyl) tobramycin 2.5H ₂ SO _{4 2} O (176 mg) is obtained.

Elemental analysis: C ₂₃ H ₄₄ N ₆ O ₁₁ ㆍ 2.5H ₂ SO ₄ ㆍ 9H ₂ O

Calculated (%): C, 27.96; H, 6.84; N, 8.51; S, 8.11

Found (%): C, 28.00; H, 6.85 N, 8.37; S, 8.25

Example 5

Preparation of 1-N- (3-hydroxypiperidine-3-carbonyl) tobramycin sulfate

In a solution of 142 mg (0.602 mmol) of 1-benzyl-3-hydroxypiperidine-3-carboxylic acid in 2 ml of dimethylformamide, 70 mg (0.602 mmol) of N-hydroxysuccinamide and 125 Mg (0.602 mmol) of dicyclohexylcarbodiimide was added and the mixture was mixed with 300 mg (0.502 mmol) of 3,2 ', 6', 3'-tetra-N-formyltobramycin for 2 hours and room temperature Stir overnight at. The precipitated dicyclohexylurea is filtered off and washed with dimethylformamide. The combined filtrate and washings are mixed with 70 ml of ethyl acetate and left to stand for 30 minutes. The precipitate was collected by filtration, washed with ethyl acetate, dissolved in water and evaporated under reduced pressure. The residue is taken up in 1 ml of water and mixed with 5 ml of concentrated methanol hydrochloride (11: 54.5). The mixture is stirred at 35 ° C. for 24 hours and neutralized with 30 ml of Amberlite IR-45.

The resin is filtered off and washed with water. The combined filtrate and washings are evaporated under reduced pressure. The residue (430 mg) was adsorbed onto a column of 30 mL Amberlite CG-50 (NH ₄ ⁺ ) and the column was washed with 400 mL water. A fraction eluted with a mixture of 500 ml of water and 500 ml of 0.4% ammonium hydroxide (one fraction: 12 ml). Evaporation of 45 to 57 under reduced pressure yielded 322 mg of residue. This was chromatographed on a column of 16.5 g of silica gel (silica gel 60 manufactured by Merck Co., Ltd.), and isopropane was eluted with a mixture of -ammonium hydroxide-chloroform (5: 1: 1) (one portion: 10 ml). Evaporation of fractions 3 to 50 under reduced pressure yielded 175 mg of 1-N- (1-benzyl-3-hydroxypiperidine-3-carbonyl) tobramycin.

This product was dissolved in a mixture of 5 ml of water and 180 mg of methanol and catalytically hydrogenated overnight at room temperature in the presence of 180 mg of 10% palladium charcoal. This catalyst was filtered off and washed with water. The combined filtrate and washings were evaporated under reduced pressure to yield 156 mg of residue. It was adsorbed onto 20 mL of Amberlite CG-50 (NH ₄ ⁺ ) and eluted with 500 mL of water and 500 mL of 0.4N ammonium hydroxide (one fraction: 8 mL). Evaporation of fractions 54 to 61 under reduced pressure yields 118 mg of 1-N- (3-hydroxypiperidine-3-carbonyl) tobramycin.

This product was dissolved in 4 mL of water, adjusted to pH 4.6 with 0.095 N sulfuric acid and mixed with ethanol. The precipitate was collected by filtration and washed with ethanol. The powder obtained is taken up in water, treated with activated charcoal and evaporated under reduced pressure to give a residue. Dehumidification until this amount yields 121 mg of 1-N- (3-hydroxy-pyrrolidine-3-carbonyl) tobramycin 2.5H ₂ SO ₄ 9H ₂ O (yield 24% ).

Example 6

1-benzyl-3-hydroxypiperidine-3-carboxylic acid

8.13 g of 1-benzyl-3-piperidone HCl BM iseline and K. Hoffman, Helv. Chim. Prepared by the method described in Acta, 37, 138 (1954), the solution was dissolved in 28.8 ml of a mixture of tetrahydrofuran-water (1: 1) and 2.99 ml (36 mmol) of concentrated hydrochloric acid. While maintaining, add 4.687 g (72 mmol) of potassium cyanide and stir the mixture for 4 hours, further mix 1.495 mL (18 mmol) of hydrochloric acid with 1.172 g (18 mmol) of potassium cyanide and stir for 1 hour, then ether Extracted. The extract was washed with water, dehydrated on magnesium sulfate and evaporated under reduced pressure. The oily residue was taken up in 14.4 ml of concentrated hydrochloric acid, heated at 80 ° C. for 1 hour, then cooled and filtered. This residue was washed with acetone. The combined filtrate and washings were evaporated under reduced pressure to give a syrupy residue, which was adjusted to pH 8 with 0.25 N aqueous ammonium hydroxide solution and washed twice with chloroform and then on 200 mL of Amberlite IR-120B (H ⁺ ) column. Was adsorbed slowly. The column was washed with 1 L of water and the eluted solution of 1.28 L of 5N aqueous ammonium hydroxide solution was evaporated under reduced pressure. Crystallization of the residue with acetone gave 1-benzyl-3-hydroxy-piperidine-3-carboxylic acid 1.3H ₂ O (melting point 103.5-104.5 ° C.) in prism. Yield 60.2%. Recrystallization of this compound with dimethylformamide gave a pure product (melting point 104-105 ° C), which was dried at 70 ° C for 48 hours on phosphorus pentachloride to give the corresponding anhydride (melting point 212-214 ° C).

Elemental Analysis: C ₁₃ H ₁₇ NO ₃

Calculated (%): C, 66.36; H, 7.28; N, 5.95

Found (%): C, 66.44; H, 7.32 N, 5.94

Claims (1)

A functional group other than the position 1 is optionally protected, and then reacted with the aminoglycoside of formula (II) and the carboxylic acid of the following formula (III) or a reactive derivative thereof and removing the functional group. Method for preparing glycoside derivatives and salts thereof.

Wherein R is hydrogen, lower alkyl, aralkyl or aralkoxycarbonyl group and n is an integer of 1 or 3. R ¹ is an aminomethyl, hydroxymethyl, 1-aminoethyl or 1-methylaminoethyl group, R ² , R ³ and R ⁶ are each hydrogen or a hydroxy group, R ⁴ is a hydroxy or amino group, and R ⁵ is amino Or a methylamino group, R ⁷ is a hydroxy or methyl group, R ⁸ is a hydrogen, hydroxymethyl, or carbamoyloxymethyl group, and the dotted line indicates the presence or absence of a double bond.