CH621558A5 - Process for the preparation of kanamycin derivatives with antibacterial activity - Google Patents

Description

The invention relates to a process for the preparation of semisynthetic derivatives of kanamycin A and B, these compounds being lN- [L - (-) - β-amino-a-hydroxypropionyl] -ó'-N-methylkanamycin A or B, lN- [ L - (-) - v-amino-a-hydroxybutyryl] -6'-N-methylkanamycin A or B and 1-N- [L - (-) - ò-amino-a-hydroxyvaleryl] -6'-N- be designated methylkanamycin A or B and the general formula IV

(IV)

621 558

correspond, in which R3 is —OH or - NH2 and R has the meanings L - (-) - y-amino-a-hydroxybutyryl, L - (-) - ß-amino-a-hydroxypropionyl or L - (-) - ò -Amino-a-hydroxy-valeryl; as well as pharmaceutically acceptable, non-toxic acid addition salts thereof.

It is known that some of the R-factor-mediated kanamycin-resistant organisms, for example E. coli K-12 R-5 and Ps. Aeruginosa GN 315, inactivate the kanamycins by 6 -N-acetylation. It is therefore an object of the present invention to produce derivatives of kanamycin A and B which are resistant to this type of enzymatic inactivation by these kanamycin A or B resistant organisms, but still retain the majority of their biological activity and spectra.

Most preferred are embodiments that result in compounds of Formula IV, wherein:

1. R is L - (-) - 7-amino-a-hydroxybutyryl and R3 is OH (IVa);

2. R is L - (-) - Y-amino-a-hydroxybutyryl and R3 is NH2 (IVb);

3. R is L - (-) - ß-amino-a-hydroxypropionyl and R3 is OH (IVc);

4. R is L - (-) - ß-amino-a-hydroxypropionyl and R3 is NH2 (IVd);

5. R is L - (-) - ò-amino-a-hydroxyvaleryl and R3 is OH (IVe); and

6. R is L - (-) - ô-amino-a-hydroxyvaleryl and R3 is NH2 (IVf); or a non-toxic pharmaceutically acceptable

acid addition salt thereof.

In the context of the present disclosure, the term “non-toxic, pharmaceutically acceptable acid addition salt” means a mono-, di-, tri- or tetra-salt which can be obtained by reacting 1 mol of compound IV with 1 to 4 mol of a non-toxic, pharmaceutically acceptable one Acid is formed. These acids include acetic acid, hydrochloric acid, sulfuric acid, maleic acid, phosphoric acid, nitric acid, hydrobromic acid, ascorbic acid, malic acid and citric acid, and all other acids commonly used to form amine-containing pharmaceuticals.

Other, most preferred embodiments result in sulfate, hydrochloride, acetate, maleate, citrate, Ascor-20 bat, nitrate or phosphate salts and in particular the monosulfate salt or the disulfate salt of compound IV.

The object on which the present invention is based is achieved by the inventive method of producing the compound of formula IV

in which R stands for L - (-) - y-amino-a-hydroxybutyryl, L - (-) - ß-amino-a-hydroxypropionyl or L - (-) - ô-amino-a-hydroxy-45 valeryl and in which R3 is the Meaning —OH or - NH2

has, solved, which is characterized in that in successive stages:

A) the compound of formula II

wherein R3 is —OH or —NH2, with an acylating agent of formula VII

621 558

OH O

I II

L - (-) - W-NH- (CH2) n-CH-C-M (VII)

where W stands for a residue which is selected from:

-

<rysn2-o-l-, ch3-c

2nd

M represents a residue selected from

V

-o-n

, -O

, 0. _N0? .

, loi o

and however preferred or

-O-

where n represents an integer from 1 to 3 in a ratio of ethyl ether, dioxane, dimethylacetamide, dimethylformamide,

nis from 0.5 mol to 1.4 mol of compound VII per mol of 6s tetrahydrofuran and propylene glycol dimethyl ether, however

Compound II, but preferably in a ratio of 0.8, preferably aqueous, tetrahydrofuran, the to 1.1 in a solvent, which is preferably selected, corresponding compound of the formula III being mixed with water and ethylene glycol dimethyl

621558

8th

arises in which n, R3 and W have the above meanings; and

B) the blocking group W from compound III preferably by methods known in the art, and preferably in the case where W is a radical of the formula

20th

qm = h2-o -'-

30th

represents, by hydrogenating the compound III with hydrogen in the presence of a metal catalyst which is preferably selected from palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, but preferably palladium and most preferably palladium-on-activated carbon, in one of water and a water-miscible solvent existing solvent system, which is preferably selected from water and dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, propylene glycol dimethyl ether or the like, but preferably 1: 1 water dioxane and preferably in the presence of a catalytic amount of glacial acetic acid, the compound being removed Formula IV is formed.

A preferred embodiment of the process for the preparation of the compounds of the formula IV is characterized by the following stages:

A) acylating the compound of formula II with an acylating agent of formula or

î î

fyV0-LH- (CH2) n-C — C - 0 -

(VII a)

? ? "fi fi \ _jCH2-0-C-NH- (CH2 ^ -C-C-0-

(VII b)

wherein N represents an integer from 1 to 3, in a solvent system of aqueous tetrahydrofuran or aqueous dimethylformamide acetone, at about room temperature, the compound of formula III

9

621558

is formed, wherein n represents an integer from 1 to 3; and

B) Hydrogenating Compound III in aqueous tetrahydrofuran in the presence of palladium on activated carbon at about atmospheric pressure to form Compound IV.

Alternatively, in step A) the acylating agent VII can be mixed in situ by mixing the compound of the formula

* ?H "

/ '\ yxii2-0-c-nh- (ch2) n-c-c-oh

with equimolar amounts of N-hydroxy-5-norbornene-2,3-dicarboximide and dicyclohexylcarbodiimide in a small amount of anhydrous tetrahydrofuran. The resulting mixture is filtered and the filtrate is added to an aqueous tetrahydrofuran mixture of 6-N-kanamycin A or B, the corresponding compound III being formed.

Compounds IV are valuable as antibacterial agents, as co-feed in animal feed, as therapeutic agents in poultry and animals, including humans, and they are particularly valuable in the treatment of infectious

30th

45

50

(ch)

I 2 "

nh I

00

1

O

O

Diseases caused by gram-positive and gram-negative bacteria.

When administered orally, the compounds IV are useful as additional treatment for preoperative sterilization of the intestine. Both the aerobic and anaerobic flora, which are sensitive to these pharmaceuticals, are reduced in the large intestine. In conjunction with suitable mechanical cleaning, they are useful in the preparation of colon operations.

Compounds IV are useful in the treatment of systemic bacterial infections when administered parenterally in the dose range of about 250 mg to about 3000 mg per day in divided doses three or four times a day. In general, the compounds are effective when administered at a dose of approximately 5.0 to 7.5 mg / kg body weight every 12 hours.

The compounds of the type of compound IV, in particular BB-K28, 142, 148, 162 and 163, all have significantly improved activities against a larger spectrum of microorganisms compared to the parent compounds from which they are derived, i.e. H. Kanamycin A and B.

Table I is shown below, which shows the minimum inhibitory concentrations (MIC) of kanamycin A and B and BB-K28, 142, 148, 162 and 163 against a variety of gram-positive and gram-negative bacteria, as determined by the Steers agar -Dilution method on Mueller-Hinton agar medium were obtained.

Table I (MIC y / ml)

organism

BB-K

BB-K

BB-K

BB-K

BB-K

Kanamycin

Kanamycin

28

142

148

162

163

A

B

Ec-LE. coli NIHJ

0.4

0.4

3.1

6.3

6.3

0.4

1.6

Ec-3 E. coli Juhl

A15 119

0.8

0.8

3.1

6.3

12.5

0.8

1.6

Ec-5 E. K-12 ML-1630

A20 363

0.8

1.6

3.1

12.5

12.5

> 100

> 100

Ec-7 E. (KM-R)

A20365

0.2

0.2

0.2

3.1

6.3

6.3

100

Ec-8 E. K-12

A 9 632

0.4

0.4

0.4

6.3

12.5

0.4

1.6

Ec-9 E. NR79 / W677

A20 664

0.4

0.4

0.4

6.3

6.3

3.1

6.3

Ec-10 E. JR35 / 0600

A20 665

0.4

0.4

0.4

3.1

6.3

50

100

621 558

10th

Table I (continued) (MIC y / ml)

organism

BB-K 28

BB-K 142

BB-K 148

BB-K 162

BB-K 163

Kanamycin A

Kanamycin B

Ec-52 E. W677

A20 684

0.4

0.8

0.4

3.1

6.3

0.8

1.6

Ec-53 E. JR66 / W677

A20 683

0.8

6.3

12.5

50

100

100

> 100

Ec-59 E.

A20 766

0.8

6.3

12.5

50

100

> 100

> 100

Ec-60 E.:

A20 898

6.3

50

> 50

100

> 100

> 100

> 100

Ec-55K-12CS2R-5

*

*

3.1

3.1

Kp-1 K. pneumoniae D-ll

0.4

0.2

0.8

0.8

1.6

0.2

1.6

Kp-8 K. Type 22

A20 680

1.6

12.5

25th

12.5

50

> 100

> 100

Sm-1 Ser. marcescens

A20 019

0.8

1.6

1.6

6.3

6.3

1.6

3.1

Pa-1 Ps. Aeruginosa D-15

3.1

3.1

3.1

6.3

12.5

12.5

25th

Pa-3 ps.

A 9 930

0.2

0.4

0.4

3.1

3.1

6.3

25th

Pa-4 ps.

H-9

25th

100

> 50

100

> 100

> 100

> 100

Pa-5 ps.

A15 150

6.3

12.5

6.3

12.5

12.5

12.5

50

Pa-15 Ps. (GM-R)

A20 717

6.3

25th

12.5

25th

25th

100

100

Pa-16 Ps. (GM-R)

A20 718

3.1

12.5

6.3

25th

50

25th

50

Pa-20 ps.

A20 325

3.1

12.5

12.5

6.3

12.5

25th

50

Pa-21 Ps.

A20 601

12.5

12.5

12.5

25th

50

50

100

Pa-23 ps.

A20 741

25th

50

50

50

100

> 100

> 100

Px-10 ps.

A20621

100

100>

> 50

> 100

> 100

Pv-L PR. vulgaris

A 9 436

0.2

0.4

0.8

0.4

3.1

Pm-1 Pr. Mirabilis

A 9 554

0.8

1.6

1.6

3.1

3.1

0.8

3.1

Pg-1 Pr. Morganii

A 9 553

0.8

0.8

1.6

6.3

6.3

0.8

3.1

Sa-2 S. aureus Smith

A15 167

0.2

0.4

0.1

1.6

1.6

0.2

0.4

Sa-4 S. FDA 209P

0.8

1.6

0.8

12.5

25th

0.8

1.6

Sa-10S. (KM-R)

A20 239

1.6

1.6

1.6

12.5

12.5

50

50

Sa-49 S.

A20 240

3.1

6.3

12.5

50

50

> 100

100

Bs-1 B Subtilis PC1-219

<

0.05

<0.05

<0.025

0.4

0.8

<0.05

0.2

M6-1 Mycob. 607

0.8

0.8

0.8

3.1

3.1

0.4

3.1

M6-2 Mycob. 607 (KM-R)

> 100

100

> 50

> 100

> 100

> 100

> 100

M6-3 Mycob. 607 (KM, SM-R)> 100

100

> 50

> 100

> 100

> 100

> 100

Mp-1 Mycob. phlei

0.2

0.4

0.4

1.6

1.6

0.8

3.1

Mr-1 Mycob. ranae

0.8

0.8

0.8

3.1

3.1

0.8

1.6

After extensive experience with kanamycin A and B and its chemical reactivity, it was expected that of the four or five primary amine functions present in kanamycin A and B, the ó'-amino function is the most reactive for steric reasons. It was therefore postulated that if the 6-amino function was converted to a 6 -N-methyl-sec-amine, its reactivity with an acylating agent would be significantly reduced, and that the 1-N-primary amine function for steric reasons would be the most reactive with the remaining primary amines. Therefore, the 6'-N-methylkanamycin A or B was acylated directly with a suitable side chain acylating agent without first blocking the other amine functions of the molecule. Control of the molar amounts of acylating agent used determines the degree of acylation that occurs at other positions.

Starting materials Preparation of L - (-) - 7-benzyloxycarbonylamino-a-hydroxybutyric acid (VI) 1. L- (-) - 7-amino-a-hydroxybutyric acid (7.4 g, 0.062 mol) becomes a solution of 5. 2 g (0.13 mol) of sodium hydroxide in 50 ml of water were added. 11.7 g (0.068 mol) of carbobenzoxychloride are added dropwise to the stirred solution at 0 to 5.degree. C. over the course of 0.5 hours and stirring of the mixture is continued for one hour at the same temperature. The reaction mixture is washed with 50 ml of ether,

adjusted to pH 2 with dilute hydrochloric acid and extracted with four 80 ml portions of ether. The ether extracts are combined, washed with a small amount of saturated sodium chloride solution, dried with anhydrous sodium sulfate and filtered. The filtrate is evaporated in vacuo and the residue obtained is crystallized from benzene, whereby 11.6 g (74%) of colorless plates with melting point 78.5 to 79.5 ° C, [cc] d -4.5 ° (c = 2, CHîOH).

50 Infrared spectrum (IR) [KBr]: IR (KBr) vc = o 1740.1690 cm-1. Nuclear Magnetic Resonance (NMR) (acetone-dö) ô (in ppm from TMS) 2.0 (2H, m), 3.29 (2H, d-d, J = 6.7 and 12 Hz). 4.16 (1H, d-d, J = 4.5 and 8 Hz), 4.99 (2H, s), 6.2 (2H, broad), 7.21 (5H, s).

55

Analysis for C12H15NO5:

C H N

60 Calculated: 56.91% 5.97% 5.53%

Found: 56.66% 5.97% 5.47%

2. N-hydroxysuccinimide ester of L - (-) - y ~ benzyloxycarbo-65 nyl-amino-a-hydroxybutyric acid (VII).

A solution of 10.6 g (0.042 mol) of compound VI and 4.8 g (0.042 mol) of N-hydroxysuccinimide * in 200 ml of ethyl acetate is cooled to 0 ° C. and then 8.6 g (0.042 mol)

11

621 558

Dicyclohexylcarbodiimide added. The mixture is kept in a refrigerator overnight. The dicyclohexyl urea that has separated is filtered off and the filtrate is concentrated to about 50 ml under reduced pressure to give colorless crystals of compound VII, which are collected by filtration; 6.4 g, melting point 121 to 122.5 ° C. The filtrate is evaporated to dryness in vacuo and the crystalline residue is washed with 20 ml of a mixture of benzene / n-hexane, an additional amount of compound VII being obtained. The overall yield is 13.4 g (92%).

[cc] d 1.5 ° (c = 2, CHCb).

IR (KBr) vc = o 1810.1755, 1740.1680 cm "1.

NMR (acetone-dö) ò (in ppm from TMS) 2.0 (2H, m), 2.83

(4H, s), 3.37 (2H, d-d, J = 6.5 and 12.5 Hz), 4.56 (1H, m),

4.99 (2H, s), 6.3 (2H, broad), 7.23 (4H, s).

Analysis for CióHisNzO ?:

C H N

Calculated: 54.85% 5.18% 8.00%

Found: 54.79% 5.21% 8.14%

54.70% 5.20% 8.12%

3. Preparation of N- (benzyloxycarbonyloxy) succinimide

N-hydroxysuccinimide ** (23 g, 0.2 mol) is in one

Solution of 9 g (0.22 mol) of sodium hydroxide dissolved in 200 ml of water. To the stirred solution is added dropwise 34 g (0.2 mol) of carbobenzoxy chloride under water cooling, and then the mixture is stirred overnight at room temperature to separate the carbobenzoxy derivative, which is collected by filtration, washed with water and air-dried. The yield is 41.1 g (82%). Recrystallization from benzene / n-hexane (10: 1) gives colorless prisms that melt at 78 to 79 ° C.

4. Preparation of 6-carbobenzoxykanamycin A.

A solution of 42.5 g (90 mmol) of kanamycin A in the form of the free base in 450 ml of water and 500 ml of dimethylformamide (DMF) is cooled below 0 ° C. and stirred vigorously. A solution of 22.4 g (90 mmol) of N- (benzyloxycarbonyloxy) succinimide in 500 ml of DMF is added dropwise to the solution over the course of about 2 hours. The mixture is stirred at -10 to 0 ° C overnight and then at room temperature for one day.

The reaction mixture is evaporated under reduced pressure below about 50 ° C. The oily residue is dissolved in a mixture of 500 ml of water and 500 ml of butanol, the mixture being filtered to remove insoluble material and separated into two layers. The butanol and the aqueous layers are treated with butanol-saturated water (500 ml X 2) or water-saturated butanol (500 mlx2), using a technique which corresponds to the countercurrent distribution. The three aqueous layers are combined and evaporated to dryness under reduced pressure to give an oily residue, part of which crystallizes on standing at room temperature. About 100 ml of methanol, which dissolves the oil, is added to the residue, including the crystals, and separated from the crystals. After adding approximately 300 ml of ethanol, the mixture is kept at room temperature overnight to give a crystalline mass which is collected by filtration. It weighs 44 g. The product contains a small amount of kanamycin A, as described by thin layer chromatography using

** G.W. Anderson et al., J. Am. Chem. Soc., 86, 1839 (1964).

n-propanol / pyridine / acetic acid / water (15: 10: 3: 12) as solvent system and ninhydrin as spray reagent.

The crude product is dissolved in 300 ml of water and chromatographed on a column (30 mm in diameter) with CG-50 ion exchange resin (NH4 + type, 500 ml). The column is rinsed with 0.1N ammonium hydroxide solution and the eluate is collected in 10 ml fractions. The desired product is contained in tubes # 10-100, while the kanamycin A is obtained from the slower moving fractions and the positional isomer (s) of the product are evidently contained in the faster moving fractions. Fractions 10-110 are combined and evaporated to dryness under reduced pressure to give 24.6 g (45%) of a colorless product, 6-carbobenzoxy-kanamycin A (II) [6'-Cbz-kanamycin A], which begins to melt at 204 ° C with discoloration and decomposes at 212 ° C with gas evolution.

[<x] D + 106 ° (c = 2, H2O).

TLC (thin layer chromatography) Rf value (silica gel F254; ninhydrin)

Solvent system 6'-Cbz-Kanamycin A Kanamycin A

n-PrOH-pyridine-AcOH-HzO 0.42 * 0.33 ** 0.15 ** 0.04

(15: 10: 3: 12)

Acetone-AcOH-H20

(20: 6: 74) 0.24 0.14

CHCb-MeOH-c •

NH4OH-H2O

(1: 4: 2: 1) 0.76 0.50

AcOMe-n-PrOH-c • NHtOH

(45: 105: 60) 0.22 *** 0.04 ***

* mainly

** fewer

*** discovered by anthrone-sulfuric acid

Thin-layer chromatography (TLC) with one of the solvent systems investigated shows that the end product is accompanied by two minor components. However, the final product is used to prepare Compound II without further purification.

5. Preparation of L - (-) - 7-amino-a-hydroxybutyric acid from ambutyrosine A or B or mixtures thereof.

Ambutyrosin A (5.0 g) [U.S. Patent 3,541,078, issued November 17, 1970] is refluxed with 160 ml of 0.5 N sodium hydroxide for 1 hour. The hydrolyzate is neutralized with 6N HCl and chromatographed on a column with CG-50 (NH4 + type). The desired L - (-) - 7-amino-a-hydroxybutyric acid is isolated by developing the column with water and removing the water by freeze-drying. The L - (-) - Y-amino-a-hydroxybutyric acid is characterized as a crystalline material with a melting point of 212.5 to 214.5 ° C [column 2, lines 31-38, US Pat. No. 3,541,078].

6. Preparation of 6-carbobenzoxykanamycin B.

To a cooled solution of 8.1 g (0.0168 mol) of kanamycin B in 120 ml of water and 80 ml of 1,2-dimethoxyethane, a solution of 4.2 g (0.0168 mol) of N- ( Benzyloxycarbonyloxy) succinimide in 40 ml 1,2-dimethoxethane. The reaction mixture is stirred overnight and evaporated under reduced pressure. The residue is dissolved in 100 ml of water and shaken twice with 50 ml of water-saturated N-butanol. The aqueous layer is separated off and adsorbed on a column with 100 ml CG-50 (NH4 + type). The column is washed with 200 ml of water, eluted with 0.05N NH4OH. The eluate is collected in 10 ml fractions. Fractions 121 to 180 are collected, evaporated and freeze-dried to give 1.58 g (15%) of the desired product. The fraction

-5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

621558

12th

NEN 1 to 120 are evaporated and chromatographed again on CG-50 (NH4 +), giving 1.21 g (12%) of the product. Melting point = 151 to 152 ° C (decomposition).

[oc] d24 + 104 ° (c = 2.5, H20) -vc = o 1710 cm-1.

Analysis for C26H43N5O12:

C H N

Calculated: 50.56% 7.02% 11.34%

Found: 50.71% 7.38% 11.48%

TLC (thin layer chromatography) (silica gel F254), Rf 0.03 in n-PrOH-pyridine-AcOH-HaO (15: 10: 3: 12). Rf 0.16 in acetone-AcOH-mO (20: 6: 74).

7. Preparation of L - (-) - y-amino-a-hydroxybutyric acid from Dl-a-hydroxy-y-phthalimidobutyric acid

A) Dehydroabietylammonium-L-cx-hydroxy-Y-phthalimido-butyrate: To a solution of 25 g (0.1 mol) of a-hydroxy-Y-phthalimidobutyric acid *** in 200 ml of ethanol is added a solution of 29 g ( 0.1 mol) of dehydroabietylamine in 130 ml of ethanol. The solution is shaken vigorously for 1 minute and left

Stand for 5 hours at room temperature; fine needles crystallize during this time. The crystals are collected by filtration, washed with 50 ml of ethanol and dried in air, giving 30.1 g (56%) of a diastereomer of the dehydro-abiethylamine salt. Melting point = 93 to 94 ° C.

[c] d + 15 ° (c = 2.5; MeOH).

Recrystallization from 300 ml of ethanol gives 23.2 g (43%) of pure product with a melting point of 94 to 95 ° C.

[a] £ + 10.8 ° (c = 2.5; MeOH).

Further recrystallization does not change the melting point or the specific rotation.

Analysis for C32H42N2O5 • H2O:

C H N

Calculated: 69.54% 8.02% 5.07%

Found: 69.58% 8.08% 5.07%

B) L - (-) - Y-amino-a-hydroxybutyric acid: 5.3 g (0.01 mol) of dehydroabiethylammonium la-hydroxy are added to a solution of 1.5 g (0.014 mol) of sodium carbonate in 40 ml of water -Y-phthalimidobutyrate and 60 ml ether. The mixture is shaken vigorously until all of the solid has dissolved. The ether layer is separated. The aqueous solution is washed twice with 20 ml portions of ether and evaporated to 15 ml under reduced pressure. 10 ml of conc. Hydrochloric acid and reflux the mixture for 10 hours. After cooling, the phthalic acid separated off is removed by filtration. The filtrate is evaporated under reduced pressure. The residue is dissolved in 10 ml of water and the solution is evaporated to dryness. This operation is repeated twice to remove excess hydrochloric acid. The remaining syrup is dissolved in 10 ml of water and filtered to remove a small amount of insoluble phthalic acid. The filtrate is adsorbed on a column with IR-120 (H +, 1 cmX35 cm), the column is washed with 300 ml of water and eluted with in ammonium hydroxide solution. Combine the ninhy-drin positive fractions 10 to 16 and evaporate under reduced pressure to give a syrup which gradually crystallizes. The crystals are triturated with ethanol, filtered and dried in a vacuum desiccator, giving 0.78 g (66%) of L - (-) - y-amino-a-hydroxybutyric acid with a melting point of 206 to 207 ° C.

[a] g> -29 ° (c = 2.5; H2O).

The IR spectrum is identical to that of the authentic sample obtained from ambutyrosine.

8. Preparation of L-β-benzyloxycarbonylamino-a-hydroxy-propionic acid XX

L-β-amino-a-hydroxypropionic acid * (8.2 g; 0.078 mol) is dissolved in a solution of 6.56 g (0.0164 mol) of sodium hydroxide and in 60 ml of water. 14.7 g (0.086 mol) of carbobenzoxychloride are added dropwise below 5 ° C. to the stirred solution. The mixture is stirred for 1 hour at room temperature, washed with 60 ml of ether and adjusted to pH 2 with dilute HCl. The precipitate is collected by filtration, washed with water and dried in air, giving 9.65 g (52%) of compound XX. The filtrate is extracted with five 100 ml portions of ether. The ether solution is washed with water, dried over sodium sulfate and evaporated to dryness in vacuo to give an additional 2.0 g (11%) of compound XX. A total of 11.65 g of Compound VI are crystallized from 500 ml of benzene / ethyl acetate (4: 1), with 9.36 g (50%) of pure compound XX having a melting point of 128.5 to 129.5 ° C.

Infrared (IR) (KBr): vc = o 1745.1690 cm-1.

[a] "0 + 2.9 ° (c 5.0; MeOH).

Nuclear Magnetic Resonance Spectra [NMR (DMSO-d6)]: ô (inppm) 3.05-3.45 (2H, m, CH2N), 4.05 (1H, dd, -O-CH-CO-), 5.03 (2H, s, ŒfeAr), 7.18 (IH, broad, NH), 7.36 (5H, s, RingH).

Analysis for C11H13NO5:

C H N

Calculated: 55.23% 5.48% 5.86%

Found: 55.34% 5.49% 5.87%

9. N-hydroxysuccinimide ester of L-β-benzyloxycarbonyl-amino-a-hydroxypropionic acid XX3

412 mg (2 mmol) of dicyclohexylcarbodiimide are added to a cooled and stirred solution of 478 mg (2 mmol) of compound XX and 230 mg (2 mmol) of N-hydroxysuccinimide in 10 ml of tetrahydrofuran (THF). The mixture is stirred at 0 to 5 ° C for 1 hour and at room temperature for 2 hours and then filtered to remove the N, N'-dicyclohexylurea. The filtrate containing the title product is used for the subsequent reaction without isolation.

10. Preparation of L-ö-benzyloxycarbonylamino-a-hydroxy-valeric acid XXII

580 mg (3.3 mmol) of carbobenzoxychloride are added dropwise to a stirred solution of 400 mg (3.0 mmol) of L-ô-amino-a-hydroxyvaleric acid * * and 250 mg (6.5 mmol) of sodium hydroxide in 25 ml of water over 30 minutes at 0 to 5 ° C. The mixture is stirred at 5 to 15 ° C. for 1 hour, washed with 25 ml of ether, adjusted to pH 2 with hydrochloric acid and extracted with three 30 ml portions of ether. The combined ether solution is shaken with 10 ml of a saturated sodium chloride solution, dried over anhydrous sodium sulfate and evaporated in vacuo to give crystals which are recrystallized from benzene, giving 631 mg (78%) of compound XXII with a melting point of 110 to 111 ° C. receives;

Infrared spectrum [IR (KBr)]: 3460.3350.1725.1685, 1535, 1280.730, 690 cm-i.

* K. Freudenberg, Ber., 47, 2027 (1914).

** S. Ohshiro et al., Yakugaku Zasshi, 87, 1184 (1967).

s

10th

15

20th

25th

30th

35

40

45

50

55

60

65

13

621558

Nuclear magnetic resonance spectrum [NMR (acetone-dö]: Ô (in ppm) 1.70 (4H, m), 4.14 (2H, q, J = 4.5 Hz), 4.19 (1H, m), 4 , 82 (2H, s), 6.2 (3H, broad), 725 (5H, s).

[a5 + 1.6 (c 10; MeOH).

Analysis for C13H ivNOs:

C H N

Calculated: 58.42% 6.41% 5.24%

Found: 58.36% 6.50% 5.27%

11. N-hydroxysuccinimide ester of L-ò-benzyloxycarbonyl-amino-a-hydroxyvaleric acid XXIII

412 mg (2.0 mmol) of N, N -dicyclohexylcarbodiimide are added to a stirred and cooled solution of 535 mg (2.0 mmol) of compound XXII and 230 mg (2.0 mmol) of N-hydroxysuccinimide in 55 ml of ethyl acetate (DCC). The mixture will

Stirred for 3 hours at room temperature and filtered to remove precipitated N, N'-dicyclohexylurea. The filtrate is evaporated in vacuo to give 780 mg (100%) viscous syrup XXIII.

IR (good): vc = o 1810.1785.1725 cm "1.

6'-N-Methylkanamycin A (BB-K 25) according to method A 1. A suspension of 618 mg 6-N-carbobenzoxykanamycin A (6th) is added dropwise to a suspension of 610 mg LÌAIH4 in 10 ml dry dioxane at 70 ° C -N-Cbz-kanamycin A) in 30 ml dry dioxane. The mixture is stirred at 70 ° C for 20 hours and then cooled to -5 to 0 ° C. About 20 ml of cold water are carefully added to the reaction mixture. After the addition has ended, the mixture is neutralized with 2N HCl and evaporated to dryness under reduced pressure. The residue is washed with a large amount of EtOH to give 536 mg of the crude product, which is dissolved in a small amount of water and chromatographed on a column of CG-50 ion exchange resin (NHt + type; 20 ml). The column is rinsed with water, 1 liter of 0.1 N NH4OH, 600 ml of 0.2 N NH4OH and finally 500 ml of 0.5 N NH4OH. Collect 10 ml fractions and subject them to the ninhydrin spot test, the disk test (B. subtilis PCI 219) and TLC (thin layer chromatography) on a silica gel plate; Solvent system, S-110 (CHCL3-MeOH-28% NH4OH-H2O = 1: 4: 2: 1). The factions

which, according to TLC, give a ninhydrin-positive and bioactive spot at RF 0.50, are combined and evaporated to dryness under reduced pressure, 137 mg (27%) BB-K 25 having a melting point of 183 to 187 ° C. (decomposition) receives.

NMR (D20): ô (ppm), 2.51 (3 H, s, 6'-N-CH3), 4.95 (1H, d,

4 Hz, 1 "-H), 5.01 (1H, d, 3 Hz, 1'-H).

Analysis for C19H38N4O11 • H2CO3:

C H N

Calculated: 42.85% 7.19% 9.99%

Found: 42.97% 7.27% 9.63%

2. A suspension of 9.0 g of 6'-N-Cbz-Kanamycin A in 200 ml of dry dioxane is added to a suspension of 8.8 g of LÌAIH4 in 100 ml of dry dioxane and the reaction mixture is stirred at 80 ° C. for 4 days. The reaction mixture is cooled to 10 ° C., carefully treated with 200 ml of water and filtered to remove insoluble material. The filtrate is neutralized with n HCl and evaporated in vacuo. The residue is washed several times with EtOH and dissolved in a small amount of water. The aqueous solution is chromatographed on a column with CG-50 (NH4 +; 200 ml), which is washed with 100 ml of water and eluted successively with 2.0 liters of 0.1N NH4OH and 1.5 liters of 0.2N NH4OH becomes. The eluate is collected in 20 ml fractions. Fractions 119 to 144 show a bioactive and ninhydrin-positive spot at Rf 0.45 in the thin-layer chromatogram (silica gel plate; CHCb-MeOH-28% NH4OH-H2O = 1: 4: 2: 1). They are combined, concentrated under reduced pressure and finally lyophilized, giving 1.139 g (16%) BB-K 25, which is identical to the product prepared according to method A- (l).

6'-N-Methylkanamycin A (BB-K 25) according to method B 7 ml trimethylchlorosilane and 14 ml hexamethyldisilazane at 70 are added to a stirred suspension of 618 mg (1 mmol) of 6-N-Cbz-kanamycin in 30 ml dry pyridine ° C too. The reaction mixture is stirred at the same temperature overnight and evaporated in vacuo. The residue is treated with dry tetrahydrofuran (THF). The insoluble material is filtered and washed with dry THF. The filtrate and washes are combined and evaporated in vacuo to give 1.567 g of the trimethylsilylated product which is dissolved in 30 ml of dry THF. The solution is added to a suspension of 758 mg of lithium aluminum hydride in 70 ml of dry THF. The mixture is refluxed for 22 hours with stirring. After cooling to about 0 ° C, the reaction mixture is carefully treated with 20 ml of ice water and filtered to remove insoluble material. The filtrate is neutralized with 6N HCl and evaporated to dryness in vacuo. The residue is washed several times with ethanol, dissolved in a small amount of water and chromatographed on a column with CG-50 (NH4 +; 50 ml). The column is washed with 50 ml of water and eluted successively with 1 liter of 0.1N NH4OH and 600 ml of 0.2N NH4OH. The eluate is collected in 20 ml fractions and monitored as described in method A (l). Fractions 47 to 72, which give a ninhydrin-positive and bioactive spot at Rf 0.50 according to TLC, are combined, evaporated under reduced pressure and lyophilized, giving 258 mg (52% from 6'-Cbz-Kanamycin A) BB -K 25 with melting point 183 to 187 ° C.

example 1

l-N- (L - (-) - Y-amino-a-hydroxybutyryl) -6'-N-methyl-kanamycin A (BB-K 28)

525 mg of N-hydroxy-succinimide ester of N-Cbz-L-7-amino-a are added to a solution of 750 mg of 6'-N-methyl-kanamycin A (BB-K 25) in 30 ml of 60% aqueous THF -hydroxybutyric acid. The reaction mixture is hydrogenated at room temperature overnight under atmospheric pressure in the presence of 500 mg of 10% palladium-on-carbon. The reaction mixture is filtered and evaporated under reduced pressure. The residue is dissolved in a small amount of water and adsorbed on a column with CG-50 (NH4 +; 70 ml). The column is washed with water and successively with 850 ml 0.1 N ammonia (tubes Nos. 1 to 43 were collected in 20 ml fractions), 1450 ml 0.2 N ammonia (tubes Nos. 44 to 115 in 20 ml fractions). Fraction) and finally 100 ml of 0.5N ammonia (tubes No. 116 to 215 in 10 ml fraction). Fractions 152 to 161, which show a bioactive and ninhydrin-positive spot at Rf 0.17 according to TLC (silica gel; CHCb-CH30H-28% NH4OH-H2O = 1: 4: 2: 1), are combined under reduced pressure s

10th

15

20th

25th

30th

35

40

45

50

55

60

65

621 558

evaporated and lyophilized to give 149 mg (16%) BB-K28 with melting point 187 to 189 ° C (decomposition).

Infrared [IR (KBr)]: vc = o 1650 cm-1.

NMR (D2O): 2.70 ppm (3H, s, N-CHs).

Analysis for: C23H45N5O13 • 2H2CO3 • 2H2O:

C H N

Calculated: 39.52% 7.03% 9.23%

Found: 39.26% 6.69% 9.69%

39.00% 6.54% 9.20%

To remove a trace of BB-K 11 isomer (3 "-N-acylated isomer), the BB-K 28 is subjected to column chromatography using a tetramine copper (TACu) type of Amberlite CG-50 ion exchange resin. BB- K 28 (73 mg) in a small amount of water and chromatographed on a column of CG-50 (TACu type; 3 ml), washed with 20 ml of water and then with 100 ml of 0.2N NH4OH and finally with 100 ml of 1.0 n NH4OH was eluted and the eluate was collected in 7 ml fractions and monitored with the ninhydrin spot test, the disk test (B. subtilis PCI 219 and Pseudomonas aeruginosa) and TLC on silica gel (S-110; ninhydrin). Fractions 21 to 24 show a ninhydrin-positive and bioactive (against the Ps. Aeru-ginosa strain) spot at Rf 0.2. They are combined and evaporated in vacuo to give 30 mg of blue powder. The blue-colored residue ( 30 mg) is dissolved in a small amount of water and adsorbed on a column with CG-50 (NÜ4 +; 3 ml), which with 20 ml of 0.2N NH4OH and 200 ml of 0.5N NH4OH is rinsed. The eluate is collected in 7 ml fractions. Fractions 19 to 23, which show a positive ninhydrin test, are combined, evaporated in vacuo and freeze-dried, giving 20 mg of pure BB-K 28 with a melting point of 187 to 189 ° C. (decomposition).

Example 2

1-N- [L - (-) - β-amino-a-hydroxypropionyl] -6'-N-methyl-kanamycin A (BB-K 162).

A mixture of 218 mg (0.912 mmol) of N-Cbz-L-iso-serine, 163 mg (0.912 mmol) of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) and 188 mg (0.912 mmol) of DCC ( Dicyclohexylcarbodiimide) in 10 ml THF is left at 5 ° C overnight and then filtered. After the filtrate is added to a solution of 441 mg (0.892 mmol) of o'-N-methyl-kanamycin A in 20 ml of 50% aqueous THF, the mixture is stirred for 5 hours at room temperature and concentrated under reduced pressure about 2 ml. The concentrate is adsorbed on a column with Amberlite CG-50 (NH4 +; 26 ml), which is washed with 40 ml of water and eluted with 500 ml of 0.1 in NH4OH. The eluate is collected in 10 ml fractions. Fractions 11 to 16 are combined and evaporated in vacuo to give 231 mg of the N-acylated product. 175 mg (40%) of the starting material, BB-K 25, are collected from the 0.3 n NHUOH eluate.

130 mg of 10% Pd on activated carbon are added to a solution of the acyl derivative in 20 ml of 50% aqueous EtOH and the mixture is hydrogenated under normal pressure at room temperature. The catalyst is filtered off and the filtrate is evaporated to remove the organic solvent. The aqueous solution obtained is subjected to column chromatography on CG-50 (NH4 +; 25 ml). The column is eluted successively with 40 ml of water, 240 ml of 0.1N NH4OH, 500 ml of 0.2N NH4OH, 300 ml of 0.4N NH4OH and the eluate is collected in 10 ml fractions. The bioactive fractions are combined and evaporated in vacuo to give 127 mg of the crude product, which is chromatographed again on a column with CG-50 (tetramine copper type; 4 ml) and with 200 ml of 0.3N NH4OH, 300 ml of 5 n NH4OH and finally with 300 ml in NH4OH. The eluate is collected in 10 ml fractions.

14

s

10th

15

20th

25th

30th

Tube No. Eluent Amount Rf Comments

4 0.3n NH4OH 27 mg 0.33 inactive

19_24 0.5nNH40H 38 mg 0.33 inactive, probably a positional isomer

50-54 L, QnNH40H 35 mg 0.37 the desired product, BB-K162

Tubes Nos. 50 to 54 are combined and evaporated to dryness, giving 35 mg (6.8%) of the desired bioactive product BB-K 162 with a melting point of 178 to 185 ° C. (decomposition).

KBr

Vç_Ql630cm 1.

Example 3

l-N- [L - (-) - ô-Amino-a-hydroxy valeryl] -6 -N-methyl-kanamycin A (BB-K 163)

A mixture of 94 mg (0.353 mmol) of N-Cbz-L-y-amino-a-hydroxyvaleric acid, 64.5 mg (0.353 mmol)

HONB and 74.5 mg (0.353 mmol) DCC in 5 ml dry THF are left overnight at 4 ° C and filtered. The filtrate is added to a solution of 175 mg (0.351 mmol) 6'-

N-methyl-kanamycin A in 10 ml 50% aqueous THF and stir the mixture at room temperature for 5 hours. The reaction mixture is hydrogenated at room temperature in the presence of 70 mg of 10% palladium on activated carbon. The catalyst is filtered off and the filtrate is evaporated to remove the THF. The aqueous concentrate obtained is chromatographed on a column with Amberlite CG-50 (NH4 + type; 20 ml) and successively with water (50 ml), 0.1 n ss NH4OH (250 ml), 0.2 n NHtOH (450 ml ) and 0.5N NH4OH (400 ml) eluted. The eluate is collected in 10 ml fractions. The bioactive fractions are combined and evaporated to dryness, giving 40 mg of crude product which is again chromatographed on a column with CG-50 (tetramine copper type; 1.5 ml) and with 10 ml of water and 100 ml of 0.5 n NH4OH is eluted. The eluate is collected in 5 ml fractions.

50

Tube No. Eluent Amount Rf Comments

7-8 0.5n NHtOH 5 mg 0.21 inactive

9 0.5n NH4OH 9 mg 0.21.0.29 a mixture of two components

10-14 0.5n NHtOH 24 mg 0.29 the desired product BB-K 163

15

621558

Tubes Nos. 10 to 14 are combined and evaporated to dryness, giving 24 mg (11%) of the desired bioactive product, BB-K 163 with a melting point of 167 to 174 ° C. (decomposition).

KBr v ^ _Ql620cm 1.

6'-N-Methyl-kanamycin B, BB-K 140 20 ml of trimethylsilyl chloride and 50 ml of hexamethyldisilazane are added to a suspension of 2.0 g (3.25 mmol) of 6-N-Cbz-kanamycin B in 100 ml of dry pyridine at 70 ° C with stirring and the mixture is stirred at the same temperature overnight. The precipitate is removed by filtration and washed with dry THF. The filtrate is combined with the washing liquids and evaporated to dryness. The oily residue is dissolved in 80 ml of dry THF. The solution is added dropwise to a stirred suspension of 4.4 g of lithium aluminum hydride in 200 ml of dry THF and the mixture is heated under reflux overnight. After cooling, the reaction mixture is carefully treated with ice water, adjusted to pH 7 with 2N HCl and evaporated to dryness under reduced pressure. After thorough washing with ethanol, the residue (2.7 g) is dissolved in 5 ml of water and chromatographed on a column with Amberlite CG-50 (NHt +; 40 ml). The column is washed with 300 ml of water and eluted with 0.1N ammonia. The eluate is collected with 10 ml fractions and monitored with ninhydrin spot test, disk test (B. subtilis PCI 219) and TLC (silica gel plate, CHCl3-CHsOH-28% NH4OH-H2O = 1: 4: 2: 1). Fractions 70 to 134, which show a ninhydrin-positive and bioactive spot at Rf 0.47 according to TLC, are combined and evaporated under reduced pressure, giving 889 mg (55%) BB-K 140 with a melting point of 177 to 181 ° C. (Decomposition).

NMR (D2O): 2.74 ppm (3H, s, N-CHs).

Analysis for CiyH.wNsOio • H2CO3:

C H N

Calculated: 42.93% 7.39% 12.52%.

Found: 42.93% 7.13% 11.86%

Example 4

l-N- [L- (-) -Y-amino-a-hydroxybutyryl] -6 -N-methyl-kanamycin B (BB-K 142)

A mixture of 253 mg (1 mmol) of N-Cbz-L-7-amino-a-hydroxy-butyric acid, 115 mg (1 mmol) of N-hydroxysuccinimide (HOSu) and 206 mg (1 mmol) of DCC is mixed for 3 hours Stirred at 5 ° C and filtered to remove the precipitate that separated during the reaction. The active ester solution obtained is added to a solution of 497 mg (1 mmol) of 6 -N-methyl-kanamycin B (BB-K 140) in 10 ml of water and the mixture is stirred at room temperature overnight. The reaction mixture is filtered and evaporated in vacuo. The residue is dissolved in 5 ml of water and adsorbed on a column with CG-50 ion exchange resin (NHt + type; 45 ml), which elutes successively with 200 ml of water, 1500 ml of 0.05N ammonia and 500 ml of 0.3N ammonia becomes. The eluate is collected in 10 ml fractions. The desired intermediate derivative is contained in fractions 58 to 72, which are eluted with 0.05 N ammonia, while 163 mg (33%) BB-K 140 are obtained from the fractions eluted with 0.3 N ammonia.

A solution of the intermediate derivative in 20 ml

50% THF is hydrogenated overnight under normal pressure in the presence of 30 mg of 10% palladium on activated carbon. The reaction mixture is filtered and concentrated to approximately 4 ml. The concentrate is adsorbed on a column with Amberlite CG-50 (NHt + type; 10 ml), which is washed with 150 ml of water and successively with 350 ml of 0.05N NH4OH, 300 ml of 0.1N NHtOH, 150 ml of 3 n NH4OH and 300 ml 0.5 n NHtOH is eluted. The eluate is collected in 15 ml fractions. Fractions 67 and 68, which have a bioactive spot at Rf 0.22 according to TLC (silica gel plate; CHCb-CH30H-28% NH4OH-H2O = 1: 4: 2: 1), are combined and on a column with CG-50 (lower part of NH4 + type 1 ml; upper part of tetramine copper type 2 ml) again chromatographed. The column is eluted with 100 ml of 0.5N NHtOH and then with 50 ml of 1.0N NH4OH and the eluate is collected in 5 ml fractions. Fractions 15 to 33 are combined and evaporated in vacuo to give 29.4 mg (5%) of the desired bioactive product, BB-K 142 with a melting point of 188 to 192 ° C. (decomposition).

IR (KBr): vc = o 1640 cm "1.

Analysis for C23H46N6O12 • 2H2CO3:

C H N

Calculated: 41.55% 6.97% 11.63%

Found: 41.52% 6.72% 10.95%

A small sample of BB-K 142 is heated at 100 ° C for 1 hour in 0.5N NaOH to give 6 -N-methyl-kanamycin B and L-HABA; this is confirmed by TLC (thin layer chromatography).

Example 5

l-N- [L- (-) -ß-amino-a-hydroxypropionyl] -6 -N-methyl-kanamycin B (BB-K 148)

A stirred mixture of 120 mg (0.5 mmol) N-Cbz-L-isoserine, 58 mg (0.5 mmol) HOSu and 103 mg (0.5 mmol) DCC in 5 ml THF is at 5 ° C overnight ditched. After the mixture was filtered, the filtrate was added to a solution of 248 mg (0.5 mmol) BB-K 140 in 5 ml water and stirred overnight. After removing the THF, the aqueous solution is adsorbed on a column with Amberlite (CG-50 (NH4 + type; 10 ml). Elution is carried out with 300 ml of 0.5 N NHtOH, followed by 300 ml of 0.1 N NHtOH , and 10 ml fractions are collected The desired intermediate derivative (68 mg) is obtained by evaporating fractions 18 to 31 obtained with 0.05N NHtOH while adding 69 mg (28%) BB-K 140 from the fractions eluted with 0.1N ammonia.

A solution of the intermediate derivative in 10 ml of water is hydrogenated overnight in the presence of 20 mg of 10% Pd-C and the reaction mixture is filtered and concentrated to approximately 5 ml. The concentrate is chromatographed on a column with Amberlite CG-50 (NH4 +; 8 ml) and eluted successively with 280 ml 0.05N NHtOH, 340 ml 0.1N NH4OH and 200 ml 0.2N NH4OH. The desired bioactive component (35 mg) is obtained from the fractions eluted with 0.2 n NHtOH. The product still shows 2 to 3 ninhydrin-positive spots and is purified by repeated chromatography on a column with CG-50 (lower part NH4 + type 1 ml; upper part tetramine copper type 2 ml), which are washed in succession with 60 ml 0, 3 n NH4OH, 110 ml 0.5 n NHtOH and 100 ml 1.0 n NHtOH is eluted. The desired product, BB-K 148, is made from fractions 40

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

621558

16

to 50 obtained which were eluted with 0.1N NH4OH.

Yield 8.1 mg (3%); Melting point 190 to 198 ° C (decomposition).

s

IR (KBr): vc = o 1630 cm-1.

Example 6

l-N- [L - (-) - ó-amino-a-hydroxyvaleryl] -

6'-N-methyl-kanamycin B 10

If, in the procedure of Example 5, the 6-N-methyl-kanamycin A used there is replaced by an equimolar amount of 6-N-methyl-kanamycin B, the title product is obtained.

Amberlite CG-50 is the trade name for the chromatographic quality of a weakly acidic cation exchange resin of a carboxylic acid polymethacrylic type.

The Cupra ammonium form of Amberlite CG-50 is produced in the following way: 10% copper II-20 sulfate solution is added to a stirred suspension of CG-50 (NH4 +) in water to obtain the copper salt of CG-50 that is filtered. The resin is washed several times with water and then treated with IN NH4OH with stirring, filtered and washed several times with water to give the deep blue cupra ammonium form of CG-50. 25th

Example 7

Preparation of the monosulfate salt of l- [L - (-) - y-amino-a-hydroxybutyryl] -6 -N-methyl-kanamycin A or B.

One mole of l- [L - (-) - y-amino-a-hydroxybutyryl] -6'-N-30 methyl-kanamycin A or B is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. 1 mol of sulfuric acid, dissolved in 500 ml of water, is added to the cooled and stirred solution. The mixture is allowed to stir for 30 minutes, after which cold 35% ethanol is added to the mixture until a precipitate occurs. The

Solids are collected by filtration and it is found that it is the desired monosulfate salt.

Example 8

Preparation of the disulfate salt of l- [L - (-) - ß-amino-a-hydroxypropionylJ-ó-N-methyl-kanamycin A or B 35 g l- [L - (-) - ß-amino-a-hydroxypropionyl] -6'-N-methyl-kanamycin A or B are dissolved in 125 ml of deionized water. The pH is lowered to 7 to 7.5 with 50% v / v sulfuric acid.

8.5 g Darco G-60 (activated carbon) are added and the mixture is slurried at ambient temperature for 0.5 hours. The charcoal is removed by suitable filtration and washed with 40 ml of water. The water wash is added to the filtrate.

The above combined filtrate / washing liquid is adjusted to pH 2 to 2.6 with 50% v / v sulfuric acid. A large amount of carbon dioxide develops. The solution is left under stirring for 20 minutes in a laboratory vacuum in order to drive off additional carbon dioxide.

8.5 g Darco G-60 are added to the degassed solution. The mixture is slurried for 0.5 hours at ambient temperature. The charcoal is removed by suitable filtration and washed with 35 ml of deionized water. The washing liquid is added to the filtrate.

The combined filtrate / washing liquid is adjusted to pH 1 to 1.3 with 50% v / v sulfuric acid. This solution is added to 600 to 800 ml of methanol (3 to 4 volumes of methanol) over a period of 10 minutes with rapid stirring. The mixture is stirred for 5 minutes at pH 1 to 1.3, passed through a sieve with a mesh size of 0.148 mm (100 mesh), stirred for 2 minutes and left to sit for 5 minutes. Most of the supernatant liquid is decanted. The remaining slurry is suitably filtered, washed with 200 ml of methanol and dried in vacuo at 50 ° C for 24 hours to give the title disulfate salt.

B

Claims (4)

00 621 558 PATENT CLAIMS 1. A process for the preparation of a compound of the formula: NH-R as well as pharmaceutically acceptable acid addition salts, characterized in that successive residues in which R is L - (-) -7-amino-a -hydroxybutyryl, L - (-) - stages: ß-amino-a-hydroxypropionyl or L - (-) - ô-amino-a-hydro-A) the compound of the formula II is xyvaleryl and R3 has the meanings —OH or -NH2 where R3 is -OH or -NH2, with an acylating agent of the formula VII OH OI II L - (-) - W-NH- (CH2) "- CH-CM j (VII) wherein W represents a radical which is selected from: 1 CH- O is formed, wherein n represents an integer from 1 to 3; and B) hydrogenating the compound of formula III in aqueous tetrahydrofuran in the presence of palladium-on-carbon at about atmospheric pressure to form the compound of formula IV. 8. Application of the method according to claim 7 to acylating agents of the formula VIII, which are obtained in step A) in situ by mixing a compound of the formula with the agent. 35 o o 11 1 0 2-0-C-NH- <CH2) Tl n f - H H 2. The method according to claim 1, characterized in that that step A) with an acylating agent of the formula ss OH O I II L - (_) - W-NH- (CH2) n-CH-C-M represents and M for wherein W is a radical of the formula / ~ \ _ch2-O X or is in a molar ratio of 0.8 to 1.1 of the acylating agent of formula VII to the compound of formula II in aqueous 65 tetrahydrofuran. 3. The method according to any one of claims 1 and 2, characterized in that a protective group W is a radical of the formula 621 558 4th 4- selected and removed in stage B) by hydrogenation. 4. The method according to claim 3, characterized in that one carries out the hydrogenation with hydrogen in the presence of a metal catalyst in a solvent system consisting of water and a water-miscible solvent. 5. The method according to any one of claims 3 and 4, characterized in that the hydrogenation with hydrogen in the presence of palladium, platinum, Raney nickel, rhodium, ruthenium or nickel in a mixture of water and dioxane, tetrahydrofuran, ethylene glycol dimethyl ether or propylene glycol dimethyl ether, carries out. 6. The method according to any one of claims 3 to 5, characterized in that one carries out the hydrogenation with hydrogen in the presence of palladium on activated carbon in a water / dioxane solvent system with a volume ratio of 1: 1 and in the presence of a catalytic amount of glacial acetic acid. 7. The method according to claim 1 for the preparation of a compound of formula IV .nhcr (IV) where R is L - (-) - y-amino-a-hydroxybutyryl, L - (-) - ß-amino-a-hydroxypropionyl or L - (-) - ó-amino-a-hydroxy-valeryl and R3 is the Meanings —OH or - NH2, characterized in that in successive 35th Stages: A) the compound of formula II with a corresponding acylating agent of the formula ? OH O ^ "~ ^ -ch2-O-C-HH- (CH2) n-c — c — 0— (VII a) or ^ A-jCH2-0-C-NH- (CH2) n-C - c- o - (VII b) where n represents an integer from 1 to 3, in an aqueous tetrahydrofuran or aqueous dimethylformamide Acetone solvent system acylated at about room temperature, the corresponding compound of formula HI 621558 HC-OH i 2n NH I 3rd M represents a residue selected from 621 558 in which n represents an integer from 1 to 3, acylated in a mol of formula II from 0.5 to 1.4 in a solvent, ratio of the compound of formula VII to the compound of 30, the corresponding compound of formula III ; h2NH-CH3 receives; and B) the protective group W is removed from the compound of the formula III, a compound of the formula IV being formed. 3 days iba o o il or QJ « 4 « with equimolar amounts of N-hydroxy-5-norbornen-2,3-dicarboximide and dicyclohexylcarbodiimide as dehydrated-45