DK149775B - ANALOGY PROCEDURE FOR PREPARING 6'-N-METHYLKANAMYCINE A AND B DERIVATIVES - Google Patents

Description

149775

The present invention relates to an analogous process for the preparation of novel derivatives of kanamycin A and B of the general formula 26, CH97-HilCH3 and AJ1.

/ vrr \ 6 "o /

CH 2 OH X

h ° 41 ^^ V y / 20 (IV) wherein k represents -OH or -NH 2 and R represents L - (-) - Y-amino-α-hydroxybutyryl, L ~ (-) -β-amino-α-hydroxypropionyl or L - (-) - δ-amino-α-hydroxyvaleryl, or a pharmaceutically acceptable non-toxic salt thereof.

From the disclosure of U.S. Patent No. 3,781,268, it is known that the 1- (L - (-) - γ-amino-ct-hydroxybutyryl) derivatives of catamycin A and B exhibit antibacterial activity against a variety of Grams. positive and Gram negative bacteria, including those resistant to kanamycin A. These known compounds contain a free amino group of the 61 carbon atom. Furthermore, it is known that certain R-factor positive kanamycin-resistant organisms, e.g. E. coli K-12 R-5 and Ps. aeruginosa GN 315 inactivates the kanamycins by 6'-N-acetylation. Surprisingly, it has been found that the introduction of a methyl group on the 6 'carbon atom of formula (IV) results in a stronger and more broad spectrum antibacterial activity.

Particularly preferred compounds of formula (IV) are according to the invention those in which 1) R represents L - (-) - γ-amino-α-hydroxybutyryl and R 3 represents OH (IVa),

ISLAND

2) R represents L - (-) - & - amino-o-hydroxypropionyl and R represents OH (iVc), and 3 3) R represents L - (-) - 6-amino-o-hydroxyvaleryl and R represents OH (IVe) ).

In the present specification, the term "non-toxic pharmaceutically acceptable acid addition salt" refers to a mono-, di-, tri- or tetra-salt formed by the interaction of 1 molecule of compound IV and 1-4 moles of a non-toxic pharmaceutically acceptable acid. Included in these acids are acetic acid, hydrochloric acid, sulfuric acid, maleic acid, phosphoric acid, nitric acid, hydrobromic acid, ascorbic acid, malic acid and cintronic acid and such other acids which are usually used to make salts of amine-containing drugs.

149775 3

The process of the invention is characterized by acylating in order A) a compound of formula H0-. CH2NH-CH3 HO-V V \

CHO0H HO-A-tsA

HO - T 2 _NH 2 B 2 wherein R 3 represents -OH or NH 2 with an acylating agent of the formula OH 0

I II

W-NH- (CH 2) n-CH-CM (VII) wherein W represents a group of the formula 0 CH-0 f ~ \ II I 3II f ~ \ // New »CH 2 -O-C-, CH 3 -C- 0-C-, o2n-nV-CH X = f NO2 o ° °

I II

or the residue of an acid halide or acid anhydride, M represents a group of the formula

ISLAND

r. ° | yN ° 2, - ° -N ^, -ί-0-C-N ^ J or 0 l | \ j \ j_o- but preferably 0 CO "· .U" φ

ISLAND ISLAND

and n represents an integer of 1-3, at a ratio of at least 0.5 mol of compound VII per ml. mole of compound II, but preferably in a ratio of at least approx. 0.5 to approx. 1.4 in a solvent, preferably selected from the group comprising a mixture of water and ethylene glycol dimethyl ether, dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran and propylene glycol dimethyl ether to prepare a compound of formula 5. CH2NH-CH3 -¾ Γ · 'Wi jrz'i — r

-Q. / HC-OH

y '^ H2) n NHj HO / I * n

tr NH

mi 3 wherein n, R and W have the above meanings, and B) removes the blocking group W from compound III in a manner known per se and, if desired, converts a obtained compound of formula (IV) into a pharmaceutically acceptable acid addition salt thereof .

Under A), the most preferred ratio is approx. 0.8 to approx.

1.1 and the reaction is preferably carried out in aqueous tetrahydrofuran. Under B), the blocking group W is removed when it has the formula 0-ch 2 -o-1 by hydrogenating compound III with hydrogen in the presence of a metal catalyst preferably selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel. but especially palladium and especially preferred palladium-on-charcoal, in a water-water miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, propylene glycol dimethyl ether or the like, but preferably 1: 1 water dioxane and preferably presence of a catalytic amount of glacial acetic acid.

It will be apparent to those skilled in the art that other agents may be used in the above process for acylating the amine functions of the intermediates of the present invention. The invention encompasses the use of all acylating agents which provide labile amine blocking groups which are commonly used in the synthesis of peptides. The labile blocking groups should be easily removable in a manner known per se. Examples of such labile blocking groups and their removal can be found in the review by A. Kapoor, J. Pharm. Sciences, 59, pages 1-27 (1970). Functional equivalents as acylating agent for primary amine groups will comprise similar carboxylic acid chlorides, bromides, acid anhydrides including mixed anhydrides and especially the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carboxylic acid, of alkyl and aryl sulfonic acids and of more hindered acids. -acetic acid. In addition, an acid azide or active ester or thio ester (for example, with p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetic acid) or the free acid itself can be coupled to the kanamycin derivative (II) after prior reaction of the free acid with Ν, Ν'-dimethyl chloroforminium chloride [cf. English 1,008,170 and Novak and Weight, Experientia XXI / 6, 360 (1965)] or by the use of enzymes or of a Ν, Ν'-carbonyldiimidazole or a Ν, Ν'-carbonylditriazole [cf. Sheehan and Hess, J. Arner. Chem. Soc., 77, 1067 (1955)] or of alkynylamine reagent [cf. R. Budjile and H. G. Viehe, Angew. Chem., International Edition 3, 582 (1964)] or by a ketium imine reagent (cf. CL Stevenes and E. Monk, J. Amer. Chem. Soc. 80, 4065 (1958)] or by an isoxazolium salt reagent [cf. RB Woodward, RA Olofson and H. Mayer, J. Amer.

Chem. Soc., 83, 1010 (1961)]. Another equivalent of the acid chloride is a corresponding azolid ie. an amide of the corresponding acid whose amide nitrogen is included in a quasi-aromatic five-membered ring containing at least two nitrogen atoms, i. imidazole, pyrazole, triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general process for preparing an azolid, Ν, Ν'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar amounts at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazantite 7 practically 9775 yield during release of carbon dioxide and 1 mole of imidazole. Dicarboxylic acids provide diimidazolides. The by-product imidazole precipitates and can be separated and the imidazolid isolated, but this is not critical. These turnovers are known per se (see United States Patent Nos. 3,079,314, 3,117,126 and 3,129,224 and British Patent Nos. 932,644, 957,570, and 959,054).

The most preferred method of preparing compounds of formula IV comprises, in order A), acylating the compound of formula II with an acylating agent of formula <3 -CH 2 -O-Lnh- (CH 2> ng - * - ° - ^ X £ j) or

^ Ϊ? H? V

wherein n is an integer of 1 to 3 in an aqueous tetrahydrofuran or aqueous dimethylformamide acetone solvent system at about room temperature to prepare the compound of formula 149775 8 HO ~ "VJH2NH'CH3 - £! = Λ

^ V \ IL

Cfl, OH HO - ^ - 7 «. \

HO-NH4-C-O

V —— ϊΟν X HC-OH

røiHdW ^ <CH2, n

V III NH

C = 0 0 wherein n is an integer of 1-3 and B) hydrogenates compound III in aqueous tetrahydrofuran in the presence of palladium-on-charcoal at about atmospheric pressure to produce compound IV.

Alternatively, in step A, the acylating agent VII may be formed in situ by mixing the compound of the formula _ OH OH r ~ \ 'in ii y-CH -OC-NH- (CH2) -C-C-OH \ = / 2 2 n with equimolar amounts of N-hydroxy-5-norbone-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 derivative to prepare the appropriate compound III.

149778 9

Compounds IV are valuable as antibacterial agents, prophylactic nutritional supplements for animal feed, therapeutic agents for poultry, mammals and humans, and are particularly valuable for treating infectious diseases caused by gram-positive and gram-negative bacteria.

Compounds IV are, by oral administration, valuable for adjunctive therapy for pre-operative bowel sterilization. Both aerobic and anaerobic flora sensitive to these drugs are reduced in the colon. Accompanied by adequate mechanical cleansing, they are valuable in preparation for colon surgery.

Compounds IV are effective in treating systemic bacterial infections by parenteral administration in a dose range of about 250 mg to approx. 3,000 mg per daily in divided doses 3 to 4 times daily. In general, the compounds are effective in administering at a dose of ca. 5.0 to approx. 7.5 mg per kg body weight every 12 hours.

The compounds of formula IV, in particular BB-K28, 142, 148, 162 and 163, all possess substantially improved activities against a broader spectrum of microorganisms than the parent compounds from which they are derived, namely kanamycin A and B.

Table I below illustrates the minimum inhibitory concentrations (MIK values) of kanamycin A and B partly and BB-K28, 142, 148, 162 and 163 against a wide variety of gram-positive and gram-negative bacteria obtained by Steer's agar dilution method of Mueller-Hinton Agar Medium.

Table II (see page 14) illustrates the minimum inhibitory concentrations (MIK values) of kanamycin A, BB-K 25, BB-K 8 (according to US Patent 3,781,268) and BB-K 28 (made according to the invention) above for a variety of aminoglycoside-resistant bacteria.

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Table II

Kanamydin A and three of its derivatives against aminoglycoside-resistant organisms _MIK in u g / ml * ______

Bristol Kanamycin

Organism-Registration-No. A BB — K25 BB — K8 BB — K28 - S. marcescens - 20460. -> 125> 32 2 4 5. marcescens 21226 22.6 4 8 1 S. marcescens 21235 63 16 32 4 S. marcescens 21974 32 5, 7 22.6 2.8 S. marcescens 21975 45 8 16 5.7 S. marcescens 22288> 125> 32 16 4 S. marcescens 22302> 125> 32 32 8 5. marcescens 22382 32 16 16 8 S. marcescens 22426> 125> 32 2.8 4 S. marcescens 22478> 125> 32 4 4 E. coli 20665> 125> 32 2 4 E. coli 20732 63 32 1 1.4 E. cloacae 21006> 125> 32 4 8 E. cloacae 21136 63 16 32 5.7 K. pneumoniae 21236> 125> 32 4 5.7

Number of sensitive strains q 3 8 15

Geometric mean of the results of two separate experiments.

MIK = minimal inhibitory concentration BB-K 25 is the 6'-N-methyl derivative of kanamycin A

BB-K8 is (the commercially available aminoglycolside amicasin) 1-N- [L - (-) - γ-amino-α-hydroxybutyric acid derivative of kanamycin A BB-K 28 is 1-N- [L- ( -) - The γ-amino-α-hydroxybutyryij-6'-N-methyl derivative of kanamycin A.

Based on the considerable experience of kanamycin A and B and its chemical reactivity, it was to be expected that for the 4 or 5 primary amine functions of kanamycin A and B, respectively, for steric reasons, it would be the 6'-amino function which was most reactive. Therefore, it was believed that the reactivity of the 6'-amino function with an acylating agent would be substantially diminished by conversion of the 6'-amino function to a 6'-N-methyl secondary amine, and that the 1-N primary amine function for steric reasons would be the most responsive among the remaining primary amine groups. Therefore, 6'-N-methylkanamycin A or B was directly acylated with a suitable side chain acylating agent without first blocking the other amine functions in the molecule. Control of the molar amounts of acylating agent used determines the degree of acylation that occurs in other positions.

Table II shows that all 15 bacterial strains are resistant (MIK i. 16 pg / ml) to kanamycin A, 12 strains are resistant to BB-K 25, and 7 strains are resistant to BB-K 8, whereas none of the 15 strains are resistant to BB-K 28.

SOURCE MATERIALS.

Preparation of L - (-) - Y-benzyloxycarbonylamino-α-hydroxybutyric acid (VI). 1 L - (-) - γ-amino-α-hydroxybutyric acid (7.4 g, 0.062 mole) was added to a solution of 5.2 g (0.13 mole) of sodium hydroxide in 50 ml of water.

To the stirred solution was added dropwise at 0 - 5 ° C over a period of 0.5 hour 11.7 g (0.068 mol) of carbobenzoxychloride and the mixture was stirred for one hour at the same temperature. The reaction mixture was washed with 50 ml of ether, the pH was adjusted to 2 with dilute hydrochloric acid and extracted with four 80 ml portions of ether. The ethereal extracts were combined, washed with a small amount of saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo and the residue was crystallized from benzene to give 11.6 g (74%) of colorless plates, m.p. 78.5 - 79-5 ° C, [a] n - 4.5 ° (c = 2, CH_OH) .

υ -i -3

Infrared (IR) [KBr]: IR (KBr) γσ = ο 1740, 1690 cm. Nuclear Magnetic Resonance (NMR) (acetone-dg) δ (in ppm from TMS) 2.0 (2H, m.), 3.29 (2H, dd, J = 6.7 and 12 Hz), 4.16 (1H, dd, J = 4.5 and 8 Hz.), 4.99 (2H, s.), 6.2 (2H, broad), 7.21 (5H, s.).

15 149775

Analysis calculated for c12H15N®5 (Percent): C, 56.91; H, 5.97; N / 5.53. Found (percent): C, 56.66; Η, 5.97; N, 5.47.

2) The N-hydroxysuccinimide ester of L - (-) - γ-benzyloxycarbonylamino-α-hydroxybutyric acid (Vil). A solution of 10.6 g (0.042 mole) of VI and 4.8 g (0.042 mole) of N-hydroxysuccinimide1 in 200 ml of ethyl acetate was cooled to 0 ° C and then 8.6 g (0-042 mole) of dicyclohexylcarbodiimide was added. The mixture was kept in the refrigerator overnight. The dicyclohexylurea which separated was filtered off and the filtrate was concentrated to ca. 50 ml under reduced pressure to give colorless VII crystals which were collected by filtration; 6.4 g, mp 121 - 122.5 ° C. The filtrate was evaporated to dryness in vacuo and the crystalline residue was washed with 20 ml of a mixture of benzene and n-hexane to give an additional amount of VII. The total yield was 13.4 g (92%). [α] D = 1.5 ° (c. = 2, CHCl 3). IR (KBr) vc = 0 1810, 1755, 1740, 1680 cm cm NM. NMR (acetone-d d) δ (in ppm from TMS) 2.0 (2H, m.), 2.83 (4H, s.), 3.37 (2H , dd, J = 6.5 and 12.5 Hz.), 4.56 (1H, m.), 4.99 (2H, s.), 6.3 (2H, broad), 7.23 (4H, s.).

Analysis calculated for cigHi8N2 ° 7 (Percent): cr 54.85; H, 5.18; N, 8.00. Found (percent): C, 64.79, 54.70; H, 5.21, 5.20; N, 8.14, 8.12.

3) Preparation of N- (benzyloxycarbonyloxy) succinimide.

2 N-hydroxysuccinimide (23 g, 0.2 mol) was dissolved in a solution of 9 g (0.22 mol) sodium hydroxide in 200 ml of water. To the stirred solution was added dropwise 34 g (0.2 mole) of carbobenzoxy chloride under water cooling, and then the mixture was stirred at room temperature overnight to separate the carbobenzoxy derivative which was collected by filtration, washed with water and air dried. Yield 41.1 g (82%). Recrystallization from benzene-n-hexane (10: 1) yielded colorless prisms melting at 78 - 79 ° C.

4) Preparation of 61-carbobenzoxycanamycin A.

A solution of 42.5 g (90 mmol) of kanamycin A as free base in 450 ml of water and 500 ml of dimethylformamide (DMF) was cooled below 0 ° C

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

2 and stirred vigorously. For the solution, set aside for a period of time for approx. 2 hours a solution of 22.4 g (90 mmol) of N- (benzyl-oxycarbonyloxy) succinimide in 500 ml of DMF. The mixture was stirred at -10 ° - 0 ° C overnight and then at room temperature for one day. The reaction mixture was evaporated under reduced pressure under ca. The oily residue was dissolved in a mixture of 500 ml of water and 500 ml of butanol and the mixture was filtered to remove insoluble material and divided into two layers. The butanol and water layers were treated with butanol-saturated water (500 ml x 2) and water-saturated butanol (500 ml x 2), respectively, using a kind of countercurrent distribution technique. The three aqueous layers were combined and evaporated to dryness under reduced pressure to give an oily residue, a portion of which crystallized on standing at room temperature.

To the residue including the crystals was added approx. 100 ml of methanol which dissolved the oil and separated it from the crystals. After adding approx. 300 ml of ethanol, the mixture was kept at room temperature overnight to give a crystalline mass which was collected by filtration. The weighed 44 g. The product contained a small amount of kanamycin A as evidenced by thin layer chromatography using n-propanol-pyridine-acetic acid water (15: 10: 3: 12) as solvent system and ninhydrin as a spray reagent.

The crude product was dissolved in 300 ml of water and chromatographed on a column (30 mm diameter) of "CG-50" ion exchange resin (NH₂ + type, 500 ml). The column was rinsed with 0.1 N ammonium hydroxide solution and the eluate was collected in 10 ml fractions. The desired product was contained in glass # 10-100, while kanamycin A was recovered from the slower fractions and the position (s) of isomers of the product appear to be contained in the faster fractions. Fractions 10-110 were combined and evaporated to dryness under reduced pressure to give 24.6 g (45%) of a colorless product 6-carbobenzoxycanamycin A (II) [61-Cbz-kanamycin A] which began to melt and stain at 204 ° C and decomposed at 212 ° C during gas evolution. [α] D + 106 ° (c. = 2, H2 O).

149775 17

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The end product was found to be accompanied by two smaller components by thin layer chromatography with one of the solvent systems tested. However, the final product was used without further purification to produce Compound II.

5) Preparation of L - (-) - γ-amino-α-hydroxybutyric acid from ambufyrosine A or B or mixtures thereof.

Ambutyrosine A (5.0 g) (U.S. Patent No. 3,541,078] was refluxed with 160 ml of 0.5 N sodium hydroxide for one hour. The hydrolyzate was neutralized with 6N HCl and chromatographed on a column of "CG-50" (NH4 + type The desired L - (-) - γ-amino-α-hydroxybutyric acid was isolated by developing the column with water and removing the water by freeze-drying. The L - (-) - γ-amino-α-hydroxybutyric acid can be characterized as a crystalline material. mp 212.5-214.5 ° C [column 2, lines 31-38 of U.S. Patent No. 3,541,078].

6) Preparation of 61-carbobenzoxycanamycin 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 was added dropwise with stirring a solution of 4.2 g (0.0168 mol) of N- (benzyloxycarbonyloxy) -succinimide in 40 ml 1. 2-dimethoxyethane. The reaction mixture was stirred overnight and evaporated under reduced pressure. The residue was dissolved in 100 ml of water and quenched twice with 50 ml of water-saturated n-butanol. The aqueous layer was separated and adsorbed on a column of 100 ml "CG-50" (NH 2 t type). The column was washed with 200 ml of water, eluted with 0.05 N NH 3 OH. The eluate was collected in 10 ml fractions. Fractions 121 - 180 were collected, evaporated and lyophilized to give 1.58 g (15%) of the desired product. Fractions 1 - 120 were evaporated and re-chromatographed on "CG-50" (NH. +) To give 1.21 g (12%) of the product. Melting point 151-152 C (dec.).

[α] D24 + 104 ° (c = 2.5, H2O). yc = o 1710 cm -1.

Analysis calculated for C 26 H 43 N 5 ° 12 (Percent): C, 50.56; H, 7.02; N, 11.34.

Found (percent): C, 50.71; H, 7.38; N, 11.48.

149775 19

Thin-layer chromatography (silica gel F254), Rf 0.03 in n-PrOH-pyridine-AcOH-H2O (15: 10: 3: 12); Rf 0.16 in acetone-AcOH-H2O (20: 6: 74).

7) Preparation of L - (-) - γ-amino-g-hydroxybutyric acid from DL-g-hydroxy-Y-phthalimide butyric acid.

A) Dehydroabietylammonium L-α-hydroxy-Y-phthalimide butyrate: 3

To a solution of 25 g (0.1 mole) of α-hydroxy-γ-phthalimide butyric acid in 200 ml of ethanol was added a solution of 29 g (0.1 mole) of dehydroabiethylamine in 130 ml of ethanol. The solution was shaken vigorously for one minute and allowed to stand at room temperature for five hours, during which fine needles crystallized. The crystals were collected by filtration, washed with 50 ml of ethanol and air dried to give 30.1 g (56%) of a diastereomer of the dehydroabiethylamine salt. Melting point 93 -94 ° C [α] 20 + 15 ° (c = 2.5, MeOH). Recrystallization from 300 ml of ethanol gave 23.2 g (43%) of the pure product, m.p. 94 - 95 ° C

[α] D 24 + 10.8 ° (c = 2.5, MeOH). Further recrystallization did not change the melting point and the specific rotation.

Analysis calculated for C32H42N2 ° 5 * H2® (percent): C, 69.54; H, 8.02; N, 5.07. Found (percent): C, 69.58; H, 8.08; N, 5.07.

B) L - (-) - γ-amino-α-hydroxybutyric acid:

To a solution of 1.5 g (0.014 mol) of sodium carbonate in 40 ml of water was added 5.3 g (0.01 mol) of dehydroabiethylammonium L-α-hydroxy-γ-phthalimide butyrate and 60 ml of ether. The mixture was shaken vigorously until all the solid had dissolved. The ether layer was separated. The aqueous solution was washed twice with 20 ml portions of ether and evaporated to 15 ml under reduced pressure. To the concentrate was added 10 ml of concentrated hydrochloric acid and the mixture was refluxed for 10 hours. After cooling, separated phthalic acid was removed by filtration. The filtrate was evaporated under reduced pressure. The residue was dissolved in 10 ml of water and the solution evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The remaining syrup was dissolved in 10 ml of water and filtered to remove a small amount of insoluble phthalic acid. The filtrate was adsorbed on a column of "IR-120" (H +, 1 cm x 35 x m), the column washed with 300 ml of water and eluted with 1 N ammonium hydroxide solution. The ninhydrin-positive fractions 10 to 16 were combined and evaporated under reduced pressure to form a syrup which crystallized gradually. The crystals were triturated with ethanol, filtered and dried in a vacuum desiccator to give 0.78 g (66%) of L - (-) - γ-amino-α-hydroxybutyric acid. Melting point 206-207 ° C [α] D -29 ° (c = 2.5, H 2 O). The IR spectrum was identical to that of an authentic sample obtained from ambutyrosine.

8) Preparation of L-g-benzyloxycarbonylamino-ct-hydroxypropionic acid XX.

UF-β-amino-α-hydroxypropionic acid (8.2 g, 0.078 mol) was dissolved in a solution of 6.56 g (0.0164 mol) of sodium hydroxide in 60 ml of water.

To the stirred solution was added dropwise 14.7 g (0.086 mole) of carbon benzoxychloride below 5 ° C. The mixture was stirred for one hour at room temperature, washed with 60 ml of ether and adjusted to pH 2 with dilute HCl. The precipitate was collected by filtration, washed with water and air dried to give 9.65 g (52%) of XX. The filtrate was extracted with five 100 ml portions of ether. The ethereal solution was washed with water, dried over sodium sulfate and evaporated to dryness in vacuo to give an additional 2.0 g (11%) of XX. A total of 11.65 g of VI was crystallized from 500 ml of benzene-ethyl acetate (4: 1) to give 9.36 g (50%) of pure XX, mp 128.5-129.5 ° C. Infrared (IR) (KBr): γc = () 1745, 1690 cm +1.5 [2.9] (c 5.0, MeOH). Nuclear Magnetic Resonance Spectrum [NMR (DMSO-dg)]: δ (in ppm) 3.05-3.45 (2H, m, CH2N), 4.05 (1H, dd, -0-CH-CO-), 5.03 (2H, s, CH 2 Ar) 7.18 (1H, broad, NH), 7.36 (5H, s, ring H).

Analysis calculated for ciiHi3NO5: C, 55.23; H, 5.48; N, 5.86.

Found: C, 55.34; H, 5.49; N, 5.87.

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

9) N-hydrosuccinimide ester of L-g-benzyloxycarbonylamino-α-hydroxypropionic acid XXI.

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

10) Preparation of L-6-benzyloxycarbonylamino-q-hydroxyvaleric acid XXII.

To a solution of 400 mg (3.0 mmol) of L-5-amino-α-hydroxyvaleric acid and 250 mg (6.5 mmol) of sodium hydroxide in 25 ml of water was added dropwise 580 mg (3.3 mmol) of carbobenzoxychloride over a period of 30 minutes at 0 The mixture was stirred for one hour at 5-15 ° C, washed with 25 ml of ether, adjusted to pH 2 with hydrochloric acid and extracted with three 30 ml portions of ether. The combined ethereal solution was shaken with 10 ml of a saturated sodium chloride solution, dried over anhydrous sodium sulfate and evaporated in vacuo to give crystals which were recrystallized from benzene to give 631 mg (78%) of XXII, mp 110-111 ° C ; infrared spectrum [IR (KBr)]: 3460, 3350, 1725, 1685, 1535, 1280, 730, 690 cm -1. Nuclear magnetic resonance spectrum [NMR (acetone-dg)]: δ (in ppm) 1.70 (4H, m ) 4.14 (2H, q, J = 4.5Hz), 4.19 (1H, m), 4.82 (2H, S), 6.2 (3H, broad), 7.25 (5H, s), [a] + 5 + 1.6 (c 10, MeOH).

Analysis calculated for: ct 58.42; H.6.41; N, 5.24.

Found: C, 58.36; H, 6.50; N, 5.27.

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

11) N-hydroxysuccinimide ester of L-6-benzyloxycarbonylamino-ct-hydroxyvaleric acid XXIII.

To a stirred and cooled solution of 535 mg (2.0 mmol) of XXII and 230 mg (2.0 mmol) of N-hydroxysuccinimide in 55 ml of ethyl acetate was added 412 mg (2.0 mmol) of Ν, Ν'-dicyclohexylcarbodiimide (DCC). The mixture was stirred for 3 hours. at room temperature and filtered to remove the precipitated Ν, Ν'-dicyclohexylurea. The filtrate was evaporated in vacuo to give 780 mg (100%) of viscous syrup XXIII. IR (neat): YC = 0 1810, 1785, 1725 cm -1.

WORKING EXAMPLES.

Example 1.

6'-methylkanamycin Å (BB-K 25) by Method A.

1) To a suspension of 610 mg of LiAlH 2 in 10 ml of dry dioxane was added dropwise at 70 ° C a suspension of 618 mg of 61-N-carbobenzoxy-kanamycin A (6'-N-Cbz-kanamycin A) in 30 ml of dry dioxane. . The mixture was stirred at 70 ° C for 20 hours and then cooled to -5-0 ° C.

To the reaction mixture was carefully added 20 ml of cold water. When the addition was complete, the mixture was neutralized with 2N HCl and then evaporated to dryness under reduced pressure. The residue was washed with a large portion of EtOH to give 536 mg of the crude product which was dissolved in a small amount of water and chromatographed on a column of "CG-50" ion exchange resin (NH 2 type, 20 ml). The column was rinsed with water, 1 liter 0.1 N NH 2 OH, 600 ml 0.2 N NH 2 OH and finally 500 ml 0.5 N NH 2 OH. 10 ml fractions were collected and subjected to ninhydrin stain test, plate substrate assay (B. subtilis PCI 219) and thin layer chromatography on silica gel plate; solvent system, S-110 (CHCl3-MeOH-28% NH4OH-H2O = 1: 4: 2: 1). The fractions giving a ninhydrin positive and bioactive spot at Rf 0.50 by thin layer chromatography were combined and evaporated to dryness under reduced pressure to give 137 mg (27%) of BB-K 25, mp 183-187 ° C (dec.) .

NMR (D20): δ (ppm), 2.51 (3H, s, δ'-Ν-Ο), 4.95 (1H, d, Hz, 1 "-H), 5.01 (1H, d, 3 Hz, l) '-H).

Analysis calculated for C19 H38 N4 O4 * H2CO3: C4-4.85; H, 7.19; N, 9.99.

Found: C, 42.97; H, 7.27; N, 9.63.

2. To a suspension of 8.8 g of LiAlH 2 in 100 ml of dry dioxane was added a suspension of 9.0 g of 6'-N-Cbz-kanamycin A in 200 ml of dry dioxane and the reaction mixture was stirred for 4 days at 80 ° C. The reaction mixture was cooled to 10 ° C, treated gently with 200 ml of water and filtered to remove insoluble material. The filtrate was neutralized with N HCl and evaporated in vacuo. The residue was washed several times with EtOH and dissolved in a small amount of water. The aqueous solution was chromatographed on a column of "CG-50" (NH₂ +, 200 mL) which was washed with 100 mL of water and eluted successively with 2.0 liters of 0.1 N NH₂ OH and 1.5 liters of 0.2 N NH₂ OH. The eluate was collected in 20 ml fractions. The fractions gave a bioactive and ninhydrin positive spot at Rf 0.45 by thin layer chromatography (silica gel plate * 01101 ^ -011-28% NH4 OH- ^ O = 1: 4: 2: 1). The fractions were combined, concentrated under reduced pressure, and finally lyophilized to give 1.139 g (16%) of BB-K 25 which was identical to the substance prepared by Process A- (1J).

Example 2.

6'-N-methylkanamycin A (BB-K 25) by Method B.

To a stirred suspension of 618 mg (1 mmol) of 6'-N-Cbz-kanamycin in 30 ml of dry pyridine was added 7 ml of trimethylchlorosilane and 14 ml of hexamethyldisilazane at 70 ° C. The reaction mixture was stirred overnight at the same temperature and evaporated in vacuo . The residue was treated with dry tetrahydrofuran (THF). The insoluble material is filtered off and washed with dry THF. The filtrate and washings were combined and evaporated in vacuo to give 1.567 g of the trimethylsilylated product which was dissolved in 30 ml of dry THF. The solution was added to a suspension of 758 mg of lithium aluminum hydride in 70 ml of dry THF. The mixture was refluxed for 22 hours with stirring. After cooling to approx. At 0 ° C, the reaction mixture was gently treated with 20 ml of ice water and filtered to remove insoluble material. The filtrate was neutralized with 6N HCl and evaporated to dryness in vacuo. The residue was washed several times with ethanol, dissolved in a small amount of water and chromatographed on a column of "CG-50" (NH₂ +, 50 ml). The column was washed with 50 ml of water and eluted successively with 1 liter of 0.1 N NH 2 OH and 600 ml 0.2 N NH 2 OH. The eluate was collected in 20 ml fractions and assayed as described under Method A- (1). Fractions 47-72 which gave a ninhydrin positive and bioactive spot at Rf 0.50 by thin layer chromatography unite U9775 24 dec, evaporated under reduced pressure and lyophilized to give 258 mg (52% from 61-Cbz-kanamycin A) of BB K 25, mp 183-187 ° C.

Example 3

1-N- (L (-) - γ-amino-g-hydroxybutyryl) -6 * -N-methyl-kanamycin A (BB-K 28).

To a solution of 750 mg of 6'-N-methylkanamycin A (BB-K 25) in 30 ml of 60% aqueous tetrahydrofuran was added 525 mg of N-hydroxysuccin imide ester of N-Cbz-L-Y-amino-α-hydroxybutyric acid. The reaction mixture was hydrogenated overnight at room temperature under atmospheric pressure in the presence of 500 mg of 10% palladium-on-charcoal. The reaction mixture was filtered and evaporated under reduced pressure. The residue was dissolved in a small amount of water and adsorbed on a column of "CG-50" (NH 2+, 70 ml). The column was washed with water and rinsed successively with 850 ml of 0.1 N ammonia (glass nos. 1-43 collected in 20 ml fractions), 1450 ml 0.2 N ammonia (glass nos. 44-115 in 20 ml fractions) and finally 100 ml 0.5 N ammonia (glass no. 116-215 in 10 ml fractions). Fractions 152 - 161 exhibiting a bioactive and ninhydrin positive spot at Rf 0.17 in thin layer chromatography (silica gel, CHCl 3 -CH 2 OH-28% NH 4 OH-1 2 O = 1: 4: 2: 1) were combined. was evaporated under reduced pressure and lyophilized to give 149 mg (16%) of BB-K 28, mp 187-189 ° (dec).

Infrared [IR (KBr)]: UC = 0 1650 cm NMR (D 2 O): 2.70 ppm (3H, s, N-CH 3).

Analysis calculated for -23 ° 5 ° 5 °] 2H 2 CH 3 '2 0: C, 39.52; H, 7.03; N, 9.23.

Found: C, 39.26, 39.00; H, 6.69, 6.54; N, 9.69, 9.20.

To remove traces of BB-K 11 type isomer (3 "-N-acylated isomer), BB-K was subjected to 28 column chromatography with tetramine copper (TACu) type" Amberlite CG-50 "ion exchange resin. BB-K 28 (73 mg) was dissolved in a small amount of water and chromatographed on a column of "CG-50" (TACu type, 3 ml). The column was washed with 20 ml of water and then eluted with 100 ml of 0.2 N NH 2 OH and finally 100 ml of 1.0 N NH 2 OH The eluate was collected in 7 ml fractions and determined by ninhydrin stain test plate substrate study (B. subtillis PCI 219 and Pseudomonas aeruginosa) and thin layer chromatography on silica gel (S-110, ninhydrin). Fractions 21-24 showed a ninhydrin positive and bioactive (opposite P. aeruginosa strain) spot at Rf 0.2, which were combined and evaporated in vacuo to give 30 mg of blue powder. The blue colored residue (30 mg) was dissolved in small amount of water and adsorbed on a column of "CG-50" (NH 2+, 3 mL) which was rinsed with 20 mL of 0.2 N NH 4 OH and 200 mL of 0.5 N NH 2 OH. s in 7 ml fractions. Fractions 19-23 showing positive ninhydrin test were combined, evaporated in vacuo and lyophilized to give 20 mg of pure BB-K 28; mp 187-189 ° C (dec.).

Example 4

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

A mixture of 218 mg (0.912 mmol) of N-Cbz-L-isoserine, 163 mg (0.912 mmol) of N-hydroxy-5-norbone-2,3-dicarboximide (HONB) and 188 mg (0.912 mmol) of dicyclohexylcarbodiimide (DCC) in 10 ml of tetrahydrofuran was kept at 5 ° C overnight and then filtered. The filtrate was added to a solution of 441 mg (0.892 mmol) of 6'-N-methylkanamycin A in 20 ml of 50% aqueous tetrahydrofuran. The mixture was stirred at room temperature for 5 hours and concentrated under reduced pressure to ca.

2 ml. The concentrate was adsorbed on a column of "Amberlite CG-50" (NH 4 +, 26 ml) which was washed with 40 ml of water and eluted with 500 ml of 0.1 N NH 4 OH. The eluate was collected in 10 ml fractions. Fractions 11-16 were combined and evaporated in vacuo to give 231 mg of the N-acylated product. From the 0.3 N NH 4 OH eluate 175 mg (40%) of the starting material BB-K 25 was obtained.

To a solution of the acyl derivative in 20 ml of 50% aqueous EtOH was added 130 mg of 10% pd on charcoal and the mixture was hydrogenated under normal pressure at room temperature. The catalyst was filtered off and the filtrate was evaporated to remove the organic solvent. The resulting aqueous solution was subjected to column chromatography on "CG-50" (NH 4 +, 25 mL). The column was successively eluted with 40 ml of water, 240 ml of 0.1 N NH 2 OH, 500 ml 0.2 N NH 2 OH, 300 ml 0.4 N NH 2 OH and the eluate was collected in 10 ml fractions. The bioactive fractions were combined and evaporated in vacuo to give 127 mg of the crude product which was chromatographed on a column of "CH-50" (tetramine copper type, 4 ml) and eluted with 200 ml of 0.3 N NH 2 OH, 300 ml. 0.5 N NH 2 OH and finally with 300 ml 1 N NH 2 OH. The eluate was collected in 10 ml fractions.

Glass No. Eluant Amount Rf Remarks 4 0.3 N NH 2 OH 27 mg 0.33 inactive 19-24 0.5 N NH.OH 38 mg 0.33 inactive, probably a position isomer 50-54 1.0 N NH.OH 35 mg 0.37 desired product BB -K162.

Glass Nos. 50-54 were combined and evaporated to dryness to give 35 mg (6.8%) of the desired bioactive product BB-K 162; mp 178-185 ° C (dec.). about 1630 cm

Example 5

1-N- [L (-) - S-amino-α-hydroxyvaleryl] -61-N-methyl-kanamycin A (BB-K 163).

A mixture of 94 mg (0.353 mmol) of N-Cbz-Ly-amino-α-hydroxy-valeric acid, 64.5 mg (0.353 mmol) of HONB and 74.5 mg (0.353 mmol) of DCC in 5 ml of dry THF was kept at 4 ° C overnight. and filtered. The filtrate was added to a solution of 175 mg (0.351 mmol) of 6'-N-methylkanamycin A in 10 ml of 50% aqueous tetrahydrofuran and the mixture was stirred at room temperature for 5 hours. The reaction mixture was hydrogenated at room temperature in the presence of 70 mg of 10% palladium-on-charcoal. The catalyst was filtered off and the filtrate was evaporated to remove THF. The resulting aqueous concentrate was chromatographed on a column of "Amb.erlite CG-50" (NH 2 + type, 20 mL), which was eluted successively with water (50 mL), 0.1 N NH 4 OH (250 mL), 0.2 N NH OH (450 ml) and 0.5 N m 2 OE (400 ml).

149775 27

The eluate was collected in 10 ml fractions. The bioactive fractions were combined and evaporated to dryness to give 40 mg of the crude product, which was re-chromatographed on a column of "CG-50" (tetramine copper type 1.5 ml) and eluted with 10 ml of water and 100 ml. 0.5 N NH 2 OH. The eluate was collected in 5 ml fractions.

Glass No. Eluant Amount Rf Remarks 7-8 0.5 N NH 4 OH 5 mg 0.21 inactive 9 0.5 N NH 2 OH 9 mg 0.21, a mixture of 0.29 two components 10-14 0.5 N NH.OH 24 mg 0.29 desired product BB-K 163rd

Glasses 10-14 were combined and evaporated to dryness to give 24 mg (11%) of the desired bioactive product, BB-K 163; mp 167 - 174 ° C (dec). 1620 cm -1.

Example 6

61-N-methylkanamycin B, BB-K 140.

To a suspension of 2.0 g (3.25 mmol) of 61-N-Cbz-kanamycin B in 100 ml of dry pyridine was added 20 ml of trimethylsilyl chloride and 50 ml of hexamethyldisilazane at 70 ° C with stirring, and stirring of the mixture was continued overnight at the same temperature. The precipitate was removed by filtration and washed with dry THF. The filtrate was combined with the washings and evaporated to dryness. The oily residue was dissolved in 80 ml of dry THF. The solution was added dropwise to a stirred suspension of 4.4 g of lithium aluminum hydride in 200 ml of dry THF and the mixture was heated under reflux overnight. After cooling, the reaction mixture was treated gently 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) was dissolved in 5 ml of water and chromatographed on a column of "Amberlite CG-50" (BH +, 40 ml). The column was washed with 300 ml of water and eluted with 0.1 N ammonia. The eluate was collected with 10 ml fractions and subjected to ninhydrin stain test, plate substrate assay (B. subtilis PCI 219) and thin layer chromatography (silica gel plate, CHCl3-CH3OH-28% NH4OH-H2O = 1: 4: 2: 1). Fractions 70-134 which gave a ninhydrin positive and bioactive spot at Rf 0.47 by thin layer chromatography were combined, evaporated under reduced pressure to give 889 mg (55%) of BB-K 140; mp 177-181 ° C (dec.).

NMR (D 2 O): 2.74 ppm (3H, s, N-CH-1).

Calcd for c19 H39 N5 O3 * H2 CO3: C, 42.93; H, 7.39; N, 12.52.

Found: C, 42.93; H, 7.13; N, 11.86.

Example 7

1-N- [L (-) - γ-amino-α-hydroxybutyryl] -61-N-methyl-kanamycin B (BB-K 142).

A mixture of 253 mg (1 mmol) of N-Cbz-LY amino-α-hydroxybutyric acid, 115 mg (1 mmol) of N-hydroxysuccinimide (HOSu) and 206 mg (1 mmol) of DCC was stirred for 3 hours at 5 ° C. C and filtered to remove the precipitate which separated during the reaction. The resulting active ester solution was added to a solution of 497 mg (1 mmol) of 6'-N-methylkanamycin B (BB-K 140) in 10 ml of water and the mixture was stirred overnight at room temperature. The reaction mixture was filtered and evaporated in vacuo. The residue was dissolved in 5 ml of water and adsorbed on a column of "CG-50" ion exchange resin (NH 4 type, 45 ml) which was eluted successively with 200 ml of water, 1500 ml of 0.05 N ammonia and 500 ml of 0.3 N ammonia. The eluate was collected in 10 ml fractions.

The desired intermediate derivative was contained in fractions 58-72 which eluted with 0.05 N ammonia, while 163 mg (33%) of BB-K 140 was obtained from fractions eluted with 0.3 N ammonia.

A solution of the intermediate derivative in 20 ml of 50% THF was hydrogenated overnight under normal pressure in the presence of 30 mg of 10% palladium-on-charcoal. The reaction mixture was filtered and concentrated to ca. 4 ml. The concentrate was adsorbed on a column of "Amberlite CG-50" (NH 2 + type, 10 mL) which was washed with 150 mL water and eluted successively with 350 mL 0.05 N NH 2 OH, 300 mL 0.1 N NH 2 OH, 150 mL 299775 29 0.3 N NH 2 OH and 300 ml 0.5 N NH 2 OH. The eluate was collected in 15 ml fractions. Fractions 67 and 68 which gave a bioactive spot at Rf 0.22 on thin layer chromatography (silica gel plate), CHCl 3 -CH 2 OH a column of "CG-50" (the lower part NH 2+ type, 1ml), the upper part tetramine copper type 2ml). The column was eluted with 100 ml of 0.5 N NH 2 OH and then with 50 ml of 1.0 N H 2 OH and the eluate was collected in 5 ml fractions. Fractions 15-33 were combined and evaporated in vacuo to give 29.4 mg (5%) of the desired bioactive product, BB-K 142; mp 188-192 ° C (del.) IR (KBr): vc = Q 1640 cm

Analysis calculated for C 23 H 46 N 6 ° 12.

C, 41.55; H, 6.97; N, 11.63.

Found: C, 41.52; H, 6.72; N, 10.95.

A small sample of BB-K was heated at 100 ° C for one hour in 0.5 N NaOH to give 6'-N-methylkanamycin B and L-HABA as confirmed by TLC.

Example 8.

1-N- [L · (-) - β-amino-g-hydroxypropionyl] -61-N-methyl-kanamycin B (BB-K 148).

A stirred mixture of 120 mg (0.5 mmol) of N-Cbz-L-isoserine, 58 mg (0.5 mmol) of HOSu and 103 mg (0.5 mmol) of DCC in 5 ml of THF was kept overnight at 5 ° C. The mixture was filtered, the filtrate was added. a solution of 248 mg (0.5 mmol) of BB-K 140 in 5 ml of water and stirred overnight. After removal of THF, the aqueous solution was adsorbed on a column of "Amberlite CG-50" (NH 2 + type, 10 ml). The elution was performed with 300 ml of 0.5 N NH 2 OH followed by 300 ml of 0.1 N NH 2 CH and 10 ml fractions were collected. The desired intermediate derivative (68 mg) was obtained by evaporation of fractions 18-31 which eluted with 0.05 N NH 2 CH, while 69 mg (28%) of BB-K 140 was obtained from fractions eluted with 0.1 N ammonia.

A solution of the intermediate derivative in 10 ml of water was hydrogenated overnight in the presence of 20 mg of 10% Pd-C, and the reaction mixture was filtered and concentrated to ca. 5 ml. The concentrate was chromatographed on a column of "Amberlite CG-50" (NH 4 +, 8 mL) and eluted successively with 280 mL 0.05 N NH 2 CH, 340 mL Q. 1 N NH 2 OH, and 200 mL 0.2 N NH 2 OH. The desired bioactive component (35 mg) was obtained from fractions eluted with 0.2 N NH 2 OH. The product which still showed two to three ninhydrin positive spots was purified by re-chromatography on a column of "CG-50" (the lowest part, NH.

type, 1 ml; the upper part of tetramine copper type (2 ml), was eluted successively with 60 ml of 0.3 N NH 2 OH, 110 ml 0.5 N NH 2 OH and 100 ml 1.0 N NH 2 OH. The desired product BB-K 148 was obtained from fractions 40-50 which were eluted with 1.0 N NH 2 OH.

Yield: 8.1 mg (3%); mp 190 - 198 ° C (dec.).

IR (KBr): vc = 01630 cm -1.

Example 9

1-N- [L - (-) - S-amino-g-hydroxyvaleryl] -6'-N-methylkanamycin N

By using an equimolar amount of 6'-N-methylkanamycin B in the method of Example 5 instead of the one using 6'-N-methylkanamycin A, the title compound is obtained.

"Amberlite CG-50" is the trade name for the chromatography quality of a slightly acidic cation exchange resin of a carboxylic-polymethacrylic type.

The cupra-ammonium form of "Amberlite CG-50" is prepared as follows: to a stirred suspension of "CG-50" (NH + +) in water was added 10% cupric sulfate solution to form the copper salt of "CG-50" , which was filtered. The resin was washed several times with water, then treated with 1N NH 4 OH with stirring, filtered and washed several times with water to form a deep blue cup frame ammonium form of "CG-50".

Example 10.

Preparation of the monosulfate salt of 1- [L - (-) - Y-amino-g-hydroxybutyryl] -6'-N-methylkanamycin A or B.

One mole of 1- [L - (-) - γ-amino-α-hydroxybutyryl] -61-N-methylkanamycin A or B was dissolved in 1-3 liters of water. The solution was filtered to remove any undissolved solids. To the cooled and stirred solution was added one mole of sulfuric acid dissolved in 500 ml of water.

1A977S

31

The mixture was stirred for 30 minutes, then cold ethanol was added to the mixture until precipitation occurred. The solids were collected by filtration and determined to be the desired monosulfate salt.

Example 11.

Preparation of the disulfate salt of 1- [L - (-) - β-amino-α-hydroxypropionyl] -6'-N-methylc anamycin A or B.

35 g of 1- [L- (-) - β-amino-α-hydroxy-propionyl] -61-N-methyl-kanamycin A or B was dissolved in 125 ml of deionized water. The pH is lowered to 7-7.5 with 50% v / v sulfuric acid.

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

This combination of filtrate and washing liquid is adjusted to pH 2-2.6 with 50% v / v sulfuric acid. A large amount of carbon dioxide is developed. The solution is allowed to stand under vacuum under stirring for 20 minutes to evaporate additional carbon dioxide.

Add 8.5 g of "Darco G-60" to the degassed solution. The mixture is slurried for 0.5 hour at ambient room temperature. The carbon is removed by appropriate filtration and washed with 35 ml of deionized water. The washing liquid is added to the filtrate.

The combination of filtrate and washing liquid is adjusted to pH value 1-1.3 with 50% v / v sulfuric acid. This solution is added with vigorous stirring over a period of 10 minutes to 600,800 ml of methanol (3-4 volumes of methanol). The mixture is stirred for 5 minutes at a pH of 1 - 1.3, passed through a 100 mesh sieve, stirred for 2 minutes, and allowed to precipitate for 5 minutes. Most of the above liquid is discarded. The remaining slurry is suitably filtered, washed with 200 ml of methanol and vacuum dried at 50 ° C for 24 hours to give the desired disulfate salt.

Claims (1)

An analogous process for the preparation of 1- (N-substituted) -6'-N-methylkanamycin A or B of the formula HO-. CH₂NHCH₂ ηοΑΖ = Λ, CH-OH) -7s \ "" _ h6 / ^ -NH "R ν 'CT (IV) wherein R represents L- (-) - γ-amino-w-hydroxybutyryl, L - (-) -β-amino - α-hydroxypropionyl or L - (-) - 6-amino-α-hydroxyvaleryl, and OR represents -OH or -NH 2, or pharmaceutically acceptable acid addition salts thereof, CHARACTERIZED by sequencing A) compound of the formula HO-. ch2nh-cb3 / ΓΑ r * R UL CHo0H HO — i- H0-— I 2 ν '^ _NH2 NHz λΓί σ li 149775. 3 wherein R represents -OH or -NH2 'with an acylene agent of the formula OH OI II W-NH- (CH2) n-CH-CM (VII) wherein W represents a labile, amino blocking group in the form of / V_CH, -0- C-, CK.-COC-, o, Nf V \ = / CH ^ WKPJI Or · 6 or the residue of an acid halide or acid anhydride, M represents a group O -o-f ^ r Λ T30 'IJ-Q - Oic> -0- o