CS228516B2 - Method for the production of guanyled aminoglykosides - Google Patents

Abstract

Novel guanylated glycosides of deoxystreptamine possibly substituted by a second carbohydrate radical and/or by an amidino or an acyl radical on one of the amine groups, which are useful as anti-bacterially active antibiotics.

Description

The present invention provides guanylated aminoglycosides, processes for their preparation, pharmaceutical compositions containing these compounds, and the use of said compounds as medicaments.

As used herein, the term "guanylated" refers to substitution with at least one amidino group.

In particular, the invention provides compounds of formula I

in which

R 1 represents a hydrogen atom or a radical of formula II, IIa, IIb, IIc or IId

(people)

228316

WITH

R 2 represents a hydrogen atom or a radical of formula III or IIIa

(III)

OH

Wherein at least one of R 1 and R 2 is hydrogen,

R3 represents a hydroxy group or a radical of the formula - NHRe,

R4 and R5 either independently represent a hydrogen atom or a hydroxy group and R and R5 represent a hydrogen atom, or R4 and R5 represent a hydrogen atom and R and R5 represent a further bond,

Re represents a hydroxy group, a radical of the formula —NHRe or a radical of the formula —N (CH3) Re,

R 7 represents a hydrogen atom or a methyl group,

Re represents a hydrogen atom or an amidino group,

R 9 represents a hydrogen atom, an amidino group or a radical of formula

O — C — X— (CH2) _n —NHR8

R 10 represents a hydrogen atom or a radical of formula IV or IVa

(IV)

R 11 represents a hydrogen atom or a radical of formula V

X represents an oxygen atom or an imino group and n is an integer of 2 to 5, wherein at least one and at most two amidino groups, and their acid addition salts, are present in the molecule.

The invention also provides a process for the production of the above compounds, characterized in that an aminoglycoside containing at least one free amino group is optionally guided together with potential or protected amino groups, whereupon any potential amino groups in the compound thus obtained are optionally converted into free amino groups and removed from the protected amino groups. protecting groups.

More particularly, the invention provides a process for the preparation of compounds of the above general formula I, characterized in that the corresponding compound of the general formula VII is guanylated

in which

R ³ is hydroxy or amino,

^One R6 represents hydroxy, amino or methylamino,

R’9 - represents a hydrogen atom or the rest of the formula

O — C — X— (CH2) n — Z

Z is amino or potential amino, and

R 1, R 2, R 1, R 2, R 4, R 5, R 7, X and n are as defined above, and wherein one or more amino groups may be protected by a suitable protecting group, optionally followed. in the product obtained, all potential amino groups present are converted to free amino groups, and all protected amino groups present are deprotected.

It is well known that the reactivity of amino groups present in aminoglycosides, such as compounds of formula VII, varies within wide limits depending on their position [see, for example, T. Naito et al., J. Antibiot. 26, 297 (1973); J. J. Wright et al., J. Antibiot. 29, 714 (1976)].

To obtain the desired guanylated product on one or two particular amino groups, it is only necessary to protect amino groups having a higher reactivity than the amino groups to be guanylated. Z mole-

In accordance with the above, disubstituted products can be obtained from starting materials containing two amino groups with approximately the same reactivity. It will be appreciated that in order to guanylate the most reactive amino group, it is not necessary to protect the other amino groups, but the protection of these groups in certain circumstances minimizes the risk of obtaining undesirable by-products and thus may lead to increased yields.

The process according to the invention can be carried out in a conventional manner known for the introduction of an amidino group, as described, for example, in Houben-Weyl, Methoden der organischen Chemie, Vol. XV / 1, p. 531 et seq. Examples of suitable guanylating agents include S-alkylisothioureas, 1-pyrazolecarbboximide dihydrochloride and 1-amido-3,5-dimethylpyrazole. The reaction may be carried out in a reaction-inert solvent such as pyridine, dimethylformamide, chloroform or mixtures thereof.

Reaction temperatures are, for example, between room temperature and an elevated temperature, such as 65-80 ° C.

Suitable protecting groups for use in the process of the present invention are those known in the art to be used for this purpose, such as benzyloxycarbonyl, tert-butyloxycarbonyl or trichloroethoxycarbonyl. These groups can be introduced and cleaved in a conventional manner, for example, analogously to those described in the Examples.

Examples of potential amino groups, such as potential amino groups represented by Z in formula VII, include azido, benzyloxycarbonylamino, tert-butyloxycarbonylamino, phthalimido, and succidimido, which can be converted to the free amino group in a conventional manner as described herein.

· Starting materials of the formula VII wherein R 9 is a residue of formula ¹

O

II -C-X-Z-CCHžjn are partially new and may be prepared for example by reacting a corresponding compound of formula VII, wherein ^one R 9 is hydrogen and the amino group is protected by an appropriate protecting group, with a compound of formula

O

Y — C — X — CCH2Jn — z o = c = N-CHCHK-Z 'in which: ·

X, Z and n are as defined above and

Y represents a leaving group, such as halogen, in particular chlorine or bromine, N-succinimidoxyl, N-phthalimidoxy, p -trophedoxy, 1-benzdtriazolyloxy or imidazdlyl.

This reaction may be carried out in a conventional manner, for example in an inert solvent such as chloroform, dimethylformamide or tetlahydrofurad, at room temperature or at elevated temperature, preferably at room temperature.

The remaining starting materials are either known or can be prepared by known methods.

The resulting compounds can be isolated and purified in a conventional manner.

The compounds of formula (I) can be converted into acid addition salts in the conventional manner and vice versa.

The compounds of formula I exhibit chemotherapeutic activity. In particular, the compounds have antimicrobial activity, as evidenced by in vitro dilution series tests and in vivo tests in mice carried out with various bacterial strains such as Staphylococcus aureus, Stearylrrcus epidermis, Streptocdccus faecalis, Pseudomonas aeruginosa, Escheri-coli. , Proteus vulgaris, Proteus · mirabilis, Proteus morganii, Shigella dysenteriae, Shigella · sonnei, Shigella flexneri, Enterobacter verogenes, Edterobartel cloacal, Alcaligenes faecalis, Klebsiella aerogenes, Klebsiella salononias, Serratia marces, Serratia marces. The above efficacy was observed in vitro at concentrations between about 0.1 and 50 µg / ml and in vivo at concentrations between about 0.4 and 100 mg / kg body weight of the test animal.

Thus, the compounds of the invention may be used as medicaments, in particular as antibacterially active antibiotics.

The recommended total daily dose is from 0.1 to 2 g and is conveniently administered in divided doses of 25 to 1500 mg two to four times a day or in 1 liter form.

Accordingly, the invention also provides a method of treating diseases or infections caused by bacteria using compounds of formula I as well as the use of compounds of formula I as chemotherapeutic agents, such as bacterially active antibiotics, and their use in the therapeutic treatment of the human or animal body. .

The compounds of the present invention may be used in the form of free bases or in the form of a commercially available acid addition salt, for example, an acid addition salt.

These salts have the same potency as the free bases.

or

228316

The compounds of the invention may be admixed with conventional chemotherapeutic diluents and carriers, and optionally other excipients, in the form of tablets, capsules or injectables.

Said compositions are also within the scope of the invention.

Examples of particular classes of compounds of the invention are the following:

(a) when R = R ‘= hydrogen

R1 = a group of formula II or IIa,

R3 = guanidino

R6 = amino or methylamino

R6 = Re = R9 = hydrogen

R a, R 5 = hydrogen or a hydroxyl group or when R together with R ‘form another bond R 1 = a group of formula IIa

R3 = guanidino

R 6 = amino or methylamino

R 1 = R 5 = R 6 = R 8 = hydrogen;

or

b) when R = R * = hydrogen

R 3 = hydroxy or amino R 1, R 5 = hydrogen, hydroxy

Re = amino, methylamino Re = hydrogen

Re = the remainder of the formula

About NH

II — С — X— (CH2) _n NH — C — NH2 or when R together with R 'form another bond R2 = hydrogen

R4 = R5 = hydrogen;

or

c) when R = R ‘= hydrogen

R 3 = hydroxy, amino R 6, R 5 = hydrogen, hydroxy

Re - guanidino

R 7 = Re = R 9 = hydrogen or when R together with R ‘form another bond R 2 = hydrogen

R4 = R5 = hydrogen; and acid addition salts thereof.

Examples of preferred classes of compounds for the above use include those derived from gentamycin or kanamycin, in particular from gentamycin C 1, C 1 _and C 2 and kanamycin A, with those compounds in which one amidino group is in the 1- position, particularly preferred. 2'- or 6 '

-N-molecules or in the ω-position of the side chain, if present as a residue of Ra, and their chemotherapeutically usable acid addition salts.

Preferred individual compounds are 2'-N-amidinogentamycin C and 2'-N-C amidinogentamycin _Ia and their chemotherapeutically acceptable acid addition salts, in particular as hydrochlorides.

The invention is illustrated by the following non-limiting examples. In these cases, all temperatures are given in degrees Celsius.

The following eluents are used for chromatography:

I = chloroform-methanol-concentrated ammonia 15: 4: 1 II = chloroform - methanol - con- centered ammonia 1: 1 : 1 III = chloroform - methanol - 25% ammonia 1: 2: 2 IV = chloroform - methanol - 25% ammonia 2: 2: 1 in = chloroform - methanol - 25% ammonia 1: 1: 1 VI = chloroform - methanol - 33% ammonia 1: 2: 2 VII = chloroform - methanol - 25% ammonia 15: 4 : 1 VIII = chloroform - methanol - 25% ammonia 70: 21: 9 IX = chloroform - methanol - 33% ammonia 15: 4 : 1 X = chloroform - methanol - 25% ammonia 2: 1: 1 XI = chloroform - methanol - con-

centered ammonia 9: 1: 0.2

XII = chloroform-methanol-concentrated ammonia 17: 3: 0.4

XIII = chloroform-methanol-25% ammonia 9: 1: 0.2

XIV = chloroform-methanol-25% ammonia 17: 3: 0.4

XV = chloroform-methanol 8: 2

XVI = chloroform-methanol-33% ammonia 17: 3: 0.4.

He did

2‘-N-amidinogentamycin C1-hydrochloride

Dissolve 2,2 g, 3,6 ', 3' -tetra-N-t-butoxycarbonylgentamycin C1 and 0.73 g of 1-pyrazolecarboxamidine hydrochloride in 50 ml of pyridine, heat the solution at 75 ° for 15 hours, then concentrate to dryness. The residue is chromatographed on a silica gel column (eluent IJ) to give a product which is pure according to thin layer chromatography (eluent I) and has R ₍ = 0.37). This product is dissolved in 5 ml of trifluoroacetic acid. 200 ml of ether are added to the solution after 5 min. of228516. The precipitated trifluoroacetate is converted to the hydrochloride on a basic ion exchange resin (Amberlite IRA 400, CP-cycle) to give the desired product which is pure according to thin layer chromatography (eluent II - lower phase) (Compound No. 1).

C-13-NMR:

NH

OF

157.3 (C), \

NH2

101.7 (1)), 97.1 (1 ‘), 84.5 (6), 77.8 (4),

75.5 (5), 70.7 (4 "), 70.0 (5 '), 68.7 (5"), 67.0 (2 "), 64.3 (3"), 58.8 (6 '), 50.8 50.4, 49.9 (1.3, 2'), 35.4 (7 '), 32.1 (8'), 28.5 (2), 23.8 (4 '), 22.9 (3), 21.9 (6'), 11.0 (7 ').

Example 1 a d 2

6'-N-amidinogentamycin C _1a -hydrochloride

Dissolve 1.7 g of 1,3,2 ‘, 3 - -“-etra-N-tert -butoxycarbonylgentamycin C 1. in 20 ml of absolute pyridine, was added to 590 mg of 1-pyrazalkarboxumidinohydride and the resulting solution was heated to 75 ° C. After 2 hours the pyridine was evaporated in vacuo and the residue was chromatographed on silica gel (eluent VII).

The product thus obtained is treated with 5 ml of trifluoroacetic acid, diluted with 20 ml of ether after 7 minutes, the precipitate is dissolved in methanol and converted to the hydrochloride on an ion exchanger (Amberlite 401S, C-cycle). The product has an Rf = 0.37 (eluent III) (Compound No. 2). '

Example 3

6, -N-umidiocmanumycin A hydrochloride

0.7 gl, 3.3 "-tri-N-terc.butoyykarbonylkanamycinu and 0.26 g of 1-pyrazolkmrboymmldlnohydrochloridu was dissolved in 30 ml of pyridine and the solution was heated for 18 hours at 70 ^c C. The solvent was removed on a rotary evaporator and the residue is chromatographed on silica gel (eluent VII). An amorphous product which, according to TLC pure and has an R _f = 0.48 (solvent IV).

This product was dissolved in 10 mL of trifluoroacetic acid and 100 mL of ether was added after 7 min. The precipitate formed was dissolved in methanol and converted to the hydrochloride on an ion exchanger (Amberlite 401S, CP-cycle). The product has an R _f = 0.13 (eluent III) (compound no. 3).

He attaches

1-C _in Nmmidinogemtumycin hydrochloride -

1.88 g of 3,2 ', 6'-tri-N-tert-butoxycarbonylgentamycin C1 _a and 730 mg of 1-pyrazolecarbo-amine hydrochloride are dissolved in 30 ml of pyridine and the solution is heated at 75 ° C for 3 hours.

The solvent was evaporated in vacuo and the residue was chromatographed on sillaagel (eluent VIII). Thin layer chromatography gave pure product Rf = 0.28 (eluent - V, lower phase).

950 mg of this product are dissolved in 3 ml of trifluoroacetic acid, after 7 minutes 50 ml of ether are added, the precipitate is filtered off, dissolved in methanol and filtered through a basic ion exchange column (Amberlite IRA, 401S, CP-cycle). Evaporation of the solvent gave the desired hydrochloride as a white amorphous powder of Rf = 0.33 (eluent III) (Compound No. 4).

C-13-NMR: '.

157.8 (N — C = N), 99.4 (1), 95.7 (Γ), 82.4 (6), 78.2 (4), 751.1 (5), 70.7 (4) "), 68.2 (5"),

66.8 (2 '), 66.7 (5'), 65.0 (3 '), 51.6 (2'), 49.7 (1.3), 43.5 (6 '), 36.0 (7 "), 31.3 (2), 26.1 (4 '), 22.0 (6"), 21.4 (3').

Example 1 a d 5

N, N -mmidogemtumycline C2-hydrochloride

In an analogous manner to that of Example 4, 1.9 g of 3,2 ‘, 6yk-tri-N-tert-butoxycarbonylgentamycin Cz and 730 mg of 1-pyrimidolcarboyamidine hydrochloride are reacted with one another. A product of Rf = 0.48 (eluent V, lower phase) is obtained.

The resulting product was deprotected in analogy to Example 4 and converted to the hydrochloride. The resulting compound was pure by thin layer chromatography and had an Rf = 0.47 (eluent III) (Compound No. 5).

C-13-NMR:

157.8 (N - C = N), 99.4 (1 '), 96.3 (1'), 82.4 (6), 78.7 (4), 75.1 - (5), 70.6 (4 "), 70.0 (5 '), 68.2 (5"), - 67.0' (2 "), 66.6 (3"), 51.6, 50.5, 49.7 (6 '. 1, 3, 2'), 35.8 (7 '), 31.3 (2), 23.0 (4'), 21.9 (6 '), 21.4 (3') 13.5 (7 ').

Example 6

NN-umidiol) gentumycin C1-hydrochloride

1.95 g of 3,2 ‘, 6‘-tri-N-tert-butoxycarbonylgentamycin C1 and 730 mg of 1-pyrazolomethyl-amine hydrochloride were reacted in a manner analogous to Example 4.

228S16

After chromatography the product is obtained, which according to TLC pure and has an R _f = 0.28 (eluant V - lower phase).

The product thus obtained is then reacted further as in Example 4 to give pure hydrochloride of R ₍ = 0.43, eluent III) according to thin layer chromatography.

C-13-NMR:

157.8 (N-CN), 99.3 (1 '), 96.2 (1'), 82.4 (6), 78.4 (4), 75.1 (5), 70.6 (6) 4 "), 70.1 (5 '), 68.2 (5"), 66.6 (2 "), 64.9 (3"), 58.6 (6'), 51.6 (2 ') ), 49.7 (1.3), 35.9 (7 "), 32.1 (8 '), 31.3 (2), 23.2, 21.4 (4', 3 ', 6" 10.9 (7 ').

Example 7

1-N- (2-guanidinoethoxycarbonyl) gentamycin C _1a (base and hydrochloride)

Dissolve 1.9 g of 1-N- (2-aminoethoxycarbonyl) -3,2 ‘, 6‘, 3 '-tetra-N-tert-butoxycarbonylgentamymycin S in 60 ml of absolute pyridine and react with 0,6 g

1-pyrazolecarboxamidine hydrochloride. After heating to 75 ° C for 15 minutes, pyridine is distilled off and the residue is purified by column chromatography. An amorphous product is obtained which is pure by TLC and has an Rf = 0.39 (eluent IX).

1.3 g of the thus obtained 1-N- (2-guanidinoethoxycarbonyl) -3,2 ', 6', 3'-tetra-N-tert-butoxycarbonylgentamycin _C1a are dissolved in 5 ml of trifluoroacetic acid and the solution is poured after 7 minutes. into 50 ml of ether. The precipitate formed is filtered off with suction, dissolved in methanol and converted to the hydrochloride salt on a basic exchanger (Amberlite IRA 401S, C1-cycle). The resulting product was pure by thin layer chromatography, Rf = 0.47 (eluent VI) and optical rotation [α] D ²⁰ = 93.6 ° (c = 1.3 in water) (Compound No. 7).

C-13-NMR (free base):

158.25 (CO, C = NH), 99.03 (Г), 98.73 (1)), 84.65 (4), 81.89 (6), 75.54 (5), 72.12 (4 "), 68.53, 68.29, 67.69 (2", 5 ', 5 "), 64.57 (3"), 64.27 (CH 2), 51.80 (2'), 50.19, 49.89 (1.3), 44.37 (6 '), 41.38 (CH 2 N), 36.95 (7 "), 35.62 (2), 27.41 (4') , 24.47 (3 '), 22.38 (6').

C-13-NMR (hydrochloride):

158.07 (CO, C-NH), 98.79 (1 '), 95.56 (1'), 81.11 (6), 78.18 (4), 75.18 (5), 70, 74 (4 "), 67.81, 66.79 (5", 2 ", 5 '), 64.93 (3"), 64.27 (СШ0), 50.97 (2'), 49.83 , 49.59 (1.3),

43.47 (6 '), 41.31 (CH 2 N), 35.86 (7 "), 31.67 (2), 26.15 (4'), 21.90 21.30 (6", 3 ' ).

Example 8

1-N- (2-guanidinoethoxycarbonyl) gentamycin C1-hydrochloride

The title compound can be obtained in an analogous manner to Example 7. The product results as a pure substance by thin layer chromatography, with an R ₍ equal to 0.53 (eluent VI) and an optical rotation [α] ο ²⁰ = 96.4 ° (c = 1.2 in water) (Compound No. 8).

Example 9

1-N- (2-aminoethoxycarbonyl) -2'-N-amidinogentamycin C 1 -hydrochloride 1 N- [2- (benzyloxycarbonylamino) ethoxycarbonyl] -3,6 ', 3'-tri-N-tert-butoxycarbonylgentamycin C 1 and O, 6 g of 1-pyrazolecarboxamide hydrochloride are dissolved in 5 ml of pyridine and the solution is heated at 70 ° C for 50 hours. The solvent was removed by rotary evaporation and the residue was chromatographed on silica gel (eluent VII). Yield according to TLC pure product _Rf = 0.20 (eluent VII).

0.65 g of this product was dissolved in 5 ml of methanol, treated with 20 ml of a 20% solution of ammonium formate in methanol / water (8: 2) and 100 mg of palladium on carbon and boiled for 5 minutes. The methanolic solution is concentrated on a rotary evaporator, the residue is dissolved in 7 ml of trifluoroacetic acid and after 7 minutes 200 ml of ether are added. The precipitate was dissolved in methanol and filtered through an ion exchange column (Amberlite IRA 301S, Cl-cycle). The filtrate is concentrated to about 10 ml and 200 ml of ether is added. To give the desired product as an amorphous powder, which according to TLC pure and has an R _f = 0.34 (solvent IV) (compound no. 9).

Example 10

1-N- (2-guanidinoethoxycarbonyl) -2‘-N-amidinogentamycin Ci hydrochloride

1.38 g of 1-N- (2-aminoethoxycarbonyl) -3,6 *, 3'-tri-N-tert-butoxycarbonylgentamycin C1 and 1.38 g of pyrazolecarboxamidine hydrochloride are dissolved in 50 ml of pyridine and the solution is heated at 70 for 40 hours. Deň: 32 ° C. After evaporation of the solvent on a rotary evaporator, the residue is chromatographed on silica gel (eluent VIII). The product has an Rf ₌ 0.40 (eluent VIII).

0.83 g of this product was dissolved in 10 ml of trifluoroacetic acid and the solution was treated in an analogous manner to Example 9, to give the desired product which, according to TLC pure and has an R _f = 0.12 (eluent III ) (Compound No. 10).

Example 11

2'-N-C amidinogentamycin _Ja - hydrochloride

2.3 g

Cia and 1.16 g of 1-pyrazolecarboxamidine hydrochloride are dissolved in 50 ml of pyridine. After 50 hours of heating. at 70 ° C, the solvent was distilled off and the residue was chromatographed on silica gel (eluent VIII). TLC gave amorphous pure product with Rf = 0.47 (eluent IV).

0.52 g of 6'-N-benzyloxycarbonyl-2'-amidinogentamycin C) is dissolved in 30 ml of methanol and 10 ml of 10% aqueous acetic acid, and the mixture is hydrogenated at 20 ° C at 0 ° C in the presence of palladium black. 3 MPa. After filtering off the catalyst, the solvent was distilled off, the residue was dissolved in methanolic hydrogen chloride (pH = 3) and the solution was filtered through ion exchange columns (Amberlite IRA 401S, Cl-cycle).

The filtrate was concentrated to 5 ml and addition of ether precipitated the desired product which, according to TLC pure and has an R _f == 0.11 (solvent IV) (compound no. 11).

C-13-NMR:

NH

OF

157.4 (C), \

NH2

101.9 (1 "), 96.6 (Γ), 84.6 (6), 77.4 (4),

75.5 (5), 70.8 (4 "), 68.8 (5"), 67.1 (2 "),

66.6 (5 ‘), 64.2 (3)), 50.6 (1.2‘), 49.8 (3),

43.7 (6 ‘), 35.6 (7)), 28.6 (2), 26.9 (4‘),

22.9 (3 ‘), 21.9 (6)).

Example 12

6'-N-amidinogentamycin Cl _a

5.4 g of gentamycin C-, and 3.6 g of 1-pyrazolecarboxamidine hydrochloride are dissolved in 120 ml of chloroform and 30 ml of pyridine and the solution is heated at 70 ° C for 20 hours. After cooling, the precipitate formed is filtered off, dissolved in methanol and reprecipitated by addition of ether. This precipitate was chromatographed on silica gel using 5% aqueous acetic acid as eluent. Thin layer chromatography gave pure product identical to the product produced in Example 2, which could be converted to the hydrochloride by the procedure described in Example 2.

Example 13

2‘-N-amidinogeintamycin Ci

0.48 g of gentamycin C1 and 0.3 g of 1-pyrazolecarboxamidine hydrochloride were added. Dissolve in 5 ml of chloroform and 5 ml of pyridine, and dissolve the solution 40. The mixture was heated at reflux for 24 hours. After cooling, the precipitate formed is filtered off and chromatographed on silica gel using 5% aqueous acetic acid. Obtained by chromatography. a thin layer of pure product identical to the product prepared in Example 1, which can be converted to the hydrochloride by the procedure described in Example 1.

The required starting material is. can be prepared as follows:

A. 1,3,6 ', 3' -tetra-N-tert-butoxycarbonylgentamycin Ci (for Example 1)

8.5 g of gentamycin C1 are dissolved in 100 ml of methanol and the solution is slowly reacted with a solution of 2.9 g of the compound of formula under cooling to 40 ° C.

O

CF 3 CSC 2 H 5 in 20 mL of methanol. After 3 hours, a solution of 17.3 g of di-tert-butyl dicarbonate in 20 ml of methanol was added dropwise, after which it was maintained for 35 hours. temperature to 20 ° C, an equal volume of 3.3% ammonia was added and after 15 hours the reaction solution was evaporated to dryness on a rotary evaporator. After silica gel column chromatography (eluent XI) is obtained according to the 'tlc pure desired product, _Rf = 0.26 (eluent XII).

B. 1,3,2 ‘, 3'-Tetra-N-tert-butoxycarbonylgentamycin Cla (for example 2)

(a) 6‘-N-benzyloxycarbonylgentamycin

Dissolve 4.5 g of gentamycin C _u in 100 ml of methanol, cool to -10 ° C and slowly add dropwise a solution of 3,1 g of N-benzylcarbonyl-5-norbornene-2,3-dicarboximide in 50 ml of methanol and 10 ml of chloroform. . After 0.5 h the solvent was evaporated in vacuo and the residue was chromatographed on silica gel. The product has an R _f = 0.37 (eluent VII).

(b) 1,3,2 ‘, 3 '-tetra-N-tert-butoxycarbonylgentamycin Cia

2.3 g of 6'-N-benzyloxycarbonylgentamycin Cia are dissolved in 50 ml of methanol and 3.6 g of di-tert-butyl dicarbonate in 30 ml of methanol are added. After four hours, the reaction is complete by thin layer chromatography. The product has an R _f = 0.58 (eluent XIII).

50 ml of a 20% ammonium formate solution and 500 mg of 10% palladium on carbon are added to the solution, the reaction mixture is boiled for 3 minutes, then filtered, the solvent is evaporated off under vacuum and the residue is dissolved in water. The solution was extracted three times with ethyl acetate, the combined extracts were dried over sodium sulfate and the solvent. is evaporated in vacuo. Thin layer chromatography gave pure product Rf = 0.25 (eluent XIII).

C. 1,3,3'-tri-N-tert-butoxycarbonylkanamycin A (for Example 3)

(a) 6'-N-benzyloxycarbonyl-1,3,3'-tri-N-tert-butoxycarbonylkanamycin A is dissolved in 50 ml of a mixture of equal parts of methanol and water, and treated with 1.27 g of di-tert-butyl dicarbonate and 1 ml of triethylamine. The reaction mixture was stirred at room temperature for 20 hours, during which time a gel precipitate formed. After dilution with water, the precipitate is filtered off. The product was pure by TLC and had an Rf = 0.42 (eluent XV).

b) 1,3,3'-tri-N-tert-butoxycarbonylcanamycin A g of the product prepared. in (a) is dissolved in a mixture of methanol and 10% aqueous acetic acid (5: 1) and hydrogenated at 20 ° C at 50 psi in the presence of palladium on carbon. After filtering off the catalyst and evaporating the filtrate, pure product of Rf = 0.77 (eluent III) is obtained by thin layer chromatography.

D. 1-N- (2-aminoethoxycarbonyl) -3,2 ‘, 6‘, 3--tetra-N-tert-butoxycarbonylgentamycin Cu (for Example 7)

a) N- (2-azidoethoxycarbonyloxy) phthalimide

To a solution of 1.74 g of 2-azidoethanol in 100 ml of ethanol-free chloroform and 10 ml of pyridine was added 5.41 g of N- (chlorocarbonyl) phthalimide. The resulting solution was stirred overnight, then stirred. It is extracted with sodium bicarbonate solution, dried over sodium sulphate and the solvent is distilled off. The crystalline residue obtained can be used directly in the next step.

b) 3,2 ‘, 6‘-tri-N-tert-butoxycarbonylgentamycin Cu

The title compound is obtained in an analogous manner to that described in Procedure E, para.

and). The product melts at 215-217 ° C and has an Rf of 0.31 (eluent XIV).

c) 1-N- (2-azidoethoxycarbonyl) -3,2,6,6-tri-N-tert-butoxycarbonylgentamycin Cla

To a solution of 11.23 g of 3,2 ', 6'-tri-N-tert-butoxycarbonylgentamycin Cla in 200 ml of ethanol-free chloro is added at room temperature. A solution of 4.56 g of N- (2-azidoethofer) was added dropwise, and after 7 hours, the resulting solution was extracted with sodium bicarbonate solution, evaporated on a rotary evaporator and the amorphous residue purified by column chromatography. R _f = 0.35 (eluent XV).

d) N, N-N- (2H-4-aminoethoxy-4 ', 3', 6 ', 3', 6 ', 3'-tetra-N-tert-butoxycarbonylgentamycin Cu

To 7.3 g of 1-N- (2-azidoethoxycarbonyl) -3,2,6-tri-N-tert-butoxycarbonylgentamycin Cu, 1.85 g of di-tert-butyl dicarbonate dissolved in 20 ml of methanol is added dropwise at room temperature. After completion of the reaction [Rf = 0.45 in chloroform / methanol (10: 1)], 200 mg of 10% polycarbonate is added and the suspension is reduced with hydrogen at room temperature.

After the reduction was complete, the catalyst was filtered off and the filtrate was evaporated on a rotary evaporator. The residue was purified by silica gel column chromatography. Thin layer chromatography gave a pure product with an Rf of 0.2 (eluent XV).

E. 1- (1 H-Aminoethoxycarbonyl) -3,2,6,6,3'-tetra-N-tert-butoxycarbonylgentamycin C 2 (for Example 8)

(a) 3,2 ‘, 6‘tri-N-tert-butoxycarbonylbutyl gentamycin C2

Dissolve 232 g of gentamycin C2 and 439 g of zinc acetate hydrate in 8 liters of isopropanol. A solution of 392 g of di-tert-butyl carbonate in isopropanol is added for 1 hour and the reaction mixture is heated at 50 ° C for 20 hours. The resulting solution was evaporated on a rotary evaporator, the residue was taken up in 10 L of 2 N ammonia and the aqueous phase was extracted six times with 1.5 L of ethyl acetate each time. The combined organic phases were washed with saturated sodium bicarbonate solution, 10% sodium bisulfate solution and water, and dried over sodium sulfate. The solvent was evaporated and the residue was triturated with acetonitrile to give a crystalline product, m.p. 254-256 ° C. TLC indicated that the product was pure and had Rf = 0.34 (eluent XIV). Additional product fractions can be obtained from the mother liquors by chromatography on silica gel (eluent XIII).

b) 1-N- (2-azidoethoxycarbonyl) 1,3,2,6,6-tri-N-tert-butoxycarbonyl-gentamycin C2

Dissolve 38.2 g of 3,2 ‘, 6‘-tri-N-tert-butoxycarbonyl) gentamycin C2 in 300 ml of chloroform and slowly add dropwise to the solution at 5.6 ° C after the addition of 5.6 g of triethylamine

0 ° C solution of 7.5 g of 2-azidoethoxycarbonyl chloride in 100 ml of chloroform. After 15 hours of reaction at 20 ° C, the reaction solution was evaporated in vacuo. A colorless foam (Rf = 0.27; eluent XV) is obtained which can be used directly in the next step.

c) 1-N- (2-azidoethoxycarbonyl) -3,2 ‘, 6‘, 3 '-tetra-N-tert-butoxycarbonylgentamycin

The product obtained in (b) is dissolved in 100 ml of chloroform, diluted with methanol to a volume of 1 liter, followed by the addition of a strongly basic ion exchanger (Gauge III, OH-cycle) and then 13 g of di-tert-butyl dicarbonate. . The resulting suspension was stirred for 15 hours, then filtered and the solvent was evaporated. A colorless foamy product with R _f = 0.9 (eluent XV), which can be - used directly in the next step.

d) 1- (2-Aminoethoxycarbonyl) -3,2 ', 6', 3 '-tetra-N-tert-butoxycarbonylgentamycin C2 g of the product prepared as described in paragraph c) are dissolved in 100 ml of a solution of 200 g of ammonium formate in 200 ml. ml of water and 800 ml of methanol are added, the solution is mixed with 300 mg of 10% palladium on carbon and the mixture is refluxed for 1.5 hours. After filtration, the solvent was evaporated and the residue was chromatographed on silica gel (eluent XVI). To afford a foamy product which by thin layer chromatography and a pure R _f = 0.2 (eluent XV).

F. 1-N- [2- (Benzyloxycarbonylamino) ethoxycarbonyl] -3,6,3,3'-tri-N-tert-butoxycarbonylgentamycin Ci (from Example 9)

(a) 3,2 ‘, 6‘-tri-N-tert-butoxycarboyylgentamycin Ci

Analogous to Part E (a), an amorphous product is obtained which is pure by TLC and has an Rf = 0.3 (eluent XIV).

(b) 3,2 ‘, 6‘-tri-N-tert-butoxycarbonyl-1-N- [2- (benzyloxycarbonyl-1 amino)] ethoxycarbonyl] gentamycin C1

31.1 g of 3,2 ', 6 ' -tri-N-t-butoxycarbonynyl-gentamycin C1 and 5 g of triethylamine are dissolved in 200 ml of chloroform and slowly mixed with a solution of 15.4 g of N- [2- (benzyloxycarbonylaminog)). ethoxycarbonyloxy] phthalimide in 100 ml of chloroform. The reaction mixture was washed several times with 20% sodium bicarbonate solution and evaporated on a rotary evaporator for 20 hours at 20 ° C. The product can be purified by chromatography on silica gel [chloroform / methanol (10: 1)]. eluent - reagent]. The product obtained had an Rf = 0.42 (eluent XV).

c) 1-N- [2- (benzyloxycarbonylamiono) ethoxycarbonyl] gentamycin C1

31.1 g 3.2 ‘, 6‘ttri-N-tert-butoxyxyarnyl-

-1-N- [2- (benzyloxycarbonylamino) ethoxycarbonyl] gentamyelin C1 is dissolved in 60 ml of trifluoroacetic acid and after 10 minutes the solution is treated with 1 liter of ether. The precipitate formed is filtered off with suction, dissolved in water and chromatographed on an ion exchange column [Amberlite CG-50 (NH4 ⁺ )] using water and 0.3 N ammonia. The ninhydrin-positive fractions were pooled and lyophilized. An amorphous foam-like product is obtained which is pure according to thin-layer chromatography and has an Rf = 0.73 (eluent V).

d) 1-N- [2- (beyzyloxycarbonylamino) ethoxycarbonyl] -2‘-N-trifluoroacetylgentamycin Ci

22.6 g of 1-N- [2- (benzyloxycarbonylamino) ethoxycarbonyl] gentamycin C1 are dissolved in 1.2 liters of methanol, and the solution is slowly treated at -10 ^DEG C. with a solution of 4.1 ml of ethyl acetate. The reaction solution was allowed to stand at 20 ° C for 20 hours, the reaction being monitored by thin layer chromatography. The reaction product had an Rf = 0. 57 (eluent VII) The resulting solution can be used directly in the next step.

e) 1-N- [2- (benzyloxycarbonylamino) ethoxycarbonyl] -3,6,3,3'-tri-N-tert-butoxycarbonylgentamycin C1

The solution prepared in (d) was treated with 23 g of di-tert-butyl dicarbonate at 20 ° C and stirred for 4 hours. The reaction solution was then diluted with 1 liter of 25% ammonia and stirred at 20 ° C for 20 hours. The reaction mixture is further diluted with water, the methanol is evaporated in vacuo and the aqueous phase is extracted several times with ethyl acetate. After washing with saturated sodium bicarbonate solution and 0.5 N hydrochloric acid, the ethyl acetate solution is evaporated on a rotary evaporator and the residue is chromatographed on silica gel (eluent XIII). The product was pure by TLC and had an Rf = 0.40 (eluent XIV).

G. 1-N- (2-Aminoethoxycarbones - 3,6 ‘, 3'-tri-N-tert-butoxycarbonylbutyl cyclic C1 (for Example 10)

1.5 g of 1-N- [2- (be-l-l-l-l-l-l-l-l-l-l-l-ethoxycarbonyl) -3,6-ethoxycarbonyl] -3,6 Dissolve 3, 3'-tri-n-tert-butoxy228516 carbonylgentamycin C1 in 10 ml of methanol, add 20 ml of a 20% solution of ammonium formate in methanol / water (8: 2) and 200 mg of palladium on carbon, and the mixture was boiled for 5 minutes.

After filtration, the reaction solution is evaporated on a rotary evaporator, the residue is taken up in water and the solution is neutralized with ammonia to pH 9. Extraction with ethyl acetate gives a product which is pure by TLC and has an _Rf = 0.23 (eluent XIV). ).

Claims (4)

SUBJECT A process for the preparation of guanyl aminoglycosides of the general formula I OF THE INVENTION (III) wherein R1 is a hydrogen atom or a radical of formula II, IIa, IIb, IIc or IId, wherein at least one of R1 and R2 is a hydrogen atom, R3 represents a hydroxy group or a radical of the formula —NHRe, Rd and Re are each independently hydrogen or hydroxy and R and R 'are hydrogen, or Rd and Rs are hydrogen and R and R * are another bond, Re represents a hydroxy group, a radical of the formula —NHR8, or a radical of the formula —N (CH3) Re, R 7 represents a hydrogen atom or a methyl group, Re represents a hydrogen atom or an amidino group, R 9 represents a hydrogen atom, an amidino group or a radical of formula ^НО \ Л ~ ° WC) ( OH OH (lib) (llc) o —C — X (CH2) _n —NHRe, R 10 represents a hydrogen atom or a radical of formula IV or IVa (IId) NH. (IV) (iVaJ R 11 represents a hydrogen atom or a radical R 2 represents a hydrogen atom or a radical of formula V of formula III or IIIa X is an oxygen atom or an amino group, and n is an integer of 2 to 5, wherein at least <one and at most two amidino groups are present, and their acid addition salts, characterized in that the corresponding compound of formula VII is guanylated in which R ³ is hydroxy or amino, ^One R6 represents hydroxy, amino or methylamino, R 8 'represents a hydrogen atom or a radical of formula O II. —C-X- (CH2) n-Z, Z is amino or potential amino, and R, R ', R 1, R 2, R 1, R 5, R 2, X and n are as defined above, and wherein one or more amino groups can be protected with a suitable protecting group, whereupon any potential amino groups present are optionally converted the amino groups and any protected amino groups present are deprotected, and the resulting product of formula (I) is optionally converted by reaction with an acid to its acid addition salt. 2. The process according to item 1, for the preparation of the compounds of the above general formula I, in which, when R and R a are each hydrogen, R 1 represents a residue of the above formula II or IIa, R 5 is guanidino, R6 is amino or methylamino, R 2, R 5 and R 8 are hydrogen atoms, R 4 and R 5 are each independently hydrogen or hydroxyl and R 7 is as in point 1, or when R and R ‘represent another bond, R 1 represents a residue of the above formula IIa, R3 is guanidino, Re is amino or - methylamino, R 4, R 5, R 6 and R 9 are each hydrogen and R2, R7, R10 and R11 are as defined in item 1, and their acid addition salts, characterized in that the compounds of formula (VII) above are used as starting materials in which: R &^lt; 3 ^& gt ^; is amino, Re ¹ represents a protected amino or methylamino group, R (9) is hydrogen and the remaining 0-symbols are as defined herein. 3. The process according to item 1, for the preparation of compounds of the above general formula I, in which, when R and R R are each hydrogen, R3 is hydroxy or amino, R4 and R5 are each hydrogen or hydroxy, Re represents an amino group or a methylamine group, R 5 represents a hydrogen atom, R 9 represents a radical of formula About NH II II. - C-X- (CH 2) n NH-C-NH 2 a R 1, R 2, R 7, R 10, R 11, X and n have the meaning as in point 1, or if R and R ‘represent a further bond, then R 2, R 3 and R 5 are hydrogen atoms and their acid addition salts, characterized in that compounds of the above-mentioned general formula (VII) in which: R ³ represents - hydroxy or protected amino, Re * represents a protected amino or methylamino group, R91 is a radical of formula O -C-X- (CH 2) _n -H 2 and the remaining general symbols are as defined herein. 4. The process according to item 1, for the preparation of compounds of the above general formula I, wherein ρ '. if R and R vždy are always hydrogen, R 3 is hydroxy or amino, R 1 and R 5 are each hydrogen or hydroxy, Re is guanidino, R 7, R 8 and R 9 are hydrogen and R 1, R 2, R 7, R 10 and R 11 have the meaning as in point 1, or if R and R * represent a further bond then R 2, R 1 and R 5 are hydrogen atoms, and acid addition salts thereof, characterized in that compounds of formula (VII) above are used as starting materials in which: R &^lt; 3 ^& gt ^; is hydroxyl or protected amino, R 6 ¹ is amino, R &^lt; 9 &gt; is hydrogen and the remaining general symbols are as defined herein. Severography, n. P., Plant 7, Most